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The growth of psychrotolerant aerobic spore-forming bacteria during refrigerated storage often results in the spoilage of fluid milk, leading to off-flavors and curdling. Because of their low toxicity, biodegradability, selectivity, and antimicrobial activity over a range of conditions, glycolipids are a novel and promising intervention to control undesirable microbes. The objective of this study was to determine the efficacy of a commercial glycolipid product to inhibit spore germination, spore outgrowth, and the growth of vegetative cells of Paenibacillus odorifer, Bacillus weihenstephanensis, and Viridibacillus arenosi, which are the predominant spore-forming spoilage bacteria in milk. For spore germination and outgrowth assays, varying concentrations (25–400 mg/L) of the glycolipid product were added to commercial UHT whole and skim milk inoculated with ∼4 log10 spores/mL of each bacteria and incubated at 30°C for 5 d. Inhibition of spore germination in inoculated UHT whole milk was only observed for V. arenosi, and only when glycolipid was added at 400 mg/L. However, concentrations of 400 and 200 mg/L markedly inhibited the outgrowth of vegetative cells from spores of P. odorifer and B. weihenstephanensis, respectively. No inhibition of spore germination or outgrowth was observed in inoculated UHT skim milk for any strain at the concentrations tested (25 and 50 mg/L). The effect of glycolipid addition on vegetative cell growth in UHT whole and skim milk when inoculated with ∼4 log10 cfu/mL of each bacteria was also determined over 21 d of storage at 7°C. Glycolipid addition at 50 mg/L was bactericidal against P. odorifer and B. weihenstephanensis in inoculated UHT skim milk through 21 d of storage, whereas 100 mg/L was needed for similar control of V. arenosi. Concentrations of 100 and 200 mg/L inhibited the growth of vegetative cells of B. weihenstephanensis and P. odorifer, respectively, in inoculated UHT whole milk, whereas 200 mg/L was also bactericidal to B. weihenstephanensis. Additional studies are necessary to identify effective concentrations for the inhibition of Viridibacillus spp. growth in whole milk beyond 7 d. Findings from this study demonstrate that natural glycolipids have the potential to inhibit the growth of dairy-spoilage bacteria and extend the shelf life of milk.

Wooden vats are used in the production of some traditional cheeses as the biofilms on wooden vat surfaces are known to transfer large quantities of microbes to cheese. However, very few studies have investigated the microbial composition of biofilms on newly developed wooden vats and how communities assemble and evolve. In the present study, the microbial communities of biofilms were characterized over the activation process on new wooden vats using amplicon sequencing of bacterial 16s rRNA and fungal internal transcribed spacer genes. Results showed that microbes from the whey effectively developed on wooden vats. Lactococcus was highly dominant throughout the vat activation process with substantial increases in the relative abundance of Acetobacter and Lactobacillus at the end of the vat activation (day 7). This was in contrast with fungal communities that stabilized early (day 1) and were dominated by Kluyveromyces. Predicted functions corresponded with the different stages of biofilm formation whereby functions associated with biofilm initiation were enriched on day 1 and those associated with growth and maturation were enriched on days 4 and 7. Microbial succession on wooden vat surfaces is expected to be reproducible based on the early onset and dominance of the deterministic process.

