Association of Escherichia coli O157:H7 with Houseflies on a Cattle Farm

The ecology of Escherichia coli O157:H7 is not well understood. The aims of this study were to determine the prevalence of and characterize E. coli O157:H7 associated with houseflies (HF). Musca domestica L. HF (n = 3,440) were collected from two sites on a cattle farm over a 4-month period and processed individually for E. coli O157:H7 isolation and quantification. The prevalence of E. coli O157:H7 was 2.9 and 1.4% in HF collected from feed bunks and a cattle feed storage shed, respectively. E. coli O157:H7 counts ranged from 3.0 × 10¹ to 1.5 × 10⁵ CFU among the positive HF. PCR analysis of the E. coli O157:H7 isolates revealed that 90.4, 99.2, 99.2, and 100% of them (n = 125) possessed the stx1, stx2, eaeA, and fliC genes, respectively. Large populations of HF on cattle farms may play a role in the dissemination of E. coli O157:H7 among animals and to the surrounding environment.     

Prevalence of Escherichia coli O157:H7 in gallbladders of beef cattle

Gallbladders and rectal contents were collected from cattle (n=933) at slaughter to determine whether the gallbladder harbors Escherichia coli O157:H7. Both gallbladder mucosal swabs and homogenized mucosal tissues were used for isolation. Only five gallbladders (0.54%) were positive for E. coli O157:H7. Fecal prevalence averaged 7.1%; however, none of the cattle that had E. coli O157:H7 in the gallbladder was positive for E. coli O157:H7 in feces. Therefore, the gallbladder does not appear to be a common site of colonization for E. coli O157:H7 in beef cattle.     

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.     

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.     

Effect of forage or grain diets with or without monensin on ruminal persistence and fecal Escherichia coli O157:H7 in cattle

Twelve ruminally cannulated cattle, adapted to forage or grain diet with or without monensin, were used to investigate the effects of diet and monensin on concentration and duration of ruminal persistence and fecal shedding of E. coli O157:H7. Cattle were ruminally inoculated with a strain of E. coli O157:H7 (10(10) CFU/animal) made resistant to nalidixic acid (Nal(r)). Ruminal and fecal samples were collected for 11 weeks, and then cattle were euthanized and necropsied and digesta from different gut locations were collected. Samples were cultured for detection and enumeration of Nal(r) E. coli O157:H7. Cattle fed forage diets were culture positive for E. coli O157:H7 in the feces for longer duration (P < 0.05) than cattle fed a grain diet. In forage-fed cattle, the duration they remained culture positive for E. coli O157:H7 was shorter (P < 0.05) when the diet included monensin. Generally, ruminal persistence of Nal(r) E. coli O157:H7 was not affected by diet or monensin. At necropsy, E. coli O157:H7 was detected in cecal and colonic digesta but not from the rumen. Our study showed that cattle fed a forage diet were culture positive longer and with higher numbers than cattle on a grain diet. Monensin supplementation decreased the duration of shedding with forage diet, and the cecum and colon were culture positive for E. coli O157:H7 more often than the rumen of cattle.     

Escherichia coli O157:H7 Shiga toxin-encoding bacteriophages: integrations,excisions, truncations,and evolutionary implications

