Family

Enterobacteriaceae

Alvin Rey Flores RMT, MT(ASCPi), MPH

Department of Medical Technology
Faculty of Pharmacy
UNIVERSITY OF SANTO TOMAS

ar flores
Generalities

• Gram Negative Enteric Coccobacilli/short plump

bacilli
• Non-sporeformers
• Facultative Anaerobes
• On BAP/CAP: large moist gray colonies
• All are gamma hemolytic except Escherichia coli
• Non capsulated except Klebsiella and Enterobacter

ar flores
Generalities

• All are motile at 37C except:

• Klebsiella, Shigella, Yersinia pestis
• Yersinia (motile at 25C)
• All are AEROGENIC except Salmonella typhi,
Yersinia, Shigella and Providencia

ar flores
Generalities

• Most members are commensal flora of the

intestinal tract except Shigella, Yersinia,
Salmonella

• All are CATALASE POSITIVE except S. dysenteriae

• All are OXIDASE NEGATIVE except Plesiomonas
• All are NITRATE REDUCERS except Photorhabdus
and Xenorhabdus, some biotypes of Pantoea
agglomerans, some species of Serratia and Yersinia
• Vector Borne: Y. pestis
ar flores
Generalities

• All are GLUCOSE fermenters

• Lactose Fermenters
• RAPID LACTOSE FERMENTERS (18-24 HOURS)
• Escherichia
• Klebsiella
• Enterobacter
• LATE LACTOSE FERMENTERS (>48 HOURS)
• Citrobacter
• Hafnia
• Yersinia enterocolitica
• Salmonella enterica subsp. arizonae
• Serratia**
• Shigella sonnei

ar flores
Generalities

Non Lactose Fermenters

 Salmonella except S. enterica subsp. arizonae
 Providencia
 Edwardsiella
 Morganella
 Proteus
 Shigella except S. sonnei
 Yersinia except Y. enterocolitica

ar flores
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
ar flores
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)
ar flores
Virulence and Antigenic Factors
• Plasmids carrying resistance genes
• Antigens
 O antigens
 H antigens
 K antigens
• K1 antigen
• Vi antigen

ar flores
Virulence and Antigenic Factors

• Pili
• Flagella
• Enterotoxins
• Enzymes
• Endotoxins

ar flores
Clinically Significant Genera

• Opportunistic Members
• True Pathogens

ar flores
Escherichia coli

• Normal flora of colon

• Common Name: “Colon Bacillus”
• Important human pathogen (UTIs, CNS
infections, sepsis, endotoxin induced shock,
and diarrheal diseases)
• Distant relative of Shigella (cross reactions)

ar flores
E. coli

Important Virulence Factors

• Pili
• Cytolysins
• Aerobactins
• O, H, K Antigens

ar flores
E. coli

• Uropathogenic Escherichia coli

• Most common cause of Urinary Tract Infections

• Primary Virulence Factor: Pili

• Other Factors: hemolysins, cytloysins, aerobactin
Key Tests:
• Indole and MR Test (+)
• LDC (+), ODC(+ mostly), ADH (- mostly)
ar flores
Diarrheogenic E. coli

• Gastrointestinal Pathogens
5 MAJOR CATEGORIES
1. ETEC
2. EIEC
3. EPEC
4. EHEC
5. EAEC
DAEC
EAEC
ar flores
ETEC

• Enterotoxigenic Escherichia coli

• A common cause of watery diarrhea among adults
and children
• Causes:
• “Traveler’s Diarrhea”/ Turista/ Montezuma’s
Revenge
• Weanling Diarrhea
• Self-limiting
• S/S Watery diarrhea, cramps, no fever or vomiting
• High Infective Dose for disease to occur (106 to 1010
organisms)

ar flores
ETEC

Virulence Factors
• Fimbriae
• Heat Labile Toxin – (cyclic adenosine
monophosphate)
• Heat Stable Toxin – (guanyl cyclase)

ar flores
EPEC

• Enteropathogenic Escherichia coli

• Infantile Diarrhea
• S/S Low grade fever, malaise, vomiting, Diarrhea,
• mucoid stools but no blood
• Capable of Adhering to intestinal cells capable of
producing lesions called “attaching and effacing
lesions”
• production of enterotoxin

ar flores
EIEC

• Enteroinvasive Escherichia coli (EIEC)

