Gram-negative rodsGram-negative rodsEnterobacteriaceaEnterobacteriacea

Clinical MicrobiologyClinical Microbiology

MESHACK JUMAMESHACK JUMA

MEDICAL MICROBIOLOGY DEPT (UONMEDICAL MICROBIOLOGY DEPT (UON((

Msc. Med Micro (UON), PGD-RM(UONMsc. Med Micro (UON), PGD-RM(UON(, (,

Bsc. Med Micro(JKUAT), Dip MLS, HND Dx MicroBsc. Med Micro(JKUAT), Dip MLS, HND Dx Micro..

Family Family EnterobacteriaceaeEnterobacteriaceae often referred to as often referred to as

“enterics“enterics””..

Bacteria

Gram positive Gram negative

Cocci Bacilli Cocci Rods

Characters of Characters of EnterobacteriaceaeEnterobacteriaceaeAll All EnterobacteriaciaeEnterobacteriaciae– Gram-negative rodsGram-negative rods

– Ferment glucose with acid production Ferment glucose with acid production

– Reduce nitrates into nitritesReduce nitrates into nitrites– Oxidase negativeOxidase negative– Catalase positiveCatalase positive

Facultative anaerobicFacultative anaerobic

All motile All motile exceptexcept Shigella, Klebsiel Shigella, Klebsiel and and YersiniaYersinia

Non-capsulated except Non-capsulated except KlebsiellaKlebsiella

Non-fastidiousNon-fastidious

Grow on bile containing media (MacConkey agar( Grow on bile containing media (MacConkey agar(

Microscopic and Colony Microscopic and Colony MorphologyMorphology

Gram negative bacilli or coccobacilliGram negative bacilli or coccobacilliNon-spore formingNon-spore formingColony morphology on BA or MH of little Colony morphology on BA or MH of little value, as they look the same, except for value, as they look the same, except for KlebsiellaKlebsiellaSelective and differential media are used Selective and differential media are used for initial colony evaluation (ex. for initial colony evaluation (ex. MacConkey, Hektoen Enteric (HE), MacConkey, Hektoen Enteric (HE), Xylose lysine deoxycholate (Xylose lysine deoxycholate (XLDXLD(( agars agars((

EnterobacteriaceaeEnterobacteriaceaeSome Some Enterobacteriaceae Enterobacteriaceae are true pathogensare true pathogens– SalmonellaSalmonella spp. spp. – ShigellaShigella spp. spp. – Yersinia Yersinia spp. spp. – Certain strains of Certain strains of E. coli E. coli (ETEC, EPEC, EIEC, EHEC( (ETEC, EPEC, EIEC, EHEC(

Most members of the Most members of the Enterobacteriaceae Enterobacteriaceae are are opportunistic or cause secondary infections of opportunistic or cause secondary infections of wounds, the urinary and respiratory tracts, and wounds, the urinary and respiratory tracts, and the circulatory system e.g. the circulatory system e.g. E. coliE. coli..Enterobacteriaceae divided into TWO main Enterobacteriaceae divided into TWO main groups according to action on LACTOSEgroups according to action on LACTOSE– Lactose Fermenters (LF(Lactose Fermenters (LF(

E. coli, Citrobacter, Klbesiella, EnterobacterE. coli, Citrobacter, Klbesiella, Enterobacter– Lactose Non-Fermenters (LNF(Lactose Non-Fermenters (LNF(

Salmonella, Shigella, Proteus, YersiniaSalmonella, Shigella, Proteus, Yersinia

Identification of Identification of EnterobacteriaceaeEnterobacteriaceae

Gram stainGram stain– All All EnterobacteriaceaeEnterobacteriaceae are Gram-negative rods are Gram-negative rods– Arranged in singleArranged in single

Identification of Identification of EnterobacteriaceaeEnterobacteriaceaeBased on Biochemical reactionsBased on Biochemical reactions

Can be grouped into threeCan be grouped into three:-:-

A) Ability to ferment carbohydrates to acid or A) Ability to ferment carbohydrates to acid or Acid and gasAcid and gas..

