Characteristion of virulence genes (Shigatoxins 1 and 2, and intimin) in Shiga toxin producing Escherichia coli (STEC) of ovine origin and an assessment of the role of these STEC in human pathogenesis
Doctor of Philosophy
University of Wollongong. Dept. of Biological Sciences
Ramachandran, Vidiya, Characteristion of virulence genes (Shigatoxins 1 and 2, and intimin) in Shiga toxin producing Escherichia coli (STEC) of ovine origin and an assessment of the role of these STEC in human pathogenesis, Doctor of Philosophy thesis, University of Wollongong. Dept. of Biological Sciences, University of Wollongong, 2002. http://ro.uow.edu.au/theses/1039
Enterohaemorrhagic Escherichia coli (EHEC), represent a subset of Shiga toxin-producing Escherichia coli (STEC), which cause diarrhoea, haemorrhagic colitis (HC) and haemorrhagic uraemic syndrome (HUS) in humans worldwide. STEC are part of the normal gastro-intestinal flora of ruminants, especially cattle and sheep, and commonly enter the food chain by the faecal contamination of carcasses at slaughter. Most studies of STEC in ruminants have focused on the bovine reservoir. This study examines the genetic characteristics of key virulence genes (stXl, SlX2 and eae) in STEC of ovine origin and a subset of STEC of human origin that possess same serotypes as those commonly recovered from sheep but not cattle.
Shiga toxins 1 and 2 are essential virulence attributes of STEC required for the induction of HUS and HC, and may play an important role in infections leading to milder gastrointestinal diseases such as diarrhoea. In this study a PCR restriction fragment length polymorphism (PCR-RFLP) assay was developed that differentiates SIXlc from other common SIXl subtypes. The SIXlc was the most common subtype identified (133 of 203; 65.5%) in STEC of ovine origin and was associated with 40 serotypes. Some serotypes, particularly 075:H8 (14 of 21 isolates) were shown to simultaneously possess both common SIXl and SIXlc subtypes. Furthermore, STEC isolates of serotypes commonly found in sheep and recovered from both symptomatic and healthy humans also contained SIXlc (12 of 34; 35.3%). These data suggest that these 12 isolates from humans may have had an ovine origin. The predominance of SIXlc among STEC isolated from ovine faeces suggests that the bacteriophage encoding this subtype preferentially inhabits the gastro-intestinal tract of sheep and/or shows a host range restricted to E. coli serotypes that colonise sheep but not cattle.
For the genetic characterisation of stX2 variants several previously published PCR-RFLP assays were used. The stX2d (stX2d-OuntJOIlllOX3a) subtypes, representing 13 serotypes, were almost exclusively identified (141 of 146; 96.6%) in STEC recovered from ovine faeces. These subtypes were predominantly associated with serotypes (05:H-, 091:H-, 0123 :Hand 0128:H2) commonly recovered from healthy sheep and rarely from healthy cattle. Furthermore, STEC isolates with serotypes predominantly associated with sheep and recovered from both symptomatic and healthy humans also contained these stX2d (11 of 21 ; 52.4%) subtypes suggesting that they probably had an ovine origin. A single isolate of serotype 091 :H21 recovered from a human with diarrhoea simultaneously possessed two stX2 variants (stX2 and stx2vhb).
Ovine STEC possessing stXlc and StX2d subtypes were never associated with the typical EHEC serogroups 026, 0103 and 0157. Recent clinical studies have demonstrated that StXlc and StX2d subtypes are rarely associated with STEC recovered from patients with HUS or He. However, examples of human STEC isolates of serotypes 05:H- and OX3:H8 associated with HUS that do possess this combination of virulence factor subtypes were identified in this study.
Intimin (lnt), encoded by the eae gene, is a well characterised outer membrane protein adhesin involved in the intimate adherence of STEC to the host epithelial membrane leading to the formation of the characteristic attaching and effacing lesions. A PCR-RFLP subtyping assay capable of simultaneously differentiating all 10 recognised intimin subtypes is reported for the first time in this study. This assay was also used to identify and type two previously unreported intimin subtypes identified as Int-£2 and Int-t2. Int-~ was the most commonly identified intimin subtype (58 of 153; 13.7%) and was associated with the greatest number ofserotypes (n=16), followed by Int-s (21 of 153; 13.7%; 7 serotypes), Int-£l (18 of 153; 11.7%; 5 serotypes), Int-y(13 of 153; 8.5%; 6 serotypes) and Int-8 (6 of 153; 4%; 5 serotypes). Intimin subtypes aI, a2, A, 8 and t1 were infrequently identified. None of the 153 eae-containing isolates simultaneously harboured more than one inti min subtype. However, intimin genes from 19 of 153 (12.4%) ovine E. coli isolates representing 16 different serotypes were untypeable suggesting an even greater variety of intimin subtypes in STEC derived from ovine faeces. Phylogenetic analyses of the C-terminal 280 amino acids (lnt280) using the Phylip package confirmed the previous division of the family of intimin proteins into the six distinct clusters represented by subtypes a, ~, y, 8, £ and 8. In addition, the inti min subtypes S, t and A submitted to GenBank, also resolved as distinct groups but their relationship to other intimin subtypes remain unclear.
The clonal relationships of ovine and human STEC isolates of serotypes (05:H-, 091 :Hand 0128:H2) were assessed by pulsed-field gel electrophoresis (PFGE) and repetitive sequence based PCR (REP and ERIC PCR). PFGE differentiated 11, 13 and 13 groups among the sixteen 05:H-, seventeen 091:H- and eighteen 0128:H2 strains respectively suggesting that STEC with these serotypes represent genetically heterogenous groups. There were no matches observed in the PFGE profiles between strains of STEC isolated from sheep and those isolated from humans. However, one 091 :H- isolate obtained from mettwurst sausage and an 091 :H- isolate from a patient with diarrhoea produced identical PFGE patterns and both the isolates were associated with a food poisoning outbreak in South Australia. This suggests that PFGE may be a useful epidemiological tool for tracing non-O 157 STEC infections. Genetic fingerprinting using REP and ERIC PCR showed a low discriminative ability for these isolates and appears unsuitable for this purpose.
Serotypes 05:H-, 091:H-, 0103:H2, 0123:H-, 0128:H2, 0157:H-1H7 and OX3:H8 have been isolated from sheep faeces and have been recovered from patients with HC and HUS. With the exception of 0157:H-1H7, the other serotypes have never been associated with outbreaks of these diseases and are only rarely recovered from sporadic cases of HC and HUS. The data obtained in this study show that sheep are a reservoir of STEC that possess Shiga toxin subtypes (stXlc and stX2d) that are rarely associated with HC and HUS. While EHEC serogroups such as 026, 0103, 0111 and 0157 are important causal serogroups in HUS, the role of other less common non-O 157 serogroups remains to be clearly defined but is probably underestimated . The subtyping assays developed in this study will play an important role in the future characterisation of STEC and will be useful tools to clarify the role ofnon-0157 STEC in human disease.