Toxinogenic Bacillus pumilus and Bacillus licheniformis from mastitic milk.

To elucidate the occurrence of heat-stable toxin-producing strains among mastitic Bacillus isolates, 100 milk samples of mastitic cows from different parts of Finland were screened. Bacillus was identified as the major organism in 23 samples. Toxinogenic Bacillus isolates identified by sperm cell motility inhibition assay were isolated from six samples. Four isolates belonged to the species Bacillus pumilus and two to Bacillus licheniformis. The toxic substances were heat-stable and soluble to methanol thus being of non-protein nature. The methanol extracted substances disrupted the sperm cell plasma membrane permeability barrier at exposure concentrations of 1-15 microg ml(-1) (B. pumilus) or 20-30 microg ml(-1) (B. licheniformis). The toxic properties of the two mastitic B. licheniformis strains were similar to those of B. licheniformis strains known to produce the lipopeptide lichenysin A and the synthetase genes lchAA, lchAB and lchAC for lichenysin were found in the mastitic strains by PCR. Toxin synthetase genes for the syntheses of lichenysin or surfactin were searched but not found in the toxic B. pumilus strains. The ribopatterns of the mastitic B. pumilus and B. licheniformis isolates were similar to those of the toxinogenic strains described earlier from food poisoning incidents and contaminated indoor air. B. licheniformis and B. pumilus survive pasteurization and other heat treatments as spores. Toxin-producing strains of these species in the dairy production chain may thus be of food safety concern.

High genetic relatedness among Mycobacterium avium strains isolated from pigs and humans revealed by comparative IS1245 RFLP analysis.

Members of the Mycobacterium avium complex cause pig mycobacteriosis and opportunistic human infections. Infections due to environmental mycobacteria are increasing in both industrial and developing countries. Mycobacterium-infected pig carcasses can pass for human consumption due to the poor specificity of meat control by visual detection at the slaughter houses. The genetic relatedness of porcine and human MAC isolates in Finland has been unknown. M. avium isolates isolated from pig organs (n=16) and clinical samples (n=13) were compared by IS1245 RFLP analysis to evaluate the similarity of the isolates obtained from human and porcine samples. Nearly identical multicopy M. avium subsp. hominissuis IS1245 RFLP fingerprints were obtained for isolates of porcine and human origin. IS1245 RFLP patterns of 38% of the porcine and human M. a. hominissuis isolates were >90% similar. The RFLP patterns of two porcine and two human isolates showed >95% similarity. The high similarity of the IS1245 RFLP patterns of the human and porcine M. a. hominissuis isolates indicates close genetic relatedness, suggesting that M. a. hominissuis is transmitted between pigs and humans, or that pigs and humans share common environmental sources of infection. Porcine and human isolates with RFLP patterns differing by only one or two bands were found, which shows that the same M. a. hominissuis strains may infect both humans and pigs.

Proliferation of mycobacteria in a piggery environment revealed by mycobacterium-specific real-time quantitative PCR and 16S rRNA sandwich hybridization.

Pig mycobacteriosis is the most common animal mycobacterial disease in Finland with a long-term average prevalence of 0.34% and temporary peaks as high as 0.85%. In the current study Mycobacterium-specific real-time qPCR and 16S rRNA sandwich hybridization were utilized for culture-independent detection and measurement of potentially infectious mycobacteria in selected piggeries. Participating herds (n=5) were selected according to prevalence of tuberculous lesions (>4%) in slaughtered carcasses. When DNA extracted from piggery bedding materials was analyzed by Mycobacterium-targeted qPCR using the SYBR green I dye for detection of amplification products, 10(5) to 10(7) cell equivalents of mycobacterial DNA were detected in unused bedding materials and 10(8) to 10(10)g(-1) dry weight in used bedding materials. When Mycobacterium-specific hybridization probes were used for detection of amplification products, 10(5) to 10(7) cell equivalents of mycobacterial DNA g(-1) dry weight were detected in unused bedding materials in four out of the five piggeries studied and up to 10(8) cell equivalents in used bedding material. The results were confirmed by the Mycobacterium-specific 16S rRNA sandwich hybridization assay. The present results show, that mycobacteria occur in organic materials commonly used on pig farms, and may proliferate in bedding materials during use. We also show that DNA- and RNA-based methods may be utilized for detection of environmental reservoirs of mycobacteria causing porcine and human infection.

Characterization of hemolytic activity of Staphylococcus aureus strains isolated from bovine mastitic milk.