B) Ability to utilize a substrate as the sole B) Ability to utilize a substrate as the sole source of carbonsource of carbon

C) Ability to enzymaticaly degrade a C) Ability to enzymaticaly degrade a compoundcompound

Identification of Identification of EnterobacteriaceaeEnterobacteriaceaeBiochemical reactionsBiochemical reactions

Oxidase testOxidase test

– All members of All members of EnterobacteriaceaeEnterobacteriaceae are oxidase negative are oxidase negative

– PseudomonasPseudomonas is oxidase positive is oxidase positive

Oxidative/Fermentation testOxidative/Fermentation test

– All members of All members of EnterobacteriaceaeEnterobacteriaceae are O+/F+ are O+/F+

– PseudomonasPseudomonas is Oxidative+ / Fermentation - is Oxidative+ / Fermentation -

Nitrate reductaseNitrate reductase

– All members of Enterobacteriaceae are nitrate reductase positiveAll members of Enterobacteriaceae are nitrate reductase positive

– PseudomonasPseudomonas is nitrate reductase negative is nitrate reductase negative

See & compare these tests for See & compare these tests for Pseudomonas spp Pseudomonas spp in the Labin the Lab

Classification of Classification of EnterobacteriaceaeEnterobacteriaceae

Enterobacteriaceae

Lactose fermentersE. coli, Citrobacter,

Klebsiella, Enterobacter

Non-lactose fermenterSalmonell, ShigellaProteus, Yersinia

There are several selective and differential media used to isolate distinguishes between LF & LNFThe most important media are:

MacConkey agarEosin Methylene Blue (EMB) agarSalmonella Shigella (SS) agarIn addition to Triple Sugar Iron (TSI) agar

Differentiation between LF & NLF byDifferentiation between LF & NLF by Growth on MacConkey agarGrowth on MacConkey agar

MacConkey Agar

Contains

Bile salts Crystal violet Lactose Neutral red

MacConkey agar is selective & differential medium for MacConkey agar is selective & differential medium for EnterobacteriaceaeEnterobacteriaceae

Inhibit growth of G+ve bacteria

Cause of selectivity

Cause of differentialpH indicatorAcidic: Pink

Lactose feremntersPink colonies

Lactose non feremnterscolorless colonies

Classification of Classification of EnterobacteriaceaeEnterobacteriaceae according to according to lactose fermentation (growth on MacConkey Agar)lactose fermentation (growth on MacConkey Agar)

Enterobacteriaceae

Lactose Fermenters Lactose Non-Fermenters

Escherichia coliKlebsiella sppEnterobacter sppCitrobacter spp

Salmonella sppSchigella sppProteus sppYersinina spp

Pink coloniesColorless colonies

AcidNeutral red

No acid

Identification of Identification of EnterobacteriaceaeEnterobacteriaceaeDifferentiation between LF & NLF byDifferentiation between LF & NLF by Growth on MacConkey agarGrowth on MacConkey agar

Method:Method:

– MacConkey agar is inoculated with tested organismMacConkey agar is inoculated with tested organism using streak plate techniqueusing streak plate technique

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

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

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

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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) agarReaction on Salmonella Shigella (SS) agarSS agar is a selective & differential medium used for isolation of SS agar is a selective & differential medium used for isolation of SalmonellaSalmonella and and ShigellaShigellaThe selective agents are The selective agents are bile saltsbile salts, and , and brilliant green dyebrilliant green dye, which , which inhibit gram-positive organismsinhibit gram-positive organismsThe medium contains only The medium contains only lactoselactose as a differential agent and thus as a differential agent and thus differentiates on the basis of lactose fermentationdifferentiates on the basis of lactose fermentationThe formation of acid on fermentation of lactose causes the neutral The formation of acid on fermentation of lactose causes the neutral red indicator to make pink coloniesred indicator to make pink coloniesNon lactose fermenting organisms are colorless on the mediumNon lactose fermenting organisms are colorless on the mediumSS agar contains SS agar contains sodium thiosulfatesodium thiosulfate and and ferric ammonium ferric ammonium citratecitrate allows the differentiation of organisms that produce H2S allows the differentiation of organisms that produce H2S– Lactose fermenters, such as Lactose fermenters, such as E. coliE. coli, have colonies which are , have colonies which are