Prior to the advent of milk pasteurization and the use of defined-strain starter cultures, the production and ripening of cheese relied on the introduction and growth of adventitious microbes from the environment. This study characterized microbial community structures throughout a traditional farmstead cheese production continuum and evaluated the role of the environment in microbial transfer. In total, 118 samples (e.g., raw milk, cheese, and environmental surfaces) were collected from milk harvesting through cheese ripening. Microbial communities were characterized based on amplicon sequencing of bacterial 16S rRNA and fungal internal transcribed spacer genes using the Illumina MiSeq platform. Results indicated that the environment in each processing room harbored unique microbial ecosystems and consistently contributed microbes to milk, curd, and cheese. The diverse microbial composition of milk was initially attributed to milker hands and cow teats and then changed substantially following overnight ripening in a wooden vat to one dominated by lactic acid bacteria, including Lactococcus lactis, Lactobacillus, and Leuconostoc, as well as fungi such as Exophiala, Kluyveromyces, and Candida. Additional microbial contributions were attributed to processing tools, but the composition of the cheese paste remained relatively stable over 60 days of ripening. In contrast, rind communities that were largely influenced by direct contact with bamboo aging mats showed a distinct succession pattern compared to the interior sections. Overall, these findings highlight the critical role of traditional tools and practices in shaping the microbial composition of cheese and broaden our understanding of processing environments as important sources of microbes in food. IMPORTANCE Throughout the 20th century, especially in the United States, sanitation practices, pasteurization of milk, and the use of commercial defined-strain starter cultures have enhanced the safety and consistency of cheese. However, these practices can reduce cheese microbial diversity. The rapid growth of the artisanal cheese industry in the United States has renewed interest in recapturing the diversity of dairy products and the microbes involved in their production. Here, we demonstrate the essential role of the environment, including the use of wooden tools and cheesemaking equipment, as sources of dominant microbes that shape the fermentation and ripening processes of a traditional farmstead cheese produced without the addition of starter cultures or direct inoculation of any other bacteria or fungi. These data enrich our understanding of the microbial interactions between products and the environment and identify taxa that contribute to the microbial diversity of cheese and cheese production.

Staphylococcus aureus is the causative agent of staphylococcal food poisoning and is a common contaminant in milk. Despite efforts to control S. aureus, recalls and outbreaks continue to occur, highlighting the need for additional interventions. This study determined the potential for protective cultures (PC) that are commercially available to producers to control S. aureus growth in raw milk and attenuate virulence by impeding staphylococcal enterotoxin (SE) production in raw milk and laboratory medium. Cultures of Hafnia alvei and Lactococcus lactis effectively inhibited S. aureus growth in raw milk to counts ~5 log CFU/mL lower than control when cocultured following a cheesemaking time and temperature profile; two cultures of Lactobacillus plantarum inhibited growth to ~1.5 log CFU/mL less than control. Cocultures of S. aureus with Lc. lactis, H. alvei and Lb. plantarum in raw milk reduced SE levels by 24.9%, 62.4%, and 76%, respectively. Lc. lactis also decreased SE production in raw milk in the absence of PC-mediated growth inhibition. Significant reductions in SE production in the absence of pathogen growth inhibition were also achieved in laboratory medium. Together, these results demonstrate the potential for PCs to inhibit S. aureus growth and impede SE production in the absence of growth inhibition.

Outbreaks of listeriosis are continually attributed to the consumption of Hispanic-style soft cheeses contaminated with Listeria monocytogenes postpasteurization. Once contaminated, L. monocytogenes can grow rapidly in cheeses like Queso Fresco (QF) even when stored at refrigeration temperatures. Several antimicrobials, including acidified calcium sulfate with lactic acid (ACSL), e-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. monocytogenes as 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. monocytogenes counts 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. monocytogenes counts to

Protective cultures (PCs) are capable of inhibiting the growth of foodborne pathogens, including Listeria monocytogenes, in food products. However, their ability to attenuate pathogen virulence in food remains uninvestigated. In this study, we used an immunomagnetic separation approach to isolate the effect of pre-exposure to commercially produced PCs in milk as a model food on the subsequent ability of L. monocytogenes to survive simulated gastrointestinal conditions and infect human Caco-2 epithelial cells. Pre-exposure to two cultures of Lactiplantibacillus plantarum reduced the ability of L. monocytogenes to survive simulated gastrointestinal conditions with counts 2.7 log CFU/mL lower than control. The ability of L. monocytogenes to invade Caco-2 cells was also reduced by 1.5 and 2.3 log CFU/mL compared to control after exposure to Lactococcus lactis subsp. lactis and a strain of Lactiplantibacillus plantarum, respectively. All Lactococcus lactis and Lactiplantibacillus plantarum cultures were able to limit the pathogen translocation across Caco-2 cells by 1.5–2.1 log CFU/mL less than that of control. When coupled with their antimicrobial activities against L. monocytogenes, these results demonstrate the ability of PCs to enhance the safety of food products by also attenuating the pathogen virulence, thereby impeding its ability to cause infection upon ingestion.

