GRAM-NEGATIVE BACILLI

Family Enterobacteriaceae

 Gram negative rods ( non-spore forming bacilli )

 Motile with peritrichous flagella except Klebsiella and Shigella
 Yersinia is also motile at 35-37C
 Facultative anaerobes
 Ferment glucose often with gas production
 Catalase positive except Shigella dysenteriae
 Oxidase negative except Plesiomonas
 Reduce nitrate to nitrite except for Erwinia and Pantoea agglomerans which has an enzyme : Nitroreductase
 Aerogenic (gas producer) except Shigella
 CPON ( CATALASE POSITIVE, OXIDASE NEGATIVE )

NOTE :

“Some spp. under Enterobactericeae inhabits the digestive colon, that’s why it is associated with diarrheal illnesses.”

“They are also differentiated based on their biochemical reactions and differences in antigenic structure”

“Since there’s a large a large diversity of genera under this family ( >15 different genera ), so they divided in into 2
groups :

1. Those who are either commonly colonizes the gastrointestinal tract or most notably associated with human
infections.
Example : Salmonella typhi ( typhoid fever ), Shigella spp. ( dysentery ), and Yersinia pestis ( plague )

2. Those who are capable of colonizing humans but are rarely associated with human infection ( environmental
habitat or animals ) or considered as OPPORTUNISTIC pathogens
Example : Citrobacter spp., Enterobacter, Klebsiella, Proteus, Serratia and more others.

“Opportunistic pathogens are more virulent once they’ve caused infection than most of the commonly associated
human infection”

Escherichia,Shigella,Salmonella,Citrobacter,Klebsiella,Enterobacter,Hafnia,Serratia, Edwardsiella, Poteus,

Providencia, Morganella, Yersinia, Erwinia

Antigenic Structure

 K antigen / Vi
 Capsular antigen
 Heat-labile
 Some Salmonella have capsular (K) antigen, referred to as Vi = Virulence
 O antigen
 Somatic/body antigen ( found in the cell wall )
 Heat-stable
 Antibodies to O antigens are predominantly IgM
 H antigen
 Located in the flagella ( flagellar antigen )
 Denatured or removed by heat or alcohol
 H antigens agglutinate with anti-H antibodies, mainly IgG

ESCHIRICHIA IMViC = + + - - TSI = A/AG H2S (-)

 E.coli strains = ETEC, EIEC, EPEC, EHEC/VTEC, EAEC, UPEC, MNEC
 Urinary Tract Infection (UTI) – 90%
 Meningitis
 Diarrheal diseases
A. ENTEROTOXIGENIC E.coli (ETEC)
 Stool appearance of patient infected : PROFUSE WATERY STOOL
 Only grow on BAP
 Produces heat labile (LT) and or heat stable (ST) enterotoxins
 (LT) resembles the structure and function of CHOLERA TOXIN
 (ST) responsible for intestinal fluid secretion by stimulating the GUANLYLATE CYCLASE
 Infection is called as your "MONTEZUMA'S REVENGE" or "TURISTA" or TRAVELER'S DIARRHEA/CHILDHOOD DIARRHEA
 GRAM-NEGATIVE BACILLI

B. ENTEROINVASIVE E.coli (EIEC)

 Invades the intestinal epithelium causing “SHIGELLA-LIKE INFECTION”
 Dysentery ( necrosis, ulceration and inflammation of the colon) usually in young children living in areas of poor
sanitation
 Watery stool with RBC WBC (neutrophil) MUCUS
C. ENTEROPATHOGENIC E.coli (EPEC)
 Noninvasive, produces no toxin
 Nosocomial, seen in newborn and infants
 WATERY DIARRHEA with MUCUS but NO BLOOD
D. ENTEROHEMORRHAGIC E.coli (EHEC)/Verotoxic E.coli (VTEC)
 Produces “SHIGA-LIKE TOXIN”
 Produces verotoxin - has a cytotoxic effect on Vero cells: vero cells are cells derived from the kidney of an african
green monkey.
 Verotoxin resembles cytotoxin of S. dysenteriae
 Hemolytic Uremic Syndrome (HUS) = most severe manifestation of EHEC (hemorrhagic colitis that can affect
kidneys and blood cells)
 Most frequently associated with serotype E.coli O157:H7
 Can grow in MacConkey but SORBITOL is used instead of lactose - only strain that is sorbitol negative (colorless
colonies)

NOTE: E.coli 0157:H7 on SMAC (Sorbitol substituted MacConkey Agar) strain that is nonfermenter of sorbitol and
colorlress growth on SMAC

