Plasmid-encoded Virulence Factors Research Articles

Differential proteome profiling of bacterial culture supernatants reveals candidates for the induction of oral immune priming in the red flour beetle.

Most organisms are host to symbionts and pathogens, which led to the evolution of immune strategies to prevent harm. Whilst the immune defences of vertebrates are classically divided into innate and adaptive, insects lack specialized cells involved in adaptive immunity, but have been shown to exhibit immune priming: the enhanced survival upon infection after a first exposure to the same pathogen or pathogen-derived components. An important piece of the puzzle are the pathogen-associated molecules that induce these immune priming responses. Here, we make use of the model system consisting of the red flour beetle (Tribolium castaneum) and its bacterial pathogen Bacillus thuringiensis, to compare the proteomes of culture supernatants of two closely related B. thuringiensis strains that either induce priming via the oral route, or not. Among the proteins that might be immunostimulatory to T. castaneum, we identify the Cry3Aa toxin, an important plasmid-encoded virulence factor of B. thuringiensis. In further priming-infection assays we test the relevance of Cry-carrying plasmids for immune priming. Our findings provide valuable insights for future studies to perform experiments on the mechanisms and evolution of immune priming.

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.

Clinical and genomic analysis of hypermucoviscous Klebsiella pneumoniae isolates: Identification of new hypermucoviscosity associated genes.

Hypermucoviscous Klebsiella pneumoniae (HmKp) poses an emerging and highly pathogenic global health threat. This study aimed to investigate the clinical and genomic characteristics of HmKp isolates to better understand the virulence mechanisms of the hypermucoviscous (HMV) phenotype. From May 2018 to August 2021, 203 non-repeat K. pneumoniae isolates causing invasive infections were collected from a hospital in Beijing, China. Isolates were divided into HmKp (n=90, 44.3%) and non-HmKp (n=113, 55.7%) groups according to string test results. Multivariate regression showed that diabetes mellitus (odds ratio [OR]=2.20, 95% confidence interval (CI): 1.20-4.05, p=0.010) and liver abscess (OR=2.93, CI 95%:1.29-7.03, p=0.012) were associated with HmKp infections. K. pneumoniae was highly diverse, comprising 87 sequence types (STs) and 54 serotypes. Among HmKp isolates, ST23 was the most frequent ST (25/90, 27.8%), and the most prevalent serotypes were KL2 (31/90, 34.4%) and KL1 (27/90, 30.0%). Thirteen virulence genes were located on the capsular polysaccharide synthesis region of KL1 strains. HmKp isolates were sensitive to multiple antibiotics but carried more SHV-type extended spectrum β-lactamase (ESBL) resistance genes (p<0.05), suggesting that the emergence of ESBL-mediated multidrug resistance in HmKp should be monitored carefully during treatment. Phylogenetic analysis disclosed that HmKp isolates were highly diverse. Comparative genomic analysis confirmed that the HMV phenotype is a plasmid-encoded virulence factor. Seventeen HmKp genes were highly associated with HmKp, and included rmpAC, 7 iron-acquisition-related genes, and pagO, which may promote liver abscess formation. This investigation provides insight into the mechanisms producing the HMV phenotype.

Efficacy of Pgp3 vaccination for Chlamydia urogenital tract infection depends on its native conformation.

Urogenital tract infections with Chlamydia trachomatis have frequently been detected among patients diagnosed with sexually transmitted infections, and such infections lead to inflammatory complications. Currently, no licensed chlamydial vaccine is available in clinical practice. We previously reported that immunization with recombinant C. trachomatis plasmid-encoded virulence factor Pgp3 provided cross-serovar protection against C. muridarum genital tract infection. Because Pgp3 is a homotrimer and human antisera only recognize the trimeric form of Pgp3, we compared the effects of the native conformation of Pgp3 (trimer) and heat-denatured Pgp3 (monomer) to determine whether the native conformation is dispensable for the induction of protective immunity against chlamydial vaginal challenge. Both Pgp3 trimer and monomer immunization induced corresponding specific antibody production, but only trimer-induced antibody recognized endogenous Pgp3, and trimer-immunized mouse splenocytes showed the highest IFN-γ production upon restimulation with the chlamydial elementary body or native Pgp3 in vitro. Importantly, only Pgp3 trimer-immunized mice showed shortened lower genital tract chlamydial shedding and decreased upper genital tract pathology. Thus, Pgp3-induced protective immunity against Chlamydia urogenital tract infection is highly dependent on the native conformation, which will guide the design of Pgp3-based polypeptides and multi-subunit chlamydial vaccines.

