RESUMEN
In this study, our objective was to evaluate the genetic stability of foodborne bacterial pathogens during serial passage in vitro and persistent in vivo carriage. Six strains of Listeria, Campylobacter, Escherichia, Salmonella, and Vibrio were serially passaged 20 times. Three colonies were picked for whole-genome sequencing (WGS) from passes P0, P5, P10, P15, and P20. In addition, isolates of Salmonella and Escherichia from three patients with persistent infections were sequenced. Genetic stability was evaluated in terms of variations detected in high-quality single-nucleotide polymorphism (hqSNP), core genome multilocus sequence typing (cgMLST), seven-gene MLST, and determinants encoding serotype, antimicrobial resistance (AMR), and virulence. During serial passage, increasing diversity was observed in Listeria, Salmonella, and Vibrio as measured by hqSNPs (from median of 0 SNPs to median of 3-5 SNPs, depending on the organism) and to a lesser extent with cgMLST (from median of 0 alleles to median of 0-5 alleles), while Escherichia and Campylobacter genomes showed minimal variation. The serotype, AMR, and virulence markers remained stable in all organisms. Isolates from persistent infections lasting up to 10 weeks remained genetically stable. However, isolates from a persistent Salmonella enterica ser. Montevideo infection spanning 9 years showed early heterogeneity leading to the emergence of one predominant genotype that continued to evolve over the years, including gains and losses of AMR markers. While the hqSNP and cgMLST variation observed during the serial passage was minimal, culture passages should be limited to as few times as possible before WGS. Our WGS data show that in vivo carriage lasting for a few weeks did not appear to alter the genotype. Longer persistent infections spanning for years, particularly in the presence of selective pressure, may cause changes in the genotype making it challenging to differentiate persistent infections from reinfections.
Asunto(s)
Genoma Bacteriano , Infección Persistente , Humanos , Tipificación de Secuencias Multilocus , Filogenia , Polimorfismo de Nucleótido Simple , Pase Seriado , Secuenciación Completa del GenomaRESUMEN
Since 1996, PulseNet has served as the national laboratory-based surveillance system for the rapid detection of outbreaks caused by foodborne bacterial pathogens in the United States. For the past two decades, pulsed-field gel electrophoresis was the gold standard subtyping method for the pathogens tracked by PulseNet. A new gold standard is now being implemented with the introduction of cost-effective whole genome sequencing (WGS) for analysis of all the organisms tracked by PulseNet. This transformation is a major undertaking that touches every functional aspect of PulseNet, including laboratory workflows, data storage, analysis management and data interpretation, and language used to communicate information (sequence profile nomenclature system). The benefits of implementing WGS go beyond improved discrimination and precision of the data; it provides an opportunity to determine strain characteristics typically obtained through resource-intensive traditional methodologies, for example, species identification, serotyping, virulence, and antimicrobial resistance profiling, all of which can be consolidated into a single WGS workflow. Such a strategy represents a major shift in the workflows currently practiced in most public health laboratories, but one that brings opportunities for streamlining surveillance activities for the network as a whole. In this study, we provide a brief summary of PulseNet's evolution the past decade along with a general description of the challenges and opportunities that lie ahead.
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Brotes de Enfermedades/prevención & control , Enfermedades Transmitidas por los Alimentos/epidemiología , Secuenciación de Nucleótidos de Alto Rendimiento , Vigilancia en Salud Pública , Salud Pública , Humanos , Cooperación Internacional , Laboratorios , Estados Unidos/epidemiologíaRESUMEN
PulseNet USA is the molecular surveillance network for foodborne disease in the United States. The network consists of state and local public health laboratories, as well as food regulatory agencies, that follow PulseNet's standardized protocols to perform pulsed-field gel electrophoresis (PFGE) and whole genome sequencing (WGS) and analyze the results using standardized software. The raw sequences are uploaded to the GenomeTrakr or PulseNet bioprojects at the National Center for Biotechnology Information. The PFGE patterns and analyzed sequence data are uploaded in real time with associated demographic data to the PulseNet national databases managed at the Centers for Disease Control and Prevention. The PulseNet databases are organism specific and provide a central storage location for molecular and demographic data related to an isolate. Sequences are compared in the databases, thereby facilitating the rapid detection of clusters of foodborne diseases that may represent widespread outbreaks. WGS genotyping data, for example, antibiotic resistance and virulence profiles, are also uploaded in real time to the PulseNet databases to improve food safety surveillance activities.
