Diabetes & Metabolic Syndrome: Clinical Research & Reviews 8 (2014) 129–132
Contents lists available at ScienceDirect
Diabetes & Metabolic Syndrome: Clinical Research & Reviews journal homepage: www.elsevier.com/locate/dsx
Original Article
Microbiological profile of bacterial pathogens from diabetic foot infections in tertiary care hospitals, Salem P. Sugandhi, D. Arvind Prasanth * Medical Microbiology Laboratory, Department of Microbiology, Periyar University, Salem 636 011, Tamil Nadu, India
A R T I C L E I N F O
A B S T R A C T
Keywords: Antimicrobial susceptibility Bacterial profile Diabetic foot ulcer Staphylococcus aureus Wagner’s grade
Background: Worldwide, diabetic foot infections are one of the most serious complications resulting in long term hospitalization among the diabetic patients. Aim: The aim of this study was to determine the microbial profile and the antibiogram pattern of the patients with diabetic foot infections. Methods: Pus samples were taken from 50 patients presenting with diabetic foot infections over a period of 10 months. The samples were processed by standard microbiological methods. Results: A total of 51 bacterial isolates were obtained from 50 patients with diabetic foot infections. The age group of these patients ranged from 30 to 80 years and the maximum number of patients were in the age group of 51–60 years. Gram negative (51%) were more prevalent than Gram positive (49%) organisms in this study. The commonest isolate was Staphylococcus aureus (41%) followed by Pseudomonas aeruginosa (35%), Enterococcus spp., (4%), Escherichia coli, (4%), Salmonella spp., (4%), Bacillus spp., (4%), Micrococcus spp., (2%), Listeria spp., (2%), Shigella spp., (2%) and Proteus spp., (2%). The antibiotic sensitivity pattern showed Meropenem, Piperacillin, Cefoperazone/Sulbactam, Piperacillin/Tazobactam and Amikacin as the most effective antimicrobial agents for the gram positive and Gram negative bacterial species. In this study, 8(44%) isolates of Gram negative bacilli were ESBL producers and 4 (19%) isolates were MRSA strains. Conclusion: The results of the study indicate that effective planning of therapy is very essential for the prevention of drug resistant organisms. ß 2014 Diabetes India. Published by Elsevier Ltd. All rights reserved.
1. Introduction Diabetic foot ulcerations are one of the leading causes of mortality and morbidity, in the developing countries [1]. The major factors that are predisposed to this condition are usually related to peripheral neuropathy and an impaired circulation, which limits the access of the phagocytes [2,3]. However, the microbial aetiology of these infections is usually complex. Many of these infections are either monomicrobial or polymicrobial in nature. In recent years, the presence of multidrug resistant organism has been reported very frequently which has further complicated the treatment regimes as well as increased the hospital stay and the cost involved in the treatment to these patients [4]. The management of these infections usually involves empirical treatment based on the antimicrobial susceptibility testing [5]. The association of multi drug resistant (MDR) pathogens with
diabetic foot ulcers increases the clinical conditions, complicate the treatment process further and possess a great challenge to the physicians or the surgeon in treating this condition [6]. Many studies have reported the bacteriology of diabetic foot infections (DFIs), but the results obtained so far have shown varying pattern based on the type and the severity of the infection, and are usually contradictory. Further there is a paucity of data with regard to the multi drug resistant organism namely ESBL-producing and the carbanemase producing organisms from this part of the country. Hence in this study an attempt has been made to study the microbiological profile of bacterial pathogens from the diabetic foot infections and determine the in vitro susceptibility pattern of these organisms to the routinely used antibiotics.
2. Materials and methods 2.1. General
* Corresponding author. Tel.: +91 9444080796. E-mail addresses: [email protected], [email protected] (D. Arvind Prasanth). http://dx.doi.org/10.1016/j.dsx.2014.07.004 1871-4021/ß 2014 Diabetes India. Published by Elsevier Ltd. All rights reserved.
A total of fifty patients with diabetes foot presenting at the outpatient department of the diabetic centre of a tertiary care
130
P. Sugandhi, D. Arvind Prasanth / Diabetes & Metabolic Syndrome: Clinical Research & Reviews 8 (2014) 129–132
hospital in Salem, Tamil nadu, India for a period of 10 months were included in this study. The institutional ethical clearance was obtained prior to the study. The informed consent was obtained from the patients prior to sampling. The clinical history of the patients was taken with regard to the age, sex, socioeconomic status, duration of diabetes; types of diabetes were recorded on a proforma exclusively meant for this study. After clinical assessment of the enrolled cases, the ulcers were graded according to the Wagner’s grade [7]. 2.2. Sample collection Two swabs were taken to collect the pus sample from deeper portions of the ulcer by making a rotatory movement with the swab. The samples were obtained using aseptic techniques to avoid contamination and were promptly transported to the laboratory in a sterile swab in ice-cold conditions.
Fig. 1. Age wise distribution pattern of patients presented in this study.
