This document discusses the identification and properties of Enterobacteriaceae, a family of Gram-negative bacteria commonly found in the intestines. Key points include: - Enterobacteriaceae are Gram-negative, non-spore forming rods that are oxidase-negative and catalase-positive. Many cause opportunistic infections in humans. - Important pathogenic genera include Escherichia coli, Salmonella, Shigella, Klebsiella, Proteus, Enterobacter, Citrobacter, and Yersinia. They can cause diseases like urinary tract infections, diarrhea, pneumonia, and sepsis. - Identification involves examining colony morphology, growth on MacConkey agar, and conducting

1. 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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2. Human diseases
 Urinary and gastrointestinal infection
 Pneumonia and meningitis
 Abscesses and wound infection
 Septicemia
 Important species and diseases
 E. coli
A. UTI
B. Diarrhoea
1. Enteropathogenic E. coli (EPEC) cause infantile enteritis
2. Enterotoxigenic (ETEC) cause travellers diarrhoea
3. Enteroinvasive (EIEC) resemble shigelloses
4. Verocytotoxigenic (VITE)
 cause haemorrhagic colitis (bloody diarrhoea)
 Haemolytic uraemia syndrome
C. Sepsis
D. Meningitis in infants (K1 antigen capsule type)
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3. 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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4. 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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5. 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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6. Important species and diseases
 Other less encountered spp
 Providencia, Hafnis and Erwinia
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7. 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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8. (-v rod) MacConkey agar
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9. Group members differentiation
1. Glucose fermentation positive
2. Cytochrome oxidase test is negative
3. 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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10. Basic principle of fermentation
Glucose Formic acid
Pyruvic acid
Acetyl COA
Acetic acid Butyric acid +Alcohol
Acetaldehyde Succinic acid Butanediol
Ethyl alcohol Propionic acid
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H2+CO2
Acetoin

11. 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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12. Process of lactose in Enterobacteriacae
lactos
e
glucose
galactose
β - galactoside bond
β – galactoside permease
glucose B-galactosidase
galactose
Glucose + galactose
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Mixed acids
glucose Pyruvic
acid

13. OPNG
O- Nitrophenol -B-galactosidase (colourless)
Enzyme ( B- galactosidase)
O – Nitrophenol
(yellow)
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14. 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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15. 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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16. 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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17. 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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18. 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
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E. coli

19. Voges proskauer Test
Acetoin Diacethyl KOH+Air +Naphthol+creatine
Red complex
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Klebsiella
E. coli

20. Citrate Test
 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)
Klebsiella,
Salmonella,
Citrobacter Escherichia
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and
Shigella