Machine Learning and Advanced Statistical Modeling Can Identify Key Quality Management Practices That Affect Postpasteurization Contamination of Fluid Milk.

ABSTRACT: Spoilage of high-temperature, short-time (HTST)- and vat-pasteurized fluid milk due to the introduction of gram-negative bacteria postpasteurization remains a challenge for the dairy industry. Although processing facility-level practices (e.g., sanitation practices) are known to impact the frequency of postpasteurization contamination (PPC), the relative importance of different practices is not well defined, thereby affecting the ability of facilities to select intervention targets that reduce PPC and provide the greatest return on investment. Thus, the goal of this study was to use an existing longitudinal data set of bacterial spoilage indicators obtained for pasteurized fluid milk samples collected from 23 processing facilities between July 2015 and November 2017 (with three to five samplings per facility) and data from a survey on fluid milk quality management practices, to identify factors associated with PPC and rank their relative importance. This ranking was accomplished using two separate approaches: multimodel inference and conditional random forest. Data preprocessing for multimodel inference analysis showed (i) nearly all factors were significantly associated with PPC when assessed individually using univariable logistic regression and (ii) numerous pairs of factors were strongly associated with each other (Cramer's V ≥ 0.80). Multimodel inference and conditional random forest analyses identified similar drivers associated with PPC; factors identified as most important based on these analyses included cleaning and sanitation practices, activities related to good manufacturing practices, container type (a proxy for different filling equipment), in-house finished product testing, and designation of a quality department, indicating potential targets for reducing PPC. In addition, this study illustrates how machine learning approaches can be used with highly correlated and unbalanced data, as typical for food safety and quality, to facilitate improved data analyses and decision making.

A practical training program for fluid milk defect judging should focus on initial training of panelists.

The sensory quality of fluid milk is of great importance to processors and consumers. Defects in the expected odor, flavor, or body of the product can affect consumer attitudes toward the product and, ultimately, willingness to purchase the product. Although many methods of sensory evaluation have been developed, defect judging is one particular method that has been used for decades in the dairy industry for evaluating fluid milk. Defect judging is a technique whereby panelists are trained to recognize and rate a standard set of fluid milk defects that originate from various sources (e.g., microbial spoilage). This technique is primarily used in processing facilities where identification of sensory defects can alert personnel to potential quality control issues in raw material quality, processing, or good manufacturing practices. In 2014-2016, a preliminary study of defective milk judging screening and training was conducted by the Milk Quality Improvement Program at Cornell University (Ithaca, NY). The study, which included 37 staff and students from the Cornell community, used prescreenings for common odors and basic tastes, followed by uniform training to select, initially train, and retrain defect judges of unflavored high temperature, short time fluid milk. Significant improvements were seen in correct identification of defect attributes following initial training for all defect attributes, with the exception of fruity/fermented. However, following retraining, significant improvements were observed in only 2 defect attributes: cooked and milk carton. These results demonstrate that initial training is important for panelists to correctly identify fluid milk defect attributes, but that subsequent retraining should be tailored toward specific attributes. This study provides a resource for dairy industry stakeholders to use to develop relevant and efficient training methods for fluid milk defect judging panels.

Cross Talk between SigB and PrfA in Listeria monocytogenes Facilitates Transitions between Extra- and Intracellular Environments.

The foodborne pathogen Listeria monocytogenes can modulate its transcriptome and proteome to ensure its survival during transmission through vastly differing environmental conditions. While L. monocytogenes utilizes a large array of regulators to achieve survival and growth in different intra- and extrahost environments, the alternative sigma factor σB and the transcriptional activator of virulence genes protein PrfA are two key transcriptional regulators essential for responding to environmental stress conditions and for host infection. Importantly, emerging evidence suggests that the shift from extrahost environments to the host gastrointestinal tract and, subsequently, to intracellular environments requires regulatory interplay between σB and PrfA at transcriptional, posttranscriptional, and protein activity levels. Here, we review the current evidence for cross talk and interplay between σB and PrfA and their respective regulons and highlight the plasticity of σB and PrfA cross talk and the role of this cross talk in facilitating successful transition of L. monocytogenes from diverse extrahost to diverse extra- and intracellular host environments.

