Microbiological Features and Risk Factors in Patients With Diabetic Foot Ulcers

Introduction. Approximately 25% of people with diabetes will experience diabetic foot ulcers (DFUs) during their lifetime. The present study was designed to evaluate the prevalence of diabetic foot infection among patients with DFUs and to identify the microorganisms isolated from the DFUs. This study also aimed to determine predisposing factors contributing to these infections at the authors’ wound care clinic at the Rajaii and Velayat University Hospitals in Qazvin, Iran. Methods. This cross-sectional descriptive study included 500 patients with diabetes mellitus between May 2011 and April 2013 at the authors’ wound care clinic in Qazvin, Iran. All demographic, clinical, and laboratory data such as age, sex, duration of diabetes (years), duration of DFUs before inclusion in the study, size of ulcer (cm²), glycosylated hemoglobin (HbA1c), and results of ulcer culture were collected for each case. The collected data were analyzed using SPSS software (SPSS Inc, Chicago, IL). A P value < 0.05 was considered significant. Results. Overall, 54 (11%) patients with infected ulcers were found in the present study. Among patients with infected DFUs, the most commonly isolated microorganism was Pseudomonas aeruginosa, which infected 35% of patients, followed by Staphylococcus aureus in 19% of patients, and methicillin-resistant S. aureus in 6% of patients. Additionally, in the multivariable logistic regression model, age > 65 years, ulcer size > 2 cm², and HbA1c > 7% were associated with the occurrence of an infection in DFUs. Conclusion. Periodic examination of the feet of patients with diabetes is necessary. In addition, to decrease the prevalence of infected DFUs, more attention to the described risk factors is recommended.

Diabetic foot ulcers (DFUs) are chronic wounds that can have a marked long-term influence on quality of life.^1,2 Approximately 25% of people with diabetes will experience a DFU during their lifetime.³ This prevalence of DFUs also has a major economic impact. Based on a European Study Group on Diabetes and the Lower Extremity (EURODIALE) study, total direct and indirect costs associated with treatment of DFUs may be as high as 10 billion euros per year.⁴ Without early diagnosing and optimal intervention, DFUs can rapidly become infected, leading to serious complications. Diabetic foot infection is defined as the disease caused by a microbial pathogen that occurs when the presence of replicating organisms is associated with tissue damage.⁵ Infection is defined as the presence of multiplying bacteria in body tissues, resulting in spreading cellular injury due to competitive metabolism, toxins, intracellular replication, or antigen-antibody response.⁶ Diabetic foot ulcer treatment is difficult because microvascular circulation in patients with diabetes is weakened, which limits the phagocytic cells, granulocyte adherence, chemotaxis, and phagocytosis in the infected area. Moreover, bacterial resistance worsens outcomes because empiric therapy may be ineffective, delaying appropriate antibiotics.

The present study was designed to evaluate the prevalence of diabetic foot infection among patients with DFUs, and to identify the microorganisms isolated from the DFUs. This study also aimed to determine predisposing factors contributing to these infections.

Study protocol. This cross-sectional descriptive study included 500 patients with diabetes mellitus between May 2011 and April 2013 at Rajaii and Velayat University Hospitals, Qazvin, Iran. Patients with DFUs who were > 18 years were eligible for inclusion. During primary examination, it was noted 61 of the 500 patients were > 18 years and had DFUs, so other patients were excluded from this study. A questionnaire that included demographic, clinical, and laboratory data such as age, sex, duration of diabetes (years), duration of DFUs before inclusion in study, size of ulcer (cm²) and glycosylated hemoglobin (HbA1c) was completed for each case.

Sampling techniques. For each patient, the DFU and surrounding skin area were cleaned using swabs soaked in 70% ethanol alcohol. Two sterile dry cotton wool swabs were used to collect samples of purulent secretions from the DFUs.

