Microbial biofilms and wound healing: an ecological hypothesis

Introduction

Microbial biofilms are structured communities of microbes that live together in an attached state, either to a surface or to each other, and are encased in a matrix of self-produced extracellular biopolymers. ¹ It is currently estimated that as much as 80% of all microbial biomass resides in a biofilm state and a majority of microbial infections are related to biofilm formation. Within biofilms microbes behave differently compared to the much better studied free-living stage, most notably with respect to antibiotic susceptibility and interactions with the host.

Most microbial biofilms related to humans are benign, or even beneficial, e.g. microbial biofilms in the vagina and the gut are essential to maintain human health. The oldest studied microbial biofilm is represented by dental plaque that was studied by Antonie van Leeuwenhoek in 1683 using the first microscope. ”From hence I conclude, that the Vinegar with which I wash my Teeth, kill'd only those Animals which were on the outside of the scurf, but did not pass thro the whole substance of it ….”. ² He, for the first time, described different types of oral bacteria (he called them animals) and observed increased tolerance of bacteria in biofilms (which he called scurf) to antimicrobial treatment. Subsequently, upon more advanced microbiological techniques, bacteria could be cultured from the oral cavity. In modern microbiology, next generation sequencing (NGS, see box 1) provides a more complete overview of microbial composition of the oral microbiome since it also identifies non-culturables. ³ The human microbiome project further illustrated the ubiquitous presence of microbes in the oral cavity, in the gut and on the skin of healthy subjects. ⁴ It is now estimated that there are 10-fold more microbial cells in and on our body than we have cells of our own. It is therefore of the utmost importance that we better understand how we are interacting with our microbes, when and why they are beneficial and when and why they cause problems. Compared to the well-studied oral microbial biofilms, studies on biofilms in chronic wounds are in their infancy. In a recent review a comparison between oral and wound biofilms has been described. ⁵ Here we discuss, mainly from a microbiological perspective, recent advances in understanding microbial biofilms in chronic wounds and how they could influence wound healing in chronic wounds. This particular part of biofilm research is still in its infancy and more research is needed to provide a solid evidence-base for anti-biofilm therapy in treatment of chronic wounds.

Box 1

Next-generation sequencing (NGS) for microbiome analysis (for more detailed information see ²¹ ). Microbial species are traditionally identified based on appearance and biochemical behavior. On a molecular level, the base-sequence of the ribosomal RNA genes are commonly used to identify species. The constant regions on the rRNA gene (most commonly the 16S rRNA gene is selected) are very stable during evolution in contrast to the variable regions that collect mutation in time. The more difference in base sequence in the variable regions, the more mutations accumulated the further back in time species differentiated. Thus, if the base sequences of the variable region is very similar, they are derived from related if not identical species. NGS allows single molecule sequencing in high amounts. Gigabase sequence data are being produced in each run. This allows not only identification of individual species, but also quantification of these species. By combining species identification with quantitative information insights into microbiome composition can be obtained. Since eukaryotes like humans do not have a 16S rRNA gene, but a very different 18S rRNA gene, small amounts of microbial DNA can be sequenced within a mixture of eukaryotic DNA, such as for instance DNA isolated from debridement samples.

Healthy biofilms; an ecological perspective

Commensal microbial biofilms are most often polymicrobial in nature and the microbes are in balance with each other and with the host (Figure 1). The endogenous balance (endo-balance) results from all positive and negative interactions at chemical, metabolic or physical levels between the various microbial species present in the biofilm. The biofilm itself interacts on the same levels with the external milieu, in this case the host. The sum of all positive and negative interactions between the members of the biofilm and the host can be termed the exogenous balance (exo-balance in Figure 1). This double balance generally does not result in pathogenic behaviour. However, environmental or host-related changes can cause disruption of this homeostasis and results in a unbalanced and potentially unhealthy biofilm. If the disruption is short, balance can be rapidly restored, but if the disruption last longer or is of a permanent nature, a new balanced situation can develop. In general, a healthy biofilm is a balanced, diverse and resilient microbial community in balance with the host. For more background on balance, resilience and health see a recent review by Krom et al. ⁶ OPEN IN VIEWER

The human skin and mucosa represents the first immunological line of defense against microbial invasion and is an effective means to prevent infections. Microbes on intact skin and mucosa of a healthy individual rarely cause an infection. However, breach of this barrier provides a “port d’entre” for microbes leading to an infection and subsequently an inflammatory response by the host. In contrast to the dry conditions of skin that are not favourable, wounds are moist and warm creating an ideal environment for many microbes. More importantly, the change from dry intact skin to damaged moist skin represents a significant host-related environmental change that will affect the biofilm balance significantly. As a result, the microbial ecosystem shifts towards a new balance, which might not be in balance with the host and therefore interferes with normal wound healing processes, resulting in extended healing time.