Protective bacterial cultures (PCs) are commercially available to producers to control undesirable microbes in foods, including foodborne pathogens such as Listeria monocytogenes. They are generally recognized as safe for consumption and many are capable of producing bacteriocins. Yet their potential to act as probiotics and confer a health benefit on the host is not known. This study investigated the ability of three commercial PCs to survive human gastrointestinal conditions and exert anti-infective properties against L. monocytogenes. Counts of two PCs of Lactiplantibacillus plantarum remained unchanged after exposure to simulated gastrointestinal conditions, whereas counts of the PC Lactococcus lactis subsp. lactis were reduced by 5.3 log CFU/mL. Cultures of Lactiplantibacillus plantarum and Lactococcus lactis subsp. lactis adhered to human Caco-2 epithelial cells at ∼ 6 log CFU/mL. This pretreatment reduced subsequent L. monocytogenes adhesion and invasion by 1–1.6 log CFU/mL and 3.8–4.9 log CFU/mL, respectively, compared to control. L. monocytogenes-induced cytotoxicity was also reduced from 29.1% in untreated monolayers to ∼ 8% in those treated with PCs. Pretreatment of Caco-2 monolayers with Lactococcus lactis subsp. lactis and one PC of Lactiplantibacillus plantarum reduced L. monocytogenes translocation by ≥ 1.2 log CFU/mL compared to control (≥ 94.5% inhibition). All PCs significantly reduced DextranFITC permeability through Caco-2 monolayers to approximately half that of control. Pretreatment with PCs also reduced L. monocytogenes-induced mortality in Caenorhabditis elegans. These findings demonstrate the potential for commercially produced PCs to exert probiotic effects in the host through protection against L. monocytogenes infection, thus providing an additional benefit to food safety beyond inhibiting pathogen growth, survival, and virulence in foods.

The documented survival of pathogenic bacteria, including Listeria monocytogenes (LM), Shiga toxin-producing Escherichia coli (STEC), and Salmonella during the manufacture and aging of some cheeses highlights the need for additional interventions to enhance food safety. Unfortunately, few interventions are compliant with the Standards of Identity for cheese. Protective bacterial cultures (PCs) represent actionable, natural interventions. However, supportive data for commercially produced PCs regarding their efficacy against pathogens and potential antagonism with each other and cheesemaking cultures are scant, thereby impeding their potential use by the cheese industry. The overall objective of this study was to identify commercially produced PCs that exert antimicrobial activity toward pathogens with minimal impact on beneficial cheese microbes. Direct antagonism and agar well diffusion assays were used to determine the impact of 10 commercially produced PCs on the growth of starter cultures and cultures of ripening bacteria and fungi. Deferred antagonism was used to evaluate the potential for antimicrobial effects against LM, STEC, and Salmonella. PCs and starter cultures were cocultured in ultrahigh-temperature-processed milk to determine the effects of coculture on starter acidification profiles when incubated according to a simulated cheesemaking temperature profile (4 h at 35°C followed by 20 h at 20°C). Compatibility assays suggest that PC antagonism is microbe and strain specific. Only one PC negatively impacted the acidification of the starters tested. PC antagonism of ripening bacteria and fungi growth varied but was consistent within species. All PCs displayed deferred inhibition of LM, STEC, and Salmonella growth, but to varying degrees. These data identify commercial PCs with potential for the control of pathogens and characterize their compatibility with cheesemaking cultures for future use by cheesemakers and investigations of their efficacy in the production of cheese. HIGHLIGHTS