As it descended from Escherichia coli O55:H7, Shiga toxin (Stx)-producing E. coli (STEC) O157:H7 is believed to have acquired, in sequence, a bacteriophage encoding Stx2 and another encoding Stx1. Between these events, sorbitol-fermenting E. coli O157:H(-) presumably diverged from this clade. We employed PCR and sequence analyses to investigate sites of bacteriophage integration into the chromosome, using evolutionarily informative STEC to trace the sequence of acquisition of elements encoding Stx. Contrary to expectations from the two currently sequenced strains, truncated bacteriophages occupy yehV in almost all E. coli O157:H7 strains that lack stx(1) (stx(1)-negative strains). Two truncated variants were determined to contain either GTT or TGACTGTT sequence, in lieu of 20,214 or 18,895 bp, respectively, of the bacteriophage central region. A single-nucleotide polymorphism in the latter variant suggests that recombination in that element extended beyond the inserted octamer. An stx(2) bacteriophage usually occupies wrbA in stx(1)(+)/stx(2)(+) E. coli O157:H7, but wrbA is unexpectedly unoccupied in most stx(1)-negative/stx(2)(+) E. coli O157:H7 strains, the presumed progenitors of stx(1)(+)/stx(2)(+) E. coli O157:H7. Trimethoprim-sulfamethoxazole promotes the excision of all, and ciprofloxacin and fosfomycin significantly promote the excision of a subset of complete and truncated stx bacteriophages from the E. coli O157:H7 strains tested; bile salts usually attenuate excision. These data demonstrate the unexpected diversity of the chromosomal architecture of E. coli O157:H7 (with novel truncated bacteriophages and multiple stx(2) bacteriophage insertion sites), suggest that stx(1) acquisition might be a multistep process, and compel the consideration of multiple exogenous factors, including antibiotics and bile, when chromosome stability is examined.     

Development of a multiplex PCR approach for the identification of Shiga toxin-producing Escherichia coli strains and their major virulence factor genes

AIMS: To develop and evaluate a multiplex PCR (mPCR) system for rapid and specific identification of Shiga toxin-producing Escherichia coli (STEC) and their main virulence marker genes. METHODS AND RESULTS: A series of mPCR assays were developed using primer pairs that identify the sequences of Shiga toxins 1 and 2 (stx1 and stx2, including the stx2c, stx2d, stx2e and stx2f variants), intimin (eaeA), and enterohaemorrhagic E. coli enterohaemolysin (ehlyA). Moreover, two additional genes (rfb O157 and fliC H7), providing the genotypic identification of the O157:H7 E. coli serotype, were detected. As an internal positive control, primers designated to amplify the E. coli 16S rRNA were included in each mPCR. All the amplified genes in the E. coli reference strains were sucessfully identified by this procedure. The method was then used for the examination of 202 E. coli isolates recovered from cattle and children. Among them, 25 (12.4%) were stx positive including the strains of O157:H7 serotype (six isolates) and O157:NM serogroup (four strains). Moreover, 20 STEC strains possessed the eaeA (intimin) and ehlyA (enterohaemolysin) genes. CONCLUSIONS: The developed mPCR-based system enabled specific detection of STEC bacteria and identification of their main virulence marker genes. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to identify STEC bacteria and the majority of their virulence gene markers, including four variants of Shiga toxin, as well as the differentiation of O157:H7 from non-O157 isolates represents a considerable advancement over other PCR-based methods for rapid characterization of STEC.     

Reduction of Escherichia coli O157:H7 shedding in cattle by addition of chitosan microparticles to feed

Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is a significant human pathogen that resides in healthy cattle. It is thought that a reduction in the prevalence and numbers of EHEC in cattle will reduce the load of EHEC entering the food chain. To this end, an intervention strategy involving the addition of chitosan microparticles (CM) to feed in order to reduce the carriage of this pathogen in cattle was evaluated. Experiments with individual Holstein calves and a crossover study found that the addition of CM to feed decreased E. coli O157:H7 shedding. In the crossover study, CM resulted in statistically significant reductions in the numbers recovered from rectal swab samples (P < 0.05) and the duration of shedding (P < 0.05). The effects of feeding CM to calves differed, indicating that the optimal levels of CM may differ between animals or that other factors are involved in the interaction between CM and E. coli O157:H7. In vitro studies demonstrated that E. coli O157:H7 binds to CM, suggesting that the reduction in shedding may result at least in part from the binding of positively charged CM to negatively charged E. coli cells. Additional studies are needed to determine the impact of CM feeding on animal production, but the results from this study indicate that supplementing feed with CM reduces the shedding of E. coli O157:H7 in cattle.     

