Comparative analysis of the diversity of aerobic spore-forming bacteria in raw milk from organic and conventional dairy farms

Bacterial contamination of raw milk can originate from different sources: air, milking equipment, feed, soil, faeces and grass. It is hypothesized that differences in feeding and housing strategies of cows may influence the microbial quality of milk. This assumption was investigated through comparison of the aerobic spore-forming flora in milk from organic and conventional dairy farms. Laboratory pasteurized milk samples from five conventional and five organic dairy farms, sampled in late summer/autumn and in winter, were plated on a standard medium and two differential media, one screening for phospholipolytic and the other for proteolytic activity of bacteria. Almost 930 isolates were obtained of which 898 could be screened via fatty acid methyl ester analysis. Representative isolates were further analysed using 16S rRNA gene sequencing and (GTG)(5)-PCR. The majority of aerobic spore-formers in milk belonged to the genus Bacillus and showed at least 97% 16S rRNA gene sequence similarity with type strains of Bacillus licheniformis, Bacillus pumilus, Bacillus circulans, Bacillus subtilis and with type strains of species belonging to the Bacillus cereus group. About 7% of all isolates may belong to possibly new spore-forming taxa. Although the overall diversity of aerobic spore-forming bacteria in milk from organic vs. conventional dairy farms was highly similar, some differences between both were observed: (i) a relatively higher number of thermotolerant organisms in milk from conventional dairy farms compared to organic farms (41.2% vs. 25.9%), and (ii) a relatively higher number of B. cereus group organisms in milk from organic (81.3%) and Ureibacillus thermosphaericus in milk from conventional (85.7%) dairy farms. One of these differences, the higher occurrence of B. cereus group organisms in milk from organic dairy farms, may be linked to differences in housing strategy between the two types of dairy farming. However, no plausible clarification was found for the relatively higher number of thermotolerant organisms and the higher occurrence of U. thermosphaericus in milk from conventional dairy farms. Possibly this is due to differences in feeding strategy but no decisive indications were found to support this assumption.     

'BITTY' CREAM: THE OCCURRENCE AND SIGNIFICANCE OF BACILLUS CEREUS SPORES IN RAW MILK SUPPLIES

SUMMARY: Methods for determining the Bacillus cereus content of milks and of rinses of dairy equipment are described and their limitations discussed. Milk samples from various sources were examined throughout the year for 'bittiness' as well as for B. cereus. The organism was not detected in all samples showing bittiness. There was a marked seasonal variation in the B. cereus index of raw milk supplies; maximum numbers were obtained from July to September and minimum numbers in April and May. Rinses of farm dairy equipment yielded few B. cereus spores but milk cans not uncommonly contained large numbers, especially in the summer months, when 10·5% of cans showed more than 5 × 10³/can. Preliminary observations on other sources of B. cereus are described and some of the problems of the control of this organism in raw milk supplies are discussed.     

Incidence of thermoduric psychrotrophs in milk produced in the west of Scotland

Thermoduric psychrotrophs were found in 27.2% of milk samples collected from 1040 farms in the west of Scotland. Of the micro-organisms isolated 85.8% were identified as belonging to the genus Bacillus and 9.9% were assigned to the coryneform group. Bacillus cereus occurred most frequently and B. licheniformis and B. coagulans also occurred in significant numbers.     

Studies on the Bacillus flora of milk and milk products

Bacillus licheniformis and B. cereus were the most commonly isolated species of Bacillus found in milk at all stages of processing. Bacillus licheniformis was ubiquitous in the farm environment and counts in raw milks heat-treated in the laboratory were higher during the winter months, whilst B. cereus was associated with cattle feed throughout the year, and tended to be more common in raw milks during the summer months. Although B. licheniformis was usually isolated in larger numbers than B. cereus, this pattern changed after raw and pasteurized milks and reconstituted milk powders were pre-incubated at ambient temperatures, and B. cereus came to dominate the Bacillus population, reaching levels associated with enterotoxin production. Investigation of the growth kinetics of strains of both species showed that B. cereus grew faster than B. licheniformis at ambient temperatures. It is suggested that post-pasteurization contamination, which is commonly blamed for spoilage of milk and milk products by B. cereus, is not necessarily the most important source of this organism.     

