A three-phase in-vitro system for studying Pseudomonas aeruginosa adhesion and biofilm formation upon hydrogel contact lenses

Abstract

BackgroundPseudomonas aeruginosa is commonly associated with contact lens (CL) -related eye infections, for which bacterial adhesion and biofilm formation upon hydrogel CLs is a specific risk factor. Whilst P. aeruginosa has been widely used as a model organism for initial biofilm formation on CLs, in-vitro models that closely reproduce in-vivo conditions have rarely been presented.ResultsIn the current investigation, a novel in-vitro biofilm model for studying the adherence of P. aeruginosa to hydrogel CLs was established. Nutritional and interfacial conditions similar to those in the eye of a CL wearer were created through the involvement of a solid:liquid and a solid:air interface, shear forces and a complex artificial tear fluid. Bioburdens varied depending on the CL material and biofilm maturation occurred after 72 h incubation. Whilst a range of biofilm morphologies were visualised including dispersed and adherent bacterial cells, aggregates and colonies embedded in extracellular polymer substances (EPS), EPS fibres, mushroom-like formations, and crystalline structures, a compact and heterogeneous biofilm morphology predominated on all CL materials.ConclusionsIn order to better understand the process of biofilm formation on CLs and to test the efficacy of CL care solutions, representative in-vitro biofilm models are required. Here, we present a three-phase biofilm model that simulates the environment in the eye of a CL wearer and thus generates biofilms which resemble those commonly observed in-situ.

A Laboratory Assessment of Factors That Affect Bacterial Adhesion to Contact Lenses

“…Bacterial adhesion during two phases of the process, two hours and 18 hours exposure of contact lenses to bacterial suspension were determined in this study. The viable bacterial numbers after 18 hours adhesion were generally higher compared with after 2 hours, an observation that agrees with some previous studies [22,38]. Combining our results with Tran et al .…”

“…Previous studies have elucidated that the initial bacterial adhesion to contact lenses increases with time, peaked at 3 to 18 hours of incubation and then remained steady, suggesting the end point of primary adhesion [ 22 , 31 , 37 ]. Bacterial adhesion during two phases of the process, two hours and 18 hours exposure of contact lenses to bacterial suspension were determined in this study.…”

“…[ 35 ] showing linear kinetics of bacterial adhesion up to 70 minutes and Randler et al . [ 22 ] investigating up to 72 hours but having incremental adhesion only up to 24 hours, illustrates that adhesion to contact lenses increases in a time dependant manner up to 18-24 hours of incubation and then viability is reduced. Perhaps, the reduction in viability is due to the bacteria entering a biofilm mode of growth, which is known to result in lower viability of cells [ 39 , 40 ] or due to biofilm dispersal that can occur when the environment nutrients are not favorable for bacteria.…”

A Laboratory Assessment of Factors That Affect Bacterial Adhesion to Contact Lenses

“…Bacterial adhesion during two phases of the process, two hours and 18 hours exposure of contact lenses to bacterial suspension were determined in this study. The viable bacterial numbers after 18 hours adhesion were generally higher compared with after 2 hours, an observation that agrees with some previous studies [22,38]. Combining our results with Tran et al .…”

“…Previous studies have elucidated that the initial bacterial adhesion to contact lenses increases with time, peaked at 3 to 18 hours of incubation and then remained steady, suggesting the end point of primary adhesion [ 22 , 31 , 37 ]. Bacterial adhesion during two phases of the process, two hours and 18 hours exposure of contact lenses to bacterial suspension were determined in this study.…”

“…[ 35 ] showing linear kinetics of bacterial adhesion up to 70 minutes and Randler et al . [ 22 ] investigating up to 72 hours but having incremental adhesion only up to 24 hours, illustrates that adhesion to contact lenses increases in a time dependant manner up to 18-24 hours of incubation and then viability is reduced. Perhaps, the reduction in viability is due to the bacteria entering a biofilm mode of growth, which is known to result in lower viability of cells [ 39 , 40 ] or due to biofilm dispersal that can occur when the environment nutrients are not favorable for bacteria.…”

Synergistic Effect of Nilavembu Choornam–Gold Nanoparticles on Antibiotic-Resistant Bacterial Susceptibility and Contact Lens Contamination-Associated Infectious Pathogenicity

“…Consistent with our study, Szczotka-Flynn et al, 2009 reported that bacterial (P. aeruginosa, S. marcescens, and S. aureus) biofilm were formed on a Silicon hydrogel CLs, which was inhibited in the presence of tested lens care solutions, while biguanide preserved care solutions were not effective against bacterial biofilms in vitro model [20]. Several studies used SEM analysis to understand bacterial adhesion and biofilm formation on CLs [32][33][34]. Consistent with our results, a cluster of microcolonies was reported using SEM analysis, and biofilm formation on CLs was evaluated (around 55%).…”

Antimicrobial Activity of Poly-epsilon-lysine Peptide Hydrogels Against Pseudomonas aeruginosa

“…Interestingly, P aeruginosa reportedly adheres at greater numbers than S aureus to CLs; however, different strains of P aeruginosa do not adhere differently. 34 , 35 We obtained similar log decreases in CFU with pɛK+ hydrogels against all P aeruginosa strains for each inocula size and time point tested. Importantly, we observed antimicrobial activity against the highly virulent exoU isolate PA39016, isolated from a patient with prolonged healing time and resistance to antibiotics.…”

