Determinative Bacteriology Flashcard Answers
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| Classification of the Streptococci, Enterococci and Streptococci like bacteria |
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Based on molecular methods, DNA-DNA hybridizations, and 16s rRNA sequencing.
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| Current classification |
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•Order “Lactobacillales” •Family “Staphylococcaceae” –Genus Gemella •Family “Lactobacillaceae” –Genus Pediococcus •Family “Carnobacteriaceae” –Genus Alloiococcus, Dolosigranulum •Family “Enterococcaceae” –Genus Enterococcus, Vagococcus, Melissococcus •Family “Leuconostocaceae” –Genus Leuconostoc, Oenococcus, Weissella •Family “Streptococcaceae” –Genus Streptococcus, Lactococcus
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| General Characteristics of the members of this Order |
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•Gram positive cocci, can be chains, tetrads or pairs. •Cell wall composition similar to other Gram positive bacteria. •Heterogenous group of bacteria, disease producers (pathogens), and industrial strains.
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| Metabolism in this Order |
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•The genera of this Order, described as to their ability to utilize glucose. –Homolactic fermentation: Glucose converted into lactic acid, no gas, with 2 ATP’s produced (net). The enzyme fructose 1,6 diphosphate aldolase is present –Heterolactic fermentation: Glucose converted into organic acids, CO2, with 1 ATP produced (net). The enzyme fructose 1,6 diphosphate is absent.
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| Genera in this Order |
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•Leuconostoc •Pediococcus •Lactococcus •Aerococcus •Gemella •Streptococcus •Enterococcus
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| Genus Leuconostoc |
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•Heterofermentative. •Pairs and chains of cocci, found on plants and in the soil. •Facultative, non motile, catalase –, gas from glucose. •vancomycinR •Grow on MRS agar. •Some reports of pathogenicity –Strain examples: Industrial cultures, used in the dairy, pickling and wine business. •L. mesenteroides •L. mesenteroides subsp. dextranicum
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| Genus Oenococcus |
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•Industrial strains used in the wine industry. •Acidophiles •Convert malic acid in wine into lactic acid and CO2. Malic acid is one of 2 predominant organic acids in wines. Thus the fermentation of it reduces acid content and flavor.
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| Genus Pediococcus |
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•Homofermentative, found on plants •Facultative, catalase-, non motile, vancomycinR. •Grow on APT agar. •Some reports of pathogenicity in patients undergoing vancomycin therapy. •Pairs/tetrads of gram positive cocci –Examples of strains: Industrial starter cultures for the meat, pickle and silage business •P. acidilactici , pediocin production •P. pentosaceus
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| Pediocin |
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•Peptide antibiotic •Post translational modification, 4629 daltons in size. Amino acids, 1-62, 19-62 is the active peptide. •Inhibits Listeria monocytogenes in foods, creates channels in their membranes. Useful in meats and soft cheeses
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| Genus Lactococcus |
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•Homofermentative, catalase -, “milk streptococci”, Lancefield group N. •Historically the “lactic acid streptococci”. •Gram positive cocci in chains. •Rare clinical isolates, considered opportunistic of low virulence. •Industrial starter cultures in the dairy business. –L. lactis, nisin production –L. cremoris –L. diacetylactis
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| Basic features of Lactococci |
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•Lactic acid bacteria (LAB), bacteria that produce lactic acid via fermentation metabolism. However (LAB) misleading, since it refers to bacteria used in the food industry. However some opportunistic bacteria in this group ferment lactic acid.
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| Characteristics of L. lactis |
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•Gram positive cocci, 38% G+C, non pathogenic food microbe. •Optimal temperature 30°C, found on plants, milk and silage. •Metabolism is fermentative, however can undergo respiration if heme is supplied in the medium. •Survival poor if grown by fermentation, good if grown by respiration •Lactococci, produce acid from glucose, fructose, mannose and N-acetylglucosamine
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| Applications of Lactococci |
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•They are used in “bioprotein” delivery, antigen or enzyme delivery in vivo. They can be taken orally and do not provoke an immune response in the host, experimental vaccine delivery system.
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| Nisin |
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•Approved as a food additive in 1969 •Lantibiotic, synthesized as a prepeptide and post translationly modified, converted into a mature peptide, the leader sequence is removed. •3500 daltons contains unusual amino acids. •Mode of action, attacks membranes of bacteria, forms channels in them. •Prevents clostridial gas formation in hard cheeses. Synergistic action with EDTA.
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| Genus Aerococcus |
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•Homofermentative, microaerophilic. •Gram positive cocci, pairs and tetrads •Isolated from the environment, water. •Cause a fatal disease in lobsters, called “gaffkemia”, opportunists in man. •In broth media, form tetrads. •A. viridans, causes gaffkemia. •A. urinae, causes UTI’s, septicemia, endocarditis in humans susceptible to penicillin, rifampin and vancomycin.
