Mikrobiologická kontaminace syrového mléka a mléčných výrobků

Angl. resum

Rep-PCR-based typing as a tool for tracking of MRSA infection origin

A performance of rep-PCR fingerprinting method with the (GTG)5 primer was explored in order to evaluate its practical application for confirmation the source of human staphylococci infection. A laboratory worker daily working with the MRSA and Staphylococcus aureus strains has been tested positively for MRSA nosocomial infection. The collection of nine MRSA strains held in the laboratory has been typed with the rep-PCR method. Analysis of the fingerprints with the unweighted pair-group method using arithmetic averages (UPGMA) clustering method revealed presence of six strain clusters with similarity lower than 85%. The fingerprint of MRSA strain infecting the human differed significantly from remaining fingerprints. This provided clear evidence that MRSA strain infecting the personal did not come from the laboratory strains collection. Our experimental results showed rep-PCR with the (GTG)5 primer as an effective tool applicable for differentiation of individual S. aureus strains. However, the reproducibility and discrimination power of the method depended on strict observance of the optimal PCR time and temperature profile and PCR composition as well

A new real-time PCR assay for rapid identification of the S. aureus/MRSA strains

The Methicillin-resistant Staphylococcus aureus (MRSA) with the livestock-associated MRSA (LA-MRSA) are of great interest to scientists and general public. The aim of our study was to present a new more rapid and reliable diagnostic method working on the RT-PCR platform applicable for monitoring of MRSA/S. aureus. The parallel testing of the S. aureus specific nuc gene sequence and the mecA gene sequence was utilised for this purpose. A collection of ten S. aureus/MRSA reference strains, fifteen genetically related non S. aureus reference strains and fifty-six environmental samples was employed for estimation of the assay performance and parameters. The environmental samples acquired in the Czech livestock farms were represented with the livestock and human nasal mucosae or skin swabs, the slaughter meat swabs and were chosen preferentially from individuals with previously confi rmed or suspected positive MRSA/S. aureus cases. The classic selective cultivation approach with the biochemical test and agar disk diffusion test was accepted as reference diagnostic method. As there were no culture positive samples that were negative using RT-PCR, our method featured with 100% sensitivity in comparison to reference method. The limit of detection allowed to identify from tens to hundreds copies of S. aureus/MRSA genome. Further, the RT-PCR assay featured with 100% inclusivity and 95% exclusivity at Cq value below 30. These parameters suggested on powerful and reliable diagnostic method with real potential of practical utilisation. We consider our method as ideal for testing of individual suspected colonies, when the results can be acquired in less than 1.5 hour

Occurrence and antimicrobial sensitivity in staphylococci isolated from goat, sheep and cow’s milk

The aim of this study was to compare the sensitivity to selected antibiotics in staphylococci isolated from goat (n = 60), sheep (n = 60) and cow’s milk (n = 120). The individual milk samples were inoculated onto Blood agar cultivated at 36 °C/24 h.The isolated species of staphylococci were identified using biochemical tests, namely STAPHYtest and identification program TNW pro 6.5. We examined the sensitivity of strains to the spectrum of antibiotics, as follows: vancomycin (VA), amoxicillin/clavulanic acid (AMC), penicillin (P), rifampicin (RD), oxacillin (OX), tetracycline (TE), erythromycin (E), chloramphenicol (C), clindamycin (DA), gentamicin (CN), ciprofloxacin (CIP), teicoplanin (TEC), cefoxitin (FOX) and novobiocin (NOV). Altogether, 97 staphylococcal isolates were obtained; 70 from cow’s milk, 11 from goat’s milk and 16 from sheep’ milk. Staphylococcus aureus was the most frequent species in milk of all animal origin tested, was detected in 54 (45%) cow’s milk, 10 (17%) goat’s and 15 (25%) sheep’ milk samples. S. lentus was identified only in goat’s and sheep’ milk whereas in cow’s milk there were representation of staphylococcal species as follows: S. haemolyticus (n = 7), S. chromogenes (n = 2), S. warneri (n = 2), S. xylosus (n = 2), S. epidermidis (n = 2) and unclassified staphylococci (n = 1). The results of S. aureus sensitivity are similar for all tested antibiotics and for all monitored milk: No resistance to vancomycin, rifampicin, chloramphenicol and teicoplanin was recorded in obtained S. aureus isolates whereas the resistance to ciprofloxacin was found out most often

Evaluation of development in indirect determination of milk fat free fatty acids in Czech Republic

