Enterobacteriacea ii biochemical reaction 2بكتريا عملي

Enterobacteriaceae (2)Biochemical reactions

ByDr. Nabil El Aila

Assistant Professor of Molecular MicrobiologyMedical Technology Department

Al -Aqsa University

Dr. Nabil El AilaDiagnostic Microbiology

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Characters of Enterobacteriaceae• All Enterobacteriaciae

– Gram-negative rods

– Ferment glucose with acid production

– Reduce nitrates into nitrites

– Oxidase negative

• Facultative anaerobic

• Motile exceptShigella and Klebsiella

• Non-capsulated except Klebsiella

• Non-fastidious

• Grow on bile containing media (MacConkey agar) Dr. Nabil El Aila

Diagnostic Microbiology

Enterobacteriaceae

• Some Enterobacteriaceae are true pathogens– Salmonella spp.– Shigella spp.– Yersinia spp.– Certain strains of E. coli (ETEC, EPEC, EIEC, EHEC)

• Most members of the Enterobacteriaceae are opportunisticor cause secondary infections of wounds, the urinary and respiratory tracts, and the circulatory system e.g. E. coli.

• Enterobacteriaceaedivided into TWO main groups according to action on LACTOSE– Lactose Fermenters (LF)

• E. coli, Citrobacter, Klbesiella, Enterobacter

– Lactose Non-Fermenters (LNF)• Salmonella, Shigella, Proteus, Yersinia Dr. Nabil El Aila


Identification of Enterobacteriaceae

• Gram stain– All Enterobacteriaceae are Gram-negative rods

– Arranged in single



Biochemical reactions

• Oxidase test

– All members of Enterobacteriaceae are oxidase negative

– Pseudomonas is oxidase positive

• O/F test

– All members of Enterobacteriaceae are O+/F+

– Pseudomonas is O+/F-

• Nitrate reductase

– All members of Enterobacteriaceae are nitrate reductase positive

– Pseudomonas is nitrate reductase negative

Classification of Enterobacteriaceae

Enterobacteriaceae

Lactose fermentersE. coli, Citrobacter,

Klebsiella, Enterobacter

Non-lactose fermenterSalmonell, ShigellaProteus, Yersinia

�There are several selective and differential media used toisolate distinguishes between LF & LNF

�The most important media are:�MacConkey agar�Eosin Methylene Blue (EMB) agar�Salmonella Shigella (SS) agar�In addition to Triple Sugar Iron (TSI) agar

Differentiation between LF & NLF by Growth on MacConkey agar

MacConkey AgarContains

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 differentialpH indicator

Acidic: Pink

Lactose feremnters

Pink colonies

Lactose non feremnters

colorless colonies

Classification of Enterobacteriaceae according to lactose

fermentation (growth on MacConkey Agar)

Enterobacteriaceae

Lactose Fermenters Lactose Non-Fermenters

Escherichia coliKlebsiella sppEnterobacter sppCitrobacter spp

Salmonella sppSchigella sppProteus sppYersinina spp

Pink colonies Colorless colonies

AcidNeutral red

No acid



Differentiation between LF & NLF by Growth on MacConkey agar

• Method:

– MacConkey agar is inoculated with tested organism using

streak plate technique

– Incubate the plate in incubator at 37 C/24 hrs

• Results:

– LF organism appears as pink colonies (e.g. E. coli)

– NLF organism appears as colorless colonies (e.g. Shigella)

Flame & Cool

Flame & Cool

Flame & Cool

1 23

45

Growth of Enterobacteriaceae on

MacConkey agar

Uninoculated plate Lactose non feremtersSalmonella, Shigella,

Proteus

Lactose feremtersE. coli, Citrobacter

Klebsiella, Enterobacter

Colorless colonies Pink colonies


Reaction on Salmonella Shigella (SS) agar• SS agar is a selective & differential medium used for isolation of

