Virulence markers and serotypes of Shiga toxin-producing Escherichia coli, isolated from cattle in Rio Grande do Sul, Brazil

AIMS: To determine the prevalence of Shiga toxin-producing Escherichia coli (STEC) and serotypes and virulence markers of the STEC isolates from beef and dairy cattle in Rio Grande do Sul, Brazil. METHODS AND RESULTS: Faecal samples from beef cattle were collected at slaughterhouses. The isolates were submitted to colony hybridization assay with specific DNA probes for stx1, stx2 and eae genes, and serotyped for the identification of O and H antigens. Thirty-nine per cent of beef cattle surveyed harboured at least one STEC strain. Among the distinct serotypes identified, 10 were shared by both beef and dairy cattle. Most of the strains isolated harboured stx2. Genotypic and phenotypic profiles allowed the identification of 34 and 31 STEC strains, isolated from beef and dairy cattle, respectively. Serotypes O10:H14, O15:H21, O96:H21, O119:H4, O124:H11, O128:H21, O137:H-, O141:H19, O159:H42, O160:H2 and O177:H11, identified in this study, have not been previously reported as STEC isolated from cattle. CONCLUSIONS: Cattle are an important reservoir of STEC strains associated with human diseases in South America. SIGNIFICANCE AND IMPACT OF THE STUDY: Determining the prevalence, genotypic profile and serotypes of STEC strains isolated from cattle enables the prediction of possible risk for public health.     

Isolation and characterization of Shiga toxin-producing Escherichia coli strains from raw milk cheeses in France

AIMS: To evaluate Shiga toxin-producing Eschericha coli (STEC) prevalence in 1039 French raw milk cheeses including soft, hard, unripened and blue mould cheeses, and to characterize the STEC strains isolated (virulence genes and serotypes). METHODS AND RESULTS: STEC strains were recovered from cheese samples by colony hybridization. These strains were then serotyped and genetically characterized. These strains (32 STEC) were then recovered from 18 of 136 stx-positive samples: 19 strains had stx2 variant genes stx(2vh-a) (n = 2), stx(2NV206) (n = 2), stx(2EDL933) (n = 4) and stx2d (n = 11). Thirty strains had the stx1 gene and one strain, the eae gene. Combinations of stx2 and stx1 genes were present in 17 (81%) of the STEC strains. Nineteen strains belonged to the O6 serogroup and the other strains belonged to the O174, O175, O176, O109, O76, O162 and O22 serogroups in decreasing frequency. CONCLUSIONS: No conclusion can be drawn at the moment concerning the potential risk to consumers because the O6:H1 serotype has already been found associated with the haemolytic uremic syndrome and almost no isolate had the eae gene. SIGNIFICANCE AND IMPACT OF THE STUDY: The large number of STEC strains recovered from the cheese samples evaluated in this study emphasizes the health risks associated with raw milk cheeses. This further emphasizes the immediate need to identify and implement effective pre- and postharvest control methods that decrease STEC carriage by dairy cattle and to eliminate contamination of their cheeses during processing.     

Animal host associated differences in Shiga toxin-producing Escherichia coli isolated from sheep and cattle on the same farm

AIMS: To investigate if cattle on the same farm as sheep are a possible risk factor for stx in sheep and to determine whether or not sheep and cattle on the same farm share the same stx pool. METHODS AND RESULTS: Faecal samples from sheep and cattle were screened for stx by polymerase chain reaction (PCR). Of these samples, 87.6 and 64.6% were stx positive in sheep and cattle, respectively. There was no difference in stx occurrence in sheep from farms with or without cattle. From stx positive samples, 118 Shiga toxin-producing Escherichia coli (STEC) isolates were recovered by a filter-hybridization method. Serotyping, PCR and pulsed-field gel electrophoresis (PFGE) showed that there was a distinct association between serotypes, stx profiles and animal species. CONCLUSIONS: Keeping animals together in pens, which enhances faecal-oral contact, is suggested as a possible explanation for the differences seen in stx occurrence. Sheep and cattle isolates are distinctly different in serotype and stx profile although isolated from the same farm, and are more related to isolates within the same serotype with the same stx profile than to isolates with different serotype from the same farm. SIGNIFICANCE AND IMPACT OF STUDY: The study supports the animal-host relationship hypothesis suggested in other studies and indicates that the STEC sheep reservoir in Norway may not pose a serious public health risk.     

