Dynamic cerebral autoregulation is preserved during orthostasis and intrathoracic pressure regulation in healthy subjects: A pilot study.

Resistance breathing may restore cardiac output (CO) and cerebral blood flow (CBF) during hypovolemia. We assessed CBF and cerebral autoregulation (CA) during tilt, resistance breathing, and paced breathing in 10 healthy subjects. Blood velocities in the internal carotid artery (ICA), middle cerebral arteries (MCA, four subjects), and aorta were measured by Doppler ultrasound in 30° and 60° semi-recumbent positions. ICA blood flow and CO were calculated. Arterial blood pressure (ABP, Finometer), and end-tidal CO2 (ETCO2) were recorded. ICA blood flow response was assessed by mixed-models regression analysis. The synchronization index (SI) for the variable pairs ABP-ICA blood velocity, ABP-MCA velocities in 0.005-0.08 Hz frequency interval was calculated as a measure of CA. Passive tilting from 30° to 60° resulted in 12% decrease in CO (p = 0.001); ICA blood flow tended to fall (p = 0.04); Resistance breathing restored CO and ICA blood flow despite a 10% ETCO2 drop. ETCO2 and CO contributed to ICA blood flow variance (adjusted R2: 0.9, p < 0.0001). The median SI was low (<0.2) indicating intact CA, confirmed by surrogate date testing. The peak SI was transiently elevated during resistance breathing in the 60° position. Resistance breathing may transiently reduce CA efficiency. Paced breathing did not restore CO or ICA blood flow.

Gram-negative postpasteurization contamination patterns of single-serve fluid milk produced in 4 different processing facilities.

An analysis of historic data on high temperature, short time (HTST) fluid milk quality showed higher total bacterial counts and lower sensory defect judging scores at d 14 postprocessing for milk packaged in single-serve containers as compared with milk packaged in half-gallon containers from the same processing facilities. As postpasteurization contamination with gram-negative bacteria is likely a major contributor to an increased spoilage risk associated with milk packaged in single-serve containers, we performed a comprehensive assessment of the microbial quality and shelf life of 265 commingled single-serve HTST fluid milk samples (including white [unflavored] skim, white [unflavored] 1%, chocolate skim, and chocolate 1%) collected over 2 visits to 4 commercial fluid milk processing facilities. Over 2 initial sampling visits, the frequency of gram-negative spoilage ranged from 14 to 79% of the product collected from the 4 facilities, with significant differences of gram-negative spoilage frequency between sampling visits, facilities (sampling visit 1, sampling visit 2, and both sampling visits combined), milk types (sampling visit 2), and filler lanes (sampling visit 2). We found no significant differences in the frequency of gram-negative spoilage between sampling time points (e.g., beginning, middle, and end of production run). Across facilities, single-serve containers of milk with gram-negative contamination showed significantly higher bacterial counts on d 7 and 14 and significantly lower sensory scores as compared with those without gram-negative contamination. Follow-up investigations, based on in-facility surveys that identified carton forming mandrels as filler components that frequently failed quality assurance ATP swab checks, found that bacterial genera, including Pseudomonas and Bacillus, isolated from single-serve milk samples were also frequently isolated from mandrels. Although interventions aimed at improving cleaning and sanitation of mandrels did not lead to significant reduction of gram-negative spoilage frequency in a comparison of 398 control and 400 intervention samples, our data still suggest that the unhygienic design of single-serve fillers is likely a root cause of gram-negative contamination of single-serve milk.

Microbacterium represents an emerging microorganism of concern in microfiltered extended shelf-life milk products.

