CHARACTERISATION OF ANTIBIOTIC RESISTANCE MECHANISMS IN GRAM-NEGATIVE ...

377 Pages·2013·20.25 MB·English

CHARACTERISATION OF ANTIBIOTIC RESISTANCE
MECHANISMS IN GRAM-NEGATIVE BACTERIA FROM
TRIPOLI AND BENGHAZI, LIBYA
By
Allaaeddin Ali El Salabi
A thesis submitted for the degree of Doctor of Philosophy
at Cardiff University
2011
Cardiff University, School of Medicine, Department of
Infection, Immunity & Biochemistry
UMI Number: U585511
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DECLARATION
This work has not previously been accepted in substance for any degree and is
not concurrently submitted in candidate for any degree.
S i g n e d ^ r r l y / f ^ ^ ? (candidate) Date .
STATEMENT 1
This thesis is being submitted in partial fulfilment of the requirements for the
degree of PhD
/
"i^m d ^ ^ (candidate) Date
STATEMENT 2
This thesis is the result of my own independent work/investigation, except
where otherwise stated. Other sources are acknowledged by explicit references
OT/j).
Signed,^ (candidate) Date .. /?:?. !!
7..
STATEMENT 3
I hereby give consent for my thesis, if accepted, to be available for
photocopying and for inter-library loan, and for the title and summary to be
made available outside organisations.
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ii
Summary
As very little information is known of the antibiotic resistance in Gram-
negative bacteria in Libya in addition to the desperate need for insight
knowledge of the antibiotic resistance in Libyan hospitals, this study was
undertaken to investigate the mechanism of antibiotic resistance in isolates
collected from clinical, non-clinical and environmental samples from Tripoli
and Benghazi, Libya. Bacterial collection include samples taken from patients
admitted to the hospitals in ICUs and other wards, they also include swabs
randomly collected from hospitals environment. These swabs were from walls,
bedsides, curtains, floors, toilets, workstations, mechanical ventilators,
stainless steel containers and instruments used in particular ICUs. This study
clearly demonstrates the emergence of MDR Gram-negative bacteria in
Tripoli and Benghazi hospitals, these MDR bacteria were clinical and non-
clinical revealing the long standing infection control problem in these
hospitals. K. pneumoniae was found as the most frequently isolated strain
being disseminated in hospitals and outside hospitals followed by E. coli. K.
pneumoniae and E. coli were detected harbouring blactx-m group 1 in
association with ISEcpl the enhancer of the p-lactamase gene movement.
More importantly, ^/«ctx-m-is in association with ISEcpl were detected carried
on conjugative plasmids of different sizes and able to move via Libyan K.
pneumoniae and E. coli to sensitive bacteria via conjugation. Some isolates of
K. pneumoniae were clonally related and were in some cases found in
different hospital revealing the outbreak of MDR K. pneumoniae in Libyan
hospitals. E. coli strains showed the emergence of more than one clone in one
hospital which indicates to the lack of hospital hygiene. Three novel sequence
types among K. pneumoniae were discovered in this study, one of which K.
pneumoniae AES817 that assigned ST511 was collected from one of Benghazi
streets and was found carrying blacrx-M-15 and ISEcpl on a plasmid of 400kb.
Characterisation of P. aeruginosa showed the emergence of clonally related
strains carrying blaym-2, one was isolated from a patient admitted to Al-Jalla
hospital in Benghazi and the other from a stainless steel container from the
same hospital but different ward, this MBL was found on a novel integron in
both strains. Interestingly, 6/<zvim-2 was found chromosomally mediated
proposing that the dissemination of this MBL might be due to mobile genetic
elements. Perhaps the most interesting finding of this study is 6/<3tmb-i which
was detected in environmental strain swabbed from the floor of Tripoli central
hospital. This MBL was unusual in terms of the similarity this gene shares
with other known MBLs and also to the discovery of this MBL carried by
environmental bacteria A. xylosoxidans, it is moreover the first MBL
discovered in Libya.
Presentations and Publications
Presentations given from this study
1- Phenotypic and Genotypic Characterisation of Clinical and non-
Clinical Gram-negative Bacteria from Benghazi-Libya. 49th
Interscience Conference on Antimicrobial Agents and Chemotherapy,
September 12-15, 2009, San Francisco.
2- Identification of Tn402, Class 1 integrons and ISCR elements among
endemic multi-drug-resistant Klebsiella pneumoniae from Benghazi-
Libya. 49th Interscience Conference on Antimicrobial Agents and
Chemotherapy, September 12-15, 2009, San Francisco.
3- Novel subclass of a Group B1 Metallo-p-lactamase, bla-\uB-u in
Clinical and non Clinical Gram-negative Bacteria from Libya. 49th
Interscience Conference on Antimicrobial Agents and Chemotherapy,
September 12-15, 2009, San Francisco.
4- The tniC-like transposon Tn5090 is commonly found in Klebsiella
pneumoniae isolates from Portugal and North Africa. 49th Interscience
Conference on Antimicrobial Agents and Chemotherapy, September
12-15, 2009, San Francisco.
V
Publications and publications in collaborations
1- Salabi, A. E., M. A. Toleman, J. Weeks, T. Bruderer, R. Frei, and T. R.
Walsh. 2010. First report of the metallo-beta-lactamase SPM-1 in
Europe. Antimicrob Agents Chemother 54:582.
