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.
Project description:Antibiotic resistance in Pseudomonas aeruginosa biofilms necessitates the need for novel antimicrobial therapy with anti-biofilm properties. Bacteriophages (phages) are recognized as an ideal biopharmaceutical for combating antibiotic-resistant bacteria especially when used in combination with antibiotics. However, previous studies primarily focused on using phages against of P. aeruginosa biofilms of laboratory strains. In the present study, biofilms of six P. aeruginosa isolated from cystic fibrosis and wound patients, and one laboratory strain was treated singly and with combinations of anti-Pseudomonas phage PEV20 and ciprofloxacin. Of these strains, three were highly susceptible to the phage, while one was partially resistant and one was completely resistant. Combination treatment with PEV20 and ciprofloxacin enhanced biofilm eradication compared with single treatment. Phage and ciprofloxacin synergy was found to depend on phage-resistance profile of the target bacteria. Furthermore, phage and ciprofloxacin combination formulation protected the lung epithelial and fibroblast cells from P. aeruginosa and promoted cell growth. The results demonstrated that thorough screening of phage-resistance is crucial for designing phage-antibiotic formulation. The addition of highly effective phage could reduce the ciprofloxacin concentration required to combat P. aeruginosa infections associated with biofilm in cystic fibrosis and wound patients.
Project description:Pseudomonas aeruginosa and Staphylococcus aureus are opportunistic pathogens and are commonly found in polymicrobial biofilm-associated diseases, namely chronic wounds. Their co-existence in a biofilm contributes to an increased tolerance of the biofilm to antibiotics. Combined treatments of bacteriophages and antibiotics have shown a promising antibiofilm activity, due to the profound differences in their mechanisms of action. In this study, 48 h old mono and dual-species biofilms were treated with a newly isolated P. aeruginosa infecting phage (EPA1) and seven different antibiotics (gentamicin, kanamycin, tetracycline, chloramphenicol, erythromycin, ciprofloxacin, and meropenem), alone and in simultaneous or sequential combinations. The therapeutic efficacy of the tested antimicrobials was determined. Phage or antibiotics alone had a modest effect in reducing biofilm bacteria. However, when applied simultaneously, a profound improvement in the killing effect was observed. Moreover, an impressive biofilm reduction (below the detection limit) was observed when gentamicin or ciprofloxacin were added sequentially after 6 h of phage treatment. The effect observed does not depend on the type of antibiotic but is influenced by its concentration. Moreover, in dual-species biofilms it was necessary to increase gentamicin concentration to obtain a similar killing effect as occurs in mono-species. Overall, combining phages with antibiotics can be synergistic in reducing the bacterial density in biofilms. However, the concentration of antibiotic and the time of antibiotic application are essential factors that need to be considered in the combined treatments.
Project description:Pseudomonas aeruginosa is a major cause of morbidity and mortality in chronically infected cystic fibrosis patients. Novel in vitro biofilm models which reliably predict the therapeutic success of antimicrobial therapies against biofilm bacteria should be implemented. The activity of fosfomycin, tobramycin, and the fosfomycin-tobramycin combination against 6 susceptible P. aeruginosa strains isolated from respiratory samples from cystic fibrosis patients was tested by using two in vitro biofilm models: a closed system (Calgary device) and an open model based on microfluidics (BioFlux). All but one of the isolates formed biofilms. The fosfomycin and tobramycin minimal biofilm inhibitory concentrations (MBIC) were 1,024 to >1,024 ?g/ml and 8 to 32 ?g/ml, respectively. According to fractional inhibitory concentration analysis, the combination behaved synergistically against all the isolates except the P. aeruginosa ATCC 27853 strain. The dynamic formation of the biofilm was also studied with the BioFlux system, and the MIC and MBIC of each antibiotic were tested. For the combination, the lowest tobramycin concentration that was synergistic with fosfomycin was used. The captured images were analyzed by measuring the intensity of the colored pixels, which was proportional to the biofilm biomass. A statistically significant difference was found when the intensity of the inoculum was compared with the intensity of the microchannel in which the MBIC of tobramycin, fosfomycin, or their combination was used (P < 0.01) but not when the MIC was applied (P > 0.01). Fosfomycin-tobramycin was demonstrated to be synergistic against cystic fibrosis P. aeruginosa strains in the biofilm models when both the Calgary and the microfluidic BioFlux systems were tested. These results support the clinical use of this combination.
