Upregulation of the PatAB Transporter Confers Fluoroquinolone Resistance to Streptococcus pseudopneumoniae

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.

Role of the Two-Component System CiaRH in the Regulation of Efflux Pump SatAB and Its Correlation with Fluoroquinolone Susceptibility

“…The efflux pump SatAB, which is equivalent to the PatAB efflux pump of Streptococcus pneumoniae , is a member of the ABC transporters, which comprise nucleotide-binding domains (NBDs) and transmembrane domains (TMDs) ( 44 , 45 ). SatAB has been previously shown to be associated with multidrug resistance, and its overexpression is a reason for S. suis FQ resistance, similar to findings for S. pneumoniae and Streptococcus pseudopneumoniae ( 20 , 24 , 46 ). In support of this role, the deletion of satAB produced an effect on the MIC value of 1 dilution (2-fold more susceptible for norfloxacin and ciprofloxacin) compared to the MIC of the WT strain, indicating that there was a difference in the sensitivity between the WT and satAB mutant strains ( Table 1 ).…”

Genomic Characterization of the Emerging Pathogen Streptococcus pseudopneumoniae

“…S4). ICE, Mega-2, and other types of resistance refer to genotypic resistance; penicillin (Pen) and co-trimoxazole (SXT) refer to phenotypic resistance (Table 3 and references 5, 16, and 58–60). NP, nasopharynx; ND, pseudogenes/truncated; NA, data not available.…”

Comparative genomic analysis of the emerging pathogen Streptococcus pseudopneumoniae: novel insights into virulence determinants and identification of a novel species-specific molecular marker

“…Roman numerals in column "ICE" refers to integration sites (Table S8). ICE, Mega-2 and "other resistances" refer to genotypic resistances; penicillin (Pen) and co-trimoxazole (SXT) refer to phenotypic resistances (Table S7) and references (5, 20, 57-59). ND, not determined due to the presence of pseudogenes/contig breaks; NA, data not available.…”