The Microbiology Laboratory Summary By Sunny Chen Topic Overview Potential bacterial candidates for this infectious scenario Possible samples taken Possible tests performed Expected results for the potential bacterial candidates ID: 913357
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Slide1
PATH 417 Case 4: One Too Many Hamburgers
The Microbiology Laboratory
Summary
By: Sunny Chen
Slide2Topic Overview
Potential bacterial candidates for this infectious scenario
Possible samples taken
Possible tests performed
Expected results for the potential bacterial candidates
Slide3Topic Overview
Potential bacterial candidates for this infectious scenario
Possible samples taken
Possible tests performed
Expected results for the potential bacterial candidates
Slide4Potential Bacterial Candidates
Possible disease:
gastroenteritis
P
ossible bacterial causes include:
Salmonella typhimurium(S. typhimurium)
Campylobacter
jejuni
(C.
jejuni
)
Escherichia coli
O157:H7
Shigella
Slide5Potential Bacterial Candidates
Possible contaminated food: ground beef (in hamburger)
Most
common bacterial
pathogens associated with this food:
Salmonella
Shiga-toxin
producing
Escherichia coli (STECs
)-
especially
E
. coli
O157:H7
Campylobacter
jejuni
Listeria monocytogenes
Staphylococcus
aureus
Slide6Possible Infectious Mechanism
grinding of
meat
bacteria
present on the surface
get
distributed throughout the meat
in the “Danger Zone” (temperatures between
4.4°C and 60°C)
bacteria multiply
why necessary to cook the meat to a safe internal temperature of 71.1 °C and to keep ground beef stored below 4.4 °C
Slide7Potential Bacterial Candidates
bacterial candidates associated with bloody diarrhea:
Escherichia coli
(particularly E. coli O157:H7)
Campylobacter
jejuni
(
C.
jejuni
)
Salmonella Typhimurium
(
S. Typhimurium
)
Shigella
sonnei
(
S.
sonnei
)
Thus, potential bacterial candidates for this case
can
include
the
above bacteria
Slide8Potential Bacterial Candidate-C.
jejuni
gram-negative
bacterium (figure on the right)
microaeroplic
, non-fermentative, and coccoid or round in shape
have flagellum to navigate in the host
Slide9Potential Bacterial Candidate-C.
jejuni
cell
wall structure: inner lipid membrane, thin peptidoglycan
layer
and an outer membrane
on the surface of the cell wall, lipopolysaccharides embedded to the outer membrane
LPS:
endotoxic
b/c can
trigger Toll-Like-Receptor 4s
Slide10Potential Bacterial Candidate-C.
jejuni
primary reservoir locale: gastrointestinal tract of animals
transmitted to humans
via:
food sources
unclean water
direct
physical contact with an infected animal
primary
symptoms:
bloody stool
abdominal pain
fever
nausea
diarrhea
vomiting
other complications involving the digestive system
Slide11Potential Bacterial Candidate-
E
. coli O157:H7
facultative anaerobic bacteria, gram-negative, rod-shaped
able to produce Shiga-toxin, can cause extensive damage to the walls of the large
intestine
hemorrhagic
colitis (diarrhea and fever followed by bloody diarrhea)/ hemolytic uremic syndrome (HUS), particularly in children and the elderly
Slide12Potential Bacterial Candidate-
E
. coli O157:H7
found in the normal flora of the gastrointestinal tract that doesn’t normally cause infections
can naturally be found in the intestinal contents of some cattle, goats, and even sheep
present in ground
beef
transmission
: usually spread through the improper preparation of beef products
Slide13Potential Bacterial Candidate-
E
. coli O157:H7
Primary symptoms:
abdominal pains
blood and watery diarrhea
vomiting
mild fevers
Slide14E. coli O157:H7 & HUS
Hemolytic uremic syndrome (HUS):
A condition that is caused by the abnormal destruction of red blood cells
red blood cells overwhelm and clog up the filtration system of the
kidneys
kidney
failure
usually develops 5-10 days after the start of bloody diarrhea caused by bacterial infections
avoid the use of antibiotics or
antimotility
agents during diarrheal illness
a
ntimotility
shown
to increase the incidence of
HUS
motility slows, the gut is exposed to the toxins for a longer period of time
antibiotic-induced injury to the bacterial membrane favors the acute release of large amounts of toxins
shown to increase the risk of full-blown HUS by 17-fold
recommendation: avoid usage except in cases of sepsis
Slide15Potential Bacterial Candidate-
S. typhimurium
a flagellated gram-negative facultative anaerobic bacilli
bacteria (right figure)
toxicity due to an outer membrane
consists largely of lipopolysaccharides (LPS)
protect the bacteria from the environment
Slide16Potential Bacterial Candidate-
S. typhimurium
found in the intestinal tract of animals
causes
two different diseases
enteric fever (
typhoid)
occurs
as the bacterium enters the bloodstream
acute
gastroenteritis
f
oodborne
infections of the gastrointestinal
tract
most common transmission method:
ground meat and the improper preparation of the meat before consumption
primary symptoms:
abdominal pain
cramps
vomiting
diarrhea
Slide17Potential Bacterial Candidate-
Shigella
(most likely
S.
