Small-colony variants (SCVs) of Staphylococcus aureus can be isolated from the chronically infected airways of patients suffering from cystic fibrosis (CF). These slow-growing morphological variants have been associated with persistent and antibiotic-resistant infections, such as osteomyelitis and device-related infections, but no information is available to date regarding the clinical significance of this special phenotype in CF lung disease. We therefore investigated the prevalence of S. aureus SCVs in CF lung disease in a 12-month prospective study and correlated the microbiological culture results with the patients' clinical data. A total of 252 patients were screened for the presence of SCVs. The prevalence rate was determined to be 17% (95% confidence interval, 10 to 25%) among S. aureus carriers. S. aureus isolates with the SCV phenotype showed significantly higher antibiotic resistance rates than those with the normal phenotype. Patients positive for SCVs were significantly older (P ‫؍‬ 0.0099), more commonly cocolonized with Pseudomonas aeruginosa (P ‫؍‬ 0.0454), and showed signs of more advanced disease, such as lower forced expiratory volume in 1 s (P ‫؍‬ 0.0148) than patients harboring S. aureus with a solely normal phenotype. The logistic regression model determined lower weight (P ‫؍‬ 0.016), advanced age (P ‫؍‬ 0.000), and prior use of trimethoprim-sulfamethoxazole (P ‫؍‬ 0.002) as independent risk factors for S. aureus SCV positivity. The clinical status of CF patients is known to be affected by multiple parameters. Nonetheless, the independent risk factors determined here point to the impact of S. aureus SCVs on chronic and persistent infections in advanced CF lung disease.

Resistance determinants that interfere with normal physiological processes in the bacterial cell usually cause a reduction in biological fitness. Fitness assays revealed that 17 of 18 in vitro-selected chromosomal mutations within the rpoB gene accounting for rifampin resistance in Staphylococcus aureus were associated with a reduction in the level of fitness. There was no obvious correlation between the level of resistance to rifampin and the level of fitness loss caused by rpoB mutations. Among 23 clinical rifampin-resistant S. aureus isolates from six countries, only seven different rpoB genotypes could be identified, whereby the mutation 481His3Asn was present in 21 (91%) of these 23 isolates. The mutation 481His3Asn, in turn, which confers low-level rifampin resistance on its own, was not shown to be associated with a cost of resistance in vitro. The restriction to distinct mutations that confer rifampin resistance in vivo, as demonstrated here, appears to be determined by the Darwinian fitness of the organisms.The selective pressure of antibiotics in general results in the emergence of resistance mechanisms on the part of bacteria (12). Hence, a direct correlation often exists between antibiotic use and the development of bacterial resistance to antibiotics over time (3). Conversely, the incidence of antibiotic resistance can be reduced by the restriction of antibiotic usage (22). This interrelation between antibiotic usage and resistance development is partly determined by the so-called biological cost of antibiotic resistance (11, 16). Since antibiotic resistance often is characterized by detriments in biological fitness, resistant bacteria can be 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 easily be overcome by the acquisition of compensatory mutations, thereby stabilizing the resistant bacteria within a given population (8,16,21).Antibiotic resistance due to chromosomal mutations that cause structural modifications in the cellular target of the drug, such as rifampin resistance in Staphylococcus aureus, can be associated with a fitness burden. The present study was aimed at 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 the level of resistance to rifampin, and (iii) the distribution of rpoB mutations in vivo in view of the possible fitness burden associated with resistance. MATERIALS AND METHODSBacteria. Fifty-four S. aureus isolates were analyzed, including 23 isolates from six countries that were rifampin resistant (Rif r ) in vivo as well as 30 mutants derived from a single susceptible isogenic reference strain that were Rif r in vitro. Twenty-six of the Rif r in vitro mutants were selected by culturing a rifampinsusceptible (Rif s ) strain on agar containing 0.25 g of ...

Linezolid resistance in Staphylococcus aureus is typically associated with mutations in the 23S rRNA gene. Here we show that the accumulation of a single point mutation, G2576T, in the different copies of this gene causes stepwise increases in resistance, impairment of the biological fitness, and cross-resistance to quinupristin-dalfopristin and chloramphenicol.

SummaryFusidic acid is a potent antibiotic against severe Gram-positive infections that interferes with the function of elongation factor G (EF-G), thereby leading to the inhibition of bacterial protein synthesis. In this study, we demonstrate that fusidic acid resistance in Staphylococcus aureus results from point mutations within the chromosomal fusA gene encoding EF-G. Sequence analysis of fusA revealed mutational changes that cause amino acid substitutions in 10 fusidic acid-resistant clinical S. aureus strains as well as in 10 fusidic acid-resistant S. aureus mutants isolated under fusidic acid selective pressure in vitro . Fourteen different amino acid exchanges were identified that were restricted to 13 amino acid residues within EF-G. To confirm the importance of observed amino acid exchanges in EF-G for the generation of fusidic acid resistance in S. aureus , three mutant fusA alleles encoding EF-G derivatives with the exchanges P406L, H457Y and L461K were constructed by sitedirected mutagenesis. In each case, introduction of the mutant fusA alleles on plasmids into the fusidic acid-susceptible S. aureus strain RN4220 caused a fusidic acid-resistant phenotype. The elevated minimal inhibitory concentrations of fusidic acid determined for the recombinant bacteria were analogous to those observed for the fusidic acid-resistant clinical S. aureus isolates and the in vitro mutants containing the same chromosomal mutations. Thus, the data presented provide evidence for the crucial importance of individual amino acid exchanges within EF-G for the generation of fusidic acid resistance in S. aureus .

No abstract