Pseudomonas fluorescens group bacterial strains are responsible for repeat and sporadic postpasteurization contamination and reduced fluid milk shelf life

Postpasteurization contamination (PPC) of high temperature, short time-pasteurized fluid milk by gram-negative (GN) bacteria continues to be an issue for processors. To improve PPC control, a better understanding of PPC patterns in dairy processing facilities over time and across equipment is needed. We thus collected samples from 10 fluid milk processing facilities to (1) detect and characterize PPC patterns over time, (2) determine the efficacy of different media to detect PPC, and (3) characterize sensory defects associated with PPC. Specifically, we collected 280 samples of high temperature, short time-pasteurized milk representing different products (2%, skim, and chocolate) and different fillers over 4 samplings performed over 11 mo at each of the 10 facilities. Standard plate count (SPC) as well as total GN, coliform, and Enterobacteriaceae (EB) counts were performed upon receipt and after 7, 10, 14, 17, and 21 d of storage at 6°C. We used 16S rDNA sequencing to characterize representative bacterial isolates from (1) test days with SPC >20,000 cfu/mL and (2) all samples with presumptive GN, coliforms, or EB. Day-21 samples were also evaluated by a trained defect judging panel. By d 21, 226 samples had SPC >20,000 cfu/mL on at least 1 d of shelf life; GN bacteria were found in 132 of these 226 samples, indicating PPC. Crystal violet tetrazolium agar detected PPC with the greatest sensitivity. Spoilage due to PPC was predominantly associated with Pseudomonas (isolated from 101 of the 132 samples with PPC); coliforms and EB were found in 27 and 37 samples with spoilage due to PPC, respectively. Detection of Pseudomonas and Acinetobacter was associated with lower flavor scores; coagulated, fruity fermented, and unclean defects were more prevalent in d-21 samples with PPC. Repeat isolation of Pseudomonas fluorescens group strains with identical partial 16S rDNA sequence types was observed in 8 facilities. In several facilities, specific lines, products, or processing days were linked to repeat product contamination with Pseudomonas with identical sequence types. Our data show that PPC due to Pseudomonas remains a major challenge for fluid milk processors; the inability of coliform and EB tests to detect Pseudomonas may contribute to this. Our data also provide important initial insights into PPC patterns (e.g., line-specific contamination), supporting the importance of molecular subtyping methods for identification of PPC sources.     

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.     

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.     

Evaluation of calf milk pasteurization systems on 6 Pennsylvania dairy farms

Waste milk has been fed to calves for many years, but concerns with bacterial contamination as well as possible transmission of diseases have discouraged widespread use of this feed. Pasteurization of waste milk is one option to reduce management risk while utilizing a valuable, low-cost, liquid feed source for calves. However, many farms currently pasteurizing waste milk lack a system to adequately monitor the efficiency of the process. A study was carried out to evaluate 6 on-farm pasteurization systems, including high-temperature, short-time pasteurizers and low-temperature, batch pasteurizers. Milk samples were taken pre- and postpasteurization as well as from the calf buckets and immediately frozen for later bacterial culture. Samples were collected twice daily for 15 d. Milk samples were examined for standard plate count (SPC), coagulase-negative staphylococci count, environmental streptococci count, coliform count, gram-negative noncoliform count, Streptococcus agalactiae count, and Staphylococcus aureus count. Before pasteurization, 68% of the samples had SPC <20,000 cfu/mL, and 39% of samples contained <100 cfu/mL of coliform bacteria. After pasteurization, 96% of samples had SPC <20,000 cfu/mL, and 92% had coliform counts <100 cfu/mL. Bacteria counts were significantly reduced by pasteurization, and pasteurized milk contained acceptable numbers of bacteria in >90% of samples. These results indicate that pasteurization can be very effective in lowering bacterial contamination of milk. However, bacteria numbers significantly increased after pasteurization and, in some cases, bacteria counts in milk fed to calves were similar to prepasteurization levels. Milk handling after pasteurization was identified as an important issue on the farms studied.     

