Jayesh J. Ahire

2,3-Dihydroxybenzoic Acid-Containing Nanofiber Wound Dressings Inhibit Biofilm Formation by Pseudomonas aeruginosa

ABSTRACT Pseudomonas aeruginosa forms biofilms in wounds, which often leads to chronic infections that are difficult to treat with antibiotics. Free iron enhances biofilm formation, delays wound healing, and may even be responsible for persistent inflammation, increased connective tissue destruction, and lipid peroxidation. Exposure of P. aeruginosa Xen 5 to the iron chelator 2,3-dihydroxybenzoic acid (DHBA), electrospun into a nanofiber blend of poly(d,l-lactide) (PDLLA) and poly(ethylene oxide) (PEO), referred to as DF, for 8 h decreased biofilm formation by approximately 75%. This was shown by a drastic decline in cell numbers, from 7.1 log10 CFU/ml to 4.8 log10 CFU/ml when biofilms were exposed to DF in the presence of 2.0 mM FeCl3 6H2O. A similar decline in cell numbers was recorded in the presence of 3.0 mM FeCl3 6H2O and DF. The cells were more mobile in the presence of DHBA, supporting the observation of less biofilm formation at lower iron concentrations. DHBA at MIC levels (1.5 mg/ml) inhibited the growth of strain Xen 5 for at least 24 h. Our findings indicate that DHBA electrospun into nanofibers inhibits cell growth for at least 4 h, which is equivalent to the time required for all DHBA to diffuse from DF. This is the first indication that DF can be developed into a wound dressing to treat topical infections caused by P. aeruginosa.

Polyethylene oxide (PEO)-hyaluronic acid (HA) nanofibers with kanamycin inhibits the growth of Listeria monocytogenes

Listeria monocytogenes is well known to cause prosthetic joint infections in immunocompromised patients. In this study, polyethylene oxide (PEO) nanofibers, containing kanamycin and hyaluronic acid (HA), were prepared by electrospinning at a constant electric field of 10kV. PEO nanofibers spun with 0.2% (w/v) HA and 1% (w/v) kanamycin had a smooth, bead-free structure at 30-35% relative humidity. The average diameter of the nanofibers was 83±20nm. Attenuated total reflectance (ATR)-Fourier transform infrared (FTIR) spectroscopy indicated that kanamycin was successfully incorporated into PEO/HA matrix. The presence of kanamycin affects the thermal properties of PEO/HA nanofibers, as shown by differential scanning calorimetry (DSC) and thermogravimetric analyses (TGA). The kanamycin-PEO-HA nanofibers (1mg; 47±3μg kanamycin) inhibited the growth of L. monocytogenes EDGe by 62%, as compared with PEO-HA nanofibers, suggesting that it may be used to coat prosthetic implants to prevent secondary infections.

Hyaluronic acid-coated poly(D,L-lactide) (PDLLA) nanofibers prepared by electrospinning and coating

Hyaluronic acid nanofibers (HA/PDLLA) were prepared by coating electrospun poly(D,L-lactide) (PDLLA) with different concentrations of HA. Highest concentration of HA deposited onto PDLLA nanofibers (17% per weight) were obtained by coating with 0.3% (w/v) HA. Coating with 0.5% (w/v) and 0.1% (w/v) HA yielded a deposit of 3.8% and 3% per weight, respectively. All nanofibers were 300–400 nm in diameter and microscopically homogeneous in structure, with traces of HA clearly visible on the surface and in-between the fibers. Examination of the nanofibers with Fourier transform infrared (FTIR) spectroscopy, thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) revealed changes in the properties of the nanofibers when coated with 0.3% (w/v) HA. Release of HA from HA/PDLLA fibers were at a linear and constant rate. One possible application for HA/PDLLA is the coating of surfaces, such as medical implants, to prevent secondary microbial infections during recovery from surgery.