Growing interest in the manufacture of extended shelf-life (ESL) milk, which is typically achieved by a high-temperature treatment called ultra-pasteurization (UP), is driven by distribution challenges, efforts to reduce food waste, and more. Even though high-temperature, short-time (HTST) pasteurized milk has a substantially shorter shelf life than UP milk, HTST milk is preferred in the United States because consumers tend to perceive UP milk as less desirable due to the "cooked" flavor associated with high-temperature processing. While ESL beyond 21 d may be possible for HTST, the survival and outgrowth of psychrotolerant aerobic spore-forming bacteria can still be a limitation to extending shelf life of HTST milk. Microfiltration (MF) is effective for reducing vegetative microorganisms and spores in raw milk, but it is unclear what the effects of membrane pore size, storage temperature, and milk type (i.e., skim vs. whole) are on the microbial shelf life of milk processed by both MF and HTST pasteurization. To investigate these factors, raw skim milk was MF using different pore sizes (0.8 or 1.2 µm), and then MF skim milk and standardized whole milk (MF skim with heat-treated [85°C for 20 s] cream) were HTST pasteurized at 75°C for 20 s. Subsequently, milk was stored at 3°C, 6.5°C, or 10°C and total bacteria counts were measured for up to 63 d. An ANOVA indicated that mean bacterial concentrations between storage temperatures were significantly different from each other, with mean maximum observed concentrations of 3.67, 5.33, and 8.08 log10 cfu/mL for storage temperatures 3°C, 6.5°C, and 10°C, respectively. Additionally, a smaller difference in mean maximum bacterial concentrations throughout shelf life was identified between pore sizes (<1 log cfu/mL), but no significant difference was attributed to milk type. An unexpected outcome of this study was the identification of Microbacterium as a major contributor to the bacterial population in MF ESL milk. Microbacterium is a psychrotolerant, thermoduric gram-positive, non-spore-forming rod with a small cell size (∼0.9 µm length and ∼0.3 µm width), which our data suggest was able to permeate the membranes used in this study, survive HTST pasteurization, and then grow at refrigeration temperatures. While spores continue to be a key concern for the manufacture of MF, ESL milk, our study demonstrates the importance of other psychrotolerant, thermoduric bacteria such as Microbacterium to these products.

Development and deployment of a supply-chain digital tool to predict fluid-milk spoilage due to psychrotolerant sporeformers.

Psychrotolerant sporeformers pose a challenge to maintaining fluid milk quality. Dynamic temperature changes along the supply chain can favor the germination and growth of these bacteria and lead to fluid milk spoilage. In this study, we aim to expand on our previous work on predicting milk spoilage due to psychrotolerant sporeformers. The key model innovations include (1) the ability to account for changing temperatures along the supply chain, and (2) a deployed user-friendly interface to allow easy access to the model. Using the frequencies and concentrations of 8 Bacillales subtypes specific to fluid milk collected in New York, the model simulated sporeformer growth in half-gallons of high-temperature, short-time (HTST) pasteurized fluid milk transported from processing facility to retail store and then to consumer. The Monte Carlo simulations predicted that 44.3% of half-gallons of milk were spoiled (defined as having a bacterial concentration >20,000 cfu/mL, a conservative estimate that represents the Pasteurized Milk Ordinance regulatory limit) after 21 d of refrigerated storage at consumer's home. Model validations showed that the model was the most accurate in predicting the mean sporeformer concentration at low temperatures (i.e., at 3°C and 4°C; compared with higher temperatures at 6°C and 10°C) within the first 21 d of consumer storage, with a root mean square error of 0.29 and 0.34 log10 cfu/mL, respectively. Global sensitivity analyses indicated that home storage temperature, facility-to-retail transportation temperature, and initial spore concentration were the 3 most influential factors for predicting milk spoilage on d 21 of shelf life. What-if scenarios indicated that microfiltration was predicted to be the most effective strategy to reduce spoilage. The implementation of this strategy (assumed to reduce initial spore concentration by 2.2 log10 cfu/mL) was predicted to reduce the percentage of spoiled milk by 17.0 percentage points on d 21 of storage and could delay the date by which 50% of half-gallons of milk were spoiled, from d 25 to 35. Overall, the model is readily deployed as a digital tool for assessing fluid milk spoilage along the supply chain and evaluating the effectiveness of intervention strategies, including those that target storage temperatures at different supply chain stages.

Consumer willingness to pay for shelf life of high-temperature, short-time-pasteurized fluid milk: Implications for smart labeling and food waste reduction.

Food waste in the United States was valued at $285 billion in 2019, representing 70% of all food surplus; dairy and eggs alone represented 15.90% of food surplus. Milk is the fifth most consumed beverage in the United States, and therefore its contribution to food waste has significant economic and environmental ramifications. Smart labels that provide precise spoilage information for fluid milk may help reduce food waste in fluid milk, but it is unclear if consumers will accept or pay for this novel technology. This paper examines consumer preferences for high temperature, short time pasteurized fluid milk shelf life and smart date labels and tests how information about the environmental impact of fluid milk food waste affects consumers' acceptance and willingness to pay. We used a choice-based conjoint study administered in an online survey, along with a between-subject experiment to measure preferences under different information treatments about the environmental impact of food waste. Our results suggest that consumers' valuations of extended shelf life and an ecolabel is positive; however, using the smart label creates disutility for consumers, thereby hindering acceptance of new labeling technology that may facilitate food waste reduction in the milk industry. These findings imply that retailers should find alternative means to enhance the communication of precise shelf life information and its role in reducing food waste.

