‘Biofilms’ delay EB wound healing

After washing wounds to remove bacteria not attached to a biofilm, sterile swabs were pressed on a 1cm² area of wound until the tissue oozed. Patients were informed that this could cause discomfort before the process and gave their consent in advance. Despite careful handling and preservation of the swabs, not enough genetic material was successfully collected or prepared to obtain genetic sequences of the bacteria that may have been present in biofilms on the wounds. This was unexpected as the method had been successful before. The researchers suggest that trying again would involve local anaesthetic and scraping or biopsy of the wound.

Four people with DEB and four people with JEB have joined the project. Samples are being collected from wounds that show no sign of infection and have not been treated with antibiotics recently. The samples are stored at -80C until they can all be analysed together. The results will be shared at the end of this one-year project.

Lead researcher:

Dr Josefine Hirschfeld is an associate professor and honorary consultant at the University of Birmingham Dental School and Hospital. She specialises in restorative dentistry with a particular focus on gum diseases and immune cells called neutrophils and their role in diseases including EB.

Co-researchers:

Dr Hadeer Ibrahim is an assistant lecturer of Dermatology at the Suez Canal University in Egypt and has been awarded a highly competitive scholarship to fund her PhD degree on EB at the University of Birmingham where she focuses her research on improving the quality of life of patients with EB of different subtypes.

Prof Iain Chapple is Head of Research for the Institute of Clinical Sciences, Head of Periodontology with the School of Dentistry and Honorary Consultant in Restorative Dentistry with Birmingham Community Health Trust.

Prof Adrian Heagerty is a Consultant Dermatologist and Honorary Professor of Dermatology at the University of Birmingham with a particular interest in EB. He leads several active research groups and heads the half-national EB service at University Hospitals Birmingham NHS Foundation Trust.

Dr Sarah Kuehne is a senior lecturer in oral microbiology and the lead of the Oral Microbiology Research Group at the University of Birmingham. Her research group holds close links to the world-renowned Institute of Microbiology and Infection (IMI) led by Prof Ian Henderson.

Collaboration:

Dr Annika Therese Kroeger, at the School of Dentistry, University of Birmingham, has extensive experience in metagenomics and transcriptomics.

Prof Moritz Kebschull, at the School of Dentistry, carried out award-winning translational research linking the clinical features and molecular foundations of periodontal and peri-implant diseases and is a co-investigator at the newly funded NIHR Biological Research Centre.

“We aim to confirm our initial exciting discovery that biofilms are present in chronic EB wounds that fail to heal, whereas they are not present in younger wounds. We will explore how common biofilms are in chronic wounds, the microbes within, and whether those differ between different types of EB. This is critically important because biofilms are resistant to traditional antibiotic treatments, and they may be a strong driver of the failure of chronic wounds to heal.

Importantly, biofilm-related diseases, such as those in the mouth (gum disease) that we have studied for over 20-years, are treated very differently to traditional wound infections. The biofilm itself must be physically disrupted and there are several simple approaches to achieving this, depending on their composition. These approaches are already in use in other diseases and could be adapted very rapidly for EB wound care, potentially transforming EB wound management. This will be part of a larger research project we are conducting, paving the road for further research on how such biofilms interact with the patient’s immune system.

In the future, we will apply different non-invasive anti-biofilm measures including use of agents to break down the biofilm matrix and the use of light therapy that will be specific to the types of microbes detected.”

– Dr Hirschfeld

Grant title: Use of highly sensitive and specific gene analytical techniques to identify microorganisms that may play a role in the chronicity of EB wounds.

Non-healing wounds in EB patients present significant morbidity and lower quality of life. Complications include: the need for protracted wound care, itch, pain, skin cancer and fusion of fingers forming so-called “mitten hands”.

We understand how and why EB blisters form, but we do not know why some blisters fail to heal for months or years despite good wound care. We believe that the underlying cause may be the presence of “biofilms” on the wound surface.

Biofilms are aggregates of different microbes living within a self-produced slimy matrix, which is attached to a surface such as skin and is often resistant to traditional treatments.

Biofilms are known to disturb wound healing and wounds harbouring biofilms frequently show no signs of infection like pus and inflammation/redness. Moreover, biofilms cannot be detected by traditional wound swabbing methods, and do not respond to regular wound care and antibiotic use. However, wounds with biofilms studied in other diseases including diabetes, burn and venous wounds, can respond well to anti-biofilm measures resulting in improved healing. Our preliminary research using different imaging techniques has shown, for the first time, the presence of biofilms on chronic wounds of different EB subtypes.

This proposal aims to confirm our observational results by genetic analysis of these biofilms to reveal the types of microbes present as well as possible differences between EB subtypes.

