Diabetes and foot infection: more than double trouble


  • Jean-Louis Richard,

    Corresponding author
    • Department of Diabetology and Nutritional Diseases, Medical Centre, University Hospital of Nîmes, Le Grau du Roi, France
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  • Jean-Philippe Lavigne,

    1. Department of Bacteriology, Carémeau University Hospital, Nîmes Cedex 9, France
    2. National Institute of Health and Medical Research, U1047, Montpellier 1 University, Faculty of Medicine, Nîmes Cedex 02, France
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  • Albert Sotto

    1. National Institute of Health and Medical Research, U1047, Montpellier 1 University, Faculty of Medicine, Nîmes Cedex 02, France
    2. Department of Tropical and Infectious Diseases, Carémeau University Hospital, Place du Professeur Robert Debré, Nîmes Cedex 9, France
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Jean-Louis Richard, Department of Diabetology and Nutritional Diseases, Medical Centre, University Hospital of Nîmes, 30240 Le Grau du Roi, France.

E-mail: jean.louis.richard@chu-nimes.fr



Infection of foot ulcers is a common, often severe and costly complication in diabetes. Many factors linked to the host, mainly immune defects, neuropathy and arteriopathy, as well as bacteria-related factors, interact in a complex way and account for the susceptibility of diabetic individuals to foot infections, the severity of such infections and difficulty to treat them.


This article reviews these factors, in the light of data from the literature and from our own results.


DFIs are not as simple as previously suggested, and new concepts must be considered, especially the virulence potential of isolates and bacterial communications through biofilms.


The development of new tools from molecular biology is a critical step to better understand and manage these infections. Copyright © 2012 John Wiley & Sons, Ltd.


basement membrane thickening


diabetic foot infection


diabetic foot ulcer


extracellular polymeric substance


functionally equivalent pathogroup


peripheral arterial disease


polymorphonuclear cell


pentose phosphate pathway


reactive oxygen species

Diabetic foot infection is a common and severe pathology. As shown in Eurodiale study, 58% of diabetic patients with a new foot ulcer had a clinically infected wound at presentation [1]. DFI is estimated to be the most common cause of diabetes-related admission in hospital and remains one of the major pathways to lower-limb amputation [2]. As reported by Lavery et al., the risk of hospitalization and lower-extremity amputation was about 56 and 155 times greater, respectively, for diabetic people who had a foot infection than for those without [3]. So, the question is why the so high prevalence and severity? This is best explained by profound disturbances in bacteria–host relationship. The aim of this article is to review host-related and bacteria-related factors involved in DFI.

Host-related disturbances

Diabetic immunopathy

For a long time, alterations of immune defences have been implicated in the susceptibility to infection of diabetic patients. Louis Pasteur was reported to acknowledge in 1895 on his deathbed that ‘the microbe is nothing, the terrain is everything’, quoting Claude Bernard's motto. A lot of immunological defects, mostly related to innate immunity, have been reported in diabetic patients [4]. The most consistent involves altered function of PMNs, specially impaired phagocytosis and bactericidal activity. Twenty years ago, it was shown that ingestion and killing of Staphylococcus aureus by PMNs were significantly reduced in diabetic patients with poor metabolic control [5]. These defects were found in the majority of studies but not in all [6]. Data regarding PMN chemotaxis and adherence are even more conflicting. Differences in patient characteristics, quality of blood glucose control and methods that authors used for testing PMN function may in part explain those discrepancies.

