Diabetic Foot Infections

Diabetic Foot Infections
INTRODUCTION
Dept. of Microbiology, Sahyadri Science College, Shivamogga
I. INTRODUCTION
1.1 HISTORY
From 2,000 years diabetes has been recognized as a devastating and deadly disease. In the first century A.D. a Greek, Aretaeus, described the destructive nature of the affliction which he named "diabetes" from the Greek word for "siphon." Eugene J. Leopold in his text Aretaeus the Cappodacian describes Aretaeus' diagnosis: "...For fluids do not remain in the body, but use the body only as a channel through which they may flow out. Life lasts only for a time, but not very long. For they urinate with pain and painful is the emaciation. For no essential part of the drink is absorbed by the body while great masses of the flesh are liquefied into urine." Physicians in ancient times, like Aretaeus, recognized the symptoms of diabetes but were powerless to effectively treat it. Aretaeus recommended oil of roses, dates, raw quinces, and gruel. And as late as the 17th century, doctors prescribed "gelly of viper's flesh, broken red coral, sweet almonds, and fresh flowers of blind nettles." In the 17th century a London physician, Dr. Thomas Willis, determined whether his patients had diabetes or not by sampling their urine. If it had a sweet taste he would diagnose them with diabetes mellitus- "honeyed" diabetes. This method of monitoring blood sugars went largely unchanged until the 20th century. Then in 1921 something truly miraculous occurred in Ontario, Canada. A young surgeon Frederick Banting, and his assistant Charles Best, kept a severely diabetic dog alive for 70 days by injecting it with a murky concoction of canine pancreas extract. With the help of Dr. Collip and Dr. Macleod, Banting and Best administered a more refined extract of insulin to Leonard Thompson, a young boy dying of diabetes. Within 24 hours, Leonard's dangerously high blood sugars had dropped to near normal levels. Until the discovery of insulin, most children diagnosed with diabetes were expected to live less than a year. In a matter of 24 hours the boy's life had been saved. News of the miracle extract, insulin, spread like wildfire across the world.
1.2. OVERVIEW OF DIABETES

Diabetes mellitus is a disorder in which blood sugar (glucose) levels are abnormally high because the body does not produce enough insulin to meet its needs. Insulin, a hormone released from the pancreas, controls the amount of sugar in the blood. When people eat or drink, food is broken down into materials, including the simple sugar glucose that the body needs to function. Sugar is absorbed into the bloodstream and stimulates the pancreas to produce insulin. Insulin allows sugar to move from the blood into the cells. Once inside the cells, it is converted to energy, which is either used immediately or stored as fat or glycogen until it is needed. The levels of sugar in the blood vary normally throughout the day. They rise after a meal and return to normal within about 2 hours after eating. Once the levels of sugar in the blood return to normal, insulin production decreases. The variation in blood sugar levels is usually within a narrow range, about 70 to 110 milligrams per deciliter (mg/dL) of blood. If people eat a large amount of carbohydrates, the levels may increase more. People older than 65 years tend to have slightly higher levels, especially after eating. If the body does not produce enough insulin to move the sugar into the cells, the resulting high levels of sugar in the blood and the inadequate amount of sugar in the cells together produce the symptoms and complications of diabetes.
1.3. TYPES OF DIABETES

Prediabetes: Prediabetes is a condition in which blood sugar levels are too high to be considered normal but not high enough to be labeled diabetes. People have prediabetes if their fasting blood sugar level is between 101 mg/dL and 126 mg/dL or if their blood sugar level 2 hours after a glucose tolerance test is between 140 mg/dL and 200 mg/dL. Identifying people with prediabetes is important because the condition carries a higher risk for future diabetes as well as heart disease. Decreasing body weight by 5 to 10% through diet and exercise can significantly reduce the risk of developing future diabetes. Type 1: In type 1 diabetes (formerly called insulin-dependent diabetes or juvenileonset diabetes), more than 90% of the insulin-producing cells of the pancreas are permanently destroyed. The pancreas, therefore, produces little or no insulin. Only about Dept. of Microbiology, Sahyadri Science College, Shivamogga 3
Diabetic Foot Infections 10% of all people with diabetes have type 1 disease. Most people who have type 1 diabetes develop the disease before age 30. Scientists believe that an environmental factorpossibly a viral infection or a nutritional factor in childhood or early adulthoodcauses the immune system to destroy the insulin-producing cells of the pancreas. A genetic predisposition may make some people more susceptible to the environmental factor. Type 2: In type 2 diabetes (formerly called non-insulin-dependent diabetes or adult-onset diabetes), the pancreas continues to produce insulin, sometimes even at higher-than-normal levels. However, the body develops resistance to the effects of insulin, so there is not enough insulin to meet the body's needs. Type 2 diabetes was once rare in children and adolescents but has recently become more common. However, it usually begins in people older than 30 and becomes progressively more common with age. About 15% of people older than 70 have type 2 diabetes. People of certain racial and ethnic backgrounds are at increased risk of developing type 2 diabetes: blacks, Native Americans, and Hispanics who live in the United States have a twofold to threefold increased risk. Type 2 diabetes also tends to run in families. Obesity is the chief risk factor for developing type 2 diabetes, and 80 to 90% of people with this disorder are overweight or obese. Because obesity causes insulin resistance, obese people need very large amounts of insulin to maintain normal blood sugar levels. Certain disorders and drugs can affect the way the body uses insulin and can lead to type 2 diabetes. High levels of corticosteroids (from Cushing's disease or from taking corticosteroid drugs) and pregnancy are the most common causes of altered insulin use. Diabetes also may occur in people with excess production of growth hormone (acromegaly) and in people with certain hormone-secreting tumors. Severe or recurring pancreatitis and other disorders that directly damage the pancreas can lead to diabetes.
1.4. DIABETIC FOOT INFECTIONS
Diabetic Foot Infections Foot infections are the most common problems in persons with diabetes. These individuals are predisposed to foot infections because of a compromised vascular supply secondary to diabetes. Local trauma and/or pressure (often in association with lack of sensation because of neuropathy), in addition to microvascular disease, may result in various diabetic foot infections that run the spectrum from simple, superficial cellulitis to chronic osteomyelitis. Infections in patients with diabetes are difficult to treat because these individuals 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. In addition, diabetic individuals can not only have a combined infection involving bone and soft tissue called fetid foot, a severe and extensive, chronic soft-tissue and bone infection that causes a foul exudate, but they may also have peripheral vascular disease that involves the large vessels, as well as microvascular and capillary disease that results in peripheral vascular disease with gangrene. Except for chronic osteomyelitis, infections in patients with diabetes are caused by the same microorganisms that can infect the extremities of persons without diabetes. Gas gangrene is conspicuous because of its low incidence in patients with diabetes, but deepskin and soft-tissue infections, which are due to gas-producing organisms, frequently occur in patients with these infections. In general, foot infections in persons with diabetes become more severe and take longer to cure than do equivalent infections in persons without diabetes.
1.5. DIABETIC FOOT INFECTION CAUSES

Several risk factors increase a person with diabetes chances of developing foot problems and diabetic infections in the legs and feet.
Footwear: Poorly fitting shoes are a common cause of diabetic foot problems.
If the patient has red spots, sore spots, blisters, corns, calluses, or consistent pain associated with wearing shoes, new properly fitting footwear must be obtained as soon as possible.