2.3. Processing of wound sample The samples were processed by inoculation on to culture media like Sheep Blood Agar (SBA), Brain Heart Infusion Agar (BHIA), Nutrient Agar (NA) and incubated at 37 8C for 24 h. The bacterial isolates grown on the media were confirmed by standard Biochemical test. 2.4. Antimicrobial susceptibility pattern The antimicrobial susceptibility testing of the bacterial isolates isolated during this study was performed as per the CLSI guidelines, 2012 [8]. The antimicrobial discs that were used in this study included Aztreonam (30 mg), Amoxyclav (30 mg), Cefpodoxime (10 mg), Cefepime (30 mg), Cefoperazone (75 mg), Cefoperazone/Sulbactam (75/10 mg), Cefixime (5 mg), Piperacillin (100 mg), Ceftazidime/Clavulanic acid (30/10 mg), Ceftriaxone (30 mg), Amikacin (30 mg), Rifampincin (5 mg), Meropenem (10 mg), Cefoxitin (30 mg), Ticarcillin/Clavulanic acid (75/ 10 mg), and Piperacillin/Tazobactam (100/10 mg) for the Gram negative bacilli. Erythromycin (15 mg), Methicillin (5 mg), Chloramphenicol (30 mg), Clindamycin (10 mg), Vancomycin (30 mg), Tetracycline (30 mg) and Ciprofloxacin (5 mg) were used to study the susceptibility patterns of the Gram positive cocci. All discs were obtained from Hi-Media Labs, Mumbai, India. 2.5. ESBL confirmatory test While performing antibiotic testing, Ceftazidime (30 mg) and Ceftazidime/Clavulanic acid (30/10 mg) discs were placed on Muller Hinton Agar (MHA) plate on which 0.5 McFarland of test organism was swabbed. Organism was considered as ESBL producer if there was 5 mm increase in zone diameter of Ceftazidime/Clavulanate disc and that of Ceftazidime disc alone. 2.6. MRSA confirmatory test Methicillin resistances of the Staphylococcus species isolated in this study was evaluated as per the CLSI guidelines, 2012 [8] by using Oxacillin (1 mcg) disc. 3. Results A total of 50 patients were presented in this study, which included 27 (54%) males and 23 (46%) females. The maximum number of patients was in the age group of 30–80 years. Most of
Fig. 2. Distribution of patients according to Wagner’s grade classification.
the patients were in the age group of 51–60 (Fig. 1). The mean age of the patients found in this study was 57 years. In this study, 35 patients presented with Grade I ulcer, 14 patients presented with Grade II ulcer, and 1 patient presented with Grade III ulcer (Fig. 2). The demographic details of the patients presenting in this study are summarized in Table 1. Out of the 50 samples, 42 (84%) samples were culture positive and 8(16%) samples were culture negative. A total of 51 bacterial isolates were obtained from 42 patients. In this study the gram positive cocci accounted for 49% which included
Table 1 Demographic detail of diabetic foot patients in this study. Demographic details
No. of patients
Age (mean) Sex Male Female Types of diabetes Type 1 Type 2 Duration of diabetics (years) 3 Size of ulcer (cm2) 4 >4 Nature of ulcer Necrotic Non necrotic
57 years
Percentage –
27 23
54 46
– 50
– 100
21 25 4
42 50 8
41 9
82 18
47 3
94 6
2 48
4 96
P. Sugandhi, D. Arvind Prasanth / Diabetes & Metabolic Syndrome: Clinical Research & Reviews 8 (2014) 129–132
131
Table 2 Microbiological profile of Bacteria isolated from diabetic foot infections. Organisms
No of isolates
Percentage
Gram positive Staphylococcus aureus Bacillus species Listeria species Micrococcus species
21 2 1 1
41 4 2 2
Gram negative Pseudomonas aeruginosa Escherichia coli Salmonella species Enterococcus species Shigella species Proteus species
18 2 2 2 1 1
35 4 4 4 2 2 Fig. 3. Types of microbial flora.
