LAB - Enterobacteriaceae Flashcards
Common genera in family of Enterobacteriaceae that are associated with human pathogenicity
- Klebsiella
- Shigella
- Salmonella
- Escherichia
- Proteus
- Citrobacter
- Serratia
- Hafnia
- Yersinia
- Enterobacter
- Providencia
- Morganella
Outside their normal habitat, opportunistic pathogens can cause some serious ____________ infections
extraintestinal
Primary intestinal pathogens
- Salmonella, Shigella, Yersinia enterocolitica, E. coli O157:H7 = true pathogens
- NOT commensal flora of GI
All Enterobacteriaceae species: (4)
- ferment glucose (OF=F)
- reduce nitrate to nitrites
- oxidase neg
- nonsporulating
Intestinal specimens (stool) for colifs
only potential pathogens are fully identified and reported
= Salmonella, Sigella, Yersinia, O157:H7)
Extraintestinal specimens (urine, wound swabs, etc.)
any Entero- may be clinically significant
- isolated on MAC
- lactose or non-lactose fermenter?
- further biochemical tests
cytochromes
iron-containing hemeproteins = components f ETC; involved in aerobic respiration
Oxidase test
- 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)
This may inhibit oxidase activity and thus should not be used when doing an oxidase test
MAC (selective media/carb fermentation)
Filter paper method (Oxidase)
- colony transferred onto a small piece of filter paper saturated with TMPD reagent
- dark purple colour = positive (rapid)
A differential medium, used as a screen test for enteric pathogens on the basis of carbohydrate fermentation, HS production, and gas production
Triple Sugar Iron (TSI)
Citrate utilization
- 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
Indole production
- 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
Spot indole
- 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
Motility test
- 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)
Medium method for motility is not useful for these organisms
strict aerobes like Pseudomonas bc will not grow in the depth of medium (use microscopic examination)
Yersinia enterocolitica motility
motile at 22C butt not at 37C
Malonate
- 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
Phenylalanine deaminase test (PAD)
- test for ability to deaminate phenylalanune to phenyl pyruvic acid by adding ferric chloride as an indicator
- Proteus-Providence (+); rest of colifs (-)
Urease activity
- 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
Methyl Red-Voges Proskauer (MR-VP)
- 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)
MR pos organisms
turning red
E. coli, Edwarsiella, Shigella, Salmonella, Citrobacter, Proteus, Klebsiella, Yersinia
MR neg organisms
turning yellow
Enterobacter, Hafnia, Serratia
VP pos organisms
pink-red
Enterobacter, Klebsiella pneumoniae, Seratia
VP neg organisms
no colour change
Scherichia, Edwarsiella, Citrobacter, Salmonella, Shigella, Yersinia, Klebsiella
MR-VP procedure
- 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
ONPG
- orthonitrophenyl-beta-galactopyranoside
- detects beta-galactosidase (cleaves lactose into glucose & galactose)
- also depends on PERMEASE ( transports lactose into cells)
ONPG Positive
- 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**
ONPG Negative
non-lactose fermenter = Salmonella spp, Shigella spp, Proteus spp, Providencia, Morganella = NO beta galactosidase
ONPG procedure
- 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
Sulphide-Indole Motility (SIM)
- example of a multi-test medium
- tests more than one aspect of their metabolism at a time
- H2S production? indole? motility?
SIM procedure
- 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
Most prompt lactose fermenters
Escherichia, Klebsiella, and Enterobacter
Analytical Profile Index (API) strips
- 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
Serological methods for ID of Shigella, Salmonella, and E. coli O157:H7
- 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
Kauffmann-White scheme
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
Serologic grouping of Shigella is based on this antigen
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
Shigella serogroups
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
delayed D-sorbitol fermentation
E. coli O157:H7
> 24 hrs
SMAC agar can be used for detection
