Int. J. Life. Sci. Scienti. Res., 1(1): 26-32, September 2015

 

Prevalence of Bacterial Infection in Patients with Diabetic Foot Lesions

 

Santosh Kumar Verma¹*, Poonam Verma¹

 

¹Jacob School of Biotechnology and Bioengineering

Sam Higginbottom Institute of Agriculture,Technology and sciences, Allahabad-211007, (U.P.) India

 

Correspondence to Author- Santosh Kumar Verma, Jacob School of Biotechnology and Bioengineering, Sam Higginbottom Institute of Agriculture,Technology and sciences, Allahabad-211007, (U.P.) India

 

ABSTRACT- Diabetic foot infections are the most common problems in persons with diabetes. Among the 50 samples, 43 (86%) showed positive results of bacterial infection. Diabetic foot lesions are divided into six grades based on the depth of the wound and extent of the tissue necrosis. Incidences of bacteria were recorded as Staphylococcus aureus (31.37%) followed by Proteus mirabilis (21.05%), Pseudomonas aeruginosa (15.79%), Streptococcus pyogenes (14.04%), Escherichia coli (7.02%), Clostridium botulinum (5.26%), Peptococcus spp. (3.50%) and Salmonella typhimurium (1.75).  The prevalence of diabetic foot infections varies according to sex, age, sugar level and economic status. Males were more susceptible to infection than females because of higher outdoor activities. Age groups of 40-50 years and fasting sugar levels of 100-150 mg/dl showed maximum incidence of bacterial infection in diabetic foot lesions. Maximum incidences of bacterial infection were found in patients of poor economic status followed by those of middle and high economic status respectively, due to lack of education about the disease and unhygienic surroundings. Except Peptococcus spp. the remaining isolates exhibited Multiple Drug Resistance (MDR). The selection of empiric antibiotic therapy depends on various factors such as infection severity, over all patient condition, medication allergies, previous antibiotic treatment, antibiotic activity, toxicity, excretion and glycemic control. Proper identification of causative agents, appropriate antibiotic therapy and management of complications of diabetic foot infections remain essential to the achievement of a successful outcome.

Key words: Diabetic foot infection and Multiple Drug Resistance

 

Introduction- Diabetes mellitus is a chronic disorder and affects large segment of population and is a major public health problem. Diabetes and foot problems are almost synchronous (Frykberg, 1998; Blazer and Heidrich, 1999; Logerfo and Coffman, 1984; Shea, 1999). The group of three problem leading on to the diabetic foot is neuropathy, vascular changes and Infections, which constitute the diabetic foot syndrome (Smith et al., 2002; Meade and Miler, 1968). Foot infections in the diabetic constitute a tremendous clinical and financial burden to the patients involved, the clinicians caring for these patients, and the community as a whole. Approximately 20% of diabetics admitted to the hospital are seen primarily for their foot problems (Levin and O’Neal, 1983). Fifty to seventy per cent of all non-traumatic amputations are performed on these diabetic patients (Gibbons and Eliopoulos, 1984).

Foot ulceration and infections are one of the leading causes of mortality and morbidity, especially in developing countries .The numbers of cases and problems associated with diabetic foot infections (DFI) have dramatically increased in recent years (Mohan and Pradeepa, 2004; Abdul et al., 1999). The main reason for this increase is the growing diabetic population in younger groups. Ulceration of the foot in diabetics is common and disabling and frequently leads to amputation of the leg. Mortality is high and healed ulcers often recur. The pathogenesis of foot ulceration is complex, clinical presentation is variable, and its management requires early expert assessment (Calhoun et al., 2002). Foot ulcers are a significant complication of diabetes which is the most common cause of no traumatic lower extremity amputations in the industrialized world. The risk of lower extremity amputation is 15 to 46 times higher in diabetics than in persons who do not have diabetes mellitus (Sarkar and Ballantyne, 2000; Fahey et al., 1991). Diabetes mellitus is recognized as an epidemic in the Asian sub-continent affecting nearly 25 millions in India alone. Diabetic foot ulcers are estimated to affect 15%of all diabetics during their lifetime and precede almost 85% of all foot amputations (Lipsky, 1999; Deresinski, 1995). Defects in host defense mechanisms have been described in the diabetic patient. Such defects include reduced leukocyte mobilization and chemotaxis and reduced phagocytic and bactericidal capacity (Mowat and Baum, 1971; Tan et al., 1975). Diabetes by virtues of its other complications like neuropathy and vasculopathy and other factors alters the musculoskeletal and soft tissue mechanics in a manner that elevates planter pressure and makes tissue damage more likely, causing nonresolving neuro-ischemic ulcers at the weight bearing sites. This is why most of the skin injuries in diabetics are seen on the planter surface, frequently at the site of highest pressure under the foot (Lipsky, 1997; Pendsey, 1994).

