Detection and Characterization of Shiga Toxigenic Escherichia coli by Using Multiplex PCR Assays for stx1, stx2, eaeA, Enterohemorrhagic E. coli hlyA, rfbO111, and rfbO157

Shiga toxigenic Escherichia coli (STEC) comprises a diverse group of organisms capable of causing severe gastrointestinal disease in humans. Within the STEC family, certain strains appear to be of greater virulence for humans, for example, those belonging to serogroups O111 and O157 and those with particular combinations of other putative virulence traits. We have developed two multiplex PCR assays for the detection and genetic characterization of STEC in cultures of feces or foodstuffs. Assay 1 utilizes four PCR primer pairs and detects the presence of stx₁, stx₂ (including variants of stx₂), eaeA, and enterohemorrhagic E. coli hlyA, generating amplification products of 180, 255, 384, and 534 bp, respectively. Assay 2 uses two primer pairs specific for portions of the rfb (O-antigen-encoding) regions of E. coli serotypes O157 and O111, generating PCR products of 259 and 406 bp, respectively. The two assays were validated by testing 52 previously characterized STEC strains and observing 100% agreement with previous results. Moreover, assay 2 did not give a false-positive O157 reaction with enteropathogenic E. coli strains belonging to clonally related serogroup O55. Assays 1 and 2 detected STEC of the appropriate genotype in primary fecal cultures from five patients with hemolytic-uremic syndrome and three with bloody diarrhea. Thirty-one other primary fecal cultures from patients without evidence of STEC infection were negative.     

Etiological Study of Diarrheal Patients in Vientiane, Lao People’s Democratic Republic

The etiological agents of diarrhea in Vientiane, Lao People’s Democratic Republic (Lao PDR), were studied in the period from October 1996 to August 1997. A total of 880 patients with diarrhea visiting medical facilities were examined for Shigella, Salmonella, diarrheagenic Escherichia coli, Vibrio, Aeromonas, Campylobacter, and rotavirus. Shigella spp., heat-stable enterotoxin (ST)-producing E. coli, and serogroup-based enteropathogenic E. coli were found to be the main organisms causing diarrhea in Vientiane, with frequencies of 16.8% (148 of 880), 17.2% (111 of 645), and 11.0% (97 of 880), respectively. Relatively low incidences were observed in the cases of Salmonella spp., (0.6%; 5 of 880), Campylobacter spp. (4.4%; 39 of 880), and rotavirus (6.1%; 9 of 148), and no isolates of V. cholerae O1 or O139 or Aeromonas were recovered. An analysis of the incidences of enteropathogens with respect to age and seasonal variations demonstrated that the frequencies of isolation of Shigella spp. and heat-labile enterotoxin-producing E. coli were significantly higher in those aged 1 to 5 years than in those younger than 1 year of age and those older than 5 years of age (P < 0.0001 and P < 0.05, respectively) and that the frequencies of isolation of Shigella spp. and ST-producing E. coli were significantly higher in the rainy season than in the dry season (P < 0.005 and P < 0.001, respectively). Almost all strains of Shigella spp. tested were resistant to ampicillin, tetracycline, and erythromycin and were susceptible to cefdinir and ofloxacin. This is the first intensive and longitudinal study to define the etiologic agents of diarrheal diseases in Lao PDR.     

Molecular Analysis of Shiga Toxigenic Escherichia coli O111:H− Proteins Which React with Sera from Patients with Hemolytic-Uremic Syndrome