If the patient has common foot abnormalities such as flat feet,bunions, or hammertoes, prescription shoes or shoe inserts may be necessary.
Nerve damage: People with long-standing or poorly controlled diabetes are at risk for having damage to the nerves in their feet. The medical term for this is peripheral neuropathy. Because of the nerve damage, the patient may be unable to feel their feet normally. Also, they may be unable to sense the position of their feet and toes while walking and balancing. With normal nerves, a person can usually sense if their shoes are rubbing on the feet or if one part of the foot is becoming strained while walking.
A person with diabetes may not properly sense minor injuries (such ascuts, scrapes, blisters), signs of abnormal wear and tear (that turn into calluses and corns), and foot strain. Normally, people can feel if there is a stone in their shoe, then remove it immediately. A person who has diabetes may not be able to perceive a stone. Its constant rubbing can easily create a sore.
Poor circulation: Especially when poorly controlled, diabetes can lead to accelerated hardening of the arteries or atherosclerosis. When blood flow to injured tissues is poor, healing does not occur properly.
Trauma to the foot: Any trauma to the foot can increase the risk for a more serious problem to develop. Infections
Athlete's foot, a fungal infection of the skin or toenails, can lead to more serious bacterial infections and should be treated promptly. Ingrown toenails should be handled right away by a foot specialist. Toenail fungus should also be treated.
Smoking: Smoking any form of tobacco causes damage to the small blood vessels in the feet and legs. This damage can disrupt the healing process and is a major risk factor for infections and amputations. The importance of smoking cessation cannot be overemphasized
1.6. DIABETIC FOOT INFECTION SYMPTOMS:
Persistent pain can be a symptom of sprain, strain, bruise, overuse, improperly fitting shoes, or underlying infection. Redness can be a sign of infection, especially when surrounding a wound, or of abnormal rubbing of shoes or socks. Swelling of the feet or legs can be a sign of underlying inflammation or infection, improperly fitting shoes, or poor venous circulation. Other signs of poor circulation include the following:
Pain in the legs or buttocks that increases with walking but improves with rest (claudication)
Hair no longer growing on the lower legs and feet Hard shiny skin on the legs Localized warmth can be a sign of infection or inflammation, perhaps from wounds that won't heal or that heal slowly. Any break in the skin is serious and can result from abnormal wear and tear, injury, or infection. Calluses and corns may be a sign of chronic trauma to the foot. Toenail fungus, athlete's foot, and ingrown toenails may lead to more serious bacterial infections.
Drainage of pus from a wound is usually a sign of infection. Persistent bloody drainage is also a sign of a potentially serious foot problem. A limp or difficulty walking can be sign of joint problems, serious infection, or improperly fitting shoes. Fever or chills in association with a wound on the foot can be a sign of a limbthreatening or life-threatening infection. Red streaking away from a wound or redness spreading out from a wound is a sign of a progressively worsening infection. New or lasting numbness in the feet or legs can be a sign of nerve damage from diabetes, which increases a persons risk for leg and foot problems
1.7. CLASSIFICATION OF INFECTION:

Skin and soft tissue infections can be divided into mild, moderate and severe. A mild infection is one which could be managed with oral antibiotic therapy and does not jeopardise the limb or life of the patient. A moderate infection is a deeper infection which may have abscess formation and may require surgical intervention and parenteral anti-
Diabetic Foot Infections microbial therapy. A severe infection is one which threatens the patients life and limb, requires admission to the hospital, surgical debridement and parenteral antimicrobial therapy. Mild Infections Deemed to be neither a limb nor life threatening processes. They are usually associated with cellulites surrounding an ulceration. A small amount of purulent material may be present at the base of the ulcer. The most likely pathogens are aerobic grampositive cocci (S. aureus, Streptococcus spp). Patients with these infections can frequently be treated as outpatients with oral antimicrobial therapy. Moderate Infections These infections are more significant and may be associated with plantar abscesses. The selected antimicrobial regimens should be effective for: staphylococci, streptococci, anaerobes and the gramnegatives. Patients who are not toxic may be treated with oral antimicrobial therapy while those with more significant infections require parenteral therapy and are best managed using the agents summarised below for severe infections. Severe Infections Patients with these processes have limb or life threatening infections requiring immediate hospitalisation, parenteral antimicrobial therapy, and consultation with a surgeon.
1.8. CONSEQUENCES
DM results in vasculopathy, nephropathy, retinopathy and neuropathy. In the lower extremity, complications arising from ischemia and/or neuropathy may include pain, discomfort, deformity (i.e., Charcot foot, claw toes, prominent metatarsal heads), ulcers, infection and may ultimately result in the amputation of a digit(s), forefoot, or an entire lower extremity. Peripheral neuropathy increases the risk of plantar ulcerations sevenfold.2Without the presence of peripheral neuropathy, it is unlikely that a person Dept. of Microbiology, Sahyadri Science College, Shivamogga 8
Diabetic Foot Infections with diabetes will develop a plantar ulceration. Important contributing factors for the development of plantar ulcerations include peripheral vascular disease, infection, and extensive or repitive trauma. Osteomyelitis may occur as a consequence of hematogenous spread, however, in the person with diabetes and the neuropathic limb, osteomyelitis is invariably due to contiguous spread from the ulceration. Unlike skin and soft tissue infections where, at the end of antimicrobial therapy, the infection is deemed cured, osteomyelitis may relapse at a later date and, therefore, not unlike a neoplasm, osteomyelitis can be deemed to be inremission versus cured. Ref: The Canadian journal of CME / April 2003
1.9. MICROFLORA
The common microflora found in diabetic foot infection are listed below: Enterobacteriaceae Pseudomonas aeruginosa Enterococcus spp. Staphylococcus aureus Streptococcus spp. Staphylococcus epidermidis Candida spp. Acinetobacter spp. Enterococcus spp. P. aeruginosa Diphtheroids Ref: The Canadian journal of CME / April 2003
1.10. ANTIBIOTIC THERAPY

