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Semmelweis University Institute of

Staphylococci and Streptococci

Dr. Béla Kocsis 2014.10.14 Gram-positiv cocci Staphylococci and Streptococci

Katalase-reaction microscopic view

negative positive

Streptococcaceae Family Micrococcaceae Family

Nitrofurantoin Susceptibility

Positiv (susceptible) Negativ (Resistant)

Staphylococcus genus Micrococcus genus Gram-positiv cocci Staphylococci

Micrococcaceae Family test

Micrococcus genus: apathogen Staphylococcus genus: coagulase positiv: S. aureus coagulase negativ: S. epidermidis, S. haemolyticus, S. saprophiticus, S. hominis

1) Microscopic morphology:

Gram positive, 1 µm cocci arranged in grape-like clusters

Staphylococcus aureus

2) Cultivation • facultative anaerob • In bouillon: homogenous turbidity • : 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 Staphylococcus aureus

3) Biochemical feature + 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

Staphylococcus aureus

3) Biochemical feature

coagulase tube test latex-agglutination Staphylococcus aureus

4) Virulence factors Staphylococcus aureus 4) Virulence factors on the bacterial cell surface polysaccharide capsule slime layer (binds 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: exoenzymes exocoagulase, fibrinolysin Clot formation and lysis of fibrin

DNase, hyalurinidase, Invasivity in different tissues phosphatase, lipase Staphylococcus aureus

4) Virulence factors:

• Toxic Shock Syndrome (TSST-1)septic state , high fever, multi organ failer

• Staphylococcus (SE) leads to diarrhoeae and vomiting, toxico-

• exfoliative toxin  split the intercellular bridges in the stratum granulosum epidermis Staphylococcus aureus

4) Virulence factors: Superantigen

Superantigens bind to 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 Staphylococcus aureus

4) Virulence factors: cytotoxins

-haemolysin - haemolysin lysis of erythrocytes - haemolysin -haemolysin

Leukocidin lysis of leukocytes

Pore forming on the cell surface

Clinial pictures

Source of infection : 5-10 % of population carry S. aures in the nose, nasopharynx Way of transmission by respitory droplets or direkt contact

Purulent on the site of infection of the , furunculus, carbunculus, woundinfections , otitis media, mastoiditis, Invasive Infections pneumonia, bakteraemia, , , ostitis, osteomyelitis, endocarditis Toxin medaited infections Gastroenteritis, TSS, Pemph. neonat, Scales Skin Syndrom Local skin infections

Impetigo Folliculitis

Furuncle Carbuncle

Deep purulent infections

• Osteomyelitis • Mediastinitis • • Meningitis, Subduralempyema, • Abscesse formation in all parenchymal organ

Fig. 8.27 – . Erythema and swelling of the left ankle joint in a young girl with staphylococcal sepsis. By courtesy of Mr. N.St.J.P. Dwyer Septic arthritis

Arthrotomia,  after Gram staining Gram positive cocci in clusters

Therapy of Staphylococcus aures infections

Antibiotic treatment β-lactam antibiotics with β-lactamase Inhibitors eg.: amoxicillin + clavulanic acid

Therapy of Staphylococcus aures infections group of antibiotics (as all β-laktams) Inhibit the synthesis (cellwall synthesis) Target molecula PBP (Pencillin Binding Protein) a transpeptidase, responsible for the cellwall synthesis Peptidoglycan of : NAM: N-acetyl-muramin acid : NAG: N-acetyl-glukosamin

NAM Ala-Glu-Lys-D-Ala-D - Ala PBP β-lactam NAG antibiotics

NAM Ala-Glu-Lys-D-Ala- D-Ala

NAG beta-lactamase (penicillinase) production

NAM Ala-Glu-Lys-D-Ala-D-Ala Resistence to beta-lactams

• Penicillin-binding Proteine (PBP) – Struktur Beta-lactamase production modifing (penicillinase production) • Resistance to all beta-lactame antibiotics: • Resistence only to – Penicillin group – – Karbapenems – monobactams – Beta-laktamase Inhibitors

MRSA: Methicillin Resistant S. aureus Therefore the treatment: Therefore the treatment is based on antibiogramm: amoxicillin + clavulanic acid Antibiotic with different target molecules : Vancomycin Penicillin Penicillinase inhibitor Mupirocin derivative

MRSA = methicillin resistent 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 Peptidoglycan of Cell wall

NAM Ala-Glu-Lys-D-Ala-D-Ala Uneffective PBP’2a NAG β-lactam Antibiotics NAM Ala-Glu-Lys-D-Ala-D-Ala

NAG

NAM Ala-Glu-Lys-D-Ala-D-Ala MRSA: methicillin resistent S. aureus in Europe 2012

Less than 1%

More than 25% VRSA = vancomycin resistent 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

Peptidoglycan of Cell wall

NAM Ala-Glu-Lys-D-Ala-D-Ala Vancomycin

NAG

NAM Ala-Glu-Lys-D-Ala-D-Ala Vancomycin

NAG

NAM Ala-Glu-Lys-D-Ala-D-Ala Vancomycin Coagulase-negativ Staphylococci Coagulase-negativ Staphylococci

