Determination and validation of D-values for Listeria monocytogenes and Shiga toxin-producing Escherichia coli in cheese milk.

Certain cheeses can be legally produced in the United States using raw milk, but they must be aged for at least 60 d to reduce pathogen risks. However, some varieties, even when aged for 60 d, have been shown to support growth of Listeria monocytogenes or survival of Shiga toxin-producing Escherichia coli (STEC). Thermization, as a subpasteurization heat treatment, has been proposed as a control to reduce the risk of pathogens in raw cheese milk while retaining some quality attributes in the cheese. However, the temperature and time combinations needed to enhance safety have not been well characterized. The objective of this research was to determine and validate decimal reduction values (D-values) for L. monocytogenes and STEC at thermization temperatures 65.6, 62.8, and 60.0°C; a D-value at 57.2°C was also determined for L. monocytogenes only. Nonhomogenized, pasteurized whole-milk samples (1 mL) were inoculated with 8-log cfu/mL L. monocytogenes or STEC (5- or 7-strain mixtures, respectively), vacuum-sealed in moisture-impermeable pouches, and heated via water bath submersion. Duplicate samples were removed at appropriate intervals and immediately cooled in an ice bath. Surviving bacteria were enumerated on modified Oxford or sorbitol MacConkey overlaid with tryptic soy agar to aid in the recovery of heat-injured cells. Duplicate trials were conducted, and survival data were used to calculate thermal inactivation rates. D65.6°C-, D62.8°C-, and D60.0°C-values of 17.1 and 7.2, 33.8 and 16.9, and 146.6 and 60.0 s were found for L. monocytogenes and STEC, respectively, and a D57.2°C-value of 909.1 s was determined for L. monocytogenes. Triplicate validation trials were conducted for each test temperature using 100 mL of milk inoculated with 3 to 4 log cfu/mL of each pathogen cocktail, A 3-log reduction of each pathogen was achieved faster in larger volumes than what was predicted by D-values (D-values were fail-safe). Data were additionally compared with published results from 21 scientific studies investigating L. monocytogenes and STEC in whole milk heated to thermization temperatures (55.0-71.7°C). These data can be used to give producers of artisanal raw-milk cheese flexibility in designing thermal processes to reduce L. monocytogenes and STEC populations to levels that are not infectious to consumers.

Toxin production by Clostridium botulinum in pasteurized milk treated with carbon dioxide.

The addition of carbon dioxide to milk at levels of <20 mM inhibits the growth of selected spoilage organisms and extends refrigerated shelf life. Our objective was to determine if the addition of CO2 influenced the risk of botulism from milk. Carbon dioxide was added to pasteurized 2% fat milk at approximately 0, 9.1, or 18.2 mM using a commercial gas-injection system. The milk was inoculated with a 10-strain mixture of proteolytic and nonproteolytic Clostridium botulinum spore strains to yield 10(1) to 10(2) spores/ml. Milk was stored at 6.1 or 21 degrees C for 60 or 6 days, respectively, in sealed glass jars or high-density polyethylene plastic bottles. Milk stored at 21 degrees C curdled and exhibited a yogurt-like odor at 2 days and was putrid at 4 days. Botulinal toxin was detected in 9.1 mM CO2 milk at 4 days and in all treatments after 6 days of storage at 21 degrees C. All toxic samples were grossly spoiled based on sensory evaluation at the time toxin was detected. Although botulinal toxin appeared earlier in milk treated with 9.1 mM CO2 compared to both the 18.2 mM and untreated milk, gross spoilage would act as a deterrent to consumption of toxic milk. No botulinal toxin was detected in any treatment stored at 6.1 degrees C for 60 days. At 6.1 degrees C, the standard plate counts (SPCs) were generally lower in the CO2-treated samples than in controls, with 18.2 mM CO2 milk having the lowest SPC. These data indicate that the low-level addition of CO2 retards spoilage of pasteurized milk at refrigeration temperatures and does not increase the risk of botulism from treated milk stored at refrigeration or abuse temperatures.

