Characterization of Mutations in DNA Gyrase and Topoisomerase IV Involved in Quinolone Resistance of Mycoplasma gallisepticum Mutants Obtained In Vitro

Abstract

Mycoplasma gallisepticum enrofloxacin-resistant mutants were generated by stepwise selection in increasing concentrations of enrofloxacin. Alterations were found in the quinolone resistance-determining regions of the four target genes encoding DNA gyrase and topoisomerase IV from these mutants. This is the first description of such mutations in an animal mycoplasma species.Mycoplasma gallisepticum is responsible for chronic respiratory diseases of chickens and sinusitis of turkeys (17). Control by antimicrobials is sometimes necessary to minimize its transmission in case of an outbreak. M. gallisepticum is known to be susceptible to several antimicrobials (10, 17) but can develop resistance against some of the quinolones used in veterinary medicine (14). Unlike human mollicutes (3, 5, 16), mechanisms involved in fluoroquinolone resistance are unknown in veterinary mycoplasmas. Most of the reported mutations involved in quinolone resistance are concentrated in the quinolone resistance-determining regions (QRDRs) of the gyrA and parC genes of DNA gyrase and topoisomerase IV, respectively (3,16,18,19,22,23). However, some mutations in the gyrB and parE QRDRs were found to be responsible for low-level resistance to fluoroquinolones (4, 5, 13). The QRDRs of the gyrA, gyrB, and parE genes of M. gallisepticum were previously described (8,20), but reports of their implication in fluoroquinolone resistance have never been published. In this study, we report the complete sequence of the parC QRDR of M. gallisepticum and the different alterations of the four QRDRs associated with quinolone resistance in mutants selected in vitro.Two strains of M. gallisepticum, ATCC 15302, a reference strain (26), and 41-91, a field strain (15), were used in the selection. Strains were grown in Frey agar or broth medium (15). Flumequine, norfloxacin, ofloxacin, and oxolinic acid used for the MIC determinations were purchased from SigmaAldrich (Saint Quentin Fallavier, France), while danofloxacin came from Pfizer (Amboise, France), marbofloxacin came from Vétoquinol (Magny-Vernois, France), difloxacin came from Fort Dodge Animal Health (Princeton, N.J.), and enrofloxacin and ciprofloxacin came from Bayer Pharma (Puteaux, France).Selection of enrofloxacin-resistant mutants was performed by serial transfers and incubations in Frey broth medium containing subinhibitory concentrations of enrofloxacin at 37°C for 5 days. The drug concentrations were gradually increased. This process was repeated 10 times. The MICs for the cloned resistant strains were determined by an agar dilution method (6) on Frey medium. Antimicrobial concentrations ranged between 0.03 and 64 g/ml. Plate contents were incubated at 37°C in a 5% CO 2 atmosphere for 5 days.DNAs were prepared according to standard methods. Amplifications of the QRDRs of gyrA and gyrB and of parE were performed with specific primers designed from the M. gallisepticum S6 strain sequences (8) and from the M. gallisepticum A5969 strain sequence (20), respectively. The 3Ј terminal region encoding the...

Persistence of Mycoplasma synoviae in hens after two enrofloxacin treatments and detection of mutations in the parC gene

“…However, this is the first time that such an increase of resistance, associated to a ParC Ser81 Pro, is described after two in vivo treatments at the therapeutic dose. This ParC substitution was also found in quinolone-resistant mutants of M. gallisepticum obtained in vitro [31,32], or in clinical isolates of M. hominis [5] and Staphylococcus aureus [38]. Our results are in accordance with those previously obtained for M. gallisepticum, where the ParC Ser81 Pro substitution was associated with a 2-4 fold increase of the enrofloxacin MIC [32].…”

“…The M. synoviae 317 and WVU1853 strains are less susceptible to enrofloxacin (MIC = 0.25 and 0.5 µg/mL, respectively) than the M. gallisepticum ATCC 15302 and 41-91 strains (MIC = 0.03 and 0.06 µg/mL, respectively) [31]. Differences in the amino acid sequences of the genes coding for topoisomerases might explain in part the differences in enrofloxacin susceptibility between the two species.…”

“…This phenomenon has already been described for M. gallisepticum in chickens [33], but successive treatments at the therapeutic dose reduced the percentage of M. gallisepticum-infected birds. This difference might be explained by the lower susceptibility to enrofloxacin of M. synoviae 317 strain (0.25 µg/mL) in comparison with the M. gallisepticum 41-91 strain (0.06 µg/mL) [31]. Moreover, treatment on M. synoviae-infected birds was performed three weeks after inoculation, on a well established infection, whereas treatments on M. gallisepticum-infected chickens were performed one or two weeks after inoculation.…”

“…Since the QRDR of the gyrA and gyrB genes of DNA gyrase and the parC and parE genes encoding for topoisomerase IV were not available for M. synoviae, amplification of the QRDR of gyrA and gyrB were performed with primers previously chosen from the nucleotide sequence of M. gallisepticum S6 strain [12]. Amplifications of parC and parE of the M. synoviae 317 strain, were initially performed with primers chosen from M. gallisepticum [31,32]. The nucleotide sequences obtained were then used to determine new internal specific primers (Tab.…”

“…DNA gyrase and topoisomerase IV, which both are essential for bacterial viability [26]. Most reported mutations involved in fluoroquinolone resistance are concentrated in the quinolone resistance determining regions (QRDR) of the gyrA/gyrB and parC/parE genes of DNA gyrase and topoisomerase IV, respectively [29,31,32].…”