Occurrence of Escherichia coli O157 on hides of slaughtered cattle

AIMS: To obtain the first information on the occurrence of Escherichia coli O157 on hides of slaughtered cattle in Serbia. METHODS AND RESULTS: A total of 355 swabs were taken on the slaughterline from five areas of hide of each of the 71 cattle in a single commercial abattoir in Serbia. Using an ISO method incorporating enrichment and immunomagnetic separation steps, E. coli O157 was isolated from the hides of 20 animals (28 x 2%). With respect to different areas of the hides, the occurrence of the pathogen was, in decreasing order: hooves (11 x 3%), brisket (8 x 4%), rump (7 x 0%), neck (4 x 2) and flank (2 x 8%). In addition, factors that had more or less effects on the occurrence included visible dirtiness of the hide, cattle's age category, geographical origin of the animals and season. CONCLUSIONS: This study revealed the presence of E. coli O157 in the beef chain in Serbia and confirmed hide as an important potential source of related contamination of beef carcasses. Therefore, incorporation of preskinning hide decontamination treatments into HACCP-based slaughterline hygiene control measures could be very useful. SIGNIFICANCE AND IMPACT OF THE STUDY: The results will enable further optimization of necessary measures along the beef chain to reduce the E. coli O157 risks in Serbia.     

Serogroup distribution and virulence characteristics of sorbitol‐negative Escherichia coli from food and cattle stool

Aims: To (i) study the serogroup distribution and virulence characteristics of non‐sorbitol‐fermenting Escherichia coli isolates from foods of animal origin and cattle faeces and (ii) re‐examine the true sorbitol and β‐d ‐glucuronidase (GUD) reactions of sorbitol‐negative (Sor^?) strains from MacConkey sorbitol agar (SMAC) to assess their phenotypic similarity with E. coli O157. Methods and Results: One hundred and thirty Sor^?E. coli were isolated from 556 food samples and 177 cattle stool samples using cefixime tellurite–supplemented SMAC (CT‐SMAC) and chromogenic HiCrome MS.O157 agar respectively. Based on typing of somatic antigen, the isolates were classified into 38 serogroups. PCR results identified about 40% strains, belonging to O5, O8, O20, O28, O48, O60, O78, O82, O84, O101, O110, O123, O132, O156, O157, O‐rough and OUT as Shiga toxigenic. Majority of O5, O84, O101, O105, O123, O157, O‐rough and OUT strains were enterohaemolytic. Further, 39·2% and 63·1% of Sor^? isolates from CT‐SMAC fermented sorbitol in phenol red broth and hydrolysed 4‐methylumbelliferyl‐β‐d ‐glucuronide (MUG) respectively. Members of serogroups O5, O28, O32, O81, O82, O84, O101, O‐rough lacked both the sorbitol fermentation (broth test) and GUD activity and might create confusion in phenotypic identification of E. coli O157. Conclusions: Sor^?E. coli isolates from raw meat, milk, shrimp and cattle stool belonged to 38 serogroups, with E. coli O157 constituting only 14·6% of the isolates. Many of these nonclinical Sor^? strains were potentially pathogenic. Nearly 39% of these Sor^?E. coli from CT‐SMAC fermented sorbitol in broth, indicating the need for confirmation of sorbitol reaction in broth. Significance and Impacts of the Study: Classical sorbitol utilization and GUD tests are not likely definitive tests for E. coli O157. Further improvement of differential media based on these phenotypic properties is necessary for detection of pathogenic serotypes from foods and environmental samples.     

Phenotypic and genotypic characterization of sorbitol-negative or slow-fermenting (suspected O157) Escherichia coli isolated from milk samples in Lombardy region