Each wound surface was rubbed with the swabs after they were moistened with sterile physiological saline. One of the swabs was then placed into a sterile bottle containing cooked meat broth medium and labeled with the date and time of collection, patient’s name, and number of sample. Samples were incubated for 24-48 hours at 35^oC-37^oC and inspected daily for growth. The second swab was placed into a sterile plastic container, labeled with the date and time of collection, patient’s name, and number of sample. That specimen was sent to the the authors’ private laboratory with the patient’s request form. Specimens were cultivated as soon as possible (after 24-48 hours). These sampling techniques were modeled after the work of Slater and colleagues⁷ and Cheesebrough.⁸

Culturing and isolation of the specimens. If growth was found on the first specimen in the cooked meat broth medium, it was inoculated onto a Neomycin Anaerobic Blood Agar Plate (Sigma-Aldrich, Singapore) for the isolation of anaerobic bacteria such as Clostridium perfringens and Bacteroides fragilis. Additional blood agar plates were inoculated for the isolation of other anaerobic bacteria, then incubated in an anaerobic jar for 48 hours at 35^oC-37^oC and inspected daily for growth. The second specimen was cultivated after 24-48 hours onto a blood agar plate for the isolation of gram-positive and gram-negative bacteria and onto a MacConkey agar plate for the isolation of gram-negative bacteria and incubated aerobic bacteria for 24-48 hours at 35^oC-37^oC. (The second specimen was cultivated onto 2 separate plates.) Microorganisms isolated from DFUs may belong to almost any group or species. The following steps, adapted from the work of Frykberg⁹ and Vandepitte¹⁰ will provide a framework to identify any bacterial species.

Ethical approval. The study obtained approval by the ethics committee of the Qazvin University of Medical Sciences. The protocols used conform to the ethical guidelines of the 1975 Helsinki Declaration.

Statistical Analysis. The collected data were analyzed using SPSS software (version 16.0, SPSS Inc, Chicago, IL). Student’s t test, chi-square, or Fisher’s exact test were used, where appropriate, for comparing clinical data between groups. Demographic characteristics, medical data, and laboratory test data were subjected to a multivariate analysis. Multivariate analyses were performed using logistic regression model to test for an independent association with the DFUs. Continuous data are presented as mean ± standard deviation. A P value < 0.05 was considered significant.

Although 500 patients with diabetes were evaluated in this study, only 61 patients with DFUs (53 Cases with more than 1 ulcer and 5 cases with 1 ulcer) were found during the primary examination. Of these patients, 41 (67%) were male and 20 (33%) were female. The mean age was 60.6 + 5.4 years (range 28-81 years). Demographic data of the included patients is summarized in Table 1. 

A total of 54 patients had infected DFUs, while 7 patients had noninfected DFUs. From the 54 infected ulcers, 68 pathogenic microorganisms were isolated. The most commonly isolated microorganisms were the gram-negative bacteria in 45 (66%) isolates, followed by gram-positive bacteria in 21 (31%); the lowest number was found in yeast in 2 (3%) isolates. The microorganisms isolated from the DFUs are presented in Table 2. 

The comparison of demographic and clinical parameters in this study showed the majority of patients with infected ulcers were male, and that age, duration of diabetes, size of ulcer, HbA1c, and osteomyelitis were significantly higher in patients with infected ulcers (Table 3). 

In the multivariable logistic regression model, age > 65 years (OR = 1.242, 95% CI = 1.251-2.114; P = 0.001), size of ulcer > 2 cm² (OR = 1.641, 95% CI = 1.109-2.551; P = 0.041), and HbA1c > 7% (OR = 2.155, 95% CI = 1.287-2.946; P = 0.032) were associated with occurrence of an infection in DFUs (Table 4). 