Infection complicates the pathological picture of diabetic foot and plays a main role in the development of moist gangrene (Shea, 1999; Smith et al., 2002; Meade and Miler, 1968). Pseudomonas spp., Enterococcus spp. and Proteus spp. carry a special role and are responsible for continuing and extensive tissue destruction with the poor blood circulation of the foot. A high frequency of anaerobic Infection has also been reported (Bailey et al., 1985; Miler and Amyes, 1996; Forbes et al., 1998). Patients with diabetes also can have a combined infection involving bone and soft tissue called fetid foot. This extensive soft tissue and bone infection causes a foul exudate, is chronic, and usually requires extensive surgical debridement and/or amputation. In general, people with diabetes have infections that are more severe and take longer to cure than equivalent infections in other people.

The infection leads to the early development of complication even after a trivial trauma, the disease progresses and becomes refractory to antibacterial therapy (Pittet et al., 1999; Pathare et al., 1998). It is essential to assess the magnitude of bacterial infection of the lesions to avoid further complications and save the diabetic foot. Early diagnosis of micorbial infections is aimed to institute the appropriate antibacterial therapy and to avoid further complications (Bailey et al., 1985; Frykberg, 1998). However, these infections are difficult to treat because these patients have impaired microvascular circulation, which limits the access of phagocytic cells to the infected area and results in a poor concentration of antibiotics in the infected tissues. For this reason, cellulitis is the most easily treatable and reversible form of foot infections in patientswith diabetes. Deep skin and soft tissue infections also usually are curable, but they can be life threatening and result in substantial longterm morbidity (Caputo et al., 1994).

Diabetic soft-tissue infections result in significant morbidity in this population of patients. The spectrum of disease ranges from infected foot ulcers, cellulitis to chronic osteomyelitis. Infections in diabetes are often polymicrobial, involving a mixture of aerobic and anaerobic flora (Lipsky, 1997). Antibiotic therapy is often empirical and an antibiotic with anaerobic cover is often recommended (Lipsky, 2004).

Antibiotic resistance in aerobic bacteria is of global concern; however, antibiotic resistance in anaerobes is often overlooked. With reports of resistance to anaerobic antimicrobials (Aldridge et al., 2001; Teng et al., 2002), and variable antimicrobial resistance amongst anaerobic genera (Snydman et al., 2002) continued surveillance of anaerobic susceptibility patterns is vital to determine current and future trends (Brazier et al., 1999).

The present study assumes significance in the Indian context where the disease is itself detected late, there is little awareness for foot care in patients and there is a significant delay in seeking the treatment. Further, a significant population is rural and work in the fields barefoot, thus increasing the chances of further infection. In such a situation, the treating physician is left with the option of treating empirically till the culture reports are available. A rough idea of the antibiotic pattern would be a useful aid for him.

 

MATERIALS AND METHODS

Place of work- The present study entitled “Prevalence of bacterial infection in patients with diabetic foot lesions” was conducted in the Department of Microbiology and Fermentation Technology, Sam Higginbotom Institute of Agriculture, Technology and Sciences, Deemed to be- University, Allahabad.

Study Material- Foot lesions samples were collected from 50 patients, suffering from Diabetic Foot infection and treated in different hospital (Swaroop Rani Hospital, Pooja hospital, The Leprosy Mission Hospital and Toshi Pathology) of Allahabad. These patients were clinically assessed and the foot lesions are classified and graded according to Wagner grading system. In the Wagner classification system, foot lesions are divided into six grades based on the depth of the wound and extent of tissue necrosis.

 

Grade 0- Preulcer. No open lesions skin intact; may have deformities, erythematous areas of pressure or hyperkeratosis

Grade 1 - Superficial ulcer clinically not infected

Grade 2 - Deep ulcer often infected but no bone involvement

Grade 3 - Deep ulcer, abscess formation and bone involvement

Grade 4 - Localized gangrene

Grade 5 - Gangrene of whole foot

Collection of samples- Discharge from the incised lesions or ulcer was collected with sterile swabs. Pus aspirated from the abscesses and debrided necrotic materials were collected for aerobic and anaerobic culture. During the sample collection the patient Performa containing details of the patient was collected (App. 1).

Isolation- Gram stained direct smear of the specimen was examined. The specimen was inoculated on to Blood agar, MacConkey’s Agar and Thioglycollate broth (App. 3.4, 3.1 and 3.3) for aerobic and anaerobic culture and incubated at 37°C for 48 hrs.

Identification of isolates- The bacterial isolates was identified by cultural and physiological, morphological and biochemical tests according to Bergey’s manual of determinative bacteriology (Holt et al., 1984) (Table 3.1).

Cultural and physiological characteristics- The isolates were identified on the basis of different colony characteristics like colour, texture, opaque etc. on culture plate (Fig. 1).

 

Fig. 1: Cultural characteristics of diabetic foot lesion isolates

 

Morphological characteristics- A gram staining of isolated bacteria was done and observation of shape and arrangement under 100x objective microscope (Table 1).

Biochemical characteristics- Different biochemical tests were performed for the identification of the microorganism (Cappuccino et al., 2004).