Survival and detection of coliforms,Enterobacteriaceae, and gram-negative bacteria in Greek yogurt

Despite the widespread use of coliforms as indicator bacteria, increasing evidence suggests that the Enterobacteriaceae (EB) and total gram-negative groups more accurately reflect the hygienic status of high-temperature, short-time pasteurized milk and processing environments. If introduced into milk as postpasteurization contamination, these bacteria may grow to high levels and produce a wide range of sensory-related defects. However, limited information is available on the use and survival of bacterial hygiene indicators in dairy products outside of pasteurized fluid milk and cheese. The goal of this study was to (1) provide information on the survival of a diverse set of bacterial hygiene indicators in the low pH environment of Greek yogurt, (2) compare traditional and alternative detection methods for their ability to detect bacterial hygiene indicators in Greek yogurt, and (3) offer insight into optimal hygiene indicator groups for use in low-pH fermented dairy products. To this end, we screened 64 bacterial isolates, representing 24 dairy-relevant genera, for survival and detection in Greek yogurt using 5 testing methods. Before testing, isolates were inoculated into plain, 0% fat Greek yogurt (pH 4.35 to 4.65), followed by a 12-h hold period at 4 ± 1°C. Yogurts were subsequently tested using Coliform Petrifilm (3M, St. Paul, MN) to detect coliforms; Enterobacteriaceae Petrifilm (3M), violet red bile glucose agar and the D-Count (bioMérieux, Marcy-l'Étoile, France) to detect EB; and crystal violet tetrazolium agar (CVTA) to detect total gram-negative bacteria. Overall, the non-EB gram-negative isolates showed significantly larger log reductions 12 h after inoculation into Greek yogurt (based on bacterial numbers recovered on CVTA) compared with the coliform and noncoliform EB isolates tested. The methods evaluated varied in their ability to detect different microbial hygiene indicators in Greek yogurt. Crystal violet tetrazolium agar detected the highest portion of coliforms, whereas EB Petrifilm detected the highest portion of EB, as well as highest portion of total gram-negative bacteria. Additionally, the D-Count method allowed for faster detection of EB in yogurt by generating results in approximately 13 h rather than the 24 h required when using EB Petrifilm and violet red bile glucose agar. Results from this study indicate that the coliform and EB groups encompass a broad range of dairy-relevant gram-negative bacteria with the ability to survive in Greek yogurt, supporting their use as microbial hygiene indicator groups in low-pH fermented dairy products.     

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.     

A study on the prevalence of gram-negative bacteria in bulk tank milk

Bulk tank milk from 131 dairy herds in eastern South Dakota and western Minnesota were examined for coliforms and noncoliform bacteria. Coliforms were detected in 62.3% of bulk tank milk samples. Counts ranged from 0 to 4.7 log10 cfu/ml. The mean count was 3.4 log10 cfu/ml. Gram-negative noncoliform bacteria were observed in 76.3% of bulk tank milk. Counts ranged from 0 to 6.2 log10 cfu/ml. The mean count was 4.8 log10 cfu/ml. A total of 234 isolates from bulk tank milk were examined to species level; 205 isolates belonged to 28 species. Coliforms and gram-negative noncoliform bacteria accounted for 32.9 and 67.1% of the total isolates, respectively. Organisms such as Agrobacterium radiobacter, Bordetella spp., Comamonas testosteroni, Listonella damsela, Ochrobactrum anthropi, and Oligella urethralis were isolated from bulk tank milk in this study. These organisms have not been reported previously in bulk tank milk. A total of 116 isolates of Pseudomonas spp. were isolated from raw milk; 98 isolates belonged to nine Pseudomonas spp., and the remaining 18 isolates could not be identified to their species level. Pseudomonas was the most predominant genus. Pseudomonas fluorescens was the most predominant species isolated from bulk tank milk and accounted for 29.9% of all isolates examined. The results of the study suggest that counts of coliforms and noncoliform bacteria in bulk tank milk vary considerably. The isolates represent a wide variety of Gram-negative bacterial species. Examination of bulk tank milk for coliforms and noncoliform bacteria could provide an indication of current and potential problems associated with bacterial counts and milk quality.     

