Incidence and diversity of potentially highly heat-resistant spores isolated at dairy farms

The presence of highly heat-resistant spores of Bacillus sporothermodurans in ultrahigh-temperature or sterilized consumer milk has emerged as an important item in the dairy industry. Their presence is considered undesirable since they hamper the achievement of commercial sterility requirements. By using a selective 30-min heat treatment at 100 degrees C, 17 Belgian dairy farms were screened to evaluate the presence, sources, and nature of potentially highly heat-resistant spores in raw milk. High numbers of these spores were detected in the filter cloth of the milking equipment and in green crop and fodder samples. About 700 strains were isolated after the selective heating, of which 635 could be screened by fatty acid methyl ester analysis. Representative strains were subjected to amplified ribosomal DNA restriction analysis, 16S rRNA gene sequencing, percent G+C content, and DNA-DNA reassociations for further identification. The strain collection showed a remarkable diversity, with representatives of seven aerobic spore-forming genera. Bacillus licheniformis and Bacillus pallidus were the most predominant species overall. Twenty-three percent of the 603 spore-forming isolates proved to belong to 18 separate novel species. These findings suggest that the selective heating revealed a pool of unknown organisms with a higher heat-resistant character. This study showed that high spore counts can occur at the dairy farm and that feed and milking equipment can act as reservoirs or entry points for potentially highly heat-resistant spores into raw milk. Lowering this spore load by good hygienic measures could probably further reduce the contamination level of raw milk, in this way minimizing the aerobic spore-forming bacteria that could lead to spoilage of milk and dairy products. Assessment and characterization of this particular flora are of great importance to allow the dairy or food industry to adequately deal with newly arising microbiological problems.     

Incidence and Diversity of Potentially Highly Heat-Resistant Spores Isolated at Dairy Farms

The presence of highly heat-resistant spores of Bacillus sporothermodurans in ultrahigh-temperature or sterilized consumer milk has emerged as an important item in the dairy industry. Their presence is considered undesirable since they hamper the achievement of commercial sterility requirements. By using a selective 30-min heat treatment at 100°C, 17 Belgian dairy farms were screened to evaluate the presence, sources, and nature of potentially highly heat-resistant spores in raw milk. High numbers of these spores were detected in the filter cloth of the milking equipment and in green crop and fodder samples. About 700 strains were isolated after the selective heating, of which 635 could be screened by fatty acid methyl ester analysis. Representative strains were subjected to amplified ribosomal DNA restriction analysis, 16S rRNA gene sequencing, percent G+C content, and DNA-DNA reassociations for further identification. The strain collection showed a remarkable diversity, with representatives of seven aerobic spore-forming genera. Bacillus licheniformis and Bacillus pallidus were the most predominant species overall. Twenty-three percent of the 603 spore-forming isolates proved to belong to 18 separate novel species. These findings suggest that the selective heating revealed a pool of unknown organisms with a higher heat-resistant character. This study showed that high spore counts can occur at the dairy farm and that feed and milking equipment can act as reservoirs or entry points for potentially highly heat-resistant spores into raw milk. Lowering this spore load by good hygienic measures could probably further reduce the contamination level of raw milk, in this way minimizing the aerobic spore-forming bacteria that could lead to spoilage of milk and dairy products. Assessment and characterization of this particular flora are of great importance to allow the dairy or food industry to adequately deal with newly arising microbiological problems.     

Identification and characterization of psychrotolerant sporeformers associated with fluid milk production and processing

