LETTER

A recent article described rpoB mutations in 18 rifampin-resistant variants of Legionella spp. (4). One of these 18 strains, isolate 8749, an in vitro-generated high-level rifampin-resistant (MIC  256 µg/ml) variant of K26, a susceptible clinical Legionella bozemanii isolate, was shown to harbor no codon exchange in the cluster I rpoB 69-bp hot spot, the DNA region investigated.

Besides codon exchanges in cluster I (e.g., codon 507 to 533 in Escherichia coli), several mutations in at least three rpoB regions outside cluster I have been described to be associated with resistance to rifampin (1). Mutations either are located 768 bp (in Mycobacterium tuberculosis) to 1,128 bp (in Helicobacter pylori) upstream of cluster I at the beginning of rpoB (5' region; codon V146F in E. coli, V149F in H. pylori, and V176F in M. tuberculosis) or can be found in two regions 123 bp (cluster II) and 468 to 471 bp (cluster III) downstream of cluster I (Fig. 1). Mutations in cluster II are frequently associated with rifampin resistance; cluster III (R687H in E. coli and R701H in H. pylori) is suspected to confer very low levels of resistance.

View larger version (14K): [in this window] [in a new window]   FIG. 1.   Rifampin resistance-determining regions in rpoB. Isoleucine codon positions and substitutions in rpoB cluster II and the newly described tyrosine (Y) substitution in L. bozemanii and threonine (T) substitution in H. pylori are indicated.

Both the parent L. bozemanii strain K26 and the resistant variant 8749 were reinvestigated. PCR primers were designed for amplification of sections encompassing the 5' region of rpoB (bp 124 to 591) (RplL-F [forward 5'-GAAGARGCNGGYGCNGARGTDGA-3'] and L4 [reverse 5'-CAACCATGANCCNCGYTANGG-3']) and clusters I, II, and III (bp 1396 to 2174) (Rif U1 [forward 5'-CGICGIGTTCGITCVGTWGGHGA-3'] and Rif D3 [reverse 5'-ATCAGATGCAACAATTCTTTCC-3']). Base pair numbering is based on the published sequence of Legionella pneumophila (4) and the unfinished Legionella Genome Project of the Columbia Genome Center (contig LP.WG.044.06.111099). IUB code is used for mixed base sites. The PCR products were sequenced on both strands. The 5' region of rpoB (2), especially codon 159 (GTT, valine), was not changed. The only mutation detected (ATT to TAT) was located in cluster II, downstream of the cluster I hot spot. This mutation induces an isoleucine-to-tyrosine amino acid substitution at position 587 of the protein sequence. In the great majority of cluster II mutations described to be associated with rifampin resistance in other species, the corresponding codon is changed. Amino acid exchanges in different species observed in our laboratory or found in the literature are summarized in Fig. 1. Also included is a cluster II isoleucine-to-threonine exchange (ATC to ACC, I586T) newly observed in two (802-1 and 802-2) of three H. pylori DNA samples provided by Wang et al., formerly described as rifampin resistant but harboring wild-type sequence in clusters I and II (6). In the third H. pylori DNA sample (802-3) from that study, amino acid exchange V149F, corresponding to the V146F exchange in E. coli (2) and the V176F exchange in M. tuberculosis, could be observed.

In conclusion, no cluster I mutation was found in strain 8749 that could explain the high level rifamycin resistance (4). Here we show that the isolate harbors a mutation in cluster II (I587Y), inducing an amino acid substitution that is known to be associated with rifamycin resistance in other species. In all comparable cases, complete sequence analysis of the respective rpoB regions (Fig. 1) revealed mutations corresponding to the Rif-resistant phenotype of H. pylori and Legionella spp. and also M. tuberculosis in our laboratory. To our knowledge, mutations outside the cluster I rpoB hot spot in Legionellaceae have not been described so far. This is also the first description of I-to-Y and I-to-T substitutions in cluster II.