Cefixime Tellurite Sorbitol MacConkey Agar Research Articles

Characterization of Shiga Toxin-producing Escherichia coli Isolated from Cattle Around Ulaanbaatar City, Mongolia

Shiga toxin-producing Escherichia coli (STEC) are associated with severe infections including hemorrhagic colitis and hemolytic uremic syndrome in humans. Ruminants are known as reservoirs of STEC; however, no data are available on STEC in ruminants in Mongolia, where more than 5 million cattle and 25 million sheep are raised. To disclose the existence and characteristics of STEC in Mongolia, in this study, we isolated and characterized STEC from cattle in Mongolia. We collected 350 rectal swabs of cattle from 30 farms near Ulaanbaatar city and isolated 45 STEC from 21 farms. Rectal swabs were precultured with modified Escherichia coli broth and then inoculated to Cefixime-Tellurite Sorbitol MacConkey agar plate and/or CHROMagar STEC agar plate for the isolation of STEC. The isolation ratios in each farm were from 0% to 40%. Multiplex PCR for the estimation of O- and H-serotypes identified 12 O-genotypes (Og-types) and 11 H-genotypes (Hg-types) from 45 isolates; however, Og-types of 19 isolates could not be determined. Stx gene subtyping by PCR identified 2 stx1 subtypes (1a and 1c) and 4 stx2 subtypes (2a, 2c, 2d, and 2g). Forty-five isolates were divided into 21 different groups based on the Og- and Hg-types, stx gene subtypes and the existence of virulence factors, ehxA, eae, and saa, which includes several major serotypes associated with human illness such as O26:H11 and O157:H7. The most dominant isolate, OgUT:H19 [stx1a (+), stx2a (+), ehxA (+) and saa (+)], was isolated from eight farms. This is the first report on the characterization of STEC in cattle in Mongolia, and the results suggest the importance of further monitoring of STEC contamination in the food chains as well as STEC infection in humans.

Isolation, prevalence of bacteria and fungi from carcasses and different locations within Yola abattoir and antimicrobial resistance profile of Escherichia coli O157:H7

The absence of modern slaughtering facilities and an unhygienic environment in Yola abattoir may result in an outbreak of antibiotic-resistant pathogenic microorganisms, leading to economic loss and human death. Hence, this study was carried out to isolate and assess the prevalence of bacteria and fungi within the abattoir environment as well as to determine the antimicrobial resistance profile of E. coli O157:H7 from different locations and carcasses. A cross-sectional study was conducted where a simple random sampling method was used for sampling. A total of 156 samples were collected. Isolation and identification of bacteria and fungi were done using standard methods. Pseudomonas was the most dominant bacterial species with a total of 151 isolates, followed by E. coli 143 and Klebsiella with 123 isolates. The highest prevalence in fungi was recorded in Aspergillus flavus at 17.6 %, followed by Aspergillus niger at 15.1 % and Trichophyton rubrum and Penicillium that had 14.5 % respectively. The highest mean air-borne coliform count was in the Lairage, it ranged between 1.1 x 106 CFU/m3 and 1.7 x 106 CFU/m3. A hundred and forty-three E. coli were isolated out of which six (4.2 %) were characterized to be E. coli O157:H7 producing characteristic colorless colonies on cefixime tellurite sorbitol MacConkey (CT-SMAC) agar. All the six E. coli O157:H7 were resistant to at least two antibiotics, ampicillin, and amoxicillin-clavulanic acid while ciprofloxacin, trimethoprim-sulfamethoxazole, and tetracycline were effective against the six E. coli O157:H7. The MAR ranged between 0.2 and 0.7. The results obtained in this study showed that there is a need for modern facilities in Yola abattoir, training of workers on hygiene, and a hygienic environment to avoid microbial contamination and spread of antimicrobial-resistant pathogenic strains like E. coli O157:H7 and mycotoxin producer Aspergillus flavus.

Using CHROMagar™ STEC medium exclusively does not recover all clinically relevant Shiga toxin-producing Escherichia coli in Aotearoa, New Zealand.

