Survival and growth characteristics of Escherichia coli O157:H7 in pasteurized and unpasteurized Cheddar cheese whey

The objective of this study was to determine the survival and growth characteristics of Escherichia coli O157:H7 in whey. A five-strain mixture of E. coli O157:H7 was inoculated into 100 ml of fresh, pasteurized or unpasteurized Cheddar cheese whey (pH 5.5) at 10(5) or 10(2) CFU/ml, and stored at 4, 10 or 15 degrees C. The population of E. coli O157:H7 (on Sorbitol MacConkey agar supplemented with 0.1% 4-methylumbelliferyl-beta-D-glucuronide) and lactic acid bacteria (on All Purpose Tween agar) were determined on days 0, 1, 4, 7, 14, 21 and 28. At all storage temperatures, survival of E. coli O157:H7 was significantly higher (P<0.01) in the pasteurized whey compared to that in the unpasteurized samples. At 10 and 15 degrees C, E. coli O157:H7 in pasteurized whey significantly (P<0.05) increased during the first week of storage, followed by a decrease thereafter. However at the same temperatures, E. coli O157:H7 exhibited a steady decline in the unpasteurized samples from day 0. At 4 degrees C, E. coli O157:H7 did not grow in pasteurized and unpasteurized whey; however, the pathogen persisted longer in pasteurized samples. At all the three storage temperatures, E. coli O157:H7 survived up to day 21 in the pasteurized and unpasteurized whey. The initial load of lactic acid bacteria in the unpasteurized whey samples was approximately 7.0 log10 CFU/ml and, by day 28, greater than 3.0 log10 CFU/ml of lactic acid bacteria survived in unpasteurized whey at all temperatures, with the highest counts recovered at 4 degrees C. Results indicate the potential risk of persistence of E. coli O157:H7 in whey in the event of contamination with this pathogen.     

Survival of Escherichia coli O157:H7 in traditional African yoghurt fermentation

Growth and survival of a nontoxigenic strain of Escherichia coli O157:H7 (ATCC 43888) was determined in traditionally fermented pasteurized milk. Preheated milk was inoculated with 1% (v/v) of a mixed culture of Lactobacillus delbrueckii ssp. bulgaricus (NCIMB 11778) and Streptococcus salivarius ssp. thermophilus (NCIMB 110368) and incubated at 25, 30, 37 or 43 degrees C for 24 h. E. coli O157:H7 (10(5) CFU/ml) were introduced into the milk pre- and post-fermentation. Fermented milk samples were subsequently stored at either 4 degrees C (refrigerator temperature) or 25 degrees C (to mimic African ambient temperature) for 5 days. After 24 h of fermentation, the pH of the samples fermented at the higher temperatures of 37-43 degrees C decreased from 6.8 to 4.4-4.0 ( +/- 0.2) whereas at the lower temperature of 25 degrees C, the pH decreased to pH 5.0 +/- 0.1. During this period, viable counts for E. coli O157:H7 increased from 10(5) to 10(8) - 10(9) CFU/ml except in milk fermented at 43 degrees C wherein viability declined to 10(4) CFU/ml. In fermented (25-30 degrees C) milk stored at 4 degrees C for 5 days, E. coli O157:H7 viability decreased from 10(8-9) to 10(6-7) CFU/ml whereas milk fermented at 43 degrees C resulted in loss of detectable cells. In contrast, storage of fermented milk samples at 25 degrees C for 5 days eventually resulted in complete loss of viability irrespective of fermentation temperature. Stationary phase E. coli O157:H7 inoculated post-fermentation (25 and 43 degrees C) survived during 4 degrees C storage, but not 25 degrees C storage. Fermentation temperature and subsequent storage temperature are critical to the growth and survival of E. coli O157:H7 in traditional fermented products involving yoghurt starter cultures.     

