Edited by: Johnan A. R. Kaleeba, Uniformed Services University of the Health Sciences, USA
Reviewed by: Naomi Sulinger Hoyle, Eliava Phage Therapy Center, Georgia; Pallab Ghosh, Harvard Medical School, USA
*Correspondence: Jamal Ahmad
This article was submitted to Infectious Diseases, a section of the journal Frontiers in Microbiology
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The study was carried on diabetic foot patients to deduce clinical attributes, the occurrence of the range of aerobic microbial flora and to assess their comparative
Diabetes mellitus being the global epidemic of 21st century, ultimately leads to end-organ damage due to hyperglycemia, imposing a major health burden. Diabetes-associated foot ulcers, followed by infection causes substantial morbidity and dreaded complications like systemic toxicity, gangrene, and lower extremity loss. The cumulative lifetime incidence for the development of foot ulcers in diabetes is as high as 25% (Noor et al.,
For infected ulcers, a post-debridement specimen from tissue should be obtained and processed for detection of causative pathogens (Zubair et al.,
The present study was designed to characterize common bacterial microbes in diabetic foot wounds, explore the drug sensitivity pattern of isolates and analysis of changing etiology of DFUs in North Indian population. Since poor healing status is a major cause of lower extremity amputations in diabetics, the study is also aimed to uncover the potential risk factors associated with the development of non-healing ulcers in diabetics.
A prospective hospital-based study was conducted in Jawaharlal Nehru Medical College, Aligarh Muslim University, Aligarh, India during the period from July 2014 to March 2016. Ninety type 2 diabetic patients with foot ulcers were admitted to endocrinology ward. Seventy patients with infected DFUs were enrolled in the study. All the subjects gave informed consent, and clearance was obtained from the Bio-Ethical Committee (BEC), Faculty of Medicine, J.N. Medical College, Aligarh Muslim University, Aligarh.
A well-structured questionnaire was developed for a detailed history and physical examination. Subjects were clinically assessed for age, sex, body mass index (BMI), lipid profile, duration of diabetes, glycemic status, liver, and renal functionality. Patients were also clinically evaluated for presence of other comorbidities such as retinopathy (fundoscopy), nephropathy (creatinine >1.5 mg% or presence of micro- or macro-albuminuria), neuropathy (absence of perception of the Semmes—Weinstein monofilament at 2 of 10 standard planter sites on either foot), peripheral vascular disease (ischemic symptoms and intermittent claudication of rest pain, with or without absence of pedal pulses or posterior tibial pulses) and hypertension (previous medication of anti-hypertensive drug or a BP ≥ 140/90 mmHg). Clinical assessment of infection in the wound was made with the occurence of classic signs of inflammation (redness, swelling, tenderness, warmth, or pain) or purulent secretions or additional minor signs of non-purulent secretions (friable or discolored granulation tissue, undermining of wound edges, foul odor). Duration and size of ulcer were calculated by multiplying longest and widest diameters and expressed in centimeter square. Ulcers were graded using Wagner classification system, grade I (superficial ulcer or ulcer of subcutaneous tissue), grade II (ulcers extended into tendon, bone, or capsule), grade III (deep ulcer with osteomyelitis, or abscess), and grade IV (gangrene of toes). Subjects with grade 0 (uninfected lesions/ intact skin/ healed ulcers) were debarred from the study. Amputation was defined as the complete loss in the transverse anatomical plane of any part of the lower limb. Diagnosis of infection involving bone was done by either using a sterile probe in exposed bone or evidence obtained from plain radiographs or magnetic resonance imaging (MRI) in the absence of sinus tract.
Pus aspirates or soft tissue samples were collected on the day of admission after proper cleaning of the diabetic wound with saline followed by debridement of superficial tissue exudates and promptly sent to a laboratory and processed for aerobic bacterial identification as described by Gadepalli et al. (
Venous blood samples were collected after an overnight fast and centrifuged at 3000 rpm for 5 min at 4°C. Serum or plasma was immediately separated and stored in aliquots at −80°C until further analysis.
