Laboratory Identification of Family Enterobacteriaceae

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• Enterobacteriaceae/ Gram (-) Enteric Bacilli/ Coliforms

  • Largest and most medically important family of gram (-) bacteria

  • Part of normal flora of most animals and common cause of nosocomial infection

  • Overtly pathogen: Salmonella typhi, Shigella spp., Yersinia pestis

  • Ferments glucose and facultative anaerobes

  • Reduce nitrates to nitrite; Except: Pantoea and Erwinia

  • All are motile; Except: Shigella and Klebsiella

  • Yersinia are motile at RT but non-motile at 37C

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• Oxidase negative; Except : Plesiomonas shigelloides

• Formerly Vibrionaceae. However, phylogenetic studies link it with Proteus.

• Often with gas production; Except: Shigella

• Catalase positive; Except: Shigella dystenteriae

• Antigens of these group are;

  • O – somatic antigen ( Cell wall)/Lipopolysaccharide/Heat stable

  • K – capsular antigen/ polysaccharide/heat labile/ mask O antigen

  • H- flagellar antigen/ protein/heat labile

• NOTE: MALDITOF is also used but cannot differentiate Shigella from E.coli

Page 4:

• General Culture Media

• Media Inhibitory Fermentation Category

• Used Bismuth Sulfide Agar ( BSA)

• Brilliant green Glucose fermentation

• Bismuth sulfite as pH indicator

• Selective Salmonella spp.: Produce black colonies

Page 5:

• General Culture Media

• Media Inhibitory Fermentation Category

• Used Cefsulodin- irgasan- novobiocin agar (CIN)

• Mannitol fermentation

• Neutral Red as pH indicator

• Selective/ Differential Isolation of Yersinia enterocolitica

• May also isolate Plesiomonas and Aeromonas

Page 6:

• General Culture Media

• Media Inhibitory Fermentation Category

• Used Eosin/ methylene blue agar (EMB)

• Eosin Y, Methylene blue Lactose and sucrose.

• ―Sucrose is an alternate energy source for slow lactose fermenters.

• pH indicator is Eosin and Methylene blue

• Selective/ Differential Fermenters: Red/ Pink colonies

• “Escherichia coli – with blue black with greenish metallic sheen”

• Non-fermenters: Translucent/ Amber / Colorless

Page 7:

• General Culture Media

• Media Inhibitory Fermentation Category

• Used Gram- negative broth (GN)

• Deoxycholate Citrate salts Mannitol

• Enrichment Enhances the recovery of enteric pathogens from fecal specimens

• (Salmonella and Shigella)

Page 8:

• General Culture Media

• Media Inhibitory Fermentation Category

• Used Hektoen enteric agar (HEK)

• Bile salts Lactose, Salicin, and Sucrose

• pH indicator bromthymol blue

• Selective/ Differential Detection of enterics from stool or enrichment broth

• Lactose Fermenter: Bright orange or salmon pink

• Non-lactose fermenter: Green to blue green

Page 9:

• General Culture Media

• Media Inhibitory Fermentation Category

• Used MacConkey agar (MAC)

• Bile salts and crystal violet Lactose

• Neutral red as pH indicator.

• ―If replaced by Sorbitol For detection of E.coli O157:H7 (-):Sorbitol

• Selective Differential Lactose fermenter: Pink or red that may be surrounded by bile salts

• Non-lactose fermenter: Colorless or transparent

Page 10:

• General Culture Media

• Media Inhibitory Fermentation Category

• Used Salmonella- Shigella agar (SS)

• Bile salts, Sodium citrate, Brilliant green

• Lactose Neutral red is the pH indicator

• Selective Lactose fermenter: Pink to red

• For Salmonella and Shigella including mixed flora

• Salmonella: Colorless with black center

Page 11:

• Media Inhibitory Fermentation Category

• Used Xylose-lysine-deoxycholate agar (XLD)

• Sodium deoxycholate less bile salts

• Sucrose and lactose in excess

• Xylose in lower amounts. Phenol red is the pH indicator.

