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Coordination of bacterial stress response mechanisms is critical for long-term survival in harsh environments for successful host infection. The general and specific stress responses of well-studied Gram-negative pathogens like Escherichia coli are controlled by alternative sigma factors, archetypically RpoS. The deadly hospital pathogen Acinetobacter baumannii is notoriously resistant to environmental stresses, yet it lacks RpoS, and the molecular mechanisms driving this incredible stress tolerance remain poorly defined. Here, using functional genomics, we identified the transcriptional regulator DksA as a master regulator for broad stress protection and virulence in A. baumannii. Transcriptomics, phenomics and in vivo animal studies revealed that DksA controls ribosomal protein expression, metabolism, mutation rates, desiccation, antibiotic resistance, and host colonization in a niche-specific manner. Phylogenetically, DksA was highly conserved and well-distributed across Gammaproteobacteria, with 96.6% containing DksA, spanning 88 families. This study lays the groundwork for understanding DksA as a major regulator of general stress response and virulence in this important pathogen.

Klebsiella pneumoniae sequence type (ST) 17 is a global problem clone that causes multidrug-resistant (MDR) hospital infections worldwide. In 2008–2009, an outbreak of MDR ST17 occurred at a neonatal intensive care unit (NICU) in Stavanger, Norway. Fifty-seven children were colonized. We observed intestinal persistence of ST17 in all of the children for up to two years after hospital discharge. Here, we investigated the within–host evolution of ST17 in 45 of those children during long-term colonization and compared the outbreak with 254 global strains. Ninety-two outbreak-related isolates were whole-genome sequenced. They had capsule locus KL25, O locus O5 and carried yersiniabactin. During within–host colonization ST17 remained stable with few single nucleotide polymorphisms, no acquisition of antimicrobial resistance (AMR) or virulence determinants, and persistent carriage of a bla CTX-M-15-encoding IncFII(K) IncFIB(K) plasmid (pKp2177_1). The global collection included ST17 from 1993 to 2020 from 34 countries, that were from human infection (41.3%), colonization (39.3%) and respiratory specimens (7.3%), from animals (9.3%), and from the environment (2.7%). We estimate that ST17 emerged mid-to-late 19th century (1859, 95 % HPD 1763–1939) and diversified through recombinations of the K and O loci to form several sublineages, with various AMR genes, virulence loci and plasmids. There was limited evidence of persistence of AMR genes in any of these lineages. A globally disseminated sublineage with KL25/O5 accounted for 52.7 % of the genomes. It included a monophyletic subclade that emerged in the mid-1980s, which comprised the Stavanger NICU outbreak and 10 genomes from three other countries, which all carried pKp2177_1. The plasmid was also observed in a KL155/OL101 subclade from the 2000s. Three clonal expansions of ST17 were identified; all were healthcare-associated and carried either yersiniabactin and/or pKp2177_1. To conclude, ST17 is globally disseminated and associated with opportunistic hospital-acquired infections. It contributes to the burden of global MDR infections, but many diverse lineages persist without acquired AMR. We hypothesize that non-human sources and human colonization may play a crucial role for severe infections in vulnerable patients, such as preterm neonates.

International audience; The grey-headed flying fox (Pteropus poliocephalus) is an endemic Australian fruit bat, known to carry zoonotic pathogens. We recently showed they harbour bacterial pathogen Klebsiella pneumoniae and closely related species in the K. pneumoniae species complex (KpSC); however, the dynamics of KpSC transmission and gene flow within flying fox colonies are poorly understood. High-resolution genome comparisons of 39 KpSC isolates from grey-headed flying foxes identified five putative strain transmission clusters (four intra- and one inter-colony). The instance of inter-colony strain transmission of K. africana was found between two flying fox populations within flying distance, indicating either direct or indirect transmission through a common food/water source. All 11 plasmids identified within the KpSC isolates showed 73% coverage (mean) and ≥95% identity to human-associated KpSC plasmids, indicating gene flow between human clinical and grey-headed flying fox isolates. Along with strain transmission, inter-species horizontal plasmid transmission between K. pneumoniae and Klebsiella africana was also identified within a flying fox colony. Finally, genome-scale metabolic models were generated to predict and compare substrate usage to previously published KpSC models, from human and environmental sources. These models indicated no distinction on the basis of metabolic capabilities. Instead, metabolic capabilities were consistent with population structure and ST/lineage.

Infections caused by metallo-beta-lactamase-producing organisms (MBLs) are a global health threat. Our understanding of transmission dynamics and how MBLs establish endemicity remains limited. We analysed two decades ofblaIMP-4evolution in a hospital using sequence data from 270 clinical and environmental isolates (including 169 completed genomes) and identified extreme gene promiscuity across 7 Gram-negative genera, 68 bacterial strains and 7 distinct plasmid types. An initial multi-species outbreak of conserved IncC plasmids (95 genomes across 37 strains) allowed endemicity to be established through the ability ofblaIMP-4to disseminate in successful strain-genetic setting pairs we termed ‘propagators’, in particularSerratia marcescensandEnterobacter hormaechei. From this reservoir,blaIMP-4persisted through diversification of genetic settings that resulted from transfer ofblaIMP-4plasmids between bacterial hosts and of the integron carryingblaIMP-4between plasmids. Our findings provide a framework for understanding endemicity and spread of MBLs and may have broader applicability to other carbapenemase-producing organisms.

Metabolic capacity can vary substantially within a bacterial species, leading to ecological niche separation, as well as differences in virulence and antimicrobial susceptibility. Genome-scale metabolic models are useful tools for studying the metabolic potential of individuals, and with the rapid expansion of genomic sequencing there is a wealth of data that can be leveraged for comparative analysis. However, there exist few tools to construct strain-specific metabolic models at scale. Here we describe Bactabolize (github.com/kelwyres/Bactabolize), a reference-based tool which rapidly produces strain-specific metabolic models and growth phenotype predictions. We describe a pan reference model for the priority antimicrobial-resistant pathogen, Klebsiella pneumoniae (github.com/kelwyres/KpSC-pan-metabolic-model), and a quality control framework for using draft genome assemblies as input for Bactabolize. The Bactabolize-derived model for K. pneumoniae reference strain KPPR1 outperformed the CarveMe-derived model across ≥201 substrate and ≥1220 knockout mutant growth predictions. Novel draft genomes passing our systematically-defined quality control criteria resulted in models with a high degree of completeness (≥99% genes and reactions captured) and high accuracy (mean 0.97, n=10). We anticipate the tools and framework described herein will facilitate large-scale metabolic modelling analyses that broaden our understanding of diversity within bacterial species and inform novel control strategies for priority pathogens.

