Principles of Microbiology and Parasitology Lecture 2: Gram-Positive and 1 Gram-Negative Cocci 1Auemphon Mordmuang, Ph.D. (Microbiology) School of Medicine, Walailak University, E-mail: [email protected], Tel: 2875
Study OutlineI. Introduction and overview 2 : Bacteria in coccusII. Important pathogenic Gram positive-cocci Genus Staphylococcus Genus Streptococcus Genus EnterococcusIII. Important pathogenic Gram negative-cocci Genus Neisseria Genus Moraxella
Learning ObjectiveAfter lesson students should be able to : 1. Discuss and describe the bacterial genus in cocci 2. Discuss the bacterial characteristics used in laboratory identification and differentiation 3. Discuss clinical significance and pathogenicity of bacteria 4. Discuss the treatment and prevention of the bacterial infections or disease 3
Gram Positive Cocci Staphylococci Streptococci 4
StaphylococciStaphylococcus (Greek: staphyle = Bunch of grape;kokkos = grain or berry)• Gram positive, Cocci in cluster• Normal flora e.g., skin, upper respiratory tract• Facultative anaerobes• Catalase positive• Nonmotile• Resistant to dry condition and high salt concentration• The major pathogen of genus, Staphylococcus aureus (coagulase positive)• Coagulase-negative staphylococci: S.epidermidis and 5 S.saprophyticus (urinary tract infection)
Staphylococci 6
Staphylococcus aureus β-hemolysis (clearly hemolyed zone) 7 on blood agarAvailable fromhttp://www.aofoundation.org/aopictures/BALI_ARI_CID/Petri-dish.jpg[Accessed April 13, 2012]
Staphylococcus aureus Mannitol salt agar (MSA) 8Available fromhttp://farm4.static.flickr.com/3547/3397272258_36411552d3.jpg[Accessed April 13, 2012]
S. aureus infection• Host compromise is required for infection: – A break in skin or insertion of foreign body – An obstructed hair follicle (folliculitis) – A compromised immune system• Epidemiology: – Carried by healthy individuals on skin and mucous membrane – Carriers serve as source of infection (by direct contact, by contamination of fomites [objects, food]) 9
Pathogenesis• The clinical outcome depends on the virulence of the pathogen and the opposing effectiveness of the host defense mechanisms• S. aureus expresses many potential virulence factors – Cell wall virulence factors – Cytolytic exotoxins – Panton-Valentine leukocidin – Superantigen exotoxins 10
Extracellular Virulence Factors• Capsule – Very thin polysaccharide – Associated with increased resistance to phagocytosis• Protein A – A major component of the S. aureus cell wall – Binds to the Fc region of IgG >> exerting an anti-opsonin >> strongly antiphagocytic effect• Fibronectin-binding protein – Promotes binding to mucosal cells and tissue matrices• Clumping factor – Enhances clumping of the organisms in the presence of plasma 11
Cytolytic Exotoxins• α, β, γ, and δ Toxins• Attack mammalian cell (including red blood cell) membranes• α Toxin >> loss of important molecules >> osmotic lysisRabbit RBC lysed with α Toxin 12
Pathogenesis 13
Panton-Valentine leukocidin• Pore-forming toxin lyses PMNs• Production of this toxin makes strains more virulent• Produced predominantly by community- acquired methicillin-resistant S. aureus (MRSA) strains 14
Superantigen Exotoxins• Enterotoxins – Six major antigenic types: A, B, C, D, E, and G – More heat-stable than S. aureus – Cause food poisoning – Stimulates the vomiting center in the brain by binding to neural receptors in the upper GI tract• Toxic shock syndrome toxin (TSST –1) – Classic cause of toxic shock syndrome (TSS) – Referred to as staphylococcal enterotoxin F (does not cause food poisoning)• Exfoliatin (exfoliative toxin) – Causes scalded skin syndrome in children – Toxin cleaves desmoglein 1 (a component of desmosomes) >> loss of the superficial skin layer 15
Clinical Significance• Localized skin infections• Deep, localized infections• Acute endocarditis• Septicemia• Pneumonia• Nosocomial infections• Toxinoses 16
