Phylogenomic analysis of a global collection of Escherichia coli ST38: evidence of interspecies and environmental transmission?

IMPORTANCE: Extraintestinal pathogenic Escherichia coli (ExPEC) sequence type (ST) 38 is one of the top 10 human pandemic lineages. Although a major cause of urinary tract and blood stream infections, ST38 has been poorly characterized from a global phylogenomic perspective. A comprehensive genome-scale analysis of 925 ST38 isolate genomes identified two broad ancestral clades and linkage of discrete ST38 clusters with specific bla CTX-M variants. In addition, the clades and clusters carry important virulence genes, with diverse but poorly characterized plasmids. Numerous putative interhost and environment transmission events were identified here by the presence of ST38 clones (defined as isolates with ≤35 SNPs) within humans, companion animals, food sources, urban birds, wildlife, and the environment. A small cluster of international ST38 clones from diverse sources, likely representing progenitors of a hospital outbreak that occurred in Brisbane, Australia, in 2017, was also identified. Our study emphasizes the importance of characterizing isolate genomes derived from nonhuman sources and geographical locations, without any selection bias.

Genome organization and genomics in Chlamydia: whole genome sequencing increases understanding of chlamydial virulence, evolution, and phylogeny.

The genus Chlamydia contains important obligate intracellular bacterial pathogens to humans and animals, including C. trachomatis and C. pneumoniae. Since 1998, when the first Chlamydia genome was published, our understanding of how these microbes interact, evolved and adapted to different intracellular host environments has been transformed due to the expansion of chlamydial genomes. This review explores the current state of knowledge in Chlamydia genomics and how whole genome sequencing has revolutionised our understanding of Chlamydia virulence, evolution, and phylogeny over the past two and a half decades. This review will also highlight developments in multi-omics and other approaches that have complemented whole genome sequencing to advance knowledge of Chlamydia pathogenesis and future directions for chlamydial genomics.

Phylogenomics of two ST1 antibiotic-susceptible non-clinical Acinetobacter baumannii strains reveals multiple lineages and complex evolutionary history in global clone 1.

Acinetobacter baumannii is an opportunistic pathogen that is difficult to treat due to its resistance to extreme conditions, including desiccation and antibiotics. Most strains causing outbreaks around the world belong to two main global lineages, namely global clones 1 and 2 (GC1 and GC2). Here, we used a combination of Illumina short read and MinION (Oxford Nanopore) long-read sequence data with a hybrid assembly approach to complete the genome sequence of two antibiotic-sensitive GC1 strains, Ex003 and Ax270, recovered in Lebanon from water and a rectal swab of a cat, respectively. Phylogenetic analysis of Ax270 and Ex003 with 186 publicly available GC1 genomes revealed two major clades, including five main lineages (L1-L5), and four single-isolate lineages outside of the two clades. Ax270 and Ex003, along with AB307-0294 and MRSN7213 (both predicted antibiotic-susceptible isolates) represent these individual lineages. Antibiotic resistance islands and transposons interrupting the comM gene remain important features in L1-L5, with L1 associated with the AbaR-type resistance islands, L2 with AbaR4, L3 strains containing either AbaR4 or its variants as well as Tn6022::ISAba42, and L4 and L5 associated with Tn6022 or its variants. Analysis of the capsule (KL) and outer core (OCL) polysaccharide loci further revealed a complex evolutionary history probably involving many recombination events. As more genomes become available, more GC1 lineages continue to emerge. However, genome sequence data from more diverse geographical regions are needed to draw a more accurate population structure of this globally distributed clone.

Two carbapenem-resistant ST1:ST231:KL1:OCL1 Acinetobacter baumannii strains recovered in Tehran, Iran, carry AbaR31 in the chromosome and AbaR4 and TnaphA6 in a RepAci6 plasmid.