Genetic relatedness of a non-motile variant O157 enteropathogenic Escherichia coli (EPEC) strain and E. coli strains belonging to pathogenic related groups

The study was undertaken to determine the clonal relationship and the genetic diversity among Escherichia coli isolates by comparing a non-motile O157 variant with three O157:H7 EHEC isolates and one O55:H7 enteropathogenic E. coli (EPEC) strain. E. coli strains were characterized by sorbitol phenotype, multilocus enzyme electrophoresis, pulsed-field gel electrophoresis, random amplification polymorphic DNA, and the presence of specific virulence genes (stx, E-hly and LEE genes). Sorbitol fermentation was observed in O157:H- (strain 116I), O55:H7 and O157:H7 (strain GC148) serotypes. stx1 or stx2 and E-hly genes were only detected among O157:H7 isolates. LEE typing revealed specific allele distribution: eaegamma, tirgamma, espAgamma, espBgamma associated with EPEC O55:H7 and EHEC O157:H7 strains (B1/1 and EDL 933), eaealpha, tiralpha, espAalpha, espBalpha related to the 116I O157:H- strain and the GC148 strain presented non-typable LEE sequences. Multilocus enzyme profiles revealed two main clusters associated with specific LEE pathotypes. E. coli strains were discriminated by random amplification of polymorphic DNA-polymerase chain reaction and pulsed-field gel electrophoresis methodologies. The molecular approaches used in this study allowed the determination of the genetic relatedness among E. coli strains as well as the detection of lineage specific group markers.     

Prevalence of Escherichia coli O157 in cattle feeds in Midwestern feedlots

Comparisons of enrichment methods (with or without antibiotics and with or without a preenrichment step) using gram-negative (GN) broth or tryptic soy broth (TSB) were conducted with feeds inoculated with Escherichia coli O157:H7. TSB was more sensitive than GN broth, and TSB with a preenrichment step followed by TSB with antibiotics was more sensitive than plain TSB enrichment, in detecting E. coli O157 in inoculated feeds. Feed samples were collected from feed bunks from 54 feedlots to determine the prevalence of E. coli O157 in cattle feeds. TSB preenrichment followed by TSB with antibiotics and the standard GN broth enrichment were used for each feed sample. All samples underwent immunomagnetic separation and were plated onto sorbitol MacConkey agar with cefixime and potassium tellurite. Identification of E. coli O157 was based on indole production, positive latex agglutination for O157 antigen, API 20E test strip results, PCR for the eaeA gene, and the presence of at least one Shiga toxin. E. coli O157 was detected in 52 of 504 feed samples (10.3%) by using GN broth enrichment and in 46 of 504 feed samples (9.1%) by using TSB followed by TSB supplemented with cefixime and vancomycin. E. coli O157 was detected in 75 of 504 feed bunk samples (14.9%) by one or both methods. There was no correlation between E. coli O157 prevalence and generic coliform counts in feeds. The prevalence of E. coli O157 in cattle feed warrants further studies to increase our knowledge of the on-farm ecology of E. coli O157 in order to develop strategies to prevent food-borne disease in humans.     

Prevalence, antibiotic susceptibility, and diversity of Escherichia coli O157:H7 isolates from a longitudinal study of beef cattle feedlots

Prevalence, antibiotic susceptibility, and genetic diversity were determined for Escherichia coli O157:H7 isolated over 11 months from four beef cattle feedlots in southwest Kansas. From the fecal pat (17,050) and environmental (7,134) samples collected, 57 isolates of E. coli O157:H7 were identified by use of bacterial culture and latex agglutination (C/LA). PCR showed that 26 isolates were eaeA gene positive. Escherichia coli O157:H7 was identified in at least one of the four feedlots in 14 of the 16 collections by C/LA and in 9 of 16 collections by PCR, but consecutive positive collections at a single feedlot were rare. Overall prevalence in fecal pat samples was low (0.26% by C/LA, and 0.08% by PCR). No detectable differences in prevalence or antibiotic resistance were found between isolates collected from home pens and those from hospital pens, where antibiotic use is high. Resistant isolates were found for six of the eight antibiotics that could be used to treat E. coli infections in food animals, but few isolates were multidrug resistant. The high diversity of isolates as measured by random amplification of polymorphic DNA and other characteristics indicates that the majority of isolates were unique and did not persist at a feedlot, but probably originated from incoming cattle. The most surprising finding was the low frequency of virulence markers among E. coli isolates identified initially by C/LA as E. coli O157:H7. These results demonstrate that better ways of screening and confirming E. coli O157:H7 isolates are required for accurate determination of prevalence.     

