Antibiotic resistance in global clone 2 Acinetobacter baumannii from Singapore

362 Pages·2017·7.7 MB·English

Antibiotic resistance in global clone 2 Acinetobacter
baumannii from Singapore

Grace Annie Bell Blackwell

A thesis submitted in the fulfilment of the requirements for the degree of
Doctor of Philosophy

Faculty of Science
School of Life and Environmental Science
The University of Sydney
NSW, Australia

2018
Declaration
This is to certify that to the best of my knowledge; the content of this thesis is my own work.
This thesis has not been submitted for any degree or other purposes.

I certify that the intellectual content of this thesis is the product of my own work and contains
no material previously published or written by another person, unless an appropriate reference
is stated in the text.

Grace Blackwell

I
ACKNOWLEDGEMENTS
First and foremost, I would like to thank my supervisor Professor Ruth Hall. The mentorship
she has provided for me over the past four years has been invaluable, particularly the time she
has spent helping me interpret data, solve problems and critique my writing. Without her, this
thesis would not have been possible and I feel privileged to have been one of her students. I
wish to acknowledge the past and present members of the Hall laboratory for their assistance
and support, and for making the lab a fun environment. I would particularly like to thank Steven
and Mohammad for always being available to answer any questions I had, and Robert who was
a constant companion through my PhD. I would also like to thank Chris, Carol, Stephanie,
Jade, Isabella and Rebecca for making the time spent at the lab enjoyable. Furthermore, I would
like to thank the staff and fellow students in G.08 who contributed to this building having such
a wonderful environment.

I would like to thank my parents, Frances and George. Their support and encouragement
through my entire education, not just my PhD, was invaluable and it would not have been
possible to get through it without them. I would also like to mention my siblings, Thomas,
Clare, Emily, Patrick and Isabel, who without your impact in my life, I would not be who I am
today. Finally, I would like to thank my partner Michael for all the love, support,
encouragement, proof-reading and patience he has provided, particularly during the writing of
this thesis.

II
PUBLICATIONS

Publications generated during the course of this study

Papers:
1. Blackwell, G.A., Nigro, S.J., Hall, R.M. Evolution of AbGRI2-0, the progenitor of the
AbGRI2 resistance island in global clone 2 of Acinetobacter baumannii. Antimicrobial
Agents and Chemotherapy, 2015; 60(3), 1421-1429. PMID: 26666934

2. Blackwell, G.A., Hamidian, M., Hall, R.M. IncM plasmid R1215 is the source of
chromosomally located regions containing multiple antibiotic resistance genes in the
globally disseminated Acinetobacter baumannii GC1 and GC2 clones. mSphere, 2016;
1(3), e00117-16. PMID: 27303751

3. Blackwell, G.A., Holt, K.E., Bentley, S.D., Hsu, L.Y., Hall, R.M. Variants of AbGRI3
carrying the armA gene in extensively antibiotic resistant Acinetobacter baumannii
from Singapore. Journal of Antimicrobial Chemotherapy, 2017, 72(4), 1031-1039.
PMID: 28073968

4. Blackwell, G.A., Hall, R.M. The tet39 determinant and the msrE-mphE genes in
Acinetobacter plasmids are each part of discrete modules flanked by inversely-oriented
pdif (XerC-XerD) sites. Antimicrobial Agents and Chemotherapy, 2017
doi:10.1128/AAC.00780-17. PMID: 28533235

III
GenBank database submissions:
1. Blackwell G.A., Holt K.E., Pickard D. and Hall R.M. Acinetobacter baumannii strain
RUH134 KL9 capsule biosynthesis locus, genomic resistance island AbGRI1-1
(Tn6166), genomic resistance island AbGRI2-0a, genomic resistance island AbGRI2-
0b, and OCL1 outer-core biosynthesis locus. Update to GenBank accession number:
JN247441; bases 48,558 to 60,783; 60,884 to 88,292; 39,633 bp. Submitted
30/06/2015.
2. Blackwell G.A. and Hall, R.M. Serratia marcescens strain NCTC 50331 plasmid
R1215, complete sequence. GenBank accession number: KU315015; 95,855 bp.
Submitted 17/12/2015.
3. Blackwell G.A. and Hall R.M. Acinetobacter baumannii strain SGH0701 genomic
resistance island AbGRI3, complete sequence. GenBank accession number:
KX011025; bases 30477 to 58194; 27,717 bp. Submitted 31/03/2016.
4. Blackwell G.A. and Hall R.M. Acinetobacter baumannii strain SGH0908 genomic
resistance island AbGRI3, complete sequence. GenBank accession number:
KX011026; 13,418 bp. Submitted 31/03/2016.
5. Blackwell G.A. and Hall R.M. Acinetobacter baumannii strain SGH1111 genomic
resistance island AbGRI3, complete sequence. GenBank accession number:
KX011027; 21,973 bp. Submitted 31/03/2016.
6. Blackwell G.A. and Hall R.M. Acinetobacter baumannii strain SGH0823 plasmid
pS30-1, complete sequence. GenBank accession number: KY617771; 18,234 bp.
Submitted 16/02/2017.

IV
AUTHORSHIP ATTRIBUTION STATEMENT

Chapter 6 of this thesis is published as:

Blackwell, G.A., Holt, K.E., Bentley, S.D., Hsu, L.Y., Hall, R.M. Variants of AbGRI3
carrying the armA gene in extensively antibiotic resistant Acinetobacter baumannii from
Singapore. Journal of Antimicrobial Chemotherapy, 2017, 72(4), 1031-1039.
I performed the experimental work, analysed the data, prepared figures and tables and wrote
the drafts of the MS.

