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11.1 Enterobacteriaceae Quick Notes

This document discusses the Enterobacteriaceae family of bacteria, including their characteristics, methods of isolation and identification, and some common genera and species. Key points covered include their normal habitat in the human GI tract, biochemical testing used to identify them, and some important pathogens within the family.

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32 views5 pages

11.1 Enterobacteriaceae Quick Notes

This document discusses the Enterobacteriaceae family of bacteria, including their characteristics, methods of isolation and identification, and some common genera and species. Key points covered include their normal habitat in the human GI tract, biochemical testing used to identify them, and some important pathogens within the family.

Uploaded by

colleges660
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© © All Rights Reserved
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Enterobacteriaceae

• A very large family found normally in Human GI tract


• Gram negative rods
– Glucose fermenters
– Oxidase negative
– Most reduce nitrates to nitrites
– Most are motile with peritrichous flagella
– Facultative anaerobes
– Grow well on BAP (dull, gray colonies with variable hemolysis)
• Cause infections of almost every area of body

Serologic Characteristics
• O: somatic antigen (cell wall)
– Heat stable
– Lipopolysaccharide of cell wall—endotoxin
• K: envelope antigen (capsular)
– Heat labile
– Covers O (inhibits agglutination)—boil
• Vi: Salmonella typhi only
• H: flagellar antigen (found only in motile members)
– Heat labile
– Used to serotype within species

Isolation
• For all cultures but stools:
– BAP and Mac (or EMB)
• BAP:
– Proteus may swarm
– Klebsiella pneumoniae is mucoid

• Stools:
– Mac (or EMB) and selective agar (HE or SS) and enrichment broth (GN, selenite)
– Plus BAP, CIN (if Yersinia suspected), SMAC (if E. coli O157:H7), TCBS (if Vibrio), Campy
(if Campylobacter)
• Urines: BAP/Mac combo

Identification
• After growth on Mac, do oxidase test.
• If oxidase negative, do biochemical tests.
– Carbohydrate fermentation
– TSI agar
– IMViC
– Urease
– Deaminase reactions
– Decarboxylase reactions
– ONPG
– H2S
– Motility

Escherichia coli
• Normal flora of human lower GI tract
• Lactose positive, IMViC ++--
• Infections:
– UTI (90% of UTIs)
– E-coli O157 – media is Sorbitol MacConkey Agar (SMAC)- diarrhea without leukocytes

Shigella
• Lactose negative and nonmotile
• Four serogroups based on O antigens: (A) S. dysenteriae,(B) S. flexneri, (C) S. boydii, (D) S.
sonnei
• Causes bacillary dysentery (humans only natural host for Shigella)
• shigellosis - diarrhea with many leukocytes
• ONPG differentiates S. sonnei +, S. flexneri -
• clear H2S – ve colony on selective media HE, SS, XLD

Klebsiella-Enterobacter-Serratia-Hafnia Group
• VP positive, lactose positive
• Klebsiella (pneumoniae): IMViC --++
-Klebsiella oxytoca indole +ve

VP positive, nonmotile, urease positive, capsule


– Causes pneumonia

• Enterobacter: motile IMViC --++


– Opportunists: UTI, respiratory and wound infections
• Serratia: Dnase positive, motile
– (marcescens): pneumonia and septicemia in immunocompromised
– ½ pigmented red

Salmonella
• Complex genus (2200 serotypes—based on O and H typing)
• Cause enteric fevers—ingestion of fecally contaminated food/water– and gastroenteritis
• Lactose negative, H2S positive, motile, lysine positive

• clear H2S +ve colony on selective media HE, SS, XLD

Proteeae
• Lactose negative, H2S positive, phenylalanine deaminase positive
• Proteus:
– Urease positive and may swarm
– Wound infections and UTIs; pneumonia and septicemia
Citrobacter
• Lysine negative and citrate pos
• C. freundii and C. diversus
– UTIs and respiratory tract infections
– nosocomial

Edwardsiella
• E. tarda: turtles, snakes and reptiles
– Source of GI infection
– Wound infections and abscesses
– H2S positive, lactose negative, motile

Yersinia –

• Y. pestis (plague) zoonotic disease

• Y. enterocolitica (enterocolitis from eating pigs) Nonmotile at 37 C and motile at 25 C

Eosin-methylene blue (EMB)


• Selective and differential medium
– Inhibits gram positive organisms and allows growth of the more hardy gram negative rods
– Lactose fermenters: blue-black (green metallic sheen --E. coli)
– Nonlactose fermenters: colorless colonies