sonnei
in North America)
Gram-negative
facultative anaerobic,
nonspore
-forming,
nonmotile
, rod-shaped bacteria
species classified into four serogroups:
S.dysenterae
S.flexneri
S.boydii
S.sonnei
(shown here)
Slide18Potential Bacterial Candidate-
Shigella
(most likely S.
sonnei
in
North America)
Disease caused: shigellosis
affects the gastrointestinal tract
associated with the symptoms:
bloody diarrhea
fever
stomach cramps
abdominal pains
Molecular cause: Shiga toxin produced
causes inflammatory response to the enteric cell wall and necrotic cell death of the colonic epithelium
Infection due to consumption of infected food or liquids
Slide19Topic Overview
Potential bacterial candidates for this infectious scenario
Possible samples taken
Possible tests performed
Expected results for the potential bacterial candidates
Slide20Possible Samples Taken
Stool
Blood
Urine
Details summarized in the table coming next
Slide21Possible Samples Taken
Sample
When to Collect
How much to Collect
Method of Collection
Storage
Transportation
Stool
As soon as the onset of diarrhea or bloody stool
Whole stools or several rectal swabs
Whole stool
collected
in a sterile plastic container with a tight, re-sealable
lid
Rectal swabs
can
be done rectally or on whole
stool
The
swabs
are inserted 1 to 1.5 inches into the rectum and rotated gently, then placed into
the transport medium
Whole stool that is not processed within 2 hours of collection should be refrigerated at
4°C
A
portion of the stool can be frozen at -15°C for antigen testing and
PCR
Specimens are refrigerated for transport using ice by overnight
mail
Frozen
stool samples are transported using dry
ice
Slide22Possible Samples Taken
Sample
When to Collect
How much to Collect
Method of Collection
Storage
Transportation
Blood
During the onset of gastroenteritis symptoms
3mL for children; 15mL for adults
A tourniquet is applied and a sterile needle is used for the puncture
site
Withdrawn
blood is collected in labelled
tubes
The tubes are inverted several times to ensure proper mixing
Collection tubes may contain anticoagulants, gels or other additives in preparation for laboratory
testing
Samples
are stored in temperatures between 4 to
25°C
Unspun serum should be refrigerated for transport using
ice
Spun
serum can be frozen for shipping by using dry
ice
Slide23Possible Samples Taken
Sample
When to Collect
How much to Collect
Method of Collection
Storage
Transportation
Urine
First passed urine in the morning or midstream catch any time of day, during the onset of symptoms
10 to 50mL
Collected in sterile plastic container with a
lid
Specimens refrigerated at 4-6°C
Samples are centrifuged for 5 to 10 minutes, then the supernatant is discard and the sediment is re-suspended in a transport
medium
Samples
should be refrigerated during transported and processed within 48
hours
Slide24Importance of Microbiology Laboratory
critical in deducing the pathogen responsible for the patient’s symptoms and diagnosis of the disease
essential that the correct bacterial pathogen is isolated from stool cultures for the management of and development of a treatment plan for patients
important in determining what antibiotics to use
some strains of bacteria are resistant to specific types of antibiotics
some types of antibiotics may be more effective against certain bacteria
urine and blood
samples: have important
role in the diagnosis of the disease
blood test: allows physicians to understand the patient’s overall health status by determining their renal function, electrolytes, overall blood cell
values
Help to determine
if
the bacteria have spread to
the systemic
circulation
urine test:
provides
information
of whether the
bacteria have spread to other sites
Slide25Topic Overview
Potential bacterial candidates for this infectious scenario