Economic and environmental analysis of processing plant interventions to reduce fluid milk waste.

With the increased awareness about the economic and environmental impact of food waste, many interventions along food supply chains have been proposed to mitigate food waste. Even though interventions used to target food waste usually revolve around logistics and operations management, we highlight a unique solution to address this issue, specifically for fluid milk. We target the intrinsic quality of fluid milk by evaluating interventions that will extend the product shelf life. We used data from a previous fluid milk spoilage simulation model, collected price and product information from retail stores, conducted an expert elicitation, and used hedonic price regressions to determine the private and social gains to the dairy processing plant when implementing 5 different interventions to extend shelf life. Our data suggest that the value of each additional day of shelf life is approximately $0.03 and indicate that increasing periodic equipment cleaning is the most cost-effective strategy for processing plants to achieve fluid milk shelf-life improvements, both from a firm's economic standpoint and from an environmental standpoint. Importantly, the approaches reported here will be valuable to help individual firms to generate customized facility and firm specific assessments that identify the most appropriate strategies for extending the shelf life of different dairy products.

Shelf-life storage temperature has a considerably larger effect than high-temperature, short-time pasteurization temperature on the growth of spore-forming bacteria in fluid milk.

In the absence of postpasteurization contamination, psychrotolerant, aerobic spore-forming bacteria that survive high-temperature, short-time (HTST) pasteurization, limit the ability to achieve HTST extended shelf-life milk. Therefore, the goal of the current study was to evaluate bacterial outgrowth in milk pasteurized at different temperatures (75, 85, or 90°C, each for 20 s) and subsequently stored at 3, 6.5, or 10°C. An initial ANOVA of bacterial concentrations over 14 d of storage revealed a highly significant effect of storage temperatures, but no significant effect of HTST. At d 14, average bacterial counts for milk stored at 3, 6.5, and 10°C were 1.82, 3.55, and 6.86 log10 cfu/mL, respectively. Time to reach 1,000,000 cfu/mL (a bacterial concentration where consumers begin to notice microbially induced sensory defects in fluid milk) was estimated to be 68, 27, and 10 d for milk stored at 3, 6.5, and 10°C, respectively. Out of 95 isolates characterized with rpoB allelic typing, 6 unique genera, 15 unique species, and 44 unique rpoB allelic types were represented. The most common genera identified were Paenibacillus, Bacillus, and Lysinibacillus. Nonmetric multidimensional scaling identified that Bacillus was significantly associated with 3 and 10°C, whereas Paenibacillus was consistently found across all storage temperatures. Overall, our data show that storage temperature has a substantially larger effect on fluid milk shelf life than HTST and suggests that abuse temperatures (e.g., storage at 10°C) allow for growth of Bacillus species (including Bacillus cereus genomospecies) that do not grow at lower temperatures. This indicates that stringent control of storage and distribution temperatures is critical for producing extended shelf-life HTST milk, particularly concerning new distribution pathways for HTST pasteurized milk (e.g., electronic commerce), and when enhanced control of spores in raw milk is not feasible.

Butyric acid-producing bacterial spore levels in conventional raw milk vary by farm.

Anaerobic butyric acid-producing sporeforming bacteria (BAB) are important microbial contaminants in raw milk that may lead to premature spoilage of certain cheeses during aging. A study was conducted to determine the baseline levels of these spores in raw milk from 7 conventional Northeast United States dairy farms over a 1-yr period. The overall mean BAB concentration was 1.79 log10 most probable number per liter with spore levels differing significantly by farm. A post-hoc farm management practices survey was conducted to determine if there was an association between farm practices on BAB levels in raw milk from these farms. Survey questions included variables related to bedding, milking preparation procedures, teat and udder cleanliness scoring, holding area cleaning procedures, and udder clipping or flaming frequency. Each variable was fitted with a linear mixed-effects model, which revealed no significant association between farm-level factors and the initial BAB concentrations in raw milk; this finding was likely due to the small sample size in this study. To demonstrate the usefulness of our data beyond the initial baseline levels of BAB spores in raw milk, we used this data set to calculate minimum number of individual samples that would be needed to be collected in future studies, which was determined to be 96 to 126 samples, to evaluate the association between farm-level factors and BAB spore concentrations in raw milk. Overall, this study provides dairy industry stakeholders with baseline data on BAB spore levels in raw milk, along with a demonstration on how these data could be used in future studies to calculate sample sizes needed to assess the effect of farm management practices on BAB levels in raw milk.