Project description:Biofilms consist of groups of bacteria attached to surfaces and encased in a hydrated polymeric matrix. Bacteria in biofilms are more resistant to the immune system and to antibiotics than their free-living planktonic counterparts. Thus, biofilm-related infections are persistent and often show recurrent symptoms. The metal chelator EDTA is known to have activity against biofilms of gram-positive bacteria such as Staphylococcus aureus. EDTA can also kill planktonic cells of Proteobacteria like Pseudomonas aeruginosa. In this study we demonstrate that EDTA is a potent P. aeruginosa biofilm disrupter. In Tris buffer, EDTA treatment of P. aeruginosa biofilms results in 1,000-fold greater killing than treatment with the P. aeruginosa antibiotic gentamicin. Furthermore, a combination of EDTA and gentamicin results in complete killing of biofilm cells. P. aeruginosa biofilms can form structured mushroom-like entities when grown under flow on a glass surface. Time lapse confocal scanning laser microscopy shows that EDTA causes a dispersal of P. aeruginosa cells from biofilms and killing of biofilm cells within the mushroom-like structures. An examination of the influence of several divalent cations on the antibiofilm activity of EDTA indicates that magnesium, calcium, and iron protect P. aeruginosa biofilms against EDTA treatment. Our results are consistent with a mechanism whereby EDTA causes detachment and killing of biofilm cells.
Project description:Microarray analysis was used to identify changes in the level of transcription of genes in P. aeruginosa drip flow biofilms in response to ciprofloxacin and tobramycin exposure. This data was evaluated and used to select strains that carry transposon mutations in genes that might play a role in antibiotic tolerance of biofilms. The strains were evaluated for defects in biofilm tolerance. Four drip flow biofilm conditions with three replicates each: (1) baseline controls at 72 hours, (2) tobramycin treated for 12 hours past baseline, (3) ciprofloxacin treated for 12 hrs past baseline, and (4) no treatment for 12 hrs past baseline.
Project description:Cystic Fibrosis (CF) airways disease involves complex polymicrobial infections where different bacterial species can interact and influence each other and/or even interfere with the whole community. To gain insights into the role that interactions between Pseudomonas aeruginosa in co-culture with Staphylococcus aureus, Inquilinus limosus, and Stenotrophomonas maltophilia may play in infection, the reciprocal effect during biofilm formation and the response of dual biofilms toward ciprofloxacin under in vitro atmospheres with different oxygen availabilities were evaluated. Biofilm formation kinetics showed that the growth of S. aureus, I. limosus, and S. maltophilia was disturbed in the presence of P. aeruginosa, under both aerobic and anaerobic environments. On the other hand, under aerobic conditions, I. limosus led to a decrease in biofilm mass production by P. aeruginosa, although biofilm-cells viability remains unaltered. The interaction between S. maltophilia and P. aeruginosa positively influenced dual biofilm development by increasing its biomass. Compared with monocultures, biomass of P. aeruginosa+ S. aureus biofilms was significantly reduced by reciprocal interference. When grown in dual biofilms with P. aeruginosa, ciprofloxacin was less effective against S. aureus, I. limosus, and S. maltophilia, with increasing antibiotic doses leading to drastic inhibitions of P. aeruginosa cultivability. Therefore, P. aeruginosa might be responsible for the protection of the whole dual consortia against ciprofloxacin activity. Based on the overall data, it can be speculated that reciprocal interferences occur between the different bacterial species in CF lung, regardless the level of oxygen. The findings also suggest that alterations of bacterial behavior due to species interplay may be important for disease progression in CF infection.
Project description:Within wounds, microorganisms predominantly exist as biofilms. Biofilms are associated with chronic infections and represent a tremendous clinical challenge. As antibiotics are often ineffective against biofilms, use of dispersal agents as adjunctive, topical therapies for the treatment of wound infections involving biofilms has gained interest. We evaluated in vitro the dispersive activity of D-amino acids (D-AAs) on biofilms from clinical wound isolates of Staphylococcus aureus and Pseudomonas aeruginosa; moreover, we determined whether combinations of D-AAs and antibiotics (clindamycin, cefazolin, oxacillin, rifampin, and vancomycin for S. aureus and amikacin, colistin, ciprofloxacin, imipenem, and ceftazidime for P. aeruginosa) enhance activity against biofilms. D-Met, D-Phe, and D-Trp at concentrations of ? 5 mM effectively dispersed preformed biofilms of S. aureus and P. aeruginosa clinical isolates, an effect that was enhanced when they were combined as an equimolar mixture (D-Met/D-Phe/D-Trp). When combined with D-AAs, the activity of rifampin was significantly enhanced against biofilms of clinical isolates of S. aureus, as indicated by a reduction in the minimum biofilm inhibitory concentration (MBIC) (from 32 to 8 ?g/ml) and a >2-log reduction of viable biofilm bacteria compared to treatment with antibiotic alone. The addition of D-AAs was also observed to enhance the activity of colistin and ciprofloxacin against biofilms of P. aeruginosa, reducing the observed MBIC and the number of viable bacteria by >2 logs and 1 log at 64 and 32 ?g/ml in contrast to antibiotics alone. These findings indicate that the biofilm dispersal activity of D-AAs may represent an effective strategy, in combination with antimicrobials, to release bacteria from biofilms, subsequently enhancing antimicrobial activity.