Emerging Themes in the Molecular Pathogenesis of Enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) are ubiquitous diarrheal pathogens that thrive in areas lacking basic human needs of clean water and sanitation. These genetically plastic organisms cause tremendous morbidity among disadvantaged young children, in the form of both acute diarrheal illness and sequelae of malnutrition and growth impairment. The recent discovery of additional plasmid-encoded virulence factors and elucidation of their critical role in the molecular pathogenesis of ETEC may inform new approaches to the development of broadly protective vaccines. Although the pathogens have been closely linked epidemiologically with nondiarrheal sequelae, these conditions remain very poorly understood. Similarly, while canonical effects of ETEC toxins on cellular signaling promoting diarrhea are clear, emerging data suggest that these toxins may also drive changes in intestinal architecture and associated sequelae. Elucidation of molecular events underlying these changes could inform optimal approaches to vaccines that prevent acute diarrhea and ETEC-associated sequelae.

Comparative genomic analysis of a Shiga toxin-producing Escherichia coli (STEC) O145:H25 associated with a severe pediatric case of hemolytic uremic syndrome in Davidson County, Tennessee, US

BackgroundShiga toxin-producing E. coli (STECs) are foodborne pathogens associated with bloody diarrhea and hemolytic uremic syndrome (HUS). Although the STEC O157 serogroup accounts for the highest number of infections, HUS-related complications and deaths, the STEC non-O157, as a group, accounts for a larger proportion of STEC infections and lower HUS cases. There is limited information available on how to recognize non-O157 serotypes associated with severe disease. The objectives of this study were to describe a patient with STEC non-O157 infection complicated with HUS and to conduct a comparative whole genome sequence (WGS) analysis among the patient’s STEC clinical isolate and STEC O157 and non-O157 strains.ResultsThe STEC O145:H25 strain EN1I-0044-2 was isolated from a pediatric patient with diarrhea, HUS and severe neurologic and cardiorespiratory complications, who was enrolled in a previously reported case-control study of acute gastroenteritis conducted in Davidson County, Tennessee in 2013. The strain EN1I-0044-2 genome sequence contained a chromosome and three plasmids. Two of the plasmids were similar to those present in O145:H25 strains whereas the third unique plasmid EN1I-0044-2_03 shared no similarity with other STEC plasmids, and it carried 23 genes of unknown function. Strain EN1I-0044-2, compared with O145:H25 and O157 serogroup strains shared chromosome- and plasmid-encoded virulence factors, including Shiga toxin, LEE type III secretion system, LEE effectors, SFP fimbriae, and additional toxins and colonization factors.ConclusionsA STEC O145:H25 strain EN1I-0044-2 was isolated from a pediatric patient with severe disease, including HUS, in Davidson County, TN. Phylogenetic and comparison WGS analysis provided evidence that strain EN1I-0044-2 closely resembles O145:H25, and confirmed an independent evolutionary path of STEC O145:H25 and O145:H28 serotypes. The strain EN1I-0044-2 virulence make up was similar to other O145:H25 and O157 serogroups. It carried stx2 and the LEE pathogenicity island, and additional colonization factors and enterotoxin genes. A unique feature of strain EN1I-0044-2 was the presence of plasmid pEN1I-0044-2_03 carrying genes with functions to be determined. Further studies will be necessary to elucidate the role that newly acquired genes by O145:H25 strains play in pathogenesis, and to determine if they may serve as genetic markers of severe disease.

