Epidemiology and characterization of Staphylococcus epidermidis isolates from humans, raw bovine milk and a dairy plant.

Geographically related Staphylococcus epidermidis isolates from human patients (n=30), dairy farms (farmers and individual raw milk from cattle, n=36) and a dairy plant (n=55) were examined for epidemiological relatedness by pulsed-field gel electrophoresis and, using in vitro methods, for the ability to produce biofilm and antimicrobial resistance. Methicillin-resistant isolates (MRSE) were also identified and characterized. Isolates from farmers and dairy cattle were found to be genetically related, while isolates from human patients were highly diverse. Some dairy plant isolates (18.2%) were closely related to those from dairy farms. Biofilm production and resistance to antimicrobial agents were most typical for isolates from human patients, of which 76.7% were MRSE. Methicillin resistance was also widespread in farm-related isolates (61.1%). This study indicates the possible transmission of S. epidermidis between cattle and farmers. Dairy products were not proven to be an important source of either human infections or methicillin-resistant strains.

The biofilm-positive Staphylococcus epidermidis isolates in raw materials, foodstuffs and on contact surfaces in processing plants.

Isolates from the "farm to fork" samples (182 isolates from 2779 samples) were examined genotypically (icaAB genes) and phenotypically (in vitro biofilm formation, typical growth on Congo red agar; CRA) with the aim to assess the risk of penetration of virulent strains of Staphylococcus epidermidis into the food chain. The contamination of meat and milk products was significantly higher in comparison with raw materials. Contamination of contact surfaces in the meat-processing plants was significantly lower than that of contact surfaces in the dairy plants. The ica genes (which precondition the biofilm formation) were concurrently detected in 20 isolates that also showed a typical growth on CRA. Two ica operon-negative isolates produced biofilm in vitro but perhaps by an ica-independent mechanism. The surfaces in the dairy plants and the milk products were more frequently contaminated with ica operon-positive strains (2.3 and 1.2 % samples) than the other sample types (0-0.6 % samples).

[Microbial biofilms in the food industry]. / Mikrobiální biofilmy v potravinárském prumyslu.

Biofilms are microbial communities whose architecture includes microorganisms, biotic substances produced by these microorganisms and attached organic and inorganic substances from the environment. They pose a serious problem in human medicine. Microbial biofilm communities are also cause for concern in the food industry since pathogenic microorganisms released from the biofilm may contaminate food and raw materials for food production. Not only the microbial community as a whole but also particular cells exhibit increased resistance to sanitation measures and disinfectants which makes it difficult to remove the biofilm or to inactivate particular built up microorganisms. Mainly model studies and molecular genetic and microscopy methods can contribute to better understanding of this issue, and thus to prevention and inactivation of microbial communities on food contact surfaces of equipment in the food production plants. Such studies would be of benefit to both health care and food processing and production.

Antimicrobial resistance of Enterococcus spp. isolates from raw beef and meat products.

E. faecalis (67%) and E. faecium (13.7%) were most frequently isolated among enterococci that contaminate cooled and frozen processed meat, follow-up heat-treated meat products and unheated fermented dry salami. Most isolates of both species were resistant to cephalothin (95 and 83 %) and clindamycin (77 and 67%, respectively). Furthermore, E. faecalis and E. faecium isolates were resistant to erythromycin (44 and 72%), tetracycline (34.5 and 17.4%), and streptomycin (13.3 and 4.3%, respectively). Only a few of the isolates were resistant to ampicillin, ampicillin-sulbactam, chloramphenicol, and vancomycin while all isolates were susceptible to gentamicin, penicillin, and teicoplanin. During the production of heat-treated meat products, numbers of resistant isolates increased in spite of the decreasing enterococcal contamination of the samples. An opposite situation was found in the production of fermented dry salami.

A combined AFLP-multiplex PCR assay for molecular typing of Escherichia coli strains using variable bacterial interspersed mosaic elements.

The original method for molecular typing of E. coli strains was developed using the polymorphism in chromosomal sequences of bacterial interspersed mosaic elements (BIMEs) detected by multiplex PCR and analysed by AFLP assay. The applicability of the method in the epidemiology of E. coli was tested on a group of 524 strains of human and veterinary origin. In the studied group 18 different genotypes were detected. Significant differences were found in the frequencies of the genotypes among various groups of strains, suggesting the method could be a promising tool in the epidemiology of E. coli.

Beef carcass contamination in a slaughterhouse and prevalence of resistance to antimicrobial drugs in isolates of selected microbial species.

Meat contaminating bacteria may be the direct cause of foodborne diseases and represent a potential cause for the drug resistance of human pathogenic agents. The prevalence and resistance to 17 antimicrobial drugs of isolates of selected bacterial species were investigated in 70 swabs of beef carcasses and 70 subsequent samples of beef meat. Molecular techniques (coagulase gene typing Staphylococcus aureus and original gene typing Escherichia coli) were used in the differentiation of isolates. Carcasses were already contaminated after evisceration, least frequently with S. aureus strains (7.5% of samples), most frequently with coagulase-negative staphylococci strains (52.2% of samples). During carcass processing, contamination with resistant or polyresistant strains of S. aureus and E. coli significantly increased (P<0.01). Gene typing isolates of S. aureus and E. coli indicated that the strains probably originated in the processing plant.

