Enterobacteriaceae

Enterobacteriaceaeد.وليد خالد سعدون

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جراثيم / د.وليد / طب عام ثالث
نظري 28/2/2017
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ENTEROBACTERIACEAE are a big family of gram negative bacilli containing 31 genera and 139 species.

Some are regular pathogens and others are normal flora .

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General Characters of Enterobacteriaceae

• All Enterobacteriaceae are:
• Gram-negative bacilli
• Ferment glucose with acid production
• Oxidase negative
• Catalase positive
• Reduce nitrate to nitrite





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• Facultative anaerobic

• Non-capsulated except Klebsiella
• Motile except Shigella and Klebsiella
• Grow on bile salts containing media (MacConkey agar)
•


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-Some Enterobacteriaceae are pathogenic like :

Salmonella spp.
Shigella spp.
Yersinia spp.
Certain strains of E. coli

-While most members of the Enterobacteriaceae are opportunistic or cause secondary infections of wounds, urinary tract ,respiratory tract , and the circulatory system.
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-Enterobacteriaceae are divided into TWO main groups according to their ability to ferment LACTOSE :

-Lactose Fermenters (LF)

E. coli, Klbesiella, Citrobacter, Enterobacter

-Non-Lactose Fermenters (NLF)

Salmonella, Shigella, Proteus, Yersinia
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METHODS OF IDENTIFICATION

we Can identify enterobacteriaceae by:
1-GRAM STAIN
2-CULTURE MEDIA
• A-MacConkey agar
• B-Salmonella Shigella (SS) agar
• C-Eosin Methylene Blue (EMB) agar
• D-Triple Sugar Iron (TSI) agar
3-BIOCHEMICAL REACTIONS
A- IMViC REACTIONS
B- UREASE TEST
4-MOTILITY TEST


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1-GRAM STAIN

-All Enterobacteriaceae are Gram-negative bacilli, motile except Shigella and Klebsiella and non-capsulated except Klebsiella




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2-CULTURE MEDIA

There are several selective and differential media used to isolate and distinguish between
members of family Enterobacteriaceae.

The most important media are:

• A-MacConkey agar
• B-Salmonella Shigella (SS) agar
• C-Eosin Methylene Blue (EMB) agar
• D-Triple Sugar Iron (TSI) agar
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A-MacConkey agar
- MacConkey (Mac) agar is a selective and differential medium used for the isolation of enterobacteriaceae from stool , urine and other specimens.




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-This medium contains bile salts, crystal violet dye , lactose and neutral red (pH indicator ).

_pH indicator is a chemical substance, its color changes when the pH of the medium is changed.

_Neutral red is a pH indicator, its color becomes pink when the pH decrease ( i.e. the acidity of the medium increase).
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-MacConkey agar is selective medium because crystal violet dye and bile salts inhibit the growth of Gram-positive organisms.

-It is also differential medium because lactose differentiates enterobacteriaceae into lactose fermenters and non-lactose fermenters according to their ability to ferment lactose.

-lactose fermenters will produce bright pink colonies.

While non-lactose fermenters will produce transparent (colorless) colonies.
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Differentiation between LF & NLF by growth on MacConkey agar

MacConkey Agar

Contains

Bile salts
Crystal violet

Lactose

Neutral red

MacConkey agar is selective & differential medium for Enterobacteriaceae

Inhibit growth of G+ve bacteria

Cause of selectivity

Cause of differential
pH indicator
Acidic: Pink

Lactose fermenters

Pink colonies
Non -lactose fermenters
colorless colonies

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Classification of Enterobacteriaceae according to lactose fermentation (growth on MacConkey Agar)
Enterobacteriaceae
Lactose Fermenters
Non -lactose fermenters

Escherichia coli

Klebsiella spp
Enterobacter spp
Citrobacter spp


Salmonella spp
Schigella spp
Proteus spp
Yersinina spp
Pink colonies

Colorless colonies

Acid

Neutral red

No acid
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B-Salmonella Shigella (SS) agar

• SS agar is a selective & differential medium used for isolation of Salmonella and Shigella



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• Salmonella Shigella(SS) agar contains:

• 1-Bile salts and brilliant green dye.
• 2-Lactose and neutral red(pH indicator)
• 3-Sodium thiosulphate and ferric ammonium citrate.
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• The selective agents are bile salts, and brilliant green dye, which inhibit the growth of gram positive organisms
• Lactose is the differential agent because it differentiates enterobacteriaceae into lactose fermenters and non fermenters, when lactose fermenters will produce pink colonies while non lactose fermenters will produce colorless colonies
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• SS agar also contains sodium thiosulfate and ferric ammonium citrate that allow to differentiate organisms that produce H2S as Salmonella producing black center in the colony.

