Stx)-producing Escherichia Coli O157 Research Articles

Differences in the Shiga Toxin (Stx) 2a Phage Regulatory Switch Region Influence Stx2 Localization and Virulence of Stx-Producing Escherichia coli in Mice.

Shiga toxin (Stx)-producing Escherichia coli (STEC) is a major cause of foodborne illness globally, and infection with serotype O157:H7 is associated with increased risk of hospitalization and death in the U.S. The Stxs are encoded on a temperate bacteriophage (stx-phage), and phage induction leads to Stx expression; subtype Stx2a in particular is associated with more severe disease. Our earlier studies showed significant levels of RecA-independent Stx2 production by STEC O157:H7 strain JH2010 (stx2astx2c), even though activated RecA is the canonical trigger for stx-phage induction. This study aimed to further compare and contrast RecA-independent toxin production in Stx2-producing clinical isolates. Deletion of recA in JH2010 resulted in higher in vitro supernatant cytotoxicity compared to that from JH2016ΔrecA, and the addition of the chelator ethylenediaminetetraacetic acid (EDTA) and various metal cations to the growth medium exacerbated the difference in cytotoxicity exhibited by the two deletion strains. Both the wild-type and ΔrecA deletion strains exhibited differential cytotoxicity in the feces of infected, streptomycin (Str)-treated mice. Comparison of the stx2a-phage predicted protein sequences from JH2010 and JH2016 revealed low amino acid identity of key phage regulatory proteins that are involved in RecA-mediated stx-phage induction. Additionally, other STEC isolates containing JH2010-like and JH2016-like stx2a-phage sequences led to similar Stx2 localization, as demonstrated by JH2010ΔrecA and JH2016ΔrecA, respectively. Deletion of the stx2a-phage regulatory region in the wild-type strains prevented the differential localization of Stx2 into the culture supernatant, a finding that suggests that the stx2a-phage regulatory region is involved in the differential ΔrecA phenotypes exhibited by the two strains. We hypothesize that the amino acid differences between the JH2010 and JH2016 phage repressor proteins (CIs) lead to structural differences that are responsible for differential interaction with RecA. Overall, we discovered that non-homologous stx2a-phage regulatory proteins differentially influence RecA-independent, and possibly RecA-dependent, Stx2 production. These findings emphasize the importance of studying non-homologous regulatory elements among stx2-phages and their influence on Stx2 production and virulence of STEC isolates.

Shiga Toxin (Stx) Phage-Encoded Lytic Genes Are Not Required for the Mouse Virulence of O157:H7 Escherichia coli Stx2-Producing Clinical Isolates.

Shiga toxin (Stx)-producing Escherichia coli (STEC) is a major cause of foodborne diarrheal illness in the United States and globally, and serotype O157:H7 is frequently associated with STEC outbreaks and sporadic cases in the United States. Severe systemic diseases associated with STEC are mediated by Stx types, particularly subtype Stx2a, encoded on inducible bacteriophages. We previously identified two STEC O157:H7 clinical isolates, JH2010 and JH2012, that exhibit a large difference in virulence in a streptomycin (Str)-treated mouse model. In this study, we aimed to identify a genetic basis for the difference in virulence between those strains. Comparison of the stx2a phage sequences showed that JH2012 lacks the lytic genes S and R on the phage genome. We also demonstrated that compared to JH2012 cultures, cultures of JH2010 released more Stx2 into the supernatant and were more sensitive to bacterial lysis during growth with ciprofloxacin (Cip), an inducer of stx phages. We therefore generated an stx2a phage SR deletion mutant strain of JH2010 to determine if those genes were responsible for the high virulence of that strain. We found that deletion of the SR genes from the stx2a phage in JH2010, and another O157:H7 strain, JH2016, resulted in increased cellular retention of Stx2, but there was no difference in virulence compared to the wild-type strains. Our results indicate that the stx2a phage SR genes are involved in Stx2 localization and phage-mediated cell lysis in vitro but that they are not required in wild-type STEC strains for virulence in a mouse model. IMPORTANCE The release of Stx from STEC has been thought to be tied to phage-mediated lysis of the host bacterial cell. In this study, we found that the stx2a phage lytic genes are not required for the virulence of pathogenic O157:H7 clinical isolates in a murine model of STEC infection or for release of Stx2a into the supernatant of bacterial cultures. These results point to an alternate mechanism for Stx2a release from STEC strains.

