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1.
Shiga toxin-producing Escherichia coli (STEC) is a food-borne pathogen that may be responsible for severe human infections. Only a limited number of serotypes, including O26:H11, are involved in the majority of serious cases and outbreaks. The main virulence factors, Shiga toxins (Stx), are encoded by bacteriophages. Seventy-four STEC O26:H11 strains of various origins (including human, dairy, and cattle) were characterized for their stx subtypes and Stx phage chromosomal insertion sites. The majority of food and cattle strains possessed the stx1a subtype, while human strains carried mainly stx1a or stx2a. The wrbA and yehV genes were the main Stx phage insertion sites in STEC O26:H11, followed distantly by yecE and sbcB. Interestingly, the occurrence of Stx phages inserted in the yecE gene was low in dairy strains. In most of the 29 stx-negative E. coli O26:H11 strains also studied here, these bacterial insertion sites were vacant. Multilocus sequence typing of 20 stx-positive or stx-negative E. coli O26:H11 strains showed that they were distributed into two phylogenetic groups defined by sequence type 21 (ST21) and ST29. Finally, an EspK-carrying phage was found inserted in the ssrA gene in the majority of the STEC O26:H11 strains but in only a minority of the stx-negative E. coli O26:H11 strains. The differences in the stx subtypes and Stx phage insertion sites observed in STEC O26:H11 according to their origin might reflect that strains circulating in cattle and foods are clonally distinct from those isolated from human patients.  相似文献   

2.
Escherichia coli O26:H11 strains were able to outgrow O157:H7 companion strains in planktonic and biofilm phases and also to effectively compete with precolonized O157:H7 cells to establish themselves in mixed biofilms. E. coli O157:H7 strains were unable to displace preformed O26:H11 biofilms. Therefore, E. coli O26:H11 remains a potential risk in food safety.  相似文献   

3.
Shiga toxin-producing Escherichia coli (STEC) O111:NM is an important serotype that has been incriminated in disease outbreaks in the United States. This study characterized cattle STEC O111:NM for virulence factors and markers by PCR. Major conclusions are that STEC O111:NM characterized in this study lacks stx2 and the full spectrum of nle gene markers, and it has an incomplete OI-122.  相似文献   

4.
Cattle are an important reservoir of Shiga toxin-producing Escherichia coli (STEC) O26, O111, and O157. The fate of these pathogens in bovine feces at 5, 15, and 25°C was examined. The feces of a cow naturally infected with STEC O26:H11 and two STEC-free cows were studied. STEC O26, O111, and O157 were inoculated into bovine feces at 101, 103, and 105 CFU/g. All three pathogens survived at 5 and 25°C for 1 to 4 weeks and at 15°C for 1 to 8 weeks when inoculated at the low concentration. On samples inoculated with the middle and high concentrations, O26, O111, and O157 survived at 25°C for 3 to 12 weeks, at 15°C for 1 to 18 weeks, and at 5°C for 2 to 14 weeks, respectively. Therefore, these pathogens can survive in feces for a long time, especially at 15°C. The surprising long-term survival of STEC O26, O111, and O157 in bovine feces shows that such feces are a potential vehicle for transmitting not only O157 but also O26 and O111 to cattle, food, and the environment. Appropriate handling of bovine feces is emphasized.  相似文献   

5.
Escherichia coli O157:H7, a zoonotic human pathogen for which domestic cattle are a reservoir host, produces a Shiga toxin(s) (Stx) encoded by bacteriophages. Chromosomal insertion sites of these bacteriophages define three principal genotypes (clusters 1 to 3) among clinical isolates of E. coli O157:H7. Stx-encoding bacteriophage insertion site genotypes of 282 clinical and 80 bovine isolates were evaluated. A total of 268 (95.0%) of the clinical isolates, but only 41 (51.3%) of the bovine isolates, belonged to cluster 1, 2, or 3 (P < 0.001). Thirteen additional genotypes were identified in isolates from both cattle and humans (four genotypes), from only cattle (seven genotypes), or from only humans (two genotypes). Two other markers previously associated with isolates from cattle or with clinical isolates showed similar associations with genotype groups within bovine isolates; the tir allele sp-1 and the Q933W allele were under- and overrepresented, respectively, among cluster 1 to 3 genotypes. Stx-encoding bacteriophage insertion site typing demonstrated that there is broad genetic diversity of E. coli O157:H7 in the bovine reservoir and that numerous genotypes are significantly underrepresented among clinical isolates, consistent with the possibility that there is reduced virulence or transmissibility to humans of some bovine E. coli O157:H7 genotypes.  相似文献   

6.
7.

Background

Shiga toxin-producing Escherichia coli (STEC) O157:H7 is the causal agent for more than 96,000 cases of diarrheal illness and 3,200 infection-attributable hospitalizations annually in the United States.

Materials and Methods

We defined a confirmed case as a compatible illness in a person with the outbreak strain during 10/07/2011-11/30/2011. Investigation included hypothesis generation, a case-control study utilizing geographically-matched controls, and a case series investigation. Environmental inspections and tracebacks were conducted.

