Influence of agitation, inoculum density, pH, and strain on the growth parameters of Escherichia coli O157:H7--relevance to risk assessment.

Foods may differ in at least two key variables from broth culture systems typically used to measure growth kinetics of enteropathogens: initial population density of the pathogen and agitation of the culture. The present study used nine Escherichia coli O157:H7 strains isolated from beef and associated with human illness. Initial kinetic experiments with one E. coli O157:H7 strain in brain-heart infusion (BHI) broth at pH 5.5 were performed in a 2 x 2 x 3 factorial design, testing the effects of a low (ca. 1-10 colony-forming units [CFU]/ml) or high (ca. 1000 CFU/ml) initial population density, culture agitation or no culture agitation, and incubation temperatures of 10, 19, and 37 degrees C. Kinetic data were modeled using simple linear regression and the Baranyi model. Both model forms provided good statistical fit to the data (adjusted r(2)>0.95). Significant effects of agitation and initial population density were identified at 10 degrees C but not at 19 or 37 degrees C. Similar growth patterns were observed for two additional strains tested under the same experimental design. The lag, slope, and maximum population density (MPD) parameters were significantly different by treatment. Further tests were conducted in a 96-well microtiter plate system to determine the effect of initial population density and low pH (4.6-5.5) on the growth of E. coli O157:H7 strains in BHI at 10, 19, and 37 degrees C. Strain variability was more apparent at the boundary conditions of growth of low pH and low temperature. This study demonstrates the need for growth models that are specific to food products and environments for plausible extrapolation to risk assessment models.

Heat resistance and fatty acid composition of Listeria monocytogenes: effect of pH, acidulant, and growth temperature.

The objective of this study was to determine the influence of pH, acidulant, and growth temperature history on the heat resistance and fatty acid composition of Listeria monocytogenes Scott A. Cells were grown to late exponential phase (OD600 = 0.6) at 10, 19, or 37 degrees C in brain heart infusion broth acidified to pH 5.4 or 7 with either acetic or lactic acid. Thermal death times at 60 degrees C subsequently were determined by using a submerged-coil heating apparatus. The surviving cell population was enumerated by spiral-plating heated samples onto tryptic soy agar supplemented with 0.6% yeast extract and 1% sodium pyruvate. The thermal resistance of cells cultured at a particular temperature was significantly lower (P < 0.05) when lactic acid was used to acidify the medium of pH 5.4. Regardless of acid identity, D values significantly decreased (P < 0.05) with increased growth temperature when the pH of the growth medium was 5.4, whereas D values significantly increased (P < 0.05) with increased temperature at pH 7. At pH 5.4 adjusted with lactic acid, D values were 1.30, 1.22, and 1.14 min for cells grown at 10, 19, and 37 degrees C, respectively. At pH 5.4 adjusted with acetic acid, L. monocytogenes failed to grow at 10 degrees C; the D values were 1.32 and 1.22 min when the cells were grown at 19 and 37 degrees C, respectively. At pH 7, the D values were 0.95, 1.12, and 1.28 min with lactic acid and 0.83, 0.93, and 1.11 min with acetic acid at 10, 19, and 37 degrees C, respectively. The most abundant fatty acids (44 to 82%) were branched-chain saturated fatty acids (anteiso-and iso-C15:0 and iso-C17:0) regardless of pH, acidulant, or growth temperature. However, there was an increase in C15:0 isomers at the expense of iso-C17:0 when the growth temperature was lowered from 37 to 10 degrees C. While variable changes in longer-chain fatty acids were found, the percentage of longer-chain (C16 and C18) fatty acids was greatest when L. monocytogenes was grown at 37 degrees C regardless of pH or acidulant. This study demonstrates that the heat resistance of L. monocytogenes depends upon its growth conditions.

Heat shock and thermotolerance of Escherichia coli O157:H7 in a model beef gravy system and ground beef.

Duplicate beef gravy or ground beef samples inoculated with a suspension of a four-strain cocktail of Escherichia coli O157:H7 were subjected to sublethal heating at 46 degrees C for 15-30 min, and then heated to a final internal temperature of 60 degrees C. Survivor curves were fitted using a linear model that incorporated a lag period (TL), and D-values and 'time to a 4D inactivation' (T4D) were calculated. Heat-shocking allowed the organism to survive longer than non-heat-shocked cells; the T4D values at 60 degrees C increased 1.56- and 1.50-fold in beef gravy and ground beef, respectively. In ground beef stored at 4 degrees C, thermotolerance was lost after storage for 14 h. However, heat-shocked cells appeared to maintain their thermotolerance for at least 24 h in ground beef held to 15 or 28 degrees C. A 25 min heat shock at 46 degrees C in beef gravy resulted in an increase in the levels of two proteins with apparent molecular masses of 60 and 69 kDa. These two proteins were shown to be immunologically related to GroEL and DnaK, respectively. Increased heat resistance due to heat shock must be considered while designing thermal processes to assure the microbiological safety of thermally processed foods.