Chapter 10 of this thesis is published as:

Blackwell, G.A., Hall, R.M. The tet39 determinant and the msrE-mphE genes in
Acinetobacter plasmids are each part of discrete modules flanked by inversely-oriented pdif
(XerC-XerD) sites. Antimicrobial Agents and Chemotherapy, 2017 doi:10.1128/AAC.00780-
17.
I performed the experimental work, analysed the data, prepared figures and tables and wrote
the drafts of the MS.

Grace Blackwell, 27/10/17

As supervisor for the candidature upon which this thesis is based, I can confirm that the
authorship attribution statements above are correct.

Prof. Ruth Hall, 27/10/17

V
ABSTRACT
Extensively and pan antibiotic resistant Acinetobacter baumannii belonging to the global clone
GC2 present a significant challenge in the treatment of infections worldwide. This study aimed
to analyse the causes of antibiotic resistance in a selection of 20 extensively antibiotic resistant
GC2 isolates from Singapore. One isolate was from 1996 and others were from 2004-2011. All
carried multiple antibiotic resistance genes including the carbapenem resistance gene oxa23.

To determine the relationships between these isolates the structures of the AbGRI1, AbGRI2
and AbGRI3 resistance islands were examined, along with ISAba1 locations and MLST
profiles (Oxford scheme). Plasmid diversity was also examined. Most isolates were ST208 or
a single locus variant and one was a triple locus variant. All had ISAba1 upstream of ampC
(resistance to third generation cephalosporins) and a version of AbGRI1 and AbGRI2.
However, AbGRI3 including the armA gene (resistance to all clinically relevant
aminoglycosides) was present in 15 isolates.

Shared features allowed evolutionary lineages to be inferred. The 1996 ST208 isolate shared
many features with most Australian isolates including AbGRI2-1, and 11 of 12 conserved
ISAba1 positions, and hence is closely related to their progenitor. Four other isolates had
AbGRI2-1 but ISAba1 in only 8 conserved positions. The remaining 15 isolates contain an
AbGRI3 variant and an IS26-derived deletion variant of AbGRI2-1. They share 3 of the
conserved ISAba1s and 2 more ISAba1 positions specific to the AbGRI3 lineage. Six AbGRI3
variants were detected. One was associated with 6 extra ISAba1 positions.

RepAci6 plasmids of 2 known backbone types and 2 novel types were found in the collection;
most were transfer proficient. Of the 14 plasmids, 2 were cryptic, 4 carried aphA6 (amikacin
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resistance) in TnaphA6, 10 had oxa23 in Tn2006 in AbaR4 and one had oxa23 in Tn2008. Two
novel transposons were also identified. All AbGRI3 isolates had a small RepAci1 plasmid with
no mobilisation genes and one carrying Tn2006 was mobilised by a RepAci6 plasmid enabling
a shared putative oriT to be found.

The tet39 tetracycline resistance determinant and the macrolide resistance gene pair msrE-
mphE were found in a novel plasmid in one isolate. These resistance genes are in dif modules,
bounded by inversely-oriented XerC-XerD binding sites, termed pdif sites. Numerous dif
modules containing many different orfs, including various toxin/antitoxin systems, were
identified in Acinetobacter plasmids. Two types of dif modules, C and D modules, that usually
alternate were defined.

VII
ABBREVIATIONS

Units

bp Base pair
kb Kilo base
Mb Mega base
M Molar
Mol Moles
U Units of activity (of enzyme)
v/v Volume per volume
w/v Weight per volume

Terms

aa Amino acid
AbaR A. baumannii resistance island
AbGRI A. baumannii genomic resistance island
CDS Calibrated Dichotomous Sensitivity
CLSI Clinical and Laboratory Standards Institute
CR Common region
5`-CS 5`-conserved segment
3`-CS 3`-conserved segment
DNA Deoxyribonucleic acid
dNTP Deoxynucleotide triphosphate
DR Direct repeat
EC European clone
GC Global clone
EDTA Ethylenediaminetetraacetic acid
EtBr Ethidium bromide
HGT Horizontal gene transfer
ICU Intensive care unit
In Integron
IS Insertion sequence
IR Inverted repeat
LB Luria Bertani
MAR Multiply antibiotic resistant
MARR Multiple antibiotic resistance region
MIC Minimum inhibitory concentration
MLST Multi-locus sequence typing
MQ Milli-Q water
NCBI National Centre for Biotechnology information
nt Nucleotide
orf Open reading frame
PCR Polymerase chain reaction
PFGE Pulse-field gel electrophoresis
RI Resistance island
SDS Sodium dodecyl sulfate
SNP Single nucleotide polymorphism
sp. Species
ST Sequence type
TBE Tris-borate EDTA
TE Tris-EDTA
Tn Transposon
Tris Tris(hydroxymethyl)aminomethane
UV Ultra-violet
V Volts
VIII
Antibiotics

IPM Imipenem
MER Meropenem
Dor Doripenem
Ap Ampicillin
SAM Ampicillin/sulbactam
TIM Ticarcillin-clavulanic acid
TZP Piperacillin-tazobactam
CTX Cefotaxime
CAZ Ceftazidime
CRO Ceftriaxone
FEP Cefepime
Sm Streptomycin
Sp Spectinomycin
Su Compound sulphonamides
Tc Tetracycline
Mc Minocycline
Dc Doxycycline
Tp Trimethoprim
Cl Chloramphenicol
Fl Florfenicol
Km Kanamycin
Nm Neomycin
Gm Gentamicin
Ne Netilmicin
Ak Amikacin
Tm Tobramycin
Nx Nalidixic acid
CIP Ciprofloxacin
LVX Levofloxacin
Rif Rifampicin
CLAV Clavulanic acid

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