Growth of strains of the major non-O157 Shiga toxin-producing Escherichia coli serogroups is not different from growth of Escherichia coli O157:H7 in neutral broth (pH 7.4) and acidified broth (pH 5.6) at 10°C.

Understanding the survival and growth of non-O157 Shiga toxin-producing Escherichia coli (STEC) strains under cold temperatures may be important for protecting public health. The aim of this study was to compare the growth of three strains of each of the major non-O157 STEC serogroups (O26, O45, O103, O111, O121, and O145) with the growth of six O157:H7 STEC strains in broth at 10°C. Brain heart infusion broth (BHIB; pH 7.4) was inoculated with a single strain of stationary-phase STEC culture to produce a starting inoculum of ∼10(6) CFU/ml and stored at 10°C for up to 96 h (three trials per strain). Populations over time were fitted to the Baranyi and Roberts model, and lag-phase duration (LPD) and growth rate were calculated for each strain per trial. Average LPD ranged from 9.2 to 32.8 h for non-O157 STEC and from 10.5 to 17.2 h for O157 STEC. One strain of O26 STEC had a significantly longer LPD (P < 0.05) than did the other strains (32.8 h); otherwise, no significant differences were noted (P > 0.05). Growth rate ranged from 0.031 to 0.060 log CFU/ml/h for non-O157 STEC strains and from 0.034 to 0.046 log CFU/ml/h for O157 STEC strains. No significant difference in growth rate was noted among strains in BHIB at pH 7.4 and 10°C. In subsequent trials, growth of a single strain of each of the non-O157 STEC serogroups was compared with growth of four acid-tolerant O157 STEC strains in BHIB acidified to pH 5.6 with lactic acid. Acidification generally increased LPD and decreased the growth rate for strains, although the effect was variable and not significant. These findings suggest that growth patterns for strains of non-O157 STEC are similar to those for strains of O157 STEC in neutral and pH 5.6 BHIB at 10°C. Further research is needed to determine whether strains behave similarly in meat systems.

Lethality of home-style dehydrator processes against Escherichia coli O157:H7 and salmonella serovars in the manufacture of ground-and-formed beef jerky and the potential for using a pathogen surrogate in process validation.

Ground-and-formed beef jerky can be made easily at home with ground beef and kits that include spice, cure, and jerky-forming equipment. Ground beef poses inherent risks of illness due to Escherichia coli O157:H7 and Salmonella contamination, making adequate pathogen lethality important in jerky manufacturing. We evaluated the effectiveness of drying regimes at eliminating E. coli O157:H7 and Salmonella in seasoned ground-and-formed beef jerky manufactured with three home-style dehydrators and one small commercial unit. Inoculated jerky strips were dried for up to 12 or 24 h in a home-style or the commercial unit, respectively, with target drying temperatures ranging from 51.7 degrees C (125 degrees F) to 71.1 degrees C (160 degrees F). Pathogen lethality varied with seasoning, temperature, and drying time (n = 288 samples). Lethality against E. coli O157:H7 ranged from 1.5 log CFU (Jerky Xpress, 57.2 degrees C [135 degrees F], 4 h) to 6.4 log CFU (Gardenmaster, 68.3 degrees C [155 degrees F], 12 h), and varied with seasoning. Lethality against Salmonella ranged from 1.7 log CFU (Jerky Xpress, 57.2 degrees C [135 degrees F], 4 h) to 6.0 log CFU (Gardenmaster, 68.3 degrees C [155 degrees F], 12 h), and also varied with seasoning. There was a > or =5-log CFU reduction in both pathogens in 0, 10, and 27 % of samples at 4, 8, and 12 h, respectively. Heating jerky for 10 min at 135 degrees C (275 degrees F) 4 or 6 h postdrying increased lethality, on average, 2.99 log CFU for Salmonella and 3.02 log CFU for E. coli O157:H7. The use of a lactic acid bacterium culture (Pediococcus spp.) as a pathogen surrogate accurately predicted safety in 28 % of samples containing E. coli O157:H7 and 78% of Salmonella-inoculated samples.

Survival of Escherichia coli O157:H7 in ground beef after sublethal heat shock and subsequent isothermal cooking.

