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Streptococcus, Enterococcus and other Gram-positive cocci Doç Dr Nevriye Gönüllü Streptococcus Gram positive Grow pattern pairs, chains Most species are facultatively anaerobes Some grow only in atmosphere enhanced with CO2 (capnophilic growth) Nutritional requirements are complex Blood, serum enriched media “Catalase-negative” Classification of common streptococcal pathogens Biochemical: S.pyogenes S.agalactiae S.dysgalactiae S.anginosus group S.bovis Viridans group S.pneumoniae Serologic/Hemolysis: Β/β C,G/ β A,C,F,G/β D/ α Nongroupable/α Nongroupable/α Streptococcus Classification is complicated 1. 2. 3. 3 different schemes are used Lancefield groupings according to serologic properties (A-H, K-M, O-V) Hemolytic patterns: β, α & γ hemolysis Biochemical properties Streptococci and their diseases S. pyogenes (group A) Pharyngitis, scarlet fever, pyoderma, erysipelas, cellulitis, necrotizing fasciitis, streptococcal toxic shock syndrome, bacteremia, rheumatic fever, glomerulonephritis Streptococci and their diseases S. agalactiae (group B) Neonatal infections (meningitis, pneumoniae, bacteremia) Urinary tract infections Amnionitis, Endometritis Wound infections Streptococcus pyogenes/physiology & structure Spherical cocci Form short (clinical specimen) or long chains (liquid media) Growth on enriched-blood agar media White colonies 1-2 mm with large zones of β-hemolysis Encapsulated strains mucoid Basic structure in the cell wall is peptidoglycan as Gram+ group and type specific antigens Streptococcus pyogenes/physiology & structure Group specific carbohydrate Within the cell wall 10 % of the dry weight A dimer of N-acetylglucosamine and rhamnose Is used to classify group A streptococci and distinguish them from others Streptococcus pyogenes/physiology & structure Type specific proteins M protein major type-specific protein associated with virulent streptococci 2 polypeptide chains 1. Highly conserved among all group A streptococci 2. Responsible for the antigenic variability >100 serotypes 3. Are subdivided into class I (share exposed antigens) and class II molecules (do not have exposed shared antigens) 4. Only bacteria with class I M proteins cause rheumatic fever Type specific proteins T protein (trypsin-resistant): useful epidemiologic marker Usefull when bacteria fail to express the M protein Structure unknown Epidemiologic classification based on identification of specific M and T types by agglutination with M- or T-specific antibodies Will be replaced by sequencing the emm gene that encodes the M protein Streptococcus pyogenes/physiology & structure Other cell surface components: components of the cell wall M-like proteins Lipoteichoic acid F protein Capsule Capsule An outer hyaluronic acid capsule (repeating molecules of glucuronic acid and Nacetylglucosamine) Antigenically indistinguishable from hyaluronic acid in mammalian connective tissues Functions: to prevent phagocytosis Encapsulated strains are responsible for severe systemic infections Streptococcus pyogenes/Pathogenesis&Immunity Virulence of S. pyogenes The ability of the bacteria to adhere to the surface of the host cells Invade into the epithelial cells Avoid opsonization & phagocytosis Produce a variety of toxins & enzymes Streptococcus pyogenes/Pathogenesis&Immunity Adherence to host cells is mediate by 10 different bacterial antigens The most important: lipoteichoic acid M proteins F protein Invasion into epithelial cells is mediate by M protein and F protein Streptococcus pyogenes/Pathogenesis&Immunity Pyrogenic exotoxins (Streptococcal pyrogenic exotoxin, Spes), originally called erythrogenic toxins Produced by lysogenic strains, similar to the toxin produced in Corynebacterium diphteriae Four immunologically distinct heat-labile toxins (SpeA, SpeB, SpeC and SpeF) Superantigens (IL-1, IL-2, IL-6, TNF-β/α Responsible for the streptococcal toxic shock syndrome Streptococcal pyrogenic exotoxin, (Spes) Act as superantigens, interacting with macrophages and helper T cells Secreted cytokines mediate important effects, including shock and the organ failure seen in patients with streptococcal toxic shock syndrome Responsible for the rash observed in scarlet fever Streptococcus pyogenes/Pathogenesis&Immunity Streptolysin S & O Streptolysine S Streptolysine O lyse erythrocytes leukocytes platelets responsible for β-hem. lyse erythrocytes leukocytes platelets antibodies are formed against ASO test Streptolysin S & O Streptolysin S: Oxygen-stable