(kjsat)kjsat.com/files/current_issue.pdf6 maryam seyyedi noosh abad introduction helicobacter pylori...

Vol. 4, №1, 2020 ISSN 2520-6133

KHAZAR JOURNAL OF

SCIENCE AND TECHNOLOGY (KJSAT)

Hamlet Isayev/Isaxanli *

Editor-in-Chief

Khazar University, Azerbaijan

Editorial Board

Abdul Latif Bin

Ahmad University Sains Malaysia

Andrey Kuznetsov

North Carolina State University,

USA

Yuri Bazilevs

University of California, San Diego,

USA

Jinhu Lü

Academy of Mathematics and Systems

Science, Chinese Academy of Sciences, China

Chunhai Fan

Shanghai Institute of Applied Physics, Chinese

Academy of Sciences, China

Shaher Momani

Mutah University, Jordan

Davood Domiri Ganji

Babol Noshirvani University of Technology,

Iran

Bernd Nilius

KU Leuven, Belgium

Mehrorang Ghaedi

Yasouj University,

Iran

Asaf Salamov

Lawrence Berkeley National Laboratory,

USA

Madjid Eshaghi Gordji

Semnan University,

Iran

Kenji Takizawa

Waseda University, Japan

Tasawar Hayat

Quaid-I-Azam University, Islamabad,

Pakistan

Tayfun Tezduyar

Rice University, USA

Lei Jiang

Institute of Chemistry, Chinese Academy of

Sciences, China

T. Nejat Veziroglu

University of Miami,

USA

*Also H. Issakhanly, H. Isakhanly, H. Isakhanli, H.A.Isayev, G.A.Isayev, or G.A.Isaev due to

differences in transliteration. I use Hamlet Isayev in mathematics and science fields and Hamlet

Isakhanli/Isaxanli in humanities.

Copyright © Khazar University Press 2020

All Rights Reserved

Managing Editors

Khazar University, Azerbaijan

Oktay Gasymov Karim Gasymov

Ilham Shahmuradov Panah Muradov

Abolfazl Movafagh Irada Khalilova

Javid Ojaghi Seyyed Abolghasem Mohammadi

Mahammad Nuriyev Asaf Omarov

Rasul Moradi Rovshan Abbasov

Siala Rustamova Saida Sharifova

Mohammad Sadegh Madadi Saeed Koboli

Khazar University

41 Mehseti str., Baku AZ1096

Republic of Azerbaijan

Tel: (99412) 4217927

Website: www.kjsat.com

KHAZAR UNIVERSITY PRESS

http://www.kjsat.com/

Biological Characteristics of Schmallenberg Virus - an Overview 5

Contents

Shalala Zeynalova, Asaf Omarov

Biological Characteristics of Schmallenberg Virus - an Overview........................... 5

Aghayeva Saltanat, Mammadov Ayaz, Rolfs Arndt, Skrahina Volha,

Rasulov Elkhan, Guliyeva Elvira

Molecular Analysis of Glucose-6-Phosphate Dehydrogenase

Gene Mutations in Azerbaijan Republic .................................................................. 18

Ilaha Orujova, Ajdar Asadov, Shafiqa Alakberzade, Agil Orujov

Assessment of the Diagnostic Value of İL-10 and Lactoferrin by BIRADS

Categories in Breast Neoplasms ............................................................................. 25

Lala Huseynova, Ziba Nasibova

Molecular-Genetic Research of Early Epileptic Encephalopathy

and Cystic Fibrosis Disease in Population of Azerbaijan ....................................... 33

Ima Amani, Majid Baseri Salehi, Fahimeh Nemati Mansour

Frequency of Prevalence of Klebsiella pneumoniae in Clinical Samples and the

Evaluation of the Role of Efflux Pump in Determining Antibiotic Resistance ........ 41

Hawa Adli

Cytokines and Peri-Implant Disease ....................................................................... 65

Shadar Alizada, Rauf Guliev, Ruhangiz Mammadova,

Mohammad Zaefizadeh

System Perspective Analysis for Molecular and Genetic Source

of Salt Tolerance in Cotton...................................................................................... 70

Ramil Sadigov

Classification of Grey-Cinnamon (Chestnut) Soils with Nutrient Source of the

Shamkirchay Reservoir and Analysis of Morphogenetic Diagnostic Indices .......... 84

Masoud Mehrizadeh

Estimation of PVT Properties Using Artificial Neural Networks

and Comparison of Results with Experimental Data .............................................. 97

Information to Contributors .............................................................................. 111

Khazar Journal of Science and Technology Volume 4 №1 2020, 5-17

© Khazar University Press 2020 DOI: 10.5782/2520-6133.2020.4.1.5

5

Biological Characteristics of Schmallenberg Virus -

an Overview

Shalala Zeynalova1*, Asaf Omarov2

1Azerbaijan, Ministry of Agriculture, 3-rd Biosafety Level Central Reference Laboratory; 2Life Sciences Department, Khazar University

*Corresponding author: [email protected]

Abstract

Schmallenberg virus disease was originally reported in Germany in 2011. The new disease

was diagnosed in the UK in January 2012. Currently, little information has been collected

about the pathogen of the Schmallenberg virus. The virus genetically belongs to the

Bunyaviridae family (Orthobunyavirus genus and Simbu serogroup). The clinical

symptoms of acute SBV infection are unspecific in sheep and goats. This study

characterized the origin, emergence, transmission, spread in Europe and Azerbaijan,

clinical signs, and the diagnosis of this virus. Currently, the Shmallenberg disease is known

to be a serious threat to Veterinary Public Health.

Keywords: Schmallenberg virus, trans missive infections, spread, Azerbaijan

Introduction

Structure of the virus

Schmallenberg Virus (SBV) is an infectious disease of ruminants. The virus

belongs to the Orthobunyavirus genus of the Bunyaviridae family, the Simbu

serogroup. Besides the Orthobunyavirus genus, the Bunyaviridae family has more

than 350 viruses divided into five genera based on serological, morphological, and

biochemical characteristics: Nairovirus, Phlebovirus, Hantavirus, and Tospovirus

with additional unclassified viruses (Charles, 1994; ICTVI, 2006). Although

viruses in the Bunyaviridae family share a tripartite RNA genome in common, they

significantly differ in their geographic distribution, disease characteristics, and

transmission mode (Bishop & Beaty, 1988; Charles, 1994; Horne & Dana, 2014).

Viruses within the Orthobunyavirus, Nairovirus, and Phlebovirus genera are

transmitted by hematophagous arthropods, whereas hantaviruses are transmitted

among rodents, and tospoviruses are transmitted between plants by non-

hematophagous thrips (Horne & Dana, 2014; ICTVI, 2006).

mailto:[email protected]

6 Shalala Zeynalova, Asaf Omarov

A little information has been collected on the pathogen of the Schmallenberg virus,

at the moment. These are a single-stranded, negative-sense, tripartite RNA

genomes with viral genes on one of three segments: the negative sense large (L)

segment, coding for the RNA-dependent RNA polymerase for transcription and

replication; the negative sense or ambisense (tospovirus) medium (M) segment,

which encodes the Gn and Gc viral glycoproteins and a non-structural protein

(NSm). Surface proteins are involved in attachment, cell fusion, and

hemagglutination; therefore, they contain important determinants of virulence.

Neutralizing antibodies are directed against the epitopes of the surface

glycoproteins G1 and G2, while antibodies to the glycoprotein G2 have protective

properties (Fischer et al., 2013; Coupeau et al., 2013). The composition of virionic

lipids corresponds to the composition of the cell membranes, which is used for

culturing the virus (Hofmann et al., 2012; Martinelle et al., 2017).

Figure1. Phylogenetic relationship between the Schmallenberg virus and orthobunyaviruses

of the Simbu, Bunyamwera, and California serogroups, adapted from Hoffmann et al.

(2012).


The most famous representatives of this group are the Crimean-Congolese

hemorrhagic fever virus, the Nairobi disease virus, the Akabane disease virus, Rift

Valley fever virus (Saeed et al., 2001; Goller et al., 2012; Kirkland, 2015). A part

of the viruses of this family is susceptible to humans, and a fatal outcome is

possible. Bunyaviruses are thermosensitive, i.e. they are quickly inactivated at a

temperature of 56 ° C, die when irradiated with ultraviolet rays, sensitive to fat

solvents and detergents, acids (Yanase et al., 2012; Elliott, 2009; Sanders et al.,

2011).

Spread of Shmallenberg virus

This group of bunyaviruses is more often allocated to the African continent where

they were found, and also in the countries of Asia and Australia. However, some of

them have been circulating in Europe for several decades (Elliott, 2009; Elbers et

al., 2011; Stokes et al., 2015). The first reports of unidentified disease of dairy

cattle were obtained from farms located in the Netherlands and Germany (in the

city of Schmallenberg were discovered three dairy cows with unexplored

symptoms) in August 2011 (Tarlinton et al., 2012; De Regge et al., 2013; Wernike

et al., 2014; Berhanu et al., 2018). This new virus named after a German city

where infects ruminant artiodactyls (cattle, sheep, goats) has been detected in 7 EU

countries: Germany, the Netherlands, Great Britain, France, Luxembourg, Italy,

and Belgium (Hofmann et al., 2012; SVE, 2014; Gubbins et al., 2014; Brülisauer et

al., 2017). There is a tendency towards the territorial and quantitative spread of

infection, both in the number of dysfunctional sites and in the number of infected

animals in these countries (ProMED, 2012; Tarlinton et al., 2012; Briese et al.,

2013; Bilk et al., 2012).

According to the source, TSN FEDERAL state reporting system, as of March 14,

2012, 944 confirmed cases of Schmallenberg virus were detected in livestock

enterprises in Germany in federal lands, of which 124 cases were detected in

livestock enterprises where cattle are kept, 780 cases in sheep farms and 40 - goat-

breeding enterprises (according to publications of the Friedrich Lefler German

Institute) (Lievaart-Peterson et al., 2012; Conraths et al., 2013).

The high SBV within-flock seroprevalence (up to 98.03%) in geographic areas

having reported SBV outbreaks in late 2011 and 2012 was expected to limit the

reemergence of the virus in 2012 (Wernike et al., 2014; EFSA, 2014)


Figure 2. Geographical spread of SBV in Europe

The occurrence of Shmallenberg diseases in Azerbaijan

In 2012, there was an unexpected increase of abortions in cattle and sheep in

Azerbaijan, which was unrelated to infections with Brucella or Chlamydia. Due to

the similarities of the overall symptomology observed, the then just recently

described Schmallenberg virus was suspected as a potential cause. This

surveillance study was hence launched to determine if the Schmallenberg virus had

made it to Azerbaijan and to monitor the situation (Gubbins et al., 2014; Zeynalova

& Vatani, 2019).

2012/13 October – June 2013/14 July – June


2014/15 July - June 2015/16 July - June

2016/17 July – June 2017/18 July - January

Figure 3. Spread of Shmallenberg disease in Azerbaijan (Zeynalova et al., 2019).

The first wave of Schmallenberg virus infections was detected in 2012/2013 and

limited mainly to the southern part of the country. In the second and bigger wave in

2013/2014, cases were found throughout most of the country (Zeynalova et al.,

2019).

Susceptible animals, transmission routes

It is proved that the transmission of Bunyavirus viruses is carried out by vectors.

Most of these vectors live in Asia or Africa, which include biting midges or

mosquitoes. After the appearance of the SBV virus in 2011, biting midges of the

species Culicoides Obsoletus coming from the genus Ceratopogonid Culicoides

were identified as SBV vectors. Studies have shown that the viral genome was

present in different species of Culicoides (Culicoides dewulfi, Culicoides

chiopterus, Culicoides punctatus, etc.) (Labuda, 1991; Rasmussen et al., 2012).


The virus was isolated in the salivary glands of woodlice in Belgium. In laboratory

conditions, virus RNAs were detected in biting midges Culicoides Obseletus

collected in Denmark in 2011 (Davies & Daly, 2013; De Regge et al., 2014; SVE,

2014). The absence of β-actin mRNA in ruminants and high titers of the virus in

these samples indicate the replication of the virus in the insect organism, that is,

Culicoides punctatus biting midges are biological carriers of the Schmallenberg

disease virus (Figure 4) (Burgin et al., 2013; EMS, 2014; Collins et al., 2017).

Biting midges of the genus Culicoides are involved in the transmission of several

viruses of the Simbu serogroup. They were recognized as the main carriers of

Bluetongue virus in northern and central Europe during the outbreak in 2006

(White et al., 2017; Nick, 2017; Rossel, 2018). Some members of the Simbu

serogroup are zoonoses (Oropouche virus). Since the Schmallenberg virus has

appeared recently, the transmission of the disease to humans has not yet been

studied. There is no evidence of human morbidity, although studies conducted at

the Dutch National Institute for Health and the Environment do not exclude the

possibility of human infection with the Schmallenberg virus. At present, there are

two ways of infection of animals: First is horizontal - with bites of blood-sucking

insects of midges and mosquitoes. Second is vertically - from the mother to the

fetus during fetal development (transplacental), accordingly in pregnant animals,

infection leads to the birth of fetuses with congenital deformities (De Regge et al.,

2013; Luttikholt et al., 2014; Afonso et al., 2014; ProMED, 2014). The third route

of infection should not be ruled out - iatrogenic, by veterinary specialists during

veterinary prophylactic (vaccinations, chemotherapeutic treatments, subcutaneous,

intramuscular injections, etc.) and diagnostic measures (taking blood, scrapings).

However, no signs of horizontal transmission (from animal to animal) were found.

Many questions remain regarding the pathogenesis of SBV infection in pregnant

animals, their transmission by the embryo and / or gametes, and the dynamics of

the virus to and within the fetus (Lehmann et al., 2012; Hoffmann et al., 2012;

Briese et al., 2013; Poskin et al., 2014).

Figure 4. Culicoides punctatus


Clinical signs

Symptoms of the disease in cattle: in adult animals, fatigue, loss of appetite, fever,

diarrhea, premature birth are first observed, part of the livestock die, milk yield

decreases sharply, abortions occur in the last half of pregnancy and cases of

stillbirths (Lievaart-Peterson et al., 2012; Davies & Daly, 2013; Kauffold et al.,

2017). In sheep and goats, SBV infection remains subclinical. The nonspecific

febrile syndrome was recorded in the summer and fall of 2011 in adult dairy cows

in the Netherlands and Germany (Garigliany et al., 2012; EFSA, 2014; White et

al., 2017). The disease is more severe in sheep and goats: there is a higher

percentage of death, depletion of animals, damage to the reproductive organs in the

maternal population. Sacral cleft of the spine was observed in two SBV-positive

stillborn lambs in 2013 (De Regge et al., 2013; Brülisauer et al., 2017). In newborn

animals, blindness, dropsy of the chest and abdominal cavities, paralysis, swelling

in the subcutaneous tissue, and pathology of the lower jaw are noted. Such

offspring, as a rule, die immediately after birth, the percentage of deaths varies

from 20 to 50% in herds infected with the virus. An autopsy revealed common

malformations of the central nervous system (CNS), including hydranencephaly,

porencephaly, lissencephaly, hydrocephalus, cerebellar and cerebral hypoplasia,

and micromyelia (Tsuda et al., 2004; Hofmann et al., 2012; Van den Brom et al.,

2012; Bilk et al., 2012).

Figure 5. Clinical signs of Shmallenberg disease in the aborted fetuses


Laboratory diagnosis and prophylaxis of disease

Laboratory diagnostics

Diagnostics is carried out employing the PCR test (test - a system for determining

the nucleic acid fragments of pathogens by polymerase chain reaction (PCR) in

biological material). The test is extremely effective in identifying difficulties to

cultivate cultures, pathogens that are not cultivated, latent forms of microorganisms

in chronic and latent forms of infection. The use of PCR diagnostics is a highly

effective method for determining intracellular parasites and viruses with high

antigenic variability. The method can identify a pathogen fragment in the material

from the animal, environmental objects (soil, water, etc.) (Lehmann et al., 2012;

Veronesi et al., 2013). A significant drawback of this reaction is its high cost; the

test takes a lot of time; it is carried out only in laboratory conditions with the

appropriate equipment. DEFRA (UK Department of the Environment, Food, and

Agriculture), as well as several institutes in Europe, are looking for a simple, easy-

to-use, inexpensive test to determine the virus or its fragments. The virus itself was

found in the intestinal contents of animals, the brain of sick and dead animals,

blood samples, samples of intraperitoneal fluid, and blood-sucking insects

(Rasmussen et al., 2012; McGrath & More, 2018).

The first indirect enzyme-linked immunosorbent assay (ELISA) to detect

Shmallenberg-specific antibodies in serum or milk samples became commercially

available shortly after the emergence of SBV (Elbers et al., 2012; Burgin et al.,

2013; Berhanu et al., 2018). The most known kits are: the indirect ELISA kit for

the detection of anti-Schmallenberg virus (SBV) antibodies in ruminant serum and

plasma from multiple species, including cattle, sheep, and goats; competitive

ELISA for the detection of antibodies directed against the Schmallenberg virus

nucleoprotein in serum or plasma from multiple species (Humphries & Burr, 2012;

Van der Poel et al., 2014; Bréard et al., 2013; Poskin et al., 2015; Collins et al.,

2017).

Based on the available official data, the European Union does not consider

vaccination mandatory during the spread of the disease. Currently, there is no

information on the transmission of the virus with meat and milk and the

vaccination against the virus is not required, however the ruminants that give birth

to non-viable calves are considered as immunized. In the case of disease, the

troupe of stillborn animals and afterbirth should be disposed of in accordance with

local (national) regulations (EFSA, 2014; Zeynalova et al., 2019).

So, despite some information, the Schmallenberg disease virus has not been

sufficiently studied. It is still not clear how the virus occurs in Europe. The

https://www.ncbi.nlm.nih.gov/pubmed/?term=Poskin%20A%5BAuthor%5D&cauthor=true&cauthor_uid=26472116


evidence presented indicates that a lot of laboratory research and genetic tests are

needed.

Abbreviations

SBV – Shmallenberg Virus

TSN- Animal Disease Reporting System

RNA- Ribonucleic acid

ELISA- Enzyme-linked Immunosorbent Assay

PCR- Polymerase chain reaction

DEFRA- Department for Environment, Food and Rural Affairs

References

Afonso, A., Abrahantes, J.C., Conraths, F., Veldhuis, A., Elbers, A., Roberts, H., & et

al. (2014). The Schmallenberg virus epidemic in Europe—2011–2013. Prev Vet Med.

116:391–403.

Bilk, S., Schulze, C., Fischer, M., Beer, M., Hlinak, A., & Hoffmann, B. (2012). Organ

distribution of Schmallenberg virus RNA in malformed newborns. Vet Microbiol.

159:236–8

Bishop, D.H., & Beaty, B.J. (1988). Molecular and biochemical studies of the evolution,

infection, and transmission of insect bunyaviruses. Philos. Trans. R. Soc. Lond. Series

B Biol. Sci. 321:463–483.

Bréard, E., Lara, E., Comtet, L., & et al. (2013). Validation of a commercially available

indirect elisa using a nucleocapside recombinant protein for detection of

Schmallenberg virus antibodies. PLoS One. 8(1): e53446.

Briese, T., Calisher, C.H., & Higgs, S. (2013). Viruses of the family Bunyaviridae:

Virology. 446: 207–216.

Charles, J.A. (1994). Akabane virus. Vet Clin North Am Food Anim Pract. Nov. 10(3),

525‐54.

Berhanu, S., Gelagay, A., Endrias, Z.G., Eystein, S. & Kassahun, A. (2018).

Seroprevalence of Schmallenberg virus in dairy cattle in Ethiopia. Acta Tropica. 178:

61-67.

Brülisauer, F., Scholes, S., Caldow, G.L., Rocchi, M., Dagleish, M.P., & Chianini, F.

(2017). Role of Schmallenberg virus infection in congenital malformations in

ruminants in Scotland in spring 2017. Vet Rec. 181:341–3 British Medical Journal

Publishing Group

Burgin, L.E., Gloster, J., Sanders, C., Mellor, P.S., Gubbins, S., & Carpenter, S.

(2013). Investigating incursions of bluetongue virus using a model of longdistance

Culicoides biting midge dispersal. Transbound Emerg Dis. 60(3):263–272.


Collins, Á.B., Barrett, D.J., Doherty, M.L., McDonnell, M., & Mee, J.F. (2017).

Significant reemergence and recirculation of Schmallenberg virus in previously

exposed dairy herds in Ireland in 2016. Transbound Emerg Dis. 64:1359–63.

Collins, Á.B., Barrett, D., Doherty, M.L., Larska, M., & Mee, J.F. (2017). Post-

epidemic Schmallenberg virus circulation: parallel bovine serological and Culicoides

virological surveillance studies in Ireland (2016). BMC Vet Res 12, 234.

Conraths, F.J., Kamer, D., Teske, K., Hoffmann, B., Mettenleiter, T.C., & Beer, M.

(2013). Reemerging Schmallenberg virus infections, Germany, 2012. Emerg Infect

Dis. 19(3):513–514.

Coupeau, D., Claine, F., Wiggers, L., Kirschvink, N., & Muylkens, B. (2013). In vivo

and in vitro identification of a hypervariable region in Schmallenberg virus. J Gen

Virol. 94(6):1168–1174.

Davies, P., & Daly, J. (2013). SBV transmission. Vet Rec. 172:509–10 British Medical

Journal Publishing Group.

De Regge, N., Madder, M., Deblauwe, I., & et al. (2014). Schmallenberg virus circulation

in Culicoides in Belgium in 2012: field validation of a real time RT-PCR approach to

assess virus replication and dissemination in midges. PLoS One. 9(1): e87005

De Regge, N., van den Berg, T., Georges, L., & Cay, B. (2013). Diagnosis of

Schmallenberg virus infection in malformed lambs and calves and first indications for

virus clearance in the fetus. Vet Microbiol. 162(2–4):595–600.

EFSA- European Food Safety Authority. (2014). Schmallenberg virus: state of the art.

EFSA. (2014). Schmallenberg virus: State of art. EFSA J. 12(5): 3681.

Elbers, A.R., Loeffen, W.L., Quak, S., & et al. (2012). Seroprevalence of Schmallenberg

virus antibodies among dairy cattle, the Netherlands, winter 2011–2012. Emerg Infect

Dis. 18(7):1065–1071.

Elbers, A.R., Koenraadt, C.J., & Meiswinkel, R. (2015). Mosquitoes and Culicoides

biting midges: vector range and the influence of climate change. Rev Sci Tech.

34(1):123–37.

Elbers, A.R., Meiswinkel, R., van Weezep, E., Sloet, O. & et al. (2011). Schmallenberg

virus in Culicoides spp. biting midges, the Netherlands, 2011. Emerg Infect Dis.

19(1):106–109.

Elliott, R.M. (2009). Bunyaviruses and climate change. Clin. Microbiol. Infect. 15:510–

517.

EMS - The emergence of Schmallenberg virus across Culicoides communities and

ecosystems in Europe. 2014. Prev Vet Med. 116:360–9.

Fischer, M., Schirrmeier, H., Wernike, K., Wegelt, A., Beer, M., & Hoffmann, B.

(2013). Development of a pan-Simbu real-time reverse transcriptase PCR for the

detection of Simbu serogroup viruses and comparison with SBV diagnostic PCR

systems. Virol J. 10:327.

Fischer, M., Hoffmann, B., Goller, K.V., Hoper, D., Wernike, K., & Beer, M. (2013). A

mutation ‘hot spot’ in the Schmallenberg virus M segment. J Gen Virol. 94 (6):1161–

1167.


Garigliany, M.M., Bayrou, C., Kleijnen, D., Cassart, D., & Desmecht, D. (2012).

Schmallenberg virus in domestic cattle, Belgium, 2012. Emerg Infect Dis. 18:1512–4

Centers for Disease Control and Prevention.

Goller, K.V., Hoper, D., Schir, M.H, Mettenleiter, T.C., & Beer, M. (2012).

Schmallenberg virus as possible ancestor of Shamonda virus. Emerg Infect Dis.

18(10):1644–1646.

Goller, K., & et al. (2012). Schmallenberg disease in sheep or goats: Past, present, and

future. Vet Microbiol. 181, 147‐153.

Gubbins, S., Richardson, J., Baylis, M., Wilson, A.J., & Abrahantes, J.C. (2014).

Modelling the continental-scale spread of Schmallenberg virus in Europe: Approaches

and challenges. Prev Vet Med. 116, 404–411.

Hoffmann, B., Scheuch, M., Höper, D., & et al. (2012). Novel orthobunyavirus in cattle,

Europe, 2011. Emerg Infect Dis. 18(3):469–472.

Hofmann, B., Scheuch, M., & et al. (2012). Schmallenberg disease in sheep or goats:

Past, present and future. Vet Microbiol. 181, 147‐153.

Hofmann, B., Scheuch, M., Höper, D., Jungblut, R., Holsteg, M., Schirrmeier, H.,

Eschbaumer, M., Humphries, D. & Burr, P. (2012). Schmallenberg virus milk

antibody ELISA. Vet Rec. 171(20):511–512.

Horne, K.M., & Dana, L. (2014). Vanlandingham, Bunyavirus-Vector Interactions.

Viruses. 6(13): 4373-4397.

ICTVI - International Committee on Taxonomy of Viruses Index of viruses-

Bunyaviridae, 2006.

Kauffold, J., Vahlenkamp, T.W., Hoops, M. (2017). Schmallenberg virus in Europe-a

review.

Kirkland, P.D. (2015). Akabane virus infection. Rev Sci Tech. Aug; 34(2), 403‐410.

Labuda, M. (1991). Arthropod vectors in the evolution of bunyaviruses. Acta Virol. 98–

105.

Lehmann, K., Werner, D., Hoffmann, B., & Kampen, H. (2012). PCR identification of

culicoid biting midges (Diptera, Ceratopogonidae) of the Obsoletus complex including

putative vectors of bluetongue and Schmallenberg viruses. Parasit Vectors. 5:213.

Lievaart-Peterson, K., Luttikholt, S.J.M., van den Brom, R., & Vellema, P. (2012).

Schmallenberg virus infection in small ruminants – First review of the situation and

prospects in Northern Europe. Small Rumin Res. 106:71.

Luttikholt, S., Veldhuis, A., van den Brom, R., & et al. (2014). Risk factors for

malformations and impact on reproductive performance and mortality rates of

Schmallenberg virus in sheep flocks in the Netherlands. PLoS One. 9(6): e100135.

Martinelle, L., Poskin, A., Dal Pozzo, F., Mostin, L., Van Campe, W., Cay, A.B., & et

al. (2017). Three different routes of inoculation for experimental infection with

Schmallenberg virus in sheep. Transbound Emerg Dis. 64:305– 8.

McGrath, G., & More, S.J. (2018). O’Neill R. Hypothetical route of the introduction of

Schmallenberg virus into Ireland using two complementary analyses. Vet Rec 182,

226–226.


Nick, D.R. (2017). Akabane, Aino and Schmallenberg virus- where do we stand and what

do we know about the role of domestic ruminant hosts and culicoides vectors in virus

transmission and overwintering. Cur. Opin. Virol., 27: 15-30.

