RESUMO
Spore-forming pathogens have a unique capacity to thrive in diverse environments, and with temporal persistence afforded through their ability to sporulate. Their prevalence in diverse ecosystems requires a One Health approach to identify critical reservoirs and outbreak-associated transmission chains, given their capacity to freely move across soils, waterways, foodstuffs and as commensals or infecting pathogens in human and animal populations. Among anaerobic spore-formers, genomic resources for pathogens including C. botulinum, C. difficile, and C. perfringens enable our capacity to identify common and unique factors that support their persistence in diverse reservoirs and capacity to cause disease. Publicly available genomic resources for spore-forming pathogens at NCBI's Pathogen Detection program aid outbreak investigations and longitudinal monitoring in national and international programs in public health and food safety, as well as for local healthcare systems. These tools also enable research to derive new knowledge regarding disease pathogenesis, and to inform strategies in disease prevention and treatment. As global community resources, the continued sharing of strain genomic data and phenotypes further enhances international resources and means to develop impactful applications. We present examples showing use of these resources in surveillance, including capacity to assess linkages among clinical, environmental, and foodborne reservoirs and to further research investigations into factors promoting their persistence and virulence in different settings.
Assuntos
Infecções por Clostridium , Saúde Única , Humanos , Infecções por Clostridium/microbiologia , Infecções por Clostridium/epidemiologia , Animais , Clostridium/genética , Clostridium/isolamento & purificação , Clostridium/classificação , Surtos de Doenças/prevenção & controle , Genômica/métodos , Toxinas Bacterianas/genéticaRESUMO
Spore-forming pathogens have a unique capacity to thrive in diverse environments, and with temporal persistence afforded through their ability to sporulate. These behaviors require a One Health approach to identify critical reservoirs and outbreak-associated transmission chains, given their capacity to freely move across soils, waterways, foodstuffs, and as commensals or infecting pathogens in human and veterinary populations. Among anaerobic spore-formers, genomic resources for pathogens including C. botulinum, C. difficile, and C. perfringens enable our capacity to identify common and unique factors that support their persistence in diverse reservoirs and capacity to cause disease. Publicly available genomic resources for spore-forming pathogens at NCBI's Pathogen Detection program aid outbreak investigations and longitudinal monitoring in national and international programs in public health and food safety, as well as for local healthcare systems. These tools also enable research to derive new knowledge regarding disease pathogenesis, and to inform strategies in disease prevention and treatment. As global community resources, the continued sharing of strain genomic data and phenotypes further enhances international resources and means to develop impactful applications. We present examples showing use of these resources in surveillance, including capacity to assess linkages among clinical, environmental, and foodborne reservoirs and to further research investigations into factors promoting their persistence and virulence in different settings.
RESUMO
Clostridioides difficile (CD) is a sporulating and toxin-producing nosocomial pathogen that opportunistically infects the gut, particularly in patients with depleted microbiota after antibiotic exposure. Metabolically, CD rapidly generates energy and substrates for growth from Stickland fermentations of amino acids, with proline being a preferred reductive substrate. To investigate the in vivo effects of reductive proline metabolism on C. difficile's virulence in an enriched gut nutrient environment, we evaluated wild-type and isogenic ΔprdB strains of ATCC43255 on pathogen behaviors and host outcomes in highly susceptible gnotobiotic mice. Mice infected with the ΔprdB mutant demonstrated extended survival via delayed colonization, growth and toxin production but ultimately succumbed to disease. In vivo transcriptomic analyses demonstrated how the absence of proline reductase activity more broadly disrupted the pathogen's metabolism including failure to recruit oxidative Stickland pathways, ornithine transformations to alanine, and additional pathways generating growth-promoting substrates, contributing to delayed growth, sporulation, and toxin production. Our findings illustrate the central role for proline reductase metabolism to support early stages of C. difficile colonization and subsequent impact on the pathogen's ability to rapidly expand and cause disease.
