Origins of the E. coli strain causing an outbreak of hemolytic-uremic syndrome in Germany.

BACKGROUND: A large outbreak of diarrhea and the hemolytic-uremic syndrome caused by an unusual serotype of Shiga-toxin-producing Escherichia coli (O104:H4) began in Germany in May 2011. As of July 22, a large number of cases of diarrhea caused by Shiga-toxin-producing E. coli have been reported--3167 without the hemolytic-uremic syndrome (16 deaths) and 908 with the hemolytic-uremic syndrome (34 deaths)--indicating that this strain is notably more virulent than most of the Shiga-toxin-producing E. coli strains. Preliminary genetic characterization of the outbreak strain suggested that, unlike most of these strains, it should be classified within the enteroaggregative pathotype of E. coli. METHODS: We used third-generation, single-molecule, real-time DNA sequencing to determine the complete genome sequence of the German outbreak strain, as well as the genome sequences of seven diarrhea-associated enteroaggregative E. coli serotype O104:H4 strains from Africa and four enteroaggregative E. coli reference strains belonging to other serotypes. Genomewide comparisons were performed with the use of these enteroaggregative E. coli genomes, as well as those of 40 previously sequenced E. coli isolates. RESULTS: The enteroaggregative E. coli O104:H4 strains are closely related and form a distinct clade among E. coli and enteroaggregative E. coli strains. However, the genome of the German outbreak strain can be distinguished from those of other O104:H4 strains because it contains a prophage encoding Shiga toxin 2 and a distinct set of additional virulence and antibiotic-resistance factors. CONCLUSIONS: Our findings suggest that horizontal genetic exchange allowed for the emergence of the highly virulent Shiga-toxin-producing enteroaggregative E. coli O104:H4 strain that caused the German outbreak. More broadly, these findings highlight the way in which the plasticity of bacterial genomes facilitates the emergence of new pathogens.

Contribution of urease to colonization by Shiga toxin-producing Escherichia coli.

Shiga toxin-producing Escherichia coli (STEC) is a food-borne pathogen with a low infectious dose that colonizes the colon in humans and can cause severe clinical manifestations such as hemolytic-uremic syndrome. The urease enzyme, encoded in the STEC chromosome, has been demonstrated to act as a virulence factor in other bacterial pathogens. The NH(3) produced as urease hydrolyzes urea can aid in buffering bacteria in acidic environments as well as provide an easily assimilated source of nitrogen that bacteria can use to gain a metabolic advantage over intact microflora. Here, we explore the role of urease in STEC pathogenicity. The STEC urease enzyme exhibited maximum activity near neutral pH and during the stationary-growth phase. Experiments altering growth conditions performed with three phylogenetically distinct urease-positive strains demonstrated that the STEC ure gene cluster is inducible by neither urea nor pH but does respond to nitrogen availability. Quantitative reverse transcription-PCR (qRT-PCR) data indicate that nitrogen inhibits the transcriptional response. The deletion of the ure gene locus was constructed in STEC strain 88-0643, and the ure mutant was used with the wild-type strain in competition experiments in mouse models to examine the contribution of urease. The wild-type strain was twice as likely to survive passage through the acidic stomach and demonstrated an enhanced ability to colonize the intestinal tract compared to the ure mutant strain. These in vivo experiments reveal that, although the benefit STEC gains from urease expression is modest and not absolutely required for colonization, urease can contribute to the pathogenicity of STEC.

