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Transcript
Micro: Lecture 17: Gram-Positive Bacteria Study Objectives •List the medically important Gram-positive bacteria. •Discuss the classification of streptococci. •Discuss the role of each virulence factor in disease. •Describe the pathogenesis, epidemiology, diseases, diagnosis and treatment of each of the organisms. •Identify the major characteristics of each organism (morphology, virulence factors, spore formation, cell wall composition and any other unique characteristics…). •Define PPD and discuss its significance in diagnosis of M. tuberculosis. Pathogenic Gram-Positive Bacteria •Coccus (round) −Staphylococcus −Streptococcus •Bacillus (Rod) −Bacillus −Clostridium −Corynebacterium −Listeria −Mycobacterium Staphylococcus Characteristics •Facultative Anaerobes (can use O2); Nonmotile; Irregular clusters •Grows best aerobically •Found in soil, water and skin of humans •Catalase-positive (Streptococcus are catalase-negative) •Three clinically important species: −S. aureus most virulent (coagulase-positive) −S. epidermidis (agent of opportunistic infections associated with indwelling equipment (catheters, prosthetics, …) −S. saprophyticus (agent of UTI) Epidemiology •Transmission – Coagulase-Positive Autoinfection (carrier), Direct contact (person with lesion), Contaminated food (person with lesion) or Fomite – an inanimate object (Survive long periods of drying); Coagulase-Negative Autoinfection (infections associated with implanted catheters and prosthetic devices; UTI) −About 30% of individuals carry S. aureus in the anterior nares −Coagulase-negative species are normal flora of skin, nares and ear canals 3 Medically Important Species of Staphylococcus •Staphylococcus aureus (Most common) –Causes most Staph infections in humans –Coagulase + –Mannitol fermenter (Will turn yellow) –Novobiocin sensitive •Staphylococcus epidermidis –Normal flora, an opportunisitc pathogen –Coagulase – –No mannitol fermentation –Novobiocin sensitive •Staphylococcus saprophyticus (Most opportunistic) –Opportunistic, causes UTI –Coagulase –Mannitol fermenter –Novobiocin resistant Differentiation of the Main Species of Staphylococci Characteristic S. aereus S. epidermidis S. saprophyticus Color of Colonies Often yellow White White to pale grey Hemolysis Most isolates A few isolates Non-hemolytic Coagulase production Yes No No Mannitol fermentation Yes No Yes Novobiocin Sensitive Sensitive Resistant Staphylococcus Pathogenesis •Coagulase −Initiates fibrin polymerization on surface of bacteria (protect from phagocytosis) −Clumping factor permits binding to fibrinogen and fibrin •Hyaluronidase / Staphylokinase (enzymes) −Permits invasion of tissues and dissolves fibrin clots formed by coagulase, respectively •Alpha-Toxin (a-toxin) −Inserts into cell membranes resulting in lysis of cells (also a hemolysin) •Beta-, Delta-, Gamma-Hemolysins −b-hemolysin degrades sphingomyelin lysing numerous cells −d-hemolysin dissociates into subunits to disrupt cell membranes −g-hemolysin components combine with PV proteins (LukS/LukF) that lyse WBC •Leukocidins (cytotoxin) −Two-component toxins which lyse WBC by forming pores •*Exfoliative Toxins A, B (detroy cell-cell adhesion) −A = heat stable (resists boiling for 20min); B = heat labile −Dissolve epidermal mucopolysaccharide matrix (desquamation); Also function as Superantigen •*Toxic Shock Syndrome Toxin (TSST-1) −Superantigen (can cause anaphylaxis) toxic shock syndrome; directly cytotoxic (?); In 20% of isolates •*Enterotoxins (multiple types – infects GI tract) −Heat-stable Superantigens resistant to gut enzymes (just 25mg induces vomiting) Virulence Determinants of S. aureus (Box A: use for cell adhesion; Protein A: Igs bind) Staphylococcus aureus Clinical Findings •Furuncle (Boils) & Carbuncle −Infected patient is often a carrier (anterior nares) −Lesions of a hair follicle, sebaceous gland or sweat gland −Resolves upon spontaneous drainage of pus −Multiple boils form a carbuncle which can spread to the blood •Impetigo −Production of exfoliative toxins produce large blisters in superficial