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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 [...]

An atypical, Stx2-producing, pathogenic Escherichia coli O157:[H.sup.-] 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 inflhC 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.

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. By comparing the intensities of a selected protein to marker proteins, or internal standards, a relative ratio is obtained. This ratio can then be used to determine the relative amount of protein expression between cellular extracts. The validity of this approach is described for a standard protein mixture, as well as, E. coli cells that were known to differentially express green fluorescent protein.

Research reveals that a genetic element composed of a 15,891-base pair putative pathogenicity island in bovine Staphylococcus aureus, strain RF122 encodes toxic shock syndrome toxin, staphylococcal enterotoxin C, and another enterotoxin-like protein suggesting gene linkage. Results further point out that the genetic element modulates the bovine immune response.

Pseudomonas aeruginosa is a pathogenic bacteria that exhibits resistance to a wide variety of antibacterial agents. Alginate is a linear, acetylated polymer encoded by the alginate biosynthetic gene cluster. Mutational and recombinant analysis of the alginate lyase gene showed that alginate production can be restored by complementation in trans with a plasmid containing all the genes inactivated by the insertion. Another protein, AlgX, was also identified as an important component in alginate production.

The alginate lyase structural gene is located inside the alginate biosynthetic gene cluster between algG and algA at 35 minutes on the Pseudomonas aeruginosa chromosome. The location was revealed by observations that alginate lyase production was regulated by AlgB, which also controls the alginate biosynthetic gene cluster and is stimulated by a high concentration of salt. These genetic studies on P. aeruginosa are in accordance with the resemblance of the structure of genes in the alginate cluster to that of an operon.

Abstract 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. Results In this study, a novel and rapid polymerase chain reaction (PCR)-based intergenic spacer (IGS)-typing system for vibrios was developed that is based on the well-known IGS regions located between the 16S and 23S rRNA genes on the bacterial chromosome. The system was optimized to resolve heteroduplex formation as well as to take advantage of capillary gel electrophoresis technology such that reproducible analyses could be achieved in a rapid manner. System validation was achieved through testing of 69 archetypal Vibrio strains, representing 48 Vibrio species, from which an 'IGS-type' profile database was generated. These data, presented here in several cluster analyses, demonstrated successful differentiation of the 69 type strains showing that this PCR-based fingerprinting method easily discriminates bacterial strains at the species level among Vibrio. Furthermore, testing 36 strains each of V. parahaemolyticus and V. vulnificus, important food borne pathogens, isolated from a variety of geographical locations with the IGS-typing method demonstrated distinct IGS-typing patterns indicative of subspecies divergence in both populations making this technique equally useful for intraspecies differentiation, as well. Conclusion This rapid, reliable and efficient IGS-typing system, especially in combination with 16S rRNA gene sequencing, has the capacity to not only discern and identify vibrios at the species level but, in some cases, at the sub-species level, as well. This procedure is particularly well-suited for preliminary species identification and, lends itself nicely to epidemiological investigations providing information more quickly than other time-honoured methods traditionally used in these types of analyses.

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

The analysis of 100 bagged lettuce and spinach samples have indicated mean total bacterial counts of 7.0 [log.sub.10] CFU/g and a broad range of =11,000/g coliforms are found in 55 samples and generic Escherichia coli bacteria is detected in 16 samples, but no Escherichia coli count has exceeded 10 MPN/g.

Produce isolates of the Escherichia coli Ont:H52 serotype carried Shiga toxin 1 and stable toxin genes but only expressed Stx1. The Ont:H52 strains shares similar pulsed-field gel electrophoresis profiles and an identical but distinctive sequence type, indicating that they are a unique Shiga-toxigenic Escherichia coli (STEC) clonal group.

Research has been conducted on Cyclospora and Eimeria species. The authors describe the nested-PCR protocol and MAMA PCR concepts applied for detection of these species, and report the development of the multiplex PCR assay for the differentiation between Cyclospora and Eimeria species.

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 β-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. [ABSTRACT FROM AUTHOR]

Escherichia coli serotype O157:H7 do not exhibit β-glucuronidase (GUD) activity but carry the gusA gene (uidA) that encodes for GUD. In trans-complementation, the gusA gene cloned from the GUD-positive variant strain 493–89 effectively restored GUD activity in O157:H7 strain 35150. Comparison of gusA sequences from the GUD-negative 35150 strain to that of 493–89 revealed several base mutations, including a guanosine (G) dinucleotide insertion that caused a frameshift in the 35150 gusA gene and introduced a predicted premature termination codon. This explains the absence of GUD activity in O157:H7. A 35150 gusA construct from which the G-G insertion was deleted restored activity in GUD-negative O157:H7 transformants. The G-G insertion was present in all GUD-negative O157:H7 strains but was absent in their GUD-positive variants. The G-G insertion that produced the characteristic GUD-negative phenotype to O157:H7 strains appeared later than the other gusA mutations in the evolutionary emergence of O157:H7. [ABSTRACT FROM AUTHOR]

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. Phylogenies were constructed from the concatenated sequences of the six housekeeping genes using maximum-parsimony, maximum-likelihood and neighbour-joining algorithms. Genetic recombination was evaluated using the incongruence length difference test, Split decomposition and measuring overall compatibility of sites. This combined technical approach provided evidence that SD Vibrio strains are largely genetically homologous to their shallow-water counterparts. Moreover, the analyses conducted support the existence of extensive horizontal gene transfer between these two groups, supporting the idea of a single panmictic population structure among vibrios from two seemingly distinct, marine environments. [ABSTRACT FROM AUTHOR]

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 0157 expression. A 1,310-bp insertion, homologous to 1S629, was observed within its gne gene. trans complementation with a functional gne gene from O157:H7 restored O157 antigen expression in MA6. [ABSTRACT FROM AUTHOR]