VALIDATION OF HEATING CONDITIONS IN PRODUCTION OF DIRECT ACIDIFIED BEEF SUMMER SAUSAGE FOR ELIMINATION OF ESCHERICHIA COLI O157:H7

The effectiveness of a typical production process for eliminating Escherichia coli O157:H7 in directly acidified all‐beef summer sausage was evaluated for formulations of different fat contents (approximately 8 and 17%) and types of direct acidulant (encapsulated citric or lactic acid). Raw batter inoculated with E. coli O157:H7 to an initial level of ca. 7.4 log cfu/g was stuffed into 64‐mm casings and processed according to a thermal processing schedule used by a small commercial processor for directly acidified summer sausage products (maximum internal product temperature of 70C, followed by cold showering). For all‐beef summer sausage, log reductions ranged from 5.3 to 5.5 cfu/g when product reached 64.4C (148F) internal temperature (IT) and 70C (158F) IT, and from 6.3 to 6.5 log cfu/g reductions when product reached 37.8C (100F) IT after thermal processing and cold showering. No differences in E. coli O157:H7 counts were observed for products with different fat or acid contents.     

ABILITY OF COLICIN V TO CONTROL ESCHERICHIA COLI O157:H7 IN GROUND BEEF

Culture counts of Escherichia coli O157:H7 showed that colicin V caused rapid death in buffered (pH 7) cell suspensions at 30C. A decrease in optical density of cell suspensions indicated that colicin V lysed cells. Decreased optical densities were proportional to increased colicin V concentrations. Colicin V was also effective in killing E. coli O157:H7 in ground beef at 4, 20 and 30C. Initial rates of killing were directly proportional to temperature.     

EFFECT OF MARINADE AND DRYING TEMPERATURE ON INACTIVATION OF ESCHERICHIA COLI O157:H7 ON INOCULATED HOME DRIED BEEF JERKY

Beef slices were inoculated (5.7–7.5 log CFU/cm²) with a 4-strain composite of E. coli O157:H7, stored (4C, 24 h), marinated (4C, 24 h), dried for 10 h at 62.5C or 68.3C, and stored for 90 days at 21C. Unmarinated beef slices dried for 10 h at 62.5C were used to determine the relative contribution of the marinate versus temperature treatment in the 62.5C trials. Samples were analyzed (bacterial enumeration with selective and nonselective agar media, pH, and a_w) following inoculation, marinating, at 4, 6, 8 and 10 h of drying, and after 30, 60 and 90 days of storage. Marination resulted in slight changes in bacterial populations (−0.3 to + 0.6 log CFU/cm²), but did not enhance bacterial reduction during drying. For all treatments, most bacterial reductions occurred in the first 4 h of drying, with little reduction thereafter. After 10 h of drying, bacterial reductions were 3.2–3.4 log CFU/cm² for unmarinated beef slices dried at 62.5C. Reductions of 2.2 and 3.0–4.6 log CFU/cm² were achieved in marinated jerky slices dried at 62.5C and 68.3C, respectively. No treatment resulted in the recommended 5-log reduction at the end of 10 h drying. However, bacteria did become undetectable by direct plating (<10 CFU/cm²) following 30 days of storage in all treatments except the unmarinated beef slices plated on tryptic soy agar (TSA). Additional work is needed to develop procedures for adequate destruction of E. coli O157:H7 during drying of beef jerky.     

EFFECTS OF CLOVE AND TEA TREE OILS ON ESCHERICHIA COLI O157:H7 IN BLANCHED SPINACH AND MINCED COOKED BEEF

The effectiveness of essential oil application in foods is the result of factor associations such as composition and storage temperatures. The aim was to evaluate the effectiveness of clove and tea tree essential oils to control Escherichia coli O157:H7 on blanched spinach and minced cooked beef exposed at 8 and 20C For both essential oils, the highest concentrations (three and four times minimal inhibitory concentrations) were needed to restrict O157:H7 populations in blanched spinach and minced cooked beef, respectively. The antimicrobial action of these essential oils was dependent on the oil concentration, the food composition and the storage temperature. The inhibition of pathogen and native microorganisms was more important at high temperatures than at low temperatures. Preliminary findings in actual foods suggest that clove and tea tree oils could be used as potential biopreservatives capable of controlling foodborne pathogens when foods are exposed to abusive temperatures.     

QUANTITATIVE DETECTION OF ESCHERICHIA COLI O157:H7 IN GROUND BEEF BY IMMUNOMAGNETIC SEPARATION AND POLYMERASE CHAIN REACTION

A quantitative assay for viable Escherichia coli O157:H7 in ground beef based on immunomagnetic separation (IMS) and the polymerase chain reaction (PCR) was developed. Bacterial cells inoculated into ground beef, were captured by immunomagnetic beads (IMB) after a 6 h non-selective enrichment. The percent capture of the target cells was consistent for the inoculation levels of 0.7 to 70 colony-forming-units (CFU)/g. Captured bacteria were lysed with PCR buffer containing 0.2 mg/mL proteinase K at 65°C for 30 min. DNA sequences of Shiga-like toxin 1 and 2 (Stx 1 and 2) were amplified independently. Log-linear relationships were observed between CFU of E. coli O157:H7 inoculated into ground beef and the integrated fluorescent image of the PCR products with Stx 1 and 2 primers after enrichment. The quantitative range was between 0.7 to 70 CFU/g.     

