Rapid and simultaneous quantitation of Escherichia coli 0157:H7, Salmonella, and Shigella in ground beef by multiplex real-time PCR and immunomagnetic separation

The objective of this study was to establish a multiplex real-time PCR for the simultaneous quantitation of Escherichia coli O157:H7, Salmonella, and Shigella. Genomic DNA for the real-time PCR was extracted by the boiling method. Three sets of primers and corresponding TaqMan probes were designed to target these three pathogenic bacteria. Multiplex real-time PCR was performed with TaqMan Universal PCR Master Mix in an ABI Prism 7700 Sequence Detection System. Final standard curves were calculated for each pathogen by plotting the threshold cycle value against the bacterial number (log CFU per milliliter) via linear regression. With optimized conditions, the quantitative detection range of the real-time multiplex PCR for pure cultures was 10(2) to 10(9) CFU/ml for E. coli O157:H7, 10(3) to 10(9) CFU/ml for Salmonella, and 10(1) to 10(8) CFU/ml for Shigella. When the established multiplex real-time PCR system was applied to artificially contaminated ground beef, the detection limit was 10(5) CFU/g for E. coli O157:H7, 10(3) CFU/g for Salmonella, and 10(4) CFU/g for Shigella. Immunomagnetic separation (IMS) was further used to separate E. coli O157:H7 and Salmonella from the beef samples. With the additional use of IMS, the detection limit was 10(3) CFU/g for both pathogens. Results from this study showed that TaqMan real-time PCR, combined with IMS, is potentially an effective method for the rapid and reliable quantitation of E. coli 0157:H7, Salmonella, and Shigella in food.     

GROWTH OF ESCHERICHIA COLI O157:H7 AND SALMONELLA SEROVARS ON RAW BEEF, PORK, CHICKEN, BRATWURST AND CURED CORNED BEEF: IMPLICATIONS FOR HACCP PLAN CRITICAL LIMITS

Small amounts (10–25 g; 6.3–20.8 cm² inoculated area) of raw ground beef, intact beef, pork and chicken (dark and white meat),and bratwurst and cured corned beef were inoculated with Salmonella serovars and Escherichia coli O157:H7, refrigerated 24 h at 5C, and then held either at 10C (± 1C) for up to 8 h or at room temperature (22C ± 2C) for up to 2 h. Except for a 0.2 log CFU increase in Salmonella serovars in ground beef during 2 h at room temperature, pathogens did not grow. Results of trials with commercial amounts of beef, pork, chicken, ground beef and bratwurst exposed to 10C for 8 h or 22C for 2 h also showed no pathogen growth. Potential critical limits for processing of previously refrigerated raw meat products are exposure temperatures between 5 and 10C for not more than 8 h or between 5 and 22C for not more than 2 h.     

Simultaneous detection of Escherichia coli O157:H7, Salmonella, and Shigella in apple cider and produce by a multiplex PCR

With three pairs of primers, a multiplex PCR assay was established for the simultaneous detection of Escherichia coli 0157:H7, Salmonella, and Shigella. Under the optimized conditions, the assay yielded a 252-bp product from E. coli O157:H7, a 429-bp product from Salmonella Typhimurium, and a 620-bp product from Shigella flexneri, respectively. When the DNA extraction of multiple target organisms was included in the same reaction, two or three corresponding amplicons of different sizes were observed. In the specificity test, 10 E. coli O157:H7 strains and one E. coli O157:NM strain showed the expected 252-bp amplicon. Seven other E. coli strains yielded no signal. Additionally, the 429-bp amplicon was produced from 20 Salmonella strains covering 16 serotypes, whereas the 620-bp amplicon was generated from 11 Shigella strains covering 4 species. No nonspecific amplification was observed with DNA from 48 other bacterial strains. Following a 24-h enrichment, the developed assay could concurrently detect the three pathogens at initial inoculation levels of approximately 8 x 10(-1) CFU/g (or CFU/ml) in apple cider, cantaloupe, lettuce, tomato, and watermelon and 8 x 10(1) CFU/g in alfalfa sprouts. The whole procedure can be easily completed within 30 h. The multiplex PCR assay can potentially be a simple, rapid, and efficient tool for presumptive and simultaneous screening of apple cider and produce for contamination by E. coli O157:H7, Salmonella, and/or Shigella.     

