New In Vitro Model To Study the Effect of Human Simulated Antibiotic Concentrations on Bacterial Biofilms

Abstract

A new in vitro pharmacokinetic/pharmacodynamic simulator for bacterial biofilms utilizing flow cell technology and confocal laser scanning microscopy is described. The device has the ability to simulate the changing antibiotic concentrations in humans associated with intravenous dosing on bacterial biofilms grown under continuous culture conditions. The free drug concentrations of a single 2-g meropenem intravenous bolus dose and first-order elimination utilizing a half-life of 0.895 h (elimination rate constant, 0.776 h ؊1 ) were simulated. The antibacterial activity of meropenem against biofilms of Pseudomonas aeruginosa PAO1 and three clinical strains isolated from patients with cystic fibrosis was investigated. Additionally, the effect of meropenem on PAO1 biofilms cultured for 24 h versus that on biofilms cultured for 72 h was examined. Using confocal laser scanning microscopy, rapid biofilm killing was observed in the first hour of the dosing interval for all biofilms. However, for PAO1 biofilms cultured for 72 h, only bacterial subpopulations at the periphery of the biofilm were affected, with subpopulations at the substratum remaining viable, even at the conclusion of the dosing interval. The described model is a novel method to investigate antimicrobial killing of bacterial biofilms using human simulated concentrations.

Antibiotic treatment of biofilm infections

“…For instance, as conceptually, biofilms may be considered as independent compartment with particular pharmacokinetics, Cao et al (21) studied the PK/PD of tobramycin in seaweed alginate-embedded biofilms providing a model for testing new treatment strategies. In another recent work, a new in vitro PK/PD simulator using flow cell technology and CLSM has been employed on P. aeruginosa laboratory and clinical strain biofilms to study the antibacterial activity of meropenem (76). Results from this work showed that a single 2 g meropenem bolus dose effectively killed thin and young biofilms but not mature ones, confirming the important implications on antibiotic dose selection in acute vs chronic biofilm infections, such as infections in CF patients and orthopedic implant infections.…”

The high persister phenotype ofPseudomonas aeruginosais associated with increased fitness and persistence in cystic fibrosis airways

“…This fitness test should be carried out in conditions as close as possible to those in which the Hip variants had been selected. We therefore simulated antibiotic treatment of CF patients in a recently developed biofilm Pharmacokinetic/Pharmacodynamic system, in which the bacteria are challenged with antibiotics in much the same way as in patients (24). We chose this model because P. aeruginosa often appears as biofilms in lungs of CF patients (25), because biofilms have been shown to harbor increased levels of persister cells (26), and because our model mimics the bacterial exposure to ciprofloxacin treatment as described for CF patients (24, 27).…”

“…We therefore simulated antibiotic treatment of CF patients in a recently developed biofilm Pharmacokinetic/Pharmacodynamic system, in which the bacteria are challenged with antibiotics in much the same way as in patients (24). We chose this model because P. aeruginosa often appears as biofilms in lungs of CF patients (25), because biofilms have been shown to harbor increased levels of persister cells (26), and because our model mimics the bacterial exposure to ciprofloxacin treatment as described for CF patients (24, 27). The isolates were chosen based on the criteria that 1) the respective times of isolation since their first detection in the CF lungs were similar, 2) they had similar MIC values for ciprofloxacin, 3) similar growth rates, and 4) belong to the same clone type.…”

“…Stepanyan et al assessed the fitness tradeoffs of two Hip strains (one hospital strain and one pilH mutant) and two Lop P. aeruginosa strains via competitive growth of paired Hip-Lop strains at a 1:1 ratio under periodic treatment with ofloxacin in Mueller-Hinton broth; Hip strains showed higher fitness under treatment conditions and the reverse in control conditions (29). In our study, we were able to test genotypically similar isolates from a similar point in infection with other selected traits in alignment in order to characterize the selective advantage of the Hip phenotype in a biofilm under clinically-replicating antibiotic exposure (24). In two independent Lop/Hip strain pairs assessed in a PD/PK flow chamber, we show that Hip cells survived ciprofloxacin treatment far better than Lop isolates, and we further find that this survival is potentially reliant on biofilm architecture.…”

“…For fitness experiments, we used a PK/PD biofilm model system combined with confocal laser-scanning microscopy. This system simulates the changing antibiotic concentrations in CF patients during intravenous dosing in addition to retaining a similar profile of antibiotic decay as the one taking place in CF patients (24). First, Hip and Lop isolates were differentially tagged with a yellow fluorescent protein (YFP) or cyan fluorescent protein (CFP) respectively (13).…”