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Medical Microbiology- Second Class- Medical Lab. Techniques Dept. 1 Medical Microbiology Lecture Notes Second Class Medical Laboratory Techniques Department (15-11) Medical...

Medical Microbiology- Second Class- Medical Lab. Techniques Dept. 1 Medical Microbiology Lecture Notes Second Class Medical Laboratory Techniques Department (15-11) Medical Microbiology- Second Class- Medical Lab. Techniques Dept. 2 11-12. Chemotherapy and Antibiotic Resistance Introduction Early death or severe lifelong debilitation from scarlet fever, diphtheria, tuberculosis, meningitis, and many other bacterial diseases. Any chemical used in treatment, relief, or prophylaxis ‫الوقاية‬of disease is defined as a chemotherapeutic drug or agent.  Antimicrobial Chemotherapy (Anti-infective Chemotherapy) is the chemical treatment of a disease.  Two types of chemotherapeutic agents are synthetic drugs (chemically prepared in the laboratory) and antibiotics (substances produced naturally by bacteria and fungi that inhibit the growth of bacteria).  Paul Ehrlich introduced an arsenic-containing chemical called salvarsan to treat syphilis (1910).  Alexander Fleming observed that the Penicillium fungus inhibited the growth of a bacterial culture. He named the active ingredient penicillin (1928).  Researchers are tackling the problem of drug-resistant microbes. Chemotherapeutic drug or agent is defined as any chemical used in treatment, relief, or prophylaxis of disease. Antimicrobial drug (also termed anti-infective drugs) are a special class of compounds capable even in high dilutions of destroying or inhibiting microorganisms. Antibiotics are substances produced by the natural metabolic processes of some microorganisms (bacteria and fungi) that can inhibit or destroy other microorganisms. Narrow-spectrum antimicrobial drugs affect a small range of microbes. Broad-spectrum drugs affect a wider range of microbes. Names of treatments that cause the outright death of microbes have the suffix -cide, meaning kill. A biocide, or germicide, kills microorganisms. Other treatments only inhibit the growth and multiplication of bacteria; their names have the suffix -stat or -stasis, meaning to stop or to steady, as in bacteriostasis. Sepsis, from the Greek for decay or putrid, indicates bacterial contamination. Aseptic means that an object or area is free of pathogens. A sterilizing agent is called a sterilant. Liquids or gases can be sterilized by filtration. Disinfectant: is a chemical substance or compound used to inactivate or destroy microorganisms on inert surfaces. When this treatment is directed at living tissue, it is called antisepsis, and the chemical is then called an antiseptic. Disinfection: It usually refers to the destruction of vegetative (non–endospore- forming) pathogens, which is not the same thing as complete sterility. Medical Microbiology- Second Class- Medical Lab. Techniques Dept. 3 As a series of stages, this process can be visualized more clearly: (1) A specific route is used to administer the drug to the host. There are four main routes of delivery: oral, circulatory, muscular, and cutaneous. (2) Body fluids dissolve the medication. (3) The medication is administered to the affected area (extracellular or intracellular). (4) The drug destroys the infectious agent or inhibits its growth. (5) Ideally without causing harm to the host's organs, the drug is finally expelled or broken down by them. The best antimicrobial drugs have low toxicity to humans and lack other side effects such as (drug resistance, allergy, and disruption of natural flora). Selecting a drug for therapy is based upon the microbe’s sensitivity to the drug, the drug’s toxicity, and the health of the patient. (Drugs may cause allergies and disrupt the host’s normal flora) Some idea of the effectiveness of a chemotherapeutic agent against a pathogen can be obtained from the minimal inhibitory concentration (MIC). The MIC is the lowest concentration of a drug that prevents growth of a particular pathogen. On the other hand, the minimal lethal concentration (MLC) is the lowest drug concentration that kills the pathogen. A cidal drug generally kills pathogens at levels only two to four times more than the MIC, whereas a static agent kills at much higher concentrations, if at all. Classification of Antibiotics according to effects on bacteria Bactericidal (Bacteriocidal) which kill bacteria and cause no growth and division. Bacteriostatic which stop the growth of bacteria without killed it. Classification of Antibiotics according to nature - natural drugs - semi-synthetic drugs - synthetic drugs Characteristics of the Ideal Antimicrobial Drug  Selectively toxic to the microbe but nontoxic to host cells.  