Chromogenic Agar Medium for Detection and Isolation of Escherichia coli Serogroups O26, O45, O103, O111, O121, and O145 from Fresh Beef and Cattle Feces (original) (raw)
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Journal of food protection, 2016
The isolation and quantification of non-O157 Shiga toxin-producing Escherichia coli (STEC) from cattle feces are challenging. The primary objective of this study was to evaluate the performance of selected agar media in an attempt to identify an optimal medium for the detection and quantification of non-O157 STEC in cattle feces. Comparison studies were performed using CHROMagar STEC, Possé differential agar (Possé), Possé modified by the reduction or addition of antimicrobials, STEC heart infusion washed blood agar with mitomycin C (SHIBAM), and SHIBAM modified by the addition of antimicrobials. Fourteen STEC strains, two each belonging to serogroups O26, O45, O103, O111, O121, O145, and O157, were used to test detection in inoculated fecal suspensions at concentrations of 10(2) or 10(3) CFU/g. One STEC strain from each of these seven serogroups was used to estimate the concentration of recovered STEC in feces inoculated at 10(3), 10(4), or 10(5) CFU/g. Significantly more suspensio...
Evaluation of detection methods for non-O157 Shiga toxin-producing Escherichia coli from food
International Journal of Food Microbiology, 2016
Shiga toxin-producing Escherichia coli (STEC) remains a major foodborne pathogen of concern across the globe. Rapid detection and isolation of this pathogen is of great importance for public health reasons. In this study the detection and isolation of four non-O157 STEC strains (O26, O103, O111, O145) from different artificially contaminated matrices, namely ground (minced) beef, cattle carcass swab, lettuce mix and sprouted soy beans, were evaluated. Low amounts of STEC were used (0.25-1.40 cfu/g) to spike the samples. All samples were enriched in parallel in Buffered Peptone Water (BPW) and Brila broth. After enrichment, detection was performed using real-time PCR (qPCR), and isolation using two chromogenic agar media, CHROMagar™ STEC and ChromID™ EHEC. Inoculation on the agar media was performed either directly after enrichment or after the use of an acid treatment procedure. Furthermore, the use of this procedure was also tested on naturally contaminated food products, using 150 stx-positive samples. Although the qPCR Cycle Threshold (Ct) values were lower after enrichment in Brila broth, no significant differences in recovery were observed between both enrichment broths. Both agar media were equally suitable for the isolation of STEC, although a significantly higher recovery was obtained when using both agar media in parallel. For samples with a Ct value above 25, an acid treatment step prior to isolation ensured a significant improvement in the recovery of STEC due to the reduction in background microbiota. This acid treatment procedure proved especially useful for the isolation of STEC from sprouted soy bean samples.
Current Trends in Detecting Non-O157 Shiga Toxin–Producing Escherichia coli in Food
Foodborne Pathogens and Disease, 2013
Non-O157 Shiga toxin-producing Escherichia coli (non-O157 STEC) strains are increasingly recognized as important foodborne pathogens worldwide. Together with E. coli O157:H7, six additional STEC serogroups (O26, O45, O103, O111, O121, and O145) are now regulated as adulterants in certain raw beef products in the United States. However, effective detection and isolation of non-O157 STEC strains from food matrices remain challenging. In the past decade, great attention has been paid to developing rapid and reliable detection methods for STEC in general (targeting common virulence factors) and specific STEC serogroups in particular (targeting serogroup-specific traits). This review summarizes current trends in detecting non-O157 STEC in food, including culture, immunological, and molecular methods, as well as several novel technologies.
Rapid isolation and detection of Escherichia coli O157:H7 by use of rainbow agar O157 and PCR assay
The Southeast Asian journal of tropical medicine and public health, 2000
This study has evaluated the use of a commercially available Rainbow agar O157 and polymerase chain reaction (PCR) assays for the detection of Shiga-like toxin producing Escherichia coli and to serotype E. coli O157:H7 from raw meat. The Rainbow agar O157 was found to be selective and sensitive for the screening of the E. coli O157 from artificially and naturally contaminated meat samples. Shiga-like toxin producing E. coli were identified with two primer pairs that amplified fragments of the SLT-I (384 bp) and SLT-II (584 bp). E. coli O157:H7 was serotyped with a primer pair specified for the H7 flagellar gene, which amplify specific DNA fragments (625 bp) from all E. coli O157:H7 strains. The use of Rainbow agar O157 described allows for the presumptive isolation of E. coli O157 in 24 hours. Identification and confirmation of the presumptive isolates as E. coli O157:H7 by PCR assays require additional 6-8 hours. The above-mentioned screening and identification procedures should pr...
