Bovine immune response to shiga-toxigenic Escherichia coli O157:H7

Although cattle develop humoral immune responses to Shiga-toxigenic (Stx+) Escherichia coli O157:H7, infections often result in long-term shedding of these human pathogenic bacteria. The objective of this study was to compare humoral and cellular immune responses to Stx+ and Stx- E. coli O157:H7. Three groups of calves were inoculated intrarumenally, twice in a 3-week interval, with different strains of E. coli: a Stx2-producing E. coli O157:H7 strain (Stx2+ O157), a Shiga toxin-negative E. coli O157:H7 strain (Stx- O157), or a nonpathogenic E. coli strain (control). Fecal shedding of Stx2+ O157 was significantly higher than that of Stx- O157 or the control. Three weeks after the second inoculation, all calves were challenged with Stx2+ O157. Following the challenge, levels of fecal shedding of Stx2+ O157 were similar in all three groups. Both groups inoculated with an O157 strain developed antibodies to O157 LPS. Calves initially inoculated with Stx- O157, but not those inoculated with Stx2+ O157, developed statistically significant lymphoproliferative responses to heat-killed Stx2+ O157. These results provide evidence that infections with STEC can suppress the development of specific cellular immune responses in cattle, a finding that will need to be addressed in designing vaccines against E. coli O157:H7 infections in cattle.     

Metabolic and genetic profiling of clinical O157 and non-O157 Shiga-toxin-producing Escherichia coli

A collection of clinical Shiga-toxin-producing Escherichia coli (STEC) strains, mainly belonging to serotypes O26, O103, O111, O145 and O157, was characterised by a polyphasic approach including molecular serotyping, PCR-based detection of virulence factors (stx1, stx2, eae, EHEC-hlyA, saa, katP, espP), carbohydrate fermentation profiles using API50 tests and random amplification of polymorphic DNA (RAPD) fingerprinting. An RAPD protocol based on the combination of 2 primers resulted in sufficiently complex patterns enabling discrimination to the serotype level. Moreover, carbohydrate fermentation profiles obtained after evaluating up to 50 different carbohydrates led to separation of different STEC serotypes. Virulence typing results confirm the association of Shiga toxins and intimin subtypes with specific serotypes and clinical diagnosis. Clinical diagnosis of strains did not correlate with either RAPD profiles or carbohydrate fermentation patterns.     

The Non-O157 Shiga-Toxigenic (Verocytotoxigenic) Escherichia coli; Under-Rated Pathogens

Following a brief review of the ecology of Escherichia coli in general, the role of Shiga-Toxigenic (Verocytotoxigenic) E. coli (STEC) as pathogens is addressed. While STEC belonging to the serogroup O157 have been extensively studied and shown to be involved in many cases and outbreaks of human disease, the importance of STEC belonging to other serogroups has not been recognized as much. This review addresses the problems associated with these pathogens, demonstrating that increasing the awareness of them is a major part of the problem. This review then demonstrates how widespread isolations especially from food animals and human disease have been, discussing in particular STEC belonging to serogroups O8, O26, O103, O111, O113 and O128. The animal host-specificity of these STEC is also reviewed. In conclusion some methods of improving isolation of these pathogens is addressed.     

Prolonged and mixed non-O157 Escherichia coli infection in an Australian household

An Australian family was identified through a Public Health follow up on a Shiga-toxigenic Escherichia coli (STEC) positive bloody diarrhoea case, with three of the four family members experiencing either symptomatic or asymptomatic STEC shedding. Bacterial isolates were submitted to stx sequence sub-typing, multi-locus variable number tandem repeat analysis (MLVA), multi-locus sequence typing (MLST) and binary typing. The analysis revealed that there were multiple strains of STEC being shed by the family members, with similar virulence gene profiles and the same serogroup but differing in their MLVA and MLST profiles. This study illustrates the potentially complicated nature of non-O157 STEC infections and the importance of molecular epidemiology in understanding disease clusters.     