Heat shock of Escherichia coli O157:H7 in broth media reportedly leads to enhanced survival during subsequent heating in broth medium or ground beef. Survival of E. coli O157:H7 during slow cooking thus may be enhanced by prior exposure to sublethal heat shock conditions, thereby jeopardizing the safety of slow-cooked products such as beef roasts. This study examined the effect of heat shocking E. coli O157:H7-inoculated lean (6 to 9% fat) ground beef on the survival of the pathogen in the same ground beef during a subsequent 4-h, 54.4 degrees C cooking process. Six different combinations of heat shock temperature (47.2, 48.3, or 49.4 degrees C) and time (5 or 30 min) were applied to a five-strain cocktail of microaerophilically grown cells in 25 g of prewarmed ground beef, which was followed by cooking at 54.4 degrees C. Temperature during a 30-min heat shock treatment did not significantly affect E. coli O157:H7 survival during subsequent isothermal cooking (P > 0.05). Survival after a 5-min heat shock was higher when the heat shock temperature was 48.3 or 49.4 degrees C (P < 0.05) than when it was 47.2 degrees C. The D-values at 54.4 degrees C (130 degrees F) (D54.4-value) of the process significantly increased only when cells were exposed to a heat shock combination of 5 min at 49.4 degrees C. Mean (n = 3 trials) reductions in E. coli O157:H7 during the 4-h, 54.4 degrees C isothermal cooking process ranged from 4.3 to 7.5 log CFU/g. Heating E. coli O157:H7-contaminated beef at the high end of the sublethal temperature range for 5 min could increase survival of E. coli O157:H7 during subsequent slow-cooking processes.

Factors associated with Salmonella prevalence on pork carcasses in very small abattoirs in Wisconsin.

The U.S. Department of Agriculture has expressed concern over Salmonella prevalence on pork carcasses. Our objectives were to survey the prevalence of Salmonella on pork carcasses in very small Wisconsin abattoirs, and identify processing conditions and indicator bacteria levels associated with reduced Salmonella prevalence. During April to July 2007, sponge samples were obtained from 181 pork carcasses at 10 Wisconsin abattoirs before carcass washing (carcass half A), and after washing and chilling and before fabrication (carcass half B). Each sample was categorized by whether the carcass was skinned, by wash-water temperature (7 to 43 degrees C), and the duration (1 or 2 days), temperature, and percent relative humidity of chilling. Sponge samples were analyzed qualitatively for Salmonella and quantitatively for Escherichia coli, coliforms, Enterobacteriaceae, and aerobic plate count (APC). Salmonella prevalences on skinned and unskinned prewash carcasses were 11.7 and 8.3%, respectively. Corresponding values for chilled carcasses were 32.0 and 19.5% for 1-day chilled carcasses, and 11.4 and 14.7% for 2-day chilled carcasses. Lower Salmonella prevalence on prewash carcasses was significantly related to lower prewash carcass APC levels (odds ratio = 7.8 per change of 1.0 log CFU/cm2), while lower Salmonella prevalence on chilled carcasses was significantly related to 2-day chilling (odds ratio = 5.2), and chilled-carcass levels of coliforms, Enterobacteriaceae, and APC (odds ratio = 1.5 to 1.9 per change of 1.0 log CFU/cm2). Salmonella prevalence on chilled pork carcasses in very small Wisconsin plants could be reduced by chilling carcasses 2 days before fabrication and improving carcass-handling hygiene.

Viability of multi-strain mixtures of Listeria monocytogenes, Salmonella typhimurium, or Escherichia coli O157:H7 inoculated into the batter or onto the surface of a soudjouk-style fermented semi-dry sausage.

The fate of Listeria monocytogenes, Salmonella typhimurium, or Escherichia coli O157:H7 were separately monitored both in and on soudjouk. Fermentation and drying alone reduced numbers of L. monocytogenes by 0.07 and 0.74 log(10)CFU/g for sausages fermented to pH 5.3 and 4.8, respectively, whereas numbers of S. typhimurium and E. coli O157:H7 were reduced by 1.52 and 3.51 log(10)CFU/g and 0.03 and 1.11 log(10)CFU/g, respectively. When sausages fermented to pH 5.3 or 4.8 were stored at 4, 10, or 21 degrees C, numbers of L. monocytogenes, S. typhimurium, and E. coli O157:H7 decreased by an additional 0.08-1.80, 0.88-3.74, and 0.68-3.17 log(10)CFU/g, respectively, within 30 days. Storage for 90 days of commercially manufactured soudjouk that was sliced and then surface inoculated with L. monocytogenes, S. typhimurium, and E. coli O157:H7 generated average D-values of ca. 10.1, 7.6, and 5.9 days at 4 degrees C; 6.4, 4.3, and 2.9 days at 10 degrees C; 1.4, 0.9, and 1.6 days at 21 degrees C; and 0.9, 1.4, and 0.25 days at 30 degrees C. Overall, fermentation to pH 4.8 and storage at 21 degrees C was the most effective treatment for reducing numbers of L. monocytogenes (2.54 log(10)CFU/g reduction), S. typhimurium (> or =5.23 log(10)CFU/g reduction), and E. coli O157:H7 (3.48 log(10)CFU/g reduction). In summary, soudjouk-style sausage does not provide a favorable environment for outgrowth/survival of these three pathogens.