skin −Often affects young children (face and limbs) −Macule that develops into pustules (rupture and crust) −Can occur as wound infection following surgery •Scalded Skin Syndrome (SSS) −AKA Ritters Disease −Production of exfoliative toxins that cause erythema (redness of skin) and epidermal desquamation at remote sites from staphylococcal infection −Face, axilla, groin affected first then all parts of body possible −Most common in neonates and children <5y Clinical Findings – 1 Infections •Toxic Shock Syndrome −High fever, vomiting, diarrhea, sore throat and myalgia −~48h can progress to shock with evidence of renal and hepatic damage −Rash may develop followed by desquamation at a deeper level than SSS −Originally associated with use of tampons in 1980s…growth of bacteria on tampon and release of TSST-1 into blood −Also occur in men and children with staphylococcal wound infections −TSS-Staphylococcus found in vagina, on tampons, in wounds, localized infections or throat (never the blood) •Staphylococcal Food Poisoning −Incubation 1-6h −Ingestion of enterotoxin-contaminated food −Nausea, vomiting and diarrhea (no fever)…few hours duration −Salted meats, custard pastries, ice cream, salad bar foods,… −Toxins are heat-stable so reheating food does not inactivate toxins (cannot taste in food) −Antibiotics are not useful Toxic Shock Syndrome Toxin-1 •A superantigen toxin that results in release cytokines causing fever, capillary leakage, circulatory collapse and shock •Superantigen toxins also prevent the development of a normal immune response of Diagnosis •Culture −Pus/surface swab, blood, sputum specimen inoculation onto Mannitol Salt agar (MSA) Successful growth, Gram stain (all are positive), catalase test, coagulase test −MSA contains 7.5% NaCl which inhibits growth of other normal flora −Catalase differentiates from Streptococcus −Coagulase differentiates more virulent S. aureus from other species −Microdilution / Disk Diffusion susceptibility tests should be done Prevention & Treatment •To prevent recurrent infections remove contaminated fomites •Use of soaps to reduce flora levels and antimicrobials to eliminate nasal carriage •Prophylaxis prior to surgery reduces incidences of infection •Methicillin-susceptible strain = Dicloxacillin −Alt = A Cephalosporin, Vancomycin, Clindamycin •MRSA = Vancomycin (+Gentamicin + Rifampin*) −Alt = Linezolid, Daptomycin, Tigecycline, A Fluoroquinolone −*Rifampin recommended if prosthetic valve or hardware present Methicillin-Resistant Staphylococcus aureus MRSA •<10% of S. aureus strains susceptible to Penicillin but many still susceptible to penicillin-derivatives •Late 80s a nosocomial S. aureus strain demonstrated resistance to penicillin-derivatives (methicillin)…ten years later a genetically distinct community-acquired (CA) methicillin-resistant strain appeared •In both cases, MRSA expressed a new resistance gene on a plasmid, mecA •CA-MRSA genetic distinction resided in the production of new types of PV leukocidins (more virulent) •Vancomycin employed to treat MRSA patients has led to generation of vancomycin-resistant MRSA (VRSA) •Several strains of VRSA have been detected since 2002 (most express a plasmid with vanA gene cluster*) •Development of such resistant strains appears to derive from resistance generated in Enterococcus that is transmitted to Staphylococcus through conjugation Streptococcus Characteristics--------------------Alpha: no hemolyse; Beta: has hemolyse •Facultative Anaerobes; Nonmotile •Blood agar is preferred because satisfies growth requirements and can differentiate groups based upon hemolysis patterns •Catalase-negative (Staphylococcus catalase-positive) •Classification by Group-Specific Surface Carbohydrate: −Group A (S. pyogenes) −Group B (S. agalactiae) −Group C (S. dysgalactiae) −Group D (S. bovis, Enterococcus) −Non-Groupable (S. pneumoniae, Viridans) •Classification by Hemolysis Pattern: −Alpha (Group D and Non-groupable) −Beta (Groups A, B, C, F, G) −Non-hemolytic (Some Group D and Non-Groupable) Epidemiology •Transmission – Direct contact, Fomites or Contaminated respiratory