Rapid detection and characterization of postpasteurization contaminants in pasteurized fluid milk.

Microbial spoilage of pasteurized fluid milk is typically due to either (1) postpasteurization contamination (PPC) with psychrotolerant gram-negative bacteria (predominantly Pseudomonas) or (2) growth of psychrotolerant sporeformers (e.g., Paenibacillus) that have the ability to survive pasteurization when present as spores in raw milk, and to subsequently grow at refrigeration temperatures. While fluid milk quality has improved over the last several decades, continued reduction of PPC is hampered by the lack of rapid, sensitive, and specific methods that allow for detection of PPC in fluid milk, with fluid milk processors still often using time-consuming methods (e.g., Moseley keeping quality test). The goal of this project was to utilize a set of commercial fluid milk samples that are characterized by a mixture of samples with PPC due to psychrotolerant gram-negative bacteria and samples with presence and growth of psychrotolerant sporeforming bacteria to evaluate different approaches for rapid detection of PPC. Comprehensive microbiological shelf-life characterization of 105 pasteurized fluid milk samples obtained from 20 dairy processing plants showed that 60/105 samples reached bacterial counts >20,000 cfu/mL over the shelf-life due to PPC with gram-negative bacteria. Among these 60 samples with evidence of gram-negative PPC spoilage over the shelf-life, 100% (60/60) showed evidence of contamination with noncoliform, non-Enterobacteriaceae (EB) gram-negative bacteria (e.g., Pseudomonas), 20% (12/60) showed evidence of contamination with coliforms, and 7% (4/60) showed evidence of contamination with noncoliform EB. Among the remaining 45 samples, 28 showed levels of gram-positive bacteria above 20,000 cfu/mL and the remaining 17 samples did not exceed 20,000 cfu/mL over the shelf-life. Evaluation of the same set of 105 samples using 6 different approaches {all possible combinations of 2 different enrichment protocols (13°C or 21°C for 18 h) and 3 different plating media [crystal violet tetrazolium agar, EB Petrifilm (3M, St. Paul, MN), and Coliform Petrifilm]} showed that enrichment at 21°C for 18 h, followed by plating on crystal violet tetrazolium agar provided for the most sensitive, accelerated detection of samples that reached >20,000 cfu/mL due to PPC with psychrotolerant gram-negatives (70% sensitivity). These results show that tests still required and traditionally used in the dairy industry (e.g., coliform testing) are not suitable for monitoring for PPC. Rather, approaches that allow for detection of all gram-negative bacteria are essential for improved detection of PPC in fluid milk.

Alles AA ，Wiedmann M ，Martin NH 《-》

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.

Reichler SJ ，Trmčić A ，Martin NH ，Boor KJ ，Wiedmann M ... -  《-》

Short communication: Coliform Petrifilm as an alternative method for detecting total gram-negative bacteria in fluid milk.

Postpasteurization contamination (PPC) of fluid milk remains a challenge for some dairy processors. Pseudomonas is the most common contaminant of fluid milk after pasteurization, and therefore methods to detect PPC should be inclusive of Pseudomonas and other gram-negative contaminants (e.g., coliform bacteria). Our objective was to compare the ability of 3M (St. Paul, MN) coliform and Enterobacteriaceae (EB) Petrifilm to detect total gram-negative bacteria with that of the standard method, crystal violet tetrazolium agar. To that end, we evaluated coliform Petrifilm, EB Petrifilm, and crystal violet tetrazolium agar to detect gram-negative bacteria in naturally contaminated samples of fluid milk. A total of 92 observations derived from shelf-life testing of 33 milk samples from 5 different processing facilities were evaluated for (1) presence of coliforms on coliform Petrifilm at both 24 and 48 h of incubation; (2) presence of any growth, regardless of gas production, on coliform Petrifilm at both 24 and 48 h of incubation; (3) presence of EB on EB Petrifilm at both 24 and 48 h of incubation; (4) presence of any growth, regardless of gas or acid production, on EB Petrifilm at both 24 and 48 h of incubation; and (5) presence of gram-negative bacteria on crystal violet tetrazolium agar after 48 h of incubation. Sensitivity and specificity analysis of results indicated that compared with the standard method (i.e., crystal violet tetrazolium agar), the method that performed the best, based on balanced accuracy (i.e., the average of sensitivity and specificity), was coliform Petrifilm evaluated for the presence of any growth after 48 h of incubation (sensitivity = 0.787; specificity = 0.839). This method can be easily adopted by the dairy industry as many processing facilities already test for coliforms using coliform Petrifilm. Improving the ability of processors to detect PPC will improve the quality of the fluid milk supply.

Rojas A ，Murphy SI ，Wiedmann M ，Martin NH ... -  《-》

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.

Martin NH ，Boor KJ ，Wiedmann M 《-》

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.

Hervert CJ ，Martin NH ，Boor KJ ，Wiedmann M ... -  《-》