Systematic review of the Listeria monocytogenes σB regulon supports a role in stress response, virulence and metabolism.

Aim: Among the alternative sigma factors of Listeria monocytogenes, σB controls the largest regulon. The aim of this study was to perform a comprehensive review of σB-regulated genes, and the functions they confer. Materials & methods: A systematic search of PubMed and Web of Knowledge was carried out to identify members of the σB regulon based on experimental evidence of σB-dependent transcription and presence of a consensus σB-dependent promoter. Results: The literature review identified σB-dependent transcription units encompassing 304 genes encoding different functions including stress response and virulence. Conclusion: Our review supports the well-known roles of σB in virulence and stress response and provides new insight into novel roles for σB in metabolism and overall resilience of L. monocytogenes.

Scientific Integrity Principles and Best Practices: Recommendations from a Scientific Integrity Consortium.

A Scientific Integrity Consortium developed a set of recommended principles and best practices that can be used broadly across scientific disciplines as a mechanism for consensus on scientific integrity standards and to better equip scientists to operate in a rapidly changing research environment. The two principles that represent the umbrella under which scientific processes should operate are as follows: (1) Foster a culture of integrity in the scientific process. (2) Evidence-based policy interests may have legitimate roles to play in influencing aspects of the research process, but those roles should not interfere with scientific integrity. The nine best practices for instilling scientific integrity in the implementation of these two overarching principles are (1) Require universal training in robust scientific methods, in the use of appropriate experimental design and statistics, and in responsible research practices for scientists at all levels, with the training content regularly updated and presented by qualified scientists. (2) Strengthen scientific integrity oversight and processes throughout the research continuum with a focus on training in ethics and conduct. (3) Encourage reproducibility of research through transparency. (4) Strive to establish open science as the standard operating procedure throughout the scientific enterprise. (5) Develop and implement educational tools to teach communication skills that uphold scientific integrity. (6) Strive to identify ways to further strengthen the peer review process. (7) Encourage scientific journals to publish unanticipated findings that meet standards of quality and scientific integrity. (8) Seek harmonization and implementation among journals of rapid, consistent, and transparent processes for correction and/or retraction of published papers. (9) Design rigorous and comprehensive evaluation criteria that recognize and reward the highest standards of integrity in scientific research.

Emerging needs and opportunities in foodborne disease detection and prevention: From tools to people.

A variety of technological advances have tremendously improved the ability of surveillance systems to detect and prevent foodborne disease cases and outbreaks. Molecular subtyping methods and surveillance systems, including PFGE and, more recently, whole genome sequencing (WGS) have been particularly important advances, but the responsible food vehicle and causative agent are still only conclusively determined in a small fraction of outbreaks. Microbial foodborne disease cases continue to take a considerable public health toll, primarily in developing countries. According to recent WHO estimates, at least 600 million cases of foodborne illness and 420,000 associated deaths occur each year; the true numbers are likely significantly higher. This review summarizes the current and anticipated global impact of improved technologies for foodborne disease surveillance and proposes key areas that will require particular attention, including the need for training activities, public-private partnerships supporting food safety, and appropriate food safety policy frameworks. The manuscript places particular focus on the development of WGS tools for surveillance of Listeria monocytogenes because this technology represents one of the most disruptive food safety technologies introduced over the last 10 years, which has revolutionized routine surveillance of L. monocytogenes in several countries. As such, it provides valuable insights into how technological advances can improve foodborne illness surveillance and illustrates the training, policy and infrastructure needs created by introduction of disruptive novel technologies. Moreover, WGS can help identify new sources of foodborne outbreaks and inform risk assessments, thereby providing valuable insights for risk-based policies aimed at preventing future foodborne illness.