Poskin, A., Martinelle, L., Mostin, L., & et al. (2014). Dose-dependent effect of

experimental Schmallenberg virus infection in sheep. Vet J. 201(3): 419–422.

Poskin, A., Verite, S., Comtet, L., Van der Stede, Y., Cay, B., & De Regge, N. (2015).

Persistence of the protective immunity and kinetics of the isotype specific antibody

response against the viral nucleocapsid protein after experimental Schmallenberg

virus infection of sheep. Vet Res. 46:119 BioMed Central.

ProMED-mail. (2012). Schmallenberg virus – Europe (34): decline, update.

ProMED-mail. (2014). Schmallenberg virus – Europe (35): Netherlands, Germany,

bovine.

Rasmussen, L.D., Kristensen, B., Kirkeby, C., & et al. (2012). Culicoids as vectors of

Schmallenberg virus. Emerg Infect Dis. 18(7):1204–1206.

Rossi, S., Viarouge, C., Faure, E., Gilot-Fromont, E., Gache, K., Gibert, P.,

Verheyden, H., Hars, J., Klein, F., Maillard, D., & et al. (2015). Exposure of

wildlife to the Schmallenberg virus in France (2011–2014): Higher, faster, stronger

(than Bluetongue). Transbound.Emerg. Dis., 64: 354-363.

Rosselkhoznadzor/Epizootic Situation/Schmallenberg Disease. 2018

Saeed, M.F., LiL., W.H., Weaver, S.C., & Barrett, A.D. (2001). Phylogeny of the Simbu

serogroup of the genus Bunyavirus. J. Gen. Virol. 82(21): 73-81.

Sanders, C.J., Shortall, C.R., Gubbins, S., & et al. (2011). Influence of season and

meteorological parameters on flight activity of Culicoides biting midges. J Appl Ecol.

48(6):1355–1364.

Scholte, E.J., Mars, M.H., Braks, M., & et al. (2014). No evidence for the persistence of

Schmallenberg virus in overwintering mosquitoes. Med Vet Entomol. 2014; 28:110–

115.

Stokes, J.E., Baylis, M., & Duncan, J.S. (2015). Paper A freedom from disease study:

Schmallenberg virus in the south of England.

SVE- Schmallenberg virus epidemic in Europe-2011-2013. (2014). Prev Vet Med.

116:391–403.

Tarlinton, R., Daly, J., Dunham, S. & Kydd, J. (2012). Review-The challenge of

Schmallenberg virus emergence in Europe. Vet. J. 194(1): 10-18.

Tsuda, T., Yoshida, K., Ohashi, S., Yanase, T., Sueyoshi, M., Kamimura, S., Misumi,

K., Hamana, K., Sakamoto, H., & Yamakawa, M. (2004). Arthrogryposis,

hydranencephaly and cerebellar hypoplasia syndrome in neonatal calves resulting

from intrauterine infection with Aino virus. Vet. Res.35: 531-538.

Van den Brom, R., Luttikholt, S.J.M., Lievaart-Peterson, K., Peperkamp, N.H.M.T.,

Mars, M.H., van der Poel, W.H.M., & et al. (2012). Epizootic of ovine congenital

malformations associated with Schmallenberg virus infection. Tijdschr Diergeneeskd.

137:106–11.

Van der Poel, W.H., Cay, B., Zientara, S., & et al. (2014). Limited interlaboratory

comparison of Schmallenberg virus antibody detection in serum samples. Vet Rec.

2014;174(15):380.

https://www.ncbi.nlm.nih.gov/pubmed/?term=Poskin%20A%5BAuthor%5D&cauthor=true&cauthor_uid=26472116

https://www.ncbi.nlm.nih.gov/pubmed/?term=Verite%20S%5BAuthor%5D&cauthor=true&cauthor_uid=26472116

https://www.ncbi.nlm.nih.gov/pubmed/?term=Comtet%20L%5BAuthor%5D&cauthor=true&cauthor_uid=26472116

https://www.ncbi.nlm.nih.gov/pubmed/?term=Van%20der%20Stede%20Y%5BAuthor%5D&cauthor=true&cauthor_uid=26472116

https://www.ncbi.nlm.nih.gov/pubmed/?term=Cay%20B%5BAuthor%5D&cauthor=true&cauthor_uid=26472116

https://www.ncbi.nlm.nih.gov/pubmed/?term=De%20Regge%20N%5BAuthor%5D&cauthor=true&cauthor_uid=26472116


Veronesi, E., Henstock, M., Gubbins, S., & et al. (2013). Implicating Culicoides biting

midges as vectors of Schmallenberg virus using semi-quantitative RT-PCR. PLoS

One. 8 (3): 57747.

Wernike, K., Silaghi, C., Nieder, M., Pfeffer, M. and Beer, M. (2014). Dynamics of

Schmallenberg virus infection within a cattle herd in Germany in 2011. Epidemiol.

Infect. 142: 1501-1504.

White, S.M., Sanders, C.J., Shortall, C.R., & Purse, B.V. (2017). Mechanistic model for

predicting the seasonal abundance of Culicoides biting midges and the impacts of

insecticide control. Parasit Vectors. 10:162.

Yanase, T., Kato, T., & Aizawa, M. (2012). Genetic reassortment between Sathuperi and

Shamonda viruses of the genus Orthobunyavirus in nature: implications for their

genetic relationship to Schmallenberg virus. Arch Virol. 157(8):1611–1616.

Zeynalova, Sh., & M.Vatani. (2019). Schmallenberg virus in Azerbaijan 2012-2018,

Springer, Archives of Virology, 164(7):1877-1881.



18

Molecular Analysis of Glucose-6-Phosphate

Dehydrogenase Gene Mutations in Azerbaijan

Republic

Aghayeva Saltanat1*, Mammadov Ayaz1,4, Rolfs Arndt2,

Skrahina Volha2, Rasulov Elkhan3, Guliyeva Elvira1 1Genetic Resources Institute of ANAS; 2Centogene AG, Rostock, Germany;

3Azerbaijan State Advanced Training Institute for Doctors named after A.Aliyev;

4Western Caspian University *Correspondin author: [email protected]

Abstract

Genetic screening of school children in Masalli region in Azerbaijan Republic identified 23

school children with G6PD enzyme activity different deficiency (from 0 up to 60%

activity). Biochemical studies were done/performed for school children with activity

efficiency on enzyme preparations from erythrocytes. As to WHO Guidelines, enzymatic

preparations were related to the following classes: 2nd class – 13 boys, 3rd class – 6 school

boys, 4th class – 4 of them. DNA molecular analysis, isolated from blood of the index

patient, was classified as the 2nd class of G6PD enzyme deficiency and has shown the

substitution of Guanine nucleotide with Adenine in position 1178. As a result of the

mutation in protein in the position 393, substitution of amino acids Arginine with Histidine

[G6PD,1178 (G-A) Arg393His] takes place.

Keywords: G6PD, biochemical polymorphism, enzyme preparation, abnormal variant,

gene, molecular-genetic analysis, mutation, nucleotide, amino acid.

Introduction

Glucose-6-phosphate dehydrogenase (G6PD: EC 1.1.1.49) is defined with gene

high polymorphism. Enzyme has been identified more than 400 biochemical

variants and one fourth of them differ with endemicity. One part of the G6PD

enzyme abnormal variants is endemic for the certain ethnic group, and another part

is specific to another ethnic group. One group of people with enzymatic deficiency

gets hemolytic crisis from some medicines and another group of people gets from

beans (favism) with food (Fuji et al., 1984; Xu et al., 1999; Zuo et al., 1999).

Molecular Analysis of Glucose-6-Phosphate Dehydrogenase Gene Mutations

in Azerbaijan Republic 19

Biochemical variants are mainly asymptomatic clinically. Big portion of

biochemical variants results in hemolytic anemias under the influence of

chemicals. And small portion of variants leads to severe chronic non-sphericytic

anemia (Huseynova et al., 2019; Aghayeva et al., 2018; Beutler, 1998).

The G6PD enzyme gene is located on the X chromosome and is transferred from

heterozygous mother to a son. Since one of the two X-sex chromosomes is

inactivated in women, the difference is observed in the clinic of heterozygotes. In

case non-affected enzyme gene becomes inactive, the affected gene mainly appears

in erythrocytes and the clinic manifestations are the same as in male hemizygote

patients (Aghayeva et al., 2019; Boytler, 1981; Hirono et al., 1995; Zuo et al.,

1999).

According to the data of World Health Organization (1997), around 100 million of

the world population manifests G6PD enzyme deficiency (WHO, 1997; Du et al.,

1988).

For Azerbaijan Republic population, study of G6PD enzyme deficiency started in

the 70s of the last century. Studies were satisfied with only enzymatic activity

studies (Aghayeva et al., 2019; Boytler, 1981).

The last century 70s’ studies undertaken for Masalli area population revealed

G6PD enzyme activity deficiency high frequencies (20-23%). Thus, Masalli area

was not a random choice when starting our studies (Beutler, 1994; Krasnopolskaya

& Shatskaya, 1987).

The goal of our studies was to research on G6PD gene molecular genetics and

G6PD enzyme physic-chemical characteristics for the index patient from pupils

living in Masalli area chosen from people with abnormal G6PD activities.

Material and Methods

Material was collected during screening from pupils from Arabkendi, Gullutepe,

Tekle, Chakhirli, Bedalan towns of Masalli area and pupils of 7-11 grades from

Masalli downtown. As a result of screening of 276 pupils, we found 23 boys with

inherited hemizygous type of G6PD enzyme.

Material collected for biochemical studies was venous blood samples in tubes with

EDTA anticoagulant (Huseynova et al., 2019; Aghayeva et al., 2018; Beutler,

1998).

Aghayeva Saltanat, Mammadov Ayaz, Rolfs Arndt,

20 Skrahina Volha, Rasulov Elkhan, Guliyeva Elvira

G6PD enzyme activity was identified with modified fluorescence method. To make

the analysis accurate and to identify the inheritance type, we got their parents and

family members involved into the study. Totally, there were 302 blood samples

processed (Aghayeva et al., 2019; Beutler, 1998).

Purification of enzyme preparations and their classification were done according to

the WHO standardized methods (WHO, 1988; Krasnopolskaya et al., 1977;

Krasnopolskaya et al., 1985).

Results and Discussion

As a result of 23 pupils’ screening on G6PD enzyme, different proportion of

enzyme deficiency (activity 0-60%) was identified. According to the data of World

Health Organization (WHO) in 1967, G6PD enzyme activity deficiency (based on

the lack) was divided into 5 classes: the 1st class – chronic non-spherocytic

anemia; the 2nd class – acute deficiency of the enzyme (lower than 10%); the 3rd

class – enzyme medium deficiency (activity 10-60%); the 4th class – very mild

deficiency of the enzyme (60%) and the 5th class – lower range of normal

enzymatic activity.

Our studies have revealed enzyme activity which complies with the WHO 2nd, 3rd

and 4th classes: 13 persons in the 2nd class, 6 boys in the 3rd and 4 pupils in the

4th classes.

Results of the genetic screening of G6PD enzyme carried in Masalli area are

presented in Table 1. 276 male pupils and 24 their family members were checked

up in five towns. G6PD enzyme phenotypic frequencies and gene frequencies were

presented. High results were provided for Masalli town school (11.11% and 0.1111

in d.f.). The lower results were for Arabkendiand Terle town pupils (5.56% and

0.0555 in d.f.). For the total area enzymatic deficiency frequency was 8,33%, and

gene frequency was equal to 0.0833.

G6PD enzyme deficit in Masalli regional center was classified as 2, 3 and 4

classes, whereas in Gullutepe and Bedalan towns, only the 2nd class was presented,

in Arabkendi and Chakhirli - the 3rd class, and in Tekle both the 2nd and 3rd

classes were found. In Bedalan town, hemizygote inheritance type for the enzyme

was identified in F.N. index patient’s 24 family members, and 6 different male

persons were found with hemizygote inheritance type.



Table 1. Results of the G6PD enzyme genetic screening of school pupils in Masalli area

Place

Amount

of

patients

Affected

patients

Phenotypic

frequency

(%)

Gene

frequency

(in decimal

fraction)

Enzyme deficiency

based classes

Regional

center Masalli

72 8 11,11 0.1111 2 pupils – class 2

2 pupils-7 class 3

4 pupils - class 4

Gullutepe

village

38 4 10.53 0.1053 3 pupils - class 2

1 pupil - class 2

Arabkendi

village

42 3 5.56 0.0555 3 pupils - class 3

Tekle village 54 3 5.56 0.0555 2 pupils - class 2

1 pupil - class 3

Chakhirli

village

30 2 7.14 0.0714 2 pupils - class 3

Bedalan

village

40 3 7.50 0.0750 3 pupils - class 2

F.N.(index

patient) for

family

members

24 6 25.0 0.2500 6 persons – class 2

Complying to the 2nd class requirements, 24 family members of the school pupil

F.N. (index patient) from Bedalan town were screened, and 6 of them showed acute

enzyme activity deficit (lower than 10%).

Table 2 presents physic-chemical characteristics of enzyme preparations made

from blood samples of F.N. index patient and his six family members with the

enzyme deficit in Bedalan town of Masalli area.

Table 2. Mutation variant of G6PD enzyme in school pupils from Bedalan village

Var

ian

t

nam

e

G6

PD

acti

vit

y

(%)

EP

-

mo

bil

ity

Km

G6

F

mk

mo

l

2d

G6

F

uti

liza

tio

n

pH

op

tim

um

Th

erm

ost

a

bil

ity

Cli

nic

man

ifes

tati

on

Bedalan 6.0-8.0 85-90 21.3-24.5 78.6-80.0 8.0-9.0 Weak low Mild

anemia

In enzyme preparations, the PH-optimum bifase variant was noted within the limits

of the PH-optimum indicator norm (pH 7,5-8,5)

Electrophoretic mobility showed norm. All samples manifested lower than norm

indications according to Michaelis-Menten constant (Km) (21.3-24.5 mkm) for G6P



substrates. For 2dG6P substrate analogue, there was high utilization range (78,6-

80,0).

So, Bedalan variant is classified as a variant with enzyme low activity (6.0-9.0%

from norm), for G6P substrate has low range of Km binding (24.4 mkm), for 2dG6P

substrate analogue has high utilization degree (up to 80% of G6P substrate), and

manifests mild anemia.

Figure 1 presents a schematic picture of X-sex chromosome structure and G6PD

locus on it (q28).

Figure 1. Scheme of X-sex chromosome structure and G6PD locus (q28).

Gene of G6Pdenzyme is located on the long shoulder of X-sex chromosome in

subtelomer Xq28 site. The gene of the enzyme Q6FD is located in the subtelomer

Xq28 part of the long shoulder of the X-sex chromosome

Firstly, cDNA based on mRNA of G6PD enzyme was synthesized by M. Persico in

1981. In 1986, T.Takizawa created the cDNA library using liver cells cloning. The

human q6fd enzyme gene has a size of 13 kbas, consisting of 12 exons and 18

introns. The exons sizes vary from 12 bp up to 236 bp. The intron sizes vary

between 97 bp and 11 kb. The Promotor part of the gene plays the role of TATA

box prior to 20 nucleotide bases starting from the part of 202 position at

ATTAAAT 51-terminal. In the promotore part, hexonucleotide sequence

GGGCGGG is repeated 3 times, and complementary to that, CCCGCCC is

repeated 6 times. These nucleotide sequences are usually located on 12 to 400

nucleotide bases apart from sequence ATTAAAT. GGGCGGG sequence bases are

located on a distance of 70kbas from 51-terminal of intron 1. Transcription level is

equilibrated with CAAT nucleotide sequence and is located at the distance of 220

nucleotide bases at the 51-terminal rich in GC nucleotide sequences. ATTG

nucleotide sequence is located at the position 411.

In 1991, Chen studied 20114 bp sequences involving G6PD enzyme gene. Amino

acid sequences were predicted using the G6PD enzyme gene sequence.

Fusco et al. (2012) observed Alu repeat gene at non-involved 51-translation part for

3 times. 12 Alu genes are located in the biggest intron 2. Capellini and Fiorelli



(2008) identified that enzyme G6PD consists of 515 amino acid bases (Aghayeva

et al., 2018; Du et al., 1988; Hirono et al., 1995; Xu et al., 1999; Zuo et al., 1999).

In blood of F.N. (index patient), mutation of G6PD enzyme gene was identified as

a result of DNA molecular analysis. Guanine nucleotide substitution with Adenine

nucleotide was identified in position 1178. Mutation resulted in substitution of

Arginine amino acid with Histidine amino acid in position 393.

For the first time, Filosa et al. (1992) found that the substitution of Guanine with

Adenine nucleotide happens in position 1178, and this new enzyme mutation was

referred as G6PD Portici. The authors related this new G6PD enzyme mutation to

the second group according to WHO classification.

Thus, as a result of genetic screening of pupils in Masalli area, 23 male pupils were

identified with different G6PD enzyme deficit (in 0-60% range). Complying to

WHO requirements, the identified enzyme deficiencies were shared into three

classes as to their biochemical characteristics: 13 pupils –to the 2nd class, 6 pupils

– to the 3rd class, and 4 pupils – to the 4th class. Complying to the 2nd class

requirements, 24 family members of the school pupil F.N. (index patient) from

Bedalan town were screened, and 6 of them showed acute enzyme activity deficit

(lower than 10%). DNA molecular analysis of G6PD gene obtained from the blood

sample of F.N. index patient identified substitution of Guanine nucleotide with

Adenine nucleotide in position 1178. This mutation resulted in the protein with

Arginine-to-Histidine [G6PD, 1178 (G-A) Arg393His] substitution in the position

393 [G6PD, 1178 (G-A) Arg393His].

Conclusion

So, G6PD enzyme deficiency for Masalli area has shown the following values:

8.33% for phenotypic frequency and 0.0833 (d.f.) for gene frequency. According to

the WHO requirements, and according to the biochemical characteristics of the

identified enzyme deficiency, findings were related to three classes: 13 pupils to

the 2nd class, 6 pupils to the 3rd class, and 4 pupils to the 4th. Molecular analysis

of G6PD gene identified substitution of Guanine nucleotide with Adenine

nucleotide in position 1178. As a result of the mutation in protein in the position

393, there was substitution of amino acids Arginine with Histidine [G6PD, 1178

(G-A) Arg393His].

Refrences



Aghayeva, S.A., Mammadov, A.M., Mamedbeyli, А.K., & Rasulov, E.M. (2019).

Combination of two genetic disturbances: G6PD enzyme deficiency and Duchenne

muscular dystrophy in one patient. Human Genome and Health, 2nd International

Conference Tbilisi, Georgia. 21 p.

Aghayeva, S.A., Huseynova, L.S., Kichibekov, B.R., Aliyeva, K.A., & Khalilov, R.I.

(2018). Inherited metabolic disease phenylketonuria and deficiency of g6pdenzyme in a

family study. Deutscher Wissenschaftsherold, German Science Herald. No. 2, 34-36.

Beutler, E. (1998). The genetics of glucose-6-phosphatedehydrogenase deficiency.

Seminars in Hematol. 27(3), 137-164.

Beutler, E. (1994). G6PD deficiency. Blood. No. 84, 3615-3636.

Boytler, E. (1981). Violation of red blood cell metabolism and hemolytic anemia.

Medicine. 256 p.

Du, C.S., Xu, Y.K., & Wu, Q.L. (1988). Glukoze-6-phosphatedehydrogenase variants and

polymorphic frequency in Guangdog. China. Hum.Genet. 60, 385-388.

Fuji, H., Miwa, S., Takegawa, S., Takahashi, K., & et al. (1984). Two new variants of

glucoze-6-phosphatedehydrogenase found Japan. Hum. Genet. 66(2), 276-278.

Hirono, A., Ishii, A., Kere, N. & et al. (1995). Molecular analysis of glucoze-6-

phosphatedehydrogenase variants in the Solomon Islands. Amer. J. Hum. Genet. 56(5).

Huseynova, L.S., Aghayeva, S.A., Mammadova, S.N., & Mahmudova, P.A. (2019).

Molecular genetic studies of the diseases Duchenne muscular dystrophy, Phenylketonuria

and Familial Mediterranean fever in the population of the Azerbaijan Republic.

Sylwan,163(5).

Krasnopolskaya, X.D., & Shatskaya, T.L. (1987). Distribution of Gd alleles in some

ethnic group in the USSR. Hum.Genet.75(3), 258-263.

Krasnopolskaya, K.D., Shatskaya, T.A., Filippov, I.K. & et al. (1977). Genetic

heterogeneity of G6PD deficiency: study of mutant Gd- alleles in the Sheki region of the

Azerbaijan SSR. Genetics. 12(8),1454-1461.

Krasnopolskaya, K.D., Yakovlev, S.A., Smirnova, O.A., & Prytkov, A.N. (1985). The

pattern of distribution of Gd- alleles in Azerbaijan. Message IV. The frequency and

polymorphism of erythrocyte G6FD deficiency in the village of Kobe, Absheron district.

Genetics. 21(3), 487-492.

Trujillano, D., Bertoli-Avella, A.M., & Rolfs, R. (2017).Clinical exome sequencing:

results from 2819 samples reflecting 1000 families. Eur. J. Hum. Genet. 25(2), 176–182.

WHO. (1988). Scientific group. Standardization of research methods G6FD erythrocytes.

No. 366. Geneva. 48 p.

Xu, W., Westwood, B., Bartsocas, C.S., Malcorra-Azpiazu, J.J., & et al. (1999). G6PD

mutations and haplotypes in various ethnic groups. Blood. 85 (1), 257-263.

Zuo, L., Chen, E., Du, C.S., & et al. (1999). Genetic study of Chinese G6PD variants by

direct PCR sequencing. Blood. 76, 51p.

http://europepmc.org/search/?scope=fulltext&page=1&query=AUTH:%22Trujillano%20D%22

http://europepmc.org/search/?scope=fulltext&page=1&query=AUTH:%22Bertoli-Avella%20AM%22

http://europepmc.org/search/?scope=fulltext&page=1&query=AUTH:%22Rolfs%20A%22



25

Assessment of the Diagnostic Value of IL-10 and

Lactoferrin by BIRADS Categories in Breast

Neoplasms

Ilaha Orujova*, Ajdar Asadov, Shafiqa Alakberzade,

Agil Orujov Department of Biochemistry and Diagnostic radiology and radiotherapy, Azerbaijan

Medical University, Baku, Azerbaijan *Corresponding author: Ilaha Orujova

Absrtact

Breast cancer is the most common cancer among women around the world. The

real way to improve the results of treatment of breast tumours is an early, and in

some cases, preclinical diagnosis. This problem can only be solved if complex

diagnostic methods are used. The aim of our work is to determine the change in the

level of IL-10 and lactoferrin in the blood serum of patients with breast neoplasms

corresponding to the BIRADS categories. The sensitivity, specificity, diagnostic

values of these biochemical parameters and the correlation between them were also

studied. According to the results of our studies, increasing levels of BIRADS

would lead to a raise in the levels of IL-10 and lactoferrin in blood serum in

patients with breast tumours. The results showed that lactoferrin had a high

diagnostic value compared to IL-10.

Keywords: Breast tumors, IL-10, lactoferrin, BIRADS

Introduction

Breast cancer (BC) is a leader in the structure of oncological diseases and mortality

rate among female population. Survival after treatment of breast cancer, taking into

account the quality of life and the total cost of treatment, Taking the life quality

and treatment high cost into account, the survival of breast cancer treatment

directly depends on the stage of the oncological process. Over the course of

decades, X-ray mammography has been considered almost the main both screening

and refinement method in the diagnosis of breast diseases (Amiraslanov &

26 Ilaha Orujova, Ajdar Asadov, Shafiqa Alakberzade, Agil Orujov

Gaziyev, 2010). Mammography well differentiates pathological processes in the

mammary glands with a large amount of adipose tissue on the background of

involution. The age-related features of the architectonics of the mammary gland

make the results of mammography in young women with a glandular structure of

the mammary glands less convincing. To date, the possibilities of ultrasound

diagnostics in mammology are no longer disputed.

An ultrasound examination (ultrasound) of the mammary glands is a safe,

affordable and highly informative method of breast examination, which is used

both for preventive examination and for detection of various diseases of the

mammary glands in the presence of relevant complaints (Rumack et al., 2011;

Zabolotskaya & Zabolotsky, 1997). To standardize the assessment of the results of

mammography, magnetic resonance imaging (MRI) and ultrasound according to

the degree of risk of malignant tumours of the mammary gland, the classification

or BIRADS scale was proposed by the American Society of Radiology (American

College of Radiology - ACR) in the late of nineties of the last century. The

BIRADS classification is primarily aimed at facilitating interpretation of complex

diagnostic cases in detection of neoplasms and differential diagnosis with breast

cancer (American College of Radiology, 2003).

With the use of modern instrumental diagnostic methods in clinical practice, the

potential of medicine in early detection of tumours has been improved and means

of laboratory diagnostics have significantly increased in recent years.

It is known that the pathogenetic mechanisms of the onset and progression of

tumour growth are the application of protein mediators - cytokines, chemokines

and growth factors. Cytokines are secreted by both lymphoid and tumour cells,

affecting many different target cells, and the cytokine network is the most

important regulatory mechanism of intercellular interactions (Muftuoglu,1993). On

the one hand, cytokines manifest themselves as factors of tumour progression

through activating the angiogenesis and migration of tumour cells. They change

function of target cells and are involved in the mechanism of evading tumour cells

from the immune surveillance system. On the other hand, cytokines can be the

main mediators of antitumor immunity. The concentration and balance of the levels

of cytokines and their antagonists contribute to the enhancement or inhibition of

the growth of breast cancer. Changes in the relative concentration of some

cytokines can directly and indirectly stimulate breast cancer (Kazakov et al., 2015)

Currently, in science and medicine, much attention is paid to antimicrobial peptides

(AMP). The data of numerous publications devoted to the study of AMP as

molecular factors of the innate immunity system indicate that these substances

Assessment of the Diagnostic Value of IL-10 and Lactoferrin by BIRADS Categories in

Breast Neoplasms 27

have significant therapeutic potential and can be considered as candidates for the

role of not only antimicrobial drugs, but also antitumor drugs of a new type. AMP

are characterized by a variety of mechanisms of cytotoxic action, which can lead to

both necrosis and apoptosis of target cells. The basis of these effects is selective

interaction with the membranes of tumour cells, similar in a number of structural

and physiological characteristics to the membranes of microorganisms. It has also

been found that AMP can inhibit tumour growth through disrupting formation of its

vascular network. Like antimicrobial activity, the antitumor effect of AMP can be

enhanced by modulating the body's protective functions. The described properties

of AMP give hope for development of new drugs based on which they/we can

overcome the resistance of tumour cells (Balandin et al., 2016).

The aim of our work was to assess the level of cytokine İL-10 and lactoferrin (LF)

by BİRADS categories, to evaluate sensitivity, specificity and diagnostic value of

these biochemical parameters, as well as the correlation between them.

