Virulence-associated Plasmid Research Articles

Genomic analysis of <i>Klebsiella pneumoniae</i> strains virulence and antibiotic resistance

Recently, Klebsiella pneumoniae strains have become widespread both in community-acquired infectious processes and in nosocomial infections. There are two pathotypes of K. pneumoniae: classical (cKp) and hypervirulent (hvKp). Representatives of any pathotype are prone to acquire and further transmit genetic factors of antibiotic resistance and virulence. This combination accounts for severity of the infectious process. Therefore, information about whether the strain belongs to either pathotype can help in prescribing proper therapy. Since there is no consensus upon hypervirulence marker, we attempted to find the most significant combinations of genetic markers of virulence and antibiotic resistance in K. pneumoniae strains. The study was aimed to conduct a genomic analysis of virulence and antibiotic resistance of K. pneumoniae clinical isolates. Materials and methods. There were examined 85 strains of K. pneumoniae isolated from diverse clinical material samples from patients in large St. Petersburg hospitals. In our work, we used classical bacteriological methods, including determination of the hypermucoviscous type using the “string test”, the mass spectrometric method (MALDI-ToF MS) for identifying bacteria, molecular methods for studying markers of virulence and antibiotic resistance (multilocus sequence typing, genome sequencing of K. pneumoniae strains). Results. Among the studied K. pneumoniae strains, the most common carbapenemase genes were OXA-48 (18.7%) and NDM-1 genes — 17.3% of strains; in 6.7% of strains, NDM-1 and OXA-48 genes were found simultaneously. The percentage of strains with β-lactamase genes CTX-M-15 was 54.7%, OXA-1 — 17.3%, TEM-1D — 13.3%, and in 17.3% of cases the OXA-1 and TEM-1D genes were simultaneously present in bacterial strains. Quinolone resistance genes were found in 68.4% of strains. The most common genes were qnrS1 (40% of strains) and qnrB1 (22.7%). Phenotypic antimicrobial susceptibility testing showed that 23.5% and 64.7% strains were resistant to colistin and carbapenems, respectively. 32.9% K. pneumoniae strains, isolated in patients with phlegmon, pneumonia, sepsis, and peritonitis, had a hypermucoid phenotype. The most common sequence types were: ST395 (24.3%), ST23 (17.6%) and ST512 (9.5%). 8% and 25.3% of strains belonged to capsule types K1 and K2, respectively. The polyketide synthesis locus ybt, which characterizes virulent strains, was detected in 69.3% isolates, and the clb locus was present in 10.7% of strains. In 73.3% and 14.7% strains, the plasmid-associated virulence loci iuc and iro were identified, which encode the biosynthesis of the siderophores aerobactin and salmochelin. We described 44 cases (58.7% of strains) of genotypic convergence of virulence and antibiotic resistance, as shown by simultaneously detected the aerobactin (iuc) locus and β-lactamase or carbapenemase genes. Thus, identification of hypervirulence may provide valuable information for the clinical management of patients with hvKp infections. Therefore, it is is obviously necessary to develop comprehensive diagnostic test for simultaneous screening of multidrug-resistant hypervirulent K. pneumoniae strains.

Plasmid-mediated virulence in Chlamydia.

Chlamydia trachomatis infection of ocular conjunctiva can lead to blindness, while infection of the female genital tract can lead to chronic pelvic pain, ectopic pregnancy, and/or infertility. Conjunctival and fallopian tube inflammation and the resulting disease sequelae are attributed to immune responses induced by chlamydial infection at these mucosal sites. The conserved chlamydial plasmid has been implicated in enhancing infection, via improved host cell entry and exit, and accelerating innate inflammatory responses that lead to tissue damage. The chlamydial plasmid encodes eight open reading frames, three of which have been associated with virulence: a secreted protein, Pgp3, and putative transcriptional regulators, Pgp4 and Pgp5. Although Pgp3 is an important plasmid-encoded virulence factor, recent studies suggest that chlamydial plasmid-mediated virulence extends beyond the expression of Pgp3. In this review, we discuss studies of genital, ocular, and gastrointestinal infection with C. trachomatis or C. muridarum that shed light on the role of the plasmid in disease development, and the potential for tissue and species-specific differences in plasmid-mediated pathogenesis. We also review evidence that plasmid-associated inflammation can be independent of bacterial burden. The functions of each of the plasmid-encoded proteins and potential molecular mechanisms for their role(s) in chlamydial virulence are discussed. Although the understanding of plasmid-associated virulence has expanded within the last decade, many questions related to how and to what extent the plasmid influences chlamydial infectivity and inflammation remain unknown, particularly with respect to human infections. Elucidating the answers to these questions could improve our understanding of how chlamydia augment infection and inflammation to cause disease.