OBJECTIVES: To analyse the context of genes conferring antibiotic resistance in two carbapenem-resistant Acinetobacter baumannii isolates recovered in Tehran, Iran. METHODS: The antibiotic resistance phenotype for 28 antibiotics was determined using disc diffusion. The whole genome sequences of ABH008 and ABS200 were determined using the Illumina HiSeq X Ten platform. Resistance genes were identified using ResFinder and multilocus sequence types were determined using the Oxford and Institut Pasteur schemes. RESULTS: Isolates ABH008 and ABS200, recovered in 2012 and 2013, respectively, in two different Tehran hospitals, belong to the common global clone 1 lineage, ST1IP and ST231OX. They are resistant to sulfamethoxazole, tetracycline, gentamicin, amikacin, third-generation cephalosporins and carbapenems. Despite being isolated in different hospitals, phylogenetic analysis indicated they are closely related. Consistent with this, both isolates carry catA1, sul1, aacC1 and aadA1 in a novel variant of the AbaR3-type resistance island, named AbaR31. Both isolates are resistant to amikacin and carbapenems owing to aphA6 and oxa23, respectively. The oxa23 gene is located in the AbaR4 resistance island, and aphA6 in TnaphA6, and both mobile elements are in an ∼90 kbp plasmid encoding the putative RepAci6 replication initiation protein. Resistance to third-generation cephalosporins is due to the acquisition by homologous recombination of a 5 kb DNA segment that contains ISAba1-ampC from a ST623 strain. CONCLUSIONS: The resistance gene complements of ABH008 and ABS200 were found in AbaR31 and a plasmid that encodes RepAci6. The close genetic relationship of ABH008 and ABS200, despite each being recovered from different hospitals, indicates transmission between the two hospitals.

Dual RNA-seq analysis of in vitro infection multiplicity and RNA depletion methods in Chlamydia-infected epithelial cells.

Dual RNA-seq experiments examining viral and bacterial pathogens are increasing, but vary considerably in their experimental designs, such as infection rates and RNA depletion methods. Here, we have applied dual RNA-seq to Chlamydia trachomatis infected epithelial cells to examine transcriptomic responses from both organisms. We compared two time points post infection (1 and 24 h), three multiplicity of infection (MOI) ratios (0.1, 1 and 10) and two RNA depletion methods (rRNA and polyA). Capture of bacterial-specific RNA were greatest when combining rRNA and polyA depletion, and when using a higher MOI. However, under these conditions, host RNA capture was negatively impacted. Although it is tempting to use high infection rates, the implications on host cell survival, the potential reduced length of infection cycles and real world applicability should be considered. This data highlights the delicate nature of balancing host-pathogen RNA capture and will assist future transcriptomic-based studies to achieve more specific and relevant infection-related biological insights.

Analysis of Complete Genome Sequence of Acinetobacter baumannii Strain ATCC 19606 Reveals Novel Mobile Genetic Elements and Novel Prophage.

Acinetobacter baumannii isolate ATCC 19606 was recovered in the US prior to 1948. It has been used as a reference and model organism in many studies involving antibiotic resistance and pathogenesis of A. baumannii, while, until recently, a complete genome of this strain was not available. Here, we present an analysis of the complete 3.91-Mbp genome sequence, generated via a combination of short-read sequencing (Illumina) and long-read sequencing (MinION), and show it contains two small cryptic plasmids and a novel complete prophage of size 41.2 kb. We also characterised several regions of the ATCC 19606 genome, leading to the identification of a novel cadmium/mercury transposon, which was named Tn6551. ATCC 19606 is an antibiotic-sensitive strain, but a comparative analysis of all publicly available ST52 strains predicts a resistance to modern antibiotics by the accumulation of antibiotic-resistance genes via plasmids in recent isolates that belong to this sequence type.

Chromatin accessibility dynamics of Chlamydia-infected epithelial cells.

