Role of Bacteria in Pathogenesis of Lower Leg Ulcers

Abstract

Lower leg ulcer is a circumscribed necrosis of epidermis, skin, and occasionally also muscular fascia, tendon, or even underlying bone with sluggish granulation and delayed covering by keratinocytes migrating from the ulcer margins. Following skin microinjury the colonization of denuded surface by local skin and floating down perineal bacterial flora takes place. The predisposing factors are venous stasis with excess capillary filtrate, high tissue fluid pressure, erythrocyte extravasation, hemosiderosis and fibrosis, or ischemia in atherosclerosis and diabetes with decreased arterial supply of nutrients, or lymph stasis with excess tissue fluid, high tissue fluid pressure and fibrosis, or excessive fat deposition in the subcutis in pathological obesity with excess tissue fluid and high tissue fluid pressure. The common denominator, irrespective of predisposing factors, is colonization of the denuded surfaces by bacteria. Although bacteria may not necessarily be the primary etiological factor, they certainly are responsible for progression of ulcer or delayed healing.

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9.1 Introduction

Lower leg ulcer is a circumscribed necrosis of epidermis, skin, and occasionally also muscular fascia, tendon, or even underlying bone with sluggish granulation and delayed covering by keratinocytes migrating from the ulcer margins. Following skin microinjury the colonization of denuded surface by local skin and floating down perineal bacterial flora takes place. The predisposing factors are venous stasis with excess capillary filtrate, high tissue fluid pressure, erythrocyte extravasation, hemosiderosis and fibrosis, or ischemia in atherosclerosis and diabetes with decreased arterial supply of nutrients, or lymph stasis with excess tissue fluid, high tissue fluid pressure and fibrosis, or excessive fat deposition in the subcutis in pathological obesity with excess tissue fluid and high tissue fluid pressure. The common denominator, irrespective of predisposing factors, is colonization of the denuded surfaces by bacteria. Although bacteria may not necessarily be the primary etiological factor, they certainly are responsible for progression of ulcer or delayed healing.

9.2 Hypothesis

There are several questions to be answered before we can achieve a progress in healing of ulcers. They are as follows: (1) Why does ulcer occur more frequently in the calf and foot than in the upper limbs or elsewhere, (2) is it local microtrauma that initiates ulcer development, (3) why does a rapid colonization of ulcer by skin residential bacteria develop so fast, (4) do perineal bacteria contribute to the ulcer colonization, (5) do the normally saprophytic skin bacteria become virulent once they colonize the denuded surface, (6) why do the colonizing bacteria proliferate rapidly increasing the bacteria cell mass, (7) which bacterial strains dominate, (8) are there dormant bacteria in calf subcutaneous tissue (persisters), (9) does a local cellular memory to bacterial antigens exist stimulating immediate recruitment of granulocytes and macrophages, and (10) is there autoimmune reaction to own granulocyte and tissue debris and insufficient granulocyte autophagy of incorporated bacteria?

This chapter will be specifically devoted to the factors predisposing for bacterial colonization and subsequent proliferation of bacteria in lower leg venous, ischemic, diabetic, and lymphedema ulcers.

9.3 Lower Limb Skin Bacteria

Human skin has been considered to harbor a complex microbial ecosystem, with transient, short-term resident and long-term resident biota, based on the consistency with which they are isolated. Staphylococcus, Micrococcus, Corynebacterium, Brevibacteria, Propionibacteria, and Acinetobacter species are, among others, regularly cultivated from normal skin. Staphylococcus aureus, Streptococcus pyogenes, and Pseudomonas aeruginosa may be transient colonizers, especially in pathological conditions [1, 2] . These strains colonize any denuded skin surface of lower limbs, among them ulcers.

9.4 Venous Ulcers

Venous insufficiency of the lower limbs is characterized by venous blood hypertension in the upright position and subsequent development of chronic edema, with elevated tissue fluid pressure and its adverse effects on fibroblasts producing and depositing collagen. Skin becomes fibrotic. Even a minor injury may denude the surface and create port of entry for bacteria dwelling on the epidermis or in the sweat and sebaceous glands. The exact mechanism of formation of ulcer in limbs with venous insufficiency remains unclear. A large defect in skin and subcutis develops. This precludes healing by wound contraction and leaves space for the granulation tissue. However, it remains unclear why covering by the keratinocytes encroaching from the ulcer edge is so sluggish and often does not occur at all. Keratinocytes remain morphologically and functionally normal. They synthesize cytokines and chemokines necessary in the healing process [3] . Vascularization of granulation tissue is abundant. However, the granulation surface is covered by granulocytes and cellular debris. This may be the effect of bacterial colonization and chemoattraction of granulocytes lysing granulation cells. An autoimmune mechanism to own destructed cells maintaining the inflammatory process cannot be excluded.

