5^th Animal Health and Veterinary Medicine Congress

Biography

Biography: Juan Tatay-Dualde

Abstract

Mycoplasma capricolum subsp. capricolum (Mcc) is one of the contagious agalactia (CA) causatives agents. Owing to the lack of other effective methods for control and prevention, antimicrobials are the most commonly selected treatment against this microorganism, being quinolones widely used. Quinolones interact with the bacterial DNA gyrase and topoisomerase IV, both implicated in DNA replication. DNA gyrase is compound by subunits A and B which are codified by gyrA and gyrB genes. Besides, topoisomerase IV is codified by parC and parE genes, and all of them (gyrA, gyrB, parC and parE) are known as the Quinolone Resistance Determining Regions (QRDR). Prior studies have demonstrated the influence of those genes in the phenotypic resistance to quinolones in several species of Mycoplasma spp. Nevertheless, molecular mechanisms involved quinolone resistances in Mcc are still unclear. Therefore, the aim of this study was to assess changes at the QRDR region in connection with quinolones resistance. For that purpose, enrofloxacin, marbofloxacin and moxifoxacin were used to select in vitro resistant mutant. The study was carried out with two Mcc field isolates (Cap1 and Cap4) and the reference strain, California Kid (CK) (NCTC 10154) by consecutively culture at subinhibitory antimicrobial concentration. MIC values were assessed between each step and when they increased, gyrA, gyrB, parC and parE were studied. Our results showed that when MIC values increased, changes were detected in all the studied genes but parE. Thus, we found 4 amino acid changes at gyrA (Ser83Ile, Cys84Thr, Ala119Thr, His154Arg), 3 amino acid changes at gyrB (Asp426Gly, Arg447Lys, Asn465Lys) and 4 amino acid changes at parC (Asp79His, Ser80Ile/Asn, Asp84Asn, Glu150Lys). However, the amino acid and position changed differed according to the strain and antimicrobial used. Moreover, the MICs values from which we were able to detect mutations varied according to de antimicrobial used. The different changes attributable to each mutation on the MIC value may be due to different effects in the structure of the proteins and consequent changes in the affinity of quinolone. Indeed, previous studies with Ureaplasma spp, demonstrated structural changes due to amino acid changes at positions 83 and 84 of the parC gene. Besides, except for the reported change at gyrA gene position 154, all the mutations observed in our study meant changing the polarity or charge of amino acids. These changes could explain protein conformational changes and thus lower affinity with quinolones.