RESUMEN
We report the draft genome sequences of the collection referred to as the Escherichia coli DECA collection, which was assembled to contain representative isolates of the 15 most common diarrheagenic clones in humans (http://shigatox.net/new/). These genomes represent a valuable resource to the community of researchers who examine these enteric pathogens.
Asunto(s)
Diarrea/microbiología , Infecciones por Escherichia coli/microbiología , Escherichia coli/genética , Genoma Bacteriano , Secuencia de Bases , Preescolar , Escherichia coli/clasificación , Escherichia coli/aislamiento & purificación , Femenino , Humanos , Lactante , Masculino , Datos de Secuencia MolecularRESUMEN
Single nucleotide polymorphisms (SNPs) in stable genome regions provide durable measurements of species evolution. We systematically identified each SNP in concatenations of all backbone ORFs in 7 newly or previously sequenced evolutionarily instructive pathogenic Escherichia coli O157:H7, O157:H(-), and O55:H7. The 1,113 synonymous SNPs demonstrate emergence of the largest cluster of this pathogen only in the last millennium. Unexpectedly, shared SNPs within circumscribed clusters of organisms suggest severely restricted survival and limited effective population sizes of pathogenic O157:H7, tenuous survival of these organisms in nature, source-sink evolutionary dynamics, or, possibly, a limited number of mutations that confer selective advantage. A single large segment spanning the rfb-gnd gene cluster is the only backbone region convincingly acquired by recombination as O157 emerged from O55. This concatenomic analysis also supports using SNPs to differentiate closely related pathogens for infection control and forensic purposes. However, constrained radiations raise the possibility of making false associations between isolates.
Asunto(s)
Escherichia coli O157/metabolismo , Secuencia de Bases , Simulación por Computador , Bases de Datos de Ácidos Nucleicos , Escherichia coli O157/genética , Escherichia coli O157/patogenicidad , Escherichia coli O157/efectos de la radiación , Genoma Bacteriano/genética , Modelos Genéticos , Datos de Secuencia Molecular , Polimorfismo de Nucleótido Simple/genética , Recombinación Genética/genéticaRESUMEN
BACKGROUND: Escherichia coli is one of the best studied organisms in all of biology, but its phylogenetic structure has been difficult to resolve with current data and analytical techniques. We analyzed single nucleotide polymorphisms in chromosomes of representative strains to reconstruct the topology of its emergence. RESULTS: The phylogeny of E. coli varies according to the segment of chromosome analyzed. Recombination between extant E. coli groups is largely limited to only three intergroup pairings. CONCLUSIONS: Segment-dependent phylogenies most likely are legacies of a complex recombination history. However, E. coli are now in an epoch in which they no longer broadly share DNA. Using the definition of species as organisms that freely exchange genetic material, this recombinational dormancy could reflect either the end of E. coli as a species, or herald the coalescence of E. coli groups into new species.
Asunto(s)
Escherichia coli/clasificación , Escherichia coli/genética , Filogenia , Recombinación Genética , Datos de Secuencia MolecularRESUMEN
Escherichia coli O157:H7, a toxin-producing food and waterborne bacterial pathogen, has been linked to large outbreaks of gastrointestinal illness for more than two decades. E. coli O157 causes a wide range of clinical illness that varies by outbreak, although factors that contribute to variation in disease severity are poorly understood. Several recent outbreaks involving O157 contamination of fresh produce (e.g., spinach) were associated with more severe disease, as defined by higher hemolytic uremic syndrome and hospitalization frequencies, suggesting that increased virulence has evolved. To test this hypothesis, we developed a system that detects SNPs in 96 loci and applied it to >500 E. coli O157 clinical strains. Phylogenetic analyses identified 39 SNP genotypes that differ at 20% of SNP loci and are separated into nine distinct clades. Differences were observed between clades in the frequency and distribution of Shiga toxin genes and in the type of clinical disease reported. Patients with hemolytic uremic syndrome were significantly more likely to be infected with clade 8 strains, which have increased in frequency over the past 5 years. Genome sequencing of a spinach outbreak strain, a member of clade 8, also revealed substantial genomic differences. These findings suggest that an emergent subpopulation of the clade 8 lineage has acquired critical factors that contribute to more severe disease. The ability to detect and rapidly genotype O157 strains belonging to such lineages is important and will have a significant impact on both disease diagnosis and treatment guidelines.
