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

<p>Objectives:Pseudomonas aeruginosa is one of the most problematic opportunistic pathogens.<br>
It is resistant to many currently used antibiotics, making it difficult to treat, and resistance may<br>
transfer to other P. aeruginosa strains. The organism can acquire resistance through horizontal<br>
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<br>
influence biofilm associated bacteria (e.g., P. aeruginosa) in hot tubs and whirlpools. There<br>
appears to be no study relating to P. aeruginosa`sinteraction with free-living amoebae and its<br>
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<br>
from water in hot tubs and whirlpools and to demonstrate if the P. aeruginosa isolates are<br>
competent in the uptake of extracellular DNA leading to the transfer of antibiotic resistance,<br>
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,<br>
against 36 antibiotic agents, including penicillins, cell wall inhibitors containing extended-<br>
spectrum beta-lactamases (ESBLs), protein synthesis inhibitors, carbapenems,</p>

<p>fluoroquinolones, cephalosporins, folic acid synthesis inhibitors, nitrofurans, monobactams<br>
and aminoglycosides. The resistance was phenotypically assessed using Kirby-Bauer’s disk<br>
diffusion method, and the results were interpreted according to the Clinical &amp; Laboratory</p>

<p>Standards Institute (CLSI) guidelines. The prevalence and frequency of antimicrobial<br>
resistance genes were investigated by PCR.</p>

<p>The study also examined transformation of naked plasmid extracellular DNA carrying<br>
antibiotic resistance genes into recipient P. aeruginosa isolates from water in hot tubs and<br>
whirlpools. In addition, using fluorescent microscopy, the experiment examined in -situ<br>
interactions of P. aeruginosa with two FLAs - Acanthamoebae polyphaga and Willaertia<br>
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<br>
all 45 strains possessing a common backbone of resistance to four antibiotics (ampicillin,<br>
cloxacillin, mecillinam and cephalothin). All strains were resistant to at least three classes of<br>
antibiotics, designating them as multidrug resistant strains. Some hot tub strains were resistant<br>
to as many as 10 antibiotics. Worryingly, five isolates were shown to possess carbapenem<br>
resistance (ertapenem and doripenem). Among the 36 antibiotics tested, 21 antibiotics are<br>
clinically relevant, and all the isolates were susceptible to those 21 antibiotics except for one<br>
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<br>
pumps (ampC, mexC1,2, mexC3,4), QS (lasl, lasR), T3SS (popB), and T6SS (tssC1). The study<br>
found most of the P. aeruginosa isolates (89.13%) had effector protein gene exoY, and fewer<br>
had another two effector protein genes exoS (54.35%) and exoU (34.78%). The biofilm-<br>
associated gene ndvB was identified in all P. aeruginosa isolates, but no isolate contained<br>
NDM, the β-lactam biofilm gene.</p>

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

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