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Methicillin-resistant Staphylococcus aureus (MRSA) infections cause substantive morbidity and mortality in neonates. Using publicly available resources from the National Center of Biotechnology Information (NCBI) and Food and Drug Administration's (FDA) GalaxyTrakr pipeline, we illustrate the dynamics of MRSA colonization and infection in neonates. Over 217 days of prospective surveillance, analyses revealed concurrent MRSA transmission chains affecting 11 of 17 MRSA-colonized patients (65%), with two clusters that demonstrated intervals of more than a month among the appearance of isolates. All MRSA infected neonates (n = 3) showed previous colonization with the infecting strain. GalaxyTrakr clustering of the NICU strains, in the context of 21,521 international isolates deposited in NCBI's Pathogen Detection Resource, revealed NICU isolates to be distinct from adult MRSA strains seen locally and internationally. Clustering of the NICU strains within an international context enhanced the resolution of strain clusters and supported the rule-out of suspected, local transmission events within the NICU. Analyses also identified sequence type 1535 isolates, emergent in the Middle East, carrying a unique SCCmec with fusC and aac(6')-Ie/aph(2'')-1a that provided a multidrug-resistant phenotype. NICU genomic pathogen surveillance, leveraging public repositories and outbreak detection tools, supports rapid identification of cryptic MRSA clusters, and can inform infection prevention interventions for this vulnerable patient population. Results demonstrate that sporadic infections in the NICU may be indicative of hidden chains of asymptomatic transmission best identified with sequenced-based approaches.
Assuntos
Infecção Hospitalar , Staphylococcus aureus Resistente à Meticilina , Infecções Estafilocócicas , Humanos , Recém-Nascido , Staphylococcus aureus Resistente à Meticilina/genética , Infecções Estafilocócicas/epidemiologia , Unidades de Terapia Intensiva Neonatal , Estudos Prospectivos , Controle de Infecções/métodos , Surtos de Doenças/prevenção & controle , Genômica , Infecção Hospitalar/epidemiologiaRESUMO
We present predictive models for comprehensive systems analysis of Clostridioides difficile, the etiology of pseudomembranous colitis. By leveraging 151 published transcriptomes, we generated an EGRIN model that organizes 90% of C. difficile genes into a transcriptional regulatory network of 297 co-regulated modules, implicating genes in sporulation, carbohydrate transport, and metabolism. By advancing a metabolic model through addition and curation of metabolic reactions including nutrient uptake, we discovered 14 amino acids, diverse carbohydrates, and 10 metabolic genes as essential for C. difficile growth in the intestinal environment. Finally, we developed a PRIME model to uncover how EGRIN-inferred combinatorial gene regulation by transcription factors, such as CcpA and CodY, modulates essential metabolic processes to enable C. difficile growth relative to commensal colonization. The C. difficile interactive web portal provides access to these model resources to support collaborative systems-level studies of context-specific virulence mechanisms in C. difficile.
Assuntos
Clostridioides difficile , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clostridioides , Clostridioides difficile/genética , Regulação Bacteriana da Expressão Gênica , Redes e Vias Metabólicas/genética , Análise de SistemasRESUMO
Leveraging systems biology approaches, we illustrate how metabolically distinct species of Clostridia protect against or worsen Clostridioides difficile infection in mice by modulating the pathogen's colonization, growth, and virulence to impact host survival. Gnotobiotic mice colonized with the amino acid fermenter Paraclostridium bifermentans survive infection with reduced disease severity, while mice colonized with the butyrate-producer, Clostridium sardiniense, succumb more rapidly. Systematic in vivo analyses revealed how each commensal alters the gut-nutrient environment to modulate the pathogen's metabolism, gene regulatory networks, and toxin production. Oral administration of P. bifermentans rescues conventional, clindamycin-treated mice from lethal C. difficile infection in a manner similar to that of monocolonized animals, thereby supporting the therapeutic potential of this commensal species. Our findings lay the foundation for mechanistically informed therapies to counter C. difficile disease using systems biology approaches to define host-commensal-pathogen interactions in vivo.