• Shiga-like
• S/S differ greatly from EPEC and ETEC
• Targets: Colonic epithelial cells
• Dysentery, invasion and destruction of intestinal
wall
• Fever, severe abdominal cramps, malaise and
watery diarrhea
• Shigella like symptoms
• MOT: Fecal Oral Route

ar flores
EHEC

• Enterohemorrhagic Escherichia coli/ Shiga-Toxin

E.coli
• Most Important Strain: O157:H7
• Implicated in an outbreak of hemorrhagic
diarrhea, colitis and hemolytic uremic syndrome
(HUS)
• S/S: watery to bloody diarrhea and colitis with no
WBCs in stools
• Virulence Factors:
• Cytotoxins
• Verotoxin I
• Verotoxin II
Culture: SMAC (sorbitol negative colonies)

ar flores
ar flores
Enteroadherent E. coli

• Implicated in diarrhea and UTIs

• Two Types:
• Diffusely Adherent E. coli
• Uropathogenic DAEC Strains: cystitis, acute
pyelonephritis
• Enteroaggregative E. coli
• Watery Diarrhea, vomiting, deyhdration,
coccasional abdominal pain

ar flores
E.coli

• Extraintestinal Infections
• MNEC
• Meningitis/Sepsis Associated E.coli
• Neonatal Meningitis
• Capsular K1 antigen

• Other Escherichia species

• Escherichia hermanii, E. vulneris – yellow
pigmented organism
• E. albertii – diarrhea among children

ar flores
Klebsiella

• Normal Habitat: GI Tract of humans and animals

• Found in environment
• Causes Opportunistic and Nosocomial infections
• UTI, pneumonia, septicemia, liver abscess, and wound
infections
• CAPSULATED
• NON MOTILE
Key Tests:
• Indole, MR (-)
• VP, Citrate (+)
• LDC (+), ODC and ADH (-)
ar flores
K. pneumoniae
• most commonly isolated
• Common Name: Friedlander’s Bacillus/Bacillus
capsulatus
• Causes:
• lower respiratory tract infections
• currant jelly like sputum
• Lung Abscess; hemoptysis; hemorrhage
• UTIs and wound infections, liver abscess,
bacteremia
• High case of antimicrobial resistance
ar flores
K. pneumoniae

• Emergence of hypervirulent clinical variant of

K. pneumoniae (hvKp)
• Able to cause infection among healthy ambulatory
patients
• Unusual sites of infection (liver, eyes, CSF)
• Metastatic spread
• Production of hypermucoviscous colonies

ar flores
n d Rao u t el l a

Escherichia
vulneris

Escherichia
hermannii

parison of t he colony morphology of Esch-

a yellow -pigment ed Escherichia hermannii
AC) agar. Escherichia vulneris may also
pigment ed colony, but t he yellow is more FIGURE 19-3 M ucoid appearance of Klebsiella pneumoniae
mannii. (Court esy Jean Barnishan.) on M acConkey (M AC) agar.

tahir99  VRG
ar flores
Other Klebsiella species

Klebsiella oxytoca
• Produces infections similar to K. pneumoniae
• Also causes antibiotic associated hemorrhagic colitis
• Characteristic Biochemical Test: Indole (+)

Klebsiella pneumoniae subsp. Ozaenae

• Recovered from nasal secretions and cerebral abscess
• Causes atrophic rhinitis (ozena)
• Presence of plasmid mediated ESBLs

ar flores
Other Klebsiella species

Klebsiella pneumoniae subsp rhinoscleromatis

• Isolated from patients with rhinoscleroma

ar flores
Enterobacter

• Habitat: Intestinal Tract of Humans and

animals; found in the environment
• Cause nosocomial and opportunistic infections
• MOTILE
• Biochemical Test:
• ODC (+); LDC (+) except E. gergoviae and E. cloacae

ar flores
Enterobacter

Most Common Species:

• E. cloacae
• E. aerogenes
• Isolated from wounds, urine, blood, CSF
• Opportunistic infections

ar flores
Other Enterobacter species

E. gergoviae – recovered from respiratory samples

Cronobacter sakazakii (Enterobacter sakazakii)

• Causes meningitis and bacteremia among neonates
• Distinct Feature: Yellow colonies
E. cancerogenus (E. taylorae)
• Causes osteomyelitis after traumatic wounds
• Key Test: Lactose (-) but ONPG (+)
• On MacConkey: colonies with purple centers after
extended incubation

ar flores
umoniae subsp. pneumoniae
E. cloacae E. aerogenes K. pneumoniae subsp. pneumoniae

Sign %+ (% +) Sign %+ (% +) Sign %+ (% +)

+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)

d 13 (8) + 96.7 + 97.2 (0.9)

− 4.1 (1.5) + 93.4 + 92.5 (1.5)
− or + 30 + 95 + 95
zation) − 0 − 0 + or (+) 88.9 (8.9)

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.

FIGURE 19-5 M ucoid, yellow -pigment ed colonies of Crono-

bact er sakazakii grow ing on brain–heart inf usion agar. (Cour-
t esy Jean Barnishan.)
ow -pigment ed Pant oea agglomerans on
(SBA). (Court esy Jean Barnishan.)

ar flores
Pantoea agglomerans

• Formerly Enterobacter agglomerans

• Septicemia (healthcare settings) IV fluids, parenteral
nutrition
• Traumatic injury from contaminated soil
• Wound infections, septic arthritis, osteomyelitis
• Key Test: Yellow pigments
• ODC, LDC, ADH (-)

• Update: Another new species: P. ananatis

ar flores
 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

FIGURE 19-5 M ucoi

bact er sakazakii grow
t esy Jean Barnishan.)
FIGURE 19-4 Yellow -pigment ed Pant oea agglomerans on
sheep blood agar (SBA). (Court esy Jean Barnishan.)

ar flores
Serratia

• Opportunistic pathogens associated with outbreaks

in health care settings (bacteremia, urinary,
respiratory tract infections)
Key Biochemical Tests
• ONPG (+) (except S. fonticola)
• DNAse (+)
• Lipase (+)
• Gelatinase (+)
Production of Prodigiosin pigment (2-methyl-3-amyl-
6-methoxyprodigiosene)
ar flores
Serratia

Serratia marcescens – most significant

• Indole (neg); MR(V); VP(+); Citrate(+)
• LDC and ADH (+)
• DNAse (+)
Serratia rubidaea
Serratia plymuthica

Serratia odorifera – “rotten potato odor”

ar flores
Serratia

ar flores
Hafnia alvei

• Linked to an emerging cause of

gastroenteritis; isolated in stool cultures
• Also Linked to hemolytic uremic syndrome,
graft versus host disease, sepsis
Key Biochemical Tests:
• Delayed positive citrate reaction
• Lactose and Sucrose (-)
• Lipase, Gelatinase, DNAse (-)
• Odor: Strong Scent of Feces
ar flores
Proteus, Providencia,
Morganella

• Tribe Proteeae
• Normal flora of the GI Tract; found in the
environment
• Opportunistic Pathogens; Noscomial
Infections

• Distinct Biochemical Test: Deaminase (+)

ar flores
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
ar flores
 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

FIGURE 19-7 Example of Prot eus mirabilis sw arming on sheep

blood agar (SBA). (Court esy Kimberly Walker and R. Abe
Baalness.)