Psychrotolerant spore-forming bacteria represent a major challenge to the goal of extending the shelf life of pasteurized dairy products. The objective of this study was to identify prominent phylogenetic groups of dairy-associated aerobic sporeformers and to characterize representative isolates for phenotypes relevant to growth in milk. Analysis of sequence data for a 632-nucleotide fragment of rpoB showed that 1,288 dairy-associated isolates (obtained from raw and pasteurized milk and from dairy farm environments) clustered into two major divisions representing (i) the genus Paenibacillus (737 isolates, including the species Paenibacillus odorifer, Paenibacillus graminis, and Paenibacillus amylolyticus sensu lato) and (ii) Bacillus (n = 467) (e.g., Bacillus licheniformis sensu lato, Bacillus pumilus, Bacillus weihenstephanensis) and genera formerly classified as Bacillus (n = 84) (e.g., Viridibacillus spp.). When isolates representing the most common rpoB allelic types (ATs) were tested for growth in skim milk broth at 6°C, 6/9 Paenibacillus isolates, but only 2/8 isolates representing Bacillus subtypes, grew >5 log CFU/ml over 21 days. In addition, 38/40 Paenibacillus isolates but only 3/47 Bacillus isolates tested were positive for β-galactosidase activity (including some isolates representing Bacillus licheniformis sensu lato, a common dairy-associated clade). Our study confirms that Paenibacillus spp. are the predominant psychrotolerant sporeformers in fluid milk and provides 16S rRNA gene and rpoB subtype data and phenotypic characteristics facilitating the identification of aerobic spore-forming spoilage organisms of concern. These data will be critical for the development of detection methods and control strategies that will reduce the introduction of psychrotolerant sporeformers and extend the shelf life of dairy products.     

The Microflora of Steam Sterilized Milking Equipment

S ummary : The classification of 1,466 bacteria, isolated on Yeastrel-milk agar incubated at 30° from 68 rinses of steam sterilized dairy equipment taken at five farms, showed that micrococci were dominant, with corynebacteria and aerobic sporeforming rods frequent, representatives of these three groups constituting 73% of the isolates. The microflora was characterized by the dominance of organisms which were relatively inactive in milk. Typical milk souring organisms such as lactic streptococci and coli-aerogenes organisms were rarely isolated, but anaerogenic Gram-negative rods resembling Achromobacter, Pseudomonas and Flavobacterium were not uncommon on milking machine clusters and on a cooling unit. The glass recorder jars contained a relatively high proportion of sporeformers.     

Comparison of types and antimicrobial susceptibility of Staphylococcus from conventional and organic dairies in west-central Minnesota, USA

Aims:  To assess whether conventional and organic dairy management practices are associated with differences in the susceptibility of Staphylococcus to antimicrobial agents.
Methods and Results:  Staphylococcus was isolated from milk samples collected from conventional and organic dairies in west-central Minnesota. Isolates were categorized as (1) coagulase-positive, (2) novobiocin-sensitive coagulase-negative or (3) novobiocin-resistant coagulase-negative. Novobiocin-resistant coagulase-negative Staphylococcus (CNS) was more common on conventional farms and novobiocin-sensitive CNS predominated the isolates from organic farms. Overall, a larger proportion of isolates from organic rather than conventional farms were susceptible to erythromycin, pirlimycin and tetracycline. However, for pirlimycin and tetracycline, different patterns of susceptibility were observed among Staphylococcus categories.
Conclusion:  In this study, organic dairy management was associated with more overall antimicrobial susceptibility among Staphylococcus than was conventional management. However, different patterns of susceptibility among Staphylococcus categories suggest that multiple management practices, including some unrelated to antimicrobial use, may contribute to the observed differences in susceptibility.
Significance and Impact of the Study:  This study adds to our understanding of the implications of dairy management choices.     

Incidence and characterization of Bacillus cereus isolates contaminating dairy products.

A total of 293 dairy products purchased from local markets were examined to determine the incidence of and characterize Bacillus cereus. Isolations were made on mannitol-egg yolk-polymyxin B agar medium and confirmed by several staining and biochemical tests. B. cereus occurred in 17% of fermented milks, 52% of ice creams, 35% of soft ice creams, 2% of pasteurized milks and pasteurized fruit- or nut-flavored reconstituted milks, and 29% of milk powders, mostly in fruit- or nut-flavored milk mixes. The average population of B. cereus in these dairy products was 15 to 280 CFU/ml or CFU/g (range, 5 to 800). The characteristics of these B. cereus isolates in terms of heat resistance, biochemical reactions, and antibiotic susceptibility were similar to previously reported data except for a higher utilization of sucrose. Some isolates were especially resistant to carbenicillin, nalidixic acid, streptomycin, and tetracycline. The MICs for the isolates were also determined. All of the tested isolates lysed rabbit erythrocytes; 98% showed verotoxicity, 68% showed cytotonic toxicity for CHO cells, and 3 of 11 selected isolates that showed strong hemolysin activity killed adult mice.     

