Tracking spore-forming bacterial contaminants in fluid milk-processing systems

The presence of psychrotolerant Bacillus species and related spore formers (e.g., Paenibacillus spp.) in milk has emerged as a key biological obstacle in extending the shelf life of high-temperature, short-time pasteurized fluid milk beyond 14 d. A recently developed rpoB DNA sequence-based subtyping method was applied to characterize spoilage bacteria present in raw milk supplies for 2 processing plants, and to assess transmission of these organisms into pasteurized products. Thirty-nine raw milk samples and 11 pasteurized product samples were collected to represent the processing continuum from incoming truck loads of raw milk to packaged products. Milk samples were held at 6°C for up to 16 d and plated for bacterial enumeration at various times throughout storage. Among the 88 bacterial isolates characterized, a total of 31 rpoB allelic types representing Bacillus and Paenibacillus spp. were identified, including 5 allelic types found in both raw milk and finished product samples. The presence of the same bacterial subtypes in raw and commercially pasteurized milk samples suggests that the raw milk supply represents an important source of these spoilage bacteria. Extension of the shelf life of high-temperature, short-time pasteurized fluid milk products will require elimination of these organisms from milk-processing systems.     

Short communication: Bacterial ecology of high-temperature, short-time pasteurized milk processed in the United States

To determine the microbial ecology of pasteurized milk within the United States, 2% fat pasteurized fluid milk samples were obtained from 18 dairy plants from 5 geographical areas representing the Northeast, Southeast, South, Midwest, and West. Of the 589 bacterial isolates identified using DNA sequence-based subtyping methods, 346 belonged to genera characterized as gram-positive endospore-forming bacteria (i.e., Bacillus and Paenibacillus). Of the 346 gram-positive endospore-forming bacteria isolated in the present study, 240 were classified into 45 allelic types identical to those previously identified from samples obtained in New York State, indicating the widespread presence of these microbes in fluid milk production and processing systems in the United States. More than 84% of the gram-positive spore-forming isolates characterized at d 1, 7, and 10 were of the genus Bacillus, whereas more than 92% of isolates characterized at d 17 of shelf life were of the genus Paenibacillus, indicating that the predominant gram-positive spoilage genera shifts from Bacillus spp. to Paenibacillus spp. during refrigerated storage.     

High temperature, short time pasteurization temperatures inversely affect bacterial numbers during refrigerated storage of pasteurized fluid milk

The grade A Pasteurized Milk Ordinance specifies minimum processing conditions of 72°C for at least 15 s for high temperature, short time (HTST) pasteurized milk products. Currently, many US milk-processing plants exceed these minimum requirements for fluid milk products. To test the effect of pasteurization temperatures on bacterial numbers in HTST pasteurized milk, 2% fat raw milk was heated to 60°C, homogenized, and treated for 25 s at 1 of 4 different temperatures (72.9, 77.2, 79.9, or 85.2°C) and then held at 6°C for 21 d. Aerobic plate counts were monitored in pasteurized milk samples at d 1, 7, 14, and 21 postprocessing. Bacterial numbers in milk processed at 72.9°C were lower than in milk processed at 85.2°C on each sampling day, indicating that HTST fluid milk-processing temperatures significantly affected bacterial numbers in fluid milk. To assess the microbial ecology of the different milk samples during refrigerated storage, a total of 490 psychrotolerant endospore-forming bacteria were identified using DNA sequence-based subtyping methods. Regardless of processing temperature, >85% of the isolates characterized at d 0, 1, and 7 postprocessing were of the genus Bacillus, whereas more than 92% of isolates characterized at d 14 and 21 postprocessing were of the genus Paenibacillus, indicating that the predominant genera present in HTST-processed milk shifted from Bacillus spp. to Paenibacillus spp. during refrigerated storage. In summary, 1) HTST processing temperatures affected bacterial numbers in refrigerated milk, with higher bacterial numbers in milk processed at higher temperatures; 2) no significant association was observed between genus isolated and pasteurization temperature, suggesting that the genera were not differentially affected by the different processing temperatures; and 3) although typically present at low numbers in raw milk, Paenibacillus spp. are capable of growing to numbers that can exceed Pasteurized Milk Ordinance limits in pasteurized, refrigerated milk.     

