Molecular screening and characterization of Shiga toxin-producing Escherichia coli in retail foods

Shiga toxin-producing Escherichia coli (STEC) strains are one of the most important recently emerged groups of food borne pathogens. This study investigated the prevalence of molecular markers for STEC and characteristics of E. coli O157 isolates from foods sold at retail markets in Wuhan, China. A total of 489 samples (350 meat products and 139 raw vegetables) were purchased from 22 large scale markets between July of 2011 and September of 2013. The meat samples consisted of frozen chicken products, raw pork, raw beef, frozen fish products and processed duck products. The raw vegetable samples consisted of lettuce, bok choy, radish, spinach, cucumber, and tomato. Shiga toxin genes (stx1 and stx2) and an O-group marker of the seven main pathogenic STEC serogroups (O157, O26, O45, O103, O111, O121, and O145) were detected in the samples by using PCR. 100% agreement was obtained between the results of the PCR targeting for wzy_O157 and the PCR targeting for rfbE_O157 gene. The result demonstrated that PCR assay targeting for wzy_O157 gene can be employed as an effective screening method for E. coli O157 in food sample. In the study, E. coli O157 and non-O157 STEC were detected in 55 (11.2%) and 75 (15.3%) samples by PCR screening, respectively. There was significant difference in the occurrence of STEC contamination between supermarkets (19/127, 15.0%) and open markets (111/362, 30.7%) (P < 0.05). Out of 489 samples, 5 samples carried O45, 1 sample carried O145 and 1 sample carried O111. Markers for O103, O26 and O121 were not detected. This result differed from other reports. Immunomagnetic separation based cultivation technique was used to isolate E. coli O157 from 27 food samples collected in 2013. Finally 7 E. coli O157 isolates were obtained. Among the 7 isolates, the prevalent stx genotype was stx_1a and stx_2a. Four E. coli O157 strains exhibited toxic effects on Vero cells, while 3 isolates had no detectable cytotoxicity effects even though they contained stx genes. All E. coli O157 isolates were sensitive to the 12 antimicrobials tested except for roxithromycin. There are some inconsistencies between the PCR screening and culture results. Characteristics of STEC isolates should be evaluated and considered for monitoring STEC contamination in foods.     

Occurrence,serotypes and virulence genes of non-O157 Shiga toxin-producing Escherichia coli in fresh beef,ground beef,and beef burger

This study determined the prevalence, serotypes and virulence genes distribution of non-O157 Shiga toxin-producing Escherichia coli in meat products collected from butchers shops and supermarkets in Mansoura city, Egypt. We have characterized 18 non-O157 STEC strains among the identified 100 E. coli isolates recovered from the examined 87 meat product samples. The prevalence of non-O157 STEC strains in fresh beef, ground beef and beef burger samples were 11.1% (3/27), 16.7% (5/30), and 33.3% (10/30), respectively. The eighteen non-O157 STEC isolated strains were serotyped into seven (38.9%) O111:H8, six (33.3%) O26:H11, two (11.1%) O111:H–, and one (5.56%) for each of O55:H7, O126:H5 and O128:H2. PCR assays for different virulence genes showed that nine (50%), eleven (61.1%), and nine (50%) strains carry stx1, stx2, and eae genes, respectively. The distribution of shiga toxin genes among the isolated strains indicated that seven (38.9%) strains harbored stx1 only, nine (50%) strains harbored stx2 only, and two (11.1%) strains harbored both stx1 and stx2. The eae gene was present in association with five (27.8%), three (16.7%), and one (5.6%) strains that harbored stx1 only, stx2 only, and both stx1 and stx2, respectively. This study concluded that the examined meat products, particularly beef burger, consumed in Egypt are considerably contaminated with a variety of non-O157 STEC serotypes, and hence consumption of such products may constitute a potential health risk for consumers.     

