RÉSZLETES BAKTERIOLÓGIA - Isemmelweis.hu/mikrobiologia/files/2014/10/FoD_04a.pdf ·...
Transcript of RÉSZLETES BAKTERIOLÓGIA - Isemmelweis.hu/mikrobiologia/files/2014/10/FoD_04a.pdf ·...
DETAILED
BACTERIOLOGY
Classification of bacteria
• Gram positive / Gram negative *
• Shape: coccus, rod (even or curved),
coccobacillus, spirochets
• Spore forming / non spore forming
• Aerobic / anaerobic
• Intracellular (maybe facultatively IC)
* Difficult to stain by Gram:
– Mycobacterium, Actinomyces (unevenly), Mycoplasma
(no cell wall!)
Taxonomy - I• Gram + coccus: Staphylococcus, Streptococcus
• Gram – coccus: Neisseria, Moraxella, Kingella
• Gram + rod:
spore froming: Bacillus, Clostridium
non spore forming: Corynebacterium, Listeria,
Erysipelothrix, Gardnerella
((Mycobacterium))
• Gram – rod:
enteral bacteria: E. coli, Salmonella, Shigella, Klebsiella,
Enterobacter, Serratia, Proteus, Morganella, Providencia,
Citrobacter, Yersinia
other fermenters: Vibrio, Aeromonas, Pesteurella
non fermenters: Pseudomonas, Acinetobacter, Steno-
trophomonas, Burkholderia
Taxonomy - II• Gram- coccobacillus: Haemophilus, Brucella, Francisella,
Bordetella, Legionella
• Intracellular (Gram - coccobacillus): Chlamydia (different cell wall); Mycoplasma, Ureaplasma (no cell wall!); Rickettsia, Coxiella, Ehrlichia, Bartonella
• Spiral: Helicobacter, Campylobacter;
Treponema, Borrelia, Leptospira
• Anaerobic:
Gram + rod: Clostridium (spore forming), Actinomyces, Mobiluncus, Lactobacillus (non spore froming)
Gram + coccus: Peptococcus, Peptostreptococcus
Gram – rod: Bacteroides, Fusobacterium, Prevotella, Porphyromonas
Gram – coccus: Veilonella
PYOGENIC COCCI
Gram + Gram –
Micrococcaceae family Streptococcaceae family Neisseriaceae family
Micrococcus Streptococcus Neisseria
Moraxella
S. pyogenes (A) Kingella
Staphylococcus S. agalactiae (B)
S. pneumoniae N. meningitidis
Enterococcus (D) N. gonorrhoeae
viridant group
S. aureus coagulase negative
staphylococci
(CNS)
Gram-positive cocci
Staphylococci,
Streptococci
Staphylococci
Staphylococcus aureus Coagulase-negative
staphylococci (CNS)
Staphylococcus epidermidis
St. saprophyticus
St. haemolyticus
St. hominis
St. lugdunensis
etc. (~40 species)
Gram staining of all species
• Most are harmless and reside
normally on the skin and
mucous membranes of
humans and other organisms
Cultivation
• very easy to grow
• facultative anaerobic
• big, round, slimy colonies
• resistant to
• heat (50 oC 30 min),
• drying,
• salt (9% NaCl)
• survive in hospitals !
Pigment production
Staphylococcus epidermidis:
white pigment
Staphylococcus aureus:
golden pigment
(staphyloxanthin)
-hemolysis
• S. aureus (+ S. haemolyticus + S. lugdunensis):
strong -hemolysis
Catalase test
• Aim: to differentiate Staphylococci
and Streptococci
• performance: adding a few drops of
H2O2 to the culture, strong bubbling
if positive
• H2O2 H2O + O2
Catalase +: Staphylococci
Catalase -: Streptococci
S. aureus virulence factors
I. cell-bound factors
• Polysaccharide capsule
• Protein A
– acts as an Fc receptor binding Fc portion of IgG
– inhibition pf phagocytosis
– biofilm formation!
