Microbiological quality of raw milk used for small-scale artisan cheese production in Vermont: effect of farm characteristics and practices.

This study 1) evaluated the overall milk quality and prevalence of 4 target pathogens (Listeria monocytogenes, Staphylococcus aureus, Salmonella spp., and Escherichia coli O157:H7) in raw milk used for small-scale artisan cheesemaking and 2) examined specific farm characteristics and practices and their effect on bacterial and somatic cell counts (SCC). Raw milk samples were collected weekly from 21 artisan cheese operations (6 organic) in the state of Vermont that manufactured raw-milk cheese from cow (12), goat (5), or sheep (4) milk during the summer of 2008. Individual samples were examined for standard plate counts (SPC), coliform counts (CC), and SCC. Samples were also screened for target pathogens both quantitatively and qualitatively by direct plating and PCR. Overall, 86% of samples had SPC <10,000 cfu/mL, with 42% <1,000 cfu/mL. Additionally, 68% of samples tested were within pasteurized milk standards for coliform bacteria under the United States' Grade A Pasteurized Milk Ordinance at <10 cfu/mL. Log(10) SPC and CC did not differ significantly among species. Similarly, method of sample delivery (shipped or picked up), farm type (organic or conventional), and duration of milking (year-round or seasonal) did not have significant effects on farm aggregated mean log(10) SPC, CC, or SCC. Strong positive correlations were observed between herd size and mean log(10) SPC and between log(10) SPC and CC as well as SCC when data from all animal species were combined. Although SCC for cow milk were significantly lower than those for goat and sheep milk, 98, 71, and 92% of cow, sheep, and goat milk samples, respectively, were within the compliance limits of the United States' Grade A Pasteurized Milk Ordinance for SCC. Fourteen of the 21 farms (67%) were positive for Staph. aureus, detected in 38% of samples at an average level of 20 cfu/mL. Neither L. monocytogenes, E. coli O157:H7, or Salmonella spp. were detected or recovered from any of the 101 samples tested. Our results indicate that the majority of raw milk produced for small-scale artisan cheesemaking was of high microbiological quality with no detectable target pathogens despite the repeat sampling of farms. These data will help to inform risk assessments that evaluate the microbiological safety of artisan and farmstead cheeses, particularly those manufactured from raw milk.

Listeria monocytogenes: a continuing challenge.

As a leading cause of death from a foodborne pathogen, Listeria monocytogenes continues to cause sporadic cases and outbreaks of illness. The most recent of these outbreaks in the United States involved consumption of hot dogs, with 101 cases of illness and 21 deaths reported to the Centers for Disease Control and Prevention for the years 1998-1999. Epidemiologic analysis determined that contamination levels in hot dogs were remarkably low (0.3 CFU [colony-forming units] L monocytogenes serotype 4b/g). That same year, manufacturers of hot dogs and luncheon meats collectively recalled more than 500,000 pounds of product owing to possible Listeria contamination. This article, through focus on issues such as reexamination of zero-tolerance policies, improvements in detection and enumeration procedures, the impact of epidemiologic innovations, and measures needed to further reduce the incidence of listeriosis will highlight why L monocytogenes remains a continuing challenge for the food industry.

Mathematically modeling the repair of heat-injured Listeria monocytogenes as affected by temperature, pH, and salt concentration.

Heat-injured cells of Listeria monocytogenes were inoculated into Listeria repair broth (LRB) adjusted to various pH levels (4.2, 5.0, 6.6, 8.0 and 9.6) and salt concentrations (0.5%, 2.5%, 5.0%, 7.5% and 10.0% w/v) at controlled temperatures (4, 10, 22, 37 and 43 degrees C) in a complete factorial manner (5(3)). Repair of the injured microorganisms was evaluated using selective and non-selective plating media. The Gompertz parameters, which were generated by fitting the equation with the bacterial counts, were used to calculate the repair percentage as a function of time from which the repair time was estimated. All growth curves fit the Gompertz equation well (R(2) > or = 0.972). A first-order model described the repair trend closely (R2 = 0.989 +/- 0.011). Heat-injured Listeria could fully repair in LRB only under 63 of 125 conditions tested during 21 days of incubation. Refrigeration temperature was the most effective means to prevent the repair of heat-injured Listeria. The minimum temperature required for repair increased with an increase in NaCl concentration. The pH ranges at which the repair could occur were narrower at 4 and 10 degrees C than those at higher temperature. The repair was observed in media containing 10% NaCl between temperatures of 22 and 43 degrees C at pH 6.6.

