Characterization of lsa(D), a Novel Gene Responsible for Resistance to Lincosamides, Streptogramins A, and Pleuromutilins in Fish Pathogenic Lactococcus garvieae Serotype II.

Abstract

Aims: Fish pathogenic Lactococcus garvieae serotype II has been isolated from cultured fish species in Japan. This study aimed to investigate the molecular mechanisms of lincomycin (LCM)-resistant L. garvieae serotype II and assess the molecular basis for lincosamides-streptogramins A-pleuromutilins (LSAP)-resistant phenotype. Results: We identified a novel lsa(D)-encoded 497-aa ATP-binding cassette F (ABC-F) protein in the LSAP-resistant strains. Amino acid identities of 41.25-54.73% were obtained between the deduced amino acids from Lsa(D) and other Lsa-type ABC-F proteins. Furthermore, comparative analysis revealed that the allele of lsa(D) with single point mutation at 233 aa position (TGG → TAG; tryptophan→premature termination codon [PTC]) in LSAP-sensitive strains. The minimum inhibitory concentrations of antimicrobials against the lsa(D) complementary strain and lsa(D)-disrupted mutant confirmed that lsa(D) conferred the LSAP-resistant phenotype. The reverse transcription-polymerase chain reaction could not detect the noncoding region of lsa(D) allelic variant in the LSAP-sensitive strains. Additionally, the PTC (TAG) in LCM-sensitive strains was replaced by TGG, CAG, or TAT in the laboratory-induced revertant mutants. Conclusions: The novel lsa(D) conferred the LSAP-resistant phenotype in clinically LCM-resistant L. garvieae serotype II strains. However, the allele of lsa(D) gene containing the PTC was found in L. garvieae serotype II, resulting in the LSAP-susceptible phenotype.