Western blot analysis was used to assess the reactivity of convalescent-phase sera from patients who were associated with an outbreak of hemolytic-uremic syndrome (HUS) caused by fermented sausage contaminated with Shiga toxin-producing Escherichia coli (STEC). The predominant STEC isolated from HUS patients belonged to serotype O111:H⁻, and reactivity to O111:H⁻ whole-cell lysates, treated or untreated with proteinase K, was examined. As expected, all five serum samples demonstrated a marked anti-lipopolysaccharide response, but several protein bands were also immunoreactive, particularly one with an apparent size of 94 kDa. One convalescent-phase serum sample was subsequently used to screen an O111:H⁻ cosmid bank and 2 of 900 cosmid clones were found to be positive, both of which contained a similar DNA insert. Western blot analysis of one of these clones identified three major immunoreactive protein bands of approximately 94, 70, and 50 kDa. An immune response to the three proteins was detectable with all five convalescent-phase serum samples but not with healthy human serum. Immunoreactive 94- and 50-kDa species were produced by a deletion derivative of the cosmid containing a 7-kb STEC DNA insert. Sequence analysis of this region indicated that it is part of the locus for enterocyte effacement, including the eaeA gene which encodes intimin. The deduced amino acid sequence of the O111:H⁻ intimin was 88.6% identical to intimin from O157:H7 STEC, and the most divergent region was the 200 residues at the carboxyl terminus, which were only 75% identical. Such variation may be antigenically significant as serum from a HUS patient infected only with the O111:H⁻ STEC reacted with intimin from an enteropathogenic E. coli O111 strain, as well as several other eaeA-positive STEC isolates, but not with an eaeA-positive STEC belonging to serotype O157:H⁻. Sera from two of the other HUS patients also failed to react with intimin from this latter strain. However, intimin from O157:H⁻ STEC did react with serum from a patient infected with both O111:H⁻ and O157:H⁻ STEC.     

Molecular Characterization of Human Atypical Sorbitol-Fermenting Enteropathogenic Escherichia coli O157 Reveals High Diversity

Alongside the well-characterized enterohemorrhagic Escherichia coli (EHEC) O157:H7, serogroup O157 comprises sorbitol-fermenting typical and atypical enteropathogenic E. coli (EPEC/aEPEC) strains that carry the intimin-encoding gene eae but not Shiga toxin-encoding genes (stx). Since little is known about these pathogens, we characterized 30 clinical isolates from patients with hemolytic uremic syndrome (HUS) or uncomplicated diarrhea with respect to their flagellin gene (fliC) type and multilocus sequence type (MLST). Moreover, we applied whole-genome sequencing (WGS) to determine the phylogenetic relationship with other eae-positive EHEC serotypes and the composition of the rfbO157 region. fliC typing resulted in five fliC types (H7, H16, H34, H39, and H45). Isolates of each fliC type shared a unique ST. In comparison to the 42 HUS-associated E. coli (HUSEC) strains, only the stx-negative isolates with fliCH7 shared their ST with EHEC O157:H7/H⁻ strains. With the exception of one O157:H⁻_fliCH16 isolate, HUS was exclusively associated with fliCH7. WGS corroborated the separation of the fliCH7 isolates, which were closely related to the EHEC O157:H7/H⁻ isolates, and the diverse group of isolates exhibiting different fliC types, indicating independent evolution of the different serotypes. This was also supported by the heterogeneity within the rfbO157 region that exhibited extensive recombinations. The genotypic subtypes and distribution of clinical symptoms suggested that the stx-negative O157 strains with fliCH7 were originally EHEC strains that lost stx. The remaining isolates form a distinct and diverse group of atypical EPEC isolates that do not possess the full spectrum of virulence genes, underlining the importance of identifying the H antigen for clinical risk assessment.     

Diarrheagenic Escherichia coli and Shigella strains isolated from children in a hospital case-control study in Hanoi, Vietnam

This case-control study detected and characterized Shigella and diarrheagenic Escherichia coli (DEC) types among Vietnamese children less than 5 years old. In 249 children with diarrhea and 124 controls, Shigella spp. was an important cause of diarrhea (P < 0.05). We used multiplex PCR and DNA probes to detect enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAggEC), enteropathogenic E. coli (EPEC), attaching and effacing E. coli (A/EEC), verocytotoxin-producing E. coli (VTEC), and enterotoxigenic E. coli (ETEC). The prevalences of DEC in the diarrhea and control groups were 25.7 and 10.5%, respectively. In 62 children with diarrhea, 64 DEC strains included 22 EAggEC (8.8%), 2 EIEC (0.8%), 23 A/EEC (9.2%), 7 EPEC (2.8%), and 10 ETEC strains (4.0%). Among controls, 13 DEC strains included 5 EAggEC strains (4.0%), 7 A/EEC strains (5.6%), and 1 EPEC strain. The characterization of DEC by serotypes, antimicrobial susceptibility patterns, virulence genes, and pulsed-field gel electrophoresis showed the occurrence of many different and highly heterogenic DEC subtypes, but common serotypes were found among ETEC, EIEC and EPEC, respectively. Serotyping was used to distinguish between A/EEC and EPEC. However, A/EEC, EPEC, and EAggEC were isolated at high frequency from both cases and controls. Further in-depth studies are needed to better understand important virulence factors of DEC, especially A/EEC, EPEC, and EAggEC.     