Species loss within a microbial community can increase resource availability and spur adaptive evolution. Environmental shifts that cause species loss or fluctuations in community composition are expected to become more common, so it is important to understand the evolutionary forces that shape the stability and function of the emergent community. Here we study experimental cultures of a simple, ecologically stable community of Saccharomyces cerevisiae and Lactobacillus plantarum, in order to understand how the presence or absence of a species impacts coexistence over evolutionary timescales. We found that evolution in coculture led to drastically altered evolutionary outcomes for L. plantarum, but not S. cerevisiae. Both monoculture- and co-culture-evolved L. plantarum evolved dozens of mutations over 925 generations of evolution, but only L. plantarum that had evolved in isolation from S. cerevisiae lost the capacity to coexist with S. cerevisiae. We find that the evolutionary loss of ecological stability corresponds with fitness differences between monoculture-evolved L. plantarum and S. cerevisiae and genetic changes that repeatedly evolve across the replicate populations of L. plantarum. This work shows how coevolution within a community can prevent destabilising evolution in individual species, thereby preserving ecological diversity and stability, despite rapid adaptation.

Klebsiella pneumoniaesequence type (ST) 17 is a global problem clone that causes multidrug-resistant (MDR) hospital infections worldwide. In 2008-2009, an outbreak of MDR ST17 occurred at a neonatal intensive care unit (NICU) in Stavanger, Norway. Fifty-seven children were colonised. We observed intestinal persistence of ST17 in all of the children for up to two years after hospital discharge. Here, we investigated the within-host evolution of ST17 in 45 of those children during long-term colonisation and compared the outbreak with 254 global strains. Ninety-two outbreak-related isolates were whole-genome sequenced. They had capsule locus KL25, O locus O5 and carried yersiniabactin. During within-host colonisation ST17 remained stable with few single nucleotide polymorphisms, no acquisition of antimicrobial resistance (AMR) or virulence determinants, and persistent carriage of ablaCTX-M-15-encoding IncFII(K) IncFIB(K) plasmid (pKp2177_1). The global collection included ST17 from 1993-2020 from 34 countries, that were from human infection (41.3%), colonisation (39.3%) and respiratory specimens (7.3%), from animals (9.3%), and from the environment (2.7%). We estimate that ST17 emerged mid-to-late 19th century (1859, 95% HPD 1763-1939) and diversified through recombinations of the K and O loci to form several sublineages, with various AMR genes, virulence loci and plasmids. There was limited evidence of persistence of AMR genes in any of these lineages. A globally disseminated sublineage with KL25/O5 accounted for 52.7% of the genomes. It included a monophyletic subclade that emerged in the mid-1980s, which comprised the Stavanger NICU outbreak and 10 genomes from three other countries, which all carried pKp2177_1. The plasmid was also observed in a KL155/OL101 subclade from the 2000s. Three clonal expansions of ST17 were identified, all were healthcare-associated and carried either yersiniabactin and/or pKp2177_1. To conclude, ST17 is globally disseminated and associated with opportunistic hospital-acquired infections. It contributes to the burden of global MDR infections, but many diverse lineages persist without acquired AMR. We hypothesise that non-human sources and human colonisation may play a crucial role for severe infections in vulnerable patients, such as preterm neonates.Impact statementKlebsiella pneumoniaeis an opportunistic pathogen that frequently causes hospital-associated multidrug-resistant (MDR) infections. Infections withK. pneumoniaestrains that are resistant to third generation cephalosporins and/or carbapenems are considered a major public health threat, as there are limited treatment options available. Some MDRK. pneumoniaeclones, including ST307 and ST258, are global problem clones because they disproportionately contribute to the burden of MDR infections and are common causes of such infections and/or outbreaks in hospitals around the world. Here we describe another such clone, ST17, which caused an MDR outbreak in our neonatal intensive care unit, affecting 57 children. We found that this clone underwent minor within-host evolution during two years of long-term gastrointestinal colonisation after hospital discharge. We then investigated the evolutionary history of ST17 globally, as it had not previously been studied. When we compared ST17 isolates from around the world with the isolates from our hospital, we discovered that whilst ST17 with antimicrobial resistance has contributed to outbreaks in other neonatal care units, it also frequently causes infections or colonises humans and non-human sources without any antimicrobial resistance.Data summaryThe genome sequences generated in this study have been deposited at the European Nucleotide Archive under BioProject PRJEB36392. The BioSample accession numbers and associated metadata for the genomes are available in Table S1. The completed annotated genome assembly of Kp2177 is available in GenBank under accession numbersCP075591(chromosome),CP075592(pKp2177_1),CP075593(pKp2177_2) andCP075594(pKp2177_3). The global dataset of 300 ST17 genomes is available for interactive viewing in Microreact athttps://microreact.org/project/kpst17.

The Klebsiella pneumoniae species complex (KpSC) is a set of seven Klebsiella taxa that are found in a variety of niches and are an important cause of opportunistic health care–associated infections in humans. Because of increasing rates of multi-drug resistance within the KpSC, there is a growing interest in better understanding the biology and metabolism of these organisms to inform novel control strategies. We collated 37 sequenced KpSC isolates isolated from a variety of niches, representing all seven taxa. We generated strain-specific genome-scale metabolic models (GEMs) for all 37 isolates and simulated growth phenotypes on 511 distinct carbon, nitrogen, sulfur, and phosphorus substrates. Models were curated and their accuracy was assessed using matched phenotypic growth data for 94 substrates (median accuracy of 96%). We explored species-specific growth capabilities and examined the impact of all possible single gene deletions using growth simulations in 145 core carbon substrates. These analyses revealed multiple strain-specific differences, within and between species, and highlight the importance of selecting a diverse range of strains when exploring KpSC metabolism. This diverse set of highly accurate GEMs could be used to inform novel drug design, enhance genomic analyses, and identify novel virulence and resistance determinants. We envisage that these 37 curated strain-specific GEMs, covering all seven taxa of the KpSC, provide a valuable resource to the Klebsiella research community.