Enterohaemorrhagic Escherichia coli O157 and non-O157 serovars differ in their mechanisms for iron supply

Clinical isolates of enterohaemorrhagic Escherichia coli, both O157 and non-O157 serotypes, were investigated for siderophore production, for growth promotion by haem and esculetin in iron-restricted conditions, for production of enterohaemolysin and esculin hydrolase, and for the presence of the chuA and ehx genes by PCR. As expected, all the strains produced enterobactin, but the prevalence of other factors varied among the serovars tested. None of the O157 and O26 strains produced aerobactin or "colibactin", whereas among other enterohaemorrhagic E. coli non-O157 serovars the frequencies of aerobactin and "colibactin" production were similar to those of commensal E. coli strains. The ability to use ferric esculetin for growth in iron-limited media was markedly more prevalent among non-O157 serovars and less prevalent among O157 strains compared with commensal E. coli strains. Almost all O157, O26 and O103 strains expressed enterohaemolysin, compared with only 50% of other non-O157 strains. Similarly, almost all O157 and O26 strains utilised haem as a host iron source; the frequency of haem use by other non-O157 strains was generally lower and variable among serovars, such that none of the O103:H2 isolates tested used haem as an iron source. The gene chuA, which encodes the haem transport protein ChuA and which is prevalent in O157:H7 strains, was only rarely noted among non-O157 serovars of enterohaemorrhagic E. coli, even among isolates that could use haem as an iron source. Overall our data demonstrate that O157:H7 and non-O157 serovars, in particular O26:H(-)/H11 and O103:H2, use distinctly different strategies for obtaining iron, and suggest two evolutionary distinct lines of enterhaemorrhagic E. coli.     

Comparative pathogenicity of Escherichia coli O157 and intimin-negative non-O157 Shiga toxin-producing E coli strains in neonatal pigs

We compared the pathogenicity of intimin-negative non-O157:H7 Shiga toxin (Stx)-producing Escherichia coli (STEC) O91:H21 and O104:H21 strains with the pathogenicity of intimin-positive O157:H7 and O157:H(-) strains in neonatal pigs. We also examined the role of Stx2d-activatable genes and the large hemolysin-encoding plasmid of O91:H21 strain B2F1 in the pathogenesis of STEC disease in pigs. We found that all E. coli strains that made wild-type levels of Stx caused systemic illness and histological lesions in the brain and intestinal crypts, whereas none of the control Stx-negative E. coli strains evoked comparable central nervous system signs or intestinal lesions. By contrast, the absence of intimin, hemolysin, or motility had little impact on the overall pathogenesis of systemic disease during STEC infection. The most striking differences between pigs inoculated with non-O157 STEC strains and pigs inoculated with O157 STEC strains were the absence of attaching and effacing intestinal lesions in pigs inoculated with non-O157:H7 strains and the apparent association between the level of Stx2d-activatable toxin produced by an STEC strain and the severity of lesions.     

Virulence inhibition by zinc in shiga-toxigenic Escherichia coli

Previously, our laboratories reported that zinc inhibited expression of several important virulence factors in enteropathogenic Escherichia coli (EPEC) and reduced EPEC-induced intestinal damage in vivo. Since EPEC is genetically related to Shiga-toxigenic E. coli (STEC), we wondered whether the beneficial effects of zinc extended to STEC as well. Treatment options for STEC infection are very limited, since antibiotics tend to exacerbate disease via enhanced toxin production, so a safe intervention for this infection would be welcome. In this study, we report that in STEC strains zinc inhibits adherence to cultured cells as well as expression of EHEC secreted protein A (EspA). In addition, zinc inhibits the expression of Shiga toxin (Stx) at both the protein and the RNA level. Zinc inhibits basal and antibiotic-induced Stx production and inhibits both Stx1 and Stx2 by ≥90% at a concentration of 0.4 mM zinc. Rabbit EPEC strains were selected for acquisition of Stx-encoding bacteriophages, and these rabbit STEC strains (designated RDEC-H19A and E22-stx2) were used to test the effects of zinc in vivo in ligated rabbit intestinal loops. In vivo, zinc reduced fluid secretion into loops, inhibited mucosal adherence, reduced the amount of toxin in the loops, and reduced STEC-induced histological damage (villus blunting). Zinc has beneficial inhibitory effects against STEC strains that parallel those observed in EPEC. In addition, zinc strongly inhibits Stx expression; since Stx is responsible for the extraintestinal effects of STEC infection, such as hemolytic-uremic syndrome (HUS), zinc might be capable of preventing severe sequelae of STEC infection.     

