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Enterobacteriaceae Si Teste Biochimice
Enterobacteriaceae Si Teste Biochimice
• Intestinal infections:
– Faeces: collection close to onset / depending on pathogenesis of
infection
– Transport media: Stuart, Cary-Blair, Amies
Collection of urine
When?:
- in the morning (first miction)
How?:
- clean uro-genital area
- eliminate first flow
- collect middle flow in
sterile container
• Principle: detection of
– pH changes produced by utilization of substrates /
– colour / other changes produced by specific by-
products
Family Enterobacteriaceae
Biochemical tests (continued)
TSI (triple sugar iron) agar – assessment of bacterial
capacity to:
a. metabolize lactose and/or sucrose
b. conduct fermentation to produce acid
c. produce gas during fermentation
d. generate H2S
TSI agar:
sucrose, lactose, glucose + mehyl red + ferrous
sulfate
IF:
• only glucose fermented →acid production in the butt of tube →
yellow, but insufficient acid to affect the methyl red in the slant
• If bacterium forms H2S, this chemical will react with the iron to form
ferrous sulfide = black precipitate in the butt (black butt)
TSI agar (continued)
• R = red = no fermentation
(obligate aerobe)
• Y = yellow = some
fermentation (facultative
anaerobe)
• YG = fermentation + gas
• Extraenteral infections:
– Urinary, respiratory, wounds & burns, sepsis , meningitis, etc.
*Verotoxins (Shiga-like toxins)
• Extraenteral infections:
– Urinary, respiratory, wounds & burns, sepsis , meningitis, etc.
E.coli – Urinary tract infections (UTI)
• Interpretation of results:
• Under 10,000 germs/mL = nonsignificant bacteriuria
(probably contamination from lower urethra)
• 10,000–100,000 germs/mL = nonconclusive; repeat test
• Over 100,000 germs/mL = UTI
• Clinical significance:
– Food poisoning
– Systemic infections (the germs cross the intestinal barrier) –
”enteric fevers”
• Secimens:
– Blood for blood culture (best collected during 1st week; percent
of isolation decreases to 25% in the 4th week)
– Bone marrow (in late stages) – lower patient compliance
– Faeces for coproculture (low isolation during 1st week; percent
increases progressively to ~75% in 4th week); also collected
from suspected chronic carriers
– Urine for culture (same isolation curve as for faeces)
Salmonella typhi (+ paratyphi)
- Bacteriological diagnosis - continued
Blood culture:
• inoculated media examined daily for 7-10 days; negative
result only if liquid media remain sterile for 10 days!
• Gram stained smear from turbid tubes: Gram negative
bacilli
• Subculture on agar slant
• Identification based on:
– colonial characters,
– biochemical tests,
– antigenic structure – slide agglutination with anti-O and anti-H
sera (Kauffmann-White classification)
Salmonella typhi (+ paratyphi)
- Bacteriological diagnosis - continued
Coproculture:
• Both in diseased patients and in chronic carrires
• Inoculation in liquid enrichment media (favour
multiplication of salmonellae and inhibit other microbial
flora) e.g.