Multiplex PCR for simultaneous detection of Salmonella spp., Listeria monocytogenes, and Escherichia coli O157:H7 in meat samples

A multiplex PCR method was developed for simultaneous detection of Salmonella spp., Listeria monocytogenes, and Escherichia coli O157:H7 in meat samples. DNA detection sensitivity for this method was 10(3) CFU/ml for each pathogen. When this protocol was used for the detection of each of the above pathogenic bacteria in spiked pork samples, 1 cell per 25 g of inoculated sample could be detected within 30 h. In the samples of naturally contaminated meat, Salmonella spp., L. monocytogenes, and E. coli O157:H7 were detected over the same time period. Excellent agreement was obtained for the results of multiplex PCR and the conventional culture method, which suggests that the multiplex PCR is a reliable and useful method for rapid screening of meat products for Salmonella spp., L. monocytogenes, and E. coli O157:H7 contamination.     

Survival and growth of Escherichia coli O157:H7, Yersinia enterocolitica and Salmonella enteritidis on decontaminated and untreated meat

Decontamination of meat or carcasses may have an effect in reducing the number of pathogens. Recontamination with other pathogens during cutting or packaging may, however, result in higher growth on decontaminated than on untreated meat due to the lack of competing non-pathogenic microorganisms. In this study we compared the growth of pathogens during storage at 10°C (worst case condition) on untreated meat and meat that had been decontaminated by steam vacuuming combined with spraying with 0.2 M lactic acid. Salmonella enteritidis inoculated on chicken multiplied quickly and reached log 7 cfu per cm(2) after 4 days of aerobic storage at 10°C, but growth was not significantly higher on decontaminated than on untreated chicken. The number of Yersinia enterocolitica inoculated on decontaminated pork skin reached log 9 cfu per cm(2) after 5 days of aerobic storage at 10°C. Overall, growth on vacuum-packed decontaminated and untreated pork under the same conditions was not significantly different, although there tended to be less growth on the untreated samples. The number of Escherichia coli O157:H7 on decontaminated beef increased by nearly 3 log cycles after 5 days of aerobic storage at 10°C compared to only a 1 log cycle increase on untreated beef. For the vacuum-packed beef, growth of E. coli O157:H7 on the fresh meat was very slow, while there was about a 3 log increase on the decontaminated beef. A higher average growth on the decontaminated beef was also found in an experiment with a very low inoculum (27 cfu per cm(2)). During storage of vacuum-packed samples there was multiplication of E. coli O157:H7 on the decontaminated beef, but virtually none on the untreated beef. This study shows that multiplication of S. enteritidis on chicken and Y. enterocolitica on pork skin was not significantly higher on decontaminated compared to untreated meat. The increased multiplication of E. coli O157:H7 on decontaminated beef, especially when vacuum-packed, gives cause for concern. Preventive measures might be a strict HACCP approach to the handling of the decontaminated meat before packaging or use of a protective culture of lactic acid bacteria.     

Predicting pathogen growth during short-term temperature abuse of raw pork, beef, and poultry products: use of an isothermal-based predictive tool