Novel Real-Time PCR Method To Detect Escherichia coli O157:H7 in Raw Milk Cheese and Raw Ground Meat

Raw milk, raw milk cheeses, and raw ground meat have been implicated in Escherichia coli O157:H7 outbreaks. Developing methods to detect these bacteria in raw milk and meat products is a major challenge for food safety. The aim of our study was to develop a real-time PCR assay to detect E. coli O157:H7 in raw milk cheeses and raw ground meat. Well-known primers targeting a mutation at position +93 of the uidA gene in E. coli O157:H7 were chosen, and a specific TaqMan-minor groove binder probe was designed. This probe targets another mutation, at position +191 of the uidA gene in E. coli O157:H7. The first step in the study was to evaluate the specificity of this probe with 156 different O157:H7/NM strains and 48 non-O157:H7/NM strains of E. coli. The sensitivity of the method was evaluated by pre- and postinoculation of cheeses and meat enrichments with different E. coli O157:H7 strains. All the E. coli O157:H7 isolates tested were positive, and none of the other bacteria were detected. Our results indicate that this method is sensitive enough to detect 10(2) E. coli O157:H7 isolates per ml of cheese or meat enrichment broth (24 h at 41.5° C) and is more sensitive than the International Organization for Standardization reference method. We can conclude that this new real-time PCR protocol is a useful tool for rapid, specific, and sensitive detection of E. coli O157:H7 in raw milk and raw ground meat products.     

Survival of Salmonella, Escherichia coli O157:H7, and Listeria monocytogenes on Inoculated Almonds and Pistachios Stored at -19, 4, and 24° C

The survival of Salmonella, Escherichia coli O157:H7, and Listeria monocytogenes was determined on almonds and pistachios held at typical storage temperatures. Almond kernels and inshell pistachios were inoculated with four- to six-strain cocktails of nalidixic acid-resistant Salmonella, E. coli O157:H7, or L. monocytogenes at 6 log CFU/g and then dried for 72 h. After drying, inoculated nuts were stored at -19, 4, or 24°C for up to 12 months. During the initial drying period after inoculation, levels of all pathogens declined by 1 to -log CFU/g on both almonds and pistachios. During storage, moisture content (4.8%) and water activity (0.4) of the almonds and pistachios were consistent at -19°C; increased slowly to 6% and 0.6, respectively, at 4°C; and fluctuated from 4 to 5% and 0.3 to 0.5 at 24°C, respectively. Every 1 or 2 months, levels of each pathogen were enumerated by plating; samples were enriched when levels fell below the limit of detection. No reduction in population level was observed at -19 or 4°C for either pathogen, with the exception of E. coli O157:H7-inoculated almonds stored at 4°C (decline of 0.09 log CFU/g/month). At 24°C, initial rates of decline were 0.20, 0.60, and 0.71 log CFU/g/month on almonds and 0.15, 0.35, and 0.86 log CFU/g/month on pistachios for Salmonella, E. coli O157:H7, and L. monocytogenes, respectively, but distinct tailing of the survival curves was noted for both E. coli O157:H7 and L. monocytogenes.     

Influence of lactic acid bacteria on survival of Escherichia coli O157:H7 in inoculated Minas cheese during storage at 8.5 degrees C

Minas cheese is a typical Brazilian fresh cheese, manufactured by addition of rennin and CaCl2 to milk, followed by draining the curd. The intrinsic characteristics of this product make it favorable for growth of pathogens, including Escherichia coli O157:H7. The influence of the addition of a commercial mesophilic type O lactic culture to this product on the growth of this pathogen during storage at 8.5 degrees C was evaluated. Eight different formulations of Minas cheese were manufactured using raw or pasteurized milk and with or without salt and lactic culture. Individual portions of each formulation were transferred to sterile plastic bags and inoculated with E. coli O157:H7 to yield ca. 10(3) or 10(6) CFU/g. After blending by hand massaging the bags, samples were stored at 8.5 degrees C for up to 14 days. E. coli O157:H7 was counted after 1, 2, 7, and 14 days of storage using 3M Petrifilm Test Kit-HEC. Counts in samples without added lactic culture showed a 2-log increase in the first 24 h and remained constant during the following 14 days. Counts in samples with added lactic culture showed a 0.5-log increase in the first 24 h, followed by a decrease. These variations were statistically significant (P < 0.05). No significant variations (P > 0.05) were obtained for cheese samples manufactured with pasteurized or raw milk, with or without salt. Results indicate that the addition of type O lactic culture may be an additional safeguard to well-established good manufacturing practices and hazard analysis and critical control point programs in the control of growth of E. coli O157:H7 in Minas cheese.     