Siderophore production and utilization by milk spoilage Pseudomonas species

Many bacteria respond to potentially growth-limiting availability of iron by producing low-molecular-weight iron chelators (siderophores). The aim of this work was to examine the siderophores synthesized and utilized by Pseudomonas spp. implicated in milk spoilage. Twenty isolates of Pseudomonas spp. previously shown to have significant milk spoilage potential were tested for the ability to produce siderophores. Of these, 14 produced pyoverdin and 2 of these also produced pyochelin; 1 produced only pyochelin; 1 produced only salicylate; 2 produced non-pyoverdin, hydroxamate-containing siderophore; and 2 produced chrome azurol sulfonate reactive material that was neither pyoverdin nor pyochelin. There was considerable diversity among the pyoverdins produced. All isolates were shown to utilize iron complexed with exogenous pyoverdin, but usage of particular exogenous pyoverdins differed among isolates. Interference with the iron-uptake systems of the Pseudomonas spp. may be a means by which food spoilage can be slowed, and the pyoverdin system would appear to be a potential target. However, given the diversity of pyoverdins produced and utilized, and the presence of other siderophores, successful interference with bacterial iron acquisition in this context may be challenging.     

Enterotoxin-producing Staphylococcus aureus genotype B as a major contaminant in Swiss raw milk cheese

The objective of this study was to characterize Staphylococcus aureus isolates from Swiss raw milk cheeses that had been found to be contaminated with coagulase-positive staphylococci and to estimate the frequency of the various genotypes, in particular the mastitis-associated Staph. aureus genotype B (GTB). The isolates were also tested for staphylococcal enterotoxin (SE) genes and other virulence factors. From 623 coagulase-positive staphylococci isolated from 78 contaminated raw milk cheeses, 609 were found to be Staphylococcus aureus. Genotyping of all Staph. aureus isolates was performed by PCR amplification of the 16S–23S rRNA intergenic spacer region, as this method was used previously to differentiate between mastitis subtypes associated with their clinical outcome. In total, 20 different genotypes were obtained and the 5 most frequently occurring genotypes were distributed in 6.4% or more of the samples. The enterotoxin-producing Staph. aureus GTB, known for its high contagiousness and increased pathogenicity in Swiss mastitis herds, was found to be the most abundant subtype at the sample level (71.8%) as well as among the isolates (62.0%). A subset of 107 isolates of the different genotypes were analyzed for the presence of SE genes and revealed 9 different SE gene patterns, with sed being most frequently detected and 26% being PCR-negative for SE genes. Almost all isolates of the major contaminant GTB contained the SE gene pattern sed, sej, ser, with half of them additionally carrying sea. Production of SE in vitro was consistent with the SE genes detected in most of the cases; however, some isolated GTB did not produce SEA. Staphylococcus aureus Protein A (spa) typing revealed 30 different subtypes and most GTB isolates belonged to the bovine spa type t2953; GTB/t2953 was linked among other subtypes to SE production in cheese and staphylococcal intoxication cases. Furthermore, 1 of the 623 isolates was a methicillin-resistant Staph. aureus, which was an seh-carrying Staph. aureus spa type tbl 0635 (non-GTB). We conclude that control and reduction of enterotoxigenic Staph. aureus GTB in dairy herds in Switzerland will not only prevent economic losses at the farm level but also improve the safety of raw milk cheeses; distribution of methicillin-resistant Staph. aureus via raw milk cheese is of less concern.     