E. ENTEROAGGREGATIVE E.coli (EAEC)

 Probably invloves biding by pili ( adhere to host cells )
 ST-like and Hemolysin-like toxins
 Actual pathogenic mechanism not known
 Watery diarrhea
 Diarrhea in HIV-infected and AIDS patients in the USA
F. UROPATHOGENIC E.coli (UPEC)
 Major cause of E.coli-associated community acquired UTI
 Four main UPEC strains (A,B1,B2 and D)
 The strains contain a variety of Pathogenicity islands (PAI)
G. Meningitis/sepsis-associated E.coli (MNEC)
 Positive for K1 antigen
 Neonatal meningitis that results in high morbidity and mortality

KLEBSIELLA IMViC = - - + + TSI = A/AG H2S (-)

 Klebsiella spp. ( K.pneumoniae subsp. pneumonia, K.rhinoscleromatis, K.oxytoca, K.ozaenae, and K.variicola) are
inhabitants of nasopharynx and gastrointestinal tract.
 Exhibit mucoid growth, large polysaccharide capsule
 Klebsiella pneumonia also known as Friedlander’s bacillus. Encapsulated and appears as mucoid colonies that
tend to string. It is also increasingly isolated from community-acquired pyogenic live abscesses worldwide and
demonstrate a hypervirulent (hypermucoviscous) phenotype (hvKP)
 Klebsiella ozanae – causes purulent sinus infection
 Klebsiella rhinoscleromatis – causes granuloma of the nose and nasopharynx
 Positive for K1 antigen
 Klebsiella oxytoca is the only Indole positive

ENTEROBACTER IMViC = - - + + TSI = A/AG H2S (-)

 Enterobacter spp ( E.aerogenes, E. asburiae, E.cancerogenus, Enterobacter cloacae subsp. cloacae,
E.hormaechei, E.kobei, E. ludgwigii) motile lactose fermenters that produces mucoid colonies.
 Opportuistic infections: UTI, RT and wound infections
 Moat predominant isolate is E.cloacae
 Produces yellow pigmentation that intensifies at 25C (Enterobacter sakazakii)

LDC ODC ADH

K.pneimoniae + - -
K.oxytoca + - -
E.aerogenes + + -
E.cloacae - + +
 GRAM-NEGATIVE BACILLI

P.agglomerans - - -

SERRATIA (+ DNase, Gelatinase, Lipase, ONPG,Ornitrophrnyl galactoside)

 Serratia spp. ( S.marcescens subsp. marcescens, S.marcescens subsp. marcescens biogroup 1, S.liquefaciens,
S.rubidea, S.odoriferous biogroup 1 and 2 ) are motile, slow lactose fermenters.
 S.marcescens and S.rubidea produce a red piment (PRODIGIOSIN)
 S.odorifera produces a rancid potato-like odor
 Common opportunistic pathogen in hospitalized patients

SALMONELLA
 Salmonella ( all serotypes ) are facultative anaerobic, motile, gram-negative rods commonly isolated in the
intestines of humans and animals.
 Sources of infection : water, milk and dairy products, shellfish from contaminated sea/water, dried or frozen
eggs, meat and meat products and household pets.
 2 primary species :
 Salmonella enterica ( human pathogen ) divided into 6 subsp.
 subsp. enterica, subsp. salamae, subsp. arizonae, subsp. diarizonae, subsp. houtenae, subsp.
indica
 Salmonella bongori ( animal pathogen )
 Salmonella enterica subsp. enterica can be further divided into serotypes:
1. Salmonella serotype Typhi ( typhoid fever ) – No diarrhea and vomiting. Symptoms are often
headache, abdominal cramping, constipation, and high fever.
 Causes enteric fever (typhoid fever):
 S. paratyphi A (serogroup A)
 S. paratyphi B (serogroup B)
 S.cholerasius (serogroup C) can cause bacteremia
 S.typhi (serogroup D)
 S. typhi serotype typhi (most important cause) has a “BLACK COLONIES WITH METALLIC
SHEEN” appearance in Bismuth Sulfate Agar (BSA)

2. Salmonella serotype Enteritidis ( associated with infections acquired from the ingestion of eggs or
chicken. Gastroenteritis is accompanied by diarrhea (PEA-SOUP STOOL), fever, and abdominal cramps.

 Enterocolitis/gastroenteritis – most common manifestation of salmonella infection (S.typhimurium)

 Widal’s Test (Tube dilution agglutination test)
 At least two serum specimens, obtained at intervals of 7-10 days, are needed to prove a rise in antibody
titer.
 Serial dilutions of unknown serum are tested against antigens from representative salmonellae
 High or rising titer of O ≥ 1:160 suggest that active infection is present
 High or rising titer of H ≥ 1:160 suggests infection past or immunization
 High titer of antibody to Vi antigen occurs in some CARRIERS

