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Year : 2019  |  Volume : 6  |  Issue : 1  |  Page : 7-11

Bacteriological profile of diabetic foot ulcers

Department of Microbiology, Institute of Medical Sciences and SUM Hospital, S'O'A University, Bhubaneswar, Odisha, India

Date of Submission02-Dec-2017
Date of Decision23-Apr-2018
Date of Acceptance18-Jun-2018
Date of Web Publication14-Feb-2019

Correspondence Address:
Bichitrananda Swain
Plot No. 171, District Centre, Chandrasekharpur, Bhubaneswar - 751 016, Odisha
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/cjhr.cjhr_117_17

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Background: Foot infections are the major complications of diabetes mellitus and lead to the development of gangrene and ultimately amputation of the limb. Proper diagnosis of the causative agents and their antibiotic susceptibility play a significant role in preventing adverse prognosis of diabetic foot. Aim: The study aims to establish the bacteriological profile of diabetic foot ulcers as well as to note resistance pattern of the implicated organisms. Materials and Methods: One hundred and forty-eight patients having diabetic foot ulcers of Wagner's Grade 1 or above were included in this study. Debrided tissue, pus, or swabs from the base of the ulcers were subjected to aerobic and anaerobic culture. The organisms were identified, and further antibiotic sensitivity of the aerobic bacteria was conducted by Kirby–Bauer's disc diffusion method. Results: Two hundred and forty aerobic and 21 anaerobic bacteria were isolated from these ulcers; Staphylococcus aureus and Bacteroides spp. are the most common aerobic and anaerobic bacteria isolated, respectively. Of the S. aureus, 77.8% were methicillin resistant, while 42.1% of the Gram-negative Enterobacteriaceae were extended-spectrum beta-lactamase (ESBL) positive. Klebsiella spp. was the highest ESBL producer whereas Acinetobacter spp. was the highest metallo-beta-lactamase producer. Linezolid, teicoplanin, and vancomycin were the most sensitive drugs for Staphylococcus spp. Gram-negative isolates were mostly sensitive to cefoperazone-sulbactam and imipenem. Pseudomonas spp. was mostly sensitive to imipenem and piperacillin-tazobactam, whereas Acinetobacter spp. was sensitive to netilmicin and levofloxacin. Conclusion: As diabetic ulcers are often infected by multidrug-resistant bacteria, a knowledge of the common bacterial pathogens implicated as well as their sensitivity pattern helps the clinician to choose the proper antibiotic for a timely treatment.

Keywords: Diabetic ulcers, extended-spectrum beta-lactamase, metallo-beta-lactamase, methicillin-resistant Staphylococcus aureus, Wagner's grading

How to cite this article:
Otta S, Debata NK, Swain B. Bacteriological profile of diabetic foot ulcers. CHRISMED J Health Res 2019;6:7-11

How to cite this URL:
Otta S, Debata NK, Swain B. Bacteriological profile of diabetic foot ulcers. CHRISMED J Health Res [serial online] 2019 [cited 2022 Jul 4];6:7-11. Available from: https://www.cjhr.org/text.asp?2019/6/1/7/252284

  Introduction Top

Diabetes mellitus, a chronic disease, is affecting a large segment of population. About 12%–25% of diabetics across the world have a lifetime risk of developing foot ulcers, thus contributing to a major public health issue.[1],[2] Rate of amputation of a limb is estimated to be forty times greater in infected nonhealing ulcer in diabetics than the patients of trauma. Infections precede in >60% cases of foot amputations.[3]Peripheral sensory and motor neuropathy leading to deformities, macro- and microangiopathy leading to ischemia, and infection are the major etiologies of diabetic foot.

[4] Foot problems are largely preventable, and successful treatment depends on the correct evaluation of the patient, diagnosis, and proper management of infection.

