Introduction: Pseudomonas aeruginosa is the main cause of mortality of many cystic fibrosis (CF) patients. Inherited resistance is not the only reason antibiotic treatment fails. In fact, in CF lung P. aeruginosa is able to grow as biofilm communities resistant to antibiotics and host response. P. aeruginosa can be killed by an abundance of phages isolated from a variety of sources, including sewage. While there are clear limitations to the use of phage therapy per se for treating bacterial infections, there is increasing evidence that bacterial viruses may be an effective adjunct to antibiotic treatment. In this investigation, we evaluated the efficacy of selected phages, tested alone and in combination with antibiotics, against biofilm formed by P. aeruginosa strains from CF patients. Materials and Methods: Five phages (Φ4, Φ9, Φ14, Φ17, Φ19), isolated from a local sewage treatment plant, were assayed against 24h-preformed biofilms by 33 P. aeruginosa strains collected from CF patients at different stages of infection (first, early, chronic). The five most effective phages were also tested in combination with antibiotics (tobramycin, TOB; amikacin, AK; meropenem, MPM), each tested at the MIC value, as assessed by broth microdilution technique. Control (CTRL) biofilms were exposed neither phage nor antibiotic. The dispersion of biofilm caused by exposure to phages, antibiotics or their combinations was spectrophotometrically measured by crystal violet stain. Results: All strains were able to form biofilm, although striking differences in biofilm biomass formation were observed (OD492 range: 0.055- 5.459). No relationship was observed among biofilm formation and stage of infection. Phages Φ14 and Φ19 exhibited higher efficacy against preformed biofilms, causing significant (at least 30% vs CTRL; p < 0.05) dispersion in 13.6% and 12.5% of cases, respectively. TOB and AK caused biofilm dispersion in 3 out of 5 (60%) cases, whereas MPM in 80%. Phages showed higher activity than antibiotics in 2 cases for TOB (Φ4, Φ9), and once (Φ4) for AK. Exposure to both phage and antibiotic always caused a dispersion comparable to phage or antibiotic tested alone. Exposure to antibiotic caused significant increase in biofilm biomass by strains Pa12 (TOB) and Pa21 (AK). Conclusions: The dispersion of preformed P. aeruginosa biofilm is phage- and strain-dependent. Some phages showed higher activity compared to antibiotics commonly used for treatment of CF lung infections. The phage-antibiotic combinations tested could not cause a biofilm dispersion higher than either agent alone. Further work is ongoing to test other combinations.

Efficacy of phages and antibiotic-phage combinations against biofilm by Pseudomonas aeruginosa from cystic fibrosis patients

Arianna Pompilio;Cristina Geminiani;Valentina Crocetta;Giovanni Di Bonaventura
2017

Abstract

Introduction: Pseudomonas aeruginosa is the main cause of mortality of many cystic fibrosis (CF) patients. Inherited resistance is not the only reason antibiotic treatment fails. In fact, in CF lung P. aeruginosa is able to grow as biofilm communities resistant to antibiotics and host response. P. aeruginosa can be killed by an abundance of phages isolated from a variety of sources, including sewage. While there are clear limitations to the use of phage therapy per se for treating bacterial infections, there is increasing evidence that bacterial viruses may be an effective adjunct to antibiotic treatment. In this investigation, we evaluated the efficacy of selected phages, tested alone and in combination with antibiotics, against biofilm formed by P. aeruginosa strains from CF patients. Materials and Methods: Five phages (Φ4, Φ9, Φ14, Φ17, Φ19), isolated from a local sewage treatment plant, were assayed against 24h-preformed biofilms by 33 P. aeruginosa strains collected from CF patients at different stages of infection (first, early, chronic). The five most effective phages were also tested in combination with antibiotics (tobramycin, TOB; amikacin, AK; meropenem, MPM), each tested at the MIC value, as assessed by broth microdilution technique. Control (CTRL) biofilms were exposed neither phage nor antibiotic. The dispersion of biofilm caused by exposure to phages, antibiotics or their combinations was spectrophotometrically measured by crystal violet stain. Results: All strains were able to form biofilm, although striking differences in biofilm biomass formation were observed (OD492 range: 0.055- 5.459). No relationship was observed among biofilm formation and stage of infection. Phages Φ14 and Φ19 exhibited higher efficacy against preformed biofilms, causing significant (at least 30% vs CTRL; p < 0.05) dispersion in 13.6% and 12.5% of cases, respectively. TOB and AK caused biofilm dispersion in 3 out of 5 (60%) cases, whereas MPM in 80%. Phages showed higher activity than antibiotics in 2 cases for TOB (Φ4, Φ9), and once (Φ4) for AK. Exposure to both phage and antibiotic always caused a dispersion comparable to phage or antibiotic tested alone. Exposure to antibiotic caused significant increase in biofilm biomass by strains Pa12 (TOB) and Pa21 (AK). Conclusions: The dispersion of preformed P. aeruginosa biofilm is phage- and strain-dependent. Some phages showed higher activity compared to antibiotics commonly used for treatment of CF lung infections. The phage-antibiotic combinations tested could not cause a biofilm dispersion higher than either agent alone. Further work is ongoing to test other combinations.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11564/721778
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