 

Carbon Source (+ve= Acid positive; (G) = Gas positive; -ve= Acid negative)

TSI (A=acid production; K=alkaline reaction; G=gas production; H₂S=sulfur reduction)

 

Antibiotic Susceptibility Test- Antibacterial susceptibility testing was performed by Kirby Bauer’s disc diffusion method according to National Committee for Clinical Laboratory Standards guidelines (NCCLS, 2002).

 

Statistical analysis- The data recorded during the course of investigation were statistically analysed by using chi square (χ²) test, correlation, t-test and conclusion was drawn (Fisher, 1950).

 

RESULTS AND DISCUSSION

Prevalence of different lesions in diabetic foot lesions- In the present study total 50 patients of diabetic foot lesion were studied for the presence of bacteria in their pus samples.  Of them 50 pus samples, 43 (86%) showed positive results of bacterial infection. The polymicrobial infection rate was low (20.93%) in this study. There were more monomicrobial cultures than polymicrobial cultures (34 vs. 9) in this study with an average 1.33 pathogen isolated from diabetic foot lesion. This rate of isolated pathogen per lesion was low compare to the studies of Raja (2007) the low prevalence of polymicrobial infection and low rate of isolated pathogen per lesion may be attributable to lack of severity of most infection and low virulence of isolated organism in this study.

All diabetic foot were classified and grouped according to Wagner grading system. In the modified Wagner classification system, foot lesions are divided into six grades based on the depth of the wound and extent of the tissue necrosis. In the present study all patients had ulcer graded 0-3 in the Wagner classification. 23(53.49%) of our patients presented with preulcer (Grade 0) ,15(34.88%) with superficial ulcers (Grade I), 3(6.98%) with deep ulcer but no bone involvement (Grade II) and 2(4.64%) deep ulcer with bone involvement (Grade III).Grade IV Grade V were absent.

While considering the bacterial infection in the diabetic patients studied under Wagner grade maximum incidence was recorded in grade 0. Gram positive tend to occurs higher as compare to Gram negative bacteria. Subsequently, grade I patients were found to be colonized with higher incidental rate of Gram negative as compare Gram positive. Further grade II and III also found to be infected by different bacterial pathogens. Since none of the samples studied included grade IV and V of Wagner grade, the bacterial incidence in these criteria could not be recorded (Table 2 and Fig. 2). Similarly higher incidence of gram positive organism in grade I and gram negative in grade II was also recorded in the study of Raymudo and Mendoza (2002).

Among the different organisms isolated from diabetic foot lesions, maximum incidence were recorded against Staphylococcus aureus (31.37%) followed by Proteus mirabilis (21.05%), Pseudomonas aeruginosa (15.79%), Streptococcus pyogenes (14.04%), Escherichia coli (7.02%), Clostridium botulinum (5.26%), Peptococcus spp. (3.50%) and Salmonella typhimurium (1.75) (Fig. 2). On analyzing the data the difference was found to be statistically significant. However slitly higher incidence of S. aureus was reported in Sharma et al. (2006) and Alavi et al. (2007). The incidence of Streptococcus (14.3%) observed in the present study is comparable with the findings of Raymudo and Mendoza. (2002). Wheat et al. (1986) reported that low-virulence organisms such as S.aureus, Streptococcus viridans, Staphylococcus epidermidis, Enterococci and certain Gram-negative bacteria caused two-thirds of mild diabetic foot infections. Most of the patient’s studied (53.49%) had Grade I ulcers which are usually uncomplicated. This may be the reason for our low isolation of anaerobes. Anaerobic organisms flourish in deep seated infections. This indicates that with an increasing grade of ulcer, the anaerobic conditions are produced as a result of increase in the depth of the wound and decrease in peripheral blood flow, leading to higher rate of infections by anaerobes. Low isolation rates of anaerobes could be due to improper sampling and unnecessary delay in transportation of samples to the microbiology laboratory as well as previous treatment of patients with multiple antibiotics. The predominance of gram positive in cases that require major amputation may be due to the high proportion of Staphylococcus aureus. Such bacteria have high Pathogenicity and cause severe tissue damage because of the production of extracellular enzymes and toxins.

 

 

Fig. 2: Incidence of different bacterial flora in diabetic foot lesions

 

Table 2:  Distribution of bacterial infection in diabetic patients of different Wagner grade

 

