Ординатура / Офтальмология / Английские материалы / Basic Sciences in Ophthalmology_Velayutham_2009
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Peptidoglycan consists of alternating strands of N-acetyl muramic acid and N- acetyl glucosamine.
In addition to these structures gram-positive cell wall has some special components called teichoic and teichuronic acids.
Gram-negative cell wall is a complex structure and thinner than that of gram-positive cell. Outer membrane is made of phospholipids bilayer, proteins and lipopolysaccharides (LPS - Endotoxin).
Growth and Nutrition of Bacteria
The most important elements necessary for synthesis of bacterial structural components (Carbohydrate, lipid, protein, nucleic acid) are hydrogen, oxygen, carbon and nitrogen. In addition to these elements, phosphorus and sulphur are also required for bacterial growth.
Many bacteria produce toxins, enzymes and pigments. Toxins and enzymes play important role in pathogenicity. Toxins are of two types:
1.Exotoxins: Exotoxins are usually heat labile proteins secreted extracellularly by certain species of bacteria which diffuse into the surrounding medium. It is soluble in water and culture medium. Production of exotoxin is a physiological process and compatible with normal life of bacteria. It can be separated from culture by filteration. Exotoxins are potent in minute quantity. They are highly antigenic and can be converted into nontoxic toxoid by addition of formalin. Toxoids are nontoxic but antigenic, i.e. retain the ability to produce antibodies (antitoxins).
2.Endotoxins: Endotoxins are heat labile lipopolysaccharide-protein complexes which form structural components of the cell wall of gramnegative bacteria and liberated only on cell lysis or death of bacteria. Endotoxin is not separable from the elaborating microorganisms and their culture filtrate is nontoxic while the dead bacteria are equally toxic. Endotoxins are poor antigens and active only in relatively large doses.
STERILIZATION AND DISINFECTION
Sterilization is a process by means of which an article, surface or medium is made free from all living microorganisms including spores.
Disinfection is a process of destruction of vegetative forms of pathogenic organisms which are capable of producing infection.
There are different methods of sterilization:
1.Physical methods:
(a)Sun light (Natural source)
(b)Dry heat (Hot air-oven temp. 165 degree centigrade for one hour) - sterilizing metals, glass, cotton etc.
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(c)Moist heat (Autoclave temp. 121 degree centigrade for 20 minutes) - sterilizing dressings, bin, culture medium etc.
(d)Filteration - HEPA filters in Laminar hood and seitz and membrane filters for sterilizing serum.
(e)UV radiation - in Laminar hoods and Bio-safety cabinets
(f)Ionising radiation - gamma radiation for disposable plastics
2.Gaseous methods
(a)Ethylene oxide - for disposable syringes and IV sets
(b)Formaldehyde - For sterilizing the operation theatres
3.Chemical methods
(a)Glutarldehyde - 2% (cidex) for endoscopes and cystoscopes
Following disinfectants are used widely:
1.Aldehyde:
(a)Glutaraldehyde - for sterilizing Bronchoscopes
(b)Formaldehyde - Wards, operation theatres and Instruments
2.Oxidising agents:
(a)Hydrogen peroxide - 3-6% kills most of the organisms
3.Halogens: Bacteriocidal, used for water sterilization
Sodium hypochlorite 5% can be used for
Disinfecting HIV and Hepatitis virus
(b) Iodine: such as povidone iodine for topical applications
4.Alcohols: Ethanol (Absolute alcohol) esp. 70 % and
Iso-propyl alcohol are effective on skin.
The other disinfectants like phenol (cresol or Lysol ), Chlorhexidine (savlon), Chloroxylenol (dettol) are also effective in killing the microorganisms.
Cultivation of Bacteria
Bacteria needs a culture medium for in vitro growth. There are plenty of culture medium available for cultivating the bacteria. The specimen to be cultivated is inoculated on a suitable medium and incubated at 37°C for 1824 hours.
The constituents of a basal culture media include water, electrolytes like sodium chloride, peptone (partially digested proteins from animal or plant source), Meat extract (lab lemco), yeast extract and agar.