Stx genotypes and antimicrobial resistance profiles of Shiga toxin-producing Escherichia coli strains isolated from human infections, cattle and foods in Brazil

A total of 107 Shiga toxin-producing Escherichia coli strains (STEC) isolated from different origins in S?o Paulo, Brazil, and belonging to different serotypes were characterized regarding stx subtypes and susceptibility to antimicrobial agents. Most of the human STEC strains harbored stx1 (85.7%), while stx2, associated or not to stx1, was identified preferentially in the animal and food strains. None of the STEC strains carried stx1c. Some genotypes occurred exclusively among strains of bovine origin as stx2c, stx1+2+2c (16.5% each), and stx2d (0.9%), whereas stx2+2c2vha) was only identified among the O157:H7 human strains. Moreover, the stx(2c2vhb) subtype was found more frequently among bovine than human strains (39% vs. 4.8%). The highest frequencies of susceptibility to antimicrobial agents were observed among bovine (87%) and food (100%) STEC strains, while 47.6% of the human isolates were resistant to at least one drug. Multiresistance occurred among O111 STEC strains from human and bovine origin. The antimicrobials to which resistance was most frequently observed were tetracycline (90%) and streptomycin (75%) among human strains, and also sulphazotrin (88%) in animal strains. A few serotypes were commonly identified among STEC strains isolated from diverse sources in Brazil, but in general the strains presented distinct stx subtypes and/or antimicrobial resistance profiles.     

Sensitivity of Shiga toxin-producing Escherichia coli (STEC) strains for colicins under different experimental conditions

Twenty Escherichia coli strains producing well-characterised colicins were tested for their inhibitory activity against five Shiga toxin-producing E. coli (STEC) strains using different media under aerobic and anaerobic conditions. The five STEC strains used were of serotype O26, O111, O128, O145 and O157:H7 which are frequently isolated serotypes associated with disease in humans. The main route of infection for humans is through the eating of badly cooked or handled beef. The major reservoir for STEC strains in cattle is the rumen. To mimic the situation in the rumen of cattle, overlay assays were also performed under anaerobic conditions in the presence of 30% rumen fluid. Colicins E1, E4, E8-J, K and S4 are most active against STEC strains under anaerobic conditions in the absence or presence of rumen fluid. These colicins will be used in future experiments with the aim to eradicate the presence of STEC in cattle.     

Subtyping of Shiga toxin 2 variants in human-derived Shiga toxin-producing Escherichia coli strains isolated in Japan

Shiga toxin 2 (Stx2) variants have been found to exhibit not only antigenic divergence, but also differences in toxicity for tissue culture cells and animals. To clarify whether all or just a subset of Stx2 variants are important for the virulence of Shiga toxin-producing Escherichia coli, we designed PCR primers to detect and type all reported variants. We classified them into four groups according to the nucleotide sequences of the Stx2 family; for example, group 1 (G1) contains VT2vha and group 2 (G2) contains VT2d-Ount. The 120 strains of Shiga toxin-producing E. coli used in this study were isolated from humans in Japan between 1986 and 1999. Among the four variant groups, the G1 gene only was detected in 23 of the 120 clinical strains (19.2%) and all belonged to the O157 serotype. G1 is considered the most important Stx2 variant group in terms of human pathogenicity. A multiplex PCR that can detect the stx1, stx2, and G1 genes was developed as a means of rapid and easy typing to better understand the roles of the different types of Stx.     

Shiga toxin-producing Escherichia coli and atypical enteropathogenic Escherichia coli strains isolated from healthy sheep of different populations in São Paulo, Brazil