Fatal Clostridium sordellii-mediated hemorrhagic and necrotizing gastroenteropathy in a dog: case report

BackgroundCanine hemorrhagic gastroenteritis (also canine gastrointestinal hemorrhagic syndrome) is commonly associated with Clostridium perfringens, although in some cases the etiology remains unclear. This report describes a fatal acute hemorrhagic and necrotizing gastroenteropathy in a dog associated with Clostridium sordellii, a bacterial species never before identified as the etiological agent of hemorrhagic and necrotizing gastroenteropathy in dogs.Case presentationA fully vaccinated, eight-year-old, female neutered Labrador presented with a history of vomiting without diarrhea. Clinical examination revealed pink mucous membranes, adequate hydration, normothermia, and normocardia. The dog was discovered deceased the following day. Post-mortem examination showed moderate amounts of dark red, non-clotted fluid within the stomach that extended into the jejunum. Discoloration was noted in the gastric mucosa, liver, lungs, and kidneys, with small petechial hemorrhages present in the endocardium over the right heart base and thymic remnants. Histological analysis demonstrated that the gastric fundic mucosa, the pyloric region, small intestine, and large intestine exhibited superficial coagulative necrosis and were lined with a layer of short Gram-positive rods. Anaerobic culture of the gastric content revealed C. sordellii as the dominant bacterial species and neither Salmonella spp., Campylobacter spp., C. perfringens, nor C. difficile were isolated. Unexpectedly, whole genome sequencing of the C. sordellii isolate showed that it lacked the main plasmid-encoded virulence factors typical of the species, indicating that the genetic determinants of pathogenicity of this strain must be chromosomally encoded. Further phylogenetic analysis revealed it to be genetically similar to C. sordellii isolates associated with gastroenteric disease in livestock, indicating that the infection may have been acquired from the environment.ConclusionsThis case demonstrates that C. sordellii can associate with a canine hemorrhagic and necrotizing gastroenteropathy in the absence of C. perfringens and illustrates the benefits of using bacterial whole genome sequencing to support pathological investigations in veterinary diagnostics. These data also update the molecular phylogeny of C. sordellii, indicating a possible pathogenic clade in the environment that is distinct from currently identified clades.

A virulence gene typing scheme for Photobacterium damselae subsp. piscicida, the causative agent of fish photobacteriosis, reveals a high prevalence of plasmid-encoded virulence factors and of type III secretion system genes

Fish photobacteriosis caused by Photobacterium damselae subsp. piscicida (Pdp) constitutes one of the major diseases affecting marine aquaculture worldwide. Although several virulence factors have been described in this pathogen, its pathobiology is still poorly understood. Two virulence factors, the exotoxin AIP56 and the siderophore piscibactin, are encoded by plasmids. Notably, presence of genes of a type III secretion system (T3SS) has been recently reported for the first time in this subspecies. However, the prevalence of these virulence genes in the populations of Pdp remains unstudied. Here, we have designed a PCR-based typing scheme for the detection of plasmid (pPHDP10)-borne aip56 gene encoding AIP56 exotoxin, plasmid (pPHDP70)-borne irp2 and frpA genes encoding piscibactin biosynthesis and transport proteins respectively, and for detection of the T3SS genes vscD and vcrD. The analysis of 103 isolates between 1963 and 2015, from different host species and geographical locations, has revealed that aip56 is highly prevalent, with only 6 isolates negative for aip56. The virulence plasmid pPHDP70 was present in 89 European and 2 Japanese isolates, refuting previous observations of a linkage of this plasmid exclusively to European strains. Most notably, 74 isolates tested positive for the T3SS genes, and their presence in photobacteriosis outbreaks could be traced back to 1980. Remarkably, we noticed that spontaneous rifampicin-resistant Pdp colonies underwent the loss of the T3SS genes vscD and vcrD, concomitantly with the loss of additional T3SS gene markers and of the putative plasmid-related genes parAB and traC, suggesting that the Pdp T3SS system is encoded within an unstable plasmid. This study unveils an unsuspected high prevalence of a putative plasmid-borne T3SS in this pathogen, and provides evidence that photobacteriosis outbreaks can be caused by distinct Pdp genotypes.

