Genome Sequencing Links Persistent Outbreak of Legionellosis in Sydney (New South Wales, Australia) to an Emerging Clone of Legionella pneumophila Sequence Type 211.

The city of Sydney, Australia, experienced a persistent outbreak of Legionella pneumophila serogroup 1 (Lp1) pneumonia in 2016. To elucidate the source and guide public health actions, the genomes of clinical and environmental Lp1 isolates recovered over 7 weeks were examined. A total of 48 isolates from human cases and cooling towers were sequenced and compared using single-nucleotide polymorphism (SNP)-based core-genome multilocus sequencing typing (MLST) and pangenome approaches. All three methods confirmed phylogenetic relatedness between isolates associated with outbreaks in the Central Business District (CBD) in March and May and those in suburb 1. These isolates were designated the "main cluster" and consisted of isolates from two patients from the CBD March outbreak, one patient and one tower isolate from suburb 1, and isolates from two cooling towers and three patients from the CBD May outbreak. All main cluster isolates were sequence type 211 (ST211), which previously has only been reported in Canada. Significantly, pangenome analysis identified mobile genetic elements containing a unique type IV A F-type secretion system (T4ASS), which was specific to the main cluster, and cocirculating clinical strains, suggesting a potential mechanism for increased fitness and persistence of the outbreak clone. Genome sequencing enabled linking of the geographically dispersed environmental sources of infection among the spatially and temporally coinciding cases of legionellosis in a highly populated urban setting. The discovery of a unique T4ASS emphasizes the role of genome recombination in the emergence of successful Lp1 clones.IMPORTANCE A new emerging clone has been responsible for a prolonged legionellosis outbreak in Sydney, Australia. The use of whole-genome sequencing linked two outbreaks thought to be unrelated and confirmed the outliers. These findings led to the resampling and subsequent identification of the source, guiding public health actions and bringing the outbreak to a close. Significantly, the outbreak clone was identified as sequence type 211 (ST211). Our study reports this ST in the Southern Hemisphere and presents a description of ST211 genomes from both clinical and environmental isolates. A unique mobile genetic element containing a type IV secretion system was identified in Lp1 ST211 isolates linked to the main cluster and Lp1 ST42 isolates that were cocirculating at the time of the outbreak.

Culture-independent genomics of a novel chlamydial pathogen of fish provides new insight into host-specific adaptations utilized by these intracellular bacteria.

Several Chlamydiales families are associated with epitheliocystis, a common condition of the fish gill epithelium. These families share common ancestors with the Chlamydiaceae and environmental Chlamydiae. Due to the lack of culture systems, little is known about the biology of these chlamydial fish pathogens. We investigated epitheliocystis in cultured Orange-spotted grouper (Epinephelus coioides) from North Queensland, Australia. Basophilic inclusions were present in the gills of 22/31 fish and the presence of the chlamydial pathogen in the cysts was confirmed by in situ hybridization. Giant grouper (Epinephelus lanceolatus) cultured in the same systems were epitheliocystis free. 16S rRNA gene sequencing revealed a novel member of the Candidatus Parilichlamydiaceae: Ca. Similichlamydia epinephelii. Using metagenomic approaches, we obtained an estimated 68% of the chlamydial genome, revealing that this novel chlamydial pathogen shares a number of key pathogenic hallmarks with the Chlamydiaceae, including an intact Type III Secretion system and several chlamydial virulence factors. This provides additional evidence that these pathogenic mechanisms were acquired early in the evolution of this unique bacterial phylum. The identification and genomic characterization of Ca. S. epinephelii provides new opportunities to study the biology of distantly-related chlamydial pathogens while shining a new light on the evolution of pathogenicity of the Chlamydiaceae.

HapFlow: visualizing haplotypes in sequencing data.

SUMMARY: HapFlow is a python application for visualizing haplotypes present in sequencing data. It identifies variant profiles present and reads and creates an abstract visual representation of these profiles to make haplotypes easier to identify. AVAILABILITY AND IMPLEMENTATION: HapFlow is freely available (under a GPL license) for download (for Mac OS X, Unix and Microsoft Windows) from github (http://mjsull.github.io/HapFlow). CONTACT: apolking@usc.edu.au.