Monte Carlo simulation model predicts bactofugation can extend shelf-life of pasteurized fluid milk,even when raw milk with low spore counts is used as the incoming ingredient

Bacterial spores from raw milk that survive the pasteurization process are responsible for half of all the spoilage of fluid milk. Bactofugation has received more attention as a nonthermal method that can reduce the presence of bacterial spores in milk and with it the spoilage of fluid milk. The objective of this work was to determine the effectiveness of bactofugation in removing spores from raw milk and estimate the effect the spore removal could have on shelf-life of fluid milk. The study was conducted in a commercial fluid milk processing facility where warm spore removal was performed using one-phase bactofuge followed by warm cream separation and high temperature, short time pasteurization. Samples from different stages of fluid milk processing with and without the use of bactofuge were tested for total plate count, mesophilic spore count, psychrotolerant spore count (PSC), and somatic cell count. Results were evaluated to determine the count reductions during different stages of fluid milk processing and compare counts in fluid milk processed with and without bactofugation. Bactofugation on average reduced the total plate count by 1.81 ± 0.72 log cfu/mL, mesophilic spore count by 1.08 ± 0.71 log cfu/mL, PSC by 0.86 ± 0.59 log cfu/mL, and somatic cell count by 135,881 ± 43,942 cells/mL. Psychrotolerant spore count in final pasteurized skim milk processed with and without bactofugation was used to predict the shelf-life of the pasteurized skim milk using the Monte Carlo simulation model. Although PSC in the initial raw milk was already low (?0.63 ± 0.47 log cfu/mL), the predicted values from the simulation model showed that bactofugation would extend the shelf-life of pasteurized skim milk by approximately 2 d. The results of this study will directly help fluid milk processors evaluate the benefits of using bactofugation as an intervention in their plants, and also demonstrate the benefits of using mathematical modeling in decision making.     

Strategies for raw sheep milk storage in smallholdings: Effect of freezing or long-term refrigerated storage on microbial growth

We assessed the effects of freezing and refrigeration over long periods on the microbiological quality of sheep milk. The raw milk was frozen in 1-L plastic bags or 5-L milk buckets and, after 1 mo, thawed at 7 or 25°C. We evaluated these samples immediately after thawing (d 0) and after 1 d of storage at 7°C. Furthermore, we stored fresh raw milk at 7°C for 10 d in the same packages and in a bulk milk cooler at 4°C (adding 10% of fresh raw milk daily). The total bacterial, total psychrotolerant, and proteolytic psychrotolerant counts were evaluated before and after thawing (for previously frozen milk) and daily (for refrigerated milk). The frozen-thawed milks showed no significant increase in bacterial counts immediately after thawing for all samples (<0.7 log cfu/mL), but only the samples packaged in 1-L bags and thawed at 7°C remained microbiologically adequate after 1 d of storage. Findings of the refrigerated samples were modeled using a modified Gompertz equation, obtaining a lag phase of around 0.5 (5-L bucket), 2.6 (1-L bag), and 7.0 (bulk milk cooler) d for total bacterial and total psychrotolerant counts. Maximum growth rates (µ_max) were 1.0 and 1.0 (5-L bucket), 1.2 and 1.3 (1-L bag) and 3.0 and 1.5 (bulk milk cooler) ln(cfu/mL) per day for total bacteria and total psychrotolerant counts, respectively. Compared with total bacteria and total psychrotolerant bacteria, psychrotolerant proteolytic bacteria grew slowly, reaching unacceptable counts only after 9 to 10 d of storage. The studied methods are interesting alternatives for preserving raw sheep milk on smallholdings.     