A computer-based tool (available at: www.wisc.edu/foodsafety/meatresearch) was developed for predicting pathogen growth in raw pork, beef, and poultry meat. The tool, THERM (temperature history evaluation for raw meats), predicts the growth of pathogens in pork and beef (Escherichia coli O157:H7, Salmonella serovars, and Staphylococcus aureus) and on poultry (Salmonella serovars and S. aureus) during short-term temperature abuse. The model was developed as follows: 25-g samples of raw ground pork, beef, and turkey were inoculated with a five-strain cocktail of the target pathogen(s) and held at isothermal temperatures from 10 to 43.3 degrees C. Log CFU per sample data were obtained for each pathogen and used to determine lag-phase duration (LPD) and growth rate (GR) by DMFit software. The LPD and GR were used to develop the THERM predictive tool, into which chronological time and temperature data for raw meat processing and storage are entered. The THERM tool then predicts a delta log CFU value for the desired pathogen-product combination. The accuracy of THERM was tested in 20 different inoculation experiments that involved multiple products (coarse-ground beef, skinless chicken breast meat, turkey scapula meat, and ground turkey) and temperature-abuse scenarios. With the time-temperature data from each experiment, THERM accurately predicted the pathogen growth and no growth (with growth defined as delta log CFU > 0.3) in 67, 85, and 95% of the experiments with E. coli 0157:H7, Salmonella serovars, and S. aureus, respectively, and yielded fail-safe predictions in the remaining experiments. We conclude that THERM is a useful tool for qualitatively predicting pathogen behavior (growth and no growth) in raw meats. Potential applications include evaluating process deviations and critical limits under the HACCP (hazard analysis critical control point) system.     

Evaluation of universal preenrichment broth for growth of heat-injured pathogens.

Universal preenrichment broth (UPB) was developed to enable enrichment of injured foodborne pathogens of different genera simultaneously in lieu of having to undergo separate simultaneous enrichment cultures for subsequent detection or isolation of each pathogen. Enrichment conditions in UPB for growth of injured pathogens to populations that will enable pathogen detection by rapid immuno-based or polymerase chain reaction (PCR)-based assays have not been defined. Hence, studies were done to determine recovery and growth rates of heat-injured Escherichia coli O157:H7, Salmonella enterica ser. Typhimurium, Salmonella enterica ser. Enteritidis. and Listeria monocytogenes in UPB. Bacterial cells were heat injured in tryptic phosphate broth at 57.2 degrees C and inoculated at populations of ca. 0.17 to 63 injured cells per ml with raw ground beef, fresh chicken, lettuce, and environmental sponge samples. Enrichment cultures were sampled at 1, 2, 3, 4, 5, 6, and 24 h at 37 degrees C postinoculation, and pathogens were enumerated on appropriate selective media. Results revealed that recovery and growth of pathogens during the first 6 h of enrichment were not sufficient to ensure adequate numbers of bacteria (> 10(3) CFU/ ml) for detection by most immunoassays or PCR assays. Cells often required 3 to 4 h for recovery before growth was initiated. Salmonella Typhimurium, Salmonella Enteritidis, E. coli O157:H7, or L. monocytogenes cell populations in enrichment cultures with ground beef or lettuce at 6 h were 0.5 to 2.9 log10 CFU/ml. At 24 h of incubation, cell counts of enrichment samples for the three pathogens from all food and environmental sponge samples ranged from 4.0 to 8.3 log10 CFU/ml. Enrichment in UPB at 37 degrees C of foods or environmental sponge samples containing heat-injured cells of Salmonella Typhimurium, Salmonella Enteritidis, E. coli O157:H7, or L. monocytogenes reliably provides at 24 h of incubation-but not at 6 h-sufficient cell populations for detection by rapid immunoassay or PCR assay procedures that can detect at least 4 log10 CFU/ml. These results raise questions regarding the sensitivity of rapid detection methods that employ an abbreviated enrichment protocol of 6 h or less.     

Reduction of Escherichia coli O157:H7 and Salmonella in ground beef using lactic acid bacteria and the impact on sensory properties