The Listeria monocytogenes Bile Stimulon under Acidic Conditions Is Characterized by Strain-Specific Patterns and the Upregulation of Motility, Cell Wall Modification Functions, and the PrfA Regulon.

Listeria monocytogenes uses a variety of transcriptional regulation strategies to adapt to the extra-host environment, the gastrointestinal tract, and the intracellular host environment. While the alternative sigma factor SigB has been proposed to be a key transcriptional regulator that facilitates L. monocytogenes adaptation to the gastrointestinal environment, the L. monocytogenes' transcriptional response to bile exposure is not well-understood. RNA-seq characterization of the bile stimulon was performed in two L. monocytogenes strains representing lineages I and II. Exposure to bile at pH 5.5 elicited a large transcriptomic response with ~16 and 23% of genes showing differential transcription in 10403S and H7858, respectively. The bile stimulon includes genes involved in motility and cell wall modification mechanisms, as well as genes in the PrfA regulon, which likely facilitate survival during the gastrointestinal stages of infection that follow bile exposure. The fact that bile exposure induced the PrfA regulon, but did not induce further upregulation of the SigB regulon (beyond that expected by exposure to pH 5.5), suggests a model where at the earlier stages of gastrointestinal infection (e.g., acid exposure in the stomach), SigB-dependent gene expression plays an important role. Subsequent exposure to bile induces the PrfA regulon, potentially priming L. monocytogenes for subsequent intracellular infection stages. Some members of the bile stimulon showed lineage- or strain-specific distribution when 27 Listeria genomes were analyzed. Even though sigB null mutants showed increased sensitivity to bile, the SigB regulon was not found to be upregulated in response to bile beyond levels expected by exposure to pH 5.5. Comparison of wildtype and corresponding ΔsigB strains newly identified 26 SigB-dependent genes, all with upstream putative SigB-dependent promoters.

Symposium review: Effect of post-pasteurization contamination on fluid milk quality.

Fluid milk quality in the United States has improved steadily over the last 2 decades, in large part due to the reduction in post-pasteurization contamination (PPC). Despite these improvements, some studies suggest that almost 50% of fluid milk still shows evidence of PPC with organisms that are able to grow at 6°C, even though PPC may be much less frequent in some facilities. Several gram-negative bacteria, when introduced as PPC, can grow rapidly at refrigeration temperatures around 6°C and can lead to bacterial levels above 20,000 cfu/mL (the regulatory limit for bacterial numbers in fluid milk in the United States) and spoilage that can be detected sensorially within 7 to 10 d of processing. Importantly, however, storage temperature can have a considerable effect on microbial growth, and fluid milk stored at 4°C and below may show considerably delayed onset of microbial growth and spoilage compared with samples stored at what may be considered mild abuse (6°C and above). Notable organisms that cause PPC and grow at refrigeration temperatures include psychrotolerant Enterobacteriaceae and coliforms, as well as Pseudomonas. These organisms are known to produce a variety of enzymes that lead to flavor, odor, and body defects that can ultimately affect consumer perception and willingness to buy. Detecting PPC in high temperature, short time, freshly pasteurized fluid milk can be challenging because PPC often occurs sporadically and at low levels. Additionally, indicator organisms typically used in fluid milk (i.e., coliforms) have been shown to represent only a fraction of the total PPC. Recent studies indicate that coliforms account for less than 20% of the total gram-negative organisms introduced into fluid milk after pasteurization. In contrast, Pseudomonas, which is not a coliform and therefore is not detected using coliform media, is the most commonly isolated genus in PPC fluid milk. To reduce PPC, processors must (1) use testing methods that can detect both coliforms and non-coliform gram-negatives (i.e., Pseudomonas) to understand true contamination rates and patterns, and (2) establish cleaning and sanitation protocols and employee and management behaviors that target persistent and transient PPC organisms.