ar flores
Providencia

• Causes UTIs, diarrhea among travelers and

children, nosocomial infections
Species
P. stuartii - burn units
P. rettgeri – UTI, diarrhea
Key Tests
• Indole, MR (+)
• Citrate (+)
• Urease (+) for P. rettgeri
ar flores
Morganella morganii

• Implicated in UTIs, neonatal sepsis

• Motile but does not swarm
Key Tests
• LDC, Citrate, Lactose (-)

ar flores
Edwardsiella tarda

• Only recognized human pathogen

• Opportunistic Pathogen
• Causes bacteremia, wound infections (aquatic
incidents)
Key Biochemical Tests
• Urease (-), LDC (+), H2S (+) Indole (+), Does
not utilize Citrate

ar flores
Citrobacter

• Formerly under Salmonellae

• Normal Flora of GI Tract
• Causes nosocomial infections, UTIs,
pneumonia, meningitis
• Key Biochemical Tests
• Utilize Citrate, ONPG, Methyl Red (+)
• Maybe mistaken for Salmonella

ar flores
Citrobacter

• C. freundii – UTIs, pneumonia, diarrhea,

intraabdominal abscess, endocarditis among
drug users

• C. koseri – neonatal meningitis; brain abscess

ar flores
True Pathogens

• Primary Intestinal Pathogens

• Not a part of the normal flora of humans

Salmonella – GI illnesses
Shigella – GI Illnesses

Yersinia – zoonotic; transmitted by wild animals

and ticks
ar flores
True Pathogens: Salmonella

• Inhabit the GI Tract of animals

• Previously, there are 3 biochemically discrete
species:
• S. enteritidis,
• S. choleraesuis,
• S. typhi
Currently, only two are recognized:
• S. enterica
• S. bongori
ar flores
Salmonella

Key Biochemical Tests

• NLF except S. enterica subsp. Arizonae
• Indole (-), VP(-), Deaminase and Urease (-)
• H2S positive except Salmonella Paratyphi A
• Motile except: Salmonella Pullorum & S.
Gallinarum

ar flores
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

ar flores
Salmonella

Virulence Factors
• Fimbriae and most especially enterotoxins are
implicated as significant virulence factors
• Ability to traverse intestinal mucosa

MOT: ingestion of contaminated water or food

(poultry, dairy products), containing animal
feces; transmitted human to human (carrier
state)
ar flores
 + − +
− − +
+ − +
− + +
− + +
+ − +

Salmonella +
+
−
+
+
+

ification.  In Murray PR, et al, editors: Manual of clinical microbiology, ed 9,

ositive; +, ≥90% of strains positive; V, 10% to 89% of strains positive.

• Antigenic Structures
Classification based on Kauffman-White
H Ag
> O, H and K (Vi Ag)
Vi Ag Ag serotyping
O Ag

FIGURE 19-8 Ant igenic st ruct ures of salmonellae used in sero-

ar flores
logic t yping.
Salmonella

• S. serotype Cholerasuis: non typhoidal fever

w/ or w/o gastroenteritis
• S. serotype Typhi: typhoid fever
• Humans are the only reservoir
• S. serotype Paratyphi: paratyphoid fever w/ or
w/o gastroenteritis
• S. serotype Typhimurium: food poisoning (dairy
products)

ar flores
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
ar flores
Salmonella

Typhoid Fever (Enteric Fever)

Caused by Salmonella Typhi
No animal reservoir; transmitted by carriers,
contaminated food
• Invades intestinal epithelial cells and invades
lymph nodes and disseminates to other organs
• Facultative Intracellular Parasite

ar flores
Salmonella

1st week: fever, dull frontal headache,

malaise, body pain, constipated (“scybalous”
stool)
2nd and 3rd week: intestinal tract, “rose spots”
appear; diarrheic stools;

ar flores
ar flores
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

Carrier State: harbored in the gall bladder

• “Typhoid Mary”
ar flores
ar flores
ar flores
Shigella

• Closely related to Escherichia

• Humans are the only reservoir
• Biochemically inert members of Family
Enterobacteriaceae
• Causes shigellosis/bacillary dysentery
(enterocolitis)

ar flores
Shigella

Key Tests
• Non-motile
• Not aerogenic
• H2S (-)
• LDC (-)
• Does not utilize citrate
• Does not Ferment Xylose
• NLF except S. sonnei

ar flores
Shigella

Virulence Factors
• Shiga-toxin
• Endotoxin
• Neurotoxin
• Invasive property
Antigens
• All possess O antigens, some species possess K
antigen
ar flores
Shigella

