Synergistic Antibiofilm Efficacy of Thymol and Piperine in Combination with Three Aminoglycoside Antibiotics against Klebsiella pneumoniae Biofilms

Abstract

Background. Thymol and piperine are two naturally occurring bioactive compounds with several pharmacological activities. In this study, their antibiofilm potential either alone or in combination with three aminoglycoside antibiotics was evaluated against a biofilm of Klebsiella pneumoniae. Methods. Determination of antimicrobial susceptibility was performed using the broth microdilution method. Biofilm formation was evaluated by the microtiter plate method. Antibiofilm activity was determined using 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium-bromide (MTT) assay. The combination studies were performed by the checkerboard microdilution method. Results. The minimum biofilm inhibitory concentration (MBIC) of streptomycin was reduced by 16- to 64-fold when used in combination with thymol, while the MBIC of kanamycin was reduced by 4-fold when combined with piperine. The minimum biofilm eradication concentration (MBEC) values of streptomycin, amikacin, and kanamycin were, respectively, 16- to 128-fold, 4- to 128-fold, and 8- to 256-fold higher than the planktonic minimum inhibitory concentration (MIC). Thymol combined with streptomycin or kanamycin showed synergic effects against the preformed biofilm with 16- to 64-fold reduction in the minimum biofilm eradication concentration values of each antibiotic in combination. Piperine acted also synergically with kanamycin with an 8- to 16-fold reduction in the minimum biofilm eradication concentration values of kanamycin in combination. Conclusion. The association of thymol with antibiotics showed a strong synergistic effect both in the inhibition of biofilm formation and the destruction of the preformed biofilm of K. pneumoniae. This study suggests that a combination of thymol with streptomycin, amikacin, or kanamycin could be a promising alternative therapy to overcome the problem of K. pneumoniae biofilm-associated infections.

Antibacterial and Anti-Biofilm Activities of Essential Oil Compounds against New Delhi Metallo-β-Lactamase-1-Producing Uropathogenic Klebsiella pneumoniae Strains

“…Our results received for thymol are similar to those obtained by other authors. For example, Bisso Ndezo et al [ 30 ] analysed the effect of thymol against four strains of K. pneumoniae isolated from urine. The authors evaluated the MIC and MBC values of thymol against bacteria, which ranged from 0.064–0.256 mg/mL and 0.256–0.512 mg/mL, respectively.…”

Antibiofilm Synergistic Activity of Streptomycin in Combination with Thymol-Loaded Poly (Lactic-co-glycolic Acid) Nanoparticles against Klebsiella pneumoniae Isolates

“…Based on the kinetic metabolic activity of biofilm formation in a previous study [ 10 ], the biofilm biomass of K. pneumoniae isolates was determined with a colorimetric method based on safranin dye [ 21 ]. Briefly, 100 µ L of bacterial inoculum (1.5 × 10 6 CFU/mL) and 100 µ L of MHB supplemented with 2% glucose were added to a 96-well flat-bottomed polystyrene plate.…”

“…In another study, thymol significantly inhibited 88% of methicillin-resistant Staphylococcus aureus (MRSA) biofilm formation at 100 μ g/mL, reduced the surface adherence of MRSA, and enhanced the antibacterial and biofilm eradication efficiency of rifampicin against MRSA [ 9 ]. In addition, our recent studies reported a synergistic antibiofilm effect between thymol and aminoglycosides against K. pneumoniae and Salmonella enterica biofilms [ 10 , 11 ]. The association of thymol with antibiotics showed a strong synergistic effect both in the inhibition of biofilm formation and the destruction of the preformed biofilm of K. pneumoniae , with the minimum biofilm inhibitory concentration (MBIC) and the minimum biofilm eradication concentration (MBEC) values of streptomycin being reduced by 16- to 64-fold [ 10 ].…”

“…In addition, our recent studies reported a synergistic antibiofilm effect between thymol and aminoglycosides against K. pneumoniae and Salmonella enterica biofilms [ 10 , 11 ]. The association of thymol with antibiotics showed a strong synergistic effect both in the inhibition of biofilm formation and the destruction of the preformed biofilm of K. pneumoniae , with the minimum biofilm inhibitory concentration (MBIC) and the minimum biofilm eradication concentration (MBEC) values of streptomycin being reduced by 16- to 64-fold [ 10 ].…”

“…In our previous study, we demonstrated that thymol could act synergistically with streptomycin to inhibit the biofilm formation and eradicate the preformed biofilm of K. pneumoniae [ 10 ]. We also showed that thymol could be encapsulated into PLGA nanoparticles to overcome issues related to its low water solubility and high volatility [ 18 ].…”