Synergistic Activity of Fosfomycin, Ciprofloxacin, and Gentamicin Against Escherichia coli and Pseudomonas aeruginosa Biofilms

Abstract

Gram-negative (GN) rods cause about 10% periprosthetic joint infection (PJI) and represent an increasing challenge due to emergence of antimicrobial resistance. Escherichia coli and Pseudomonas aeruginosa are among the most common cause of GN-PJI and ciprofloxacin is the first-line antibiotic. Due to emergence of fluoroquinolone resistance, we evaluated in vitro the activity of fosfomycin, ciprofloxacin, and gentamicin, alone and in combinations, against E. coli and P. aeruginosa biofilms. Conventional microbiological tests and isothermal microcalorimetry were applied to investigate the anti-biofilm activity of the selected antibiotics against standard laboratory strains as well as clinical strains isolated from patients with prosthetic joint associated infections. The biofilm susceptibility to each antibiotic varied widely among strains, while fosfomycin presented a poor anti-biofilm activity against P. aeruginosa. Synergism of two-pair antibiotic combinations was observed against different clinical strains from both species. Highest synergism was found for the fosfomycin/gentamicin combination against the biofilm of E. coli strains (75%), including a gentamicin-resistant but fosfomycin-susceptible strain, whereas the gentamicin/ciprofloxacin combination presented synergism with higher frequency against the biofilm of P. aeruginosa strains (71.4%). A hypothetical bacteriolysis effect of gentamicin could explain why combinations with this antibiotic seem to be particularly effective. Still, the underlying mechanism of the synergistic effect on biofilms is unknown. In conclusion, combinatorial antibiotic application has shown to be more effective against biofilms compared to monotherapy. Further in vivo and clinical studies are essential to define the potential treatment regimen based on our results.

Keywords: Escherichia coli; Pseudomonas aeruginosa; antibiotic activity; antibiotic resistance; biofilm-associated infection; clinical isolates; isothermal microcalorimetry; synergism.

Figure 1

Microcalorimetry analysis of planktonic bacteria exposed to serial antibiotic concentrations for 24 h. Numbers represent concentrations (in μg/ml) of fosfomycin (A,B), ciprofloxacin (C,D), and gentamicin (E,F) against E. coli ATCC 25922 (left column) and P. aeruginosa ATCC 27853 (right column). Circled values represent the MHIC. GC, growth control; NC, negative control. Data of a representative experiment are reported.

Figure 2

Microcalorimetry analysis of biofilm bacteria after exposure to serial antibiotic concentrations for 24 h. Numbers represent concentrations (in μg/ml) of fosfomycin (A,B), ciprofloxacin (C,D), and gentamicin (E,F) against E. coli ATCC 25922 (left column) and P. aeruginosa ATCC 27853 (right column). Circled values represent the MBBC. GC, growth control; NC, negative control. Data of a representative experiment are reported.

Figure 3

Evaluation of synergistic activity of paired antibiotics by IMC against E. coli ATCC 25922 (A,C,E) and P. aeruginosa ATCC 27853 (B,D,F) biofilms. Numbers represent concentrations (in μg/ml). Circled values represent the MBBC. GC, growth control; NC, negative control; FOS, fosfomycin; GEN, gentamicin; CIP, ciprofloxacin. Data of a representative experiment are reported.