  Materials and Methods Top

In the present study, 148 diabetic patients with ulcers of Wagner's Grade 1 or above who consented for the work were included in the study. Local examination of the foot and ulcer was done, and grading was carried out as per Wagner's system. Biochemical, hematological, serological, as well as radiological profiles of the patients were noted. Ulcer surfaces were rinsed with sterile normal saline, and swabs were collected from base of the ulcer after debriding the superficial exudates.[5] Pus if present was aspirated using a sterile syringe. The debrided necrotic tissue,[6] pus, or swab was put into preheated Robertson's Cooked Meat medium and incubated anaerobically in McIntosh–Filde's jar at 37°C. This was further subcultured in anaerobic condition on neomycin blood agar plate. For aerobic culture, the samples were inoculated on predried plates of blood agar and MacConkey agar along with a nutrient broth. The colonies grown on the plates after overnight incubation at 37°C were identified using a standard protocol. Anaerobes isolated were identified to the genus level only. Antimicrobial susceptibility testing of aerobic isolates was done using Kirby–Bauer's disc diffusion method on Muller-Hinton agar plates. For Staphylococcus isolates' methicillin resistance was done by disc diffusion technique using Mueller-Hinton agar containing 4% NaCl with a 1 μg of oxacillin disc. Vancomycin resistance was preliminarily detected by 30 μg vancomycin disc and was further confirmed by HiComb MIC test from HiMedia. Extended-spectrum beta-lactamase (ESBL) testing was done using both the ceftazidime (30 μg) and ceftazidime-clavulanic acid (30/10 μg) discs. Metallo-beta-lactamase (MBL) production was detected using 10 μg of imipenem along with a second disc containing 10 μl of 0.5 M ethylenediaminetetraacetic acid placed at a 15 mm distance.

  Results Top

In this study of the total 148 diabetic patients presenting with ulcers, 106 (71.6%) were males and 42 (28.4%) were females. Most of the patients (45.9%) belonged to the age group of 51–60 years. Apart from four cases of type 1 diabetes mellitus, all others were of type 2 variety. Majority of the cases presented with ulcer within 6–10 years of being diagnosed as a diabetic, whereas 8.2% of cases presented even after 20 years of diagnosis. Most of the patients came to this hospital only after 2–4 weeks of the development of foot ulcer. Half of these patients were having a combination therapy of insulin and oral hypoglycemic agents. However, 17.6% of cases had either left treatment or were irregularly taking the medications leading to an uncontrolled blood sugar. When the ulcers were graded as per Wagner's system, Grade 2 ulcers were the most predominant (31.08%) in the present study. This was closely followed by Grade 3 and Grade 4 ulcers (27.2% each). Thus, gangrenous ulcers (Grade 4 and Grade 5) constituted 29.9% of all the ulcers.

Of the 148 cases, 92 (62.2%) were monomicrobial, 40 (27.0%) were polymicrobial, and only 16 (10.8%) cases were sterile on culture. About 33% of Grade 1 ulcers did not reveal any growth of organisms, the percentage of which is drastically reduced as the grade of ulcer increased. Percentage of ulcers with polymicrobial etiology increased as the grade of ulcer increases such that 50% of gangrenous ulcers were polymicrobial [Table 1].
Table 1: Distribution of number of organisms isolated in different Wagner's grade

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A total number of aerobes and anaerobes isolated from 148 ulcer were 240 (91.9%) and 21 (8.1%), respectively. A number of organisms per ulcer in this study were 1.76. A number of aerobic and anaerobic organisms isolated per sample increased with the rise in grade of ulcer. Anaerobic organism isolation was highest in the ulcers of Grades 4 and 5. Only aerobic bacteria were isolated in 111 ulcers, while in 21 ulcers, anaerobic organisms were present in addition to aerobic microbes. None of the ulcers had pure anaerobic organisms [Table 2].
Table 2: Variation of number of organisms isolated according to grade of ulcer

