listeria: how i learned to stop worrying and love the ......listeria monocytogenes was chosen for...

Brendan Jackson, MD, MPHLCDR US Public Health Service

Division of Foodborne, Waterborne, and Environmental DiseasesCenters for Disease Control and Prevention

National Center for Emerging and Zoonotic Infectious DiseasesDivision of Foodborne, Waterborne, and Environmental Diseases

Listeria: How I Learned to Stop Worrying and Love the Sequencer

With apologies to Mr. Kubrick

Do Cases Represent an Outbreak?

Find Related Cases byPulsed-Field Gel Electrophoresis (PFGE)

=

Limitation: Genetically Unrelated Isolate Might Appear Same by PFGE

=?

Limitation: Genetically Related Isolate Might Appear Different By PFGE

=

?

Listeria WGS Pilot Project

Started September 2013 Goal: Sequence all Listeria monocytogenes isolates Near real-time (<1 week for patient isolates)

Public Health Agency of Canada

National Center for Biotechnology Information

Presenter

Presentation Notes

To reinvigorate listeriosis surveillance and control, a diverse group of agencies, including those listed below, began a whole-genome sequencing (or WGS) pilot project. Our goal is to improve detection and investigation of outbreaks by combinding WGS with epidemiologic data.

Why Listeria monocytogenes? Illness is rare but serious, costly,

and commonly outbreak associated

Current subtyping methods (e.g., pulsed-field gel electrophoresis) are not ideal

Listeria genome is fairly small and relatively easy to sequence and analyze

Strong epidemiologic surveillance (Listeria Initiative)

Strong regulatory component

Presenter

Presentation Notes

Listeria monocytogenes was chosen for this project because it is rare but serious, costly, and commonly outbreak associated, and—critically—controllable. In terms of technology, current subtyping methods are not ideal, whereas the Listeria genome is fairly small and relatively easy to sequence and analyze. Finally, we have both strong epidemiologic surveillance through the Listeria Initiative and a strong regulatory component.

Listeria WGS Analysis

Kmer

High-quality single nucleotide polymorphism (hqSNP)

Whole-genome multilocussequence typing (wgMLST)

SNPs wgMLSTallele

differences

1 1

gene 1 gene 2 gene 3

Relationship Between SNPs and wgMLST Alleles

Presenter

Presentation Notes

Consolidate multiple workflows

SNPs wgMLSTallele

differences

1 1

2 2



Presenter

Presentation Notes


SNPs wgMLSTallele

differences

1 1

2 2

5 2



Presenter

Presentation Notes


Listeria Cluster Metrics Before and After WGS

14

0 2 613

19

6 5 4

2021

6 93

98

0

20

40

60

80

100

120

No. of clustersdetected

No. of clustersdetected sooneror only by WGS

No. of outbreakssolved

(food sourceidentified)

Median no. ofcases per

cluster

No. of caseslinked to food

source

Pre-WGS (Sept 2012–Aug 2013)

WGS Year 1 (Sept 2013–Aug 2014)

WGS Year 2 (Sept 2014–Aug 2015)

How WGS Influenced Investigations Show that apparent PFGE clusters are not single-source

outbreaks or are pseudo-clusters

Refine outbreak case definitions by excluding cases

Show that isolates with different PFGE patterns are related

Increase confidence in the link between human and product isolates

Characterize the ecology of long-term pathogen reservoirs in the food chain

BREAKING UP PFGE CLUSTERS

PFGE Cluster Possibly Associated with Uncommon Deli Meats, 2013

0

1

2

3

Week of Isolation

Num

ber o

f Iso

late

s

92% (12/13)reported eating deli meat

(incl. liverwurst, head cheese)

Presenter

Presentation Notes


WGS for PFGE Cluster

wgMLST (<All Characters>)

100

959085807570 Id

PNUSAL000156PNUSAL000250PNUSAL000200PNUSAL000146PNUSAL000428PNUSAL000079PNUSAL000029PNUSAL000309PNUSAL000350

63[50-73]

46[26-59]

242[26-274]

251[26-525]

240[26-525]

Allele median[min-max] differences


100

959085807570 Id

PNUSAL000156PNUSAL000250PNUSAL000200PNUSAL000146PNUSAL000428PNUSAL000079PNUSAL000029PNUSAL000309PNUSAL000350

63[50-73]

46[26-59]

242[26-274]

251[26-525]

240[26-525]

Allele median[min-max] differences

CLUSTER

REFINING CASE DEFINITIONS

Presenter

Presentation Notes


2013 Outbreak Linked to French-Style Soft Cheese

WGS Refines Case Definition (Same PFGE)“How Close is Close?”