All pus samples were Gram-stained and the bacteria were isolated by inoculation of specimens on a set of selective and non-selective media such as blood agar, MacConkey agar, and nutrient agar (Hi Media, Mumbai, India), and were incubated at 37°C overnight. Isolated organisms were identified on the basis of culture characteristics, colony morphology, and biochemical reactions as per the standard protocols (Collee et al.,
Antimicrobial susceptibility testing of aerobic isolates was done by using Mueller-Hinton agar using Kirby-Bauer disk diffusion method as recommended by Clinical and Laboratory Standards Institute (
Plasma/ serum obtained at the day of admission were processed for estimation of HbA1c by ion- exchange high-performance liquid chromatography (Bio-Rad D-10, India) and glucose estimation by colorimetric assays. Serum lipid analysis (triglycerides, total cholesterol, LDL- cholesterol, HDL-cholesterol, VLDL-cholesterol, and phospholipids) was done using commercially available kits (Avantor, U.S.A) according to manufacturer guidelines (Allain et al.,
Quantitative variables were represented as means ± standard deviation and categorical data as a percentage (%). Student's
Out of 90 type 2 diabetic patients with DFUs admitted to endocrinology ward, seventy had infected DFUs. Patients were grouped depending upon healing and non-healing state of ulcer following routine examination based on healing signs/symptoms. Routine clinical examination of ulcers was done up-to 2 months from the day of admission to evaluate the healing status. Non- healing lesions were clinically diagnosed on the basis of absence of epithelialization, angiogenesis, and formation of granulation tissues. Patients with non-healing wounds were significantly older than those without it (
N | 27 | 43 | ||
Male (%) | 22 (78.5%) | 29 (82.5%) | NS | |
Age (yrs) | 50.2±10.8 | 55.9±11.6 | < |
|
Diabetes duration (yrs) | 7.6±5.5 | 7.4±5.1 | NS | |
BMI (kg/m2) | 25.5±5.2 | 31.1±3.1 | < |
|
HbA1c (%) | 9.1±2.0 | 11±3.6 | < |
|
FPG (mg/dL) | 170±44.2 | 200.7±58.6 | < |
|
Se. Cr (mg/dL) | 1.4±0.8 | 1.2±0.5 | NS | |
TC (mg/dL) | 164.2±22.4 | 170.2±29.43 | NS | |
TAGs (mg/dL) | 148.3±38.9 | 183.1±54.8 | < |
|
HDL (mg/dL) | 47.6±10.1 | 39.4±6.6 | < |
|
LDL (mg/dL) | 71.6±11.3 | 78.0±6.1 | < |
|
<4 cm2 | 13 (48.1%) | 12 (27.9%) | < |
|
≥4 cm2 | 14 (51.8%) | 31 (72%) | ||
Ulcer duration (months) | 1.2±0.7 | 1.7±0.6 | < |
|
1 | 1 (3.7%) | 2 (4.6%) | NS | |
2 | 11 (40.7%) | 13 (30.2%) | NS | |
3 | 5 (18.5%) | 12 (27.9%) | NS | |
4 | 8 (29.6%) | 16 (37.2%) | NS | |
Hypertension | 11 (39.2%) | 18 (45.7%) | NS | |
Retinopathy | 20 (71.4%) | 29 (77.1%) | NS | |
Neuropathy | 16 (39%) | 12 (24.3%) | < |
|
Nephropathy | 24 (85.3%) | 32 (95.4%) | NS | |
Peripheral vascular disease | 15 (53.5%) | 29 (78.3%) | < |
|
Osteomyelitis | 12 (35.7%) | 31 (78.3%) | < |
|
8 (29.6%) | 25 (58.1%) | < |
||
Minor | 8 (29.6%) | 22 (51.1%) | NS | |
Major | 0 (0%) | 3 (6.9%) |
Among the subjects, the mean duration of infected ulcer was 1.45 ± 0.6 months. Size of ulcer ≥4 cm2 was observed in 45 (64.3%) cases and <4 cm2 in 25 (35.7%) subjects. Surgical amputation was done in 33 (47.1%) subjects, of which 3 (4.3%) underwent major amputation, and 30 (42.85%) were subjected to minor amputation. Diabetic foot wounds of non-healing category were significantly long-standing than healing wounds (