Multiplex fluorogenic real-time PCR for detection and quantification of Escherichia coli O157:H7 in dairy wastewater wetlands

Surface water and groundwater are continuously used as sources of drinking water in many metropolitan areas of the United States. The quality of water from these sources may be reduced due to increases in contaminants such as Escherichia coli from urban and agricultural runoffs. In this study, a multiplex fluorogenic PCR assay was used to quantify E. coli O157:H7 in soil, manure, cow and calf feces, and dairy wastewater in an artificial wetland. Primers and probes were designed to amplify and quantify the Shiga-like toxin 1 (stx1) and 2 (stx2) genes and the intimin (eae) gene of E. coli O157:H7 in a single reaction. Primer specificity was confirmed with DNA from 33 E. coli O157:H7 and related strains with and without the three genes. A direct correlation was determined between the fluorescence threshold cycle (C(T)) and the starting quantity of E. coli O157:H7 DNA. A similar correlation was observed between the C(T) and number of CFU per milliliter used in the PCR assay. A detection limit of 7.9 x 10(-5) pg of E. coli O157:H7 DNA ml(-1) equivalent to approximately 6.4 x 10(3) CFU of E. coli O157:H7 ml(-1) based on plate counts was determined. Quantification of E. coli O157:H7 in soil, manure, feces, and wastewater was possible when cell numbers were >/=3.5 x 10(4) CFU g(-1). E. coli O157:H7 levels detected in wetland samples decreased by about 2 logs between wetland influents and effluents. The detection limit of the assay in soil was improved to less than 10 CFU g(-1) with a 16-h enrichment. These results indicate that the developed PCR assay is suitable for quantitative determination of E. coli O157:H7 in environmental samples and represents a considerable advancement in pathogen quantification in different ecosystems.     

Enumeration of Salmonella and Escherichia coli O157:H7 in ground beef, cattle carcass, hide and faecal samples using direct plating methods

AIM: To develop and validate high throughput methods for the direct enumeration of viable and culturable Salmonella and Escherichia coli O157:H7 in ground beef, carcass, hide and faecal (GCHF) samples from cattle. METHODS AND RESULTS: The hydrophobic grid membrane filtration (HGMF) method and the spiral plate count method (SPCM) were evaluated as rapid tools for the estimation of pathogen load using GCHF samples spiked with known levels of Salmonella serotype Typhimurium. Validation studies showed that for a single determination of each sample type the low end of the detection limits were approx. 2.0 x 10(0) CFU g(-1) for ground beef, 5.0 x 10(-1) CFU (100 cm(2))(-1) for Salmonella and 8.0 x 10(-1) CFU (100 cm(2))(-1) for E. coli O157:H7 on carcasses, 4.0 x 10(1) CFU (100 cm(2))(-1) for hide and 2.0 x 10(2) CFU g(-1) for faecal samples. In addition, ground beef (n = 609), carcass (n = 1520) and hide (n = 3038) samples were collected from beef-processing plants and faecal samples (n = 3190) were collected from feed-lot cattle, and these samples were tested for the presence of Salmonella and E. coli O157:H7 by enrichment and enumeration methods. CONCLUSIONS: The direct enumeration methods described here are amenable to high throughput sample processing and were found to be cost-effective alternatives to other enumeration methods for the estimation of Salmonella and E. coli O157:H7, in samples collected during cattle production and beef processing. SIGNIFICANCE AND IMPACT OF THE STUDY: Use of the methods described here would allow for more routine testing and quantification data collection, providing useful information about the effectiveness of beef processing intervention strategies.     