Isolation and genetic characterization of a coinfection of non-O157 Shiga toxin-producing Escherichia coli

A coinfection of O177:NM and O55:H7 Shiga toxin-producing Escherichia coli (STEC) was identified for a child with acute bloody diarrhea and hemolytic uremic syndrome by using culture and serotype-specific molecular reagents. The profile of O157-related genetic islands revealed that the O55:H7 isolate was highly similar to O157 STEC whereas the O177:NM isolate lacked several fimbrial O islands and non-locus-of-enterocyte-effacement effector determinants. However, both STEC serotypes are known to cause serious disease, and the significant repertoire of virulence determinants in both strains made it impossible to determine their individual contributions to the clinical symptoms.     

TccP2 of O157:H7 and non-O157 enterohemorrhagic Escherichia coli (EHEC): challenging the dogma of EHEC-induced actin polymerization

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 and enteropathogenic E. coli (EPEC) trigger actin polymerization at the site of bacterial adhesion by inducing different signaling pathways. Actin assembly by EPEC requires tyrosine phosphorylation of Tir, which subsequently binds the host adaptor protein Nck. In contrast, Tir(EHEC O157) is not tyrosine phosphorylated and instead of Nck utilizes the bacterially encoded Tir-cytoskeleton coupling protein (TccP)/EspF(U), which mimics the function of Nck. tccP is carried on prophage CP-933U/Sp14 (TccP). Typical isolates of EHEC O157:H7 harbor a pseudo-tccP gene that is carried on prophage CP-933 M/Sp4 (tccP2). Here we report that atypical, beta-glucuronidase-positive and sorbitol-fermenting, strains of EHEC O157 harbor intact tccP and tccP2 genes, both of which are secreted by the LEE-encoded type III secretion system. Non-O157 EHEC strains, including O26, O103, O111, and O145, are typically tccP negative and translocate a Tir protein that encompasses an Nck binding site. Unexpectedly, we found that most clinical non-O157 EHEC isolates carry a functional tccP2 gene that encodes a secreted protein that can complement an EHEC O157:H7 DeltatccP mutant. Using discriminatory, allele-specific PCR, we have demonstrated that over 90% of tccP2-positive non-O157 EHEC strains contain a Tir protein that can be tyrosine phosphorylated. These results suggest that the TccP pathway can be used by both O157 and non-O157 EHEC and that non-O157 EHEC can also trigger actin polymerization via the Nck pathway.     

Genetic diversity of O157:H7 and non-O157 verocytotoxigenic Escherichia coli from Argentina inferred from multiple-locus variable-number tandem repeat analysis (MLVA)

Although serotype O157:H7 has been implicated in most cases of haemolytic-uraemic syndrome (HUS), there is growing concern about non-O157 serotypes of verocytotoxigenic Escherichia coli (VTEC). Multiple-locus variable-number tandem repeat analysis (MLVA) has been focused on the specific typing of O157:H7 isolates, but recently, a generic MLVA assay for E. coli and Shigella has been developed. We performed a study of the polymorphism in 7 generic VNTR loci both in VTEC O157:H7 and non-O157 isolates from Argentina, in order to asses the ability of the method to type this group of isolates and to get insight into their genetic diversity. Sixty-four isolates from cattle, patients with diarrhoea, and contaminated food belonging to 8 different serotypes were studied. All of them could be typed by this method and revealed 41 different MLVA genotypes. The MLVA dendrogram showed 2 main clusters which corresponded to O157:H7 and non-O157, respectively. Our results confirm the suitability of this MLVA method for analyzing VTEC isolates belonging to several serotypes, both O157:H7 as well as non-O157, highlight the genetic variability of the O157:H7 serotype and the need of additonal research in order to find more VNTR loci that could allow a higher discrimination among non-O157 VTEC.     

