Determinative Bacteriology Test Answers – Flashcards
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| Group B Streptococci |
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•GBS, commonly colonize the lower gastrointestinal tract and vaginal epithelium of healthy adults, but they remain a potentially devastating pathogen to susceptible infants. •Because the newborn is deficient in host defenses, ie, phagocytes, complement and specific antibody; therefore an environment exists for potential problems •The first strains of GBS were studied by Lancefield, these strains were isolated from cows with mastitis, and was a concern mainly in the dairy industry. •First report of GBS in humans appeared in 1938. In the 1960’s reports of hemolytic GBS among newborns. In the 1970’s GBS eclipsed E. coli as the leading cause of newborn sepsis. Today colonization of GBS in mother a risk factor for neonates.
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| Group B Streptococcus: |
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•Gram positive cocci, Example species: Streptococcus agalactiae •Beta hemolytic •BacitracinR •SXTR •Hippurate hydrolysis + •CAMP + •Growth in 6.5 % NaCl, variable •Bile esculin – •PYR -
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| Bacitracin Susceptibility |
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•Disk test performed on Blood agar medium. •Look for a zone or no zone around the disk. •Group B streptococci, are resistant, no zone around the disk.
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| Sulfamethoxazole-Trimethoprim SXT test |
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•This test distinguishes groups A and B streptococci from other streptococci. •Both Groups A and B are resistant to SXT. •Disk test done on agar surface.
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| Hippurate hydrolysis |
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•Group B streptococci are able to hydrolyze hippurate to glycine and benzoic acid. •Broth test contains hippurate, incubated with cells, centrifuge cells, collect supernatant, add ferric chloride, ppt means benzoate present. Or add ninhydrin to check for glycine, deep blue color.
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| Identification of Group B Streptococcus |
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•Camp Test –Use a beta hemolytic S. aureus 25923. –Group B Streptococci, produce a protein called “CAMP Factor”, that interacts with the beta hemolysin produced and secreted by S. aureus to cause enhanced hemolysis. This appears as an arrowhead. –Presumptive for Group B streptococci.
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| Bile Esculin Test |
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•Accomplished in Bile Esculin medium. •Tests the ability of certain streptococci to grow in the presence of 1-4% Bile salts and hydrolyze esculin to esculetin. Which in the presence of Fe++ forms a black complex. •Group B streptococci are negative.
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Growth in 6.5 % NaCl (GBS) |
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•Group B streptococci are variable.
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| Identification tests for Group B streptococci |
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•All of the previous including growth in Lim broth •Optochin(ethyl hydrocupreine hydrochloride) susceptibility, a disk test done on blood agar. However must measure the zones of inhibition. A zone of 14 mm or greater around the 6 mm disk indicates susceptibilty. Group B streptococci are resistant.
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| Lim Broth |
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•Todd Hewitt Broth supplemented with 10μg/ml nalidixic acid, 15μg/ml colistin, 10mg/ml yeast extract. •Todd Hewitt broth: use 30g/liter of –Beef heart, 500g –Neopeptone, 20g –Dextrose, 2g –Sodium chloride, 2g –Disodium phosphate, 0.4g –Sodium carbonate, 2.5g
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| Pyrrolidonyl arylamidase(pyr) test |
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•Broth containing pyrrolidonyl-naphthylamide is incubated with the bacterium for 4 hrs. If pyr is hydrolyzed, free naphthylamide is detected with a diazo dye, red color. •Group B is negative.
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| Virulence Factors Group B, β-hemolytic streptococci |
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Capsule sialic acid C5a peptidase β hemolysin Lipoteichoic acid C antigen Cell surface penicillin binding protein Hyaluronic acid lyase
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| GBS Capsule |
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•Capsule: (CPS) 9 types, the polysaccharide capsular antigens are designated Ia, Ib, II, III, IV, V, VI, VII, VIII. These capsules are composed of glucose, galactose, N-acetylglucosamine, sialic acid. The types are comprised of differing arrangements of these carbohydrates. Most predominant infections are the result of types: Ia, Ib, II, III and V.
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| GBS Sialic acid |
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• The presence of sialic acid on GBS creates a surface that does not activate the alternative complement pathway, therefore a virulence factor on GBS
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| GBS C5a peptidase |
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•C5a peptidase: C5a is a complement component, that is produced by alveolar epithelial cells, and acts as an attractant for inflammatory cells. Therefore the C5a peptidase cleaves the C5a at the C terminus and interfers with C5a mediated neutrophil chemotaxis.
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GBS β hemolysin
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•β hemolysin seen as a virulence factor in pulmonary infections. This creates pores in pulmonary alveolar epithelial cells, suggesting a role in neonatal infections.
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| GBS Lipoteichoic acid |
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•Lipoteichoic acid: facilitates adherence as a first step in infection.
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| GBS C antigen |
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•C antigen: cell surface protein that mediates the internalization of these organisms in cervical endothelial cells.
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GBS Cell surface penicillin binding protein |
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•Cell surface penicillin binding protein, enables streptococcal cells to resist intracellular killing by phagocytic cells.