SHIGELLA
 Shigella spp. ( S.dysenteriae, S.flexneri, S.boydii, and S. sonnei) nonmotile, lysine decarboxylase negative, citrate
negative, malonate negative, and H2S negative, non-lactose fermenter.
 All strains ferment glucose without gas production, EXCEPT: S.flexneri and S.boydii
 Natural habitat of shigellae is limited to the intestinal tracts of humans and other primates, where they produce
bacillary dysentery
 All shigella are capable of causing dysentery. However, S.dysenteriae 1 produces the most severe forms of illness
and may lead to hemolytic uremic syndrome (HUS)
 Shigellosis may begin as watery diarrhea, fever, and abdominal cramps.
 DYSENTERY = STOOLS WITH BLOOD OR MUCUS

GROUP TYPE CATALASE ONPG MANNITOL

S.dysenteriae A - - -
S.flexneri B + - +
S.boydii C + - +
S.sonnei D + +. Late lactose ferm. +
 GRAM-NEGATIVE BACILLI

CITROBACTER
 Causes UTI and sepsis
 Organisms resemble Salmonella but are OPNG positive and LDC negative
 Citrobacter spp. (C.freundii, S.braakii, C.koseri or formerly known as C.diversus)
 C.freundii (H2S +), C.koseri (H2S -)

PROTEUS
 Proteus spp. ( P. mirabilis, P.vulgaris, P.penneri)
 Normal inhabitants of gastrointestinal tract. Motile, non-lactose fermenters.
 Can deaminate phenylalanine.
 Produce infections in humans only when they leave the intestinal tract : UTI, bacteremia, pneumonia,
nosocomial infections. Can also cause renal calculi = Staghorn (Struvite/Triple phosphate)
 P.mirabilis and P.vulgaris exhibit “swarming motility/swarming appearance
 Proteus has a distinct odor referred to as a “chocolate cake/burnt chocolate”
 Certain strains share specific polysaccharides with some rickettsia and are agglutinated by sera from patients
with rickettsial diseases. (Weil-Felix Test)
 Serology :
Weil – Felix Test
• Classic test that uses CROSS – REACTING Proteus vulgaris and Proteus mirabilis anitgens to diagnose a
rickettsial infection
• Clinically sgnificant titer: >320
Reagent/Antigen: OX-2, OX-19 from P. vulgaris
: OX-K from P. mirabilis

PROVIDENCIA
 Normal inhabitants of gastrointestinal tract. Motile, non-lactose fermenters.
 Can deaminate phenylalanine.
 most commonly associated with urinary tract infections and isolated from the feces of children with diarrhea.

EDWARDSIELLA
 Isolated from the environment and many cold-blooded animals including reptiles, freshwater and aquarium fish,
frogs, anf turtles.
 Edwardsiella tarda – rarely pathogenic to humans, but, in patients with certain risk factors, it can lead to
severe, disseminated infections.

YERSINIA
 Nonmotile at 37C but motile at room temperature except Yersinia pestis.
 Yersinia spp. ( Y.pestis, Y. enterocolitica, and Y. pseudotuberculosis)
 catalase-, oxidase-, and indole-positive; non– lactose fermenting
 Exhibit BIPOLAR STAINING
 Yersinia pestis –
 Plague/bubonic plague – infection of wild rodents transmitted from one rodent to another and
occasionally by the bite of fleas. (Xenopsylla cheopsis = rat flea/vector)
 Produced pandemics of black death with millions of fatalities
 Broth cultures exhibit a characteristics “stalactite pattern” in which clumps of cells adhere to
one side of the tube
 Inclusion called as : Bipolar bodies (safety pins appearance)
 Stain : Wayson stain
 Yesinia enterocolitica
 Most common cause of death with bacterial contaminated units (blood bag)
 Characterized by fever, diarrhea and abdominal pain
 Bactermia
 Cultured on CEFSULODIN- IRGASAN- NOVOBIOCIN (CIN) AGAR : exhibit “Bull’s eye colonies”
 Yersinia pseudotuberculosis
 causes a food-borne infection characterized by self-limited mesenteric lymphadenitis that
mimics appendicitis.
 GRAM-NEGATIVE BACILLI

HAFNIA ALVEI
 (formerly Enterobacter hafniae)
 motile non–lactose fermenter. Infections are associated with consumption of contaminated food such as meat
and dairy products.
 Most infections with H. alvei are identified in patients with severe underlying disease (e.g., malignancies) or after
surgery or trauma

MORGANELLA
 Morganella spp. ( M.morganii subsp. morganii, M. morganii subsp. sibonii)
 Often associated with stool specimens collected from patients with symptoms of diarrhea.
 No clear clinical association exists when these organisms are isolated.

