Enzymatic quorum quenching increases antibiotic susceptibility of multidrug resistant Pseudomonas aeruginosa

Abstract

Background and objectives: There is increasing emergence of multidrug resistant Pseudomonas aeruginosa (MDRPA) strains and drug resistance is positively-correlated with biofilm-forming ability. Since about 10% of P. aeruginosa genome is controlled by quorum sensing (QS), alteration in its antibiotic susceptibility by targeting QS was the focus of the present study.

Materials and methods: One day biofilms of PAO1 and three urinary tract infection MDRPA isolates (PA2, PA8 and PA18) were formed in 96-well microtiter plate. Biofilms were exposed to concentration gradient of ciprofloxacin and gentamicin to obtain Minimum Biofilm Eradication Concentration (MBEC) by direct enumeration method. Susceptibility of 24 h biofilms was evaluated by treatment with ciprofloxacin and gentamicin per se and in combination with lactonase. The effect was also examined on 72 h biofilms by Scanning Electron Microscopy.

Results: Lactonase treatment did not have any effect on growth of the selected strains but 73.42, 69.1, 77.34 and 72.5% reduction of biofilm was observed after lactonase (1 unit) treatment, respectively. Antibiotics in combination with lactonase (0.3 units) resulted in an increased susceptibility of the biofilm forms by>3.3, 4, 5 and 1.5 folds of MBEC, for ciprofloxacin and>6.67, 12.5, 6 and>2.5 folds, for gentamicin respectively, which could be due to the disruption of biofilm by lactonase treatment as shown by scanning electron microscopy. Also there was significant reduction (p<0.001) in virulence factor production by the strains.

Conclusion: Lactonase treatment increased antibiotic susceptibility of the biofilms of MDRPA isolates underscoring the potential of quorum quenching in antimicrobial therapeutics.

Keywords: AHL Lactonase; Antibiotic Susceptibility; Biofilm; Multidrug resistance; Pseudomonas aeruginosa; antimicrobial therapeutics.

Fig. 1

Specific biofilm forming index of P. aeruginosa PAO1 strain and clinical isolates±standard deviations.

Fig. 2A

Percent antibiotic resistance by different P. aeruginosa strains: 1) PAO1, 2) PA2, 3) PA8, 4) PA11, 5) PA18, 6) G1, 7) G9, 8) G10, 9) G13, 10) 2663, 11) 3633, 12) 3751, 13) 3849, 14) 3878, 15) 3882, 16) 4250, 17) 4287, 18) 4299, 19) 4303.fig. 2B. Percent strain resistance to different antibiotics.

Fig. 3

Effect of lactonase on lactones producted by P. aeruginosa PAO1 strain and three UTI clinical isolates.

Figs. 4a-d

Effect of lactonase treatment on antibiotic resistance of P. aeruginosa strains a) PAO1 wild-type b) PA2 c) PA8 d) PA18.

Fig. 5 (a-f)

SEM of biofilm formed by PA2 isolate of P. aeruginosa on glass surface using Scanning Electron Microscope (Model JSM6100-JEOL). Fig. a) 24 h biofilm; b, c) 72 h biofilm; d) gentamicin (100 µg/ml) treated 72 h biofilm; e) lactonase (0.3 units) treated 72 h biofilm; f) gentamicin (100 µg/ml) and lactonase (0.3 units) treated 72 h biofilm

Fig. 6

Effect of lactonase on virulence factors of P. aeruginosa PAO1 strain and three selected UTI clinical isolates (PA2, PA8 and PA18.)