Salmonella and Shigella• The selective agents are bile salts, and brilliant green dye, which inhibit

gram-positive organisms• The medium contains only lactoseas a differential agent and thus

differentiates on the basis of lactose fermentation• The formation of acid on fermentation of lactose causes the neutral red

indicator to make pink colonies• Non lactose fermenting organisms are colorless on the medium• SS agar contains sodium thiosulfateand ferric ammonium citrate

allows the differentiation of organisms that produce H2S– Lactose fermenters, such as E. coli, have colonies which are pink– Shigella appears transparent or amber– Salmonella appears transparent or amber with black centers due to

H2S production

LactoseLactose fermenter

AcidNeutral red

Pink colonies

Ferrous sulfideBlack precipitate

H2S + Ferric ammonium citrate

Identification of EnterobacteriaceaeDifferentiation between LF & NLF by Growth on SS agar

• Method:

– SS agar is inoculated with tested organism using

streak plate technique

– Incubate the plate in incubator at 37 C/24 hrs

Flame & Cool

Flame & Cool

Flame & Cool

1 2

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A Klebsiella pneumoniaeB Escherichia coliC Salmonella spD Proteus mirabilisE Ps. aeruginosa

Both are lactose fermenters

Both Salmonella sp. & Proteus product H2S

Pseudomonas colonies are nearly colorless

Growth of Enterobacteriaceae on SS agar

Reaction on Triple Sugar Iron (TSI) Agar• TSI contains

– Enzymatic Digest of Casein, Enzymatic Digest of Animal Tissue,

and Yeast Enriched Peptone provide the nitrogen, carbon, and

vitamins required for organismgrowth.

– Three different types of sugars

• Glucose (1 part)

• Lactose (10 part)

• Sucrose (10 part)

– Phenol red (acidic: Yellow)

• TSI dispensed in tubes with equal butt & slant

Reaction on Triple Sugar Iron (TSI) Agar

• Principle

– To determine the ability of an organism to attack a specific

carbohydrateincorporated into a basal growth medium, with

or without the production of gas, along with the determination

of possible hydrogen sulphideproduction.

• When the carbohydrates are fermented, acid production is detectedby the Phenol Red pH indicator.

• SodiumThiosulfateis reduced to hydrogen sulfide, and hydrogensulfide reacts with an iron salt yielding the typical black iron sulfide.

• Ferric Ammonium Citrate is the hydrogen sulfide (H2S) indicator. SodiumChloride maintainsthe osmotic balanceof the medium. Agar is the solidifying agent

Reaction on TSI

• Method:

– Inoculate TSI medium with an organism by

inoculating needle by stabbing the butt and

streaking the slant

– Incubate at 37°C for 24 hours


Example

Result

Reaction on TSI

H2SSlant

color

Butt

color

Non fermenter

e.g.

Pseudomonas

Alk/Alk/-

(No action on sugars)NegativeRedRed

LNF

e.g. ShigellaA/Alk/-

(Glucose fermented without H2S)

Negative

RedYellow

LNF

e.g. Salmonella &

Proteus

A/Alk/+ (Glucose fermented

with H2S)

Positive

black in

buttRedYellow

LF

e.g. E. coli,

Klebsiella,

Enterobacter

A/A/-

(three sugars are

fermented)

NegativeYellowYellow

Result

EMBSSMacConkey

O/FNitrate reductase

OxidaseGram stain

Metallic sheen

LFLFO+/F++ve-ve-ve rodE. coli

DarkLFLFO+/F++ve-ve-ve rodsCitrobacter

DarkLFLFO+/F++ve-ve-ve rodsKlebsiella

DarkLFLFO+/F++ve-ve-ve rodsEnterobacter

ColorlessNLF/H2S

NLFO+/F++ve-ve-ve rodsSalmonella

ColorlessNLFNLFO+/F++ve-ve-ve rodsShigella

ColorlessNLF/H2S

NLFO+/F++ve-ve-ve rodsProteus

Summary of morphology, cultural characteristics, and biochemical reactions of Enterobacteriaceae