PANTOEA SPP.
 Pantoea spp. ( P.agglomerans and P. ananatis)
 Appear as a yellow-pigmented colony and are lysine-, arginine-, and ornithine-negative
 Indole-positive and mannitol-, raffinose-, salicin-, sucrose-, maltose-, and xylose-negative.
 Associated with traumatic injury from objects contaminated with soil (e.g., wound infections, septic arthritis,
and osteomyelitis)

PLESIOMONAS SHIGELLOIDES
 A fresh water inhabitant that is transmitted to humans by ingestion of contaminated water or by exposure of
disrupted skin and mucosal surfaces.
 Can cause gastroenteritis, most commonly in children
 Disease presentation varies from acute watery to subacute chronic diarrhea and invasive, dysentery-like
diarrhea
 it is among the few species of clinically relevant bacteria that decarboxylate lysine, ornithine, and arginine.

LABORATORY

 Enterobacteriaceae are typically isolated from a variety of sources in combination with other more fastidious
organisms
 Fecal specimens should be collected from patients preferably within the first 4 days of illness and before
administration of antimicrobials.
 All Enterobacteriaceae have similar microscopic morphology; therefore, Gram staining is not significant for the
presumptive identification of Enterobacteriaceae. (coccobacilli or straight rods with rounded ends)
 Yersinia spp. demonstrate a bipolar staining that resembles a closed safety pin when stained with methylene
blue or Wayson stain.
 Klebsiella granulomatis can be visualized in scrapings of lesions stained with Wright or Giemsa stain.

1. Growth on media : BAP/CA/EMB/MAC/HEA/SSA

 Enterobacteriaceae produce similar growth on blood and chocolate agars; colonies are large, gray, and smooth.
 Colonies of Klebsiella or Enterobacter may be mucoid because of their polysaccharide capsule.
 E. coli is often beta-hemolytic on blood agar, but most other genera are nonhemolytic.
 As a result of motility, P. mirabilis, P. penneri, and P. vulgaris “swarm” on blood and chocolate agars. Swarming
results in the production of a thin film of growth on the agar surface.
 Colonies of Y. pestis on 5% sheep blood agar are pinpoint at 24 hours but exhibit a rough, cauliflower
appearance at 48 hours.
 Broth cultures of Y. pestis exhibit a characteristic “stalactite pattern” in which clumps of cells adhere to one side
of the tube.
 Y. enterocolitica produces bull’s-eye colonies (dark red or burgundy centers surrounded by a translucent border;
on CIN agar at 48 hours. However, because most Aeromonas spp. produce similar colonies on CIN agar, it is
important to perform an oxidase test to verify that the Bull’s-eye colony of Yersinia enterocolitica (arrow) on
cefsulodin-irgasan-novobiocin (CIN) agar. organisms are Yersinia spp. (oxidase negative).

2. Triple Sugar Iron Agar test ( TSI )

Whenever you see the name of this test i.e. ‘Triple Sugar Iron Agar‘, you have to remember that it’s a test
that has three sugar (lactose, sucrose, and glucose) and also iron; and it contains agar as solidifying
agent (TSI is a semi-solid media having slant and butt).
 GRAM-NEGATIVE BACILLI

 Sugars : 10 parts lactose, 10 parts sucrose, 1 part glucose

 pH indicator: phenol red
 H2S indicator: Sodium thiosulfate ( it is reduced to SO2 and H2S ) H2S reacts with ferrous sulfate to produce black
ferric sulfide

 0.1% glucose: If only glucose is fermented, only enough acid is produced to turn the butt
yellow. The slant will remain red.
 1.0 % lactose/1.0% sucrose: If lactose or sucrose or both sugars are fermented, a large amount
of acid will produce which turns both butt and slant yellow. So the appearance of yellow color in
both slant and butt indicates that the isolate has the ability to ferment lactose or sucrose or both.
 Iron (ferrous sulfate): Indicator of H2S formation
 Phenol red: Indicator of acidification (It is yellow in acidic conditions and red under alkaline
conditions).
 It also contains peptone which acts as a source of nitrogen (remember that whenever peptone is
utilized under aerobic condition, ammonia is produced).
Interpretation/Principle of TSI Agar Test

1. If lactose (or sucrose) is fermented, a large amount of acid is produced, which turns the phenol
red indicator yellow both in the butt and in the slant. Some organisms generate gases, which
produce bubbles/cracks in the medium.
2. If lactose is not fermented but the small amount of glucose is, the oxygen-deficient butt will be
yellow (remember that butt has comparatively more glucose than slant i.e. more media and more
glucose), but on the slant, the acid produced (less acid produces in slant as media in slant is less)
will be oxidized to carbon dioxide and water by the organism and the slant will be red (alkaline or
neutral pH).
3. If neither lactose/sucrose nor glucose is fermented, both the butt and the slant will be red. The
slant can become a deeper red-purple (more alkaline) as a result of the production of ammonia
from the oxidative deamination of amino acids (remember peptone is a major constituent of TSI
agar).
4. if H2S is produced, the black color of ferrous sulfide is seen.