Antibiotic choice is primarily dependent on causative pathogen and epidemiology. However, treatment with antibiotics often needs to be commenced before culture and sensitivity results are available. Thus initial treatment is empirical. It is based on the presumed pathogen. The following antibiotics are used to treat the infections caused by most common pathogens. For,
1. Staphylococcus aureus - primarily, Penicillinase-resistant penicillin (e.g. flucloxacillin) is used. As an alternative Doxycycline, or Clindamycin are used. 2. Meticillin-resistant Staphylococcus aureus primarily, Vancomycin, Teicoplanin are used. As an alternative Rifampicin with either Trimethoprim, or Doxycycline, or Fusidic acid is used. Even Co-trimoxazole and Linezolid are also used. 3. Beta-haemolytic streptococcus Amoxicillin is used as a primary antibiotic. And Clindamycin is used as an alternative. 4. Enterococcus- primarily, Amoxicillin or Co-amoxiclav are used. As an alternative, Vancomycin or Linezolid are used. 5. Pseudomonas (gram-negative bacillus) - Quinolones (e.g. high dose ciprofloxacin) is used as a primary antibiotic. Piperacillin-tazobactam, or Meropenem are used as an alternative antibiotic. For anaerobic pathogens, used as an alternative antibiotic. Ref: The Diabetic Foot Journal Vol 12 No 2 2009 Metronidazole is used primarily and Clindamycin is
The antibiotics are also given on the basis of the scenario of the infection. It is explained in the following table: Scenario Mild to moderate, localized cellulitis (outpatient) Moderate to severe cellulitis (inpatient) Nafcillin (Unipen) or oxacillin Primary antibiotic Dicloxacillin (Pathocil) Alternative antibiotic Cephalexin (Keflex); amoxicillin/clavulanate potassium (Augmentin); oral clindamycin (Cleocin Cefazolin (Ancef); ampicillin/sulbactam (Unasyn); clindamycin IV; vancomycin Moderate to severe Ampicillin / sulbactam (vancocin) Ticarcillin/clavulanate (Timentin); 10
Diabetic Foot Infections cellulitis with ischemia or significant local necrosis piperacillin/tazobactam (Zosyn); clindamycin plus ciprofloxacin (Cipro); ceftazidime (Fortaz) or cefepime (Maxipime) or cefotaxime (Claforan) or ceftriaxone (Rocephin) plus metronidazole (Flagyl); cefazolin (for Staphylococcus Life- or limbthreatening infection Ticarcillin/clavulanate or piperacillin/tazobactam, with or without an aminoglycoside aureus); nafcillin (Unipen); oxacillin Clindamycin plus ciprofloxacin or tobramycin (Nebcin); clindamycin plus ceftazidime or cefepime or cefotaxime or ceftriaxone; imipenem/cilastin (Primaxin) or meropenem (Merrem); vancomycin plus aztreonam (Azactam) plus metronidazole; vancomycin plus cefepime; ceftazidime plus metronidazole
1.11. COMPLICATIONS OF ANTIBIOTIC THERAPY:

Obtaining a proper wound culture specimen allows the clinician to define the pathogens involved and their antibiotic susceptibility. Unfortunately, results of cultures are generally not available for at least 23 days. Thus, most antibiotic therapy for infections is selected empirically . By the time culture results arrive, they are usually too late to influence the initial antibiotic choice. Many clinicians, therefore, feel compelled to select an antibiotic regimenthat will cover most of the likely bacteria for all but the mildest infections, leading to an unnecessarily broad spectrum of therapy. This over prescribing increases the likelihood of adverse drug effects, drives antibiotic resistance, and increases the cost of treatment. To the contrary, some clinicians conclude that it is not worthwhile to obtain cultures, believing that the belated results are unhelpful. This approach, however, makes it difficult to properly select an alternative regimen if the patient is failing to respond to the initial agent(s).
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Diabetic Foot Infections The increasing incidence of antibiotic resistant pathogens as causes of diabetic foot infections makes selecting empiric antibiotic therapy more difficult. Those who treat these patients are well aware of the growing problem of methicillin resistant Staphylococcus aureus (MRSA), which is now frequently acquired in the community and in various types of health care facilities .In addition, in many clinics (especially in developing countries), highly resistant strains of gram negative bacilli, especially Pseudomonas aeruginosa, are an increasing problem . Similarly, Enterococcus species are often isolated from diabetic foot wounds, especially in patients recently treated with antibiotics. It is often unclear whether some of these latter, less-virulent isolates, unlike S. aureus, represent true pathogens that require targeted therapy An additional dilemma facing practitioners is that current guidelines recommend only treating diabetic foot wounds that are clinically infected, i.e., those with purulent secretions or with signs or symptoms of inflammation. Unfortunately, wounds in patients with peripheral neuropathy or peripheral arterial disease may fail to show classic symptoms of inflammation, which makes diagnosis difficult .Thus, it may be difficult to know which wounds require antibiotic therapy.
1.12. ROLE OF PLANT EXTRACTS IN DIABETIC FOOT INFECTION

