Strains and compounds
Klebsiella pneumoniae reference strain (MTCC 432: K. pneumoniae-1) was procured from Microbial Type Culture Collection (MTCC) Chandigarh, India and E. coli MG1655 was a kind gift from Dr. Aswin Sai Narayan Seshasayee NCBS, Bangalore. The clinical isolates of Klebsiella pneumoniae and Escherichia coli were obtained from Sundaram Medical Foundation (SMF), Chennai, India. The K. pneumoniae isolates are designated as (BC936, E474, BC1415, U2016, BC1994, BC2412, U3866) and the E. coli isolates as (U3176 and U3790). All the antibiotics, media and chemicals employed in the study were purchased from Sigma Aldrich, USA, Alfa-Aesar, USA or HiMedia, India. The plant metabolites used as test compounds was from a natural product library which include caffeic acid, naringin, naringenin, arjunolic acid, ursolic acid, acetyl shikonin, β-methylacrylshikonin, chrysin, chrysophenol, ventilone, ventiloquinone, emodin and physcion. Stocks for the test compounds were freshly prepared in DMSO and stored at − 20 °C for further use.
All the test compounds were screened for their minimum inhibitory concentration (MIC) by microbroth 2-fold-dilution method to check for the antimicrobial efficacy against the E. coli and K. pneumoniae strains as reported earlier (Andrews and Andrews 2001). Similarly, the susceptibility pattern of other clinical isolates towards other antibiotics was also analyzed.
Synergy and modulation of antibiotic resistance
To understand the combinatorial activity, plant metabolites and antibiotics were used in combination at different concentrations by checkerboard assay against reference and clinical isolates as reported earlier (Lowrence et al. 2016). The Fractional inhibitory concentration (FIC) index was calculated and if FIC values are < 0.5, the interaction is synergistic, 0.5–2.0, interaction is additive and > 2, the interaction is antagonistic (Odds 2003). The colistin potentiating ability of ursolic acid against colistin resistant isolates of E. coli and K. pneumoniae was evaluated. Ursolic acid at sub-MIC concentration was used along with varying concentrations of colistin and incubated for 18–24 h at 37 °C. The fold reduction in MIC (MIC reversal) of colistin when combined with ursolic acid was determined as modulation factor as reported earlier (Lowrence et al. 2016; Sundaramoorthy et al. 2018).
Real time efflux study
To analyze the efflux pump inhibitory activity of ursolic acid against Klebsiella pneumoniae and Escherichia coli, real time efflux studies were performed using ethidium bromide as a substrate. The cells were de-energized, EtBr was added and then glucose was added to re-energize the cells, which would activate efflux. Resulting fluorescent intensity was measured with Ex 360 nm and Em 590 nm as reported earlier (Sundaramoorthy et al. 2018). The increase in fluorescent intensity of EtBr was taken as a measure of efflux inhibition activity.
Time kill assay
Bactericidal effect of ursolic acid in combination with colistin was evaluated against the XDR clinical isolates U3790 and BC936 by time kill assay (Belley et al. 2008; Grillon et al. 2016). Early log phase cells were subjected to following treatments, viz., colistin (4 μg/ml) and colistin (4 μg/ml) + ursolic acid (40 µM). Untreated culture was maintained as a growth control. The samples were withdrawn at different time intervals (0, 1, 2, 3, 4, 5 and 24 h), serially diluted and plated on agar plates. The plates were incubated at 37 °C for 24 h and from plate counts, Colony forming units (CFU)/ml was calculated and bactericidal effect of combination was discerned.
Membrane permeability and integrity assay
The ability of ursolic acid to permeabilize outer membrane of Enterobacteriaceae was assessed by 1-N-phenylethylamine (NPN) uptake assay as reported earlier (Helander and Mattila-Sandholm 2000). NPN exhibits enhanced fluorescence in phospholipid environment. Since the outer membrane (OM) of Gram negative bacteria affords steric hindrance to hydrophobic molecules and prevents NPN entry due to LPS, increased NPN fluorescence due to treatment, indicates enhanced OM permeability. Briefly, cells were grown to mid-log phase collected and washed with 5 mM HEPES buffer containing 0.2% glucose at pH 7.5 and resuspended in an equal volume of the same buffer. NPN was added at a concentration of 0.5 mM, this was immediately followed by addition of ursolic acid. Fluorescence due to NPN was measured (Ex 350 and Em 420 nm) using spectrofluorimeter (JASCO FP-8500, Jasco, Tokyo, Japan). NPN in buffer and NPN in buffer along with cells were maintained as controls.
