rhs genes are potential markers for multilocus sequence typing of Escherichia coli O157:H7 strains.

DNA sequence-based molecular subtyping methods such as multilocus sequence typing (MLST) are commonly used to generate phylogenetic inferences for monomorphic pathogens. The development of an effective MLST scheme for subtyping Escherichia coli O157:H7 has been hindered in the past due to the lack of sequence variation found within analyzed housekeeping and virulence genes. A recent study suggested that rhs genes are under strong positive selection pressure, and therefore in this study we analyzed these genes within a diverse collection of E. coli O157:H7 strains for sequence variability. Eighteen O157:H7 strains from lineages I and II and 15 O157:H7 strains from eight clades were included. Examination of these rhs genes revealed 44 polymorphic loci (PL) and 10 sequence types (STs) among the 18 lineage strains and 280 PL and 12 STs among the 15 clade strains. Phylogenetic analysis using rhs genes generally grouped strains according to their known lineage and clade classifications. These findings also suggested that O157:H7 strains from clades 6 and 8 fall into lineage I/II and that strains of clades 1, 2, 3, and 4 fall into lineage I. Additionally, unique markers were found in rhsA and rhsJ that might be used to define clade 8 and clade 6. Therefore, rhs genes may be useful markers for phylogenetic analysis of E. coli O157:H7.

Efficacy of electrolyzed oxidizing water for the microbial safety and quality of eggs.

During commercial processing, eggs are washed in an alkaline detergent and then rinsed with chlorine to reduce dirt, debris, and microorganism levels. The alkaline and acidic fractions of electrolyzed oxidizing (EO) water have the ability to fit into the 2-step commercial egg washing process easily if proven to be effective. Therefore, the efficacy of EO water to decontaminate Salmonella Enteritidis and Escherichia coli K12 on artificially inoculated shell eggs was investigated. For the in vitro study, eggs were soaked in alkaline EO water followed by soaking in acidic EO water at various temperatures and times. Treated eggs showed a reduction in population between > or = 0.6 to > or =2.6 log10 cfu/g of shell for S. Enteritidis and > or =0.9 and > or =2.6 log10 for E. coli K12. Log10 reductions of 1.7 and 2.0 for S. Enteritidis and E. coli K12, respectively, were observed for typical commercial detergent-sanitizer treatments, whereas log10 reductions of > or =2.1 and > or =2.3 for S. Enteritidis and E. coli K12, respectively, were achieved using the EO water treatment. For the pilot-scale study, both fractions of EO water were compared with the detergent-sanitizer treatment using E. coli K12. Log10 reductions of > or = 2.98 and > or = 2.91 were found using the EO water treatment and the detergent-sanitizer treatment, respectively. The effects of 2 treatments on egg quality were investigated. EO water and the detergent-sanitizer treatments did not significantly affect albumen height or eggshell strength; however, there were significant affects on cuticle presence. These results indicate that EO water has the potential to be used as a sanitizing agent for the egg washing process.

Optimization of iron supplementation for enhanced detection of Salmonella Enteritidis in eggs.

Mixed raw egg contents were inoculated with approximately 10 CFU of Salmonella Enteritidis and supplemented with 0 to 7 mg of FeSO4 per g of egg contents. Egg contents were then incubated at 37 degrees C, and Salmonella Enteritidis colonies were enumerated for up to 106 h. Iron supplementation significantly enhanced the growth of Salmonella Enteritidis. Within the first 24 h of incubation, the optimum iron level for Salmonella Enteritidis growth in egg contents was between 0.2 and 2 mg of FeSO4 per g of egg contents. After 24 h of incubation at 37 degrees C. Salmonella Enteritidis counts in eggs supplemented with 0.5 mg of FeSO4 per g of egg contents consistently reached approximately 1 x 10(9) CFU/ml, whereas Salmonella Enteritidis counts in eggs without iron supplementation varied from less than 5 CFU/ml to 8.4 x 10(6) CFU/ml. A 3 by 3 factorial design was used to study the effect of type of preenrichment and level of iron supplementation on the growth of Salmonella Enteritidis in egg contents. No significant differences in Salmonella Enteritidis counts between preenrichment and nonpreenrichment treatments were observed when egg contents were supplemented with 0.5 mg of FeSO4 per g of egg contents. It was concluded that preenrichment was not necessary for isolation of Salmonella Enteritidis from eggs. The effect of iron supplementation on the sensitivity of detection by the direct plating method was investigated. The direct plating method detected a significantly higher percentage of Salmonella Enteritidis in raw egg contents supplemented with 0.5 mg of FeSO4 per g of egg contents (90%) than in raw egg contents without iron supplementation (63.3%).

