LESSON 11.

NON-ENTERIC
MEDT 14 | CLIN. BACTERIOLOGY (LEC)
GASTROINTESTINAL
Jonnel P. Andaya || 3rd Year PATHOGENS AND NON-
Transcribed by: Ybanez, Trisha Mae C. | De los Reyes,
John Dominic M.
FERMENTATIVE GRAM
NEGATIVE
essential in the prevention of the spread of
OUTLINE: Aeromonas infections. Oral fluid electrolyte
I. INTRODUCTION substitution is employed in the prevention of
dehydration, and broad-spectrum antibiotics are
II. VIBRIO SPP.
used in severe Aeromonas outbreaks. This
A. V. cholerae review presents an overview of emerging
B. V. parahaematicus Aeromonas infections and proposes the need
C. V. vulnificus for actions necessary for establishing adequate
D. V. alginolyticus prevention measures against the infections.
E. Laboratory Diagnosis  Campylobacter spp.
III. AEROMONAS SPP. o Campylobacter species represent one of the
most common causes of bacterial diarrheal
IV. CAMPYLOBACTER SPP.
illness worldwide. According to the United
A. C. Jejuni States Centers for Disease Control, there are
B. C. Fetus subsp fetus about 1.3 million cases of Campylobacter
C. Laboratory Diagnosis infection each year in the United States alone.
V. HELICOBACTER SPP. This leads to an economic cost between $1.3 to
A. H. pylori 6.8 billion dollars annually in the United States.
B. H. cinaedi & H. fenneliae Campylobacter infection is associated with the
consumption of raw milk, undercooked poultry,
C. Laboratory Diagnosis
and contaminated water. Patients typically
VI. NON-FERMENTATIVE GRAM NEGATIVE experience a self-limited diarrheal illness lasting
BACILLI 5 to 7 days. Immunocompromised and elderly
A. General Characteristics patients are at the highest risk for morbidity,
B. Oxidative - Fermentative (OF) Test mortality and prolonged illness. Despite having
C. Pseudomonas spp. treatment and eradication modalities in place in
animal reservoirs, there has been a dramatic
D. Acinetobacter spp.
increase of cases in developed and
E. Stenotophomonas maltophilia underdeveloped regions of the world.
F. Burkholderia spp.
VII. OTHER NON-FERMENTATIVE GRAM NEGATIVE VIBRIO SPP.
BACILLI  Gram-negative, short, curved, asporongenous rods
A. Alcaligenes faecalis (non-spore forming)
B. Oligella spp.  Monotrichous flagella but some may produce
C. Moraxella lacunata peritrichous flagella especially when grown in solid
D. Chromobacterium violaceum medium.
E. Shewanella putrefaciens

INTRODUCTION
 Vibrio spp.
o Infections caused by pathogenic Vibrios remain
a severe threat to the public. Most of these
infections result from the consumption of
undercooked seafood products or contaminated  Most are halophilic bacteria (requires high salt
water. Also, person-to-person transmission has concentration) except: V. cholerae and V. mimicus
been documented. These infections are (can grow in environments with 0% NaCl conc.)
classified into cholera and noncholera types.
 Facultative anaerobes
Vibrio cholerae infections can be fatal if not
properly managed. Noncholera infections range
from self-limiting gastroenteritis to severe life-
threatening septicaemia and necrotizing fasciitis.
V. cholerae and V. parahaemolyticus are mostly
associated with human infections. However,
other Vibrios, such as V. alginolyticus, V.
harveyi, V. anguillarum, V. mimicus, V.
metschnikovii, V. vulnificus, and V. fluvialis,
which have been detected, particularly in marine
environments, are now considered as emerging  Species:
human pathogens. 1. V. cholerae
 Aeromonas spp. 2. V. vulnificus
o Aeromonas species are ubiquitous bacteria in 3. V. parahemolyticus
terrestrial and aquatic milieus. They are 4. V. metschnikovii
becoming renowned as enteric pathogens of 5. V. hollisae
serious public health concern as they acquire a 6. V. alginolyticus
number of virulence determinants that are linked 7. V. damsel
with human diseases, such as gastroenteritis,
 Common isolates:
soft-tissue, muscle infections, septicemia, and
1. V. cholerae OI and non-OI strain
skin diseases. Proper sanitary procedures are
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2. V. parahaemolyticus o In Blood agar, colonies can be alpha or beta

3. V. vulnificus hemolytic
4. V. alginolyticus
 Considered not to be part of human indigenous normal
flora (microbiota)
 Commonly found in brackish water, marine water, and
salt water because it is their source of high salt
concentration.
 Isolated in: algae, plankton, fish and shellfish
 Mode of transmission: Ingestion of contaminated
foods (raw or undercooked sea food)
o It may cause diarrhea, vomiting, gastroenteritis,  Susceptibility test: susceptible to 150-ug vibriostatic
dehydration after ingestion. Some patients may O/129 disk in Millon Hinton Agar.
develop self-limiting signs and symptoms while
others may develop severe infections after
ingestion of contaminated foods with this
organism.