Asunto(s)
Brotes de Enfermedades , Escherichia coli O157/clasificación , Escherichia coli O157/patogenicidad , Síndrome Hemolítico-Urémico/epidemiología , Filogenia , Algoritmos , Intervalos de Confianza , Escherichia coli O157/genética , Variación Genética , Genoma Bacteriano , Genotipo , Hospitalización , Humanos , Oportunidad Relativa , Polimorfismo de Nucleótido Simple/genética , Análisis de Secuencia de ADN , Toxina Shiga/genética , Spinacia oleracea/microbiología , Factores de Tiempo , Estados Unidos/epidemiología , VirulenciaRESUMEN
Escherichia albertii has been associated with diarrhea in humans but not with disease or infection in animals. However, in December 2004, E. albertii was found, by biochemical and genetic methods, to be the probable cause of death for redpoll finches (Carduelis flammea) in Alaska. Subsequent investigation found this organism in dead and subclinically infected birds of other species from North America and Australia. Isolates from dead finches in Scotland, previously identified as Escherichia coli O86:K61, also were shown to be E. albertii. Similar to the isolates from humans, E. albertii isolates from birds possessed intimin (eae) and cytolethal distending toxin (cdtB) genes but lacked Shiga toxin (stx) genes. Genetic analysis of eae and cdtB sequences, multilocus sequence typing, and pulsed-field gel electrophoresis patterns showed that the E. albertii strains from birds are heterogeneous but similar to isolates that cause disease in humans.
Asunto(s)
Animales Domésticos/microbiología , Animales Salvajes/microbiología , Aves/microbiología , Infecciones por Enterobacteriaceae/veterinaria , Escherichia , Animales , Pollos/microbiología , Patos/microbiología , Electroforesis en Gel de Campo Pulsado , Endotoxinas/genética , Infecciones por Enterobacteriaceae/microbiología , Escherichia/genética , Pinzones/microbiología , Gansos/microbiología , Genes Bacterianos/genética , Datos de Secuencia Molecular , Passeriformes/microbiología , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN , Factores de Virulencia/genéticaRESUMEN
Alternative sigma factor 54 (RpoN) is an important regulator of stress resistance and virulence genes in many bacterial species. In this study, we report on the gene expression alterations that follow rpoN inactivation in Escherichia coli O157 : H7 strain Sakai (Sakai rpoN : : kan), and the influence of RpoN on the acid resistance phenotype. Microarray gene expression profiling revealed the differential expression of 103 genes in SakairpoN : : kan relative to Sakai. This included the growth-phase-dependent upregulation of genes required for glutamate-dependent acid resistance (GDAR) ( gadA, gadB, gadC and gadE), and the downregulation of locus of enterocyte effacement (LEE) genes, which encode a type III secretion system. Upregulation of gad genes in SakairpoN : : kan during exponential growth correlated with increased GDAR and survival in a model stomach system. Complementation of SakairpoN : : kan with a cloned version of rpoN restored acid susceptibility. Genes involved in GDAR regulation, including rpoS (sigma factor 38) and gadE (acid-responsive regulator), were shown to be required for the survival of SakairpoN : : kan by the GDAR mechanism. This study describes the contribution of rpoN to acid resistance and GDAR gene regulation, and reveals RpoN to be an important regulator of stress resistance and virulence genes in E. coli O157 : H7.
Asunto(s)
Ácidos/metabolismo , Escherichia coli O157/patogenicidad , Proteínas de Escherichia coli/metabolismo , Fosfoproteínas/metabolismo , ARN Polimerasa Sigma 54/metabolismo , Escherichia coli O157/genética , Escherichia coli O157/crecimiento & desarrollo , Proteínas de Escherichia coli/genética , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosfoproteínas/genética , ARN Polimerasa Sigma 54/genética , Regulón , VirulenciaRESUMEN
The Escherichia coli O157 : H7 TW14359 strain was implicated in a multi-state outbreak in North America in 2006, which resulted in high rates of severe disease. Similarly, the O157 : H7 RIMD0509952 (Sakai) strain caused the largest O157 : H7 outbreak to date. Both strains were shown to represent divergent phylogenetic lineages. Here we compared global gene expression patterns before and after epithelial cell exposure, as well as the ability to adhere to and invade epithelial cells, between the two outbreak strains. Epithelial cell assays demonstrated a 2.5-fold greater adherence of the TW14359 strain relative to Sakai, while whole-genome microarrays detected significant differential expression of 914 genes, 206 of which had a fold change >/=1.5. Interestingly, most locus of enterocyte effacement (LEE) genes were upregulated in TW14359, whereas flagellar and chemotaxis genes were primarily upregulated in Sakai, suggesting discordant expression of these genes between the two strains. The Shiga toxin 2 genes were also upregulated in the TW14359 strain, as were several pO157-encoded genes that promote adherence, including type II secretion genes and their effectors stcE and adfO. Quantitative RT-PCR confirmed the expression differences detected in the microarray analysis, and expression levels were lower for a subset of LEE genes before versus after exposure to epithelial cells. In all, this study demonstrated the upregulation of major and ancillary virulence genes in TW14359 and of flagellar and chemotaxis genes in Sakai, under conditions that precede intimate bacterial attachment to epithelial cells. Differences in the level of adherence to epithelial cells were also observed, implying that these two phylogenetically divergent O157 : H7 outbreak strains vary in their ability to colonize, or initiate the disease process.