Agar is derived from sea weed (algae). It has carbohydrates and fiber. It gives the solidifying effect to the solid media.
Simple or basal media: Basal media includes peptone water, Nutrient broth, Nutrient agar. Peptone water consists of 1% peptone and 0.5% sodium chloride and distilled water at a pH of 7.4 to 7.5
1.Nutrient broth is prepared from adding 1% meat extract to peptone water.
2.Nutrient agar is prepared by adding 2% agar to the nutrient broth.
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Enriched medium: For the growth of certain fastidious bacteria, 5% sheep blood can be added to the Nutrient Agar (Blood agar). Other substances like cysteine, haemin can be added to chocolate agar (charred blood agar). Enrichment medium: Certain liquid media which favours the growth of the particular bacteria and suppress the growth of commensals, e.g. Selenite F broth, tetrathionate broth for Isolation of Salmonellae.
Selective medium: Selective media are media that contain additives that enhance the growth of the desired organism by inhibiting other organism. DCA (Deoxy cholate citrate agar for Salmonella and Shigella spp.), LJ (Lowenstein Jensen ) agar for Mycobacteria and TCBS (Thiosulphate, Citrate, Bile salt, Sucrose) for Vibrios.
Differential media: MacConkey agar for enterobactericeae. This contains lactose to differentiate lactose fermentors (E. coli) from non-lactose fermentors (proteus)
Transport medium: Used for transporting specimens, e.g. Stuart's medium for Gonococci and VR (Venkatraman Ramakrishnan) medium for Vibrios.
Identification of the bacteria after cultivation requires an array of biochemical tests to confirm.
The important and basic tests include catalase (Streptococcus are positive). Oxidase (Pseudomonas are positive), sugar fermentation reactions (e.g. Glucose, lactose etc.), Indole formation (E. coli is positive), Citrate utilization (Klebsiella is positive), coagulase (Staphylococcus is positive), Methyl red test (MR), Voges-Proskauer test (VP), urease (Helicobacter pylori + ve) and hydrogen sulphide production (Proteus).
SYSTEMATIC BACTERIOLOGY
Staphylococcus
Staphylococcus are gram-positive cocci that occur in grape like clusters. They are the commonest cause of localised suppurative lesion in man. Staphylococci were first observed in human pyogenic lesions by Von Reckling Lausen in 1871. Staphylococci are classified into 2 groups based on coagulase production. Staph. aureus which is coagulase positive, mannitol fermenting are pathogenic. They produce toxins. Most strain from golden yellow colonies.
Staph. epidermidis (Staph. albus) which is coagulase negative, mannitol non-fermenting are usually saprophytes. They do not produce toxins.Most strain form white colonies, though some may be pigment (Staph. citreus).
Morphology
They are spherical cocci approximately 1µ in diameter arranged in grape like cluster. Cluster formation is due to cells dividing sequentially in three perpendicular planes. They may also found singly or in pairs and in short
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chain. They are nonmotile, non-sporing and non-capsulated. Sometimes capsules are demonstrated in young culture. They readily stain with aniline dyes and are uniformly gram-positive. The genus staphylococci has atleast 20 species.
Culture
They grow on nutrient agar incubated at 370°C for 24 hours. They form small white or golden brown colonies which are opaque. They can also be cultures on blood agar to demonstrate the type of hemorrhage. 5% Sheep blood is used to prepare blood agar plates. They grow in Maonkey's medium and produce small pink colonies due to fermentation of Lactose. Other media particular for Staphylococci are salt-milk agar (8-10 % of NaCl), Ludlam's medium.
Biochemistry
Staphylococci ferment a good number of sugar producing acid but no gas. They are catalase positive unlike Streptococci. They hydrolyse urea reduce nitrates to nitrites, liqueify gelatin and are MR and VP positive but indole negative. Phosphotase is produced.