Transcriptome changes in response to temperature in the fish pathogen Photobacterium damselae subsp. damselae: Clues to understand the emergence of disease outbreaks at increased seawater temperatures.

The marine bacterium Photobacterium damselae subsp. damselae (Pdd) is a generalist and facultative pathogen that causes disease in a wide range of marine animals including fish species of importance in aquaculture. Disease outbreaks in fish farms have been correlated with an increased water temperature during summer months. In this study, we have used RNA sequencing to analyze the transcriptome of Pdd RM-71 cultured at two different temperatures, which simulated temperature conditions experienced during free swimming lifestyle at mid latitudes in winter months (15°C) and during outbreaks in aquaculture in warm summer months (25°C). The enhanced bacterial growth of Pdd observed at 25°C in comparison to 15°C suggests that an elevated seawater temperature contributes to the build-up of a sufficient bacterial population to cause disease. In comparison to growth at 15°C, growth at 25°C resulted in the upregulation of genes involved in DNA synthesis, nutrient uptake, chemotaxis, flagellar motility, secretion systems and antimicrobial resistance. Plasmid-encoded virulence factors, which include a putative adhesin/invasin OmpU, a transferrin receptor and a serum resistance protein, were also upregulated. Transcription factor RpoS, genes involved in cold shock response, modulation of cell envelope and amino acid metabolism, as well as genes of yet unknown function were downregulated at 25°C. Notably, the gene encoding damselysin cytotoxin (Dly) was among the most highly transcribed genes at the two assayed temperatures, at levels comparable to the most highly expressed housekeeping genes. This study contributes to our understanding of the regulatory networks and biology of a generalist marine bacterial pathogen, and provides evidence that temperature regulates multiple physiological and virulence-related functions in Pdd.

Chlamydial plasmid-encoded virulence factor Pgp3 interacts with human cathelicidin peptide LL-37 to modulate immune response

We have previously reported that Chlamydia trachomatis plasmid-encoded Pgp3 is able to neutralize anti-chlamydial activity of human cathelicidin peptide LL-37 by binding to and forming stable complex with LL-37. Besides its microbicidal activity, LL-37 also modulates immune response, including inducing cytokine/chemokine production in fibroblast/epithelial cells and recruitment of inflammatory cells. We now report that LL-37 was significantly induced in the genital tracts of women diagnosed positive for C. trachomatis. Both the LL-37-stimulated IL-6/8 production in human endometrial epithelial cells and the LL-37-induced neutrophil chemotaxis were blocked by Pgp3. Interestingly, although Pgp3 itself alone could not induce cytokines in epithelial cell cells, it did so in neutrophils. Importantly, the Pgp3 proinflammatory activity in neutrophils was significantly enhanced by forming complex with LL-37 although LL-37 alone failed to induce cytokine production in neutrophils. Thus, we have demonstrated that Pgp3 can modulate the proinflammatory activities of LL-37 on epithelial cells by forming stable complex with LL-37 but the Pgp3's own proinflammatory activity on myeloid cells is enhanced by forming the same complex. We hypothesize that Chlamydia may use Pgp3 to both block detrimental inflammation for improving its own fitness in the genital tract epithelial tissue and activate myeloid cell-mediated inflammation for potentially promoting spreading between the hosts, the latter of which may inevitably contribute to the development of inflammatory sequelae such as tubal fibrosis.