Enteropathogenic (EPEC) and Shigatoxigenic Escherichia coli (STEC) in broiler chickens and derived products at different retail stores

Enteropathogenic (EPEC) and Shigatoxigenic Escherichia coli (STEC) are foodborne pathogens that cause potentially fatal infant diarrhea and hemolytic uremic syndrome, respectively. We investigated the presence of intimin and Shiga toxin encoding genes, as indicators of EPEC and STEC presence in cloacae and chicken products. The analyzed products were hamburgers, giblets and carcasses obtained from poultry and butcher shops. EPEC contamination predominated over STEC contamination in cloacae and chicken products, although some differences were detected when the kind of food or shop was taken into account. In particular, among chicken hamburgers we found a greater proportion of EPEC than STEC-positive samples at poultry shops, while in butcheries STEC was predominant. This finding could suggest cross contamination during handling at butcheries. The results indicate that it is necessary to improve hygienic measures both during slaughtering and manipulation of chicken products at retail stores, to provide a safe product to consumers.     

Comparison of the RAPID-B® flow cytometer and the BAX® system for the detection of non-O157 shiga toxin-producing Escherichia coli (STEC) in beef products

The beef industry continues to be interested in reliable rapid detection technologies for shiga toxin-producing Escherichia coli (STEC). Current rapid technologies require several hours of pre-enrichment and additional time on the rapid technology instrument. A flow cytometer-based system (RAPID-B^®) has been shown to improve the turn-around for results with a more rapid pre-enrichment requiring only 6.5 h pre-enrichment for a 25 g and 8.5 h for a 375 g sample, followed by an additional 30 min time to achieve final results using the screening technology. The purpose of this study was to validate the RAPID-B^® technology for non-O157 STEC detection as compared to the USDA-FSIS reference method which utilizes the BAX^® system. A total of 180 STEC isolates from various sources and 20 non-STEC strains were used to evaluate specificity and sensitivity using the RAPID-B^® flow cytometer. Also, three different weights (25, 325 and 375 g) of beef trim and ground beef samples were spiked with each STEC to verify detection sensitivity of BAX^® system and RAPID-B^® flow cytometer. For both methods, samples were confirmed by culturing using the USDA-FSIS reference method regardless of the screening result. The RAPID-B^® flow cytometer showed that 180 isolates were all positive and the 20 non-STEC strains were all negative. For spiked beef samples, overall detection sensitivity was the same for both the BAX^® system and RAPID-B^® flow cytometer. When detection sensitivity was based on sample weight, there was no differences in 25 and 375 g samples between RAPID-B^® flow cytometer and USDA-FSIS reference method. The RAPID-B^® system yielded the same sensitivity as the reference method with a decrease of over 10 h of pre-enrichment time and 3 h of rapid screening detection time. In conclusion, the RAPID-B^® flow cytometer based on whole cell detection generated similar results as BAX^® system therefore the RAPID-B^® flow cytometer system could be a valuable rapid method for the detection of non-O157 STEC in beef products.     

Formalin-fixed cells as an internal standard approach for the detection and quantitative assessment of Shiga toxin-producing Escherichia coli (STEC)

Several Shiga toxin-producing Escherichia coli (STEC) are considered adulterants in raw, non-intact ground meat, and numerous detection assays for these microorganisms have been investigated. The objective of this research was to determine the efficacy of formalin-fixed STEC cells as a potential means to calibrate various detection assays. Eight and ten percent formalin-fixed cells were compared with quantitative PCR (qPCR), and the results suggest that the ten percent formalin fixed cell populations were more stable for quantitative assays. These results were based on the approximation of cells/mL by means of phase contrast microscopy. Fixed cells were spiked into ground beef samples, and the recovery rate for each STEC serotype was determined by qPCR. Additionally, the log cells/mL of spiked samples were determined by qPCR and flow cytometry. The log cells/mL evaluated for each assay were determined to not be statistically significant, which suggests that formalin fixed cells could be used across a wide range of detection platforms (immuno-based versus molecular). Commercial detection kits could potentially incorporate formalin-fixed cells as a more standardized positive control to calibrate detection platforms simultaneously as well as among laboratories.     