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| GBS Hyaluronic acid lyase |
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•Hyaluronic acid lyase: may help to spread infection by breakdown of this component in placental tissues, fetal tissue and amniotic fluids.
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| Clinical significance of Group B β hemolytic streptococci |
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•Major cause of disease in neonatal and perinatal (period shortly before and after birth) periods. Females, have the organism in their rectum and vagina, 10-35%. Up to 60% of females will carry the organism intermittently. Colonization of the vagina may reflect contamination from the rectum. •The presence of Group B strep in the vagina of the mother at the time of birth can lead to infection of the neonate.
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| Pathogenesis of Group B streptococci |
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Meningitis: Group B streptococci are the leading cause of neonatal meningitis in the US during the first 4 months of life. Neonatal sepsis, two forms: Maternal carriage is a risk factor. Maternal antibodies to CPS of GBS is low in these females. –Early onset, occurs in utero or within 5 days of birth. Mortality rate 50%, 2.9 cases/1,000 births –Late onset, occurs 10 days to 4 months. Mortality rate of 20%. Possibly spread via breast feeding from GBS infected mastitis in mothers.
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| Group B streptococci General Info |
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•Normal habitat: pharynx, vagina and stool. •Example species: Streptococcus agalactiae, is a beta hemolytic streptococcus, first recognized in 1938, as a cause of “childbed fever”. •In the 1970’s leading cause of neonatal meningitis as well as postpartum endometritis. •1990’s 3-4 cases per 100,000 cases of invasive S.agalactiae, in non pregnant adults.
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| GBS disease in nonpregnant adults |
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•Most common infections caused by GBS in adults are skin, soft tissue and bone infections. Usually these infections are complications of chronic diabetes. Patients with indwelling catheters are at high risk for GBS bacteremia. Pneumonia is a severe form of GBS disease with high mortality
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| Treatment for Group B streptococcal disease |
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•Penicillin G plus an aminoglycoside. •Heavily colonized mother’s are treated intrapartum to prevent colonization of their newborns.
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| GBS vaccines |
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•Early clinical trials gave mixed results, vaccines based on specific capsules from strains need to be made more immunogenic. Conjugate vaccines, (polysaccharide-protein) seem to offer the best protection.
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| Group C streptococci |
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•Gram positive cocci •Lancefield grouping •Beta or alpha hemolysis •Normal habitat: Pharynx, vagina and skin •Pathogenesis •Example species: S. equi and S. dysgalactiae. •S. dysgalactiae subsp. equisimilis most human isolate, recovered from carriers and those within exudative pharynigitis and tonsillitis. •S. equi causes a respiratory infection in horses called “strangles”. High fever, mucopurulent nasal discharge and lymph node abscesses.
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| Identification of Group C streptococci |
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•Beta hemolysis •Bacitracin variable •SXTS •CAMP negative •Hippurate negative •PYR negative •Bile esculin negative •Growth in 6.5 % NaCl negative •OptochinR
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| Group D streptococci |
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•All posses a common group D cell wall antigen •They grow in the presence of Bile esculin •Two groups: –Enterococci –Non-enterococci The term “enterococcus”, dates to the late 1800’s. The enterococci were given genus status in 1984.
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| Enterococci |
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•Various hemolytic reactions •SXTR •Growth in 6.5% NaCl and 40% bile salts •Growth at 10°C and 45°C •Hippurate variable •Hydrolysis of pyrrolidonyl arylamidase •Growth on KF agar. •Normal habitat: large bowel •Pathogensis: UTI,endocarditis •Ex species: E. faecalis, E. facium and E.durans
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| Enterococci general info |
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•These organisms are normal residents of the GI and biliary tracts and lower numbers of the vagina and urethra. Found in human feces 105 to 107 /g. •Most common cause of nosocomial UTI and wound infections, rarely cause pneumonia and meningitis. Involved with 15-20% endocarditis. •Third most common cause of nosocomial bacteremias. •Resistant to penicillin, cephalosporins and acquisition of high level vancomycin resistance, makes them dangerous in serious superinfections in patients receiving antibiotics. •VDE, vancomycin-dependent enterococci, first described in 1994, enterococci that only grow in the presence of vancomycin. A problem with long term usage.
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| Infections caused by enterococci |
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•E. faecalis and E. faecium, account for 95% of all enterococci infections. They are now the leading cause of surgical site infections. Diseases caused by these strains is compounded by their intrinsic and acquired antibiotic resistance.
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| Virulence factors, enterococci |
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•Some strains of E. faecalis produce: –Cytolysin/hemolysin, acts on RBC’s. –Aggregation substance, plasmid encoded protein that promotes clumping. –Extracellular surface protein, helps the microbe evade antibodies. –Lipoteichoic acid, constitute the group D Ag, induces interferon production. –Coccolysin, an extracellular metalloendopeptidase which inactivates a vasoactive peptide.
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| Unusual molecules produced by enterococci |
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•Phermones: Plasmid encoded, small peptide that promotes conjugation between enterococci. •Bacteriocin AS-48: a wide spectrum antimicrobial peptide produced by these bacteria.
