INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

Rifampin, a semisynthetic derivative of the rifamycins, is active against both gram-positive and gram-negative bacteria. It has principally been advocated for use in the treatment of tuberculosis but has nevertheless found use in therapy for infections caused by staphylococci (28), legionellae (29), and brucellae (1) and as a prophylactic treatment for meningococcus (25) and Haemophilus influenzae (34) carriers. Rifampin is widely used in South Africa for the treatment of tuberculosis in children. The majority of young children have colonization with pneumococci in their nasopharynx, a fact which results in the bacteria being indirectly exposed to the selective pressures of the antimicrobial agent. Rifampin is, however, not routinely used in the treatment of pneumococcal infections, although it may be used in combination with ceftriaxone or cefotaxime for cephalosporin-resistant pneumococcal meningitis (16, 18). Its mechanism of action is based on its ability to bind to and inactivate the bacterial DNA-dependent RNA polymerase, a mode of action not shared by any other antibiotic in use (10, 35).

The RNA polymerase molecule consists of five subunits, namely, ₂'. Rifampin binds directly to the  subunit of the polymerase (38), and bacterial resistance to rifampin has been shown to be due to mutations occurring in the rpoB gene. Mutations to rifampin resistance result in alterations of rifampin binding and render the organism highly resistant. These mutations occur at a frequency of approximately 10⁶ to 10⁸ in naturally occurring cells (19, 28).

Bacterial resistance to rifampin has been investigated extensively with Escherichia coli (15), Mycobacterium tuberculosis (4, 17, 31, 32), M. smegmatis (12, 22), M. leprae (13), and Neisseria meningitidis (5). The mutations within the rpoB gene appear to cluster into three distinct areas and are generally numbered according to the E. coli protein coordinates. The principal clusters, I (amino acids 505 to 532) and II (amino acids 560 to 572), harbor most of the mutations, while a single mutation at position 687 defines cluster III (11, 14, 15, 38). The discovery of numerous other mutations (23) has made the delineation of the central rifampin resistance region difficult. In E. coli, the majority of the mutations map to clusters I and II, while in other species, rifampin resistance appears to be due to cluster I mutations.

Until recently, little was known about the mechanism of rifampin resistance in pneumococci. We thus investigated rifampin resistance among South African clinical strains of Streptococcus pneumoniae.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Bacterial strains. Twenty-four rifampin-resistant and 8 rifampin-susceptible isolates were randomly selected from a group of clinical isolates collected from 1987 to 1996 and kept at the South African Institute for Medical Research, a reference center for laboratories in South Africa. S. pneumoniae R6, an unencapsulated laboratory strain, was also included in this study. All isolates were serotyped by use of the Quellung reaction (8) with specific antisera from the Staten Seruminstitut (Copenhagen, Denmark). Serogroups, serotypes, and antibiotic data for the isolates are presented in Table 1.

Susceptibility testing. The MICs were determined by the agar dilution method according to the guidelines of the National Committee for Clinical Laboratory Standards (26). Antibiotic plates were made by use of Mueller-Hinton agar supplemented with 5% horse blood.

DNA isolation. Chromosomal DNA was extracted by the salt lysis method of Ausubel et al. (3). DNA was precipitated at 37°C for 10 min with 0.6 volume of a solution containing 20% polyethylene glycol (PEG) and 2.5 M NaCl. DNA was resuspended in water containing 20 µg of RNase A per ml and stored at 70°C for further use.

PCR. The PCR primers were designed from the S. pneumoniae rpoB gene sequence obtained from the TIGR database (contig no BSP131). The primers rpoBF1 (5'-GACAATGAAGTCTTGACACC-3') (nucleotides 1144 to 1163; S. pneumoniae coordinates) and rpoBR1 (5'-CGTGACAACACCTGTTTC-3') (nucleotides 1486 to 1503) were designed to flank clusters I and II. For isolates which carried no mutations within this central region, the primers rpoBF2 (5'-CCTGAAACTGGAGAAATCTTG-3') (nucleotides 721 to 741), rpoBR2 (5'-TGTTACAGGACGGATATTGAT-3') (nucleotides 1174 to 1194), rpoBF3 (5'-GTTCAAACACCATACCGTAAG-3') (nucleotides 1456 to 1176), and rpoBR3 (5'-CAATGAACCATCTTCACGACG-3') (nucleotides 1906 to 1926) were designed to amplify the regions upstream and downstream of clusters I and II.

PFGE. Pulsed-field gel electrophoresis (PFGE) for S. pneumoniae was carried out by the method of Lefevre and coworkers (21). Separation of the SmaI restriction fragments was achieved by electrophoresis for 21 h at 200 V, and visualization was done with UV illumination after ethidium bromide staining.