Influence of sodium chloride on growth of lactic acid bacteria and subsequent destruction of Escherichia coli O157:H7 during processing of Lebanon bologna.

Due to undesirable quality changes, Lebanon bologna is often processed at temperatures that do not exceed 48.8 degrees C (120 degrees F). Therefore, it is important to study parameters that influence the destruction of Escherichia coli O157:H7 in Lebanon bologna. The objective of the present study was to determine the influence of curing salts (NaCl and NaNO2) on the destruction of E. coli O157:H7 during Lebanon bologna processing. Fermentation to pH 4.7 at 37.7 degrees C reduced populations of E. coli O157:H7 by approximately 0.3 log10, either in the presence or absence of curing salts. Subsequent destruction of E. coli O157:H7 during heating of fermented product to 46.1 degrees C was significantly reduced by the presence of 3.5% NaCl and 156 ppm NaNO2, compared to product without curing salts (P < 0.01). The presence of a higher level of NaCl (5%) in Lebanon bologna inhibited the growth of lactic acid bacteria (LAB), which yielded product with higher pH (approximately 5.0) and significantly reduced the destruction of E. coli O157:H7 even further (P < 0.05). Lower concentrations of NaCl (0, 2.5%) yielded Lebanon bologna with higher LAB counts and lower pHs, compared to product with 5% NaCl. When lactic acid was used to adjust pH in product containing different levels of NaCl, it was determined that low pH was directly influencing destruction of E. coli O157:H7, not NaCl concentration.

Optimizing detection of heat-injured Listeria monocytogenes in pasteurized milk.

Optimal conditions for the detection of heat-injured cells of Listeria monocytogenes in modified Pennsylvania State University (mPSU) broth were determined using a response surface design generated by a computer program, EChip. Different combinations of incubation temperatures and lithium, magnesium, and D-serine concentrations were evaluated to determine the optimum conditions for the detection of heat-injured L. monocytogenes in filter-sterilized whole milk inoculated with selected problematic background microflora. A concentration of 212 mM lithium chloride completely inhibited the growth of Enterococcus faecium while permitting recovery and detection of L. monocytogenes. A concentration of 15.8 mM MgSO4 was found to be optimum for the recovery and detection of L. monocytogenes. A concentration of 140.2 mM D-serine was found to completely inhibit the germination of Bacillus subtilis var. globii spores but not recovery and detection of L. monocytogenes. Under optimum concentrations of LiCl, MgSO4, and D-serine and in the absence of background microflora, the effect of incubation temperature on percentage detection was described by a second-order polynomial model, and 28 degrees C was determined to be optimal. In the presence of background microflora, the effect of incubation temperature on percentage detection of heat-injured cells was described by a third-order polynomial model, and 30 degrees C was found to be optimal. Optimizing the levels of highly specific and selective agents, nutrients, and incubation temperature in one recovery enrichment system dramatically increased the Listeria/background microflora ratio. This resulting medium, optimized PSU (oPSU) broth, greatly improved the detection of heat-injured and nonheat-injured L. monocytogenes by both conventional and molecular methods (Oxoid's Listeria Rapid Test, Gen-Probe's Accuprobe Listeria monocytogenes Culture Identification Test, and Qualicon's BAX for screening Listeria monocytogenes).