Invited review: Redefining raw milk quality-Evaluation of raw milk microbiological parameters to ensure high-quality processed dairy products.

Raw milk typically has little bacterial contamination as it leaves the udder of the animal; however, through a variety of pathways, it can become contaminated with bacteria originating from environmental sources, the cow herself, and contact with contaminated equipment. Although the types of bacteria found in raw milk are very diverse, select groups are particularly important from the perspective of finished product quality. In particular, psychrophilic and psychrotolerant bacteria that grow quickly at low temperatures (e.g., species in the genus Pseudomonas and the family Enterobacteriaceae) and produce heat-stable enzymes, and sporeforming bacteria that survive processing hurdles in spore form, are the 2 primary groups of bacteria related to effects on processed dairy products. Understanding factors leading to the presence of these important bacterial groups in raw milk is key to reducing their influence on processed dairy product quality. Here we examine the raw milk microbiological parameters used in the contemporary dairy industry for their utility in identifying raw milk supplies that will perform well in processed dairy products. We further recommend the use of a single microbiological indicator of raw milk quality, namely the total bacteria count, and call for the development of a whole-farm approach to raw milk quality that will use data-driven, risk-based tools integrated across the continuum from production to processing and shelf-life to ensure continuous improvement in dairy product quality.

How Can AI Help Improve Food Safety?

With advances in artificial intelligence (AI) technologies, the development and implementation of digital food systems are becoming increasingly possible. There is tremendous interest in using different AI applications, such as machine learning models, natural language processing, and computer vision to improve food safety. Possible AI applications are broad and include, but are not limited to, (a) food safety risk prediction and monitoring as well as food safety optimization throughout the supply chain, (b) improved public health systems (e.g., by providing early warning of outbreaks and source attribution), and (c) detection, identification, and characterization of foodborne pathogens. However, AI technologies in food safety lag behind in commercial development because of obstacles such as limited data sharing and limited collaborative research and development efforts. Future actions should be directed toward applying data privacy protection methods, improving data standardization, and developing a collaborative ecosystem to drive innovations in AI applications to food safety.

Consumer perceptions of QR code technology for enhanced fluid milk shelf-life information provision in a retail setting.

There is increasing awareness of the impact of food waste and the large role best-by or sell-by dates play in consumer food waste. To address this issue, predictive models have been developed that can not only provide more accurate best-by dates for fluid milk but could also be used to dynamically predict shelf-life (e.g., based on distribution data such as storage temperatures) and adjust prices for products closer to the end of shelf-life. However, limited information is available on strategies to communicate this type of information to consumers. Here we assessed the consumer acceptance of (1) quick response (QR) code technology to communicate product shelf-life and (2) shelf-life dependent pricing based on QR codes by offering both half-gallon fluid milk with traditional printed best-by dates and identical products with QR codes to convey best-by dates over an 8-wk time period in a retail setting. Overall, 62% of half-gallon containers sold over this time frame featured QR codes and 48% of QR code scans were linked to subsequent sales, suggesting the possibility of substantial consumer acceptance of novel technologies to display and communicate best-by dates. Preliminary data based on a small number of sales also showed that consumers did purchase QR code-labeled products offered at a reduced price due to limited remaining shelf-life. Our data suggest that at least some consumer segments would adopt QR code-based shelf-life labels, which presents an opportunity to better manage and communicate "best-by" dates and use dynamic pricing strategies to reduce food waste that occurs when an end-of-shelf-life product is either not sold or is discarded by consumers. Overall, QR codes represent a strategic opportunity for the dairy industry to achieve greater sustainability and to foster stronger connections with customers through enhanced provision of information that highlights sustainability practices implemented across the whole supply chain.

Development of a risk assessment model to predict the occurrence of late blowing defect in Gouda cheese and evaluate potential intervention strategies.

