Predicting Antibiotic-Associated Virulence of Pseudomonas aeruginosa Using an ex vivo Lung Biofilm Model

Abstract

Background Bacterial biofilms are known to have high antibiotic tolerance which directly affects clearance of bacterial infections in people with cystic fibrosis (CF). Current antibiotic susceptibility testing methods are either based on planktonic cells or do not reflect the complexity of biofilms in vivo . Consequently, inaccurate diagnostics affect treatment choice, preventing bacterial clearance and potentially selecting for antibiotic resistance. This leads to prolonged, ineffective treatment. Methods In this study, we use an ex vivo lung biofilm model to study antibiotic tolerance and virulence of Pseudomonas aeruginosa . Sections of pig bronchiole were dissected, prepared and infected with clinical isolates of P. aeruginosa and incubated in artificial sputum media to form biofilms, as previously described. Then, lung-associated biofilms were challenged with antibiotics, at therapeutically relevant concentrations, before their bacterial load and virulence were quantified and detected, respectively. Results The results demonstrated minimal effect on the bacterial load with therapeutically relevant concentrations of ciprofloxacin and meropenem, with the latter causing an increased production of proteases and pyocyanin. A combination of meropenem and tobramycin did not show any additional decrease in bacterial load but demonstrated a slight decrease in total proteases and pyocyanin production. Conclusion In this initial study of six clinical isolates of P. aeruginosa showed high levels of antibiotic tolerance, with minimal effect on bacterial load and increased proteases production, which could negatively affect lung function. Thus, the ex vivo lung model has the potential to be effectively used in larger studies of antibiotic tolerance in in vivo -like biofilms, and show how sub optimal antibiotic treatment of biofilms may potentially contribute to exacerbations and eventual lung failure. We demonstrate a realistic model for understanding antibiotic resistance and tolerance in biofilms clinically and for molecules screening in anti-biofilm drug development.

Transcriptome Analysis of Pseudomonas aeruginosa Biofilm Infection in an Ex Vivo Pig Model of the Cystic Fibrosis Lung

“…Following investigation of quorum sensing gene expression and the implication on efflux pump expression, we aimed to determine whether growth in the EVPL model—with no prior exposure to antimicrobials—resulted in differing expression of antimicrobial resistance (AMR) associated genes. We have previously shown that high levels of antibiotic tolerance are evident in the EVPL model ( 16 ) and clinical infection typically demonstrates much higher resistance to antimicrobial treatment than is predicted in vitro . We looked at the expression of 52 P. aeruginosa PA14 AMR genes, predicted using the Comprehensive Antibiotic Resistance Database (CARD) ( 26 ), in each contrast.…”

“…We have developed an ex vivo pig lung (EVPL) model of the CF lung environment that replicates key phenotypic aspects of P. aeruginosa chronic biofilm infection ( 13 – 16 ). We aimed to assess the P. aeruginosa transcriptome as biofilm infection establishes in the EVPL model, to determine the extent to which the model accurately replicates P. aeruginosa gene expression during human CF infection.…”

Transcriptome analysis ofPseudomonas aeruginosabiofilm infection in anex vivopig model of the cystic fibrosis lung

“…shown that high levels of antibiotic tolerance are evident in the EVPL model [16] and clinical infection typically demonstrates much higher resistance to antimicrobial treatment than is predicted in vitro. We looked at the expression of 52 P. aeruginosa PA14 AMR genes, predicted using the Comprehensive Antibiotic Resistance Database (CARD) [27], in each contrast.…”

“…We have developed an ex vivo pig lung (EVPL) model of the CF lung environment that replicates key phenotypic aspects of P. aeruginosa chronic biofilm infection [13][14][15][16]. We aimed to assess the P. aeruginosa transcriptome as biofilm infection establishes in the EVPL model, to determine the extent to which the model accurately replicates P. aeruginosa gene expression during human CF infection.…”

Speciality Grand Challenge for “Biofilms”

“…While there is some utility to pursue the development of non-mammalian models of biofilm-infection such as Drosophila melanogaster , Galleria mellonella or Danio rerio (Zebrafish) ( Lebeaux et al., 2013 ), identification of ex vivo models that recreate the infectious environment should be encouraged. Among recently developed ex vivo models are, for example, an ex vivo pig lung biofilm model used for understanding antibiotic tolerance of P. aeruginosa biofilms ( Hassan et al., 2020 ) or an ex vivo murine skin biopsy model to characterize B. burgdoferi biofilms, the etiological agent of Lyme disease ( Torres et al., 2020 ). Last but not least with the development of reconstituted organs such as organoids or organ-on-a-chip, one could anticipate that adaptations of these models to the understanding of the physiopathology of biofilm infections, interactions of biofilms with the immune response or biofilm behaviors in the presence of antimicrobials would provide important information relevant to biofilms in infectious contexts ( Jimi et al., 2017 ; Choi et al., 2020 ; Yuan et al., 2020 ).…”