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 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.

Fig 1

Melt curve (A) and amplification plot (B) of the MAMA-MS-rrl assay developed for the determination of macrolide and lincomycin susceptibility. The heterozygous MYCS-63 (genotype Het; purple line) displayed bimodal melting peak at the specific melting temperatures of genotype L (MYCS-64; dark blue line; Tm 77.27°C) and genotype H (MYCS-65; red line; Tm 82.48°C) M. synoviae isolates (A). The no-template negative control (light blue line) was not amplified (B), but induced artifact-associated fluorescence (A). Fig 1A.: y-axis: derivative reporter, the negative first-derivative of the normalized fluorescence generated by the reporter during PCR amplification; x-axis: temperature melt curve; Fig 1B.: y-axis: difference between the normalized reporter (Rn) value (normalized fluorescence) of the reaction and the Rn value of the baseline signal generated by the instrument; x-axis: number of cycles.

Fig 2. Determination of macrolide susceptibility by the developed MAMA-MS-rplV-1 assay.

Amplification products of the genotype L (MYCS-78; dark blue line; Tm 75.48°C) and genotype H (MYCS-79; red line; Tm 79.35°C) M. synoviae isolates showed specific melting temperatures. The no-template negative control (light blue line) displayed melting peak at non-specific temperature (light blue line; Tm 84.42°C). y-axis: derivative reporter, the negative first-derivative of the normalized fluorescence generated by the reporter during PCR amplification; x-axis: temperature melt curve.

Fig 3. Determination of fluoroquinolone susceptibility by the developed MAMA-MS-gyrA assay.

Amplification products of the genotype L (MYCS-23; dark blue line; Tm 74.14°C) and genotype H (MYCS-27; red line; Tm 78.31°C) M. synoviae isolates showed specific melting temperatures. Clinical sample H1 displayed bimodal curve with a peak at the specific melting temperature of genotype L (Tm 74.02°C) and an additional peak at non-specific temperature (Tm 81.28°C). The no-template negative control (light blue line) was not amplified. y-axis: derivative reporter, the negative first-derivative of the normalized fluorescence generated by the reporter during PCR amplification; x-axis: temperature melt curve.