Infection of the soft tissue and related clinical challenges

Keith Cutting

Wound infection is a major impediment to healing and therefore precision in terms of diagnosis and management is of vital importance. The microorganisms (bacterial and fungal) that cause infection are generally categorised in terms of their physiological state as either, planktonic or biofilm. Planktonic refers to the free-floating plankton-like state as opposed to the attached or sessile form, biofilm. Both phenotypes can be pathogenic, cause infection and contribute individually to the burden of healthcare, the implications of which include an increase in patient morbidity and the use of human, material and financial healthcare resources.

The fact that microbes exist in two phenotypes (planktonic and biofilm) confounds the construction of an uncomplicated definition of infection that can be universally accepted. None the less, Wolcott and colleagues have proposed - The detrimental colonisation of a host-organism by a foreign species¹ and this provides not only a working definition of infection but also an opportunity to explore the challenges that exist in the diagnosis and treatment of wound infection. The differences between planktonic and biofilm infection will be discussed, and how these variances in phenotypical behaviour impact on diagnosis and treatment options. 

Planktonic microbes

Robert Koch when seeking to understand what precisely causes human infection studied bacteria in pure culture. His resultant declaration that ‘one bacterial species is responsible for a given infection’ became the dominant view and continues to guide standard working laboratory practices.

The free floating or planktonic microbe is commonly studied under standard laboratory conditions, where four phases of growth have been identified.  These phases consist of: a lag phase where the microbe adapts to the growth conditions; a log phase where there is exponential growth; a stationary phase where the population growth is limited by one of three factors i.e. essential nutrient depletion, build up of metabolites or end products, and where there is exhaustion of biological space; and a death phase where microbes die and the population declines.²

Planktonic species are able to reproduce approximately every 15 minutes, gain nutrients from the environment, excrete their waste³ and thrive in the moist and nutrient-rich environment provided by wounds.⁴ When acute infection occurs the presentation is rapid resulting in tissue destruction followed by resolution when appropriate antimicrobial therapy is used. The up-regulation of virulence factors and the expression of bacterial proteases cause tissue lysis upon which the planktonic species feed.⁵ Planktonic microbes are therefore highly predatory in nature where the expression of virulence results in an aggressive attack on the host leading to cellular death and the degradation and utilisation of cellular material. Such acute infection is visually characterised by the classical Celcian signs of inflammation, rubor, tumor, calor and donor (redness, swelling, heat and pain) and that coincide with the arrival of inflammatory cells (neutrophils and macrophages) that pour into the site that is under attack.¹

Biofilm

Biofilms, groups of micro-organisms that stick together, can be pathogenic and may provide an explanation for many of the features found in chronic wounds including; elevated protease levels, impaired host-defences, decreased expression of tissue inhibitors of metalloproteases, elevated pro-inflammatory cytokines and resistance/tolerance to antimicrobials.⁶

Although Gristina et al are attributed with the first recorded observation of wound associated biofilms⁷ the roots of biofilmology science appear to lie in the late 1970s with the publication of an article entitled How bacteria stick.⁸ Up until this point, investigation into biofilm and formation of the glycocalyx, now known as the extracellular polymeric substance (EPS) and often characterised as slime, commenced in the 1960s with investigation of the opportunistic pathogen Streptococcus mutans, and the discovery of carbohydrate fibres that surround bacteria in aquatic systems.⁸ The glycocalyx/EPS had up until this point remained an unknown quantity due to standard microbiology laboratory culturing of individual (planktonic) bacterial strains⁸. The EPS of wound biofilms is generally composed of polymeric sugars, microbial/host DNA, microbial proteins and host molecules (fibrinogen)⁹ and this allows the microbes that are encased within the EPS to demonstrate robust resistance to biocides and host immune cells. Biofilm bacteria, often associated with being present in chronic wounds¹⁰ are protected not only from the effects of humoral chemical and cellular attack but also from antibiotics and antiseptics with the consequence that they are difficult to expunge. ^{6, 11} It has been estimated that 80% of human infections, including but not limited to, endocarditis, Crohn’s disease and chronic wounds are caused by biofilm encased microbes.^12,13

