UNIVERSITY OF SANTO TOMAS | MEDICAL TECHNOLOGY

DIAGNOSTIC MICROBIOLOGY | ENTEROBACTERIACEAE

GENERA AND SPECIES TO BE CONSIDERED

֍ TRIBE ESCHERICHIAE o Hafnia alvei

o Escherichia coli o Serratia marcesens
o Shigella sonnei o Enterbacter aerogenes
o Shigella dysenteriae o Pantoeae agglomerans
o Shigella fleneri o Cronobacter sakazakii
o Shigella boydii ֍ TRIBE PROTEAE
֍ TRIBE EDWARDSIELLEAE o Proteus vulgaris
o Edwardsiella tarda o Proteus mirabilis
֍ TRIBE SALMONELLEAE o Providentia stuarti
o Salmonella enterica o Morganella morganii
o Salmonella bongori ֍ TRIBE CITROBACTERIACEAE
֍ TRIBE KLEBSIELLAE o Citrobacter freundii
o Klebsiella pneumoniae subsp. o Citrobacter koseri
pneumoniae ֍ TRIBE YERSINIAE
o Klebsiella pneumoniae subsp. ozonae o Yersinia pestis
o Klebsiella oxytoca o Yersinia enterocolitica
o Klebsiella granulomatis ֍ Plesiomonas shigelloides

GENERAL CHARACTERISTICS

1. All are Oxidase Negative except Plesiomonas spp.

a. Plesiomonas shigelloides reactions may resemble Aeromonas (both are Catalase and Oxidase Positive) they
may be differentiated using DNAse wherein Plesiomonas would test negative while Aeromonas would test
positive
2. Most are Catalase Positive except Shigella dysenteriae type 1
3. Most are Motile except
a. Yersinia pestis
b. Yersinia enterocolitica at 37 degrees Celsius
c. Klebsiella spp.
d. Shigella spp.
4. Most are Nitrate Reducers except Photorabdus spp. and Xenorhabdus spp.
5. All are able to Ferment Glucose.
6. Antigenic Strucutre
a. H antigen : Flagellar Antigen : Heat labile
b. K antigen : Capsular Antigen : Heat labile; remove by boiling to expose the somatic antigen
c. O antigen : Somatic Antigen : Heat Stable

BIOCHEMICAL TESTS SUMMARY

INDOLE METHYL RED VP CITRATE UREA H2S

Plesiomonas spp. Proteus spp. Klebsiella spp. Salmonella enteritidis Klebsiella spp. Salmonella spp.
Proteus vulgaris Shigella spp. Enterobacter spp. Providencia spp. E. cloacae E. tarda
Providencia spp. Escherichia coli Serratia spp. Enterobacter spp. C. koseri C. freundii
Citrobacter koseri Providencia spp. Hafnia alvei Klebsiella spp. Y. enterocolotica C. braakii
Escherichia coli Morganella morganii Citrobacter spp. Serratia Proteus spp.
Morganella morganii Edwardsiella tarda Proteus mirabilis RAPID : PPM
Edwardsiella tarda Citrobacter spp. Serratia spp. Providencia rettgeri
Klebsiella oxytoca Salmonella spp. Proteus spp.
Ewingella americana Ewingella americana M. morganii
Serratia odoriferans 2
Y. enterocolitica

LACTOSE FERMENTATION

A. RAPID LACTOSE FERMENTERS

a. Escherichia coli – however there are some strains that are NLF and must be distnguished from Shigella spp.
b. Klebsiella spp.
c. Enterobacter spp.
B. LATE LACTOSE FEREMENTERS / ONPG +
a. Citrobacter spp.
i. C. koserii provides a varying result
b. Serratia spp.
c. Salmonella serotype Paratyphi
d. Salmonella serotype Typhi
e. Salmonella serotype Typhimurium
f. Salmonella arizonae
g. Shigella sonnei
C. NON LACTOSE FERMENTERS
a. Shigella spp.
b. Salmonella enteritidis
c. Proteus spp.
d. Providencia spp.
e. Morganella morganii
f. Hafnia alvei
g. Yersinia spp.

