Bacterial Growth Guideline: Reassessing its Clinical Relevance in Wound Healing

  From a microbiological perspective, successful wound healing is dependent on the hosts' ability to maintain control over the micro-organisms that inevitably colonize wound tissue.

Since the mid-1960s, research has shown that microbial (or, more specifically, bacterial) numbers have a significant impact on wound healing; there is widespread perception that the presence of 100,000 or more bacterial cells per gram of wound tissue is a key determinant in delayed wound healing and infection. Today, this 10⁵ guideline has a significant influence on wound care practice, and quantitative tissue biopsy or superficial swab samples are routinely used to determine whether a wound is infected and/or unlikely to heal. But is it really that simple? Are micro-organisms so predictable, irrespective of type? Should the diagnosis of wound infection be based primarily on microbiological findings rather than clinical observations? If the 10⁵ guideline is reliable, can it be applied to both acute and chronic wounds?

Micro-organisms and Man

  In normal life, an essential and mutually beneficial relationship exists between micro-organisms and man. The vast numbers of micro-organisms that naturally colonize the human body (approximately 10¹⁵ cells) are critical to health because they protect the host from opportunistic pathogens through a process known as colonization resistance.¹ On the skin, relatively small populations of resident bacteria such as Staphylococcus epidermidis and skin diphtheroids help prevent colonization by more pathogenic bacteria such as Staphylococcus aureus. In the human colon (large intestine), extremely dense populations of predominantly anaerobic bacteria (involving more than 400 different species in total) form a stable, symbiotic relationship with the host, despite the fact that many of these micro-organisms are potentially pathogenic.

  If the resident flora of the human body is disturbed, the health status of the host will likely be affected. One example is antibiotic-associated diarrhea. Oral administration of antibiotics such as ampicillin and clindamycin suppresses the resident gut flora; subsequently, unrestricted toxins are produced by an anaerobic bacterium (Clostridium difficile), causing inflammation and necrosis of the gut wall. In this situation, a change in environmental conditions favors growth of, and virulence expression in, C. difficile - factors that, collectively, are critical to the onset of infection.

Micro-organisms and Wounds

  Wound tissue is susceptible to contamination by micro-organisms (predominantly bacteria) from the external environment. Obvious sources include periwound skin (normal resident skin flora) and micro-organisms introduced following traumatic injury (nonresident exogenous flora). However, the majority of wound contaminants are derived from the mucosal surfaces of the host (endogenous flora), particularly the oral cavity and gut. In these sites, microbial colonization is dense and diverse and involves both anaerobic and aerobic bacteria (anaerobes outnumber aerobes by a factor of up to 1,000 to one² ). Wounds in close proximity to the mouth and gut harbor microbial populations consistent with the resident flora of these sites,^3-6 and the microbiology of acute and chronic wounds at these sites is complex.⁷ Detailed microbiological analyses have shown that anaerobes constitute approximately 30% of the total microbial population in noninfected venous leg ulcers^8-10; this proportion increases to approximately 50% in clinically infected venous leg ulcers.⁸

  Because wounds often become colonized by micro-organisms from a variety of sources, the resulting polymicrobial ecosystem is unique and complex. Within this ecosystem, an often dense microbial population is likely to consist of mixed aerobic and anaerobic bacteria, many of which may be antibiotic-resistant and/or pathogenic. Consequently, this polymicrobial cocktail not only provides an infection threat to the wound, but also presents a serious cross contamination risk, particularly in the hospital environment (see Figure 1).

  For micro-organisms to become problematic in a wound, local conditions need to favor their proliferation. Essentially, the presence of moisture, nutrition, and a low oxygen tension encourage the growth of a diversity of micro-organisms. When introduced into a wound, micro-organisms flourish in this favorable but abnormal habitat (particularly if local circulation is compromised), and their relationship with the host may change from symbiotic to pathogenic in order to out-compete other colonizing micro-organisms and resist clearance by the host's immune system. This may involve bacteria increasing the production of specific enzymes or toxins, producing cell adhesion and cell-protecting components (eg, biofilms), or interacting with other bacteria in order to gain a competitive advantage over the host. The density and diversity of wound microflora are likely to influence communication strategies within and among species that subsequently dictate the expression of virulence factors essential for microbial survival. The net effect is a probable increase in microbial pathogenicity; hence, presenting a greater challenge to the host.

