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

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.

Keywords: Pseudomonas aeruginosa; antibiotic-bacteriophage combination; bacteriophages; biofilm-associated infection; dual-species biofilm; isothermal microcalorimetry; methicillin-resistant Staphylococcus aureus; scanning electron microscopy.

FIGURE 1

Distribution pattern of bacterial populations over the time. Number of viable cells (in log10 CFU/mL) of P. aeruginosa and MRSA on mono- and dual-species biofilms formed after 3 h (A), 6 h (B), 12 h (C) and 24 h (D). Data are reported as CFU/mL mean ± standard deviation of at least three independent experiments. Figure 2 | Dual-species biofilm formed by MRSA (ATCC 43300) and P. aeruginosa (ATCC 27853) on porous glass beads after 24 h of incubation. Image (B) is a close-up from (A). Numbers 1 and 2 indicate a MRSA bacterium and a P. aeruginosa bacterium, respectively, whereas three and four point out a water channel and the extracellular polymeric matrix of the biofilm correspondingly.

FIGURE 2

SEM analysis of dual-species biofilm formed by MRSA (ATCC 43300) and P. aeruginosa (ATCC 27853) on porous glass beads after 24h of incubation. Image (b) is a close-up from (a). Numbers 1 and 2 indicate a MRSA bacterium and a P. aeruginosa bacterium respectively, whereas 3 and 4 point out a water channel and the extracellular polymeric matrix of the biofilm correspondingly.

FIGURE 3

Microcalorimetry analysis of planktonic MRSA ATCC 43300 (A) and P. aeruginosa ATCC 27853 (B) cells treated with two-fold increasing concentrations of ciprofloxacin (left column, numbers represent concentrations in mg/L of antibiotic) or with phages (right column). A circled value represents the MHIC, defined as the lowest concentration of antimicrobial able to suppress the metabolic heat production of planktonic bacteria. GC, growth control (dashed line); NC, negative control. Data of a representative experiment are reported.

FIGURE 4

Microcalorimetry analysis of (A,B) mono- and (C) dual-species MRSA (ATCC 43300) and P. aeruginosa (ATCC 27853) biofilms treated with two-fold increasing concentrations of ciprofloxacin (left column, numbers represent concentrations in mg/L of antibiotic) or with phages (right column). Each curve shows the heat produced by viable bacteria present in the biofilm after 24 h of antibiotic or phage treatment. A circled value represents the MBEC, defined as the lowest concentration of antibiotic that strongly reduced the viability of biofilm cells leading to the absence of heat flow production from treated beads when incubated during 48 h in fresh medium and no colonies after sonication and plating. GC, growth control (dashed line); NC, negative control. Data of a representative experiment are reported.

FIGURE 5

Effect of PYO and Sb-1 phage preparations on viability of biofilm-embedded mono and mix bacteria populations. S. aureus/P. aeruginosa mono- (A,B) and dual-species (C) biofilms formed on porous glass beads were exposed to phages. Data are reported as log10 CFUs/mL mean ± standard deviation of at least three independent experiments. Percent of cell reduction of treated samples compared to untreated samples was calculated as: percent reduction = [(A–B)/A]×100, where A is the mean number of viable bacteria of the growth control and B is the mean number of viable bacteria after exposure to PYO or PYO+Sb-1.

FIGURE 6

Evaluation of MRSA ATCC 43300/P. aeruginosa ATCC 27853 dual-species biofilm viability after simultaneous exposure during 24 h to ciprofloxacin at increasing doses (0.5–64 mg/L) plus (A) PYO or (B) PYO+Sb-1 monitored by microcalorimetry. Numbers represent antibiotic concentrations (in mg/L). GC, growth control (dashed line); NC, negative control.

FIGURE 7

Evaluation of MRSA ATCC 43300/P. aeruginosa ATCC 27853 dual-species biofilm viability after staggered exposure to phages and ciprofloxacin monitored by microcalorimetry. Each curve shows the heat produced by viable bacteria present in biofilms pretreated for 3 h (A,B), 6 h (C,D), 12 h (E,F) and 24 h (G,H) with PYO (graphs on the left) or PYO+Sb-1 (graphs on the right) followed by exposure to ciprofloxacin at increasing doses (0.5–64 mg/L) for 24 h. Numbers above curves represent antibiotic concentrations (in mg/L). Circled values represents the MBEC, defined as the lowest concentration of antibiotic that strongly reduced the viability of biofilm cells leading to the absence of heat flow production from treated beads when incubated during 48 h in fresh medium and no colonies after sonication and plating. GC, growth control (dashed line); NC, negative control.

FIGURE 8

SEM analysis of S. aureus/P. aeruginosa dual-species biofilms grown on porous glass beads for 24 h without treatment (a) and after exposure to 24 h monotherapy with (b) ciprofloxacin (1 mg/L); (c) PYO; or (d) pyo+sb-1.

FIGURE 9

SEM analysis of S. aureus/P. aeruginosa dual-species biofilm grown on porous glass beads for 24 h and treated with a combinatorial therapy of (a) simultaneous exposure to PYO and ciprofloxacin (1 mg/L, 24 h); (b) staggered exposure to PYO (12 h) followed by ciprofloxacin (1 mg/L, 24 h); (c) simultaneous exposure to PYO+Sb-1 and ciprofloxacin (1 mg/L, 24 h); or (d) staggered exposure to PYO+Sb-1 (12 h) followed by ciprofloxacin (1 mg/L, 24 h).