Molecular Characterization of Antibacterial Resistant Genes in Escherichia coli Isolated from ...

164 Pages·2017·1.91 MB·English

Ministry of Higher Education and Scientific Research
University of Al-Qadisiyah / College of Medicine
Department of Microbiology

Molecular Characterization of Antibacterial
Resistant Genes in Escherichia coli Isolated
from Clinical Samples

A Thesis
Submitted to the Council of the
College of Medicine / University of Al-Qadisiyah
in Partial Fulfillment of the Requirements for
The Degree of Master of Science
in Medical Microbiology

By

Alaa Hamza Jaber Al-Jelehawy
B.Sc. Laboratory Investigation / Kufa University (2014)
Supervised by
Assistant Professor Assistant Professor
Assistant
Dr. Ibtisam H. Al-Azawi Dr. Aqeel R. Hassa

October 2016 A.D. Muharram Al-Haram 1438 A.
ميحرلا نحمرلا للها مسب


ًاميظَع َكْيَلَع للها لُـُْضَف َنَاَكَو مُلَْعَت نُُْكَُْت مَلاَم َكملََّعَو

ميظعلا يلعلا للها قدص
ءاسنلا ةروس
)111( ةيلآا
Dedication

This work is dedicated

To

Al-Imam Ali (Aleh Alsalam)
To

My dear country (Iraq)

And to

The spirit of my father

Alaa 2016
Acknowledgements
In the name of God the most merciful compassionate, the peace
and the mercy upen our Messenger prophet Mohammed and his sanctified
household. All thanks to God for granting me with will, strength, and
patience with which this research had been accomplished.
I would like to express my gratitude to the deanery of College of
Medicine and the headship of Department of Microbiology for the
support and facilities I received.
It is a pleasure to express my deep appreciation to my supervisors:
Assistant Professor Dr. Ibtisam H. Al-Azawi and Assistant Professor
Dr. Aqeel R. Hassan, for their valuable guidance, assistance, streanth,
and motivation throughout the cource of preparing my thesis, during the
progress of the present work.
I am grateful to the Department of Biology, Faculty of Sciences,
University of Kufa for providing me with facilities to complete this
research.
I'd like to express my sincere thanks and appreciation to Assist
Prof. Dr. Ahmed Abduljabbar Jaloob Al-Janaby for his support and
advice.
I'd like to express my deep appreciation to Dr. Arshad Nori Al-
Dujaili, Head of the Department of Biology, and to Dr.Mohammed
Alzeyadi for his support and advice.
I would like to thank Prof. Dr. Ali Muhsin Al-Mohana
Department of Microbiology / College of Medicine / Kufa University for
his valuable advices.
I would like to thank Prof. Dr. Abdulkareem A. Mahmood. /
College of Medicine / Kufa University for his valuable advices.
Last but not the the least, I am indeed grateful to my family for
their understanding, and their great help in accomplishing my study.
To all, please accept my truthful thanks.

