Bacteriological Profile of Aerobic and Anaerobic Isolates of Trophic Ulcer in Leprosy: A Study from Eastern India.

INTRODUCTION: Trophic ulcer is a dreaded complication of leprosy. Secondary infection compounds the damage to the already neglected ulcer. AIMS: To find out the bacterial pathogens in the isolates from trophic ulcers of leprosy and to find the drug sensitivity of the aerobic isolates so as to start a suitable antibiotic therapy.
METHODOLOGY: An institution-based, cross-sectional study done over a period of 2 years. Swab was taken from the deeper part of the ulcer. It was put on a suitable culture media. Bacteriological profile was determined and antibiogram was done subsequently.
RESULTS: Sixty patients with trophic ulcer secondary to leprosy were screened, among which all were screened for aerobic isolates and 38 were screened for anaerobic isolates. Among the aerobic isolates, 88% of patients were culture-positive. The most common organism was Staphylococcus aureus (37.7%), followed by Pseudomonas aeruginosa (22.64%), Proteus mirabilis (15.09%), Escherischia coli (13.2%), Klebsiella (9.43%). Maximum overall sensitivity was seen with amikacin (93.1%) and linezolid (89.65%). Maximum overall resistance was noted with cotrimoxazole (58.62%) and coamoxiclav (51.72%). Among the 38 patients cultured for anaerobic isolates, 17 were culture-positive for anaerobic organisms. Isolates showing Peptococcus were 6 (15.7%), purely Peptostreptococcus were 4 (10.5%), purely bacteroides were 3 (7.8%), and mixed growths were 4 (10.5%).
CONCLUSION: Secondary bacterial infection is quite common in leprosy trophic ulcers. The most common organism was Staphylococcus aureus. Isolates were mostly sensitive to amikacin and linezolid and resistant to cotrimoxazole and coamoxiclav. Anaerobic isolates were not uncommon, with Peptococcus being the most common among them.

Introduction

Trophic ulcers are one of the dreaded complications of leprosy. Owing to complete anesthesia of the affected part, the patient is completely unaware of the damage occurring due to trauma to the vulnerable pressure-prone areas of his body such as bony prominences of lateral malleoli, elbow, and heel of the hand (pisiform bone) leading to chronic nonhealing ulcers. The foot is the most common area of the body which is prone to develop ulcers due to cracks and fissures and trauma from external sources and also due to internal injuries caused by walking.[1] Furthermore, complications occur by superadded bacterial infections which are one of the major causes of its chronicity.

In such cases, knowledge of the most common bacteria infecting such an ulcer is useful in a clinical setup and in the field (where culture facilities are not available) in starting a treatment empirically and preventing progression of the condition which may lead even to amputation of the limb.

The relative lack of information about the most common aerobic and anaerobic bacteria infecting a trophic ulcer in leprosy in India has prompted us to undertake this study. This study aimed to find the bacterial pathogen (if any) in aerobic and anaerobic isolates from trophic ulcers of leprosy to demonstrate the drug sensitivity of the aerobic isolate(s) so as to start a suitable antibiotic therapy.

Methodology

This was an institution-based, cross-sectional study done over a period of 2 years with a sample size of 60 patients.

Inclusion criteria

Patients with leprosy with trophic ulcers

Informed consent (if <18 years age, then from guardian).

Exclusion criteria

Already on antibiotics

Comorbidities such as diabetes and other causes of trophic ulcers

Grossly contaminated ulcer.

Collection of sample

The ulcer was cleaned with normal saline. The slough was removed and the samples were collected from the deeper part of the ulcer with sterile bacterial loop. For aerobic culture, the material was transported by a sterile test tube, and for anaerobic culture, the material was put in Stuart's transport medium.

Culture and antibiotic sensitivity

For aerobic isolates, the sample was inoculated on MacConkey and nutrient agar for culture. Antibiogram was obtained by Kirby–Bauer disc diffusion technique and National Committee of Clinical Laboratory Standards guidelines.[2] For anaerobic culture, the material was put in blood agar with neomycin. It was immediately transferred to gas pack system to maintain anaerobic environment. It was incubated at 37°C for 2–3 days.

Antibiotic sensitivity for anaerobic culture could not be done due to insufficient logistic support.

Results

A total of 60 patients with trophic ulcer secondary to leprosy were screened, of which all patients were screened for aerobic isolates and 38 patients were screened for anaerobic isolates. Among these, around 88% were positive for aerobic isolates and around 45% were culture-positive for anaerobic isolates. Seven patients (12%) showed no growth.

The most common organism in aerobic isolates was Staphylococcus aureus (37.7%), followed by Pseudomonas aeruginosa (22.64%), Proteus mirabilis (15.09%), Escherischia coli (13.2%), and Klebsiella (9.43%). Mixed growth was noted in 15% of cases [Figure 1].