Asunto(s)
Brotes de Enfermedades , Infecciones por Escherichia coli/microbiología , Escherichia coli O157/patogenicidad , Regulación Bacteriana de la Expresión Génica , Animales , Adhesión Bacteriana/genética , Bovinos , Línea Celular , Células Epiteliales/microbiología , Infecciones por Escherichia coli/epidemiología , Infecciones por Escherichia coli/genética , Escherichia coli O157/genética , Escherichia coli O157/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Perfilación de la Expresión Génica , Humanos , Datos de Secuencia Molecular , América del Norte/epidemiología , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Toxina Shiga II/genética , Toxina Shiga II/metabolismo , Especificidad de la Especie , Virulencia/genéticaRESUMEN
Enterotoxigenic Escherichia coli (ETEC) is a common cause of diarrhea among children living in and among travelers visiting developing countries. Human ETEC strains represent an epidemiologically and phenotypically diverse group of pathogens, and there is a need to identify natural groupings of these organisms that may help to explain this diversity. Here, we sought to identify most of the important human ETEC lineages that exist in the E. coli population, because strains that originate from the same lineage may also have inherited many of the same epidemiological and phenotypic traits. We performed multilocus sequence typing (MLST) on 1,019 ETEC isolates obtained from humans in different countries and analyzed the data against a backdrop of MLST data from 1,250 non-ETEC E. coli and eight ETEC isolates from pigs. A total of 42 different lineages were identified, 15 of which, representing 792 (78%) of the strains, were estimated to have emerged >900 years ago. Twenty of the lineages were represented in more than one country. There was evidence of extensive exchange of enterotoxin and colonization factor genes between different lineages. Human and porcine ETEC have probably emerged from the same ancestral ETEC lineage on at least three occasions. Our findings suggest that most ETEC strains circulating in the human population today originate from well-established, globally widespread ETEC lineages. Some of the more important lineages identified here may represent a smaller and more manageable target for the ongoing efforts to develop effective ETEC vaccines.
Asunto(s)
Análisis por Conglomerados , Escherichia coli Enterotoxigénica/clasificación , Escherichia coli Enterotoxigénica/genética , Infecciones por Escherichia coli/microbiología , Infecciones por Escherichia coli/veterinaria , Evolución Molecular , Adulto , Animales , Técnicas de Tipificación Bacteriana , Niño , Preescolar , Dermatoglifia del ADN , ADN Bacteriano/química , ADN Bacteriano/genética , Escherichia coli Enterotoxigénica/aislamiento & purificación , Proteínas de Escherichia coli/genética , Transferencia de Gen Horizontal , Humanos , Análisis de Secuencia de ADN , Porcinos , Factores de Virulencia/genéticaRESUMEN
Escherichia coli O157:H7 strains can be classified into different genotypes based on the presence of specific Shiga toxin-encoding bacteriophage insertion sites. Certain O157:H7 genotypes predominate among human clinical cases (clinical genotypes), while others are more frequently found in bovines (bovine-biased genotypes). To determine whether inherent differences in gene expression explain the variation in infectivity of these genotypes, we compared the expression patterns of clinical genotype 1 strains with those of bovine-biased genotype 5 strains using microarrays. Important O157:H7 virulence factors, including locus of enterocyte effacement genes, the enterohemolysin, and several pO157 genes, showed increased expression in the clinical versus bovine-biased genotypes. In contrast, genes essential for acid resistance (e.g., gadA, gadB, and gadC) and stress fitness were upregulated in bovine-biased genotype 5 strains. Increased expression of acid resistance genes was confirmed functionally using a model stomach assay, in which strains of bovine-biased genotype 5 had a 2-fold-higher survival rate than strains of clinical genotype 1. Overall, these results suggest that the increased prevalence of O157:H7 illness caused by clinical genotype 1 strains is due in part to the overexpression of key virulence genes. The bovine-biased genotype 5 strains, however, are more resistant to adverse environmental conditions, a characteristic that likely facilitates O157:H7 colonization of bovines.
