Using Bacteriophages as a Trojan Horse to the Killing of Dual-Species Biofilm Formed by Pseudomonas aeruginosa and Methicillin Resistant Staphylococcus aureus.

Tamta Tkhilaishvili,Carsten Perka,Andrej Trampuz,Mercedes Gonzalez Moreno,Lei Wang

doi:10.3389/fmicb.2020.00695

Abstract

Pseudomonas aeruginosa and Staphylococcus aureus are pathogens able to colonize surfaces and form together a mixed biofilm. Dual-species biofilms are significantly more resistant to antimicrobials than a monomicrobial community, leading to treatment failure. Due to their rapid bactericidal activity, the self-amplification ability and the biofilm degrading properties, bacteriophages represent a promising therapeutic option in fighting biofilm-related infections. In this study, we investigated the effect of either the simultaneous or staggered application of commercially available phages and ciprofloxacin versus S. aureus/P. aeruginosa dual-species biofilms in vitro. Biofilms were grown on porous glass beads and analyzed over time. Different techniques such as microcalorimetry, sonication and scanning electron microscopy were combined for the evaluation of anti-biofilm activities. Both bacterial species were susceptible to ciprofloxacin and to phages in their planktonic form of growth. Ciprofloxacin tested alone against biofilms required high concentration ranging from 256 to >512 mg/L to show an inhibitory effect, whereas phages alone showed good and moderate activity against MRSA biofilms and dual-species biofilms, respectively, but low activity against P. aeruginosa biofilms. The combination of ciprofloxacin with phages showed a remarkable improvement in the anti-biofilm activity of both antimicrobials with complete eradication of dual-species biofilms after staggered exposure to Pyophage or Pyophage + Staphylococcal phage for 12 h followed by 1 mg/L of ciprofloxacin, a dose achievable by intravenous or oral antibiotic administration. Our study provides also valuable data regarding not only dosage but also an optimal time of antimicrobial exposure, which is crucial in the implementation of combined therapies.

Highlights

Many common infectious diseases can be initiated by a single pathogen or virulence factor, others can be attributed to a polymicrobial origin (Peters et al, 2012)
The evaluation of the bacteria adhered to the beads over time showed a considerably higher concentration of Methicillin-resistant S. aureus (MRSA) cells at 3 and 6 h of incubation in dual-species biofilms compared to P. aeruginosa cells (Figure 1A,B), whereas at 12 h of incubation the concentration of P. aeruginosa increased substantially (Figure1C) and at 24 h of incubation the concentration of P. aeruginosa showed values comparable to those from MRSA (Figure 1D)
S. aureus and P. aeruginosa are two bacterial pathogens commonly isolated in mixed-species biofilm infections (Hotterbeekx et al, 2017)

Summary

Introduction

Many common infectious diseases can be initiated by a single pathogen or virulence factor, others can be attributed to a polymicrobial origin (Peters et al, 2012). Treatment is often complicated due to the synergies of polymicrobial biofilms on limiting the effectiveness of antibiotics (Wolcott et al, 2013). The lack of effective therapies against polymicrobial biofilm infections is a pressing need for the development of new antimicrobial strategies. There are only limited studies investigating the activity of phages against polymicrobial biofilms (Sillankorva et al, 2010; Kay et al, 2011; Chhibber et al, 2015; Oliveira et al, 2018; Melo et al, 2019) and just recently Akturk et al (2019) evaluated the simultaneous and staggered administration of a P. aeruginosa-targeting monophage and conventional antibiotics on S. aureus/P. aeruginosa dualspecies biofilms

Methods

Results

Conclusion