Venous leg ulcer healing time is increased with each subsequent bacterial strain identified in the ulcer. A retrospective study

Abstract

Objective

The aim of the study was to analyze bacterial flora in venous leg ulcers, empiric and targeted antibiotic therapy, and factors influencing healing time.

Materials and methods

Data from 30 patients with venous leg ulcers were retrospectively analyzed. Collected data included: sociodemographic data, wounds information, number of comorbidities, results of the microbiological examination, and empiric and targeted antibiotic therapy. To compare the empiric and targeted treatment in terms of their compatibility, the group of patients was divided into the four subgroups: NEA (no empiric antibiotics), NA (no antibiotics), ETA+ (compatibility of empiric and targeted antibiotic), ETA− (no compatibility of empiric and targeted antibiotic).

Results

The average ulcer healing time was 163.4 ± 97.1 (range 51.0 to 426.0) days and increased by 28 days with each additional bacterial strain in the ulcer (p = 0.041). Healing time did not differ between the four groups. Staphylococcus aureus and Pseudomonas aeruginosa were the most frequent bacteria. Amoxicillinium/acidum clavulanicum was the most common empirical antibiotic and amoxicillinium/acidum clavulanicum, and levofloxacinum was the most common targeted antibiotic.

Conclusions

Venous leg ulcer healing time was prolonged with each subsequent bacterial strain in the ulcer, and it was independent from systemic antibiotic therapy.

Keywords

Chronic venous insufficiency microbiology leg ulcers wound infection venous ulceration

Introduction

A venous leg ulcers (VLU) is a full-thickness skin defect that is usually located in the area of the medial malleolus and it results from chronic venous disorders (CVD).¹ CVD represent a variety of chronic venous insufficiencies (CVIs), with clinical manifestations ranging from edema to venous ulceration of the lower extremities.²

The risk factors for CVD include older age, family history, female sex, pregnancy, obesity, standing for a long time, deep or superficial vein thrombosis.³ The main pathophysiological mechanism of CVD is venous hypertension. Venous dilatation resulting from the hypertension damages the vein walls and venous valves, which could result in the development of venous reflux.⁴ A low physical activity level or sedentary lifestyle worsens these changes because of reduced muscular pumping activity in the lower extremities. Hemodynamic changes are transferred to microcirculation, which triggers local inflammatory and proteolytic processes that lead to impairing micro- and macro-circulatory flow.^2,3 Clinical manifestations of these changes include venous wall and valve remodeling, varicose formation, edema, and leg ulceration.^2,3 Leg ulcers are initially a superficial wound, which may become larger in the case of negligence.^4,5 Venous ulceration does not show a tendency towards spontaneous healing because of disturbed venous drainage.¹ The clinical description of CVD, according to the Clinical, Etiological, Anatomical, and Pathophysiological (CEAP) classiﬁcation system, ranges from 0 to 6.³ The term CVI refers to more advanced CVD, stages C3–C6.³ The clinical signs could be associated with symptoms such as pain, aching, cramps, or burning.⁶

Active leg ulcers are susceptible to microbial invasion. Bacterial infection could lead to delayed healing, cellulitis, enlargement of the ulcer size, and even systemic illness. Recognition and treatment of an infected venous leg ulcer is essential as part of the overall treatment that is recommended for patients with CVD.⁷ The initial method in for CVD treatment includes compression therapy with specialist bandages or ready-to-use compression products, such as pantyhose or stockings.^1,8 Multilayer compression therapy is the most common and recommended. In includes the following two types: two-layer compression composed of a protective pad and a short stretch bandage, and 4-layer compression composed of a protective pad, a crepe bandage, a compression bandage, and a fixing bandage.^1,9 Before using compression therapy, ankle-brachial index (ABI) is recommended to exclude lower limb ischemia.¹⁰ A group of experts from the European Wound Management Association (EWMA) developed guidelines for the local management of wounds, which recommends TIME (where T is tissue debridement; I is infection and inflammation control; M is moisture balance; and E is edges, epidermization, and stimulation) to treat chronic wounds. The TIME procedure comprises thorough wound cleaning, necrotic tissue debridement, wound disinfection and dressing, which will provide appropriate conditions for wound healing.¹ The Polish Wound Management Association (PWMA) guidelines for the presence of clinical signs of infection recommend collecting scrapings from the wound for microbiological testing, using antibacterial dressing, proper antiseptic, and antibiotic therapy.¹¹

Venous leg ulcers constitute a global health problem with a large socioeconomic impact. It is estimated that active and healed ulcers occur in approximately 2% of the population and are mostly observed in persons between 60 and 80 years of age.¹² The risk of ulceration increases with age.¹³ The duration of an ulceration exceeds 9 months in approximately 50% of patients and recurrences are observed in about two-thirds of patients with this condition.¹⁴ The direct cost of treating venous disease in the US is $3 billion annually.³

Objective

The aim of the study was microbiological analysis of venous leg ulcers and analysis of empiric and targeted antibiotic therapy, and to investigate the factors that influence healing time.