AIMS: To investigate phenotypic and genotypic aspects of sorbitol-negative or slow-fermenting Escherichia coli, suspected to belong to O157 serogroup, isolated in Italy. METHODS AND RESULTS: Milk samples originating from goats and cows were screened for the presence of E. coli O157 with cultural methods. Sorbitol-negative or slow-fermenting strains were subjected to phenotypic characterization, antibiotic resistance profiles, PCR reactions for detection of toxins (stx(1) and stx(2)) and intimin (eae(GEN) and eae(O157)) genes and clustering by pulsed field gel electrophoresis (PFGE). Only one strain revealed to be O157. Susceptibility to 11 antibiotics highlighted the high resistance to tetracycline (50%), sulfonamide and streptomycin (33%). The stx(2) gene was detected in two strains; only the strain identified as O157 exhibited an amplicon for both eae genes. PFGE identified seven distinct XbaI macrorestriction patterns at a similarity level of 41%. CONCLUSIONS: The use of sorbitol fermentation as cultural method is not sufficient for STEC discrimination while PCR assay proved to be a valuable method. SIGNIFICANCE AND IMPACT OF THE STUDY: The study reports presence of Shiga toxin-producing E. coli in raw milk, signalling a potential risk for humans.     

Isolation of Escherichia coli O157:H7 strains that do not produce Shiga toxin from bovine, avian and environmental sources

AIMS: To determine the potential for naturally occurring Shiga toxin-negative Escherichia coli O157 to acquire stx(2) genes. METHODS AND RESULTS: Multiple E. coli O157:H7 isolates positive for eae and ehxA, but not for stx genes, were isolated from cattle, water trough sediment, animal bedding and wild bird sources on several Ohio dairy farms. These isolates were experimentally lysogenized by stx(2)-converting bacteriophage. CONCLUSIONS: Shiga toxin-negative strains of E. coli O157 are present in multiple animal and environmental sources. SIGNIFICANCE AND IMPACT OF THE STUDY: Shiga toxin-negative strains of E. coli O157 present in the food production environment are able to acquire the stx genes, demonstrating their potential to emerge as new Shiga toxin-producing E. coli strains.     

Direct PCR detection of Escherichia coli O157:H7

AIMS: This paper reports a simple, rapid approach for the detection of Shiga toxin (Stx)-producing Escherichia coli (STEC). METHODS AND RESULTS: Direct PCR (DPCR) obviates the need for the recovery of cells from the sample or DNA extraction prior to PCR. Primers specific for Stx-encoding genes stx1 and stx2 were used in DPCR for the detection of E. coli O157:H7 added to environmental water samples and milk. CONCLUSIONS: PCR reactions containing one cell yielded a DPCR product. SIGNIFICANCE AND IMPACT OF THE STUDY: This should provide an improved method to assess contamination of environmental and other samples by STEC and other pathogens.     

Prevalence of Escherichia coli O157 and O157:H7-infecting bacteriophages in feedlot cattle feces

AIM: To estimate the distribution and prevalence of both Escherichia coli O157 and O157:H7-infecting bacteriophages within a 50,000 head commercial beef feedlot. METHODS AND RESULTS: Escherichia coli O157 was detected in approximately 27% of the individual samples, distributed across seven of the 10 pens screened. In a simple initial screen to detect O157:H7-infecting phages, none were detected in any pen or individual sample. In contrast, after a series of enrichment procedures O157:H7-infecting phages were detected in every pen and in the majority of the samples from most pens; virulent bacteriophages active against E. coli O157:H7 were detected post-enrichment from 39/60 (65%) of the feedlot samples, and 58/60 (approximately 97%) contained phage that infected E. coli B or O157:H7. CONCLUSIONS: The data we present here indicates that we may be grossly underestimating the prevalence of O157:H7-infecting phages in livestock if we simply screen samples and that enrichment screening is required to truly determine the presence of phages in these ecosystems. SIGNIFICANCE AND IMPACT OF THE STUDY: Our data suggest that O157:H7-infecting phages may play a role in the ecology and transient colonization of cattle by E. coli O157:H7. Further, this and previous data suggest that before starting in vivo pathogen eradication studies using phage or any other regime, test animals should be enrichment screened for phage to avoid erroneous results.     

