Mycoplasma flocculare is genetically closely related to M. hyopneumoniae, the etiologic agent of porcine enzootic pneumonia, and is frequently isolated with this second species. In this article, we report on the development of the first multilocus sequence typing (MLST) scheme for M. flocculare, based on three genes (adk, rpoB and tpiA). In total, 5022 bp of sequence were analyzed. MLST was used to characterize seven M. flocculare isolates and the reference strain. Eight distinct sequence types were defined, showing the great intraspecies variability of M. flocculare, and the high discriminatory power of the new typing method. The relative contribution of recombinations to the genomic evolution of M. flocculare was revealed by calculating the index of association (I: 0.0185). This MLST scheme is now available for the acquisition of new knowledge on M. flocculare epidemiology via an online database comprising the DNA sequences of each allele, available at http://pubmlst.org/mflocculare/.
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