Occurrence and quantification of Shiga toxin-producing Escherichia coli from food matrices
Aim: The objective of the study was to detect Shiga toxin-producing Escherichia coli (STEC) and develop a quantitative polymerase chain reaction (qPCR) assay to quantify the bacterial DNA present in different food matrices. Materials and Methods: A total of 758 samples were collected during a period from January 2015 to December 2016 from Kozhikode, Thrissur, and Alappuzha districts of Kerala. The samples consisted of raw milk (135), pasteurized milk (100), beef (132), buffalo meat (130), chevon (104), beef kheema (115), and beef sausage (42). All the samples collected were subjected to isolation and identification of STEC by conventional culture technique. Confirmation of virulence genes was carried out using PCR. For the quantification of STEC in different food matrices, a qPCR was standardized against stx1 gene of STEC by the construction of standard curve using SYBR green chemistry. Results: The overall occurrence of STEC in raw milk (n=135), beef (n=132), buffalo meat (n=130), chevon (n=104), and beef kheema (n=115) samples collected from Kozhikode, Thrissur, and Alappuzha districts of Kerala was 19.26%, 41.6%, 16.92%, 28.85%, and 41.74%, respectively. PCR revealed the presence of stx 1 and stx 2 genes in 88.46 and 83.64 and 30.77 and 40.00% of STEC isolates from raw milk and beef samples, respectively, while 100% of the STEC isolates from buffalo beef and beef kheema samples carried stx 1 gene. Real-time qPCR assay was used to quantify the bacterial cells present in different food matrices. The standard curve was developed, and the slopes, intercept, and R 2 of linear regression curves were −3.10, 34.24, and 0.99, respectively. Conclusion: The considerably high occurrence of STEC in the study confirms the importance of foods of animal origin as a vehicle of infection to humans. In the present study, on comparing the overall occurrence of STEC, the highest percentage of occurrence was reported in beef kheema samples. The study shows the need for rigid food safety measures to combat the potential pathogenic effects of harmful bacteria throughout the production chain from production to consumption.
Shiga toxin-producing Escherichia coli in food: Incidence, ecology, and detection strategies
Food Control, 2016
Commensal Escherichia coli are commonly utilized for investigating the genetic and biochemical requirements of microorganisms, and have served in a wide variety of applications. Pathogenic E. coli known as Shiga toxin (Stx)-producing E. coli (STEC) are associated with various food products including ground beef. These pathogens are present in a wide range of environments, and have caused numerous foodborne outbreaks and recalls. These outbreaks and the increased awareness of STEC have led to certain STEC serotypes to be declared adulterants in non-intact raw meat. Various STEC detection methods have been investigated, and numerous cultural and molecular-based detection methods continue to be modified to meet regulatory requirements. However, STEC serotypes may possess certain characteristics that lead to bias in the likelihood of a certain serotype being detected in an assay. Understanding the characteristics of these STEC serotypes will provide means for optimizing the detection platforms, and as a result limit foodborne illness and recalls caused by STEC due to enhanced cultural and molecular detection capabilities. 7 Gonzalez et al., (1998) observed higher numbers of E. coli in the feces of cattle that were fed diets consisting of higher amounts of grain. Additionally, when cattle diets were switched from a 90% grain diet to a hay diet, these authors observed nearly fold fewer E. coli after only five days from the diet alteration (Diez-Gonzalez et al., 1998). They also concluded that grain was more efficient with regard to cattle performance, and that strictly forage-based diets were unlikely to become commonplace in American cattle feed. However, they suggested that switching from a grain to forage diet prior to slaughter could potentially limit E. coli transmission from farm-to-slaughter (Diez-Gonzalez et al., 1998). Following their research on the influence of diets and the prevalence of E. coli O157:H7 in sheep, Kudva et al., (1997) suggested that ruminants fed a grain based diet exhibited less E. coli O157:H7 shedding compared to ruminants fed a forage based diet. These authors also suggested that dietary changes could create a more favorable environment for E. coli O157:H7 in ruminants (Kudva et al., 1997), while Hovde et al., (1999) observed differences in the duration of STEC shedding by cattle fed grain and forage based diets. E. coli O157:H7 (10 10 CFU) was administered into the rumen of cattle via a gastric tube, and these cattle were fed either grain or forage based diets. Fresh fecal samples were obtained every 3 to 4 days, and cultured for E. coli O157:H7. These authors were able to culture E. coli O157:H7 from forage fed cattle roughly 10 times longer than grain fed cattle-forage fed cattle contained E. coli O157:H7 for an average of 39 days (alfalfa hay) and an average of 42 days (grass hay), while grain fed cattle contained E. coli O157:H7 for an average of only 4 days (Hovde et al., 1999). Additionally, unlike Diez-Gonzalez et al., (1998) observations on increased recovery of acid-resistant E. coli from grain fed cattle, these authors found no differences in acid resistance of E. coli O157:H7 fed grain and forage based diets (Hovde et al., 1999). These contrasting results of E. coli and STEC levels in
Screening of food raw materials for the presence of Shiga toxin-producing Escherichia coli O91:H21
Letters in Applied Microbiology, 2009
Aims: To provide information on the prevalence and detection, in foods, of Shiga toxin-producing Escherichia coli (STEC) O91:H21. Methods and Results: Seven hundred fifteen minced beef meats and 205 raw milk samples were analysed by stx-specific PCR-ELISA. Samples positive for stx were subsequently tested for the presence of wzy-O91, fliC-H21 and the adhesin-encoding gene saa. For minced meat, 16 (2AE2%) and 11 (1AE5%) samples were found positive for (stx, wzy-O91, fliC-H21) and (stx, wzy-O91, fliC-H21, saa) combinations, respectively. For raw milk, seven (3AE4%) samples were found positive for the (stx, wzy-O91, fliC-H21) combination but none of these contained saa. Two STEC O91:H21 saa-positive strains and three STEC O91 H21-and saa-negative strains were isolated by colony hybridization. Conclusions: A low prevalence of potentially pathogenic STEC O91:H21 in food products was found using a combination of PCR assays targeting stx, wzy-O91, fliC-H21 and saa. Significance and Impact of the Study: The PCR-based approach described here represents a valuable method for rapid screening of food samples contaminated by STEC O91:H21.