Listeria monocytogenes can form persistent biofilms on food processing surfaces, resulting in cross-contamination of food products, including milk and milk products. Natural glycolipids are a promising intervention to control undesirable microbes due to their antimicrobial activity and low toxicity. This study aimed to determine the antimicrobial activity of glycolipids to control L. monocytogenes biofilms as well as in milk and on Queso Fresco. Application of a natural glycolipid product significantly reduced biofilm-associated L. monocytogenes on both polystyrene and stainless steel at concentrations as low as 45 mg/L. When added to UHT skim milk, a concentration of 1000 mg/L inhibited L. monocytogenes growth through 7 days of storage at 7 °C, and application of 1300 and 1500 mg/L reduced counts to levels below the limit of enumeration at day 21. In contrast, 2000 mg/L were necessary to inhibit growth through 7 days in whole milk. Glycolipid solutions at concentrations ≥10% reduced L. monocytogenes counts on Queso Fresco through 7 days when applied as a dip. Overall, natural glycolipids have potential as a natural alternative for the removal of biofilms and as an antimicrobial to control L. monocytogenes in milk and milk products with short shelf lives.

Hydrogen peroxide (H2O2) has a long history of use as an antimicrobial in the dairy industry to preserve raw milk. The effect of H2O2 on the survival of Listeria monocytogenes on the surface of Queso Fresco (QF) and the degradation of H2O2 on cheese during storage and in dip solutions following successive treatments were determined. Treatment of QF in 10% H2O2 for 5 s significantly reduced L. monocytogenes by 2.27 log cfu g−1 by 30 min and to ∼0.5 log cfu g−1 at 24 h post-treatment with no growth thereafter. Residual H2O2 levels on cheese decreased from 3200 mg L−1 at 30 min to levels below the limit of quantification at day 21. H2O2 concentrations in dip solutions did not change through 100 successive treatments. These data further support the application of H2O2 as an effective post-lethality treatment to control L. monocytogenes on high-moisture cheese with potential for commercial application.

SUMMARYListeria monocytogeneshas been the most common microbial cause of cheese-related recalls in both the United States and Canada in recent years. SinceL. monocytogenesis inactivated by pasteurization, the majority of these cases have been linked to environmental and cross-contamination of fresh-soft, soft-ripened, and semi-soft cheeses. Cross-contamination of foods withL. monocytogenesis a continuous risk throughout the food supply chain and presents unique challenges for subsequent illness and outbreak investigations. Reports on outbreaks of listeriosis attributed to cross-contamination downstream from primary processing help highlight the critical role of epidemiological investigation coupled with coordinated molecular subtyping and surveillance in the recognition and investigation of complex foodborne outbreaks. Despite their complexity, environmental sampling throughout the supply chain coupled with improved genotyping approaches and concomitant analysis of foodborne illness epidemiological exposure data are needed to help resolve these and similar cases more rapidly and with greater confidence.

Cheeses made from unpasteurized milk are preferred by many consumers for their complex flavors and aromas among other attributes. However, the potential presence and survival of foodborne pathogens remain a concern. There is, therefore, a need for developing practical and science-based preventive strategies to enhance the safety of these products. The use of commercially produced protective bacterial cultures (PC) can be a practical approach. This study examined the potential of commercially produced PCs when used both individually or in combinations to control Listeria monocytogenes and Salmonella, as well as O157:H7 and non-O157:H7 shiga toxin-producing Escherichia coli (STEC) in growth medium and raw milk. The antimicrobial activity of nine individual PCs and 28 combinations thereof was first determined in growth medium using the cell-free supernatants (CFS). Following the determination of median inhibitory concentrations of individual PCs and their combinations, fractional inhibitory concentration indexes were calculated to classify combinations as synergistic, additive, or antagonistic. Overall, L. monocytogenes was inhibited by lower concentrations of CFS compared to the Gram-negative pathogens. Combinations of PCs with synergistic antimicrobial activity against each pathogen were identified. The efficacy of individual PCs was further determined in raw milk following an incubation time and temperature profile similar to that of cheesemaking and ripening. L. monocytogenes growth was inhibited to counts of ~6 log and ~3 log CFU/mL lower than control when cocultured with PCs of Lactococcus lactis or Lactobacillus plantarum, respectively. Hafnia alvei inhibited the growth of STEC O157, non-O157 STEC, and Salmonella enterica in coculture to ~2 log, ~3.5 log, and ~3 log CFU/mL lower than pathogen controls, respectively. Potential synergistic activity was noted for the combination of Lc. lactis and Pediococcus acidilactici against L. monocytogenes when cocultured in raw milk. This study provides data for the efficacy of commercially produced PCs in controlling the growth of pathogens, suggesting that the use of PCs is a promising strategy for enhancing food safety including the safety of raw milk products such as cheese.

