Irrigation method matters: Contamination and die-off rates of Escherichia coli on dry bulb onions after overhead and drip irrigation in Washington State (2022-2023).

Two U.S. outbreaks of salmonellosis in 2020 and 2021 were epidemiologically linked to red onions. The 2020 outbreak investigation implicated production agricultural water as a likely contamination source. Field trials were designed to investigate prevalence and survival of Escherichia coli (surrogate for Salmonella) on dry bulb onions after application of contaminated irrigation water at the end of the growing period. Irrigation water was inoculated at 3 log most probable number (MPN)/100 mL (2022 and 2023) or 5 log MPN/100 mL (2023, drip only) with a cocktail of rifampin-resistant E. coli and applied with the final irrigation (0.4 acre-inch/0.4 hectare-cm) to onions. Onion bulbs (40 or 80) were sampled immediately after irrigation and throughout field curing (4 weeks) and E. coli was enumerated using a MPN method. For drip irrigation, at 3 log MPN/100 mL E. coli was detected on 13% of onions at 24 h but not detected at 0 h; at 5 log MPN/100 mL for drip irrigation applied to saturated soil, E. coli was detected in 63% of onions at 0 h. Prevalence significantly (P<0.05), decreased after 7 d of curing with cell densities of 1-1,400 MPN/onion. At the end of field curing in 2023, 1/80 onions had detectable E. coli (2.04 MPN/onion). E. coli was detected in a significantly smaller percentage of onions (2022: 13%; 2023: 68%) after a contaminated drip irrigation event compared to overhead irrigation (98-100%; P<0.05). After overhead irrigation E. coli was detected in onions (1-1,000 MPN/onion) on day 0. Prevalence decreased significantly (P <0.05) after 7 d of field curing in both years (2022: 15%; 2023: 7%). E. coli was not detected on Calibra onions (80/year) at the end of field curing in either year but was detected at <12 MPN/onion in 2.5-3.75% of onions (n=80) for other cultivars. These data confirm limited contamination risk associated with drip irrigation water quality and begin to quantify contamination risks associated with overhead irrigation of dry bulb onions.

Optimized Ultraviolet-C Processing Inactivates Pathogenic and Spoilage-Associated Bacteria while Preserving Bioactive Proteins, Vitamins, and Lipids in Human Milk.

Holder pasteurization (HoP) enhances donor human milk microbiological safety but damages many bioactive milk proteins. Though ultraviolet-C irradiation (UV-C) can enhance safety while better preserving some milk proteins, it has not been optimized for dose or effect on a larger array of bioactive proteins. We determined the minimal UV-C parameters that provide >5-log reductions of relevant bacteria in human milk and how these treatments affect an array of bioactive proteins, vitamin E, and lipid oxidation. Treatment at 6000 and 12 000 J/L of UV-C resulted in >5-log reductions of all vegetative bacteria and bacterial spores, respectively. Both dosages improved retention of immunoglobulin A (IgA), IgG, IgM, lactoferrin, cathepsin D, and elastase and activities of bile-salt-stimulated lipase and lysozyme compared with HoP. These UV-C doses caused minor reductions in α-tocopherol but not γ-tocopherol and no increases in lipid oxidation products. UV-C treatment is a promising approach for donor human milk processing.

Characterizing Spoilage of Coconut-based Creamers: A Multifaceted Approach to Identify Problematic Bacteria and Their Potential Sources in a New Product Category.