This has never been studied, it will involve the use of new techniques and will be a multi-disciplinary collaborative project. This will help us develop new therapies in a more targeted way, as we will have strong indications as to why wounds fail to heal. This will lead to a more personalised approach to therapy, resulting in better wound management strategies and help proper healing of chronic wounds in EB patients.

We are in the process of samples collection. Samples are being collected from patients with EB who have acute/chronic wounds that show no signs of infection and have not been treated with antibiotics recently. We are aiming at collecting samples from 5 patients with dystrophic EB (DEB) and at least 3 with junctional EB (JEB). Sample collection from this rare skin condition is challenging, and our inclusion/exclusion criteria need to be applied. However, so far, we managed to recruit 4 DEB and 4 JEB patients. Samples are kept in -80C freezer for later processing once all samples are collected. (From progress report April 2024.)

Non-healing wounds in EB patients present significant morbidity and lower quality of life. Complications include: the need for protracted wound care, itch, pain, skin cancer and fusion of fingers forming so-called “mitten hands”. We understand how and why EB blisters form, but we do not know why some blisters fail to heal for months or years despite good wound care. We believe that the underlying cause may be the presence of “biofilms” on the wound surface.

Biofilms are aggregates of different microbes living within a self-produced slimy matrix, which is attached to a surface such as skin and is often resistant to traditional treatments. Biofilms are known to disturb wound healing and wounds harbouring biofilms frequently show no signs of infection as pus and inflammation/redness. Moreover, biofilms cannot be detected by traditional wound swabbing methods, and do not respond to regular wound care and antibiotic use. However, wounds with biofilms studied in other diseases including diabetes, burn and venous wounds, can respond well to anti-biofilm measures resulting in improved healing.

We aimed to confirm our initial exciting discovery that biofilms are present in chronic EB wounds that fail to heal, whereas they are not as common in younger wounds. We aimed to explore how common biofilms are in chronic wounds, the microbes within, and whether those differ between different types of EB. This is critically important because biofilms are resistant to traditional antibiotic treatments, and they may be a strong driver of the failure of chronic wounds to heal. Importantly, biofilm-related diseases, such as those in the mouth (gum disease) that we have studied for over 20-years, are treated very differently to traditional wound infections. The biofilm itself must be physically disrupted and there are several simple approaches to achieving this, depending on their composition. These approaches are already in use in other diseases and could be adapted very rapidly for EB wound care, potentially transforming EB wound management. This is part of a larger research project we are conducting, paving the road for further research on how such biofilms interact with the patient’s immune system.

People with EB attending at both Solihull and Birmingham dental hospital, Birmingham, UK as part of their regular medical care were recruited. Patients recruited were selected with the following criteria: being older than 18 years old with no other chronic medical conditions, not immunocompromised and not receiving topical or systemic antibiotics for at least 2 weeks prior to sample collection and who have wounds showing no signs of infection e.g., pus, redness or inflammation with a duration of either less more than 3 months resembling chronic wounds or less than 2 weeks resembling acute ones as a control. After obtaining informed written consents per the study ethics approval, wounds were irrigated with sterile saline to get rid of floating bacteria. Then a sterile swab was used to obtain wound surface sample through sampling a 1 cm2 area with pressure till tissue oozes. Patients were warned beforehand that this can cause discomfort. However, that was important to get a representative sample. The swab was then placed in a sterile tube with a liquid that preserves the genetic material, transmitted in a cold media of dry ice and transported to the lab in the same day to be kept in –80C freezer till later processing. For sample processing, swabs were thawed gradually, prepared for RNA extraction using a kit per the manufacturer’s protocol. Extracted RNA was then sent for analysis and library preparation to a company that failed to obtain data out of it due to the very small amount of cells and contained genetic material collected with our non-invasive wound sampling method. The samples were then retrieved back and sent to a different company after having a long discussion with their experts on our samples’ nature. Unfortunately, the second company could not obtain data either.

This outcome was unforeseen as the kits and sample collection tools used have been used for obtaining such data and our team has good experience in using them, obtained through previous similar projects for RNA extraction. Besides, similar methods for collecting data on wounds microbiome have been used by other researchers. To take this research question forward for future research, wound biopsies or careful wound scraping instead of wound swabs are advised as an alternative for wound surface sample collection. This may require some form of local anaesthesia to make this procedure more comfortable for people affected by EB. The study question and hypothesis are still valid and reaching an outcome would be beneficial in redirecting the way we see EB wounds and tailoring proper management plan that could accelerate the healing process, minimize wound chronicity with its associated symptoms of pain, itchiness and long-term complications as scar, contracture, deformity and skin cancer. (From final report 2025.)