Short CommunicationReinforcement of the bactericidal effect of ciprofloxacin on Pseudomonas aeruginosa biofilm by hyperbaric oxygen treatment
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.
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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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