The opportunistic intracellular bacterial pathogen Rhodococcus equi elicits type I interferon by engaging cytosolic DNA sensing in macrophages

Rhodococcus equi is a major cause of foal pneumonia and an opportunistic pathogen in immunocompromised humans. While alveolar macrophages constitute the primary replicative niche for R. equi, little is known about how intracellular R. equi is sensed by macrophages. Here, we discovered that in addition to previously characterized pro-inflammatory cytokines (e.g., Tnfa, Il6, Il1b), macrophages infected with R. equi induce a robust type I IFN response, including Ifnb and interferon-stimulated genes (ISGs), similar to the evolutionarily related pathogen, Mycobacterium tuberculosis. Follow up studies using a combination of mammalian and bacterial genetics demonstrated that induction of this type I IFN expression program is largely dependent on the cGAS/STING/TBK1 axis of the cytosolic DNA sensing pathway, suggesting that R. equi perturbs the phagosomal membrane and causes DNA release into the cytosol following phagocytosis. Consistent with this, we found that a population of ~12% of R. equi phagosomes recruits the galectin-3,-8 and -9 danger receptors. Interestingly, neither phagosomal damage nor induction of type I IFN require the R. equi's virulence-associated plasmid. Importantly, R. equi infection of both mice and foals stimulates ISG expression, in organs (mice) and circulating monocytes (foals). By demonstrating that R. equi activates cytosolic DNA sensing in macrophages and elicits type I IFN responses in animal models, our work provides novel insights into how R. equi engages the innate immune system and furthers our understanding how this zoonotic pathogen causes inflammation and disease.

Rhodococcus equi Infections in Goats: Characterization of Virulence Plasmids.

Rhodococcus equi is an uncommon cause of systemic pyogranulomatous infections in goats with macroscopic similarities to caseous lymphadenitis caused by Corynebacterium pseudotuberculosis. Caprine cases have previously been reported to be caused by avirulent R. equi strains. Six cases of R. equi infection in goats yielding 8 R. equi isolates were identified from 2000 to 2017. Lesions varied from bronchopneumonia, vertebral and humeral osteomyelitis, and subcutaneous abscesses, to disseminated infection involving the lungs, lymph nodes, and multiple visceral organs. Isolates of R. equi from infected goats were analyzed by polymerase chain reaction for R. equi virulence-associated plasmid ( vap) genes. Seven of 8 isolates carried the VapN plasmid, originally characterized in bovine isolates, while 1 isolate lacked virulence plasmids and was classified as avirulent. The VapN plasmid has not been described in isolates cultured from goats.

ПАТОМОРФОЛОГИЧЕСКИЕ ИЗМЕНЕНИЯ ПЕЧЕНИ БЕЛЫХ МЫШЕЙ ПРИ ЭКСПЕРИМЕНТАЛЬНОЙ ЧУМНОЙ ИНФЕКЦИИ, ВЫЗВАННОЙ YERSINIA PESTIS С РАЗНЫМ ПЛАЗМИДНЫМ СОСТАВОМ