Beta (beta) and delta (delta)-hemolysin of Staphylococcus aureus strains were cultured in vitro in milk lactoserum (whey) prepared from both healthy and mastitis bovine milk. Production of beta- and delta-hemolysins were detected in 12 out of 50 strains studied. The association between N-acetyl-beta-D-glucosaminidase (NAGase) activity, plasmin activity (PL) and trypsin inhibitory capacity (TIC), known as inflammatory indicators for mastitis, and hemolytic activity were also studied. Mastitic milk decreased directly the lytic effect of both beta-and delta-hemolysins of S. aureus on hemolytical blood agar plates. S. aureus in healthy milk samples produced more beta-hemolysin (3 times) and delta-hemolysin (2 times) when compared to S. aureus supernatants in milk from infected quarters. Furthermore, beta- and delta-hemolysis correlated negatively with TIC and NAGase and PL activities. Addition of reduced glutathione (GSH) or beta-mercaptoethanol into the artificial medium enhanced hemolysins activity.

Evaluation of the antibacterial and hemolytic activities of Latvian herbal preparation.

Three extracts originating from a combination of various Latvian plant species were tested for their antibacterial activities by evaluating growth delays using a fully automated microturbidimetric method. Ten different human and bovine strains of the genera Staphylococcus and Micrococcus were used as test microorganisms. The inhibitory effect in vitro was defined as the difference between the growth rate without herbs and the growth rate in the presence of an extract. Among the tested strains, Staphylococcus aureus was found sensitive to all 3 extracts. However, extract I was the most effective in slowing the growth of all strains tested. Using appropriate tester strains it should be possible to set up a broad-range microtubidimetry assay for individual herb screening in vitro. The hemolytic effects of the individual extracts on human erythrocytes were also studied at different concentrations. Two of the herbal extracts had minimal lytic effects on eurocaryotic cells. An additional hemolysis test was conducted in the presence of coenzyme Q10 (CoQ10) as a free radical scavenger: CoQ10 had no effect on the hemolytic reaction.

Characterization of Sphingomonas isolates from Finnish and Swedish drinking water distribution systems.

Sphingomonas species were commonly isolated from biofilms in drinking water distribution systems in Finland (three water meters) and Sweden (five water taps in different buildings). The Sphingomonas isolates (n = 38) were characterized by chemotaxonomic, physiological and phylogenetic methods. Fifteen isolates were designated to species Sphingomonas aromaticivorans, seven isolates to S. subterranea, two isolates to S. xenophaga and one isolate to S. stygia. Thirteen isolates represented one or more new species of Sphingomonas. Thirty-three isolates out of 38 grew at 5 degrees C on trypticase soy broth agar (TSBA) and may therefore proliferate in the Nordic drinking water pipeline where the temperature typically ranges from 2 to 12 degrees C. Thirty-three isolates out of 38 grew at 37 degrees C on TSBA and 15 isolates also grew on blood agar at 37 degrees C. Considering the potentially pathogenic features of sphingomonas, their presence in drinking water distribution systems may not be desirable.

Significance of apoptosis and its relationship to antioxidants after ochratoxin A administration in mice.

A study of the appearance of liver apoptosis after ochratoxin A (OTA) administration was performed in male mice. Administration of OTA twice a week for one or two weeks period results in the occurrence of apoptosis in mice"s liver. The presence of intracellular apoptosis bodies was detected at two weeks after toxin treatment. Light microscopic examination demonstrated the presence of eosinophilic globules, often containing apoptotic bodies. They were found within the cytoplasm of intact hepatic cells. The number of apoptotic bodies was further enhanced at two weeks, resulting in 8 fold increases in liver over the control values. No evidence of cell necrosis could be observed by histological and biochemical analysis at one week. However, centrilobular necrosis was evident at two weeks. The ability of the combined antioxidants: Coenzyme Q 10 (CoQ 10), L-carnitine, Zn, Mg, N-acetyl cysteine, vitamin C, vitamin E and selenium or tamoxifen to intervene in apoptosis induced in livers of mice by OTA was also investigated. The inhibition by these scavengers was more clear in mice treated with OTA for one week than those mice treated for two weeks. Treatment with tamoxifen, known in restoration of tumor suppressor function and on induction of programmed cell death (apoptosis), after OTA administration, had no significant inhibition effect on the incidence of apoptotic bodies in liver. Because hepatic glutathione represents the major defence against toxic liver injury, we studied the activity of tissue reduced glutathione (GSH), known to inhibit apoptosis. Our finding showed that two weeks after treatment, OTA caused a decrease of the GSH activity. However, treatment of mice with the combined antioxidants could enhance hepatic antioxidant/detoxification system, as indicated by increase in hepatic reduced glutathione level. In the light of these results, apoptosis was observed after two weeks of OTA administration. We also suggest that use of the combined antioxidants may be of interest in conditions were certain toxin-mediated forms of cell death and/or apoptosis contribute significantly to toxicity.