Foodborne Pathogens and Disease, 2011
Six Shiga toxin-producing Escherichia coli (STEC) serogroups, which include O26, O45, O103, O111, O121, and O145, are responsible for the majority of non-O157 STEC infections in the United States, representing a growing public health concern. Cattle and other ruminants are reservoirs for these pathogens; thus, food of bovine origin may be a vehicle for infection with non-O157 STEC. Methods for detection of these pathogens in animal reservoirs and in food are needed to determine their prevalence and to develop intervention strategies. This study describes a method for detection of non-O157 STEC in ground beef, consisting of enrichment in modified tryptic soy broth at 428C, followed by real-time multiplex polymerase chain reaction (PCR) assays targeting stx 1 , stx 2 , and eae genes and the wzx gene in the O-antigen gene clusters of the six serogroups, and then immunomagnetic separation (IMS) followed by plating onto Rainbow Ò Agar O157 and PCR assays for confirmation of isolates. All ground beef samples artificially inoculated with 1-2 and 10-20 CFU=25 g of ground beef consistently gave positive results for all of the target genes, including the internal amplification control using the multiplex real-time PCR assays after enrichment in modified tryptic soy broth for a total of 24 h (6 h at 378C and 18 h at 428C). The detection limit of the real-time multiplex PCR assays was *50 CFU per PCR. IMS for O26, O103, O111, and O145 was performed with commercially available magnetic beads, and the IMS beads for O45 and O121 were prepared using polyclonal antiserum against these serogroups. A large percentage of the presumptive colonies of each serogroup picked from Rainbow Agar O157 were confirmed as the respective serogroups; however, the percent recovery of STEC O111 was somewhat lower than that of the other serogroups. This work provides a method for detection and isolation in ground beef and potentially other foods of non-O157 STEC of major public health concern.
Journal of Food Protection, 1996
Shiga-like toxin (SLT)-producing Escherichia coli (SLTEC) is the leading cause of acute renal failure among children. SLTEC are most commonly ingested from contaminated food, and because cattle are a major reservoir, ground beef and milk have been a significant source of contamination associated with multiperson outbreaks. While serotype O157:H7 has been principally identified in the United States there are many other SLTEC serotypes associated with human disease. We have therefore examined the utility of an enzyme immunoassay (EIA) for Shiga-like toxins as a means of detecting the presence of low levels of multiple SLTEC serotypes in ground beef and milk. In the present study we demonstrated that it is possible to detect low levels (approximately 1 SLTEC per g of ground beef) in both small-scale (2 g of beef per 5 ml) and standard large-scale (25 g of beef per 225 ml) food microbial cultures. The EIA was also capable of allowing detection of SLTEC in nonspiked retail ground beef sa...
Foodborne Pathogens and Disease, 2008
To detect various serotypes of Shiga toxin-producing Escherichia coli (STEC) in food, methods independent of serotyping are needed. We established procedures to isolate STEC using a rapid and sensitive loopmediated isothermal amplification (LAMP) assay targeting the Shiga toxin (ST) gene and a method of plating LAMP assay positive dilutions onto media for the selection of E. coli. After incubation, suspensions of a colony or some colonies were tested in the LAMP assay. Positive suspensions were diluted and plated onto selective media. The procedure was repeated. Finally, LAMP positive colonies were confirmed as STEC and serotype. As a result of surveillance in beef in 2005-2007, 11 of 720 samples (1.5%) tested positive for the ST gene by LAMP assay. Serotype O8, O128, and O-untypeable STEC were isolated from the samples by the newly established procedure. It was demonstrated that the procedure was effective for detecting STEC independent of serotype.