Ciprofloxacin-Eluting Nanofibers Inhibits Biofilm Formation by Pseudomonas aeruginosa and a Methicillin-Resistant Staphylococcus aureus

Pseudomonas aeruginosa and Staphylococcus aureus are commonly associated with hospital-acquired infections and are known to form biofilms. Ciprofloxacin (CIP), which is normally used to treat these infections, is seldom effective in killing cells in a biofilm. This is mostly due to slow or weak penetration of CIP to the core of biofilms. The problem is accentuated by the release of CIP below MIC (minimal inhibitory concentration) levels following a rapid (burst) release. The aim of this study was to develop a drug carrier that would keep CIP above MIC levels for an extended period. Ciprofloxacin was suspended into poly(D,L-lactide) (PDLLA) and poly(ethylene oxide) (PEO), and electrospun into nanofibers (CIP-F). All of the CIP was released from the nanofibers within 2 h, which is typical of a burst release. However, 99% of P. aeruginosa PA01 cells and 91% of S. aureus Xen 30 cells (a methicillin-resistant strain) in biofilms were killed when exposed to CIP-F. CIP levels remained above MIC for 5 days, as shown by growth inhibition of the cells in vitro. The nanofibers were smooth in texture with no bead formation, as revealed by scanning electron and atomic force microscopy. A single vibration peak at 1632 cm-1, recorded with Fourier transform infrared spectroscopy, indicated that CIP remained in crystal form when incorporated into PDLLA: PEO. No abnormalities in the histology of MCF-12A breast epithelial cells were observed when exposed to CIP-F. This is the first report of the inhibition of biofilm formation by CIP released from PDLLA: PEO nanofibers.

Copper-Containing Anti-Biofilm Nanofiber Scaffolds as a Wound Dressing Material

Copper particles were incorporated into nanofibers during the electrospinning of poly-D,L-lactide (PDLLA) and poly(ethylene oxide) (PEO). The ability of the nanofibers to prevent Pseudomonas aeruginosa PA01 and Staphylococcus aureus (strain Xen 30) to form biofilms was tested. Nanofibers containing copper particles (Cu-F) were thinner (326 ± 149 nm in diameter), compared to nanofibers without copper (CF; 445 ± 93 nm in diameter). The crystalline structure of the copper particles in Cu-F was confirmed by X-ray diffraction (XRD). Copper crystals were encapsulated, but also attached to the surface of Cu-F, as shown scanning transmission electron microscopy (STEM) and transmission electron microscopy (TEM), respectively. The copper particles had no effect on the thermal degradation and thermal behaviour of Cu-F, as shown by thermogravimetric analysis (TGA) and differential scanning calorimeter (DSC). After 48 h in the presence of Cu-F, biofilm formation by P. aeruginosa PA01 and S. aureus Xen 30 was reduced by 41% and 50%, respectively. Reduction in biofilm formation was ascribed to copper released from the nanofibers. Copper-containing nanofibers may be incorporated into wound dressings.

Surfactin-loaded polyvinyl alcohol (PVA) nanofibers alters adhesion of Listeria monocytogenes to polystyrene

Surfactin-loaded polyvinyl alcohol (PVA) nanofibers were spun using gravity electrospinning. Scanning electron microscopy (SEM) images showed that nanofibers spun with surfactin are free from bead formation and uniform in diameter. The average nanofiber diameters were decreased (273±39nm, 259±39nm and 217±33nm) with increasing levels of surfactin (0.5, 1.0 and 1.5%, w/v) into PVA (10%, w/v). The 10% (w/v) PVA had average fiber diameter of 303±33nm. Atomic force microscopy (AFM) analysis showed that fibers spun with surfactin are not smooth as PVA fibers. The surface average roughness (Sa) estimated for surfactin loaded nanofibers (0.5%: 19.0nm, 1.0%: 20.4nm and 1.5%: 20.7nm) was higher as compared with PVA (10%:15.8nm). Scanning transmission electron microscopy (STEM) showed no matrix differences between PVA and surfactin-loaded PVA nanofibers. Fourier transform infrared (FTIR) microscopy revealed uniform distribution of surfactin in PVA. Based on differential scanning calorimetry (DSC) analyses, surfactin decreased the crystallinity of PVA during spinning. No antimicrobial activity was detected against methicillin-resistant Staphylococcus aureus (MRSA) strain Xen 30, Listeria monocytogenes EDGe, Escherichia coli Xen 14, and Pseudomonas aeruginosa PA01. However, the adhesion of L. monocytogenes to polystyrene in presence of surfactin-loaded nanofibers decreased significantly (OD595: 0.012±0.001) as compared with control (OD595: 0.022±0.002), suggesting that these nanofibers may be used in wound dressings or in the coating of prosthetic devices to prevent biofilm formation and secondary infections.