Late blowing defect (LBD) is an important spoilage issue in semi-hard cheese, with the outgrowth of Clostridium tyrobutyricum spores during cheese aging considered to be the primary cause. Although previous studies have explored the microbial and physicochemical factors influencing the defect, a risk assessment tool that allows for improved and rational management of LBD is lacking. The purpose of this study was to develop a predictive model to estimate the probability of LBD in Gouda cheese and evaluate different intervention strategies. The spore concentration distribution of butyric acid bacteria (BAB) in bulk tank milk was obtained from 8 dairy farms over 12 mo. The concentration of C. tyrobutyricum from raw milk to the end of aging was simulated based on Gouda brined for 2 d in saturated brine at 8°C and aged at 13°C. Predicted C. tyrobutyricum concentrations during aging and estimated concentration thresholds in cheese at onset of LBD were used to predict product loss due to LBD during a simulated 1-yr production. With the estimated concentration thresholds in cheese ranging from 4.36 to 4.46 log most probable number (MPN)/kg of cheese, the model predicted that 9.2% (±1.7%) of Gouda cheese showed LBD by d 60; cheeses predicted to show LBD at d 60 showed a mean pH of 5.39 and were produced with raw milk with a mean BAB spore count of 143 MPN/L. By d 90, 36.1% (±3.4%) of cheeses were predicted to show LBD, indicating that LBD typically manifests between d 60 and 90, which is consistent with observations from the literature and the cheese industry. Sensitivity analysis indicated that C. tyrobutyricum maximum growth rate as well as concentration threshold in cheese at onset of LBD are the most important variables, identifying key data needs for development of more accurate models. The implementation of microfiltration or bactofugation of raw milk (assumed to show 98% efficiency of spore removal) in our model prevented occurrence of LBD during the first 60 d of aging. Overall, our findings provide a framework for predicting the occurrence of LBD in Gouda as well as other cheeses and illustrate the value of developing digital tools for managing dairy product quality.

Development of a Monte Carlo simulation model to predict pasteurized fluid milk spoilage due to post-pasteurization contamination with gram-negative bacteria.

Psychrotolerant gram-negative bacteria introduced as post-pasteurization contamination (PPC) are a major cause of spoilage and reduced shelf life of high-temperature, short-time pasteurized fluid milk. To provide improved tools to (1) predict pasteurized fluid milk shelf life as influenced by PPC and (2) assess the effectiveness of different potential interventions that could reduce spoilage due to PPC, we developed a Monte Carlo simulation model that predicts fluid milk spoilage due to psychrotolerant gram-negative bacteria introduced as PPC. As a first step, 17 gram-negative bacterial isolates frequently associated with fluid milk spoilage were selected and used to generate growth data in skim milk broth at 6°C. The resulting growth parameters, frequency of isolation for the 17 different isolates, and initial concentration of bacteria in milk with PPC, were used to develop a Monte Carlo model to predict bacterial number at different days of shelf life based on storage temperature of milk. This model was then validated with data from d 7 and 10 of shelf life, collected from commercial operations. The validated model predicted that the parameters (1) maximum growth rate and (2) storage temperature had the greatest influence on the percentage of containers exceeding 20,000 cfu/mL standard plate count on d 7 and 10 (i.e., spoiling due to PPC), which indicates that accurate data on maximum growth rate and storage temperature are important for accurate predictions. In addition to allowing for prediction of fluid milk shelf life, the model allows for simulation of "what-if" scenarios, which allowed us to predict the effectiveness of different interventions to reduce overall fluid milk spoilage due to PPC through a set of proof-of-concept scenario (e.g., frequency of PPC in containers reduced from 100% to 10%; limiting distribution temperature to a maximum of 6°C). Combined with other models, such as previous models on fluid milk spoilage due to psychrotolerant spore-forming bacteria, the data and tools developed here will allow for rational, digitally enabled, fluid milk shelf life prediction and quality enhancement.

Phylogeographic Clustering Suggests that Distinct Clades of Salmonella enterica Serovar Mississippi Are Endemic in Australia, the United Kingdom, and the United States.

