A Streamlined Study on Chitosan-Zinc Oxide Nanomicelle Properties to Mitigate a Drug-Resistant Biofilm Protection Mechanism

Abstract

The nosocomial multidrug resistant bacteria (MDR), are rapidly circulating from water surfaces to humans away from the clinical setting, forming a cyclical breeding ground of resistance, causing worldwide infections, and thus requiring urgent responses. The combination of chitosan and zinc oxide (CZNPs), with proven bactericidal effects on some MDRs, was further studied to set the stage for a broad-spectrum in vivo utilization of CZNPs. Toward ensuring CZNPs' uniformity and potency, when it faces not only biofilms but also their extracellular polymeric substances (EPS) defense mechanism, the size, zeta potential, and polydispersity index (PDI) were determined through dynamic light scattering (DLS). Furthermore, the efficacy of CZNPs was tested on the inhibition of MDR Gram-negative Escherichia coli BAA-2471 and Gram-positive Enterococcus faecium 1449 models, co-cultured in an Alvatex 3D fiber platform as a biofilm-like structure. The Biotek Synergy Neo2 fluorescent microplate reader was used to detect biofilm shrinkage. The biofilm protection mechanism was elucidated through detection of EPS using 3D confocal and transmission electronic microscopy. Results indicated that 200 μl/mL of CZNPs, made with 50 nm ZnO and 10,000 Da chitosan (N = 369.1 nm; PDI = 0.371; zeta potential = 22.8 mV), was the most promising nanocomposite for MDR biofilm reduction, when compared to CZNPs enclosing ZnO, 18 or 100 nm. This study depicts that CZNPs possess enough potency and versatility to face biofilms' defense mechanism in vivo.

Evaluation of bactericidal effects of silver hydrosol nanotherapeutics against Enterococcus faecium 1449 drug resistant biofilms

“…The TEM image of E. faecium 1449 revealed a coccus-shaped bacterium expressing pili ( Figure 5 ). This observation is consistent with our previous report ( Limayem et al., 2020 ). Representative TEM images in Figure 6 demonstrated the death of E. faecium 1449 cells in all treatment samples following the Ag NP treatment at various concentrations for 3 hours.…”

“…The inert polystyrene Alvatex strata 3D scaffold inserts (ReproCELL Europe Ltd., Glasgow, UK) that mimic the structural in vivo conditions were utilized to grow E. faecium 1449 biofilms as previously described ( Limayem et al., 2020 ). E. faecium 1449 biofilm matrices composed of extracellular polymeric substances (EPS) were treated with Ag NPs at various concentrations for 24 hours.…”

“…The stained biofilms were then individually imaged with the confocal laser scanning microscope. To assess the EPS formation, E. faecium 1449 biofilms were stained with a combination of fluorescein isothiocyanate isomer I (FITC) and calcofluor white M2R (CFW) dyes for the visualization of protein (green fluorescence signals) and β-polysaccharide components of EPS (blue fluorescence signals) using confocal microscopy as previously described ( Limayem et al., 2020 ).…”

“…where ratios of viable and dead bacterial cells are defined as determined at an excitation/emission of 470/510 nm and 535/617 nm, respectively ( Limayem et al., 2020 ).…”

“…Nosocomial infections from multidrug resistant (MDR) Enterococcus alone comprise a serious threat to public health, registering an estimate of 54,500 hospitalizations and 5,400 deaths annually [ ]. MDR bacteria like Enterococci have allowed isolates to survive multiple generations of anti-gram-positive therapeutics ( Miller et al., 2014 ), requiring novel nanotherapeutics including metal oxides such as the chitosan delivering zinc oxide nanocomposites (CZNPs) ( Limayem et al., 2020 ) or silver (Ag) hydrosol ( Sim et al., 2018 ). Vancomycin-resistant E. faecium (VRE ) are increasingly becoming a threat as a nosocomial infection ( Johnston and Jaykus, 2004 ; Puchter et al., 2018 ).…”