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Among the pathogens isolated by aerobic culture, most predominant organisms were Staphylococcus aureus (30%), followed by Pseudomonas aeruginosa (11.7%) and Escherichia coli (10%) [Table 3].
Table 3: Different types of organisms isolated from the diabetic ulcers

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Most of the S. aureus were sensitive to vancomycin (91.5%), teicoplanin (91.1%), and linezolid (90%). Among the aminoglycosides, netilmicin was the most sensitive drug (81.8%). They showed 87.5% and 71.8% sensitivity to levofloxacin and piperacillin-tazobactam, respectively. Nearly 77.8% of S. aureus were methicillin-resistant S. aureus (MRSA). Coagulase-negative Staphylococcus was more susceptible to the antibiotics than S. aureus and showed highest sensitivity to vancomycin and cefoperazone-sulbactam. All the Enterococcus spp. isolated were sensitive to vancomycin, netilmicin, and levofloxacin, while 87% of them were sensitive to piperacillin-tazobactam and 75% to amoxicillin-clavulanic acid.

In the present study, imipenem was the most useful antibiotic for the treatment of isolates for members of Enterobacteriaceae family. These isolates were mostly sensitive to piperacillin-tazobactam, levofloxacin, and netilmicin. Amoxicillin-clavulanic acid and cephalosporins were the most resistant antibiotics.

Nonfermenters (Pseudomonas spp. and Acinetobacter spp.) showed a higher degree of resistance to imipenem than those of Enterobacteriaceae. Pseudomonas spp. were usually sensitive to piperacillin-tazobactam (86.6%) and ceftazidime-clavulanic acid (71.4%), whereas Acinetobacter spp. was mostly sensitive to netilmicin (60%).

Highest degree of production of ESBL and MBL was shown by Klebsiella spp. and Acinetobacter spp., respectively [Table 4].
Table 4: Sensitivity pattern of various organisms

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  Discussion Top

In the present study, most of the patients (45.9%) belonged to the age group of 51–60 years; the males outnumbering females and patients mostly developed ulcers within 5 years of the detection of diabetes mellitus. This may be due to peripheral neuropathy, peripheral vasculopathy, and outdoor occupation in males.[7] Studies have reported male sex as a significant risk factor for nonhealing ulcer.[8] It has also been stated that male diabetic patients with multidrug-resistant Gram-negative bacilli-infected foot ulcers have poor glycemic control and have higher mortality than their female counterparts.[9] An alarming fact was that 5.4% of the included patients were diagnosed as type 2 diabetics only after having a foot ulcer. This may be due to the lack of knowledge among the population regarding the symptoms of the disease and proper foot care.

In this study, 62.2% of wound cultures showed monomicrobial flora and 27.1% had polymicrobial flora. This is similar to other studies.[10],[11] The higher incidence of monomicrobial flora in this study than few other works[12],[13] is probably due to the higher prevalence of mild and superficial ulcers. It has been postulated that the uninfected ulcers are generally colonized by polymicrobial flora.[14] However, in this study, the cases included had signs of infection of the ulcers, and the samples were collected by curetting from base of the ulcers rather than superficial swabs. This may be another cause for high percentage of singular yield of organisms from the ulcers. Almost 10.8% of ulcers had no bacterial growth which may be due to the prior treatment with broad-spectrum antibiotics in most of the cases (94.6%).

In our study, the number of organisms per ulcer is 1.76. This correlated well with those of Zubair et al.[10] and Raja et al.[11] However, other studies[5],[12],[15],[16] show higher number of isolates per ulcer. The number of organisms per ulcer varies significantly with the grade of ulcer, time of presentation after development of ulcer, as well as the sampling techniques. Prior antibiotic therapy may have also influenced the outcome in our study.

