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Lab Identification of Enterobacteriacae

General properties Gram negative rod Some are major components of intestinal NF Widely distributed Oxidase negative and Catalase positive Ferment glucose rather than oxidized Some are motile Don't form spore Grow well on MacConkey agar Reduce nitrate to nitrite

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Human diseases

Urinary and gastrointestinal infection Pneumonia and meningitis Abscesses and wound infection Septicemia Important species and diseases E. coliA. UTI B. Diarrhoea1. Enteropathogenic E. coli (EPEC) cause infantile enteritis2. Enterotoxigenic (ETEC) cause travellers diarrhoea3. Enteroinvasive (EIEC) resemble shigelloses 4. Verocytotoxigenic (VITE) cause haemorrhagic colitis (bloody diarrhoea) Haemolytic uraemia syndrome C. SepsisD. Meningitis in infants (K1 antigen capsule type)

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Important species and diseases

Shigellae Bloody and pus diarrhoea Shigellae dysenteriae and S. sonnei, S. flexneri Salmonella spp Salmonella thyphi (typhoid fever) Bacteremia and enterocolitis Klebsiella spp K. pneumoniae K. oxytaca Pneumoniae, UTI and bacteremia Hospital acquired infections

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Important species and diseases

Proteus spp UTI, bacteremia Kidney stone P. mirabilis and P. vulgaris Enterobacter spp UTI, nosocomial pathogen E. aerogenes (capsulated) E. cloacae and E. agglomerans

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Important species and diseases

Serratia spp Opportunistic hospital pathogen Neonatal bacteremia and pneumonia S. marcescens and S. liquifaciens Citrobacter spp UTI C. freundii Yersinia spp Y. pestis (plague) Y. enterolitica

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Important species and diseases

Other less encountered spp Providencia, Hafnis and Erwinia

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Laboratory diagnosis

Presumptive identification

1. Gram stained preparation (-v rod or cocco bacilli)

2. Colony morphology on blood agar Large Gray, dry, Mucoid or swarming

3. Red colonies on MacConkey agar Indicate organism can form acid

4. Biochemical tests for species identification

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(-v rod) MacConkey agar

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Group members differentiation

1. Glucose fermentation positive2. Cytochrome oxidase test is negative3. Nitrate is reduced to nitrite Carbohydrate utilization Not all carbohydrates are sugar Fermentation term is also misused Fermentation is an oxidation-reduction Take place in an anaerobic environment Substrate serve as final (H) acceptor Process detected by PH indicators Acidification may result from other pathway All bacteria metabolize carbohydrates are facultative anaerobic Utilization may not be under strictly anaerobic conditions

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Basic principle of fermentation

Pyruvic acid

Acetaldehyde Succinic acid Butanediol

Glucose Formic acid

Propionic acidEthyl alcohol

H2+CO2

Acetoin

Acetyl COA

Acetic acid Butyric acid +Alcohol

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B- Galactosidase and PNPG test

To detect galactosidase enzyme Useful for lactose fermenters such as Some E. coli strain from Shigella sonnei Some strains of Citrobacter and S. arizonae (ONPG +V) from Salmonella spp (ONPG –v) The test measure enzyme only

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lactose

glucose

galactose

β - galactoside bond

β – galactoside permease

galactose

glucose B-galactosidase

Glucose + galactose

glucose Pyruvic acid

Mixed acids

Process of lactose in Enterobacteriacae

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OPNG

O- Nitrophenol -B-galactosidase (colourless)

Enzyme ( B- galactosidase)

O – Nitrophenol (yellow)

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Cytochrome Oxidase test

Do not use steel or nichrome wire loop Tetramethyl - p-phenylenediamine when it takes the electron from the last

element (cytochrome oxidase) in the electron transport chain.

Positive result give blue colour (10-20 second)

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Detection of carbohydrates fermentation

Media ingredients Trypticase, NaCl,

phenol red, carbohydrate, distilled water and PH adjusted to 6.8

Fermentation indicated by yellow colour and gas formation by Durham tube

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Kliger Iron agar or Triple sugar iron agar

Media contain two or three sugar

Contain ferric PH indicator (phenol red) Nitrogen source To differentiate lactose

from non lactose Fermenter (KIA)

Fermentation of sucrose (TSIA)

Detection gas Detection of hydrogen

sulphide production

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Indol production

ability of the organism to split indole from the amino acid tryptophan

Tryptophanase enzyme Kovac's reagent (isoamyl

alcohol, p-Dimethylaminobenzaldehyde, concentrated hydrochloric acid) to the culture broth

Results: Positive result red color

(occurring within a few seconds) Negative result yellow color

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Methyl Red Test

MR test is a quantitative test for acid production, requiring positive organism to produce strong acids (lactic, acetic, formic) from glucose via the mixed acid fermentation pathway

only those organism that can maintain low PH of about ph 4-4.5 can be called

methyl red – positive organisms that are MR (+) are

always VP (-)

Klebsiella

E. coli

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Voges proskauer Test

Acetoin Diacethyl KOH+Air + Naphthol+creatine

Red complex

Klebsiella

E. coli

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a test depends upon the ability of the organism, to utilize citrate as the sole source of carbon and energy growth

Simmon’s Citrate Agar Kosher’s Media bromthymol blue, a pH indicator

with a range of 6.0 to 7.6 uninoculated Simmon's

citrate agar has a pH of 6.9, so it is an intermediate green color (neutral pH)

Growth of bacteria in the media leads to development of a Prussian blue color at more alkaline pH's (around 7.6) (positive citrate)

Citrate Test

Klebsiella, Salmonella, Citrobacter Escherichia

and Shigella