Analysis of multiply antibiotic-resistant Acinetobacter baumannii belonging to Global Clone 1

269 Pages·2014·12.7 MB·English

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Analysis of multiply antibiotic-resistant
Acinetobacter baumannii belonging to
Global Clone 1

Mohammad Hamidian

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

School of Molecular Bioscience
The University of Sydney
NSW, Australia
May 2014
Declaration

The thesis presented here contains no material which has been submitted or accepted for any other
degree or qualification at any university or institution. To the best of my knowledge this thesis is
original and contains no material previously published or written by another person, unless an
appropriate reference is stated in the text.

Mohammad Hamidian
16/05/2014

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ACKNOWLEDGMENTS
First and foremost, I want to thank my supervisor Professor Ruth Hall. It has been a great
privilege to be her Ph.D. student. I would like to thank her for the many hours she spent
helping me interpreting my data, improving my writing skills and teaching me how to think
and how to present my data. She patiently provided the vision, encouragement and advise
necessary for me to proceed throughout my PhD.
I would like to express my debt of gratitude to the past and present members of the Hall Lab
for their scientific collaborations, supports and for being more than a colleague over the
course of my PhD. Amy, Jeremy, and Virginia who helped me beginning this project and
Sashi and Steven for the countless helps throughout this project. A special thank to Chris
who helped a lot with the proof reading of this thesis. I would also like to thank Robert,
Johanna, Matthew and Grace who made this journey more pleasant.
I want to thank my parents, which I owe them everything. I would also like to thank my
wonderful wife, Mehrnoosh, whose love and encouragement was my driving force all along
this project and allowed me finishing this journey. Finally, I would like to dedicate this
thesis to her and also want to thank her for her patience putting up with my bad moods
whilst writing this thesis.

Mohammad Hamidian
16/05/2014
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PUBLICATIONS

Publications generated during the course of this study.

Papers:

1. Hamidian M, Holt KE, Pickard D, Dougan G, Hall RM. A GC1 Acinetobacter baumannii
isolate carrying AbaR3 and the aminoglycoside resistance transposon TnaphA6 in a
conjugative plasmid. J Antimicrob Chemother. 2014 Apr;69(4):955-8.

2. Hamidian M, Wynn M, Holt KE, Pickard D, Dougan G, Hall RM. Identification of a marker
for two lineages within the GC1 clone of Acinetobacter baumannii. J Antimicrob
Chemother. 2014 Feb;69(2):557-8.

3. Hamidian M, Hall RM. Tn6168, a transposon carrying an ISAba1-activated ampC gene and
conferring cephalosporin resistance in Acinetobacter baumannii. J Antimicrob Chemother.
2014 Jan;69(1):77-80.

4. Hamidian M, Hall RM. ISAba1 targets a specific position upstream of the intrinsic ampC
gene of Acinetobacter baumannii leading to cephalosporin resistance. J Antimicrob
Chemother. 2013 Nov;68(11):2682-3.

5. Hamidian M, Hancock DP, Hall RM. Horizontal transfer of an ISAba125-activated ampC
gene between Acinetobacter baumannii strains leading to cephalosporin resistance. J
Antimicrob Chemother. 2013 Jan; 68(1): 244-5.

6. Hamidian M, Nigro SJ, Hall RM. Variants of the gentamicin and tobramycin resistance
plasmid pRAY are widely distributed in Acinetobacter. J Antimicrob Chemother. 2012 Dec;
67(12): 2833-6.

7. Post V, Hamidian M, Hall RM. Antibiotic-resistant Acinetobacter baumannii variants
belonging to global clone 1. J Antimicrob Chemother. 2012 Apr; 67(4): 1039-40.

8. Hamidian M, Hall RM. AbaR4 replaces AbaR3 in a carbapenem-resistant Acinetobacter
baumannii isolate belonging to global clone 1 from an Australian hospital. J Antimicrob
Chemother. 2011 Nov; 66(11): 2484-91.

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GenBank database submissions:

1. Hamidian, M., Holt, K., Pickard, D., Dougan, G. and Hall, R. Conjugative plasmids of
Acinetobacter carrying the aminoglycoside resistance transposon TnaphA6. GenBank
accession number: KCF669606; 70, 100 bp (Submitted 13-SEP-2013).

2. Hamidian, M., Wynn, M., Holt, K., Pickard, D., Dougan, G. and Hall, R. Acinetobacter
baumannii strain WM98 multiple antibiotic resistance region AbaR0. GenBank accession
number: KF483599; 65,329 bp (Submitted 01-AUG-2013).

3. Hamidian, M. and Hall, R. M. ISAba1 targets a specific position upstream of the intrinsic
ampC. GenBank accession number: KF030679; 2,739 (Submitted 10-MAY-2013).

4. Hamidian, M. and Hall, R. M. ISAba1 targets a specific position upstream of the intrinsic
ampC. GenBank accession number: KF030678; 2,738 (Submitted 10-MAY-2013).

