New enterovirulent Escherichia coli serogroup 64474 showing antigenic and genotypic relationships to Shigella boydii 16.

Studies based on the analysis of housekeeping genes indicate that Escherichia coli and all Shigella species, except for Shigella boydii type 13, belong to a single species. This study analysed the phenotypic and genotypic characteristics of 23 E. coli strains isolated in different countries from faecal specimens taken from children with diarrhoea. Strains were identified using the VITEK system and typed with rabbit sera obtained against 186 somatic and 53 flagellar E. coli antigens and against 45 Shigella somatic antigens. Biochemical analysis of these strains showed a typical E. coli profile with a defined reaction against both E. coli O179 and S. boydii 16 somatic antisera. Agglutination assays for flagellar antigens showed a response against H2 in 7 (30 %) strains, H10 in 2 (9 %) strains, H32 in 12 (52 %) strains and H34 in 2 (9 %) strains, demonstrating 4 serotypes associated with this new somatic antigen 64474. A serum against one of these E. coli strains (64474) was prepared. Absorption assays of S. boydii 16 and E. coli 64474 antisera with E. coli O179 antigen removed the agglutination response against this O179 antigen completely, while the agglutination titres against both S. boydii 16 and E. coli 64474 remained the same. Four (17 %) E. coli strains showed antimicrobial resistance to piperacillin only, one (4 %) to piperacillin and trimethoprim/sulfamethoxazole, one (4 %) to ciprofloxacin, nitrofurantoin and piperacillin, and two (9 %) strains were resistant to ciprofloxacin, norfloxacin, ofloxacin, piperacillin and trimethoprim/sulfamethoxazole. With regards to PCR assays, one (4 %) of the strains was positive for Shigella gene ipaH, one (4 %) for ipaA, two (9 %) for ipaB, one (4 %) for ipaD, two (9 %) for sepA and three (13 %) for ospF. The rfb gene cluster in the E. coli strains was analysed by RFLP and compared with the gene cluster obtained from S. boydii 16. The rfb-RFLP patterns for all 23 E. coli strains were similar to those obtained for S. boydii 16. The results from PCR tests to detect rfb genes wzx (encoding O unit flippase) and wzy (encoding polymerase) belonging to a cluster related to the biosynthesis of the S. boydii 16-specific O antigen were positive in 21 (91 %) and 22 (96 %) of the strains, respectively. PCR assays to detect E. coli virulence genes were also performed. These assays detected enterotoxigenic E. coli genes ltA1 in 12 of the strains (52 %), st1a in 4 (17 %), cfa1 in 6 (26 %), cs1 in 1 (4 %), cs3 in 3 (13 %), cs13 in 9 (39 %) and cs14 in 5 (22 %) of the strains. Results from the PFGE analyses confirmed the wide geographical distribution of these strains suggesting that 64474 : H2, 64474 : H10, 64474 : H32 and 64474 : H34 are new serotypes of E. coli strains with a defined virulence capacity, and share a common O antigen with S. boydii 16.

The expression of lipopolysaccharide by strains of Shigella dysenteriae, Shigella flexneri and Shigella boydii and their cross-reacting strains of Escherichia coli.

Strains of Shigella dysenteriae, Shigella flexneri and Shigella boydii express lipopolysaccharides, that enable the serotyping of strains based on their antigenic structures. Certain strains of S. dysenteriae, S. flexneri and S. boydii are known to share epitopes with strains of Escherichia coli; however, the lipopolysaccharide profiles of the cross-reacting organisms have not been compared by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) lipopolysaccharides profiling. In the present study, type strains of these bacteria were examined using SDS-PAGE/silver staining to compare their respective lipopolysaccharide profiles. Strains of S. dysenteriae, S. boydii and S. flexneri all expressed long-chain lipopolysaccharide, with distinct profile patterns. The majority of strains of Shigella spp., known to cross-react with strains of E. coli, had lipopolysaccharide profiles quite distinct from the respective strain of E. coli. It was concluded that while cross-reacting strains of Shigella spp. and E. coli may express shared lipopolysaccharide epitopes, their lipopolysaccharide structures are not identical.