CLINICALLY SIGNIFICANT ISOLATES

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DIAGNOSTIC MICROBIOLOGY | ENTEROBACTERIACEAE
֍ Escherichia coli
• Characteristics
 Motility differentiates it from Shigella spp. (nonmotile)
 Lactose Fermenters; in EMB Agar, its colonies produce a greenish metallic sheen
 Some strains are NLF (Inactive E.coli)
 IMVC (++--)
 Indole and Methyl Red positive
 Produces Gas from D-Glucose Fermentation
• Most common cause of community-acquired UTI
• Enteroinvasive E. coli (EIEC)
 Produces Shigella-like infection
• Enterohemorrhagic E. coli (EHEC)
 Aka Verotoxin-Producing E. coli (VTEC)
 May cause HUS
 Important Strain : E.coli O157:H7  unable to forment sorbitol
• Enterotoxigenic E. coli (ETEC / VTEC)
 Produce a heat labile toxin similar to Cholera Toxin
 Cause of Traveler’s Diarrhea / Montezuma’s Revenge
• Enteropathogenic E. coli (EPEC)
 Non-invasive, non-toxin producing E. coli
• Enteroaggregative E. coli (EAEC)
 Adhere to epithelial cells in a stacking fashion
• Diffuse-adhesive E. coli (DAEC)
 Adhere to cells in a diffused pattern
֍ Shigella spp.
• Characteristics
 Non-Motile
 Differentiates it from E. coli and Salmonella
 NLF except S. dysenteriae (Late Fermenter)
 IMVC (-+--)
 + Only in Methyl Red
 Gas production is uncommon
 S. flexneri serotype 6 and S. boydii serotype 14 produces gas from glucose
 LDC Negative
 Aids in differentiation of Shigella (LDC -) from E. coli and Salmonella (LDC +)
o Because all of them are MR positive
 Citrate Negative
 Malonate Negative
 H2S Negative
 Differentiates it from Salmonella
• NOTE : ODC TEST AND MANNITOL FERMENTATION
 Shigella sonnei is ODC+ while other species of Shigella are negative.
 Shigella dysenteriae is unable to ferment mannitol unlike the other Shigella spp.
• Subgroups
 Group A : S. dysenteriae
 Group B : S. flexneri
 Group C : S. boydii
 Group D : S. sonnei
• Diseases Associated
 Shigellosis
 Bacillary Dysentery
 Blood, mucus, pus in stool
 HUS – S. dysenteriae 1
֍ Salmonella spp.
• Salmonella enteritidis (Human Pathogen)
 subsp. Arizonae : Late Lactose Fermenter
 subsp. Enterica – subdivided into serotypes
 Serotype Typhi – primary identifiable serotype; Typhoid Fever ; no animal reservoir
hosts
 Serotype Enteritidis – infections associated with poultry products
 Serotype Paratyphi and Serotype Choleraesius – Paratyphoid
 subsp. Salmae
 subsp. Diarizonae
 subsp. Houtenae
 subsp. Indica
• Salmonella bongori (Animal Pathogen)
• Characteristics
 Motile
 NLF except S. arizonae
 IMVC : MR +
 Negative Indole
 Negative VP
 Citrate + only in S. enteritidis
 H2S +
 Main similarity with Citrobacter spp.