• Clumping factor
– masking fibrin coat
• Teichoic acid (CWTA)
Protein A
Coagulase test -I
• Tube coagulase test: inoculating the bacteria
into citrate treated rabbit plasma, coagulation
occurs in a few hours (exocoagulase)
Coagulase +: Staphylococcus aureus
Coagulase -: all other Staphylococci (“CNS”)
-+
Coagulase test -II
• Clumping test - fibrinogen-coated particles
endocoagulase binds fibrinogen
(“Staphaurex”, „Pastorex”)
• performance: mixing bacteria with fibrinogen-bound
latex particles on microscope slide clumping of
bacteria (in a few seconds)
S. aureus virulence factors
II. Enzymes for invasion and spread
• Hyaluronidase
– breaks down proteoglycans in connective tissue
• Fibrinolysin (=staphylokinase)
– lyses fibrin clots (similar to streptokinase)
• Lipase
– degradation of fats and oils (facilitation of colonization of sebaceous glands)
• Nuclease (DNAse)
• Hemolysins (alpha, beta, gamma, delta)
– they are hemolytic toxins that destroy red blood cells, neutrophils, macrophages, and platelets
• Leukocidins
– destroy leukocytes (is the cause of necrotic lesions involving the skin or mucosa, including necrotic hemorrhagic pneumonia)
– Panton-Valentine leukocidin (PVL)
• present in all CA-MRSA
• no hemolytic activity
S. aureus virulence factors
III. Cytotoxins
S. aureus virulence factors IV. Exotoxins
• TSST-1 (toxic shock syndrome toxin)
– Superantigen!
– often associated with tampon use
– localised infection (vagina or wound), but toxin can
go through mucosa and can lead to MOF
– characteristic rash (sunburn like) usually seen at
onset
S. aureus virulence factors
IV. Exotoxins
• Exfoliative toxin
– Superantigen!
– SSSS = staphylococcal scalded skin syndrome (Ritter disease)
– protease activity of the exfoliative toxins causes peeling of the skin
– no inflammation (important in diagnosis!)
– mainly in small children
S. aureus virulence factors
IV. Exotoxins
• Enterotoxins (A-F)
– Superantigens!
– heat stable exotoxins which cause food
poisoning, resulting in nausea, vomiting and
diarrhoea
– resistant to cooking (100 oC, 30 min)
– C, D: dairy products
impetigo
furuncle
Staphylococcus aureus skin infections
• Folliculitis
– If on eye: hordeolum
• Furuncle
• Carbuncle
– bacteremia, fever
• Impetigo
folliculitis
hordeolum
Staphylococcus aureus skin infections
• Cellulitis
– infection of the deeper layers of skin and the tissues beneath
• Bullous impetigo
– Localised form of SSSS
Bullous impetigo
• Wound infections
– burns, postoperational, trauma
– from the skin flora
Staphylococcus aureus skin infections
Invasive infections
• Osteomyelitis– hematogenous spread or superinfection of trauma
• Endocarditis– following heart surgery (esp. valve replacement)
– 50% letality
• Pneumonia– predominantly affects people with underlying lung
disease including those on mechanical ventillation
• Meningitis– accounts for 1-9% of cases of bacterial meningitis
• Sepsis– half of all cases of inpatient sepsis
– in immunocompromised patients
Antibiotic therapy
• Skin infections: local treatment
• High resistance rates !!
• Antibiogram must be prepared
• Resistance to penicillin G: ~ 90%
• Methicillin resistance (MRSA):
– varies from country to country
– resistance to all beta-lactams & others
MRSA• Treatment of MRSA infections:
– Vancomycin !