Characterization of inducible stx2-positive Escherichia coli O157:H7/H7- strains isolated from cattle in France

Aims:  To quantify the variability of the Shiga toxin 2 (Stx2) production by a panel of stx2 -positive Escherichia coli O157:H7/H7- isolates from healthy cattle before and after induction with enrofloxacin.
Methods and Results:  ProSpecT^® ELISA was used to quantify the Stx2 production by stx2 -positive E. coli O157:H7/H7- isolates in native conditions (basal level) or after induction with enrofloxacin. Whereas only 15·2% of the E. coli O157:H7/H7- strains studied displayed significant amounts of detectable Stx2 without induction, most of them were shown to be inducible, and at various levels, in presence of subinhibitory concentrations of enrofloxacin.
Conclusions:  We demonstrated the capability of a highly elevated proportion of stx2 -positive, but constitutively Stx2 -negative, E. coli O157:H7/H7- isolates from healthy cattle to produce significant levels of Shiga toxin Stx2 in presence of subtherapeutic concentrations of enrofloxacin, an antibiotic of the fluoroquinolones family only licensed for veterinary use.
Significance and Impact of the Study:  This study documents the risk that bovine-associated Shiga toxin producing E. coli isolates may become more frequently pathogenic to humans as a side-effect of the increasing use of veterinary fluoroquinolones in the oral treatment of food animals like cattle or poultry.     

Characterization of antimicrobial resistance and seasonal prevalence of Escherichia coli O157:H7 recovered from commercial feedlots in Alberta,Canada

Aims: To characterize antimicrobial resistance (AMR) and determine the seasonal prevalence of Escherichia coli O157:H7 isolated from commercial feedlots. Methods and Results: Escherichia coli O157:H7 were isolated from faecal and oral samples collected at monthly intervals from three commercial feedlots over a 12‐month period. A total of 240 isolates were characterized using pulsed‐field gel electrophoresis (PFGE) technique. A subset of 205 isolates was analysed for AMR using Sensititre system and AMR genes (tet, sul and str) by PCR. Seven PFGE clusters (≥90% Dice similarity) were identified, and two clusters common to all three feedlots were recovered year‐round. The majority of isolates (60%) were susceptible to all antimicrobials and were closely related (P < 0.001), whereas isolates with unique AMR patterns were not related. The prevalences of AMR from feedlots A, B and C were 69%, 1% and 38%, respectively. Resistance to tetracycline (69%) and sulfisoxazole (68%) was more prevalent in feedlot A than other two feedlots. The presence of strA and strB genes was linked in the majority of isolates, and tet(A) and tet(B), and sul1 and sul2 genes were present individually. Escherichia coli O157:H7 were genetically diverse during summer and fall, and strains from winter and spring months were more closely related. Conclusions: Antimicrobial resistance was more common in E. coli O157:H7 obtained from two of the three commercial feedlots, and the phenotypic expression of resistance was correlated with the presence of resistant genes. A highly diverse E. coli O157:H7 population was found during summer and fall seasons. Significance and Impact of the Study: Information would help understanding the dynamics of AMR in E. coli O157:H7 from commercial feedlots.     

Isolation and characterization of Escherichia coli O157 from retail beef and bovine feces in Thailand

Antibody to Escherichia coli O157 lipopolysaccharide was detected in the sera of healthy individuals more frequently in Southern Thailand than in Japan. The result suggested possible exposure of Thai people to E. coli O157. E. coli O157:H7 or O157:H(-) was isolated from four of 95 retail beef and one of 55 bovine feces samples collected in Southern Thailand by enrichment culture followed by immunomagnetic bead separation. Four of the five strains carried the stx(2) gene alone or in combination with the stx(1) gene. The strains were shown to be genetically distinct by an arbitrarily primed PCR method.     

Identification of a Shiga-toxin type I variant containing an IS1203-like element, from Shiga-toxin producing Escherichia coli O157:H7

We found two Shiga toxin producing Escherichia coli O157:H7 strains isolated from humans carrying the stx(1) gene with an IS1203-like element (designated as IS1203v(1)). The IS1203v(1) was inserted into the coding region of the A subunit 7 bp upstream from the TGA termination codon, resulting in a loss of two amino acid residues (Ser-Ser) from its C terminus. Toxicity of the Stx1 was confirmed by Vero cell assay. IS1203v(1) hardly affected the stx(1) gene in either its expression or the toxicity of its product.     

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.     