Bacterial killing by PMN is related to both nonoxidative and oxidative mechanisms [7]. The former reflects the action of potent antimicrobial peptides within the cytoplasmic azurophil granules, such as bactericidal/permeability-increasing protein, defensins or cathepsin G. On the other hand, the oxidative pathway depends on oxidants whose production is related to activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase on plasma membrane, leading to production of ROS as superoxide anion and hydrogen peroxide. Moreover, myeloperoxidase from azurophil granule allows the formation of highly toxic hypochlorous acid from hydrogen peroxide and chlorine ion. Activation of the oxidative metabolism, known as the respiratory burst, critically depends on the NADPH supplied by the PPP (also known as hexose monophosphate shunt). Abnormal bactericidal activity in diabetic patients seems to be mainly due to defects in oxidative mechanisms as shown by the in vitro nitroblue tetrazolium reduction test and chemiluminescence technique [5, 8, 9]. In these studies, the resting oxidative activity of PMN from diabetic patients was shown to be equal to or higher than in nondiabetic controls, but the respiratory burst was severely impaired. It is worth noting that two of these studies used S. aureus as target bacteria [5, 8], a frequent and serious pathogen in DFI. So, although the resting production of ROS was reported as normal or even increased in the basal state [10], the respiratory burst was shown to be severely impaired; this defect might account for the decreased bacterial killing and hence for the susceptibility of diabetic people to infection. Of note, it was recently reported that respiratory burst activity was reduced in PMN from type 2 diabetic patients with DFU [11]. As oxidative pathways critically depend on NADPH availability, exclusively supplied by the PPP, it has been suggested that depletion of NADPH may account for PMN killing defects in diabetic patients. Excessive activity of the polyol pathway in hyperglycaemic state is a potential candidate for the NADPH depletion because the polyol pathway is a great consumer of NADPH; other mechanisms have been suggested, such as a low activity of insulin-dependent enzymes in the PPP due to insulin resistance [12, 13] or zinc deficiency [11]. Moreover, reduction of myeloperoxidase [12, 14] as well as superoxide dismutase [13] activity in PMN from diabetic patients could possibly decrease halogenation of bacterial proteins and contribute to defective bacterial killing.

Impaired monocyte/macrophage functions (chemotaxis and phagocytosis) have been also described in diabetic patients, but their pathogenic mechanism and clinical significance remain unclear [6]. Various disturbances of cellular innate immunity have been reported in diabetic people, including a low serum level of complement factor 4 (C4) and abnormal production of cytokines by monocytes, but these abnormalities seem to play a minor role, if any, in susceptibility of diabetic patients to infections [6]. Regarding adaptive immunity, no consistent data have been reported.

Interestingly, in diabetic mice, an S. aureus hind paw infection that is equivalent to the most common skin and soft-tissue infections occurring in diabetic people has been developed [15, 16]. In this model, both nonobese (type 1) and obese db/db (type 2) diabetic mice were shown to clear infection more slowly than nondiabetic control mice. In vitro bacterial killing of blood from db/db mice was significantly lower than that of nondiabetic mice. Moreover, neutrophils from db/db mice exhibited a poor respiratory burst after incubation with S. aureus compared with nondiabetic mice. This decreased respiratory burst was also shown in nonobese diabetic mice, and although PMN infiltration was more pronounced in db/db mice compared with the nondiabetic counterpart, the inflammatory response was decreased in nonobese diabetic versus nondiabetic mice. Finally, S. aureus hind paw infection was cleared more rapidly in insulin-controlled versus frankly hyperglycaemic nonobese diabetic mice. So, a persistent hyperglycaemic state appears to have a deleterious effect on innate immunity, favouring susceptibility and chronicity to skin and soft-tissue infection of S. aureus in diabetic mice. Nevertheless, there may be some differences between type 1 and type 2 diabetes, as shown by a decreased inflammatory response in type 1 compared with an enhanced response in type 2 diabetic mice.

To conclude on diabetic immunopathy, a number of studies have been carried out about its effect on infection. Most of them have shown defects of innate immunity, especially in microbicidal activity of PMN, which are more evident in the presence of frank hyperglycaemia. However, there is no consensus regarding the cause, extent and clinical consequences of such abnormalities.