 Biochemical test:
o All species ferments glucose
o All are non-lactose except to V. vulnificus
o All species are oxidase-positive and nitrate
 Associated infections: reduction positive except for V. metschnikovii
1. Cholera – Vibrio cholerae O139 o Motility test: Broth- polar sheathed flagella ;
2. Gastroenetritis – Vibrio cholerae non-O1 and Solid media – peritrichous, unsheated flagella
Vibrio cholerae non-O139
3. Necrotizing fasciitis

4. Severe fatal septicemia – V. vulnificus VIBRIO CHOLERAE

 Most important and clinical significant specie of Vibrio
spp.
 Primary disease: “Cholera” – caused by V. cholerae
O1 strain and V. cholerae O139 strain. However, there
are strains that are not associated with cholera, V.
cholerae non-O1 strain but it can cause gastroenteritis.
 Motility: “Rapid darting motility” or “Shooting-star
motility”
 Virulence factor: Choleragen (cholera toxin)
o Cholera toxin – similar with the structure of
labile toxin which is found in enterotoxic E.
 Culture: coli. The only difference is that cholera toxin
o In Mac Conkey agar, colonies are Non is composed of two subunits called CTX-A
Lactose Fermenter except only to Vibrio and CTX-B, the reason why it is called “AB
vulnificus which is lactose fermenter toxin”.
o A subunit – responsible for active infection
o B subunit - not antigenic but is responsible
for the binding with the receptor of the
enterocytes.
 Has a pili that will attach to the intestinal lining and will
secrete its toxin, Cholera toxin. And then endocytosis
will occur. Will enter the enterocyte cells and will
attach to adenine cyclase and will produce too much
amount of cyclic adenosine monophosphate. This
o In Chocolate agar, colonies are smooth, adenosine monophosphate will activate the channel to
opaque and iridescent with greenish hue have many profusions of water in lumen and
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electrolytes. It is the reason why cholera patients often
have severe diarrhea.
 It may develop self-limiting signs and symptoms and
some may develop severe infections such as losing of
electrolytes and dehydration. It could lead to muscle
cramps, metabolic acidosis, hypovolemic shock, and
due to electrolyte imbalance, it may cause cardiac
arrhythmia.
 High mortality rate if patient is not given replacement
electrolytes immediately.
 Hallmark: Rice watery stool (10-30 times per day). As
time goes by, stool becomes colorless and odorless.
 Antigenic structure: Somatic O and flagellar H.
o It is important to identify specific strains.
o Example: V. cholerae O1 (Somatic O1 strain)
V. cholerae O139 (Somatic O139 strain)
 Biochemical tests:
o Ornithine Lysine Carboxylase: positive
o Oxidase test: positive
o Indole test: positive
o String test: positive
 Provide sample in slide and drop
sodium desoxycholate in the slide
and mix it using wire loop. Once you
lift the wire loop and produce string-
like thread, it is positive.
 Principle: Once you add sodium
desoxycholate to the sample in slide,
it could lyse the membrane of
bacteria. Once the membrane of the
bacteria is lysed all DNA will be
released and it could form mucoid
consistency in the sample and form
string-like thread.
 Biogroups of epidemic Vibrio cholera: grouped
based on their specific variation in biologic behavior.
a. Classical V. cholerae - Vogues Proskauer (-);
does not agglutinates chicken RBCs; susceptible
to polymixin B (50ug)
b. El tor V. cholerae - Vogue Proskauer (+);
agglutinate chicken RBC; and resistant to
polymixin B
 Serotypes of Vibrio cholerae: grouped based on
their cell surface antigen. Can be identified using
certain antibodies.
a. Vibrio cholerae subgroups are Vibrio cholera O1,
Vibrio cholera O139 and Vibrio cholera non-O1
b. Vibrio cholera O1 serotypes: Ogawa (A, B),
Inaba (A, C) and Hikojima ( A, B, C)
 Potent enterotoxin: Cholera toxin, zot toxin, and ace
toxin
 Colonies in BAP are smooth and medium-to large-
sized with a greenish hue.
 V. cholerae O1 is common cause of cholera (Somatic
O1 strain)
 Cholera
o Acute diarrheal infection that mainly spread
through contaminated water source
o Acquired by ingestion of contaminated and
improperly preserved food like shellfish, milk
and ice cream.
o Hallmark of cholera: “Rice watery stool” (10
to 30 times of defecation per day)
o V. cholerae O1 is the common cause of
epidemic cholera while the non-O1 strain
causes gastroenteritis
 Choleragen
o Protein toxin that mainly produced by Vibrio
cholera O1 strain.
o Stimulates the hypersecretion of water and
chloride ions and inhibit sodium ion
absorption, which result in a fluid loss of 10 to
15 liters and electrolytes
o All antigens of this toxin are poorly
immunogenic leading to recurring infections.
VIBRIO PARAHAEMATICUS
 Associated with the second most common cause of
gastroenteritis among Vibrio species
 Etiologic agent of “Summer diarrhea” outbreak in
Japan year 1950s
 Leading cause of pandemic infection: Vibrio
parahaemolyticus serotype O3:K6
 Mode of Transmission: Ingestion of contaminated
seafood like oysters, scallops, crabs, lobsters, shrimps,
and sardines
 Virulence factor: Heat stable hemolysin
 Pathogenicity: Hemolysin lyses Human RBC
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 Selective medium: Wagatsuma agar (high- salt

