Profile and Antibiotic Susceptibility of Bacterial Pathogens Associated With Diabetic Foot Ulcers From a Rural Area

Objective. This cross-sectional study assesses the profile and antibiotic susceptibility of aerobic bacterial pathogens associated with diabetic foot ulcers (DFUs). Materials and Methods. Two swab samples from 140 DFUs with various Wagner grades were processed for identification using routine culture methods and antimicrobial susceptibility by Kirby-Bauer disc diffusion method. Results. A total of 125 (89.29%) samples were found to be positive for bacteria on culture. A higher incidence of positive culture (94.32%) was found in individuals with a blood sugar level > 200 mg/dL. The highest number of culture-positive cases was observed in Wagner grade 2 DFUs (45%). Overall infection was monomicrobial in 83.20% (104) and polymicrobial in 16.80% (21) of samples. Staphylococcus aureus (21.09%) and Pseudomonas aeruginosa (19.05%) were the most common isolates. Linezolid (100%) and imipenem (75.70%) were the most effective antimicrobial agents against gram-positive and gram-negative isolates, respectively. Conclusions. The results show an overall increase in bacterial resistance to antimicrobial agents and emphasize the importance of an antimicrobial susceptibility pattern in the selection of appropriate antibiotic(s) to institute the rational antibiotic therapy. 

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Diabetes mellitus is a chronic disorder affecting a large portion of the global population.The global burden of diabetes is projected to increase from 246 million people to more than 380 million by the year 2025.¹ Diabetic foot is one of the most notable complications. About 15% of individuals with diabetes mellitus are prone to develop an ulcer, and between 10% to 30% of patients with diabetic foot ulcers (DFUs) will eventually progress to an amputation.¹ If not treated in a timely manner, a diabetic foot may progress to osteomyelitis or gangrene, which can lead to septicemia, amputation, and death.¹ Further, the risk of ulceration in the feet of individuals with diabetes from rural areas is increased; less education, low socioeconomic status, foot moisture changes, barefoot walking, and lack of adequate facilities for diabetes care are important contributing factors.^2,3 

Infections in individuals with diabetes are difficult to manage because of impaired microvascular circulation, which limits phagocytic cell and antibiotic concentration in the infected area. Optimal management of diabetic foot infections  can potentially reduce the incidence of infection-related morbidities, duration of hospitalization, incidence of major limb amputation, and the cost of management.⁴ Proper bacterial identification and appropriate antibiotic therapy are essential for a successful outcome.⁴

Diabetic foot infections are often mismanaged particularly in regard to antibiotics. The wide-spread, indiscriminate, and haphazard use of antibiotics precipitate the emergence of multidrug-resistant bacteria strains. Hence, a detailed knowledge of causal organisms and their susceptibility patterns play a key role in the management of diabetic foot infections.⁵

The present study assesses the profile and antibiotic susceptibility of aerobic bacterial pathogens associated with DFUs. 

In this cross-sectional study, 140 confirmed cases of DFUs of various Wagner grades belonging to different age groups and both sexes were included. Exclusion criteria consisted of patients currently on antibiotic treatment or Wagner grade 0 DFUs.

Two swabs were collected from the lowest depth of the ulcer. One swab was used for preparation of smear for Gram staining and the other for aerobic bacteria isolation. The specimens were processed for direct microscopy and isolation of causal organisms by using 5% sheep blood agar and MacConkey agar. The growth was studied after overnight incubation at 37°C, and each colony was processed further for identification using standard procedures.⁶ The antimicrobial susceptibility of each isolate was performed by Kirby-Bauer disc diffusion technique.⁷ For susceptibility testing of gram-positive bacteria, the following were used: penicillin (10 U), cefoxitin (30 µg), erythromycin (15 µg), clindamycin (2 µg), cotrimoxazole (25 µg), tetracycline (30 µg), ciprofloxacin (5 µg), levofloxacin (5 µg), ofloxacin (5 µg), gentamicin (10 µg), linezolid (30 µg), and vancomycin (30 µg). However, for gram-negative bacteria, amikacin (30 µg), gentamicin (10 µg), cefepime (30 µg), ceftriaxone (30 µg), ceftazidime (30 µg), ciprofloxacin (5 µg), cotrimoxazole (25 µg), levofloxacin (5 µg), tetracycline (30 µg), piperacillin/tazobactam (10 µg), meropenem (10 µg), and imipenem (10 µg) were used as per Clinical and Laboratory Standards Institute guidelines.⁸

Statistical analysis
Chi-square test was performed. 