Salmonella enterica serovar Mississippi is the 2nd and 14th leading cause of human clinical salmonellosis in the Australian island state of Tasmania and the United States, respectively. Despite its public health relevance, relatively little is known about this serovar. Comparison of whole-genome sequence (WGS) data of S. Mississippi isolates with WGS data for 317 additional S. enterica serovars placed one clade of S. Mississippi within S. enterica clade B ("clade B Mississippi") and the other within section Typhi in S. enterica clade A ("clade A Mississippi"), suggesting that these clades evolved from different ancestors. Phylogenetic analysis of 364 S. Mississippi isolates from Australia, the United Kingdom, and the United States suggested that the isolates cluster geographically, with U.S. and Australian isolates representing different subclades (Ai and Aii, respectively) within clade A Mississippi and clade B isolates representing the predominant S. Mississippi isolates in the United Kingdom. Intraclade comparisons suggested that different mobile elements, some of which encode virulence factors, are responsible for the observed differences in gene content among isolates within these clades. Specifically, genetic differences among clade A isolates reflect differences in prophage contents, while differences among clade B isolates are due to the acquisition of a 47.1-kb integrative conjugative element (ICE). Phylogenies inferred from antigenic components (fliC, fljB, and O-antigen-processing genes) support that clade A and B Mississippi isolates acquired these loci from different ancestral serovars. Overall, these data support that different S. Mississippi phylogenetic clades are endemic in Australia, the United Kingdom, and the United States. IMPORTANCE The number of known so-called "polyphyletic" serovars (i.e., phylogenetically distinct clades with the same O and H antigenic formulas) continues to increase as additional Salmonella isolates are sequenced. While serotyping remains a valuable tool for reporting and monitoring Salmonella, more discriminatory analyses for classifying polyphyletic serovars may improve surveillance efforts for these serovars, as we found that for S. Mississippi, distinct genotypes predominate at different geographic locations. Our results suggest that the acquisition of genes encoding O and H antigens from different ancestors led to the emergence of two Mississippi clades. Furthermore, our results suggest that different mobile elements contribute to the microevolution and diversification of isolates within these two clades, which has implications for the acquisition of novel adaptations, such as virulence factors.

Identification, subtyping, and tracking of dairy spoilage-associated Pseudomonas by sequencing the ileS gene.

Pseudomonas spp. are important spoilage bacteria that negatively affect the quality of refrigerated fluid milk and uncultured cheese by generating unwanted odors, flavors, and pigments. They are frequently found in dairy plant environments and enter dairy products predominantly as postpasteurization contaminants. Current subtyping and characterization methods for dairy-associated Pseudomonas are often labor-intensive and expensive or provide limited and possibly unreliable classification information (e.g., to the species level). Our goal was to identify a single-copy gene that could be analyzed in dairy spoilage-associated Pseudomonas for preliminary species-level identification, subtyping, and phenotype prediction. We tested 7 genes previously targeted in a Pseudomonas fluorescens multilocus sequence typing scheme for their individual suitability in this application using a set of 113 Pseudomonas spp. isolates representing the diversity of typical pasteurized milk contamination. For each of the 7 candidate genes, we determined the success rate of PCR and sequencing for these 113 isolates as well as the level of discrimination for species identification and subtyping that the sequence data provided. Using these metrics, we selected a single gene, isoleucyl tRNA synthetase (ileS), which had the most suitable traits for simple and affordable single-gene Pseudomonas characterization. This was based on the number of isolates successfully sequenced for ileS (113/113), the number of unique allelic types assigned (83, compared with 50 for 16S rDNA), nucleotide and sequence diversity measures (e.g., number of unique SNP and Simpson index), and tests for genetic recombination. The discriminatory ability of ileS sequencing was confirmed by separation of 99 additional dairy Pseudomonas spp. isolates, which were indistinguishable by 16S rDNA sequencing, into 28 different ileS allelic types. Further, we used whole-genome sequencing data to demonstrate the similarities in ileS-based phylogenetic clustering to whole-genome-based clustering for 27 closely related dairy-associated Pseudomonas spp. isolates and for 178 Pseudomonas type strains. We also found that dairy-associated Pseudomonas within an ileS cluster typically shared the same proteolytic and lipolytic activities. Use of ileS sequencing provides a promising strategy for affordable initial characterization of Pseudomonas isolates, which will help the dairy industry identify, characterize, and track Pseudomonas in their facilities and products.

The Majority of Typhoid Toxin-Positive Salmonella Serovars Encode ArtB, an Alternate Binding Subunit.

