The Genetic Epidemiology of Antibiotic Resistance Professor Neil Woodford Antimicrobial Resistance & Healthcare Associated Infections (AMRHAI) Reference Unit Crown copyright
The forensics of AMR Resistance involves emergence of mutations spread of resistance genes spread of resistant strains Genes Gene carriers IS, In, Tn, plasmids Tracking and characterizing the resistant strains their resistance genes Host species Strains, clones, phylogenetic groups, virulence traits, co-resistance Patients Hospital / community setting; risk factors 2 Sydney, 20th November 2014 Crown Copyright
The molecular epidemiology of resistance The resistant isolates molecular methods define strains relevant to hospital epidemiology 1. inter-patient or inter-hospital spread of strains 2. identifying new strains with the same resistance 3. on a national & global scale, the strains are highly diverse Their resistance genes / elements horizontal transfer of transposons or plasmids fundamental units of resistance characterization needed for the bigger picture origins and evolution of resistance 3 Sydney, 20th November 2014 Crown Copyright
Can you distinguish TEM-1 from CTX-M-15? Enzyme Examples Confer resiatance to Penicilllinases ESBLs pampc TEM-1/-2, SHV-1, OXA- 1/-30 TEM-, SHV-, OXA-, CTX- M, VEB, PER CMY, ACC, DHA, FOX, MOX, ENT / EBC Penicillins; early cephalosporins; overexpression affects penicillininhibitor combinations All generation cephalosporins (not cephamycins) 3 rd gen cephs (not 4 th ); cephamycins Carbapenems remain active against producers of these enzymes and are increasingly used for treatment 4 Sydney, 20th November 2014 Crown Copyright
Multiple escape events for the CTX-M family K. georgiana- related CTX-M-9 group K. georgiana- related K. ascorbata- related K. ascorbata- related The most successful variant: CTX-M-15 5 Sydney, 20th November 2014 Crown Copyright
followed by spread and global domination 6 Sydney, 20th November 2014 Crown Copyright Hawkey and Jones. JAC 2009; 64 (Suppl. 1), i3-i10
Travel destination influences risk and type of resistance 7 Sydney, 20th November 2014 Crown Copyright Ostholm-Balkhed et al. JAC 2013; 68: 2144-53
ST131 dominates among ESBL-positive E. coli 8 Sydney, 20th November 2014 Crown Copyright
Geom. mean MICs (mg/l), CTX-M-15 +ve E. coli Epidemic A Other CTX-M- 15 Cefotaxime 37.3 93.2 Ceftazidime 2.9 23.0 Pip/taz 20.1 13.2 Ciprofloxacin 17.5 6.7 Trimethoprim 256 9.6 Gentamicin 1.1 28.6 Amikacin 9.0 18.2 Nitrofurantoin 8 7.3 9 Sydney, 20th November 2014 Crown Copyright Woodford et al., JAC 2004, 54, 735
Disruption of ISEcp1-bla CTX-M in ST131 strain A Most strains : ISEcp1 bla CTX-M-15 400-bp Strain A : ISEcp1 IS26 bla CTX-M-15 IS26 between the bla gene and its usual promoter lower cephalosporin MICs 10 Sydney, 20th November 2014 Crown Copyright
Bacteria don t keep resistance genes to themselves www.biotoon.com 11 Sydney, 20th November 2014 Crown Copyright
Neat packages of multi-resistance Antibiotic classes Aminoglycosides Genes aac6 -Ib-cr aada5 Mechanism Modify drug β-lactams bla CTX-M-15 bla OXA-1 Destroy drug bla TEM-1 Chloramphenicol catb4 Modify drug Macrolides mph(a) Efflux Fluoroquinolones aac6 -Ib-cr Modify drug Sulfonamides suli By-pass Trimethoprim dhfr XVII By-pass Tetracycline tet(a) Efflux 12 Sydney, 20th November 2014 Crown Copyright Woodford, Carattoli et al. AAC
IncFII plasmids are spreading CTX-M-15 globally (Canada) UK Strain D UK Strain A 13 Sydney, 20th November 2014 Crown Copyright Woodford & Carattoli, AAC, 2009: 53: 4472-82
but not all CTX-M plasmids in ST131 confer MDR UK strain C 96 kb, IncI1 encodes CTX-M-3 and TEM-1 no other resistance genes 14 Sydney, 20th November 2014 Crown Copyright Woodford & Carattoli, unpublished
Closely related CTX-M ESBLs have different origins related genes usually on distinct plasmid types CTX-M-3 : IncL/M (Poland), IncA/C, IncHI2, IncI1 CTX-M-15 (Asp240Gly) : IncF (IA, IB, II) ISEcp1-bla CTX-M-3 /-15 linking sequences often vary 48-bp (CTX-M-15) or 128-bp (CTX-M-3) separate escape events, rather than mutation after escape global CTX-M-15 explosion repeated mutation of bla CTX-M-3 it s horizontal plasmid spread and clonal expansion 15 Sydney, 20th November 2014 Crown Copyright
