View Article PDF

Acinetobacter baumannii is a gram-negative coccobacillus that has emerged as a multi-drug resistant (MDR) cause of hospital acquired infection in many locations worldwide.[[1,2]] Infections primarily affect vulnerable patients in the intensive care setting and are associated with high crude mortality.[[1]] The emergence of MDR A. baumannii has occurred in association with regional and international spread of hospital adapted clones.[[3–5]] Carbapenem antimicrobials were the primary treatment option for MDR A. baumannii strains. However, the emergence of carbapenem resistance in these global lineages, mediated primarily by acquired OXA carbapenemases, has severely restricted treatment options.[[3,6]]

In the Pacific/Oceania regions, carbapenem resistant A. baumannii have been reported from French Polynesia, New Caledonia and Australia since the early- to mid-2000s.[[7–9]] In 2015–2017 an outbreak occurred in a Fijian neonatal intensive care unit, with invasive infection associated with a crude mortality of 86%.[[10]] MDR A. baumannii is not endemic in New Zealand hospitals, with sporadic cases identified primarily in individuals with a history of overseas hospitalisation. A single outbreak has been reported in New Zealand, with an MDR, but carbapenem sensitive, strain that affected an Auckland hospital in 1998–1999.[[11]] However, in recent times we have experienced an increase in their detection, associated with prior hospitalisation in neighbouring Pacific Island countries and territories (PICT).

The limited antimicrobial treatment options and propensity of carbapenem resistant A. baumannii to cause hospital outbreaks poses a threat to healthcare in New Zealand and other PICT. Enhancing our understanding of the local epidemiology of carbapenem resistant A. baumannii may assist with mitigation strategies. Utilising the high discriminatory capacity of whole genome sequence (WGS) molecular epidemiology we aim to contextualise local New Zealand isolates within the global epidemiology, establish the local relationship between isolates, considering especially those with links to PICT, and explore potential routes of dissemination in the Pacific regions.

Methods

This was a retrospective descriptive study. We identified carbapenem resistant A. baumannii complex isolates with putative acquired OXA carbapenemase genes from laboratory records at Auckland District Health Board (ADHB) and the Antimicrobial Resistance (AMR) Reference Laboratory at the Institute of Environmental Science and Research (ESR) from January 2010 to April 2018. This is believed to have captured the majority of cases in New Zealand over this period as all laboratories were encouraged to send carbapenem resistant A. baumannii isolates to ESR for further characterisation. Basic demographic information (age, gender, ethnicity) and epidemiological metadata, such as a documented history of overseas hospitalisation, were retrieved from clinical records where possible. Study approval was obtained from the New Zealand Health and Disability Commission ethics committee (reference number 18/NTB/24). Funding was provided by the A+ Trust Microbiology Education and Research Fund.

Isolates were grown on sheep blood agar and identified to species-complex by MALDI-ToF MS (BioMerieux). Susceptibility to meropenem, piperacillin-tazobactam and colistin, was determined using Sensititre (ThermoFisher Scientific) microbroth dilution method. Meropenem and colistin mean inhibitory concentrations (MICs) were interpreted as per EUCAST Acinetobacter breakpoints; and piperacillin-tazobactam MICs were interpreted as per CLSI Acinetobacter breakpoints (EUCAST breakpoints not being available for Acinetobacter versus piperacillin-tazobactam).[[12,13]] Susceptibility to ciprofloxacin, cotrimoxazole, gentamicin, and amikacin were determined using disc diffusion as per EUCAST.[[12]] Ceftazidime susceptibility was determined by disc diffusion as per CLSI (EUCAST breakpoints not being available for Acinetobacter versus ceftazidime).[[13]]

DNA was extracted from each isolate utilising the QIAamp DNA mini-Kit (QIAGEN). Unique dual indexed libraries were prepared using the Nextera XT DNA sample preparation kit (Illumina). Libraries were sequenced on the Illumina NextSeq 500 with 150-cycle paired end chemistry as described by the manufacturer’s protocols. Bioinformatic analysis was performed using the Nullarbor[[14]] v2 pipeline. Briefly, Trimmomatic[[15]] v0.36 was used to remove adaptors and low-quality bases and reads. Kraken[[16]] v1.0 was used to perform in silico species detection and assess the paired-end read-sets for contamination. Short-reads were assembled de novo using SPAdes[[17]] v3.12.0 and the resultant contigs were annotated using Prokka[[18]] v1.14. Multi-locus sequence type (MLST) was determined using MLST[[19]] v2.11 with the A. baumannii Pasteur scheme[[20]] (cpn60, fusA, gltA, pyrG, recA, rplB, rpoB) downloaded in July 2018. OXA carbapenemase genes were identified using the Resfinder[[21]] database in Abricate[[22]] v0.8.2. Mash[[23]] v2.1 was used to create a distance matrix from k-mer hashes, and QuickTree[[24]] v2.3 was used to construct a neighbour joining tree for exploratory analysis of the relationship among isolates. A core-genome (defined as sequences found in >99% of isolates) maximum likelihood tree was then inferred for MLST ST2 isolates with IQ-TREE[[25]] v1.6.5, using the A. baumannii reference genome under NCBI accession NC_021729, with core-genome SNPs identified using Snippy[[26]] v4.0, and probable recombinant sites removed using Gubbins[[27]] v2.3.1. Reads for each sequenced isolate (AB1-20, AB22-34) have been deposited in NCBI under Bioproject accession PRJNA855258.

Results

Thirty-three distinct carbapenem resistant A. baumannii complex isolates were identified from 32 persons (cases) between January 2010 and April 2018. Twenty-four of 33 (73%) isolates were identified since January 2015 (Figure 1), and 23 (70%) were identified in the Auckland region (Figure 1A). Eighteen of the 26 (69%) isolates with available data were identified in clinical specimens, while eight (31%) were identified in MDR organism screens alone. Cases had a median age of 56 years (range <1 to 77) and 18 of 32 (56%) were female. Eight of 30 (27%) cases, with available ethnicity data, were reported as Pākehā/NZ European; four (13%) as Māori; four (13%) as Fijian; four (13%) as Samoan; three (10%) as Indian; three (10%) as other European; two (7%) as Fijian Indian; one as Chinese (3%); and one (3%) as other Asian. Twenty-one of 32 (66%) cases had an identifiable history of “recent” overseas hospitalisation; 14 were direct hospital to hospital transfers, five were hospitalised overseas in the preceding four weeks, and in two cases the exact timeframe could not be identified. Ten of these 21 (48%) persons had been hospitalised in Fiji, four (19%) in Samoa, and one (5%) in each of French Polynesia, China, India, Korea, Thailand, Greece and Romania. One of these persons carried two distinct strains. Two further cases had a strong epidemiological link (hospitalised in same ward in New Zealand) to imported cases (Fiji and French Polynesia respectively); giving 24 (73%) of 33 isolates an identifiable link to overseas hospitalisation (Figure 1B).

Thirty-one (94%) of the 33 A. baumannii complex isolates were identified (from sequence data) as A. baumannii sensu stricto (AB1-16, 18–20, 22–25, 27–34) and two (6%) as A. pittii (AB26, 17). Twenty-three (74%) of the 31 A. baumannii sensu stricto were ST 2 (AB1-10, 13, 16, 19, 22–24, 27–29, 31–34), three (10%) were ST 25 (AB11, 12, 14), two (6%) were ST 1 (AB18, 25), and there was one (3%) each of ST 103 (AB15), ST 107 (AB20), and ST 164 (AB30). None of the 31 A. baumannii sensu stricto were susceptible to meropenem (as per selection criteria); three (10%) were susceptible to ceftazidime; none were susceptible piperacillin-tazobactam; one (3%) was susceptible to gentamicin; five (16%) were susceptible to amikacin; three (10%) were susceptible to cotrimoxazole; three (10%) were susceptible to ciprofloxacin; and 29 (94%) were suspectable to colistin. None of the A. pitti were susceptible to meropenem or piperacillin-tazobactam but were susceptible to all other antimicrobials tested. Twenty-nine (94%) of the 31 A. baumannii sensu stricto carried a bla{{OXA-23}} gene and two (6%) carried bla{{OXA-40-like}} genes. Both A. pittii isolates carried a bla{{OXA-40-like}} gene.

