Influence of modified atmospheric storage, lactic acid, and NaCl on survival of sublethally heat-injured Listeria monocytogenes

The effect of package atmosphere on survival of uninjured and sublethally heat-injured Listeria monocytogenes, inoculated onto tryptose phosphate agar containing 0.85% lactic acid and 2% NaCl (TPALAS) was investigated. Inoculated TPALAS plates were packaged in air, 100% N2 (N2), 30% CO2-70% N2 (CO2-N2), and vacuum and stored at 4 and 20 degrees C for up to 31 days. Recovery of L. monocytogenes from TPALAS was influenced by the injury status (i.e., injured and uninjured) of the inoculum, storage atmosphere (air, N2, CO2-N2, and vacuum), storage temperature (4 and 20 degrees C), and recovery media [tryptose phosphate agar (TPA) and modified Oxford agar (MOX)] (P <0.05). Overall, storage at 4 degrees C supported greater survival than storage at 20 degrees C (P< 0.05). Uninjured L. monocytogenes stored at 4 degrees C was recovered on TPA better than sublethally heat-injured L. monocytogenes stored at 40 degrees C (P < 0.05). Recovery of sublethally heat-injured L. monocytogenes stored at 4 degrees C followed the order N2 > CO2-N2 > air > vacuum (P < 0.05), whereas recovery of uninjured L. monocyrogenes stored at 4 degrees C followed the order N2 > CO2-N2 > vacuum > air (P < 0.05). Air and vacuum atmospheres supported greater survival of uninjured and heat-injured L. monocytogenes than N2 and CO2-N2 atmospheres at 20 degrees C (P < 0.05). Recovery of sublethally heat-injured L. monocytogenes stored at 20 degrees C followed the order vacuum > air> CO2-N2 = N2 (P <0.05), whereas recovery of uninjured L. monocytogenes stored at 20 degrees C followed the order vacuum > air> CO2-N2 > N2 (P<0.05). Uninjured L. monocytogenes stored under N2 at 4 degrees C was recovered best, whereas sublethally heat-injured L. monocytogenes stored under N2 at 20 degrees C was recovered poorest (P < 0.05). Factors such as package atmosphere and storage temperature, involved in the production, storage, and distribution of fermented foods must be thoroughly evaluated when determining strategies for control and detection of L. monocytogenes in such products.     

Growth and recovery of selected gram-negative bacteria in reconditioned wastewater.

Previous reports indicate that Escherichia coli O157:H7, Salmonella spp., and Vibrio cholerae can grow in nutrient-limited, reconditioned wastewater over the temperature range of 4 to 46 degrees C when the biological oxygen demand of this water is <2, while its coliform growth response (CGR) is >2. In the current study, we investigated the growth response of Vibrio parahaemolyticus, Shigella spp., Vibrio vulnificus, and Pseudomonas aeruginosa in water samples with a CGR of >2 over the temperature range of 4 to 50 degrees C. Both the nonselective media, tryptic soy agar, and the selective media used to identify the pathogen were used for their recovery. The selective media were thiosulfate-citrate-bile-sucrose (TCBS), MacConkey agar (MAC), and Pseudomonas isolation agar (PIA) for the Vibrio, Shigella, and Pseudomonas spp., respectively. V. parahaemolyticus numbers declined rapidly after surviving for 6 days under the nutrient-limiting growth conditions. Shigella spp. did not grow but survived for >28 days at 4 to 25 degrees C. V. vulnificus grew over the narrow temperature range of 12 to 21 degrees C and survived for >21 days at the higher and lower temperature ranges. P. aeruginosa survived and grew during the 14-day test period at 13 to 35 degrees C. Recovery on the nonselective agar gave statistically (P > 0.05) higher numbers than the respective selective media commonly used for these pathogens. These results indicate that caution should be used in attempting direct recoveries using selective media of the four gram-negative bacteria species used in this study from the nutrient-limited water environment.     