Prevalence and clinical manifestations of shiga toxin-producingEscherichia coli infections in Austrian children

The prevalence and clinical manifestations of infections associated with Shiga toxin-producingEscherichia coli (STEC) among Austrian children were assessed. Stool samples from 280 pediatric patients were analyzed by enzyme immunoassay (EIA) for the presence of free fecal Shiga toxin (Stx) 1 and 2, and by culture on sorbitol MacConkey agar. Specimens testing positive by the EIA were subjected to a cytotoxicity assay, polymerase chain reaction analysis, and a colony hybridization test. Direct culture on MacConkey agar demonstrated the presence of threeEscherichia coli O157:H7-positive stools. These were also positive by EIA and by the DNA-based methods. An additional six samples were positive by EIA, and in four of these, non-O157 STEC of serotypes O111H^–, O146: H^–, and O113:H53 could be isolated. Analysis of stools for a variety of enteric pathogens demonstrated that STEC was the third most common bacterial pathogen. The clinical manifestations of STEC infections were difficult to distinguish from those of infections caused by other enteric pathogens, as most patients presented with watery diarrhea. The median age of children with STEC infections was 27.6 months (range, 7 months to 5.75 years); children withSalmonella orCampylobacter infections were younger on average, while those withRotavirus infections were older. This study demonstrated that althoughEscherichia coli O157:H7 could be identified with the same sensitivity by both EIA or agar-based methods, the identification of non-O157 STEC strains was enhanced by the use of EIA followed by colony hybridization. Analysis of overnight cultures from 53 STEC isolates revealed that all strains producing Stx1, Stx2, or Stx2c reacted in the EIA. However, culture supernatants from Stx2e-producingEscherichia coli O101 were negative in the EIA. Despite this disadvantage, the EIA is easy to perform and time efficient and can be recommended as a screening test for non-O157 STEC in children with diarrhea.     

Results of screening for verocytotoxin-producingEscherichia coli in faeces in Belgium

During an 18-month period all stools submitted to a microbiology laboratory in Belgium for culture were screened for VerocytotoxinproducingEscherichia coli (VTEC) serotype O157. In the stool samples from 3940 patients, eight (0.2 %) VTEC O157 strains were isolated, seven of which were O157:H7. Additional screening for other serotypes of VTEC in 332 selected stool samples yielded four more strains (serotypes O2:K1:H6, O111:H^–, O117:K1:H7 and OC70/86:H^–). The 0.3 % isolation rate for all VTEC was comparable to that forShigella spp. Eight children under 30 months and two adults suffered from uncomplicated gastroenteritis. A 5-month-old child and a 41-year-old woman presented with hemolytic uremic syndrome a few days after onset of a diarrheal episode.     

Clonal Diversity of Chilean Isolates of Enterohemorrhagic Escherichia coli from Patients with Hemolytic-Uremic Syndrome, Asymptomatic Subjects, Animal Reservoirs, and Food Products

To determine clonal relationship among Chilean enterohemorrhagic Escherichia coli (EHEC) strains from different sources (clinical infections, animal reservoirs, and food), 54 EHEC isolates (44 of E. coli O157, 5 of E. coli O111, and 5 of E. coli O26) were characterized for virulence genes by colony blot hybridization and by pulsed-field gel electrophoresis (PFGE). By colony blotting, 12 different genotypes were identified among the 44 E. coli O157 isolates analyzed, of which the genetic profile stx₁⁺ stx₂⁺ hly⁺ eae⁺ was the most prevalent. All human O157 strains that were associated with sporadic cases of hemolytic-uremic syndrome (HUS) carried both the stx₁ and stx₂ toxin-encoding genes and were eaeA positive. Only 9 of 13 isolates from human controls were stx₁⁺ stx₂⁺, and 8 carried the eaeA gene. Comparison of profiles obtained by PFGE of XbaI-digested genomic DNA showed a great diversity among the E. coli O157 isolates, with 37 different profiles among 39 isolates analyzed. Cluster analysis of PFGE profiles showed a wide distribution of clinical isolates obtained from HUS cases and asymptomatic individuals and a clonal relationship among O157 isolates obtained from HUS cases and pigs. Analysis of virulence genes showed that a correlation exists among strains with the genotype stx₁⁺ stx₂⁺ eae⁺ and pathogenic potential. A larger difference in the PFGE restriction patterns was observed among the EHEC strains of serogroups O26 and O111. These results indicate that several different EHEC clones circulate in Chile and suggest that pigs are an important animal reservoir for human infections by EHEC. Guidelines have been proposed for better practices in the slaughter of animals in Chile.     