Correlation Co-efficient(r) = - 0.898, t (cal)5% = 9.25 > t (tab)5% = 8.61 (P=0.0004),  S=  Significant  Grade 0 - Preulcer. No open lesions skin intact; may have deformities, Grade 1 - Superficial ulcer clinically not infected. Grade 2 - Deep ulcer often infected but no bone involvement. Grade 3 - Deep ulcer, abscess formation and bone involvement. Grade 4 - Localized gangrene. Grade 5 - Gangrene of whole foot.	Total=57(100%)	50						Total Samples
		43(86%)						Positive Patients
		5	4	3	2	1	0	Wagner Grade
		0	0	2(4.64%)	3(6.98%)	15(34.88%)	23(53.49%)	Diabetes (%)
	18(31.57%)	0(0%)	0(0%)	1(33.33%)	1(20%)	4(26.67%)	12(35.29%)	Staphylococcus aureus	Gram Positive	Bacterial Incidence (%)
	8(14.04%)	0(0%)	0(0%)	0(0%)	0(0%)	2(13.33%)	6(17.65%)	Streptococcus  pyogenes
	2(3.50%)	0(0%)	0(0%)	1(33.33%)	1(20%)	0(0%)	0(0%)	Peptococcus spp.
	3(5.26%)	0(0%)	0(0%)	1(33.33%)	2(40%)	0(0%)	0(0%)	Clostridium  botulinum
	12(21.05%)	0(0%)	0(0%)	0(0%)	0(0%)	3(20%)	9(26.47%)	Proteus  mirabilis	Gram Negative
	9(15.79%)	0(0%)	0(0%)	0(0%)	0(0%)	5(33.33%)	4(11.76%)	Pseudomonas aeruginosa
	4(7.02%)	0(0%)	0(0%)	0(0%)	1(20%)	1(6.67%)	2(5.88%)	Escherichia coli
	1(1.75%)	0(0%)	0(0%)	0(0%)	0(0%)	0(0%)	1(2.94%)	Salmonella  typhimurium

 

Antibiotic susceptibility of the isolates- The antimicrobial susceptibility patterns of the Gram positive bacteria isolated from Diabetic Foot Lesions against some antimicrobial agents were shown in Table 3. In the present study maximum organisms were sensitive to Ciprofloxacin, Ofloxacin, Gentamycin, Imipenem and Chloramphenicol and resistant to Cefuroxime, Erythromycin and Piperacillin. Except Peptococcus spp. other Gram positive bacteria were Multiple Drug Resistant (MDR).

The antimicrobial susceptibility pattern of the Gram negative bacteria is shown in Table 4. Most of the organisms were sensitive to Gentamycin and Imipenem, intermediate to Erythromycin and resistant to Penicillin G, Oxacillin, Vancomycin Ampicillin and Co-trimoxazole. All Gram negative bacteria were Multiple Drug Resistant (MDR).

Similarly Staphylococcus aureus showed good sensitivity to ciprofloxacin as the similar results were reported previously by Alavi et at. (2007). Finding of Clostridium species were highly susceptible to norfloxacin, gentamycin, erythro­mycin, chloramphenicol, ofloxacin, and ciprofloxacin recorded by Orji et at. (2009). Edo and Eregie (2007) observed that ciprofloxacin, gentamicin and perfloxacin were effective against Gram-positive. All the aerobes were sensitive to Amikacin and gentamicin reported in the study of Anandi et al. (2004). Ozer et al. (2010) found that chloramphenicol was the most effective agents against Gram-positives. Ampicillin showed resistant against Proteus mirabilis, Pseudomonas aeruginosa, and Escherichia coli recorded by Khoharo et al. (2009). Sapico (1985) observed that Chloramphenicol as impressive antibiotics against our anaerobic isolates (Fig. 3–10).

No single antimicrobial agent can cover all of the possible organisms isolated from diabetic foot infections. The present study findings illustrate that antimicrobial therapy needs to be selected based on antimicrobial sensitivity patterns of isolates.

 

Table 3: Antibiotic susceptibility patterns of Gram positive isolates

 

S. No.	Name of Isolates	Antibiotics
		Ciprofloxacin(5µg)	Ofloxacin(5µg)	Ampicillin(30µg)	Gentamycin(10µg)	Cefuroxime(30µg)	Erythromycin(15µg)	Imipenem(10µg)	Piperacillin(100µg)	Chloramphenicol(30µg)
1.	Staphylococcus aureus	+++	+++	+++	+++	-	-	-	-	-
2.	Streptococcus pyogenes	-	-	-	++	-	++	+++	-	-
3.	Peptococcus spp.	+++	-	++	+++	+++	-	+++	+++	+++
4.	Clostridium botulinum	+++	+++	-	+++	++	-	+++	-	+++

 Sensitive= +++, Intermediate= ++, Resistance= -

 

Table 4: Antibiotic susceptibility patterns of Gram negative isolates

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       

S.No.	Name of Isolates	Antibiotics
		Penicillin G(10unit)	Oxacillin(5µg)	Erythromycin(15µg)	Vancomycin(30 µg)	Gentamycin(10 µg)	Cefuroxime(30µg)	Imipenem(10µg)	Ampicillin(10µg)	Co-trimoxazole(25µg)
1.	Proteus mirabilis	-	-	-	-	+++	-	+++	-	-
2.	Pseudomonas aeruginosa	-	-	++	-	+++	++	+++	-	-
3.	Escherichia coli	-	-	++	-	+++	+++	+++	-	-
4.	Salmonella typhimurium	-	-	-	-	+++	-	+++	-	+++