Use of microfiltration to improve fluid milk quality

The objectives of the research were to determine the growth characteristics of bacteria in commercially pasteurized skim milk as a function of storage temperature; to determine the efficacy of a microfiltration and pasteurization process in reducing the number of total bacteria, spores, and coliforms in skim milk; and to estimate the shelf life of pasteurized microfiltered skim milk as a function of storage temperature. For the first objective, commercially pasteurized skim milk was stored at 0.1, 2.0, 4.2, and 6.1 degrees C. A total bacterial count >20,000 cfu/mL was considered the end of shelf life. Shelf life ranged from 16 d at 6.1 degrees C to 66 d at 0.1 degrees C. Decreasing storage temperature increased lag time and reduced logarithmic growth rate of a mixed microbial population. The increased lag time for the mixed microbial population at a lower storage temperature was the biggest contributor to longer shelf life. For the second objective, raw skim milk was microfiltered at 50 degrees C using a Tetra Alcross M7 Pilot Plant equipped with a ceramic Membralox membrane (pore diameter of 1.4 microm). The 50 degrees C permeate was pasteurized at 72 degrees C for 15 s, and cooled to 6 degrees C. Bacterial counts of raw skim milk were determined by standard plate count. Bacterial counts of microfiltered and pasteurized microfiltered skim milk were determined using a most probable number method. Across 3 trials, bacterial counts of the raw milk were reduced from 2,400, 3,600, and 1,475 cfu/mL to 0.240, 0.918, and 0.240 cfu/mL, respectively, by microfiltration. Bacterial counts in the pasteurized microfiltered skim milk for the 3 trials were 0.005, 0.008, and 0.005 cfu/mL, respectively, demonstrating an average 5.6 log reduction from the raw count due to the combination of microfiltration and pasteurization. For the third objective, pasteurized microfiltered skim milk was stored at each of 4 temperatures (0.1, 2.0, 4.2, and 6.1 degrees C) and the total bacterial count was determined weekly over a 92-d period. At 6 time points in the study, samples were also analyzed for noncasein nitrogen and the decrease in casein as a percentage of true protein was calculated. After 92 d, 50% of samples stored at 6.1 degrees C and 12% of samples stored at 4.2 degrees C exceeded a total bacterial count of 20,000 cfu/mL. No samples stored at 0.1 or 2.0 degrees C reached a detectable bacterial level during the study. When the bacterial count was <1,000 cfu/mL, shelf life was limited because sufficient proteolysis had occurred at 32 d at 6.1 degrees C, 46 d at 4.2 degrees C, 78 d at 2.0 degrees C, and >92 d at 0.1 degrees C to produce a detectable off-flavor in skim milk produced from a raw milk with a 240,000 somatic cell count.     

Effect of modified atmosphere packaging on the persistence and expression of virulence factors of Escherichia coli O157:H7 on shredded iceberg lettuce

Fresh-cut leafy greens contaminated with Escherichia coli O157:H7 have caused foodborne outbreaks. Packaging conditions, coupled with abusive storage temperatures of contaminated lettuce, were evaluated for their effect on the potential virulence of E. coli O157:H7. Shredded lettuce was inoculated with 5.58 and 3.98 log CFU E. coli O157:H7 per g and stored at 4 and 15°C, respectively, for up to 10 days. Lettuce was packaged under treatment A (modified atmosphere packaging conditions used for commercial fresh-cut produce, in gas-permeable film with N(2)), treatment B (near-ambient air atmospheric conditions in a gas-permeable film with microperforations), and treatment C (high-CO(2) and low-O(2) conditions in a gas-impermeable film). E. coli O157:H7 populations from each treatment were determined by enumeration of numbers on MacConkey agar containing nalidixic acid. RNA was extracted from packaged lettuce for analysis of expression of virulence factor genes stx(2), eae, ehxA, iha, and rfbE. E. coli O157:H7 populations on lettuce at 4°C under all treatments decreased, but most considerably so under treatment B over 10 days. At 15°C, E. coli O157:H7 populations increased by at least 2.76 log CFU/g under all treatments. At 15°C, expression of eae and iha was significantly greater under treatment B than it was under treatments A and C on day 3. Similarly, treatment B promoted significantly higher expression of stx(2), eae, ehxA, and rfbE genes on day 10, compared with treatments A and C at 15°C. Results indicate that storage under near-ambient air atmospheric conditions can promote higher expression levels of O157 virulence factors on lettuce, and could affect the severity of E. coli O157:H7 infections associated with leafy greens.     

