Investigation into Formation of Lipid Hydroperoxides from Membrane Lipids in Escherichia coli O157:H7 following Exposure to Hot Water.

Although studies have shown antimicrobial treatments consisting of hot water sprays alone or paired with lactic acid rinses are effective for reducing Escherichia coli O157:H7 loads on beef carcass surfaces, the mechanisms by which these interventions inactivate bacterial pathogens are still poorly understood. It was hypothesized that E. coli O157:H7 exposure to hot water in vitro at rising temperatures for longer time periods would result in increasing deterioration of bacterial outer membrane lipids, sensitizing the pathogen to subsequent lactic acid application. Cocktails of E. coli O157:H7 strains were subjected to hot water at 25 (control) 65, 75, or 85 °C incrementally up to 60 s, after which surviving cells were enumerated by plating. Formation of lipid hydroperoxides from bacterial membranes and cytoplasmic accumulation of L-lactic acid was quantified spectrophotometrically. Inactivation of E. coli O157:H7 proceeded in a hot water exposure duration- and temperature-dependent manner, with populations being reduced to nondetectable numbers following heating of cells in 85 °C water for 30 and 60 s (P < 0.05). Lipid hydroperoxide formation was not observed to be dependent upon increasing water temperature or exposure period. The data suggest that hot water application prior to organic acid application may function to increase the sensitivity of E. coli O157:H7 cells by degrading membrane lipids.

A survey of nitrate and nitrite concentrations in conventional and organic-labeled raw vegetables at retail.

A national survey of the nitrate ( NO3(-)) and nitrite ( NO2(-)) concentrations in raw and highly consumed vegetables available at retail in the United States was conducted. A total of 194 samples of fresh broccoli, cabbage, celery, lettuce, and spinach categorized as conventional or organic by label were collected from 5 major cities in different geographic regions of the United States and analyzed to determine NO3(-) and NO2(-) concentrations. There were no differences in the mean NO2(-) values of conventional compared with organic vegetables taken from the 5 metropolitan areas. However, significant differences in mean pairwise comparisons between some conventional and organic vegetables for NO3(-) content were observed. The mean NO2(-) concentration of both conventional and organic vegetables ranged between 0.1 and 1.2 mg/kg of fresh weight (FW) with the exception of conventional spinach that contained 8.0 mg/kg FW. Mean NO3(-) contents of conventional broccoli, cabbage, celery, lettuce, and spinach were 394, 418, 1496, 851, and 2797 mg/kg FW, respectively, while their organic-labeled counterparts averaged 204, 552, 912, 844, and 1318 mg/kg FW. In most cases, organic vegetables were numerically lower in NO3(-) content than their conventional counterparts. Based on survey results, the finding that low NO3(-) levels were observed in some organic vegetables in different cities may warrant further study to determine if true differences exist, due to production practices, seasonal differences, and the magnitudes of those differences. Furthermore, the geographic differences in NO3(-) content of vegetables may flaw estimates of daily NO2(-) and NO3(-) exposure.

Effects of lactic acid and commercial chilling processes on survival of Salmonella, Yersinia enterocolitica, and Campylobacter coli in pork variety meats.

Current industry chilling practices with and without the application of 2% L-lactic acid were compared for their effectiveness at reducing levels of Salmonella, Yersinia enterocolitica, and Campylobacter coli on pork variety meats. Pork variety meats (livers, intestines, hearts, and stomachs) were inoculated individually with one of the three pathogens and subjected to five different treatment combinations that included one or more of the following: water wash (25°C), lactic acid spray (2%, 40 to 50°C), chilling (4°C), and freezing (-15°C). Samples were analyzed before treatment, after each treatment step, and after 2, 4, and 6 months of frozen storage. Results showed that when a lactic acid spray was used in combination with water spray, immediate reductions were approximately 0.5 log CFU per sample of Salmonella, 0.8 log CFU per sample of Y. enterocolitica, and 1.1 log CFU per sample of C. coli. Chilling, both alone and in combination with spray treatments, had little effect on pathogens, while freezing resulted in additional 0.5-log CFU per sample reductions in levels of Salmonella and Y. enterocolitica, and an additional 1.0-log CFU per sample reduction in levels of C. coli. While reductions of at least 1 log CFU per sample were observed on variety meats treated with only a water wash and subsequently frozen, samples treated with lactic acid had greater additional reductions than those treated with only a water spray throughout frozen storage. The results of this study suggest that the use of lactic acid as a decontamination intervention, when used in combination with good manufacturing practices during processing, causes significant reductions in levels of Salmonella, Y. enterocolitica, and C. coli on pork variety meats.