Putting it all together in the real world: ESBLs in nursing homes in Northern Ireland 40% patients colonized 134 / 135 isolates phylogenetic group B2, ST131 1) Strain spread: Strain A with CTX-M-15 on IncFII/FIA plasmid pek499 2) Plasmid spread: 3 strains with IncI1 plasmids (pek204-like) 3) Gene / Transposon spread: CTX-M-3 encoded by IncI1, IncFIA-B, IncN, and IncY plasmids 16 Sydney, 20th November 2014 Crown Copyright
Transposition has facilitated spread of bla CTX-M-3 to other plasmids ISEcp1-mediated mobilization of: bla CTX-M-3 only bla CTX-M-3 and bla TEM-1 Latter structure is identical to structures on IncFIA, IncFIA-FIB, IncN and IncY plasmids in Belfast 17 Sydney, 20th November 2014 Crown Copyright Dhanji et al. JAC 2011; 66: 297-303
Hospital antibiotic sales (kg; IMS data) 3000 Carbapenems 2500 2000 1500 1000 500 0 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 use of carbapenems new selective pressures..., but what consequences? 18 Sydney, 20th November 2014 Crown Copyright
Acquired carbapenemases Class Carbapenemase Enterobacteriaceae Non-fermenters A (non-metallo) KPC +++ + BIC, GES, IMI, NMC, SME + +/- B (metallo) IMP*, VIM* +++ +++ NDM +++ ++ AIM, DIM, GIM, SIM, SPM, TMB - ++ D (non-metallo) OXA-48-like +++ +/- OXA-23, -40, -58, -143, - 235 +/- +++ IMP- & VIM- types are integron-associated IMP-types described first, but have been overtaken by other types 19 Sydney, 20th November 2014 Crown Copyright
Strain dynamics: a dominant international KPC +ve K. pneumoniae clone, but... ST258 ST258 ST258 One KPC outbreak: 11, 25, 27. 248, (258 - Col-R) 321. 468. 490, 491 plus Enterobacter + E. coli 20 Sydney, 20th November 2014 Crown Copyright Nordmann et al. TLID 2009; 9: 228 36
A KPC cluster: spread of strains and plasmids Isolate Patient Bacterial ID VNTR profile KPC Plasmid n=6 A, B, C, D, E, H K. pneumoniae 6, 4, 2, 0, -, 2, 2, 3, 1 pkpqil-d2 3 C E. cloacae n.a. pkpqil-d2 7 F Klebsiella oxytoca n.a. pkpqil-d2 8 G Citrobacter freundii n.a. Not pkpqil-like* 9 G K. pneumoniae 1, -, 4, 1, -, 1, 3, 5, 1 Not pkpqil-like* Patients A, B, C, D, E & H spread of the same K. pneumoniae strain Patients C and F spread of pkpqil plasmid into two other species Patient G Separate introduction (distinct strains and plasmids) 21 Sydney, 20th November 2014 Crown Copyright
International plasmid epidemic : OXA-48 in Klebsiella, Enterobacter and E. coli OXA-181 (c. 7kb) OXA-48 (c. 62 kb) OXA-48 22 Sydney, 20th November 2014 Crown Copyright
OXA-48 and its known relatives: (ex-shewanella spp.) OXA Shewanella piezotolerans WP3 OXA Shewanella baltica OS223 OXA Shewanella putrefaciens CN-32 OXA-181 Shewanella xiamenensis S12 OXA-163 OXA-48 OXA-162 OXA-54 Shewanella oneidensis MR-1 OXA-55 Shewanella algae KB-1 OXA Shewanella loihica PV-4 OXA Shewanella pealeana ATCC 700345 OXA Shewanella halifaxensis HAW-EB4 A 23 Gram-negative Sydney, 20th November genus 2014 isolated Crown Copyright from lake sediments
All this yet we still can t answer some questions H104600421 H104600425 E. coli K. pneumoniae E. coli + K. pneumoniae, same patient 15000 10000 8144 7126 6108 5000 4072 3054 2036 1636 1018 506 similar NDM plasmids Do they indicate: transfer in the patient, or that patient was colonized by two strains already possessing related plasmids? 24 Sydney, 20th November 2014 Crown Copyright
Whole genome sequencing and resistance prediction Will it solve our problems, or just create new ones? Need an iterative catalogue of all resistance determinants By species? Need algorithm(s) for fast processing Will need high sensitivity and specificity low numbers of very major errors (resistant, but no R gene found so called susceptible) and major errors (susceptible, but R gene found and so called resistant) Will need to be validated to be equivalent or superior to routine susceptibility testing 25 Sydney, 20th November 2014 Crown Copyright
Concordance of genotypic vs. phenotypic antibiograms for E. coli (n=74) 26 Sydney, 20th November 2014 Crown Copyright J. Antimicrob. Chemother. (2013)
Can WGS eventually (ever?) replace some AST? All rapid tests for mechanisms = surrogates for rapid AST Absence of a resistance mechanism doesn t confirm susceptibility cannot indicate appropriate empiric therapy Presence of a resistance mechanism used to infer likely resistance indicates potentially inappropriate empiric therapy (Confirming susceptibility = prime criterion for appropriate therapy) Challenge is to replace surveillance in e.g. surveys first Likely to be problematic antibiotic classes with poor concordance 27 Sydney, 20th November 2014 Crown Copyright