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The aim of this research was to develop multiplex polymerase chain reaction assays for the detection of aminoglycoside ( strA ,  strB ), sulphonamide ( sulI ,  sulII ), tetracycline ( tetA ,  tetB ,  tetK ,  tetM ,  tetO ), macrolide and lincosamide ( msrA ,  ermA ,  ermB ,  ermC ,  mefA/E ) genes of resistance in mastitis pathogens ( Escherichia coli ,  Staphylococcus aureus ,  Streptococcus uberis ,  Streptococcus agalactiae  and  Streptococcus dysgalactiae ). Applying the established assays, we investigated the distribution of antibiotic resistance genes in the above mentioned species isolated from milk samples in the Czech Republic. Each assay consisted of seven pairs of primers. Six of them amplified fragments of antibiotic resistance genes and one pair a fragment of a species specific gene. Polymerase chain reaction conditions were optimized to amplify seven gene fragments simultaneously in one reaction. In total, 249 isolates were used, among which 111 were positive for  E. coli , 52 for  S. aureus  and 86 for  Streptococcus  spp. The majority (60.2%) of bacteria carried at least one antibiotic resistance gene and 44.6% were multidrug-resistant. The designed multiplex polymerase chain reaction assays may be applied as diagnostic method to replace or complement standard techniques of antibiotic susceptibility testing in the mentioned pathogens.

Detection of selected antibiotic resistance genes using multiplex PCR assay in mastitis pathogens in the Czech Republic