Results

We identified 58 cases in 10 states; 67% were hospitalized and 6.4% developed hemolytic uremic syndrome. Any romaine consumption was significantly associated with illness (matched Odds Ratio (mOR) = 10.0, 95% Confidence Interval (CI) = 2.1–97.0). Grocery Store Chain A salad bar was significantly associated with illness (mOR = 18.9, 95% CI = 4.5–176.8). Two separate traceback investigations for romaine lettuce converged on Farm A. Case series results indicate that cases (64.9%) were more likely than the FoodNet population (47%) to eat romaine lettuce (p-value = 0.013); 61.3% of cases reported consuming romaine lettuce from the Grocery Store Chain A salad bar.

Conclusions

This multistate outbreak of STEC O157:H7 infections was associated with consumption of romaine lettuce. Traceback analysis determined that a single common lot of romaine lettuce harvested from Farm A was used to supply Grocery Store Chain A and a university campus linked to a case with the outbreak strain. An investigation at Farm A did not identify the source of contamination. Improved ability to trace produce from the growing fields to the point of consumption will allow more timely prevention and control measures to be implemented.  相似文献   

8.
Novel and as yet rare non-O157 Shiga toxin (Stx)-producing Escherichia coli (STEC) serotypes are emerging in Europe. Two different sorbitol-fermenting STECs, O100:H- carrying the virulence gene stx2 and O127:H40 carrying stx1 and eae genes (found in two related subjects), were isolated from patients’ stool samples. Non-O157 STEC infections in humans are currently under-diagnosed. This report highlights the need for, and importance of, screening for Shiga toxins or serotypes other than just O157.  相似文献   

9.
Isogenic strains of Escherichia coli O157:H7, missing either stx2 or the entire Stx2-encoding phage, were compared with the parent strain for their abilities to colonize sheep. The absence of the phage or of the Shiga toxin did not significantly impact the magnitude or duration of shedding of E. coli O157:H7.  相似文献   

10.
To determine the prevalence of Shiga toxin (Stx)-producing Escherichia coli (STEC) in slaughter animals in Dhaka, Bangladesh, we collected rectal contents immediately after animals were slaughtered. Of the samples collected from buffalo (n = 174), cows (n = 139), and goats (n = 110), 82.2%, 72.7%, and 11.8% tested positive for stx1 and/or stx2, respectively. STEC could be isolated from 37.9%, 20.1%, and 10.0% of the buffalo, cows, and goats, respectively. STEC O157 samples were isolated from 14.4% of the buffalo, 7.2% of the cows, and 9.1% of the goats. More than 93% (n = 42) of the STEC O157 isolates were positive for the stx2, eae, katP, etpD, and enterohemorrhagic E. coli hly (hlyEHEC) virulence genes. STEC O157 isolates were characterized by seven recognized phage types, of which types 14 (24.4%) and 31 (24.4%) were predominant. Subtyping of the 45 STEC O157 isolates by pulsed-field gel electrophoresis showed 37 distinct restriction patterns, suggesting a heterogeneous clonal diversity. In addition to STEC O157, 71 STEC non-O157 strains were isolated from 60 stx-positive samples from 23.6% of the buffalo, 12.9% of the cows, and 0.9% of the goats. The STEC non-O157 isolates belonged to 36 different O groups and 52 O:H serotypes. Unlike STEC O157, most of the STEC non-O157 isolates (78.9%) were positive for stx1. Only 7.0% (n = 5) of the isolates were positive for hlyEHEC, and none was positive for eae, katP, and etpD. None of the isolates was positive for the iha, toxB, and efa1 putative adhesion genes. However, 35.2% (n = 25), 11.3% (n = 8), 12.7% (n = 9), and 12.7% (n = 9) of the isolates were positive for the lpfO113, saa, lpfAO157/01-141, and lpfAO157/OI-154 genes, respectively. The results of this study provide the first evidence that slaughtered animals like buffalo, cows, and goats in Bangladesh are reservoirs for STEC, including the potentially virulent STEC strain O157.  相似文献   

11.
Serotyping is the foundation of pathogenic Escherichia coli diagnostics; however, few laboratories have this capacity. We developed a molecular serotyping protocol that targets, genetically, the same somatic and flagellar antigens of E. coli O26:H11 used in traditional serotyping. It correctly serotypes strains untypeable by traditional methods, affording primary laboratories serotyping capabilities.  相似文献   

12.
The main pathogenic enterohemorrhagic Escherichia coli (EHEC) strains are defined as Shiga toxin (Stx)-producing E. coli (STEC) belonging to one of the following serotypes: O157:H7, O26:H11, O103:H2, O111:H8, and O145:H28. Each of these five serotypes is known to be associated with a specific subtype of the intimin-encoding gene (eae). The objective of this study was to evaluate the prevalence of bovine carriers of these “top five” STEC in the four adult cattle categories slaughtered in France. Fecal samples were collected from 1,318 cattle, including 291 young dairy bulls, 296 young beef bulls, 337 dairy cows, and 394 beef cows. A total of 96 E. coli isolates, including 33 top five STEC and 63 atypical enteropathogenic E. coli (aEPEC) isolates, with the same genetic characteristics as the top five STEC strains except that they lacked an stx gene, were recovered from these samples. O157:H7 was the most frequently isolated STEC serotype. The prevalence of top five STEC (all serotypes included) was 4.5% in young dairy bulls, 2.4% in young beef bulls, 1.8% in dairy cows, and 1.0% in beef cows. It was significantly higher in young dairy bulls (P < 0.05) than in the other 3 categories. The basis for these differences between categories remains to be elucidated. Moreover, simultaneous carriage of STEC O26:H11 and STEC O103:H2 was detected in one young dairy bull. Lastly, the prevalence of bovine carriers of the top five STEC, evaluated through a weighted arithmetic mean of the prevalence by categories, was estimated to 1.8% in slaughtered adult cattle in France.  相似文献   