Polyphenol oxidase activity as a potential intrinsic index of adequate thermal pasteurization of apple cider.

In response to increasing concerns about microbial safety of apple cider, the U.S. Food and Drug Administration has mandated treatment of cider sufficient for a 5-log reduction of the target pathogen. Pasteurization has been suggested as the treatment most likely to achieve a 5-log reduction, with Escherichia coli O157:H7 as the target pathogen. Regulators and processors need a reliable method for verifying pasteurization, and apple cider polyphenol oxidase (PPO) activity was studied as a potential intrinsic index for thermal pasteurization. The effect of pasteurization conditions and apple cider properties on PPO activity and survival of three pathogens (E. coli O157:H7, Salmonella, and Listeria monocytogenes) was studied using a Box-Behnken response surface design. Factors considered in the design were pasteurization conditions, i.e., hold temperature (60, 68, and 76 degrees C), preheat time (10, 20, 30 s), and hold time (0, 15, 30 s), pH, and sugar content ((o)Brix) of apple cider. Response surface contour plots were constructed to illustrate the effect of these factors on PPO activity and pathogen survival. Reduction in PPO activity of at least 50% was equivalent to a 5-log reduction in E. coli O157:H7 or L. monocytogenes for cider at pH 3.7 and 12.5 (o)Brix. Further studies, however, are needed to verify the relationship between PPO activity and pathogen reduction in cider with various pH and (o)Brix values.

Influence of ovine milk in mixture with bovine milk on the quality of reduced fat Muenster-type cheese.

Reduced fat Muenster-type cheeses were manufactured from a mixture of bovine skim milk and ovine whole milk and from bovine milk only (control). Cheeses were evaluated at 15, 30, 60, 90, 120, and 180 d of age for numbers and type of microflora, casein hydrolysis, and amounts of free fatty acids. alpha(s1)-Casein degradation was similar for both cheeses during the aging period, but beta-casein degradation proceeded at a faster rate in the control cheese. The total amounts of free fatty acids remained constant throughout the ripening time; however, the cheeses produced with bovine/ovine milk yielded a significantly larger amount of caprylic (C8:0) and capric (C10:0) acids compared with the bovine milk cheeses. Lactobacilli increased during the aging period, while the populations of lactic acid bacteria, yeast and molds, and lipolytic organisms did not increase. Both cheeses had comparable cheese flavor intensity, but the bovine/ovine milk cheese had a greater occurrence of off flavors. The bovine/ovine milk cheeses were firmer than the bovine cheeses throughout the aging period.

Validation of apple cider pasteurization treatments against Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes.

Time and temperature pasteurization conditions common in the Wisconsin cider industry were validated using a six-strain cocktail of Escherichia coli O157:H7 and acid-adapted E. coli O157:H7 in pH- and degrees Brix-adjusted apple cider. Strains employed were linked to outbreaks (ATCC 43894 and 43895, C7927, and USDA-FSIS-380-94) or strains engineered to contain the gene for green fluorescent protein (pGFP ATCC 43894 and pGFP ATCC 43889) for differential enumeration. Survival of Salmonella spp. (CDC 0778. CDC F2833, and CDC H0662) and Listeria monocytogenes (H0222, F8027, and F8369) was also evaluated. Inoculated cider of pH 3.3 or 4.1 and 11 or 14 degrees Brix was heated under conditions ranging from 60 degrees C for 14 s to 71.1 degrees C for 14 s. A 5-log reduction of nonadapted and acid-adapted E. coli O157:H7 was obtained at 68.1 degrees C for 14 s. Lower temperatures, or less time at 68.1 degrees C, did not ensure a 5-log reduction in E. coli O157:H7. A 5-log reduction was obtained at 65.6 degrees C for 14 s for Salmonella spp. L. monocytogenes survived 68.1 degrees C for 14 s, but survivors died in cider within 24 h at 4 degrees C. Laboratory results were validated with a surrogate E coli using a bench-top plate heat-exchange pasteurizer. Results were further validated using fresh unpasteurized commercial ciders. Consumer acceptance of cider pasteurized at 68.1 degrees C for 14 s (Wisconsin recommendations) and at 71.1 degrees C for 6 s (New York recommendations) was not significantly different. Hence, we conclude that 68.1 degrees C for 14 s is a validated treatment for ensuring adequate destruction of E. coli O157:H7, Salmonella spp., and L. monocytogenes in apple cider.