Studies were conducted to determine whether four strains of lactic acid bacteria (LAB) inhibited Escherichia coli O157: H7 and Salmonella in ground beef at 5 degrees C and whether these bacteria had an impact on the sensory properties of the beef. The LAB consisted of frozen concentrated cultures of four Lactobacillus strains, and a cocktail mixture of streptomycin-resistant E. coli O157:H7 and Salmonella were used as pathogens. Individual LAB isolates at 10(7) CFU/ml were added to tryptic soy broth containing a pathogen concentration of 10(5) CFU/ml. Samples were stored at 5 degrees C, and pathogen populations were determined on days 0, 4, 8, and 12. After 4 days of storage, there were significant differences in numbers of both pathogens exposed to LAB isolates NP 35 and NP 3. After 8 and 12 days of storage, all LAB reduced populations of both pathogens by an average of 3 to 5 log cycles. A second study was conducted in vacuum-packaged fresh ground beef. The individual LAB isolates resulted in an average difference of 1.5 log cycles of E. coli O157:H7 after 12 days of storage, and Salmonella populations were reduced by an average of 3 log cycles. Following this study, a mixed concentrated culture was prepared from all four LAB and added to ground beef inoculated with pathogen at 10(8) CFU/g. After 3 days of storage, the mixed culture resulted in a 2.0-log reduction in E. coli O157:H7 compared with the control, whereas after 5 days of storage, a 3-log reduction was noted. Salmonella was reduced to nondetectable levels after day 5. Sensory studies on noninoculated samples that contained LAB indicated that there were no adverse effects of LAB on the sensory properties of the ground beef. This study indicates that adding LAB to raw ground beef stored at refrigeration temperatures may be an important intervention for controlling foodborne pathogens.     

Antimicrobial and antioxidant activities of natural extracts in vitro and in ground beef

Inhibition of Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes by grape seed extract (ActiVin) and pine bark extract (Pycnogenol) and the effect of these natural extracts on the oxidative stability of raw ground beef were studied. In an agar dilution test, the MICs of ActiVin and Pycnogenol were determined to be 4.0 mg/ml for 4.43 log CFU per plate of E. coli O157:H7 and 4.0 mg/ml for 4.38 log CFU per plate of L. monocytogenes. In an inhibition curve test, populations of E. coli O157:H7, Salmonella Typhimurium, and L. monocytogenes fell to below the detection limit (10 CFU/ml) after 16 h of incubation. The numbers of E. coli O157:H7, L. monocytogenes, and Salmonella Typhimurium declined by 1.08, 1.24, and 1.33 log CFU/g, respectively, in raw ground beef treated with 1% Pycnogenol after 9 days of refrigerated storage. ActiVin (1%) and oleoresin rosemary (1%) resulted in an approximately 1-log CFU/g reduction in the populations of all three pathogens after 9 days. The addition of 1% ActiVin and Pycnogenol contributed to the maintenance of an acidic pH of 5.80 and 5.58, respectively, in raw ground beef. Compared to the control, all treatments increased in L* (lightness), with the exception of ActiVin. ActiVin and oleoresin rosemary had the highest a* (redness) and b* (yellowness) values, respectively. ActiVin most effectively retarded lipid oxidation, followed by Pycnogenol. The results suggest that these natural extracts have potential to be used with other preservative methods to reduce pathogenic numbers, lipid oxidation, and color degradation in ground beef.     

Antimicrobial activity of cetylpyridinium chloride washes against pathogenic bacteria on beef surfaces

Cetylpyridinium chloride (CPC), a water-soluble, neutral pH, colorless compound, is widely used in oral hygiene products to inhibit bacteria responsible for plaque. Previously, researchers have demonstrated that CPC not only reduces Salmonella typhimurium on poultry but also prevents cross-contamination. To determine the effectiveness of CPC against pathogens associated with lean and adipose beef surfaces, several spray-washing experiments (862 kPa, 15 s, 35 degrees C) with 1% (wt/vol) CPC were conducted. On lean beef surfaces, CPC immediately reduced 5 to 6 log10 CFU/cm2 of Escherichia coli O157:H7 and Salmonella typhimurium to virtually undetectable levels (0 log10 CFU/cm2), as well as after 35 days of refrigerated (4 degrees C), vacuum-packaged storage. On adipose beef surfaces, 5 log10 CFU/cm2 Salmonella typhimurium and E. coli O157:H7 were reduced immediately (>2.5 log10 CFU/cm2) with 1% CPC; by day 35 the reduction was <1.3 log10 CFU/cm2. Further plate overlay analyses indicated that the effectiveness of CPC against pathogens on adipose surfaces was not hampered by the presence of meat components or fatty acids. Additional chemical and microbiological analyses of 1% CPC-treated beef surfaces subjected to a secondary water wash (following contact times of 0, 5, 10, 15, or 30 min) or grinding did reduce pathogenic bacteria and CPC levels. However, residual CPC levels following any of the treatments were considered excessive for human consumption. Despite the residual levels, this study is the first to demonstrate the effect of CPC on pathogenic bacteria associated with beef surfaces immediately after treatment and also after long-term, refrigerated, vacuum-packaged storage.     