5. Hamidian, M. and Hall, R. M. A transposon carrying an ISAba1-activated ampC gene
conferring to in cephalosporin resistance in Acinetobacter baumannii. GenBank accession
number: KC118540; 7,664 bp (Submitted 29-APR-2013).

6. Hamidian, M. and Hall, R. M. Tn6168, a transposon carrying an ISAba1-activated ampC
gene and conferring cephalosporin resistance in Acinetobacter baumannii. GenBank
accession number: KF002790; 1,513 bp (Submitted 26-APR-2013).

7. Hamidian, M. and Hall, R. M. AbaR26, an AbaR3-type resistance island in an Australian
global clone 1 (GC1) isolate. GenBank accession number: KC665626; 21,160 bp
(Submitted 19-FEB-2013).

8. Hamidian, M. and Hall, R. M. Horizontal transfer of ISAba1-activated ampC genes leading
to in cephalosporin resistance in Acinetobacter baumannii global clone 1. GenBank
accession number: KC118541 2,740 bp (Submitted 30-OCT-2012).

9. Hamidian, M., Hancock, D. P. and Hall, R. M. Horizontal transfer of an ISAba125-activated
ampC gene between Acinetobacter baumannii strains leading to cephalosporin resistance.
GenBank accession number: JQ684178; 2,647 bp (Submitted 17-FEB-2012).

10. Hamidian,M., Nigro,S.J. and Hall,R.M. Variants of the gentamicin and tobramycin
resistance plasmid pRAY are widely distributed in Acinetobacter. GenBank accession
number: JQ904627; 6,078 bp (Submitted 05-APR-2012).

11. Hamidian, M. and Hall, R. M. AbaR4 replaces AbaR3 in a carbapenem-resistant
Acinetobacter baumannii isolate belonging to global clone 1 from an Australian hospital.
GenBank accession number: JN107991; 17,632 bp (Submitted 10-JUN-2011).

12. Hamidian, M. and Hall, R. M. AbaR3-type resistance islands in Australian global clone 1
(GC1) Isolates. GenBank accession number: JN409449; 3,932 bp (Submitted 27-Jun-2011).

13. Hamidian, M. and Hall, R. M. AbaR3-type island in European GC1 isolates. GenBank
accession number: JN968482: 3,917 bp (Submitted 27-OCT-2011).

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GenBank entries updated by adding new sequences:

1. Hamidian, M., Hancock, D.P. and Hall, R. M. Horizontal transfer of an ISAba125-activated
ampC gene between Acinetobacter baumannii strains leading to cephalosporin resistance.
GenBank accession number: FJ172370; 13,449 bp added (Updated 02-MAR-2012).

2. Hamidian, M. and Hall, R. M. Resistance to cephalosporins in Acinetobacter baumannii due
to replacement of the chromosomal ampC gene by an IS activated ampC gene. GenBank
accession number: GQ406245; 2,535 bp added (Updated 08-FEB-2012).

3. Hamidian, M. and Hall, R. M. Resistance to cephalosporins in Acinetobacter baumannii due
to replacement of the chromosomal ampC gene by an IS activated ampC gene. GenBank
accession number: GQ406246; 1,507 bp added (Updated 08-FEB-2012).

4. Hamidian, M. and Hall, R. M. Resistance to cephalosporins in Acinetobacter baumannii due
to replacement of the chromosomal ampC gene by an IS activated ampC gene. GenBank
accession number: HM590877; 2,743 bp added (Updated 14-DEC-2011).

5. Hamidian, M., Nigro, S. J. and Hall, R. M. Variants of the gentamicin and tobramycin
resistance plasmid pRAY are widely distributed in Acinetobacter. GenBank accession
number: JF343535; 5,141 bp added (Updated 24-APR-2012).

6. Hamidian, M. and Hall, R. M. ISAba1 targets a specific position upstream of the intrinsic
ampC gene of Acinetobacter baumannii leading to cephalosporin resistance. GenBank
accession number: JN968483; 2,733 bp added (Updated 13-MAY-2013).

7. Hamidian, M. and Hall, R. M. ISAba1 targets a specific position upstream of the intrinsic
ampC gene of Acinetobacter baumannii leading to cephalosporin resistance. GenBank
accession number: JN247441; 1,544 bp added (Updated 13-MAY-2013).