Serodiagnosis Using Microagglutination Assay during the Food-Poisoning Outbreak in Japan Caused by Consumption of Raw Beef Contaminated with Enterohemorrhagic Escherichia coli O111 and O157

A microagglutination (MA) assay to identify antibodies to Escherichia coli O111 and O157 was conducted in sera collected from 60 patients during a food-poisoning outbreak affecting 181 patients in Japan which was caused by the consumption of contaminated raw beef. Enterohemorrhagic E. coli (EHEC) O111:H8 and/or O157:H7 was isolated from the stools of some of the patients, but the total rate of positivity for antibodies to O111 (45/60, 75.0%) was significantly higher than that for antibodies to O157 (10/60, 16.7%). The MA titers of antibodies to O111 measured in patients with hemolytic-uremic syndrome and bloody diarrhea were higher than those measured in patients with only diarrhea. In patients from whose stool no isolates of E. coli O111 and O157 were obtained, the positive antibody detection rates were 12/19 (63.2%) for O111 and 2/19 (10.5%) for O157, and the MA titers of antibodies to O111 measured were higher than those to O157. Similarly, the MA titers of antibodies to O111 were significantly higher than those to O157, regardless of the other groups, including groups O111, O111 and O157, and O157. These serodiagnosis results suggest that EHEC O111:H8 stx₂ played a primary role in the pathogenesis of this outbreak. Furthermore, our findings suggest that the isolates from the patients'' stool specimens were not always the major causative pathogen in patients with multiple EHEC infections, because the sera from patients from whose stools only O157 was isolated were positive for antibodies to O111. Measuring antibodies to E. coli O antigen is helpful especially in cases with multiple EHEC infections, even with a non-O157 serotype.     

Non-O157:H7 Stx2-Producing Escherichia coli Strains Associated with Sporadic Cases of Hemolytic-Uremic Syndrome in Adults

From August 1996 to May 1997, six verotoxin-producing Escherichia coli (VTEC) strains were isolated from stool specimens of adults suffering from hemolytic-uremic syndrome (HUS). All the isolates were stx₂ positive and belonged to different serotypes: O6:H4, O91:H10, O91:H21, O rough:H16, OX3:H−, and O nontypeable:H−. The enterohemolysin (Ehly)-encoding genes were detected in two isolates, and none of the isolates harbors the intimin (Eae)-encoding gene. These findings suggest that stx₂-positive non-O157:H7 VTEC is a major cause of HUS in adults and that several sources of pathogens are responsible for local endemic infections.     

Immunological Characterization of Escherichia coli O157:H7 Intimin γ1

Portions of the intimin genes of Escherichia coli O157:H7 strain E319 and of the enteropathogenic E. coli O127:H6 strain E2348/69 were amplified by PCR and cloned into pET-28a(+) expression vectors. The entire 934 amino acids (aa) of E. coli O157:H7 intimin, the C-terminal 306 aa of E. coli O157:H7 intimin, and the C-terminal 311 aa of E. coli O127:H6 intimin were expressed as proteins fused with a six-histidine residue tag (six-His tag) in pET-28a(+). Rabbit antisera raised against the six-His tag-full-length E. coli O157:H7 intimin protein fusion cross-reacted in slot and Western blots with outer membrane protein preparations from the majority of enterohemorrhagic and enteropathogenic E. coli serotypes which have the intimin gene. The E. coli strains tested included isolates from humans and animals which produce intimin typesα (O serogroups 86, 127, and 142), β1 (O serogroups 5, 26, 46, 69, 111, 126, and 128), γ1 (O serogroups 55, 145, and 157), γ2 (O serogroups 111 and 103), and (O serogroup 103) and a nontypeable intimin (O serogroup 80), results based on intimin type-specific PCR assays. Rabbit antisera raised against the E. coli O157:H7 C-terminal fusion protein were much more intimin type-specific than those raised against the full-length intimin fusion protein, but some cross-reaction with other intimin types was also observed for these antisera. In contrast, the monoclonal antibody Intγ1.C11, raised against the C-terminal E. coli O157 intimin, reacted only with preparations from intimin γ1-producing E. coli strains such as E. coli O157:H7.     