Beverage innovation is a growing trend with a reliance on comanufacturing relationships to launch products quickly. A recent comanufacturing relationship is the utilization of dairy processing facilities to process plant-based beverages using high-temperature short-time (HTST) pasteurization. While the shelflife of HTST bovine milk is well established at 21 days, retailers are expecting new refrigerated beverages to achieve a 60-day shelflife. Little is known about the microbial stability of these new beverages, particularly those with complex formulations. Our objective was to identify bacterial taxa leading to the spoilage of four coconut-based creamers and their potential sources (raw ingredients or packaging). We used a multifaceted approach including plate counting and 16S rRNA metabarcoding to monitor microbial growth in products throughout shelflife (60 d, 4 °C), and cold enrichment (7 °C, 11 d) of ingredients and packaging. Nearly all product units (25/26) had elevated microbial loads (>4.3 log CFU/mL) prior to the 60-d target, with early spoilage detected at 21 d. Key spoilage taxa included Pseudomonas, Streptococcus, Aerococcus, Paenibacillus, Sphingomonas, and Oceanobacillus. Pseudomonas were responsible for "early" product spoilage (21-32 d), whereas Oceanobacillus were important in products with very "late" spoilage (60-62 d). All key spoilage taxa were identified in cold enrichments of multiple units of waxboard cartons. Paenibacillus was the dominant bacterium in 47% (10/21) of product units. In addition to carton samples, Paenibacillus was also identified in one raw ingredient (mushroom extract). Metabarcoding identified Listeria sensu stricto as a dominant taxon in three individual product units from three distinct production lots. Listeria was also found in 31% (5/16) of cold enrichments of individual cartons. Taxa responsible for spoilage of plant-based beverages were identified as well as demonstrating packaging as an important contamination source.

Before and After: Evaluation of Microbial and Organic Loads in Produce Handling and Packing Operations with Diverse Cleaning and Sanitizing Procedures.

Inadequate cleaning and/or sanitation (C/S) of food contact surfaces (FCSs) has been frequently reported during Produce Safety Rule inspections; however, limited data are available evaluating the effectiveness of C/S processes in produce operations. Different C/S practices were evaluated in four fresh produce operations for their efficacy in reducing microbial and organic loads on various FCSs. Microbial (aerobic plate counts; APC) and organic (ATP) loads were quantified during production, after cleaning, and after sanitizing, if applicable. Operations included: a berry packinghouse (BerryPK; wet cleaning), a blueberry harvest contractor (BerryHC; cleaning + sanitizing, C+S), and two mixed vegetable packinghouses (MixedV1; C+S, and MixedV2; rinsing + sanitizing, R+S). Following wet cleaning, significant reductions in APCs (p < 0.05) were seen on high-density polyethylene (HDPE) storage trays (n = 50) in BerryPK (3.1 ± 0.9 to 2.5 ± 0.7 log CFU/100 cm2). In BerryHC, a greater reduction in APCs was seen on HDPE harvest buckets (n = 25) following C+S (3.8 ± 0.5 to 1.1 ± 0.4 log CFU/100 cm2), compared to wet cleaning only in BerryPK. Stainless steel and conveyor belt FCSs (n = 16) in MixedV1 were sampled, and a significant reduction in APCs (p < 0.05) was observed when comparing in-use (4.8 ± 1.3 log CFU/100 cm2) to post-C+S (3.9 ± 0.7 log CFU/100 cm2). When similar FCSs (n = 17) were sampled in MixedV2, R+S also led to significant reduction in APCs (3.3 ± 0.6 to 1.9 ± 0.6 log CFU/100 cm2) (p < 0.05). ATP testing in fresh produce settings yielded inconsistent results, with no correlation between organic and bacterial loads detected during production (R2 = 0.00) across four operations, and weak correlations observed after cleaning (R2 = 0.18) and after sanitation (R2 = 0.33). The results from this study provide the foundational basis for future research on practical and effective C/S methods tailored to the produce industry.

High-Pressure Processing of Human Milk: A Balance between Microbial Inactivation and Bioactive Protein Preservation.