13.
Twelve cluster groups of Escherichia coli O26 isolates found in three cattle farms were monitored in space and time. Cluster analysis suggests that only some O26:H11 strains had the potential for long-term persistence in hosts and farms. As judged by their virulence markers, bovine enterohemorrhagic O26:H11 isolates may represent a considerable risk for human infection.Shiga toxin (Stx)-producing Escherichia coli (STEC) strains comprise a group of zoonotic enteric pathogens (42). In humans, infections with some STEC serotypes result in hemorrhagic or nonhemorrhagic diarrhea, which can be complicated by hemolytic-uremic syndrome (HUS) (49). These STEC strains are also designated “enterohemorrhagic E. coli” (EHEC). Consequently, EHEC strains represent a subgroup of STEC with a high pathogenic potential for humans. Strains of the E. coli serogroup O26 were originally classified as enteropathogenic E. coli due to their association with outbreaks of infantile diarrhea in the 1940s. In 1977, Konowalchuk et al. (37) recognized that these bacteria produced Stx, and 10 years later, the Stx-producing E. coli O26:H11/H− strains were classified as EHEC. EHEC O26 strains constitute the most common non-O157 EHEC group associated with diarrhea and HUS in Europe (12, 21, 23, 24, 26, 27, 55, 60). Reports on an association between EHEC O26 and HUS or diarrhea from North America including the United States (15, 30, 33), South America (51, 57), Australia (22), and Asia (31, 32) provide further evidence for the worldwide spread of these organisms. Studies in Germany and Austria (26, 27) on sporadic HUS cases between 1996 and 2003 found that EHEC O26 accounted for 14% of all EHEC strains and for ∼40% of non-O157 EHEC strains obtained from these patients. A proportion of 11% EHEC O26 strains was detected in a case-control study in Germany (59) between 2001 and 2003. In the age group <3 years, the number of EHEC O26 cases was nearly equal to that of EHEC O157 cases, although the incidence of EHEC O26-associated disease is probably underestimated because of diagnostic limitations in comparison to the diagnosis of O157:H7/H− (18, 34). Moreover, EHEC O26 has spread globally (35). Beutin (6) described EHEC O26:H11/H−, among O103:H2, O111:H, O145:H28/H−, and O157:H7/H−, as the well-known pathogenic “gang of five,” and Bettelheim (5) warned that we ignore the non-O157 STEC strains at our peril.EHEC O26 strains produce Stx1, Stx2, or both (15, 63). Moreover, these strains contain the intimin-encoding eae gene (11, 63), a characteristic feature of EHEC (44). In addition, EHEC strains possess other markers associated with virulence, such as a large plasmid that carries further potential virulence genes, e.g., genes coding for EHEC hemolysin (EHEC-hlyA), a catalase-peroxidase (katP), and an extracellular serine protease (espP) (17, 52). The efa1 (E. coli factor for adherence 1) gene was identified as an intestinal colonization factor in EHEC (43). EHEC O26 represents a highly dynamic group of organisms that rapidly generate new pathogenic clones (7, 8, 63).Ruminants, especially cattle, are considered the primary reservoir for human infections with EHEC. Therefore, the aim of this study was the molecular characterization of bovine E. coli field isolates of serogroup O26 using a panel of typical virulence markers. The epidemiological situation in the beef herds from which the isolates were obtained and the spatial and temporal behavior of the clonal distribution of E. coli serogroup O26 were analyzed during the observation period. The potential risk of the isolates inducing disease in humans was assessed.In our study, 56 bovine E. coli O26:H11 isolates and one bovine O26:H32 isolate were analyzed for EHEC virulence-associated factors. The isolates had been obtained from three different beef farms during a long-term study. They were detected in eight different cattle in farm A over a period of 15 months (detected on 10 sampling days), in 3 different animals in farm C over a period of 8 months (detected on 3 sampling days), and in one cow on one sampling day in farm D (Table (Table1)1) (28).

TABLE 1.