Acid adaptation and temperature effect on the survival of E. coli O157:H7 in acidic fruit juice and lactic fermented milk product.

In this study, two strains of Escherichia coli O157:H7, (ATCC 43889 and ATCC 43895) were acid adapted at pH 5.0 in tryptic soy broth (TSB) for 4 h. Commercial products of mango juice (pH 3.2), asparagus juice (pH 3.6), Yakult--a diluted milk fermented drink (pH 3.6), and low-fat yoghurt (pH 3.9) were inoculated with acid-adapted or nonadapted cells of E. coli O157:H7. Survival of the inoculated E. coli O157:H7 in these commercial food products during storage at 25 or 7 degrees C was examined. It was found that although survival of the acid-adapted and nonadapted E. coli O157:H7 ATCC 43895 in asparagus juice during storage at 7 degrees C did not show marked difference, in general, acid adaptation and low temperature enhanced the survival of E. coli O157:H7 in both the commercial fruit juices tested. On the contrary, acid adaptation reduced the survival of both the strains of the test organism in Yakult and low-fat yoghurt stored at 7 degrees C. Besides, E. coli O157:H7 ATCC 43895 survived longer than ATCC 43889 in all the products examined, regardless of the storage temperature and acid adaptation.     

Inactivation of microorganisms in milk and apple cider treated with ultrasound

Nonthermal technologies are emerging as promising alternatives to heat treatment for food processing. Ultrasound, defined as sound waves with a frequency greater than 20 kHz, has proven bactericidal effects, especially when combined with other microbial-reduction strategies such as mild heating. In this study, ultrasound treatment (sonifier probe at 20 kHz, 100% power level, 150 W acoustic power, 118 W/cm2 acoustic intensity) with or without the effect of mild heat (57 degrees C) was effective at reducing microbial levels in raw milk, Listeria monocytogenes levels inoculated in ultrahigh-temperature milk, and Escherichia coli O157:H7 in apple cider. Continuous flow ultrasound treatment combined with mild heat (57 degrees C) for 18 min resulted in a 5-log reduction of L. monocytogenes in ultrahigh-temperature milk, a 5-log reduction in total aerobic bacteria in raw milk, and a 6-log reduction in E. coli O157:H7 in pasteurized apple cider. Inactivation regressions were second-order polynomials, showing an initial period of rapid inactivation, eventually tailing off. Results indicate that ultrasound technology is a promising processing alternative for the reduction of microorganisms in liquid foods.     

Survival of Escherichia coli O157:H7 and Campylobacter jejuni in bottled purified drinking water under different storage conditions

Survival of Escherichia coli O157:H7 and Campylobacter jejuni that were separately inoculated into bottled purified drinking water was investigated during storage at 22, 4, and -18 °C for 5, 7, and 2 days, respectively. Two inoculation levels were used, 1 and 10 CFU/ml (10(2) and 10(3) CFU/100 ml). In samples inoculated with 10(2) CFU/100 ml, C. jejuni was not detectable (>2-log reduction) after storage under the conditions specified above. E. coli O157:H7 was detected on nonselective and selective media at log reductions of 1.08 to 1.25 after storage at 22 °C, 1.19 to 1.56 after storage at 4 °C, and 1.54 to 1.98 after storage at -18 °C. When the higher inoculation level of 10(3) CFU/100 ml was used, C. jejuni was able to survive at 22 and 4 °C, with 2.25- and 2.17-log reductions, respectively, observed on nonselective media. At these higher inoculation levels, E. coli O157:H7 was detectable at 22, 4, and -18 °C, with log reductions of 0.76, 0.97, and 1.21, respectively, achieved on nonselective media. Additionally, E. coli O157:H7 showed significant differences in culturability (P<0.05) on the nonselective and selective culture media under the different storage conditions, with storage at -18 °C for 2 days being the treatment most inhibiting. The percentage of sublethal injury of E. coli O157:H7 ranged from ～33 to 75%, indicating that microbial examination of bottled water must be done carefully, otherwise false-negative results or underestimation of bacterial numbers could pose a health risk when low levels of pathogens are present.     