Survey of residual nitrite and nitrate in conventional and organic/natural/uncured/indirectly cured meats available at retail in the United States.

A survey of residual nitrite (NO(2)(-)) and nitrate (NO(3)(-)) in cured meats available at retail was conducted to verify concentrations in conventional (C) products and establish a baseline for organic/natural/uncured/indirectly cured (ONC) products. In this study, 470 cured meat products representing six major categories were taken from retail outlets in five major metropolitan cities across the United States. Random samples representing both C and ONC type products were analyzed for NO(2)(-) and NO(3)(-) content (ppm) using an ENO-20 high-performance liquid chromatography system equipped with a reverse phase column. Generally, there were no differences in NO(2)(-) concentrations between C and ONC meat categories, but a few ONC products surveyed in certain cities were lower in NO(3)(-) content. Pairwise comparisons between cities indicated that NO(2)(-) and NO(3)(-) contents of all C type products were not appreciably different, and the same was true for most ONC products. Numerical NO(2)(-) values were less variable than NO(3)(-) concentrations within each meat product category. NO(2)(-) concentrations were similar to those previously reported by Cassens ( Cassens , R. G. Residual nitrite in cured meat . Food Technol. 1997a , 51 , 53 - 55 ) in 1997. Residual NO(2)(-) and NO(3)(-) values in this study were numerically lower than those reported by NAS ( National Academy of Sciences . The Health Effects of Nitrate, Nitrite, and N-Nitroso Compounds ; National Academy Press : Washington, DC , 1981 ) in 1981. Data from this survey provide a benchmark of NO(2)(-) and NO(3)(-) concentrations for ONC products available at retail.

Dispersion and survival of Escherichia coli O157:H7 and Salmonella Typhimurium during the production of marinated beef inside skirt steaks and tri-tip roasts.

To determine the depth of pathogen dispersion and the ability of pathogens to survive in enhanced beef products and spent marinade, beef inside skirt steaks and tri-tip roasts were vacuum tumbled with two commercial marinades. The marinades were inoculated with Escherichia coli O157:H7 and Salmonella Typhimurium, resulting in an approximate count of 5.2 log CFU/ml. Both inside skirt steaks and tri-tip roasts were vacuum tumbled for 1 h and sampled immediately after tumbling (day 0), or were vacuum packaged, stored (ca. 4°C), and sampled on days 7 and 14. Samples of the spent marinade were taken after tumbling (day 0) and on days 3 and 7. For both marinades, Salmonella Typhimurium and E. coli O157:H7 were dispersed throughout the inside skirt steaks during vacuum tumbling. Although Salmonella Typhimurium and E. coli O157:H7 for the skirt steaks were still detectable after 14 days of storage, the log values were lower than those on days 0 and 7. For the tri-tip roasts, the pathogen distribution varied, depending on the thickness of the roasts, and pathogens were detectable on days 0, 7, and 14. The spent marinade sampled on days 0, 3, and 7 showed that the pathogens survived at refrigerated temperatures. Because pathogens can transfer to the interior of beef inside skirt steaks and tri-tip roasts when vacuum tumbled with contaminated marinade and survived during refrigerated storage, establishments should consider the potential food safety risks associated with reuse of marinade during the production of vacuum-tumbled beef products.

Clostridium difficile in retail meat and processing plants in Texas.

