Steven R. Monday

Genetic Diversity among Clonal Lineages within Escherichia coli O157:H7 Stepwise Evolutionary Model

Molecular characterization and subtyping show genetic diversities within clonal complexes.

Specificity of PCR and Serological Assays in the Detection of Escherichia coli Shiga Toxin Subtypes

ABSTRACT 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.

Genetic Analysis for Virulence Factors in Escherichia coli O104:H21 That Was Implicated in an Outbreak of Hemorrhagic Colitis

ABSTRACT Isolates of enterohemorrhagic Escherichia coli (EHEC) of serotype O104:H21 implicated in a 1994 outbreak of hemorrhagic colitis in Montana were analyzed for the presence of trait EHEC virulence markers. By using a multiplex PCR that specifically amplifies several genes, the O104:H21 strains were found to carry only the Shiga toxin 2 gene (stx2) and to express Stx2. They did not have the eaeA gene for γ-intimin, which is typically found in O157:H7, or the α- or β-intimin derivatives, which are common in other EHEC and enteropathogenic E. coli serotypes. Results of the multiplex PCR also indicated that the ehxA gene for enterohemolysin was absent from O104:H21. This, however, was not consistent with the results of a phenotypic assay that showed them to be hemolytic or a PCR analysis with another set ofehxA-specific primers, which indicated the presence ofehxA. To resolve this discrepancy, the ehxAregion in O104:H21 and O157:H7 strains, to which the multiplex PCR primers anneal, was cloned and sequenced. Comparison of the sequences showed that the upstream primer binding site in the ehxAgene of O104:H21 was not identical to that of O157:H7. Specifically, there were several base mutations, including an A-to-G substitution at the 3′ end of the primer binding site. These base mutations are presumably not unique to O104:H21, since other enterohemolytic serotypes were also not detected with the ehxA primers used in the multiplex PCR. Comparison of the ehxA sequences of O104:H21 strains with those of other Stx-producing E. colistrains showed that they more closely resembled those of O8:H19 strains, which have cluster II ehxA genes, than those of O157:H7 strains, which have cluster I ehxA sequences. By modifying the upstream ehxA primer, the multiplex PCR was able to detect ehxA genes in both O157:H7 and O104:H21 strains.

Microbiological Quality of Bagged Cut Spinach and Lettuce Mixes

ABSTRACT Analysis of 100 bagged lettuce and spinach samples showed mean total bacterial counts of 7.0 log10 CFU/g and a broad range of <4 to 8.3 log10 CFU/g. Most probable numbers (MPN) of ≥11,000 /g coliforms were found in 55 samples, and generic Escherichia coli bacteria were detected in 16 samples, but no E. coli count exceeded 10 MPN/g.

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 strains 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. Either complementation with a functional flhC or repair of this mutation restored H7 antigen expression and motility. PCR analysis of several nonmotile E. coli O157 strains from various geographical sources confirmed that the 12-bp flhC deletion is found only in the cluster of German SF O157 strains, providing a potentially useful marker by which these atypical strains can be identified. The loss of motility via mutations in the flhDC operon that we observed in the German SF O157 strains is consistent with a similar phenomenon currently observed in a significant subset of other important gram-negative pathogens.

Identification of a Protein Biomarker Unique to the Pandemic O3:K6 Clone of Vibrio parahaemolyticus

ABSTRACT 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 organisms 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 organisms pathogenicity has not yet been observed. We have described a proteomics-based method to distinguish individual V. parahaemolyticus strains on the basis of their protein profiles and identified a specific protein that is characteristic of the pandemic O3:K6 strain and its clonal derivatives. In the pandemic clone of V. parahaemolyticus, a histone-like DNA-binding protein, HU-α, has a C-terminal amino acid sequence different from those of other strains of V. parahaemolyticus. Upon further study, it was discovered that the gene encoding this protein has a 16-kbp insert at the 3′ terminus of the open reading frame for this protein. By using the protein sequence of the unique biomarker for the pandemic clone of V. parahaemolyticus, it was possible to rationally design specific PCR-based probes and assays that permit the rapid and precise identification of pandemic strains of V. parahaemolyticus.

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. The beta- and epsilon-eae-bearing O157:H16 strains shared approximately 90% pulsed-field gel electrophoresis (PFGE) similarity and were distinct from the other strains that had other eae alleles. Interestingly, an epsilon-eae O157:H16 strain isolated from meat in France shared PFGE similarity to the O157:H16 strains from water in the United States. Multilocus sequence typing showed that there is clonal diversity within the O157 serogroup, as some O157:non-H7 strains clustered with EPEC clonal groups, while others clustered within the ST-171 group of diverse strains and serotypes that had not previously included any strains from the O157 serogroup. Clonal analysis also showed that none of the eae-positive O157:non-H7 strains we examined were closely related to the pathogenic O157:H7 serotype.

Produce Isolates of the Escherichia coli Ont:H52 Serotype That Carry both Shiga Toxin 1 and Stable Toxin Genes

ABSTRACT 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.

Prolonged course of toxic shock syndrome associated with methicillin-resistant Staphylococcus aureus enterotoxins G and I

Toxic shock syndrome (TSS), which may be hfe-threatening, is defined by clinical and laboratory evidence of fever, rash, hypotension, and multisystem abnormalities in the absence of other causes. Most cases are nonmenstrual in origin. I-* Whereas Staphylococcus aureus TSS toxin-l causes the vast majority of menstrual cases, it causes only about half of other cases.” Staphylococcal enterotoxins (SEs) were implicated in nonmenstrual TSS cases shortly after the role of TSS toxin-l was elucidated, and currently, SEs that are reported to be associated with TSS are SEs A, B, C, D, G, H, and I, with SE-B most commonly reported and SE-G and SE-I most recently reported, albeit the latter two with scant clinical details.2s4-8 The following case associated with SE-G and SE-I illustrates the consequences of these immunomodulatory pyrogenic toxin superantigens over a prolonged course, as well as pitfalls in their diagnosis and management.

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

ABSTRACT 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.