Improved microbiological assay procedures for dihydrostreptomycin residues in milk and dairy products.

Because dihydrostreptomycin can remain as a slowly removed antibiotic residue in dairy animals and because of the need for more sensitive procedures with which to provide information concerning antibiotic residues in food products, procedures were developed in more sensitive assays of dihydrostreptomycin in milk and some representative dairy products. The cleanup procedures that aided these improvements were (i) precipitation of milk proteins by acidification and (ii) centrifugation to extract cheeses and to remove physical barriers to diffusion in the cylinder plate assay. In addition, a thin, single seeded assay layer was used to maximize diffusion. Levels of dihydrostreptomycin as low as 0.06 microgram/ml in milk and 0.2 to 0.4 microgram/g in cheeses were measurable; these levels were some fourfold more sensitive than those presently recommended by the Food and Drug Administration.     

An automated method for the detection of staphylococcal heat stable deoxyribonuclease in dairy products

An automated sandwich immunoassay with specific polyclonal antibodies for the detection of Staphylococcus aureus thermostable nuclease (DNase) is described. To evaluate this assay, different quantities of purified S. aureus nuclease were added to dairy products. Additionally, staphylococcal counts and nuclease activity of milk samples inoculated with S. aureus were determined. Different extraction procedures were performed and compared. The results indicated that the automated test was a reliable method for detecting DNase activity in milk products. The procedure was completed in 2 h and detected 1 ng of DNase ml-1. Detection of the DNase was especially useful in cheeses and could be used to confirm positive enterotoxin results.     

Assessing and analysing contamination of a dairy products processing plant by Staphylococcus aureus using antibiotic resistance and PFGE

A dairy product processing plant was studied for 2.5 years to examine contamination with Staphylococcus aureus and try to correlate the source of contamination. Cultures were submitted to an antibiotic susceptibility test (AST) and characterised by Pulsed-field Gel Electrophoresis (PFGE) analysis. Results showed that 35.2% (19/51) of food handlers were asymptomatic carriers of S. aureus, and that 90.4% (19/21) of raw milk sampled was contaminated. Staphylococcus aureus was isolated from only 10 samples among more than 3200 investigated dairy products. No S. aureus contamination was found on machinery. The AST analysis demonstrated sensitivity of tested S. aureus to oxacillin, cephalothin, vancomycin, gentamicin, and sulfamethoxazole/trimethoprim. AST analysis generated eight different phenotypic profiles, but did not allow us to identify the source of contamination in seven of ten final products. PFGE analysis proved to be a sensitive method as it generated 42 different DNA banding profiles among the 48 S. aureus investigated, demonstrating a lack of predominance of endemic strains in the plant, contrary to suggestions raised by antibiotic resistance typing. Based on PFGE genotyping, S. aureus strains isolated from four contaminated final products were similar to four S. aureus isolated from raw milk. Five final products contained S. aureus different from all other strains collected, and one showed similarity to a strain isolated from a food handler. These results suggest contamination by raw milk as the main source of contamination of the final dairy products.     

Phenotypic typing, technological properties and safety aspects of Lactococcus garvieae strains from dairy environments