1. Alkaline slant/no change in butt (K/NC) i.e Red/Red = glucose, lactose, and sucrose non-fermenter
2. Alkaline slant/alkaline butt (K/K) i.e Red/Red = glucose, lactose, and sucrose non-fermenter
3. Alkaline slant/acidic butt (K/A); Red/Yellow = glucose fermentation only, gas (+ or -), H2S (+ or -)
4. Acidic slant/acidic butt (A/A); Yellow/Yellow = glucose, lactose and/or sucrose fermenter gas (+ or -), H2S (+ or -).
 GRAM-NEGATIVE BACILLI

Name of the organism Slant Butt Gas H2S

Acid (A) Acid (A) Pos (+) Neg (-)
Escherichia, Klebsiella, Enterobacter
Alkaline (K) Neg (-) Neg (-)
Shigella, Serratia Acid (A)
Alkaline (K)
Salmonella, Proteus Acid (A) Pos (+) Pos (+)
Pseudomonas Alkaline (K) Alkaline (K) Neg (-) Neg (-)

3. Lysine Iron Agar ( LIA )

Lysine Iron Agar (LIA) is a combination medium used for the differentiation of gram-negative bacilli (enterics)
based on decarboxylation or deamination of lysine and the formation of hydrogen sulfide (H2S). In
combination with Triple Sugar Iron (TSI) agar, LIA is used to identify members of Salmonella and Shigella.

 pH indicator : bromcresol purple

 H2S indicator : ferric ammonium citrate

Interpretation/Principle of LIA Agar Test

Lysine iron agar contains an aerobic slant and an anaerobic butt. The test organism is stabbed in the butt and streaked
on the slant in a fishtail streak. The tube is then tightly capped and incubated for 18 to 24 hours before reading the results.

In the anaerobic butt, organisms capable of glucose fermentation produce acid, resulting in yellow color. If the organism
produces lysine decarboxylase, it removes CO2 from L-lysine and forms cadaverine, an alkaline
product. Cadaverine neutralizes the organic acids formed by glucose fermentation, and the butt of the medium reverts
to the original alkaline state (purple). The purple color throughout indicates lysine decarboxylation. The purple color in
the slant with a yellow (acidic) butt indicates no lysine decarboxylation.

Deamination reactions require the presence of oxygen. Therefore, any evidence of deamination will be seen only in the
slant. If the organism produces lysine deaminase, the resulting deamination reaction will produce compounds that react
with the ferric ammonium citrate and a coenzyme, flavin mononucleotide (FMN), forming a burgundy (dark red color) on
the slant. A red slant with a yellow (acidic) butt indicates lysine deamination.

Hydrogen sulfide (H2S) is produced in LIA by the anaerobic reduction of thiosulfate. Ferric ions in the medium react with
the H2S to form a black precipitate in the butt.

A: Alkaline slant and alkaline butt

(K/K)
B: Alkaline slant/alkaline butt, H2S
positive (K/K, H2S)
C: Alkaline slant/acid butt (K/A)
D: Red slant/acid butt (R/A)
E: Uninoculated tube

Color Result Interpretation

Purple slant/purple Alkaline slant/alkaline Lysine deaminase negative; Lysine decarboxylase

butt butt (K/K) positive

Purple slant/yellow Alkaline slant/acid butt Lysine deaminase negative; Lysine decarboxylase
butt (K/A) negative; Glucose fermentation

Red slant/ yellow Red slant/acid butt Lysine deaminase positive; Lysine decarboxylase
butt (R/A) negative; Glucose fermentation
 GRAM-NEGATIVE BACILLI

Black precipitate H2S production Sulfur reduction

4. IMViC

The IMViC test is a series of four different biochemical tests used in identifying and differentiating bacteria, especially
the members of Enterobacteriaceae . The name IMViC is an acronym for the four tests: Indole Test, Methyl Red (MR)
Test, Voges-Proskauer (VP) Test, and Citrate Utilization Test . These tests are used to study the physiological
characteristics of bacteria from the family Enterobacteriaceae, especially Escherichia and Enterobacter .
The letter “i” after ‘V’ is only for the rhyming purpose, it does not indicate any test.

 Indole = Detects : Tryptophanase

Medium : Tryptone Broth, SIM
Indicator : KOVAC’S or Ehrlich’s reagent
Positive result : RED RING

 MRVP test = Medium : MRVP Broth or Peptone glucose Broth

a) Methyl Red (Mixed acid production)
- organisms that produce enough acid will overcome the neutralizing effect of the buffer.
-Positive result : RED
b) Voges Proskauer
- detects: ACETOIN
- reagent :
- positive result : RED
“MOST ENTEROBACTERIACEAE, give OPPOSITE MR and VP reactions”

 Citrate utilization = determine if an organism can utilize citrate as sole source of carbon
Medium : Simon’s Citrate Agar (SCA)
pH indicator : bromthymol blue
positive result : Growth and intense Blue

