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'Novel technologies to study the resistome'
- 1. Willem van Schaik Institute of Microbiology and Infection University of Birmingham, United Kingdom w.vanschaik@bham.ac.uk twitter.com/WvSchaik Novel technologies to study the resistome This presentation can be downloaded from www.slideshare.net/WillemvanSchaik1
- 2. The natural role of antibiotics Antibiotics and antibiotic resistance Products of >2 billion years of evolution “Antibiotics and antibiotic resistance are everywhere, but the environment, most importantly due to human and animal use, selects”
- 3. Centers for Disease Control and Prevention
- 4. Mechanisms of antibiotic resistance Lewis, 2013. Nature Rev Drug Disc. 12:1371 Resistance through mutation Resistance through horizontal gene transfer antibiotic resistance genes
- 5. abx the gut resistome repertoire of antibiotic resistance genes in the gut microbiome The human microbiome
- 6. The resistome of critically ill patients receiving intensive antibiotic therapy Buelow et al. 2015, JAC 69:2215; Buelow, Bello González et al., 2017. BioRxiv 102467 http://en.wikipedia.org/wiki/Intensive_care_unit
- 7. Methods to study the resistome Culture-based approaches o Selective culture of antibiotic-resistant bacteria Culture-independent methods o Metagenomic shotgun sequencing o Functional metagenomics o High-throughput quantitative PCR Van Schaik, 2015. Philos Trans R Soc Lond B Biol Sci. 370(1670): 20140087.
- 8. Methods to study the resistome Culture-based approaches o Selective culture of antibiotic-resistant bacteria Culture-independent methods o Metagenomic shotgun sequencing o Functional metagenomics o High-throughput quantitative PCR Van Schaik, 2015. Philos Trans R Soc Lond B Biol Sci. 370(1670): 20140087.
- 10. Culture-based approaches ’80% of 16S rRNA gene sequences from species that have not been cultivated’Eckburg et al., Science, 2005. 308:1635
- 11. Culture-based approaches “Culture of faeces under 212 different conditions, yielded 174 new species” Lagier et al., 2012. Clin Microbiol Infect 18:1185 Browne et al., 2016. Nature 533:543 Used a single medium (YCFA) Compared sequence reads of colonies on plate with sequence reads of DNA isolated from faeces
- 12. Culture-based approaches Rettedal et al., 2014. Nature Comm 5:4714 Stool sample of healthy subject Plated on modified Gifu anaerobe agar supplemented with single antibiotics Identify growing (tolerant) colonies
- 13. Methods to study the resistome Culture-based approaches o Selective culture of antibiotic-resistant bacteria Culture-independent method o Metagenomic shotgun sequencing o Functional metagenomics o High-throughput quantitative PCR
- 14. Methods to study the resistome Culture-based approaches o Selective culture of antibiotic-resistant bacteria Culture-independent method o Metagenomic shotgun sequencing o Functional metagenomics o High-throughput quantitative PCR
- 15. Metagenomic shotgun sequencing Computationally complex, involve bioinformaticians Analysis pipelines MG-RAST: metagenomics.anl.gov EBI Metagenomics: www.ebi.ac.uk/metagenomics ResFinder: https://cge.cbs.dtu.dk/services/ResFinder/
- 16. Forslund et al., 2014. BioEssays. 36:316 human Pal et al., 2016. Microbiome 4:54
- 17. 32-year old male, traffic trauma Intensive prophylactic antibiotic therapy erythromycin flucoxacillin vancomycin cefazolin Buelow et al., 2014. J. Antimicrob Chemother. 69:2215
- 18. Rich diversity of antibiotic resistance genes, even in absence of selection 1.0 0.56 0.13 β-lactams aminoglycosides macrolides tetracyclines glycopeptides sulphonamides chloramphenicols trimethoprim Day 4 Low High Undetectable Day4 Day14 Day16 Day28 Day313 Metagenomic shotgun sequencing Buelow et al., 2014. J. Antimicrob Chemother. 69:2215 Selection for resistance during ICU stay, particularly for aminoglycosides (used in prophylactic antibiotic therapy)
- 19. Metagenomic shotgun sequencing Limitations of sequencing Expensive (~€400 for 5 Gbp of data on HiSeq 2500) assemblies frequently only cover resistance gene no information on genetic context of resistance gene no information on organism harboring resistance gene aph(2”)-Ib ?