Possible samples taken
Possible tests performed
Expected results for the potential bacterial candidates
Slide26Possible Tests Performed
Stool culture
laboratory will ‘grow’ (culture) the pathogens present in the stool sample to determine whether a pathogen is present
if so, identify (at least narrow down) the possible pathogen based on the media conditions and the characteristics demonstrated
sample processed
selected for portions that may contain blood or mucus
ensures the highest number surviving of pathogens for culture
Slide27Possible Tests Performed
Stool
culture (Conti)
plated on different media for identification purposes:
MacConkey (MAC) agar for general recovery of gram negative rod shaped bacteria
Selective and differential medium used to isolate gram negative bacteria and determine if they are fermenting or non-fermenting
A selective/differential medium designed for the recovery of
Salmonella
E.g. Bismuth sulfite agar
A medium designed for the recovery of
Campylobacter
E.g. Campy blood agar
A medium designed for the recovery of
E. coli O157:H7
and/or enrichment broth for testing for the presence of Shiga toxin
E.g. MacConkey agar with sorbitol (SMAC) or
cefixime
-tellurite SMAC (CT-SMAC)
A medium designed for the recovery of
Shigella
E.g.
HardyChrom
Salmonella
Shigella
(SS
)
Note: Any blood
samples/urine samples
would
undergo
similar culturing protocol as the stool sample
Slide28Possible Tests Performed—
Different
Media
Medium
Composition
Principle
MacConkey (MAC) agar
• Pancreatic digest of gelatin and peptones
provide the essential nutrients, vitamins and nitrogenous factors required by bacteria
•
NaCl
-
maintains
osmotic balance
• Agar
solidifies medium
• Lactose monohydrate
a fermentable source of
C,H,and
Os
• Crystal violet and bile salts
inhibit
the growth of most gram positive bacteria
• Neutral red
a pH indicator
A selective and differential
medium
Used to isolate gram-negative bacteria by:
inhibit growth of gram-positive bacteria
differentiate between fermenting (pink colonies) and non-fermenting (colourless colonies) gram-negative bacteria
Fermentation causes a local pH
decrease
causes
the indicator to turn pink
Slide29Possible Tests Performed-MAC Agar
Slide30Possible Tests Performed—
Different
Media
Medium
Composition
Principle
Bismuth
sulfite
agar
• Beef extract and peptone
provide nitrogen
• Dextrose
energy source
• Disodium phosphate
buffering agent
• Ferrous sulfate
detects H2S production
• Bismuth sulfite indicator and brilliant green
inhibit the growth of gram-positive bacteria and members of the coliform group
(e.g. E. coli)
• Agar
solidifies
the
medium
A
highly selective/differential
medium
for
Salmonella
sp. including typhoid fever causing
serotypes
Bismuth sulfite indicator and brilliant green inhibit
growth of
gram-positive and coliform bacteria
(e.g.
E
.
coli
)
to allow
Salmonella
to grow freely
Most
Salmonella
enterica
subspecies are capable of producing
H
2
S
(
detected by ferrous sulfate in the medium
)
- iron precipitated
formation of colonies that have
a brown/black colour with a metallic sheen
- Other bacteria would have inhibited growth/
different colored colonies (likely greenish)
Slide31Possible Tests Performed-Bismuth
Sulfite
Agar
Slide32Possible Tests Performed
—
Different
Media
Medium
Composition
Principle
Campy blood agar
• Casein and me
a
t peptone
provide nutrients for Campylobacter growth
• Sodium
provides electrolytes and maintains osmotic balance
• Dextrose
provides an energy source
Y
east
extract
provides essential vitamin B
• Sheep’s blood
provides growth factors
•
Cephalothin
, amphotericin B, trimethoprim, vancomycin, and
polymyxin
B
inhibit the growth of
Enterobacteriaceae
,
staphylococcus
, and yeast
A
highly selective
medium
for
Campylobacter
sp.
