Microbiological pro ile and Drug Resistant Organism’s pattern in Diabetic Foot Ulcer Patients at Tertiary Care Hospital Puducherry

Type II diabetes mellitus is a signi icant health problem that developed globally. This study was carried out on patients with diabetic foot ulcer (DFU) to assess the bacterial and fungal lora, susceptibility, and drug-resistant isolates and devises an empiric antimicrobial therapy. Clinical data and patient samples were collected from 300 diabetic foot ulcer patients between September 2014, and September 2016 and samples were processed as per CLSI guidelines. Most of the pathogenic isolate recovered according to the Wagner classi ication system in DFU. The most commonly found isolates in our Study was Pseudomonas aeruginosa (22%), Staphylococcus aureus (15%)Escherichia coli (11%) followed by others. Antimicrobial resistance appears in aerobic, anaerobic as well as candida isolates in our study. Our results show most gramnegative bacteria were sensitive to colistin and tigecycline, and 44% of Gramnegative bacteria were ESBL producers, and among 20%of the gram-negative isolates were Multidrug resistant (MDR) organisms. Proper diagnosis of the causative agents, surveillance monitoring on the susceptibility of the isolates and determining the drugs for the empirical treatment of diabetic foot ulcers will prevent prolonged hospital stay and amputation.


INTRODUCTION
Foot ulcer infections in diabetic patients are signi icant complications. 69.2 million peoples were nationwide affected, and globally 415 million people having diabetes ( International Diabetes Federation, 2015). Diabetes is a multifactorial disease in which various factors act in an elaborate man-ner (Walker and Colledge, 2013). The effect of diabetes includes neuropathy, peripheral vascular disease and poor glycolic control. According to the severity of diabetic foot ulcer; there are four classi ication systems, Wagner's, PEDIS, which are used worldwide (Chadwick et al., 2013). Wagner Classi ication System for Foot Ulcers was described in Table 1. Diabetic Patients possess a greater incidence of several common infections, including pulmonary, Urinary Tract infections. It is the most common bacterial infections noticed in patients with diabetes mellitus in clinical practice and the main reason for hospital admittance (Al-Salihi and Jumaah, 2013). Pathogenic bacteria mostly colonize these ulcers, and infection is facilitated by immunological de icits related to diabetes (Geerlings, 1999) rapidly progressing to deeper tissues, increasing the presence of necrotic tissue, rendering amputation inevitable (Lipsky et al., 2005). Diabetic patients frequently require minor or major amputations (15-27%), which contribute to high morbidity among diabetic patients, but is also associated with severe clinical depression and increased mortality rates. Mainly ESBL producing Gram-negative bacilli and MDR Gram-negative bacteria isolates lead to severe infection and also prone to amputation of major or minor below the knee and below great ankle toe, metatarsal (Murali et al., 2014).
Initially, antimicrobials are selected empirically for the treatment of DFU infections. With a declining number of novel antibiotics being developed and impetuous use of available antibiotics, antibiotic resistance has become a universal issue in healthcare institution (Chen et al., 2018). Aim of the present study was to determine microbiological causes of diabetic foot infections and Antibiotic drug-resistant pattern of the isolates.

MATERIALS AND METHODS
Total of 300 samples was collected from diabetic patients with foot ulcers admitted to Sri Lakshmi Narayana Institute of Medical Sciences between September 2014 and September 2016. This study was conducted after obtaining approval from the institutional human ethical committee of SLIMS. After obtaining consent from the patients who are interested in participating were included. Extensive Gangrene involving whole Foot Samples were collected from the patients after the debridement of the ulcer base. Before the sample, sterile normal saline followed by gentle rubbing of the foot wounds and tissue abrasion with 70% alcohol, to avoid contamination. Pus swab and tissue specimen were sent to Microbiology laboratory for sample processing, isolation and identi ication of microorganisms according to CLSI guidelines. The tissue samples were homogenized and inoculated on blood agar, and MacConkey agar was incubated for 24-48 hours at 37 • C under aerobic condition.
Isolation of anaerobic bacteria was incubated in an anaerobic chamber at 37 • C and examined at 48 hours and 96 hours. For isolation of fungal organisms specimen was inoculated in Sabouraud dextrose agar 25-30ºC at 48 hours. All isolates were Processes for Grams staining and biochemical tests and antibiotic sensitivity was done using Kirby Bauer's disc diffusion method. The antifungal susceptibility testing of yeast isolates was carried out using the disk diffusion method as per M44-A CLSI guidelines.
Of the 24 anaerobic isolates, 8 (33.3%) were resistant to clindamycin, followed by 6 (25%) to penicillin and 6 (25%) to cefoxitin. Imipenem and metronidazole were sensitive to all anaerobes in Table 9. Out of 35 isolates of C. albicans, 18 was resistant to luconazole, 16 were resistant to amphotericin B,12 was resistant to voriconazole, and 14 were resistance to Itraconazole. Of the 21aa isolates of Non-Candida albicans ssp, 11 were resistant to amphotericin B, 9 were resistant to Itraconazole, 6 were resistant to luconazole and voriconazole described in Table 10.
A foot ulcer is one of the debilitating complications in diabetics. Nearly half of all lower extremity amputations are diabetes-related (Yerat and Rangasamy, 2015). Antibiotic therapy for diabetic foot infections is started empirically following likely causative organism. The de initive treatment is later modi ied according to bacterial culture and sensitivity report. In this study, the age group of the patients ranged from 35 to 85 years. Most of the patients were in the age group 45-65 years, and most of the diabetic foot ulcer patients are men 63% compared to females 37%. Like our study, males were infected more than female was seen in other studies (Chakraborty and Mukherjee, 2015;Gopi et al., 2017). Most of the isolates in our Study from Wagner grading system II and III in DFU Similarly reported a maximum number of isolates in diabetic foot ulcer patients in Wagner grade II and III (Hefni et al., 2013).
The Enterobacteriaceae family was resistant to most of the antibiotics tested, except colistin and tigecycline. High resistance rates to Cipro loxacin and Levo loxacin in our present study correlates with other studies (Siami et al., 2001). High-Level resistant patterns like VRE, MRSA, and ESBL, our study shows (33%) VRE, (61%) MRSA and (40%) ESBL. Percentage resistance rates are comparable with other studies (Amini et al., 2013). This high antimicrobial resistance among diabetic Foot infecting bacteria may be due to several factors including previous antibiotic usage and its frequency and length of hospitalization stay.

CONCLUSIONS
Our present study shows Gram-negative bacteria are playing a signi icant role in diabetic foot ulcer infection. Surveillance and monitoring the antibiotic susceptibility of the isolates will be helpful in the empirical treatment of diabetic ulcers. Active infection control team and Clinical pharmacists should continuously monitor the prevalent organisms and prepare their antibiograms, periodically and inform the clinicians. This will help to reduce the cost by unnecessary usage of resistant antibiotics.