Screening of Novel Genes Involvedin Biofilm Mediated resistance to Antibiotics in MDR Pseudomonas aeruginasa

Abstract

The role of biofilm in the pathogenesis of some chronic human infection is now widely accepted. Pseudomonas aeruginosa is a model organism for biofilm study, which seems to have complex regulatory circuit that controls biofilm formation. Biofilm forming physiology of P. aeruginosa should have some genetic implication. In this study, the potential of biofilm formation in P. aeruginosa, was assessed and susceptibility to selected carbapenem antibiotic was determined. The potential regulatory gene was identified using homology searches and used in the study for determining the role of this gene in biofilm formation using PCR amplification and cloning. Assessment of biofilm formation by 8 confirmed Pseudomonas isolates were done using normal polystyrene microtitre plate (non-tissue coated), using crystal violet staining and read at 551nm. The AST was carried following Clinical and Laboratory Standard Institute protocol in Muller Hilton Agar plate using 10 mcg disc of imipenem and meropenem antibiotics. BLASTp program identified PA1961 gene as putative regulatory gene of the family LysR- type transcriptional regulator in P. aeruginosa for which primer was designed using PrimerBLAST tool of NCBI. PCR was used to determine whether presence or absence of the gene was related to biofilm formation physiology. Out of 8 isolates, two different isolates, 1 isolate showed consistent biofilm formation using cut-off (Mean OD of negative control + 3X SD of negative control) and 1 isolate showed antibiotic resistance to both IPM and MRP, making 12.5%(12.5% biofilm former and 12.5% antibiotic resistance), which is considerably low as compared to previous studies. Next we aimed to draw if there is any relationship of biofilm formation with antibiotic resistance. We identified PA1961 gene using homology search and expected its presence only in biofilm forming carbapenem resistant isolates. In contrary the gene was found to be present in all isolates, including the positive control (PAO1) and the result was inconclusive in drawing direct relationship of biofilm formation with antibiotic resistance in Pseudomonas. We could have done protein analysis for the same gene to be sure of this genes role in biofilm formation. But since it was a surface protein, protein quantification would not provide be done. Further study with the increase in number of fresh isolates, stringent control (inclusion of negative control strain) and robust bioinformatics analysis will provide conclusive result for the identification of novel genes involved in biofilm mediated antibiotic resistance in MDR pathogens. Keywords: Biofilm, Antibiotic resistance, Carbapenem, P. aeruginosa, LysR-type transcriptional regulator, Antibiotic susceptibility test.