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ABSTRACT
A. baumannii resistant to multiple antibiotics challenges the treatment of infections caused by
this organism. Multiply-antibiotic resistant (MAR) isolates mainly belong to two major global
clones GC1 and GC2, and emergence of MAR strains is mainly due to the global spread of these
clones. The objective of this study was to analyze the genetic basis of antibiotic resistance in
Australian GC1 isolates.
Twenty six MAR GC1 isolates were recovered from different Australian hospitals in Sydney,
Melbourne, Adelaide and Brisbane between 1997 to 2011. The isolates were examined to determine
how closely they were related to one another. This included examining resistance to 23 antibiotics
and the causes of the resistance phenotypes observed. All but one were resistant to ciprofloxacin
and nalidixic acid, and all but one isolate were resistant to gentamicin, kanamycin and streptomycin
and sulphonamides. More than half of the isolates were resistant to 3rd generation cephalosporins
but only five isolates were resistant to carbapenems.
Molecular typing revealed that the GC1 collection consisted of diverse isolates but two
outbreak strains from a Sydney hospital were identified. The outbreak strains all belonged to
ST109/231 (Oxford Scheme) but there were 2 groups based on their shared properties. These strains
were recovered between 2006-2011 in the spinal injury unit of one of the Sydney hospitals
indicating that they have persisted in the hospital environment. MLST analysis showed that more
than half of the remaining 12 Australian GC1 isolates belonged to ST109/231, and the remainder
were all SLV (ST347 and ST247) and DLV (ST126) of ST109/231 differing either in gpi or the gpi
and gyrB alleles respectively.
MAR GC1 isolates include an AbaR3-type island, which usually includes several antibiotic
and heavy metal resistance determinants, in the comM gene. An AbaR island of this type was found
in all but one of the Australian GC1 isolates. Amongst the isolates with an AbaR3-type island, only
four isolates from different Australian hospitals were found to contain AbaR3 and the remaining
isolates contained distinct variants, some of which had not been seen before. The loss of the AbaR
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segments had largely occurred by IS26-mediated deletion. Homologous recombination between two
Tn6018 copies removing the entire multiply antibiotic resistance region was also found in one
isolate. A deletion in the 5'-CS of the class 1 integron was also identified in some AbaR islands.
This separated the islands into two groups, one with the deletion and the other without. The AbaR
island in an isolate from a Sydney hospital, which was named AbaR0, lacked the deletion but was
otherwise identical to AbaR3. AbaR0 was proposed to be the ancestral island as it includes an intact
5'-CS and also contains an additional orfP in the integron cassette array. The structure of AbaR
islands was also a property that could group the isolates as the ones with the same AbaR were
closely related.
One carbapenem resistant isolate recovered in Sydney in 2008 was found to carry oxa23 in
the AbaR4 island in the comM gene instead of an AbaR0/AbaR3-type island. This isolate belongs to
a separate lineage that has derived from its antibiotic susceptible ancestor, found in WGS database
of GenBank, by incorporating AbaR4 and becoming carbapenem resistant.
In Australian isolates, resistance to third generation cephalosporins was due to the activation
of the ampC gene by an ISAba1 providing it with a new promoter. In three Australian ST126
(Oxford Scheme) isolates, the ISAba1-ampC structure was found to be part of an ISAba1 bounded
transposon, Tn6168. Several other isolates included an ampC sequence distinct from the standard
GC1 sequence. Comparison of the draft genome of these isolates to the standard GC1 sequence
without the IS next to ampC, revealed sequence patches of 30 kb in a single isolate and 2.4 kb in
one group of 8 strains. In the later case, the source of sequence was found to be ST25 strains
(Institut Pasteur). In one isolate from Greece activation of ampC was found via insertion of
ISAba125 next to the start of ampC.
Plasmids were found to play an important role in carbapenem and tobramycin resistance in
Australian isolates. In two carbapenem resistant isolates AbaR4 was found in an 86 kb conjugative
plasmid. Variants of a 6 kb plasmid named pRAY*, which carries the aadB gene were found to be
responsible for tobramycin resistance.
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Together, the results of this study showed the variety of mobile elements, genomic islands,
plasmids, transposons and insertion sequences, that incorporate antibiotic resistance genes in
Australian GC1 isolates. AbaR islands were found to carry genes conferring resistance to
kanamycin, gentamicin, streptomycin and sulphonamides. IS26-mediated deletions and homologous
recombination between Tn6018 copies in the AbaR islands were shown to be responsible for
generating new variants. AbaR0, the ancestor of AbaR islands was found and all of the variants
seen so far could be derived from it. Moreover, structure of AbaR islands was found to be an
extremely useful tool to type the strains.
Carbapenem resistance was rare in Australian GC1 strains. Three strains carried the oxa23
carbapenem resistance gene in an AbaR4 island, which includes a backbone related to the backbone
of AbaR islands. In one isolate, AbaR4 was found where AbaR0/AbaR3 type islands are usually
found. This indicates that transposon backbone of AbaR4 can target the same position as
AbaR0/AbaR3. The other two strains carried AbaR4 in a conjugative plasmid, and the oxa23 gene
can be potentially disseminated by this plasmid into strains of different types. The small plasmid
pRAY* was responsible for introducing the tobramycin resistance gene aadB into GC1s. Hence,
this study shows the significance of plasmids incorporating additional determinants in GC1s. The
most unexpected finding was horizontal transfer of DNA segments that contain ISAba1-ampC
generating resistance to third generation cephalosporins.
Overall, Australian GC1 isolates included a diverse collection. However, two outbreak GC1
strains were identified in a single ward of one of the Sydney hospitals. Outbreak strains persisted
for a period of time and were then replaced by a GC2 strain.

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