1. Medically Important Bacteria Gram Positive Cocci

2. IntroductionGram-Positive Start by remembering that there are 6 classic gram positive bugs that cause disease in humans, and basically every other organism is gram-negative. Of the gram-positives, 2 are cocci, and the other 4 are rod-shaped (bacilli). The 2 gram-positive cocci both have the word coccus in their names: 1) Streptococcus forms strips of cocci. 2) Staphylococcus forms clusters of cocci. 2

3. Two of the 4 gram-positive rods produce spores (spheres that protect a dormant bacterium from the harsh environment). They are: 3) Bacillus 4) Clostridium The last 2 gram-positive rods do not form spores: 5) Corynebacterium 6) Listeria, which surprisingly has endotoxin- surprising because ALL other organisms with endotoxin are gram-negative. 3

4. Gram-Negative Of the gram-negative organisms, there is only one group of gram-negative cocci. It is actually a diplococcus (looks like 2 coffee beans kissing): Neisseria. There is also just 1 group of spiral-shaped organisms: the Spirochetes. This group includes the bacterium Treponema pallidum, which causes syphilis. The rest are gram-negative rods or pleomorphic. 4

5. Exceptions: 1) Mycobacteria are weakly gram-positive but stain better with a special stain called the acid- fast stain. This special group includes organisms that cause tuberculosis and leprosy. 2) Spirochetes have a gram-negative cell wall but are too small to be seen with the light microscope and so must be visualized with a special darkfield microscope. 3) Mycoplasma do not have a cell wall. They only have a simple cell membrane, so they are neither gram positive nor gram-negative 5

6. 6 Medically Important Cocci GPC –Streptococcus –Staphylococcus EnterococcusGNC –Neisseria –Moraxella –Branhamella

7. Streptococci Characters of Streptococci –Gram positive cocci –1 µ m in diameter –Chains or pairs –Usually capsulated –Non motile –Non spore forming –Facultative anaerobes –Fastidious –Catalase negative (Staphylococci are catalase positive)

8. Classification of Streptococci Streptococci can be classified according to: –Oxygen requirements Anaerobic (Peptostreptococcus) Aerobic or facultative anaerobic (Streptococcus) –Serology (Lanciefield Classification) –Hemolysis on Blood Agar (BA)

9. Serology: Lanciefield Classification Streptococci classified into many groups from A-K & H-V One or more species per group Classification based on C- carbohydrate antigen of cell wall –Groupable streptococci A, B and D (more frequent) C, G and F (Less frequent) –Non-groupable streptococci S. pneumoniae (pneumonia) viridans streptococci –e.g. S. mutans –Causing dental caries Streptococci Group A S. pyogenes Group B S. agalactiae Group C S. equisimitis Group D Enterococcus Other groups (E-U) Lanciefield classification

11. Classification of Streptococci Based on Hemolysis on Blood Agar Hemolysis on BA –  -hemolysis Partial hemolysis Green discoloration around the colonies e.g. non-groupable streptococci (S. pneumoniae & S. viridans) –  -hemolysis Complete hemolysis Clear zone of hemolysis around the colonies e.g. Group A & B (S. pyogenes & S. agalactiae) –  -hemolysis No lysis e.g. Group D (Enterococcus spp) Streptococci  -hemolysis  -hemolysis  -hemolysis

12. Hemolysis on Blood agar  -hemolysis  -hemolysis  -hemolysis

13. Group A streptococci Include only S. pyogenes Group A streptococcal infections affect all ages peak incidence at 5-15 years of age 90% of cases of pharyngitis

14. Pathogenesis and Virulence Factors Structural components –M protein M, which interferes with opsonization and lysis of the bacteria –Lipoteichoic acid & F protein adhesion –Hyaluronic acid capsule, which acts to camouflage the bacteria Enzymes –Streptokinases –Deoxynucleases –Anti-C5a peptidase Pyrogenic toxins that stimulate macrophages and helper T cells to release cytokines Streptolysins –Streptolysin O lyse red blood cells, white blood cells, and platelets –Streptolysin S facilitate the spread of streptococci through tissues

15. Disease caused by S. pyogenes Suppurative –Non-Invasive Pharyngitis ( “ strep throat ” )-inflammation of the pharynx Skin infection, Impetigo –Invasive Scarlet fever-rash that begins on the chest and spreads across the body Pyoderma-confined, pus-producing lesion that usually occurs on the face, arms, or legs Necrotizing fasciitis-toxin production destroys tissues and eventually muscle and fat tissue Non Suppurative –Rheumatic fever: Life threatening inflammatory disease that leads to damage of heart valves muscle –Glomerulonephritits Immune complex disease of kidney inflammation of the glomeruli and nephrons which obstruct blood flow through the kidneys

16. Impetigo folliculitiscellulitis Pharyngitis

17. Scarlet fever-rash Pyoderma Necrotizing fasciitis

18. Differentiation between  -hemolytic streptococci The following tests can be used to differentiate between  -hemolytic streptococci –Lanciefield Classification –Bacitracin susceptibility Test Specific for S. pyogenes (Group A) –CAMP test Specific for S. agalactiae (Group B)

19. Bacitracin sensitivity Principle: –Bacitracin test is used for presumptive identification of group A –To distinguish between S. pyogenes (susceptible to B) & non group A such as S. agalactiae (Resistant to B) –Bacitracin will inhibit the growth of gp A Strep. pyogenes giving zone of inhibition around the disk Procedure: –Inoculate BAP with heavy suspension of tested organism –Bacitracin disk (0.04 U) is applied to inoculated BAP –After incubation, any zone of inhibition around the disk is considered as susceptible

20. CAMP test Principle: –Group B streptococci produce extracellular protein (CAMP factor) –CAMP act synergistically with staph.  -lysin to cause lysis of RBCs Procedure: –Single streak of Streptococcus to be tested and a Staph. aureus are made perpendicular to each other –3-5 mm distance was left between two streaks –After incubation, a positive result appear as an arrowhead shaped zone of complete hemolysis –S. agalactiae is CAMP test positive while non gp B streptococci are negative

21. CAMP test