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Outbreaks of listeriosis are continually attributed to the consumption of Hispanic-style soft cheeses contaminated with Listeria monocytogenespostpasteurization. Once contaminated, L. monocytogenescan grow rapidly in cheeses like Queso Fresco (QF) even when stored at refrigeration temperatures. Several antimicrobials, including acidified calcium sulfate with lactic acid (ACSL), ɛ-polylysine (EPL), hydrogen peroxide (HP), lauric arginate ethyl ester (LAE), and sodium caprylate (SC), have demonstrated antilisterial activity in food. The objectives of this study were to determine the efficacy of these antimicrobials used individually and in combination to control L. monocytogenesas surface contaminants on QF and to identify additive and synergistic interactions. Cheeses were surface inoculated at ∼4 log CFU/g, dipped in antimicrobial solutions, vacuum packaged, and then stored at 7°C for 35 days. L. monocytogenescounts were determined 24 h after application of the antimicrobials and then weekly throughout storage. Dip treatments in a 5% (v/v) HP solution reduced L. monocytogenescounts to <0.5 log CFU/g within 24 h with no increase in counts through day 35. Dip treatments in LAE at 2 and 5% alone and in combination with EPL at 10% produced initial reductions in pathogen counts (1.5 to 1.8 CFU/g) but did not inhibit pathogen growth compared with the sterile water control. Dip applications of ACSL at 25% also produced an initial ∼1.5-log reduction in L. monocytogenescounts followed by regrowth. Application of SC at 10% alone and in combination with either EPL or LAE inhibited growth to <1 log CFU/g through 21 days of storage. The combination of ACSL+SC worked synergistically to inhibit the growth of L. monocytogeneson QF to <1 log CFU/g through 35 days. These data indicate that HP alone and treatments containing EPL, LAE, or ACSL in combination with SC are promising postlethality treatments and process controls for L. monocytogeneson QF through a 21-day shelf life.

The use of antimicrobial compounds can be an effective approach to control Listeria monocytogenesin ready-to-eat foods, but it can also be limited by cost, restrictions on concentrations in foods, and potential changes to organoleptic properties. Combinatorial approaches that produce additive or synergistic effects allow for reductions in individual antimicrobial concentrations while achieving the same level of control. The present study determined the MIC and MBC of an antimicrobial compound when used alone or in binary combinations against L. monocytogenesin growth media adjusted to pH values 7.4 and 5.5 and characterized interactions as synergistic, additive, or antagonistic. Inhibitory and bactericidal concentrations were defined as changes in L. monocytogenescounts of ≤1.0 or ≥3.0 log CFU/mL compared with the starting inoculum, respectively. Individually, lauric arginate (LAE), hydrogen peroxide (HP), and ɛ-polylysine (EPL) inhibited L. monocytogenesgrowth at the lowest concentrations when applied alone in broth adjusted to pH 7.4. Similarly, LAE, EPL, and HP had the lowest MBCs in broth adjusted to both pH levels. The inhibitory efficacy of both caprylic acid and sodium caprylate (SC) increased at the lower pH, with reductions in MICs of >98%. In total, 35 and 19 additive or synergistic inhibitory and bactericidal combinations were identified at pH values 7.4 and 5.5, respectively. Combinations of acidified calcium sulfate with lactic acid (ACSL) and SC were among the most synergistic inhibitory groupings at both pH levels, whereas EPL+LAE were the most effective bactericides at pH 7.4. Combinations of SC with EPL or ACSL were also among the most effective bactericides at pH 5.5. These data serve as a foundation for developing more effective antimicrobial approaches for the control of L. monocytogenesin foods with different pH levels.