Differentiation of Enterococcus spp. by cell membrane fatty acid methyl ester profiling, biotyping and ribotyping.

AIMS: Gas chromatographic analysis of cell membrane fatty acid methyl esters (FAME), biochemical profiling (biotyping) and EcoRI restriction endonuclease profiling of DNA containing ribosomal RNA sequences (ribotyping) were compared for differentiation of Enterococcus spp. METHODS AND RESULTS: FAME profiling, biotype profiling and ribotyping of 41 strains from retail Swiss-type cheeses and five strains from culture collections resulted in 17, 25 and 26 groups, respectively, with only two pairs of strains having the same FAME group, biotype profile and ribogroup. CONCLUSION: Substantial overlap occurred in groupings assigned by the three methods. SIGNIFICANCE AND IMPACT OF THE STUDY: Differentiation of Enterococcus spp. strains increases if multiple methods are used.

Influence of salting procedure on the composition of Muenster-type cheese.

Muenster-type cheeses were salted with a traditional saturated brine solution or by direct addition of salt to the curd. Cheeses were evaluated at 0, 30, 60, 90, 120, and 180 d of age for numbers and type of microflora, casein hydrolysis, and amounts of free fatty acids. No significant differences were found in the populations of starter, lactobacilli, or yeast for the brine- and direct-salted cheeses. The amounts of free fatty acids liberated were similar for both cheeses. The hydrolysis of alpha s1-casein was complete at 90 d of age, whereas only 40% of the beta-casein was hydrolyzed at 180 d of age. The inner layer of the brine-salted cheeses had the highest number of starter microorganisms, followed by the middle and outer layers, respectively. The salt concentrations were similar in the three layers after 4 mo of age. Results of this study showed that comparable Muenster-type cheese could be produced with either of the salting procedures. With direct salt addition to curd, a 59% reduction was observed in salt emissions from the Muenster manufacturing process.

Efficacy of novel organic acid and hypochlorite treatments for eliminating Escherichia coli O157:H7 from alfalfa seeds prior to sprouting.

This study investigated novel two-step organic acid/hypochlorite treatments as alternatives to 20000 ppm active chlorine (from calcium hypochlorite) for eliminating Escherichia coli O157:H7 from alfalfa seeds prior to sprouting. Commercially available alfalfa seeds were inoculated with a five-strain E. coli O157:H7 mixture and dried to attain ca. 10(6) CFU/g of seeds. Seeds then underwent one of several soak treatments including: (1) 5% (v/v) lactic acid for 10 min at 42 degrees C; (2) 5% acetic acid (v/v) for 10 min at 42 degrees C; (3) 2.5% lactic acid for 10 min at 42 degrees C followed by 2000 ppm active chlorine (from calcium hypochlorite) for 15 min at 25 degrees C; (4) 5% lactic acid for 10 min at 42 degrees C followed by 2000 ppm active chlorine for 15 min at 25 degrees C; or (5) 20000 ppm active chlorine for 15 min at 25 degrees C. Each treatment reduced numbers of inoculum cells by about 6.0 log10 CFU/g as determined by plating on Sorbitol MacConkey agar (SMac). Plating on non-selective brain heart infusion agar (BHI) showed that treatments 1-4 reduced counts by 2.3-4.1 log10 CFU/g, thus indicating a large proportion of injured cells. Successive lactic acid and hypochlorite treatments (3 and 4) were more lethal than either organic acid alone (1 and 2). No surviving cells were detected on SMac or BHI following treatment with 20000 ppm active chlorine (treatment 5). Regardless of the previous treatment, E. coli O157:H7 counts increased to 10(7)-10(8) CFU/g during sprouting. Germination of seeds was not adversely affected by any of the treatments (germination > 90%). Results of this study show that: (a) non-lethal cell injury must be considered when evaluating intervention treatments against E. coli O157:H7 on alfalfa seeds; (b) reductions of 2-4 log10 CFU/g can be attained without using 20000 ppm active chlorine; (c) successive lactic acid and hypochlorite treatments have greater lethality than organic acid treatments alone; and (d) none of the treatments tested can prevent regrowth of surviving E. coli O157:H7 during sprouting.