Materials and methods

Data from 30 patients with non-healing venous ulcers (VLUs), who attended the Wound Care Clinic (WCC, outpatients) in Warsaw, Poland between 2014 and 2018 were retrospectively analyzed. The inclusion criteria were as follows: unilateral and solitary active leg ulcer with local signs of infection, no antibiotic therapy at baseline, microbiological examination during treatment, and complete wound healing. Thirty patients met these criteria among 100 patients who were considered for the study. The etiology of venous ulcers was confirmed by physical examination, which showed the presence of venous insufficiency features (e.g. swelling of the lower limbs, brown skin lesions within the shin), which was confirmed by Doppler ultrasound. The ischemic etiology was excluded using the ABI. According to Wells Model the clinical probability of deep vein thrombosis in our patients was low.

In each patient included in the analysis, the material for microbiological examination (scrapings from soft tissues) was collected during the first visit, before starting empiric antibiotic therapy. The collection of the material was preceded by cleaning the wound from biofilm, thorough washing with physiological saline and drying with a sterile gauze pad. Wound scrapings were collected with a new, sterile surgical spoon into a sterile tube. All microbiological procedures were performed in the microbiological laboratory in collaboration with WCC. Various patient specimens were cultured according to standard microbiological procedures. Isolates were identified using a mass spectrometer (MALDI Biotyper). Susceptibility testing was performed using the Vitek 2 or disc diffusion method and the results were interpreted according to EUCAST (European Committee on Antimicrobial Susceptibility Testing) recommendations.¹⁵ To detect carbapenemase production, the combination disk test was performed and results were confirmed using the biochemical CarbaNP test.^16,17 Production of extended spectrum beta-lactamases (ESBLs) was examined using a modified double disc synergy test (DDST).¹⁸ All tests were recommended by EUCAST.¹⁹

All patients underwent the standard procedure of wound debridement and disinfection using antimicrobial liquids and topical antibacterial dressings and two- or four-layer compression therapy were applied. Surgical treatment of the veins was not applied during the ulcer treatment process.

Data collected included age, sex, wound duration (time between the development of the wound and the initiation of treatment at the WCC, i.e., less than 1 month, l–6 months, over 1 year), comorbidities (i.e. type II diabetes mellitus [DM], cardiac diseases, hypertension, venous thrombosis, atherosclerosis), were generalized to “number of comorbidities”. Due to the low number of individual diseases, separate analyses were not possible. The data also concerned the presence of obesity that was diagnosed using the body mass index classifications, the type and the number of bacterial strains in the ulcer, and empirical and targeted antibiotic therapy. Compatibility between empirical and targeted antibiotic was analyzed and the duration of antibiotic therapy and the duration of healing were determined.

To compare the empiric and targeted treatment in terms of their compatibility, the group of patients was divided into the following subgroups:

NEA (no empiric antibiotic), which is the group without empiric antibiotics but with targeted antibiotics;

NA (no antibiotic), which is the group that did not use empirical and targeted antibiotic or did not use targeted antibiotic but had empirical antibiotic;

ETA (empiric-targeted antibiotic), which is the group, in which empirical and targeted antibiotics were used, including:

ETA+ (compatibility of empiric and targeted antibiotic), which is the group in which the antibiotic compatibility occurred and

ETA− (no compatibility of empiric and targeted antibiotic), which is the group in which there was no compatibility of targeted or empirical antibiotics.

All patients no topical antibiotics were used.

Ethical considerations

The Ethics Committee at the Medical University of Warsaw, Poland approved the experimental protocol (No. AKBE/149/17). Written consent was obtained from the management of the WCC to use data from patients’ records. Patient data were fully anonymized.

Statistical analysis

The variables were analyzed using the following basic descriptive statistics: cardinality (N), arithmetic mean, median, minimum (min), maximum (max), lower (Q1) and upper (Q3) quartile, and standard deviation (SD). For categorical variables, rates and interest are presented, and Pearson’s correlation (r) coefficients were used.

For intergroup comparisons, the Mann–Whitney U-test (for two groups) and the Kruskal–Wallis test (for three or more groups) with the Dunn post-hoc test and the Holm correction for multiple testing were used. The value of significance was set at 0.05.

The bootstrap method power analysis was also performed.