Diabetic neuropathy

Autonomic dysfunction

Involvement of autonomic nerve fibres is a common and frequently underestimated component of diabetic polyneuropathy [17]. The cutaneous microcirculation has a unique anatomical arrangement. The arterial supply and venous drainage are organized as two main horizontal plexuses in the dermis: a lower deep plexus at the dermal–subcutaneous junction and an upper superficial plexus in the papillary dermis. This latter plexus gives rise to a nutritive capillary loop in each dermal papilla [18]. Between these two plexuses are arteriovenous anastomoses that are highly innervated and low-resistance vascular structures that are normally in a constricted state by sympathetic tone. Autonomic dysfunction leads to a peripheral sympathetic denervation that in turn is responsible for the loss of the vasoconstrictor tone and opening of arteriovenous shunt [19]. Arteriovenous shunting can result in a misdistribution of blood flow bypassing the nutritive capillary bed (nutritive capillary ischemia) [20]. Moreover, arteriovenous shunting increases venous and then tissue pressure, a phenomenon similar to the compartment syndrome, resulting in a relative deep ischemia and in a reduced tissue delivery of oxygen and nutrients [21].

Autonomic failure can also cause anhidrosis, favouring the occurrence of cracks and fissures on the feet that are portals of entry for pathogens, leading to infected ulcers [17].

C-fibre dysfunction and nerve axon reflex

Unmyelinated C-fibres not only are the postganglionic axons of the autonomic nervous system but also are involved in the perception of pain. Stimulation of C-nociceptive fibres generates a local nerve axon reflex, giving rise to the neurogenic inflammation that is the occurrence of inflammatory symptoms resulting from release of neuropeptides from sensory nerve terminals [22]. Nerve axon reflex consists in antidromic release of proinflammatory agents from collateral C-fibre axons in response to stimulation of afferent terminals [23]. In this process, polymodal capsaicin-sensitive sensory C-fibres are of utmost importance because they represent 70% of all C-fibres in the skin and exhibit transmembrane cation channels (vanilloid receptors or transient receptor potential vanilloid), allowing the release of neuropeptides [24]. Various neuropeptides are produced and released by C-polymodal nociceptors, including substance P and calcitonin gene-related peptides, which were shown to modulate inflammation and immunity by complex mechanisms through connection with specialized cells such as dendritic and mast cells, release of cytokines and modulation of T-cell activation [23-25]. Nerve axon reflex can be assessed by different methods; using laser Doppler imager flare technique, Krishnan and Rayman [26] showed a reduced response to skin heating in type 2 diabetic patients with clinical neuropathy compared with type 2 diabetic people without neuropathy and nondiabetic control subjects. So, it is conceivable that an impaired neurogenic inflammation due to C-fibre dysfunction could explain the frequent lack of local and systemic inflammatory signs even in severe DFI. This contributes along with autonomic dysfunction to enhance susceptibility to infection of diabetic neuropathic foot ulcers, as recently emphasized by Schaper et al. [27].

Loss of protective sensation (LOPS)

Because sensorimotor neuropathy is responsible for the loss of protective sensation in the foot, DFUs are prone to chronicity and thereby have a major risk to be infected.

The various mechanisms by which diabetic neuropathy might enhance susceptibility for infection and worsen its severity are summarised in Figure 1.

Figure 1.

Role of neuropathy in diabetic foot infection

Figure 2.

Interactions between metabolic, anatomical and bacteriological factors in diabetic foot infection