2- Chouchani, C., R. Marrakchi, and A. El Salabi. 2011. Evolution of beta-
lactams resistance in Gram-negative bacteria in Tunisia. Crit Rev
Microbiol 37:167-177.
3- Chouchani, C., R. Marrakchi, L. Ferchichi, A. El Salabi, and T. R.
Walsh. 2011. VIM and IMP metallo-beta-lactamases and other extended-
spectrum beta-lactamases in Escherichia coli and Klebsiella pneumoniae
from environmental samples in a Tunisian hospital. Apmis 119:725-732.
4- Chouchani, C., A. El Salabi, R. Marrakchi, L. Ferchichi, and T. R.
Walsh.Characterization of IncA/C conjugative plasmid harbouring
blajEM-52 and blacrx-u-is extended-spectrum p-lactamases in clinical
isolates of Escherichia coli in Tunisia (accepted).
5- Allaaeddin El Salabi, Pardha Saradhi Borra, Mark A. Toleman, 0rjan
Samuelsen and Timothy R. Walsh Genetic and biochemical
characterization of a novel metallo-p-lactamase, TMB-1, from a
Achromobacter xylosoxidans strain isolated from Tripoli, Libya
(submitted)
6- Allaaeddin El Salabi, Mark A. Toleman, Ahmed Matmati, Chedly
Chouchani and Timothy R. Walsh blawu-2 positive Pseudomonas
aeruginosa isolated from operating apparatus and patients in Tripoli,
Libya (submitted)
7- Allaaeddin El Salabi, Mark A. Toleman, Abdulazizi Zorgani and
Timothy R. Walsh. Molecular characterization of antibiotic resistance
mechanisms in K. pneumoniae isolated from Tripoli and Benghazi
hospitals (in progress)
8 Allaaeddin El Salabi, Mark A. Toleman, Asma Alramli and Timothy R.
-
Walsh. Molecular characterization of antibiotic resistance mechanisms
in E. coli collected from Tripoli and Benghazi hospitals (in progress)
vi
Acknowledgements
My thanks go first and foremost to Allah, who created me to discover how great he is and
increase my faith in him.
I would like to thank the following people without whose help the completion of this thesis
would not have been possible.
To my supervisor Prof Timothy Rutland Walsh, who taught me how innovation could be
achieved and continued to support and encourage the spirit of scientific discovery in me.
To my advisor Dr Mark Alexander Toleman for his valuable advice and support, who taught
me the molecular genetics of bacteria.
To Mrs Janis Weeks for her unending help and support, with a smile if nothing else.
To the staff at Reference Centre for Detection of Antimicrobial Resistance, Department of
Microbiology and Infection Control, University Hospital of North Norway for their valuable
advice and help with the TMB-1 purification.
To Dr Henry Ryley for his help with the PFGE pictures analysis.
To Dr Amanda Tonks for her encouragement and support
To Dr Mandy Wootton and staff at her laboratory for their valuable help with the
identification of Libyan isolates
To all staff within section of Medical Microbiology, School of Medicine, University hospital
of Wales for their lovely support
To my favourite brother Fakhrieddin El Salabi, who was waiting to share with me the
moments of completion my studies and passed away before the completion of my PhD.
To all my family members for their irrepressible support and encouragement with which I
could not have even begun this.
To Prof Salha Ben-Gwirif for her encouragement and support and the spirit of Dr Fathi
Belied, who taught me the basics of microbiology.
To my friend Ahmed Gawhari and his wife Neven, for their lovely support and help.
To my friend Othman Boaisha for his kind encouragement and support
To staff of Faculty of Public Health, University of Benghazi for their support
To Ranya, my wife for, her support, patience and endurance
Finally to Amina Benour my mother, whom I love, who supported me, prayed for me, looked
after me and was very close to me even at a distance, her help and encouragement was
unbelievable.
This thesis is dedicated to
the spirit o f my father, the
spirit o f my brother and
to the new Libya
LIST OF FIGURES
Fig. 1.1 Chemical structure of daptomycin.............................................. 6
Fig. 1.2 Chemical structure of oxazolidinone radezolid................... 6
Fig. 1.3 Chemical structure of the fluoroquinolone delafloxacin 6
Fig. 1.4 Chemical structure of the aminoglycoside ACHN-490 ....... 7
Fig. 1.5 Chemical structure of the tetracycline omadacycline 7
Fig. 1.6 Chemical structure of the Avibactam NXL-104....................... 7
Fig. 1.7 Cell wall envelopes of Gram-positive and Gram-negative
bacteria.................................................................................... 8
Fig. 1.8 N-formimodoyl-thienamycin............................................... 15
Fig. 1.9 Inhibition of protein synthesis by aminoglycosides 19
Fig. l.li Inhibition of cell wall synthesis by p-lactams.................... 20
Fig. 1.1 Inhibition of DNA synthesis by quinolones........................ 20
Fig. l.i: Global emergence of MBLs................................................... 32
Fig. l.i: The emergence of CTX-M type ESBLs in Europe 33
Fig. 1.1- The occurrence of K. pneumoniae resistant to 3rd generation
cephalosporins in Europe.................................................. 36
Fig. 1.1: The occurrence of P. aeruginosa resistant to carbapenems
in Europe.................................................................................. 37