Abstract
Biofilm formation conferring pathogenicity is a survival strategy for Pseudomonas aeruginosa. P. aeruginosa’s virulence may differ due to differences in host-microbe interactions and the growth environment. The epithelial cell line within the respiratory system and the keratinocytes on the skin form the first physical barrier of defence. P. aeruginosa spp. biofilm formation and virulence factor secretion with and without quorum quenching (QQ) treatment was studied in co-culture using A549 and HaCaT cell lines; pyocyanin and rhamnolipid productions and elastolytic activity as virulence factors were quantified by independent assays. Biofilm formation was evaluated under dynamic conditions by quantifying total carbohydrates, alginate, proteins and eDNA. A sandwich ELISA was performed to study IL-8 secretion by the epithelial cells. The difference in gene expression of the quorum sensing (QS) and virulence factors between strains during individual and combination treatments was analysed by qPCR. Combination treatment by farnesol and tyrosol was more effective against P. aeruginosa biofilms when grown in co-cultures. The strain RBHi was found to be 3 to 4 times more virulent compared to PAO1 and NCTC 10,662, respectively, and combination treatment was more effective against RBHi strain when grown in co-culture with A549 cell line. The addition of quorum quenchers (QQs) individually and in combination reduced IL-8 secretion by A549 cells. Relative mRNA expression showed upregulation of the QS genes and virulence factors. Co-culture of P. aeruginosa and HaCaT cell line showed a general decrease in gene expression, especially in the case of P. aeruginosa RBHi when treated with farnesol and tyrosol combination.Key points• Differentiating the interactions of biofilm formed by different phenotypes of P. aeruginosa, NCTC 10,662 (non-mucoid), PAO1 (semi mucoid) and RBHi (heavily mucoid).• Biofilm formed by these P. aeruginosa strains on two commonly afflicted tissues represented by A549 (lung) and HaCaT (skin) cell lines.• Anti-biofilm/anti-virulence roles of quorum quenchers, tyrosol and farnesol in co-cultures.
Highlights
The Gram-negative bacterium, Pseudomonas aeruginosa, is a well-known opportunistic pathogen that causes genetically inherited human cystic fibrosis (CF)
This study focuses on farnesol and tyrosol and assesses their ability to inhibit, as fungal quorum sensing (QS) molecules, the QS process in P. aeruginosa, and as quorum quenching (QQ) when grown on mammalian cell lines
P. aeruginosa sp. adherence pattern to A549 and HaCaT cell lines with and without QQ mediated by farnesol and tyrosol
Summary
The Gram-negative bacterium, Pseudomonas aeruginosa, is a well-known opportunistic pathogen that causes genetically inherited human cystic fibrosis (CF). Literature has documented that in such a scenario, a primary selective phenotypic change is the production of alginate that envelops the bacteria (mucoid). This results in sticky mucus, most notably in the respiratory tract of CF patients making them susceptible to chronic lung infections and the high rate of morbidity and mortality associated with CF (Bhagirath et al 2016). The presence of non-mucoid, mucoid, and strongly mucoid strains of P. aeruginosa highlights the variety of genetic variation in this pathogenic bacterium (Workentine et al 2013).
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