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| Genus Gemella |
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•Homofermentative •Gram positive cocci, single / pairs •6 species: G. haemolysans, appears as diplococci with flattened sides. Usually part of the upper respiratory tract. G. morbillorum, found in the human respiratory tract and GI tract. •Catalase - , infrequent clinical isolation. Susceptible to a variety of antimicrobial agents.
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| Genus Streptococcus |
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•Homofermentative, facultative anaerobes. Growth usually stimulated by CO2. •Gram positive cocci, pairs / chains, catalase -, oxidase -. •General identification methods. •Streptococci are also classified via –Hemolysis (alpha, beta and gamma) –Carbohydrate cell wall surface antigens
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Genus Streptococcus: Hemolysis based division |
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–Beta hemolysis (clear), the result of two distinct hemolysins. •Streptolysin O, antigenic and oxygen sensitive. ASO test. Binds to cholesterol in RBC’s •Streptolysin S, non antigenic and oxygen stable. Binds to phospholipids in RBC’s -Alpha hemolysis, incomplete lysis, green color on RBC plate. Loss of hemoglobin into the agar results in the green discoloration. -Gamma hemolysis, misnomer since there is no hemolysis.
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| Genus Streptococcus: Carbohydrate surface antigen grouping |
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–Lancefield method (1930’s), acid, hot water extraction. Limited to large clinical labs. Today rapid kits, carrier coupled with specific antistreptococcal antibodies to the various groups. Human groups (A, B, C, F and G) are based on cell wall polysacchrides. Group D and enterococci are based on cell wall lipoteichoic acids. Today there are 20 groups A to V omitting I and J. These group specific antigens are covalently linked to the peptidoglycan and are composed of a branched polymer of L-rhamnose or glycerol teichoic acid linked to hexosamine.
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Streptococcus pyogenes (Group A, GAS) virulence factors:
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–M protein –Capsule –Streptolysin O –DNase B,A,C & D –Hyaluronidase –Proteinases –NADase –Streptokinase –Erythrogenic toxin –Lipoteichoic acid (LTA)
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| M protein |
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•> 100 types •Acid and heat stable, trypsin labile. •They are composed of 2 polypeptide chains complexed together in a α helical coiled coil configuration. •Anchored in cell membrane, projects from the cell surface, “fuzz like”, MW 40,000 to 80,000 daltons. •Appear as fibrils on the surface of the bacterial cell. C terminal is in the cell membrane, N terminal extends beyond the cell surface. •Strains rich in M protein are resistant to phagocytosis and intracellular destruction by PMN’s. The M protein interfers with opsonization of the bacteria by the alternative Complement pathway. •Considered a major virulence factor in Group A streptococci. •A mediator of attachment to pharyngeal and skin surfaces. Cross reactivity of anti M protein to human cardiac tissue.
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| Lipoteichoic acid (LTA) |
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•Surface exposed molecule covalently linked to the cytoplasmic membrane glycolipid that extends through the cell wall and capsule. LTA along with M protein mediates oral and skin epithelial attachment and subsequent colonization.
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| Capsule |
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•Some group A strains possess a capsule composed of hyaluronic acid, which is made up of β(1→4) linked units of glucuronic acid and N-acetylglucosamine. •Similar to the ground substance of connective tissue, therefore makes the strain non immunogenic. •It exerts antiphagocytic activity. •Inhibits complement activation, prevents deposition of C3b on the bacterial surface, thereby conferring resistance to phagocytosis. •The capsule seems critical to mucosal colonization in the pharyngeal area
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| Streptolysin O (SLO) |
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•A hemolysin produced by Group A, oxygen labile. •Primarily responsible for Beta hemolysis seen on blood plates. •MW 50,000 to 75,000 daltons. Molecule lysis RBC’s and slits holes in PMN’s. •Destroys adjacent cells and tissue, contributes to the spread of the bacterium from the local site. •SLO binds to cholesterol in eukaryotic membranes, where it form multiple transmembrane pores. •Antibody titer to streptolysin O is an indicator of recent Group A infection. Used to test for Rheumatic fever.
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| Streptolysisn S (SLS) |
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•Another hemolytic exotoxin made by GAS. •This is the hemolysin produce on Blood agar in the lab. This hemolysin is nonimmunogenic, and is oxygen stable.
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| DNase, B,A,C and D |
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•DNA degradation, titer against DNase B, helpful for determination of prior Group A infection. •Molecules have a MW of 25,000 to 30,000 daltons. •Enzymes degrade viscous DNA in thick pus from PMN’s.
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| Hyaluronidase |
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•Produced by Group A, depolymerizes the ground substance of connective tissue, facilitates the spread of the organism through tissues. •How the enzyme degrades capsular hyaluronic acid of the organism and without increasing susceptibility to increased phagocytosis remains an enigma.
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| Streptokinase |
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•Produced by Group A, hydrolyze fibrin clots, prevent the formation of fibrin barriers. Dissolves clots. •Thus eliminates a effective local barrier, helps spread the organism from local sites. •Recent studies have shown that this protein is important for the establishment and spread of GAS in skin infections.
