16. Genome sequencing to control bacterial infections Flashcards
What is the current method of bacterial infection diagnosis?
Culture and observation based methods
How does culture based diagnosis of bacterial infection work?
- Collect a patient sample: Blood or urine.
- Grow the sample on selective media to select for specific bacteria.
- Then do phenotypic analysis using gram staining or more likely MALDI TOF.
- The bacteria is then identified.
- Then do antibiotic susceptibility testing on the bacteria which can take weeks.
How long does culture based diagnosis of bacterial infections take?
- 48-72 hours.
- Long for slower growing bacteria
What can be added onto the end of culture-based diagnosis of bacterial infections?
- Molecular typing of the bacteria
- This is done for surveillance or tracking in outbreaks.
What is AST?
Antibiotic susceptibility testing
How could Whole Genome Sequencing be used to diagnose bacterial infections?
What databases could WGS be compared to for bacterial diagnosis?
- Databases that contain all previously sequenced bacteria.
- They can contain sequence metadata.
- They can also compare the genome to all known AMR genes
What are the advantages of using sequence databases in WGS for diagnosis of bacterial infections?
- The databases and information is shared nationally and internationally.
- This is important for molecular tracking.
- This includes tracking outbreaks like COVID-19.
What information can WGS for bacterial diagnosis provide?
- Identity of the species
- The relatedness of the strains/infections.
- The resistance genes present
- The virulence genes present
Why is knowing the relatedness of infections important?
- It is important for outbreak control and molecular tracking.
Why is knowing the presence of specific virulence factors important in infection?
- They can determine the outcome of infection.
- They can determine what treatment is given.
- Eg TSST in S. aureus (only present in about 4% of isolates)
What are the benefits of using WGS in bacterial diagnosis?
- WGS comparisons of bacteria have the highest possible discriminatory power.
- WGS can be used to show the presence of AMR genes and pathogenicity genes.
- Metagenomic next-generation sequencing allows for culture-free identification of a range of pathogens in mixed samples.
Why does WGS have a very high discriminatory power?
- It is directly comparing the genome.
- This means it can detect differences of 1 nucleotide.
- Different SNPs and what they mean can be identified.
How can WGS be used to detect the presence of AMR or pathogenicity genes?
- By using targeted Next-Generation Sequencing.
- It uses data from whole genome sequencing and the antibiotic resistome of a bacterial pathogen.
- This is fed into a machine learning algorithm.
- This can then predict the antimicrobial susceptibility of a pathogen on par with routine culture-based approaches.
What can metagenomic next-generation sequencing be used to do?
- Culture free identification of a range of pathogens in complex polymicrobial samples.
- These samples could be clinical or environmental.
- It is hypothesis free so it is searching for any and all possible pathogens.
- It could be useful for detecting rare pathogens or pathogens that are difficult to culture.
What are the benefits of WGS compared to culture based methods for bacterial diagnosis?
- WGS is much quicker.
- It is more accurate
- It provides more information about the pathogen.
What is the potential steps in WGS sequencing for bacterial diagnosis?
- Extract the DNA and sequence.
- Bioinformatic analysis
- AMR gene detection
- Give the correct antibiotics
- Use to monitor outbreaks
What is Staphylococcus aureus?
- A gram-positive opportunistic pathogen.
- Causes a huge health and economic burden.
- Multidrug resistant
- Colonises the human nasopharynx and the intestine.
- Colonisation is a risk factor for disease.
- The 1st S. aureus genome sequenced in 2001. It took 5 years and millions of dollars
How was WGS tested experimentally for AMR detection in S. aureus?
- 501 S. aureus isolates were sequenced.
- Then interrogated using BLASTn to find known resistance genes within the sequence.
- Resistance to 12 commonly used antimicrobial agents carried on either the chromosome or plasmid.
- These results were then compared to the results of traditional culture-based AST.
How accurate is WGS for S. aureus AMR detection experimentally?
- 87% of the genome sequenced prediction were accurate to the AST results.
- This is good however it needs to be optimised for the incorrect 13%.
How was the WGS for S. aureus AMR detection improved during the study?
- For the 13% of isolates that didn’t match the AST they were tested again.
- The assays were repeated and their genome sequences manually gone through.
- The biggest error was that WGS predicted susceptibility to penicillin, but AST showed it was resistant.
Why is low coverage a problem when using WGS for AME detection?
- It can show isolates as susceptible to penicillin or others when they are resistant.