BACKGROUND: Donor human milk banks use Holder pasteurization (HoP; 62.5°C, 30 min) to reduce pathogens in donor human milk, but this process damages some bioactive milk proteins. OBJECTIVES: We aimed to determine minimal parameters for high-pressure processing (HPP) to achieve >5-log reductions of relevant bacteria in human milk and how these parameters affect an array of bioactive proteins. METHODS: Pooled raw human milk inoculated with relevant pathogens (Enterococcus faecium, Staphylococcus aureus, Listeria monocytogenes, Cronobacter sakazakii) or microbial quality indicators (Bacillus subtilis and Paenibacillus spp. spores) at 7 log CFU/mL was processed at 300-500 MPa at 16-19°C (due to adiabatic heating) for 1-9 min. Surviving microbes were enumerated using standard plate counting methods. For raw milk, and HPP-treated and HoP-treated milk, the immunoreactivity of an array of bioactive proteins was assessed via ELISA and the activity of bile salt-stimulated lipase (BSSL) was determined via a colorimetric substrate assay. RESULTS: Treatment at 500 MPa for 9 min resulted in >5-log reductions of all vegetative bacteria, but <1-log reduction in B. subtilis and Paenibacillus spores. HoP decreased immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin G, lactoferrin, elastase and polymeric immunoglobulin receptor (PIGR) concentrations, and BSSL activity. The treatment at 500 MPa for 9 min preserved more IgA, IgM, elastase, lactoferrin, PIGR, and BSSL than HoP. HoP and HPP treatments up to 500 MPa for 9 min caused no losses in osteopontin, lysozyme, α-lactalbumin and vascular endothelial growth factor. CONCLUSION: Compared with HoP, HPP at 500 MPa for 9 min provides >5-log reduction of tested vegetative neonatal pathogens with improved retention of IgA, IgM, lactoferrin, elastase, PIGR, and BSSL in human milk.

Evaluating Paenibacillus odorifer for its potential to reduce shelf life in reworked high-temperature, short-time fluid milk products.

Rework is a common practice used in the dairy industry as a strategy to help minimize waste from processing steps or errors that might otherwise render the product unsaleable. Dairy processors may rework their high-temperature, short-time (HTST) fluid milk products up to code date (21 d) at a typical dilution rate of ≤20% rework into ≥80% fresh raw milk. Bacterial spores present in raw milk that can survive pasteurization and grow at refrigeration temperatures are often responsible for milk spoilage. However, the potential impact of growth and thermal resistance of organisms in reworked product has not been investigated. Our objective was to characterize growth, sporulation, and thermal resistance of Paenibacillus odorifer under conditions representative of extreme storage conditions (time and temperature) of reduced fat (2%) and chocolate milk to evaluate whether product containing rework would have a reduced shelf life. Commercial UHT-pasteurized 2% milk and chocolate milk were independently inoculated with 4 strains of P. odorifer at 1 to 2 log cfu/mL and stored at 4°C and 7°C for 30 d. Changes in P. odorifer cell densities were determined by standard serial dilution with spread plating on tryptic soy agar with yeast extract and incubation at 25°C for 48 h. Spore counts were determined following thermal treatment at 80°C for 12 min. Thermal resistance of a cocktail of P. odorifer in milk was determined after treatments at 63°C for 30 min and 72°C for 15 s. Strains of P. odorifer grew rapidly at 7°C and reached a maximum cell density of ~8 log cfu/g in both 2% and chocolate milk within 12 d. All strains grew more slowly at 4°C and had not reached maximum cell density by 21 d. With extreme temperature abuse (25°C, 24 h), P. odorifer will sporulate in milk; however, thermally resistant subpopulations, including spores, did not develop in milk at 4°C until after stationary phase was achieved (>24 d). Vegetative cells of P. odorifer were verified to be sensitive to pasteurization (>7 log reduction); therefore, P. odorifer would not be expected to contribute to reduced shelf life of fluid milk products containing rework, even with extended storage before rework.

Resistome characterization of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli isolated from wastewater treatment utilities in Oregon.