Materials and Methods

In this work, we presented the results of a study of 92 patients undergoing

examination for breast cancer at the Oncology Clinic of the Azerbaijan Medical

University for the period from 2014 to 2017. The age of patients ranged from 18 to

79 years. All patients underwent an ultrasound examination with a combination of

dopplerography and mammography. An ultrasound examination has been

performed by using the MINDRAY D70 device (China) and a mammography was

conducted by using the SIEMENS MAMMOMAT INSPIRATION device

(Germany). Out of all examined patients, 48 women were malignant and 28-benign

breast tumours. 16 apparently healthy women have been included into the control

group. All women have been divided into 4 groups according to the BIRADS

category: BIRADS1, BIRADS2, BIRADS4, BIRADS5.

In the blood of all women included in the study, serum levels of IL-10 and LF have

been studied. The concentration of the studied parameters has been determined by

enzyme-linked immunosorbent assay on a STAT Fax 303 Plus apparatus (USA)

using a reagent kit of the VEKTOR – BEST firm (Russia) to define cytokines and

Cloud-clone (China) for LF.

The ELISA method is a highly sensitive and highly specific immunodiagnostic

method, with the help of which a qualitative and quantitative determination of

various substances with the properties of antigen, hapten (defective antigen) or

antibody is carried out. The ELISA method is widely used to diagnose infectious


and non-infectious diseases in humans and animals and can also be used to confirm

the quality of biological drugs. The principle of ELISA consists in the specific

interaction of an antigen and an antibody, creation of an Ag/Ab complex and a

conjugate, and determination of a resulting complex according to the degree of

colour by using a substrate mixture.

To calculate the sensitivity, specificity and diagnostic value of tumour markers and

cytokines a “cut of point” has been determined in SOCS-20 by using ROC analysis

in advance and statistical parameters calculated in binary classification with respect

to this point in MS EXCEL-2013.

Results and discussion

IL-10 is an anti-inflammatory cytokine. During infection, it inhibits the activity of

Th1 cells, NK cells, and macrophages, all of which are required for optimal

pathogen clearance but also contribute to tissue damage. In consequence, IL-10 can

both impede pathogen clearance and ameliorate immunopathology (Couper et al.,

2008). As a result of our studies, it was revealed that in women included in the

BIRADS 1 category, the level of IL-10 ranged from 2.8-9.5 ng/ml, averaging 6.13

ng/ml (Table 1). In the BIRADS 2 group, the level of this indicator was 3.8-16.2

ng/ml, an average of 8.77 ng/ml (p <0.05). In patients included in the BIRADS 4

category, the level of IL-10 averaged 6.13 ng/ml, min and max levels were

respectively 5.4-19.1 ng/ml (p <0.001). In the BIRADS 5 category, the level of IL-

10 ranged between 7.5-23.2 ng/ml, averaging 14.78 ng/ml (p <0.001).

Table 1. The level of IL-10 in breast tumours according to BIRADS classification

According to our data, in women included in the BIRADS 2 group, in comparison

with the control group, the level of İL-10 increased 1.43 times, and in the BIRADS

N Mean

Std.

Deviation

Std.

Error

95% Confidence

Interval for Mean

Min. Max.

Lower

Bound

Upper

Bound

Il-

10

B1 16 6,13 2,20 0,55 4,96 7,30 2,8 9,5

B2 19 8,77 3,56 0,82 7,05 10,48 3,8 16,2

B4 26 10,47 4,34 0,85 8,72 12,22 5,4 19,1

B5 31 14,78 4,73 0,85 13,04 16,51 7,5 23,2

Total 92 10,82 5,09 0,53 9,76 11,87 2,8 23,2


Breast Neoplasms 29

4 and BIRADS 5 groups the level of this cytokine was even higher and increased

1.7 and 2.4 times respectively.

Figure 1. The level of IL-10 in neoplasms of the breast according to the

classifications BIRADS

Interleukin-10, which has an important coordinated role in breast carcinogenesis

(Sheikhpour & Mohiti, 2014), is an anti-inflammatory cytokine that regulates the

immune response and inhibits the pro-inflammatory functions of antigen-

presenting cells (APCs) through expression of antagonizing costimulatory

molecules. Its low expression is associated with poor survival outcome. The role of

IL-10 in breast cancer is controversial. Interleukin -10 has both pro- and anti-tumor

effects. IL-10 mRNA expression is seen in more than 50% of tumor samples. Also,

greater IL-10 protein concentrations are seen in serum of breast cancer patients

than in that of healthy individuals and this is associated with poor clinical

outcomes. Interleukin-10 promoted proliferation and metastasis of tumor cells and

inhibited T-cell proliferation and function (Li et al., 2014; Fernandez, 2006;

Sheikhpour et al., 2018).

We also determined the level of LF in the blood serum of patients with breast

neoplasms according to the BIRADS categories (Table 1). In the BIRADS group 1,

the LF level ranged from 0.9-5.8 ng/ml, averaging 3.26 ng/ml. In women included

in the BIRADS 2 category, the average level of this indicator was 5.81 ng/ml, the

interval of variation was 1.9-9.2 ng / ml. In the BIRADS 4 min and max categories,

the indicators were 5.1 and 18.8 ng / ml, respectively, the average level was 10.24

ng/ml (p<0.001). In patients included in the BIRADS 5 group, the LF value ranged

between 8.2-42.7 ng/ml, the average level was 30.84 ng/ml (Figure 2).

https://www.ncbi.nlm.nih.gov/pubmed/?term=Sheikhpour%20E%5BAuthor%5D&cauthor=true&cauthor_uid=30324115


As compared with the control group, in women included in the BIRADS 2 group,

the LF value increased 1.78 times, and in the BIRADS4 and BIRADS 5 groups the

level of this indicator increased 3.14 and 9.5 times, respectively.

Table 2. LF level in case of breast neoplasms according to BIRADS classification.

N Mean

Std.

Deviation

Std.

Error

95% Confidence

Interval for Mean

Min

imu

m

Max

imu

m

Lower

Bound

Upper

Bound

LF B1 16 3,26 1,37 0,34 2,53 3,99 0,9 5,8

B2 19 5,81 2,41 0,55 4,65 6,97 1,9 9,2

B4 26 10,24 3,66 0,72 8,76 11,72 5,1 18,8

B5 31 30,84 9,41 1,69 27,39 34,29 8,2 42,7

Total 92 15,05 12,97 1,35 12,37 17,74 0,9 42,7

Figure 2. LF level in case of breast neoplasms according to BIRADS classification

Lactoferrin (LF) has a direct effect on cell growth, due to which it can act as a

regulatory element in protecting the body from the development of tumours and

metastases. The protein has a direct effect on the growth of tumour cells, which is

confirmed by the slowdown or lack of splicing of its mRNA in cancer cells. LF

blocks transition from G1 to S stage of the cell cycle (Borzenkova et al., 2010). It

adversely affects cell proliferation, causing changes in the expression and/or

activity of important regulatory cell cycle proteins. By activating the signaling

pathway of fatty acid synthase in cancer cells, the protein induced apoptosis. In

addition, LF stimulates the production and/or activation of various immune cells


Breast Neoplasms 31

including lymphocytes and normal killer cells, and also increases the sensitivity of

target cells to lysis by killer cells (Balandin, 2016; Naidu, 2010).

In our work, we also evaluated the sensitivity, specificity, and diagnostic value of

IL-10 and LF. According to our data, the sensitivity of IL-10 in assessing the

malignancy of a breast tumour was 60.4 ± 7.1%, and specificity was 78.6 ± 7.8%.

The predictive usefulness of a positive result was 82.9 ± 6.4%, the predictive

usefulness of a negative result was 53.7 ± 7.8%. The likelihood ratio of a positive

result was 2.82 and was assessed as mediocre, and a negative result of 0.50 and

assessed as unsuitable. The total diagnostic weight of the test was 67.1 ± 5.4%.

In the course of studies, it was found that the sensitivity of the LF in assessing the

malignancy of a breast tumour was 83.3±5.4%, and the specificity was–

85.7±6.6%. The predictive usefulness of a positive result was 90.9±4.3%, the

predictive usefulness of a negative results was 75.0±7.7%. The likelihood ratio of a

positive result was 5.83 and a negative result of 0.19 and both parameters were

rated as good. The total diagnostic weight of the test was 84.2±4.2%.

Table 2. The sensitivity and specificity of the cytokines IL-10 and LF in breast

tumors

Se, % Sp, % pPV, % nPV, % LR+ LR-

IL-10 60,4±7,1 78,6±7,8 82,9±6,4 53,7±7,8 2,82 0,50

LF 83,3±5,4 85,7±6,6 90,9±4,3 75,0±7,7 5,83 0,19

It should also be noted that in the course of our studies, a statistically significant

positive correlation was observed between IL-10 and lactoferrin (ρ=0.645,

p˂0.001).

Taking into account the results of our study we can conclude that these data may

serve in the diagnosis and monitoring of breast cancer treatment.

References

Amiraslanov, A.T. (2010). Oncology. A.T Amiraslanov, A.Y.Gaziyev, Baku: "Education",

912 p.

American College of Radiology. (2003). BI-RADS Breast imaging reporting abd data

system. Breast imaging atlas: mammography, breast ultrasound, breast MR-imaging.

Virginia. Reston. 268 p.

Balandin, S.V., Emelyanova, A.A., Kalashnikova, M.B., Kokryakov, V.N., Shamova,

O.V., & Ovchinnikova, T.V. (2016). Molecular mechanisms of anti-tumor action of

natural antimicrobic peptides. Bioorganic chemistry.42(6), 633–648.


Borzenkova, N.V., Balabushevich, N.G., & Larionova, N.I. (2010). Lactoferrin: Physico-

chemical properties, biological functions, delivery systems, drugs and biologically

active additives. Biopharmaceutical journal. 2(3).

Carol, M.R, Stephanie, R., Wilson, J., William, C., & Deborah, L. (2011). Diagnostic

Ultrasound, Elsevier/Mosby. 2031.

Couper, K.N., Blount, D.G., & Riley, E.M. (2008) IL-10: The master regulator of

Immunity of infection. J. Immunol. 180 (9), 5771-5777.

Fernandez, L., Alverez, G.R., Carmen, A.P.M., & Alcocer-Gonzalez, J. (2006).

Relationship between IL-10 and tumor markers in breast cancer patients. Breast.

15:482-489.

Muftuoglu, E. (1993). Cytokines. Immunology, Izmir: Saray Medical Bookstores, 77-97.

Kazakov, O.V., Orlov, N.B., Poveshenko, O.V., Kim, I.I., & Bondarenko, N.A. (2015).

Serum cytokines as markers of oncogenesis and treatment efficacy in experimental rat

mammary tumors. Basic research. 1 (8), 1664-1670.

Li, Y., Yu, H., Jiao, S., &Yang, J. (2014). Prognostic value of IL-10 expression in tumor

tissues of breast cancer patients. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 30(5):517-

20. [PubMed].

Naidu, A.S. (2000). Lactoferrin: natural, multifunctional, antimicrobial. Florida: CRC

Press, 86 p.

Sheikhpour, E., Noorbakhsh, P., Foroughi, E., Soudabeh, F., Nasiri, R.,

& Neamatzadeh, H. (2018). A Survey on the Role of Interleukin-10 in Breast

Cancer: A Narrative Rep Biochem Mol Biol. 7(1): 30–37.

Sheikhpour, R., & Mohiti, J. (2014). The effect of progesterone on p53 in T47D cell line.

Urmia J Med Sci. 25(10), 954-960.

Zabolotskaya, N.V., & Zabolotsky, V.S. (1997). Ultrasound mammography. Training

atlas. M. Strom,102 p.

https://www.google.az/search?hl=ru&tbo=p&tbm=bks&q=inauthor:%22Carol+M.+Rumack%22

https://www.google.az/search?hl=ru&tbo=p&tbm=bks&q=inauthor:%22Stephanie+R.+Wilson%22

https://www.google.az/search?hl=ru&tbo=p&tbm=bks&q=inauthor:%22J.+William+Charboneau%22

https://www.google.az/search?hl=ru&tbo=p&tbm=bks&q=inauthor:%22Deborah+Levine%22

https://www.ncbi.nlm.nih.gov/pubmed/24796749

https://www.ncbi.nlm.nih.gov/pubmed/?term=Sheikhpour%20E%5BAuthor%5D&cauthor=true&cauthor_uid=30324115

https://www.ncbi.nlm.nih.gov/pubmed/?term=Noorbakhsh%20P%5BAuthor%5D&cauthor=true&cauthor_uid=30324115

https://www.ncbi.nlm.nih.gov/pubmed/?term=Foroughi%20E%5BAuthor%5D&cauthor=true&cauthor_uid=30324115

https://www.ncbi.nlm.nih.gov/pubmed/?term=Nasiri%20R%5BAuthor%5D&cauthor=true&cauthor_uid=30324115

https://www.ncbi.nlm.nih.gov/pubmed/?term=Neamatzadeh%20H%5BAuthor%5D&cauthor=true&cauthor_uid=30324115

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6175593/



33

Molecular-Genetic Research of Early Epileptic

Encephalopathy and Cystic Fibrosis Disease in

Population of Azerbaijan

Lala Huseynova1*, Ziba Nasibova2

1Azerbaijan Medical University 2Genetic Resources Institute of NAS

Corresponding author: [email protected]

Abstract

The purpose of our research was to conduct an analysis of an Azerbaijani patient diagnosed

with early epileptic encephalogatia for the first time using modern molecular genetic

methods, to study the genetics of the disease. In order to research of molecular genetic

study of early epileptic encephalopathy, we screened the SPTAM1 gene among children

with similar diagnoses. For the first time in a patient diagnosed with epileptic

encephalopathy, this associated identity of SPTAN1 has been identified, with no indication

that the mutation has been localized. Among the new alcoholic diseases in the Republic of

Azerbaijan are the centers for the treatment of early epileptic encephalopathy and health

disease SPTAM1 gene screening. Five mutations for CFTR gene for the first time was

identified in Azerbaijan population. They are as follows: Phe508del, 965,(T>C), 1000

(G>T), 1210-1211 (T>G) and 328 (G>C). Gene frequencies were equal to: Phe508del

(68,75%), in two 965,T>C (12,5%) and in each of – 1000 G>T (6,25%), 1210-1211,T>G

(6,25%) and 328,G>C (6,25%). We were first to describe mutation 965, T>C (Leu322Pro).

Among children under study, cystic fibrosis diagnosed kids’ amount consisted 5,37%.

Keywords: inherited diseases, mutation, gene, protein, nucleotide, amino acid

Introduction

Epileptic encephalopathies (EE) are a group of progressing diseases of different

etiology which is expressed as neurocognitive deficit and epileptiform activity on

the electroencephalogram. EE make 15% of all epilepsy forms in childhood age

and up to 40% of all epileptic onsets in their first 3 years of life (Zhu et al., 2017).

10 syndromic forms of EE are outlined (Saitsu et al., 2010; Stabach & Morrow,

1997). Genetic factors play special role in pathologies development in around 70-

80% patients and not less than 40% of all idiopathic epilepsies have got monogenic


https://pubmed.ncbi.nlm.nih.gov/?term=Zhu+X&cauthor_id=29186148

34 Lala Huseynova, Ziba Nasibova

nature (Writzl et al., 2012). 35 genes responsible for EE occurrence are identified

and the search is still continued. Severe genetic heterogeneity of early EEs is

showed, 16 of which are inherited autosome-dominant, 13 –autosome –recessive, 4

– X-linked recessive and 2 – X-linked dominant (Hamdan et al., 2012; Nonoda et

al., 2013).

Goal of our researches is modern molecular genetic diagnostics study of one

patient with epileptic encephalopathy diagnosis from Azerbaijani family.

As to WHO information, there more than 6000 inherited diseases that have been

already studied. The major part of identified genetic diseases consist of monogene

natured ones. It is understood from their names that one gene is mutated and causes

monogene natured inherited disease. There are around 5000 monogene inherited

diseases (Trujillano et al., 2017; Riordan et al., 1989).

Cystic fibrosis is the widest spread monogene natured inherited disease. Cystic

fibrosis is autosome recessive inritance type disease (Bailey, 2013).

Frequency of the disease (homozyous form) is 1:2500-5000 in newborns.

Heterozygous carriers are born as 1:25-30 (McKusick, 2002).

In 80’s of the last century, gene (CFTR) structure of cystic fibrosis disease was

identified and studied by means of molecular-genetic methods. Gene CFTR locates

on the long arm of the seventh chromosome (7q31). The size of the gene is 190 kb

and covers 27 exons. CFTR gene takes part in synthesis of transmembrane protein

sized 170 kDa (Bailey, 2013).

For now more than 700 mutations of CFTR gene have been identified, and most of

them are rarely encounted. Inheritance type for cystic fibrosis is autosome-

recessive (Vissers et al., 2016).

Pathogenic variants in the CFTR gene are causative for cystic fibrosis, an

autosomal recessive disorder. Cystic fibrosis is a multisystem disease affecting

epithelia of the respiratory tract, exocrine pancreas, intestine, hepatobiliary system,

and exocrine sweat glands. Morbidities include progressive obstructive lung

disease with bronchiectasis, frequent hospitalizations for pulmonary disease,

pancreatic insufficiency and malnutrition, recurrent sinusitis and bronchitis, and

male infertility. Pulmonary disease is the major cause of morbidity and mortality in

cystic fibrosis. Meconium ileus occurs at birth in 15%-20% of newborns with

cystic fibrosis. More than 95% of males with cystic fibrosis are infertile (OMIM®:

219700; GeneReviews - PMID: 20301428) (Bailey, 2013).


Molecular-Genetic Research of Early Epileptic Encephalopathy and

Cystic Fibrosis Disease in Population of Azerbaijan 35

70% of all identified mutations of CFTR gene present one and the same mutation.

In exon 11 in place of protein biosynthesis the mutation between nucleotides 1521-

1523 causes deletion of Phenialanine in position 508. Since the synthesized protein

is not structurally normal, it cannot come out from endomplazmatic reticulum and,

changes are observed in its tolerance and activity (Riordan et al., 1989).

All types of mutations of CFTR gene are identified as point mutations, manor and

major deletions, transversions, inversions et cetera. Around half of all identified

mutations are missense mutations (Nonoda et al., 2013; Riordan et al., 1989).

The following mutations consist 76% of CFTR gene mutations: del121kb, delf508,

del1501507, 1677delTA, 2143delT, 2184insA, 394delTT, 3821delT, G542X,

W1282X, N1303K, L138ins, R334W and 3849+10kb C→T (Rose & Uhl, 2010;

Stabach & Morrow, 1997).

Altogether, protein synthesized by CFTR gene regulates chloride channels’

activities which are located in apical membranes of epithelial cells. Disease

damages function of lungs and pancreas. Diagnostic sign is increase of clorides and

sodium values in kid’s sweat. Only 2% of patients have got normal values of

chlorides in sweat with tipical features of the disease. In these cases, molecular-

genetic methods are used to identify mutation in CFTR gene (McKusick, 2002).

CFTR gene mutations causing cystic fibrosis inherited disease have not been

identified for Azerbaijan Republic population.

Thus, we put up a goal to study CFTR gene mutations for population of Azerbaijan

Republic, using molecular-genetic method complex.

Materıal and Methods

A child born in 2019 was diagnosed with early epileptic encephalogatia at the

Azerbaijan Medical University's Teaching Therapeutic Clinic. During the

compilation of the genealogy, it was determined that the patient's parents, as well

as his parents, were in the marriage of close relatives. Thus, the fathers of the

patient's parents are brothers. Genetic analysis of the patient and his parents was

performed. For analysis, venous blood was soaked in three different DBS cards

(Dry blood spot), and after drying, it was sent to the German CENTEGENE

laboratory in a special envelope for genetic analysis. Direct sequencing of the

SPTAN1 gene was performed by the Sanger method. Using the method, it was

possible to test an existing mutation within the SPTAN1 gene. The Whole Exome


Sequencing method (CentoXome®) was used. The method was developed in

CENTOGENE laboratory (Rostock, Germany).

Missens mutation of the SPTAN1 gene in a 9-month-old child diagnosed with early

epileptic encephalopathy using a combination of modern molecular genetic

methods: quanine nucleotide replacement with adenine nucleotide has been

identified in gene 2908 (SPTAN1 2908G> A). As a result of the mutation, the

amino acid glutamine was replaced by the amino acid lysine at position 970 of the

protein (Glu970Lys). According to the recommendations of Centogene Laboratory

and ACMG (American College of Medical Genetics), the mutation was classified

as grade 3 according to the degree of importance.

Examination has been provided for 149 children-patients who applied to Scientific

Research Pediatry Institute under Ministry of Health, Republic Children’s Hospital

and polyclinic departments in the different areas. For every patient 1 ml venouz

blood has been sampled into a tube with EDTA anticoagulant solution. Later on it

was absorbed to special DBS (dried blood spots) cards and dried up for an hour at

room temperature, only then has been sent to the laboratory for further analysis.

Study has been carried out at “Laboratory science” Chair atthe Azerbaijan State

Doctors’ Advanced Training Institute after A.Aliyev and CENTOGENE laboratory

in Rostock, Germany.

A part of analysis were tested on ROTOR-GENE apparatus. To do that, a panel of

6 mutations of CFTR gene: delF508, W1282X, N1303K, delT2143, 3849+10kb

C→T və del2,3-21kb) was used.

The different part of analyses havs been carried out by means of fluorimetric

methods, liquid chromatography and mass-spectrometry.

Results

Missens mutation of the SPTAN1 gene in a 9-month-old child diagnosed with early

epileptic encephalopathy using a combination of modern molecular genetic

methods: quanine nucleotide replacement with adenine nucleotide has been

identified in gene 2908 (SPTAN1 2908G> A). As a result of the mutation, the

amino acid glutamine was replaced by the amino acid lysine at position 970 of the

protein (Glu970Lys). According to the recommendations of Centogene Laboratory

and ACMG (American College of Medical Genetics), the mutation was classified

as grade 3 according to the degree of importance.



The following table presents results of 8 cystic fibrosis patients identified during

149 children patients were tested for CFTR gene mutations.

Patient F.A. is double heterozygote according to two different mutations

(compound form). The first nucleotide substitution has taken place in the exon 4 of

CFTR gene in position 328. Guanine is changed with Cytosine. As a result of this

mutation, protein synthesized changes in position 110 Asparagine aminoacid with

Histedine aminoacid (328 G>C, 110 Asp>His).

The second change in CFTR gene happened in the exon 8. In the position 1000of

the exon Cytosine nucleotide was substituted with Thymine nucleotide, and in the

result protein biosynthesized got change of Arginine amnoacid with Triptophan

aminoacid in position 334 (1000 C>T, 334 Arg>Trp). In father of the patient F.A.

328 G>C (110 Asp>His) CFTR gene mutation was found in heterozyous state, in

his mother - 1000 C>T (334 Arg>Trp) mutation also in heterozygous state.

Five patients - D.S., A.T., A.N., R.F. and B.A. had got the same deletion in exon

11 of CFTR gene genin between 1221-1223 nucleotides in hetero- and

homozygogous form. As a result of the mutation, biosynthesis of protein shows

deletion of Phenylalanine aminoacid (Phe508del).

B.D. patienthas got homozygous form of CFTR gene mutation as a result of change

of Thymine nucleotide with Cytosine nucleotide in position 965 (965, T>C/965, T

C). During protein biosynthesis, Leucine aminoacid was substituted wit Proline

aminoacid in position 322. Prior to us mutation 965, T>C has not been described

in Azerbaijan.

Some of mutations Phe508del (patients A.T.,A.N., R.E., B.A.) identifications have

been done with ROTOR-GENE apparatus. Other part of mutations has been

identified through directly sequencing.

Only one of 5 identified mutations (Phe508del) is the mostly spread type. The rest

four mutations: 328(G>A), 965(T>C), 1210-1211(T>G) and 1000(G>T) are not the

often encountered and treated as rare mutations of CFTR gene.

Identified mutations have been found in intron 9 and exons 4, 8, 9 and 11.

Eight patients have been examined, and out of 16 CFTR genes: 11 identified

Phe508del mutated gene (68,75%), two manifested 965,T>C mutated gene

(12,5%), the rest each one gene revealed – 1210-1211,T>G (6,25%), 1000, G>T

(6,25%) and 328,G>C (6,25%) mutations.


Among tested patients frequency of index patients with cystic fibrosis was 5,37%.

If showing in fractions, then CFTR gene frequency was 0,0537.

Table 1. Results of molecular genetic studies of CFTR gene

Patient Gene mutation Protein

mutation

Genotype Method

D.Kh. 1521-1523 exon 11

1210-1211T>G intron 9

Phe508del Compound Direct

sequencing

D.S. 1521-1523del exon 11 Phe508del Homozygote Direct

sequencing

F.A. 328 G>A exon 4

1000 G>T exon 8

Asp>110His

Arg>334Trp

Compound Direct

sequencing

B.D. 965 T>C Leu322Pro Homozygote Direct

sequencing

A.T. 1521-1523del exon 11 Phe508del Homozygote Genetic panel

A.N. 1521-1523del exon 11 Phe508del Homozygote Genetic panel

R.E. 1521-1523del exon 11 Phe508del Homozygote Genetic panel

B.A. 1521-1523del exon 11 Phe508del Homozygote Genetic panel

Thus, as a result of screening done in Baku city of Azerbaijan Republic children

hospitals, eight children-patients with cystic fibrosis diagnosis were identified for 5

mutations of CFTR gene. One mutation of these, i.e. 965 (T>C) has not been

found and described prior to us.

To prophylaxy cystic fibrosis in the Republic, it is recommended to carry out

genetic screening of newborns, to consult genetically risky families and to provide

prenatal diagnosis of fetus during the following pregnancies.

Conclusıon

1. Missens mutation of the SPTAN1 gene in a 9-month-old child diagnosed with

early epileptic encephalopathy using a combination of modern molecular

genetic methods: quanine nucleotide replacement with adenine nucleotide has

been identified in gene 2908 (SPTAN1 2908G> A).

2. As a result of the mutation, the amino acid glutamine was replaced by the

amino acid lysine at position 970 of the protein (Glu970Lys).

3. For the first time five mutations of CFTR gene: Phe508del, 965,(T>C), 1000

(G>T), 1210-1211 (T>G) and 328 (G>C) have been identified.



4. Gene frequencies for mutations are equal: Phe508del (68,75%), in two

965,T>C (12,5%) and for each one – 1000 G>T (6,25%), 1210-1211,T>G

(6,25%) and 328,G>C (6,25%).Mutasiyaları gen tezlikləri bırabərdi:

5. Mutation 965, T>C (Leu322Pro) has not been described prior to us.

6. Among all examined children, frequency of cystic fibrosis was 5,37%.

Reference

Bailey, S.D. (2013). Variation at the NFATC2 locus increases the risk of thiazolidinedione-

induced edema in the Diabetes REduction Assessment with ramipril and rosiglitazone

Medication (DREAM) study. DREAM investigators Diabetes care 6,12-16.

De Ligt, J., Willemsen, M.H., van Bon, B.W., Kleefstra, T., Yntema, H.G., & Kroes, T.

(2012). Diagnostic exome sequencing in persons with severe intellectual disability. N

Engl J Med. 367(20):1921–9.

Hamdan, F.F., Saitsu, H., Nishiyama, K., Gauthier, J., Dobrzeniecka, S., Spiegelman,

D., Lacaille, J.-C., Decarie, J.-C., & et al. (2012). Identification of a novel in-frame

de novo mutation in SPTAN1 in intellectual disability and pontocerebellar

atrophy. Europ. J. Hum. Genet. 20: 796-800.