Inactivation of Salmonella typhimurium and Escherichia coli O157:H7 in apple juice by a combination of nisin and cinnamon

Pasteurized apple juice with nisin (0, 25, 50, 100, and 200 ppm, wt/vol) and cinnamon (0 and 0.3%, wt/vol) was inoculated with Salmonella Typhimurium and Escherichia coli O157:H7 at 10(4) CFU/ml and stored at 5 and 20 degrees C. Counts on tryptic soy agar (TSA), selective medium (xylose Lysine desoxycholate agar for Salmonella Typhimurium, and MacConkey sorbitol agar for E. coli O157:H7), and thin agar layer (TAL) were determined at 1 h and 1, 3, 7, and 14 days. The TAL method (selective medium overlaid with TSA) was used for recovery of sublethally injured cells. The pathogens were gradually inactivated by the acidic pH of apple juice. Nisin and cinnamon greatly contributed to the inactivation. The killing effect was more marked at 20 degrees C, with counts in all treated samples being undetectable by direct plating in 3 days for Salmonella Typhimurium and 7 days for E. coli O157:H7. Thus, several factors influenced the decrease in counts: low pH, addition of nisin and cinnamon, and storage temperature. The TAL method was as effective as TSA in recovering injured cells of the pathogens. The combination of nisin and cinnamon accelerates death of Salmonella Typhimurium and E. coli O157:H7 in apple juice and so enhances the safety of the product.     

Survival and recovery of viable but nonculturable Listeria monocytogenes cells in a nutritionally depleted medium

Survival of a desiccated five-strain Listeria monocytogenes mixture during storage in sand at 4 degrees C for 2 months was determined using the acridine orange direct count method with novobiocin and plate counts. Samples of inoculated sand were taken every 2 weeks, incubated at 37 degrees C for 6 h, stained with acridine orange, and then examined with a fluorescence microscope. Elongated viable but nonculturable cells were most frequently observed during weeks 2 and 4. At weeks 6 and 8, most of the cells either remained viable or were dead. In each microscopic field, only one or two viable but nonculturable cells were observed among hundreds of other viable culturable cells, indicating that L. monocytogenes does not generally become viable but nonculturable. Therefore, viable but nonculturable cells are not a concern when plating environmental samples or desiccated L. monocytogenes cells on nonselective media. Tryptic soy agar with 0.6% (wt/vol) yeast extract (TSAYE) and Columbia agar were used as nonselective plate count media. Modified Oxford agar and TSAYE + 5% (wt/vol) sodium chloride were used as the selective plate count media. The effects of aerobic or anaerobic incubation and media supplementation with 0.1% or 1% (wt/vol) sodium pyruvate were tested to optimize recovery of desiccated cells. Nonselective media showed better recovery when TSAYE and Columbia agar contained 0.1% (wt/vol) pyruvate and were incubated aerobically. These two culture methods were equally effective (P > 0.05) for recovering desiccated L. monocytogenes cells.     

Thermal resistance of heat-, cold-, and starvation-injured Salmonella in irradiated comminuted Turkey

To investigate the effects of sublethal stress on Salmonella thermal inactivation kinetics, an eight-strain Salmonella cocktail was subjected to heat shock (30 min at 54 degrees C), cold shock (2 h at 4 degrees C), and starvation stress (10 days in phosphate buffer at 4 degrees C), harvested by centrifugation, and inoculated into irradiated comminuted turkey. Immediately after stressing, the Salmonella cocktails contained 89.1% heat-injured, 44.7% cold-injured, and 67.7% starvation-injured cells, as determined by plating on selective and nonselective media. D60 degrees C-values for the heat-shocked cocktail (0.64 min on Trypticase soy agar containing 0.6% yeast extract [TSAYE], 0.35 min on xylose lysine desoxycholate [XLD] agar) were higher (P < 0.05) than those for the unshocked control (0.41 min on TSAYE, 0.17 min on XLD), whereas D60 degrees -values for the cold-shocked cocktail (0.38 min on TSAYE, 0.17 min on XLD) were not significantly different from those for the control. Starved cells had the same D60 degrees C-value on TSAYE as did the unshocked cocktail, but the D60 degrees C-value on XLD was significantly lower (0.14 min). Although starvation and cold shock were not thermally protective, heat shock increased thermal resistance, indicating that product history and the physiological state of the Salmonella cells should be considered when developing and validating thermal processes. D60 degrees C-values observed on selective media were significantly lower than those observed on nonselective media for all stress treatments and for the control. Therefore, nonselective culture media should be used to assess the response of microorganisms to a thermal challenge when developing performance standards for lethality.     