Resistance
Staphylococci are more resistant of non-sporing bacteria. Dried on threads, they retain their viability for 3-6 months. They may withstand 600°C for 30 minutes. Some strains grown in the presence of 10-15% NaCl. Phenol and Mercury per chloride kills them. Staphylococci are uniformly resistant to lysozyme. They were originally sensitive to sulphonamides, penicillin and other antibiotics, but they develop drug resistive. So readily that most strain, especially from the hospital environment are now resistant to the commonly used antibiotic. Resistance may be due to Betalactamase production, e.g. penicillin. G, Ampicillin, Ticarcillin and similar drugs. Now many strains of S. aureus are even resistant to oxacillin and methicillin. They are coined as MRSA (Methicillin resistant Staphylococci aureus) and they are sensitive to vancomycin and teicoplanin.
Antigen Structure
Staphylococci contains protein, antigenic polysaccharides. Capsular antigen are found in mucoid strain. Peptidoglycan polysaccharide elicits the production of interleukin - 1 (endogenous progeny ) and is responsible for the end toxic activity. Protein A is a cell wall component. It binds to the fact portion of Gig bound to protein A is free to combine with a specific antigen. This is called coagglutination and is used in diagnostic lists.
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Bacteriophage Typing
Staphylococci may be typed, based on their susceptibility to bacteriophage. The reference center for Staphylococcal phage typing in India is located in the Department of Microbiology, Maulana Azad Medical College, New Delhi. According to the information available, phage type 52/52A/80/81 is prevalent in most parts of India.
Toxic and Virulence Factors
Staph. aureus produce a number of exotoxins. The toxins are haemolysin, Leucocidin, Hyaluronidase fibrinolysin (streptokinase), nucleases, lipases. Pyogenic exotoxin is produced by Staphylococcus strain causing toxic shock syndrome.
Enterotoxin
This toxin is responsible for the manifestation of staphylococcal food poisoning, nausea, vomiting, diarrhea within six hours of consuming contaminated food.
Exfoliate Toxin
This epidermolytic toxin leads to a variety of exfoliate skin diseases including generalized exfoliation (Reiter's syndrome), toxic epidermal necrolysis, localized bullous impetigo and generalized scarlatini form eruption. This clinical picture is called Staphylococcal scalded skin syndrome.
Coagulase
Staph. aureus has the property of clotting human plasma. This is due to the enzyme coagulase, which along with an activator, the coagulase reacting factor (CRF) present in plasma, converts fibrinogen to fibrin. Coagulase promotes the virulence of the organism by inhibiting phagocytosis.
Pathogenicity
Staphylococci cause the majority of acute pyogenic lesions in man. Staphylococcal diseases may be classified as cutaneous and deep infections, acute toxaemia including food poisoning, exfoliative diseases and the toxic shock syndrome. Cutaneous lesions are furuncles, styes, boils, abscesses. carbuncles, impetigo and pemphigus neonatorum. Hospital-cross infections (Nosocominal ) are common.
The commonest deep infection is acute osteomyelitis. In the respiratory track, it causes tonsillitis, pharyngitis, sinusitis and pneumonia. Hematogenous spread may lead to meningitis, endocarditis, renal abscesses and brain abscesses Staphylococcal food poisoning results when food is contaminated with
enterotoxins is consumed. The symptoms start within 6 hours.
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Toxic shock syndrome is characterized by acute onset of high fever, hypotension, vomiting, diarrhea and scarlatiniform rash.
Epidemiology
Staphylococcal disease may follow endogenous or exogenous infection. The modes of transmission may be by contact, direct or through fomites, by dust or by air-borne droplets. They are the commonest cause of hospital acquired infections and postoperative wound infections, especially the MRSA strains.
Laboratory Diagnosis
The specimens to be collected are pus, sputum, faeces, blood, nasal swab (for carriers ), CSF and urine (pylonephritis ).
1.Direct microscopy. Gram stain. Group like clusters of Gram-positive cocci seen.
2.Culture of the organism can be done on blood agar plates, Nutrient agar and selective medium like salt-milk agar.The colonies on NA are small, opaque with golden brown color. On BA, beta-hemolysis seen. Smear from the culture are examined and subjected for coagulase test and other biochemical tests such as mannitol fermentation.