Typing of E. coli O26 isolates
Sampling day, source, and isolateSerotypeVirulence profile by:
fliC PCR-RFLPstx1 genestx2 geneStx1 (toxin)Stx2 (toxin)Subtype(s)
efa1 genebEHEC-hlyA genekatP geneespP genePlasmid size(s) in kbCluster
stx1/stx2eaetirespAespB
Day 15
    Animal 6 (farm A)
        WH-01/06/002-1O26:H11H11++stx1ββββ+/++++110, 127
        WH-01/06/002-2O26:H11H11++stx1ββββ+/++++110, 127
        WH-01/06/002-3O26:H11H11++stx1ββββ+/++++110, 127
    Animal 8 (farm A)
        WH-01/08/002-2O26:H11H11++stx1ββββ+/++++110, 127
    Animal 26 (farm A)
        WH-01/26/001-2O26:H11H11++stx1ββββ+/++++130, 127
        WH-01/26/001-5O26:H11H11++stx1ββββ+/++++110, 127
        WH-01/26/001-6O26:H11H11++stx1ββββ+/++++110, 127
        WH-01/26/001-7O26:H11H11++stx1ββββ+/−+++110, 127
Day 29
    Animal 2 (farm A)
        WH-01/02/003-1O26:H11H11++stx1ββββ+/++++110, 126
        WH-01/02/003-2O26:H11H11++stx1ββββ+/++++110, 126
        WH-01/02/003-5O26:H11H11++stx1ββββ+/++++110, 126
        WH-01/02/003-6O26:H11H11++stx1ββββ+/+++110, 126
        WH-01/02/003-7O26:H11H11++stx1ββββ+/++++110, 126
        WH-01/02/003-8O26:H11H11++stx1ββββ−/++++110, 126
        WH-01/02/003-9O26:H11H11++stx1ββββ+/++++1106
        WH-01/02/003-10O26:H11H11++stx1ββββ+/++++1106
    Animal 26 (farm A)
        WH-01/26/002-2O26:H11H11++stx1ββββ+/++++130, 125
        WH-01/26/002-5O26:H11H11++stx1ββββ+/++++130, 125
        WH-01/26/002-8O26:H11H11++stx1ββββ+/++++130, 125
        WH-01/26/002-9O26:H11H11++stx1ββββ+/++110, 125
        WH-01/26/002-10O26:H11H11++stx1ββββ+/++++130, 125
Day 64
    Animal 20 (farm A)
        WH-01/20/005-3O26:H11H11++stx1ββββ+/+130, 2.52
Day 78
    Animal 29 (farm A)
        WH-01/29/002-1O26:H11H11++stx1ββββ+/−+130, 12, 2.54
        WH-01/29/002-2O26:H11H11++stx1ββββ+/++++130, 12, 2.54
        WH-01/29/002-3O26:H11H11++stx1ββββ+/++++130, 12, 2.54
        WH-01/29/002-4O26:H11H11++stx1ββββ+/++++130, 12, 2.54
        WH-01/29/002-5O26:H11H11++stx1ββββ+/++130, 12, 2.54
Day 106
    Animal 27 (farm A)
        WH-01/27/005-2O26:H11H11++stx1ββββ+/−+++145, 110, 123
        WH-01/27/005-5O26:H11H11++stx1ββββ+/++++130, 12, 2.55
        WH-01/27/005-6O26:H11H11++stx1ββββ+/+130, 12, 2.55
Day 113
    Animal 7 (farm C)
        WH-04/07/001-2O26:H11H11++++stx1/stx2ββββ+/+++55, 35, 2.511
        WH-04/07/001-4O26:H11H11++++stx1/stx2ββββ+/++++5512
        WH-04/07/001-6O26:H11H11++++stx1/stx2ββββ+/++++5512
Day 170
    Animal 22 (farm C)
        WH-04/22/001-1O26:H11H11++stx1ββββ+/++++110, 12, 6.312
        WH-04/22/001-4O26:H11H11++stx1ββββ+/++++110, 12, 6.312
        WH-04/22/001-5O26:H11H11++stx1ββββ+/++++110, 12, 6.312
Day 176
    Animal 14 (farm D)
        WH-03/14/004-8O26:H11H11++stx1ββββ+/+++11010
Day 218
    Animal 27 (farm A)
        WH-01/27/009-1O26:H11H11++++stx1/stx2ββββ+/++++110, 129
        WH-01/27/009-2O26:H11H11++++stx1/stx2ββββ+/++++110, 129
        WH-01/27/009-3O26:H11H11++++stx1/stx2ββββ+/++++110, 128
        WH-01/27/009-8O26:H11H11++++stx1/stx2ββββ+/++110, 128
        WH-01/27/009-9O26:H11H11++++stx1/stx2ββββ+/++++110, 129
Day 309
    Animal 29 (farm A)
        WH-01/29/010-1O26:H11H11++stx1ββββ+/++++110, 35, 124
        WH-01/29/010-2O26:H11H11++stx1ββββ+/++130, 55, 358
        WH-01/29/010-3O26:H11H11++stx1ββββ+/++++130, 35, 128
Day 365
    Animal 8 (farm C)
        WH-04/08/008-6O26:H11H11++stx1ββββ+/++++110, 5512
Day 379
    Animal 9 (farm A)
        WH-01/09/016-2O26:H32H32++stx1/stx2−/−145, 130, 1.81
    Animal 27 (farm A)
        WH-01/27/014-3O26:H11H11++stx1ββββ+/++++110, 129
        WH-01/27/014-4O26:H11H11++stx1ββββ+/++++110, 129
        WH-01/27/014-5O26:H11H11++stx1ββββ+/++++110, 128
Day 407
    Animal 29 (farm A)
        WH-01/29/013-4O26:H11H11++stx1ββββ+/++++110, 12, 2.58
        WH-01/29/013-7O26:H11H11++stx1ββββ+/++++110, 12, 2.58
Day 478
    Animal 27 (farm A)
        WH-01/27/017-1O26:H11H11++++stx1/stx2ββββ+/++++110, 128
        WH-01/27/017-5O26:H11H11++++stx1/stx2ββββ+/++++110, 128
        WH-01/27/017-6O26:H11H11++++stx1/stx2ββββ+/++++1108
        WH-01/27/017-7O26:H11H11++++stx1/stx2ββββ+/++++1108
        WH-01/27/017-10O26:H11H11+++stx1ββββ+/++++130, 12, 2.58
Open in a separate windowastx1/stx2, gene stx1 or stx2.befa1 was detected by two hybridizations (with lifA1-lifA2 and lifA3-lifA4 probes). +/+, complete gene; +/− or −/+, incomplete gene; −/−, efa1 negative.The serotyping of the O26 isolates was confirmed by the results of the fliC PCR-restriction fragment length polymorphism (RFLP) analysis performed according to Fields et al. (25), with slight modifications described by Zhang et al. (62). All O26:H11 isolates showed the H11 pattern described by Zhang et al. (62). In contrast, the O26:H32 isolate demonstrated a different fliC RFLP pattern that was identical to the H32 pattern described by the same authors. It has been demonstrated that EHEC O26:H11 strains belong to at least four different sequence types (STs) in the common clone complex 29 (39). In the multilocus sequence typing analysis for E. coli (61), the tested five EHEC O26:H11 isolates (WH-01/02/003-1, WH-01/20/005-3, WH-01/27/009-9, WH-03/14/004-8, and WH-04/22/001-1) of different farms and clusters were characterized as two sequence types (ST 21 and ST 396). The isolates from farms A and C belong to ST 21, the most frequent ST of EHEC O26:H11 isolates found in humans and animals (39), but the single isolate from farm D was characterized as ST 396.Typing and subtyping of genes (stx1 and/or stx2, eae, tir, espA, espB, EHEC-hlyA, katP, and espP) associated with EHEC were performed with LightCycler fluorescence PCR (48) and different block-cycler PCRs. To identify the subtypes of the stx2 genes and of the locus of enterocyte effacement-encoding genes eae, tir, espA, and espB, the PCR products were digested by different restriction endonucleases (19, 26, 46). The complete pattern of virulence markers was detected in most bovine isolates examined in our study. An stx1 gene was present in all O26 isolates. In addition, an stx2 gene was found in nine O26:H11 isolates in farm A and in three isolates of the same type in farm C, as well as in the O26:H32 isolate. Both Stx1 and Stx2 were closely related to families of Stx1 and Stx2 variants or alleles. EHEC isolates with stx2 genes are significantly more often associated with HUS and other severe disease manifestations than isolates with an stx1 gene, which are more frequently associated with uncomplicated diarrhea and healthy individuals (13). In contrast to STEC strains harboring stx2 gene variants, however, STEC strains of the stx2 genotype were statistically significantly associated with HUS (26). The stx2 genotype was found in all O26 isolates with an stx2 gene, while the GK3/GK4 amplification products after digestion with HaeIII and FokI restriction enzymes showed the typical pattern for this genotype described by Friedrich et al. (26). The nucleotide sequences of the A and B subunits of the stx2 gene of the selected bovine O26:H11 isolate WH-01/27/017-1 (GenBank accession no. EU700491) were identical to the stx2 genes of different sorbitol-fermenting EHEC O157:H− strains associated with human HUS cases and other EHEC infections in Germany (10) and 99.3% identical in their DNA sequences to the stx2 gene of the EHEC type strain EDL933, a typical O157:H7 isolate from an HUS patient. A characteristic stx1 genotype was present in all O26 isolates. The nucleotide sequences of the A and B subunits of the stx1 gene of the tested bovine O26:H11 isolate WH-01/27/017-1 (GenBank accession no. EU700490) were nearly identical to those of the stx1 genes of the EHEC O26:H11 reference type strains H19 and DEC10B, which had been associated with human disease outbreaks in Canada and Australia. Nucleotide exchanges typical for stx1c and stx1d subtypes as described by Kuczius et al. (38) were not found. All bovine O26:H11 strains produced an Stx1 with high cytotoxicity for Vero cells tested by Stx enzyme-linked immunosorbent assay and Vero cell neutralization assay (53). The Stx2 cytotoxicity for Vero cells was also very high in the O26:H11 isolates.Not only factors influencing the basic and inducible Stx production are important in STEC pathogenesis. It has been suggested that the eae and EHEC-hlyA genes are likely contributors to STEC pathogenicity (2, 3, 13, 50). Ritchie et al. (50) found both genes in all analyzed HUS-associated STEC isolates. In all O26:H11 isolates we obtained, stx genes were present in combination with eae genes. Only the O26:H32 isolate lacked an eae gene. To date, 10 distinct variants of eae have been described (1, 19, 