Targeting Pre-Formed Biofilms of the Notorious Pseudomonas aeruginosa using Anidulafungin and Antibacterial Agents in in-vitro and ex-vivo Urinary Tract Infection Models

Abstract

Background: A crucial problem in patient care is the ability of certain bacteria, including Pseudomonas aeruginosa, to form biofilms on implanted and indwelling medical devices. P. aeruginosa biofilms play a role in protecting the bacterial community, increasing antimicrobial resistance, and making the bacterium among the most threatening nosocomial pathogens. Hence, it is important to develop therapeutic interventions that would not only inhibit biofilm formation, but also be able to eradicate the pre-formed ones. Several studies investigated the presence of 1,3β-D-glucan, a major component of fungal cell wall, in P. aeruginosa biofilms. Moreover, previous studies at the department of Experimental Pathology, Immunology and Microbiology at the American University of Beirut shed light on the importance of an Echinocandin, known as Micafungin, in inhibiting the formation of biofilms in case of P. aeruginosa. Here we aimed to investigate the effectiveness of the combination therapy that involves Anidulafungin, another Echinocandin, with either Colistin, Gentamicin, or Ciprofloxacin in eradicating pre-formed P. aeruginosa biofilms in in-vitro and ex-vivo urinary tract infection models.

Methods: A P. aeruginosa PAN14 clinical strain was screened for its susceptibility against a panel of six antimicrobial agents (Colistin, Gentamicin, Ciprofloxacin, Tazocin, Ceftazidime and Cefepime). The Minimal Biofilm Inhibitory Concentration (MBIC) and the Minimal Biofilm Eradication Concentration (MBEC) were evaluated for the antimicrobials alone and in combination with Anidulafungin. PAN14 biofilms were grown on catheter sections under static and flow conditions, and on ECV304 epithelial cells to further assess the efficacy of the combination therapy. Crystal Violet stain was performed to ensure the presence of biofilms, and Scanning Electron Microscopy (SEM) in order to visualize any morphological changes that took place following treatment.

Results: Serial broth microdilution assay revealed that PAN14 planktonic cells display susceptibility to all tested antimicrobials. Moreover, combining variable concentrations of Anidulafungin to Colistin, Gentamicin, and Ciprofloxacin lead to a decrease in the MBIC and MBEC values. Crystal Violet stain ensured the presence of biofilms on catheter sections, and SEM images revealed complete eradication of PAN14 preformed biofilms in case of combining Anidulafungin with Colistin or Gentamicin, but not with Ciprofloxacin. PAN14 biofilms developed successfully on ECV304 cells for further assessment of the eradication effect of the combination therapy.

Conclusion: This study enabled us to assess the efficacy of combining Anidulafungin with certain antibacterial agents in eradicating pre-formed biofilms, and would therefore pave the way for further assessments of Anidulafungin as well as other Echinocandins in vivo and in potential clinical trials.