Human infections with verocytotoxin-producing Escherichia coli O157--10 years of E. coli O157 serodiagnosis.

From 1997 to 2007, the Laboratory of Enteric Pathogens (LEP), Health Protection Agency, UK, received sera from 2148 patients for testing for antibodies to the LPS of verocytotoxin-producing Escherichia coli (VTEC) O157. A total of 676 (31.5 %) sera had antibodies binding the LPS of E. coli O157 and the majority of patients were below the age of 10 years, a trend observed for both males and females. Antibody-positive patients had haemolytic uraemic syndrome (HUS) in 79.3 % of cases and most of these presented with the atypical (D-) form of HUS. Nine patients were shown to have antibodies to the LPS of E. coli belonging to serogroups O26 (4), O103 (2), O111 (1) and O145 (2) and one patient had antibodies to the somatic antigens of both E. coli O26 and O103. The serodiagnosis of infections with E. coli O157 and other VTEC continues to be an important adjunct to bacteriology. Where clinicians suspect the involvement of a VTEC in disease, patients' sera should be submitted to the LEP for analysis without delay.

Serodiagnosis of Salmonella enterica serovar Typhi and S. enterica serovars Paratyphi A, B and C human infections.

The aim of this study was to evaluate an immunoassay for the detection of human serum antibodies to the LPS and flagellar antigens of Salmonella Typhi and Salmonella Paratyphi A, B and C, and to the Vi capsular polysaccharide of S. Typhi and S. Paratyphi C. A total of 330 sera were used; these originated from 15 patients who were culture-positive for S. Typhi and 15 healthy controls, together with 300 sera submitted to the Laboratory of Enteric Pathogens for Salmonella serodiagnosis. By SDS-PAGE/immunoblotting, all 15 sera from culture-positive patients had serum antibodies to the 9,12 LPS antigens and 10 had antibodies to the 'd' flagellar antigens. Of the 300 reference sera, 22 had antibodies to the 9,12 LPS antigens, one to the 1,4,5,12 LPS antigens and 12 to the 6,7 LPS antigens. Only two sera had antibodies to flagellar antigens, one of which bound to the 'b' and the other to the 'd' antigen. An ELISA was developed that successfully detected serum antibodies to the Vi capsular polysaccharides, but because of the kinetics of serum antibody production to the Vi, these antibodies may be of limited value in the serodiagnosis of acute infection with S. Typhi and S. Paratyphi C. The immunoassays described here provide a sensitive means of detecting serum antibodies to the LPS, flagellar and Vi antigens of S. Typhi and S. Paratyphi, and constitute a viable replacement for the Widal assay for the screening of sera. The Salmonella serodiagnosis protocols described here are the new standard operating procedures used by the Health Protection Agency's National Salmonella Reference Centre based in the Laboratory of Enteric Pathogens, Colindale, UK.

A novel serovar of Shigella dysenteriae from patients with diarrhoea in Bangladesh.

Every year, around 3 % of isolates from patients with diarrhoea at Dhaka Hospital, ICDDR,B, are identified as Shigella-like organisms (SLOs) based on their activity in biochemical tests. These isolates do not react with any of the current Shigella antisera including all existing and provisional serotypes. Among these SLOs, a unique cluster of seven isolates with an identical plasmid profile was found and these isolates were further characterized by phenotypic and genotypic techniques. All were nonlactose fermenters, with an identical biochemical pattern typical of Shigella dysenteriae. They were classified as invasive since they harboured the 140 MDa invasive plasmid, were able to bind Congo red, produced keratoconjunctivitis in the guinea pig eye, and were positive by PCR for the ipaH gene and Shigella enterotoxin 2 [ShET-2] gene. All isolates were resistant to ampicillin, tetracycline and sulfamethoxazole-trimethoprim but were susceptible to mecillinam, nalidixic acid, ceftriaxone and ciprofloxacin. Six of the isolates were identical in DNA pattern by PFGE with the seventh exhibiting a closely related pattern; both patterns were distinguishable from all other Shigella and Escherichia coli patterns. An antiserum prepared against one of the isolates reacted with all isolates and did not cross-react with other Shigella and E. coli serotype reference strains. It is therefore proposed that these isolates represent a new provisional serovar of S. dysenteriae, type strain KIVI 162.