Nucleotide sequence analysis. Single-stranded PCR products were prepared for sequencing with streptavidin-coated magnetic particles (Boehringer). Briefly, a standard amplification reaction (100 µl) was performed with a 5'-biotinylated forward primer (rpoBF1, rpoBF2, or rpoBF3) and an unlabelled reverse primer. Nonincorporated dNTPs and primers were removed by precipitation with PEG as described previously (3), and the pellet was resuspended in 40 µl of Tris-EDTA buffer. Single-stranded DNA bound to the magnetic beads was prepared according to the manufacturer's recommendations. The unlabelled DNA was salt precipitated from the supernatant and resuspended in 20 µl of water.

Cloning of PCR products. The amplified PCR products of the rpoB gene were PEG precipitated (3). Blunt-end ligations into SmaI (Boehringer)-digested expression vector pGEM-7Zf(+) were performed. The ligations were carried out with 12-µl reaction volumes containing 0.1 µg of SmaI-restricted vector, 0.3 µg of PCR product, 8% PEG 6000; 4.34 mM ATP, 5 U of SmaI, and 2 U of T4 DNA ligase (United States Biochemicals) in ligation buffer provided by the manufacturer. The mixtures were subjected to cycling overnight at between 10 and 30°C for 30 s by the method described by Lund et al. (24). Competent E. coli JM109 cells were electrotransformed with purified ligated DNA as described previously (6). Transformants were selected for -complementation on Luria-Bertani agar containing 50 µg of ampicillin per ml, 20 mg of -galactosidase per ml, and 100 mM isopropyl--D-thiogalactopyranoside (IPTG).

Transformations. Transformations were performed with competent S. pneumoniae R6 as detailed by Tomasz and Hotchkiss (33). Thawed competent cells were incubated with 1 µg of cloned PCR product per ml at 30°C for 30 min. The cells were allowed to express resistance at 37°C for 60 min before plating of 100 µl of cells onto selection media. The plates were incubated for 48 h at 37°C. DNA-free controls were also tested in order to confirm that any growth observed was the result of transformation and not mutation. Cells were also plated on antibiotic-free medium for the determination of viable counts. The MICs for transformants were determined as described above.

Nucleotide sequence accession numbers. The accession numbers AJ236789, AJ236790, AJ236791, and AJ236792 have been assigned to the sequences submitted to the EMBL database.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

PCR amplification and sequence analysis. The amino acid numbering used in this study is that of the S. pneumoniae sequence obtained from the TIGR database. The corresponding E. coli numbering has been inserted in some instances to allow for alignment with previously identified mutations.

View larger version (30K): [in this window] [in a new window]   FIG. 1.   Nucleic acid substitutions occurring in the 23 rifampin-resistant strains containing mutations. The codon numbering is that of the S. pneumoniae sequence obtained from the TIGR database. Cluster I () and cluster III () are indicated above the amino acid residues. The shaded codons correspond to the E. coli Asp516 and His526 amino acids. Mutations that have been reported previously are indicated by asterisks (14, 16, 30). The mutations observed appear in the boxes; the numbers represent the number (percentage) of strains in which they occur.

Transformations. The significance of the mutations observed was confirmed by transformation. The rpoBF1-rpoBR1 PCR product containing cluster I mutations and the fragment obtained with rpoBF3-rpoBR3 were able to transform rifampin-susceptible R6 to resistance. The mean transformation efficiencies were 6.3 × 10⁴ and 1.4 × 10⁴, respectively. The MICs for the transformants obtained with the cluster I product were equivalent to the MICs for the donor, while the downstream fragment was only able to transform S. pneumoniae R6 so that the MIC was 16 µg/ml.

Genetic relatedness of isolates. The observation that many of the rpoB gene sequences obtained were identical led us to investigate the clonal relationship of the isolates. PFGE patterns (Fig. 2) were obtained for isolates representative of the different mutation groups. Contrary to expectations, the PFGE patterns of the isolates were very different. Isolates 85991, 8085, and 17 were related on the basis of their PFGE pattern, but none of the other isolates were of clonal origin.

View larger version (125K): [in this window] [in a new window]   FIG. 2.   PFGE separation of the SmaI restriction fragments of clinical isolates of S. pneumoniae. Lane Mw, lambda phage molecular weight ladder; lanes A to F, reference clones 9V, 6B, Spanish 23F, Tennessee 23F, and South African 19A and 6B, respectively; lanes G to Q, rifampin-resistant isolates 46471, 13897, 1412, 42, 2, 55, 36023, 18078, 85991, 8085, and 17, respectively; lanes R to T, rifampin-susceptible isolates 620, 111, and N1503, respectively.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Rifampin resistance among South African clinical isolates of S. pneumoniae appears to involve mutations within the  subunit of the rpoB gene, as is the case in numerous other bacteria.