We had a lower isolation of anaerobic bacteria (8.1%) than other studies[6],[11],[16] in which it can be up to 51.56%. Rate of isolation of anaerobes varies as per the method of sampling, prior antibiotic therapy, and type of wound. It has been postulated that, in the superficial grades (Wagner 1 and 2), aerobic bacteria (Staphylococcus spp., Streptococcus spp., and Enterobacteriaceae) are the predominant pathogens, while anaerobic bacteria add up in Wagner's Grade 3–5 ulcers.[17] In our study, most of the anaerobes were isolated from Grade 4 to Grade 5 ulcer cases. In this work, most common anaerobic isolates were Bacteroides followed by Peptostreptococcus. This correlates well with other studies[15],[18] where Bacteroides spp. have been postulated as the most prevalent anaerobe associated with diabetic ulcers.

Gram-positive aerobic bacteria were more frequently isolated in comparison to Gram negatives. This is in agreement with several works,[19],[20] but few other studies[16],[21],[22] show Gram-negative isolates as the most predominant aerobic infection in diabetic foot ulcers. Environmental factors, such as sanitary habits and use of water for perianal wash after defecation leading to contamination of the hands by fecal flora, have been proposed to influence the pattern of organisms isolated in the developing countries, particularly in the rural setup.[23] Predominance of lower grade as well as recently developed ulcers in this study may also be a contributing factor.

Sensitivity pattern of the microbes in diabetic foot ulcers is often heralded by the presence of multidrug-resistant strains. The presence of MDR organisms is the only significant independent predictor of glycemic control.[16] About 77.8% of S. aureus isolated in the present study were methicillin resistant which may vary from 10.6% to 71.4% in various studies.[6],[10],[16],[23] Seven strains of vancomycin-resistant S. aureus were found in the present study. Similarly, ESBL production was noted in 42.1% of Gram-negative isolates. Klebsiella spp. was the highest ESBL producer. Acinetobacter spp. was the highest MBL producer. Citrobacter spp. although had low rate of ESBL production (46.1%) was highly resistant (83.4%) to cephalosporins, thus indicating other mechanism of resistance. The MBL enzymes which hydrolyze all beta-lactam drugs and carbapenems were commonly associated with Acinetobacter spp. Association of MBL-producing strains in diabetic ulcers can lead to the high incidence of treatment failure.

In our study like many others,[21],[22],[23] linezolid, teicoplanin, and vancomycin were the most sensitive drugs for Staphylococcus spp. which is probably due to the high prevalence of MRSA strains. Other antibiotics such as piperacillin-tazobactam, cefoperazone-sulbactam, netilmicin, and levofloxacin were moderately effective for Gram-positive coverage.

The present study like few others[22] noted the isolates of Enterobacteriaceae family to be the most sensitive to cefoperazone-sulbactam and imipenem, while ofloxacin, gentamicin, amoxicillin-clavulanic acid, and cefotaxime were the most resistant antibiotics. Pseudomonas spp. similar to previous works[16],[21] showed highest sensitivity to imipenem and piperacillin-tazobactam, but Acinetobacter spp. were the most notorious strains showing a very low degree of sensitivity to almost all the drugs being used.

  Conclusion Top

Treatment of diabetic ulcers frequently involves the use of an empiric antibiotic. The severity of wound and local antimicrobial susceptibility pattern often determine the choice of empiric treatment. In our study, S. aureus was the most predominant bacterial cause. The type and number of infecting organisms vary as per the grade of ulcer. In Grades 4 and 5 ulcers, it is important to include the treatment for anaerobic organisms as well. There is an increasing percentage of multidrug resistance organisms associated with these ulcers which dims the prognosis. Linezolid can be used for empiric therapy for lower grade ulcers while imipenem, linezolid, and metronidazole can be used for higher grade ulcers. Nevertheless, proper knowledge of foot care is essential to prevent ulceration as well as for the early diagnosis of diabetic foot.


We are thankful to the S'O'A University, Kalinga Nagar, Bhubaneswar, Odisha, for giving permission to perform this research work.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

  References Top

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  [Table 1], [Table 2], [Table 3], [Table 4]

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