I&I investigating infections 1 Flashcards
What is blood agar?
Blood agar is agar enriched with sheep or horse blood
What does blood agar allow?
Allos more fastidious organisms to grow
What is an example of a pathogen that grows very well in blood agar?
Chocolate agar is a chocolate-brown coloured agar created by heating blood agar
What does the heating process to make chocolate agar release and why may it be necessary?
The heating process releases lots of micronutrients from the blood, which may be necessary for certain organisms to grow
What pathogen grows well in chocolate agar?
Neisseria meningitidis
What are the steps involved in the microbial investigation of suspected bacterial meningitis?
-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
What is done with the CSF sample collected for suspected meningitis?
-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
How does CSF look like?
Gin clear
How does CSF look like in meningitis?
In meningitis, the CSF becomes cloudy due to a high WBC count and the presence of protein
What colour do gram negative bacteria stain?
Gram negative bacteria stain pink/red
What colour do gram positive bacteria stain?
Gram positive bacteria stain blue
What shape are cocci bacteria?
Cocci are round bacteria
What shape are diplococci bacteria?
Diplococci are round bacteria that exist in pair
What shapes are bacilli bacteria?
Bacilli are rod shaped bacteria
Why is microscopy alone not enough to identify gram-negative bacilli?
-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
Why must we take into consideration the source of sample when collecting a sample and what can we do?
-Site where sample is taken from may have large numbers of commensals
–Hence we use differential growth selection where we use differential growth media
How do you differentiate between commensals and commensals that become pathogenic?
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
Why is all positive gram stain not always indicative for disease?
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
What is alpha haemolytic bacteria and how does it appear on blood agar plate?
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
What is beta haemolytic bacteria and how does it appear on blood agar plate?
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
What does non-haemolytic bacteria release and how does it appear on blood agar?
Non-haemolytic bacteria do not release any substances that affect the blood agar
What may bacteria gain entry into in cases of bacterial meningitis and what can this cause?
In a proportion of cases of bacterial meningitis, the bacteria gain entry into the blood and cause bacteraemia and septicaemia
How are bloods cultured?
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)
How do blood culture bottles appear pre inoculation?
Pre-inoculation (before the blood sample is put in), the bottles have a clear appearance
What indicator is present at the bottom of blood culture bottles and what does this detect?
-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
How do we investigate the cause of the bacteraemia
-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
How does S. pneumoniae appear on a blood culture after its on an agar plate?
An example of this is S. pneumoniae, which is 𝝰-haemolytic and forms ‘draughtsman’ colonies, which are shaped like draughts pieces
How could we confirm the presence of s.pneumoniae and what would this show?
-To confirm the presence of S. pneumoniae, we could gram-stain the colony
-This would show large numbers of gram-positive diplococci
What further tests would we carry out to confirm the identity of an organism?
- Microbiological phenotype
- Biochemical tests
- MALDI-TOF-MS
- Immunological
How would we carry out biochemical tests to identify an organism?
We can use antibiotics or different agar plates to eliminate different species and narrow down our options
What is MALDI-TOF-MS?
a form of mass spectrometry used to identify different bacteria
What immunological methods would we use to confirm the identity of an organism?
we can use targeted Ab to tag species specific Ag
What does the bile solubility test for S.pneumoniae involve?
-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
What do we see with gut streptococcus when its bile solubility is tested?
With a gut streptococcus, we will not see this clear zone as the bacterium must be resistant to bile
What type of test do we carry out to distinguish between N.meningitidis and other gram-negative non-haemolytic diplococci?
To distinguish N. meningitidis from other gram-negative non-haemolytic diplococci, we must carry out an oxidase test
What does an oxidase test to identify N.meningitids involve and what does this test show if it is present?
-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
What are the steps involved in antibiotic testing?
-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
What do we measure when carrying out antibiotic sensitivity testing and how do we do this?
-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
How do we test for meningitis on a patient that has already been given IM or IV penicilin?
Ag detection test to look for dead bacteria
What does a PCR detect in Ag detection tests and what are the pros and cons?
detects bacteria specific DNA. It is very sensitive but complex and time consuming
What do latex agglutination tests involve in Ag detection tests? what is a pro?
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
What antibiotics may be used for the prophylaxis of meningococcal disease?
-Rifampacin. Given as 4 doses over 2 days
-Ciprofloxacin in a single dose
Why is penicillin not used for prophylaxis of meningococcal disease?
Penicillin is not used as it is the antibiotic of choice to treat the disease
What are the drawbacks of rifampacin?
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)
What is the policy for antibiotics for prophylaxis of meningococcal disease?
The policy is that there should be no overlap between drugs for prophylaxis and treatment
What are clusters(outbreaks) of disease defined as?
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
