staphylococci and streptococci - semmelweis...

Staphylococci and Streptococci

Dr. Béla Kocsis

2014.10.14

Semmelweis University Institute of Medical Microbiology

Gram-positiv cocci Staphylococci and Streptococci

Katalase-reaction

negative positive

Micrococcaceae Family

Nitrofurantoin Susceptibility

Positiv (susceptible)

Staphylococcus genus

Negativ (Resistant) Micrococcus genus

Streptococcus genus

Streptococcaceae Family

microscopic view

Gram-positiv cocci Staphylococci

Micrococcaceae Family

Micrococcus genus: apathogen Staphylococcus genus:

coagulase positiv: S. aureus

coagulase negativ: S. epidermidis, S. haemolyticus, S. saprophiticus, S. hominis

Coagulase test

Staphylococcus aureus

1) Microscopic morphology: Gram positive, 1 µm cocci arranged in grape-like clusters


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


3) Biochemical feature 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

latex-agglutination


3) Biochemical feature

coagulase tube test


4) Virulence factors

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)


4) Virulence factors on the bacterial cell surface

exocoagulase, fibrinolysin

DNase,

hyalurinidase,

phosphatase,

lipase


4) Virulence factors: exoenzymes

Clot formation and lysis of fibrin

Invasivity in different tissues


• Toxic Shock 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


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

4) Virulence factors: Superantigen exotoxin


-haemolysin

- haemolysin

- haemolysin

-haemolysin

Leukocidin lysis of leukocytes

Pore forming on the cell surface

lysis of erythrocytes

4) Virulence factors: cytotoxins

Clinial pictures

Purulent infections on 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

Impetigo

Folliculitis

Furuncle Carbuncle

Local skin infections

• Osteomyelitis

• Mediastinitis

• Peritonitis

• Meningitis, Subduralempyema,

• Abscesse formation in all parenchymal organ

Deep purulent infections

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

Septic arthritis

Arthrotomia, pus 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

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



beta-lactamase (penicillinase) production

Resistence to beta-lactams

Beta-lactamase production

(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:

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

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

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

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

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 hemolysis

• Biochemical features

– Katalase +

– Koagulase -

– Mannit -


• Belongs to 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

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, 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

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

Streptococci

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

Classification of the Streptococcus 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

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 1m in diameter arranged in long chains

• capsule is composed of hyaluronic acid


2) Cultivation: demanding bacteria (vitamin B)

blood agar media:

-haemolysis 1 mm, circular, tiny needletip colonies

S. pyogenes on blood agar


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. 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

5) Clinical pictures by 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

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

Tonsillitis follicularis Impetigo contagiosa

Ignaz Semmelweis

Ignaz Semmelweis demonstrated 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


Nekrotising fasciitis

Erysipel


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: papilla hyperthrophy on the tounge

Exanthems on the skin

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.

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

Enterococcus genus

1) Microscopic morphology: Gram

postive cocci (elongated) 1m 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

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

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”

Fig. 2.21 Pneumococcal pneumonia. Preparation of sputum showing

predominance of pneumococci mostly as lanceolate diplococci. Gram

stain. By courtesy of Dr. J.R. Cantey



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


• Can not 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


Clinical picture:

lobar pneumonia

sinusitis, otitis media bacteriaemia, meningitis

ulcus serpens corneae (eye infection)

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


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

staphylococci and streptococci - semmelweis...

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