Ethics 
The study protocol was approved by the Ethics Committee of the Maharashtra Institute of Medical Sciences & Research Medical College, Latur, India. Written informed consent was obtained from all patients.

Of the 140 patients studied, the highest number of DFUs were observed in the group of patients aged 61 to 70 years (45, 32.14%) followed by those of 51 to 60 years (37, 26.43%). Mean patient age was 59.72 ± 11.75 years. Men were more commonly affected (112, 80.00%), and the sex ratio (M:F) was 4:1 (Table 1). The number of DFUs in each Wagner grade classification were as follows: grade 2 (63 cases, 45.00%), grade 3 (32 cases, 22.86%), grade 1 (24 cases, 17.14%), grade 4 (13 cases, 9.29%), and grade 5 (8 cases, 5.71%).

A total of 125 (89.29%) samples were positive on culture while 15 (10.71%) were sterile on culture. Of the 88 patients who had a blood sugar level (BSL) > 200 mg/dL, 83 showed a greater number of culture-positive samples (94.32%) than the 52 cases with a BSL ≤ 200 mg/dL showing culture positivity in 42 cases (80.77%). The difference between the culture-sterile and culture-positive samples among the 2 BSL groups was statistically significant (P < .05). Patients with a BSL > 200 mg/dL had a greater number of isolates (100; 1.20 isolates/case) than patients with a BSL ≤ 200 mg/dL (42; 1.00 isolates/case). The difference between the mean isolates of patients with a BSL ≤ 200 mg/dL and those with a BSL > 200 mg/dL was statistically significant (P < .05). Monomicrobial etiology was found to be more common with 104 (83.20%) samples than polymicrobial etiology with 21 (16.80%). The percentage of polymicrobial infections increased from grade 1 (0%) to grade 5 (27.20%). 

A total of 147 aerobic bacterial isolates were obtained. Of the 147 isolates, 40 (27.21%) were gram-positive cocci and 107 (72.79%) were gram-negative bacilli. The most common isolate was Staphylococcus aureus (21.09%) followed by Pseudomonas aeruginosa (19.05%), Klebsiella oxytoca (17.01%), and K pneumoniae (14.97%) (Table 2). 

Gram-positive aerobes showed 100% susceptibility to linezolid and 97.5% resistance to penicillin. Gentamicin (76.32%) and vancomycin (70.00%) were other antimicrobial agents effective against gram-positive bacteria (Table 3). 

Against gram-negative bacteria, imipenem (75.70%) was found to be the most effective antimicrobial agent followed by amikacin (46.73%). A majority of the gram-negative bacteria were resistant to gentamicin, cotrimoxazole, tetracycline, levofloxacin, and other antimicrobial agents tested (Table 4).  

Diabetic foot ulcers are considered the most threatening and disabling complication of diabetes. An initial trivial lesion may progress to chronic, nonhealing wounds or gangrene that may need amputation of the toe, foot, or leg. Successful treatment of DFUs depends on a correct evaluation of the patient, isolation of pathogens, and identification of their antibiotic susceptibility patterns.

In the present study, the largest number of DFUs were observed in the age group of 61 to 70 years (32.14%) and the mean patient age was 59.72 ± 11.75 years. These findings are similar to previous studies.^9-12 In developing countries, the majority of people with diabetes are in the age group of 45 to 64 years¹³; DFUs in this age group can be caused by the compounding effects of neuropathy, peripheral vascular disease, and atherosclerosis associated with diabetes.¹⁴ In the present study, men were more commonly affected (80%) than women (20%). This finding agrees with previous studies.^10,12,15 The increased occurrence of DFUs in men may be attributed to smoking and outdoor activities. 