A total of 87 aerobic isolates were identified from 70 ulcer specimens, averaging 1.2 species per lesion. Fifty-three (75.7%) had monomicrobial infection, and polymicrobial etiology was observed in 17 (24.3%) cases. In our study Gram-negative bacilli were predominant (75.9%) than Gram-positive cocci (24.1%). In terms of relative abundance,
Subjects with non-healing ulcers during hospital stay had a predominance of
5 (5.7%) | 12 (13.8%) | |
5 (5.7%) | 11 (12.6%) | |
3 (3.4%) | 12 (13.8%) | |
9 (10.3%) | 4 (4.6%) | |
8 (9.2%) | 3 (3.4%) | |
5 (5.7%) | 5 (5.7%) | |
2 (2.3%) | 3 (3.4%) |
Based on antibiotic susceptibility pattern, 62.5% strains of
Amikacin | 14 (87.5%) | 2 (12.5%) | 5 (100%) | 0 (0%) | 19 (90.5%) | 2 (9.5%) |
Erythromycin | 10 (62.5%) | 6 (37.5%) | 4 (80%) | 1 (20%) | 14 (66.7%) | 7 (33.3%) |
Ciprofloxacin | 11 (68.75%) | 5 (31.25%) | 4 (80%) | 1 (20%) | 15 (71.4%) | 6 (28.6%) |
Gentamicin | 10 (62.5%) | 6 (37.5%) | 4 (80%) | 1 (20%) | 14 (66.7%) | 7 (33.3%) |
Levofloxacin | 6 (37.5%) | 10 (62.5%) | 4 (80%) | 1 (20%) | 10 (47.6%) | 11 (52.4%) |
Oxacillin | 11 (68.75%) | 5 (31.25%) | 5 (100%) | 0 (0%) | 16 (76.2%) | 5 (23.8%) |
Vancomycin | 16 (100%) | 0 (0%) | 5 (100%) | 0 (0%) | 21 (100%) | 0 (0%) |
In
Amikacin | 13 (76.5%) | 4 (23.5%) | 9 (69.2%) | 4 (30.8%) | 5 (45.4%) | 6 (54.5%) | 3 (30%) | 7 (70%) | 30 (58.8%) | 21 (41.2%) |
Cefoperazone | 3 (17.6%) | 14 (82.35%) | 4 (30.8%) | 9 (69.2%) | 2 (18.2%) | 9 (81.8%) | 3 (30%) | 7 (70%) | 15 (29.4%) | 22 (43.1%) |
Cefixime | 6 (35.3%) | 11 (64.7%) | 2 (15.4%) | 11 (84.6%) | 10 (90.9%) | 1 (9.0%) | 6 (60%) | 4 (40%) | 24 (47.0%) | 27 (52.9%) |
Cefepime | 6 (35.3%) | 11 (64.7%) | 10 (76.9%) | 3 (23.1%) | 4 (36.4%) | 7 (63.6%) | 5 (50%) | 5 (50%) | 25 (49.0%) | 26 (51.0%) |
Cefetriaxone | 6 (35.3%) | 11 (64.7%) | 10 (76.9%) | 3 (23.1%) | 2 (18.2%) | 9 (81.8%) | 4 (40%) | 6 (60%) | 22 (43.1%) | 29 (56.9%) |
Gentamicin | 5 (29.4%) | 12 (70.6%) | 6 (46.15%) | 7 (53.8%) | 4 (36.4%) | 7 (63.6%) | 4 (40%) | 6 (60%) | 19 (37.2%) | 32 (62.7%) |
Levofloxacin | 5 (29.4%) | 12 (70.6%) | 10 (76.9%) | 3 (23.1%) | 6 (54.5%) | 5 (45.4%) | 4 (40%) | 6 (60%) | 25 (49.0%) | 26 (51.0%) |
Tobramycin | 5 (29.4%) | 12 (70.6%) | 6 (46.15%) | 7 (53.8%) | 6 (54.5%) | 5 (45.4%) | 3 (30%) | 7 (70%) | 20 (39.2%) | 30 (58.8%) |
Cefeperazone + Sulbactam | 8 (47.0%) | 9 (52.9%) | 12 (92.3%) | 1 (7.7%) | 5 (45.4%) | 6 (54.5%) | 6 (60%) | 4 (40%) | 31 (60.8%) | 20 (39.2%) |
Among the isolates of
Amikacin | 5 (33.3%) | 10 (66.7%) |
Ceftazidime | 4 (26.7%) | 11 (73.3%) |
Cefepime | 4 (26.7%) | 11 (73.3%) |
Levofloxacin | 6 (40.0%) | 9 (60.0%) |
Sparfloxacin | 4 (26.7%) | 11 (73.3%) |
Tobramycin | 5 (33.3%) | 10 (66.7%) |
Piperacillin | 8 (53.3%) | 7 (46.7%) |
Piperacillin + Tazobactam | 12 (80.0%) | 3 (20.0%) |
Imipenem | 15 (100%) | 0 (0%) |
This study presents a comprehensive clinical and microbiological contour of infected DFUs in hospitalized patients. Patients with non-healing ulcers were older and had poor glycemic status than those with healing wounds. Impaired insulin secretion/action causes a halt in the uptake of glucose, leading to reduced activity of fibroblasts, and polymorphonuclear neutrophils causing poor healing (Rosenberg,
