Biofilm inhibition of multidrug-resistant Pseudomonas aeruginosa using green-synthesized silver nanoparticles and colistin

Abstract

We aimed to investigate the synergistic effects of colistin and green-synthesized silver nanoparticles on the biofilm formation and expression of Quorum Sensing regulated and related genes in clinical isolates of P. aeruginosa. Ten clinical P. aeruginosa isolates collected from patients with burn wound infections were investigated. The antibiotic sensitivity pattern of the isolates was determined using disk diffusion and microbroth dilution tests. The silver nanoparticles (AgNPs) were synthesized using propolis and characterized. The microtiter plate method and scanning electron microscopy (SEM) were used to evaluate the synergistic effects of colistin and silver nanoparticles combination (AgNPs@CL) on the inhibition of biofilm formation. The effect of AgNPs@CL on the expression of genes controlled by QS was evaluated using RT-PCR. All isolates were strong biofilm formers. Confronting AgNPs@CL, all isolates were either synergistic or additive and effectively decrease the minimum inhibitory concentration (MIC) and minimum biofilm inhibitory concentration (MBIC) values of Carbapenem-Resistant P. aeruginosa (CRPA) isolates. The SEM analysis corroborated the enhanced biofilm inhibition observed with the combined treatment compared to individual AgNPs or colistin treatments. When exposed to AgNPs@CL, the expression levels of lasI, lasR, rhlI, rhlR, pelA, and pslA genes significantly decreased in P. aeruginosa ATCC 27,853 and clinical isolate No. #354, which displayed synergistic activity. In contrast, with additive activity, clinical isolate No. #30 showed no significant decrease. Targeting critical components of QS could effectively inhibit biofilm production. The results of our study suggest AgNPs@CL as an auxiliary to antibiotic therapy.

Keywords: Pseudomonas aeruginosa; Biofilms; Colistin; Drug synergism; Quorum sensing.

Fig. 1

(A) Five isolates highlighted in yellow box are clonally close. The pink highlighted box encloses five clinical isolates along with P. aeruginosa ATCC 27,853, which are genetically distant. Three isolates enclosed in boxes were chosen for SEM and RT-PCR analysis. (B) Minimum Spanning Tree illustrates isolates’ distribution based on their distance in loci. Five green circles partitioned in pink with thick connecting lines are distinct in only two loci. Each circle belongs to an isolate. Dotted lines between circles demonstrate distances in more than two loci among the isolates. All concentrations are in µg/mL.

Fig. 2

AgNPs characterization. (A) TEM photograph at 100 nm displays polymorphic AgNPs. (B) Histogram displaying the size distribution of nanoparticles analyzed through ImageJ software based on their diameter. The majority of the nanoparticles had a diameter of 60–80 nm. (C) XRD pattern of the peaks observed at 2θ at 38.39, 44.69, 64.79, and 77.79 corresponded to Miller indices (111), (200), (220), and (311). (D) The FTIR spectrum of AgNPs displays intense absorption bands.

Fig. 3

(A) The maximum absorbance (λ max) of AgNPs were recorded as 500–550 nm in UV-Vis spectrophotometer. (B) The surface charge of AgNPs was measured to be-22.3 Mv. (C) Cell viability value of AgNPs. When L929 cells exposed to 37.5 and 75 µg/mL of AgNPs, the percentage of cells that remained alive were 55.63 and 50.22, respectively. The symbol * indicates a statistically significant difference between the 37.5 µg/mL and control negative groups (P-values < 0.05). (D) MTT cytotoxicity of AgNPs at 37.5 µg/mL and 75 µg/mL.

Fig. 4

(A) Antimicrobial susceptibility pattern of isolate No. #354 determined by disk diffusion method. (B) The microtitre plate assay was used to evaluate the biofilm production capacity of the isolates.

Fig. 5

(A) Isolates under AgNPs, Colistin (CL), and AgNPs@CL treatment. The first left column depicts Un-Treated samples. Biofilm mass mitigation during single and combined treatments is evident in all three isolates (5000× magnification). (B.1) Biofilm cement surrounds the sessile cells in the developing Un-Treated biofilm. (B.2) Biofilm treated at sub-FBIC concentration of AgNPs@CL suggest extreme cellular damages. Pierced membrane (yellow dashed circle), cell shrinkage (red dashed circle), cell leakage (white dotted circle), and membrane blebs (green dashed circle) together with obvious biofilm mass mitigation in a scanty microscopic field (20,000× magnification). The lines observed in the SEM images are likely due to the deformation of the polystyrene substrate under pressure or chemical interactions during biofilm preparation. These artifacts do not affect the integrity of the biofilm itself or the interpretation of the treatment effects observed.

Fig. 6

The heatmap clustering illustrates the log2-fold changes of P. aeruginosa ATCC 27,853, isolates Nos. #354, and #30 normalized to two reference genes under the scheduled treatments. The numbers in parentheses indicate the level of suppression in percentage. Both rows and columns are clustered using Euclidean distance and Ward linkage. The suppression range varies from purple (little) to red (marked). Dendrogram A classifies isolates into three clusters based on the suppression level confronting scheduled treatments.