Compensatory Evolution of pbp Mutations Restores the Fitness Cost Imposed by β-Lactam Resistance in Streptococcus pneumoniae

Abstract

The prevalence of antibiotic resistance genes in pathogenic bacteria is a major challenge to treating many infectious diseases. The spread of these genes is driven by the strong selection imposed by the use of antibacterial drugs. However, in the absence of drug selection, antibiotic resistance genes impose a fitness cost, which can be ameliorated by compensatory mutations. In Streptococcus pneumoniae, β-lactam resistance is caused by mutations in three penicillin-binding proteins, PBP1a, PBP2x, and PBP2b, all of which are implicated in cell wall synthesis and the cell division cycle. We found that the fitness cost and cell division defects conferred by pbp2b mutations (as determined by fitness competitive assays in vitro and in vivo and fluorescence microscopy) were fully compensated by the acquisition of pbp2x and pbp1a mutations, apparently by means of an increased stability and a consequent mislocalization of these protein mutants. Thus, these compensatory combinations of pbp mutant alleles resulted in an increase in the level and spectrum of β-lactam resistance. This report describes a direct correlation between antibiotic resistance increase and fitness cost compensation, both caused by the same gene mutations acquired by horizontal transfer. The clinical origin of the pbp mutations suggests that this intergenic compensatory process is involved in the persistence of β-lactam resistance among circulating strains. We propose that this compensatory mechanism is relevant for β-lactam resistance evolution in Streptococcus pneumoniae. For many years, pneumococcal infections have been usually treated with β-lactams. However, the rapid emergence of β-lactam resistance has complicated the antimicrobial treatment of these infections in the last two decades. The emergence and stability of antibiotic resistance is a complex biological process driven by different factors, such as the volume of antibiotic used. Furthermore, many studies on the effect of a reduction in β-lactam consumption have reported a sustained resistance level to S. pneumoniae, suggesting that other factors contribute to the persistence of β-lactam resistance. By horizontal gene transfer, S. pneumoniae is able to acquire genes from resistant strains or the commensal streptococci, which confer β-lactam resistance. Here, we show that when certain resistance genes are acquired individually, an important cost results in the bacterial fitness. However, some clinical strains which have acquired genes that increase β-lactam resistance can also compensate the fitness cost imposed by this resistance, thereby producing a selective advantage and raising the potential spreading of β-lactam resistance. We suggest that pbp1a and pbp2x mutant alleles are acquired for their compensatory effect on fitness in addition to their contribution in developing higher β-lactam resistance levels, and that this process may occur even in the absence of antibiotics.