The serodiagnosis of human infections with Yersinia enterocolitica and Yersinia pseudotuberculosis.

The techniques of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting were evaluated for the serodiagnosis of human infections with Yersinia enterocolitica and Yersinia pseudotuberculosis. Lipopolysaccharide (LPS) was prepared from strains comprising four serogroups of Y. enterocolitica and five serogroups of Y. pseudotuberculosis, tested against 200 sera submitted to the Laboratory of Enteric Pathogens for routine serodiagnosis, and shown to contain antibodies to Yersinia LPS by agglutination. Forty four sera were found to contain antibodies that bound to one of the LPS preparations used in the immunoassay. Thirty five of the sera contained antibodies to the LPS of Y. enterocolitica O3, whilst three contained antibodies to the LPS of Y. enterocolitica O5, 27 and Y. enterocolitica O9 LPS respectively. Two sera had antibodies to the LPS of Y. pseudotuberculosis II and a single serum contained antibodies to Y. pseudotuberculosis IV. The SDS-PAGE-immunoblotting procedure described proved to be a reliable procedure for the serodiagnosis of infections with Y. enterocolitica and Y. pseudotuberculosis.

Designation of O174 and O175 to temporary O groups OX3 and OX7, and six new E. coli O groups that include Verocytotoxin-producing E. coli (VTEC): O176, O177, O178, O179, O180 and O181.

Two temporary Escherichia coli O group strains OX3 and OX7 are given permanent status as O174 and O175, respectively. Both these test strains were originally isolated from cases of human diarrhoea. Whereas the O174 strain is negative for known virulence genes, the O175 strain is positive with the probe derived from the CVD432 plasmid associated with the aggregative adherence phenotype, the Enteroaggregative heat-stable enterotoxin 1 gene (astA) and daaC (F1845 afimbrial adhesin) associated with the diffuse adherence (DA) phenotype. Additionally, six E. coli strains are established as antigenic test strains for six new O groups, designated O176, O177, O178, O179, O180 and O181. All six strains produced Verocytotoxin and were positive for vtx1, vtx2, or both genes. Additional virulence genes associated with diarrhoeal disease in humans were found in four of the strains. O176 and O177 strains were isolated from calves, O178 and O181 strains from meat, the O179 strain was from human bloody diarrhoea, and the O180 strain from swine. Preliminary data on the occurrence and epidemiology of these eight new O groups amongst groups of diarrhoeagenic E. coli are reviewed.

Analysis of saliva for antibodies to the LPS of Escherichia coli O157 in patients with serum antibodies to E. coli O157 LPS.

The salivary antibody response to the Escherichia coli O157 LPS antigen was assessed in 44 patients with serum antibodies binding to the LPS of E. coli O157. Saliva from 477 controls was also examined to assess the specificity of the immunoassay used. Twenty of the 44 patients had salivary antibodies to E. coli O157 LPS, giving the salivary antibody test a sensitivity of 0.45 and a predictive positive value for seropositivity of 1.00. The presence of these antibodies appeared not to relate to the time interval between serum sampling and saliva sampling. None of the 477 volunteers had salivary antibodies binding to the LPS of E. coli O157 alone; however, 15 had antibodies which bound non-specifically to both O157 LPS and BSA.

Vero cytotoxin-producing Escherichia coli O157 gastroenteritis in farm visitors, North Wales.