Treatment
As the drug resistance is so common in staphylococci, appropriate antibiotic should be chosen after antibiotic sensitivity tests. Penicillin is effective. If resistant , methicillin and cloxacillin are effective against pencillinase producing strain. For MRSA, Vancomycin and teicoplanin can be used.
Coagulase Negative Staphylococci
Staphylococcus epidermidis
These are coagulase negative staphylococci which are part of the normal flora. They may act as opportunistic pathogens causing septicemia, cystitis, UTI endocarditis and colonise the prosthetic valves of the heart.
Staphylococcus saprophyticus
This coagulase negative Staphylococcus has become clinically important as a common cause of acute urinary tract infection and is resistant to Novobiocin.
Micrococci
These are gram-positive cluster forming cocci which differ from staphylococci in fermenting sugars. They occur in tetrads or eight. They are saprophytes that may rarely cause opportunistic infection.
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STREPTOCOCCI
Introduction
Streptococci are gram-positive cocci arranged in chains. They are important human pathogen causing pyogenic infection. They are also responsible for non-suppurative lesion like acute rheumatic fever and glomerulonephritis.
Classification
The Streptococci are classified based on their hemolytic properties.
α. Alpha-hemolytic streptococci produce a greenish discoloration with partial hemolysis around the colonies
β. Beta-hemolytic streptococci produce a sharply defined, clear, colorless zone of hemolysis.
γ. Gamma or non-hemolytic streptococci produce no change in the blood agar medium.
Most of the pathogenic streptococci (Streptococcus pyogenes) fall into the beta group and are called the hemolytic streptococci. The alpha streptococci are commensals of the throat (Str. viridans). The gamma streptococci include the fecal streptococci (Str. faecalis or enterococcus ).
The hemolytic streptococci were classified by Lancefield (1933) serologically into groups based on the nature of a carbohydrate (c) antigen on the cell wall. These are known as Lancefield groups, 19 of which have been identified so far and namedA-U (without I & J). The great majority of hemolytic streptococci that produce infections belong to group A (Str. pyogenes). These may be further divided into types based on the protein (M,T & R) antigen present on the cell surface (Griffith typing ). About eighty types Str. pyogenes have been recognized so far.
Streptococci Pyogenes
They are spherical cocci having 0.5 - 1.0 µ in size. Gram positive and appear as chains.
Culture
It is an aerobe and facultative anaerobe, growing best at a temperature of 370°C (22–42°C). It is a fastidious organism which grows in culture medium containing blood or serum.
On blood agar, after incubation for 24 hours, the colonies are small (0.5 - 1.00 mm ), circular, semi-transparent with an area of clear hemolysis around them. Growth and hemolysis are promoted by 10% CO2. Strain with well marked capsules produce mucoid colonies. Biochemically they ferment most of the sugars produced acid but no gas. They are catalase negative and are not soluble in 10% bile, unlike Pneumococci.
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Resistance
They are easily destroyed by heat. It is resistant to crystal violet. It is susceptible to penicillin and may antibiotics and unlike Staphylococci does not develop resistance to antibiotics.
Antigenic Structure
The cell wall is composed of an outer layer containing protein and lipoteichoic acid, a middle layer of group specific carbohydrate and an inner layer of peptidoglycan (mucoprotein) is responsible for cell wall rigidity. It has also some biological properties such as pyogenic and thrombolytic activity.
Toxins and Virulence Factors
Streptococcus pyogenes forms several exotoxins and enzymes which contribute to its virulence.
Hemolysins
Streptococci produce two hemolysis, Streptolysin 'O' and 'S' Streptolysin O is antigenic and antistreptolysin regularly appear in sera following Streptococcal infections. Estimation of ASO titer (Anti-streptolysin O) is a serological procedure of the retrospective diagnosis of infection with Str. pyogenes an ASO tube of >200 IU is significant.