The maximum likelihood phylogenetic tree (based on analysis of 281,823 SNPs) of the 23 ST 2 A. baumannii sensu stricto is shown in Figure 2. There were three genomic clusters of closely related isolates separated by <15 single nucleotide polymorphisms (SNPs) on pairwise analysis; in contrast to thousands of SNPs between each respective cluster and other ST 2 strains; These clusters included, 1) 12 isolates identified predominantly in the Auckland Region between 2015 and 2018; 2) three isolates identified in Canterbury between 2015 and 2018; and 3) two isolates from Auckland in 2011. Clusters 1, 2, and 3 accounted for 36%, 9%, and 6% of the 33 carbapenem resistant A. baumannii complex isolates in this study (Figure 1C).

View Figures 1 & 2.

Discussion

This study describes the epidemiology of carbapenem resistant A. baumannii in New Zealand from 2010 to 2018. The majority of cases were identified in the Auckland city region, reflecting perhaps a relatively larger population size, regional differences in population demographics, receipt of medical repatriations, and/or provision of tertiary services to other PICT. The majority of cases also had an identifiable history of recent overseas hospitalisation; in particular, hospitalisation in Fiji or Samoa. An increase in cases associated with these countries has occurred since 2015 in temporal association with an outbreak in Fiji.[[10]] This association contrasts with other MDR Gram-negative bacilli in New Zealand, such as carbapenemase producing Enterobacterales, which are typically associated with healthcare contact or travel in South and South-East Asia.[[28]]

A. baumannii ST 2 was the most common ST identified. ST 2 is a globally distributed hospital adapted clone that is commonly associated with outbreaks, including in Fiji.[[3,10]] Using WGS based molecular epidemiology we identified three clusters of closely related isolates among the 23 ST 2. Cluster 1 consisted of 12 isolates that were identified at three different laboratories in the Auckland Region and one laboratory in the Waikato between 2015 and 2018. Nine (75%) of the cases had a history of recent hospitalisation in either Fiji (5) or Samoa (4). Of the remaining three cases, one is presumed to represent transmission in the New Zealand hospital setting, another was of Samoan ethnicity, while the final case had no demographic or epidemiological data available. Cluster 1 isolates were resistant to all antimicrobials tested except colistin and all carried the carbapenemase gene bla{{OXA-23}}. The close epidemiological, phylogenetic, and temporal relationship of the Cluster 1 isolates indicate recent trans-national spread of a single strain between healthcare facilities in Fiji, Samoa and New Zealand. We hypothesise Cluster 1 to be the same strain responsible for the 2015–2017 outbreak in Fiji but did not have isolates available to allow testing of this hypothesis.[[10]] Cluster 2 consisted of three ST 2 isolates identified in Canterbury between 2015 and 2018. The earliest case had a history of hospitalisation in South Korea. Their close phylogenetic relationship and common location suggests local transmission. Cluster 3 consisted of two isolates identified in an Auckland hospital in 2011. One case had a history of hospitalisation in French Polynesia with the second case strongly linked by epidemiological and now genomic data to in-hospital transmission in New Zealand.

In addition to the isolates described in Cluster 1, a further five unrelated cases were associated with recent hospitalisation in Fiji. These included four A. baumannii sensu stricto isolates: one ST 1 from 2011, one ST 107 from 2014, one non-clustered ST 2 from 2017, and one ST 25 from 2017; as well as a single A. pittii from 2016. This suggests there have been multiple strains of carbapenem resistant Acinetobacter introduced into Fijian hospitals and/or circulation of the transposons bearing carbapenem resistance genes over the past decade, the latter which could be the subject of a future study. In contrast, all four isolates associated with hospitalisation in Samoa were part of ST 2 cluster 1. The ST 2 strain associated with hospitalisation in French Polynesia from 2011 was not closely related to any isolates associated with Fiji or Samoa suggesting a separate introduction into the Pacific. The acquired bla{{OXA-23}} gene was the most common carbapenemase gene identified. The clonal nature of a significant proportion of the isolates in this study narrows bla{{OXA}} diversity; however, the predominance of bla{{OXA-23}} is consistent with reports from the Asia-Oceania regions.[[29]]

Increasing numbers of carbapenem resistant A. baumannii have been identified in New Zealand since 2015. This has occurred in association with the transnational spread of a ST 2 strain between Fiji, Samoa and New Zealand.

With a known propensity to cause hospital outbreaks and limited antimicrobial treatment options, carbapenem resistant A. baumannii poses a potentially escalating threat to safe healthcare delivery in New Zealand and other PICT. The major risk factor for carbapenem resistant A. baumannii infection/colonisation in the New Zealand setting is recent hospitalisation overseas; including in PICT that historically have been considered low risk for MDR gram-negative organisms. Hospitals require systematic processes to identify high risk individuals at presentation so appropriate microbiological screening can be performed and transmission-based infection control precautions implemented.

Summary

Abstract

Aim

Carbapenem resistant Acinetobacter baumannii have limited treatment options and a propensity to cause hospital outbreaks. In recent years an increase in their detection has been observed in New Zealand. This study aimed to describe the molecular epidemiology of these isolates.

Method

This study utilised carbapenem resistant A. baumannii complex isolates identified across New Zealand between January 2010 to April 2018. Whole genome sequence analysis and associated demographic information was used to contextualise local isolates within the global epidemiology and establish the relationship between isolates.

Results

Thirty-three carbapenem resistant A. baumannii complex isolates (31 A. baumannii sensu stricto) were identified. Twenty-four (73%) were from January 2015 onwards. Twenty-four (73%) had an identifiable epidemiological link to overseas hospitalisation. Twenty-three (74%) of 31 A. baumannii sensu stricto were sequence type (ST) 2 (Pasteur scheme). Phylogenetic analysis identified three ST2 clusters. The largest cluster, of 12 isolates, was from 2015 onwards; with nine (75%) associated with recent hospitalisation in Fiji or Samoa.

Conclusion

Increasing numbers of carbapenem resistant A. baumannii are being identified in New Zealand. Our data show that this is in large part associated with transnational spread of a single A. baumannii sensu stricto ST 2 strain between Fiji, Samoa and New Zealand.

Author Information

Matthew R Blakiston: Clinical Microbiologist, Microbiology Department, LabPlus, Auckland District Health Board. Auckland, New Zealand. Mark B Schultz: Bioinformatics Specialist, Microbiological Diagnostic Unit Public Health Laboratory, Peter Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, Australia. ORCID: 0000-0002-7689-6531. Indira Basu: Scientific officer. Microbiology Department, LabPlus, Auckland District Health Board. Auckland, New Zealand. Susan A Ballard: Principal Scientist, Microbiological Diagnostic Unit Public Health Laboratory, Department of Microbiology and Immunology, The Peter Doherty Institute of Infection and Immunity, The University of Melbourne, Melbourne, Australia. Deborah Williamson: Clinical Microbiologist, Royal Melbourne Hospital and Microbiological Diagnostic Unit Public Health Laboratory, Melbourne, Australia. Sally Roberts: Clinical Microbiologist, Microbiology Department, LabPlus, Auckland District Health Board. Auckland, New Zealand.

Acknowledgements

The authors acknowledge Helen Heffernan from the Institute of Environmental Science and Research Limited (ESR) for provision of isolates; and Susan Taylor (Counties Manukau Health), Joshua Freeman (Canterbury DHB), Chris Mansell (Waikato DHB), and Hanna-Sofia Anderrsson (Midcentral DHB) for provision of demographic and epidemiological information on cases.

Correspondence

Matthew Blakiston: Microbiology Department, LabPlus, PO Box 110031, Auckland City Hospital, Auckland, 1148, New Zealand. Ph: 022 642 6039.