McKusick, A. (2002). Mendelian inheritance in man. Tenth edition, London, p. 2115.

Nonoda, Y., Saito, Y., Nagai, S., Sasaki, M., Iwasaki, T., Matsumoto, N., Ishii, M. &

Saitsu, H. (2013). Progressive diffuse brain atrophy in West syndrome with marked

hypomyelination due to SPTAN1 gene mutation. Brain Dev. 35: 280-283.

Rose, J.E., & Uhl, G.R. (2010). Personalized smoking cessation: interactions between

nicotine dose, dependence and quit-success genotype score. (PMID: 20379614)

Molecular medicine (Cambridge, Mass.). 7, 9, 10-13.

Riordan, J.R., Rommens, J.M., Kerem, B., Alon, N., Rozmahel, R., Grzelczak, Z.,

Zielenski, J., Lok, S., Plavsic, N., & Chou, J.L. (1989). Identification of the cystic

fibrosis gene: cloning and characterization of complementary

DNA. Science. 245 (4922): 1066–73.

Saitsu, H., Tohyama, J., Kumada, T., Egawa, K., Hamada, K., Okada, I., Mizuguchi,

T., Osaka, H., Miyata, R., & Furukawa, T. (2010) Dominant-negative mutations in

alpha-II spectrin cause West syndrome with severe cerebral hypomyelination, spastic

quadriplegia, and developmental delay. Am. J. Hum. Genet. 86: 881-891.

Stabach, P.R. & Morrow, J.S. (1997) Site-directed mutagenesis of alpha II spectrin at

codon 1175 modulates its mu-calpain susceptibility. Biochemistry, 4, 21-24.

Tohyama, J., Akasaka, N., Osaka, H., Maegaki, Y., Kato, M., Saito, N., Yamashita, S.

& Ohno, K. (2008). Early onset West syndrome with cerebral hypomyelination and

reduced cerebral white matter. Brain Dev. 30: 349-355, 2008.

Trujillano, D., Bertoli-Avella, A.M., & Rolfs, A. (2017). Clinical exome sequencing:

results from 2819 samples reflecting 1000 families. Eur J Hum Genet. Feb; 25(2):

176–182.

Vissers, L.E., Gilissen, C. & Veltman, J.A. (2016). Genetic studies in intellectual

disability and related disorders. Nat Rev Genet. 17(1):9–18

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=search&Dopt=b&term=20379614


http://europepmc.org/search/?scope=fulltext&page=1&query=AUTH:%22Bertoli-Avella%20AM%22

http://europepmc.org/search/?scope=fulltext&page=1&query=AUTH:%22Rolfs%20A%22


Writzl, K., Primec, Z.R., Strazisar, B.G., Osredkar, D., Pecaric-Meglic, N., Kranjc,

B.S., Nishiyama, K., Matsumoto, N. & Saitsu, H. (2012). Early onset West

syndrome with severe hypomyelination and coloboma-like optic discs in a girl with

SPTAN1 mutation. Epilepsia 53: e106-e110, 2012. Note: Electronic Article.

Zhu, X., Padmanabhan, R., Copeland, B., Bridgers, J., & Ren, Z. (2017). A Case-

Control Collapsing Analysis Identifies Epilepsy Genes Implicated in Trio Sequencing

Studies Focused on De Novo Mutations. Genet. 29:13(11).

https://pubmed.ncbi.nlm.nih.gov/?term=Zhu+X&cauthor_id=29186148

https://pubmed.ncbi.nlm.nih.gov/?term=Padmanabhan+R&cauthor_id=29186148

https://pubmed.ncbi.nlm.nih.gov/?term=Copeland+B&cauthor_id=29186148

https://pubmed.ncbi.nlm.nih.gov/?term=Bridgers+J&cauthor_id=29186148

https://pubmed.ncbi.nlm.nih.gov/?term=Ren+Z&cauthor_id=29186148



41

Frequency of Prevalence of Klebsiella pneumoniae in

Clinical Samples and the Evaluation of the Role of

Efflux Pump in Determining Antibiotic Resistance

Ima Amani1, Majid Baseri Salehi2*,

Fahimeh Nemati Mansour3

1Department of Microbiology, Science and Research Branch, Islamic Azad University,

Tehran Medical Branch; 2Department of Microbiology, Science and Research Branch,

Islamic Azad University, Kazerun, Iran; 3Department of Biotechnology, Science and

Research Branch, Islamic Azad University, Tehran Medical Branch *Corresponding author: [email protected]

Abstract

In this study 193 Klebsiella pneumoniae were isolated from urinary tract infections, ulcers,

sputum and blood. Initially, Mac agar medium was used to isolate the bacterium, and for

each suspected isolate, pink and aqueous colonies were stained and biochemical tests of

catalase, oxidase, TSI, IMVIC Test and urase were performed. Confirmation of the isolates

using 16SrRNA sequencing Some isolates are evaluated. Then all isolates evaluated for

sensitivity to antibiotics such as Ampicillin, Amoxicillin clavulanate, Piperacillin,

Cefoxitin, Cefuroxime Imipenem, Tetracyclines, Nitrofurantoin, Polymyxin B Colistin,

they use disk diffusion test. In the process, the presence of the acr efflux pump gene is

confirmed by using an specific primer namely Acr primer, and finally, using phenylalanine-

arginine- beta naphthylamide inhibitor, the relationship between antibiotic resistance and

efflux pump function is evaluated. Overall, 50.2% of the collected samples contained

Klebsiella. Thus, 193 of 384 clinical specimens contained Klebsiella. Of the 193 positive

samples, the groin lesions had the highest percentage and the abscess had lowest percentage

of Klebsiella infection, although Klebsiella was significantly separated from the throat,

sputum, catheter and foley. Antibiotics, cefazolin, ceftazidime, ciprofloxacin,

Chloramphenicol, tetracycline had higher antibacterial activity. Results were analyzed by

Whonet 5 and SPSS software.

Keywords: Klebsiella pneumoniae, Enterobacteriaceae, efflux pump, drug

resistance

42 Ima Amani, Majid Baseri Salehi, Fahimeh Nemati Mansour

Introduction

Klebsiella, a gram-negative bacterium, belongs to the family Enterobacteriaceae.

This bacterium was isolated by Carl Friedlander from the lung of a patient with

pneumonia and is called Bacillus Friedlander, a severe and deadly pneumonia

agent. Like other immobilized Klebsiella species and produces a polysaccharide

capsule (Mphba, 2005).

Morphology and cultivation characteristics

The members of the genus Klebsiella are shorter and thicker than other members of

the Enterobacteriaceae family and are about 1–2 μm long and 0.8–0.8 μm wide.

They are seen as placenta or single and sometimes resemble diplobacillus

pneumococcus. It produces a large capsule in environments that contain

carbohydrates and no nitrogen. Klebsiella strains have type 1, 2 and 3 fimbriae.

This bacterium disappears for 30 minutes at 55C (Mphba, 2005; Hsueh et al.,

1977).

These organisms are positive for urease, lysine decarboxylase and negative for tests

of ornithine decarboxylase, arginine dehydrolase, and indole production. The VP

test is positive. This organism is acidic in the TSI medium, without hydrogen

sulfide gas production as well as pyrolysis. Growth of this bacterium, although

optional anaerobic, is weak under absolute anaerobic conditions. Lack of ability to

lysis red blood cells on blood agar medium. The optimum growth temperature of

this organism is 37 ° C (Forbes et al., 2007).

Almost all grow in citrate and molar Hinton containing KCNs and their G + C is

52-58%. These organisms are part of the natural microflora of the intestine,

stomach, and human respiratory tract, and about one-third of them are carriers of

this bacterium. These bacteria are one of the most important causes of nosocomial

infections and cause a wide range of infections, including septicemia, pneumonia,

urinary tract infection, meningitis, and purulent abscesses (especially liver

abscesses) (Mphba, 2005).

Klebsiella pneumoniae clinical isolates are divided into four groups based on

nucleotide changes in the gyrA, parC and rpoB genes, including KP I, A-KP II, KP

II-B, KP III (Alves et al., 2006).

Klebsiella is the only genus in the Enterobacteriaceae family that has the ability to

stabilize nitrogen in the air and convert it to ammonia and amino acids. Another


Evaluation of the Role of Efflux Pump in Determining Antibiotic Resistance 43

metabolic product of this bacterium is 1,3-propanediol, which is produced under

microaerophilic conditions (Huang et al., 2002).

Pathogenic factors

Five factors have been proposed for Klebsiella pathogenesis, including capsular

antigen (as the most important virulence factor in Klebsiella pneumoniae),

adhesives, lipopolysaccharide, siderophore, Klebsiella pneumoniae (Struve et al.,

2009; Campos et al., 2004).

Typing methods of Klebsiella pneumoniae

Klebsiella pneumoniae typing methods are either based on phenotypic

characteristics called phenotyping or based on genotypic characteristics called

genotyping. Phenotyping, which includes serotyping, biotyping, bacteriocin typing,

phagotyping, and antibiograms, can generally be said to lack complete

differentiation, reproducibility, and typing. Genotyping examines the differences

and similarities of DNA sequences that are called Mass-Sequencing Technologies

if they look at specific parts of DNA, and Whole Genome Sequencing, if they look

at the entire DNA sequence. Some Fingerprinting methods are currently considered

as the most suitable methods for genotyping microorganisms for epidemiological

purposes. These include PFGE, ribotyping and some PCR-based typing techniques

such as RAPD, AFLP, VNTR. All of these methods use an electric field to separate

DNA fragments (Shakil et al., 2008).

Pathogenicity of Klebsiella pneumoniae

The strains of Klebsiella pneumoniae that cause pneumonia are usually capsular

serotypes 1 and 2. The cases are in people of middle age or older who have

underlying medical problems, such as chronic bronchopulmonary disease, diabetes,

or alcoholism. Klebsiella pneumonia in about 25 to 75% of patients produces a

thick bloody sputum that does not smell disgusting. In this pneumonia, necrosis

and abscess are more likely than other bacterial pneumonia and blood cultures are

positive in 25% of patients. Klebsiella pneumoniae strains are particularly resistant

to many drugs and generally cause more severe disease than streptococcal

pneumonia. Even with proper treatment, the mortality rate of Klebsiella pneumonia

is 40 to 60 percent. Klebsiella pneumoniae is one of the causes of urinary tract


infections, wound infections, bacteremia and meningitis (Cortés et al., 2002).

Pneumonia caused by Klebsiella spp often causes necrotic destruction of alveolar

spaces, produce cavity formation and pale bloody sputum. The organism's ability to

cause disease due to decreased host defense is due to complex and prolonged

surgical procedures as well as increased use of different drugs (Lin et al., 2004). In

humans Klebsiella species are found on the skin, pharynx or digestive tract. The

bacterium is found in sterile wounds, urine, and may be considered normal flora of

the small intestine and bile ducts (Campos et al., 2004). Klebsiella is transmitted

from one patient to another through contaminated medical devices, contaminated

hands of hospital staff, and blood products, while areas causing infection by

Klebsiella spp., Surgical wounds, peritoneum, catheter insertion sites, Urinary

ducts, gastrointestinal and biliary tracts (Wilson’s, 2006). Klebsiella pneumoniae is

the cause of urinary tract infections, neonatal arthritis, meningitis, wound

infections, nosocomial pneumonia, bacteremia, septicemia, and soft tissue

infections (Lin et al., 2004). Hospital-acquired pneumonia is a severe disease with

high mortality associated with rapid invasion, high fever, bloody sputum and

visible abscesses on chest radiographs. The mortality rate is about 25 to 50%

(Campos et al., 2004). Klebsiella pneumoniae cause syndrome which is the reason

for septicemia with liver abscesses, which brings about 10 to 40% of deaths. K1

capsular serotype is the dominant serotype of Klebsiella pneumoniae in the

development of hepatic abscesses (Williams et al., 1983). Various underlying

diseases including diabetes, alcohol overuse, bile duct disease, malignancies, liver

cirrhosis, kidney disease, intra-abdominal infections, history of steroids, history of

abdominal surgery and history of antibiotic use (Roberts et al., 1989), all are risk

factors for liver abscess infection due to the K1 capsular serotype in Klebsiella

pneumoniae (Cbat, 1989). Klebsiella pneumoniae as a common pathogen causing

bacterial endophthalmitis of intrinsic origin associated with liver abscess in Asia.

Klebsiella pneumoniae K1 capsular serotype has the ability to cause central

nervous system infection as a complication of purulent liver abscesses in patients.

Klebsiella pneumoniae is also associated with chronic diarrhea in HIV-positive

people (Hbjak et al., 1987).

Antibiotic Resistance Mechanisms in Klebsiella

Antibiotics are natural products or synthetic organic compounds that usually inhibit

or destroy certain bacteria at low concentrations. Antibiotics are divided into 5

categories in terms of function. 1) Cell wall synthesis inhibitor 2) Protein synthesis

inhibitor 3) Interference with nucleic acid synthesis 4) Inhibition of metabolic

pathway 5) Degradation of membrane structure.



Resistance to antibiotics

Resistance to antibiotics is applied in two ways. 1. Genetic dependent 2. Non-

genetic dependent. Genetically dependent resistance is either dependent on the

chromosome or the plasmid (Izard et al., 1981).

Drug resistance dependent on Klebsiella efflux pump

Nowadays one of the mechanisms of drug resistance in bacteria is the existence of

efflux pump in bacteria. efflux pumps are transfer protein like toxins and

antibiotics from the cell to the environment (Izard et al., 1981). In recent years,

Klebsiella drug-resistant strains have been widely observed, and resistance to

quinolones is generally one of the most important object. On the other hand, the

mechanism of resistance to quinolone antibiotics reports the presence of efflux

pumps in these bacteria (Bagley et al., 1981). A common feature of all efflux

systems is the ability to remove a variety of antibacterial substances such as

antibiotics, biocides, dyes, cleaners, fatty acids and organic solvents (Ferragut et

al., 1983). Efflux pumps reduce the concentration of drug inside the cell and create

resistant strains of bacteria by increasing the lowest inhibitory concentration

(MIC). In bacteria, there are five families of efflux pumps that are divided into two

groups according to the energy acquisition method for substrate transport: first-

class pumps that use ATP energy for transport and only include ABC pumps or

(cascade-dependent proteins to ATP). Second-class pumps use the gradient energy

of the proton concentration to transport the substrates (Sakazaki et al., 1989). The

five major classes of efflux pumps are the MFS or Major facilitator superfamily,

the SMR or Small multidrug resistant family, the RND or Resistant nodulation

division family, the MATE or Multidrug and toxic efflux family, and the ABC or

ATP binding cassette family (Gavini et al., 1983). Efflux pumps are either single or

triple. One-part efflux pumps direct the protein out of the bacterial cell after protein

absorption, while three-part efflux pumps excrete it directly after the drug is

absorbed (Ferragut et al., 1983; Drancourt et al., 2001; Alves et al., 2006).

Multidrug resistance (MDR) is a bacterial pathogenic ability to withstand deadly

doses of structurally diverse drugs capable of eradicating non-resistant strains. MDR

is recognized as a major threat to human health by the World Health Organization.

Of the four general mechanisms that cause antibiotic resistance, including target

transformation, drug inactivation, permeability reduction, and increased efflux

activity, efflux pump removal, which acts as an important MDR mechanism.

The efflux pump can not only remove a wide range of antibiotics due to its multi-

substrate property, but also provide additional resistance mechanisms that reduce

intracellular antibiotic concentration and increase mutation accumulation. The


over-expression of multi-drug efflux pumps is increasingly indicative of clinical

drug resistance. On the other hand, the evidence gathered indicates that efflux

pumps have physiological function in bacteria and that their expression is in tune

with different types of environmental and physiological signals (Bsgfg, 2006).

Mechanism of drug secretion by efflux pump

Efflux pumps are prominent in terms of their high drug efficacy and broad

substrate properties, due to their multidrug resistance. The fact that all bacterial

genomes contain the efflux pump gene, particularly in stressful situations,

development, pathogenesis and contagion, their expression is closely monitored by

a number of local and global transcription regulators. It has been suggested that

drug efflux pumps have physiological function. The evidence gathered suggests

that efflux pumps actually play a general role in detoxification in different

physiological processes of bacteria.

Although efflux genes are distributed throughout bacterial genomes, with the

exception of a few native efflux systems, most of them are under strict control by

different transcriptional regulators and their role is to facilitate the adaptation of

bacteria to specific stimuli.

Given the capacity of efflux pumps for a wide range of indirect structural

chemicals, it is predictable that over-expression of efflux pumps may lead to

unintended withdrawal of metabolites or other signaling molecules, resulting in

deleterious effects, affects cellular physiology.

Therefore, expression of efflux pumps is usually well regulated and expressed

only at low levels under normal and normal growth conditions. Although the

composition and performance of MDR, efflux pumps are relatively conserved in

different species, their regulatory mechanism is significantly different.

Given the high importance of Klebsiella pneumoniae multidrug resistance in the

treatment of infectious diseases as well as the strong role of oqxAB efflux pumps

in the development of this type of resistance, we aimed to investigate the frequency

of efflux pump-dependent antibiotic resistance in isolates. Clinical Klebsiella

pneumoniae isolated from patients referred to Shiraz hospitals.

Literature review

In a study by Ziqing Deng and colleagues in 2014 on the efflux pump, they

concluded that the efflux pump can not only remove a wide range of antibiotics due

to its Poly-substrate property, but also gain mechanisms. Additional resistance is

also found, which reduces intracellular antibiotic concentration and increases



mutation accumulation. Overexpression of multi-drug efflux pumps is increasingly

associated with clinical drug resistance. On the other hand, the evidence gathered

indicates that efflux pumps have physiological function in bacteria and that their

expression is in tune with different types of environmental and physiological

signals (Sun et al., 2014).

In 2007 According to study by the Department of Microbiology and Immunology

at Kingston University, the efflux pump is found in almost all bacterial species, and

the genes of this type of proteins are put on chromosomes or plasmids. Membrane

scattered regions, energy sources and substrates, bacterial efflux pumps are divided

into five families: RND family, large facilitator family (MFS), ATP family

(adenosine triphosphate), family (ABC), small multiple family (SMR) (Poole,

2007; Piddock, 2006).

In a 2013 study by Jadwiga and Anna, apart from the RND family found only in

gram-negative bacteria, the output systems of the other four families MFS, ABC,

SMR and MATE are widely used in both bacteria. Gram positive and negative are

distributed (Handzlik et al., 2013).

In a 2004 study by Nikaido, Efflux pumps, which are single-component

transducers or multicomponent systems, include not only an inner membrane

receptor but also an outer membrane channel and a pre-plasmatic adapter protein

similar to the efflux pumps such as RND (Li & Nikaido, 2004).

According to a 2006 study by the University of Birmingham, RND family pumps

are widely used, due to tripartiite compounds that directly release various drugs

from the cytosol or periplasmic space out of the bacterial cells. It is known with

significant antibiotic resistance, such as AcrB in Escherichia coli, Salmonella

typhimurium, and MexB in Pseudomonas aeruginosa. In gram-positive bacteria,

efflux pumps are clinical members of the MFS family (Piddock, 2006).

In 2013 at Rakuno Gakuen University in Sapporo, Japan on 50 clinical specimens

obtained on E.coli, isolated from human clinical specimens and canine feces, show

a strong correlation in overexpression of AcrAB pumps with It is highly resistant to

fluoroquinolone (Sato et al., 2013).

In another study conducted by Dr. Pakzad et al. (2013) at the University of Ilam on

the Klebsiella pneumoniae strain, 52 of the 40 patients who were hospitalized at

Shahid Motahari Hospital in Tehran, all 40 isolates of ciprofloxacin, tetracycline,

Ceftazidime and gentamicin showed high levels of AcrAB efflux pump, especially

in ciprofloxacin-resistant strains (Pakzad et al., 2013).


Also, a 2012 study by Wayne State University in the United States reported a

relationship between over-expression of the efflux pump with appropriate clinical

MDR in Gram-positive bacteria. Among the several hundred clinical specimens of

S. aureus isolated by Christos et al., It was found that overexpression of the pump

gene was geographically widespread. These strains were predominantly resistant to

methicillin and their resistance was correlated with norA and mepA expression

(Kosmidis et al., 2013).

According to the findings, at the University of Birmingham in 2006, the Efflux

pump is prominent in terms of its high drug delivery efficiency and its broad

substrate properties, due to its multidrug resistance (Piddock, 2006).

In 2015, Jing Sun et al., Realized that all bacterial genomes contained the efflux

pump gene, Espicially under stressful conditions, development, pathogenicity, and

contagion, their expression being closely monitored by a number of Local and

global transcriptional regulators have suggested that medicinal efflux pumps have

physiological function. Evidence was collected that efflux pumps actually play a

general detoxification role in different physiological processes of bacteria (Zou et

al., 2015).

According to a study done at the Department of Microbial Biotechnology in

Madrid in 2004, although efflux genes are distributed everywhere in bacterial

genomes, with the exception of a few autophagy systems, most of them are under

strict control by different transcriptional regulators. And their role is to facilitate

the adaptation of bacteria to specific stimuli (Alonso et al., 2004).

According to a 2009 study by the School of Microbiology and Infectious Diseases

at the University of Saka, Japan, given the efflux pump's capacity for a wide range

of indirect structural chemicals, it is predictable that over-expression of the efflux

pumps may be Lead to the unwanted secretion of metabolites or other signaling

molecules, which in turn has deleterious effects on cellular physiology. Therefore,

expression of efflux pumps is usually well regulated and expressed only at low

levels under normal growth conditions. Although the composition and performance

of MDR efflux pumps are relatively conserved in different species, their regulatory

mechanism is significantly different (Nishino et al., 2009).

Methodology

In this study, SPSS software version 20 “Statistical Package for the Social

Sciences” and the statistical program, Student's t-distribution and Chi Square were

used to analyze the data.



Collection of clinical specimens

The following statistical formula was used to determine sample size:

z pqn

d=

2

2

where in;

z: The value of the standard normal variable is 95% and is 1.96

p: A proportion of people in the community who had the desired attribute were

considered to be 0.5.

q: A proportion of people in the community who did not have the desired attribute

were considered to be 0.5.

d: And the error value is equal to 0.05.

Due to the above relation, sample size is calculated as follows: 384

A total of 384 specimens, from those who had clinical symptoms, the sampling was

done randomly included pharyngitis patients swabs, sinus secretions of patients

with chronic sinusitis, secretion of middle ear infections patients, pulmonary

secretions of patients with respiratory failure who were admitted to the intensive

care unit, and samples of urinary tract infections, in Dena and Namazi hospital

were collected. The sampling period lasted from 3 months from May 2019 to July

2019

Phenotypic isolation and identification of clinical specimens

Klebsiella pneumoniae isolates are Gram-negative bacilli, oxidase negative and

lactose positive which in acidic and yellow environment in TSI medium acidic and

yellow which are associated with gas production. In terms of endolysis and MR test

negative and VP reaction and citrate test positive. These organisms are positive for

the lysine decarboxylase test and are generally immobilized and their urea

hydrolysis test is also positive.

Preservance Preservation of Klebsiella strains

After identifying the isolates for long-term storage, the bacteria were first cultured

in vials containing TSB medium and incubated at 37 ° C with 20% sterile glycerol


and then incubated until the tests were performed. The culture was stored in a -70 °

C freezer (BMB, 2012).

Confirmation of the identification of Klebsiella by molecular method

To confirm the phenotypic identification, 5 isolates were selected by chance and

evaluated using 16SrRNA primer.

Determination of susceptibility of Klebsiella isolates to antibiotics by disk

diffusion method

The test was performed using the Kirby Bauer standard method according to the

International Laboratory Standards Institute (CLSI) guidelines. Muller Hinton Agar

medium, antibiotic discs with standard table were used for this test. Muller Hinton

agar medium was prepared and its pH was adjusted from 7.2 to 7.4. The plates

were incubated at 35 ° C for 24 h to control infection. Containers containing

antibiotic discs of ampicillin, cefazolin, piperacillin, tobramycin and imipenem

were then transferred from a -20 ° C freezer (long-term storage) to a 4-degree

refrigerator (short-term storage) (MAST UK antibiotic discs were purchased). A

few minutes before the test, the disc containers were kept in the laboratory to reach

room temperature. Next, a standard microbial suspension was prepared for the test.

Since the strains that were not cultured for more than 24 hours were used for the

preparation of the suspension, the samples were cultured on simple gel medium the

day before antibiogram. They were then incubated at 35 ° C for 24 h. Transfer

some of the colon to a tube containing 2 ml of sterile physiological serum, and after

mixing with the mixer, the resulting suspension turbidity was adjusted to half-

McFarland's turbidity. The sterile cotton swab was then immersed in the prepared

bacterial suspension and then squeezed onto the lateral wall of the test tube to drain

off the excess fluid, then cultured on a Muller Hinton agar medium by rotating the

culture angle, for three times. 15 minutes after the inoculation of the antibiotic

discs that were brought to room temperature, they were placed on a plate at a

distance of at least 2.25 cm from each other as well as from the edge of the plate.

Plates were incubated at 35 ° C for 24 hours. Then, using the ruler, the diameter of

the growth zone around each disk was measured and the corresponding results

were recorded in the prepared forms.

Table 1. Features of Klebsiella

Antibiotics Klebsiella

Antibiogram

Sensitive

to

Resistance

to

Intermediate

to

Cefazolin 13% 88% -

Ceftriaxon 23% 77% -

Cefixime 22% 78% _



Nalidixic acid 18% 82% -

Tobramycin 33% 50% 17%

Pipracillin _ 100% _

Cefepime 63% 38% -

Imipenem 100% - -

Meropenem 45% 54.00% _

Chloramphenicol 81% 18% -

Ampicillin - 100% -

Nitrofurantoin 66% 33% _

Cefotaxim 26.08% 74% -

DNA extraction for genotypic identification and determination of efflux pump

genes

DNA extraction was performed in this study using the Total Extraction Kit, a

product of Sinaagen. The basis of DNA extraction using the kit was that the high

concentration of salt binds DNA to the matrix with a silica filter. Reversibly, under

conditions of low salt concentration, such as in the wash solution or Tris-HCL 10

mM, the DNA is extracted from the silica filter and inserted into the above

solutions. The ributinase enzyme in this kit digests the RNA and protein together

with the DNA, making the resulting DNA readily available for polymerase chain

reaction or PCR.

The steps of DNA extraction

For this test, 5 ml of the bacterial suspension containing 1.5 108 108 bacteria per

ml were centrifuged at 300 rpm for 5 minutes. The solution was then discarded and

the precipitate was washed with PBS. This operation was repeated twice.

Subsequently, the addition of 10µl of periclase buffer and 10µl of ributinase was

added and slowly vortexed for a few seconds. The test tubes were then incubated at

55 ° C for 30 min. From the above sample, 10µl was added to a 1.5 sterile

Ependorf tube. Then 400µl of Laisse buffer solution was added and vortexed for 20

seconds at high speed. Afterwards, 300µl of the solution was added to the tube and

vortexed at high speed for 20 seconds. After completion of this step, the sample

was transferred to the spin column and centrifuged at 1 g for 1 minute. The spin

column was transferred to a new collecting tube, and 400µL of the wash buffer was

added and centrifuged for 1 minute at 13,000 rpm. This operation was repeated

twice.