三种食源性致病菌多重PCR检测方法的建立

目的:建立同时快速检测沙门氏菌、志贺氏菌和肠出血性大肠杆菌O157∶H7的多重PCR方法。方法:根据沙门氏菌的invA基因、志贺氏菌的ipaH基因及肠出血性大肠杆菌O157∶H7的uidA基因设计3对引物,通过单因素实验、L9(34)正交实验优化反应体系,并对多重PCR扩增的敏感性进行分析。结果:3对引物能特异性扩增出495、620、252bp的目的片段;在最优多重PCR反应体系下,多重PCR检测3种致病菌的灵敏度达104CFU/mL;将该法应用于人工污染实验,可在5h内得到准确、稳定的检测结果。结论:该方法操作简单、检测特异性和灵敏度较高,能够实现对沙门氏菌、志贺氏菌和肠出血性大肠杆菌O157∶H7 3种食源性致病菌的快速监控和诊断。      

Antimicrobial effect of herb extracts against Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella typhimurium associated with beef

The effects of plant extracts against pathogenic bacteria in vitro are well known, yet few studies have addressed the effects of these compounds against pathogens associated with muscle foods. A series of experiments was conducted to determine the effectiveness of a commercially available, generally recognized as safe, herb extract dispersed in sodium citrate (Protecta One) or sodium chloride (Protecta Two) against Escherichia coli O157:H7, Salmonella typhimurium, and Listeria monocytogenes associated with beef. In the first experiment, E. coli O157:H7, Salmonella typhimurium, and L. monocytogenes inoculated onto beef and subjected to surface spray treatments with 2.5% solutions of Protecta One or Protecta Two were not affected by immediate application (day 0) of the herbal extracts. However, after 7 days of storage at 4 degrees C, E. coli O157:H7 was reduced by >1.3 log10 CFU/cm2 by Protecta Two; L. monocytogenes was reduced by 1.8 and 1.9 log10 CFU/cm2 by Protecta One and Protecta Two, respectively; Salmonella typhimurium was not reduced >0.3 log10 CFU/cm2 by either extract by day 7. In the second experiment, 2.5% Protecta Two (wt/vol or wt/wt) added to inoculated lean and adipose beef trim, processed, and packaged as ground beef chubs (80% lean, 20% adipose), did not reduce pathogen populations >0.5 log10 CFU/g up to 14 days at 4 degrees C. In the third experiment, surface spray treatments of beef with 2.5% lactic acid or 2.5% solutions of Protecta One or Protecta Two, vacuum packaged, and stored up to 35 days at 4 degrees C did reduce E. coli O157:H7, L. monocytogenes, and Salmonella Typhimurium slightly. These studies suggest that the use of herb extracts may afford some reductions of pathogens on beef surfaces; however, the antimicrobial activity may be diminished in ground beef by adipose components.     

