Exposure of Pseudomonas aeruginosa to Cinnamaldehyde Selects Multidrug Resistant Mutants

Abstract

Cinnamaldehyde (CNA), the main component of cinnamon essential oil, is one of the most active plant compounds against nosocomial pathogen Pseudomonas aeruginosa. Exposure of wild-type strain PA14 (MIC 700 µg/mL) for 5 to 10 days to fixed (900 µg/mL) or increasing (from 900 to 1400 µg/mL) concentrations of this natural antibacterial resulted in emergence of resistant mutants CNA-A1 to A3, and CNA-B1 to B7, respectively. Genome sequencing experiments showed that each of CNA-A1 to A3 mutants differed from PA14 by one SNP, and a slight increase in CNA resistance level (from 700 to 900 µg/mL). By comparison, mutants B1 to B7 were more resistant (up to 1100 µg/mL); each of them harbored multiple SNPs (from 24 to 39) likely as a consequence of alteration of DNA mismatch repair gene mutS. Of the ten mutants selected, eight contained mutations in gene nalC, which indirectly downregulates expression of the operon that codes for multidrug efflux system MexAB-OprM, and showed increased resistance (up to 16-fold versus PA14) to antibiotic molecules exported by the pump, including ß-lactams and fluoroquinolones. Of the six mutants with the highest CNA resistance, five were no longer motile because of alteration of genes flgJ, fliE and/or pilJ genes. Altogether, our data show that P. aeruginosa is able to adapt to strong electrophilic molecules such as CNA by upregulating its intrinsic efflux pump MexAB-OprM, and through less well-characterized pleiotropic changes. Whether multidrug-resistant mutants can emerge in patients using cinnamon essential oil as self-medication needs to be assessed further.

Keywords: Pseudomonas aeruginosa; antibiotic resistance; cinnamaldehyde; efflux; essential oils.

Figure 1

Impact of genes mexAB deletion on resistance to CNA. Genes mexAB were deleted in PA14 (CNA MIC before deletion equal to 700 µg/mL), and CNA-resistant nalC mutants CNA-B1, B3, and B5 (1000 µg/mL), and CNA-B4, B6, B7 (1100 µg/mL). The inactivated mutants (∆AB) and their progenitors were spotted onto the surface of an MHA plate containing 500 µg/mL CNA.

Figure 2

Motility and initial surface attachment of CNA-resistant mutants. (A) Swarming motility: the mutations found in genes responsible for flagellar structure/assembly, pili formation, and biosynthesis of biofilm matrix are indicated. (B) Absorbance at 600 nm is representative of sessile bacteria stained by crystal-violet in microplates (mean values resulting from 10 replicates; Student’s t-test, *** p-value < 0.001).

Figure 3

Increased resistance of PA14∆fliE to CNA. Serial Log₁₀ dilutions of strain PA14 and its derivative mutants PA14∆fliE and PA14∆nalC all calibrated at 0.5 McFarland were spotted onto the surface of an MHA plate containing 600 µg/mL CNA.