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Bases de Datos como Asunto , Brotes de Enfermedades/prevención & control , Enfermedades Transmitidas por los Alimentos/epidemiología , Laboratorios , Salud Pública , Bases de Datos Factuales , Electroforesis en Gel de Campo Pulsado , Humanos , Vigilancia en Salud Pública , Estados Unidos/epidemiología , Secuenciación Completa del GenomaAsunto(s)
Brotes de Enfermedades , Microbiología Ambiental , Vivienda para Animales , Infecciones por Salmonella/epidemiología , Salmonella enterica/aislamiento & purificación , Animales , Humanos , Michigan/epidemiología , Servicios Postales , Aves de Corral , Infecciones por Salmonella/microbiología , Salmonella enterica/genética , Serogrupo , Estados Unidos/epidemiologíaAsunto(s)
Infecciones Bacterianas/epidemiología , Brotes de Enfermedades , Enfermedades Transmitidas por los Alimentos/epidemiología , Vigilancia de la Población , Salud Pública/métodos , Infecciones Bacterianas/microbiología , Infecciones Bacterianas/prevención & control , Brotes de Enfermedades/prevención & control , Infecciones por Enterobacteriaceae/epidemiología , Infecciones por Enterobacteriaceae/prevención & control , Escherichia coli/genética , Escherichia coli/aislamiento & purificación , Escherichia coli/patogenicidad , Enfermedades Transmitidas por los Alimentos/microbiología , Enfermedades Transmitidas por los Alimentos/prevención & control , Humanos , Listeria/genética , Listeria/aislamiento & purificación , Listeria/patogenicidad , Listeriosis/epidemiología , Listeriosis/prevención & control , Vigilancia de la Población/métodos , Salud Pública/tendencias , Salmonella/genética , Salmonella/aislamiento & purificación , Salmonella/patogenicidad , Estados Unidos , Secuenciación Completa del GenomaRESUMEN
Non-O157 Shiga toxin-producing Escherichia coli (STEC) are foodborne pathogens of growing concern worldwide that have been associated with several recent multistate and multinational outbreaks of foodborne illness. Rapid and sensitive molecular-based bacterial strain discrimination methods are critical for timely outbreak identification and contaminated food source traceback. One such method, multiple-locus variable-number tandem repeat analysis (MLVA), is being used with increasing frequency in foodborne illness outbreak investigations to augment the current gold standard bacterial subtyping technique, pulsed-field gel electrophoresis (PFGE). The objective of this study was to develop a MLVA assay for intra- and inter-serogroup discrimination of six major non-O157 STEC serogroups-O26, O111, O103, O121, O45, and O145-and perform a preliminary internal validation of the method on a limited number of clinical isolates. The resultant MLVA scheme consists of ten variable number tandem repeat (VNTR) loci amplified in three multiplex PCR reactions. Sixty-five unique MLVA types were obtained among 84 clinical non-O157 STEC strains comprised of geographically diverse sporadic and outbreak related isolates. Compared to PFGE, the developed MLVA scheme allowed similar discrimination among serogroups O26, O111, O103, and O121 but not among O145 and O45. To more fully compare the discriminatory power of this preliminary MLVA method to PFGE and to determine its epidemiological congruence, a thorough internal and external validation needs to be performed on a carefully selected large panel of strains, including multiple isolates from single outbreaks.