Verifying apple cider plant sanitation and hazard analysis critical control point programs: choice of indicator bacteria and testing methods.

The objectives of this study were (i) to evaluate the survival of coliforms, Escherichia coli, and enterococci in refrigerated apple cider; (ii) to develop simple and inexpensive presumptive methods for detection of these bacteria; (iii) to perform a field survey to determine the prevalence of these bacteria on apples and in apple cider; and (iv) based on our results, to recommend the most useful of these three indicator groups for use in verifying apple cider processing plant sanitation and hazard analysis critical control point (HACCP) programs. Eight of 10 coliform strains (5 E. coli, 1 Enterobacter aerogenes, and 2 Klebsiella spp.) inoculated into preservative-free apple cider (pH 3.4, 13.3(o) Brix) survived well at 4 degrees C for 6 days (< or = 3.0 log10 CFU/ml decrease). Of 21 enterococci strains (Enterococcus faecalis, E. faecium, and E. durans), only 2 E. durans and 3 E. faecium strains survived well. Simple broth-based colorimetric methods were developed that detected the presence of approximately 10 cells of coliforms or enterococci. In three field studies, samples of unwashed apples (drops and picked), washed apples, and freshly pressed cider were presumptively analyzed for total coliforms, E. coli, and enterococci using qualitative and/or quantitative methods. Drop apples were more likely than picked apples to be contaminated with E. coli (26.7% vs. 0%) and enterococci (20% vs. 0%). Washing had little effect on coliform populations and in one field study was associated with increased numbers. Total coliform populations in cider ranged from < 1 CFU/ml to > 738 most probable number/ml, depending on the enumeration method used and the sample origin. E. coli was not recovered from washed apples or cider, but enterococci were present on 13% of washed apple samples. The qualitative coliform method successfully detected these bacteria on apples and in cider. Based on its exclusively fecal origin, good survival in apple cider, and association with drop apples, we conclude that E. coli is the most useful organism for verifying apple cider sanitation and HACCP programs.

Differentiation of lactate-fermenting, gas-producing Clostridium spp. isolated from milk.

Endospores of Clostridium spp. capable of producing gas in a lactate-containing medium were enumerated from 14 pasteurized milk samples from Wisconsin cheese plants. Concentrations of endospores of lactate-fermenting, gas-producing Clostridium spp. were between 5.0 x 10(-2) and 1.7 x 10(0) MPN ml(-1). Concentrations of presumptive C. tyrobutyricum endospores (defined by subterminal endospore position and lactate dehydrogenase activity) were lower, not exceeding 2.0 x 10(-2) MPN ml(-1). Based on subterminal endospore position, lactate dehydrogenase activity, and a carbohydrate fermentation profile identical to C. tyrobutyricum strain ATCC 25755, five isolates (Ct) were initially characterized as C. tyrobutyricum, a known cause of late-blowing in high-pH cheeses. Twenty-eight other isolates (Cx) produced gas from lactate, but differed from ATCC 25755 in either endospore position, lactate dehydrogenase activity or carbohydrate fermentation profile. When inoculated at high concentrations in Gouda cheese, strain ATCC 25755, two Ct isolates and 18 Cx isolates tested produced gas during ripening. Among the five Ct isolates obtained and two reference strains confirmed as C. tyrobutyricum, there were four qualitatively different volatile organic acid byproduct profiles. Each of the two confirmed C. tyrobutyricum reference strains and five Ct isolates had distinct quantitative cell membrane fatty acid (CMFA) profiles. The Cx isolates represented 14 different volatile organic acid byproduct profiles and each isolate had a unique CMFA profile. Pulsed field gel electrophoresis (PFGE) of DNA from the two confirmed reference C. tyrobutyricum strains, four Ct and three Cx isolates, showed a low degree of relatedness. The results of this study suggest that a heterogeneous group of lactate-fermenting, gas-producing Clostridium spp. may be found in milk. Gas chromatographic analysis of volatile organic acid byproducts or CMFA, and PFGE of DNA are highly discriminating methods for differentiating Clostridium spp. that may cause late blowing in high-pH cheeses.