Sorbitol MacConkey Agar Research Articles

Assessment of commercial chromogenic solid media for the detection of non-O157 Shiga toxin-producing Escherichia coli (STEC)

Detection of Shiga toxin-producing Escherichia coli (STEC) has evolved significantly since the introduction of sorbitol-MacConkey agar. This study compares four chromogenic media (CHROMagar™ STEC, Rainbow® O157 agar, CHROMagar™ O157, and Colorex® O157) in their identification of non-O157 STEC. When 161 non-O157 STEC were directly inoculated onto each medium, detection rates on CHROMagar™ STEC, Rainbow® O157 agar, CHROMagar™ O157 and Colorex® O157 were 90%, 70%, 3.7% and 6.8%, respectively. Tellurite minimal inhibitory concentrations (MICs) correlated with growth on CHROMagar™ STEC as 20 of 22 isolates with poor or no growth had MICs ≤1μg/mL. Stool spiking experiments revealed that CHROMagar™ STEC had the highest recovery of the six most common non-O157 STEC, ranging from 30% (in mucoid stool) to 98% (in watery stool). When using clinical stool samples, CHROMagar™ STEC had a sensitivity, specificity, positive predictive value, and negative predictive value of 84.6%, 87%, 13.9%, and 99.6%, respectively.

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.

Stable Flies (Stomoxys calcitrans L.) from Confined Beef Cattle Do Not Carry Shiga-Toxigenic Escherichia coli (STEC) in the Digestive Tract.

Stable flies (Stomoxys calcitrans L.) are very common around confined and pastured cattle, and due to their painful bites they are very important animal pests. Cattle are asymptomatic reservoirs of foodborne pathogens, Escherichia coli O157:H7 and other Shiga-toxigenic E. coli serotypes (STEC). In the present study, the potential of stable flies to carry STEC in a beef cattle feedlot was assessed. Stable flies (n = 180) were collected over 3 summer months and processed individually for STEC-8 that included the serotype O157 and seven non-O157 serotypes (O26, O45, O103, O104, O111, O121, and O145). Isolation and detection of STEC was based on direct plating as well as the enrichment/immunomagnetic separation approach. Modified Posse agar (mP) was used for culturing non-O157 serotypes and sorbitol MacConkey agar with cefixime and potassium tellurite (CT-SMAC) for E. coli O157. Multiplex polymerase chain reactions were used for differentiation of individual serotypes and detection of virulence genes (stx1, stx2, eae, and ehxA). Of 180 stable flies, 67 (37.2%) carried enterics on mP (mean: 3.6 ± 1.05 × 10(6) colony-forming units [CFU]/fly) and 55/180 (30.5%) were positive for bacteria on CT-SMAC (mean: 1.2 ± 1.08 × 10(4) CFU/fly). However, stable flies positive for E. coli serotypes of interest were very rare (prevalence: 1.1%). The three serotype-positive isolates, two E. coli O26 and one E. coli O45, were recovered from two flies and neither of them harbored the virulence genes. We conclude that stable flies likely do not play a role as a biological vector and/or reservoir of STEC-8 in cattle feedlots.

Detection, Virulence Gene Assessment and Antibiotic Resistance Pattern of O157 Enterohemorrhagic Escherichia coli in Tabriz, Iran.