Significant results are also presented in point or bar charts showing the mean with standard deviation (box and whiskers diagram).

The calculations were made using a statistical program R²⁰ (ver. 3.5.3) using the tidyverse²¹ and ggplot2²² packages.

Results

General information

The study group enrolled 23 women (76.7%) and seven men (23.3%). The average aged of the participants was 74.7 ± 9.8 (range 60.0 to 93.0) years. The ulcer was predominantly present for “over 1 year” (N = 17; 56.6%). Five patients (16.7%) were obese. The average number of comorbidities was 1.6 ± 1.4 (range 0 to 6).

Bacteriological analysis

The analysis involved of 30 scrapings from the ulcers, in which 62 bacterial strains were detected. The most common bacterial strain was Staphylococcus aureus and Pseudomonas aeruginosa. The results of the microbiological tests are shown in Table 1.

Table 1.

Types of bacterial strains detected in microbiological tests of ulcer scrapings from patients with VLUs (% of all strains, N = 62).

Bacteria	N	%
Staphylococcus aureus	18	29.0
Pseudomonas aeruginosa	11	17.7
Enterococcus faecalis	8	12.9
Escherichia coli	5	8.1
Proteus mirabilis	4	6.5
Enterobacter cloacae	2	3.2
Staphylococcus epidermidis	2	3.2
Citrobacter braakii, Citrobacter braakii, Citrobacter koseri, Klebsiella oxytoca, Klebsiella pneumoniae, Proteus hauser, Proteus vulgaris, Staphylococcus agalactiae, Staphylococcus haemolyticus, Staphylococcus lugduensis, Staphylococcus simulans, Streptococcus agalactiae, Streptococcus b-hem.gr.C	1, each	1.6, each

The most common combination of 2 bacteria strain were: Staphylococcus aureus and Pseudomonas aeruginosa (7 patient), Enterococcus faecalis and Pseudomonas aeruginosa (5 patient), Enterococcus faecalis and Staphylococcus aureus (5 patient), Enterococcus faecalis and Proteus mirabilis (3 patient). The most common combination of 3 bacteria strain was: Staphylococcus aureus, Pseudomonas aeruginosa and Enterococcus faecalis (2 patient).

In almost half of the cases, the patients had one bacteria strain (43.3%). In one case (3.3%), the presence of bacteria in the ulcer was excluded. The number of bacteria strains isolated from each wound in 5 of cases (16.7%) was two, in 6 of cases (20.0%) was three, in four of cases (13.3%) was four, and in one of cases (3.3%) was five.

Analysis of antibiotic therapy

The most commonly used antibiotic was amoxicillinium/acidum clavulanicum (35.9% of the total; 50.0% of empirical antibiotics and 31.0% of targeted antibiotics). Both empirical and targeted antibiotics were administered per os. The antibiotic doses (recommended in SPS – Summary of Product Characteristics) are selected individually for each case, taking into account the severity of the infection, age, weight and renal function of the patient and after consultation with a clinical pharmacologist. Antibiotics used as empirical and targeted therapy are show in Table 2.

Table 2.

Summary of antibiotic use (% of all antibiotics for subgroup, N = 10 for empiric, N = 29 for targeted).

Type	Name	N	%
Empiric antibiotic	Amoxicillinium/Acidum clavulanicum	5	50.0
	Clindamycinum	2	20.0
	Levofloxacinum	2	20.0
	Ciprofloxacinum	1	10.0
Targeted antibiotic	Amoxicillinium/Acidum clavulanicum	9	31.0
	Levofloxacinum	7	24.1
	Ciprofloxacinum	6	20.7
	Clindamycinum	3	10.3
	Ampicillinum/Sulbactam	1	3.4
	Cefalexinum	1	3.4
	Doxycyclinum	1	3.4
	Tobramycinum	1	3.4

The empiric antibiotics were used in monotherapy, but in five cases of targeted therapy, a combination of antibiotics were used. This combinations were: amoxicillinium/acidum clavulanicum and levofloxacinum, amoxicillinium/acidum clavulanicum and ciprofloxacinum, amoxicillinium/acidum clavulanicum and doxycyclinum, ampicillinum/sulbactam and ciprofloxacinum and the last ciprofloxacinum and clindamycinum.

Fifteen patients had no empiric antibiotic therapy, and in the group with antibiotic therapy, compatibility between empirical and targeted therapy was observed in four cases (Table 3).

Table 3.

Antibiotic therapy use subgroup results.