5. Malonate utilization = Determine if an organism can utilize maloinate as sole source of carbon
Positive : blue (Salmonella)
Negative : green or yellow (Shigella)

6. Urease test = detects urease enzyme

Medium : Christensen’s Urea afar or Stuart’s Urea broth
pH indicator : Phenol red
Positive: RED
RAPID UREASE PRODUCERS : ________________________

SLOW UREASE PRODUCERS : __________________________

7. ONPG
Rapid test to detect : B-galactosidase
Substrate ONPG (o-nitrophenyl-beta-D-galactopyranoside)
Positive: YELLOW

8. Decarboxylase
Enzyme that removes the carboxyl group (COOH) from an amino acid
Uses decarboxylase basal medium with indicator (bromcresol purple) + sugar (glucose) + amino acid (lysine, ornithine,
arginine) + control tube with no amino acid
Medium : Moller’s medium
Positive : PURPLE to YELLOW

9. Phenylalanine deaminase
Enzyme that removes amino group (NH2) from an amino acid
Medium : Phenylalanine agar, overnight culture + 10% FeCl3
Positive : GREEN SLANT and FLUID

10. Gelatin Liquefaction

Determine if an organism can breakdown gelatin into amino acid
Positive: liquefaction
 GRAM-NEGATIVE BACILLI

IMViC REACTIONS
Escherichia
I M V C
E.coli + + - -

Klebsiella
I M V C
K.pneumoniae - - + +
K.oxytoca + V + +
K.ozaenae - + - V

Enterobacter
I M V C
E.aerogenes - - + +
E.cloacea - - + +
E.agglomerans V V V V

Serratia
I M V C
S.marcescens - V + +
S.liquefaciens - + + +

Hafnia
I M V C
H.alvei - V V -

Salmonella
I M V C
Most serotypes - + - +

Shigella
I M V C
ABC V + - -
D - + - -

Citrobacter
I M V C
C.freundii V + - V
C.diversus/koseri + + - +

Proteus
I M V C
P.mirabilis - + V V
P.vulgaris + + - V
P.penneri - + - -

Providencia
I M V C
P.rettgeri + + - +
P.stuartii + + - +

Morganella
I M V C
M.morganii subsp. + + - -
Morganii

Edwardsiella
I M V C
E.tarda + + - -

Yersinia
I M V C
Y.eneterocolitica V + - -
 GRAM-NEGATIVE BACILLI

Y.frederiksenii + + - V
Y.intermedia + + - -
OTHER GRAM-NEGATIVE BACILLI

Pseudomonas aeruginosa
 The most commonly encountered gram-negative sp. that is not a member of family Enterobactericeae and is
uncommon member of normal human microbiota.
 Obligate aerobe
 Motile and rod shaped
 Sweet or grape-like or corn-tako like odor
 Grows well at 37C-42C, its growth at 42C will differentiate it from other Pseudomonas sp.
 Oxidase positive
 Grape-like or “corn-tortilla-like” odor resulting from production of 2-aminoacetophenone
 Does not ferment carbohydrate : TSI is K/K
 Produces blue-green pigment : PYOCYANIN = blue
PYOVERDIN/FLUOROSCEIN = green
 Produces infection of wound and burns, giving rise to a blue-green pus
 Meningitis, UTI, pneumonia, sepsis
 Mild otitis externa/media : “SWIMMER’S EARS”
 Skin lesion : “Ecthyma Gangrenosum”
 “Hot tub or Whirlpool Dermatitis”
 Resistant to a number of disinfectants and has been responsible for serious nosocomial infetctions
 Keratitis associated with contact lense’s case solution/ liquid”
 Respiratory infection in patient with CYSTIC FIBROSIS/CGD (P.cepacia/ B.cepacia)

Burkholderia mallei
 Formerly P.mallei
 Causes “GLANDER’S DISEASE” a disease of horses and similar animals transmissible to humans
 Horses – pulmonary involvement
 Human – fatal, begin as ulcer of skin and mucous membrane followed by lymphagitis and sepsis

Burkholderia pseudomallei
 Formerly P.pseudomallei
 Causes “MELIOIDOSIS” , an endemic glander’s like disease of animals and humans
 “Viatnamese Time Bomb Disease”
 Medium : ASHDOWN MEDIUM

Vibrio
 Facultative anaerobe : “comma shaped”
 Motile : monotrichous
 Oxidase positive ( diff. from Enterobacteriaceae)
 Halophilic, except : V. cholera and V. mimicus
 Vibrio cholerae - O1 & O139, strains that caused pandemic spread of disease (Cholera)
CHOLERA-TOXIN (CT)
“Rice water stools” = hallmark of cholera
Medium : APW/TCBS

String test : regeant 0.5% Sodium deoxycholate

Positive : lyses vibrio cells
 Vibrio cholerae serogroups : Inaba – Philippines
Ogawa – India
Hikojima – Japan