Figure 1

Growth pattern on aerobic isolates

Staphylococcus aureus was maximally sensitive to amikacin (100%), imipenem (100%), linezolid (100%), and gentamycin (85%). Pseudomonas aeruginosa was maximally sensitive to piperacillin + tazobactam (91.6%), cefoprazone + sulbactam (83.3%), amikacin (83.3%), and imipenem (83.3%). Escherischia coli was maximally sensitive to cefoperazone + sulbactam (100%), amikacin (100%), imipenem (85.7%), and linezolid (85.7%). Proteus mirabilis was maximally sensitive to cefoperazone + sulbactam (75%), amikacin (75%), and linezolid (75%) [Table 1].

Table 1

Sensitivity pattern of common aerobic isolates

Antibiotics	Staphylococcus aureus (n=20)	Pseudomonas aeruginosa (n=12)	Escherischia coli (n=7)	Klebsiella spp. (n=5)	Proteus mirabilis (n=8)
Amikacin	20	10	7	5	6
Linezolid	20	9	6	5	6
Gentamycin	17	6	5	4	3
Cefoperazone + sulbactam	18	10	7	5	6

Staphylococcus aureus showed maximum resistance to cotrimoxazole (55%) and coamoxiclav (45%). Pseudomonas aeruginosa too showed maximum resistance to cotrimoxazole (75%) and coamoxicalv (75%). Escherischia coli showed maximum resistance to amoxiclav (71.4%) and ciprofloxacin (57.1%). Proteus mirabilis showed maximum resistance to cotrimoxazole (75%), ciprofloxacin (75%), ceftazidime (75%), and cefepime (75%) [Table 2].

Table 2

Resistance pattern of common aerobic isolates

Antibiotics	Staphylococcus aureus (n=20)	Pseudomonas aeruginosa (n=12)	Escherischia coli (n=7)	Klebsiella spp. (n=5)	Proteus mirabilis (n=8)
Cotrimoxazole	11	9	3	3	6
Coamoxiclav	9	9	5	1	4
Cefepime	7	9	3	3	6
Cefixime	4	8	3	2	5

Maximum overall sensitivity was seen with amikacin (93.1%) and linezolid (89.65%). Maximum overall resistance was noted with cotrimoxazole (58.62%) and coamoxiclav (51.72%).

Among the 38 patients screened for anaerobic isolates, culture was positive in 17 (44.7%) patients. Among them, Peptococcus was the most common single isolate (15.7%), followed by Peptostreptococcus (10.5%) and bacteroides (7.8%), whereas mixed growth was seen in 4 (10.5%) cases [Figure 2].

Figure 2

Isolates on anaerobic culture

Discussion

Consecutive sixty patients of leprosy with trophic ulcer attending the leprosy outpatient department of the Department of Dermatology, Venereolgy and Leprosy of a tertiary care center of eastern India were studied. The patients were thoroughly examined according to the predetermined case record form, and the pus obtained from the trophic ulcer site was subjected to aerobic culture in 60 patients and, additionally, anaerobic culture was done in 38 patients. After the isolation of the organism, a suitable antibiogram was obtained by Kirby–Bauer disc diffusion technique to a set of specific antibiotics for the aerobic isolates. The antibiotic sensitivity for the anaerobic isolates could not be done because of lack of logistical support. Nevertheless, our study is one of the few studies to have done anaerobic culture on trophic ulcers ever published. The results obtained have been discussed below and are compared with the relevant literature wherever available [Tables 3–5].

Table 3

Comparison of culture patterns of different studies

Study	Aerobic isolates (total)	Anaerobic isolates (total)
Sharma et al.	51.1%	Not done
Ferriera et al. (n=30)	100%	Not done
Raja et al. (n=194)	100%	Not done
Majumdar et al. (n=56)	96.4%	Not done
George et al. (n=108)	63.8%	67.5%
Our study n=60 (aerobic) n=38 (anaerobic)	88.3%%	44.7%

Table 5

Comparison of culture growth of different studies for anaerobic isolates

Study	Peptococcus	Peptostreptococcus	Bacteroids	Mixed	No growth
George et al. (n=108)	Anaerobic cocci-27.7%		23.1%	63.8%	32.4%
Our study (n=38)	15.7%	10.5%	7.8%	10.5%	55.2%

Sharma et al. found that the most frequent bacterial isolates from trophic ulcers due to diabetes were Staphylococcus aureus (38.4%), Pseudomonas aeruginosa (17.5%), and Proteus (14%) in their study.[3]

Ferreira et al. reported that the most frequent isolates were Staphylococcus aureus (36.2%), Proteus mirabilis (15.5%), Enterobacter aerogenes (8.6%), Escherichia coli, Morganella morganii, and Pseudomonas aeruginosa (13.3%).[4]

Tiendrebeogo et al. reported Staphylococcus aureus as the most frequent bacterium isolated from such patients.[5]

In our study, the most common bacteria to be isolated were Staphylococcus aureus (37.7%), Pseudomonas aeruginosa (22.6%), Proteus mirabilis (15.09%), and Escherischia coli (13.2%) which correlated very well with the above studies.

