Enterobacteriaceae

Lab Thirteen:- ________________ Medical Microbiology
Prepared by: Luma J. Witwit



Enterobacteriaceae

The enteric bacteria members of the family Enterobacteriaceae are the most commonly isolated normal or pathogenic microbes accounting for the most frequent cause of diarrhea and associate illnesses as well as over 50% of nosocomial infections(including gastroenteritis, respiratory and urinary tract infections). They are gram negative bacilli group that are non-spore-forming ) frequently motile ( with the exception of Shigella and Klebsiella sp.). facultative anaerobes(oxidaes negative ) that ferment glucose, reduce nitrates to nitrite, and break down peroxide( catalase positive). Although their normal habitat is the intestinal tract of humans and other animals, they may also be found in soil, water, and decaying material.

Based on their ability to ferment lactose, the enterics can be further subdivided into Coli forms(normal as well as opportunistic enterics that ferment lactose within 48 hours) for example: Escherichia coli, Klebsiella, Enterobacter, Citrobacter, Non-Coli forms (normal or pathogenic enterics that either fail to ferment lactose or do so with extremely slow kinetics) for example: opportunistic: Proteus, Providencia , Edwardsiella, Hafnia and Serratia.

Pathogenic: Salmonella , Shigella , Yesinia . The various genera of enteric bacilli can be differentiated by using selective and differential media and biochemical tests .

Specimen
Urine , stools , blood, pus, spinal fluid, sputum, soil and water .

Lab diagnosis
1- Gram stain ( gram negative bacilli )
2- Streaking on differential media

a) E.M.B ( Eosine Methylene Blue )

Which gave excellent differentiation of E. coli and Klebsiella aeroginosa , The E-coli colonies usually show a dark center and have a greenish metallic sheen , colonies of Klebsiella aeroginosa are usually much larger than typical E. coli , pink , mucoid.


b) MacConkey agar

It gives amore clear – cut differential between the enteric pathogens lactose fermenter and non lactose fermenter .

The colonies of coli form bacteria are red in color and may be surrounded by a zone of precipitated bile . This reaction is due to the action of the acids , produced by fermentation of lactose , The colonies of non lactose fermenter bacilli do not greatly alter the appearance of the medium . these colonies give an alkaline reaction , are uncolored and transparent.

3- IMVC test :-

a) Indol test

Certain bacteria such E .coli have the ability split the amino acid typtophane into indol and pyruvic acid , indol which indicate by adding the kovacs regent after inoculating the tube contain peptone water (1%) for 18-24 at 37C .

b) Methyl-Red test ( Mixed acid fermentation ) A number of gram negative intestinal bacteria can differentiated on the basis of the end product produce when ferment glucose in MR-VP medium for (24-48 h) at 37C produce larger amounts of lactic , acetic , succinic and formic plus CO2 , H2 and ethanol , these organisms are generally great gas producers, too because they produce the enzyme formic hydrogenase , which splits formic acid into equal parts CO2 , H2 .

Indicator dropping bottle of methyl red. add three – four drops of methyl red to tube which was inoculated with E.coli the tube should became red immediately.

c) Voges – Proskaur test ( Butanediol fermentation )

A negative methyl red test may indicate that these organisms being test produced a lot of( 2,3 butanediol) and ethanol instead of acids .

Indicator consist of alpha-naphthol + KOH 40% to detect acetyl methyl carbinol .


d) Citrate Utilization

The ability of some organisms to utilize citrate as a source of carbon . Simmons citrate agar is used to detect this ability .
Indicator is bromothymol blue in this media will change from green to blue .

e) Triple Sugar Iron Agar (TSI)

This is a most useful differential tube medium in the study of the gram negative intestinal microorganisms ( Salmonella , Shigella ) it contain at least two sugars glucose and lactose . some contain a third sugar sucrose , it also contain phenol red to indicate when fermentation occurs , in addition of iron salts for detection of H¬2S .

Reading results :-

1- Red slant and yellow bottom with or without gas (K/A) indicates fermentation of glucose sugar .

2- Yellow slant and bottom with or without gas formation indicates fermentation of the lactose and glucose ( A/A )

3- No change indicates that no fermentation of all sugars in this media.

4- Urease test :-

The test used for detection of Proteus and other Gram negative intestinal bacteria which are capable of splitting urea by producing urease enzyme .


Urease hydrolyze urea to ammonia and carbon dioxide .
Alkalinization of the urine by ammonia can cause magnesium phosphate and calcium phosphate to be come super saturated and crystallize out of solution to form stones .

5- Motility test :-
The test differentiate between motile bacteria from non motile bacteria by using the sterile medium is inoculated by stabbing through the center of the medium and is incubated at the proper temp for 24-48 hours .