Biofilms and bacterial imbalances in chronic wounds: anti‐Koch

Abstract

Microbial imbalances and synergistic relationships between bacteria in medically important biofilms are poorly researched. Consequently, little is known about how synergy between bacteria may increase the net pathogenic effect of a biofilm in many diseases and infections, including chronic wounds. Microbial synergy in chronic wounds may increase virulence and pathogenicity, leading to enhanced tissue degradation, malodour and in some cases, an impairment of the host immune response. Microbial synergy and growth within a biofilm provide a competitive advantage to the microorganisms cohabiting in a wound, thereby promoting their survival and tolerance and resistance to antimicrobial agents. The aim of this article was to provide greater insight into microbial imbalances found within wound biofilms and the significance they may have on non healing and infected wounds. We also present two possible hypotheses which could explain the role microorganisms play in non healing chronic wounds and offer possible strategies for combating harmful and detrimental biofilms.

INTRODUCTION

It is widely accepted and well documented that both acute and chronic wounds are more susceptible to infection when the microbial load within the wound bed reaches a ‘critical level’, a term often referred to as ‘critical colonisation’1, 2. However, speculation continues as to whether this certain critical level [i.e. >10⁵ colony‐forming units (cfu) per gram of tissue] of microorganisms in a wound impairs healing 3, 4, 5, 6, 7, 8. In addition, conjecture exists over the exact role that specific pathogens play in both non healing and infected chronic wounds 9, 10, 11, 12, 13, 14, 15.

As well as microbial numbers and individual wound pathogens, the significance of specific bacterial combinations and interactions has also been deemed important in many infections and diseases 16, 17, including acute and chronic wounds 18, 19, 20.

It is the aim of this article to highlight the significance of microbial imbalances and the importance of interactions between microorganisms found within wounds and outline microbial hypotheses that we considered relevant to chronic wound management strategies.

MICROBIAL NUMBER AND IMBALANCE IN DELAYING WOUND HEALING

In 1964, Bendy (18) first proposed that microbial numbers have an important role to play in non healing and infected wounds. Within Bendy's study, it was reported that healing in decubitus ulcers occurred only when the bacterial load in the wound fluid was <10⁶ cfu/ml. In addition, Breidenbach and Trager (21) showed that microbial levels of ≥10⁴ cfu/g of tissue were responsible for delayed wound healing. In 1969, Robson and Heggers (22) reported that infections in acute and chronic wounds existed when a microbial load of >10⁵ cfu/g of tissue was present. Despite these initial findings, recent research has shown that a correlation between exact microbial numbers and wound healing does not exist to the degree previously reported. For example, Robson (23) documented that healing of wounds occurred even in the presence of high numbers of bacteria. A large number of reported studies correlating microbial numbers with wound healing have often not considered the fact that bacteria are randomly distributed within wound tissue. Consequently, determining the true microbial density of a wound is often very subjective. This further questions the value of microbial numbers in assessing the risk of infection and progression of a wound to healing. This concept is supported by the findings of Schneider et al. (24) who evaluated the microbial distribution in tissue taken from seven pressure ulcers. Schneider et al. showed that variability of microbial counts existed in single‐tissue biopsy specimens, concluding that bacterial numbers had limited value in predicting an optimal time required for wound closure. Similar findings have also been reported by Sapico et al. (25) and Davies et al. (26). Comparison between chronic wound studies is difficult because variations exist, particularly in the sampling methods used, the different wound types sampled as well as uncontrollable exogenous and/or endogenous factors (12).

The number of microorganisms has without doubt a role to play in the wound‐healing process; however, microbial numbers alone should not be interpreted in isolation when wound infection is predicted, a situation alluded to by Majewski et al. (27).

SPECIES OF BACTERIA: A CAUSE OF WOUND INFECTION?

A number of chronic wound studies have focused on determining associations between specific culturable microbes and delayed wound healing and infection. It is well documented that bacteria such as Staphylococcus aureus, Pseudomonas aeruginosa and β‐haemolytic streptococci produce very destructive virulence factors 3, 28, 29. The role these bacteria play in delayed healing and infection is not disputed. However, with β‐haemolytic streptococci for example, although they are associated with tissue destruction, their role and relevance in wound healing remains unclear (14).

Many different species of bacteria have been cultured from chronic wounds including Klebsiella sp., Enterococcus sp., Proteus sp. and Enterobacter cloacae 30, 31, 32, 33, 34, 35. In addition to these, non sporing Gram‐negative anaerobic bacteria are often found in abundance in chronic wounds (36). Anaerobic bacteria that are commonly cultured have included Peptostreptococcus, Bacteroides, Prevotella, Finegoldia, Peptoniphilus and Porphyromonas species 32, 36, 37, 38, 39.

Anaerobic bacteria are known to express adhesion factors, tissue‐damaging exoenzymes and anti‐phagocytic factors, and these bacteria can grow as biofilms. It is perhaps not surprising, therefore, that anaerobic bacteria are known to impair the wound‐healing process 40, 41, 42.