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DIAGNOSTIC MICROBIOLOGY | ENTEROBACTERIACEAE
o In XLD : Citrobacter has black center but the colonies are yellow (LDC-)
(Salmonella would have a Red colored colony with black center due to
fermentation of Xylose with a subsequent + LDC reaction)
 Use KCN Medium to break similarity (Salmonella are unable to grow in KCN) or LDC
rxn (Salmonella is + to LDC, Citrobacter is negative)
 Salmonella Paratyphi A does not produce H2S
 Citrate +
 Another main similarity of Salmonella and Citrobacter
 LDC +
֍ Citrobacter spp.
• Characteristics
 Motile
 Indole Negative except C. koserii (diversus)
 MR positive
 Citrate +
 H2S + except C. koserii
 LDC Negative
 Primary difference of Salmonella and Citrobacter
 Able to Growth in KCN
• Citrobacter koserii
 Indole + and H2S Negative unlike the other Citrobacter organisms (Indole – H2S +)
• Citrobacter freundii
• Citrobacter braakii
֍ Yersinia spp.
• Bipolar staining / safety-pin appearance on Wasyson stained smears
• Y. pestis – transmitted by rat flea – Agent of Bubonic Plague
 Stalactite pattern of growth on broth
 Nonmotile at any temperature
• Y. enterocolitica
 Psychrophile
 Main Contaminant in Whole Blood Units / Packed Red Blood Cells causing Transfusion-Related
Sepsis
 Motile only at 25 degrees Celsius.
֍ Edwardsiella tarda
• NLF H2S Producer
• Motile
• Primarily found in waters harboring fish and turtles
֍ Proteus spp.
• Characteristic SWARMING COLONIES on Culture Media
• Burn chocolate odor
• Proteus vulgaris
 The Indole + species
• P. mirabilis
 The Citrate + species
• H2S + on TSI only
֍ Providencia spp.
• P. stuartii
 MAINLY UREASE NEGATIVE UNLIKE OTHER PROVIDENCIA!
• P. rettgerii
֍ Morganella morganii
• Citrate Negative
 Differentiates it from Proteus mirabilis and Providencia (+)
֍ Klebsiella spp.
• Klebsiella pneumoniae subsp. pneumoniae
• Klebsiella pneumonia subsp. ozonae
• Klebsiella oxytoca
 INDOLE + Klebsiella organism
• Klebsiella granulomatis
 DONOVANOSIS
 Culutre – grows only on egg yolk based special media or human monocytes
֍ Enterobacter spp.
• Enterobacter aerogenes : LDC +
• Enterobacter cloacae : ADH +
֍ Cronobacter sakazakii
• Produces yellow pigment
• ODC +
• ADH +
• Neonatal infections are primarily due to the organism’s ability to utilize sialic acid found in breast milk
֍ Pantoea agglomerans
• Triple Decarboxylate Negative Oragnism
֍ Hafnea alvei
• Mostly opportunistic
֍ Serratia spp.
• DNAse and Gelatinase +
• Serratia marcesens

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DIAGNOSTIC MICROBIOLOGY | ENTEROBACTERIACEAE
• Serratia odoriferans
֍ Ewingella americana
• MRVP +
• Biochemically inactive
֍ Plesiomonas spp.
• Oxidase +
• Indole +
• MR +
• Pleomorphic filamentous

CULTURE MEDIA FOR ISOLATION AND PRESUMPTIVE IDENTIFICATION

֍ MAC-CONKEY AGAR
• PURPOSE : isolation of gram-negative bacilli, observe lactose fermentation
• COMPOSITION
 Lactose
 Crystal Violet – inhibits gram-positive organisms
 Bile Salts – inhibits other gram-negative organisms
 Neutral Red – pH indicator
• COLONIAL CHARACTERISTICS
 Lactose Fermenting Colonies : Red-Pink
 NLF Colonies : Yellow
֍ EOSIN-METHYLENE BLUE AGAR
• PURPOSE : Lactose Fermentation, isolation of gram negative bacilli
• COMPO SITION
 EMB are both the indicators and inhibitors
 Eosin
 Methylene Blue
• Growth characteristics
 E. coli – greenish metallic sheen
 LF – pink to purple colonies
֍ HEKTOEN-ENTERIC AGAR
• PURPOSE : Isolation of Intestinal Enteric Pathogens
• COMPOSITION
 Lactose
 Sucrose
 Bromthymol Blue – pH indicator
 Ferric Ammonium Citrate
• PRINCIPLE
 Lactose and Sucrose Fermenters make pH acidic  Yellow/Orange Colonies
 Nonfermenters – no change in pH  Green Colonies
 H2S producers  Black Center
• COLONIAL CHARACTERISTICS
 Salmonella  green with black center
 Shigella  green
֍ Xylose-Lysine-Desoxycholate Agar
• Purpose : Isolation of Intestinal Enteric Pathogens
• Composition
 Lactose
 Sucrose
 Xylose
 Bile Salts Inhibitor
 Phenol Red
 Sodium Thiosulfate + Ferric Ammonium Chloride
• Principle
 Fermentation of Xylose lowers the pH  yellow color
 H2S production  Black Center
 Nonfermenters of Xylose  red colonies
 LDC Rxn turns pH alkaline  Red
• Growth Patterns
 Salmonella spp.  Red with Black Center except Paratyphi A
 Shigella spp.  Red, no Black Center [NXF! NEG LDC]
 E. coli  Red
 Citrobacter freundii  Yellow with Black Center
 Citrobacter koserii, Proteus, Providencia, Morganella  Yellow no black center
֍ Salmonella-Shigella Agar
• Purpose : Isolation of Salmonella and Shigella species
• Composition
 Sodium thiosulfate and ferric ammonium citrate
 Neutral Red
 Lactose
• Growth Pattern
 Salmonella  colorless with black center
 Shigella  colorless
֍ SELENITE-F BROTH / Gram-Negative Broth
• Enrichment of Salmonella organisms