Escherichia coli O157:H7 in environments of culture-positive cattle

Outbreaks of Escherichia coli O157:H7 disease associated with animal exhibits have been reported with increasing frequency. Transmission can occur through contact with contaminated haircoats, bedding, farm structures, or water. We investigated the distribution and survival of E. coli O157:H7 in the immediate environments of individually housed, experimentally inoculated cattle by systematically culturing feed, bedding, water, haircoat, and feed bunk walls for E. coli O157:H7 for 3 months. Cedar chip bedding was the most frequently culture-positive environmental sample tested (27/96 or 28.15%). Among these, 12 (44.0%) of positive bedding samples were collected when the penned animal was fecal culture negative. Survival of E. coli O157:H7 in experimentally inoculated cedar chip bedding and in grass hay feed was determined at different temperatures. Survival was longest in feed at room temperature (60 days), but bacterial counts decreased over time. The possibility that urine plays a role in the environmental survival of E. coli O157:H7 was investigated. Cedar chip bedding moistened with sterile water or bovine urine was inoculated with E. coli O157:H7. Bedding moistened with urine supported growth of E. coli O157:H7, whereas inoculated bedding moistened with only water yielded decreasing numbers of bacteria over time. The findings that environmental samples were frequently positive for E. coli O157:H7 at times when animals were culture negative and that urine provided a substrate for E. coli O157:H7 growth have implications for understanding the on-farm ecology of this pathogen and for the safety of ruminant animal exhibits, particularly petting zoos and farms where children may enter animal pens.     

Escherichia coli with anti-O157:H7 activity isolated from bovine colon

AIMS: To isolate bacteria from bovine gastrointestinal tract and investigate their inhibitory effect on Escherichia coli O157:H7 in vitro. METHODS AND RESULTS: A total of 2400 bacterial colonies were isolated from cattle colonic mucous membrane. Thirteen strains demonstrated the ability to inhibit the growth of E. coli O157:H7. From these, seven were screened for the presence of virulence factors as: stx(1), stx(2), ehxA, eae, st1a and lt1 by polymerase chain reaction. The selected bacteriocin-producing bacteria showed susceptibility to most of the antibiotics used. CONCLUSIONS: The strains of E. coli isolated, which exhibit inhibitory activity on E. coli O157:H7 growth by the production of inhibitory substances, may be useful in the control of this pathogen in reservoirs. An important characteristic of these strains was the absence of any of the virulence factors assayed and the susceptibility to most of the antibiotics used for Gram-negative bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: These microorganisms might be used as probiotic bacteria to reduce the carriage of E. coli O157:H7 in cattle, thus limiting the contamination of carcasses at slaughter and subsequently the contamination of foods and the transfer of this pathogen to man.     

Experimental and field studies of Escherichia coli O157:H7 in white-tailed deer

Studies were conducted to evaluate fecal shedding of Escherichia coli O157:H7 in a small group of inoculated deer, determine the prevalence of the bacterium in free-ranging white-tailed deer, and elucidate relationships between E. coli O157:H7 in wild deer and domestic cattle at the same site. Six young, white-tailed deer were orally administered 10(8) CFU of E. coli O157:H7. Inoculated deer were shedding E. coli O157:H7 by 1 day postinoculation (DPI) and continued to shed decreasing numbers of the bacteria throughout the 26-day trial. Horizontal transmission to an uninoculated deer was demonstrated. Although E. coli O157:H7 bacteria were recovered from the gastrointestinal tracts of deer necropsied from 4 to 26 DPI, attaching and effacing lesions were not apparent in any deer. Results are similar to those of inoculation studies in calves and sheep. In field studies, E. coli O157 was not detected in 310 fresh deer fecal samples collected from the ground. It was detected in feces, but not in meat, from 3 of 469 free-ranging deer in 1997. In 1998, E. coli O157 was not detected in 140 deer at the single positive site found in 1997; however, it was recovered from 13 of 305 dairy and beef cattle at the same location. Isolates of E. coli O157:H7 from deer and cattle at this site differed with respect to pulsed-field gel electrophoresis patterns and genes encoding Shiga toxins. The low overall prevalence of E. coli O157:H7 and the identification of only one site with positive deer suggest that wild deer are not a major reservoir of E. coli O157:H7 in the southeastern United States. However, there may be individual locations where deer sporadically harbor the bacterium, and venison should be handled with the same precautions recommended for beef, pork, and poultry.     

