Download Staphylococci and Streptococci

Semmelweis University
Institute of Medical Microbiology
Staphylococci and Streptococci
Dr. Béla Kocsis
2014.10.14
Gram-positiv cocci
Staphylococci and Streptococci
Katalase-reaction
microscopic view
positive
negative
Streptococcaceae Family
Micrococcaceae Family
Nitrofurantoin Susceptibility
Streptococcus genus
Positiv (susceptible)
Negativ (Resistant)
Staphylococcus genus
Micrococcus genus
Gram-positiv cocci
Staphylococci
Micrococcaceae Family
Micrococcus genus:
apathogen
Coagulase test
Staphylococcus genus:
coagulase positiv: S. aureus
coagulase negativ:
S. epidermidis, S. haemolyticus,
S. saprophiticus, S. hominis
Staphylococcus aureus
1) Microscopic morphology:
Gram positive, 1 µm cocci
arranged in grape-like clusters
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
Staphylococcus aureus
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
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 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: exoenzymes
exocoagulase, fibrinolysin
Clot formation and lysis of fibrin
DNase,
hyalurinidase,
phosphatase,
lipase
Invasivity in different tissues
Staphylococcus aureus
4) Virulence factors: exotoxins
• 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
Staphylococcus aureus
4) Virulence factors: Superantigen exotoxin
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
- haemolysin
-haemolysin
Leukocidin
lysis of erythrocytes
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 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
Local skin infections
Impetigo
Folliculitis
Furuncle
Carbuncle
Deep purulent infections
•
•
•
•
•
Osteomyelitis
Mediastinitis
Peritonitis
Meningitis, Subduralempyema,
Abscesse formation in all parenchymal organ
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
Peptidoglycan of Cell wall : NAM: N-acetyl-muramin acid
: NAG: N-acetyl-glukosamin
NAM
NAG
Ala-Glu-Lys-D-Ala-D-Ala
PBP
β-lactam
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
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
Penicillin
derivative
Penicillinase inhibitor
Therefore the treatment is based on
antibiogramm:
Antibiotic with different target
molecules :
Vancomycin
Linezolid
Mupirocin
Clindamycin
Daptomycin
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
NAG
NAM
Ala-Glu-Lys-D-Ala-D-Ala
PBP’2a
Ala-Glu-Lys-D-Ala-D-Ala
NAG
NAM
Ala-Glu-Lys-D-Ala-D-Ala
Uneffective
β-lactam
Antibiotics
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
Ala-Glu-Lys-D-Ala-D-Ala
Vancomycin
Ala-Glu-Lys-D-Ala-D-Ala
Vancomycin
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 -
Staphylococcus epidermidis
• 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
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, 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 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: 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 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. 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
Impetigo contagiosa
Tonsillitis follicularis
Childbed fever (puerperal fever)
by S. pyogenes
Ignaz
Ignaz Semmelweis
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)
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:
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.
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: 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
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
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”
Streptococcus pneumoniae
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
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
greater refractiveness
around the bacteria by
microscope
S. pneumoniae
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