Sensitive= +++, Intermediate= ++, Resistance= -

 

 

Fig. 3: Antibiotic susceptibility of Pseudomonas aeruginosa

Fig. 4: Antibiotic susceptibility of Proteus mirabilis

  

Fig.  5: Antibiotic susceptibility of Clostridium botulinum    

 

Fig.  6: Antibiotic susceptibility of Staphylococcus aureus

 

Fig.  7:  Antibiotic susceptibility of Peptococcus spp

 

 

Fig.  8: Antibiotic susceptibility of Streptococcus pyogenes

 

 

Fig.  9: Antibiotic susceptibility of Salmonella typhimurium

 

Fig. 10:  Antibiotic susceptibility of Escherichia coli

 

CONCLUSIONS- In the study we concluded that the Maximum incidence of foot lesions were observed in the category of Wagner grade 0 (53.49%) followed by grade 1 (34.88%), grade 2 (6.98%) and grade 3 (4.64%). Significant difference in incidence of Saphylococcus aureus were found to occur (31.54%) followed by Proteus mirabilis (21.05%), Pseudomonas aeruginosa (15.79%), Streptococcus pyogenes (14.04%), Escherichia coli (7.02%), Clostridium botulinum (5.26%), Peptococcus spp. (3.50%) and Salmonella typhimurium (1.75%) (P<0.05).

Age groups of 40-50 years tend to show maximum incidence of bacterial infection in diabetic foot lesions followed by the diabetic patients within the age group of 50-60 years. Male patients were found to be more susceptible to bacterial infection as compare to female.

Patients with sugar level of 100-150 mg/dl at fasting were found to show more bacterial infection followed by patients who fill in the category of 150-200 mg/dl and 200-250 mg/dl. Least evidence was observed in patients with fasting sugar level 250-300 or 300-350 mg/dl and the difference was found to be statistically significant (P<0.05). Lower economic status patients (53.49%) were found to suffer more followed by medium economic status (39.53%). However less evidence was recorded among high economic status (0.70%) patients (p<0.05). However non-significant difference of diabetic foot on the bacterial infection was recorded. The isolates were found to possess Multiple Drug Resistance (MDR) except Peptococcus spp.

Diabetic foot infections are generally polymicrobial. Hence a higher incidence of different pathogenic microbes was observed in patients suffering from diabetic foot lesions. Further different factors like sex (male), sugar level (100-150 mg/dl) and economic status were found to have significant effect on the incidence of bacterial infection. Proper education regarding foot wear and foot care is strongly recommended in such patients. The selection of empiric antibiotic therapy depends on various factors such as infection severity, over all patient condition, medication allergies, previous antibiotic treatment, antibiotic activity, toxicity, excretion and glycemic control. Proper identification of causative agents, appropriate antibiotic therapy and management of complication of diabetic foot infections remain essential to the achievement of a successful outcome.

 

REFERENCES

1.      Abdul, H., Arshad, W. and Sariq, M. 1999. Mortality in diabetes mellitus-data from the developing regions of the world. Diabetes Research and Clinical Practice. 43(1): 67-74.

2.      Akbar, N. and Bilal, N. 2004. The Sweet Foot Relation of Glycemic Control with Diabetic Foot Lesions. International Journal of Pathology. 2(2): 90 - 93.

3.      Akhter,T., Baqai, R. And Aziz, M. 2010. Antibacterial effect of nsaids on clinical isolates of urinary tract infection and diabetic foot infection. Pakistan Journal of Pharmaceutical Sciences. 23 (1): 108 - 113.

4.      Alavi, S.  M., Khosravi, A. D., Sarami, A., Dashtebozorg, A. and Montazeri, E. A. 2007. Bacteriologic study of diabetic foot ulcer. Pakistan Journal of Medical Sciences.  23 (5): 681 - 684.

5.      Aldridge, K. E., Ashcraft, D., Cambre, K., Pierson, C. L., Jeenkins, S. G. and Rosenblatt, J. E. 2001. Multicenter survey of the changing in vitro antimicrobial susceptibilities of clinical isolates of Bacteroides fragilis Group, Prevotella, Fusobacterium, Porphyromonas and Peptostreptococcus spp. Antimicrobial Agents and Chemotherapy. 45(4): 1238 - 1243.

6.      Anandi, C., Alaguraja, D., Natarajan, V., Ramanathan, M., Subramaniam, C.S., Thulasiram, M. and Sumithra, S. 2004. Bacteriology of Diabetic Foot Lesions. Indian Journal of Medical Microbiology. 22 (3):175-178

7.      Ata, A., Lee, J., Bestle, S. L., Desemone, J. and Stain, S. C. 2010. Postoperative Hyperglycemia and Surgical Site Infection in General Surgery Patients. Archives of Surgery. 145(9): 858-864. 

8.      Aziz, K. M. A. 2010. Association between high risk foot, retinopathy and hba1c in saudi diabetic population. Pakistan Journal of Physiology. 6 (2): 22 - 28.