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Pyrogenic exotoxins
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•SPE A, B, C and D. , called “erythrogenic toxin”. •Cause the rash of scarlet fever. •The spe B gene is encoded chromosomally in all Group A strains, others are encoded on a bacteriophage
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| Binding of streptococcal adhesions to host cell receptors |
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•This results in intracellular signaling events in target cells. The M protein binds specific receptors on human keratinocytes, (skin cells). A streptococcal surface enzyme (enolase) functions as a plasminogen receptor.
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| Diseases associated with Streptococcus pyogenes |
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•Supprative (pus forming) –Strep throat –Scarlet fever –Impetigo •Nonsuppurative (non pus forming) –Bright’s disease –Rheumatic fever –Necrotizing fascitis
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| Septic sore throat and Scarlet fever |
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•Both diseases occur in all age groups, most often in young children. •Initiated by droplet nuclei, or in a susceptible host by factors that upset the host-parasite equilibrium. •The bacteria attach to the pharyngeal mucosa and colonize, The M protein and capsule, main factors in preventing phagocytosis. Streptolysins S, and O destroy PMN’s. •Strep throat, acute pharyngitis, yellow gray exudate, and fever. •Scarlet fever, if the organism produces a pyrogenic exotoxin (erythrogenic toxin), diffuse reddening of the skin and a rash prominent on the trunk, neck and extremities.
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| Impetigo |
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•Disease of the skin in newborns, same mechanisms seen in strep throat and the IgG Fc receptors play a role in inhibiting phagocytosis. •The disease initiates on the face but may involve the eyes, nose, lips and limbs. These Group A strains are often nephritogenic, causing acute hemorrhagic glomerulonephritis.
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| Erysipelas |
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•All age groups. •A disease of the skin and subcutaneous tissue, occurs on the face or lower extremities, characterized by a fiery red advancing erythema.
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| Cellulitis, Lymphangitis |
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•All age groups. •Skin infection organisms travel along the lymphatic channels and the result is a purulent inflammation.
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| Pathogenesis and Clinical Manifestations of Group A infections |
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•Primary diseases of Mucous membrane surfaces, skin and subcutaneous tissues. (Septic sore throat and Scarlet fever), (Impetigo, Erysipelas, Cellulitis, Lymphangitis). •Primary diseases of the female genital tract, (Puerperal sepsis). •Dissemination by direct extension, from pharynx to produce abscesses in the sinuses, middle ear, meninges and to the lungs. This could result in septicemia, acute endocarditis and joint involvement. With puerperal sepsis a severe systemic shocklike disease is observed. The case fatality rate is always high when dissemination occurs. Acute invasive form, high mortality rate. •Late sequelae, these are the nonsuppurative diseases acute rheumatic fever and acute glomerulonephritis (Bright’s disease). These occur at some period of time after onset of acute group A disease.
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| Necrotizing fasciitis |
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•Caused by Group A streptococci. “Flesh eating bacteria”, impacts 500-1,500 people/yr in the U.S. Fever, severe pain, swelling and destruction of muscle tissue.
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| Puerperal Sepsis |
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•A disease of the uterine endometrium, usually initiated in a susceptible host during or after delivery of a newborn from droplet nuclei from another source or endogenous activation do to imbalance of the host parasite relationships.
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| Rheumatic fever |
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•Occurs most commonly in young children during fall and winter, and can only occur when preceded by pharyngitis caused by any Group A streptococcal serotypes. The disease typically occurs 1 to 5 weeks from 0.1-3% of untreated pharyngitis patients. The mechanism is believed to be based on cross-reactive epitopes among M proteins and target tissues, including human cardiac tissue, and the binding of anti-M protein to the cardiac tissue. •2 theories as to the clinical picture. •1. Lysis of bacteria releases cell wall components. Humoral response to bacterial antigens. Antibodies cross react to heart tissue. Complement activation causes damage to heart. •2. Bacterial toxins, Streptolysin O, enter circulation. Direct damage to heart valve.
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| Acute glomerulonephritis |
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•Occurs most commonly in children, post pharyngitis or impetigo. It involves any of the 12 serotypes of Group A strep that are nephritogenic strains. In untreated patients (1-15%), 1-5 weeks post onset of initial disease. Diseasse is due to immune complex deposits on the glomerular basement membrane. Major clinical manifestations are renal damage, hypertension, edema, proteinuria and hemauria
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| Changing disease patterns of Streptococcus pyogenes |
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•Time Prevalent disease •18-19thC Puerperal fever, fasciitis in war wounds, blood infections •19-early 20 Scarlet, rheumatic fevers, wound infections •1940’s Rheumatic, scarlet fevers disappear. •Today Some scarlet fever , Invasive systemic, toxic shock like syndrome, fasciitis, nosocomial wound, pneumonia, endocartditis
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| Identification of Streptococcus pyogenes |
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•Gram stain •Hemolysis •Catalase reaction •Bacitracin •SXT •Precipitin test group antigen •ASO titer •Anti DNase titer •CAMP reaction •Growth in 6.5 % NaCl •Bile esculin
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