Prevalence and trends of bacterial contamination in fresh fruits and vegetables sold at retail in Canada

In recent years, fresh fruits and vegetables have been linked to numerous foodborne illness outbreaks in different regions of the world, including in Canada. In light of rising concerns over the microbial safety of these commodities, the Canadian Food Inspection Agency conducted retail surveys to obtain information on the occurrence of bacterial pathogens in a wide range of produce available in the Canadian marketplace (local vs. imported, organic vs. conventional). Samples (n = 31,329) were collected across Canada over four years (2009–2013) and consisted of leafy vegetables (n = 12,073), leafy herbs (n = 6032), green onions (n = 3381), cantaloupes (n = 3230), tomatoes (n = 4837) and berries (n = 1776). These samples were analysed in ISO 17025-accredited laboratories for various bacterial pathogens (Salmonella, Escherichia coli O157, Shigella, Campylobacter and Listeria monocytogenes), as well as for generic E. coli, an indicator of fecal contamination. The Wilson confidence interval was used to determine the prevalence of the different micro-organisms in the commodities investigated. Control charts and seasonal indices, statistical tools adapted here to explore the large amount of data collected for each commodity, were used to identify potential adverse events or trends in bacterial contamination. The prevalence of bacterial contamination observed during this study in the six commodities examined was generally very low, with prevalence intervals ranging from [0, 0.08%] in tomatoes to [0.79, 1.30%] in leafy herbs. Most of the samples that were reported as “positive for bacterial contamination” had elevated (>100 CFU or MPN/g) levels of generic E. coli, but did not have detectable levels of the bacterial pathogens investigated. Of the samples that did have detectable levels of bacterial pathogens, the only bacteria that were both detected and isolated were Salmonella and L. monocytogenes. Despite the overall low prevalence of contamination seen in most produce, a notable seasonal trend was observed in the leafy vegetable group, where higher bacterial contamination rates were confirmed in the summer in organic as opposed to conventional products. These findings provide valuable baseline information that can support food safety decisions, and confirm that the vast majority of fresh fruits and vegetables available on the Canadian market are safe in terms of bacteriological hazards.     

Fat removal with hydrolyzed corn starch for real-time qPCR detection of Salmonella enterica in ground beef in 4.5 hours without enrichment

The rapid detection of low number of Salmonella in ground beef ideally requires an effective and economic molecular assay. The molecular analysis for the detection of Salmonella in ground beef by the polymerase chain reaction requires efficient methodology for extraction of targeted cells and effective removal of PCR inhibitors from the sample. The efficacy of hydrolyzed corn starch for the removal of fat along with the use of activated charcoal coated with milk proteins to remove PCR inhibitors were assessed. Salmonella enterica ser. Enteritidis was detected by real-time quantitative PCR at a level of 1 CFU/g of ground beef in 25 g samples containing 7%, 15% or 27% fat without enrichment. This study documents that partially hydrolyzed corn starch functions as effectively as beta-cyclodextrin for selective removal of fat.     

Prevalence,molecular identification and antimicrobial resistance profile of Salmonella serovars isolated from retail beef products in Mansoura,Egypt