– Others: linezolid, TMP-SMX, clindamycin, tertacycline, quinupristin/dalfopristin (Synercid), aminoglycosides
MRSA rates
MRSA % of invasive isolates in Hungary:
(data of the National Center for Epidemiology)
2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013
4.7 9.0 14.9 16.7 19.9 25.1 23.6 23.0 28.6 30.1 27.0 25.1 24.0
Staphylococcus aureus carriage
• frequently found in normal flora of skin and nasal mucosa
• Carriage rates:– ~20% are long term carriers
– ~30% intermittently
– higher rates among • hospital staff and patients
• ekzema patients
• farmers
• where:– nasal and throat mucosa
– perinasal skin, hands
– perianal skin
Carrying MRSA
• Prevention of spread:
– Hand hygiene!
– Environmental cleaning / disinfection
• iodine, hexachlorophene
– Personal protective equipments
– Isolation of patients
– Regular screening of medical staff
• Decolonisation:
– Intranasal mupirocin
– Resistance is developing!
(Hungary, 2012: 4,5%)
Staphylococcus epidermidis
• Normal flora of the skin, facultative
(opportunistic) pathogen
• Mainly nosocomial infections!! Related to foreign
body:
– Prosthetic joints
– Prosthetic heart valves
– Sepsis from i.v. devices
– UTIs related to catheters
• Frequent skin contaminant in hemocultures!
• Th: vancomycin
Removal of an infected
prosthetic joint shows a
bacterial biofilm on an
infected arthroplasty.
A prosthetic joint with the
presence of antibiotic beads
TO PREVENT INFECTION
Other coagulase negative staphylococci
• S. saprophyticus:
– second frequent in UTIs after E. coli, esp. in young
women
– differentiation from S. epid: novobiocin resistance
– Th: ciprofloxacin
• S. hominis
– Skin flora; opportunistic pathogen
• S. haemolyticus
– Skin flora; opp. pathogen; inserted devices
• S. lugdunensis
– agressive endocarditis
S. epidermidis (S) and S. saprophyticus (R)
with novobiocin disc
Streptococci
Streptococcus pyogenes
Gram staining
Streptococcus pyogenes
Streptococci
• Streptococcus pyogenes
• Streptococcus agalactiae
• Streptococcus pneumoniae
• viridant streptococci
• Enterococcus genus
Grouping of streptococci
• Lancfield grouping (cell wall polysaccharide):
– S. pyogenes = A (GAS)
– S. agalactiae = B (GBS)
– Enterococci = D
• Based on hemolysis:
– β-hemolytic: S. pyogenes, S. agalactiae
– α-hemolytic: S. pneumoniae, viridant streptococci
Streptococcus pyogenes
• Gram-positive coccus,
arranged in chains
• smal, pin-point colonies
• fastidious!
• strong β-hemolysis
S. pyogenes infections
I. Respiratory tract
• pharyngitis („Strep throat”)
• tonsillitis follicularis
II. Skin infections
Erysipelas
severe impetigo
• erysipelas
• phlegmone
• impetigo
• cellulitis
II. Skin infections - 2
Flame-like spread
necrotising fasciitis
“flesh eating bacterium”
Other severe, acute,
pyogenic infections
• otitis media, sinusitis
• meningitis
• puerperal fever
Scarlat fever
Raspberry tongue
Peeling after scarlat fever
• rush oll over the body
• toxin of S. pyogenes in the throat
Post-streptococcal diseases
• rheumatic fever
• glomerulonephritis
• rheumatoid arthritis
ASO-titer!
• allergic origin
• a few weeks after the
acute infection
Treatment and prevention
• penicillin !!
