Abstract
ABSTRACTMultidrug-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.
Highlights
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
PYO is recognized as a virulence factor (VF), because this molecule is helpful to P. aeruginosa colonization in several ways, including cytotoxicity toward host ciliated epithelial cells [17], immune cells [18], and competing microbes [19]
Given that in patients with P. aeruginosa lung infections, airway epithelial cells are in a position to encounter relatively high concentrations of both pseudomonal phenazines [15] and the inhaled antibiotics colistimethate sodium (CMS) and formed colistin [14], we asked whether such an encounter poses a potential hazard
Summary
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. 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. In two randomized clinical trials [12, 13], inhalation of CMS in the forms of a nebulized solution and powder caused repeated treatment-related adverse events in the respiratory system of 35% and 82% of CF patients, respectively It is not currently known by which mechanism(s) inhaled CMS may produce pulmonary side effects. Given that in patients with P. aeruginosa lung infections, airway epithelial cells are in a position to encounter relatively high concentrations of both pseudomonal phenazines [15] and the inhaled antibiotics CMS and formed colistin [14], we asked whether such an encounter poses a potential hazard. We demonstrate that CMS can interact with PYO by way of producing a synergistically enhanced cytotoxic effect against airway epithelial cells in vitro and identify inhibitors capable of modulating this cytotoxicity
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