National Reference Laboratory- the Irish Experience Rosemarie - - PowerPoint PPT Presentation

WGS as a surveillance tool in the National Reference Laboratory- the Irish Experience

Rosemarie Slowey 25th April 2019

State Veterinary Service

Introduction of WGS in the AMR NRL

• Began in 2016
• Illumina Miseq – shared with NRLs for

Campylobacter, Salmonella, Listeria, S. aureus and E. coli as well as virology division

• Aim: implementation of WGS as a routine typing

tool

• Initial setup – postdoc from University College

Dublin, support from Illumina

WGS Workflow

DNA Extraction

• Ultraclean microbial DNA isolation kit
• MagNa Pure (Gram negative)

Library prep • Nextera XT → Flex kit Sequencing • V3 kit Assembly

• Bionumerics

Analysis

• Bionumerics, Blast, CGE tools

Staff

• 2 Laboratory analysts, 1 research
• fficer
• Training from Illumina, Applied

Maths, EURLs To date 10 Campylobacter, 35 Enterococcus, 250 E. coli and 220 Salmonella sequenced

Initial Challenges

• Clustering related to DNA extraction

method for Gram negatives

• Issues with Qubit calibration
• IT security issues – FTP, Basespace,

CGE access

• New server- data storage
• Computer upgrades required
• Bionumerics affected by network

speed

Why do we use WGS?

• 1. To validate results of AST testing before

inclusion in EUSR/ national one health report

• 2. To assess trends and screen for emerging

genotypes

• 3. To contribute towards enhanced AMR

surveillance (National Action Plan)

• 4. To provide more robust data to inform

stakeholders-colleagues formulating policy, prescribing vets and farmers

To validate results of AST testing

High IMI/ ETR/TEM MIC blaCMY-2 or Amp C promoter region mutations ESBL genes present in all 13 isolates 11x bla CTX-M- 1 2 x bla SHV-2

• S. Orion

CTX, Caz Res No ESBL genes Identified No ESBL genes Identified in 4

• f 156

presumptive positive ESBL E. coli

EUSR 2016 - prevalence of ESBL-producing E. coli from broilers

To assess trends

Proportion of broiler caecalsamples positive for presumptive ESBL- producing E. coli

ESBLs in broiler E. coli isolates

blaSHV-12 isolates blaCTX-M-1 isolates

BlaCTX-M-1 and BlaSHV-12 predominated, with diverse ST-types

blaCTX-M-1(n=69) blaSHV-12 (n=20) IncI1 94 50 ST-3 84 ST26 or 36 35 IncF 74 65 F43:A:B- 15

ESBLs in broiler E. coli isolates ctd.

blaCTX-M-1

• 50% CTX-M-15+ Sul2+Tet(A)
• 57% aadA5/ dfrA17 class 1

integron BlaSHV-1

• 66%- QnrS1

No O1/ O2/O78 strains 37% of 2018 isolates EAST-1 positive % isolates containing IncI1 and IncF Plasmids

Current situation

EUSR 2017 - prevalence of ESBL-producing E. coli from fattening pigs

To screen for emerging genotypes

• Prevalence of ESBL-

producing E. coli lower in pigs (18%)

• Prevalence of blaCTX-

M-15 in pig E. coli increased from 2015 to 2017

• No ST131
• blaCTX-M-15

associated with ISEcp1

• Variable resistance

gene combinations

• IncF, IncY, Col

plasmids

To provide more robust data to inform stakeholders-case study

History : Beef suckler herd of 50 - 60 cows and calves lost 11 cows and 31 calves between 2015-17 Presentation : enteritis in young calves, post caesarean infections unresponsive to antimicrobial treatment Post- mortem findings- MDR E. coli isolated from internal organs and faeces Was this the cause of mortality? Treatment options?

WGS Results ST-10 – WGS Virulence factors: F17a ST-88 with mutations in the AmpC promoter region Virulence Factors: EAST-1, CS31A ,

microcin M part of colicin H, siderophore receptors, extracellular serine protease and increased serum survival, adherence protein and long polar fimbrae.

Phenotype 1 isolate 3 Isolates Amp-C Phenotype

AMP-CHL-CIP-GEN-NAL-SMX-TET-TMP AMP-CTX-CAZ-CIP-CHL-GEN-NAL-SMX-TET-TMP

Where has WGS been most useful in our lab?

• Rapid confirmation of genotypes from assemblies
• More efficient than AST testing of less common and

fastidious bacteria (Pasteurella multocida)

• Less time consuming than individual ESBL PCRs +

Sanger sequencing

• Additional information- not just AMR
• Provides information on what resistance genes are-

and are not circulating

The Challenges

• QC of data without

bioinformatician

• What to do with “grey hits”?
• Still takes 7 working days from

culture to assembled files

• Failed runs
• Extra costs- software and

contracts

Entry N50 NrContigs AvgDeNovoCover Length 1 69596 161 73.0 2875005 2 30240 450 53.9 5468620 3 15971 983 35.7 5369509 4 5063 2772 34.5 6172247 5 54598 213 89.2 2712757

Final Thoughts

• WGS EQAS like GMI PT will be

essential

• No hits ≠ no resistance genes
• Support will be required from

EURL

• Amount of information can be
• verwhelming

Acknowledgements

Thanks to my colleagues in the AMR NRL and the veterinary laboratory service, DAFM.