Asunto(s)
Enfermedades de los Bovinos/microbiología , Infecciones por Escherichia coli/microbiología , Infecciones por Escherichia coli/veterinaria , Escherichia coli O157/genética , Perfilación de la Expresión Génica , Animales , Bovinos , Colifagos/genética , Escherichia coli O157/clasificación , Escherichia coli O157/aislamiento & purificación , Proteínas de Escherichia coli/biosíntesis , Proteínas de Escherichia coli/genética , Jugo Gástrico/microbiología , Genotipo , Humanos , Viabilidad Microbiana , Modelos Teóricos , Análisis de Secuencia por Matrices de Oligonucleótidos , Profagos/genética , Estómago/microbiología , Factores de Virulencia/biosíntesis , Factores de Virulencia/genéticaRESUMEN
Integrating laterally acquired virulence genes into the backbone regulatory network is important for the pathogenesis of Escherichia coli O157:H7, which has captured many virulence genes through horizontal transfer during evolution. GadE is an essential transcriptional activator of the glutamate decarboxylase (GAD) system, the most efficient acid resistance (AR) mechanism in E. coli. The full contribution of GadE to the AR and virulence of E. coli O157:H7 remains largely unknown. We inactivated gadE in E. coli O157:H7 Sakai and compared global transcription profiles of the mutant with that of the wild type in the exponential and stationary phases of growth. Inactivation of gadE significantly altered the expression of 60 genes independently of the growth phase and of 122 genes in a growth phase-dependent manner. Inactivation of gadE markedly downregulated the expression of gadA, gadB, and gadC and of many acid fitness island genes. Nineteen genes encoded on the locus of enterocyte effacement (LEE), including ler, showed a significant increase in expression upon gadE inactivation. Inactivation of ler in the DeltagadE strain reversed the effect of gadE deletion on LEE expression, indicating that Ler is necessary for LEE repression by GadE. GadE is also involved in downregulation of LEE expression under conditions of moderately acidic pH. Characterization of AR of the DeltagadE strain revealed that GadE is indispensable for a functional GAD system and for survival of E. coli O157:H7 in a simulated gastric environment. Altogether, these data indicate that GadE is critical for the AR of E. coli O157:H7 and that it plays an important role in virulence by downregulating expression of LEE.
Asunto(s)
Escherichia coli O157/genética , Proteínas de Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Regulón , Factores de Transcripción/metabolismo , Ácidos/metabolismo , Sitios de Unión , Infecciones por Escherichia coli/microbiología , Escherichia coli O157/metabolismo , Escherichia coli O157/patogenicidad , Proteínas de Escherichia coli/genética , Silenciador del Gen , Humanos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Unión Proteica , Factores de Transcripción/genética , VirulenciaRESUMEN
Transmission of group B Streptococcus (GBS) from mothers to neonates during childbirth is a leading cause of neonatal sepsis and meningitis. Although subtyping tools have identified specific GBS phylogenetic lineages that are important in neonatal disease, little is known about the genetic diversity of these lineages or the roles that recombination and selection play in the generation of emergent genotypes. Here, we examined genetic variation, selection, and recombination in seven multilocus sequence typing (MLST) loci from 94 invasive, colonizing, and bovine strains representing 38 GBS sequence types and performed DNA sequencing and PCR-based restriction fragment length polymorphism analysis of several putative virulence genes to identify gene content differences between genotypes. Despite the low level of diversity in the MLST loci, a neighbor net analysis revealed a variable range of genetic exchange among the seven clonal complexes (CCs) identified, suggesting that recombination is partly responsible for the diversity observed between genotypes. Recombination is also important for several virulence genes, as some gene alleles had evidence for lateral gene exchange across divergent genotypes. The CC-17 lineage, which is associated with neonatal disease, is relatively homogeneous and therefore appears to have diverged independently with an exclusive set of virulence characteristics. These data suggest that different GBS genetic backgrounds have distinct virulence gene profiles that may be important for disease pathogenesis. Such profiles could be used as markers for the rapid detection of strains with an increased propensity to cause neonatal disease and may be considered useful vaccine targets.