Survival of bacterial pathogens in pasteurized process cheese slices stored at 30 degrees C.

Six lots of commercial pasteurized process cheese slices were evaluated for the ability to support the growth of four foodborne pathogens, Listeria monocytogenes, Staphylococcus aureus, Salmonella serotypes, and Escherichia coli O157:H7, during 4 days of storage at 30 degrees C. Individual cheese slices were inoculated separately with each pathogen to yield ca. 10(3) CFU/g. Slices were packaged in sterile plastic sample bags and stored at 30 degrees C for up to 96 h. Population of Salmonella serotypes and Escherichia coli O157:H7 decreased an average of 1.3 and 2.1 log10 CFU/g, respectively, by 36 h and Salmonella serotypes decreased an additional 0.6 log10 CFU/g during the remaining 60 h. Populations of Listeria monocytogenes also decreased, although to a lesser extent, exhibiting approximately a 0.6-log10 CFU/g reduction in 96 h. Staphylococcus aureus levels remained relatively constant during the testing period, and were below levels that support detectable enterotoxin production. The process cheese slices tested allowed survival but did not support rapid growth of S. aureus, whereas populations of L. monocytogenes, E. coli O157:H7, and Salmonella serotypes decreased during the 96-h storage at 30 degrees C.

Growth and penetration of Salmonella enteritidis, Salmonella heidelberg and Salmonella typhimurium in eggs.

Eggs and egg dishes are important vehicles for Salmonella infections. Salmonella enteritidis, Salmonella typhimurium and Salmonella heidelberg, which can be isolated from chicken ovaries and feces, have been implicated in approximately 50% of the foodborne salmonellosis outbreaks in the United States. In this study, the growth of these three organisms, inoculated into yolks and albumen, was compared at 4, 10 and 25 degrees C. Regardless of whether 10(2) cfu/g or 10(4) cfu/g was inoculated into the yolk or albumen, populations of all strains increased 3 logs or more in number in one day when incubated at 25 degrees C. Maximum numbers of Salmonella ranged from 10(8) to 10(10) cfu/g. All strains grew at 10 degrees C, but peak numbers were lower and occurred later than those at 25 degrees C. Populations of the three Salmonella strains inoculated into eggs stored at 4 degrees C grew sporadically; in some test groups populations declined. The potential for Salmonella in contaminated feces to establish in the interior of eggs was examined by monitoring shell penetration. At 25 degrees C, all three Salmonella strains penetrated the shell in 3 days, but at 4 degrees C, only S. typhimurium was found in one membrane sample. When hatchery conditions were simulated by incubating eggs at 35 degrees C for 30 min followed by storage at 4 degrees C, penetration was enhanced. Penetration was observed by day 1-3 when eggs were exposed to 10(4) cfu Salmonella/g feces. Increasing the inoculum to 10(6) cfu/g feces resulted in 50-75% of the contents of eggs to be contaminated by day 1. All Salmonella-positive samples were detected by enrichment. Results of this study indicate that S. enteritidis, S. typhimurium, or S. heidelberg present in feces can penetrate to the interior of eggs and grow during storage.

Fate of Listeria monocytogenes and pediococcal starter cultures during the manufacture of chicken summer sausage.