36, 45, 47). Some serotypes were closely associated with a particular intimin variant: the O157 serogroup was linked to γ-eae, the O26 serogroup to β-eae, and the O103 serogroup to ɛ-eae (4, 19, 20, 58). Our study confirms these associations. All bovine O26:H11 isolates were also typed as members of the β-eae subgroup. A translocated intimin receptor gene (tir gene) and the type III secreted proteins encoded by the espA and espB genes were found in all 56 O26:H11 isolates but not in the O26:H32 isolate. These other tested locus of enterocyte effacement-associated genes belonged to the β-subgroups. These results are in accord with the results of China et al. (19), who detected the pathotypes β-eae, β-tir, β-espA, and β-espB in all investigated human O26 strains. Like the eae gene, the EHEC-hlyA gene was found in association with severe clinical disease in humans (52). Aldick et al. (2) showed that EHEC hemolysin is toxic (cytolytic) to human microvascular endothelial cells and may thus contribute to the pathogenesis of HUS. In our study, the EHEC-hlyA gene was detected in 50 of the 56 bovine E. coli O26:H11 isolates which harbored virulence-associated plasmids of different sizes (Table (Table1).1). The presence of virulence-associated plasmids corresponded to the occurrence of additional virulence markers such as the espP and katP genes (17). The katP gene and the espP gene were detected in 49 and 50 of the 56 O26:H11 isolates, respectively. The espP gene was missing in six of the seven bovine O26:H11 isolates in which the katP genes were also absent. Both genes were not found in the O26:H32 isolate (Table (Table1).1). Although we found large plasmids of the same size in O26:H11 isolates, they lacked one or more of the plasmid-associated virulence factors (Table (Table1).1). Two DNA probes were used to detect the efa1 genes by colony hybridization. (DNA probes were labeled with digoxigenin [DIG] with lifA1-lifA2 and lifA3-lifA4 primers [14] using the PCR DIG probe synthesis kit [Roche Diagnostics, Mannheim, Germany]; DIG Easy Hyb solution [Roche] was used for prehybridization and hybridization.) Positive results with both DNA probes were obtained for 52 of 56 E. coli O26:H11 isolates. A positive signal was only found in three isolates with the lifA1-lifA2 DNA probe and in one isolate with the lifA3-lifA4 probe. An efa1 gene was not detected in the O26:H32 isolate (Table (Table11).We also analyzed the spatial and temporal behavior of the O26:H11/H32 isolates in the beef herds by cluster analysis (conducted in PAUP* for Windows version 4.0, 2008 [http://paup.csit.fsu.edu/about.html]). This was performed with distance matrices using the neighbor-joining algorithm, an agglomerative cluster method which generates a phylogenetic tree. The distance matrices were calculated by pairwise comparisons of the fragmentation patterns produced by genomic typing through pulsed-field gel electrophoresis analysis with four restriction endonucleases (XbaI, NotI, BlnI, and SpeI) and the presence or absence of potential virulence markers (Fig. (Fig.11 and Table Table1).1). To this end, the total character difference was used, which counts the pairwise differences between two given patterns. During a monitoring program of 3 years in four cattle farms (29), different O26:H11 cluster groups and one O26:H32 isolate were detected in three different farms. The genetic distance of the O26:H32 isolate was very high relative to the O26:H11 isolates. Therefore, the O26:H32 isolate was outgrouped. The O26:H11 isolates of each farm represented independent cluster groups. The single isolate from farm D fitted better to the isolates from farm C than to those from farm A. This finding is in accord with the geographical distance between the farms. The fact that the farms were located in neighboring villages may suggest that direct or indirect connections between the farms were possible (e.g., by person contacts or animal trade). However, the isolates from farm C and farm D belonged to different sequence types (ST 21 and ST 396), which may argue against a direct connection. Interestingly, O26:H11 isolates with and without stx2 genes were detected in the same clusters. This phenomenon was observed in both farm A and farm C. In farm A, the isolates with additional stx2 genes were found in animal 27 and were grouped in clusters 8 and 9 (day 218). An stx2 gene was repeatedly found (four isolates) in the same animal (animal 27). The isolates grouped in cluster 8 on a later day of sampling (day 478). All other O26:H11 isolates grouped in the same clusters and obtained from the same animals (27 and 29) on different sampling days lacked an stx2 gene. Also, the isolates obtained from animal 27 on previous sampling days, which grouped in clusters 3 and 5, exhibited no stx2 genes. In farm C, the three isolates with additional stx2 genes obtained from animal 7 grouped in clusters 11 and 12. An stx2 gene was absent from all other O26:H11 isolates grouped in the same cluster 12 on later sampling days, and no other isolates of cluster 11 were found later on. However, we detected members of many clusters over relatively long periods (clusters 5, 8, and 9 in farm A and cluster 12 in farm C), but members of other clusters were only found on single occasions. This patchy temporal pattern is apparently not a unique property of O26:H11, as we found similar results for cluster groups of other EHEC serotypes of bovine origin (28). The isolates grouped in the dominant cluster 8 were found on 5 of 9 sampling days over a period of 10 months. In contrast, we found the members of clusters 4, 5, 9, and 12 only on two nonconsecutive sampling days. The period during which isolates of these groups were not detected was particularly long for cluster 4 (231 days). We also observed the coexistence of different clusters over long periods in the same farm and in the same cattle (clusters 8 and 9), while one of the clusters dominated. Transmission of clusters between cattle was also observed. These results suggest that some of the EHEC O26:H11 strains had the potential for a longer persistence in the host population, while others had not. The reasons for this difference are not yet clear. Perhaps the incomplete efa1 gene found in isolates of clusters which were only detected once might explain why some strains disappeared rapidly. Efa1 has been discussed as a potential E. coli colonization factor for the bovine intestine used by non-O157 STEC, including O26 (54, 56). The O165:H25 cluster detected during a longer period in farm B may have disappeared after it had lost its efa1 gene (28). The precise biological activity of Efa1 in EHEC O26 is not yet known, but it has been demonstrated that the molecule is a non-Stx virulence determinant which can increase the virulence of EHEC O26 in humans (8).Open in a separate windowFIG. 1.Neighbor-joining tree of bovine E. coli O26:H11/H32 strains based on the restriction pattern obtained after digestion with XbaI, NotI, BlnI, and SpeI.We distinguished 12 different clusters, but complete genetic identity was only found in two isolates. The variations in the O26:H11 clusters may be due to increasing competition between the bacterial populations of the various subtypes in the bovine intestine or to potential interactions between EHEC O26:H11 and the host.The ephemeral occurrence of additional stx2 genes in different clusters and farms may be the result of recombination events due to horizontal gene transfer (16). The loss of stx genes may occur rapidly in the course of an infection, but the reincorporation by induction of an stx-carrying bacteriophage into the O26:H11 strains is possible at any time (9, 40). Nevertheless, an additional stx2 gene may increase the dangerousness of the respective EHEC O26:H11 strains. While all patients involved in an outbreak caused by an EHEC O26:H11 strain harboring the gene encoding Stx2 developed HUS (41), the persons affected by another outbreak caused by an EHEC O26:H11 strain that produced exclusively Stx1 had only uncomplicated diarrhea (60).In conclusion, our results showed that bovine O26:H11 isolates can carry virulence factors of EHEC that are strongly associated with EHEC-related disease in humans, particularly with severe clinical manifestations such as hemorrhagic colitis and HUS. Therefore, strains of bovine origin may represent a considerable risk for human infection. Moreover, some clusters of EHEC O26:H11 persisted in cattle and farms over longer periods, which may increase the risk of transmission to other animals and humans even further.  相似文献   