mannitol medium)

VIBRIO VULNIFICUS
 Lactose fermenter species of Vibrio
 Second to Vibrio cholerae as the cause of severe
Vibrio associated infections. It can also cause
gastroenteritis.
 If there is an open wound exposed to warm saltwater
with V. vulnificus, it will cause wound infection,
especially in immunocompromised patients, it may
develop septicemia.  String test:
 Associated with severe fatal septicemia. o Differentiate Vibrio from Aeromonas:
 Mode of transmission: Ingestion of contaminated Aeromonas is negative and Vibrio is
raw oysters and fish. positive
 If an open wound came in contact with contaminated
warm saltwater, it can cause wound infection.

VIBRIO ALGINOTICUS
 Not commonly isolated and considered as the least
pathogenic to humans
 Medium requires 1% to 10% NaCl because it is
strictly halophiles
o Reagent: 0.5% sodium desoxycholate
 Associated infections: Extraintestinal infections such
o (+) Result: The lysis of cells release DNA,
as eye, ear and wound infection.
which can then have pulled up into a viscous

string using an inoculating loop.
 Vibriostatic test (Susceptibility test)- O/129:
o It is used to separate vibrios (susceptible)
from other oxidase-positive glucose
fermenters like aeromonads (resistant)
o This test uses 150 ug vibriostatic agent
LABORATORY DIAGNOSIS O/129 (2,4-diamino-6,7-diisopropylpteridine)
 Specimen: Rectal swab, stool, pus and tissue
 Transport medium: Cary – Blair Medium- sample
should be collected and transported only using this
medium
 Culture media:
o Culture media:
 Alkaline peptone water (pH 8.5) –
enrichment medium to enhance the
recovery rate of Vibrio spp. Swabs
can directly inoculated in alkaline  Biochemical Tests:
peptone water. It promotes the o TSI reaction: A/A, (-) gas, (-) H2S
replication of Vibrio prior to o LIA reaction: K/K
subculture into TCBS. o Vibrio cholerae: (+) citrate and indole
 Thiosulfate-citrate-bile salts- o V. vulnificus: (+) indole and cellobiose
sucrose (TCBS) – a differential o V. mimicus: (+) indole
medium
 Mac Conkey Agar- most Vibrio spp. AEROMONAS SPP.
are non-lactose fermenter.  Species are glucose fermenters
 Blood Agar Plate – most Vibrio spp.
requore 0.5% of NaCl exc. V.
cholerae and V. mimicus. However,
V. alginolyticus can tolerate up to
10% NaCl.
o Colonial growth in TCBS agar:
 TCBS inhibits gram positive
bacteria
 pH indicator: Thymol blue and
parathymol blue  Microscopy: Gram negative straight rods
 Sucrose fermenter: (Yellow  Motile with single polar flagellum (monotrichous) and
colonies at on TCBS) – V. cholera, facultative anaerobes
V. alginolyticus and V. metschnikovii
 Non-sucrose fermenters (Green
colonies on TCBS): Vibrio mimicus,
Vibrio vulnificus, Vibrio
parahaemolyticus and Vibrio damsel
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 Can typically grow at 4 degrees Celsius and 42 TEST Aeromonas Pleisiomonas Vibrio
degrees Celsius (Wound (Diarrheal (Wound
 Considered not to be part of human indigenous normal infection; Disease; infection;
flora (microbiota) Diarrheal Gastroenteritis) Gastroenteritis)
Disease)
 Habitat: found in chlorinated water, freshwater and
estuarine Oxidase + + + except
 Etiologic agent of “Red leg” disease in amphibians Vibrio
 Like ETEC (Enterotoxigenic E. coli), species of metschnikovii
Aeromonas may also cause Traveler’s diarrhea
 Associated extraintestinal infections are septicemia, Vibriost Resistant Susceptible Susceptible
Meningitis, keratitis, and wound infections atic
O/129
(150 ug)