Data of histological study of pathomorphological changes in liver of white mice with experimental plague are represented in the article. An important element in evaluation and prediction of the pathological process in the liver is the analysis of hepatocyte karyokinesis, the mitotic index calculation, the counting number of atypical mitosis and the degree of organ regeneration ability. Dynamics of these liver changes at infection process in experimental animals caused by Yersinia pestis plasmid variants was investigated in this work. The variability of liver injuries was shown depending on plasmid-associated virulence of the infection agent. It was proved that the Y. pestis strain carrying three basic pYP (6 mDa), pYV (45 mDa), pYT (61 mDa) plasmids caused the most severe systemic disease with lethal outcome as a rule. Lack of species-specific plasmids and especially virulence and pathogenicity plasmids reduced the rate of infection process generalization and the ability of the pathogen to initiate pathological changes incompatible with the microorganism life. Therefore, all morphological liver changes in experimental plague infection are directly dependent on plasmid profile of the pathogen. The mechanism of toxicity included the damaging effect of the toxin to the cellular structures and failure of the metabolic processes in the organism. Acuity of intoxication and the development of pathological process can be estimated by exploring changes in liver morphology.

Rescue of an intracellular avirulent Rhodococcus equi replication defect by the extracellular addition of virulence-associated protein A.

Rhodococcus equi is a facultative intracellular bacterium that can escape from bactericidal mechanisms associated with phagocytosis. Virulence-associated protein A (VapA), encoded on a virulence-associated plasmid, is essential for intracellular survival in macrophages, but its function is not known. Here, we show that the extracellular addition of recombinant glutathione S-transferase (GST)-VapA fusion protein rescued the intracellular replication defect of a mutant lacking the vapA gene. Furthermore, the virulence-plasmid-cured strain could also multiply to nearly wild-type levels by the addition of GST-VapA. The present data suggest that VapA can alter the intraphagocytic environment, thereby affecting its suitability for the growth of R. equi.

Morphogenesis of Experimental Infection Caused by Plasmid Variants of Yersinia pseudotuberculosis.

The dynamics of pathomorphological changes in response to infection with plasmid variants of Yersinia pseudotuberculosis was studied in experimental animals. Variability of cell injuries in pseudotuberculosis histopathology depended on the plasmid-associated virulence of the infection agent. Infection with highly virulent two-plasmid strain pYV48:pVM82 MDa and Y. pseudotuberculosis strain with low virulence with the only plasmid pVM82 MDa led to the development of cell destruction (necrosis and apoptosis) in the target organs. Apoptosis predominated in response to infection by plasmid variant pVM82 MDa with low virulence.

Draft Genome Sequences of Four Salmonella enterica Strains Isolated from Turkey-Associated Sources.

We report the draft genomes of four Salmonella enterica isolates evaluated for the contribution of plasmids to virulence. Strains SE163A, SE696A, and SE710A carry plasmids demonstrated to facilitate plasmid-associated virulence, while SE819 is less virulent and has been used as a recipient for conjugation experiments to assess plasmid-encoded virulence mechanisms.

Phylotyping and ColV plasmid-associated virulence genotyping of E. coli isolated from broiler chickens with colibacillosis in Iran

Avian pathogenic Escherichia coli (APEC) strains are responsible for significant losses to the poultry industry in many parts of the world. Although, many serotypes of E. coli are non pathogen, a relatively few number of serotypes carry virulence genes which make them damaging for chicken and other poultry. Previously, high prevalence of virulence genes carried on plasmid and/or chromosomal DNA has demonstrated in APECs obtained from different countries. The aim of the present study is characterization of E. coli isolated from broiler chickens with pericarditis and femoral head necrosis (FHN) associated with colibacillosis based on the phylogenetic typing and ColV plasmid-carried virulence genotyping. In present study, 290 E. coli isolates from pericardial sac (n = 120) and lesions of femur (n = 100) of chickens exhibiting clinical and necropsy findings of colibacillosis from poultry farms in Fars province, Iran, besides 70 isolates from the intestinal content of apparently healthy birds were phylotyped and examined for the presence of seven virulence genes (ompT, hly, iss, iutA, iron, tsh, and cvaC) carried on ColV plasmid by multiplex PCR. Phylotyping analysis showed that the most of the APEC isolates fell into the phylogenetic group A and subgroup A1. Also, virulence genotyping indicated that ColV plasmid-carried virulence genes were significantly higher in APEC compared to avian fecal E. coli (AFEC). Based on the results of this study, having several virulence genes of ColV plasmid and belonging to the phylogenetic group A might provide useful characteristics for the better identifying Iranian APECs. However, the link between avian E. coli pathogenicity and different phylotypes and genotypes needs more future works involving a larger number of samples to be confirmed.