Correspondence Email

mattbl@adhb.govt.nzurl

Competing Interests

Nil.

1) Peleg A, Seifert H, Paterson D. Acinetobacter baumannii: Emergence of a successful pathogen. Clin Microbiol Rev. 2008; 21:538-82.

2) Antunes C, Visca P, Towner K. Acinetobacter baumannii: evolution of a global pathogen. Pathog Dis. 2014; 71:292-301.

3) Karah N, Sundsford A, Towner K, Samuelson O. Insights into the global molecular epidemiology of carbapenem non-susceptible clones of Acinetobacter baumannii. Drug Resist Updat. 2012; 15:237-47

4) Zarilli R, Pournaras S, Giannouli M, Tsakris A. Global evolution of multi-drug resistant Acinetobacter baumannii clonal lineages. Int J Antimicrob Agents. 2013; 41:11-19

5) Diancourt L, Passet V, Nemec A, et al. The population structure of Acinetobacter baumannii: expanding multi-resistant clones from an ancestral susceptible genetic pool. PLoS One. 2010; 5 (4):e10034

6) Higgins P, Dammhayn C, Hackel M, Seifert H. Global spread of carbapenem resistant Acinetobacter. J Antimicrob Chemother. 2010; 65: 233-38.

7) Naas T, Levy M, Hirshauer C, et al. Outbreak of Carbapenem-Resistant Acinetobacter baumannii producing the carbapenemase OXA-23 in a tertiary care centre of Papeete, French Polynesia. J Clin Microbiol. 2005; 43: 4826-29

8) Le Hello S, Falcot V, Lacassin F, et al. Molecular epidemiology of carbapenem-resistant Acinetobacter baumannii in New Caledonia. Clin Microbio Infection. 2008; 14:977-81.

9) Peleg A, Franklin C, Bell J, Spelman D. 2006. Emergence of carbapenem resistance in Acinetobacter baumannii recovered from blood cultures in Australia. Infect control Hosp Epidemiol. 2006; 27: 759-61

10) Ministry of Health and Medical Services (Fiji) [Internet]. Acinetobacter baumannii outbreak in NICU at the colonial war memorial hospital Suva, Fiji, December 2016 - July 2017. Technical Report. [Available from: https://www.health.gov.fj/wp-content/uploads/2017/09/ACINETOBACTER-BAUMANNII-JULY-2017.pdf].

11) Roberts S, Findley R, Lang S. Investigation of an outbreak of multi-drug resistant Acinetobacter baumannii in an intensive care unit. J Hosp Infect. 2011; 48:228-232.

12) The European Committee on Antimicrobial Susceptibility Testing. Breakpoint tables for interpretation of MICs and zone diameters. Version 8.1. http://www.eucast.org.

13) Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing; 28[[th]] Ed. CLSI supplement M100.

14) Seeman T, Gonsalves de Silva A, Bulach D, Schultz M, Kwong J, Howden B. Nullarbor Github. http://:github.com/tseeman/nullabor.

15) Bolger A, Lohe M, Lohse, Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;20:2113-2120.

16) Wood DE, Salzberg SL. Kraken: ultrafast metagenomic sequence classification using exact alignments. Genome Biology 2014, 15. https://doi.org/10.1186/gb-2014-15-3-r46.

17) Bankevich A, Nurk S, Antipov D. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Bio. 2012; 19:445-477.

18) Seemann T. Prokka: rapid prokaryotic genome annotation. Bioinformatics. 2014;30:2068-2069.

19) Seemann T. mlst, Github https://github.com/tseemann/mlst.

20) Jolley K, Maiden: Scalable analysis of the bacterial genome variation at the population level. BMC bioinformatics. 2010; 11. https://doi.org/10.1186/1471-2105-11-595.

21) Zankari E, Hasman H, Cosentino, et al. Identification of acquired antimicrobial resistance genes. J Antimicrob Chemother. 2012;67:2640-2644.

22) Seemann T. Abricate, Github https://github.com/tseemann/abricate.

23) Ondov B, Treagan T, Melsted P, et al. Mash: fast genome and metagenome distance estimation using MinHash. Genome Biol. 2016;17. https://doi.org/10.1186/s13059-016-0997-x.

24) Howe, K. QuickTree. Gitbub. https://github.com/khowe/quicktree.

25) Nguyen L, Schmidt H, von Haeseler A, Minh B. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol Biol Evol. 2015;32: https://doi.org/10.1093/molbev/msu300.

26) Seeman T. Snippy. Github https://github.com/tseemann/snippy.

27) Croucher N, Page A, Conner T. Rapid phylogenetic analysis of large samples of recombinant bacterial whole genome sequences using Gubbins. Nucleic Acids Res. 2015;43. https://doi.org/10.1093/nar/gku1196.

28) Blakiston M, Heffernan H, Roberts S, Freeman J. The clear and present danger of carbapenemase-producing Enterobacteriaceae (CPE) in New Zealand: time for a national response plan. N Z Med J. 2017; 130: 72-79.

29) Kamolvit W, Sidjabat H, Paterson D. Molecular epidemiology and mechanisms of carbapenem resistance of Acinetobacter spp. in Asia and Oceania. Microb Drug resist. 2015; 21:424-43.

For the PDF of this article,
contact nzmj@nzma.org.nz

View Article PDF

Acinetobacter baumannii is a gram-negative coccobacillus that has emerged as a multi-drug resistant (MDR) cause of hospital acquired infection in many locations worldwide.[[1,2]] Infections primarily affect vulnerable patients in the intensive care setting and are associated with high crude mortality.[[1]] The emergence of MDR A. baumannii has occurred in association with regional and international spread of hospital adapted clones.[[3–5]] Carbapenem antimicrobials were the primary treatment option for MDR A. baumannii strains. However, the emergence of carbapenem resistance in these global lineages, mediated primarily by acquired OXA carbapenemases, has severely restricted treatment options.[[3,6]]

In the Pacific/Oceania regions, carbapenem resistant A. baumannii have been reported from French Polynesia, New Caledonia and Australia since the early- to mid-2000s.[[7–9]] In 2015–2017 an outbreak occurred in a Fijian neonatal intensive care unit, with invasive infection associated with a crude mortality of 86%.[[10]] MDR A. baumannii is not endemic in New Zealand hospitals, with sporadic cases identified primarily in individuals with a history of overseas hospitalisation. A single outbreak has been reported in New Zealand, with an MDR, but carbapenem sensitive, strain that affected an Auckland hospital in 1998–1999.[[11]] However, in recent times we have experienced an increase in their detection, associated with prior hospitalisation in neighbouring Pacific Island countries and territories (PICT).

The limited antimicrobial treatment options and propensity of carbapenem resistant A. baumannii to cause hospital outbreaks poses a threat to healthcare in New Zealand and other PICT. Enhancing our understanding of the local epidemiology of carbapenem resistant A. baumannii may assist with mitigation strategies. Utilising the high discriminatory capacity of whole genome sequence (WGS) molecular epidemiology we aim to contextualise local New Zealand isolates within the global epidemiology, establish the local relationship between isolates, considering especially those with links to PICT, and explore potential routes of dissemination in the Pacific regions.

Methods

This was a retrospective descriptive study. We identified carbapenem resistant A. baumannii complex isolates with putative acquired OXA carbapenemase genes from laboratory records at Auckland District Health Board (ADHB) and the Antimicrobial Resistance (AMR) Reference Laboratory at the Institute of Environmental Science and Research (ESR) from January 2010 to April 2018. This is believed to have captured the majority of cases in New Zealand over this period as all laboratories were encouraged to send carbapenem resistant A. baumannii isolates to ESR for further characterisation. Basic demographic information (age, gender, ethnicity) and epidemiological metadata, such as a documented history of overseas hospitalisation, were retrieved from clinical records where possible. Study approval was obtained from the New Zealand Health and Disability Commission ethics committee (reference number 18/NTB/24). Funding was provided by the A+ Trust Microbiology Education and Research Fund.