Growth + + v (Not all are

in media halophilic
with 0% bacteria; V.
 Culture NaCl cholerae & V.
mimicus grow in
o In Blood agar colonies is large, raised, white,
0% NaCl)
and opaque
o In Mac Conkey agar colonies is pink color Growth - - +
o In Colistin Nalidixic Acid Agar colonies in media (Non- (Non- (Cholerae and
have a “bulls – eye” appearance with 6% halophilic) halophilic mimicus can
o Biochemical test: (+) oxidase and catalase NaCl still grow even
test though they are
non-halophilic.)
 Groups of Aeromonas Fermentation Test
o Mesophilic group
 Aeromonas hydrophila complex Aeromonas Pleisiomonas Vibrio
 Aeromonas veronii
 Aeromonas caviae complex Glucose + + v
o Psychrophilic group - a non-motile group that
grows best at 22 degrees Celsius to 25 Inositol - + -
degrees Celsius; includes the species
Aeromonas salmonicida (fish pathogen) Mannitol v - +

Sucrose v - v
 Characteristics of Aeromonas
o Aeromonas caviae is the most common
isolate for GI infection
o A. hydrophila and A. veronii are the common CAMPYLOBACTER SPP.
isolates in HUS (Hemolytic Uremic Syndrome)  Motile, single polar flagellum (small, curved, or S
o VIbriostatic O/129 test: Resistant shaped rods) and oxidase positive
o Inositol fermentation: Negative
o String test: Negative
o Positive indole test: Aeromonas caviae,
Aeromonas hydrophila and Aeromonas
veronii
o TSI reaction
 A/A, (-) gas, (-) H2S for Aeromonas
caviae
 A/A, (+) gas, (+) H2S for Aeromonas
hydrophila and Aeromonas veronii
o Aeromonas hydrophilia (which actually
means “water loving”) causes gastroenteritis
and cellulitis
 Microaerophillic except to Campylobacter rectus and
 Differential Characteristics of Aeromonas,
Campylobacter curvus (both obligate or striclty
Plesiomonas, and Vibrio
anaerobes)
o Plesiomonas are Enterobactarium
Gram Negative with Polar Flagella
o Plesiomonas shigelloides - one most clinically
significant
o Share Common Features/Infections and
Biochemical Tests
o All gram negative catalase positive
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 Most recognized antecedent cause of Guillain- Barre
Syndrome because many afflicted patients are found
positive for its antibodies
o Autoimmune disease; After recovery of
infection from C jejuni. The body produces
antibodies which attack Lipopolysaccharides
of the bacteria in which have the same
structure to gangliosides of our body’s
peripheral nerve cells - attacking the myelin
sheath, nodes of ranvier, & neuromuscular
junction. This results to acroparestesia,
symptoms of Guillain-Barre Syndrome,
tingling of both sides of the body from feet to
diaphragm
 May infect animals such as cattle and swine and
cause sterility and abortion
 Culture: Colonies are gray, flat, glistening, and
irregular with “tailing effect” along the streak line or
“runny spreading growth”
 Species:
o Camplylobacter jejuni
o Campylobacter coli
o Campylobacter fetus subsp. fetus
o Campylobacter sputorum
o Campylobacter concisus
o Campylobacter curvus
o Campylobacter rectus
 Enteric Campylobacter are
o Campylobacter jejuni
o Campylobacter coli
o Campylobacter lari
 Campylobacter lari is isolated in
blood culture sample
 Microscopy:
o Faint staining, Gram negative, small, curved
or S-shaped rods
o Old culture may appear cocobacillary.
o Enteric campylobacter appear as long spirals
or are seagull wing-shaped
CAMPYLOBACTER JEJUNI
 Asaccharolytic, fastidious and slow growing organisms
 Darting motility but unable to grow in media with high
salt concentration
 Most common cause of bacterial gastroenteritis
 Mode of transmission: Ingestion of contaminated foods
such as chicken and turkey
 Its optimal temperature for growth is 42 degrees
Celsius
 Infective dose: >10,000 organisms
CAMPYLOBACTER FETUS SUBSP FETUS
 Causes bacteremia and occasionally associated with
gastrointestinal illness
LABORATORY DIAGNOSIS
 Specimen: Fecal, rectal swab and blood
 Rectal swab is a less preferred specimen for the
isolation of enteric campylobacters
Microscopy
 Recommended counterstain: Carbolfuchsin
 Hanging drop preparation: Exhibit a darting motility
 If safranin is used as a secondary stain, it should be
applied for two to three minutes
 To observe the typical motility, organisms should be
suspended in TSB
Culture
 Selective media: Campy-BAP, Butzler agar, Skirrow’s
agar, and charcoal cefoperazone desoxycholate agar
(CCDA)
 Transport medium: Cary-Blair medium
 Butszler and Skirrow’s media are both enriched and
have added antibiotics
o Butszler - contains cefoperazone, rifampicin,
and amphotericin b
o Skirrow’s agar - vancomycin, trimethoprim
and polymyxin b
 CCDA contains antimicrobial agents and is selective
for Campylobacter species
 In a blood culture. A two-week incubation may be
needed since the natural turbidity affects “detection”;
thus, blind cultures may also be necessary.
 Campylobacter can be detected effectively by a CO2
monitoring system
HELICOBACTER SPP.
 Found in gastrointestinal tract of mammals and birds
 Motile by monopolar or multi-bipolar flagella
 Most species have strong urease activity and are
microaerophiles
 Microscopy: Gram negative, helical(S-shaped) rods
that resemble Campylobacter
 Culture: CAP- colonies are gray and translucent.
 Biochemical test:
o (+) oxidase and catalase
 Mode of transmission:
o Oral-oral route and fecal-oral route
 Species:
o H.pylori,
o H. cinaedi,
o H. fenneliae,
o H. rappini (formerly Flexispira rappini)
HELICOBACTER PYLORI
 Most clinically significant specie of Helicobacter
 Major cause of: Gastric carcinoma, peptic ulcer and
type B gastritis
 Found in the mucous layer of the antrum and fundus
of the stomach but does not penetrate the gastric
epithelium
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 It binds with the Lewis antigen (part of the blood group Differential Characteristics of Campylobacter and
antigen) and the monosaccharide sialic acid Helicobacter
o Lewis Antigen is expressed by columnar
epithelium and also of H. pylori - molecular
Biochemical/ Campylobacter Helicobacter
mimicry. Thus, when a bacterium is found
and an antibody is produced, both H. pylori Susceptibility Test jejuni pylori
and stomach cells’ Lewis antigen are
attacked causing Gastric carcinoma, peptic Catalase test + +
ulcer, and type B gastritis.
o Columnar cell in stomach produces mucous Nitrate reduction + V*
to protect the stomach lining from acids. H.
pylori can survive in acid since it is strong
urease positive, it can convert urea into C02 Urease -- +
and Ammonia. Ammonium can neutralize the
acidity of the stomach and can attach to the Hippurate hydrolysis + --
stomach lining. From there, H. pylori can
excrete exotoxin that can cause inflammatory
response or apoptosis that can cause Indoxyl Acetate + --
ulceration of the stomach hydrolysis
 Primary habitat: Human gastric mucosa
 Mode of transmission: Susceptibility to:
Resistant Susceptible
o Oral-oral route fecal-oral route  Cephalothin
 Biochemical test:
o Strong urease producer (vital in the survival
 Nalidixic acid Susceptible Resistant
and growth in the gastric mucosa)
(30mcg)
 Diagnostic test:
o (+) urea breath (sensitive and specific test)
Growth at
HELICOBACTER CINAEDI & HELICOBACTER FENNELIAE
⮚ 15 °C -- --
 These species have been isolated from the blood of
patients with bacteremia and recovered from the blood
of homosexual males with or without HIV ⮚ 25 °C -- --