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DIAGNOSTIC MICROBIOLOGY | ENTEROBACTERIACEAE
֍ BISMUTH SULFITE
• Highly selective for Salmonella
• Useful in endemic or epidemic cases
֍ CEFSULODIN-IRGASAN-NOVOBIOCIN AGAR
• Purpose : Isolation of Yersinia enterocolitica
• Colonial Characteristics : Bull’s Eye Colonies
 Aeromonas are also able to grow and exhibit the same colonial morphology. Hence it is important
to test the colonies for oxidase to confirm whether or not the colony is Yersinia (oxidase negative)
֍ Brilliant Green Agar
• Purpse : Isolation of NLF
• Composition
 Brilliant Green
• Growth Characteristics
 Proteus and Salmonella  red/pink colonies
֍ TRIPLE SUGAR IRON AGAR SLANT
• PURPOSE : OBSERVE SUGAR FERMENTATION AND H2S PRODUCTION
• COMPOSITION
 Lactose, Sucrose, Glucose [ 10 : 10 : 1]
 Phenol Red
 Ferrous Sulfate, Sodium Thiosulfate
• PRINCIPLE
 Fermentation of Glucose is observed on the butt  yellowing of the butt due to acid pH
• Gas Production  cracked butt, lifted butt
 Fermentation of Lactose and Sucrose observed on the slant  yellowing of the slant due to acid pH
 H2S production  blackens the media
• GROWTH PATTERNS
 STRATEGY : WHEN IT IS H2S + IT IS OF MINIMAL IMPORTANCE TO DETERMINE
WHETHER IT IS A/A OR K/A.

A/A K/A
+ H2S Salmonella arizonae GAS + : Proteus mirabilis, Proetus vulgaris
Proteus mirabilis GAS V : Salmonella spp., E. tarda
Proteus vulgaris GAS - : Citrobacter freundii,
Citrobacter freundii
- H2S GAS + : EKE GAS + : Klebsiella, Citrobacter koserii
E. coli, Klebsiella, Enterobacter Gas V : Hafnia, Serratia, Providencia, Morganella
GAS – : Serratia GAS - : Plesiomonas, Shigella, Yersinia

֍ LYSINE-IRON AGAR SLANT

• PURPOSE : OBSERVE GLUCOSE FERMENTATION, DECARBOXYLATION, DEAMINATION, AND
H2S PRODUCTION
• COMPOSITION
 Lysine
 Peptones
 0.1% Gluvose
 Bromcresol Purple
 Ferric Ammonium Citrate
• PRINCIPLE
 Slant  observe deamination
 Butt  turns yellow due to glucose fermentation, observe decarboxylation  butt becomes purple
 LDC  Decarboxylation of Lysine yields Cadaverine  pH alkaline in butt = purple
 Lysine Deaminase  Deamination of Lysine  pH alkaline in slant = red
• a compound is formed that, in the presence of ferric ammonium citrate and a coenzyme,
flavin mononucleotide, forms a burgundy color on the slant, hence the Proteus spp. will
not produce a black ppt from its typical H2S production because the ferric ammonium
citrate is used for the deamination
• GROWTH PATTERNS
 K/K H2S+  Salmonella spp.
• LDC +
• Deaminase Negative
 K/A H2S +  Citrobacter freundii
• LDC –
• Deaminase –
 K/A H2S -  Citrobacter koserii, Shigella spp.
 R/A  Proteus, Providencia Morganella
• LDC –
• Deaminase +

BIOCHEMICAL TESTS

֍ OXIDASE
• Detects presence of Oxidase which catalyzes transfer of electrons to oxygen through aerobic bacterial
respiration systems
• SUBSTRATE : tetramethyl-p-phenyldiamine dihydrochloroide
• END-PRODUCT : Indophenol
• + result : violet color production

