Study on bacteriological proﬁle and antibiotic susceptibility in diabetic foot infection in a teaching hospital, Telangana

Background: Diabetic foot is one of the most signiﬁcant complications of diabetes, Chronic infections are caused by Enterococci, various Enterobacteriaceae obligate anaerobes, Pseudomonas aeruginosa. Aim of the study: To study bacteriological proﬁle and antibiotic susceptibility in diabetic foot infection. Materials and Methods: Cross sectional study was done in 110 diabetic foot infection cases in the Department of Microbiology, Institute of Karimnagar, Telangana. The grading of diabetic foot ulcers was done according to Wagner’s Classiﬁcation system. The samples were collected from the exudates and ulcers. Gram stain was done and standard protocol for culture and sensitivity was followed for all the cases. Results: A total of 110 cases were studied. The patient age ranged from 35 to 75 years and the male to female ratio was 2:1. Out of 110 isolates, 72.7% isolates were Gram negative bacilli with P. aeruginosa 31.8% being the predominant followed by E.coli 27.2%, K.pneumoniae 10%, P.mirabilis 4.5%, S.aureus 12.7% was the predominant isolate followed by Enterococci spp 4.5% and Coagulase negative staphylococci (CONS) 4.5% and S. pyogenes were 5.4%. Conlcusion: Both Gram positive cocci and Gram negative bacilli can cause diabetic foot infections and this study showed a preponderance of Gram negative bacilli. Early culture and sensitivity test of the bacterial isolates helps in guiding the treatment plan This is an Open Access (OA) journal, and articles are distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms.


Introduction
Diabetic foot infection (DFI) is one of the most significant and devastating complications of diabetes, and is defined as a foot affected by ulceration that is associated with neuropathy and/or peripheral arterial disease of the lower limb in a patient with diabetes. The prevalence of diabetic foot ulceration in the diabetic population is 4-10%; the condition is more frequent in older patients. Two types of diabetes mellitus exist: type 1 and type 2 (formerly known as insulin and non-insulin diabetes mellitus). Type 2 diabetes(T2DM) is the most common type of diabetes Chronic infections are caused by Enterococci, various Enterobacteriaceae, obligate anaerobes, Pseudomonas aeruginosa, and sometimes, other non-fermentative gramnegative rods. 7 Gram-negative bacilli, mainly of Enterobacteriaceae, are found in patients with chronic or previously treated infections. Pseudomonas species is usually found in wounds that have been soaked or treated with wet dressings or hydrotherapy. Enterococci are obtained from patients who have previously received a cephalosporin. Obligate anaerobic species are isolated from wounds with ischemic necrosis or that involve deeper tissues. Antibiotic-resistant organisms, especially methicillin resistant S. aureus, are found from patients who have previously received antibiotic therapy; they are often acquired during the previous hospitalization. 8 The risk factors for diabetic foot infection (DFI) are presence of wounds that have penetrated to the bone, wounds with a duration of >30 days, recurrent wounds, wounds with traumatic etiology and the presence of peripheral arterial disease. Neuropathy and history of previous amputation are significant risk factors for infection. The risk of infection is more in walking barefoot. Positive history of hospitalization and amputation are greater in patients with DFI compared to those without. 9 Regular foot examination, patient education, simple hygienic practices, provision of appropriate footwear, and prompt treatment of minor injuries can decrease ulcer occurrence by 50% and eliminate the need for major amputation in nonischemic limbs. 10,11

Aim of the study
To study bacteriological profile and antibiotic susceptibility in diabetic foot infections.

Materials and Methods
This was a cross-sectional study done in the department of Microbiology, at Prathima Institute of Medical Sciences, Nagunuru, Karimnagar, Telangana over a period of 21 months from June 2018 to February 2020.
There were no ethical issues involved in the study. Informed consent was obtained from all the participants included in the study.

Methodology
Demographic characteristics were noted in detail including age, gender, history of present illness, past history regarding the duration of diabetes, history of any drug intake, allergies.
The patients were examined clinically, and the grading of diabetic foot ulcers was done according to Wagner's Classification and the University of Texas Wound Classification System. 12

Wagner's classification of diabetic foot ulcers
Grade 0: no ulcer in a high-risk foot. Grade 1: superficial ulcer involving the full skin thickness but not underlying tissues. Grade 2: deep ulcer, penetrating down to ligaments and muscles, but no bone involvement or abscess formation. Grade 3: deep ulcer with cellulitis or abscess formation, often with osteomyelitis. Grade 4: localized gangrene. Grade 5: extensive gangrene involving the whole foot.
Under all aseptic precautions samples were collected from the affected site using sterile cotton swabs.
The samples were obtained from the deeper portion of the ulcers using two sterile swabs.
One swab was used for Gram-staining, and another was used for inoculation of culture and sensitivity.
The samples after collection were transported within 2 hours to the microbiology laboratory.
All the Gram-stained smears were examined for Grampositive and Gram-negative bacteria. Standard protocol for culture and sensitivity was followed by inoculating the swab onto different culture plates such as blood agar, chocolate agar, and McConkey's agar medium. Overnight incubation at 37 • C of the inoculated plates was done and examined for the growth next day. Based on Gram-staining and colony morphology, bacterial isolates were identified, and biochemical reactions were performed for confirmation.
The antibiotic susceptibility testing was done by Kirby-Bauer disk diffusion method as per CLSI guidelines. 13,14 According to recommendations of Clinical Laboratory Standard Institute (CLSI) for antibiotic sensitivity testing and based on the size of inhibition zone around the disc, three forms of Sensitive (S), Intermediate (I) and Resistant (R) pattern were interpreted.
All the data collected was entered into the master chart and excel sheet and subjected to further analysis.
Microsoft Excel applications performed sufficient data entry and mathematical analysis. Categorical variables were expressed as frequencies and percentages. The comparison of normally distributed continuous variables between the groups was performed using Student's t test. For all statistical tests, a p value less than 0.05 was taken to indicate a significant difference. Microsoft word and Excel have been used to generate graphs, tables.

