Identification,and Genetic Environment Of Novel Lnu(G)Gene Conferring Resistance To Lincomycin

Lincosamide antibiotics,extracted from fermentation broth of Streptomyces,are highly lipophilic basic compounds.Lincosamide antibiotics mainly includes lincomycin,clindamycin and pirlimycin.Lincosamides can prevent the extension of peptide chain and inhibit the synthesis of protein by acting on the 23S rRNA in the 50S subunit of the ribosome of the susceptible organism,These antibiotics are active against many gram-positive cocci and anaerobes.However,with the increasing use of lincosamide antibiotics in human and veterinary clinic,The antibiotic resistance in bacteria has appeared.Resistance to lincosamides are mainly divided into two categories:The first is modification of the intracellular target of the antibiotics by N6 dimethylation of adenine in 23S rRNA,which confers cross-resistance to macrolide,lincosamide,and streptogramin B type antibiotics(MLSB phenotype).This alteration of the antibiotic target site is catalyzed by an rRNA methyltransferase encoded by erm genes.The second is the lincosamide adenylating enzyme,which can cause the bacterial resistance by inactivating lincosamide.leading to lincosamide inactivation caused by bacterial resistance.Since the first report of a lincosamide nucleotidyltransferase lnu(A)(formly linA),a number of Inu genes have been described in various bacteria of human or animal origin(http://faculty.washington.edu/marilynr/),including lnu(B),lnu(C),lnu(D),lnu(E)and lnu(F),The presence of these resistance genes indicated that the resistance of bacteria to antibiotics was common,and it also suggested that there might be other types of lincomycin resistance genes existing in bacteria.In this study,based on the sequenced whole genome of swine Enterococcus faecalis E531,we identified a novel gene conferring resistance to lincomycin,named Inu(G),by using sequence analysis,gene cloning,susceptbility testing,UPLC-MS/MS,reverse PCR,I-Ceul-PFGE,and southern blot.The amino acid sequence between Inu(G)and Inu(A)-Inu(F)revealed the different degree of homology(31%～76%).E.faecalis JH2-2 harboring pAM401-lnu(G)showed a 4-fold increase in the MICs of lincomycin,compared to E.faecalis JH2-2 or E.faecalis JH2-2 harboring empty vector pAM401 only.UPLC-MS/MS experiment demonstrated thelnu(G)enzyme catalyzed adenylyation of lincomycin.Reverse PCR indicated the lnu(G)gene was embedded into a novel Tn554 family transposon,designated Tn6260.Compared with other transposons of the Tn554 family,this transposon also has tnpA,tnpB,and tnpC genes.I-CeuI-PFGE and southern blot analysis showed that Tn6260 is located on the chromosome.The distribution of Inu(G)gene in enterococci of swine origin showed that the association between lnu(G)and transposon Tn6260 promoted horizontal transmission of lnu(G)at some degree.In summary,we identified a novel gene conferring resistance to lincomycin,named Inu(G),whose product can catalyze adenylyation of lincomycin,which can increase 4-fold times in the MICs of lincomycin.In addition,lnu(G)is carried by the transposon Tn6260,located on the chromosome.