Measurement of antibacterial activities of T-2 toxin, deoxynivalenol, ochratoxin A, aflatoxin B1 and fumonisin B1 using microtitration tray-based turbidimetric techniques.

Various mycotoxins were tested for their antibacterial activity by evaluating growth delays using a fully automated microturbidmetric method. Ten different strains of the genera Escherichia, Streptococcus, Staphylococcus, Yersinia, Salmonella, Erysipelothrix and Lactobacillus were used as test micro-organisms. T-2 toxin, deoxynivalenol (DON), ochratoxin A (OTA), aflatoxin B1 (AFB1) and fumonisin B1 (FB1) were used as representative mycotoxins. The inhibitory effect in vitro was defined as the difference between the growth rate without mycotoxins and the growth rate in the presence of a mycotoxin. Among the tested strains, Streptococcus agalactiae was found to be sensitive to all the toxins, with the exception of OTA. T-2 toxin and FB1 were the most effective in slowing down the growth of Staphylococcus aureus. AFB1 affected the growth of Yersinia enterocolitica. The growth rate of Escherichia coli and Salmonella infantis was decreased by FB1. Among the bacterial strains used in this study, only the growth of Erysipelothrix rhusiopathiae was inhibited by OTA. Thus, using appropriate tester strains it should be possible to set up a broad-range microtubidimetry assay for individual mycotoxin screening in vitro. We concluded that the microtitration technique provides a rapid, convenient and high-throughput capacity system to analyse bacteria-mycotoxin interactions.

Effects of tamoxifen, melatonin, coenzyme Q10, and L-carnitine supplementation on bacterial growth in the presence of mycotoxins.

The involvement of toxic oxygen intermediates in the bacteriostatic effects of mycotoxins (T-2 toxin, deoxynivalenol, ochratoxin A, aflatoxin B1, and fumonisin B1) was investigated by producing bacterial growth curves using turbidimetry assays in the presence and absence of oxygen radical-scavenging substances. The strains used in this study included Escherichia coli (FT 101), Streptococcus agalactiae (FT 311, FT 313, FT 315), Staphylococcus aureus (FT 192), Yersinia enterocolitica (FT 430), Salmonella infantis (FT 431), Erysipelothrix rhusiopathiae (FT 432), Lactobacillus plantarum (FT234) and Lactobacillus casei (FT 232). Tamoxifen, melatonin, l-carnitine and coenzyme Q10 were used as radical scavengers against oxygen toxicity to the strains studied. Tamoxifen was the most effective in inhibiting bacterial growth when used at a high concentration, whereas melatonin and l-carnitine were less effective. A combination of l-carnitine and coenzyme Q10 provided better protection against oxygen toxicity caused by the mycotoxins growth than they did individually. It was concluded that oxygen radicals are involved in the killing of bacteria and that there is endogenous formation of toxic oxygen products by mycotoxins. The objective of this study was to determine whether the antioxidants were able to counteract the toxic effects of the mycotoxins. The data obtained indicate that bacterial growth can be inhibited especially by T-2 toxin, aflatoxin B1 and ochratoxin A and that this effect can be partially counteracted by antioxidants such as coenzyme Q10 plus l-carnitine.

Binding of Staphylococcus aureus to milk fat globules increases resistance to penicillin-G.

The susceptibility to penicillin-G of Staphylococcus aureus strains that cause mastitis was tested in milk and in Iso-sensitest broth (ISB): The minimal inhibitory concentrations (MIC) of beta-lactamase-positive strains in milk were 10-100-fold those in ISB, whereas the MIC of beta-lactamase-negative strains in milk were some 10-fold those in ISB; beta-lactamase production was induced by milk in beta-lactamase-positive strains. Much of the increase in resistance to penicillin-G caused by milk can be attributed to milk fat globules; the increase in resistance was related to the binding capacity of the bacteria to milk fat globules as well as to capsule formation by the bacteria. It appears that the binding of the staphylococci to the fat globules and bacterial capsule formation resulted in a biofilm type of growth. In this case, the staphylococci behaved differently from the planktonic type of growth in artificial broth medium in which antibiotic susceptibility testing is usually carried out.