The present study was undertaken to determine the prevalence of Salmonella in 270 raw meat samples (90 each of fresh beef, ground beef, and beef burger) purchased on nine occasions from various supermarkets and butchers' shops in Mansoura city, Egypt. Using conventional biochemical identification, Salmonella species were recovered from 23.3% (21/90), 20% (18/90), and 12.2% (11/90) of fresh beef, ground beef and beef burger samples, respectively with an overall prevalence of 18.5% (50/270) among all the meat products examined. Higher prevalence were obtained based on molecular identification, by detecting gyrB and invA genes, which verified the presence of Salmonella species in 30.0% (27/90), 26.7% (24/90), and 16.7% (15/90) of fresh beef, ground beef, and beef burger samples, respectively with an overall prevalence of 24.4% (66/270) among all the meat products tested. Of the 2635 presumptive colonies tested, 228 were biochemically identified as Salmonella, while 272 were molecularly identified as Salmonella, which were all positive for the enterotoxin (stn) virulent gene. Of the 272 serologically tested strains, 266 were serologically identified into six Salmonella serovars, while 6 strains were untypable. Salmonella Typhimurium and Salmonella Enteritidis were the most prevalent serovars with an incidence of 38.2% (104/272) and 34.6% (94/272), respectively. The other four serovars identified were Salmonella Haifa, Salmonella Muenster, Salmonella Virchow, and Salmonella Anatum were detected at lower prevalences of 11% (30/272), 7.4% (20/272), 4% (11/272) and 2.6% (7/272), respectively. Interestingly, the antimicrobial susceptibility testing indicated that all of the 100 Salmonella serovars tested were multidrug resistant (resistant to three or more antibiotics). Our findings demonstrated that the retail beef products tested were widely contaminated with multidrug-resistant Salmonella and such contamination may constitute a major public health concern.     

Development of a real-time PCR procedure for quantification of viable Escherichia coli in populations of E. coli exposed to lactic acid,and the acid tolerance of verotoxigenic E. coli (VTEC) from cattle hides

The aims of this study were to develop a real-time PCR procedure for determining the effects on Escherichia coli of treatment for decontaminating beef carcasses with lactic acid solution, and determining if there were differences in the acid tolerance of E. coli generally and verotoxigenic E. coli (VTEC). Suspensions of E. coli were incubated with 4% lactic acid at pH 3.6. The numbers of surviving E. coli at different incubation times were determined from plate counts and from quantification by real-time PCR of the uidA gene in DNA preparations. The numbers of viable E. coli progressively declined, by about 4 log units during incubation for 6 h. The mean cycle threshold (Ct) values for uidA in DNA from samples collected at different times and treated or not treated with propidium monoazide (PMA) before DNA extraction were similar. Treatment with 1% sodium deoxycholate (SD) before PMA treatment resulted in an increase of >6 Ct when the reduction in viable cell number was around 1 log. When E. coli incubated with 4% lactic acid solutions of pH 2.4, 2.8, 3.2 or 3.6 were resuscitated in half strength brain heart infusion (BHI) for 2 h before treatments with SD and PMA, the slope of the plot relating Ct values to the numbers of viable E. coli was 1.85 Ct log cfu⁻¹ with the correlation coefficient (R²) being 0.80. The findings indicate that while the membranes of E. coli inactivated by 4% lactic acid were largely impermeable to PMA, the membranes of both dead and injured cells were rendered permeable to PMA by treatment with 1% SD. Resuscitation in BHI restored the membrane barrier properties of the injured cells. Treatment with lactic acid resulted in increases in Ct values of 4.1, 3.7, 2.5 and 1.5 for the uidA, stx₁, stx₂ and eae genes, respectively; and the increases in Ct values for the latter two genes were significantly different (p < 0.05) from that for the uidA gene. This indicates that VTEC carrying stx₂ and/or eae were more acid resistant than other E. coli. Thus, caution should be exercised when using generic E. coli as an indicator for VTEC for assessment of the antimicrobial efficacy of organic acid decontaminating treatments at abattoirs.     