Linear plasmids are extrachromosomal DNA elements that have been found in a small number of bacterial species. To date, the only linear plasmids described in the family Enterobacteriaceae belong to Salmonella , first found in Salmonella enterica Typhi. Here, we describe a collection of 12 isolates of the Klebsiella pneumoniae species complex in which we identified linear plasmids. Screening of assembly graphs assembled from public read sets identified linear plasmid structures in a further 13 K . pneumoniae species complex genomes. We used these 25 linear plasmid sequences to query all bacterial genome assemblies in the National Center for Biotechnology Information database, and discovered an additional 61 linear plasmid sequences in a variety of Enterobacteriaceae species. Gene content analysis divided these plasmids into five distinct phylogroups, with very few genes shared across more than two phylogroups. The majority of linear plasmid-encoded genes are of unknown function; however, each phylogroup carried its own unique toxin–antitoxin system and genes with homology to those encoding the ParAB plasmid stability system. Passage in vitro of the 12 linear plasmid-carrying Klebsiella isolates in our collection (which include representatives of all five phylogroups) indicated that these linear plasmids can be stably maintained, and our data suggest they can transmit between K. pneumoniae strains (including members of globally disseminated multidrug-resistant clones) and also between diverse Enterobacteriaceae species. The linear plasmid sequences, and representative isolates harbouring them, are made available as a resource to facilitate future studies on the evolution and function of these novel plasmids.

Linear plasmids are extrachromosomal DNA elements that have been found in a small number of bacterial species. To date, the only linear plasmids described in the family

Linear plasmids are extrachromosomal DNA that have been found in a small number of bacterial species. To date, the only linear plasmids described in the Enterobacteriaceae family belong to Salmonella, first found in Salmonella Typhi. Here, we describe a collection of 12 isolates of the Klebsiella pneumoniae species complex in which we identified linear plasmids. We used this collection to search public sequence databases and discovered an additional 74 linear plasmid sequences in a variety of Enterobacteriaceae species. Gene content analysis divided these plasmids into five distinct phylogroups, with very few genes shared across more than two phylogroups. The majority of linear plasmid-encoded genes are of unknown function, however each phylogroup carried its own unique toxin-antitoxin system and genes with homology to those encoding the ParAB plasmid stability system. Passage in vitro of the 12 linear plasmid-carrying Klebsiella isolates in our collection (which include representatives of all five phylogroups) indicated that these linear plasmids can be stably maintained, and our data suggest they can transmit between K. pneumoniae strains (including members of globally disseminated multidrug resistant clones) and also between diverse Enterobacteriaceae species. The linear plasmid sequences, and representative isolates harbouring them, are made available as a resource to facilitate future studies on the evolution and function of these novel plasmids.Significance as a BioResource to the communityThis study provides the first report of linear plasmids identified within the Klebsiella pneumoniae species complex and the first report in Enterobacteriaceae besides Salmonella.We present the first comparative analysis of linear plasmid sequences in Enterobacteriaceae, however whilst this family is highly clinically significant, the functional and/or evolutionary importance of these plasmids is not yet clear. To facilitate future studies to address these questions, we have publicly deposited (i) the collection of linear plasmid sequence data; (ii) isolates representative of each of the distinct linear plasmid phylogroups.Data SummaryThe authors confirm all supporting data, code and protocols have been provided within the article or through supplementary informationWhole genome sequence reads from Klebsiella pneumoniae isolates sequenced in this study have been deposited in NCBI SRA under the accession numbers listed in Table S1.Representative annotated sequences of one linear plasmid per phylogroup have been deposited in FigShare, doi 10.26180/16729126.A copy of all linear plasmid sequences that we assembled from publicly available genome sequence reads are available in FigShare, doi 10.26180/16531365. Read accessions for these are given in Table S1.Eleven representative K. pneumoniae isolates harbouring linear plasmids described in this study have been deposited with the National Collection of Type Cultures (NCTC) and are available for purchase under the NCTC accession numbers listed in Table S1.K. pneumoniae 1194/11 (representative of phylogroup B) has been deposited in the Microorganisms Collection Center, Adolfo Lutz Institute, São Paulo, Brazil. To request strain 1194/11 (IAL 3063, SISGEN ABBF09B), contact: Microorganisms Collection CenterCulture Collection LaboratoryInstituto Adolfo Lutz, Sao Paulo State Department of HealthAddress: Av Dr Arnaldo, 351, 10 floor, room 1020Phone number: +55 11 3068-2884Zip code 01246-000, São Paulo, BrazilE-mail: colecaoial@ial.sp.gov.brAlignments of terminal inverted repeat sequences for each phylogroup can be found in Data S1, available on FigShare, doi 10.26180/16531371.

The Grey-headed flying fox (Pteropus poliocephalus) is an endemic Australian fruit bat, known to carry pathogens with zoonotic potential. We recently showed these bats harbour the bacterial pathogens Klebsiella pneumoniae and closely related species in the K. pneumoniae species complex (KpSC). However, the dynamics of Klebsiella transmission and gene flow within flying fox colonies were not explored and remain poorly understood.Here we report a high-resolution genomic comparison of 39 KpSC isolates from Greyheaded flying foxes. Illumina whole genome sequences (n=39) were assembled de novo and the Kleborate genotyping tool was used to infer sequence types (STs). Oxford Nanopore sequences were generated for 13 isolates (one for each distinct ST) in order to generate high-quality completed reference genomes. Read mapping and variant calling was used to identify single nucleotide variants (SNVs) within each ST, using the relevant reference genome. In silico genome-scale metabolic models were generated to predict and compare substrate usage to 59 previously published KpSC models for isolates from human and environmental sources, which indicated no distinction on the basis of metabolic capabilities.High-resolution genome comparisons identified five putative strain transmission clusters (four intra- and one inter-colony, n=2-15 isolates each, ≤25 pairwise SNVs). Inter-colony transmission of Klebsiella africana was found between two flying fox populations located within flying distance. The 13 completed genomes harboured 11 plasmids, all of which showed 37-98% coverage (mean 73%) and ≥95% identity to those previously reported from human-associated KpSC. Comparison of plasmids from different flying fox associated KpSC indicated an interspecies horizontal plasmid transmission between K. pneumoniae and K. africana for a 98 kbp plasmid, pFF1003.These data indicate that KpSC are able to transmit directly via flying fox populations or indirectly via a common source, and that these isolates can harbour plasmids with similarity to those found in human derived KpSC, indicating gene flow is occurring between isolates from Grey-headed flying fox KpSC and human clinical isolates.