• Selective Differential Yellow: Fermentation of the excess carbohydrates to produce acid and absence of lysine Decarboxylase

• Colorless or red: produced by organisms that do not ferment any of the sugars.

• Yellow to red: fermentation of xylose (yellow), but because it is in small amounts, it is used up quickly, and the organisms switch to decarboxylation of lysine

Page 12:

• Colony characteristics of Lactose Fermenters

• Encapsulated Klebsiella pneumoniae “mucoid colonies

• MacConkey Agar

Page 13:

• E. Coli and Ent. aerogenes

• E. Coli and K. pneumoniae Colony characteristics of Lactose Fermenters

• MacConkey Agar

Page 14:

• Eosin Methylene Blue Greenish Metallic Sheen (E. coli) Colony characteristics of Lactose Fermenters

• Eosin Methylene Blue

Page 15:

• Colony characteristics of Lactose Fermenters

• MacConkey Agar EMB Agar

Page 16:

• Laboratory Identification

Page 17:

• Gram Stain ! Gram Negative

Page 18:

• Oxidase ! Negative

• Test for the position of cytochrome C to produce a dark-blue end product when reacted with Microdase Reagent.

Page 19:

• Nitrate Reduction ! Positive

• Test the ability of the organism to reduce nitrate to nitrite

• Nitrate broth with the organism is tested for presence of gas and reduction of nitrate

Page 20:

• Triple Sugar Iron (TSI)

• Differentiates gram (-) organisms by their ability to ferment glucose, lactose and sucrose and to produce hydrogen sulphide.

• 1 part glucose, 10 parts sucrose, 10 parts lactose

• Read at 18-24 hrs

• pH indicator is phenol red and H2S indicator is Ferrous sulfate

• Interpretation of TSI

Page 21

• TSI Results and Interpretation

  • Nonfermenters: alkaline slant and alkaline deep = K/K

  • Nonlactose fermenters (glucose fermenters): alkaline slant (red) and acid deep (yellow) = K/A

  • Lactose and Sucrose Fermenters: Acid slant (yellow) and Acid deep (yellow) = A/A

  • H2S production: black precipitate in the medium/Butt is acidic

  • Kliger's iron agar: similar medium but only has two sugars; Glucose and Lactose

  • P. vulgaris / S. marsescens/ Yersinia enterocolitica are A/A on TSI but only K/A on KIA

Page 22

• Nonfermenters: alkaline slant and alkaline deep = K/K

• Nonlactose fermenters (glucose fermenters): alkaline slant (red) and acid deep (yellow) = K/A

• Lactose and Sucrose Fermenters: Acid slant (yellow) and Acid deep (yellow) = A/A

• H2S production: black precipitate in the medium/Butt is acidic

Page 23

• Enterobacteriaceae on TSI

  • Lactose Fermenter: A/A

  • Late Lactose Fermenter

  • Non Lactose Fermenter

  • Escherichia

  • Enterobacter

  • Klebsiella

  • Salmonella arizonae

  • Citrobacter

  • Serratia

  • Salmonella

  • Shigella

  • Edwardsiella

  • Morganella

  • Providencia

  • Proteus

  • Yersinia

  • Hafnia

Page 24

• IMViC (Indole + Methyl Red + Vogues Proskauer + Citrate)

• Enterics have opposite MRVP reaction

• Reading time must be within 24hrs

Page 25

• Indole

  • Detects an organism's ability to produce the enzyme tryptophanase and deaminase tryptophan to indole, pyruvic acid, and ammonia.

  • Detected by the addition of Ehrlich's reagent or Kovac's reagent.

  • Positive color is red or pink.

  • Spot Indole (Kovac Reagent) is used for colonies from SBA or CAP and positive is blue color.

  • SIM (Sulfide Indole Motility) is also used for indole. TTC (Triphenyl Tetrazolium Chloride) is added for visualization in which colorless turns red with growth.