- 20. Screen for phenotypes Functional metagenomics Library in E. coli Adapted from Allen et al., 2010. Nature Rev Microbiol 8:251 long insert library fosmids (30 – 40 kbp inserts) labour-intensive detailed information on gene context short insert library general cloning vector (2 – 5 kbp inserts) more high-throughput than long inserts limited information on gene context Sommer et al., 2009. Science 325:1128 Pehrsson et al., 2016. Nature 533:212 Potential to functionally identify novel antibiotic resistance genes Allen et al., 2009. ISME J 3:243 Udikovic-Kolic et al., 2014. PNAS 111:15202
- 21. Resistance gene Plasmid-associated (conjugation/mobilization) Plasmid-associated Phage-associated IS Element Functional metagenomics Resistance genes are associated with mobile genetic elements… …that are carried by anaerobic gut commensals Buelow et al., 2014. J. Antimicrob Chemother. 69:2215 Mobile genetic elements predicted by ACLAME, ISfinder Origins of clones predicted by CARMA3
- 22. n = 1 follow-up study 10 healthy volunteers 10 ICU-patients, acutely admitted no history of hospitalisation or antibiotic use stay ≥10 days
- 23. The gut resistome nanolitre-scale qPCRs (Fluidigm Biomark) 88 samples x 96 targets
- 24. Primers for 81 resistance genes 16S rRNA: for relative quantification Most common resistance genes in gut microbiota Forslund et al., 2013. Genome Res. 23:1163; Hu et al., 2014. Nat. Commun 4:2151 Clinically relevant resistance genes ESBLs, carbapenemases, mecA, vancomycin resistance genes The gut resistome Buelow, Bello Gonzalez et al., BioRxiv 102467 (in revision)
- 25. Buelow, Bello Gonzalez et al., BioRxiv 102467 (in revision) ICU patients healthy subjects
- 26. The gut resistome aac(6’)-Ii, ermC: aminoglycoside, macrolide resistance in Enterococcus qacA: biocide resistance (efflux) in Gram-positive cocci tetQ: tetracycline resistance in Bacteroides Buelow, Bello Gonzalez et al., BioRxiv 102467 (in revision)
- 27. Methods to study the resistome Culture-based approaches o Selective culture of antibiotic-resistant bacteria Culture-independent methods o Metagenomic shotgun sequencing o Functional metagenomics o High-throughput quantitative PCR o Long-read sequencing Van Schaik, 2015. Philos Trans R Soc Lond B Biol Sci. 370(1670): 20140087.
- 28. Methods to study the resistome Culture-based approaches o Selective culture of antibiotic-resistant bacteria Culture-independent methods o Metagenomic shotgun sequencing o Functional metagenomics o High-throughput quantitative PCR o Long-read sequencing Van Schaik, 2015. Philos Trans R Soc Lond B Biol Sci. 370(1670): 20140087.
- 29. DNA sequencing - Nanopore Sequencer is €2000 Flow cell €500 – 1000 High error rates but rapid improvements Long reads Up to 900 kbp High output 5 Gbp/run Can now directly sequence DNA, RNA
- 30. 1 airport trolley = 1 one lab Quick et al. 2016. Nature 530:228-232.
- 31. clone DNA in vectors and plate library on plates with antibiotics sequence inserts of resistant clones using MinION 36 – 78 hours
- 32. Clones plated on spectinomycin Sequencing on MinION Resistome mapping using Nanopore sequencing Co-localization of aadA (aminoglycoside resistance) and dfrA (trimethoprim resistance)
- 33. Long-read sequencing of metagenomes Porecamp 2015 University of Birmingham DNA sample of UMCU sewage 1785 reads, 3.6 Mbp data Average: 2.0 kb read Analysis with ResFinder Standard settings but 50% ID 2 reads with resistance genes Picture: @IrisKolder Rogers, unpublished data
- 34. 3.9 kbp read with aadA24 and sul1 Enterobacterial plasmid, 85% identity (~15% error rate in read) Rogers, unpublished data
- 35. 2.2 kbp read with erm(B) Clostridium transposon Tn6194, 87% identity Nanopore sequencing has the potential to map the resistome link resistance genes with mobile genetic elements Key factor: DNA quality and fragment lengthRogers, unpublished data
- 36. Many approaches to study the resistome Not a single right one Balance between cost and speed High-throughput resistome profiling by qPCR: diagnostic tool? Conclusions This presentation can be downloaded from www.slideshare.net/WillemvanSchaik1
- 37. Elena Buelow Marc Bonten Rob Willems Evelien Oostdijk Maaike van Mourik Iris Braat Els Oosterink Wouter de Steenhuijsen Piters Jumamurat Bayjanov Eline Majoor & undergraduate students Collaborators Funding EvoTAR: Evolution and Transfer of Antibiotic Resistance Priority Medicines AMR Vidi Teresita Bello Gonzalez Susana Fuentes Leo Lahti Dennis Versluis Mark van Passel Hauke Smidt Brian Jones Lesley Ogilvie Marco Maria D’Andrea Heike Schmitt