Whole genome sequencing of S. pneumoniae ciprofloxacin-resistant strains generated in vitro and from the field

The second-generation fluoroquinolone ciprofloxacin is a bactericidal antibiotic targeting DNA topoisomerase IV and DNA gyrase. Resistance to ciprofloxacin in Streptococcus pneumoniae mainly occurs through the acquisition of mutations in the quinolone-resistance-determining-region of the ParC and GyrA targets. A role in low-level ciprofloxacin resistance has also been attributed to efflux mediated by the overexpression of the major facilitator PmrA and the heterodimeric ABC transporter PatA/PatB. To look into ciprofloxacin resistance at a genome wide scale and to discover additional mutations implicated in resistance, we performed whole genome sequencing of a S. pneumoniae isolates selected for resistance to ciprofloxacin in vitro and of a clinical isolate displaying low-level ciprofloxacin resistance. Gene disruption and DNA transformation experiments with PCR fragments harboring the mutations identified in the genome of the in vitro S. pneumoniae R6M2B mutant revealed that the bulk its resistance was due to QRDRs mutations in parC and gyrA and to the overexpression of the ABC transporters PatA and PatB. In contrast, no QRDR mutations were identified in the genome of the clinical S. pneumoniae isolate 60827 and ciprofloxacin susceptibility assays performed in the presence of the efflux pump inhibitor reserpine revealed that its resistance originated solely from efflux. Interestingly, the genome sequence of S. pneumoniae 60827 revealed mutations in the coding region of patA and patB that we implicated in resistance. Finally, a mutation in the NAD(P)H-dependent glycerol-3-phosphate dehydrogenase identified in S. pneumoniae 60827 is shown to protect against reactive oxygen species which are the main death effector of bactericidal antibiotics like fluoroquinolones.