Comparison of the incidence of Listeria on equipment versus environmental sites within dairy processing plants.

This study was undertaken to compare the incidence of Listeria contamination of processing equipment with that of the general dairy processing environment. A total of 378 sponge samples obtained from 21 dairy plants were analyzed for Listeria using three different enrichment media. Use of extended microbiological analysis allowed us to identify 26 Listeria positive sites which would have not been identified had a single test format been employed. Eighty (80) of 378 sites (21.2%) were identified as Listeria positive. Listeria innocua was isolated from 59 of the 80 (73.8%) positive samples, L. monocytogenes was identified in 35 (43.8%) of the positive samples, and L. seeligeri was isolated from 5 (6.3%) of the Listeria positive samples. Positive equipment samples were obtained from 6 of the 21 (28.6%) plants and 19 of the 21 (90.5%) plants had positive environmental sites. Seventeen of the 215 (7.9%) samples from equipment were positive for Listeria species. Eleven of these sites, including 3 holding tanks, 2 table tops, 3 conveyor/chain systems, a pasta filata wheel, a pint milk filler and a brine pre-filter machine, were positive for L. monocytogenes. Nineteen of the 21 (90.5%) plants had positive environmental sites. Sixty-three of the 163 (41.1%) samples from environmental sites were Listeria positive and 24 were positive for L. monocytogenes. Two-tailed student t-test analysis of the mean frequencies indicated that the level of contamination was significantly higher (p < 0.001) in 'environmental' (49.7%) as opposed to 'equipment' samples (7.0%). Our study indicates that environmental contamination with Listeria does not necessarily translate into contamination of equipment within the same plant, and that greater emphasis needs to be placed on the cleaning and sanitizing of the plant environment.

Combined secondary enrichment of primary enrichment broths increases Listeria detection.

The efficacy of combining dual primary enrichment cultures into a single secondary broth was evaluated for detecting Listeria in naturally contaminated meats and environmental samples obtained from dairy processing plants. A total of 336 samples were tested using University of Vermont modified Listeria enrichment broth (UVM) and Listeria repair broth containing selective agents (LRBS) as primary enrichment media. Eighty samples (23.8%) yielded Listeria by at least one method. Neither primary enrichment broth was significantly better (P>0.05) than the other in identifying Listeria-positive samples. UVM media, when used as a primary enrichment broth, identified 66 Listeria-positive samples, while the use of LRBS as a primary enrichment broth identified 65 Listeria-positive samples. Listeria detection improved significantly (P<0.01) when two primary enrichment media were used for sample analysis. It is not clear whether this improvement was due to simply replicating the primary enrichment or to the particular pair of primary enrichment media used. The use of a dual secondary enrichment procedure was better (P<0.05) than the use of either individual primary enrichment medium alone. The overall rate of recovery increased from 81.3 to 82.5% for single secondary enrichment to 93.8% using a dual secondary enrichment technique. Analysis of results obtained when combining two independent isolation methods versus combining two primary enrichment media into one single secondary enrichment broth indicated that there was no significant difference (P>0.05) in either procedure. Inoculum size (0.1 ml versus 0.2 ml) did not have an effect on the overall rate of recovery. The procedure developed increased the sensitivity of testing while decreasing the potential workload associated with an increase in enrichment procedures.

Incidence and seasonal variation of Listeria species in bulk tank goat's milk.

Four hundred and fifty raw goat's milk samples obtained from the bulk tanks of 39 goat farms were analyzed for Listeria spp. over a 1-year period. Modified versions of the U.S. Department of Agriculture Food Safety and Inspection Service (USDA-FSIS) and Food and Drug Administration (FDA) protocols were used for recovery of Listeria. Overall, 35 (7.8%) samples yielded Listeria spp. with Listeria monocytogenes identified in 17 of the 35 (3.8%) Listeria-positive samples. Listeria innocua was detected in 26 (5.8%) samples. Eight milk samples contained both L. monocytogenes and L. innocua. Milk samples from 18 of the 39 (46.2%) farms were positive for Listeria at least once during this 1-year study. The modified USDA-FSIS method, which used Listeria repair broth rather than University of Vermont (UVM) broth for primary enrichment followed by a 4-h nonselective incubation period, yielded more Listeria-positive samples (77.1%) than the FDA method (51.4%). All L. monocytogenes isolates belonged to serotypes 1 (62.6%) or 4 (37.4%). Moreover, five different Listeria ribotypes were identified from 34 selected L. monocytogenes isolates, 2 of which were deemed to be of clinical importance. Listeria isolation rates were markedly higher during winter (14.3%) and spring (10.4%) as compared to autumn (5.3%) and summer (0.9%) with these trends similar to those previously reported for cow's milk.