Predicting Wound Infection

  All wounds are at risk of infection because they are colonized with micro-organisms. However, the probability of infection is dependent on the relationships between the colonizing micro-organisms and the host.^11-13

  Micro-organisms. Two microbial-related factors need to be considered with respect to wound infection - the contamination level (ie, numbers of micro-organisms, otherwise termed microbial density, load, dose, or bioburden) and the net pathogenic effect (virulence).

  In 1965, Bendy et al¹⁴ first reported that healing in pressure ulcers was directly related to the number of colonizing bacteria. In a controlled trial comparing topical antibiotic and nonantibiotic treatments, wound healing progressed only if the total count from superficial swab samples was less than 10⁶ colony forming units (cfu)/mL of wound fluid. Subsequent investigations undertaken by Robson et al^15-17 demonstrated that healing in pressure ulcers and closure of surgical wounds could be predicted by quantifying bacteria in biopsied tissue. This early work formed the basis of the 10⁵ guideline, the now widely accepted concept that a level of bacterial growth > 100,000 viable organisms per gram of tissue is necessary to cause wound infection and can be used to diagnose infection.¹¹ Although other key factors predisposing to wound infection and the importance of host-microbial balance are well recognized, today's wound care practitioners are influenced primarily by the 10⁵ guideline,¹⁸ and treatment is based on the bacterial count in deep or superficial tissue. However, for the appropriate management of microbially challenged wounds (eg, heavily colonized, critically colonized, and clinically infected), practitioners should have a more balanced awareness of the broader issues relating to micro-organisms and wounds.

  The relationship between micro-organisms and host are quite different in acute and chronic wounds and therefore should be addressed separately.

  Acute wounds and the 10⁵ guideline. Undoubtedly, the probability of wound infection and/or delayed healing increases as the level of contamination increases. This applies particularly to surgical and traumatic wounds where high doses of mixed micro-organisms are likely the key factor preventing wound progression and healing. In a clean surgical wound, the host inflammatory response would be expected to eliminate any contaminating micro-organisms as part of the normal wound healing process. In gastrointestinal surgery involving breach of mucosal surfaces and contamination of the wound with fecal microflora, the mixed and heavy microbial onslaught is more difficult to control; hence, prophylactic antibiotics are usually administered before surgery to supplement the host's inflammatory response.

  Clinical research has shown the importance of the microbial load in determining the probability of surgical wound sepsis and the appropriate timing for delayed closure. In a controlled clinical and microbiological trial on wounds following surgery for perforated or gangrenous appendix, Raahave et al¹⁹ reported that wound sepsis was more likely to occur when the median mixed aerobe-anaerobe count in wounds before closure was 4.6 x 10⁵ cfu/cm². Although this study emphasizes the importance of the bacterial load with respect to wound sepsis, contamination in these postsurgical abdominal wounds involved mixed aerobic-anaerobic bacteria that are capable of working in synergy. This means that if two or more bacteria are able to work together in unison, their net pathogenic effect is greater than if the same organisms worked independently of each other. In this study, the two predominant wound isolates were Escherichia coli and Bacteroides fragilis; Brook²⁰ reported the important pathogenic role of B. fragilis in the presence of E. coli (referred to as the "helper" bacterium). Similarly, clinical signs of infection were demonstrated when mixed doses of E. coli and B. fragilis were inoculated into guineapig partial-thickness wounds (approximately 4.5 x 10⁴ cfu of each bacterium), but not when the bacteria were inoculated separately in much higher doses (approximately 9 x 10⁴ cfu).²¹ Although these two bacteria are natural cohabitants in the gut, when introduced into a new environment such as the appendix or a postsurgical wound, their combined effect can be devastating. Synergy between these two bacteria is likely to involve E. coli reducing the oxygen tension in the wound to facilitate the growth of B. fragilis, and B. fragilis subsequently producing substances that compromise host inflammatory cell activity.²¹

  Acquiring a tissue sample for quantitative analysis is not a burdensome task for the surgeon to undertake during surgery, and the resulting microbiological data are likely to be useful in determining the appropriate time for closure of a wound or predicting the risk of sepsis. However, if a surgical or traumatic wound is infected, clinical signs (eg, inflammation, pain, edema, and suppuration) should prompt early treatment, irrespective of the microbial load. Microbiologists working in wound care have argued that tissue biopsies are not required to determine the bacterial load in acute wounds because a correlation exists between quantitative tissue biopsy and semi-quantitative superficial swab analyses.^22,23

