Ulceration in the lower extremity may be due to infectious agents. Diverse groups of microbes including bacteria, viruses, parasites, and fungi have been implicated [41].

Chronic leg ulcers are defined as those that show no tendency to heal after 3 months of appropriate treatment or are still not fully healed at 12 months.

The interaction between ulcer and bacteria can be stratified into four levels: contamination, colonization, critical colonization, and infection [11], while contamination and colonization by microbes are not believed to inhibit healing, the line between colonization and infection can be difficult to define.

The term “critical colonization” has been used to describe the stage at which bacteria begin to adversely affect wound healing [11]. Moreover, the underlying pathogenesis of chronic wounds may result in wounds of different etiologies being differently affected by bacteria [12–14].

Chronic wounds by their very nature may not always display the classic symptoms of infection (pain, erythema, edema, heat, and purulence), and it has been suggested that an expanded list, including signs specific to secondary wounds (such as serous exudate plus concurrent inflammation, delayed healing, color of granulation tissue, foul odor, and wound breakdown) be employed to identify infection [15].

Microbiologically, a critical bacterial load, synergic relationships between bacterial species, and the presence of specific pathogens have all been proposed as indicators of infection. The presence of microbes per se is not indicative of wound infection.

The possibility that a critical microbial load might directly affect the healing outcome in both acute and chronic wounds has been considered for several decades, with a direct relationship first being demonstrated by Bendy et al. [16] in 1964. Since then, work carried out by Robson [17] and others has led to the widely held opinion that nonhealing is associated with a bacterial load of more than 10⁵ bacteria per gram of tissue.

The concept of bacterial synergy which recognizes the importance of interspecies interactions has been purported to occur in chronic wounds through studies such as that by Bowler and Davies [18]. They found the growth and pigmentation of some Gram-negative anaerobes to be enhanced by some facultative bacteria through the provision of an essential, unidentified growth factor. Furthermore, they found significantly greater numbers of anaerobes in infected ulcers compared with noninfected ones.

With regard to specific pathogens, beta-hemolytic streptococci [17, 19] S. aureus [12], Enterobacteriaceae [12], and Pseudomonas species [12, 20] have all been implicated as having potentially adverse effects on wound healing. The impact of these species may vary in different settings, for example, over 60 % of arterial and diabetic ulcers colonized with S. aureus develop an infection compared with only 20 % of venous ulcers similarly colonized [12].

In summary, microorganisms are identified in the deep tissue of all chronic wounds, yet the role they play and the impact of specific species on wound longevity are not clear. The distinction between infected and colonized wounds has to be considered on a clinical basis and not by microbiological analysis only due to the universal colonization of chronic wounds [21]. Microbial analysis can be of benefit when considered in concert with clinical observations to confirm causative organisms and their sensitivities [22] and so enable refinement of antibiotic regimens [21].

The microflora of leg and foot ulcers is usually polymicrobial, and recent studies using molecular techniques have emphasized the complex ecology of these wounds [23, 24]. Using conventional techniques, the mean number of bacterial species per ulcer has been found to range from 1.6 up to 4.4 [25–28]. Hansson et al. [29] observed that 86 % of ulcers with no clinical signs of infection contained more than one bacterial species.

Staphylococcus aureus and coagulase-negative Staphylococcus have been the predominant organisms isolated. S. aureus has been reported in frequencies varying from 43 % of infected leg ulcers to 88 % of noninfected leg ulcers [29], whereas Staphylococcus epidermidis has been reported in 14 % of venous ulcer specimens [30] and 20.6 % of diabetic foot ulcers (DFUs) [27]. Pseudomonas aeruginosa is another frequently identified organism and has been found in 7–33 % of ulcers [12, 26, 29]. A number of other aerobic species have also been reported, including Escherichia coli [18, 27, 29–31], Enterobacter cloacae, Klebsiella species, Streptococcus species, Enterococcus species [28, 29] and Proteus species [31]. This is by no means an exhaustive list, but is illustrative of the range of aerobic bacteria that exist in chronic wounds.

In addition to aerobes, anaerobic organisms are frequently identified in wounds, albeit with considerable variation. Trengove et al. [20] found obligate anaerobes in one-quarter of chronic leg ulcer samples, while Ge et al. [31] found they constituted only 6 % of DFU wound isolates.

However, a focused study by Bowler and Davies [18] found anaerobes in 73 % of noninfected leg ulcers and 82 % of infected leg ulcers. The most common isolates found in both the infected and noninfected leg ulcers were Peptostreptococcus species and pigmented and nonpigmented Prevotella/Porphyromonas species [18]. Finegoldia magna (previously classified as Peptostreptococcus magnus) was found by Hansson et al. [29] to be present in 19.6 % and Peptoniphilus asaccharolyticus in 9.8 % of noninfected venous leg ulcers. Kontiainen and Rinne [28] found that clinical swabs sent for analysis, presumably from infected or assumed infected wounds, yielded obligate anaerobic rods (mainly Bacteroides species) from 12 % of ulcers and anaerobic cocci (Peptostreptococcus) from 8 %. Ge et al. [31] found Bacteroides, Peptostreptococcus, and Prevotella species to be the most frequently isolated obligate anaerobes in mild or moderately infected DFUs. The continuity of the microbial profile of chronic wounds over time is unclear from the limited literature that has examined this issue. Hansson et al. [29] considered the microflora of chronic wounds to be a relatively stable entity having found that 90 % of ulcers that were followed for 4 months, or until healing, contained at least one resident organism that was isolated from all monthly swabs. Furthermore, Gilchrist and Reed [32] considered chronic wounds to have stable microbial populations, following the observation that once a species was present, it generally remained so under hydrocolloid dressings, with the exception of the transient appearance of P. aeruginosa. However, closer examination of their data shows that 85 % of wounds acquired new aerobes and 45 % new anaerobes over the 8 week of study period. Trengove et al. [20] logged the occurrence of new bacterial groups appearing in wounds after initial swabs had been taken. They found at least one new bacterial group present in subsequent swabs in 82 % of patients and thus concluded that the microbial populations of chronic wounds alter over time.