LETTER

Rifampin is an antibiotic that interacts specifically with the -subunit of DNA-dependent RNA polymerase (RpoB) encoded by the rpoB gene (2). Rifampin resistance (Rif^r) is usually due to changes in the amino acid sequence of the target site resulting in reduced affinity of RpoB for rifampin. The majority of Escherichia coli Rif^r mutations are located in two highly conserved regions of RpoB encompassing amino acid residues 507 to 533 (Rif cluster I) and 563 to 572 (Rif cluster II) (2). Substitutions in these clusters, particularly at "hot spots" such as the residue cognate to E. coli S531, are responsible for acquired Rif^r in several species of bacteria. Less common mechanisms of Rif^r include mutations in RpoB outside Rif clusters I and II, decreased membrane permeability, efflux, and enzymatic modification of the antibiotic.

Several species of free-living and host-associated spirochetes of the genera Spirochaeta, Leptospira, and Treponema that were isolated without rifampin are resistant to relatively high concentrations of this antibiotic (MICs, 50 to >200 µg/ml) (5). Rif^r is widespread among spirochetes, and rifampin sensitive strains have not been isolated (5, 10). Studies conducted by Leschine and Canale-Parola (5) with purified Spirochaeta aurantia RpoB suggested that spirochetal Rif^r may be due to a low affinity of RpoB for rifampin. Alekshun et al. (1) proposed that an N at the RpoB residue cognate to E. coli S531 is the primary molecular determinant of naturally occurring Rif^r in Borrelia burgdorferi and possibly in other spirochetes. Furthermore, Lee et al. (4) observed an N531 in the RpoB of 22 Borrelia reference strains. Our analysis of the complete amino acid sequences of Treponema pallidum strain Nichols (AE001205) and Leptospira biflexa (AF150880) RpoB confirmed the presence of an N531 in both organisms. Additional amino acid substitutions associated with Rif^r were not present inside or outside Rif clusters I and II. These observations prompted us to examine the Rif clusters of several Rif^r host-associated Treponema spp.

Treponema pallidum Street strain 14, an erythromycin-resistant clinical isolate, was grown by testicular cultivation in rabbits as previously described (8). T. denticola (ATCC 35405) and T. phagedenis (Reiter) were grown as previously described (7). T. socranskii subsp. socranskii (ATCC 35536) and T. medium G7201 were grown in NOS medium as previously described (6). Four clonal isolates of bovine papillomatous digital dermatitis (PDD)-associated Treponema were grown as previously described (9). Genomic DNA was extracted from each of the Treponema spp. as previously described (8). The complete rpoB of T. pallidum Street strain 14 was PCR amplified using primers (forward, 5'-CGGCGTCTCCCCTGTGTG-3'; reverse, 5'-ATTGTCTCAGGCTTTTTCAC-3') based on Nichols strain nucleotide sequences flanking rpoB (AE001205). An approximately 2.3-kb internal fragment of rpoB was amplified from each of the cultivable Treponema spp. using PCR primers (forward, 5'-CGTTCGCCTGGTGTTATC-3'; reverse, 5'-AGACCCTTGTTTCCGTGG-3') based on the preliminary sequence of T. denticola rpoB (The Institute for Genomic Research, http: //www.tigr.org). Gel-purified PCR amplicons were cloned and both DNA strands were sequenced as previously described (8).

A deduced amino acid sequence alignment representing the RpoB region containing Rif clusters I and II for each of the Treponema spp. is presented in Fig. 1. The corresponding sequences of rifampin-sensitive E. coli and Staphylococcus aureus and Rif^r L. biflexa and B. burgdorferi are shown for comparison. Although substitutions in Rif cluster I residues 508, 518, and 531 are present in all of the Treponema spp., only the N531 substitution correlates with Rif^r. While not commonly observed in Rif^r bacteria, an N531 substitution is associated with high-level resistance in Mycobacterium celatum, an organism that is naturally Rif^r (3). Substitutions in Rif cluster II are not present in any of the Treponema spp.

View larger version (44K): [in this window] [in a new window]   FIG. 1.   Deduced amino acid sequence alignment of Treponema spp. RpoB region containing Rif clusters I and II with the corresponding sequences of wild-type rifampin-sensitive E. coli (AE000472) and S. aureus (X64172) and naturally Rifr L. biflexa (AF150880) and B. burgdorferi (L48488). Numbering is based on the E. coli RpoB sequence. Rif clusters I and II are indicated. Residues where amino acid substitutions are known to correlate with Rifr are boldfaced. Arrow, N531 substitution that is present in all Treponema spp. and correlates with Rifr. Abbreviations: Eco, E. coli; Sau, S. aureus, Lbi, L. biflexa; Bbu, B. burgdorferi; Tpa, T. pallidum Street strain 14; Tde, T. denticola 35405; Tme, T. medium G7201; Tso, T. socranskii subsp. socranskii; Tph, T. phagedenis Reiter; PDD1, PDD-associated Treponema isolate 1-9185MED; PDD2, isolate 9-3379; PDD3, isolate 7-2009; PDD4, isolate 2-1498. The amino acid sequence of T. pallidum Street strain 14 is identical to that of T. pallidum strain Nichols (AE001205) in the RpoB region shown.

Our results support the hypothesis of Alekshun et al. (1) that N531 is primarily responsible for spirochetal Rif^r. However, some variations in the level of Rif^r among cultivable spirochetes, including Treponema spp., have been reported (5, 10). It is possible that additional mechanisms such as membrane permeability or mutations occurring in RpoB outside Rif clusters I and II are responsible for such observations. Further studies are required to elucidate this. Finally, our results also support the use of rifampin as a selective agent for the isolation of Treponema spp. from human and animal specimens (5).

The sequences of the 198-bp region of rpoB from the Treponema spp. have been deposited in GenBank under accession numbers AF389072 to AF389080.