Culture-independent genomic characterisation of Candidatus Chlamydia sanzinia, a novel uncultivated bacterium infecting snakes.

BACKGROUND: Recent molecular studies have revealed considerably more diversity in the phylum Chlamydiae than was previously thought. Evidence is growing that many of these novel chlamydiae may be important pathogens in humans and animals. A significant barrier to characterising these novel chlamydiae is the requirement for culturing. We recently identified a range of novel uncultured chlamydiae in captive snakes in Switzerland, however, nothing is known about their biology. Using a metagenomics approach, the aim of this study was to characterise the genome of a novel chlamydial taxon from the choana of a captive snake. In doing so, we propose a new candidate species in the genus Chlamydia (Candidatus Chlamydia sanzinia) and reveal new information about the biological diversity of this important group of pathogens. RESULTS: We identified two chlamydial genomic contigs: a 1,113,073 bp contig, and a 7,504 bp contig, representing the chromosome and plasmid of Ca. Chlamydia sanzinia strain 2742-308, respectively. The 998 predicted coding regions include an expanded repertoire of outer membrane proteins (Pmps and Omps), some of which exhibited frameshift mutations, as well as several chlamydial virulence factors such as the translocating actin-recruitment phosphoprotein (Tarp) and macrophage inhibition potentiator (Mip). A suite of putative inclusion membrane proteins were also predicted. Notably, no evidence of a traditional chlamydial plasticity zone was identified. Phylogenetically, Ca. Chlamydia sanzinia forms a clade with C. pneumoniae and C. pecorum, distinct from former "Chlamydophila" species. CONCLUSIONS: Genomic characterisation of a novel uncultured chlamydiae from the first reptilian host has expanded our understanding of the diversity and biology of a genus that was thought to be the most well-characterised in this unique phylum. It is anticipated that this method will be suitable for characterisation of other novel chlamydiae.

Comparative genomic analysis of human Chlamydia pneumoniae isolates from respiratory, brain and cardiac tissues.

Chlamydia pneumoniae is an obligate intracellular bacterium implicated in a wide range of human diseases including atherosclerosis and Alzheimer's disease. Efforts to understand the relationships between C. pneumoniae detected in these diseases have been hindered by the availability of sequence data for non-respiratory strains. In this study, we sequenced the whole genomes for C. pneumoniae isolates from atherosclerosis and Alzheimer's disease, and compared these to previously published C. pneumoniae genomes. Phylogenetic analyses of these new C. pneumoniae strains indicate two sub-groups within human C. pneumoniae, and suggest that both recombination and mutation events have driven the evolution of human C. pneumoniae. Further fine-detailed analyses of these new C. pneumoniae sequences show several genetically variable loci. This suggests that similar strains of C. pneumoniae are found in the brain, lungs and cardiovascular system and that only minor genetic differences may contribute to the adaptation of particular strains in human disease.

Genetic diversity in the plasticity zone and the presence of the chlamydial plasmid differentiates Chlamydia pecorum strains from pigs, sheep, cattle, and koalas.