9.4.1 Do Bacteria Colonizing Ulcers Originate From Foot, Calf, and Perineal Skin?

In order to answer the question whether foot and ulcer flora may be similar to that of perineum, we cultured skin smears (Tables 9.1 and 9.2) and compared bacterial phenotypes. Out of 17 toe web isolates, 13 were of the same phenotype as perineal isolates.

Table 9.1 Numerical prevalence of bacterial isolates from perineal skin (n = 15 patients)

Table 9.2 Numerical frequency of bacterial isolates from toe web skin (n = 12 patients)

Could bacteria be present in and around the varicose veins?

The exact etiology of varicose vein formation and development of ulcer is still full of questions. Are these two entities linked with each other? Could dormant persister bacteria in leg subcutaneous tissue be responsible for vein wall destruction and subsequently ulcer formation?

We tried to detect bacteria in the varicose veins and subcutis using two techniques. Biopsy material was homogenized and cultured in routine media, and in another method, it was placed on bacteriological culture plates and observed for 3 weeks. In this last technique, tissue environment for bacteria was preserved and plate contained erythrocytes (iron). This mimicked a normal tissue situation. In addition, bacterial 16sRNA was identified in specimens [4] .

9.4.2 Bacterial Isolates in Varicose Veins

In our studies, varicose veins specimen stage 4 (CEAP classification) revealed presence of bacterial isolates in 40 %, whereas controls taken from healthy cadaveric organ donors contained live bacteria in only 4 % (Table 9.3). Disinfected skin specimens from the sites of varicectomy showed presence of microbes in 4 %. The dominant isolates from vein specimens were Staphylococci, preponderantly coagulase negative; however, in a few cases Enterococcus faecium was also detected. Staphylococci were highly sensitive to antibiotics except of penicillin (Table 9.4). Thirty-three percent of isolates were methicillin resistant. The 16sRNA was detected in 69 % of specimens, evidently higher than the percentage of live bacterial cells.

Bacterial culture on the Hemoline plates revealed microbes migrating from the outer aspect of varices, adjacent fat but not muscles (Fig. 9.1).

Fig. 9.1

Fragments of tissue harvested from ischemic upper calf. (1) Bone marrow, (2) popliteal vein, (3) subcutaneous fat, (4) popliteal artery, (5) fat adjacent to artery, (6) skin bacteria migrate from subcutis. Confluent bacterial colonies of coagulase-negative Staphylococci formed around the specimens. Interestingly, bone marrow contained hemolytic bacteria

Table 9.3 Bacteriology of varices of great saphenous veins (GSV)

Table 9.4 Sensitivity to antibiotics of bacterial isolates from varicose fragments of the great saphenous vein

9.4.3 Bacterial Isolates on Ulcers

Bacterial phenotypes on ulcer exudate remain similar to those identified on adjacent skin; however, the numerical distribution of strains becomes different (Table 9.5). Strains of Gram-negative Bacilli dominate over others, and the number of colonies is tripled (Tables 9.6 and 9.7). This may be the result of more favorable environmental conditions on the granulation tissue for some strains or less favorable for others. Interestingly, bacteria cultured from the ulcer surface revealed increasing resistance to antibiotics compared with the flora taken from normal leg skin (Tables 9.8 and 9.9).

Table 9.5 Numerical frequency of bacterial isolates from varicose ulcer (n = 56 patients)

Table 9.6 Prevalence of bacterial strains isolated from leg varicose ulcers and calf skin surface of normal subjects

Table 9.7 Frequency of bacteria isolates on perineal skin, calf skin, toe web, and varicose ulcer (in %)

Table 9.8 Sensitivity to antibiotics of bacterial isolates from 18 varicose ulcers and leg skin of 30 normals

Table 9.9 Sensitivity to antibiotics of bacterial isolates from 18 leg varicose ulcers and calf skin of 30 normal controls

Our studies clearly showed that bacterial colonization and superimposed infections are common in venous leg ulcers and contribute to poor wound healing. Necrotic tissue is laden with bacteria, while devitalized tissue impairs the body’s ability to fight infection and serves as a pabulum for bacterial growth [5].

9.5 Can Bacterial Flora Be Eradicated or at Least Attenuated?

9.5.1 Systemic Antibiotics

A recent Cochrane Review of 22 randomized control trials of systemic and topical antibiotics and antiseptics for venous ulcer treatment found no evidence that routine use of oral antibiotics improves healing rates. No between-group differences were detected in terms of complete healing for comparisons: antibiotics given according to antibiogram versus usual care, ciprofloxacin versus standard care/placebo, trimethoprim versus placebo, ciprofloxacin versus trimethoprim, and amoxicillin versus topical povidone-iodine [6].