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.     

Guidelines for monitoring bulk tank milk somatic cell and bacterial counts

This study was conducted to establish guidelines for monitoring bulk tank milk somatic cell count and bacterial counts, and to understand the relationship between different bacterial groups that occur in bulk tank milk. One hundred twenty-six dairy farms in 14 counties of Pennsylvania participated, each providing one bulk tank milk sample every 15 d for 2 mo. The 4 bulk tank milk samples from each farm were examined for bulk tank somatic cell count and bacterial counts including standard plate count, preliminary incubation count, laboratory pasteurization count, coagulase-negative staphylococcal count, environmental streptococcal count, coliform count, and gram-negative noncoliform count. The milk samples were also examined for presence of Staphylococcus aureus, Streptococcus agalactiae, and Mycoplasma. The bacterial counts of 4 bulk tank milk samples examined over an 8-wk period were averaged and expressed as mean bacterial count per milliliter. The study revealed that an increase in the frequency of isolation of Staphylococcus aureus and Streptococcus agalactiae was significantly associated with an increased bulk tank somatic cell count. Paired correlation analysis showed that there was low correlation between different bacterial counts. Bulk tank milk with low (<5000 cfu/mL) standard plate count also had a significantly low level of mean bulk tank somatic cell count (<200,000 cells/mL), preliminary incubation count (<10,000 cfu/mL), laboratory pasteurization count (<100 cfu/mL), coagulase-negative staphylococci and environmental streptococcal counts (<500 cfu/mL), and noncoliform count (<200 cfu/mL). Coliform count was less likely to be associated with somatic cell or other bacterial counts. Herd size and farm management practices had considerable influence on somatic cell and bacterial counts in bulk tank milk. Dairy herds that used automatic milking detachers, sand as bedding material, dip cups for teat dipping instead of spraying, and practiced pre-and postdipping had significantly lower bulk tank somatic cell and/or bacterial counts. In conclusion, categorized bulk tank somatic cell and bacterial counts could serve as indicators and facilitate monitoring of herd udder health and milk quality.     

Short communication: Diagnostic accuracy of the Petrifilm culture system for identifying colostrum with excessive bacterial contamination in Quebec dairy herds

The objective of this study was to validate the diagnostic accuracy of the Petrifilm culture system (3M, St. Paul, MN) for identifying colostrum with excessive bacterial contamination. An observational cross-sectional study was conducted between October 2015 and February 2016. Two colostrum aliquots were collected during the first meal of 332 calves (33 commercial Holstein dairy farms) in Quebec, Canada. One aliquot per calf was used to quantify the total bacteria count and the total coliform count using standard bacteriological laboratory testing (reference test). These results were dichotomized to identify colostrum with excessive bacterial contamination [aerobic count plate (AC) >100,000 cfu/mL; coliform count plate (CC) >10,000 cfu/mL]. The Petrifilm system was used to quantify both aerobic and coliform contamination of the other colostrum aliquot from each calf. As such, AC and CC were used according to the manufacturer's recommendations. The area under the curve of the receiver operating characteristic curve of AC and CC compared with the laboratory were 0.83, and 0.95, respectively. Using the optimal threshold of >24,000 cfu/mL for AC results, the Petrifilm system had a sensitivity (Se) of 69%, specificity (Sp) of 86%, and a kappa value of 0.54. Using the optimal threshold of >4,000 cfu/mL for CC results, the Petrifilm system had a Se of 93%, Sp of 90%, and kappa value of 0.64. Overall, these results suggest that the Petrifilm system is an appropriate alternative for identifying colostrum with excessive bacterial contamination.     

