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MLSCI 242 > LAB - Enterobacteriaceae > Flashcards

LAB - Enterobacteriaceae Flashcards

1
Q

Common genera in family of Enterobacteriaceae that are associated with human pathogenicity

A
  • Klebsiella
  • Shigella
  • Salmonella
  • Escherichia
  • Proteus
  • Citrobacter
  • Serratia
  • Hafnia
  • Yersinia
  • Enterobacter
  • Providencia
  • Morganella
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2
Q

Outside their normal habitat, opportunistic pathogens can cause some serious ____________ infections

A

extraintestinal

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3
Q

Primary intestinal pathogens

A
  • Salmonella, Shigella, Yersinia enterocolitica, E. coli O157:H7 = true pathogens
  • NOT commensal flora of GI
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4
Q

All Enterobacteriaceae species: (4)

A
  • ferment glucose (OF=F)
  • reduce nitrate to nitrites
  • oxidase neg
  • nonsporulating
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5
Q

Intestinal specimens (stool) for colifs

A

only potential pathogens are fully identified and reported

= Salmonella, Sigella, Yersinia, O157:H7)

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6
Q

Extraintestinal specimens (urine, wound swabs, etc.)

A

any Entero- may be clinically significant

  • isolated on MAC
  • lactose or non-lactose fermenter?
  • further biochemical tests
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7
Q

cytochromes

A

iron-containing hemeproteins = components f ETC; involved in aerobic respiration

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8
Q

Oxidase test

A
  • colour from oxidation of colourless TMPD (interacts with ETC) at the level of cytochrome c = dark purple indophenol
  • pos depends on the presence of cytochrome c (bacteria whose ETC has cytochrome c oxidase)
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9
Q

This may inhibit oxidase activity and thus should not be used when doing an oxidase test

A

MAC (selective media/carb fermentation)

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10
Q

Filter paper method (Oxidase)

A
  • colony transferred onto a small piece of filter paper saturated with TMPD reagent
  • dark purple colour = positive (rapid)
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11
Q

A differential medium, used as a screen test for enteric pathogens on the basis of carbohydrate fermentation, HS production, and gas production

A

Triple Sugar Iron (TSI)

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12
Q

Citrate utilization

A
  • certain organisms can use citrate as their sole source of carbon
  • pos = growth -> blue due to alkaline end products produced (bromothymol blue)
  • useful for differentiation of lactose fermenters
  • Kleb and Enterobacter utilize citrate but E. coli does not
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13
Q

Indole production

A
  • bacteria capable of hydrolyzing tryptophan (in peptone water) to form indole
  • detected by colour rxn with dimethylamino benzaldehyde (Kovac’s)
  • tube test could check for motility before adding Kovac’s by performing a wet mount
  • differentiation of lactose fermenters: E. coli pos and Enterobacter + Klebsiella neg
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14
Q

Spot indole

A
  • spot test = more sensitive than tube
  • can’t check for motility first
  • useful for organisms that won’t grow in peptone water (anaerobes)
  • colony transferred ti filter paper; saturated ith spot indole reagent (p-dimethylaminocinnanaldehyde); blue/green is pos
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15
Q

Motility test

A
  • directly test by microscopic examination of “hanging drop” or wet prep of organisms (peptone water before Kovac’s)
  • indirectly by observing growth in a semi-solid motility medium
  • motile organisms (facultative anaerobes) spread from line of inoculum = diffuse pattern
  • non-motile organism growth is confined to inoculum line
  • Tetrazolium salts = aid in reading; changes to red (red on stab line only if non-motile)
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16
Q

Medium method for motility is not useful for these organisms

A

strict aerobes like Pseudomonas bc will not grow in the depth of medium (use microscopic examination)

17
Q

Yersinia enterocolitica motility

A

motile at 22C butt not at 37C

18
Q

Malonate

A
  • utilize malonate as a source of carbon?
  • end products = acetate and CO2
  • alkaline pH so bromthymol blue changes from green to blue (alkaline) in a positive test
19
Q

Phenylalanine deaminase test (PAD)

A
  • test for ability to deaminate phenylalanune to phenyl pyruvic acid by adding ferric chloride as an indicator
  • Proteus-Providence (+); rest of colifs (-)
20
Q