Isolation of Campylobacter jejuni from raw milk

Campylobacter jejuni was isolated from raw milk by a method that can routinely detect less than or equal to 1 organism per ml. This procedure was used in a survey of 195 separate farms and showed a 1.5% incidence of C. jejuni in milk from bulk tanks.     

Isolation of Campylobacter jejuni from raw milk.

Campylobacter jejuni was isolated from raw milk by a method that can routinely detect less than or equal to 1 organism per ml. This procedure was used in a survey of 195 separate farms and showed a 1.5% incidence of C. jejuni in milk from bulk tanks.     

Psychrotrophic strains of Bacillus cereus producing enterotoxin

In investigations on three outbreaks of Bacillus cereus food poisoning in Spain and The Netherlands, the causative strains grew within a temperature range of 4-37 degrees C, but not at 43 degrees C. Such psychrotrophic types were found to occur in various dairy products (including ca 25% of 35 samples of pasteurized milk) and some mousses and cook/chill meals. Growth of and enterotoxin production by psychrotrophic B. cereus could be prevented by temperatures below 4 degrees C and pH-values not exceeding 5.0.     

Comparative study of the frequency and flagellar serotype flora of Bacillus thuringiensis in soils and silkworm-breeding environments

Two environments, soils and silkworm ( Bombyx mort )-breeding farms, in Kumamoto Prefecture, Japan, were explored for the comparative analysis of the frequency and the flagellar (H) serotype flora of Bacillus thuringiensis. This organism was found in 39.3% of the sericulture farms and 18.5% of the silkworm litter samples. The frequency of B. thuringiensis was 4.5% among the populations of the Bacillus cereus/B. thuringiensis group in silkworm-breeding insectaries. The predominant H serotype detected in silkworm litters was 4ac (35.6%), followed by serotypes 3abc (20.0%), 8ab (13.3%), 4ab (13.3%) and 7 (2.2%). The majority (87.1%) of sericulture-derived isolates exhibited larvicidal activity against the silkworm and/or the mosquito, Aedes aegypti. Of soil samples examined, 8.1% contained B. thuringiensis. The frequency of B. thuringiensis colonies was 0.6% among soil populations of the B. cereus/B. thuringiensis group. Of 22 soil isolates, 14 were assigned to five H serotypes: 10, 17, 18, 21 and 29. Most soil isolates showed no insecticidal activity against B. mori and A. aegypti , but two, belonging to H serotype 10 and an undescribed serotype, exhibited moderate larvicidal activity against A. aegypti.     

Psychrotrophic strains of Bacillus cereus producing enterotoxin

In investigations on three outbreaks of Bacillus cereus food poisoning in Spain and The Netherlands, the causative strains grew within a temperature range of 4·37†C, but not at 43†C. Such psychrotrophic types were found to occur in various dairy products (including ca 25% of 35 samples of pasteurized milk) and some mousses and cook/chill meals. Growth of and enterotoxin production by psychrotrophic B . cereus could be prevented by temperatures below 4†C and pH-values not exceeding 5·0.     

Variation in carbon footprint of milk due to management differences between Swedish dairy farms