The

The Klebsiella pneumoniae species complex (KpSC) is a set of seven Klebsiella taxa which are found in a variety of niches, and are an important cause of opportunistic healthcare-associated infections in humans. Due to increasing rates of multi-drug resistance within the KpSC, there is a growing interest in better understanding the biology and metabolism of these organisms to inform novel control strategies. We collated 37 sequenced KpSC isolates isolated from a variety of niches, representing all seven taxa. We generated strain-specific genome scale metabolic models (GEMs) for all 37 isolates and simulated growth phenotypes on 511 distinct carbon, nitrogen, sulphur and phosphorus substrates. Models were curated and their accuracy assessed using matched phenotypic growth data for 94 substrates (median accuracy of 96%). We explored species-specific growth capabilities and examined the impact of all possible single gene deletions on growth in 145 core carbon substrates. These analyses revealed multiple strain-specific differences, within and between species and highlight the importance of selecting a diverse range of strains when exploring KpSC metabolism. This diverse set of highly accurate GEMs could be used to inform novel drug design, enhance genomic analyses, and identify novel virulence and resistance determinants. We envisage that these 37 curated strain-specific GEMs, covering all seven taxa of the KpSC, provide a valuable resource to the Klebsiella research community.

Infection of wMel Wolbachia in Aedes aegypti imparts two signature features that enable its application for biocontrol of dengue. First, the susceptibility of mosquitoes to viruses such as dengue and Zika is reduced. Second, a reproductive manipulation is caused that enables wMel introgression into wild-type mosquito populations. The long-term success of this method relies, in part, on evolution of the wMel genome not compromising the critical features that make it an attractive biocontrol tool. This study compared the wMel Wolbachia genome at the time of initial releases and 1–7 years post-release in Cairns, Australia. Our results show the wMel genome remains highly conserved up to 7 years post-release in gene sequence, content, synteny and structure. This work suggests the wMel genome is stable in its new mosquito host and, therefore, provides reassurance on the potential for wMel to deliver long-term public-health impacts.

Species loss within a microbial community can increase resource availability and spur adaptive evolution. Environmental shifts that cause species loss or fluctuations in community composition are expected to become more common, so it is important to understand the evolutionary forces that shape the stability and function of the emergent community. Here we study experimental cultures of a simple, ecologically stable community of Saccharomyces cerevisiae and Lactobacillus plantarum, in order to understand how the presence or absence of a species impacts coexistence over evolutionary timescales. We found that evolution in coculture led to drastically altered evolutionary outcomes for L. plantarum, but not S. cerevisiae. Both monoculture- and co-culture-evolved L. plantarum evolved dozens of mutations over 925 generations of evolution, but only L. plantarum that had evolved in isolation from S. cerevisiae lost the capacity to coexist with S. cerevisiae. We find that the evolutionary loss of ecological stability corresponds with fitness differences between monoculture-evolved L. plantarum and S. cerevisiae and genetic changes that repeatedly evolve across the replicate populations of L. plantarum. This work shows how coevolution within a community can prevent destabilising evolution in individual species, thereby preserving ecological diversity and stability, despite rapid adaptation.

Background Third-generation cephalosporin-resistant Gram-negatives (3GCR-GN) and vancomycin-resistant enterococci (VRE) are common causes of multi-drug resistant healthcare-associated infections, for which gut colonisation is considered a prerequisite. However, there remains a key knowledge gap about colonisation and infection dynamics in high-risk settings such as the intensive care unit (ICU), thus hampering infection prevention efforts. Methods We performed a three-month prospective genomic survey of infecting and gut-colonising 3GCR-GN and VRE among patients admitted to an Australian ICU. Bacteria were isolated from rectal swabs (n = 287 and n = 103 patients ≤2 and > 2 days from admission, respectively) and diagnostic clinical specimens between Dec 2013 and March 2014. Isolates were subjected to Illumina whole-genome sequencing (n = 127 3GCR-GN, n = 41 VRE). Multi-locus sequence types (STs) and antimicrobial resistance determinants were identified from de novo assemblies. Twenty-three isolates were selected for sequencing on the Oxford Nanopore MinION device to generate completed reference genomes (one for each ST isolated from ≥2 patients). Single nucleotide variants (SNVs) were identified by read mapping and variant calling against these references. Results Among 287 patients screened on admission, 17.4 and 8.4% were colonised by 3GCR-GN and VRE, respectively. Escherichia coli was the most common species (n = 36 episodes, 58.1%) and the most common cause of 3GCR-GN infection. Only two VRE infections were identified. The rate of infection among patients colonised with E. coli was low, but higher than those who were not colonised on admission (n = 2/33, 6% vs n = 4/254, 2%, respectively, p = 0.3). While few patients were colonised with 3GCR- Klebsiella pneumoniae or Pseudomonas aeruginosa on admission (n = 4), all such patients developed infections with the colonising strain. Genomic analyses revealed 10 putative nosocomial transmission clusters (≤20 SNVs for 3GCR-GN, ≤3 SNVs for VRE): four VRE, six 3GCR-GN, with epidemiologically linked clusters accounting for 21 and 6% of episodes, respectively (OR 4.3, p = 0.02). Conclusions 3GCR-E. coli and VRE were the most common gut colonisers. E. coli was the most common cause of 3GCR-GN infection, but other 3GCR-GN species showed greater risk for infection in colonised patients. Larger studies are warranted to elucidate the relative risks of different colonisers and guide the use of screening in ICU infection control.

Assembly of bacterial genomes from long-read data (generated by Oxford Nanopore or Pacific Biosciences platforms) can often be complete: a single contig for each chromosome or plasmid in the genome. However, even complete bacterial genome assemblies constructed solely from long reads still contain a variety of errors, and different assemblies of the same genome often contain different errors. Here, we present Trycycler, a tool which produces a consensus assembly from multiple input assemblies of the same genome. Benchmarking using both simulated and real sequencing reads showed that Trycycler consensus assemblies contained fewer errors than any of those constructed with a single long-read assembler. Post-assembly polishing with Medaka and Pilon further reduced errors and yielded the most accurate genome assemblies in our study. As Trycycler can require human judgement and manual intervention, its output is not deterministic, and different users can produce different Trycycler assemblies from the same input data. However, we demonstrated that multiple users with minimal training converge on similar assemblies that are consistently more accurate than those produced by automated assembly tools. We therefore recommend Trycycler+Medaka+Pilon as an ideal approach for generating high-quality bacterial reference genomes.Data availabilitySupplementary figures, tables and code can be found at: github.com/rrwick/Trycycler-paperReads, assemblies and reference sequences can be found at: bridges.monash.edu/articles/dataset/Trycycler_paper_dataset/14890734

