Bacteriology lab Flashcards
What is meant by best-guess diagnosis
The initial diagnosis of infection is based on the principles of making an informed best-guess clinical diagnosis.
Present illness: date of onset, symptoms, signs (esp. rashes)
Past history: previous infections particularly with resistant organism e.g. MRSA, hospitalizations, travel, antimicrobial use.
Use these to inform what you want the lab to look for- important when taking samples from non-sterile sites
State some microbiological possibilities for Billy’s case
Infectious Diarrhoea Endocarditis (SBE) -sub-acute bacteria Syphilis - STI causing skin rash Toxoplasma- lymphadenopathy and reactivation in immunocompromised Tuberculosis Brucellosis - unpasteurised milk Melliodosis
State the common diagnostic techniques used in microbiology
Culture i) Sterile sites (blood/CSF) ii) Non sterile sites
Serology
Molecular Techniques
Antimicrobial Susceptibility Testing
Why is culture still used over molecular methods
Culture still used – to analyse sensitivity
Molecular methods- need to know mechanism of resistance – but don’t need to know for cultures
Whole genome sequencing- to correlate with phenotype
Describe the difference between sterile and non-sterile sites
Sterile- should be no bacteria- bone- csf, blood
Non sterile- commensal bacteria
Non-sterile- only report what may be relevant from clinical details and travel history- look for your differential
Describe serology
Serology- look for anitbodies in immunitry
Evidence of infection- when organisms are difficult to grow or molecular tests not sensitive enough- i.e syphilis
Describe how skin is not a sterile site
Rosebury estimates that 50 million individual bacteria live on the average square centimeter (5x107/cm2) of human skin
[5x107/cm2 x 20,000 cm2/person = 1011 bacteria]
He describes the skin surface of our bodies as akin to a “teeming population of people going Christmas shopping.”
What bacteria is commensal in the nasopharynx
Streptococci
What’s important to remember about microbial susceptibility testing
- Antimicrobial susceptibility testing – used to test AB resistance but takes a long time
Describe the conditions that blood cultures are kept in.
Place into plastic bottles with growth medium
Machine agitates bottle (to ensure homogeneous spread of nutrients), warm it- 37 degrees- to increase growth
Rich broth and favourable conditions promote the growth of bacteria
Describe how a positive result is detected by the machine
Aerobic and anaerobic
Paediatric blood culture- small amounts of blood- strep
Indicator- bacteria produce toxic products- which will change colour- machine detects colour change
Beads to absorb antibiotics- to maxmise growth
What should be done once a positive blood result is flagged
Flag positive after 16-20 hours- then take from machine – sesntivity test
Agar plates- susceptibility testing
Antibotics can give false negatives- no growth- so place pus there – patient had no antibiotics- no growth
Send samples before you start patient on antibiotics
As you spred out- diluted antibiotic– so you get growth
You should then go gram-testing
Summarise the key features of gram negative and gram positive bacteria
Gram +ve = thick wall, purple stain, retains dye.
Gram –ve = thin wall, pink stain, loses dye and outer LPS layer
Many AB target the cell wall but may not be able to get past the outer membrane in gram- bacteria – e.g. vancomycin works only on +ve.
o Gram POSITIVE = skin and soft tissue.
o Gram NEGATIVE = abdomen and urinary tract
What are two components of the bacterial cell wall
NAM=N-acetylglucosamine NAG=N-acetylmuramic acid
Summarise staphylococci
Gram+ cocci are the most common bacterium.
Staphylococci often form clumps and look like bunches of grapes as they bud divide.
You can do a coagulase test to test between coagulase± staphylococci:
o Coagulase + = Staphylococci aureus.
o Coagulase - = Common skin microbes.
Prosthetic materials are prone to biofilm formation by staph. Infections which can be very bad – e.g. in hip replacements.
Describe the morphology of gram + cocci
Gram positive- retain crystal violent niadine gets trapped- deep purple
Shape- identification
Clumps- staphylococci- grapes
Name two antibiotics that can be used to treat staphylococcus
Methicillin or vancomycin
What is the purpose of the coagulase test
Two groups- coagulase test (but other ways)
Shows ability to form a clot in horse plasma
Lots of coagulase negative in skin- so blood cultures- will see gram positive cocci which are coagulase negative- so test differentiates between virulent can commensal/contaminant staph cooci
Compare the virulence of coagulase positive and negative Staphylococci
Staphylococcus aureus (including Methicillin Resistant Staphylococcus aureus, MRSA)
Severe infections eg: skin/soft tissue, endocarditis, osteomyelitis
Coagulase Negative Staphylococci
Skin commensals of low pathogenic potential. Can infect prosthetic material causing line, pacemaker infections and endocarditis
How can we kill coagulase negative staphylococci when takin blood sample
Let alcohol dry to kill commensal stapy cocci
Summarise streptococci
Streptococci generally form chains in the gram stain.
On blood agar, streptococci separate into two groups:
o Alpha haemolysis – incomplete haemolysis, turns green.
E.G. Streptococcus pneumoniae.
o Beta haemolysis – complete haemolysis, clears agar.