Diagnostic laboratories in Aotearoa, New Zealand (NZ) refer cultures from faecal samples positive for Shiga toxin genes to the national Enteric Reference Laboratory for isolation of Shiga toxin-producing Escherichia coli (STEC) for epidemiological typing. As there was variation in the culture media being referred, a panel of 75 clinical isolates of STEC, representing 28 different serotypes, was used to assess six commercially available media and provide guidance to clinical laboratories. Recommendations were subsequently tested for a 3-month period, where STEC isolations and confirmations were assessed by whole genome sequencing analysis against the culture media referred. CHROMagar™ STEC (CH-STEC; CHROMagar Microbiology, Paris, France) or CH-STEC plus cefixime-tellurite sorbitol MacConkey agar was confirmed inferior to CH-STEC plus blood agar with vancomycin, cefsulodin, and cefixime (BVCC). The former resulted in fewer STEC types (n=18) being confirmed compared to those from a combination of CH-STEC and BVCC (n=42). A significant (P<.05) association with an STEC's ability to grow on CH-STEC and the presence of the ter gene cluster, and eae was observed. Culturing screen positive STEC samples onto both CH-STEC and BVCC ensures a consistently higher recovery of STEC from all clinical samples in NZ than CH-STEC alone.

Serological identification, virulence factors, antibiogram and plasmid profile of Escherichia coli serotypes in raw milk and pasteurized milk products

Milk is an excellent medium that supports microbial growth with resultant spoilage of the products. Thus, this study determined the antibiogram, presumptive location of antibiotic resistance markers (ARM), and plasmid DNA molecular weight of pathogenic Escherichia coli serotypes in raw milk (n = 76) and pasteurized milk products (n = 77). Quality of raw milk and pasteurized milk products was evaluated using Methylene Blue Reduction Test (MBRT). Occurrence of E. coli in the samples was determined by Cefixime tellurite-sorbitol MacConkey agar and latex agglutination kits. The virulence factors, antibiotic susceptibility, location of ARM, and plasmid DNA molecular weight of E. coli serotypes were assayed using bacteriological media, disc diffusion, acridine orange and agarose gel electrophoresis, respectively. Based on the MBRT, 2.6% samples were graded as excellent, 9.8% samples were of good quality, while 22.2% samples were of a very poor quality as decolourization of methylene blue occurred in ˂ 20 min. E. coli 0157 and E. coli 0145 had the highest and lowest percentage of occurrence, respectively; ≤ 29 E. coli serotypes were caseinase, amylase and lecithinase producers. E. coli serotypes were highly sensitive to Ciprofloxacin and Gentamycin with percentage sensitivity ranging from 74.6 % to 81.4 %; ≤ 50.0 % E. coli 0125 were sensitive to Ofloxacin; between 33.3 % and 50.0 % E. coli 0103 were resistant to Nalidixic Acid and Ofloxacin; ≥ 53.7 % E. coli serotypes were multi-drug resistant; 19.0 % had their entire ARM located on the plasmids, while 50.0 % had their ARM residing both on the plasmids and chromosomes with plasmids sizes ranging from 0.54 kb to 43.5 kb. The occurrences of pathogenic E. coli in the milk samples indicated the need to enlighten milkers on the hygienic practices to adopt during milking and as well showed that post-pasteurization contamination of milk products should be avoided.

Detection of Escherichia coli O157: H7 and Non-O157 Strains in Meat, Human Stool, Abattoir Wastewater and Hygiene Practices among Meat Peddlers in Port Harcourt, Nigeria

Aim: To determine the prevalent rates of Escherichia coli O157: H7 and non-O157 strains in of isolates from meat, human stool and abattoir waste water, collected from selected sources in Port Harcourt, Nigeria.&#x0D; Study Design: Case-controlled study.&#x0D; Place and Duration of Study: Selected places in Port Harcourt, Rivers State, Nigeria, between November, 2020 to November, 2021.&#x0D; Methodology: Three hundred and forty -nine (349) samples were analyzed, 80 meat and 63 waste waters from five abattoirs cited in the city, 46 meat samples from five selected sites sold by roadside butchers, 109 patient stool samples and 30 stool samples from food sellers, in addition to 20 stool samples from healthy subjects and 1 commercial bottled water which served as control samples. Tryptone soya broth (TSB) as an enrichment media, selective agar media namely; Eosin methylene blue agar, cefixime tellurite-sorbitol MacConkey agar (CT-SMAC), Chromagar STEC were used to isolate STEC from the samples and serology done with O157 latex agglutinating kit to confirm E. coli O157:H7 serogroup.&#x0D; Results: The results showed that Isolation rates obtained for E. coli O157:H7 and non O157 respectively were: abattoir meat 11(13.8%);13(16.3%), roadside meat 5(10.9%); 10(21.7%), clinical stool 7(6.4%); 27(24.7%), food sellers’ stool 4(13.3%);6(20%), waste abattoir water 2(3%); 14(22.2%). Food sellers and abattoir effluents were found to be potential sources of STEC dissemination in Port Harcourt. The data obtained were statistically analyzed using chi- square which showed that there was no significant difference (p &lt; 0.05) in the rate of isolation of E. coli O157: H7 and non- O157 strain from the samples.&#x0D; Conclusion: The detection of E.coli O157:H7 and non-O157 strains in raw meat (food), environment and clinical samples in Port Harcourt may give rise to a potential widespread public health hazard if strict adherence to proper hygiene management are not place to regulate food processing in other to reduce contamination and foodborne infections.