BackgroundBacterial genomes follow a U-shaped frequency distribution whereby most genomic loci are either rare (accessory) or common (core); the union of these is the pan-genome. The alignable fraction of two genomes from a single species can be low (e.g. 50-70%), such that no single reference genome can access all single nucleotide polymorphisms (SNPs). The pragmatic solution is to choose a close reference, and analyse SNPs only in the core genome. Given much bacterial adaptability hinges on the accessory genome, this is an unsatisfactory limitation.ResultsWe present a novel pan-genome graph structure and algorithms implemented in the software pandora, which approximates a sequenced genome as a recombinant of reference genomes, detects novel variation and then pan-genotypes multiple samples. The method takes fastq as input and outputs a multi-sample VCF with respect to an inferred data-dependent reference genome, and is available at https://github.com/rmcolq/pandora.Constructing a reference graph from 578 E. coli genomes, we analyse a diverse set of 20 E. coli isolates. We show pandora recovers at least 13k more rare SNPs than single-reference based tools, achieves equal or better error rates with Nanopore as with Illumina data, 6-24x lower Nanopore error rates than other tools, and provides a stable framework for analysing diverse samples without reference bias. We also show that our inferred recombinant VCF reference genome is significantly better than simply picking the closest RefSeq reference.ConclusionsThis is a step towards comprehensive cohort analysis of bacterial pan-genomic variation, with potential impacts on genotype/phenotype and epidemiological studies.

Funder: KEH is supported by a Senior Medical Research Fellowship from the Viertel Foundation of Australia RJB through a UKRI MRC New Investigator Research Grant (held by KSB, MR/R020787/1), Shigella sonnei is the most common agent of shigellosis in high-income countries, and causes a significant disease burden in low- and middle-income countries. Antimicrobial resistance is increasingly common in all settings. Whole genome sequencing (WGS) is increasingly utilised for S. sonnei outbreak investigation and surveillance, but comparison of data between studies and labs is challenging. Here, we present a genomic framework and genotyping scheme for S. sonnei to efficiently identify genotype and resistance determinants from WGS data. The scheme is implemented in the software package Mykrobe and tested on thousands of genomes. Applying this approach to analyse >4,000 S. sonnei isolates sequenced in public health labs in three countries identified several common genotypes associated with increased rates of ciprofloxacin resistance and azithromycin resistance, confirming intercontinental spread of highly-resistant S. sonnei clones and demonstrating the genomic framework can facilitate monitoring the spread of resistant clones, including those that have recently emerged, at local and global scales.

Salmonella enterica serovar Hvittingfoss is an important foodborne serotype of Salmonella, being detected in many countries where surveillance is conducted. Outbreaks can occur, and there was a recent multistate foodborne outbreak in Australia. S. Hvittingfoss can be found in animal populations, though a definitive animal host has not been established. Six species of birds were sampled at Roebuck Bay, a designated Ramsar site in northwestern Australia, resulting in 326 cloacal swabs for bacterial culture. Among a single flock of 63 bar-tailed godwits (Limosa lapponica menzbieri) caught at Wader Spit, Roebuck Bay, in 2018, 17 (27%) were culture positive for Salmonella. All other birds were negative for Salmonella. The isolates were identified as Salmonella enterica serovar Hvittingfoss. Phylogenetic analysis revealed a close relationship between isolates collected from godwits and the S. Hvittingfoss strain responsible for a 2016 multistate foodborne outbreak originating from tainted cantaloupes (rock melons) in Australia. While it is not possible to determine how this strain of S. Hvittingfoss was introduced into the bar-tailed godwits, these findings show that wild Australian birds are capable of carrying Salmonella strains of public health importance. IMPORTANCESalmonella is a zoonotic pathogen that causes gastroenteritis and other disease presentations in both humans and animals. Serovars of S. enterica commonly cause foodborne disease in Australia and globally. In 2016-2017, S. Hvittingfoss was responsible for an outbreak that resulted in 110 clinically confirmed human cases throughout Australia. The origin of the contamination that led to the outbreak was never definitively established. Here, we identify a migratory shorebird, the bar-tailed godwit, as an animal reservoir of S. Hvittingfoss. These birds were sampled in northwestern Australia during their nonbreeding period. The presence of a genetically similar S. Hvittingfoss strain circulating in a wild bird population, 2 years after the 2016-2017 outbreak and ∼1,500 km from the suspected source of the outbreak, demonstrates a potentially unidentified environmental reservoir of S. Hvittingfoss. While the birds cannot be implicated in the outbreak that occurred 2 years prior, this study does demonstrate the potential role for wild birds in the transmission of this important foodborne pathogen.

Shigella species are specialised lineages of Escherichia coli that have converged to become human-adapted and cause dysentery by invading human gut epithelial cells. Most studies of Shigella evolution have been restricted to comparisons of single representatives of each species; and population genomic studies of individual Shigella species have focused on genomic variation caused by single nucleotide variants and ignored the contribution of insertion sequences (IS) which are highly prevalent in Shigella genomes. Here, we investigate the distribution and evolutionary dynamics of IS within populations of Shigella dysenteriae Sd1, Shigella sonnei and Shigella flexneri. We find that five IS (IS1, IS2, IS4, IS600 and IS911) have undergone expansion in all Shigella species, creating substantial strain-to-strain variation within each population and contributing to convergent patterns of functional gene loss within and between species. We find that IS expansion and genome degradation are most advanced in S. dysenteriae and least advanced in S. sonnei; and using genome-scale models of metabolism we show that Shigella species display convergent loss of core E. coli metabolic capabilities, with S. sonnei and S. flexneri following a similar trajectory of metabolic streamlining to that of S. dysenteriae. This study highlights the importance of IS to the evolution of Shigella and provides a framework for the investigation of IS dynamics and metabolic reduction in other bacterial species., Author summary Originally classified as a genus due to shared disease features, Shigella species actually represent distinct lineages of Escherichia coli that have become human-restricted dysentery pathogens via convergent evolution. A hallmark of this host-adaptation is the accumulation of insertion sequences (IS), which interrupt genes and cause deletions or genome rearrangements. Due to technical limitations in identifying IS insertions from short-read data, our understanding of the contribution of IS to the evolution of Shigella and other host- restricted bacteria has relied on analyses of either single genome representatives, or small numbers of genomes, and there are no large-scale population genomic studies exploring IS dynamics within bacterial species. Here we explore IS variation within and between Shigella species, revealing their historical and ongoing contributions to evolutionary convergence and metabolic streamlining.