A 100-Year Review: Microbiology and safety of milk handling.

Microbes that may be present in milk can include pathogens, spoilage organisms, organisms that may be conditionally beneficial (e.g., lactic acid bacteria), and those that have not been linked to either beneficial or detrimental effects on product quality or human health. Although milk can contain a full range of organisms classified as microbes (i.e., bacteria, viruses, fungi, and protozoans), with few exceptions (e.g., phages that affect fermentations, fungal spoilage organisms, and, to a lesser extent, the protozoan pathogens Cryptosporidium and Giardia) dairy microbiology to date has focused predominantly on bacteria. Between 1917 and 2017, our understanding of the microbes present in milk and the tools available for studying those microbes have changed dramatically. Improved microbiological tools have enabled enhanced detection of known microbes in milk and dairy products and have facilitated better identification of pathogens and spoilage organisms that were not known or well recognized in the early 20th century. Starting before 1917, gradual introduction and refinement of pasteurization methods throughout the United States and many other parts of the world have improved the safety and quality of milk and dairy products. In parallel to pasteurization, others strategies for reducing microbial contamination throughout the dairy chain (e.g., improved dairy herd health, raw milk tests, clean-in-place technologies) also played an important role in improving microbial milk quality and safety. Despite tremendous advances in reducing microbial food safety hazards and spoilage issues, the dairy industry still faces important challenges, including but not limited to the need for improved science-based strategies for safety of raw milk cheeses, control of postprocessing contamination, and control of sporeforming pathogens and spoilage organisms.

Home Alone: Elimination of All but One Alternative Sigma Factor in Listeria monocytogenes Allows Prediction of New Roles for σB.

Among Listeria monocytogenes' four alternative σ factors, σB controls the largest regulon. As σB-dependent transcription of some genes may be masked by overlaps among regulons, and as some σB-dependent genes are expressed only under very specific conditions, we hypothesized that the σB regulon is not yet fully defined. To further extend our understanding of the σB regulon, we used RNA-seq to identify σB-dependent genes in an L. monocytogenes strain that expresses σB following rhamnose induction, and in which genes encoding the other alternative sigma factors have been deleted. Analysis of RNA-seq data with multiple bioinformatics approaches, including a sliding window method that detects differentially transcribed 5' untranslated regions (UTRs), identified 105 σB-dependent transcription units (TUs) comprising 201 genes preceded by σB-dependent promoters. Of these 105 TUs, 7 TUs comprising 15 genes had not been identified previously as σB-dependent. An additional 23 genes not reported previously as σB-dependent were identified in 9 previously recognized σB-dependent TUs. Overall, 38 of these 201 genes had not been identified previously as members of the L. monocytogenes σB regulon. These newly identified σB-dependent genes encode proteins annotated as being involved in transcriptional regulation, oxidative and osmotic stress response, and in metabolism of energy, carbon and nucleotides. In total, 18 putative σB-dependent promoters were newly identified. Interestingly, a number of genes previously identified as σB-dependent did not show significant evidence for σB-dependent transcription in our experiments. Based on promoter analyses, a number of these genes showed evidence for co-regulation by σB and other transcriptional factors, suggesting that some σB-dependent genes require additional transcriptional regulators along with σB for transcription. Over-expression of a single alternative sigma factor in the absence of all other alternative sigma factors allowed us to: (i) identify new σB-dependent functions in L. monocytogenes, such as regulation of genes involved in 1,2-propanediol utilization (LMRG_00594-LMRG_00611) and biosynthesis of pyrimidine nucleotides (LMRG_00978-LMRG_00985); and (ii) identify new σB-dependent genes involved in stress response and pathogenesis functions. These data further support that σB not only regulates stress response functions, but also plays a broad role in L. monocytogenes homeostasis and resilience.

Short communication: Pseudomonas azotoformans causes gray discoloration in HTST fluid milk.