BACKGROUND: Chlamydia pecorum is a globally recognised pathogen of livestock and koalas. To date, comparative genomics of C. pecorum strains from sheep, cattle and koalas has revealed that only single nucleotide polymorphisms (SNPs) and a limited number of pseudogenes appear to contribute to the genetic diversity of this pathogen. No chlamydial plasmid has been detected in these strains despite its ubiquitous presence in almost all other chlamydial species. Genomic analyses have not previously included C. pecorum from porcine hosts. We sequenced the genome of three C. pecorum isolates from pigs with differing pathologies in order to re-evaluate the genetic differences and to update the phylogenetic relationships between C. pecorum from each of the hosts. METHODS: Whole genome sequences for the three porcine C. pecorum isolates (L1, L17 and L71) were acquired using C. pecorum-specific sequence capture probes with culture-independent methods, and assembled in CLC Genomics Workbench. The pairwise comparative genomic analyses of 16 pig, sheep, cattle and koala C. pecorum genomes were performed using several bioinformatics platforms, while the phylogenetic analyses of the core C. pecorum genomes were performed with predicted recombination regions removed. Following the detection of a C. pecorum plasmid, a newly developed C. pecorum-specific plasmid PCR screening assay was used to evaluate the plasmid distribution in 227 C. pecorum samples from pig, sheep, cattle and koala hosts. RESULTS: Three porcine C. pecorum genomes were sequenced using C. pecorum-specific sequence capture probes with culture-independent methods. Comparative genomics of the newly sequenced porcine C. pecorum genomes revealed an increased average number of SNP differences (~11 500) between porcine and sheep, cattle, and koala C. pecorum strains, compared to previous C. pecorum genome analyses. We also identified a third copy of the chlamydial cytotoxin gene, found only in porcine C. pecorum isolates. Phylogenetic analyses clustered porcine isolates into a distinct clade, highlighting the polyphyletic origin of C. pecorum in livestock. Most surprising, we also discovered a plasmid in the porcine C. pecorum genome. Using this novel C. pecorum plasmid (pCpec) sequence, a) we developed a pCpec screening assay to evaluate the plasmid distribution in C. pecorum from different hosts; and b) to characterise the pCpec sequences from available previously sequenced C. pecorum genome data. pCpec screening showed that the pCpec is common in all hosts of C. pecorum, however not all C. pecorum strains carry pCpec. CONCLUSIONS: This study provides further insight into the complexity of C. pecorum epidemiology and novel genomic regions that may be linked to host specificity. C. pecorum plasmid characterisation may aid in improving our understanding of C. pecorum pathogenesis across the variety of host species this animal pathogen infects.

Culture-independent genome sequencing of clinical samples reveals an unexpected heterogeneity of infections by Chlamydia pecorum.

Chlamydia pecorum is an important global pathogen of livestock, and it is also a significant threat to the long-term survival of Australia's koala populations. This study employed a culture-independent DNA capture approach to sequence C. pecorum genomes directly from clinical swab samples collected from koalas with chlamydial disease as well as from sheep with arthritis and conjunctivitis. Investigations into single-nucleotide polymorphisms within each of the swab samples revealed that a portion of the reads in each sample belonged to separate C. pecorum strains, suggesting that all of the clinical samples analyzed contained mixed populations of genetically distinct C. pecorum isolates. This observation was independent of the anatomical site sampled and the host species. Using the genomes of strains identified in each of these samples, whole-genome phylogenetic analysis revealed that a clade containing a bovine and a koala isolate is distinct from other clades comprised of livestock or koala C. pecorum strains. Providing additional evidence to support exposure of koalas to Australian livestock strains, two minor strains assembled from the koala swab samples clustered with livestock strains rather than koala strains. Culture-independent probe-based genome capture and sequencing of clinical samples provides the strongest evidence yet to suggest that naturally occurring chlamydial infections are comprised of multiple genetically distinct strains.

Comparative genomics of koala, cattle and sheep strains of Chlamydia pecorum.

BACKGROUND: Chlamydia pecorum is an important pathogen of domesticated livestock including sheep, cattle and pigs. This pathogen is also a key factor in the decline of the koala in Australia. We sequenced the genomes of three koala C. pecorum strains, isolated from the urogenital tracts and conjunctiva of diseased koalas. The genome of the C. pecorum VR629 (IPA) strain, isolated from a sheep with polyarthritis, was also sequenced. RESULTS: Comparisons of the draft C. pecorum genomes against the complete genomes of livestock C. pecorum isolates revealed that these strains have a conserved gene content and order, sharing a nucleotide sequence similarity > 98%. Single nucleotide polymorphisms (SNPs) appear to be key factors in understanding the adaptive process. Two regions of the chromosome were found to be accumulating a large number of SNPs within the koala strains. These regions include the Chlamydia plasticity zone, which contains two cytotoxin genes (toxA and toxB), and a 77 kbp region that codes for putative type III effector proteins. In one koala strain (MC/MarsBar), the toxB gene was truncated by a premature stop codon but is full-length in IPTaLE and DBDeUG. Another five pseudogenes were also identified, two unique to the urogenital strains C. pecorum MC/MarsBar and C. pecorum DBDeUG, respectively, while three were unique to the koala C. pecorum conjunctival isolate IPTaLE. An examination of the distribution of these pseudogenes in C. pecorum strains from a variety of koala populations, alongside a number of sheep and cattle C. pecorum positive samples from Australian livestock, confirmed the presence of four predicted pseudogenes in koala C. pecorum clinical samples. Consistent with our genomics analyses, none of these pseudogenes were observed in the livestock C. pecorum samples examined. Interestingly, three SNPs resulting in pseudogenes identified in the IPTaLE isolate were not found in any other C. pecorum strain analysed, raising questions over the origin of these point mutations. CONCLUSIONS: The genomic data revealed that variation between C. pecorum strains were mainly due to the accumulation of SNPs, some of which cause gene inactivation. The identification of these genetic differences will provide the basis for further studies to understand the biology and evolution of this important animal pathogen.