Occurrence of Bacillus sporothermodurans and other aerobic spore-forming species in feed concentrate for dairy cattle

AIMS: To determine the aerobic spore composition and presence of Bacillus sporothermodurans spores in feed concentrate for dairy cattle. METHODS AND RESULTS: Six feed concentrate samples from five different farms were analysed. High levels of spores (up to 10(6) spores g(-1)) were found. Identification of 100 selected isolates was obtained by a combination of fatty acid methyl esters analysis, amplified ribosomal DNA restriction analysis and 16S rDNA sequencing. Ninety-seven isolates could be identified to the species level or assigned to a phylogenetic species group. Most of the isolates obtained after a heat treatment of 10 min at 80 degrees C were identified as members of the B. subtilis group (32 isolates), B. pumilus (25 isolates), B. clausii (eight isolates) and B. licheniformis (eight isolates). The isolates with very heat-resistant spores, obtained after a heat treatment of 30 min at 100 degrees C, were identified as members of the B. subtilis group (five isolates), B. sporothermodurans (three isolates), B. amyloliquefaciens (one isolate), B. oleronius (one isolate) and B. pallidus (one isolate). Bacillus cereus was present in each feed concentrate sample and was isolated using a selective mannitol egg yolk polymyxin agar medium. CONCLUSIONS: Feed concentrate for dairy cattle contains known as well as as yet unknown species of Bacillus and related genera with properties relevant to the dairy sector. SIGNIFICANCE AND IMPACT OF THE STUDY: The results formulate the hypothesis that feed concentrate can be a contamination source of spores, including those of B. sporothermodurans, for raw milk at the farm level.     

Incidence and characterization of Bacillus cereus isolates contaminating dairy products

A total of 293 dairy products purchased from local markets were examined to determine the incidence of and characterize Bacillus cereus. Isolations were made on mannitol-egg yolk-polymyxin B agar medium and confirmed by several staining and biochemical tests. B. cereus occurred in 17% of fermented milks, 52% of ice creams, 35% of soft ice creams, 2% of pasteurized milks and pasteurized fruit- or nut-flavored reconstituted milks, and 29% of milk powders, mostly in fruit- or nut-flavored milk mixes. The average population of B. cereus in these dairy products was 15 to 280 CFU/ml or CFU/g (range, 5 to 800). The characteristics of these B. cereus isolates in terms of heat resistance, biochemical reactions, and antibiotic susceptibility were similar to previously reported data except for a higher utilization of sucrose. Some isolates were especially resistant to carbenicillin, nalidixic acid, streptomycin, and tetracycline. The MICs for the isolates were also determined. All of the tested isolates lysed rabbit erythrocytes; 98% showed verotoxicity, 68% showed cytotonic toxicity for CHO cells, and 3 of 11 selected isolates that showed strong hemolysin activity killed adult mice.     

Comparison of prevalence and antimicrobial susceptibilities of Campylobacter spp. isolates from organic and conventional dairy herds in Wisconsin

The prevalence and antimicrobial susceptibilities of Campylobacter spp. isolates from bovine feces were compared between organic and conventional dairy herds. Thirty organic dairy herds, where antimicrobials are rarely used for calves and never used for cows, were compared with 30 neighboring conventional dairy farms, where antimicrobials were routinely used for animals for all ages. Fecal specimens from 10 cows and 10 calves on 120 farm visits yielded 332 Campylobacter isolates. The prevalence of Campylobacter spp. in organic and conventional farms was 26.7 and 29.1%, and the prevalence was not statistically different between the two types of farms. Campylobacter prevalence was significantly higher in March than in September, higher in calves than in cows, and higher in smaller farms than in large farms. The rates of retained placenta, pneumonia, mastitis, and abortion were associated with the proportion of Campylobacter isolation from fecal samples. The gradient disk diffusion MIC method (Etest) was used for testing susceptibility to four antimicrobial agents: ciprofloxacin, gentamicin, erythromycin, and tetracycline. Two isolates were resistant to ciprofloxacin, and none of isolates was resistant to gentamicin or erythromycin. Resistance to tetracycline was 45% (148 of 332 isolates). Tetracycline resistance was found more frequently in calves than in cows (P = 0.042), but no difference was observed between organic and conventional farms. When we used Campylobacter spp. as indicator bacteria, we saw no evidence that restriction of antimicrobial use on dairy farms was associated with prevalence of resistance to ciprofloxacin, gentamicin, erythromycin, and tetracycline.     