WGS analysis by Enteric Diseases Laboratory Branch, CDC

2013L-55432013L-5598

2013L-56702013L-57472013L-5743

2013L-53742013L-5418

2013L-51212013L-5449

2013L-55352013L-5687

2013L-5688

Median: 80SNPs

Median: 9.5 SNPs

Duplicate isolates All isolates shown part of

a PFGE-defined Listeriacluster investigation

Varying levels of relatedness within the PFGE cluster

Can fine-tune analyses by focusing on cases at varying levels of genetic relatedness

Median: 33 SNPs

Presenter

Presentation Notes

In this example, all isolates shown were part of a PFGE-defined cluster investigation. In many investigations of PFGE-defined clusters, we suspect that some cases aren’t truly linked to the others, but it can be hard to sort out the signal (or those cases truly linked to a common food source) amidst the noise, or the background unrelated cases. Here, we could fine-tune our analyses by focusing on cases at varying levels of genetic relatedness, though the question always is “how close is close?” In this investigation, all cases in red were from a single state.

Epidemiology and Exposure Data Still Needed


2013L-55432013L-5598

2013L-56702013L-57472013L-5743

2013L-53742013L-5418

2013L-51212013L-5449

2013L-55352013L-5687

2013L-5688

Median: 80SNPs

Median: 9.5 SNPs

Duplicate isolates

Median: 33 SNPsNone of these patients had eaten kale

Isolate from kale

Isolate from kale fairly closely related to other isolates

None of the patients had eaten kale, however

Although more specific, WGS data still need to be interpreted in the context of all the evidence

Presenter

Presentation Notes

We learned that the isolate in black, which is fairly closely related to isolates in red and blue, was a food isolate from kale. This got us excited… so we checked on whether any of the patients had eaten kale. None had. This was a reminder that simply comparing food and clinical isolates by WGS is not sufficient to establish a link; epidemiology and exposure data are still needed. This cluster too remains unsolved.

TYING PFGE PATTERNS TOGETHER

Identified Listeriosis Cluster Not Evident by PFGE

5 cases, 3 PFGE patterns, 3 states All patients originally from

Eastern Europe

8 hqSNPs13.5 hqSNPs

2013 isolate

2 hqSNPs

68 hqSNPsNearest neighbor


State 1 isolateState 2 isolateState 1 isolate

State 3 isolateState 1 isolate

State 1State 1State 1State 2State 3

Presenter

Presentation Notes

I’ll provide a few examples of clusters detected and investigations changed by WGS. In this example, sequencing detected a cluster not evident by PFGE since there were 5 cases with 3 PFGE patterns (shown at right in green, yellow, and red) from 3 states. The SNP tree below shows that these five isolates were highly genetically related, and epidemiologic data supported this link since all 5 patients turned out to be from Eastern Europe. Unfortunately, we were only able to obtain limited food exposure data from several of these patients and were unable to solve this cluster.

Examples from Outbreaks Linked to Ice Cream and Middle Eastern-Style Soft Cheese


100

9998

cdc_id

2015L-62872015L-66282014L-63882014L-64172015L-64342013L-55772013L-57182014L-66322013L-54222013L-57482013L-51862015L-61032015L-63282015L-61232014L-66082013L-53462012L-55102013L-51532014L-61822012L-51912010L-21562014L-62392015L-64582015L-65872013L-5195

SourceSite

BLOODBloodBloodBloodBloodBloodBloodBLOODBloodBloodCSFBloodBloodStoolFluidBloodParacentesis BloodBloodBloodBlood, NOSBloodBloodBloodCSFQueso Fresco

14[4-26]

15[4-39]

