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

Katinka Bekő,Zsuzsa Kreizinger,Salvatore Catania,Miklós Gyuranecz,Anneke Feberwee,Orsolya Saller,Cécile Yvon,Iddya Karunasagar

doi:10.1371/journal.pone.0241647

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 102−104 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.

Highlights

Mycoplasma synoviae infection occurs worldwide, leading to infectious synovitis or respiratory diseases in chickens and turkeys, and it can be related to eggshell apex abnormalities in chickens as well
Specification of the designed mismatch amplification mutation assays (MAMAs) was performed on DNAs extracted from pure cultures of the 92 previously examined M. synoviae strains [9]. Genotype calls of these strains were compared with their whole genome sequences (SRA accession numbers: PRJNA634246 and PRJNA63425; GenBank accession numbers: CP011096 and KP704286)
As the amplicons of these species had melting temperatures around the average Tm of genotype L M. synoviae strains (77.3 ̊C), excluding the presence of cross-reacting Mycoplasma species by a universal Mycoplasma polymerase chain reaction (PCR) in the doubtful cases is recommended for the reliable determination of macrolide and lincomycin susceptibility of M. synoviae isolates

Summary

Introduction

Mycoplasma synoviae infection occurs worldwide, leading to infectious synovitis or respiratory diseases in chickens and turkeys, and it can be related to eggshell apex abnormalities in chickens as well. Prudent use of antibiotics in the management of M. synoviae infection can be improved greatly by the determination of antibiotic susceptibility prior to the treatment. Most common method of antibiotic susceptibility testing is the determination of minimal inhibitory concentration (MIC) values in vitro by broth or agar microdilution. Interpretation of the results is difficult, because neither standard breakpoints of susceptible, intermediate and resistant categories to antimicrobial agents, nor internationally harmonized testing conditions have been defined yet concerning avian Mycoplasma species. The results of in vitro antibiotic susceptibility tests can only predict the expected in vivo efficacy of the antibiotics [2], and the microdilution tests are very labor-intensive and time-consuming methods, as they require previous isolation and pure culture of the bacterium [3]. A rapid and cost-effective method is the detection of resistance-associated mutations by molecular biological assays [5, 6]

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