Seasonal variation of Shiga toxin-encoding genes (stx) and detection of E. coli O157 in dairy cattle from Argentina

Aims:  To study the seasonal variation of Shiga toxin-encoding genes ( stx ) and to investigate the presence of Shiga toxin-producing Escherichia coli (STEC) O157 in cattle belonging to five dairy farms from Argentina.
Methods and Results:  Rectal swab samples were collected from 360 dairy cows in each season and 115 and 137 calves in autumn and in spring, respectively. The stx were investigated by multiplex PCR and it was used as the indicator for STEC. Samples positives for stx were tested by PCR for eae-γ1 of E. coli O157 and then subjected to IMS (immunomagnetic separation). In positive animals significant differences in the prevalence of stx between warm and cold seasons were detected. In warm seasons, stx1  +  stx2 increased and stx1 decreased, independently of the animal category. The prevalence of STEC O157 in cows and calves were 0·2% and 0·8%, respectively.
Conclusions:  This work provides new data about the occurrence of stx and STEC O157 in dairy herds from Argentina and suggests a relationship between the type of stx and season of year.
Significance and Impact of Study:  The detection of STEC O157 and the seasonality of stx and its types provide an opportunity to improve control strategies designed to prevent contamination of food products and transmission animal-person.     

Occurrence and molecular characteristics of antimicrobial resistance of Escherichia coli from broilers,pigs and meat products in Thailand and Cambodia provinces

Nine hundred and forty‐one samples were collected in Sa Keao, Thailand (n = 554) and Banteay Meanchey, Cambodia (n = 387) from July 2014 to January 2015. A total of 667 Escherichia coli isolates (381 isolates from Sa Keao and 286 isolates from Banteay Meanchey) were obtained and examined for antimicrobial susceptibility, class 1 integrons, ESBL genes and horizontal transfer of resistance determinants. Prevalence of E. coli in pig and broiler carcass samples from slaughterhouses and fresh markets was 36–85% in Sa Keao and 11–69% in Banteay Meanchey. The majority of these isolates were multidrug resistant (75.3%). Class 1 integrons were common in both Thai (47%) and Cambodian (62%) isolates, of which four resistance gene cassette arrays including aadA1, dfrA1‐aadA1, dfrA12‐aadA2 and aadA2‐linF were identified. Class 1 integrons in two broiler isolates from Sa Keao (dfrA12‐aadA2) and one broiler isolate from Banteay Meanchey (dfrA1‐aadA1) were horizontally transferable. Sixteen isolates were confirmed to be ESBL‐producing strains with ESBL gene bla_CTX‐M‐15, broad spectrum β‐lactamase gene bla_TEM‐1 and the AmpC gene bla_CMY‐2 being detected. The bla_TEM‐1 gene was most prevalent and located on a conjugative plasmid.     

Clonal turnover of enterohemorrhagic Escherichia coli O157:H7 in experimentally infected cattle

A total of 401 enterohemorrhagic Escherichia coli (EHEC) O157:H7 isolates from two experimentally infected calves were analyzed using molecular biological methods. Genetic differences detected by pulsed-field gel electrophoresis were observed between the inoculated and recovered strains as early as 1 day post inoculation. The loss of the inoculated clone was observed in one calf. Replication and dissemination of the EHEC O157:H7 strains that mutated in cattle may result in the diversification of this organism among cattle populations.     

Fate of Escherichia coli O157 and detection of stx phage during fermentation of maize, an animal feedstuff

AIMS: The fate of inoculated Escherichia coli O157, stx phages and the physico-chemical properties of maize were studied during laboratory-scale fermentation by naturally occurring lactic acid bacteria. METHODS AND RESULTS: Before fermentation, chopped maize was inoculated with 6.2 log(10) CFU g(-1) of a five-isolate mixture of E. coli O157. After fermentation, the silage contained 70.6 g kg(-1) dry matter (DM) lactic acid and 12.8 g kg(-1) DM acetic acid and was pH 4.0. Levels of E. coli O157 fell rapidly, and none of the five isolates could be recovered from the fermenting maize after 8 days. Using a resuscitation step did not consistently enhance recovery of E. coli O157. Stx phages were not isolated from the fermenting maize at any time. Pulsed-field gel electrophoresis (PFGE) genotyping showed that E. coli O157 2975 and 64a/01 survived better than the other three isolates studied. Escherichia coli O157 isolate 1474/00 was particularly sensitive to the laboratory procedures used to harvest the inocula and contaminate the maize. Some colonies recovered during the fermentation had one to three band alterations compared with the initial PFGE genotypes. SIGNIFICANCE AND IMPACT OF THE STUDY: None of the five different E. coli O157 genotypes survived maize fermentation. Fermentation of maize produces an animal feedstuff that is unlikely to contain E. coli O157 or stx phages.     

