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

Abstract

Purpose: Our purpose was to test glycyrrhizin (GLY) effects and ciprofloxacin interactions on multidrug resistant (MDR) isolates of Pseudomonas aeruginosa in vitro and in vivo in a mouse model of keratitis.

Methods: A Hardy-disk tested antibiotic sensitivity of isolates MDR9 (nonocular) and B1045 (ocular). GLY MIC (both isolates) and ciprofloxacin was determined spectrophotometrically. A live/dead assay using confocal microscopy and plate count, tested GLY effects on bacterial permeabilization/viability. Proteomics profiled bacterial efflux pumps (MDR9 vs. PAO1); RT-PCR comparatively tested GLY effects on their mRNA expression levels. The activity of efflux pumps was tested using ethidium bromide (EB); and scanning electron microscopy (SEM) visualized the effects of GLY treatment of bacteria. A combination of GLY and ciprofloxacin was tested in C57BL/6 mice (begun 18 hours after infection) and disease scored, photographed and MPO and plate counts done.

Results: MDR9 was resistant to 6/12 and B1045 to 7/12 antibiotics (both to ciprofloxacin). MIC GLY for MDR9 was 40 mg/mL and 15 mg/mL for B1045. Ciprofloxacin MIC (32 μg/mL) was reduced 2-fold to 16 μg/mL when ciprofloxacin and GLY were combined. GLY altered bacterial membrane permeability and reduced viability. Proteomics revealed increased efflux pumps in MDR9 versus PAO1; GLY reduced their mRNA expression levels and EB suggested decreased activity. In C57BL/6 mice, treatment with GLY and ciprofloxacin versus ciprofloxacin, significantly reduced clinical scores, plate count, and MPO.

Conclusions: GLY decreases MDR by: altering bacterial parameters, including viability and efflux pump activity. In vivo, it increases the effectiveness of ciprofloxacin, reducing ocular disease, plate count, and MPO activity.

Figure 1

MIC of GLY and ciprofloxacin. Treatment of either isolate with GLY (A, B) or MDR9 with ciprofloxacin (C) inhibited visible bacterial growth in a concentration dependent manner. Visible growth was completely inhibited by GLY at 40 mg/mL (MDR9), 15 mg/mL (B1045) and by ciprofloxacin at 32 μg/mL. GLY reduced MIC of ciprofloxacin for MDR9 2-fold (D).

Figure 2

After 18 hours GLY (concentrations shown) treatment of MDR9, confocal microscopy (A–D) showed bacteria as either red/permeabilized or green/live. Viable plate count after 18 (E) or 6 (F) hours incubation with GLY showed reduced log₁₀ cfu.

Figure 3

Scanning EM after 18 hours GLY (20 mg/mL) in vitro. Most GLY-treated bacteria (A–D) are swollen, clumped, and show membrane “holes.” A few appeared normal (C, D) and similar to controls (E, F). Magnification: (A) 4000; (B) 45,000; (C) 10,000; (D) 45,000; (E) 4500; (F) 45,000; (A, C, E) Scale bar: 1 μm. (B, D, F) Scale bar: 100 nm.

Figure 4

Proteomics: MDR9 versus PAO1. (A, B) Volcano plots; (B, C) efflux pumps significantly increased in MDR9 versus PAO1.

Figure 5

Effect of GLY (20 mg/mL or 7.5 mg/mL, 3 hours) on RND efflux pump mRNA expression for MDR9 (A–D) and B1045 (E–H). Ethidium bromide plate (I): PAO1-positive fluorescence control, MDR9 without GLY (no fluorescence) and after 18 hours treatment with 20 mg/mL GLY, fluorescence similar to control. Mean colony fluorescence (J).

Figure 6

GLY potentiates ciprofloxacin. Shown for clinical score (A) slit-lamp photo (compare ciprofloxacin + GLY [E] with PBS [B]; GLY [C] and ciprofloxacin [D]); bacterial plate count at 3 and 5 days p.i. (F), and MPO (G) at 5 days p.i.