Minimizing the level of Bacillus cereus spores in farm tank milk

In a year-long survey on 24 Dutch farms, Bacillus cereus spore concentrations were measured in farm tank milk (FTM), feces, bedding material, mixed grass and corn silage, and soil from the pasture. The aim of this study was to determine, in practice, factors affecting the concentration of B. cereus spores in FTM throughout the year. In addition, the results of the survey were used in combination with a previously published modeling study to determine requirements for a strategy to control B. cereus spore concentrations in FTM below the MSL of 3 log₁₀ spores/L. The B. cereus spore concentration in FTM was 1.2 ± 0.05 log₁₀ spores/L and in none of samples was the concentration above the MSL. The spore concentration in soil (4.9 ± 0.04 log₁₀ spores/g) was more than 100-fold higher than the concentration in feces (2.2 ± 0.05 log₁₀ spores/g), bedding material (2.8 ± 0.07 log₁₀ spores/g), and mixed silage (2.4 ± 0.07 log₁₀ spores/g). The spore concentration in FTM increased between July and September compared with the rest of the year (0.5 ± 0.02 log₁₀ spores/L difference). In this period, comparable increases of the concentrations in feces (0.4 ± 0.03 log₁₀ spores/g), bedding material (0.5 ± 0.05 log₁₀ spores/g), and mixed silage (0.4 ± 0.05 log₁₀ spores/g) were found. The increased B. cereus spore concentration in FTM was not related to the grazing of cows. Significant correlations were found between the spore concentrations in FTM and feces (r = 0.51) and in feces and mixed silage (r = 0.43) when the cows grazed. The increased concentrations during summer could be explained by an increased growth of B. cereus due to the higher temperatures. We concluded that year-round B. cereus spores were predominantly transmitted from feeds, via feces, to FTM. Farmers should take measures that minimize the transmission of spores via this route by ensuring low initial contamination levels in the feeds (<3 log₁₀ spores/g) and by preventing growth of B. cereus in the farm environment. In addition, because of the extremely high B. cereus spore concentrations in soil, the contamination of teats with soil needs to be prevented.     

Identification of dairy farm management practices associated with the presence of psychrotolerant sporeformers in bulk tank milk

Some strains of sporeforming bacteria (e.g., Bacillus spp. and Paenibacillus spp.) can survive pasteurization and subsequently grow at refrigeration temperatures, causing pasteurized fluid milk spoilage. To identify farm management practices associated with different levels of sporeformers in raw milk, a bulk tank sample was obtained from and a management and herd health questionnaire was administered to 99 New York State dairy farms. Milk samples were spore pasteurized [80°C (176°F) for 12 min] and subsequently analyzed for most-probable number and for sporeformer counts on the initial day of spore pasteurization (SP), and after refrigerated storage (6°C) at 7, 14, and 21 d after SP. Management practices were analyzed for association with sporeformer counts and bulk tank somatic cell counts. Sixty-two farms had high sporeformer growth (≥3 log cfu/mL at any day after SP), with an average sporeformer count of 5.20 ± 1.41 mean log₁₀ cfu/mL at 21 d after SP. Thirty-seven farms had low sporeformer numbers (<3 log cfu/mL for all days after SP), with an average sporeformer count of 0.75 ± 0.94 mean log₁₀ cfu/mL at 21 d after SP. Farms with >25% of cows with dirty udders in the milking parlor were 3.15 times more likely to be in the high category than farms with ≤10% of milking cows with dirty udders. Farms with <200 cows were 3.61 times more likely to be in the high category than farms with ≥200 cows. Management practices significantly associated with increased bulk tank somatic cell count were a lack of use of the California mastitis test at freshening and >25% of cows with dirty udders observed in the milking parlor. Changes in management practices associated with cow cleanliness may directly ensure longer shelf life and higher quality of pasteurized fluid milk.     

