Biological Cost of Rifampin Resistance from the Perspective of Staphylococcus aureus

Resistance determinants that interfere with normal physiologicalprocesses in the bacterial cell usually cause a reduction inbiological fitness. Fitness assays revealed that 17 of 18 invitro-selected chromosomal mutations within the rpoB gene accountingfor rifampin resistance in Staphylococcus aureus were associatedwith a reduction in the level of fitness. There was no obviouscorrelation between the level of resistance to rifampin andthe level of fitness loss caused by rpoB mutations. Among 23clinical rifampin-resistant S. aureus isolates from six countries,only seven different rpoB genotypes could be identified, wherebythe mutation 481His $->$ Asn was present in 21 (91%) of these 23 isolates.The mutation 481His $->$ Asn, in turn, which confers low-level rifampinresistance on its own, was not shown to be associated with acost of resistance in vitro. The restriction to distinct mutationsthat confer rifampin resistance in vivo, as demonstrated here,appears to be determined by the Darwinian fitness of the organisms.

INTRODUCTION

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Introduction

The selective pressure of antibiotics in general results inthe emergence of resistance mechanisms on the part of bacteria(12). Hence, a direct correlation often exists between antibioticuse and the development of bacterial resistance to antibioticsover time (3). Conversely, the incidence of antibiotic resistancecan be reduced by the restriction of antibiotic usage (22).This interrelation between antibiotic usage and resistance developmentis partly determined by the so-called biological cost of antibioticresistance (11, 16). Since antibiotic resistance often is characterizedby detriments in biological fitness, resistant bacteria canbe decimated selectively by a decreased use of antibiotics (10).It appears, however, that resistance never completely disappears.This feature may be owing to the fact that the loss of biological(Darwinian) fitness on the part of resistant bacteria can easilybe overcome by the acquisition of compensatory mutations, therebystabilizing the resistant bacteria within a given population(8, 16, 21).

Antibiotic resistance due to chromosomal mutations that causestructural modifications in the cellular target of the drug,such as rifampin resistance in Staphylococcus aureus, can beassociated with a fitness burden. The present study was aimedat analyzing (i) the biological cost of RNA polymerase (rpoB)mutations conferring rifampin resistance on S. aureus, (ii)the relationship between the cost of rpoB mutations and thelevel of resistance to rifampin, and (iii) the distributionof rpoB mutations in vivo in view of the possible fitness burdenassociated with resistance.

MATERIALS AND METHODS

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Materials and Methods

Bacteria. Fifty-four S. aureus isolates were analyzed, including 23 isolatesfrom six countries that were rifampin resistant (Rif^r) in vivoas well as 30 mutants derived from a single susceptible isogenicreference strain that were Rif^r in vitro. Twenty-six of theRif^r in vitro mutants were selected by culturing a rifampin-susceptible(Rif^s) strain on agar containing 0.25 µg of rifampin perml. The other four Rif^r in vitro mutants were obtained by culturingmutant 1 on agar containing 10 µg of rifampin per ml.

Genotyping. All clinical MRSA isolates were analyzed by pulsed-field gelelectrophoresis (PFGE) as described previously (24). Mutationsconferring rifampin resistance were determined by partial DNAsequencing of the rpoB gene, including resistance clusters Iand II, as described previously (25).

Fitness assay. The relative fitnesses of the Rif^s parental strain and the Rif^rin vitro mutants that evolved were determined by paired competitionexperiments. The parental strain and the Rif^r mutant strainwere grown to an optical density at 600 nm of 0.6; 1 ml of eachculture was mixed and diluted 10^-5 in 5 ml of Luria-Bertanimedium to create a mixed culture of Rif^r and Rif^s bacteria thatwas incubated at 37°C for 20 h. The number of viable Rif^rand Rif^s cells was determined at the beginning and end of theexperiment by plating aliquots of the culture on medium containingeither 0.125 µg of rifampin per ml or no drug. The numberof generations (G) of the mutant and isogenic parental strainthat occurred in the mixed broth was calculated as describedby Billington et al. (4): G = (log B - log A)/log 2, where Ais the number of CFU per milliliter at time zero and B is thenumber of CFU per milliliter at the end of the culture period.The relative fitness of each strain was determined from theratio of the number of generations from Rif^r to Rif^s strain.

Statistics. Statistical analysis was performed by the one-sample t test.

RESULTS

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Results

Eighteen different rpoB genotypes could be identified with respectto the in vitro mutations in S. aureus conferring rifampin resistance(Fig. 1). Only one mutational change, 481His $->$ Asn, which was capableof conferring low-level resistance, was not associated witha reduction in the level of fitness (Table 1). In contrast,all other mutants responded to the burden of resistance by meansof a distinct decrease in Darwinian fitness. In fact, the biologicalcost of resistance differed markedly among these mutants, rangingfrom little loss of fitness (mutant 17) to an immense loss offitness (mutant 7) (Table 1).

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FIG. 1. Amino acid substitutions in RpoB of rifampin-resistant S. aureus strains. The codon numbering is that of the S. aureus rpoB sequence (1). Mutations identified in vivo and in vitro are shown below and above the wild-type sequence, respectively.

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TABLE 1. Relative fitness of rifampin-resistant S. aureus in vitro mutants

Interestingly, no relationship was detected between the costof the rpoB mutation and the level of resistance (MIC) to rifampin(Fig. 2).

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FIG. 2. Correlation between rifampin MIC and fitness of S. aureus mutants.