A comparative study between overlay method and selective-differential media for recovery of stressed Enterobacter sakazakii cells from infant formula

This study compares the performance of different selective-differential media with the overlay method for recovery of stressed cells of Enterobacter sakazakii from infant formula milk (IFM). Five different selective-differential media were used in this study: OK medium, violet red bile agar (VRBA), Druggan-Forsythe-Iversen agar (DFI), Enterobacteriaceae enrichment (EE) agar, and fecal coliform agar (FCA). Tryptic soy agar supplemented with 0.1% sodium pyruvate (TSAP) was used as a control. The overlay method involved applying a thin layer (8ml) of each of the selective media onto TSAP after spreading a sample onto TSAP. Reconstituted IFM was inoculated by ca 1x10(7)CFU/ml of a mixture of four strains of E. sakazakii and subjected to different stress conditions: heat (55 degrees C for 10min), a freeze-thaw cycle (-20 degrees C for 24h, thawed at room temperature, frozen again at -20 degrees C, and thawed), acidic pH (pH 3.56 for 15min), alkaline pH (pH 11.04 for 15min), and desiccation (E. sakazakii was inoculated onto powdered IFM at a level of ca 1x10(6)CFU/g, held at 21 degrees C, water activity of the inoculated product was 0.29 and examined at 0, 15, and 30d). No major differences were noticed between the control (TSAP) and the overlay methods. However, the overlay method recovered significantly higher numbers of stressed E. sakazakii cells compared to selective-differential media. Also, the selective-differential media exhibited some variability in terms of their capabilities to recover stressed cells of E. sakazakii. Among all the examined selective-differential media, DFI performed better for recovering stressed E. sakazakii cells. This study suggests that the overlay method may serve as a potential alternative to direct selective plating for best recovery of E. sakazakii from IFM.     

Evaluation of direct plating methods to enumerate Alicyclobacillus in beverages

Ten agar media were evaluated for their suitability to support spore germination and colony development by six strains of Alicyclobacillus acidoterrestris, three strains of Alicyclobacillus acidocaldarius, and one strain of Alicyclobacillus cycloheptanicus. The influence of plating method (pour versus spread), incubation temperature (43 degrees C and 50 degrees C), and incubation time (up to 10 days) on colony development were determined. K agar, Alicyclobacillus medium (ALI agar), and Bacillus acidoterrestris thermophilic (BAT) agar recovered the highest numbers of spores. Orange serum agar and Hiraishi glucose yeast extract agar were the least suitable. Overall, surface plating was superior to pour plating and, with the exception of one strain of A. acidocaldarius which grew better at 50 degrees C, incubation of K agar, ALI agar, and BAT agar plates at 43 degrees C or 50 degrees C resulted in recovery of equivalent numbers of spores. Essentially all viable spores were detected on media incubated for 3 days at 43 degrees C. The ability of one strain of each Alicyclobacillus species to grow in ten non-carbonated commercially manufactured beverages at 30 degrees C and 43 degrees C was markedly affected by the composition of the beverages. Results show that surface plating samples on BAT agar, followed by incubating plates at 43 degrees C for 3 days provide the most suitable conditions to enumerate ten strains of three species of Alicyclobacillus most commonly responsible for spoilage of beverages.     

Survival of Escherichia coli O157:H7 in frozen foods: impact of the cold shock response

The survival of Escherichia coli O157:H7 strains in both frozen foods and trypticase soy broth (TSB) was investigated following cold shocking at 10 degrees C for 1.5 h. Using both trypticase soy agar (TSA) and violet red bile agar (VRBA) as recovery media, it was demonstrated that survival levels between cold shocked (CS) and non-cold shocked (NS) E. coli in ground beef or pork were not significantly different (P < or = 0.05). In contrast, cold shocking E. coli in either milk, whole egg or sausage resulted in a significant(P < or = 0.05) enhancement in survival. For milk, survival levels of CS E. coli, by 28 days of frozen storage, were 1.89 and 1.66 log10 cfu/ml higher on TSA and VRBA, respectively, when compared to NS cells. In egg these values were 0.64 and 1.31, while in sausage, values of 0.74 and 1.19 were obtained. In TSB (pH 7) survival of CS E. coli for some strains was about 3 log10 cfu/ml higher when compared to NS cells. Acidification of TSB (pH 5), however, appeared to negate the protective effects of the cold shock treatment. In milk, increasing the differential between the growth and cold shock temperatures resulted in higher numbers of survivors. In this regard the growth-cold shock temperature protocol giving optimum protection was 37-10 degrees C. In contrast, growth of E. coli at 20 degrees C followed by cold shocking at 10 degrees C did not result in any significant freeze protection. In addition, increased protection due to cold shocking was correlated with the appearance of a novel protein appearing at pI 4.8 following isoelectric focusing analysis, thus demonstrating an alteration of protein synthesis.     

