Characterisation of STEC strains • Mario RSM Souza et al

Memórias do Instituto Oswaldo Cruz, 2010

Thirty-eight strains of Shiga toxin-producing Escherichia coli (STEC) were characterised in terms of biochemical properties, enterohaemolysin production and plasmid carriage. A wide variation in the biochemical properties was observed among the STEC, with 14 distinct biotypes identified. Biotype 1 was the most common, found in 29% of the strains. Enterohaemolysin production was detected in 29% of the strains. Most of the bacterial strains (95%) carried one or more plasmids and considerable heterogeneity in size and combinations was observed. Seven distinct plasmid profiles were identified. The most common profile, characterised by the presence of a single plasmid of ~90 kb, was found in 50% of these strains. These data indicate extensive diversity among STEC strains. No correlation was found among biotype, serotype, enterohaemolysin production and plasmid profile.

2005

A total of 42 Shiga toxin-producing (STEC) strains from slaughtered healthy cattle in Switzerland were characterized by phenotypic and genotypic traits.The 42 sorbitol-positive, non-O157 STEC strains belonged to 26 O:H serotypes (including eight new serotypes) with four serotypes (O103:H2, O113:H4, O116:H-, ONT:H-) accounting for 38.1% of strains. Out of 16 serotypes previously found in human STEC (71% of strains), nine serotypes (38% of strains) were serotypes that have been associated with hemolytic–uremic syndrome (HUS). Polymerase chain reaction (PCR) analysis showed that 18 (43%) strains carried the stx1 gene, 20 strains (48%) had the stx2 gene, and four (9%) strains had both stx1 and stx2 genes. Of strains encoding for stx2 variants, 63% were positive for stx2 subtype. Enterohemolysin (ehxA), intimin (eae), STEC autoagglutinating adhesin (saa) were detected in 17%, 21%, and 19% of the strains, respectively. Amongst the seven intimin-positive strains, one possessed intimin type β1 (O5:H-), one intimin γ1 (O145:H), one intimin γ2/θ, (O111:H21), and four intimin ɛ (O103:H2).The strains belonged to 29 serovirotypes (association between serotypes and virulence factors). O103:H2 stx1eae-ɛ ehxA, O116:H- stx2, and ONT:H- stx2c were the most common accounting for 29% of the strains. Only one strain (2.4%) of serovirotype O145:H- stx1stx2eae-γ1ehxA showed a pattern of highly virulent human strains. This is the first study providing characterization data of bovine non-O157 STEC in Switzerland, and underlining the importance of the determination of virulence factors (including intimin types) in addition to serotypes to assess the potential pathogenicity of these strains for humans.

FEMS Microbiology Letters, 2007

The distribution of virulence markers related to cytolethal distending toxin-V (CDT-V), subtilase cytotoxin (SubAB), the enterohemorrhagic Escherichia coli factor for adherence (Efa1), the adhesin similar to IrgA (Iha), the long polar fimbriae (LpfO113), the autoagglutinating adhesin (Saa), and the protein required for full expression of adherence of O157:H7 Sakai strain (ToxB) was investigated in 121 Shiga toxin-producing E. coli (STEC) strains isolated in Brazil. STEC strains were isolated from human infections (n = 49), cattle (n = 68) and ground meat samples (n = 4). Overall, the lpfA O113 , iha, efa1, saa, and toxB sequences were observed in 89.2%, 87.6%, 47.1%, 43%, and 13.2% of the strains, respectively. The genes efa1 (96.6%) and toxB (27%) were only identified among eae-positive strains, while saa (83.8%), cdt-V (12.9%), and subAB (48.4%) just occurred in eaenegative STEC strains. STEC strains harboring cdt-V and subAB were for the first time described in the South American subcontinent. In addition, the simultaneous presence of cdt-V and subAB has not been previously reported, nor the presence of subAB in STEC O77, O79, O105, O174, and O178 serogroups. A diversity of virulence profiles was observed among the STEC strains studied. The most prevalent profile observed among eae-positive STEC strains mainly isolated from humans was eae efa1 iha lpfA O113 , whereas iha lpfA O113 saa ehxA subAB prevailed among eae-negative STEC strains, mostly isolated from cattle and foods.