Two formulations of chicken summer sausages [100% hand deboned chicken meat (HDCM) and 85% HDCM and 15% chicken hearts (HDCM-CH)] were prepared with a nonpediocin-producing (PED-) Pediococcus acidilactici starter culture and inoculated with 10(4) or 10(7) cfu of a five-strain mixture of Listeria monocytogenes/g of batter. Sausages were fermented to pH 5.0 (11 h), cooked to an internal temperature of 66.5 C, cold-showered, and stored at 4 C (60 days) and 30 C (7 days). For both formulations and inoculation levels, L. monocytogenes populations decreased 1.3 to 1.8 log10 cfu/g by the end of fermentation. No L. monocytogenes organisms were recovered from sausages (by enrichment) following the cook and shower or storage at 4 or 30 C. In contrast, P. acidilactici increased .7 to 1.2 log10 cfu/g during fermentation, and < 10(2) cfu/g remained after the cook and shower and storage at 4 and 30 C. In a second set of experiments, sausages (HDCM) were prepared with a PED- or a pediocin-producing (PED+) P. acidilactici starter culture and challenged with the L. monocytogenes mixture (10(7) cfu/g). The PED- culture reduced numbers of L. monocytogenes 1.2 log10 cfu/g during fermentation, whereas L. monocytogenes numbers declined 2.6 log10 cfu/g in the presence of the PED+ culture. Although acid production by both starter cultures was equivalent, greater inhibition of L. monocytogenes by the PED+ compared with the PED- starter culture was attributed to in situ production of pediocin. Pediococcal starter cultures and proper cooking eliminated L. monocytogenes from sausages and established that PED+ cultures provide an additional hurdle against poultry-related listeriosis.

The effects of diacetate with nitrite, lactate, or pediocin on the viability of Listeria monocytogenes in turkey slurries.

The antilisterial effects of sodium diacetate (0.1, 0.3 and 0.5%) alone or in combination with sodium nitrite (30 ppm), sodium lactate (2.5%) or pediocin (5000 arbitrary units/ml) were evaluated in slurries (25% meat in sterile deionized H2O) prepared from vacuum-packaged, ready-to-eat turkey breast meat and challenged with Listeria monocytogenes. In the absence of food additives, counts of L. monocytogenes increased from 4.5 log10 cfu/ml to ca. 8 log10 cfu/ml within 1 day at 25 degrees C and within 14 days at 4 degrees C. Similarly, the pathogen grew to ca. 8 log10 cfu/ml within 1 d at 25 degrees C and within 28 days at 4 degrees C in slurries containing nitrite or lactate. In the presence of pediocin, after an initial decrease of 0.9 log10 cfu/ml, numbers of the pathogen reached ca. 8 log10 cfu/ml within 5 days at 25 degrees C and within 28 days at 4 degrees C. However, 0.3 and 0.5% diacetate in turkey slurries were listericidal at 4 and 25 degree C, respectively. In the presence of nitrite with diacetate, there was no appreciable difference in growth of L. monocytogenes compared with diacetate alone. Antilisterial activity was potentiated in treatments containing lactate with 0.3% diacetate at 25 degrees C and lactate with 0.1% diacetate at 4 degrees C, compared to similar treatments containing diacetate or lactate alone. A listericidal effect (ca. 7 log10 cfu/ml difference compared to slurries without additives) was observed in treatments containing pediocin with 0.5% diacetate at 25 degrees C and pediocin with 0.3% diacetate at 4 degrees C. The pH of slurries containing 0.3 or 0.5% diacetate was 5.5 and 5.2, respectively, whereas nitrite (pH 6.2), lactate (pH 6.3) or pediocin (pH 6.2) in slurries had a negligible effect on pH compared to the control (pH 6.2). The increased antilisterial activity in slurries with diacetate in combination with other additives was due to synergistic effects and not just pH. Thus, sodium diacetate alone can be used to delay growth of L. monocytogenes in turkey, and an additional level of safety can be achieved using diacetate in combination with sodium lactate or pediocin.

Determination of virulence of different strains of Listeria monocytogenes and Listeria innocua by oral inoculation of pregnant mice.