droplets (GAS); In utero or during birth (GBS); Direct contact with nasal secretions or Contaminated respiratory droplets (S. pneumoniae or Pneumococcus) Main Pathogenic Groups of Streptococci Characteristics Used to Differentiate Major Groups of Streptococci Streptococcus pyogenes (GAS) Pathogenesis •M Protein / Lipoteichoic Acids / Protein F (on outside of bacteria) −Adherence to nasopharynx and skin epithelia −M protein and C5a peptidase (will degrade) block phagocytosis and PMN recruitment, respectively •Streptodornase / Streptokinase −DNase and Fibrinolysin to break up Neutrophil Extracellular Traps (NET) which are networks of granule protein (fibrin) and chromatin to capture pathogens so PMN antimicrobials can kill microbes •Pyrogenic Exotoxin (Erythrogenic Toxin) A, B, C −Superantigens toxic shock syndrome −Pyrogenic Exotoxin A is produced by Streptococcus carrying lysogenic phage −Streptococcal TSS and Scarlet Fever •Diphosphopyridine Nucleotidase −Lyse WBC •Streptolysin O / Streptolysin S −(O) Anaerobic Hemolysin that is rapidly inactivated in the presence of oxygen −(S) Aerobic Hemolysin; induced upon bacteria exposure to serum −Both damage tissue cells and lyse phagocytes Group A Streptococcal Virulence Factors Exotoxins Streptococcus pyogenes (GAS) Clinical Findings •Streptococcal Pharyngitis (Strep Throat) −Sore throat, fever, headache (tonsil, soft palate, uvula red, swollen, covered with yellow exudate)…1wk duration (any age…5-15y) •Impetigo −Small vesicle surrounded by erythema on face or lower extremities…enlarges over few days…develops into pustule…breaks to form crusted lesion (2-5y) •Erysipelas −Spreading area of erythema/edema (face), pain, fever, lymphadenopathy…previous history of streptococcal pharyngitis •Streptococcal TSS −Involves any site of GAS infection…myalgia, chills, severe pain at infected site −Necrotizing fasciitis and myonecrosis, nausea/vomiting, diarrhea…hypotension, shock, organ failure (bacteremia) •Scarlet Fever −Buccal mucosa, temples, cheeks are deep red (pale area around mouth and nose) −Tongue covered with yellow-white exudate and red papillae (strawberry tongue) −“Sandpaper” rash on d2 (chestextremities) •Acute Rheumatic Fever (ARF) / Acute Glomerulonephritis (AGN) −(ARF) Fever, carditis, chorea (involuntary movement disorder), arthritis…3wks following “strep throat”…recurrent attacks upon S. pyogenes encounter can result in damage to the heart (heart failure) −(AGN) 3-6wks after “strep throat” or skin infection…lesions of glomeruli Diagnosis •Ag Detection −Rapid detection kit for Group A Carbohydrate (must follow with culture as only 90-95% sensitive…goal to avoid ARF) •Culture −Throat swab, pus, sputum, tissue specimen inoculation onto Blood agar Beta hemolysis −Also test susceptibility to Bacitracin (GAS is susceptible while others are not) Prevention & Treatment •Penicillin G or V −Alt = (if allergic) Erythromycin, A Cephalosporin, Clindamycin •Treatment within 10d of symptoms onset prevents ARF (although does not prevent AGN)…goal is to remove the Ag so as not to develop crossreactive Igs Streptococcus agalactiae (GBS) Characteristics •Leading cause of sepsis and meningitis in the first few days of life •Normal resident of the GI tract…can spread to the vagina (10-30% of women) •During pregnancy and delivery…GBS may gain access to the amniotic fluid or colonize the newborn as it passes through the birth canal •About 2 cases/1,000 births in US •GBS exposed to mucus membranes and quickly spreads to the blood (lungs, CNS) •GBS capsule binds serum Factor H (binds to host cell glycosaminoglycans or GAG and degrades C3b) to prevent alternate pathway of complement activation •As long as mom has the IgG against GBS, the newborn is protected by the classical pathway of complement •Onset is first few days of life Presents as respiratory distress, fever, lethargy, irritability, hypotension…pneumonia (common) and meningitis (5-10%)…~20% mortality (if CNS infection…20-30% permanent brain damage) •Late-onset develops at 1-3mo •Diagnosis by detection of group B Ag in blood and culture on Blood agar •Prophylactic therapy of