Probing antimicrobial resistance and sanitizer tolerance themes and their implications for the food industry through the Listeria monocytogenes lens.

The development of antibiotic resistance is a serious public health crisis, reducing our ability to effectively combat infectious bacterial diseases. The parallel study of reduced susceptibility to sanitizers is growing, particularly for environmental foodborne pathogens, such as Listeria monocytogenes. As regulations demand a seek-and-destroy approach for L. monocytogenes, understanding sanitizer efficacy and its uses are critical for the food industry. Studies have reported the ability of L. monocytogenes to survive in sanitizer concentrations 10-1000 times lower than the manufacturer-recommended concentration (MRC). Notably, data show that at MRC and when applied according to the label instructions, sanitizers remain largely effective. Studies also report that variables such as the presence of organic material, application time/temperature, and bacterial attachment to surfaces can impact sanitizer effectiveness. Due to the lack of standardization in the methodology and definitions of sanitizer resistance, tolerance, and susceptibility, different messages are conveyed in different studies. In this review, we examine the diversity of definitions, terminology, and methodologies used in studies examining L. monocytogenes resistance and susceptibility to antimicrobials. Research available to date fails to demonstrate "resistance" of L. monocytogenes to recommended sanitizer treatments as prescribed by the label. As such, sanitizer tolerance would be a more accurate description of L. monocytogenes response to low sanitizer concentrations (i.e., sub-MRC). Conservative use of word "resistance" will reduce confusion and allow for concise messaging as sanitizer research findings are communicated to industry and regulators.

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.

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.

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.

Genomic characterization of Listeria monocytogenes recovered from dairy facilities in British Columbia, Canada from 2007 to 2017.

Listeria monocytogenes is a foodborne pathogen of concern in dairy processing facilities, with the potential to cause human illness and trigger regulatory actions if found in the product. Monitoring for Listeria spp. through environmental sampling is recommended to prevent establishment of these microorganisms in dairy processing environments, thereby reducing the risk of product contamination. To inform on L. monocytogenes diversity and transmission, we analyzed genome sequences of L. monocytogenes strains (n = 88) obtained through the British Columbia Dairy Inspection Program. Strains were recovered from five different dairy processing facilities over a 10 year period (2007-2017). Analysis of whole genome sequences (WGS) grouped the isolates into nine sequence types and 11 cgMLST types (CT). The majority of isolates (93%) belonged to lineage II. Within each CT, single nucleotide polymorphism (SNP) differences ranged from 0 to 237 between isolates. A highly similar (0-16 SNPs) cluster of over 60 isolates, collected over 9 years within one facility (#71), was identified suggesting a possible persistent population. Analyses of genome content revealed a low frequency of genes associated with stress tolerance, with the exception of widely disseminated cadmium resistance genes cadA1 and cadA2. The distribution of virulence genes and mutations within internalin genes varied across the isolates and facilities. Further studies are needed to elucidate their phenotypic effect on pathogenicity and stress response. These findings demonstrate the diversity of L. monocytogenes isolates across dairy facilities in the same region. Findings also showed the utility of using WGS to discern potential persistence events within a single facility over time.

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.

Attachment and Survival of Escherichia coli O157:H7 on In-Shell Hazelnuts.

The multistate Escherichia coli (E. coli) O157:H7 outbreak associated with in-shell hazelnuts highlights the pathogen's ability to involve non-traditional vehicles in foodborne infections. Furthermore, it underscores significant gaps in our knowledge of pathogen survivability and persistence on nuts. Therefore, this study investigated the ability of E. coli O157:H7 to attach and survive on in-shell hazelnuts. In-shell hazelnuts were inoculated with a four-strain mixture of E. coli O157:H7 at 7.6 log colony forming units (CFU)/nut by wet or dry inoculation, stored at ambient conditions (24 ± 1 °C; 40% ± 3% relative humidity (RH) and sampled for twelve months. For the attachment assay, in-shell hazelnuts were inoculated and the adherent population was enumerated at 30 s-1 h following inoculation. Irrespective of the inoculation method, ~5 log CFU of adherent E. coli O157:H7 was recovered from the hazelnuts as early as 30 s after inoculation. Conversely, pathogen survival was significantly reduced under dry inoculation with samples being enrichment negative after five months of storage (p < 0.05). On the other hand, wet inoculation led to a significantly longer persistence of the pathogen with ~3 log CFU being recovered from the in-shell nuts at 12 months of storage (p < 0.05). These results indicate that E. coli O157:H7 can survive in significant numbers on in-shell hazelnuts when stored under ambient conditions.

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.