Attenuation of quorum sensing genes in Pseudomonas aeruginosa BY Piper betle leaf extract / Saiedatul Akmal

Faculty of Chemical and Process Engineering Technology

Thesis (Doctor of Philosophy) -- Universiti Malaysia Pahang – 2024

Includes bibliographical references

The antimicrobial resistance demonstrated by Pseudomonas aeruginosa is alarming the healthcare system. The conventional antibiotics used to treat P. aeruginosa infection have lost their effectiveness due to the resistance of this pathogen to multiple antimicrobials. With the declining number of approved new antimicrobials, the threat posed by P. aeruginosa is exacerbated further. In this study, Piper betle leaf extract was investigated for its potential as an alternative therapeutic agent to treat P. aeruginosa infection as opposed to antibiotics. The adopted approach in this study manipulates the communication network of P. aeruginosa, known as the quorum sensing (QS) system, to identify phytochemicals in P. betle leaf extract possessing anti-QS activity that adversely affect the pathogenesis in P. aeruginosa without killing the pathogen. The effect of the identified phytochemicals on the expression of the QS genes (lasI, lasR, rhlI, and rhlR) in P. aeruginosa was investigated via real-time quantitative polymerase chain reaction (qPCR) analysis. Consequently, the effect of the identified phytochemicals on the virulence factors (motility, biofilm, elastase, protease, pyocyanin, and pyoverdine) production by P. aeruginosa was further investigated to validate and support the gene expression analysis. Both minimal inhibitory concentration (MIC) and planktonic growth analyses ruled out antibiotic effect of P. betle leaf extract toward P. aeruginosa. Gas chromatography–mass spectrometry (GC–MS) analysis of a fraction of P. betle leaf extract revealed a group of fatty acids (FAs), that comprised 65% of the sample size. They are identified as hexadecenoic acid (C16H30O2) and octadecanoic acid (C18H36O2) of saturated FAs, and 12-octadecadienoic acid (C18H34O2) and 7-octadecenoic acid (C18H34O2) of unsaturated FAs. These FAs significantly reduced the expression level of QS genes of interest by almost 50% in treated-P. aeruginosa. Corresponding to the attenuated QS genes expression level, the motility of P. aeruginosa was adversely affected with swimming motility of the treated group exhibited 80% reduction in the growth diameter. Biofilm formation was distorted in treated-P. aeruginosa where it formed in clumps rather than a confluent formation, with 37% reduction in biofilm biomass. Meanwhile, all tested virulent factors showed significant reduction in production including elastase (31.10% ± 0.05 reduction), protease (46.76% ± 0.06 reduction), pyocyanin (53.57% ± 0.03 reduction) and pyoverdine (50.45% ± 0.05 reduction). In conclusion, the anti-QS activity demonstrated by the identified FAs is unrestricted to antibiotic effect of the P. betle leaf extract, but rather resulted from the interference on the pathogen intercellular communication mechanism. This is evidenced by the attenuated expression of the QS genes in P. aeruginosa due to the treatment with the FAs and consequently, the adversely affected virulence factors production. These FAs offers an alternative mechanism of action for therapeutic agents as opposed to conventional antibiotics that are prone to resistance acquisition by bacterial pathogens. Through this innovative approach, the discovery of alternative therapeutic agents is now feasible and should pave the way for a sustainable and economically viable therapy options for bacterial infection in the healthcare system.