Rhodococcus equi Infections in Dogs.

Five cases of Rhodococcus equi infection in dogs were identified from 2003 to 2014. Three of the dogs had severe, internal lesions attributable to R. equi that have not been previously described: endophthalmitis, endocarditis, and suppurative pleuropneumonia. Isolates from 4 of the dogs were analyzed by polymerase chain reaction for Rhodococcus virulence-associated plasmid (vap) genes. One isolate was vapA-positive, 2 lacked a virulence plasmid, and 1 carried the novel vapN-associated plasmid (pVAPN) recently characterized in bovine isolates. The pVAPN plasmid has not been described in isolates cultured from companion animals. Four of the dogs either were receiving immunosuppressive drugs or had endocrinopathies. R. equi has the potential to cause significant infections in dogs, and immunocompromised animals should be considered at risk for infection.

Hybrid Shiga Toxin-Producing and Enterotoxigenic Escherichia sp. Cryptic Lineage 1 Strain 7v Harbors a Hybrid Plasmid.

Hybrid isolates of Shiga toxin-producing Escherichia coli (STEC) and enterotoxigenic E. coli (ETEC) encoding heat-stable enterotoxin (ST) are being reported with increasing frequency from a variety of sources. However, information regarding the plasmids that these strains harbor is scarce. In this study, we sequence and characterize a plasmid, p7v, from the STEC/ETEC hybrid strain 7v. Whole-genome phylogenetic analyses of STEC/ETEC hybrid strains and prototype E. coli isolates of other pathotypes placed 7v in the Escherichia sp. cryptic lineage 1 (CL1) clade. The complete plasmid, p7v, was determined to be 229,275 bp and encodes putative virulence factors that are typically carried on STEC plasmids as well as those often carried on ETEC plasmids, indicating that the hybrid nature of the strain extends beyond merely encoding the two toxins. Plasmid p7v carries two copies of sta with identical sequences, which were discovered to be divergent from the sta sequences found in the prototype human ETEC strains. Using a nomenclature scheme based on a phylogeny constructed from sta and stb sequences, the sta encoded on p7v is designated STa4. In silico analysis determined that p7v also encodes the K88 fimbria, a colonization factor usually associated with porcine ETEC plasmids. The p7v sequence and the presence of plasmid-encoded virulence factors are compared to those of other STEC/ETEC CL1 hybrid genomes and reveal gene acquisition/loss at the strain level. In addition, the interrogation of 24 STEC/ETEC hybrid genomes for identification of plasmid replicons, colonization factors, Stx and ST subtypes, and other plasmid-encoded virulence genes highlights the diversity of these hybrid strains. Hybrid Shiga toxin-producing Escherichia coli/enterotoxigenic Escherichia coli (STEC/ETEC) strains, which have been isolated from environmental, animal, and human clinical samples, may represent an emerging threat as food-borne pathogens. Characterization of these strains is important for assessing virulence potential, aiding in the development of pathogen detection methods, and understanding how the hybrid strains evolve to potentially have a greater impact on public health. This study represents, to our knowledge, both the first characterization of a closed plasmid sequence from a STEC/ETEC hybrid strain and the most comprehensive phylogenetic analysis of available STEC/ETEC hybrid genomes to date. The results demonstrate how the mobility of plasmid-associated virulence genes has resulted in the creation of a diverse plasmid repertoire within the STEC/ETEC hybrid strains.

Genomic Insights into a New Citrobacter koseri Strain Revealed Gene Exchanges with the Virulence-Associated Yersinia pestis pPCP1 Plasmid.