Chronic (biofilm) infections differ from planktonic (acute) infections by virtue of the fact that they endure, sometimes lasting for years or even decades.¹ Their ‘behaviour’ is not predatory as seen in their planktonic counterpart but more of a parasitic nature where they ‘live off’ the host while at the same time avoid invoking a host immune response⁴. Biofilm has a number of channels to down-regulate virulence and avoid significant harm to the host. This allows the biofilm continuing utilisation of fluid and nutrients from the host.¹⁴

Whereas acute infection is easily recognised through the manifestation of the Celcian signs any visual cues that may relate to the presence of biofilm have yet to be agreed.  Currently, the clinical diagnosis of wound biofilm has not surpassed the point where the clinician’s suspicion of their presence has been raised.

Diagnosing wound infection

The definition of infection The detrimental colonisation of a host-organism by a foreign species¹ leads us to acknowledge that the mere presence of microbes in a wound is not indicative of concurrent infection - the literature informs us that the majority of wounds are colonised¹⁵. The detrimental component of colonisation refers to the microbes ability to overwhelm host defences, utilise host resources and prejudice healing¹.

The diagnosis of wound infection is a clinical one and although qualitative and/or quantitative sampling may take place the role of such sampling is to identify bacterial/fungal presence and to determine sensitivities/resistance to empirical treatment rather than as a diagnostic tool. At this point it is important to note that the vast majority of terrestrial and aquatic environmental bacteria cannot be identified using traditional culture-based methods.¹⁶ In medicine, the cultures of middle ear aspirates are positive in only 20-40% of otitis media cases.¹⁷
It is known that bacteria in the basilar region of a biofilm show no metabolic activity. These cells are potentially viable but non-culturable.¹⁸ Not all infected wounds will demonstrate the classical Celcian signs so this necessitated the development of secondary or subtle signs of infection.¹⁹

•  Bridging of the epithelium or soft tissue
• Delayed healing (compared with normal rate for site/condition)
• Discolouration
• Friable granulation tissue that bleeds easily
• Malodour
• Pocketing at the base of the wound
• Unexpected wound pain/tenderness
• Wound breakdown

Cutting²⁰ andGardner ²¹ have conducted validation exercises on these secondary criteria. Cutting²⁰ found that thirty-nine of the 40 decisions (97.5%) made by the researcher on the infected status of the wounds were corroborated by the wound swab culture. The Gardner²¹ results show that the signs specific to secondary wounds are more accurate than the classical signs of infection as indicators of infection. The only sign that did not demonstrate validity was pocketing of the wound base.

Latterly, criteria for wound infection have been developed for six wound types and these indicate there are subtle differences in criteria found in six types of wounds²². Although these latter criteria have yet to undergo validation they incorporate all of the criteria that were described in the secondary signs of infection published in 1994 with the addition of a few ‘new’ criteria. It is interesting to note that whereas the Celcian signs utilise four features of the inflammatory response, the subtle signs of infection consist of a range of physical or sensory wound features that although likely to be associated with concurrent inflammation do not mirror a conventional inflammatory response but indicate an interruption to the orderly sequence of healing. 

This lack of clarity in what constitutes a wound infection is most probably related to the fact that microbes exist in two phenotypes (planktonic and biofilm). The phenotypical conduct of the two forms (planktonic/predatory and biofilm/parasitic)²³ results in a variance of virulence expression. To put this into context the planktonic bacteria pose the threat of invading other areas of tissue while biofilm encased microbes may: produce virulence factors such as lipopolysaccharide, continually release planktonic cells and incorporate bacterial DNA into the biofilm matrix.¹ Recent evidence indicates that biofilm infections are polymicrobial in nature and that biofilm is the phenotypical dominant form found in infectious diseases, including chronic wounds.¹

It has been proposed that four conditions need to be met in order to determine whether an infection is caused by biofilm.²⁴ These are: the pathogens are surface associated or adherent to a substrate, the bacteria are clustered and encased in a matrix of bacterial or host constituents, the infection is localised (not spreading), and the infection resists antibiotic treatment despite the demonstration of planktonic susceptibility.