Bactericidal effects of Lactobacillus reuteri and allyl isothiocyanate on Escherichia coli O157:H7 in refrigerated ground beef

Two naturally occurring antimicrobial agents were tested in packages of refrigerated ground beef for their ability to reduce the viability of Escherichia coli O157:H7 during storage. Allyl isothiocyanate (AITC) and Lactobacillus reuteri were tested separately and together for their action against a cocktail of five strains of E. coli O157:H7 in ground beef held at 4 degrees C for 25 days. Ground beef prepared from whole, raw inside round beef roasts was inoculated with low (3 log CFU/g) or high (6 log CFU/g) levels of the E. coli O157:H7 mixture. The beef was treated with AITC (about 1,300 ppm), L. reuteri, or both, along with 250 mM of glycerol per kg of meat at two levels (3 and 6 log CFU/g) and according to a design that yielded 8 controls plus 10 different treatments. Samples were analyzed for E. coli O157:H7 survivors, numbers of total bacteria, and lactic acid bacteria on days 0 to 25 at 5-day intervals. L. reuteri at both input levels with glycerol killed E. coli O157:H7 at both inoculated levels before day 20. AITC completely eliminated E. coli O157:H7 at the low-inoculum level (3 log CFU/g) and reduced viability >4.5 log CFU/g at the high-inoculum level (6 log CFU/g) by the end of the storage period. The combination of L. reuteri and AITC did not yield an additive effect against E. coli O157:H7 viability. L. reuteri in the presence of glycerol was highly effective against E. coli O157:H7 in ground beef during refrigerated storage (4 degrees C) in modified atmosphere packages. Sensory testing is planned to evaluate effects of treatments.     

Validation of the use of organic acids and acidified sodium chlorite to reduce Escherichia coli O157 and Salmonella typhimurium in beef trim and ground beef in a simulated processing environment

A study was conducted to determine if acidified sodium chlorite (1,200 ppm) and acetic and lactic acids (2 and 4%) were effective in reducing foodborne pathogens in beef trim prior to grinding in a simulated processing environment. The reduction of Salmonella Typhimurium and Escherichia coli O157:H7 at high (4.0 log CFU/g) and low (1.0 log CFU/g) inoculation doses was evaluated at various processing steps, including the following: (i) in trim just after treatment application, (ii) in ground beef just after grinding, (iii) in ground beef 24 h after refrigerated storage, (iv) in ground beef 5 days after refrigerated storage, and (v) in ground beef 30 days after frozen storage. All antimicrobial treatments reduced the pathogens on the trim inoculated with the lower inoculation dose to nondetectable numbers in the trim and in the ground beef. There were significant reductions of both pathogens in the trim and in the ground beef inoculated with the high inoculation doses. On the trim itself, E. coli O157:H7 and Salmonella Typhimurium were reduced by 1.5 to 2.0 log cycles, with no differences among all treatments. In the ground beef, the organic acids were more effective in reducing both pathogens than the acidified sodium chlorite immediately after grinding, but after 1 day of storage, there were no differences among treatments. Overall, in the ground beef, there was a 2.5-log reduction of E. coli O157:H7 and a 1.5-log reduction of Salmonella Typhimurium that was sustained over time in refrigerated and frozen storage. Very few sensory differences between the control samples and the treated samples were detected by a consumer panel. Thus, antimicrobial treatments did not cause serious adverse sensory changes. Use of these antimicrobial treatments can be a promising intervention available to ground beef processors who currently have few interventions in their process.     

A new 7-plex PCR assay for simultaneous detection of shiga toxin-producing Escherichia coli O157 and Salmonella Enteritidis in meat products

A 7-plex PCR assay was developed to achieve an effective detection and identification of serotype Enteritidis of Salmonella spp. and shiga toxin-producing type of Escherichia coli O157 in meat products. Six DNA sequences in the invA and sdfI genes of Salmonella Enteritidis as well as rfbE, eae, stx1, and stx2 genes of E. coli O157:H7 were employed to design primers. The multiplex PCR assay could specifically and sensitively detect and identify target pathogens. Applying the assay to meat samples, the multiplex PCR assay was able to simultaneously detect and identify the two pathogens at a sensitivity of three CFU/10 g raw meats after simple 16 h enrichment in buffered peptone water. Further applying in 21 retail meat samples revealed that two samples were positive for non-shiga toxin producing E. coli O157, one sample was positive for Stx2 producing E. coli O157 and five samples were positive for Salmonella enterica Enteritidis. Taken together, the 7-plex PCR assay is a rapid and reliable method for effectively screening single or multiple pathogens occurrences in various meat products, and could also identify the Salmonella enterica Enteritidis from all Salmonella spp. and shiga toxin producing type from all E. coli strains. Considering as a non expensive screening tool, the multiplex PCR assay has a great potential in complement for food stuff analysis by conventional microbiological tests.     

