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Enterobacteriaceae
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Generalities
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Generalities
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Generalities
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Generalities
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Generalities
• Deaminase Positive:
• Proteus, Providencia, Morganella
• H2S Positive:
• Salmonella, Proteus, Citrobacter and Edwardsiella
• Voges Proskauer (+)
• “SHEK” group
• Urease Producers (Rapid/Late)
• Rapid Urease Producers
• Proteus, Providencia rettgeri, Morganella morganii
• Late Urease Producers
• Citrobacter, Klebsiella, Enterobacter, Yersinia, Serratia
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Taxonomic Classification
• CLASSIFIED into TRIBES based on biochemical
characteristics
1. Escherichiaeae (Escherichia, Shigella)
2. Edwardsielleae (Edwardsiella)
3. Salmonelleae (Salmonella)
4. Citrobacteriaceae (Citrobacter)
5. Klebsielleae (Klebsiella, Enterobacter, Pantoea,
Cronobacter, Hafnia, Serratia)
6. Proteeae (Proteus, Providencia, Morganella)
7. Yersinieae (Yersinia)
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Virulence and Antigenic Factors
• Plasmids carrying resistance genes
• Antigens
O antigens
H antigens
K antigens
• K1 antigen
• Vi antigen
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Virulence and Antigenic Factors
• Pili
• Flagella
• Enterotoxins
• Enzymes
• Endotoxins
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Clinically Significant Genera
• Opportunistic Members
• True Pathogens
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Escherichia coli
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E. coli
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E. coli
• Gastrointestinal Pathogens
5 MAJOR CATEGORIES
1. ETEC
2. EIEC
3. EPEC
4. EHEC
5. EAEC
DAEC
EAEC
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ETEC
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ETEC
Virulence Factors
• Fimbriae
• Heat Labile Toxin – (cyclic adenosine
monophosphate)
• Heat Stable Toxin – (guanyl cyclase)
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EPEC
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EIEC
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EHEC
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Enteroadherent E. coli
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E.coli
• Extraintestinal Infections
• MNEC
• Meningitis/Sepsis Associated E.coli
• Neonatal Meningitis
• Capsular K1 antigen
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Klebsiella
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n d Rao u t el l a
Escherichia
vulneris
Escherichia
hermannii
tahir99 VRG
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Other Klebsiella species
Klebsiella oxytoca
• Produces infections similar to K. pneumoniae
• Also causes antibiotic associated hemorrhagic colitis
• Characteristic Biochemical Test: Indole (+)
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Other Klebsiella species
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Enterobacter
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Enterobacter
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Other Enterobacter species
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umoniae subsp. pneumoniae
E. cloacae E. aerogenes K. pneumoniae subsp. pneumoniae
+w or − 65 − 2 + 95.4 (0.1)
+ 95 + 97 − 0
− 0 + 98 + 98 (6.3)
+ 97 (2) − 0 − 0
ase + 96 (1.3) + 98 (0.8) − 0
(+) 0 (94.2) (+)or− 0 (61.2) − 0 (0.2)
− or + 21.7 (1.3) + 94.2 d 84.4 (0.3)
WH: Edwards and Ewing’s identification of Enterobacteriaceae, ed 4, East Norwalk, CT, 1986, Appleton & Lange.
n 1 or 2 days; (+), positive reaction after ≥3 days (decarboxylase tests: 3 or 4 days); −, ≥90% no reaction in 30 days; + or −, most
strains negative; − or +, most strains negative, some cultures positive; d, different reactions, +, (+), −; +w, weakly positive reaction.