A solid agar overlay method for recovery of heat-injured Listeria monocytogenes

A solid agar overlay method was developed for recovery of heat-injured Listeria monocytogenes. Presolidified nonselective tryptic soy agar with 0.6% yeast extract (TSAYE, 2% agar) was overlaid on top of solidified modified Oxford agar (MOX). Heat injury of L. monocytogenes was conducted at 58 degrees C for 6 min in a jacketed flask filled with tryptic soy broth. Both noninjured and heat-treated L. monocytogenes cells were plated onto TSAYE, MOX, and TSAYE-MOX plates. No significant differences (P > 0.05) in recovery were found among the three media for noninjured bacterial cells. Recovery of heat-injured L. monocytogenes cells on TSAYE-MOX overlay plates was equivalent to that on the nonselective TSAYE medium, whereas recovery on the selective MOX medium was significantly lower (P < 0.05) compared with both TSAYE and the overlay plates. There were no significant differences (P > 0.05) among the overlay plates prepared 0, 2, 4, 6, 8, 16, and 24 h prior to plating heat-injured bacterial cells. The TSAYE-MOX overlay also allowed differentiation of L. monocytogenes from a mixture of four other types of foodborne pathogens. This solid agar overlay method for recovery of heat-injured L. monocytogenes cells is less time-consuming and less complicated than the conventional overlay-underlay technique and the double overlay modification of the thin agar layer method and may allow for greater laboratory plating efficiencies.     

Improving recovery of Salmonella enterica serovar typhimurium DT104 cells injured by heating at different water activity values

This study describes the evaluation of potentially more sensitive methods for the recovery of Salmonella cells injured by heating (54 to 60 degrees C) at different water activity values (0.65 to 0.90, reduced using equal portions of glucose and fructose). These methods included gradual rehydration, the use of diluting media with added solutes or blood, the addition of blood to plating agar, and the use of different incubation temperatures and times. Gradual rehydration of cells that had been challenged at low water activity (0.65 and 0.70) and high temperature markedly improved recovery, measured as a >50% increase in the time to obtain a 3-log10 reduction in cell numbers, compared to dilution into media with a high water activity. Adding sucrose, glycerol, or blood to the diluting media (maximal recovery diluent) did not improve recovery, but a plating agar containing blood recovered approximately 38% more cells than nutrient agar. Prolonged incubation of agar plates allowed recovery of injured Salmonella cells that presumably had extended lag periods, with significantly higher recovery rates after 48 h incubation at 37 degrees C than after 24 h (P = 0.05). This work highlights that by recovering Salmonella using a method specific to the nature of the injury, a better prediction of food safety and the success of food processing can be made.     