Asunto(s)
ADN Bacteriano/análisis , ADN Bacteriano/genética , Infecciones por Escherichia coli/microbiología , Repeticiones de Minisatélite , Tipificación Molecular/métodos , Escherichia coli Shiga-Toxigénica/genética , Técnicas de Tipificación Bacteriana/métodos , Electroforesis en Gel de Campo Pulsado , Proteínas de Escherichia coli/genética , Humanos , Serogrupo , Toxina Shiga/biosíntesis , Toxina Shiga/genética , Escherichia coli Shiga-Toxigénica/clasificación , Escherichia coli Shiga-Toxigénica/aislamiento & purificaciónRESUMEN
The objective of this study was to develop a canonical, parsimoniously-informative SNP panel for subtyping Shiga-toxin producing Escherichia coli (STEC) O157:H7 that would be consistent with epidemiological, PFGE, and MLVA clustering of human specimens. Our group had previously identified 906 putative discriminatory SNPs, which were pared down to 391 SNPs based on their prevalence in a test set. The 391 SNPs were screened using a high-throughput form of TaqMan PCR against a set of clinical isolates that represent the most diverse collection of O157:H7 isolates from outbreaks and sporadic cases examined to date. Another 30 SNPs identified by others were also screened using the same method. Two additional targets were tested using standard TaqMan PCR endpoint analysis. These 423 SNPs were reduced to a 32 SNP panel with the almost the same discriminatory value. While the panel partitioned our diverse set of isolates in a manner that was consistent with epidemiological data and PFGE and MLVA phylogenies, it resulted in fewer subtypes than either existing method and insufficient epidemiological resolution in 10 of 47 clusters. Therefore, another round of SNP discovery was undertaken using comparative genomic resequencing of pooled DNA from the 10 clusters with insufficient resolution. This process identified 4,040 potential SNPs and suggested one of the ten clusters was incorrectly grouped. After its removal, there were 2,878 SNPs, of which only 63 were previously identified and 438 occurred across multiple clusters. Among highly clonal bacteria like STEC O157:H7, linkage disequilibrium greatly limits the number of parsimoniously informative SNPs. Therefore, it is perhaps unsurprising that our panel accounted for the potential discriminatory value of numerous other SNPs reported in the literature. We concluded published O157:H7 SNPs are insufficient for effective epidemiological subtyping. However, the 438 multi-cluster SNPs we identified may provide the additional information required.
Asunto(s)
Escherichia coli O157/genética , Polimorfismo de Nucleótido Simple , Escherichia coli Shiga-Toxigénica/genética , Infecciones por Escherichia coli/microbiología , HumanosRESUMEN
This chapter describes the procedure of generating pulsed-field gel electrophoresis (PFGE) profiles (DNA fingerprints) of Shiga toxin-producing Escherichia coli O157:H7 (STEC O157) and non-O157 STEC strains within 48 h, based on the standardized laboratory protocol developed by the Centers for Disease Control and Prevention, USA. The protocol describes the preparation of agarose plugs containing STEC O157 and non-O157 STEC cells, the digestion of bacterial DNA in the plugs using restriction endonuclease enzymes, and the electrophoresis conditions to generate the characteristic PFGE profiles of STEC O157 and non-O157 STEC isolates.
Asunto(s)
Electroforesis en Gel de Campo Pulsado/métodos , Infecciones por Escherichia coli/microbiología , Escherichia coli O157/genética , Escherichia coli Shiga-Toxigénica/genética , Dermatoglifia del ADN , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , Infecciones por Escherichia coli/genética , Escherichia coli O157/aislamiento & purificación , Escherichia coli O157/patogenicidad , Serotipificación , Escherichia coli Shiga-Toxigénica/aislamiento & purificación , Escherichia coli Shiga-Toxigénica/patogenicidadRESUMEN
Shiga toxin-producing Escherichia coli (STEC) are a common cause for food-borne diarrheal illness outbreaks and sporadic cases. Here, we report the availability of the draft genome sequences of 228 STEC strains representing 32 serotypes with known pulsed-field gel electrophoresis (PFGE) types and epidemiological relationships, as well as 12 strains representing other diarrheagenic E. coli pathotypes.
RESUMEN
Salmonella enterica serotype Tennessee strain CDC07-0191 was isolated from the 2006-2007 multistate food-borne outbreak linked to peanut butter in the United States. Here we report a high-quality draft assembly of the genome sequence of this strain, derived from a patient. This is the first reported high-quality draft genome sequence for S. enterica serotype Tennessee, which will enable in-depth studies of its transmission and virulence.
RESUMEN
Cronobacter (formerly known as Enterobacter sakazakii) is a genus comprising seven species regarded as opportunistic pathogens that can be found in a wide variety of environments and foods, including powdered infant formula (PIF). Cronobacter sakazakii, the major species of this genus, has been epidemiologically linked to cases of bacteremia, meningitis in neonates, and necrotizing enterocolitis, and contaminated PIF has been identified as an important source of infection. Robust and reproducible subtyping methods are required to aid in the detection and investigation, of foodborne outbreaks. In this study, a pulsed-field gel electrophoresis (PFGE) protocol was developed and validated for subtyping Cronobacter species. It was derived from an existing modified PulseNet protocol, wherein XbaI and SpeI were the primary and secondary restriction enzymes used, generating an average of 14.7 and 20.3 bands, respectively. The PFGE method developed was both reproducible and discriminatory for subtyping Cronobacter species.