Identification and characterisation of Escherichia coli strains of O157 and non-O157 serogroups containing three distinct Shiga toxin genes

Three Shiga toxin (Stx)-producing Escherichia coli (STEC) strains from patients with diarrhoea were identified, each of which contained three distinct stx genes (stx1, stx2 and stx2c). The strains belonged to the serotypes O52:H19, O75:H- and O157:H- and harboured eae and EHEC-hly sequences. Colony-blot immunoassay was used to demonstrate that both major types of Stx were expressed. The association of stx genes with either phage or phage DNA was demonstrated in all three strains. Isolated phage DNA from all strains contained stx1 sequences, but stx2 sequences were found only in phage DNA of two of these strains. The presence of three distinct stx genes may enhance the virulence of STEC strains and should be monitored. The observations demonstrate not only the potential of stx genes to spread within different serotypes, but also their capacity to accumulate within a single strain.     

Urease genes in non-O157 Shiga toxin-producing Escherichia coli: mostly silent but valuable markers for pathogenicity

The distribution of ureC was investigated among 294 Escherichia coli isolates, comprising 72 strains from the E. coli standard reference collection (ECOR), 62 strains from the diarrhoeagenic E. coli (DEC) collection, and 160 clinical isolates of Shiga toxin-producing E. coli (STEC). The ureC gene was more frequent among STEC isolates harbouring eae than among those lacking eae (p < 0.0001). All clinical STEC isolates of serogroups O111 and O145 contained ureC, but only two of 294 isolates expressed urease activity. The silencing of urease expression could not be linked to a stop codon in ureD. The frequent occurrence of ure genes in eae-positive STEC isolates makes them valuable markers for virulence.     

非O157产志贺毒素大肠杆菌分离株亚碲酸钾抗性水平及抗性基因簇分析

目的 了解我国部分地区非O157产志贺毒素大肠杆菌(Shiga toxin-producing Escherichia coli,STEC)分离株的亚碲酸钾抗性水平、抗性基因簇及其关系.方法 使用平皿法检测亚碲酸钾抗性水平,采用PCR方法检测亚碲酸钾抗性基因簇.结果 在所检测的39株非O157 STEC中,仅有5株菌亚碲酸钾抗性水平介于128～512 μg/ml,同时携带亚碲酸钾抗性基因簇(terABCDE).另有2株菌的亚碲酸钾抗性水平为8 μg/ml,3株菌为2 μg/ml,其余29株菌均＜1 μg/ml,且这34株菌terABCDE均阴性.结论 大多数非O157 STEC分离株对亚碲酸钾敏感,在使用含亚碲酸钾的选择性培养基分离非O157 STEC时应慎重.     

Microevolution of epidemiological highly relevant non-O157 enterohemorrhagic Escherichia coli of serogroups O26 and O111

Enterohemorrhagic Escherichia coli (EHEC) are a cause of bloody diarrhea, hemorrhagic colitis (HC) and the potentially fatal hemolytic uremic syndrome (HUS). While O157:H7 is the dominant EHEC serotype, non-O157 EHEC have emerged as serious causes of disease. In Germany, the most important non-O157 O-serogroups causing one third of EHEC infections, including diarrhea as well as HUS, are O26, O103, O111 and O145. Interestingly, we identified EHEC O-serogroups O26 and O111 in one single sequence type complex, STC29, that also harbours atypical enteropathogenic E. coli (aEPEC). aEPEC differ from typical EHEC merely in the absence of stx-genes. These findings inspired us to unravel a putative microevolutionary scenario of these non-O157 EHEC by whole genome analyses.Analysis of single nucleotide polymorphisms (SNPs) of the maximum common genome (MCG) of 20 aEPEC (11 human/ 9 bovine) and 79 EHEC (42 human/ 36 bovine/ 1 food source) of STC29 identified three distinct clusters: Cluster 1 harboured strains of O-serogroup O111, the central Cluster 2 harboured only O26 aEPEC strains, while the more heterogeneous Cluster 3 contained both EHEC and aEPEC strains of O-serogroup O26. Further combined analyses of accessory virulence associated genes (VAGs) and insertion sites for mobile genetic elements suggested a parallel evolution of the MCG and the acquisition of virulence genes. The resulting microevolutionary model suggests the development of two distinct EHEC lineages from one common aEPEC ancestor of ST29 by lysogenic conversion with stx-converting bacteriophages, independent of the host species the strains had been isolated from.In conclusion, our cumulative data indicate that EHEC of O-serogroups O26 and O111 of STC29 originate from a common aEPEC ancestor and are bona fide zoonotic agents. The role of aEPEC in the emergence of O26 and O111 EHEC should be considered for infection control measures to prevent possible lysogenic conversion with stx-converting bacteriophages as major vehicle driving the emergence of EHEC lineages with direct Public Health consequences.     

