Irrespective of whether wound care is practised in an acute or chronic setting, improved outcomes are possible if treatment plans take into consideration the wound microbiome, the role played by biofilm in recurrent infections, and microbial virulence mechanisms. 

Delayed healing and wound infection continue to be a significant cause of human suffering.

The use of topical antimicrobial agents and systemic antibiotics for wound infection should be based upon sound diagnostic and stewardship principles, and every effort made to reduce selective pressure and the long-term development of resistant pathogens.    

Wound bioburden and the transition to infection 

Normal wound healing is characterised by the successful completion of four distinct but overlapping phases – haemostasis, inflammation, proliferation, and remodelling.  

All wounds are colonised by multiple species of bacteria; however, provided the microbial  bioburden is controlled by normal inflammatory and immune regulation, colonisation alone will not delay, and is in fact, beneficial to the wound healing process.3 

Bacteria in wounds can be ‘planktonic’ (eg mobile, non-attached), or ‘sessile’ (eg immobile, attached) – the latter within a biofilm. Wound biofilms stimulate a chronic inflammatory response which directly contributes to delayed healing. Integral to the ‘biofilm life cycle’, bacteria and biofilm fragments are dispersed to other parts of the wound bed which then go on to form new biofilm.   

Colonisation: the presence of microorganisms that undergo limited proliferation without evoking a host response, while healing is not impeded or delayed. Sources of these microorganisms may be the normal flora of the skin, the urogenital and gastrointestinal tracts, or exogenous (other people) and the environment.

Traditionally, a bacterial load of ≥1×105 CFU/g (colony-forming units per gram of wound tissue) has been used as the threshold for diagnosing wound infection. However, in non-healing wounds, this threshold may not be appropriate because the relationship between bacterial load and infection is not straightforward.  

Bacterial burdens below this threshold may even delay healing, particularly in patients with impaired immune defences, or when particularly virulent bacteria are involved. Conversely, some wounds with a bioburden well above 105 CFU/g may heal without antimicrobial intervention.3,6  

Furthermore, wound swabs may not be representative of the microbiology of the wound, as they will not sample bacteria below the wound surface or may not identify the species responsible for impaired healing. Although a biopsy is likely to produce a more representative sample of the bacterial profile (through the full thickness of the wound), it is invasive, requires specific skills, and may cause further complications. 

Figure 1. The wound infection continuum4   


Advanced age, obesity, malnutrition, long-term steroid use, organ dysfunction and underlying auto-immune disease are all examples of comorbid conditions that increase the risk of progression to infection due to reduced efficacy of the immune system. Local wound factors such as poor perfusion, slough, foreign bodies, undermining, or tunnelling prevent the body from launching an effective immune response.  

Individuals with impaired leucocyte chemotaxis (the movement of leukocytes to damaged tissue eg diabetics) or those who are immunocompromised; are at risk for increased bioburden in their wounds. Transition from colonisation to infection is determined not only by the density of the bioburden, but also by:   

  • The types and virulence of the micro-organisms present 
  • Synergy between different bacterial species  
  • The ability of the host to mount an immune response.  

Overt wound infection is usually characterised by the classic signs and symptoms that occur because of the immune response to bacteria (I.e., pain, heat, swelling, redness, and purulent exudate). However, in patients with diabetes or other immune modifying conditions, peripheral vascular disease, neuropathy, or advanced age, wound assessment for infection can be challenging because these classic features may not be present. More subtle or ‘covert’ symptoms of wound infection include delayed healing, dull or friable hypertrophic granulation tissue, pocketing; changes in the characteristics or amount of exudate, and malodour. 3,4,5,6,7,8,9  

The role of biofilm and bacterial virulence  

Microorganisms co-exist in organised and symbiotic communities, secreting a protective extracellular polymeric substance (EPS) known as biofilm which aids their irreversible attachment to the wound surface. Mature biofilm is a significant feature of non-healing and chronic wounds.2,4,7,8,9 

This highly protective polysaccharide layer does not allow the body’s immune system to detect the presence of the microorganisms within it, so they effectively evade the immune response (eg antibodies and neutrophil phagocytosis), and can withstand oxygen, nutrient, and moisture deprivation, altered pH, and the impact of antiseptics and antibiotics.    