Staphylococcus aureus 41%, Micrococcus spp., 2%, Listeria spp., 2% and Bacillus spp., 4%. The Gram-negative bacilli accounted for 51% which included Pseudomonas aeruginosa 35%, Escherichia coli 4%, Salmonella spp., 4%, Shigella spp., 2%, Enterococcus spp., 4% and Proteus spp., 2%. The Gram negative organisms were isolated in greater number than the Gram positive organisms (Table 2). The microbial flora of bacterial isolates was summarized in Fig. 3. Methicillin-resistant S. aureus (MRSA) were reported in 4 (19%) cases and extended spectrum of Beta Lactamase (ESBL) isolates in 8 (44%) cases. In our study, Meropenem, Piperacillin, Cefoperazone/Sulbactam, Piperacillin/Tazobactam, Ticarcillin/ Clavulanic acid and Amikacin were the most effective antimicrobial agents for the Gram negative bacilli, while Amikacin, Tetracycline, Ciprofloxacin and Vancomycin were the most effective antimicrobial agents for the Gram positive organisms. The resistant pattern of these isolated is summarized in Tables 3 and 4. 4. Discussion The diabetic foot ulceration is one of the most important complications of diabetic patients leading to amputation and requires extreme care during treatment. However, clinicians have a greater role to play as it involves early diagnosis, prompt therapy and proper management. For the effective management of these infections, knowledge on the risk factors and the microorganism involved is very essential. Hence, this study was performed to evaluate the factors involved in this condition pertaining to this region. In this study the diabetic foot infection was significantly higher in males (54%) than in females (46%) and the patients in the age group of 51–60 years were most significantly infected with diabetic foot infections. The findings of this study are similar to the one reported by Umasankari et al. [9] and
Bentkover et al. [10], which had a higher predominance of male (85%) and people in the age group of 50–80 years as the most affected by foot infections. The diabetic foot infections are mostly polymicrobial. But in our study there were a higher percentage of mono microbial infections findings similar to the study of Dhanasekaran et al. [11] and Tiwari et al. [12]. Gram negative organisms were present in greater number than Gram positive organisms which correlated with the study of Sotto et al. [13]. In this study P. aeruginosa was the predominant bacterial species reported similar to the one observed by Citron et al. [14]; Sivanmaliappan et al. [15], which stated P. aeruginosa as the predominant isolate and this organisms should not be ignored and considered as contaminant as it results in sepsis leading to amputation. The results of this study were compatible with the findings of Lipsky et al. [16]; Gu et al. [17] and Warren et al. [18], which reported S. aureus as the predominant isolates. Esposito et al. [19] revealed that skin and soft tissue infections include decubitus ulcers, infected vascular or diabetic foot ulcers, had a high frequency of colonization with antimicrobialresistant organisms, including Methicillin-resistant S. aureus (MRSA), Vancomycin-resistant enterococci (VRE), penicillinresistant pneumococci (PRP), Extended spectrum beta-lactamase-producing Gram negative organisms (ESBL). In this study the presence of Methicillin-resistant S. aureus (MRSA) was also reported in 19% cases and Extended spectrum of Beta Lactamase (ESBL) in 44% cases. Most of the Gram positive cocci were found to be highly sensitive to Amikacin, Tetracycline and Vancomycin. Meropenem, Piperacillin, Cefoperazone/Sulbactam, Piperacillin/Tazobactam and Amikacin were the most effective antimicrobial agents for the Gram negative bacterial species. Abdulrazaka et al. [20] study stated that Metronidazole, Imipenem, Meropenem, Piperacillin and Clindamycin were the most effective antibiotics against the bacteriodes species.
Table 3 Antibiotic resistance pattern of gram positive organism from diabetic foot infections. Name of the organism
Total no of isolates
Antibiotics (% of resistance) *
OX
AK
C
CD
T
CIP
MET
VA
8 (38.09%) – – –
4 (19.04%) – – –
5 (23.8%) – – –
7 (33.3%) – – –
11 (52.38%) – – –
5 (23.8%) – – –
5 (23.8%) – – –
8 (38.09%) – – –
– – – –
E
Staphylococcus aureus Micrococcus species Bacillus species Listeria species *
21 1 2 1
E – Erythromycin; OX – Oxacillin; M – Methicillin; C – Chloramphenicol; CD – Clindamycin; V – Vancomycin; T – Tetracycline; CIP – Ciprofloxacin.
(100%)
(100%) (50%)
(100%) (50%) (100%) (100%)
AT – Aztreonam; AMC – Amoxyclav; CPD – Cefpodoxime; CPM – Cefepime; CPZ – Cefoperazone; CFS – Cefoperazone/Sulbactam; CFM – Cefixime; PI – Piperacillin; CAC – Ceftazidime/Clavulanic acid; CTR – Ceftriaxone; AK – Amikacin; RIF – Rifampincin; MRP – Meropenem; CX – Cefoxitin; TCC – Ticarcillin/Clavulanic acid; PIT – Piperacillin/Tazobactam.
*
4 (22%) – – – – – 4 (22%) – – – – – 16 (88%) 1 (50%) 2 (100%) 2 (100%) 1 (100%) 1 (100%) 10 (55%) – 2 (100%) 2 (100%) – 1 (100%) 2 (11%) – – – – – 18 2 2 2 1 1 Pseudomonas aeruginosa Escherichia coli Salmonella species Enterococcus species Shigella species Proteus species
4 (22%) – – 1 (50%) – –
4 – 2 1 1 1
(22%)
7 1 2 2 1 1
(38%) (50%) (100%) (100%) (100%) (100%)
14 (77%) 1 (50%) 2 (100%) 1 (50%) – –
2 (11%) – – 1 (50%) – –
13 (72%) 1 (50%) 2 (100%) 2 (100%) 1 (100%) –
4 – 2 1 – 1
(22%)
8 (44%) – – – – –
4 (22%) – 1(50%) 1 (50%) – –
15 (83%) 1 (50%) 2 (100%) 2 (100%) 1 (100%) 1 (100%)
AK PI CFM CPM MRP CPD TCC CAC CPZ AMC PIT CX AT CTR CFS *
Antibiotics (% of resistance)
Total no of isolates Name of the organism
Table 4 Antibiotic resistance pattern of Gram negative organism from diabetic foot infections.