Despite efforts to control Listeria monocytogenes in dairy processing environments, contamination and subsequent outbreaks of listeriosis continue to occur. The ability of L. monocytogenes to grow during refrigerated storage necessitates strategies to prevent contamination, reduce pathogen numbers, and limit growth during storage. The objective of this study was to determine the efficacy of edible antimicrobial coatings to control L. monocytogenes on Queso Fresco (QF) when applied before (pre-coated; PC) or after (pre-inoculated; PI) surface contamination. Coating solutions were formulated to contain 2% chitosan and either 5% hydrogen peroxide (HP), 5% lauric arginate (LAE), 25% acidified calcium sulfate with lactic acid (ACSL), or combinations of 10% sodium caprylate (SC) with either LAE or ACSL. Fresh QF samples (25g) were inoculated with L. monocytogenes at ~4 log CFU/g prior to, or following, antimicrobial coating application. Cheeses were then vacuum packaged and stored at 7°C for 35 days with weekly enumeration of L. monocytogenes. Aside from ACSL and LAE+SC, there was no effect of coating application timing (PC vs. PI) on the change in L. monocytogenes counts over time. Chitosan coatings without additional antimicrobials were more effective than controls but did not inhibit L. monocytogenes growth beyond 7 days. Coatings containing HP at 5% were equally effective when applied before or after L. monocytogenes inoculation, significantly reducing L. monocytogenes counts by more than 3 log CFU/g and inhibiting growth through 35 days of storage. Coatings formulated with ACSL at 25% were more effective when applied to pre-inoculated cheeses but neither application produced significant reduction in L. monocytogenes counts or inhibited growth. Although LAE coatings were more effective than ACSL, neither were more effective than chitosan coatings alone. The addition of SC to ACSL and LAE coatings enhanced their antimicrobial activity as ACSL+SC and LAE+SC coatings reduced L. monocytogenes counts by >1 log CFU/g after 24 hours and were listeristatic through 28 and 35 days, respectively. The identification of listericidal and listeristatic edible antimicrobial coating applications that are effective when applied before or after contamination events identifies a new approach for the control of L. monocytogenes on fresh cheese.

Cheese brines are often used for prolonged periods, with adjustments made only to pH and salt content. Pathogens, including Salmonella enterica Typhimurium, Escherichia coli O157:H7, and Listeria monocytogenes, have been shown to survive long periods in model and commercial brines under common brining conditions. The objective of this study was to determine the survival of L. monocytogenes in model cheese brines, with and without whey added at 2%, when acidified to a pH of 2 using food-grade acids. Survival in untreated brines over a 6-month period was also assessed. Cultures of L. monocytogenes were propagated to induce salt and acid tolerance prior to inoculation at ∼6 log CFU/mL into model brines (pH 5.2, 20% NaCl). Following a week-long adaption period at 12°C, inoculated brines were acidified to pH 2.0 within 15 min using either hydrochloric, acetic, citric, or lactic acid, held at that pH for up to 24 h, and neutralized prior to enumeration and enrichment. Overall, each acid treatment was capable of achieving ≥5-log reductions in L. monocytogenes counts within 135 min at pH 2. Hydrochloric acid required the lowest volume to achieve treatment pH and was the most effective treatment in the absence of whey. However, it was the least effective in the presence of whey. Acetic acid produced rapid inactivation in both brines but required impractical volumes of acid to reach the treatment pH. Citric acid was similarly effective in both brines but was the second least effective in terms of time to achieve a ≥5-log reduction. Although only slight and insignificant differences were observed, lactic acid appears to be the more practical and promising approach for the inactivation of L. monocytogenes in cheese brines by producing the most rapid inactivation in the presence and absence of whey. Acidification as a preventive control for L. monocytogenes could increase adoption of brine treatments by small-scale cheese producers, thereby reducing food safety risks.