The history of infectious diseases raised the plague as one of the most devastating for human beings. Far too often considered an ancient disease, the frequent resurgence of the plague has led to consider it as a reemerging disease in Madagascar, Algeria, Libya, and Congo. The genetic factors associated with the pathogenicity of Yersinia pestis, the causative agent of the plague, involve the acquisition of the pPCP1 plasmid that promotes host invasion through the expression of the virulence factor Pla. The surveillance of plague foci after the 2003 outbreak in Algeria resulted in a positive detection of the specific pla gene of Y. pestis in rodents. However, the phenotypic characterization of the isolate identified a Citrobacter koseri. The comparative genomics of our sequenced C. koseri URMITE genome revealed a mosaic gene structure resulting from the lifestyle of our isolate and provided evidence for gene exchanges with different enteric bacteria. The most striking was the acquisition of a continuous 2 kb genomic fragment containing the virulence factor Pla of the Y. pestis pPCP1 plasmid; however, the subcutaneous injection of the CKU strain in mice did not produce any pathogenic effect. Our findings demonstrate that fast molecular detection of plague using solely the pla gene is unsuitable and should rather require Y. pestis gene marker combinations. We also suggest that the evolutionary force that might govern the expression of pathogenicity can occur through the acquisition of virulence genes but could also require the loss or the inactivation of resident genes such as antivirulence genes.

Chlamydiaceae Genomics Reveals Interspecies Admixture and the Recent Evolution of Chlamydia abortus Infecting Lower Mammalian Species and Humans.

Chlamydiaceae are obligate intracellular bacteria that cause a diversity of severe infections among humans and livestock on a global scale. Identification of new species since 1989 and emergence of zoonotic infections, including abortion in women, underscore the need for genome sequencing of multiple strains of each species to advance our knowledge of evolutionary dynamics across Chlamydiaceae. Here, we genome sequenced isolates from avian, lower mammalian and human hosts. Based on core gene phylogeny, five isolates previously classified as Chlamydia abortus were identified as members of Chlamydia psittaci and Chlamydia pecorum. Chlamydia abortus is the most recently emerged species and is a highly monomorphic group that lacks the conserved virulence-associated plasmid. Low-level recombination and evidence for adaptation to the placenta echo evolutionary processes seen in recently emerged, highly virulent niche-restricted pathogens, such as Bacillus anthracis. In contrast, gene flow occurred within C. psittaci and other Chlamydiaceae species. The C. psittaci strain RTH, isolated from a red-tailed hawk (Buteo jamaicensis), is an outlying strain with admixture of C. abortus, C. psittaci, and its own population markers. An average nucleotide identity of less than 94% compared with other Chlamydiaceae species suggests that RTH belongs to a new species intermediary between C. psittaci and C. abortus. Hawks, as scavengers and predators, have extensive opportunities to acquire multiple species in their intestinal tract. This could facilitate transformation and homologous recombination with the potential for new species emergence. Our findings indicate that incubator hosts such as birds-of-prey likely promote Chlamydiaceae evolution resulting in novel pathogenic lineages.

Phenotypic and Genotypic Characterization of Salmonella enterica Serovar Enteritidis Isolates Associated with a Mousse Cake-Related Outbreak of Gastroenteritis in Ningbo, China.

Salmonella enterica subsp. enterica serovar Enteritidis (Salmonella Enteritidis) is a major pathogen responsible for causing the largest number of sporadic cases and outbreaks of human salmonellosis worldwide. In this study, an outbreak of Salmonella Enteritidis involving 112 cases in Ningbo, China was investigated with a combination of genotypic subtyping methods and phenotypic analysis. The pulsed-field gel electrophoresis and multilocus variable-number tandem-repeat analysis profiles showed that most of the outbreak clinical isolates (22/23) were indistinguishable from each other and were identical to the isolates obtained from implicated mousse cakes, demonstrating that this outbreak of gastroenteritis was caused by Salmonella Enteritidis-contaminated mousse cakes. Moreover, all isolates, irrespective of source, had an identical antibiotic susceptibility pattern. Five virulence-associated genes in Salmonella pathogenicity islands and the plasmid-associated virulence genes spvB/C were present in both the food and clinical isolates. Importantly, all of these isolates can survive well under low-temperature treatment, indicating that manufacturers of foodstuffs with raw ingredients (not subjected to thermal processing) should use an effective approach to prevent or eliminate the microbial hazards to public health.