15 (83%) – 2 (100%) 1 (50%) 1 (100%) 1 (100%)
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RIF
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5. Conclusion Diabetic foot infections are one of the major causes of social and economic problem. S. aureus and P. aeruginosa were found to be the most predominant bacterial pathogens from diabetic foot infections in this study. Knowledge on the antibiotic susceptibility pattern of these isolates is very crucial in the planning of appropriate treatment modalities of patients with foot infections. Hence proper management of diabetic foot infections is very essential which includes selection of appropriate therapeutic regime, good glycemic control and proper foot care. Conflict of Interest None. Acknowledgments We express our sincere thanks to the patients, doctors, the diabetes nurses, and the hospitals for making this study possible. We extend our thanks to the ethical committee for granting permission for this study and the head of the department for providing us with the facilities. References [1] Mohammad Z, Abida M, Jamal A. The clinico-bacteriology and the risk factors for diabetic foot infections with multidrug resistant micro-organisms in north India. Biol Med 2010;2(4):22–34. [2] Raja NS. Microbiology of the diabetic foot infections in a teaching hospital in Malaysia: a retrospective study of 194 cases. J Microbiol Immunol Infect 2007;40(1):39–44. [3] Ozer B, Kalachi A, Semerci E, Duran N. Infections and aerobic bacterial pathogens in diabetic foot infections. Afr J Microbiol Res 2010;4(20):2153–60. [4] Shankar EM, Mohan V, Premalatha G, Srinivasan RS, Usha AR. Bacterial aetiology of diabetic foot infections in south India. Eur J Intern Med 2005;16:567–70. [5] Sharma VK, Khakda PB, Joshi A, Sharma R. The common pathogens which were isolated from diabetic foot infections in Bir F hospital. Kathmandu Univ Med J 2006;4(3):295–301. [6] Yoga R, Khairul A, Sunitha K, Suresh C. Bacteriology of diabetic foot lesions. Med J Malays 2006;61:14–6. [7] Wagner FW. The dysvascular foot: a system of diagnosis and treatment. Foot Ankle 1981;2:64–122. [8] Clinical Laboratory Standards Institute (CLSI). 11th ed. Performance standards for antimicrobial susceptibility testing, vol. 32, 11th ed. 2012;p. 1. [9] Umasankari J, Jeya M, Sekar C. Microbiological study of diabetic foot ulcers and analysis of ESBL activity of the bacterial isolates. J Pharm Res 2012;5(4):2329–32. [10] Bentkover JD, Champion AH. Economic evaluation of alternative methods of treatment for diabetic foot ulcers patients. Wound Care Clin 1999;5:207–15. [11] Dhanasekaran G, Sastry G, Viswanathan M. Microbial pattern of soft tissue infections in diabetic patients in south India. Asian J Diabetes 2003;5:8–10. [12] Tiwari S, Pratyush DD, Dwivedi A, Gupta SK, Rai M, Singh SK. Microbiological and clinical characteristics of diabetic foot infections in northern India. J Infect Dev Ctries 2012;6(4):329–32. [13] Sotto A, Richard JL, Combescure C, Jourdan N. Beneficial effects of implementing guidelines on microbiology and costs of infected diabetic foot ulcers. Diabetologia 2010;53:2249–55. [14] Diane Citron M, Ellie Goldstein JC, Vreni Merriam C, Benjamin Lipsky A, Murray Abramson A. Bacteriology of moderate-to-severe diabetic foot infections and in vitro activity of antimicrobial agents. J Clin Microbiol 2007;45(9):2819–28. [15] Sivanmaliappan TS, Sevanan M. Antimicrobial susceptibility patterns of Pseudomonas aeruginosa from diabetes patients with foot ulcer. Int J Microbiol 2011;4. [16] Benjamin Lipsky A, Anthony Berendt R, Gunner Deery H, John Embil M, Warren Joseph S, Adolf Karchmer W, et al. Diagnosis and treatment of diabetic foot infections. Clin. Infect. Dis. 2004;39:885–910. [17] Gu J, Li H, Li M, Vuong C, Otto M, Wen Y, Gao Q. Bacterial insertion sequence IS256 as a potential molecular marker to discriminate invasive strains from commensal strains of Staphylococcus epidermidis. J. Hosp. Infect. 2005;61:342–8. [18] Warren YA, Tyrrell KL, Citron DME, Goldstein J. Clostridium aldenense sp. nov. and Clostridium citroniae sp. nov. isolated from human clinical infections. J Clin Microbiol 2006;44:2416–22. [19] Esposito S, Leone S, Noviello S, Lanniello F, Fiore M. Antibiotic resistance in long-term care facilities. New Microbiol. 2007;30:326–31. [20] Adel Abdulrazaka, Ibrahim Bitarb Z, Ayesh Al-Shamalic A, Ahmed Mobasherc L. Bacteriological study of diabetic foot infections. J Diabetes Complic 2005; 19:138–41.
