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Chlamydia trachomatis is the world's most prevalent bacterial sexually transmitted infection and leading infectious cause of blindness, yet it is one of the least understood human pathogens, in part due to the difficulties of in vitro culturing and the lack of available tools for genetic manipulation. Genome sequencing has reinvigorated this field, shedding light on the contemporary history of this pathogen. Here, we analyze 563 full genomes, 455 of which are novel, to show that the history of the species comprises two phases, and conclude that the currently circulating lineages are the result of evolution in different genomic ecotypes. Temporal analysis indicates these lineages have recently expanded in the space of thousands of years, rather than the millions of years as previously thought, a finding that dramatically changes our understanding of this pathogen's history. Finally, at a time when almost every pathogen is becoming increasingly resistant to antimicrobials, we show that there is no evidence of circulating genomic resistance in C. trachomatis., Funding agencies:Sanger Institute through the Wellcome Trust 098051 Australian National Health and Medical Research Council 1060768

A recent surveillance and outbreak report published in Eurosurveillance by Petrovay et al. on the ‘Emergence of the lymphogranuloma venereum L2c genovariant, Hungary, 2012 to 2016’ [1] provides an observation of the first European cases of a genotype of Chlamydia trachomatis associated with severe haemorrhagic proctitis. The authors of this paper diagnosed the strains as lymphogranuloma venereum (LGV)- associated and performed partial sequencing of the ompA gene (ca 1,070 bp), which is a standard typing method for C. trachomatis. The ompA gene sequence obtained was compared with those from reference isolates, and reported to be 100% concordant with the ompA sequence belonging to an L2-D recombinant strain described in 2011 [2]. This strain was named ‘L2c’, as it was found to possess a chimeric genome, not because it has a novel ompA-genotype. We would like to point out that the ompA gene sequence of this L2-D recombinant strain, and by implication those of the Hungarian isolates, is identical to that of archetypal L2 strains, for example the reference strain L2/434 [3].

Chlamydia suis is an endemic pig pathogen, belonging to a fascinating genus of obligate intracellular pathogens. Of particular interest, this is the only chlamydial species to have naturally acquired genes encoding for tetracycline resistance. To date, the distribution and mobility of the Tet-island is not well understood. Our study focused on whole genome sequencing of 29 C. suis isolates from a recent porcine cohort within Switzerland, combined with data from USA tetracycline-resistant isolates. Our findings show that the genome of C. suis is very plastic, with unprecedented diversity, highly affected by recombination and plasmid exchange. A large diversity of isolates circulates within Europe, even within individual Swiss farms, suggesting that C. suis originated around Europe. New World isolates have more restricted diversity and appear to derive from European isolates, indicating that historical strain transfers to the USA have occurred. The architecture of the Tet-island is variable, but the tetA(C) gene is always intact, and recombination has been a major factor in its transmission within C. suis. Selective pressure from tetracycline use within pigs leads to a higher number of Tet-island carrying isolates, which appear to be lost in the absence of such pressure, whereas the loss or gain of the Tet-island from individual strains is not observed. The Tet-island appears to be a recent import into the genome of C. suis, with a possible American origin.

The Planctomycetes-Verrucomicrobia-Chlamydiae (PVC) bacterial superphylum constitutes a broad range of organisms with an intriguing array of ultrastructural morphologies, including intracellular membranes and compartments and their corresponding complex genomes encoding these forms. The phylum Chlamydiae are all obligate intracellular bacteria and, although much is already known of their genomes from various families and how these regulate the various morphological forms, we know remarkably little about what is likely the deepest rooting clade of this phylum, which has only been found to contain pathogens of marine and fresh water vertebrates. The disease they are associated with is called epitheliocystis; however, analyses of the causative agents is hindered by an inability to cultivate them for refined in vitro experimentation. For this reason, we have developed tools to analyse both the genomes and the ultrastructures of bacteria causing this disease, directly from infected tissues. Here we present structural data for a member of the family Ca. Similichlamydiaceae from this deep-rooted clade, which we have identified using molecular tools, in epitheliocystis lesions of gilthead seabream (Sparus aurata) in Greece. We present evidence that the chlamydial inclusions appear to develop in a perinuclear location, similar to other members of the phylum and that a chlamydial developmental cycle is present, with chlamydial forms similar to reticular bodies (RBs) and elementary bodies (EBs) detected. Division of the RBs appeared to follow a budding process, and larger RBs with multiple condensed nucleoids were detected using both transmission electron microscopy (TEM) and by focused-ion beam, scanning electron microscopy (FIB-SEM). As model hosts, fish offer many advantages for investigation, and we hope by these efforts to encourage others to explore the biology of fish pathogens from the PVC.