Prevalence and antibiotic resistance profile of Shiga-toxigenic Escherichia coli O157 (STEC) from retailed miscellaneous meat and fish types in Abuja, Nigeria

Most Escherichia coli strains are harmless intestinal bacteria of animals, but some are implicated in food infection/poisoning especially Shiga toxin (or Vero toxin) producing E. coli (STEC) due to consumption of meat. This study was conducted to determine the prevalence and antibiotic resistance profile of Shigatoxigenic Escherichia coli O157 (STEC) from retailed miscellaneous fish and meat types in Abuja, Federal Capital Territory, Nigeria. A total of 256 meat and fish consisting of cow muscles, intestines, rumen-sacs, livers and tails, cat-fish, frozen fish (mackerel and herrings) were examined. Escherichia coli were isolated by enrichment culture cefixime-tellurite sorbitol MacConkey agar (CT-SMAC), morphological, biochemical, serotype latex agglutination and disk diffusion methods. Of the 256 samples, 138 (53.9%) were contaminated with E. coli and 28 (21.7%) E. coli strains were positive for Shigatoxigenic Escherichia coli O157 (STEC). Meat muscles had the highest prevalence of STEC (7.41%) among meat samples, followed by rumen-sacs (6.0%), intestines (5.77%), tails (4.0%), and the prevalence of STEC in Fish includes Cat-fish intestine (26.7%), skin (21.4%), Mackerel intestine (26.7%), skin (14.3%), and Herrings skin (15.4%), gill (7.1%). All the STEC assessed indicated multi-drug resistance, with the isolates showing 100% resistant to ampicilin, and erythromycin, nitrofurantoin (95.7%), amoxicilin clavulanic acid (84.3%), sulphamethaxazole/trimethoprim (75%), streptomycin (75%), tetracycline (66.17%), and gentamycin (53.6%). The isolates were susceptible to ciprofloxacin (66.7%), Cefoxitin (66.7%), amikacin (39.3%), and chloramphenicol (35.7%). The implication of STEC in this study suggests that contaminated meat types are sold to consumers and can result to serious foodborne hazards. Prescription of ciprofloxacin and cefoxicilin are recommended against this organism. Application of good hygienic procedures in meat and fish handling processes and proper boiling before consumption can mitigate the risk of infection due to resistance STEC strains.

Occurrence of Multi-Drug Resistant Escherichia Coli and Escherichia Coli O157:H7 in Meat and Swab Samples of Various Contact Surfaces at Abattoir and Butcher Shops in Jimma Town, Southwest District of Ethiopia.

BackgroundRaw meat is one of the commonly consumed traditional diets in Ethiopia. However, unhygienic processing and distribution practices are risky for contamination of meat leading to human infection. This study was conducted to assess the presence of multi-drug resistant E. coli with special emphasis on E. coli O157:H7 from meat of cattle and swab samples at abattoir houses and butcher shops in Jimma town, Southwest district of Ethiopia.MethodologyA cross-sectional descriptive study was conducted from April to July, 2018. The isolation and identification processes passed through enrichment of samples with modified tryptone soy broth (mTSB), streaked onto MacConkey agar and Cefixime-tellurite sorbitol MacConkey agar, biochemical testing (indole and TSI), followed by latex agglutination testing.ResultsOut of 505 samples, 102 (20.2%) and 27 (5.4%) were positive for E. coli and E. coli O157:H7, respectively. Of these, 55 (19.3%) and 47 (21.4%) of E. coli and 17 (6.0%) and 10 (4.5%) of E. coli O157:H7 were isolated from the abattoir and butcher shop samples, respectively. A significant difference in the occurrences was observed among sample sources. Antimicrobial susceptibility test results showed that, 92.2% to 96.1% of E. coli and 85.5% to 96.3% of E. coli O157:H7 were susceptible to third generation cephalosporin, ciprofloxacin, gentamycin, kanamycin, streptomycin, and chloramphenicol. About 91.2% and 97.1% of E. coli and 88.9% and 92.6% of E. coli 0157:H7 were resistant to ampicillin and erythromycin, respectively. A total of 57 (44.2%) E. coli and E. coli O157:H7 isolates were resistant to three or more classes of antibiotics. All abattoir and butcher shop workers did not have any formal education or training certificates on food safety, and unhygienic practices were also observed.ConclusionThe presence of E. coli and E. coli O157:H7 including multi-drug resistant isolates in raw meat highlights how the current meat processing and distribution practice was unhygienic. Therefore, strategies in the prevention and control of food-borne infections that could be caused by multi-drug resistant strains will depend greatly on hygienic processing and distribution practices of meat.

