Abstract
We selectively characterized three isolates from Pseudomonas aeruginosa keratitis patients and how glycyrrhizin (GLY) affected them. Type III toxins were determined using polymerase chain reaction (PCR). Minimum Inhibitory Concentration (MIC) of GLY and assays for its effects on: time kill, bacterial permeability, and biofilm/adhesion were done. In vivo, C57BL/6 (B6) mice were treated topically with GLY after G81007 infection. Clinical score, photography with a slit lamp and RT-PCR were used to assess treatment effects. Isolates expressed exoS and exoT, but not exoU. MIC for all isolates was 40 mg/mL GLY and bacteriostatic effects were seen for G81007 after treatment using time kill assays. From viability testing, GLY treatment significantly increased the number of permeabilized bacteria (live/dead assay). Isolates 070490 and G81007 formed more biofilms compared with R59733 and PAO1 (control). GLY-treated bacteria had diminished biofilm compared with controls for all isolates. GLY reduced adherence of the G81007 isolate to cultured cells and affected specific biofilm associated systems tested by reverse transcription PCR (RT-PCR). In vivo, after G81007 infection, GLY treatment reduced clinical score and messenger RNA (mRNA) expression of IL-1β, TNF-α, CXCL2 and HMGB1. This study provides evidence that GLY is bacteriostatic for G81007. It also affects biofilm production, adherence to cultured cells, and an improved keratitis outcome.
Highlights
Infection with Pseudomonas aeruginosa (P. aeruginosa), an opportunistic, Gram-negative bacterium, is often associated with microbial keratitis, especially in extended-wear contact lens users [1,2,3].Pseudomonas keratitis develops rapidly and elicits an acute inflammatory response in cornea, which contributes to eradication of the bacterium
HMGB1 with is a an target for we demonstrated that reducing improved the keratitis disease outcome invasive glycyrrhizin (GLY), the[21]
Since G81007 produced the greatest amount of biofilm of the three isolates, A549 lung epithelial cells and transformed human corneal epithelial cells (HCET) were used to test the ability of the isolate cells and transformed human corneal epithelial cells (HCET) were used to test the ability of the isolate to adhere to these cells
Summary
Infection with Pseudomonas aeruginosa (P. aeruginosa), an opportunistic, Gram-negative bacterium, is often associated with microbial keratitis, especially in extended-wear contact lens users [1,2,3]. P. aeruginosa may possess one or more of the genes exoS, exoT and exoU, which code for the cytotoxins ExoS, ExoT, and ExoU, secreted through the type III secretion system These cytotoxins promote bacterial evasion of the host immune response, the dissemination of the organism from the infection site, and inhibition of DNA synthesis, leading to host cell death [6,7,8,9]. HMGB1 with is a an target for we demonstrated that reducing improved the keratitis disease outcome invasive glycyrrhizin (GLY), the[21]. GLY treatment viability, adhesion to lung and human corneal epithelial cells, and biofilm formation in vitro, as well bacterial growth, viability, adhesion to lung and human corneal epithelial cells, and biofilm formation as reducing keratitis and pro-inflammatory molecules in an in vivo animal model. In vitro, as well as reducing keratitis and pro-inflammatory molecules in an in vivo animal model
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