Initial characterization of the nascent polypeptide-associated complex in yeast

The three subunits of the nascent polypeptide-associated complex (alpha, beta1, beta3) in Saccharomyces cerevisiae are encoded by three genes (EGD2, EGD1, BTT1). We found the complex bound to ribosomes via the beta-subunits in a salt-sensitive manner, in close proximity to nascent polypeptides. Estimation of the molecular weight of the complex of wild-type cells and cells lacking one or two subunits revealed that the composition of the complex is variable and that as yet unknown proteins might be included. Regardless of the variability, a certain balance of the subunits has to be maintained: the deletion of one subunit causes downregulation of the remaining subunits at physiological growth temperature. Cells lacking both beta-subunits are unable to grow at 37 degrees C, most likely due to a toxic effect of the alpha-subunit. Based on in vitro experiments, it has been proposed that the function of mammalian nascent-polypeptide associated complexes (NAC) is to prevent inappropriate targeting of non-secretory nascent polypeptides. In vivo, however, the lack of NAC does not cause secretion of signal-less invertase in yeast. This result and the lack of a drastic phenotype of cells missing one, two or three subunits at optimal conditions (28 degrees C, YPD-medium) suggest either the existence of a substitute for NAC or that cells tolerate or 'repair' the damage caused by the absence of NAC.     

Low prevalence of Listeria monocytogenes in human stool

Listeria monocytogenes is a foodborne pathogen that is found widely in the environment and in a variety of ready-to-eat foods, yet human invasive infection is relatively rare (five cases per million people annually in the United States). Despite wide exposure to this organism, little is known about the prevalence of L. monocytogenes in human stool, and it is not known whether human fecal dispersal contributes to human foodborne transmission. We cultured 827 stool specimens (well formed and loose-watery) from individuals from four large metropolitan areas of New York state for L. monocytogenes and found only 1 (0.12%) positive specimen. L. monocytogenes was also isolated from the blood of the person with the single positive specimen, and the two isolates were indistinguishable by molecular subtyping (both were ribotype DUP-1042B). This provides further evidence that human L. monocytogenes fecal carriage among persons with and without diarrheal disease is remarkably low. Unlike the case for other foodborne pathogens (e.g., Salmonella), human shedders probably do not contribute significantly to L. monocytogenes contamination of foods. However, we observed a single individual with invasive listeriosis that shed the pathogen in feces, indicating the potential for fecal dispersal of L. monocytogenes from persons with listeriosis.     

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.     

International Life Sciences Institute North America Listeria monocytogenes strain collection: development of standard Listeria monocytogenes strain sets for research and validation studies

Research and development efforts on bacterial foodborne pathogens, including the development of novel detection and subtyping methods, as well as validation studies for intervention strategies can greatly be enhanced through the availability and use of standardized strain collections. These types of strain collections are available for some foodborne pathogens, such as Salmonella and Escherichia coli. We have developed a standard Listeria monocytogenes strain collection that has not been previously available. The strain collection includes (i) a diversity set of 25 isolates chosen to represent a genetically diverse set of L. monocytogenes isolates as well as a single hemolytic Listeria innocua strain and (ii) an outbreak set, which includes 21 human and food isolates from nine major human listeriosis outbreaks that occurred between 1981 and 2002. The diversity set represents all three genetic L. monocytogenes lineages (I, n = 9; II, n = 9; and III, n = 6) as well as nine different serotypes. Molecular subtyping by EcoRI automated ribotyping and pulsed-field gel electrophoresis (PFGE) with AscI and ApaI separated the 25 isolates in the diversity set into 23 ribotypes and 25 PFGE types, confirming that this isolate set represents considerable genetic diversity. Molecular subtyping of isolates in the outbreak set confirmed that human and food isolates were identical by ribotype and PFGE, except for human and food isolates for two outbreaks, which displayed related but distinct PFGE patterns. Subtype and source data for all isolates in this strain collection are available on the Internet and are linked to the PathogenTracker database (www.pathogentracker.com), which allows the addition of new, relevant information on these isolates, including links to publications that have used isolates from this collection. We have thus developed a core L. monocytogenes strain collection, which will provide a resource for L. monocytogenes research and development efforts with centralized Internet-based data curation and integration.     

Growth and persistence of Listeria monocytogenes isolates on the plant model Arabidopsis thaliana

While the majority of human listeriosis cases appear to be linked to consumption of processed ready-to-eat foods (e.g., deli meats), a few listeriosis outbreaks have been linked to consumption of contaminated vegetables. In this study, we assessed four isolates representing the major Listeria monocytogenes lineages for their abilities to attach to and grow on Arabidopsis thaliana, a well-characterized plant model. When plants were dipped for 5min into 3ml of water containing 8.8logCFU of L. monocytogenes and rinsed repeatedly, L. monocytogenes was recovered from the leaves at densities from 1.52 to 2.17logCFU/cm(2). Ten days after exposure, bacterial numbers had increased over initial numbers by 2.60-2.95logCFU/cm(2). Using L. monocytogenes expressing GFP, bacteria were visualized in the intercellular spaces of A. thaliana leaves, suggesting internalization through stomata. These data indicate that L. monocytogenes can rapidly attach to and multiply on plant surfaces and colonize intercellular spaces in A. thaliana leaves where it may be protected from sanitation treatments. When A. thaliana seeds were exposed to L. monocytogenes, between 4.23 and 4.57logCFU/cm(2) were recovered from leaves 7 days post-germination, suggesting that contaminated seeds can produce contaminated plants. Overall, our study demonstrates that prevention of L. monocytogenes contamination of plants throughout growing stages is critical, consistent with recommendations for other produce-transmitted foodborne pathogens.     