Currently, there is no one statistically significant study that is able to conclusively and consistently show a relationship between clinical outcome and the microbial composition of a chronic wound.

The use of molecular techniques applied to chronic wound microbiology has recently enhanced our understanding of the microbiology of chronic wounds, biofilms and their implications to non healing and infected wounds 35, 36, 43, 44, 45, 46, 47, 48. In fact, use of molecular techniques has showed that many microorganisms found in both acute and chronic wounds, exist in a ‘viable but non culturable’ state. This is important as such organisms can still play a significant role in non healing and infections but their presence has previously been unrecognised 35, 46, 49, 50.

Using advanced molecular techniques, Dowd et al. (36) identified an array of different bacteria colonising diabetic foot ulcers, venous leg ulcers and pressure ulcers. Bacteria identified included Staphylococcus, Pseudomonas, Peptoniphilus, Enterobacter, Stenotrophomonas, Finegoldia and Serratia spp. Further findings showed differences in bacterial populations for each wound type and highlighted the limitations of traditional culturable techniques which greatly underestimate the true microbial diversity of a wound.

Despite advances being made with the use of modern molecular microbiological techniques, consensus remains that common wound pathogens, such as S. aureus 31, 51, P. aeruginosa 12, 39, 52 and β‐haemolytic streptococci, specifically cause delayed healing and infection in chronic wounds (23). However, based on the evidence to date, the presence of particular species of microorganisms should not be considered in isolation or as being essential for wound infection to occur or for healing to be impaired.

The accumulation of a complex biofilm, composed of a diverse array of microbes, that is a ‘community’, is often considered to be responsible for delayed healing and wound infection. This concept has been supported by a number of recent publications 47, 48, 50, 53, 54, 55, 56, 57, 58, 59, 60, 61. As a result, it is the collective ‘pathogenic effect' of the biofilm rather than individual planktonic bacteria that may be the underlying reason for delayed chronic wound healing.

The biofilm/community hypothesis contrasts with certain historical medical theories related to disease and infection, in particular, the ‘Germ Theory’ and ‘Koch’s postulates' (Robert Koch, 1843–1910). At this juncture, it is worth reflecting on Koch's postulates which, based on Rivers' translation (62), are as follows:

• 1The parasite occurs in every case of the disease in question and under circumstances which can account for the pathological changes and clinical course of the disease.
• 2It occurs in no other disease as a fortuitous and non pathogenic parasite.
• 3After being fully isolated from the body and repeatedly grown in pure culture, it can induce the disease anew.

The principles of Koch's postulates were initially established based on the etiology of anthrax and tuberculosis. However, these principles have since been generalised to other diseases including wound infections and delayed healing. This is evident when we consider the application of very specific topical antibiotics, once considered a routine practice, in the management of infected wounds. On this basis alone, the theory behind Koch's postulates should not be viewed in its entirety and applied to wound infections. Despite this, many physicians and wound care practitioners still continue to adopt the principles of Koch's postulates when searching for answers relating to why chronic wounds fail to heal or the reasons behind infections. Undoubtedly, within the era of modern wound care, an improved understanding of chronic wound microbiology is warranted, particularly now as microbiology is making a paradigm shift towards ‘biofilmology’ and the area of sociomicrobiology 35, 39, 54, 58, 63, 64.

BIOFILMS IN CHRONIC WOUNDS

Biofilms are communities of microorganisms, often attached to a surface or to each other and embedded within a matrix of extracellular polymeric substances (EPS) (65). The sequential stages involved in the formation of a biofilm in a wound include the initial conditioning of the wound surface, irreversible adhesion of pioneering microorganisms via microbial adhesions and wound bed receptors, coadhesion of secondary and tertiary colonisers and the development of a matrix of EPS which encases the attached or ‘sessile’ bacteria.

EPS has a significant role to play in wound healing. It is composed of a complex combination of polysaccharides, proteins, glycoproteins and DNA. However, its composition is dictated by the residing microorganisms and the external environment, bathing the biofilm. Functionally, the EPS is significant to biofilm maturation and promotes, among other mechanisms, inherent resistance of the biofilm to both antimicrobials and host immune responses.

Microorganisms residing within a biofilm are phenotypically different to their free floating or planktonic counterparts. This was showed in a study by Whiteley et al. (66) who reported that some 73 additional genes are expressed by P. aeruginosa when grown as a biofilm compared with its planktonic counterpart. Modulation of gene expression is considered important to bacterial survival and maintenance once a microorganism is attached to a surface. This critical control of gene expression is thought to aid and prepare biofilm microorganisms for rapid adaptation during external adverse conditions, a concept referred to as ‘biological insurance’(67).