pinkpink– ShigellaShigella appears transparent or amber appears transparent or amber– SalmonellaSalmonella appears transparent or amber with black centers appears transparent or amber with black centers

due to H2S productiondue to H2S production

Lactose Lactose fermenter Acid Neutral red Pink coloniesFerrous sulfideBlack precipitate

H2S + Ferric ammonium citrate

Identification of Identification of EnterobacteriaceaeEnterobacteriaceaeDifferentiation between LF & NLF byDifferentiation between LF & NLF by Growth on SS agarGrowth on SS agar

Method:Method:

– SS agar is inoculated with tested organismSS agar is inoculated with tested organism using using

streak plate techniquestreak plate technique

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

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AA.. Klebsiella pneumoniaeKlebsiella pneumoniaeBB.. Escherichia coliEscherichia coliCC: : Salmonella spSalmonella sp..DD: : Proteus mirabilisProteus mirabilis EE: : Ps. aeruginosaPs. aeruginosa . .

BothBoth are lactose fermentersare lactose fermenters

BothBoth Salmonella sp. & Proteus Salmonella sp. & Proteus product H2Sproduct H2S

PseudomonasPseudomonas colonies are nearly colorlesscolonies are nearly colorless

Growth of Enterobacteriaceae on SS agar

Growth of Growth of EnterobacteriaceaeEnterobacteriaceae on on EMB agarEMB agar

Coli-type colonies are very dark, almost black e.g. E. coli

Reaction on Triple Sugar Iron (TSI) AgarReaction on Triple Sugar Iron (TSI) Agar

TSI containsTSI contains

– Three different types of sugarsThree different types of sugars

Glucose (0.1%)Glucose (0.1%)

Lactose (1%)Lactose (1%)

Sucrose (1%)Sucrose (1%)

– Phenol red (acidic: Yellow)Phenol red (acidic: Yellow)

TSI dispensed in tubes with equal butt & slantTSI dispensed in tubes with equal butt & slant

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

carbohydrate incorporated into a basal growth medium, with or carbohydrate incorporated into a basal growth medium, with or

without the production of gas, along with the determination of without the production of gas, along with the determination of

possible hydrogen sulphide production. possible hydrogen sulphide production.

Reaction on TSIReaction on TSI

Method:Method:– Inoculate TSI medium with an organism by Inoculate TSI medium with an organism by

inoculating needle by stabbing the butt and inoculating needle by stabbing the butt and streaking the slantstreaking the slant

– Incubate at 37°C for 24 hoursIncubate at 37°C for 24 hours

Reaction on TSIResult

Example

Butt color Slant color H2S

Yellow RedNegative A/Alk/-

(Glucose fermented)LNF

e.g. Shigella

Yellow RedPositive

black in buttA/Alk/+

(Glucose fermented with H2S)LNF e.g. Salmonella &

Proteus

Yellow Yellow NegativeA/A/-

(three sugars are fermented)LF e.g. E. coli,

Klebsiella,Enterobacter

Red Red NegativeAlk/Alk/-

(No action on sugars)Non fermenter e.g.

Pseudomonas

Results

Practicle WorkPracticle Work

Gram stainGram stain

Oxidase testOxidase test

Indole testIndole test

O/F test *O/F test *

Nitrate reductase test*Nitrate reductase test*

Growth on MacConkey’s agarGrowth on MacConkey’s agar

Growth on EMB agar*Growth on EMB agar*

Growth on SS agarGrowth on SS agar

Reaction on TSIReaction on TSI