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| Non-enterococci |
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•Group D streptococci •Failure to grow in 6.5% NaCl •SXTS •PYR negative •Various hemolysis reactions •Example species: S.bovis, S. equinus •S. bovis, 2 biotypes observed, “1” is mannitol +, “2” is mannitol -. Group 2 were human isolates, Group 1 were cattle isolates.
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| Group F streptococci |
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•Anginosus group •Gram positive cocci •Normal habitat: oral cavity, throat •Demonstrate all 3 types of hemolysis. •Pathogenesis: sinus infections, deep tissue abscesses, endocarditis. •Ex species: S. anginosus •Same identification profile as the Group C
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| Group G streptococci |
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•Gram positive cocci •Normal habitat: mouth, throat, G.I. tract, and skin flora. •Many types of infections •Beta hemolytic
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| Group K streptococci |
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•Ex species: S.salivarius
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| Lancefield Group “NONE” streptococci |
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•Gram positive cocci •Alpha hemolytic •Negative for hippurate, growth in 6.5% NaCl, and CAMP •Ex species: S. pneumoniae •Leading cause of community acquired pneumonia •Penicillin sensitive •ID in lab: Quellung Reaction, Bile Salts and Optochin susceptibility
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| Virulence factors Streptococcus pneumoniae |
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•Polysaccharide capsule (remember S and R studies by Griffith in mice, proving DNA) •Teichoic acids and lipoteichoic acids •Pneumolysin •Autolysin •Neuraminidase enzymes •Hyaluronidase •IgA proteases •PspA (pneumococcal surface protein)
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| S. pnuemoniae Polysaccharide capsules |
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•Polysaccharide capsules: 90 types, 23 account for bacteremia / meningitis. Long polymers of repeating units of 2-7 monosaccharides different impacts on the host.
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| S. pneumoniae Teichoic acids |
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•Teichoic acids: cell wall associated attached to the peptidoglycan.
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| S. pneumoniae Lipoteichoic acids |
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•Lipoteichoic acids: linked to the membrane, repeating units of glucose, NAG, ribitol 5-P.
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| S. pneumoniae Pneumolysin |
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| Pneumolysin: 53,000 daltons, cytotoxic protein, interacts with cholesterol in cell membranes, it also inhibits bactericidal activity of phagocytic cells. |
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| S. pneumoniae Autolysin |
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•Autolysin: separates daughter cells after cell division, also breaks down bacteria to help spread pneumolysin and α hemolysin.
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| S. pneumoniae Neuraminidase |
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•Neuraminidase: cleave terminal sialic acid from cell surfaces to expose NAG-galactose moieties that mediate bacterial cell attachment.
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| S. pneumoniae Hyaluronidase |
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•Hyaluronidase: facilitates spread of the organism.
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| S. pneumonia IgA1 proteases |
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•IgA1 proteases: inactivates sIgA to facilitate colonization.
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S. pneumoniae PspA
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•PspA: surface protein, function unknown.
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| Pneumococcal vaccines |
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•2 vaccines produced: pure polysaccharide vaccines and polysaccharide-protein conjugate vaccines. •Comprised of 23 capsular serotypes. •Given to: people 65 yrs or older, persons 2-64 yrs who have chronic illness, those with sickle cell, people living in high risk environments “nursing homes”, immuno compromised individuals.
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| Clinical significance |
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•S. pneumoniae, major cause of community acquired bacterial pneumonia. •The microbe may be harbored in the upper respiratory tract of 5-10% of adults, with higher carriage rates in closed populations. •Most serious infections occur in adults > 65 yrs of age, and infants < 3 yrs of age. Mortality rate approaches 70% among treated patients older than 70. •In the host the microbe gains access to the alveolar spaces by aspiration and may result in lobar pneumonia. •Approximately 500,000 cases/yr in the US.
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| Viridans streptococci |
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•Heterogeneous group of streptococci, “oral streptococci”, grouped on the basis of 16s rRNA, since they do not contain a “C” carbohydrate. •Alpha or non hemolytic on sheep’s blood. •Members of this group are harbored in the nose and throat of >95% of humans, and with the exception of S. mutans, do not cause a disease in the “normal” host. •Clinically causes 30-40% of all subacute bacterial endocarditis, usually have preexisting valve problems. S.oralis, S.mitis, S. mutans, S. salivaris. Usually from dental manipulation in patients with these problems. •S. mutans, have glucosyltranferases, that hydrolyze sucrose in the diet, connect the glucose moieties and form dental plaque. They produce a large amount of acid, role in dental carries.
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| Identification of the Viridans streptococci |
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•Hemolysis alpha or none •Bacitracin variable •SXTS •CAMP negative •Hippurate hydrolysis variable •PYR negative •Bile esculin variable •Growth in 6.5% NaCl negative •OptochinR
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| Anaerobic streptococci |
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•Genus Peptostreptococcus, most significant pathogen in the group. Normal flora of the mouth, intestinal tract and vagina.
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