Isolates were grown on sheep blood agar and identified to species-complex by MALDI-ToF MS (BioMerieux). Susceptibility to meropenem, piperacillin-tazobactam and colistin, was determined using Sensititre (ThermoFisher Scientific) microbroth dilution method. Meropenem and colistin mean inhibitory concentrations (MICs) were interpreted as per EUCAST Acinetobacter breakpoints; and piperacillin-tazobactam MICs were interpreted as per CLSI Acinetobacter breakpoints (EUCAST breakpoints not being available for Acinetobacter versus piperacillin-tazobactam).[[12,13]] Susceptibility to ciprofloxacin, cotrimoxazole, gentamicin, and amikacin were determined using disc diffusion as per EUCAST.[[12]] Ceftazidime susceptibility was determined by disc diffusion as per CLSI (EUCAST breakpoints not being available for Acinetobacter versus ceftazidime).[[13]]

DNA was extracted from each isolate utilising the QIAamp DNA mini-Kit (QIAGEN). Unique dual indexed libraries were prepared using the Nextera XT DNA sample preparation kit (Illumina). Libraries were sequenced on the Illumina NextSeq 500 with 150-cycle paired end chemistry as described by the manufacturer’s protocols. Bioinformatic analysis was performed using the Nullarbor[[14]] v2 pipeline. Briefly, Trimmomatic[[15]] v0.36 was used to remove adaptors and low-quality bases and reads. Kraken[[16]] v1.0 was used to perform in silico species detection and assess the paired-end read-sets for contamination. Short-reads were assembled de novo using SPAdes[[17]] v3.12.0 and the resultant contigs were annotated using Prokka[[18]] v1.14. Multi-locus sequence type (MLST) was determined using MLST[[19]] v2.11 with the A. baumannii Pasteur scheme[[20]] (cpn60, fusA, gltA, pyrG, recA, rplB, rpoB) downloaded in July 2018. OXA carbapenemase genes were identified using the Resfinder[[21]] database in Abricate[[22]] v0.8.2. Mash[[23]] v2.1 was used to create a distance matrix from k-mer hashes, and QuickTree[[24]] v2.3 was used to construct a neighbour joining tree for exploratory analysis of the relationship among isolates. A core-genome (defined as sequences found in >99% of isolates) maximum likelihood tree was then inferred for MLST ST2 isolates with IQ-TREE[[25]] v1.6.5, using the A. baumannii reference genome under NCBI accession NC_021729, with core-genome SNPs identified using Snippy[[26]] v4.0, and probable recombinant sites removed using Gubbins[[27]] v2.3.1. Reads for each sequenced isolate (AB1-20, AB22-34) have been deposited in NCBI under Bioproject accession PRJNA855258.

Results

Thirty-three distinct carbapenem resistant A. baumannii complex isolates were identified from 32 persons (cases) between January 2010 and April 2018. Twenty-four of 33 (73%) isolates were identified since January 2015 (Figure 1), and 23 (70%) were identified in the Auckland region (Figure 1A). Eighteen of the 26 (69%) isolates with available data were identified in clinical specimens, while eight (31%) were identified in MDR organism screens alone. Cases had a median age of 56 years (range <1 to 77) and 18 of 32 (56%) were female. Eight of 30 (27%) cases, with available ethnicity data, were reported as Pākehā/NZ European; four (13%) as Māori; four (13%) as Fijian; four (13%) as Samoan; three (10%) as Indian; three (10%) as other European; two (7%) as Fijian Indian; one as Chinese (3%); and one (3%) as other Asian. Twenty-one of 32 (66%) cases had an identifiable history of “recent” overseas hospitalisation; 14 were direct hospital to hospital transfers, five were hospitalised overseas in the preceding four weeks, and in two cases the exact timeframe could not be identified. Ten of these 21 (48%) persons had been hospitalised in Fiji, four (19%) in Samoa, and one (5%) in each of French Polynesia, China, India, Korea, Thailand, Greece and Romania. One of these persons carried two distinct strains. Two further cases had a strong epidemiological link (hospitalised in same ward in New Zealand) to imported cases (Fiji and French Polynesia respectively); giving 24 (73%) of 33 isolates an identifiable link to overseas hospitalisation (Figure 1B).

Thirty-one (94%) of the 33 A. baumannii complex isolates were identified (from sequence data) as A. baumannii sensu stricto (AB1-16, 18–20, 22–25, 27–34) and two (6%) as A. pittii (AB26, 17). Twenty-three (74%) of the 31 A. baumannii sensu stricto were ST 2 (AB1-10, 13, 16, 19, 22–24, 27–29, 31–34), three (10%) were ST 25 (AB11, 12, 14), two (6%) were ST 1 (AB18, 25), and there was one (3%) each of ST 103 (AB15), ST 107 (AB20), and ST 164 (AB30). None of the 31 A. baumannii sensu stricto were susceptible to meropenem (as per selection criteria); three (10%) were susceptible to ceftazidime; none were susceptible piperacillin-tazobactam; one (3%) was susceptible to gentamicin; five (16%) were susceptible to amikacin; three (10%) were susceptible to cotrimoxazole; three (10%) were susceptible to ciprofloxacin; and 29 (94%) were suspectable to colistin. None of the A. pitti were susceptible to meropenem or piperacillin-tazobactam but were susceptible to all other antimicrobials tested. Twenty-nine (94%) of the 31 A. baumannii sensu stricto carried a bla{{OXA-23}} gene and two (6%) carried bla{{OXA-40-like}} genes. Both A. pittii isolates carried a bla{{OXA-40-like}} gene.

The maximum likelihood phylogenetic tree (based on analysis of 281,823 SNPs) of the 23 ST 2 A. baumannii sensu stricto is shown in Figure 2. There were three genomic clusters of closely related isolates separated by <15 single nucleotide polymorphisms (SNPs) on pairwise analysis; in contrast to thousands of SNPs between each respective cluster and other ST 2 strains; These clusters included, 1) 12 isolates identified predominantly in the Auckland Region between 2015 and 2018; 2) three isolates identified in Canterbury between 2015 and 2018; and 3) two isolates from Auckland in 2011. Clusters 1, 2, and 3 accounted for 36%, 9%, and 6% of the 33 carbapenem resistant A. baumannii complex isolates in this study (Figure 1C).

View Figures 1 & 2.

Discussion

This study describes the epidemiology of carbapenem resistant A. baumannii in New Zealand from 2010 to 2018. The majority of cases were identified in the Auckland city region, reflecting perhaps a relatively larger population size, regional differences in population demographics, receipt of medical repatriations, and/or provision of tertiary services to other PICT. The majority of cases also had an identifiable history of recent overseas hospitalisation; in particular, hospitalisation in Fiji or Samoa. An increase in cases associated with these countries has occurred since 2015 in temporal association with an outbreak in Fiji.[[10]] This association contrasts with other MDR Gram-negative bacilli in New Zealand, such as carbapenemase producing Enterobacterales, which are typically associated with healthcare contact or travel in South and South-East Asia.[[28]]

A. baumannii ST 2 was the most common ST identified. ST 2 is a globally distributed hospital adapted clone that is commonly associated with outbreaks, including in Fiji.[[3,10]] Using WGS based molecular epidemiology we identified three clusters of closely related isolates among the 23 ST 2. Cluster 1 consisted of 12 isolates that were identified at three different laboratories in the Auckland Region and one laboratory in the Waikato between 2015 and 2018. Nine (75%) of the cases had a history of recent hospitalisation in either Fiji (5) or Samoa (4). Of the remaining three cases, one is presumed to represent transmission in the New Zealand hospital setting, another was of Samoan ethnicity, while the final case had no demographic or epidemiological data available. Cluster 1 isolates were resistant to all antimicrobials tested except colistin and all carried the carbapenemase gene bla{{OXA-23}}. The close epidemiological, phylogenetic, and temporal relationship of the Cluster 1 isolates indicate recent trans-national spread of a single strain between healthcare facilities in Fiji, Samoa and New Zealand. We hypothesise Cluster 1 to be the same strain responsible for the 2015–2017 outbreak in Fiji but did not have isolates available to allow testing of this hypothesis.[[10]] Cluster 2 consisted of three ST 2 isolates identified in Canterbury between 2015 and 2018. The earliest case had a history of hospitalisation in South Korea. Their close phylogenetic relationship and common location suggests local transmission. Cluster 3 consisted of two isolates identified in an Auckland hospital in 2011. One case had a history of hospitalisation in French Polynesia with the second case strongly linked by epidemiological and now genomic data to in-hospital transmission in New Zealand.