• Subgroups (based on O antigen groups)

A - S. dysenteriae
• endotoxin, enterotoxin, neurotoxin,
invasiveness
B - S. flexneri (Strong’s bacillus)
C - S. boydii (Newcastle Manchester)
D - S. sonnei (Duval’s)

ar flores
Shigella

• MOT: fecal-oral, contaminated food/water

• <10 yrs are most affected, <1yrs most
susceptible
• Low Infective Dose
• Manifestations: fever chills, cramps, diarrhea,
pain and tenesmus 24-48 hrs after ingestion
>Intestines then to colon
> Bloody, with pus, and mucoid stools
ar flores
Yersinia

• Zoonotic
• 3 Species
• Yersinia pestis
• Yersinia pseudotuberculosis
• Yersinia enterocolitica
• Safety Pin Appearance
• Non-motile at 37C
• Test: Indole (-); MR(+); Urease (+) except Y.
pestis
ar flores
Yersinia

• LPS Composition
• Y. pestis – lacks O-specific polysaccharide chain
• May posses Lipid A- LOS
• Y. pseudotuberculosis and Y. enterocolitica –
possess complete O-polysaccharide

ar flores
Yersinia pestis

• Zoonotic; Disease of Rodents

• Transmitted to humans by fleas
• Facultative Intracellular Parasite

• Causes: Plague/Bubonic Plague/Black Death

Virulence Factors:
Fraction1, V (protein), W (lipoprotein)
Symptoms: high fever and buboes
ar flores
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;

Culture: can grow in BAP, CAP, TSA, CIN

• Fried Egg Colonies
• Prefers to grow at 25-30C
ar flores
ar flores
ar flores
Yersinia enterocolitica

• Most commonly isolated species

• Reservoir: Pigs
• Causes Yersinosis
• S/S: mimics appendicitis, enteritis, arthritis,
erythema nodosum, septicemia (px with
hemochromatosis)
• MOT: ingestion of contaminated food (pork
meat, milk)

ar flores
Yersinia enterocolitica

• Survival in cold temperatures (4C) (cold

enrichment)
• Most common contaminant in blood products
Key Characteristic
• Culture: Cefsulodin Irgasan Novobiocin Agar
• Bull’s Eye Colonies

ar flores
ar flores
Yersinia pseudotuberculosis

• Pathogen of rodents (guinea pigs), birds,

rabbits
• Rare in humans
• Animals: S/S Caseous Swellings
(Pseudotubercles)
• Humans: mesenteric lymphadenitis,
septicemia

ar flores
Oxidase (+): Plesiomonas
shigelloides

• Oxidase (+) member of Enterobacteriaceae

• Motile (polar flagellum)
• Not a normal flora of GI
• Habitat: Freshwater and estuarine waters
• Causes: Gastroenteritis (cholera like illness)
• Ingestion of uncooked shellfish
•

ar flores
P. shigelloides

• Share some biochemical features with

Shigella
• Key Tests: Unable to grow in 6% NaCl;
glucose fermenter; oxidase (+)

ar flores
Laboratory Diagnosis of
Enterics

Alvin Rey Flores, RMT, MPH

Department of Medical Technology
UST Faculty of Pharmacy
Specimen Collection

• Specimens: Stool, Rectal Swabs, Blood, Urine,

Wounds, Sputum, etc.
• Transport Media: Cary-Blair, Amies or Stuart
Media
• Gram Stain: Gr (-) Coccobacilli
• Plesiomonas: pleomorphic rods
• Wayson Stain: Safety pin for Y. pestis
• Growth: 35-37C; Facultative Anaerobe
• For K. granulomatis: direct examination of
Wright of Giemsa stained scrapings of lesion
•

ar flores
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

FIGURE 6-3 Quant it at ive isolat ion t echnique.