Diabetic angiopathy

Peripheral arterial disease

Disease of large artery in the lower limb is a well-known independent risk factor for DFI. In a case-control study, Peters et al. [28] reported a 5.5-fold increased risk for DFI in patients with PAD compared with those free of PAD. From a cohort study of 247 diabetic patients with DFU followed up during a 2-year period, Lavery et al. [3], using a stepwise logistic regression analysis, found that PAD was associated with a twofold increased risk for infection. More recently, in the Eurodiale study, both infection and PAD were reported in 31% of patients referred for a new DFU [29]. Moreover, in this group, the odds ratio for nonhealing was the highest, higher than for the group with only PAD, suggesting that PAD and DFI may act synergistically for delaying healing. Ischemia-related tissue necrosis evidently favours infection of the wound, making gangrene from dry to wet. In addition, it was suggested that PAD might worsen microcirculation failure, but that remains a subject of debate (see the succeeding part of this article). PAD not only favours occurrence of infection but also makes the infection more severe as mentioned earlier. This may be due to not only a poor delivery of oxygen and nutrients in the infected ischemic wound but also a poor antibiotic tissue penetration as shown by Raymakers et al. [30].


Structurally, thickening of the capillary BMT is the hallmark of the diabetic microangiopathy [31]. Although BMT does not lead to capillary occlusion [32], it may act as a permeability barrier, impeding the nutrients and activated leukocytes to be delivered in the tissues [31, 32] and thereby enhancing susceptibility for and severity of infection.

Functionally, microangiopathy is mainly characterized by impaired ability of capillaries to vasodilate in response to injury [33]. Part of this impaired hyperaemic response may be due to the rigidity of the thickened basement membrane [32, 33]. Other factors play a significant role, especially endothelial dysfunction, which appears to be partially linked to neuropathy [32]. The effect of PAD on microcirculation is a subject of discussion [32, 34]. It is worth noting that maximal hyperaemic response to heating of foot skin was shown to be reduced in diabetic patients with neuropathy compared with those without complication and with normal control individuals, but there was no difference in neuropathic patients whether they had PAD or not. The same results were obtained in the response to iontophoresis of acetylcholine. On the other hand, the response to iontophoresis of sodium nitroprusside was lower in neuropathic diabetic patients with PAD than in those without PAD. This suggests that microvascular dysfunction may mainly be associated with neuropathy, whereas PAD acts preferentially on endothelium-independent vasodilatation [35]. Impairment of microcirculation renders the foot functionally ischemic and thus may contribute to DFI.

So, arteriopathy has a dual effect on infection as PAD (macroangiopathy) and is responsible for an ‘organic’ ischemia, whereas microcirculation failure (microangiopathy) results in a functional ischemia.

Anatomy of the foot

The anatomy of the foot is unique because of its division into compartments, although some controversies exist about the exact boundaries of these compartments [36]. In the plantar aspect, the plantar aponeurosis is the most superficial fascia. Its central portion is the thickest and spans from the medial calcaneal tubercle to the bases of the proximal phalanges in a fan-like manner. This plantar fascia is the inferior boundary of the three plantar compartments separated each other by intermuscular septa. The medial compartment is roofed by the inferior aspect of the first metatarsal bone and is bounded laterally by a septum that runs longitudinally from the calcaneus to the medial side of the first metatarsal head. The lateral compartment is roofed by the fifth metatarsal bone and is bounded medially by a septum from the calcaneus to the fifth metatarsal head. Between those two compartments is the central compartment roofed by the inferior aspect of the second to fourth metatarsal bone. The interosseous compartments between metatarsal heads are located at the dorsal aspect of the foot and roofed by the dorsal subcutaneous structures [37]. Communications exist between the central and the neighbouring compartments as well as between the plantar compartments and the dorsal space. Moreover, the plantar aspect of the foot communicates with the deep posterior and peroneal compartment of the leg [36, 38]. These plantar compartments are an easy way for infection to spread proximally towards the heel and the lower limb. In addition, by direct communication or in perforating intermuscular septa, infection can spread from a compartment to the adjacent one.

One the other hand, compartments form a close confined space. As a consequence of infection, oedema can develop and increases the pressure in the compartment, which, when exceeding the hydrostatic pressure, results in ischemia, tissue necrosis and small artery thrombosis, a phenomenon known as ‘compartment syndrome’ [36-39]. Interestingly, it had been suggested that compartment pressure might be elevated in neuropathic diabetic patients possibly because of accumulation of sorbitol [39].