Raja concluded that antimicrobial susceptibility results in his study showed that Gram-negative bacteria were sensitive to imipenem and amikacin, while vancomycin showed good activity against Gram-positive bacteria.[6] Ramani et al. found in their study that the aerobic bacteria were most sensitive to gentamycin.[7]

Majumdar et al. observed that out of 56 samples studied, aerobic bacterial growth was noted in 54 cases. Staphylococcus aureus, Escherischia coli, Proteus sp., and Pseudomonas sp. were isolated in 32, 16, 22, and 4 cases, respectively. No growth of organism was found in two cases. Mixed growth (more than one organism) was noticed in 20 (36%) samples. Chloramphenicol and gentamycin were the two drugs that showed efficacy to the extent of 75%–100% and 25%–100%, respectively in in vitro studies.[8]

Our study showed Staphylococcus aureus to be maximally sensitive to gentamycin (85%), amikacin (100%), imipenem (100%), and linezolid (100%); Pseudomonas aeruginosa to be maximally sensitive to piperacillin + tazobactam (91.6%), cefoprazone + sulbactam (83.3%), amikacin (83.3%), and imipenem (83.3%); Escherischia coli to be maximally sensitive to cefoperazone + sulbactam (100%), amikacin (100%), imipenem (85.7%), and linezolid (85.7%). Proteus mirabilis was maximally sensitive to cefoperazone + sulbactam (75%), amikacin (75%), and linezolid (75%).

Martínez-Gómez et al. found in their study that nearly 30% of Escherischia coli strains were resistant to amoxicillin/clavulanic acid and ciprofloxacin.[9]

Tiendrebeogo et al. found that Staphylococcus aureus and Pseudomonas which were the most common organisms isolated were resistant to many antibiotics such as tetracycline, penicillin, and cotrimoxazole.[5]

Ebenezer et al. concluded that cotrimoxazole and tetracycline were of little value in the treatment of neuropathic plantar ulcers.[10] Our study correlated well with the above studies. Staphylococcus aureus showed maximum resistance to cotrimoxazole (55%) and coamoxiclav (45%). Pseudomonas aeruginosa too showed maximum resistance to cotrimoxazole (75%) and coamoxicalv (75%). Escherischia coli showed maximum resistance to amoxiclav (71.4) and ciprofloxacin (57.1%). Proteus mirabilis showed maximum resistance to cotrimoxazole (75%), ciprofloxacin (75%), ceftazidime (75%), and cefepime (75%).

George et al. reported that materials from 108 trophic ulcers from leprosy cases were studied bacteriologically. Four cases showed growth of pure anaerobes and 69 showed mixed growth of aerobic and anaerobic bacteria. The predominant anaerobes were fusobacteria (41), anaerobic cocci (30), and bacteroides (25). Clostridia were isolated only in 10 cases.[11]

According to our study, among the 17 anaerobic culture-positive cases, those isolates showing single isolate of Peptococcus were 6 (15.7%), Peptostreptococcus were 4 (10.5%), bacteroides were 3 (7.8%), whereas mixed growth was seen in 4 (10.5%) cases. Due to lack of logistic support, antibiotic sensitivity of the anaerobic organisms could not be performed.

Conclusion

Secondary bacterial infection is quite common in leprosy trophic ulcers. The most common organisms are Staphylococcus aureus, Pseudomonas aeruginosa, and Proteus mirabilis. According to our study, isolates were mostly sensitive to amikacin and linezolid and resistant to cotrimoxazole and coamoxiclav. Amikacin and linezolid are the best drugs for empirical therapy at present in areas where culture facilities are not available, so as to curtail the duration of morbidity. Early treatment will ensure maximum limb salvage and prevent further complications, thus improving the quality of life of the patient.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

Table 4

Comparison of culture growth of different studies for aerobic isolates

Study	Staphylococcus aureus	Pseudomonas aeruginosa	Escherischia coli	Klebsiella spp.	Proteus spp.
Sharma et al.	38.4%	17.5%	-	-	14%
Ferriera et al. (n=30)	36.2%	13.3%	13%	-	15.5%
Raja et al. (n=194)	44%	25%	9%	15%	28%
Majumdar et al. (n=56)	59.2%	7.4%	29.6%	-	40.7%
Our study (n=60)	37.7%	22.6%	13.2%	9.4%	15.09%