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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 ?g/mL) was reduced 2-fold to 16 ?g/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.

SUBMITTER: Hazlett LD

PROVIDER: S-EPMC6944246 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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

Hazlett Linda D LD Ekanayaka Sandamali A SA McClellan Sharon A SA Francis Rebecca R

Investigative ophthalmology & visual science 20190701 8

<h4>Purpose</h4>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.<h4>Methods</h4>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 perme ...[more]

PMID: 31311033

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Project description:The increasing prevalence of multi-drug-resistant (MDR) strains of Pseudomonas aeruginosa among critically ill humans is of significant concern. In the current study, we show that MDR clinical isolates of P. aeruginosa representing three distinct genotypes that display high virulence against intestinal epithelial cells, form novel appendage-like structures on their cell surfaces. These appendages contain PstS, an extracellular phosphate binding protein. Using anti-PstS antibodies, we determined that the PstS-rich appendages in MDR strains are involved in adherence to and disruption of the integrity of cultured intestinal epithelial cell monolayers. The outer surface-expressed PstS protein was also identified to be present in P. aeruginosa MPAO1, although to a lesser degree, and its role in conferring an adhesive and barrier disruptive phenotype against intestinal epithelial cells was confirmed using an isogenic DeltaPstS mutant. Formation of the PstS rich appendages was induced during phosphate limitation and completely suppressed in phosphate-rich media. Injection of MDR strains directly into the intestinal tract of surgically injured mice, a known model of phosphate limitation, caused high mortality rates (60%-100%). Repletion of intestinal phosphate in this model completely prevented mortality. Finally, significantly less outer surface PstS was observed in the MPAO1 mutant DeltaHxcR thus establishing a role for the alternative type II secretion system Hxc in outer surface PstS expression. Gene expression analysis performed by RT-PCR confirmed this finding and further demonstrated abundant expression of pstS analogous to pa5369, pstS analogous to pa0688/pa14-55410, and hxcX in MDR strains. Taken together, these studies provide evidence that outer surface PstS expression confers a highly virulent phenotype of MDR isolates against the intestinal epithelium that alters their adhesive and barrier disrupting properties against the intestinal epithelium.

Dataset Information

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

Publications

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

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