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Pantoea agglomerans
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D-tartrate,
Jordan’s − or + 30 + 95
Sodium alginate (utilization) − 0 − 0
Modified from Ewing WH: Edwards and Ewing’s identification of Enterobacteriaceae, ed 4, East Norwalk, C
+, ≥90% positive within 1 or 2 days; (+), positive reaction after ≥3 days (decarboxylase tests: 3 or 4 days);
cultures positive, some strains negative; − or +, most strains negative, some cultures positive; d, different re
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Serratia
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Serratia
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Hafnia alvei
• Tribe Proteeae
• Normal flora of the GI Tract; found in the
environment
• Opportunistic Pathogens; Noscomial
Infections
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Proteus
Proteus mirabilis
Proteus vulgaris
Proteus penneri : H2S (-)
Causes
• UTIs, wound, ear, infections, nosocomial
infections, kidney stone formation
Key Characteristics:
• Swarming Colonies
• Burnt Chocolate Odor
• Differentiated by Indole Test and ODC Test
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Methyl red + + + + + + +
Voges-Proskauer − − or + − − − − −
Simmons citrate − + or (+) d + + + −
Christensen urea + + or (+) + − − or + + +
H2S (TSI) − (70% ) + + − − − −
Ornithine decarboxylase − + − − − − +
Phenylalanine deaminase + + + + + + +
Acid produced from
Sucrose + d + d d d −
Mannitol − − − − d + −
Salicin − − d − − d −
Adonitol − − − + − + −
Rhamnose − − − − − + or − −
Maltose + − + − − − −
Xylose + + + or (+) − − − or + −
Arabitol − − − − − + −
Swarms + + + − − − −
Modified from Washington J: Laboratory procedures in clinical microbiology, ed 2, New York, 1981, Springer-Verlag.
H2S, Hydrogen sulfide; TSI, triple-sugar iron agar; +, ≥90% positive reaction within 1 or 2 days; −, no reaction (≥90%) in 30 days; − or +, most strains
negative, some cultures positive; + or (+), most reactions occur within 1 or 2 days, some are delayed; d, different reactions, + or −, most cultures positive,
some strains negative.
Pro v i d en ci a
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Providencia
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Edwardsiella tarda
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Citrobacter
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Citrobacter
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True Pathogens
Salmonella – GI illnesses
Shigella – GI Illnesses
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S. enterica has 6 subspecies:
• importantly, S. enterica subsp. enterica
• S. enterica subsp. enterica serotype Typhi
• S. enterica subsp. enterica serotype Enteritidis
• Other subspecies:
• S. enterica subsp. salamae
• S. enterica subsp. arizonae
• S. enterica subsp. diarizonae
• S. enterica subsp. houtenae
• S. enterica subsp. indica
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Salmonella
Virulence Factors
• Fimbriae and most especially enterotoxins are
implicated as significant virulence factors
• Ability to traverse intestinal mucosa
Salmonella +
+
−
+
+
+
• Antigenic Structures
Classification based on Kauffman-White
H Ag
> O, H and K (Vi Ag)
Vi Ag Ag serotyping
O Ag
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Salmonella
Disease States
Gastroenteritis (Enterocolitis)
• Usually caused by S. Typhimurium
• Food poisoning (poultry and dairy products)
• Ingestion of poultry, dairy products, peanut butter
other foodstuffs
• Invasion of epithelial cells of intestinal tract
• Cholera-Like Toxin
• S/S Watery Diarrhea, abdominal pain, vomiting,
fever
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Salmonella
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Salmonella
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Salmonella
Bacteremia (Sepsis)
• Most often caused by Salmonella Choleraesuis
• Usually seen among patients with underlying
diseases or a children with enterocolitis
• Common sequelae: salmonella osteomyelitis
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Shigella
Key Tests
• Non-motile
• Not aerogenic
• H2S (-)
• LDC (-)
• Does not utilize citrate
• Does not Ferment Xylose
• NLF except S. sonnei
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Shigella
Virulence Factors
• Shiga-toxin
• Endotoxin
• Neurotoxin
• Invasive property
Antigens
• All possess O antigens, some species possess K
antigen
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Shigella
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Shigella
• Zoonotic
• 3 Species
• Yersinia pestis
• Yersinia pseudotuberculosis
• Yersinia enterocolitica
• Safety Pin Appearance
• Non-motile at 37C
• Test: Indole (-); MR(+); Urease (+) except Y.
