Usage of antimicrobials on 60 dairy farms in Northern Germany and characterization of Methicillin-resistant Staphylococcus aureus (MRSA) and extended spectrum beta-lactamases producing Escherichia coli (ESBLs-producing E. coli) isolated from bulk tank milk samples

The objectives of this study were to gain insight into the usage of antimicrobials on dairy farms in Germany and into the presence of MRSA and ESBLs-producing E. coli in bulk tank milk samples. To this end a questionnaire was performed among herd managers of 60 farms (herd size from 25 to 3,000 animals) in Northern Germany, who were participating in the study on a voluntary basis. Bulk tank milk samples were obtained at a single occasion and analysed for the presence of MRSA and ESBLs-producing E. coli. In addition MRSA isolates from milk from the strain collection of the National Reference Laboratory for coagulase positive staphylococci incl. S. aureus (NRL Staph) were characterized by multiplex PCR, SCCmec typing, spa typing, and DNA micro array analysis. Herd managers reported that lameness, metritis and mastitis were regarded as the most important health problems of cows. Neonatal calf diarrhea and the bovine respiratory disease complex (BRD) were reported as the most important disorders in calves. Betalactams, tetracyclines, macrolides, sulfonamides, fluoroquinolones, aminoglycosides, phenicols, and polypeptides were the antimicrobial drug classes that were administered to diseased animals on the dairy farms. A third generation cephalosporin - ceftiofur - which is registered for use in digital phlegmona was the antimicrobial most frequently administered to lame cows. Amoxicillin, tetracyclines and ceftiofur were preferentially used in the treatment of metritis. BRD in calves was mostly treated with florfenicol or macrolides, whereas fluoroquinolones were most frequently administered to diarrheic calves. The herd managers reported that on average 2.8% of their cows were treated for clinical mastitis per month. Milk samples were sent for bacteriological examination in case of clinical mastitis, or, less frequently, from cows with elevated somatic cell counts (SCC). The most frequently mentioned antimicrobial agents that have been used in the treatment of cows with mastitis were cefquinome, penicillin, and the fixed drug combination cefalexin+kanamycin and ampicillin+cloxacillin. On the majority of farms (85% of the farms) a routine procedure at drying off is applied, which includes the intra mammary administration of antimicrobials. Cloxacillin and cefquinome and the fixed drug combination penethamate hydriodide + benethamine penicillin + framycetinsulfate were the antimicrobial drugs mainly used for dry cow therapy. In total 36 MRSA isolates from bovine milk were characterized. Five isolates originated from the bulk tank samples obtained during the farm visits and 31 isolates originated from the strain collection of the NRL Staph. All isolates were confirmed to be MRSA by multiplex PCR and DNA micro array analysis. Two different spa types were identified, namely t011 (22 isolates) and t034 (14 isolates). Among those, 33 carried SCCmec type V, 2 strains had SCCmec type III, and only one strain had SCCmec type IVa. All MRSA strains were phenotypically resistant to oxacillin and tetracyclines. None of the strains was resistant to mupirocin, vancomycin, or linezolid. In total 16 resistance patterns were detected, and the most common resistance pattern was TET-OXA alone. Multidrug resistance (MDR) to a range of three to seven antimicrobial agents was found in 72.2% of the isolates. The most predominant multidrug resistance pattern was TET-ERYCLI-OXA-QUI/DAL. All MRSA strains carried more than one antimicrobial resistance gene, and 18 different antimicrobial resistance gene patterns were identified. The most common genotypic resistance patterns were mecA-blaZ-blaI-blaR-tetM-tetEfflux-tetK and mecAblaZ-blaI-blaR-tetM-tetEfflux-ermA. All 36 MRSA strains carried the genes mecA, tetM, and tetEfflux, and tested negative for vanA, vanB, vanZ (vancomycin), msrA, mefA, mpbBM (macrolides), linA, cfr (lincosamides), vatA, vatB, vga, vgb (streptogramin), aphA (aminoglycoside), dfrA (trimethoprim), and cat (chloramphenicol). In the present study, all strains were tested negative for genes encoding for enterotoxins, genes encoding for toxic shock syndrome toxins (tst1, tst-RF122), Pantone-Valentine Leukocidin (PVL), leukocidins (lukM/lukF-P83, lukD, lukE, lukY-var2), and hemolysin Beta (hlb). In addition, all strains harbored lukX and lukY-var1 (leukocidins/haemolysin toxin family protein), hla (haemolysin alpha), un-truncated hlb (haemolysin beta), hld (haemolysin delta), and lukF, lukS, hlgA (haemolysin gamma). ESBLs-producing E. coli were isolated from bulk tank milk samples from 30% of the farms. Detection of ESBLs-producing E. coli was associated with the occurrence of metritis and dystocia on the farms. The use of antimicrobials in the treatment of cows following dystocia and in the treatment of umbilical infections and arthritis in calves was also associated with the presence of ESBLs-producing E. coli in bulk tank milk. All ESBLsproducing E. coli strains were resistant to ampicillin, cephalothin, ticarcillin, piperacillin, ceftiofur, and cefpodoxime. None of the strains were found resistant to florfenicol, spectinomycin, amoxicillin + clavulanic acid, ceftazidime, and cefoxitin. In conclusion, the results of the questionnaire demonstrate that fluoroquinolones, oxacillin and cloxacillin as well as 3rd and 4th generation cephalosporins are administered to milking cows and their offspring as initial treatment for the most frequently occurring diseases on dairy farms. Testing bulk tank milk seems a suitable tool to monitor the presence of MRSA and ESBLs-producing E. coli in milk. The presence of the latter bacteria in milk underlines the need to heat treat milk before consumption.



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