Functional and phylogenetic analysis of ureD in Shiga toxin-producing Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) is a food-borne pathogen that can cause severe health complications and utilizes a much lower infectious dose than other E. coli pathotypes. Despite having an intact ure locus, ureDABCEFG, the majority of EHEC strains are phenotypically urease negative under tested conditions. Urease activity potentially assists with survival fitness by enhancing acid tolerance during passage through the stomach or by aiding with colonization in either human or animal reservoirs. Previously, in the EHEC O157:H7 Sakai strain, a point mutation in ureD, encoding a urease chaperone protein, was identified, resulting in a substitution of an amber stop codon for glutamine. This single nucleotide polymorphism (SNP) is observed in the majority of EHEC O157:H7 isolates and correlates with a negative urease phenotype in vitro. We demonstrate that the lack of urease activity in vitro is not solely due to the amber codon in ureD. Our analysis has identified two additional SNPs in ureD affecting amino acid positions 38 and 205, in both cases determining whether the encoded amino acid is leucine or proline. Phylogenetic analysis based on Ure protein sequences from a variety of urease-encoding bacteria demonstrates that the proline at position 38 is highly conserved among Gram-negative bacteria. Experiments reveal that the L38P substitution enhances urease enzyme activity; however, the L205P substitution does not. Multilocus sequence typing analysis for a variety of Shiga toxin-producing E. coli isolates combined with the ureD sequence reveals that except for a subset of the O157:H7 strains, neither the in vitro urease-positive phenotype nor the ureD sequence is phylogenetically restricted.

Identification of Bdellovibrio bacteriovorus HD100 Bd0714 as a Nudix dGTPase.

Bdellovibrio bacteriovorus bacteria are predatory organisms that attack other gram-negative bacteria. Here, we report that Bd0714 is a Nudix dGTPase from B. bacteriovorus HD100 with a substrate specificity similar to that of Escherichia coli MutT and complements an E. coli mutT-deficient strain. We observed different transcription levels of the gene throughout the predator life cycle.

Vibrio cholerae flagellins induce Toll-like receptor 5-mediated interleukin-8 production through mitogen-activated protein kinase and NF-kappaB activation.

Vaccine reactogenicity has complicated the development of safe and effective live, oral cholera vaccines. Delta ctx Vibrio cholerae mutants have been shown to induce inflammatory diarrhea in volunteers and interleukin-8 (IL-8) production in cultured intestinal epithelial cells. Bacterial flagellins are known to induce IL-8 production through Toll-like receptor 5 (TLR5). Since the V. cholerae genome encodes five distinct flagellin proteins, FlaA to FlaE, with homology to conserved TLR5 recognition regions of Salmonella FliC, we hypothesized that V. cholerae flagellins may contribute to IL-8 induction through TLR5 and mitogen-activated protein kinase (MAPK) signaling. Each purified recombinant V. cholerae flagellin induced IL-8 production in T84 intestinal epithelial cells and also induced nuclear factor kappa B (NF-kappaB) activation in HEK293T/TLR5 transfectants, which was blocked by cotransfection with a TLR5 dominant-negative construct, demonstrating TLR5 specificity. Supernatants derived from Delta flaAC and Delta flaEDB mutants induced IL-8 production in HT-29 intestinal epithelial cells and in HEK293T cells overexpressing TLR5, whereas Delta flaABCDE supernatants induced significantly less IL-8 production, demonstrating the contribution of multiple flagellins in IL-8 induction. NF-kappaB activation by Delta flaABCDE supernatants was partially restored by flaA or flaAC complementation. Western analysis confirmed the presence of V. cholerae flagellins in culture supernatants. Purified recombinant V. cholerae FlaA activated the MAPKs p38, c-jun N-terminal kinase (JNK), and extracellular regulated kinase (ERK) in T84 cells. FlaA-induced IL-8 production in T84 cells was inhibited by the p38 inhibitor in combination with either the JNK or ERK inhibitors. Collectively, these data suggest that V. cholerae flagellins are present in culture supernatants and can induce TLR5- and MAPK-dependent IL-8 secretion in host cells.

Genome Sequences of Listeria monocytogenes Serotype 4b Variant Strains Isolated from Clinical and Environmental Sources.

Listeria monocytogenes strains that show a novel PCR serotyping profile (IVb-v1) have been reported recently. Here, we announce the draft genome sequences of five L. monocytogenes IVb-v1 strains isolated from the United States and Australia that harbor a 6.3-kb DNA cassette characteristic of serotype 1/2a strains.

Comparative genomics and stx phage characterization of LEE-negative Shiga toxin-producing Escherichia coli.