Additionally, pathogenic bacteria within biofilms release a wide variety of highly virulent proteases (enzymes that inactivate or break down proteins) and exotoxins to aid the process of infection, which adversely affect growth factors and other susceptible tissue proteins necessary for healing. 2,3,4,5,8

Biofilm: A structured community of microbes with genetic diversity and variable gene expression (phenotype) that creates specific behaviours and defences used to produce chronic infections. Biofilms are characterised by significant tolerance to immune cells and antimicrobial agents while remaining protected from host immunity. 2,3,4,6,7,8,9 

Bacterial proteases are regarded as the most important of all bacterial virulence factors in the establishment of infection.5   

They interfere with immune function in several ways, including breaking down antibodies, impeding immune cell function by suppressing chemotaxis, preventing phagocytosis, and hindering immune cell communication.  

Bacterial proteases induce an excessive and prolonged inflammatory response, which in turn, increases host protease production and interferes with normal protease regulatory mechanisms. This contributes to the vicious circle of delayed healing in which chronic wounds often become trapped. (Refer to figure 2.) 

 Pseudomonas aeruginosa, Staphylococcus aureus, Proteus mirabilis and Enterococcus faecalis are particularly virulent bacterial species that are commonly implicated in wound infection. Pseudomonas aeruginosa is well known for using ‘quorum sensing’ (complex chemical signalling) to coordinate an invasive infective process through the formation of biofilm, swarming motility, and to rapidly increase its numbers.4,5  

Signs of biofilm formation:4


  • Failure of appropriate antimicrobial therapy. 
  • Recurrence of delayed healing or infection on cessation of antimicrobial therapy. 
  • Delayed healing despite optimal wound management and health support. 
  • Increased exudate.  
  • Low-level chronic inflammation. 
  • Low-level erythema. 
  • Poor granulation/friable hyper granulation. 
  • Secondary or ‘covert’ signs of infection. 

The patient’s immune defences are inadequate to eradicate biofilm but recognise its presence with the inappropriate over-recruitment of neutrophils and pro-inflammatory cytokines. Cytokines are specialised ‘signalling molecules’ that stimulate the inflammatory process and the excessive production of of matrix metalloproteinases (MMPs).  

 MMPs are released by macrophages, endothelial cells, and epidermal cells.  

Their normal function is to break down proteins in the ‘remodelling’ of the extracellular matrix (ECM) as healing progresses. However, an oversupply of MMPs will also result in the destruction  of growth factors.  

Therefore, if the rate of extracellular matrix (ECM) degradation matches or exceeds the rate of ECM production, healing will slow down or regress. This then leads to tissue destruction and increased capillary permeability, which in turn, increases exudate levels and provides nutrition for the biofilm. 

Figure 2. Cullen’s circle – the vicious cycle of delayed wound healing 5

Microbiological culture – to swab or not to swab?

Microbiological culture should not be undertaken routinely or without substantial cause.1,4,5,6,7,8 

Only clinically infected wounds should be cultured and preferably by obtaining tissue by curettage or biopsy. If this expertise is not available, the Levine technique for superficial sampling is believed to give the most representative microbiological culture result.4,8 

This is because fluid is expressed from the wound bed, thereby sampling a greater concentration of microorganisms from both the surface and slightly below the surface of the wound. 

  • Wound sampling is an aseptic procedure! Hand hygiene and the wearing of sterile gloves is required. 
  • Collect the specimen before topical or systemic antibiotics are initiated. 
  • The wound bed should be irrigated with sterile normal saline solution beforehand to remove debris and superficial contaminants. 
  • Obtain a swab culture from a viable wound bed – do not swab slough and avoid the edges of the wound.  
  • Pre-moisten the swab with sterile normal saline solution to obtain an optimal yield. 
  • Roll the tip of the swab over 1-2cm area of viable tissue with enough force to produce exudate from the wound bed. 
  • Include relevant patient risk related details and any previous antimicrobial therapy on the laboratory request form. 
  • Ensure the specimen reaches the laboratory within four hours! 

 A semi-quantitative culture result is an estimate of the number of CFUs of bacteria present, where results are usually expressed as + (scanty) ++ (moderate) or +++ (heavy, profuse) growth. The use of a semi-quantitative swab culture is of less value in guiding wound care decisions than a quantitative swab culture since the true bacterial burden of the wound tissue cannot be determined. 