Infections resistant to broad spectrum antibiotics due to the emergence of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae is of global concern. This study characterizes the resistome (i.e., entire ecology of resistance determinants) of 11 ESBL-producing Escherichia coli isolates collected from eight wastewater treatment utilities across Oregon. Whole genome sequencing was performed to identify the most abundant antibiotic resistance genes including ESBL-associated genes, virulence factors, as well as their sequence types. Moreover, the phenotypes of antibiotic resistance were characterized. ESBL-associated genes (i.e., blaCMY, blaCTX, blaSHV, blaTEM) were found in all but one of the isolates with five isolates carrying two of these genes (four with blaCTX and blaTEM; one with blaCMY and blaTEM). The ampC gene and virulence factors were present in all the E. coli isolates. Across all the isolates, 31 different antibiotic resistance genes were identified. Additionally, all E. coli isolates harbored phenotypic resistance to beta-lactams (penicillins and cephalosporins), while 8 of the 11 isolates carried multidrug resistance phenotypes (resistance to three or more classes of antibiotics). Findings highlight the risks associated with the presence of ESBL-producing E. coli isolates in wastewater systems that have the potential to enter the environment and may pose direct or indirect risks to human health.

Water Application Method Influences Survival or Growth of Escherichia coli on Bulb Onions during Field Curing.

ABSTRACT: The impact of water application method on bacterial survival at or after the final irrigation was evaluated in bulb onions during commercially relevant field drying (curing). A three-strain rifampin-resistant cocktail of Escherichia coli was introduced to onions via a single overhead spray application in two separate trials (5.22 [trial 1] or 2.40 [trial 2] log CFU per onion) 2 to 3 days after the final irrigation. Onions were lifted from the soil 8 days after spray inoculation and, in some cases, foliage was removed (topping); onions remained in the field for an additional ca. 2 weeks (total ca. 3 weeks of curing). E. coli populations declined on the onions in the first 4 h after spray inoculation. E. coli was recovered from 38 (48%) or 28 (35%) of 80 whole-onion enrichments at the end of curing in trials 1 or 2, respectively. Topping did not significantly impact the percentage of E. coli-positive onions detected at the end of curing. From 8 h to 21 days, E. coli populations on positive onions ranged from 1 CFU per onion to 7 log CFU per onion in both trials, representing a potential risk of E. coli growth with overhead application of contaminated water at the end of onion production. In trial 2, additional rows of onions were inoculated via a 22-cm subsurface or surface drip irrigation line (1.94 log CFU/mL for 2.5 h). E. coli was detected in 0 (subsurface) and 4 (surface) of 50 whole-onion enrichments 3 h after the initiation of drip irrigation. Positive onions were detected at days 1 (4 of 50) and 7 (1 of 50) with subsurface drip inoculation, and at days 1 (7 of 50), 7 (2 of 50), and 14 (2 of 50) with surface drip inoculation. E. coli was not detected in whole-onion enrichments at the end of curing when inoculated by subsurface (0 of 50) or surface (0 of 50) drip irrigation. Application of contaminated water through drip irrigation, when coupled with field curing, results in low rates of contamination of bulb onions at the time of harvest.

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.

Nonstarter Bacterial Communities in Aged Cheddar Cheese: Patterns on Two Timescales.

The aim of this study was to investigate the temporal stability of microbial contamination during cheddar cheese production by examining patterns of nonstarter bacteria in 60-day aged cheddar collected from the start and end of 30 consecutive production days. Further, we explored the source of these temporal microbial variations by comparing microbial communities in the aged cheese to those on food contact surfaces from a piece of cheesemaking equipment previously identified as a major source of nonstarter bacteria in the same processing environment. 16S rRNA metabarcoding and culture-based sequencing methods identified two Streptococcus sequence variants significantly associated with the end of the production day in both the aged cheese and the cheese processing environment. Closer inspection of these sequence variants in the aged cheese over the 40-day sampling period revealed sinusoidal-like fluctuations in their relative ratios, which appeared to coincide with the Lactococcus starter rotation schedule. These results demonstrate that the microbial composition of finished cheese can vary according to the timing of processing within a production day. Further, our results demonstrate that time-of-day microbial differences in cheese can result from bacterial growth on food contact surfaces and that the composition of these microbial differences is subject to change day-to-day and may be linked to routine changes in the Lactococcus starter culture. IMPORTANCE Long production schedules used in modern cheese manufacturing can create circumstances that support the growth of microorganisms in the cheese processing environment. This work demonstrates that this growth can lead to significant changes in the microbial quality of aged cheese produced later in the production day. Further, we demonstrate that the dominant bacteria associated with these microbial changes throughout production are subject to change between days and might be influenced by specific cheese manufacturing practices. These findings improve understanding of microbial contamination patterns in modern food manufacturing facilities, thereby improving our ability to develop strategies to minimize quality losses due to microbial spoilage.