Poly-l-lysine-functionalized magnetic beads combined with polymerase chain reaction for the detection of Staphylococcus aureus and Escherichia coli O157:H7 in milk

Rapid and credible detection of pathogens is essential to prevent and control outbreaks of foodborne diseases. In this study, a poly-l-lysine-functionalized magnetic beads (PLL-MB) strategy combined with a PCR assay was established to detect Staphylococcus aureus. We also detected Escherichia coli O157:H7 to further verify the strategy for gram-negative bacteria detection. Poly-l-lysine has strong positive charges because of its amino groups, which can conjugate with the carboxyl of carboxyl magnetic beads. Furthermore, it can be used to combine with bacteria through electrostatic adsorption. Under optimum conditions, the developed PLL-MB complexes showed 90% capture efficiency in phosphate-buffered saline and 85% capture efficiency in milk for S. aureus detection. The limit of detection of the PLL-MB-PCR assay was 10² cfu/mL (1.8 × 10² cfu/mL for S. aureus and 7 × 10² cfu/mL for E. coli O157:H7) in phosphate-buffered saline and milk samples. The whole assay can be performed within 4 h. The proposed strategy showed a lower limit of detection when compared with the conventional PCR assay without enrichment. In addition, this method exhibited the advantages of a high-efficient, cost-efficient, and simple operation, indicating its potential applications in foodborne pathogen detection.     

Microbial contamination of harvested colostrum on Czech dairy farms

The objective of this study was to perform a quantitative and qualitative evaluation of microbial contamination of harvested colostrum on 39 Czech dairy farms. The study identified the proportion of colostrum samples that met the recommended goals for total plate count (TPC), total coliform count (TCC), and gram-negative noncoliform count (NCC), and evaluated the effect of the farm, breed, parity, season of the year, time of calving, and colostrum volume on these 3 microbiological parameters. Colostrum samples from cows (n = 1,241; 57.6% from Czech Fleckvieh, and 42.4% from Holstein breed) were collected on dairy farms between autumn of 2015 and spring of 2017. The samples were collected after the first milking directly from milking buckets. In 155 out of 1,241 colostrum samples (26 farms, 6 samples each, except 1 farm), the species of microorganisms obtained by culture were determined, and the findings were classified into 4 groups according to the probable source of contamination as follows: (1) normal inhabitants of bovine skin and mucosa, (2) fecal contaminants, (3) environmental contaminants, and (4) potential gram-positive mammary pathogens. Our results showed heavy microbial contamination of collected colostrum samples (TPC median = 408,000 cfu/mL; TCC median = 200 cfu/mL; NCC median = 80 cfu/mL). Only 28.4% of samples met the requirement for TPC (<100,000 cfu/mL), 88.2% for TCC (<10,000 cfu/mL), and 86.0% for NCC (<5,000 cfu/mL). Among the tested factors, we found that farm had a significant effect on all 3 microbiological parameters, volume of colostrum had an effect on TPC (the highest TPC in <3.0 L of colostrum), and season had an effect on TCC and NCC (higher TCC in summer than in autumn and winter; the highest NCC in summer and higher in autumn than in spring and winter). Our results showed that most microbes isolated from colostrum belonged to normal inhabitants of bovine skin and mucosa, fecal, or environmental contaminants (i.e., 82.6%, 81.9%, and 75.5% of colostrum samples, respectively). Potential gram-positive mammary pathogens were found in 13.5% of samples. Escherichia coli was isolated from 9.0% of colostrum samples, and Streptococcus uberis and Streptococcus parauberis were each isolated from 5.2% of samples. Our study showed high microbial contamination of colostrum collected on dairy farms. Therefore, better hygiene and sanitation around colostrum harvest should be addressed by farmers.     