In addition to the isolates described in Cluster 1, a further five unrelated cases were associated with recent hospitalisation in Fiji. These included four A. baumannii sensu stricto isolates: one ST 1 from 2011, one ST 107 from 2014, one non-clustered ST 2 from 2017, and one ST 25 from 2017; as well as a single A. pittii from 2016. This suggests there have been multiple strains of carbapenem resistant Acinetobacter introduced into Fijian hospitals and/or circulation of the transposons bearing carbapenem resistance genes over the past decade, the latter which could be the subject of a future study. In contrast, all four isolates associated with hospitalisation in Samoa were part of ST 2 cluster 1. The ST 2 strain associated with hospitalisation in French Polynesia from 2011 was not closely related to any isolates associated with Fiji or Samoa suggesting a separate introduction into the Pacific. The acquired bla{{OXA-23}} gene was the most common carbapenemase gene identified. The clonal nature of a significant proportion of the isolates in this study narrows bla{{OXA}} diversity; however, the predominance of bla{{OXA-23}} is consistent with reports from the Asia-Oceania regions.[[29]]

Increasing numbers of carbapenem resistant A. baumannii have been identified in New Zealand since 2015. This has occurred in association with the transnational spread of a ST 2 strain between Fiji, Samoa and New Zealand.

With a known propensity to cause hospital outbreaks and limited antimicrobial treatment options, carbapenem resistant A. baumannii poses a potentially escalating threat to safe healthcare delivery in New Zealand and other PICT. The major risk factor for carbapenem resistant A. baumannii infection/colonisation in the New Zealand setting is recent hospitalisation overseas; including in PICT that historically have been considered low risk for MDR gram-negative organisms. Hospitals require systematic processes to identify high risk individuals at presentation so appropriate microbiological screening can be performed and transmission-based infection control precautions implemented.

Summary

Abstract

Aim

Carbapenem resistant Acinetobacter baumannii have limited treatment options and a propensity to cause hospital outbreaks. In recent years an increase in their detection has been observed in New Zealand. This study aimed to describe the molecular epidemiology of these isolates.

Method

This study utilised carbapenem resistant A. baumannii complex isolates identified across New Zealand between January 2010 to April 2018. Whole genome sequence analysis and associated demographic information was used to contextualise local isolates within the global epidemiology and establish the relationship between isolates.

Results

Thirty-three carbapenem resistant A. baumannii complex isolates (31 A. baumannii sensu stricto) were identified. Twenty-four (73%) were from January 2015 onwards. Twenty-four (73%) had an identifiable epidemiological link to overseas hospitalisation. Twenty-three (74%) of 31 A. baumannii sensu stricto were sequence type (ST) 2 (Pasteur scheme). Phylogenetic analysis identified three ST2 clusters. The largest cluster, of 12 isolates, was from 2015 onwards; with nine (75%) associated with recent hospitalisation in Fiji or Samoa.

Conclusion

Increasing numbers of carbapenem resistant A. baumannii are being identified in New Zealand. Our data show that this is in large part associated with transnational spread of a single A. baumannii sensu stricto ST 2 strain between Fiji, Samoa and New Zealand.

Author Information

Matthew R Blakiston: Clinical Microbiologist, Microbiology Department, LabPlus, Auckland District Health Board. Auckland, New Zealand. Mark B Schultz: Bioinformatics Specialist, Microbiological Diagnostic Unit Public Health Laboratory, Peter Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, Australia. ORCID: 0000-0002-7689-6531. Indira Basu: Scientific officer. Microbiology Department, LabPlus, Auckland District Health Board. Auckland, New Zealand. Susan A Ballard: Principal Scientist, Microbiological Diagnostic Unit Public Health Laboratory, Department of Microbiology and Immunology, The Peter Doherty Institute of Infection and Immunity, The University of Melbourne, Melbourne, Australia. Deborah Williamson: Clinical Microbiologist, Royal Melbourne Hospital and Microbiological Diagnostic Unit Public Health Laboratory, Melbourne, Australia. Sally Roberts: Clinical Microbiologist, Microbiology Department, LabPlus, Auckland District Health Board. Auckland, New Zealand.

Acknowledgements

The authors acknowledge Helen Heffernan from the Institute of Environmental Science and Research Limited (ESR) for provision of isolates; and Susan Taylor (Counties Manukau Health), Joshua Freeman (Canterbury DHB), Chris Mansell (Waikato DHB), and Hanna-Sofia Anderrsson (Midcentral DHB) for provision of demographic and epidemiological information on cases.

Correspondence

Matthew Blakiston: Microbiology Department, LabPlus, PO Box 110031, Auckland City Hospital, Auckland, 1148, New Zealand. Ph: 022 642 6039.

Correspondence Email

mattbl@adhb.govt.nzurl

Competing Interests

Nil.

1) Peleg A, Seifert H, Paterson D. Acinetobacter baumannii: Emergence of a successful pathogen. Clin Microbiol Rev. 2008; 21:538-82.

2) Antunes C, Visca P, Towner K. Acinetobacter baumannii: evolution of a global pathogen. Pathog Dis. 2014; 71:292-301.

3) Karah N, Sundsford A, Towner K, Samuelson O. Insights into the global molecular epidemiology of carbapenem non-susceptible clones of Acinetobacter baumannii. Drug Resist Updat. 2012; 15:237-47

4) Zarilli R, Pournaras S, Giannouli M, Tsakris A. Global evolution of multi-drug resistant Acinetobacter baumannii clonal lineages. Int J Antimicrob Agents. 2013; 41:11-19

5) Diancourt L, Passet V, Nemec A, et al. The population structure of Acinetobacter baumannii: expanding multi-resistant clones from an ancestral susceptible genetic pool. PLoS One. 2010; 5 (4):e10034

6) Higgins P, Dammhayn C, Hackel M, Seifert H. Global spread of carbapenem resistant Acinetobacter. J Antimicrob Chemother. 2010; 65: 233-38.

7) Naas T, Levy M, Hirshauer C, et al. Outbreak of Carbapenem-Resistant Acinetobacter baumannii producing the carbapenemase OXA-23 in a tertiary care centre of Papeete, French Polynesia. J Clin Microbiol. 2005; 43: 4826-29

8) Le Hello S, Falcot V, Lacassin F, et al. Molecular epidemiology of carbapenem-resistant Acinetobacter baumannii in New Caledonia. Clin Microbio Infection. 2008; 14:977-81.

9) Peleg A, Franklin C, Bell J, Spelman D. 2006. Emergence of carbapenem resistance in Acinetobacter baumannii recovered from blood cultures in Australia. Infect control Hosp Epidemiol. 2006; 27: 759-61

10) Ministry of Health and Medical Services (Fiji) [Internet]. Acinetobacter baumannii outbreak in NICU at the colonial war memorial hospital Suva, Fiji, December 2016 - July 2017. Technical Report. [Available from: https://www.health.gov.fj/wp-content/uploads/2017/09/ACINETOBACTER-BAUMANNII-JULY-2017.pdf].

11) Roberts S, Findley R, Lang S. Investigation of an outbreak of multi-drug resistant Acinetobacter baumannii in an intensive care unit. J Hosp Infect. 2011; 48:228-232.

12) The European Committee on Antimicrobial Susceptibility Testing. Breakpoint tables for interpretation of MICs and zone diameters. Version 8.1. http://www.eucast.org.

13) Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing; 28[[th]] Ed. CLSI supplement M100.

14) Seeman T, Gonsalves de Silva A, Bulach D, Schultz M, Kwong J, Howden B. Nullarbor Github. http://:github.com/tseeman/nullabor.

15) Bolger A, Lohe M, Lohse, Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;20:2113-2120.

16) Wood DE, Salzberg SL. Kraken: ultrafast metagenomic sequence classification using exact alignments. Genome Biology 2014, 15. https://doi.org/10.1186/gb-2014-15-3-r46.

17) Bankevich A, Nurk S, Antipov D. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Bio. 2012; 19:445-477.

18) Seemann T. Prokka: rapid prokaryotic genome annotation. Bioinformatics. 2014;30:2068-2069.

19) Seemann T. mlst, Github https://github.com/tseemann/mlst.

20) Jolley K, Maiden: Scalable analysis of the bacterial genome variation at the population level. BMC bioinformatics. 2010; 11. https://doi.org/10.1186/1471-2105-11-595.

21) Zankari E, Hasman H, Cosentino, et al. Identification of acquired antimicrobial resistance genes. J Antimicrob Chemother. 2012;67:2640-2644.

22) Seemann T. Abricate, Github https://github.com/tseemann/abricate.

23) Ondov B, Treagan T, Melsted P, et al. Mash: fast genome and metagenome distance estimation using MinHash. Genome Biol. 2016;17. https://doi.org/10.1186/s13059-016-0997-x.

24) Howe, K. QuickTree. Gitbub. https://github.com/khowe/quicktree.

25) Nguyen L, Schmidt H, von Haeseler A, Minh B. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol Biol Evol. 2015;32: https://doi.org/10.1093/molbev/msu300.

26) Seeman T. Snippy. Github https://github.com/tseemann/snippy.

27) Croucher N, Page A, Conner T. Rapid phylogenetic analysis of large samples of recombinant bacterial whole genome sequences using Gubbins. Nucleic Acids Res. 2015;43. https://doi.org/10.1093/nar/gku1196.

28) Blakiston M, Heffernan H, Roberts S, Freeman J. The clear and present danger of carbapenemase-producing Enterobacteriaceae (CPE) in New Zealand: time for a national response plan. N Z Med J. 2017; 130: 72-79.

29) Kamolvit W, Sidjabat H, Paterson D. Molecular epidemiology and mechanisms of carbapenem resistance of Acinetobacter spp. in Asia and Oceania. Microb Drug resist. 2015; 21:424-43.

For the PDF of this article,
contact nzmj@nzma.org.nz

View Article PDF

Acinetobacter baumannii is a gram-negative coccobacillus that has emerged as a multi-drug resistant (MDR) cause of hospital acquired infection in many locations worldwide.[[1,2]] Infections primarily affect vulnerable patients in the intensive care setting and are associated with high crude mortality.[[1]] The emergence of MDR A. baumannii has occurred in association with regional and international spread of hospital adapted clones.[[3–5]] Carbapenem antimicrobials were the primary treatment option for MDR A. baumannii strains. However, the emergence of carbapenem resistance in these global lineages, mediated primarily by acquired OXA carbapenemases, has severely restricted treatment options.[[3,6]]

In the Pacific/Oceania regions, carbapenem resistant A. baumannii have been reported from French Polynesia, New Caledonia and Australia since the early- to mid-2000s.[[7–9]] In 2015–2017 an outbreak occurred in a Fijian neonatal intensive care unit, with invasive infection associated with a crude mortality of 86%.[[10]] MDR A. baumannii is not endemic in New Zealand hospitals, with sporadic cases identified primarily in individuals with a history of overseas hospitalisation. A single outbreak has been reported in New Zealand, with an MDR, but carbapenem sensitive, strain that affected an Auckland hospital in 1998–1999.[[11]] However, in recent times we have experienced an increase in their detection, associated with prior hospitalisation in neighbouring Pacific Island countries and territories (PICT).

The limited antimicrobial treatment options and propensity of carbapenem resistant A. baumannii to cause hospital outbreaks poses a threat to healthcare in New Zealand and other PICT. Enhancing our understanding of the local epidemiology of carbapenem resistant A. baumannii may assist with mitigation strategies. Utilising the high discriminatory capacity of whole genome sequence (WGS) molecular epidemiology we aim to contextualise local New Zealand isolates within the global epidemiology, establish the local relationship between isolates, considering especially those with links to PICT, and explore potential routes of dissemination in the Pacific regions.

Methods

This was a retrospective descriptive study. We identified carbapenem resistant A. baumannii complex isolates with putative acquired OXA carbapenemase genes from laboratory records at Auckland District Health Board (ADHB) and the Antimicrobial Resistance (AMR) Reference Laboratory at the Institute of Environmental Science and Research (ESR) from January 2010 to April 2018. This is believed to have captured the majority of cases in New Zealand over this period as all laboratories were encouraged to send carbapenem resistant A. baumannii isolates to ESR for further characterisation. Basic demographic information (age, gender, ethnicity) and epidemiological metadata, such as a documented history of overseas hospitalisation, were retrieved from clinical records where possible. Study approval was obtained from the New Zealand Health and Disability Commission ethics committee (reference number 18/NTB/24). Funding was provided by the A+ Trust Microbiology Education and Research Fund.

Isolates were grown on sheep blood agar and identified to species-complex by MALDI-ToF MS (BioMerieux). Susceptibility to meropenem, piperacillin-tazobactam and colistin, was determined using Sensititre (ThermoFisher Scientific) microbroth dilution method. Meropenem and colistin mean inhibitory concentrations (MICs) were interpreted as per EUCAST Acinetobacter breakpoints; and piperacillin-tazobactam MICs were interpreted as per CLSI Acinetobacter breakpoints (EUCAST breakpoints not being available for Acinetobacter versus piperacillin-tazobactam).[[12,13]] Susceptibility to ciprofloxacin, cotrimoxazole, gentamicin, and amikacin were determined using disc diffusion as per EUCAST.[[12]] Ceftazidime susceptibility was determined by disc diffusion as per CLSI (EUCAST breakpoints not being available for Acinetobacter versus ceftazidime).[[13]]

DNA was extracted from each isolate utilising the QIAamp DNA mini-Kit (QIAGEN). Unique dual indexed libraries were prepared using the Nextera XT DNA sample preparation kit (Illumina). Libraries were sequenced on the Illumina NextSeq 500 with 150-cycle paired end chemistry as described by the manufacturer’s protocols. Bioinformatic analysis was performed using the Nullarbor[[14]] v2 pipeline. Briefly, Trimmomatic[[15]] v0.36 was used to remove adaptors and low-quality bases and reads. Kraken[[16]] v1.0 was used to perform in silico species detection and assess the paired-end read-sets for contamination. Short-reads were assembled de novo using SPAdes[[17]] v3.12.0 and the resultant contigs were annotated using Prokka[[18]] v1.14. Multi-locus sequence type (MLST) was determined using MLST[[19]] v2.11 with the A. baumannii Pasteur scheme[[20]] (cpn60, fusA, gltA, pyrG, recA, rplB, rpoB) downloaded in July 2018. OXA carbapenemase genes were identified using the Resfinder[[21]] database in Abricate[[22]] v0.8.2. Mash[[23]] v2.1 was used to create a distance matrix from k-mer hashes, and QuickTree[[24]] v2.3 was used to construct a neighbour joining tree for exploratory analysis of the relationship among isolates. A core-genome (defined as sequences found in >99% of isolates) maximum likelihood tree was then inferred for MLST ST2 isolates with IQ-TREE[[25]] v1.6.5, using the A. baumannii reference genome under NCBI accession NC_021729, with core-genome SNPs identified using Snippy[[26]] v4.0, and probable recombinant sites removed using Gubbins[[27]] v2.3.1. Reads for each sequenced isolate (AB1-20, AB22-34) have been deposited in NCBI under Bioproject accession PRJNA855258.