Application of Whole Genome Sequencing to Understand Diversity and Presence of Genes Associated with Sanitizer Tolerance in Listeria monocytogenes from Produce Handling Sources.

Recent listeriosis outbreaks linked to fresh produce suggest the need to better understand and mitigate L. monocytogenes contamination in packing and processing environments. Using whole genome sequencing (WGS) and phenotype screening assays for sanitizer tolerance, we characterized 48 L. monocytogenes isolates previously recovered from environmental samples in five produce handling facilities. Within the studied population there were 10 sequence types (STs) and 16 cgMLST types (CTs). Pairwise single nucleotide polymorphisms (SNPs) ranged from 0 to 3047 SNPs within a CT, revealing closely and distantly related isolates indicative of both sporadic and continuous contamination events within the facility. Within Facility 1, we identified a closely related cluster (0-2 SNPs) of isolates belonging to clonal complex 37 (CC37; CT9492), with isolates recovered during sampling events 1-year apart and in various locations inside and outside the facility. The accessory genome of these CC37 isolates varied from 94 to 210 genes. Notable genetic elements and mutations amongst the isolates included the bcrABC cassette (2/48), associated with QAC tolerance; mutations in the actA gene on the Listeria pathogenicity island (LIPI) 1 (20/48); presence of LIPI-3 (21/48) and LIPI-4 (23/48). This work highlights the potential use of WGS in tracing the pathogen within a facility and understanding properties of L. monocytogenes in produce settings.

Impact of biosolids amendment and wastewater effluent irrigation on enteric antibiotic-resistant bacteria - a greenhouse study.

Reuse of wastewater effluent and biosolids in agriculture is essential to sustainable water and nutrient resource management practices. Wastewater and biosolids, however, are reportedly the recipients, reservoirs, and sources of antibiotic-resistant enteric pathogens. While decay rates of fecal bacterial indicators in soil are frequently studied, very few studies have reported on the persistence of the antibiotic-resistant sub-populations. Little is known about how multi-drug resistance phenotypes of enteric bacteria in agricultural soil change over time. In this study, germinated carrot seeds were planted in soil that received biosolids amendment and/or wastewater effluent irrigation in a greenhouse setting. We quantified total and antibiotic-resistant fecal bacterial indicators (Escherichia coli and enterococci) weekly in soil and total E. coli at harvest (day 77) on carrots. Antibiotic susceptibility of 121 E. coli and 110 enterococci collected isolates were determined. E. coli or enterococci were not recovered from the soil without biosolids amendment regardless of the irrigation water source. After biosolids amendment, soil E. coli and enterococci concentrations increased more than 3 log10 CFU/g-TS within the first week, declined slowly over time, but stayed above the detection limit (0.39 CFU/g-TS) over the entirety of the study. No statistical difference was found between effluent wastewater or water irrigation in soil total and antibiotic-resistant E. coli and enterococci concentrations or carrots E. coli levels. Soil antibiotic-resistant E. coli and enterococci decayed significantly faster than total E. coli and enterococci. Moreover, the prevalence of multi-drug resistant (resistance to three or more antibiotics) E. coli declined significantly over time, while almost all collected enterococci isolates showed multi-drug resistance phenotypes. At harvest, E. coli were present on carrots; the majority of which were resistant to ampicillin. The survival of antibiotic-resistant enteric bacteria in soil and on harvested carrots indicates there are transmission risks associated with biosolids amendment use in root crops.

Population dynamics of coliforms in a commercial Cheddar cheese production facility.