Microbicidal rather than microbistatic  Relatively soluble and functions even when highly diluted in body fluids.  Remains potent long enough to act and is not broken down or excreted prematurely.  Not subject to the development of antimicrobial resistance.  Complements or assists the activities of the host’s defenses Remains active in tissues and body fluids.  Readily delivered to the site of infection.  Not excessive in cost. Medical Microbiology- Second Class- Medical Lab. Techniques Dept. 4  Does not disrupt the host’s health by causing allergies or predisposing the host to other infections. Properties of Some Common Antibacterial Drugs Medical Microbiology- Second Class- Medical Lab. Techniques Dept. 5 Antimicrobial Activity Can Be Measured by Specific Tests..... Practical lecture There are different modes of action of antimicrobial drugs:  Inhibition of cell wall synthesis.  Inhibition of protein synthesis.  Inhibition of nucleic acid replication and transcription.  Injury of plasma membrane.  Inhibition of essential metabolite synthesis. Tortora, 561 Bacterial cell (according to the species) develops different types of resistance, and this issue considered the problem of the world. The resistant mechanisms are:  Blocking the antibiotic entry.  Inactivation by enzymes.  Alteration of the target molecule.  Efflux of antibiotic. Medical Microbiology- Second Class- Medical Lab. Techniques Dept. 6 Tortora, 580 13. Immunity and vaccination Immunity is the ability of the human body to tolerate the presence of material indigenous to the body and to eliminate foreign substances. This discriminatory ability to eliminate foreign substances is performed by a complex system of interacting cells called the immune system. The immune system develops a defense against antigens, which are substances that can stimulate the immune system. This defense is known as the immune response and usually involves the production of: Protein molecules (immunoglobulins or antibodies, the major component of humoral immunity) by B-lymphocytes (B-cells) Specific cells, including T-lymphocytes (also known as cell-mediated immunity). Types of Immunity There are two basic mechanisms for acquiring immunity: passive and active. Passive Immunity Passive immunity is protection by antibody or antitoxin produced by one animal or human and transferred to another. Passive immunity provides immediate protection against infection, but that protection is temporary. The antibodies will degrade during a period of weeks to months, and the recipient will no longer be protected. Active Immunity Active immunity is protection produced by a persons own immune system. The immune system is stimulated by an antigen to produce antibody-mediated and cell- mediated immunity. Unlike passive immunity, which is temporary, active immunity usually lasts for many years, often for a lifetime. Another way to produce active immunity is by vaccination (An easy, secure, and reliable method of preventing hazardous infections before you are exposed to them). Medical Microbiology- Second Class- Medical Lab. Techniques Dept. 7 Vaccines (Vaccines train your immune system to create antibodies, just as it does when it’s exposed to a disease) contain antigens that stimulate the immune system to produce an immune response that is often similar to that produced by the natural infection. With vaccination, however, the recipient is not subjected to the disease and its potential complications. Vaccines reduce risks of getting a disease by working with your body’s natural defenses to build protection. When you get a vaccine, your immune system responds It: Recognizes the invading germ, such as the virus or bacteria. Produces antibodies. Antibodies are proteins produced naturally by the immune system to fight disease. Remembers the disease and how to fight it. If you are then exposed to the germ in the future, your immune system can quickly destroy it before you become unwell. Most vaccine preparations contain one of the following antigenic stimulants. killed whole bacterial cells or inactivated viruses. live, attenuated bacterial cells or viruses. antigenic components of cells or viruses. genetically engineered microbes or microbial antigens. Killed microorganisms Killed vaccines have the advantage over attenuated m. in that they pose no risk of vaccine associated infection. Killed