Growth characteristics of Shiga toxin-producing Escherichia coli (STEC) stressed by chlorine,sodium chloride,acid, and starvation on lettuce and cantaloupe

Shiga toxin-producing Escherichia coli (STEC) is a major foodborne pathogen causing serious illnesses and hospitalizations in the United States. Bacteria that are exposed to environmental stresses during food processing may exhibit different growth patterns in the subsequent growth environment. The purpose of this study was to examine the effect of environmental stresses on the growth of O15H and non-O157 STEC in lettuce or cantaloupe. Strains of O157:H7 and non-O157 STEC (O26:H11, O103:H1, O104:H4, and O145:NM) were subjected to four selected stresses including 2 ppm of chlorine, a_w of 0.97 (osmotic stress), and pH 5 (acid stress) at 22 °C for 24 h, or starvation (lack of nutrients) at 22 °C for 15 d. A cocktail mix of stressed or non-stressed (control) O157 or non-O157 at 3 log CFU/g (control or stressed) was inoculated on lettuce or cantaloupe and incubated at 10 and 22 °C for four weeks. While there were significant differences (p < 0.05) in the growth of stressed and unstressed cells of non-O157 STEC, no difference was observed in the growth of stressed and unstressed O157 STEC cells. Osmotic-stressed non-O157 STEC had significantly higher cell populations than control with 2 log difference (9.0 vs. 6.8 log CFU/g) at 10 °C on lettuce and 1 log difference (9.3 vs. 8.3 log CFU/g) at 22 °C on cantaloupe after 4 weeks. Acid-stressed non-O157 STEC had significantly higher cell populations than control at 10 °C after 4 weeks with >1 log difference (7.7 vs. 6.3 log CFU/ml) on cantaloupe. Starvation-stressed non-O157 STEC showed significantly higher cell populations than control with 1 log difference (8.4 vs. 7.2 log CFU/g) at 22 °C on cantaloupe after 4 weeks. The results indicated that osmotic, acid, or starvation stress may enhance the growth of non-O157 STEC on lettuce or cantaloupe and lead to a greater safety risk.     

Development of a controlling method for Escherichia coli O157:H7 and Salmonella spp. in fresh market beef by using polylysine and modified atmosphere packaging

Escherichia coli O157:H7 and Salmonella spp. often contaminate fresh beef. In Japan, an E coli outbreak caused by raw beef made 181 people ill and 5 individuals dead in 2011. Responding to this outbreak, an effective sterilization method for fresh beef is expected to be developed. In this study, ε-polylysine combined with CO₂-packaging method was examined for controlling these pathogens in fresh beef. At an incubation temperature of 4 °C, approximately 4.3 log and 2.4 log reduction in bacterial numbers were observed after 7-day incubation for E. coli O157:H7 and Salmonella, respectively, in ε-polylysine-added beef. When effectiveness of CO₂-packaging combined with ε-polylysine was investigated, CO₂ did not have additional inhibiting effect on bacterial growth compared to only-ε-polylysine-treated samples when incubated at 4 °C. However, effectiveness of CO₂ was observed when incubated at 10 °C where approximately 2.9 log and 4.4 log reduction in E. coli cell numbers were observed in only-ε-polylysine-treated samples and polylysine- and CO₂-treated group, respectively, and approximately 1.7 log and 3.5 log reduction in Salmonella cell numbers were observed in only-ε-polylysine-treated samples and polylysine and CO₂-treated group, respectively. This study confirmed that ε-polylysine or ε-polylysine combined with CO₂ packaging are effective in preventing foodborne diseases caused by raw beef.     

Radiation D10 values for Salmonella Typhimurium LT2 and an Escherichia coli cocktail in pecan nuts (Kanza cultivar) exposed to different atmospheres