Lactose

Lactose fermentation

Acid

Neutral red
Pink colonies
Ferrous sulfide
Black precipitate
H2S + Ferric ammonium citrate+ Sodium thiosulfate




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• As a result , the appearance of enterobacteriaceae on SS agar will be as follow:

- Non lactose fermenters, such as Shigella produce colorless colonies.
- Lactose fermenters, such as E. coli, produce pink colonies.
• - Salmonella produce colorless colonies with black centers due to H2S production.


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• C-Eosin Methylene Blue (EMB) agar

-It is selective and differential medium for the isolation and differentiation of Enterobacteriaceae

-EMB agar contains eosin Y and methylene blue dyes which inhibit gram positive bacteria and serve as differential indicators in response to the fermentation of lactose.


-Lactose fermenters produce blue-black colonies while Salmonella and Shigella colonies are colorless.

-E.coli colonies may show a characteristic green metallic sheen due to rapid fermentation of lactose.
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D-Triple Sugar Iron (TSI) agar

• It differntiates enterobacteriaceae according to:
• 1-Lactose fermentation.
• 2-H2S production.
• 3-Gas production.
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• Triple sugar iron agar (TSI): This medium consist of

• Three sugars=(1gm glucose,10gm lactose, 10gm sucrose).
• Ferrus sulphate.
• Phenol red(pH indicator)(which is red and become yellow when the medium is acidic)
-TSI agar has slant and butt
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-After inoculation of Enterobacteriaceae into TSI agar ,at first, All Enterobacteriaceae will ferment glucose producing acid changing the color of the butt from red to yellow ( because more glucose is present in the butt than in the slant ) while the slant remain red .

-Then if the bacteria is lactose fermenter it will ferment lactose also changing the color of the slant also to yellow.

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-So lactose fermenters cause yellowish discoloration of both the butt and slant like E.coli and Klebsiella

-While in non-lactose fermenters only the butt will be yellow while the slant is red like Shigella.

Lactose fermenters

Non-lactose fermenters

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-If the bacteria produce H2S (like Salmonella) it will
cause black discoloration in the TSI agar, and if it is gas producer so it will produce bubbles or crack in
the medium.
- So the TSI agar reaction will be read as follow:
Reaction in the slant (K or A) / Reaction in the butt (K or A) + H2S ( if present ) + Gas ( if present )
E.coli= A/A
Salmonella = K/A+H2S





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(A)- IMViC REACTIONS(B)- Urease Test

• IMViC reactions are set of four useful reactions that distinguish between members of the Enterobacteriaceae family based on their metabolic by-products.
• The four reactions are: Indole test, Methyl Red test, Voges Proskauer test and Citrate utilization test. The letter “i” is only used for spelling purpose.
3-BIOCHEMICAL REACTIONS
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INDOLE TEST

Principle:-
Indole test is used to detect bacteria that can produce indole from amino acid tryptophan using the enzyme typtophanase.

Tryptophane Tryptophanase Indole + Pyurvic acid + NH3

(amino acids)


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-Production of indole is indicated by the addition of Kovac’s reagent (which contains para-dimethyl aminobenzaldehyde, alcohol and concentrated HCl)

-Indole reacts with the aminobenzaldehyde in the Kovac’s reagent giving a red color ring at the top of the reaction.
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• Tryptophane Tryptophanase Indole + Pyurvic acid + NH3

(amino acids) Kovac’s Reagent


Red color ring in the top




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Procedure:-

-Inoculate the medium with the bacteria and incubate for 48 hours at 37 C . Add 5 drops of Kovac’s Reagent and shake gently , formation of red ring indicate a positive reaction

Medium:-

Peptone water ( with double concentration of peptone ).
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(1)

(2)
(3)
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Example:-

Escherichia coli: Positive;
Klebsiella pneumoniae: Negative
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METHYL RED (MR) TEST

Principle:-It is used to detect the ability of an organism to produce large amounts of acid (pH ≤ 4.4) from glucose fermentation.

Medium :-

Glucose phosphate peptone water

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• Methyl red is a pH indicator which is red in color at a pH of 4.4 or less.

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• Procedure:-

• The bacterium to be tested is inoculated into glucose phosphate peptone water which contains glucose and a phosphate buffer and incubated at 35C for 4 days.
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-The pH of the medium is then tested by the addition of 5 drops of MR reagent.