How three adventitious lactic acid bacteria affect proteolysis and organic acid production in model Portuguese cheeses manufactured from several milk sources and two alternative coagulants

Model cheeses were manufactured according to a full factorial experimental design to help shed light on the individual and combined roles played by 3 native lactic acid bacteria (Lactococcus lactis ssp. lactis, Lactobacillus brevis, and Lactobacillus plantarum) upon proteolysis and organic acid evolution in cheese. The model cheeses were manufactured according to a generally representative Portuguese artisanal protocol, but the (ubiquitous) adventitious microflora in the cheesemaking milk were removed via sterilization before manufacture; therefore, the specific effects of only those lactic acid bacteria selected were monitored. In addition, 2 types of coagulant (animal and plant) and 3 types of cheesemaking milk (cow, sheep, and goat) were assessed to determine their influence on the final characteristics of the model cheeses. The nature of the coagulant appeared to be essential during the first stage of proteolysis as expected, whereas the contribution of those bacteria to the pools of total free AA and organic acids was crucial afterward. This was especially so in terms of the differences observed in the metabolisms of lactic acid (in the case of Lactococcus spp.) as well as acetic and citric acids (in the case of Lactobacillus spp.).     

Predictive modeling of Bacillus cereus spores in farm tank milk during grazing and housing periods

The shelf life of pasteurized dairy products depends partly on the concentration of Bacillus cereus spores in raw milk. Based on a translation of contamination pathways into chains of unit-operations, 2 simulation models were developed to quantitatively identify factors that have the greatest effect on the spore concentration in milk. In addition, the models can be used to determine the reduction in concentration that could be achieved via measures at the farm level. One model predicts the concentration when soil is the source of spores, most relevant during grazing of cows. The other model predicts the concentration when feed is the main source of spores, most relevant during housing of cows. It was estimated that when teats are contaminated with soil, 33% of the farm tank milk (FTM) contains more than 3 log₁₀ spores/L of milk. When feed is the main source, this is only 2%. Based on the predicted spore concentrations in FTM, we calculated that the average spore concentration in raw milk stored at the dairy processor during the grazing period is 3.5 log₁₀ spores/L of milk and during the housing period is 2.1 log₁₀ spores/L. It was estimated that during the grazing period a 99% reduction could be achieved if all farms minimize the soil contamination of teats and teat cleaning is optimized. During housing, reduction of the concentration by 60% should be feasible by ensuring spore concentrations in feed below 3 log₁₀ spores/g and a pH of the ration offered to the cows below 5. Implementation of these measures at the farm level ensures that the concentration of B. cereus spores in raw milk never exceeds 3 log₁₀ spores/L.     

Effect of applying bacterial inoculants containing different types of bacteria to corn silage on the performance of dairy cattle

This study examined the effect of applying different bacterial inoculants to corn silage at the time of ensiling on the performance of lactating dairy cows. Corn plants were harvested at 35% dry matter (DM), chopped, and ensiled in 2.4-m-wide bags after application of (1) no inoculant (CON); (2) Biotal Plus II (B2) containing Pediococcus pentosaceus and Propionibacteria freudenreichii; (3) Buchneri 40788 (BUC) containing Lactobacillus buchneri; or (4) Buchneri 500 (B500) containing Pediococcus pentosaceus and L. buchneri. All inoculants were supplied by Lallemand Animal Nutrition (Milwaukee, WI). Each of the 4 silages was included in separate total mixed rations consisting of 44% corn silage, 50% concentrate, and 6% alfalfa hay (DM basis). Fifty-two lactating Holstein cows were stratified according to milk production and parity and randomly assigned at 22 d in milk to the 4 dietary treatments. Cows were fed for ad libitum consumption and milked twice daily for 49 d. Dietary treatment did not affect intakes (kg/d) of DM (20.0), crude protein (CP; 3.7), neutral detergent fiber (NDF; 5.7), or acid detergent fiber (ADF; 3.6), or digestibility (%) of DM (73.9) or CP (72.4). However, NDF digestibility was lower in cows fed B2 compared with those fed other diets (45.3 vs. 53.0%). Consequently, cows fed B2 had lower digestible NDF intake (kg/d) than those fed other diets (2.5 vs. 3.0 kg/d). Dietary treatment did not affect milk yield (32.3 kg/d), efficiency of milk production (1.61), concentrations of milk fat (3.18%) and protein (2.79%), or yields of milk fat (1.03 kg/d) and protein (1.26 kg/d). Inoculant application to corn silage did not affect milk yield or feed intake of cows.     