Etiological diagnosis of post-diarrheal hemolytic uremic syndrome (HUS): humoral response contribution.

Hemolytic uremic syndrome (HUS) is characterized by microangiopathic hemolysis, thrombocytopenia, and thrombus formation leading to tissue injury. HUS is classified according to its etiology as post-diarrheal or atypical HUS. Differential diagnosis of both entities continues to be a challenge for pediatric physicians. The aim was to improve the rapid etiological diagnosis of post-diarrheal HUS cases based on the detection of Shiga toxin (Stx)-producing Escherichia coli (STEC) infection by screening of stx1/stx2 and rfbO157 in cultured stools by multiplex PCR, and the additional detection of anti-lipopolysaccharide (anti-LPS) O157, O145, and O121 antibodies by Glyco-iELISA test. In addition, we studied patients' relatives to detect circulating pathogenic strains that could contribute to HUS diagnosis and/or lead to the implementation of measures to prevent dissemination of familial outbreaks. This study describes the diagnosis of 31 HUS patients admitted to Hospital Municipal de Niños Prof Dr Ramón Exeni during the 2017-2020 period. Stool PCR confirmed the diagnosis of STEC associated with HUS in 38.7% of patients (12/31), while anti-LPS serology did in 88.9% (24/27). In those patients in which both methods were carried out (n = 27), a strong association between the results obtained was found. We found that 30.4% of HUS patients had at least one relative positive for STEC. We could identify 96.3% (26/27) of HUS cases as secondary to STEC infections when both methods (genotyping and serology) were used. The results demonstrated a high circulation of STEC in HUS families and the prevalence of the STEC O157 serotype (83%) in our pediatric cohort. A higher-resolution version of the Graphical abstract is available as Supplementary information.

Phylogeny, Prevalence, and Shiga Toxin (Stx) Production of Clinical Escherichia coli O157 Clade 2 Strains Isolated in Shimane Prefecture, Japan.

This study investigated the genetic and pathogenic variation of the subgroups of clade 2 strains of Shiga toxin (Stx)-producing Escherichia coli (STEC) O157. A total of 111 strains of STEC O157 isolated in Shimane prefecture, Japan, were classified in clade 2 (n = 39), clade 3 (n = 16), clade 4/5 (n = 3), clade 7 (n = 14), clade 8 (n = 17), and clade 12 (n = 22) by single-nucleotide polymorphism analysis and lineage-specific polymorphism assay-6. These results showed a distinct difference from our previous study in which clade 3 strains were the most prevalent strains in three other prefectures in Japan, indicating that the clade distribution of O157 strains was different in different geographic areas in Japan. Phylogenetic analysis using insertion sequence (IS) 629 distribution data showed that clade 2 strains formed two clusters, designated 2a and 2b. Stx2 production by cluster 2b strains was significantly higher than by cluster 2a strains (P < 0.01). In addition, population genetic analysis of the clade 2 strains showed significant linkage disequilibrium in the IS629 distribution of the strains in clusters 2a and 2b (P < 0.05). The ΦPT values calculated using the IS629 distribution data indicated that strains in clusters 2a and 2b were genetically different (P < 0.001). Cluster 2b strains are a highly pathogenic phylogenetic group and their geographic spread may be a serious public health concern.

Analysis Shiga Toxin-Encoding Bacteriophage in a Rare Strain of Shiga Toxin-Producing Escherichia coli O157:H7 stx2a/stx2c.