Background:Shiga toxin-producing Escherichia coli (STEC) is a food-borne pathogen and infection with this organism causes illnesses such as bloody diarrhea, hemorrhagic colitis and hemolytic-uremic syndrome.Objectives:Considering the lack of any information about the prevalence rate and the antibiotic resistance pattern of O157:H7 serotype in Tabriz, finding answers to the above mentioned subjects was among the goals of this study.Materials and Methods:Two hundred E. coli strains from diarrheal or non-diarrheal stools of outpatients and hospitalized cases in Tabriz Imam Reza hospital were isolated between September and December 2014 using MacConkey agar and standard biochemical tests and then cultured on sorbitol MacConkey agar. The sorbitol-negative isolates were confirmed as the O157 serotype using O157 antisera. A multiplex polymerase chain reaction (PCR) method was used for the detection of stx-1, stx-2, eae, and mdh genes and the antibiotic resistance pattern of these isolates was determined using Kirby-Bauer method and clinical and laboratory standards institute (CLSI) standards.Results:Of the isolates 11 (5.5%) were sorbitol-negative, which were later analyzed by multiplex PCR and the results revealed that 2 (18.18%) isolates contained the stx-1 gene, 10 (90.91%) contained the stx-2 gene, and 5 (45.45%) contained the eae gene. The stx-2 and eae genes were the most commonly encountered virulence factors. All or most of the isolates were susceptible to ceftazidime (100%), gentamicin (100%), ciprofloxacin (100%), nalidixic acid (90.9%), trimetoprim sulfamethoxazole (90.9%), chloramphenicol (90.9%), ampicillin (81.8%), and cephalothin (72.7%). On the contrary, moderate susceptibility of the isolates to doxycycline (54.5%) was observed.Conclusions:Due to the low frequency of STEC O157 and the high susceptibility rates of the isolates to the tested antibiotics in this study, STEC O157 has not become a major problem in Tabriz yet, but comprehensive microbiological surveillance programs that provide early warning and limit the scale of possible outbreaks would be essential.

Isolation of Multidrug-Resistant Escherichia coli O157 from Goats in the Somali Region of Ethiopia: A Cross-Sectional, Abattoir-Based Study.

Toxigenic Escherichia coli (E. coli) are an important cause of gastroenteritis in developing countries. In Ethiopia, gastroenteritis due to food-borne disease is a leading cause of death. Yet, there is no surveillance for E. coli O157 and little is known about the carriage of this pathogen in Ethiopia’s livestock. This study aimed to assess the prevalence and levels of antimicrobial resistance of E. coli O157 in goat meat, feces, and environmental samples collected at a large abattoir in the Somali region of Ethiopia. The samples were enriched in modified tryptone broth containing novobiocin, and plated onto sorbitol MacConkey agar. Isolates were confirmed using indole test and latex agglutination. Antimicrobial susceptibility testing was conducted using the disk diffusion method. A total of 235 samples, including 93 goat carcass swabs, 93 cecal contents, 14 water, 20 hand, and 15 knife swabs were collected. Overall, six (2.5%) samples were contaminated with E. coli O157 of which two (2.1%) were isolated from cecal contents, three (3.2%) from carcass swabs, and one (7.1%) from water. All isolates were resistant to at least two of the 18 antimicrobials tested. Two isolates (33.3%) were resistant to more than five antimicrobials. Abattoir facilities and slaughter techniques were conducive to carcass contamination. This study highlights how poor hygiene and slaughter practice can result in contaminated meat, which is especially risky in Ethiopia because of the common practice of eating raw meat. We detect multi-resistance to drugs not used in goats, suggesting that drugs used to treat human infections may be the originators of antimicrobial resistance in livestock in this ecosystem. The isolation of multidrug-resistant E. coli O157 from goats from a remote pastoralist system highlights the need for global action on regulating and monitoring antimicrobial use in both human and animal populations.

Investigation into the Efficacy of Bdellovibrio bacteriovorus as a Novel Preharvest Intervention To Control Escherichia coli O157:H7 and Salmonella in Cattle Using an In Vitro Model