Oral antibiotics may be indicated in patients with venous ulcer and inflammation of the surrounding tissues. It should be treated with systemic Gram-positive bactericidal antibiotics.

9.5.2 Topical Antibiotics and Antiseptics

Topically applied antimicrobials can be effective. Cadexomer iodine: more participants were healed when given cadexomer iodine compared with standard care [6, 7]. No between-group differences in complete healing were detected when cadexomer iodine was compared with the following: hydrocolloid dressing, paraffin gauze dressing, dextranomer, and silver-impregnated dressings. Povidone iodine: no between-group differences in complete healing were detected when povidone-iodine was compared with the following: hydrocolloid, moist or foam dressings according to wound status, and growth factor. Silver-based preparations: no between-group differences in complete healing were detected when 1 % silver sulfadiazine ointment was compared with standard care/placebo and tripeptide copper complex, when different brands of silver-impregnated dressings were compared, or when silver-impregnated dressings were compared with non-antimicrobial dressings [6].

Other topical antibiotics: more ulcers healed at four weeks when treated with an enzymatic cleanser (a nonantibiotic preparation) compared with a chloramphenicol-containing ointment. No between-group differences in complete healing were detected for framycetin sulfate ointment versus enzymatic cleanser, chloramphenicol ointment versus framycetin sulfate ointment, mupirocin ointment versus vehicle, and topical antibiotics given according to antibiogram versus an herbal ointment [6] .

9.6 Arterial Ischemic Ulcer

Arterial ischemic ulcers develop in the calf or dorsum of the foot in cases with obstruction of large arteries after an incidental trauma, insect bite, and skin scratching. They should be differentiated from the diabetic ulcers by other location and lack of diabetes symptoms. They are usually painful with intensive peri-ulcer skin inflammation [8] . Restoration of flow is crucial to infection control in arterial ulcers and must be addressed first. However, the colonizing bacterial flora hampers healing.

We were trying to identify the source of bacteria in this type of ulcers. They could originate from the surface of adjacent skin and/or from the preexisting dormant perister forms in the deep soft tissues. In our studies we harvested, in nondiabetic patients, fragments of arteries, muscles, and lymphatics from lower limbs, amputated because of uncontrollable rest pain in multilevel arterial obstructions without peripheral necrosis [9]. In over 50 % arterial specimens contained live bacteria (Table 9.10).

9.6.1 Bacterial Isolates in Arterial Walls

In group I of 60 ischemic limbs specimens of tibial and popliteal arteries contained bacterial cells in 60.6 % and femoral arteries in 30.8 % In the healthy femoral arteries, microbial cells were isolated in 11 % (ischemic vs. controls, p <0.05). The Gram-positive bacteria were sensitive to all antibiotics but penicillin. Enterococcus was sensitive to vancomycin (Table 9.11).

Optical evaluation of colonies formed from migrating bacteria on Hemoline plates revealed dominance of Staphylococci (Fig. 9.2). They were present in arteries, muscles, and subcutis. Single colonies of highly pathogenic bacteria, as quoted above, could also be seen in some specimens. Interestingly, microbes were also present in the bone marrow.

Fig. 9.2

Fragments of varicose great saphenous vein and adjacent tissues. (1) Fat adhering to the vein, (2) varix placed with external wall on the plate, (3) muscle, (4) varix placed with intima on the plate. Confluent colonies of coagulase-negative Staphylococci formed around the specimens

9.6.2 Microbial DNA in Arterial Wall

In group I, out of 60 samples of tibial, popliteal, and femoral arteries, the 16 s RNA gene was detected in 70 %. In 25 normal femoral arteries revealed presence of 16sRNA in 21 %.

Table 9.10 Prevalence of various bacterial strains in arteries of the lower limbs in patients with critical lower bacteria limb ischemia undergoing amputation (no toe and foot necrosis) (n = 60)

Table 9.11 Sensitivity of Gram-negative bacterial isolates from femoral and popliteal artery wall to antibiotics in patients with acute leg ischemia undergoing reconstruction or amputation (in % of specimens) [9]

	Pseudomonas	Proteus mirabilis	Acinetobacter	Citrobacter
Amo/penicill. gr.a	0	50	0	0
Amox/clav. ac	0	75	0	0
Piper + tazobactam	75	100	50	–
Ticarcillin	75	50	0	0
Ctx/caphalo. 3 g	50	100	50	0
Ceftriaxone	0	100	50	–
Ceftazidime	0	100	50	–
Aztreonam	75	100	0	–
Imipenem	75	100	50	–
Ceftazidime 1	0	100	50	–
Cotrimoxazole	0	50	0	–
Tobramycin	75	50	50	–
Amikacin	100	100	50	–
Gentamicin	75	50	50	100
Netilmicin	75	50	50	100
Pef/quinolones 2 g	60	50	50	100
Ciprofloxacin	100	100		100

The bacterial strains detected in deep tissues of ischemic limbs could be the main source of microbes potentially colonizing totally ischemic regions and bring about formation of ulcer or even necrosis of limb fragments.

