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

Abstract

The molecular mechanisms of resistance to fluoroquinolones, tetracyclines, an aminocyclitol, macrolides, a lincosamide, a phenicol, and pleuromutilins were investigated in Mycoplasma bovis. For the identification of mutations responsible for the high MICs of certain antibiotics, whole-genome sequencing of 35 M. bovis field isolates and 36 laboratory-derived antibiotic-resistant mutants was performed. In vitro resistant mutants were selected by serial passages of M. bovis in broth medium containing subinhibitory concentrations of the antibiotics. Mutations associated with high fluoroquinolones MICs were found at positions 244 to 260 and at positions 232 to 250 (according to Escherichia coli numbering) of the quinolone resistance-determining regions of the gyrA and parC genes, respectively. Alterations related to elevated tetracycline MICs were described at positions 962 to 967, 1058, 1195, 1196, and 1199 of genes encoding the 16S rRNA and forming the primary tetracycline binding site. Single transversion at position 1192 of the rrs1 gene resulted in a spectinomycin MIC of 256 g/ml. Mutations responsible for high macrolide, lincomycin, florfenicol, and pleuromutilin antibiotic MICs were identified in genes encoding 23S rRNA. Understanding antibiotic resistance mechanisms is an important tool for future developments of genetic-based diagnostic assays for the rapid detection of resistant M. bovis strains. KEYWORDS antibiotic resistance, cattle, Mycoplasma bovisA ntibiotics are among the most important therapeutic tools in the veterinary and human medicine, but their use is limited since resistance tends to evolve in pathogenic bacteria. The microorganisms are exposed to selective pressure by the use of antimicrobials in medicine and agriculture, favoring the development, survival, and spread of resistant clones (1).Mycoplasma spp. are members of the class Mollicutes and comprise the simplest life form that can replicate independently from the host (2). Mycoplasma spp. have no cell wall and they have a limited number of metabolic pathways. The greatly reduced genome size and coding capacity of Mycoplasma spp. makes them a good model for genetic studies. Mycoplasma spp. are fast-evolving bacteria with several human and animal pathogens; however, their importance is often underestimated (2). Mycoplasma bovis is a major cause of calf pneumonia, mastitis and arthritis, and it is responsible for significant economic losses (3). Adequate housing and appropriate antibiotic treatment

Upregulation of PD-L1 Expression by Prostaglandin E2 and the Enhancement of IFN-γ by Anti-PD-L1 Antibody Combined With a COX-2 Inhibitor in Mycoplasma bovis Infection

“…M. bovis is a highly contagious pathogen, and there are no effective vaccines owing to its immunosuppressive effects. Although antibiotics are potentially effective against M. bovis, strains resistant to antibiotics have recently been emerging and spreading (37,38). Therefore, a novel strategy for the control of M. bovis infection is required.…”

Relationship between Antimicrobial Susceptibility and Multilocus Sequence Type of Mycoplasma bovis Isolates and Development of a Method for Rapid Detection of Point Mutations Involved in Decreased Susceptibility to Macrolides, Lincosamides, Tetracyclines, and Spectinomycin

“…These five isolates which possessed rrs C1192A showed significantly low susceptibility to spectinomycin (P Ͻ 0.0001) (spectinomycin MICs, from 0.5 to 8 g/ml [normal isolates] to 256 to 1,024 g/ml [isolates with C1192A]) ( Fig. 4) (9).…”

“…Hence, it is desirable to develop a method for quick and easy assessment of the antimicrobial susceptibility of the causal strain. In M. bovis or Mycoplasma californicum, specific mutations located in the 16S rRNA gene (rrs), 23S rRNA gene (rrl), DNA gyrase gene (gyrA), and topoisomerase IV gene (parC) were found in strains with low susceptibility to various antimicrobial agents (7)(8)(9). Reconfirming the universality of the roles of these mutations and exploiting them may lead to the development of a method that can compensate for the drawbacks of conventional methods (7).…”

“…We investigated a variety of mutation points that are suggested to be involved in decreased susceptibility to antimicrobial agents in Japan and reconfirmed their roles in antimicrobial susceptibility. Subsequently, we attempted to establish a probe-based genetic method for the rapid detection of these mutations (9). Table S1 in the supplemental material).…”

Development of Molecular Methods for Rapid Differentiation of Mycoplasma gallisepticum Vaccine Strains from Field Isolates

“…M. gallisepticum genomic DNAs of vaccine strains 6/85 and ts-11 were extracted from 5 ml of logarithmic-phase broth cultures using a QIAamp DNA minikit (Qiagen, Inc.). Next-generation sequencing was performed on an Ion Torrent platform (New England BioLabs, Hitchin, United Kingdom), as previously described (27,28). Reads were mapped to M. gallisepticum strain R low (GenBank accession number AE015450.2) as a reference genome and annotated using the Geneious software version 10.2.3 (29).…”