Characterization of cytolethal distending toxin genes and expression in shiga toxin-producing Escherichia coli strains of non-O157 serogroups

We identified cytolethal distending toxin and its gene (cdt) in 17 of 340 non-O157 Shiga toxin-producing Escherichia coli (STEC) strains (serotypes O73:H18, O91:H21, O113:H21, and O153:H18), all of which were eae negative. cdt is either chromosomal and homologous to cdt-V (serotypes O73:H18, O91:H21, and O113:H21) or plasmidborne and identical to cdt-III (serotype O153:H18). Among eae-negative STEC, cdt was associated with disease (P = 0.003).     

Two distinct cytotoxic activities of subtilase cytotoxin produced by shiga-toxigenic Escherichia coli

Subtilase cytotoxin (SubAB) is a recently identified AB5 subunit toxin produced by Shiga-toxigenic Escherichia coli. The A subunit is thought to be a subtilase-like, serine protease, whereas the B subunit binds to the toxin receptor on the cell surface. We cloned the genes from a clinical isolate; the toxin was produced as His-tagged proteins. SubAB induced vacuolation at concentrations greater than 1 microg/ml after 8 h, in addition to the reported cytotoxicity induced at a ng/ml level after 48 h. Vacuolation was induced with the B, but not the A, subunit and was dependent on V-type ATPase. The cytotoxicity of SubAB at low concentrations was associated with the inhibition of protein synthesis; the 50% inhibitory dose was approximately 1 ng/ml. The A subunit, containing serine 272, which is thought to be a part of the catalytic triad of a subtilase-like serine protease, plus the B subunit was necessary for this activity, both in vivo and in vitro. SubAB did not cleave azocasein, bovine serum albumin, ovalbumin, or synthetic peptides. These data suggest that SubAB is a unique AB toxin: first, the B subunit alone can induce vacuolation; second, the A subunit containing serine 272 plus the B subunit inhibited protein synthesis, both in vivo and in vitro; and third, the A subunit proteolytic activity may have a strict range of substrate specificity.     

Antimicrobial resistance of Shiga toxin (verotoxin)-producing Escherichia coli O157:H7 and non-O157 strains isolated from humans, cattle, sheep and food in Spain

A total of 722 Shiga toxin-producing Escherichia coli (STEC) isolates recovered from humans, cattle, ovines and food during the period from 1992 to 1999 in Spain were examined to determine antimicrobial resistance profiles and their association with serotypes, phage types and virulence genes. Fifty-eight (41%) out of 141 STEC O157:H7 strains and 240 (41%) out of 581 non-O157 STEC strains showed resistance to at least one of the 26 antimicrobial agents tested. STEC O157:H7 showed a higher percentage of resistant strains recovered from bovine (53%) and beef meat (57%) than from human (23%) and ovine (20%) sources, whereas the highest prevalence of antimicrobial resistance in non-O157 STEC was found among isolates recovered from beef meat (55%) and human patients (47%). Sulfisoxazole (36%) had the most common antimicrobial resistance, followed by tetracycline (32%), streptomycin (29%), ampicillin (10%), trimethoprim (8%), cotrimoxazole (8%), chloramphenicol (7%), kanamycin (7%), piperacillin (6%), and neomycin (5%). The multiple resistance pattern most often observed was that of streptomycin, sulfisoxazole, and tetracycline. Ten (7%) STEC O157:H7 and 71 (12%) non-O157 strains were resistant to five or more antimicrobial agents. Most strains showing resistance to five or more antimicrobial agents belonged to serotypes O4:H4 (4 strains), O8:H21 (3 strains), O20:H19 (6 strains), O26:H11 (8 strains eae-beta1), O111:H- (3 strains eae-gamma2), O118:H- (2 strains eae-beta1), O118:H16 (5 strains eae-beta1), O128:H- (2 strains), O145:H8 or O145:H- (2 strains eae-gamma1), O157:H7 (10 strains eae-gamma1), O171:H25 (3 strains), O177:H11 (5 strains eae-beta1), ONT:H- (3 strains/1 eae-beta1) and ONT:H21 (2 strains). Interestingly, most of these serotypes, i.e., those indicated in bold) were found among human STEC strains isolated from patients with hemolytic uremic-syndrome (HUS) reported in previous studies. We also detected, among non-O157 strains, an association between a higher level of multiple resistance to antibiotics and the presence of the virulence genes eae and stx(1). Moreover, STEC O157:H7, showed an association between certain phage types, PT21/28 (90%), PT23 (75%), PT34 (75%), and PT2 (54%), with a higher number of resistant strains. We conclude that the high prevalence of antimicrobial resistance detected in our study is a source of concern, and cautious use of antibiotics in animals is highly recommended.     

