PATH 417 Case 4: One Too Many Hamburgers - PowerPoint Presentation

Slide1

PATH 417 Case 4: One Too Many Hamburgers

The Microbiology Laboratory

Summary

By: Sunny Chen

Slide2

Topic Overview

Potential bacterial candidates for this infectious scenario

Possible samples taken

Possible tests performed

Expected results for the potential bacterial candidates

Slide3

Topic Overview

Potential bacterial candidates for this infectious scenario

Possible samples taken

Possible tests performed

Expected results for the potential bacterial candidates

Slide4

Potential Bacterial Candidates

Possible disease:

gastroenteritis

P

ossible bacterial causes include:

Salmonella typhimurium(S. typhimurium)

Campylobacter

jejuni

(C.

jejuni

)

Escherichia coli

O157:H7

Shigella

Slide5

Potential 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

Slide6

Possible 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

Slide7

Potential 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

Slide8

Potential 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

Slide9

Potential 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

Slide10

Potential 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

Slide11

Potential 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

Slide12

Potential 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

Slide13

Potential Bacterial Candidate-

E

. coli O157:H7

Primary symptoms:

abdominal pains

blood and watery diarrhea

vomiting

mild fevers

Slide14

E. 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

Slide15

Potential 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

Slide16

Potential 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

Slide17

Potential 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)

Slide18

Potential 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

Slide19

Topic Overview

Potential bacterial candidates for this infectious scenario

Possible samples taken

Possible tests performed

Expected results for the potential bacterial candidates

Slide20

Possible Samples Taken

Stool

Blood

Urine

Details summarized in the table coming next

Slide21

Possible 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

Slide22

Possible 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

Slide23

Possible 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

Slide24

Importance 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

Slide25

Topic Overview

Potential bacterial candidates for this infectious scenario

Possible samples taken

Possible tests performed

Expected results for the potential bacterial candidates

Slide26

Possible 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

Slide27

Possible 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

Slide28

Possible 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

Slide29

Possible Tests Performed-MAC Agar

Slide30

Possible 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)

Slide31

Possible Tests Performed-Bismuth

Sulfite

Agar

Slide32

Possible 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

Slide33

Possible Tests Performed-Campy Blood Agar

Slide34

Possible 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

Slide35

Possible Tests Performed

—

SMAC or CT-SMAC

SMAC with

E. Coli

O157

Slide36

Possible 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)

Slide37

Possible Tests Performed

—

SS

Slide38

Side 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)

Slide39

Side 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)

Slide40

Side 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)

Slide41

Possible 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

Slide42

Possible 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

Slide43

Possible 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

Slide44

Possible 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

:

Slide45

Possible 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

Slide46

Possible 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

Slide47

Possible 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

Slide48

Possible 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

Slide49

Possible Tests

P

erformed—

Additional Tests

Gram staining

Slide50

Possible 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

Slide51

Possible 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

Slide52

Possible Tests

P

erformed—

Additional Tests

Oxidase Test

Slide53

Possible 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)

Slide54

Possible Tests

P

erformed—

Additional Tests

Catalase Test

Slide55

Possible 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

Slide56

Possible 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

Slide57

Possible 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

Slide58

Possible 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

Slide59

Possible 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

Slide60

Possible Tests

P

erformed—

Additional Tests

Antibiotic Sensitivity Test

Slide61

Possible 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

Slide62

Possible Tests

P

erformed—

Additional Tests

Agglutination Test

Slide63

Topic Overview

Potential bacterial candidates for this infectious scenario

Possible samples taken

Possible tests performed

Expected results for the potential bacterial candidates

Slide64

Expected 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

Slide65

Expected 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

Slide66

Expected 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

Slide67

Side 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

Slide68

Image 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

Slide69

Image 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

Slide70

Thank you!