Assuntos
Clostridiales/fisiologia , Clostridioides difficile/patogenicidade , Infecções por Clostridium/microbiologia , Infecções por Clostridium/terapia , Clostridium/fisiologia , Simbiose , Aminoácidos/metabolismo , Animais , Arginina/metabolismo , Butiratos/metabolismo , Ceco/metabolismo , Ceco/microbiologia , Clostridiales/crescimento & desenvolvimento , Clostridioides difficile/genética , Clostridioides difficile/fisiologia , Clostridium/crescimento & desenvolvimento , Fermentação , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Redes Reguladoras de Genes , Vida Livre de Germes , Camundongos , Índice de Gravidade de Doença , Biologia de Sistemas , VirulênciaRESUMO
Escherichia coli O145 serogroup is one of the big six non-O157 Shiga toxin producing E. coli (STEC) that causes foodborne illnesses in the United States and other countries. Cattle are a major reservoir of STEC, which harbor them in their hindgut and shed in the feces. Cattle feces is the main source of hide and subsequent carcass contaminations during harvest leading to foodborne illnesses in humans. The objective of our study was to determine the virulence potential of STEC O145 strains isolated from cattle feces and hide samples. A total of 71 STEC O145 strains isolated from cattle feces (n = 16), hide (n = 53), and human clinical samples (n = 2) were used in the study. The strains were subjected to whole genome sequencing using Illumina MiSeq platform. The average draft genome size of the fecal, hide, and human clinical strains were 5.41, 5.28, and 5.29 Mb, respectively. The average number of genes associated with mobile genetic elements was 260, 238, and 259, in cattle fecal, hide, and human clinical strains, respectively. All strains belonged to O145:H28 serotype and carried eae subtype γ. Shiga toxin 1a was the most common Shiga toxin gene subtype among the strains, followed by stx2a and stx2c. The strains also carried genes encoding type III secretory system proteins, nle, and plasmid-encoded virulence genes. Phylogenetic analysis revealed clustering of cattle fecal strains separately from hide strains, and the human clinical strains were more closely related to the hide strains. All the strains belonged to sequence type (ST)-32. The virulence gene profile of STEC O145 strains isolated from cattle sources was similar to that of human clinical strains and were phylogenetically closely related to human clinical strains. The genetic analysis suggests the potential of cattle STEC O145 strains to cause human illnesses. Inclusion of more strains from cattle and their environment in the analysis will help in further elucidation of the genetic diversity and virulence potential of cattle O145 strains.
Assuntos
Escherichia coli/genética , Escherichia coli/patogenicidade , Fezes/microbiologia , Sequenciamento Completo do Genoma , Animais , Sequência de Bases , Sistemas CRISPR-Cas/genética , Bovinos , Farmacorresistência Bacteriana/genética , Escherichia coli/isolamento & purificação , Tamanho do Genoma , Genoma Bacteriano , Humanos , Filogenia , Plasmídeos/genética , Prófagos/genética , Virulência/genéticaRESUMO
Escherichia coli O103, harbored in the hindgut and shed in the feces of cattle, can be enterohemorrhagic (EHEC), enteropathogenic (EPEC), or putative non-pathotype. The genetic diversity particularly that of virulence gene profiles within O103 serogroup is likely to be broad, considering the wide range in severity of illness. However, virulence descriptions of the E. coli O103 strains isolated from cattle feces have been primarily limited to major genes, such as Shiga toxin and intimin genes. Less is known about the frequency at which other virulence genes exist or about genes associated with the mobile genetic elements of E. coli O103 pathotypes. Our objective was to utilize whole genome sequencing (WGS) to identify and compare major and putative virulence genes of EHEC O103 (positive for Shiga toxin gene, stx1, and intimin gene, eae; n = 43), EPEC O103 (negative for stx1 and positive for eae; n = 13) and putative non-pathotype O103 strains (negative for stx and eae; n = 13) isolated from cattle feces. Six strains of EHEC O103 from human clinical cases were also included. All bovine EHEC strains (43/43) and a majority of EPEC (12/13) and putative non-pathotype strains (12/13) were O103:H2 serotype. Both bovine and human EHEC strains had significantly larger average genome sizes (P < 0.0001) and were positive for a higher number of adherence and toxin-based virulence genes and genes on mobile elements (prophages, transposable elements, and plasmids) than EPEC or putative non-pathotype strains. The genome size of the three pathotypes positively correlated (R2 = 0.7) with the number of genes carried on mobile genetic elements. Bovine strains clustered phylogenetically by pathotypes, which differed in several key virulence genes. The diversity of E. coli O103 pathotypes shed in cattle feces is likely reflective of the acquisition or loss of virulence genes carried on mobile genetic elements.