Transfer of the Virulence‐Associated Protein A‐Bearing Plasmid between Field Strains of Virulent and AvirulentRhodococcus equi

Virulent and avirulent isolates of Rhodococcus equi coexist in equine feces and the environment and are a source of infection for foals. The extent to which plasmid transfer occurs among field strains is ill-defined and this information is important for understanding the epidemiology of R. equi infections of foals. To estimate the frequency of transfer of the virulence plasmid between virulent and avirulent strains of R. equi derived from foals and their environment. None. In vitro study; 5 rifampin-susceptible, virulent R. equi isolates obtained from clinically affected foals or air samples from a farm with a history of recurrent R. equi foal pneumonia were each mixed with 5 rifampin-resistant, avirulent isolates derived from soil samples, using solid medium, at a ratio of 10 donor cells (virulent) per recipient cell. Presumed transconjugates were detected by plating on media with rifampin and colony immunoblotting to detect the presence of the virulence-associated protein A. Three presumed transconjugates were detected among 2,037 recipient colonies, indicating an overall estimated transfer frequency of 0.15% (95% CI, 0.03–0.43%). All 3 transconjugates were associated with a single donor and 2 recipient strains. Genotyping and multiplex PCR of presumed transconjugates demonstrated transfer of the virulence-associated protein A-bearing plasmid between virulent and avirulent R. equi. Transfer of the virulence plasmid occurs with relatively high frequency. These findings could impact strategies to control or prevent R. equi through environmental management.

Complete Nucleotide Sequences of Virulence-Resistance Plasmids Carried by Emerging Multidrug-Resistant Salmonella enterica Serovar Typhimurium Isolated from Cattle in Hokkaido, Japan

In the present study, we have shown that virulence-resistance plasmids from emerging multidrug-resistant isolates of Salmonella enterica serovar Typhimurium were derived from a virulence-associated plasmid, essential for systematic invasiveness of S. Typhimurium in mice (pSLT), through acquisition of a large insert containing a resistance island flanked by IS1294 elements. A bla CMY-2-carrying plasmid from a cefotaxime-resistant isolate comprised a segment of Escherichia coli plasmid pAR060302 and the replication region (IncFIB) of a virulence-resistance plasmid. These results provide insights into the evolution of drug resistance in emerging clones of S. Typhimurium.

Experimental Infections of the Reservoir Species Peromyscus leucopus with Diverse Strains of Borrelia burgdorferi, a Lyme Disease Agent

ABSTRACTThe rodent Peromyscus leucopus is a major natural reservoir for the Lyme disease agent Borrelia burgdorferi and a host for its vector Ixodes scapularis. At various locations in northeastern United States 10 to 15 B. burgdorferi strains coexist at different prevalences in tick populations. We asked whether representative strains of high or low prevalence differed in their infections of P. leucopus. After 5 weeks of experimental infection of groups with each of 6 isolates, distributions and burdens of bacteria in tissues were measured by quantitative PCR, and antibodies to B. burgdorferi were evaluated by immunoblotting and protein microarray. All groups of animals were infected in their joints, ears, tails, and hearts, but overall spirochete burdens were lower in animals infected with low-prevalence strains. Animals were similar regardless of the infecting isolate in their levels of antibodies to whole cells, FlaB, BmpA, and DbpB proteins, and the conserved N-terminal region of the serotype-defining OspC proteins. But there were strain-specific antibody responses to full-length OspC and to plasmid-encoded VlsE, BBK07, and BBK12 proteins. Sequencing of additional VlsE genes revealed substantial diversity within some pairs of strains but near-identical sequences within other pairs, which otherwise differed in their ospC alleles. The presence or absence of full-length bbk07 and bbk12 genes accounted for the differences in antibody responses. We propose that for B. burgdorferi, there is selection in reservoir species for (i) sequence diversity, as for OspC and VlsE, and (ii) the presence or absence of polymorphisms, as for BBK07 and BBK12.