Efficacy of on-farm use of ultraviolet light for inactivation of bacteria in milk for calves

Ultraviolet light is being employed for bacterial inactivation in milk for calves; however, limited evidence is available to support the claim that UV light effectively inactivates bacteria found in milk. Thus, the objective of this observational study was to investigate the efficacy of on-farm UV light treatment in reducing bacteria populations in waste milk used for feeding calves. Samples of nonsaleable milk were collected from 9 Pennsylvania herds, twice daily for 15 d, both before and after UV light treatment (n = 60 samples per farm), and analyzed for standard plate count, coliforms, noncoliform, gram-negative bacteria, environmental and contagious streptococci, coagulase-negative staphylococci, Streptococcus agalactiae, Staphylococcus aureus count, and total solids percentage, and log reduction and percentage log reduction were calculated. Data were analyzed using the mixed procedure in SAS. In all bacteria types, samples collected after UV treatment contained significantly fewer bacteria compared with samples collected before UV treatment. Weighted least squares means for log reduction (percentage log reduction) were 1.34 (29%), 1.27 (58%), 1.48 (53%), 1.85 (55%), 1.37 (72%), 1.92 (63%), 1.07 (33%), and 1.67 (82%) for standard plate count, coliforms, noncoliform, gram-negative bacteria, environmental and contagious streptococci, Strep. agalactiae, coagulase-negative staphylococci, and Staph. aureus, respectively. A percentage log reduction greater than 50% was achieved in 6 of 8 bacteria types, and 43 and 94% of samples collected after UV treatment met recommended bacterial standards for milk for feeding calves. Based on these results, UV light treatment may be effective for some, but not all bacteria types found in nonsaleable waste milk. Thus, farmers should take into account the bacteria types that may need to be reduced when considering the purchase of a UV-treatment system.     

Identification and characterization of elevated microbial counts in bulk tank raw milk

The bacterial composition of bulk tank milk from 13 farms was examined over a 2-wk period to characterize sudden elevations in the total bacterial count referred to as "spikes." Bulk tank milk samples collected at each pick-up were analyzed for standard plate count, Petrifilm aerobic count, somatic cell count, gram-negative organisms, and streptococci. Twenty standard plate count spikes were observed: 12 associated with streptococci, 4 associated with gram-negative organisms, 2 associated with streptococci and gram-negative organisms, and 2 that were not definitively characterized. Spikes ranged from 14,000 to 600,000 cfu/ml. Streptococcus uberis was isolated as the predominant organism from 11 spikes, and Escherichia coli was isolated from 4 spikes. Statistical analysis of total bacterial counts indicated a high correlation (r = 0.94) between standard plate counts and Petrifilm aerobic count. Regression analysis of standard plate counts and Petrifilm aerobic counts yielded the equation log10 (standard plate count) = 0.73 + 0.85log10 (Petrifilm aerobic count), indicating that the correlation, although strong, is not one to one. In a related pilot study, triplicate bulk tank milk samples were collected and analyzed for total bacterial count and presumptive streptococcus, gram-negative, and staphylococcus counts. Two-way ANOVA of these triplicate data indicated a lack of significant variation among the triplicate samples, suggesting that one sample can reliably gauge the microbial status of the entire bulk tank.     

Shelf life of pasteurized microfiltered milk containing 2% fat

The goal of this research was to produce homogenized milk containing 2% fat with a refrigerated shelf life of 60 to 90 d using minimum high temperature, short time (HTST) pasteurization in combination with other nonthermal processes. Raw skim milk was microfiltered (MF) using a Tetra Alcross MFS-7 pilot plant (Tetra Pak International SA, Pully, Switzerland) equipped with Membralox ceramic membranes (1.4 μm and surface area of 2.31 m²; Pall Corp., East Hills, NY). The unpasteurized MF skim permeate and each of 3 different cream sources were blended together to achieve three 2% fat milks. Each milk was homogenized (first stage: 17 MPa, second stage: 3 MPa) and HTST pasteurized (73.8°C for 15 s). The pasteurized MF skim permeate and the 3 pasteurized homogenized 2% fat milks (made from different fat sources) were stored at 1.7 and 5.7°C and the standard plate count for each milk was determined weekly over 90 d. When the standard plate count was >20,000 cfu/mL, it was considered the end of shelf life for the purpose of this study. Across 4 replicates, a 4.13 log reduction in bacteria was achieved by MF, and a further 0.53 log reduction was achieved by the combination of MF with HTST pasteurization (73.8°C for 15 s), resulting in a 4.66 log reduction in bacteria for the combined process. No containers of MF skim milk that was pasteurized after MF exceeded 20,000 cfu/mL bacteria count during 90 d of storage at 5.7°C. The 3 different approaches used to reduce the initial bacteria and spore count of each cream source used to make the 2% fat milks did not produce any shelf-life advantage over using cold separated raw cream when starting with excellent quality raw whole milk (i.e., low bacteria count). The combination of MF with HTST pasteurization (73.8°C for 15 s), combined with filling and packaging that was protected from microbial contamination, achieved a refrigerated shelf life of 60 to 90 d at both 1.7 and 5.7°C for 2% fat milks.     

