Altered outer membrane protein profiles of Pseudomonas aeruginosa highly susceptible to the efflux pump inhibitor Phe-Arg-b-naphthylamide

Abstract number: 1733_379

Campo-Esquisabel A.B., Martí S., Sierra J.M., Martinez-Martinez L., Vila J.

Objective: Multidrug effllux systems play an important role in resistance of P. aeruginosa (Pae) to several antibiotics. Phe-Arg-b-naphthylamide (PAN) is usually able to inhibit these systems. When evaluating the relevance of efflux in some clinical isolates of Pae we noticed that some of them were inhibited by low concentration of PAN alone. We have evaluated OMP expression in Pae highly susceptible to PAN.

Methods: Three Pae clinical isolates (HUMV1, HUMV2 and HUMV3) for which MIC of FEP was at least 2 times higher than that of CAZ were selected among a collection of 20 isolates with a similar phenotype. MICs of PAN and of FEP in the absence and in the presence of 20 mg/l of PAN were determined by reference microdilution (CLSI guidelines). The study of clonal relation between the isolates was performed by pulsed-field gel electrophoresis (PFGE) digested with SpeI. Fractions of enriched cell envelope proteins from the three organisms grown in media without or with subMIC of PAN profiles were characterised by SDS-PAGE. Proteins were further analysed by MALDI-TOF mass spectometry after in-gel trypsin digestion.

Results: MICs (mg/l) of FEP for HUMV1, HUMV2 and HUMV3 were 64, 32 and 16 mg/l, respectively. MIC of FEP+PAN for HUMV3 was 4 mg/l, but HUMV1 and HUMV2 did not grow in the presence of 20 mg/l of PAN. In fact, MICs of PAN alone for HUMV1 and HUMV2 were 4 and 2 mg/l, respectively. The PFGE profile for HUMV1 and HUMV3 was identical, and their corresponding protein profiles differed in three proteins identified as: major outer membrane lipoprotein I (present in HUMV3, absent in HUMV1), 30S ribosomal protein S16 (reduced expression in HUMV1) and lisozyme (present in HUMV1, absent in HUMV3). Major outer membrane lipoprotein I was also absent in the PAN-susceptible HUMV2 isolate.

Conclusions: Hypersusceptibility of two P. aeruginosa strains to the efflux pump inhibitor Phe-Arg-b-naphthylamide was linked to loss of major outer membrane lipoprotein I.