Asunto(s)
Cronobacter/clasificación , Tipificación Molecular/métodos , Análisis del Polimorfismo de Longitud de Fragmentos Amplificados , Animales , Cronobacter/genética , Cronobacter/aislamiento & purificación , Cronobacter/metabolismo , Cronobacter sakazakii/clasificación , Cronobacter sakazakii/genética , Cronobacter sakazakii/aislamiento & purificación , Cronobacter sakazakii/metabolismo , Enzimas de Restricción del ADN , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Electroforesis en Gel de Campo Pulsado , Infecciones por Enterobacteriaceae/diagnóstico , Infecciones por Enterobacteriaceae/microbiología , Alimentos en Conserva/microbiología , Enfermedades Transmitidas por los Alimentos/diagnóstico , Enfermedades Transmitidas por los Alimentos/microbiología , Humanos , Leche/microbiología , Reproducibilidad de los Resultados , Vibrio cholerae/clasificación , Vibrio cholerae/genética , Vibrio cholerae/aislamiento & purificación , Vibrio cholerae/metabolismo , Yersinia pestis/clasificación , Yersinia pestis/genética , Yersinia pestis/aislamiento & purificación , Yersinia pestis/metabolismoRESUMEN
Shigella flexneri is one of the agents most frequently linked to diarrheal illness in developing countries and often causes outbreaks in settings with poor hygiene or sanitary conditions. Travel is one of the means by which S. flexneri can be imported into developed countries, where this pathogen is not commonly seen. A robust and discriminatory subtyping method is needed for the surveillance of S. flexneri locally and regionally, and to aid in the detection and investigation of outbreaks. The PulseNet International network utilizes standardized pulsed-field gel electrophoresis (PFGE) protocols to carry out laboratory-based surveillance of foodborne pathogens in combination with epidemiologic data. A multicenter validation was carried out in nine PulseNet laboratories located in North and South America, Europe, and Asia, and it demonstrated that a new protocol is highly robust and reproducible for subtyping of S. flexneri. This protocol, already approved for PulseNet laboratories, applies NotI and XbaI as primary and secondary restriction enzymes, respectively, under electrophoresis conditions of initial switch time of 5 s to final switch time of 35 s, at 6 volts/cm.
Asunto(s)
Técnicas de Tipificación Bacteriana , ADN Bacteriano/metabolismo , Shigella flexneri/clasificación , Técnicas de Tipificación Bacteriana/normas , ADN Bacteriano/química , Dinamarca , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Disentería Bacilar/diagnóstico , Disentería Bacilar/microbiología , Electroforesis en Gel de Campo Pulsado , Hong Kong , Medio Oriente , América del Norte , Control de Calidad , Reproducibilidad de los Resultados , Shigella flexneri/aislamiento & purificación , Shigella flexneri/metabolismo , América del Sur , Factores de TiempoRESUMEN
Salmonella enterica subsp. enterica serovar Enteritidis is a major cause of food-borne salmonellosis in the United States. Two major food vehicles for S. Enteritidis are contaminated eggs and chicken meat. Improved subtyping methods are needed to accurately track specific strains of S. Enteritidis related to human salmonellosis throughout the chicken and egg food system. A sequence typing scheme based on virulence genes (fimH and sseL) and clustered regularly interspaced short palindromic repeats (CRISPRs)-CRISPR-including multi-virulence-locus sequence typing (designated CRISPR-MVLST)-was used to characterize 35 human clinical isolates, 46 chicken isolates, 24 egg isolates, and 63 hen house environment isolates of S. Enteritidis. A total of 27 sequence types (STs) were identified among the 167 isolates. CRISPR-MVLST identified three persistent and predominate STs circulating among U.S. human clinical isolates and chicken, egg, and hen house environmental isolates in Pennsylvania, and an ST that was found only in eggs and humans. It also identified a potential environment-specific sequence type. Moreover, cluster analysis based on fimH and sseL identified a number of clusters, of which several were found in more than one outbreak, as well as 11 singletons. Further research is needed to determine if CRISPR-MVLST might help identify the ecological origins of S. Enteritidis strains that contaminate chickens and eggs.