Thin agar layer method for recovery of heat-injured Listeria monocytogenes

A thin agar layer (TAL) method was developed to recover heat-injured Listeria monocytogenes. Modified Oxford medium (MOX), a selective plating medium, inhibits heat-injured L. monocytogenes from growing, whereas tryptic soy agar (TSA), a nonselective medium, does not. In order to facilitate recovery of heat-injured L. monocytogenes cells while providing selectivity of isolation of L. monocytogenes from other bacteria in the sample, a unique TAL procedure was developed by overlaying 5 ml of nonselective medium (TSA) onto prepoured and solidified MOX medium in an 8.5-cm-diameter petri dish. The injured L. monocytogenes repaired and started to grow in the TSA during the first few hours after incubation of the plate. During the resuscitation of injured cells, the selective agents from MOX diffused to the TSA top layer to inhibit other microorganisms. L. monocytogenes showed a typical reaction (black colonies) on TAL after 24 h of incubation at 37 degrees C. The recovery rate for heat-injured L. monocytogenes with the TAL method was compared with those rates associated with TSA, MOX, and the traditional overlay method (OV; pouring selective agar on top of resuscitated cells on TSA agar after 3 h incubation). Milk and 0.1% peptone water that were inoculated with L. monocytogenes (4 to 5 log CFU/ml) were heated for 15 min at 55 degrees C. L. monocytogenes was enumerated on TSA, MOX, OV, and TAL media and procedures. No significant difference occurred among TSA, OV, and TAL (P > 0.05) in terms of enumeration of heat-injured L. monocytogenes, but these media recovered significantly higher numbers than did MOX agar (P < 0.05)-in both samples. The TAL method involves only one step, whereas OV is a more cumbersome two-step procedure.     

Isolation of Yersinia enterocolitica from foods.

Many selective enrichment and plating media for the isolation of Yersinia enterocolitica from foods are described. However, at present no single isolation procedure is available for the recovery of all pathogenic strains of Yersinia enterocolitica. Cold enrichment in phosphate-buffered saline plus 1% sorbitol and 0.15% bile salts (PBSSB) and two-step enrichment with tryptone soy broth (TSB) and bile oxalate sorbose (BOS) broth are very efficient methods for the recovery of a wide spectrum of serotypes of Y. enterocolitica. Enrichment in irgasan ticarcillin chlorate (ITC) broth was found to be the most efficient method for the recovery of strains of serotype 0:3, which is the most common clinical serotype of Y. enterocolitica in Europe. Post-enrichment alkali treatment often results in higher isolation rates. Cefsulodin irgasan novobiocin (CIN) agar and Salmonella-Shigella deoxycholate calcium chloride (SSDC) agar are the most commonly used plating media. For the recovery of serotype 0:8 strains, the common clinical isolates in North America, enrichment in BOS and plating on CIN seems the most efficient procedure. Selection of the proper enrichment procedure will depend on the bio/serotypes of Yersinia spp. sought and on the type of food to be examined. The use of more than one medium for both enrichment and plating will result in higher recovery rates of Yersinia spp. from foods. Parallel use of the following two isolation procedures is recommended. (1) Enrichment in ITC for 2 days at 24 degrees C; plating on SSDC agar (2 days at 30 degrees C). (2) Pre-enrichment in TSB for 1 day at 24 degrees C; enrichment in BOS for 5 days at 24 degrees C; alkali treatment (mixing 0.5 ml enriched broth with 4.5 ml of 0.5% KOH in 0.5% NaCl for 5 s); plating on CIN agar (2 days at 24 degrees C).     

Survival of Salmonella enteritidis phage type 30 on inoculated almonds stored at -20, 4, 23, and 35 degrees C

To evaluate the survival of Salmonella on raw almond surfaces, whole almond kernels were inoculated with Salmonella Enteritidis phage type (PT) 30 collected from a 24-h broth culture or by scraping cells from an agar lawn. Kernels inoculated with lawn-collected cells to 8, 5, 3, and 1 log CFU per almond after a 24-h drying period were stored for 161 days at 23 +/- 3 degrees C. Calculated rates of reduction were similar for the four inoculum levels (0.22, 0.28, 0.29, and 0.22 log CFU/month, respectively). Kernels inoculated to 7.1 or 8.0 log CFU per almond after drying were stored for 171 or 550 days, respectively, at selected temperatures, including -20 +/- 2 degrees C, 4 +/- 2 degrees C, 23 +/- 3 degrees C, and 35 +/- 2 degrees C. No significant reductions of Salmonella were observed during storage at -20 and 4 degrees C over 550 days. At 35 degrees C, a biphasic survival curve was observed, with calculated reductions of 1.1 log CFU/month from days 0 to 59 and no significant reduction from days 59 to 171. At 23 degrees C, reductions of 0.18 and 0.30 log CFU/month were calculated for 171 and 550 days of storage, respectively. When combined with data from the study of inoculum levels, an overall average calculated reduction at 23 degrees C was 0.25 +/- 0.05 log CFU/month. Significantly greater reductions were observed during the 24-h drying period when broth-collected cells were used as the inoculum, suggesting that cells collected from agar lawns were more resistant to drying. However, after initial drying, the rates of reduction at 23 degrees C did not differ significantly between the inoculum preparation methods. Salmonella Enteritidis PT 30 survives for long periods on almond kernels under a variety of commonstorage conditions.     