In December 2015, six cases of Shiga toxin (Stx)-producing Escherichia coli (STEC) O157:H7 stx2a/stx2c phage type (PT) 24 were identified by the national gastrointestinal disease surveillance system at Public Health England (PHE). Frozen grated coconut imported from India was implicated as the vehicle of infection. Short and long read sequencing data were interrogated for genomic markers to provide evidence that the outbreak strain was from an imported source. The outbreak strain belonged to a sub-lineage (IIa) rare in domestically acquired infection in the United Kingdom, and indicative of an imported strain. Phylogenetic analysis identified the most closely related isolates to the outbreak strain were from cases reporting recent travel not to India, but to Uganda. Phylo-geographical signals based on travel data may be confounded by the failure of local and/or global monitoring systems to capture the full diversity of strains in a given country. This may be due to low prevalence strains circulating in-country under the surveillance radar, or a recent importation event involving the migration of animals and/or people. Comparison of stx2a-encoding prophage harbored by the outbreak strain with publicly available stx2a-encoding prophage sequences revealed that it was most closely related to stx2a-encoding prophage acquired by STEC O157:H7 that caused the first outbreak of STEC-hemolytic uremic syndrome (HUS) in England in 1982–83. Animal and people migration events may facilitate the transfer of stx2a-encoding prophage from indigenous STEC O157:H7 to recently imported strains, or vice versa. Monitoring the global transmission of STEC O157:H7 and tracking the exchange of stx2a-encoding phage between imported and indigenous strains may provide an early warning of emerging threats to public health.

Genetic and Functional Analyses of Virulence Potential of an Escherichia coli O157:H7 Strain Isolated From Super-Shedder Cattle.

Shiga toxin (Stx)-producing Escherichia coli (STEC) O157:H7 is an enteric pathogen that causes life-threatening disease in humans, with cattle being major natural reservoirs. A group of STEC O157:H7 with a dramatic combination of high virulence potentials and super-shedder bovine origin have been isolated. Here, an STEC O157:H7 isolate, JEONG-1266, was analyzed by comparative genomics, stx genotyping, and phenotypic analyses. The phylogenetic typing and whole-genome comparison consistently showed that JEONG-1266 is genetically close to EC4115 (one of 2006 Spinach outbreak isolates) and SS17 (an isolate from super-shedder cattle) strains, all of which belong to lineage I/II and Clade 8. Both lineage I/II and Clade 8 are known to be mostly associated with clinical strains with high virulence and severe clinical symptoms. Further, JEONG-1266, like EC4115 and SS17, harbors stx2a/stx2c genes, and carries Stx-encoding prophages, specifically the φstx2a-γ subtype. Possession of the φstx2a-γ subtype of Stx-encoding prophages and production of Stx2a have been shown to be a key signature associated with hypervirulent STEC O157:H7 strains. In silico virulence typing elucidated JEONG-1266, EC4115, and SS17 shared a highly conserved profile of key virulence genes at the nucleotide sequence level. Consistently, phenotypic data showed that JEONG-1266 expressed a high level of Stx2 toxins and had the full capacity of adhesion in vitro. Taken together, our study suggests that JEONG-1266 may represent an emerging STEC O157:H7 group, which are hypervirulent strains that originate from super-shedders, that can be a threat to food safety and public health.

Genomic Characterization of β-Glucuronidase-Positive Escherichia coli O157:H7 Producing Stx2a.

Among Shiga toxin (Stx)–producing Escherichia coli (STEC) O157:H7 strains, those producing Stx2a cause more severe diseases. Atypical STEC O157:H7 strains showing a β-glucuronidase–positive phenotype (GP STEC O157:H7) have rarely been isolated from humans, mostly from persons with asymptomatic or mild infections; Stx2a-producing strains have not been reported. We isolated, from a patient with bloody diarrhea, a GP STEC O157:H7 strain (PV15-279) that produces Stx2a in addition to Stx1a and Stx2c. Genomic comparison with other STEC O157 strains revealed that PV15-279 recently emerged from the stx1a/stx2c-positive GP STEC O157:H7 clone circulating in Japan. Major virulence genes are shared between typical (β-glucuronidase–negative) and GP STEC O157:H7 strains, and the Stx2-producing ability of PV15-279 is comparable to that of typical STEC O157:H7 strains; therefore, PV15-279 presents a virulence potential similar to that of typical STEC O157:H7. This study reveals the importance of GP O157:H7 as a source of highly pathogenic STEC clones.

