Construction of stable fluorescent laboratory control strains for several food safety relevant Enterobacteriaceae.

Using naturally-occurring bacterial strains as positive controls in testing protocols is typically feared due to the risk of cross-contaminating samples. We have developed a collection of strains which express Green Fluorescent Protein (GFP) at high-level, permitting rapid screening of the following species on selective or non-selective plates: Escherichia coli O157:H7, Shigella sonnei, S. flexneri, Salmonella enterica subsp.

Comparative Sequence Analysis of Multidrug-Resistant IncA/C Plasmids from .

Determinants of multidrug resistance (MDR) are often encoded on mobile elements, such as plasmids, transposons, and integrons, which have the potential to transfer among foodborne pathogens, as well as to other virulent pathogens, increasing the threats these traits pose to human and veterinary health. Our understanding of MDR among has been limited by the lack of closed plasmid genomes for comparisons across resistance phenotypes, due to difficulties in effectively separating the DNA of these high-molecular weight, low-copy-number plasmids from chromosomal DNA. To resolve this problem, we demonstrate an efficient protocol for isolating, sequencing and closing IncA/C plasmids from sp.

Staphylococcal Enterotoxin Type R Pseudogene Presence in Reference and Outbreak Strains.

Novel staphylococcal enterotoxins (SEs) expressed by Staphylococcus aureus strains have been described throughout the years, among these being the SE protein SER. To further characterize this toxin, this research used 13 S. aureus strains previously determined to contain the SE type R (ser) gene.

Plasmids from Shiga Toxin-Producing Escherichia coli Strains with Rare Enterohemolysin Gene (ehxA) Subtypes Reveal Pathogenicity Potential and Display a Novel Evolutionary Path.

Unlabelled: Most Shiga toxin-producing Escherichia coli (STEC) strains associated with severe disease, such as hemolytic-uremic syndrome (HUS), carry large enterohemolysin-encoding (ehxA) plasmids, e.g., pO157 and pO103, that contribute to STEC clinical manifestations.

Tracing Origins of the Salmonella Bareilly Strain Causing a Food-borne Outbreak in the United States.

Background: Using a novel combination of whole-genome sequencing (WGS) analysis and geographic metadata, we traced the origins of Salmonella Bareilly isolates collected in 2012 during a widespread food-borne outbreak in the United States associated with scraped tuna imported from India.

Methods: Using next-generation sequencing, we sequenced the complete genome of 100 Salmonella Bareilly isolates obtained from patients who consumed contaminated product, from natural sources, and from unrelated historically and geographically disparate foods. Pathogen genomes were linked to geography by projecting the phylogeny on a virtual globe and produced a transmission network.

Complete Sequences of Six IncA/C Plasmids of Multidrug-Resistant Salmonella enterica subsp. enterica Serotype Newport.

Multidrug-resistant (MDR) Salmonella enterica subsp. enterica serotype Newport has been a long-standing public health concern in the United States. We present the complete sequences of six IncA/C plasmids from animal-derived MDR S.

Comparative genomic analysis and virulence differences in closely related salmonella enterica serotype heidelberg isolates from humans, retail meats, and animals.

Salmonella enterica subsp. enterica serovar Heidelberg (S. Heidelberg) is one of the top serovars causing human salmonellosis.

Genetic and phylogenetic evidence for horizontal gene transfer among ecologically disparate groups of marine Vibrio.

Vibrio represents a diverse bacterial genus found in different niches of the marine environment, including numerous genera of marine sponges (phylum Porifera), inhabiting different depths and regions of benthic seas, that are potentially important in driving adaptive change among Vibrio spp. Using 16S rRNA gene sequencing, a previous study showed that sponge-derived (SD) vibrios clustered with their mainstream counterparts present in shallow, coastal ecosystems, suggesting a genetic relatedness between these populations. Sequences from the topA, ftsZ, mreB, rpoD, rctB and toxR genes were used to investigate the degree of relatedness existing between these two separate populations by examining their phylogenetic and genetic disparity.

Vibrio caribbeanicus sp. nov., isolated from the marine sponge Scleritoderma cyanea.