Epidemiological Investigation, Serotypes and Distribution of Verocytotoxigenic &lt;i&gt;Escherichia coli&lt;/i&gt; (VTEC) in Raw Milk and Milk Products in Uyo, Nigeria

Food borne diseases are of great concern globally especially in the developing countries where poor sanitation is applied during collection and processing of milk from animals. The epidemiological investigation, serotypes and distribution of verocytotoxin (VTI and VT2)- producing Escherichia coli in raw milk and milk products were determined using structured questionnaire, Cefixime tellurite-sorbitol MacConkey agar, agglutination kits and VTEC-RPLA Toxin detection Kit. Out of 27 milkers, 7.4 % had primary education, 22.2 % washed the milk utensils with cold water and soap, 11.1 % washed their hands before milking, while 7.4 % milkers washed the udder of the animals before milking. All the yoghurts had the product names; 85.7 % had NAFDAC numbers; 80.0% had Batch Numbers, while 71.4 % had Manufacturer s’ Addresses. The unpasteurized milk samples had E. coli 0157 and non 0157 E. coli counts (CFU.ml-1) ranging from 4.0 x 102 to 1.7 x 103 and 6.0 x 102 to 2.0 x 103 , respectively, while E. coli 0157 and non 0157 E. coli counts of milk products were between 1.0 x 102 and 1.0 x 103 CFU.ml-1. E. coli 0157 had the highest percentage occurrence (38.3%), while E. coli 0145 had the lowest percentage occurrence (2.1%). More than 38.3% of the E. coli serotypes produced VT2, while ≥ 12.8% were VT1 producers. The occurrence of VTEC in the unpasteurized milk shows that the milkers should be enlightened on the necessary sanitary practices to adopt during milking and also post-pasteurization contamination of milk products should be avoided.&#x0D; Key Words: Verotoxigenic, Escherichia coli, Milk, Yoghurt, Nono, Serotypes.

Detection of Shiga-Toxin Producing E.coli (STEC), Enteropathogenic E.coli (EPEC) And Enterotoxigenic E.coli (ETEC) From Animals By M-PCR

This research investigated the presence of virulence genes encoding F41, K99, eae, Stx1, Stx2 and STa and the antimicrobial resistance of animal Escherichia coli (E. coli) isolates. Clinical isolates (n:233) were evaluated from faecal samples of cattle, sheep, goats, horses, cats and dogs collected between the years of 2010 to 2015 from Turkey. Enterohaemorrahic E.coli (EHEC) O157:H7 was detected by using cefixime tellurite sorbitol MacConkey agar (CT-SMAC) and Wellcollex E. coli (Remel®). The Kirby-Bauer disc diffusion test was performed to detect the resistance pattern of the isolates to ampicillin, Amoxycillin/clavulanic acid, enrofloxacin, ceftiofur, trimethoprim/sulfamethoxazole and tetracycline. The results showed that 40% of the ruminant isolates were identified as Shiga-toxin producing Escherichia coli (STEC). Enterotoxigenic E. coli (ETEC) was detected in samples from cattle (0.9%) and sheep (12%). Enteropathogenic E.coli (EPEC) was detected in samples from cattle (0.9%) and dogs (11.4%). EHEC O157:H7 was not detected any of the isolates. Among all E. coli isolates that carried at least one virulence gene, 8 (19%) were resistant to more than three antimicrobials, 7 (16.7%) were resistant to at least one antimicrobial and 27 (64.3%) were susceptible to all antimicrobials.