Prevalence of Salmonellae, Listeria monocytogenes, and fecal coliforms in bulk tank milk on US dairies

The objective of this study was to determine the prevalence of Salmonella, Listeria monocytogenes, and fecal coliforms in bulk tank milk in the United States. As part of the NAHMS Dairy 2002 survey, 861 bulk tank milk samples were collected from farms in 21 states. Milk was directly plated on selective agars for direct bacterial enumeration and was enriched in selective broths to increase detection sensitivity. Somatic cell counts (SCC) and standard plate counts (SPC) were also determined. Coliforms were detected in 95% (818 of 860) of the samples, and the average SCC was 295,000 cells/mL. Twenty-two samples (2.6%) were culture-positive for Salmonella, and 9 serotypes were identified: Montevideo (n = 7), Newport (n = 4), Muenster (n = 2), Meleagridis (n = 2), Cerro (n = 2), 44:Z36 (Z38) (n = 2), Dublin (n = 1), Anatum (n = 1), and 9, 12:nonmo-tile (n = 1). Listeria monocytogenes was isolated from 56 (6.5%) samples, and serotyping of these isolates yielded 5 serotypes (1/2a, 1/2b, 3b, 4b, and 4c). Of the L. monocytogenes isolates, 93% were serotypes 1/2a, 1/2b, and 4b, the most common human clinical isolates. Regional differences in L. monocytogenes and Salmonella prevalence were observed, but more studies are needed to determine the validity of these differences. There were no apparent relationships between SCC or SPC and incidence of Salmonella or L. monocytogenes. Although the prevalence of L. monocytogenes and Salmonella was low, these pathogens represent a potential risk to consumers of raw milk and raw milk products.     

Microbiological spoilage of vacuum and modified atmosphere packaged Vietnamese Pangasius hypophthalmus fillets

This study investigated the identity, growth and metabolite production of micro-organisms causing spoilage of Pangasius hypophthalmus fillets packaged in air, vacuum and modified atmospheres (MAP) (MAP 1: 50%CO(2)-50%N(2) and MAP 2: 50%CO(2)-50%O(2)) during storage at 4 °C. Based on the time it took for psychrotrophic total colony counts to exceed 7 log cfu g(-1), the shelf life of the fillets packaged in air, vacuum, MAP 1 and MAP 2 was estimated to be 7, 10, 12 and 14 days respectively. The longest lag phases were observed in the samples packaged in MAP 2 (50%CO(2)-50%O(2)). In the fillets packaged in air and under vacuum, the dominant flora identified by partial 16S rDNA sequencing at the end of the shelf life generally consisted of Gram-negative bacteria mostly belonging to the genera Serratia and Pseudomonas. In contrast, lactic acid bacteria (Carnobacterium maltaromaticum and Carnobacterium divergens) and Brochothrix thermosphacta were identified as the dominant spoilage flora in the samples packaged under the two MAPs investigated. By means of solid-phase microextraction gas chromatography mass spectrometry (SPME GC-MS) analysis, volatile organic compounds in the headspace of the samples at the end of the shelf life were identified for each packaging condition. Based on these results, a selective ion flow tube mass spectrometry (SIFT-MS) method was developed to quantify the production of volatile metabolites during storage of the fillets. The results of these analyses indicated that several compounds contributed to the bacterial spoilage of Pangasius fillets e.g., ethanol, 2,3-butanediol, diacetyl, acetoin, ethyl acetate, acetic acid and sulfur compounds. It also emerged that the production of these compounds was dependent on the packaging condition applied.     