Free fatty acids (FFAs) in fat are important indicator of raw milk quality. Result reliability of FFAs is important. Aim was to verify MIR–FT (mid infrared spectroscopy with Fourier’s transformations) method for its calibration to determine FFAs, time stability of MIR–FT FFA calibration and calibration levelling in laboratory networks. Reference (RE) milk samples (1 set = 8) were prepared according to CSN 57 0533 (FFAs in mmol.100g−1 of fat). MIR–FT instruments were: 1 LactoScope FTIR (DE); 2 Bentley FTS (BE); 2 MilkoScan FT 6000 (FO). 3 calibrations of MIR–FT (5) in 3 laboratories were performed. Bulk milk samples came from 4 herds and 2 breeds. These 4 samples were used for calibration in native and modified form. Modification increased FFAs by cca 100%. Calibration set had 8 samples. 1 between calibration interval was checked monthly by proficiency testing (PT). PT set had 10 samples. 5 samples were with native milk and 5 had modified fat content, lower and higher. Maximal value of difference variability for calibration quality validation is x (sd of difference MIR–FT and RE) plus 1.64 multiple of sd (on 95% level), 1.0613 mmol.100g−1. Mean validation correlation coefficient (r) between MIR–FT and RE results was 0.802 ± 0.082 (P < 0.001), from 0.666 to 0.945. Minimal value at calibration is x minus 1.64 multiple of sd (0.668). Correlations between MIR–FT results were higher by 8.4% (0.869 (P < 0.001) > 0.802). Example PT with 10 and 5 milk samples had similar results of r 0.887 and 0.953 (P < 0.001 and P < 0.05). There is possibility to construct a levelling programme for calibrated instruments. Some equation between PT reference and instrumental values could correct MIR–FT results for their better comparability

Analyse of relationships between freezing point and selected indicators of udder health state among cow, goat and sheep milk

Milk freezing point (MFP) is important quality indicator. Aim was to analyse the relationships of MFP to selected udder health milk indicators (MIs) by comparison between cows (reference), goats and sheep. Bulk milk samples came from 3 herds of Czech Fleckvieh (B, n 93) and 1 goat herd and sheep flock (White short-haired, W, n 60; Tsigai, C, n 60). Animal nutrition was performed under the typical country conditions. MIs which were investigated: DM, dry matter; SNF, solid non fat; L, lactose (all in %); SCC, somatic cell count (103 ml−1); EC, electrical conductivity (mS cm−1); MFP (°C); Na and K (in mg kg−1). W MFP was −0.5544 ± 0.0293, B −0.5221 ± 0.0043 and C −0.6048 ± 0.0691 °C. The B MFP was related to L (−0.36; P < 0.01), W was not related to L (−0.07; P > 0.05) and C was related to L (0.40; P < 0.01). These facts could be explainable by worse SCC geometric averages for used W (3,646 103 ml−1) and C (560 103 ml−1) milk as compared to B (159 103 ml−1). Only 0.5 and 10.5% of variations in MFP were explainable by variations in DM and SNF in B, 32.7 and 12.8% in W but already 49.4 and 45.0% in C. Higher C values were caused by high MFP variability, 11.8% (C) versus 0.8% (B). There is possible to derive the more reliable MFP qualitative limits for more efficient monitoring rules of milk quality problems in B, W and C

Interspecies and seasonal differences of retinol in dairy ruminant´s milk

Milk is an essential source of macronutrients and among lipophilic vitamins is significant source of retinol. The contribution of milk to the reference daily intake for retinol varies from 11% to 16%, worldwide. The most consumed dairy products are fresh, dehydrated and condensed milk in which the amonuts of retinol are not modified to those of in whole milk. Retinol is essential to ensure a good functionality of the immune system and plays a critical role in vision, reproduction, cell differentiation as well as growth and development and is found only in animal tissues. The aim of our study was to evaluate the interspecies differences in the retinol concentration of whole raw bovine, caprine and ovine milk and to observe seasonal variation of retinol in bulk tank milk samples. Samples of raw milk were colleceted on different farms in the Czech Republic between 2013 and 2014. Retinol was measured by ultra high performance liquid chromatography with UV detection (325 nm) in isocratic mode after alkaline saponification with methanolic potassium hydroxide solution and liquid-liquid extraction into non polar organic solvent of whole raw milk. To avoid vitamin losses or degradation during the procedure, antioxidants were added to the sample extraction media. Our results indicate significant interspecies differences between bovine and ovine milk and caprine and ovine milk. Concentration of retinol is very similar in bovine and caprine milk 0.96 &plusmn;0.11 mg/L, 0.94 &plusmn;0.25 mg/L, respectively. The mean concentration in sheep&acute;s milk is 1.75 &plusmn;0.24 mg/L. The seasonal variation of retinol in raw bovine milk was detected as high significant, with the highest concentration during winter. These results contribute to the nutrition evaluation of milk in the Czech Republic and indicate, that the sheep&acute;s milk is the best source of retinol among the milks of ruminants kept in the Czech Republic, however it is not used in its fluid form for human consumption.<!--[endif] --