Results

Thirty-three distinct carbapenem resistant A. baumannii complex isolates were identified from 32 persons (cases) between January 2010 and April 2018. Twenty-four of 33 (73%) isolates were identified since January 2015 (Figure 1), and 23 (70%) were identified in the Auckland region (Figure 1A). Eighteen of the 26 (69%) isolates with available data were identified in clinical specimens, while eight (31%) were identified in MDR organism screens alone. Cases had a median age of 56 years (range <1 to 77) and 18 of 32 (56%) were female. Eight of 30 (27%) cases, with available ethnicity data, were reported as Pākehā/NZ European; four (13%) as Māori; four (13%) as Fijian; four (13%) as Samoan; three (10%) as Indian; three (10%) as other European; two (7%) as Fijian Indian; one as Chinese (3%); and one (3%) as other Asian. Twenty-one of 32 (66%) cases had an identifiable history of “recent” overseas hospitalisation; 14 were direct hospital to hospital transfers, five were hospitalised overseas in the preceding four weeks, and in two cases the exact timeframe could not be identified. Ten of these 21 (48%) persons had been hospitalised in Fiji, four (19%) in Samoa, and one (5%) in each of French Polynesia, China, India, Korea, Thailand, Greece and Romania. One of these persons carried two distinct strains. Two further cases had a strong epidemiological link (hospitalised in same ward in New Zealand) to imported cases (Fiji and French Polynesia respectively); giving 24 (73%) of 33 isolates an identifiable link to overseas hospitalisation (Figure 1B).

Thirty-one (94%) of the 33 A. baumannii complex isolates were identified (from sequence data) as A. baumannii sensu stricto (AB1-16, 18–20, 22–25, 27–34) and two (6%) as A. pittii (AB26, 17). Twenty-three (74%) of the 31 A. baumannii sensu stricto were ST 2 (AB1-10, 13, 16, 19, 22–24, 27–29, 31–34), three (10%) were ST 25 (AB11, 12, 14), two (6%) were ST 1 (AB18, 25), and there was one (3%) each of ST 103 (AB15), ST 107 (AB20), and ST 164 (AB30). None of the 31 A. baumannii sensu stricto were susceptible to meropenem (as per selection criteria); three (10%) were susceptible to ceftazidime; none were susceptible piperacillin-tazobactam; one (3%) was susceptible to gentamicin; five (16%) were susceptible to amikacin; three (10%) were susceptible to cotrimoxazole; three (10%) were susceptible to ciprofloxacin; and 29 (94%) were suspectable to colistin. None of the A. pitti were susceptible to meropenem or piperacillin-tazobactam but were susceptible to all other antimicrobials tested. Twenty-nine (94%) of the 31 A. baumannii sensu stricto carried a bla{{OXA-23}} gene and two (6%) carried bla{{OXA-40-like}} genes. Both A. pittii isolates carried a bla{{OXA-40-like}} gene.

The maximum likelihood phylogenetic tree (based on analysis of 281,823 SNPs) of the 23 ST 2 A. baumannii sensu stricto is shown in Figure 2. There were three genomic clusters of closely related isolates separated by <15 single nucleotide polymorphisms (SNPs) on pairwise analysis; in contrast to thousands of SNPs between each respective cluster and other ST 2 strains; These clusters included, 1) 12 isolates identified predominantly in the Auckland Region between 2015 and 2018; 2) three isolates identified in Canterbury between 2015 and 2018; and 3) two isolates from Auckland in 2011. Clusters 1, 2, and 3 accounted for 36%, 9%, and 6% of the 33 carbapenem resistant A. baumannii complex isolates in this study (Figure 1C).

View Figures 1 & 2.

Discussion

This study describes the epidemiology of carbapenem resistant A. baumannii in New Zealand from 2010 to 2018. The majority of cases were identified in the Auckland city region, reflecting perhaps a relatively larger population size, regional differences in population demographics, receipt of medical repatriations, and/or provision of tertiary services to other PICT. The majority of cases also had an identifiable history of recent overseas hospitalisation; in particular, hospitalisation in Fiji or Samoa. An increase in cases associated with these countries has occurred since 2015 in temporal association with an outbreak in Fiji.[[10]] This association contrasts with other MDR Gram-negative bacilli in New Zealand, such as carbapenemase producing Enterobacterales, which are typically associated with healthcare contact or travel in South and South-East Asia.[[28]]

A. baumannii ST 2 was the most common ST identified. ST 2 is a globally distributed hospital adapted clone that is commonly associated with outbreaks, including in Fiji.[[3,10]] Using WGS based molecular epidemiology we identified three clusters of closely related isolates among the 23 ST 2. Cluster 1 consisted of 12 isolates that were identified at three different laboratories in the Auckland Region and one laboratory in the Waikato between 2015 and 2018. Nine (75%) of the cases had a history of recent hospitalisation in either Fiji (5) or Samoa (4). Of the remaining three cases, one is presumed to represent transmission in the New Zealand hospital setting, another was of Samoan ethnicity, while the final case had no demographic or epidemiological data available. Cluster 1 isolates were resistant to all antimicrobials tested except colistin and all carried the carbapenemase gene bla{{OXA-23}}. The close epidemiological, phylogenetic, and temporal relationship of the Cluster 1 isolates indicate recent trans-national spread of a single strain between healthcare facilities in Fiji, Samoa and New Zealand. We hypothesise Cluster 1 to be the same strain responsible for the 2015–2017 outbreak in Fiji but did not have isolates available to allow testing of this hypothesis.[[10]] Cluster 2 consisted of three ST 2 isolates identified in Canterbury between 2015 and 2018. The earliest case had a history of hospitalisation in South Korea. Their close phylogenetic relationship and common location suggests local transmission. Cluster 3 consisted of two isolates identified in an Auckland hospital in 2011. One case had a history of hospitalisation in French Polynesia with the second case strongly linked by epidemiological and now genomic data to in-hospital transmission in New Zealand.

In addition to the isolates described in Cluster 1, a further five unrelated cases were associated with recent hospitalisation in Fiji. These included four A. baumannii sensu stricto isolates: one ST 1 from 2011, one ST 107 from 2014, one non-clustered ST 2 from 2017, and one ST 25 from 2017; as well as a single A. pittii from 2016. This suggests there have been multiple strains of carbapenem resistant Acinetobacter introduced into Fijian hospitals and/or circulation of the transposons bearing carbapenem resistance genes over the past decade, the latter which could be the subject of a future study. In contrast, all four isolates associated with hospitalisation in Samoa were part of ST 2 cluster 1. The ST 2 strain associated with hospitalisation in French Polynesia from 2011 was not closely related to any isolates associated with Fiji or Samoa suggesting a separate introduction into the Pacific. The acquired bla{{OXA-23}} gene was the most common carbapenemase gene identified. The clonal nature of a significant proportion of the isolates in this study narrows bla{{OXA}} diversity; however, the predominance of bla{{OXA-23}} is consistent with reports from the Asia-Oceania regions.[[29]]

Increasing numbers of carbapenem resistant A. baumannii have been identified in New Zealand since 2015. This has occurred in association with the transnational spread of a ST 2 strain between Fiji, Samoa and New Zealand.

With a known propensity to cause hospital outbreaks and limited antimicrobial treatment options, carbapenem resistant A. baumannii poses a potentially escalating threat to safe healthcare delivery in New Zealand and other PICT. The major risk factor for carbapenem resistant A. baumannii infection/colonisation in the New Zealand setting is recent hospitalisation overseas; including in PICT that historically have been considered low risk for MDR gram-negative organisms. Hospitals require systematic processes to identify high risk individuals at presentation so appropriate microbiological screening can be performed and transmission-based infection control precautions implemented.

Summary

Abstract

Aim

Carbapenem resistant Acinetobacter baumannii have limited treatment options and a propensity to cause hospital outbreaks. In recent years an increase in their detection has been observed in New Zealand. This study aimed to describe the molecular epidemiology of these isolates.