Objectives mcr-9.1 is a newly described mobile colistin resistance gene. We have noted its presence in multiple species of carbapenem-resistant Enterobacterales (CRE) from our institution. We aimed to determine the clinical features, genomic context and phenotypic impact of mcr-9.1 carriage in a series of patients between 2010 and 2019. Methods We identified 32 patients with mcr-9.1-carrying CRE isolates (mCRE) and collected demographic, antimicrobial exposure and infection data. Whole-genome sequencing (including short and long reads) was performed on 32 isolates. We assessed sequence similarity of mcr-9.1-harbouring plasmids, then compared our findings with plasmids for which sequence data were publicly available. Results There was no colistin exposure in patients prior to isolation of mCRE. mcr-9.1 was identified on IncHI2 plasmids across four different bacterial species and was co-located with blaIMP-4 in 23/30 plasmids studied. mCRE isolates did not demonstrate phenotypic colistin resistance, either at baseline or following sublethal colistin exposure, thus showing that mcr-9.1 alone is not sufficient for resistance. Publicly available sequence data indicated the presence of carbapenemase genes in 236/619 mcr-9.1-carrying genomes (38%). IncHI2 plasmids carrying mcr-9.1 and carbapenemase genes were detected in genomes from North America, Europe, North Africa, Asia and Oceania. Conclusions Spread of mcr-9.1 in CRE from our institution was driven by IncHI2 ‘superplasmids’, so termed because of their large size and their prolific carriage of resistance determinants. These were also detected in global CRE genomes. Phenotypic colistin resistance was not detected in our isolates but remains to be determined from global mCRE.

Knowledge of mobile genetic elements that capture and disseminate antimicrobial resistance genes between diverse environments, particularly across human&ndash, animal boundaries, is key to understanding the role anthropogenic activities have in the evolution of antimicrobial resistance. Plasmids that circulate within the Enterobacteriaceae and the Proteobacteria more broadly are well placed to acquire resistance genes sourced from separate niche environments and provide a platform for smaller mobile elements such as IS26 to assemble these genes into large, complex genomic structures. Here, we characterised two atypical Z/I1 hybrid plasmids, pSTM32-108 and pSTM37-118, hosting antimicrobial resistance and virulence associated genes within endemic pathogen Salmonella enterica serovar Typhimurium 1,4,[5],12:i:-, sourced from Australian swine production facilities during 2013. We showed that the plasmids found in S. Typhimurium 1,4,[5],12:i:- are close relatives of two plasmids identified from Escherichia coli of human and bovine origin in Australia circa 1998. The older plasmids, pO26-CRL125 and pO111-CRL115, encoded a putative serine protease autotransporter and were host to a complex resistance region composed of a hybrid Tn21-Tn1721 mercury resistance transposon and composite IS26 transposon Tn6026. This gave a broad antimicrobial resistance profile keyed towards first generation antimicrobials used in Australian agriculture but also included a class 1 integron hosting the trimethoprim resistance gene dfrA5. Genes encoding resistance to ampicillin, trimethoprim, sulphonamides, streptomycin, aminoglycosides, tetracyclines and mercury were a feature of these plasmids. Phylogenetic analyses showed very little genetic drift in the sequences of these plasmids over the past 15 years, however, some alterations within the complex resistance regions present on each plasmid have led to the loss of various resistance genes, presumably as a result of the activity of IS26. These alterations may reflect the specific selective pressures placed on the host strains over time. Our studies suggest that these plasmids and variants of them are endemic in Australian food production systems.

Salmonella enterica serotype Kentucky can be a common causative agent of salmonellosis, usually associated with consumption of contaminated poultry. Antimicrobial resistance (AMR) to multiple drugs, including ciprofloxacin, is an emerging problem within this serotype. We used whole-genome sequencing (WGS) to investigate the phylogenetic structure and AMR content of 121 S. e nterica serotype Kentucky sequence type 198 isolates from five continents. Population structure was inferred using phylogenomic analysis and whole genomes were compared to investigate changes in gene content, with a focus on acquired AMR genes. Our analysis showed that multidrug-resistant (MDR) S. enterica serotype Kentucky isolates belonged to a single lineage, which we estimate emerged circa 1989 following the acquisition of the AMR-associated Salmonella genomic island (SGI) 1 (variant SGI1-K) conferring resistance to ampicillin, streptomycin, gentamicin, sulfamethoxazole and tetracycline. Phylogeographical analysis indicates this clone emerged in Egypt before disseminating into Northern, Southern and Western Africa, then to the Middle East, Asia and the European Union. The MDR clone has since accumulated various substitution mutations in the quinolone-resistance-determining regions (QRDRs) of DNA gyrase (gyrA) and DNA topoisomerase IV (parC), such that most strains carry three QRDR mutations which together confer resistance to ciprofloxacin. The majority of AMR genes in the S. e nterica serotype Kentucky genomes were carried either on plasmids or SGI structures. Remarkably, each genome of the MDR clone carried a different SGI1-K derivative structure; this variation could be attributed to IS26-mediated insertions and deletions, which appear to have hampered previous attempts to trace the clone’s evolution using sub-WGS resolution approaches. Several different AMR plasmids were also identified, encoding resistance to chloramphenicol, third-generation cephalosporins, carbapenems and/or azithromycin. These results indicate that most MDR S. e nterica serotype Kentucky circulating globally result from the clonal expansion of a single lineage that acquired chromosomal AMR genes 30 years ago, and has continued to diversify and accumulate additional resistances to last-line oral antimicrobials. This article contains data hosted by Microreact.

The increasing incidence and emergence of multi-drug resistant (MDR) Acinetobacter baumannii has become a major global health concern. Colistin is a historic antimicrobial that has become commonly used as a treatment for MDR A. baumannii infections. The increase in colistin usage has been mirrored by an increase in colistin resistance. We aimed to identify the mechanisms associated with colistin resistance in A. baumannii using multiple high-throughput-sequencing technologies, including transposon-directed insertion site sequencing (TraDIS), RNA sequencing (RNAseq) and whole-genome sequencing (WGS) to investigate the genotypic changes of colistin resistance in A. baumannii. Using TraDIS, we found that genes involved in drug efflux (adeIJK), and phospholipid (mlaC, mlaF and mlaD) and lipooligosaccharide synthesis (lpxC and lpsO) were required for survival in sub-inhibitory concentrations of colistin. Transcriptomic (RNAseq) analysis revealed that expression of genes encoding efflux proteins (adeI, adeC, emrB, mexB and macAB) was enhanced in in vitro generated colistin-resistant strains. WGS of these organisms identified disruptions in genes involved in lipid A (lpxC) and phospholipid synthesis (mlaA), and in the baeS/R two-component system (TCS). We additionally found that mutations in the pmrB TCS genes were the primary colistin-resistance-associated mechanisms in three Vietnamese clinical colistin-resistant A. baumannii strains. Our results outline the entire range of mechanisms employed in A. baumannii for resistance against colistin, including drug extrusion and the loss of lipid A moieties by gene disruption or modification.