Infection by Escherichia coli and Shigella species are among the leading causes of death due to diarrheal disease in the world. Shiga toxin-producing E. coli (STEC) that do not encode the locus of enterocyte effacement (LEE-negative STEC) often possess Shiga toxin gene variants and have been isolated from humans and a variety of animal sources. In this study, we compare the genomes of nine LEE-negative STEC harboring various stx alleles with four complete reference LEE-positive STEC isolates. Compared to a representative collection of prototype E. coli and Shigella isolates representing each of the pathotypes, the whole genome phylogeny demonstrated that these isolates are diverse. Whole genome comparative analysis of the 13 genomes revealed that in addition to the absence of the LEE pathogenicity island, phage-encoded genes including non-LEE encoded effectors, were absent from all nine LEE-negative STEC genomes. Several plasmid-encoded virulence factors reportedly identified in LEE-negative STEC isolates were identified in only a subset of the nine LEE-negative isolates further confirming the diversity of this group. In combination with whole genome analysis, we characterized the lambdoid phages harboring the various stx alleles and determined their genomic insertion sites. Although the integrase gene sequence corresponded with genomic location, it was not correlated with stx variant, further highlighting the mosaic nature of these phages. The transcription of these phages in different genomic backgrounds was examined. Expression of the Shiga toxin genes, stx(1) and/or stx(2), as well as the Q genes, were examined with quantitative reverse transcriptase polymerase chain reaction assays. A wide range of basal and induced toxin induction was observed. Overall, this is a first significant foray into the genome space of this unexplored group of emerging and divergent pathogens.

Development of a novel genetic system to create markerless deletion mutants of Bdellovibrio bacteriovorus.

Bdellovibrio bacteriovorus is a species of unique obligate predatory bacteria that utilize gram-negative bacteria as prey. Their life cycle alternates between a motile extracellular phase and a growth phase within the prey cell periplasm. The mechanism of prey cell invasion and the genetic networks and regulation during the life cycle have not been elucidated. The obligate predatory nature of the B. bacteriovorus life cycle suggests the use of this bacterium in potential applications involving pathogen control but adds complexity to the development of practical genetic systems that can be used to determine gene function. This work reports the development of a genetic technique for allelic exchange or gene inactivation by construction of in-frame markerless deletion mutants including the use of a counterselectable marker in B. bacteriovorus. A suicide plasmid carrying the sacB gene for counterselection was used to inactivate the strB gene in B. bacteriovorus HD100 by an in-frame deletion. Despite the inactivation of the strB gene, B. bacteriovorus was found to retain resistance to high concentrations of streptomycin. The stability of a plasmid for use in complementation experiments was also investigated, and it was determined that pMMB206 replicates autonomously in B. bacteriovorus. Development of this practical genetic system now facilitates the study of B. bacteriovorus at the molecular level and will aid in understanding the regulatory networks and gene function in this fascinating predatory bacterium.

Global survey of diversity among environmental saltwater Bacteriovoracaceae.

Halophilic Bacteriovorax (Bx), formerly known as the marine Bdellovibrio, are Gram-negative, predatory bacteria found in saltwater systems. To assess their genetic diversity and geographical occurrence, the small subunit rRNA (ssu-rRNA) gene sequences were analysed from 111 marine, salt lake and estuarine isolates recovered from 27 locations around the world. Phylogenetic analysis of these isolates using Geobacter as the outgroup revealed eight distinct ribotype clusters each with at least two isolates. Each cluster was composed of isolates with >or= 96.5% similarity in ssu-rRNA sequences. Three single isolate outliers were observed. Many of the Bx ribotypes were widely dispersed among different types of ecosystems (e.g. cluster III was recovered from the Great Salt Lake, the Atlantic Ocean, Pacific Ocean, Chesapeake Bay and gills of aquarium fish). However, cluster V was only recovered from a single ecosystem, estuaries. Cluster V was originally detected in the Chesapeake Bay and subsequently in the Pamlico Sound/Neuse River system. Principal coordinate analysis revealed that the sequences of the isolates from different environments were distinct from each other. The results of this study reveal the saltwater Bx to be phylogenetically and environmentally more diverse than was previously known.