AIMS: To characterize Lactococcus garvieae strains of dairy origin and to determine their technological properties and safety for their possible use in starter culture preparation. METHODS AND RESULTS: Forty-seven L. garvieae isolates, recovered from two artisanal Italian cheeses were studied, in comparison with 12 fish isolates and the type strain of the species. Phenotypic typing revealed that the strains could be differentiated on the basis of their ecological niche of origin in lactose positive strains (all isolated from dairy sources) and lactose negative strains (all isolated from fish). Furthermore, the strains exhibited a high degree of physiological variability, showing the presence of 26 different biotypes. The strains possessed moderate acidifying and proteolytic activities and did not produce bacteriocins. A safety investigation revealed that all strains were sensitive to vancomycin and moderately resistant to kanamycin; some biotypes were tetracycline resistant. Production of biogenic amines or presence of genes encoding virulence determinants occurred in some isolates. CONCLUSIONS: The prevalence of L. garvieae in some artisanal Italian cheeses can be linked to the typicity of the products. Although in a few cases an antimicrobial resistance or a presence of virulence determinants may imply a potential hygienic risk, most of the strains showed positive properties for their possible adjunction in a starter culture preparation, to preserve the natural bacterial population responsible for the typical sensorial characteristics of the traditional raw milk cheeses. SIGNIFICANCE AND IMPACT OF THE STUDY: L. garvieae strains can be considered an important part of the microbial population associated with the natural fermentation of artisanal Italian cheeses. A deepened characterization of the strains may aid in understanding the functional and ecological significance of their presence in dairy products and in selecting new strains for the dairy industry.     

Evaluation of the TECRA Escherichia coli O157 visual immunoassay for tests on dairy products

The TECRA visual immunoassay was compared with an enrichment plating procedure according to the United States Food and Drug Administration procedures, for the detection of Escherichia coli in dairy foods. Both methods were used to detect E. coli O157 which had been inoculated into cheeses, milk powders, caseins and whey products. A centrifugation step was needed to test whey products according to the TECRA method. The results from both test methods were in complete agreement.     

Detection of Mycobacterium avium ssp. paratuberculosis in cheese, milk powder and milk using IS900 and f57-based qPCR assays

Aims: To develop a quantitative PCR assay for sensitive and specific detection of Mycobacterium avium ssp. paratuberculosis (Map) in a range of dairy products. Methods and Results: TaqMan^® assays were designed to target the IS900 and f57 genetic elements of Map. Both real‐time PCR assays were integrated with the Adiapure^® Map DNA extraction kit and assessed separately for the detection/quantification of Map in spiked milk, Cheddar cheese and milk powder. Assays were validated against Cheddar cheese samples containing known concentrations of Map. The IS900 qPCR assay was significantly more sensitive than the assay based on the f57 primer/probe. At a threshold cycle value of 38, limits of detection (LOD) for the IS900 qPCR assay were 0·6 CFU ml^?1, 2·8 CFU g^?1 and 30 CFU g^?1 for artificially contaminated pasteurized milk, whole milk powder and Cheddar cheese, respectively. The respective LOD’s for the f57 assay were 6·2 CFU ml^?1, 26·7 CFU g^?1 and 316 CFU g^?1. Conclusion: The integrated Adiapure^® extraction – IS900 real time assay described is a sensitive, quantitative method for the detection of Map in dairy products. This is the first study to consider qPCR as a quantitative estimation of Map‐DNA in cheese and whole milk powder. Significance and Impact of the Study: The assay developed allows sensitive detection and quantification of Map DNA in a range of dairy products which is valuable for the screening and surveillance of this potential zoonotic organism.     

[3H] dihydrostreptomycin accumulation and binding to ribosomes in Rhizobium mutants with different levels of streptomycin resistance.

Rhizobium trifolii B1, a symbiotic nitrogen fixer, is sensitive to streptomycin (10 microgram/ml) and spontaneously produces spheroplast-like forms during cultivation. Streptomycin-resistant mutants selected with high doses of antibiotic (1,000 microgram/ml) showed pleiotropic changes, including loss of spheroplast formation and infectivity to plants, whereas mutants selected with low doses of streptomycin (10 to 100 microgram/ml) retained properties of parent strain B1 (I. Zelazna-Kowalska, Acta Microbiol. Pol., in press). The present studies revealed that strain B1 and its mutant with a high level of streptomycin resistance, B1 strH, accumulated the antibiotic at similar rates. Mutant B1 strL, with a low level of streptomycin resistance (up to 100 microgram/ml), accumulated the antibiotic at a lower rate. Ribosomes isolated from strains B1 and B2 strL bound [3H]dihydrostreptomycin, whereas those from strain B1 strH did not. These observations indicate that, in R. trifolii B1, mutation to a high level of streptomycin resistance affects ribosomal structure, whereas low-level resistance involves a change in membrane permeability.     

