I&I investigating infections 1

Question 1

What is blood agar?

Answer 1

Blood agar is agar enriched with sheep or horse blood

Question 2

What does blood agar allow?

Answer 2

Allos more fastidious organisms to grow

Question 3

What is an example of a pathogen that grows very well in blood agar?

Answer 3

Chocolate agar is a chocolate-brown coloured agar created by heating blood agar

Question 4

What does the heating process to make chocolate agar release and why may it be necessary?

Answer 4

The heating process releases lots of micronutrients from the blood, which may be necessary for certain organisms to grow

Question 5

What pathogen grows well in chocolate agar?

Answer 5

Neisseria meningitidis

Question 6

What are the steps involved in the microbial investigation of suspected bacterial meningitis?

Answer 6

-With suspected bacterial meningitis, we’d take a blood sample, CSF sample and a throat swab
-We’d then carry out several tests on each sample

Question 7

What is done with the CSF sample collected for suspected meningitis?

Answer 7

-With the CSF, we’d start by measuring protein and glucose levels
-We’d then smear it on a glass slide and look for the presence of any bacterial cells (remember CSF is a sterile body site, so no cells should be present)
-Gram staining is the next step to help us identify any bacteria
-We’d also do a CSF WBC count to see whether leukocytes are infiltrating the CSF to try and fight an infection

Question 8

How does CSF look like?

Answer 8

Gin clear

Question 9

How does CSF look like in meningitis?

Answer 9

In meningitis, the CSF becomes cloudy due to a high WBC count and the presence of protein

Question 10

What colour do gram negative bacteria stain?

Answer 10

Gram negative bacteria stain pink/red

Question 11

What colour do gram positive bacteria stain?

Answer 11

Gram positive bacteria stain blue

Question 12

What shape are cocci bacteria?

Answer 12

Cocci are round bacteria

Question 13

What shape are diplococci bacteria?

Answer 13

Diplococci are round bacteria that exist in pair

Question 14

What shapes are bacilli bacteria?

Answer 14

Bacilli are rod shaped bacteria

Question 15

Why is microscopy alone not enough to identify gram-negative bacilli?

Answer 15

-It’s worth noting that all gram-negative bacilli (e.g. E. coli) appear the same, so microscopy alone can’t identify them
-Further tests are required to definitively identify a gram-negative bacillus

Question 16

Why must we take into consideration the source of sample when collecting a sample and what can we do?

Answer 16

-Site where sample is taken from may have large numbers of commensals
–Hence we use differential growth selection where we use differential growth media

Question 17

How do you differentiate between commensals and commensals that become pathogenic?

Answer 17

A key example of this is neutrophils. If they surround a bacterium that is usually considered a commensal, chances are it is pathogenic in that particular case

Question 18

Why is all positive gram stain not always indicative for disease?

Answer 18

This is another key consideration because a positive gram stain will not always be diagnostic for disease, as the pneumococcus may just be part of the normal flora of that body site

Question 19

What is alpha haemolytic bacteria and how does it appear on blood agar plate?

Answer 19

𝝰-haemolytic bacteria (e.g. S. pneumoniae) release molecules that oxidise the Hb in RBCs, therefore on a blood agar plate they are surrounded by a green/brown-tinged ring

Question 20

What is beta haemolytic bacteria and how does it appear on blood agar plate?

Answer 20

β-haemolytic bacteria (e.g. S. pyogenes) release a haemolysin toxin, which causes complete lysis of the blood agar, leading to the presence of clear zones around the colonies

Question 21

What does non-haemolytic bacteria release and how does it appear on blood agar?

Answer 21

Non-haemolytic bacteria do not release any substances that affect the blood agar

Question 22

What may bacteria gain entry into in cases of bacterial meningitis and what can this cause?

Answer 22

In a proportion of cases of bacterial meningitis, the bacteria gain entry into the blood and cause bacteraemia and septicaemia

Question 23

How are bloods cultured?

Answer 23

It is cultured both aerobically (in bottles with silver tops) and anaerobically (in bottles with purple tops) to ensure all possible bacteria may be detected (i.e. aerobes and anaerobes)

Question 24

How do blood culture bottles appear pre inoculation?

Answer 24

Pre-inoculation (before the blood sample is put in), the bottles have a clear appearance

Question 25

What indicator is present at the bottom of blood culture bottles and what does this detect?