pestis
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Yersinia
• LPS Composition
• Y. pestis – lacks O-specific polysaccharide chain
• May posses Lipid A- LOS
• Y. pseudotuberculosis and Y. enterocolitica –
possess complete O-polysaccharide
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Yersinia pestis
Virulence Factors:
Fraction1, V (protein), W (lipoprotein)
Symptoms: high fever and buboes
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Yersinia pestis
Bubonic/Glandular Form
• Most common
• S/S high fever, headaches and buboes,
hemorrhages (blackish discoloration)
Septicemic Form – proliferation in blood;
Pneumonic Form - secondary to septicemic form;
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Yersinia enterocolitica
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Yersinia pseudotuberculosis
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Oxidase (+): Plesiomonas
shigelloides
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P. shigelloides
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Laboratory Diagnosis of
Enterics
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Cultures
Stool Cultures
• Rectal Swabs placed in transport media
• Subculture to Enrichment Broths to enhance
growth of pathogens
• Inoculate to selective and differential culture
media
• Streaking: Clock Method
• Inoculate first to the least inhibitory medium
Cultures
Stool Cultures
• Incubate plates
• Colonies tested for Oxidase
• Biochemical Tests (TSI, LIA); then Indole, MR,
VP, Citrate, SIM, Urease, LDC, ODC, ADH
Cultures
Urine Cultures
• Perform Urine Culture if Urinalysis reveals
many PMNs and presence of bacteria
• Urine specimen is mixed well and a calibrated
loop (0.01 or 0.001ml) is dipped and streaked
on BAP and MAC using quantitative isolation
• Incubate; then perform Gram Stain
• Count the number of colonies on BAP to
determine CFU/ml
✓ Ca
Performing
Specimen is placed on plate
1 information
with 1:100 or 1:000 L loop
steps that m
in the Case
Colonies growing
• Call the
2 Cross-streaks after 18-24 hour
• Docume
incubation
• Request
Cult ur
A/A
A/AG +
K/A
K/AG
K/AG+
K/A+
K/K
Lysine Iron Agar
LIA
K/K
R/A
K/K+
K/A
K/A+
IMViC
Decarboxylase
Phenylalanine Deaminase
Gelatin Hydrolysis
SIM
Urease
DNASE
ONPG
MUG test
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CHA PTER 9 Biochemical Ident if icat ion of Gram-Negat ive Bact eria 193
Spot indole Tryptophanase Organism from blood agar or any Positive reaction identifies Escherichia
tryptophan-containing medium is placed coli, Proteus vulgaris; aids in
on a swab and reagent is added; identification of anaerobes
hydrolysis of tryptophan to indole is
indicated by production of blue to
blue-green color on addition of DMAC
ONPG β-Galactosidase Ester linkage of orthonitrophenyl moieties Determines lactose fermentation (yellow
to various carbohydrates; hydrolysis color) in slow lactose fermenters;
results in release of yellow differentiates Neisseria lactamica
ortho-nitrophenol from pathogenic Neisseria spp.
Oxidase Cytochrome-c oxidase Blue compound is produced when Differentiation of nonfermenters; aids in
tetramethyl-para-phenylenediamine identification of Neisseria,
reacts with cytochrome c Aeromonas, Vibrio, and
Campylobacter spp.
Catalase Catalase Breakdown of hydrogen peroxide into Differentiation of staphylococci from
oxygen and water, resulting in rapid streptococci and of Listeria spp. from
production of bubbles streptococci
Bile solubility Autocatalyzes colony in the presence of the Presumptive identification of
surfactant sodium deoxycholate (bile Streptococcus pneumoniae in
salt) sputum, blood, and CSF cultures
PYR PYR Hydrolysis of amide substrate with Identification of group A streptococci;
formation of free β-naphthylamide, differentiates Enterococcus from
which combines with DMAC to form a group D streptococci
bright red color
Rapid urease Urease Rapid hydrolysis of urea by urease releases Screening test for Cryptococcus,
ammonia; alkalinity causes phenol red Proteus, and Klebsiella spp. and
indicator to change from yellow to red Yersinia enterocolitica
Rapid hippurate hydrolysis Hippuricase Enzymatic hydrolysis of hippurate visualized Speciation of Streptococcus agalactiae,
by addition of ninhydrin Campylobacter jejuni, and Listeria
(triketohydrindine) spp.