14.
Shiga toxin-producing Escherichia coli (STEC) strains belonging to serotypes O157:H7, O26:H11, O103:H2, O111:H8, and O145:H28 are known to be associated with particular subtypes of the intimin gene (eae), namely, γ1, β1, ε, θ, and γ1, respectively. This study aimed at evaluating the usefulness of their detection for the specific detection of these five main pathogenic STEC serotypes in cattle feces. Using real-time PCR assays, 58.7% of 150 fecal samples were found positive for at least one of the four targeted eae subtypes. The simultaneous presence of stx, eae, and one of the five O group markers was found in 58.0% of the samples, and the five targeted stx plus eae plus O genetic combinations were detected 143 times. However, taking into consideration the association between eae subtypes and O group markers, the resulting stx plus eae subtype plus O combinations were detected only 46 times. The 46 isolation assays performed allowed recovery of 22 E. coli strains belonging to one of the five targeted STEC serogroups. In contrast, only 2 of 39 isolation assays performed on samples that were positive for stx, eae and an O group marker, but that were negative for the corresponding eae subtype, were successful. Characterization of the 24 E. coli isolates showed that 6 were STEC, including 1 O157:H7, 3 O26:H11, and 2 O145:H28. The remaining 18 strains corresponded to atypical enteropathogenic E. coli (aEPEC). Finally, the more discriminating eae subtype-based PCR strategy described here may be helpful for the specific screening of the five major STEC in cattle feces.  相似文献   

