Uptake of and Resistance to the Antibiotic Berberine by Individual Dormant, Germinating and Outgrowing Bacillus Spores as Monitored by Laser Tweezers Raman Spectroscopy.

Shiwei Wang,Milomir Suvira,Yong-Qing Li,Jing Yu,Peter Setlow

doi:10.1371/journal.pone.0144183

Shiwei Wang, Milomir Suvira + Show 3 more

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https://doi.org/10.1371/journal.pone.0144183

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Abstract

Berberine, an alkaloid originally extracted from the plant Coptis chinensis and other herb plants, has been used as a pharmacological substance for many years. The therapeutic effect of berberine has been attributed to its interaction with nucleic acids and blocking cell division. However, levels of berberine entering individual microbial cells minimal for growth inhibition and its effects on bacterial spores have not been determined. In this work the kinetics and levels of berberine accumulation by individual dormant and germinated spores were measured by laser tweezers Raman spectroscopy and differential interference and fluorescence microscopy, and effects of berberine on spore germination and outgrowth and spore and growing cell viability were determined. The major conclusions from this work are that: (1) colony formation from B. subtilis spores was blocked ~ 99% by 25 μg/mL berberine plus 20 μg/mL INF55 (a multidrug resistance pump inhibitor); (2) 200 μg/mL berberine had no effect on B. subtilis spore germination with L-valine, but spore outgrowth was completely blocked; (3) berberine levels accumulated in single spores germinating with ≥ 25 μg/mL berberine were > 10 mg/mL; (4) fluorescence microscopy showed that germinated spores accumulated high-levels of berberine primarily in the spore core, while dormant spores accumulated very low berberine levels primarily in spore coats; and (5) during germination, uptake of berberine began at the time of commitment (T1) and reached a maximum after the completion of CaDPA release (Trelease) and spore cortex lysis (Tlysis).

Highlights

Gram-positive spore-forming bacteria of various Bacillus and Clostridium species have long been of significant research interest, since spores of some of these species cause food spoilagePLOS ONE | DOI:10.1371/journal.pone.0144183 December 4, 2015Berberine Uptake in Individual Bacillus Spores
When berberine was combined with 20 μg/mL of the multidrug resistance pumps (MDRs) inhibitor INF55, colony formation from spores was inhibited completely at 25 μg/mL berberine (Fig 1A), suggesting that MDRs play a role in protecting B. subtilis against berberine
Previous work has shown that berberine blocks growth of both Gram negative and Gram positive bacteria including B. subtilis, and the mechanisms of berberine action include both binding to nucleic acids as well as inhibiting cell division by binding to the crucial cell division protein FtsZ [11,12,13,14,19]

Summary

Introduction

Gram-positive spore-forming bacteria of various Bacillus and Clostridium species have long been of significant research interest, since spores of some of these species cause food spoilage. This commitment step is likely coincident with the beginning of rapid release of monovalent cations and initiation of slow release of 15–30% of the spore core’s large pool of the 1:1 chelate of pyridine-2,6-dicarboxylic acid (DPA) with divalent cations, predominantly Ca2+ (CaDPA) This slow CaDPA release is followed by rapid release of all remaining CaDPA, degradation of the spores’ peptidoglycan cortex by cortex-lytic enzymes (CLEs) leading to the swelling of the spore core and much water uptake leading to initiation of metabolism [5]. Laser tweezers Raman spectroscopy (LTRS) in combination with fluorescence and DIC microscopy were used to analyze the location, levels and uptake kinetics of berberine in individual dormant and germinated B. subtilis spores. The effects of combinations of berberine and an MDR inhibitor, INF55, on spore germination and outgrowth were examined

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