The incidence and severity of disease associated with toxigenic Clostridium difficile have increased in hospitals in North America from the emergence of newer, more virulent strains. Toxigenic C. difficile has been isolated from food animals and retail meat with potential implications of transfer to human beings. The objective of the present study was to determine the prevalence of C. difficile in pork from sausage manufacturing plants and retail meat in Texas. Twenty-three C. difficile isolates were detected from 243 meat samples (9.5%) from 3 sausage-manufacturing plants and 5 retail meat outlets from 2004 to 2009. Twenty-two isolates were positive for toxins A, B, and binary toxin, and were characterized as toxinotype V, PFGE type-NAP7, or "NAP7-variant." Susceptibilities to 11 antimicrobial agents in the current study were similar to those reported previously for toxinotype V isolates, although the results suggested somewhat reduced resistance than reported for other meat, animal, or human clinical toxinotype V isolates.

Evaluation of Escherichia coli O157:H7 translocation and decontamination for beef vacuum-packaged subprimals destined for nonintact use.

The translocation of Escherichia coli O157:H7 as well as the impact of water washing and partial or complete surface trimming as possible pathogen reduction strategies were evaluated for vacuum-packaged beef subprimals destined for nonintact use. Cap-on and cap-off beef top sirloin butts were inoculated with two levels of E. coli O157:H7: a high-inoculum level of approximately 10(4) CFU/cm(2) and a low-inoculum level of approximately 10(2) CFU/cm(2). Following inoculation, the subprimals were vacuum packaged and stored for 0, 14, or 28 days. Upon removal from storage, the following sites were evaluated: exterior of the bag, purge, the inoculation site on the subprimal, the area adjacent to the inoculation site, and the surface opposite from the inoculation site. The following treatments then were applied: water wash, water wash followed by full-surface trimming, water wash followed by partial-surface trimming, full-surface trimming, full-surface trimming followed by water wash, partial-surface trimming, and partial-surface trimming followed by water wash. For both high- and low-inoculated top sirloin butts, contamination of adjacent and opposite surfaces was found after vacuum packaging. Of the treatments applied, water washing alone was the least effective for both high- and low-inoculated subprimals. Full trimming, with or without a water wash, proved to be the most effective treatment used to reduce E. coli O157:H7 to nondetectable levels.

Alternative cooling procedures for large, intact meat products to achieve stabilization microbiological performance standards.

Achieving the U. S. Department of Agriculture, Food Safety and Inspection Service (USDA-FSIS) stabilization microbiological performance standards for cooling procedures proves to be challenging for processors of large, whole-muscle meat products. This study was conducted to determine if slower cooling times than those provided by USDA-FSIS guidance will comply with the performance standard for Clostridium perfringens. Large (9 to 12 kg) cured bone-in hams (n = 110) and large (8 to 13 kg) uncured beef inside rounds (n = 100) were used. Stabilization treatments extended times to reduce internal product temperature from 54.4 to 26.7°C (hams and rounds) and from 26.7 to 7.2°C (for hams) and 26.7 to 4.4°C (for rounds). Control treatments, defined by current USDA-FSIS Appendix B guidelines, and a "worst-case scenario" treatment, in which products were cooled at room temperature (approximately 22.8°C) until internal product temperature equilibrated, were used. For both hams and rounds, stabilization showed less than 1-log growth of C. perfringens for all treatments, with the exception of the worst-case scenario for rounds. As expected for products cooled at room temperature, there was >1-log growth of C. perfringens reported for rounds, and the addition of curing ingredients to hams had an inhibitory effect on the growth of C. perfringens. The results demonstrate that industry may have increased flexibility associated with cooling large, whole-muscle cuts while still complying with the required stabilization microbiological performance standards.

Synergistic inhibition of Listeria monocytogenes in vitro through the combination of octanoic acid and acidic calcium sulfate.

