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Home , Insertion sequence, Plasmid

Transposable elements and their role in the spread of in

Adam Roberts Liverpool School of tropical Medicine [email protected] @GCAGATGCAATG Definitions

• is a segment of DNA able to excise from a and re-insert • This insertion may be at the same site or at a different site in the same, or in a different . Heritable consequences of insertion • (MGEs)

Haas & Rak, 2002 Ciric, et al.,2014

Salyers, Insertionet al., 1995Plasmids sequences Davies, et al., 2000

Conjugative transposons Composite transposons Insertion sequences • Biologically these are the simplest types of MGE • Consist of a and its binding site.

Transposase gene

IR IR Insertion sequences • Over 1500 different ISs discovered to date in almost 300 eubacterial and archaeal species

Siguier et al., 2006 Insertion sequences • Some bacterial cells may have hundreds of copies of the same insertion sequence

How can a accommodate so many copies? Composite transposons

An insertion sequence is present at each end of a composite transposon

The ISs move together and therefore can lead to the transposition of the intervening DNA

IS IS

The intervening DNA often contains resistance enabling selection in Translocatable Units (Tus) • Putative novel mobile genetic element. • No replicase gene found on the TUs. • Often carry antibiotic resistance genes. Translocatable Units & Composite Transposons

• If the two IS elements of composite transposons are similar • It will match the structures that could give rise to TUs.

TU IS1216 elements

• IS element in IS6 family. • Commonly found in bacteria. • Usually associated with antibiotic resistance genes • IS1216 composite transposons were found to be excised and formed as a TU.

Ciric, L. et al. Antibiotic and antiseptic resistance genes are linked on a novel Ciric, L. et al. Minocycline resistance in an oral Streptococcus infantis isolate is encoded by mobile genetic element: Tn6087. J Antimicrob Chemother 66 2235-9 (2011) tet(S) on a novel small, low copy number . FEMS Microbiol Lett. 352:106-15. (2014) Result-IS1216 composite transposon PCR

• DNA primers amplifying outward from IS1216 were designed and used for PCR on oral IS1216F1 metagenomic DNA.

• Multiple PCR products with the size between 200 to 4,000 bp were found.

IS1216R1 Result-IS1216 composite transposon PCR

• 4 different clones were identified to be amplified from IS1216 composite transposon.

Sample Predicted Structure

qrg-∆hyp IS1216 (1321 bp) Qrg confers

qrg-hyp resistance IS1216 to (1772 bp) antiseptics Tn6087 (Ciric, et al., 2011)

Result-IS1216 composite transposon PCR

Sample Predicted Structure

uspA-∆orf16 IS1216 (984 bp)

uspA-orf16-orf17 IS1216 (3313 bp) Plasmid pIL5 uspA= (Gorecki et al. 2011) universal stress A

The other composite transposons

IS elements found in IS elements commonly IS elements in IS6 family Streptococcus spp. found on Tns or plamids

IS861 IS26 IS3 IS1161 IS240 IS256 IS1167 IS257 IS1485 IS1381 IS1548 • Another IS257 composite transposons was identified. • Containing kanamycin nucleotidyltransferase Result-TU verification PCR

• As composite transposons have a potential to excise into UCS, the four structures found by IS1216 PCR and the one from IS257 PCR, therefore, had the potential to form a TU. • To verify this, outward reading PCR was carried out. Result-UCS verification PCR

• Sample qrg-∆hyp IS1216 and uspA-orf16-orf17 IS1216 were confirmed as UCS, as the amplicons with expected size were found and IS1216 were found in sequencing data.

1182bp 1075bp

Tansirichaiya, S. et al. PCR-based detection of composite transposons and translocatable units from oral metagenomic DNA. FEMS Microbiol Lett. 363(18). pii: fnw195 What about clinical relevance? mcr-1 mediated resistance

Liu, Y.-Y. et al. Emergence of plasmid-mediated colistin resistance Liang, B. et al. Transferable Plasmid-Borne mcr-1 in a Colistin-Resistant mechanism mcr-1 in and human beings in China: a microbiological flexneri Isolate. Applied Environ Microbiol. 84. e02655-17 (2018) and molecular biological study. Lancet Infect. Dis. 16, 161–168 (2016).