Presence of Shiga toxin-producing Escherichia coli O157:H7 in living layer hens

AIMS: To evaluate the presence of Shiga toxin-producing strains of Escherichia coli (STEC) of the O157:H7 serotype in living layer hens so as to analyse the role of this avian species as potential reservoir. METHODS AND RESULTS: Cloacal swabs were collected between November 2004 and November 2005 from four intensive management layer hen farms and analysed for STEC O157:H7 by immunomagnetic separation methods and multiplex polymerase chain reaction for stx1 and/or stx2, the E. coli attaching and effacing (eae) and hly genes. STEC was detected in 26 of the 720 samples. CONCLUSIONS: The layer hens analysed were shown to carry STEC O157:H7. The presence of this bacterium in living layer hen farms investigated did not result in any detectable increase in gastrointestinal disease in this species. SIGNIFICANCE AND IMPACT OF THE STUDY: Living layer hens are a novel potential reservoir of E. coli O157:H7.     

Intermittent and persistent shedding of Escherichia coli O157 in cohorts of naturally infected calves

AIMS: We conducted two short-term studies of cohorts of naturally infected calves to determine the prevalence and concentrations of Escherichia coli O157 shed in faeces. METHODS AND RESULTS: Two cohorts of calves were sampled; in the first study 14 calves were sampled up to five times a day for 5 days; in the second study a group of 16 separate calves were sampled once or twice a day for 15 days. All cattle within the two cohorts shed E. coli O157 at some point during the respective studies. In 18% of samples, E. coli O157 could only be isolated using immunomagnetic separation after an enrichment period, suggesting concentrations <250 CFU g(-1). The highest concentrations recorded were 6.7 x 10(5) and 1.6 x 10(6) CFU g(-1) for studies 1 and 2 respectively. CONCLUSIONS: Persistent, high shedders (shedding >10(3) CFU g(-1)) were evident in both studies but, in the majority of calves, the pathogen was isolated intermittently. SIGNIFICANCE AND IMPACT OF THE STUDY: The variable patterns of shedding have important implications for the design of appropriate sampling protocols and for gaining meaningful estimates of parameters used in mathematical models of transmission.     

玉溪市大肠埃希菌O157菌株的流行率、表型与毒力因子特征

目的:检测并分析大肠埃希菌O157(Escherichia coli O157)在玉溪市奶牛、猪、鸡和腹泻病人中的带菌率及其特征,为E.coli O157感染症的防治和该菌分离鉴定技术的建立提供科学依据.方法:将玉溪市最大的一个奶牛场、1个猪屠宰场、2个生禽市场和市医院检验科作为监测点,采集牛、猪、鸡、腹泻病人粪便标本数分别为70、250、350和400例,用免疫磁性分离法和免疫色层技术进行E.coli O157菌株的分离培养,纯化菌株经3种鉴别培养基培养以及法国生物梅里埃公司全自动微生物鉴定系统VITEK 32 GNI 或肠杆菌科鉴定系统API 20 E试条、血清学、噬菌体型、亚碲酸盐抗性、聚合酶链反应等检测与分析.结果:牛、猪、鸡、腹泻病人E.coli O157的带菌率分别为1.4%、2.0%、0和1.2%,分离到4株E.coli O157∶H7、6株E.coli O157∶Hund和1株E.coli O157∶HNM,认识了11株E.coli O157的形态学、生物化学、血清学、噬菌体型、亚碲酸盐抗性、毒力因子等特征.结论:单一动物群或人群可同时携带多株E.coli O157,甚至同一个体也会携带2株E.coli O157;E.coli O157菌株间的表型与毒力因子特征存在一定差异,基因指纹技术可确认并证实菌株间的差异性.