Alterations in the Quinolone Resistance-Determining Regions and Fluoroquinolone Resistance in Clinical Isolates and Laboratory-Derived Mutants of Mycoplasma bovis: Not All Genotypes May Be Equal

Abstract

Mycoplasma bovis is considered a major contributor to respiratory diseases in young cattle. Resistant M. bovis isolates have increasingly been reported worldwide due to extensive use of antimicrobials to treat bovine pneumonia. The frequency of isolates resistant to fluoroquinolones varies considerably from one country to another. The MICs of isolates collected in France have only increased from "very low" to "low." The present study was conducted to investigate whether alterations in the quinolone resistance-determining regions (QRDRs) could account for this slight modification in susceptibility. No correlation between QRDR alterations and increased MICs was evidenced in clinical isolates. In addition, all clinical isolates were subtyped, and the tendencies of the different sequence types to develop resistance through mutations in QRDRs under selective pressure in vitro were examined. In vitro, 3 hot spots for mutations in QRDRs (position 83 in GyrA and positions 80 and 84 in ParC) were associated with a high level of resistance when cumulated. We showed that the point mutations in the QRDRs observed in vitro were different (in location and selection rapidity) between the different subtypes. Our in vitro observations were corroborated by the recent detection of a clinical isolate highly resistant to fluoroquinolones (MIC > 16 g/ml) and belonging to the subtype which easily accumulates QRDR alterations in vitro. The current increased prevalence of this subtype in clinical isolates highlights the urgent need to control fluoroquinolone usage in veterinary medicine. Mycoplasma bovis is a wall-less bacterium responsible for severe infections in cattle, including pneumonia, mastitis, arthritis, and otitis (1). In young cattle, it is now recognized as a major contributor to economic losses associated with bovine respiratory diseases (BRD). The infection pressure of BRD usually peaks 2 to 3 weeks after calves are mingled in fattening units following transportation from their respective birth farms (2). Since no efficient vaccines are available and licensed for use outside the United States (3), efforts to control M. bovis infections often rely on antimicrobial treatments, administered either prophylactically or in the early stages of the disease. Antimicrobials used for the treatment or prevention of BRD usually include broad-spectrum cephalosporins (cefquinome and ceftiofur), extended-spectrum fluoroquinolones (enrofloxacin, danofloxacin, and marbofloxacin), florfenicol, and long-lasting macrolides (tulathromycin, gamithromycin, and tildipirosin) (4). This extensive use of antibiotics has predictably resulted in an increase in resistant isolates over time. In France, it was recently shown that contemporary M. bovis strains had become significantly less susceptible than archival strains to 9 of the 12 antimicrobials tested (5). With regard to fluoroquinolones, the decrease in susceptibility was limited (only 1 dilution of the MIC), and no highly resistant isolates were observed in a set of more than 90 strains. This wa...

Development of in vitro resistance to fluoroquinolones in Pseudomonas aeruginosa

“…Additionally, we detected replacement of serine at position 457 by cysteine in parE, which was similar to clinical results showing an altered arginine residue [12]. However, we did not identify a parC mutation unlike previous clinical results from sequencing both clinical isolates and laboratory derived mutations of Mycoplasma bovis, which showed different abilities in the development of resistance [13]. We speculate that the parC mutation could be related to the drug combination or hospital environment associated with its identi cation.…”

Mutations Associated with Decreased Susceptibility to Seven Antimicrobial Families in Field and Laboratory-Derived Mycoplasma bovis Strains

“…All M. bovis strains in the present study with high fluoroquinolone MICs (Ն10 g/ml) contained at least one substitution in both gyrA and parC genes. Earlier described mutation hotspots in GyrA and ParC of naturally and artificially selected resistant M. bovis strains have been identified between amino acid positions 81 to 87 and positions 78 to 84, respectively (10,11,21,22). However, the SNP (G244A) causing increased MIC values to fluoroquinolones at position 82 (Asp¡Asn) in GyrA protein of M. bovis is described for the first time.…”

“…*, Altered positions related to high MICs to fluoroquinolones already described in M. bovis isolates (10,11,21,22 the rrs1 and rrs2 alleles of field strains with spectinomycin MICs of Յ4 g/ml but not in the reference strain PG45 (Table 2). Macrolides, lincosamides, phenicols, and pleuromutilins.…”

Development of In Vitro resistance to fluoroquinolones in Pseudomonas aeruginosa

“…showing different abilities in the development of resistance [13]. We speculate that the parC mutation could be related to the drug combination or the hospital environment, which is why we used the in vitro model to investigate the mechanisms of P.aeruginosa resistance to overcome the uncertainty of clinical treatment.…”