Contents lists available at ScienceDirect
Diabetes & Metabolic Syndrome: Clinical Research & Reviews journal homepage: www.elsevier.com/locate/dsx
Original Article
Microbiological profile of bacterial pathogens from diabetic foot infections in tertiary care hospitals, Salem P. Sugandhi, D. Arvind Prasanth * Medical Microbiology Laboratory, Department of Microbiology, Periyar University, Salem 636 011, Tamil Nadu, India
A R T I C L E I N F O
A B S T R A C T
Keywords: Antimicrobial susceptibility Bacterial profile Diabetic foot ulcer Staphylococcus aureus Wagner’s grade
Background: Worldwide, diabetic foot infections are one of the most serious complications resulting in long term hospitalization among the diabetic patients. Aim: The aim of this study was to determine the microbial profile and the antibiogram pattern of the patients with diabetic foot infections. Methods: Pus samples were taken from 50 patients presenting with diabetic foot infections over a period of 10 months. The samples were processed by standard microbiological methods. Results: A total of 51 bacterial isolates were obtained from 50 patients with diabetic foot infections. The age group of these patients ranged from 30 to 80 years and the maximum number of patients were in the age group of 51–60 years. Gram negative (51%) were more prevalent than Gram positive (49%) organisms in this study. The commonest isolate was Staphylococcus aureus (41%) followed by Pseudomonas aeruginosa (35%), Enterococcus spp., (4%), Escherichia coli, (4%), Salmonella spp., (4%), Bacillus spp., (4%), Micrococcus spp., (2%), Listeria spp., (2%), Shigella spp., (2%) and Proteus spp., (2%). The antibiotic sensitivity pattern showed Meropenem, Piperacillin, Cefoperazone/Sulbactam, Piperacillin/Tazobactam and Amikacin as the most effective antimicrobial agents for the gram positive and Gram negative bacterial species. In this study, 8(44%) isolates of Gram negative bacilli were ESBL producers and 4 (19%) isolates were MRSA strains. Conclusion: The results of the study indicate that effective planning of therapy is very essential for the prevention of drug resistant organisms. ß 2014 Diabetes India. Published by Elsevier Ltd. All rights reserved.
1. Introduction Diabetic foot ulcerations are one of the leading causes of mortality and morbidity, in the developing countries [1]. The major factors that are predisposed to this condition are usually related to peripheral neuropathy and an impaired circulation, which limits the access of the phagocytes [2,3]. However, the microbial aetiology of these infections is usually complex. Many of these infections are either monomicrobial or polymicrobial in nature. In recent years, the presence of multidrug resistant organism has been reported very frequently which has further complicated the treatment regimes as well as increased the hospital stay and the cost involved in the treatment to these patients [4]. The management of these infections usually involves empirical treatment based on the antimicrobial susceptibility testing [5]. The association of multi drug resistant (MDR) pathogens with
diabetic foot ulcers increases the clinical conditions, complicate the treatment process further and possess a great challenge to the physicians or the surgeon in treating this condition [6]. Many studies have reported the bacteriology of diabetic foot infections (DFIs), but the results obtained so far have shown varying pattern based on the type and the severity of the infection, and are usually contradictory. Further there is a paucity of data with regard to the multi drug resistant organism namely ESBL-producing and the carbanemase producing organisms from this part of the country. Hence in this study an attempt has been made to study the microbiological profile of bacterial pathogens from the diabetic foot infections and determine the in vitro susceptibility pattern of these organisms to the routinely used antibiotics.
2. Materials and methods 2.1. General
* Corresponding author. Tel.: +91 9444080796. E-mail addresses: [email protected], [email protected] (D. Arvind Prasanth). http://dx.doi.org/10.1016/j.dsx.2014.07.004 1871-4021/ß 2014 Diabetes India. Published by Elsevier Ltd. All rights reserved.
A total of fifty patients with diabetes foot presenting at the outpatient department of the diabetic centre of a tertiary care
130
P. Sugandhi, D. Arvind Prasanth / Diabetes & Metabolic Syndrome: Clinical Research & Reviews 8 (2014) 129–132
hospital in Salem, Tamil nadu, India for a period of 10 months were included in this study. The institutional ethical clearance was obtained prior to the study. The informed consent was obtained from the patients prior to sampling. The clinical history of the patients was taken with regard to the age, sex, socioeconomic status, duration of diabetes; types of diabetes were recorded on a proforma exclusively meant for this study. After clinical assessment of the enrolled cases, the ulcers were graded according to the Wagner’s grade [7]. 2.2. Sample collection Two swabs were taken to collect the pus sample from deeper portions of the ulcer by making a rotatory movement with the swab. The samples were obtained using aseptic techniques to avoid contamination and were promptly transported to the laboratory in a sterile swab in ice-cold conditions.
Fig. 1. Age wise distribution pattern of patients presented in this study.