Genome analysis and CRISPR typing of Salmonella enterica serovar Virchow.

BACKGROUND: Salmonella enterica subsp. enterica serovar Virchow has been recognized as a significant health burden in Asia, Australia and Europe. In addition to its global distribution, S. Virchow is clinically significant due to the frequency at which it causes invasive infections and its association with outbreaks arising from food-borne transmission. Here, we examine the genome of an invasive isolate of S. Virchow SVQ1 (phage type 8) from an outbreak in southeast Queensland, Australia. In addition to identifying new potential genotyping targets that could be used for discriminating between S. Virchow strains in outbreak scenarios, we also aimed to carry out a comprehensive comparative analysis of the S. Virchow genomes. RESULTS: Genome comparisons between S. Virchow SVQ1 and S. Virchow SL491, a previously published strain, identified a high degree of genomic similarity between the two strains with fewer than 200 single nucleotide differences. Clustered Regularly Interspaced Palindromic Repeats (CRISPR) regions were identified as a highly variable region that could be used to discriminate between S. Virchow isolates. We amplified and sequenced the CRISPR regions of fifteen S. Virchow isolates collected from seven different outbreaks across Australia. We observed three allelic types of the CRISPR region from these isolates based on the presence/absence of the spacers and were able to discriminate S. Virchow phage type 8 isolates originating from different outbreaks. A comparison with 27 published Salmonella genomes found that the S. Virchow SVQ1 genome encodes 11 previously described Salmonella Pathogenicity Islands (SPI), as well as additional genomic islands including a remnant integrative conjugative element that is distinct from SPI-7. In addition, the S. Virchow genome possesses a novel prophage that encodes the Type III secretion system effector protein SopE, a key Salmonella virulence factor. The prophage shares very little similarity to the SopE prophages found in other Salmonella serovars suggesting an independent acquisition of sopE. CONCLUSIONS: The availability of this genome will serve as a genome template and facilitate further studies on understanding the virulence and global distribution of the S. Virchow serovar, as well as the development of genotyping methods for outbreak investigations.

A Novel Marine Mammal Coxiella burnetii-Genome Sequencing Identifies a New Genotype with Potential Virulence.

The obligate intracellular bacterial pathogen Coxiella burnetii has been identified in a few species of marine mammals, some of which are showing population declines. It has been hypothesized that C. burnetii in marine mammals is a distinct genotype that varies significantly from the typical terrestrial genotypes. It appears to lack an IS1111. Isolates originating from Australian marine animals have a distinctly non-Australian profile of multiple-locus variable-number tandem-repeat analysis (MLVA). Extracted Coxiella DNA of Australian fur seal placental origin was sequenced using the Novaseq platform. Illumina 150 bp paired-end reads were filtered and trimmed with Trimgalore. The microbial community present in the sequenced genome was evaluated with Kraken and Bracken software using the NCBI database. A phylogenetic analysis was performed using 1131 core genes. Core genes were identified using Panaroo and inputted into Iqtree to determine the maximum-likelihood tree. A second phylogenetic tree was created using Rickettsiella grylii and using seven housekeeping genes. Results were compared with the C. burnetii Nine Mile RSA439 virulent genome. This new Australian marine mammal isolate of Coxiella (PG457) appears to be a novel genotype that lacks IS1111 and has a distinct MLVA signature (ms26, ms27, ms28, ms30, and ms31). The presence of genes for multiple virulence factors appears to give this genotype sufficient pathogenicity for it to be considered a possible causative agent of abortion in Australian fur seals as well as a potential zoonotic risk.

Genome sequence of the emerging pathogen Aeromonas caviae.