One feature that is common to all chronic wounds irrespective of aetiology is that of delayed healing. If the underlying cause (repeated trauma, pressure, ischaemia, venous hypertension et cetera) of the wound is managed correctly then it is reasonable to assume that the delay to healing is a consequence of the presence of biofilm infection. In all chronic wounds an excess of neutrophils can be found.^{25, 26} This indicates the presence of a persistent inflammatory state. Although there is evidence that biofilm presence and chronic inflammation co-exist²⁷ a comprehensive overview of the precise relationship between inflammation and healing has yet to be described.²⁸ We do not yet know the answer to the question - is inflammation an inevitable result of the host response, or a consequence of the colonising biofilm?

Treating wound infection

The difficulties in diagnosing wound infection complicates the prescribing of safe and effective treatment. Planktonic cells lack colony defences and are, therefore, susceptible to antimicrobials that support resolution of the infection. In those instances when the antimicrobials are ineffective the immune defences are overwhelmed and the host succumbs to the infectious process.
With perhaps the exception of acute infections where multi-resistant organisms are found, treatment is comparably uncomplicated where the administration of antibiotic treatment in accordance with local guidelines usually leads to prompt resolution of the infection.

The treatment of chronic wound infection is more complex. In the absence of clinical criteria for biofilm infection and with the knowledge that: biofilm encased microbes cannot be cultured in the laboratory, the polymicrobial nature of biofilm communities, together with the reality of bacterial synergy, makes targeting treatment towards the offending populations difficult.

A range of treatment options to control biofilm has been proposed including: physical/chemical debridement, antibiotics, anti-inflammatories, anti-adhesion, quorum-sensing disruption and antimicrobial peptides.²⁹However, not all of these options are available to clinicians whose practice is in a day-to-day community or hospital setting. In addition, many treatments are instigated in the absence of diagnostic information and sometimes based on what can be called ‘hope for the best’ outcome.  Those treatment options that are readily available and are included in the consensus document “Recommendation for the management of biofilm” ³⁰ will now be discussed.

Debridement

The biofilm matrix appears to offer a robust defence against a number of topical agents and wound dressings and as a consequence the physical debridement of biofilm appears to be an essential approach.³¹Debridement of the wound surface results in the release of microbes from the biofilm upon which they revert to planktonic phenotype and become vulnerable to biocidal agents.³² An advantage of surgical debridement is alteration of the wound anatomy thereby removing niches where biofilm can attach and proliferate.⁶ If sharp/surgical debridement is not possible then other means of debridement e.g. larvae or autolytic should be employed.

Antibiotics

Antibiotics are commonly, the treatment strategy of choice for bacterial infections. Their mode of action is bacteriostatic (preventing cell division) or bactericidal (cell killing). Unfortunately, they increase selective pressure, the natural selection process that promotes one group of organisms over another by killing those that are susceptible and the promotion of resistance through the survivors.

Sessile bacteria however, have a slow metabolic rate and are insensitive to bactericidal antibiotics. In addition, the EPS matrix protects biofilm bacteria from antibiotic onslaught and opsonisation and phagocytic defence mechanisms.³³ Thus, biofilm encased bacteria are distinctly recalcitrant to antibiotic intervention when compared to planktonic cells³⁴. The minimum bactericidal concentration (MBC) of antibiotics needed to treat biofilm may be up to 1000 times greater when compared to planktonic bacteria³⁵. Although antibiotics are unable to eradicate biofilm populations they have a positive adjunctive role to antiseptics and debridement methods.³⁶ The selection of the most appropriate antibiotic to treat biofilm is a key challenge and should be compliant with stewardship guidelines.

