INTRODUCTION

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Rifampin and isoniazid remain the two most important drugs for the treatment of tuberculosis, and resistance to either drug represents a serious impediment to successful therapy. Resistance to rifampin is associated with mutations in the gene coding for the beta subunit of RNA polymerase (rpoB). Early studies examined the rate of rifampin resistance mutations in Mycobacterium tuberculosis (3). More recently, the sequencing of the rpoB gene in M. tuberculosis and the development of direct sequencing of PCR products have allowed determination of the actual mutations (11, 16), and there has been extensive analysis of mutations in the rpoB gene of rifampin-resistant patient isolates of M. tuberculosis (2, 4-7, 9-11, 13-17). Telenti et al. demonstrated that at least 95% of rifampin-resistant isolates have mutations in rpoB and that the mutations are clustered in an 81-bp region (16). Because RNA polymerase is an essential enzyme, there must be a limited number of possible mutations that confer rifampin resistance and retain polymerase activity, and this is reflected in the very low rate of mutation to resistance. About 40 distinct point mutations and in-frame insertions and deletions in rpoB have been identified in M. tuberculosis isolates. Point mutations in two codons, those encoding Ser₅₃₁ and His₅₂₆, are seen in about 70% of rifampin-resistant clinical isolates, with Ser₅₃₁-to-Leu (TCG-to-TTG) mutations being by far the most common. We hypothesized that the various point mutations (single base changes) occur at about the same frequencies and that the finding of predominant mutations in clinical isolates is likely a result of specific mutations having a selective advantage, at the phenotypic level, for proliferation in humans following rifampin therapy. To explore this question, we isolated independent spontaneous rifampin-resistant mutant versions of well-characterized M. tuberculosis laboratory strain H37Rv by plating 100 separate cultures onto rifampin-containing medium. We then identified the rpoB mutations in the resulting 64 mutants. Contrary to our expectations, the distribution of mutations mirrored that seen in patient isolates.

	MATERIALS AND METHODS

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Isolation of rifampin-resistant mutants. M. tuberculosis H37Rv from our laboratory stock was cultured in Middlebrook 7H9 broth at 37°C for 32 days to an optical density at 600 nm of 0.83, corresponding to approximately 10⁸ CFU/ml. The culture was diluted and inoculated into 500 ml of 7H9 broth to yield a concentration of about 10³ CFU/ml, and 5-ml aliquots were dispensed into 100 culture tubes. The tubes were incubated at 37°C for 32 days with daily shaking. Approximately 2 ml of culture containing most of the cell mass was pipetted from the bottom of each tube and transferred to a sterile screw-cap microcentrifuge tube. The tube was centrifuged for 30 s, the supernatant was aspirated, and the bacterial pellet was suspended in 1 ml of 0.5% Tween 80 in water. The tube was centrifuged, and the supernatant was aspirated, leaving a small amount of liquid. The pellet was suspended, and the entire volume was spread onto one quadrant of a plate containing Middlebrook 7H10 agar with 1 µg of rifampin/ml. The plates were incubated for 4 weeks at 37°C. Colonies from these plates were subcultured in 7H9 broth without Tween 80 containing 2 µg of rifampin/ml.

Amplification and sequencing of the rpoB gene. Genomic DNA was isolated using a mechanical cell disruption procedure. Briefly, 1 ml of a 7H9 broth culture was added to a 1.5-ml screw-cap microcentrifuge tube containing approximately 250 mg of siliconized zirconia and silica beads (0.1 mm in diameter), 200 µl of chloroform, and 300 µl of Tris-EDTA buffer. This mixture was vigorously agitated for 2 min using a Mickle cell disrupter (Brinkman Instruments, Inc., Westbury, N.Y.) and then centrifuged at 10,000 rpm in an Eppendorf model 5917R microcentrifuge for 5 min. The aqueous phase, containing genomic DNA, was collected and stored at 4°C.

