From the perspective of the human population, a biological cost is associated with antibiotic resistance insofar as, first, infections due to resistant pathogens constitute increasing causes of morbidity and mortality worldwide and, second, the financial burden for the development of new and potent antibiotics must be borne by society (3
). In addition, however, there is the price associated with antibiotic resistance from the perspective of the bacterium itself, to the extent that resistance determinants may interfere with the normal physiological process in the cell and hence cause a reduction in the level of biological fitness (2
In point of fact, several recent studies have shown that isolates with resistance genotypes are less fit than their sensitive counterparts in the absence of antibiotic selective pressure, thereby indicating a considerable cost of resistance (5
). The biological cost of antibiotic resistance, however, can be considerably diminished and even compensated for by evolutionary changes within the bacterial genome (6
). Hence, bacteria often do pay a metabolic price, such as reduced growth rate, reduced invasiveness, or loss of virulence, for the acquisition of drug resistance in the short term; but their adaptation to the physiological cost is likely to foster the stable maintenance of resistance in the long term.
Our study aimed to investigate the intrinsic detriments in biological fitness associated with RNA polymerase (rpoB
) mutations that confer rifampin resistance in S. aureus
. Three principal findings emerged. First, the competition assays of in vitro-selected Rifr
mutants with their Rifs
isogenic counterpart revealed that only one rpoB
genotype displayed no fitness burden, whereas the other mutations were associated in some cases with a considerable loss of fitness. It must be kept in mind, however, that growth defects in vitro do not always necessarily go along with growth defects in vivo. Nonetheless, Moorman and Mandell (17
) also showed that rifampin-resistant strains of S. aureus
display growth defects in vitro and in some cases display reduced virulence in an animal model. Second, no relationship between the magnitude of the biological cost and the level of resistance to rifampin could be detected. Third, the variation in frequency of rpoB
mutations conferring resistance to rifampin in S. aureus
in vivo appears to be a function of the Darwinian fitness of the organism. Indeed, the 481His→Asn mutation, which was not associated with a loss of fitness in vitro, was shown to be prevalent in 91% of the Rifr
clinical S. aureus
isolates tested. Moreover, since the in vivo isolates that display the 481His→Asn mutation were obtained from six countries and revealed different genetic backgrounds by macrorestriction analysis, it seems very unlikely that the high prevalence of this “no-cost” mutation can be explained by the clonal spread of a rifampin-resistant ancestral strain. As such, it seems likely that, in vivo, a functional restriction on RNA polymerase and subsequent bacterial fitness limit the expression of the full range of theoretical mutants defined in vitro. Interestingly, high-level resistance within clinical isolates was mainly attributable to double mutations within rpoB
, including the mutational change 481His→Asn. This in turn is indicative of a resistance-mediating function on the part of the additional mutation, since the 481His→Asn mutational change on its own confers only low-level resistance. Regarding the two isolates that exhibited three mutational changes within rpoB
in vivo, however, it still remains a matter of speculation as to whether both of the additional mutations, besides the 481His→Asn mutation, contribute to resistance.
These findings demonstrate that the ability of resistance determinants to survive in bacteria is due not only to compensatory mutations that substantially diminish the biological cost of antibiotic resistance, as shown by others (2
), but also to the selection among 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 elucidated in vitro for the rpsL
gene, which is responsible for resistance to high concentrations of streptomycin in Salmonella enterica
serovar Typhimurium (5
). Moreover, rpsL
mutations responsible for streptomycin resistance in clinical isolates of Mycobacterium tuberculosis
were shown to be the same as those with no cost in experiments performed with Salmonella
serovar Typhimurium (8
Extrapolation of these findings makes it tempting to speculate that resistance will never disappear completely because there is no evolutionary disadvantage to being resistant once adaptation has taken place, i.e., by acquisition of compensatory mutations, or when fitness has not been diminished, i.e., by selection of no-cost mutations. Consequently, policies of decreased antibiotic usage intended to reduce resistance might not be as successful as originally anticipated. Finally, strict hygienic and antibiotic treatment regimens for prevention of the spread of resistance together with continued drug development are required if the human population wishes to stay one step ahead of antibiotic resistance.