Population dynamics and bidirectional transfer of Listeria monocytogenes and Shiga toxin-producing Escherichia coli during cheese production in wooden vats.

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, the safety of using wooden vats for cheese production remains controversial as the porous structure of wood provides an irregular surface that may protect any attached pathogen cells from cleaning and sanitation processes. On the other hand, the absence of pathogens in wooden vats has been reported in multiple studies and wooden materials have not been associated with foodborne illness outbreaks. The present study determined the survival of Listeria monocytogenes and Shiga toxin-producing Escherichia coli (STEC) during the production of an uncooked pressed cheese in wooden vats as well as their ability to transfer to the wood and then to milk used in subsequent batches of cheese production in the absence of formal cleaning. Results from the study indicate that pathogens inoculated in milk grew during production of the uncooked cheese, but showed limited ability to colonize the wooden vats and contaminate subsequent batches. These results suggest that the risks of using wooden vats to produce cheese is low if the milk is of high microbiological quality.

The impact of environmental conditions and milk type on microbial communities of wooden vats and cheeses produced therein.

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. Variability in microbial communities on wooden vats could lead to inconsistent cheese production. In the present study, the influences of environmental conditions and milk type (raw or heat-treated) on the microbial composition of vat biofilms and cheeses made in the vats were studied using amplicon sequencing of bacterial 16S rRNA and fungal internal transcribed spacer genes. Results showed that the microbial composition of biofilms was influenced by environmental conditions but not the milk type used in cheese production. The microbial composition of cheeses can be further affected by bacterial contributions from milk and the selective forces of environmental conditions. Results of this study suggest that controlling environmental conditions could maintain a more consistent microbial composition of biofilms on wooden vats and resulting cheeses. The use of wooden vats coupled with heat-treated milk at one or more stages of cheese production might be a viable approach to produce cheese with high microbial diversities and reduce risks of undesirable microbes related to food safety and quality.

Anti-infective properties of the protective culture Hafnia alvei B16 in food and intestinal models against multi-drug resistant Salmonella.

Salmonella enterica is a ubiquitous and multi-host pathogen that causes significant morbidity and mortality worldwide. Outbreaks of foodborne salmonellosis continue to occur, highlighting the need for additional interventions. The present study investigated the potential for the commercial protective culture Hafnia alvei B16 to provide enhanced protection against multi-drug resistant strains of S. enterica serovars Typhimurium and Newport by attenuating their virulence when cocultured in milk (as a model food) and broth, and by protecting intestinal epithelial cells from pathogen infection in vitro. Exposure to HA in milk inhibited the subsequent adhesion of S. Typhimurium by 95.23%, whereas the invasion capacity of both serovars was reduced when cocultured with HA in broth and milk. The inhibition of invasion by S. Typhimurium and S. Newport was greater when cocultured in milk (86.95% and 86.58%, respectively) compared to broth (51.64% and 79.88%, respectively). Exposure to HA in both media decreased the expression of virulence genes in S. Typhimurium and S. Newport. Pre-treatment of Caco-2 cells with HA reduced invasion of S. Newport by 89.68% compared to control. These data demonstrate the potential for HA to enhance food safety by attenuating Salmonella virulence and protecting against pathogen invasion of intestinal epithelial cells.

Characterization of microbial community assembly on new wooden vats for use in cheese production.

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.

Probiotic potential of commercial dairy-associated protective cultures: In vitro and in vivo protection against Listeria monocytogenes infection.

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 âˆ¼ 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 âˆ¼ 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.

Composition, Succession, and Source Tracking of Microbial Communities throughout the Traditional Production of a Farmstead Cheese.

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.

Antimicrobial effects of a bioactive glycolipid on spore-forming spoilage bacteria in milk.

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.

Application of bioactive glycolipids to control Listeria monocytogenes biofilms and as post-lethality contaminants in milk and cheese.

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.

The effect of protective cultures on Staphylococcus aureus growth and enterotoxin 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.

Compatibility of Commercially Produced Protective Cultures with Common Cheesemaking Cultures and Their Antagonistic Effect on Foodborne Pathogens.

ABSTRACT: 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.

Effect of modified atmosphere packaging on the growth of spoilage microorganisms and Listeria monocytogenes on fresh cheese.

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 <1 log10 cfu/g through 14 d and ∼1.5 log10 cfu/g by d 21. Mean L. monocytogenes counts increased to levels significantly higher than inoculation (d 0) on cheeses stored in MAP2 and MAP3 on d 21, on d 28 for MAP4, and on d 35 for cheeses stored under MAP5 conditions. Overall, significant treatment × time interactions were observed between air, vacuum, and MAP1 when each was compared with MAP2, MAP3, MAP4, and MAP5. These data demonstrate that packaging fresh cheese under modified atmospheres containing CO2 may be a promising approach to extend shelf life while limiting L. monocytogenes growth during cold storage.

Acidification of Model Cheese Brines To Control Listeria monocytogenes.

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.

Control of Listeria monocytogenes in whole milk using antimicrobials applied individually and in combination.

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), ε-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.

Efficacy of Antimicrobials Applied Individually and in Combination for Controlling Listeria monocytogenes as Surface Contaminants on Queso Fresco.

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), ε-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 <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. monocytogenes counts 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. monocytogenes on 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. monocytogenes on QF through a 21-day shelf life.

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

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.

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 $ 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.

Microbiological Quality and Safety Issues in Cheesemaking.

As the manufacture of cheese relies in part on the select outgrowth of microorganisms, such conditions can also allow for the multiplication of unwanted contaminants. Milk ultimately becomes contaminated with microorganisms originating from infection, the farm environment, and feedstuffs, as well as milking and processing equipment. Thus, poor sanitation, improper milk handling, and animal health issues can result in not only decreased yield and poor quality but also sporadic cases and outbreaks of dairy-related disease. The entry, establishment, and persistence of food-borne pathogens in dairy processing environments also present a considerable risk to products postprocessing. Food safety management systems coupled with regulatory policies and microbiological standards for milk and milk products currently implemented in various nations work to reduce risk while improving the quality and safety of cheese and other dairy products. With that, cheese has enjoyed an excellent food safety record with relatively few outbreaks of food-borne disease considering the amount of cheese produced and consumed worldwide. However, as cheese production and consumption continue to grow, we must remain vigilant in ensuring the continued production of safe, high-quality cheese.

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.

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.

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.