Asunto(s)
Proteínas Bacterianas/genética , Variación Genética , Recombinación Genética , Selección Genética , Streptococcus agalactiae/genética , Streptococcus agalactiae/patogenicidad , Factores de Virulencia/genética , Técnicas de Tipificación Bacteriana , Análisis por Conglomerados , Dermatoglifia del ADN , ADN Bacteriano/genética , Genotipo , Humanos , Filogenia , Polimorfismo de Longitud del Fragmento de Restricción , Análisis de Secuencia de ADN , Infecciones Estreptocócicas/microbiología , Streptococcus agalactiae/aislamiento & purificaciónRESUMEN
In addition to causing diarrhea, Escherichia coli O157:H7 infection can lead to hemolytic-uremic syndrome (HUS), a severe disease characterized by hemolysis and renal failure. Differences in HUS frequency among E. coli O157:H7 outbreaks have been noted, but our understanding of bacterial factors that promote HUS is incomplete. In 2006, in an outbreak of E. coli O157:H7 caused by consumption of contaminated spinach, there was a notably high frequency of HUS. We sequenced the genome of the strain responsible (TW14359) with the goal of identifying candidate genetic factors that contribute to an enhanced ability to cause HUS. The TW14359 genome contains 70 kb of DNA segments not present in either of the two reference O157:H7 genomes. We identified seven putative virulence determinants, including two putative type III secretion system effector proteins, candidate genes that could result in increased pathogenicity or, alternatively, adaptation to plants, and an intact anaerobic nitric oxide reductase gene, norV. We surveyed 100 O157:H7 isolates for the presence of these putative virulence determinants. A norV deletion was found in over one-half of the strains surveyed and correlated strikingly with the absence of stx(1). The other putative virulence factors were found in 8 to 35% of the O157:H7 isolates surveyed, and their presence also correlated with the presence of norV and the absence of stx(1), indicating that the presence of norV may serve as a marker of a greater propensity for HUS, similar to the correlation between the absence of stx(1) and a propensity for HUS.
Asunto(s)
Brotes de Enfermedades , Escherichia coli O157/genética , Escherichia coli O157/patogenicidad , Enfermedades Transmitidas por los Alimentos/epidemiología , Enfermedades Transmitidas por los Alimentos/microbiología , Genoma Bacteriano , Spinacia oleracea/microbiología , ADN Bacteriano/análisis , Síndrome Hemolítico-Urémico/etiología , Polimorfismo Genético , Toxina Shiga II/genética , VirulenciaRESUMEN
BACKGROUND: Evolutionary analyses of enterohemorrhagic Escherichia coli (EHEC) have identified two distantly related clonal groups: EHEC 1, including serotype O157:H7 and its inferred ancestor O55:H7; and EHEC 2, comprised of several serogroups (O26, O111, O118, etc.). These two clonal groups differ in their virulence and global distribution. Although several fully annotated genomic sequences exist for strains of serotype O157:H7, much less is known about the genomic composition of EHEC 2. In this study, we analyzed a set of 24 clinical EHEC 2 strains representing serotypes O26:H11, O111:H8/H11, O118:H16, O153:H11 and O15:H11 from humans and animals by comparative genomic hybridization (CGH) on an oligoarray based on the O157:H7 Sakai genome. RESULTS: Backbone genes, defined as genes shared by Sakai and K-12, were highly conserved in EHEC 2. The proportion of Sakai phage genes in EHEC 2 was substantially greater than that of Sakai-specific bacterial (non-phage) genes. This proportion was inverted in O55:H7, reiterating that a subset of Sakai bacterial genes is specific to EHEC 1. Split decomposition analysis of gene content revealed that O111:H8 was more genetically uniform and distinct from other EHEC 2 strains, with respect to the Sakai O157:H7 gene distribution. Serotype O26:H11 was the most heterogeneous EHEC 2 subpopulation, comprised of strains with the highest as well as the lowest levels of Sakai gene content conservation. Of the 979 parsimoniously informative genes, 15% were found to be compatible and their distribution in EHEC 2 clustered O111:H8 and O118:H16 strains by serotype. CGH data suggested divergence of the LEE island from the LEE1 to the LEE4 operon, and also between animal and human isolates irrespective of serotype. No correlation was found between gene contents and geographic locations of EHEC 2 strains. CONCLUSION: The gene content variation of phage-related genes in EHEC 2 strains supports the hypothesis that extensive modular shuffling of mobile DNA elements has occurred among EHEC strains. These results suggest that EHEC 2 is a multiform pathogenic clonal complex, characterized by substantial intra-serotype genetic variation. The heterogeneous distribution of mobile elements has impacted the diversification of O26:H11 more than other EHEC 2 serotypes.
