Verocytotoxin-producing Escherichia coli O128ab:H2 bacteremia in a 27-year-old male with hemolytic-uremic syndrome.

Verocytotoxin-producing Escherichia coli (VTEC) strains of serotype O128ab:H2 were isolated from blood and stool of a 27-year-old male presenting diarrhea-associated hemolytic-uremic syndrome complicated by bacteremia. This report once again illustrates the pathogenic potential of a non-O157 VTEC strain carrying a virulence profile previously associated with mild disease.

Incidence and virulence determinants of verocytotoxin-producing Escherichia coli infections in the Brussels-Capital Region, Belgium, in 2008-2010.

The incidence of verocytotoxin-producing Escherichia coli (VTEC) was investigated by PCR in all human stools from Universitair Ziekenhuis Brussel (UZB) and in selected stools from six other hospital laboratories in the Brussels-Capital Region, Belgium, collected between April 2008 and October 2010. The stools selected to be included in this study were those from patients with hemolytic-uremic syndrome (HUS), patients with a history of bloody diarrhea, patients linked to clusters of diarrhea, children up to the age of 6 years, and stools containing macroscopic blood. Verocytotoxin genes (vtx) were detected significantly more frequently in stools from patients with the selected conditions (2.04%) than in unselected stools from UZB (1.20%) (P = 0.001). VTEC was detected most frequently in patients with HUS (35.3%), a history of bloody diarrhea (5.15%), or stools containing macroscopic blood (1.85%). Stools from patients up to the age of 17 years were significantly more frequently vtx positive than those from adult patients between the ages of 18 and 65 years (P = 0.022). Although stools from patients older than 65 years were also more frequently positive for vtx than those from patients between 18 and 65 years, this trend was not significant. VTEC was isolated from 140 (67.9%) vtx-positive stools. One sample yielded two different serotypes; thus, 141 isolates could be characterized. Sixty different O:H serotypes harboring 85 different virulence profiles were identified. Serotypes O157:H7/H- (n = 34), O26:H11/H- (n = 21), O63:H6 (n = 8), O111:H8/H- (n = 7), and O146:H21/H- (n = 6) accounted for 53.9% of isolates. All O157 isolates carried vtx2, eae, and a complete O island 122 (COI-122); 15 also carried vtx1. Non-O157 isolates (n = 107), however, accounted for the bulk (75.9%) of isolates. Fifty-nine (55.1%) isolates were positive for vtx1, 36 (33.6%) were positive for vtx2, and 12 (11.2%) carried both vtx1 and vtx2. Pulsed-field gel electrophoresis revealed wide genetic diversity; however, small clusters of O157, O26, and O63:H6 VTEC that could have been part of unidentified outbreaks were identified. Antimicrobial resistance was observed in 63 (44.7%) isolates, and 34 (24.1%) showed multidrug resistance. Our data show that VTEC infections were not limited to patients with HUS or bloody diarrhea. Clinical laboratories should, therefore, screen all stools for O157 and non-O157 VTEC using selective media and a method for detecting verocytotoxins or vtx genes.

Multicenter evaluation of a sequence-based protocol for subtyping Shiga toxins and standardizing Stx nomenclature.

When Shiga toxin-producing Escherichia coli (STEC) strains emerged as agents of human disease, two types of toxin were identified: Shiga toxin type 1 (Stx1) (almost identical to Shiga toxin produced by Shigella dysenteriae type 1) and the immunologically distinct type 2 (Stx2). Subsequently, numerous STEC strains have been characterized that express toxins with variations in amino acid sequence, some of which confer unique biological properties. These variants were grouped within the Stx1 or Stx2 type and often assigned names to indicate that they were not identical in sequence or phenotype to the main Stx1 or Stx2 type. A lack of specificity or consistency in toxin nomenclature has led to much confusion in the characterization of STEC strains. Because serious outcomes of infection have been attributed to certain Stx subtypes and less so with others, we sought to better define the toxin subtypes within the main Stx1 and Stx2 types. We compared the levels of relatedness of 285 valid sequence variants of Stx1 and Stx2 and identified common sequences characteristic of each of three Stx/Stx1 and seven Stx2 subtypes. A novel, simple PCR subtyping method was developed, independently tested on a battery of 48 prototypic STEC strains, and improved at six clinical and research centers to test the reproducibility, sensitivity, and specificity of the PCR. Using a consistent schema for nomenclature of the Stx toxins and stx genes by phylogenetic sequence-based relatedness of the holotoxin proteins, we developed a typing approach that should obviate the need to bioassay each newly described toxin and that predicts important biological characteristics.

Virulence profiling and disease association of verocytotoxin-producing Escherichia coli O157 and non-O157 isolates in Belgium.

