Tentative title: Differential effect on swarming and biofilm formation of Pseudomonas aeruginosa HRW.1-S3 in presence of glucose and gentamicin. Rationale of the study Swarming Biofilm Decision making - might be crucial for survival of bacterial colony, under different stresses like nutrient deficiency and antibiotic treatment. If we could understand the cellular and molecular networks involved in different stress conditions of swarming behaviours in Pseudomonas aeruginosa isolates, It can be used as a tool to regulate defensive behaviours of the bacteria and thus enable less biofilm production Help us to deal with the threats posed by this pathogenic bacteria and their associated risk factors Why Pseudomonas aeruginosa is being used for this study? • Pseudomonas aeruginosa, in general, is an avid biofilm former and becoming resistant to antibiotics and has been implicated in diseases like cystic fibrosis, skin infections etc. • In 2017, multidrug-resistant Pseudomonas aeruginosa caused an estimated 32,600 infections among hospitalized patients and 2,700 estimated deaths in the United States. (AR threats report) • Pseudomonas aeruginosa is known to affect immunocompromised patients like those suffering from Cystic Fibrosis and AIDS and can cause peritonitis, sepsis, and bacteremia. This infections are recalcitrant in nature thus making it more deadly and their new ways of avoiding antibiotics are an area of great concern. Why the study of swarming and biofilm in presence of glucose and antibiotics is important? • Glucose is the preferred carbon source for most living organism and it has been reported there are conflicting reports on effect of glucose on biofilm formation. • Pseudomonas aeruginosa have a tendency to form biofilms under antibiotic treatment thus escaping their action. Upon retraction of the stress, they again get released causing chronic infections. • Swarming are associated with biofilms and their relation can be investigated to know the molecular mechanism lying and thus any way to reduce biofilm formation and its threat can be accomplished. Experiments done • Swarming of HRW.1-S3 in presence of differential glucose. (N=3) • MIC of Pseudomonas aeruginosa HRW.1-S3 in presence of gentamicin. (N=3) • Swarming of HRW.1-S3 in presence of both differential glucose and gentamicin. (N=3) • Scanning electron microscopy of swarming of HRW.1-S3 under differential glucose and gentamicin. (N=1) • Biofilm formation of HRW.1-S3 in presence of differential glucose. (N=3) • Biofilm formation of HRW.1-S3 in presence of both differential glucose and gentamicin. (N=3) • Confocal laser scanning microscopy of biofilm formed under differential glucose and gentamicin. (N=3) • EPS study of the biofilm formed under differential glucose and gentamicin. (N=3) • Scanning electron microscopy of the biofilm formed under differential glucose and gentamicin. (N=1) Experiments done • Reactive oxygen species generation in both biofilm and planktonic cells of HRW.1-S3 in presence of both differential glucose and gentamicin. (N=2) • Microbial adhesion assay of HRW.1-S3 in presence of differential glucose and gentamicin. (N=3) ***Basic follow up experiments (Swarming in NA, lower glucose percentage) are also done. Findings from the study • It is evident from the studies that Pseudomonas aeruginosa HRW.1-S3 swarming motility has an inverse relation with glucose percentage, whereas there is a positive correlation with glucose percentage in case of biofilm. • In the biofilm assays, higher biofilm load was evident with the introduction of higher dose of glucose and antibiotics. • The increased biofilm load is further verified by CLSM and SEM studies followed by EPS quantification. ROS and adhesive properties of the bacteria also seem to play an important role as they increased with increasing biofilm load. • In the case of swarming, it is seen that with the introduction of antibiotic at lower concentration; PA start to swarm whereas at higher doses of antibiotics this swarming pattern gets reduced signifying their change of state from more motile to sessile state. • Overall, at lower doses of gentamicin, swarming and biofilm load both are increasing whereas at higher doses of gentamicin swarming is getting reduced whereas biofilm load is increasing, signifying that the relation between swarming and biofilm is completely conditional and it may vary depending upon the microenvironment and its constituents. Findings from the study • Another conclusion which can be drawn is that at lower doses of gentamicin, due to lesser stress the bacteria are able to swarm, but upon receiving higher doses of gentamicin these swarmer cells tend to become more restricted in their movement and are trying to form biofilms by accumulating more cells in a confined space and forming a matrix. Pending Experiments • Repetition of SEM. • Repetition of ROS assay. • TEM for flagella visualization. • LPS isolation and visualization • Molecular work
