A novel phenicol exporter gene, fexB, found in enterococci of animal origin

Abstract

Objectives: To investigate two porcine Enterococcus isolates for the genetic basis of phenicol resistance and to determine the location and the genetic environment of the novel resistance gene.Methods: A total of 391 isolates with reduced florfenicol susceptibility (MIC ≥16 mg/L), obtained from 557 nasal swabs of individual pigs, were screened by PCR for the known florfenicol resistance genes. Isolates that were negative in these PCRs were analysed for their species assignment and antimicrobial susceptibility. Plasmids were extracted and subjected to transformation and conjugation assays. Restriction fragments of the phenicol resistance plasmids were cloned and sequenced. The sequences obtained were analysed and compared with sequences deposited in the databases. Results:The two isolates, Enterococcus faecium EFM-1 and Enterococcus hirae EH-1, exhibited MICs of chloramphenicol and florfenicol of 64 mg/L and carried a new phenicol resistance gene, designated fexB. This gene codes for a phenicol exporter of 469 amino acids organized in 14 transmembrane domains. The fexB gene was located on the 35 kb pEFM-1 from E. faecium and on the 25.3 kb pEH-1 from E. hirae, respectively. Both plasmids were non-conjugative. The fexB gene was found to be embedded in virtually the same genetic environment of 14.8 kb in both plasmids. Conclusion:To the best of our knowledge, this is the first report of the new florfenicol exporter gene fexB. Based on its plasmid location, horizontal transfer from the enterococci to other bacteria is possible.

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“…Along with ARG109, other residues such as GLN112 and GLY113 were also conserved in FexB protein. Motif-B was found to be important in the transfer of motif-B antiporters along with addition of protonation status with substrate binding in MdfA [13] and we predict a similar function for motif-B in FexB protein.…”

“…It is the only motif with membrane-embedded basic residue among MFS members which is also termed as 3D motif-B [13]. We observed that motif-B (R 109 mfQ 112 G 112 ) was also conserved in FexB protein (Fig.1) and is located on helix-5 (Fig.…”

“…The FexB protein has 469 amino acid sequence length and consists of 14 trans membrane domains ( 14 ability towards Clm [13]. Therefore, it can be predicted that the mutation of ALA30 and SER93 residues in FexB protein may affect its binding towards Clm.…”

“…MdfA orthologs [13].It is located in rocker helix of Transmembrane5(TM5) ("GX6GX3GPX2GPX2 G") and is also called as antiporter motif [41]. We identified the "I 139 LGIIA 144 GCIGVGTAGG 154 P 1 55 IFG 158 " consensus sequence as motif-C in FexB that is present on helix-6 region (Fig.…”

“…The fexB gene was located on the plasmids in both Enterococcus species and codes for FexB protein that consists of 469 amino acids with 14 transmembrane domains. FexB protein confers resistance to Clm and Ff and shares amino acid similarity of 56.1% with FexA and 15.6% with PexA protein sequences [13]. The structural data on FexB protein has not been reported till date.…”