SURVIVAL OF SALMONELLA TYPHIMURIUM, ESCHERICHIA COLI O157:H7 AND LISTERIA MONOCYTOGENES DURING STORAGE ON BEEF SANITIZED WITH ORGANIC ACIDS

Sterile beef tissue was inoculated with either Salmonella typhimurium, Escherichia coli O157:H7 or Listeria monocytogenes Scott A and washed with 23C distilled water, 1% lactic acid or 1% acetic acid. The washed tissue was subjected to simulated dry chilling or spray chilling followed by storage at 5C. The washed tissue was stored at 5C for up to 21 days at 26% relative humidity, and total bacterial populations were determined by plating on nonselective and selective agars. There was no significant difference in the surviving populations of S. typhimurium, Escherichia coli O157:H7, or L. monocytogenes after storage, irrespective of chilling method. The surviving populations of bacteria were significantly lower on acid washed adipose tissue, when compared to the comparable water washed tissue. These results indicate that although injury and recovery of pathogenic bacteria may occur as a result of organic acid carcass sanitizing treatments, there was no practical significance of this phenomenon after 3 days of storage.     

INFLUENCE OF STARVATION, TEMPERATURE, AND pH ON CULTURABILITY OF ESCHERICHIA COLI O157:H7

In farm and food processing environments Escherichia coli O157:H7 can survive for days to months. In this study, we investigated the influence of starvation, pH (pH 4 or 7), temperature (4, 10 and 22C), and exposure to chlorine (50, 100, 150, 200 ppm) on culturability of E. coli O157:H7. The culturable cell population in PBS decreased to an undetectable level at 4C and 10C, but not at 22C during the experimental period. Cells suspended in PBS adjusted to pH 4 lost culturability more rapidly than at pH 7. Culturable cells were not detectable after exposure to chlorine, however, viable cell populations in the range of 1 – 2 log remained stable for 5 days. During loss of culturability two distinct morphological cell populations emerged; typical rod shaped and coccoid shaped cells. In this study, independent of exposure to chlorine, conditions of low temperature and low pH had the greatest influence on entry of E. coli O157:H7 into a VNC state.     

SENSITIVE AND RAPID DETECTION OF ESCHERICHIA COLI O157:H7 IN GROUND BEEF BY NESTED PCR INCORPORATING IMMUNOMAGNETIC SEPARATION

A protocol for detection of low numbers of E. coli O157:H7 in ground beef by nested PCR incorporating immunomagnetic separation (IMS) was developed. The protocol enabled detection of 24 colony-forming-units (CFU) in 10 g of seeded ground beef without enrichment cultivation. Differential centrifugation was used for maximally recovering the target CFU. Partial digestion of the resulting cell pellet with proteinase K at 37°C was used for the removal of beef tissue, which was required for the proper function of IMS. Within the range of 24 to 2400 CFU/10 g, a log linear relationship between the numbers of inoculated CFU and the integrated intensity of the nested PCR products was obtained with both shiga-like toxin (SLT) 1 and 2 primer pairs.     

DETECTION OF THE VIABLE BUT NONCULTURABLE STATE IN ESCHERICHIA COLI O157:H7

Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is recognized as a frequent cause of gastroenteritis ranging from mild to severe bloody diarrhea. The Shiga-like toxin produced by EHEC can result in hemolytic uremic syndrome, now the major cause of acute kidney failure in children in the United States. Inadequately cooked beef is most commonly implicated in the transmission of EHEC, although only a small fraction of cattle appear to harbor the organism. In several studies EHEC positive herds were detected only in the summer months correlating with the occurrence of human infections. Numerous E. coli strains have been shown to enter a viable but nonculturable (VBNC) state as a result of environmental stresses, including low temperature. Using traditional plating methods and the BacLight Molecular Probe, we monitored EHEC strains incubated in river water (RW) and artificial sea water (ASW) at temperatures of 5C and 25C for their entry into the VBNC state. EHEC strains remained culturable for over 40 days in both ASW and RW incubated at 25C. In ASW, these levels were higher than a non-EHEC control. At 5C, the number of culturable EHEC cells dropped gradually in both RW and ASW. Using the BacLight Molecular Probe, we were able to demonstrate that these cells, though not culturable, were viable indicating entry into the VBNC state. Our study suggests that temperature and not salinity is the primary signal for entry into this state .     

