Bactericidal effect of colistin on planktonic Pseudomonas aeruginosa is independent of hydroxyl radical formation

Abstract

The bactericidal effect of several major types of antibiotics has recently been demonstrated to be dependent on the formation of toxic amounts of hydroxyl radicals (OH) resulting from oxidative stress in metabolically active cells. Since killing by the antimicrobial peptide colistin does not require bacterial metabolic activity, we tested whether the bactericidal effect of colistin depends on the formation of OH. In Pseudomonas aeruginosa cultures, OH-mediated killing by ciprofloxacin was demonstrated by decreased bacterial survival and induction of 3′-(p-hydroxyphenyl) fluorescein (HPF) fluorescence. OH-mediated killing by ciprofloxacin was further confirmed by rescue of cells and reduction of HPF fluorescence due to prevention of OH accumulation by scavenging with thiourea, by chelating with dipyridyl, by decreasing metabolism as well as by anoxic growth. In contrast, no formation of OH was seen in P. aeruginosa during killing by colistin, and prevention of OH accumulation could not rescue P. aeruginosa from killing by colistin. These results therefore demonstrate that the bactericidal activity of colistin on P. aeruginosa is not dependent on oxidative stress. In conclusion, antimicrobial peptides that do not rely on OH formation should be considered for treatment of Gram-negative bacteria growing at low oxygen tension such as in endobronchial mucus and paranasal sinuses in cystic fibrosis patients, in abscesses and in infectious biofilm.

Introduction

Polymyxin E (colistin) is a bactericidal lipopeptide antibiotic active against Gram-negative bacteria. The mechanisms of action of the cationic colistin include disruption of the cytoplasmic membrane through interaction with cationic binding sites on the cell surface lipopolysaccharides, which destabilises the outer membrane and promotes its own uptake [1]. However, solid data to support disruption of the cytoplasmic membrane of Gram-negative bacteria by polymyxins at clinically relevant concentrations remain to be established. Owing to its nephrotoxicity, use of colistin in patients with cystic fibrosis (CF) has been restricted to inhalation treatment of lung infections caused by Pseudomonas aeruginosa. However, colistin has resurfaced as a last-line treatment option for multidrug-resistant organisms such as P. aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae as well as for carbapenemase-producing Enterobacteriaceae in other infections [2].

Recently, a common mechanism of killing by bactericidal antibiotics has been suggested to involve the formation of harmful hydroxyl radicals (OH) through the Fenton reaction. According to this model, transient hyperoxidation by the respiratory chain of NADH from the tricarboxylic acid (TCA) cycle leads to production of superoxide (O₂⁻) causing release of iron from iron–sulphur clusters resulting in substrates for generation of toxic amounts of OH by the Fenton reaction. Consequently, it is suggested that killing by bactericidal antibiotics depends on the bacterial metabolic state, i.e. the TCA cycle [3]. According to the proposed underlying mechanism, both O₂⁻ and H₂O₂ formation are involved in the production of OH [3]. But while O₂⁻ and H₂O₂ can be enzymatically eradicated by superoxide dismutases, catalases and peroxidases, no known enzyme is able to catalyse the cellular detoxification of OH, which may induce lethal oxidative lesions on proteins, lipids and DNA [4]. Killing by colistin, however, is reduced by bacterial metabolic activity [5], [6], suggesting that the bactericidal activity of colistin does not necessitate formation of OH by hyperoxidation of NADH from the TCA cycle. In addition, it may be presumed that formation of OH requires the presence of molecular oxygen (O₂), which emphasises the need to consider the type and location of infection carefully when selecting optimal antibiotic treatment. In particular, the infected mucus of CF patients contains anaerobic zones [7], which are mainly due to oxygen consumption by the summoned neutrophils [8]. Furthermore, decreased mucosal O₂ tension prevails in the paranasal sinuses of CF patients, which represent a gateway for infection in the lung [9]. Based on this knowledge, we were interested in investigating: (i) the role of reactive oxygen species (ROS) formation during killing of P. aeruginosa by colistin; (ii) the effect on bacterial killing of ROS modulators such as thiourea for scavenging of OH [10] and chelation of ferrous iron with dipyridyl in order to prevent accumulation of OH through Fenton reactions [11]; and (iii) the effect of limitation of nutrients and O₂ on OH formation and bacterial killing during antibiotic treatment. Therefore, we have constructed bacterial killing curves and estimated OH formation during antibiotic treatment in aerobic and anaerobic conditions as well as during nutrient limitation. For comparison, ciprofloxacin was used as an antipseudomonal drug, with the effect depending on oxidative stress in metabolically active cells [3].