Observations and Results
Total of 110 patients with diabetic foot infection were studied. In the present study age distribution ranged from 35 years to 75 years. Majority of the patients with diabetic foot infection were among 55-65 years constituting 39% (43/110). Table 1 3

Type of diabetes in cases with diabetic foot ulcers
There were 15 (13.6%) cases of Type 1 diabetes mellitus and 95 (86.3%) cases of Type 2 diabetes mellitus.

Risk factors associated with diabetic foot ulcers
Some of the risk factors observed were as follows: Hypertension was present in 45 (40.9%) cases, history of smoking was seen in 26 (23.6%) cases, history of trauma was present in 7 (6.3%) cases, alcohol intake was seen in 32 (29%) cases.

Clinical presentations associated with diabetic foot ulcers
In the present study, vasculopathy was the commonest clinical presentation associated with foot ulcers and was seen in 41 (37.2%) cases followed by neuropathy seen in 30 (27.2%) cases. History of steroid therapy was present in 10 (9%) cases, HIV positivity was seen in 19 (17.2%) cases and 10 (9%) cases were immunocompromised.

Treatment history for diabetes mellitus
In the present study, 54.5% (60/110) of diabetic foot ulcer patients were on oral hypoglycemic drugs, 27.2% (30/110) patients were not on any medications and 18.1% (20/110) patients were on insulin injections.

Number of organisms isolated from diabetic foot ulcer
In the present study, 75 (68.1%) cases showed single organism growth and 35 (31.8%) cases showed growth of multiple organisms.

Bacterial isolates
In the present study, Gram-negative growth was seen in 80 (72.7%) cases and Gram-positive bacterial isolates were present in 30 (27.2%) cases.

Comparative studies related to Age distribution
In the present study, the patients age ranged from 35-75 years. Majority of the patients with diabetic foot were among 55-65 years constituting 39% and next common age group was among 46-55 years ie, about 32.7%. Our findings were compared with other studies. Shashanka R et al 15 in their study observed maximum number of patients (54%) in the age group of 56-65 years and the second most common age group was between 45 and 55 years (22%). Otta S et al 16 noted most of their patients (45.9%) among 51-60 years. In the study by Hefni AH et al 17 diabetic foot infections were highest among the age group of 51-60 years, followed by 41-50 years age group. In the study by Shanmugam P et al 18 maximum number of patients (20%) was in the age group of 60 to 65 years followed by 50 to 55 years (18%).

Comparative studies related to gender wise distribution
In the present study, males (67.2%) were commonly affected by diabetic foot ulcers when compared to females (32.7%). Similar findings were observed in Otta S et al study 16 where, among l 148 diabetic patients presenting with ulcers, 106 (71.6%) were males and 42 (28.4%) were females. Whereas, a study conducted by Hefni AH et al 17 37 were males and 38 were females with an almost equal male to female ratio.
In the present study, Grade 2 ulcers were the most predominant constituting 41.8% cases. Our finings are similar to those reported by Hefni AH et al 17 where they also noted Grade II ulcers as the most predominant type of ulcers. Table 5

Comparative studies related to Antibiotic sensitivity
In the present study, Pseudomonas aeruginosa, which was the most commonly isolated bacteria showed 100% sensitivity to meropenem and 93% to imipenem and 80% sensitivity to piperacillin-tazobactam, 90% to Cefaperazone + sulbactum. In a study conducted by Shashanka R et al 15 also, Pseudomonas aeruginosa, was the most prevalent bacteria and showed 100% sensitivity to imipenem and meropenem and 75% sensitivity to piperacillin-tazobactam. In a study done, in 2011, it was showed that P. aeruginosa strains were 83.3% sensitive to cefotaxime. Our findings did not compare well with these results. 17 Otta S et al 16 observed 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 extended spectrum beta lactamase (ESBL) and metallobeta lactamase (MBL) was shown by Klebsiella spp. and Acinetobacter species respectively.
In our study, all the strains of staphylococci which were isolated were 100% sensitive to teicoplanin, linezolid. They were 95% sensitive to vancomyicin and 80% to Piperacillin + tazobactum. CONS showed 100% sensitivity to vancomyicin and Piperacillin + tazobactum.    Staphylococcus was more susceptible to the antibiotics than S. aureus and showed highest sensitivity to vancomycin and cefoperazone-sulbactam.

Conclusion
Both Gram positive cocci and Gram negative bacilli can cause diabetic foot infections and this study showed a preponderance of Gram negative bacilli. Early culture and sensitivity test of the bacterial isolates helps in guiding the treatment plan by instituting appropriate antibiotics. Knowledge on the antibiotic susceptibility pattern of the isolates from diabetic foot infections is crucial for planning the appropriate treatment of these cases, prior to getting the susceptibility reports from the laboratory. These observations are important, especially for patient management and development of empirical antibiotic guidelines.

Limitations
The limitation for this study is that the validated diabetic foot infection tool classification was not used as it is not been practiced routinely in our centre. Despite that, there is no difference in the management of our diabetic foot infections in compare to the practice that has been published elsewhere.