colonized women has dramatically reduced transmissions •Penicillin G −Alt = Ampicillin, A Cephalosporin, Vancomycin Streptococcus pneumoniae Pathogenesis •Choline Binding Protein −Binds phosphocholines of bacterial cell wall with carbohydrates of nasopharynx epithelia •Capsule −Prevents C3b deposition on bacterial cell surface −Blocks phagocytosis •Pneumolysin −Produced but not secreted −Production of peroxides during growth induces autolysins −Autolysins degrade peptidoglycan thereby lysing the bacteria −Pneumolysin is released: oDirectly cytotoxic to endothelial cells…(permits dissemination into the bloodstream) oImpairs ciliary action (paralyzed) oDirectly suppresses phagocytic activity oSuppresses local inflammatory immune response ( anti-inflammatory cytokines) oTriggers platelet activation (DIC can occur from concurrent vascular leakage due to endothelial damage in lungs) oPneumococcal pneumonia does not elicit extensive damage to the lung tissue as in other infections such as influenza (local immunosuppression) Streptococcus pneumoniae Clinical Findings •Infections most common in young (<2y) and old (>60y) •Pneumococcal Pneumonia −A leading cause of pneumonia in world (500,000 cases/y in US) −5 million children die each year worldwide −Shaking chills, high fever, cough (tinged with blood), chest pain…5-10d duration −Higher incidence in those over 50y •Pneumococcal Meningitis −One of the 3 leading causes of bacterial meningitis (N. meningitidis, H. influenzae) −Meningitis may develop solely or follow pneumonia or otitis media −Headache, stiff neck, fever, photophobia, irritability •Other Infections −Otitis Media oMost frequent cause with millions of cases/y −Sinusitis −Bacteremia Endocarditis, Arthritis, Peritonitis Diagnosis •Microscopy −Gram stain of sputum (bronchial lavage), CSF, blood Gram-positive Diplococci •Culture −Sputum, CSF, blood specimen inoculation onto Blood agar Gram stain, susceptibility to Optochin (ethylhydrocupreine…S. pneumoniae is susceptible while others are not) Prevention & Treatment •Pneumococcal Conjugate Vaccine (PCV13, Prevnar 13®) −Children/Adults <18y…4 doses (2, 4, 6, 12-15mo) or 1-2 doses if >2y −13 capsule Ags conjugated to CRM197 mutant diphtheria toxin •Pneumococcal Polysaccharide Vaccine (PPSV23, Pneumovax®) −All adults >65y −2y-64y with health condition (heart disease, AIDS,…) −88% of serotypes (23 capsule Ags) •Penicillin G or V (if susceptible) −Alt = (if allergic) Erythromycin, A Cephalosporin •Ceftriaxone (if intermediate resistance to penicillin G) •Vancomycin (if high level of resistance to penicillin G) Bacillus anthracis Characteristics •Aerobic or Facultative Anaerobes; Nonmotile; Spore-forming •All other species are low-virulence saprophytes found in air, soil, water •Protein capsule •Koch used this bacteria to work out Koch’s postulates; Pasteur developed vaccine for sheep, goats and cows using attenuation methods Epidemiology •Anthrax primarily a disease of horses, sheep and cattle who acquire it from spores of B. anthracis contaminating their pastures •Ideal biowarfare agent since spores of B. anthracis have long-life, stability and require few spores to produce infections (respiratory is most severe) •Transmission – Direct contact with spores (through skin), Ingestion of spores (rare) or Inhalation of spores •B. cereus and B. subtilis cause infections of the eye, soft tissues and lung associated with immunosuppression, trauma, indwelling catheters or contaminated medical equipment •B. cereus also can produce an entertoxin (cAMP) and cause diarrhea Pathogenesis •Protein Capsule −Adherence to tissues (?) −Effective blockade of phagocytic uptake •Anthrax Toxin −Protective Antigen (PA) oBinds to ATR on host cell surface oHost furin (membrane protease) cleaves PA into monomer …heptamerization of PA induces clustering of ATR on lipid rafts…bind either EF or LF and are endocytosed into host cell −Edema Factor (EF) oEF functions as an adenylate cyclase to cAMP −Lethal Factor (LF) oProtease that cleaves MAPKK pathway proteins and induces TNF secretion…induces cell death of macrophages and endothelial cells Clinical