1 research outputs found
An integrated model system to gain mechanistic insights into biofilm-associated antimicrobial resistance in Pseudomonas aeruginosa MPAO1
open access articlePseudomonas aeruginosa MPAO1 is the parental strain of the widely utilized transposon mutant collection for this important clinical
pathogen. Here, we validate a model system to identify genes involved in biofilm growth and biofilm-associated antibiotic
resistance. Our model employs a genomics-driven workflow to assemble the complete MPAO1 genome, identify unique and
conserved genes by comparative genomics with the PAO1 reference strain and genes missed within existing assemblies by
proteogenomics. Among over 200 unique MPAO1 genes, we identified six general essential genes that were overlooked when
mapping public Tn-seq data sets against PAO1, including an antitoxin. Genomic data were integrated with phenotypic data from an
experimental workflow using a user-friendly, soft lithography-based microfluidic flow chamber for biofilm growth and a screen with
the Tn-mutant library in microtiter plates. The screen identified hitherto unknown genes involved in biofilm growth and antibiotic
resistance. Experiments conducted with the flow chamber across three laboratories delivered reproducible data on P. aeruginosa
biofilms and validated the function of both known genes and genes identified in the Tn-mutant screens. Differential protein
abundance data from planktonic cells versus biofilm confirmed the upregulation of candidates known to affect biofilm formation,
of structural and secreted proteins of type VI secretion systems, and provided proteogenomic evidence for some missed MPAO1
genes. This integrated, broadly applicable model promises to improve the mechanistic understanding of biofilm formation,
antimicrobial tolerance, and resistance evolution in biofilms