A Gram-negative, oxidase-positive, catalase-negative, facultatively anaerobic, motile, curved rod-shaped bacterium, strain N384(T), was isolated from a marine sponge (Scleritoderma cyanea; phylum Porifera) collected from a depth of 795 feet (242 m) off the west coast of Curaçao. On the basis of 16S rRNA gene sequencing, strain N384(T) was shown to belong to the genus Vibrio, most closely related to Vibrio brasiliensis LMG 20546(T) (98.8% similarity), Vibrio nigripulchritudo ATCC 27043(T) (98.

Specificity of PCR and serological assays in the detection of Escherichia coli Shiga toxin subtypes.

Specificity analysis for stx or Stx subtypes in Escherichia coli showed that the PCR assays we tested did not detect stx(1d) and stx(2f), and some also missed stx(2b) and stx(2g). Most of the serological assays examined did not detect Stx2c, Stx2e, Stx2f, and Stx2g, and some strain-to-strain variation in reactivity was observed for Stx2b.

Virulence characterization of Shiga-toxigenic Escherichia coli isolates from wholesale produce.

The 13 Shiga-toxigenic Escherichia coli (STEC) strains isolated from wholesale spinach and lettuce consisted mostly of serotypes that have not been implicated in illness. Among these strains, however, were two O113:H21 that carried virulence genes common to this pathogenic serotype (stx(2), ehxA, saa, and subAB), suggesting that their presence in ready-to-eat produce may be of health concern.

Population dynamics of Vibrio spp. associated with marine sponge microcosms.

Vibrio is a diverse genus of marine-associated bacteria with at least 74 species and more expected as additional marine ecospheres are interrogated. This report describes a phylogenetic reconstruction of Vibrio isolates derived from one such unique ecosystem, marine sponges (Phylum Porifera) collected from depths of 150 to 1242 feet. 16S rRNA gene sequencing along with molecular typing of 16S-23S rRNA intergenic spacer regions clustered many sponge-associated Vibrio (spp) with current known species.

Prevalence, characterization and clonal analysis of Escherichia coli O157: non-H7 serotypes that carry eae alleles.

We examined O157:non-H7 strains isolated from various sources and geographical locations and found 15/57 strains to carry eae alleles, including alpha, beta, epsilon and kappa/delta, suggesting that these strains may be prevalent. All strains were serologically and genetically confirmed to be O157, but none were the H7 serotype or carried any trait virulence factors of the Escherichia coli O157:H7 serotype. Genetic H typing of the eae-positive strains showed that the alpha-eae-bearing strain was H45, while the beta- and epsilon-eae strains were H16 and the kappa/delta-eae strains were H39.

PCR-based method for targeting 16S-23S rRNA intergenic spacer regions among Vibrio species.

Background: The genus Vibrio is a diverse group of Gram-negative bacteria comprised of 74 species. Furthermore, the genus has and is expected to continue expanding with the addition of several new species annually. Consequently, it is of paramount importance to have a method which is able to reliably and efficiently differentiate the numerous Vibrio species.

Genetic analysis for the lack of expression of the O157 antigen in an O Rough:H7 Escherichia coli strain.

The O-antigen (rfb) operon and related genes of MA6, an O rough:H7 Shiga-toxigenic Escherichia coli strain, were examined to determine the cause of the lack of O157 expression. A 1,310-bp insertion, homologous to IS629, was observed within its gne gene. trans complementation with a functional gne gene from O157:H7 restored O157 antigen expression in MA6.

Genetic diversity among clonal lineages within Escherichia coli O157:H7 stepwise evolutionary model.

Escherichia coli O157:H7 variants were examined for trait mutations and by molecular subtyping to better define clonal complexes postulated on the O157:H7 evolution model. Strains of beta-glucuronidase-positive, sorbitol-negative O157:H7 isolated in United States and Japan were identical to A5 clonal strain and shared sequence type (ST)-65 by multilocus sequence typing (MLST); thus, they belong in A5. However, these strains exhibited pulsed-field gel electrophoresis (PFGE) profile differences that suggested genomic divergence between populations.

