Genotyping Mycoplasma hyorhinis by multi-locus sequence typing and multiple-locus variable-number tandem-repeat analysis

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

“…In this study, 55 M. hyorhinis isolates from previous studies (Bek} o et al, 2019;F€ oldi et al, 2020) or collected in 2019-2020 and the type strain NCTC 10130 (GenBank Accession number: LS991950.1) were used. DNA extracted from the original clinical specimens of 15 clinical isolates was also examined (Supplementary Table 1).…”

“…The initial minimal inhibitory concentration (MIC) of each strain was defined as the lowest concentration of the antibiotic that completely inhibited the growth at the time of colour change in the growth control. Since there are no official breakpoints available for the antibiotic susceptibility testing of M. hyorhinis, previously published breakpoints for M. hyopneumoniae or other porcine respiratory pathogens were used (Hannan et al, 1997;Felde et al, 2020;Vilar o et al, 2020). MIC values ≥4 mg/mL of tylosin, tylvalosin, gamithromycin and lincomycin were considered high (Hannan et al, 1997;Felde et al, 2020), while for tilmicosin and tulathromycin higher breakpoints were suggested, thus MIC values ≥16 mg/mL were considered high (Vilar o et al, 2020).…”

“…The 23S rRNA and 50S ribosomal protein L4 and L22 coding genes were previously connected to macrolide and lincomycin resistance in Mycoplasma species (Kobayashi et al, 2005;Sulyok et al, 2017;Bek} o et al, 2020a;Felde et al, 2020). These genes were aligned and analysed in Geneious Prime software version 2019.2.1 (Kearse et al, 2012) to detect SNPs.…”

“…One of the allele-specific primers is marked with an additional 15-to 20-bp-long GC clamp that increases the melting temperature and the size of the amplicon as well. The temperature shift is detected on a real-time PCR platform in the presence of an intercalating fluorescent DNA dye (melt-MAMA) and the size difference is revealed in agarose gel electrophoresis (agarose-MAMA) (Birdsell et al, 2012;Sulyok et al, 2018;Bek} o et al, 2020b;Felde et al, 2020). Primers were designed based on the whole genome sequences of M. hyorhinis field isolates and the type strain (NCTC 10130) in the Geneious Prime software version 2019.2.1 (Kearse et al, 2012).…”

“…A point mutation A2059G (according to Escherichia coli numbering) at the V domain of the 23S rRNA was found in in vitro mutants and field isolates as well. In addition, point mutations between nucleotide positions 2058 and 2062 (E. coli numbering) were connected to macrolide and lincosamide resistance in several Mycoplasma species (Sulyok et al, 2017;Chernov et al, 2018;Gautier-Bouchardon, 2018;Bek} o et al, 2020a;Felde et al, 2020).…”