Carriage of four bacterial pathogens by beef cattle in Northern Ireland at time of slaughter

AIMS: To determine the prevalence of four bacterial zoonotic pathogens in beef cattle at time of slaughter in Northern Ireland (NI), in order to assess their potential for reducing beef safety. METHODS AND RESULTS: Faeces were collected postmortem from beef cattle (n =220) at seven EU registered abattoirs. Standard enrichment culturing methods were employed, plus immunomagnetic enrichment in the case of Escherichia coli O157:H7. Campylobacter spp. were found in 52 samples (24.8%), Listeria monocytogenes in 10 (4.8%), E. coli O157:H7 in 2 (0.9%) whilst Salmonella spp. were isolated from six out of 200 samples (3.0%). Five salmonellas were Salmonella Chandans and one was Salmonella Liverpool. CONCLUSIONS: Campylobacter spp. were the most frequently isolated pathogen, despite being relatively rare in beef. Genotyping showed the campylobacters to be very diverse, indicating cattle encounter campylobacters from many sources. The remaining three pathogens, which are associated with meats, occurred at relatively low frequencies, especially E. coli O157:H7. The Salmonella serovars found rarely infect humans. SIGNIFICANCE AND IMPACT OF THE STUDY: The low prevalence of E. coli O157:H7 in NI beef cattle was confirmed and the reasons for this merit further study. The four pathogens should have little impact on beef quality.     

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.     

Isolation of potentially pathogenic Escherichia coli O157:H7 from the Ganges River

Escherichia coli serotype O157:H7 was detected among bacteria collected from the Ganges River. O157:H7 isolates tested positive for stx(1), stx(2), and eae gene sequences. Identification of potentially pathogenic isolates from extensively used source water indicates that O157:H7 may be a significant but as yet underacknowledged public health concern in India.     

Real-time PCR for the detection of Escherichia coli O157:H7 in dairy and cattle wastewater

AIMS: Developing and evaluating a rapid real-time polymerase chain reaction (PCR) method for the identification of Escherichia coli O157:H7 in cattle and dairy wastewater samples produced from mozzarella cheese factories, without pre-enrichment step before DNA extraction. METHODS AND RESULTS: Wastewater samples were collected from a dairy farm producing mozzarella cheese and located in Puglia (south of Italy). Plate count and other microbial assays were performed 1 h after sampling. Wastewater samples were artificially inoculated with 10(4), 10(7) and 10(8) cells ml(-1) of E. coli O157:H7, strain EDL933. PCR protocols for stx1, stx2 and eae genes were first tested on pure DNA extracted from type strains, in order to optimize the amplification conditions and reagent concentration before real-time PCR experiments. Three specific fragments of ca 106, 150 and 200 bp corresponding to genes eae, stx1 and stx2, respectively, were obtained. Real-time PCR experiments were performed with DNA extracted from dairy and manure wastewater samples inoculated with 10(4), 10(7) and 10(8) colony-forming units (CFU) ml(-1) of E. coli O157:H7 strain EDL 933. The sensitivity limit of the assay was 10(-1) pg microl(-1) for eae, stx2 and 16SrRNA, and 1 pg microl(-1) for stx1 gene respectively. CONCLUSIONS: A real-time PCR protocol has been developed and used in order to identify potential pathogens in dairy wastewater, in which previous methods (including standard PCR) failed to work. SIGNIFICANCE AND IMPACT OF THE STUDY: Cattle and dairy wastewater samples produced from mozzarella cheese factories may harbour verocytotoxin-producing E. coli. The availability of rapid and sensitive molecular methods may be useful to monitor the persistence of verocytotoxin-producing E. coli in general and to assess the effectiveness of wastewater treatments.