EnterobacteriaceaeEnterobacteriaceae

Bkochemical testsBkochemical tests

Enterobacteriaceae

Opportunistic pathogensEscherichia coli

Klebsiella pneumoniaeEnterobacter aerogenes

Serratia marcescensProteus spp.

Providencia spp.Citrobacter spp.

Obligate pathogensSalmonella spp.

Shigella spp.Yersinia spp.

Some E. coli strains

Sepsis

Meningitis

UTI

Diarrhea

Pneumonia

Klebsiella

K. pneumoniae and K. oxytoca are the most commonly isolated.Can cause community-acquired primary lobar pneumonia

(frequently involves necrotic destruction of alveolar space), and infections of wound, soft tissue, and urinary tract.

Risk factors for pneumonia: alcoholism; compromised pulmonary function.

*In Taiwan: liver abscess is commonly seen in infection of diabetes patients by K. pneumoniae .

K. granulomatis may cuase granuloma inguinale, a sexually transmitted disease, in some countries.

K. rhinoscleromatis: granulomatous disease of the nose.K. ozaenae: chronic atrophic rhinitis.

Other opportunistic Enterobacteriaceae

IMViC TestIMViC Test

IIndole, ndole, MMethyl Red, ethyl Red, VVoges-Prosakaur, oges-Prosakaur, CCitrate (IMViC) Tests:itrate (IMViC) Tests:– The following four tests comprise a series of The following four tests comprise a series of

important determinations that are collectively important determinations that are collectively called the IMViC series of reactions called the IMViC series of reactions

– The IMViC series of reactions allows for the The IMViC series of reactions allows for the differentiation of the various members of differentiation of the various members of EnterobacteriaceaeEnterobacteriaceae..

IIMViC: MViC: IIndole testndole test PrinciplePrinciple

Certain microorganisms can metabolize Certain microorganisms can metabolize tryptophan by tryptophanasetryptophan by tryptophanase

The enzymatic degradation leads to the The enzymatic degradation leads to the formation of pyruvic acid, indole and formation of pyruvic acid, indole and

ammoniaammonia The presence of indole is detected by The presence of indole is detected by

addition of Kovac's reagent.addition of Kovac's reagent.

Tryptophaneamino acids

Tryptophanase Indole + Pyurvic acid + NH3

Kovac’s Reagent

Red color in upper organic layer`

IIMViC: MViC: IIndole testndole test

Method:Method: Inoculate Inoculate tryptone water with the tested with the tested

microorganism microorganism Incubate at 37°C for 24 hours Incubate at 37°C for 24 hours After incubation interval, add 1 ml After incubation interval, add 1 ml

Kovacs reagent, shake the tube gently and , shake the tube gently and read immediatelyread immediately

IIMViC: MViC: IIndole testndole test

Result:Result: A bright pink color in the top A bright pink color in the top

layer indicates the presence layer indicates the presence of indoleof indole

The absence of color means The absence of color means that indole was not produced that indole was not produced i.e. indole is negativei.e. indole is negative

Special Features:Special Features: Used in the differentiation of Used in the differentiation of

genera and species. e.g. genera and species. e.g. E. coliE. coli (+) from (+) from KlebsiellaKlebsiella (-). (-).

Positive teste.g. E. coli

Negative teste.g. Klebsiella

IIMVMViC testiC testMMethyl Red-ethyl Red-VVoges Proskauer (MR-VP) Testsoges Proskauer (MR-VP) Tests

Glucose

Acidic pathway

Mixed acids pH less than

4.4Methyl Redindicator

Red color

Principle

MR positiveE. coli

Or Neutral pathway

Acety methyl carbinol(ACETOIN)

Barrit’s ABarrit;s B

Pink colorVP positiveKlebsiella

IIMVMViC test: iC test: MMRRVVP testP test

Inoculate the tested organism into Inoculate the tested organism into One tubeOne tube of of MRVP broth broth

Incubate the tubes at 37°C for 24 hoursIncubate the tubes at 37°C for 24 hours

AFTER INCUBATION:  Pour 1/3 of the suspension into a clean AFTER INCUBATION:  Pour 1/3 of the suspension into a clean

nonsterile tube:  nonsterile tube: 

Run the MR test in the tube with 2/3, and the VP test in the Run the MR test in the tube with 2/3, and the VP test in the

open tube with 1/3. open tube with 1/3.

– For methyl red:  Add 6-8 drops of methyl red reagent. For methyl red:  Add 6-8 drops of methyl red reagent.

– For Voges-Proskauer:  Add 12 drops of Barritt's A (For Voges-Proskauer:  Add 12 drops of Barritt's A (--naphthol), mix, 4 drops of Barritt's B (40% KOH), mix naphthol), mix, 4 drops of Barritt's B (40% KOH), mix

– Let sit, Let sit, undisturbedundisturbed, for at least 1hour, for at least 1hour

Method

IIMVMViC test: iC test: MMR/R/VVP testP testResults

Methyl Red test Voges-Proskauer test

Red: Positive MR (E. coli)

Yellow or orange: Negative MR (Klebsiella)

Pink: Positive VP (Klebsiella)

No pink: Negative VP (E. coli)

Citrate Utilization TestCitrate Utilization TestPrinciple:

Citrate Na2CO3

Alkaline,↑pH

Blue colourBromothymol blue

Simmone’s Citrate media

Positive test: Klebsiella, Enterobacter, Citrobacter

CO2 + Na + H2OPyruvate

Positive test

Negative test: E. coli

Contains Citrate as a sole of C source

Citrate Utilization TestCitrate Utilization Test

Incubate at 37°C for 24 hours.Incubate at 37°C for 24 hours.

Method Method

Streak a Streak a Simmon's Citrate agarSimmon's Citrate agar slant with slant with

the organism the organism

Citrate Utilization TestCitrate Utilization Test

Examine for growth (+)Examine for growth (+) Growth on the medium Growth on the medium

is accompanied by a is accompanied by a rise in pH to change rise in pH to change the medium from its the medium from its initial green color to initial green color to

deep bluedeep blue

ResultResult

PositiveKlebsiella, Enterobacter

NegativeE. coli

Urease TestUrease Test Urea agar contains urea and phenol redUrea agar contains urea and phenol red

Urease is an enzyme that catalyzes the conversion of Urease is an enzyme that catalyzes the conversion of urea to CO2 and NH3urea to CO2 and NH3

Ammonia combines with water to produce ammonium Ammonia combines with water to produce ammonium hydroxide, a strong base which ↑ pH of the medium. hydroxide, a strong base which ↑ pH of the medium.

↑ ↑ in the pH causes phenol red r to turn a deep pink. in the pH causes phenol red r to turn a deep pink. This is indicative of a positive reaction for ureaseThis is indicative of a positive reaction for urease

Urea UreaseCO2 + NH3

H2ONH4 OH ↑ in pH

Phenol Red

PinkPositive test

Streak a urea agar tube with the organism incubate at 37°C for 24 h

Method

PrinciplePrinciple

Urease TestUrease Test

If color of medium turns If color of medium turns from yellow to pink from yellow to pink indicates positive test. indicates positive test. ProteusProteus give positive give positive reaction after 4 h while reaction after 4 h while KelebsiellaKelebsiella and and EnterobacterEnterobacter gave gave positive results after 24 hpositive results after 24 h

Result

Positive test Negative test