A pregnant mouse model was developed to follow the course of infection after peroral inoculation with six different strains of Listeria monocytogenes and one strain of Listeria innocua. Tissues were sampled and analyzed by microbiologic and histologic methods for 5 days postinoculation. In gnotobiotic pregnant BALB/c mice, L. monocytogenes Scott A (SA), serotype 4b, colonized the gastrointestinal tract, translocated to the livers and spleens of mice by day 1 postinoculation, and multiplied in these tissues until day 4. Infection of the placental tissues occurred by days 3 and 4 and was followed by infection of the fetuses. Little damage of colonic and cecal tissues was evident by histologic examination. Livers and spleens showed a cellular immune response; a similar immune response was not detected in the placentas or fetuses. A rough variant of L. monocytogenes SA which was as virulent as the parent strain in mice when injected intraperitoneally was less virulent perorally and did not consistently infect the fetuses. L. monocytogenes ATCC 19113, serotype 3a, did not colonize the gastrointestinal tract, nor was it isolated from any internal tissue. L. monocytogenes strains of serotypes 1/2a and 1/2b behaved like the SA strain in this mouse model. L. innocua colonized the gastrointestinal tract and translocated to the livers and spleens but did not survive in these organs and rapidly disappeared without infecting placental and fetal tissues. In comparison with gnotobiotic mice, conventional pregnant mice inoculated with L. monocytogenes strains showed less consistent infection. These results suggest that the gnotobiotic pregnant mouse is a useful model for detecting differences in virulence relating to colonization, invasiveness, and uteroplacental infection which cannot be detected by intraperitoneal inoculation of mice.

Genomic analysis of Pediococcus starter cultures used to control Listeria monocytogenes in turkey summer sausage.

The pulsed-field technique of clamped homogeneous electric field electrophoresis was employed to characterize and size genomic DNA of three pediocin-producing (Ped+) and two non-pediocin-producing (Ped-) strains of Pediococcus acidilactici. Comparison of genomic fingerprints obtained by digestion with the low-frequency-cleavage endonuclease AscI revealed identical restriction profiles for four of the five strains analyzed. Summation of results for 10 individually sized AscI fragments estimated the genome length to be 1,861 kb for the four strains (H, PAC1.0, PO2, and JBL1350) with identical fingerprints. Genomic analysis of the pediocin-sensitive, plasmid-free strain P. acidilactici LB42 with the unique fingerprint revealed nine AscI fragments and a genome length of about 2,133 kb. Ped- (JBL1350) and Ped+ (JBL1095) starter cultures (one each) were used to separately prepare turkey summer sausage coinoculated with a four-strain Listeria monocytogenes mixture (ca. 10(5) CFU/g). The starter cultures produced equivalent amounts of acid during fermentation, but counts of L. monocytogenes were reduced to a greater extent in the presence of the Ped+ starter culture (3.4 log10 unit decrease) than in the presence of the Ped- starter culture (0.9 log10 unit decrease). Although no listeriae were recovered from sausages following the cook/shower, appreciable pediocin activity was recovered from sausages prepared with the Ped+ strain for at least 60 days during storage at 4 degrees C. The results of this study revealed genomic similarities among pediococcal starter cultures and established that pediocins produced during fermentation provide an additional measure of safety against listerial proliferation in turkey summer sausage.

Fate of Escherichia coli O157:H7 as affected by pH or sodium chloride and in fermented, dry sausage.

The influence of pH adjusted with lactic acid or HCl or sodium chloride concentration on survival or growth of Escherichia coli O157:H7 in Trypticase soy broth (TSB) was determined. Studies also determined the fate of E. coli O157:H7 during the production and storage of fermented, dry sausage. The organism grew in TSB containing less than or equal to 6.5% NaCl or at a pH of 4.5 to 9.0, adjusted with HCl. When TSB was acidified with lactic acid, the organism grew at pH 4.6 but not at pH 4.5. A commercial sausage batter inoculated with 4.8 x 10(4) E. coli O157:H7 per g was fermented to pH 4.8 and dried until the moisture/protein ratio was less than or equal to 1.9:1. The sausage chubs were then vacuum packaged and stored at 4 degrees C for 2 months. The organism survived but did not grow during fermentation, drying, or subsequent storage at 4 degrees C and decreased by about 2 log10 CFU/g by the end of storage. These studies reveal the importance of using beef containing low populations or no E. coli O157:H7 in sausage batter, because when initially present at 10(4) CFU/g, this organism can survive fermentation, drying, and storage of fermented sausage regardless of whether an added starter culture was used.