- The S. aureus isolates were reported as susceptible as no BlaZ gene was detected in bioinformatic analysis.
- This was because the BlaZ gene was missed due to low coverage and its small sequence contigs.
- If you have low coverage of a gene, you cannot be sure of its identity and confirm its presence/
- This then requires resequencing or manually going through the sequences to confirm the presence of the gene.
What does low coverage mean?
- In optimal sequences each ~1000bp will be sequenced 30-50 times so you can be sure what the bases/gene you think it is.
- For low coverage sequences you get like 5-10 reads for that gene.
- This means you cannot be sure what that gene is.
- In S. aureus areas of low coverage have higher CG content.
What was the result of the optimisation of WGS for S. aureus AMR detection?
- The overall sensitivity was 97%.
- The overall specificity was 99%.
- Both compared to standard AST.
- It is as good as AST.
What are the limitations of using WGS for AMR detection?
- Unknown and unidentified resistance mechanisms can cause errors as bioinformatic databases are only as good as the information you give it.
- As in silico resistance typing is a query based method, it cannot identify novel resistance mechanisms when traditional AST can.
- Any novel resistance mechanisms need to be functionally validated using culture based methods.
What is Mycobacterium tuberculosis?
- M. tb is a Mycobacteriaceae, acid-fast bacilli.
- It has a unique and complex cell envelope.
- It causes Tuberculosis which is a chronic bacterial infection.
- It causes more deaths worldwide than any other disease.
- Approx 2 billion people worldwide are infected with M. tb but most people don’t develop active TB.
How is M. tuberculosis transmitted?
- It is very transmissible.
- It is transmitted in aerosol droplets which can infect the lungs and other organs.
What is the unique composition of the M. tuberculosis cell envelope?
- The core contains peptidoglycan, arabinogalactan and mycolic acid layers.
- It also has a waxy outer membrane.
- This makes M. tb hard to treat.
- It is also associated with the slow growth of M. tb.
What infection is tuberculosis associated with?
- It is associated with people living with a weakened immune system particularly AIDS.
- WHO estimates 8.7 million new cases of TB and 1.5 million deaths due to TB in 2014.
- 430,000 of these deaths occurred in patients co-infected with HIV.
What is active TB?
- The active infection of M. tb
- 75% is pulmonary TB.
- Symptoms include chest pain, bloody cough, prolonged cough
- Can be fatal without treatment.
- Can develop granulomas.
Why is tuberculosis treatment difficult?
- It is a very slowing growing bacteria this makes it difficult to diagnose and treat.
- Treatment takes 6-12 months and requires multiple drugs to be effective.
- Luckily this treatment has a 90% cure rate if followed properly.
- Compliance with this treatment can be low due to its length.
- Drug resistance is also a problem
Why is resistance a problem in tuberculosis?
- New strains of M. tb have displayed resistance to all antibiotics.
- Extensively drug resistance tuberculosis (XDR-TB) are resistant to rifampicin, isoniazid, at least 1 fluoroquinolone and 1 3rd line drug like linezolid.
What is driving the spread of resistance in M. tb?
- Weak healthcare systems
- Poor antibiotic stewardship
- Incorrect treatment which amplifies resistance
- Poor education surrounding transmission and treatment.
What are some examples of 1st line treatments for tuberculosis?
- Rifamycins
- Rifampin
- Isoniazid
- Ethambutol
- Streptomycin
What are some examples of 2nd line treatments for tuberculosis?
- Fluoroquinolones.
- Aminoglycosides.
- Ethionamide
What are some examples of 3rd line treatments for tuberculosis?
- Linezolid
- Amoxicillin-clavulanate
- Meropenem
What are the traditional methods used to diagnose M. tuberculosis?
- LJ slants and Ziehl-Neelsen staining
- Mycobacterial growth incubator tube
- IFNy release assays
Diagnosis of M. tb: LJ slants and Ziehl-Neelsen staining
- Tb colonies are grown on Lowenstein - Jensen media on a slant.
- This cultured for 2 weeks at 37 degrees.
- Ziehl-Neelsen staining stains the outer envelope of TB.
- It can take upto 2-3 weeks to see colonies then more to do antibiotic susceptibility testing.
- It is time consuming.
- It cannot detect low bacterial load of TB infection.
Diagnosis of M. tb: Mycobacterial growth incubator tube (MGIT)
- It is a culture system that monitors oxygen production.