Cattle are an important reservoir for the foodborne pathogens Salmonella and Escherichia coli O157:H7; they frequently harbor these microorganisms in their digestive tracts and shed them in their feces. Thus, there is potential for contamination of cattle hides and, subsequently, carcasses. Interventions aimed at reducing or eliminating pathogen shedding preharvest will also reduce the likelihood of beef product contamination by these pathogens. Therefore, this study used an in vitro model to evaluate Bdellovibrio bacteriovorus, a gram-negative microorganism that preys upon other gram-negative microorganisms, as a preharvest intervention to control Salmonella and E. coli O157:H7. Rumen fluid and feces were inoculated with pansusceptible or antimicrobial-resistant strains of one pathogen. Control samples were treated with HEPES buffer, whereas experimental samples were exposed to HEPES buffer plus B. bacteriovorus. Salmonella and E. coli O157:H7 populations were quantified at 0, 24, 48, and 72 h. The most-probable-number (MPN) technique, followed by streaking onto xylose lysine Tergitol 4 agar, was used to determine Salmonella populations, whereas spread plating onto sorbitol MacConkey agar supplemented with cefixime and tellurite was employed to enumerate E. coli O157:H7. B. bacteriovorus reduced pansusceptible Salmonella in cattle feces by 2.02 Log MPN/g (P = 0.0005) and antimicrobial-resistant Salmonella by 3.79 (P < 0.0001) and 2.24 (P = 0.0013) Log MPN/g after 24 and 48 h, respectively, in comparison to control samples. Significant reductions were not observed for E. coli O157:H7 in rumen or feces. These data suggest that further investigation into B. bacteriovorus efficacy as a preharvest intervention to control Salmonella in cattle is warranted.

English

This study was conducted at the livestock/wildlife interface areas of national parks and traditional cattle grazing areas. The main aim of the study was to establish the occurrence of Escherichia coli serogroups isolated from Kafue lechwe antelopes (Kobus leche kafuensis) and pastoral cattle. A total of 593 faecal samples from lechwe (232) and pastoral cattle (361) were conveniently picked from the grazing pastures in the interface areas and cultured for E. coli. From each faecal sample, two or three presumptive E. coli isolates were picked to constitute 1,283 isolates with 574 from lechwe and 708 from cattle. Some of these isolates were found to be similar to Shiga toxin-producing E. coli O157 on CHROMagar and sorbitol MacConkey agar. Only 18 E. coli isolates from Kafue lechwe antelopes were grouped into eight serogroups while 32 from pastoral cattle were grouped into 16 serogroups. The most prevalent type-able serogroups from lechwe antelopes and pastoral cattle were O125 (5.8%) and O29 (4.2%), respectively. On further analysis by fermentation of various sugars, some isolates showed a similar pattern suggesting that the strains were indistinguishable. Key words: Escherichia coli, diarrheagenic, Shiga toxin, Kobus leche kafuensis, pastoral cattle.

Characterization of enteropathogenic and Shiga toxin-producing Escherichia coli in cattle and deer in a shared agroecosystem.

Shiga toxin-producing Escherichia coli (STEC) is an important foodborne pathogen. Cattle are suggested to be an important reservoir for STEC; however, these pathogens have also been isolated from other livestock and wildlife. In this study we sought to investigate transmission of STEC, enterohemorrhagic E. coli (EHEC) and enteropathogenic E. coli (EPEC) between cattle and white-tailed deer in a shared agroecosystem. Cattle feces were collected from 100 animals in a Michigan dairy farm in July 2012, while 163 deer fecal samples were collected during two sampling periods (March and June). The locations of deer fecal pellets were recorded via geographic information system mapping and microsatellite multi-locus genotyping was used to link the fecal samples to individual deer at both time points. Following subculture to sorbitol MacConkey agar and STEC CHROMagar, the pathogens were characterized by serotyping, stx profiling, and PCR-based fingerprinting; multilocus sequence typing (MLST) was performed on a subset. STEC and EHEC were cultured from 12 to 16% of cattle, respectively, and EPEC was found in 36%. Deer were significantly less likely to have a pathogen in March vs. June where the frequency of STEC, EHEC, and EPEC was 1, 6, and 22%, respectively. PCR fingerprinting and MLST clustered the cattle- and deer-derived strains together in a phylogenetic tree. Two STEC strains recovered from both animal species shared MLST and fingerprinting profiles, thereby providing evidence of interspecies transmission and highlighting the importance of wildlife species in pathogen shedding dynamics and persistence in the environment and cattle herds.