Biotypes (Biovars)
CLASSICAL EL TOR
Red Cell hemolysis Negative Positive
VP Negative Positive
Polymyxin B Susceptible Resistant
Agglutination with chicken RBCs Negative Positive

 V.alginolyticus – blood, eye, and ear infection

Wound nad ear infection associated with marine environment
Halophilic
Sucrose fermentation positive; TCBS ( yellow colonies )
 V. parahemolyticus – gastroenteritis, usually associated with contaminated seafoods
Halophilic
Sucrose fermentation negative; TCBS ( green colonies )
 GRAM-NEGATIVE BACILLI

 V. mimicus - gastroenteritis and ear infections associated with marine environment

Nonhalophilic
Sucrose fermentation negative; TCBS ( green colonies )
 V. vulnificus - Septicemia and wound infections involving marine environment
Halophilic
TCBS ( green or yellow colonies )

Haemophilus
 Nonmotile, non-sporeforming
 Facultative anaerobes
 Most species are oxidase and catalase positive
 Preferred incubation : 35-37C / 5-7% CO2
 Requires protoporphyrin IX ( X factor and V factor )
 except for Haemophilus ducreyi, Haemophilus spp. normally inhabit the upper respiratory tract of humans.
 H.influenzae – “Pfeiffer’s bacillus/ Bacillus influenzae”
Six serotypes ( a, b, c, d, e and f) ; most frequently encountered serotype infection is “b”
Encapsulated strains are pathogenic
Main cause of meningitis in children < 5 years old
Associated with respiratory conditions including epiglottitis
Required X & V
 H. aegypticus – “ Koch-Week’s bacillus”
Resembles H.influenzae biotype III
Causes : PINK EYE CONJUNCTIVITIS
Required X & V
 H.ducreyi – “SMALLEST PATHOGENIC BACILLI”
Causative agent of “chancroid or soft chancre” , venereal disease characterized by
painful ulcers in the genetalia
Direct examination : short bacilli in a “ school of fish arrangement” / “ railroad track
pattern”
Required X factor only

SPECIES REQUIREMENT FOR BETA D-ALA

X FACTOR V FACTOR HEMOLYSIS
H.influenzae + + - -
H.parainfluenzae - + - +
H.haemolyticus + + + -
H.parahaemolyticus - + + +
H. aegypticus + + - -
H. aphrophilus - - - +
H. paraphrophilus - + - +
H.ducreyi + - - -

Campylobacter
 Characteristic : S – shaped or “Sea Gull’s wings”
 Microaerophilic : 5% O2, 10% CO2, 85% Nitrogen
 Microaerophilic and capnophilic, motile, gram negative rods that are associated with gastritis and diarrhea
 Spp. ( C.jejuni, C. coli )
 Campylobacter jejuni = one of the most common cause of food poisoning in USA
 Optimum temperature for growth : 42 – 43 C
 Motility : DARTING MOTILITY (single polar flagellum)
 Culture media : CAMPY’S and SKIRROW’S
 Take note :
CAMPYLOBACTER JEJUNI and CAMPYLOBACTER COLI = HUMAN PATHOGEN ( gastritis and diarrhea )
CAMPYLOBACTER JEJUNI = also associated with GULLAN-BARRE SYNDROME
CAMPYLOBACTER FETUS = ANIMAL PATHOGEN ( abortion in animals / no growth @42C)

Helicobacter
 Characteristic : S – shaped or “Sea Gull’s wings”
 Associated with PEPTIC ULCER
 Natural habitat is the human stomach, where the organism is found in the mucus secreting cells
 Current evidence may suggest a role between H.pylori and peptic and duodenal ulcers
 Histology staining and culture of biopsies obtained from the stomach or duodenum are recommended for the
identification.
 Microaerophilic : 35-37C, motile ( 4-6 polar flagella )
 Has an increase urease production ; responsible for survival in the stomach
 GRAM-NEGATIVE BACILLI

 Take note:
CAMPYLOBACTER = 42C
HELICOBACTER = 35-37C

Bordetella
 Bordetella spp. are aerobic and nonmotile EXCEPT, B.bronchiseptica ( perotrichous)
 B. pertussis Causative agent of : WHOOPING COUGH

Three stages of pertussis ( Whooping cough )

1. Catarrhal : geneal flulike symptoms
2. Paroxysmal : repetitive coughing episodes
3. Convalescent : recovery phase

Ideal specimen : NASOPHARYNGEAL SWAB

Culture medium : Bordet-Gengou
: Regan-Lowe (Charcoal Sephalexin ) much preferred
Colonies : mercury droplet

 B. parapertussis :causes pertussis like syndrome ( mild form )

 B. bronchiseptica : inhabits respiratory tract of canines (kennel’s cough),infrequently responsible for chronic
respiratory tract infections in humans who are immunocompromised .