Subjects with non-healing lesions had a high prevalence of neuropathy and peripheral vascular disease. Loss of sensation of peripheral arteries leads to recurrent ulcers with susceptibility to develop multidrug-resistance isolates due to heavy prior treatment with antimicrobials, thus causing delayed healing. Inadequate blood flow caused due to thickening of arteries of lower extremities creates an ischemic environment and compromised mobility causing prolonged periods of unrelieved pressures on the extremities. This results in increased shearing force applied to the skin and underlying tissues leading to decrease in oxygen tension and eventual tissue necrosis (Defloor,
In the present study we observed that out of 87 aerobic bacteria isolated from 70 pus samples, an average of 1.2 organisms was found. These findings are similar to those reported by Viswanathan et al. (
In the present study,
All strains of
Our study has shown that amongst the Enterobacteriace group, amikacin showed good antimicrobial activity against
Based on our result and what is documented in literature so far, it is amply clear that there is no antibiotic which can cover all isolates, and therefore, a combination of drugs has to be recommended to overcome the extensive multidrug-resistance. The emergence of resistant strains in DFIs is a major hurdle to our efforts to prevent limb loss as the infection is most common cause complicating the diabetic foot pathogenesis. Even if the pathogen is susceptible to one particular antimicrobial, the drug is unlikely to accomplish therapeutic concentration at the site of infection due to hindrance caused by deranged host immune responses, virulence factors, such as proteases, hemolysins, and collagenases that cause inflammation and impede wound healing contribute to the chronicity of the infection (Bowler and Davies,
It must be considered that the present study is a prospective study but with highly significant results. A more comprehensive multi-centric study covering a diversity of population along with previous hospitalization details and prior antibiotic exposure is warranted to cover a wide array of microbial range. This information will form a substantive baseline data to elucidate the causes of high prevalence of non-healing ulcers amongst the Indian population.
This study demonstrates the existence of a diversity of organisms in DFUs. The emerging dominance of Gram-negative aerobes replacing Gram-positive bacteria in infected diabetic foot wounds may impose a serious health burden in healing of ulcers. Therefore, antimicrobial prescribed should be broadened covering both Gram-negative and Gram-positive pathogens to improve the healing status in diabetic foot. Antimicrobial prescribed should also target multidrug-resistant strains which are a compounding trouble in treatment of diabetic foot infection and associated complications. In addition, a proper clinical analysis, including the presence of neuropathy and vascular disease, history of ulcerations should be routinely done in diabetics.
SN performed experimental work along with statistical calculations. JA and IP conceived the hypothesis provided the overview during experimental work and helped in preparation of this manuscript into the final form. MO was involved in standardization of clinical methodologies and manuscript drafting.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
SN is grateful to the Jawaharlal Nehru Memorial Fund, New Delhi, India, for the award of a Ph.D. scholarship. We are also thankful to Prof. Alim Naqvi and Dr. Braj Raj Singh, Centre of Excellence in Material Science (Nanomaterials), Z.H. College of Engineering and Technology, Aligarh Muslim University, Aligarh for helping in microbial analysis and manuscript drafting.