ID: Streptococci and Enterococci Flashcards
What are the three classifications of streptococci
Alpha haemolytic
Beta haemolytic
Gamma haemolytic
What are the basic characteristics for streptococci
Could be alpha, beta or gamma haemolyisis
KOH negative
Catalase negative*
Oxidase negative
What tests would you carry out for streptococci?
(6)
Lancefield grouping (b) (g)
Bacitracin (b)
Optochin (a)
MacConkey (g)
Bile Aesculin (g)
Vancomycin (g)
What are the basic characteristic test results for enterococci?
(4)
Cocci*
Gram positive*
Aerobe*
Catalase negative *
What’s the most important step when identifying streptococci/enterococcus?
If you have a gram positive, aerobe, catalase negative cocci, you need to be able to identify haemolysis
Only certain tests carried out for each type of haemolysis
Gamma haemolysis usually means an enterococcus
What are the results of the preliminary ID tests for streptococci?
Cocci
Gram positive
Catalase negative
What are the five clinically significant strains of streptococci?
Strep pneumonia
Strep pyogenes
Strep viridans
Strep mutans
Strep agalactiae
How can you tell a strep is strep pneumoniae from how is appears on a blood agar plate and under a microscope
Alpha haemolysis
Draughtsman colonies or mucoid colonies
Lanceolate -> flame shaped cocci
What strep strains have alpha haemolysis
S pneumoniae (most likely)
S viridians
What strep strains have beta haemolysis
S pyogenes (most likely)
S agalactiae
What strep strains have gamma haemolysis
S. bovis however any gram positive cocci which are catalase negative and don’t have haemolysis are more than likely enterococcus than strep
What four tests are used to speciate streptococci?
Optochin susceptibility
Bacitracin susceptibility
MacConkey agar -> bile susceptibility
Lancefield grouping (classifies B-haemolytic strep)
What is the only test needed to speciate alpha-haemolytic strep; S. pneumoniae and S viridans
Optochin susceptibility test
What is the principle behind the optochin susceptibility test
(2)
This test detects an organism’s susceptibility to the chemical optochin
Optochin tests the fragility of the bacterial cell membrane and causes S. pneumoniae to lyse due to changes in surface tension, as shown by a zone of inhibition around the optochin disc
How do you carry out an optochin susceptibility test
(7)
Ask for blood agar, optochin disk x3, bijuo jar of sterile water and a swab x3
Divide a blood agar into three section, half for your test and two quarters for positive and negative control
Prepare a suspension of test organism and controls using a reduced volume of sterile water
Lawn inoculate your test and controls
Place one optochin disc in centre of test inoculum and the controls
Incubate overnight at 37 degrees
Examine for a zone of inhibitions
What is your positive control for optochin susceptibility, i.e which strep species is susceptible to optochin?
Streptococcus pneumoniae
What is your negative control for optochin susceptibility, i.e. which strep species is resistant to optochin
Streptococcus viridans
What three tests must be carried out for B-haemolytic strep
Lancefield grouping
Bacitracin susceptibility test
Ability to grow on MacConkey agar
What is the principle behind the lancefield grouping test
(4)
The majority of pathogenic strep possess specific carbohydrate antigens, which permit the classification of strep into groups
Strep group antigens can be extracted from bacterial cells and their presence demonstrated with latex particles previously coated with group-specific antibodies
These latex particles will agglutinate in the presence of homologous antigen, but will remain in smooth suspension if absent
The use of enzymatic extraction procedure considerably shortens the time required for antigen extraction and much improves the antigen yield
What is the Lancefield grouping test kit called
Oxoid Streptococcal Grouping Kit
How do you prepare your bacteria for the Lancefield agglutination test?
(4)
Dispense 0.4ml of Oxoid Streptococcus Extraction Enzyme into a labelled test tube
Select 2-5 test colonies and emulsify in the enzyme preparation
Incubate for 10 minutes at 37 degrees Celsius in a water bath -> make sure to shake vigorously at 5 mins in
Remove and allow to cool to room temp
How do you carry out the Lancefield grouping test
(6)
Ask for a reaction card, sticks, latex reagent A, B, C, D, F and G,
Ask for S. pyogenes or Enterococcus control?