Enhanced virulence of the Escherichia coli O157:H7 spinach-associated outbreak strain in two animal models is associated with higher levels of Stx2 production after induction with ciprofloxacin.

Shiga toxin (Stx)-producing Escherichia coli (STEC) causes hemorrhagic colitis and the hemolytic-uremic syndrome (HUS). STEC strains may produce Stx1a and/or Stx2a or variants of either toxin. A 2006 spinach-associated outbreak of STEC O157:H7 resulted in higher hospitalization and HUS rates than previous STEC outbreaks. The spinach isolate, strain K3995, contains both stx2a and stx2c. We hypothesized that the enhanced virulence of K3995 reflects the combination of stx2 alleles (carried on lysogenic phages) and/or the amount of Stx2 made by that strain. We compared the virulence of K3995 to those of other O157:H7 isolates and an isogenic Stx2 mutant in rabbits and mice. We also measured the relative levels of Stx2 produced from those strains with or without induction of the stx-carrying phage. Some rabbits infected with K3995 exhibited intestinal pathology and succumbed to infection, while none of those infected with O157:H7 strain 2812 (Stx1a(+) Stx2a(+)) died or showed pathological signs. Rabbits infected with the isogenic Stx2a mutant K3995 stx2a::cat were not colonized as well as those infected with K3995 and exhibited no signs of disease. In the streptomycin-treated mouse model, more animals infected with K3995 died than did those infected with O157:H7 strain 86-24 (Stx2a(+)). Additionally, K3995 produced higher levels of total Stx2 and toxin phage DNA in cultures after phage induction than did 86-24. Our results demonstrate the greater virulence of K3995 compared to other O157:H7 strains in rabbits and mice. We conclude that this enhanced virulence is linked to higher levels of Stx2 expression as a consequence of increased phage induction.

Serotype, Shiga Toxin (Stx) Type, and Antimicrobial Resistance of Stx-Producing Escherichia coli Isolated from Humans in Shizuoka Prefecture, Japan (2003^|^ndash;2007)

The serotype, Shiga toxin (Stx) type, and antimicrobial resistance patterns of 138 Stx-producing Escherichia coli (STEC) strains isolated from humans between 2003 and 2007 in Shizuoka Prefecture, Japan were characterized. The predominant O serogroups of the STEC isolates were O157, O26, and O111. Antimicrobial susceptibility testing of the STEC isolates showed that 31 of the 138 isolates (22.5%) were resistant to antibiotics. Compared to the results reported in the previous studies, a higher rate of STEC O157 isolates were susceptible to all the antimicrobial agents used in this study. However, antimicrobial susceptibility data from this study showed that antimicrobial resistance patterns have increased by 6 compared to the survey performed by Masuda et al. between 1987 and 2002 (Jpn. J. Food Microbiol., 21, 44-51, 2004). This indicates that STEC isolates have evolved to show a variety of antimicrobial resistance patterns. It is important to consider the population of isolates showing decreased susceptibility to clinically relevant drugs such as ciprofloxacin (CPFX) and fosfomycin (FOM). All the 3 STEC isolates resistant to nalidixic acid showed low susceptibility to CPFX (MIC, 0.25-0.5 μg/ml). In addition, a decreased susceptibility to FOM was clearly observed in the E. coli O26 isolates. Our findings also showed that 1 STEC O26 strain could possibly be a chromosomal AmpC β-lactamase hyperproducer. These results suggest that antimicrobial therapy may be less effective in patients with non-O157 STEC infections than in those with STEC O157 infections.