FEMS Microbiology Letters, 2005

This study reports the phenotypic and genotypic characterization of 144 Shiga toxin-producing Escherichia coli (STEC) strains isolated from urban sewage and animal wastewaters using a Shiga toxin 2 gene variant (stx 2 )-specific DNA colony hybridization method. All the strains were classified as E. coli and belonged to 34 different serotypes, some of which had not been previously reported to carry the stx 2 genes (O8:H31, O89:H19, O166:H21 and O181:H20). Five stx 2 subtypes (stx 2 , stx 2c , stx 2d , stx 2e and stx 2g ) were detected. The stx 2 , stx 2c , stx 2d and stx 2e subtypes were present in urban sewage and stx 2e was the only stx 2 subtype found in pig wastewater samples. The stx 2c and stx 2g were more associated with cattle wastewater. One strain was positive for the intimin gene (eae) and five strains of serotypes were positive for the adhesin encoded by the saa gene. A total of 41 different seropathotypes were found. On the basis of occurrence of virulence genes, most non-O157 STEC strains are assumed to be low-virulence serotypes. (C. García-Aljaro). www.fems-microbiology.org FEMS Microbiology Letters 246 (2005) 55-65

Journal of Clinical Microbiology, 2004

We have investigated 677 Shiga toxin-producing Escherichia coli (STEC) strains from humans to determine their serotypes, virulence genes, and clinical signs in patients. Six different Shiga toxin types (1, 1c, 2, 2c, 2d, and 2e) were distributed in the STEC strains. Intimin (eae) genes were present in 62.6% of the strains and subtyped into intimins ␣1, ␤1, ␥1, , , and . Shiga toxin types 1c and 2d were present only in eae-negative STEC strains, and type 2 was significantly (P < 0.001) more frequent in eae-positive STEC strains. Enterohemorrhagic E. coli hemolysin was associated with 96.2% of the eae-positive strains and with 65.2% of the eae-negative strains. Clinical signs in the patients were abdominal pain (8.7%), nonbloody diarrhea (59.2%), bloody diarrhea (14.3%), and hemolytic-uremic syndrome (HUS) (3.5%), and 14.3% of the patients had no signs of gastrointestinal disease or HUS. Infections with eae-positive STEC were significantly (P < 0.001) more frequent in children under 6 years of age than in other age groups, whereas eae-negative STEC infections dominated in adults. The STEC strains were grouped into 74 O:H types by serotyping and by PCR typing of the flagellar (fliC) genes in 221 nonmotile STEC strains. Eleven serotypes (accounted for 69% of all STEC strains. We identified 41 STEC strains belonging to 31 serotypes which had not previously been described as human STEC. Twenty-six of these were positive for intimins ␣1 (one serotype), ␤1 (eight serotypes), (two serotypes), and (three serotypes). Our study indicates that different types of STEC strains predominate in infant and adult patients and that new types of STEC strains are present among human isolates.

Global Veterinaria, 2009

Shiga toxin-producing Eschericia coli are diverse organisms inducing sever gastrointestinal and systematic diseases recognized worldwide. Ten serogroups were isolated, morphologically identified and biochemically characterized from 281 samples (fecal swabs and internal organs) of diarrheic field cases collected from cattle and ostrich private farms. Haemolysis, enterocytotoxicity, pathogenicity and antibiogram patterns were determined. All isolates were Stxs producers according to the cytopathic effects noticed in inoculated Vero cells monolayers with CD activity titers calculated 32 (O1 and bovine O157), 8 (O8 and O25), 64 (O20), 50 4 (O78) and 128 (O127, O146, O153, Ostrich O157) CD /100 µl. Dominant protein bands with apparent 50 molecular masses of 106 to109 kDa, 80-83 kDa, 54-56 kDa, 41 kDa, 37 kDa, 32 kDa, 26-25 kDa, 22 kDa and 7.8 kDa were separated by SDS-PAGE of E coli cultures supernatants. These bands were not uniformly expressed nor equally presented by all serotypes under study. In addition, 346 bp stx2 specified sequence were amplified by PCR in five serotypes only. None of the isolates were positive for stx1 coding sequences. It is clearly noticed that some serogroups are more virulent than others. Virulence properties depend on less characterized factors than Stx1 and Stx2 which has been proved from profiles of secretory proteins. It was concluded that STEC virulence is multifactorial process. However, Shiga toxins production, in particular Stx2, is a minimum requirement. Other secreted virulence proteins such as EspA (26-25 kDa), EspB (37 kDa), EspD (41 kDa), EspE (80-83 kDa), EspP (106-109 kDa) and Clostridium difficile-like toxins (32 kDa) are putative candidates involved in disease production their contribution in pathogenicity of isolated strains should be identified on both structural and functional levels.