An outbreak of Vero cytotoxin-producing Escherichia coli O157 (VTEC O157) gastroenteritis in visitors to an open farm in North Wales resulted in 17 primary and 7 secondary cases of illness. E. coli O157 Vero cytotoxin type 2, phage type 2 was isolated from 23 human cases and environmental animal fecal samples. A case-control study of 16 primary case-patients and 36 controls (all children) showed a significant association with attendance on the 2nd day of a festival, eating ice cream or cotton candy (candy floss), and contact with cows or goats. On multivariable analysis, only the association between illness and ice cream (odds ratio [OR]=11.99, 95% confidence interval [CI] 1.04 to 137.76) and cotton candy (OR=51.90, 95% CI 2.77 to 970.67) remained significant. In addition to supervised handwashing, we recommend that foods on open farms only be eaten in dedicated clean areas and that sticky foods be discouraged.

Production of serum antibodies that recognise epitopes located on the R3 region of Escherichia coli core lipopolysaccharides by patients infected with strains of enterohaemorrhagic E. coli.

Antibody-antigen cross-reactions were examined with sera from patients with Escherichia coli O157 infection and lipopolysaccharide (LPS) purified from a range of enterohaemorrhagic E. coli (EHEC) including those belonging to serogroups O26, O103, O111, O145 and O157. Six of 10 patients infected with an O157 EHEC produced serum antibodies that cross-reacted with common LPS-core epitopes, which were expressed by 23 of 33 strains of EHEC examined. These common LPS-core epitopes were also present on strains of E. coli O26 which did not produce verocytotoxin. These cross-reacting antibodies did not influence the basic immunoblotting procedures used for the routine serodiagnosis of infections with E. coli O157.

The kinetics of antibody production to antigens of Escherichia coli O157 in a pregnant woman with haemolytic uraemic syndrome.

Sequential blood samples taken from a pregnant woman with haemolytic uraemic syndrome caused by verocytotoxin (VT)-producing Escherichia coli O157 were used to examine the kinetics of serum antibody production to E. coli O157 lipopolysaccharide (LPS), intimin and the conserved region of the translocated intimin receptor (Tir-M). Umbilical cord blood and two samples of blood from the newborn baby were also examined for antibodies to these antigens. In the mother, antibodies of the IgM class, specific for E. coli O157 LPS, were produced in the initial stages of the infection, reaching a peak at 9 days after onset of diarrhoea and subsiding 3 days later. High levels of IgG class antibodies, specific for E. coli O157 LPS, were detected 8 days after the onset of diarrhoea and were present at high titres on day 18. Serum antibodies of the IgA class to E. coli O157 LPS were not detected. Antibodies binding to Tir-M were detected 8 days after the onset of diarrhoea and high titres of these antibodies were still present on day 18. Serum antibodies to intimin were not detected in the mother and no antibodies to any of the antigens tested were detected in either the baby's blood or cord blood. This study describes for the first time the kinetics of serum antibody production during pregnancy, to selected antigens expressed by E. coli O157.

Characteristics and association with disease of two major subclones of Shiga toxin (Verocytotoxin)-producing strains of Escherichia coli (STEC) O157 that are present among isolates from patients in Germany.

Shiga toxin (Verocytotoxin) producing E. coli (STEC) O157 were isolated from 168 patients living in different parts of Germany. Most isolates were from sporadic cases and seven small outbreaks with STEC O157 were identified. The 168 strains were examined for phenotypic and genotypical traits in order to identify major types of STEC O157 occurring in Germany. Phage typing (PT) revealed PT8 (n = 54) and PT2 (n = 48) strains as most frequent (60.7%) among the isolates. Carriage of the stx(2) gene by STEC O157 was closely associated with hemolytic uremic syndrome (100%) and with bloody diarrhea (61.7%). The stx(2) gene was frequent in PT88, PT47 (both 100%), PT2 (91.5%) and PT4 (87.5%) strains and more rarely (33.3%) found in strains belonging to the other PTs. PT8 and PT2 strains formed two groups which differed from each other in their motility, stx-genotypes and the severity of the illness they caused. Pulsed-field gel electrophoresis of PT2 and PT8 strains and hybridization of XbaI digested DNA with stx(1) and stx(2) specific gene probes revealed similarities among epidemiologically unrelated strains belonging to the same PT. The results indicate that STEC O157 PT2 and PT8 strains form two distinct subclones which are dominating in Germany and other European countries.