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Cloning and characterization of a novel macrolide efflux gene, mreA, from Streptococcus agalactiae.

ABSTRACT: A strain of Streptococcus agalactiae displayed resistance to 14-, 15-, and 16-membered macrolides. In PCR assays, total genomic DNA from this strain contained neither erm nor mef genes. EcoRI-digested genomic DNA from this strain was cloned into lambda Zap II to construct a library of S. agalactiae genomic DNA. A clone, pAES63, expressing resistance to erythromycin, azithromycin, and spiramycin in Escherichia coli was recovered. Deletion derivatives of pAES63 which defined a functional region on this clone that encoded resistance to 14- and 15-membered, but not 16-membered, macrolides were produced. Studies that determined the levels of incorporation of radiolabelled erythromycin into E. coli were consistent with the presence of a macrolide efflux determinant. This putative efflux determinant was distinct from the recently described Mef pump in Streptococcus pyogenes and Streptococcus pneumoniae and from the multicomponent MsrA pump in Staphylococcus aureus and coagulase-negative staphylococci. Its gene has been designated mreA (for macrolide resistance efflux).

SUBMITTER: Clancy J

PROVIDER: S-EPMC164195 | biostudies-other | 1997 Dec

REPOSITORIES: biostudies-other

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Cloning and characterization of a novel macrolide efflux gene, mreA, from Streptococcus agalactiae.

Clancy J J Dib-Hajj F F Petitpas J W JW Yuan W W

Antimicrobial agents and chemotherapy 19971201 12

A strain of Streptococcus agalactiae displayed resistance to 14-, 15-, and 16-membered macrolides. In PCR assays, total genomic DNA from this strain contained neither erm nor mef genes. EcoRI-digested genomic DNA from this strain was cloned into lambda Zap II to construct a library of S. agalactiae genomic DNA. A clone, pAES63, expressing resistance to erythromycin, azithromycin, and spiramycin in Escherichia coli was recovered. Deletion derivatives of pAES63 which defined a functional region on ...[more]

PMID: 9420045

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Project description:The mreA gene from Streptococcus agalactiae COH31 gamma/delta, resistant to macrolides and clindamycin by active efflux, has recently been cloned in Escherichia coli, where it was reported to confer macrolide resistance (J. Clancy, F. Dib-Hajj, J. W. Petitpas, and W. Yuan, Antimicrob. Agents Chemother. 41:2719--2723, 1997). Cumulative data suggested that the mreA gene was located on the chromosome of S. agalactiae COH31 gamma/delta. Analysis of the deduced amino acid sequence of mreA revealed significant homology with several bifunctional flavokinases/(flavin adenine dinucleotide (FAD) synthetases, which convert riboflavin to flavin mononucleotide (FMN) and FMN to FAD, respectively. High-performance liquid chromatography experiments showed that the mreA gene product had a monofunctional flavokinase activity, similar to that of RibR from Bacillus subtilis. Sequences identical to those of the mreA gene and of a 121-bp upstream region containing a putative promoter were detected in strains of S. agalactiae UCN4, UCN5, and UCN6 susceptible to macrolides. mreA and its allele from S. agalactiae UCN4 were cloned on the shuttle vector pAT28. Both constructs were introduced into E. coli, where they conferred a similar two- to fourfold increase in the MICs of erythromycin, spiramycin, and clindamycin. The MICs of a variety of other molecules, including crystal violet, acriflavin, sodium dodecyl sulfate, and antibiotics, such as certain cephalosporins, chloramphenicol, doxycycline, nalidixic acid, novobiocin, and rifampin, were also increased. In contrast, resistance to these compounds was not detected when the constructs were introduced into E. faecalis JH2-2. In conclusion, the mreA gene was probably resident in S. agalactiae and may encode a metabolic function. We could not provide any evidence that it was responsible for macrolide resistance in S. agalactiae COH31 gamma/delta; broad-spectrum resistance conferred by the gene in E. coli could involve multidrug efflux pumps by a mechanism that remains to be elucidated.

Dataset Information

Cloning and characterization of a novel macrolide efflux gene, mreA, from Streptococcus agalactiae.

Publications

Cloning and characterization of a novel macrolide efflux gene, mreA, from Streptococcus agalactiae.

Similar Datasets

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