• Belong to the normalflora of the skin and mucosalayers

• Fakultative Pathogens

S. epidermidis S. hominis S. hemolyticus S. saprophyticus

Staphylococcus epidermidis

• Morphology: Gram-positve cocci in grape-like clusters • Cultivation: white pigment without

• Biochemical features – Katalase + – Koagulase - – Mannit - Staphylococcus epidermidis

• Belongs to the normalflora of the skin • On the intact skin  causes no infection

• On plastic instruments formation – exopolysaccharide – Matrixproteins (Fibrin, Fibrinogen)

Attachment , colonisation Bloodstream infection

Staphylococcus epidermidis

Therapy: plastic devices should 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 saprophyticus

1) Microscopic morphology: Gram-positive cocci grape-like clusters 2) Cultivation: no hemolysis on blood agar 3) Biochemical : coagulase negative, 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 Staphylococcus haemolyticus

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 Streptococci

Gram positive cocci : Streptococcus genus

Morphology: Gram positive cocci 1m in diameter arranged in chains Cultivation: demanding bacteria blood agar media (-, -, - haemolysis) 1 mm in diameter roundish, tiny needletip colonies Biochemical feature: catalase negative Classification of the Streptococcus genus

1. Haemolysis: a) - haemolysis: , 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  infections “A” group: S. pyogenes “B” group: S. agalactiae “D” group: faecalis

Classification of the Streptococcus 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

Streptococcus pyogenes

1) Microscopic morphology: Gram positive cocci 1m in diameter arranged in long chains

• capsule is composed of hyaluronic acid

Streptococcus pyogenes

2) Cultivation: demanding bacteria (vitamin B) blood agar media: -haemolysis 1 mm, circular, tiny needletip colonies

S. pyogenes on blood agar Streptococcus pyogenes

3) Biochemical features:

Antigen structure: Lancfield group “A”

according to M protein it is grouped in serotypes Streptococcus pyogenes 4) Virulance factors 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. S and O (haemolysin): anti-streptolysin O titer – confirming rheumatic fever!

IV. Exoenzymes: hyaluronidase (,,spreading factor”) DNase streptokinase (cleaves plasminogen to plasmin promoting fibronolysis

5) Clinical pictures by S. pyogenes

I. Purulent infections: II. Toxin mediated infections: III. Complications: mediated by Scarlate fever, Post-streptococcal S. pyogenes bacterium TSST diseases: typ2 and typ 3 hypersensitive reactions 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”)

Streptococcus pyogenes 5) Clinical pictures

Impetigo contagiosa Tonsillitis follicularis

Childbed fever (puerperal fever) by S. pyogenes

Ignaz Semmelweis demonstrated that childbed fever (puerperal fever), caused by streptococcal infections, Ignaz Semmelweis 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) Streptococcus pyogenes 5) Clinical pictures

Nekrotising fasciitis

Erysipel Streptococcus pyogenes 5) Clinical pictures

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 Scarlet fever Strawberry tongue: Exanthems papilla hyperthrophy on on the skin the tounge III. Poststreptococcal diseases (complications of 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 III. Poststreptococcal diseases (complications) Immunity: Antibacterial: you can have 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.

Streptococcus agalactiae 1) Microscopic morphology: Gram positive cocci 1m in diameter arranged in chains 2) Cultivation: blood agar media: -haemolysis (narrow) 1 mm in diameter circular, tiny needletip colonies diagnostic antibiotic: (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: Enterococcus genus

1) Microscopic morphology: Gram postive cocci (elongated) 1m in diameter arranged in short chains

Antigen structure: Group D Lancfield type

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 Enterococcus faecalis, Enterococcus faecium Clinical pictures: enteric cocci : present in the intestine (normal flora)  facultative pathogen of bile tract and urinary tract nosocomial infection after surgery Intestinal trauma /perforation  sepsis, peritonitis

Treatment: natural resistance and sulfonamid!

Th.: synergistic combination: ampicillin + gentamycin Th: vancomycin  increased level of resistance to glycopeptid :  VRE: vancomycin resistant Enterococci

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

Peptostreptococci

Anaerobic Streptococci! • Normal flora 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

Streptococcus pneumoniae

1) Microscopic morphology: Gram positive diplococci, ,,flame-shaped” or ,,lancet-shaped”

Streptococcus pneumoniae

Fig. 2.21 Pneumococcal pneumonia. Preparation of sputum showing predominance of pneumococci mostly as lanceolate diplococci. . By courtesy of Dr. J.R. Cantey 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 Streptococcus pneumoniae

• Can not be grouped with the Lancefield technique!

• based on capsule – 91 serotypes

• ,,quellung”-reaction (German ,,swelling”): anticapsular antibody plus pneumococci Quellung reaction of S. pneumoniae greater refractiveness around the bacteria by

Streptococcus pneumoniae

Source of infection: 5-10% of population carry S. pneumoniae in nose, throat

Clinical picture: lobar pneumonia sinusitis, otitis media bacteriaemia, meningitis ulcus serpens corneae (eye infection) Streptococcus pneumoniae

Treatment: high penicillin 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 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