a whole new world - aphl · – sarah kogut, emily lutterloh, eleanor adams, rosalie giardina,...

June 6, 2019

A whole new world:Public health impact applying WGS and bioinformatic analysis in the AR Lab Network

Kimberlee Musser, PhDChief of Bacterial DiseaseWadsworth Center, NYSDOH

Regional AR Lab Network Laboratories

Acquired Carbapenemasegenes:

blaKPCblaNDM

blaVIMblaIMPblaOXA-48

(CP= carbapenemase producing)

CRPACarbapenem-Resistant

Pseudomonas aeruginosa

CRECarbapenem-Resistant

Enterobacteriaceae

CP-CRECP-CRPA

https://www.cdc.gov/drugresistance/pdf/16_264313-e_capers_ar_prevention_program_508.pdf

https://www.cdc.gov/vitalsigns/pdf/2018-04-vitalsigns.pdf

AR Lab Network –Northeast 2018 TestingCRE Isolate Submission

• 834 Tested• 542 identified as CP-CRE (65%)

– 479 KPC– 46 NDM – 21 OXA-48 like– 1 KPC/NDM– 1 NDM/OXA-48

CRPA Isolate Submission• 1336 Tested• 55 identified as CP-CRPA (4%)

– 29 VIM – 15 KPC – 3 OXA-2/OXA50 like– 2 NDM– 2 IMP – 1 IMP/NDM

CRE/CRPA Colonization Screening

Cepheid XpertCarba-R

Why whole-genome sequencing (WGS)?

Real-time PCR

• Detects 10 AR genes• Doesn’t identify:

– novel or emerging mechanisms of resistance

– AR genes to the variant level (example: blaKPC-2 vs blaKPC-5)

Whole-genome Sequencing

Identified isolates with up to 25 AR genes conferring resistance to 11 different classes of antibiotics

• Identify all AR genes in genome– Database includes over 5,500– Identify emerging and novel AR

genes

• Outbreak investigations– Identify transmission events between patients– Where is the AR gene located?

Antibiotic Resistance WGS at Wadsworth: Pipeline Overview

Wolfgang Haas, PhDNYS EID Fellow

If the data above suggests the isolates may be related:Mutation Event Analysis What are the genetic differences between the isolates?

Illumina Sequencing Reads

de novo assembly & quality control

Are the bacteria similar?

(MultilocusSequence Typing)

What antibiotic resistance genes are

present?

Where are the antibiotic resistance

genes located?Plasmid?

Emily Snavely, PhDAR Fellow

Total submitted for WGS: ~1,100Total analyzed with pipeline: ~850

PresenterPresentation Notes

Supplemental Reporting through LIMS

Gene Variant

MLST

WGS-determined Carbapenemase Gene Variants

• KPC (carbapenemase) is endemic • >2000 long term care facilities in the NYC metropolitan

area• Facilities face numerous challenges that can impact

infection control – Staffing shortage, turnover, and training– Limited private rooms– Inconsistent infection control procedures

• Facilities can also be involved with Candida auris

How can WGS assist in focusing finite Epi resources?

WGS in Endemic Settings: Epi Context

PresenterPresentation NotesGo in person and review charts because know what cases are actually linked. Help epi focus.

AR Lab Network: 4 WGS Investigations

NE Region

Wadsworth WGS Investigation

1- CP-CRPA Outpatient Urology Clinic2- CP-CRE – No transmission identified?3- CP-CRE- It’s complicated!4- Plasmid transmission

1- CP-CRPA Outpatient Urology Clinic

• The SNP analysis determined that 18 of the blaVIM-2 containing CP-CRPA isolates had SNP differences (0-20) suggesting they may share a common source (dark green)

• A subcluster of 8/18 isolates were 0-4 SNPs different from each other, indicating they were very closely related

• SNP analysis informed the epidemiological investigation

• 2018 CRPA analysis by WGS • Identified 26 blaVIM-2 gene variant• The WGS predicted MLST ST 111 for 23/26

• Epi investigation identified 14 who received care at the same outpatient urology clinic

• Male, private residence, no specific hospital connections identified

• Onsite visit identified practices inconsistent with standards of infection control

SNP Analysis of 25 blaVIM-2 gene variant isolates

September 2018: Infection Preventionists notified DOH of an outpatient urology practice common among cases

Index case: 72-year-old male with respiratory failure admitted for pneumonia (No foreign travel)

Pathogen: Klebsiella pneumoniae-OXA-48 from woundInvestigation Details:Long term care facility (LTCF): • 40 bed unit that care for ventilated residents • Not on contact precautions (CP)• Single point prevalence survey (PPS) - 37 residents

2- CP-CRE – No transmission identified?