Pseudomonas species are well recognized as dairy product spoilage organisms, particularly due to their ability to grow at refrigeration temperatures. Although Pseudomonas-related spoilage usually manifests itself in flavor, odor, and texture defects, which are typically due to production of bacterial enzymes, Pseudomonas is also reported to cause color defects. Because of consumer complaints, a commercial dairy company shipped 4 samples of high temperature, short time (HTST)-pasteurized milk with distinctly gray colors to our laboratory. Bacterial isolates from all 4 samples were identified as Pseudomonas azotoformans. All isolates shared the same partial 16S rDNA sequence and showed black pigmentation on Dichloran Rose Bengal Chloramphenicol agar. Inoculation of one pigment-producing P. azotoformans isolate into HTST-pasteurized fluid milk led to development of gray milk after 14 d of storage at 6°C, but only in containers that had half of the total volume filled with milk (∼500 mL of milk in ∼1,000-mL bottles). We conclusively demonstrate that Pseudomonas can cause a color defect in fluid milk that manifests in gray discoloration, adding to the palette of color defects known to be caused by Pseudomonas. This information is of considerable interest to the dairy industry, because dairy processors and others may not typically associate black or gray colors in fluid milk with the presence of microbial contaminants but rather with product tampering (e.g., addition of ink) or other inadvertent chemical contamination.

Stochastic and Differential Activation of σB and PrfA in Listeria monocytogenes at the Single Cell Level under Different Environmental Stress Conditions.

During host infection, the foodborne pathogen Listeria monocytogenes must sense and respond to rapidly changing environmental conditions. Two transcriptional regulators, the alternative sigma factor B (σB) and the Positive Regulatory Factor A (PrfA), are key contributors to the transcriptomic responses that enable bacterial survival in the host gastrointestinal tract and invasion of host duodenal cells. Increases in temperature and osmolarity induce activity of these proteins; such conditions may be encountered in food matrices as well as within the host gastrointestinal tract. Differences in PrfA and σB activity between individual cells might affect the fate of a cell during host invasion, therefore, we hypothesized that PrfA and σB activities differ among individual cells under heat and salt stress. We used fluorescent reporter fusions to determine the relative proportions of cells with active σB or PrfA following exposure to 45°C heat or 4% NaCl. Activities of both PrfA and σB were induced stochastically, with fluorescence levels ranging from below detection to high among individual cells. The proportion of cells with active PrfA was significantly higher than the proportion with active σB under all tested conditions; under some conditions, nearly all cells had active PrfA. Our findings further support the growing body of evidence illustrating the stochastic nature of bacterial gene expression under conditions that are relevant for host invasion via food-borne, oral infection.

The Evolving Role of Coliforms As Indicators of Unhygienic Processing Conditions in Dairy Foods.

Testing for coliforms has a long history in the dairy industry and has helped to identify raw milk and dairy products that may have been exposed to unsanitary conditions. Coliform standards are included in a number of regulatory documents (e.g., the U.S. Food and Drug Administration's Grade "A" Pasteurized Milk Ordinance). As a consequence, detection above a threshold of members of this method-defined, but diverse, group of bacteria can result in a wide range of regulatory outcomes. Coliforms are defined as aerobic or facultatively anaerobic, Gram negative, non-sporeforming rods capable of fermenting lactose to produce gas and acid within 48 h at 32-35°C; 19 genera currently include at least some strains that represent coliforms. Most bacterial genera that comprise the coliform group (e.g., Escherichia, Klebsiella, and Serratia) are within the family Enterobacteriaceae, while at least one genus with strains recognized as coliforms, Aeromonas, is in the family Aeromonadaceae. The presence of coliforms has long been thought to indicate fecal contamination, however, recent discoveries regarding this diverse group of bacteria indicates that only a fraction are fecal in origin, while the majority are environmental contaminants. In the US dairy industry in particular, testing for coliforms as indicators of unsanitary conditions and post-processing contamination is widespread. While coliforms are easily and rapidly detected, and are not found in pasteurized dairy products that have not been exposed to post-processing contamination, advances in knowledge of bacterial populations most commonly associated with post-processing contamination in dairy foods has led to questions regarding the utility of coliforms as indicators of unsanitary conditions for dairy products. For example, Pseudomonas spp. frequently contaminate dairy products after pasteurization, yet they are not detected by coliform tests. This review will address the role that coliforms play in raw and finished dairy products, their sources and the future of this diverse group as indicator organisms in dairy products.