Chlamydia are Gram-negative, obligate intracellular bacterial pathogens responsible for a broad spectrum of human and animal diseases. In humans, Chlamydia trachomatis is the most prevalent bacterial sexually transmitted infection worldwide and is the causative agent of trachoma (infectious blindness) in disadvantaged populations. Over the course of its developmental cycle, Chlamydia extensively remodels its intracellular niche and parasitises the host cell for nutrients, with substantial resulting changes to the host cell transcriptome and proteome. However, little information is available on the impact of chlamydial infection on the host cell epigenome and global gene regulation. Regions of open eukaryotic chromatin correspond to nucleosome-depleted regions, which in turn are associated with regulatory functions and transcription factor binding. We applied formaldehyde-assisted isolation of regulatory elements enrichment followed by sequencing (FAIRE-Seq) to generate temporal chromatin maps of C. trachomatis-infected human epithelial cells in vitro over the chlamydial developmental cycle. We detected both conserved and distinct temporal changes to genome-wide chromatin accessibility associated with C. trachomatis infection. The observed differentially accessible chromatin regions include temporally-enriched sets of transcription factors, which may help shape the host cell response to infection. These regions and motifs were linked to genomic features and genes associated with immune responses, re-direction of host cell nutrients, intracellular signalling, cell-cell adhesion, extracellular matrix, metabolism and apoptosis. This work provides another perspective to the complex response to chlamydial infection, and will inform further studies of transcriptional regulation and the epigenome in Chlamydia-infected human cells and tissues.

Escherichia coli ST8196 is a novel, locally evolved, and extensively drug resistant pathogenic lineage within the ST131 clonal complex.

The H30Rx subclade of Escherichia coli ST131 is a clinically important, globally dispersed pathogenic lineage that typically displays resistance to fluoroquinolones and extended spectrum ß-lactams. Isolates EC233 and EC234, variants of ST131-H30Rx with a novel sequence type (ST) 8196, isolated from unrelated patients presenting with bacteraemia at a Sydney Hospital in 2014 are characterised here. EC233 and EC234 are phylogroup B2, serotype O25:H4A, and resistant to ampicillin, amoxicillin, cefoxitin, ceftazidime, ceftriaxone, ciprofloxacin, norfloxacin and gentamicin and are likely clonal. Both harbour an IncFII_2 plasmid (pSPRC_Ec234-FII) that carries most of the resistance genes on an IS26 associated translocatable unit, two small plasmids and a novel IncI1 plasmid (pSPRC_Ec234-I). SNP-based phylogenetic analysis of the core genome of representatives within the ST131 clonal complex places both isolates in a subclade with three clinical Australian ST131-H30Rx clade-C isolates. A MrBayes phylogeny analysis of EC233 and EC234 indicates ST8196 share a most recent common ancestor with ST131-H30Rx strain EC70 isolated from the same hospital in 2013. Our study identified genomic hallmarks that define the ST131-H30Rx subclade in the ST8196 isolates and highlights a need for unbiased genomic surveillance approaches to identify novel high-risk MDR E. coli pathogens that impact healthcare facilities.

An Integrated Proteomic and Transcriptomic Analysis Reveals the Venom Complexity of the Bullet Ant Paraponera clavata.

A critical hurdle in ant venom proteomic investigations is the lack of databases to comprehensively and specifically identify the sequence and function of venom proteins and peptides. To resolve this, we used venom gland transcriptomics to generate a sequence database that was used to assign the tandem mass spectrometry (MS) fragmentation spectra of venom peptides and proteins to specific transcripts. This was performed alongside a shotgun liquid chromatography-mass spectrometry (LC-MS/MS) analysis of the venom to confirm that these assigned transcripts were expressed as proteins. Through the combined transcriptomic and proteomic investigation of Paraponera clavata venom, we identified four times the number of proteins previously identified using 2D-PAGE alone. In addition to this, by mining the transcriptomic data, we identified several novel peptide sequences for future pharmacological investigations, some of which conform with inhibitor cysteine knot motifs. These types of peptides have the potential to be developed into pharmaceutical or bioinsecticide peptides.

Genomic profiling of Escherichia coli isolates from bacteraemia patients: a 3-year cohort study of isolates collected at a Sydney teaching hospital.