Infected DFUs are normally caused by microorganisms. Late onset diagnosis and weak response to antibiotic treatment is due to the high numbers of microorganisms colonized in the foot. The general objective of this study was to detect pathogenic microorganisms in patients with infected DFUs. Overall, 54 out of 500 (11%) patients with infected ulcers were found in the present study. In this study, bacteria and fungi that cause diabetic foot infection were studied and the results showed that all patients with infected DFUs had 1 or more pathogenic microorganisms. Similar findings were also reported by El-Tahawy,¹¹ Abdulrazak and colleagues,¹² and Sharma and coauthors.¹³ The main reasons for these infections were hyperglycemia and foot complications.

The common microorganisms that were isolated from the diabetic foot infections were P. aeruginosa (35%), S. aureus (19%), E. coli (16%), Klebsiella spp. (6%), methicillin-resistant Staphylococcus aureus (MRSA) (6%), and Candida albicans (3%).

These results showed gram-negative bacilli were the dominant pathogens found in 66% of DFU cases, while gram-positive bacteria were isolated from 31% of the cases and yeasts isolated from 3% of the cases (Table 2). These rates of isolated pathogens were comparable to previous studies.^11-13

The increase in ratio of gram-negative bacteria in this study may be because patients were immunocompromised and highly susceptible to hospital-acquired infections, either after colonization with environmental strains or following invasive surgery, and the bacteria exerted pathogenic effects by producing endotoxin.¹⁴

Patients who had received prolonged, inappropriate, or broad-spectrum antibiotics, or who had long hospital stays, chronic wounds, or surgical procedures were more likely to have infected wounds and bacterial colonizations. S. aureus infections can develop due to exposure of contaminated hands of caregivers rather than to the overuse of antibiotics.¹⁵ On the other hand, a variety of virulence factors, including lipopolysaccharide, exotoxin A, leukocidin, extracellular slime, proteases, phospholipase, and several other enzymes make P. aeruginosa the most clinically significant bacteria among patients with DFUs.¹⁶

The comparison of demographic and clinical parameters in this study showed males had significantly more infected ulcers than females. These findings are consistent with the previous study by Kajetan and colleagues.¹⁷

Diabetes mellitus leads to impaired immunologic defense.¹⁸ In addition, older age may create immunosuppressive conditions. This combination may lead to an increased risk for infected DFUs. A longer duration of diabetes may have a deteriorating effect on an infected DFUs. Size of ulcer is one of the main elements of length of ulcer healing; large ulcer size may be indicative of a long period of healing.

The most widely used clinical test to estimate blood glucose control is measurement of HbA1c, which reflects mean blood glucose over the entire 120-day life span of the red blood cell. It correlates best with the patient’s mean blood glucose over the previous 8 to 12 weeks. A target HbA1c should be determined individually, but a level close to 7% seems to be acceptable for the majority of people with type 2 diabetes.¹⁹ Good glycemic control reduces the risk of microvascular complication with type 2 diabetes.²⁰

The multivariable logistic regression model in this study showed an HbA1c >7% is a major risk factor for the development of infected DFUs, demonstrating the relationship between microvascular complication and HbA1c > 7%.

Overall, 54 (11%) out of 500 patients with infected ulcers were found in the present study. The most commonly isolated microorganisms among patients with infected DFUs were P. aeruginosa (35%), S. aureus (19%), and MRSA (6%).This study also showed the number of males, age, duration of diabetes, size of ulcer, HbA1c, and osteomyelitis were all significantly higher in patients with infected ulcers. In the multivariable logistic regression model, age > 65 years, size of ulcer > 2 cm², and HbA1c > 7% were associated with occurrence of an infection in DFUs. The authors suggest more attention to these risk factors in patients with diabetes.

Affiliations: ¹Department of Vascular Surgery, Qazvin University of Medical Sciences, Qazvin, Iran; and ²Department of Pathology, Qazvin University of Medical Sciences, Qazvin, Iran

Correspondence:
Simin Samani, MD
Assistant Professor of Pathology
Qazvin University of Medical Sciences
Clinical Research Develop Unit
Qazvin, Iran
researcheran@gmail.com

Disclosure: The authors disclose no financial or other conflicts of interest.