So, the anatomical characteristics of the foot make the infectious process more severe, promoting the spread of infection and aggravating the tissue damage.

Bacteria-related disturbances

Intuitively, density and virulence of pathogens could play a role to account for severity of DFI.

Density of pathogens

In 1964, Bendy et al. [40] first proposed that the number of bacteria plays a critical role for the development of infection. Thereafter, some authors suggested that a bacterial density equal to or higher than 104 or 105 g−1 (or cm−3) of tissue may be required to cause wound infection [41]. Finally, the concept of critical colonization emerged as a stage in which bacterial density is not sufficient to trigger infection but may suffice to surpass host defence without overt generalized immunological reaction. Critical colonization thus appears to be a precursor state of infection and has been implicated in delayed healing. In agreement with this concept, it has been recently shown by Xu et al. [42] that in neuropathic DFU, a high bacterial load was associated with a worse rate of wound healing and there was a significant inverse relationship between colony-forming unit (CFU) count from exudates and wound healing rate. However, how relevant is this concept in clinical practice? Ten years ago, Dow et al. [43] had already observed that β-haemolytic streptococci at 102 CFU g−1 of tissue were able to induce tissue damage, whereas a count greater than 105 CFU of less pathogenic organisms was of little significance. In the same way, Robson and Heggers [44] documented that wound healing may occur despite a high number of bacteria. Moreover, this concept, relying mostly on studies in acute wounds, is difficult to define in either clinical or microbiological terms and does not take into account the presence of specific pathogens. From a practical point of view, diagnosis of critical colonization should require to systematically perform a tissue biopsy for every wound, whose interpretation may be questionable because of heterogeneity of bacteria distribution within the wound tissue. Finally, it remains questionable that use of bacterial colony count may help the clinician to decide antibiotic therapy. Therefore, critical colonization appears as a debatable and unpractical concept.

Bacterial virulence

Testing the virulence power of bacteria seems to be a more promising way to characterize infection and to differentiate it from colonization. Our studies focused on S. aureus, the most prevalent pathogen isolated from DFU, using several techniques, including DNA microarray-based genotyping, multiplex polymerase chain reaction (PCR) and Caenorhabditis elegans and zebrafish killing assays.

DNA microarray is a technique from molecular biology able to detect genes related to virulence and antibiotic resistance [45]. Using this method, we were able to show that virulence and resistance genes were far more often present in clinically infected DFU than in uninfected wounds. Hence, 20 of 22 S. aureus isolates from uninfected ulcers were free of virulence genes, whereas these genes were detected in all but one of the 85 strains from infected ulcers [46]. Multiplex PCR technique enabled us to screen a larger number of virulence genes. With this technique, we showed that the prevalence rate of ten genes was significantly higher in strains from infected ulcers than in those from noninfected ulcers [47]. Moreover, by logistic regression analysis, a combination of five genes (sea, sei, hlgv, cap5 and lukDE) allowed to differentiate infected from noninfected DFU with a good sensitivity (0.98) and specificity (0.87). In a more recent study, we used a new generation of DNA microarray that covered 334 different genes for genotyping 195 S. aureus isolated as the sole pathogen in DFU. We found that a single gene (lukDE) may suffice to differentiate uninfected from infected ulcers (personal data). Moreover, lukDE was able to predict the outcome of grade 1 DFU. Finally, this new DNA microarray generation allowed to compare the strains, determine their clonality and thus predict more specifically the colonizing or infecting profile of S. aureus.