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.

Isolation EnterohemorrhagicEscherichia coli from gall bladder of buffaloes and determine its sensitivity for antibiotics

Enterohemorrhagic Escherichia coli (EHEC) has been associated with hemorrhagic colitis, a severe form of diarrhea, and with hemolytic uremic syndrome, This study was preformed to find EHEC in gall bladder of buffaloes , (150 gall bladder) samples were collected from slaughter house and cultured on MacConky agar then identified by culture on CefiximTellurite Sorbitol-MacConkey, agar then tested with several types of antibiotics and serotyping of isolates to determine EHEC, in addition to conjucation methods were preformed .Results showed that 13 isolates of E.coli were obtained and 6 strainS were grown on CefiximTellurite Sorbitol-MacConkey agar and positive for specific antisera of (EHEC O157:H7), and all strains were only sensitive for nitrofurane, When conjucation done all recipent cell were resistant for antibiotics: (ampicillin,streptomycin,sulfonamide,trimthoprine,tetracycline,chloramphenicol,gentamycin and amikacin).

Modification of methods for detection of Escherichia coli O157:H7 on produce

To establish the best detection method for Escherichia coli O157:H7 on produce, the sensitivity, specificity, and recovery values of 3 selective media [sorbitol MacConkey agar containing cefixime and tellurite (CTSMAC), sorbitol MacConkey agar containing cefixime, tellurite, and 5-bromo-4-chloro-3-indoxyl-β-glucuronide (CTB-SMAC), and CHROMagar O157 were compared. Sample preparation methods (pummeling, sonication, and hand-shaking) were compared to determine the optimum procedure for qualitative analysis from cherry tomato. The selectivity and recovery rates of stressed cell of the 3 tested media were in the order of CHROMagar O157>CTBSMAC>CT-SMAC. The recovery rates of hand-shaken tomatoes (10/10) were higher than rates of sonicated (8/10) and pummeled tomatoes (6/10) for inoculated cherry tomatoes in modified E. coli (mEC) broth enriched with novobicin. CHROMagar O157 was the optimum agar medium for detection of E. coli O157:H7. Cherry tomatoes should be treated with hand-shaking to enhance recovery for qualitative analysis.

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.

Kinetic Behavior of Escherichia coli on Various Cheeses under Constant and Dynamic Temperature.

In this study, we developed kinetic models to predict the growth of pathogenic Escherichia coli on cheeses during storage at constant and changing temperatures. A five-strain mixture of pathogenic E. coli was inoculated onto natural cheeses (Brie and Camembert) and processed cheeses (sliced Mozzarella and sliced Cheddar) at 3 to 4 log CFU/g. The inoculated cheeses were stored at 4, 10, 15, 25, and 30°C for 1 to 320 h, with a different storage time being used for each temperature. Total bacteria and E. coli cells were enumerated on tryptic soy agar and MacConkey sorbitol agar, respectively. E. coli growth data were fitted to the Baranyi model to calculate the maximum specific growth rate (μmax; log CFU/g/h), lag phase duration (LPD; h), lower asymptote (log CFU/g), and upper asymptote (log CFU/g). The kinetic parameters were then analyzed as a function of storage temperature, using the square root model, polynomial equation, and linear equation. A dynamic model was also developed for varying temperature. The model performance was evaluated against observed data, and the root mean square error (RMSE) was calculated. At 4°C, E. coli cell growth was not observed on any cheese. However, E. coli growth was observed at 10°C to 30°C with a μmax of 0.01 to 1.03 log CFU/g/h, depending on the cheese. The μmax values increased as temperature increased, while LPD values decreased, and μmax and LPD values were different among the four types of cheese. The developed models showed adequate performance (RMSE = 0.176–0.337), indicating that these models should be useful for describing the growth kinetics of E. coli on various cheeses.

