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- 1. Staphylococci and Streptococci Dr.Béla Kocsis 2014.10.14 Semmelweis University Institute of Medical Microbiology
- 2. Gram-positiv cocci Staphylococci andStreptococci Katalase-reaction negative positive Micrococcaceae Family Nitrofurantoin Susceptibility Positiv (susceptible) Staphylococcus genus Negativ (Resistant) Micrococcus genus Streptococcus genus Streptococcaceae Family microscopic view
- 3. Gram-positiv cocci Staphylococci Micrococcaceae Family Micrococcusgenus: apathogen Staphylococcus genus: coagulase positiv: S. aureus coagulase negativ: S. epidermidis, S. haemolyticus, S. saprophiticus, S. hominis Coagulase test
- 4. Staphylococcus aureus 1) Microscopicmorphology: Gram positive, 1 µm cocci arranged in grape-like clusters
- 5. Staphylococcus aureus 2) Cultivation •facultative anaerob • In bouillon: homogenous turbidity • agar plate: 2-3 mm in diameter, circular, golden yellow colonies • pigment in non diffusable, fat soluble stains only the colonies • S. aureus AU referes to gold • On blood agar: -haemolysis • selective cultivation method: 7.5% NaCl
- 6. Staphylococcus aureus 3) Biochemicalfeature catalase + coagulase + Exocoagulase (free coagulase): • enzyme produced and released by the S. aureus, • binds to serum factor immunoglobulin, this complex can convert fibrinogen to fibrin • detecting: coagulase tube test Endocoagulase: „clumping factor” (bound coagulase): • on the bacterial surface, • direct convertion of fibrinogen to fibrin • detecting: slide agglutination, latex-agglutination
- 7. latex-agglutination Staphylococcus aureus 3) Biochemicalfeature coagulase tube test
- 8.
- 9. polysaccharide capsule slime layer(binds bacteria to catheters, grafts) teicholic acid, lipoteicholic acid (mediates the attachment of staphylococci to mucosal surfaces) adhesive proteins (collagene-, laminin-binding protein) clumping factor: endocoagulase mimikry by the fibrin layer macrophages can not reach them protein A (unique affinity for binding to the Fc fragment of immunoglobulin, prevents antibody-mediated immune clearance of S.aureus) Staphylococcus aureus 4) Virulence factors on the bacterial cell surface
- 10. exocoagulase, fibrinolysin DNase, hyalurinidase, phosphatase, lipase Staphylococcus aureus 4)Virulence factors: exoenzymes Clot formation and lysis of fibrin Invasivity in different tissues
- 11. Staphylococcus aureus • ToxicShock Syndrome Toxin (TSST-1)septic state , high fever, multi organ failer • Staphylococcus enterotoxin (SE) leads to diarrhoeae and vomiting, toxico-infection • exfoliative toxin split the intercellular bridges in the stratum granulosum epidermis 4) Virulence factors: exotoxins
- 12. Staphylococcus aureus Superantigens bindto T helper on the T cell receptor V β site leads to proliferation of T cells and overproduction of cytokins: TNF- β, IFN- γ, IL-2. The patients get into septic state : hypotension, shock, mulit-organ-failer 4) Virulence factors: Superantigen exotoxin
- 13. Staphylococcus aureus -haemolysin - haemolysin -haemolysin -haemolysin Leukocidin lysis of leukocytes Pore forming on the cell surface lysis of erythrocytes 4) Virulence factors: cytotoxins
- 14. Clinial pictures Purulent infectionson the site of infection of the skin folliculitis, furunculus, carbunculus, woundinfections , otitis media, mastoiditis, mastitis Invasive Infections pneumonia, bakteraemia, sepsis, meningitis, ostitis, osteomyelitis, endocarditis Toxin medaited infections Gastroenteritis, TSS, Pemph. neonat, Scales Skin Syndrom Source of infection : 5-10 % of population carry S. aures in the nose, nasopharynx Way of transmission by respitory droplets or direkt contact
- 15.