organisms often provide a weak or short lived immune response. Some vaccines such as polio and typhoid vaccines, are available both in live and killed versions. Live pathogens When live pathogens are used they are attenuated (weakened) to preclude clinically. consequences of infection. Attenuated microbes reproduce in the recipient typically leading to a more robust and long-lasting immune response than can be obtained through vaccination with killed organisms. Microbial extracts Instead of using whole O. vaccine can be composed of antigen molecules (often those located on the surface of the M. ex flagella, fimbriae). Extracted from the pathogen or prepared by recombination DNA technique. The efficacy of these vaccines varies. In some instances, the vaccine antigen is present on all strains of the organism, and the vaccine, thus protected against by all strains. Toxoids These are derivatives of bacterial exotoxins produced by chemically altering the natural toxin or by engineering bacteria to produce harmless variants of the toxin. Medical Microbiology- Second Class- Medical Lab. Techniques Dept. 8 Vaccines containing toxoid are used when the pathogenicity of the O. is a result of the secreted toxins. Depending on the specific vaccine, administration is generally via intramuscular or subcutaneous. Classification of Vaccines There are two basic types of vaccines. Their characteristics are different and determine how each type is used. 1. Live, attenuated, and 2. Inactivated. 1. Live, Attenuated Vaccines Live vaccines are derived from “wild” viruses or bacteria. These wild viruses or bacteria are attenuated (weakened) in a laboratory, usually by repeated culturing. For example, the measles virus used as a vaccine today was isolated from a child with measles disease in 1954. Almost 10 years of serial passage using tissue culture media were required to transform the wild virus into the attenuated vaccine virus. 2. Inactivated Vaccines Inactivated vaccines are not live and cannot replicate. These vaccines cannot cause disease, even in an immunodeficient person. Inactivated antigens are less affected by circulating antibody than are live antigens, so they may be given when antibody is present in the blood (e.g., in infancy or following receipt of antibody-containing blood products). Classification of Vaccines according to source of production: Bacterial Vaccines Vaccines with more specialized indications are described below: Anthrax (Bacillus anthracis), Cholera (Vibrio cholerae), Typhoid fever (Salmonella typhi), Plague (Yersinia pestis). Viral Vaccines Hepatitis A, Hepatitis B, Varicella zoster, Polio, Influenza, Measles, mumps and rubella, Human papilloma virus vaccine,Triple vaccine Medical Microbiology- Second Class- Medical Lab. Techniques Dept. 9 14. Staphylococci The most clinically important species of Staphylococci include Staphylococcus aureus, S. epidermidis and S. saprophyticus. They are Gram-positive cocci; usually arranged in clusters; non-motile; catalase positive; non-sporing; grow over a wide temperature range (10–42 ˚C), with an optimum of 37 ˚C; aerobic and facultative anaerobs (except Staphylococcus aureus subsp. anaerobius and Staphylococcus saccharolyticus, which are obligate anaerobes); grow on simple media. Classification 1. Colonial morphology: S. aureus colonies are grey to golden yellow; S. epidermidis and S. saprophyticus colonies are white. Staphylococci may produce haemolysins, resulting in haemolysis on blood agar. 2. Coagulase test: S. aureus possesses the enzyme coagulase, which acts on plasma to form a clot. Other staphylococci (e.g. S. epidermidis and S. saprophyticus) do not possess this enzyme and are often termed, collectively, ‘coagulase-negative staphylococci or non S. aureus Staphylococci’ (CoNS). There are three methods to demonstrate the presence of coagulase: (a) tube coagulase test: diluted plasma is mixed with a suspension of the bacteria; after incubation, clot formation indicates S. aureus (b) slide coagulase test: a more rapid and simple method in which a drop of plasma is added to a suspension of staphylococci on a glass slide; visible clumping indicates the presence of coagulase. (c) latex agglutination test: cells are mixed with coated latex particles; visible agglutination provides simultaneous detection of staphylococci containing coagulase and/or protein A. 3. Deoxyribonuclease (DNAase) production: S. aureus possesses an enzyme, DNAase, which depolymerises and hydrolyses DNA; other staphylococci rarely possess this enzyme. 