Rapid detection of Salmonella in milk by combined immunomagnetic separation-polymerase chain reaction assay

During the past few years, milk has presented a risk of Salmonella contamination; it has been implicated as the cause in several outbreaks of salmonellosis. Because conventional detection methods require 5 to 7 d for completion and involve several subcultivation stages followed by biochemical and serological tests, rapid and sensitive methods have been sought, mainly at the DNA level. Therefore, a study including milk samples was conducted to evaluate the performance of a combination of 2 techniques—immunomagnetic separation and polymerase chain reaction (PCR)—for the detection of Salmonella. The 16-, 14-, 12-, 10-, and 8-h nonselective pre-enrichment steps before immunomagnetic separation and the high-pure DNA preparation method before PCR were used in a combined assay. Milk samples, which were found to be Salmonella-negative by a reference method, were first inoculated with Salmonella Enteritidis. Next, the shortest pre-enrichment time that is required for detection of 1 or 10 cfu of Salmonella/mL by combined immunomagnetic separation-PCR assay was found by using 16-, 14-, 12-, 10-, and 8-h incubation periods. The detection limit using a 16-, 14-, or 12-h nonselective pre-enrichment was 1 to 10 cfu/mL. However, the sensitivity decreased to 10¹ and 10² cfu/mL, respectively, when 10- and 8-h pre-enrichments were used. This assay, in conjunction with a 12-h pre-enrichment, proved to be rapid (overall 16 h) and sensitive (1-10 cfu/mL) for the detection of Salmonella in milk samples and promising for routine use in the detection of Salmonella in milk.     

Non-culture-based identification of mastitis-causing bacteria by MALDI-TOF mass spectrometry

The purpose of this study was to evaluate the detection limit of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for direct identification, without previous microbiological culture, of bovine mastitis-causing bacteria from milk samples. Milk samples (n = 15) were experimentally contaminated with Staphylococcus aureus, Streptococcus uberis, Streptococcus agalactiae, Streptococcus dysgalactiae, and Escherichia coli to have bacterial counts ranging from 10³ to 10⁹ cfu/mL. These contaminated milk samples were subjected to a preparation protocol for bacterial ribosomal protein extraction using the MALDI Sepsityper kit (Bruker Daltonik, Bremen, Germany), which allowed MALDI-TOF MS coupled with Biotyper software (Bruker Daltonik) to identify bacterial fingerprints based on intact ribosomal proteins. The ability of MALDI-TOF MS to correctly identify bacterial strains from experimentally contaminated milk (without previous microbiological culture) depended on the bacterial count of the samples and on the species of the bacteria evaluated. Adequate identification at the bacterial species level (score ≥2.0) directly from milk samples required bacterial counts in the following ranges: ≥10⁶ cfu/mL of Staph. aureus, ≥10⁷ cfu/mL of E. coli, and ≥10⁸ cfu/mL of Strep. agalactiae, Strep. dysgalactiae, and Strep. uberis. We concluded that direct identification of mastitis-causing pathogens is possible for Staph. aureus, E. coli, Strep. agalactiae, Strep. dysgalactiae, and Strep. uberis, but correct identification depended on the bacterial count in the milk samples.