Comparison of growth kinetics for healthy and heat-injured Listeria monocytogenes in eight enrichment broths.

Detection of Listeria in food products is often limited by performance of enrichment media used to support growth of Listeria to detectable levels. In this study, growth curves were generated using healthy and heat-injured Listeria monocytogenes strain F5069 in three nonselective and five selective enrichment broths. Nonselective enrichment media included the current Food and Drug Administration Bacteriological Analytical Manual Listeria enrichment broth base (BAM), Listeria repair broth (LRB), and Trypticase soy broth. Selective enrichment media included BAM with selective agents and LRB with selective agents, BCM L. monocytogenes preenrichment broth, Fraser broth, and UVM-modified Listeria enrichment broth. The Gompertz equation was used to model the growth of L. monocytogenes. Gompertz parameters were used to calculate exponential growth rate, lag-phase duration (LPD), generation time, maximum population density (MPD), and time required for repair of injured cells. Statistical differences (P < 0.05) in broth performance were noted for LPD and MPD when healthy and injured cells were inoculated into the broths. With the exception of Fraser broth, there were no significant differences in the time required for the repair of injured cells. Results indicate that the distinction between selective and nonselective broths in their ability to grow healthy Listeria and to repair sublethally injured cells is not solely an elementary issue of presence or absence of selective agents.

Comparison of 3M Petrifilm environmental Listeria plates against standard enrichment methods for the detection of Listeria monocytogenes of epidemiological significance from environmental surfaces.

Environmental monitoring using sensitive methods for detection and elimination of harborage sites of Listeria monocytogenes is key to the control of this organism. The 3M Petrifilm Environmental Listeria (EL) Plate-a no enrichment method-was compared with the USDA/FSIS, modified USDA/FSIS (mUSDA), and ISO methods for detection/recovery of L. monocytogenes on 4 environmental surfaces (brick, dairy board, stainless steel, and epoxy resin). The efficacy of 3 sampling devices including the Microbial-Vac system((R)), environmental sponge, and 3M Quick swab in recovering epidemiologically significant strains of uninjured and sublethally injured L. monocytogenes from environmental surfaces was evaluated. Environmental surfaces were inoculated with Listeria to obtain final cell densities of approximately 10 to 100 CFU/100 cm(2). The surfaces were then sampled and processed. For all methods, percent recovery (% samples where Listeria was detected) was significantly higher (P < 0.05) for uninjured cells (75% to 100%) compared to injured cells (58.9% to 81.1%). The Petrifilm EL Plate method efficiently recovered both low level and injured Listeria populations from environmental test surfaces when used in conjunction with environmental sponge and the 3M Quick swab sampling. The mUSDA method was superior to all other methods for recovering both uninjured (100% recovery) and injured L. monocytogenes (80.8% to 81.1% recovery). Sponges and swabs were equally effective in recovering uninjured and injured Listeria and were significantly different (P < 0.05) from the Microbial-Vac system. The findings indicate that both mUSDA and Petrifilm EL Plate methods can be used for the detection of potentially injured Listeria on food processing environmental surfaces.

Impact of nitrite on detection of Listeria monocytogenes in selected ready-to-eat (RTE) meat and seafood products.