Asunto(s)
Hibridación Genómica Comparativa , Escherichia coli Enterohemorrágica/genética , Genoma Bacteriano , Animales , Técnicas de Tipificación Bacteriana , ADN Bacteriano/genética , Escherichia coli Enterohemorrágica/clasificación , Genes Bacterianos , Variación Genética , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Filogenia , Profagos/genéticaRESUMEN
Most Shiga toxin-producing Escherichia coli (STEC) infections that are associated with severe sequelae such as hemolytic uremic syndrome (HUS) are caused by attaching and effacing pathogens that carry the locus of enterocyte effacement (LEE). However, a proportion of STEC isolates that do not carry LEE have been associated with HUS. To clarify the emergence of LEE-negative STEC, we compared the genetic composition of the virulence plasmids pO113 and pO157 from LEE-negative and LEE-positive STEC, respectively. The complete nucleotide sequence of pO113 showed that several plasmid genes were shared by STEC O157:H7. In addition, allelic profiling of the ehxA gene demonstrated that pO113 belongs to a different evolutionary lineage than pO157 and that the virulence plasmids of LEE-negative STEC strains were highly related. In contrast, multilocus sequence typing of 17 LEE-negative STEC isolates showed several clonal groups, suggesting that pathogenic LEE-negative STEC has emerged several times throughout its evolution.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Fosfoproteínas/metabolismo , Escherichia coli Shiga-Toxigénica/patogenicidad , Técnicas de Tipificación Bacteriana , ADN Helicasas/genética , Escherichia coli O157/genética , Escherichia coli O157/patogenicidad , Proteínas de Escherichia coli/genética , Proteínas Hemolisinas/genética , Síndrome Hemolítico-Urémico/microbiología , Humanos , Fosfoproteínas/genética , Filogenia , Plásmidos/genética , Análisis de Secuencia de ADN , Escherichia coli Shiga-Toxigénica/genética , Transactivadores/genética , Virulencia/genéticaRESUMEN
Group B streptococci (GBS), a leading cause of neonatal sepsis and meningitis, are transferred to neonates from colonized mothers during childbirth. Prior studies using multilocus sequence typing (MLST) have found specific GBS clones (e.g., sequence type 17 [ST-17]) to be associated with neonatal disease in several geographic locations. Few population-based studies, however, have been conducted to determine the frequency of disease caused by specific GBS clones. MLST was used to assess the genetic diversity of 192 GBS strains from neonates and young children identified by population-based surveillance in Alberta, Canada, from 1993 to 2002. Comparisons were made to 232 GBS strains collected from colonized pregnant women, and all strains were characterized for one of nine capsule (cps) genotypes. A total of 47 STs were identified, and more than 80% of GBS strains were represented by 7 STs that have been shown to predominate in other populations. ST-17 and ST-19 were more prevalent in strains causing early onset disease (EOD) and late onset disease (LOD) than from pregnant women, whereas STs 1, 12, and 23 were more common in pregnant women. In addition, ST-17 strains and close relatives more frequently caused meningitis than sepsis and LOD versus EOD in this population of neonates. Further research is required to better understand why strains belonging to the ST-17 phylogenetic lineage are more likely to cause both LOD and meningitis and may provide clues into the pathogenesis of these conditions.
Asunto(s)
Dermatoglifia del ADN/métodos , Meningitis Bacterianas/epidemiología , Sepsis/epidemiología , Infecciones Estreptocócicas/epidemiología , Streptococcus agalactiae/clasificación , Streptococcus agalactiae/aislamiento & purificación , Adolescente , Canadá/epidemiología , Niño , Preescolar , Análisis por Conglomerados , Femenino , Genotipo , Humanos , Lactante , Recién Nacido , Meningitis Bacterianas/microbiología , Epidemiología Molecular , Polimorfismo Genético , Embarazo , Complicaciones Infecciosas del Embarazo/microbiología , Sepsis/microbiología , Análisis de Secuencia de ADN , Infecciones Estreptocócicas/microbiología , Streptococcus agalactiae/genéticaRESUMEN
Probe hybridization array typing (PHAT) is a previously validated, high-throughput, highly discriminatory binary typing method based on the presence or absence of genetic material. To increase the utility of PHAT, we identified a refined PHAT probe set using 24 known and potential Escherichia coli virulence genes, by which groups similar to multilocus sequence typing (MLST) clonal groups (CGs) could be determined. We PHAT typed 1,132 E. coli isolates, representing at least 62 MLST CGs and diverse disease states, using a "library-on-a-slide" microarray format. Using 24 PHAT probes, all 62 MLST CGs in the representative E. coli collection were distinguished. For major CGs, PHAT correctly classified all sequence types within CG7 and CG17 but misclassified between one and four sequence types for CG13, CG14, CG23, CG38, and CG58, giving an overall sensitivity and specificity of 80.4 and 98.7%, respectively. After application of the PHAT classification to the whole collection, MLST validation of the PHAT probe classification resulted in sensitivities from 0.0 to 100.0% and specificities from 75.0 to 100.0% for individual CGs and an overall sensitivity and specificity of 64.7 and 88.3%, respectively. The refined PHAT probe set is capable of classifying isolates into groups in a manner similar to major clonal complexes of MLST, indicating coevolution between the chromosomal background and the flexible gene pool. Further refinement is needed to distinguish between closely related groups. For analysis of large bacterial collections, PHAT is a relatively time- and cost-efficient method and is ideal for a first level of analysis.
