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Shivaji Sisinthy, Ranjith Konduri, Arunasri Anakala, Jahnabi Ramchiary, S S Prakash Jogadhenu, Savitri Sharma; Gene targets in ocular pathogenic Escherichica coli for mitigation of biofilm formation to overcome antibiotic resistance. Invest. Ophthalmol. Vis. Sci. 2019;60(9):2541.
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To ascertain whether mhpA (3-3-hydroxyphenylpropanoate hydroxylase), mhpB (2,3-dihydroxyphenylpropionate 1,2-dioxygenase), ryfA (non coding RNA), tolA (inner membrane protein), bdcR (regulatory gene of bdcA), ytfR (ABC transporter ATP-binding protein) and mdtO (multidrug efflux system) known to be up regulated in expression in the biofilm phase in ocular pathogenic E. coli L- 1216/2010 (Ranjit et al. 2017) are required for biofilm formation. Only ryfA and tolA have been reported earlier to be required for biofilm formation.
Four transmembrane genes ytfR, mdtO, ryfA, and tolA and three metabolic genes mhpA, mhpB, bdcR based on their increased expression in the biofilms were speculated to be involved in biofilm formation. To establish functionality of the 7 genes, gene-specific knockout mutants were generated by homologous recombination in ocular E. coli, according to method described earlier. Mutants were validated by sequencing and Real time PCR. Mutants following validation were monitored for biofilm formation by XTT method and confocal microscopy. The antibiotic susceptibility of the mutants was also ascertained.
1. Biofilm formation was inhibited in five mutants (ΔbdcR, ΔmhpA, ΔmhpB, ΔtolA and ΔryfA) and thickness of biofilm reduced from 17. 2 µm in the control to 1.5 to 4.8 µm in the mutants.2. Mutants ΔytfR and ΔmdtO retained the potential to form biofilm.3. Complementation of the mutants with the wild type gene restored biofilm formation potential in all mutants except in ΔmhpB.4. The 5 mutants which lost their ability to form biofilm did not exhibit any change in their sensitivity to Ceftazidime, Cefuroxime, Ciprofloxacin, Gentamycin, Cefotaxime, Sulfamethoxazole, Imipenem, Erytromycin and Streptomycin in the planktonic phase compared to wild type ocular E. coli.5. Mutant ΔmdtO was the only mutant with altered MIC to Sulfamethoxazole, Imipenem, Erytromycin and Streptomycin both in the planktonic and biofilm phase.
First report of the involvement of the metabolic genes mhpA, mhpB and bdcR in biofilm formation in E. coli. In addition 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, tolA and ryfA .
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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