The impact of sodium nitrite (NaNO2) on detection and recovery of Listeria monocytogenes from select ready-to-eat (RTE) foods including smoked salmon, smoked ham, beef frankfurters, and beef bologna was assessed. Nitrite-containing (NC; 100 to 200 ppm NaNO2) or nitrite-free (NF) foods were inoculated with a 5-strain cocktail of L. monocytogenes by immersion into Butterfield's buffer solution containing 5.4 to 7.4 x 10(3) L. monocytogenes per milliliter. Inoculated products were vacuum-packaged and stored at 5 degrees C. A weekly comparative analysis was performed for presence of L. monocytogenes using 5 detection methods on products held at 5 degrees C for up to 8 wk. L. monocytogenes initially present at <100 CFU/g during the first 2 wk of storage increased throughout the study, attaining final populations of approximately 1 x 10(4) to 1 x 10(5) CFU/g. Lactic acid bacteria predominated throughout the study in all products. Exposure to NaNO2 (100 to 200 ppm) resulted in 83% to 99% injury to the L. monocytogenes strains tested. The genetic-based BAX System (DuPont Qualicon, Wilmington, Del., U.S.A.) and modified USDA/FSIS methods detected 98% to 100% of Listeria-positive food samples and were consistently superior to and significantly different (P < 0.05) from conventional cultural methods in recovering Listeria from NC samples. Data show that nitrite-induced injury adversely affects detection and recovery of L. monocytogenes from NC food, confirming earlier findings that nitrite-induced injury masks L. monocytogenes detection in NC RTE food products. Nitrite-injured Listeria can subsequently repair upon nitrite depletion and grow to high levels over extended refrigerated storage.

Concerns of microbial pathogens in association with dairy foods.

Recent outbreaks of foodborne disease linked to Salmonella, Listeria, and Yersinia have highlighted consumer awareness of microbiological problems in the food supply. Such outbreaks affirm the need for improved testing, environmental monitoring, and epidemiological surveillance. This paper reviews the entry of microbial pathogens into foods, with an emphasis on dairy products, by examining the contribution of the processing environment to microbial contamination. Numerous surveys, including a recent audit of dairy processing plants in Vermont, have revealed common foci of environmental contamination by Listeria and Yersinia persistent within dairy processing environments. With respect to dairy products, the bacterial pathogens discussed in this manuscript share a common source, raw milk. Characteristics possessed by Salmonella, Listeria, and Yersinia are compared and contrasted. In the case of Listeria, this bacterium's role as a newly emerged foodborne pathogen is discussed. Finally, the economic consequences associated with foodborne disease are highlighted, and future prospects related to foodborne illness are presented.

Historical perspectives on methodology to detect Listeria monocytogenes.

While recognized as a causative agent of illness in animals and humans for some time, the foodborne role of Listeria monocytogenes is a new and emerging one. This review briefly summarizes the historical developments in methodology used to detect the presence of L. monocytogenes. Although clinical procedures exist, these procedures do not consider isolation of Listeria from heavily contaminated environments. Federal agencies such as the U.S. Food and Drug Administration and the U.S. Department of Agriculture have defined protocols for the isolation of Listeria from dairy and meat products, respectively. Each of these protocols, and current problems common to all methods for the isolation of Listeria from food products, are discussed. Finally, future challenges with respect to improvement in our abilities to recognize, isolate, and rapidly identify Listeria in foods are presented.

Method for flow cytometric detection of Listeria monocytogenes in milk.

This report describes a method for the detection of Listeria monocytogenes in raw milk by flow cytometric analysis of fluorescently labeled bacterial populations. The use of immunofluorescence in combination with measures of DNA content by propidium iodide labeling and size by light scattering enabled specific identification of L. monocytogenes from Streptococcus faecalis, Streptococcus agalactiae, Streptococcus uberis, Staphylococcus epidermidis, and Staphylococcus hyicus. Additional specific resolution of L. monocytogenes populations was achieved through selective enrichment of raw milk in Listeria enrichment broth. These procedures should permit the rapid screening of milk and other food samples for L. monocytogenes and eliminate many of the short-comings associated with conventional fluorescent-antibody procedures.

Development of a repair-enrichment broth for resuscitation of heat-injured Listeria monocytogenes and Listeria innocua.