Salmonella enterica serotype Kentucky (S. Kentucky) can be a common causative agent of salmonellosis, usually associated with consumption of contaminated poultry. Antimicrobial resistance (AMR) to multiple drugs, including ciprofloxacin, is an emerging problem within this serotype. We used whole-genome sequencing (WGS) to investigate the phylogenetic structure and AMR content of 121 S. Kentucky ST198 isolates from five continents. Population structure was inferred using phylogenomic analysis and whole genomes were compared to investigate changes in gene content, with a focus on acquired AMR genes. Our analysis showed that multidrug resistant (MDR) S. Kentucky isolates belonged to a single lineage, which we estimate emerged circa 1989 following the acquisition of the AMR-associated Salmonella genomic island 1 (variant SGI1-K) conferring resistance ampicillin, streptomycin, gentamicin, sulfamethoxazole, and tetracycline. Phylogeographic analysis indicates this clone emerged in Egypt before disseminating into Northern, Southern and Western Africa, then to the Middle East, Asia and the European Union. The MDR clone has since accumulated various substitution mutations in the quinolone resistance determining regions (QRDR) of DNA gyrase (gyrA) and DNA topoisomerase (parC), such that most strains carry three QRDR mutations which together confer resistance to ciprofloxacin. The majority of AMR genes in the S. Kentucky genomes were carried either on plasmids or SGI structures. Remarkably, each genome carried a different SGI1-K derivative structure; this variation could be attributed to IS26-mediated insertions and deletions, which appear to have hampered previous attempts to trace the clone's evolution using sub-WGS resolution approaches. Several different AMR plasmids were also identified, encoding resistance to chloramphenicol, third-generation cephalosporins, carbapenems, and/or azithromycin. These results indicate that most MDR S. Kentucky circulating globally result from the clonal expansion of a single lineage that acquired chromosomal AMR genes 30 years ago, and has continued to diversify and accumulate additional resistances to last-line oral antimicrobials.

© 2019 Oxford University Press. All rights reserved. Objectives: Recent reports indicate the emergence of a new carbapenemase-producing Klebsiella pneumoniae clone, ST307. We sought to better understand the global epidemiology and evolution of this clone and evaluate its association with antimicrobial resistance (AMR) genes. Methods: We collated information from the literature and public databases and performed a comparative analysis of 95 ST307 genomes (including 37 that were newly sequenced). Results: We show that ST307 emerged in the mid-1990s (nearly 20 years prior to its first report), is already globally distributed and is intimately associated with a conserved plasmid harbouring the blaCTX-M-15 ESBL gene and several other AMR determinants. Conclusions: Our findings support the need for enhanced surveillance of this widespread ESBL clone in which carbapenem resistance has occasionally emerged.

Acinetobacter baumannii is a common causative agent of hospital-acquired infections and a leading cause of infection in burns patients. Carbapenem resistant A. baumannii is considered a major public health threat and has been identified by the World Health Organization as the top priority organism requiring new antimicrobials. The most common mechanism for carbapenem resistance in A. baumannii is via horizontal acquisition of carbapenemase genes. In this study, we sampled 20 A. baumannii isolates from a patient with extensive burns, and characterized the evolution of carbapenem resistance over a 45-day period via Illumina and Oxford Nanopore sequencing. All isolates were multi-drug resistant, carrying two genomic islands that harboured several antibiotic resistance genes. Most isolates were genetically identical and represent a single founder genotype. We identified three novel non-synonymous substitutions associated with meropenem resistance: F136L and G288S in AdeB (part of the AdeABC efflux pump) associated with an increase in meropenem MIC to ≥8 μg/mL; and A515V in FtsI (PBP3, a penicillin-binding protein) associated with a further increase in MIC to 32 μg/mL. Structural modelling of AdeB and FtsI showed that these mutations affected their drug binding sites and revealed mechanisms for meropenem resistance. Notably, one of the adeB mutations arose prior to meropenem therapy but following ciprofloxacin therapy, suggesting exposure to one drug whose resistance is mediated by the efflux pump can induce collateral resistance to other drugs to which the bacteria has not yet been exposed.DATA SUMMARYAll raw genome sequences, including Illumina paired end short reads and Oxford Nanopore long reads, have been deposited in the SRA under project PRJNA396979. Individual accessions for each strain are provided in Table S1.The annotated genome assembly for strain A2, the reference genome for the founder genotype, has been submitted to GenBank under accession CP024124 (chromosome) and CP024125 (plasmid).Hybrid assemblies for strains A1, A3, A8, A13, A15, A17 and A20 are available in FigShare, doi: 10.4225/49/5987e14e9b530 (note they were not deposited in GenBank as they differ from A2 by only 1-4 SNPs as indicated).IMPACT STATEMENTSAcinetobacter baumannii is a highly drug resistant pathogen that is frequently found within intensive care units (ICUs) and especially impacts patients with severe burns. While several studies have examined the global population structure of A. baumannii, few have investigated within-host evolution of A. baumannii in direct response to antibiotic treatment in a single patient. Here, we analysed the genetic evolution of A. baumannii isolated from a patient with severe burns over the course of their stay in ICU. The A. baumannii population on this patient was highly drug resistant, carrying two distinct genomic islands encoding resistance to several antibiotics but not carbapenems. The bacterial population comprised four distinct subclades, two of which had evolved carbapenem resistance over the course of antibiotic treatment through novel mutations in genes associated with drug binding. One subclade was also transmitted to another patient on the ward. While carbapenem resistance is common in A. baumannii, this is generally attributed to horizontally transferred carbapenemase genes. These data provide evidence for carbapenem resistance arising in vivo via non-synonymous substitutions during a single infection episode, demonstrating carbapenem resistance can emerge in genetic isolation in response to exposure to carbapenems and other drugs.