Macrorestriction analysis by PFGE of chromosomal DNA of 23 rifampin-resistantin vivo isolates from six countries revealed 19 different genotypes(Fig. 3). Remarkably, only seven different rpoB genotypes couldbe demonstrated in vivo, whereby the 481His $->$ Asn mutation, whichprevailed in 91% of isolates, was not demonstrably associatedwith a cost of resistance in vitro (Fig. 1; Table 2). Whereaslow-level resistance in vivo is attributable solely to thissingle mutation at amino acid position 481, high-level resistancein vivo generally is traceable to multiple mutations, includingthe 481His $->$ Asn mutation, thereby indicating a step-by-step mechanismin resistance development.

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FIG. 3. SmaI macrorestriction patterns and corresponding dendrogram from representatives of the S. aureus PFGE genotypes (indicated by capital letters).

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TABLE 2. In vivo mutations conferring rifampin resistance on S. aureus isolates of various PFGE genotypes

DISCUSSION

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Discussion

From the perspective of the human population, a biological costis associated with antibiotic resistance insofar as, first,infections due to resistant pathogens constitute increasingcauses of morbidity and mortality worldwide and, second, thefinancial burden for the development of new and potent antibioticsmust be borne by society (3, 14). In addition, however, thereis the price associated with antibiotic resistance from theperspective of the bacterium itself, to the extent that resistancedeterminants may interfere with the normal physiological processin the cell and hence cause a reduction in the level of biologicalfitness (2, 16, 18).

In point of fact, several recent studies have shown that isolateswith resistance genotypes are less fit than their sensitivecounterparts in the absence of antibiotic selective pressure,thereby indicating a considerable cost of resistance (5, 7,9, 23). The biological cost of antibiotic resistance, however,can be considerably diminished and even compensated for by evolutionarychanges within the bacterial genome (6, 13, 15, 19, 20). Hence,bacteria often do pay a metabolic price, such as reduced growthrate, reduced invasiveness, or loss of virulence, for the acquisitionof drug resistance in the short term; but their adaptation tothe physiological cost is likely to foster the stable maintenanceof resistance in the long term.

Our study aimed to investigate the intrinsic detriments in biologicalfitness associated with RNA polymerase (rpoB) mutations thatconfer rifampin resistance in S. aureus. Three principal findingsemerged. First, the competition assays of in vitro-selectedRif^r mutants with their Rif^s isogenic counterpart revealed thatonly one rpoB genotype displayed no fitness burden, whereasthe other mutations were associated in some cases with a considerableloss of fitness. It must be kept in mind, however, that growthdefects in vitro do not always necessarily go along with growthdefects in vivo. Nonetheless, Moorman and Mandell (17) alsoshowed that rifampin-resistant strains of S. aureus displaygrowth defects in vitro and in some cases display reduced virulencein an animal model. Second, no relationship between the magnitudeof the biological cost and the level of resistance to rifampincould be detected. Third, the variation in frequency of rpoBmutations conferring resistance to rifampin in S. aureus invivo appears to be a function of the Darwinian fitness of theorganism. Indeed, the 481His $->$ Asn mutation, which was not associatedwith a loss of fitness in vitro, was shown to be prevalent in91% of the Rif^r clinical S. aureus isolates tested. Moreover,since the in vivo isolates that display the 481His $->$ Asn mutationwere obtained from six countries and revealed different geneticbackgrounds by macrorestriction analysis, it seems very unlikelythat the high prevalence of this "no-cost" mutation can be explainedby the clonal spread of a rifampin-resistant ancestral strain.As such, it seems likely that, in vivo, a functional restrictionon RNA polymerase and subsequent bacterial fitness limit theexpression of the full range of theoretical mutants definedin vitro. Interestingly, high-level resistance within clinicalisolates was mainly attributable to double mutations withinrpoB, including the mutational change 481His $->$ Asn. This in turnis indicative of a resistance-mediating function on the partof the additional mutation, since the 481His $->$ Asn mutational changeon its own confers only low-level resistance. Regarding thetwo isolates that exhibited three mutational changes withinrpoB in vivo, however, it still remains a matter of speculationas to whether both of the additional mutations, besides the481His $->$ Asn mutation, contribute to resistance.

These findings demonstrate that the ability of resistance determinantsto survive in bacteria is due not only to compensatory mutationsthat substantially diminish the biological cost of antibioticresistance, as shown by others (2, 6, 15), but also to the selectionamong resistance alleles favoring those that impose the lowest(if any) biological costs, as proposed here.

Another example of a no-cost resistance mutation has been elucidatedin vitro for the rpsL gene, which is responsible for resistanceto high concentrations of streptomycin in Salmonella entericasubsp. enterica serovar Typhimurium (5). Moreover, rpsL mutationsresponsible for streptomycin resistance in clinical isolatesof Mycobacterium tuberculosis were shown to be the same as thosewith no cost in experiments performed with Salmonella serovarTyphimurium (8).

Extrapolation of these findings makes it tempting to speculatethat resistance will never disappear completely because thereis no evolutionary disadvantage to being resistant once adaptationhas taken place, i.e., by acquisition of compensatory mutations,or when fitness has not been diminished, i.e., by selectionof no-cost mutations. Consequently, policies of decreased antibioticusage intended to reduce resistance might not be as successfulas originally anticipated. Finally, strict hygienic and antibiotictreatment regimens for prevention of the spread of resistancetogether with continued drug development are required if thehuman population wishes to stay one step ahead of antibioticresistance.

ACKNOWLEDGMENTS

We thank Denia Franck and Marita Parschau for technical assistance.

This work was supported by grants from the Marie Christine Heldand Erika Hecker Foundation.

FOOTNOTES

* Corresponding author. Mailing address: Institut für Medizinische Mikrobiologie, Klinikum der Johann Wolfgang Goethe-Universität, Paul-Ehrlich-Str. 40, 60596 Frankfurt am Main, Germany. Phone: 49 69 6301 6438. Fax: 49 69 6301 5767. E-mail: Wichelhaus{at}em.uni-frankfurt.de.

REFERENCES

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