Biochemistry and Pharmacology - Research Publications
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ItemGenome Sequence of Acinetobacter baumannii Strain D36, an Antibiotic-Resistant Isolate from Lineage 2 of Global Clone(AMER SOC MICROBIOLOGY, 2015-12-17)Multiply antibiotic-resistant Acinetobacter baumannii isolate D36 was recovered in Australia in 2008 and belongs to a distinct lineage of global clone 1 (GC1). Here, we present the complete 4.13 Mbp genome sequence (chromosome plus 4 plasmids), generated via long read sequencing (PacBio).
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ItemGenome Sequence of Acinetobacter baumannii Strain A1, an Early Example of Antibiotic-Resistant Global Clone 1(AMER SOC MICROBIOLOGY, 2015-03-12)Acinetobacter baumannii isolate A1 was recovered in the United Kingdom in 1982 and belongs to global clone 1 (GC1). Here, we present its complete 3.91-Mbp genome sequence, generated via a combination of short-read sequencing (Illumina), long-read sequencing (PacBio), and manual finishing.
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ItemGenome-scale rates of evolutionary change in bacteria(MICROBIOLOGY SOC, 2016-11)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 approximately 10-5 to 10-8 nucleotide substitutions per site year-1. This variation was negatively associated with sampling time, 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.
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ItemInferring demographic parameters in bacterial genomic data using Bayesian and hybrid phylogenetic methods(BMC, 2018-06-19)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.
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ItemEvolution of carbapenem resistance in Acinetobacter baumannii during a prolonged infection(MICROBIOLOGY SOC, 2018-03)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 multidrug resistant, carrying two genomic islands that harboured several antibiotic-resistance genes. Most isolates were genetically identical and represented 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-1; and A515V in FtsI (PBP3, a penicillin-binding protein) associated with a further increase in MIC to 32 µg ml-1. 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 bacterium has not yet been exposed.
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ItemISMapper: identifying transposase insertion sites in bacterial genomes from short read sequence data(BIOMED CENTRAL LTD, 2015-09-03)BACKGROUND: Insertion sequences (IS) are small transposable elements, commonly found in bacterial genomes. Identifying the location of IS in bacterial genomes can be useful for a variety of purposes including epidemiological tracking and predicting antibiotic resistance. However IS are commonly present in multiple copies in a single genome, which complicates genome assembly and the identification of IS insertion sites. Here we present ISMapper, a mapping-based tool for identification of the site and orientation of IS insertions in bacterial genomes, directly from paired-end short read data. RESULTS: ISMapper was validated using three types of short read data: (i) simulated reads from a variety of species, (ii) Illumina reads from 5 isolates for which finished genome sequences were available for comparison, and (iii) Illumina reads from 7 Acinetobacter baumannii isolates for which predicted IS locations were tested using PCR. A total of 20 genomes, including 13 species and 32 distinct IS, were used for validation. ISMapper correctly identified 97 % of known IS insertions in the analysis of simulated reads, and 98 % in real Illumina reads. Subsampling of real Illumina reads to lower depths indicated ISMapper was able to correctly detect insertions for average genome-wide read depths >20x, although read depths >50x were required to obtain confident calls that were highly-supported by evidence from reads. All ISAba1 insertions identified by ISMapper in the A. baumannii genomes were confirmed by PCR. In each A. baumannii genome, ISMapper successfully identified an IS insertion upstream of the ampC beta-lactamase that could explain phenotypic resistance to third-generation cephalosporins. The utility of ISMapper was further demonstrated by profiling genome-wide IS6110 insertions in 138 publicly available Mycobacterium tuberculosis genomes, revealing lineage-specific insertions and multiple insertion hotspots. CONCLUSIONS: ISMapper provides a rapid and robust method for identifying IS insertion sites directly from short read data, with a high degree of accuracy demonstrated across a wide range of bacteria.
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ItemRepeated local emergence of carbapenem-resistant Acinetobacter baumannii in a single hospital ward(MICROBIOLOGY SOC, 2016-03)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.
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ItemEvidence of microevolution of Salmonella Typhimurium during a series of egg-associated outbreaks linked to a single chicken farm(BMC, 2013-11-19)BACKGROUND: The bacterium Salmonella enterica serovar Typhimurium (S. Typhimurium) is one of the most frequent causes of foodborne outbreaks of gastroenteritis. Between 2005-2008 a series of S. Typhimurium outbreaks occurred in Tasmania, Australia, that were all traced to eggs originating from a single chicken farm. We sequenced the genomes of 12 isolates linked to these outbreaks, in order to investigate the microevolution of a pathogenic S. Typhimurium clone in a natural, spatiotemporally restricted population. RESULTS: The isolates, which shared a phage type similar to DT135 known locally as 135@ or 135a, formed a clade within the S. Typhimurium population with close similarity to the reference genome SL1334 (160 single nucleotide polymorphisms, or SNPs). Ten of the isolates belonged to a single clone (<23 SNPs between isolate pairs) which likely represents the population of S. Typhimurium circulating at the chicken farm; the other two were from sporadic cases and were genetically distinct from this clone. Divergence dating indicated that all 12 isolates diverged from a common ancestor in the mid 1990 s, and the clone began to diversify in 2003-2004. This clone spilled out into the human population several times between 2005-2008, during which time it continued to accumulate SNPs at a constant rate of 3-5 SNPs per year or 1x10-6 substitutions site-1 year-1, faster than the longer-term (~50 year) rates estimated previously for S. Typhimurium. Our data suggest that roughly half of non-synonymous substitutions are rapidly removed from the S. Typhimurium population, after which purifying selection is no longer important and the remaining substitutions become fixed in the population. The S. Typhimurium 135@ isolates were nearly identical to SL1344 in terms of gene content and virulence plasmids. Their phage contents were close to SL1344, except that they carried a different variant of Gifsy-1, lacked the P2 remnant found in SL1344 and carried a novel P2 phage, P2-Hawk, in place SL1344's P2 phage SopEϕ. DT135 lacks P2 prophage. Two additional plasmids were identified in the S. Typhimurium 135@ isolates, pSTM2 and pSTM7. Both plasmids were IncI1, but phylogenetic analysis of the plasmids and their bacterial hosts shows these plasmids are genetically distinct and result from independent plasmid acquisition events. CONCLUSIONS: This study provides a high-resolution insight into short-term microevolution of the important human pathogen S. Typhimurium. It indicates that purifying selection occurs rapidly in this population (≤ 6 years) and then declines, and provides an estimate for the short-term substitution rate. The latter is likely to be more relevant for foodborne outbreak investigation than previous estimates based on longer time scales.