Identification of Virulence-Associated Plasmids in Rhodococcus equi in Humans with and without Acquired Immunodeficiency Syndrome in Brazil

Virulence of Rhodococcus equi strains from 20 humans in Brazil was investigated by using a polymerase chain reaction to characterize isolates as virulent (VapA), intermediately virulent (VapB), and avirulent. Nine isolates were obtained from human immunodeficiency virus (HIV)-positive patients, six from HIV-negative patients, and five from patients of unknown status. Five isolates were VapB positive, four were VapA positive, and eleven were avirulent. Among the nine isolates from HIV-positive patients, five contained VapB plasmids and two contained VapA plasmids. Five VapB-positive isolates had the type 8 virulence plasmid. Eleven of the patients had a history of contact with livestock and/or a farm environment, and none had contact with pigs.

Targeted enrichment of ancient pathogens yielding the pPCP1 plasmid ofYersinia pestisfrom victims of the Black Death

Although investigations of medieval plague victims have identified Yersinia pestis as the putative etiologic agent of the pandemic, methodological limitations have prevented large-scale genomic investigations to evaluate changes in the pathogen's virulence over time. We screened over 100 skeletal remains from Black Death victims of the East Smithfield mass burial site (1348-1350, London, England). Recent methods of DNA enrichment coupled with high-throughput DNA sequencing subsequently permitted reconstruction of ten full human mitochondrial genomes (16 kb each) and the full pPCP1 (9.6 kb) virulence-associated plasmid at high coverage. Comparisons of molecular damage profiles between endogenous human and Y. pestis DNA confirmed its authenticity as an ancient pathogen, thus representing the longest contiguous genomic sequence for an ancient pathogen to date. Comparison of our reconstructed plasmid against modern Y. pestis shows identity with several isolates matching the Medievalis biovar; however, our chromosomal sequences indicate the victims were infected with a Y. pestis variant that has not been previously reported. Our data reveal that the Black Death in medieval Europe was caused by a variant of Y. pestis that may no longer exist, and genetic data carried on its pPCP1 plasmid were not responsible for the purported epidemiological differences between ancient and modern forms of Y. pestis infections.

Unity in Variety--The Pan-Genome of the Chlamydiae

Chlamydiae are evolutionarily well-separated bacteria that live exclusively within eukaryotic host cells. They include important human pathogens such as Chlamydia trachomatis as well as symbionts of protozoa. As these bacteria are experimentally challenging and genetically intractable, our knowledge about them is still limited. In this study, we obtained the genome sequences of Simkania negevensis Z, Waddlia chondrophila 2032/99, and Parachlamydia acanthamoebae UV-7. This enabled us to perform the first comprehensive comparative and phylogenomic analysis of representative members of four major families of the Chlamydiae, including the Chlamydiaceae. We identified a surprisingly large core gene set present in all genomes and a high number of diverse accessory genes in those Chlamydiae that do not primarily infect humans or animals, including a chemosensory system in P. acanthamoebae and a type IV secretion system. In S. negevensis, the type IV secretion system is encoded on a large conjugative plasmid (pSn, 132 kb). Phylogenetic analyses suggested that a plasmid similar to the S. negevensis plasmid was originally acquired by the last common ancestor of all four families and that it was subsequently reduced, integrated into the chromosome, or lost during diversification, ultimately giving rise to the extant virulence-associated plasmid of pathogenic chlamydiae. Other virulence factors, including a type III secretion system, are conserved among the Chlamydiae to variable degrees and together with differences in the composition of the cell wall reflect adaptation to different host cells including convergent evolution among the four chlamydial families. Phylogenomic analysis focusing on chlamydial proteins with homology to plant proteins provided evidence for the acquisition of 53 chlamydial genes by a plant progenitor, lending further support for the hypothesis of an early interaction between a chlamydial ancestor and the primary photosynthetic eukaryote.