Answer 25

-On the bottom of the bottle, there is an indicator that detects CO2 production
-If something grows after inoculation with blood, it will respire and CO2 will be produced, changing the colour of the indicator

Question 26

How do we investigate the cause of the bacteraemia

Answer 26

-Once we know we have cultured something from a blood sample, we will take some of the sample and inoculate agar plates with it
-We then culture them overnight (for ~18 hours) and look at the colonies present
-Sometimes, we can use the morphology of the colony to identify the bacterium

Question 27

How does S. pneumoniae appear on a blood culture after its on an agar plate?

Answer 27

An example of this is S. pneumoniae, which is 𝝰-haemolytic and forms ‘draughtsman’ colonies, which are shaped like draughts pieces

Question 28

How could we confirm the presence of s.pneumoniae and what would this show?

Answer 28

-To confirm the presence of S. pneumoniae, we could gram-stain the colony
-This would show large numbers of gram-positive diplococci

Question 29

What further tests would we carry out to confirm the identity of an organism?

Answer 29

1. Microbiological phenotype
2. Biochemical tests
3. MALDI-TOF-MS
4. Immunological

Question 30

How would we carry out biochemical tests to identify an organism?

Answer 30

We can use antibiotics or different agar plates to eliminate different species and narrow down our options

Question 31

What is MALDI-TOF-MS?

Answer 31

a form of mass spectrometry used to identify different bacteria

Question 32

What immunological methods would we use to confirm the identity of an organism?

Answer 32

we can use targeted Ab to tag species specific Ag

Question 33

What does the bile solubility test for S.pneumoniae involve?

Answer 33

-We use an optochin disc, which contains optochin – a substitute for the bile salt deoxycholate
-With S. pneumonia, we will see a clear zone of lysis around the disc

Question 34

What do we see with gut streptococcus when its bile solubility is tested?

Answer 34

With a gut streptococcus, we will not see this clear zone as the bacterium must be resistant to bile

Question 35

What type of test do we carry out to distinguish between N.meningitidis and other gram-negative non-haemolytic diplococci?

Answer 35

To distinguish N. meningitidis from other gram-negative non-haemolytic diplococci, we must carry out an oxidase test

Question 36

What does an oxidase test to identify N.meningitids involve and what does this test show if it is present?

Answer 36

-This involves squirting an oxidase reagent (tetramethyl -phenylenediamine HCl) onto to colonies
-If the colonies are N. meningitidis, the regent will turn blue due to the presence of oxidase

Question 37

What are the steps involved in antibiotic testing?

Answer 37

-We start by plating the organism that we cultured onto an antibiotic susceptibility test
-On the agar, we have discs that contain different antibiotics
-The antibiotic diffuses from the disc into the agar
-If the bacterium is sensitive to the antibiotic, it will be killed and therefore won’t grow in that area
-This leads to the appearance of zones of inhibition around each effective disc

Question 38

What do we measure when carrying out antibiotic sensitivity testing and how do we do this?

Answer 38

-When carrying out antibiotic sensitivity testing, we measure the MIC (minimum inhibitory concentration) of a drug against a particular organism
-To do this, we use specialised strips that contain a gradient of antibiotics

Question 39

How do we test for meningitis on a patient that has already been given IM or IV penicilin?

Answer 39

Ag detection test to look for dead bacteria

Question 40

What does a PCR detect in Ag detection tests and what are the pros and cons?

Answer 40

detects bacteria specific DNA. It is very sensitive but complex and time consuming

Question 41

What do latex agglutination tests involve in Ag detection tests? what is a pro?

Answer 41

involve using a panel of specific Ab attached to latex particles to agglutinate the bacterial Ag and latex particles together, giving a visible result.
-Very rapid

Question 42

What antibiotics may be used for the prophylaxis of meningococcal disease?

Answer 42

-Rifampacin. Given as 4 doses over 2 days
-Ciprofloxacin in a single dose

Question 43

Why is penicillin not used for prophylaxis of meningococcal disease?

Answer 43

Penicillin is not used as it is the antibiotic of choice to treat the disease

Question 44

What are the drawbacks of rifampacin?

Answer 44

The drawbacks of rifampicin are that it drives resistance, interacts with oral contraceptives, it overlaps with TB and has limited availability (in which case ceftriaxone can be used)

Question 45

What is the policy for antibiotics for prophylaxis of meningococcal disease?

Answer 45

The policy is that there should be no overlap between drugs for prophylaxis and treatment

Question 46

What are clusters(outbreaks) of disease defined as?

Answer 46

This is defined as being two confirmed cases with the same serogroup (e.g. N. meningitidis group B), within a defined group (e.g. school, college, work place), arising within a four week period