LABORATORY DIAGNOSIS
 Specimens are gastric biopsy tissue, urine, feces and ⮚ 42 °C + V
dental plaque
 Gastric tissue is the best specimen for the culture for
the culture of H. pylori NON - FERMENTATIVE GRAM NEGATIVE BACILLI
 Tissue specimens should be maintained at 4 degrees
Celsius and processed within 2 hours of collection INTRODUCTION
 Urine specimen is utilized for ammonia testing
Pseudomonas spp.
● Pseudomonas is a type of bacteria (germ) that is found
Gram Stain commonly in the environment, like in soil and in water. Of
 0.1% basic fuchsin counterstain enhances morphology the many different types of Pseudomonas, the one that
 Stains for biopsy specimens. Warthin-Starry stain, most often causes infections in humans is called
silver stain, or Giemsa Stain Pseudomonas aeruginosa, which can cause infections in
the blood, lungs (pneumonia), or other parts of the body
Culture after surgery.
 Culture media: CAP, MTM, Skirrow’s agar, and
Acinetobacter spp.
Brucella agar with 5% sheep’s blood
● Members of the genus Acinetobacter are ubiquitous, free
 Transport media: Stuart medium,Cysteine brucella
living, small aerobic Gram negative cocco-bacilli that prefer
broth with 20% glycerol, and isotonic saline with 4%
moist environment and can be easily obtained from soil,
glucose water, food and sewage. They are usually considered to be
 A gastric tissue biopsy specimen should be placed in a opportunistic pathogens, and of recent have been reported
Stuart medium to cause a number of outbreaks of nosocomial infections in
 Helicobacter may require more than five days of hospitalized patients like septicaemia, pneumonia, wound
incubation in a capnophilic environment sepsis, endocarditis, meningitis and urinary tract infection
(UTI). Although acknowledged to be an opportunist in
Other tests hospitalised patients, community acquired infections are
 Nucleic amplification (polymerase chain reaction or reported and they can cause suppurative infections in
PCR) is a sensitive method for detecting H. pylori virtually every organ system.
 H. pylori is susceptible to metronidazole
 Susceptibility test: Agar dilution using MHA with 5%
Stenotrophomonas maltophilia
aged (> two weeks) sheep’s blood, incubated at
● Stenotrophomonas maltophilia is increasingly recognized
microaerophilic condition, and read after three days.
as an important cause of nosocomial infection. Infection
occurs principally, but not exclusively, in debilitated and
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immunosuppressed individuals. Management of S.

