3.30 Mon Lectures: The pyogenic cocci (1) 4.1 Wed Lectures: The pyogenic cocci (2) 4.6 Lectures: Enterobacteriaceae,Vibrios,Mycobacteria helicobacter pylori，Corynebacterium (1) Lab: Care and use of the microscope 4.8 Lectures: Enterobacteriaceae,Vibrios,Mycobacteria helicobacter pylori，Corynebacterium (2) 4.9 Thur 3.30 Mon Lectures: The pyogenic cocci 4.1 Wed Lectures: Enterobacteriaceae,Vibrios,Mycobacteria helicobacter pylori，Corynebacterium (1) 4.6 Lab: Care and use of the microscope 4.8 Lectures: Enterobacteriaceae,Vibrios,Mycobacteria helicobacter pylori，Corynebacterium (2) 4.9 Thur The labs in the next week are canceled, will be shifted to the following week. Labs will extend to 5.25 and 5.28.

Zooming in pyogenic coccus: disease and pathogenesis

Pyogenic coccus

Pyogenic coccus

Major pathogenic species Staphylococcus (the staphylococcus) Staphylococcus aureus Staphylococcus epidermidis Staphylococcus saprophyticus Streptococcus (the streptococcus) Streptococcus pyogenes Streptococcus pneumoniae (the pneumococcus) Neisseria (the neisseria) Neisseria meningitidis (the meningococcus) Neisseria gonorrhoeae (the gonococcus)

Staphylococcus aureus Diseases Pathogenesis Roles of bacterial virulent factors Diagnosis Methods for bacteria classification Treatment and prevention Issues

Bacteria morphology Staphylococcus : grape-like irregular clusters Streptococcus : chains or pairs Neisseria : kidney-shaped diplococci

Bacteria culture: pigmentation The bacteria produces liposoluble pigments which make the colonies appear in certain color: Staphylococcus aureus golden yellow Staphylococcus epidermidis white Staphylococcus saprophyticus white or lemon yellow

Bacteria culture: mannitol fermentation Biochemical reactions: for example, Staphylococcus aureus is capable of using sugar mannitol (甘露醇) as a food source and will produce acidic byproducts of fermentation that will lower the pH of the media. Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae and Streptococcus pyogenes on mannitol salt agar plates (containing 7.5% NaCl, mannitol and phenol red).

Diseases I. Suppurative infections (化脓性感染) II. Toxicoses (毒素性疾病)

I. Suppurative infections Local infections (contained at the local) Superficial skin lesions such as sore (疮), furuncle (疖) and stye (睑腺炎，麦粒肿) Systemic infections (spread through the blood stream) Septicemia (败血症) Pyemia (脓血症): spread to different organs to cause pneumonia (肺炎), mastitis (乳腺炎), phlebitis (静脉炎), meningitis (脑膜炎), urethritis (尿道炎) and abscesses (脓肿)

II. Toxicoses Food poisoning A gastrointestinal illness. Symptoms include nausea, vomiting, stomach cramps, and diarrhea. Toxic shock syndrome (TSS,毒性休克综合征) Characterized by a sudden onset of fever, chills, vomiting, diarrhea, muscle aches and rash. It can rapidly progress to severe and intractable hypotension and multisystem dysfunction. Commonly occurs in women. Scalded skin syndrome (SSS, 烫伤样皮肤综合症) Caused by staphylococcal skin infection. The skin blisters and peels off as though burned. Additional symptoms are fever, chills, and weakness. commonly occurs in infants.

Pathogenesis Cell wall virulent factors Exotoxins Staphylococcal protein A (SPA) Coagulase Exotoxins Staphylolysin (cytolytic) Leukocidin (cytolytic) enterotoxin (superantigen) toxic shock syndrome toxin 1 (superantigen) Exfoliatin (superantigen)

Staphylococcal protein A (SPA) Surface bound (in the cell wall) or free proteins. Bind to Fc portion of IgG from human, mouse and guinea pig (but not to that from rabbit), and thus inhibit Fc receptor-mediated phagocytosis by macrophages.

Inhibition of phagocytosis Fc receptor SPA IgG

Agglutination test In this test, anti-Staphylococcus aureus IgG can bind SPA using Fc fragment and the bacteria through Fab to from complexes that can been seen with eyes.

Coagulase Bound (in the cell wall): catalyzes fibrinogen (纤维蛋白原) into fibrin (纤维蛋白). Cause coating of the bacteria with fibrin and thus inhibit phagocytosis and killing mediated by serum components. Free: secreted and turns into staphylothrombin after activation by cofactors in the plasma, which catalyzes fibrinogen into fibrin. Cause coagulation of the plasma and helps to restrain infections at the local.

Coagulase test and CNS A test of coagulation of human or rabbit plasma in the presence of anticoagulant (citrate or heparin). Coagulase-negative staphylococci (CNS) used to be thought as non-pathogenic, however, they have become a major source of hospital-acquired infections: Staphylococcus epidermidis Staphylococcus saprophyticus

Cytolytic exotoxins: staphylolysin and leukocidin Attack mammalian cell membranes, cause abscess and tissue necrosis. Often referred to as hemolysins. Staphylolysins: Four types: , ,  and  Staphylolysin ,  and  can lyse erythrocytes (the basis for hemolytic reaction); toxic to many other cells such as white blood cells and muscle cells Staphylolysin : close to leukocidin. Leukocidin: lyse macrophages and neutrophils.

Hemolysis test Bacteria are grown on sheep blood agar plates (containing 5% sheep blood): α- hemolytic reaction, partial hemolysis with a green coloration： Streptococcus pneumoniae β- hemolytic reaction, complete hemolysis： Staphylococcus aureus Streptococcus pyogenes γ - hemolytic reaction, no hemolysis: Staphylococcus epidermidis most strains Staphylococcus saprophyticus

Superantigens Superantigens can bind both to class II MHC molecules (but not in the peptide cleft) and also to a relatively conserved region of the TCR βchain. This leads to non-specific activation of 2-20% of all T cells (vs. 0.01%), and massive unregulated cytokine release.

Superantigen exotoxins: enterotoxin, TSST-1 , and exfoliatin Enterotoxin, 9 serotypes, responsible for staphylococcal food poisoning. TSST-1, close to enterotoxin, cause toxic shock syndrome (TSS). Exfoliatin, 2 serotypes, cause staphylococcal scalded skin syndrome (SSS).

Diagnosis Pus abscesses and superficial skin lesions Sputum lower respiratory tract infections Blood septicemia, pyemia Food/feces or vomit food poisoning Mid-stream urine urethritis

Kidney-shaped diplococci mannitol fermentation S. aureus CNS Streptococcus Neisseria Gram staining + - bacteria morphology irregular clusters Chains or pairs Kidney-shaped diplococci colony pigmentation Golden white mannitol fermentation hemolysis test β-hemolytic γ -hemolytic coagulase test positive negative agglutination test Use serum samples from patients.

Treatment and prevention Antibiotics MRSA refers to methicillin (甲氧苯青霉素)-resistant Staphylococcus aureus, and most MRSA strains are also multiple drug resistant Chromosomal mutation Plasmid-conferred CNS, eg. Staphylococcus epidermidis Biofilm formation Exotoxin neutralizing antibodies No vaccines available currently

Summary Major pyogenic cocci Identification of Staphylococcus aureus and related bacteria (diagnosis) Diseases and molecular mechanisms Drug resistance and mechanisms