Assuntos
Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Fezes/microbiologia , Genômica/métodos , Sequências Repetitivas Dispersas , Fatores de Virulência/genética , Animais , Bovinos , Escherichia coli/classificação , Escherichia coli/isolamento & purificação , Escherichia coli/patogenicidade , Variação Genética , Humanos , FilogeniaRESUMO
Primary virulence factors of Pseudomonas syringae pv. tomato DC3000 include the phytotoxin coronatine (COR) and a repertoire of 29 effector proteins injected into plant cells by the type III secretion system (T3SS). DC3000 derivatives differentially producing COR, the T3SS machinery and subsets of key effectors were constructed and assayed in leaves of Nicotiana benthamiana. Bacteria were inoculated by the dipping of whole plants and assayed for population growth and the production of chlorotic spots on leaves. The strains fell into three classes. Class I strains are T3SS+ but functionally effectorless, grow poorly in planta and produce faint chlorotic spots only if COR+ . Class II strains are T3SS- or, if T3SS+ , also produce effectors AvrPtoB and HopM1. Class II strains grow better than class I strains in planta and, if COR+ , produce robust chlorotic spots. Class III strains are T3SS+ and minimally produce AvrPtoB, HopM1 and three other effectors encoded in the P. syringae conserved effector locus. These strains differ from class II strains in growing better in planta, and produce chlorotic spots without COR if the precursor coronafacic acid is produced. Assays for chlorotic spot formation, in conjunction with pressure infiltration of low-level inoculum and confocal microscopy of fluorescent protein-labelled bacteria, revealed that single bacteria in the apoplast are capable of producing colonies and associated leaf spots in a 1 : 1 : 1 manner. However, COR makes no significant contribution to the bacterial colonization of the apoplast, but, instead, enables a gratuitous, semi-quantitative, surface indicator of bacterial growth, which is determined by the strain's effector composition.
Assuntos
Aminoácidos/metabolismo , Indenos/metabolismo , Nicotiana/microbiologia , Folhas de Planta/microbiologia , Pseudomonas syringae/metabolismo , Pseudomonas syringae/patogenicidade , Sistemas de Secreção Tipo III/metabolismo , Pseudomonas syringae/genética , VirulênciaRESUMO
The enterohemorrhagic pathotype represents a minor proportion of the Escherichia coli O103 strains shed in the feces of cattle. We report here the genome sequences of 43 strains of enterohemorrhagic E. coli (EHEC) O103:H2 isolated from feedlot cattle feces. The genomic analysis will provide information on the genetic diversity and virulence potential of bovine EHEC O103.