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This study was designed to determine the efficacy of probiotics mixture in the control of Shiga-toxin Escherichia coli (STEC) O157:H7 in experimentally infected lambs. Fifteen Yankassa breed of lambs aged between 3-4 weeks old were used. The lambs were divided into three groups of five lambs each (n=5).&nbsp; Group A: Neither probiotics nor STEC O157:H7 were administered (control), Group B: lambs were administered viable STEC O157:H7 cells at 6 x 108CFU/ml&nbsp; together with daily administration of probiotics&nbsp; mixture at 4.5 x108 CFU/ml, Group C: lambs were administered only viable STEC O157:H7 cells at 6x108 CFU/ml without probiotics. Faecal samples from all the experimental lambs were screened for the presence of STEC O157:H7 before the commencement of this study using Tryptone soy broth (TSB) as an enrichment media and Cefixine-tellurite sorbitol MacConkey agar (CT-SMAC) as a selective media. Following oral inoculation of the lambs with STEC cells, faecal samples were collected once weekly for six weeks, for STEC O157:H7 isolation and enumeration. STEC O157:H7 was confirmed by its reaction with O157 and H7 anti-sera (Wellcomexr). STEC O157:H7 was not shed by lambs in group A (control). However, Group B lambs administered a mixture of probiotics shed significantly lower (P&lt;0.05) counts of STEC cells in the six weeks post-infection than Group C lambs that received only STEC cells without probiotics. In conclusion, the use of probiotics mixture significantly (P&lt;0.05) reduced the faecal shedding of STEC O157:H7. It was therefore recommended that probiotics should be administered to lambs to help control these pathogens. Key words: Shiga-toxin producing Escherichia coli O157:H7, probiotics mixture, cefixime-tellurite Sorbitol MacConkey Agar, tryptone soy broth, anti-sera. &nbsp

Improving the Enrichment and Plating Methods for Rapid Detection of Non-O157 Shiga Toxin–Producing Escherichia coli in Dairy Compost

A culture method to detect non-O157 Shiga toxin-producing Escherichia coli (STEC) was optimized in this study. The finished dairy compost with 30% moisture content was inoculated with a cocktail of six non-O157 STEC serovars at initial concentrations of 1 to 100 CFU/g. Afterward, non-O157 STEC cells in the inoculated dairy compost were enriched by four methods, followed by plating onto cefixime-tellurite sorbitol MacConkey agar supplemented with 5 mg/liter novobiocin (CTNSMAC) and modified Rainbow agar containing 5 mg/liter novobiocin, 0.05 mg/liter cefixime trihydrate, and 0.15 mg/liter potassium tellurite (mRBA). Immunomagnetic bead separation (IMS) was used to compare the cell concentration of individual non-O157 STEC serotypes after enrichment. There was no significant difference (P > 0.05) between CTN-SMAC and mRBA for non-O157 STEC enumeration. The single-step selective enrichment recovered ca. 0.54 log CFU/g more cells (ca. 0.41 log CFU/g for compost-adapted cells) (P < 0.05) compared with the two-step enrichment. Furthermore, the duration of the process to detect non-O157 STEC from dairy compost by selective enrichment, followed by IMS, was optimized. Among six non-O157 STEC serotypes, serotypes O111, O45, and O145 reached the highest cell density after enrichment in dairy compost, and the cell populations reached 7.3, 7.4, and 7.8 log CFU/g within 16 h of incubation, respectively. In contrast, without an enrichment step, the IMS detection limit of individual non-O157 STEC serovars ranged from 3.15 to 4.15 log CFU/g in dairy compost. These results demonstrate that low levels of non-O157 STEC can be detected within 2 days from dairy compost by using a culture method with an optimized enrichment procedure followed by IMS.

Detection of Verocytotoxigenic &amp;lt;i&amp;gt;Escherichia coli&amp;lt;/i&amp;gt; O157 Serotype in Dairy Products in Abuja, Nigeria

Ruminants are recognized as healthy carriers of Verocytotoxigenic Escherichia Coli (VTEC) organisms and as such most dairy products may provide these bacteria with favourable conditions for their growth. A cross sectional study was conducted to detect the occurrence of VTEC O157 in dairy products in the Federal Capital Territory, Abuja, Nigeria. Raw milk, Nunu and yoghurt were analyzed using standard cultural and biochemical procedures to isolate typical E. coli. Isolated E. coli samples were sub-cultured into plates of sorbitol MacConkey and Cefixime Tellurite-Sorbitol MacConkey agar. Isolates that are sorbitol negative were further characterized using commercially procured latex agglutination test kits. A total of 367 samples were analyzed out of which 3 tested positive for VTEC O157 (108 of raw milk-1 (0.93%); 127 of Nunu-2 (1.57%) and 132 of yoghurt-none). There was no significant association (p > 0.05) between season and infection with VTEC O157 in dairy products. Dairy products remained a potential vehicle for VTEC O157 infection.

