Diabetes mellitus being the global epidemic of 21^st 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., 2015). More than 85% of lower extremity amputations in patients with diabetes are preceded by foot ulcers (Pecoraro et al., 1990). Diabetic peripheral neuropathy and peripheral arterial disease are the key etiologic agents in foot ulceration. These may act together with other factors such as biomechanical, immunological imbalances, microvascular disease, foot deformities, hindered joint mobility, and increased susceptibility to infection thereby representing diabetic foot ulcers (DFUs) as a major social, medical and economic problem of developing countries (Eneroth et al., 1999). Infection is diagnosed on the presence of ≥2 classical signs of inflammation (erythema, edema, and purulence). Spanning the spectrum from simple, superficial cellulitis, microbial flora can lead to chronic osteomyelitis, and dreaded systemic toxicity along with gangrenous limbs resulting in lower extremity amputation (Lipsky et al., 2004). Often in association with lack of sensation, stiffened arteries, compromised host immune response, and recurrence of ulcers especially harbored by multidrug-resistant organisms places diabetic foot patients at higher risk of non-healing ulcers. Earlier studies have shown that most diabetic foot infections are polymicrobial with a predominance of Gram-positive cocci especially S. aureus, and Streptococci (Sapico et al., 1984; Jones et al., 1985). However, recent studies indicate the dominance of Gram-negative pathogens in the monomicrobial state, particularly members of family Enterobacteriace and Pseudomonas (Tiwari et al., 2011; Turhan et al., 2013). The prolonged course of antimicrobials for uninfected lesion or healed wounds, long hospital stay, and surgical measures may predispose patients to colonization and infection with drug-resistant pathogens and associated adverse outcomes. Multidrug-resistant organisms like methicillin-resistant Staphylococcus aureus (MRSA), metallo-beta-lactamases (MBL), extended-spectrum beta-lactamases (ESBL) producers, and ampicillin-resistant beta-lactamases (AmpC) producers further complicate the aura making lower extremity loss more common (Dang et al., 2003; Tascini et al., 2006; Kandemir et al., 2007; Richard et al., 2008). Proper wound management requires early analysis of infection and prompt initiation of appropriate antimicrobial therapy.

For infected ulcers, a post-debridement specimen from tissue should be obtained and processed for detection of causative pathogens (Zubair et al., 2010). Antibiotic susceptibility testing is a prerequisite for the management of infections which can help to make better therapeutic choices.

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.

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, 1990).

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, 1999).

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. (2002), where cultures yielded an average of 1.2 isolates per lesion, and were lower than the previous studies of Zubair et al. (2011), Gadepalli et al. (2006), and Shahid et al. (2009), with the reported average rate of isolation between 1.57, 2.3, and 5.8 respectively. The decrease in the number of isolates (1.2 organisms per lesion) in the present study in comparison to previously cited reports, could be because of widespread use of broad-spectrum antibiotics inclusive of piperacillin/tazobactam by the Primary Health Care physicians without obtaining proper pus culture before initiating antibiotic. Unlike Western countries reporting Gram-positive aerobes as predominant pathogens (Wang et al., 2010; Roberts and Simon, 2012), there has been a changing trend in the microorganisms causing DFI, with Gram-negative bacteria replacing Gram-positive bacteria in India (Ramakant et al., 2011; Umadevi et al., 2011). Interestingly, we have also observed the predominance of Gram-negative organisms in DFUs in our study and the same has also been reported by Mehta et al. (2014) and Shankar et al. (2005). However, previous studies reported the dominance of Gram-positive aerobes in diabetic foot infection (Mantey et al., 2000; Fejfarová et al., 2002). The ratio of Gram-negative to Gram-positive aerobes in our study was 2.9, which is lower than reported by Gadepalli et al. (2006). The prevalence of this kind of discrepancy in findings, could be because of geographical variations, age-sex composition, grading of ulcers and study settings included in the analysis.

In the present study, E. coli (19.5%), and S. aureus (18.3%) were the most predominating Gram-negative and Gram-positive isolates respectively. These findings are similar to those reported by Kandati et al. (2015) and Girish and Kumar (2011), from Southern India. Among non- healing ulcers, E. coli and P. aeruginosa (13.7% each), were most prevalent aerobes. This raises a serious concern for aggressive patterns of Gram-negative aerobes and their role in the poor healing of diabetic foot wounds.

All strains of S. aureus including MRSA were sensitive to vancomycin, which is similar to the findings of Reghu et al. (2016), Kandati et al. (2015), and Mehta et al. (2014), but against the findings of Girish and Kumar (2011).

Our study has shown that amongst the Enterobacteriace group, amikacin showed good antimicrobial activity against E. coli, followed by Klebsiella species, Citrobacter species, and Proteus species. Amikacin can, therefore, be considered as a better choice of drug in infection caused by these organisms. Our findings on the sensitivity of amikacin for Enterobacteriace are in tune with Girish and Kumar (2011), who reported the highest sensitivity of amikacin to Citrobacter species (100%), followed by Proteus species (99%), E. coli (86%) and Klebsiella species (72%). Our study also indicates that Pseudomonas infection can respond better to imipenem, which is corroborated by the earlier reports quoting imipenem, the best sensitive drug to P. aeruginosa (Mantey et al., 2000; Bansal et al., 2008).

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, 1999; Von Eiff et al., 2002).

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.