In pigs, Chlamydia suis has been associated with respiratory disease, diarrhea and conjunctivitis, but there is a high rate of inapparent C. suis infection found in the gastrointestinal tract of pigs. Tetracycline resistance in C. suis has been described in the USA, Italy, Switzerland, Belgium, Cyprus and Israel. Tetracyclines are commonly used in pig production due to their broad-spectrum activity and relatively low cost. The aim of this study was to isolate clinical C. suis samples in cell culture and to evaluate their antibiotic susceptibility in vitro under consideration of antibiotic treatment on herd level. Swab samples (n = 158) identified as C. suis originating from 24 farms were further processed for isolation, which was successful in 71% of attempts with a significantly higher success rate from fecal swabs compared to conjunctival swabs. The farms were divided into three treatment groups: A) farms without antibiotic treatment, B) farms with prophylactic oral antibiotic treatment of the whole herd consisting of trimethoprime, sulfadimidin and sulfathiazole (TSS), or C) farms giving herd treatment with chlortetracycline with or without tylosin and sulfadimidin (CTS). 59 isolates and their corresponding clinical samples were selected and tested for the presence or absence of the tetracycline resistance class C gene [tet(C)] by conventional PCR and isolates were further investigated for their antibiotic susceptibility in vitro. The phenotype of the investigated isolates was either classified as tetracycline sensitive (Minimum inhibitory concentration [MIC] < 2 μg/ml), intermediate (2 μg/ml ≤ MIC < 4 μg/ml) or resistant (MIC ≥ 4 μg/ml). Results of groups and individual pigs were correlated with antibiotic treatment and time of sampling (beginning/end of the fattening period). We found clear evidence for selective pressure as absence of antibiotics led to isolation of only tetracycline sensitive or intermediate strains whereas tetracycline treatment resulted in

Obligate intracellular chlamydial bacteria of the Planctomycetes-Verrucomicrobia-Chlamydiae (PVC) superphylum are important pathogens of terrestrial and marine vertebrates, yet many features of their pathogenesis and host specificity are still unknown. This is particularly true for families such as the Waddliacea which, in addition to epithelia, cellular targets for nearly all Chlamydia, can infect and replicate in macrophages, an important arm of the innate immune system or in their free-living amoebal counterparts. An ideal pathogen model system should include both host and pathogen, which led us to develop the first larval zebrafish model for chlamydial infections with Waddlia chondrophila. By varying the means and sites of application, epithelial cells of the swim bladder, endothelial cells of the vasculature and phagocytosing cells of the innate immune system became preferred targets for infection in zebrafish larvae. Through the use of transgenic zebrafish, we could observe recruitment of neutrophils to the infection site and demonstrate for the first time that W. chondrophila is taken up and replicates in these phagocytic cells and not only in macrophages. Furthermore, we present evidence that myeloid differentiation factor 88 (MyD88) mediated signaling plays a role in the innate immune reaction to W. chondrophila, eventually by Toll-like receptor (TLRs) recognition. Infected larvae with depleted levels of MyD88 showed a higher infection load and a lower survival rate compared to control fish. This work presents a new and potentially powerful non-mammalian experimental model to study the pathology of chlamydial virulence in vivo and opens up new possibilities for investigation of other members of the PVC superphylum.

Aquaculture is a burgeoning industry, requiring diversification into new farmed species, which are often at risk from infectious disease. We used a mesocosm technique to investigate the susceptibility of sharpsnout seabream (Diplodus puntazzo) larvae to potential environmental pathogens in seawater compared to control borehole water. Fish exposed to seawater succumbed to epitheliocystis from 21 days post hatching, causing mortality in a quarter of the hosts. The pathogen responsible was not chlamydial, as is often found in epitheliocystis, but a novel species of the γ-proteobacterial genus Endozoicomonas. Detailed characterisation of this pathogen within the infectious lesions using high resolution fluorescent and electron microscopy showed densely packed rod shaped bacteria. A draft genome sequence of this uncultured bacterium was obtained from preserved material. Comparison with the genome of the Endozoicomonas elysicola type strain shows that the genome of Ca. Endozoicomonas cretensis is undergoing decay through loss of functional genes and insertion sequence expansion, often indicative of adaptation to a new niche or restriction to an alternative lifestyle. These results demonstrate the advantage of mesocosm studies for investigating the effect of environmental bacteria on susceptible hosts and provide an important insight into the genome dynamics of a novel fish pathogen.

Many bacterial pathogens are specialized, infecting one or few hosts, and this is often associated with more acute disease presentation. Specific genomes show markers of this specialization, which often reflect a balance between gene acquisition and functional gene loss. Within Salmonella enterica subspecies enterica, a single lineage exists that includes human and animal pathogens adapted to cause infection in different hosts, including S. enterica serovar Enteritidis (multiple hosts), S. Gallinarum (birds), and S. Dublin (cattle). This provides an excellent evolutionary context in which differences between these pathogen genomes can be related to host range. Genome sequences were obtained from ∼ 60 isolates selected to represent the known diversity of this lineage. Examination and comparison of the clades within the phylogeny of this lineage revealed signs of host restriction as well as evolutionary events that mark a path to host generalism. We have identified the nature and order of events for both evolutionary trajectories. The impact of functional gene loss was predicted based upon position within metabolic pathways and confirmed with phenotyping assays. The structure of S. Enteritidis is more complex than previously known, as a second clade of S. Enteritidis was revealed that is distinct from those commonly seen to cause disease in humans or animals, and that is more closely related to S. Gallinarum. Isolates from this second clade were tested in a chick model of infection and exhibited a reduced colonization phenotype, which we postulate represents an intermediate stage in pathogen-host adaptation.