Aims:  Sheep are important carriers of Shiga toxin-producing Escherichia coli (STEC) in several countries. However, there are a few reports about ovine STEC in American continent.
Methods and Results:  About 86 E. coli strains previously isolated from 172 healthy sheep from different farms were studied. PCR was used for detection of stx ₁, stx ₂, eae, ehxA and saa genes and for the identification of intimin subtypes. Restriction fragment length polymorphism (RFLP)–PCR was performed to investigate the variants of stx ₁ and stx ₂, and the flagellar antigen ( fli C) genes in nonmotile isolates. Five isolates were eae ⁺ and stx ⁻, and belonged to serotypes O128:H2/β-intimin (2), O145:H2/γ, O153:H7/β and O178:H7/ε. Eighty-one STEC isolates were recovered, and the stx genotypes identified were stx _1c stx _2d-O118 (46·9%), stx _1c (27·2%), stx _2d-O118 (23·4%), and stx _1c stx _2dOX3a (2·5%). Pulsed-field gel electrophoresis (PFGE) revealed 27 profiles among 53 STEC and atypical enteropathogenic Escherichia coli (EPEC) isolates.
Conclusions:  This study demonstrated that healthy sheep in São Paulo, Brazil, can be carriers of potential human pathogenic STEC and atypical EPEC.
Significance and Impact of the Study:  As some of the STEC serotypes presently found have been involved with haemolytic uraemic syndrome (HUS) in other countries, the important role of sheep as sources of STEC infection in our settings should not be disregarded.     

Rapid culture-free identification and molecular typing of Shiga toxin-producing Escherichia coli by PCR-RFLP

Using culture-independent technology, PCR-RFLP were used to identify and type STEC in the stool of a patient with HUS. Fecal PCR-RFLP patterns were identical to those of the STEC O157:H7 isolated from the patient.     

Distribution of virulence profiles related to new toxins and putative adhesins in Shiga toxin-producing Escherichia coli isolated from diverse sources in Brazil

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 eae-negative 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.     

Shiga toxin-producing Escherichia coli and enteropathogenic Escherichia coli in healthy goats in India: occurrence and virulence properties

AIMS: To describe the occurrence and virulence gene pattern of shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) in healthy goats of Jammu and Kashmir, India. METHODS AND RESULTS: A total of 220 E. coli strains belonging to 60 different 'O' serogroups was isolated from 206 local (nonmigratory) and 69 migratory goats. All the 220 strains were screened for the presence of stx(1), stx(2), eaeA and hlyA genes. Twenty-eight E. coli (75.6%) strains from local and nine (24.3%) strains from migratory goats belonging to 18 different serogroups showed at least presence of one virulence gene studied. Twenty-eight strains (16.47%) (belonging to 13 different serogroups) from local goats carried stx(1) gene alone or in combination with stx(2) gene, while as only one strain (2%) from migratory goats possessed stx(2) gene alone. Interestingly in the present study none of the STEC strains carried eaeA gene. Similarly, none of the strains from local goats possessed eaeA and none of the migratory goats possessed stx(1) gene. Eight strains (16%) (belonging to four different serogroups) from migratory goats carried eaeA gene. Twenty-five (14.7%) and seven (14%) strains from local and migratory goats harboured hlyA gene respectively. CONCLUSIONS: Healthy goats of Jammu and Kashmir state serve as a reservoir of STEC and EPEC. Further studies in this direction are needed to work out whether or not they are transmitted to humans in this part of world. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first report of isolation of STEC and EPEC strains from healthy goats in Jammu and Kashmir State of India, which could be a source of infection to humans.     

Inhibition of growth of Shiga toxin-producing Escherichia coli by nonpathogenic Escherichia coli

During routine quality control testing of diagnostic methods for Shiga toxin-producing Escherichia coli (STEC) using stool samples spiked with STEC, it was observed that the Shiga toxin could not be detected in 32 out of 82 samples tested. Strains of E. coli isolated from such stool samples were shown to be responsible for this inhibition. One particular isolate, named E. coli 1307, was intensively studied because of its highly effective inhibitory effect; this strain significantly reduced growth and Shiga toxin levels in coculture of several STEC strains regardless of serovar or Shiga toxin type. The probiotic E. coli Nissle 1917 inhibited growth and reduced Shiga toxin levels in STEC cultures to an extent similar to E. coli 1307, but commensal E. coli strains and several other known probiotic bacteria (enterococci, Bacillus sp., Lactobacillus acidophilus ) showed no, or only small, inhibitory effects. Escherichia coli 1307 lacks obvious fitness factors, such as aerobactin, yersiniabactin, microcins and a polysaccharide capsule, that are considered to promote the growth of pathogenic bacteria. We therefore propose strain E. coli 1307 as a candidate probiotic for use in the prevention and treatment of infections caused by STEC.     