Survival of Escherichia coli O157:H7, Salmonella typhimurium and Listeria monocytogenes in and on vacuum packaged Lebanon bologna stored at 3.6 and 13.0 degrees C.

Escherichia coli O157:H7, Salmonella Typhimurium, or Listeria monocytogenes was spread onto the surface of Lebanon bologna luncheon slices using sterile glass rods. The inoculated slices were stacked and vacuum packaged. The packages were stored at 3.6 or 13 degrees C. The foodborne pathogens. E. coli O157:H7, Salmonella Typhimurium, or L. monocytogenes were reduced in Lebanon bologna during storage at 3.6 or 13 degrees C. The higher storage temperature (13.0 degrees C) resulted in significantly faster destruction of E. coli O157:H7 and L. monocytogenes, compared to storage at refrigeration temperature (3.6 degrees C) (P < 0.005). E. coli O157:H7 was the most resistant to destruction among the three foodborne pathogens. A linear destruction of E. coli O157:H7 occurred only after an initial lag period. Storage temperature did not have a significant effect on the rate of destruction of Salmonella Typhimurium. Foodborne pathogens inoculated prior to fermentation did not show any enhanced survival compared to control cells (inoculated after fermentation) during storage of the Lebanon bologna at 3.6 degrees C.

Validation of a 5-log10 reduction of Listeria monocytogenes following simulated commercial processing of Lebanon bologna in a model system.

Recently, numerous product recalls and one devastating outbreak that claimed 21 lives were attributed to Listeria monocytogenes in ready-to-eat meat products. Consequently, the Food Safety and Inspection Service published a federal register notice requiring manufacturers of ready-to-eat meat and poultry products to reassess their hazard analysis and critical control point plans for these products as specified in 9 CFR 417.4(a). Lebanon bologna is a moist, fermented ready-to-eat sausage. Because of undesirable quality changes. Lebanon bologna is often not processed above 48.9 degrees C (120 degrees F). Therefore, the present research was conducted to validate the destruction of L. monocytogenes in Lebanon bologna batter in a model system. During production, fermentation of Lebanon bologna to pH 4.7 alone significantly reduced L. monocytogenes by 2.3 log10 CFU/g of the sausage mix (P < 0.01). Heating the fermented mix to 48.9 degrees C in 10.5 h destroyed at least 7.0 log10 CFU of L. monocytogenes per g of sausage mix. A combination of low pH (5.0 or lower) and high heating temperatures (> or =43.3 degrees C, 115 degrees F) destroyed more than 5 log10 CFU of L. monocytogenes per g of sausage mix during the processing of Lebanon bologna. In conclusion, an existing commercial process, which was validated for destruction of Escherichia coli O157:H7, was also effective for the destruction of more than 5 log10 CFU of L. monocytogenes.

Comparison of different enrichment broths and background flora for detection of heat-injured Listeria monocytogenes in whole milk.

Various primary enrichment broths, including University of Vermont medium (UVM), Listeria enrichment broth (LEB), modified LEB, and aerobic and anaerobic L-PALCAMY, were compared with aerobic and anaerobic Pennsylvania State University (PSU) broths for the detection of severely heat-injured (62.8 degrees C for 5, 10, or 15 min; no colony appearance after heat injury on aerobic Trypticase soy agar containing 0.6% yeast extract and modified Oxford medium) Listeria monocytogenes Scott A. Anaerobic conditions were produced by adding L-cysteine and then purging the headspace with N2. The effect of uninjured background flora (10(3) CFU/ml of Enterococcus faecium) on frequency of detection was examined. Anaerobic PSU broth resulted in the lowest false-positive rate and the highest frequency of detection of severely heat-injured L. monocytogenes compared with UVM, LEB, and modified LEB (P < 0.05). The presence of E. faecium significantly enhanced the detection of heat-injured (10 min at 62.8 degrees C) L. monocytogenes in aerobic and anaerobic PSU and aerobic and anaerobic L-PALCAMY broths (P < 0.05). The highest concentration of uninjured E. faecium (>10(6) CFU/ml) inhibited the detection of heat-injured L. monocytogenes (P < 0.05). A heat-resistant, LiCl-tolerant Lactobacillus isolate from raw milk increased the rate of both false-positive and false-negative reactions.