Letters in Applied Microbiology, 2007

The expression of Stx1 and ⁄ or Stx2 genes characterizes Shiga toxin-producing Escherichia coli (STEC) strains, and their products function as major virulence factors. The only way to identify all types of STEC, in any kind of test sample, is the detection of these toxins produced by the bacteria or of the genes associated with Shiga toxin (Stx) production . described three categories of E. coli producing Shiga toxin, comprising trace-, low-to moderate-, and high-level producers of the toxin, and since this description, several distinct types of culture or enrichment media have been employed in STEC diagnosis in order to achieve detectable levels of Stx synthesis.

BMC Microbiology, 2008

Background: Shiga toxin-producing Escherichia coli (STEC) is an important cause of bloody diarrhoea (BD), non-bloody diarrhoea (NBD) and the haemolytic uraemic syndrome (HUS). In Argentina and New Zealand, the most prevalent STEC serotype is O157:H7, which is responsible for the majority of HUS cases. In Australia, on the other hand, STEC O157:H7 is associated with a minority of HUS cases. The main aims of this study were to compare the phenotypic and genotypic characteristics of STEC O157 strains isolated between 1993 and 1996 from humans in Argentina, Australia and New Zealand, and to establish their clonal relatedness. Results: Seventy-three O157 STEC strains, isolated from HUS (n = 36), BD (n = 20), NBD (n = 10), or unspecified conditions (n = 7) in Argentina, Australia and New Zealand, were analysed. The strains were confirmed to be E. coli O157 by biochemical tests and serotyping. A multiplex polymerase chain reaction (PCR) was used to amplify the stx 1 , stx 2 and rfb O157 genes and a genotyping method based on PCR-RFLP was used to determine stx 1 and stx 2 variants. This analysis revealed that the most frequent stx genotypes were stx 2 /stx 2c (vh-a) (91%) in Argentina, stx 2 (89%) in New Zealand, and stx 1 /stx 2 (30%) in Australia. No stx 1-postive strains were identified in Argentina or New Zealand. All strains harboured the eae gene and 72 strains produced enterohaemolysin (EHEC-Hly). The clonal relatedness of strains was investigated by phage typing and pulsed-field gel electrophoresis (PFGE). The most frequent phage types (PT) identified in Argentinian, Australian, and New Zealand strains were PT49 (n = 12), PT14 (n = 9), and PT2 (n = 15), respectively. Forty-six different patterns were obtained by XbaI-PFGE; 37 strains were grouped in 10 clusters and 36 strains showed unique patterns. Most clusters could be further subdivided by BlnI-PFGE. Conclusion: STEC O157 strains isolated in Argentina, Australia, and New Zealand differed from each other in terms of stx-genotype and phage type. Additionally, no common PFGE patterns were found in strains isolated in the three countries. International collaborative studies of the type reported here are needed to detect and monitor potentially hypervirulent STEC clones.

Japanese journal of infectious diseases, 2009

Shiga toxin-producing Escherichia coli (STEC) include O157:H7 and non-O157 serotypes. The public health impact of STEC infections is high because of their ability to cause severe infections. We characterized our STEC strains isolated from diarrheal and asymptomatic persons in northern and southwest Iran. The 29 STEC strains were examined for the presence of virulence genes by polymerase chain reaction (PCR) and their H type was analyzed by PCR-restriction fragment length polymorphism (RFLP) of the fliC gene. Moreover, the adherence properties of these strains were checked by HeLa cell adherence assay. The presence of non-O157 isolates under these conditions was again verified. The stx1 gene was present in 93% of the isolates, and the gene encoding intimin (eae) was not found to be present among the isolates. Almost all of the STEC isolates, with the exception of three, were non-adherent upon tissue culture assay. The serogrouping revealed the presence of seven different O types amon...

Applied and Environmental Microbiology, 2017

Shiga toxin-producing Escherichia coli (STEC) strains of the O91:H21 serotype have caused severe infections, including hemolytic-uremic syndrome. Strains of the O91 serogroup have been isolated from food, animals, and the environment worldwide but are not well characterized. We used a microarray and other molecular assays to examine 49 serogroup O91 strains (environmental, food, and clinical strains) for their virulence potential and phylogenetic relationships. Most of the isolates were identified to be strains of the O91:H21 and O91:H14 serotypes, with a few O91:H10 strains and one O91:H9 strain being identified. None of the strains had the eae gene, which codes for the intimin adherence protein, and many did not have some of the genetic markers that are common in other STEC strains. The genetic profiles of the strains within each serotype were similar but differed greatly between strains of different serotypes. The genetic profiles of the O91:H21 strains that we tested were identi...