Identification of O Serotypes, Genotypes, and Virulotypes of Shiga Toxin-Producing Escherichia coli Isolates, Including Non-O157 from Beef Cattle in Japan

Bovines are recognized as an important reservoir of Shiga toxin-producing Escherichia coli (STEC). Although STEC strains are significant foodborne pathogens, not all of the STEC held by cattle are pathogenic, and which type of STEC that will become epidemic in humans is unpredictable. Information about the prevalence of serotype and virulence gene distribution in beef cattle is insufficient to develop monitoring and controlling activities for a food safety and security program. Thus, this study investigated the prevalence of O157 and non-O157 STEC in Japanese beef cattle and characterized the isolates by the type of O antigen and several virulence markers to help predict the pathogenicity. In this study, 64.2% (176 of 274) of enrichment cultures of fecal samples collected from an abattoir and farms were stx1 and/or stx2 positive by PCR. STEC strains were isolated from 22.1% (39 of 176) of the positive fecal samples, and these isolates represented 17 types of O antigen (O1, O2 or O50, O5, O8, O55, O84, O91, O109, O113, O136, O150, O156, O157, O163, O168, O174, and O177). Two selective media targeting major STEC groups, cefixime-tellurite sorbitol MacConkey agar and CHROMagar O26/O157, allowed isolation of a variety of STEC strains. The most frequently isolated STEC was O113 (8 of 39), which has previously been reported as a cause of foodborne infections. Although most of the O113 STEC isolated from infected patients possessed the enterohemolysin (hlyA) gene, none of the O113 STEC cattle isolates possessed the hlyA gene. The second most common isolate was O157 (6 of 39), and all these isolates contained common virulence factors, including eae, tir, lpf1, lpf2, and hlyA. This study shows the prevalence of O157 and non-O157 STEC in Japanese beef cattle and the relationship of O antigen and virulotypes of the isolates. This information may improve identification of the source of infection, developing surveillance programs or the current understanding of virulence factors of STEC infections.

Simultaneous Immunomagnetic Separation Method for the Detection of Escherichia coli O26, O111, and O157 from Food Samples

We performed a simultaneous immunomagnetic separation (IMS) assay on Shiga toxin–producing Escherichia coli (STEC) serogroups O26, O111, and O157 with immunobeads coated with O26, O111, or O157 antibodies that were simultaneously added to an aliquot of food culture. We also compared the usefulness of CHROMagar STEC medium against various selective isolation agars designed to test for the three serogroups. Samples of sliced beef, ground beef, and radish sprout were artificially contaminated with STEC O26, O111, and O157 strains after incubation in enrichment broth and were subjected to conventional and simultaneous IMS assays. Simultaneous IMS did not affect the sensitivity of target cell detection. For STEC O26, O111, and O157 inoculated into the enriched samples of sliced beef and radish sprout, the detection ability of CHROMagar STEC was similar to or exceeded that of other isolation agars. However for STEC O157 inoculated into ground beef cultures, cefixime tellurite sorbitol MacConkey agar (CT-SMAC) was the superior detection medium. The performance of simultaneous IMS combined with CT-SMAC and CHROMagar STEC detection is similar to that of the Japanese standard method for isolating E. coli O26, O111, and O157. However, the proposed approach involves the same time, materials, and labor costs as the standard E. coli O157 reference procedure but allows detection of three E. coli serotypes rather than a single strain.

Sorbitol-Fermenting, β-Glucuronidase–Positive, Shiga Toxin–Negative Escherichia coli O157:H7 in Free-Ranging Red Deer in South-Central Spain

We investigated the prevalence of Escherichia coli O157:H7 in free-ranging red deer in south-central Spain, to assess their potential as reservoir hosts of sorbitol-fermenting (SF) E. coli O157:H7 strains, which are emerging causes of hemolytic uremic syndrome in Europe. Fecal samples from 264 hunter-harvested Iberian red deer (Cervus elaphus) were collected in 25 different game estates and examined for E. coli O157:H7 by culture and PCR. E. coli O157:H7 was detected and isolated in 4 of the 25 game estates sampled (16%) and the isolates obtained (four in total) were further phenogenotypically characterized. One of them was biochemically typical of E. coli O157:H7, that is, neither fermented sorbitol nor exhibited β-glucuronidase (GUD) activity, and carried genes encoding Shiga toxins (Stx) 1 and 2, the intimin subtype γ1, the enterohemorrhagic E. coli (EHEC)-hemolysin, and the ter gene cluster. The rest of the isolates (three of four) fermented sorbitol, exhibited GUD activity after 18-24 h incubation, and carried genes encoding the intimin subtype γ1 and the EHEC-hemolysin, although no Stx-encoding genes were detected. All these atypical isolates carried the sfp gene cluster, lacked the ter gene cluster, and were unable to grow on cefixime tellurite sorbitol MacConkey agar, which are typical features of SF E. coli O157:H7 strains isolated from patients. In total, SF, GUD-positive, Stx-negative E. coli O157:H7 strains were isolated in 3 of the 25 game estates sampled (12%), with an overall sample-level prevalence of 1.1% (3/264). Our findings indicate that free-ranging red deer may be one of the possible reservoir hosts of Stx-negative derivatives of SF E. coli O157:H7.

