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Abstract
3. Microbiology of diabetic foot
Diabetic foot infections are generally polymicrobial in nature, osteomyelitis most often a single pathogen (Kessler et al., 2005). 76% of organisms isolated from diabetic foot ulcers were Gram-negative aerobic bacilli and 24% of organisms were Gram-positive cocci. Gram-positive aerobic bacteria were found to be the predominant organisms causing diabetic foot infections in many studies but the more recent studies report Gram-negative aerobes to be the commonest organisms in diabetic foot ulcers (Gadepalli et al., 2006 and Shankar et al., 2005).
Pseudomonas aeruginosa was the organism isolated in 23% samples. Staphylococcus aureus was second commonest (21%). A number of studies have found that Staphylococcus aureus and other Gram-positive aerobes are the main causative pathogen usually isolated in more than 60% of cases. The prevalence of MRSA is as high as 20-30%(Lipsky et al., 2004).
2.1.4. Problems of antibiotic use in treatment
When considering antibiotic therapy, the efficacy, toxicity, dosage, route of administration, duration of treatment and whether a mono-or multi-agent regimen is required all need to be carefully evaluated. In sepsis, therapy is commonly commenced without the benefit of microbiological information on culture and sensitivities of the organism and empirical regimens are usually started as soon as life-threatening infection is diagnosed or suspected. The antibiotic used depends on the most likely source of infection and the organisms that commonly originate from it. It is important that a good history is obtained and physical examination undertaken to determine the most likely source of infection (Nunes et al., 2006).
2.2. Gram-positive diabetic foot bacteria
2.2.1. Staphylococcus aureus
2.2.1.1. Taxonomy of S.aureus species
The Staphylococcus genus includes at least 40 species. Of these, nine have two subspecies and one has three subspecies. At least 30 species of staphylococci have been recognized by biochemical analysis. This is especially so with DNA-DNA hybridization (Mandal, 2013). The genus Staphylococcus is in the Bacterial family Staphylococcaceae, which includes three lesser known genera, Gamella, Macrococcus and Salinicoccus. The best-known of its nearby phylogenetic relatives are the members of the genus Bacillus in the family Bacillaceae, which is on the same level as the family Staphylococcaceae. The Listeriaceae are also a nearby family Http://text book of bacteriology.net, 2008).
2.2.1.2. Physiology of Staphylococcus aureus species
Staphylococcus aureus is facultative anaerobic Gram-positive cocci which occur singly, in pairs, and irregulular clusters. S. aureus is nonmotile, non-spore forming (Turnidge et al., 2008). S. aureus reproduces asexually by binary fission. The two daughter cells do not fully separate and remain attached to one another. This is why the cells are observed in clusters. This clustering helps to distinguish staphylococci from streptococci, which usually grow in chains. Growing on solid medium colonies of S. aureus these appear as golden clumps (Mandal, 2013).
The cell wall of the bacteria contains teichoic acid. Ribitol teichoic acid (Polysaccharide A) is present in Staphylococcus aureus. Protein A uniformly coats surface of S. aureus and is usually oxidase negative. They produce an extracellular cell clumping factor, and some strains produce capsules. They are capable of prolonged survival on environmental surfaces in varying conditions (Lowy et al., 2011). Staphyloccoci are capable of generating energy by aerobic respiration, and by fermentation which yields mainly lactic acid and require complex nutrients, e.g., many amino acids and vitamins B, for growth. S. aureus is very tolerant of high concentrations of sodium chloride, up to 1.7 molar. The organisms are resistant to temperatures as high as 50°C, to high salt concentrations, and to drying. Colonies are usually large (6-8 mm in diameter), smooth, and translucent. These bacteria are Gram-positive cocci about 0.5-1.0 μm in diameter (Mandal, 2013).
In 1884, Rosenbach described the two pigmented colony types of staphylococci and proposed the appropriate nomenclature: Staphylococcus aureus (yellow) and Staphylococcus albus (white), only Staphylococcus aureus and Staphylococcus epidermidis are significant in their interactions with humans. S. aureus colonizes mainly the nasal passages, but it may be found regularly in most other anatomical locales, including the skin, oral cavity and gastrointestinal tract. S. epidermidis is an inhabitant of the skin. It has been estimated that approx. 20-30% of the general population are S. aureus carriers (Http:// text book of bacteriology.net, 2008).
2.2.1.3. Laboratory diagnostic characteristics of Staphylococcus aureus species
After sample from the lesions are taken, they can be stained with Gram stain. S. aureus is Gram positive. The organism from the clinical specimen from blood culture or pus is then streaked over solid media such as blood agar, tryptic soy agar or heart infusion agar. If the specimen is suspected to be contaminated it is plated on mannitol salt agar containing 7.5% sodium chloride (Mandal, 2013).
The major criterion for identification is the organism’s ability to clot plasma.Typical colonies are yellow to golden yellow in color, smooth, entire, slightly raised, and hemolytic on 5% sheep blood agar. However, many strains may appear dirty white and nonhemolytic. It also gives apositive mannitol fermentation and deoxyribonuclease test (Turnidge et al., 2008). Most strains ferment mannitol, and glucose under anaerobic conditions, produced aceton from glucose, grew and produced black colonies on tellurite glycine agar, required both thiamine and nicotinic acid, and did not require other vitamins or purines( Brown and Evans, 1963).
S. aureus produces coagulase which interacts with prothrombin in the blood causing plasma to coagulate by converting fibrinogen into fibrin. Blood coagulation is used to distinguish S. aureus from others members of the genus, which are collectively designated as coagulase-negative staphylococci (CoNS) (Ray and Ryan, 2004). S. aureus is catalase-positive (meaning it can produce the enzyme catalase.