High numbers of Shiga toxin-producing Escherichia coli found in bovine faeces collected at slaughter in Japan

The prevalence and concentration of Shiga toxin-producing Escherichia coli (STEC) in cattle faeces (n=605) at the time of slaughter was studied in Shimane Prefecture, Japan on a monthly basis between April 2000 and March 2001. Screening with stx-PCR determined a prevalence of 37.5%. After analysis of spread faeces and enriched samples on cefixime, tellurite and sorbitol-MacConkey agar using HCl treatment, 114 STEC strains were singly or concomitantly isolated from 97 cattle (15.9%). Of the 605 cattle, 31 (5.1%) harbored O26:H11, O111:H-, O121:H19 or O157:H7, which had the stx1 and/or stx2 and eae and hlyA genes, and 7 (23%) of these 31 cattle were high level carriers that contained these typical STEC at concentrations of 10(5)-10(8) CFU/g of faeces. The predominant serotype was O26:H11 (20 strains) and the second most frequent was O157:H7 (9 strains). Of the 605 cattle, 68 (11.2%) harboured 36 other serotypes and 6 (5.9%) of the 67 cattle were high level carriers. As a comparison between the prevalence of STEC and the faecal pH, it was demonstrated that STEC can be isolated from cattle with a wide range of faecal pH values. The presence of a high-carriage animal at the abattoir increases the potential risk of meat contamination during the slaughtering process, regardless of faecal pH.     

多重PCR-DHPLC快速检测食品中产志贺毒素大肠杆菌

应用多重PCR(motiplex PCR)结合变性高效液相色谱技术(denaturing high-performanceliquid chromatography,DHPLC)建立了快速检测食品中产志贺毒素大肠杆菌O111和O157的方法.以基因wzxO111、rfbEO157为靶基因,建立多重PCR-DHPLC方法,进行特异性和灵敏度测试,同时进行RT-PCR检测比较灵敏度.该方法具有良好特异性,可以一次PCR扩增同时检测O111、O157;灵敏度达到25 CFU/mL.129份牛肉样品中检出1例O111,3例O157阳性;74份鸡肉样品中检测出O111、O157阳性各1例,67份蔬菜样品中未检测到O111、O157.本文建立O111、O157多重PCR-DHPLC检测方法,操作简便,特异性强,适用于产志贺毒素大肠杆菌筛选检测.     

Development of a real-time PCR assay with an internal amplification control for the screening of Shiga toxin-producing Escherichia coli in foods

Aims:  To develop and evaluate a real-time PCR assay incorporating an internal amplification control (IAC) suitable for the screening of Shiga toxin (Stx)-producing Escherichia coli (STEC) in foods.
Methods and Results:  A competitive IAC was constructed and included in an stx -specific real-time PCR assay. Coupled to 18-h enrichment and automated DNA extraction, the assay could reliably detect the presence of STEC in minced meats inoculated at 10 CFU per 25 g. Its performance was evaluated on 415 minced beef and 112 raw milk cheese samples and compared with that of a PCR-ELISA method. Fifty-three minced meats and 31 cheeses were found stx -positive, giving 98·3% and 93·75% concordance, respectively, with the PCR-ELISA reference method.
Conclusions:  A highly sensitive stx -specific real-time PCR method including an IAC was developed, facilitating monitoring of false-negative results due to PCR inhibitors.
Significance and Impact of the Study:  Combined with automated DNA extraction, the stx -IAC real-time PCR assay represents a suitable method for rapid screening of STEC in foods.     

产志贺样毒素和肠毒素大肠杆菌分子流行病学

[目的]人和动物腹泻的主要病原菌为大肠杆菌,本文主要研究贵州省致腹泻大肠杆菌毒力因子的分布类型.[方法]采用PCR技术对各毒力因子的基因分布进行研究.[结果]共分离到333株大肠杆菌,其中产肠毒素大肠杆菌(ETEC)在腹泻的人、猪、牛群中占优势,分别为:人群73(n=112),猪群82(n=106),牛群18(n=115).在ETEC菌株中检测到热敏肠毒素(lt)和不耐热肠毒素(st)基因,还存在lt/st并存现象.从人、猪、牛群中还检测到产志贺样毒素大肠杆菌(STEC),其中源自猪的STEC的检出率最高.大部分STEC同时携带lt、st或lt和st同时并存.编码F18菌毛的主亚基由fedA基因编码.对所分离大肠杆菌F18菌毛进行的研究结果表明,fedA基因主要与肠毒素基因共存,与stx基因并存的类型较少,25份猪源STEC菌株中仅有4份检测到fedA基因.[结论]贵州省人群、猪群和牛群致腹泻病原菌中以带F18菌毛的ETEC为主,STEC主要分布在腹泻的猪群中.     