The ability of the divalent cations magnesium, iron, calcium and manganese; yeast extract; pyruvate; catalase; and the carbohydrates glucose, lactose, sucrose, esculin, fructose, galactose, maltose, and mannose to facilitate repair of heat-injured Listeria monocytogenes and Listeria innocua was evaluated. Listeria populations were injured by heating at 56 degrees C for 50 min. To determine the effects on repair, Trypticase soy broth (TSB) was supplemented with each medium component to be evaluated. Repair occurred to various degrees within 5 h in TSB supplemented with glucose, lactose, sucrose, yeast extract, pyruvate, or catalase. Chelex-exchanged TSB was supplemented with divalent cations; magnesium and iron cations were found to have a role in repair. Listeria repair broth (LRB) was formulated by utilizing the components that had the greatest impact upon repair. When incubated in LRB, heat-injured Listeria cells completed repair in 5 h. After the repair, acriflavin, nalidixic acid, and cycloheximide were added to LRB to yield final concentrations identical to those of the selective enrichment broths used in the procedures of the Food and Drug Administration and the U.S. Department of Agriculture. The efficacy of LRB in promoting repair and enrichment of heat-injured Listeria cells was compared with that of existing selective enrichment broths. Repair was not observed in the Food and Drug Administration enrichment broth, Listeria enrichment broth, or University of Vermont enrichment broth. The final Listeria populations after 24 h of incubation in selective enrichment media were 1.7 x 10(8) to 9.1 x 10(8) CFU/ml; populations in LRB consistently averaged 2.5 x 10(11) to 8.2 x 10(11) CFU/ml.

Flow cytometry for automated analysis of milk containing Listeria monocytogenes.

This paper describes the application of flow cytometry to the analysis of milk for the presence of Listeria monocytogenes. A total of 939 raw milk samples from 54 farms were analyzed for the presence of L. monocytogenes by cold enrichment vs flow cytometry, which incorporated a selective enrichment step. Analyzed samples consisted of milk from individual farm bulk tanks; string samples which represented milk from 25 to 40 cows; and combination samples representing milk from 200 cows. Raw milk samples were directly enriched for 24 h at 37 degrees C and analyzed by flow cytometry following fluorescence labeling of bacterial populations, or were cold enriched for 1 month at 4 degrees C. Following cold enrichment, samples were streaked to McBride's Listeria agar, and suspect colonies were biochemically identified as L. monocytogenes. L. monocytogenes was isolated from only 15 of the analyzed samples, all of which were common to one farm. Results of flow cytometry analysis yielded a 5.86% false positive rate and a 0.53% false negative rate when compared with culture procedures.

Effects of pH on distribution of Listeria ribotypes in corn, hay, and grass silage.

Listeria app, isolated from 13 of 129 (10%) corn silage samples, 21 of 76 (28%) hay silage samples, and 3 of 5 (60%) grass silage samples during a previous Vermont survey were subjected to automated ribotype (RT) analysis. The 13 positive corn silage samples contained 3 Listeria monocytogenes isolated (three RTs, including one known clinical RT) and 10 L. innocua isolates (four RTs). Similarly, 2 L. monocytogenes isolates (two RTs) and 19 L. innocua isolates (three RTs) were identified in the 21 positive hay silage samples. The three positive grass silage samples contained two L. innocua isolates (two RTs) and one isolate of L. welshimeri. One hundred seven of 129 (83%) high-quality (pH < 4.0) corn silage samples accounted for 8 of 13 Listeria isolates from corn silage, including isolates belonging to one L. monocytogenes clinical RT. In contrast, low-quality hay silage (70 of 76 [92%] samples having a pH of > or = 4.0) harbored 20 of 21 isolates, including isolates belonging to two nonclinical L. monocytogenes RTs. Poor-quality silage is readily discernible by appearance; however, these findings raise new concerns regarding the safety of high-quality (pH < 4.0) corn silage, which can contain Listeria spp., including L. monocytogenes strains belonging to RTs of clinical importance in cases of food-borne listeriosis.

Virulence of Culture Filtrate from Heat-Injured and Repaired Listeria Strains: Assay on Bovine Mammary Epithelial (MAC-T) Cells.