Relationship between stx Genotype and Stx2 Expression Level in Shiga Toxin-Producing Escherichia coli O157 Strains

To determine the expression level of Shiga toxin (Stx) 2-related toxins (Stx2 and Stx2c) produced by each of 33 Stx-producing Escherichia coli (STEC) O157 strains, stx2 and stx2c mRNAs (stx2-related mRNA) were measured using real-time PCR with primers that recognize sequences common to stx2 and stx2c. The amount of Stx2 and Stx2c protein was measured using a reversed passive latex agglutination (RPLA) kit. Expression of stx2-related mRNA was significantly higher in STEC O157 strains carrying the stx2 gene (i.e., stx2, stx1/stx2, or stx2/stx2c) than in most strains that carried the stx2c gene but not the stx2 gene (i.e., stx2c or stx1/stx2c). RPLA might not measure the precise amount of each toxin variant; nevertheless, stx2-inclusive strains had 40-fold higher mean toxin titers than did strains that carried the stx2c gene but not the stx2 gene, with the exception of 1 stx2c strain. Interestingly, 1 stx2c strain that was isolated from a patient with severe hemorrhagic diarrhea had the highest stx2-related mRNA expression and the highest toxin titer of all 33 STEC O157 strains. Taken together, these findings indicated that measurement of stx2-related mRNA expression could reflect differences in production levels of toxins among STEC strains.

Mouse model for hemolytic uremic syndrome induced by outer membrane vesicles ofEscherichia coliO157:H7

Hemolytic uremic syndrome (HUS) is characterized by acute renal failure in children and is typically complicated with thrombocytopenia and hemolytic anemia. Although mouse models of HUS have been evaluated using Shiga toxin (STx) combined with or without lipopolysaccharide (LPS), no HUS model has been tested using purified outer membrane vesicles (OMVs) from STx-producing Escherichia coli (STEC) O157:H7. Accordingly, we investigated whether OMVs of STEC O157:H7 conveying STx2 and LPS can cause HUS-like symptoms in mice inoculated intraperitoneally. Three types of OMVs differing in LPS acylation status and STx2 amount were used to compare their ability to induce HUS-like symptoms. Native OMVs (nOMV) with fully hexa-acylated LPS caused HUS-like symptoms at 72-96h after dually divided injections of 1μg nOMV per animal. However, elevated doses of modified OMVs (mOMV) carrying mainly penta-acylated LPS were required to induce similar HUS signs. Moreover, mitomycin-C-induced OMVs (mcOMV) that were enriched with STx2 induced HUS-like symptoms similar to those of nOMV at the same dose. These results suggest that the OMVs of STEC O157:H7 potentiated with STx2 and fully hexa-acylated LPS can be utilized for inducing HUS-like symptoms in mice and could be the causative material involved in the development of HUS.

Stx genotype and molecular epidemiological analyses of Shiga toxin-producing Escherichia coli O157:H7/H− in human and cattle isolates

The relationship between human diseases caused by infection with Shiga toxin (Stx)-producing Escherichia coli (STEC) O157 strains and O157 strains isolated from cattle was investigated in an area where stockbreeding is prolific. For this purpose, the stx genotypes, the molecular epidemiological characteristics of 268 STEC O157 strains including 211 human-origin strains and 57 cattle-origin strains, and clinical manifestations of 210 STEC-infected people were analyzed. Of 211 human-origin strains, 92 strains (44%) were of the stx1/stx2 genotype, and 74 strains (35%) were of the stx2c genotype. Most of the people infected with stx2c genotype strains presented no symptoms or mild symptoms such as slight diarrhea, except for 3 patients with bloody diarrhea. Of the 57 cattle-origin strains, 27 strains (47%) were of the stx2c genotype and 17 strains (30%) were of the stx1/stx2 genotype. Pulsed-field gel electrophoresis (PFGE) and insertion sequence (IS) analysis demonstrated that 11 isolates (41%) of the 27 cattle isolates of the stx2c genotype had high homology (>95% identity) with human isolates. These results suggest that some genetic patterns of the stx2c genotype strains might be preserved in cattle or their surrounding environment for several years, and during these periods, they might have opportunities to infect people through various routes. Because of the mild virulence of the stx2c genotype strains, they seemed to be transmitted asymptomatically from cattle to humans and then spread from person to person. It may be a public health concern. Further, they occasionally cause severe symptoms in humans; therefore, caution is warranted for infections by stx2c genotype O157 strains, in addition to stx2-possessing genotype O157 strains.