Isolation of Enterohaemorrhagic &lt;i&gt;Escherichia coli&lt;/i&gt; O104 Strains from Raw Meat Products in the North West Province, South Africa

Enterohaemorrhagic <i>E. coli</i> (EHEC) are Shiga toxin-producing <i>E. coli</i> strains that possess unique pathogenic properties and are characterized by certain seropathotypes that are frequently associated with outbreaks or sporadic cases of human infections. Although EHEC strains belonging to the serotype O104:H4 has rarely been associated with human diseases in the past, outbreaks of infections caused by this organism resulted to HUS and bloody diarrhoea in humans in countries that have advanced public health facilities. The aim of this study was to isolate <i>E. coli </i>O104 strains from meat samples obtained from supermarkets and retail shops in the North West Province, South Africa. A further objective was to determine the identities of the isolates using Gram staining, preliminary biochemical tests (oxidase test, Triple Sugar Iron (TSI) test, citrate utilization and Sorbitol fermentation test) and confirmatory (API 20E and PCR analysis) assays. A total of nineteen meat samples were collected from four butcheries and shops in the study area and Sorbitol MacConkey agar was used for selective isolation of bacteria. A total of 304 presumptive colonies were subjected to identification tests for <i>E. coli</i>. Large proportions (65.5% to 93%) of these isolates were oxidase negative; fermented the sugars in the TSI medium; utilized citrate and fermented sorbitol. On the contrary only a small proportion (27.3%) of the isolates produced hydrogen sulphide gas. Based on patterns obtained for the biochemical profiles of the isolates 102 (33.6%) were positively identified as <i>E. coli</i>. All the 304 <i>E. coli</i> isolates were subjected to specific PCR designed to amplify the<i>wzx</i><SUB>O104</SUB> gene fragment that facilitates identification of <i>E. coli</i> O104 strains and a total of 52 isolates were positively identified. Despite the fact that not all samples were positive for the pathogen, the presence of <i>E. coli</i> O104 strains in some of the meat samples was of great concern. These bacteria cells have the potential to cause severe health effects on consumers if present in undercooked food products.

Occurrence and characterization of Shiga toxin-producing Escherichia coli O157:H7 and other non-sorbitol-fermenting E. coli in cattle and humans in urban areas of Morogoro, Tanzania.

Escherichia coli strains such as Shiga toxin-producing E. coli (STEC), enteropathogenic E. coli, enterotoxigenic, attaching, and effacing E. coli, and enteroinvasive E. coli cause diarrhea in humans. Although other serotypes exist, the most commonly reported STEC in outbreaks is O157:H7. A cross-sectional study was conducted to isolate and characterize non-sorbitol-fermenting (NSF) E. coli O157:H7 from urban and periurban livestock settings of Morogoro, Tanzania. Human stool, cattle feces, and soil and water samples were collected. Observations and questionnaire interview studies were used to gather information about cattle and manure management practices in the study area. E. coli were isolated on sorbitol MacConkey agar and characterized by conventional biochemical tests. Out of 1049 samples, 143 (13.7%) yielded NSF E. coli. Serological and antimicrobial tests and molecular typing were performed to NSF E. coli isolates. These procedures detected 10 (7%) pathogenic E. coli including STEC (n=7), enteropathogenic E. coli (EPEC) (n=2), and attaching and effacing E. coli (A/EEC) (n=1) strains. The STEC strains had the ability to produce VT1 and different VT2 toxin subtypes that caused cytopathic effects on Vero cells. The prevalence of STEC in cattle was 1.6%, out of which 0.9% was serotype O157:H7 and the overall prevalence of diarrheagenic E. coli in cattle was 2.2%. The serotypes O157:H7, O142:H34, O113:H21, O+:H-, O+:H16, and O25:H4 were identified. One ESBL-producing isolate showed the MLST type ST131. To our knowledge, this is the first finding in Tanzania of this recently emerged worldwide pandemic clonal group, causing widespread antimicrobial-resistant infections, and adds knowledge of the geographical distribution of ST131. Cattle manure was indiscriminately deposited within residential areas, and there was direct contact between humans and cattle feces during manure handling. Cattle and manure management practices expose humans, animals, and the environment to pathogenic E. coli and other manure-borne pathogens. Therefore, there is a need to improve manure management practices in urban and periurban areas to prevent pathogen spread and associated human health risks.