Detection of Intimins α, β, γ, and δ, Four Intimin Derivatives Expressed by Attaching and Effacing Microbial Pathogens

Intimins are outer membrane proteins expressed by enteric bacterial pathogens capable of inducing intestinal attachment-and-effacement lesions. A eukaryotic cell-binding domain is located within a 280-amino-acid (Int280) carboxy terminus of intimin polypeptides. Polyclonal antiserum was raised against Int280 from enteropathogenic Escherichia coli (EPEC) serotypes O127:H6 and O114:H2 (anti-Int280-H6 and anti-Int280-H2, respectively), and Western blot analysis was used to explore the immunological relationship between the intimin polypeptides expressed by different clinical EPEC and enterohemorrhagic E. coli (EHEC) isolates, a rabbit diarrheagenic E. coli strain (RDEC-1), and Citrobacter rodentium. Anti-Int280-H6 serum reacted strongly with some EPEC serotypes, whereas anti-Int280-H2 serum reacted strongly with strains belonging to different EPEC and EHEC serotypes, RDEC-1, and C. rodentium. These observations were confirmed by using purified Int280 in an enzyme-linked immunosorbent assay and by immunogold and immunofluorescence labelling of whole bacterial cells. Some bacterial strains were recognized poorly by either antiserum (e.g., EPEC O86:H34 and EHEC O157:H7). By using PCR primers designed on the basis of the intimin-encoding eae gene sequences of serotype O127:H6, O114:H2, and O86:H34 EPEC and serotype O157:H7 EHEC, we could distinguish between different eae gene derivatives. Accordingly, the different intimin types were designated α, β, δ, and γ, respectively.     

Lipopolysaccharide as an Antigen Target for the Formulation of a Universal Vaccine against Escherichia coli O111 Strains