Growth is optimized for
Campylobacter
G
rowth of yeast,
Enterobacteriaceae
(e.g.
E.
coli
,
Salmonella
, and
staphylococcus
)
are
inhibited
Colonies should be yellowish/greyish and
non-hemolytic
Slide33Possible Tests Performed-Campy Blood Agar
Slide34Possible Tests Performed
—
Different
Media
Medium
Composition
Principle
MacConkey agar with sorbitol (SMAC) or
cefixime
-tellurite SMAC (CT-SMAC)
• Same composition as MacConkey agar
except replace lactose with sorbitol
E. coli
O157:H7
is unable to ferment
•
Allows
differentiation
of
pathogenic O157:H7
E. coli
from other serotypes
(as
it is unable to ferment sorbitol
)
• Would
be
likely done as a subculture of
E. coli
(of unknown serotype) cultured first upon regular MAC agar
• O157:H7
E. coli
colonies would be colourless
they can
’t
ferment the sorbitol (which would lower the pH around the colony and change the colour of the colony to red)
colourless colonies
Slide35Possible Tests Performed
—
SMAC or CT-SMAC
SMAC with
E. Coli
O157
Slide36Possible Tests Performed
—
Different
Media
Medium
Composition
Principle
HardyChrom
Salmonella
Shigella
(SS)
• Agar
solidifies medium
• Bile salts and sodium
deoxycholate
inhibit growth of gram-positive bacteria
Peptones
provide nutrients for bacterial growth
• Selective antimicrobial agents
inhibit other types of enteric bacteria
• Fermentable C
,
H
,
Os
allows
distingu
ish
between enteric pathogens and delayed lactose fermenters
• A pH indicator
• Ferric ammonium citrate and sodium thiosulfate
allow visualization of
H
2
S
producing colonies
• Patented chromogenic mixture
produces different colours depending on which bacterial enzymes degrade them
• A selective medium
allows differentiation between Salmonella and
Shigella
.
Color differentiation:
• Most
Salmonella
subtypes produce H
2
S
would appear as colonies with black centers
• Non-H
2
Sproducing
Shigella
would appear as teal-blue colonies
• Other bacterial species
would be completely/partially inhibited
if present, would be a different colour (due to chromogenic mixture)
Slide37Possible Tests Performed
—
SS
Slide38Side note: A table of more media and their intended use (Pt 1)
Medium
Intended use and notes
All-purpose broths
Gram-negative (GN) broth
Selective enrichment for Gram-negative rods, specifically Salmonella and Shigella (subculture after 6–8 h of incubation, not part of routine setup unless for STEC EIA), can be used for STEC EIA
Selenite F broth
Selective enrichment for Gram-negative rods, specifically Salmonella and Shigella (subculture after 18–24 h of incubation, may inhibit growth of some Shigella species) (not part of routine setup)
Organism-specific broths
Alkaline peptone water
Selective enrichment broth for Vibrio, when requested (subculture to TCBS after 24 h of incubation)
MAC broth
Can be used for STEC EIA, enrichment for Y. enterocolitica if incubated at 25°C (not part of routine setup)
All-purpose agars
Hektoen enteric (HE)
Selective medium for Gram-negative rods, differentiates lactose fermenters (yellow-orange) from nonfermenters (blue or green), H2S production can be detected (black precipitate)
MacConkey (MAC)
Selective medium for Gram-negative rods, differentiates lactose fermenters (pink) from nonfermenters (colorless)
Salmonella-shigella (SS)
Selective medium for Gram-negative rods, differentiates lactose fermenters (pink/red) from nonfermenters (colorless), H2S production can be detected (black precipitate)
Xylose-lysine-deoxycholate (XLD)
Selective medium for Gram-negative rods, differentiates lactose fermenters (yellow) from
nonfermenters
(red), H2S production can be detected (black precipitate)
Slide39Side note: A table of more media and their intended use (Pt 2)
Medium