Determining the source of Bacillus cereus and Bacillus licheniformis isolated from raw milk, pasteurized milk and yoghurt

Aims:  Strain-specific detection of Bacillus cereus and Bacillus licheniformi s in raw and pasteurized milk, and yoghurt during processing.
Methods and Results:  Randomly selected isolates of Bacillus spp. were subjected to PCR analysis, where single primer targeting to the repetitive sequence Box elements was used to fingerprint the species. The isolates were separated into six different fingerprint patterns. The results show that isolates clustered together at about the 57% similarity level with two main groups at the 82% and 83% similarity levels, respectively. Contamination with identical strains both of B. cereus and B . licheniformis in raw and pasteurized milk was found as well as contaminated with different strains (in the case of raw milk and yoghurt/pasteurized milk and yoghurt). Several BOX types traced in processed milk samples were not discovered in the original raw milk.
Conclusions:  BOX-PCR fingerprinting is useful for characterizing Bacillus populations in a dairy environment. It can be used to confirm environmental contamination, eventually clonal transfer of Bacillus strains during the technological processing of milk.
Significance and Impact of the Study:  Despite the limited number of strains analysed, the two Bacillus species yielded adequately detectable banding profiles, permitting differentiation of bacteria at the strain level and showing their diversity throughout dairy processing.     

基于高通量测序技术分析四川两个奶牛场乳汁的菌群差异

【目的】本试验测定了两个奶牛场健康乳汁和乳房炎乳汁中微生物菌群的变化,以揭示不同奶牛场之乳汁菌群的异同,评估其对乳汁代谢的影响是否相同。【方法】采用16S rRNA高通量测序技术,分别测定两个奶牛场6头健康奶牛和6头乳房炎奶牛乳汁中微生物16S rRNA V4区序列,并对菌群群落结构和多样性进行比较,分析场内及场间的乳汁菌群差异。【结果】四组乳汁样本共获得4013234条原始序列,经过滤后获得2887024条优化序列。Alpha多样性Chao指数、Ace指数、Shannon指数、Simpson指数差异均不显著(P0.05);Beta多样性四组样本均分别聚类;在场1和场2中,引起奶牛乳房炎的优势菌属分别是克雷伯氏菌属和埃希氏菌属;在2个奶牛场的健康乳汁中,场2的埃希氏菌属、葡萄球菌属的丰度显著高于场1;在2个奶牛场的乳房炎乳汁中,场2的埃希氏菌属、乳球菌属的丰度显著高于场1;2个奶牛场健康乳汁中的嗜冷菌总丰度分别为31.87%和38.72%;关联分析及功能预测分析表明,2个奶牛场健康乳汁与乳房炎乳汁优势物种之间的关系差异较大;场1无论是Level 1还是Level 2水平,均发现显著性差异的代谢通路,而场2均未发现显著性差异的代谢通路。【结论】本试验研究了两个奶牛场健康乳汁和乳房炎乳汁微生物菌群之间的异同,为两个奶牛场在乳房炎的预防工作以及原料奶在冷链运输过程中质量控制提供理论依据。     

Failure to recover Bacillus thuringiensis from the Lützow-Holm Bay region of Antarctica

A total of 65 samples, consisting of 19 floral materials and 46 soil samples, collected from a coastal area of Lu¨tzow-Holm Bay on the Antarctic sub-continent, were examined for the presence of Bacillus thuringiensis . The frequency of samples containing aerobic spore-forming bacteria was 41·5% and most of the sporeformers were not allied to the B. cereus group. Only one sample from a floating mat of blue-green algae contained B. cereus -like sporeformers. However, there was no evidence for the formation of parasporal inclusions in these isolates. The result suggests that the natural microflora of Antarctica may lack B. thuringiensis , a mesophile widely distributed in other environments.     