Survival of Escherichia coli O157:H7 in Galotyri cheese stored at 4 and 12 degrees C

Post-process contamination of fresh acid-curd cheeses with Escherichia coli O157:H7 may pose a risk considering the low infectious dose and the ability of the pathogen to survive in acidic foods. To evaluate its survival in Galotyri, a traditional Greek acid-curd cheese, portions (0.5 kg) of two commercial fresh products, one artisan (pH 3.9+/-0.1) and the other industrial (pH 3.7+/-0.1), were inoculated with approximately 3.0 or 6.5 log cfu g(-1) of a five-strain cocktail of E. coli O157:H7, including rifampicin-resistant derivatives of the strains ATCC 43895 and ATCC 51657, and stored aerobically at 4 and 12 degrees C. Survival was monitored for 28 days by plating cheese samples on tryptic soy agar with 100 mg l(-1) rifampicin (TSA+Rif), SMAC and Fluorocult E. coli O157:H7 agar media. The pathogen declined much faster (P<0.05) in the industrial as compared to the artisan cheeses at both temperatures. Thus, while E. coli O157:H7 became undetectable by culture enrichment after 14 days at 4 degrees C in industrial samples, irrespective of the inoculation level, populations of 1.4-1.9 and 4.2-5.1 log cfu g(-1) survived after 28 days in the corresponding artisan cheeses with the low and high inocula, respectively. Survival was longer and greater (P<0.05) on TSA+Rif than on SMAC and Fluorocult, indicating the presence of acid-injured cells. Interestingly, survival of E. coli O157:H7 after 14-28 days in cheeses was better at 12 degrees C than at 4 degrees C, probably due to yeasts which grew on the surface of temperature-abused cheeses. The large difference in the pathogen's inactivation between the industrial and artisan cheeses at 4 degrees C could not be associated with major differences in pH or type/concentration of organic acids, suggesting another anti-E. coli O157:H7 activity by the industrial starter. The high survival of the pathogen in artisan Galotyri under conditions simulating commercial storage should be of concern.     

Identification of bacteria crucial to histamine accumulation in pacific mackerel during storage

Bacterial growth and histamine formation in Pacific mackerel during storage at 0, 4, 15, and 25 degrees C were monitored. To identify bacterial species contributing to histamine formation, several groups of bacteria were isolated by using selective media under temperatures corresponding to the various storage conditions. Initially, low counts of bacteria were found in the gill, skin, and intestine of fresh fish, and only weak histamine formers were found in the gill. Histamine was found in the muscle when fish were stored above 4 degrees C, and aerobic plate counts reached 10(6) CFU/g. When fish became unsuitable for human consumption by abusive storage, toxicological levels of histamine were always found. The highest level of histamine formed was 283 mg/100 g in 2 days. The optimum temperature for supporting growth of prolific histamine formers was 25 degrees C. The most prolific and prevalent histamine former was Morganella morganii, followed by Proteus vulgaris, both of which were isolated on violet red bile glucose (VRBG) agar. At 15 degrees C, a significant level of histamine was still produced in fish muscle, although prolific histamine formers were less frequently detected than at 25 degrees C. The isolates on thiosulfate citrate bile salts sucrose (TCBS) agar were weak histamine formers and identified as Vibrio parahaemolyticus and Vibrio alginolyticus. At 4 degrees C, less than 57.4 mg/100 g of histamine was found in fish stored for 14 days. Most isolates were natural bacterial flora in the marine environment and identified as weak histamine formers. At 0 degrees C, neither histamine former nor histamine production was detected up to 14 days of storage.     