Occurrence and Characterization of Shiga Toxin–ProducingEscherichia coliin Raw Meat, Raw Milk, and Street Vended Juices in Bangladesh

The major objective of this study was to investigate the prevalence of Shiga toxin (Stx)-producing Escherichia coli (STEC) in different types of food samples and to compare their genetic relatedness with STEC strains previously isolated from animal sources in Bangladesh. We investigated a total of 213 food samples, including 90 raw meat samples collected from retail butcher shops, 20 raw milk samples from domestic cattle, and 103 fresh juice samples from street vendors in Dhaka city. We found that more than 68% (n = 62) of the raw meat samples were positive for the stx gene(s); 34% (n = 21) of buffalo meats and 66% (n = 41) of beef. Approximately 10% (n = 2) of the raw milk and 8% (n = 8) of the fresh juice samples were positive for stx. We isolated STEC O157 from seven meat samples (7.8%), of which two were from buffalo meats and five from beef; and no other STEC serotypes could be isolated. We could not isolate STEC from any of the stx-positive raw milk and juice samples. The STEC O157 isolates from raw meats were positive for the stx(2), eae, katP, etpD, and enterohemorrhagic E. coli hly virulence genes, and they belonged to three different phage types: 8 (14.3%), 31 (42.8%), and 32 (42.8%). Pulsed-field gel electrophoresis (PFGE) typing revealed six distinct patterns among seven isolates of STEC O157, suggesting a heterogeneous clonal diversity. Of the six PFGE patterns, one was identical and the other two were ≥90% related to PFGE patterns of STEC O157 strains previously isolated from animal feces, indicating that raw meats are readily contaminated with fecal materials. This study represents the first survey of STEC in the food chain in Bangladesh.

Prevalence of Stx Phages in Environments of a Pig Farm and Lysogenic Infection of the Field E. coli O157 Isolates with a Recombinant Converting Phage

The prevalence and nature of Shiga toxin (Stx)-producing Escherichia coli (STEC) and Stx phage were investigated in 720 swine fecal samples randomly collected from a commercial breeding pig farm in China over a 1-year surveillance period. Eight STEC O157 (1.1%), 33 STEC non-O157 (4.6%), and two stx-negative O157 (0.3%) isolates were identified. Fecal filtrates were screened directly for Stx phages using E. coli K-12 derivative strains MC1061 as indicator, yielding 15 Stx1 and 57 Stx2 phages. One Stx1 and eight Stx2 phages were obtained following norfloxacin induction of the eight field STEC O157 isolates. All Stx1 phages had hexagonal heads with long tails, while Stx2 phages had three different morphologies. Notably, most of field STEC O157 isolates released more free phages and Stx toxin after induction with ciprofloxacin. Furthermore, upon infection with the recombinant phage ΦMin27(Δstx::cat), E. coli laboratory strains produced both lysogenic and lytic phage, whereas two of the eight O157 STEC isolates produced only lysogens. The lysogens from laboratory strains produced infectious particles similar to ΦMin27. Similarly, the lysogens from the STEC O157 isolates released Stx phage too, although free ΦMin27(Δstx::cat) particles were not detected. Collectively, our results reveal that breeding pig farms could be important reservoirs for Stx phages and that residual antibacterial agents may enhance the release of Stx phages and the expression of Stx.