Localized skin infections 17 • Small, superficial abscesses involving hair follicles (folliculitis) or sweat or sebaceous glands • Infection of an eyelash follicle (external hordeolum) • Foreign bodies >> subcutaneous abscesses called furuncles (boils) • Carbuncles are larger, deeper, multiloculated skin infections • Impetigo is usually a localized, superficial, spreading crusty skin lesion generally seen in childrenAvailable from http://www.ilikeeye.com/stye/http://siamhealth.net/public_html/Health/Photo_teaching/folliculitis.htm#.VZoc4e-JheU[accessed July 6, 2015]
Furuncle (boil) 18
Staphylococcal Carbuncle 19
Deep, localized infections• May be metastatic from superficial infections or skin carriage or may result from trauma• Acute and chronic infection of bone marrow• Acute infection of joint space in children (septic joint) 20
Acute Endocarditis• Associated with intravenous drug abuse – Injection of contaminated preparations – By needles contaminated with S. aureus – The skin is not sterilized before injection >> bacteria can be introduced into soft tissues and the bloodstream 21
Toxicoses• Toxic shock syndrome – High fever, rash (resembling a sunburn, with diffuse erythema followed by desquamation), vomiting, diarrhea, hypotension, and multiorgan involvement (especially GI, renal, and/or hepatic damage) – Outbreak occurred in the late 1970s among menstruating women (use of hyperabsorbant tampons) 22
Toxicoses• Staphylococcal gastroenteritis – Often contaminated by; • A food handler • Protein rich e.g. egg salad or cream pastry • Salty, like ham • Improperly refrigerated – Heat-resistant toxins are able to withstand subsequent reheating – Enterotoxin in the food has already been formed before ingested >> short incubation period (< 6 hours) – Symptoms, such as nausea, vomiting, and diarrhea 23
Toxicoses• Scalded skin syndrome – Resulting from the action of an exfoliative toxin – Involves the appearance of superficial bullae – Toxin that attacks the intercellular adhesive of the stratum granulosum >> marked epithelial desquamation 24
Laboratory Identification• Specimens: surface swab, pus, blood, CSF• Staining: Gram stain• Culture: nutrient agar, blood agar, MSA• Biochemical tests: mannitol fermentation• Catalase test• Coagulase test: positive for S.aureus 25
Catalase Test• Catalase breaks hydrogen peroxide into water and molecular oxygen• Genus Staphylococcus are catalase-positive, but Genus Streptococcus and Enterococcus are catalase-negativeAvailable from 26http://iws2.collin.edu/dcain/CCCCD%20Micro/Catalase.jpg[Accessed April 13, 2012]
Coagulase TestCoagulase is a protein enzyme produced by several microorganisms thatenables the conversion of fibrinogen to fibrin positive negative 27
Treatment• The frequent presence of acquired antibiotic resistance• Hospital-acquired S. aureus infections are now resistant to penicillin G due to penicillinase-encoding plasmids or transposons• Require β-lactamase-resistant penicillins, such as methicillin or oxacillin• Resistant to a number of β-lactam antibiotics, such as methicillin, oxacillin and amoxicillin >> methicillin-resistant S. aureus (MRSA)• Vancomycin has been the agent of choice for empiric treatment of life-threatening MRSA• The incidence of vancomycin resistance has increased steadily• Aanltderdnaapttivoemdyrcuings>s>u?c?h?as quinupristin- dalfopristin, linezolid, 28
Prevention• There is no effective vaccine against S. aureus• Infection control procedures, such as barrier precautions and disinfection of hands and fomites 29
Coagulase-Negative Staphylococci• Staphylococcus epidermidis – Large numbers as part of the normal flora of the skin – Low virulence – A common cause of infection of implants such as heart valves and catheters – Produces an extracellular polysaccharide material called slime >> facilitates adherence to bioprosthetic material surfaces/a barrier to antimicrobial agents 30
Coagulase-Negative Staphylococci• Staphylococcus saprophyticus – A frequent cause of cystitis in women – Tends to be sensitive to most antibiotics, even penicillin G – Resistance to novobiocin (distinguished from S. epidermidis) 31