Staphylococcus aureus isolates from dairy cows and humans on a farm differ in coagulase genotype.

Staphylococcus aureus is a frequent cause of animal and human infections. The aim of the present study was to test diversity of the populations of S. aureus colonising cattle and humans sharing an infected environment. Eighty-six S. aureus isolates obtained from dairy cows, from people coming into contact with dairy cows on the farm and the other farm personnel were characterized by restriction fragment length polymorphism of the coagulase gene. Molecular analyses identified ten polymorphism types with prevalent presentation of type II in isolates from cow's milk and type IV in isolates from people coming into contact with dairy cows on the farm (the cattlemen) and the other farm personnel. Seven further genotypes were identified among the isolates from the cattlemen. The results indicate that the strains dominating in human population did not equate to the causative agents of bovine mastitis.

Prevalence of and resistance to anti-microbial drugs in selected microbial species isolated from bulk milk samples.

The prevalence of strains of Staphylococcus aureus, coagulase-negative (CN) staphylococci, Listeria monocytogenes, Escherichia coli, Enterococcus faecalis, E. faecium and Bacillus cereus, was investigated in 111 bulk milk samples. Staphylococcus aureus was isolated from 38 samples, CN staphylococci from 63 samples, E. coli from 49 samples, E. faecalis or E. faecium from 107 samples, and L. monocytogenes from two samples. Bacillus cereus was not found in any of the samples and three samples were free of any of the selected species. Sensitivity to the anti-microbial drugs amikacin, ampicillin, ampicillin + sulbactam, cephalothin (CLT), cephotaxime, clindamycin, chloramphenicol (CMP), co-trimoxazole, erythromycin (ERY), gentamicin, neomycin, norfloxacin, oxacillin, penicillin, streptomycin (STR), tetracycline (TTC) and vancomycin was tested using the standard dilution technique. Minimum inhibitory concentration (MIC) characteristics (MIC50, MIC90, MIC range) were determined for each microbial species. Resistance against one or more anti-microbial drugs was found in 93% of S. aureus, 40% of CN staphylococci, 73% of E. coli, 88% of E.faecalis, 55% of E.faecium, and one L. monocytogenes strain. Most of the strains, particularly enterococci, were resistant to STR, TTC, and ERY (MIC50 4 microg/ml). A high percentage of staphylococci were resistant to beta-lactam antibiotics. High resistance to CLT was found in 11 strains of E. coli (MIC 256 microg/ml) and strains resistant to CMP (MIC90 16 microg/ml) were detected. The highest numbers of resistance phenotypes were found in E. coil (16) and CN staphylococci (12). Eighteen identical resistance phenotypes were demonstrated in indicator bacteria (E. coli, E. faecalis, E. faecium) and pathogens (S. aureus, CN staphylococci) isolated from the same bulk milk sample. The obtained resistance data were matched against the herd owners' information on therapeutic use of the drugs. This confrontation could not explain the findings of strains resistant to ERY or CMP. Our findings are evidence of selection of resistant strains among not only pathogenic agents, but also among indicator bacteria which can become significant carriers of transmissible resistance genes.

Comparison of methods for the determination of antimicrobial resistance in Staphylococcus aureus from bovine mastitis.

The results of three standard methods (broth dilution, agar dilution, disk diffusion) and an experimental modification of the microdilution method for determination of resistance to ampicillin, cephalotin, cloxacillin, neomycin, novobiocin, penicillin and streptomycin were compared using 151 Staphylococcus aureus isolates obtained from cases of mastitis. The accuracy of the dilution methods was compared by determination of minimum inhibition concentrations (MIC, MIC50, MIC90 and modal MIC) and by assessment of the agreement within the tolerance of +/-1 dilution step in 2-fold dilution series. The results of the dilution methods were further compared with those of the reference disk diffusion method and the strains were classified as sensitive or resistant using the interpretation criteria for human strains. The comparisons indicated that MIC characteristics and the final classification as sensitive or resistant were method-dependent. Resistance to aminoglycoside antibiotics was observed more often when using broth dilution methods, especially when the broth was supplemented with lactose.

[Effectiveness of the BR-Test and the Intest in the determination of chemotherapeutic residues in raw and preserved milk]. / Ucinnost BR-testu a INTESTU pri stanovení reziduí chemoterapeutik v syrovém a prezervovaném mléce.