Longitudinal studies on Listeria in smoked fish plants: impact of intervention strategies on contamination patterns

Four ready-to-eat smoked fish plants were monitored for 2 years to study Listeria contamination patterns and the impact of plant-specific Listeria control strategies, including employee training and targeted sanitation procedures, on Listeria contamination patterns. Samples from the processing plant environment and from raw and finished product were collected monthly and tested for Listeria spp. and Listeria monocytogenes. Before implementation of intervention strategies, 19.2% of raw product samples (n = 276), 8.7% of finished product samples (n = 275), and 26.1% of environmental samples (n = 617) tested positive for Listeria spp. During and after implementation of Listeria control strategies, 19.0% of raw product samples (n = 242), 7.0% of finished product samples (n = 244), and 19.5% of environmental samples (n = 527) were positive for Listeria spp. In one of the four fish plants (plant 4), no environmental samples were positive for L. monocytogenes, and this plant was thus excluded from statistical analyses. Based on data pooled from plants 1, 2, and 3, environmental Listeria spp. prevalence was significantly lower (P < 0.05) for nonfood contact surfaces and the finished product area and for the overall core environmental samples after implementation of control strategies. Listeria prevalence for floor drains was similar before and after implementation of controls (49.6 and 54.2%, respectively). Regression analysis revealed a significant positive relationship (P < 0.05) between L. monocytogenes prevalence in the environment and in finished products before implementation of control strategies; however, this relationship was absolved by implementation of Listeria control strategies. Molecular subtyping (EcoRI ribotyping) revealed that specific L. monocytogenes ribotypes persisted in three processing plants over time. These persistent ribotypes were responsible for all six finished product contamination events detected in plant 1. Ribotype data also indicated that incoming raw material is only rarely a direct source of finished product contamination. While these data indicate that plant-specific Listeria control strategies can reduce cross-contamination and prevalence of Listeria spp. and L. monocytogenes in the plant environment, elimination of persistent L. monocytogenes strains remains a considerable challenge.     

Growth and stress resistance variation in culture broth among Listeria monocytogenes strains of various serotypes and origins

Twenty-five Listeria monocytogenes strains of various serotypes and sources, including clinical and food isolates associated with the same outbreaks, were characterized and compared based on growth rates and heat and acid death rates. Growth was monitored in tryptic soy broth supplemented with 0.6% yeast extract (TSBYE) at 4 and 30 degrees C for 32 days and 20 h, respectively. Heat and acid stress responses in TSBYE heated to 55 degrees C or acidified to pH 3.0 with lactic acid were evaluated for 240 or 120 min, respectively. Extensive variation in growth and stress resistance was observed among the tested strains. Growth rate differences were less evident at 30 than at 4 degrees C, where growth rates (log CFU per milliliter per day) ranged from 0.28 to 0.43. Thermal and acid death rates (log CFU per milliliter per minute) ranged from -0.023 to -0.052 and from -0.012 to -0.134, respectively. Serotype appeared to play a significant role (P < 0.05) only with respect to the heat resistance of the organism. Serotype 4b isolates as a group had lower heat resistance than did isolates representing all other serotypes combined. Although no clear origin-related (food versus clinical) trends were observed under the tested conditions, outbreak-related isolates of serotype 4b had lower acid death rates (higher acid resistance) (P < 0.05) than did the rest of the strains belonging to this serotype. Strain Scott A exhibited slow growth at 4 degrees C and low acid resistance, behavior that was distinct among both clinical and serotype 4b isolates. The results of this study highlight the risks associated with extrapolation to other strains of findings obtained with only one strain of L. monocytogenes. This information should be useful when test strains are to be selected for the evaluation of antimicrobial alternatives in ready-to-eat meat and other food products and when risk assessments are to be conducted.     