E.G. Group A – Streptococcus pyogenes – skin/soft tissue infection
E.G. Group B – Streptococcus agalactiae – sepsis in the young
Strep – sensisitve to penicllins and cephalosporins
What are two likely causes of gram positive cocci in chains
Divide into chains- will be enterococci or streptococic
Summarise the different types of haemolytic streptococci
Beta haemolysis (alpha,beta and gamma)
Group A beta haemoltuc strep –step pyogenes –sore throats- can also cause necrotisisng fasciitis – rheumatic fever and glomerularnephritis
Group b beta– step agalactiae- neonatal sepsis
C and G- less virulent than group A- cellulitis.
Alpha haemollysis – pneumococcus, strep pneumoniae
Summarise gram negative bacilli
Rods
More resistant than gram positive
Amoxiciloin resistan, gentamycin, aminoglycosides and quinolones
Do not take up gram stain and thus appear pink.
E.G. E. coli.
Summarise the different causes of diarrhoea
Bacteria:
Salmonella (inc S. typhi ), Shigella, Campylobacter,
E coli O157, C difficile, Cholera
Parasites:
Amoeba, Giardia, Cryptosporidium
Viruses
What is important to remember about diarrhoea
Camplyobacter- food poisoning – most common
C.diff- associated with antibotics
D and V in community- look for norovirus
No vomititng from bacteria in food poisoning
Look at report- they will rule out what hasn’t been isolated
Summarise the different investigations available for stool samples
Bacteria
Culture on agar plates.
Only Salmonella, Shigella and Campylobacter
looked for routinely.
Different pathogens have different culture
requirements.
Clostridium difficile – toxin detection or PCR for
toxin gene
Parasites
Concentration, special stains
Don’t grow parasites- different stains for different parasites
What is important to remember about stool samples
Food poisoning- self-limiting- so identifaction more important – use PCR to screen- if positive then do sensitivity
Selective agar plates- not a rich broth- trying to suppress growth- will have anbtibiotics and not all the nutrients- only want to grow pahtognes- grow at 42- campylobacter can grow- but kills everything else
Look for E.coli O157 too
Different pathogens- different culture environments
c.diff- difficult to growth- commensal in child gut — most labs don’t try and grow- so use toxin detection or molecular tests
Describe the agar for salmonella
Xylose lysine deoxycholate agar
Salmonella- hydrogen sulfide on agar- black circles- doens;t ferment xyklose – turn red- whereas most other bacteria turn yellow
Describe the agar for campylobacter
Specific growth requirements- grey watery colour
Don’t grow fast (48 hours)
Can tolerate high temps - so heat to 42 degrees to kill other bacteria
Describe the agar for Vibrio Cholerae
TCBS: vibrio cholerae turn it green
Summarise the other agar plates
Agar plates: non-selective can be used for blood, as should be sterile apart from bacteria from infection
Chocolate agar: cooked blood - releases nutrients to allow certain bacteria grow e.g. H. Influenzae
Macconkey agar: grows gram negative organisms
Blood agar: streptococci can be distinguished depending on whether perform alpha or beta haemolysis
Alpha: incomplete - turn agar green; includes Strep. Pneumoniae (pneumonia/meningitis)
Beta: complete - clears the agar; includes Strep. Pyogenes (skin and soft tissue infection) and Strep. Agalactiae (neonatal sepsis)
Summarise PPV
High rate of c.diff- PHE will investigate quality of hiospital
Pre-test probability – specificity and sensitivity
Only send tests with reasonable possibility
If result is positive- what does this mean for patient –PPV- depends on pre test possibility (prevalence)
Why we don’t send tests
Lower pre-test possibility- greater rate of false positivr– PPV will be reduced
Describe what is meant by PPV
PPV: if patient is unlikely to have a disease, and you test for it, you lower the PPV; equally, if highly likely then the more likely a positive test represents true positive
So as prevalence increases, sensitivity increases (greater percentage of true positives)
NPV (specificity) will decrease as prevalence increases- more likely to be a false negative
Describe the guidelines for sensitivity testing
British Society for Antimicrobial Chemotherapy method.
EUCAST in Europe
MIC (Minimum Inhibitory Concentration) = the lowest amount of AB required to inhibit growth of bacteria in vitro.
MIC isn’t very useful on its own so we set breakpoints which correlate MIC with clinical success as an AB.
o A bacterium with an MIC below the breakpoint means there is a good chance of success with that AB.
o A bacterium with an MIC above the breakpoint is RESISTANT.
Breakpoint = a chosen concentration of AB- based on clinical outcome- depends on pharmacodynamics, pharmacokinetics etc
Describe disc diffusion
Use a set concentration of AB in each disc and incubate for 24 hours.
The zone size is interpreted using he breakpoints in a AB table.
o Measure zone diameter to determine whether a bacterium is resistant to a AB or not.