Aeromonas caviae is a Gram-negative, motile and rod-shaped facultative anaerobe that is increasingly being recognized as a cause of diarrhea in children. Here we present the first genome sequence of an A. caviae strain that was isolated as the sole pathogen from a child with profuse diarrhea.

Whole genome sequencing based differentiation between re-infection and relapse in Indian patients with tuberculosis recurrence, with and without HIV co-infection.

INTRODUCTION: Differentiation between relapse and reinfection in cases with tuberculosis (TB) recurrence has important implications for public health, especially in patients with human immunodeficiency virus (HIV) co-infection. We compared Mycobacterial Interspersed Repeat Unit (MIRU) typing and spoligotyping with whole genome sequencing (WGS) to differentiate between relapse and reinfection in patients (HIV-positive and HIV-negative) with TB recurrence. We also assessed the value of WGS to track acquired drug resistance in those with relapse after successful treatment. METHOD: Forty-one paired M. tuberculosis isolates collected from 20 HIV-positive and 21 HIV-negative patients were subjected to WGS in addition to spoligotyping and MIRU typing. Phylogenetic and Single Nucleotide Substitution (SNP) clustering analyses were performed to determine whether recurrences were due to relapse or re-infection. RESULTS: Comparison of M. tuberculosis genomes indicated that 95% of TB recurrences in the HIV-negative cohort were due to relapse, while the majority of TB recurrences (75%) in the HIV-positive cohort was due to reinfection (P = 0.0001). New drug resistance mutations were acquired in 5/24 cases (20.8%) that experienced relapse. CONCLUSIONS: WGS provided increased resolution, but differentiation between relapse and reinfection was broadly consistent with MIRU and spoligotyping. The high contribution of reinfection among HIV infected patients experiencing TB recurrence warrants further study to explore risk factors for TB exposure.

Revealing COVID-19 transmission in Australia by SARS-CoV-2 genome sequencing and agent-based modeling.

In January 2020, a novel betacoronavirus (family Coronaviridae), named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was identified as the etiological agent of a cluster of pneumonia cases occurring in Wuhan City, Hubei Province, China1,2. The disease arising from SARS-CoV-2 infection, coronavirus disease 2019 (COVID-19), subsequently spread rapidly causing a worldwide pandemic. Here we examine the added value of near real-time genome sequencing of SARS-CoV-2 in a subpopulation of infected patients during the first 10 weeks of COVID-19 containment in Australia and compare findings from genomic surveillance with predictions of a computational agent-based model (ABM). Using the Australian census data, the ABM generates over 24 million software agents representing the population of Australia, each with demographic attributes of an anonymous individual. It then simulates transmission of the disease over time, spreading from specific infection sources, using contact rates of individuals within different social contexts. We report that the prospective sequencing of SARS-CoV-2 clarified the probable source of infection in cases where epidemiological links could not be determined, significantly decreased the proportion of COVID-19 cases with contentious links, documented genomically similar cases associated with concurrent transmission in several institutions and identified previously unsuspected links. Only a quarter of sequenced cases appeared to be locally acquired and were concordant with predictions from the ABM. These high-resolution genomic data are crucial to track cases with locally acquired COVID-19 and for timely recognition of independent importations once border restrictions are lifted and trade and travel resume.

Key Transitions in the Evolution of Rapid and Slow Growing Mycobacteria Identified by Comparative Genomics.

Mycobacteria have been classified into rapid and slow growing phenotypes, but the genetic factors that underlie these growth rate differences are not well understood. We compared the genomes of 157 mycobacterial species, representing all major branches of the mycobacterial phylogenetic tree to identify genes and operons enriched among rapid and slow growing mycobacteria. Overlaying growth phenotype on a phylogenetic tree based on 304 core genes suggested that ancestral mycobacteria had a rapid growth phenotype with a single major evolutionary separation into rapid and slow growing sub-genera. We identified 293 genes enriched among rapid growing sub-genera, including genes encoding for amino acid transport/metabolism (e.g., livFGMH operon) and transcription, as well as novel ABC transporters. Loss of the livFGMH and ABC transporter operons among slow growing species suggests that reduced cellular amino acid transport may be growth limiting. Comparative genomic analysis suggests that horizontal gene transfer, from non-mycobacterial genera, may have contributed to niche adaptation and pathogenicity, especially among slow growing species. Interestingly, the mammalian cell entry (mce) operon was found to be ubiquitous, irrespective of growth phenotype or pathogenicity, although protein sequence homology between rapid and slow growing species was low (<50%). This suggests that the mce operon was present in ancestral rapid growing species, but later adapted by slow growing species for use as a mechanism to establish an intra-cellular lifestyle.