Filament formation by Salmonella spp. inoculated into liquid food matrices at refrigeration temperatures,and growth patterns when warmed

In this study, the formation of multicellular filamentous Salmonella cells in response to low temperatures was investigated by using isolates of Salmonella enterica serovar Enteritidis PT4 and S. enterica serovar Typhimurium DT104 as the inocula. The formation of filamentous cells in two liquid food matrices at the recommended maximum temperature for refrigeration (8 degrees C) was monitored and compared with that in tryptone soya broth. Giemsa staining was performed to locate nuclear material within the filaments. Single filaments were warmed on agar at 37 degrees C, and the subsequent rate of septation was quantified. For all strains tested, > 70% of the Salmonella cells inoculated had become filamentous after 4 days in media at 8 degrees C, indicating that filamentation could occur during the shelf life of most refrigerated foods. Strains with impaired RpoS expression were able to form filaments at 8 degrees C, although these filaments tended to be shorter and less numerous. All strains also formed filamentous cells at 8 degrees C in retail milk or chicken meat extract. Filaments often exceeded 100 microm in length and appeared straight-sided under the microscope in media and in foods, and Giemsa staining demonstrated that regularly spaced nucleoids were present. This phenotype indicates that an early block in cell septation is probably responsible for filamentation. When filaments were warmed on agar at 37 degrees C, there was a rapid completion of septation, and for one filament, a >200-fold increase in cell number was observed within 4 h. There are clear public health implications associated with the filamentation of Salmonella in contaminated foods at refrigeration temperatures, especially when the possibility of rapid septation of filamentous cells upon warming is considered.     

Recovery of heat-injured Listeria innocua

Listeria innocua was subjected to thermal inactivation and the extent of heat-injured cells was quantified. Cultures were heated in liquid medium for different times, using temperatures in the range of 52.5 to 65.0 degrees C, and plated on Tryptic Soy Agar with 0.6% yeast extract (TSAYE) used as non-selective medium and on TSAYE plus 5% NaCl (TSAYE+NaCl) and Palcam agar with selective supplement (Palcam agar) as selective media. The difference observed in counts in non-selective and in selective media gave an indication of cell injury during the heat treatment. D- and z- values were calculated for all conditions considered. For each temperature, D-values obtained using non-selective recovery procedures were higher than the ones obtained using the two selective media. When comparing the selective media, it can be concluded that Palcam agar allowed recovery and growth of thermally injured cells and so it was less inhibitor than TSAYE+NaCl. Another important result was the influence of temperature on the degree of cellular injury. As temperature increases, the degree of heat-injured cells also increases, and consequently concern has to be taken with the temperature and the counting medium used in food processing studies. The results of this work clearly demonstrated that selective media used for Listeria monocytogenes enumeration/detection might not be suitable for the recovery of heat-injured cells, which can dangerously underestimate the presence of this foodborne pathogen.     

Survival and growth of Escherichia coli O157:H7 in roast beef and salami after exposure to an alkaline cleaner

Survival and growth of wild-type (EDL 933) and rpoS-deficient (FRIK 816-3) strains of Escherichia coli O157:H7 after exposure to an alkaline cleaner for 2 min and inoculating into roast beef (pH 6.3) and hard salami (pH 4.9) at low (0.003 to 0.52 CFU/g) and high (0.69 to 31.5 CFU/g) populations were determined. Roast beef was stored at 4 and 12 degrees C; salami was stored at 4, 12, and 20 degrees C. At 4 degrees C, untreated cells of both strains showed greater reductions in populations in salami than in roast beef during a 21-day storage period. Populations of treated and untreated cells recovered from roast beef and salami stored at 4 degrees C on tryptic soy agar were significantly (P < or = 0.05) higher than on sorbitol MacConkey agar, indicating that a portion of the cells was injured. Treated and untreated cells grew in roast beef at 12 degrees C. Growth of treated cells of the FRIK 816-3 strain in roast beef at 12 degrees C was significantly slower than that of the EDL 933 strain. Populations of both strains decreased at different rates in salami stored at different temperatures (20 degrees C > 12 degrees C > 4 degrees C). E. coli O157:H7 strain EDL 933 grew more rapidly at 20 degrees C in a slurry (pH 5.97) prepared from stored salami (17 days at 20 degrees C) on which Penicillium chrysogenum had grown than in a slurry (5.23) prepared from salami showing no mold growth. Within 2 to 3 days, populations were ca. 3 log CFU/ml higher in slurry made from infected salami than in control salami. Results indicate that treatment of E. coli O157: H7 with an alkaline cleaner for 2 min does not impair resuscitation and growth of surviving cells in roast beef at 12 degrees C. Cross protection of cells exposed to an alkaline cleaner against subsequent stress conditions imposed by roast beef and salami stored at 4 degrees C was not evident in either of the test strains.     

