Tipificación molecular y mecanismos de resistencia antimicrobiana en micoplasmas asociados a la agalaxia contagiosa

Resum

This doctoral thesis is presented as a compendium of publications and the articles that form part of it have the common goal of evaluate different diagnostic techniques for the detection of M. agalactiae in goat milk samples and the characterization of field isolates of contagious agalactia causing mycoplasmas by molecular typing and the study of their antimicrobial resistance mechanisms. Thus, the specific objectives and methods were: 1) To determine the lower M. agalactiae detection limits of culture and PCR techniques in experimentally inoculated goat milk samples, 2) To establish the agreement between PCR and culture techniques for the detection of M. agalactiae using mastitic milk samples retrieved from chronically infected commercial dairy herds, 3) To assess the genetic variability of different M. mycoides subsp. capri and M. capricolum subsp. capricolum field isolates with different epidemiological characteristics isolated from contagious agalactia endemic areas, 4) To evaluate the in vitro antimicrobial susceptibility of M. capricolum subsp. capricolum field isolates retrieved from Spain and Italy and 5) To study the importance of the QRDR in the acquisition of quinolone resistance, determining the specific genes that provide useful information about quinolone resistance in M. agalactiae and M. capricolum subsp. capricolum isolates. The obtained results allowed us to conclude: 1) In goat milk samples, bacterial culture technique has a lower detection limit of M. agalactiae than PCR. Therefore, culture is recommended when bulk tank milk samples are analyzed, whereas both techniques have a good agreement when detecting this pathogen in mastitic milk samples; 2) MLST schemes are a useful tool to detect genetic differences between different M. mycoides subsp. capri field isolates coming from different geographical areas. Moreover, they also allow the detection of variations between isolates retrieved from the same herd at the same time; 3) M. mycoides subsp. capri and M. capricolum subsp. capricolum show great genetic variability between field isolates coming from the same contagious agalactia endemic area. These differences are greater than those reported in other mycoplasma species such as M. agalactiae; 4) There is no connection between phylogeny and antimicrobial susceptibility in the studied M. capricolum subsp. capricolum strains; 5) Doxycycline is the most effective antimicrobial inhibiting M. capricolum subsp. capricolum growth. Besides, some of the analyzed field isolates were resistant to quinolones and macrolides; 6) The acquisition of quinolone resistance in M. capricolum subsp. capricolum and M. agalactiae is due to mutations in the genes that encode enzymes DNA gyrase and topoisomerase IV and 7) parC is the gene providing most information about quinolone susceptibility in M. capricolum subsp. capricolum and M. agalactiae.