  Chronic wounds and the 10⁵ guideline. For the day-to-day management of chronic wounds (eg, leg ulcers and pressure ulcers) where many factors influence healing, the validity of the 10⁵ guideline as a primary indicator of infection is questionable, despite reports indicating that such wounds are unlikely to heal when the bacterial load exceeds the 100,000 threshold.^11,14 This is because the guideline implies that all types of bacteria are capable of causing wound infection at a dose of 10⁵ cfu/g of tissue, despite the complexity and adaptability of bacteria in chronic wounds (the only exception to this rule is the presence of betahemolytic streptococci^11,24). However, an infecting dose is known to vary with the type of organism involved and the way in which it is presented.¹²

  Wound sampling and quantitative analysis. With awareness raised regarding the complex aerobic-anaerobic microbiology of wounds and the ability of cohabiting organisms to work in synergy, clinicians should consider what type of wound sample will generate the most meaningful data - a superficial swab or a tissue biopsy. Many argue that a deep tissue biopsy will identify a causative organism; whereas, superficial samples will only collect debris and, therefore, provide meaningless data. However, in virtually all cases, wound contamination occurs from outside of the wound; consequently, organisms that penetrate into deeper tissue will have disseminated from superficial tissue. Thorough microbiological investigation of a superficial swab sample will isolate all organisms that may be involved in delayed healing, but it will not determine the specific deeper-penetrating organism(s). With adequate clinical information regarding the wound (eg, type, location, signs of infection, and current antimicrobial therapy), the microbiologist can conduct appropriate quantitative and qualitative investigations, provide an expert opinion on the causative organism(s), and target them with antimicrobial therapy. Superficial swab samples provide a general picture of the wound microbiology; tissue biopsy samples are highly localized and may not reflect the microflora in other parts of a wound (see Figure 2). This is a topic of continued debate. Additionally, isolating only the deeper penetrating organisms and using the quantitative data to guide therapy may overlook superficial organisms that potentially influence the healing status of a wound.

  In a study of infected diabetic foot ulcers, Sapico et al²⁵ reported that less invasive micro-organisms may be synergistic with more virulent ones and play a crucial role in wound infection. Although microbial synergy is not widely recognized within the field of wound care, a clear indication of its contribution in an infected leg ulcer has been reported. In this case, S. aureus facilitated the growth and pathogenicity of a pigmenting anaerobe.²⁶ Delayed healing associated with microbial synergy also was indicated in a study of leg ulcers by Tre ngove et al²⁷; although healing was not influenced by any specific bacterium (including beta-hemolytic streptococci), the presence of four or more bacterial groups in a wound was associated with delayed healing. The potential significance of superficial bacteria in wound healing recently was recognized in a study evaluating an antimicrobial dressing in the management of 29 nonhealing chronic wounds.²⁸ Although the antimicrobial dressing reduced only the numbers of superficial bacteria (the deeper tissue counts did not change), a marked clinical improvement was observed in the majority of the wounds. Evidence suggests that micro-organisms residing in superficial wound tissue can contribute to delayed wound healing²⁸; this may not be a direct effect, but is likely to occur via polymicrobial interactions.

  Microbiology in deep and superficial tissue not withstanding, surface sampling is easier and less time-consuming to perform than tissue biopsy, and the cost is approximately one-sixth that of tissue biopsy.²⁹ In addition, surface sampling does not impose the practicing restrictions associated with tissue biopsy¹⁷ and it is less traumatic to the patient.

  Microbiological analysis of samples. Another extremely important but less known aspect of wound microbiology is the microbiological analysis. If infection is to be diagnosed on the basis of a tissue count, the quality of the sample is critical. In the laboratory, standard practice is to aseptically weigh the tissue sample, heat with flame to remove surface contamination, and homogenize the tissue using a known volume of fluid culture medium. The resulting suspension is diluted serially (10-fold) and a known volume of each dilution is plated out onto a series of agar plates to culture a variety of micro-organisms. Following appropriate incubation, organisms are counted, the dilution factors are considered, and a total count per gram of tissue is calculated. This procedure is significantly more time-consuming and expensive than semi-quantitative analysis of a superficial sample. Anaerobes are expensive and time-consuming to culture, but considering the prevalence of anaerobes in chronic wounds, they should not be (but often are) excluded in quantitative tissue biopsy counts. With this in mind, how valid is a tissue count that the practitioner receives from the laboratory? Is it appropriate to confirm that a wound is not infected based on a result that does not provide the complete microbiological picture?