The cytotoxic effect of culture filtrates from healthy, heat-stressed, and repaired Listeria monocytogenes and L. innocua on the bovine mammary epithelial cell line MAC-T was examined. Culture filtrates were collected from Listeria spp. following treatments which included: (i) 18 h of growth of Listeria at 37°C; (ii) sublethal heat treatment at 56°C for 50 minutes; (iii) repair of the injured cells at 37°C for 7 h; (iv) growth of repaired bacterial cells at 37°C for 36 h; and (v) heat injury at 56°C for 50 min of the cell population obtained after the initial repair and growth. Strains chosen for study included two genetic mutants of L. monocytogenes : a hemolysin-negative mutant, CNL 85/162 (Hly-) and a hemolysin-positive revertant, CNL 85/163 (Hly+). Culture filtrates obtained from Hly- bacteria did not prevent adhesion of the mammary epithelial cells and slightly stimulated their growth. In contrast, culture filtrates from Hly+ bacteria grown for 18 h significantly reduced the ability of MAC-T cells to adhere to the cell culture dishes, prevented the growth of those cells that were attached to the dishes, and caused cell death. Supernatants from Hly+ and Hly- following injury and during repair had no lethal effect on MAC-T cells. The effects of culture medium obtained after growth of the repaired Listeria cells on MAC-T cells were similar to those recorded for medium from the first 18 h growth for both strains, indicating that cells regain virulence potential once they have repaired and reinitiated growth. Culture filtrates obtained from L. monocytogenes Scott A showed results similar to those of Hly+, decreasing adherence and growth of MAC-T cells, while L. innocua culture filtrate had no adverse effect. The results of these experiments suggest that when injured, L. monocytogenes does not demonstrate adverse effects towards MAC-T cells. Once repair is completed and the listeria are growing, activity towards MAC-T cells is restored.

Recovery of different Listeria ribotypes from naturally contaminated, raw refrigerated meat and poultry products with two primary enrichment media.

Isolation rates for Listeria monocytogenes and the other Listeria spp. typically improve when samples are enriched in more than one primary enrichment medium. This study evaluated the abilities of two primary enrichment media, University of Vermont-modified Listeria enrichment broth (UVM) and Listeria repair broth (LRB), to recover different ribotypes of Listeria spp. from raw meat and poultry samples. Forty-five paired 25-g retail samples of ground beef, pork sausage, ground turkey, and chicken (160 samples) underwent primary enrichment in UVM and LRB (30 degrees C for 24 h) followed by secondary enrichment in Fraser broth (35 degrees C for 24 and 40 h) and plating on modified Oxford agar. After 24 h of incubation of 35 degrees C, 608 Listeria colonies from selected positive samples were biochemically confirmed as L. monocytogenes (245 isolates), L innocua (276 isolates), and L. welshimeri (89 isolates) and then ribotyped with the automated Riboprinter microbial characterization system (E. I. du Pont de Nemours & Co., Inc.). Thirty-six different Listeria strains comprising 16 L. monocytogenes (including four known clinical ribotypes), 12 L. innocua, and 8 L. welshimeri ribotypes were identified from selected positive samples (15 samples of each product type; two UVM and two LRB isolates per sample). Twenty-six of 36(13 L. monocytogenes) ribotypes were detected with both UVM and LRB, whereas 3 of 36 (1 L. monocytogenes) and 7 of 36 (3 L. monocytogenes) Listeria ribotypes were observed with only UVM or LRB, respectively. Ground beef, pork sausage, ground turkey, and chicken yielded 22 (8 L. monocytogenes), 21 (12 L. monocytogenes), 20 (9 L. monocytogenes), and 19 (11 L. monocytogenes) different Listeria ribotypes, respectively, with some Listeria ribotypes confined to a particular product. More importantly, major differences in both the number and distribution of Listeria ribotypes, including previously recognized clinical and nonclinical ribotypes of L. monocytogenes, were observed when 10 UVM and 10 LRB isolates from five samples of each product were ribotyped. When a third set of six samples per product type was examined from which two Listeria isolates were obtained by using only one of the two primary enrichment media, UVM and LRB failed to detect L. monocytogenes (both clinical and nonclinical ribotypes) in two and four samples, respectively. These findings stress the importance of using more than one primary enrichment medium and picking a sufficient number of colonies per sample when attempting to isolate specific L. monocytogenes strains during investigations of food-borne listeriosis.

Metabolic and Structural Sites of Damage in Heat- and Sanitizer-Injured Populations of Listeria monocytogenes.