Development and validation of a culture-based method suitable for monitoring environmental survival of Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium in developing countries

A non-GMO culture-based method suitable for studying the fate of Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium in an agricultural environment in developing countries was developed and validated. This method is based on the use of spontaneous rifampicin-resistant mutants of non-toxigenic variants of E. coli O157:H7 and S. Typhimurium combined with counting of colony-forming units (CFU) on selective media, i.e., Cefixime–Tellurite–Sorbitol MacConkey agar containing 100 μg/ml rifampicin, 50 μg/ml cycloheximide and 50 μg/ml nystatin for E. coli O157:H7 and Xylose–Lysine–Tergitol-4 agar containing the same antimicrobials for S. Typhimurium. Validation experiments using gfp- and ds-red-labelled derivatives of the rifampicin-resistant mutants in a non-sterile manure-soil matrix demonstrated that the new culture method was effective in the selection and recovery of the test strains without any detectable interference from background contaminants both in the short and long term. The rifampicin-resistant-based culture method designed provides a feasible low-cost option to study environmental survival of E. coli O157:H7 and Salmonella spp. in developing countries.

The occurrence of Escherichia coli O157:H7 in market and abattoir meat in Plateau State, Nigeria

Escherichia coli O157:H7 is a newly emerging pathogen frequently associated with the consumption of foods of bovine origin. The severity of the infections caused by this food borne pathogen in the young and the elderly has had a tremendous impact on human health and food industry. The present study evaluated the occurrence of E. coli O157:H7 in abattoir and market meat in seven selected sites in Plateau State, Nigeria. One hundred and forty samples of each of the various meat parts including muscle, liver, heart, kidney and intestine were collected to give a total of 700 meat samples. The samples were aseptically collected at monthly intervals from December 2008 to November 2009 after which they were transported to the laboratory for bacteriological analysis. Enrichment medium Tryptone Soya Broth (TSB) and selective agar media, namely; sorbitol MacConkey agar (SMAC)and cefixime tellurite-sorbitol MacConkey agar (CT-SMAC) were used for the isolation of E. coli O157:H7 from the various meat samples. The results of this study indicated that the intestinal samples had the highest percentage occurrence of the organism (3.39%), while muscle tissue had the lowest (0.43%). The organ meats had comparable percentage occurrence of the organism, with 1.0% for liver, 0.86% for kidney and 0.71% for heart samples. Significant difference (P 0.05) existed in the occurrence of the organism with respect to the sampling sites and the sources (abattoir and market). The presence of E. coli O157:H7 in beef should be regarded as being highly hazardous to consumers and handlers alike because of the severity of the infections associated with the organism. Thus, hygienic and good management of meats and meat environments are very essential.

P.004 Comparative evaluation of two commercial rapid tests able to detect hepatitis C virus (HCV) antibody: Flavicheck HCV versus ImmunoComb® HCV II

Numbers of Escherichia coli O157 in food may be low and sensitive techniques are therefore needed for its detection. The objectives of this study were to use carcass meat samples artificially inoculated with various strains of E. coli O157 to compare the sensitivity of enrichment in three different media and to compare immunomagnetic separation followed by culture of magnetic beads to cefixime tellurite sorbitol MacConkey agar with three immunoassays for the detection of E. coli O157 in the enrichment cultures. Duplicate 250, 25 and 2–3 CFU of each of 16 strains of E. coli O157 added to 25-g samples of beef carcass meat were used to compare the sensitivity of (1) enrichment in supplemented tryptone soya broth (sTSB), Reveal™ 8-h and Reveal™ 20-h media, and (2) immunomagnetic separation and culture to cefixime tellurite sorbitol MacConkey agar (IMS/CT-SMAC) with Reveal™, VIP™ and STAT™ immunoassays for detecting the organism. An initial inoculum of 250 CFU/25 g meat was detected in all 32 samples by IMS/CT-SMAC performed on all enrichment media and by Reveal™ performed on Reveal™ 8-h and Reveal™ 20-h media, but in only 30, 19 and 9 samples by Reveal™, VIP™ and STAT™, respectively, performed on sTSB medium. An initial inoculum of 25 CFU/25 g meat was detected in 28, 32 and 30 of 32 samples by IMS/CT-SMAC performed on sTSB, Reveal™ 8-h and Reveal™ 20-h media, respectively, and in 32 and 30 samples by Reveal™ performed on Reveal™ 8-h and Reveal™ 20-h media, but in only 22, 11 and 2 samples by Reveal™, VIP™ and STAT™, respectively, performed on sTSB medium. An initial inoculum of 2–3 CFU/25 g meat was detected in 25, 31 and 28 of 32 samples by IMS/CT-SMAC performed on sTSB, Reveal™ 8-h and Reveal™ 20-h media, respectively, and in 25 and 23 samples by Reveal™ performed on Reveal™ 8-h and Reveal™ 20-h media, but in only 14, 1 and 0 samples by Reveal™, VIP™ and STAT™, respectively, performed on sTSB medium.