Survival of Escherichia coli O157:H7 in ground-beef patties during storage at 2, -2, 15 and then -2 degrees C, and -20 degrees C

The survival of Escherichia coli O157:H7 and of a nonpathogenic control strain of E. coli was monitored in raw ground beef that was stored at 2 degrees C for 4 weeks, -2 degrees C for 4 weeks, 15 degrees C for 4 h and then -2 degrees C for 4 weeks, and -20 degrees C. Irradiated ground beef was inoculated with one E. coli control strain or with a four-strain cocktail of E. coli O157:H7 (ca. 10(5) CFU/g), formed into patties (30 to 45 g), and stored at the appropriate temperature. The numbers of the E. coli control strain decreased by 1.4 log 10 CFU/g, and pathogen numbers declined 1.9 log 10 CFU/g when patties were stored for 4 weeks at 20 degrees C. When patties were stored at -2 degrees C for 4 weeks, the numbers of the E. coli control strain and the serotype O157:H7 strains decreased 2.8 and 1.5 log 10 CFU/g, respectively. Patties stored at 15 degrees C for 4 h prior to storage at -2 degrees C for 4 weeks resulted in 1.6 and 2.7 log 10-CFU/g reduction in the numbers of E. coli and E. coli O157:H7, respectively. Storage of retail ground beef at 15 degrees C for 4 h (tempering) did not result in increased numbers of colony forming units per gram, as determined with violet red bile, MRS lactobacilli, and plate-count agars. Frozen storage (-20 degrees C) of ground-beef patties that had been inoculated with a single strain of E. coli resulted in approximately a 1 to 2 log 10-CFU/g reduction in the numbers of the control strain and individual serotype O157:H7 strains after 1 year. There was no significant difference between the survival of the control strain and the O157:H7 strains, nor was there a difference between O157:H7 strains. These data demonstrate that tempering of ground-beef patties prior to low-temperature storage accelerated the decline in the numbers of E. coli O157:H7.     

Comparative survival of Salmonella typhimurium DT 104, Listeria monocytogenes, and Escherichia coli O157:H7 in preservative-free apple cider and simulated gastric fluid

This study compared the survival of three-strain mixtures (ca. 10(7) CFU ml(-1) each) of Salmonella typhimurium DT104, Listeria monocytogenes, and Escherichia coli O157:H7 in pasteurized and unpasteurized preservative-free apple cider (pH 3.3-3.5) during storage at 4 and 10 degrees C for up to 21 days. S. typhimurium DT104 populations decreased by <4.5 log10 CFU ml(-1) during 14 days storage at 4 and 10 degrees C in pasteurized cider, and by > or =5.5 log10 CFU ml(-1) during 14 days in unpasteurized cider stored at these temperatures. However, after 7 days at 4 degrees C, the S. typhimurium DT104 populations had decreased by only about 2.5 log10 CFU ml(-1) in both pasteurized and unpasteurized cider. Listeria monocytogenes populations decreased below the plating detection limit (10 CFU ml(-1)) within 2 days under all conditions tested. Survival of E. coli O157:H7 was similar to that of S. typhimurium DT104 in pasteurized cider at both 4 and 10 degrees C over the 21-days storage period, but E. coli O157:H7 survived better (ca. 5.0 log10 CFU ml(-1) decrease) than S. typhimurium DT104 (> 7.0 log10 CFU ml(-1) decrease) after 14 days at 4 degrees C in unpasteurized cider. In related experiments, when incubated in simulated gastric fluid (pH 1.5) at 37 degrees C, S. typhimurium DT104 and L. monocytogenes were eliminated (5.5-6.0 log10 CFU ml(-1) decrease) within 5 and 30 min, respectively, whereas E. coli O157:H7 concentrations decreased only 1.60-2.80 log10 CFU ml(-1) within 2 h.     

