Abstract
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 (ΔbdcR, ΔmhpA, ΔmhpB, ΔryfA, and ΔtolA) and the thickness of biofilm reduced from 17.2 μm in the wildtype to 1.5 to 4.8 μm in the mutants. Mutants ΔytfR and ΔmdtO retained the potential to form biofilm. Complementation of the mutants with the wild type gene restored biofilm formation potential in all mutants except in ΔmhpB. The 5 mutants which lost their ability to form biofilm (ΔbdcR, ΔmhpA, ΔmhpB, ΔtolA, and ΔryfA) 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 ΔmdtO 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.
Highlights
Antimicrobial resistance (AMR) refers to the ability of microorganisms to resist the inhibitory effect of an antimicrobial agent at the minimum inhibitory concentration (MIC) of the antimicrobial agent
We demonstrated that ocular E. coli from patients with conjunctivitis, keratitis or endopthalmitis were resistant to one or more of the antibiotics tested and majority of the isolates were positive for biofilm formation
Fim genes coding for type 1 fimbriae, genes coding for the LPS lipid A moiety, yhjN coding for cellulose synthase regulator protein, were up regulated in ocular E. coli L-1216/2010 but not in E. coli ABU strain 83972 (Hancock and Klemm, 2007) and in E. coli UPEC strain CFT073 (Schembri et al, 2003)
Summary
Antimicrobial resistance (AMR) refers to the ability of microorganisms (bacteria, fungi, viruses, and parasites) to resist the inhibitory effect of an antimicrobial agent at the minimum inhibitory concentration (MIC) of the antimicrobial agent. Over the years with the development of the crystal violet method, XTT method, confocal microscopy and scanning electron microscopy it has been possible to monitor the dynamics of biofilm formation, and evaluate simultaneously the extent of AMR These studies have clearly established that biofilm confers AMR to microorganisms (Hoyle et al, 1992; Elder et al, 1995; Ranjith et al, 2017). It was observed that ocular E. coli in biofilm phase did not exhibit up regulation of the stress response encoding genes (such as cspG, cspH, pphA, ibpA, ibp, soxS, hha, and yfiD) as observed in ABU strains of E. coli These results indicate differences between the ocular and the non-ocular pathogenic E. coli in expression of genes in the biofilm phase
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