Group	N	%
NEA	15	50.0
NA	6	20.0
ETA−	5	16.7
ETA+	4	13.3

ETA+, compatibility of empiric-targeted antibiotic; ETA−, non-compatibility of empiric-targeted antibiotic; NEA, no empiric antibiotic; NA, no antibiotic, patients who had neither empiric or targeted antibiotic therapy (5), or had empiric antibiotic therapy, but did not have targeted antibiotic therapy (1).

Analysis of factors associated with healing time

The average ulcers healing time was 163.4 ± 97.1 (range 51.0 to 426.0) days. There were no differences in healing time duration between the ETA+, ETA−, and NEA groups, as shown in Table 4.

Table 4.

Comparison of individual groups of antibiotic compatibility in terms of healing time.

Parameter	ETA	ETA+	ETA−	NEA	Test	p-Value
Healing time (days)					Kruskal–Wallis	0.862
N	9	4	5	15
mean (SD)	142.4 (54.5)	130.2 (66.7)	152.2 (48.3)	171.7 (106.4)
median (min–max)	137.0 (51.0–223.0)	128.0 (51.0–214.0)	146.0 (90.0–223.0)	123.0 (61.0–424.0)
Q1–Q3	123.0–165.0	105.0–153.2	137.0–165.0	97.0–198.0

ETA, empiric-targeted antibiotic together; ETA+, compatibility of empiric-targeted antibiotic; ETA−, non-compatibility of empiric-targeted antibiotic; NEA, no empiric antibiotic; SD, standard deviation.

No significant relationship was found between wound healing time and other parameters, which is shown in Table 5.

Table 5.

Correlation between healing time and sex, presence of diabetes mellitus (DM), obesity, and wound duration (time between the development of the wound and the initiation of treatment at the WCC).

Parameter	Level	N	Mean (SD)	Median (min–max)	Q1–Q3	Test	p-Value
Sex	F	23	167.4 (105.9)	133.0 (51.0–426.0)	97.0–199.0	Mann–Whitney U-test	0.844
	M	7	150.1 (64.5)	165.0 (84.0–257.0)	91.0–181.5
DM	no	21	167.8 (91.5)	146.0 (61.0–426.0)	100.0–186.0	Mann–Whitney U-test	0.428
	yes	9	153.1 (114.4)	100.0 (51.0–424.0)	92.0–183.0
Obesity	no	25	154.6 (90.1)	133.0 (51.0–426.0)	92.0–184.0	Mann–Whitney U-test	0.303
	yes	5	207.2 (129.7)	183.0 (100.0–424.0)	117.0–212.0
Wound duration	less than 1 month	8	130.9 (68.2)	112.5 (51.0–223.0)	82.8–190.8	Kruskal–Wallis	0.588
	less 6 months	5	141.4 (54.8)	123.0 (92.0–212.0)	94.0–186.0
	over 1 year	17	185.1 (114.6)	146.0 (84.0–426.0)	100.0–184.0

Note: There was no significant correlation between these parameters and healing time.

Pearson's correlation analysis showed no significant relationships between healing time and age (p = 0.622) and between healing time and the number of comorbidities (p = 0.846).

The small groups is the limitation of the study, what is indicated in the appropriate section in the manuscript. The power analysis was performed using bootstrap method. For all tests power vary from 0,05 to 0,13.

A multifactor analysis for healing time

Multifactorial analysis was performed to evaluate the impact of the following variables: sex, age, number of comorbidities, obesity, ETA compatibility group, and number of bacteria in culture on healing time. These results showed that only the number of bacterial strains had an effect on healing time. Healing time increased by approximately 28.4 days with each subsequent bacterial strain (p = 0.041). However, attention should be paid to the poor fit of the model (explains about 11% of variation), as shown Figure 1.

Figure 1.

Healing time (days) and number of bacterial strains.

Discussion

A chronic wound is a skin lesion that develops as a result of a disease process or injury and that persisted for 4–6 weeks despite treatment.¹³ Chronic wounds constitute a challenge for physicians and nurses, but they are most problematic for the patient, mainly because the chronic wounds lower their quality of life.²³ Thus, the healing time is important. Our study showed that there is no influence of such factors as age, gender, obesity, diabetes mellitus, number of comorbidities, and wound duration on wound healing time. There are discrepancies in the data regarding healing time. Skene et al. revealed that the healing time was shorter among younger patients without signs of deep vein thrombosis, with smaller initial ulcer area and shorter duration of the ulcer.²⁴ The group that was analyzed in our study was selected from all patients with lower leg ulceration, and it consisted of patients with the topical signs of infection, which could influence the healing time. Healing time also depends on many factors, which were not analyzed in this study, including the existence of risk factors such as tobacco smoking, sedentary lifestyle, a history of thrombosis, lower extremity injury, or genetic predisposition.^8,25