Incompatibility in the fungus Podospora anserina. Are the mutations abolishing the incompatibility reaction ribosomal mutations?

Summary In the Ascomycete Podospora anserina the incompatibility reaction due to the interaction of non allelic genes exhibits some sensitivity to the antibiotic dihydrostreptomycin as well as to high levels of Magnesium. This incompatibility reaction can be suppressed or made more sensitive to the Magnesium or dihydrostreptomycin effect by mutations in the same genes mod1 and mod2. Properties of mutant strains suggest that mod1 and mod2 are ribosomal genes whose products seem to regulate, in a positive way for the first gene and in a negative way for the second gene, the translation of specific messengers, especially that of some proteolytic enzymes.     

Inhibitory activity of <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> LMG P-26358 against <Emphasis Type="Italic">Listeria innocua</Emphasis> when used as an adjunct starter in the manufacture of cheese

Lactobacillus plantarum LMG P-26358 isolated from a soft French artisanal cheese produces a potent class IIa bacteriocin with 100% homology to plantaricin 423 and bacteriocidal activity against Listeria innocua and Listeria monocytogenes. The bacteriocin was found to be highly stable at temperatures as high as 100°C and pH ranges from 1-10. While this relatively narrow spectrum bacteriocin also exhibited antimicrobial activity against species of enterococci, it did not inhibit dairy starters including lactococci and lactobacilli when tested by well diffusion assay (WDA). In order to test the suitability of Lb. plantarum LMG P-26358 as an anti-listerial adjunct with nisin-producing lactococci, laboratory-scale cheeses were manufactured. Results indicated that combining Lb. plantarum LMG P-26358 (at 108 colony forming units (cfu)/ml) with a nisin producer is an effective strategy to eliminate the biological indicator strain, L. innocua. Moreover, industrial-scale cheeses also demonstrated that Lb. plantarum LMG P-26358 was much more effective than the nisin producer alone for protection against the indicator. MALDI-TOF mass spectrometry confirmed the presence of plantaricin 423 and nisin in the appropriate cheeses over an 18 week ripening period. A spray-dried fermentate of Lb. plantarum LMG P-26358 also demonstrated potent anti-listerial activity in vitro using L. innocua. Overall, the results suggest that Lb. plantarum LMG P-26358 is a suitable adjunct for use with nisin-producing cultures to improve the safety and quality of dairy products.     

Ecology of spontaneous fermentation in one winery during 5 consecutive years

A concentration protocol based on trichloroacetic acid precipitation was evaluated and compared with the reference method using dialysis concentration. Different quantities of purified staphylococcal enterotoxins were added to pasteurized Camembert-type cheeses. Detection of enterotoxins in these cheeses was performed using an automated detection system. Raw goat milk Camembert-type cheeses involved in a staphylococcal food poisoning were also tested. Both enterotoxin extraction methods allowed detection of the lowest enterotoxin concentration level used in this study (0.5 ng g-1). Compared with the dialysis concentration method, TCA precipitation of staphylococcal enterotoxins was 'user-friendly' and less time-consuming. These results suggest that TCA precipitation is a rapid (1 h), simple and reliable method of extracting enterotoxin from food which gives excellent recovery from dairy products.     

Immunochromatographic technique for express determination of ampicillin in milk and dairy products

An immunochromatographic method for determination of β-lactam antibiotic ampicillin has been developed. The method is based on the competitive interaction between antibiotic molecules contained in the sample and protein conjugate of penicillin immobilized on a membrane for binding with specific antibodies labeled with colloidal gold, which occurs during movement of the sample to be tested and reagents along the membrane. The completion of the test system ensures control of exceeding the maximum permissible content of the antibiotic in milk and dairy products (10 ng/mL). The possibility of testing milk, raw milk, and dairy products for 10 minutes at room temperature without sample pretreatment has been demonstrated.     