A promising approach to developing a vaccine against O111 strains of diarrheagenic Escherichia coli that exhibit different mechanisms of virulence is to target either the core or the polysaccharide chain (O antigen) of their lipopolysaccharide (LPS). However, due to structural variations found in both these LPS components, to use them as antigen targets for vaccination, it is necessary to formulate a vaccine able to induce a humoral immune response that can recognize all different variants found in E. coli O111 strains. In this study, it was demonstrated that, despite differences in composition of oligosaccharide repeat units between O111ab and O111ac LPS subtypes, antibodies against one O111 subtype can recognize and inhibit the adhesion to human epithelial cells of all categories of O111 E. coli (enteropathogenic E. coli [EPEC], enterohemorrhagic E. coli [EHEC], and enteroaggregative E. coli [EAEC]) strains regardless of the nature of their flagellar antigens, mechanisms of virulence, or O111 polysaccharide subtypes. These antibodies were also able to increase the clearance of different strains of O111 E. coli by macrophages. PCR analyses of the pathways involved in O111 LPS core biosynthesis showed that all EAEC strains have core type R2, whereas typical EPEC and EHEC have core type R3. In contrast, atypical EPEC strains have core types R2 and R3. In summary, the results presented herein indicate that the O111 polysaccharide and LPS core types R2 and R3 are antigen targets for panspecific immunotherapy against all categories of O111 E. coli.Pathogenic strains of O111 Escherichia coli exist as three distinct categories of diarrheagenic organisms, namely, enteropathogenic E. coli (EPEC; typical and atypical), enterohemorrhagic E. coli (EHEC), and enteroaggregative E. coli (EAEC) (7). In developing countries, diarrhea induced by these pathogens is a serious illness that inflicts a huge health and economic burden on the population (46, 48). Despite the fact that sanitation and clean water can markedly reduce the cases of diarrhea in areas of endemicity, surveillance studies have demonstrated that in Latin America alone more than 80% of the population has no access to sewage systems or treated water (44). Different serotypes of Shiga toxin-producing E. coli pathogens (O111:H⁻, O111:H8, and O111:H2) are also a public health problem in developed countries worldwide, where they have been responsible for outbreaks of bloody diarrhea and cases of hemolytic-uremic syndrome (HUS) (4, 12, 14, 21, 28, 32, 35, 55). One of the worst outbreaks of O111 E. coli happened in August 2008 in Oklahoma, where 341 people become ill, 70 people were hospitalized, 17 people developed HUS, and 1 person died (5, 8). In addition, other pathogens such as Salmonella enterica subsp. enterica serovar Adelaide and Salmonella enterica subsp. enterica serovar 50:z:e,n,x also have the same lipopolysaccharide (LPS) polysaccharide structure as that found in O111 E. coli (29).Because of the impact that O111 E. coli strains have on public health, a lot of effort has been devoted to developing a safe, cheap, and effective vaccine to prevent diarrheagenic diseases caused by these pathogens.The best approach to constructing a vaccine capable of protecting against a wide range of different strains of O111 E. coli is to target the LPS polysaccharide chain (O antigen), since 75% of the outer membrane of all Gram-negative bacteria is covered by LPS (38, 50). This approach is supported by the fact that conjugated vaccines against polysaccharides have been used successfully against polysaccharide-encapsulated organisms such as Streptococcus pneumoniae and Haemophilus influenzae type b in clinical practice (42). However, to use the O111 polysaccharide chain as an antigen target for the construction of a universal vaccine against enteric O111 E. coli pathogens, the antigenic variation of O111 subtypes between different E. coli strains has to be taken into account (7, 33, 59). In addition, although the O111 polysaccharides that compose their capsules are identical to the ones present on their external membranes (17, 53, 54), it has been demonstrated by Goldman and coworkers that the capsules of O111 bacteria are poorly recognized by antibodies raised against O111 LPS derived from the bacterial membrane (17), indicating that immunization with capsulated bacteria induces antibody responses different from those induced by immunization with noncapsulated bacteria.In addition, the O111 E. coli strains can be either naked or capsulated, although the O111 polysaccharides that compose their capsules are identical to the ones present on their external membranes, except for the absence of a lipid A core (17, 53, 54).The LPS core can also be targeted for vaccination or immunotherapy (11, 19, 39). It is not considered a virulence factor, although its involvement in bacterial adhesion has been reported (24). Structural variations are also found in the external part of the LPS core (37), and they have to be considered in order to generate antibodies capable of identifying all antigenic variants encountered within O111 bacteria.Another element of the humoral immune response involved in clearance of pathogens is the complement system, which, independently of antibody, can be activated by pathogens in the initial stages of infection and, by itself, can kill pathogens directly. However, it is not effective in recognizing or eliminating all bacteria in samples (3, 30, 43, 45). The complement system can also promote bacterial uptake and destruction by macrophages by interacting with both the pathogen and the complement receptors present on the macrophage membrane. However, when complement activation is not enough to promote bacterial killing by macrophages, antibodies are required (25, 26, 34).To investigate whether the O111 LPS polysaccharide of E. coli is a good antigen candidate for the formulation of a universal vaccine capable of preventing infection by O111 pathogens, electrophoretic, molecular, serological, and immunological analyses were conducted in order to determine whether antibodies against O111 polysaccharides can recognize O111 EHEC, EPEC, and EAEC, can inhibit their adhesion to human epithelial cells, and can stimulate their clearance by macrophages.In addition, the compositions of the cores of 73 samples of all categories of O111 bacteria were characterized by PCR analysis of the enzymes responsible for the biosynthesis of all five types of LPS core: R1, R2, R3, R4, and K12.     

Discrimination of Enterohemorrhagic Escherichia coli (EHEC) from Non-EHEC Strains Based on Detection of Various Combinations of Type III Effector Genes

Enterohemorrhagic Escherichia coli (EHEC) strains comprise a subgroup of Shiga-toxin (Stx)-producing E. coli (STEC) and are characterized by a few serotypes. Among these, seven priority STEC serotypes (O26:H11, O45:H2, O103:H2, O111:H8, O121:H19, O145:H28, and O157:H7) are most frequently implicated in severe clinical illness worldwide. Currently, standard methods using stx, eae, and O-serogroup-specific gene sequences for detecting the top 7 EHEC serotypes bear the disadvantage that these genes can be found in non-EHEC strains as well. Here, we explored the suitability of ureD, espV, espK, espN, Z2098, and espM1 genes and combinations thereof as candidates for a more targeted EHEC screening assay. For a very large panel of E. coli strains (n = 1,100), which comprised EHEC (n = 340), enteropathogenic E. coli (EPEC) (n = 392), STEC (n = 193), and apathogenic strains (n = 175), we showed that these genetic markers were more prevalent in EHEC (67.1% to 92.4%) than in EPEC (13.3% to 45.2%), STEC (0.5% to 3.6%), and apathogenic E. coli strains (0 to 2.9%). It is noteworthy that 38.5% of the EPEC strains that tested positive for at least one of these genetic markers belonged to the top 7 EHEC serotypes, suggesting that such isolates might be Stx-negative derivatives of EHEC. The associations of espK with either espV, ureD, or Z2098 were the best combinations for more specific and sensitive detection of the top 7 EHEC strains, allowing detection of 99.3% to 100% of these strains. In addition, detection of 93.7% of the EHEC strains belonging to other serotypes than the top 7 offers a possibility for identifying new emerging EHEC strains.     

