# Preparing a Dual-Species In Vitro Biofilm Model for Testing Antibiofilm Efficacy

## TL;DR

This paper describes a new in vitro model using an artificial skin substrate to test how well wound dressings can prevent and treat dual-species bacterial biofilms.

## Contribution

The study introduces a reproducible dual-species biofilm model on an electrospun gelatin-glucose matrix for testing antibiofilm wound care products.

## Key findings

- The model supported dual-species biofilm growth up to 10⁸ CFU/Gel-Gluc after 24 hours.
- Electrospun PCL dressings with antibiotics effectively prevented and treated biofilms.
- Efficacy of E. coli treatment varied depending on the dual-species combination.

## Abstract

All wounds are contaminated, and there is a risk of developing
an infection. Furthermore, most wounds contain biofilm and are contaminated
by two bacteria, termed dual-species, or more bacteria, termed polybacterial
biofilms. New antibacterial and antibiofilm wound care products are
constantly being developed to combat this problem. There is a need
to develop more biorelevant and reproducible models to test the efficacy
of these wound care products. We used an electrospun (ES) gelatin-glucose
matrix (Gel-Gluc) as an artificial skin substrate for dual-species
biofilm formation using wound pathogens Staphylococcus
aureus, Escherichia coli, and Pseudomonas aeruginosa, combining
them in pairs. When analyzing the biofilms, selective agars were used
to differentiate various bacteria from one another while counting.
The developed method supported the growth of dual-species biofilm
that contained both bacteria up to 108 CFU/Gel-Gluc after
24 h. Over 48 h, there was a decrease in the number of S.
aureus in the biofilms. Confocal microscopy imaging allowed
monitoring of the location of bacteria in the Gel-Gluc and proved
that different species were located closely together. ES polycaprolactone
(PCL) fibrous wound dressings containing chloramphenicol (CAM) or
ciprofloxacin (CIP), or their pristine analogs, were used to test
the model. Both ES fibrous wound dressings were effective in preventing
dual-species biofilm formation. PCL-CIP fibrous dressing was also
effective in treating biofilms. The efficacy of treatment of E. coli varied in different dual-species combinations
of E. coli. The developed dual-species
biofilm model on artificial skin (Gel-Gluc) supported the successful
growth of different bacterial combinations and proved to be suitable
for testing the efficacy of ES fibrous wound dressings in preventing
and treating biofilms.