Abstract: Metronidazole is the drug of choice for anaerobic infection in diabetic foot ulcers (DFU) for a majority of clinicians. The present study was conducted to determine if Metronidazole is really making a difference in the healing of DFU. Methods. Deep tissue samples from the wound area of 61 diabetic foot patients were tested for anaerobic bacterial infection (Peptostreptococcus productus, Bacteroides, and Clostridium) by polymerase chain reaction (PCR). PCR-positive patients were randomized into 2 groups: Metronidazole and non-Metronidazole. Antibiotics for the control of infection were given in both groups as per clinical condition of patients. Treatment outcome was assessed by complete healing of the wound. Results. Out of 61 patients, PCR detected evidence of anaerobic infection in 32 (52%), while culture methods detected only 5 (8%) (Clostridium spp.), hence emphasizing the significance of the PCR technique over culture methods in detection of microbes. In this study, Clostridium was found with maximum prevalence of n (75%), followed by Bacteroides with n (53.1%), and Peptostreptococcus productus with n (40.6 %). Across all Wagner Ulcer Classification grades, Clostridium was the most prevalent anaerobe, and significantly associated with wound age and total leukocyte count. No difference was noted in wound healing in both groups at the end of 16 weeks. Conclusions. The authors propose that it is not mandatory to supplement Metronidazole in antibiotic regime for treatment of DFU.

Introduction

  Diabetic foot ulcers (DFU) are estimated to effect 15% of all individuals with diabetes during their lifetime and precede almost 85% of all foot amputations.1,2 They are the most common, disabling, and costly complications of diabetes. Diabetic foot wounds become easily infected due to several factors including suppressed immunity, inadequate blood supply, and neuropathy. Infection of a diabetic foot is one of the key contributing factors to morbidity and to a majority of lower limb amputations. It is further complicated by anaerobic pathogens resulting in delayed wound healing. The increase in resistance among anaerobic pathogens poses a problem in the choice of empiric antibiotic regimens.3 Metronidazole has become a choice among clinicians for eradication of anaerobic infection. The decision regarding management of anaerobic infection in a diabetic foot with specific therapy like Metronidazole is still a matter of debate.4   To the author’s knowledge, there are no published studies mentioning the use of PCR technique in detection of anaerobic infection in DFU, and determining efficacy of Metronidazole in treatment outcome of such foot ulcers. This study was designed primarily to detect prevalence of anaerobic infection by using PCR technique and to evaluate if Metronidazole is really making a difference in healing of DFU that detect positive for anaerobes.

Methods

  The protocol was approved by the Ethics Committee of the Department of Endocrinology and Metabolism, Banaras Hindu University (Varanasi, Uttar Pradesh, India) and informed consent was obtained from all subjects. The study enrolled diabetic foot patients visiting Sir Sundarlal Hospital, Banaras Hindu University, Varanasi, U.P, India, during the period of May 2010 to April 2011.   Deep tissue biopsy specimens were obtained from 61 patients with DFU who had an ankle brachial pressure index (ABPI) < 0.9 and a Wagner Ulcer Classification ≥ Grade 3. The specimens were collected in sterile phosphate buffered saline (PBS) from the wound sites and subsequently tested for the presence of anaerobes by PCR technique. Patients detected positive for anaerobes by PCR were assigned to either the Metronidazole or non–Metronidazole group using a randomization technique. Patients in both groups received antibiotics for the control of infection based on their clinical condition, DFU, previous culture sensitivity reports, and total leukocyte count. Both groups underwent surgical debridement and regular dressing changes. They also received insulin for glycemic control. Wound healing was assessed by reduction in wound size during subsequent follow-up visits on weeks 4, 8, 12, and 16. Complete healing was determined by the criterion of the Wound Healing Society as complete epithelialization of wound surface with absence of drainage.   Collected data was analyzed with appropriate statistical tests (Fisher’s exact test, chi-square test, and Student’s t test) using SPSSv16.0. (SPSS Inc, Chicago, IL, United States) and significance was tested at P < 0.05. Total genomic DNA of biopsy samples were extracted employing a Fast Tissue-to-PCR Kit, as per the instructions of the manufacturer (Fermentas Inc, Glen Burnie, MD, United States). The 16S rDNA PCR product sizes specific for genera Bacteroides, Peptostreptococcus productus, and Clostridium were 950 bp, 270 bp, and 619 bp, respectively, and were amplified using genus specific primer.5 Amplification was performed in a PTC-100 Thermal Cycler (MJ Research, Inc, Waltham, MA, United States). The PCR reaction mix included 1.5 U of Taq DNA polymerase (Bangalore Genei, Bangalore, India), 1X PCR buffer with 2.5 mM MgCl2, 25 pmol each of the forward and reverse primers (Integrated DNA Technologies, Inc, Coralville, IA, United States), 125 µM each of the dNTPs, and 4 µl of tissue extract DNA in a total volume of 50 µl. The amplification conditions were as follows: 1 cycle of 94ºC for 5 minutes; followed by 30 cycles of 94º C for 30 seconds; annealing for 1 minute (annealing temperatures for Bacteroides, Peptostreptococcus productus and Clostridium were 52º C, 51º C, and 53º C, respectively); 72º C for 1 minute; final extension at 72º C for 8 minutes; and finally, cool down to 4º C. Polymerase chain reaction products were checked for expected sizes on 1.5% agarose gel using a gel documentation unit (BioRad Laboratories, Hercules, CA, United States).