Metabiotic associations of molds and Salmonella Poona on intact and wounded cantaloupe rind

Salmonella Poona, a serotype rarely implicated in human infections, has recently caused several cantaloupe-associated outbreaks of salmonellosis. Metabiotic associations of molds and foodborne pathogens on produce have been reported. We tested proteolytic activity and measured changes in the pH of cantaloupe rind caused by growth of Alternaria alternata, Cladosporium cladosporioides, Epicoccum nigrum, Geotrichum candidum, and Penicillium expansum. Survival and growth characteristics of Salmonella Poona co-infected with each mold on the surface rind and in wounded rind tissue as affected by temperature were determined. C. cladosporioides, G. candidum, and P. expansum, but not A. alternata and E. nigrum, showed proteolytic activity on agar media containing gelatin and/or casein, with concurrent increases in pH, thus favoring survival and growth of salmonellae. Intact and mechanically wounded tissue of cantaloupe rinds were inoculated with a five-strain mixture of S. Poona and/or test mold. Five inoculation schemes were used: mold only, S. Poona only, mold and S. Poona simultaneously, mold then S. Poona 3 days later, and S. Poona then mold 3 days later. The pH of cantaloupe rinds inoculated with molds and stored at 20 degrees C for 14 days was significantly higher (P < or =0.05) than on day 0. Only the pH of rinds inoculated with C. cladosporioides or G. candidum was significantly higher (P < or =0.05) on day 21 than on day 0, when cantaloupes were stored at 4 degrees C. An initial population of S. Poona increased from 3.3 log(10) cfu/sample (ca. 7 cm(2)) of cantaloupe rind to populations as high as 9.5 log(10) cfu/sample during storage at 20 degrees C for up 14 days, regardless of co-inoculation with molds. Populations of S. Poona decreased or remained constant at 4 degrees C for up to 21 days. Results demonstrate that persistence and growth of S. Poona on intact, wounded, and decaying cantaloupe rind are not markedly affected by the presence of molds.     

Fate of Escherichia coli O157:H7 and Listeria monocytogenes in strawberry juice and acidified media at different pH values and temperatures

Survival and growth of Escherichia coli O157:H7 and Listeria monocytogenes in strawberry juice and acidified media at different pH levels (pH 3.4 to 6.8) and temperatures were studied. Sterile strawberry juice (pH 3.6) and acidified trypticase soy broth (TSB) media (pH 3.4 to 6.8) were inoculated with approximately 6.7 log CFU/ml E. coli O157:H7 or 7.3 log CFU/ ml L. monocytogenes, incubated for 3 days at 4 and 37 degrees C. Bacterial levels were determined after 2 h, 1 day, and 3 days using surface plating nonselectively on tryptic soy agar and selectively on sorbitol MacConkey agar for E. coli O157:H7 or modified Oxford agar for L. monocytogenes. A spectrophotometer (660 nm) was also used to study growth inhibition of L. monocytogenes in different TSB and strawberry juice media (pH 3.4 to 7.3). E. coli O157:H7 survived well at pH values of 3.4 to 6.8 at 4 degrees C, but the number of injured cells increased as pH decreased and incubation time increased. At 37 degrees C, E. coli O157:H7 was inactivated at pH of < or = 3.6 but could grow at pH 4.7. L. monocytogenes was quickly injured at pH of < or = 4.7 within 2 h of storage at 4 degrees C and then was slightly and gradually inactivated as storage time increased. L. monocytogenes survived well at pH 6.8 at 4 degrees C and grew well at 37 degrees C. Growth of L. monocytogenes at 37 degrees C was inhibited in TSB by 1% citric acid and 0.5% malic acids at pH 3.4 or by 50% strawberry juice at pH 4.7. Bacterial injury and inactivation appeared to be induced by the acids in strawberry juice. The acids, pH value, temperature, and time were important factors for bacterial survival, inactivation, and growth in the media tested.