Plants have anchored to the mother earth long before man has set his feet and it is said that god hadendowed them with materials for survival of man and animal long before these creatures were made by him. The world health organization (WHO) estimates that about 80% of the population is still depends upon these herbal medicines for their treatment of diseases due to easy availability, economic and less side effects when compared to allopathic system of medicines. Nearly 2000 of natural drugs are mentioned in Indian Materia Medica that have reported various pharmacological activities, out of these 1600 are from plant origin.2 Herbal remedies have formed the basis of traditional medicine for millennia, and have formed the root of modern pharmacology. While science from roughly the 1880's onwards has striven to isolate the active compounds found in medicinal herbs, the list is ever growing. Wound infections are most common in developing countries, such as Sub-Saharan African and South Asian countries, than in developed countries. Current estimates indicate that nearly 6 million people suffer from chronic wounds worldwide.
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Diabetic Foot Infections The prevalence of chronic wounds in the community was reported as 4.5 per 1000 population, whereas that of acute wounds was nearly double, at 10.5 per 1,000 agents for wound healing, and largely preferred because of their widespread availability and effectiveness as crude preparations. Due to the present stress filled life a lot of people are developing diabetes at a very younger age. Wound healing is impaired in diabetic patients with infection or hyperglycemia. The heat shock proteins (HSPs), originally identified as heat-inducible gene products, are a highly conserved family of proteins that respond to a wide variety of stress. Wounding induces HSPs, particularly in the epidermis. In the initial phase of wound healing there is an inflammatory response, followed by organization of the fibrin-rich exudates and subsequent re-epithelialisation and formation of granulation tissue. The wound bed contains abundant inducible HSP70 which contributes to protein homeostasis and cell survival within the healing wound. HSP functions are compromised under conditions of diabetes. Both type 1 and type 2 diabetes are characterized by an increased risk for the development of micro vascular and macro vascular complications. In diabetes, endogenous defence systems are overwhelmed, causing various types of stress. Uncontrolled oxidative stress represents a characteristic feature of diabetes. Among the other important conditions related to diabetes are dyslipidemia, modification of proteins and lipids, and perturbations in the tissue antioxidant defence network15. In traditional medicine plants are generally used for treatment of various diseases and abnormalities in the body. The below lists of the plants are used as traditional medicine for the treatment of wounds. Plant Achalypha indica L. Achyranthes Adhatoda vasica Aloe vera Alternanthera brasiliana Alternanthera brasiliana Alternanthera sessilis Linn. Argemone mexicana. Arrabidaea chica Verlot Berberis aristata Blumea lacera. Borassus flabellifer L. Bryophyllum pinnatum Calendula officinalis Centella asiatica Part used Not mentioned Leaves Not mentioned Not mentioned Leaves Kuntz Leaves Leaves Not mentioned Not mentioned Not mentioned Not mentioned Fruit Leaf Not mentioned Leaves 13
Diabetic Foot Infections Citrus reticulate Citrus sinensis Coleus aromaticus Cordia dichotoma Coronopus didymus. Curcuma longa Eleusine coracana Euphorbia neriifolia Evolvulus numularius Linn. Ficus asperifolia Gentiana lutea Gossypium arboretum Grewia tiliaefolia. Hamelia patens Hippophae rhamnoides Hypericum perforatum L. Hyptis suaveolens Ipomoea Batatas. Juglans nigra. Kalanchoe pinnata Lam Madhu ghrita Mesquite Prosopis Moringa oleifera Musa sapientum var. paradisiacal Naravelia zeylanica Ocimum sanctum Linn. Paspalum scrobiculatum Piper betle Portulaca oleracea Linn. Prosopis juliflora DC. Pueraria tuberose Punica granatum Quercus infectoria. Rhizopus arrhizus Rubia cordifolia Saba florida Salvia splendens Saussurea lappa Sesamum indicum L Sphaeranthus indicus Sphagnum holocellulose Tephrosia purpurea Tephrosia purpurea (Linn.) Terminalia arjuna Terminalia bellirica Roxb. Terminalia bellirica Roxb. Thespesia populnea Toddalia asiatica Linn. Tridax procumbens Verbena officinalis Vernonia arborea Essential Oil Peel Leaves Forst Fruits Not mentioned Rhizome whole grain fiour Leaf Not mentioned Aqueous extract Rhizome Aqueous extract Not mentioned Not mentioned Not mentioned Not mentioned Leaves Tuberous root Leaves Leaf Not mentioned Leaves Aqueous leaves Not mentioned Leaves Not mentioned whole grain flour Leaf Not mentioned Leaf juice Not mentioned Peals Galls Not mentioned Not mentioned Leaf Leaves Root SEED Not mentioned Not mentioned Not mentioned Not mentioned Stem bark Fruits Fruits Leave Stem bark Whole plant Not mentioned Not mentioned 14
Diabetic Foot Infections Vernonia arborea Buch Aqueous and methanoolic extracts of bark
1.13. OBJECTIVES:
1. Sampling of diabetic foot infection. 2. Isolation of bacteria. 3. Biochemical characterization of the organisms. 4. Comparison of herbal and allopathic drugs as treatment measure.
MATERIALS AND METHODS
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II. MATERIALS AND METHODS: 2.1 MATERIALS

Table No 1: Antibiotics used Sl No 1 2 3 4 5 6 7 8 9 10 11 12 Antibiotics Used Amikacin Gentamycin Kanamycin Vancomycin Erythromycin Ampicillin Methicillin Penicillin Polymyxin-B Tetracycline Chloramphenicol Streptomycin Spectrum of Antibiotic + + + + + + and + and Class of antibiotic Amino glycoside Amino glycoside Amino glycoside Glycopeptides Macrolides Penicillin Penicillin Penicillin Polypeptide Tetracycline Synthetic Drug Amino glycoside
Table No 2: Plant Material Selected Sl No 1 Plants name Gymnema Sylvester Plant part used Leaves has an Significance important place among such
antidiabetic medicinal herbs and it boosts 2 Hyptis suaveolens Leaves your insulin level It has a very good antimicrobial activity and it is effective against staphylococcus auers and 3 4 Polyanlthia longifolia Butea monosperma Leaves Leaves pseudomonas aerogenosa plant is used for skin disease, fever, diabetes and hypertension. It has very good antibacterial property and used tumors and its juice is used for night 5 Momordica charantia 6 Murraya koenigii Leaves Leaves blindness. It is a very good medicinal food for diabetes and also a African traditional medicine and also a antiviral and anticancerous property. They are much valued as an anti-diabetic, antioxidant, antimicrobial, anti-inflammatory, hepatoprotective, anti-hypercholesterolemic.
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Diabetic Foot Infections 7 Azadirachta indica Leaves They also contain iron. In diabetic foot infection it is very help ful in fungal infection between the toes and also in 8 Centella asiatica Leaves healing the ulcers specifically Centella is a mild adaptogen, is mildly antibacterial, antiviral, anti-inflammatory, nervine and antiulcerogenic, 9 Aegle marmelos Leaves anxiolytic,
vulnerary. It is very much used in peptic ulcers,ulcers and inflammation and also used in respiratory disorders. The leaf extracts were tested for possible antibacterial activity in the disk assay using eighteen (18) human pathogenic bacteria
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Sclerodendron inermae
Leaves
2.2. METHODOLOGY
2.2.1. Sampling Hospital in Tarikere was selected for collection of diabetic foot infection samples. The samples were collected in sterile viales with the normal saline. The sterilized swab in normal saline was swabbed around the patients wound infection and again that swab is dipped in the normal saline and brought back to the laboratory for further processing.
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2.2.2. Patients History Table No 3: Patients History Sl No 1 2 3 4 5 6 7 Patients name Veena Eshwarapa Balen R Deepak Shashi kumar Manjunath Lokesh Patient age 24 59 34 32 42 48 56 Type of wound Foot Toes Foot Foot Foot Feet Toes
2.2.3. Isolation and characterization of isolates. A) Plating on general media and selective media by streak plate method. The collected foot infection samples were taken and to get the discrete colonies, quadrant streaking was done.
1. Sterile nutrient agar media is poured into sterile petriplates and allowed for
solidification.
2. Then the sample is taken and inoculated them by streaking on the plate. 3. The streaking begins to touching a loop full of sample at one edge of the agar
media and this edge pointed is called primary streak.

4. The primary steak is followed by 2-3 parallel streaks, right angle to first streak
and continued in the same way.

5. So the final lines should not be coming with the primary streak. 6. Then the plates were taken and incubated at 37 degree for 24hours.
7. For the selective media also we followed the same procedure. The media taken was selective for some of the bacteria. B) Gram staining For the characterization of the colonies which we had got we had done the gram staining.
1. A clean glass slide was taken a thin smear of bacterial culture was prepared and
heat fixed.
2. Smear was flooded with crystal violet and maintained for 1min then the smear is
washed with water.
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3. The stained smear was subjected to the action of moderent like grams iodine and
retained for 1 min.