Streptolysin S is responsible for the hemolysis seen around streptococcal colonies on the surface of blood agar plates. The other virulence factors enzyme include erythrogenic toxin, Streptokinase streptodornase, hyaluronidase and proteinase.
Pathogenicity
Respiratory Infections
The primary site of invasion of the human body by Str. pyogenes is the throat. Sore throat is the commonest of streptococcal infections. It may be localized to tonsils or pharynx.
Scarlet fever is a special variety of sore throat where infection is caused by the strain producing erythrogenic toxin.
Skin Infection
Str. pyogenes produce a variety of suppurative infection of the skin. For example, erysipelas and impetigo.
Non-suppurative Complication
Str. pyogenes infections lead to two important non-suppurative sequelae— Acute rheumatic fever and acute glomerulonephritis. The antigen of streptococci cross react with the glomerular antigen and heart tissue antigen.
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Epidemiology
Streptococcal infection of the respiratory trait are more frequent in children 5-8 years of age. Crowding is an important factor in the transmission of infection.
Laboratory Diagnosis
The diagnosis is established by Gram stain of the pus, CSF or throat swab. Cultivation of the organism can be done on blood agar plates incubated at 37°C under 5-10% CO2. The fluorescent antibody technique has been employed for the rapid identification of group A streptococci.
ASO titres are useful in the diagnosis of acute rheumatic fever and glomerulonephritis.
Prophylaxis
The indication for prophylaxis in streptococcal infection is only in the prevention of rheumatic fever. This is achieved by a long-term administration of penicillin in children who have developed early signs of rheumatic fever.
Treatment
All beta hemolytic group and streptococci are sensitive to penicillin G. in patients allergic to penicillin, erythromycin or Cephalexin may be used. Fluroquinolones such as ciprofloxacin, and ofloxacin are also effective.
The Viridians Group: (Str. viridians)
This group consists of Streptococci that produce alpha hemolysis on blood agar. They are the commensals of the mouth and throat. Generally they are non-pathogenic, but in persons with predisposing factors, such as valvular disease of the heart. They produce subacute bacterial endocarditis. Tooth extraction in such persons is dangerous and should be done under antibiotic— They are susceptible to penicillin.
Group G Streptococci (Enterococci)
Enterococci usually appears in pairs. They are predominantly present in intestine, genital tract and saliva.
They may be associated with urinary tract infection, subacute bacterial endocarditis, septicemia and peritonitis. They are sensitive to penicillin.
PNEUMOCOCCUS
Pneumococcus (Str. pneumoniae)
Pneumococci are gram-positive lanceolate diplococci which resemble the viridans streptococci. Pneumococci possesses a polysaccharide capsule which confers virulence to the organism. Pneumococci are normal inhabitants of the upper respiratory tract. They are the most prevalent simple bacterial agent in
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pneumonia and in other media in children. They can also cause sinusitis, bronchitis, meningitis, and septicemia.
Morphology
Pneumococci are typically small (1µ) slightly elongated cocci presenting a flame shaped or lanceolate appearance. They are capsulated. They are strained by Gram stain and the capsule may be demonstrated by Indian ink staining (Fig. 33.5).
Fig. 33.5: Capsulated diplococci (Pneumococci) from sputum (Gram's stain)
Culture
Pneumococci have complete growth requirements and grow only in enriched media. Growth is improved by 5—10% CO2.
On blood agar, after incubation for 18 hours, the colonies are small (0.5 - 1 mm) dome-shaped and glistening with an area of greenish discoloration (alpha hemolysis ) around them.
Biochemically, the ferment several sugars forming acid only. Pneumococci are bile soluble and they are catalase and oxidase negative.
Resistance
Pneumococci are delicate organisms that are readily destroyed by heat. They are sensitive to penicillin and other antibiotics. The sensitivity of Pneumococci to optochin useful in differentiating them from Streptococcus.
Antigenic Structure
The most important antigen of the pneumococci is the type specific capsular polysaccharide. Typing may be carried out by agglutination of the cocci with the type specific antigen.