Xavier, B. B. et al. Identification of a novel plasmid-mediated colistin-resistance gene, mcr-2, in , Belgium, June 2016. Eurosurveillance 21, 30280 (2016). Yin, W. et al. Novel plasmid-mediated colistin resistance gene mcr-3 in Escherichia coli. MBio 8, e00543–17 (2017).

Carattoli, A. et al. Novel plasmid-mediated colistin resistance mcr-4 gene in Salmonella and Escherichia coli, Italy 2013, Spain and Belgium, 2015 to 2016. Eurosurveillance 22, 30589 (2017). Borowiak, M. et al. Identification of a novel transposon-associated phosphoethanolamine gene, mcr-5, conferring colistin resistance in d-tartrate fermenting Salmonella entericasubsp. enterica serovar Paratyphi B. J. Antimicrob. Chemother. 72, 3317–3324 (2017). Wang, R. et al. The global distribution and spread of the mobilized colistin resistance gene mcr-1. Nature Comms. 9:1179 (2018) Metagenomic screens for novel resistance genes Metagenomic Samples • Oral Metagenomic C7C4 resistant clone

Un HindIII digested digested

15 14 10 8 8 (vector)

4 4 3.7 3 3

BlastN: Neisseria meningitidis strain M25074 genome BlastX: MULTISPECIES: enoyl-[acyl-carrier-protein] reductase [Neisseria] fabI in triclosan resistant clones • 9 other triclosan resistant clones end sequenced • Primers for fabI designed from that species • PCR on triclosan resistance confirmed they all contain fabI from a variety of different species;

Triclosan resistant clone number Source of fabI

C7C4 Neisseria meningitidis A5A12, M8G5 Campylobacter concisus A5B12 Campylobacter gracilis E4E3, L5F10, X5F8 Prevotella sp. N9E7, R2G11 Haemophilus parainfluenzae H4E9 Porphyromonas sp.

Tansirichaiya S, Reduced Susceptibility to Antiseptics Is Conferred by Heterologous Housekeeping Genes. Microb Drug Resist doi: 10.1089/mdr.2017.0105.(2017). Genes not normally involved in resistance may, when expressed in heterologous hosts, give rise to resistance phenotypes.

Does it occur in nature?

Virtually any gene can find its way onto a mobile genetic element. resistance screen The motif has the pattern G-x(4)-GK-[TS], where G, K, T and S denote glycine, lysine, threonine and serine residues respectively, and x denotes any amino acid. Binds the phosphate on ATP. Tetracycline resistance screen Strain Tetracycline Minocycline Tigecycline

E. coli EPI300 2 μg/ml 1 μg/ml 0.5 μg/ml pHSG396

PS9.3 32 μg/ml 1 μg/ml 8 μg/ml

PS9.4 2 μg/ml 1 μg/ml 0.5 μg/ml

PS9.5 2 μg/ml 1 μg/ml 0.5 μg/ml

PS9.6 32 μg/ml 1 μg/ml 8 μg/ml

PS9.7 32 μg/ml 1 μg/ml 8 μg/ml

Designated tetAB(60)

Reynolds LJ, et al. Efflux in the Oral Metagenome: The Discovery of a Novel Tetracycline and Tigecycline ABC Transporter. Front Microbiol. 7:1923. doi: 10.3389/fmicb.2016.01923. (2016)

Conclusions

Insertion sequences are responsible for the mobilisation of many (any?) gene which flank their insertion sites.

Once mobilised onto a composite transposon, and challenged with a suitable selective pressure, they will facilitate the transfer into multiple replicons.

Should analysis include a survey of the agents of dissemination (ISs) as well as resistance genes?

Functional metagenomic analysis for novel resistance genes can give an indication into what might emerge given a new selective landscape.

Had we spotted mcr-1 earlier could we have brought in colistin bans in use earlier?