4. The smear is washed with water and treated with decolourizing agent like 95%
ethyl alcohol for about 10-25 seconds.

5. The alcohol washed smear is then washed with water. 6. The smear is then treated with counter stain such as saffranin and allowed for
1min.
7. Slide is washed with water blot dry and observed under microscope.
C) KOH Solubility This is the other method to characterize the bacteria whether it is gram positive or negative. 1. A clean glass slide is taken without any oil or dirt.
2. 3% of koh solution is prepared and a loop full of solution is transferred to the
slide. 3. The testing bacterial colony is picked from sterile inoculation loop and spread over the solution which was there in the glass slide. D) Biochemical Tests: i. Catalase Test:
1. A clean glass slide was taken. 2. A drop of 3% hydrogen peroxide was placed on the centre of the slide.
3. A loop full of 24 hour old bacterial culture was mixed to the drop and immediately observed. ii. Starch Hydrolysis:
1. Starch agar medium was prepared sterilized and dispensed into sterile plates and
allowed for solidification.

2. On the solidified medium streak inoculation of bacterial culture was done using
inoculation loop.
3. Then plates were kept for incubation at 37 degree for 24 hours.
iii. Gelatin hydrolysis:

1. Gelatin media was prepared, poured in to test tubes and sterilized.
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2. The bacterial culture was stab inoculated to test tubes and kept for incubation at
37 degree for 96 hours; one uninoculated tube was maintained as control. 3. After incubation the tubes were kept at low temperature (4 degree) for 15-20 min. then the tubes were observed. 2.2.4. In vitro antibiotic assay (disc diffusion method)
1. Sterilized nutrient agar media was poured into sterilized petriplate and kept for
solidification.
2. All the plates are inoculated with the test bacteria. 3. Dip a sterile cotton swab in broth culture of test bacteria. 4. Using dipped saturated swab the entire agar surface is swabbed horizontally,
vertically and around the edge of plate to ensure heavy growth of test bacteria on the surface.
5. Allow all the plates to dry for 5 min. using sterilized forecep antibiotic discs were
placed at equal distance on the agar surface.

6. Gently press the disc so that it adheres to the surface of agar. 7. Incubated all the plates at 37 degree for 24-48 hours. (Alves, 2007)
2.2.5. Herbal Extract Preparation: We had prepared the extraction of plants by cold extraction method. The procedure is like this, Plant materials were washed individually with clean sterile water and each of respective shaded dry plant material was blended into fine powder in electrical blender. The plant material concentration was prepared from 60 to 100% with the help of distilled water. This plant material was filtered using a sterile muslin cloth after which the extract obtained and stored at 4oC until required according to the method of Azu and Onyeagba, 2007.
2.2.6. In vitro antibacterial assay by well in agar method. The antibacterial assay of plant materials was done by well in agar methods. The molten nutrient agar media is poured into sterile plates and kept for solidification. After solidification 24 hours old bacterial cultures were swab inoculated on Dept. of Microbiology, Sahyadri Science College, Shivamogga 20
Diabetic Foot Infections the surface of media. Wells were made on the agar media with the help of sterilized cork borer of 9 mm diameter. 100-200 microliter of selected different plant extract was poured to well with the help of sterile pipettes. Plates were incubated at 37 o C for 24-48 hours. (alade, et. Al. 1993)
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RESULTS
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III. RESULTS 3.1 SAMPLING:

Out of 7 diabetic foot samples collected and 23 isolates of bacteria were recovered from nutrient agar media and selective media like baired parker, pseudomonas agar, coagulase mannitol agar and mannitol agar media. And after maintained the pure culture of these bacteria.
3.2 ISOLATION:
From the first, second, fifth, sixth and seventh samples we got each 1 bacterial isolate each, in the third plate we got 4 bacteria and from the fourth plate we got 2 bacteria. After isolating these organisms from nutrient agar media. Same samples when streaked on selective media 12 isolates were recovered. So totally from nutrient media and selective media we have recovered 23 isolates.
3.3 CHARACTERIZATION:
From the grams staining we have confirmed the morphology of the bacteria and also the gram positive and negative bacteria. Out of 23 isolates we had recovered 18 gram positive bacteria and 5 gram negative bacteria. In 18 gram positive isolates cocci are 11 and bacilli are 7 in number. In the gram negative isolates cocci are in 3 and other 2 are bacilli.
3.4 KOH SOLUBILITY:

From the KOH solubility test we have confirmed the 18 gram positive organisms and 5 are gram negative by showing the thread when picked up with the loop and mixed with the 30% KOH solution.
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Table No 3: Morphology of Bacterial Isolates Sl No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Sl No 1 2 3 4 5 Isolate Number Isolate no 1 Isolate no 2 Isolate no 3 Isolate no 4 Isolate no 5 Isolate no 6 Isolate no 7 Isolate no 8 Isolate no 9 Isolate no 10 Isolate no 11 Isolate no 12 Isolate no 13 Isolate no 14 Isolate no 15 Isolate no 16 Isolate no 17 Isolate no 18 Isolates Number Isolate no 19 Isolate no 20 Isolate no 21 Isolate no 22 Isolate no 23 Morphology of Gram Positive Bacteria Bacilli in chains Bacilli individual Cocci in cluster Bacilli in chains Bacilli in chain Cocci in cluster cocci single Cocci single Bacilli single Cocci single Bacilli Cocci Cocci Cocci Bacilli Cocci Cocci Diplococcic Morphology of gram negative bacteria Cocci Cocci Cocci Bacilli Bacilli in chains
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Table No 4 : Characterization of Bacterial Isolates Sl No 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Isolates Number Isolate no 1 Isolate no 2 Isolate no 3 Isolate no 4 Isolate no 5 Isolate no 6 Isolate no 7 Isolate no 8 Isolate no 9 Isolate no 10 Isolate no 11 Isolate no 12 Isolate no 13 Isolate no 14 Isolate no 15 Isolate no 16 Isolate no 17 Isolate no 18 Isolate no 19 Isolate no 20 Isolate no 21 Isolate no 22 Isolate no 23 KOH Positive + + + + + + + + + + + + + + + + + + KOH Negative + + + + +
+ Gram positive organism - Gram negative organisms
3.5. BIOCHEMICAL TESTS:

3.5.1. Catalase test In the taken 23 bacterial isolates air bubbles or effervescence was observed in 18 bacterial isolates and the remaining were negative. 3.5.2. Starch hydrolysis: Zone of hydrolysis was observed in 7 isolates and media colour was changed to dark blue and in the remaining isolates the hydrolysis was not seen. 3.5.3. Gelatin hydrolysis: 7 gelatin tubes showed liquefaction when kept at low temperature and remaining other tubes donot show liquefaction. Table No 5 : Catalase Test: Sl No Isolate Number Catalase Positive Catalase Negative
25
Diabetic Foot Infections 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Isolate number 1 Isolate number 2 Isolate number 3 Isolate number 4 Isolate number 5 Isolate number 6 Isolate number 7 Isolate number 8 Isolate number 9 Isolate number 10 Isolate number 11 Isolate number 12 Isolate number 13 Isolate number 14 Isolate number 15 Isolate number 16 Isolate number 17 Isolate number 18 Isolate number 19 Isolate number 20 Isolate number 21 Isolate number 22 Isolate number 23 + catalase positive - catalase negative Table No 6 : Starch hydrolysis: Sl No 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Isolate number Isolate number 1 Isolate number 2 Isolate number 3 Isolate number 4 Isolate number 5 Isolate number 6 Isolate number 7 Isolate number 8 Isolate number 9 Isolate number 10 Isolate number 11 Isolate number 12 Isolate number 13 Isolate number 14 Isolate number 15 Isolate number 16 Isolate number 17 Isolate number 18 Isolate number 19 Isolate number 20 Isolate number 21 Isolate number 22 Isolate number 23 Starch positive + + Starch negative + + + + + + + + + + + + + + + + + + + + + + +
26
+ Starch hydrolysed -starch not hydrolysed.
27
Table No 6 : Gelatine hydrolysis: Sl No 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Isolate number Isolate number 1 Isolate number 2 Isolate number 3 Isolate number 4 Isolate number 5 Isolate number 6 Isolate number 7 Isolate number 8 Isolate number 9 Isolate number 10 Isolate number 11 Isolate number 12 Isolate number 13 Isolate number 14 Isolate number 15 Isolate number 16 Isolate number 17 Isolate number 18 Isolate number 19 Isolate number 20 Isolate number 21 Isolate number 22 Isolate number 23 Gelatine Positive + + + + + + + Gelatine Negative -
+ Liquefaction - No liquefaction.
28
3.6. Effect of antibiotics on bacterial isolates: 3.6.1. Gram positive bacteria: Out of eighteen isolates recovered streptomycin showed maximum length of zone of inhibition compared to chloromphenicol in broad spectrum antibiotics. So streptomycin is preferable and effective. The antibiotics which we have tested particularly for gram positive bacteria are penicillin, erythromycin, amphicillin, methecillin and vancomycin. In these penicillin and erythromycin showed maximum length of zone of inhibition for isolate number 1 and other antibiotics did not showed any inhibition for isolate number 1. For isolate number 2 penicillin showed maximum length of inhibition followed by erythromycin then vancomyin other antibiotics did not showed zone of inhibition. Isolate number 3 showed maximum length of inhibition to penicillin and erythromycin followed by vancomycin other antibiotics did not showed any zone of inhibition. Isolate number 4 and 5 showed maximum length of inhibition to penicillin followed by vancomycin and all other antibiotics are minimum effective or in effective. In isolatate number 6 penicillin showed maximum length of inhibition followed by vancomycin then amphiciln other antibiotics did not show any sort of inhibition. In isolate number 7 penicillin show maximum length of zone of inhibition followed by vancomycin then amphicillin and other antibiotics did not show any inhibition. In isolate number 8 vancomycin showed maximum length of inhibition followed by penicillin and amphicillin other antibiotics did not showed any zone of inhibition. In isolate number 9 penicillin showed maximum length of zone of inhibition followed by vacomycin then amphicillin and other antibiotics didnt showed any sort of inhibition. In isolate number 10 erythromycin showed maximum length of zone of inhibition followed by vacomycin then penicillin other antibiotics didnt showed any type of zone of inhibition. Isolate number 11 showed maximum length of inhibition to erythromycin followed by vancomycin then penicillin and other antibiotics didnt show any sort of zone of inhibition. Isolate number 12 showed maximum length of inhibition to erythromycin followed by amphicillin then to vancomycin and other antibiotics didnt show inhibition to any antibiotics. Isolate number 13 showed maximum number of inhibition to penicillin followed by vancomycin then to erythromycin then to amphicillin other antibiotics did not showed any sort of zone of inhibition. Isolate number 14 showed zone of inhibition only to penicillin and to all other antibiotics did not show any sort of
29
Diabetic Foot Infections zone of inhibition. Isolate number 15 and 17 showed zone of inhibition only to erythromycin and to all other antibiotics it didnt showed any sort of zone of inhibition. Isolate number 16 showed maximum length of zone of inhibition to erythromycin followed by penicillin and all other antibiotics didnt show any sort of zone of inhibition. Isolate number 18 showed maximum number of inhibition to vancomycin followed by penicillin and erythromycin,all other antibiotics didnt showed any sort zone of inhibition. 3.6.2. Effect of antibiotics on these isolates Gram negative bacteria: Out of five gram negative isolates recovered streptomycin showed maximum length of zone of inhibition, but chloromphenicol did not show any effect on these isolates, so comparing both of these broad spectrum antibiotics streptomycin is preferable and effective. The antibiotics which we have tested particularly for gram negative bacteria other than broad spectrum antibiotics like amikacin, gentamycin, tetracycline and polymixin B, gentamycin showed the highest length of zone of inhibition against the taken gram negative isolates than any other antibiotics which we have tested followed by amikacin then tetracycline and polymixin B. so gentamycin is preferable and effective for gram negative bacteria.
30
Table No 8 : Effect of Antibiotics on these Isolates Sl. No 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Isolate numbers Isolate no 1 Isolate no 2 Isolate no 3 Isolate no 4 Isolate no 5 Isolate no 6 Isolate no 7 Isolate no 8 Isolate no 9 Isolate no 10 Isolate no 11 Isolate no 12 Isolate no 13 Isolate no 14 Isolate no 15 Isolate no 16 Isolate no 17 Isolate no 18 38 39 40 40 34 38 14 39 40 33 26 0 33 22 19 27 19 18 32 32 20 22 0 36 24 20 17 0 13 23 14 10 Antibiotics tested Zone of inhibition in mm
Streptomycin erythromycin chloromphenicol Penicillin amphicillin methicillin vancomycin
30 0 0 0 0 More than 40 17 23 0 0 0 31 0 27
30 38 20 33 40 28 39 14 34 10 11 0 27 17 0 14 0 10
0 0 0 0 11 13 18 14 12 0 0 18 13 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
o 16 18 19 22 19 27 20 16 27 17 17 23 0 0 0 0 21
LESS GROWTH AROUND THE DISK
31
Table No 9: Effect of antibiotics on gram negative bacteria. Sl No 1 2 3 4 5 Isolate Numbers Isolate no 19 Isolate no 20 Isolate no 21 Isolate no 22 Isolate no 23 0 27 38 31 32 0 0 0 0 0 Antibiotics tested Zone of inhibition in mm
Streptomycin Chloromphenicol amikacin gentamycin tetracycline polymixin B
16 18 21 26 24
20 20 25 27 28
15 11 14 18 13
15 13 15 17 15
3.7. Effects of plant extracts on these isolates: For isolate no 1 Aegle marmelos showed maximum length of inhibition followed by Azadirocht indica and Momordica charantia. For isolate no 2 Aegle marmelos showed maximum length of inhibition followed by Butea monospeerma and Sclerodendron inermea and all other show minimum inhibition. For isolate no 3 Aegle marmelos showed maximum length of inhibition followed by Momordica charantia all other plants extract showed less growth. For Isolate no 4 Sclerodendron inermea showed the maximum length of zone of inhibition followed by Aegle marmelos all other showed minimum inhibition for all other plants extract. For isolate no 5 Aegle marmelos showed maximum length of inhibition followed by Gymnema sylvester and other plant extract showed minimum inhibition. For isolate no 6 Gymnema sylvester showed maximum length of zone of inhibition followed by Momordica charantia and other plant extract showed minimum inhibition. For isolate no 7 Polyanlthia longifolia showed maximum length of inhibition followed by Aegle marmelos and Sclerodendron inermea and other showed minimum inhibition. For isolate no 8 Butea monospeerma showed maximum length of zone of inhibition followed by Aegle marmelos and all other showed minimum inhibition. For isolate no 9 Momordica charantia showed maximum length of zone of inhibition followed Aegle marmelos and other plant extracts showed minimum inhibition. For isolate no 10 Butea monospeerma showed maximum length of zone of inhibition followed by Azadirocht indica then Aegle marmelos and all other plant extracts showed minimum inhibition. For isolate 11 Aegle marmelos showed maximum length of zone of inhibition followed by Azadirocht indica and all other plant extracts showed minimum inhibition. For isolate no 12 Polyanlthia longifolia and Azadirocht indica showed maximum length of zone of inhibition followed by Gymnema sylvester and all other plants shows minimum inhibition. For isolate no 13 Aegle marmelos showed maximum length of zone of inhibition followed by Murraya koenigii and all other showed minimum
32