As a biofilm matures, the synthesis of EPS is up‐regulated and this helps to develop the complex architecture of the biofilm. EPS is considered to act as a scaffold for the biofilm which is able to sequester and retain nutrients, enzymes and metal ions (e.g. iron), all of which are known to sustain the biofilm and maintain its stability, thus aiding microbial survival (55). Unlike biofilms which naturally occur in regions of the human body, such as on skin, teeth, gastrointestinal and vaginal mucosa, the presence of biofilms in a chronic wound is not considered natural, and as such, wounds have never been considered to have their own ‘indigenous microbiota’(68). However, for many wounds that have remained open for decades, it could be hypothesised that long‐term non healing wounds may in fact have their own indigenous microbiota. Nevertheless, it is interesting to speculate that a biofilm in a chronic wound exists in a state of commensalism or mutualism with the host. It is possible that, over time, the biofilm and wound exist in a state of homeostasis 69, 70. This theory may seem more plausible to accept in individuals who are immunocompromised.

Homeostasis or a balanced microbial population is perhaps better described as a climax community where the biofilm has reached a state of equilibrium. Such a climax microbial community is reached following synergistic, antagonistic and mutualistic interactions between the many bacteria found within the wound bed. A good example of this would be the relationship between facultative and strictly anaerobic species within the biofilm. It is likely that as a biofilm develops towards its climax community, the activity of facultative anaerobes can create anaerobic regions within the biofilm which support the growth of strictly anaerobic organisms. Clearly, in such cases, this synergistic relationship between the relative species influences the composition of the climax community. The formation of the microbial climax is further aided by the communication systems bacteria use when attaching to a surface and when growing as part of a community 71, 72. Such a communication system serves to coordinate gene expression and therefore coordinates function and activities of biofilm organisms, aiding microbial stability and long‐term survival 71, 72.

Microorganisms encased within the biofilm have been reported to be randomly distributed and yet still functionally organised into niches, with each niche considered to have its own specific function and role. Heterogeneity within the biofilm is considered to be fundamental to the ecological stability of the wound biofilm and therefore its recalcitrance. Depending on exogenous and endogenous factors, development of the microbial ecosystem in a chronic wound may initially be under constant dynamic flux. Consequently, any physiological and biochemical changes in the wound bed will alter relative microbial competiveness within the wound biofilm. This may lead to the enrichment of less predominant, yet potentially more pathogenic bacteria, which then become problematic by becoming the dominant biofilm species. Such a community shift may be affected by numerous factors including pH, temperature, type of administered wound dressing or antimicrobial, and the host immune response.

When the dynamics of the wound biofilm community alter, infection can occur. By using targeted therapies against specific bacteria in the wound, or adjusting factors that influence community structure, it might be possible to convert a pathogenic biofilm into a commensal one. Such a strategy may aid in combating infection and promoting wound healing.

WOUND‐MICROBIAL BIOFILM HYPOTHESES

In conjunction with the theory above and the discussion at the beginning of this article, there are two hypotheses which could have bearing on the use of targeted biofilm management strategies. Such hypotheses were first described for plaque‐mediated diseases (73). The first hypothesis is the ‘specific bacterial hypothesis' and the second is the ‘non specific bacterial’ or ‘community hypothesis'. A better understanding of these hypotheses may help us direct future strategies for combating ‘harmful’ biofilms known to cause chronic wound infections.

The specific bacterial hypothesis suggests that despite a complex microbial diversity present in a wound, only a few species of bacteria are actually involved in delayed wound healing and therefore contribute to the infection process. Contrary to this hypothesis is the non specific bacterial which suggests that any infection in the wound is because of the overall composition and functioning ‘unit’ of the biofilm per se. The non specific hypothesis considers that the complex heterogeneous microflora plays a role in infection and disease and does not consider individual pathogenic bacteria alone as causing delayed healing. While both hypotheses are fundamentally complex, they may be significant to chronic wound infections and therefore healing. Both theories are yet to be proven in chronic wound microbiology, but wound care practitioners should be aware of each hypothesis and take these into consideration as part of an ongoing wound management strategy.

CONCLUSIONS

The chronic wound bed is known to be composed of a community of microorganisms within a biofilm which has properties ‘greater than the sum of its constituent members' (74). Conditions within the chronic wound biofilm which can affect its balance include the interactions that occur with other microorganisms, as well as changes in pH, temperature, nutrient levels and the host's immune response.

Enhanced growth of opportunistic pathogenic microorganisms within the wound biofilm represents an ecological shift in the wound microbiology resulting in an alteration of the once stable climax community. This microbiological shift will alter the homeostasis of the biofilm leading to potential overgrowth of problematic opportunistic wound pathogens. Such an ecological shift could result in the decreased growth of suppressive non pathogenic competing bacteria. Consequently, better control measures that will prevent this ecological shift to a predominantly ‘pathogenic biofilm’ which might delay wound healing are necessary.

It is fundamental to further our understanding of chronic wound healing from a biofilm concept approach as we begin to appreciate that many disease aetiologies are now more complex than once thought in medicine, that is the ‘Koch’s postulates' approach. A better understanding of the significance of certain bacterial combinations in chronic wounds and delayed wound healing is necessary. This may help direct effective wound management strategies using probiotics. Potential biomarkers known to indicate an alteration in wound biofilm homeostasis may assist in the development of management strategies directed specifically towards harmful biofilms, while at the same time aiding the enhancement of the growth of helpful biofilms which may improve wound healing.