Original research

TE

Detection of Anaerobic Infection

in Diabetic Foot Ulcer Using
PCR Technique and the Status of
Metronidazole Therapy on Treatment
Outcome

PL

From the 1Department of

Endocrinology & Metabolism,
Institute of Medical Sciences,
Banaras Hindu University, Varanasi,
India; 2School of Biotechnology,
Banaras Hindu University, Varanasi,
India; 3Department of General
Surgery, Institute of Medical
Sciences, Banaras Hindu University,
Varanasi, India

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.

WOUNDS 2012;24(10):283288

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Nitin Aherrao, MD;1 Shailesh K. Shahi, MSc;2 Awanindra

Dwivedi, MSc;1 Ashok Kumar, PhD;2 Sanjeev Gupta, MS;3
Surya Kumar Singh, DM1

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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

iabetic 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
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Keypoints

Methods

TE

PL

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
This study was designed to detect prevalence of
anaerobic infection using PCR technique, and to
evaluate if Metronidazole is making a difference in
healing of DFU that detect positive for anaerobes.

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 (Fishers exact test, chi-square test, and Students 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
M each of the dNTPs, and 4 l of tissue extract DNA in a
total volume of 50 l. The amplification conditions were
as follows: 1 cycle of 94C for 5 minutes; followed by 30
cycles of 94C for 30 seconds; annealing for 1 minute (annealing temperatures for Bacteroides, Peptostreptococcus
productus and Clostridium were 52 C, 51C, and 53C,
respectively); 72C for 1 minute; final extension at 72C
for 8 minutes; and finally, cool down to 4C. 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).

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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 authors 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.

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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 nonMetronidazole 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

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Keypoints

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.
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.

PL

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Aherrao et al

Figure 2. Amplification of Bacteroides specific fragment

Lanes 1-5: 1-DF3, 2-DF4, 3-DF7, 4-DF8, and 5-DF10.
M-100bp ladder.

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

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Figure 1. Amplification of Clostridium specific amplicon

Lanes 1-5: 1-DF2, 2-DF4, 3-DF7, 4-DF8, and 5-DF9.
M- 100bp ladder.

Figure 3. Amplification of Peptostreptococcus productus Lanes 1-5: 1-DF2, 2-DF4, 3-DF9, 4-DF10, and
5-DF13. M-50bp ladder.

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

Keypoints

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, Fishers exact probability).
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Table 1. Comparison of baseline characteristics and the healing outcome of Metronidazole and nonMetronidazole treatment groups.

Distribution of age

Distribution of Sex

Non-Metronidazole
group
(Mean 2 SD)
(n = 15)

55.29 9.11

51.67 9.14

4 (23.5%)

3 (20%)

Female
Male

Distribution of Wagner Ulcer Classification grade

12 (80%)

1.000
(Fishers Exact
probability)

Grade 4

8 (47.1%)

5 (33.3%)

Grade 5

3 (17.6%)

3 (20%)

24.76 + 2.14

24.35 + 2.41

0.62

7.71 5.03

5.67 3.87

0.21

69.53 61.51

89.00 48.26

0.33

0.70 0.23

0.75 0.33

0.64

130.59 13.39

136.00 8.28

0.19

Diastolic

79.41 9.66

81.87 6.61

0.42

Hb level (g/l)

78.39 15.0

82.13 14.24

0.48

(17.99 9.36) x 109

(16.81 6.15) x 109

0.68

177.47 85.79

144.38 61.94

0.23

27.33 4.22

0.58

Duration of DMa (years)

Duration of DFb (days)

Ankle brachial pressure index

Blood pressure (mm Hg)

0.43

Total cell count (cells/L)

11

Creatinine (mol/L)

12

Albumin (g/L)

28.41 6.28

13

Calcium (mmol/L)

1.88 0.19

1.86 0.20

0.75

14

HbA1c (proportion of Hb)

0.12 0.02

0.11 0.02

0.24

15

Healing outcome of the 2 groups

13 (76.5%)

12 (80%)

4 (23.5%)

3 (20%)

1.000
(Fishers Exact probability)

10

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Body mass index

Systolic

7 (46.7%)

PL

6 (35.3%)

0.27

Grade 3

13 (76.4%)

Statistical
significance
(P value)

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Metronidazole group
(Mean 2 SD)
(n = 17)

Characteristics

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Serial No.

Healed
Non-healed
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Aherrao et al

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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 studys 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.

N
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PL

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)

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.
The strength of this studys 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.

Discussion

Keypoints

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statistically significant difference in the treatment outcome in both groups (P = 1, Fishers exact probability).

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Conclusion

8.

Acknowledgements

2.

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3.

Lipsky BA. Evidence-based antibiotic therapy of diabetic foot infections. FEMS Immunol Medical Microbiol. 1999;26:267-276.
Deresinski S. Infections in the diabetic patient: Strategies for the clinician. Infectious Disease Reports.
1995;1(1): 1-12.
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.
Lofmark S, Edlund C, Nord CE Metronidazole is still
the drug of choice for treatment of anaerobic infections. Clin Infect Dis. 2010;50:1623.
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.
Candel Gonzalez FJ, Alramadan M, Matesanz M, et
al.Infections in diabetic foot ulcers. Eur J Intern Med.
2003;14:341-343.
Chincholikar DA, Pal RB. Study of fungal and bacterial
infections of the diabetic foot. Indian J Pathol Microbiol. 2002;45:15-22.

1.

References

PL

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.

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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;13:351353.
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.

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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.

4.

5.

6.

7.

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