Two food isolates of Listeria monocytogenes (strains ATCC 51414 and F5027) were sublethally injured by exposure to heat (56°C for 20 min) or to a chlorine sanitizer (Antibac, 100 ppm for 2 min). Percent injury following treatment ranged from 84% to 99%. Injured Listeria were repaired in Listeria repair broth (LRB) at 37°C. Comparison of the repair curves generated by each method indicated that the time for repair was greater for sanitizer-injured cells (14 h) than for heat-injured cells (5 h). Sites of injury were determined by repairing heat- and sanitizer-treated Listeria in LRB supplemented with one of the following inhibitors: rifampicin (10 and 20 µg/ml), chloramphenicol (5 µg/ml), cycloserine D (10 and 20 µg/ml), and carbonyl cyanide m-chlorophenyl-hydrazone(CCCP) (2.5 µg/ml). In both heat- and sanitizer-injured populations, a total inhibition of repair was seen following incubation with rifampicin, chloramphenicol and CCCP. These results clearly indicate a requirement for mRNA, protein synthesis, and oxidative phosphorylation for repair to occur. The cell wall is not a site of damage since cycloserine D had no effect on repair of heat- or sanitizer-injured Listeria . Investigation of damage to the cell membrane showed that stress caused by sublethal heat or sanitizer did not allow proteins or nucleotides to leak into the medium. The recognition of injury and repair in Listeria will lead to improved methods of detection and ultimately to control strategies which prevent outgrowth of this organism in foods.

Comparative recovery of uninjured and heat-injured Listeria monocytogenes cells from bovine milk.

The standard selective enrichment protocols of the Food and Drug Administration (FDA) and U.S. Department of Agriculture (USDA) were compared with an experimental nonselective broth enrichment (NSB) protocol and variations of the standard cold-enrichment (CE) protocol for the recovery of heat-injured Listeria monocytogenes. Bacterial cells (10(7)/ml) were suspended in sterile milk and heated at 71.7 degrees C in a slug-flow heat exchanger for holding times ranging from 1 to 30 s. Surviving cells were determined (50% endpoint) by the given protocols, and the following D values were obtained: NSB, D = 2.0 +/- 0.5 s; FDA, D = 1.4 +/- 0.3 s; USDA, D = 0.6 +/- 0.2 s; CE, D less than or equal to 1.2 s. The respective direct-plating media used in these enrichments were also analyzed for recovery, and the following D values were calculated from the enumeration of surviving cells; NSB, D = 2.7 +/- 0.8 s; FDA, D = 1.3 +/- 0.4 s; USDA, D = 0.7 +/- 0.2 s. The low levels of heat-injured L. monocytogenes cells which were detected at inactivation endpoints on the optimal nonselective media (25 degrees C for 7 days) failed to recover and multiply during experimental CEs (4 degrees C for 28 days). Initial inactivation experiments in which raw whole milk was used as the heating menstruum gave much lower recoveries with all protocols. The detectable limits for uninjured cells that were suspended in raw milk were similar (0.35 to 3.2 cells per ml) for the standard CE, FDA, and USDA protocols.(ABSTRACT TRUNCATED AT 250 WORDS)

Thermal inactivation of Listeria monocytogenes within bovine milk phagocytes.

Thermal resistance of intracellular and freely suspended Listeria monocytogenes that was associated with a milkborne outbreak of listeriosis was studied by using the sealed tube and slug flow heat exchanger methods. Test temperatures for the former method were 57.8, 62.8, 66.1, and 68.9 degrees C (136, 145, 151, and 156 degrees F, respectively); whereas those for the latter method were 66.1, 68.9, 71.7, and 74.4 degrees C (151, 156, 161, and 166 degrees F, respectively). The heating menstruum was sterile, whole milk. The intracellular inoculum was generated from an in vitro phagocytosis reaction by using endotoxin-induced bovine milk phagocytes. The phagocyte population consisted of 88% neutrophils, 8% macrophages, and 4% lymphocytes. Neutrophils harbored the majority of intracellular L. monocytogenes. The mean level of infectivity in the phagocyte population was 43%, and there were 26.1 +/- 19.3 bacteria per cell (10(4) viable cells per ml of test milk). Initial bacterial counts for the freely suspended and intracellular experiments (the latter was based on a sonically disrupted sample) were 10(6) L. monocytogenes cells per ml. Heat-stressed bacteria were recovered by direct plating in parallel with recovery from an enrichment broth; both methods gave comparable results. The predicted D62.8 degrees C (145 degrees F) value for intracellular sealed tube studies was 53.8 s (ZD = 5.6 degrees C [10.0 degrees F]), indicating a safe 33.4 D margin of inactivation for vat pasteurization (62.8 degrees C for 30 min).(ABSTRACT TRUNCATED AT 250 WORDS)