Shiga toxin-producing Escherichia coli O157 in beef and chicken burgers, and chicken carcasses in Buenos Aires, Argentina

We describe the isolation and characterization of Shiga toxin (Stx)-producing Escherichia coli (STEC) O157:H7 from cooked and uncooked beef and chicken burgers and from chicken carcasses collected during sampling procedures in 2001 and 2002 in Buenos Aires City, Argentina. Of the 24 STEC O157:H7 strains isolated, 20 were recovered from 19 (6.8%) out of 279 samples of beef and chicken burgers, and 4 strains from 4 (10.3%) out of 39 chicken carcasses. The samples were analyzed following the USDA/FSIS 2002 method. The prevalent stx genotype was stx 2 and stx 2c (12 strains, 50%). All strains were characterized as eae and ehxA-positive. By XbaI-PFGE, the strains yielded 10 different patterns. Eighteen out of 24 strains were grouped in four clusters: #1 (4 strains, AREXHX01.0043), #2 (4 strains, AREXHX01.0022), #3 (8 strains, AREXHX01.0139), and #4 (2 strains, AREXHX01.0200). Identical strains by phage typing, stx genotyping and PFGE were detected in uncooked and cooked beef and chicken burgers in different restaurants, which had been collected on the same or different sampling dates. These findings help to underline the importance of STEC O157 detection in meat products, to improve active surveillance, and to define control strategies in order to prevent new cases of STEC infection.

Prevalence and Genetic Characterization of Shiga Toxin-Producing Escherichia coli Isolates from Slaughtered Animals in Bangladesh

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 stx(1) and/or stx(2), 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 stx(2), eae, katP, etpD, and enterohemorrhagic E. coli hly (hly(EHEC)) 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 stx(1). Only 7.0% (n = 5) of the isolates were positive for hly(EHEC), 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 lpf(O113), saa, lpfA(O157/01-141), and lpfA(O157/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.

Relationship between pathogenicity for humans and stx genotype in Shiga toxin-producing Escherichia coli serotype O157

To examine the reason why people infected with Shiga toxin (Stx) producing Escherichia coli (STEC) O157 strains develop varying clinical manifestations, 65 STEC O157 isolates originating from 64 different occurrences of infection in Miyazaki Prefecture in 2001-2003 and their 79 infected individuals were analyzed by stx genotyping, quantitative analysis of reversed passive latex agglutination (RPLA), genomic DNA analysis using pulsed-field gel electrophoresis (PFGE), and clinical manifestations. The isolates were found to carry the following stx genes: stx2vha alone (60.0%), stx1/stx2 (27.7%), stx1/stx2vha (6.1%), stx2 alone (3.1%), and stx2/stx2vha (3.1%). No strain carried the stx1 gene alone. STEC strains carrying stx2 were more frequently associated with clinical manifestations of hemolytic-uremic syndrome (HUS) or bloody diarrhea than those carrying stx2vha. Clusters of PFGE banding patterns were correlated well with the stx genotypes. We conclude that stx genotype is one of the important factors of clinical outcome of STEC O157 infection and that pathogenicity for humans was higher in the stx2 genotype strains than in the stx2vha genotype strains, as reported previously by other researchers. Further, we newly found that four clusters identified by PFGE using restriction enzyme XbaI, stx genotypes and clinical manifestations were well correlated with each other.