3- CP-CRE- It’s complicated!

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Index Case (K. pneumoniae OXA-48)

KPC

Colonization Screening:• No New OXA-48• 14 KPC positive

Index case: 90 year old male with respiratory failure and admitted with sepsis (No foreign travel)

Pathogen: K. pneumoniae - NDM from blood and urineInvestigation Details:ACF “3”: 4 visits last several months• Private room/CP for C. difficileLTCF “C”: • 40 bed unit overflow care for ventilator/tracheostomy residents • Not on CP (4 roommates)• 5 Serial PPS (initial 3 as rings around index)

3- CP-CRE- It’s complicated!

Multiple carbapenemase-resistance mechanisms are present in isolates recovered

Facility C• Index case:

– NDM-1 K. pneumoniae • 105 total dual-rectal swabs received

– 45 real-time PCR positive swabs • 5 NDM positive• 34 KPC positive• 6 NDM and KPC positive

• 21 swabs sent for culturing– 15 swabs – at least 1 CP-CRE recovered

NDM-1 mutation events analysis- 5 of 7 areRelated to the index case

Key for Presentations

NYS IDRIsolateIDWGS AnalysisNYS IDRIsolate

MLSTCarbapenemase Variant

IDR1800050136-01-02A-12K. pneumoniae101OXA-48IDR1800050111-01-04A-1

IDR1800050112-01-04A-2K. pneumoniae392KPC-2IDR1800050112-01-04A-2











IDR1800050114-01-02A-3P. aeruginosaNon-typeableKPC-5IDR1800050142-01-03A-13

IDR1800052816-01-03B-5E. coli131/ 43KPC-2IDR1800052807-01-04B-1

IDR1800052807-01-04B-1K. pneumoniae392KPC-2 IDR1800052810-01-05B-2

IDR1800052815-01-02 B-4E. cloacae536KPC-3IDR1800052813-01-04B-3

IDR1800052813-01-04B-3K. pneumoniae45KPC-3IDR1800052815-01-02 B-4

IDR1800052810-01-05B-2K. pneumoniae258KPC-3IDR1800052816-01-03B-5




IDR1800052825-01-04B-9K. pneumoniaeNon-typeableKPC-3IDR1800052825-01-04B-9

IDR1800055385-01-02C-1K. pneumoniaeNon-typeableNDM-1IDR1800055385-01-02C-1






IDR1900001675-01-00*C-11K. pneumoniaeNon-typeableNDM-1IDR1900001556-01-02C-7

IDR1900003143-01-02C-17K. pneumoniae258KPC-2IDR1900001561-01-02C-8

IDR1800055385-01-23C-2S. marcescensNo MLSTKPC-3IDR1900001564-01-02C-9

IDR1900003132-01-04C-14E. coli95 / 1KPC-3IDR1900001565-01-02C-10

IDR1800055388-01-02C-4K. pneumoniae45KPC-3IDR1900001675-01-00*C-11






IDR1900006114-01-04 C-18K. pneumoniae258KPC-3IDR1900003143-01-02C-17

IDR1900003141-01-03C-16K. pneumoniae15KPC-3IDR1900006114-01-04 C-18

Sheet1

IsolateIDWGS AnalysisIsolateIDWGS AnalysisIsolateIDWGS AnalysisIsolateIDWGS Analysis

MLSTCarbapenemase VariantMLSTCarbapenemase VariantMLSTCarbapenemase VariantMLSTCarbapenemase Variant

A-12K. pneumoniae101OXA-48A-12K. pneumoniae101OXA-48B-5E. coli131/ 43KPC-2C-1K. pneumoniaeNon-typeableNDM-1

A-2K. pneumoniae392KPC-2A-2K. pneumoniae392KPC-2B-1K. pneumoniae392KPC-2C-3K. pneumoniaeNon-typeableNDM-1