Sensitivities and Specificities of Premier E. coli O157 and Premier EHEC Enzyme Immunoassays for Diagnosis of Infection with Verotoxin (Shiga-Like Toxin)-Producing Escherichia coli

This study describes the performance of two rapid enzyme immunoassays, Premier E. coli O157 and Premier EHEC (Meridian Diagnostics Inc., Cincinnati, Ohio) for the detection in stools of Escherichia coli O157 and verotoxins (Shiga-like toxins), respectively. Both tests were performed on stools from 876 children presenting to eight emergency departments with diarrhea. Standard culture, including E. coli O157:H7 isolation, was performed, and paired sera were taken for anti-O157-lipopolysaccharide antibody determination. Stools from patients enrolled in the study, and those yielding discordant results, were sent to a reference laboratory for repeat testing and further investigation, including cytotoxicity and non-O157 verotoxin-producing E. coli culture. Results were classified as field results (obtained in the eight site laboratories) and resolved results (obtained after repeat testing in the central laboratory). The “gold standard” for sensitivity of both tests and for specificity of Premier E. coli O157 was isolation of E. coli O157:H7 or a fourfold anti-O157 antibody rise. Specimens positive by the Premier EHEC test and negative for E. coli O157 culture were examined for non-O157 verotoxin-producing E. coli. The field sensitivity of Premier E. coli O157 was 86%, that of Premier EHEC was 89%, and the specificity of Premier E. coli O157 was 98%. Ten of 13 discordant Premier E. coli O157 results were reassigned as true results after repeat testing. Ten non-O157 verotoxin-producing E. coli isolates were recovered from Premier EHEC-positive, E. coli O157 culture-negative stools. Only one specimen gave an unequivocally false-positive Premier EHEC result. Both tests are highly sensitive and are specific if correctly performed. The Premier EHEC test will be particularly valuable as a practical routine test for the detection of non-O157 verotoxin-producing E. coli.Approximately 8 to 10% of children with diarrhea caused by Escherichia coli strains which are capable of producing verotoxins (VTs) (Shiga-like toxins) develop hemolytic-uremic syndrome (HUS) 5 to 10 days after the onset of symptoms (4). Antigenically distinct verotoxins (VT1 and VT2) can cause endothelial injury in vitro. Because endothelial injury is thought to precipitate the microangiopathy that results in HUS, it has been hypothesized that HUS results from endothelial injury caused by systemic spread of these toxins (6, 8–10). No intervention has proven effective in preventing HUS. The efficacy of an oral synthetic toxin receptor analog (SYNSORB Pk) in preventing systemic spread of VT, and subsequent development of HUS, is currently being evaluated in a phase 3 randomized controlled trial. In an earlier phase 2 trial of the same agent, patients were recruited by clinical criteria in order to begin treatment as early as possible in the disease process. Once enrolled, many had to be excluded from the analysis of the efficacy of the intervention because laboratory tests did not confirm the presence of verotoxin-producing E. coli (VTEC). To improve the efficiency of enrolling children with true VTEC infection in the phase 3 trial, two enzyme immunoassays (EIAs) were performed on all potentially eligible subjects with diarrhea. The first test (Premier E. coli O157) detects the presence of O157 antigen in stool, and the second (Premier EHEC) detects the presence of VT1 and VT2. One difficulty with the clinical evaluation of the Premier EHEC test is that the logical “gold standard” would be the stool cytotoxicity test, but this test is not widely available, and specimen transport under appropriate conditions to a laboratory that can perform it may be difficult to organize, so that the sensitivity of the enterohemorrhagic E. coli (EHEC) test has to be evaluated by using E. coli O157:H7 isolation as a surrogate gold standard. This paper reports our experience with these tests and partially addresses this problem.     