Results

  Out of 61 patients, 32 (52%) had evidence of anaerobic infection as detected by PCR, while only 5 (8%) were detected to have anaerobes by conventional culture methods. In the present study of 32 cases, Clostridium was found with maximum prevalence of 24 (75%), followed by Bacteroides with 17 (53.1%), and Peptostreptococcus producutus with 13 (40.6%) [Figures 1a, 1b, and 1c]. In 13.9 (43.5%) of the cases, only 1 anaerobe was detected, whereas 13.9 (43.5%) and 4 (12.5%) cases were found to have infection with any combination of 2, or with all 3, anaerobes, respectively. There was a statistically significant association of Clostridium infection with duration of diabetic foot and total leukocyte count (P = 0.029 and P = 0.008, respectively). On the other hand, no significant correlation was observed between the pattern of anaerobic infection and age; sex; duration of diagnosis with diabetes mellitus; or levels of hemoglobin, serum calcium, albumin, creatinine, and hemoglobin A1c. The patients randomized into 2 groups had similar clinical and biochemical characteristics at baseline (Table 1). At the end of 16 weeks of followup, in the 17-patient Metronidazole group, 13 (76.5%) were found to have complete healing of the wound, whereas in the 15-patient non-Metronidazole group, 12 (80%) had complete healing. There was no statistically significant difference in the treatment outcome in both groups (P = 1, Fisher’s exact probability).

Discussion

  There is a need for continuous surveillance of resistant bacteria to provide the basis for empirical therapy and reduce the risk of complications. This study was completed to determine prevalence of anaerobes in diabetic foot infections and actual consistency of effects of Metronidazole in healing of DFU. Diabetic foot infections are usually polymicrobial in nature due to aerobic bacteria (Staphylococcus spp., Streptococcus sp., Enterobacteriaceae), anaerobic bacteria (Bacteroides spp., Clostridium spp., Peptostretococcus spp.) and fungi.6,7   Conventional culture methods have long been used to identify bacteria in diabetic foot wounds. Culture results usually reveal a single organism,8 and sometimes even fail to demonstrate organisms, despite other clinical evidences of infection. Unfortunately, results of cultures are generally not available for at least 2-3 days.9 Polymerase chain reaction methods have made it possible to detect most species of pathogens in the wound in a matter of hours rather than days. Polymerase chain reaction is also able to detect much smaller concentrations of microorganisms than standard cultures and establish involvement of multiple organisms.10 Also, previous antibiotic therapy is much less likely to cause false-negative results with PCR than with the standard culture. Thus, being more reliable than the traditional culture method, PCR can be routinely used and could revolutionize how clinicians utilize antimicrobials against increasingly diverse and resistant pathogens.   The clinical characteristics of patients with anaerobic foot infections do not differ significantly from those without, except that their prevalence increases with a higher Wagner Ulcer Classification Grade. Thus, a high index of suspicion is needed for diabetic foot infections classified as a Wagner Grade ≥ 3.3. Clostridium was the most frequently identified anaerobe across all Wagner grades, followed by Bacteroides and Peptostreptococcus productus, whereas earlier studies showed higher prevalence of   Peptostreptococcus spp.3 An association was noted between Clostridium infection, duration of DFU, and total leukocyte count; the study findings suggested that advancing age of a wound increased the risk of Clostridium infection; however, the authors did not observe any kind of association of the other 2 anaerobes (ie, Bacteriodes and Peptostreptococcus productus) with patients’ clinical or biochemical characteristics. It was found that, at the end of 16 weeks, both groups reflected equivalent outcomes (ie, both had comparable healing of their ulcers, irrespective of the 2 treatment strategies given).   The authors interpret the results to mean Metronidazole is not mandatory for treatment of DFU with anaerobic infection, assuming antibiotics given for control of the aerobic infection are effective over the anaerobes in the wound. With this primary study, the authors propose that Metronidazole does not need to supplement an antibiotic regime for the treatment of DFU. The plausible reasons for the aforementioned findings could be the potency of empirical antibiotics being high enough to eradicate all kinds of infection, including anaerobes, or the formation of an unfavorable environment for growth of anaerobes.   Other likely explanations could be improvement of the immune system after eradication of aerobic bacteria, or development of resistance among anaerobic isolates against Metronidazole.3 Surgical debridement may also have resulted in elimination of anaerobes. The strength of this study’s observation relies on the fact that the authors only randomized cases into 2 groups that were detected positive for anaerobic infection by PCR and did not differ in terms of clinical and biochemical characteristics at baseline; thus, ascertainment bias should not be a concern in this data. In the study, evaluations performed using a conventional method demonstrated limited anaerobic bacteria. In contrast, the PCR technique could detect the maximum number of anaerobes. Although the study is small, this data could serve as the basis for therapeutic recommendations for DFU infections.