Comparison of different reducing agents for enhanced detection of heat-injured Listeria monocytogenes.

The effect of different reducing agents (L-cysteine, Oxyrase, and Enterococcus faecium) and their combinations on the detection of heat-injured (62.8 degrees C, 7.5 min or 10 min) Listeria monocytogenes was examined. The incorporation of L-Cysteine (0.5 g/liter) yielded higher percentage detection than any of the other reducing agents (P < 0.05). The combination of Oxyrase (10 U/ml) and E. faecium (10(3) CFU/ml) synergistically enhanced the detection of L. monocytogenes heat-injured for 10 min at 62.8 degrees C (P < 0.05). Simultaneous addition of L-cysteine (0.5 g/liter) and Oxyrase (10 U/ml) significantly lowered the recovery of heat-injured L. monocytogenes (P < 0.05). Higher activities of Oxyrase (50 U/ml) inhibited the detection of heat-injured L. monocytogenes (P < 0.05). The rates of depletion of relative percentage O2 were in the order: L-cysteine (0.5 g/liter; 6.63%/ min) > Oxyrase (10 U/ml; 5.00%/min) > E. faecium (10(8) CFU/ml; 1.66%/min) > E. faecium (10(3) CFU/ml; 0.20%/min). The final levels of redox potential (Eh) achieved were -110.5 mV, -100 mV, -83.5 mV, and -25 mV for E. faecium (10(8) CFU/ml), L-cysteine, Oxyrase, and E. faecium (10(3) CFU/ml), respectively.

Development of a simple recovery-enrichment system for enhanced detection of heat-injured Listeria monocytogenes in pasteurized milk.

A simple anaerobic recovery-enrichment system, semisolid Penn State University (ssPSU) broth, that enhances recovery of heat-injured Listeria monocytogenes, was rapidly achieved in 10-ml screw-capped tubes by adding Bacto-agar (2.5 g/liter) and L-cysteine (0.5 g/liter) to Penn State University broth. Glucose was removed from the formulation for ssPSU broth to prevent the growth of thermoduric lactobacilli. Ferric ammonium citrate was added to ssPSU broth to detect esculin hydrolysis and to indicate the presumptive presence of L. monocytogenes. Replacement of phosphate buffer with 3-[N-morpholino]propanesulfonic acid (MOPS) buffer and addition of magnesium sulfate (15 mM) enhanced recovery and detection of L. monocytogenes heat treated at 62.8 degrees C for 20 min. D-Serine, at a concentration of 150 mM, was found to inhibit germination of Bacillus spp. spores but did not inhibit severely heat-injured L. monocytogenes. Finally, ssPSU broth was modified (to mPSU broth) to contain the following: (i) Bacto-agar, 2.5 g/liter; (ii) ferric ammonium citrate, 0.5 g/liter; (iii) MOPS buffer, pH 7.0; (iv) D-serine, 13.7 g/liter; (v) D-alanine, 11.6 g/liter; and (iv) magnesium sulfate, 1.81 g/liter. Incubation temperature significantly affected the recovery and detection of severely heat-injured L. monocytogenes. L. monocytogenes that were heat challenged in filter-sterilized whole milk at 62.8 degrees C for 20, 25, and 30 min could not be detected at incubation temperatures > or = 30 degrees C but were consistently detected after incubation at 25 degrees C for 174, 199, and 330 h, respectively. Heat-injured cells of L. monocytogenes that were added to various commercial brands of pasteurized whole milk were also detected using mPSU broth. When clostridial spores (10(4) spores per ml) were added to filter-sterilized milk containing either heat-injured or non-heat-injured L. monocytogenes, only the latter could be detected in mPSU broth. The mPSU broth system requires no purging with nitrogen gas to create anaerobic conditions and permits recovery, growth, and detection of L. monocytogenes in one vessel in the presence of thermoduric background microflora commonly found in pasteurized milk.