RESUMO
Escherichia coli serotype O157:H7 is a zoonotic food- and waterborne bacterial pathogen that causes a high hospitalization rate and can cause life-threatening complications. Increasingly, E. coli O157:H7 infections appear to originate from fresh produce. Ruminants, such as cattle, are a prominent reservoir of E. coli O157:H7 in the United States. California is one of the most agriculturally productive regions in the world for fresh produce, beef, and milk. The close proximity of fresh produce and cattle presents food safety challenges on a uniquely large scale. We performed a survey of E. coli O157:H7 on 20 farms in California to observe the regional diversity and prevalence of E. coli O157:H7. Isolates were obtained from enrichment cultures of cow feces. Some farms were sampled on two dates. Genomes from isolates were sequenced to determine their relatedness and pathogenic potential. E. coli O157:H7 was isolated from approximately half of the farms. The point prevalence of E. coli O157:H7 on farms was highly variable, ranging from zero to nearly 90%. Within farms, generally one or a few lineages were found, even when the rate of isolation was high. On farms with high isolation rates, a single clonal lineage accounted for most of the isolates. Farms that were visited months after the first visit might have had the same lineages of E. coli O157:H7. Strains of E. coli O157:H7 may be persistent for months on farms.IMPORTANCE This survey of 20 cow-calf operations from different regions of California provides an in depth look at resident Escherichia coli O157:H7 populations at the molecular level. E. coli O157:H7 is found to have a highly variable prevalence, and with whole-genome sequencing, high prevalences in herds were found to be due to a single lineage shed from multiple cows. Few repeat lineages were found between farms in this area; therefore, we predict that E. coli O157:H7 has significant diversity in this area beyond what is detected in this survey. All isolates from this study were found to have pathogenic potential based on the presence of key virulence gene sequences. This represents a novel insight into pathogen diversity within a single subtype and will inform future attempts to survey regional pathogen populations.
Assuntos
Doenças dos Bovinos/microbiologia , Infecções por Escherichia coli/veterinária , Escherichia coli O157/isolamento & purificação , Animais , California/epidemiologia , Bovinos , Doenças dos Bovinos/epidemiologia , Infecções por Escherichia coli/epidemiologia , Infecções por Escherichia coli/microbiologia , Escherichia coli O157/classificação , Escherichia coli O157/genética , Fezes/microbiologia , Genômica , FilogeniaRESUMO
Enteropathogenic Escherichia coli (EPEC) pathotype represents a minor proportion of E. coli O103 strains shed in the feces of feedlot cattle. The draft genome sequences of 13 strains of EPEC O103 are reported here. The availability of the genome sequences will help in the assessment of genetic diversity and virulence potential of bovine EPEC O103.
RESUMO
BACKGROUND: Effector proteins are translocated into host cells by plant-pathogens to undermine pattern-triggered immunity (PTI), the plant response to microbe-associated molecular patterns that interferes with the infection process. Individual effectors are found in variable repertoires where some constituents target the same pathways. The effector protein AvrPto from Pseudomonas syringae has a core domain (CD) and C-terminal domain (CTD) that each promotes bacterial growth and virulence in tomato. The individual contributions of each domain and whether they act redundantly is unknown. RESULTS: We use RNA-Seq to elucidate the contribution of the CD and CTD to the suppression of PTI in tomato leaves 6 h after inoculation. Unexpectedly, each domain alters transcript levels of essentially the same genes but to a different degree. This difference, when quantified, reveals that although targeting the same host genes, the two domains act synergistically. AvrPto has a relatively greater effect on genes whose expression is suppressed during PTI, and the effect on these genes appears to be diminished by saturation. CONCLUSIONS: RNA-Seq profiles can be used to observe relative contributions of effector subdomains to PTI suppression. Our analysis shows the CD and CTD multiplicatively affect the same gene transcript levels with a greater relative impact on genes whose expression is suppressed during PTI. The higher degree of up-regulation versus down-regulation during PTI is plausibly an evolutionary adaptation against effectors that target immune signaling.