4. Protein A detection: S. aureus possesses a cell-wall antigen, protein A; antibodies to proteinA agglutinate S. aureus but no other staphylococci. 5. Novobiocin sensitivity: useful for differentiating between species of coagulase- negative staphylococci; S. saprophyticus is novobiocin resistant and S. epidermidis is sensitive. Medical Microbiology- Second Class- Medical Lab. Techniques Dept. 10 Morphology and identification 1. On microscopy, S. aureus is seen as typical Gram- positive cocci in ‘grape-like’ clusters. 2. It is both coagulase and DNAase positive. 3. Other biochemical tests can be performed for full identification. 4. Resistant enough to survive drying, heat, and other harsh environmental conditions. S. aureus colonies on a blood agar plate 5. Growth is enhanced in the presence of O2 and CO2. (2–3mm diameter). 6. Its nutrient requirements can be satisfied by routine laboratory media. 7. High salt tolerance (7.5–10%), called Halophilic bacteria. 8. Catalase positive The catalase enzyme convert hydrogen peroxide (H2O2) into water and oxygen. 9. Coagulase positive (Coagulase is an enzyme that cause plasma to clot). 10.Pencillinase degrad penicillin. 11.Blood agar plate growing Staphylococcus aureus. Some strains show two zones of hemolysis. The relatively clearer inner zone is caused by α_-toxin, whereas the outer zone is fuzzy and appears only if the plate has been Medical Microbiology- Second Class- Medical Lab. Techniques Dept. 11 refrigerated. This outer zone is the β_, or “hot-cold,” hemolysin that shows up when the plate is refrigerated. 12.Cultured on blood agar and mannitol salt agar 24hr/37∘C 13.diagnosis by biochemical tests API Staph. 14.Muller-Hinton agar used for sensitivity test. Pathogenicity S. aureus causes disease because of its ability to adhere to cells, spread in tissues and form abscesses, produce extracellular enzymes and exotoxins, combat host defenses and resist treatment with many antibiotics. Pathogenicity factors produced by S. aureus Associated infections  Skin: boils, impetigo, furuncles, wound infections, staphylococcal scalded skin syndrome;  Subcutaneous and Systemic infections: 1. Respiratory: pneumonia, lung abscesses, exacerbations of chronic lung disease; 2. Skeletal: most common cause of osteomyelitis and septic arthritis; Medical Microbiology- Second Class- Medical Lab. Techniques Dept. 12 3. Invasive: bloodstream infection, infective endocarditis, deep abscesses (brain, liver, spleen), toxic shock syndrome; 4. Gastrointestinal: toxin-mediated food poisoning; 5. Device related: indwelling catheters, prosthetic joints and heart valves. Laboratory diagnosis  Microscopic detection of the microorganism in clinical samples (pus from wound or burn, watery diarrhea, etc.)  direct exam stained by Gram stain (Direct isolation from the infected site or blood cultures).  Detection of serum antibodies to staphylococcal haemolysin and DNAase. Treatment and prevention Antimicrobial agents, such as flucloxacillin, remain the first-line treatment for sensitive strains of S.aureus. Cephalosporins, nafcillin, sulfa drugs, vancomycin can be used. S. epidermidis  S. epidermidis is both coagulase and DNAase negative and is present in large numbers on the human skin and mucous membranes.  S. epidermidis is a cause of bacterial endocarditis. It is also a major cause of infections of implanted devices such as cerebrospinal shunts.  The microorganism colonizes implanted devices by attaching firmly onto artificial surfaces.  Some strains also produce a slime layer (glycocalyx), which appears to facilitate adhesion and protect the microorganism from antibiotics and host defenses.  S. epidermidis one of the most frequently isolated microorganisms from blood cultures.  S. epidermidis occasionally causes urinary tract infections, particularly in catheterized patients. S. saprophyticus S. saprophyticus is both coagulase and DNAase negative and is frequently associated with urinary tract infections in sexually active young women, occasionally resulting in severe cystitis with hematuria. How to differentiate between S. epidermidis and S. saprophyticus? Medical Microbiology- Second Class- Medical Lab. Techniques Dept. 13 15. Streptococci 1. Gram-positive cocci (spherical, but they can also appear ovoid or rod like especially in actively dividing young cultures), 2. Non-spore forming, non-motile (except for flagellated strains), 3. facultative anaerobes, 4. characteristically form pairs or chains during growth. 5. They can form capsules and slime layers. 