Membrane integrity assay
Compromise in cell membrane integrity due to treatment with ursolic acid was assessed as reported previously (Marks et al. 2013). Briefly, cells after treatment were collected at different time points (0, 1, 2, 3 and 4 h), pelleted at 13,250 rcf for 5 min. The release of DNA and proteins, due to loss of inner membrane integrity, was quantified by measuring absorbance at 260 nm and 280 nm respectively using UV–Vis Spectrophotometer (Evolution 201, Thermoscientific, USA). Treatment with 0.5% Triton X 100 was used as a positive control.
Membrane potential assay
The effect of ursolic acid alone/with colistin in perturbing membrane potential was evaluated using DiSc3, a cationic membrane permeabilizing dye. Intact bacterial cells accumulate the dye in the lipid bilayer, resulting in quenching of fluorescence. When the membrane gets depolarized, dye gets released to the surrounding aqueous phase and fluorescence gets enhanced (Te Winkel et al. 2016). The fluorescent intensity (Ex 610 ± 5 nm and Em 660 ± 5 nm) of buffer with DiSc3 (1 μM) was measured initially. Mid log cells were added, which reduces the fluorescent intensity due to accumulation of dye in cells. Colistin, ursolic acid and colistin with ursolic acid treatments were given and the resulting variation in fluorescence intensity due to various treatments was quantified using spectrofluorimeter (JASCO FP-8500, Jasco, Tokyo, Japan).
Release of reactive oxygen species (ROS) from XDR E. coli and K. pneumoniae clinical isolates, in the presence of colistin and ursolic alone and in combination was discerned using fluorophore Dichloro-dihydro-fluorescein diacetate (DCFH-DA) and fluorescence of ROS induced dichlorofluorescein (DCF) formation was quantified using a fluorescence spectrophotometer (JASCO FP-8500, JASCO, Tokyo, Japan) (Ex 485 nm and Em 538 nm).
Colistin accumulation studies
In order to visualize the intracellular accumulation of colistin within the cells, colistin was conjugated using the fluorophore dansyl chloride as reported earlier (Soon et al. 2011). Intracellular accumulation of dansyl chloride conjugated colistin in response to different treatments viz., colistin-dansyl chloride, colistin-dansyl chloride + ursolic acid, colitin-dansyl chloride + CCCP was evaluated using fluorescent microscopic imaging (Nikon eclipse Ni-U, Nikon, Tokyo, Japan), to discern effect of various treatments on colistin accumulation.
Fish toxicity studies
All experiments were performed in compliance with applicable national and/or institutional guidelines for the care and use of animals (Animal Biosafety Level 2). Adult zebrafish (Danio rerio), either male/female, measuring 4 to 5 cm in length, weighing approx. 300 mg, were purchased from a local aquarium in Thanjavur, India. Animal acclimatization was performed as reported earlier (Westerfield 1995). To evaluate the effect of ursolic acid on brain and liver enzyme profiles of zebrafish, a total of 10 fish were exposed to 32 mg/L of the respective compounds for 48 h. At the end of exposure (48 h), fish were sacrificed (anesthetized by 150 mM MS-222 and euthanized by decapitation), skin removed and the liver/brain from two fish from the same group were pooled and homogenized in ice-cold buffer (Tris–HCl, 0.1 M, pH 7.4). The homogenate was centrifuged (10,000×g, 10 min, 4 °C) and supernatant used for all analyses in duplicates. Protein was estimated by the method of Lowry et al. (1951). Estimation of carboxyl esterase was essentially as described by Argentine and James (Argentine and James 1995) and acetylcholinesterase (AChE) activity was measured by Edmann’s degradation. Histopathology of ursolic acid injected fish was performed to analyze any histopathological alterations. The fish were sacrificed and fixed with 10% formalin. Thin sections were made after embedding process, stained with hematoxylin–eosin and viewed and imaged using a bright field microscope (Nikon Eclipse Ni-U, Japan).
Intramuscular infection of zebrafish (n = 6) with colistin resistant Klebsiella pneumoniae BC936 and Escherichia coli U3790 strains corresponding to OD of 0.2 (~ 1 × 106 CFU/ml) was performed as reported earlier (Neely et al. 2002) with slight modifications. 2 h post infection, compounds viz., ursolic acid/colistin alone and ursolic acid +colistin combination were administered via intramuscular injection as a single dose. 48 h post treatment, fish were euthanized, decapitated, muscle tissue was dissected, minced, serial diluted and plated onto LB agar to discern colony counts after 24 h of incubation. Based on cell counts, graph was plotted and ability of ursolic acid alone and in combination with colistin to reduce bacterial bioburden in infected muscle tissue was estimated.