maltophilia-associated infection is problematic because
many strains of the bacterium manifest resistance to
multiple antibiotics

Burkholderia spp.
● The genus Burkholderia contains organisms that are
important causes of human, animal and plant disease, as
well as organisms useful in promoting plant growth and
bioremediation. The type species, Burkholderia cepacia, is
important in all of these activities. Originally identified as a
plant pathogen that caused soft rot in onions (Burkholder,
1950), B. cepacia has emerged in the last 20 years as an
opportunistic pathogen in nosocomial infections,
particularly in individuals with cystic fibrosis (CF) or chronic
granulomatous disease (Gilligan, 1991; Govan et al., PSEUDOMONAS SPP.
1996b; LiPuma, 1998; Speert et al., 1994).
● Genus is the most commonly isolated non – fermentative
bacilli
GENERAL CHARACTERISTICS ● Are obligate aerobic, non-sporeforming, and motile with
● Not indigenous microbiota of the human and considered as polar flagella
opportunistic pathogens ● Primary plate media are MAC and BAP from which they
● Habitat: water, soil, vegetables, food, and hospital surface usually grow
● Can withstand disinfectants such as chlorhexidine and ● Can be isolated from cosmetics, swimming pool, hot tubs,
quarternary ammonium compounds and inner parts of the shoes
● Can be isolated from nebulizer, dialysate fluids, salines, ● Fluorescent group:
catheters, and other hospital devices (e.g. surgical o P. aeruginosa
instruments) hence, appropriate time of sterilization and o P. Fluorescens
application of sterilants are essential o P. Putida
● Some can tolerate high temperature (e.g. Pseudomonas o P. veronii
aeruginosa) o P. monteilii
● Most species produce oxidase o P. mosselii
● Metabolism: primarily respiratory and never fermentative - ● Biochemical test:
require oxygen to generate energy/ATP o (+) oxidase
● They ferment carbohydrates by oxidative method (non- o (+) catalase
lactose fermenters) and not by enzymatic reaction, and o Non - lactose fermenter
produce very weak acid end products ● TSIA reaction: K/K, (-) gas, (-) H2S
● Acid production is absent in oxidative-fermentative (OF)
media if it is overlaid with mineral oil - simply because they PSEUDOMONAS AERUGINOSA (AGENT OF BLUE PUS)
are non-fermentative, but are oxidative instead.
● Most commonly isolated species of Pseudomonas at
clinical specimens/most clinically significant
OXIDATIVE - FERMENTATIVE (OF) TEST ● Not a member of Enterobacteriaceae but most commonly
● Two (2) ways on how bacteria utilize carbohydrates encountered gram negative bacterium
o Fermentation ● Exhibit optimal growth at 42 degrees Celsius and
⮚ bacteria can utilize carbohydrates with or composed of monotrichous flagella
without oxygen ● In BAP, colonies are flat, metallic sheen; can produce
o Oxidation diffusible pigments which can either be blue-green or red
⮚ bacteria require oxygen brown color colonies and have a “grape-like” or “corn
● For the purpose of differentiating Pseudomonas from tortilla” odor; organisms produce beta hemolytic pattern in
Enterobacteriaceae this media and sometimes mucoid (observed in patient with
● Determines the ability of organisms to utilize a substrate, Cystic fibrosis)
thereby producing acid by-products in the presence or ● Virulence factors are Endotoxin (lipopolysaccharide), pili,
absence of oxygen alginate, capsule, and exotoxins (lecithinase, elastase, and
● Semi-solid medium: OF medium with glucose protease)
● Oxygen barrier: Mineral oil ● Aerobic bacteria and are opportunistic pathogens where
● Procedure: Two OF tubes is utilized; one of the tubes is there is higher risk of infection among
overlaid with mineral oil. immunocompromised individuals
● pH indicator: Andrade’s acid fuchsin, bromcresol purple,
bromthymol blue, and phenol red Mode of Transmission
● Positive (+) Result: o Ingestion of contaminated food or water
o Open tube: exhibit a yellow color o Use of contaminated devices and contact to open skin
o Closed tube: exhibits a green/blue-green color wounds
● Hugh and Leifson medium is an OF medium with 1% o Contact with contaminated contact lens’ rinse solution
carbohydrates and bromthymol blue as pH indicator o May also found in hot tubs and whirlpool baths - they
can tolerate and survive in high temperature

