Graphene Oxide affects Staphylococcus aureus and Pseudomonas aeruginosa dual species biofilm in Lubbock Chronic Wound Biofilm model

Abstract

Chronic wound management becomes a complex procedure because of the persistence of forming biofilm pathogens that do not respond to antimicrobial treatment. The aim of this paper is to detect the Graphene Oxide-GO effect on Staphylococcus aureus and Pseudomonas aeruginosa dual species wound biofilm in Lubbock Chronic Wound Biofilm-LCWB model. LCWB is a recognized model that mimics the spatial microbial colonization into chronic wounds and reproduces the wound and its clot. Staphylococcus aureus PECHA 10 and P. aeruginosa PECHA 4, are the pathogens used in the study. The GO effect on both in forming and mature biofilms, is detected by the evaluation of the CFU/mg reduction, the cell viability and ultrastructural analysis of the treated LCWBs. Graphene Oxide, at 50 mg/l, shows a significant antibiofilm effect in forming and mature LCWBs. In particular, during the biofilm formation, GO reduces the S. aureus and P. aeruginosa growth of 55.05% ± 4.73 and 44.18% ± 3.91 compared to the control. In mature biofilm, GO affects S. aureus and P. aeruginosa by reducing their growth of 70.24% ± 4.47 and 63.68% ± 17.56, respectively. Images taken by SEM show that GO display a disaggregated microbial effect also disrupting the fibrin network of the wound-like biofilm framework. In conclusion, GO used against microorganisms grown in LCWB, displays a significant inhibitory action resulting in a promising tool for potential application in wound management.

Prebiotic Combinations Effects on the Colonization of Staphylococcal Skin Strains

“…Anonymized Staphylococcus aureus 815, S. aureus PECHA 10, S. epidermidis 317 and S. epidermidis MDG1 clinical strains, coming from the private collection of the Bacteriology Laboratory of Pharmacy Department, were used in this study. Staphylococcus aureus 815 [ 17 , 18 , 19 ], S. aureus PECHA 10 [ 20 ] and S. epidermidis 317 [ 21 ] were previously characterized for their biofilm-related properties.…”

“…The images were recorded at excitation/emission wavelengths of 485/498 nm for SYTO 9 and 535/617 nm for propidium iodide. The analysis of viable cells in the planktonic phase and viable and clustered cells in the sessile phase was determined by examining ten random fields of view for each sample, and each slide was controlled by three blinded microbiologists [ 20 , 23 , 24 ].…”

Antimicrobial Peptide L18R Displays a Modulating Action against Inter-Kingdom Biofilms in the Lubbock Chronic Wound Biofilm Model

“…The effect of L18R peptide was evaluated on the LCWB model following a previously described methodology [ 19 ]. Briefly, bacteria were cultured in TSB and incubated at 37 °C overnight in aerobic conditions, then were standardized to OD 600 = 0.125 and diluted 1:10 for S. aureus PECHA 10 and 1:100 for P. aeruginosa PECHA 4, to obtain 10 6 CFU/mL and 10 5 CFU/mL, respectively.…”

“…After 48 h of incubation, the mature biofilms were harvested from the glass tubes, the pipette tips were removed, and the biofilm biomass was washed two times with sterile PBS. After biovolume (V = π × r 2 × h) and biomass weight determination, LCWBs were placed on Petri dishes containing Bolton broth (Oxoid, Milan, Italy) with 1.5% bacteriological agar to produce the “wound bed” for the chronic wound biofilm model [ 11 , 19 , 20 ].…”

“…After placing the mature biofilm on the “wound bed”, LCWBs were treated with an amount of L18R peptide (at a final concentration of 100 μg/mL), amikacin (AMK, as treatment control, at a final concentration of 64 μg/mL), and PBS (untreated control) with a volume that depended on each LCWB biovolume (V = π × r 2 × h) [ 19 ]. Amikacin was chosen both because it is used in antibiotic treatment for patients with chronic wounds [ 21 ] and because it was previously tested in the LCWB model [ 19 , 22 ]. The untreated and treated LCWB biovolumes were compared.…”