- Growth of TB = fluorescence
- This is expensive
- However contamination is common and can massively alter results and fluoresce is non-specific.
Diagnosis of M. tb: IFNy release assays
- Collect blood from potential TB patients.
- T cell stimulation with specific TB antigens
- Measure IFNy release in response to these antigens.
What are the limitations of IFNy release assays?
- It is expensive
- BCG vaccinated patients can produce false positives.
- It produces cross-reactivity positives with non-tuberculosis mycobacteria.
- It cannot differentiate between active or latent TB.
What is molecular detection of M. tb?
- The WHO recommends the Xpert MTB/RIF assay for initial TB test.
- It is automated, integrated cartridge-based molecular assay to detect M. tb and identify rifampicin resistance directly from sputum.
- Sample into collection vessels and add reagents into cartridges.
- Uses Real-time PCR to amplify an M. tb specific sequence of the rpoB gene.
- This is then probed with molecular beacons for mutations with rif resistance.
- It can provide results in 2 hours.
- It gives no strain identity and only 1 AMR gene.
What is rifampicin?
A first line antibiotic for M. tb infection.
How can WGS be used for M. tb AMR detection?
- It normally takes around 2 months to do AST on M. tb.
- Rapid WGS may replace current methods of identifying and typing M. tb.
- It offers the ultimate molecular resolution important for surveillance, outbreak investigation and AMR detection.
What is an example of using WGS for M. tb AMR detection?
- A patient infected with XDR-TB and positive MGIT culture
- WGS and phylogenetic tree showed the patient was infected with 2 distantly related strains of M. tb.
- The genetic basis for phenotype resistance to 9 drugs when normal AST could have only detected a few of these.
Can WGS be used for AMR detection in M. tb?
- WGS reduces the time to diagnose XDR-TB from weeks to days.
- It cannot completely replace AST.
- This is because of the lack of understanding of the genetic basis of AMR.
How has WGS for M. tb AMR detection been proven in trials?
- 176 genetically diverse M. tb isolates were examined for resistance to 11 anti-tuberculosis drugs.
- Results using culture AST was compared to the WGS.
- Overall, the sensitivity and specificity of the WGS AST were 86.6% and 94.5% but differs depending on antibiotic use.
- It was really good for Ethambutol but not as good for detecting streptomycin resistance.
- WGS is good for molecular tracking and surveillance.
What are the limitations of using WGS for M. tb AMR detection?
- It is difficult to classify resistance when the raise in MIC still lies within the treatment window.
- Certain regions of the genome have low coverage so it is difficult to detect SNPs.
- It is restricted to the knowledge we have of the genetic basis of resistance.
How was WGS used to identify the source of an MRSA outbreak in a neonatal ICU?
- Prior to WGS outbreaks were hard to distinguish.
- Multiple patients in a neonatal ICU were part of an MRSA outbreak.
- Isolates from this outbreak were compared to other outbreak patients, non outbreak MRSA patients and MRSA patients from the wider hospital to work out where the outbreak came from.
- Mapping of the genome sequencing of the MRSA isolates showed 2 distinct groups only separated by 102 SNPs.
What did WGS of the MRSA outbreak in neonatal ICU show?
- 2 groups of MRSA.
- Group 1 contained all the isolates associated with the NICU outbreak. They were very closely related.
- Group 2 was all the other isolates that were not considered to be part of the outbreak and differed from group 1 by at least 136 SNPs suggesting no transmission between these patients.
- WGS sequencing confirmed which patients were involved in the outbreak.
Why was isolate 6C from the NICU MRSA outbreak more distantly related to the other?
- It carries a mutation in a DNA region that leads to increased mutation frequency.
- It has a lot more mutations then the other outbreak isolates.
What is the future of using WGS to track outbreaks?
- It could be used in real time and not just retrospectively.
- This means quick identification of the source and decolonisation and resolving of the issue.
- This would massively improve patient care and lead to savings for the healthcare system.
What is Escherichia coli?
- A gram-negative, versatile bacterium.
- It has a high degree of genome plasticity so it is naturally competent.
- It is an important member of the normal intestinal microflora of humans and other mammals.
- Pathogenic variants can cause enteric disease and extra intestinal diseases.
- They can be facultative or obligate pathogens.
- It has a major health and economic burden and lots of AMR.
What are the 6 pathotypes of E. coli?
