Development of a molecular biological assay for the detection of markers related to decreased susceptibility to macrolides and lincomycin in Mycoplasma hyorhinis

Abstract

The control of Mycoplasma hyorhinis infection relies mainly on antimicrobial therapy. However, the antibiotic susceptibility testing of the bacteria is usually not performed before applying the treatment, and thus therapeutic failures are not uncommon. In the case of M. hyorhinis, several antibiotic-resistance-related single nucleotide polymorphisms (SNPs) are known but assays for their detection have not been described yet. The aims of the present study were to investigate macrolide- and lincomycin-resistance-related SNPs in Hungarian M. hyorhinis isolates and to develop mismatch amplification mutation assays (MAMA) to detect the identified resistance markers. Minimal inhibitory concentrations (MIC) of different drugs and whole genome sequences of 37 M. hyorhinis isolates were used to find the resistance-related mutations. One MAMA assay was designed to detect the mutation of the 23S rRNA gene at nucleotide position 2058 (Escherichia coli numbering). For further evaluation, the assay was challenged with 17 additional isolates with available MIC data and 15 DNA samples from clinical specimens. The genotypes of the samples were in line with the MIC test results. The developed assay supports the practice of targeted antibiotic usage; hence it may indirectly reduce some bacterial resistance-related public health concerns.

Antimicrobial susceptibility profiles of Mycoplasma hyorhinis strains isolated from five European countries between 2019 and 2021

“…The macrolide and lincomycin susceptibility of the M . hyorhinis isolates collected in the present study was also tested by a MAMA [ 10 ] and, based on the total number of examined isolates (76 isolates in the present study in addition to the published isolates), the true detection rate of the assay for tylosin, tulathromycin and lincomycin is 123/126 (97.62%) and 122/126 (96.83%) for tilmicosin and tylvalosin. Based on the information of this molecular biological assay in most of the isolates, a single nucleotide polymorphism of the 23S rRNA gene contributes to evade the effect of macrolides and lincomycin, as this SNP (A2066G; numbering according to the M .…”

“…The mismatch amplification mutation assay was performed based on the primer sequences and conditions described earlier [ 10 ] using a Bio-Rad C1000 Touch ™ Thermal Cycler, CFX96 ™ Real-Time System (Bio-Rad Laboratories Inc., USA). The 23S rRNA sequencing primers Mhr-D5-1F and Mhr-D5-1R published earlier [ 7 ] were used to amplify a 192 bp long product which was sequenced on an ABI Prism 3100 automated DNA sequencer (Applied Biosystems, USA).…”

“…A single nucleotide polymorphism (SNP) at position A2066G (numbering based on the 23S rRNA gene of the M . hyorhinis type strain NCTC10130) has been identified as a molecular marker for macrolide and lincomycin resistance [ 7 , 10 ]. A mismatch amplification mutation assay (MAMA) was developed to detect this SNP, which provides a convenient and cost-effective method to characterize macrolide and lincomycin susceptibility of the isolates.…”

“…A mismatch amplification mutation assay (MAMA) was developed to detect this SNP, which provides a convenient and cost-effective method to characterize macrolide and lincomycin susceptibility of the isolates. With the MAMA assay, genotypes associated with high (H) or low (L) minimal inhibitory concentration (MIC) values can be distinguished based on the melting temperatures of the amplicons in a real-time PCR platform [ 10 ].…”

Evaluation of a Method for Standardized Antimicrobial Susceptibility Testing with Mycoplasma hyorhinis Field Isolates

“…The whole genome sequence of the type strain M. hyorhinis DSM 25591 showed the presence of a G2057A (Escherichia coli numbering) transition, which is known to confer resistance to 14-membered macrolides [27]. Földi and coworkers recently identified an A2058G transition via a mismatch amplification mutation assay in M. hyorhinis field isolates [47]. Moreover, an A2059G transition has been described in Japanese M. hyorhinis isolates [19].…”