We cloned a gene, bexA, that codes for a multidrug efflux transporter from the chromosomal DNA of Bacteroides thetaiotaomicron ATCC 29741 by using an Escherichia coli ⌬acrAB ⌬acrEF mutant as a host. Although the initial recombinant construct contained other open reading frames, the presence of bexA alone was sufficient to confer to the E. coli host elevated levels of resistance to norfloxacin, ciprofloxacin, and ethidium bromide. Disruption of bexA in B. thetaiotaomicron made the strain more susceptible to norfloxacin, ciprofloxacin, and ethidium bromide, showing that this gene is expressed in this organism and functions as a multidrug efflux pump. The deduced BexA protein sequence was homologous to the protein sequence of Vibrio parahaemolyticus NorM, a multidrug efflux transporter, and thus, BexA belongs to the multidrug and toxic compound extrusion (MATE) family.Members of the Bacteroides fragilis group are anaerobic bacteria of the highest clinical relevance, and B. fragilis and Bacteroides thetaiotaomicron are often isolated from patients with suppurative anaerobic infections. They are gram-negative, obligately anaerobic organisms with a broad spectrum of recognized resistance to antimicrobial agents (23), including aminoglycosides, most of the penicillins and cephalosporins, and fluoroquinolones (except for a few recently developed compounds such as trovafloxacin and clinafloxacin) (2, 3).Active multidrug efflux processes, usually involving secondary transporters belonging to the major facilitator superfamily, small multidrug resistance family, and resistance-nodulationdivision (RND) superfamily, are now known to be important, especially in the baseline or intrinsic resistance of many bacteria to antimicrobial agents (16,21). More recently, a new family, the multidrug and toxic compound extrusion (MATE) family, has been discovered (4), but its contribution to drug resistance has been known only for a few isolated cases (12, 13).We found previously that at least a portion of the remarkable norfloxacin resistance (MICs, 16 to 32 g/ml) of B. fragilis was attributed to active efflux of this agent (12). To elucidate the efflux mechanism, we attempted to clone the genes responsible for norfloxacin efflux from the chromosomal DNAs of B. fragilis and B. thetaiotaomicron using as the host an Escherichia coli mutant strain with defects in multidrug efflux pumps. Here we report on the cloning and sequencing of a gene, bexA, that is involved in the efflux of norfloxacin, ciprofloxacin, and ethidium bromide from B. thetaiotaomicron. Interestingly, the BexA transporter is a member of the MATE family. MATERIALS AND METHODSBacterial strains and growth conditions. B. fragilis ATCC 25285 and B. thetaiotaomicron ATCC 29741 5482 and were grown in general anaerobic GAM broth (Nissui, Tokyo, Japan) and supplemented Trypticase soy broth (sTSB) (12) in an anaerobic chamber.For cloning, two host strains were used. They were E. coli DH5␣ [K-12 supE44 ⌬lacU169 (80 lacZ⌬M15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1] (26) and AG102AX [K-...

Inorganic polyphosphates [Poly(P)] are often distributed in osteoblasts. We undertook the present study to verify the hypothesis that Poly(P) stimulates osteoblasts and facilitates bone formation. The osteoblast-like cell line MC 3T3-E1 was cultured with Poly(P), and gene expression and potential mineralization were evaluated by reverse-transcription polymerase chain-reaction. Alkaline phosphatase activity, von Kossa staining, and resorption pit formation analyses were also determined. The potential role of Poly(P) in bone formation was assessed in a rat alveolar bone regeneration model. Poly(P) induced osteopontin, osteocalcin, collagen 1alpha, and osteoprotegerin expression and increased alkaline phosphatase activity in MC 3T3-E1 cells. Dentin slice pit formation decreased with mouse osteoblast and bone marrow macrophage co-cultivation in the presence of Poly(P). Promotion of alveolar bone regeneration was observed locally in Poly(P)-treated rats. These findings suggest that Poly(P) plays a role in osteoblastic differentiation, activation, and bone mineralization. Thus, local poly(P) delivery may have a therapeutic benefit in periodontal disease.

These data demonstrate that constitutive bmeB expression is prevalent in B. fragilis. At least seven BmeB efflux pumps are functional in transporting antimicrobials and have overlapping substrate profiles, and at least four confer intrinsic resistance.

Sixteen homologs of multidrug resistance efflux pump operons of the resistance-nodulation-cell division (RND) family were found in the Bacteroides fragilis genome sequence by homology searches. Disruption mutants were made to the mexB homologs of the four genes most similar to Pseudomonas aeruginosa mexB. Reverse transcription-PCR was conducted and indicated that the genes were transcribed in a polycistronic fashion and that the promoter was upstream of bmeA (the mexA homolog). One of these disruption mutants (in bmeB, the mexB homolog) was more susceptible than the parental strain to certain cephems, polypeptide antibiotics, fusidic acid, novobiocin, and puromycin. The gene for this homolog and the adjacent upstream gene, bmeA, were cloned in a hypersensitive Escherichia coli host. The resultant transformants carrying B. fragilis bmeAB were more resistant to certain agents; these agents also had lower MICs for the B. fragilis bmeB disruption mutants than for the parental strain. The putative efflux pump operon is composed of bmeA, bmeB, and bmeC (a putative outer membrane channel protein homologous with OprM). Addition of the efflux pump inhibitors, carbonyl cyanide m-chlorophenylhydrazone (a proton conductor that eliminates the energy source) and Phe-Arg ␤-naphthylamide (MC-207,110) (the first specific inhibitor described for RND pumps in P. aeruginosa), resulted in lowered MICs in the parental strain but not in the bmeB disruption mutant, indicating that the bmeB pump is affected by these inhibitors. This is the first description of RND type pumps in the genus Bacteroides.Bacteroides fragilis, an anaerobic gram-negative rod, is an opportunistic pathogen that can cause significant mortality in infections resulting from abdominal trauma or surgery (7,19). Although it accounts for only 0.5% of the enteric flora, it is the Bacteroides species most frequently isolated from patients with intra-abdominal infections and/or bacteremia (in which mortality reached 45% if inactive therapy was given). It often presents a serious problem for therapy, since it is resistant to many antibiotics, including most of the penicillins, cephalosporins, and the quinolones (1,3,19,20,25).Gram-negative bacteria including B. fragilis are usually more resistant to a large number of antibiotics and other noxious agents than are gram-positive bacteria. Clinically significant levels of antibiotic resistance are caused by interplay between the efficient outer membrane (OM) permeability barrier, ubiquitous periplasmic ␤-lactamases, and recently recognized multidrug resistance (MDR) efflux pumps (17). These pumps have broad substrate specificity and may act synergistically with the permeability barrier to result in significant intrinsic resistance to many antimicrobials. These pumps expel the antimicrobial from the cell into the surrounding space, and the antimicrobials then have to pass through the OM permeability barrier to regain entry to the cell (18). Thus, the MDR pumps can effect significant resistance even when their transporter activi...