 The quantitative culture technique provides a more accurate count of the number of colony- forming units expressed logarithmically per millilitre of exudate or per gram of tissue (eg 105 CFU/mL). Quantitative cultures assist clinicians in determining the threshold above which the bacterial burden of a culture may be of clinical significance. Quantitative results should also be comparable from laboratory to laboratory. 

The association between pathogens and patients at risk

Obtaining meaningful specimen results from chronic wounds such as pressure or lower limb ulcers is problematic, primarily because of their aetiology, ischaemia, physical location (commensal flora may interfere with the culture result), and wound duration.  

 The combination of exudate and slough underneath occlusive dressings induces potent autolytic debridement and stimulates microbial proliferation, while the possibility of urinary or faecal contamination should always be considered. 

 If a bone culture is taken for suspected osteomyelitis, prior liaison with the medical microbiologist is recommended, as it will likely reveal a different culture result (eg fewer microorganisms) which will improve the sensitivity and specificity of the culture result. 

Figure 3. The association between pathogens and patient risk profiles

 Practice points

  • There is a fluctuating continuum in the microbiological ‘lifecycle’ of complicated and chronic wounds – ‘stalling’ of the healing process is suggestive of a high bioburden and biofilm activity. 
  • Recognition of, and proactive management of biofilms is integral to wound bed preparation and the management of infection.  
  • There is no indication for routinely performing wound cultures in the absence of clinical signs of infection.  
  • All wounds are colonised – the presence of microorganisms in a wound does not in itself define an infection, and treatment based on culture results alone is not warranted. 
  • If a drug-resistant pathogen has previously been isolated, or the patient has received prolonged or broad-spectrum antibiotic therapy, it is recommended that the Medical Microbiologist be consulted.  
  • Supporting laboratory investigations for the diagnosis of wound infection requiring antimicrobial therapy include a Full Blood Count (FBC) and differential white cell count, renal function (urea, creatinine), glycated haemoglobin (HbA1c), erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), Procalcitonin and blood culture (for pyrexia >38ºC). 
  • The patient is a key team member, and should also be involved in decision making, to ensure that they have insight into and comply with their treatment and follow-up. 
  • The principles of antimicrobial stewardship (AMS) are integral to the wound infection continuum, biofilm-based wound care, and the judicial use of antimicrobial agents.


  1. British Society for Antimicrobial Chemotherapy in collaboration with ESGAP/ESCMID (2018). ANTIMICROBIAL STEWARDSHIP: From Principles to Practice. 
  2. Clinton A, Carter T. Review: Chronic Wound Biofilms: Pathogenesis and Potential Therapies. Lab Medicine Fall 2015; 46:277-284 
  3. Edwards R, Harding K. Bacteria and wound healing. Current Opinion in Infectious Diseases 2004; 17(2):91-6 
  4. International Wound Infection Institute (IWII). Wound infection in clinical practice. Wounds International 2016. 
  5. Läuchli S, Swanson T, Serena T, Harding K. The use of a point-of-care test for bacterial protease activity in chronic wounds. Wounds International 2015; Vol 6 (4). 
  6. Lipsky BA, Dryden M, Gottrup F, Nathwani D et al (2016). Antimicrobial stewardship in wound care: A Position Paper from the British Society for Antimicrobial Chemotherapy and European Wound Management Association. Antimicrob Chemother 2016; 71: 3026–3035. 
  7. Murphy C, Atkin L, Swanson T, Tachi M, Tan YK, Vega de Ceniga M, Weir D, Wolcott R. International consensus document. Defying hard-to-heal wounds with an early antibiofilm intervention strategy: wound hygiene. J Wound Care 2020; 29 (Suppl 3b):S1–28. 
  8. Schultz G, Bjarnsholt T, James GA, Leaper DJ et al (2017). Consensus guidelines for the identification and treatment of biofilm in chronic nonhealing wounds. Wound Rep Reg 2017; 25: 744-757. The Wound Healing Society. 
  9. Wounds UK (2020). Best Practice Statement: Antimicrobial stewardship strategies for wound management. Wounds UK, London.