– Leifson (nutrient broth with acid sodium selenite);
– Muller-Kauffmann (broth with tetrationate and bile)
• Incubate overnight at 37°C
• Reinoculate on selective solid media (nutrients + sugars
+ pH indicator + substances which inhibit other germs)
Salmonella typhi (+ paratyphi)
- Bacteriological diagnosis - continued
Coproculture: (continued)
Colonial characters on selective solid media:
• Wilson-Blair (high selectivity; indicator: brilliant green)
– Black, flat colonies, 1-2 mm, metallic halo
• S-S (selective for Salmonella and Shigella):
– Fine, semitransparent colonies, with black centre (H 2S)
• Hektoen enteric agar :
– Fine, green colonies, with black centre (H2S)
• MacConkey (medium selectivity):
– Semitransparent, lactose-negative (colourless) colonies
Salmonella on S-S agar: fine, semitransparent
colonies, black centre (H2S)
Salmonella on MacConkey agar
• Semitransparent, lactose-
negative (colourless)
colonies
Salmonella on agar with lactose and bile salts:
lactose negative (colourless) colonies with black
centre (H2S)
Salmonella on Hektoen agar
• black centre
colonies (H2S)
Salmonella – Coproculture - continued
Procedure:
• agar plates flooded with liquid culture of the bacterial
isolate; remove excess liquid; leave culture film to dry;
• inoculate set of phage suspensions onto plate surface;
incubate overnight at 37°C
• Interpretation: phage lysis reactions recorded and
compared to a set of standards (developed by Public
Health England, formerly PHLS, Colindale, England)
Phage typing Salmonella enteritidis
Salmonella typhi
- Serological diagnosis -
The Widal test:
• Principle: reaction between antibodies in patient serum
and specific antigens of S. typhi → clumping
(agglutination) visible to the naked eye
• easy to perform BUT less reliable than bacteriology:
– cross-reactivity with other Salmonella species,
– the test cannot distinguish between a current infection
and a previous infection or vaccination status
• Still used in low resource areas with high
prevalence of typhoid fever (endemic areas e.g.
India, Pakistan)
Genus Shigella
Clinical significance:
• dysentery – diarrhoea with multiple stools with mucus
and blood + general symptoms: dehydration, fever,
abdominal pain + neurologic symptoms (neurotoxin
secreted by Shigella shiga)
Common characters:
• Gram negative bacilli, nonmotile, nonsporulating
Genus Shigella - classification
Common characters:
• Gram negative, short bacilli,
rounded ends, nonsporulating,
arranged in diplo (in pairs) on
the long axis, enacpsulated
Isolation:
• Specimens from normally sterile sites (blood, CSF, etc):
– Nutrient broth + reinoculation on blood agar
• Specimens from highly contaminates sites (e.g. faeces):
– Media for enterobacteria: MacConkey, XLD
• Identification:
– Blood agar – large, white-grey colonies, mucous, aspect of
”pouring culture” – in time the colour changes to brown
(”chameleoning” phenomenon)
– MacConkey – large, mucoid colonies, red/pink (lactose positive)
– in time colour changes to yellow (lactose negative) –
“chameleoning” by alkalinisation of the medium
Klebsiella – blood agar (non hemolytic
mucoid colonies)
Klebsiella: Mucous colonies
Klebsiella – pink colonies (Lactose positive)
on MacConkey agar
Klebsiella colonies on MacConkey: colour starts to
change - Lactose positive (red/pink) colonies start
to change to lactose negative (yellow) –
alkalinisation
Klebsiella – identification – continued
Biochemical characters
Genus Proteus
• 4 species:
– P.vulgaris, P.mirabilis, P.penneri, P. myxofaciens
• Common characters:
– Gram negative, short bacilli, rounded ends, high polymorphism,
high motility (peritrichous cili), nonsporulating, nonencapsulated
• Habitat:
– soil, trash, sewage, altered meat, etc. – involved in putrefaction
processes
• Clinical significance:
– comensal of human and animal digestive flora;
– facultatively pathogenic: UTI, otitis, synusitis, meningitis, sepsis
(community or hospital acquired infections)
Genus Proteus
• Collection of specimens:
– urine, faeces, pus, sputum, CSF, blood, etc
• Direct microscopy:
– only for naturally sterile specimens e.g. CSF
– PMNs + Gram negative bacilli, noncharacteristic arrangement +
filamentous bacilli (high polymorphism of Proteus spp)
• Isolation and identification:
– Blood agar: swarming phenomenon (concentric growth waves
invading the entire plate after overnight incubation); invades
other bacterial colonies; no isolated colonies
– Selective media: round colonies, same colour as the
medium/transparent, black centre (”cat‘s eye”) – H2S production
Proteus
• Swarming
phenomenon on
tryptic soy agar
Genera Morganella and Providencia