Dennis J. D'amico

Inactivation of Microorganisms in Milk and Apple Cider Treated with Ultrasound

Nonthermal technologies are emerging as promising alternatives to heat treatment for food processing. Ultrasound, defined as sound waves with a frequency greater than 20 kHz, has proven bactericidal effects, especially when combined with other microbial-reduction strategies such as mild heating. In this study, ultrasound treatment (sonifier probe at 20 kHz, 100% power level, 150 W acoustic power, 118 W/cm2 acoustic intensity) with or without the effect of mild heat (57 degrees C) was effective at reducing microbial levels in raw milk, Listeria monocytogenes levels inoculated in ultrahigh-temperature milk, and Escherichia coli O157:H7 in apple cider. Continuous flow ultrasound treatment combined with mild heat (57 degrees C) for 18 min resulted in a 5-log reduction of L. monocytogenes in ultrahigh-temperature milk, a 5-log reduction in total aerobic bacteria in raw milk, and a 6-log reduction in E. coli O157:H7 in pasteurized apple cider. Inactivation regressions were second-order polynomials, showing an initial period of rapid inactivation, eventually tailing off. Results indicate that ultrasound technology is a promising processing alternative for the reduction of microorganisms in liquid foods.

Low Incidence of Foodborne Pathogens of Concern in Raw Milk Utilized for Farmstead Cheese Production

Overall milk quality and prevalence of four target pathogens in raw milk destined for farmstead cheesemaking was examined. Raw milk samples were collected weekly from June to September 2006 from 11 farmstead cheese operations manufacturing raw milk cheese from cows, goats, and sheeps milk. Samples were screened for Listeria monocytogenes, Staphylococcus aureus, Salmonella, and Escherichia coli O157:H7 both quantitatively (direct plating) and qualitatively (PCR). Overall, 96.8% of samples had standard plate counts of < 100,000 CFU/ml, 42.7% of which were < 1,000 CFU/ml. Although no federal standards exist for coliforms in raw milk, 61% of samples tested conformed to pasteurized milk standards under the U.S. Pasteurized Milk Ordinance (PMO) at < 10 CFU/ml. All cow and sheep milk samples and 93.8% of goat milk samples were within the limits dictated by the PMO for somatic cell counts. Of the 11 farms, 8 (73%) produced samples that were positive for S. aureus, which was detected in 34.6% (46 of 133) of milk samples. L. monocytogenes was isolated from three milk samples (2.3%), two of which were from the same farm. E. coli O157:H7 was recovered from one sample of goats milk for an overall incidence of 0.75%. Salmonella was not recovered from any of the 133 samples. The findings of this study suggest that most raw milk intended for farmstead cheesemaking is of high microbiological quality with a low incidence of pathogens. These data will help inform risk assessments associated with the microbiological safety of farmstead cheeses, particularly those manufactured from raw milk.

Behavior of Escherichia coli O157:H7 during the manufacture and aging of Gouda and stirred-curd Cheddar cheeses manufactured from raw milk.

This study was conducted to examine the fate of Escherichia coli O157: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. coli O157: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. coli O157: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. coli O157:H7 in Gouda or stirred-curd Cheddar cheese manufactured from raw milk contaminated with low levels of this pathogen.

60-day aging requirement does not ensure safety of surface-mold-ripened soft cheeses manufactured from raw or pasteurized milk when Listeria monocytogenes is introduced as a postprocessing contaminant.

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 monocytogenes on cheeses manufactured from raw or pasteurized milk and held for > 60 days at 4 degrees 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. monocytogenes at approximately 0.2 CFU/ cm2 (low level) or 2 CFU/cm2 (high level), ripened, wrapped, and held at 4 degrees C. Listeria populations 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. monocytogenes introduced 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.

Enhanced detection of Listeria spp. in farmstead cheese processing environments through dual primary enrichment, PCR, and molecular subtyping.

The incidence and ecology of Listeria spp. 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 Listeria spp. by using three detection/isolation protocols. The use of dual enrichment methods, which allowed for the recovery of injured Listeria spp. (mUSDA), identified more Listeria species-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 Listeria spp., 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. monocytogenes isolates differentiated almost equally into ribotypes of lineages I and II. Of most concern was the persistent and widespread contamination of a plant with L. monocytogenes DUP-1042B, a ribotype previously associated with multiple outbreaks of listeriosis. Our results suggest that the extent of contamination by Listeria spp., 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 Listeria spp. control and monitoring programs with a focus on ribotypes of epidemiological significance.

Detection, isolation, and incidence of Listeria spp. in small-scale artisan cheese processing facilities: a methods comparison.

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 Listeria spp. 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 Listeria spp., including Listeria monocytogenes. These results, however, did not differ significantly from those obtained with Listeria repair broth or Oxoid 24 Listeria enrichment broth. When full methods were considered, the use of dual enrichment methods identified the most Listeria spp.-positive samples, whereas the BAX System PCR Assay for the Genus Listeria 24E provided the most rapid results (approximately 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 Listeria spp., 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 Listeria niches and that certain ribotypes are particularly persistent, inhabiting environments and specific sites for over 2 years. Overall, our results suggest that the extent of Listeria spp. contamination, notably L. monocytogenes, in small-scale artisan cheese processing environments is low.

Characterization of Staphylococcus aureus strains isolated from raw milk utilized in small-scale artisan cheese production.

Staphylococcus aureus is 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. aureus strains 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. aureus strains 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. aureus is 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.

Comparing the Behavior of Multidrug-Resistant and Pansusceptible Salmonella during the Production and Aging of a Gouda Cheese Manufactured from Raw Milk

Outbreaks of salmonellosis have been linked to the consumption of cheese, and emerging multidrug-resistant (MDR) strains of Salmonella may 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 Salmonella strains 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 Salmonella Newport and Salmonella Typhimurium 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 Salmonella strains. Salmonella counts 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

Synergistic Antimicrobial Combinations Inhibit and Inactivate Listeria monocytogenes in Neutral and Acidic Broth Systems

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 Salmonella strains may not pose a greater threat to cheese safety than do non-MDR Salmonella strains.

Listeria monocytogenes cross-contamination of cheese: risk throughout the food supply chain.

The use of antimicrobial compounds can be an effective approach to control Listeria monocytogenes in 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. monocytogenes in 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. monocytogenes counts of ≤1.0 or ≥3.0 log CFU/mL compared with the starting inoculum, respectively. Individually, lauric arginate (LAE), hydrogen peroxide (HP), and ε-polylysine (EPL) inhibited L. monocytogenes growth 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. monocytogenes in foods with different pH levels.