Interaction of Bacterial Phenazines with Colistimethate in Bronchial Epithelial Cells

Abstract

Multidrug-resistant bacterial infections are being increasingly treated in clinics with polymyxins, a class of antibiotics associated with adverse effects on the kidney, nervous system, or airways of a significant proportion of human and animal patients. Although many of the resistant pathogens display enhanced virulence, the hazard of cytotoxic interactions between polymyxin antibiotics and bacterial virulence factors (VFs) has not been assessed, to date. We report here the testing of paired combinations of four Pseudomonas aeruginosa VF phenazine toxins, pyocyanin (PYO), 1-hydroxyphenazine (1-HP), phenazine-1-carboxylic acid (PCA), and phenazine-1-carboxamide (PCN), and two commonly prescribed polymyxin drugs, colistin-colistimethate sodium (CMS) and polymyxin B, in three human airway cell lines, BEAS-2B, HBE-1, and CFT-1. Cytotoxicities of individual antibiotics, individual toxins, and their combinations were evaluated by the simultaneous measurement of mitochondrial metabolic, total transcriptional/translational, and Nrf2 stress response regulator activities in treated cells. Two phenazines, PYO and 1-HP, were cytotoxic at clinically relevant concentrations (100 to 150 μM) and prompted a significant increase in oxidative stress-induced transcriptional activity in surviving cells. The polymyxin antibiotics arrested cell proliferation at clinically achievable (<1 mM) concentrations as well, with CMS displaying surprisingly high cytotoxicity (50% effective dose [ED₅₀] = 180 μM) in BEAS-2B cells. The dose-response curves were probed by a median-effect analysis, which established a synergistically enhanced cytotoxicity of the PYO-CMS combination in all three airway cell lines; a particularly strong effect on BEAS-2B cells was observed, with a combination index (CI) of 0.27 at the ED₅₀ PCA, PCN, and 1-HP potentiated CMS cytotoxicity to a smaller extent. The cytotoxicity of CMS could be reduced with 10 mM N-acetyl-cysteine. Iron chelators, while ineffective against the polymyxins, could rescue all three bronchial epithelial cell lines treated with lethal PYO or CMS-PYO doses. These findings suggest that further evaluations of CMS safety are needed, along with a search for means to moderate potentially cytotoxic interactions.

Keywords: colistimethate sodium; cytotoxicity; phenazines; polymyxins; pyocyanin; synergism.

FIG 1

Cytotoxicity of polymyxins and P. aeruginosa phenazines in BEAS-2B cells. The mitochondrial metabolic activity was evaluated fluorimetrically by measuring rates of resazurin reduction. Transcriptional/translational activity was assessed by the determination of destabilized GFP in cell lysates. The relative activity of the stress-responsive transcriptional factor Nrf2 was calculated by normalizing the luminescence readings with the GFP fluorescence values from the same wells. The open and closed circles correspond to 12-h (plus 12-h recovery) and 24-h drug exposure schedules. The error bars are standard deviations (SDs) for at least 3 biological samples. Conversion formulae for the drug concentration are as follows: 1 μM equals 1.4 mg/liter colistin sulfate, 1.45 mg/liter polymyxin B sulfate, or 1.75 mg/liter colistimethate sodium.

FIG 2

Combination indexes (CI) calculated for different levels of cytotoxicity effects. Each indicated drug combination was tested against BEAS-2B, HBE-1, and CFT-1 cells with the 24-h schedule. Cellular survival/proliferation was assayed with resazurin. The CI values were determined for 50%, 75%, and 90% cell inhibition effects; in each curve, these values are depicted by the left, central, and right points, respectively. The gray area indicates the additivity range, arbitrarily established from the “self-versus-self” combination data (CMS-CMS and PYO-PYO in Table 2). The error bars are SDs for at least 3 experiments.

FIG 3

Isobolographic depiction of interactions in selected polymyxin-phenazine combinations following the 24-h drug exposure schedule. The isobole labels represent surviving fractions of cells treated with the combinations. A linear shape of the isoboles indicates additivity, while bending of the isoboles toward the origin point signifies a synergistic interaction; the opposite bend for the CST-PYO combination versus BEAS-2B cells suggests weak antagonism between the drugs.

FIG 4

Activity of the Nrf2 transcription factor in BEAS-2B cells in response to 24-h exposures to combinations of CST-PYO (A), CMS-PYO (B), and PMB-PYO (C). The contour labels and the background color denote fold increases in luminescence relative to the values for untreated control wells.

FIG 5

Effects of antioxidants on cytotoxicity of pyocyanin (PYO), colistimethate (CMS), and the CMS-PYO and CMS–1-hydroxyphenazine (1-HP) combinations in three human bronchial epithelial cell lines, as measured after a 24-h exposure in the resazurin assay. Gray bars correspond to antioxidants that are capable of reducing reactive oxidants, and slanted patterned bars correspond to strong iron chelators. Specific concentrations of the cytotoxic agents are 400 μM PYO (A), 600 μM PYO (E and I), 600 μM CMS (B), 1 mM CMS (F and J), 300 μM CMS–200 μM PYO (C), 500 μM CMS–200 μM PYO (G and K), and 300 μM CMS–100 μM 1-HP (D, H, and L). Specific concentrations of antioxidants are 10 mM N-acetyl-cysteine (NAC), 50 μM tyrphostin AG490 (AG490), 100 μM chlorogenic acid (ChlA), 200 μM deferasirox (Dfx), 100 μM EUK134 (cell-permeable catalase/SOD mimic), 50 μM o-phenanthroline (o-Phen), and 100 μM resveratrol (Resv). The error bars are SDs for 3 experiments. Statistical significance between the “no-inhibitor” and antioxidant effects was probed by analysis of variance; for clarity, Ø denotes statistical nonsignificance (P ≥ 0.05).

FIG 6

The transcriptional activity reporter construct. A block of eight antioxidant/electrophile response elements, the sites for binding the cellular stress master regulator Nrf2, is followed by a minimal cytomegalovirus (mCMV) promoter that is followed by a Brazilian click beetle green luciferase gene. In addition, the EF-1 promoter provides continuous transcription of destabilized green fluorescent protein and puromycin resistance genes. A pair of flanking insulators (Ins) protects the construct from epigenetic silencing (25), while the piggyBac transposon inverted terminal repeats (ITR) provide a clean insertion of the reporter into transcriptionally active regions of cellular chromatin.