Bacillus wiedmannii sp. nov., a psychrotolerant and cytotoxic Bacillus cereus group species isolated from dairy foods and dairy environments.

A facultatively anaerobic, spore-forming Bacillus strain, FSL W8-0169T, collected from raw milk stored in a silo at a dairy powder processing plant in the north-eastern USA was initially identified as a Bacillus cereus group species based on a partial sequence of the rpoB gene and 16S rRNA gene sequence. Analysis of core genome single nucleotide polymorphisms clustered this strain separately from known B. cereus group species. Pairwise average nucleotide identity blast values obtained for FSL W8-0169T compared to the type strains of existing B. cereus group species were <95 % and predicted DNA-DNA hybridization values were <70 %, suggesting that this strain represents a novel B. cereus group species. We characterized 10 additional strains with the same or closely related rpoB allelic type, by whole genome sequencing and phenotypic analyses. Phenotypic characterization identified a higher content of iso-C16 : 0 fatty acid and the combined inability to ferment sucrose or to hydrolyse arginine as the key characteristics differentiating FSL W8-0169T from other B. cereus group species. FSL W8-0169T is psychrotolerant, produces haemolysin BL and non-haemolytic enterotoxin, and is cytotoxic in a HeLa cell model. The name Bacillus wiedmannii sp. nov. is proposed for the novel species represented by the type strain FSL W8-0169T (=DSM 102050T=LMG 29269T).

Resilience in the Face of Uncertainty: Sigma Factor B Fine-Tunes Gene Expression To Support Homeostasis in Gram-Positive Bacteria.

Gram-positive bacteria are ubiquitous and diverse microorganisms that can survive and sometimes even thrive in continuously changing environments. The key to such resilience is the ability of members of a population to respond and adjust to dynamic conditions in the environment. In bacteria, such responses and adjustments are mediated, at least in part, through appropriate changes in the bacterial transcriptome in response to the conditions encountered. Resilience is important for bacterial survival in diverse, complex, and rapidly changing environments and requires coordinated networks that integrate individual, mechanistic responses to environmental cues to enable overall metabolic homeostasis. In many Gram-positive bacteria, a key transcriptional regulator of the response to changing environmental conditions is the alternative sigma factor σ(B) σ(B) has been characterized in a subset of Gram-positive bacteria, including the genera Bacillus, Listeria, and Staphylococcus Recent insight from next-generation-sequencing results indicates that σ(B)-dependent regulation of gene expression contributes to resilience, i.e., the coordination of complex networks responsive to environmental changes. This review explores contributions of σ(B) to resilience in Bacillus, Listeria, and Staphylococcus and illustrates recently described regulatory functions of σ(B).

Development and Validation of Pathogen Environmental Monitoring Programs for Small Cheese Processing Facilities.