9.      Bailey, T. S., Yu, H. M. and Rayfield, E. J. 1985. Patterns of foot examination in a diabetic clinic. American Journal of Medicine. 78(3): 371 - 374.

10.  Blazer, K. and Heidrich, M. 1999. Diabetic gangrene of the foot. Journal of Chirurg. 70 (7): 831 - 844.

11.  Brazier, J. S., Stubbs, S. L. and Duerden, B. I. 1999. Metronidazole resistance among clinical isolates belonging to the Bacteroides fragilis Group: time to be conserned. Journal of Antimicrobial Chemotherapy. 44(1): 580 - 601.

12.  Calhoun, J. H., Overgaard, K. A. and Stevens, C. M. 2002. Diabetic foot ulcer and infections: Current concepts. Advance Skin Wound Care. 15(1): 31 - 42; quiz 44 - 35.

13.  Cappuccino, J. G. and Sherman, N. 2004. Microbiology A Laboratory Manual, Part-5, In: Biochemical Activities of Microorganism, 6^th Ed. Chapter-21 to 32.Publishedby Person Education (Singapore) Pte. Ltd. Indian Branch, 482 F.I.E. Patpareganj Delhi 110092 India. 133 - 195.

14.  Caputo, G. M., Cavanagh, P. R., Ulbrecht, J. S., Gibbons, G. W. and Karchmer, A. W. 1994. Assessment and management of foot disease in patients with diabetes.The New England Journal of Medicine. 331(13): 854 – 60.

15.  Citron, D. M., Goldstein, E.J. C. Merriam, C. V. Lipsky, B. A. and Abramson, M. A. 2007. Bacteriology of Moderate-to-Severe Diabetic Foot Infections and In Vitro Activity of Antimicrobial Agents. Journal of Clinical Microbiology.45 (9): 2819–2828.

16.  Catherine Amalia S. Colayco, M.D., Myrna T. Mendoza, M.D., Marissa M. Alejandria, M.D. and Concepcion F. Ang, R.M.T. 2002. Microbiologic and Clinical Profile of Anaerobic Diabetic Foot Infections. Philippine Journal of Microbiology and  Infectious  Diseases  31(4):151-160.

17.  Dhanwal, D., Kumar, P., and Nehru, R. 2003. Clinical and investigative profile of patients with diabetic foot from north India. Paper presented at: 18^th International Diabetes Federation Congress. August 24-29; Paris France.

18.  Deresinski, S. 1995. Infections in diabetic patient’s strategies for clinicians. Infectious Disease Reports .1(1): 1.

19.  El-Tahawy, A. T. 2000. Bacteriology of diabetic foot infections. Saudi Medical Journal. 21 (4): 344 - 347.

20.  Edo, A. E. and Eregie, A. 2007. Bacteriology of diabetic foot ulcers in Benin City, Nigeria.  Mera: Diabetes International Federation. 21 - 23.

21.  Fahey, T., Sadaty, A. and Jones, W. 1991. Diabetic impaires the late inflammatory response to wound healing. Journal of Surgical Research. 50(4): 308 - 313.

22.  Fernandes, L. F., Pimenta, F. C. and Fernandes, F. F. 2007. Isolation and susceptibility profile of bacteria in diabetic foot and venous stasis ulcer of patients admitted to the emergency room of the main university hospital in the state of Goias, Brazil. Journal of Vascular Brasileiro. 6 (3): 211 - 217.

23.  Fisher R. A., 1950. A handbook of Agricultural Statistics. Achal Prakashan Mandir, Publisher: Kanpur. 46 - 92.

24.  Forbes, B. F., Sahn D. F. and Weist, A. S. 1998. Anaerobic bacteriology Laboratory consideration chapter 59 sections 12^th In Bailey and Scott’s Diagnostic Microbiology.10^th ed published by Mosby. 12(59): 696 - 710.

25.  Frykberg, R. G. 1998. Diabetic foot ulcer: current concepts. Journal of Foot and Ankle Surgery. 37 (5): 440 - 446.

26.  Gaur, D. S., Varma, A. and Gupta P. 2007. Diabetic Foot in Uttaranchal. Journal of Medical Education and Research. 9 (1): 18 – 20.

27.  Gibbons, G. W. and Eliopoulos, G. M. 1984. Infection of the diabetic foot In: Kozak et al. (eds). Management of Diabetic Foot Problems.Phila: W.B.Saunders Company. 97 - 102.

28.  Holt, J. G., Bergey, D. H., Krieg, N .R. 1984. Bergey’s Manual of Systematic Bacteriology, Vol1& 2, Williams and Wilkins, Baltimore, USA.164 - 1160.

29.  Islam, M. A., Talukdar, Y., Sayeed M. A. and Rokeya B. 2003. Biophysical characteristic of diabetic patients with foot ulcer in a Bangladeshi population. Paper presented at: 18^th International Diabetes Federation Congress. August 24-29; Paris France.

