How Genome Sequencing Will Help Control Bacterial Infections Flashcards

Question

What does the molecular detection of M. tb entail

Answer 1

- Xpert MTB/RIF assays as the initial test for TB - It is automated, integrated, cartridge-based real-time PCR assay - detects M. tb and identifies rifampicin resistance directly from sputum through presence of rpoB gene - This is then probed with molecular beacons (special DNA probes that light up) to detect presence of M. tb DNA and rpoB mutations

Answer 2

This gene codes for the RNA polymerase beta subunit, a key target of rifampicin. Mutations in a specific region of rpoB are strongly associated with rifampicin resistance.

Answer 3

- Results in around 2 hours form a sample input to result - High sensitivity and specificity even in HIV co-infected individuals - Minimal lab infrastructure needed

Answer 4

Primarily detects rifampicin resistance, so additional tests may be needed to confirm MDR-TB or other drug resistances. More expensive than microscopy or culture.

Answer 5

1) Major time-saver for diagnosis and treatment decisions 2) High-resolution insight into drug resistance 3) Uncovers complex infections 4) Supports public health surveillance

Answer 6

Traditional diagnosis of M. tb, including drug susceptibility testing (AST), takes 1–2 months due to the slow growth rate of the bacterium. WGS dramatically shortens this time — results can be available in a few days after culture is positive (e.g., from a MGIT culture). Faster diagnosis = faster, more targeted treatment → crucial for preventing disease progression and transmission.

Answer 7

WGS identifies the exact mutations in the genome that confer resistance to antibiotics (e.g., mutations in rpoB, katG, gyrA, etc.). This is especially important for XDR-TB (resistant to at least isoniazid, rifampicin, a fluoroquinolone, and a second-line injectable).

Answer 8

WGS revealed that the patient had two distantly related Beijing strains — this can’t be detected with standard tests. This level of strain typing helps in outbreak investigations, tracking transmission chains, and understanding pathogen evolution.

Answer 9

Ultimate molecular resolution = highly accurate identification and typing of strains. Useful in global and local surveillance programs for TB control. Helps health systems detect emerging resistance patterns or hypervirulent strains.

Answer 10

Not all genetic resistance mechanisms are known. Some phenotypic resistance occurs without a known genetic mutation - So phenotypic AST is still needed to confirm susceptibility, especially for new drugs or unexplained resistance. MIC ambiguity Low genome coverage areas Dependence on known mutations

Answer 11

When MIC is near the therapeutic threshold, even WGS may not reliably say whether a strain is resistant or susceptible — this supports the earlier point about WGS needing AST confirmation.

Answer 12

These regions may hide important SNPs (mutations) that WGS can’t confidently call — again aligning with the earlier case where blaZ was missed due to short contigs.

Answer 13

WGS can only detect resistance if we already know the relevant genetic mutations — so it can’t identify novel or rare resistance mechanisms. This echoes the earlier limitation: “a query-based method cannot recognize novel variants.”

Answer 14

The Neonatal MRSA Outbreak Investigation

Answer 15

Infants are vulnerable to infection Before WGS, MRSA strains from the same general genetic lineage were hard to tell apart using traditional typing methods - which made it difficult to know if patients were part of the same outbreak or not

Answer 16

They used Whole Genome Sequencing (WGS) to analyze: 7 isolates from infants linked to the outbreak. 7 additional MRSA isolates from the same hospital that were not obviously linked to the outbreak They mapped the genome sequences of 10 of these isolates to a reference MRSA strain Then they performed phylogenetic analysis to look at how similar the isolates were.

Answer 17

Two genetically distinct groups were found: 1) Group 1 - Outbreak group - Contained all 7 outbreak-associated isolates that were all similar to each other, differing by very few SNPs, suggesting person-to-person transmission within the NICU 2) Group 2 - Non-outbreak group - These differed by at least 136 SNPs which means its too genetically distant to be part of the same transmission chain

Answer 18

WGS confirmed the outbreak cluster by showing the NICU-associated isolates were nearly identical. It ruled out transmission for the other 3 MRSA isolates, which traditional methods may not have been able to do.

Answer 19

Quickly identify the source of infection Guide infection control, such as decolonisation strategies Prevent unnecessary quarantines or treatments Lead to faster containment and fewer infections

Answer 20

WGS could be a routine part of hospital surveillance. Offers better patient care, reduced hospital stays, and lower healthcare costs. Helps distinguish between colonization and actual outbreaks — which traditional diagnostics often struggle with.

Answer 21

Gram-negative, versatile bacterium with high degree of genome plasticity (amenable to natural and random genetic alteration) Important member of the normal intestinal microflora of humans and other mammals Variants can be facultative or obligate pathogens Major health and economic burden and AMR

Answer 22

Six pathotypes cause enteric disease (diarrhoea or dysentery) and extra-intestinal diseases (UTIs and meningitis)

Answer 23

A large severe outbreak occurred in Germany in May-June 2011 where more than 4,000 people developed bloody diarrhoea and 850 people developed Hemolytic Uremic Syndrome - a life threatening kidney complication.

Answer 24

EHEC O104:H4 - a rare hybrid strain of E. coli It showed traits of enteroaggregative E. coli (EAEC) known for mild, persistent diarrhoea It also had gene for the Shiga toxin which causes the Hemolytic Uremic Syndrome (HUS) Carried ESBLs genes

Answer 25

Sequenced the outbreak strain and compare it to other E. coli and Shigella strains Analysed the core genomes to build a phylogenetic tree showing evolutionary relationships

Answer 26

The outbreak strain belonged to the EAEC lineage but recently acquired: - A shiga toxin phage: the genetic element that produces Shiga toxin - Antibiotic resistance plasmids such as TEM-1 and CTX-M

Answer 27

The strain was not a typical EHEC, nor a regular EAEC - it was a new hybrid: Enteroaggregative Hemorrhagic E. coli (EAHEC) It combined the adhesive properties of EAEC (making it "sticky") with the toxin production of EHEC (making it dangerous), and added antibiotic resistance - leading to a highly virulent, hard-to-treat strain.

Answer 28

WGS provided the resolution needed to: - Trace the origin and evolution of the outbreak strain. - Discover that the strain acquired Shiga toxin genes recently. - Clarify outbreak sources - Identify new pathotypes - Guide public health interventions - And improve preparedness for emerging hybrid pathogens

Answer 29

Focus: Within host evolution Case: Single patient colonised with methicillin-susceptible S. aureus Outcome: Transition from nasal colonisation to fata bacteraemia

Answer 30

8 mutations were identified between the colonising strains and the strain that caused fatal bacteraemia. They were protein truncating mutations (premature stop codons) in regulatory genes like rsp which increased intracellular persistence

Answer 31

WGS can detect subtle mutations over time, helping trace the genetic changes that transform a commensal strain into a lethal pathogen - information often missed by traditional methods

Answer 32

It demonstrates that even with minimal genetic variation within a host can result in a major outcomes, highlighting the importance WGS in diagnostics, infection prediction, and personalised medicine