Characterization of a novel macrolide efflux gene, mef(B), found linked to sul3 in porcine Escherichia coli.
OBJECTIVE
The aim of this study was to characterize a putative novel macrolide efflux gene located in the vicinity of sul3 in porcine Escherichia coli.


METHODS
Five sul3-encoding E. coli isolates of porcine origin were investigated by plasmid characterization and random amplification of polymorphic DNA (RAPD) PCR. Unknown DNA adjacent to the sul3 genes was amplified using a PCR approach, followed by sequencing of the fragments. The putative macrolide efflux gene was cloned into pK18. The cloned gene was characterized by susceptibility testing by Etest in the presence and absence of efflux inhibitors.


RESULTS
Five sul3-encoding isolates, demonstrated to be unrelated by RAPD PCR, were characterized. The immediate genetic context of sul3 in five isolates was identical to that in plasmid pVP440, and in all cases, sul3 was associated with class 1 integrons. In three isolates, an open reading frame (orf2) encoding a putative protein with 38% amino acid identity to Mef(A) was found, while the two remaining isolates contained a fragment of orf2 truncated by IS26 insertion. In three of the isolates, this DNA region was demonstrated to be located on non-conjugative plasmids. When the complete orf2 was cloned, it conferred high-level resistance to erythromycin and azithromycin, and the resistance property could be partially inhibited using the efflux inhibitor Phe-Arg beta-naphthylamide dihydrochloride. The gene was named mef(B).


CONCLUSIONS
A new macrolide efflux protein, Mef(B), with 38% amino acid identity to Mef(A), has been characterized and represents the second member of the mef family of genes.
Liu
Jiahui
J
Department of Cellular and Molecular Medicine, Bristol Centre for Antimicrobial Research, University of Bristol, University Walk, Bristol, UK.
Keelan
Paula
P
Bennett
Peter M
PM
Enne
Virve I
VI
D000818:Animals; D000900:Anti-Bacterial Agents / Q000378:metabolism* / Q000494:pharmacology; D017963:Azithromycin / Q000378:metabolism / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics*; D015373:Bacterial Typing Techniques; D003001:Cloning, Molecular; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D024881:Drug Resistance, Bacterial; D004917:Erythromycin / Q000378:metabolism / Q000494:pharmacology; D004926:Escherichia coli / Q000145:classification / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D023061:Gene Order; D005838:Genotype; D032023:Integrons; D018942:Macrolides / Q000378:metabolism* / Q000494:pharmacology; D026901:Membrane Transport Proteins / Q000235:genetics*; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D010957:Plasmids; D019105:Random Amplified Polymorphic DNA Technique; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013552:Swine / Q000382:microbiology
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D018942:Macrolides; D026901:Membrane Transport Proteins; D004917:Erythromycin; D017963:Azithromycin