2.3. Processing of wound sample The samples were processed by inoculation on to culture media like Sheep Blood Agar (SBA), Brain Heart Infusion Agar (BHIA), Nutrient Agar (NA) and incubated at 37 8C for 24 h. The bacterial isolates grown on the media were confirmed by standard Biochemical test. 2.4. Antimicrobial susceptibility pattern The antimicrobial susceptibility testing of the bacterial isolates isolated during this study was performed as per the CLSI guidelines, 2012 [8]. The antimicrobial discs that were used in this study included Aztreonam (30 mg), Amoxyclav (30 mg), Cefpodoxime (10 mg), Cefepime (30 mg), Cefoperazone (75 mg), Cefoperazone/Sulbactam (75/10 mg), Cefixime (5 mg), Piperacillin (100 mg), Ceftazidime/Clavulanic acid (30/10 mg), Ceftriaxone (30 mg), Amikacin (30 mg), Rifampincin (5 mg), Meropenem (10 mg), Cefoxitin (30 mg), Ticarcillin/Clavulanic acid (75/ 10 mg), and Piperacillin/Tazobactam (100/10 mg) for the Gram negative bacilli. Erythromycin (15 mg), Methicillin (5 mg), Chloramphenicol (30 mg), Clindamycin (10 mg), Vancomycin (30 mg), Tetracycline (30 mg) and Ciprofloxacin (5 mg) were used to study the susceptibility patterns of the Gram positive cocci. All discs were obtained from Hi-Media Labs, Mumbai, India. 2.5. ESBL confirmatory test While performing antibiotic testing, Ceftazidime (30 mg) and Ceftazidime/Clavulanic acid (30/10 mg) discs were placed on Muller Hinton Agar (MHA) plate on which 0.5 McFarland of test organism was swabbed. Organism was considered as ESBL producer if there was 5 mm increase in zone diameter of Ceftazidime/Clavulanate disc and that of Ceftazidime disc alone. 2.6. MRSA confirmatory test Methicillin resistances of the Staphylococcus species isolated in this study was evaluated as per the CLSI guidelines, 2012 [8] by using Oxacillin (1 mcg) disc. 3. Results A total of 50 patients were presented in this study, which included 27 (54%) males and 23 (46%) females. The maximum number of patients was in the age group of 30–80 years. Most of
Fig. 2. Distribution of patients according to Wagner’s grade classification.
the patients were in the age group of 51–60 (Fig. 1). The mean age of the patients found in this study was 57 years. In this study, 35 patients presented with Grade I ulcer, 14 patients presented with Grade II ulcer, and 1 patient presented with Grade III ulcer (Fig. 2). The demographic details of the patients presenting in this study are summarized in Table 1. Out of the 50 samples, 42 (84%) samples were culture positive and 8(16%) samples were culture negative. A total of 51 bacterial isolates were obtained from 42 patients. In this study the gram positive cocci accounted for 49% which included
Table 1 Demographic detail of diabetic foot patients in this study. Demographic details
No. of patients
Age (mean) Sex Male Female Types of diabetes Type 1 Type 2 Duration of diabetics (years) 3 Size of ulcer (cm2) 4 >4 Nature of ulcer Necrotic Non necrotic
57 years
Percentage –
27 23
54 46
– 50
– 100
21 25 4
42 50 8
41 9
82 18
47 3
94 6
2 48
4 96
P. Sugandhi, D. Arvind Prasanth / Diabetes & Metabolic Syndrome: Clinical Research & Reviews 8 (2014) 129–132
131
Table 2 Microbiological profile of Bacteria isolated from diabetic foot infections. Organisms
No of isolates
Percentage
Gram positive Staphylococcus aureus Bacillus species Listeria species Micrococcus species
21 2 1 1
41 4 2 2
Gram negative Pseudomonas aeruginosa Escherichia coli Salmonella species Enterococcus species Shigella species Proteus species
18 2 2 2 1 1
35 4 4 4 2 2 Fig. 3. Types of microbial flora.
