Development of molecular assays for the rapid and cost-effective determination of fluoroquinolone, macrolide and lincosamide susceptibility of Mycoplasma synoviae isolates

Abstract

Mycoplasma synoviae infection occurs worldwide, leading to considerable economic losses in the chicken and turkey industry due to infectious synovitis, respiratory diseases and eggshell apex abnormalities. Control programs against M . synoviae infection are based on eradication, vaccination and medication with antimicrobial agents. Prudent use of antibiotics can be improved greatly by the determination of antibiotic susceptibility prior to the treatment. However, the conventional broth or agar microdilution is very labor-intensive and time-consuming method. Thus, there is an increasing need for rapid antimicrobial susceptibility tests in order to guide antibiotic therapy more effectively. The aim of this study was to develop mismatch amplification mutation assays (MAMAs) to detect resistance-associated mutations in M . synoviae . M . synoviae strains with previously determined minimal inhibitory concentrations (MICs) and whole genomes (n = 92) were used for target selection and assay specification. For the evaluation of the developed assays, 20 clinical samples and an additional 20 M . synoviae isolates derived from these specimens were also included in this study. MIC values of these 20 isolates were determined by broth microdilution method. Five MAMAs were designed to identify elevated MICs of fluoroquinolones, while three MAMAs were developed to detect decreased susceptibility to macrolides and lincomycin. The sensitivity of the MAMA tests varied between 10 2 −10 4 template copy number/reaction depending on the assay. Clinical samples showed identical genotype calls with the M . synoviae isolates derived from the corresponding specimens in each case. Supporting the results of conventional in vitro sensitivity tests, our approach provides a feasible tool for diagnostics. Rapidity, robustness and cost-effectiveness are powerful advantages of the developed assays. Supporting prudent antibiotic usage instead of empirical treatment, the use of this method can reduce significantly the economic impact of M . synoviae in the poultry industry and decrease bacterial resistance-related public health concerns.

Integrating the Human and Animal Sides of Mycoplasmas Resistance to Antimicrobials

“…SNP genotyping using Melt Analysis of Mismatch Amplification Mutation Assays (Melt-MAMA) has also been proposed for detection of resistance-associated mutations in animal mycoplasmas, but it is usually based on in-house tests, that have never been validated on a large scale [113,114]. For M. bovis, a good correlation between meltingprofiles and MICs has been noted for several antimicrobials [113], but not for M. synoviae, due to the existence of numerous non-hotspot mutations in the target genes [114]. The method is not applicable on all the antimicrobials and is highly dependent on the PCR machine used.…”