6. Catalase negative (important test to differentiate them from Staphylococci) 7. They have a peroxidase system for inactivating hydrogen peroxide (which allows their survival even in the presence of oxygen). 8. fastidious in nutrition and require enriched media for cultivation. 9. Colonies are usually small, non-pigmented, and glistening. 10. Most members of the genus are quite sensitive to drying, heat, and disinfectants. Classification of Streptococci The classification of streptococci into major categories has been based on (1) colony morphology and hemolytic reactions on blood agar. (2) serologic specificity of the cell walls group-specific substance (Lancefield antigens) and other cell wall or capsular antigens. (3) biochemical reactions and resistance to physical and chemical factors. (4) ecologic features. (5) molecular genetics have replaced phenotypic methods. A. Hemolysis 1. Complete disruption of erythrocytes with clearing of the blood around the bacterial growth is called β-hemolysis (the human-pathogenic species Streptococcus pyogenes, S. agalactiae). 2. Incomplete lysis of erythrocytes with reduction of hemoglobin and the formation of green pigment is called α- hemolysis (Streptococcus pneumoniae). 3. Non-hemolytic- Streptococci (sometimes called γ- Streptococci grown in blood culture showing Gram-positive cocci in chains. hemolysis) (Streptococcus mutans and Streptococcus bovis). B. Group-Specific Substance (Lancefield Classification) This carbohydrate is contained in the cell wall of many streptococci and forms the basis of serologic grouping into Lancefield groups A–H and K–U. Medical Microbiology- Second Class- Medical Lab. Techniques Dept. 14 C. Capsular Polysaccharides The antigenic specificity of the capsular polysaccharides is used to classify some streptococci. D. Biochemical Reactions Biochemical tests include sugar fermentation reactions, tests for the presence of enzymes, and tests for susceptibility or resistance to certain chemical agents. STREPTOCOCCUS PYOGENES - important human pathogen. - typically produces large (1 cm in diameter) zones of β-hemolysis around colonies greater than 0.5 mm in diameter. - hydrolysis of L-pyrrolidonyl-β-naphthylamide. (PYR)+ve - susceptible to bacitracin - Streptococci is Gram-positive. - Most group-A strains produce capsules composed of hyaluronic acid. - The hyaluronic acid capsule likely plays a greater role in virulence. -S. pyogenes cell wall contains proteins (M, T, R antigens), carbohydrates (group specific), and peptidoglycans. - Hair like pili consist partly of M protein and are covered with lipoteichoic acid. (Another type-specific molecule is the M-protein, of which about 80 different subtypes exist. This substance is the main component of fimbriae, the spiky surface projections that contribute to virulence by resisting phagocytosis and improving adherence). Culture Growth of streptococci tends to be poor on solid media or in broth unless enriched with blood or tissue fluids. Nutritive requirements vary widely among different species. Growth and hemolysis are aided by incubation in 10% CO2. Most pathogenic hemolytic streptococci grow best at 37°C. Most streptococci are facultative anaerobes and grow under aerobic and anaerobic conditions. Group A β-hemolytic streptococci (S. pyogenes) after growth overnight on a 10-cm plate with 5% sheep blood agar. The small (0.5–1 mm diameter) white colonies are surrounded by diffuse zones of β-hemolysis 7–10 mm in diameter. (Courtesy of H Reyes. Medical Microbiology- Second Class- Medical Lab. Techniques Dept. 15 Antigenic structure (M Protein) This substance is a major virulence factor of S. pyogenes. M protein is a filamentous structure anchored to the cell membrane that penetrates and projects from the streptococcal cell wall. Enzymes and Toxins A. Streptokinase (Fibrinolysin) Streptokinase is produced by many strains of group-A β-hemolytic streptococci. It transforms the plasminogen of human plasma into plasmin, an active proteolytic enzyme that digests fibrin and other proteins, allowing the bacteria to escape from blood clots. B. Deoxyribonucleases Streptococcal deoxyribonucleases A, B, C, and D degrade DNA (DNases) and similar to streptokinase facilitate the spread of streptococci in tissue by liquefying pus. C. Hyaluronidase Hyaluronidase splits hyaluronic acid, an important component of the ground substance of connective tissue. Thus, hyaluronidase aids in spreading infecting microorganisms (spreading factor). D. Pyrogenic Exotoxins (Erythrogenic Toxin) Pyrogenic exotoxins are elaborated by S. pyogenes. There are three antigenically