Association of Yersinia enterocolitica with the manufacture of cheese and occurrence in pasteurized milk.

Raw milk in southern Ontario frequently contains Yersinia enterocolitica. The potential for transmission of this organism by cheese manufactured from unpasteurized milk was evaluated by examination of milk and cheese curd samples from cheese manufacturing plants and finished cheddar and Italian cheeses. The incidence of Y. enterocolitica was lower in cheese curd samples (9.2%) than in raw milk (18.2%). Most of the curd samples showed a positive phosphatase test, indicating production from raw milk. One curd sample yielded Y. enterocolitica after 4 weeks of storage at 4 degrees C but was negative after 8 weeks. All samples of cheddar and Italian cheeses, most of which showed a positive phosphatase test, were negative for Y. enterocolitica. One out of 265 samples (0.4%) of pasteurized fluid dairy products contained Y. enterocolitica.     

Conditional dihydrostreptomycin resistance in Bacillus subtilis

Mutants resistant to dihydrostreptomycin were isolated and genetically analyzed in Bacillus subtilis. Two new classes of mutants distinct from the ribosomal strA locus were found. One class, strB, was located between metC3 and ura-1 on the chromosome. The second class, strC, mapped in the spore gene region close to the spoA locus. Both mutant classes were resistant to dihydrostreptomycin during growth but sensitive to the antibiotic during sporulation. Resuspension sporulation experiments with a strB mutant showed that sensitivity to the antibiotic was acquired early in the sporulation process. The germination and outgrowth of strB spores was sensitive to the antibiotic until growth commenced, whereupon the culture was resistant. Thus the mutants are sensitive to dihydrostreptomycin during both sporulation and germination but resistant during the growth phase.     

Focus on the supramolecular structure of milk fat in dairy products

Bovine fat is dispersed in raw milk as natural milk fat globules, with an average diameter of 4 microm, which are enveloped in a biological membrane, the milk fat globule membrane (MFGM). However, dairy processes modify the supramolecular structure and the surface composition of milk fat. Thus, milk fat is present in many dairy products under various forms. In this study, we focused on the fact that natural milk fat globules are rarely consumed in their native state, i.e. in fresh raw milk. In most drinking milks, fat globules are homogenised in order to avoid their rising at the surface of the products. Furthermore, fat globules are heat treated to avoid the growth of micro-organisms. As a consequence of the technological process applied, the volume-weighted average diameter of fat globules in drinking milks is in the range 0.2-0.5 microm. Homogenisation of fat globules led to the partial disruption of the MFGM and to the adsorption of milk proteins. Moreover, this study showed that in cheeses, milk fat can be dispersed as (i) fat globules with the MFGM, (ii) aggregates of fat globules, (ii) homogenised fat globules, (iii) free fat and (iv) a combination of different phases and structures. The knowledge of the supramolecular structure of milk fat in dairy products is of primary importance regarding its technological, sensorial and nutritional properties.     

Fermentation of plant-based dairy alternatives by lactic acid bacteria

Ethical, environmental and health concerns around dairy products are driving a fast-growing industry for plant-based dairy alternatives, but undesirable flavours and textures in available products are limiting their uptake into the mainstream. The molecular processes initiated during fermentation by lactic acid bacteria in dairy products is well understood, such as proteolysis of caseins into peptides and amino acids, and the utilisation of carbohydrates to form lactic acid and exopolysaccharides. These processes are fundamental to developing the flavour and texture of fermented dairy products like cheese and yoghurt, yet how these processes work in plant-based alternatives is poorly understood. With this knowledge, bespoke fermentative processes could be engineered for specific food qualities in plant-based foods. This review will provide an overview of recent research that reveals how fermentation occurs in plant-based milk, with a focus on how differences in plant proteins and carbohydrate structure affect how they undergo the fermentation process. The practical aspects of how this knowledge has been used to develop plant-based cheeses and yoghurts is also discussed.     