It has been hypothesized that inhibition of foodborne pathogens can be enhanced by using antimicrobials in combination. A broth dilution assay was devised to determine whether inhibition of Listeria monocytogenes exposed to the combination of the fatty acid octanoic acid (OCT) and the organic acid-containing antimicrobial acidic calcium sulfate (ACS) was enhanced compared with the inhibition of the pathogen exposed to either antimicrobial applied singly. MICs for OCT and ACS were 25.00 µg/g and 1.56 ml/liter, respectively, for all strains of the pathogen tested. Fractional inhibitory concentrations (FICs) from the combination exposures were calculated for use in characterizing the antimicrobial interaction as antagonistic, additive indifferent, or synergistic with respect to L. monocytogenes inhibition. Combining OCT and ACS resulted in observed synergistic inhibition of L. monocytogenes; isobolograms for all strains curved toward the origin, and FIC indices (FIC(I)s) were <1.0. Future investigations of the antimicrobial combination should focus on determining the mechanism of action of combined antimicrobials and the levels of antimicrobials required for pathogen inhibition on the surfaces of ready-to-eat meats.

Inhibition of Listeria monocytogenes by food antimicrobials applied singly and in combination.

Combining food antimicrobials can enhance inhibition of Listeria monocytogenes in ready-to-eat (RTE) meats. A broth dilution assay was used to compare the inhibition of L. monocytogenes resulting from exposure to nisin, acidic calcium sulfate, ε-poly-L-lysine, and lauric arginate ester applied singly and in combination. Minimum inhibitory concentrations (MICs) were the lowest concentrations of single antimicrobials producing inhibition following 24 h incubation at 35 °C. Minimum bactericidal concentrations (MBCs) were the lowest concentrations that decreased populations by ≥3.0 log(10) CFU/mL. Combinations of nisin with acidic calcium sulfate, nisin with lauric arginate ester, and ɛ-poly-L-lysine with acidic calcium sulfate were prepared using a checkerboard assay to determine optimal inhibitory combinations (OICs). Fractional inhibitory concentrations (FICs) were calculated from OICs and were used to create FIC indices (FIC(I)s) and isobolograms to classify combinations as synergistic (FIC(I) < 1.00), additive/indifferent (FIC(I)= 1.00), or antagonistic (FIC(I) > 1.00). MIC values for nisin ranged from 3.13 to 6.25 µg/g with MBC values at 6.25 µg/g for all strains except for Natl. Animal Disease Center (NADC) 2045. MIC values for ε-poly-L-lysine ranged from 6.25 to 12.50 µg/g with MBCs from 12.50 to 25.00 µg/g. Lauric arginate ester at 12.50 µg/g was the MIC and MBC for all strains; 12.50 mL/L was the MIC and MBC for acidic calcium sulfate. Combining nisin with acidic calcium sulfate synergistically inhibited L. monocytogenes; nisin with lauric arginate ester produced additive-type inhibition, while ε-poly-L-lysine with acidic calcium sulfate produced antagonistic-type inhibition. Applying nisin along with acidic calcium sulfate should be further investigated for efficacy on RTE meat surfaces.

Survival of Listeria monocytogenes in Postpasteurized Precooked Beef Roasts.

The potential for Listeria monocytogenes to survive various times and temperatures of postpasteurization in precooked beef roasts was investigated. Precooked eye of round roasts were inoculated with 109 cells of L. monocytogenes per package prior to packaging in cook-in bags and postpasteurization. Four groups of roasts (n = 54) each containing 27 uninoculated and 27 inoculated roasts were allotted to four pasteurization treatments consisting of two different exposure temperatures (91°C, 96°C) and two different dwell times (3 min, 5 min). Equal numbers of inoculated and uninoculated roasts from each treatment were then stored at 4 and 10°C. Triplicate samples of inoculated and uninoculated roasts were sampled on day 1, 4, 8, and 12 for product stored at 10°C and 1, 8, 14, 28, and 56 d for product stored at 4°C. Survivors were encountered for every treatment employed. The lethality of the treatment was directly related to an increase in dwell time and postpasteurization temperature. Treatment 4 (96°C, 5 min) was found to decrease the L. monocytogenes population the greatest (p < 0.05) and also maintained a lower (p < 0.05) count from its initial inoculum level during storage at both 4 and 10°C.