Ribosome clearance by FusB-type proteins mediates resistance to the antibiotic fusidic acid.

Resistance to the antibiotic fusidic acid (FA) in the human pathogen Staphylococcus aureus usually results from expression of FusB-type proteins (FusB or FusC). These proteins bind to elongation factor G (EF-G), the target of FA, and rescue translation from FA-mediated inhibition by an unknown mechanism. Here we show that the FusB family are two-domain metalloproteins, the C-terminal domain of which contains a four-cysteine zinc finger with a unique structural fold. This domain mediates a high-affinity interaction with the C-terminal domains of EF-G. By binding to EF-G on the ribosome, FusB-type proteins promote the dissociation of stalled ribosome⋅EF-G⋅GDP complexes that form in the presence of FA, thereby allowing the ribosomes to resume translation. Ribosome clearance by these proteins represents a highly unusual antibiotic resistance mechanism, which appears to be fine-tuned by the relative abundance of FusB-type protein, ribosomes, and EF-G.

Delayed development of linezolid resistance in Staphylococcus aureus following exposure to low levels of antimicrobial agents.

The development of resistance to linezolid (LZD) in gram-positive bacteria depends on the mutation of a single 23S rRNA gene, followed by homologous recombination and gene conversion of the other alleles. We sought to inhibit this process in Staphylococcus aureus using a range of antibacterial agents, including some that suppress recombination. A model for the rapid selection of LZD resistance was developed which allowed the selection of LZD-resistant mutants with G2576T mutations in all five copies of the 23S rRNA gene following only 5 days of subculture. The emergence of LZD-resistant isolates was delayed by exposing cultures to low concentrations of various classes of antibiotics. All antibiotic classes were effective in delaying the selection of LZD-resistant mutants and, with the exception of fusidic acid (FUS) and rifampin (RIF), prolonged the selection window from 5 to approximately 15 days. Inhibitors of DNA processing were no more effective than any other class of antibiotics at suppressing resistance development. However, the unrelated antimicrobials FUS and RIF were particularly effective at preventing the emergence of LZD resistance, prolonging the selection window from 5 to 25 days. The enhanced suppressive effect of FUS and RIF on the development of LZD resistance was lost in a recA-deficient host, suggesting that these drugs affect recA-dependent recombination. Furthermore, FUS and RIF were shown to be effective inhibitors of homologous recombination of a plasmid into the staphylococcal chromosome. We suggest that RIF or FUS in combination with LZD may have a role in preventing the emergence of LZD resistance.

A fusidic acid-resistant epidemic strain of Staphylococcus aureus carries the fusB determinant, whereas fusA mutations are prevalent in other resistant isolates.

Fusidic acid-resistant epidemic Staphylococcus aureus strains causing impetigo bullosa have been reported in Scandinavia. We show that these strains form part of a European epidemic clonotype that carries the fusB determinant. In contrast, resistance to fusidic acid in a collection of nonepidemic strains resulted primarily from mutations in fusA.

The role of mutators in the emergence of antibiotic-resistant bacteria.

Bacteria contain a number of error prevention and error correction systems that maintain genome stability. However, strains exhibiting elevated mutation frequencies have recently been reported amongst natural populations of pathogenic Escherichia coli, Salmonella enterica, Pseudomonas aeruginosa, Neisseria meningitidis, Helicobacter pylori and Streptococcus pneumoniae. The majority of naturally occurring, strong mutators contain defects in the methyl-directed mismatch repair (MMR) system, with mutations in mutS predominating. MMR-deficient strains possess superior genetic backgrounds for the selection of some antibiotic-resistance mutations since mutation frequencies up to 1000-fold higher than normal strains have been reported, and resistance levels achieved in mutators can be greater than those arising in non-mutator hosts. MMR is a major constraint to interspecies recombination events. Removal of this barrier, as in the case of MMR defective mutators, also enhances the frequency of horizontal gene transfer, which is an important mechanism of acquired drug resistance in bacteria. Permanent global mutator status is associated with loss of fitness as mutators accumulate deleterious mutations more frequently than non-mutators. Fitness limitations of mutators may be overcome simply by the high bacterial cell densities that can be achieved during acute infection or by the adoption of transient mutator status. Mutators are a risk factor during the treatment of bacterial infections as they appear to enhance the selection of mutants expressing high- and low-level antibiotic resistance and have the capacity to refine existing plasmid-located resistance determinants.

Biological properties of novel antistaphylococcal quinoline-indole agents.

The antibacterial properties of novel quinoline-indole (QI) agents were examined. QI agents demonstrated potent bactericidal activities against Staphylococcus aureus, killing by lytic and nonlytic mechanisms. S. aureus mutants resistant to a lytic QI agent (SEP 155342) and a nonlytic QI agent (SEP 118843) arose at frequencies of 1.4 x 10(-9) and 1.2 x 10(-8), respectively, by selection at four times the MICs. Mutants resistant to QI agent SEP 155342 were unstable, but mutants resistant to QI agent SEP 118843 displayed stable resistance. Mutants resistant to QI agent SEP 118843 were not cross resistant to other inhibitors, including QI agent SEP 155342. Addition of QI agents SEP 118843 and SEP 155342 at four times the MIC caused nonspecific inhibition of several macromolecular biosynthetic pathways in S. aureus. Within 10 min, QI agents SEP 118843 and SEP 155342 both interfered with bacterial membrane integrity, as measured by uptake of propidium iodide. Agents from the two classes of the QI agents probably kill staphylococci by separate mechanisms which, nevertheless, both involve interference with cytoplasmic membrane function. Precise structure-activity relationships for the division of QI agents into two classes could not be determined. However, lytic activity was often associated with substitution of a basic amine at position 4 of the quinoline nucleus, whereas compounds with nonlytic activity usually contained an aromatic ring with or without a methoxy substituent at position 4. Nonlytic QI agents such as SEP 118843 may possess selective activity against the prokaryotic membrane since this compound failed to lyse mouse erythrocytes when it was added at a concentration equivalent to four times the MIC for S. aureus.

Insertional inactivation of mutS in Staphylococcus aureus reveals potential for elevated mutation frequencies, although the prevalence of mutators in clinical isolates is low.

Populations defective in mismatch repair that exhibit elevated mutation frequencies to antibiotic resistance have been reported amongst pathogenic Gram-negative bacteria. Whether such mutators occur widely in clinical isolates of Gram-positive species, and in important pathogens such as Staphylococcus aureus, is unknown. Insertional inactivation of the mutS gene of S. aureus RN4220 by targeted plasmid integration produced a strain with mutation frequencies for antibiotic resistance up to 78-fold greater than those exhibited by RN4220, thereby providing proof of the concept that staphylococcal mutators could arise. Subsequently, 493 clinical S. aureus isolates were examined for the presence of mutators. However, no strain exhibited a > or =10-fold increase in mutation frequency compared with laboratory strain 8325-4. Detailed phenotypic and genotypic analysis of vancomycin-intermediate S. aureus strain Mu50 was performed, since the published genome sequence of this organism suggests that mutS is inactive as a result of a frameshift. However, elevated mutation frequencies were not observed in Mu50, and re-sequencing of a portion of mutS from this strain indicated that this gene was intact. Transient increases in mutation frequency during the stationary phase of growth occur in other bacteria, although no such increases were observed in S. aureus. We conclude that neither permanent increases in the basal mutation frequency, nor transient increases in mutation frequency under starvation, are likely to play a significant role in the development of antibiotic resistance in S. aureus.