Antimicrobial efficacy against Pseudomonas aeruginosa biofilm formation in a three-dimensional lung epithelial model and the influence of fetal bovine serum

Abstract

In vitro models that mimic in vivo host-pathogen interactions are needed to evaluate candidate drugs that inhibit bacterial virulence traits. We established a new approach to study Pseudomonas aeruginosa biofilm susceptibility on biotic surfaces, using a three-dimensional (3-D) lung epithelial cell model. P. aeruginosa formed antibiotic resistant biofilms on 3-D cells without affecting cell viability. The biofilm-inhibitory activity of antibiotics and/or the anti-biofilm peptide DJK-5 were evaluated on 3-D cells compared to a plastic surface, in medium with and without fetal bovine serum (FBS). In both media, aminoglycosides were more efficacious in the 3-D cell model. In serum-free medium, most antibiotics (except polymyxins) showed enhanced efficacy when 3-D cells were present. In medium with FBS, colistin was less efficacious in the 3-D cell model. DJK-5 exerted potent inhibition of P. aeruginosa association with both substrates, only in serum-free medium. DJK-5 showed stronger inhibitory activity against P. aeruginosa associated with plastic compared to 3-D cells. The combined addition of tobramycin and DJK-5 exhibited more potent ability to inhibit P. aeruginosa association with both substrates. In conclusion, lung epithelial cells influence the efficacy of most antimicrobials against P. aeruginosa biofilm formation, which in turn depends on the presence or absence of FBS.

Non-hierarchical, RhlR-regulated acyl-homoserine lactone quorum sensing in a cystic fibrosis isolate ofPseudomonas aeruginosa

“…To address this question, we incubated an in vivo -like three-dimensional (3D) lung epithelial cell culture model (A549 cell line) [42] with either PAO1, E90, or engineered QS transcription-factor mutants. The 3D lung cell model possesses several advantages over the standard A549 monolayer as an infection model, including increased production of mucins, formation of tight junctions and polarity, decreased expression of carcinoma markers, and physiologically relevant cytokine expression and association of P. aeruginosa with the epithelial cells [42,43]. Following an incubation period of 24 hours, we measured cell death of the 3-D cell cultures via cytosolic lactate dehydrogenase (LDH) release.…”

“…Infection studies were performed on cultures grown for 11 to 14 days in the RWV. Thereafter, 3-D cell cultures were equally distributed in a 48-well plate at a concentration of 2.5 × 10 5 cells/well (250 μL volume), and infected with the different strains at a targeted multiplicity of infection of 30:1 as described previously[43]. All infection studies were performed in the above-described cell culture medium, with the exception that no FBS was added given the interference of serum compounds with QS signaling [57].…”

Design and Assessment of Anti-Biofilm Peptides: Steps Toward Clinical Application

“…In biomatrices, these peptides were found to have increased antimicrobial activity against Escherichia coli, reducing bacterial loads by up to 3 log-fold at concentrations that were not effective in MHB [51]. P. aeruginosa biofilms, when grown in a 3D epithelial cell model mimicking the human lung, exhibited decreased susceptibility to the peptide antibiotic colistin and D-enantiomeric peptide DJK-5 in the presence of fetal bovine serum compared to serum-free conditions [50]; intriguingly, the combination of DJK-5 with tobramycin substantially reduced the formation of P. aeruginosa biofilms in this 3D model. Recently, de Breij et al [24] designed an LL-37-derived peptide, SAAP-148, with bactericidal and anti-biofilm activity at 12.8 μM against P. aeruginosa and Staphylococcus aureus in 50% human plasma.…”

“…To overcome the lack of transferability from in vitro to in vivo activity, research on anti-microbial agents needs to address the influence of host conditions on biofilm structures and peptide activity [2,45,50]. The introduction of physiologically relevant host materials into antimicrobial bioassays can partially address this issue, but we have to be wary of the influence of host molecules on traditional detection methods such as optical density and fluorescence.…”

Modeling Host-Pathogen Interactions in the Context of the Microenvironment: Three-Dimensional Cell Culture Comes of Age

“…It was the first technology used to develop 3-D models for infection studies with bacterial (Salmonella) and viral (rhinovirus) pathogens (11,51). A range of RWV-derived 3-D models have been developed using cell lines, stem cells, and/or primary cells, including small and large intestine (11,80,141,143,145,146,152,(168)(169)(170)(171)(172)(173)(174)(175)(176)(177), lung (144,147,(178)(179)(180)(181)(182), liver (148,153,174,183,184), bladder (8,(185)(186)(187), reproductive tissue (149)(150)(151)(188)(189)(190), heart (191)(192)(193), prostate (142,186,194), pancreas (195,<...>…”