Short Communication
Reinforcement of the bactericidal effect of ciprofloxacin on Pseudomonas aeruginosa biofilm by hyperbaric oxygen treatment

https://doi.org/10.1016/j.ijantimicag.2015.12.005Get rights and content

Highlights

  • Recent data suggest that lack of O2 adds to bacterial tolerance to antibiotics.

  • We have developed a biofilm model with large zones of oxygen depletion.

  • Hyperbaric oxygen treatment (HBOT) results in reoxygenation of the biofilm model.

  • HBOT caused increased killing of Pseudomonas aeruginosa biofilm by ciprofloxacin.

  • HBOT may reinforce the activity of ciprofloxacin against P. aeruginosa biofilm.

Abstract

Chronic Pseudomonas aeruginosa lung infection is the most severe complication in cystic fibrosis patients. It is characterised by antibiotic-tolerant biofilms in the endobronchial mucus with zones of oxygen (O2) depletion mainly due to polymorphonuclear leucocyte activity. Whilst the exact mechanisms affecting antibiotic effectiveness on biofilms remain unclear, accumulating evidence suggests that the efficacy of several bactericidal antibiotics such as ciprofloxacin is enhanced by stimulation of the aerobic respiration of pathogens, and that lack of O2 increases their tolerance. Reoxygenation of O2-depleted biofilms may thus improve susceptibility to ciprofloxacin possibly by restoring aerobic respiration. We tested such a strategy using reoxygenation of O2-depleted P. aeruginosa strain PAO1 agarose-embedded biofilms by hyperbaric oxygen treatment (HBOT) (100% O2, 2.8 bar), enhancing the diffusive supply for aerobic respiration during ciprofloxacin treatment. This proof-of-principle study demonstrates that biofilm reoxygenation by HBOT can significantly enhance the bactericidal activity of ciprofloxacin on P. aeruginosa. Combining ciprofloxacin treatment with HBOT thus clearly has potential to improve the treatment of P. aeruginosa biofilm infections.

Introduction

Pseudomonas aeruginosa persists and grows in biofilms in the endobronchial mucus in cystic fibrosis (CF) patients [1], and the endobronchial secretions of CF patients with chronic P. aeruginosa lung infection exhibit a stratified microenvironment that imposes physiological constraints on the pathogens [2], [3], [4]. Polymorphonuclear leukocytes (PMNs) accumulating around P. aeruginosa biofilm aggregates can induce intense oxygen (O2) consumption during their respiratory burst and the formation of nitric oxide, thereby impeding aerobic respiration of P. aeruginosa [5], [6]. However, P. aeruginosa can adapt to such PMN-induced microenvironmental changes via anaerobic respiration by denitrification in the anoxic zones of endobronchial secretions [3], [4]. A PMN-imposed restriction of O2 availability is further verified by a positive correlation between PMN density and slow growth of P. aeruginosa in infected CF lungs [7], where hypoxia, anaerobic respiration and fermentation cause slower growth by P. aeruginosa than under normoxic conditions [8], [9].

The tolerance of P. aeruginosa against the active host response and intense antibiotic treatment in the CF lungs is believed to be enhanced by the formation of biofilm [10] and limited O2 availability due to bacterial consumption [11], but the significance of O2 availability in the environment surrounding the biofilm, including the role of PMNs, remains to be studied in detail.

Several bactericidal antibiotics such as β-lactams, aminoglycosides and fluoroquinolones target bacterial aerobic respiration [12], [13], [14]. Their efficacy may thus be highly sensitive to the availability of O2 and is has been suggested that the metabolic status of pathogenic bacteria can affect their susceptibility towards bactericidal antibiotics [11], [12], [13]. It is well known that hyperbaric oxygen treatment (HBOT) of planktonic P. aeruginosa can enhance the efficacy of antibiotic treatment [15], [16]. In this study, we established an agar-embedded P. aeruginosa biofilm model in order to immobilise P. aeruginosa in zones of anoxia resembling the observations of microbial activity in the anoxic parts of sputum from CF patients [3], [4].

This model was used to investigate whether anoxic agar-embedded P. aeruginosa biofilms resilient to ciprofloxacin could be sensitised by HBOT alleviating O2 limitation and increasing the diffusive O2 supply and thus aerobic respiration in the biofilm.

Section snippets

Bacterial strain, media and antibiotics

Wild-type P. aeruginosa strain PAO1 was obtained from the Pseudomonas Genetic Stock Centre (http://www.pseudomonas.med.ecu.edu). The minimum inhibitory concentration (MIC) of the strain was 0.125 mg/L as determined by Etest (bioMérieux, Ballerup, Denmark). The strain was grown in Luria–Bertani (LB) broth [5 g/L yeast extract (Oxoid, Roskilde, Denmark), 10 g/L tryptone (Oxoid) and 10 g/L NaCl (Merck, Rahway, NJ), pH 7.5], incubated overnight at 37 °C and shaken at 170 rpm. The bactericidal antibiotic

Effect of hyperbaric oxygen treatment on the survival of Pseudomonas aeruginosa biofilms during ciprofloxacin treatment

Significantly less bacteria survived 4 h of treatment with ciprofloxacin when HBOT was applied (P = 0.0008), whereas no effect of HBOT on survival was observed after 2 h of ciprofloxacin treatment, indicating a time-dependent increased killing by supplemental HBOT (Fig. 1). Compared with normoxic conditions, HBOT did not affect the density of P. aeruginosa in untreated biofilms, and prolongation of HBOT from 2 h to 4 h did not increase the density of P. aeruginosa in untreated biofilm (Fig. 1).

Reoxygenation of Pseudomonas aeruginosa biofilms by hyperbaric oxygen treatment during ciprofloxacin treatment

The

Discussion

Lung mucus in chronically infected CF patients harbours stratified microhabitats, wherein bacterial biofilm aggregates are surrounded by PMNs creating localised O2 depletion, and where P. aeruginosa can survive without O2 owing to its ability to grow, albeit more slowly, on denitrification [3]. The O2 exposure of P. aeruginosa biofilms can thus modulate their growth rate and thus potentially their susceptibility to antibiotic treatment. In this study, we have demonstrated that reoxygenation of

Acknowledgement

The authors are indebted to Senior Hyperbaric Supervisor Michael Bering Sifakis in assisting with chamber support and maintenance.

Funding: Financial support was provided by a UC-CARE (University of Copenhagen–Center for Antimicrobial Research) grant to MKo; and the Danish Research Council for Independent Research|Technology and Production Sciences to MKü.

Competing interests: None declared.

Ethical approval: Not required.

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    Citation Excerpt :

    The distinct growth rates of bacteria in acute and chronic lung infections may provide important clues for advancing antibiotic treatment. The knowledge that the effect of antibiotic treatment of bacterial biofilm is enhanced when the growth rate of the bacteria is accelerated by supplemental oxygen [24–28] could serve a strategy for improving treatment of chronic lung infections, where clinical cure is complicated and rare. In addition, supplemental nutrients or metal traces may induce oxidative stress-mediated enhancement of the activity of antibiotics against biofilm [29–34].

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