Examining biofilm formation and copper susceptibility testing methods in Pseudomonas aeruginosa sink drain isolates to study the role of the GI-7 genomic island

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Doucet, Ali N.
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Copper is an antimicrobial metal used in sink drains that is becoming ineffective due to copper tolerance of species such as Pseudomonas aeruginosa enhanced by the presence of the genomic island GI-7. Studies of copper tolerance associated with GI-7 are hindered by the lack of accurate planktonic and biofilm testing methods to determine copper susceptibility, and a lack of knowledge regarding the prevalence of this sequence amongst Canadian Pseudomonas isolates. In chapter 3, we developed a new deep well biofilm device that provided increased surface area for biofilm biomass formation. This method can be used to screen antimicrobial susceptibility in a 96-well high throughput format. The increased surface area allowed for greater biofilm biomass per mm2 as compared to the standard device and revealed plastic preferences in biofilm biomass formation by P. aeruginosa and Escherichia coli. Both devices produced different minimum biofilm eradication (MBEC) values for benzalkonium chloride and bleach, yet both were suitable for biofilm cultivation. In chapter 4, we examined 2467 genome sequenced Pseudomonas isolates collected from a 2017-2019 Ontario hospital intensive care unit study. Isolates from patients and hospital rooms with regular and copper sink drains were collected and used to explore if GI-7 presence was associated with copper sink drains. This analysis revealed that GI-7 is widely spread across clinical and environmental isolates of certain multi-locus sequence types, but statistical analyses did not show significant associations between GI-7 and copper sink drains due limited sampling from copper sink drains. Some correlation trends amongst GI-7 isolates were noted and discussed. Finally, phenotypic characterization of P. aeruginosa GI-7 copper tolerance through planktonic and biofilm culturing methods was examined with various P. aeruginosa strains. Copper salt addition to media caused lethal acidification that inhibited both planktonic and biofilm culture growth, regardless of GI-7 presence. To overcome this, we modified a previously used copper water exposure method to eliminate the effects of copper acidity and verify copper tolerance differences between susceptible PAO1 and PA-8 strains with and without GI-7. Overall, our findings offer improved techniques for studying copper tolerance and exploring the role of GI-7 in Canadian hospital Pseudomonas isolates.
pseudomonas, pseudomonas aeruginosa, copper, antimicrobial resistance, copper tolerance, sink drain, biofilm