15[4-101]

wg M LS T (< A ll C ha ra c te rs > )1

00

90

80

70

60

50

40

30

20

10

12.5[0-18]

2571

Allele median[min-max] differences at node

PFG E-AscI-pattern

G X6A16.0026G X6A16.0026G X6A16.0061G X6A16.0020G X6A16.0282

PFG E-ApaI-pa ttern

G X6A12.0227G X6A12.0227G X6A12.0026G X6A12.0227G X6A12.0355

Ice Cream

Soft Cheese

LINK BETWEEN HUMAN AND PRODUCT ISOLATES

Presenter

Presentation Notes


0

1

2

3

4

1 8 15 22 29 6 13 20 27 3 10 17 24 31

June July August

Num

ber o

f Cas

es

Week of isolation in 2014

States:AL, IL (4), MA, TN

Cluster Epi

• 6/7 (86%) male • Ages 40-85 years• Two deaths• LI forms for 2 patients • Both ate queso fresco• Matching cheese and

environmental isolates noted from Indiana from 2011

July 2014: PFGE Cluster of Listeriosis Found by PulseNet

Presenter

Presentation Notes

This next example is from an recent investigation. In this cluster there were 9 cases with matching PFGE patterns from across the country, but WGS showed that 5 isolates (shown in yellow) were closely related. Four of these patients lived metropolitan Chicago and a 5th lived a few hours away in Michigan. Some of the patients had died, limiting the food history that could be obtained. It was suspicious, however, that 2 of 2 patients asked had eaten sprouts, which are not commonly reported by listeriosis patients. In mid August, we learned that FDA had isolated Listeria with indistinguishable PFGE patterns from sprouts and a production facility in Chicago. Thirteenth most common pattern in PulseNet (~1% of isolates, all human).

hqSNP PhylogenyAug 13, 2014

CA__M14X00709|02/27/2014|2014L-6114

IN__14REF0128|11/17/2013|2013L-5692

IL__131878|09/20/2013|2013L-5579

IL__132446|11/02/2013|2013L-5672

IL__125821|06/12/2013|2013L-5282

IL__134857|07/11/2014|2014L-6385

MA_14EN4302|06/15/2014|2014L-6389

CT__339767001|08/28/2013|2013L-5500

MA_13EN6104|08/06/2013|2013L-5430

MA_13EN7591|missing|2013L-5572

CT__322656001|07/04/2013|2013L-5352

PA__M13016330001A|09/09/2013|2013L-5517

PA__M13017889001A|10/10/2013|2013L-5564

IL__124338|03/28/2013|2013L-5149

IL__134808|06/18/2014|2014L-6325

IL__134856|07/11/2014|2014L-6384

IL__134803|06/07/2014|2014L-6304

IL__124895|04/23/2013|2013L-5175

0.02

1a

Isolate before June 2014 (GX6A16.0319|GX6A12.0246)Cluster 1407MLGX6-1 (GX6A16.0319|GX6A12.0246)

Isolate before June 2014 (GX6A16.0319|GX6A12.0244)

Phylogeny by EDLB

38 [29-47]

46.5 [34-58]

74

2.5 [0-7]

36

New England

clade

Excluded clade

(mostly IL)

PA clade

Presenter

Presentation Notes

This slide from earlier in our investigation shows multiple clades in different colors and their corresponding hqSNP distances. Each of these clades cluster by geography quite well. Three of the four isolates within the current cluster are located at the bottom of the tree on clade 1bi and are highlighted in yellow. (there are two other IL isolates from 2013 that were investigated in the cluster last year; food exposure information is lacking for these cases) You’ll notice in peach two PA cases that are close together. This leads us to believe that the remaining two isolates with pending WGS from PA are likely not related to the current cluster. In pink there is a cluster of cases from New England And at the top you’ll see in green that mostly includes isolates from IL; there are is an IL isolate that was identifed by PFGE within this cluster and as you can see it is distant from remaining IL isolates.