15.
Shiga toxin (Stx)-producing Escherichia coli (STEC) are recognized as important human pathogens of public health concern. Many animals are the sources of STEC. In this study we determined the occurrence and characteristics of the STEC in yaks (Bos grunniens) from the Qinghai-Tibetan plateau, China. A total of 728 yak fecal samples was collected from June to August, 2012 and was screened for the presence of the stx1 and stx2 genes by TaqMan real-time PCR after the sample was enriched in modified Tryptone Soya Broth. Of the 138 (18.96%) stx1 and/or stx2-positive samples, 85 (61.59%) were confirmed to have at least 1 STEC isolate present by culture isolation, from which 128 STEC isolates were recovered. All STEC isolates were serotyped, genotyped by pulsed-field gel electrophoresis (PFGE) and characterized for the presence of 16 known virulence factors. Fifteen different O serogroups and 36 different O:H serotypes were identified in the 128 STEC isolates with 21 and 4 untypable for the O and H antigens respectively. One stx1 subtype (stx1a) and 5 stx2 subtypes (stx2a, stx2b, stx2c, stx2d and stx2g) were present in these STEC isolates. Apart from lpfAO157/OI-141, lpfAO157/OI-154, lpfAO113, katP and toxB which were all absent, other virulence factors screened (eaeA, iha, efa1, saa, paa, cnf1, cnf2, astA, subA, exhA and espP) were variably present in the 128 STEC isolates. PFGE were successful for all except 5 isolates and separated them into 67 different PFGE patterns. For the 18 serotypes with 2 or more isolates, isolates of the same serotypes had the same or closely related PFGE patterns, demonstrating clonality of these serotypes. This study was the first report on occurrence and characteristics of STEC isolated from yaks (Bos grunniens) from the Qinghai-Tibetan plateau, China, and extended the genetic diversity and reservoir host range of STEC.  相似文献   