It was observed that 75% of the rifampin-resistant isolates studied contained cluster I mutationsboth Asp₄₁₅-Glu (equivalent to E. coli Asp₅₁₆) and His₄₂₅-Asn (equivalent to E. coli His₅₂₆) or His₄₂₅-Asn alone. Numerous mutations at both of these locations have been reported previously, both for E. coli (15) and, more recently, while this study was being conducted, for S. pneumoniae (7). The substitution Asp₄₁₅-Asn has been reported to confer low-level resistance (MIC, 8 µg/ml) in S. pneumoniae (7) on its own. We report for the first time the presence in S. pneumoniae of an Asp₄₁₅-Glu substitution, a mutation reported previously to occur in M. tuberculosis and shown to be involved in the resistance phenotype (17, 32). The second cluster I mutation, His₄₂₅-Asn, has been reported for S. pneumoniae (7), N. meningitidis (5), and M. tuberculosis (31, 32). It is interesting to note that a large proportion of the isolates studied contained both mutations, an observation made previously for M. tuberculosis (36). The presence of both Asp₄₁₅-Glu and His₄₂₅-Asn mutations does not, however, appear to contribute to higher MICs, and the results obtained suggest that the presence of His₄₂₅-Asn alone is sufficient to confer high-level resistance to rifampin. These results are consistent with other findings in that mutations at the Asp₅₁₆ and His₅₂₆ codons lead to high-level rifampin resistance in both E. coli and M. tuberculosis (15, 27, 36). The predominance of the His₄₂₅-Asn mutation in the rifampin-resistant isolates used in this study suggests that this mutation plays an important role in the selection of antibiotic resistance, as has been alluded to previously by Carter and colleagues (5).

Resistant isolates that did not contain mutations in cluster I or cluster II have been reported previously for S. pneumoniae (7) and M. tuberculosis (17, 36), but further investigation of their nucleotide sequences was not attempted. Sequence analysis of the six isolates that lacked cluster I or cluster II mutations in this study revealed the presence of eight mutations, Arg₅₂₃-Lys, Glu₅₂₆-Ala, Iso₅₃₄-Val, Asn₅₄₉-Ser, Iso₅₅₀-Ser, Asn₅₉₅-Glu, Gln₅₉₇-Lys, and Tyr₆₀₀-Phe, downstream of cluster II, in a region which we now designate S. pneumoniae cluster III. This area is distinct from the E. coli cluster III region (15). The presence of all eight mutations in all six isolates lends support to the assumption that this cluster of mutations involved in rifampin resistance was acquired by homologous recombination of transformed DNA. The MIC for the transformants of these isolates was 16 µg/ml, lower than that for the parent strain, suggesting that these changes may operate in conjunction with other, as-yet-unidentified mutations within rpoB or elsewhere to confer resistance to rifampin. This idea is consistent with the findings for E. coli (30), where the cluster III mutation Arg₆₈₇-His confers only very low levels of rifampin resistance, suggesting that it might be located at the periphery of the region of RNA polymerase that binds to rifampin. Genetic evidence also suggests that cluster I (the region around E. coli Arg₅₂₉) and cluster III (the region around E. coli Arg₆₈₇) are in close proximity in the native protein, suggesting that these two regions of the polypeptide are both involved in forming the binding site for rifampin. A similar scenario may be envisaged to occur in S. pneumoniae, with cluster I (positions 406 to 434) and cluster III (positions 523 to 600) interacting to form the antibiotic binding site. Such a scenario may account for the very high MIC observed for isolate 55 (256 µg/ml), which was found to contain mutations at His₄₂₅ and in the region from Arg₅₂₃ to Tyr₆₀₀.

One isolate, 13897 (MIC, 64 µg/ml), was sequenced from Lys₂₄₇ to Tyr₆₂₇ (sequence not shown) and was found to contain no mutations within this region. It is also conceivable that mutations that could play a role in conferring resistance exist elsewhere in rpoB. Substitutions of amino acids 146 and 687, located outside clusters I and III, have been described for E. coli (15, 23). Further characterization of the rpoB gene from isolate 13897 is currently in progress.

Several alternatives have been proposed to account for the lack of mutations observed in rpoB in rifampin-resistant strains. These include the modification of rifampin (2, 37), alterations in drug uptake or efflux mechanisms (9, 20), and changes in antibiotic permeability or metabolism (17). It has also been suggested that mutations in other subunits of the polymerase could contribute to rifampin resistance, perhaps by altering the conformation of the rifampin binding region of the  subunit (12). Although these mechanisms have only been hinted at for organisms other than S. pneumoniae, we cannot rule out the possibility that they may play a role in rifampin resistance in pneumococci.

Sequence analysis revealed the possibility that some isolates may be of clonal origin, but the PFGE fingerprints show that these isolates are in fact unrelated to each other and reference clones. It appears, therefore, that rifampin resistance among South African clinical isolates is not due to the clonal spread of resistant strains.

These molecular studies support the idea that rifampin-resistant strains could be selected by a mutational event occurring among susceptible strains. The shared sequence of mutations identified for the first time in cluster III may indicate a single transformational event that has occurred among a variety of strains.

Our study has documented a second area of mutations in the rpoB gene that confers rifampin resistance in S. pneumoniae and suggests that other mechanisms have yet to be clarified.