Outbreaks of salmonellosis have been linked to the consumption of cheese, and emerging multidrug-resistant (MDR) strains of Salmonellamay be more virulent and more tolerant than less resistant strains to stresses encountered in food production, which may enhance the survival of these resistant strains in cheese. This study was conducted to compare the behavior of MDR and pansusceptible Salmonellastrains during the manufacture and aging of Gouda cheese and compare pathogen recovery via several rapid and traditional methods. Cheeses were manufactured from raw milk inoculated with a six-strain cocktail of either MDR or susceptible SalmonellaNewport and SalmonellaTyphimurium at initial levels of ~ 20 CFU/ml. Samples of milk, whey, curd, and finished cheese were analyzed using eight enrichment and detection protocols. Overall, changes in pathogen levels observed throughout manufacture and aging did not differ significantly between MDR and susceptible Salmonellastrains. Salmonellacounts increased significantly during manufacture to a mean of 734 CFU/g on day 1 followed by a significant decrease over 60 days of aging to <1 CFU/g. Although levels fell and stayed below the direct plating detection limit of ≥5 CFU/g after 54 days on average, viable cells remained detectable after enrichment for an average of 210 ± 40 days. The International Organization for Standardization methods with and without PCR detection provided the most accurate results, and the remaining methods, notably those with selective primary incubation, produced results that disagreed significantly with the true result. Overall, our findings suggest that MDR Salmonellastrains may not pose a greater threat to cheese safety than do non-MDR Salmonellastrains.

Staphylococcus aureusis an important agent of bacterial mastitis in milking animals and of foodborne intoxication in humans. The purpose of this study was to examine the genetic and phenotypic diversity, enterotoxigenicity, and antimicrobial resistance of S. aureusstrains isolated from raw milk used for the production of artisan cheese in Vermont. Cross-tabulations revealed that the 16 ribotypes identified among the 90 milk isolates examined were typically associated with a specific animal species and that more than half of these ribotypes were unique to individual farms. In general, specific EcoRI ribotypes were commonly associated with specific phenotypical characteristics, including staphylococcal enterotoxin production or the lack thereof. Limited antimicrobial resistance was observed among the isolates, with resistance to ampicillin (12.51%) or penicillin (17.04%) most common. Two isolates of the same ribotype obtained from the same farm were resistant to oxacillin with 2% NaCl. More than half (52.22%) of isolates produced toxin, and 31 of the 32 isolates solely produced staphylococcal enterotoxin type C. Although these data demonstrate that S. aureusstrains found in raw milk intended for artisan cheese manufacture are capable of enterotoxin production, staphylococcal enterotoxin C is not typically linked to foodborne illness. Because S. aureusis a common contaminant of cheese, an understanding of the ecology of this pathogen and of the antimicrobial susceptibility and toxigenicity of various strains will ultimately contribute to the development of control practices needed to enhance the safety of artisan and farmstead cheese production.

This study was conducted to examine the fate of Escherichia coliO157:H7 during the manufacture and aging of Gouda and stirred-curd Cheddar cheeses made from raw milk. Cheeses were manufactured from unpasteurized milk experimentally contaminated with one of three strains of E. coliO157:H7 at an approximate population level of 20 CFU/ml. Samples of milk, whey, curd, and cheese were collected for enumeration of bacteria throughout the manufacturing and aging process. Overall, bacterial counts in both cheese types increased almost 10-fold from initial inoculation levels in milk to approximately 145 CFU/g found in cheeses on day 1. From this point, counts dropped significantly over 60 days to mean levels of 25 and 5 CFU/g in Cheddar and Gouda, respectively. Levels of E. coliO157:H7 fell and stayed below 5 CFU/g after an average of 94 and 108 days in Gouda and Cheddar, respectively, yet remained detectable after selective enrichment for more than 270 days in both cheese types. Changes in pathogen levels observed throughout manufacture and aging did not significantly differ by cheese type. In agreement with results of previous studies, our results suggest that the 60-day aging requirement alone is insufficient to completely eliminate levels of viable E. coliO157:H7 in Gouda or stirred-curd Cheddar cheese manufactured from raw milk contaminated with low levels of this pathogen.