Epidemiological typing of Bacillus spp. isolated from food.

Biotypes, fatty acid profiles, and restriction fragment length polymorphisms of a PCR product (PCR-RFLP of the cereolysin AB gene) were compared for 62 isolates of the Bacillus cereus group. Eleven isolates originated from various foods, and 51 isolates were obtained from pasteurized milk which had been processed by two different dairies. The isolates were clustered into 6 biotypes, 10 fatty acid groups, or 7 PCR-RFLP clusters. Isolates with mesophilic or psychrotrophic characteristics were preferentially distributed into specific fatty acid or PCR-RFLP groups (P = 0.004). Unique fatty acid clusters were predominantly found in milk samples of each dairy (P < 0.0001), suggesting that certain dairy plants may harbor plant-specific B. cereus which might constantly contribute to postpasteurization contamination.     

Factors contributing to the seasonal variation of Bacillus spp. in pasteurized dairy products

The seasonal variation in the spoilage of pasteurized products, especially double cream, by spore-forming bacteria was due to a number of factors. By far the most important was the seasonal variation in the types of organisms isolated from raw milks. Psychrotrophic spore-formers predominated in the summer-autumn months and these strains were able to germinate rapidly and grow in refrigerated dairy products. There was evidence that the concentration of one or more factors which promoted germination of psychrotrophic strains of Bacillus spp. in milk was higher during the summer than in the winter. This again may contribute to seasonal differences in spoilage by spore-forming bacteria. Post-heat treatment contamination by spores of Bacillus spp. may also be more prevalent in the summer-autumn period and evidence was obtained that spores associated with post-pasteurization contamination could germinate and grow more rapidly than those introduced into the product from the raw material. Thus, the increased spoilage of pasteurized products by Bacillus spp. observed in the June to October period may be due to a combination of factors. The relative contribution that each makes is not easily resolved.     

Comparison of Prevalence and Antimicrobial Susceptibilities of Campylobacter spp. Isolates from Organic and Conventional Dairy Herds in Wisconsin

The prevalence and antimicrobial susceptibilities of Campylobacter spp. isolates from bovine feces were compared between organic and conventional dairy herds. Thirty organic dairy herds, where antimicrobials are rarely used for calves and never used for cows, were compared with 30 neighboring conventional dairy farms, where antimicrobials were routinely used for animals for all ages. Fecal specimens from 10 cows and 10 calves on 120 farm visits yielded 332 Campylobacter isolates. The prevalence of Campylobacter spp. in organic and conventional farms was 26.7 and 29.1%, and the prevalence was not statistically different between the two types of farms. Campylobacter prevalence was significantly higher in March than in September, higher in calves than in cows, and higher in smaller farms than in large farms. The rates of retained placenta, pneumonia, mastitis, and abortion were associated with the proportion of Campylobacter isolation from fecal samples. The gradient disk diffusion MIC method (Etest) was used for testing susceptibility to four antimicrobial agents: ciprofloxacin, gentamicin, erythromycin, and tetracycline. Two isolates were resistant to ciprofloxacin, and none of isolates was resistant to gentamicin or erythromycin. Resistance to tetracycline was 45% (148 of 332 isolates). Tetracycline resistance was found more frequently in calves than in cows (P = 0.042), but no difference was observed between organic and conventional farms. When we used Campylobacter spp. as indicator bacteria, we saw no evidence that restriction of antimicrobial use on dairy farms was associated with prevalence of resistance to ciprofloxacin, gentamicin, erythromycin, and tetracycline.     

Factors contributing to the seasonal variation of Bacillus spp. in pasteurized dairy products

The seasonal variation in the spoilage of pasteurized products, especially double cream, by spore-forming bacteria was due to a number of factors. By far the most important was the seasonal variation in the types of organisms isolated from raw milks. Psychrotrophic spore-formers predominated in the summer-autumn months and these strains were able to germinate rapidly and grow in refrigerated dairy products. There was evidence that the concentration of one or more factors which promoted germination of psychrotrophic strains of Bacillus spp. in milk was higher during the summer than in the winter. This again may contribute to seasonal differences in spoilage by spore-forming bacteria. Post-heat treatment contamination by spores of Bacillus spp. may also be more prevalent in the summer-autumn period and evidence was obtained that spores associated with post-pasteurization contamination could germinate and grow more rapidly than those introduced into the product from the raw material. Thus, the increased spoilage of pasteurized products by Bacillus spp. observed in the June to October period may be due to a combination of factors. The relative contribution that each makes is not easily resolved.     