The occurrence of Staphylococcus aureus on a farm with small-scale production of raw milk cheese

In recent years, the small-scale production of raw milk products has increased in Norway, and there is some concern that such foods may pose a risk of staphylococcal food poisoning to consumers. The aim of the study was to evaluate potential sources of contamination of raw milk cheese with Staphylococcus aureus on a bovine dairy farm with small-scale production. Samples for bacteriological analyses (n = 144) were collected from the animals, the environment, processing equipments, from humans, and from cheeses at various stages of production. Staphylococcus aureus was isolated from 10 of 11 cows, the farmer, equipment, the environment, and the cheese. Seventy-five Staph. aureus isolates were genotyped by pulsed-field gel electrophoresis, tested for enterotoxin (SE) production by reversed passive latex agglutination, for SE genes by multiplex polymerase chain reaction, and for penicillin resistance by the cloverleaf method. Five different pulsotypes were identified and SE gene fragments were identified in 11 isolates, but no isolates produced SE or were penicillin resistant. Staphylococcus aureus was found throughout the farm, and appeared to be spread with the milk to the environment, equipment, and to products. One pulsotype dominated and was identified from most sample sites on the farm. Raw milk products are vulnerable to contamination with Staph. aureus. Strategies to reduce the occurrence of Staph. aureus in bulk milk are of particular importance on farms where milk is used for raw milk products.     

An improved method to culture Staphylococcus aureus from bovine milk

Staphylococcus aureus is an important udder pathogen often associated with subclinical mastitis in dairy cows. Identification of Staph. aureus-positive udder quarters and cows is an important part of control programs to reduce spread of Staph. aureus within and between dairy herds. Therefore, accurate and easy-to-perform culturing methods of Staph. aureus in milk are needed. In the present study, 8 methods for isolation of Staph. aureus in bovine milk samples were investigated. The methods involved different culturing volumes, enrichment, incubation, and freezing processes as well as sedimentation and use of the Mastistrip cassette (SVA, Uppsala, Sweden). Three different sets of milk samples were collected, and 6, 5, and 4 methods were used in each subset of samples. Our results indicate an increased probability of detecting Staph. aureus in milk samples when a simple incubation step (37°C for 18 h) without additives was included before culturing. Using this incubation method, the number of Staph. aureus-positive udder quarters and cows increased by 50 and 29%, respectively, compared with using the standard method of direct culturing of 10 μL of milk. The improved method may be especially useful for detection of low concentrations of Staph. aureus in milk; for example, when screening herds for Staph. aureus.     

A mixed-species microarray for identification of food spoilage bacilli

Failure of food preservation is frequently caused by thermostable spores of members of the Bacillaceae family, which show a wide spectrum of resistance to cleaning and preservation treatments. We constructed and validated a mixed-species genotyping array for 6 Bacillus species, including Bacillus subtilis, Bacillus licheniformis, Bacillus pumilus, Bacillus sporothermodurans, Bacillus cereus and Bacillus coagulans, and 4 Geobacillus species, including Geobacillus stearothermophilus, Geobacillus thermocatenulatus, Geobacillus toebii and Geobacillus sp., in order to track food spoilage isolates from ingredient to product. The discriminating power of the array was evaluated with sets of 42 reference and 20 test strains. Bacterial isolates contain a within-species-conserved core genome comprising 68-88% of the entire genome and a non-conserved accessory genome comprising 7-22%. The majority of the core genome markers do not hybridise between species, thus they allow for efficient discrimination at the species level. The accessory genome array markers provide high-resolution discrimination at the level of individual isolates from a single species. In conclusion, the reported mixed-species microarray contains discriminating markers that allow rapid and cost-effective typing of Bacillus food spoilage bacteria in a wide variety of food products.     

Combined pH and high hydrostatic pressure effects on Lactococcus starter cultures and Candida spoilage yeasts in a fermented milk test system during cold storage

