Characterization of Three Ocular Clinical Isolates of P. aeruginosa: Viability, Biofilm Formation, Adherence, Infectivity, and Effects of Glycyrrhizin

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.

Glycyrrhizin Use for Multi-Drug Resistant Pseudomonas aeruginosa: In Vitro and In Vivo Studies

“…This trend in antibiotic resistant bacterial infections is of serious global concern, fueled by the lack of effective antibiotics available and few being developed. 3 GLY, derived from Glycyrrhiza glabra, is antimicrobial and anti-inflammatory 6 ; it has many additional effects 6 and has shown promise against non-MDR isolates 7,8,16,19,20 and our current work shows it is effective against MDR isolates. Clinically, GLY also has been successfully used to treat blepharitis 28 and hepatitis patients 29,30 with no toxicity.…”

“…The MIC was assigned to the concentration of GLY that resulted in an absorbance reading of zero. 20 MIC ciprofloxacin (1-32 lg/mL) for MDR9 was measured with and without GLY (20 mg/mL) similarly.…”

“…Samples were photographed using a Leica TCS-SP8 (Leica Microsystems, Buffalo Grove, IL, USA) and the average percentage of live (green) bacteria was quantitated as described. 20,21 Live (green) or dead/permeabilized (red) bacteria were counted in four 40lm 2 areas of four representative micrographs (n ¼ 16 areas/ group), averaged and expressed as percent total live bacteria. 20…”

“…20,21 Live (green) or dead/permeabilized (red) bacteria were counted in four 40lm 2 areas of four representative micrographs (n ¼ 16 areas/ group), averaged and expressed as percent total live bacteria. 20…”

“…For each pellet (1.5 mL of each bacterial culture/ pellet) total RNA was isolated (RNA STAT-60; Tel-Test, Friendswood, TX, USA) per the manufacturer's instructions and as reported before. 19,20 A total of 1 lg of each RNA sample was reverse transcribed using Moloney-murine leukemia virus (M-MLV) reverse transcriptase (Invitrogen, Carlsbad, CA, USA) to produce a cDNA template PCR. cDNA products for each were diluted 1:25 with DEPC-treated water and a 2-ll aliquot of diluted cDNA was used for the real-time RT-PCR reaction.…”