- 23. • Osteomyelitis • Mediastinitis •Peritonitis • Meningitis, Subduralempyema, • Abscesse formation in all parenchymal organ Deep purulent infections
- 26. Fig. 8.27 –Septic arthritis. Erythema and swelling of the left ankle joint in a young girl with staphylococcal sepsis. By courtesy of Mr. N.St.J.P. Dwyer
- 27. Septic arthritis Arthrotomia, pus after Gram staining Gram positive cocci in clusters
- 34. Therapy of Staphylococcusaures infections Antibiotic treatment β-lactam antibiotics with β-lactamase Inhibitors eg.: amoxicillin + clavulanic acid
- 35. Therapy of Staphylococcusaures infections Penicillin group of antibiotics (as all β-laktams) Inhibit the peptidoglycan synthesis (cellwall synthesis) Target molecula PBP (Pencillin Binding Protein) a transpeptidase, responsible for the cellwall synthesis β-lactam antibiotics Peptidoglycan of Cell wall : NAM: N-acetyl-muramin acid : NAG: N-acetyl-glukosamin NAM NAG NAM NAG NAM PBP Ala-Glu-Lys-D-Ala-D-Ala Ala-Glu-Lys-D-Ala-D-Ala Ala-Glu-Lys-D-Ala-D-Ala beta-lactamase (penicillinase) production
- 36. Resistence to beta-lactams Beta-lactamaseproduction (penicillinase production) • Resistence only to Penicillin group • Penicillin-binding Proteine (PBP) – Struktur modifing • Resistance to all beta-lactame antibiotics: – Penicillins – Cephalosporins – Karbapenems – monobactams – Beta-laktamase Inhibitors MRSA: Methicillin Resistant S. aureus Therefore the treatment: amoxicillin + clavulanic acid Penicillinase inhibitor Penicillin derivative Antibiotic with different target molecules : Vancomycin Linezolid Mupirocin Clindamycin Daptomycin Therefore the treatment is based on antibiogramm:
- 37. MRSA = methicillinresistent S. aureus Methicillin belongs Penicillin group of antibiotics (β-laktam) Inhibtion of Peptidoglycan synthesis Target molecula PBP (Pencillin Binding Protein) Target molecule mutation PBP’2a modified target conferes resistance to all β-lactam antibiotics Ala-Glu-Lys-D-Ala-D-Ala Ala-Glu-Lys-D-Ala-D-Ala Ala-Glu-Lys-D-Ala-D-Ala Uneffective β-lactam Antibiotics Peptidoglycan of Cell wall NAM NAG NAM NAG NAM PBP’2a
- 38. MRSA: methicillin resistentS. aureus in Europe 2012 Less than 1% More than 25%
- 39. VRSA = vancomycinresistent S. aureus Vancomycin effective agent against MRSA Inhibition of Cell wall synthesis Targetmolecule is D-Alanin in Murein Targer modification (D-Ala-lactate) leads to resistance VRSA Ala-Glu-Lys-D-Ala-D-Ala Ala-Glu-Lys-D-Ala-D-Ala Ala-Glu-Lys-D-Ala-D-Ala Vancomycin Vancomycin Vancomycin Peptidoglycan of Cell wall NAM NAG NAM NAG NAM
- 40.
- 41. Coagulase-negativ Staphylococci • Belongto the normalflora of the skin and mucosalayers • Fakultative Pathogens S. epidermidis S. hominis S. hemolyticus S. saprophyticus
- 42. Staphylococcus epidermidis • Morphology:Gram-positve cocci in grape-like clusters • Cultivation: white pigment without hemolysis • Biochemical features – Katalase + – Koagulase - – Mannit -
- 43. Staphylococcus epidermidis • Belongsto the normalflora of the skin • On the intact skin causes no infection • On plastic instruments biofilm formation – exopolysaccharide – Matrixproteins (Fibrin, Fibrinogen) Attachment , colonisation Bloodstream infection
- 44. Therapy: plastic devicesshould be removed Therapy based on antibiogram S. epidermidis resistance to beta-lactams: MRSE : methicillin resistant S. epidermidis Resistance to other group of antibiotics too: vancmomycin, linezolid Staphylococcus epidermidis
- 45. Staphylococcus saprophyticus 1) Microscopicmorphology: Gram-positive cocci grape-like clusters 2) Cultivation: no hemolysis on blood agar 3) Biochemical : coagulase negative, novobiocin resistent, urease positive! Clinical features: Belongs to the skin normalflora mainly on the genitals cystitist („honeymoon cystitis”) in young sexualle active women S. saprophyticus can bind to the uroepithel and by the urease activity NH3 will irritate the mucosalayer
- 46. Staphylococcus haemolyticus Staphylococcus hominis 1)Microscopic morfology: Gram-positiv cocci grape-like clusters 2) cultivation: white colonies weak or no hemolysis 3) Biochemical features : novobiocin susceptible Belong to the normal flora of the skin : Nosocomial pathogen biofilm production on catheter, canuls, plastic devices, tubes of intubation Mucus layer damages help the invasion to the bloodstream bacteraemia and sepsis
- 47.