The importance of the bacterial virulence to induce infection was confirmed by killing assays using C. elegans and zebrafish as host models. C. elegans is a transparent and mostly hermaphrodite nematode, about 1 mm in length with a limited number of cells, which feeds mainly on environmental bacteria in soil and E. coli OP50 in the laboratory. This nematode has been proposed as a useful animal model to study host–pathogen interactions and assess the virulence potential of microorganisms [48], and the C. elegans killing assay has been validated to study the virulence of S. aureus[49]. The test is based on ingestion of S. aureus by the nematodes, leading ultimately to the death of the worms. Because many of the virulence factors used by S. aureus to induce disease in humans are also required for full pathogenicity in nematodes, the percentage of killed worms along the time (worm survival curve) when exposed to different S. aureus strains is an indirect marker of virulence capacity. We found that for all but one S. aureus strain isolated from infected DFU, the time required to kill 50% of the worms (LT50) was shorter than 2 days, whereas for all but one S. aureus strain isolated from uninfected (grade 1) ulcers, LT50 was longer than 3 days [47]. These encouraging results are currently being validated using another animal model, the zebrafish (personal data). In the latter, innate immune system is close to that of humans [50] and a model of infection by S. aureus has already been established [51]. The transparency of the embryos and use of fluorescent bacteria make it possible to follow infections in real time.

Bacterial interactions: the role of biofilms

Bacterial interactions are currently considered important in many infectious diseases, including acute and chronic wounds [52]. Bacteria may exist as planktonic or free-floating cells or sessile attached cells, contributing to form biofilms. According to Donlan and Costerton [53], biofilm is a (polymicrobial) sessile community characterized by cells that are irreversibly attached to a substratum or interface or to each other, and these cells are encased in a matrix of EPSs that they have produced. These cells exhibit an altered phenotype with respect to growth rate and gene transcription. Attachment of ‘pioneering’ bacteria is the first step towards biofilm development. Then, microbial cells begin to proliferate, forming small aggregates or microcolonies, and other bacterial colonizers coadhere within the biofilm under construction. This step generates chemical signals (auto-inducers), allowing cell-to-cell communication essential for biofilm formation, including EPS production, synergistic or antagonist interactions between cells and changes in bacterial phenotypic characteristics. This communication by auto-inducers is known as quorum sensing and gives information about cell density. So, when the number of cells has reached a critical threshold (the quorum), bacteria start to produce EPS and some genes get upregulated. In the mature biofilm, bacteria are embedded in EPS matrix and form three-dimensional mushroom-like structures containing water channels. Dispersal of bacterial cells from biofilm can be a passive phenomenon, such as erosion by fluid shear or an active one, known as ‘seeding dispersal’, and secondary to quorum-sensing-mediated death and lysis of cell subpopulation within the biofilm [52-54]. Biofilms are claimed to be involved in a number of infectious diseases such as endocarditis, prostatitis, periodontitis, otitis media as well as in infection from medical devices. Biofilms have been found in osteomyelitic bone [55], a common complication of DFU [56]. More recently, James et al. [57], using light and scanning electron microscopy, reported that 60% of chronic wounds exhibited biofilms as opposed to 6% of acute wounds; of note, 13 of 50 chronic wounds that the authors analyzed were DFUs and 10 (77%) contained biofilm. According to Potera [58], 65% of human infections involve biofilms, but the presence of biofilms is not necessarily detrimental, occurring naturally in regions of the human body such as teeth, gastrointestinal tract or vaginal mucosa, and might be even protective against infection [52, 59].