Epidemiology of Danish Aeromonas salmonicida subsp. salmonicida in Fish Farms Using Whole Genome Sequencing

Furunculosis, a serious infection caused by the bacterium Aeromonas salmonicida subsp. salmonicida is common in sea-reared rainbow trout production in Denmark. Developing an effective control strategy requires knowledge of the epidemiology, as well as the genomic and virulent variability of the Danish A. salmonicida subsp. salmonicida isolates. To obtain this, the genomes of 101 A. salmonicida subsp. salmonicida, including 99 Danish isolates, one Scottish strain and the type strain NCIMB 1102, were sequenced using the Illumina HiSeq platform. Isolates were de novo assembled, examined for presence of plasmids, virulence and iron acquisition proteins, genomic islands, and antibiotic resistance genes. Single Nucleotide Polymorphisms were aligned and subjected to Bayesian temporal phylogenetic and maximum likelihood tree reconstruction using the published genome of A. salmonicida subsp. salmonicida A449 as reference. Bayesian temporal phylogenetic reconstruction suggests that four major introductions of A. salmonicida subsp. salmonicida into Denmark have occurred. The introductions correlate with the freshwater and subsequent seawater expansion of rainbow trout production. Initial transmission of the bacterium could have been from seawater to freshwater or vice versa, and most minor clades include a mixture of strains from different fresh- and seawater farms. Genomic variation of A. salmonicida subsp. salmonicida mostly appeared to be associated with their plasmids and plasmid encoded virulence factors. Nine A. salmonicida subsp. salmonicida isolates harbored worldwide known antibiotic resistance genes against several antibiotics and there is an indication that 33% of the isolates contained the genomic island AsaGEI1b. These findings not only support the usefulness of whole genome sequencing for genetic studies of homogeneous bacteria in general, but provide novel information about the Danish A. salmonicida subsp. salmonicida population, with implications for vaccine development in efforts to better protect Danish rainbow trout in the future.

Comparative genomic analysis and characterization of incompatibility group FIB plasmid encoded virulence factors of Salmonella enterica isolated from food sources

BackgroundThe degree to which the chromosomal mediated iron acquisition system contributes to virulence of many bacterial pathogens is well defined. However, the functional roles of plasmid encoded iron acquisition systems, specifically Sit and aerobactin, have yet to be determined for Salmonella spp. In a recent study, Salmonella enterica strains isolated from different food sources were sequenced on the Illumina MiSeq platform and found to harbor the incompatibility group (Inc) FIB plasmid. In this study, we examined sequence diversity and the contribution of factors encoded on the IncFIB plasmid to the virulence of S. enterica.ResultsWhole genome sequences of seven S. enterica isolates were compared to genomes of serovars of S. enterica isolated from food, animal, and human sources. SeqSero analysis predicted that six strains were serovar Typhimurium and one was Heidelberg. Among the S. Typhimurium strains, single nucleotide polymorphism (SNP)-based phylogenetic analyses revealed that five of the isolates clustered as a single monophyletic S. Typhimurium subclade, while one of the other strains branched with S. Typhimurium from a bovine source. DNA sequence based phylogenetic diversity analyses showed that the IncFIB plasmid-encoded Sit and aerobactin iron acquisition systems are conserved among bacterial species including S. enterica. The IncFIB plasmid was transferred to an IncFIB plasmid deficient strain of S. enterica by conjugation. The transconjugant SE819::IncFIB persisted in human intestinal epithelial (Caco-2) cells at a higher rate than the recipient SE819. Genes of the Sit and aerobactin operons in the IncFIB plasmid were differentially expressed in iron-rich and iron-depleted growth media.ConclusionsMinimal sequence diversity was detected in the Sit and aerobactin operons in the IncFIB plasmids present among different bacterial species, including foodborne Salmonella strains. IncFIB plasmid encoded factors play a role during infection under low-iron conditions in host cells.

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.

Plasmid-Encoded Pgp5 Is a Significant Contributor to Chlamydia muridarum Induction of Hydrosalpinx.