Asunto(s)
ADN Bacteriano/genética , Secuencias Invertidas Repetidas , Tipificación Molecular/métodos , Salmonella enteritidis/clasificación , Salmonella enteritidis/genética , Factores de Virulencia/genética , Animales , Pollos , Análisis por Conglomerados , ADN Bacteriano/química , Brotes de Enfermedades , Huevos , Microbiología Ambiental , Microbiología de Alimentos , Genotipo , Humanos , Datos de Secuencia Molecular , Infecciones por Salmonella/microbiología , Salmonelosis Animal/microbiología , Salmonella enteritidis/aislamiento & purificación , Análisis de Secuencia de ADN , Estados Unidos/epidemiologíaRESUMEN
Salmonella enterica subsp. enterica is the leading cause of bacterial food-borne disease in the United States. Molecular subtyping methods are powerful tools for tracking the farm-to-fork spread of food-borne pathogens during outbreaks. In order to develop a novel multilocus sequence typing (MLST) scheme for subtyping the major serovars of S. enterica subsp. enterica, the virulence genes sseL and fimH and clustered regularly interspaced short palindromic repeat (CRISPR) loci were sequenced from 171 clinical isolates from nine Salmonella serovars, Salmonella serovars Typhimurium, Enteritidis, Newport, Heidelberg, Javiana, I 4,[5],12:i:-, Montevideo, Muenchen, and Saintpaul. The MLST scheme using only virulence genes was congruent with serotyping and identified epidemic clones but could not differentiate outbreaks. The addition of CRISPR sequences dramatically improved discriminatory power by differentiating individual outbreak strains/clones. Of particular note, the present MLST scheme provided better discrimination of Salmonella serovar Enteritidis strains than pulsed-field gel electrophoresis (PFGE). This method showed high epidemiologic concordance for all serovars screened except for Salmonella serovar Muenchen. In conclusion, the novel MLST scheme described in the present study accurately differentiated outbreak strains/clones of the major serovars of Salmonella, and therefore, it shows promise for subtyping this important food-borne pathogen during investigations of outbreaks.
Asunto(s)
Tipificación de Secuencias Multilocus/métodos , Salmonella enterica/genética , Virulencia/genética , Alelos , Análisis por Conglomerados , ADN Bacteriano/genética , Electroforesis en Gel de Campo Pulsado , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Polimorfismo Genético/genética , Salmonella enterica/clasificaciónRESUMEN
The PulseNet Methods Development and Validation Laboratory began a re-evaluation of the standardized pulsed-field gel electrophoresis (PFGE) protocols with the goal of optimizing their overall performance and robustness. Herein, we describe a stepwise evaluation of the PulseNet-standardized PFGE protocol for Listeria monocytogenes that led to the modification of several steps which significantly improved the overall appearance and reproducibility of the resulting PFGE data. These improvements included the following: (1) reducing the cell suspension concentration, (2) increasing lysozyme incubation temperature from 37 degrees C to 56 degrees C, and (3) decreasing the number of units of restriction enzymes AscI and ApaI. These changes were incorporated into a proposed protocol that was evaluated by 16 PulseNet participating laboratories, including 2 international participants. Results from the validation study indicated that the updated L. monocytogenes protocol is more robust than the original PulseNet-standardized protocol established in 1998 and this resulted in the official adoption of the new protocol into the PulseNet system in the spring of 2008. The modifications not only represent an improvement to the protocol but also describe procedural improvements that could be potentially applied to the PFGE analysis of other Gram-positive organisms.