• no vaccination
• scarlat fever chemoprophylaxis: penicillin
Streptococcus agalactiae
• Formerly only in animals (bovine mastitis)
• Since 1970’s: neonatal sepsis, meningitis No. 1
• In newborns:
– early manifestation (EOD)
• within 6 days
• risk groups (e.g. low weight)
• meningits, sepsis, pneumonia
– later manifestation (LOD)
• within 4 months
• meningitis, bacteremia, osteoarthritis
Str. agalactiae adult infections
• In pregnant women:
– 20-35% asymptomatic vaginal carriage
– newborns aquire in 50-75%
– screening in 35-37. week of pregnancy
– chemoprophylaxis: i.v. ampicillin during birth
• In non-pregnant adults: mainly >60 y
– skin- and bone infections, bacteremia, urosepsis, pneumonia
CAMP test
CAMP +: Streptococcus agalactiae
Str. agalactiae
St. aureus
• aim: Str. agalactiae diagnostics
• performance: Str. agalactiae and St. aureus
enhance each other’s β–haemolysis if
inoculated in a form of crossing lines
bacitracin sensitivity:
S. pyogenes: S
S. agalactiae: R
Streptococcus pneumoniae„pneumococcus”
• lobar pneumonia
• CAP (= comunity acquired pneumonia)
No.1. causative agent!
Other pneumococcal infections
• meningitis !!
• otitis media, sinusitis !!
• (peritonitis)
• ulcus serpens corneae
• Age specificity: <5 years and >60 years
• WHO: >1 million children die every year
Laboratory detection
• microscope: Gram + diplococci
• culture: blood agar, 5% CO2
incubator
– α-hemolysis, autolytic colonies
– optochin sensitive
– bile soluble
• capsule detection
– with antibody (capsule swelling test,
agglutination)
– capsular staining
Optochin sensitivity
Pneumococcal vaccines
• Pneumovax 23– Polysaccharide vaccine (MSD)
– for adults or >5y children
– 23 capsular antigens (=serotypes)
• Prevenar-13– < 2 y children (at 2, 4, 15 months)
– conjugated vaccine
– 13 serotypes:
4, 6B, 9V, 14, 18C, 19F, 23F, 1, 5, 7F, 3, 6A, 19A
Total: 94 capsular antigens
(= 94 serotypes)
Pneumococcus asymptomatic carriage
• Carriers are the major sources of infection
• very frequent in small children attending DCCs,
cca. 30-40% carriage rate (peaks at ~3 y)
• decreases in adulthood
• seasonal changes
• different serotypes, sometimes multiple strains
• carriage duration: weeks -- months
• conjugate vaccines influence carriage as well
Enterococcus genus
Enterococcus on E67 medium
• normal intestinal flora
• microscope: in pairs or short chains
• culture: non-fastidious
• resistant to bile (40%), salt, high T (60oC 30 min)
• hemolysis: might be α, β or γ
• esculin hydrolysis
Enterococcal infections
• Nozokomial infections!– cystitis (chateter-associated)
– septis, endocarditis (venous canule)
– peritonitis, abdominal abscess
– 2nd most frequent Gram+ after staphylococci
• E. faecalis, E. faecium
• High-level resistance!
- vancomycin R (VRE)
- antibiogram must be prepared
- reservoir of resistance genes!!
Viridant streptococci
• S. mutans, S. mitis, S. sanguis, S. oralis, S.
salivarius
• α-hemolysis
• infections:
– subacute bacterial endocarditis (heart valves)
– due to major oral surgery, tooth extraction
– caries !
Caries (Streptococcus mutans)
Dental plaque
• Dental Caries is the destruction of the enamel, dentin or cementum of teeth due to bacterial activities.
• Caries are initiated by direct demineralization of the enamel of teeth due to lactic acid and other organic acids which accumulate in dental plaque.
• Lactic acid bacteria in the plaque produce lactic acid from the fermentation of sugars and other carbohydrates in the diet of the host.
• Streptococcus mutans and Streptococcus sanguis are most consistently been associated with the initiation of dental caries.
Peptostreptococci
• normal flora of oral cavity, intestine
andvagina
• obligate anaerobe!
• produce stinking gases
• infections: abscesses (lung, brain)
• treatment: metronidazole, clindamycin