Queso Fresco has a limited shelf life and has been shown to support the rapid growth of Listeria monocytogenes during refrigerated storage. In addition to improving quality and extending shelf life, modified atmosphere packaging (MAP) has been used to control the growth of pathogenic microorganisms in foods. The objectives of this study were to determine the effects of MAP conditions on the survival and growth of spoilage microorganisms and L. monocytogenes during storage of Queso Fresco manufactured without starter cultures. For L. monocytogenes experiments, cheeses were surface inoculated at ∼4 log10 cfu/g before packaging. Inoculated and uninoculated (shelf life experiments) cheeses were placed in 75-µm high-barrier pouches, packaged under 1 of 7 conditions including air, vacuum, or combinations of N2 and CO2 [100% N2 (MAP1), 30% CO2:70% N2 (MAP2), 50% CO2:50% N2 (MAP3), or 70% CO2:30% N2 (MAP4), 100% CO2 (MAP5)], and stored at 7°C. Samples were removed weekly through 35 d of storage. Listeria monocytogenes counts were determined for inoculated samples. Uninoculated samples were assayed for mesophilic and psychrotolerant counts, lactic acid bacteria, coliforms, and yeast and mold. In general, cheeses packaged under conditions consisting of higher contents of CO2 had lower pH levels during storage compared with those stored in conditions with lower levels or no CO2 at all. Similarly, the antimicrobial efficacy of MAP in controlling spoilage microorganisms increased with increasing CO2 content, whereas conditions consisting of 100% N2, vacuum, or air were less effective. Mean L. monocytogenes counts remained near inoculation levels for all treatments at d 1 but increased ∼2 log10 cfu/g on cheeses packaged in air, vacuum, and 100% N2 (MAP1) conditions at d 7 and an additional ∼1.5 log10 cfu/g at d 14 where they remained through 35 d. In contrast, treatments consisting of 70% CO2 (MAP4) and 100% CO2 (MAP5) limited increases in mean L. monocytogenes counts to

Dairy product recalls and dairy-related illnesses are often the result of contamination with Listeria monocytogenes, which can occur throughout the dairy production and supply chains. The use of antimicrobial compounds is one practical approach for controlling pathogen survival and growth in foods. The goal of this study was to use fluid milk as a model system to identify listeristatic or listericidal treatments that show promise for application in fluid milk and for further evaluation in other dairy products (e.g., cheese). Caprylic acid (CA), e-polylysine (EPL), hydrogen peroxide, lauric arginate (LAE), and sodium caprylate (SC) were added individually or in combination to whole milk inoculated with L. monocytogenes at ˜4 log10 cfu/mL. Samples were stored at 7°C for 21 d, and L. monocytogenes counts were determined weekly. Inhibitory concentrations of LAE (800 mg/L) and EPL (100-400 mg/L), as well as SC and CA (3,200 mg/L each), were identified. The addition of EPL at 800 mg/L reduced L. monocytogenes counts by >3 log10 cfu/mL from initial inoculation levels after 21 d. Addition of hydrogen peroxide to milk reduced counts by >3 log10 cfu/mL from initial inoculation within 24 h (400 and 800 mg/L) or by d 7 (200 mg/L). Although the combinatory treatments of EPL + CA, EPL + LAE, and LAE + SC were characterized as indifferent, EPL + SC worked synergistically to reduce L. monocytogenes populations in milk over 21 d. Overall, these data identify potential antimicrobial treatments to control L. monocytogenes in milk and serve as a foundation for the continued development of antimicrobial controls for L. monocytogenes in dairy products.

