AUTHOR=Ranjith Konduri , Ramchiary Jahnabi , Prakash Jogadhenu S. S. , Arunasri Kotakonda , Sharma Savitri , Shivaji Sisinthy 

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

JOURNAL=Frontiers in Microbiology

VOLUME=10

YEAR=2019

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2019.01308

DOI=10.3389/fmicb.2019.01308

ISSN=1664-302X

ABSTRACT=<p>The present work is an attempt to establish the functionality of genes involved in biofilm formation and antibiotic resistance in an ocular strain of <italic>Escherichia coli</italic> (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 <italic>E. coli</italic>. The genes that were mutated included three transmembrane genes <italic>ytfR</italic> (ABC transporter ATP-binding protein), <italic>mdtO</italic> (multidrug efflux system) and <italic>tolA</italic> (inner membrane protein), <italic>ryfA</italic> coding for non-coding RNA and three metabolic genes <italic>mhpA</italic> (3-3-hydroxyphenylpropionate 1,2-dioxygenase), <italic>mhpB</italic> (2,3-di hydroxyphenylpropionate 1,2-dioxygenase), and <italic>bdcR</italic> (regulatory gene of <italic>bdcA</italic>). 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<italic>bdcR</italic>, Δ<italic>mhpA</italic>, Δ<italic>mhpB</italic>, Δ<italic>ryfA</italic>, and Δ<italic>tolA</italic>) 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<italic>ytfR</italic> and Δ<italic>mdtO</italic> 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<italic>mhpB</italic>. The 5 mutants which lost their ability to form biofilm (Δ<italic>bdcR</italic>, Δ<italic>mhpA</italic>, Δ<italic>mhpB</italic>, Δ<italic>tolA</italic>, and Δ<italic>ryfA</italic>) 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 <italic>E. coli</italic>. But Δ<italic>mdtO</italic> 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 <italic>mhpA</italic> and <italic>mhpB</italic> and <italic>bdcR</italic> (regulatory gene of <italic>bdcA</italic>) in biofilm formation in ocular <italic>E. coli</italic>. In addition we provide evidence that <italic>tolA</italic> and <italic>ryfA</italic> are required for biofilm formation while <italic>ytfR</italic> and <italic>mdtO</italic> are not required. Mitigation of biofilm formation to overcome antibiotic resistance could be achieved by targeting the genes <italic>bdcR, mhpA, mhpB, ryfA</italic>, and <italic>tolA</italic>.</p>