Gram Negative Rods
Enterobacteriaceae (Coliforms)
API 20E Test Strip
All Negative
All Positive
The fast way to run many tests on an organism is to inoculate either an APT 20E test
strip ($12 each) or an Enterotube ($45 each). The organism is placed into each well
and the results are read two days later.
API 20E Test Record Sheets
Enterotube: more expensive than API-20E media
Symbols that indicate test
results:
• A: acid has formed. This usually indicates
a sugar nutrient was broken down.
• ALK: means the media is alkaline after the
test. This usually also indicates a negative
test, so we just abbreviate it to “K”.
Therefore:
• K: means the test was negative
• G: means gas formed
• You could also perform each of those tests
in individual test tubes or plates, as shown
in the following slides.
Thio Broth: Determines oxygen
requirements
This test is done to see if the organism
is aerobic (growth only at the top),
anaerobic (growth only at the butt), or
facultative (growth throughout). A
facultative organism can live with or
without air, so that is considered a
virulence factor.
Thio Broth
Not shown:
anaerobic; growth
only at the butt
Aerobic
Facultative
Litmus milk
• This is done to see if the organism has the enzyme
lactase. If it does, it will ferment lactose, the milk
sugar. The waste product will be an acid, so it will
change the pH indicator from bluish to pink.
• If gas is produced during this process, gas bubble will
also appear. Some organisms ferment lactose and
produce gas, and some ferment lactose without
making gas.
• Since milk also contains a protein called casein, this
test will also determine if the organism has the enzyme
caseinase to break that protein down. If it breaks the
protein down only partially, curds (solid milk) will form.
If it can break the milk protein all the way down to
amino acids, a brown ring appears at the top.
Litmus Milk Test
P
A
A/C
A/C
G
A
A
K
White (R) means litmus was reduced and became clear.
Pink (A) means it fermented lactose and is acidic.
Bluish (K) means the pH is basic, so lactose was not fermented
Curds formed (C)
Gas in the curd (G)
Brown ring (P) means the casein protein in the milk was broken down (proteolysis)
Litmus Milk
A/C
G
Motility Test
Positive
Triple Sugar Iron (TSI) Slants
A = acid (sugar was fermented)
K = negative result
G = gas formation
H2S = sulfur was used, iron was reduced to black color
K/AG
K/K
K/K
K/A
H2S
A/AG
A/AG
MR Test: pH indicator
Negative
Positive
(acid present)
VP Test
Negative
Positive
IMViC (+ + - -)
E. coli
Indole
Positive
MR
Positive
VP
Negative
Citrate
Negative
IMViC (- - + +)
Enterobacter
Indole
Negative
MR
Negative
VP
Positive
Citrate
Positive
IMViC (+ - - -)
Indole
Positive
MR
Negative
VP
Negative
Citrate
Negative
Urea Test
Positive
Negative
Negative
Citrate
Positive
Negative
Antibiotic
Sensitivity
Antibiotic Sensitivity
Organism is
resistant to the
antibiotic
Organism is
susceptible to
the antibiotic
Antibiotic Sensitivity:
Check zone diameters on given table
Organism is
resistant to
the antibiotic
Organism is
susceptible
to the
antibiotic
Organism
has
intermediate
resistance to
the antibiotic
Positive Coagulase Test:
Differentiates S. Aureus from S epidermidis
Catalase
Oxidase Test
Citrobacter freundii
Citrobacter freundii
• Uses citrate as their sole source of carbon.
• These bacteria can be found almost
everywhere in soil, water, wastewater, etc.
It can also be found in the human
intestine.
• They are rarely the source of illnesses,
except for infections of the urinary tract
and infant meningitis.
Citrobacter freundii
Motility: negative
MR: positive
+
- -
+
-
+
-
Escherichia coli
Non-motile species
Motile species
Escherichia coli
Escherichia coli
• One of the main species of bacteria living in the lower
intestines of mammals, known as gut flora.
• There are about 1 trillion excreted in the feces daily.
• However, the bacteria are not confined to this
environment, and specimens have also been located, for
example, on the edge of hot springs.
• The E. coli strain O157:H7 is one of hundreds of strains
of the bacterium that causes illness in humans.
• E. coli are unable to sporulate. Thus, treatments which
kill all active bacteria, such as pasteurization or simple
boiling, are effective for their eradication, without
requiring the more rigorous sterilization which also
deactivates spores.
Escherichia coli
• E. coli can generally cause several intestinal and extraintestinal infections such as urinary tract infections,
meningitis, peritonitis, septicemia and pneumonia.
• Although it is more common in females due to the
shorter urinary tract, urinary tract infection is seen in both
males and females. Poor toilet habits can predispose to
infection (doctors often advise women to "wipe front to
back, not back to front") but other factors are also
important (pregnancy in women, prostate enlargement in
men).
• If E. coli bacteria escape the intestinal tract through a
perforation (a hole or tear, for example from an ulcer, a
ruptured appendix, or a surgical error) and enter the
abdomen, they usually cause peritonitis that can be fatal
without prompt treatment. However, E. coli are extremely
sensitive to such antibiotics as streptomycin or
gentamycin.
Escherichia coli
• Certain strains of E. coli, such as Escherichia
coli O157:H7, produce a toxin and can cause
food poisoning usually associated with eating
cheese and contaminated meat (contaminated
during or shortly after slaughter or during
storage or display).
• The usual countermeasure is cooking suspect
meat "well done"; the alternative of careful
inspection of slaughtering and butchering
methods (to make sure that the animal's colon is
removed and not punctured).