Asunto(s)
Técnicas de Tipificación Bacteriana/métodos , Escherichia coli/clasificación , Escherichia coli/genética , Hibridación de Ácido Nucleico/métodos , Análisis de Secuencia de ADN/métodos , Análisis por Conglomerados , ADN Bacteriano/genética , Genotipo , Humanos , Masculino , Sensibilidad y EspecificidadRESUMEN
Escherichia coli O157:H7 has caused serious outbreaks of food-borne illness via transmission in a variety of food vehicles, including unpasteurized apple juice, dried salami, and spinach. To understand how this pathogen responds to the multiple stresses of the food environment, we compared global transcription patterns before and after exposure to model apple juice. Transcriptomes of mid-exponential- and stationary-phase cells were evaluated after 10 min in model apple juice (pH 3.5) using microarrays probing 4,886 open reading frames. A total of 331 genes were significantly induced upon exposure of cells to model apple juice, including genes involved in the acid, osmotic, and oxidative stress responses as well as the envelope stress response. Acid and osmotic stress response genes, including asr, osmC, osmB, and osmY, were significantly induced in response to model apple juice. Multiple envelope stress responses were activated as evidenced by increased expression of CpxR and Rcs phosphorelay-controlled genes. Genes controlled by CpxR (cpxP, degP, and htpX) were significantly induced 2- to 15-fold upon exposure to apple juice. Inactivation of CpxRA resulted in a significant decrease in survival of O157:H7 in model apple juice compared to the isogenic parent strain. Of the 331 genes induced in model apple juice, 104 are O157-specific genes, including those encoding type three secretion effectors (espJ, espB, espM2, espL3, and espZ). Elucidating the response of O157:H7 to acidic foods provides insight into how this pathogen is able to survive in food matrices and how exposure to foods influences subsequent transmission and virulence.
Asunto(s)
Bebidas/microbiología , Escherichia coli O157/fisiología , Regulación Bacteriana de la Expresión Génica , Malus/microbiología , Escherichia coli O157/efectos de los fármacos , Proteínas de Escherichia coli/biosíntesis , Perfilación de la Expresión Génica , Análisis de Secuencia por Matrices de Oligonucleótidos , Estrés Fisiológico , Regulación hacia ArribaRESUMEN
Extended multilocus sequence typing (MLST) analysis of atypical Escherichia isolates was used to identify five novel phylogenetic clades (CI to CV) among isolates from environmental, human, and animal sources. Analysis of individual housekeeping loci showed that E. coli and its sister clade, CI, remain largely indistinguishable and represent nascent evolutionary lineages. Conversely, clades of similar age (CIII and CIV) were found to be phylogenetically distinct. When all Escherichia lineages (named and unnamed) were evaluated, we found evidence that Escherichia fergusonii has evolved at an accelerated rate compared to E. coli, CI, CIII, CIV, and CV, suggesting that this species is younger than estimated by the molecular clock method. Although the five novel clades were phylogenetically distinct, we were unable to identify a discriminating biochemical marker for all but one of them (CIII) with traditional phenotypic profiling. CIII had a statistically different phenotype from E. coli that resulted from the loss of sucrose and sorbitol fermentation and lysine utilization. The lack of phenotypic distinction has likely hindered the ability to differentiate these clades from typical E. coli, and so their ecological significance and importance for applied and clinical microbiology are yet to be determined. However, our sampling suggests that CIII, CIV, and CV represent environmentally adapted Escherichia lineages that may be more abundant outside the host gastrointestinal tract.