The spin column was again inserted into a new collection tube and this time 30µL

of buffer was added and then incubated at 65 ° C for 3–5 minutes. Finally, the

sample was centrifuged at 13,000 rpm for 1 minute to wash the DNA.

Determination of extracted DNA purity:

Optical absorption of the DNA extracted at 260 and 280 nm was measured by a

nanodrop machine. If the resulting number is equal to or greater than 1.8 ng / μl,

the extraction steps are well performed.

The primers used in the present study

16SrRNA-F5´-AGA GTT TGA TCM TGG CTC AG-3´

16SrRNA-R 5´-CGG TTA CCT TGT TAC GAC TT-3´

OqxA-F 5′-GCGTCTCGGGATACATTGAT-3′

OqxA-R 5′-GGCGAGGTTTTGATAGTGGA-3′

OqxB-F 5′-CTGGGCTTCTCGCTGAATAC-3′

OqxB-R 5′-CAGGTACACCGCAAACACTG-3′

PCR reaction was performed using positive and negative controls. The standard

strain of Klebsiella pneumoniae ATCC 700603 was used for positive control and

the distilled water without DNA was used for negative control.

Polymerase Chain Reaction and Test Procedures

In order to increase the efficiency, accuracy and ease of carrying out the

polymerase chain reaction, the Master Kit made by Synagen was used. This kit

contains all the ingredients needed to carry out the reaction, with the exception of

DNA template and primers. Polymerase Chain Reaction was performed in a

volume of 12.5 µL and in 0.2 ml sterile microtubes.

The following equation can be used to calculate the melting temperature or

Annealing Temperature:

Formula for calculating Ta: Ta = 0.3 x Tm(primer) + 0.7 Tm (product) – 14.9

1. Tm(primer) = Melting temperature of the primers



2. Tm(product) = Melting temperature of the product

Primer Tm calculation: Tm = 4(G + C) + 2(A + T) =°C

It should be noted that all primers used in this study were extracted from validated

sources. Therefore, no gradient temperature program was required to perform PCR.

Electrophoresis

In this method, the products of the polymerase chain reaction were electrophoresed

and the bands formed on the gel, the presence or absence of the desired gene in the

isolates genome was evaluated.

Solutions and buffers used in electrophoresis:

Buffer {(X50) TAE} (X 502) Tris-acetate EDTA

Use of Whonet software to analyze the effect of antibiotics on clinical isolates

Whonet is a software program designed to inform antimicrobial resistance based on

WHO laboratory evaluation of infections.

This software is a tool for evaluating microbial epidemiology, selecting antibiotics,

spreading disease and identifying problems in laboratory tests. In the present study

Whonet version 5.6 software was used.

Phenotypic Evaluation of efflux Pumps in Klebsiella pneumoniae Isolates

Using Cartwheel (Ethidium Bromide)

For this purpose ethidium bromide solution was used and 3 different dilutions of

1/2, 1/4 and 1/8 were prepared using sterile distilled water. When working with

ethidium bromide solution, safety conditions such as gloves, masks, goggles and

hoods were observed. Then 4 g of the powder was dissolved and autoclaved in 200

ml of distilled water, followed by removal of the culture medium to autoclave

when the temperature reached 40 ° C using a sample of one cc of each sample.

Diluted ethidium bromide dilutions were poured into a 10 cm sterile plate and then

added to the culture medium and stirred to mix with ethidium bromide solution,

then allowed to completely solidify. Klebsiella specimens isolated by sterile loops

were drawn as a straight line. Plates were incubated in a 37 ° C incubator for 24

hours after which the results were recorded and documented by UVTEC gel

apparatus.


Determination of dependence of antibiotic resistance on efflux pump in

Klebsiella clinical isolates

At this stage of the experiment, initially isolates are all resistant to ciprofloxacin,

tetracycline and chloramphenicol. MIC test or minimum inhibitory concentration

were calculated, then the resistant strains were cultured independently in Muller

Hinton liquid medium overnight. In 10 tubes containing 1 ml of Muller Hinton's

liquid medium, different dilutions of antibiotics and then bacterial suspension were

added. Minimum inhibitory concentration was measured after 24 hours with or

without turbidity.

Similarly, after the Muller Hinton liquid medium was cooled, 100 mg / lt of PAβN

(phenylalanine-arginine beta naphthylamide inhibitor) was added.

Statistics Analysis

In this study, the results were analyzed using SPSS software version 20. Analytical

methods in the present study including descriptive analysis (Cross tab) and

determination of significant relationships with Parametric and Nonparametric

methods including Chi-square were studied. Finally, the significance level of

statistical relationships was evaluated at p <0.05.

Data analysis and conclusion

Phenotypic and Genotypic Identification and Determination of Klebsiella

Occurrence in Clinical Specimens

Overall, 50.2% of the collected samples contained Klebsiella by the

bacteriological, cultural and biochemical tests. Thus, 193 of 384 clinical specimens

contained Klebsiella. Of the 193 positive samples, inguinal had the highest

percentage and abscess the lowest percentage of Klebsiella infection, although

Klebsiella was significantly removed from the throat, sputum, and foley catheter.

Overall, 18.65% of clinical samples in both Dena and Namazi hospitals contained

Klebsiella, which, given the ethical issues and commitments that were considered

at the time of sampling, indicated the incidence of this bacterium in the listed

hospitals separately was avoided. On the other hand, phenotypic identification of

Klebsiella isolates showed that all isolates belonged to the pneumoniae species. To

confirm the phenotypic identification, 5 strains were selected by chance and



evaluated using 16SrRNA primer, all of which confirmed the phenotypic

identification.

Table 2. Klebsiella’s resistance or sensitive

Clinical

Sample No Kl. Percentage Resistance Sensitive

Determination

of antibiotic

resistance

spectrum and

their

percentage

Trachea 50 9 18% -

XDR (18)

MDR(46)

Urine 63 10 15.87%

MDR(20)

Sputum 6 1 16.6%

-

Nasal

Swab

16 2 12.5%

-

Inguinal 17 6 35.3%

MDR(50)

Throat 20 5 25%

MDR(20)

Abscess 9 1 11.11% MDR(100)

Foley

catheter

12 2 16.6%

MDR(100)

The results shown in the table show the highest antibiotic resistance in strains

isolated from trachea samples and then inguinal and foley catheter and the lowest

antibiotic resistance in strains isolated from urine, sputum and nasal swabs.

However, the highest sensitivity was evaluated in Klebsiella strains isolated from

urine and then sputum. On the other hand, all Klebsiella strains were susceptible to

cefazolin antibiotic and partially and not all Klebsiella strains isolated to

chloramphenicol, tetracycline and cefipime antibiotics.

As previously described, resistance to at least one agent in three or more groups of

antibiotics defined for MDR, resistance to at least one agent in all antibiotic groups

except one or two XDR groups, and resistance to all antibiotic agents in all. Groups

are considered PDRs. Therefore, the results of the present study showed that only

XDR was observed in strains isolated from the trachea, while the rest of the clinical

specimens contained the MDR Klebsiella pneumoniae strains except sputum and

nasal swab samples.


Antibiotic effect analysis by using Whonet software

The results of data analysis using Whonet software showed that Klebsiella clinical

isolates were the most resistant to cefepamin and ceftriaxon antibiotics and the

most susceptible to nitrofurans and chloramphenicol.

Figure 1. Percentage of resistance of Klebsiella isolates to antibiotics

Figure 2. Percentage of Klebsiella strains resistance to antibiotics



Figure 3. Percentage of susceptibility of Klebsiella strains to antibiotics

Genotypic evaluation of Efflux pump in Klebsiella pneumoniae isolates using

oqxAB genes

Following the results of the Cartwheel test for phenotypic identification of the

efflux pump in isolated Klebsiella, it was shown that some Klebsiella strains were

unable to maintain this dye after exposure to ethidium bromide. However, some

Klebsiella strains were able to maintain this color. Therefore, it was concluded that

strains that were not able to maintain dye were likely to contain an efflux pump to

remove antibiotics from the cell.

The results of genotypic evaluation of efflux pump in isolated Klebsiella showed

that all strains in the phenotypic test likely contained efflux pump gene containing

oqxAB genes.

The results showed that antibacterial, cefazolin, ceftazidime, ciprofloxacin,

chloramphenicol, tetracycline had more antibacterial activity after blocking the

efflux pump inhibitor. These antibiotics were antibiotics whose resistance to

Klebsiella pneumoniae isolates was dependent on the efflux pump as their growth

inhibitory region was changed from the original antibiogram. Results were

analyzed using Whonet 5 and SPSS v22 software. According to the Whonet

software, the horizontal axis in these graphs shows the bacterial growth inhibition

diameter and the vertical plot in percent of the isolation growth inhibition diameter.


Figure 4. OqxA gene electrophoresis gel band size: 200 pairs

Figure 5. OqxB gene electrophoresis gel band size: 150 pairs

Evaluation of dependence of antibiotic resistance of Klebsiella isolates on

efflux pump

The results of evaluation of antibiotic resistance of Klebsiella isolates to efflux

pump showed that addition of efflux pump inhibitor (phenylalanine-arginine- beta-

naphthylamide) to culture medium increased susceptibility of Klebsiella isolates to

ciprofloxacin and tetracycline antibiotics was Resistance to other antibiotics

dependent on the efflux pump was not evaluated.



Statistical analysis of data

In the present study, the relationship between resistance of Klebsiella pneumoniae

to antibiotics and presence and absence of efflux pump genes was examined by

chi-squared test. The results showed that there was a significant relationship

between the antibiotic resistance of Klebsiella isolates to ciprofloxacin and

tetracycline antibiotics with P value <0.05. Chi-squared test was used for statistical

analysis and the results showed a significant relationship with P value <0.05

between isolates resistant to some antibiotics such as ciprofloxacin and tetracycline

and presence of oqxAB genes.

Discussion

Nowadays The high prevalence of Klebsiella pneumoniae in the hospital

environment today has limited the options available to treat infections caused by

this bacterium. However, antibiotic control politics are important along with the

implementation of infection control measures. In a 2013 study by Derakhshan et al.

In Tehran. Antibiotic susceptibility testing was performed for 116 clinical isolates

of Klebsiella pneumoniae strain. The results showed that the antibiotic resistance of

gentamicin, amikacin is 60% and 37%, which is higher than the resistance of these

drugs in the present study (Liu, 2012). In a study conducted by Pakzad et al. (2013)

in Tehran. Antibiotic susceptibility testing was performed for 52 Klebsiella

pneumoniae isolates isolated from burn samples. The results showed that

gentamicin resistance was 73% which is higher than the resistance of these drugs in

the present study (Pakzad et al., 2013). In a 2011 study by Galani et al in Greece,

antibiotic susceptibility testing was performed on 14 Klebsiella pneumoniae

isolates with high MIC for amikacin, gentamicin, which had a 48.2% and 8.1%

frequency of resistance to amikacin and gentamicin, respectively (Galani et al.,

2012).

A study by Ma et al. (2008) in Taiwan. Antibiotic susceptibility testing was

performed for 235 Klebsiella pneumoniae isolates and the gentamicin and amikacin

antibiotic resistance were 94.4% and 91.1%, respectively. In a study conducted by

Habeeb et al. (2013) in Pakistan, of the 25 isolates of Klebsiella pneumoniae

producing beta lactamase with a wide range of 60% rmtB gene expression was

reported. Results showed that rmtB gene expression in these strains was very high

at this hospital in Pakistan. In another study the 55 isolates of Klebsiella

pneumoniae resistant to several drugs, cefotaxime, ciprofloxacin and amikacin

were observed (Yang et al., 2011). A study by Kang et al. (2008) at the Korea

Teaching Hospital between 40 Klebsiella pneumoniae strains with high levels of

aminoglycosides, including amikacin, tobramycin, gentamicin and kanamycin, 28


isolates containing armA and 9 isolates containing rmtB. Multiplex PCR assay

was performed for simultaneous detection of 7 aminoglycoside resistance genes in

Enterobacteriaceae (Hu et al., 2013). Thirty isolates of Klebsiella pneumoniae

resistant to gentamicin, tobramycin and amikacin contained rmtB and armA genes

(Yang et al., 2011). A study by Wang et al. (2007) in Korea from 7127

Enterobacteriaceae isolates, 463 isolates were highly resistant to amikacin (6.5%).

218 isolates carried the armA and rmtB genes. Of the 218 isolates, 153 strains

contained armA and 51 isolates contained rmtB (Kang et al., 2009). A study by Yu

et al. (2008) in China of the 337 Klebsiella pneumoniae isolates, 39 isolates had

very high resistance to gentamicin, amikacin, and tobramycin (MIC≥ 256 μg / ml)

(Yu et al., 2009). Wu et al. (2009) in Shanghai determined that among 202

Klebsiella pneumoniae isolates, 35 were resistant to gentamicin and amikacin.

Feyzabadi et al. (2010) reported that antibiotic susceptibility testing was performed

on 89 clinical samples of Klebsiella pneumoniae. Results showed that the

resistance to gentamicin, amikacin were 34.8% and 16.9%, respectively, which is

lower than the drug resistance in the present study. In this study, resistance to

amikacin was higher than gentamicin. A 2013 study of Klebsiella pneumoniae

specimens collected from various locations such as Taiwan-Australia-Argentina-

Belgium-Turkey-South Africa found that 100% of the isolated isolates of the

OqxAB efflux pump were present (Alexander & Rietschel, 2001). The results of

Saadatian Farivar study on Klebsiella pneumoniae isolates isolated from Tehran

hospitals showed that 96% of samples had OqxAB efflux pump (Nassif &

Sansonetti, 1986). In a descriptive study conducted by Hashemi et al. (2014) they

concluded that the prevalence of OqxA and OqxB pumps was 50% and in

ciprofloxacin-resistant strains a 2/3 times increase in OqxAB efflux pump was

observed. Also, studies conducted in 2013 and 2015 showed the presence of both

OqxA and OqxB genes was 60.2 percent and in the next study in 2015 the presence

of OqxA 56.7 percent and OqxB, 54.6 percent were reported. Beta-lactamase-

producing enterobacteria are among the major emerging pathogens in nosocomial

infections (Joppa et al., 2006).

There is a high prevalence of beta-lactamase-producing Klebsiella pneumoniae in

hospitals. As the treatment options available are limited, antibiotic control politics

along with the implementation of infection control measures are important.

In another study conducted on the Klebsiella pneumoniae strain, 52 patients who

were burned and hospitalized at Shahid Motahari Hospital in Tehran showed all 40

isolates resistant to ciprofloxacin, tetracycline, ceftazidime and gentamicin, with

high levels of efflux pump. AcrAB was especially expressed in ciprofloxacin-

resistant strains (Alves et al., 2006).



Conclusion

The results of the present study showed that resistance to Klebsiella bacteria

isolated from clinical specimens showed a high level of resistance to antibiotics.

However, the mechanism of resistance in these bacteria cannot be largely related to

the role of the efflux pump. However, based on the findings, resistance to

tetracycline and ciprofloxacin antibiotics is dependent on the efflux pump function.

On the other hand, the results of this study showed that antibiotic resistance in

Klebsiella pneumoniae strains isolated from clinical specimens was very high, so

physicians should be careful in prescribing the drug to be the best option. Use

medication for the patient. Antibiogram testing for all isolates at the hospital

should also be able to identify antibiotic resistance mechanisms, especially those

associated with efflux pumps, and inform physicians. However, alternative drugs to

reduce the bacterial resistance of this bacterium should be considered.

References

Alexander, C., & Rietschel, E.T. (2001). Invited review: bacterial lipopolysaccharides and

innate immunity. Journal of endotoxin research. 7(3):167-202.

Alonso, A., Morales, G., Escalante, R., Campanario, E., Sastre, L., & Martinez, J.L.

(2004). Overexpression of the multidrug efflux pump SmeDEF impairs

Stenotrophomonas maltophilia physiology. Journal of Antimicrobial Chemotherapy.

53(3):432-4.

Alves, M.S., da Silva Dias, R.C., de Castro, A.C.D., Riley, L.W., & Moreira, B.M.

(2006). Identification of clinical isolates of indole-positive and indole-negative

Klebsiella spp. Journal of clinical microbiology. 44(10):3640-6.

Bagley, S.T., Seidler, R.J., & Brenner, D.J. (1981). Klebsiella planticola sp. nov.: a new

species of Enterobacteriaceae found primarily in nonclinical environments. Current

Microbiology. 6(2):105-9.

BMB, N. (2012). Bacteriology. Navid shiraz press. 30 p.

Bsgfg, P. (2006). The Genus Enterobacter. 3.

Campos, M.A., Vargas, M.A., Regueiro, V., Llompart, C.M., Albertí, S., &

Bengoechea, J.A. (2004). Capsule polysaccharide mediates bacterial resistance to

antimicrobial peptides. Infection and immunity. 72(12):7107-14.

Cbat J.M. (1987). Role of polysaccharide and complement in susceptibility of Klebsiella

pneumonia to nonimmune serum. InfectImmune. (Role of polysaccharide and

complement in susceptibility of Klebsiella pneumonia to nonimmune serum).

Cortés, G., Borrell, N., de Astorza, B., Gómez, C., Sauleda, J., & Albertí, S. (2002).

Molecular analysis of the contribution of the capsular polysaccharide and the

lipopolysaccharide O side chain to the virulence of Klebsiella pneumoniae in a murine

model of pneumonia. Infection and immunity. 70(5): 2583-90.


Drancourt, M., Bollet, C., Carta, A., & Rousselier, P. (2001). Phylogenetic analyses of

Klebsiella species delineate Klebsiella and Raoultella gen. nov., with description of

Raoultella ornithinolytica comb. nov., Raoultella terrigena comb. nov. and Raoultella

planticola comb. nov. International journal of systematic and evolutionary

microbiology. 51(3): 925-32.

Feizabadi, M.M., Delfani, S., Raji, N., Majnooni, A., Aligholi, M., Shahcheraghi, F., &

et al. (2010). Distribution of bla TEM, bla SHV, bla CTX-M genes among clinical

isolates of Klebsiella pneumoniae at Labbafinejad Hospital, Tehran, Iran. Microbial

drug resistance. 16(1):49-53.

Ferragut, C., Izard, D., Gavini, F., Kersters, K., De Ley, J., & Leclerc, H. (1983).

Klebsiella trevisanii: a new species from water and soil. International Journal of

Systematic and Evolutionary Microbiology. 33(2):133-42.

Forbes, B.A. & Weissfeld, A.S. (2007). Enterobacteriaceae. Bailey & Scott's Diagnostic

Microbiology. 12.

Galani, I., Souli, M., Panagea, T., Poulakou, G., Kanellakopoulou, K., & Giamarellou,

H. (2012). Prevalence of 16S rRNA methylase genes in Enterobacteriaceae isolates

from a Greek university hospital. Clinical Microbiology and Infection. 18(3): E52-E4.

Gavini, F., Izard, D., Grimont, P., Beji, A., Ageron, E., & Leclerc, H. (1983). Priority of

Klebsiella planticola Bagley, Seidler, and Brenner 1982 over Klebsiella trevisanii

Ferragut, Izard, Gavini, Kersters, DeLey, and Leclerc. International Journal of


Habeeb, M.A., Haque, A., Nematzadeh, S., Iversen, A., & Giske, C.G. (2013). High

prevalence of 16S rRNA methylase RmtB among CTX-M extended-spectrum β-

lactamase-producing Klebsiella pneumoniae from Islamabad, Pakistan. International

journal of antimicrobial agents. 41(6): 524-6.

Handzlik, J., Matys, A., & Kieć-Kononowicz, K. (2013). Recent advances in multi-drug

resistance (MDR) efflux pump inhibitors of Gram-positive bacteria S. aureus.

Antibiotics. 2(1):28-45.

Hashemi, A., Fallah, F., Erfanimanesh, S., Hamedani, P., Alimehr, S., & Goudarzi, H.

(2014). Detection of β-lactamases and outer membrane porins among Klebsiella

pneumoniae strains isolated in Iran. Scientifica.

Hbjak, J. (1987). Clinical and pathogenic Microbiology. 5, (Clinical and pathogenic

Microbiology).

Hsueh, C., Chin, J., Yu, Y., Chen, H., Li, W., Shen, M. & et al. (1977). Genetic analysis

of the nitrogen fixation system in Klebsiella pneumoniae. Scientia Sinica. 20(6): 807-

17.

Huang, H., Gong, C.S., & Tsao, G.T. (2002). Production of 1, 3-propanediol by

Klebsiella pneumoniae. Biotechnology for Fuels and Chemicals. Springer. 687-98.

Izard, D., Ferragut, C., Gavini, F., Kersters, K., De Ley, J., & Leclerc, H. (1981).

Klebsiella terrigena, a new species from soil and water. International Journal of


Joppa, B., Cole, S., & Gallagher, S. (2006). Pulsed-Field Electrophoresis for Separation

of Large DNA. 2.



Kang, H.Y., Kim, J., Seol, S.Y., Lee, Y.C., Lee, J.C., & Cho, D.T. (2009).

Characterization of conjugative plasmids carrying antibiotic resistance genes encoding

16S rRNA methylase, extended-spectrum beta-lactamase, and/or plasmid-mediated

AmpC beta-lactamase. The Journal of Microbiology. 47(1):68-75.

Kosmidis, C., Schindler, B.D., Jacinto, P.L., Patel, D., Bains, K., Seo, S.M., & et al.

(2012). Expression of multidrug resistance efflux pump genes in clinical and

environmental isolates of Staphylococcus aureus. International journal of

antimicrobial agents. 40(3): 204-9.

Li, X.Z., & Nikaido, H. (2004). Efflux-mediated drug resistance in bacteria. Drugs.

64(2):159-204.

Lin, J.C., Chang, F.Y., Fung, C.P., Xu, J.Z., Cheng, H.P., Wang, J.J., & et al. (2004).

High prevalence of phagocytic-resistant capsular serotypes of Klebsiella pneumoniae

in liver abscess. Microbes and infection. 6(13): 1191-8.

Liu, Y.G., & Chen, Y. (2007). High-efficiency thermal asymmetric interlaced PCR for

amplification of unknown flanking sequences. Biotechniques. 43(5):649-56.

Ma, L., Lin, C.J., Chen, J.H., Fung, C.P., Chang, F.Y., Lai, Y.K., & et al. (2009).

Widespread dissemination of aminoglycoside resistance genes armA and rmtB in

Klebsiella pneumoniae isolates in Taiwan producing CTX-M-type extended-spectrum

β-lactamases. Antimicrobial agents and chemotherapy. 53(1):104-11.

Mphba, H.M. (2005). Citrobacter klebsiella enterobacter serratia and other

Enterobacteriaceae. 2005;10.

Nassif, X., & Sansonetti, P.J. (1986). Correlation of the virulence of Klebsiella

pneumoniae K1 and K2 with the presence of a plasmid encoding aerobactin. Infection

and Immunity. 54(3):603-8.

Nishino, K., Nikaido, E., & Yamaguchi, A. (2009). Regulation and physiological function

of multidrug efflux pumps in Escherichia coli and Salmonella. Biochimica et

Biophysica Acta (BBA)-Proteins and Proteomics. 1794(5): 834-43.

Pakzad, I., Karin, M.Z., Taherikalani, M., Boustanshenas, M., & Lari, A.R. (2013).

Contribution of AcrAB efflux pump to ciprofloxacin resistance in Klebsiella

pneumoniae isolated from burn patients. GMS hygiene and infection control. 8(2).

Piddock, L.J. (2006). Multidrug-resistance efflux pumps? not just for resistance. Nature

Reviews Microbiology. 4(8):629.

Piddock, L.J. (2006). Clinically relevant chromosomally encoded multidrug resistance

efflux pumps in bacteria. Clinical microbiology reviews. 19(2):382-402.

Poole, K. (2007). Efflux pumps as antimicrobial resistance mechanisms. Annals of

medicine. 39(3):162-76.

Roberts, I.S. (1989). SFK. Interactions between bacterial surfaces and phagocyte plasma

membranes. Portland Press Limited.

Sakazaki, R., Tamura, K., Kosako, Y., & Yoshizaki, E. (1989). Klebsiella

ornithinolytica sp. nov., formerly known as ornithine-positiveKlebsiella oxytoca.

Current Microbiology. 18(4): 201-6.

Shakil, S., Khan, R., Zarrilli, R., & Khan, A.U. (2008). Aminoglycosides versus

bacteria–a description of the action, resistance mechanism, and nosocomial

battleground. Journal of biomedical science. 15(1):5-14.


Sato, T., Yokota, S., Okubo, T., Ishihara, K., Ueno, H., Muramatsu, Y., & et al. (2013).

Contribution of the AcrAB-TolC efflux pump to high-level fluoroquinolone resistance

in Escherichia coli isolated from dogs and humans. Journal of Veterinary Medical

Science. 75(4): 407-14.

Struve, C., Bojer, M., & Krogfelt, K.A. (2009). Identification of a conserved

chromosomal region encoding Klebsiella pneumoniae type 1 and type 3 fimbriae and

assessment of the role of fimbriae in pathogenicity. Infection and immunity.

77(11):5016-24.

Sun, J., Deng, Z., Yan, A. (2014). Bacterial multidrug efflux pumps: mechanisms,

physiology and pharmacological exploitations. Biochemical and biophysical research

communications. 453(2):254-67.

Wilson’s, T. (2006). Microbiology and Microbial Infections. Enterobacteriaceas.

10(Microbiology and Microbial Infections).

Williams, P., Lambert, P.A., Brown, M.R., & Jones, R.J. (1983). The role of the O and

K antigens in determining the resistance of Klebsiella aerogenes to serum killing and

phagocytosis. Microbiology. 129(7): 2181-91.

Wu, Q., Zhang, Y., Han, L., Sun, J., & Ni, Y. (2009). Plasmid-mediated 16S rRNA

methylases in aminoglycoside-resistant Enterobacteriaceae isolates in Shanghai,

China. Antimicrobial agents and chemotherapy. 53(1):271-2.

Yang, J., Ye, L., Wang, W., Luo, Y., Zhang, Y., & Han, L. (2011). Diverse prevalence

of 16S rRNA methylase genes armA and rmtB amongst clinical multidrug-resistant

Escherichia coli and Klebsiella pneumoniae isolates. International journal of

antimicrobial agents. 38(4): 348-51.

Yu, F., Wang, L., Pan, J., Yao, D., Chen, C., Zhu, T., & et al. (2009). Prevalence of 16S

rRNA methylase genes in Klebsiella pneumoniae isolates from a Chinese teaching

hospital: coexistence of rmtB and armA genes in the same isolate. Diagnostic

microbiology and infectious disease. 64(1):57-63.

Zou, T., Lum, C.T., Lok, C.N., Zhang, J.J., Che, C.M. (2015). Chemical biology of

anticancer gold (III) and gold (I) complexes. Chemical Society Reviews. 44(24):8786-

801.