Alaa 2016

Summary:
This study was designed to identify the distribution of some genes
responsible for antibacterial resistance in Escherichia coli isolated from
different clinical samples by using phenotypic methods and genotypic
technique (by monoplex PCR). A total of 400 samples were collected
from urine (n=100), stool (n=75), and blood (n=45) from patients
admitted to the Al-Hamza Hospital in Al-Diwaniya City and Al-Sadder
medical City in Al-Najaf Governorate-Iraq.
The results demonstrated that there were 220 specimens (55%)
were diagnosed as gram negative bacteria. From total of 220 specimens
there were 49 isolates (22.27%) identified as E. coli by cultural,
biochemical characteristics and Vitek-2 system.
The susceptibility of E. coli isolates towards 14 type of antibiotics
were tested using disk diffusion method. The results showed that all
isolates of E. coli (100%) were resistant to cefotaxime, 39 (79.59%)
isolates were multidrug resistant, 9 isolates (18.36%) were extensive drug
resistant. This study proved that 47 isolates (95.91%) were resistant to
ampicillin and tobramycin, 41isolates (83.67%) were resistant to
amoxicillin+clavulanic acid. The results of the present study
demonstrated that 39 isolates (79.59%) and 38 isolates (77.55%) of E.coli
were resistant to ceftriaxone and ceftazidime respectively. The current
study showed that 34 isolates of E. coli (69.38%) were resistant to
azteronam, 26 isolates (53.06%) were resistant to nalidixic acid, 21
isolates (42.85%) were resistant to ciprofloxacin & gentamycin, 17
isolates (34.69%) were resistant to cefoxitin, 8 isolates (16.32%) were
resistant to chloramphenicol, 6 isolates (12.24%) and 2 isolates (4.08%)
were resistant to nitrofurantoin & imipenem respectively.
On the other hand, all of E. coli isolates (49) were detected as the
potential ESBL-producers by using confirmatory method (double disk
synergy test), the results showed that out of 49 isolates of E. coli
examined in this study, ESBL were detected in only one isolates (2.04%).
Polymerase Chain Reaction has been used to detect of some genes
encoding for antimicrobial resistance in E. coli isolates. Regarding genes
that responsible for ESBL enzymes (bla , bla and bla ), the
CTX-M OXA TEM
current results proved that bla genes have highest rate (100%)
TEM
followed by bla and bla (91.83 %) for each.
CTX-M OXA
Finally, present study showed some plasmid mediated quinolones
resistance genes (qnrA, qnrB and qnrS). Results demonstrated that these
genes have the same percentage (100%).
List of contents
Numbers Title page
Summery 1-11
List of contents V11-111
List of tables V11-
V111
List of figures V111
List of abbreviation IX-X

Chapter One
Introduction and Literature Review
1 Introduction 1
1.1 Aim of study 3
1.2 Literature Review 4
1.2.1 Taxonomy and Description 4
1.2.2 Growth culture 5
1.2.3 Pathogenicity 5
1.2.3.1 Enterotoxigenic E. coli (ETEC) 6
1.2.3.2 Enteropathogenic E. coli (EPEC) 7
1.2.3.3 Enteroinvasive E. coli (EIEC) 8
1.2.3.4 Eenterohemorrhagic E. coli (EHEC) 8
1.2.3.5 Enteroaggregative E. coli (EAggEC) 9
1.2.3.6 Diffusely adherent E. coli (DAEC) 11
1.2.4 Antibiotics resistance 11
1.2.4.1 Genetics of bacterial antibiotics resistance 13
1.2.4.1.1 Antibiotic resistance via mutations 13
Antibiotic resistance via horizontal gene
1.2.4.1.2 13
transfer
1.2.5 β-lactam antibiotics 14
1.2.5.1 Mechanism of β-lactams action 14
1.2.6 β-lactamase inhibitors 14
1.2.7 β- Lactamase 16
1.2.7.1 Classification of β-lactamases 16
1.2.7.2 Mechanism of action of β-lactamases 16
1.2.7.3 Type of β-Lactamase 17
1.2.7.3.1 Extended spectrum β-lactamases 17
1.2.7.3.2 Carbapenemases 19
1.2.7.3.3 AmpC β-lactamases 19
Molecular Mechanisms of Action of
1.2.8 01
Quinolones
1.2.9 Plasmid-Mediated Quinolone Resistance 01
Chapter Two

Materials and Methods
2 Materials and methods 03
2.1 Materials 03
2.1.1 Instruments and Equipments 03
2.1.2 Biological and Chemical Materials 04
2.1.3 Culture Media 05
2.1.4 Antibiotics 06
Antibiotic
2.1.4.1 06
discs
standard Strain
2.1.5 07
Bacterium
0.1.6 PCR Materials 07
2.1.6.1 Master Mix 07
PCR Amplification PMQR
2.1.6.2 08
Primers
2.1.6.3 PCR Amplification ESBLs Primers 08
2.2 Methods 09
2.2.2 Preparation of Buffers and Solutions 09
McFarland (0.5) Turbidity
2.2.2.1 09
Standard
2.2.2.2 Normal Saline solution 09
Phosphate buffer
2.2.2.3 09
solution(PBS)
2.2.2.4 Urea solution 31
2.2.2.5 EDTA Solutions for disks preparation 31
2.2.3 β-lactam Antibiotic Solutions 31
2.2.4 Solutions used for β-lactamase detection 31
2.2.5 Solutions Used in DNA Extraction 31
2.2.5.1 Tris-EDTA Buffer (TE) Buffer 31
2.2.5.2 Salt-Tris-EDTA (STE) Buffer 31