Nonimmunogenic Produced in the presence of serum Streptolysin O: Oxygen-labile Anti-ASO test useful for documenting recent group A streptococcal infection Antigenically related to toxins produced by:S.pneumoniae, C.tetani, C.perfringens Streptococcus pyogenes/Pathogenesis&Immunity Streptokinases Two forms: A & B Mediate the cleavage of plasminogen, releasing the protease plasmin that Cleaves fibrin and fibrinogen, resulting in the lysis of clots and fibrin deposits Lyse blood clots, fibrin deposits, facilitate the rapid spread of bacteria Anti-streptokinase antibodies: useful marker for infection Streptococcus pyogenes/Pathogenesis&Immunity Deoxyribonucleases Four immunologically distinct: DNAases A to D Not cytolytic Depolymerase free DNA in pus: reduce viscosity of the abscess, facilitate spread Developed antibodies: important markers of cutaneous infections Other enzymes C5a peptidase disrupts C5a (activate phagocyting cells) Hyaluronidase (spreading factor) diphosphopyridine nucleotidase (DPNase): role in pathogenesis is unknown Epidemiology Colonize the oropharynx of healthy children and young adults Carriage 15-20% Other streptococcal species (S.anginosus, S.intermedius, S.constelatus) can carry group-specific A antigen: do not cause pharyngitis Is transient (bacteriocins produced by other streptococci suppress the growth) Epidemiology Disease caused by recently acquired strains Primarily infect children between 5-15 years Spread from person to person through respiratory droplets Crowding, classrooms, daycare facilities Particularly during the winter months Epidemiology/ Soft-tissue infections Pyoderma, erysipelas, cellulitis are preceded by skin colonization after which the organisms are introduced into the tissues through a break in the skin Clinical diseases Suppurative streptococcal disease Nonsuppurative streptococcal disease Suppurative streptococcal disease Pharyngitis Pyoderma Erysipelas Cellulitis Necrotizing fasciitis Streptococcal toxic shock syndrome Other suppurative diseases bakteremia Pharyngitis Develops 2 to 4 days after exposure to the pathogen Sore throut, fever, malaise, and headache Posterior pharynx erythematous With exudate Cervical lymphadenopathy Scarlet fever Complication of pharyngitis Strain lysogenized by a temperate bacteriophage Stimulates production of a pyrogenic exotoxin Rash, Pastia’lines Strawberry tongue Pyoderma Purulent (“pyo“) infection of the skin (“derma“) Infects exposed areas (face, legs) Introduced into the subcutaneous tissue Through a break in the skin Erysipelas Erythros “red“; pella “skin“ Involved skin area is typically raised Young children and older adults Cellulitis Involves the skin and deeper subcutaneous tissues Distinction of infected skin is not as clear Local and systemic signs Necrotizing fasciitis Also called streptococcal gangrene Occurs deep in the tissue Extensive destruction of muscle and fat “flesh-eating bacteria“ Gangrene and systemic symptoms Strept.toxic shock syndrome Other M serotypes 1 or 3 Prominent hyaluronic acid capsules Pyrogenic exotoxins: SpeA and SpeC Multisystem toxicity Shock and organ failure (kidney, lungs, liver) Puerperal sepsis Pneumonia Bacteremia in patients with necrotizing fasciitis or toxic shock syndrome Mortality 40% Nonsuppurative streptococcal disease Rheumatic fever Acute glomerulonephritis Rheumatic fever Nonsuppurative complication Involvement of the heart valves Migratory arthritis Associated with streptococcal pharyngitis If antibiotic prophylaxis is not used Diagnosis is made on the basis of clinical findings and findings of a recent S.pyogenes infection: culture results Detection of group A Ag Elevation of ASO, antiDNase B, antihyaluronidase antibodies Acute glomerulonephritis Acute inflammation of the renal glomeruli Edema, hypertension, hematuria Specific nephritogenic strains are associated with this disease Streptococcus pyogenes/IDENTIFICATION Microscopy: gram +cocci in pairs and chains Antigen detection: use antibodies that react with the group-specific carbohydrate in the bacterial wall Enzyme immunassay (EIA) or bound to latex particles Sensitivity is low (90%): all negative results must be confirmed by culture Streptococcus pyogenes/IDENTIFICATION Culture: specimens must be obtained from the posterior oropharynx or skin lesions Nutritionally enriched agar media with sheep blood Selective media (media with “bactrim”) Prolonged incubation Identification Susceptibility to bacitracin L-pyrrolidonyl arylamidase (PYR): hydrolyzes L-pyrrolidonyl-β-naphtylamide