Asunto(s)
Electroforesis en Gel de Campo Pulsado/métodos , Electroforesis en Gel de Campo Pulsado/normas , Listeria monocytogenes/clasificación , Dermatoglifia del ADN , ADN Bacteriano/análisis , Laboratorios , Listeria monocytogenes/genética , Muramidasa/metabolismo , Reproducibilidad de los ResultadosRESUMEN
The PulseNet USA subtyping network recently established a standardized protocol for multiple-locus variable-number tandem repeat analysis (MLVA) to characterize Shiga toxin-producing Escherichia coli O157. To enable data comparisons from different laboratories in the same database, reproducibility and high quality of the data must be ensured. The aim of this study was to test the robustness and reproducibility of the proposed standardized protocol by subjecting it to a multilaboratory validation process and to address any discrepancies that may have arisen from the study. A set of 50 strains was tested in 10 PulseNet participating laboratories that used capillary electrophoresis instruments from two manufacturers. Six out of the 10 laboratories were able to generate correct MLVA types for 46 (92%) or more strains. The discrepancies in MLVA type assignment were caused mainly by difficulties in optimizing polymerase chain reactions that were attributed to technical inexperience of the staff and suboptimal quality of reagents and instrumentation. It was concluded that proper training of staff must be an integral part of technology transfer. The interlaboratory reproducibility of fragment sizing was excellent when the same capillary electrophoresis platform was used. However, sizing discrepancies of up to six base pairs for the same fragment were detected between the two platforms. These discrepancies were attributed to different dye and polymer chemistries employed by the manufacturers. A novel software script was developed to assign alleles based on two platform-specific (Beckman Coulter CEQ8000 and Applied Biosystems Genetic Analyzer 3130xl) look-up tables containing fragment size ranges for all alleles. The new allele assignment method was validated at the PulseNet central laboratory using a diverse set of 502 Shiga toxin-producing Escherichia coli O157 isolates. The validation confirmed that the script reliably assigned the same allele for the same fragment regardless of the platform used to size the fragment.
Asunto(s)
ADN Bacteriano/análisis , Electroforesis Capilar/normas , Infecciones por Escherichia coli/microbiología , Escherichia coli O157/clasificación , Escherichia coli O157/genética , Alelos , Secuencia de Bases , Fragmentación del ADN , Electroforesis Capilar/instrumentación , Electroforesis Capilar/métodos , Escherichia coli O157/metabolismo , Microbiología de Alimentos , Humanos , Laboratorios/normas , Filogenia , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Toxinas Shiga/biosíntesis , Secuencias Repetidas en TándemRESUMEN
The pandemic spread of Vibrio parahaemolyticus is an international public health issue. Because of the outbreak potential of the organism, it is critical to establish an internationally recognized molecular subtyping protocol for V. parahaemolyticus that is both rapid and robust as a means to monitor its further spread and to guide control measures in combination with epidemiologic data. Here we describe the results of a multicenter, multicountry validation of a new PulseNet International standardized V. parahaemolyticus pulsed-field gel electrophoresis (PFGE) protocol. The results are from a composite analysis of 36 well-characterized V. parahaemolyticus isolates from six participating laboratories, and the isolates represent predominant serotypes and various genotypes isolated from different geographic regions and time periods. The discriminatory power is very high, as 34 out of 36 sporadic V. parahaemolyticus strains tested fell into 34 distinguishable PFGE groups when the data obtained with two restriction enzymes (SfiI and NotI) were combined. PFGE was further able to cluster members of known pandemic serogroups. The study also identified quality measures which may affect the performance of the protocol. Nonadherence to the recommended procedure may lead to high background in the PFGE gel patterns, partial digestion, and poor fragment resolution. When these quality measures were implemented, the PulseNet V. parahaemolyticus protocol was found to be both robust and reproducible among the collaborating laboratories.
Asunto(s)
Técnicas de Tipificación Bacteriana/normas , ADN Bacteriano/genética , Electroforesis en Gel de Campo Pulsado/normas , Epidemiología Molecular/normas , Vibrio parahaemolyticus/clasificación , Vibrio parahaemolyticus/genética , Técnicas de Tipificación Bacteriana/métodos , Análisis por Conglomerados , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Genotipo , Humanos , Epidemiología Molecular/métodosRESUMEN
Infections caused by foodborne bacterial pathogens continue to be a major public health issue around the world. During the past decade, pulsed-field gel electrophoresis (PFGE) has become the gold standard for molecular subtyping and source tracking of most foodborne bacteria. Owing to problems inherent in PFGE technology, new methods have been developed focusing on DNA sequence-based subtyping. This review discusses the feasibility of using multilocus sequence typing, multiple-locus variable-number tandem repeat analysis, single nucleotide polymorphisms, microarrays, whole genome sequencing and mass spectrometry for subtyping foodborne bacterial pathogens.