A new 7-plex PCR assay for simultaneous detection of shiga toxin-producing Escherichia coli O157 and Salmonella Enteritidis in meat products

A 7-plex PCR assay was developed to achieve an effective detection and identification of serotype Enteritidis of Salmonella spp. and shiga toxin-producing type of Escherichia coli O157 in meat products. Six DNA sequences in the invA and sdfI genes of Salmonella Enteritidis as well as rfbE, eae, stx1, and stx2 genes of E. coli O157:H7 were employed to design primers. The multiplex PCR assay could specifically and sensitively detect and identify target pathogens. Applying the assay to meat samples, the multiplex PCR assay was able to simultaneously detect and identify the two pathogens at a sensitivity of three CFU/10?g raw meats after simple 16?h enrichment in buffered peptone water. Further applying in 21 retail meat samples revealed that two samples were positive for non-shiga toxin producing E. coli O157, one sample was positive for Stx2 producing E. coli O157 and five samples were positive for Salmonella enterica Enteritidis. Taken together, the 7-plex PCR assay is a rapid and reliable method for effectively screening single or multiple pathogens occurrences in various meat products, and could also identify the Salmonella enterica Enteritidis from all Salmonella spp. and shiga toxin producing type from all E. coli strains. Considering as a non expensive screening tool, the multiplex PCR assay has a great potential in complement for food stuff analysis by conventional microbiological tests.     

Evaluation of peroxyacetic acid as a potential pre-grinding treatment for control of Escherichia coli O157:H7 and Salmonella Typhimurium on beef trimmings

Peroxyacetic acid was evaluated in four separate trials for ability to reduce populations of Escherichia coli O157:H7 and Salmonella serotype Typhimurium on fresh beef trim. Trial 1 examined the effectiveness of peroxyacetic acid on individual pieces of fresh beef trim. Trial 2 evaluated the efficacy of peroxyacetic acid at low levels of contamination on batches of fresh beef trim. Trial 3 studied a washing effect of water. Lastly, Trial 4 compared the effectiveness of peroxyacetic acid to lactic acid. At various inoculation levels, peroxyacetic acid reduced populations of both pathogens by approximately 1.0log(10)CFU/cm(2) on fresh beef trim. Trial 3 showed that approximately half of the reductions found in Trials 1 and 2 were due to a washing effect of the water dip. In addition, as shown in Trial 1, increases in concentrations (>200ppm) did not significantly increase log(10) reductions of both pathogens. Following a water dip in Trial 4, peroxyacetic acid caused a reduction of 0.7log(10)CFU/cm(2) in E. coli O157:H7 and 1.0log(10)CFU/cm(2) in Salmonella Typhimurium, whereas lactic acid caused a reduction of 1.3log(10)CFU/cm(2) in E. coli O157:H7 and 2.1log(10)CFU/cm(2) in S. Typhimurium following the water dip. These results show that peroxyacetic acid was not more effective than 2% l-lactic acid in reducing pathogens on fresh beef trim.     

A national survey of the microbiological quality of retail raw meats in Australia

