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.

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, stx1 and/or stx2, 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.

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.

The gram-negative bacterium Vibrio cholerae causes cholera, an acute diarrheal disease characterized by voluminous rice water stools and rapid dehydration. V. cholerae produces an ADP-ribosylating enterotoxin called cholera toxin (CT), encoded by ctxA and ctxB, that activates the host cell enzyme adenylate cyclase, resulting in profuse diarrhea in humans (15). The preeminence of CT as a major virulence factor of cholera was confirmed in volunteer studies, making this toxin a prime target in the development of cholera vaccine strains (21,23). While the first generation of ⌬ctxA and ⌬ctxAB V. cholerae vaccine strains did not cause cholera, they nevertheless caused significant reactogenicity in individuals in the form of mild to moderate diarrhea (23). Although the reason for the reactogenicity of the ⌬ctx vaccine strains is unknown, two hypotheses were introduced as possible explanations (22). First, V. cholerae may produce additional unidentified enterotoxins that were previously undetectable because of the dominant effects of CT. Second, adherence of V. cholerae during colonization of the proximal small intestine may cause alterations in small intestine function, resulting in diarrhea. Although further efforts to improve live oral cholera vaccines have focused on deleting newly characterized toxins, the reactogenicity in these strains was not markedly diminished (9, 49, 52). This observation supports the latter hypothesis.While cholera is not generally considered an inflammatory disease, there is evidence of inflammation in cholera patients and from human and animal vaccine studies. For instance, immune cell infiltration and activation have been observed in patients with cholera (10,24,33,34). More recently, Qadri et al. reported neutrophil infiltration into the lamina propria along with an increase in inflammatory mediators, such as tumor necrosis factor alpha, in adults and children during the acute stage of V. cholerae O1 and O139 serogroup infections (35,36). In human volunteer studies, the fecal lactoferrin levels induced by the reactogenic ⌬ctx vaccine strain CVD110 were markedly higher than those induced by the wild-type CT-expressing El Tor strain (44). The lactoferrin levels induced by CVD110 were comparable to elevated levels found in volunteers who ingested Shigella, the prototypic inflammatory enteric pathogen (30). Lactoferrin is a major component of polymorphonuclear leukocytes and is secreted by most mucosal membranes during inflammatory responses. Its presence in feces is indicative of leukocyte infiltration and intestinal inflammation. In a rabbit model, elevated levels of interleukin-1␤ (IL-1␤) and IL-8 were observed following infection with two different reactogenic ⌬ctx V. cholerae vaccine strains (E. C. Boedecker and J. B. Kaper, unpublished observations). IL-8 production has also been reported to contribute to the recruitment of neutrophils following infection with the enteric pathogens Salmonella spp. and Escherichia coli (4, 25, 42). Reacto-* Corresponding author. Mailing address:

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.

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.

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. Shiga toxin-producing Escherichia coli (STEC) is an enteric pathogen that colonizes the colon in humans and causes a variety of symptoms ranging from mild diarrhea to bloody diarrhea or the more severe renal pathology of hemolytic uremic syndrome, sometimes followed by death (17). In the first two STEC genomes sequenced, those of the O157:H7 EDL933 strain and O157:H7 Sakai strain, a genomic island containing, among other genes, ureDABCEFG, a cluster of genes coding for the enzyme urease and its accessory proteins, was identified (12, 39). Subsequently, many other STEC strains, including a wide variety of serotypes, have been found to carry the ure gene cluster (9,35,36,46). Urease, which hydrolyzes the substrate urea to form CO 2 and two molecules of NH 3 (29), has been demonstrated to contribute to virulence in several other bacterial species (1, 5-8, 25, 43). The NH 3 produced raises the pH of the cytoplasm, thereby buffering the bacteria under acidic environmental conditions. In addition, the NH 3 provides the bacterial cell with an easily assimilated source of nitrogen. Despite the fact that STEC has been recognized as an emergent pathogen since 1982 (42), virtually no information about the regulation of the ure genes and how they contribute to pathogenicity in this important pathogen is available.Neither STEC strain EDL933 nor the Sakai strain...

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.