Staphylococcus aureus 41%, Micrococcus spp., 2%, Listeria spp., 2% and Bacillus spp., 4%. The Gram-negative bacilli accounted for 51% which included Pseudomonas aeruginosa 35%, Escherichia coli 4%, Salmonella spp., 4%, Shigella spp., 2%, Enterococcus spp., 4% and Proteus spp., 2%. The Gram negative organisms were isolated in greater number than the Gram positive organisms (Table 2). The microbial flora of bacterial isolates was summarized in Fig. 3. Methicillin-resistant S. aureus (MRSA) were reported in 4 (19%) cases and extended spectrum of Beta Lactamase (ESBL) isolates in 8 (44%) cases. In our study, Meropenem, Piperacillin, Cefoperazone/Sulbactam, Piperacillin/Tazobactam, Ticarcillin/ Clavulanic acid and Amikacin were the most effective antimicrobial agents for the Gram negative bacilli, while Amikacin, Tetracycline, Ciprofloxacin and Vancomycin were the most effective antimicrobial agents for the Gram positive organisms. The resistant pattern of these isolated is summarized in Tables 3 and 4. 4. Discussion The diabetic foot ulceration is one of the most important complications of diabetic patients leading to amputation and requires extreme care during treatment. However, clinicians have a greater role to play as it involves early diagnosis, prompt therapy and proper management. For the effective management of these infections, knowledge on the risk factors and the microorganism involved is very essential. Hence, this study was performed to evaluate the factors involved in this condition pertaining to this region. In this study the diabetic foot infection was significantly higher in males (54%) than in females (46%) and the patients in the age group of 51–60 years were most significantly infected with diabetic foot infections. The findings of this study are similar to the one reported by Umasankari et al. [9] and
Bentkover et al. [10], which had a higher predominance of male (85%) and people in the age group of 50–80 years as the most affected by foot infections. The diabetic foot infections are mostly polymicrobial. But in our study there were a higher percentage of mono microbial infections findings similar to the study of Dhanasekaran et al. [11] and Tiwari et al. [12]. Gram negative organisms were present in greater number than Gram positive organisms which correlated with the study of Sotto et al. [13]. In this study P. aeruginosa was the predominant bacterial species reported similar to the one observed by Citron et al. [14]; Sivanmaliappan et al. [15], which stated P. aeruginosa as the predominant isolate and this organisms should not be ignored and considered as contaminant as it results in sepsis leading to amputation. The results of this study were compatible with the findings of Lipsky et al. [16]; Gu et al. [17] and Warren et al. [18], which reported S. aureus as the predominant isolates. Esposito et al. [19] revealed that skin and soft tissue infections include decubitus ulcers, infected vascular or diabetic foot ulcers, had a high frequency of colonization with antimicrobialresistant organisms, including Methicillin-resistant S. aureus (MRSA), Vancomycin-resistant enterococci (VRE), penicillinresistant pneumococci (PRP), Extended spectrum beta-lactamase-producing Gram negative organisms (ESBL). In this study the presence of Methicillin-resistant S. aureus (MRSA) was also reported in 19% cases and Extended spectrum of Beta Lactamase (ESBL) in 44% cases. Most of the Gram positive cocci were found to be highly sensitive to Amikacin, Tetracycline and Vancomycin. Meropenem, Piperacillin, Cefoperazone/Sulbactam, Piperacillin/Tazobactam and Amikacin were the most effective antimicrobial agents for the Gram negative bacterial species. Abdulrazaka et al. [20] study stated that Metronidazole, Imipenem, Meropenem, Piperacillin and Clindamycin were the most effective antibiotics against the bacteriodes species.
Table 3 Antibiotic resistance pattern of gram positive organism from diabetic foot infections. Name of the organism
Total no of isolates
Antibiotics (% of resistance) *
OX
AK
C
CD
T
CIP
MET
VA
8 (38.09%) – – –
4 (19.04%) – – –
5 (23.8%) – – –
7 (33.3%) – – –
11 (52.38%) – – –
5 (23.8%) – – –
5 (23.8%) – – –
8 (38.09%) – – –
– – – –
E
Staphylococcus aureus Micrococcus species Bacillus species Listeria species *
21 1 2 1
E – Erythromycin; OX – Oxacillin; M – Methicillin; C – Chloramphenicol; CD – Clindamycin; V – Vancomycin; T – Tetracycline; CIP – Ciprofloxacin.
(100%)
(100%) (50%)
(100%) (50%) (100%) (100%)
AT – Aztreonam; AMC – Amoxyclav; CPD – Cefpodoxime; CPM – Cefepime; CPZ – Cefoperazone; CFS – Cefoperazone/Sulbactam; CFM – Cefixime; PI – Piperacillin; CAC – Ceftazidime/Clavulanic acid; CTR – Ceftriaxone; AK – Amikacin; RIF – Rifampincin; MRP – Meropenem; CX – Cefoxitin; TCC – Ticarcillin/Clavulanic acid; PIT – Piperacillin/Tazobactam.
*
4 (22%) – – – – – 4 (22%) – – – – – 16 (88%) 1 (50%) 2 (100%) 2 (100%) 1 (100%) 1 (100%) 10 (55%) – 2 (100%) 2 (100%) – 1 (100%) 2 (11%) – – – – – 18 2 2 2 1 1 Pseudomonas aeruginosa Escherichia coli Salmonella species Enterococcus species Shigella species Proteus species
4 (22%) – – 1 (50%) – –
4 – 2 1 1 1
(22%)
7 1 2 2 1 1
(38%) (50%) (100%) (100%) (100%) (100%)
14 (77%) 1 (50%) 2 (100%) 1 (50%) – –
2 (11%) – – 1 (50%) – –
13 (72%) 1 (50%) 2 (100%) 2 (100%) 1 (100%) –
4 – 2 1 – 1
(22%)
8 (44%) – – – – –
4 (22%) – 1(50%) 1 (50%) – –
15 (83%) 1 (50%) 2 (100%) 2 (100%) 1 (100%) 1 (100%)
AK PI CFM CPM MRP CPD TCC CAC CPZ AMC PIT CX AT CTR CFS *
Antibiotics (% of resistance)
Total no of isolates Name of the organism
Table 4 Antibiotic resistance pattern of Gram negative organism from diabetic foot infections.