A-10K. pneumoniae392KPC-2A-10K. pneumoniae392KPC-2B-4E. cloacae536KPC-3C-6K. pneumoniaeNon-typeableNDM-1





A-11K. pneumoniae1481KPC-2A-11K. pneumoniae1481KPC-2B-8K. pneumoniae258KPC-3C-17K. pneumoniae258KPC-2

A-1K. pneumoniae258KPC-3A-1K. pneumoniae258KPC-3B-9K. pneumoniaeNon-typeableKPC-3C-2S. marcescensNo MLSTKPC-3

A-13K. pneumoniae258KPC-3A-13K. pneumoniae258KPC-3C-14E. coli95 / 1KPC-3

A-7K. pneumoniae258KPC-3A-7K. pneumoniae258KPC-3C-4K. pneumoniae45KPC-3

A-8K. pneumoniae258KPC-3A-8K. pneumoniae258KPC-3C-10K. pneumoniae45KPC-3

A-3P. aeruginosaNon-typeableKPC-5A-3P. aeruginosaNon-typeableKPC-5C-5K. pneumoniae258KPC-3

B-5E. coli131/ 43KPC-2C-8K. pneumoniae258KPC-3

B-1K. pneumoniae392KPC-2C-12K. pneumoniae258KPC-3

B-4E. cloacae536KPC-3C-15K. pneumoniae258KPC-3



B-6K. pneumoniae258KPC-3



B-9K. pneumoniaeNon-typeableKPC-3

C-1K. pneumoniaeNon-typeableNDM-1







C-17K. pneumoniae258KPC-2

C-2S. marcescensNo MLSTKPC-3

C-14E. coli95 / 1KPC-3









KPC-3

NYS IDRIDWGS AnalysisNYS IDRIDWGS AnalysisNYS IDRIDWGS Analysis

MLSTCarbapenemase VariantMLSTCarbapenemase VariantMLSTCarbapenemase Variant

IDR1800050136-01-02Klebsiella pneumoniae101OXA-48IDR1800050111-01-04Klebsiella pneumoniae258KPC-3IDR1900003141-01-03Klebsiella pneumoniae15KPC-3

IDR1800050112-01-04Klebsiella pneumoniae392KPC-2IDR1800050142-01-03Klebsiella pneumoniae258KPC-3IDR1800052813-01-04Klebsiella pneumoniae45KPC-3



IDR1800050120-01-21Klebsiella pneumoniae392KPC-2IDR1800052815-01-02 Enterobacter cloacae536KPC-3 IDR1800052810-01-05Klebsiella pneumoniae258KPC-3


IDR1800050127-01-02Klebsiella pneumoniae1481KPC-2 IDR1800052810-01-05Klebsiella pneumoniae258KPC-3IDR1800050122-01-02Klebsiella pneumoniae258KPC-3




IDR1800050122-01-02Klebsiella pneumoniae258KPC-3IDR1800052825-01-04Klebsiella pneumoniaeNon-typeableKPC-3IDR1800052821-01-03Klebsiella pneumoniae258KPC-3

IDR1800050126-01-02Klebsiella pneumoniae258KPC-3IDR1800055385-01-23Serratia marcescensNo MLSTKPC-3IDR1800052822-01-02Klebsiella pneumoniae258KPC-3

IDR1800050114-01-02Pseudomonas aeruginosaNon-typeableKPC-5IDR1900003132-01-04Escherichia coli95 / 1KPC-3IDR1800055389-01-02Klebsiella pneumoniae258KPC-3

IDR1800052816-01-03Escherichia coli131/ 43KPC-2IDR1800055388-01-02Klebsiella pneumoniae45KPC-3IDR1900001561-01-02Klebsiella pneumoniae258KPC-3


IDR1800052815-01-02 Enterobacter cloacae536KPC-3IDR1800055389-01-02Klebsiella pneumoniae258KPC-3IDR1900003140-01-02Klebsiella pneumoniae258KPC-3

IDR1800052813-01-04Klebsiella pneumoniae45KPC-3IDR1900001561-01-02Klebsiella pneumoniae258KPC-3IDR1900006114-01-04 Klebsiella pneumoniae258KPC-3

IDR1800052810-01-05Klebsiella pneumoniae258KPC-3IDR1900003130-01-06Klebsiella pneumoniae258KPC-3IDR1800052825-01-04Klebsiella pneumoniaeNon-typeableKPC-3