16.
Escherichia coli O157:H7 genotypes in the bovine reservoir may differ in virulence. The proportion of clinical genotypes among cattle isolates was weakly (P = 0.054) related to the international incidence of E. coli O157:H7-associated hemolytic-uremic syndrome, varied among clinical isolates internationally, and also differed along the putative cattle-hamburger-clinical case transmission chain.  相似文献   

17.
To identify Shiga toxin-producing Escherichia coli genes associated with severe human disease, a genomic subtraction technique was used with hemolytic-uremic syndrome-associated O91:H21 strain CH014 and O6:H10 bovine strains. The method was adapted to the Shiga toxin-producing E. coli genome: three rounds of subtraction were used to isolate DNA fragments specific to strain CH014. The fragments were characterized by genetic support analysis, sequencing, and hybridization to the genome of a collection of Shiga toxin-producing E. coli strains. A total of 42 fragments were found, 19 of which correspond to previously identified unique DNA sequences in the enterohemorrhagic E. coli EDL933 reference strain, including 7 fragments corresponding to prophage sequences and others encoding candidate virulence factors, such a SepA homolog protein and a fimbrial usher protein. In addition, the subtraction procedure yielded plasmid-related sequences from Shigella flexneri and enteropathogenic and Shiga toxin-producing E. coli virulence plasmids. We found that lateral gene transfer is extensive in strain CH014, and we discuss the role of genomic mobile elements, especially bacteriophages, in the evolution and possible transfer of virulence determinants.  相似文献   

18.
Variations in time and space of a clonal group of Escherichia coli O165:H25 on a cattle farm were monitored. The virulence marker pattern (stx genes, eae gene, hlyEHEC gene, katP gene, espP gene, efa gene) suggests that E. coli O165:H25 of bovine origin may represent a risk for human infection.  相似文献   

19.
《PloS one》2012,7(11)
In May of 2011, an enteroaggregative Escherichia coli O104:H4 strain that had acquired a Shiga toxin 2-converting phage caused a large outbreak of bloody diarrhea in Europe which was notable for its high prevalence of hemolytic uremic syndrome cases. Several studies have described the genomic inventory and phylogenies of strains associated with the outbreak and a collection of historical E. coli O104:H4 isolates using draft genome assemblies. We present the complete, closed genome sequences of an isolate from the 2011 outbreak (2011C–3493) and two isolates from cases of bloody diarrhea that occurred in the Republic of Georgia in 2009 (2009EL–2050 and 2009EL–2071). Comparative genome analysis indicates that, while the Georgian strains are the nearest neighbors to the 2011 outbreak isolates sequenced to date, structural and nucleotide-level differences are evident in the Stx2 phage genomes, the mer/tet antibiotic resistance island, and in the prophage and plasmid profiles of the strains, including a previously undescribed plasmid with homology to the pMT virulence plasmid of Yersinia pestis. In addition, multiphenotype analysis showed that 2009EL–2071 possessed higher resistance to polymyxin and membrane-disrupting agents. Finally, we show evidence by electron microscopy of the presence of a common phage morphotype among the European and Georgian strains and a second phage morphotype among the Georgian strains. The presence of at least two stx2 phage genotypes in host genetic backgrounds that may derive from a recent common ancestor of the 2011 outbreak isolates indicates that the emergence of stx2 phage-containing E. coli O104:H4 strains probably occurred more than once, or that the current outbreak isolates may be the result of a recent transfer of a new stx2 phage element into a pre-existing stx2-positive genetic background.  相似文献   

20.
A detailed analysis of the molecular epidemiology of non-O157:H7 Shiga toxin-producing Escherichia coli (STEC) was performed by using isolates from sporadic cases of hemolytic-uremic syndrome (HUS), animal reservoirs, and food products. The isolates belonged to the O91 and OX3 serogroups and were collected in the same geographical area over a short period of time. Five typing methods were used; some of these were used to explore potentially mobile elements like the stx genes or the plasmids (stx2-restriction fragment length polymorphism [RFLP], stx2 gene variant, and plasmid analyses), and others were used to study the whole genome (ribotyping and pulsed-field gel electrophoresis [PFGE]). The techniques revealed that there was great diversity among the O91 and OX3 STEC strains isolated in central France. A close relationship between strains of the same serotype having the same virulence factor pattern was first suggested by ribotyping. However, stx2-RFLP and stx2 variant analyses differentiated all but 5 of 21 isolates, and plasmid analysis revealed further heterogeneity; a unique combination of characteristics was obtained for all strains except two O91:H21 isolates from beef. The latter strains were shown by PFGE to be the most closely related isolates, with >96% homology, and hence may be subtypes of the same strain. Overall, our results indicate that the combination of stx2-RFLP, stx2 variant, and plasmid profile analyses is as powerful as PFGE for molecular investigation of STEC diversity. Finally, the non-O157:H7 STEC strains isolated from HUS patients were related to but not identical to those isolated from cattle and food samples in the same geographical area. The possibility that there are distinct lineages of non-O157:H7 STEC, some of which are more virulent for humans, should be investigated further.  相似文献   

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