We have previously shown that the plasmid-encoded Pgp3 is a major virulence factor for C. muridarum induction of hydrosalpinx. We now report that Pgp5 also plays a significant role in the development of hydrosalpinx following C. muridarum induction. Pgp5 deficiency was introduced via either in-frame deletion (CM-Δpgp5) or premature stop codon installation (CM-pgp5S). Mice infected with either CM-Δpgp5 or CM-pgp5S developed hydrosalpinges at significantly reduced levels with an incidence rate of <40% and a mean severity score of 2 or less. In contrast, 80% or more mice developed hydrosalpinx with a severity score of >3 when mice were infected with Pgp5-sufficient C. muridarum (plasmid-competent wild type or plasmid-free C. muridarum transformed with a full plasmid or depleted of pgp7 gene). The attenuated pathogenicity of the Pgp5-deficient C. muridarum correlated with a significantly reduced level of ascending infection in the oviduct tissue despite the similar overall shedding courses between mice infected with Pgp5-deficeint versus sufficient C. muridarum. Furthermore, in the oviducts of mice infected with Pgp5-deficient C. muridarum, significantly lower levels of inflammatory cell infiltration and cytokine production were detected. Thus, Pgp5 is a significant plasmid-encoded virulence factor for C. muridarum pathogenicity in the upper genital tract.

The Pathogen-annotated Tracking Resource Network (PATRN) system: A web-based resource to aid food safety, regulatory science, and investigations of foodborne pathogens and disease

Investigation of foodborne diseases requires the capture and analysis of time-sensitive information on microbial pathogens that is derived from multiple analytical methods and sources. The web-based Pathogen-annotated Tracking Resource Network (PATRN) system (www.patrn.net) was developed to address the data aggregation, analysis, and communication needs important to the global food safety community for the investigation of foodborne disease. PATRN incorporates a standard vocabulary for describing isolate metadata and provides a representational schema for a prototypic data exchange standard using a novel data loading wizard for aggregation of assay and attribution information. PATRN currently houses expert-curated, high-quality “foundational datasets” consisting of published experimental results from conventional assays and next generation analysis platforms for isolates of Escherichia coli, Listeria monocytogenes, and Salmonella, Shigella, Vibrio and Cronobacter species. A suite of computational tools for data mining, clustering, and graphical representation is available. Within PATRN, the public curated data repository is complemented by a secure private workspace for user-driven analyses, and for sharing data among collaborators. To demonstrate the data curation, loading wizard features, and analytical capabilities of PATRN, three use-case scenarios are presented. Use-case scenario one is a comparison of the distribution and prevalence of plasmid-encoded virulence factor genes among 249 Cronobacter strains with similar attributes to that of nine Cronobacter isolates from recent cases obtained between March and October, 2010–2011. To highlight PATRN's data management and trend finding tools, analysis of datasets, stored in PATRN as part of an ongoing surveillance project to identify the predominant molecular serogroups among Cronobacter sakazakii isolates observed in the USA is shown. Use-case scenario two demonstrates the secure workspace available for private users to upload and analyze sensitive data, and for collating cross-platform datasets to identify and validate congruent datapoints. SNP datasets from WGS assemblies and pan-genome microarrays are analyzed in a combinatorial fashion to determine relatedness of 33 Salmonella enterica strains to six strains collected as part of an outbreak investigation. Use-case scenario three utilizes published surveillance results that describe the incidence and sources of O157:H7 E. coli isolates associated with a produce pre-harvest surveillance study that occurred during 2002–2006. In summary, PATRN is a web-based integrated platform containing tools for the management, analysis and visualization of data about foodborne pathogens.

Tracing genomic variations in two highly virulent Yersinia enterocolitica strains with unequal ability to compete for host colonization