Bacteriophage Types and Antibiotic Sensitivity of Staphylococci from Bovine Milk and Human Nares

The number, phage types, and antibiotic sensitivity of coagulase-positive staphylococci from grade A raw milk samples produced on 40 farms in the Athens, Ga., milkshed were determined. Counts of mannitol-positive staphylococci in milk ranged from 100 to 3,580 per milliliter, with an arithmetic mean of 1,047. Examination of the nares of 48 dairymen on 34 of the farms also revealed that 13 (27%) were carriers of coagulase-positive staphylococci. Isolates from milk (412) and from nares (39) were tested against the Coles, Seto-Wilson, and International phage sets and 87, 68, and 56%, respectively, proved typable. Nine isolates were not typable. Each of the 33 phages used lysed one or more of the isolates. Staphylococcal phage types per milk sample ranged from 0 to 5, 0 to 7, and 0 to 8, with arithmetic means of 1.9, 2.3, and 2.3, respectively. Of the 13 narial carriers, 7 harbored staphylococci of one or more of the same phage types as those isolated from the milk at the respective farms. Randomly selected isolates were tested against high and low concentrations of 12 common antibiotics. All were either moderately sensitive or resistant to polymixin B. Over 30% were moderately sensitive or resistant to dihydrostreptomycin and penicillin individually. With but few exceptions, all isolates were sensitive to chlortetracycline, bacitracin, carbomycin, chloramphenicol, erythromycin, neomycin, novobiocin, oxytetracycline, and tetracycline individually.     

新疆发酵乳品中乳酸菌的分离鉴定及其耐药性

目的对新疆传统发酵乳品中乳酸菌进行分离鉴定并检测其耐药性。方法利用传统形态学鉴定法和生化鉴定等方法对新疆发酵乳中乳酸菌进行鉴定,采用纸片扩散法对分离鉴定的菌进行耐药性分析。结果从新疆发酵乳品中共分离出8株乳酸菌,经鉴定分别为瑞士乳杆菌(Lactobacillus helveticus)、植物乳杆菌(Lactobacillus plantarum)、马乳酒样乳杆菌(Lactobacillus kefianofaciens)、乳酸乳球菌(Lactococcus lactis)、副干酪乳杆菌(Lactobacillus paracasei)、副干酪乳杆菌类坚韧亚种(Lactobacillus paracasei subsp.tolerans)、哈尔滨乳杆菌(Lactobacillus harbinensis)、希氏乳杆菌(Lactobacillus hilgardii),并且发现8株乳酸菌对万古霉素、庆大霉素、阿莫西林、多西环素、环丙沙星、阿奇霉素、头孢他啶、头孢孟多具有一定敏感性。结论新疆发酵乳品中以乳杆菌居多,对常见抗生素具有一定的敏感性。     

Monoclonal antibody based enzyme-linked immunosorbent assay for aminoglycoside antibiotic kanamycin in foodstuff

Monoclonal antibodies to aminoglycoside antibiotic kanamycin (KM) were raised as a result of mice complex immunization with glutaraldehyde conjugates BSA with KM, tobramycin (TM) and gentamicin. Using antibodies an indirect competitive enzyme-linked immunosorbent assay was developed. This method allows to determine antibiotic up to 1.2 ng/ml in water solutions, milk and eggs and up to 2.5 ng/ml in honey. The recovery rate from these products spiked with KM was 83, 84 and 96% respectively. The assay of KM based on homologous and heterologous solid-phase conjugates were estimated. The cross-reactivity with TM could vary from 7 to 54%. The same indexes for of amikacin were more constant and reached 7-8%. The other aminoglycosides showed no inhibitory activity.     