Chronic wounds: pathophysiology

The function of wound healing is to re-establish skin integrity, which is important to prevent infection and maintain fluid homeostasis. Wound healing usually follows three distinct stages (for a recent review see ⁷ and references therein). Initially the inflammatory phase, followed by a granulation phase and finally an epithelialisation phase, all have clear clinical appearances (Figure 2). Chronic wounds, such as venous ulcers, do not resolve because they remain in a state of chronic inflammation and do not reach epithelialisation phase. In other words, while “normal” wounds progress to the next phase of wound healing, the ulcer resides in a particular wound phase, i.e. either the inflammation or granulation phase, and does not progress to wound closure. This causes long-term problems with increased infection risks and sub-optimal fluid-homeostasis. In addition to seriously decreasing the quality of life of the patient, chronic wounds represent a great financial burden for healthcare systems worldwide. Chronic wounds are common in the adult population with prevalence of around 1% and increases to 3–5% in the population over 65 years of age ( ⁷ and references therein). It can therefore be expected that chronic wounds will increase with the aging of the global population. OPEN IN VIEWER

Healing of venous ulcers is notoriously difficult and reports have shown that 25–50% of these wounds persist for longer than 1 year.^8,9 A better understanding of the different possible reasons explaining the inability of venous ulcers to heal is desperately needed. Many systemic and local factors may contribute to the observed state of chronic pathological inflammation in venous ulcers, but chronic venous insufficiency is one of the key factors, obviously. On the microcirculatory level, local hypoxia has been observed as a result of venous hypertension and stasis. ¹⁰ Low oxygen pressure at the cellular level is a trigger of pathological inflammation in itself, resulting in a hostile wound environment. Ongoing tissue injury due to pathologic inflammation, bacterial colonisation, and tissue hypoxia may cause absence of healing or even enlargement of the venous ulcer.

When the balance is tipped

Biofilms in chronic wounds are understudied, possibly because they do not resemble the archetype microbial biofilm on inert or biological surfaces.^11,12 This has led to a long controversy about the existence of biofilms in chronic wound. Over the past couple of years several excellent reviews have appeared that describe the structure and microbial composition of biofilms in chronic wounds and the consequences for treatment are becoming clear.^11,13–16 Antibiotic resistance, or general non-responsiveness to treatment with antimicrobials prevents successful treatment of infections and new approaches are required involving biofilm testing to find effective antimicrobial (anti-biofilm) therapy. ¹⁵ The onset of molecular methods to identify microbes within wounds has illustrated the understudied role of anaerobic bacteria and fungi in chronic wound infections.^17,18 Anaerobic bacteria might proliferate under hypoxic conditions, such as present in venous ulcerations. In addition, in the oral cavity, anaerobic bacteria are related to chronic infections and are characterized by increased proteolytic activity. Anaerobosis is related to the endo-balance since aerobic bacteria effectively utilize available oxygen resulting in hypoxic conditions deeper in the biofilm. Increased proteolytic activity is a result of the exo-balance as host tissue damage and increased gingival crevicular fluid flow result in elevated availability of (serum) proteins. In analogy, the hypoxic conditions, either induced by colonisation by aerobic bacteria or a result of host action, in venous ulcers could induce anaerobic bacteria. The high levels of host derived proteins selects for proteolytic bacteria. These anaerobic proteolytic bacteria have gone largely unnoticed in the past since anaerobic culturing is not part of the standard microbiological diagnostics for chronic wound infections.

Based on the new insights on microbial ecology in wounds we proposed two hypotheses related to chronic wounds. These two fundamentally different hypothetical mechanisms link biofilm formation to the inability of a wound to heal (Figure 3). The first hypothesis is dependent on biofilm formation (endo-balance) and has been extensively described by Bjarnsholt et al. ¹⁹ Microbial biofilm infections are less susceptible to the immune system and simultaneously cause a prolonged immune response, which together prevents wound healing. ²⁰ In addition, proteolytic activity of the anaerobic bacteria interferes with an effective immune response (similar to their activity in chronic periodontitis) and therewith prevents successful microbial clearance resulting in continuous infections and delayed wound healing. Finally, increased oxygen consumption by bacteria could induce hypoxic conditions. Local hypoxia and high protein levels could further select for anaerobic proteolytic bacteria and also preventing effective wound healing. ¹⁰ OPEN IN VIEWER

The second hypothesis is based on the host (exo-balance). Due to some underlying deficiencies such as diabetes, malnutrition, cancer etc., the wound does not heal. Due to continuous absence of skin integrity, such chronic wounds are more prone to re-infection by skin-derived flora and microbial biofilms get the chance to develop. Biofilm formation might even be stimulated by a low-level immunological response as harsh environmental conditions, such as the high concentrations of reactive oxygen species originating from immunecells, are know to stimulate biofilm formation. As a consequence of biofilm formation microbial clearance is even more difficult.

All chronic wounds are characterised by continuous recruitement of immunecells that release enzymes, free oxygen radicals, etc. all of which cause damage to the host. The question is whether this is caused by or it is the cause of microbial colonisation.

Future perspectives

In sharp contrast to biofilm infections in the oral cavity the limited information on biofilms in chronic wounds prevents a clear-cut answer to the question: “what is the most successful treatment therapy?”. More fundamental and clinical research is needed to discriminate the causes and consequences of microbial biofilm formation in (delaying) wound healing. Depending on the hypothesis followed, aggressive antimicrobial therapy could be the way to go, alternatively, stimulation of wound healing would prevent re-infection of the wound and enable wound-closure. Based on the (unexpected) presence of anaerobic bacteria in wounds, therapy that prevents (local) hypoxia from developing seem promising. This “sword” cuts on both sides as it would inhibit growth of anaerobic proteolytic bacteria and prevent necrosis from taking place, both effects would stimulate wound healing. ¹⁰

Either way, the next couple of years will yield more insights into the microbial ecology of chronic wound (endo-balance) and the way the microbial flora and the host interact (exo-balance). These insights will provide better treatment options that are desperately needed in light of the expected increase, related to aging population, in patients with chronic wounds and the limitations put on health care budgets world-wide.

Conflict of interest

All the authors have no conflict of interest and nothing to disclose.

Funding

Dr. Krom is supported by a grant from the University of Amsterdam for research into the focal point “Oral Infections and Inflammation”.