To identify mitigation options to reduce greenhouse gas (GHG) emissions from milk production (i.e. the carbon footprint (CF) of milk), this study examined the variation in GHG emissions among dairy farms using data from previous CF studies on Swedish milk. Variations between farms in these production data, which were found to have a strong influence on milk CF, were obtained from existing databases of 1051 dairy farms in Sweden in 2005. Monte Carlo (MC) analysis was used to analyse the impact of variations in seven important parameters on milk CF concerning milk yield (energy-corrected milk (ECM) produced and delivered), feed dry matter intake (DMI), enteric CH4 emissions, N content in feed DMI, N-fertiliser rate and diesel used on farm. The largest between-farm variations among the analysed production data were N-fertiliser rate (kg/ha) and diesel used (l/ha) on farm (CV = 31% to 38%). For the parameters concerning milk yield and feed DMI, the CV was approximately 11% and 8%, respectively. The smallest variation in production data was found for N content in feed DMI. According to the MC analysis, these variations in production data led to a variation in milk CF of between 0.94 and 1.33 kg CO2 equivalents (CO2e)/kg ECM, with an average value of 1.13 kg CO2e/kg ECM. We consider that this variation of ±17%, which was found to be based on the used farm data, would be even greater if all Swedish dairy farms were included, as the sample of farms in this study was not totally unbiased. The variation identified in milk CF indicates that a potential exists to reduce GHG emissions from milk production on both the national and farm levels through changes in management. As milk yield and feed DMI are two of the most influential parameters for milk CF, feed conversion efficiency (i.e. units ECM produced/unit DMI) can be used as a rough key performance indicator for predicting CF reductions. However, it must be borne in mind that feeds have different CF due to where and how they are produced.     

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。     

Development of a fluorogenic probe-based PCR assay for detection of Bacillus cereus in nonfat dry milk

A fluorogenic probe-based PCR assay was developed and evaluated for its utility in detecting Bacillus cereus in nonfat dry milk. Regions of the hemolysin and cereolysin AB genes from an initial group of two B. cereus isolates and two Bacillus thuringiensis isolates were cloned and sequenced. Three single-base differences in two B. cereus strains were identified in the cereolysin AB gene at nucleotides 866, 875, and 1287, while there were no species-consistent differences found in the hemolysin gene. A fluorogenic probe-based PCR assay was developed which utilizes the 5'-to-3' exonuclease of Taq polymerase, and two fluorogenic probes were evaluated. One fluorogenic probe (cerTAQ-1) was designed to be specific for the nucleotide differences at bases 866 and 875 found in B. cereus. A total of 51 out of 72 B. cereus strains tested positive with the cerTAQ-1 probe, while only 1 out of 5 B. thuringiensis strains tested positive. Sequence analysis of the negative B. cereus strains revealed additional polymorphism found in the cereolysin probe target. A second probe (cerTAQ-2) was designed to account for additional polymorphic sequences found in the cerTAQ-1-negative B. cereus strains. A total of 35 out of 39 B. cereus strains tested positive (including 10 of 14 previously negative strains) with cerTAQ-2, although the assay readout was uniformly lower with this probe than with cerTAQ-1. A PCR assay using cerTAQ-1 was able to detect approximately 58 B. cereus CFU in 1 g of artificially contaminated nonfat dry milk. Forty-three nonfat dry milk samples were tested for the presence of B. cereus with the most-probable-number technique and the fluorogenic PCR assay. Twelve of the 43 samples were contaminated with B. cereus at levels greater than or equal to 43 CFU/g, and all 12 of these samples tested positive with the fluorogenic PCR assay. Of the remaining 31 samples, 12 were B. cereus negative and 19 were contaminated with B. cereus at levels ranging from 3 to 9 CFU/g. All 31 of these samples were negative in the fluorogenic PCR assay. Although not totally inclusive, the PCR-based assay with cerTAQ-1 is able to specifically detect B. cereus in nonfat dry milk.     