Salmonella enterica encodes a wide array of virulence factors. One novel virulence factor, an A2B5 toxin known as the typhoid toxin (TT), was recently identified among a variety of S. enterica serovars. While past studies have shown that some serovars encode both the TT (active subunits CdtB and PltA and binding subunit PltB) and a second binding subunit (ArtB), these serovars were thought to be the exception. Here, we show that genes encoding the TT are detected in more than 100 serovars representing distinct phylogenetic lineages of S. enterica subsp. enterica, although clade B and section Typhi are significantly more likely to encode TT genes than serovars from other clades. Furthermore, we show that 81% of these TT-positive serovars also encode artB, suggesting that the cooccurrence of both toxin binding subunits is considerably more common than previously thought. A combination of in silico modeling, bacterial two-hybrid system screening, and tandem affinity purification (TAP) of toxin subunits suggests that ArtB and PltB interact in vitro, at least under some growth conditions. While different growth conditions yielded slightly higher transcript abundances of artB and pltB, both genes had their highest relative transcript abundances when Salmonella was grown under low-Mg2+ conditions, suggesting that ArtB and PltB may compete for inclusion in the TT. Together, our results suggest that ArtB likely plays an important and previously underappreciated role in the biology of the TT produced by typhoidal and nontyphoidal SalmonellaIMPORTANCE While previous reports had suggested that the typhoid toxin (TT) could potentially use ArtB as an alternate binding subunit, this was thought to play a minor role in the evolution and biology of the toxin. In this study, we establish that both TT genes and artB are widespread among Salmonella enterica subsp. enterica, suggesting that TT likely plays a broader role in Salmonella virulence that extends beyond its proposed role in typhoid fever. Furthermore, our data suggest the selective maintenance of both toxin binding subunits, which may compete for inclusion in the holotoxin. Last, our data support the importance of characterizing diverse nontyphoidal Salmonella (NTS) serovars, as the presence of classically defined typhoidal virulence factors among NTS serovars continues to challenge the typhoid-nontyphoid Salmonella paradigm.

A standard set of testing methods reliably enumerates spores across commercial milk powders.

Contamination of dairy powders with sporeforming bacteria is a concern for dairy processors who wish to penetrate markets with stringent spore count specifications (e.g., infant powders). Despite instituted specifications, no standard methodology is used for spore testing across the dairy industry. Instead, a variety of spore enumeration methods are in use, varying primarily by heat-shock treatments, plating method, recovery medium, and incubation temperature. Importantly, testing the same product using different methodologies leads to differences in spore count outcomes, which is a major issue for those required to meet specifications. As such, we set out to identify method(s) to recommend for standardized milk powder spore testing. To this end, 10 commercial milk powders were evaluated using methods varying by (1) heat treatment (e.g., 80°C/12 min), (2) plating method (e.g., spread plating), (3) medium type (e.g., plate count milk agar), and (4) incubation time and temperature combinations (e.g., 32°C for 48 h). The resulting data set included a total of 48 methods. With this data set, we used a stepwise process to identify optimal method(s) that would explain a high proportion of variance in spore count outcomes and would be practical to implement across the dairy industry. Ultimately, spore pasteurized mesophilic spore count (80°C/12 min, incubated at 32°C for 48 h), highly heat resistant thermophilic spore count (100°C/30 min, incubated at 55°C for 48 h), and specially thermoresistant spore enumeration (106°C/30 min, incubated at 55°C for 48 h) spread plating on plate count milk agar were identified as the optimal method set for reliable enumeration of spores in milk powders. Subsequently, we assessed different powder sampling strategies as a way to reduce variation in powder spore testing outcomes using our recommended method set. Results indicated that 33-g composite sampling may reduce variation in spore testing outcomes for highly heat resistant thermophilic spore count over 11-g and 33-g discrete sampling, whereas there was no significant difference across sampling strategies for specially thermoresistant spore enumeration or spore pasteurized mesophilic spore count. Finally, an interlaboratory study using our recommended method set and a modified method set (using tryptic soy agar with 1% starch) among both university and industry laboratories showed increased variation in spore count outcomes within milk powders, which not only was due to natural variation in powders but also was hypothesized to be due to technical errors, highlighting the need for specialized training for technicians who perform spore testing on milk powders. Overall, this study addresses challenges to milk powder spore testing and recommends a method set for standardized spore testing for implementation across the dairy industry.

Invited review: Controlling dairy product spoilage to reduce food loss and waste.

