• Author(s) / Creator(s)
  • 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.

    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`sinteraction with free-living amoebae and its
    general ecology from the water system in the hot tubs and whirlpools.

    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.

    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 & Laboratory

    Standards Institute (CLSI) guidelines. The prevalence and frequency of antimicrobial
    resistance genes were investigated by PCR.

    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.

    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.

    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.

    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 nineto 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.

    Conclusions: This study'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.

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  • Type of Item
    Research Material
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    Attribution-NonCommercial 4.0 International