Characterization and Epidemiologic Subtyping of Shiga Toxin–ProducingEscherichia coliStrains Isolated from Hemolytic Uremic Syndrome and Diarrhea Cases in Argentina

Argentina has a high incidence of hemolytic uremic syndrome (HUS); 12.2 cases per 100,000 children younger than 5 years old were reported in 2002. Shiga toxin (Stx)-producing Escherichia coli (STEC) is the primary etiologic agent of HUS, and STEC O157 is the predominant serogroup isolated. The main objective of the present work was to establish the phenotypic and genotypic characteristics of the STEC strains in general isolated from Argentine children during a prospective study and the clonal relatedness of STEC O157:H7 strains using subtyping techniques. One hundred and three STEC strains isolated from 99 children were included. The phenotypic and genotypic features were established, and a polymerase chain reaction-restriction fragment length polymorphism (PCRRFLP) was performed to determine stx2 variants. The clonal relatedness of E. coli O157 isolates was established by phage typing and pulsed-field gel electrophoresis (PFGE). The 103 STEC strains belonged to 18 different serotypes, and 59% were of serotype O157:H7. Stx2 was identified in 90.3%, and stx1 in 9.7%. Among the 61 STEC O157 strains, 93.4% harbored the stx2/stx2vh-a genes; PT4 (39.3%) and PT2 (29.5%) were the predominant phage types. Using PFGE with the enzyme XbaI, a total of 41 patterns with at least 80% similarity were identified, and seven clusters with identical profiles were established. Some of the clusters were further split by PFGE using BlnI as the second enzyme. Isolates with indistinguishable PFGE patterns were with one exception also indistinguishable by phage typing and stx genotyping. These findings confirmed that some isolates were genetically related. However, no epidemiological linkages were identified. STEC strains with different genotypes and belonging to diverse serotypes were isolated in Argentina. Some STEC O157 strains could not be distinguished by applying subtyping techniques such as PFGE and phage typing.

Serological methods for the detection of STEC infections.

AbstractThe detection of antibodies to Shiga toxin (Stx)-producing Escherichia coli (STEC) antigens serves varied purposes: (1) the etiologic diagnosis of acute hemolytic uremic syndrome (HUS) and hemorrhagic colitis (HC) in the clinical laboratory; (2) epidemiological investigations; (3) the study of immune responses in STEC-mediated diseases, immunization trials, and animal models. Although the isolation of STEC from the feces of a patient with HC or HUS is generally sufficient evidence for its etiological role in these diseases, it may fail because of a number of circumstances. For example, a timely stool specimen may not be available, the primary laboratory may be unaware of the clinical diagnosis or apply inadequate isolation methods, or the patient may have received suppressive antibiotics. Moreover, when patients present with HUS, usually 5-7d after the onset of diarrhea, the excretion rate of STEC organisms is already substantially diminished. Among E. coli isolates from patients with HUS and HC, STEC O157:H7 predominates. However, so-called non-O157:H7 STEC serotypes are emerging both as causes of outbreaks and sporadic HUS and diarrhea, especially in Europe, Australia and South America. The clinical features of non-O157 STEC infections closely resemble those of prototypic E. coli O157:H7 disease (1,2). The microbiological diagnosis of non-O157:H7 STEC strains is complicated by the lack of easily detectable biochemical or growth characteristics and large serotype diversity. Serological techniques offer a complementary, culture-independent diagnostic approach. They are indispensable for epidemiological and immunization studies.KeywordsHemolytic Uremic SyndromeShiga ToxinNeutralization AssayACHN CellHemorrhagic ColitisThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Adult nondiarrhea hemolytic uremic syndrome associated with shiga toxin escherichia coli O157:H7 bacteremia and urinary tract infection

About 15% of children with Shiga toxin (Stx) producing Escherichia coli (STEC) primarily of serotype O157:H7, gastrointestinal infection, and watery or bloody diarrhea, may develop hemolytic uremic syndrome (D+ HUS). Usually D+ HUS is not complicated by bacteremia and patients recover spontaneously without antibiotic treatment. We report here an adult case of a STEC O157:H7 urinary tract infection complicated by bacteremia and HUS that was not preceded by diarrhea (D- HUS). Cases of D- HUS need to be carefully examined for foci other than the gastrointestinal tract, and patients with E coli bacteremia should receive early antibiotic treatment as would any patient with sepsis. Am J Kidney Dis 41:E4. © 2003 by the National Kidney Foundation, Inc.