Page 26

• MRVP (Methyl Red and Vogues Proskauer)

• Based on either the mixed acid fermentation pathway or the butylene glycol pathway.

• Detects the end of glucose fermentation.

• Two fractions: One for MR and another for VP

• Organisms under mixed acid fermentation produce acid that will convert the methyl red to red color (<pH 4.5)

• Orange is conclusive, if conclusive after 48hrs of incubation. Continue incubation up to an additional three days.

Page 27

• VP measures acetoin or acetylmethylcarbinol

• The addition of 40% potassium hydroxide followed by a-napthol will produce a red complex (neutral pH) which indicates a positive test.

• Coblentz method may also be used using alpha napthol + potassium hydroxide + Creatine

Page 28

• Citrate

• Determines the ability of an organism to utilize sodium citrate as a sole source of carbon from pyruvate.

• Ammonium salts are the nitrogen source and use of these will liberate ammonia producing alkaline pH.

• Bromthymol blue indicator turns the medium from green to Prussian blue which is a positive reaction.

• False negative if cap not loose.

• If Christensen citrate medium is used, phenol red as pH indicator and organic nitrogen. At alkaline pH, it changes to yellow to pink.

Page 29

• Lactose Fermenter: Citrate Utilization Test

• MVI

• C

• E.coli

• Klebsiella

• Enterobacter

• +/-

• -/+

• -/+

• +-/+

• -/+

• +/+

Page 30

• Urease

• Organisms that produce urease will hydrolyze urea to ammonia.

• Ammonia reacts to form ammonium carbonate which increases the pH.

• Detected by phenol red which turns bright pink.

• Christensen urea agar is generally the preferred medium.

• Slow urease (after 4hrs): Klebsiella, Enterobacter, Yersinia, Serratia, Citrobacter

• Rapid urease (<4hrs): Proteus, Providencia, Morganella (PPM)

Page 31

• Gelatin hydrolysis

• Bacteria that produce gelatinases breakdown gelatin into amino acids.

• Inoculated and incubated at room temp.

• If incubation at 35C it should be chilled in a refrigerator before reading the results.

• Liquefaction is the positive reaction

Page 32

• Hydrogen Sulfide

• Bacterium utilizes sodium thiosulfate as a sulfur source to form H2S, a colorless gas.

• It combines with indicator ferrous sulfate to form a black color.

• Used to differentiate Salmonella (+) from Shigella (-)

• Can be found in certain agar such as SIM, HEA, TSI, and LIA

Page 33

• Phenylalanine deaminase

• Deamination of phenylalanine (removal of an amine group) results in the production of phenylpyruvic acid.

• Following overnight incubation of a 10% Ferric Chloride to an inoculated slant, a green color indicates a positive reaction.

• If tryptophan is used, produces a brown color.

• Proteus, Providencia, and Morganella are positive for this (PPM)

Page 34

• Decarboxylase (Moeller) and Dihydrolase

• Decarboxylation (removal of carboxyl-COOH group) of lysine, ornithine, and arginine (dihydrolase) may be detected by inoculating media with a specific amino acid and glucose as a carbohydrate source.

• Semisolid agar tubes are inoculated by stabbing. An acid pH and anaerobic environment must be present.

• Yellow color indicates glucose fermentation and results in an acid pH that activates the decarboxylase enzyme.

• A positive test caused by alkaline pH shift which is purple due to amines.

• Media are Moeller decarboxylase medium, MIO, or Lysine Iron Agar.

• Violet color = presence of decarboxylase (pH indicator is bromcresol purple)

Page 35

• Decarboxylation & Dihydrolase Reactions

• Lysine (Amino Acid) -> Lysine decarboxylase -> Cadaverine + CO2

• Ornithine -> Ornithine decarboxylase -> Putrescine

• Arginine -> Arginine dihydrolase -> Citrulline

Page 36

• B-galactosidase / ortho-nitrophenyl-B-D-galactopyranoside (ONPG)

  • Late or slow lactose fermenters may appear as non fermenter on primary media.