Role of bacterial association and penetration on destruction of Escherichia coli O157:H7 in beef tissue by high pH.

This study was undertaken to determine if association with collagen enables Escherichia coli O157:H7 to resist high-pH treatments and to determine the effects of high pH on the survival of E. coli O157:H7 within different layers of beef tissue. E. coli O157:H7 was inoculated onto purified bovine type I collagen on 12-mm2 circular glass coverslips, plain 12-mm2 circular glass coverslips (control), and 12-mm2 irradiated (cobalt-60) lean beef tissue. The rates of destruction of E. coli O157:H7 inoculated on coverslips in pH 10.5 NaHCO3-NaOH buffer at 35 degrees C were determined at various sampling times. E. coli O157:H7 cells associated with collagen and treated in the same manner were also examined using scanning electron microscopy to determine if association with collagen enabled the organism to resist high-pH treatments. The inoculated tissue was treated in pH 13.0 NaHCO3-NaOH buffer at 25 degrees C, and penetrating cells of E. coli O157:H7 were recovered using a cryostat technique. There was no significant difference (P < 0.05) between the rates of destruction of collagen-associated E. coli O157:H7 and non-collagen-associated E. coli O157:H7 following exposure to high-pH treatments. Scanning electron micrographs showed that collagen-associated E. coli O157:H7 cells appeared physically damaged by exposure to high-pH treatments, and association of E. coli O157:H7 to collagen did not increase the resistance of the organism to destruction by high-pH rinses. No significant differences were seen between 20 ml of NaHCO3-NaOH buffer at pH 13.0 (treatment) and 20 ml of distilled water at pH 7.0 (control) when E. coli O157:H7 cells were recovered in beef tissue at depths of up to 2,000 microm (P < 0.05). The ability of E. coli O157:H7 to penetrate beef tissue may be an important factor in reducing the effectiveness of high-pH treatments in killing this organism on beef tissue. This finding should be considered in the future when designing treatments to decontaminate beef carcasses.

Development and validation of a dynamic growth model for Listeria monocytogenes in fluid whole milk.

Listeria monocytogenes, a psychrotrophic microorganism, has been the cause of several food-borne illness outbreaks, including those traced back to pasteurized fluid milk and milk products. This microorganism is especially important because it can grow at storage temperatures recommended for milk (< or =7 degrees C). Growth of L. monocytogenes in fluid milk depends to a large extent on the varying temperatures it is exposed to in the postpasteurization phase, i.e., during in-plant storage, transportation, and storage at retail stores. Growth data for L. monocytogenes in sterilized whole milk were collected at 4, 6, 8, 10, 15, 20, 25, 30, and 35 degrees C. Specific growth rate and maximum population density were calculated at each temperature using these data. The data for growth rates versus temperature were fitted to the Zwietering square root model. This equation was used to develop a dynamic growth model (i.e., the Baranyi dynamic growth model or BDGM) for L. monocytogenes based on a system of equations which had an intrinsic parameter for simulating the lag phase. Results from validation of the BDGM for a rapidly fluctuating temperature profile showed that although the exponential growth phase of the culture under dynamic temperature conditions was modeled accurately, the lag phase duration was overestimated. For an alpha0 (initial physiological state parameter) value of 0.137, which corresponded to the mean temperature of 15 degrees C, the population densities were underpredicted, although the experimental data fell within the narrow band calculated for extreme values of alpha0. The maximum relative error between the experimental data and the curve based on an average alpha0 value was 10.42%, and the root mean square error was 0.28 log CFU/ml.

