Synergistic effect of electrolyzed oxidized water (EO) and peroxyacetic acid on plasmid-mediated quinolone resistance genes of Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa displays major biofilm formation, food spoilage, and significant losses for the food industry. It extremely exhibits inherent resistance to various antibiotics, including quinolones, which can spread via plasmids. The present study investigates the prevalence of biofilm-forming P. aeruginosa in meat products and assesses its antibiotic resistance patterns. Evaluating the effectiveness of electrolyzed-oxidized water (EOW) and per-acetic acid (PAA) alone or in a combination against P. aeruginosa biofilm formed on stainless steel (SS) surfaces. Besides, the effect on quinolone-resistance (qnr) gene expression level using real-time PCR. P. aeruginosa was isolated from 24.6% of the total tested samples (37/150), with a significant variation observed regarding the contamination levels of the tested products. 36.3% of the isolates demonstrated robust biofilm production, with 82.3% displaying multi-drug resistance (MDR). Isolates revealed complete susceptibility to amikacin (100%) and gentamycin (82%). Quinolone resistance was observed in 23% and 17% of the isolates for ciprofloxacin and levofloxacin, respectively. 80% (4/5) among the confirmed isolates were enclosed the plasmid qnr genes. The genes pslA and gyrA were detected in 40% and 60%, respectively. EOW, particularly when combined with PAA, reveals strong antibacterial activity against P. aeruginosa and greatly decreases its count. Moreover, it can eliminate its biofilm within 20 min of exposure and significantly decrease the expression levels of quinolone-resistant genes. In conclusion, PAA and EOW are effective agents for biofilm control on SS surfaces, particularly when used in combined form, and could be more effective in combating resistant bacterial infections and controlling the spread of PMQR.

Keywords: P. aeruginosa; Per-acetic acid; Plasmid-mediated quinolone resistance (PMQR) genes. Electrolyte oxidized water (EOW).

Fig. 1

Mosaic plot with coloured cases for the observed frequencies in the incidence rate of p. aeruginosa

Fig. 2

(a): Resistance rate of p. aeruginosa isolates to different antibiotics. (b): The violin plot showed variation in multiple antibiotic resistance in isolates. (c): Spearman’s correlation plot among different antibiotic-resistant phenotypes and biofilm production. Correlation coefficients are shown as colours on the scale (positive: blue, negative: red). AMC: amoxicillin/clavulanate, CAZ: ceftazidime, CTX: cefotaxim, MEM: meropenem, CIP ciprofloxacin, LEV: levofloxacin, GN: gentamicin, AK: Amikacin and SXT: sulfamethoxazole /trimethoprim

Fig. 3

Agarose gel electrophoresis for uniplex conventional PCR amplification of (A): amplification of 16SrRNA gene for identification of P. aeruginosa isolates at 956 bp, (B): amplification of pslA gene at 656 bp, (C): amplification of qnrA gene at 580 bp, (D): amplification of qnrB gene at 264 bp, (E): amplification of qnrS at 428 bp and (F): amplification of gyrA gene at 358 bp. Lane L: ladder, Lane 1–5 for samples

Fig. 4

(a)- Effect of different treatment on P. aeruginosa colony count, (b)- Effect of contact time of treatment on P. aeruginosa count with initial count (control–ve group) = 7.59 log CFU/cm². (c)- Effect of different treatments on P. aeruginosa biofilm count and, (d)- Effect of contact time of treatment on P. aeruginosa biofilm count with initial count (control–ve group) = 9.44 log CFU/cm². G1:treated with 0.1% PAA; G2:treated with 0.3% PAA; G3:treated with EOW; G4:treated with a combined mix of EOW + 0.1% PAA. G5:treated with a combined mix of EOW + 0.3% PAA. * = means a significance difference, ** means moderate significance difference, **** means a highly significance difference

Fig. 5

Fold change of the relative gene expression levels of (pslA, qnrA, qnrB, and qnrS) concerning the effect of three different treatment groups in comparison to control. Group 1: treated with Per-acetic acid (PAA) 0.1%, Group 2: treated with EOW (pH, 2.1), Group 3: treated with mixed (EOW + PAA 0.1%)