Negative detection of residues of inhibitory substances has become one of the standard quality traits of raw milk also in the Czech Republic. Hence a requirement appeared in 1991 to evaluate the usability of two commercially produced microtitration tests for this purpose. The tests of BR-TEST (Enterotox) and INTEST (Mlékárenský prumysl) were performed in weekly intervals for the time longer by a week than is the declared expiration, that means seven replications in BR-TEST and five replications in INTEST. Compact and stripped microplates (STRIPS) designated BR-TEST "AS", BR-TEST 12x8 "AS" and INTEST C-96 type and INTEST 0-96/6x16 were used for the tests. The principle of inhibition demonstration in the concentration series of selected drugs was applied. The drugs were chosen with respect to the frequency of their use for clinical treatments in the CR. Tab. II shows the used drugs and concentration series. The following parameters were tested: detection limits, reproducibility, effect of milk preservation, and the declared expiration. Tab. III shows detection limits. It is apparent the BR-TEST detection responses to streptomycin and chloramphenicol were lower in BR-TEST than in INTEST in our experiments. The responses to penicillin, oxacillin and oxytetracycline were lower in some cases, and equal in other ones. On the other hand, the INTEST detection response to sulfadimidin was markedly lower in comparison with BR-TEST. Tab. IV summarizes the range of detection limits in milk with Heeschen's reagent. In our experiments the detection response of BR-TEST to the tested chemotherapeutics was lower in some cases, in other cases it was the same as in INTEST.(ABSTRACT TRUNCATED AT 250 WORDS)

[Determination of protein A in Staphylococcus aureus strains from a collection]. / Stanovení proteinu A u sbírkových kmenu Staphylococcus aureus.

The author used the haemagglutination method to estimate A protein in 153 strains of Staphylococcus aureus from collections. Protein A was detected in 66% of coagulase positive strains deposited in the course of time in the collection in 1983-1988. All strains were kept in skimmed milk at -20 degrees C. For evaluation of the reaction a semiquantitative scale was proposed. The author observed a diminished capacity to form protein A with the time of storage of the strains. In strains deposited for a shorter time in the collection (only three years) the percentage of positive results rose to 92.5%. In coagulase negative strains protein A was not detected.

[Influencing superovulation in cattle with serum gonadotropin antibody]. / Ovlivnení superovalace u skotu protilátkou proti sérovému gonadotropinu.

The effectiveness of GnRH (Luliberin) and goat anti-PMSG serum prepared at the Veterinary Research Institute, Brno, was studied during the superovulation of heifers treated with PMSG and Estrumate. The use of GnRH did not exert any positive influence on superovulation, as compared with the control groups,. when anti-PMSG serum was administered five hours from the determined beginning of oestrus (46-53 hours from the first administration of Estrumate), the length of oestrus was reduced in comparison with the control group (25.8 and 51.3 h), the number of 22.3). The tested heifers produced 17.8 ova, on an average, out of which (4.1 and 22.3). The tested heifers produced 17.8 ova, on an average, out of which 12.5 were good-quality embryos. The control heifers produced 6.9 ova including 2.9 embryos. The obtained results provide convincing evidence of the promising results of the use of goat anti-PMSG serum during the superovulation cattle.

Superovulation in cattle: Effect of goat anti-PMSG serum.

Sixteen heifers were superovulated using 5 000 i.u. PMSG on days 9 to 11 of the oestrous cycle (day 1 of the experiment) followed by two injections of 500 mug Estrumate 48 and 54 hours later. Eight of them were injected with goat anti-PMSG serum 5 hours after the first signs of oestrus were observed. Compared with the control group, the treatment with anti-PMSG serum resulted in a shorter heat period (25.8 vs. 51.3 hours), a higher mean number of ovulations (22.1 vs. 18.0) and a lower number of follicles over 10 mm in diameter (4.1 vs. 22.3). The mean numbers of eggs recovered in the experimental and control groups were 17.8 and 6.9, of which 70.2 and 42.0 per cent, respectively, were viable embryos. The concentrations of progesterone and 17-beta oestradiol in the blood plasma showed no significant differences between the experimental and control animals. A higher oestradiol in the control group on day 9 of the experiment was in keeping with the histological picture of the target organs and with a significantly higher number of follicles at slaughter on days 12 to 14 of the experiment.

[Effect of chorionic gonadotropin administration on the concentration of testosterone in the blood of boars]. / Vliv aplikace choriového gonadotropinu na koncentraci testosteronu v krbi kancu.

In intact boars, intravenous administration of 100 to 6000 I. U. chorionic gonadotropin elicited a pronounced rise in the plasma level of testosterone, depending on the time interval from the administration of chorionic gonadotropin and on the size of the administered dose. In the blood of castrated boars such an application of choronic gonadotropin did not influence the concentration of testosterone. It was inferred that the rise in the levels of blood testosterone in boars after administration of chorionic gonadotropin was specific for testicular incretion and indicated the endocrine reserve of the testes. There is a discussion concerning the importance of the functional testing of the incretion capacity of testes for differential diagnosis different forms o incretion hypogonadism.