Brucella
 From milk / dairy products
 Causative agent of : BRUCELLOSIS / UNDULANT FEVER / MALTA FEVER
 Small, nonmotile, aerobic, coccobacilli or short rods
 Organisms are normal flora of the urinary tract and gastrointestinal tracts of sheep/goat (B.melitensis), cattles
(B.abortus), pigs (B.suis), and dogs (B.canis)
 Human acquire infections through the ingestion of contaminated animal products, including meats and milk; farmers
can directly acquire infections through direct animal contact
 Take note :
Milk born disease to man = disease from cow is pass to man/human
Milk born disease to cow = disease from man is pass to cow

 Specimen : BLOOD & BONE MARROW

 Culture for Brucella : CASTAÑEDA BOTTLES = should be maintain for 3-4 weeks
Biphasic medium ( agar + broth )

Legionella
 L. pneumophila : causative agent of LEGIONNAIRE’S DISEASE / PONTIAC FEVER
( pneumonia ) ( mild form of Legionnaire’s )

 L. micdadei : causative agent of PITTSBURGH PNEUMONIA

 L. bozemanni : causative agent of WIGA’S AGENT OF PNUEMONIA
 Sources : WATER
Natural source : pound or creeks
Artificial source : AIRCON and HEATING SYSTEM
 Stain : DIETERLE or WARTHIN-STARRY STAIN
 GRAM-NEGATIVE BACILLI

 Naturally found in both natural and artificial water sources; bacteria have been found in pounds, creeks and
streams, wet soil, water-cooling towers for air-conditioning and heating systems, shower heads, and plumbing
systems.
 Not usually detectable directly in clinical material by gram stain; the use of 0.1% fuchsin substituted for safranin in
the Gram-stain procedure may enhance the visibility of the organisms.
 Specimen : Sputum / Bronchial washing (BW)
 Culture : BCYE and FEELEY-GORMAN

Pasteurella
 P.multocida (agent of PASTEURELLOSIS) = wound infection from animal bite or animal scratch
 The organism is carried in the oral cavity and respiratory and gastrointestinal tracts of cats and dogs
 Also causative agent of shipping fever in cattles, a hemorrhagic septicemia
 Humans may acquired the infection following contact with domestic animals that harbor the bacterium; most
frequently the route of infection is from the bite or scratch of an infected animal, usually a cat.
 Wound infections are the most common type of Pasteurella infection

Francisella
 Faintly staining, coccobacilli that are non-motile and obligately aerobeic
 Causative agent of TULAERMIA = disease of rodents, primarily in rabbits
 Individuals who handle the infected animal, acquire this zoonosis through direct contact with blood or through
animal bite or scratch
 Can also be inhaled or or acquired indirectly through insect vectors primarily ticks
 Culture : BLOOD GLUCOSE CYSTINE MEDIUM

Gardnerella
 Formerly known as Haemophilus vaginalis; Corynebacterium vaginalis
 G. vaginalis ( associated with bacterial vaginosis )
 Grram-variable to gram-negative bacillus
 Bacterial vaginosis (BV) is characterized by foul-smelling, grayish, vaginal discharge
 Amsel and Nugent scoring system are used to diagnose BV
 Cytology/Pap’s : Clue cells ( Squamous epithelial cells studded with bacteria)
 Whiff of Sniff test : Vaginal discharge + 10% KOH = positive if there’s Fishy amine like odor
 Organism can be isolated on : HUMAN BLOOD BILAYER TWEEN MEDIUM (HBT)

Sterptobacillus moniliformis
 Normal flora of rodents
 Agent of : ( RATBITE FEVER / ANIMAL BITE OR SCRATCH )
( HAVERHILL FEVER ) from ingestion of contaminated milk

Capnocytophaga
 Capnophilic, fusiform or filamentous bacilli
 Characteristic motility : GLIDING MOTILITY

Calymmatobacterium granulomatis
 Agent of ( GRANULOMA INGUINALE / DONOVANOSIS )
 Encapsulated, pleomorphic mostly observed in vacuoles in large mononuclear cells
 Group of microorganisms are seen within mononuclear cells; this pathognomonic entity is known as DONOVAN
BODY
 Closely related to Klebsiella
 Based on DNA-DNA hybridization, it has been recently proposed that the name of this organism be change to
Klebsiella granulomatis

Eikenella corrodens
 Fastidious, capnophilic rod; part of the gingival and bowel flora
 CORRODING BACTERIUM
 Characrteristic in agar : corroding/corrodes / pits
 Produces bleach like odor

Chromobacterium violaceum
 Found in soil and water, has the ability to produce violacein ( purple pigment )

Cardiobacterium hominis
 Normally found in the upper respiratory tract and is isolated as a rare cause of endocarditis