MotilityUreaseCitrateVPMRIndoleTSI

Motile-ve-ve-ve+ve+veA/A/-E. coli

Motile-ve+ve-ve+ve+veA/A/-Citrobacter freundii

Non motile

+ve+ve+ve-ve-veA/A/-Klebsiella pneumoniae

Motile+ve+ve+ve-ve-veA/A/-Enterobacter cloacae

Motile-ve+ve-ve+ve-veA/Alk/+Salmonella typhi

Non motile

-ve-ve-ve+ve-veA/Alk/-Shigella boydii

MotileSwarwing

+ve+ve-ve+ve-veA/Alk/+Proteus mirabilis

Summary of morphology, cultural characteristics, and biochemical reactions of Enterobacteriaceae

Lysine Iron Agar (LIA)• Lysine iron agar (LIA) slants test organisms for the ability to

deaminate lysine or decarboxylate lysine.Lysine deaminationis an aerobicprocess which occurs on the slant of the media.Lysine decarboxylation is an anaerobicprocess which occurs in the buttof the media.

• LIA slants contain lysine, glucose, peptones, bromcresol purple (pH indicator), sodium thiosulfate and ferric ammonium citrate. If the organism has the ability to decarboxylate lysine, it produces an amine end-product which reacts with the pH indicator to give a purple color in the butt of the tube.

(Negative decarboxylation: yellow butt).

• If the organism has the ability to deaminate lysine, the ammonia produced will react with the ferric ammonium citrate to produce a dark red color on the slant of the tube. (Negative deamination: purple slant). Organisms which produce hydrogen sulfide gas will exhibit a black precipitate in the butt of the tube.

Lysine Iron Agar (LIA)• This agar is used as a diagnostic test for salmonellae.

• Salmonellae are the only group of Enterobacteriaceae that regularly

decarboxylate lysin(by lysine decarboxylase) and produce large

amounts of hydrogen sulphide.

• Bacteria that decarboxylate lysine cause an alkaline reaction (purple

colour) throughout the medium.

• Those that do not, produce an alkaline slant and an acid butt

(yellow) due to fermentation of glucose.

• Some bacteria like proteus speciesmay deaminate the lysine and

produce a red slant and acid butt.

• Production of hydrogen sulphidecauses a blackening in the

medium due to formation of ferrous sulphide. The indicator is

bromcresol purple.

Tube 1: Positive decarboxylation (butt), negative deamination (slant)Tube 2: Negative decarboxylation (butt), positive deamination (slant)


Uninoculated medium Alkaline/alkaline/H2S:

Salmonella.

3. Alkaline/yellow/H2S:

Citrobacter.

Lysine Iron Agar (LIA)

COLLECTION, TRANSPORT, AND EXAMINATIONOF FAECES ( STOOL SPECIMENS )

Possible pathogens

Gram Positive Gram Negative

• Clostridium perfringens Shigella species

type A and C Salmonella species

• Clostridium difficile Campylobacter species

Bacillus cereus ( toxin ) Escherichia coli

• Staphylococcus aureus(toxin) ( ETEC,EIEC,EPEC)

Vibrio cholerae 01

Other Vibrio species

Yersinia enterocolitica

• Viruses : mainly rotaviruses, adenoviruses, coxsackieviruses, echoviruses, and

polioviruses.

• Fungi : Candida albicans .Dr. Nabil El Aila


Parasites :Eggs : Amoebae :

Ascaris lumbricoides Entamoeba histolytica

Hookworm Flagellates:Trichuris trichiura Giardia lamblia

Schistosoma species Trichomonas hominis

Hymenolepis species Ciliates :Diphylobothrium latum Taenia species

Entrobius vermicularis Balantidium coli,

Larvae:Strongyloides stercoralisCysts :Entamoeba histolytica

Giardia lamblia

Balantidium coli

Isospora belli ( oocysts )

CommensalsThe normal microbial flora of the gastrointestinal tract is greatly influenced by diet. Microorganisms, which may form part of this normal flora, include:Gram Positive Gram NegativeEnterococci Escherichia coli*Anaerobic streptococci Proteus*Lactobacilli Enterobacter*Clostridia Hafnia *

Citrobacter*Providencia*

Morganella*Serratia*Klebsiella *Bacteroides speciesPseudomonas aeruginosa

• These genera belong to the family Enterobacteriaceae. Enterobacteria are often described as coliforms.