Advances in Clinical Sample Preparation for Identification and Characterization of Bacterial Pathogens Using Metagenomics.

Whole genome sequencing (WGS) plays an increasing role in communicable disease control through high-resolution outbreak tracing, laboratory surveillance and diagnostics. However, WGS has traditionally relied on microbial culture in order to obtain pathogen specific DNA for sequencing. This has severely limited the application of whole genome sequencing on pathogens with fastidious culturing requirements. In addition, the widespread adoption of culture-independent diagnostic tests has reduced availability of cultured isolates for confirmatory testing and surveillance. These recent developments have created demand for the implementation of techniques enabling direct sequencing of microbial genomes in clinical samples without having to culture an isolate. However, sequencing of specific organisms from clinical samples can be affected by high levels of contaminating DNA from the host and other commensal microorganisms. Several methods have been introduced for selective lysis of host cells and/or separate specific organisms from a clinical sample. This review examines the different approaches for sample preparation that have been used in diagnostic and public health laboratories for metagenomic sequencing.

Genome-wide analysis of Streptococcus pneumoniae serogroup 19 in the decade after the introduction of pneumococcal conjugate vaccines in Australia.

The decline in invasive pneumococcal disease (IPD), following the introduction of the 7-valent pneumococcal conjugate vaccination (PCV-7), was tempered by emergence of non-vaccine serotypes, particularly 19A. In Australia, three years after PCV-7 was replaced by PCV-13, containing 19A and 19F antigens, serogroup 19 was still a prominent cause of IPD in children under five. In this study we examined the evolution of serogroup 19 before and after introduction of paediatric vaccines in New South Wales (NSW), Australia. Genomes of 124 serogroup 19 IPD isolates collected before (2004) and after introduction of PCV-7 (2008) and PCV-13 (2014), from children under five in NSW, were analysed. Eleven core genome sequence clusters (cgSC) and 35 multilocus sequence types (ST) were identified. The majority (78/124) of the isolates belonged to four cgSCs: cgSC7 (ST199), cgSC11 (ST320), cgSC8 (ST63) and cgSC9 (ST2345). ST63 and ST2345 were exclusively serotype 19A and accounted for its predominantly intermediate penicillin resistance; these two clusters first appeared in 2008 and largely disappeared after introduction of PCV-13. Serogroup 19 was responsible for the highest proportion of vaccine failures in NSW. Relatively low immunogenicity of serogroup 19 antigens and Australia's three-dose vaccine schedule could affect the population dynamics of this serogroup.

Molecular characterisation of the Chlamydia pecorum plasmid from porcine, ovine, bovine, and koala strains indicates plasmid-strain co-evolution.

Background. Highly stable, evolutionarily conserved, small, non-integrative plasmids are commonly found in members of the Chlamydiaceae and, in some species, these plasmids have been strongly linked to virulence. To date, evidence for such a plasmid in Chlamydia pecorum has been ambiguous. In a recent comparative genomic study of porcine, ovine, bovine, and koala C. pecorum isolates, we identified plasmids (pCpec) in a pig and three koala strains, respectively. Screening of further porcine, ovine, bovine, and koala C. pecorum isolates for pCpec showed that pCpec is common, but not ubiquitous in C. pecorum from all of the infected hosts. Methods. We used a combination of (i) bioinformatic mining of previously sequenced C. pecorum genome data sets and (ii) pCpec PCR-amplicon sequencing to characterise a further 17 novel pCpecs in C. pecorum isolates obtained from livestock, including pigs, sheep, and cattle, as well as those from koala. Results and Discussion. This analysis revealed that pCpec is conserved with all eight coding domain sequences (CDSs) present in isolates from each of the hosts studied. Sequence alignments revealed that the 21 pCpecs show 99% nucleotide sequence identity, with 83 single nucleotide polymorphisms (SNPs) shown to differentiate all of the plasmids analysed in this study. SNPs were found to be mostly synonymous and were distributed evenly across all eight pCpec CDSs as well as in the intergenic regions. Although conserved, analyses of the 21 pCpec sequences resolved plasmids into 12 distinct genotypes, with five shared between pCpecs from different isolates, and the remaining seven genotypes being unique to a single pCpec. Phylogenetic analysis revealed congruency and co-evolution of pCpecs with their cognate chromosome, further supporting polyphyletic origin of the koala C. pecorum. This study provides further understanding of the complex epidemiology of this pathogen in livestock and koala hosts and paves the way for studies to evaluate the function of this putative C. pecorum virulence factor.

