No abstract

A well-designed and properly validated primer produces a specific and efficient qPCR assay. The primer’s melting temperature, G-C content, its size, as well as the amplicon’s size are the principal considerations while designing the primer for this study and it was in accordance with the MIQE guidelines. Subsequently, the designed primer was evaluated prior to the analysis to verify its efficiency and precision for the relative quantification of the gene transcription analysis. The validity of a qPCR assay is reliant on three elements; the qPCR efficiency (E) and the R2 as well as the slope, that were derived from constructed standard curve. The resulted E for the genes lasI, lasR, rhlI, rhlR and rplS are 92%, 93%, 96%, 92% and 94%, respectively. While the R2 and the slope value for these genes are 0.9991 and -3.523 for the lasI, 0.9991 and -3.501 for the lasR, 0.9989 and -3.434 for the rhlI, 0.9999 and -3.535 for the rhlR, and 0.9935 and -3.487 for the rplS. Melt curve analyses carried out post qPCR assay resulted in amplification of a single product. Resulted E, R2 and slope for all studied genes fell between the acceptable range, validating the use of designed primer for further analysis in the changes in transcription level of quorum sensing genes in treated Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen that orchestrate its disease manifestation via a complex communication system known as quorum sensing (QS). Under the regulation of this complex network of QS system, is a myriad of virulence factors including the flagella and pili that contribute to its motility. To establish an infection in a host, P. aeruginosa must first attach itself to host cells with the assistance of flagella and pili and it would be a mere impossible for P. aeruginosa to initiate and establish an infection in the host without those features. In this study, we investigated the motility of P. aeruginosa under the influence of Piper betle leaves extract by growing the bacteria on a semi solid medium and compared the growth spread of the pathogen against the untreated sample. There are three types of motilities that were being investigated, which were swimming, swarming and twitching. In all three assays, a fraction of ethyl acetate extract of P. betle leaves were found to reduce the motility of P. aeruginosa with greatest defect in swimming at more than 80% reduction as compared to the untreated sample. Meanwhile, the fraction partially decreased the swarming and twitching of P. aeruginosa by 58% and 50% reduction respectively, in comparison to the untreated sample. The exhibited defect in the motility of P. aeruginosa proves the anti-QS property of the fraction of ethyl acetate extract of P. betle leaves and could be developed as a potential therapeutic agent against the pathogen.

Anti-quorum sensing property has been a popular alternative approach over bactericidal/bacteriostatic property in combating bacterial infection while simultaneously tackling the dilemma of antibiotic resistance. We presented pyoverdin assay as an initial screening to qualitatively determine the anti-quorum sensing activity in Piper betle by measuring the loss of absorbance at wavelength 630 nm. Growth of Pseudomonas aeruginosa was proven to be unaffected by the presence of the P. betle leaves extract. The regression value (R2) of the quorum sensing (QS) activity in untreated supernatant of P. aeruginosa was 0.9636 and we presented the QS activity in fold-change, normalized to untreated sample for a fair comparison between batch of assays. We further assessed the QS activity in the extracts of P. betle leaves and found the QS activity of P. aeruginosa grown in the presence of ethyl acetate extract at 200 μg/ml was reduced to 0.6-fold. As the concentrations went lower, higher fold of QS activity was observed, suggesting that P. betle leaves extract is demonstrating anti-QS activity at a higher concentration. Further fractionate of ethyl acetate crude extracts resulted in three fractionates with high anti-QS activity with >50% reduction in QS activity and five fractionates with intermediate anti-QS activity. The use of pyoverdin assay to qualitatively portray the anti-QS activity could shorten the lengthiness of extracting and measuring the signaling molecule yet, produces reliable information to screen for anti-QS activity and guide for further fractionation and purification of bioactive compound.