distinct streptococcal pyrogenic exotoxins (Spe): A, B, and C. The streptococcal pyrogenic exotoxins have been associated with streptococcal toxic shock syndrome and scarlet fever. E. Hemolysins The β-hemolytic group A S. pyogenes elaborates two hemolysins (streptolysins) that not only lyse the membranes of erythrocytes but also damage a variety of other cell types. Streptolysin O -protein. - hemolytically active in the reduced state (available– SH groups) but rapidly inactivated in the presence of oxygen. - responsible for some of the hemolysis seen when growth occurs in cuts made deep into the medium in blood agar plates. - combines quantitatively with anti-streptolysin O (ASO), an antibody that appears in humans after infection with any streptococci that produce streptolysin O. -This antibody blocks hemolysis by streptolysin O. Streptolysin S - agent responsible for the hemolytic zones around streptococcal colonies growing on the surface of blood agar plates. Medical Microbiology- Second Class- Medical Lab. Techniques Dept. 16 - elaborated in the presence of serum—hence the name streptolysin S. -not antigenic. Most isolates of S. pyogenes produce both of these hemolysins. Up to 10% produce only one. Pathogenesis and Clinical Findings From the lymphatics, the infection can extend to the bloodstream. 1. Erysipelas—If the portal of entry is the skin, erysipelas results. Lesions are raised and characteristically red. 2. Cellulitis—Streptococcal cellulitis is an acute, rapidly spreading infection of the skin and subcutaneous tissues. 3. Necrotizing fasciitis (streptococcal gangrene)—There is extensive and very rapidly spreading necrosis of the skin, tissues, and fascia. Medical Microbiology- Second Class- Medical Lab. Techniques Dept. 17 4. Puerperal fever—If the streptococci enter the uterus after delivery, puerperal fever develops, which is essentially a septicemia originating in the infected wound (endometritis). 5. Bacteremia or sepsis—Infection of traumatic or surgical wounds with streptococci results in bacteremia, which can rapidly be fatal. Local Infection 1. Streptococcal sore throat—The most common infection caused by β-hemolytic S. pyogenes is streptococcal sore throat or pharyngitis. S. pyogenes adheres to the pharyngeal epithelium by means of lipoteichoic acid-covered surface pili and by means of hyaluronic acid in encapsulated strains. S. pyogenes infection of the upper respiratory tract does not usually involve the lungs. 2. Streptococcal pyoderma—Local infection of superficial layers of skin, especially in children, is called impetigo. It consists of superficial vesicles that break down and eroded areas whose denuded surface is covered with pus and later is encrusted. Medical Microbiology- Second Class- Medical Lab. Techniques Dept. 18 Streptococcal Toxic Shock Syndrome, and Scarlet Fever Fulminant ‫خاطف‬, invasive S. pyogenes infections with streptococcal toxic shock syndrome are characterized by shock, bacteremia, respiratory failure, and multiorgan failure. Death occurs in about 30% of patients. Pyrogenic exotoxins A–C also cause scarlet fever in association with S. pyogenes pharyngitis or with skin or soft tissue infection. Post-streptococcal Diseases (Rheumatic Fever, Glomerulonephritis) 1. Acute glomerulonephritis—This sometimes develops 1–5 weeks (mean 7days) after S. pyogenes skin infection (pyoderma, impetigo) or pharyngitis. 2. Rheumatic fever—This is the most serious sequela of S. pyogenes because it results in damage to heart muscle and valves. Certain strains of group A streptococci contain cell membrane antigens that cross-react with human heart tissue antigens. Laboratory Diagnosis—Practical lecture. Streptococcus pneumoniae -is a member of the S. mitis group. -Gram-positive diplococci, often lancet shaped or arranged in chains. -possessing a capsule of polysaccharide that permits typing with specific antisera. - In sputum or pus, single cocci or chains are also seen. - With age, the organisms rapidly become Gram-negative and tend to lyse spontaneously. -Lysis of pneumococci occurs in a few minutes when ox bile (10%) or sodium deoxycholate (2%) is added to a broth culture or suspension of organisms at neutral pH. (Viridans streptococci do not lyse and are thus easily differentiated from pneumococci). -On solid media, the growth of pneumococci is inhibited around a disk of optochin; viridans streptococci are not inhibited by optochin. Medical Microbiology- Second Class- Medical Lab. Techniques Dept. 19 - capsule swelling test, or quellung reaction is very important