Conclusion

  This study demonstrated the significance of a molecular technique like PCR in detection of microbes in DFU. This finding could have further implications on the diagnosis and management of DFU in a cost-effective manner. It was also observed that in a DFU, antibiotics given for the control of aerobic infection were sufficient for the control of anaerobes as well. Metronidazole is not necessary for the eradication of anaerobic infection in DFU. This would reduce the cost of foot care in resource-constrained areas.

Acknowledgements

  The authors would like to thank Dr. Sameer Prabhakar from the Department of General Surgery for support with biopsy sample collection, and Dr. Archisman Mohapatra from the Department of Preventive and Social Sciences, Institute of Medical Science, Banaras Hindu University for support with statistical analysis of data. The authors also wish to express their gratitude to the Department of Biotechnology, Banaras Hindu University, and to the patients who participated in the study.

References

1. Lipsky BA. Evidence-based antibiotic therapy of diabetic foot infections. FEMS Immunol Medical Microbiol. 1999;26:267-276. 2. Deresinski S. Infections in the diabetic patient: Strategies for the clinician. Infectious Disease Reports. 1995;1(1): 1-12. 3. Colayco CAS, Mendoza MT, Alejandria MM, Ang CF. Microbiologic and clinical profile of anaerobic diabetic foot infections. Phil J Microbiol Infect Dis. 2002;31:151-160. 4. Lofmark S, Edlund C, Nord CE Metronidazole is still the drug of choice for treatment of anaerobic infections. Clin Infect Dis. 2010;50:16–23. 5. Rekha R, Rizvi MA , Jaishree P. Designing and validation of genus-specific primers for human gut flora study. Electron J Biotechnol. 2006;9:505-511. 6. Candel Gonzalez FJ, Alramadan M, Matesanz M, et al.Infections in diabetic foot ulcers. Eur J Intern Med. 2003;14:341-343. 7. Chincholikar DA, Pal RB. Study of fungal and bacterial infections of the diabetic foot. Indian J Pathol Microbiol. 2002;45:15-22. 8. Shankar EM, Mohan V, Premalatha G, Srinivasan RS, Usha AR. Bacterial etiology of diabetic foot infections in South India. Europ Journ of Intern Med. 2005;16:567-570. 9. Lipsky BA. Empirical therapy for diabetic foot infections: are there clinical clues to guide antibiotic selection. Clin Microbiol Infect. 2007;13:351–353. 10. Singh SK, Gupta K, Tiwari S, et al. Detecting aerobic bacterial diversity in patients with diabetic foot wounds using ERIC-PCR: A preliminary communication. Int J Low Extrem Wounds. 2009;8:203-208. Nittin Aherrao, MD; Awanindra Dwivedi, MSc; and Surya Kumar Singh, DM are from the Department of Endocrinology & Metabolism, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India. Shailesh K. Shahi, MSc; and Ashok Kumar, PhD are from the School of Biotechnology, Banaras Hindu University, Varanasi, India. Sanjeev Gupta, MS is from the Department of General Surgery, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India. Address correspondence to: Prof S. K Singh Department of Endocrinology & Metabolism, Institute of Medical Sciences Banaras Hindu University Varanasi – 221005 Uttar Pradesh, India sksingh.endocrinebhu@gmail.com