Winter feeding systems and dairy cow breed have an impact on milk composition and flavour of two Protected Designation of Origin French cheeses

This study investigates the effects of two feeding systems and two dairy cow breeds on milk yield and composition, physical and sensorial properties of Camembert and Pont-l’Evêque cheeses. The experiment consisted of a 2 × 2 factorial arrangement of treatments. A low energy grass diet with only 15% of concentrate (LowGS) was compared with a high-energy maize silage diet with 30% concentrate (HighMS). Thirty-four Holstein (Ho) and 34 Normande (No) cows in early lactation were assigned to one of two feeding systems for a 6-week period. Cows on the LowGS feeding system had lower milk yield, fat and protein content. In both feeding systems, No cows had lower milk yields but higher milk protein contents than Ho cows. The LowGS feeding system altered milk fatty acid (FA) composition by reducing saturated FA. Breed had only a small impact on milk FA. Concerning milk coagulating properties, only the firmness was reduced by the LowGS feeding and the Ho breed. The effects of breed and feeding system on the protein content of cheeses were more marked in Camembert cheese than in Pont-l’Evêque cheese. However, the Camembert cheese from Ho-LowGS was, in fact, characterized especially by lower protein content. LowGS feeding system and No breed produced more yellow cheeses. Feeding systems had limited effects on the firmness of Camembert and Pont-l’Evêque cheeses measured by penetrometry. In sensory analysis, Ho breed and LowGS feeding produced a Camembert cheese with a more melting texture in the mouth due to the increase of spreadability index and of proteolysis. The type of cheese also had an influence: the effects were more important on Camembert cheese than on Pont-l’Evêque cheese. Only the Ho-LowGS treatment produced a very specific Camembert cheese different from the others. The feeding systems and breed of dairy cow have no determinant effect on PDO (protected designation of origin) Camembert and Pont-l’Evêque cheeses, especially regarding taste. In this kind of trial, despite the effects of feeding systems and breed on milk composition, the role of cheese ripening and microbiology appears to be of considerable importance.     

Distribution of antimicrobial‐resistant lactic acid bacteria in natural cheese in Japan

To determine and compare the extent of contamination caused by antimicrobial‐resistant lactic acid bacteria (LAB) in imported and domestic natural cheeses on the Japanese market, LAB were isolated using deMan, Rogosa and Sharpe (MRS) agar and MRS agar supplemented with six antimicrobials. From 38 imported and 24 Japanese cheeses, 409 LAB isolates were obtained and their antimicrobial resistance was tested. The percentage of LAB resistant to dihydrostreptomycin, erythromycin, and/or oxytetracycline isolated from imported cheeses (42.1%) was significantly higher than that of LAB resistant to dihydrostreptomycin or oxytetracycline from cheeses produced in Japan (16.7%; P = 0.04). Antimicrobial resistance genes were detected in Enterococcus faecalis (tetL, tetM, and ermB; tetL and ermB; tetM) E. faecium (tetM), Lactococcus lactis (tetS), Lactobacillus (Lb.), casei/paracasei (tetM or tetW), and Lb. rhamnosus (ermB) isolated from seven imported cheeses. Moreover, these E. faecalis isolates were able to transfer antimicrobial resistance gene(s). Although antimicrobial resistance genes were not detected in any LAB isolates from Japanese cheeses, Lb. casei/paracasei and Lb. coryniformis isolates from a Japanese farm‐made cheese were resistant to oxytetracycline (minimal inhibitory concentration [MIC], 32 µg/mL). Leuconostoc isolates from three Japanese farm‐made cheeses were also resistant to dihydrostreptomycin (MIC, 32 to > 512 µg/mL). In conclusion, the present study demonstrated contamination with antimicrobial‐resistant LAB in imported and Japanese farm‐made cheeses on the Japanese market, but not in Japanese commercial cheeses.