releasing βnaphthylamide Antibody detection:antibodies against streptolysin O (the ASO test); appear 3-4 weeks after the exposure Anti-DNase B test if glomerulonephritis is suspected Treatment Penisilin Erythromycin or oral cephlosporin Newer macrolides Drainage and surgical debridement Rheumatic fever: Longterm antibiotic prophylaxis Streptococcus agalactiae (Group B) The only species that carries the group B antigen physiology & structure Gr + cocci Short or long chains (indistinguishable from S. pyogenes) Buttery colonies, narrow zone of β-hemolysis Subdividing The B antigen group specific Capsular polysaccharides type-specific C protein (surface protein) Streptococcus agalactiae/IDENTIFICATION Microscopy Culture: Readily grow on a nutritionally enriched medium Large colonies β-hemolysis may be absent selective broth medium with antibiotics Identification Preliminary identification (+) CAMP test, hydrolysis of hippurate Pathogenesis Genital colonization with group B streptococci has been associated with increased risk of premature delivery Serotypes Ia, III and V are most commonly associated with colonization and disease Colonize lower gastrointestinal tract and genitourinary tract 60% of infants born become colonized with their mothers’ organisms Early/Late –onset disease Septicemia and meningitis in newborns Early: infants younger than 7 days Late: 1 week-3 months In adults:urinary tract infections, wound infections, skin and soft-tissue infections Diagnosis Antigen detection: latex agglutination Culture: enriched medium Beta-hemolysis Nucleic acid-based tests: PCR Identification: CAMP test or hydrolysis of hippurate Treatment Penisilin: needs greater MIC Tolerance to penicilin has beem reported Combination of penicillin and aminoglycoside in serious infections or vancomycin in penicillin allergy Prevention: all pregnant women should be screened for colonization at 35-37 weeks of gestation Other beta-hemolytic streptococci Group C, F and G are most commonly associated with human disease 2 species of particular importance S. anginosus S. dysgalactiae Posses group A, C, F or G capsular polysaccaride Abscess formation, bacteremia Viridans Streptococci A heterogeneous colection of α-hemolytic and nonhemolytic streptococci Their name is derived from viridis (Latin for green) Produce a green pigment on blood agar media More than 20 species; are classified into five subgroups: Viridans Streptococci: subgroups Anginosus: S.anginosus, S.intermedius Mitis: S.mitis, S.pneumoniae, S.sanguis Mutans:S.mutans, S.sobrinus Salivarius: S.salivarius, S.vestibularis Bovis:S.bovis, S.equinus Ungrouped: S.suis Viridans Streptococci: culture Require complex media supplemented with blood products and an incubation atmosphere at 5-10 % CO2. Some strains are “nutritionally deficient streptococci“ : Can grow only in the presence of exogenously supplied pyridoxal (the active form of vitamin B6) These organisms can grow in blood cultures, but cannot grow when subcultured (unless pyridoxal supplemented media are used). Viridans Streptococci: colonization Colonize the oropharinx, gastrointestinal tract, genitourinary tract Rarely found on the skin surface, because fatty acids are toxic to them Viridans Streptococci: infections Associated with dental caries, subacute endocarditis, and suppurative intraabdominal infections Subacute bacterial endocarditis is associated with S.gordonii, S.mitis, S.mutans, S.oralis, S.sanguis Antibiotic resistance In the past they were highly susceptible to penicillin (MIC <0.1µg/ml) Moderately and highly resistant strains have become common Treatment: Penicillin+aminoglycoside Broad spectrum cephalosporin+vancomycin Streptococcus pneumoniae Isolated independently by Pasteur and Steinberg more than 100 years ago Is still a leading cause of morbidity and mortality Encapsulated, Gr + coccus Oval or lancet-shaped cells, in pairs or short chains α-hemolytic: production of pneumolysin , an enzyme that degrades hemoglobin producing a green product Streptococcus pneumoniae Can grow only on enriched media (with blood products) Catalase (-) Polysaccharide capsule is used for the serologic classification: 90 serotypes Capsular polysaccharides are used in vaccines Streptococcus pneumoniae: cell wall Peptidoglycan layer is typical of gram-positive cocci Teichoic acid, rich in galactosamine and choline C polysaccharide (CRP): precipitates a serum globulin fraction (C-reactive protein) F antigen: lipid-bound teichoic acid in the bacterial cytoplasmic membrane