Comparison of Methods for Detection and Isolation of Cold- and Freeze-Stressed Escherichia coli O157:H7 in Raw Ground Beef

A comparison was made of the relative efficiencies of three enrichment media, RapidChek Escherichia coli O157:H7 enrichment broth (REB), R&F broth (RFB), and modified E. coli broth containing novobiocin (mEC+n), and four selective plating media for detection of cold- and freeze-stressed E. coli O157:H7 in raw ground beef. Ground beef (25 g) was inoculated with E. coli O157:H7 at ≤0.5 and ≤2 CFU/g, and samples were then enriched immediately or were stored at 4°C for 72 h or at −20°C for 2 weeks and then enriched. After 8 or 20 h of enrichment, the cultures were plated onto R&F E. coli O157: H7 chromogenic plating medium, cefixime-tellurite sorbitol MacConkey agar, CHROMagar O157, and Rainbow agar O157 and tested using the RapidChek E. coli O157 lateral flow immunoassay and a multiplex PCR assay targeting the E. coli O157: H7 eae, stx1, and stx2 genes. Recovery of E. coli O157:H7 on the four agar media was 4.0 to 7.9 log CFU/ml with the REB enrichment, 1.4 to 7.4 log CFU/ml with RFB, 1.7 to 6.7 log CFU/ml with mEC+n incubated at 42°C, and 1.3 to 3.3 log CFU/ml from mEC+n incubated at 35°C. The percentages of positive ground beef samples containing nonstressed, cold-stressed, and freeze-stressed E. coli O157:H7 as obtained by plating, the immunoassay, and the PCR assay were 97, 88, and 97%, respectively, with REB, 92, 81, and 78%, respectively, with RFB, 97, 58, and 53%, respectively, with mEC+n incubated at 42°C, and 22, 31, and 25%, respectively, with mEC+n incubated at 35°C. Logistic regression analyses of the data indicated significant main effects of treatment, type of medium, enrichment time, inoculum concentration, and detection method. In particular, a positive result was 1.1 times more likely to occur after 20 h of enrichment than after 8 h, 25 times more likely with RFB and REB than with mEC+nat35°C, 3.7 times more likely with an initial inoculum of ≤2.0 CFU/g than with ≤0.5 CFU/g, 2.5 to 3 times more likely using freeze-stressed or nonstressed bacteria than with cold-stressed bacteria, and 2.5 times more likely by plating than by the immunoassay or the PCR assay. REB had better overall performance for enrichment of cold- and freeze-stressed E. coli O157:H7 present in ground beef than did the other media examined.

Presence of Shiga toxin-producing E. coli O157:H7 in a survey of wild artiodactyls

This study was carried out to evaluate the role of wild artiodactyls as reservoirs of Escherichia coli O157:H7 for livestock and humans. Retroanal mucosal swabs samples from 206 red deer ( Cervus elaphus), 20 roe deer ( Capreolus capreolus), 6 fallow deer ( Dama dama) and 11 mouflon ( Ovis musimon), collected during the hunting season (autumn–winter) in South-western Spain, were screened. Samples were pre-enriched in modified buffered peptone water, concentrated by an immunomagnetic separation technique and cultured onto selective cefixime tellurite sorbitol MacConkey agar. Polymerase chain reaction (PCR) was used to detect the presence of genes coding O157 and H7 antigens and the virulence factors verocytotoxin, intimin and enterohaemolysin. Three E. coli O157:H7 isolates were obtained from red deer (1.5%). Two of them showed inability to ferment sorbitol and lack of β- d-glucuronidase (GUD) activity, however, the other strain investigated was an atypical sorbitol-fermenting E. coli O157:H7 with GUD + activity. This is the first report pointing to red deer as a reservoir of E. coli O157:H7 in Spain.