Epidemiological links

Question 1

list the order of microbial taxonomy

Answer 1

1. domain- bacteria
2. kingdom
3. phylum- proteobacteria
4. class- gamma proteobacteria
5. order- enterobacteriales
6. family- enterobacteriacea
7. genus- escherichia
8. species- escherichia coli

Question 2

what is meant by microbial strain in a taxonomic sense

Answer 2

a strain is made up of the descendants of a single isolation in pure culture and is usually made up of a succession of cultures ultimately derived from an initial single colony

Question 3

what is the definition in nature of microbial strain

Answer 3

a strain is an isolate or group of isolates that can be distinguished from other isolates of the same genus and species by phenotypic characteristics or genotypic characteristics or both

Question 4

how can we differentiate microorganisms below species level

Answer 4

1. phenotypic typing methods
   - antibiotic resistance typing
   - phage typing (staphylococcus aureus)
   - serotyping (salmonella)
2. genetic typing methods- genotyping
   - electrophoretic methods (PCR fingerprinting)
   - sequence base typing (single gene sequencing)
   - other methods (SNPs and microsatillite analysis )

Question 5

what are phenotypic typing methods based on

Answer 5

based on analysis of phenotypic characteristics

Question 6

what are genetic typing methods based on

Answer 6

based on analysis of genetic characteristics

Question 7

what are the reagents needed in PCR

Answer 7

1. DNA template extracted from culture or sample
2. DNA polymerase
3. nucleotide bases (dNTPs)
4. primers flanking the region of interest
5. water
6. polymerase buffer

Question 8

what is PCR

Answer 8

exponential amplification of nucleic acids by a series of alternating heating and cooling steps

Question 9

what does PCR stand for

Answer 9

polymerase chain reaction

Question 10

describe the process of amplification of nucleic acids

Answer 10

1. analysis of amplified DNA fragments typically involves an electrophoresis in agarose or polyacrylamide gel
2. fragments are separated according to their sizes
3. gels are visualised under UV light

Question 11

give examples of application of genotyping methods

Answer 11

1. outbreak of health care associated Burkholderia bacteremia and infection attributed to contaminated sterile gel used for central line insertion
2. whole genome analysis of Exserohilum rostrum from an outbreak of fungal meningitis

Question 12

describe the outbreak of BCC

Answer 12

1. a group of gram - aerobic bacilli
2. patients with BCC are typically unwell, with multiple comorbidities, including being immunocompromised
3. common sources of infection are usually contaminated IV fluids, contaminated ventilation equipment or contaminated fomites
4. this outbreak is attributed to contaminated ultrasound gel
5. molecular typing of the isolates used several techniques including a MLST method

Question 13

what does MLST stand for

Answer 13

multi locus sequence typing

Question 14

what is MLST

Answer 14

an unambiguous procedure for characterising microbial isolates, using the sequences of internal fragments of usually 7 house keeping genes
- approx 450-500bp internal fragments of each gene are used, as these can be accurately sequenced on both strands using an automated DNA sequencer

Question 15

what occurs for each housekeeping gene in MLST

Answer 15

For each housekeeping gene, the different sequences present within a microbial species are assigned as distinct alleles

Question 16

what occurs for each microbial isolate in MLST

Answer 16

For each microbial isolate, the alleles at the end of each loci define the allelic profile or sequence type

Question 17

what is each isolate characterised by in MLST

Answer 17

characterised by a series of 7 numbers which correspond to the alleles at the 7 housekeeping loci

Question 18

what is the prevelance of the allelic profiles in MLST

Answer 18

1. most bacterial species have sufficient variation within housekeeping genes to provide many alleles per locus
   - allowing billions of distinct allelic profiles to be distinguished using 7 housekeeping loci
2. the allelic profiles of isolates can easily be compared to those in a large central database via the internet

Question 19

explain the link between MLST and the B.cenocepacia outbreak

Answer 19

1. MLST typing demonstrated that B.cenocepacia isolates from patients linked to the use of the ultrasound gel and an isolate grown from the gel were highly genetically related to each other
2. most isolates related to the infection shared the same MLST sequence type
3. the ultrasound gel was identified as the most likely source of the infection outbreak
4. within 24 hours of being notified, the therapeutic goods administration contacted the distributor and manufacturer, issued a notice of recall and classified the event as life threatening

Question 20

describe the outbreak of fungal meningitis

Answer 20

1. more than 13000 people were exposed to contaminated methylprednisolone and 750 developed fungal infections
2. vast majority of infections were caused by E.rostratum , a saprobic mould found in soil
3. WGST was used to investigate if the fungal strains identified from patients and lots of methylprednisolone were the same or different
4. 2 next generation sequencing platforms were used

Question 21

what does WGST stand for

Answer 21

whole genome sequence typing

Question 22

what did the whole genome analysis find in the outbreak of fungal meningitis

Answer 22

1. showed that most outbreak isolates had identical genomes
2. no differences found between strains isolated from patients and lots of methylprednisolone
3. data demonstrated that the outbreak strains were clonal and suggested a single common source
4. isolates from the 2 different methylprednisolone lots were indistinguishable suggesting that the source of contamination was persistent