Associated Infections

o Gram negative bacillary bacteremia - blood infection

due to gram negative bacilli
o Swimmer’s ear (otitis externa) - itchiness of the ear,
red and inflamed, pus is draining from the ear
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o Ecthyma gangrenosum - lesions characterized by  Catalase positive

hemorrhagic pustules that evolve into necrotic black  Obligate aerobes
ulcer  It can be found in environment, in soil, and it can
o Jacuzzi syndrome (necrotizing skin rash) - itchy spot contaminate hospital equipment such as catheters and
on skin that becomes bumpy red rash especially in humidifiers.
areas previously covered with swimsuit  Mode of Transmission: Nosocomial (hospital acquired
o Etiologic cause of cystic fibrosis (genetic disorder). infection)
Pseudomonas set in after mucoid secretions which  Opportunistic pathogen
clog airways in the lungs.  Multi-drug resistant
o Causes hospital acquired infections that are related to Associated Infections:
antibiotic resistance  Urinary Tract Infection (UTI)
 Pneumonia
Pigments produced by Pseudomonas aeruginosa
 Endocarditis
o Pyoverdin- yellow-green or yellow-brown pigment
 Meningitis
o Pyocyanin: blue pigment (only produce by
 Cellulitis
P.aeruginosa)
 Wound infection
o Pyocyanin: blue pigment (only produce by
Cultivation:
P.aeruginosa)
o Pyomelanin: brown or black pigment  It may also appear as gram-positive bacilli from blood
culture bottles
Distinguishing characteristics of P. aeruginosa  MAC colonies exhibit purple color
o (+) growth of 42 degrees Celsius  Partially lactose fermenter in MacConkey
o (+) acetamide and citrate utilization (both reactions  Besides from MacConkey Agar other selective
produce blue color) medium available:
o (+) gluconate production o CHROM Agar (colonies are red)
o (+) arginine dehydrolase (ADH) Biochemical Tests:
 Catalase (+)
 Oxidase (-)
PSEUDOMONAS FLUORESCENS & PSEUDOMONAS PUTIDA Other Spp.:
 A. baumannii - glucose-oxidizing, non-hemolytic
● Can be isolated form contaminated blood products, strains (common isolate)
cosmetics, hospital equipment, urine, and respiratory  A. lwoffii - non-glucose-oxidizing, non-hemolytic
specimens. strains (common isolate)
● Can grow at 4 degrees Celsius and associated to  A. haemolyticus - non-glucose-oxidizing, hemolytic
transfusion-associated septicemia strains
● P. fluorescens is positive gelatin hydrolysis test (Differential
test) STENOTROPHOMONAS MALTOPHILIA
General Characteristics:
LABORATORY DIAGNOSIS
 Maltophilia is came from Greek words “maltbum”
● Specimens: Blood, wound discharge, sputum, and sterile which means malt and “philia” which means “affinity”
fluids that collectively means “affinity to malt”
● Gram stain: Straight and slender Gram negative rods  Contaminate blood-drawing equipment and
● Biochemical Test: disinfectants.
o TSI Reaction: K/N or K/K (Alkaline slant/neutral butt)  Microscopy: short to medium sized, gram-negative,
o Motility Test: Positive straight rods
o (+) oxidase  Third to the most isolated non-fermentative, gram-
o (-) H2S production negative bacillus
● Phage Typing  Motile
o this test allows the identification of bacteria by testing  Strictly aerobes
their vulnerability of to bacterial viruses (bacteriophage)  Can grow at 42 degrees Celsius
Associated Infections:
Culture Media  Endocarditis
o BAP, CAP, MAC  Bacteremia
o Sellers medium - promotes pigment production  Wound infections
o Cetrimide agar (cetrymethyl ammonium bromide) - a Cultivation:
differential and selective medium that also enhances
 In BAP – colonies exhibit lavender-green to light
the pigment production (pyoverdin and pyocyanin) of P.
purple with distinct odor similar to ammonia
aeruginosa
 In MAC – colonies are blue color
o Irgasan (2,4,4-trichloro-2’hydroxydiphenylether)
Biochemical Tests:
o C390 (9-chloro-9 [4-diethylaminophenyl]-10
 Catalase (+)
phenylacridan)
o Pseudomonas fluorescent group has yellow-green or  Oxidase (-)
yellow-brown colonies  Esculin (+)
 Gelatin hydrolysis (+)
 Dnase test (+)
ACINETOBACTER SPP.
General Characteristics:  LDC (+)