Survival of Escherichia coli O157:H7 in set yogurt as influenced by the production of an exopolysaccharide, colanic acid

Previous studies conducted in our laboratory revealed that Escherichia coli O157:H7 cells capable of producing colanic acid (CA), the acidic polysaccharide of mucoid slime, had increased tolerance to sublethal heat and the extreme pH of microbiological culture media. This study was undertaken to determine the effect of CA on the fate of E. coli O157:H7 during the processing and storage of an acid food: yogurt. Pasteurized and homogenized whole milk was inoculated with a wild-type E. coli O157:H7, its CA-deficient mutant, or a mixture (1:1) of the two strains. Set yogurt was processed from the contaminated milk and stored at 4 degrees and 15 degrees C for 3 weeks. Samples of milk and yogurt were withdrawn during processing and storage and analyzed for total plate counts and populations of E. coli O157:H7 and starter cultures. The results showed that E. coli O157:H7 survived longer in yogurt stored at 15 degrees C than at 4 degrees C. Cells of E. coli O157:H7 deficient in CA production died off more rapidly than those of the parent strain. This suggests that CA plays a role in protecting cells of E. coli O157:H7 from stress during the processing and storage of set yogurt.     

Growth and survival of Escherichia coli O157:H7 during the manufacture and ripening of raw goat milk lactic cheeses

The behaviour of Escherichia coli O157:H7 was studied during the manufacture and ripening of raw goat milk lactic cheeses. Cheese was manufactured from raw milk in the laboratory and inoculated with E. coli O157:H7 to a final concentration of 10, 100 and 1000 cfu ml(-1). E. coli O157:H7 was counted by CT-SMAC (Mac Conkey Sorbitol Agar with cefixim and tellurite) and O157:H7 ID throughout the manufacturing and ripening processes. When the milk was inoculated with 10, 100 or 1000 cfu ml(-1), counts decreased to less than 1 log(10) g(-1) in curds just prior to moulding. However, viable E. coli O157:H7 were found in cheeses throughout processing, and even after 42 days of ripening. Results indicate that E. coli O157:H7 survives the lactic cheese manufacturing process. Thus, the presence of low numbers of E. coli O157:H7 in milk destined for the production of raw milk lactic cheeses can constitute a threat to the consumer.     

Survival of Salmonella typhimurium and Escherichia coli O157:H7 in cheese brines

Survival of Salmonella typhimurium and Escherichia coli O157:H7 was studied in model brines and brine from three cheese plants. Three strain mixtures of S. typhimurium and E. coli O157:H7 (10(6) CFU/ml) were inoculated separately into 23% model brine with or without added pasteurized whey (2%) and as a combined inoculum into the commercial brines. The model brines were incubated at 8 and 15 degrees C for 28 days, and the commercial brines at 4 and 13 degrees C for 35 days. Populations of both pathogens in the model brine + whey decreased slowly over 28 days (1.0-2.0 log CFU/ml) with greater survival at 8 degrees C than at 15 degrees C. Corresponding decreases in model brine without whey were 1.9-3.0 log CFU/ml, with greater survival at 8 degrees C than at 15 degrees C. Both S. typhimurium and E. coli O157:H7 survived significantly better (P < 0.05) at 4 degrees C than at 13 degrees C in two of the commercial brines. The survival of each pathogen in the commercial brines at 13 degrees C was significantly influenced by brine pH. Both pathogen populations decreased most rapidly in commercial brines during the first week of storage (2.5-4.0 and 2.3-2.8 log CFU/ml for S. typhimurium and E. coli O157:H7, respectively) with significant recovery (ca. 0.5 log CFU/ml increase) often occurring in the second week of storage. Counts changed little thereafter. Overall, E. coli O157:H7 survived better than S. typhimurium, with differences of 0.1-1.2 log CFU/ml between the two pathogens. Results of this study show that cheese brine could support the survival of contaminating S. typhimurium and E. coli O157:H7 for several weeks under typical brining conditions.     