Method

This study utilised carbapenem resistant A. baumannii complex isolates identified across New Zealand between January 2010 to April 2018. Whole genome sequence analysis and associated demographic information was used to contextualise local isolates within the global epidemiology and establish the relationship between isolates.

Results

Thirty-three carbapenem resistant A. baumannii complex isolates (31 A. baumannii sensu stricto) were identified. Twenty-four (73%) were from January 2015 onwards. Twenty-four (73%) had an identifiable epidemiological link to overseas hospitalisation. Twenty-three (74%) of 31 A. baumannii sensu stricto were sequence type (ST) 2 (Pasteur scheme). Phylogenetic analysis identified three ST2 clusters. The largest cluster, of 12 isolates, was from 2015 onwards; with nine (75%) associated with recent hospitalisation in Fiji or Samoa.

Conclusion

Increasing numbers of carbapenem resistant A. baumannii are being identified in New Zealand. Our data show that this is in large part associated with transnational spread of a single A. baumannii sensu stricto ST 2 strain between Fiji, Samoa and New Zealand.

Author Information

Matthew R Blakiston: Clinical Microbiologist, Microbiology Department, LabPlus, Auckland District Health Board. Auckland, New Zealand. Mark B Schultz: Bioinformatics Specialist, Microbiological Diagnostic Unit Public Health Laboratory, Peter Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, Australia. ORCID: 0000-0002-7689-6531. Indira Basu: Scientific officer. Microbiology Department, LabPlus, Auckland District Health Board. Auckland, New Zealand. Susan A Ballard: Principal Scientist, Microbiological Diagnostic Unit Public Health Laboratory, Department of Microbiology and Immunology, The Peter Doherty Institute of Infection and Immunity, The University of Melbourne, Melbourne, Australia. Deborah Williamson: Clinical Microbiologist, Royal Melbourne Hospital and Microbiological Diagnostic Unit Public Health Laboratory, Melbourne, Australia. Sally Roberts: Clinical Microbiologist, Microbiology Department, LabPlus, Auckland District Health Board. Auckland, New Zealand.

Acknowledgements

The authors acknowledge Helen Heffernan from the Institute of Environmental Science and Research Limited (ESR) for provision of isolates; and Susan Taylor (Counties Manukau Health), Joshua Freeman (Canterbury DHB), Chris Mansell (Waikato DHB), and Hanna-Sofia Anderrsson (Midcentral DHB) for provision of demographic and epidemiological information on cases.

Correspondence

Matthew Blakiston: Microbiology Department, LabPlus, PO Box 110031, Auckland City Hospital, Auckland, 1148, New Zealand. Ph: 022 642 6039.

Correspondence Email

mattbl@adhb.govt.nzurl

Competing Interests

Nil.

1) Peleg A, Seifert H, Paterson D. Acinetobacter baumannii: Emergence of a successful pathogen. Clin Microbiol Rev. 2008; 21:538-82.

2) Antunes C, Visca P, Towner K. Acinetobacter baumannii: evolution of a global pathogen. Pathog Dis. 2014; 71:292-301.

3) Karah N, Sundsford A, Towner K, Samuelson O. Insights into the global molecular epidemiology of carbapenem non-susceptible clones of Acinetobacter baumannii. Drug Resist Updat. 2012; 15:237-47

4) Zarilli R, Pournaras S, Giannouli M, Tsakris A. Global evolution of multi-drug resistant Acinetobacter baumannii clonal lineages. Int J Antimicrob Agents. 2013; 41:11-19

5) Diancourt L, Passet V, Nemec A, et al. The population structure of Acinetobacter baumannii: expanding multi-resistant clones from an ancestral susceptible genetic pool. PLoS One. 2010; 5 (4):e10034

6) Higgins P, Dammhayn C, Hackel M, Seifert H. Global spread of carbapenem resistant Acinetobacter. J Antimicrob Chemother. 2010; 65: 233-38.

7) Naas T, Levy M, Hirshauer C, et al. Outbreak of Carbapenem-Resistant Acinetobacter baumannii producing the carbapenemase OXA-23 in a tertiary care centre of Papeete, French Polynesia. J Clin Microbiol. 2005; 43: 4826-29

8) Le Hello S, Falcot V, Lacassin F, et al. Molecular epidemiology of carbapenem-resistant Acinetobacter baumannii in New Caledonia. Clin Microbio Infection. 2008; 14:977-81.

9) Peleg A, Franklin C, Bell J, Spelman D. 2006. Emergence of carbapenem resistance in Acinetobacter baumannii recovered from blood cultures in Australia. Infect control Hosp Epidemiol. 2006; 27: 759-61

10) Ministry of Health and Medical Services (Fiji) [Internet]. Acinetobacter baumannii outbreak in NICU at the colonial war memorial hospital Suva, Fiji, December 2016 - July 2017. Technical Report. [Available from: https://www.health.gov.fj/wp-content/uploads/2017/09/ACINETOBACTER-BAUMANNII-JULY-2017.pdf].

11) Roberts S, Findley R, Lang S. Investigation of an outbreak of multi-drug resistant Acinetobacter baumannii in an intensive care unit. J Hosp Infect. 2011; 48:228-232.

12) The European Committee on Antimicrobial Susceptibility Testing. Breakpoint tables for interpretation of MICs and zone diameters. Version 8.1. http://www.eucast.org.

13) Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing; 28[[th]] Ed. CLSI supplement M100.

14) Seeman T, Gonsalves de Silva A, Bulach D, Schultz M, Kwong J, Howden B. Nullarbor Github. http://:github.com/tseeman/nullabor.

15) Bolger A, Lohe M, Lohse, Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;20:2113-2120.

16) Wood DE, Salzberg SL. Kraken: ultrafast metagenomic sequence classification using exact alignments. Genome Biology 2014, 15. https://doi.org/10.1186/gb-2014-15-3-r46.

17) Bankevich A, Nurk S, Antipov D. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Bio. 2012; 19:445-477.

18) Seemann T. Prokka: rapid prokaryotic genome annotation. Bioinformatics. 2014;30:2068-2069.

19) Seemann T. mlst, Github https://github.com/tseemann/mlst.

20) Jolley K, Maiden: Scalable analysis of the bacterial genome variation at the population level. BMC bioinformatics. 2010; 11. https://doi.org/10.1186/1471-2105-11-595.

21) Zankari E, Hasman H, Cosentino, et al. Identification of acquired antimicrobial resistance genes. J Antimicrob Chemother. 2012;67:2640-2644.

22) Seemann T. Abricate, Github https://github.com/tseemann/abricate.

23) Ondov B, Treagan T, Melsted P, et al. Mash: fast genome and metagenome distance estimation using MinHash. Genome Biol. 2016;17. https://doi.org/10.1186/s13059-016-0997-x.

24) Howe, K. QuickTree. Gitbub. https://github.com/khowe/quicktree.

25) Nguyen L, Schmidt H, von Haeseler A, Minh B. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol Biol Evol. 2015;32: https://doi.org/10.1093/molbev/msu300.

26) Seeman T. Snippy. Github https://github.com/tseemann/snippy.

27) Croucher N, Page A, Conner T. Rapid phylogenetic analysis of large samples of recombinant bacterial whole genome sequences using Gubbins. Nucleic Acids Res. 2015;43. https://doi.org/10.1093/nar/gku1196.

28) Blakiston M, Heffernan H, Roberts S, Freeman J. The clear and present danger of carbapenemase-producing Enterobacteriaceae (CPE) in New Zealand: time for a national response plan. N Z Med J. 2017; 130: 72-79.

29) Kamolvit W, Sidjabat H, Paterson D. Molecular epidemiology and mechanisms of carbapenem resistance of Acinetobacter spp. in Asia and Oceania. Microb Drug resist. 2015; 21:424-43.

Contact diana@nzma.org.nz
for the PDF of this article

Subscriber Content

The full contents of this pages only available to subscribers.
Login, subscribe or email nzmj@nzma.org.nz to purchase this article.

LOGINSUBSCRIBE