The microbiological quality and safety of cheese begin with milk. Its abundance of nutrients, high moisture, and near-neutral pH make it an excellent growth medium not only for the microorganisms that are used for making cheese but for adventitious microorganisms, including those that spoil milk and cheeses and those that cause illness in humans, as well.

Development of science-based interventions in raw milk cheese production is challenging due to the large diversity of production procedures and final products. Without an agreed upon categorization scheme, science-based food safety evaluations and validation of preventive controls would have to be completed separately on each individual cheese product, which is not feasible considering the large diversity of products and the typically small scale of production. Thus, a need exists to systematically group raw milk cheeses into logically agreed upon categories to be used for food safety evaluations. This paper proposes and outlines one such categorization scheme that provides for 30 general categories of cheese. As a base for this systematization and categorization of raw milk cheese, we used Table B of the US Food and Drug Administration's 2013 Food Code, which represents the interaction of pH and water activity for control of vegetative cells and spores in non-heat-treated food. Building on this table, we defined a set of more granular pH and water activity categories to better represent the pH and water activity range of different raw milk cheeses. The resulting categorization scheme was effectively validated using pH and water activity values determined for 273 different cheese samples collected in the marketplace throughout New York State, indicating the distribution of commercially available cheeses among the categories proposed here. This consensus categorization of cheese provides a foundation for a feasible approach to developing science-based solutions to assure compliance of the cheese processors with food safety regulations, such as those required by the US Food Safety Modernization Act. The key purpose of the cheese categorization proposed here is to facilitate product assessment for food safety risks and provide scientifically validated guidance on effective interventions for general cheese categories. Once preventive controls for a given category have been defined, these categories would represent safe havens for cheesemakers, which would allow cheesemakers to safely and legally produce raw milk cheeses that meet appropriate science-based safety requirements (e.g., risk to human health equivalent to pasteurized milk cheeses).

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.

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.

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.

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 cow's, goat's, and sheep's 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

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.

Listeria selective enrichment media are designed to enhance the isolation of the organism and increase the chances of detection. Drawbacks include the requirements for prolonged sample incubation (48 to 72 h) and manual addition of selective agents, which may be a source of contamination. Modified Listeria recovery broth (mLRB) is a proprietary enrichment medium formulated to facilitate the recovery of injured cells; its selective agents are incorporated into a format that allows delayed release until 6 h of incubation. We evaluated the change in cell populations over time for acid- and nitrite-injured Listeria monocytogenes in mLRB with the selective agents added manually at 0 h (mLRBS0) and 6 h (mLRBS6). Recovery of injured cells in mLRB plus time-delayed tablets (mLRBTD) was also compared with that in enrichment media recommended by the U.S. Department of Agriculture (University of Vermont broth), the U.S. Food and Drug Administration (buffered Listeria enrichment broth), and the International Organization for Standardization (demi-Fraser broth). Nitrite- or acid-injured Listeria at approximately 10 CFU/ml were inoculated into each broth medium, and Listeria populations were enumerated at various times from 12 to 48 h of incubation at 37°C. Analysis of variance revealed that acid-injured Listeria populations in mLRBS6 at 24 h were significantly higher (P < 0.05) than those in mLRBS0; however, the differences in populations on these two media were not significant for nitrite-injured cells. Cell populations of four strains of Listeria inoculated into mLRBTD were significantly higher at 24 h than when those strains were enriched in buffered Listeria enrichment broth, demi-Fraser broth, and University of Vermont broth. Comparison between artificially contaminated milk and meat samples with a four-strain cocktail of Listeria resulted in cell populations that were significantly higher (P < 0.05) at 24 h on mLRBTD for contaminated meat than on mLRB for contaminated milk. Delivery of selective agents via time-delayed release tablets into mLRB maximizes recovery of acid- and nitrite-injured Listeria and saves analyst time during food sample analysis.

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.

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