Systemic administration of antibiotics is indicated in cases with acute inflammatory changes. The duration of therapy with high doses should depend on the systemic symptoms. Subsequently, taking into account presence of bacteria in deep tissues, low doses should be given even for months. Topical administration of antibiotics has not been proved effective; however, antimicrobials can be applied as in the venous ulcers.

9.7 Diabetic Foot Ulcer

Ischemia in diabetics results from atherosclerosis of the leg vessels, often bilateral, multi-segmental, and distal, involving arteries below the knee. Considerable data support the observation that 10⁵ organisms/g of tissue are necessary for infection and to allow invasive sepsis for most types of bacteria [10–12] .

The immune system is impaired in diabetic patients, and we should be aware of factors that increase the risk of infection for neuro-ischemic ulcers including the long-term ulceration (>30 days), presence of loss of sensation, and comorbidities associated with immunosuppression, renal insufficiency, long-term steroid use, etc. [13].

Bacteria colonize the diabetic ulcers. They evoke a local host inflammatory reaction preventing healing. However, they may also cause inflammation of tissues surrounding the ulcer with systemic symptoms.

We studied the bacterial flora from the edges of diabetic ulcers or fistulae in Wagner’ stage IV (Table 9.12). In contrast to the venous ulcers, bacterial flora contained Gram-negative Bacilli and few Cocci. They revealed high resistance to most antibiotics (Tables 9.13 and 9.14).

Table 9.12 Bacterial isolates from the bottom of diabetic foot ulcer (n = 25pts)

Table 9.13 Sensitivity to antibiotics of Gram-negative bacteria from foot ulcer of diabetic patients (in%)

Table 9.14 Sensitivity to antibiotics of Gram-positive bacteria from foot ulcer of diabetic patients

Systemic treatment with antibiotics is mandatory in diabetic ulcer with acute inflammation of the ulcer surrounding tissues. The topical administration of antibiotics has not been proved effective. The other antimicrobial therapy remains the same as for the venous ulcer.

9.8 Lower Limb Lymphedema Ulcer

Ulcer development is one of the complications in chronic lymphedema. Although not frequent, it is a very serious condition [14] . Ulcers are usually formed in the lower parts of calf or dorsum of the foot. Denuded skin surface is colonized by skin flora. Oozing of lymph precludes covering of the surface by ulcer edge keratinocytes. In addition, microbes present in the lymphedematous tissues and lymph enhance the local host immune reaction [15]. Patients with obstructive lymphedema suffer from recurrent attacks of dermatolymphangioadenitis caused by bacteria present in a dormant state in the stagnant tissue fluid and lymph (Tables 9.15 and 9.16) (Fig. 9.3). This microflora is responsible for nonhealing of ulcers and poor healing after the debulking surgery. Interestingly, the detected bacteria are sensitive to most antibiotics (Tables 9.17 and 9.18).

Fig. 9.3

Lymph sample from lower limb during acute dermatolymphangioadenitis was spread on the plate. Multiple colonies of Staph. aureus and coagulase-negative Staphylococci, few Enterococci

Lymphatic ulcer should be treated by debulking of the fragment of ulcerated tissue. Systemic antibiotics should be given perioperatively and for as long as wound healing is not completed. Thereafter, long-term penicillin (Penidure) should be given in a dose of 1,200,000 u., i.m., every 3 weeks for 1 year or longer, to prevent recurrence of ulceration and dermatolymphangioadenitis attacks.

Table 9.15 Prevalence of bacterial isolates from specimens obtained from lower limb tissues, lymph and lymphatics, and nodes of patients with secondary lymphedema. In parentheses are values from 30 healthy volunteers

Table 9.16 Numerical prevalence of bacteria isolated from specimens obtained from lymphedematous legs

Table 9.17 Sensitivity to antibiotics of bacterial isolates from skin surface, surgical skin incision, lymph and lymph nodes in 54 European patients with lymphedema of lower limbs and 30 normal control

Table 9.18 Sensitivity to antibiotics of bacterial isolates from skin surface, surgical skin incisions, lymph and lymph nodes of 54 European patients with secondary lymphedema of the lower limbs and 30 normal controls