Distribution ofEscherichia colistrains harbouring Shiga toxin-producingE. coli(STEC)-associated virulence factors (stx1, stx2, eae, ehxA) from very young calves in the North Island of New Zealand

The objective of this study was to determine the distribution of Shiga toxin-producing Escherichia coli (STEC) virulence markers (stx1, stx2, eae, ehxA) in E. coli strains isolated from young calves aged fewer than 7 days (bobby calves). In total, 299 recto-anal mucosal swabs were collected from animals at two slaughter plants and inoculated onto tryptone bile X-glucuronide and sorbitol MacConkey agar supplemented with cefixime and potassium tellurite. Isolates were analysed using multiplex polymerase chain reaction to detect stx1, stx2, eae and ehxA genes. The most common combination of virulence markers were eae, ehxA (n = 35) followed by eae (n = 9). In total, STEC and atypical enteropathogenic E. coli (aEPEC) were isolated from 8/299 (2·6%) and 37/299 (12·3%) calves, respectively. All the isolates could be assigned to 15 genotype clusters with >70% similarity cut-off using XbaI pulsed-field gel electrophoresis. It may be concluded that healthy calves from the dairy industry are asymptomatic carriers of a diverse population of STEC and aEPEC in New Zealand.

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.

Detection of Escherichia coli O157:H7 and O104:H4 in patients with diarrhea in Northern Lebanon and characterization of fecal E.coli producing ESBL and carbapenemase genes

Background. While most strains of Escherichia coli (E. coli) are harmless, some are causing intestinal infections of varying severity. Then Shiga toxin-producing E. coli (STEC)/ enterohemorrhagic E. coli strains can be associated with fatal clinical manifestations. Of these E. coli Serotypes O157: H7 and O104: H4 were responsible for worldwide epidemics causing thousands of intestinal infections and dozens of deaths.The aim of this research is to investigate the prevalence of E. coli O157: H7Â and O104: H4 in the diarrheal stools of 242 Lebanese patients.Materials and methods. This study includes 242 E. coli strains isolatedfrom fecal specimens of patients with diarrhea between February2013 and May 2014 in the microbiology department of Nini HospitalLaboratory in Tripoli - North Lebanon. All specimens were inoculatedon sorbitol MacConkey agar. Sorbitol negative strains were investigated for detection of stx1, stx2 and eae genes using real-time PCR. All carbapenem-resistant strains and ESBL producers were investigated by PCR for presence of KPC, IMI, NMC-A, EMS, GHG, VIM, NDM, IMP,OXA-48, blaTEM, blaCTX-M, blaSHV, blaOXA, blaGES and blaPER..Results. A total of 14 sorbitol negative strains were detected. The search for stx1, stx2 and eae genes showed the presence of a single positive strain for E. coli O157: H7. Out of 242 E.coli strains, 48 (19.8%) were ESBL-positive, 4 (1.6%) were resistant to ertapenem, and all were negative for stx2 genes, The blaCTX-M gene was the most frequentamong ESBL positive strains (85%), followed by the blaTEM gene (50%). One strain had the blaNDM-1 gene, another had the blaOXA-48 gene and 2 strains were probably resistant due to impermeability.Conclusion. The results of this study demonstrate rarely presence of enterohemorrhagic E. coli, but shows the frequent presence of multidrug resistant E.coli in the intestinal flra of North Lebanese patients. Therefore, it is important to search for MDR E.coli in the intestinal flra of patients who are going to be treated with major operations or those admitted to intensive care units

