MULTIDRUG-RESISTANT PSEUDOMONAS AERUGINOSA – A PROBLEM IN HOT TUBS AND WHIRLPOOLS IN CANADA
Creators: Mufty, Md Murad
Publisher: name: University of Alberta Library
Year: 2022

<p>Objectives: Pseudomonas aeruginosa is one of the most problematic opportunistic pathogens. It is resistant to many currently used antibiotics, making it difficult to treat, and resistance may transfer to other P. aeruginosa strains. The organism can acquire resistance through horizontal gene transfer but is thought to be incompetent in natural transformation. </p>

<p>Free-living amoebae (FLA) are a critical group of the microbial protozoa community that influence biofilm associated bacteria (e.g., P. aeruginosa) in hot tubs and whirlpools. There appears to be no study relating to P. aeruginosa`s interaction with free-living amoebae and its general ecology from the water system in the hot tubs and whirlpools. </p>

<p>Therefore, this study aimed to investigate the antibiotic resistance profiles of P. aeruginosa from water in hot tubs and whirlpools and to demonstrate if the P. aeruginosa isolates are competent in the uptake of extracellular DNA leading to the transfer of antibiotic resistance, and whether FLA may promote early uptake of the extracellular DNA.</p>

<p>Methods: 45 P. aeruginosa isolates from water in hot tubs and whirlpools were assayed, against 36 antibiotic agents, including penicillins, cell wall inhibitors containing extended-spectrum beta-lactamases (ESBLs), protein synthesis inhibitors, carbapenems, fluoroquinolones, cephalosporins, folic acid synthesis inhibitors, nitrofurans, monobactams and aminoglycosides. The resistance was phenotypically assessed using Kirby-Bauer’s disk diffusion method, and the results were interpreted according to the Clinical &amp; Laboratory Standards Institute (CLSI) guidelines. The prevalence and frequency of antimicrobial resistance genes were investigated by PCR.</p>

<p>The study also examined transformation of naked plasmid extracellular DNA carrying antibiotic resistance genes into recipient P. aeruginosa isolates from water in hot tubs and whirlpools. In addition, using fluorescent microscopy, the experiment examined in-situ interactions of P. aeruginosa with two FLAs - Acanthamoebae polyphaga and Willaertia magna on the transformation of the extracellular plasmid DNA to P. aeruginosa isolates. </p>

<p>Results: All the tested P. aeruginosa isolates were resistant to at least six antibiotics; and with all 45 strains possessing a common backbone of resistance to four antibiotics (ampicillin, cloxacillin, mecillinam and cephalothin). All strains were resistant to at least three classes of antibiotics, designating them as multidrug resistant strains.  Some hot tub strains were resistant to as many as 10 antibiotics. Worryingly, five isolates were shown to possess carbapenem resistance (ertapenem and doripenem).  Among the 36 antibiotics tested, 21 antibiotics are clinically relevant, and all the isolates were susceptible to those 21 antibiotics except for one carbapenem (doripenem), for which only one isolate was resistant to this drug.</p>

<p>All the tested P. aeruginosa isolates expressed genes present for porin (oprL, oprD), efflux pumps (ampC, mexC1,2, mexC3,4), QS (lasl, lasR), T3SS (popB), and T6SS (tssC1). The study found most of the P. aeruginosa isolates (89.13%) had effector protein gene exoY, and fewer had another two effector protein genes exoS (54.35%) and exoU (34.78%). The biofilm-associated gene ndvB was identified in all P. aeruginosa isolates, but no isolate contained NDM, the β-lactam biofilm gene. </p>

<p>The research also demonstrated that P. aeruginosa were competent at taking up plasmid DNA containing antibiotic resistance genes from their environment, and stably incorporating this DNA into their cellular metabolism. Transformation was observed after 49 days of monoculture with plasmid DNA.  In contrast, when FLA were present in the culture, transformation occurred in only nine to 14 days, albeit only a single experiment was performed, and therefore some uncertainty remains as to whether predatory amoeba may induce greater rates of transformation in P. aeruginosa. </p>

<p>Conclusions: This study&#39;s results indicate a high frequency of multi-class and multi-drug resistance in P. aeruginosa isolated from hot tubs in Alberta. The observation that carbapenem-resistant P. aeruginosa were found in hot tubs is of critical concern. Nevertheless, this study demonstrated that hot tub isolates were mostly susceptible to the currently used antibiotics recommended for treatment of P. aeruginosa infections. The study observed that the P. aeruginosa isolates from hot tubs and whirlpools could acquire extracellular plasmid DNA containing antibiotic resistance genes through transformation. The results also suggest that the presence of FLAs within the same niche as P. aeruginosa may potentially prompt extracellular plasmid DNA transformation in P. aeruginosa.</p>