MUG β-Glucuronidase Hydrolysis of substrate to fluorescent Presumptive identification of E. coli and
compound MUG, which fluoresces blue Streptococcus anginosus group;
under long-wave UV light enterohemorrhagic E. coli is negative
LAP LAP LAP hydrolyzes substrate to leucine and Presumptive identification of catalase-
α-naphthylamine, which reacts with negative, gram-positive cocci
DMAC to form a red color
CSF, Cerebrospinal fluid; DMAC, p-dimethylaminocinnamaldehyde; LAP, leucine aminopeptidase; MUG, 4-methylumbelliferyl-β-D-glucuronide; ONPG,
orthonitrophenyl galactoside; PYR, L-pyrrolidonyl-β-naphthylamide; UV, ultraviolet.
194 PA RT I Int roduct ion t o Clin ical M icrobiology
TABLE 9-3 Commercially Available Manual and Automated Systems for Microbial Identification
M anual
API bioMérieux1 Carbohydrate utilization/ Enterobacteriaceae, other GN bacilli,
chromogenic substrate Staphylococcus spp., Streptococcus
spp., Enterococcus spp., Neisseria
spp., GP bacilli, yeast, anaerobes
Crystal E/NF BD Diagnostic Systems2 Carbohydrate utilization/ Enterobacteriaceae, other GN bacilli,
chromogenic substrate Neisseria spp., Haemophilus spp.,
GP cocci, GP bacilli
Enterotube II BD Diagnostic Systems Carbohydrate utilization/ Enterobacteriaceae, other GN bacilli
chromogenic substrate
Microplate Biolog4 Carbohydrate utilization GP organisms, GN organisms, yeasts,
anaerobes
Gonochek EY Laboratories6 Chromogenic substrate Neisseria/Moraxella spp.
Micro-ID (RapID) Remel3 Carbohydrate utilization/ Enterobacteriaceae, other GN bacilli,
chromogenic substrate Neisseria spp., Haemophilus spp.,
streptococci, enterococci, yeast,
anaerobes, UTI, GP bacilli
MicroScan (TouchScan) Siemens Healthcare Diagnostics5 Carbohydrate utilization/ GP organisms, GN organisms, UTI,
chromogenic substrate Haemophilus spp., Neisseria spp.
Oxi-Ferm II BD Diagnostic Systems Carbohydrate utilization Nonfermenter GN bacteria
Plate Remel Chromogenic substrate Neisseria/Moraxella spp., Escherichia
coli, Streptococcus spp., yeasts
Uni-N/F-Tek Remel Carbohydrate utilization Nonfermenters, yeast
Automated
BD Phoenix BD Diagnostic Systems Carbohydrate utilization/ Enterobacteriaceae, other GN bacilli,
chromogenic substrate/ GP
fluorogenic substrate
MicroScan (Autoscan, WalkAway) Siemens Healthcare Diagnostics Carbohydrate utilization/ Enterobacteriaceae, other GN bacilli,
chromogenic substrate/ Neisseria spp., Haemophilus spp.,
fluorogenic substrate streptococci, enterococci,
staphylococci, GP bacilli, yeast,
anaerobes
Sensititre TREK Diagnostic Systems7 Carbohydrate utilization/ GN organisms, GP organisms,
chromogenic substrate anaerobes
Sherlock Microbial Identification System MIDI8 Fatty acid analysis of GN organisms, GP organisms,
microbial cells Mycobacterium spp., yeasts
Vitek (AMS) bioMérieux Carbohydrate utilization/ Enterobacteriaceae, other GN bacilli,
chromogenic substrate Neisseria spp., Haemophilus spp.,
streptococci, enterococci,
staphylococci, GP bacilli, yeast,
anaerobes
API, Analytical Profile Index; AMS, AutoMicrobic System; GN, gram-negative; GP, gram-positive; UTI, urinary tract infection.
1
Hazelwood, MO.
2
Sparks, MD.
3
Serologic Grouping
• Shigella
• Salmonella