In the present study, 89.29% of cases had a positive culture while 10.71% of cases were sterile on culture. These results are consistent with Mukkunath et al⁹ and Akhi et al,¹⁶ who also reported sterile culture rates of 10%, but are not in concordance with Umadevi et al¹⁵ (2.9%) and Tiwari et al (21%).¹⁷ In the present study, patients who had a BSL > 200 mg/dL showed a greater number of culture-positive samples (83, 94.32%) and had a greater number of isolates (100; 1.20 isolates/case). These findings are in agreement with Patil and Patil.¹⁸ The higher culture-positive rates in patients with a BSL > 200 mg/dL may be attributed to significant diminution in intracellular bactericidal activity of leukocytes in patients with poorly controlled diabetes.

In the present study, the majority of cases were Wagner grade 2 DFUs (45%), and the percentage of polymicrobial infections increased from grade 1 (0%) to grade 5 (27.2%). Similar patterns of infection have been reported in earlier studies.^9,19,20 An increase in the DFU grade correlated with an increase in depth of ulcer and chronicity, leading to polymicrobial infections.

In the present study of 147 isolates, 27.21% were gram-positive cocci and 72.79% were gram-negative bacilli. These findings are consistent with earlier studies.^{15,17-20,21-24} The pattern of isolation of S aureus (21.09%) as the most predominant aerobic isolate followed by P aeruginosa (19.05%) is in agreement with Gadepalli et al,¹² Sridhar et al,²⁴ and Chopdekar et al.²⁵ This may be attributed to the common occurrence of these organisms in the surrounding environment and as a part of normal skin flora. 

In the present study, gram-positive aerobes showed 100% susceptibility to linezolid and 97.5% resistance to penicillin. Linezolid has been reported to be 100% effective against gram-positive isolates from DFUs in previous studies,^16,22 showing that it currently is the most effective antimicrobial agent against gram-positive isolates because of its restricted use. Penicillin was an ineffective antimicrobial agent against gram-positive isolates, indicating that this antibiotic has lost its clinical efficacy in the treatment of infections caused by gram-positive cocci and should be omitted from the empirical treatment. Against gram-negative bacteria, imipenem (75.70%) was found to be an effective antimicrobial agent. However, other antimicrobial agents, including meropenem, ceftazidime, gentamicin, levofloxacin, and cotrimoxazole, were less effective against gram-negative isolates.These findings are in concordance with previous reports.^{5,11,15,16,19,22,26,27} 

The overall observation shows there is a predominance of S aureus in the present study, which contrasts studies from urban areas that show either Escherechia coli¹⁷or P aeruginosa^28,29 as the predominant bacteria associated with DFUs. In the present study, both gram-positive and gram-negative isolates showed differential susceptibility patterns, which is different from the antibiotic susceptibility pattern of the isolates of earlier studies from urban areas.^12,28 The majority of the isolates from the present study were found to be resistant to most of the antibiotics, usually prescribed on an empirical basis. 

Diabetic foot infections, especially infections involving deeper tissue, may be associated with anaerobic infections. The authors’ objective to evaluate aerobic infections alone, without attempting to isolate anaerobic bacteria, is a limitation of this study. 

The results of present study show there is an overall increase in the percentage of antibiotic-resistant strains in rural areas. This could be attributed to the fact that many ulcers are chronic and likely treated by multiple general practitioners with a variety of antibiotics, leading to inadequate treatment and development of resistant strains. The results also show there is a vast difference in the susceptibility patterns of the present study and earlier reports, indicating the susceptibility pattern changes from place to place. In addition, the results emphasize the importance of studying the antimicrobial susceptibility pattern on a more individual basis, as the standard antibiotic susceptibility pattern may not hold true for every area or hospital. 

Authors: Kalpana Jaju, MD; Asha Pichare, MD; Milind Davane, MD; and Basavraj Nagoba, PhD

Affiliation: Department of Microbiology, Maharashtra Institute of Medical Sciences & Research (MIMSR) Medical College, Latur, India 

Correspondence: Basavraj Nagoba, PhD, Assistant Dean, Research & Development, MIMSR Medical College, Latur – 413 531 (M.S.) India; dr_bsnagoba@yahoo.com

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