Effects of sodium citrate plus sodium diacetate and buffered vinegar on Escherichia coli O157:H7 and psychrotrophic bacteria in brine-injected beef

The objective of this research was to examine the effects of sodium citrate plus sodium diacetate or buffered vinegar on Escherichia coli O157:H7 and psychrotrophic bacteria when incorporated in brine solutions for injected beef. Two experiments were conducted in which 30 top rounds and 30 top sirloins were injected (110%) to contain (i) 0.5% sodium chloride and 0.4% sodium tripolyphosphate as the control (CNT); (ii) CNT with a 1% solution of 80% sodium citrate plus 20% sodium diacetate (SC + D); or (iii) CNT with 2% buffered vinegar (VIN) in the final product. For the E. coli challenge, muscles were surface inoculated to target 6 log CFU/cm(2). After injection and 10 days of storage in a vacuum package (4°C), one half of each muscle was sampled raw and the other half was cooked to an internal temperature of 60°C with a 12-min hold. For raw samples, a significant reduction of 0.6 and 1.0 log CFU/g of E. coli O157:H7 was observed in both SC + D- and VIN-injected top rounds and sirloins, respectively. All cooked samples were E. coli O157:H7 negative. For psychrotrophic analysis, subprimals were injected and vacuum packaged for 10 days at 0 ± 1°C. After 10 days of storage, steaks were fabricated and placed in aerobic display (4 ± 1°C) for 1, 7, 14, and 21 days. Psychrotrophic organism growth was restricted in SC + D and VIN samples when compared with CNT on all days except day 1. Sodium citrate plus sodium diacetate or buffered vinegar may improve the safety and shelf life of multineedle brine-injected beef.     

Survival of Salmonella enterica, Escherichia coli O157:H7, and Listeria monocytogenes on inoculated walnut kernels during storage

The survival of single strains or cocktails of Salmonella, Escherichia coli O157:H7, and Listeria monocytogenes was evaluated on walnut kernels. Kernels were separately inoculated with an aqueous preparation of the pathogens at 3 to 10 log CFU/g, dried for 7 days, and then stored at 23°C for 3 weeks to more than 1 year. A rapid decrease of 1 to greater than 4 log CFU/g was observed as the inoculum dried. In some cases, the time of storage at 23°C did not influence bacterial levels, and in other cases the calculated rates of decline for Salmonella (0.05 to 0.35 log CFU/g per month) and E. coli O157:H7 (0.21 to 0.86 log CFU/g per month) overlapped and were both lower than the range of calculated declines for L. monocytogenes (1.1 to 1.3 log CFU/g per month). In a separate study, kernels were inoculated with Salmonella Enteritidis PT 30 at 4.2 log CFU/g, dried (final level, 1.9 log CFU/g), and stored at -20, 4, and 23°C for 1 year. Salmonella Enteritidis PT 30 declined at a rate of 0.10 log CFU/g per month at 23°C; storage time did not significantly affect levels on kernels stored at -20 or 4°C. These results indicate the long-term viability of Salmonella, E. coli O157:H7, and L. monocytogenes on walnut kernels and support inclusion of these organisms in hazard assessments.     