Comparison of the Difco EZ Coil rapid detection system and Petrifilm test kit-HEC for detection of Escherichia coli O157:H7 in fresh and frozen ground beef.

The Difco EZ Coli Rapid Detection System was compared to the 3M Petrifilm method for detection of Escherichia coli O157:H7 in raw ground beef. Raw meatballs (25 g) were inoculated with 10 to 15 cells of Escherichia coli O157:H7, stored for various times and at different temperatures, and then stomached for 2 min in 225 ml of EZ Coli enrichment broth, which was then incubated at 42 degrees C for 18 to 24 h. A 1-ml sample of the enrichment broth was loaded into the top of the detector tips and the remaining EZ Coli broth held at 35 degrees C before streaking onto MacConkey sorbitol agar and tryptic soy agar with yeast extract. A duplicate set of meatballs were tested using the 3M Petrifilm Test Kit-HEC for hemorrhagic Escherichia coli O157:H7. In this method raw meatballs (25 g) were enriched for 6 h in modified EC broth containing novobiocin at 37 degrees C prior to inoculation of the Petrifilm E. coli Count Plates, which were incubated at 42 degrees C for 18 h. The immunoblot ELISA was performed following this incubation. Presumptive positive isolates from both methods were confirmed using Oxoid E. coli Latex Agglutination and Difco Pasco ID Tripanels. Both methods permitted detection of 10 to 15 cells of E. coli O157:H7 per ml (i) immediately following inoculation, (ii) after 3 days of refrigerated storage at 8 degrees C, and (iii) after 30 days in frozen storage at -20 degrees C. The Difco EZ Coli Detection System proved to be a simpler and faster screening method with identification of negative and presumptive positive samples within 15 to 18 h.

Destruction of Escherichia coli O157:H7 and Salmonella typhimurium in Lebanon bologna by interaction of fermentation pH, heating temperature, and time.

Fermented meats have caused food-borne illness due to enterohemorrhagic Escherichia coli. Consumption of Lebanon bologna was epidemiologically associated wit a recent outbreak of salmonellosis. The present study was conducted to determine the effect of pH (after the fermentation step), final heating temperature, and time on destruction of E. coli O157:H7 and Salmonella typhimurium in Lebanon bologna. Raw Lebanon bologna mix was inoculate with either of the pathogens (ca.10(8) CFR/g and fermented for 12 h at 80 degrees F (26.7 degrees C) and then at 100 degrees F (37.8 degrees C) unit the pH reached wither 5.2 or 4.7. The mix was then heated to 110, 115, or 120 degrees F (43.3, 46.1, or 48.9 degrees C). The bologna was sampled at various times, decimally diluted, and plated on either McConkey sorbitol agar or XLD agar to enumerate E. coli O157:H7 and S. typhimurium, respectively. Fermentation alone reduced populations of both pathogens by < 2 log units and heating alone reduced populations of E. coli O157:H7 by < 3 log units. A combination of fermenting to either pH 5.2 or 4.7, followed by heating at 110 degrees F (43.3 degrees C) for 20h, 115 degrees F (46.1 degrees C) for 10 h, or 120 degrees F (48.9 degrees C) for 3 h reduced populations of both pathogens by > 7 log units. Overall S. typhimurium cells were either equally or significantly less resistant (P < 0.01) than cells of E. coli O157:H7. Significantly interactions (P < 0.01) among the three factors for the destruction of E. coli O157:H7 were observed. A process-specific regression equation was developed to predict the destruction of E. coli O157:H7 in Lebanon bologna.

The role of defeathering in the contamination of turkey skin by Salmonella species and Listeria monocytogenes.