Persistence of Shiga toxin-producing Escherichia coli O26 in cow slurry

AIMS: The main objective of this study was to evaluate the growth and survival of Shiga toxin-producing Escherichia coli (STEC) O26 in cow slurry; this serogroup is regarded as an important cause of STEC-associated diseases. METHODS AND RESULTS: Four STEC were examined by polymerase chain reaction (PCR) to determine whether they harbour key virulence determinants and also by pulsed-field gel electrophoresis (PFGE) to obtain overview fingerprints of their genomes. They were transformed with the pGFPuv plasmid and were separately inoculated at a level of 10(6) CFU ml(-1) in 15 l of cow slurry. All STEC O26 strains could be detected for at least 3 months in cow slurry without any genetic changes. The moisture content of the slurry decreased over time to reach a final value of 75% while the pH increased from 8.5 to 9.5 units during the last 50 days. CONCLUSION: STEC O26 strains were able to survive in cow slurry for an extended period. SIGNIFICANCE AND IMPACT OF THE STUDY: Long-term storage of waste slurry should be required to reduce the pathogen load and to limit environmental contamination by STEC O26.     

A prospective study on Shiga toxin-producing Escherichia coli in children with diarrhoea in Paraná State, Brazil

Aims:  To examine stool specimens from children with diarrhea from Paraná State, southern Brazil, for presence of STEC.
Methods and Results:  A PCR screening assay for stx genes was used to examine a loopful of confluent colonies of 306 stool samples cultures. In six (1.96%) of them, DNA fragments of the expected size were observed, and the presence of stx was confirmed by DNA sequencing. Then up to 100 single colonies from each of the six stool cultures were analyzed using the same PCR protocol. However, stx -positive colonies were found only in two of the cultures. The E. coli strains belonged to serotypes O69:H11 and O178:H19, and presented genotypes stx ₁ eae ehxA and stx ₁ respectively. Shiga toxin production was confirmed using the VTEC Screen Seiken. Except ampicillin, they were susceptible to all the antimicrobials tested.
Conclusions:  These results show that STEC may be an important cause of diarrhea in children of Paraná State, and that they are present in low numbers in stools. The strains belonged to serotypes not commonly found associated with STEC and probably present low virulence.
Significance and Impact of Study:  These results indicate that molecular methods are required to diagnosis of STEC infections.     

Detection, isolation and characterization of Shiga toxin-producing Escherichia coli in retail-minced beef using PCR-based techniques, immunoassays and colony hybridization

AIMS: To provide information on detection of Shiga toxin-producing Escherichia coli (STEC) in retail-minced beef using an approach combining (i) PCR-based techniques and automated immunoassay for stx screening and detection of the five major serogroups associated with human infection, and (ii) immunomagnetic separation (IMS) and colony hybridization assays for bacterial strain isolation. METHODS AND RESULTS: Twenty-seven out of 164 minced beef samples were stx-positive by PCR-ELISA, nine of which were also positive by real-time PCR for at least one marker of the five main serogroups tested (O26, O103, O111, O145 and O157). Two E. coli O103 stx-negative strains were isolated from two out of 10 IMS and nine STEC strains that did not belong to the five main serogroups were isolated by colony hybridization. CONCLUSIONS: PCR techniques are applicable for rapid screening of samples containing both an stx gene and an O-group marker of the five main pathogenic STEC serogroups. Isolation of STEC strains belonging to the main non-O157 serogroups remains difficult. SIGNIFICANCE AND IMPACT OF THE STUDY: This study presents an evaluation of a multi-faceted approach for the detection of the most frequently reported human pathogenic STEC serogroups. The advantages and limits of this strategy are presented.