Abstract
We characterized the mechanism of fluoroquinolone-resistance in two isolates of Streptococcus pseudopneumoniae having fluoroquinolone-efflux as unique mechanism of resistance. Whole genome sequencing and genetic transformation experiments were performed together with phenotypic determinations of the efflux mechanism. The PatAB pump was identified as responsible for efflux of ciprofloxacin (MIC of 4 μg/ml), ethidium bromide (MICs of 8–16 μg/ml) and acriflavine (MICs of 4–8 μg/ml) in both isolates. These MICs were at least 8-fold lower in the presence of the efflux inhibitor reserpine. Complete genome sequencing indicated that the sequence located between the promoter of the patAB operon and the initiation codon of patA, which putatively forms an RNA stem-loop structure, may be responsible for the efflux phenotype. RT-qPCR determinations performed on RNAs of cultures treated or not treated with subinhibitory ciprofloxacin concentrations were performed. While no significant changes were observed in wild-type Streptococcus pneumoniae R6 strain, increases in transcription were detected in the ciprofloxacin-efflux transformants obtained with DNA from efflux-positive isolates, in the ranges of 1.4 to 3.4-fold (patA) and 2.1 to 2.9-fold (patB). Ciprofloxacin-induction was related with a lower predicted free energy for the stem-loop structure in the RNA of S. pseudopneumoniae isolates (−13.81 and −8.58) than for R6 (−15.32 kcal/mol), which may ease transcription. The presence of these regulatory variations in commensal S. pseudopneumoniae isolates, and the possibility of its transfer to Streptococcus pneumoniae by genetic transformation, could increase fluoroquinolone resistance in this important pathogen.
Highlights
Streptococcus pneumoniae is a main human pathogen causing community-acquired pneumonia, meningitis, bacteremia, and otitis media (Obaro, 2000)
The MICs of isolates 5305 and CipR71 to FQs and some dyes in the presence and absence of the efflux pump inhibitor reserpine were compared to three efflux-negative control strains: S. pneumoniae R6, S. pseudopneumoniae CCUG48465, and the S. pseudopneumoniae type strain CCUG49455T
The two S. pseudopneumoniae clinical isolates, which showed 8- to 32-fold higher MICs of these drugs in the absence of reserpine than the control strains, essentially behave like the control strains in the presence of the pump inhibitor (Table 3)
Summary
Streptococcus pneumoniae (the pneumococcus) is a main human pathogen causing community-acquired pneumonia, meningitis, bacteremia, and otitis media (Obaro, 2000). FQ-resistance in this bacterium occurs mainly by the alteration of their DNA topoisomerases This can occur either by point mutation or by intraspecific (Stanhope et al, 2005), or interspecific recombination with the genetically related streptococci of the mitis group, commensal microorganisms of the oral cavity (Ferrándiz et al, 2000; Balsalobre et al, 2003; Stanhope et al, 2005). Compensation of this cost in isolates carrying recombinant topoisomerase genes (Balsalobre et al, 2011) would envisage a future spread of these resistant isolates These recombinant isolates are originated by genetic interchange with chromosomal DNA of other streptococci of the mitis group. Recombination and the spread of a few international clones are central factors generating antibiotic resistance in S. pneumoniae
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