Since the plasmid-mediated polymyxin resistance gene mcr-1 was first reported in Escherichia coli and Klebsiella pneumoniae in China, only one mcr-1-positive isolate of Shigella sonnei, containing inactivated mcr-1, has been reported worldwide. In this study, 1650 historical S. sonnei strains isolated from 2003–2015 in China were screened for the mcr-1 gene. Antimicrobial susceptibilities and resistance genes of mcr-1-positive isolates were determined, and the transferability of polymyxin resistance by plasmid conjugation was investigated. Pulsed-field gel electrophoresis (PFGE), plasmid profiles and Southern blotting were used to analyse genetic relationships and plasmid characteristics, and mcr-1-positive plasmids were sequenced. Six mcr-1-positive S. sonnei isolates from Shanghai (2010–2012) with polymyxin B resistance (MICs 4–8 μg/mL) were identified. Four of these exhibited multidrug resistance, including resistance to azithromycin and third-generation cephalosporins, and co-harboured blaCTX-M-14, mph(A) and blaTEM on different plasmids. mcr-1-positive plasmids shared highly similar IncI2 backbones that resembled reference plasmids, although some differences were observed, including various and abundant insertion sequences/patterns (IS1294, IS1 and ISApl1) and a diverse recombination shufflon region. mcr-1 in S. sonnei may date back to mid-2006. Here we report for the first time the presence of active mcr-1 in multidrug-resistant S. sonnei in China, which has existed since at least 2010. This study highlights the diverse mobile genetic elements on mcr-1-harboring plasmids, potentially resulting in high rates of mcr-1 horizontal transfer among Enterobacteriaceae. These findings emphasise the importance of continuous national and international surveillance of mcr-1-positive Shigella and changes in antibiotic resistance patterns.

Background Recent developments in sequencing technologies make it possible to obtain genome sequences from a large number of isolates in a very short time. Bayesian phylogenetic approaches can take advantage of these data by simultaneously inferring the phylogenetic tree, evolutionary timescale, and demographic parameters (such as population growth rates), while naturally integrating uncertainty in all parameters. Despite their desirable properties, Bayesian approaches can be computationally intensive, hindering their use for outbreak investigations involving genome data for a large numbers of pathogen isolates. An alternative to using full Bayesian inference is to use a hybrid approach, where the phylogenetic tree and evolutionary timescale are estimated first using maximum likelihood. Under this hybrid approach, demographic parameters are inferred from estimated trees instead of the sequence data, using maximum likelihood, Bayesian inference, or approximate Bayesian computation. This can vastly reduce the computational burden, but has the disadvantage of ignoring the uncertainty in the phylogenetic tree and evolutionary timescale. Results We compared the performance of a fully Bayesian and a hybrid method by analysing six whole-genome SNP data sets from a range of bacteria and simulations. The estimates from the two methods were very similar, suggesting that the hybrid method is a valid alternative for very large datasets. However, we also found that congruence between these methods is contingent on the presence of strong temporal structure in the data (i.e. clocklike behaviour), which is typically verified using a date-randomisation test in a Bayesian framework. To reduce the computational burden of this Bayesian test we implemented a date-randomisation test using a rapid maximum likelihood method, which has similar performance to its Bayesian counterpart. Conclusions Hybrid approaches can produce reliable inferences of evolutionary timescales and phylodynamic parameters in a fraction of the time required for fully Bayesian analyses. As such, they are a valuable alternative in outbreak studies involving a large number of isolates. Electronic supplementary material The online version of this article (10.1186/s12862-018-1210-5) contains supplementary material, which is available to authorized users.

Estimating the rates at which bacterial genomes evolve is critical to understanding major evolutionary and ecological processes such as disease emergence, long-term host-pathogen associations, and short-term transmission patterns. The surge in bacterial genomic data sets provides a new opportunity to estimate these rates and reveal the factors that shape bacterial evolutionary dynamics. For many organisms estimates of evolutionary rate display an inverse association with the time-scale over which the data are sampled. However, this relationship remains unexplored in bacteria due to the difficulty in estimating genome-wide evolutionary rates, which are impacted by the extent of temporal structure in the data and the prevalence of recombination. We collected 36 whole genome sequence data sets from 16 species of bacterial pathogens to systematically estimate and compare their evolutionary rates and assess the extent of temporal structure in the absence of recombination. The majority (28/36) of data sets possessed sufficient clock-like structure to robustly estimate evolutionary rates. However, in some species reliable estimates were not possible even with “ancient DNA” data sampled over many centuries, suggesting that they evolve very slowly or that they display extensive rate variation among lineages. The robustly estimated evolutionary rates spanned several orders of magnitude, from 10−6 to 10−8 nucleotide substitutions site-1 year-1. This variation was largely attributable to sampling time, which was strongly negatively associated with estimated evolutionary rates, with this relationship best described by an exponential decay curve. To avoid potential estimation biases such time-dependency should be considered when inferring evolutionary time-scales in bacteria.

We recently reported a dramatic increase in the prevalence of carbapenem-resistant Acinetobacter baumannii infections in the intensive care unit (ICU) of a Vietnamese hospital. This upsurge was associated with a specific oxa23-positive clone that was identified by multilocus VNTR analysis. Here, we used whole-genome sequence analysis to dissect the emergence of carbapenem-resistant A. baumannii causing ventilator-associated pneumonia (VAP) in the ICU during 2009-2012. To provide historical context and distinguish microevolution from strain introduction, we compared these genomes with those of A. baumannii asymptomatic carriage and VAP isolates from this same ICU collected during 2003-2007. We identified diverse lineages co-circulating over many years. Carbapenem resistance was associated with the presence of oxa23, oxa40, oxa58 and ndm1 genes in multiple lineages. The majority of resistant isolates were oxa23-positive global clone GC2; fine-scale phylogenomic analysis revealed five distinct GC2 sublineages within the ICU that had evolved locally via independent chromosomal insertions of oxa23 transposons. The increase in infections caused by carbapenem-resistant A. baumannii was associated with transposon-mediated transmission of a carbapenemase gene, rather than clonal expansion or spread of a carbapenemase-harbouring plasmid. Additionally, we found evidence of homologous recombination creating diversity within the local GC2 population, including several events resulting in replacement of the capsule locus. We identified likely donors of the imported capsule locus sequences amongst the A. baumannii isolated on the same ward, suggesting that diversification was largely facilitated via reassortment and sharing of genetic material within the localized A. baumannii population.