A national survey of the microbiology of meat (ground beef and diced lamb) at the retail level in Australia was undertaken. For ground beef samples (n = 360), the mean aerobic plate count (APC) was 5.79 log CFU/g, and Escherichia coli was detected in 17.8% of samples; the mean population for these positive samples was 1.49 log CFU/g. Enterobacteriaceae were detected in 96.9% of samples (mean for positive samples, 3.01 log CFU/g), and coagulase-positive staphylococci were detected in 28.1% of samples (mean for positive samples, 2.18 log CFU/g). For diced lamb samples (n = 360), the mean APC was 5.71 log CFU/g, and E. coli was detected in 16.7% of samples (mean for positive samples, 1.67 log CFU/g). Enterobacteriaceae were detected in 91.1% of samples (mean for positive samples, 2.85 log CFU/g), and coagulase-positive staphylococci were detected in 22.5% of samples (mean for positive samples, 2.34 log CFU/g). Salmonella was recovered from 4 (1.1%) of the 360 ground beef samples (isolates were Salmonella Typhimurium phage types), and E. coli O157 was recovered from 1 (0.3%) of 357 samples; Campylobacter and Clostridium perfringens were not recovered from any of the 91 and 94 samples tested, respectively. Salmonella was recovered from 2 (0.6%) of the 360 diced lamb samples (serovars were Salmonella Infantis and Salmonella Typhimurium), Campylobacter was recovered from 1 (1.1%) of 95 samples, and C. perfringens was recovered from 1 (1.1%) of 92 samples.     

Microbial evaluation of Spanish potato omelette and cooked meat samples in University restaurants

The focus of this study was to evaluate the microbial quality of Spanish potato omelette and cooked meat samples including pork loin, chicken croquettes, long pork sausage, chicken breast, and meatballs from University restaurants. Microbiological analyses of Spanish potato omelette and cooked meat samples resulted in aerobic plate counts from <1.00 to 2.90 and from <1.00 to 6.04 log10 CFU g(-1), respectively. Total coliforms ranged from <3 to 43 most probable number (MPN) g(-1) and from <3 to >2,400 MPN g(-1) for Spanish potato omelette and meat products, respectively. Escherichia coli, coagulase-positive staphylococci, and Lancefield group-D streptococci were detected in 1.7%, 3.5%, and 12.9% of Spanish potato omelette samples, respectively. For cooked meat samples, 8.8%, 7.6%, and 24.6% contained E. coli, coagulase-positive staphylococci, and Lancefield group-D streptococci, respectively. E. coli O157:H7, Salmonella spp., and Shigella spp. were not detected. Klebsiella pneumoniae, Klebsiella oxytoca, Citrobacter freundii, Enterobacter cloacae, and Serratia spp. were isolated from Spanish potato omelette samples. For cooked meat samples, C. freundii, E. cloacae, and Aeromonas hydrophila were detected. The results suggest that some handling practices should require more attention, and as a consequence, a hazard analysis and critical control point program should be developed and implemented.     

Effect of food matrix and cell growth on PCR-based detection of Escherichia coli O157:H7 in ground beef

The purpose of this work was (i) to investigate the feasibility of a previously reported upstream processing method for PCR template preparation to facilitate the detection of Escherichia coli O157:H7 from ground beef and (ii) to assess the impact of cell growth (no growth in the matrix versus growth in the matrix) on molecular detection limits. Two food matrices (autoclaved and raw ground beef) were evaluated in all studies. For no-growth experiments, 10-g meat samples were inoculated with 10(2) to 10(7) CFU/g E. coli O157:H7 and then homogenized. The homogenates were processed to remove large particulates and inhibitors using a two-phase upstream processing method consisting of two sequential centrifugation steps, the second of which used titanous hydroxide to facilitate bacterial immobilization. After upstream processing, sample concentrates were extracted for DNA isolation and amplified by PCR. For growth experiments, 10-g meat samples were inoculated at 1 CFU of E. coli O157:H7 per gram, allowed to grow to 10(2) to 10(7) CFU/g, and then processed for PCR assay. Cell recoveries after upstream processing ranged from 15.9 to 77.6% and were not facilitated by the use of titanous hydroxide, as compared with a saline control (P > 0.05). Bacterial cell recovery and PCR detection limits were similar when comparing autoclaved ground beef and raw ground beef, but cell recoveries were highly variable for raw ground beef samples in which E. coli O157:H7 cells were allowed to grow before processing for detection. Overall, PCR detection limits approximated 10(3) CFU/g of ground beef for all treatments. These results indicate that use of model food systems may not always provide an accurate replication of real-world conditions when evaluating PCR detection limits.