0

1

2

3

4

1 8 15 22 29 6 13 20 27 3 10 17 24 31

June July August

Num

ber o

f Cas

es

Week of isolation in 2014

Highly-related by WGS

Less related by WGS

Cluster Epidemic Curve

IL ILIL IL MI

Presenter

Presentation Notes

This next example is from an recent investigation. In this cluster there were 12 cases with matching PFGE patterns from across the country, but WGS showed that 5 isolates (shown in yellow) were closely related. Four of these patients lived metropolitan Chicago and a 5th lived a few hours away in Michigan. Some of the patients had died, limiting the food history that could be obtained. It was suspicious, however, that 2 of 2 patients asked had eaten sprouts, which are not commonly reported by listeriosis patients. In mid August, we learned that FDA had isolated Listeria with indistinguishable PFGE patterns from sprouts and a production facility in Chicago.

Counties where cases are located

Suspicious, but running out of leads

September 4: A Crack in the Case

Isolates uploaded with matching PFGE pattern from mung bean sprouts and sprout irrigation water

Collected as part of routine FDA inspection

Where was the facility?

Exposure Data

LI forms 1 Illinois patient ate sprouts Other patient interviewed with version of questionnaire that didn’t

include sprouts; now unreachable 1/1 sprout consumption vs. 35/760 (5%) historical

cases OR = 20.1 (95% CI, 1.06–infinity) But one case?

Information from Michigan

Patient denies sprout consumption, but reports eating pad thai at several Asian-style restaurants

At least two serve mung bean sprouts

87[87-87]

5[2-6]5[2-54]

55[2-69]

64[2-191]

Environmental IsolateSprout

Allele Median [min-max]

Whole-genome Multilocus Sequence Typing (wgMLST)

100

999897969594

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

TennesseePennsylvaniaIllinoisIllinois

IllinoisIllinoisPennsylvaniaMassachusettsAlabamaIllinois

TennesseePennsylvania

IllinoisIllinoisEnvironmental IsolateSproutIllinoisIllinois

PennsylvaniaMassachusetts

AlabamaIllinois

0.02

68 hqSNPs

1 ± 1 hqSNPs [0-3]

58 hqSNPs [0-72]65.5 hqSNPs [54-72]

275 hqSNPs


hqSNP Analysis

Patient Isolates Virtually Identical to Sprout Isolates by WGS

Isolate from MI also highly-related (not

shown)

Presenter

Presentation Notes

WGS of the sprout isolates confirmed our suspicions, showing that they were very closely related to the nearby human cases. Note here how the 2 methods, the gene-based wgMLST and the SNP method, produced very similar phylogenetic trees.

Listeria monocytogenes Outbreak Linked to Mung Bean Sprouts

Despite limited epi data (2/2 consuming sprouts), WGS results and inspection findings enough to take action

CDC and states recommended that consumers not eat and retailers not buy sprouts from the producer

Ultimately the company agreed to permanently cease operations

Federal government later filed lawsuit to prevent firm from resuming production

Presenter

Presentation Notes

This next example is from an recent investigation. In this cluster there were 12 cases with matching PFGE patterns from across the country, but WGS showed that 5 isolates (shown in yellow) were closely related. Four of these patients lived metropolitan Chicago and a 5th lived a few hours away in Michigan. Some of the patients had died, limiting the food history that could be obtained. It was suspicious, however, that 2 of 2 patients asked had eaten sprouts, which are not commonly reported by listeriosis patients. In mid August, we learned that FDA had isolated Listeria with indistinguishable PFGE patterns from sprouts and a production facility in Chicago.

Caution: Isolates in an Outbreak Are Not Always Highly Related

100

9590858075706560555045

4 [0–13]

5 [0–14]

1563 [0–1696]

Allele differences at node: median [min–max]

