Abstract
Pseudomonas aeruginosa is an ESKAPE pathogen associated with difficult-to-treat burn wound and surgical-site infections. This study aimed to characterise an extensively drug resistant (XDR) P. aeruginosa isolate (designated PAW1) and to investigate the antibiofilm and antipersister effect of acetic acid on PAW1. PAW1 was identified using biotypic (VITEK) and genotypic (16S rDNA) analysis. Minimum inhibitory concentration (MIC) and disc susceptibility testing showed high level resistance against all antibiotics from classes including beta lactams, cephems, carbapenems and fluoroquinolones. It was therefore identified as extensively drug resistant (XDR), showing resistance to all antibiotics except for, aminoglycoside (gentamicin and netilmicin) and lipopeptides (polymyxin B). Time kill assays showed antibiotic tolerant, persister cell formation in presence of 100X MICs of gentamicin and polymyxin B. Other virulence traits such as ability to produce lipase, protease, haemolysin, and siderophores and to form biofilms were additional factors which may contribute to its pathogenicity. PAW1 showed promising susceptibility against acetic acid with MIC and minimum biofilm inhibitory concentration of 0.156% (v/v). Percent viability of PAW1 was dependent on dose and treatment time of acetic acid. 0.625% acetic acid treatment of 5 minutes was effective in killing >90% planktonic cells showing lesser toxicity to L929 cells (IC50 = 0.625%). Biofilm disruption caused due to acetic acid was also dose dependent, showing 40.57% disruption after treatment with 0.625% acetic acid for 5 minutes. FESEM imaging and live dead staining of planktonic and biofilm forms of PAW1 confirmed that acetic acid treatment caused 19.04% of cell shrinkage and disruption of extracellular matrix resulting in killing of cells. Antipersister activity of acetic acid was demonstrated by showing complete killing of PAW1 at 4X MIC. Overall, this study characterised an XDR isolate P. aeruginosa showing resistance and tolerance to various antibiotics. Antipersister and antibiofilm effect of acetic acid demonstrates the importance of forgotten topical agents as an effective strategy to treat XDR pathogens.
Highlights
The acronym “ESKAPE” denotes six hospital associated pathogens: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. with formidable and ever-present adversary associated with broad spectrum of infections in humans [1, 2]
P. aeruginosa is one of the ESKAPE members associated with secondary wound infections which are difficult to treat owing to the multidrug resistant (MDR) nature of this pathogen further narrowing down the usage of existing antibiotics, topical antiseptics and disinfectants [3]
P. aeruginosa used in the present study was an isolate from a recalcitrant sternal wound infection of a diabetic patient who was put on negative pressure wound therapy
Summary
The acronym “ESKAPE” denotes six hospital associated pathogens: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. with formidable and ever-present adversary associated with broad spectrum of infections in humans [1, 2]. P. aeruginosa has virulence traits such as biofilm forming ability, siderophore production, production of extracellular enzymes and other proteins responsible for pathogenicity [4, 5] which add to the arsenal available to cause infections. P. aeruginosa is known to enter a persister state during which a sub-population of cells remains metabolically inactive, tolerating the presence of antibiotics [6]. This persister state is defined by the ability of an organism to survive high doses of an antibiotic despite being sensitive to it at lower concentrations. Biofilm forming ability and persister state are a major cause of the recurrent infections caused by this pathogen [8,9,10,11]
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