Microbiological quality of bagged cut spinach and lettuce mixes.

Analysis of 100 bagged lettuce and spinach samples showed mean total bacterial counts of 7.0 log(10) CFU/g and a broad range of < 4 to 8.3 log10 CFU/g.

Produce isolates of the Escherichia coli Ont:H52 serotype that carry both Shiga toxin 1 and stable toxin genes.

Produce isolates of the Escherichia coli Ont:H52 serotype carried Shiga toxin 1 and stable toxin genes but only expressed Stx1. These strains had pulsed-field gel electrophoresis profiles that were 90% homologous to clinical Ont:H52 strains that had identical phenotypes and genotypes. All Ont:H52 strains had identical single nucleotide polymorphism profiles that are suggestive of a unique clonal group.

A top-down proteomics approach for differentiating thermal resistant strains of Enterobacter sakazakii.

Thermal tolerance has been identified as an important factor relevant to the pathogenicity of Enterobacter sakazakii in human neonates. To identify a biomarker specific for this phenotypic trait, intact protein expression profiles of 12 strains of E. sakazakii were obtained using liquid chromatography mass spectrometry.

Identifying new PCR targets for pathogenic bacteria using top-down LC/MS protein discovery.

We have investigated the use of a top-down liquid chromatography/mass spectrometric (LC/MS) approach for the identification of specific protein biomarkers useful for differentiation of closely related strains of bacteria. The sequence information derived from the protein biomarker was then used to develop specific polymerase chain reaction primers useful for rapid identification of the strains. Shiga-toxigenic Escherichia coli (STEC) strains were used for this evaluation.

Identification of a protein biomarker unique to the pandemic O3:K6 clone of Vibrio parahaemolyticus.

The present method of characterizing Vibrio parahaemolyticus strains involves serotyping or detection methods based on assessment of the presence or absence of genes thought to be markers of an organism's pathogenicity. It is unclear whether these assays detect all pathogenic V. parahaemolyticus strains since a clear correlation between the presence of a particular gene and the organism's pathogenicity has not yet been observed.

A 12-base-pair deletion in the flagellar master control gene flhC causes nonmotility of the pathogenic German sorbitol-fermenting Escherichia coli O157:H- strains.

An atypical, Stx2-producing, pathogenic Escherichia coli O157:H(-) strain has been isolated with increasing frequency from hemolytic uremic syndrome patients in Germany. The lack of the H7 antigen coupled with the strain's ability to ferment sorbitol and express beta-glucuronidase have complicated its detection and identification. In this study, we have determined that the loss of motility in these German sorbitol-fermenting (SF) O157 strains is due to a 12-bp in-frame deletion in flhC that is required for transcriptional activation of genes involved in flagellum biosynthesis.

Relative quantitation of intact proteins of bacterial cell extracts using coextracted proteins as internal standards.

A method for quantitating protein expression using LC/MS of whole proteins is described. This method is based on the fact that some proteins present in cells are abundant universal proteins whose expression levels exhibit little variation. This method demonstrates that these coextracted proteins can be used as internal standards to which the other proteins in the sample can be compared.

Use of endogenous host plasmids for generation of Escherichia coli O157:H7 and Shigella sonnei strains that stably express the green fluorescent protein.

The gfp gene was manipulated from a commercially available, high copy vector into endogenous plasmids of Escherichia coli O157:H7 and Shigella sonnei to yield stable GFP strains that required neither high copy number for visualization nor antibiotics for stable maintenance of the phenotype. The GFP phenotype of these strains remained stable after repeated passages in media and conditions that enhance plasmid instability and loss from bacterial cells. These results demonstrate the utility of the endogenous plasmids in selectively marking bacteria without altering host cellular function or biochemical properties.

Targeting single-nucleotide polymorphisms in the 18S rRNA gene to differentiate Cyclospora species from Eimeria species by multiplex PCR.

Cyclospora cayetanensis is a coccidian parasite that causes protracted diarrheal illness in humans. C. cayetanensis is the only species of this genus thus far associated with human illness, although Cyclospora species from other primates have been named.