Culture Media

• BAP and CAP

• Gram Negative Broth, Selenite Broth,
Tetrathionate Broth
• MacConkey Agar, Eosin Methylene Blue Agar,
Sorbitol MacConkey Agar
• Hektoen Enteric Agar, Xylose Lysine
Desoxycholate Agar, Salmonella Shigella Agar
• Bismuth Sulfite Agar, Cefsulodin Irgasan
Novobiocin
MacConkey
Hektoen Enteric Agar
Salmonella on XLD
SSA
S. typhi on BSA
TCBS
Biochemical Tests

• All Enterics are capable of fermenting

GLUCOSE
• All LACTOSE fermenters are also GLUCOSE
Fermenters
• Enzymes Important to Metabolize and Take up
Lactose
• Beta-Galactoside permease (Lactose Permease)
• Entry of lactose through cell wall
• Beta-galactosidase
• Breaks down lactose into glucose and galactose
Triple Sugar Iron
TSI

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

ar flores
 CHA PTER 9 Biochemical Ident if icat ion of Gram-Negat ive Bact eria 193

TABLE 9-2 Rapid Biochemical Tests Performed on Isolated Colonies

Test Bacterial Enzyme Mode of Action Applications

Spot indole Tryptophanase Organism from blood agar or any
tryptophan-containing medium is placed
on a swab and reagent is added;
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
to various carbohydrates; hydrolysis
results in release of yellow
ortho-nitrophenol
Oxidase Cytochrome-c oxidase Blue compound is produced when
tetramethyl-para-phenylenediamine
reacts with cytochrome c
Catalase Catalase Breakdown of hydrogen peroxide into
oxygen and water, resulting in rapid
production of bubbles
Bile solubility Autocatalyzes colony in the presence of the
surfactant sodium deoxycholate (bile
salt)
PYR PYR Hydrolysis of amide substrate with
formation of free β-naphthylamide,
which combines with DMAC to form a
bright red color
Rapid urease Urease Rapid hydrolysis of urea by urease releases
ammonia; alkalinity causes phenol red
indicator to change from yellow to red
Rapid hippurate hydrolysis Hippuricase Enzymatic hydrolysis of hippurate visualized
by addition of ninhydrin
(triketohydrindine)
MUG β-Glucuronidase Hydrolysis of substrate to fluorescent
compound MUG, which fluoresces blue
under long-wave UV light
LAP LAP LAP hydrolyzes substrate to leucine and
α-naphthylamine, which reacts with
DMAC to form a red color
Positive reaction identifies Escherichia
coli, Proteus vulgaris; aids in
identification  of anaerobes
Determines lactose fermentation (yellow
color) in slow lactose fermenters;
differentiates Neisseria lactamica
from pathogenic Neisseria spp.
Differentiation of nonfermenters; aids in
identification  of Neisseria,
Aeromonas, Vibrio, and
Campylobacter spp.
Differentiation of staphylococci from
streptococci and of Listeria spp. from
streptococci
Presumptive identification  of
Streptococcus pneumoniae in
sputum, blood, and CSF cultures
Identification  of group A streptococci;
differentiates Enterococcus from
group D streptococci
Screening test for Cryptococcus,
Proteus, and Klebsiella spp. and
Yersinia enterocolitica
Speciation of Streptococcus agalactiae,
Campylobacter jejuni, and Listeria
spp.
Presumptive identification  of E. coli and
Streptococcus anginosus group;
enterohemorrhagic E. coli is negative
Presumptive identification  of catalase-
negative, gram-positive cocci

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

Name Manufacturer Principle Organisms Identified

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