Polymicrobial Ventilator-Associated Pneumonia: Fighting In Vitro Candida albicans-Pseudomonas aeruginosa Biofilms with Antifungal-Antibacterial Combination Therapy

Abstract

The polymicrobial nature of ventilator-associated pneumonia (VAP) is now evident, with mixed bacterial-fungal biofilms colonizing the VAP endotracheal tube (ETT) surface. The microbial interplay within this infection may contribute for enhanced pathogenesis and exert impact towards antimicrobial therapy. Consequently, the high mortality/morbidity rates associated to VAP and the worldwide increase in antibiotic resistance has promoted the search for novel therapeutic strategies to fight VAP polymicrobial infections. Under this scope, this work aimed to assess the activity of mono- vs combinational antimicrobial therapy using one antibiotic (Polymyxin B; PolyB) and one antifungal (Amphotericin B; AmB) agent against polymicrobial biofilms of Pseudomonas aeruginosa and Candida albicans. The action of isolated antimicrobials was firstly evaluated in single- and polymicrobial cultures, with AmB being more effective against C. albicans and PolyB against P. aeruginosa. Mixed planktonic cultures required equal or higher antimicrobial concentrations. In biofilms, only PolyB at relatively high concentrations could reduce P. aeruginosa in both monospecies and polymicrobial populations, with C. albicans displaying only punctual disturbances. PolyB and AmB exhibited a synergistic effect against P. aeruginosa and C. albicans mixed planktonic cultures, but only high doses (256 mg L-1) of PolyB were able to eradicate polymicrobial biofilms, with P. aeruginosa showing loss of cultivability (but not viability) at 2 h post-treatment, whilst C. albicans only started to be inhibited after 14 h. In conclusion, combination therapy involving an antibiotic and an antifungal agent holds an attractive therapeutic option to treat severe bacterial-fungal polymicrobial infections. Nevertheless, optimization of antimicrobial doses and further clinical pharmacokinetics/pharmacodynamics and toxicodynamics studies underpinning the optimal use of these drugs are urgently required to improve therapy effectiveness and avoid reinfection.

Fig 1

Therapeutic effect of AmB (a) and PolyB (b) against 24 h-old single- and dual-species biofilms formed by P. aeruginosa and C. albicans. *P<0.05 indicates a statistically different reduction in comparison with the respective control (corresponding to 0 mg L^-1).

Fig 2. Effect of AmB and PolyB, combined at different concentrations, against 24 h-old dual-species biofilms formed by P. aeruginosa and C. albicans.

*P<0.05 indicates statistically different reduction in comparison with the respective control (corresponding to 0 mg L^-1).

Fig 3

Time-kill kinetics obtained for the combinatorial activity of AmB (at 0.0156 mg L^-l) and PolyB (at 256 mg L^-l) against dual-species biofilms of P. aeruginosa and C. albicans (a) and epifluorescence images from mixed C. albicans and P. aeruginosa biofilms 24 h post-treatment discriminated by PNA FISH assay (b) and stained with LIVE/DEAD® staining system (c).