Efflux Might Participate in Decreased Susceptibility to Oxytetracycline in Contagious Agalactia-Causative Mycoplasma spp.

SIMPLE SUMMARY: Contagious agalactia is a multi-faceted disease affecting small ruminants worldwide. It is caused by four different Mycoplasma (sub)-species. In the absence of highly efficient vaccines, its control relies mostly on antibiotic treatment. Tetracyclines are one of the main families used, as they are cheap and often ensure clinical recovery, if not microbial clearance. However, some isolates have shown lowered susceptibilities even without the mutations in the target gene known to result in resistance. We suspected that an active efflux mechanism could be responsible for such lower-susceptibility phenotypes. Using various techniques, we demonstrated that most of the strains we studied did exhibit a capacity to actively extrude various substances including tetracyclines. This might contribute to low resistance profiles. ABSTRACT: Contagious agalactia is associated with mastitis, keratoconjunctivitis, arthritis, pneumonia, and septicemia in small ruminants in countries with large dairy industries worldwide. The causative agents belong to four (sub)species of the Mycoplasma genus that have remained essentially susceptible to antimicrobials, including to the widely-used tetracycline family. However, some clinical isolates have been detected that show increased minimum inhibitory concentrations of tetracyclines, although they do not harbor the mutation in the 16SrRNA gene usually associated with resistance. The present work aimed to assess whether efflux pumps, infrequently described in mycoplasmas, could participate in the observed moderate loss of susceptibility. General efflux mechanisms were measured (i) using the fluorescence property of ethidium bromide when accumulated intracellularly and intercalated in the mycoplasma genomes, its active extrusion resulting in a temperature-dependent decrease in fluorescence and (ii) monitoring the growth inhibition of mycoplasmas by subinhibitory concentrations of tetracycline with or without reserpine, a known inhibitor of efflux in other bacteria. Both methods revealed non-specific efflux phenomena in most of the isolates tested, although their efficacy was difficult to quantify. This property could contribute to the acquisition of mutations conferring resistance by maintaining intracellular concentrations of tetracyclines at subinhibitory levels.