Characterization of Enterohemorrhagic Escherichia coli O111 and O157 Strains Isolated from Outbreak Patients in Japan

In April and May 2011, there was a serious food-poisoning outbreak in Japan caused by enterohemorrhagic Escherichia coli (EHEC) strains O111:H8 and O157:H7 from raw beef dishes at branches of a barbecue restaurant. This outbreak involved 181 infected patients, including 34 hemolytic-uremic syndrome (HUS) cases (19%). Among the 34 HUS patients, 21 developed acute encephalopathy (AE) and 5 died. Patient stool specimens yielded E. coli O111 and O157 strains. We also detected both EHEC O111 stx₂ and stx-negative E. coli O111 strains in a stock of meat block from the restaurant. Pulsed-field gel electrophoresis (PFGE) and multilocus variable-number tandem-repeat analysis (MLVA) showed that the stx-negative E. coli O111 isolates were closely related to EHEC O111 stx₂ isolates. Although the EHEC O157 strains had diverse stx gene profiles (stx₁, stx₂, and stx₁ stx₂), the PFGE and MLVA analyses indicated that these isolates originated from a single clone. Deletion of the Stx2-converting prophage from the EHEC O111 stx₂ isolates was frequently observed during in vitro growth, suggesting that strain conversion from an EHEC O111 stx₂ to an stx-negative strain may have occurred during infection.     

Antibiogram of E. coli serotypes isolated from children aged under five with acute diarrhea in Bahir Dar town

Background

Diarrheal disease and its complications remain a major cause of morbidity and mortality in children. The prevalence and antibiogram of E. coli as causative agents of diarrhea vary from region to region, and even within countries in the same geographical area.

Objectives

To determine the serotype and antimicrobial susceptibility of E. coli in children under-five years of age.

Methods

A cross-sectional study was conducted among 422 children with diarrhea from December 2011 to February 2012. Identification of E. coli and antimicrobial susceptibility testing were done following standard procedures.

Results

The overall isolation rate of E. coli was 48.3%. Poly 2 sero-groups, poly 3 sero-groups, poly 4 sero-groups and E. coli O157:H7 accounted for 80 (39.2%), 40 (19.6%), 25 (12.3%), and 59 (28.9%) of the isolates, respectively. Poly 2 sero-groups, constituting isolates belonging to enteropathogenic E. coli were the most commonly isolated serotypes. E. coli exhibited high levels of antimicrobial resistance to ampicillin (86.8%), tetracycline (76%) and cotrimoxazole (76%). Low levels of resistance to ciprofloxacin (6.9%) and norfloxacin (9.3%) were documented.

Conclusion

High prevalence of diarrheagenic E. coli compounded by alarming antimicrobial resistances is a serious public health problem. Regular determination of antibiogram and public education are recommended.     

Usability and Performance of CHROMagar STEC Medium in Detection of Shiga Toxin-Producing Escherichia coli Strains

The performance and usability of CHROMagar STEC medium (CHROMagar Microbiology, Paris, France) for routine detection of Shiga toxin-producing Escherichia coli (STEC) strains were examined. The ability of the medium to selectively propagate STEC strains differing by their serotypes and virulence genes was studied with a collection of diarrheagenic E. coli isolates (n = 365) consisting of 49 different serotypes and with non-STEC and other bacterial isolates (n = 264). A total of 272 diarrheagenic E. coli (75.0%) isolates covering 24 different serotypes grew on CHROMagar STEC. The highest detection sensitivities were observed within the STEC serogroups O26 (90.0%), O111 (100.0%), O121 (100.0%), O145 (100.0%), and O157 (84.9%), and growth on CHROMagar STEC was highly associated with the presence of the tellurite resistance gene (terD). The specificity of the medium was 98.9%. In addition, CHROMagar STEC was used in parallel with a Shiga toxin-detecting immunoassay (Ridaquick Verotoxin/O157 Combi; R-biopharm, Darmstadt, Germany) to screen fecal specimens (n = 47) collected from patients suffering from hemorrhagic diarrhea. Positive growth on CHROMagar STEC was confirmed by the Premier EHEC enzyme immunoassay (Meridian Bioscience, Inc., Cincinnati, OH), and discrepant results between the two screening methods were confirmed by stx gene-detecting PCR. All 16 of the 47 stool samples that showed positive growth on CHROMagar STEC were also positive in the confirmatory tests. CHROMagar STEC proved to be an interesting option for STEC screening, allowing good detection sensitivity and specificity and permitting strain isolation for further outbreak investigations when required.