The detection of coliforms in young cheese is a potential indication of undesirable microbial growth within the processing environment. The aim of this study was to investigate sources and conditions that lead to the intermittent detection of coliforms (1-3 log cfu/g) in young Cheddar cheese at a single commercial facility. Analysis of historical production data, in combination with iterative investigative sampling events, was performed to determine coliform levels in milk, whey, curd, and surfaces at the beginning, middle, and end of the production day. After sanitation, conveyor belt pieces from the draining and matting conveyor (DMC) were collected and evaluated for bacterial survivors using culture-based methods and scanning electron microscopy. Production data analysis indicated that cheese produced later in the production day (≥16 h) was significantly more likely to test positive for coliforms than cheese made earlier in the production day (<12 h). Enumeration of coliforms in raw and heat-treated milk demonstrated that the subpasteurization thermal treatment (67-70°C, 26-28 s) was effective at reducing, but not eliminating, coliforms. Repeated sampling identified the DMC, particularly the drain belt and belt 1, as a critical area that supported coliform growth during the production day. Coliform levels in whey entering the weir maintained a level of <1 cfu/mL throughout production; however, coliform levels in whey below the drain belt increased from <1 cfu/mL at midday (8 h) to 5.04 log cfu/mL by the end of the production day (~18 h). Routine sanitation inside the DMC resulted in undetectable coliform levels on easily accessible surfaces. However, enrichment and scanning electron microscopy of belt sections revealed pockets of viable coliforms and other bacteria in cracks and defects in conveyor belts, indicating that sanitation did not eliminate all viable bacteria. Low levels of coliforms are present in heat-treated milk and survive sanitation in the DMC and could serve as the initial seed for high levels of coliforms at the end of the production day.

The Cheese Production Facility Microbiome Exhibits Temporal and Spatial Variability.

A primary goal of modern cheese manufacturing is consistent product quality. One aspect of product quality that remains poorly understood is the variability of microbial subpopulations due to temporal or facility changes within cheese production environments. Therefore, our aim was to quantify this variability by measuring day-day and facility-facility changes in the cheese facility microbiome. In-process product (i.e., milk and cheese) and food-contact surfaces were sampled over the course of three production days at three cheese manufacturing facilities. Microbial communities were characterized using 16S rRNA metabarcoding and by plating on selective growth media. Each facility produced near-identical Cheddar cheese recipes on near-identical processing equipment during the time of sampling. Each facility also used a common pool of Lactococcus starter cultures which were rotated daily as groups of 4-5 strains and selected independently at each facility. Diversity analysis revealed significant facility-facility and day-day differences at each sample location. Facility differences were greatest on the food contact surfaces (i.e., draining-matting conveyor belts), explaining between 25 and 41% of the variance. Conversely, daily differences within each facility explained a greater proportion of the variance in the milk (20% vs. 12%) and cheese (29% vs. 20%). Further investigation into the sources of these differences revealed the involvement of several industrially relevant bacteria, including lactobacilli, which play a central role in flavor and texture development during Cheddar cheese ripening. Additionally, Streptococcus was found to contribute notably to differences observed in milk samples, whereas Acinetobacter, Streptococcus, Lactococcus, Exiguobacterium, and Enterobacteriaceae contributed notably to differences on the food contact surfaces. Facility differences in the cheese were overwhelmingly attributed to the rotation of Lactococcus starter cultures, thus highlighting circumstances where daily microbial shifts could be misinterpreted and emphasizing the importance of repeated sampling over time. The outcomes of this work highlight the complexity of the cheese facility microbiome and demonstrate daily and facility-facility microbial variations which might impact cheese product quality.

Relative performance of commercial citric acid and quaternary ammonium sanitizers against Listeria monocytogenes under conditions relevant to food industry.