Inactivation of sanitizer-injured Escherichia coli O157:H7 on baby spinach using X-ray irradiation

Chemical sanitizers are widely used to minimize cross-contamination during commercial flume washing of leafy greens. However, in addition to microbial inactivation, sanitizer exposure can also lead to sublethal injury. This study assessed the potential of sanitizer-induced cell injury to enhance resistance of Escherichia coli O157:H7 on baby spinach when X-ray irradiation was subsequently used as a microbial inactivation strategy. A 3-strain E. coli O157:H7 cocktail was exposed to a peroxyacetic acid-based sanitizer (PAS) at 5.2 ppm, a chlorine-based sanitizer (CS) at 5.2 ppm, both of which are commonly used in flume washing systems, or to a quaternary ammonium-based sanitizer (QAS) at 18 ppm to obtain 86–99% injury. Pre-irradiated, round-cut (2.54 cm diameter) baby spinach leaves were dip-inoculated by immersion in the injured cocktail for 5 min and then irradiated in Whirl-pak bags at doses of up to 0.063 kGy using a low-energy X-ray irradiator (Rayfresh Foods, Ann Arbor, MI). Healthy and injured survivors were respectively quantified by plating appropriate dilutions on Sorbitol MacConkey Agar (SMAC) overlayed with trypticase soy agar containing 0.6% yeast extract and SMAC. On inoculated spinach, E. coli O157:H7 injury decreased from 86–99 to 66, 63, and 1% for PAS-, CS-, and QAS-treated cells, respectively. D10-values for PAS-, QAS-, and CS-injured E. coli O157:H7 on baby spinach were 0.014 ± 0.000, 0.022 ± 0.001, and 0.024 ± 0.001 kGy, respectively. Prior exposure to PAS significantly (P < 0.05) enhanced E. coli O157:H7 susceptibility to X-ray irradiation, while exposure to CS significantly (P < 0.05) reduced susceptibility. These results suggest that PAS may be preferred for irradiated baby spinach.

Quantifying Transfer Rates of Salmonella and Escherichia coli O157:H7 between Fresh-Cut Produce and Common Kitchen Surfaces

Cross-contamination between foods and surfaces in food processing environments and home kitchens may play a significant role in foodborne disease transmission. This study quantifies the cross-contamination rates between a variety of fresh-cut produce and common kitchen surfaces (ceramic, stainless steel, glass, and plastic) using scenarios that differ by cross-contamination direction, surface type, produce type, and drying time/moisture level. A five-strain cocktail of rifampin-resistant Salmonella was used in transfer scenarios involving celery, carrot, and watermelon, and a five-strain cocktail of rifampin-resistant Escherichia coli O157:H7 was used in transfer scenarios involving lettuce. Produce or surface coupons were placed in buffer-filled filter bags and homogenized or massaged, respectively, to recover cells. The resulting solutions were serially diluted in 0.1% peptone and surface plated onto tryptic soy agar with 80 μg/ml rifampin and bismuth sulfite agar with 80 μg/ml rifampin for Salmonella or sorbitol MacConkey agar with 80 μg/ml rifampin for E. coli O157:H7. When the food contact surface was freshly inoculated, a high amount (>90%) of the inoculum was almost always transferred to the cut produce item. If the inoculated food contact surfaces were allowed to dry for 1 h, median transfer was generally >90% for carrots and watermelon but ranged from <1 to ∼70% for celery and lettuce. Freshly inoculated celery or lettuce transferred more bacteria (<2 to ∼25% of the inoculum) compared with freshly inoculated carrots or watermelon (approximately <1 to 8%). After 1 h of drying, the rate of transfer from inoculated celery, carrot, and lettuce was <0.01 to ∼5% and <1 to ∼5% for watermelon. Surface moisture and direction of transfer have the greatest influence on microbial transfer rates.