RAPID DETECTION OF GERMINATING BACILLUS CEREUS CELLS USING FLUORESCENT IN SITU HYBRIDIZATION

Methods for the specific detection of Bacillus spores are needed in many situations such as the recognition of food poisoning. This study presents an experimental design in order to find the best combination of germination conditions leading to a rapid and detectable fluorescent in situ hybridization (FISH) signal from Bacillus cereus spores present in pure cultures and milk samples.
B. cereus ATCC 14579 and HER 1414 were incubated in 20 different growth media by using a combination of various germinants such as sugars, amino acids and dipicolinic acid. Also, three different germination factors were tested: incubation temperature, inoculum concentration and a heat shock treatment. Permeabilization procedure and hybridization time were optimized on the best germination condition found. B. cereus -specific FISH probes were validated under the optimized condition and in detection of spiked B. cereus spores in 1% ultra heat-treated milk samples. FISH-labeled cells were detected by using flow cytometry, and the results were confirmed by fluorescence microscopy. The optimal condition allows the detection of B. cereus spores in less than 2 h. Overall, a ninefold reduction in total time for detection was achieved when comparing with previous works. Therefore, the permeabilization and hybridization optimizations mentioned in this study are major improvements for the detection time of B. cereus spores.

PRACTICAL APPLICATIONS

By using the optimized conditions of germination/outgrowth, permeabilization and hybridization, the detection of 10³ cfu/mL of Bacillus cereus spores using fluorescent in situ hybridization is possible within 2 h in milk sample.     

Campylobacter enteritis associated with consumption of unpasteurised milk.

In October and November 1978 two outbreaks of enteritis occurred in the north of England. Symptoms lasted two to over eight days but in no case necessitated admission to hospital. Faecal specimens from most of the patients were found to contain thermophilic Campylobacter sp. Inquiry disclosed that all patients had consumed unpasteurised milk from local farms. Examination of rectal swabs from the cattle concerned and milk socks yielded strains of Campylobacter sp indistinguishable from those isolated from the patients. It was therefore concluded that, since campylobacters are not known to be excreted in milk, faecal contamination of the milk had probably occurred and had led to these outbreaks. Evidence suggests that thermophilic Campylobacter sp is an occasional contaminant of milk. So long as unpasteurised milk continues to be distributed further outbreaks will probably occur.     

Rearing goat kids away from their dams 1. A survey to understand rearing methods

Despite an estimated global goat population of over one billion, little is known about methods being used to feed milk to artificially reared kids (reared away from their dams) and how kids are weaned from these systems. Quantifying and characterising current methods utilised on farms will enable future targeted research to investigate best practice methods for milk feeding and weaning of artificially reared kids. A recall-based survey that investigated on-farm kid-rearing practices (focusing on the milk feeding and weaning stages) was distributed via social media, and regional goat organisations across multiple countries. A total of 242 responses from 16 countries were collected and geographically grouped. Responses that could not be grouped sufficiently were removed (nine responses from eight countries). A total of 233 responses from eight countries (United States of America (USA) 72; United Kingdom (UK) 71; Australia 33; Canada 23; New Zealand 20; European Union (EU) 14), were analysed. Most farms (217; 93%) bred their own kids. The most common milk feeding method was bottle feeding, used on 135 farms (57.9%), followed by ad libitum feeding used by 72 (30.9%). A relationship between number of kids reared and feeding system was identified, χ²(3, N = 233) = 89.605, P < 0.001, with farms rearing > 100 kids more likely to feed milk ad libitum. A total of 170 farms (72.9%) were weaned based on a target age and 85 (36.4%) on a target weight, 53 (22.7%) used both and 45 (19.3%) neither. Target weaning ages and weights varied across countries; the median age was 84 days (interquartile range (IQR) 56–84), and the median weight was 16 kg (IQR 15–18). A difference was found between milk feeding systems for weaning method (Χ²(2, N = 232) = 63.797, P ≤ 0.001), with kids most likely to be abruptly weaned from ad libitum systems (or gradually weaned from bottle feeding). Abrupt weaning was used by 67 farms (28.8%), and gradual weaning was used by 165 (71.1%). Gradual weaning strategies included reducing milk quantity (150 farms; 93% of farms providing detail) and diluting milk (six farms; 4%). A total of 169 (72.5%) supplied enrichment that met the survey’s definition; items to climb on/hide in were most common, provided by 157 farms (92.8%). Findings suggest differing practices in smaller-scale bottle-fed versus larger-scale ad libitum milk systems, likely reflecting differing system needs. This highlights a requirement for welfare-focused research in kids reared artificially in order to identify and communicate best practices to ensure on-farm welfare is optimised within each system.     