  The relevance of a specific number of micro-organisms in a wound also needs to be challenged because microbial synergy can reduce the infectious dose. Smith et al³⁰ demonstrated that the lethal dose of the anaerobe, Fusobacterium necrophorum, was reduced from < 10⁶ cfu to >10 cfu when administered subcutaneously to mice in combination with a sublethal dose of E. coli. Clearly, infection is significantly influenced by the types of bacteria and their interactions and does not rely on numbers alone. For this reason, the view that the mere presence of organisms in a wound is less important than the level of bacterial growth¹¹ must be put into perspective. Kerstein³¹ specifically stated that quantitative microbiology alone cannot predict the risk of wound sepsis and that no single, perfect predictor of wound infection exists.

  Qualitative microbiology. The presence of a specific pathogen does not necessarily mean that a wound is infected. S. aureus is the most prevalent bacterium in wounds, and although recognized as a potential pathogen, a correlation between clinical infection and its presence in chronic wounds is lacking.^8,10 Similarly, although beta-hemolytic streptococci are potentially highly virulent (producing virulence factors such as hyaluronic capsule, M protein, streptolysin, streptokinase, and hyaluronidase³²) and are a key etiologic agent in aggressive soft tissue infections, they are not always associated with infection in chronic wounds.^8,27 Many anaerobes commonly found in wounds are also potentially pathogenic³³; therefore, they should not be excluded from investigation in the laboratory. If a wound is malodorous, the presence of anaerobes is highly probable; this also applies to samples showing no growth following aerobic incubation. Although anaerobes are often slower to culture in the laboratory, a series of simple tests can usually determine the key groups (clostridia, anaerobic streptococci, and pigmenting and non-pigmenting Gram-negative anaerobes) within 48 to 72 hours.²⁶

  The literature indicates that quantitative microbiology (ie, the 10⁵ guideline) is increasingly emphasized and accepted when diagnosing infection. However, when the potential variability in quality and analysis of tissue samples is taken into account, the potential importance of superficial micro-organisms and polymicrobial synergy, the microbiological methods used, the types of micro-organisms investigated, and the validity and value of the quantitative biopsy in diagnosing chronic wound infection are questionable. Similarly, qualitative microbiology cannot be used to diagnose infection or to identify a causative organism in polymicrobial wounds because more than one organism may be involved. Wound infection must be based primarily on clinical signs (eg, increasing pain, friable granulation tissue, foul odor, and wound breakdown³⁴) and subsequent qualitative microbiology can confirm that empirical antibiotic treatment had been selected appropriately.

  In summary, wound care practitioners are encouraged to diagnose infection and base their treatment decisions on a highly specific quantitative microbiological assessment that has debatable clinical validity. For the day-to-day management of chronic wounds, the benefits of tissue biopsy are unlikely to outweigh the practical and cost-associated implications. With adequate communication between the practitioner and the microbiologist, a semi-quantitative/qualitative swab taken with adequate justification (ie, clinical signs of infection or a nonhealing wound without clinical signs of infection) can be used to generate clinically relevant and timely data. A collective evaluation and appreciation of the numbers, interactions, and virulence potential of micro-organisms in complex wounds is critical to effective wound management.

  The host. In 1880, Louis Pasteur stated, "The germ is nothing. It is the terrain in which it is found that is everything."³⁵ In other words, the local environment is a critical factor in infection.

  Wound tissue is potentially a favorable terrain for micro-organisms due to the presence of moisture, nutrition, and warmth. In the event of tissue contamination, a host inflammatory response designed to inactivate and remove potentially dangerous micro-organisms is provoked. The polymorphonuclear neutrophil (PMN) is a host inflammatory cell that possesses highly potent antimicrobial mechanisms; consequently, the presence and activity of this cell type in a wound environment is essential to healing. The presence of PMNs at the wound site (and also other factors that influence wound healing, such as oxygen and nutrients) is dependent on an adequate blood supply to the local tissue. Surgical wounds in the anus exemplify the importance of tissue perfusion; despite their susceptibility to gross bacterial contamination, these wounds rarely become infected.³⁶ In contrast, the reduced availability of essential blood components in ischemic wounds severely impairs healing. When deprived of oxygen, cells involved in wound healing die, leading to the accumulation of devitalized tissue that provides an ideal environment for the growth of a diversity of micro-organisms. Additionally, ischemia and a dry environment interfere with delivery and activity of PMNs in wound tissue, compromising the clearance of wound microflora. Polymorphonuclear neutrophil function can be further impaired by pathophysiological conditions (eg, disease and chemotherapy) and in patients with diabetes, the probability of infection in foot ulcers is five times greater than in non-diabetic patients.³⁷