Whereas the association of verocytotoxin-producing Escherichia coli (VTEC) O157:H7 with the hemolytic uremic syndrome (HUS) is well established, the medical importance of many non-O157 serotypes remains unclear. Using polymerase chain reaction (PCR), we have investigated the distribution of the pathogenicity island O island 122 (OI-122) and other virulence genes in VTEC belonging to seropathotypes (SPT) A through D, and assessed their association with human disease. Two hundred sixty-five VTEC isolated from human stools comprising 52 O157 (of which 14 associated with HUS) and 213 non-O157 isolates (of which 19 associated with HUS) were studied. A complete OI-122 (COI-122) was detected in all O157, but in only 35 (16.4%) of non-O157 strains. A progressive decrease in the frequency of COI-122 was observed from SPT A through D, with a concomitant increase in the frequencies of incomplete and absent OI-122. We focused on the variable virulence profiles of the non-O157 serotypes and found that COI-122 was also more frequently present in isolates associated with HUS (p=0.001). The individual genes vtx2, eae, espP, as well as the OI-122-associated genes sen, nleB, nleE, and the efa gene cluster were significantly more often present in non-O157 VTEC associated with HUS. Non-O157 isolates carrying the combined virulence profile vtx2-nleE-efa showed the strongest association with HUS (p<0.0001). Molecular risk assessment by determination of virulence profiles of individual isolates may be useful in the identification of highly virulent non-O157 strains. We showed that the detection of a specific gene combination could assist in identifying non-O157 VTEC isolates that pose a serious public health concern.

Virulence profiling and quantification of verocytotoxin-producing Escherichia coli O145:H28 and O26:H11 isolated during an ice cream-related hemolytic uremic syndrome outbreak.

In September-October 2007, a mixed-serotype outbreak of verocytotoxin-producing Escherichia coli (VTEC) O145:H28 and O26:H11 occurred in the province of Antwerp, Belgium. Five girls aged between 2 and 11 years developed hemolytic uremic syndrome, and seven other coexposed persons with bloody diarrhea were identified. Laboratory confirmation of O145:H28 infection was obtained for three hemolytic uremic syndrome patients, one of whom was coinfected with O26:H11. The epidemiological and laboratory investigations revealed ice cream as the most likely source of the outbreak. The ice cream was produced at a local dairy farm using pasteurized milk. VTEC of both serotypes with indistinguishable pulsed-field gel electrophoresis patterns were isolated from patients, ice cream, and environmental samples. Quantitative analysis of the ice cream indicated concentrations of 2.4 and 0.03 CFU/g for VTEC O145 and O26, respectively. Virulence typing revealed that the repertoire of virulence genes carried by the O145:H28 outbreak strain was comparable to that of O157 VTEC and more exhaustive as compared to the O26:H11 outbreak strain and nonrelated clinical strains belonging to these serotypes. Taken together, these data suggest that O145:H28 played the most important role in this outbreak.

Antimicrobial resistance testing of verocytotoxin-producing Escherichia coli and first description of TEM-52 extended-spectrum ß-lactamase in serogroup O26.

We have investigated the antimicrobial resistance of verocytotoxin-producing Escherichia coli (VTEC) strains isolated from humans, animals, food, and the environment in Belgium. Resistance was more frequent in non-O157 strains from humans than in O157 strains from humans or other sources, and among non-O157 VTEC strains, intimin-positive strains were more resistant than intimin-negative strains. We also report the first VTEC strain producing an IncI1 extended-spectrum ß-lactamase encoded by plasmid-borne bla(TEM-52); this ß-lactamase was previously associated with Salmonella enterica and E. coli isolates from different origins.

Integron characterization and typing of Shiga toxin-producing Escherichia coli isolates in Belgium.

The presence of integrons and the antibiotic susceptibility profiles of STEC strains isolated in Belgium were analysed. The collection contained 306 strains, of which 225 were human isolates and 81 originated from different food or animal sources. Integrons were detected by PCR in 7.5% of the tested isolates and all were class 1 integrons. The integron-positive strains all belonged to the human collection. By RFLP, five different types (A, B, C, D, E) were distinguished. The antibiotic-resistance gene cassettes were identified by sequencing representatives of the five different types. Two types of gene cassettes were found in different combinations, one encoding resistance to streptomycin/spectinomycin and the other encoding resistance to trimethoprim. One of the gene cassettes present was the rarely detected aadA23, which was now apparently for the first time reported in Western Europe. Susceptibility profiling of the strains for 11 antibiotics was done by standard disc diffusion assays. Among the 23 integron-positive strains, 17 different antibiotic susceptibility profiles were found. In the 283 integron-negative strains, 24 different antibiotic susceptibility profiles were observed. The majority of these strains were susceptible to all tested antibiotics (n=218, 77.0%). The integron-positive strains were significantly more resistant to eight of the eleven tested antibiotics compared to the integron-negative strains (P<0.05). PFGE profiles of integron-positive strains within selected serogroups did not cluster together.

Occurrence of non-sorbitol fermenting, verocytotoxin-lacking Escherichia coli O157 on cattle farms.

Escherichia coli O157 is often associated with hemorrhagic colitis and the hemolytic uremic syndrome (HUS). The verocytotoxins are considered to be the major virulence determinants. However, vt-negative E. coli O157 were recently isolated from patients with HUS. Several transmission routes to humans are described, but cattle feces are the primary source from which both the food supply and the environment become contaminated with E. coli O157. In a prevalence study performed on dairy, beef, mixed dairy/beef and veal farms in the summer of 2007, vt-negative isolates were detected on 11.8% (8/68) of the positive farms. From these eight farms, a total of 43 sorbitol-negative E. coli O157:H7 were collected. On five farms, only strains negative for the vt genes were present whereas both vt-negative and vt-positive strains could be detected on three other farms. Further characterization revealed that all isolates carried the eaeA and hlyA genes. Pulsed-field gel electrophoresis (PFGE) of all isolates resulted in nine different PFGE types and within the vt-negative strains, four different genotypes were identified, indicating that certain genetic clones are widespread over the cattle population.