Clinical consequences of biofilm are important. First, sessile bacteria show increased resistance to antibiotic agents. Thus, it was reported that the minimal inhibitory concentration and minimal bactericidal may be up to 100- to 1000-fold higher for the sessile form of the bacteria compared with their planktonic counterparts [60]. Several mechanisms are put forward to account for this resistance [53, 54, 59]: EPS might constitute a chemical and/or physical barrier against penetration of some antibiotics. Modification of the micro-environment may also alter efficacy of antibiotic agents. Proximity of cells favours horizontal gene transfers and hence transfers of resistance gene. Sessile cells might also have upregulation of resistance gene. Because the biofilm is polymicrobial, antibiotic sensitivity is heterogeneous, with some species being susceptible to a given agent but others not. Finally, because biofilm-associated cells grow more slowly than planktonic cells, it has been proposed that they take up antibiotics more slowly. This phenomenon is more evident for the cells at the base of the biofilm, known as persister cells. As mentioned earlier, sessile bacteria are more virulent than their planktonic counterpart because of upregulation of genes encoding for virulence factors and horizontal gene transfer between bacteria. Another consequence of biofilm formation is resistance of bacteria to the immune system possibly due to decreased penetration of phagocytes through the EPS and/or defective opsonization impeding engulfment of bacteria by phagocytic cells [54, 61]. As a whole, those three characteristics (resistance to antibiotic agents, expression of virulence factors and resistance to the host immune system) may explain the frequent severity, chronicity and recidivism of DFI; moreover, the detachment of cells from the biofilm may facilitate the spread of infection notably to the bone.

The fundamental question is why are some biofilms ‘healthy’ and others able to induce infection. Two hypotheses are currently under discussion [52]. The first, the ‘specific bacterial hypothesis’, suggests that only a few species of bacteria within the heterogeneous polymicrobial biofilm may be involved in the infectious process. Conversely, the ‘nonspecific bacterial hypothesis’ (or ‘community hypothesis’) considers the bacterial composition of biofilm as a whole such as ‘a functional unit’ and does not take account of individual pathogenic bacteria alone. Recently, the concept of FEPs was developed by Dowd et al. [62]. According to this hypothesis, certain bacterial species considered as nonpathogenic when alone or species not capable of maintaining a chronic infection on their own may co-aggregate symbiotically in a pathogenic biofilm and act synergically to cause a chronic infection. Preliminary data supporting this concept have been published based on new biomolecular techniques that allow to identify various clusters in chronic DFU made up of particular species. A nonrandom distribution pattern of bacteria existed in the wounds. For example, a quantitative analysis of the distance of bacterial aggregates to the wound surface showed that the aggregates of Pseudomonas aeruginosa were located significantly deeper in the wound bed than those of S. aureus[63]. This particular distribution of P. aeruginosa and S. aureus may explain the under-representation of P. aeruginosa and over-representation of S. aureus in chronic wounds by conventional culturing of wound swab samples. Anaerobes are present in most of FEPs, and distribution of the various pathogens in those multispecies biofilms is nonrandom; aerobic bacteria are localized in the upper surface where oxygen content is relatively high, whereas anaerobes are localized more deeply in niches created by oxygen consumption by aerobes. Biofilm and FEP concept may explain the delayed healing of chronic wounds, why cultures from chronically infected diabetic foot wounds are often polymicrobial and why anaerobes are frequently isolated when sampling, transport and culture methods are optimal. A complex sequence of events takes place during the formation of these biofilms, which includes random bacterial settlement of early colonizers, an increased competition among the present species and a niche differentiation resulting in very heterogeneous biofilms. This may also account for the resistance of some DFIs to a single antibiotic agent. In practice, the role of biofilms in DFI may encourage sharp debridement to remove biofilm bacteria and to obtain in-depth specimens for bacteriological culture. High prevalence of anaerobes in pathological biofilm requires using suitable culture media to isolate them. Because species traditionally considered as low virulent can be aggressive if they are part of an FEP, they should be viewed cautiously. Finally, techniques of molecular biology have to be developed to identify bacterial populations that are difficult to isolate by the usual culture techniques.

In conclusion, DFI is a complex pathology (Figure 2) involving to a varying extent the host and the bacteria. As a whole, this may explain why DFIs are frequent, often severe and difficult to treat. Finally, classic dogma such as Koch's postulates or the single-pathogen paradigm must be revisited in the light of new data from molecular biology.


This work was supported by grants from the French Society of Diabetes (ALFEDIAM grant 2008), Pfizer, the Languedoc-Roussillon Region (Chercheur d'avenir Grant 2009) and the National Institute of Health and Medical Research.

Conflict of interest

None declared.