Bacterial Identification

1. Bacterial Identification

3. streptococci • Gram-positive cocci • Facultatively anaerobic • Occur in pairs and chains of varying length. • Catalase negative • They are usually classified based on their hemolytic properties on blood agar and according to their serologic groups. • Hemolysis refers to is the lysis of the red blood cells in the agar surrounding bacterial colonies and is a result of bacterial enzymes called hemolysins.

4. Types of hemolysis • Beta hemolysis refers to colorless zone surrounding the colony, where a complete lysis of the red blood cells. • Alpha hemolysis appears as a zone of partial hemolysis surrounding the colony, often accompanied by a greenish discoloration of the agar. • Gamma reaction refers to no hemolysis or discoloration of the agar surrounding the colony.

7. Lancefield system • Many of the streptococci can also be classified under the Lancefield system. • In this case, they are divided into a number of distinct serologic groups on the basis of carbohydrate antigens in their cell wall. • These antigenic groups are designated by the letters A through T. • Lancefield serologic groups A, B, C, D, F, and G are the ones that normally infect humans. • Not all pathogenic streptococci can be identified by Lancefield typing (e.g., Streptococcus pneumoniae).

8. Beta hemolytic streptococci • Group AStreptococcus pyogenes: have somevirulence factors like: • Lipoteichoic acid (which allows adherence to target host cells). • M-protein (which resists phagocytic engulfment). • Tissue damaging toxins such as leukocidin (kills leukocytes) and erythrogenic toxin (damages endothelium). • Tissue damaging enzymes such as proteinase, hyaluronidase, and Dnase (thought to be especially important in infections of the skin and soft tissues as well as the rapid spread of the organism through the lymphatics).

9. Isolation and identification • Usually isolated on Blood agar. • There are two streptococcal hemolysins, • streptolysin S and streptolysin O. • Streptolysin O can be inactivated by oxygen so more distinct hemolysis can be seen by stabbing the agar several times. • Only Streptococcus pyogenes among the group A beta streptococci is sensitive to bacitracin, as shown by a zone of inhibition

11. B B Bacitracin Sensitive Bacitracin Resistant S.Pyogenes Non group A β-hemolytic Streptococci

12. Group B Streptococcus agalactiae • Usually show a small zone of beta hemolysis on Blood agar, although some strains are non-hemolytic. • They are often found in the genital and intestinal tracts of healthy adults with from 5% to 40%, depending on the population. • These organisms cause neonatal meningitis and septicemia after transmission from the normal vaginal flora of the mother.

13. Isolation and identification • The organism can be identified on the basis of beta hemolysis. • Hydrolysis of hippurate and CAMP reaction • CAMP is an abbreviation for the names of the four individuals who originally described the test. • Group B streptococci produce a factor that increases beta hemolysis of anS. aureusindicator strain.

14. CAMP Test Streptococcus agalactiae secretes a protein called the "CAMP” factor and when it comes into contact with a beta hemolysin produced by a specific strain of Staphylococcus aureus causes a synergistic, enhanced hemolysis. The test is performed by streaking the S. aureus strain down an agar plate containing sheep blood. Then placing the unknown beta hemolytic strep perpendicular to the staph streak but not touching. After overnight incubation if the strep is a group B strep, the hemolysins from the staph and strep will synergistically produce a large "arrowhead" of hemolysis.

16. Hippurate Test • The organism hydrolyzes hippurate to its basic components, benzoic acid and the amino acid glycine. • Detection of the glycine is accomplished by oxidation with ninhydrin which produces a deep purple color end product. • The hippurate test is also used to identify unrelated bacterial species like Campylobacter jejuni, Gardnerella vaginalis and Listeria monocytogenes.

18. Group D Streptococci • Streptococcus bovis and Enterococcus faecalis. • Both are normal flora of the intestinal tract. • Both organisms are among the most common organisms causing endocarditis. • Most group D streptococci show alpha hemolysis or no hemolysis on blood agar, although some strains are beta hemolytic.

19. Isolation and identification • Streptococcus [Enterococcus] faecalis broth (SFB): • selective medium for the detection of fecal streptococci (group D) and enterococci from water, milk and other materials. • Growth of all other cocci is inhibited. • Fermentation of glucose is indicated by a color change in the broth. • Sodium azide is the inhibitory agent. • Bromcresol purple is the indicator.

20. Uninoculated tube of SF broth. pH indicator is violet at a neutral pH. Enterococcus faecalisgrowing in SF broth. Note yellow-brown color indicating acid

21. Bile Esculin agar • Selective medium for the detection of fecal streptococci (group D) and enterococci. • Tests ability of the organism to hydrolyze esculin to esculetin. • The esculetin reacts with ferric ammonium citrate to form a blackish precipitate. • Oxgall (bile) is inhibitory agent. • Ferric citrate is indicator

23. Streptococcus pneumoniae • Gram-positive usually appearing as a diplococcus, but occasionally appearing singularly or in short chains. • Alpha-hemolytic bacterium. • Fastidious, growing best in 5% carbon dioxide. • Pneumococci are frequently found as normal flora of the nasopharynx of healthy carriers.

24. Optochin sensitive S.pneumoniae Optochin resistant Viridans Streptococci Isolation and identification • Isolation on Blood agar (They are surrounded by a zone of alpha hemolysis). • Optochin sensitivity

27. Staphylococci

28. Staphylococci are often found in the human nasal cavity (and on other mucous membranes) as well as on the skin. • They are gram-positive cocci and occur singly, in pairs, in short chains, and most commonly, in irregular grape-like clusters. • The staphylococci are strongly catalase positive. • Reduce nitrates to nitrites. • Generally tolerate relatively high concentrations of sodium chloride (7.5-10%). • There are five species of staphylococci commonly associated with clinical infections: • Staphylococcus aureus. • S. epidermidis. • S. haemolyticus. • S. saprophyticus.

30. Staphylococcus aureus • Gram-positive, cluster-forming coccus. • Nonmotile, nonsporeforming and facultative anaerobe • Fermentation of glucose produces mainly lactic acid ferments mannitol (distinguishes from S. epidermidis). • Catalase positive and coagulase positive. • Given golden yellow colony on agar. • Normal flora of humans found on nasal passages, skin and mucous membranes. • Pathogen of humans, causes a wide range of suppurative infections, as well as food poisoning and toxic shock syndrome.

31. Virulence factors • Leukocidin (kills leukocytes). • Alpha and delta toxins (damage tissue membranes). • Microcapsules (resist phagocytic engulfment and destruction). • Coagulase and protein A (both help resist phagocytic engulfment). • Some strains also produce TSST-1 (toxic shock syndrome toxin-1) and cause toxic shock syndrome.

32. Isolation and Identification • Blood agar with a novobiocin: • Hemolysis: Usually beta. • Pigment: Often creamy gold. • Mannitol fermentation on Mannitol Salt agar (MSA) • Staphylococci are able to tolerate the high salt concentration found in mannitol Salt agar and thus grow readily. • If mannitol is fermented, the acid produced turns the phenol red pH indicator from red (alkaline) to yellow (acid). • Production of deoxyribonuclease (DNase) on DNase agar: Positive • Cell wall clumping factor: Positive