In Vitro Analysis of Tobramycin-Treated Pseudomonas aeruginosa Biofilms on Cystic Fibrosis-Derived Airway Epithelial Cells

Abstract

P. aeruginosa forms biofilms in the lungs of individuals with cystic fibrosis (CF); however, there have been no effective model systems for studying biofilm formation in the CF lung. We have developed a tissue culture system for growth of P. aeruginosa biofilms on CF-derived human airway cells that promotes the formation of highly antibiotic-resistant microcolonies, which produce an extracellular polysaccharide matrix and require the known abiotic biofilm formation genes flgK and pilB. Treatment of P. aeruginosa biofilms with tobramycin reduced the virulence of the biofilms both by reducing bacterial numbers and by altering virulence gene expression. We performed microarray analysis of these biofilms on epithelial cells after treatment with tobramycin, and we compared these results with gene expression of (i) tobramycin-treated planktonic P. aeruginosa and (ii) tobramycintreated P. aeruginosa biofilms on an abiotic surface. Despite the conservation in functions required to form a biofilm, our results show that the responses to tobramycin treatment of biofilms grown on biotic versus abiotic surfaces are different, as exemplified by downregulation of genes involved in Pseudomonas quinolone signal biosynthesis specifically in epithelial cell-grown biofilms versus plastic-grown biofilms. We also identified the gene PA0913, which is upregulated by tobramycin specifically in biofilms grown on CF airway cells and codes for a probable magnesium transporter, MgtE. Mutation of the PA0913 gene increased the bacterial virulence of biofilms on the epithelial cells, consistent with a role for the gene in the suppression of bacterial virulence. Taken together, our data show that analysis of biofilms on airway cells provides new insights into the interaction of these microbial communities with the host.

Co-culture Models of <em>Pseudomonas aeruginosa</em> Biofilms Grown on Live Human Airway Cells

“…Specifically, the Static Co-culture Biofilm Model and the Flow Cell Co-culture Biofilm Model take advantage of the ability of P. aeruginosa to interact with and bind to the epithelial surface of the lung. Using these models, we have shown that the response of the co-culture biofilms to antibiotic treatment is unique and that these models are more likely to accurately reflect the infectious state 1,5 . In this regard, we have reported that the resistance to tobramycin increases by >25-fold when P. aeruginosa biofilms are grown on airway cells compared to biofilms grown on abiotic surfaces such as glass 5 .…”

Inquiry‐based examination of chemical disruption of bacterial biofilms

“…The biofilm assay allows the students to isolate biological communities (in each well of the 96-well plate) and evaluate changes in the community following addition or removal of certain chemical factors. Modified versions of this assay have proven useful in analyzing gene regulation/ changes following different stimuli in a research setting [6,12]. This laboratory exercise offers the educator a unique platform to introduce valuable biological concepts in an inquiry-based activity that, as demonstrated, results in a significant increase in student learning.…”

“…Since the discovery of biofilms, researchers have been trying to elucidate the impact of biofilm formation on human health [5]. Although our primary research focus is on the formation and persistence of biofilms in the cystic fibrosis airway by analyzing the connection between gene regulation and pathogenicity elicited during antibiotic treatment [6,7], biofilms are found ubiquitously in nature. It is becoming increasingly clear that bacteria form biofilms as a normal way of existing in many different environments [8].…”

Global regulator Anr represses PlcH phospholipase activity in Pseudomonas aeruginosa when oxygen is limiting

“…P. aeruginosa persists in the CF lung in a biofilm-like mode of growth , and data suggested that P. aeruginosa experiences oxygen limitation and induction of Anr in vivo (Hassett et al, 2009;Jackson et al, 2013;Petrova et al, 2012;Williamson et al, 2012;Worlitzsch et al, 2002). As P. aeruginosa forms robust biofilms in the CFBE airway epithelial co-culture model system and as Anr is important in this process (Anderson et al, 2008;Jackson et al, 2013), we sought to determine if Anr regulated plcH expression in this system. In order to assess plcH regulation in co-culture, P. aeruginosa PAO1 WT and Danr strains were co-incubated with a confluent monolayer of CFTRDF508 (CFBE41o 2 ) homozygous human airway epithelial cells for 6 h to allow for biofilm formation (Anderson et al, 2008).…”

“…As P. aeruginosa forms robust biofilms in the CFBE airway epithelial co-culture model system and as Anr is important in this process (Anderson et al, 2008;Jackson et al, 2013), we sought to determine if Anr regulated plcH expression in this system. In order to assess plcH regulation in co-culture, P. aeruginosa PAO1 WT and Danr strains were co-incubated with a confluent monolayer of CFTRDF508 (CFBE41o 2 ) homozygous human airway epithelial cells for 6 h to allow for biofilm formation (Anderson et al, 2008). Monolayer-associated cells were enumerated and, as published previously (Jackson et al, 2013), it was found that anr mutant cocultures contained threefold fewer cells associated with the airway epithelium compared with WT (Fig.…”