INACTIVATION OF ESCHERICHIA COLI O157:H7 ON INOCULATED ALFALFA SEEDS WITH OZONATED WATER UNDER PRESSURE1

Alfalfa seeds inoculated with Escherichia coli O157:H7 (～10⁵ CFU/g) were subjected to low hydrostatic pressure. Seeds immersed in ozonated water at 4C were held at 8 and 12‐psi ozone pressure for 2, 4, 8, 16, 32, and 64 min. Alternatively, seeds were continuously sparged with ozone for up to 64 min and then held at 12 psi for 5 min. Controls consisted of sparging and pressurization with air. Thirty‐two minute treatments of continuous ozone sparging followed by pressurization of seeds at 12 psi for 5 min were repeated with the addition of four surfactants (Tween 20, Tween 80, SPAN 20, and SPAN 80) in the treatment water. Enumeration of E. coli O157:H7 on treated, untreated, and control seeds was done on tryptic soy agar supplemented with 50 μg/mL of nalidixic acid. The reduction in population of E. coli O157:H7 on seeds treated with the 8 and 12 psi hydrostatic pressure in ozonated water ranged from 0.74 ?1.56 log₁₀ CFU/g and 0.72 – 1.62 log₁₀ CFU/g, respectively. Control treatments carried out with air pressurization of seeds resulted in maximum population reductions of 1.55 log₁₀ and 1.83 log₁₀ CFU/g for 8 and 12 psi, respectively. For seeds treated with continuous ozone sparging (2 – 64 min) followed by pressurization at 12 psi for 5 min, the highest reduction was 2.03 log₁₀ CFU/g. Reductions were, however, not significantly different (P > 0.05) from control treatment (with air) which reduced the populations by 0.57 – 2.19 log₁₀ CFU/g. The presence of surfactants during continuous sparging of water followed by pressurization at 12 psi was not beneficial. None of the treatments adversely affected the germination of the seeds.     

BEHAVIOR OF LISTERIA MONOCYTOGENES AND ESCHERICHIA COLI O157:H7 IN FRESH-SLICED CACTUS-PEAR FRUIT

Storage experiments were conducted to follow the behavior of Listeria monocytogenes and Escherichia coli O157:H7, deliberately inoculated on fresh‐cut cactus‐pear fruits before packaging under modified and control atmosphere and stored at four different temperatures (4, 8, 12 and 20C). L. monocytogenes was able to proliferate during storage at different temperature both in control and modified atmosphere. By comparing the sanitary‐risk values with those of shelf life, it is possible to conclude that the storage of cactus‐pear samples at temperatures greater than 4C, both in control and in modified atmospheres, could lead to a significant health‐time risk, and that this is strictly affected by temperature. E. coli O157:H7 was able to proliferate only in the sample stored at 4 and 8C in both package atmospheres. On the contrary, this species was completely suppressed at the higher temperatures. In our study, E. coli O157:H7 appeared to be much less suited for survival on the surface of the fruit than L. monocytogenes.     

GROWTH OF ESCHERICHIA COLI O157:H7 AND SALMONELLA SEROVARS ON RAW BEEF, PORK, CHICKEN, BRATWURST AND CURED CORNED BEEF: IMPLICATIONS FOR HACCP PLAN CRITICAL LIMITS

Small amounts (10–25 g; 6.3–20.8 cm² inoculated area) of raw ground beef, intact beef, pork and chicken (dark and white meat),and bratwurst and cured corned beef were inoculated with Salmonella serovars and Escherichia coli O157:H7, refrigerated 24 h at 5C, and then held either at 10C (± 1C) for up to 8 h or at room temperature (22C ± 2C) for up to 2 h. Except for a 0.2 log CFU increase in Salmonella serovars in ground beef during 2 h at room temperature, pathogens did not grow. Results of trials with commercial amounts of beef, pork, chicken, ground beef and bratwurst exposed to 10C for 8 h or 22C for 2 h also showed no pathogen growth. Potential critical limits for processing of previously refrigerated raw meat products are exposure temperatures between 5 and 10C for not more than 8 h or between 5 and 22C for not more than 2 h.     

CONCURRENT DETECTION OF ESCHERICHIA COLI O157:H7 AND SALMONELLA FROM A SINGLE ENRICHMENT IN 24 H

The objective of this study was to determine if a single assay protocol could result in the concurrent detection of Escherichia coli O157:H7 and Salmonella from a single sample grown in a single enrichment in 24 h. Twenty-five and 375 g of ground beef nonfat dry milk, and dry pet food samples were seeded with low (10 cfu/sample) and high (100 cfu/sample) levels ofE. coli O157:H7 and Salmonella cultures and incubated at 35 and 41C for 18 h for nonselective preenrichment. Incubated samples were analyzed by immunomagnetic separation (IMS) following a 6 h incubation for selective enrichment at 37C using M-broth and enzyme linked immumosorbent assay (ELISA). Depending on the food samples and the inoculation level, the minimum concurrent detection level of E. coli O157:H7 and Salmonella was <1 cfu/g in the samples at the competitor flora level of 10⁵ cfu/g or less in ground beef samples, but in other cases of higher competitor loads and low target inoculations E. coli O157:H7 could not be detected in the presence of the Salmonella.