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.     

Evaluation of methods for recognising strains of the Bacillus cereus group with food poisoning potential among industrial and environmental contaminants

Toxin production, biochemical properties and ribotypes of Bacillus cereus group (B. cereus, B. thuringiensis, B. mycoides) strains originating from industrial and environmental sources (n = 64), from food poisoning incidents (n = 22) and from reference sources (n = 7) were analysed. Forty ribotypes were found among the 93 strains. Eleven strains from food poisoning incidents produced emetic (mitochondrio) toxin, as determined by the boar spermatozoa toxicity test. These strains possessed closely similar ribotypes which were rare among strains of other origins. Sperm toxin producing (cereulide positive) strains did not hydrolyse starch and did not produce haemolysin BL, as determined by the reverse passive latex agglutination test. Sixteen different ribotypes were found among B. cereus strains from board machines (n = 16) and from packaging board (n = 16), indicating many different sources of B. cereus contamination in board mills. Strains originating from packaging board had predominantly different ribotypes from those of dairy and dairy product originating strains. Nine (53%) out of 17 strains from a single dairy process shared the same ribotype whereas strains from milk and milk products from different dairies had different ribotypes indicating that B. cereus group populations were dairy specific. Twenty-two percent of strains isolated from the paperboard industry on non-selective medium were lecithinase negative, including enterotoxin producing strains. This stresses the importance of other detection methods not based on a positive lecithinase reaction.     

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.     

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.     

An appraisal of carbon footprint of milk from commercial grass-based dairy farms in Ireland according to a certified life cycle assessment methodology

Purpose

Life cycle assessment (LCA) studies of carbon footprint (CF) of milk from grass-based farms are usually limited to small numbers of farms (<30) and rarely certified to international standards, e.g. British Standards Institute publicly available specification 2050 (PAS 2050). The goals of this study were to quantify CF of milk from a large sample of grass-based farms using an accredited PAS 2050 method and to assess the relationships between farm characteristics and CF of milk.

Materials and methods

Data was collected annually using on-farm surveys, milk processor records and national livestock databases for 171 grass-based Irish dairy farms with information successfully obtained electronically from 124 farms and fed into a cradle to farm-gate LCA model. Greenhouse gas (GHG) emissions were estimated with the LCA model in CO₂ equivalents (CO₂-eq) and allocated economically between dairy farm products, except exported crops. Carbon footprint of milk was estimated by expressing GHG emissions attributed to milk per kilogram of fat and protein-corrected milk (FPCM). The Carbon Trust tested the LCA model for non-conformities with PAS 2050. PAS 2050 certification was achieved when non-conformities were fixed or where the effect of all unresolved non-conformities on CF of milk was?<?±5 %.

Results and discussion

The combined effect of LCA model non-conformities with PAS 2050 on CF of milk was <1 %. Consequently, PAS 2050 accreditation was granted. The mean certified CF of milk from grass-based farms was 1.11 kg of CO₂-eq/kg of FPCM, but varied from 0.87 to 1.72 kg of CO₂-eq/kg of FPCM. Although some farm attributes had stronger relationships with CF of milk than the others, no attribute accounted for the majority of variation between farms. However, CF of milk could be reasonably predicted using N efficiency, the length of the grazing season, milk yield/cow and annual replacement rate (R ²?=?0.75). Management changes can be applied simultaneously to improve each of these traits. Thus, grass-based farmers can potentially significantly reduce CF of milk.

Conclusions

The certification of an LCA model to PAS 2050 standards for grass-based dairy farms provides a verifiable approach to quantify CF of milk at a farm or national level. The application of the certified model highlighted a wide range between the CF of milk of commercial farms. However, differences between farms’ CF of milk were explained by variation in various aspects of farm performance. This implies that improving farm efficiency can mitigate CF of milk.