Escherichia coli
• Escherichia coli O157:H7 is the strain is believed to be
associated with the 2006 United States E. coli outbreak
linked to fresh spinach. Severity of the illness varies
considerably; it can be fatal, particularly to young
children, the elderly or the immunocompromised
• E. coli are susceptable to amoxicillin, cephalosporins,
aminoglycosides, streptomycin, or gentamycin.
• Antibiotic resistance is a growing problem. Some of this
is due to overuse of antibiotics in humans, but some of it
is probably due to the use of antibiotics as growth
promoters in food animals.
Escherichia coli
-
-
Enterobacter aerogenes
Enterobacter aerogenes
• Several strains of the these bacteria are
pathogenic and cause opportunistic infections in
immunocompromised (usually hospitalized)
hosts.
• The urinary and respiratory tract are the most
common sites of infection.
• Enterobacter can be distinguished from other
Gram neg rods by virtue of being a 'fast
fermenter' of lactose (as are E. coli and
Klebsiella).
Enterobacter
Methyl Red: negative
++
Klebsiella pneumoniae
Klebsiella pneumoniae
• This is a nonmotile, encapsulated, lactosefermenting, and facultatively anaerobic
organism.
• It is found in the normal flora of the mouth,
skin, and intestines.
• It naturally occurs in the soil and about
30% of strains can fix nitrogen in
anaerobic conditions.
Klebsiella pneumoniae
• K. pneumoniae can cause bacterial pneumonia, typically
due to aspiration by alcoholics, though it is more
commonly implicated in hospital-acquired urinary tract
and wound infections, particularly in
immunocompromised individuals.
• Klebsiella ranks second to E. coli for urinary tract
infections in older persons.
• Feces are the most significant source of patient infection,
followed by contact with contaminated instruments.
• Klebsiella is resistant to ampicillin but susceptible to
aminoglycosides and cephalosporins.
Klebsiella pneumoniae
Proteus vulgaris
Proteus vulgaris
• Responsible for many human urinary tract
infections.
• Proteus species do not ferment lactose,
are oxidase negative, and urease positive;
some species are motile.
Proteus vulgaris
Providencia stuartii
Providencia stuartii
• This organism is motile.
• They are opportunistic pathogens in
humans and can cause urinary tract
infections, particularly in patients with
long-term indwelling urinary catheters or
extensive severe burns.
• Some strains are sensitive to ampicillin.
Providencia stuartii
IMViC: + + - +
ODC: ONPG: +
H2S: Mannitol: -
Salmonella enterica
Salmonella enterica
Salmonella enterica
• This organism causes typhoid fever and
foodborne illness.
• Salmonella species are motile and
produce hydrogen sulfide.
• They usually do not ferment lactose.
Salmonella enterica
• Food poisoning from Salmonella is usually from
poultry and raw eggs and from food that has
been cooked or frozen, and not eaten straight
away.
• The overuse of antibiotics in both the poultry and
beef industries have created a strain of
salmonella which is potentially resistant to
antibiotics.
• It can also be caught by handling reptiles, such
as iguanas.
Salmonella enterica
• The prevention of Salmonella as a food
illness involves effective sanitizing of food
contact surfaces.
• Alcohol has proven to be an effective
topical sanitizer against Salmonella.
Salmonella enterica
Shigella somnei
Shigella somnei
• This organism is non-motile, non-spore
forming. It is H2S positive.
• It is the causative agent of human
shigellosis.
• Shigella infection is typically via ingestion
(fecal–oral contamination).
• 10 bacterial cells can be enough to cause
an infection.
Shigella somnei
• The stool may contain blood, mucus, or pus (eg
dysentery).
• Shigella cause dysentery that results in the
destruction of the epithelial cells of the intestine.
• Some strains produce an enterotoxin similar to
E. coli O157:H7
• Symptoms usually begin two to four days after
ingestion. Symptoms usually last for several
days, but can last for weeks.
• Severe dysentery can be treated with ampicillin.
Shigella somnei
•
•
•
•
•
•
•
IMViC: - + - - or + + - ODC: + or LDC: Urease: H2S: Sucrose: TSI: K/A
Serratia marcescens
Serratia marcescens
• This organism is involved in nosocomial
infections, particularly urinary tract infections and
wound infections.
• It is motile.
• Due to its preference for damp conditions, it is
commonly found growing in bathrooms
(especially on tile grout), where it manifests as a
pink discoloration.
• Once established, complete eradication of the
organism is often difficult, but can be
accomplished by application of a bleach-based
disinfectant.
Serratia marcescens
• S. marcescens can cause conjunctivitis
and tear duct infections.
• It is common in the respiratory and urinary
tracts of adults and the gastrointestinal
system of children.
Serratia marcescens
•
•
•
•
IMViC: - + + + or - - + +
LDC: +
ARA: ORN: +
Lactose Negative
• Perform an Indole test:
– Positive
•
•
•
•
Proteus vulgaris (H2S negative)
Providencia stuartii (H2S positive)
E. coli (Citrate negative)
Citrobacter freundii (H2S positive, Citrate positive)
– Negative
• Perform a urease test (all should be negative)
• Perform the MR-VP test
Urease negative
• Perform a motility test
– Positive
• Salmonella enterica (H2S positive)
• Serratia marcescens (H2S negative)
– Negative
• Shigella somnei
MR-VP Tests
• +/– Citrobacter freundii
• -/+
– Klebsiella pneumoniae (non-motile)
– Enterobacter aerogenes (motile)