Abstract
Streptococcus pneumoniae is a leading cause of respiratory tract infections, meningitis, and acute otitis media. The emergence of S. pneumoniae strains with resistance to the ß-lactam and macrolide antibiotics has complicated the treatment of pneumococcal infections and created a need for new agents. Recently developed compounds within the fluoroquinolone group of antimicrobials have shown potential as anti-pneumococcal antibiotics. Although fluoroquinolone resistance is rare in most areas (i.e. <2%), it is feared that increased use of these agents could accelerate the development of resistance. Fluoroquinolone resistance in S. pneumoniae is mediated by a combination of at least two mechanisms: (1) amino acid substitutions within the quinolone resistance determining regions (QRDRs) of DNA gyrase and/or DNA topoisomerase IV, and (2) drug efflux via unknown transporter proteins. Although previous studies have examined the selection of these resistance mechanisms to varying degrees, a more rigorous and extensive investigation was warranted. The current research aimed to compare the ability of the fluoroquinolones ciprofloxacin and moxifloxacin to select resistance over three mutational steps, correlate changes in fluoroquinolone susceptibility with mutations in the QRDRs, examine the role of efflux using phenotypic tests, and investigate the expression of several putative efflux pumps.|The newer fluoroquinolone moxifloxacin was more active in vitro against S. pneumoniae than ciprofloxacin and selected fewer resistant mutants. The potency of moxifloxacin against first-step mutants of S. pneumoniae is a property that may help Molecular Characterization of Fluoroquinolone Resistance in Streptococcus pneumoniae prevent the emergence of resistance. The minimum inhibitory concentration (MIC) of moxifloxacin was < 1 pg/mL in 13 of 14 moxifloxacin-selected first-step mutants. Therefore, the MIC of moxifloxacin for the majority of selected mutants remained within the susceptible range (susceptible breakpoint, MIC < 1 pg/mL), suggesting that both the parental strain and a potential first-step mutant would be inhibited with moxifloxacin treatment.|Ciprofloxacin and moxifloxacin were found to exert very different selection pressures that yielded different types of mutants when fluoroquinolone resistance was selected. Ciprofloxacin selected first-step mutants harboring amino acid alterations in DNA topoisomerase IV. In contrast, moxifloxacin selected first-step mutants with amino acid changes in DNA gyrase, suggesting that these two fluoroquinolones have different primary targets. The dissimilarity in the mutants selected by ciprofloxacin and moxifloxacin became more pronounced during the third selectional step. While most moxifloxacin third-step mutants developed an additional QRDR mutation, the majority of ciprofloxacin-selected mutants did not develop additional QRDR changes. This suggests that exposure to high concentrations of ciprofloxacin selected an alternative mechanism, such as the increased production of drug efflux pumps. This hypothesis was supported by the results of phenotypic tests using the efflux substrate, ethidiuin bromide. After three exposures to ciprofloxacin, 86% of the resulting mutants showed at least a 4-fold increase in ethidium bromide MIC, indicating the possible role of efflux in the reduced susceptibility to ciprofloxacin that could not be correlated with QRDR mutations. On the other hand, none of the mutants selected with moxifloxacin exhibited reduced susceptibility to ethidium bromide, suggesting that moxifloxacin had a reduced potential for selecting mutant phenotypes suggestive of enhanced efflux.|Based upon the increases in ethidium bromide MICs for the majority of ciprofloxacin- selected mutants, it was hypothesized that these mutants were overexpressing one or more drug efflux pumps. The expression of 12 putative drug efflux genes was investigated in suspected efflux mutants using Real Time RT-PCR. The expression of two novel efflux genes, patA and patB, was significantly increased in mutants selected with ciprofloxacin, suggesting that the increased expression of these genes could contribute to fluoroquinolone resistance. In addition, the increased expression of putative ABC-transporter gene, spr 1183, and the decreased expression of the putative permease gene, spr 1734, was observed in an efflux mutant, suggesting that several mechanisms other than QRDR mutations are involved in mediating fluoroquinolone resistance in S. pneumoniae.|RT-PCR was used to demonstrate that pat A and patB are co-transcribed on a single mRNA transcript, suggesting that the products of patA and patB form a single heterodimeric efflux pump and are coordinately regulated, perhaps from a single promoter upstream of patA. Because promoter mutations are often involved in increasing the efficiency of gene transcription, it was hypothesized that mutations existed within the promoter region of patA that contributed to its overexpression. DNA sequence analysis revealed that a point mutation was present within the 5' untranslated region (5’ UTR) of patA in a ciprofloxacin-selected efflux mutant. To determine if the mutation modified the secondary structure of the 5’ UTR in the efflux mutant, the RNA secondary structure of patA was analyzed using an RNA-folding program, but no differences were observed. Because overexpression of this pump was selected after ciprofloxacin treatment and therefore may play a role in fluoroquinolone resistance, further experiments are warranted to elucidate the mechanism of overexpression.|In conclusion, with fewer drugs available for treating S. pneumoniae infections, it is imperative to identify novel therapeutic targets and design strategies for inhibiting resistance mechanisms. Uncovering the molecular basis of fluoroquinolone resistance in S. pneumoniae is an important step that may ultimately lead to the development of resistance mechanism inhibitors and agents with improved potency. However, until these drugs are available, it is critical that clinicians use the fluoroquinolones that will minimize the selection of resistance, thereby preserving the utility of these antimicrobials against S. pneumoniae.