Multilocus sequence typing of Mycoplasma hyorhinis strains identified by a real-time TaqMan PCR assay

Abstract

A real-time TaqMan PCR assay based on the gene encoding the protein p37 was developed to detect Mycoplasma hyorhinis. Its specificity was validated with 29 epidemiologically unrelated M. hyorhinis strains (28 field strains and one reference strain) and other mycoplasma species or with other microorganisms commonly found in pigs. The estimated detection limit of this qPCR assay was 125 microorganism equivalents/μl. The same 29 epidemiologically unrelated M. hyorhinis strains and four previously fully sequenced strains were typed by two portable typing methods, the sequencing of the p37 gene and a multilocus sequence typing (MLST) scheme. The first method revealed 18 distinct nucleotide sequences and insufficient discriminatory power (0.934). The MLST scheme was developed with the sequenced genomes of the M. hyorhinis strains HUB-1, GDL-1, MCLD, and SK76 and based on the genes dnaA, rpoB, gyrB, gltX, adk, and gmk. In total, 2,304 bp of sequence was analyzed for each strain. MLST was capable of subdividing the 33 strains into 29 distinct sequence types. The discriminatory power of the method was >0.95, which is the threshold value for interpreting typing results with confidence (D=0.989). Population analysis showed that recombination in M. hyorhinis occurs and that strains are diverse but with a certain clonality (one unique clonal complex was identified). The new qPCR assay and the robust MLST scheme are available for the acquisition of new knowledge on M. hyorhinis epidemiology. A web-accessible database has been set up for the M. hyorhinis MLST scheme at http://pubmlst.org/mhyorhinis/.

FIG 1

Genetic relationships between 33 M. hyorhinis strains, as estimated by clustering analysis of 18 sequences (Seq) of the p37 gene. The phylogram was constructed using tools at www.phylogeny.fr and the one-click mode.

FIG 2

Genetic relationships between 33 M. hyorhinis strains, as estimated by clustering analysis of 29 sequence types (ST) revealed by MLST. The phylogram was constructed using tools at www.phylogeny.fr and the one-click mode.

FIG 3

BURST analysis of M. hyorhinis STs. Clonal complex 1 includes ST1 (ancestral genotype), ST2, ST13, and ST17 (descended from single-locus variant genotypes) and ST7, ST24, and ST16 (descended from double-locus variant genotypes). The assigned ancestral genotype is within the central ring (ST1). ST11 and ST22 overlap.