65

Cytokines and Peri-Implant Disease

Hawa Adli Life Sciences Department, Khazar University

[email protected]

Abstract

The aim of this review was to summarize data from studies making inquiries on levels of

the cytokines in pathogenesis of peri-implant diseases. Here we reviewed the recent

developments on IL-1β, IL-10, IL-17, IL-21, and IL-33. We highlighted recent advances

during the last few years in this area and discussed their roles in immune regulation in

patients with peri-implant disease.Our results revealed that identifying the levels of these

cytokines (IL-1β, IL-10, IL-17, IL-21, and IL-33) may help to predict the diagnosis of peri-

implant diseases. From a safety perspective, this study emphasized the need to consider the

impact of these interleukin on peri-implant diseases.

Keywords: Peri-implant diseases, cytokines, interleukins

Introduction

Dental implantation is a surgical process of alloplastic material insertion into the

hard and soft tissues of maxillary and mandibular jaws (Petkovic-Curcin et al.,

2011). The immune reaction of organism against any artificial material into the

body is going to protect the organism against pathogens and injuries, and these

reactions will lead to complete recovery of the tissues and accepting implants or

break the implant functionality and cause implant failure, so that, in worst

conditions the implant should be removed (Petkovic-Curcin et al., 2011;

Kondyurina & Kondyurin, 2020). Peri-implant lesions have shown to contain a

wide range of inflammatory cell infiltrate, with greater proportions of plasma cells,

neutrophils and macrophages (Schincaglia et al., 2016).

Cytokines are small messenger proteins which are mostly secreted by immune cells

and act as key modulators of immune response by playing a strategic role in

differentiation, maturation, and activation of various cells types. (Lipiäinen et al.,

2015; Su et al., 2012) Depending on specific local environments, cytokines exhibit


66 Hawa Adli

either pro-inflammatory effects or anti-inflammatory effects or both (Su et al.,

2012).

Pro-inflammatory cytokines are immune-regulatory cytokines that promote

inflammation (Proinflammatory cytokines, 2020). They are mainly produced by

activated macrophages and involved in up-regulation of inflammatory process.

Major pro-inflammatory cytokines include IL-1β, IL-6, and TNF-α (Proinflam-

matory cytokines list, 2020). The anti-inflammatory cytokines are a sequence of

immune-regulatory molecules that control the Pro-inflammatory response. The

typical anti-inflammatory cytokines are interleukin (IL)-1 receptor antagonist, IL-4,

IL-10, IL-11, and IL-13 (Anti-inflammatory cytokines, 2020). In order to maintain

health, there should be a balance between pro-inflammatory and anti-inflammatory

cytokines (Inflammatory cytokine, 2020). Imbalances between these two usually

obstruct the resolution of inflammation and, instead, lead to tissue destruction

(Duarte et al., 2016). Inflammatory cytokines can perform as a predictive and

preventive biological tool as well (Farhad et al., 2019).

The expression of cytokines is affected by genetic, epigenetic, microbiota

heterogeneity and environmental factors (Teixeira et al., 2016). Peri-implant

mucositis and peri- implantitis are two main inflammatory processes affecting

tissues around the functional dental implant and due to inflammatory disorders, the

process may cause implant loss. Nevertheless, evaluating the inflammatory

cytokine levels could be a tool for early diagnose, prevention and therapy of peri-

implant disease (Severino et al., 2016).

In this sense, saliva is an abundant biological fluid and it’s used as a diagnostic tool

for a variety and range of diseases. More than 3000 proteins and peptides have

been characterized by recent proteomic approaches in human saliva involved in

different biological functions in the oral cavity (Bhattarai et al., 2018; Ventura et

al., 2018). Thus, salivary cytokines could be important to analyze the changes in

peri-implant diseases (Fonseca et al., 2012). Moreover, saliva samples are easy to

be collected in a noninvasive manner and at low-price, but in few articles, authors

examined salivary biomarkers to investigate the presence and progression of the

peri-implant disease (Teixeira et al., 2016).

The present study was designed to determine the importance of IL-1β, IL-10, IL-

17, IL-21, IL-33 as biochemical markers for early diagnosis and early prevention of

peri-implant diseases.

Interleukin 1 beta (IL-1β) - IL-1β is a pro-inflammatory cytokine present in

healthy peri-implant tissues in certain concentration. The progression of gingival

Cytokines and Peri-Implant Disease 67

inflammation is due to the consequence of IL-1β and IL-6 activities, which cause a

significant tissue damage by osteoclasts activation (Petkovic-Curcin et al., 2011).

Interleukin 10, 17 and 21 (IL-10, IL-17 and IL-21) - IL‐10 and IL‐17 are among

the new cytokines that have not yet been extensively investigated in prior

researches (Farhad et al., 2019). IL‐10 is a human cytokine with potent anti-

inflammatory properties. Thereby, IL‐10 prevents host damaging by limiting

immune response to pathogens. IL‐17 is a pro-inflammatory cytokine that plays

both protective and tissue destruction role in immune system. The main role of IL-

17 against infection is to influence on neutrophils and activate macrophages in the

infectious origin (Farhad et al., 2019). The occurrence of peri-implant disease is

the result of a dysregulated host immune response to periodontal bacteria. A

number of existing studies have recently focused on presence and function of IL-17

in these diseases (Farhad et al., 2019; Lira-Junior et al., 2019). According to the

result of a study conducted in 2014, concentrations of IL-17 reduced as a

consequence of the progression of gingivitis to periodontitis means that in the

presence of higher depth of dental pocket the level of IL-17 was decreased

(Johnson et al., 2004). In another comparative study between mucositis and peri-

implantitis sites reported in 2016, a significant increase has been found in IL-21

levels in mucositis than peri-implantitis according to author’s judgment that was

the first study to investigate the expression of IL-21 in peri-implant disease

(Teixeira et al., 2016).

Interleukin 33 (IL-33) - The IL-33 is a member of the IL-1 family and plays a

central role in immune responses where has recently demonstrated that the

Production of IL-33 by induction of RANKL is Associated with periodontal

disease. Therefore, by increasing the levels of IL-33 the tissue destruction may

increase in mucositis and peri-implantitis (Severino et al., 2016).

Currently, the frequent diagnostic tools for peri-implant disease are radiographic

parameters and clinical signs including probing depth, bleeding on probing,

suppuration, degree of implant mobility and bone loss (Duarte et al., 2016).

Though, it is not easy to perform all of these diagnostic methods or might not be

sufficient enough to differentiate the peri-implant disease onset, activity and

progression; That is why, there is a need of an investigative effort to identify the

levels of different cytokines which may help to predict more accurate diagnosis,

activity and progression of peri-implant disease. Particularly few studies have been

carried out and explored the role of interleukins in the pathogenesis of peri-implant

diseases (Farhad et al., 2019).

https://en.wikipedia.org/wiki/Interleukin_1

68 Hawa Adli

Conclusion

The available evidence suggests that levels of certain interleukins (IL-1β, IL-10,

IL-17, IL-21, and IL-33) may aid in the diagnosis and early prevention of peri-

implant diseases when used in combination with other biomarkers and approaches.

Consequently, further research is needed to obtain determinative results in this

regard.

References

Anti-inflammatory cytokines. (2020). https://www.sinobiological.com/Anti-

inflammatory-cytokines.html

Bhattarai, K., Kim, H., & Chae, H. (2018). Compliance with Saliva Collection Protocol

in Healthy Volunteers: Strategies for Managing Risk and Errors. International Journal

of Medical Sciences, 15(8), 823-831.

Duarte, P., Serrão, C., Miranda, T., Zanatta, L., Bastos, M., & Faveri, M. (2016).

Could cytokine levels in the peri-implant crevicular fluid be used to distinguish

between healthy implants and implants with peri-implantitis? A systematic

review. Journal of Periodontal Research, 51(6), 689-698.

Farhad, S. Z., Rezazadeh, F., & Mohammadi, M. (2019). Interleukin - 17 and

Interleukin-10 as Inflammatory and Prevention Biomarkers in Periimplant

Diseases. International journal of preventive medicine, 10, 137.

Fonseca, F., Junior, M., Lourenço, E., de Moraes Teles, D., & Figueredo, C. (2012).

Cytokines expression in saliva and peri-implant crevicular fluid of patients with peri-

implant disease. Clinical Oral Implants Research, 25(2).

Inflammatory cytokine. (2020). https://en.wikipedia.org/wiki/Inflammatory_cytokine

Johnson, R., Wood, N., & Serio, F. (2004). Interleukin-11 and IL-17 and the Pathogenesis

of Periodontal Disease. Journal of Periodontology, 75(1), 37-43.

Kondyurina, I., & Kondyurin, A. (2020). Foreign body reaction (immune respond) for

artificial implants can be avoided. rom https://arxiv.org/pdf/1905.02500.pdf

Lipiäinen, T., Peltoniemi, M., Sarkhel, S., Yrjönen, T., Vuorela, H., Urtti, A., &

Juppo, A. (2015). Formulation and Stability of Cytokine Therapeutics. Journal of

Pharmaceutical Sciences, 104(2), 307-326.

Lira-Junior, R., Teixeira, M., Lourenço, E., Telles, D., Figueredo, C., & Boström, E.

(2019). CSF-1 and IL-34 levels in peri-implant crevicular fluid and saliva from patients

having peri-implant diseases. Clinical Oral Investigations.

Petkovic-Curcin, A., Matic, S., Vojvodic, D., Stamatovic, N., & Todorovic, T. (2011).

Cytokines in pathogenesis of peri-implantitis. Vojnosanitetski Pregled, 68(5), 435-440.

Proinflammatory cytokines. (2020). Retrieved 5 January 2020, from

https://www.sinobiological.com/Proinflammatory-cytokines.html

Proinflammatory cytokines list. (2020).

https://www.sinobiological.com/Proinflammatory-cytokines-list.html

https://www.sinobiological.com/Anti-inflammatory-cytokines.html

https://www.sinobiological.com/Anti-inflammatory-cytokines.html

https://en.wikipedia.org/wiki/Inflammatory_cytokine

https://arxiv.org/pdf/1905.02500.pdf

https://www.sinobiological.com/Proinflammatory-cytokines.html

https://www.sinobiological.com/Proinflammatory-cytokines-list.html

Cytokines and Peri-Implant Disease 69

Schincaglia, G., Hong, B., Rosania, A., Barasz, J., Thompson, A., & Sobue, T. (2016).

Clinical, Immune, and Microbiome Traits of Gingivitis and Peri-implant

Mucositis. Journal of Dental Research, 96(1), 47-55.

Severino, V., Beghini, M., de Araújo, M., de Melo, M., Miguel, C., Rodrigues, W., &

de Lima Pereira, S. (2016). Expression of IL-6, IL-10, IL-17 and IL-33 in the peri-

implant crevicular fluid of patients with peri-implant mucositis and peri-

implantitis. Archives of Oral Biology, 72, 194-199.

Su, D., Lu, Z., Shen, M., Li, X., & Sun, L. (2012). Roles of Pro- and Anti-Inflammatory

Cytokines in the Pathogenesis of SLE. Journal of Biomedicine and

Biotechnology, 2012, 1-15.

Teixeira, M., Lira-Junior, R., Telles, D., Lourenço, E., & Figueredo, C. (2016). Th17-

related cytokines in mucositis: is there any difference between peri-implantitis and

periodontitis patients? Clinical Oral Implants Research, 28(7), 816-822.

Ventura, T., Ribeiro, N., Dionizio, A., Sabino, I., & Buzalaf, M. (2018). Standardization

of a protocol for shotgun proteomic analysis of saliva. Journal of Applied Oral

Science, 26(0).



70

System Perspective Analysis for Molecular and

Genetic Source of Salt Tolerance in Cotton

Shadar Alizada1*, Rauf Guliev1, Ruhangiz Mammadova2,

Mohammad Zaefizadeh3 1Baku State University; 2Genetic Resources Institute of ANAS;

3Islamic Azad University, Ardabil Branch *Corresponding author: [email protected]

Abstract

Sources of tolerance salinity stress in plants are the results of sets of the simple effect

and interaction among genes. A systematic perspective at the polygenic trait process

of resistance to environmental stresses, including salinity stress, and the interactions

among genes and their inheritance, may provide new insights into the process of

integrating beneficial genes into genotypes. In this study, we aimed interaction of

candidate genes include NHX1, TPS1, ERF2, SOD, CIPK, PP2C. Salinity was

selected and their correlation was calculated using their pathway. The results showed

that the effect of antioxidant gene alignment including SOD, the most direct effect

and the genes most effective in regulating sodium / potassium channels and

antiporters were the second most effective factor. The relationship between these two

groups of genes and their protein activity was positive and highly significant (Pvalue

<0.001). This result showed that, strengthening of antioxidant systems in cotton

either directly or indirectly through environmental induction or through trans

activators can be effective in salt stress tolerance of genotypes. If we ignore the

effects of environmental induction regulated by agro-ecophysiological conditions,

most transcriptional factors study focused on ERF1. It was through binding to CIS

elements that were effective in resisting to salt stress.

Keywords: pathway studio, cotton, salt stress, genetic tolerance


System Perspective Analysis for Molecular and Genetic Source of Salt

Tolerance in Cotton 71

Introduction

Three categories of soil affected by salt was classified in: saline soils, alkaline soils

and salt-alkaline soils (Novo et al., 2006). Saline soils comprise excessive amount of

neutral salts, which include NaCl and Na2SO4, as a major part, resulting in salt

stress. NaOH and Na2CO3 are responsible for the saline and alkalization of soils by

creating a high pH value, with a destructive effect on plants growth (Shi et al., 2005).

Stress resulted from alkaline soils causes several issues of osmotic pressure stress,

diferent types of ionic injuries and high pH stress. Plants under salt-alkaline

conditions sufer from both salt stress caused by excessive salt ions and alkaline stress

caused by high pH (Zhang et al., 2013). There are different genes for biological

processing, cellular component and molecular function in the cotton under salt stress

(Figure 1).

In general, most of the genes and proteins related to Na+ stress (treated with NaCl)

and high pH (treated with NaOH) are also involved in the pathways against Na2CO3

stress. High pH leads to oxygen deprivation stress, which causes cotton organs

nigrities. High pH also increases the synthesis of pectin-related enzymes, that

strengthens cell wall to defense damage of high pH. In the process of the hydrolysis

of ATPase, extra H+ produced help to neutralize OH− within the cytoplasm

(Figure 2).

Besides, genes and proteins related with ion homeostasis under Na+ stress were also

found in our study, such as protein kinases, transcription and transporters. These

genes and proteins have been reported in previous studies. MYBs that regulate genes

of the anthocyanin pathway were up-regulated under Na2CO3 stress, which always

cause leaves to turn red or orange in apple (Min et al., 2016). Here, our study

provides some candidate genes, particularly responding to high pH and Na+ stresses.

For instance, Hexokinase (HXK) acted as a sugar sensor in eukaryotic cells (Min et

al., 2016), being found to be up-regulated under high pH stress, which indicates that

genes encoding HXK may be related to high pH stress (Bingeli et al., 2018).

There are several different genes on the Na+ tolerance mechanism and other stress

resistant, which had complicity interaction between.

72 Shadar Alizada, Rauf Guliev, Ruhangiz Mammadova, Mohammad Zaefizadeh

Figure 1. Different genes for biological processing, cellular component and molecular

function in the cotton under salt stress (Bingeli et al., 2018)



Figure 2. Model of the regulatory networks to Na+ stress and high pH [Bingeli et al., 2018]

Trehalose-6-phosphase synthase (TPS)

Trehalose, a non-reducing disaccharide, is composed of two glucose molecules that

are connected by α, α-1, 1- glycosidic linkage and exist in bacteria, fungi, algae and

plants (Elbein et al., 2001). TPS was synthesized in the high osmolarity cell

condition. Trehalose protects bioactive substances and cell structures, such as

proteins, nucleic acids, and biological membranes, under adverse environmental

stresses, such as drought, freezing, oxidation, high salt, high temperature and low

temperature (Garg et al., 2002). Trehalose synthesis in plants is a two-step process:

first, TPS catalyzes UDP-glucose and glucose-6-phosphate to generate trehalose-6-

phosphate (T6P); second, trehalose-6- phosphate phosphatase catalyzes the

dephosphorylation of trehalose-6-phosphate to trehalose. The structure of TPS

proteins in plants contains two domains: TPS and TPP; however, many studies have

shown that the TPP domain in TPS proteins appears to have lost enzymatic activity

during evolution (Vandesteene et al., 2010). The 3d structure of TPS on the figure 3

was shown.


Figure 3. The 3D structure of Trehalose-6-phosphase synthase (TPS)

A TPS gene was cloned named as AtTPS1 which had the trehalose-6-phosphase

synthetase function from higher plants (Blazquez et al., 1998). The AtTPS1 mutant

TPS1 was a recessive embryonic lethal gene [Eastmond et al, 2002]. Even so,

AtTPS1 played an important role in the process of vegetative growth and transition to

flowering (WGomes et al., 2006). Studies showed that TPS expression levels in

cotton increased under drought stress (Kosmas et al., 2006) and TPS genes in maize

were also found upregulated in response to both salt and temperature stress (Jian et

al., 2010). OsTPS1 might enhance the abiotic stress tolerance of rice by increasing

the trehalose and proline content (Li et al., 2011). Many studies have suggested that

TPSs play a vital role in plants adjusting to environmental stresses.

Profiling of Protein Phosphatases (PP2C):

The protein kinases (PKs) and protein phosphatases (PPs) are known to regulate the

protein function, and are the fundamental molecular mechanism, by reversing protein

phosphorylation during cellular signaling (Hamna et al., 2019). Thus, it is involved

in many biological processes, such as signal transduction, development, and

environmental stimuli. The PKs phosphorylate largely serine (Ser), threonine (Thr),

and tyrosine (Tyr), whereas PPs can reverse this function by eliminating the

phosphate group (Li et al., 2014). Mg2+-dependent protein phosphatase (PP2C) is

evolutionarily conserved from Archaea to higher plants that pointedly modulate

stress signaling pathways and reverse the stress-induced PK cascades to complex



environmental stimuli (Cao et al., 2016). From various literature, several key stress-

responsive protein kinases genes have been extensively studied and proven to

respond in diverse stress conditions including biotic and abiotic factors (Boudsocq et

al., 2004).

In Arabidopsis, several members of PP2c such as PLL4 and PLL5 (POL-like gene)

are known to adjust leaf development, though with no obvious functions within the

meristem (Song et al., 2005). Cotton, as an oil crop and an important source of

natural textile fiber, plays a crucial role in agriculture and industry all around the

world. However, its production is mainly constrained due to various abiotic and

biotic stress conditions. The release of Gossypium whole-genome data in four

different cotton species, such as Gossypium arboreum (Li et al., 2014), Gossypium

barbadense (Yuan et al., 2015), Gossypium hirsutum (Zhang et al., 2015) and

Gossypium raimondii (Wong et al., 2012) and their publicly available database

allows us to comprehensively characterize the PP2C gene family based on

bioinformatic tools.

Figure 4. The 3D structure of Profiling of Protein Phosphatases (PP2C)

Following the gene structure organization analysis, conserved protein motifs, and cis-

elements, we traced the duplication gene pairs and their evolutionary divergence that

likely resulted in the widespread extension of the PP2C gene family. The protein

phosphatase (PP2C) gene family, known to participate in cellular processes, is one of

the momentous and conserved plant-specific gene families that regulate signal

transduction in eukaryotic organisms. Recently, PP2Cs were identified in


Arabidopsis and various other crop species, but analysis of PP2C in cotton is yet to

be reported.

NHX1

Protein of NHX1 with molecular function potassium and sodium antiporter activity

contend potassium ion transmembrane transport source and regulation of intracellular

pH. Sodium ion import across plasma membrane can be established resistance to salt

stress in plant.

Plants have developed a complex mechanism to prevent damages caused by

environmental changes. ABA is a pivotal element in this mechanism. ABA functions

in seed germination inhibition, growth regulation, fruit abscission, and stomatal

closure (Raghavendra et al., 2010). These better salt-tolerant accessions could be

selected as parents to accelerate the progress of cotton tolerance breeding by

molecular design (Rai et al., 2011).

Figure 5. The 3D structure of NHX1

Salt tolerance is a genetically and physiologically complex trait controlled by

multiple small effect genes (Flowers, 2004). With the fast development of SNP

arrays and next-generation sequencing technology, GWAS is becoming a novel and

effective method for determining useful genes in crop plants. Association analysis

has been successfully used in mining candidate genes of important agronomic traits

https://www.ebi.ac.uk/QuickGO/term/GO:0071805






in cotton, such as fiber quality, yield and its components, and Verticillium wilt

resistance (Wang et al., 2017). The cotton accessions consisting of diverse

germplasms worldwide showed large variations in RSR and STL under salt stress. A

total of 10 and 15 SNPs significantly associated with RSR and STL were identified,

respectively, of which the two SNPs i46598Gh and i47388Gh on D09 were

simultaneously associated with the two traits (Sun et al., 2018).

CBL-interacting protein kinase (CIPK)

A few studies have indicated that the CIPKs function in plant development (Held et

al., 2011), nutrient uptake (Qingchen et al., 2017), and pollen tube elongation (Zhou

et al., 2015). The CIPKs perform major functions in stress responses. The first

characterized CBL-CIPK network is the SOS network. Plasma membrane-localized

SOS3 (AtCBL4) recruits SOS2 (AtCIPK24). Subsequently, the CBL-CIPK complex

activates a NaC/HC antiporter SOS1 (AtNHX7), enhances sodium export, and

promotes salt tolerance (Qiu et al., 2002). The atcipk3 mutant shows a hypersensitive

phenotype when treated with salt and ABA (Kim et al., 2007). The atcipk23 mutant

exhibits enhanced drought tolerance by regulating leaf transpiration (Cheong et al.,

2007), whereas the atcipk21 mutant presents impaired salt and osmotic tolerance

(Pandey et al., 2015). Similar CBL-CIPK networks are also found in other plant

species including wheat. TaCIPK25 negatively regulated salt tolerance in transgenic

wheat (Jin et al., 2016). The over expression of the constitutively activated form of

BnCIPK6 enhances salt and low-KC tolerances, as well as ABA sensitivity in

Arabidopsis (Chen et al., 2012).

ETHYLENE RESPONSE FACTORS

Cotton is native to tropical and subtropical zones and is relatively resistant to stress

(Zhou et al., 2016). Its long growth period makes it suffer from a variety of abiotic

stresses that reduce production or quality. During the reproductive growth stage,

yield reduction and fiber quality compromises are inescapable when drought stress

conditions override the plant’s protective mechanisms (Loka & Oosterhuis, 2014).

Cotton is also susceptible to salt and cold stresses during seed germination (Shannon

& Francois, 1976). Therefore, improving the comprehensive tolerance of cotton to

numerous stresses for instance drought, salt, and extreme temperature is very fruitful

and vital for cotton breeding. ROS are oxygen-containing substances of metabolites

and their derivatives are generated through oxidation in plants directly or indirectly

(Mettler et al., 2011). ROS play a role in stress signal transduction, but excessive


active oxygen oxidation harms the plants. Additionally, fourteen genes were

identified as responsive to stresses, such as heat, dehydration and phosphate stress.

Examples of these genes are late embryogenesis-abundant protein 2, late

embryogenesis-abundant protein and dehydrin. (Zhou et al., 2016).

Figure 6. Ethylene-activated signaling pathway

Ethylene response factors (ERFs) are transcription factors that play crucial roles

in plant immunity (Na Song et al., 2019). In Arabidopsis, several ERFs have

been identified as important regulators in Botrytis resistance, such as ORA59,

ERF1, and RAP2.2 (Zhao et al., 2012). Most ERFs are able to bind specifically

to DNA sequences containing a GCC (GCC box) and/or a dehydration-

responsive element/C-repeat (DRE/CRT box, A/GCCGAC) (Na Song et al.,

2019).




Figure 7. The 3D structure of ERF2 (Source: PDB)

Figure 8. The relationship between genes and proteins to effect on the cotton salt tolerance

stress

The analysis of the relationship between genes and proteins to effect on the cotton

salt tolerance stress with pathway studio showed that, ABA-induced AtNHX1

expression was also decreased in abi1-1 but not in abi2-1. Accumulation of JA after

inoculation with different Pst strains. Activities of enzymes involved in scavenging

of reactive oxygen species such as SOD and ascorbate peroxidase also increased

during leaf maturation and showed significant fluctuations in mature leaves. After

inoculation with Pst / avrRpt2, the accumulation of SA in pad4 mutants was similar


to that in WT plants, in accordance with the observation that pad4 does not display an

enhanced susceptibility to this pathogen strain. Although total SOD activity slightly

increased in chloroplasts of salt-treated Lem plants, differentiation between SOD

types revealed that only stromal Cu/ZnSOD activity increased (Mittova et al., 2000).

References

Bailey-Serres, J., & Mittler, R. (2006). The roles of reactive oxygen species in plant cells.

Plant Physiol 141: 311.

Blazquez, M.A, Santos, E., Flores, C.L., Martinez-Zapater, J.M., Salinas, J., & Gancedo,

C. (2016). Isolation and molecular characterization of the Arabidopsis TPS1 gene,

encoding trehalose-6-phosphate synthase. Plant J. 13(5):685–9.

Boudsocq, M., Barbier-Brygoo, H., & Lauriere, C. (2004). Identification of nine sucrose

nonfermenting 1-related proteinkinases 2 activated by hyperosmotic and saline stresses

in Arabidopsis thaliana. J. Boil. Chem. 279,41758–41766.

Cao, J., Min, J., Peng, L., & Chu, Z. (2016). Genome-wide identification and

evolutionary analyses of the PP2C gene family with their expression profiling in

response to multiple stresses in Brachypodium distachyon. BMC Genomics. 17: 175.

Chen, L., Ren, F., Zhou, L., Wang, Q.Q., Zhong, H., & Li, X.B. (2012).The Brassica napus

calcineurin B-Like 1/CBL-interacting protein kinase 6(CBL1/CIPK6) component is

involved in the plant response to abiotic stressand ABA signalling. J. Exp. Bot. 63,

6211–6222.

Cheong, Y.H., Pandey, G.K., Grant, J.J., Batistic, O., Li, L., Kim, B.G., & et al. (2007).

Two calcineurin B-like calcium sensors, interacting with protein kinaseCIPK23, regulate

leaf transpiration and root potassium uptake in Arabidopsis.Plant J. 52, 223–239.

Eastmond, P.J., van Dijken, A.J.H., Spielman, M., Kerr, A., Tissier, A.F., Dickinson,

H.G., Jones, J.D.G., Smeekens, S.C., & Graham, I.A. (2002). Trehalose-6-phosphate

synthase 1, which catalyses the first step in trehalose synthesis, is essential for

Arabidopsis embryo maturation. Plant J. 2002;29(2):225–35.

Elbein, A.D, Pan, Y.T., Pastuszak, I., & Carroll, D. (2003). New insights on trehalose: a

multifunctional molecule. Glycobiology. 2003;13(4):17.

Flowers, T.J. (2002). Improving crop salt tolerance. J. Exp. Bot. 55, 307–319.doi:

10.1093/jxb/erh003

Garg AK, Kim JK, Owens TG, Ranwala AP, Do Choi Y, Kochian LV, Wu RJ. Trehalose

accumulation in rice plants confers high tolerance levels to different abiotic stresses.