The combined effects of high pressure processing (HPP) and pH on the glycolytic and proteolytic activities of Lactococcus lactis subsp. lactis, a commonly used cheese starter culture and the outgrowth of spoilage yeasts of Candida species were investigated in a fermented milk test system. To prepare the test system, L. lactis subsp. lactis C10 was grown in UHT skim milk to a final pH of 4.30 and then additional samples for treatment were prepared by dilution of fermented milk with UHT skim milk to pH levels of 5.20 and 6.50. These milk samples (pH 4.30, 5.20 and 6.50) with or without an added mixture of two yeast cultures, Candida zeylanoides and Candida lipolytica (10⁵ CFU mL⁻¹ of each species), were treated at 300 and 600 MPa (≤20 °C, 5 min) and stored at 4 °C for up to 8 weeks. Continuing acidification by starter cultures, as monitored during storage, was substantially reduced in the milk pressurised at pH 5.20 where the initial titratable acidity (TA) of 0.40% increased by only 0.05% (600 MPa) and 0.10% (300 MPa) at week 8, compared to an increase of 0.30% in untreated controls. No substantial differences were observed in pH or TA between pressure-treated and untreated milk samples at pH 4.30 or 6.50. The rate of proteolysis in milk samples at pH values of 5.20 and 6.50 during storage was significantly reduced by treatment at 600 MPa. Treatment at 600 MPa also reduced the viable counts of both Candida yeast species to below the detection limit (1 CFU mL⁻¹) at all pH levels for the entire storage period. However, samples treated at 300 MPa showed recovery of C. lipolytica from week 3 onwards, reaching 10⁶–10⁷ CFU mL⁻¹ by week 8. In contrast, C. zeylanoides did not show any recovery in any of the pressure-treated samples during storage.     

Antibacterial activity of the extracts from the fruit rinds of Garcinia cowa and Garcinia pedunculata against food borne pathogens and spoilage bacteria

The crude hexane and chloroform extracts from the fruit rinds of Garcinia cowa and Garcinia pedunculata were studied for their antibacterial activity against some foodborne pathogens and spoilage bacteria such as Bacillus cereus, Bacillus coagulans, Bacillus subtilis, Staphylococcus aureus and Escherichia coli. The minimum inhibitory concentrations (MICs) of the extracts determined by the agar dilution method were ranging from 15 to 500 μg/ml and 300 to 1250 μg/ml for G. cowa and G. pedunculata, respectively. However, the hexane and chloroform extracts from the fruit rinds of G. cowa exhibited marked inhibitory effect against all the test organisms and were more effective than that of G. pedunculata extracts. The antibacterial activity of all the extracts was more pronounced against the tested Gram-positive bacteria than the tested Gram-negative bacterium. Furthermore, this study is the first report on the in vitro antibacterial activity of extracts from the fruit rinds of G. cowa and G. pedunculata.     

Molecular Subtyping and Tracking of Listeria monocytogenes in Latin-Style Fresh-Cheese Processing Plants

Latin-style fresh cheeses, which have been linked to at least 2 human listeriosis outbreaks in the United States, are considered to be high-risk foods for Listeria monocytogenes contamination. We evaluated L. monocytogenes contamination patterns in 3 Latin-style fresh-cheese processing plants to gain a better understanding of L. monocytogenes contamination sources in the manufacture of these cheeses. Over a 6-mo period, 246 environmental samples were collected and analyzed for L. monocytogenes using both the Food and Drug Administration (FDA) method and the Biosynth L. monocytogenes detection system (LMDS). Finished cheese samples from the same plants (n = 111) were also analyzed by the FDA method, which was modified to include L. monocytogenes plating medium (LMPM) and the L. monocytogenes confirmatory plating medium (LMCM) used in the LMDS method. Listeria monocytogenes was detected in 6.3% of cheese and 11.0% of environmental samples. Crates, drains, and floor samples showed the highest contamination rates, with 55.6, 30.0, and 20.6% L. monocytogenes positive samples, respectively. Finished products and food contact surfaces were positive in only one plant. The FDA method showed a higher sensitivity than the LMDS method for detection of L. monocytogenes from environmental samples. The addition of LMPM and LMCM media did not further enhance the performance of the FDA method for L. monocytogenes detection from finished products. Molecular subtyping (PCR-based allelic analysis of the virulence genes actA and hly and automated ribotyping) was used to track contamination patterns. Ribotype DUP-1044A, which had previously been linked to a 1998 multistate human listeriosis outbreak in the United States, was the most commonly identified subtype (20/36 isolates) and was isolated from 2 plants. This ribotype was persistent and widespread in one factory, where it was also responsible for the contamination of finished products. We hypothesize that this ribotype may represent a clonal group with a specific ability to persist in food processing environments. While previous listeriosis outbreaks were linked to Latin-style fresh cheeses made from unpasteurized milk, the presence of this organism in pasteurized cheese products illustrates that persistent environmental contamination also represents an important source of finished product contamination.     