-Development of red color is taken as positive, while yellow color indicates MR negative organism.
Example:-
• Eschericihia coli: Positive;
• Klebsiella pneumoniae: Negative
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Positive red color (e.g E.coli)

Negative yellow color (e.g klebsella)
Methyl Red (MR) test
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Methyl Red (MR)

Positive red color (e.g E.coli)
Negative yellow color (e.g klebsella)



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VOGES PROSKAUER (VP) TEST

Principle:-
It is used to detect bacteria that produce Acetyl-methyl carbinol (acetoin) during glucose fermentation.
If acetoin is present, it is oxidized in the presence of air and KOH to diacetyl acetoin.

Acetoin + 40%KOH Diacetyl acetoin

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• Diacetyl aceton then reacts with guanidine components of peptone in which bacteria is inoculated in the presence of alphanaphthol to produce red color.
alphanaphthol
Diacetyl + Guanidine Red Color aceton
Medium :-
Glucose phosphate peptone water


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Procedure:-
-Inoculate the bacteria into glucose phosphate broth and incubate for at least 48 hours.
-0.2 ml of 40% KOH is added to the broth and shaken.
-Then 0.6 ml of alpha-naphthol is added to the test broth and it is shaken.
-The tube is allowed to stand for 15 minutes.
Appearance of red color is considered as a positive test.
Examples: Escherichia coli: Negative
Klebsiella pneumoniae: Positive
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Vogus proskour (VP)

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CITRATE UTILIZATION TEST

Principle: This test detects the ability of an organism to utilize citrate as the sole source of carbon and ammonium salts as the sole source of nitrogen .


Medium :-
Simmone’s Citrate media




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• Procedure:-

• Bacteria to be tested is inoculated on a medium (Simmone’s Citrate media) containing sodium citrate, ammonium salt and a pH indicator bromothymol blue (it is green in color normally but become blue in alkaline pH).
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Simmone’s Citrate media

sodium citrate +ammonium salts

sodium carbonate + ammonium hydroxide

Alkaline,↑pH
Bromothymol blue
Blue colour

Positive test

Utilization
Utilization of sodium citrate and ammonium salts in the medium by the bacteria will produce sodium carbonate and ammonium hydroxide respectively . Both of them are alkaline compounds that will raise the pH in the media changing the color of bromothymol blue from green to blue .

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Examples: Escherichia coli: Negative;

Klebsiella pneumoniae: Positive






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• Citrate utilization

• VP
• MR
• Indole
• Bacterium
• -
• -
• +
• +
• E. coli
• +
• +
• -
• -
• K.pneumoniae
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(B)- Urease Test
Principle:

-To detect organisms that produce urease which is an enzyme that catalyzes the conversion of urea to CO2 and NH3.

-Urea agar contains urea and phenol red(pH indicator)

-We inoculate the organism in urea agar. if the organism produce urease then urease will convert urea to CO2 and NH3(ammonia) which will increase the pH of the media causes phenol red to turn into a deep pink color. This is indicative of a positive reaction for urease
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Urea

CO2 + NH3

↑ in pH

Phenol Red
Pink
Positive test
Urease

• -Positive test is indicated by changing the color of the medium from yellow to pink.

• -Proteus give positive reaction after 4 hr.s while Kelebsiella and Enterobacter give positive results after 24 hr.s
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4-MOTILITY TEST

Semisolid medium

• -This test is based on the presence of the bacterial flagella.
• If the bacteria have flagellum, the semisolid medium will be cloudy and the test will be positive.
• -All enterobacteriaceae are motile except Shigella and Kliebsella
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1-GRAM STAIN

2-CULTURE MEDIA
• A-MacConkey agar
• B-Salmonella Shigella (SS) agar
IDENTIFICATION of enterobacteriaceae






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• C-Eosin Methylene Blue (EMB) agar

• D-Triple Sugar Iron (TSI) agar









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3-BIOCHEMICAL REACTIONS

A- IMViC REACTIONS

B- UREASE TEST

4-MOTILITY TEST












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Summary of morphology, cultural characteristics,
and biochemical reactions of Enterobacteriaceae
• EMB
• SS
• MacConkey
• Nitrate reductase
• Oxidase
• Gram stain
• Metallic sheen
• LF
• LF
• +ve
• -ve
• -ve rod
• E. coli
• Dark
• LF
• LF
• +ve
• -ve
• -ve rods
• Citrobacter
• Dark
• LF
• LF
• +ve
• -ve
• -ve rods
• Klebsiella
• Dark
• LF
• LF
• +ve
• -ve
• -ve rods
• Enterobacter
• Colorless
• NLF/H2S
• NLF
• +ve
• -ve
• -ve rods
• Salmonella
• Colorless
• NLF
• NLF
• +ve
• -ve
• -ve rods
• Shigella
• Colorless
• NLF/H2S
• NLF
• +ve
• -ve
• -ve rods
• Proteus
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THANK YOU VERY MUCH

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