Streptococci• Gram-positive cocci in chain• Nonmotile• Catalase-negative• Facultative anaerobic 32
Classification of Streptococci• Hemolytic properties on blood agar: – β-hemolysis – α-hemolysis – γ-hemolysis 33
34
Group A Streptococci (Streptococcus pyogenes) β-hemolysis on blood agar• The most clinically important member of this group• Can invade apparently intact skin or mucous membranes• Some strains cause postinfectious sequelae: – Rheumatic fever – Acute glomerulonephritis• Does not survive well in the environment 35
Structure 36
Structure• Capsule: – Hyaluronic acid, identical to that found in human connective tissue >> not recognized as foreign >> nonimmunogenic – Antiphagocytic• Components of cell wall – M protein • Not infectious in the absence of M protein • Highly variable >> over 80 different antigenic types • Antiphagocytic • Interferes with complement binding – Protein F • Mediates attachment to fibronectin in the pharyngeal epithelium 37
Extracellular products 38
Epidemiology• Reservoir: – The skin – Mucous membranes of the human host• Spread person to person by: – Respiratory droplets – Skin contact – especially in crowded environments such as classrooms and children’s play areas 39
Pathogenesis• S. pyogenes cells attach to the pharyngeal mucosa via actions of protein F, lipoteichoic acid, and M protein• Grow and secrete toxins >> causing damage to surrounding cells >> invading the mucosa >> eliciting an inflammatory response with attendant influx of white cells, fluid leakage, and pus formation• Possibly resulting in septicemia and/or seeding of distant sites 40
Clinical significance• Cellulitis• Acute pharyngitis or pharyngotonsilitis• Impetigo• Erysipelas• Puerperal sepsis• Necrotizing fasciitis (streptococcal gangrene)• Streptococcal toxic shock syndrome• Post-streptococcal sequelae 41
CellulitisSpecifically affects the dermis and subcutaneous fat 42
Acute pharyngitis or pharyngotonsilitis• Associated with severe, purulent inflammation of the posterior oropharynx and tonsillar areas• Scarlet fever: a sunburnlike rash develops on the neck, trunk, and extremities in response to the release of pyrogenic exotoxin Available from http://www.gponline.com/exclusive-gps- alert-scarlet-fever-levels-highest-1982/infections-an4d3- infestations/article/1341995 [Accessed July 6, 2015]
Impetigo• Begins on any exposed surface (most commonly, the legs)• Typically affecting children• Cause severe and extensive lesions on the face and limbsHighly contagious infection of the superficial layers of the epiderm44is
Erysipelas• A fiery red, advancing erythema, especially on the face or lower limbs• Affecting all age groups More superficial than cellulitis 45
Necrotizing fasciitis (streptococcal gangrene)• Extensive and very rapidly spreading necrosis of the skin, tissues, and fascia• Termed flesh eating bacteria 46
Streptococcal Toxic Shock Syndrome• Mediated by the production of streptococcal pyrogenic exotoxins that function as superantigens• Patients may initially present with flulike symptoms, followed shortly by necrotizing soft tissue infection, shock, acute respiratory distress syndrome, and renal failure 47
Post-Streptococcal Sequelae• Acute rheumatic fever– Autoimmune disease occurs 2 to 3 weeks after theinitiation of pharyngitis– Caused by crossreactions between antigens of the heartand joint tissues, and the streptococcal antigen(especially the M protein epitopes)– Characterized by fever, rash, carditis, and arthritis• Acute glomerulonephritis– This rare, occurs as soon as 1 week after impetigo orpharyngitis– Antigen-antibody complexes on the basement membraneof the glomerulus initiate the disease 48
Post-streptococcal GNAvailable from http://www.unckidneycenter.org/images/glomcap.jpg 49[Accessed April 13, 2012]
Laboratory Identification• Microscopy• Culture• Latex agglutination• Bacitracin disk test: sensitive 50
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