Sodium lactate delays toxin production by Clostridium botulinum in cook-in-bag turkey products.

Comminuted raw turkey, containing 1.4% sodium chloride, 0.3% sodium phosphate, and 0 (control), 2.0, 2.5, 3.0, or 3.5% sodium lactate, was inoculated with a 10-strain mixture of proteolytic type A and B Clostridium botulinum spores. The inoculated turkey was vacuum packaged and cooked by immersion in heated water to an internal temperature of 71.1 degrees C. Samples were incubated at 27 degrees C for up to 10 days. Five samples per treatment were examined for botulinal toxin at specific intervals. Sodium lactate exhibited an antibotulinal effect which was concentration dependent. Processed turkey containing 0, 2.0, 2.5, 3.0, or 3.5% sodium lactate was toxic after 3, 4 to 5, 4 to 6, 7 or 7 to 8 days, respectively. Subsequent studies with a broth medium revealed that lactate, not the sodium ion, was the principal factor in delaying botulinal-toxin formation.

Fate of Listeria monocytogenes in processed meat products during refrigerated storage.

The fate of Listeria monocytogenes during refrigerated storage was determined on several processed meat products, including ham, bologna, wieners, sliced chicken, sliced turkey, fermented semidried sausage, bratwurst, and cooked roast beef. The meats were surface inoculated with a five-strain mixture of less than or equal to 200 or ca. 10(5) L. monocytogenes cells per package, vacuum packaged, and stored at 4.4 degrees C. Survival or growth of listeriae was determined for up to 12 weeks of storage or until the product was spoiled. The organism survived but did not grow on summer sausage, grew only slightly on cooked roast beef, grew well on some wiener products but not on others, grew well (10(3) to 10(5) CFU/g increase within 4 weeks) on ham, bologna, and bratwurst, and grew exceptionally well (10(3) to 10(5) CFU/g increase within 4 weeks) on sliced chicken and turkey. The rate of growth depended largely upon the type of product and the pH of the product. Growth was most prolific on processed poultry products. The organism generally grew well on meats near or above pH 6 and poorly or not at all on products near or below pH 5. These results indicate the importance of preventing postprocessing contamination of L. monocytogenes in a variety of ready-to-eat meat products.

Survival of Listeria monocytogenes in milk during high-temperature, short-time pasteurization.

Milk from cows inoculated with Listeria monocytogenes was pooled for 2 to 4 days and then heated at 71.7 to 73.9 degrees C for 16.4 s or at 76.4 to 77.8 degrees C for 15.4 s in a high-temperature, short-time plate heat exchanger pasteurization unit. L. monocytogenes was isolated from milk after heat treatment in six of nine pasteurization trials done at 71.7 to 73.9 degrees C and in none of three trials done at 76.4 to 77.8 degrees C. An average of 1.5 to 9.2 L. monocytogenes cells was seen in each milk polymorphonuclear leukocyte before heat treatment in 11 of 12 pasteurization trials. Noticeable degradation of leukocytes with intracellular listeria was detected in unpasteurized milk after 3 days of storage at 4 degrees C, and by 4 days of storage leukocytes had deteriorated to cellular debris, suggesting that holding unpasteurized milk refrigerated for 4 or more days would eliminate a protective effect leukocytes may provide for increasing heat resistance of L. monocytogenes. Results indicate that under the conditions of this study, L. monocytogenes can survive the minimum high-temperature, short-time treatment (71.7 degrees C, 15 s) required by the U.S. Food and Drug Administration for pasteurizing milk.