S. pneumoniae / diseases Pneumoniae Meningitis Sinusitis Otitis media Bacteremia Streptococcus pneumoniae/Pathogenesis&Immunity The disease manifestations are caused primarily by the host response to infection rather than the production of organism-specific toxic factors Streptococcus pneumoniae/Pathogenesis&Immunity Colonization and migration S.pn colonizes the oropharynx Can spread to the lungs, paranasal sinuses, middle ear, blood stream By means of: Surface protein adhesins secretory IgA (sIgA) protease Pneumolysin: a cytotoxin similar to the streptolysin O in S.pyogenes Streptococcus pneumoniae/Pathogenesis&Immunity Tissue destruction Mobilization of inflamatory cells characteristic of pneumococcal infections Teichoic acid and peptidoglycan fragments: activate alternative complement pathway producing C5a Pneumolysin: activates the classic complement pathway, resulting in prodution of C3a and C5a H2O2 production: lead to tissue damage Phosphorylcholin: binds phosphodiesteraseactivating factor, allowing bacteria to enter host cells Streptococcus pneumoniae/Pathogenesis&Immunity Phagocytic survival Survive phagocytosis because of the antiphagocytic protection afforded by its capsule Pneumolysin mediates suppression of the phagocytosis The virulence is a direct result of this capsule Epidemiology Common inhabitant of the throat and nasopharynx in healthy peaple A 5-75% incidence of such carriage has been reported Colonization more common in children than in adults Occurs at approximately 6 months of age Epidemiology Pneumococcal disease occurs when organisms colonizing the nasopharynx and oropharynx spread to distal locci: The lungs (pneumonia) Paranasal sinuses (sinusitis) Ears (otitis media) Meninges (meningitis) Bacteremia Epidemiology A common cause of bacterial pneumonia Meningitis Sinusitis Otitis media Bacteremia Streptococcus pneumoniae/IDENTIFICATION Microscopy Culture: Lancet-shaped, Gr (+) diplococci, unstained capsule (Gram stain with “quellung” reaction) Enriched supplemented medium with blood Selective medium with gentamicin Identification Bile solubility test Optochin (ethylhydrocupreine dihydrochloride) Quellung reaction Polyvalent anticapsular antibodies are mixed with the bacteria Streptococcus pneumoniae-Antibiotic resistance Resistance to multiple antibiotics, including penicillin was reported Mechanism: Decreased affinity of the antibiotic for the penicillin-binding proteins present in the bacterial cell wall Enterococcus The enterococci (“enteric cocci”) were previously classified as group D streptococci They possess the group D cell wall antigen (glycerol teichoic acid) Are distinct from other group D streptococci (nonenterococcal group D streptococci e.g., Streptococcus bovis) 1984: the new genus Enterococcus Enterococcus Most frequently isolated & most commonly responsible for human disease with streptococcus among gram-positive cocci 16 species in the genus E. faecalis & E. faecium are most commonly isolated Enterococcus/physiology & structure They can not be differentiated from S. pneumoniae in microscope Facultatively anaerobic Optimal growth temp.= 35 oC (10oC to 45oC) White, large colonies on blood agar (after 24h) Nonhemolytic (or α or β-hemolysis) Grow in the presence of 6.5 % NaCl, tolerate 40 % bile salts, hydrolyse esculin Enterococcus/Pathogenesis&Immunity Are commensal with limited potential for causing disease Do not possess toxins Cannot avoid being engulfed & killed by phagocytic cells BUT, Cause Serious Disease Enterococcus/Pathogenesis&Immunity Virulence factors Adhesive factors Secrete extracellular proteins (cytolysin) Bacteriocins Inherently resistant to many antibiotics Epidemiology Are enteric bacteria Recovered in feces collected from humans and animals E.g., E.faecalis 107 per gram feces E. faecium are less frequently E.gallinarum and casseliflavus: colonize the intestine in small numbers; resistant to vancomycin; can be confused with the more important species Enterococcus/Clinical Diseases Can cause life-threatening infections One of the most feared nosocomial pathogens 10% of all nos. infct. Most commonly involved sites Urinary tract Blood stream A sever complication: endocarditis( following bacteremia) Enterococcus/Laboratory diagnosis Grow readily on nonselective media Resemble S. pneum. Differentiation resistant to optochin don’t dissolve when exposed to bile hydrolyze PYR pyrolidonyl-β-naphthylamide Other Catalase-negative Gram positive cocci Leuconostoc (VaR) Pediococcus (VaR) Lactococcus Aerococcus (air coccus)