  • Lacks permease that allows lactose to enter the cell but has B-galactose which splits lactose.

  • Forms yellow compound as a positive result.

  • Used to differentiate Citrobacter (+) from Salmonella (-).

Page 37

• DNAse

  • Bacterial endonucleases that cleave phosphodiester bonds in DNA resulting in smaller subunits of polynucleotide.

  • Streaked in a medium with 0.2% DNA and incubated for 24hrs at 35C.

  • Following incubation, 1N HCL is added to the surface of the medium.

  • Unhydrolyzed DNA is insoluble in HCl and forms a precipitate.

  • A positive reaction is the formation of a clear zone (halo) around the inoculum.

  • Used to differentiate Serratia (+) to Escherichia coli.

Page 38

• Motility

  • Can be detected by microscopic examination of a wet mount.

  • In addition, semi-solid with agar concentration of less than 4% may be used.

  • Following incubation overnight, movement away from the stab or a hazy appearance means a motile bacterium (SIM).

Page 39

• Acetate Utilization Test

  • Uses acetate as the sole source of carbon.

  • Sodium acetate in the medium resulting in alkaline pH in which green to blue.

  • Used to differentiate E. coli (+) from Shigella (-).

Page 40

• Fermentation medium

  • Based on fermentation of carbohydrate into basal medium.

  • Andrade formula to differentiate enteric from corynebacterium.

  • Bromcresol purple to differentiate enterococcus from streptococcus.

  • Durham tube for gas production.

  • Peptone medium with Andrade (Enteric and Coryneform).

    • Positive: pink with or without gas.

    • Negative: Growth no color (straw).

  • BHI + BCP (Streptococcus to Enterococcus).

    • Positive: Growth of Yellow color.

    • Negative: Growth of Purple color.

Page 41

• Malonate Utilization

  • Sodium malonate as the sole source of carbon.

  • Bromthymol blue as a pH indicator.

  • Ammonium sulfate as a nitrogen source.

  • (+) result increased alkalinity from green to blue.

  • To differentiate Salmonella (+) from Citrobacter (-).

Page 42

• Lysine iron agar

  • Based on decarboxylation or deamination of lysine and formation of H2S (Lysine + glucose + Fe ammonium citrate + sodium thiosulfate).

  • Bromcresol purple as a pH indicator.

  • Aerobic slant and anaerobic butt.

  • Medium turns yellow if acid, but if lysine is decarboxylase to cadaverine, it would neutralize acid turning it back to purple.

  • If lysine occurs with a compound due to ferric ammonium citrate and flavin mononucleotide, it produces a burgundy slant.

Page 43

• Interpretations of LIA

  • K/K (Purple/Purple): lysine decarboxylation and no fermentation of glucose.

  • K/A (Purple/Yellow): glucose fermentation only.

  • R/A (Red/Yellow) – lysine deamination and glucose fermentation.

  • NOTE: Proteus does not blacken the medium.

Page 44

• Part 2: Organisms under Family Enterobacteriaceae

Page 45

• Escherichia coli

  • Most common cause of UTI.

  • #2 cause of neonatal meningitis.

  • Greenish metallic sheen on EMB.

  • On TSI, A/A with gas but (-) H2S.

  • Normal flora of GIT.

  • (+) for B-glucuronidase using 4-methylumberlliferyl-B-glucuronide (MUG) under long wavelength of ultraviolet light.

  • Do not isolate from media with dyes (EMB).

Page 46

• Escherichia coli

Page 47

• Sorbitol MacConkey (SMac)

Page 48

• Edwardsiella

  • Edwardsiella tarda is associated with wound infection and bacteremia.

  • K/A + gas + HEAVY H2S.

  • IMVIC Reaction of ++-- similarly resembles E.coli.

Page 49

• Genus: Shigella

  • Nonmotile.

  • On TSI, K/A with no gas.

  • (+) KCN broth in which Salmonella is (-).

  • Do not hydrolyze urea.