 Non-fermentive gram-negative bacillus  Antimicrobial test – Broth microdilution and E-test
 Non-motile
 Under microscope it is mistaken as Neisseria because BURKHOLDERIA SPP.
they are coccobacillus that vary in size and appear as
pleomorphic General Characteristics:
 Oxidase negative  Generally non-pathogenic
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 Motile by polar flagella except B. mallei
 Mode of Transmission: contact with heavily
contaminated medical devices
 It grows well in BAP, CAP and MAC
 Microscopy: Medium-sized, gram-negative, straight
rods
Biochemical Tests:
 Catalase (+)
 Oxidase (+)
Other Spp.:
 Burkholderia cepacia
 Burkholderia pseudomallei
 Burkholderia mallei
 Burkholderia gladioli
BURKHOLDERIA CEPACIA
 Found and isolated in anesthetics, nebulizers, detergents,
and disinfectants
 Culture media: grows well at BAP and MAC but may lose
its viability on the former in three to four days
 In BAP, colonies exhibit a non-wrinkled yellow or yellow-
green color
 Biochemical tests are weak –positive oxidase reaction, (+)
LDC and ONPG
 Associated infection is pneumonia among patient with
cystic fibrosis
BURKHOLDERIA MALLEI
 Etiologic agent of glanders or farcy disease which is a
severe infection that affects horses and donkeys
 Can be potential bioterrorism agent
 Only non-motile member of the genus
 Variable growth on MAC
 In BAP, colonies are non-pigmented
 Oxidase production is variable
BURKHOLDERIA PSEUDOMALLEI
 Vietnamese Time Bomb Disease
 Etiologic agent of melioidosis, which is a glanders-like
disease
 Motile with a polar tuft of flagella
 Found or isolated from muddy soil and rice paddies
 High risk to infection are farmers and soldiers
 Mode of transmission: inhalation of contaminated debris or
direct inoculation through damaged skin or mucous
membranes
 Associated signs and symptoms can be mistaken with
tuberculosis
 Microscopy: gram-negative bacteria with presence of
bipolar bodies
 Culture: Ashdown medium with colistin - colonies are dry,
wrinkled, and have a deep pink color.
OTHER NON - FERMENTATIVE GRAM-NEGATIVE
BACILLI
ALCALIGENES FAECALIS
 It is an obligate aerobic -Motile peritrichous gram negative
bacillus
 Found and isolated in soil and water, as well as in moist
hospital environment
 Mode of transmission: exposure to contaminated medical
devices and solutions
 Grows well on MAC
 In BAP - colonies are feather edge, non-pigmented, alpha
hemolytic and have a fruity odor which similar to that of
apples or strawberries.
 Biochemical test: oxidase (+); grows in 6.5 % NaCl broth
OLIGELLA SPP.
 Aerobic rod that may appear coccoid in shape
 It may colonize the distal urethra and cause a serious and
active infection
 Microscopy: small, paired, gram negative bacilli or
coccobacilli
 Culture:
o In BAP colonies are small, opaque, and white
o No growth in MAC
 Biochemical tests are catalase test (+); oxidase (+); PAD
test positive; gas producer (+) and assaccharolytic
MORAXELLA LACUNATA
 Non-motile, assacharolytic and obligate aerobes
 Etiologic agents of blepharoconjuctivitis or angular
conjunctivitis
 Microscopy: Gram negative with coccobacillary to bacillary
forms
 Culture:
o In BAP, colonies are small and pit the agar
o Do not grow in MAC
 Biochemical test: (+) catalase and oxidase (+)
CHROMOBACTERIUM VIOLACEUM
 Only species in the genus Chromobacterium
 It is motile with polar flagella
 Opportunistic pathogen that causes neutrophil deficiency to
immunocompromised patients
 Grows on MAC at 42 degrees Celsius
 Microscopy: May appear as curved bacilli
 Culture: Colonies exhibit violet pigmentation (violacein
pigment)
 Biochemical test: Variable oxidase (v)
SHEWANELLA PUTREFACIENS
 Found and isolated from water, dairy products, petroleum
gas, and other environmental source
 Strongly H2S (+), motile and saccharolytic bacteria
 Associated infections: ocular infection, otitis media, and
septicemia
 Gram negative rods
 In BAP, colonies are mucoid and greenish (including the
medium)
 Biochemical test: (+) Oxidase and ornithine decarboxylase
(+)
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