Diabetic Foot Infections inhibition. For isolate no 14 only Centella asiatica showed maximum length of zone of inhibition and all other showed less growth. For isolate no 15 Aegle marmelos showed maximum length of zone of inhibition followed by Sclerodendron inermea and all other plant extract showed minimum inhibition. For isolate no 16 Aegle marmelos showed maximum length of zone of inhibition followed by Azadirocht indica then Sclerodendron inermea and all other plants extract showed minimum inhibition. For isolate no 17 Aegle marmelos showed maximum length of zone of inhibition followed by Gymnema sylvester and all other plants extract showed minimum inhibition. For isolate no 18 Aegle marmelos showed maximum length of zone of inhibition followed by Polyanlthia longifolia and Hyptis suaveolens and all other plants extract showed minimum inhibition. For isolate no 19 Aegle marmelos showed maximum length of zone of inhibition followed by Sclerodendron inermea and all other plants extract showed minimum inhibition. For isolate no 20 only Aegle marmelos showed the inhibition all other plants extract showed less growth on the media. For isolate no 21 Aegle marmelos showed maximum length of zone of inhibition followed by Polyanlthia longifolia and Momordica charantia and all other plant extracts showed minimum inhibition. For isolate no 22 Aegle marmelos and Polyanlthia longifolia showed maximum length of zone of inhibition and all other plants extracts showed minimum inhibition. For isolate no 23 Aegle marmelos and Momordica charantia showed maximum length of zone of inhibition followed by Polyanlthia longifolia, Butea monospeerma and Murraya koenigii and all other plant extract showed minimum length of inhibition. On comparing the inhibition of plant extract with the antibiotics our plants crude extracts showed maximum length of zone of inhibition so if we purify our extract it may show more inhibition than that of the antibiotics.
33
Table no 10 : Effect of plant extract on these isolates Sl no 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Isolates number Isolate no 1 Isolate no 2 Isolate no 3 Isolate no 4 Isolate no 5 Isolate no 6 Isolate no 7 Isolate no 8 Isolate no 9 Isolate no 10 Isolate no 11 Isolate no 12 Isolate no 13 Isolate no 14 Isolate no 15 Isolate no 16 Isolate no 17 Isolate no 18 Isolate no 19 Isolate no 20 Isolate no 21 Isolate no 22 Isolate no 23 PL 13 14 17 20 13 13 13 14 12 13 14 13 17 15 HS 15 15 14 0 13 12 11 10 13 12 10 12 BM 19 19 11 17 14 13 10 15 Type of plants used Zone of inhibition in mm GS AM MC AI SI 19 12 15 21 12 13 16 17 12 13 17 13 11 19 15 16 12 21 13 16 14 16 14 12 16 16 12 15 18 12 12 13 15 16 17 14 15 12 13 18 14 18 11 13 13 20 16 15 15 18 14 12 20 13 10 17 14 16 18 14 21 18 13 13 16 16 11 14
MK 15 13 13 14 15 16 10 12 14 15
CA 12 13 10 12 12 12
LESS GROWTH AROUND THE WELL
34
PL- Polyanthia longifolia HS- Hyptis suaveolens BM- Butea monosperma GS- Gymnema sylvester AM- Aegele marmelos MC- Momordica charanta AI- Azadirachta indica SI- Sclerodendron inermae MK- Murraya koenigii CA- Centella asiatica
35
DISCUSSION
36
IV. DISCUSSION
Diabetic foot infections involves series of complications associated with the microbial flora, which are established in the infections at different stages leading to mild, moderate and severe infections controlling these microbial population is a risky task, as they are found to be drug resistant and polymicrobial. So recent decades researchers are oriented towards isolating these pathogens, characterize them and suggest a best antibiotic therapy (Ref et. al. 2009). Present work on diabetic foot infections was carried out with the interaction of isolating bacterial pathogens from foot infection samples of different age grouped diabetic patients. All the clinical samples have yielded, positive results by showing bacterial isolates. These were characterized by morphological and biochemical tests. Similar work has also been carried out by (Ref. et. al. 2008) where isolation of anaerobic bacteria has also been done. Drug resistant flora like S. aureus, hemolytic srepotococci and P. aeruginosa were the frequently encountered bacterial pahtgens in most of the diabetic foot infection samples tested. (Ref et al. 2004). In the present project we too had obtained the similar results as we had recovered MRSA and P. aeruginosa from the samples. We had even attempted for evaluating the efficiency of herbal extracts on these resistant isolates. Among the tested extract Aegle marmelos, Polyanathia longifolia and Sclerodendron inermae have shown successful inhibition of these isolates. In a study conducted by (Ref et. al. 2011) effect of plant extracts on the foot infection isolates are also carried out. But the present work was successful in tracing out the extracts which are effective on methicillin resistant Staphylococci and P. aeruginosa. So extraction of the active components from these extracts and their application have to be carried out in future studies.
37
SUMMARY
38
V. SUMMARY
Diabetic foot infections are the common problems in persons suffering from diabetes. These foot ulcers know to contain a variety of microflora. The organisms were screened by using the samples collected from the different diabetic foot care centre. They were isolated by using common nutrient agar media and different selective media. Then each isolates were subjected for different biochemical tests for characterization. The isolates were then subjected for antibiotic sensitivity test by using different antibiotics, by following Kirby-Bauers disc diffusion method. Antimicrobial activity of different plants leaf extracts was done from the plants namely, Polyanthia longifolia, Hyptis suaveolens, Butea monosperma, Gymnema Sylvester, Aegele marmelos, Momordica charanta, Azadirachta indica, Sclerodendron inermae, Murraya koenigii, Centella asiatica. The antimicrobial activity of these leaf extracts was tested by following agar-well diffusion method. A comparative assessment between commercially available antibiotics and traditional plant extract was done.
39
CONCLUSION
40
VI. CONCLUSION
Diabetic foot ulcers contain a variety of microflora, which inhibit the wound healing capacity of individuals immune system. The presence of these organisms further leads to gangrene and May also leads to the removal of infected part from the body, if not treated properly in the initial stage. When the microbial activity of different antibiotics was done, we observed maximum zone of inhibition by streptomycin which is a broad spectrum antibiotic. In case of gram positive bacteria antibiotics such as penicillin, erythromycin showed a moderate zone of inhibition followed by this, vancomycin and ampicillin showed minimum zone of inhibition. The other antibiotic like chloermphenicol and methecillin does not showed any sort of inhibition. In case of gram negative bacteria, gentamycin is the only antibiotic which showed maximum inhibition. Among the plant extracts used Aegle marmelos and polyanthia longifolia showed maximum zone of inhibition. The other plants like Azardiraclita indica, Murraya koenigii, Momordica charantia showed moderate zone of inhibition. Sclerodendron inerrmae showed least zone of inhibition other plant does not showed inhibition. By the present study, we can say that, Aegle marmelos, polyanthia longifolia, excellent in inhibiting the growth of bacteria when compared to commercial antibiotics. So these plants can be used in the treatment of diabetic foot ulcers.
41
REFERENCE
42
VII. REFERENCE
1.
Andrew. S. Powlsn and Anthony P Coll, 2010, the treatment of diabetic foot infection, journal of antimicrobial chemotherapy.
2.
Barton, P. & Parslow, N. (1996). Diabetic/neuropathic leg ulcer. In Wound care:a comprehensive guide for community nurses (pp. 59-69).
3.
Brazier JS, Stubbs SL, Duerden BI. Metronidazole resistance among clinical isolates belonging to the Bacteroides fragilis group: time to be concerned? J Antimicrob Chemother 1999;44:580-1.
4.
Cefalu WT, Waldman S, Ryder S (2007). Pharmacotherapy for the treatment of patients with type 2 diabetes mellitus: rationale and specific agents. Clin. Pharmacol. Ther. 81(5): 636-649
5.
Dahmen, R., Haspels R., Koomen, B., & Hoeksma, A.F. (2001). Therapeutic Footwear for the neuropathic foot: An algorithm. Diabetes Care, 24 (4), 705-9.
6.
Game FL, Jeffcoate WJ (2008) Primarily management of osteomyelitis of the foot in diabetes. Diabetologia 51: 9627.
7.
Hardik P. Trivedi, Nimish L Pathak, Mahendra G Gavaniya, Anjana K Patel, Dr Harshkanth .D. Trivade and Nitin M Panchal, 2011, Alge marmelous suppresses diabetes, international journal of pharmaceutical research and development.
8.
Lipsky BA. Osteomyelitis of the foot in diabetic patients. Clin Infect Dis 1997;25:1318-26.
9.
Mokabberi R, Ravakhah K (2007). Emphysematous urinary tract infections: diagnosis, treatment and survival (case review series). Am. J. Med. Sci. 333(2)pp:111-116.
43
10.
Naila R, Saadia SA, Samreen R, James L, Waheed MA, Tahir S, Paul JT (2009). High dose thiamine therapy for people with type 2 diabetes and microalbuminuria: a randomised, double-blind, placebo controlled study. Diabetologia. Springer Berlin / Heidelberg. 52(2): pp 208-212.
11.
Sandhya. S, Saikumar. P, Vinod K.R, David Banji and Kumar K ,2011, plants as potent diabetic and wound healing agents , journal of drugs and medicines.
12.
Stess RM, Jensen SR, Mirmiran R, 1997, The role of dynamic plantar pressures in diabetic foot ulcers. Diabetes Care ; 20: 855-8.
13.
Turner R, Cull C, Holman R (1996). United Kingdom Prospective Diabetes Study 17: a 9-year update of a randomized, controlled trial on the effect of improved metabolic control on complications in noninsulin-dependent diabetes mellitus. Ann. Intern. Med.
14.
Vichayanrat A, Lueseangdang L, Pitimana-aree S, Vanasang S, Inthuprapa M, Tandhanand S, 1979, Diabetic foot ulcer. Siriaj Hosp Gaz pp: 883-97.
15.
Yadlapalli N G, Vaishnav A, Sheehan P, 2002, Conservative Treatment of Diabetic Foot Ulcers Complicated by Osteomyelitis Wounds.
44
PHOTOGRAPHS
45
VIII. PHOTOGRAPHS: 8.1 ISOLATES
8.2 ORGANISMS
46
Microscope view of gram Positive cocci
Microscope view of gram Negative bacilli
8.3 ANTIBIOTIC SENSITIVITY PHOTOS
47
48
8.4 ANTIBACTERIAL ACTIVITY OF PLANT EXTRACTS
ISO No. 1
ISO No. 2
ISO No. 3
ISO No. 4
49
IX APPENDIX 9.1 MEDIA COMPOSITION:

Nutrient Agar Media Ingredient Peptone Beef extract Sodium chloride Distilled water pH 7 Mannitol Salt Agar Ingredients Peptic digest animal tissue Pancreatic digest of casein Beef extract Sodium chloride d-mannitol phenol red agar pH 7.4 Coagulase Mannitol Salt Agar Ingredients Brain heart infusion Casein enzymic hydrolystase Papaic digest of soyabean meal Sodium chloride Mannitol Bromo cresol purple Agar pH 7.4 Composition 5g 10.50g 3.50g 3.50g 10g 0.02g 14.50g Composition 5g 5g 1g 75g 10g 0.025g 15g Composition 5g 5g 3g 1000ml
50
Diabetic Foot Infections Baired Parker Agar Ingredients Pancreatic digest of casein Beef extract Yeast extract Glycine Sodium pyruvate Lithium chloride Agar pH 6.8 Composition 10g 5g 1g 12g 10g 5g 20g
Pseudomonas Agar Base Ingredients Pancreatic digest of gelatin Casein enzymic hydrolysate Potassium sulphate Magnesium chloride Agar pH 7.1 Composition 16g 10g 10g 1g 11g
51
Starch Agar Media Ingredients Starch Peptone Beef extract Agar Distill water pH 7 Composition 20g 5g 3g 15g 1000 ml
Gelatin Media Ingredients Peptone Beef extract Gelatin Distill water pH 6.8 Composition 5g 3g 5g 1000 ml
52

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Diabetic Foot Infections

Dept. of Microbiology, Sahyadri Science College, Shivamogga