in the diagnosis of this bacterium. S. pneumoniae in sputum are seen as lancetshaped Gram-positive diplococci. Degenerating nuclei of polymorphonuclear cells are the large darker irregular red shapes (arrow). Mucus and amorphous debris are present in the background. Original magnification ×1000. Culture Pneumococci form small round colonies, at first domeshaped and later developing a central depression with an elevated rim. Other colonies may appear glistening because of capsular polysaccharide production. Pneumococci are α-hemolytic on blood agar. Growth is enhanced by 5–10% CO2. Quellung Reaction When pneumococci of a certain type are mixed with specific antipolysaccharide serum of the same type—or with polyvalent antiserum—on a microscope slide, the capsule swells markedly, and the organisms agglutinate by crosslinking of the antibodies. Medical Microbiology- Second Class- Medical Lab. Techniques Dept. 20 Disease production Pneumococci produce disease through their ability to multiply in the tissues. The virulence of the organism is a function of its capsule, which prevents or delays ingestion by phagocytes. ENTEROCOCCI Enterococci are Gram-positive, catalase-negative, facultative anaerobic bacteria that are usually oval-shaped and are arranged in pairs or short chains; occasionally, single organisms can be seen as well. The enterococci possess the group D group-specific substance and were therefore previously classified as group D streptococci. Enterococcus faecalis and Enterococcus faecium being the two species most commonly isolated from clinical specimens. Enterococci are part of the normal enteric microbiota. Morphology and Identification Enterococcus species grow readily on nonselective media, such as sheep-blood agar and chocolate agar. Enterococci are usually non-hemolytic, but are occasionally α- hemolytic, or rarely β-hemolytic. Enterococcus species are resistant to optochin and colonies do not dissolve when exposed to bile, whereas S. pneumoniae is optochin- susceptible and bile soluble. Furthermore, enterococci are PYR positive, grow in the presence of bile, hydrolyze esculin (bile-esculin positive), and in contrast to non- enterococcal group D streptococci, enterococci grow well in 6.5% NaCl. Enterococci grow well at a wide temperature range between 10°C and 45°C. Pathogenesis and Pathology Most enterococcal infections appear to arise from the endogenous flora via translocation from their major colonization site (GI tract). In general, virulence is mediated by two major properties, including (intrinsic) antimicrobial resistance of enterococci as well as their ability to adhere to cells and tissues and form biofilms. Several potential virulence factors have been identified and may play a role in the pathogenesis of enterococcal infections. These virulence factors include surface Medical Microbiology- Second Class- Medical Lab. Techniques Dept. 21 adhesion proteins, membrane glycolipids, secreted toxins (eg, cytolysin and hemolysin), secreted proteases (eg, gelatinase), and extracellular superoxide. Clinical Findings In hospitalized patients, the most common sites of infection are the urinary tract, burn and surgical wounds, biliary tract, and blood. Urinary tract infections (UTIs) are by far the most common form of enterococcal infections, and are frequently associated with indwelling catheters, instrumentation, or structural abnormalities of the genitourinary tract. Intra-abdominal and pelvic infections. Bacteremia and endocarditis are also common forms of enterococcal infections, and are frequently associated with metastatic abscesses and high mortality rates. Infections of the respiratory tract (eg, pneumonia, otitis, and sinusitis) and/or the central nervous system (eg, meningitis) have been described, but occur rarely. Two forms of enterococcal meningitis have been described: spontaneous and postoperative meningitis. Spontaneous meningitis is typically a community- associated infection in patients with severe comorbidities (eg, diabetes, chronic renal failure, and immunosuppression), whereas postoperative meningitis is a hospital- acquired infection (eg, associated with ventricular shunt devices, CNS electrodes). Neonatal infections due to enterococci have also been described. Enterococci are part of the normal adult vaginal microbiota, and can therefore be acquired by neonates during vaginal delivery. Neonatal enterococcal infections are typically late-onset sepsis, pneumonia, but other infections such as UTIs, surgical site infections, and meningitis have also been described.