A Laboratory Assessment of Factors That Affect Bacterial Adhesion to Contact Lenses

“…Bacterial adhesion during two phases of the process, two hours and 18 hours exposure of contact lenses to bacterial suspension were determined in this study. The viable bacterial numbers after 18 hours adhesion were generally higher compared with after 2 hours, an observation that agrees with some previous studies [22,38]. Combining our results with Tran et al .…”

“…Previous studies have elucidated that the initial bacterial adhesion to contact lenses increases with time, peaked at 3 to 18 hours of incubation and then remained steady, suggesting the end point of primary adhesion [ 22 , 31 , 37 ]. Bacterial adhesion during two phases of the process, two hours and 18 hours exposure of contact lenses to bacterial suspension were determined in this study.…”

“…[ 35 ] showing linear kinetics of bacterial adhesion up to 70 minutes and Randler et al . [ 22 ] investigating up to 72 hours but having incremental adhesion only up to 24 hours, illustrates that adhesion to contact lenses increases in a time dependant manner up to 18-24 hours of incubation and then viability is reduced. Perhaps, the reduction in viability is due to the bacteria entering a biofilm mode of growth, which is known to result in lower viability of cells [ 39 , 40 ] or due to biofilm dispersal that can occur when the environment nutrients are not favorable for bacteria.…”

Synergistic Effect of Nilavembu Choornam–Gold Nanoparticles on Antibiotic-Resistant Bacterial Susceptibility and Contact Lens Contamination-Associated Infectious Pathogenicity

“…Consistent with our study, Szczotka-Flynn et al, 2009 reported that bacterial (P. aeruginosa, S. marcescens, and S. aureus) biofilm were formed on a Silicon hydrogel CLs, which was inhibited in the presence of tested lens care solutions, while biguanide preserved care solutions were not effective against bacterial biofilms in vitro model [20]. Several studies used SEM analysis to understand bacterial adhesion and biofilm formation on CLs [32][33][34]. Consistent with our results, a cluster of microcolonies was reported using SEM analysis, and biofilm formation on CLs was evaluated (around 55%).…”

Antimicrobial Activity of Poly-epsilon-lysine Peptide Hydrogels Against Pseudomonas aeruginosa

“…Interestingly, P aeruginosa reportedly adheres at greater numbers than S aureus to CLs; however, different strains of P aeruginosa do not adhere differently. 34 , 35 We obtained similar log decreases in CFU with pɛK+ hydrogels against all P aeruginosa strains for each inocula size and time point tested. Importantly, we observed antimicrobial activity against the highly virulent exoU isolate PA39016, isolated from a patient with prolonged healing time and resistance to antibiotics.…”

A Laboratory Assessment of Factors That Affect Bacterial Adhesion to Contact Lenses

“…Bacterial adhesion during two phases of the process, two hours and 18 hours exposure of contact lenses to bacterial suspension were determined in this study. The viable bacterial numbers after 18 hours adhesion were generally higher compared with after 2 hours, an observation that agrees with some previous studies [22,38]. Combining our results with Tran et al .…”

“…Previous studies have elucidated that the initial bacterial adhesion to contact lenses increases with time, peaked at 3 to 18 hours of incubation and then remained steady, suggesting the end point of primary adhesion [ 22 , 31 , 37 ]. Bacterial adhesion during two phases of the process, two hours and 18 hours exposure of contact lenses to bacterial suspension were determined in this study.…”

“…[ 35 ] showing linear kinetics of bacterial adhesion up to 70 minutes and Randler et al . [ 22 ] investigating up to 72 hours but having incremental adhesion only up to 24 hours, illustrates that adhesion to contact lenses increases in a time dependant manner up to 18-24 hours of incubation and then viability is reduced. Perhaps, the reduction in viability is due to the bacteria entering a biofilm mode of growth, which is known to result in lower viability of cells [ 39 , 40 ] or due to biofilm dispersal that can occur when the environment nutrients are not favorable for bacteria.…”

Synergistic Effect of Nilavembu Choornam–Gold Nanoparticles on Antibiotic-Resistant Bacterial Susceptibility and Contact Lens Contamination-Associated Infectious Pathogenicity

“…Consistent with our study, Szczotka-Flynn et al, 2009 reported that bacterial (P. aeruginosa, S. marcescens, and S. aureus) biofilm were formed on a Silicon hydrogel CLs, which was inhibited in the presence of tested lens care solutions, while biguanide preserved care solutions were not effective against bacterial biofilms in vitro model [20]. Several studies used SEM analysis to understand bacterial adhesion and biofilm formation on CLs [32][33][34]. Consistent with our results, a cluster of microcolonies was reported using SEM analysis, and biofilm formation on CLs was evaluated (around 55%).…”

Antimicrobial Activity of Poly-epsilon-lysine Peptide Hydrogels Against Pseudomonas aeruginosa

“…Interestingly, P aeruginosa reportedly adheres at greater numbers than S aureus to CLs; however, different strains of P aeruginosa do not adhere differently. 34 , 35 We obtained similar log decreases in CFU with pɛK+ hydrogels against all P aeruginosa strains for each inocula size and time point tested. Importantly, we observed antimicrobial activity against the highly virulent exoU isolate PA39016, isolated from a patient with prolonged healing time and resistance to antibiotics.…”