In vitro evaluation of gene expression and adherence of Staphylococcus epidermidis and Staphylococcus aureus by using RT-PCR after disinfection of ocular prosthesis acrylic resins

Abstract

The disinfection of ocular prosthesis aims to control microbial biofilm formation and to prevent the inflammation and infection of the conjunctival endogenous microbiota associated with Staphylococcus epidermidis and Staphylococcus aureus. Biofilm development on the ocular prosthesis may induce frequent ocular bacteremia and antibiotics resistance. The gene expression of icaA/icaD of S. epidermidis, and bap of S. aureus is related to the bacterial adherence and biofilm formation. The aim of this study will be to assess the adherence and to detect/quantify the expression of these genes in planktonic and biofilm cultures developed on acrylic resin surfaces of S. epidermidis and S. aureus, after treatment with three disinfectant solutions. A total of 144 samples will be fabricated (1.0 cm in diameter and 0.3 cm in thickness). Half of the samples will be used for bacterial adherence assay, and the others will used for gene expression assay. For each 72 samples, half will be inoculated with S. epidermidis, and half will be inoculated with S. aureus for biofilm development. Samples will be divided into four groups (9 per group): distilled water (control) (G1); treated with neutral soap (G2); treated with Opti-Free (G3); and treated with 4% chlorhexidine (G4). Microbiological analysis will be performed in triplicate in three independent experiments. Biofilm and planktonic cultures will be collected at three different points corresponding to biofilm development stages: initial (24h), intermediate (48h) and mature (72h). The number of colonies will be verified based on the number of CFU/mL after disinfection treatments. The total RNA of these samples will be obtained and the gene expression for mRNA of icaA/icaD and bap will be quantified by Quantitative Real-Time PCR after disinfection treatments. Gene expression will be normalized to housekeeping genes (gmk, 16S) to determine the relative level of expression. Data will be subjected to statistical analysis and evaluated according to the appropriate statistical test. (AU)