Electron beam irradiation can help prevent outbreaks due to consumption of raw nuts. However, their high-fat content makes irradiation difficult and methods to reduce the dose required for decontamination (D₁₀-value) are needed; in addition, accurate D₁₀-values are currently lacking. Our objectives were (1) to determine the effect of irradiation set-up on the D₁₀-values of the populations of an Escherichia coli cocktail (BAA-1427, BAA-1428, BAA-1430) and Salmonella Typhimurium LT2 inoculated in pecans and (2) to assess the effect of Modified Atmosphere Packaging (MAP) conditions on the D₁₀-values.Pecan halves [Carya illinoinensis (Wangenh.) K. Koch cv. Kanza] were inoculated either in the dorsal grooves or on their flat surface and irradiated at room temperature using a 1.35-MeV e-beam accelerator at 0.2, 0.4, 0.6, and 0.8 kGy under vacuum-packaging (VP), air-packaging (AP), 100% nitrogen-packaging (NP), and 100% oxygen-packaging (OP). There were no (P > 0.05) differences in D₁₀-values between the microorganisms under the different atmospheres. The D₁₀-values obtained when pecans were irradiated under vacuum were higher (P < 0.05) for both microorganisms. Irradiation under nitrogen atmosphere seems a promising alternative because it will help delay the onset of rancidity, when compared to irradiation under atmospheric or 100%-oxygen conditions. The D₁₀-values obtained when the microorganisms were inoculated on the pecans' flat surface were 44–13% higher than when inoculated within the dorsal grooves, because the grooves absorbed higher doses. This finding illustrates the importance of proper irradiation set-up when treating raw, unshelled pecans, for accurate calculation of the D₁₀-values of selected microorganisms.     

A novel reference real-time PCR assay for the relative quantification of (game) meat species in raw and heat-processed food

In order to increase their profit, food producers may be tempted to replace expensive meat species by cheaper ones. Due to their selectivity and sensitivity, PCR based methods are frequently applied to identify and quantify meat species to detect food adulteration. Quantification of meat species in highly processed food products is, however, a difficult task. When we used a previously published reference system to relatively quantify the deer content in heat treated foodstuffs, we obtained recoveries that were substantially >100%. In the present study we aimed to improve the applicability of the reference system, targeting a 97 bp fragment of the myostatin gene, to heat treated products by reducing the length of the target sequence. The novel reference system, targeting a 70 bp fragment of the myostatin gene, was found to amplify the target region in 27 mammals and poultry species. Meat mixtures as well as raw and heat treated model sausages were analysed to demonstrate the applicability of the novel reference system for relatively quantifying the game meat content in processed food products. With the novel reference system, the bias introduced by heat treatment was largely eliminated.     

Suitability of ccRSM as a tool to predict inactivation and its kinetics for Escherichia coli,Staphylococcus aureus and Pseudomonas fluorescens in homogenized milk treated by manothermosonication (MTS)

The application of ultrasound (US) technology (i.e. sonication) has been used as an alternative to thermal technologies to reduce the microbial population of foods and beverages. This approach can be used on its own or combined with pressure and temperature (i.e. manothermosonication (MTS)). Microbial inactivation kinetics by sonication remains unclear. In the present study, a central composite response surface model (ccRSM) was used in order to study the effects of temperature (20–52 °C), acoustic intensity (60–120 W/cm²) and treatment time (40–240 s) at a constant pressure (225 kPa) by MTS processing on microbial inactivation. Reductions of up to c. 1.6 log CFU/ml were achieved for Escherichia coli and Pseudomonas fluorescens. Lower inactivation values were reported for Staphylococcus aureus (1.05 log CFU/ml). These inactivation values were achieved using conditions of 36 °C, 90 W/cm² and 240 s. Inactivation of E. coli by MTS (R² = 0.90) was described by an exponential curve, whereas inactivation of S. aureus and P. fluorescens by MTS (R² ≥ 0.73) were described by a linear trend. The coefficient of determination values were obtained following validation between the theoretical model and the experimental values. Statistical analysis showed that treatment time was the factor with greatest influence on microbial inactivation for all three microorganisms. The present study confirms the suitability of using ccRSM as a tool for investigating and predicting the inactivation of E. coli, S. aureus and P. fluorescens as a function of temperature, acoustic power and treatment time, and may also be useful for predicting inactivation of other microorganisms commonly found in raw milk.