Molecular characterisation of bacterial contamination in semi-final gelatine extracts, using denaturing gradient gel electrophoresis

Contamination of gelatine may affect the safety and/or quality of its applications. Characterisation of bacterial isolates from semi-final gelatine batches revealed thermotolerant, aerobic, endosporeforming contaminants. In this paper, bacterial contamination in gelatine batches is analysed without previous isolation, by means of denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA sequences. V9 and V6-V8 regions of the 16S rDNA gene were found more suitable for this purpose than V1 or V3 regions. Bacillus fumarioli, Bacillus licheniformis, members of the 'Bacillus cereus group', Bacillus subtilis, Bacillus shackletonii, Brevibacillus borstelensis and Brevibacillus agri were detected.     

Phytoestrogens and Their Metabolites in Bulk-Tank Milk: Effects of Farm Management and Season

Phytoestrogens have structures similar to endogenous steroids and may induce or inhibit the response of hormone receptors. The objectives of the present study were to compare the effects of long-term vs. short-term grassland management in organic and conventional dairy production systems, compare organic and conventional production systems and assess seasonal variation on phytoestrogen concentrations in bulk-tank milk. The concentrations of phytoestrogens were analyzed in bulk-tank milk sampled three times in two subsequent years from 28 dairy farms: Fourteen organic (ORG) dairy farms with either short-term or long-term grassland management were paired with 14 conventional (CON) farms with respect to grassland management. Grassland management varied in terms of time since establishment. Short-term grassland management (SG) was defined as establishment or reseeding every fourth year or more often, and long-term grassland management (LG) was defined as less frequent establishment or reseeding. The proportion of red clover (Trifolium pretense L.) in the herbage was positively correlated with milk concentrations of the mammalian isoflavone equol. Therefore, organically produced bulk-tank milk contained more equol than conventionally produced milk, and milk from ORG-SG farms had more equol than milk from ORG-LG farms. Milk produced during the indoor-feeding periods had more equol than milk produced during the outdoor feeding period, because pastures contained less red clover than fields intended for silage production. Organically produced milk had also higher concentrations of the mammalian lignan enterolactone, but in contrast to equol, concentrations increased in the outdoor-feeding periods compared to the indoor-feeding periods. There were no indications of fertility problems on ORG-SG farms who had the highest red clover proportions in the herbage. This study shows that production system, grassland management, and season affect milk concentrations of phytoestrogens. However, compared to soy products, milk concentrations of phytoestrogens are low and future studies are required to investigate if the intake of phytoestrogens from dairy products has physiological effects in humans.     

L-forms of aerobic spore-forming bacteria isolated from urologic patients

New data are presented on the ability of different aerobic spore-forming bacteria isolated from the organism of urological patients to produce L-forms of these microorganisms in the presence of penicillin and ampicillin. Bacillus cereus is shown to be the most resistant to these antibiotics.     

Detection of Hippurate Hydrolase among Bacillus Species by Thin Layer Chromatography and other Methods

S ummary . The ability of 111 isolates of 33 species of Bacillus to hydrolyze hippurate into benzoic acid and glycine was tested. All, other than the strains classified as B. badius, B. cereus var. mycoides, B. cereus var. thuringiensis, B. macquariensis, B. medusa, B. pacificus, B. psychrosaccharolyticus and some B. macerans and B. polymyxa strains, were positive by thin layer chromatography (TLC). Of the 4 methods compared (hippurate agar, the sulphuric acid method, precipitation with ferric chloride and TLC), only the TLC procedure allowed a rapid and definite detection of the hydrolytic product, benzoic acid. The use of hippurate hydrolase as an additional diagnostic aid in the differentiation of aerobic spore-forming bacilli is discussed. Other characters were used to compare some recently described Bacillus spp. The combination B. cereus subsp. medusa n. subsp. is proposed.