Karakterizacija mehanizama rezistencije na antibiotike i molekularna tipizacija izolata Escherichia coli poreklom od goveda i svinja
Escherichia coli isolates from milk samples of cows with clinical mastitis and from pigs were used for molecular typing, to determine antimicrobial resistance and to characterize resistance genes and mobile genetic elements. Seven isolates from cow milk and 15 isolates from pigs were included in the study. All of the isolates originated from farms located in the Autonomous Province of Vojvodina except for one isolate which originated from a pig raised in a farm located in the district of Mačva. Applying Pulsed-Field Gel Electrophoresis (PFGE) and Random Amplified Polymorphic DNA (RAPD) analysis it was established that all of the isolates were genetically distinguishable. The phylogenetic analysis was done by multiplex PCR method with three primers for identification of two genes and one DNA fragment. This method enables the classification of E. coli isolates into four phylogenetic groups (A, B1, D and B2) depending on the amplicon combination obtained. Seventeen E. coli isolates belong to phylogenetic group A, three isolates belong to group B1 and two isolates were classified in group D. All of the isolates (Nos 22) were multidrug resistant as resistance was found to >3 antibiotics. Resistance to fluoroquinolones which are important antibiotics in human medicine was found in 13 isolates. In all of these isolates single or multiple mutation were found in the topoisomerase genes. In isolates 1M-13, 2M-13, 3M-13, 2S-13, four amino acid substitutions were found i.e. Ser83Leu i Asp87Asn on gyrA gene, Ser80Ile on parC gene and Ser458Ala on parE gene. In isolate 7S-13 identical mutations were found on gyrA and parC genes but in parE gene amino acid substitution Ser448Thr was found. In isolates 3S-13, 6S-13, 14S-14 and 15S-14, the same amino acid substitutions were found on gyrA and parC genes as well except for the isolate 6S- 13, where on parC gene amino acid substitution Glu84Lys was found. Single point mutation was found on gyrA gene in isolates 4M-13, 1S-13, 5S-13, 12S-13 with the minimal inhibitory concentration to ciprofloxacin of 0.5 to 0.125 mg/L. Isolates 4M-13, 1S-13 and 12S-13 had mutation Ser83Leu, while isolate 5S-13 had mutation Asp87Asn and MIC to CIP was 0.062 mg/L. Plasmid mediated resistance was identified in one isolate of E. coli from cow milk (3M- 13) and one isolate from a pig (7S-13) and it was encoded by aac6’Ib-cr gene while in one isolate from a pig (16S-14) the qnrS gene was identified. The blaCTX-M-1 gene conferring resistance to extended-spectrum cephalosporins was found in one isolate from cow milk (7M-14) and one isolate from a pig (7S-13). The strA and strB pair of genes, encoding streptomycin inactivating enzymes were found in 17 isolates. The blaTEM-1 gene was found to confer resistance to ampicillin in 16 E. coli isolates, while tetA and tetB genes were found in all isolates resistant to tetracycline (22 isolates). Genes encoding integrase 1 (int1) were found in 14 E. Coli isolates, while genes encoding integrase 2 (int2) were found in two E. coli isolates. Integrons class 1 was detected in 12 E. Coli isolates and integron class 2 in one isolate from a pig. Gene cassette in integrons was analyzed. The cassette aadA1 was found in two E. coli isolates (4M-13, 1S-13), while the drfA17-aadA5 gene cassette array was found in isolates 3S-13, 7S-13, 14S-14 and 15S- 14. Another cassette array dfrA1-aadA1 was identified in isolates number 6M-13, 5S-13, 11S-13. In E. coli isolate 1M-13 a gene cassette array aadA2-linF was found in integron 1 element, while single cassette aadA23 was found in isolate 13S-14. The E. coli isolate 16S-14 both integron 1 and integron 2 were identified. In integron 1 gene cassette aadA23 was found, while in integron 2 a gene cassette array aadA1-sat2 was detected. Conjugative plasmids were identified in 16 isolates (73%) among which multidrug resistant conjugative plasmid (resistance to >3 antibiotics) was found in ten isolates. In conclusion, numerous E.coli isolates confer resistances to antibiotics which are used in human medicine. The high resistance to ciprofloxacin in E. coli isolates from cow milk and pigs can be explained by the overuse of antibiotics, especially fluoroquinolones, in livestock industry in Serbia.