Administration of ertapanem has been shown to improve outcomes in diabetic foot ulcers³⁷ possibly through the suppression of multiple biofilm species.³⁸ The targeting of treatment towards 1. specific subpopulations e.g. MRSA³⁹ or 2. keystone organisms in the bacterial community can produce good outcomes.⁴⁰  An additional strategy is to use antibiotics that insert into the cell wall e.g. daptomycin and linezolid have shown to impact on some biofilm infections.^41,42

Antiseptics

Antiseptics, like antibiotics are also bacteriostatic or bactericidal in action. They are not selective and therefore can be toxic to mammalian cells depending on their concentration. Their advantage is that they are broad spectrum in action, rarely select for resistance and are applied topically thus avoiding reliance on access to the wound via the blood supply. In poorly perfused or ischaemic wounds this is a distinct advantage.⁴³ A range of popular topical antiseptics are available and include:

• Engineered/modified honey
• Honey - medical grade
• Iodine - povidone and cadexomer
• Octenidine dihydrochloride
• Polyhexamethidine biguanide - PHMB
• Silver - ionic, metallic, nanocrystaline

Chlorhexidine has not been included in the list above as although it is considered to be relatively safe the results from a variety of studies are unable to demonstrate discernible value for its use in open wounds.⁴⁴ 

Engineered/modified honey is a wound antiseptic dressing that shows good antimicrobial activity that is dependent on the generation of reactive oxygen species.⁴⁵ It has been shown to be effective in reducing and preventing line site colonisation⁴⁶ and to inhibit biofilm formation.⁴⁷ Honey has been found effective against multi-resistant microbes⁴⁸ and possess anti-biofilm activity.⁴⁹

Povidone/Cadexomer iodine - Povidone iodine has been shown to reduce bacterial counts and inflammation⁵⁰ and to be effective against biofilm.⁵¹ Cadexomer iodine is effective against multi-resistant organisms⁵² and also possesses anti-biofilm activity.^{53, 54} 

Octenidine hydrochloride is effective against multi-resistant micro-organisms⁵⁵ and can rapidly inactivate planktonic and biofilm cells of S. aureus, MRSA and VRSA.⁵⁶ Polyhexamethidine biguanide PHMB, is a broad spectrum antiseptic with low toxicity, high tissue compatibility and is able to block microbial attachment and remove biofilm.⁵⁷

Silver - ionic, metallic, nanocrystaline. The active agent in silver based products is ionic silver. This has been shown to be effective against wound microbes.^{58, 59} Silver efficacy against biofilms is mixed.  One study showed ionic silver efficacy against P. aeruginosa mature biofilms⁶⁰ whereas measurable differences between five commercially available dressing in antimicrobial efficacy were found in vitro.⁶¹

Utilising debridement, together with antibiotics and antiseptics as concurrent interventions complies with the concept of biofilm-based wound care (BBWC). This strategy has been reported to be effective in a cohort of patient with critical limb ischaemia.⁶² BBWC provides a framework for managing wounds where biofilm infection is thought to be the cause of delayed healing. 

It would be unwise to solely rely on the use of traditional antimicrobial methods (antibiotics) in the management of wound infection. Advanced therapies for effectively managing microbial load need to be introduced. One such intervention is that of gas plasma where ionised gas delivers reactive oxygen species/reactive nitrogen species  to the wound and is effective against bacteria, fungi and viruses and is a recognised anti-biofilm agent and does not select for antibiotic resistance.⁶³

Conclusions

Clinicians are regularly confronted with soft tissue infection challenges.  An inquiring mind together with extensive experience is required if accuracy in clinical diagnoses is to be achieved. Treatment of infection is equally challenging, the clinician has to consider the intricacies of acute and chronic infection treatment strategies. Management of biofilm infection is in the early stages of development and more research is needed so that effective and readily available interventions can be easily applied and deliver effective treatment.