- 48. Gram positive cocci: Streptococcus genus Morphology: Gram positive cocci 1m in diameter arranged in chains Cultivation: demanding bacteria blood agar media (-, -, - haemolysis) 1 mm in diameter roundish, tiny needletip colonies Biochemical feature: catalase negative
- 49. Classification of theStreptococcus genus 1. Haemolysis: a) - haemolysis: Streptococcus pyogenes, S. agalactiae b) -haemolysis: S. pneumoniae c) non haemolytic: S. lactis, Enterococci 2.Lancefield grouping: according to the polysaccharide “C” in the cell wall serogroups: A, B, C, D, F, G human infections “A” group: S. pyogenes “B” group: S. agalactiae “D” group: Enterococcus faecalis
- 50. Classification of theStreptococcus genus 3. “M” protein in the cell wall: serotypes • S. pyogenes > 90 serotypes • in certain diseases different serotypes are characteristic: • e.g.: serotype 10 – scarlet fever; • serotype 2, 4, 12, 49 – acut glomerulonephritis • (nephritogen strains) 4. 16 S rRNA sequence coding DNA sequence: • 6 clusters: anginosus, pyogen, mitis, salivarius, bovis, mutans
- 51. Streptococcus pyogenes 1) Microscopicmorphology: Gram positive cocci 1m in diameter arranged in long chains • capsule is composed of hyaluronic acid
- 52. Streptococcus pyogenes 2) Cultivation:demanding bacteria (vitamin B) blood agar media: -haemolysis 1 mm, circular, tiny needletip colonies S. pyogenes on blood agar
- 53. Streptococcus pyogenes 3) Biochemicalfeatures: Antigen structure: Lancfield group “A” according to M protein it is grouped in serotypes
- 54. Streptococcus pyogenes 4) Virulancefactors I. On the cell surface: lipoteicholic acid, F-protein, capsule II. Exotoxin: erythrogenic toxin – scarlet fever (capillar toxin) Spe A, B, C, F – streptococcal pyrogenic exotoxin III. Streptolysin S and O (haemolysin): anti-streptolysin O titer – confirming rheumatic fever! IV. Exoenzymes: hyaluronidase (,,spreading factor”) DNase streptokinase (cleaves plasminogen to plasmin promoting fibronolysis
- 55. 5) Clinical picturesby S. pyogenes I. Purulent infections: mediated by S. pyogenes bacterium II. Toxin mediated infections: Scarlate fever, TSST III. Complications: Post-streptococcal diseases: typ2 and typ 3 hypersensitive reactions
- 56. 5) Clinical pictures I.Pyogenic infections (mediated by the bacterium) • pharyngitis, • tonsillitis follicularis, • otitis media, sinusitis • meningitis, • pneumonia, endocarditis • puerperal fever (Semmelweis) • Impetigo, • erysipel • myositis • necrotising fasciitis (“fleish-eating bacterium”)
- 57. Streptococcus pyogenes 5) Clinicalpictures Tonsillitis follicularis Impetigo contagiosa
- 61. Ignaz Semmelweis Ignaz Semmelweisdemonstrated that childbed fever (puerperal fever), caused by streptococcal infections, was transmitted to patients by doctor’s hands Pioneer of antisepsis in obstetrics Women giving birth in hospitals by medical students and physicians were 4x more likely to contract puerperal fever compared to those by midwives Handwashing with chlorin water (leach powder) Childbed fever (puerperal fever) by S. pyogenes
- 62. Streptococcus pyogenes 5) Clinicalpictures Nekrotising fasciitis Erysipel
- 63. Streptococcus pyogenes 5) Clinicalpictures II. Toxin mediated diseses • Scarlet fever: mediated by erythrogen toxin, which can destroy the endothel cell of capillaries – red rash • Can not be formed into toxoid! NO vaccination • 2 days after the infection exanthems on the skin and throat
- 64. Scarlet fever Strawberry tongue: papillahyperthrophy on the tounge Exanthems on the skin
- 65. III. Poststreptococcal diseases (complicationsof a S. pyogenes infection) 1.Rheumatic fever: Typ 2 hypersensitive reaction: surface anigen of the heart muscle is similar to the Str. pyogenes antigen(M-protein) antibodies bound to the heart muscle • inflammatory changes in the heart (pancarditis) • endocarditis: damage of heart valves 2. Acute glomerulonephritis (GN): immuncomplex mediated Immunkomplexes in joints: polyarthritis Immunkomplexes in the glomerulus : nephritis • Typ 3 hypersensitive reaction: immuncomplexes bind to the glomerulus basalmembrane glomerulonephritis • Hypertonia and oedema 3. Erythema nodosum: • subcutan nodles, immuncomplex mediated
- 66.
- 67. Immunity: Antibacterial: you canhave tonsillitis follicularis more than once (several serotypes) Antitoxical: you acquire scarlet fever only once (erythrogenic toxin has the same structure in all the strains) Treatment: penicillin (natural susceptible to penicillin), • macrolid (if the patient has penicillin allergy) • complications should be prevented.