Urease activity

A
  • splits urea into CO2 and ammonia
  • phenol red turns deep cerise due to alkalinity
  • conventional = Christensen’s urea agar (sterile buffered nutrient agar base containing phenol red indicator)
  • Stuart urea broth is more buffered
21
Q

Methyl Red-Voges Proskauer (MR-VP)

A
  • two tests in one
  • both detect acid production from fermentation of glucose
  • positive MR = more complete catabolism of glucose to highly acidic end products (lactic, acetic, succinic, formic acids) than VP pos (2,3-butanediol and ethanol following mixed-acid fermentation)
22
Q

MR pos organisms

A

turning red

E. coli, Edwarsiella, Shigella, Salmonella, Citrobacter, Proteus, Klebsiella, Yersinia

23
Q

MR neg organisms

A

turning yellow

Enterobacter, Hafnia, Serratia

24
Q

VP pos organisms

A

pink-red

Enterobacter, Klebsiella pneumoniae, Seratia

25
Q

VP neg organisms

A

no colour change

Scherichia, Edwarsiella, Citrobacter, Salmonella, Shigella, Yersinia, Klebsiella

26
Q

MR-VP procedure

A
  • inoculate broth and incubate
  • remove 1 mL of broth and place in sterile tube; 3-4 drops of methyl red; pos is distinct red at top, neg is yellow layer
  • add VP reagent (Barritts alpha-naphthol + 40% KOH) to OG broth; pos will be pink to red
27
Q

ONPG

A
  • orthonitrophenyl-beta-galactopyranoside
  • detects beta-galactosidase (cleaves lactose into glucose & galactose)
  • also depends on PERMEASE ( transports lactose into cells)
28
Q

ONPG Positive

A
  • lactose fermenter = E. coli, Kleb spp., Enterobacter spp. = produce B-galactosidase and permease
  • late lactose fermenter = Citrobacter spp, Arizona spp = only B-galactosidase so slow

** yellow in few minutes vs, 24 hours**

29
Q

ONPG Negative

A

non-lactose fermenter = Salmonella spp, Shigella spp, Proteus spp, Providencia, Morganella = NO beta galactosidase

30
Q

ONPG procedure

A
  • broth = organism taken from medium with high lactose conctn; inoculated in ONPG broth; split beta-galactoside bond, releasing o-nitrophenol = yellow-coloured compound
  • disk-method = medium with high lactose -> dense suspension -> ONPG disk added = yellow if pos
31
Q

Sulphide-Indole Motility (SIM)

A
  • example of a multi-test medium
  • tests more than one aspect of their metabolism at a time
  • H2S production? indole? motility?
32
Q

SIM procedure

A
  • stab SIM medium (2/3), then incubate)
  • H2S if black
  • indole if red ring on surface of deep after adding Kovac’s
  • motility if fan away from streak
33
Q

Most prompt lactose fermenters

A

Escherichia, Klebsiella, and Enterobacter

34
Q

Analytical Profile Index (API) strips

A
  • ID limited number of gram neg colifs
  • test strips have wells containing dehydrated substrates to detect enzymatic activity
  • bacterial suspension used to rehydrate wells; strips incubated
  • colour change (may have to add reagent, etc.)
  • negs and pos = profile number and compare to online codebook
35
Q

Serological methods for ID of Shigella, Salmonella, and E. coli O157:H7

A
  • O (somatic antigen) - heat-stabile antigen located in cell wall, resistant to alcohol
  • H (flagellar) - heat-labile; resists formalin
  • K (capsular) - heat-labile polysaccharide; K1 of E. coli, Vi of S. Typhi; may mask O antigen
36
Q

Kauffmann-White scheme

A

serotyping scheme for Salmonella sp.
- developed upon examining rxns of different suspensions of this organism with crude antisera raised against the other antigenic group strains

37
Q

Serologic grouping of Shigella is based on this antigen

A

O

  • is agglutination fails with O antisera, the suspension is heated to remove capsular antigen and is retested with O antisera
  • may belong to serogroups A, B, C, or D which may have multiple serotypes
38
Q

Shigella serogroups

A

A - S. dysenteriae

B - S. flexneri

C - S. boydii

D - S. sonnei

** A-C physiologically similar; A,B,D = major bacterial cause of GI disease

39
Q

delayed D-sorbitol fermentation

A

E. coli O157:H7
> 24 hrs
SMAC agar can be used for detection

MLSCI 242 (39 decks)

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