Intended use and notes
Highly selective/differential agars
Bismuth sulfite
Isolation of Salmonella, including Salmonella Typhi (black on this medium)
Brilliant green
Isolation of Salmonella (red, pink, or white with red halo on this medium), inhibits Salmonella Typhi and Salmonella Paratyphi
Blood agar with ampicillin
Isolation of Aeromonas (not part of routine setup unless specifically requested)
Campy Blood
Isolation of Campylobacter
Campy CVA
Isolation of Campylobacter
Campylosel
Isolation of Campylobacter
Cefsulodin-Irgasan-novobiocin (CIN)
Isolation of Yersinia enterocolitica or Aeromonas (deep red center and transparent margin [bull's eye appearance] on this medium) (not part of routine setup)
Charcoal selective
Isolation of Campylobacter
Charcoal-cefoperazone-deoxycholate agar (CCDA)
Isolation of Campylobacter
CHROMagar Salmonella
Isolation of Salmonella (mauve-rose on this medium)
Slide40Side note: A table of more media and their intended use (Pt 3)
Medium
Intended use and notes
CHROMagar O157
Isolation of O157 STEC (mauve on this medium)
CHROMagar STEC
Isolation of 6 most common STEC serogroups (mauve on this medium)
Cycloserine-cefoxitin-egg yolk/cycloserine-cefositin-fructose
Isolation of Clostridium difficile (not part of routine setup unless requested)
HardyChrom SS
Isolation of Salmonella (black on this medium) and Shigella (teal on this medium)
nositol-brilliant green-bile salt
Isolation of P. shigelloides (white to pink on this medium) (not part of routine setup unless requested)
MacConkey agar with sorbitol (SMAC) or cefixime-tellurite SMAC (CT-SMAC)
Isolation of E. coli O157 (colorless on this medium)
Thiosulfate-citrat e-bile salts-sucrose (TCBS)
Isolation of Vibrio (not part of routine setup unless requested), V. cholerae is yellow on this medium, V. parahaemolyticus is green on this medium, some vibrios are inhibited
CHROMagar O157
Isolation of O157 STEC (mauve on this medium)
CHROMagar STEC
Isolation of 6 most common STEC serogroups (mauve on this medium)
Cycloserine-cefoxitin-egg yolk/cycloserine-cefositin-fructose
Isolation of Clostridium difficile (not part of routine setup unless requested)
HardyChrom SS
Isolation of Salmonella (black on this medium) and
Shigella
(teal on this medium)
Slide41Possible Tests
P
erformed—
Secondary Biochemical Tests
Slanted media test
used to subculture suspicious colonies for pathogen identity confirmation
involved the classic “3-tube” biochemical test
triple sugar iron agar (TSI)
lysine iron agar (LIA)
Christensen urea
Slide42Possible Tests
P
erformed—
Triple Sugar Iron Agar (TSI)
Involves 3 sugars: lactose, sucrose, and glucose
determine the fermentation preferences of the bacteria
contains “butt” (bottom) and “slant” (top) regions
Represent poorly or richly oxygenated regions
Contain pH indicator for detection of fermentation
Phenol red indicator turns yellow for acidic environment
Slide43Possible Tests
P
erformed—
Triple Sugar Iron Agar (TSI)
Reaction Principles
lactose or sucrose is
fermented
both
butt and slant turns yellow, gas may be generated, bubbles/crackles observed
only
small amount of glucose is
fermented
butt
(more media, more glucose)turns yellow , slant (less media, less glucose, acid oxidized by the organisms) remains red
n
one
of the sugars are
fermented
both
the butt and slant would be red
H
2
S
produced
indicator
turns black for presence of ferrous sulfide
Slide44Possible Tests
P
erformed—
Triple Sugar Iron Agar (TSI)
Slant/Butt Description
Color Presentation (Slant/Butt)
Interpretation
Alkaline slant/no change in butt (K/NC)
Red/Red
glucose, lactose and sucrose non-fermenter
Alkaline slant/Alkaline butt (K/K)
Red/Red
glucose, lactose and sucrose non-fermenter
Alkaline slant/acidic butt (K/A)