Ask for (pre-mixed?) enzyme (and test) extract??? -> don’t know if this will be done for you previously or you will have to do it yourself
Add 1 drop of latex reagent A, B, C, D, F, G to a circle on your reaction card
Look for agglutination
Carry out this test using the positive control first so you know the reagents are working
What does a Lancefield group A indicate?
Streptococcus pyogenes
What does a Lancefield group B indicate?
Streptococcus agalactiae
What does Lancefield group C indicate?
Streptococcus equi (most probable)
Streptococcus epidemicus
Streptococcus equisimilis
What does Lancefield group D indicate?
Streptococcus bovis
Or the more probable Enterococcus -> E. faecalis
What does Lancefield group F indicate?
Streptococcus anginosus
What does Lancefield group G indicate?
Streptococcus canis
Streptococcus dysgalactiae
Which are the most significant Lancefield grouping?
(4)
Group A = Strep pyogenes
Group B = Strep agalactiae
Group C = Strep equi or strep equisimilis
Group D = Enterococcus faecalis or Strep bovis
What is the principle behind Bacitracin susceptibility
(2)
Used to speciate group A, Beta haemolytic strep i.e. to confirm S. pyogenes
Group A strep are sensitive to bacitracin while other groups of strep are resistant
How do you carry out a Bacitracin susceptibility test?
(6)
Ask for blood agar, x3 bijou jars full of sterile water, and x3 bacitracin discs
Divide a blood agar plate into three sections, one for test, two for controls
Prepare a suspension of test and controls using a reduced volume of sterile water
Lawn inoculum of test and controls
Place one bacitracin disc in the centre of each inoculum
Incubate overnight at 37 degrees
What is your positive control for bacitracin, which organism is susceptible for bacitracin?
Strep pyogenes is susceptible
What is your negative control for bacitracin, which organism is resistant for bacitracin?
Use an enterococcus e.g. E.Coli
What is the principle behind MacConkey agar?
(5)
A selective and differential culture medium
Crystal violet and bile salts make it selective for Gram-negative and enteric (normally found in the intestinal tract) bacteria
Differential based on lactose fermentation.
Lactose fermenters turn red or pink on MacConkey agar, and nonfermenters do not change color.
The media detects lactose fermentation by enteric bacteria with the pH indicator neutral red.
What is your positive control for MacConkey agar?
Enterococci e.g. E. faecalis
What is your negative control for MacConkey agar?
Streptococcus e.g. S. pyogenes
What is a positive result for MacConkey?
Growth
What is a negative result for MacConkey?
No growth
Which Lancefield group will grow on MacConkey
Group D -> Enterococcus
What should you do if none of your tests confirm species of Strep?
Say what it isnt
Say what it could be
Suggest biochemical testing
Which are the only Strep you can ID?
(4)
Strep pyogenes (Lancefield A)
Strep pneumoniae (positive optochin)
Strep viridans (negative optochin)
Strep agalactiae possibly -> if B-haemolytic strep but not Lancefield A
What tests do you carry out to speciate enterococcus?
(5)
MacConkey Agar (confirm enterococcus and not strep)
Lancefield (Group D confirms enterococcus and not strep)
Chromogenic VRE Medium
Bile Aesculin Agar
Vancomycin
What is the principle behind bile aesculin agar?
(4)
A selective and differential agar used to identify members of the Enterococcus genus
Bile salts are selective for Enterococcus
Aesculin is differential -> enterococcus hydrolyse aesculin to form aesculetin and dextrose
Aesculetin combines with ferric citrate in the medium to produce insoluble iron salts, resulting in the blackening of the medium which is a positive result
How should you carry out a bile susceptibility test?
Ask for a bile aesculin agar plate, E.Coli + control, Strep pyogenes negative control
Spot inoculate
Incubate at 45 degrees !!!!
What is a positive bile aesculin test?
Blackening of the medium
What are the three clinically significant species of Enterococcus
E. faecalis
E. faecium
E. durans
Explain how to use chromogenic VRE medium
This isn’t in the lab note???
Chromogenic Vancomycin Resistant Enterococcus medium
Specific and selective chromogenic medium for the detection and differentiation of Enterococcus faecium and E. faecalis showing acquired Vancomycin resistance (VRE
E. faecalis = bluish green
E. faecium = violet
What should you do if you ID an enterococcus?
Suggest biochemical testing
Suggest vancomycin susceptibility as some strains are resistant
Suggest chromogenic VRE Medium