Comparative evaluation of culture- and BAX polymerase chain reaction-based detection methods for Listeria spp. and Listeria monocytogenes in environmental and raw fish samples

Two commercial polymerase chain reaction (PCR)-based Listeria detection systems, the BAX for Screening/Listeria monocytogenes and the BAX for Screening/Genus Listeria, and a culture-based detection system, the Biosynth L. monocytogenes Detection System (LMDS), were evaluated for their ability to detect L. monocytogenes and Listeria spp. in raw ingredients and the processing environment. For detection of L. monocytogenes from raw fish, enrichment was performed in Listeria enrichment broth (LEB), followed by plating on both Oxford agar and LMDS L. monocytogenes plating medium (LMPM). Detection of Listeria and L. monocytogenes from environmental samples was performed using LMDS enrichment medium, followed by plating on both Oxford agar and LMPM. A total of 512 environmental samples and 315 raw fish were taken from two smoked fish processing facilities and screened using these molecular and cultural Listeria detection methods. The BAX for Screening/L monocytogenes was used to screen raw fish and was 84.8% sensitive and 100% specific. The BAX for Screening/Genus Listeria was evaluated on environmental samples and had 94.7% sensitivity and 97.4% specificity. In conjunction with enrichment in LEB, LMPM had a sensitivity and specificity for detection of L. monocytogenes from raw fish of 97.8 and 100%, respectively. Use of LMDS enrichment medium followed by plating on LMPM allowed for sensitivity and specificity rates of 94.8 and 100%, respectively, for detection of L. monocytogenes from environmental samples. We conclude that both the BAX systems and the use of LMPM allow for reliable and rapid detection of Listeria spp. and L. monocytogenes. While the BAX systems provide screening results in about 3 days, the use of LMPM allows for L. monocytogenes isolation in 4 to 5 days.     

Psychrotolerant spore-former growth characterization for the development of a dairy spoilage predictive model

Psychrotolerant spore-forming bacteria represent a major challenge regarding microbial spoilage of fluid milk. These organisms can survive most conventional pasteurization regimens and subsequently germinate and grow to spoilage levels during refrigerated storage. To improve predictions of fluid milk shelf life and assess different approaches to control psychrotolerant spore-forming bacteria in the fluid milk production and processing continuum, we developed a predictive model of spoilage of fluid milk due to germination and growth of psychrotolerant spore-forming bacteria. We characterized 14 psychrotolerant spore-formers, representing the most common Bacillales subtypes isolated from raw and pasteurized milk, for ability to germinate from spores and grow in skim milk broth at 6°C. Complete growth curves were obtained by determining total bacterial count and spore count every 24 h for 30 d. Based on growth curves at 6°C, probability distributions of initial spore counts in bulk tank raw milk, and subtype frequency in bulk tank raw milk, a Monte Carlo simulation model was created to predict spoilage patterns in high temperature, short time-pasteurized fluid milk. Monte Carlo simulations predicted that 66% of half-gallons (1,900 mL) of high temperature, short time fluid milk would reach a cell density greater than 20,000 cfu/mL after 21 d of storage at 6°C, consistent with current spoilage patterns observed in commercial products. Our model also predicted that an intervention that reduces initial spore loads by 2.2 Log₁₀ most probable number/mL (e.g., microfiltration) can extend fluid milk shelf life by 4 d (end of shelf life was defined here as the first day when the mean total bacterial count exceeded 20,000 cfu/mL). This study not only provides a baseline understanding of the growth rates of psychrotolerant spore-formers in fluid milk, it also provides a stochastic model of spoilage by these organisms over the shelf life of fluid milk, which will ultimately allow for the assessment of different approaches to reduce fluid milk spoilage.     

Longitudinal assessment of dairy farm management practices associated with the presence of psychrotolerant Bacillales spores in bulk tank milk on 10 New York State dairy farms

The ability of certain spore-forming bacteria in the order Bacillales (e.g., Bacillus spp., Paenibacillus spp.) to survive pasteurization in spore form and grow at refrigeration temperatures results in product spoilage and limits the shelf life of high temperature, short time (HTST)-pasteurized fluid milk. To facilitate development of strategies to minimize contamination of raw milk with psychrotolerant Bacillales spores, we conducted a longitudinal study of 10 New York State dairy farms, which included yearlong monthly assessments of the frequency and levels of bulk tank raw milk psychrotolerant spore contamination, along with administration of questionnaires to identify farm management practices associated with psychrotolerant spore presence over time. Milk samples were first spore pasteurized (80°C for 12 min) and then analyzed for sporeformer counts on the initial day of spore pasteurization (SP), and after refrigerated storage (6°C) for 7, 14, and 21 d after SP. Overall, 41% of samples showed sporeformer counts of >20,000 cfu/mL at d 21, with Bacillus and Paenibacillus spp. being predominant causes of high sporeformer counts. Statistical analyses identified 3 management factors (more frequent cleaning of the bulk tank area, the use of a skid steer to scrape the housing area, and segregating problem cows during milking) that were all associated with lower probabilities of d-21 Bacillales spore detection in SP-treated bulk tank raw milk. Our data emphasize that appropriate on-farm measures to improve overall cleanliness and cow hygiene will reduce the probability of psychrotolerant Bacillales spore contamination of bulk tank raw milk, allowing for consistent production of raw milk with reduced psychrotolerant spore counts, which will facilitate production of HTST-pasteurized milk with extended refrigerated shelf life.