BackgroundYersinia enterocolitica is a gastrointestinal foodborne pathogen found worldwide and which especially affects infants and young children. While different bioserotypes have been associated with varying pathogenicity, research on Y. enterocolitica is mainly conducted on the highly virulent mouse-lethal strains of biotype 1B and serotype O:8. We demonstrate here that two Y. enterocolitica bioserotype 1B/O:8 strains, 8081 and WA-314, display different virulence and fitness properties in a mouse model. In vivo co-infection experiments revealed that strain WA-314 overcomes strain 8081 in the colonization of spleen and liver. To trace the reasons of this incongruity, we present here the first high-quality sequence of the whole genome of strain WA-314 and compare it to the published genome of strain 8081.ResultsRegions previously accepted as unique to strain 8081, like the YAPI and YGI-3 genomic islands, are absent from strain WA-314, confirming their strain-specificity. On the other hand, some fitness- and bacterial competition-associated features, such as a putative colicin cluster and a xenobiotic-acyltransferase-encoding gene, are unique to strain WA-314. Additional acquisitions of strain WA-314 are seven prophage-like regions. One of these prophages, the 28-kb P4-like prophage YWA-4, encodes a PilV-like protein that may be used for adhesion to and invasion of the intestinal cells. Furthermore, a putative autotransporter and two type 1 fimbrial proteins of strain WA-314 show a sequence similarity <50% with the orthologous proteins in strain 8081. The dissimilar sequences of these proteins indicate possible different functions or interaction modes, reflecting the specific adhesion properties of Y. enterocolitica strains 8081 and WA-314 and thus the different efficiency of host colonization. Further important differences were found in two pYV plasmid-encoded virulence factors, YopM and YscP. The impact of these differences on virulence is discussed.ConclusionsOur study emphasizes that the virulence of pathogens can be increased, by acquiring new genes and/or improving the function of essential virulence proteins, resulting in permanently hyper-virulent strains. This work also highlights the importance of addressing genetic and phenotypic variations among closely related bacterial strains, even those belonging to the same bioserotype.

Comparative Genomics and stx Phage Characterization of LEE-Negative Shiga Toxin-Producing Escherichia coli

Infection by Escherichia coli and Shigella species are among the leading causes of death due to diarrheal disease in the world. Shiga toxin-producing E. coli (STEC) that do not encode the locus of enterocyte effacement (LEE-negative STEC) often possess Shiga toxin gene variants and have been isolated from humans and a variety of animal sources. In this study, we compare the genomes of nine LEE-negative STEC harboring various stx alleles with four complete reference LEE-positive STEC isolates. Compared to a representative collection of prototype E. coli and Shigella isolates representing each of the pathotypes, the whole genome phylogeny demonstrated that these isolates are diverse. Whole genome comparative analysis of the 13 genomes revealed that in addition to the absence of the LEE pathogenicity island, phage-encoded genes including non-LEE encoded effectors, were absent from all nine LEE-negative STEC genomes. Several plasmid-encoded virulence factors reportedly identified in LEE-negative STEC isolates were identified in only a subset of the nine LEE-negative isolates further confirming the diversity of this group. In combination with whole genome analysis, we characterized the lambdoid phages harboring the various stx alleles and determined their genomic insertion sites. Although the integrase gene sequence corresponded with genomic location, it was not correlated with stx variant, further highlighting the mosaic nature of these phages. The transcription of these phages in different genomic backgrounds was examined. Expression of the Shiga toxin genes, stx1 and/or stx2, as well as the Q genes, were examined with quantitative reverse transcriptase polymerase chain reaction assays. A wide range of basal and induced toxin induction was observed. Overall, this is a first significant foray into the genome space of this unexplored group of emerging and divergent pathogens.

Genome Sequences and Phylogenetic Analysis of K88- and F18-Positive Porcine Enterotoxigenic Escherichia coli

Porcine enterotoxigenic Escherichia coli (ETEC) continues to result in major morbidity and mortality in the swine industry via postweaning diarrhea. The key virulence factors of ETEC strains, their serotypes, and their fimbrial components have been well studied. However, most studies to date have focused on plasmid-encoded traits related to colonization and toxin production, and the chromosomal backgrounds of these strains have been largely understudied. Here, we generated the genomic sequences of K88-positive and F18-positive porcine ETEC strains and examined the phylogenetic distribution of clinical porcine ETEC strains and their plasmid-associated genetic content. The genomes of porcine ETEC strains UMNK88 and UMNF18 were both found to contain remarkable plasmid complements containing known virulence factors, potential novel virulence factors, and antimicrobial resistance-associated elements. The chromosomes of these strains also possessed several unique genomic islands containing hypothetical genes with similarity to classical virulence factors, although phage-associated genomic islands dominated the accessory genomes of these strains. Phylogenetic analysis of 78 clinical isolates associated with neonatal and porcine diarrhea revealed that a limited subset of porcine ETEC lineages exist that generally contain common toxin and fimbrial profiles, with many of the isolates belonging to the ST10, ST23, and ST169 multilocus sequencing types. These lineages were generally distinct from existing human ETEC database isolates. Overall, most porcine ETEC strains appear to have emerged from a limited subset of E. coli lineages that either have an increased propensity to carry plasmid-encoded virulence factors or have the appropriate ETEC core genome required for virulence.