Good, as you can test resistance to multiple antibiotics at the same time
Describe gradient MICs
Grow bacteria with antibiotic- place gradient strip
where clear zone ends on strip -determines the MIC
Summarise the potential causes of billy’s diarrhoea
Infectious diarrhoea does not usually persist
for 4 months
Don’t usually get systemic disease (but can
with typhoid, amoebic abscess)
Intestinal helminths don’t cause significant
diarrhoea (but could cause rashes, fever,
eosinophilia)
Long-Lasting diarrhoea- think of Chron’s
Describe the nature of the bacteraemia in SBE
SBE- multiple blood cultures- pulse of bacteraemia- before febrile- take blood
SBE- continues bacteriaema – endovascular source- to capture evidence of consistent bacteraemia
What are the key features of SBE
Rash
Fever
maybe weight loss
blood culture- consistent bacteraemia
What are the features of syphillis and toxoplasma
Rash
Fever
Lymph node involvement
Serum for antibodies- hard to grow
Describe the key features of TB
Fever
Weight Loss
Culture IFN-y - host response
Describe Brucellosis
Maybe rash
Lymph nodes
Fever
Serum for antibodies and blood culture
What is the effect of culture on sensitivity
Cultutere- enrichment so more sensitive
What is important to remember about sepsis
Don’t delay antibiotics whilst you wait for test results
Describe seroconversion
Convalescent sample 10-14 days later- negative acute- positive conalaescent sample- due to seroconversion- shows acute disease- makes it less clinically useful- because of time lag- need to treat patient.
Acute phase -IgM
Secondary exposure - IgG
So, if you see negative IgG on first sample, but positive on convalescent sample- had and acquired immunity to infection.
What is important to remember about microbiology
Always include clinical information including travel history on requests.
Contact Infectious Diseases or Microbiology early if you require advice. Try and send samples for culture prior to starting antibiotics if possible.
What was done next for Billy
What tests now?
GP does skin punch biopsy
Summarise what we look for in non-sterile sites
Non-sterile sites: specific pathogens only
Diarrhoea: stool sample looking for Salmonella, Shigella, Campylobacter, E. Coli, C. Diff., cholera rather than all bacteria
Summarise the typical investigations in a microbiology lab
Most microbiology samples are cultured on agar plates, which takes time: For organisms to multiply sufficiently usually requires 24-48 hours (some need longer incubation: e.g. TB, brucella, actinomycetes)
To culture again for antibiotic sensitivities: another 24 hours
Microscopy direct under the microscope – urine various stains (Gram, Ziehl-Nielsen (ZN), etc.) – pus, tissue fluids fluorescence, with conjugated antibodies to specific antigens
Direct antigen detection (particle agglutination tests, ELISA)
Molecular probes and amplification (PCR, etc.)
Serology: looking for antibodies as evidence of infection/immunity
Describe the optimal time for the collection of specimens
In the acute phase of illness and before staring antimicrobials
Collection from proper site, avoiding contamination by normal flora
Prompt transport to lab since micro-organisms multiply in transit
Adequate quantity and appropriate number of specimens
Acute sera and Convalescent sera (paired), for rising antibody titres
Summarise the microbiological examination of urine
Bedside: Naked eye – clear, cloudy, haemorrhagic. note: although these are NOT microbiological investigations dipstick tests for nitrites, leucocytes, blood, protein, bilirubin, ketones may provide indication of there being an infection. Nitrites strongly suggest bacteriuria as many species of gramnegative bacteria convert nitrates to nitrites.
Microscopy: WBC (pyuria suggests infection), RBC (may also indicate tumour/microemboli/trauma), epithelial cells (suggest the specimen has been contaminated during collection), crystals, casts.
Culture on MacConkey agar (urine should be sterile so any microbial growth is potentially significant in an appropriately taken sample)
Quantitative colony count for “significant” bacteruria (>105 bacteria/ml)
Antibiotic sensitivity testing of bacteria that grow
Summarise the microbiological examination of feaces
Naked eye, consistency, blood stained, colour, presence of worms
Microscopy: ova, cysts, parasites
Culture on inhibitory media – e.g. deoxycholatecitrate agar (DCA), selenite (Faeces contains 1012-14 bacteria per gram, so selective media are used to suppress background ‘flora’ organisms)
Certain organisms such as Vibrio cholerae are not looked for routinely therefore it is important adequate clinical information is provided on the request to allow the appropriate laboratory investigations to be carried out.
Toxin detection (Clostridium difficile)
Special stains, e.g. for cryptosporidia
Describe some useful stains
Gram stain of CSF, joint fluid, purulent exudates
ZN/auramine stain of e.g. sputum, for TB
FTA (fluorescent treponemal antibody) for antibodies to T. pallidum
Describe direct antigen detection
Meningococcal antigen in CSF
C. difficile toxin in faeces
Legionella and Pneumococcal antigen in urine
Describe PCR
Chlamydia in genital specimens
Rapid PCR for MRSA
Summarise the results for Billy
Microscopy of stool for parasites, especially giardia, amoeba (which cause diarrhoea but not rashes) and for higher parasites (may cause rashes but rarely diarrhoea). Stool culture for the common bacterial pathogens – salmonella, campylobacter, shigella. Stool result often negative.
Rash/skin lumps. Often viral aetiology. Also, infected insect bites, syphilis, gonococcal infection, typhoid, endocarditis, systemic parasites. ? Take skin biopsy – prolonged cultures for TB, fungi.