The analysis of milk components and pathogenic bacteria isolated from bovine raw milk in Korea

Bovine mastitis can be diagnosed by abnormalities in milk components and somatic cell count (SCC), as well as by clinical signs. We examined raw milk in Korea by analyzing SCC, milk urea nitrogen (MUN), and the percentages of milk components (milk fat, protein, and lactose). The associations between SCC or MUN and other milk components were investigated, as well as the relationships between the bacterial species isolated from milk. Somatic cell counts, MUN, and the percentages of milk fat, protein, and lactose were analyzed in 30,019 raw milk samples collected from 2003 to 2006. The regression coefficients of natural logarithmic-transformed SCC (SCCt) on milk fat (−0.0149), lactose (−0.8910), and MUN (−0.0096), and those of MUN on milk fat (−0.3125), protein (−0.8012), and SCCt (−0.0671) were negative, whereas the regression coefficient of SCCt on protein was positive (0.3023). When the data were categorized by the presence or absence of bacterial infection in raw milk, SCCt was negatively associated with milk fat (−0.0172), protein (−0.2693), and lactose (−0.4108). The SCCt values were significantly affected by bacterial species. In particular, 104 milk samples infected with Staphylococcus aureus had the highest SCCt (1.67) compared with milk containing other mastitis-causing bacteria: coagulase-negative staphylococci (n = 755, 1.50), coagulase-positive staphylococci (except Staphylococcus aureus; n = 77, 1.59), Streptococcus spp. (Streptococcus dysgalactiae, n = 37; Streptococcus uberis, n = 12, 0.83), Enterococcus spp. (n = 46, 1.04), Escherichia coli (n = 705, 1.56), Pseudomonas spp. (n = 456, 1.59), and yeast (n = 189, 1.52). These results show that high SCC and MUN negatively affect milk components and that a statistical approach associating SCC, MUN, and milk components by bacterial infection can explain the patterns among them. Bacterial species present in raw milk are an important influence on SCC in Korea.     

Short communication: growth characteristics of Streptococcus uberis in UHT-treated milk

Streptococcus uberis is an important environmental pathogen associated with bovine mastitis as well as with high total bacterial numbers in bulk tank milk. This study was conducted to determine whether S. uberis reproduction is likely to contribute to high bacterial numbers in bulk tank milk. Four S. uberis raw milk isolates were individually inoculated into UHT-treated milk and incubated at 4.4 or 7 degrees C for up to 5 d to simulate appropriate cooling; at 10 degrees C for 5 d to simulate marginally inadequate cooling; at 21 or 25 degrees C for 7 h to simulate ambient temperatures; or at 32 degrees C for 7 h to simulate elevated temperature conditions. None of the S. uberis isolates grew at either 4.4 or 7 degrees C. Streptococcus uberis growth at 10 degrees C appeared to be ribotype-specific. Although ribotype 116-520-S-1 isolates did not grow at 10 degrees C, ribotype 116-520-S-2 isolate numbers increased up to 3.5 log10 cfu/mL within 5 d. Generation times were calculated as 2.7 +/- 0.1 h, 2.1 +/- 0.1 h, and 1.0 +/- 0.1 h for 116-520-S-1 isolates and 1.8 +/- 0.4 h, 1.3 +/- 0.3 h, and 0.8 +/- 0.1 h for 116-520-S-2 isolates at 21, 25, and 32 degrees C, respectively. Our results suggest that high numbers of S. uberis in bulk tank milk are more likely to reflect high numbers of S. uberis shed by mastitic cows, rather than multiplication of these organisms under cooling conditions required for production of Grade A milk.