Red/Yellow
glucose fermentation only, gas (+ or -), H2S (+ or -)
Acidic slant/acidic butt (A/A)
Yellow/Yellow
glucose, lactose and/or sucrose fermenter gas (+ or -), H2S (+ or -)
Expected Results
:
Slide45Possible Tests
P
erformed—
Lysine Iron Agar
(LIA)
also contains slant and butt regions
tests the ability of bacteria to
deaminate
lysine or
decarboxylate
lysine
occur in the slant or butt of the media respectively
Slide46Possible Tests
P
erformed—
Lysine Iron Agar
(LIA)
Reaction Principles
production of
decarboxylate
lysine= creation of a product
reacts with the
bromcresol
purple
indicator
purple
butt, slant remains purple
negative decarboxylation=yellow butt, purple slant
Deamination of lysine=production of ammonia
reacts
with the ferric ammonium citrate
dark red slant, yellow butt
Negative deamination=purple slant
Presence of H
2
S
black precipitate in the butt
Slide47Possible Tests
P
erformed—
Lysine Iron Agar
(LIA)
Slant/Butt Description
Color Presentation (Slant/Butt)
Interpretation
Alkaline slant/Alkaline butt (K/K)
Purple/Purple
negative lysine deamination (slant),positive lysine decarboxylation (butt)
Acidic slant/Alkaline butt (A/K)*
Yellow/ Purple
positive lysine deamination (slant), positive lysine decarboxylation (butt)
Red slant/Acidic butt (R/A)
Red/ Yellow
positive lysine deamination (slant), negative lysine decarboxylation (butt)
Acidic slant/Acidic butt (A/A)
Yellow/Yellow
positive lysine deamination (slant), negative lysine decarboxylation (butt)
Expected Results:
Tube 1
:
K/K
Tube
2
:
R/A
*
There might be a mistake on the wiki generated
Slide48Possible Tests
P
erformed—
Additional Tests
Gram staining
A common microbiology lab technique
used to differentiate between gram-positive
(blue/purple
) and gram-negative (red/pink) cells
involves staining bacterial cells with crystal violet (a water soluble dye)+ add Gram’s iodine solution
iodine forms insoluble complex with crystal violet
followed by
decolorization
-decolorizer dehydrates the peptidoglycan layer
Gram-Positive: crystal violet-iodine complex is trapped within the bacteria, dye is retained, can’t be counter-stained
Gram-Negative: crystal violet-iodine complex is lost (dye is lost), counter stained to be red/pink
Slide49Possible Tests
P
erformed—
Additional Tests
Gram staining
Slide50Possible Tests
P
erformed—
Additional Tests
Oxidase Test
determines if the bacteria has the cytochrome C oxidase enzyme
responsible for catalyzing electron transport to electron acceptors as a part of the bacteria’s respiratory chain
test involves catalyzing a redox reaction
turns the reagent (
tertramethyl
-p-
phenylene
-diamine
dihydrochloride
) from colorless to a deep purple
Different methods:
dry filter paper method
direct plate method
swab method
impregnated oxidase strip method
test tube method
Slide51Possible Tests
P
erformed—
Additional Tests
Oxidase Test
General Principle:
introduce the colony to be tested to the reagent
observe for color change
Expected Results
Positive=color change to purple
Negative=no/delayed color change (remain colorless)
Wet filter paper method protocol
Soak a strip of filter paper in 1% solution of reagent
Rub a speck of the culture onto the filter paper
Observe for colour change, where positive is deep purple in 5-10s, delayed positive is purple in 10-60s, and negative is colourless or colour change after 60s
Slide52Possible Tests
P
erformed—
Additional Tests
Oxidase Test
Slide53Possible Tests
P
erformed—
Additional Tests
Catalase Test
tests bacteria for the presence of enzyme catalase
catalyzes the breakdown of H
2
O
2
into H
2
O and O
2
introduce the bacteria to H
2
O
2
Positive=rapid bubbling observed (O
2
produced)
Negative=no bubbling observed