Asunto(s)
Bacterias/clasificación , Bacterias/genética , Técnicas de Tipificación Bacteriana/métodos , Microbiología de Alimentos , Bacterias/aislamiento & purificación , Técnicas de Tipificación Bacteriana/tendencias , Electroforesis en Gel de Campo Pulsado , Repeticiones de Minisatélite , Análisis de Secuencia por Matrices de Oligonucleótidos , Polimorfismo de Nucleótido Simple , Análisis de Secuencia de ADNRESUMEN
Crohn's disease (CD) and ulcerative colitis (UC) are idiopathic inflammatory conditions of the gut. Our goal was to investigate if invasive Escherichia coli strains were present in patients with inflammatory bowel disease (IBD). Bacterial strains were isolated from biopsy material obtained from normal controls, and patients with a clinical diagnosis of CD and UC. Invasive bacteria were characterized by gentamicin protection assay and biochemical profiling (Api-20E). Strains were characterized by induction of cytokine expression in epithelial and macrophage cell cultures, measurement of epithelial barrier function, and confocal microscopy. Of all invasive bacterial strains in CD 98.9% were identified as E. coli as opposed to 42.1% in UC and 2.1% in normal controls. Epithelial invasion in vitro was significantly higher for CD-associated E. coli (8.4%, +/-5.5 of initial inoculum (I/O)) in comparison to UC (2.5%, +/-0.4 I/O), but highest for strains from inflamed CD tissue (11.3%, +/-4.3 I/O). Both, CD and UC E. coli strains induced high mean TNF-alpha expression in macrophage cell lines (2604.8 pg/10(5) cells, +/-447.4; 2,402.6 pg/10(5) cells, +/-476.3, respectively), but concentrations were significantly higher for isolates from inflamed CD tissue (3071.3 pg/10(5) cells, +/-226.0). Invasive E. coli from IBD tissue induced similar concentrations of interleukin (IL)-8 in epithelial cell cultures, but strains from inflamed CD tissue induced significantly less epithelial IL-8 (674.1 pg/10(5) cells, +/-58.0 vs 920.5 pg/10(5) cells, +/-94.6). IBD-associated E. coli strains significantly decreased transepithelial resistance, induced disorganization of F-actin and displacement of ZO-1, and E-cadherin from the apical junctional complex (AJC). In comparison to normal controls and UC, E. coli are more prevalent in CD, are highly invasive, and do not encode for known effector proteins. E. coli strains from IBD patients regulate cytokine expression and epithelial barrier function, two pathological features of IBD.
Asunto(s)
Colitis Ulcerosa/microbiología , Enfermedad de Crohn/microbiología , Citocinas/metabolismo , Escherichia coli/patogenicidad , Mucosa Intestinal/microbiología , Células CACO-2 , Estudios de Casos y Controles , Colitis Ulcerosa/metabolismo , Enfermedad de Crohn/metabolismo , Escherichia coli/genética , Humanos , Mucosa Intestinal/metabolismo , PermeabilidadRESUMEN
Infections by Shiga toxin-producing Escherichia coli O157:H7 (STEC O157) are the predominant cause of bloody diarrhea and hemolytic uremic syndrome in the United States. In silico comparison of the two complete STEC O157 genomes (Sakai and EDL933) revealed a strikingly high level of sequence identity in orthologous protein-coding genes, limiting the use of nucleotide sequences to study the evolution and epidemiology of this bacterial pathogen. To systematically examine single nucleotide polymorphisms (SNPs) at a genome scale, we designed comparative genome sequencing microarrays and analyzed 1199 chromosomal genes (a total of 1,167,948 bp) and 92,721 bp of the large virulence plasmid (pO157) of eleven outbreak-associated STEC O157 strains. We discovered 906 SNPs in 523 chromosomal genes and observed a high level of DNA polymorphisms among the pO157 plasmids. Based on a uniform rate of synonymous substitution for Escherichia coli and Salmonella enterica (4.7x10(-9) per site per year), we estimate that the most recent common ancestor of the contemporary beta-glucuronidase-negative, non-sorbitolfermenting STEC O157 strains existed ca. 40 thousand years ago. The phylogeny of the STEC O157 strains based on the informative synonymous SNPs was compared to the maximum parsimony trees inferred from pulsed-field gel electrophoresis and multilocus variable numbers of tandem repeats analysis. The topological discrepancies indicate that, in contrast to the synonymous mutations, parts of STEC O157 genomes have evolved through different mechanisms with highly variable divergence rates. The SNP loci reported here will provide useful genetic markers for developing high-throughput methods for fine-resolution genotyping of STEC O157. Functional characterization of nucleotide polymorphisms should shed new insights on the evolution, epidemiology, and pathogenesis of STEC O157 and related pathogens.