Functional Analysis of Pneumococcal Drug Efflux Pumps Associates the MATE DinF Transporter with Quinolone Susceptibility

The pneumococcal chromosome encodes about 140 transporters, many of which are predicted to be involved in efflux. In order to critically evaluate pneumococcal efflux, a series of transporter mutants were constructed, and their phenotypes were assayed by disk diffusion, microdilution drug susceptibility testing (MIC testing), growth of cultures at sub-MIC concentrations, and phenotype microarray analysis. Mutants with mutations in seven ATP binding cassette (ABC) transporters, three multiantimi-crobial extrusion (MATE) family efflux pumps, and one major facilitator superfamily (MFS) transporter were obtained in Strep-tococcus pneumoniae strain DP1004. The susceptibility of these 11 mutants to over 250 different substances was compared to that of the parent strain. Of the tested transporters, only the ABC transporter PatAB (SP2073-5) presented a clear multidrug re-sistance (MDR) profile, as the mutant showed significantly increased susceptibility to ethidium bromide, acriflavine, and ber-berine. Among the other transporters analyzed, the mutants devoid of the MATE efflux pump SP2065 exhibited reduced suscep-tibility to novobiocin, and those with mutations of the MATE family DinF transport system (SP1939) exhibited increased susceptibility to moxifloxacin, ciprofloxacin, and levofloxacin. This change in quinoloneMIC was found to be independent from the competence-mediated effect of quinolones on the cinA-recA-dinF operon. Furthermore, the dinFmutant, in contrast to the parental strain, allowed selection for quinolone-resistant mutants when exposed to moxifloxacin. These data confirm the clear MDR profile of the PatAB ABC transporter and suggest for the MATEDinF a phenotype associated with quinolone susceptibil-ity, particularly for moxifloxacin