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 (LS_AP)-resistant phenotype.

Results:

We identified a novel lsa(D)-encoded 497-aa ATP-binding cassette F (ABC-F) protein in the LS_AP-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 LS_AP-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 LS_AP-resistant phenotype. The reverse transcription–polymerase chain reaction could not detect the noncoding region of lsa(D) allelic variant in the LS_AP-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 LS_AP-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 LS_AP-susceptible phenotype.