- EPEC - enteropathogenic E coli
- EHEC - enterohemorrhagic E coli
- ETEC - enterotoxigenic e coli
- EAEC - enteroaggregative E coli
- EIEC - Enteroinvasive E coli
- DAEC - diffuse aherent E coli
What is the only E coli pathotype that is a facultative pathogen?
DAEC
What was the EHEC O104:H4 outbreak?
- A large outbreak of diarrhoea and haemolytic-uremic syndrome (HUS) in germany.
- Caused by E coli.
- > 4000 cases and 50 deaths.
- Outbreak has a plasmid characteristic of EAEC and a plasmid encoding ESBLs.
- Historically these EAEC don’t cause HUS.
- Isolates from the outbreak were indistinguishable from previous strains using non-WGS methods.
What did WGS examination of the O104:H4 show?
- The strain was EAEC that had acquired genes for Shiga toxin and antibiotic resistance.
- It was compared to 53 E coli and Shigella genomes to understand its origin.
- the EAEC O104:H4 formed a distinct cluster in the phylogenetic tree.
- Genetic similarity between the Shiga toxin-encoding outbreak strains and EAEC without Shiga suggests that Shiga toxin acquisition was a recent event.
- Presence of O104:H4 strains within EAEC clade confirms that the outbreak strain is not a prototypical EHEC that has acquired some virulence features of EAEC
What are the origins of EHEC O104:H4?
- O104:H4 contains pAA from EAEC which is important for virulence due to the AAF operon.
- It contains Shiga but lost LEE from EHEC .
- O104:H4 also obtained a resistance plasmid with TEM and CTX-M from somewhere else.
- This led to the suggestion of a new E coli pathotype. Entero-aggragative-haemorrhagic E coli. (EAHEC)
What is the AAF operon?
- Aggregative adhesion fimbrial operon.
- Hallmark of EAEC strains
- Important for bacteria adhesion.
What else can WGS be used to investigate?
Host evolutionary dynamic between colonising and pathogenic strains.
How has WGS been used to explore host evolutionary dynamics in MSSA?
- The authors used WGS to uncover genetic changes that accompany the transition from the nasal carriage to fatal bacteraemia.
- Data is from a single patient. 6 nasal samples and 1 blood culture.
- The patient died following chemotherapy and this infection.
What did WGS sequencing of MSSA samples show regarding host evolutionary dynamics?
- Used maximum likelihood trees based on the sequences.
- They were all MSSA and all strains were very similar.
- This shows there was little variation so it is a homogenous population arising from a single population of MSSA.
- This origin was the population of MSSA in the nasopharynx.
- There were just 8 mutations accompanying the transition of asymptomatic MSSA to fatal bloodstream infection.
- Most of these mutations were protein truncations that caused disease progression.
- This included a premature stop codon in the virulence regulator AraC.
What was the difference in virulence between the nasal MSSA isolates and bacteraemia MSSA?
- It was measured using T cell death.
- High T cell death = high toxin production.
- The fatal bacteraemia MSSA has lower virulence production than the nasal bacteria
- Through genomic interrogation and GWAS, this was identified as due to a mutation in rsp
What is rsp?
A transcriptional repressor
What do mutation in rsp in MSSA cause?
- Natural mutations in rsp cause reduced toxicity but increased ability to form abscesses.
- The toxicity is measured by observing RBC break down in the presence or absence of the gene.
- rsp mutant mice survive longer than wild-type mice.
- However there was no difference in the immune infiltration of the organs of the mice which is unusual if the mice survive longer.
- The mutation causes prolonged residence of MSSA in cells and more dissemination through the body.
- It is also thought to upregulate immune subversion proteins.
What can rsp inactivation cause in terms of type of MSSA infection?
- Reduced toxicity and virulence
- Increased dissemination and immune subversion.
- This makes the MSSA more suited to bacteraemia as opposed to nasal colonisation.
Is WGS a viable method to replace culture based methods?
- Currently they are not efficient or cost effective to replace culture based technologies.
- The exception to this is for slow-growing bacteria like M. tuberculosis.
How can WGS improve M. tuberculosis treatment?
- WGS can rapidly improve TB diagnosis.
- Determine AMR genes
- It ensures that patients get the correct antibiotic treatment faster.
What could WGS be really crucial for?
- Bacterial surveillance, tracking transmission and identifying sources of outbreaks.
- It can enhance our understanding of fundamental pathobiology and the evolution of microorganisms.
What needs to happen to make WGS a major tool in bacterial diagnosis?
The technology needs to become faster, more accurate and more cost effective.