IDR1800052818-01-02Klebsiella pneumoniae258KPC-3IDR1900003140-01-02Klebsiella pneumoniae258KPC-3

IDR1800052821-01-03Klebsiella pneumoniae258KPC-3IDR1900006114-01-04 Klebsiella pneumoniae258KPC-3PavillionSilvercrestPalm Gardens

IDR1800052822-01-02Klebsiella pneumoniae258KPC-3IDR1900003141-01-03Klebsiella pneumoniae15KPC-3

IDR1800052825-01-04Klebsiella pneumoniaeNon-typeableKPC-3

IDR1800055385-01-02Klebsiella pneumoniaeNon-typeableNDM-1






IDR1900001675-01-00*Klebsiella pneumoniaeNon-typeableNDM-1

IDR1900003143-01-02Klebsiella pneumoniae258KPC-2

IDR1800055385-01-23Serratia marcescensNo MLSTKPC-3

IDR1900003132-01-04Escherichia coli95 / 1KPC-3







IDR1900006114-01-04 Klebsiella pneumoniae258KPC-3


Citrobacter freundii - KPC

C. Freundii - KPC

4- Plasmid transmission

Potential Contacts

K. Pneumoniae - KPC E. Coli - KPC

KPC-2 Plasmid A

KPC-2 Plasmid B

WGS (MinION) Results

Bacteria or plasmids can spread throughout a healthcare facility.

• Only method to confirm plasmid-mediated transmission events

• What AR plasmids are circulating in our region?

– How diverse are the plasmids within our region?

– Transmission dynamics of antibiotic resistance

– Identify novel plasmids

Opportunities to expand AR knowledge: Long-read Sequencing (Nanopore MinION)

PresenterPresentation NotesSome mechanisms of resistance are very widespread and repetitive. Identify isolates with both chromosomal and plasmid copies of AR genes

Conclusions• Antibiotic resistance is one of the

biggest public health challenges of our time

• Detecting transmission in Northeast is complicated by the widespread dissemination of KPC– WGS provides the resolution

necessary to detect recent transmission events

• WGS is a valuable tool to inform epidemiologic investigations

Acknowledgements• Wadsworth Center, NYSDOH

– Bacteriology Laboratory– Elizabeth Nazarian– Emily Snavely– Navjot Singh – Wolfgang Haas– Kara Mitchell– Janine Bodnar– Christine Wagner– Phil Weeber– Shannon Morris– Kailee Cummings– Joseph Shea– Nadine Peinovich– Shirley Kelly-Parson– Alyssa Sossei This research was supported in part by an appointment to the Antimicrobial Resistance Fellowship Program administered by the Association of Public Health Laboratories (APHL) and funded by the CDC. This work was supported by the New York State

Department of Health and by Cooperative Agreement Number NU50CK000423 funded by the Centers for Disease Control and Prevention (CDC).

• Applied Genomics Technology Core• Bioinformatics Core • CDC• APHL• NYSDOH Healthcare Epidemiology and

Infectious Disease – Sarah Kogut, Emily Lutterloh, Eleanor Adams, Rosalie

Giardina, Monica Quinn, Ron Jean Denis– CDC, Belinda Ostrowsky

• Northeast Public Health Labs• NYC PHL

– Jennifer Rakeman, Ying Lin

• Clinical Laboratory Submitters• Facilities

Wadsworth Center ARLN Team

Slide Number 1Regional AR Lab Network LaboratoriesSlide Number 3Slide Number 4AR Lab Network –Northeast 2018 TestingSlide Number 6Why whole-genome sequencing (WGS)?Antibiotic Resistance WGS at Wadsworth: Pipeline OverviewSlide Number 9Slide Number 10Slide Number 11AR Lab Network: 4 WGS Investigations1- CP-CRPA Outpatient Urology Clinic2- CP-CRE – No transmission identified?Slide Number 15Slide Number 16Slide Number 17Multiple carbapenemase-resistance mechanisms are present in isolates recovered4- Plasmid transmissionSlide Number 20ConclusionsAcknowledgements

a whole new world - aphl · – sarah kogut, emily lutterloh, eleanor adams, rosalie giardina,...

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