Persistence of Mycoplasma hyopneumoniae in experimentally infected pigs after marbofloxacin treatment and detection of mutations in the parC gene

Abstract

The ability of Mycoplasma hyopneumoniae to persist despite fluoroquinolone treatments was investigated with pigs. Groups of specific-pathogen-free pigs were experimentally infected with M. hyopneumoniae strain 116 and treated with marbofloxacin at the therapeutic dose (TD) or half of the therapeutic dose (TD/2) for 3 days. Results showed that, despite tissue penetration of marbofloxacin, particularly in the trachea and the tracheal secretions, the treatments did not have any influence on M. hyopneumoniae recovery from tracheal swabs. Mycoplasmas were also isolated from inner organs and tissues such as liver, spleen, kidneys, and bronchial lymph nodes. Recontamination of pigs via environment could not explain mycoplasma persistence after medication, as decontamination of pigs and allocation to a new disinfected environment did not have any significant effect on the phenomenon. A significant decrease in the susceptibility level to marbofloxacin of 12 mycoplasma clones reisolated after the treatments (TD/2 and TD) was observed. Two point mutations were found in the ParC quinolone resistance-determining region (QRDR) of DNA topoisomerase IV (Ser80-->Phe and Asp84-->Asn), and one point mutation was observed just behind the QRDR of ParC (Ala116-->Glu). This is the first time that mutations in a gene coding for topoisomerase IV have been described for M. hyopneumoniae after in vivo marbofloxacin treatments in experimentally infected pigs. However, development of resistance is not sufficient to explain M. hyopneumoniae persistence in vivo since (i) marbofloxacin concentrations were above the marbofloxacin MIC of the wild-type strain and (ii) mycoplasmas reisolated after a single injection of marbofloxacin did not display an increased marbofloxacin MIC.

FIG. 1.

M. hyopneumoniae reisolation from tracheal swabs in infected untreated pigs and in infected pigs treated at TD and TD/2 for three consecutive days with marbofloxacin (from day 27 to day 29).

FIG. 2.

Time-kill curves of marbofloxacin against (a) the M. hyopneumoniae 116 wild-type strain and (b) one clone isolated on day 34 from an M. hyopneumoniae 116-infected pig treated at the therapeutic dose of marbofloxacin for 3 days (from day 27 to day 29) (limit of detection, log₁₀ CFU/ml = 3; 200 CFU/ml).