Assuntos
Proteínas de Bactérias/metabolismo , Doenças das Plantas/microbiologia , Imunidade Vegetal/genética , Pseudomonas syringae/patogenicidade , Solanum lycopersicum/genética , Transcriptoma , Regulação para Baixo , Regulação da Expressão Gênica de Plantas , Solanum lycopersicum/microbiologia , Análise de Sequência de RNA , Regulação para CimaRESUMO
Bacterial flagellin is perceived as a microbe (or pathogen)-associated molecular pattern (MAMP or PAMP) by the extracellular pattern recognition receptors, FLS2 and TLR5, of plants and mammals respectively. Flagellin accidently translocated into mammalian cells by pathogen type III secretion systems (T3SSs) is recognized by nucleotide-binding leucine-rich repeat receptor NLRC4 as a pattern of pathogenesis and induces a death-associated immune response. The non-pathogen Pseudomonas fluorescens Pf0-1, expressing a Pseudomonas syringae T3SS, and the plant pathogen P. syringae pv. tomato DC3000 were used to seek evidence of an analogous cytoplasmic recognition system for flagellin in the model plant Nicotiana benthamiana. Flagellin (FliC) was secreted in culture and translocated into plant cells by the T3SS expressed in Pf0-1 and DC3000 and in their ΔflgGHI flagellar pathway mutants. ΔfliC and ΔflgGHI mutants of Pf0-1 and DC3000 were strongly reduced in elicitation of reactive oxygen species production and in immunity induction as indicated by the ability of challenge bacteria inoculated 6 h later to translocate a type III effector-reporter and to elicit effector-triggered cell death. Agrobacterium-mediated transient expression in N. benthamiana of FliC with or without a eukaryotic export signal peptide, coupled with virus-induced gene silencing of FLS2, revealed no immune response that was not FLS2 dependent. Transiently expressed FliC from DC3000 and Pectobacterium carotovorum did notinduce cell death in N. benthamiana, tobacco or tomato leaves. Flagellin is the major Pseudomonasâ MAMP perceived by N. benthamiana, and although flagellin secretion through the plant cell wall by the T3SS may partially contribute to FLS2-dependent immunity, flagellin in the cytosol does not elicit immune-associated cell death. We postulate that a death response to translocated MAMPs would produce vulnerability to the many necrotrophic pathogens of plants, such as P. carotovorum, which differ from P. syringae and other (hemi)biotrophic pathogens in benefitting from death-associated immune responses.
Assuntos
Sistemas de Secreção Bacterianos , Flagelina/metabolismo , Imunidade Inata , Nicotiana/imunologia , Pseudomonas fluorescens/metabolismo , Pseudomonas syringae/metabolismo , Agrobacterium/genética , Pectobacterium carotovorum/genética , Pectobacterium carotovorum/metabolismo , Pseudomonas fluorescens/genética , Pseudomonas syringae/genética , Receptores Imunológicos/metabolismo , Transformação GenéticaRESUMO
Pseudomonas syringae pv. tomato DC3000 produces the phytotoxin coronatine, a major determinant of the leaf chlorosis associated with DC3000 pathogenesis. The DC3000 PSPTO4723 (cmaL) gene is located in a genomic region encoding type III effectors; however, it promotes chlorosis in the model plant Nicotiana benthamiana in a manner independent of type III secretion. Coronatine is produced by the ligation of two moieties, coronafacic acid (CFA) and coronamic acid (CMA), which are produced by biosynthetic pathways encoded in separate operons. Cross-feeding experiments, performed in N. benthamiana with cfa, cma, and cmaL mutants, implicate CmaL in CMA production. Furthermore, analysis of bacterial supernatants under coronatine-inducing conditions revealed that mutants lacking either the cma operon or cmaL accumulate CFA rather than coronatine, supporting a role for CmaL in the regulation or biosynthesis of CMA. CmaL does not appear to regulate CMA production, since the expression of proteins with known roles in CMA production is unaltered in cmaL mutants. Rather, CmaL is needed for the first step in CMA synthesis, as evidenced by the fact that wild-type levels of coronatine production are restored to a ΔcmaL mutant when it is supplemented with 50 µg/ml l-allo-isoleucine, the starting unit for CMA production. cmaL is found in all other sequenced P. syringae strains with coronatine biosynthesis genes. This characterization of CmaL identifies a critical missing factor in coronatine production and provides a foundation for further investigation of a member of the widespread DUF1330 protein family.