Prospective genomic characterization of the German enterohemorrhagic Escherichia coli O104:H4 outbreak by rapid next generation sequencing technology.

An ongoing outbreak of exceptionally virulent Shiga toxin (Stx)-producing Escherichia coli O104:H4 centered in Germany, has caused over 830 cases of hemolytic uremic syndrome (HUS) and 46 deaths since May 2011. Serotype O104:H4, which has not been detected in animals, has rarely been associated with HUS in the past. To prospectively elucidate the unique characteristics of this strain in the early stages of this outbreak, we applied whole genome sequencing on the Life Technologies Ion Torrent PGM™ sequencer and Optical Mapping to characterize one outbreak isolate (LB226692) and a historic O104:H4 HUS isolate from 2001 (01-09591). Reference guided draft assemblies of both strains were completed with the newly introduced PGM™ within 62 hours. The HUS-associated strains both carried genes typically found in two types of pathogenic E. coli, enteroaggregative E. coli (EAEC) and enterohemorrhagic E. coli (EHEC). Phylogenetic analyses of 1,144 core E. coli genes indicate that the HUS-causing O104:H4 strains and the previously published sequence of the EAEC strain 55989 show a close relationship but are only distantly related to common EHEC serotypes. Though closely related, the outbreak strain differs from the 2001 strain in plasmid content and fimbrial genes. We propose a model in which EAEC 55989 and EHEC O104:H4 strains evolved from a common EHEC O104:H4 progenitor, and suggest that by stepwise gain and loss of chromosomal and plasmid-encoded virulence factors, a highly pathogenic hybrid of EAEC and EHEC emerged as the current outbreak clone. In conclusion, rapid next-generation technologies facilitated prospective whole genome characterization in the early stages of an outbreak.

Structural Insights into Inhibition of Bacillus anthracis Sporulation by a Novel Class of Non-heme Globin Sensor Domains

Pathogenesis by Bacillus anthracis requires coordination between two distinct activities: plasmid-encoded virulence factor expression (which protects vegetative cells from immune surveillance during outgrowth and replication) and chromosomally encoded sporulation (required only during the final stages of infection). Sporulation is regulated by at least five sensor histidine kinases that are activated in response to various environmental cues. One of these kinases, BA2291, harbors a sensor domain that has ∼35% sequence identity with two plasmid proteins, pXO1-118 and pXO2-61. Because overexpression of pXO2-61 (or pXO1-118) inhibits sporulation of B. anthracis in a BA2291-dependent manner, and pXO2-61 expression is strongly up-regulated by the major virulence gene regulator, AtxA, it was suggested that their function is to titrate out an environmental signal that would otherwise promote untimely sporulation. To explore this hypothesis, we determined crystal structures of both plasmid-encoded proteins. We found that they adopt a dimeric globin fold but, most unusually, do not bind heme. Instead, they house a hydrophobic tunnel and hydrophilic chamber that are occupied by fatty acid, which engages a conserved arginine and chloride ion via its carboxyl head group. In vivo, these domains may therefore recognize changes in fatty acid synthesis, chloride ion concentration, and/or pH. Structure-based comparisons with BA2291 suggest that it binds ligand and dimerizes in an analogous fashion, consistent with the titration hypothesis. Analysis of newly sequenced bacterial genomes points to the existence of a much broader family of non-heme, globin-based sensor domains, with related but distinct functionalities, that may have evolved from an ancestral heme-linked globin.