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

Date Published: January 23, 2017

Publisher: Public Library of Science

Author(s): Maria E. Rodrigues, Susana P. Lopes, Cláudia R. Pereira, Nuno F. Azevedo, Anália Lourenço, Mariana Henriques, Maria O. Pereira, Joy Sturtevant.


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.

Partial Text

Ventilator-associated pneumonia (VAP) is a respiratory infectious disease, now recognized as having a polymicrobial nature. VAP occurs 48–72 hours after endotracheal intubation and has an associated estimated mortality of 10–40% [1]. The starting-point for VAP development is the presence of an endotracheal tube (ETT), which allows the leakage of contaminated oropharyngeal secretions down to the lungs and is prone to microbial colonization [2]. A wide spectrum of pathogens is able to attach the ETT surface. Pseudomonas aeruginosa stands out in these infections, ranking for higher fatality rates [3], mainly due to its ability to develop biofilms resilient to antibiotic therapy. Isolation of fungal species, such as Candida albicans, is also common from tracheal secretions, but usually leads only to the colonization of the airways, rather than causing pneumonia in critically-ill patients [4, 5]. However, the risk of VAP due to infection by P. aeruginosa is facilitated and markedly increased in patients displaying C. albicans tracheobronchial colonization [6, 7]. Both P. aeruginosa and C. albicans have tendency to form resistant polymicrobial biofilms, playing extensive ecological roles in nosocomial infections, such as VAP [8, 9]. Co-infection by both species has also been well documented, with ample evidence supporting the multifaceted bacterial-fungal and/or bacterial/fungal-host interactions [10–22]. So far, no reliable methods are currently available to detect ETT’s biofilms while the patient remains on invasive mechanical ventilation. Additionally, only few preventive and therapeutic strategies to reduce ETT biofilm formation and VAP have been tested in clinical settings [23–26].

The growing evidence that most respiratory infections are developed throughout complex processes involving several pathogens [59] could partially explain the lack of response to conventional therapeutic regimens that primarily target single causative agents instead of all members in the consortia. So, it is strongly suggested that the antimicrobial therapy outcome in mixed-species infections may be severely impacted by the number, type and interplay of microorganisms within the polymicrobial communities. In light of the recent reports regarding complex interactions between P. aeruginosa and C. albicans in VAP [60–62], the present study intended to understand how these species respond to individual and combined antimicrobial therapy using antibiotic and antifungal agents.

The role of polymicrobial biofilms in infectious diseases, such as VAP, is of utmost importance and will probably direct novel therapies that target the multiplicity of species within the consortia by avoiding the enhanced pathogenesis that results from interactions among the causative microbes of such infections. The increased incidence of drug resistant fungi and bacteria in polymicrobial consortia has resulted, in part, from the intense use of antifungals and antibiotics in clinical settings. For therapies to be maximally effective, however, anti-biofilm therapeutic interventions will be required. Here, combination of an antibiotic agent (PolyB) and an antifungal agent (AmB) had shown a potential synergy therapeutic effect against polymicrobial communities involving P. aeruginosa and C. albicans. However, for clinical application purposes, such combination should be optimized (e.g. antimicrobial concentrations, and timing of administration) to avoid reinfection.




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