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There are several anecdotal reports of insertion sequence (IS) element inactivation of verotoxin genes among enterohaemorrhagic Escherichia coli of the serotype O157:H7, a pathogen causing severe gastrointestinal disease in infected humans. These insertions can be expected to drastically reduce the virulence of the bacteria. IS element inactivation has been shown to be reversible in model systems, suggesting the possibility of spontaneous restoration of virulence. In this study, traditional and high-throughput sequencing was used to characterise three patterns of IS629 inactivation of verotoxin 2 genes in EHEC O157:H7, caused by insertion or insertion followed by partial deletion. At least one of the patterns of inactivation appears to have persisted several years among cattle O157:H7, indicating it has no major effect on fitness in the animal reservoir. Digital PCR was used to directly quantify the reversal rates of the insertional inactivation of a selected isolate under laboratory conditions. Inserts were found to be absent from in the order of 1/105 of individual genomes, with significantly higher loss frequencies observed in cultures under nutrient-poor conditions. We conclude that strains with this type of inactivation found in food or animal samples should be considered a threat to human health, and may pose a challenge for PCR-based detection methods. [ABSTRACT FROM AUTHOR]

It has been earlier hypothesized that lysogenic infection with Stx-encoding phages influences protein expression in the bacterial host, and therefore, some differentially expressed proteins could affect survival characteristics and pathogenicity. We compared the protein expression profiles of the host MG1655 and lysogens by 2D electrophoresis. Four different genes identified were all related to Fe/S subunit production, namely, nfuA, fdoH, sdhB and ftnA. To explore the role of nfuA in the biology of Stx prophage lysogeny, gene knockout experiments and phage lysogenic conversion were performed. The inactivation of nfuA caused the prophage to enter its lytic life cycle, especially under an iron-depleted condition. A similar activity was also detected in the Escherichia coli O157:H7 strain from which the Stx phage Min 27 was originally isolated. NfuA might be the positive regulator of genes controlling lysogenic cycle such as cI, cII and cIII since their transcriptional level was significantly reduced in nfuA deletion mutant as shown by qRT-PCR. We conclude that NfuA is essential for maintenance of Stx phage lysogeny in host's genetic reservoir under iron-deficient condition. [ABSTRACT FROM AUTHOR]

During the establishment of Escherichia coli O157:H7 infection, its capacity to adhere to host intestinal epithelial cells is the critical first step in pathogenesis. It also has the capability to form biofilms, and because both are surface activities, we sought to gain insight into a potential linkage between biofilm formation and adherence to epithelial cells. We conducted an adherence assay with 51 biofilm-negative mutants and two human epithelial cell lines, T84 and HEp2. Our results show that unlike wild-type cells, biofilm-negative mutants adhere poorly to epithelial cells. Some adhesin-negative mutants were fully competent in biofilm formation, however. Thus, biofilm-forming activity in E. coli O157:H7 EDL933 is required for adherence to T84 and HEp2 cells, but it is not sufficient. [ABSTRACT FROM AUTHOR]

Escherichia coli hemolysin, also termed α-hemolysin, is a virulence factor for extraintestinal E. coli. Hemolysin operon ( hlyCABD) transcription is inhibited by the nucleoid-associated protein, H-NS. This inhibition is stronger at lower growth temperature. This study investigated transcription of the homologous EHEC hemolysin (enterohemolysin) operon ( ehxCABD) in EHEC O157:H7 strain EDL933. We examined the influence of H-NS, the σ factor RpoS, and the small RNA DsrA, which is known to inhibit H-NS function and to stimulate RpoS synthesis. During growth at 30 °C, DsrA overexpression increased ehxA transcription in the wild type but not in an hns deletion mutant. During growth at 37 °C, DsrA overexpression increased ehxA transcription independent of hns genotype. This indicates that DsrA influences ehxCABD operon transcription by two different routes, one (at lower temperature) at least partially dependent on H-NS, and one (at higher temperature) independent of H-NS. An rpoS deletion mutant expressed nondetectable levels of ehxA mRNA regardless of growth temperature or DsrA overexpression, indicating that the RpoS σ factor is essential for ehxCABD operon expression. [ABSTRACT FROM AUTHOR]

Thirty-eight Shiga toxin-producing Escherichia coli (STEC) O157:H7/H− strains isolated from human infections, cattle and foods in Brazil and in some other Latin American countries were compared with regard to several phenotypic and genotypic characteristics. The genetic relatedness of the strains was also determined by pulsed-field gel electrophoresis (PFGE). Similar biochemical behaviour was identified, regardless of the origin and country of the strains. Most (89.5%) strains were sensitive to the antimicrobial agents tested, but resistance to at least one drug was observed among bovine strains. Although a diversity of stx genotypes was identified, most (77.8%) of the human strains harboured stx2 or stx2 stx2c(2vha), whereas stx2c(2vha) prevailed (64.2%) among strains isolated from cattle. stx1 and stx1 stx2c(2vha) were the genotypes identified less frequently, and occurred exclusively among strains isolated from food and cattle, respectively. Despite differences in the stx genotypes, all strains carried eae-γ, efa1, ehx, iha, lpfO157 and toxB sequences. Many closely related subgroups (more than 80% of similarity) were identified by PFGE, and the presence of a particular O157:H7 STEC clone more related to human infections in Brazil, as well as a common origin for some strains isolated from different sources and countries in Latin America can be suggested. [ABSTRACT FROM AUTHOR]

We found frequent IS1 integration nearby the stx2 gene during in vitro mutagenesis of an stx2 variant, stx2vhd. To examine the possibility that such insertions have been contributing to generate new stx2 variants, we screened 86 strains of Escherichia coli O157:H7 isolated in Japan for variations in the ca. 4-kb region flanking the stx2 locus using PCR methods. Two major classes were identified based on the PCR amplicon size. DNA sequence analysis revealed that the stx2 subtype of the two classes were stx2 (referred to as stx2-EDL933) and stx2vhd. IS1203v insertions were found in three stx2vhd-positive strains and two stx2-EDL933-positive strains, and no other insertions were found. These results suggest that the DNA sequences surrounding the stx2 genes are preferably integrated by IS1203v in wild-type Shiga toxin-producing E. coli strains. [Copyright &y& Elsevier]

It has been earlier hypothesized that lysogenic infection with Stx-encoding phages influences protein expression in the bacterial host, and therefore, some differentially expressed proteins could affect survival characteristics and pathogenicity. We compared the protein expression profiles of the host MG1655 and lysogens by 2D electrophoresis. Four different genes identified were all related to Fe/S subunit production, namely, nfuA, fdoH, sdhB and ftnA. To explore the role of nfuA in the biology of Stx prophage lysogeny, gene knockout experiments and phage lysogenic conversion were performed. The inactivation of nfuA caused the prophage to enter its lytic life cycle, especially under an iron-depleted condition. A similar activity was also detected in the Escherichia coli O157:H7 strain from which the Stx phage Min 27 was originally isolated. NfuA might be the positive regulator of genes controlling lysogenic cycle such as cI, cII and cIII since their transcriptional level was significantly reduced in nfuA deletion mutant as shown by qRT-PCR. We conclude that NfuA is essential for maintenance of Stx phage lysogeny in host's genetic reservoir under iron-deficient condition., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)