  • Do not produce H2S.

Page 50

• Shigella spp.

  • Most communicable of bacterial diarrhea (dysentery).

  • No other reservoir aside from humans.

  • Primarily found in crowded or substandard conditions.

  • Closely related to E.coli on a molecular basis.

  • Exotoxin (Shiga Toxin) of Shigella dysenteria that is capable of producing GI mucosal damage or dysentery/invasive gastroenteritis. If cytokine is involved, it may lead to Hemolytic uremic disease.

Page 51

• S. dystenteriae/ Shiga Bacillus

• S. flexneri/ Strong’s Bacillus

• S. boydii/ New Manchester’s Bacillus

• S. sonnei Duval’s bacillus

• Mannitol Ferment - + + +

• ONPG V - V +

• ODC - - - +

• Serogroup A B C D

Page 52

• Genus: Salmonella

  • Usually do not ferment lactose.

  • Negative for indole, (+)MR, (-)VP, PAD, and Urease (-).

  • Major producer of H2S (Except: S. paratyphi A).

  • Motile, EXCEPT: S. pullorum and S. gallinarum.

  • LDC positive, Except: Salmonella paratyphi A.

  • On TSI: K/A + gas and on LIA K/K.

  • Do not grow in a medium with potassium cyanide (KCN).

Page 53

• Serogroups of Salmonella (A-D = Somatic antigen) (Vi = Flagellar antigen)

• Salmonella paratyphi A Serogroup A

• Salmonella paratyphi B Serogroup B

• Salmonella cholerasuis - Bacteremia Serogroup C1

• Salmonella typhi/ Ebert’s bacillus - Enteric fever/ Typhoid Fever Serogroup D and Vi

Page 54

• Specimen

  • Blood – first week of the disease.

  • Urine – positive after the second week.

  • Stool – second or third week of enteric fever but the first week in enterocolitis.

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• Genus: Citrobacter

  • Previously known as BETHESDA-BALLERUP GROUP

  • Resembles Salmonella but ONPG (+) and LDC negative

  • On plates, may resemble E.coli but biochemically resembles Salmonella

  • C. freundii – can be isolated in diarrheal stool culture, known to cause extraintestinal which is acquired in hospital setting

  • C. koseri (formerly C. diversus) – case of nursery outbreaks of neonatal meningitis and brain abscesses.

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• Citrobacter Indole ODC Malonate

  • C. braaki: V Positive, Negative

  • C. freundii: V Negative, Negative

  • C. koseri: Positive, Positive, Positive

Page 57:

• Klebsiella – Enterobacter – Pantoea – Cronobacter – Hafnia – Serratia

  • Usually Indole (-)

  • MR (-), VP (+)

  • Large amount of gas on TSI

  • Most grow on Citrate and Potassium Cyanide

  • None produce H2S

  • Few hydrolyzes urea

Page 58:

• Genus: Klebsiella

  • With very mucoid and tendency to coalesce

  • Encapsulated (Polysaccharide) that is (+) Neufeld Quellung

  • On String test (+)

  • On TSI, A/A +gas

Page 59:

• Indole VP LDC

  • K. pneumoniae: -, +, +

  • K.oxytoca: +, +, +

  • K. rhinoscleromatis: -, +, +

Page 60:

• Genus: Enterobacter

  • All are A/A + gas on TSI, Except E. taylorae K/A + gas

  • Motile and ODC positive

  • Associated with UTI and wound infection

  • Enterobacter cloacae and Enterobacter aerogenes most common isolate

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• Genus: Enterobacter Lactose LDC ADH ODC Urease Yellow Pigment

  • E. cloacae: +, +, -, +, -, -

  • E. aerogenes: +, -, +, +, V, -

  • E. gergoviae: V, +, -, +, +, -

  • E. sakazakii: +, -, +, +, -, + (at RT)

  • E. taylorae: -, -, +, +, -, -

Page 62:

• Genus: Hafnia

  • Late lactose fermenter

  • On TSI, K/A or A/A with gas

Page 63:

• Genus: Serratia

  • Serratia marsescens produced red pigment (prodigiosin) when incubated at RM which is associated with community acquired endocarditis among drug users

  • (+) for ONPG

  • On TSI, K/A or A/A with gas

  • Ability to produce extracellular DNAse

  • Resistance with wide range of antibiotics

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• ADDITIONAL:

  1. S. marcescens: assoc. with Pneumonia and septicemia

     • Free-living in soil and water

     • Able to resist cephalotin and colistin

     • Chromogenic at room temp.