Pathogen environmental monitoring programs (EMPs) are essential for food processing facilities of all sizes that produce ready-to-eat food products exposed to the processing environment. We developed, implemented, and evaluated EMPs targeting Listeria spp. and Salmonella in nine small cheese processing facilities, including seven farmstead facilities. Individual EMPs with monthly sample collection protocols were designed specifically for each facility. Salmonella was detected in only one facility, with likely introduction from the adjacent farm indicated by pulsed-field gel electrophoresis data. Listeria spp. were isolated from all nine facilities during routine sampling. The overall Listeria spp. (other than Listeria monocytogenes ) and L. monocytogenes prevalences in the 4,430 environmental samples collected were 6.03 and 1.35%, respectively. Molecular characterization and subtyping data suggested persistence of a given Listeria spp. strain in seven facilities and persistence of L. monocytogenes in four facilities. To assess routine sampling plans, validation sampling for Listeria spp. was performed in seven facilities after at least 6 months of routine sampling. This validation sampling was performed by independent individuals and included collection of 50 to 150 samples per facility, based on statistical sample size calculations. Two of the facilities had a significantly higher frequency of detection of Listeria spp. during the validation sampling than during routine sampling, whereas two other facilities had significantly lower frequencies of detection. This study provides a model for a science- and statistics-based approach to developing and validating pathogen EMPs.

Clonal Clustering Using 10-Gene Multilocus Sequence Typing Reveals an Association Between Genotype and Listeria monocytogenes Maximum Growth Rate in Defined Medium.

We used a 10-gene (10G) multilocus sequence typing scheme to investigate the diversity and phylogenetic distribution of 124 Listeria monocytogenes strains across major lineages, major serotypes, and seven epidemic clones that have been previously associated with outbreaks. The 124 isolates proved to be diverse, with a total of 81 sequence types (10G-STs) belonging to 13 clonal complexes (CCs), where all STs of the same CC differ from one another in up to 3 of the 10 alleles (named as 10G-triple-locus-variant-clonal-complexes [10G-TLV-CCs]). Phenotypic characterization for 105 of the 124 strains showed that L. monocytogenes had variable maximum growth rate (µ(max)) in a defined medium at 16°C, and classification by lineage or serotype was not able to reflect the genetic basis for the difference of this phenotype. Among the six major 10G-TLV-CCs, 10G-TLV-CC4 that included lineage I strains had significantly lower µ(max) (Tukey honestly significant difference adjusted [adj.] p < 0.05) compared to 10G-TLV-CC1 and 10G-TLV-CC3 that both comprised lineage II strains, indicating a distinct difference in growth of these L. monocytogenes isolates under nutrient-limited conditions among some of the CCs. However, the other three (10G-TLV-CC2, 6, and 10) of the six major 10G-TLV-CCs containing either lineage I or lineage II strains did not show significantly different µ(max) compared to the others (adj. p < 0.05). Our findings highlighted the importance of using molecular typing methods that can be used in evolutionary analyses as a framework for further understanding the phenotypic characteristics of subgroups of L. monocytogenes.

Spore populations among bulk tank raw milk and dairy powders are significantly different.

To accommodate stringent spore limits mandated for the export of dairy powders, a more thorough understanding of the spore species present will be necessary to develop prospective strategies to identify and reduce sources (i.e., raw materials or in-plant) of contamination. We characterized 1,523 spore isolates obtained from bulk tank raw milk (n=33 farms) and samples collected from 4 different dairy powder-processing plants producing acid whey, nonfat dry milk, sweet whey, or whey protein concentrate 80. The spores isolated comprised 12 genera, at least 44 species, and 216 rpoB allelic types. Bacillus and Geobacillus represented the most commonly isolated spore genera (approximately 68.9 and 12.1%, respectively, of all spore isolates). Whereas Bacillus licheniformis was isolated from samples collected from all plants and farms, Geobacillus spp. were isolated from samples from 3 out of 4 plants and just 1 out of 33 farms. We found significant differences between the spore population isolated from bulk tank raw milk and those isolated from dairy powder plant samples, except samples from the plant producing acid whey. A comparison of spore species isolated from raw materials and finished powders showed that although certain species, such as B. licheniformis, were found in both raw and finished product samples, other species, such as Geobacillus spp. and Anoxybacillus spp., were more frequently isolated from finished powders. Importantly, we found that 8 out of 12 genera were isolated from at least 2 different spore count methods, suggesting that some spore count methods may provide redundant information if used in parallel. Together, our results suggest that (1) Bacillus and Geobacillus are the predominant spore contaminants in a variety of dairy powders, implying that future research efforts targeted at elucidating approaches to reduce levels of spores in dairy powders should focus on controlling levels of spore isolates from these genera; and (2) the spore populations isolated from bulk tank raw milk and some dairy powder products are significantly different, suggesting that targeting in-plant sources of contamination may be important for achieving low spore counts in the finished product. These data provide important insight regarding the diversity of spore populations isolated from dairy powders and bulk tank raw milk, and demonstrate that several spore genera are detected by multiple spore count methods.