• Fungi: Candida species and yeasts.• Other: Mycoplasma and a variety of protozoa.

Collection of stool sample

• Stool specimens are usually collected in a clean, dry disinfectant free

bedpan or suitable wide necked container.

• The container need not be sterile, ask the patient to avoid

contaminating the stool with urine.

• Transfer a portion of the specimen especially which contains mucus,

pus or bloodinto a clean dry leak-proof container.

• A diarrhealstool usually gives good results.

• Label the specimen, on the container not lid, and send it with a request

form to reach the laboratory within 1 hour.

• Stool passed into the toilet bowel must not be used for culture.

• No toilet paper should be placed in the bed pan or specimen container,

which may contain bismuththat interferes with the laboratory tests.

Collection of stool sample

• If it is not possible to obtain stool specimen, a rectal swab may be used

to obtain the sample by inserting a cotton swab into the anus beyond

the anal sphincter for about 10 seconds, carefully rotate the swab and

withdraw.

• If delayover 18-24 hours is suspected, the specimen should be mixed

with an equal volume of buffered glycerol saline. Also you can insert

the swab in a container of sterile cary - Blair transport medium.

• Salmonella, Shigella , Vibrio and Yersenia species survive well in cary-

Blair medium for up to 48 hours but Campylobacter for up to 6 hours.

• If cholera is suspected transfer about 1ml of specimen into 10ml of

sterile alkaline peptone water , label and send to the microbiology

department within 8 hours of collection.Dr. Nabil El Aila


Suspected organisms

1- E-coli (infant &NLF)2- salmonella & shigella3- campylobactorjejuni most common human pathogen

indol&citrate (+)urease (- )oxidase (+) nalidixic acid (S) cephalo thin (R)


Suspected organisms

• 4- V.choleraeserogroup -01

catalse (+)oxidase (+)motile (+)lactose (NLF)sensitive to(dry – sunlight – acid pH)TCBS (thiosulfate citrate bile sucrose

Agar –yallow colonies)

Stool or Rectal swab culture*MacConky plate (NLF)

*XLD agar*HE

*selenite F broth/ GN broth

*TCBS (y.colores)*alkaline peptone water (APW)

*Selective media for campylobactor(skirrow&blaser with suplements)campylobactor microaerophilic

(42C°-O2 5%-Co210%-N2 85%-48hr)

Direct Culture SS agar

Stool/Rectal swab

GN/Selenite broth

Subculture Hecktoen

Check for suspected colonies

Incubate Overnight 37C°



Confirmation using Biochemical and serological reaction

Detection of Salmonella & Shigella

Salmonella on SS-agar: Salmonella on Hektoen agar:

Salmonella on XLD Gram stain of Salmonella

Bichemical reactions of Salmonella on Triple Sugar Iron


Bichemical reactions of Salmonella on API20E

Salmonella on SS-agar: Salmonella on Hektoen agar:

Salmonella on XLD Gram stain of Salmonella

Bichemical reactions of Salmonella on Triple Sugar Iron


Shigella on SS-agar:Shigella on Hektoen agar:

Shigella on XLDGram stain of Shigella



API20E

Direct Blood agar

Stool/Rectal swab

Alkaline peptone water

Subculture TCBSCheck for suspected colonies




Confirmation using Biochemical and serological reaction

Detection of VibrioCholera

Direct TCBS


Vibrio cholerae TCBSDr. Nabil El Aila


Health & Medicine

Enterobacteriacea ii biochemical reaction 2بكتريا عملي