Essential oils reduce Escherichia coli O157:H7 and Salmonella on spinach leaves

The efficacy of cinnamaldehyde and Sporan for reducing Escherichia coli O157:H7 and Salmonella on spinach leaves was investigated. Spinach leaves were inoculated with a five-strain cocktail of Salmonella or E. coli O157:H7, air dried for ca. 30 min, and then immersed in a treatment solution containing 5 ppm of free chlorine, cinnamaldehyde, or Sporan (800 and 1,000 ppm) alone or in combination with 200 ppm of acetic acid (20%) for 1 min or with water (control). After spin drying, treated leaves were analyzed periodically during 14 days of storage at 4°C for Salmonella, E. coli O157:H7, total coliforms, mesophilic and psychrotrophic bacteria, and yeasts and molds. Treatment effects on color and texture of leaves also were determined. Sporan alone (1,000S), Sporan plus acetic acid (1,000SV), and cinnamaldehyde-Tween (800T) reduced E. coli O157:H7 by more than 3 log CFU/g (P < 0.05), and 1,000SV treatment reduced Salmonella by 2.5 log CFU/g on day 0. E. coli O157:H7 and Salmonella populations on treated spinach leaves declined during storage at 4°C. The 1,000SV treatment was superior to chlorine and other treatments for reducing E. coli O157:H7 during storage. Saprophytic microbiota on spinach leaves increased during storage at 4°C but remained lower on leaves treated with Sporan (800S) and Sporan plus acetic acid (1,000SV) than on control leaves. The color and texture of Sporan-treated leaves were not significantly different from those of the control leaves after 14 days. Sporan plus acetic acid (1,000SV) reduced E. coli O157:H7 and Salmonella on baby spinach leaves without adverse effects on leaf color and texture.     

Inactivation of Shiga toxin-producing Escherichia coli in single-strength lemon and lime juices containing preservatives

The objective of this study was to determine the inactivation of non-O157 Shiga toxin-producing Escherichia coli (STEC) serotypes in comparison with O157 STEC in commercially produced, shelf-stable lemon and lime juices. The present validation tests confirmed that storage of the juices containing preservatives at room temperatures (22°C) for 3 days (72 h) ensures a >6-log reduction of O26, O45, O103, O111, O121, O145, and O157 STEC. These results demonstrate that non-O157 STEC had survival abilities comparable to those of E. coli O157:H7 strains in acidic food products such as lemon and lime juices (pH 2.5 ± 0.1); therefore, the storage conditions deemed to inactivate E. coli O157:H7 similarly inactivate the non-O157 serotypes.     

Effect of storage temperature and duration on the behavior of Escherichia coli O157:H7 on packaged fresh-cut salad containing romaine and iceberg lettuce

This study investigated the impact of storage temperature and duration on the fate of Escherichia coli O157:H7 on commercially packaged lettuce salads, and on product quality. Fresh-cut Romaine and Iceberg lettuce salads of different commercial brands were obtained from both retail and wholesale stores. The packages were cut open at one end, the lettuce salad inoculated with E. coli O157:H7 via a fine mist spray, and resealed with or without an initial N(2) flush to match the original package atmospheric levels. The products were stored at 5 and 12 °C until their labeled "Best If Used By" dates, and the microbial counts and product quality were monitored periodically. The results indicate that storage at 5 °C allowed E. coli O157:H7 to survive, but limited its growth, whereas storage at 12 °C facilitated the proliferation of E. coli O157:H7. There was more than 2.0 log CFU/g increase in E. coli O157:H7 populations on lettuce when held at 12 °C for 3 d, followed by additional growth during the remainder of the storage period. Although there was eventually a significant decline in visual quality of lettuce held at 12 °C, the quality of this lettuce was still fully acceptable when E. coli O157:H7 growth reached a statistically significant level. Therefore, maintaining fresh-cut products at 5 °C or below is critical for reducing the food safety risks as E. coli O157:H7 grows at a rapid, temperature-dependent rate prior to significant quality deterioration. PRACTICAL APPLICATION: Specific information regarding the effect of temperature on pathogen growth on leafy greens is needed to develop science-based food safety guidelines and practices by the regulatory agencies and produce industry. Temperature control is commonly thought to promote quality of leafy greens, not safety, based at least partially on a theory that product quality deterioration precedes pathogen growth at elevated temperatures. This prevalent attitude results in temperature abuse incidents being frequently overlooked in the supply chain. This study demonstrates that human pathogens, such as E. coli O157:H7, can grow significantly on commercially packaged lettuce salads while the product's visual quality is fully acceptable. Packaged fresh-cut salads are marketed as "ready-to-eat" while lacking an effective pathogen kill step during their preparation. Thus, maintaining storage temperature at 5 °C or below is critical to prevent pathogen proliferation and mitigate food safety risks should pathogen contamination inadvertently occur during crop growth or postharvest fresh-cut processing.