The study of bacterial populations using whole-genome sequencing is of considerable scientific and clinical interest. However, obtaining bacterial genomic information is not always trivial: the target bacteria may be difficult to culture or uncultured, and they may be found within samples containing complex mixtures of other contaminating microbes and/or host cells, from which it is very difficult to derive robust sequencing data. Here we describe our procedure to generate sufficient DNA for whole-genome sequencing from clinical samples and without the need for culture, as successfully used on the difficult-to-culture, obligate intracellular pathogen Chlamydia trachomatis. Our protocol combines immunomagnetic separation (IMS) for targeted bacterial enrichment with multiple displacement amplification (MDA) for whole-genome amplification (WGA), which is followed by high-throughput sequencing. Compared with other techniques that might be used to generate such data, IMS-MDA is an inexpensive, low-technology and highly transferable process that provides amplified genomic DNA for sequencing from target bacteria in under 5 h, with little hands-on time.

The use of whole-genome sequencing as a tool for the study of infectious bacteria is of growing clinical interest. Chlamydia trachomatis is responsible for sexually transmitted infections and the blinding disease trachoma, which affect hundreds of millions of people worldwide. Recombination is widespread within the genome of C. trachomatis, thus whole-genome sequencing is necessary to understand the evolution, diversity, and epidemiology of this pathogen. Culture of C trachomatis has, until now, been a prerequisite to obtain DNA for whole-genome sequencing; however, as C trachomatis is an obligate intracellular pathogen, this procedure is technically demanding and time consuming. Discarded clinical samples represent a large resource for sequencing the genomes of pathogens, yet clinical swabs frequently contain very low levels of C trachomatis DNA and large amounts of contaminating microbial and human DNA. To determine whether it is possible to obtain whole-genome sequences from bacteria without the need for culture, we have devised an approach that combines immunomagnetic separation (IMS) for targeted bacterial enrichment with multiple displacement amplification (MDA) for whole-genome amplification. Using IMS-MDA ill conjunction with high-throughput multiplexed Illumina sequencing, we have produced the first whole bacterial genome sequences direct from clinical samples. We also show that this method can be used to generate genome data from nonviable archived samples. This method will prove a useful tool in answering questions relating to the biology of many difficult-to-culture or fastidious bacteria of clinical concern.

Chlamydia trachomatis is responsible for both trachoma and sexually transmitted infections, causing substantial morbidity and economic cost globally. Despite this, our knowledge of its population and evolutionary genetics is limited. Here we present a detailed phylogeny based on whole-genome sequencing of representative strains of C. trachomatis from both trachoma and lymphogranuloma venereum (LGV) biovars from temporally and geographically diverse sources. Our analysis shows that predicting phylogenetic structure using ompA, which is traditionally used to classify Chlamydia, is misleading because extensive recombination in this region masks any true relationships present. We show that in many instances, ompA is a chimera that can be exchanged in part or as a whole both within and between biovars. We also provide evidence for exchange of, and recombination within, the cryptic plasmid, which is another key diagnostic target. We used our phylogenetic framework to show how genetic exchange has manifested itself in ocular, urogenital and LGV C. trachomatis strains, including the epidemic LGV serotype L2b.

Udgivelsesdato: 2009-Jan, Bacterial infections of the lungs of cystic fibrosis (CF) patients cause major complications in the treatment of this common genetic disease. Burkholderia cenocepacia infection is particularly problematic since this organism has high levels of antibiotic resistance, making it difficult to eradicate; the resulting chronic infections are associated with severe declines in lung function and increased mortality rates. B. cenocepacia strain J2315 was isolated from a CF patient and is a member of the epidemic ET12 lineage that originated in Canada or the United Kingdom and spread to Europe. The 8.06-Mb genome of this highly transmissible pathogen comprises three circular chromosomes and a plasmid and encodes a broad array of functions typical of this metabolically versatile genus, as well as numerous virulence and drug resistance functions. Although B. cenocepacia strains can be isolated from soil and can be pathogenic to both plants and man, J2315 is representative of a lineage of B. cenocepacia rarely isolated from the environment and which spreads between CF patients. Comparative analysis revealed that ca. 21% of the genome is unique in comparison to other strains of B. cenocepacia, highlighting the genomic plasticity of this species. Pseudogenes in virulence determinants suggest that the pathogenic response of J2315 may have been recently selected to promote persistence in the CF lung. The J2315 genome contains evidence that its unique and highly adapted genetic content has played a significant role in its success as an epidemic CF pathogen.