30.  Khoharo, H. K., Ansari, S. and Qureshi, F. 2009. Diabetic foot ulcers; common isolated pathogens and in vitro antimicrobial activity. Professional Medical Journal.16   (1): 53 - 60.

31.  Levin, M. E. and O`Neal, L. W. 1983. Preface. In: Kevin and O`Neal (eds).The Diabetic Foot, 3rd edition.St_Louis:C.V.Mosby Company.11.

32.  Lipsky, B. A. 1997. Osteomyelitis of the foot in diabetic patients.Clinical Infectious Diseases. 25(6): 1318 - 1326.

33.  Lipsky, B. A. 1999. Evidence based antibiotic therapy of diabetic foot infection. Federation of European Materials Societies Immunology and Medical Microbiology. 26: 267 - 276.

34.  Lipsky, B. A. 2004. Medical treatment of diabetic foot infections. Clinical Infectious Diseases. 39 (2): 104 - 114.

35.  Little, J.R. and Kobarjashi, G.S. 1993. Pathogenesis and management of diabetic foot lesions. In: Levin, ME, O’Neal LW. eds. Infection of the diabetic foot. 5th ed., St. Louis, C.V. Mosby Company. 17.

36.  Logerfo, F. W. and Coffman, J. D. 1984. Current concepts. Vascular and micro vascular diseases of the foot in diabetes.The New England Journal of Medicine. 311(25): 1615 - 1619.

37.  Manikandan, P., Selvam, K. P., Shobana, C. S., Ravikumar, K., Rajaduraipandi, K., Narendran, V. and Manoharan, C. 2008. Corneal ulcer of bacterial and fungal etiology from diabetic patients at a tertiary care eye hospital, Coimbatore, south India. Medicine and Biology.15 (2): 59 – 63.

38.  Meade, J. W. and Miller, C. B. 1968. Major infections of the foot. Medical Times. 96: 154 - 165.

39.  Miler, R. S. and Amyes, S. G. B. 1996. Laboratory control of antimicrobial therapy. Chapter 8 In Mackie and MC Cartney Practical medical Microbiology.edi.by Collee JG, Frases AG, Marmion BP Simmons a (14^th edition published by Churchill Livingston).151 - 178.

40.  Mohan, V. and Pradeepa, R. 2004. Epidemic of type 2 diabetes in developing nations. Current Medical Literature. 21(1): 69 - 76.

41.  Mordi, R. M. and Momoh, M. I. 2008. Incidence of Proteus species in wound infections and their sensitivity pattern in the University of Benin Teaching Hospital. African Journal of Biotechnology. 8 (5): 725 - 730.

42.  Mowat, A. G. and Baum, J. 1971. Chemotaxis of polymorph nuclear leucocytes from patients with diabetes mellitus. New England Journal of Medicine. 284(12): 621 - 627.

43.  Murugan, S., Mani, K. R. and Uma, D. P. 2008. Prevalence of Methicillin Resistant Staphylococcus aureus among Diabetes Patients with Foot Ulcers and their Antimicrobial Susceptibility Pattern. Journal of Clinical and Diagnostic Research. (2): 979 - 984.

44.  Nather, A., Chionh, S. B., Chan, Y. H., Chew, J. L. L., Lin, C. B., Neo, S. H. and Sim, E. Y. 2008. Epidemiology of diabetic foot problems and predictive factors for limb loss. Journal of Diabetic Complications 22(2): 77–82.

45.  Ng, L. S., Kwang, L. L., Yeow, S. C. and Tan, T. Y.  2008. Anaerobic Culture of Diabetic Foot Infections: Organisms and Antimicrobial Susceptibilities. Annals Academy of Medicine Singapore. 37(11): 936 - 939.                       

46.  Orji, F. A., Nwachukwu, N. C. and Udora E. C. 2009. Bacteriological evaluation of diabetic ulcers in Nigeria. African Journal of Diabetes Medicine .17(2):19 - 21.

47.  Ozer, B., Kalaci, A., Semerci, E., Duran, N., Davul, S. and Yanat A. N. 2010. Infections and aerobic bacterial pathogens in diabetic foot. African Journal of Microbiology Research. 4 (20): 2153 - 2160.

48.  Pathare, N. A., Bal, A., Talvalkar, G. V. and Antani, D. V. 1998. Diabetic foot infections a study of microorganisms associated with the different Wagner grades. Indian Journal of Pathology and Microbiology. 41 (4): 437 - 441.

49.  Paul, S., Barai, L., Jahan, A. and Haq, A. 2009. A bacteriological study of diabetic foot infection in an urban tertiary care hospital of Dhaka city. Journal of Ibrahim Medical College.   3(2): 50-54.

50.  Pecoraro, R.E., Ahroni, J.H. and Borko, E.J. 1991. Chronology and determinants of tissue repair in diabetic lower extremity ulcers. Diabetes. 40:1305 -1313.