This study was undertaken to determine whether the incidence of either Salmonella spp. or Listeria monocytogenes on turkeys at three commercial processors could be related to the type of defeathering system: 1) conventional, 58 C common bath scald; 2) kosher, 7 C common bath scald; or 3) steam-spray, 62 C nonimmersion scald. Flocks were sampled before defeathering, after defeathering, and after chill at each facility. The incidence of Salmonella-positive turkeys significantly increased subsequent to conventional defeathering (10 positive out of 14) as compared with before defeathering (3/14). The number of Salmonella-positive carcasses following kosher (0/14) and steam-spray (2/14) defeathering were similar to the number of Salmonella-positive carcasses found prior to defeathering (1/14 and 3/14, respectively). The incidence of Salmonella-positive carcasses following chill was slightly lower, but not significantly different than the number of Salmonella-positive carcasses found immediately following defeathering at all processors (8/14, 0/14, 1/14 for conventional, kosher, and steam-spray processors, respectively). Although L. monocytogenes was detected on turkeys sampled before chilling (2/10, kosher) and after chilling (8/14, kosher; 1/14, conventional), no L. monocytogenes was detected on turkeys at any of the processors prior to the evisceration process. Flocks with high aerobic plate counts prior to processing were more likely to contain Salmonella-positive birds throughout processing. Aerobic plate counts of all flocks were similar after chill whether or not Salmonella spp. and L. monocytogenes were detected.

Effect of type of defeathering system on Salmonella cross-contamination during commercial processing.

The cross-contamination effects of three commercial defeathering systems were compared using turkeys from a single Salmonella-positive flock (< or = 15% cloacal-positive). Single or "common" flocks were used to control flock-to-flock variability. Thirty birds were mechanically defeathered in each system as the first flock of the day and compared with 30 hand-defeathered (control) birds. Three trials, each using a different common flock, were completed. In Trial 1, the incidence of Salmonella-positive birds decreased following mechanical defeathering at all three processors. The incidence of Salmonella-positive carcasses in test flocks increased following steam-spray (approximately 100%) and kosher (approximately 50%) defeathering in Trials 2 and 3, whereas no increase in Salmonella-positive carcasses resulted from conventional defeathering. The decrease in the number of Salmonella-positive birds as a result of defeathering observed in Trial 1, as compared to increases observed in Trials 2 and 3, may be related to the selection of feather-contaminated (Trial 1) vs intestinal-colonized (Trials 2 and 3) turkeys. Surface temperature of the carcasses and length of time required to defeather were monitored within each system. It is hypothesized that the increases in the number of Salmonella-positive birds following steam-spray and kosher defeathering in Trials 2 and 3 were a result of skin surface changes occurring during the defeathering process, which allowed increased adherence or entrapment of Salmonella spp. on or within remaining skin layers.

Penetration of Salmonella enteritidis into Eggs Subjected to Rapid Cooling.

Eggs were cooled to 0°C using two different cooling rates, natural convection, and forced convection at an air speed of 30.5 m/min. Upon rapid cooling using forced convection and when brought back to room temperature, eggs were more prone to penetration by Salmonella enteritidis (strain PS8NSR). Eggs cooled using forced convection had 100% penetration by PS8NSR; eggs cooled using natural convection had 91.3% penetration; and uncooled eggs had 48% penetration. Scanning electron microscopy revealed that shells of both cooled and uncooled eggs had microscopic cracks; however, cracks were more numerous and larger in shells of cooled eggs.

A novel strictly anaerobic recovery and enrichment system incorporating lithium for detection of heat-injured Listeria monocytogenes in pasteurized milk containing background microflora.