Environmental sampling, focused on environmental sites with a high probability of contamination, was conducted in eight artisan cheese processing facilities. Samples (n= 236) from 86 food contact surfaces and 150 non–food contact surfaces were examined for the presence of Listeriaspp. by comparing the efficacy of three different primary enrichment media used in five detection and isolation methods. University of Vermont broth was the most sensitive primary enrichment medium for the detection of Listeriaspp., including Listeria monocytogenes. These results, however, did not differ significantly from those obtained with Listeriarepair broth or Oxoid 24 Listeriaenrichment broth. When full methods were considered, the use of dual enrichment methods identified the most Listeriaspp.–positive samples, whereas the BAX System PCR Assay for the Genus Listeria24E provided the most rapid results (~30 h). Cultural results from the direct plating of secondary enrichment broths were generally in agreement with PCR results when compared within methods. Despite minor differences in efficacy, all five methods were in agreement with one another. Overall, 24 (10.7%) of the 236 environmental samples were positive for Listeriaspp., all of which were collected from non–food contact surfaces. Nine of these sites were also positive in previous sampling events, suggesting that these sites serve as Listerianiches and that certain ribotypes are particularly persistent, inhabiting environments and specific sites for over 2 years. Overall, our results suggest that the extent of Listeriaspp. contamination, notably L. monocytogenes, in small-scale artisan cheese processing environments is low.

The incidence and ecology of Listeriaspp. in farmstead cheese processing environments were assessed through environmental sampling conducted in nine different plants over a 10-week period. Environmental samples (n= 705) were examined for the presence of Listeriaspp. by using three detection/isolation protocols. The use of dual enrichment methods, which allowed for the recovery of injured Listeriaspp. (mUSDA), identified more Listeriaspecies–positive samples with higher sensitivity than the standard USDA method. The addition of PCR to the mUSDA method identified the most Listeria monocytogenes–positive samples, achieving greater sensitivity of detection while substantially reducing time. Overall, 7.5% of samples were positive for Listeriaspp., yielding 710 isolates, 253 of which were subtyped by automated ribotyping to examine strain diversity within and between plants over time. The isolation of specific ribotypes did not appear to be affected by the enrichment protocol used. Fifteen (2.1%) samples yielded L. monocytogenesisolates differentiated almost equally into ribotypes of lineages I and II. Of most concern was the persistent and widespread contamination of a plant with L. monocytogenesDUP-1042B, a ribotype previously associated with multiple outbreaks of listeriosis. Our results suggest that the extent of contamination by Listeriaspp., notably L. monocytogenes, in farmstead cheese plants is comparatively low, especially for those with on-site farms. The results of this study also identified points of control for use in designing more effective Listeriaspp. control and monitoring programs with a focus on ribotypes of epidemiological significance.

Because of renewed interest in specialty cheeses, artisan and farmstead producers are manufacturing surface-mold-ripened soft cheeses from raw milk, using the 60-day holding standard (21 CFR 133.182) to achieve safety. This study compared the growth potential of Listeria monocytogeneson cheeses manufactured from raw or pasteurized milk and held for >60 days at 4°C. Final cheeses were within federal standards of identity for soft ripened cheese, with low moisture targets to facilitate the holding period. Wheels were surface inoculated with a five-strain cocktail of L. monocytogenesat approximately 0.2 CFU/cm2(low level) or 2 CFU/cm2(high level), ripened, wrapped, and held at 4°C. Listeriapopulations began to increase by day 28 for all treatments after initial population declines. From the low initial inoculation level, populations in raw and pasteurized milk cheese reached maximums of 2.96±2.79 and 2.33±2.10 log CFU/g, respectively, after 60 days of holding. Similar growth was observed in cheese inoculated at high levels, where populations reached 4.55±4.33 and 5.29±5.11 log CFU/g for raw and pasteurized milk cheeses, respectively. No significant differences (P< 0.05) were observed in pH development, growth rate, or population levels between cheeses made from the different milk types. Independent of the milk type, cheeses held for 60 days supported growth from very low initial levels of L. monocytogenesintroduced as a postprocess contaminant. The safety of cheeses of this type must be achieved through control strategies other than aging, and thus revision of current federal regulations is warranted.