  2. S.liquifaciens

     • Able to ferment arabinose

  3. S.rubidaea

     • Also produce red pigment

  4. S.odorifera

     • Rancid potato-like odor

Page 65:

• PPM (Proteus – Providencia - Morganella)

  • Biochemically similar

  • Rapid urease and PAD (+)

  • Non-lactose fermenter (K/A + gas)

  • Motile

  • (+) MR and (+) LDC

Page 66:

• Genus: Proteus

  • Source of Weil felix for diagnosis of Ricketssia

  • #2 cause of UTI

  • May Swarm on BAP

  • Chocolate cake or burnt cake smell/ burnt gun powder/ fishy smell

  • Can cause alkaline pH of urine

  • Associated with UTI with putrid odor

  • Associated with stone formation

Page 67:

• Proteus vulgaris flagellar stain "swarming motility"

Page 68:

• Indole Test ODC Ampicillin, Cephalosporin And Chloramphenicol

  • Proteus mirabilis: - pneumonia - Septicemia - + S

  • Proteus vulgaris: - UTI + - R

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• Providencia and Morganella

  • Providencia

    • Difficult to treat

    • Presence of pungent odor

    • Citrate (+)

    • Providencia rettgeri is the only (+) urease while P. stuartii is variable

    • Associated with burned patients

  • Morganella

    • Morganella morganii is associated with UTI

    • Citrate (-) and LDC (-)

Page 70:

• Genus: Yersinia

  • BAP is routinely used but CIN is best medium for isolation

  • Yellow over orange due to weak acid production on Slant of TSI

  • With bipolar staining resembling safety pin appearance

  • ALL are NM at 37C (Y. pestis is not motile at 25-30C while the rest do)

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• Yersinia enterocolitica

  • Waterborne gastroenteritis

  • Usually transmitted from pet feces (eg, puppies), contaminated milk, or pork.

  • Causes acute diarrhea or pseudoappendicitis (right lower abdominal pain due to mesenteric adenitis or terminal ileitis).

  • Production of bull’s eye colonies on CIN

  • ODC and Urease (+)

  • ONPG (+)

  • Can survive at Cold temperature (Associated with deadly infections in blood bag)

Page 72:

• Differentiation of Yersinia spp.

  • Y. pestis: Motility at @ 25C: -, @37C: -

  • Y. enterocolitica: Motility at @ 25C: +, @37C: -

  • Y.pseudotuberculosis: Motility at @ 25C: +, @37C: -

Page 73:

• Plesiomonas shigelloides

  • Microscopy: Gram negative

  • Culture: Non-hemolytic- BAP

  • White to pink colonies- inositol brilliant green bile salt agar

  • Opaque apron like colonies – CIN

  • Growth on TCBS: -, growth on HEA: +

  • Similar with E.coli on plated media

  • Catalase +, most strains are LF, Gas -, indole +, MR +, Positive trio decarboxylase test

  • Facultative anaerobe, with polar flagella

  • Cross agglutinate with Shigella! Shigelloides

  • TSI: K/A, gas -, H2S -

Page 74:

• References:

  • Bailey and Scott’s, Diagnostic Microbiology 14th ed.

  • Rodriguez, M.T. Bacteriology, Handbook for Medical Technologists

  • Mahon, Connie Textbook of Diagnostic Microbiology 6th Ed.

  • CEU LEAPS- Clinical Bacteriology Lab Course Copy 74

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• End of Discussion.