Exercise 2 · 2019. 9. 24. · Date DTU Title MGEs harboring AMR genes may be the link between animals and humans 3 Unpublished • Inoculum doses of 10^8, 10^5 or 10^2 CFU • The

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Exercise 2Chasing MGEs

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Mobile Genetic Elements (MGEs) and AMR• Several antimicrobial resistance (AMR) genes at least in the bacterial species we examine

in the EURL-AR network are associated with MGEs• AMR linked to MGEs spreads more widely that AMR associated to chromosomal

mutations

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Partridge et al., 2018

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MGEs harboring AMR genes may be the link between animals and humans

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Unpublished

• Inoculum doses of 10^8, 10^5 or 10^2 CFU• The exogenous strain was lost within 3 days after inoculation in all mice, whereas the

CMY-2 plasmid was stably maintained within the gut microbiota for up to 19 days, suggesting that plasmid transfer and persistence was not dependent on donor survival

• The exogenous strain was shed in higher numbers by mice inoculated with higher doses, whereas the transconjugants were detected in higher numbers and persisted longer in the fecal microbiota of mice inoculated with the lowest inoculum dose (10^2 CFU)

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Insertion Sequences (IS) and composite Transposons (Tn)

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From Partridge et al., 2018

ISs• small MGEs carrying little more than one

(sometimes two) trasposase gene

• Classification based on active site key aminoacids and transposition mechanism

Composite transposon• A region bounded by two copies of the same

or related IS

Used as ‘signatures’ of AMR acquisition events

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Archetypal plasmid

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Norman A. et al. 2009. Phil. Trans. R. Soc. B

Module for:

- Initiation of replication

- Control of replication

Module for:

- Partitioning

- Stable inheritance

Module for accessory functions:

- Resistance genes

- Degradation of various compounds

Module for:

- Mating pair formation

- Coupling protein unit

- Relaxases (enzymes

‘processing’ the plasmid

DNA during conjugation)

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Plasmid typing methods

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PlasmidFinder

• If two replicons are similar, they tend to be incompatible (not able to stablyco-exist in the same bacterial cell)

• Example of terminology: IncA/C, IncI1, IncN, IncHI1, etc… and there aresubgroups (IncFI, FII-V, etc…)

• Plasmid from different Inc groups differ in their properties (narrow/broadhost range)

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http://pubmlst.org/plasmid/

IncI1 (2008)

IncF (2010)

IncHI2 (2010)

IncN (2011)

IncHI1

Plasmid Multilocus Sequence Typing (pMLST)

repI: replicase geneardA: type I restriction-modification enzymetrbA: involved in maintenance and palsmid transfersogS: DNA primasepilL: type IV pilus biogenesis

FII: the copA region of the FII repliconFIA: the region comprising the iterons and thereplication protein repE gene of the FIA repliconFIB: the replication protein repB gene of the FIBrepliconFiC: the copA region og the FIC replicon

pDLST: plasmid double locus sequence typing(smr0018 & smr0199 profiles)Multiplex PCR (bipA, smr0092, smr0183)

repN: replicase genetraJ: mobilization of plasmid during conjugationkorA: negatively regulating the synthesis of the conjugal pilus and mating pore during conjugation

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Exercise 2 – Chasing MGEsLearning outcomes:

• Describe different types of Mobile Genetic Elements (MGEs) associated with spread of antimicrobial resistance (AMR) genes;

• Understand the complexity of identifying MGEs in WGS data generated by short read technologies;

• Determine the genetic context of selected antimicrobial resistance genes;• Infer possible epidemiological scenarios based on molecular data;• Present and explain the results to a wider audience.

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Target AMR gene in this exercise• blaCTX-M-14, blaCTX-M-15, blaCTX-M-27, blaCTX-M-55

These genes code for Extended-Spectrum Beta-Lactamases (ESBLs) which hydrolyzeseveral beta-lactam antibiotics including compounds that are often used for empiricaltherapy and compounds that are considered critically important antimicrobials for human medicine

• blaCMY-2

These gene code for AmpC Beta-Lactamase (AmpC) which hydrolyzes several beta-lactamantibiotics including compounds that are often used for empirical therapy and compoundsthat are considered critically important antimicrobials for human medicine. The spectrum of beta-lactam compounds hydrolyzed by EBSLs and AmpC partially overlaps

• mcr-1This gene mediates resistance to colistin which is a last resort drug for treatment of infections by multidrug-resistant bacteria

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The basic question:• What is the genetic context of the target AMR genes in your sequences?

By genetic context I mean:• The immediate DNA surrounding the AMR gene (read: ISs)• Any possible inference on the genomic location (chromosome or plasmids? If

plasmid, which one?) of the AMR gene

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But never forget that this analysis is a mean to answerthe real question:

Can we hypothesize an exchange of the target AMR gene between the two strains?

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Instructions• READ THE MANUAL!• Each group has two sequences• Find the contig with the target AMR gene• Define if that contig is likely part of a plasmid or chromosome (using BLAST at NCBI)• Detect the immediate genetic context of the beta-lactam resistance gene (using ISFinder)• Make an annotation/ drawing

There are many commercial and public software for performing the steps above.Let’s try to use some freely available ones

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Possible critical points in the exercise

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Visualization and Annotation of Open Reading Frames

The double stranded DNA can be ”read” in 6 ways:• Three on the positive strand• Three on the negative strand

Let’s look at the positive strand:

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Possible critical points in the exercise

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Visualization and Annotation of Open Reading Frames

The double stranded DNA can be ”read” in 6 ways:• Three on the positive strand• Three on the negative strand

Let’s look at the positive strand:

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Possible critical points in the exercise

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Visualization and Annotation of Open Reading Frames

The double stranded DNA can be ”read” in 6 ways:• Three on the positive strand• Three on the negative strand

Let’s look at the positive strand:

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Possible critical points in the exercise

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Visualization and Annotation of Open Reading Frames

The double stranded DNA can be ”read” in 6 ways:• Three on the positive strand• Three on the negative strand

Let’s look at the positive strand:

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Possible critical points in the exercise

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Visualization and Annotation of Open Reading Frames

The double stranded DNA can be ”read” in 6 ways:• Three on the positive strand• Three on the negative strand

Let’s look at the positive strand:

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Possible critical points in the exercise

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Visualization and Annotation of Open Reading Frames

The double stranded DNA can be ”read” in 6 ways:• Three on the positive strand• Three on the negative strand

NO!

YES!

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Possible criticalpoints in the exercise

12 MQHRIIL…1 MQHRIIL…

Your protein is longer than the one(s) in NCBI:Really or can you ”Trim” the ORF?

1 MQHRIIL…12 MQHRIIL…

Your protein is shorter than the one(s) in NCBI:Really a partial protein or can you ”Extend” the ORF?

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Possible critical point in the exercise• If you want to verify if the target AMR gene is plasmid-

mediated, you can identify a possible plasmid that could be used as reference to align your contigs.

• For this alignment, use BLASTN at NCBI, choose ‘Align two more sequences’ and put the reference plasmid as query and all contigs from your isolate as subject.

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ISFinder https://isfinder.biotoul.fr/blast.php20

I recommend you to use ISFinder “as BLAST at NCBI hits for insertion sequences may not be easily interpretable”… what does this mean?

∆Tn2

Annotation of your sequence

Possible critical points in the exercise

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Now it’s your turn!

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