This study sought to assess the genetic variability of Escherichia coli isolated from bloodstream infections (BSIs) presenting at Concord Hospital, Sydney during 2013-2016. Whole-genome sequencing was used to characterize 81 E. coli isolates sourced from community-onset (CO) and hospital-onset (HO) BSIs. The cohort comprised 64 CO and 17 HO isolates, including 35 multidrug-resistant (MDR) isolates exhibiting phenotypic resistance to three or more antibiotic classes. Phylogenetic analysis identified two major ancestral clades. One was genetically diverse with 25 isolates distributed in 16 different sequence types (STs) representing phylogroups A, B1, B2, C and F, while the other comprised phylogroup B2 isolates in subclades representing the ST131, ST73 and ST95 lineages. Forty-seven isolates contained a class 1 integron, of which 14 carried blaCTX -M-gene. Isolates with a class 1 integron carried more antibiotic resistance genes than isolates without an integron and, in most instances, resistance genes were localized within complex resistance loci (CRL). Resistance to fluoroquinolones could be attributed to point mutations in chromosomal parC and gyrB genes and, in addition, two isolates carried a plasmid-associated qnrB4 gene. Co-resistance to fluoroquinolone and broad-spectrum beta-lactam antibiotics was associated with ST131 (HO and CO), ST38 (HO), ST393 (CO), ST2003 (CO) and ST8196 (CO and HO), a novel ST identified in this study. Notably, 10/81 (12.3 %) isolates with ST95 (5 isolates), ST131 (2 isolates), ST88 (2 isolates) and a ST540 likely carry IncFII-IncFIB plasmid replicons with a full spectrum of virulence genes consistent with the carriage of ColV-like plasmids. Our data indicate that IncF plasmids play an important role in shaping virulence and resistance gene carriage in BSI E. coli in Australia.

Early Transcriptional Landscapes of Chlamydia trachomatis-Infected Epithelial Cells at Single Cell Resolution.

Chlamydia are Gram-negative obligate intracellular bacterial pathogens responsible for a variety of disease in humans and animals worldwide. Chlamydia trachomatis causes trachoma in disadvantaged populations, and is the most common bacterial sexually transmitted infection in humans, causing reproductive tract disease. Antibiotic therapy successfully treats diagnosed chlamydial infections, however asymptomatic infections are common. High-throughput transcriptomic approaches have explored chlamydial gene expression and infected host cell gene expression. However, these were performed on large cell populations, averaging gene expression profiles across all cells sampled and potentially obscuring biologically relevant subsets of cells. We generated a pilot dataset, applying single cell RNA-Seq (scRNA-Seq) to C. trachomatis infected and mock-infected epithelial cells to assess the utility, pitfalls and challenges of single cell approaches applied to chlamydial biology, and to potentially identify early host cell biomarkers of chlamydial infection. Two hundred sixty-four time-matched C. trachomatis-infected and mock-infected HEp-2 cells were collected and subjected to scRNA-Seq. After quality control, 200 cells were retained for analysis. Two distinct clusters distinguished 3-h cells from 6- and 12-h. Pseudotime analysis identified a possible infection-specific cellular trajectory for Chlamydia-infected cells, while differential expression analyses found temporal expression of metallothioneins and genes involved with cell cycle regulation, innate immune responses, cytoskeletal components, lipid biosynthesis and cellular stress. We find that changes to the host cell transcriptome at early times of C. trachomatis infection are readily discernible by scRNA-Seq, supporting the utility of single cell approaches to identify host cell biomarkers of chlamydial infection, and to further deconvolute the complex host response to infection.

Dual RNA-Seq of Chlamydia and Host Cells.

During the infection of a host cell by a bacterial pathogen, a cascading series of gene expression changes occurs as each organism manipulates or responds to the other via defense or survival strategies. Unraveling this complex interplay is key for our understanding of bacterial virulence and host response pathways for the development of novel therapeutics. Dual RNA sequencing (dual RNA-Seq) has recently been developed to simultaneously capture host and bacterial transcriptomes from an infected cell. Leveraging the sensitivity and resolution allowed by RNA-seq, dual RNA-Seq can be applied to any bacteria-eukaryotic host interaction. We pioneered dual RNA-Seq to simultaneously capture Chlamydia and host expression profiles during an in vitro infection as proof of principle. Here we provide a detailed laboratory protocol and bioinformatics analysis guidelines for dual RNA-seq experiments focusing on Chlamydia as the organism of interest.

Bioinformatic analysis of bacteria and host cell dual RNA-sequencing experiments.