MacConkey (MAC) agar


• Selective and differential
• Used for the same thing as EMB—pick one
– Lactose fermenters: pink/red
– Nonlactose ferementers: colorless

Salmonella-Shigella agar (SS)


• Differential and moderately selective for the isolation of Salmonella and Shigella spp.
– May inhibit some Shigella
– Lactose fermenters: red/pink
– Nonlactose fermenters: H2S negative organisms produce colorless colonies; H2S positive
organisms produce colorless colonies with black centers

Hektoen Enteric Agar (HE)


• Differential and moderately selective medium used to isolate Salmonella and Shigella spp.
• Uninoculated HE is green
– Lactose fermenters: large and yellow to salmon color
– Nonlactoser fermenters: Shigella- green colonies; Salmonella= blue-green with black centers

Enrichment Broths
• Used to improve the recovery of Salmonella and Shigella spp.
• Inoculate first day with small portion of stool. Incubate for specified # hours and plate onto
selective media (HE or SS).

Carbohydrate utilization
• Fermenter: produces acids from a carbohydrate
• A single carbohydrate is added to basal medium containing pH indicator. When carbohydrate is
used, acids are formed and the color of the pH indicator changes.
• Adonitol, arabinose, dulcitol, glucose, lactose, maltose, mannitol, raffinose, salicin, sorbitol,
sucrose, xylose
• Gas produced?

ONPG test (orthonitrohphenyl-B-D-galactopyranoside)


• Detects b-galactosidase, the enzyme that cleaves lactose into glucose and galactose.
• Permease is not needed for ONPG to enter bacterial cells.
• ONPG is colorless; a yellow color appears when orthonitrophenol is split from ONPG.
• All lactose + organisms are ONPG positive; not all ONPG + organisms are lactose positive.

Hydrogen sulfide (H2S) production


• Some organisms can produce H2S by removing sulfur from sulfur-containing amino acids or
inorganic sulfur compounds. When sulfur combines with a heavy metal (iron or lead), a black
precipitate forms.
• Media that incorporate an H2S system include: SS, HE, XLD, TSI, and others.

Triple Sugar Iron Agar (TSI)


• Contains three sugars (glucose, lactose, and sucrose) plus systems to detect H2S production
• Slant: detects lactose fermentation
– If yellow- lactose fermented (A)
– If red- lactose not fermented (K)
• Butt: detects glucose fermentation
– If yellow- glucose fermented (A)
– If red- glucose not fermented (K)
• H2S production: black

Methyl Red (MR) and Voges-Proskauer (VP) tests


• Enterbacteriaceae metabolize pyruvate (glucose metabolite) through one of two pathways.
– If mixed acids are produced: methyl red pathway
– If acetoin and butylene glycol produced: VP pathway

Citrate utilization test


• Determines if an organism can use citrate as its only source of carbon
• Organisms that can do this turn the medium alkaline as they grown.
• Bromthymol blue in include in agar to detect pH change.
• Blue-color: positive
• Original green color: negative
Phenylalanine deaminase
• Some organisms can deaminate phenylalnine to phenylpyruvic acid
• Phenylpyruvic acid is detected by adding ferric chloride to the test medium
• A green color forms when ferric chloride combines with phenylpyruvic acid.

Decarboxylase tests
• Determine an organism’s ability to degrade an amino acid by removing the carboxyl group.
(produce alkaline environment)
• Organism is inoculated into medium containing glucose, pH indicator, and amino acid. Need a
control tube (lacks amino acid). Overlay with mineral oil.
• Purple test tube: positive
• Yellow test tube: negative

Urease test
• Organisms that possess the enzyme urease can hydrolyze urea to form ammonia. (alkaline
conditions)
• Detected by pH indicator
• Pink: positive
• Yellow: negative

Motility
• Hanging drop:
• Motility test medium: needle used to stab semi-solid medium.
• Whole tube turbid: motile
• Only in stab: nonmotile

Indole
• Some organisms possess the enzyme tryptophanase, which breaks down tryptophan into indole
and other end products.
• Tube method or spot test
• Red: positive
• No color change: negative

Spot test

1. Place several drops (1 -1.5 ml) of Indole Spot reagent (1% or 5% p-


methylaminobenzaldehyde if +ve red or 1% p-dimethylaminocinnamaledhyde if +ve blue
green)) on a piece of filter paper.
2. Spread an isolated pure colony (from an 18-24 hour culture) on the saturated surface of
the filter paper using a sterile loop
3. Examine immediately

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