It is assumed that bacteria are present in all chronic wounds. However, it does not mean an infection.¹¹ Studies confirmed the presence of biofilm-forming microorganisms in chronic wounds.²⁶ During biofilm production, most of the normal bacteria become pathogenic because of horizontal gene transfer from pathogenic bacteria.²⁷ The results of our investigation revealed that the ulcer healing time depends on the number of bacterial strains in the wound. The presence of each subsequent strain increased the healing time by about 28 days. Most chronic wounds contain more than one bacterial species and produce a synergetic effect that results in a previously nonvirulent bacterial species becoming virulent. The prolongation of healing time could also be connected with bacterial load in the wound, and it was already shown that the bacterial load, which forms biofilms and shows high-level resistance toward antibiotics, contributes to a delay in healing.²⁷ In a mature biofilm, bacteria grow slowly because of nutrient deficiencies, which causes bacterial resistance to antibiotics.²⁷ Bacteria in the biofilm are able to penetrate deep into the tissues, so taking a swab only from the wound surface may give false results and not reveal the real cause of the infection.²⁶ That is why it is so important to properly collect material for microbiological tests to reveal the total amount of bacterial strains in the wound, which will help to determine the optimal treatment including local treatment and/or the possible use of a systemic antibiotic.

No drugs are currently approved by the US Food and Drug Administration for the treatment of chronic venous insufficiency.²⁸ Antibiotics are recommended for an infected ulcer, but current evidence does not support that systemic antibiotics are associated with improved healing of venous ulcers.^28,29 Our analysis also showed that the healing time was independent of antibiotic therapy. Even compatibility with empiric and targeted therapy did not shorten the healing time. For chronic wounds, topical treatment is of key importance in the treatment process and if there are no general symptoms of wound infection and the only critical colonization is found to impede healing, systemic antibiotic therapy is not recommended because it does not reduce the number of bacteria in the granulation tissue.³⁰ Systemic antibiotic therapy usually supports topical biofilm removal and prevents bacteria from penetrating into healthy tissue and the bloodstream,²⁶ but it should be rationally used. According to our results, if the clinical state of patient allows it, empiric antibiotic therapy should be applied with caution, and targeted therapy, based on the susceptibility tests, is recommended. According to the National Antibiotic Protection Program in Poland, when general antibiotic therapy is clinically justified, it is recommended to use agents with a long-term effect and in high doses, such as for bone inflammations.³⁰ The duration of antibiotic therapy for soft tissue infection has not been strictly determined, and the most frequent duration is 7 days for mild infection, 10–14 days if there are symptoms of general infection, and 6–12 weeks when osteomyelitis is present.³⁰

Our study showed that the most common bacterial strains in VLU were S. aureus and P. aeruginosa. The results are consistent with Lim et al.³¹ in Australia who investigated a group of 39 patients with lower leg ulcerations and with Tzaneva et al.³² who performed a study in Bulgaria. A study conducted in the Danish population also demonstrated that S. aureus (93.5%) and P. aeruginosa (52.2%) occurred most frequently in wounds.³³

Our study showed that amoxicillinium/acidum clavulanicum was most commonly used. Similar results were presented by Tzaneva et al.³² from Bulgaria after studying a group of 110 patients with chronic wounds with a venous etiology.³⁴ The study also confirmed that S. aureus and P. aeruginosa, which were the most common causes of venous ulcer infection, are sensitive to amoxicillinium/acidum clavulanicum.^34,35

Limitations

Because of the retrospective nature of the study, we analyzed only selected factors that might influence the healing time. We did not take into account pressure therapy and local therapy introduced to our patients or the existence of the risk factors that were mentioned above due to lack of data in the documentation. The number of patients included in the study was low, and therefore, this research should be continued in a larger study group.

Conclusion

The healing time for venous leg ulcers depends on the number of bacterial strains in the wound. The presence of each subsequent strain increased the healing time by about 28 days. The healing time was independent of systemic antibiotic therapy. Because compatibility of empiric and targeted therapy did not shorten the healing time, empiric antibiotic therapy should be applied with caution, and targeted therapy, based on the susceptibility tests, is recommended among patients with clinical indications for systemic antibiotic therapy.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

Ethical approval

The ethics committee of Medical University of Warsaw, Poland approved this study (No. AKBE/149/17).

Guarantor

KK.

Contributorship

KK and KWG researched literature and conceived the study. KK collected data and wrote first draft manuscript. KGW and BCP presented the methodology, data analysis and prepared manuscript for review and editing. All authors reviewed and edited the manuscript and approved the final version of the manuscript.

ORCID iD

Karolina Kruszewska

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