- 68. 1) Microscopic morphology:Gram positive cocci 1m in diameter arranged in chains 2) Cultivation: blood agar media: -haemolysis (narrow) 1 mm in diameter circular, tiny needletip colonies diagnostic antibiotic: bacitracin (R) CAMP + 3) Antigen structure: Lancfield group “B” 4) Pathogenicy: colonisation in the vagina 5) Clinical pictures: during pregnancy: abortion during delivery the neonates can be infected: newborn pneumonia, ARDS, meningitis, sepsis (Screening of pregnant women after the 35th week of gestation!) • Treatment and prevention: ampicillin Streptococcus agalactiae
- 69. Enterococcus genus 1) Microscopicmorphology: Gram postive cocci (elongated) 1m in diameter arranged in short chains Antigen structure: Group D Lancfield type
- 70. Enterococcus faecalis, Enterococcus faecium 2)Cultivation: on blood agar greyish colonies • (sometimes green court under the colony) • selective culture media – black colonies (E67 culture media) 3) Biochemical feature: esculin (polysaccharid) hydrolysis
- 71. Enterococcus faecalis, Enterococcus faecium Clinicalpictures: enteric cocci : present in the intestine (normal flora) facultative pathogen inflammation of bile tract and urinary tract nosocomial infection after surgery Intestinal trauma /perforation sepsis, peritonitis Treatment: natural resistance cephalosporin and sulfonamid! Th.: synergistic combination: ampicillin + gentamycin Th: vancomycin increased level of resistance to glycopeptid : VRE: vancomycin resistant Enterococci
- 72. Streptococcus viridans group (S.mutans, S. mitis, S. sanguis, S. salivarius, S. milleri) heterogenous collection of - haemolytic Streptococci ,,viridae” – Latin term for green Member of the normal flora of the oral cavity. • Cultivation on blood and chocolate agar: - haemolysis • Separate from S. pneumoniae S: normal flora optochin R • clinical picture: In oral cavity: colonisation on the teeth dental plaque formation dental caries • If Streptococcus viridans enter the circulation cause subacute endocarditis
- 73. Peptostreptococci Anaerobic Streptococci! • Normalflora of the oral cavity, gastrointestinal tract. • Polymicrobic, pyogenic infections, abscess formation in the abdominal cavity, lung and brain or in the oral cavity • Treatment: metronidazol, clindamycin Abscess
- 74. Streptococcus pneumoniae 1) Microscopic morphology: Grampositive diplococci, ,,flame-shaped” or ,,lancet-shaped”
- 75. Fig. 2.21 Pneumococcalpneumonia. Preparation of sputum showing predominance of pneumococci mostly as lanceolate diplococci. Gram stain. By courtesy of Dr. J.R. Cantey Streptococcus pneumoniae
- 76. Streptococcus pneumoniae 2) Cultivation: •blood and chocolate agar - haemolysis • autolysis: ageing colonies are umbilicated 3) Biochemical features optochin sensitivity (S) separate from the viridans group 4) Virulenc factor Polysaccharide capsule
- 77. Streptococcus pneumoniae • Cannot be grouped with the Lancefield technique! • based on capsule – 91 serotypes • ,,quellung”-reaction (German ,,swelling”): anticapsular antibody plus pneumococci greater refractiveness around the bacteria by microscope Quellung reaction of S. pneumoniae
- 78. Streptococcus pneumoniae Clinical picture: lobarpneumonia sinusitis, otitis media bacteriaemia, meningitis ulcus serpens corneae (eye infection) Source of infection: 5-10% of population carry S. pneumoniae in nose, throat
- 79. Streptococcus pneumoniae Treatment: highpenicillin resistance decreased affinity of the antibiotic to PBP Therapy: macrolid, fluoroquinolones Prevention: 13-valent polysaccharide vaccine (conjugated) obligatory vaccine for new borns ( in Hungary since 2014 July) recommended: 1. children (born before 2014 July) 2. adults above 65 years of age 3. adults with chronic disease (COPD, heart failure) 4. patient after splenectomy
- 80. Vaccinations against bacterial infections 1)BCG = against Mycobacterium tuberculosis (living attenuated bacterium) 2) aP = against Bordetella pertussis (acellulare Pertusis vaccination) 3) Diphtheria = against Corynebacterium diphtheriae (toxoid) 4) Tetanus = against Clostridium tetani (toxoid) 5) Hib = against Haemophilus influenzae b capsule antigen 6) Neisseria meningitidis capsule antigen 7) Streptococcus pneumoniae capsule anigen 8) Salmonella typhi killed bacteria (polysaccharide derivative) 9) Vibrio cholerae killed bacteria