Ranjith Konduri, Ramchiary Jahnabi, Prakash Jogadhenu S S, Arunasri Kotakonda, Sharma Savitri, Shivaji Sisinthy
Jhaveri Microbiology Centre - Prof. Brien Holden Eye Research Centre, LV Prasad Eye Institute, Hyderabad, India.
Research Scholar, Manipal Academy of Higher Education, Manipal, India.
Front Microbiol. 2019 Jun 21;10:1308. doi: 10.3389/fmicb.2019.01308. eCollection 2019.
The present work is an attempt to establish the functionality of genes involved in biofilm formation and antibiotic resistance in an ocular strain of (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 . The genes that were mutated included three transmembrane genes (ABC transporter ATP-binding protein), (multidrug efflux system) and (inner membrane protein), coding for non-coding RNA and three metabolic genes (3-3-hydroxyphenylpropionate 1,2-dioxygenase), (2,3-di hydroxyphenylpropionate 1,2-dioxygenase), and (regulatory gene of ). 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, Δ, Δ, Δ, and Δ) 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 and Δ 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. The 5 mutants which lost their ability to form biofilm (Δ, Δ, Δ, Δ, and Δ) 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 . But Δ 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 and and (regulatory gene of ) in biofilm formation in ocular . In addition we provide evidence that and are required for biofilm formation while and are not required. Mitigation of biofilm formation to overcome antibiotic resistance could be achieved by targeting the genes , and .
本研究旨在确定从一名眼内炎患者玻璃体中分离并鉴定的眼内菌株（L-1216/2010）中参与生物膜形成和抗生素耐药性的基因的功能。为此，通过同源重组在眼内菌株中产生了七个单独的基因特异性敲除突变体。突变的基因包括三个跨膜基因（ABC转运蛋白ATP结合蛋白）、（多药外排系统）和（内膜蛋白）、编码非编码RNA的基因以及三个代谢基因（3-3-羟基苯丙酸1,2-双加氧酶）、（2,3-二羟基苯丙酸1,2-双加氧酶）和（的调控基因）。通过测序和逆转录PCR对突变体进行验证，并通过XTT法和共聚焦显微镜监测生物膜形成。还确定了突变体的抗生素敏感性。结果表明，五个突变体（Δ、Δ、Δ、Δ和Δ）中的生物膜形成受到抑制，生物膜厚度从野生型的17.2μm降至突变体中的1.5至4.8μm。突变体Δ和Δ保留了形成生物膜的潜力。除Δ外，用野生型基因对突变体进行互补恢复了所有突变体的生物膜形成潜力。与野生型眼内菌株相比，失去形成生物膜能力的5个突变体（Δ、Δ、Δ、Δ和Δ）在浮游生长阶段对头孢他啶、头孢呋辛酯、环丙沙星、庆大霉素、头孢噻肟、磺胺甲恶唑、亚胺培南、红霉素和链霉素的敏感性没有任何变化。但Δ是唯一在浮游生长阶段和生物膜阶段对磺胺甲恶唑、亚胺培南、红霉素和链霉素的最低抑菌浓度发生改变的突变体。这是第一份证明代谢基因和以及（的调控基因）参与眼内菌株生物膜形成的报告。此外，我们提供证据表明生物膜形成需要和，而不需要和。通过靶向基因、和可以实现减轻生物膜形成以克服抗生素耐药性。