Giving selenium supplements to dairy cows strengthens the inflammatory response to intramammary infection and induces a growth-suppressing effect on mastitis pathogens in whey.

Supplementing the feed of selenium-deficient diary cows with selenium (Se)-yeast or selenite at a level of 0.2 p.p.m. induced self-cure of subclinical mastitis; the prevalence of quarters harbouring subclinical mastitis (bacteriological criteria) decreased to about one half during the 8 week supplementation period. Three phenomena became apparent to explain the beneficial effect of selenium on mastitis: 1. The recruitment of phagocytes to the infected milk compartment of the udder was improved due to Se-supplementation; the correlation between infection and the respective inflammatory response, as indicated by the somatic cell count (SCC) and the N-acetyl-beta-D-glucosaminidase activity (NAGase) of milk, was poor in selenium-deficient cows. Selenium supplementation significantly improved the correlation; 2. Selenium supplementation induced an unspecified antibacterial activity in milk lactoserum (whey), restricting in vitro growth of the mastitis pathogens Escherichia coli, Staphylococcus aureus, Streptococcus agalactiae and Streptococcus uberis, bacterial growth rates in whey samples became inversely correlated with the respective blood glutathione peroxidase activities (GSH-Px); and 3. Selenium supplementation had an effect on redox activities and sulfhydryl activities in whey. These changes were correlated with in vitro bacterial growth rates.

Comparison of penicillin-G susceptibility testing methods of staphylococci isolated from bovine mastitis.

Penicillin-G susceptibility was analyzed on forty staphylococcal strains isolated from mastitic bovine quarters (29 coagulase positive and 11 coagulase negative) by the standard Kirby-Bauer agar diffusion method, the epsilon-Test, the Vetmic, turbidimetric MIC analysis in Iso-Sensitest Broth (ISB) and whey. Penicillinase production was tested. Parallel susceptibility tests were carried out in whole milk, whey and ISB using resazurin and triphenyltetrazolium as the indicators of bacterial activity. The traditional susceptibility testing methods (radial agar diffusion, MIC in broth culture) showed good agreement with each other and confirmed that the tests can be used interchangeably with the current breakpoint values (0.25 micrograms/ml and phi 26 mm). The tests carried out in whey showed good correlation with the traditional tests. However, the susceptibility testings in milk resulted in additional variation. Therefore, traditional susceptibility tests of Penicillin-G in artificial media are limited in estimation of bacterial susceptibility when they grow in whole milk. The relevance of this observation regarding mastitis therapy is discussed.

Automation of the resazurin reduction test using fluorometry of microtitration trays.

Microtitration plate technique and -fluorometry was applied to automate the resazurin reduction test for monitoring bacterial numbers in broth cultures and milk. The effect of resazurin and resorufin concentration, bacterial species, growth medium, pH, and redox potential on the fluorescence response was studied. The timing of the appearance of maximum fluorescence was directly related to the logarithm of the number of colony-forming units (log CFU). Fresh milk and heat-treated milk contain interfering redox systems. The technique based on microtitration plate fluorometry, when fully automated, seems to provide a high-capacity system for analyzing bacterial numbers in foodstuffs and other media.

Effect of frequent milkings on milk NAGase, plasmin, trypsin inhibitory capacity and the quality of whey as the growth medium for mastitis pathogens.

Unequal milking intervals affected milk somatic cell count and N-acetyl-beta-D-glucosaminidase (NAGase) activity. The total daily output of milk NAGase and plasmin decreased, if quarters were emptied frequently during the day. Mastitis pathogens showed stimulated growth in whey prepared from filled quarters as compared with growth in whey from quarters emptied frequently during the day. The quality of whey as growth medium for mastitis pathogens paralleled plasmin activity in respective milk samples. Adaptation of mastitis pathogens to grow in whey had an enhancing effect on bacterial growth during subsequent inoculations in whey. Bacteria probably "learn" to overcome the effect from endogenous antibacterial factors and to use nutrients present in whey.

Bacterial growth in mastitic whey in relation to bacterial association with mastitis.

The growth of mastitis pathogens (eight strains) and non-pathogenic bacteria (eight strains) was studied using microturbidometry. All bacteria grew well in whey originating from uninflamed quarters. However, the growth of bacteria which are not considered to be mastitis pathogens (Lactobacillus plantarum, Bacillus subtilis, Streptococcus lactis and Pseudomonas fluorescens) became suppressed by whey from mastitic quarters. On the contrary, the growth of Staphylococcus aureus and Escherichia coli was generally better in mastitic samples. Streptococcus agalactiae showed inconsistent results.