Project description:Enhanced tolerance of biofilm-associated bacteria to antibiotic treatments is likely due to a combination of factors, including changes in cell physiology as bacteria adapt to biofilm growth and the inherent physiological heterogeneity of biofilm bacteria. In this study, a transcriptomics approach was used to identify genes differentially expressed during biofilm growth of Pseudomonas aeruginosa. These genes were tested for statistically significant overlap, with independently compiled gene lists corresponding to stress responses and other putative antibiotic-protective mechanisms. Among the gene groups tested were those associated with biofilm response to tobramycin or ciprofloxacin, drug efflux pumps, acyl homoserine lactone quorum sensing, osmotic shock, heat shock, hypoxia stress, and stationary-phase growth. Regulons associated with Anr-mediated hypoxia stress, RpoS-regulated stationary-phase growth, and osmotic stress were significantly enriched in the set of genes induced in the biofilm. Mutant strains deficient in rpoS, relA and spoT, or anr were cultured in biofilms and challenged with ciprofloxacin and tobramycin. When challenged with ciprofloxacin, the mutant strain biofilms had 2.4- to 2.9-log reductions in viable cells compared to a 0.9-log reduction of the wild-type strain. Interestingly, none of the mutants exhibited a statistically significant alteration in tobramycin susceptibility compared to that with the wild-type biofilm. These results are consistent with a model in which multiple genes controlled by overlapping starvation or stress responses contribute to the protection of a P. aeruginosa biofilm from ciprofloxacin. A distinct and as yet undiscovered mechanism protects the biofilm bacteria from tobramycin.
Project description:In young cystic fibrosis (CF) patients, Staphylococcus aureus is typically the most prevalent organism, while in adults, Pseudomonas aeruginosa is the major pathogen. More recently, it was observed that also Streptococcus anginosus plays an important role in exacerbations of respiratory symptoms. These species are often coisolated from CF lungs, yet little is known about whether antibiotic killing of one species is influenced by the presence of others. In the present study, we compared the activities of various antibiotics against S. anginosus, S. aureus, and P. aeruginosa when grown in monospecies biofilms with the activity observed in a multispecies biofilm. Our results show that differences in antibiotic activity against species grown in mono- and multispecies biofilms are species and antibiotic dependent. Fewer S. anginosus cells are killed by antibiotics that interfere with cell wall synthesis (amoxicillin plus sulbactam, cefepime, imipenem, meropenem, and vancomycin) in the presence of S. aureus and P. aeruginosa, while for ciprofloxacin, levofloxacin, and tobramycin, no difference was observed. In addition, we observed that the cell-free supernatant of S. aureus, but not that of P. aeruginosa biofilms, also caused this decrease in killing. Overall, S. aureus was more affected by antibiotic treatment in a multispecies biofilm, while for P. aeruginosa, no differences were observed between growth in mono- or multispecies biofilms. The results of the present study suggest that it is important to take the community composition into account when evaluating the effect of antimicrobial treatments against certain species in mixed biofilms.
Project description:The coexistence between species that occurs in some infections remains hard to achieve in vitro since bacterial fitness differences eventually lead to a single organism dominating the mixed culture. Pseudomonas aeruginosa and Staphylococcus aureus are major pathogens found growing together in biofilms in disease-affected lungs or wounds. Herein, we tested and analyzed different culture media, additives and environmental conditions to support P. aeruginosa and S. aureus coexistence in vitro. We have unraveled the potential of DMEM to support the growth of these two organisms in mature cocultured biofilms (three days old) in an environment that dampens the pH rise. Our conditions use equal initial inoculation ratios of both strains and allow the stable formation of separate S. aureus microcolonies that grow embedded in a P. aeruginosa biofilm, as well as S. aureus biofilm overgrowth when bovine serum albumin is added to the system. Remarkably, we also found that S. aureus survival is strictly dependent on a well-characterized phenomenon of oxygen stratification present in the coculture biofilm. An analysis of differential tolerance to gentamicin and ciprofloxacin treatment, depending on whether P. aeruginosa and S. aureus were growing in mono- or coculture biofilms, was used to validate our in vitro coculture conditions.