Growth of Listeria monocytogenes in cold temperatures coupled with its tolerance of antimicrobials can promote its survival and persistence in food processing environments. The food industry relies heavily on cleaning and sanitation to control L. monocytogenes; therefore, it is important to understand the environmental context (i.e., temperature) on the efficacy of antimicrobials used in food industry. The minimum bactericidal concentrations (MBCs) of an "eco-friendly" citric acid-based (CAB) sanitizer and a conventional quaternary ammonium compound (CQAC) sanitizer were determined against 14 L. monocytogenes isolates at 4-30 °C. A subset of isolates (n = 3) was also exposed to sub-lethal concentrations of sanitizers to assess differences in growth behavior. CAB and CQAC were effective at manufacturer recommended concentrations in liquid assays. The MBC of CAB was significantly lower at 4 °C compared to 23 °C (p < 0.05), whereas the MBC of CQAC was unchanged between 4 °C and 23 °C. Manufacturers' recommendations for dose and duration of CAB and CQAC were unable to consistently achieve a >5-log reduction of L. monocytogenes attached to surfaces. Findings from this study demonstrate the importance of sanitizer evaluation under conditions representative of their use in the food industry.

Growth potential and biofilm development of nonstarter bacteria on surfaces exposed to a continuous whey stream.

Commercial Cheddar cheese production uses an automated, continuous production system that provides favorable conditions for specific undesirable bacterial subpopulations in certain sections of the processing system. The draining and matting conveyor (DMC) is a large, fully enclosed series of conveyor belts that separates curd and whey on the first drain belt and supports the cheddaring process in subsequent sections. In a previous study, we demonstrated that coliforms increase in the draining section of the DMC (pH 6.0-6.3, 36°C) over a typical 18-h production shift and can lead to detectable coliforms in finished cheese. Sampling at the commercial plant indicated 2 sources of very low levels of coliforms: (1) subpasteurized whey and curd entering the DMC and (2) surfaces in the DMC after sanitation. Mitigation of these sources would require different approaches. The aim of this study was to investigate whether naturally low levels of coliforms in whey could increase in the bulk liquid and attach to different surface materials within 18 h. A laboratory-scale system was created to mimic the conditions of the initial draining section of the DMC and consisted of single-pass, naturally contaminated whey (pH 6.3, 35°C) flowing through a bioreactor (1.11 L/h) containing coupons of surface types found in the DMC (stainless steel and polypropylene). Whey inside the bioreactor chamber and surface coupons were enumerated for bacterial subpopulations on selective media for planktonic and attached bacteria, respectively, at 0, 12, 15, and 18 h. Bacterial isolates were identified by 16S rDNA sequencing. Nonstarter bacteria present in the whey at 0 h included coliforms (Enterobacter), Pseudomonas, and Acinetobacter (0.80, 2.55, and 2.32 log cfu/mL, respectively), with each increasing significantly in whey (6.18, 7.00, and 5.89 log cfu/mL) and on coupons (5.20, 6.85, and 5.29 log cfu/cm2, respectively) after 18 h in the continuous flowing system. Scanning electron microscopy confirmed bacterial attachment on both surfaces, with early biofilm development evident on polypropylene coupons by 18 h. Results from this laboratory-scale study demonstrated that naturally low levels of coliforms entering the DMC in the whey could replicate within the conditions of the draining section of the DMC to the levels found in the commercial production environment.

Adaptation to a Commercial Quaternary Ammonium Compound Sanitizer Leads to Cross-Resistance to Select Antibiotics in Listeria monocytogenes Isolated From Fresh Produce Environments.

The effective elimination of Listeria monocytogenes through cleaning and sanitation is of great importance to the food processing industry. Specifically in fresh produce operations, the lack of a kill step requires effective cleaning and sanitation to mitigate the risk of cross-contamination from the environment. As facilities rely on sanitizers to control L. monocytogenes, reports of the development of tolerance to sanitizers and other antimicrobials through cross-resistance is of particular concern. We investigated the potential for six L. monocytogenes isolates from fresh produce handling and processing facilities and packinghouses to develop cross-resistance between a commercial sanitizer and antibiotics. Experimental adaptation of isolates belonging to hypervirulent clonal complexes (CC2, CC4, and CC6) to a commercial quaternary ammonium compound sanitizer (cQAC) resulted in elevated minimum inhibitory concentrations (2-3 ppm) and minimum bactericidal concentrations (3-4 ppm). Susceptibility to cQAC was restored for all adapted (qAD) isolates in the presence of reserpine, a known efflux pump inhibitor. Reduced sensitivity to 7/17 tested antibiotics (chloramphenicol, ciprofloxacin, clindamycin, kanamycin, novobiocin, penicillin, and streptomycin) was observed in all tested isolates. qAD isolates remained susceptible to antibiotics commonly used in the treatment of listeriosis (i.e., ampicillin and gentamicin). The whole genome sequencing of qAD strains, followed by comparative genomic analysis, revealed several mutations in fepR, the regulator for FepA fluoroquinolone efflux pump. The results suggest that mutations in fepR play a role in the reduction in antibiotic susceptibility following low level adaptation to cQAC. Further investigation into the cross-resistance mechanisms and pressures leading to the development of this phenomenon among L. monocytogenes isolates recovered from different sources is needed to better understand the likelihood of cross-resistance development in food chain isolates and the implications for the food industry.