The Microbial Continuum and Implications for Wound Care Practice

  The inevitable contamination of wound tissue by micro-organisms provokes a series of interrelated host and microbial responses that ultimately determine whether a wound will heal. Microbial progression leading to wound infection is believed to follow a series of stages during which the micro-organisms gain increasing control over the host. This has been referred to as an infection continuum.³⁸ If microbial progression is not interrupted, infection is the likely outcome (see Figure 3).

  Initially, contaminating micro-organisms are exposed to a new environment where they must adapt quickly to survive. Early acclimatization to the wound environment may involve bacteria producing cell-protecting components (eg, biofilms) to resist the host immune response, adhesion molecules that enable them to quickly attach to host cells, and enzymes and toxins that facilitate dissemination through tissue (virulence factors). At this point in the continuum, a normal host inflammatory response usually maintains control over the contaminants, although the dose and virulence characteristics of the micro-organisms involved cannot be overlooked. If the local conditions are compromised (eg, presence of foreign bodies, necrotic tissue, dead space, tissue hypoxia, and reduced PMN activity) micro-organisms have an opportunity to colonize tissue. If, at any stage in the continuum, progression towards wound healing is interrupted, a host-microbial balance in favor of the micro-organisms should be suspected. At this stage, the wound may be critically colonized, meaning that the micro-organisms are interfering with wound healing without inducing obvious clinical signs of infection. The presence of ischemia, slough, necrosis, increased exudate, and hypoxia and a change in wound appearance are all conditions that may alert the practitioner to a potential microbiological problem. At this point, it may be appropriate to sample the wound for microbiological analysis and administer a topical antimicrobial agent (eg, a dressing containing iodine, silver, or an antibiotic formulation) that is effective against a broad spectrum of micro-organisms to help redress the balance in favor of the host.

  Because achieving sterility in a wound is not possible, the objective must be to achieve a host-manageable bioburden (ie, a population of micro-organisms the host can control). Although topical antimicrobial agents are important in this respect, some non-medicated, moisture-retentive dressings also reduce the incidence of wound infection,^39,40 most probably by creating a moist environment that is conducive to PMN activity⁴¹ and by eliminating dry necrotic tissue and dead space that facilitate microbial growth. Consequently, the combined effects of antimicrobial activity (achieved by both endogenous and exogenous mechanisms) and a moist wound environment are likely to create optimal conditions for healing.

  Ultimately, if microbial progression is not interrupted, the net pathogenic effect is likely to exceed that of the host's natural defenses, leading to more widespread dissemination of one or more organisms and the subsequent onset of local and systemic signs of infection. A spreading infection is likely to require systemic antibiotics that target both aerobes and anaerobes and supplement the host's immune response in regaining control over the microbial challenge.

Conclusion

  In the often complex and diverse polymicrobial wound, microbial communication processes are likely to occur that enable organisms to interact and enhance their survival strategies to the detriment of the host. The density and diversity of wound microflora are likely to influence the expression of virulence factors essential for their survival and therefore should be assessed collectively. Although quantitative microbiology is relevant to the management of surgical wounds, it is only one of many host-microbial factors that may influence wound healing and should not be used in isolation to diagnose infection and guide treatment.

  Wound infection should be diagnosed primarily on the basis of clinical signs and supported by microbiological observations. Wound sampling should be restricted to clinically infected wounds and wounds without clinical signs of infection that fail to heal (they may be critically colonized).

  With appropriate dialogue between the wound care practitioner and the microbiologist, a superficial swab sample can be used to generate clinically relevant semi-quantitative and qualitative data. In treating wounds that are clinically infected or at risk of infection (eg, polymicrobial, heavily colonized, and critically colonized), the primary objective is to establish a host-manageable bioburden. This can be achieved by using antimicrobial dressings in nonhealing, noninfected wounds or those with localized infection and with systemic antibiotics that target both aerobic and anaerobic bacteria in spreading wound infections.

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