Australian human and parrot Chlamydia psittaci strains cluster within the highly virulent 6BC clade of this important zoonotic pathogen.

Chlamydia psittaci is an avian pathogen and zoonotic agent of atypical pneumonia. The most pathogenic C. psittaci strains cluster into the 6BC clade, predicted to have recently emerged globally. Exposure to infected parrots is a risk factor with limited evidence also of an indirect exposure risk. Genome sequencing was performed on six Australian human and a single avian C. psittaci strain isolated over a 9 year period. Only one of the five human patients had explicit psittacine contact. Genomics analyses revealed that the Australian C. psittaci strains are remarkably similar, clustering tightly within the C. psittaci 6BC clade suggested to have been disseminated by South America parrot importation. Molecular clock analysis using the newly sequenced C. psittaci genomes predicted the emergence of the 6BC clade occurring approximately 2,000 years ago. These findings reveal the potential for an Australian natural reservoir of C. psittaci 6BC strains. These strains can also be isolated from seriously ill patients without explicit psittacine contact. The apparent recent and global spread of C. psittaci 6BC strains raises important questions over how this happened. Further studies may reveal whether the dissemination of this important zoonotic pathogen is linked to Australian parrot importation rather than parrots from elsewhere.

Genomic Comparison of Translocating and Non-Translocating Escherichia coli.

Translocation of E. coli across the gut epithelium can result in fatal sepsis in post-surgical patients. In vitro and in vivo experiments have identified the existence of a novel pathotype of translocating E. coli (TEC) that employs an unknown mechanism for translocating across epithelial cells to the mesenteric lymph nodes and the blood stream in both humans and animal models. In this study the genomes of four TEC strains isolated from the mesenteric lymph nodes of a fatal case of hospitalised patient (HMLN-1), blood of pigs after experimental shock (PC-1) and after non-lethal haemorrhage in rats (KIC-1 and KIC-2) were sequenced in order to identify the genes associated with their adhesion and/or translocation. To facilitate the comparison, the genomes of a non-adhering, non-translocating E. coli (46-4) and adhering but non-translocating E. coli (73-89) were also sequenced and compared. Whole genome comparison revealed that three (HMLN-1, PC-1 and KIC-2) of the four TEC strains carried a genomic island that encodes a Type 6 Secretion System that may contribute to adhesion of the bacteria to gut epithelial cells. The human TEC strain HMLN-1 also carried the invasion ibeA gene, which was absent in the animal TEC strains and is likely to be associated with host-specific translocation. Phylogenetic analysis revealed that the four TEC strains were distributed amongst three distinct E. coli phylogroups, which was supported by the presence of phylogroup specific fimbriae gene clusters. The genomic comparison has identified potential genes that can be targeted with knock-out experiments to further characterise the mechanisms of E. coli translocation.

Chlamydia genomics: providing novel insights into chlamydial biology.

Chlamydiaceae are obligate intracellular pathogens that have successfully evolved to colonize a diverse range of hosts. There are currently 11 described species of Chlamydia, most of which have a significant impact on the health of humans or animals. Expanding chlamydial genome sequence information has revolutionized our understanding of chlamydial biology, including aspects of their unique lifecycle, host-pathogen interactions, and genetic differences between Chlamydia strains associated with different host and tissue tropisms. This review summarizes the major highlights of chlamydial genomics and reflects on the considerable impact these have had on understanding the biology of chlamydial pathogens and the changing nature of genomics tools in the 'post-genomics' era.