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 UNIVERSITY OF SANTO TOMAS | MEDICAL TECHNOLOGY
DIAGNOSTIC MICROBIOLOGY | ENTEROBACTERIACEAE
 + : Neisseria, Aeromonas, Pseudomonas, Campylobacter, Pasteurella, Vibrio, Gardnerella
 - : Enterobacteriaciae except Plesiomonas
֍ INDOLE
• SPOT INDOLE TEST
 Detects presence of Tryptophanase which converts Tryptophan to Indole
 Reagent : Ehrlich’s : p-dimethylaminobenzaldehyde with Ethanol
 + Result : Blue Color within 30 seconds
• TUBE INDOLE TEST
 Kovac’s Reagent – dimethylaminebenzaldehyde with HCl
 + Resut : Red Ring at interphase
 + Enterobacteriaciae : PPPCEM-EdKo
• Plesiomonas
• Providencia spp
• Proteus vulgaris
• Citrobacter koserii
• Escherichia coli
• Morganella spp.
• Edwardsiella tarda
• Klebsiella oxytoca

֍ MRVP
• Methyl Red and Voges Proskauer Test
• Methyl Red
 Detects Mixed Acid Fermentation Pathway
 + at pH 4.4 = Red
 + = PSEPMECS
 Proteus spp.
 Salmonella spp.
 Escherichia coli
 Providencia spp.
 Morganella morganii
 Edwardsiella tarda
 Citrobacter spp.
 Shigella spp.
• Voges Proskauer
 Detects Acetoin Production
 Conversion of Acid to 2,3-butanediol and Acetoin
 Addition of a-naphthol KOH
 Butylene Glycol Pathway
 + at pH 5.5
 + = KESH
 Klebsiella
 Enterobacter
 Serratia
 Hafnia

֍ CITRATE UTILIZATION TEST

• Simmon’s Citrate Agar : Butt-Slant
• Indicator : Bromthymol Blue
• Original color of agar : Green
• Bacteria that can grow produce Citrate-Permease
• Citrate is utilized and converted to pyruvate which enters the metabolic cycle
• Citrate utilziation produces Ammonia and Ammonium Hydroxide which truns the pH alkaline causing a
change of color to BLUE
• + = SPECK
 Proteus mirabilis
 Salmonella spp.
 Providencia spp.
 Enterobacter spp.
 Citrobacter spp.
 Klebsiella spp.
֍ UREA HYDROLYSIS
• Christensen’s Urease Broth (Light Orange uninoculated broth)
• Indicator : Phenol Red
• Urea under presence of urease is broken into ammonia and carbon dioxide
• + result : Magenta coloration
• Rapid Urease Producers (2-4hrs): Proteus spp., Providencia rettgeri, Morganella
• Slow Urease producers (24hrs) : SKYCE
 Serratia spp.
 Klebsiella pneumoniae
 Yersinia spp.
 Citrobacter koserii
 Enterobacter spp.
֍ DECARBOXYLASE TEST
• Base : Moeller
• + : Purple

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DIAGNOSTIC MICROBIOLOGY | ENTEROBACTERIACEAE
Arginine  Citrulline  Ornithine
•
 + = E. cloacae
• Lysine  Cadaverine
 + = EKESE
 Escherichia coli
 Klebsiella
 Enterobacter cloacae and Enterobacter aerogenes
 Salmonella
 Edwardsiella
• Ornithine  Putrescine
 + = EMEEP
• Escherichia coli
• Morganella morganii
• Enterobacter cloacae and E. aerogenes
• Edwardsiella
• Proteus mirabilis
֍ PHENYLALANINE DEAMINASE AGAR
• Detects presence of phenylalanine deaminase which converts phenylalanine to phenylpyruvic acid
• Reagent FeCl3 (10%)
• + result = Green on agar slant after adding rgt
• + = PPM (Proteus, Providencia, Morganella)