Many different methods
Test Tube method protocol
Ensure the colonies tested are 18-24hrs old, scrape several colonies up with a sterile tool (wooden stick, glass rod)
Immerse colonies in solution
Look for immediate bubbling (positive test result)
Slide54Possible Tests
P
erformed—
Additional Tests
Catalase Test
Slide55Possible Tests
P
erformed—
Additional Tests
PCR
a method of amplifying the gene of interest
general protocol:
Denaturation
– heating the mixture to 94ºC, the two strands of the DNA molecule separate into single strands
Annealing
– the mixture is cooled down to 50-70ºC, allowing primers to bind to complementary sites on the single strands
Extension – the mixture is heated to 72ºC, allowing DNA polymerase to extend a new, complementary strand from the primers
Result
–doubling of DNA at the end of every cycle
Slide56Possible Tests
P
erformed—
Additional Tests
PCR
Application in this case:
E. coli
O157:H7 possesses the genes Stx1 and Stx2 for Shiga toxin production
The other suspected pathogens of this case lack this gene
Target these genes using PCR would allow for identification of
E. coli
O157:H7 as the causative agent in this case
Slide57Possible Tests
P
erformed—
Additional Tests
ELISA(Enzyme-linked Immunosorbent Assay)
used to detect the presence of a specific antigen
An example protocol:
fix unknown antigen to an immobile surface, add antibodies specific to the antigen of interest
if the unknown antigen is the antigen of interest, binding would occur
w
ash
the plate to remove any unbound antibodies
any antibody bound to the plate after washing can be detected (they’re usually conjugated with an enzyme or some other molecules for detection)
indicative of the presence of the antigen of interest
Slide58Possible Tests
P
erformed—
Additional Tests
ELISA(Enzyme-linked Immunosorbent Assay)
Application in this case (
E. coli
O157:H7):
Antigen of interest: Shiga toxin
only antigen from
E. coli
O157:H7 would have the specific antibodies bound to it
detection of
E. coli
O157:H7
Direct ELISA shown on the right as an example of ELISA
Slide59Possible Tests
P
erformed—
Additional Tests
Antibiotic Sensitivity Test
Aka susceptibility testing, a technique used:
to test for antibiotic resistant pathogens
to determine which antibiotic treatments would be most effective for the treatment of a particular patient
a variety of procedural methods available
E.g. the disc diffusion test
Inoculate a Mueller-Hinton Agar (MHA) plate with the bacteria of interest
Apply (typically up to 12) disks containing antimicrobial agents
Incubate the plate for 16-24 hours (depending on specific tests)
Observe zones of inhibition around disks, measure the diameter of these zones
Zones are dependent on:
diffusion rate of the antimicrobial agent
susceptibility of the pathogen to the antimicrobial agent
Slide60Possible Tests
P
erformed—
Additional Tests
Antibiotic Sensitivity Test
Slide61Possible Tests
P
erformed—
Additional Tests
Agglutination Test
final test for pathogen identity confirmation
general protocol:
Place a homogenous suspension of the colony of interest in a test tube or a slide
Test against antiserum (of known antibodies) specific to pathogen of interest
Visible clumping seen within 1 min=
positive
pathogen
of interest detected
Slide62Possible Tests
P
erformed—
Additional Tests
Agglutination Test
Slide63Topic Overview
Potential bacterial candidates for this infectious scenario
Possible samples taken
Possible tests performed
Expected results for the potential bacterial candidates
Slide64Expected Results for
E.coli 0157:H7
Test
Expected Result
MacConkey plate
pink colonies (lactose-fermenting)
EMB plate
blue colonies (lactose-fermenting)
SM