	RESULTS

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The procedure for isolating a set of independent mutants was essentially the approach used in the classic fluctuation test of Luria and Delbruck (8). A dense culture of wild-type, rifampin-susceptible M. tuberculosis H37Rv was diluted to yield a 500-ml culture containing approximately 10³ CFU/ml. Results of a previous study of the frequency of mutations to rifampin resistance and our own experience indicated that no rifampin-resistant mutants should be present in the total population of 5 × 10⁵ CFU in this culture (3). The culture was aliquoted into 100 tubes, and these parallel cultures were incubated to allow growth to the high density needed to obtain rifampin-resistant mutants. Most of the cell mass from each of the 100 cultures was harvested and plated onto 7H10 agar containing rifampin. We expected this procedure to produce mutants derived from independent mutational events in each culture. Of the 100 cultures, 32 did not yield colonies. Colonies picked from four plates failed to grow on subculture in 7H9 broth containing rifampin. The average number of mutations per culture (m) can be calculated based on the number of cultures that yielded no mutants using the Poisson equation, p₀ = e^m, where p₀ is the proportion of cultures yielding no mutants. This value is 1.13 for p₀ = 0.32 or 1.02 for p₀ = 0.36, depending on which value is used for plates with no mutants. It should be noted that we plated most, but not all, of the cells in each 5-ml culture, and the number of cultures with no mutants may have been slightly lower had we recovered all mutants. Regardless, this result indicates that in most cases all colonies derived from each culture are the progeny from a single mutational event. The remaining 64 cultures yielded rifampin-resistant mutants (Table 1). Of these, 62 plates had from 1 to 11 colonies (average, 2.32 colonies), with 29 plates having only a single colony. One colony was picked from each of the plates for subculture. The remaining two plates, plates 10 and 76, had 27 and 288 colonies, respectively, and 10 colonies were picked from each of these two plates.

All of the point mutations detected in this study have been reported previously (Table 1). All 10 colonies picked from plate 76 had the same mutation, Ser₅₃₁ to Leu (TCG to TTG), consistent with the conclusion that the mutation occurred early in this culture and yielded many progeny. Similarly, 9 of the 10 colonies from plate 10 had the Ser₅₃₁-to-Leu mutation. The other colony gave a result indicating a mixture of mutants with mutations Ser₅₃₁ to Leu and His₅₂₆ to Tyr (CAC to TAC), i.e., peaks for both C and T were present at each position. For simplicity, we did not consider the latter mutant in the analysis but instead counted culture 10 as representing a Ser₅₃₁-to-Leu mutation.

Of the 64 mutants, 39 (60.9%) had the Ser₅₃₁-to-Leu mutation (Table 2). This is the most common mutation seen in patient isolates also. Two mutants had a different mutation in codon 531, resulting in a Ser₅₃₁-to-Trp change. Four mutations in codon 526 were identified in a total of 16 mutants. This is the second-most-frequent site for mutations seen in patient isolates. Four other mutations were identified: two point mutations, a three-base deletion, and a three-base insertion.

The sizes of the colonies on the rifampin plates were recorded as tiny, small, medium, and large. There was no correlation between colony size and mutation; in particular, we observed all colony sizes for Ser₅₃₁-to-Leu mutants.

	DISCUSSION

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In this study we demonstrated that the rpoB mutations most commonly seen in rifampin-resistant clinical isolates of M. tuberculosis, especially the one resulting in the Ser₅₃₁-to-Leu change, also predominate in independent strain H37Rv mutants obtained by selection in vitro on 7H10 medium containing rifampin. This was not the result we anticipated; we expected that all point mutations would occur at about the same frequency. In patients with tuberculosis, rifampin resistance evolves following treatment when the rare spontaneously resistant mutants proliferate and replace the population of sensitive bacilli that have been killed by the drug. Although mutations imparting rifampin resistance occur at low frequency, the bacillary load present in cavitary tuberculosis is sufficient to allow for their selection. Expansion of the population of resistant mutants requires several weeks, and it is reasonable to assume that if several distinct mutants are present at the onset of therapy, the more-robust mutants, perhaps those with Ser₅₃₁-to-Leu mutations, will likely predominate and ultimately be isolated from the patient's sputum. The fitness of the mutant might also affect the probability of transmission, increasing the proportion of such mutants in patients with primary resistance. Although there have been significant studies of the evolution of rifampin resistance in patients, none have dealt with the emergence of specific mutations, which has been possible only in recent years.