Proc Natl Acad Sci U S A. 99(25):15898–903.

Gomez, L.D, Baud, S., Gilday, A., Li, Y., & Graham, I.A. (2006). Delayed embryo

development in the ARABIDOPSIS TREHALOSE-6-PHOSPHATE SYNTHASE 1

mutant isassociated with altered cell wall structure, decreased cell division and starch

accumulation. Plant J. 46(1):69–84.

Held, K., Pascaud, F., Eckert, C., Gajdanowicz, P., Hashimoto, K., Corratge-Faillie, C.,

& et al. (2011). Calcium-dependent modulation and plasma membranetargeting of the

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4776448/



AKT2 potassium channel by the CBL4/CIPK6 calciumsensor/protein kinase complex.

Cell Res. 21, 1116–1130.

Hwang, J.H., Seo, D.H., Kang, B.G., Kwak, J.M., & Kim, W.T. (2015). Suppression of

Arabidopsis AtPUB30 resulted in increased tolerance to saltstress during germination.

Plant Cell Rep. 34, 277–289.

Jiang, W., Fu, F.L., Zhang, S.Z., Wu, L., & Li, W.C. (2010). Cloning and characterization

of functional trehalose-6-phosphate synthase gene in maize. J Plant Biol. 53(2):134–41.

Jin, X., Sun, T., Wang, X.T., Su, P.P., Ma, J.F., He, G.Y., & et al. (2016). Wheat

CBLinteractingprotein kinase 25 negatively regulates salt tolerance in transgenicwheat.

Sci. Rep. 6:28884.

Kim, K.N., Cheong, Y.H., Grant, J.J., Pandey, G.K., & Luan, S. (2003). CIPK3, a

calcium sensor-associated protein kinase that regulates abscisic acid and cold signal

transduction in Arabidopsis. Plant Cell. 15, 411–423.

Kim, B.G., Waadt, R., Cheong, Y.H., Pandey, G.K., Dominguez-Solis, J.R., Schultke, S.,

& et al. (2007). The calcium sensor CBL10 mediates salt tolerance byregulating ion

homeostasis in Arabidopsis. Plant J. 52, 473–484.

Kosmas, S.A, Argyrokastritis, A., Loukas, M.G, Eliopoulos, E., Tsakas, S., & Kaltsikes,

P.J. (2006). Isolation and characterization of drought-related trehalose 6-phosphate-

synthase gene from cultivated cotton (Gossypium hirsutum L.). Planta. 223(2):329–39.

Li, H.W., Zang, B.S., Deng, X.W., & Wang, X.P. (2011). Overexpression of the trehalose-

6- phosphate synthase gene OsTPS1 enhances abiotic stress tolerance in rice. Planta.

234(5):1007–18.

Li, F., Fan, G., Wang, K., Sun, F., Yuan, Y., Song, G., Li, Q., Ma, Z., Lu, C., Zou, C. &

et al. (2014). Genome sequence ofthe cultivated cotton Gossypium arboreum. Nat.

Genet. 46, 567–572.

Loka, D.A., & Oosterhuis, D.M. (2014) Water-deficit stress effects on pistil biochemistry

and leaf physiology in cotton (Gossypium hirsutum L.). S. Afr. J. Bot. 93: 131–136.

Luo, Q., Wei, Q., Wang, R., Zhang, Y., Zhang, F., He, Y., Zhou, S., Feng, J.,Yang, G. &

He, G. (2017). BdCIPK31,a Calcineurin B-LikeProtein-Interacting Protein

Kinase,Regulates Plant Response to Droughtand Salt Stress.Front. Plant Sci. 8:1184.

Valentina, M., Moshe, T., Micha, V., & Micha, G. (2002). Salt stress induces

up‐regulation of an efficient chloroplast antioxidant system in the salt‐tolerant wild

tomato species Lycopersicon pennellii but not in the cultivated species, 2002.

Mittler, R., Vanderauwera, S., Suzuki, N., Miller, G., Tognetti, V.B., Vandepoele, K., &

et al. (2011). ROS signaling: the new wave? Trends Plant Sci. 16: 300–309.

Min, Mu., Xu-Ke, Lu., Jun-Juan, W., De-Long, W., Zu-Jun, Y., Shuai, W., Wei-Li, F., &

et al. (2016). Genome-wide Identification and analysis of the stress-resistance function

of the TPS(Trehalose-6-Phosphate Synthase) gene family in cotton. BMC Genetics,

17:54.

Na, S., Lan, M., Weiguang, W., Huanhuan S., Lei, W., Ian T., & Baldwin, J. (2019). An

ERF2-like transcription factor regulates production of the defense sesquiterpene

capsidiol upon Alternaria alternata infection. Jornal of Experimental Botany, 70(20),

5895–5908.

https://onlinelibrary.wiley.com/action/doSearch?ContribAuthorStored=Mittova%2C+Valentina

https://onlinelibrary.wiley.com/action/doSearch?ContribAuthorStored=Tal%2C+Moshe

https://onlinelibrary.wiley.com/action/doSearch?ContribAuthorStored=Volokita%2C+Micha

https://onlinelibrary.wiley.com/action/doSearch?ContribAuthorStored=Guy%2C+Micha


Novo, F.G., & Bouzas, F.G. (2006). Water and nature. Te berth of life. Water Crisis: Myth

or Reality? 235–252.

Qiu, Q.S., Guo, Y., Dietrich, M.A., Schumaker, K.S., & Zhu, J.K. (2002). Regulation of

SOS1, a plasma membrane NaC/HC exchanger in Arabidopsisthaliana, by SOS2 and

SOS3. Proc. Natl. Acad. Sci. U.S.A. 99, 8436–8441.

Raghavendra, A.S., Gonugunta, V.K., Christmann, A., & Grill, E. (2010). ABA

perception and signalling. Trends Plant Sci. 15, 395–401.

Rai, M.K., Kalia, R.K., Singh, R., Gangola, M.P., & Dhawan, A.K. (2011). Developing

stress tolerant plants through in vitro selection—An overview of therecent progress.

Environ. Exp. Bot. 71, 89–98.

Shannon, M.C., & Francois, L.E. (1976). Influence of seed pretreatments on salt tolerance

of cotton during germination. Agron J. 69: 619–622.

Shi, D.C. & Wang, D.L. (2005). Efects of various salt-alkaline mixed stresses on

Aneurolepidium chinense (Trin.) Kitag. Plant Soil 271, 15–26.

Song, S.K., & Clark, S.E. (2005). POL and related phosphatases are dosage-sensitive

regulators of meristem and organ development in Arabidopsis. Dev. Boil. 285, 272–284.

Sun, Z., Li, H., Zhang, Y., Li, Z., Ke, H., Wu, L., Zhang, G., Wang, X., & Ma, Z.

(2018) Identification of SNPsand Candidate Genes Associated With Salt Tolerance at

the Seedling Stage in Cotton (Gossypium hirsutum L.) North China Key Laboratory

for Crop Germplasm Resources of Education Ministry, Key Laboratory for Crop

Germplasm Resources of Hebei Province, Hebei Agricultural University, Baoding,

China ORIGINAL RESEARCH.

Vandesteene, L., Ramon, M., Le Roy, K., Van Dijck, P., & Rolland, F.A. (2010). single

active trehalose-6-P synthase (TPS) and a family of putative regulatory TPS-like

proteins in Arabidopsis. Mol Plant. 3(2):406–19.

Wang, K., Wang, Z., Li, F., Ye, W., Wang, J., Song, G., Yue, Z., Cong, L., Shang, H.,

Zhu, S. & et al. (2012). The draft genome of a diploid cotton Gossypium raimondii.

Nat. Genet. 44, 1098–1103.

Yuan, D., Tang, Z., Wang, M., Gao, W., Tu, L., Jin, X., Chen, L., He, Y., Zhang, L.,

Zhu, L., & et al. (2015). The genome sequence of Sea-Island cotton (Gossypium

barbadense) provides insights into the allo poly ploidization and development of

superior spinnable fibres. Sci. Rep. 5, 17662.

Zentella, R., Mascorro-Gallardo, J.O., Van Dijck, P., Folch-Mallol, J., Bonini, B., Van

Vaeck, C., Gaxiola, R., Covarrubias, A.A., Nieto-Sotelo, J., Thevelein, J.M. & et al.

(2006). A Selaginella lepidophylla trehalose-6-phosphate synthase complements growth

and stress-tolerance defects in a yeast tps1 mutant. Plant Physiol.

Zhang, B., Chen, X., Lu, X., Shu, N., Wang, X., Yang, X., Wang, S., & et al. (2018).

Transcriptome Analysis of Gossypium hirsutum L. Reveals Diferent Mechanisms among

NaCl, NaOH and Na2CO3 Stress Tolerance.SCIentIfIC RepoRTS. 8:13527.

Zhang, T., Hu, Y., Jiang, W., Fang, L., Guan, X., & Chen, J. (2015). Sequencing of

allotetraploid cotton (Gossypium hirsutum L. acc. TM-1) provides a resource for fiber

improvement. Nat. Biotechnol. 2015, 33(5):531-7.



Zhang, X., Wei, L., Wang, Z. & Wang, T. (2013). Physiological and molecular features of

Puccinellia tenuifora tolerating salt and alkaline-salt stress. Journal of integrative plant

biology 55, 262–276.

Zhao, Y., Wei, T., Yin, K.Q., Chen, Z., Gu, H., Qu, L.J., & Qin, G. (2012).

ArabidopsisRAP2.2 plays an important role in plant resistance to Botrytis cinerea

andethylene responses. New Phytologist 195, 450–460.

Zhou, B., Zhang, L., Ullah, A., Jin, X., Yang, X., Zhang, X. (2016). Identification of

Multiple Stress Responsive Genes by Sequencing a Normalized cDNA Library from

Sea-Land Cotton (Gossypium barbadense L.). PLoS ONE 11(3): e0152927.

Zhou, Y.L., Sun, X.D., Yang, Y.Q., Li, X., Cheng, Y., & Yang, Y.P. (2016). Expression of

Stipa purpurea SpCIPK26 in Arabidopsis thaliana enhances saltand drought tolerance

and regulates abscisic acid signaling. Int. J. Mol. Sci.17:966.



84

Classification of Grey-Cinnamon (Chestnut) Soils

with Nutrient Source of the Shamkirchay Reservoir

and Analysis of Morphogenetic Diagnostic Indices

Ramil Sadigov Department of Petrochemical Technology and Industrial Ecology, Azerbaijan State

University of Oil and Industry,

Institute of Soil Sciences and Agrochemstry of ANAS

[email protected]; [email protected]

Abstract

The mountain grey-cinnamon (chestnut) soils which are irrigated by the Shamkirchay water

reservoir is formed mainly on the foothill, plain and partly on the mountainousborders and

orographically on the northern east slope of the Little Caucasus are situated between 40039/

- 41000/ north latitude and 45050/ - 46020/ east longitude. The classification of mountain

grey-cinnamon soils in the zone with 54263,74 ha on subtypes and analysis of the

morphogenetic diagnostic indices has been given in the article. 7 subtypes of mountain

grey-cinnamon soils are formed in the zone: separate morphological description of each

subtype was analyzed in the article. At the same time, a modern state of the main

physicochemical and fertility indices of the soils in the basin zone and information about

the researches performed in the soils inside of the Shamkir, Goy-gol and Samukh borders

have been presented. The diagnostic indices of the soil sections in the characteristic places,

morphological description and agrochemical features of the soil profiles on subtypes and

their analysis result were explanatory analyzed. Humus, total nitrogen, phosphorus and

potassium, granulometric composition (sand, dust, silt and clay fractions) environment

reaction of soil (pH) and calcareous (CaCO3) were fixed, statistically analyzed.

Keywords: water reservoir, canal, agro-ecological evolution, mountain grey-

cinnamon (chestnut), humus layer.

Introduction

One of the most global problems of the XXI century is protection of the

environment, including soil cover. Though the man plays a main role in nature-

human-nature relations, it directly and indirectly depends on nature. Air, water,

material blessings, natural resources, raw materials for industry are important


Shamkirchay Reservoir and Analysis of Morphogenetic Diagnostic Indices 85

blessings which are presented to the man by the nature. Recently, the environment

has been subjected to anthropogenic change. The high development of the modern

scientific-technical progress deepened this process (Babayev et al., 2011a;

Babayev et al., 2011; Sadig & Mammadov, 2018; Sadigov, 2013; Sadigov, 2018b).

The zones which are irrigated by the Shamkirchay water reservoir developed

historically as a farming zone. Here 6 soil types and 12 subtypes are formed in the

zone with 72279,34 ha. The information about mountain grey-cinnamon (chestnut)

soils (54263,74 ha) is given in the article.

The zone of mountain grey-cinnamon (chestnut) soils on subtypes is given in

hectare scale on the first table. The mostly spreading subtype is ordinary grey-

cinnamon (chestnut) soils which form 17063,25 ha, but the lastly spreading

subtype is fully undeveloped mountain grey-cinnamon (chestnut) soil with 4480,47

ha. The soils analyzed are presented with blue color on Table 1 in the article (Sadig

& Mammadov, 2018; Sadigov, 2013; Sadigov, 2018a).

Mountain grey-cinnamon soils spread at 200-600 meter height from sea level in the

research zone. Here the vegetation mat grass-tipsak different grassy plants and

wormwood-ephemera-grain plants spread in dry steppes. The soil forming rocks

are lime-stones, lime-stony conglomerates, tuffaceous breccia and their soft

weathering products.

The winter of the zone is dry, but the summer is subtropical climatic. The days

with the temperatures above 100 C are 210-240, but the days with the temperatures

above 50 C are 240-270.

Generally, mountain grey-cinnoman soils cover 75,07 % of the research zone.

Before us, study of the soil cover of the Little Caucasus was performed by

V.V.Dochucaev, M.M.Sibirtsev, M.P.Babayev, V.H.Hasanov, Ch.M.Jafarova,

H.M.Hajiyev, B.G.Shakuri, K.A.Alakbarov, V.R.Volobuev, M.E.Salaev,

A.A.Ibrahimov, A.M.Shikhlinsky, A.N.Izyumov and other scientist (Babayev et

al., 2011; Ministr, 1972; Mammadov, 2007; Sadigov, 2013).

Table 1. Soil types and subtypes included in the Shamkirchay

№ Name of lands Areas (ha)

On types On subtypes

1. Mountain cinnamon-meadow 725,77

2. Mountain-forest-cinnamon 2498,43

3. Mountain grey-cinnamon (chestnut) 54263,74

3.1. Fully undeveloped mountain grey-cinnamon

(chestnut)

4480,47

3.2. Anciently irrigated solonetzlike ordinary grey- 3912,56

86 Ramil Sadigov

cinnamon (chestnut)

3.3. Irrigated mountain bright grey-cinnamon (chestnut) 7603,44

3.4. Irrigated solonetzlike ordinary grey-cinnamon

(chestnut)

17063,25

3.5. Irrigated gazh mountain ordinary grey-cinnamon

(chestnut)

5086,97

3.6. Irrigated mountain ordinary grey-cinnamon

(chestnut)

6299,59

3.7. Anciently irrigated saline mountain ordinary grey-

cinnamon (chestnut)

9817,46

4. Mountain grey-cinnamon (chestnut) meadow 10522,55

4.1. Irrigated mountain grey-cinnamon (chestnut)

meadow

10522,55

5. Alluvial-meadow 683,96

5.1. Leached alluvial-meadow 546,44

5.2. Weakly developed calcareous alluvial-meadow 137,52

6. Alluvial-meadow-forest 558,75

6.1. Glayey-alluvial-meadow-forest 558,75

7. Strong high gypsum fully developed clayey-salty

rocks-soil-grounds

837,98

8. Cobblestone fine sediments of the riverbeds 2168,16

Total 72279,34

Material and methods

The material for the research was determined in 2 parts: theoretic and practical

part. So, the result of the long complex researches about classifiation, nomenclature

and diagnostics of soils in Azerbaijan has been analyzed in the theoretic part. The

alalyses were performed in the soil section. On the basic of the modern methods

used in the world experiment , the results were obtained in the practical part. The

land map was compiled on the basis od ArcCIS program and the soil types and

subtypes were concerned to the International Land Names (WRB). During the

research, the soil horizons indexing was performed. The genetic indications of

soils were adapted to the correlation with the main indices of Azerbaijan land

classifications od WRB system (Soil groups).

The main aim of the research is an investigation on subtypes of the mountain grey-

cinnoman soil type in which the Shamkirchay water reservoir is nutrient, study of

the impact of natural and antropogenic factors, regulation of the fertility indices in

these soils, adapting of morphogenetic diagnostics and soil subtypes nomenclature

to modern classification.

Mountain grey-cinnamon soils with nutrient source of Shamkirchay reservoir



widely spread in 54263,74 ha in three districs (in Shamkir, Goy-gol, Samukh

regions). Mountain grey-cinnamon soils in the same regions are taken as a research

object. Anciently, irrigated solonetzlike mountain ordinary grey cinnamon

(chestnut), irrigated solontzlike ordinary grey-cinnamon (chestnut), irrigated

mountain bright grey-cinnamon (chestnut), irrigated gazh mountain ordinary grey-

cinnamon (chestnut), irrigated mountain ordinary grey-cinnamon (chestnut),

anciently irrigated saline mountain ordinary grey-cinnamon (chestnut) soils have

been analyzed. Recently, there have been couple of analyses on some

mountaninous zones such as irrigated solonetzlike mountain.

Table 2. Classification of mountain grey-cinnamon soils of WRB (Soil groups)

№ Name of soils Clasification on WRB

1 Mountain grey-cinnamon (chestnut) Mgc

1.1 Fully undeveloped mountain grey-

cinnamon (chestnut)

Mgcfu

1.2 Anciently irrigated solonetzlike

ordinary grey-cinnamon (chestnut)

Mgcai.slo

1.3 Irrigated mountain bright grey-

cinnamon (chestnut)

Mgc1ib

1.4 Irrigated solonetzlike ordinary grey-

cinnamon (chestnut)

Mgc2i.sl

1.5 Irrigated gazh mountain ordinary

grey-cinnamon (chestnut)

Mgc2i

1.6 Irrigated mountain ordinary grey-

cinnamon (chestnut)

Mgc2i

1.7 Anciently irrigated saline mountain

ordinary grey-cinnamon (chestnut)

Mgc2a.is

The researches were conducted in mountain grey-cinnamon (chestnut) soils on

certain routes in 2015-2020. The sections were applied in the characteristic places

(definition on geigraphical coordinates). The soil section was taken from

characteristic places (one soil section on each subtype). It’s density, granulometric

composition, colour, structure, hardness and some morphological indications were

registered. The geographical coordinates of the taken soil samples were defined by

“Garmin GPS map 62s” apparatus. The taken soil was given to the laboratory of

“Agroecology and Bonitation of Soils” in the Istitute of Soil Science and

Agrochemstry of ANAS for physicochemical analyses and the required prosedures

were realized on the basis of the adopted methods (AZS ISO, 2013; Ministry,

1972; Mammadov, 2007; Sadigov, 2016).

88 Ramil Sadigov

During the field researches, the total humus was investigated by I.M.Turin’s

method, total nitrogen-by Keildal and carbonates-by Calcimeter apparatus. In the

form of CaCO3, was analyzed by the titration method, total phosporius (P) and total

potassium (K) by ICP-MS (agilent) apparatus, granulometric composition from one

leading factors was analyzed by N.A.Kaachinsky’s method. To define the

absorbing ability, the absorbed cations were fixed by D.Ivanov’s method,

hygroscopic hymidity-by thermal method (soil is dried at 0,50 temperature), the

environment reaction of soil was fixed by pH meter (in 1:5 ratio) in water solution,

ammoniac absorbed from nitrogen forms by Konvey, ammoniac solving in water

by Nesler, nitrates by Grandal-lian method. The accuracy of the results was

specified by the mathematic statistic (V.A.Dospekhov) method (Mammadov, 2007;

Sadigov, 2016).

Results

One of the important problems is an investigation of mountain grey-cinnamon

soils with nutriens source of New Shamkirchay reservoir. The mountain grey-

cinnoman soils were attracted to the agricultural circulation since ancient times and

it is in use today.

The sections in the characteristic places are concerned with the “Antropogenic

changed soils class”. Studying the water and air regime in such types of soil,

controling the change of biological activity, defining formation of the cultured

layer, observing the agroirrigation horizons formation in the profile and other

investigation problems were performed.

The research zone concerned the “accumulative carbonate section” from the

viewpoint of the section and types character of soil. Here, the soils used in

agriculture were formed in sinceancient times. During periods of flood and abundent

water, the river debris are rich in salts in some zones and nutrient on the upper layers

of soils in the zones which were irrigated with muddy water of the Shamkirchay

since ancient times. The cultural soil forming process occurs under an impact of the

river debris products on upper layers in 40-80 cm of density in these soils.

Generated morphological description of the mountain grey-cinnamon soils

historically formed in the research zone was given below:

0-20 cm: is observed with density and hardnes of gazh layer. The thickest layer

and richness of humus are aviable. The color is dark-cinnamon, porous. Air

permeability is good. It has small heaplike structure. It is medium clayey, moist



and rich in root and rootlets of plant, the insect ways activate, air and water

conductivity. The soil fissures is clearly noticed. It is clearly transitional and boils

under an influence of HCl. Depth of carbonate layer is slightly noticed.

20-50 cm: color is brownish, brown-cinnamon, large granular structural, porous. It

is medium clayey, strong miost, plant roots, insect ways are slightly observed. The

carbonate traces are clearly noticed in the soil. The large cracks are observed. It is

clearly transitional and boils under an influence of HCl. Depth on carbonate layer

is clearly noticed.

50-100 cm: color is bright cinnamon, humus layer is slightly noticed. It is heavy

clayey, granular structural, hard, little moist, gradual transitional. It has separate

tree and plant roots, and doesn’t boil under an impact of HCl. Depth of carbonate

layer is clearly noticed.

The diagnostic indices have been analyzed in order to define fertility parameters of

soils spreading in the zone. The analyses are explanatory described on Table 3.

The list of the sections applied in the characteristic places on subtypes of mountain

grey-cinnamon soils aviable in the research area is given on Table 4.

It is clear from morphological description of different soil section applied in the

research area of a result of the performed field-soil and cameral laboratorial

researches that there are differences between AUa-density of humus layer, a

quantity of nitrogen by percentage, formation Bca-layer with illuvial calcareous,

depth and hardness, their structural-aggregates, granulometric composition,

hydroscopic humidity and other morpho-diagnostic indications in different

formations and farming areas.

In Table 3, the section concerning the fully undevoloped mountain grey-cinnamon

(chestnut) soil subtype, an analysis of diagnostic indices of the 19th section fertility

parameters was analyzed. The section was fixed on geographic coordinates. X

ccordinate (the east lenght) of the 19th section was fixed 460 3/ 53,690// E, but Y

coordinate (the north width) was fixed at 400 43/ 28,922// N (Table 4). These soils

spread in 4480,47 ha area (Table 1). The humus layer density is 4,09-1,30 % along

the profile in these soils. Nitrogen is 0,291-0,161 % according to humus.

Hygroscopic humidity is 7,45-4,72 %, CO2 % 18,48-7,82 %, CaCO3 14,02-7,82 %

due to CO2, a sum of absorbed bases is 40,42-29,97 mg-ekv, pH vibrates by 7,0-

8,1. In granulometric composition of subtype, the percentage quantity less than

<0,001 mm is 28,07-32,96 %, but the percentage amount less than <0,01 mm is

56,76-65,13 %. It is clear from the granulometric analysis that these soils are

mainly medium and heavy clayey.

90 Ramil Sadigov

92 Ramil Sadigov

The analysis of diagnostic indices of the fertility parameters in the 92th section

concerning the Anciently irrigated solonetzlike mountain ordinary grey-cinnamon

(chestnut) soil subtype was performed (Table 3). These soils are 3912,56 ha in the

research zone (Table 1). The cut was fixed on geographical coordinates. X

coordinate of the Section 92 (the east lenght) is at 460 17/ 3,536// E, Y coordinate

(the north width) is at 400 48/ 6,586// N (Table 4). In Anciently irrigated

solonetzlike mountain ordinary grey-cinnamon (chestnut) soils, the humus layer

density along the profile is 4,63-1,13 %. According to humus nitrogen is 0,324-

0,157 %. Hygroscopic humidity is 7,03-5,71 %, 11,35-13,36 % with CO2, CaCO3 is

12,38-14,03 % with CO2, a sum of absorbed bases is 36,44-28,12 mg-ekv, pH 7,4-

8,0. The percentage quantity less than <0,001 mm is 25,65-30,39 %, the

percentage amount less than <0,01 mm is 58,05-65,15 %. It is clear from

granulometric analysis that these soils are medium and heavy clayey (Table 3).

The analysis of diagnostic indices of the fertility parameters in the 81th section

concerning the irrigated mountain bright grey-cinnamon (chestnut) soil subtype

was also conducted (Table 3). These soils are 7603,44 ha in the research zone

(Table 1). The cut was fixed on geographical coordinates. X coordinate of the

Section 81 (the east lenght) is at 460 12/ 35,697// E, Y coordinate (the north width)

is at 400 53/ 25,626// N (Table 4). In irrigated mountain bright grey-cinnamon

(chestnut) soils, the humus layer density along the profile is 4,84-1,60 %.

According to humus nitrogen is 0,338-0,135 %. Hygroscopic humidity is 7,53-5,52

%, 16,71-12,04 % with CO2, CaCO3 is 15,34-10,69 %, with CO2, a sum of

absorbed bases is 31,80-28,75 mg-ekv, pH 7,0-7,9. The percentage quantity less

than <0,001 mm is 28,55-25,23 %, the percentage amount less than <0,01 mm is

61,12-69,19 %. It is clear from granulometric analysis that these soils are medium

and heavy clayey (Table 3).

Irrigated solontzlike mountain ordinary grey-cinnamon (chestnut) soils

The analysis of the diagnostic indices of section 48 fertility parameters, due to this

soil subtype, was analyzed. These soils spread in 17063,25 ha zone (Table 1). The

section is fixed on geographical coordinates. X coordinate of the Section 48 (the

east lenght) is at 460 16/ 5,315//E, Y coordinate (the north width) is at 400 45/

12,766// N (Table 4). The humus layer density along the profile is 3,47-1,45 %.

Nitrogen corresponding to humus is 0,252-0,126 % in these soils. Hygroscopic

moisture is 7,01-5,60 %. 21,36-11,96 % with CO2, CaCO3 is 20,63-11,96 %, due to

CO2, a sum of absorbed bases is 39,56-35,36 mg-ekv, pH is 7,6-8,2. The

percentage quantity less than <0,001 mm is 25,71-30,50 %, the percentage amount

less than <0,01 mm is 54,89-62,16 %. It was clear that these soils are medium and



heavy clayey. Through dry residue isn’t observed on the upper layers but it is

observed towards low layers (Table 3).

