Antibacterial activity of iron oxide, iron nitride, and tobramycin conjugated nanoparticles against Pseudomonas aeruginosa biofilms

Abstract

Background: Novel methods are necessary to reduce morbidity and mortality of patients suffering from infections with Pseudomonas aeruginosa. Being the most common infectious species of the Pseudomonas genus, P. aeruginosa is the primary Gram-negative etiology responsible for nosocomial infections. Due to the ubiquity and high adaptability of this species, an effective universal treatment method for P. aeruginosa infection still eludes investigators, despite the extensive research in this area.

Results: We report bacterial inhibition by iron-oxide (nominally magnetite) nanoparticles (NPs) alone, having a mean hydrodynamic diameter of ~ 16 nm, as well as alginate-capped iron-oxide NPs. Alginate capping increased the average hydrodynamic diameter to ~ 230 nm. We also investigated alginate-capped iron-oxide NP-drug conjugates, with a practically unchanged hydrodynamic diameter of ~ 232 nm. Susceptibility and minimum inhibitory concentration (MIC) of the NPs, NP-tobramycin conjugates, and tobramycin alone were determined in the PAO1 bacterial colonies. Investigations into susceptibility using the disk diffusion method were done after 3 days of biofilm growth and after 60 days of growth. MIC of all compounds of interest was determined after 60-days of growth, to ensure thorough establishment of biofilm colonies.

Conclusions: Positive inhibition is reported for uncapped and alginate-capped iron-oxide NPs, and the corresponding MICs are presented. We report zero susceptibility to iron-oxide NPs capped with polyethylene glycol, suggesting that the capping agent plays a major role in enabling bactericidal ability in of the nanocomposite. Our findings suggest that the alginate-coated nanocomposites investigated in this study have the potential to overcome the bacterial biofilm barrier. Magnetic field application increases the action, likely via enhanced diffusion of the iron-oxide NPs and NP-drug conjugates through mucin and alginate barriers, which are characteristic of cystic-fibrosis respiratory infections. We demonstrate that iron-oxide NPs coated with alginate, as well as alginate-coated magnetite-tobramycin conjugates inhibit P. aeruginosa growth and biofilm formation in established colonies. We have also determined that susceptibility to tobramycin decreases for longer culture times. However, susceptibility to the iron-oxide NP compounds did not demonstrate any comparable decrease with increasing culture time. These findings imply that iron-oxide NPs are promising lower-cost alternatives to silver NPs in antibacterial coatings, solutions, and drugs, as well as other applications in which microbial abolition or infestation prevention is sought.

Keywords: Alginate; Antibacterial agents; Antibiotic resistance; Biofilm; Cystic fibrosis; Drug delivery; Iron-oxide nanoparticles; Magnetite; Pseudomonas aeruginosa; Zero-valent iron nanoparticles.

Fig. 1

Biofilm colony on infected tissue with viscous mucus layer characteristic of an infection in the CF respiratory tract, illustrating the inability of drugs and antibodies to penetrate the mucus and biofilm barriers and reach the target microbial colonies

Fig. 2

Purification of succinylated polyethylene glycol (PEG) through dialysis tubing in deionized (DI) water

Fig. 3

Image showing the presence of pyocyanin (blue-green) pigment produced by P. aeruginosa cultures grown on agar plates

Fig. 4

Schematic diagram of minimum inhibitory concentration (MIC) determination of tobramycin, iron-oxide NPs, tobramycin-NP conjugates, and zero-valent iron NPs in P. aeruginosa liquid cultures

Fig. 5

TEM images of iron-oxide NPs prior to alginate capping. Image A, scale bar is 50 nm, image B (insert) is a HRTEM image of crystal structure, scale bar 5 nm, and FFT of fringing with crystal indices

Fig. 6

HRTEM image of an iron nitride NP showing crystalline fringes, scale bar is 5 nm

Fig. 7

Energy dispersive x-ray spectrum of iron-oxide NPs, showing elemental composition

Fig. 8

Indexed magnetite peaks from powder x-ray diffraction pattern obtained with a Cu Kα 1.54 Å source and a monochromator

Fig. 9

XRD spectrum of iron nitride NPs taken with a Cu Kα 1.54 Å source and a monochromator

Fig. 10

DLS size-distribution histogram of iron-oxide NPs prior to polymer coating

Fig. 11

DLS size distribution showing average hydrodynamic size of iron-oxide NPs after alginate capping

Fig. 12

Comparison of hysteresis loops of nanocrystalline samples of iron oxide (red) and iron nitride (blue) of similar grain size, showing the significantly stronger magnetic properties of iron nitride. Left image shows entire hysteresis loop of iron nitride. Right image is a close up of the same, showing hysteresis loop of iron oxide

Fig. 13

Magnetization vs temperature for iron-oxide and iron-nitride NPs under zero-field cooled (lower curves) and field cooled (upper curves) conditions. The magnetization of the ferrofluid samples was measured with a DC field of 100 Oe (τ_m = 100 s) in the temperature range between 9 K and 350 K

Fig. 14

Agar cultures used for susceptibility testing. A) Agar plate with impregnated disks prior to overnight incubation. B) Image shows zone of inhibition (ZOI) halo around disk impregnated with antimicrobial agent of interest; a positive susceptibility result. C) Motility testing results in agar stab cultures after incubation; upper tube is a negative motility result and lower tube is a positive motility result

Fig. 15

Minimum inhibitory concentration (MIC) of tobramycin on P. aeruginosa colonies as a function of growth time. Note that the cutoff concentration for susceptibility of P. aeruginosa to tobramycin in liquid cultures is ≤ 4 μg/mL. Therefore, none of the cultures are susceptible to tobramycin by CLSI standards

Fig. 16

Optical density (OD) at a 600 nm wavelength for liquid cultures exposed to treatment with iron-oxide NPs, zero-valent iron, or tobramycin-conjugated iron-oxide NPs. To avoid interference, NPs were removed by magnetic separation prior to optical measurements. The calculated average error for OD measurements was ± 0.01. Specific errors, not the average error, were used to calculate statistical significance

Fig. 17

Percent bacterial inhibition vs. treatment concentration in liquid cultures in cuvette. All NP samples presented here were capped with alginate

Fig. 18

Mechanisms of cell damage and response after exposure to iron-containing NPs. Iron ions, released from NPs, can cross the membrane via either active cellular uptake or leakage through sites with reduced membrane integrity. Highly reactive hydroxyl radicals resulting from Fe²⁺ reaction with hydrogen peroxide primarily cause oxidative damage. Fe³⁺ could be reduced by NADH and, thus, regenerating Fe²⁺. OH·radicals could also cause damage to DNA, proteins and lipids. Fe²⁺ may also directly damage DNA