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The present work is an attempt to establish the functionality of genes involved in biofilm formation and antibiotic resistance in an ocular strain of  Escherichia coli  (L-1216/2010) which was isolated and characterized from the Vitreous fluid of a patient with Endophthalmitis. For this purpose, seven separate gene-specific knockout mutants were generated by homologous recombination in ocular  E. coli . The genes that were mutated included three transmembrane genes  ytfR  (ABC transporter ATP-binding protein),  mdtO  (multidrug efflux system) and  tolA  (inner membrane protein),  ryfA  coding for non-coding RNA and three metabolic genes  mhpA  (3-3-hydroxyphenylpropionate 1,2-dioxygenase),  mhpB  (2,3-di hydroxyphenylpropionate 1,2-dioxygenase), and  bdcR  (regulatory gene of  bdcA ). Mutants were validated by sequencing and Reverse transcription-PCR and monitored for biofilm formation by XTT method and confocal microscopy. The antibiotic susceptibility of the mutants was also ascertained. The results indicated that biofilm formation was inhibited in five mutants (Δ bdcR , Δ mhpA , Δ mhpB , Δ ryfA , and Δ tolA ) and the thickness of biofilm reduced from 17.2 μm in the wildtype to 1.5 to 4.8 μm in the mutants. Mutants Δ ytfR  and Δ mdtO  retained the potential to form biofilm. Complementation of the mutants with the wild type gene restored biofilm formation potential in all mutants except in Δ mhpB . The 5 mutants which lost their ability to form biofilm (Δ bdcR , Δ mhpA , Δ mhpB , Δ tolA , and Δ ryfA ) did not exhibit any change in their susceptibility to Ceftazidime, Cefuroxime, Ciprofloxacin, Gentamicin, Cefotaxime, Sulfamethoxazole, Imipenem, Erythromycin, and Streptomycin in the planktonic phase compared to wild type ocular  E. coli . But Δ mdtO  was the only mutant with altered MIC to Sulfamethoxazole, Imipenem, Erythromycin, and Streptomycin both in the planktonic and biofilm phase. This is the first report demonstrating the involvement of the metabolic genes  mhpA  and  mhpB  and  bdcR  (regulatory gene of  bdcA ) in biofilm formation in ocular  E. coli . In addition we provide evidence that  tolA  and  ryfA  are required for biofilm formation while  ytfR  and  mdtO  are not required. Mitigation of biofilm formation to overcome antibiotic resistance could be achieved by targeting the genes  bdcR, mhpA, mhpB, ryfA , and  tolA .

Gene Targets in Ocular Pathogenic Escherichia coli for Mitigation of Biofilm Formation to Overcome Antibiotic Resistance