The analysis of the diagnostic indices of section 102 fertility parameters

concerning Irrigated gazh mountain ordinary grey-cinnamon (chestnut) soil

subtype was analyzed. These soils spread in 5086,97 ha zone (Table 1). The section

is fixed on geographical coordinates. X coordinate of the Section 102 (the east

lenght) is at 450 56/ 2,709// E, Y coordinate (the north width) is at 400 57/ 14,711// N

(Table 4). The humus layer density along the profile is 2,12-0,73 %. Corresponding

to humus nitrogen is 0,167-0,131 % in these soils. Hygroscopic moisture is 7,87-

6,83 %. 18,67-12,99 % with CO2, CaCO3 is 17,30-14,71 %, due to CO2, a sum of

absorbed bases (SAB) is 41,05-29,33 mg-ekv, pH is 7,1-7,8. The percentage

quantity less than <0,001 mm is 25,43-28,57 %, the percentage amount less than

<0,01 mm is 55,73-61,71 %. It was clear that these soils are medium and heavy

clayey look like another soil subtypes. Through dry residue isn’t observed on the

upper layers but it is observed towards low layers, too (Table 3).

Table 4. List of the sections applied on mountain grey-cinnamon (chestnut) soil subtypes in

the characteristic places (fixing on geographical coordinates)

№ Name of soil subtypes Classificatio

n on WRB

Number of

section

X coordinate

(east lenght)

Y coordinate

(north width)

Mountain grey-cinnamon (chestnut) (Mgc)

1 Fully undeveloped

mountain grey-cinnamon

(chestnut)

Mgcfu

Section 19 460 3/ 53,690// E 400 43/

28,922// N

2 Anciently irrigated

solonetzlike ordinary

grey-cinnamon

(chestnut)

Mgcai.slo

Section 92 460 17/ 3,536// E 400 48/ 6,586//

N

3 Irrigated mountain bright

grey-cinnamon

(chestnut)

Mgc1ib Section 81 460 12/ 35,697// E 400 53/

25,626// N

4 Irrigated solonetzlike

ordinary grey-cinnamon

(chestnut)

Mgc2i.sl

Section 48 460 16/ 5,315// E 400 45/

12,766// N

5 Irrigated gazh mountain


(chestnut)

Mgc2i

Section 102 450 56/ 2,709// E 400 57/

14,711// N

6 Irrigated mountain


(chestnut)

Mgc2i

Section 61 460 0/ 10,346// E 400 51/

51,172// N

7 Anciently irrigated saline

mountain ordinary grey-

cinnamon (chestnut)

Mgc2a.is

Section 76 460 11/ 24,947// E 400 49/

25,428// N

94 Ramil Sadigov

Irrigated mountain ordinary grey-cinnamon (chestnut) soil subtype

The analysis of the diagnostic indices of section 61 fertility parameters concerning

to this soil subtype was analyzed. These soils spread in 6299,59 ha zone (Table 1).

The section is fixed on geographical coordinates. X coordinate of the Section 61

(the east lenght) is at 460 0/ 10,346// E, Y coordinate (the north width) is at 400 51/


Corresponding to humus nitrogen is 0,307-0,133 % in these soils. Hygroscopic

humidity is 8,04-6,40 %. 9,02-7,93 % with CO2, CaCO3 is 9,77-4,71 %, due to

CO2, a sum of absorbed bases (SAB) is 35,80-30,94 mg-ekv, pH is 7,0-7,6. The


less than <0,01 mm is 54,50-61,22 %. It was clear from granulometric composition

that these soils are medium and heavy clayey, too. Through dry residue isn’t

observed on the upper layers but it is observed towards low layers (Table 3).

Anciently irrigated saline mountain ordinary grey-cinnamon (chestnut) soil

subtype

Figure 1. Sections applied in the characteristic places of mountain grey-cinnamon soils in

the research zone



The analysis of the diagnostic indices of section 76 fertility parameters concerning

to this soil subtype was analyzed. These soils spread in 9817,46 ha zone (Table 1).

The section is fixed on geographical coordinates. X coordinate of the Section 61

(the east lenght) is at 460 11/ 24,947// E, Y coordinate (the north width) is at 400 49/


Corresponding to humus nitrogen is 0,256-0,113 % in these soils. Hygroscopic

moisture is 7,53-5,52 %. 23,09-7,85 % with CO2, CaCO3 is 18,59-7,85 %, due to

CO2, a sum of absorbed bases (SAB) is 38,63-24,23 mg-ekv, pH is 7,0-7,6. The


less than <0,01 mm is 66,95-71,11 %. It was clear from granulometric analysis that

these soils are heavy clayey and clayey. Through dry residue isn’t observed on the

upper layers but it is observed towards low layers (Table 3). The sections applied

in the characteristic zone are shown in figure 1.

Conslution

1. The main physicochemical and nutrient on upper layer of soils were analyzed

with modern methods as a result of the chemical analyses in soil samples

taken from mountain grey-cinnamon (chestnut) soils.

2. On Table 1, the soil types and subtypes including in the Shamkirchay water

reservoir, their zones (ha) are given. At the same time, the classification on

WRB (soil groups) of mountain grey-cinnamon (chestnut) soils is shown on

Table 2.

3. On Table 3, the analyses of the fertility parameters in the fertility parameters

of the sections applied in the characteristic places on mountain grey-cinnamon

(chestnut) soil subtypes was given. An analysis of the table was reflected in

the article.

4. The sections applied in the characteristic places on mountain grey-cinnamon

(chestnut) soil subtypes in the research area were shown both in table and map

forms.

5. The diagnostic indices were studied by new methods on 7 subtypes of

mountain grey-cinnamon (chestnut) soil subtypes. The ecological processes

were analyzed and important results were obtained. So, humus, nitrogen,

hygroscopic humidity, CO2, CaCO3 due CO2, a sum of absorbed bases, pH

environment of the zone, granulometric composition in 2 forms (<0,001 mm

and <0,01), dry residue AUa, A/B, Bta, B/C and C profils were studied in each

soil section (Table 3).

96 Ramil Sadigov

Refrences

AZS ISO. (2013). State Standard of the Republic of Azerbaijan. Soil quality -

laboratory methods for determining the microbiological respiration of the soil.

AZS ISO, Baku, 2013.

Babaev, M.P., Jafarova, Ch.M. & Hasanov, V.H. (2011). Modern classification

of Azerbaijan soils. Baku, “Elm”, 360 p.

Babaev, M.P., Hasanov, V.H., Jafarova, Ch.M., & Huseynova, S.M. (2011).

Morphogenetic diagnostics, nomenclature and classification of Azerbaijan

soils. Baku, “Elm”, 452 p.

Mammadov, G.S. (2007). Socio-economic and environmental bases of efficient

use of Azerbaijan's soil resources. Baku, "Elm", 854 p.

Ministry of Agriculture of Azerbaijan. State Land Management Project

Institute. (1972). Report on the soil cover and effective use of Shamkir

region. Ganja 1972. 187 p.

Sadig, M.N. & Mammadov, E.E. (2018). Characteristics of the relationship

between physical and chemical and fertility indicators of agricultural soils in

the mountainous zone. Collection of scientific works devoted to the 110th

anniversary of Hasan Aliyev "Soil Science and Agrochemistry", 23 (1-2), 130-

136.

Sadigov, R.A. (2013). Influence of erosion processes on fertility parameters of

soils in the cultivation area of the northeast slope of the Lesser Caucasus.

Philosophy doctoral thesis manuscript. 167 p.

Sadigov, R.A. (2016). The influence of the “Main Canals” of the New

Shamkirchay Reservoir on the soil and environmental conditions of the basin.

International research journals "Successes in modern science and education",

"Successes of modern science" SUCCESSES OF MODERN SCIENCE.

Included in the Higher Attestation Commission (No. 862), Agris, RSCI No.

12, T.11.

Sadigov, R.A. (2018a). A brief overview of soil-water and geological surveys in

the Shamkirchay reservoir basin and the methodology and technology of field

operations using the VEP (Vertical Electric Probing) method. Journal of

Scientific Works of AzSUU No. 1., 54-61.

Sadigov, R.A (2018b). Investigation of erosion processes in the mountain-brown

soils of the New Shamkirchay reservoir. Collection of scientific works

dedicated to the 110th anniversary of Hasan Aliyev "Soil Science and

Agrochemistry", 23 (1-2), 259-262.



97

Estimation of PVT Properties Using Artificial Neural

Networks and Comparison of Results with

Experimental Data

Masoud Mehrizadeh

Department of Petroleum Engineering, Khazar University, Baku, Azerbaijan

E-mail: [email protected]

Abstract

The importance of pressure, volume and temperature (PVT) properties, bubble

pressure, gas-to-oil solubility ratio and oil volume factor have made it necessary to

precisely determine these properties for the calculation of reservoir performance. In

the absence of laboratory measurements to determine the PVT properties of crude

oil, two methods, used commonly, include the equations of state and the

experimental relations of PVT. The equation of state is based on the information

concerned with the fluid composition details of the reservoir, which is very

expensive and time-consuming to determine. Whereas, PVT relationships are based

on data obtained from easily measured ground layers. These data include reservoir

pressure, reservoir temperature, and the specific gravity of oil and gas. Recent

Studies show that artificial neural networks have a great ability to predict the PVT

properties. Due to the training capability in neural networks, these networks were

rapidly applied in engineering and were widely used in petroleum engineering.

Estimation of porosity and permeability of reservoirs, prediction of outflow

generated by oil wells, estimation of oil recovery, prediction of asphaltene

deposition and estimation of PVT properties are the most important applications of

artificial neural networks in petroleum engineering. By preparing and collecting

more than 1000 PVT data related to southern Iran reservoirs, 577 data were

selected to be used in the project and were randomly divided into two parts, 486

data for network training and 91 data for testing. The three-layer structure (one

hidden layer with 6 neurons) was selected as the best structure and the batch back-

propagation training method as the best learning algorithm. The results of the

network were in a good agreement with experimental data, which the average

relative error using training set in estimation of the volume factor and densities of

oil were 0.557 and 0.509% respectively and using test data were 1.032 and 1.104%

respectively.


98 Masoud Mehrizadeh

Keywords: neuron, neural network, error backpropagation, neural network

application, PVT properties, supersaturated reservoirs.

Introduction

PVT properties such as oil formation volume factor )( OB and density )( O are

used extensively in almost all petroleum engineering issues, especially in the

reservoir engineering sector. For example, the application of these properties can

be seen in issues such as material balance, well testing, reservoir simulation, etc

(Al-Marhoun & Osman, 2002).

Actually, these properties should be experimentally determined in the laboratory,

but usually experimental data are not always available and require a great deal of

time and investment. There are many empirical correlations that estimate the

different PVT properties. These relationships are obtained using linear or nonlinear

regressions and sometimes predict these properties in the form of graphs. These

relationships and correlations mainly are not accurate due to using limited

empirical data extracting them. These relations and correlations are not accurate

due mainly to applying limited empirical data out of which they are extracted.

Researchers have recently used the idea of artificial neural networks to determine

the PVT properties. Applying this method, albeit of having the limitations

mentioned in the paragraph above, has an important learning advantage, which

means one can train the network and update the network whenever new data is

available.

Subject literature and previous studies

Gharbi and Elsharkawy (1999) presented a neural network model for predicting

PVT properties of Middle Eastern crude oil. The data used to train this network

(498 data) include the most collected ones to develop the model for predicting PVT

properties from Middle Eastern crude oil resources. The model can predict bubble

pressure and oil volume factor as a function of dissolved gas to oil ratio, gas

specific gravity, oil specific gravity and temperature. In this study, the network

results are compared with the results of the empirical relations for the Middle East.

The empirical relationships whose results are compared with the network results

include; Al-Marhoun, Standing and GlasØ.

Estimation of PVT Properties Using Artificial Neural Networks and

Comparison of Results with Experimental Data 99

Marhoun and Osman (2002) developed a PVT neural network to estimate bubble

point pressure. The model design uses 803 recorded data collected from Malaysia,

Middle East, Gulf of Mexico and Colombia. Network input data include gas to oil

ratio, API °, relative gas density and reservoir temperature. This model is more

accurate than other experimental models (Goda et al., 2003).

Goda et al. (2003) presented a model that predicts bubble point pressure and oil

volume factor through two interconnected neural networks. The first network has

four layers which reservoir temperature, API °, relative gas density and gas to oil

ratio are the input to first layer. Their network has two hidden layers, each has 10

neurons, that are activated by the Log Sigmoid transfer function. The output layer

has a neuron whose transfer function is of pure linear type. This network uses 160

data from the Middle East region for training and another 20 data for network

testing.

The second network also has four layers. The purpose of the network is to estimate

the oil volume factor. The input layer has five neurons for the five input data types

including reservoir temperature, API °, relative gas density, gas-to-oil ratio and

finally the bubble point pressure predicted from the first grid. Each of its two

hidden layers has 8 neurons with the Log Sigmoid transfer function. The output

layer has a neuron with the oil volume factor output data. The main difference

between this network and previous PVT predicting networks is that other networks

use four common input data to predict the oil volume factor, but in addition to the

four data, this model uses the bubble point pressure estimated by another neural

network (Moghadassi et al., 2008).

Moghadasi et al. (2008) presented a new model for predicting reservoir PVT

properties. The data set used to design this model is extracted from Perry's

Chemical Engineers' Handbook. Different training schemes such as SCG, LM and

RP have been used/applied for the back-propagation algorithm. The LM algorithm

with 60 neurons in the hidden layer with the lowest mean square error (MSE) of

0.000606 was selected as the best network (Laugier & Richon, 2003).

Neural network structure

Preparing and collecting the data required for neural network were one of the most

difficult parts of this research. After collecting more than 1000 experimental PVT

data from over 55 samples of under-saturated (Pres < Pbubble) reservoirs in Iran, 577

data on oil formation volume factor and density were selected. These data were


then randomly divided into two sections, out of which 486 were used for network

training and the rest (91) for network testing.

Reviewing numerous/wide volume of books and articles; pressure (P), temperature

(T), oil API degree, relative coefficient of dissolved gas (Rs) and specific gas

density (γg) were selected as network input parameters.

The first step to design a neural network is to determine the number of layers,

neurons of each layer and the transfer function of each layer. It is always clear that

the number of neurons in the first layer is the same as the parameters input to the

neural network and it is a function of the problem type. Therefore, the first layer in

this case study consists of 5 neurons. The first layer neurons, i.e. the input

information, transfer their values directly to the second layer.

Table 1. Overview of total data used

Number of

Points

PVT Property Minimum Maximum Mean

577 Pressure, psi 535 9300 3764.12

577 Temperature, °F 116 277 215.145

577 Solution GOR, SCF/STB 66.8 1998.39 922.305

577 S.G. of Total Gas Evolved 0.77 1.61 1.056

577 Stock Tank Oil Gravity, °API 7.24 42.89 26.99

577 FVF, bbl/STB 1.039 2.408 1.593

577 Oil Density, lb/ft3 33.14 62.085 44.368

To reduce the range of input variables in order to make more stable in proposed

neural network, all the input variables as well as the target variables according to

eq. 1 have become dimensionless and then are used in the network.

(1)

The number of neurons in the output layer is also equal to the number of target

parameters.

To determine the network structure, all possible modes have been investigated and

for each case, the Average Absolute Percent Relative Error and relative square root

)min()max(

)min(

oldold

oldoldnew

XX

XXX

−

−=



mean square error (RMS) have been calculated for train and test data. The

following equations illustrate the relationships used:

exp

exp

X

XXE

est

i

−= (2)

=n

ia En

E1

1 (3)

=n

iEn

RMS1

2/12 ]1

[ (4)

−−−=nn

est XXXXr1

exp

1

2

exp /)(1 (5)

=n

Xn

X1

exp

1 (6)

RMS or the relative root mean square error represents the distribution of data

around the deviation from zero error, and r or the Correlation Coefficient is a

measure of the success rate of the proposed relationship in reducing the standard

deviation. After investigating the results, two modes of one hidden layer with 6

neurons and two hidden layers (each of 5 neurons) were selected and different

modes were investigated on these two structures to determine the optimal state. For

instance, several transfer functions were used and the weights obtained in the

previous step were used/applied/considered as the initial weights in the next step.

Finally, the three-layer structure (one hidden layer with 6 neurons) was chosen as

the final structure with Tanh transfer function for the hidden layer and linear

transfer function for the output layer. Batch back propagation was Also identified

as the best network training method.


Figure 1. Schematic of proposed network

Table 2 Proposed Network’s Weights and Bias Values

Connections Weights Connections Weights Connections Weights

N1L1-N1L2 -1.1444 N3L1-N6L2 1.1901 B1-N5L2 0.6623

N1L1-N2L2 -0.0814 N4L1-N1L2 0.7074 B1-N6L2 0.3032

N1L1-N3L2 0.2933 N4L1-N2L2 0.4247 N1L2-N1L3 0.7084

N1L1-N4L2 0.2293 N4L1-N3L2 -0.6211 N1L2-N2L3 -0.3885

N1L1-N5L2 0.0806 N4L1-N4L2 0.7755 N2L2-N1L3 -0.5457

N1L1-N6L2 -0.3875 N4L1-N5L2 0.2433 N2L2-N2L3 -0.1033

N2L1-N1L2 0.7034 N4L1-N6L2 0.3784 N3L2-N1L3 0.3992

N2L1-N2L2 0.3061 N5L1-N1L2 -0.0271 N3L2-N2L3 0.152

N2L1-N3L2 0.176 N5L1-N2L2 0.6449 N4L2-N1L3 0.1444

N2L1-N4L2 0.7632 N5L1-N3L2 0.4846 N4L2-N2L3 -0.7827

N2L1-N5L2 0.2602 N5L1-N4L2 0.1442 N5L2-N1L3 0.3189

N2L1-N6L2 0.1391 N5L1-N5L2 0.8289 N5L2-N2L3 -0.0003

N3L1-N1L2 1.238 N5L1-N6L2 -0.5597 N6L2-N1L3 0.247

N3L1-N2L2 -0.9579 B1-N1L2 1.6673 N6L2-N2L3 -0.4815

N3L1-N3L2 0.5877 B1-N2L2 0.6059 B2-N1L3 0.2195

N3L1-N4L2 0.9374 B1-N3L2 0.1885 B2-N2L3 -0.1374

N3L1-N5L2 0.3539 B1-N4L2 0.3618



Result

After selecting the optimal structure for the neural network and its training, the

second part of the data (91 data) was used to test estimation accuracy of the

network.

Table 3 presents the error values of the network results. Figures 2, 3, 4, and 5 also

show the scatter plots comparing experimental results with those estimated by the

network:

Table 3. Network Results Error Values

Correlation

Coefficient RMS Ea (%) Emax (%) Emin (%)

0.999590131 1.020049926 0.556592298 5.84041065 9.36841E-05 )(TrainBo

0.982429677 1.047043715 0.508632407 7.098559348 0.000652503 )(Traino

0.99909827 1.480137336 1.032430012 5.138218685 0.001333529 )(TestBo

0.956828755 1.538169325 1.104391183 5.971239206 0.004537934 )(Testo

Figure 2. Comparison between experimental values of and values estimated by the network

for data used in training


Figure 3. Comparison between the experimental values and the values estimated by the

network for the data used in training

Figure 4. Comparison between experimental values and values estimated by the network

for the data used in testing



Figure 5. Comparison between experimental values and values estimated by the network for

the data used in testing

Comparison with empirical relationships

To evaluate the accuracy of the presented network compared with the empirical

relationships published, several relationships are selected and the results are

compared. To use the empirical equations and due to this fact that the samples are

under-saturated, i.e. their pressure is above the bubble pressure, the volume factor

and density are calculated in two steps. First, by/through using the empirical

relation, their value at the bubble point is determined, then by defining the

Coefficient of Isothermal Compressibility, that value is calculated at the desired

pressure: The first step goes through applying the empirical relation by which their

value at the bubble point is determined and following that by defining the

Coefficient of Isothermal Compressibility, the value is calculated at the desired

pressure.

)]([0 ppcEXPBB boob −= (6)

)]([0 boob ppcEXPBB −= (7)

As can be seen, the above relationships need to calculate the value of that property

at the bubble point, determine the bubble pressure and determine the coefficient of

isothermal compressibility. They are, therefore, used to calculate a property in the

supersaturated sample.


Many researchers have proposed empirical relationships to determine the PVT

properties of which the most reliable relations have been attempted to be used

while comparing the proposed network. (In cases where all 3 relationships were

not available from one researcher, the relationship by another researcher was

assumed to be relevant).

The relationships used are as follows (Laugier, S. & Richon, 2003; Danesh, 1998;

William, 1990):

Ahmed’s Correlations:

sssob RaRaRaPaPaPaTaTaTaFB 9

2

876

2

543

2

21 +++++++++=

)( 131211

10

a

g

aa

s APIRaF +=

4

1 105243973.4 −−=a 6

2 109063637.3 −=a

5542509.53 −=a 6

4 107603220.5 −−=a

9

5 109528992.3 −−=a 289473.166 =a

4

7 108718887.3 −=a 8

8 100703685.7 −=a

4358395.19 −=a 12869353.010 −=a

023484894.011 =a 015966573.012 =a

021946351.013 =a

)]]()([[ bobo aPEXPaPEXPDEXPBB −=

1]0025999.0588893.4[ −+= sRD

00018473.0−=a

1)0025999.0588893.4( −+−= sRB

The Al-Marhoun equation is used to calculate the bubble pressure.

))]()(([ bobo aPEXPaPEXPBEXP −=



ed

o

c

g

b

sb TRaP =

3-10×5.38088 = a 0.715082 = b

1.87784- = c 3.1437 = d

The Standing relation is used to calculate the density at the bubble point .

Standing’s Correlations: 2.1

5.0

25.1000120.09759.0

+

+= TRB

o

g

sob

( ) 4.1102.1883.0

−= a

gsb RP

The Vesquez-Beggs relation is used to calculate Co .

Glaso’s Correlations:

A

obB 100.1 +=

( )2** )(27683.0)(91329.258511.6 obob BLogBLogA −+−=

Here the Standing equation was used to calculate the bubble pressure and the

Vesquez-Beggs relation to calculate Co.

1.32657 = e

5.131

5.141

+=

APIO

P

APITRC

gs

o

+−++−=

510

61.1211802.1751433

175.15.0

25.1000147.0972.0

0136.04.62

+

+

+=

TR

R

o

g

s

gsg

ob


After computing the results of the empirical relationships, they were compared

with the results from the neural network which are shown in the following tables:

(In each case, the relative error of absolute mean, minimum error, maximum error,

and root relative squared error were compared).

Table 4. Comparison of Neural Network Error with Other Experimental Equations

to Esimate Oil Volume factor (Training Data)

Table 5. Comparison of neural network error with Ahmed empirical equation to

estimate oil density (training data)

Emax (%) Emin (%) Ea (%) RMS

ANN 7.0985 0.00065 0.508 1.047

Ahmed 14.463 0.021 4.132 4.946

Table 6. Comparison of Neural Network Error with Other Experimental Equations

to estimate Oil Volume factor (Test Data)


ANN 5.1382 0.0013 1.0324 1.480

Ahmed 17.792 0.216 7.337 8.508

Standing 16.089 0.110 3.035 4.173

Glaso 14.298 0.027 2.185 3.080

RMS Ea (%) Emin Emax (%)

ANN 1.020 0.5565 0.000094 5.8404

Ahmed 8.649 7.535 0.019 18.258

Standing 4.266 3.138 0.007 16.089

Glaso 3.034 2.291 0.003 14.298



Table 4. Comparison of neural network error with Ahmed empirical equation to

estimate oil density (test data)


ANN 5.971 0.00453 1.1043 1.538

Ahmed 13.780 0.354 4.070 4.895

The results obtained indicate the high accuracy of the proposed network with

respect to these experimental relationships.

Conclusion

1. A new model was presented to determine the oil volume factor and density for

Iran's under-saturated reservoirs. The proposed model is based on artificial

neural networks and has been developed using 577 empirical data from over

55 Iranian under-saturated samples.

2. From 577 data, 486 data were used for network training and 91 data for

network testing.

3. The proposed network has an input layer with 5 neurons representing the

network inputs and one hidden layer with 6 neurons and an output layer with 2

neurons representing the network outputs. Batch back propagation algorithm

was also used for network training.

4. The results showed that the network is capable of estimating oil volume factor

and oil density with high accuracy and provides better results than existing

empirical relationships. The average network error in determining these

properties was about 0.5% for training data and about 1% for test data.

References

Al-Marhoun, M.A., & Osman, E.A. (2002). Using Artificial Neural Networks to develop

New PVT Correlations for Saudi Crude Oils. SPE 78592.

Danesh, A. (1998). PVT and Phase Behaviour of Petroleum Reservoir Fluids. Elsevier

Science B.V., Netherlands.


Gharbi, B.C., & Elsharkawy, M. (1999). Neural Network Model for Estimating the PVT

Properties of Middle East Crude Oils, SPE Reservoir Eval. & Eng. 2(3).

Goda, H.M., Shokri, E.M., Fattah, K.A., & Sayyouh, M.H. (2003). Prediction of the

PVT Data using Neural Network Computing Theory; SPE 85650.

Laugier, S., & Richon, D. (2003). Use of artificial neural networks for calculating derived

thermodynamic quantities from volumetric property data, Fluid Phase Equilib. 210,

247-255.

Moghadassi, A.R., Parvizian, F., Hosseini, S.M., & Fazlali, A.R. (2008). A New

Approach for Estimation of PVT Properties of Pure Gases Based on Artificial Neural

Network Model.

William, D.M. (1990). The Properties of Petroleum Fluids. PennWell Publishing

Company, USA.

Information to Contributors

Khazar Journal of Science and Technology focuses on the results of original

research projects in Mathematics, Physics, Chemistry, Earth science and life

science as well as Engineering and Technology.

The journal is published in English.

The Editorial Board does not accept articles published or submitted for publication

elsewhere.

Information material, such as notifications about conferences, books, jubilees, etc.

may also be published under the information heading. Articles and other materials

published in the Khazar Journal of Science and Technology represent the opinions

of the author(s) and should not be considered to reflect the opinion of the Editorial

Board.

Specifications

Manuscripts should not exceed 25 published pages (approximately 10,000 words).

Those of a length exceeding 25 pages, nut containing very important results, can be

published by the consent of the Editorial Board. Articles may be submitted by e-

mail. Please submit your manuscript to the following addresses:

[email protected] and [email protected]

The first page should include the title of the article, the names and primary

affiliations of the author(s), an abstract of 50-250 words, and 4-7 keywords. All

figures, photographs, tables, or drawings should be numbered.

References must be in one of the following styles (depending on the subject area):

AMS, LaTeX, ACS, AIP, Vancouver system, etc. entries in the reference list

should be put in alphabetical order or in order of citation. Footnotes in the text

should be avoided if possible.

The author(s) should provide subject classification codes (AMS subject

classification numbers, the Physics and Astronomy Classification Scheme, ACM

computing Classification System or Library of Congress Classification).

The Khazar Journal of Science and Technology follows the COPE’s guidelines in

dealing with allegations of research misconduct. Khazar Journal of Science and

Technology follows the conflict of interest policy of the PNAS (Proceedings of the

National Academy of sciences, USA).


(kjsat)kjsat.com/files/current_issue.pdf6 maryam seyyedi noosh abad introduction helicobacter pylori...

Documents