Bile Salt-Stimulated Lipase Activity in Donor Breast Milk Influenced by Pasteurization Techniques.

Breast milk contains bile salt-stimulated lipase (BSSL), which significantly increases the fat digestion capacity of newborns who have limited pancreatic lipase secretion in the first few months after birth. Problematically, Holder pasteurization used in non-profit milk banks to ensure the microbiological safety of donor milk for infants, particularly preterm infants (<37 weeks gestation age), destroys milk BSSL, thus limiting infant fat absorption capacity. Alternative strategies are needed to ensure the safety of donor milk while preserving BSSL activity. Three alternative pasteurization techniques-high-pressure processing (HPP, 550 MPa, 5 min), gamma cell irradiation (IR, 2.5 Mrads) and UV-C (254 nm, 0-33,000 J/L)-were compared with Holder pasteurization (low-temperature long-time, LTLT, 62.5°C, 30 min) for retention of BSSL activity in donor breast milk. As the time required for donor milk pasteurization by UV-C in published methods was not clear, donor breast milk was spiked with seven common bacterial strains and treated by UV-C for variable time periods and the minimum UV-C dosage required to achieve a 5-log10 reduction of CFU/mL was determined. Eight thousand two hundred fifty J/L of UV-C exposure was sufficient to achieve 5-log10 reduction of each of bacterial targets, including Bacillus and Paenibacillus spores. The retention of BSSL activity was highest after HPP (retaining 62% of the untreated milk BSSL activity), followed by UV-C (16,500 J/L), IR and LTLT (35, 29, and 0.3% retention, respectively). HPP was an effective alternative to pasteurize milk with improved retention of BSSL activity compared to Holder pasteurization. Future work should investigate the effect of alternative pasteurization techniques on the entire array of bioactive components in donor breast milk and how these changes affect preterm infant health outcomes. Implementation of HPP technique at milk banks could improve donor milk-fed infant fat absorption and growth.

Prevalence of Listeria spp. in produce handling and processing facilities in the Pacific Northwest.

Listeria monocytogenes is a significant concern for the produce industry; however, there is limited information to support the practical decision-making to mitigate this risk. This study investigated the prevalence of Listeria spp. and L. monocytogenes in seven produce handling and processing (PHP) facilities in the Pacific Northwest. PHP facilities were defined as facilities that receive raw agricultural commodities and further handle, pack, wash, or process prior to distribution into the retail sector. Environmental swabs (n = 50/facility) were collected in high-risk areas (e.g., near raw product entry points) from seven PHP facilities over two visits. Listeria spp. were isolated using modified ISO 11290-1 method and speciated with Microgen® Listeria-ID. Listeria spp., including L. monocytogenes, were found in 5/7 PHP. Prevalence of Listeria spp. ranged from 2% to 26% in these five facilities. Drains, entry areas, and portable equipment consistently tested positive for Listeria spp. during active production. Two additional sampling rounds (n = 50/round) were conducted in the highest prevalence facility (Facility #1). Overall, Listeria spp. were detected in 44/150 (29.3%) swabs collected from Facility #1. This study demonstrated the high prevalence of Listeria spp. near raw product entry points across PHP facilities.