Differential surface contact killing of pristine and low EPS Pseudomonas aeruginosa with Aloe vera capped hematite (α-Fe2O3) nanoparticles

Abstract

Biogenic hematite (α-Fe₂O₃) nanoparticles (NPs) of average size <10 nm were synthesized using green approach with Aloe vera extract (ALE). The aim of the study was to assess the protective effect of extracellular polymeric substances (EPS) against antibacterial and antibiofilm activities of ALE-α-Fe₂O₃NPs in normal EPS producers (pristine) and experimentally modified (low-EPS) Pseudomonas aeruginosa (P. aeruginosa) cells and the mechanism of cell killing. Formation of ALE-α-Fe₂O₃NPs has been validated by X-ray diffraction (XRD), transmission electron microscopy (TEM), scanning electron microscopy (SEM) and Fourier-transformed infrared spectroscopy (FTIR) analysis. The FTIR data suggested the possible role OH group bearing organic compounds of ALE in metal reduction and nucleation of NPs. Gas Chromatography-Mass spectroscopy (GC-MS) analysis revealed the presence of oxime-methoxy-phenyl, ethanone 1-phenyl, hexadecanoic acid, cyclohexanol 2,6-dimethyl, tetracontane, stigmast-5-en-3-ol, cyclohexanol 2,6-dimethyl, and cyclohexasiloxane dodecamethyl on the surface of ALE-α-Fe₂O₃NPs. Cell viability assay and SEM imaging revealed significantly greater bacteriostatic and/or bactericidal effect of ALE-α-Fe₂O₃NPs in low EPS cells compared to pristine cells or bare-α-Fe₂O₃NPs. This is attributed to thinner protective layer of EPS around the low EPS cells, and higher dispersibility and stability of ALE-α-Fe₂O₃NPs. Absorption of ALE-α-Fe₂O₃NPs and bare-α-Fe₂O₃NPs on EPS surface and within EPS matrix was ascertained by atomic absorption spectroscopy (AAS). The results suggest differential internalization of ALE-α-Fe₂O₃NPs and bare-α-Fe₂O₃NPs in P. aeruginosa cells. The flow cytometry (FCM) results exhibited increased intracellular granularity in low EPS (18.94%) as compared with pristine (10.94%) cells, which signifies the greater internalization of ALE-α-Fe₂O₃NPs. Moreover, the proportionate increase in intracellular ROS generation in low EPS (20.47%) via-a-vis pristine (7.56%) cells was observed. Overall, the results elucidate that ALE-α-Fe₂O₃NPs-bacterial interaction leads to attachment of NPs to EPS surface, migration within the EPS matrix and penetration into cell, which eventually results in growth inhibition due to intracellular ROS activity. Owing to significant antibacterial and antibiofilm activities, ALE-α-Fe₂O₃NPs may serve as a good candidate for clinical management of extended spectrum beta lactamases (ESBL) positive P. aeruginosa.

Keywords: Aloe vera; Biofilm inhibition; Biogenic synthesis; Cellular uptake; Hematite nanoparticles.