Short communication: Postpasteurization hold temperatures of 4 or 6°C, but not raw milk holding of 24 or 72 hours, affect bacterial outgrowth in pasteurized fluid milk.

As fluid milk processors continue to reduce microbial spoilage in fluid milk through improved control of postpasteurization contamination and psychrotolerant sporeformer outgrowth, it is necessary to identify strategies to further improve the quality and extend the shelf life of fluid milk products that are high-temperature, short-time pasteurized. Solutions that optimize product quality, and are economically feasible, are of particular interest to the dairy industry. To this end, this study examined the effects of raw milk holding time and temperature of pasteurized milk storage over shelf life on bacterial growth. In 3 independent replicates, raw milk was stored for 24 and 72 h before pasteurization at 76°C for 25s and then incubated at 3 different storage conditions: (1) 4°C for 21d; (2) 4°C for the first 48 h, then 6°C for the duration of the 21-d shelf life; or (3) 6°C for 21d. Total bacteria counts were assessed initially and on d 7, 14, and 21. No substantial difference in bacterial growth over shelf life was observed between samples processed from raw milk held for 24 versus 72 h. A significantly lower bacterial load was seen at d 21 after pasteurization in samples held at 4°C, versus 4°C for the first 48 h followed by 6°C for the duration of the 21-d shelf life and samples held at 6°C for 21d. This work demonstrates the importance of maintaining control of the fluid milk cold chain throughout postpasteurization, transportation, and retail storage on fluid milk microbial quality.

Transcriptomic Analysis of the Adaptation of Listeria monocytogenes to Growth on Vacuum-Packed Cold Smoked Salmon.

The foodborne pathogen Listeria monocytogenes is able to survive and grow in ready-to-eat foods, in which it is likely to experience a number of environmental stresses due to refrigerated storage and the physicochemical properties of the food. Little is known about the specific molecular mechanisms underlying survival and growth of L. monocytogenes under different complex conditions on/in specific food matrices. Transcriptome sequencing (RNA-seq) was used to understand the transcriptional landscape of L. monocytogenes strain H7858 grown on cold smoked salmon (CSS; water phase salt, 4.65%; pH 6.1) relative to that in modified brain heart infusion broth (MBHIB; water phase salt, 4.65%; pH 6.1) at 7°C. Significant differential transcription of 149 genes was observed (false-discovery rate [FDR], <0.05; fold change, ≥2.5), and 88 and 61 genes were up- and downregulated, respectively, in H7858 grown on CSS relative to the genes in H7858 grown in MBHIB. In spite of these differences in transcriptomes under these two conditions, growth parameters for L. monocytogenes were not significantly different between CSS and MBHIB, indicating that the transcriptomic differences reflect how L. monocytogenes is able to facilitate growth under these different conditions. Differential expression analysis and Gene Ontology enrichment analysis indicated that genes encoding proteins involved in cobalamin biosynthesis as well as ethanolamine and 1,2-propanediol utilization have significantly higher transcript levels in H7858 grown on CSS than in that grown in MBHIB. Our data identify specific transcriptional profiles of L. monocytogenes growing on vacuum-packaged CSS, which may provide targets for the development of novel and improved strategies to control L. monocytogenes growth on this ready-to-eat food.