51.  Pendsey, S.P. 1994. Epidemological aspects of diabetic foot. International Journal of Diabetes in Developing Countries. 14(1): 37 - 38.

52.  Performance standards for antimicrobial susceptibility testing (2002).12^th informational supplement. NCCLS document M100-S12, 22No.1.Pennsylvania, USA.

53.  Pittet, D., Wyssa, B., Herter-Clevel, C., Kursteiner, K., Vaucher, J.,and Lew P.D. 1999. Outcome of diabetic foot infections treated conservatively a retrospective cohort study with long term follow up. Archives of International Medicine. 159 (8): 851 - 856.

54.  Raja, N. S. 2007. Microbiology of diabetic foot infections in a teaching hospital in Malaysia: a retrospective study of 194 cases. Journal of Microbiology, Immunology and Infection. 40(1): 39 – 44.

55.  Rathore, A. H. 2009. Diabetic foot. Professional Medical Journals 16(4): 472-474

56.  Raymundo, M. P.  And Mendoza, M. T. 2002. The Microbiologic Features and Clinical Outcome of Diabetic Foot Infections among Patients Admitted at UP-PGH. Philippine Society of Microbiology and Infectious Diseases. 31 (2): 51 - 63.

57.  Rizvi, F., Khawaja, Z. H., Mehmood, N., Zeeshan, S., Afzal, M., and Baig, A. 2009. Frequency and extent of Foot Lesion and the Susceptibility Pattern of Infective Organisms in Diabetic Foot. Asia-Pacific Microwave Conference. 3 (1). 36-40.

58.  Santos, V. P., Silveira, D. R.  and  Caffaro, R. A. 2006. Risk factors for primary major amputation in diabetic patients. Sao Paulo Medical Journal.124 (2): 66 - 70.

59.  Sapico, F. L. 1985. Foot Infections in the Diabetic: A Review of Microbiologic Aspects. Philippine Society of Microbiology and Infectious Diseases. 14 (2): 52 - 54.

60.  Sarkar, P. K. and Ballantyne, S. 2000. Management of leg ulcers. Journal of Postgraduate Medicine. 76(901): 674-682.

61.  Sharma, V.K., Khadka, P.B., Joshi, A. and Sharma, R. 2006. Common pathogens isolated in diabetic foot infection in BirHospital .Kathmandu University Medical Journal. 4(3): 295-301.

62.  Shea, K. W. 1999. Antimicrobial therapy for diabetes foot infection. A practical approach. Journal of Postgraduate Medicine. 106 (1): 85 - 86, 89 - 94.

63.  Singh, S. K. 2001.  Role of Combination of Multiple Herbal Drugs (Septilin) in the Prophylaxis of Diabetic Foot Ulcer–A Double Blind Trial.  Indian Journal of Dermatology.  46 (2): 83 - 85.

64.  Sivakumari, V. and Shanthi, G. 2009. Antibiotic Susceptibility of Common Bacterial Pathogens Isolated From Diabetic Pus. Journal of Advanced Biotechnology. 8(10): 10 - 13.

65.  Smith, J. M. B., Payne, J. E. and Berue, T. V. 2002. Diabetes foot lesions of skin and soft tissue infections of surgical importance.Chapter14. The Surgeons Guide to Antimicrobial Chemotherapy. 218 - 221.

66.  Snydman, D. R., Jacobus, N. V., MacDermott, L. A., Ruthazer, R., Goldstein, E. J. and Finemold, S. M. 2002. National survey on the susceptibility of Bacteroides fragilis Group: report and analysis of trends. Journal of Clinical Infectious Diseases.  35 (1): 126 - 134.

67.  Tan, J. S., Anderson, J. L., Watanakunakorn, C. and Dhait, J. P. 1975. Neutrophil dysfunction in diabetes mellitus. Journal of Laboratory and Clinical Medicine. 55(1): 26 - 33.

68.  Teng, L. J., Hsueh, P. R., Tsai, J. C., Liaw, S. J., Ho, S. W. and Luh, K. T. 2002.  High incidence of cefoxitin and clindamycin resistance among anaerobes in Taiwan. Antimicrobial Agent and Chemotherapy. 46(9): 2908 - 2913.

69.  VinodKumar, C. S., Srinivasa, H., Basavarajappa, K. G. and Bandekar, N. 2010. Isolation of Bacteriophages for MRSA Obtained from Diabetic Foot - A Possible Treatment Option in Infections. International Journal of Biotechnology and Biochemistry 0973-2691. 6 (5): 801 - 809.

70.  Wheat, L. J., Allen, S. D. and Henry, M. 1986. Diabetic foot infections. Bacteriological analysis. Archives of Intenal Medicine. 146(10): 1935-1940.

71.  Zubair, M., Malik, A., Ahmad, J., Rizvi, M., Farooqui, K. J., Rizvi, M. W. 2011. A study of biofilm production by gram-negative organisms isolated from diabetic foot ulcer patients. Biology and Medicine. 3 (2): 147 - 157.