15 (83%) – 2 (100%) 1 (50%) 1 (100%) 1 (100%)
P. Sugandhi, D. Arvind Prasanth / Diabetes & Metabolic Syndrome: Clinical Research & Reviews 8 (2014) 129–132
RIF
132
5. Conclusion Diabetic foot infections are one of the major causes of social and economic problem. S. aureus and P. aeruginosa were found to be the most predominant bacterial pathogens from diabetic foot infections in this study. Knowledge on the antibiotic susceptibility pattern of these isolates is very crucial in the planning of appropriate treatment modalities of patients with foot infections. Hence proper management of diabetic foot infections is very essential which includes selection of appropriate therapeutic regime, good glycemic control and proper foot care. Conflict of Interest None. Acknowledgments We express our sincere thanks to the patients, doctors, the diabetes nurses, and the hospitals for making this study possible. We extend our thanks to the ethical committee for granting permission for this study and the head of the department for providing us with the facilities. References [1] Mohammad Z, Abida M, Jamal A. The clinico-bacteriology and the risk factors for diabetic foot infections with multidrug resistant micro-organisms in north India. Biol Med 2010;2(4):22–34. [2] Raja NS. Microbiology of the diabetic foot infections in a teaching hospital in Malaysia: a retrospective study of 194 cases. J Microbiol Immunol Infect 2007;40(1):39–44. [3] Ozer B, Kalachi A, Semerci E, Duran N. Infections and aerobic bacterial pathogens in diabetic foot infections. Afr J Microbiol Res 2010;4(20):2153–60. [4] Shankar EM, Mohan V, Premalatha G, Srinivasan RS, Usha AR. Bacterial aetiology of diabetic foot infections in south India. Eur J Intern Med 2005;16:567–70. [5] Sharma VK, Khakda PB, Joshi A, Sharma R. The common pathogens which were isolated from diabetic foot infections in Bir F hospital. Kathmandu Univ Med J 2006;4(3):295–301. [6] Yoga R, Khairul A, Sunitha K, Suresh C. Bacteriology of diabetic foot lesions. Med J Malays 2006;61:14–6. [7] Wagner FW. The dysvascular foot: a system of diagnosis and treatment. Foot Ankle 1981;2:64–122. [8] Clinical Laboratory Standards Institute (CLSI). 11th ed. Performance standards for antimicrobial susceptibility testing, vol. 32, 11th ed. 2012;p. 1. [9] Umasankari J, Jeya M, Sekar C. Microbiological study of diabetic foot ulcers and analysis of ESBL activity of the bacterial isolates. J Pharm Res 2012;5(4):2329–32. [10] Bentkover JD, Champion AH. Economic evaluation of alternative methods of treatment for diabetic foot ulcers patients. Wound Care Clin 1999;5:207–15. [11] Dhanasekaran G, Sastry G, Viswanathan M. Microbial pattern of soft tissue infections in diabetic patients in south India. Asian J Diabetes 2003;5:8–10. [12] Tiwari S, Pratyush DD, Dwivedi A, Gupta SK, Rai M, Singh SK. Microbiological and clinical characteristics of diabetic foot infections in northern India. J Infect Dev Ctries 2012;6(4):329–32. [13] Sotto A, Richard JL, Combescure C, Jourdan N. Beneficial effects of implementing guidelines on microbiology and costs of infected diabetic foot ulcers. Diabetologia 2010;53:2249–55. [14] Diane Citron M, Ellie Goldstein JC, Vreni Merriam C, Benjamin Lipsky A, Murray Abramson A. Bacteriology of moderate-to-severe diabetic foot infections and in vitro activity of antimicrobial agents. J Clin Microbiol 2007;45(9):2819–28. [15] Sivanmaliappan TS, Sevanan M. Antimicrobial susceptibility patterns of Pseudomonas aeruginosa from diabetes patients with foot ulcer. Int J Microbiol 2011;4. [16] Benjamin Lipsky A, Anthony Berendt R, Gunner Deery H, John Embil M, Warren Joseph S, Adolf Karchmer W, et al. Diagnosis and treatment of diabetic foot infections. Clin. Infect. Dis. 2004;39:885–910. [17] Gu J, Li H, Li M, Vuong C, Otto M, Wen Y, Gao Q. Bacterial insertion sequence IS256 as a potential molecular marker to discriminate invasive strains from commensal strains of Staphylococcus epidermidis. J. Hosp. Infect. 2005;61:342–8. [18] Warren YA, Tyrrell KL, Citron DME, Goldstein J. Clostridium aldenense sp. nov. and Clostridium citroniae sp. nov. isolated from human clinical infections. J Clin Microbiol 2006;44:2416–22. [19] Esposito S, Leone S, Noviello S, Lanniello F, Fiore M. Antibiotic resistance in long-term care facilities. New Microbiol. 2007;30:326–31. [20] Adel Abdulrazaka, Ibrahim Bitarb Z, Ayesh Al-Shamalic A, Ahmed Mobasherc L. Bacteriological study of diabetic foot infections. J Diabetes Complic 2005; 19:138–41.