Bacterial pathogens subvert host cells by manipulating cellular pathways for survival and replication; in turn, host cells respond to the invading pathogen through cascading changes in gene expression. Deciphering these complex temporal and spatial dynamics to identify novel bacterial virulence factors or host response pathways is crucial for improved diagnostics and therapeutics. Dual RNA sequencing (dRNA-Seq) has recently been developed to simultaneously capture host and bacterial transcriptomes from an infected cell. This approach builds on the high sensitivity and resolution of RNA sequencing technology and is applicable to any bacteria that interact with eukaryotic cells, encompassing parasitic, commensal or mutualistic lifestyles. Several laboratory protocols have been presented that outline the collection, extraction and sequencing of total RNA for dRNA-Seq experiments, but there is relatively little guidance available for the detailed bioinformatic analyses required. This protocol outlines a typical dRNA-Seq experiment, based on a Chlamydia trachomatis-infected host cell, with a detailed description of the necessary bioinformatic analyses with currently available software tools.

A Laboratory Methodology for Dual RNA-Sequencing of Bacteria and their Host Cells In Vitro.

Dual RNA-Sequencing leverages established next-generation sequencing (NGS)-enabled RNA-Seq approaches to measure genome-wide transcriptional changes of both an infecting bacteria and host cells. By simultaneously investigating both organisms from the same biological sample, dual RNA-Seq can provide unique insight into bacterial infection processes and reciprocal host responses at once. However, the difficulties involved in handling both prokaryotic and eukaryotic material require distinct, optimized procedures. We previously developed and applied dual RNA-Seq to measure prokaryotic and eukaryotic expression profiles of human cells infected with bacteria, using in vitro Chlamydia-infected epithelial cells as proof of principle. Here we provide a detailed laboratory protocol for in vitro dual RNA-Seq that is readily adaptable to any host-bacteria system of interest.

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.

The Number, Organization, and Size of Polymorphic Membrane Protein Coding Sequences as well as the Most Conserved Pmp Protein Differ within and across Chlamydia Species.

Variation is a central trait of the polymorphic membrane protein (Pmp) family. The number of pmp coding sequences differs between Chlamydia species, but it is unknown whether the number of pmp coding sequences is constant within a Chlamydia species. The level of conservation of the Pmp proteins has previously only been determined for Chlamydia trachomatis. As different Pmp proteins might be indispensible for the pathogenesis of different Chlamydia species, this study investigated the conservation of Pmp proteins both within and across C. trachomatis,C. pneumoniae,C. abortus, and C. psittaci. The pmp coding sequences were annotated in 16 C. trachomatis, 6 C. pneumoniae, 2 C. abortus, and 16 C. psittaci genomes. The number and organization of polymorphic membrane coding sequences differed within and across the analyzed Chlamydia species. The length of coding sequences of pmpA,pmpB, and pmpH was conserved among all analyzed genomes, while the length of pmpE/F and pmpG, and remarkably also of the subtype pmpD, differed among the analyzed genomes. PmpD, PmpA, PmpH, and PmpA were the most conserved Pmp in C. trachomatis,C. pneumoniae,C. abortus, and C. psittaci, respectively. PmpB was the most conserved Pmp across the 4 analyzed Chlamydia species.

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.

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.

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.

Chlamydia caviae infection alters abundance but not composition of the guinea pig vaginal microbiota.

In humans, the vaginal microbiota is thought to be the first line of defense again pathogens including Chlamydia trachomatis. The guinea pig has been extensively used as a model to study chlamydial infection because it shares anatomical and physiological similarities with humans, such as a squamous vaginal epithelium as well as some of the long-term outcomes caused by chlamydial infection. In this study, we aimed to evaluate the guinea pig-C. caviae model of genital infection as a surrogate for studying the role of the vaginal microbiota in the early steps of C. trachomatis infection in humans. We used culture-independent molecular methods to characterize the relative and absolute abundance of bacterial phylotypes in the guinea pig vaginal microbiota in animals non-infected, mock-infected or infected by C. caviae. We showed that the guinea pig and human vaginal microbiotas are of different bacterial composition and abundance. Chlamydia caviae infection had a profound effect on the absolute abundance of bacterial phylotypes but not on the composition of the guinea pig vaginal microbiota. Our findings compromise the validity of the guinea pig-C. caviae model to study the role of the vaginal microbiota during the early steps of sexually transmitted infection.