Heat-injured cells of Listeria monocytogenes were recovered from heated raw milk containing noninjured Enterococcus faecium by combining a simple method for obtaining strict anaerobiosis with a novel enrichment broth, Penn State University broth (PSU broth). Strictly anaerobic conditions were rapidly achieved by adding 0.5 g of filter-sterilized cysteine per liter to PSU broth and then purging the preparation with N2 gas. Little resuscitation or growth occurred in strictly anaerobic PSU broth without lithium chloride because of overgrowth by E. faecium. The growth of E. faecium decreased dramatically with increasing LiCl concentration; LiCl concentrations of 8 and 10 g/liter were completely bacteriostatic. The mechanism of inhibition by LiCl appeared to involve competition with the divalent cations Ca2+ and Mg2+. Heat-injured L. monocytogenes consistently recovered and grew rapidly in strictly anaerobic PSU broth containing 4, 6, or 7 g of LiCl per liter. The use of strictly anaerobic PSU broth containing 7 g of LiCl per liter permitted detection of severely heat-injured L. monocytogenes in one simple recovery-enrichment step by eliminating oxygen toxicity and inhibiting the growth of background microflora, without preventing the resuscitation and subsequent growth of heat-injured L. monocytogenes. L. monocytogenes heated in raw milk at 62.8 degrees C for 10, 15, and 20 min could be consistently recovered from strictly anaerobic PSU broth enrichment cultures at 30 degrees C after 48, 96, and 144 h, respectively, and hence, use of PSU broth may result in better recovery of both injured and noninjured cells from foods than currently used U.S. Department of Agriculture and Food and Drug Administration preenrichment procedures.

Destruction of gram-negative food-borne pathogens by high pH involves disruption of the cytoplasmic membrane.

High pH has been shown to rapidly destroy gram-negative food-borne pathogens; however, the mechanism of destruction has not yet been elucidated. Escherichia coli O157:H7, Salmonella enteritidis ATCC 13706, and Listeria monocytogenes F5069 were suspended in NaHCO3-NaOH buffer solutions at pH 9, 10, 11, or 12 to give a final cell concentration of approximately 5.2 x 10(8) CFU/ml and then held at 37 or 45 degrees C. At 0, 5, 10, and 15 min the suspensions were sterilely filtered and each filtrate was analyzed for material with A260. Viability of the cell suspensions was evaluated by enumeration on nonselective and selective agars. Cell morphology was evaluated by scanning electron microscopy and transmission electron microscopy. A260 increased dramatically with pH and temperature for both E. coli and S. enteritidis; however, with L. monocytogenes material with A260 was not detected at any of the pHs tested. At pH 12, numbers of E. coli and S. enteritidis decreased at least 8 logs within 15 s, whereas L. monocytogenes decreased by only 1 log in 10 min. There was a very strong correlation between the initial rate of release of material with A260 and death rate of the gram-negative pathogens (r = 0.997). At pH 12, gram-negative test cells appeared collapsed and showed evidence of lysis while gram-positive L. monocytogenes did not, when observed by scanning and transmission electron microscopy. It was concluded that destruction of gram-negative food-borne pathogens by high pH involves disruption of the cytoplasmic membrane.

Inhibition of Aspergillus flavus and Selected Gram-positive Bacteria by Chelation of Essential Metal Cations by Polyphosphates.

A simple well-plate technique was utilized to determine the effect of various metals on the growth of microorganisms in media containing different polyphosphates. Aspergillus flavus and four gram-positive bacteria were completely inhibited by media containing 1% of various alkaline polyphosphates, whereas four gram-negative bacteria were not. Significant differences were observed between the type of polyphosphate added, the type of metal added, and the species of gram-positive bacterium inhibited. The addition of Mg2+ stimulated growth of A. flavus and Bacillus cereus in the presence of tetrasodium pyrophosphate, whereas Mn2+ permitted growth of A. flavus and Staphylococcus aureus in the presence of sodium hexametaphosphate. Iron supplementation allowed the growth of S. aureus and Listeria monocytogenes on media containing 1 % tetrasodium pyrophosphate. A method for determining the amount of calcium and magnesium in water was modified to detect free Mg2+ by replacing EDTA with phosphate. The addition of free Mg2+, but not Mg2+ chelated by tetrasodium pyrophosphate, permitted the growth of B. cereus on a medium containing tetrasodium pyrophosphate. It is speculated that polyphosphates specifically inhibited A. flavus and gram-positive bacteria by removing essential metals from cation-binding sites located within their cell walls.