Food loss and waste is a major concern in the United States and globally, with dairy foods representing one of the top categories of food lost and wasted. Estimates indicate that in the United States, approximately a quarter of dairy products are lost at the production level or wasted at the retail or consumer level annually. Premature microbial spoilage of dairy products, including fluid milk, cheese, and cultured products, is a primary contributor to dairy food waste. Microbial contamination may occur at various points throughout the production and processing continuum and includes organisms such as gram-negative bacteria (e.g., Pseudomonas), gram-positive bacteria (e.g., Paenibacillus), and a wide range of fungal organisms. These organisms grow at refrigerated storage temperatures, often rapidly, and create various degradative enzymes that result in off-odors, flavors, and body defects (e.g., coagulation), rendering them inedible. Reducing premature dairy food spoilage will in turn reduce waste throughout the dairy continuum. Strategies to reduce premature spoilage include reducing raw material contamination on-farm, physically removing microbial contaminants, employing biocontrol agents to reduce outgrowth of microbial contaminants, tracking and eliminating microbial contaminants using advanced molecular microbiological techniques, and others. This review will address the primary microbial causes of premature dairy product spoilage and methods of controlling this spoilage to reduce loss and waste in dairy products.

Transcriptional profiling of the L. monocytogenes PrfA regulon identifies six novel putative PrfA-regulated genes.

The transcriptional activator Positive Regulatory Factor A (PrfA) regulates expression of genes essential for virulence in Listeria monocytogenes. To define the PrfA regulon, the 10403S wildtype (WT) strain, a constitutively active prfA* mutant, and an isogenic ∆prfA mutant were grown under PrfA-inducing conditions in a medium containing glucose-1-phosphate and pre-treated with 0.2% activated charcoal. RNA-seq-generated transcript levels were compared as follows: (i) prfA* and WT; (ii) WT and ∆prfA and (iii) prfA* and ∆prfA. Significantly higher transcript levels in the induced WT or constitutively active PrfA* were identified for 18 genes and 2 ncRNAs in at least one of the three comparisons. These genes included: (i) 10/12 of the genes previously identified as directly PrfA-regulated; (ii) 2 genes previously identified as PrfA-regulated, albeit likely indirectly; and (iii) 6 genes newly identified as PrfA-regulated, including one (LMRG_0 2046) with a σA-dependent promoter and PrfA box located within an upstream open reading frame. LMRG_0 2046, which encodes a putative cyanate permease, is reported to be downregulated by a σB-dependent anti-sense RNA. This newly identified overlap between the σB and PrfA regulons highlights the complexity of regulatory networks important for fine-tuning bacterial gene expression in response to the rapidly changing environmental conditions associated with infection.

Interventions designed to control postpasteurization contamination in high-temperature, short-time-pasteurized fluid milk processing facilities: A case study on the effect of employee training, clean-in-place chemical modification, and preventive maintenance programs.

Postpasteurization contamination (PPC) with gram-negative bacteria adversely affects the quality and shelf-life of milk through the development of flavor, odor, texture, and visual defects. Through evaluation of milk quality at 4 large fluid milk processing facilities in the northeast United States, we examined the efficacy of 3 strategies designed to reduce the occurrence of PPC in fluid milk: (1) employee training (focusing on good manufacturing practices) alone and (2) with concurrent implementation of modified clean-in-place chemistry and (3) preventive maintenance (PM) focused on replacement of wearable rubber components. Despite increases in employee knowledge and self-reported behavior change, microbiological evaluation of fluid milk before and after interventions indicated that neither training alone nor training combined with modified clean-in-place interventions significantly decreased PPC. Furthermore, characterization of gram-negative bacterial isolates from milk suggested that specific bacterial taxonomic groups (notably, Pseudomonas sequence types) continued to contribute to PPC even after interventions and that no major changes in the composition of the spoilage-associated microbial populations occurred as a consequence of the interventions. More specifically, in 3 of 4 facilities, gram-negative bacteria with identical 16S rDNA sequence types were isolated on multiple occasions. Evaluation of a PM intervention showed that used rubber goods harbored PPC-associated bacteria and that PPC may have been less frequent following a PM intervention in which wearable rubber goods were replaced (reduction from 3/3 samples with PPC before to 1/3 samples after). Overall, our findings suggest that commonly used "broad stroke interventions" may have a limited effect on reducing PPC. Our case study also demonstrates the inherent complexities of identifying and successfully addressing sanitation problems in large and complex fluid milk processing facilities. For example, broad changes to sanitation practices without improvements in PM and sanitary equipment design may not always lead to reduced PPC. Our data also indicate that although short-term evaluations, such as pre- and post-tests for employee training, may suggest improvements after corrective and preventive actions, extensive microbial testing, ideally in combination with isolate characterization, may be necessary to evaluate return on investment of different interventions.