LAB DIAGNOSIS TECHNIQUES

֍ SPECIMEN : STOOL
• If not processed within 2 hours of collection, must be placed in transport media such as Cary-Blair medium.
• Rectal swabs are acceptable if stools cannot be collected.
• Routine Medium
 MacConkey / Eosin Methylene Blue
 Hektoen Enteric Agar / Salmonella-Shigella / Xylose-Lysine-Deoxycholate Agar
 Gram-Negative Broth / Selenite broth
 Campylobacter blood agar
 5% SBA
• Medium used upon request / suspicion
 Cefsulodin-Irgasan-Novobiocin Agar : Yersinia enterocolitica
 Sorbitol-MacConkey Agar : Verotoxic E. coli
 Thiosulfate-Citrate Bile Salts – Sucrose Agar : Vibrio
 Cycloserine-cefoxitin-fructose egg yolk agar : Clostridium difficile
 Brilliant Green / Bismuth Sulfite Agar : Salmonella typhi
• Culture Proper
 Insert swab into stool culture and smear then perform gram stain
 Insert another swab and roll it over the surface of BAP, MAC, HEA, and CampyBA
 Insert the swab into the GN broth and cap the tube
 Streak optional plates if any
 Incubate all plates, except CampyBAP, and GN broth at 35-37 degrees Celsius for 24 hours.
Incubate CampyBAP at 42 degrees Celsius in a microaerophillic environment.
 At 6-8 hours of incubation, subculture the GN broth to fresh MacConkey and HEA
 Examine plates after 24 hrs.
 Not for yeasts and Staphylococcus aureus in BAP
 Re-incubate any plates without growth for another 24 hours.
 Examine after 48 hrs including those subcultured at GN broth.
• Pathogens
 Enterotoxin-Producing
 V. cholerae
 Enterotoxigenic E. coli
 Salmonella spp.
 S. aureus
 Campylobacter jejuni
 Clostridium perfringens
 Clostridium difficile – toxin A
 Bacillus cereus – also proce emetic toxin that induces vomitting
 Cytotoxin-producing
 Enterohemorrhagic E. coli
 Shigella spp.
 Clostridium difficele – toxin B
 Neurotoxin-Producing
 Clostridium botulinum – botulinum toxin
 Shigella dysenteriae
֍ IDENTIFICATION
• Presumptive for presence of Enteric – Oxidase Test
• In MacConkey, red to pink colonies indicate lactose fermentation. NLF must further be tested for Late
Fermentation via ONPG Test.
 If your colonies are LF and are oxidase negative gram negative rods the possible organisms are
o Escherichia coli
o Klebsiella spp.
o Enterobacter

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UNIVERSITY OF SANTO TOMAS | MEDICAL TECHNOLOGY
DIAGNOSTIC MICROBIOLOGY | ENTEROBACTERIACEAE
Motility Testing will aid in ruling out Klebsiella if the microorganism is motile.

Indole Test is useful in indentifying E.coli from the other two unless K. oxytoca is

present.
 Methyl Red Rxn  Escherichia coli
 VP Rxn  Enterobacter and Klebsiella
o Differentiate through
 Motility : Klebsiella is nonmotile
 ODC : Kleb is negative for ODC
• Differentiating Enterobacter organisms
 ADH + : E. cloacae, Cronobacter sakazakii
 C. sakazakii is unable to ferment
Sorbitol and negative for urea but +
Phenylalanine Deaminase
 LDC + : E. aerogenes
• NON-LACTOSE FERMENTERS
 ONPG Testing may provide information for differentiation and identification.
 + ONPG microorganisms are typically Salmonella arizonae, Shigella sonnei, Serratia spp,
and Citrobacter
o H2S Testing + for Salmonella and Citrobacter freundii and braaki
 TSI AGAR
 H2S PRODUCTION – suggest Salmonella, Citrobacter, Proteus spp. Edwardsiella tarda
o Check for IMVC rxns
o Check for Urease Activity  suggestive for Proteus
 Non H2S Producers NLF  usually Shigella
o Check for motility - Shigella is nonmotile!
 Kleb vs Shigella
• Lactose Fermentation
• MRVP rxn
• Citrate
 Yersinia enterocolitis
• Motile at 25 degrees celsius
• Isolation via CIN – bull’s eye
o Motile
 Providencia
• Providencia stuartii is Urease negative
• R/A in LIA
• Citrate Positive
• MR +
• Rapid Urease
 Morganella
• Citrate negative
• MR +
• Rapid Urease
 Serratia
• Pigment production
• DNAse positive
• Citrate positive
• VP +
• Late Urease producer
• ONPG +