The recent paper of Billington et al. also describes the isolation of rifampin-resistant H37Rv mutants (1). They determined that Ser₅₃₁ to Leu is the most common mutation seen with in vitro-generated rifampin-resistant mutants and demonstrated that a mutant with this mutation shows the least physiological cost relative to the rifampin-susceptible parent strain and thus has a selective advantage. However, they sampled a smaller number of batch cultures than we did and, in contrast to our study, picked multiple colonies from the same cultures. Surprisingly, they detected only four mutations, Ser₅₃₁ to Leu and three mutations in the His₅₂₆ codon, among 156 colonies screened. Their results indicate either that these mutations occur at a higher frequency than other point mutations or that these mutants grow more rapidly and thus are more likely to be detected using their sampling procedure. The method used in the present study, essentially that of Luria and Delbruck (8), was intended to avoid the latter possibility. As in the classic fluctuation test, variation in the number of resistant colonies obtained from each separate culture reflects the time at which the mutation occurred. If the selection occurs at the appropriate time, most colonies derived from each culture represent progeny from a single event. In this case, the low frequency of rifampin-resistant mutants is advantageous, because few mutants are present even in dense cultures. The fact that one-third of the cultures did not yield mutants is consistent with single-mutation events in most of the cultures. If each culture contains only a single mutant there is no opportunity for competition between different mutants and enrichment of the faster-growing mutant prior to plating. Thus, the distribution of mutations with this procedure should reflect the relative rates of occurrence of the various mutations. We did not sequence all colonies from all plates, and it is possible that some selection bias occurred. Looking only at the cultures that yielded a single colony, in which there could be no bias in the selection of the colony for sequencing, 18 of 29 (62%) mutations were Ser₅₃₁ to Leu.

Two possible mechanisms could explain the higher rate of occurrence of the Ser₅₃₁-to-Leu mutants. This could result from a higher rate of occurrence of the corresponding specific base substitution, i.e., an increased error rate at that site. Studies of DNA repair in M. tuberculosis have been reviewed recently (12), but we are not aware of any data that would explain a higher frequency of the Ser₅₃₁-to-Leu mutation. An alternative explanation is that other mutations occur at the same frequency but are less successful in making the transition to expression of the mutant beta subunit (as a result of genotypic and phenotypic lag), either in the absence of rifampin (in 7H9 broth) or upon initial exposure to rifampin on plates. To impact our results, this would require a lethal effect rather than merely a reduction in growth rate. Undoubtably many mutations in rpoB are lethal, but about 40 distinct mutations have been identified in patient isolates, indicating that all of the associated mutants are sufficiently fit to proliferate in the host, grow on laboratory media, and demonstrate rifampin resistance in standard susceptibility testing procedures, such as growth on 7H10 medium at the critical concentration of 1 µg of rifampin/ml (7a). Differences in MICs of rifampin for various rpoB mutants have been demonstrated. Because of this, we used the minimum concentration of rifampin (1 µg/ml) that reliably inhibits wild-type M. tuberculosis to avoid a selective advantage for certain mutants.

Whatever the mechanism, our results indicate that the Ser₅₃₁-to-Leu mutation and the multiple mutations in codon 526 occur at a significantly higher frequency than other point mutations. The precise rate of occurrence of these mutations cannot be determined from this limited study, but the overall trend is clear. Such higher occurrence rates may contribute to the high rate at which these mutants are isolated from patients with tuberculosis, although the emergence of rifampin resistance in humans is much more complex than that reflected in the simple in vitro experiments reported here.