Shiga toxin-producing Escherichia coli in children: diagnosis and clinical manifestations of O157:H7 and non-O157:H7 infection

Shiga toxin-producing Escherichia coli (STEC), a cause of food-borne colitis and hemolytic-uremic syndrome in children, can be serotype O157:H7 (O157) or other serotypes (non-O157). E. coli O157 can be detected by culture with sorbitol-MacConkey agar (SMAC), but non-O157 STEC cannot be detected with this medium. Both O157 and non-O157 STEC can be detected by immunoassay for Shiga toxins 1 and 2. The objectives of this study were first to compare the diagnostic utility of SMAC to that of the Premier EHEC enzyme immunoassay (Meridian Diagnostics) for detection of STEC in children and second to compare the clinical and laboratory characteristics of children with serotype O157:H7 STEC and non-O157:H7 STEC infections. Stool samples submitted for testing for STEC between April 2004 and September 2009 were tested by both SMAC culture and the Premier EHEC assay at Children's Hospital Boston. Samples positive by either test were sent for confirmatory testing and serotyping at the Hinton State Laboratory Institute (HSLI). Chart review was performed on children with confirmed STEC infection. Of 5,110 children tested for STEC, 50 (0.9%) had STEC infection confirmed by culture; 33 were O157:H7 and 17 were non-O157:H7. The Premier EHEC assay and SMAC culture detected 96.0% and 58.0% of culture-confirmed STEC isolates (any serotype), respectively, and 93.9% and 87.9% of STEC O157:H7 isolates, respectively. There were no significant differences in disease severity or laboratory manifestations of STEC infection between children with O157:H7 and those with non-O157 STEC. The Premier EHEC assay was significantly more sensitive than SMAC culture for diagnosis of STEC, and O157:H7 and non-O157:H7 STEC caused infections of similar severity in children.     

Virulence genes in verocytotoxigenic Escherichia coli strains isolated from humans and cattle

Verocytotoxigenic Escherichia coli (VTEC) causing diarrhoea, haemorrhagic colitis and haemolytic-uremic syndrome usually have additional traits such as the adhesin intimin and a large plasmid that seems to increase virulence. There are, however, isolates of VTEC causing serious symptoms that do not harbour these traits. In the present study we have used PCR with primers detecting adhesin genes other than eaeA, namely fimA, papC, sfaD/sfaE and daaE. We have also used PCR to detect the genes hlyA and iutA that besides the plasmid-borne gene E-hly possibly support the bacterial access to iron. The aim of the study was to identify and compare the presence of virulence genes in VTEC isolates of human and cattle origin. The main finding was that the absence of E-hly might be compensated for by the gene iutA coding for aerobactin or hlyA coding for alpha-haemolysin as 94% of the human VTEC isolates had at least one of these genes. Interestingly, only 45% of VTEC isolated from cattle had any of these genes. We propose that this might be the reason for the relatively low incidence of symptomatic VTEC infections among humans in relation to the high number of VTEC among cattle.