Asunto(s)
Escherichia/clasificación , Escherichia/genética , Adaptación Fisiológica , Animales , Técnicas de Tipificación Bacteriana , Secuencia de Bases , Cartilla de ADN/genética , ADN Bacteriano/genética , Ecosistema , Microbiología Ambiental , Escherichia/aislamiento & purificación , Escherichia/fisiología , Escherichia coli/clasificación , Escherichia coli/genética , Escherichia coli/aislamiento & purificación , Escherichia coli/fisiología , Evolución Molecular , Tracto Gastrointestinal/microbiología , Flujo Génico , Genes Bacterianos , Humanos , Fenotipo , Filogenia , Reacción en Cadena de la Polimerasa , Factores de TiempoRESUMEN
A collection of 54 clinical and agricultural isolates of Burkholderia cenocepacia was analyzed for genetic relatedness by using multilocus sequence typing (MLST), pathogenicity by using onion and nematode infection models, antifungal activity, and the distribution of three marker genes associated with virulence. The majority of clinical isolates were obtained from cystic fibrosis (CF) patients in Michigan, and the agricultural isolates were predominantly from Michigan onion fields. MLST analysis resolved 23 distinct sequence types (STs), 11 of which were novel. Twenty-six of 27 clinical isolates from Michigan were genotyped as ST-40, previously identified as the Midwest B. cenocepacia lineage. In contrast, the 12 agricultural isolates represented eight STs, including ST-122, that were identical to clinical isolates of the PHDC lineage. In general, pathogenicity to onions and the presence of the pehA endopolygalacturonase gene were detected only in one cluster of related strains consisting of agricultural isolates and the PHDC lineage. Surprisingly, these strains were highly pathogenic in the nematode Caenorhabditis elegans infection model, killing nematodes faster than the CF pathogen Pseudomonas aeruginosa PA14 on slow-kill medium. The other strains displayed a wide range of pathogenicity to C. elegans, notably the Midwest clonal lineage which displayed high, moderate, and low virulence. Most strains displayed moderate antifungal activity, although strains with high and low activities were also detected. We conclude that pathogenicity to multiple hosts may be a key factor contributing to the potential of B. cenocepacia to opportunistically infect humans both by increasing the prevalence of the organism in the environment, thereby increasing exposure to vulnerable hosts, and by the selection of virulence factors that function in multiple hosts.
Asunto(s)
Complejo Burkholderia cepacia/patogenicidad , Caenorhabditis elegans/microbiología , Fibrosis Quística/microbiología , Variación Genética , Interacciones Huésped-Patógeno , Cebollas/microbiología , Animales , Antibiosis , Infecciones por Burkholderia/microbiología , Complejo Burkholderia cepacia/clasificación , Complejo Burkholderia cepacia/genética , Humanos , Michigan , Enfermedades de las Plantas/microbiología , Rhizoctonia/crecimiento & desarrollo , Microbiología del SueloRESUMEN
BACKGROUND: Enteropathogenic E. coli (EPEC) is a prominent cause of diarrhoea, and is characterised in part by its carriage of a pathogenicity island: the locus for enterocyte effacement (LEE). EPEC is divided into two subtypes according to the presence of bundle-forming pili (BFP), a fimbrial adhesin that is a virulence determinant of typical EPEC (tEPEC), but is absent from atypical EPEC (aEPEC). Because aEPEC lack BFP, their virulence has been questioned, as they may represent LEE-positive Shiga toxin-producing E. coli (STEC) that have lost the toxin-encoding prophage, or tEPEC that have lost the genes for BFP. To determine if aEPEC isolated from humans in Australia or New Zealand fall into either of these categories, we undertook phylogenetic analysis of 75 aEPEC strains, and compared them with reference strains of EPEC and STEC. We also used PCR and DNA hybridisation to determine if aEPEC carry virulence determinants that could compensate for their lack of BFP. RESULTS: The results showed that aEPEC are highly heterogeneous. Multilocus sequence typing revealed that 61 of 75 aEPEC strains did not belong to known tEPEC or STEC clades, and of those that did, none expressed an O:H serotype that is frequent in tEPEC or STEC strains associated with disease. PCR for each of 18 known virulence-associated determinants of E. coli was positive in less than 15% of strains, apart from NleB which was detected in 30%. Type I fimbriae were expressed by all aEPEC strains, and 12 strains hybridised with DNA probes prepared from either bfpA or bfpB despite being negative in the PCR for bfpA. CONCLUSION: Our findings indicate that clinical isolates of aEPEC obtained from patients in Australia or New Zealand are not derived from tEPEC or STEC, and suggest that functional equivalents of BFP and possibly type I fimbriae may contribute to the virulence of some aEPEC strains.