colourless colonies (does not ferment sorbitol)
Oxidase Test
positive
Catalase
positive
Gram staining
Red(Gram negative)
PCR
Positive (bacteria possess Stx1 and Stx2 genes)
ELISA
positive (produces Shiga toxin)
TSI
K/A or A/A, gas
LIA
K/A or A/A
Antibiotic Sensitivity Test
Susceptible to nitrofurantoin, ciprofloxacin, and
norflaxocin
Not susceptible tetracycline, erythromycin, and amoxicillin
Slide65Expected Results for
Campylobacter
Test
Expected Result
Campylobacter Skirrow agar
small, mucoid, flat or slightly raised, non-hemolytic translucent and creamy-grey colonies (selective media for the bacteria)
Oxidase
Positive
Catalase
Positive
Gram staining
Red (gram-negative)
PCR
negative (bacteria does not possess Stx1 and Stx2 genes)
ELISA
negative (does not produces Shiga toxin)
Antibiotic Sensitivity Test
Susceptible to erythromycin
Not susceptible to ciprofloxacin
Slide66Expected Results for
Salmonella
Test
Expected Result
MacConkey media
colorless colonies (non-lactose fermenting)
EMB
colourless colonies (non-lactose fermenting)
XLD media
red colonies with black centers (
decarboxylates
lysine-red,
H
2
S
production under alkaline conditions-black)
HE media
bluish-green colonies
(
H
2
S
production)
Oxidase
Negative
Catalase
Positive
Gram staining
Red (Gram negative)
PCR
negative (bacteria does not possess Stx1 and Stx2 genes)
ELISA
negative (does not produces Shiga toxin)
TSI
K/A,
H
2
S
,
± gas
LIA
K/K or A/A,
H
2
S
Antibiotic Sensitivity Test
Susceptible to ciprofloxacin
Not susceptible to ampicillin
Slide67Side Note: Some additional characteristics of the potential bacterial candidates
Bacterial Species
Detection Method
Microbiologic Characteristics
Campylobacter
Campylobacter
Skirrow
agar
Curved gram-negative rod
Rapidly motile
E.coli
MacConkey, EMB, or SM agar
Gram-negative rod
Lactose-producing
Salmonella
MacConkey, EMB, XLD, or HE agar
Gram-negative rod
Non-lactose H2S-producing
Lysine
decarboxylator
Slide68Image source
Slide 7-http://enfo.agt.bme.hu/
drupal
/sites/default/files/Campylobacter%20jejuni.JPG
Slide 10-https://foodlawlatest.files.wordpress.com/2014/07/e-coli-o157-h7.jpg
Slide 14-http://www.dzif.de/fileadmin/user_upload/news/zeitlos/bacteria/Salmonella_typhimurium_490.jpg
Slide 16-http://atlas.microumftgm.ro/
bacteriologie
/
bactsp
/
shigella
/
frotiu
/pic/sh_me.jpg
Slide 29-http://apchute.com/
wellmeyer
/media/McConUnin.jpg
http://www.microbiologyinfo.com/wp-content/uploads/2015/11/Morphology-on-MacConkey-Agar.jpg
Slide 31-
https://
www.dlsweb.rmit.edu.au/set/LearningObjects/FoodMicroTutes/images/S4Dscn1249.jpg
http://
kisanbiotech.img15.kr/MS_product/34133/1455159585_732591.jpg
Slide 33-
https://lh4.googleusercontent.com/-
UCdKPz8K9hU/VL-dXLvc4gI/AAAAAAAAAP8/yQCjv1KKYY8/w318-h324/1021.png
Slide 35-http://
www.oxoid.com/omd/library/fullsize/CM0813.jpg
Slide 37-http://
files.constantcontact.com/fbc79467001/84a3781c-f3c1-4c5f-af1c-906fe7fbd227.jpg
Slide 44-
http://
web.clark.edu/tkibota/images/TSI.gif
Slide 47-http://iws2.collin.edu/
dcain
/CCCCD%20Micro/LIA.jpg
Slide 49-http://laboratoryinfo.com/
wp
-content/uploads/2016/01/gram-positive-vs-gram-negative.png
Slide 52-https://www.cdc.gov/meningitis/lab-manual/images/chapt7-figure04.gif
Slide69Image source
Slide
54-http://
www.telmeds.org/wp-content/uploads/2009/10/CatalaseResults1.jpg
Slide 56-http://
www.scielo.br/img/revistas/bjm/v43n2/13f03.jpg
Slide 58-http://
www.studyread.com/wp-content/uploads/2016/09/Elisa-test-direct.jpg
Slide 60-https://www.cdc.gov/meningitis/lab-manual/images/chapt11-figure01.gif;
http://
www.lav-asoria.com/content/781927/ANTIBIOGRAMA.JPG
Slide 62-https://
www.cdc.gov/groupbstrep/images/lab-agglutinationtests-lg.jpg
Text source from the Wiki page generated
Slide70Thank you!