Findings •Cutaneous Anthrax −Incubation 2-5d after spore exposure −Erythematous papule Vesicular lesion Ulcerative lesion Scab (Black Eschar)…7-10d transition −Accompanied by mild systemic symptoms; rare cases, local edema is precursor to bacteremia and death •Pulmonary Anthrax −Historically – inhaled spores upon work in confined space with contaminated hides, hair, wool,…(Wool Sorter’s Disease) −Incubation 1-2d after spore exposure −Mild fever, nonproductive cough quickly progress to respiratory distress and cyanosis (alveoli are destroyed) −Bacteremia ensues to “seed” every organ system…95% mortality (untreated) Diagnosis •Culture −Skin lesion, Sputum, CSF, blood specimen inoculation onto Blood agar Non-hemolytic growth, Gram stain (Gram-positive bacillus) −Saprophytic species are beta-hemolytic and motile (swarming) Prevention & Treatment •Anthrax Vaccine Absorbed (AVA, Biothrax®) −Filtrate of B. anthracis culture with added aluminum hydroxide (adjuvant) −Contains PA and other immunogenic proteins −Recommended for those who may be exposed to large doses of endospores (some military, lab workers, some animal handlers) −3 initial doses (2wks apart)…then 3 booster doses (6, 12, 18mo) •Ciprofloxacin −Alt = A Tetracycline, Penicillin G, Amoxicillin Corynebacterium diphtheriae Characteristics / Epidemiology / Pathogenesis •Facultative Anaerobe; Pleomorphic (club-shaped bacillus) •60 species…found in plants and animals; colonize skin, respiratory tract, GI tract and urogenital tract (can be asymptomatic carriers) •Transmission – Contaminated respiratory droplets (remain viable for hours) •Diphtheria Toxin (DTX) −Encoded by a lysogenic phage – works when Iron is low! Then will produce the toxin! −B subunit serves as ligand for host receptor (heparin-binding epidermal growth factor) −A subunit inactivates elongation factor-2 (EF-2) −One DTX molecule can inactivate all EF-2 within the cell −Cells die following uptake of DTX Clinical Findings •Diphtheria −Begins as an “exudative pharyngitis” oSore throat, low-grade fever, malaise −Exudate on tonsils, pharynx and larynx evolves into a grayish “pseudomembrane” (necrotic epithelia embedded in fibrin, RBC and WBC…active bacteria) −Removal results in capillary damage and bleeding −Resolves within 1wk −Complications: oBreathing obstruction (dyspnea) oCardiac arrythmia oComa (5-10% mortality) Diagnosis •Culture −Swabs of nose and/or throat inoculated onto Blood agar (eliminate Streptococcus) and Cystin-Tellurite agar No hemolysis on Blood agar and growth on Tellurite agar −Tellurite inhibits growth of most URT microbes; tellurite is reduced by C. diphtheriae producing black colony coloration −PCR performed to confirm presence of DTX Prevention & Treatment •DTaP vaccine •Antimicrobials and Diphtheria Antitoxin −Antitoxin given on day of diagnosis •Erythromycin −Alt = Penicillin G Listeria monocytogenes Characteristics / Epidemiology / Pathogenesis •Facultative Anaerobe; Motile; Psychrophile (growth at 4C) •Found in intestinal tract of many animals (poultry, cattle, sheep) •5-10% of humans harbor without symptoms •Transmission – Contaminated food (unpasteurized milk, cheeses, contaminated ice cream, raw vegetables, raw meat, cold cuts) or Congenital to newborn (during labor/delivery) •Internalin −Adherence to enterocytes, M cells, phagocytes, hepatocytes, fibroblasts −Induces uptake by cells (endosome or phagosome) −Actin reorganization to move into other cells acquiring a double membrane vacuole •Listeriolysin O −Degrades endosome/phagosome/vacuole membranes to escape into cytosol and avoid lysosomal fusion Clinical Findings •Listeriosis −Nausea, abdominal pain, watery diarrhea, fever (self-limiting) −Bacteria may disseminate to other sites (cellular bacteremia)…sepsis −Tropism for CNS – meningitis −Fecal contamination at birth can transmit Listeria to newborn…neonatal sepsis Diagnosis •Culture −Blood, CSF specimen inoculated onto Mueller-Hinton agar with added sheep RBC Gram stain, Catalase test (positive), evident small zone of hemolysis −Can also inoculate PALCAM agar with food source Prevention & Treatment •Ampicillin