Thermal destruction of Escherichia coli O157:H7 in beef and chicken: determination of D- and z-values.

Thermal inactivation of a four-strain mixture of E. coli O157:H7 was determined in 90% lean ground beef, and lean ground chicken. Inoculated meat was packaged in bags which were completely immersed in a circulating water bath and held at 55, 57.5, 60, 62.5, and 65 degrees C for predetermined lengths of time. D-values, determined by linear regression, in beef were 21.13, 4.95, 3.17, 0.93 and 0.39 min, respectively (z = 6.0 degrees C). Using a survival model for non-linear survival curves, D-values in beef ranged from 20.45 min (D1; and there was no D2) at 55 degrees C to 0.16 min (D1) and 1.45 min (D2) at 65 degrees C. When E. coli O157:H7 four-strain cocktail was heated in chicken, D-values calculated by both approaches were consistently less at all temperatures. The heat resistance of E. coli O157:H7 was not altered after refrigerated or frozen storage of inoculated beef for 48 h. The results of this study will be beneficial to the food industry in designing HACCP plans to effectively eliminate E. coli O157:H7 in the meat products used in this study.

Growth of Clostridium perfringens from spore inocula in sous-vide turkey products.

Clostridium perfringens growth from a spore inoculum was investigated in vacuum-packaged, cook-in-bag ground turkey (pH 6) that included 0.3% (w/w) sodium pyrophosphate, and sodium chloride at 0, 1, 2, or 3% (w/w). The packages were processed to an internal temperature of 71.1 degrees C, ice chilled and stored at various temperatures. The total C. perfringens population was determined by plating diluted samples on tryptose-sulfite-cycloserine agar followed by anaerobic incubation at 37 degrees C for 48 h. At 28 degrees C, the addition of 3% salt in turkey was effective in delaying growth for 12 h. At 15 degrees C, growth occurred at a relatively slow rate in the presence of 1-2% salt. Vegetative cells were not observed even after 28 days of storage in the presence of 3% salt. C. perfringens growth was not observed at 4 degrees C regardless of salt levels. The D-values ranged from 23.2 min (no salt) to 17.7 min (3% salt). Cyclic and static temperature abuse of refrigerated products for 8 h did not lead to growth by C. perfringens from a spore inoculum.

Growth and Sporulation Potential of Clostridium perfringens in Aerobic and Vacuum-Packaged Cooked Beef.

Growth of Clostridium perfringens in aerobic-and anaerobic-(vacuum) packaged cooked ground beef was investigated. Autoclaved ground beef was inoculated with ~3.0-log10 CFU/g of C. perfringens , packaged and stored at various temperatures. Vegetative cells and heat-resistant spores were enumerated by plating unheated and heated (75°C for 20 min) meat samples on tryptose-sulfite-cycloserine agar. Clostridium perfringens grew to >7 logs within 12 h at 28, 37 and 42°C under anaerobic atmosphere and at 37 and 42°C under aerobic conditions. At 28°C under aerobic conditions, growth was relatively slow and total viable count increased to >6 logs within 36 h. Similarly, growth at 15°C in air was both slower and less than under vacuum. Regardless of packaging, the organism either declined or did not grow at 4, 8 and 12°C. Spores were not found at <12°C. Spores were detected as early as 8 h at 42°C under anaerobic conditions, but in general, the type of atmosphere had little influence on sporulation at ≥28°C. Temperature abuse (28°C storage) of refrigerated products for 6 h will not permit C. perfringens growth. However, cyclic and static temperature abuse of such products for relatively long periods may lead to high and dangerous numbers of organisms. Reheating such products to an internal temperature of 65°C before consumption would prevent food poisoning since the vegetative cells were killed.

Influence of Growth Temperature on Injury and Death of Listeria monocytogenes Scott A During a Mild Heat Treatment.

The growth temperature of Listeria monocytogenes has a pro-found effect on injury and death of washed cells that are suspended in phosphate buffer and exposed to 52°C for 1 h. The temperature of 52°C had low lethality for cells grown at 37 or 42°C, but there was a 103-104-fold increase in killing for cells grown at 28, 19, 10, or 5°C. There was little injury with exposure to 52°C of cells grown at 5, 10, or 19°C, but injury increased as the temperature of growth increased. When cells were grown anaerobically, lethality induced at 52°C increased as the growth temperature decreased, but there was more injury under anaerobic conditions than for aerobically grown cells. The results indicate that L. monocytogenes cells growing at low temperatures are more susceptible to heat induced death.