Patient isolatesApple and environmental isolates

Cluster 1

Cluster 2

Example of polyclonality


ECOLOGY OF PATHOGEN RESERVOIRS

Presenter

Presentation Notes


2013 Outbreak Linked to Farmstead CheeseCFSAN006121|CFSAN006123CFSAN006122CFSAN004369|CFSAN004358

CFSAN004366|CFSAN004352|

CFSAN004362CFSAN0043492013L-5214CFSAN004354CFSAN004376

CFSAN004367CFSAN004359



CFSAN004357CFSAN004364CFSAN004353CFSAN004368CFSAN004348CFSAN004351CFSAN004350


CFSAN004360CFSAN004363CFSAN004373

CFSAN004377CFSAN004374CFSAN004356

2010L-1790CFSAN004361

2013L-52832013L-5275

2013L-53572013L-5554

2013L-52232013L-5337

2013L-5298

Outbreak cases, 2013<10 high quality

SNPs

Environmental and food samples from cheese-making facility, 2010–2013

2013L-5514

Patient isolate, 2010


Isolates from 2010 virtually identical to isolates from 2013

Presenter

Presentation Notes


2013 Outbreak Linked to Mexican-Style Soft Cheese

663 hqSNPs

6 hqSNPs[2-18]

Cheese ACheese ACheese ACheese BCheese BCheese CCheese ACheese DCheese ECheese FClinical isolateClinical isolateClinical isolateClinical isolateClinical isolateClinical isolate2013 isolate

0.1

Nearest neighbor


Presenter

Presentation Notes

This outbreak provides a different sort of example. Here there were 7 cases in a single Eastern state among Hispanic persons. The health department initially tried to find a locally-produced food source without success. When WGS showed that a patient isolate from the West coast was highly related by WGS, they widened the scope of the investigation and ultimately identified the culprit cheese, leading to a recall and eventual closure of the production facility, which was found to have dreadful conditions. Listeria isolates from cheese samples were later found to be highly related to the clinical isolates.

2013–2014 Outbreak Linked to Central and South American-Style Soft Cheeses


100


13[2-22]

7[2-11]

12[4-18]

5

4[6-9]

7[4-10]

SourceSite

Environmental SwabsEnvironmental SwabsBLOODCheeseCheeseplacentaCSFCheeseBloodBloodplacentaEnvironmental SwabsEnvironmental SwabsCSFUrineBlood


13[2-22]

7[2-11]

12[4-18]

5

4[6-9]

7[4-10]

Different PFGE pattern from others


Listeriosis Linked to Recalled Stone Fruits

• July 2014 recall receives extensive media coverage

• Hundreds of inquiries from concerned clinicians and public

• Stone fruit isolates obtained from company

• 4 human isolates in 2014 with PFGE match

• Was it an outbreak?

MMWR. Mar 20, 2015.

Patients with Recurrent Listeria Infections

~70 year old man: 3 positive blood cultures,attributed to pacemaker lead infection

June 2013 Sept 2013 May 2014



June 2013 Sept 2013 May 2014

3 hqSNPs

All food removed from home



June 2013 Sept 2013 May 2014

3 hqSNPs 22 hqSNPs

23 hqSNPs




~80 year old man: 2 positive blood cultures 14 months apart; isolates 19 hqSNPs different

June 2013 Sept 2013 May 2014

3 hqSNPs 22 hqSNPs

23 hqSNPs


Listeria WGS Discussion

Helped detect more clusters and solve more outbreaks

Focused epidemiological resources

Identified new food sources of listeriosis

Epidemiologic data remain essential

WGS not just for Listeria anymore

Presenter

Presentation Notes

In summary, this project represents the first use of routine, prospective WGS-enhanced surveillance. In the WGS period, our surveillance detected more clusters, solved more outbreaks, and allowed linkage of a sporadic case to a likely food source. We were also able to identify new food sources of listeriosis, including lettuce and nectarines. Even with this advanced technology, it’s clear that epidemiology remains essential to the interpretation of subtyping results, and it must keep pace. Missing data remain a challenge and we continue to work closely with state partners to improve completeness. Finally, this work is a great example of the power of inter-agency and multidisciplinary collaboration.

For more information please contact Centers for Disease Control and Prevention

1600 Clifton Road NE, Atlanta, GA 30333Telephone: 1-800-CDC-INFO (232-4636)Visit: www.cdc.gov | Contact CDC at: 1-800-CDC-INFO or www.cdc.gov/info

The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention.

National Center for Emerging and Zoonotic Infectious DiseasesDivision of Foodborne, Waterborne, and Environmental Diseases

State, Local, and Territorial Health Departments

Advanced Molecular Detection Initiative

Food and Drug Administration

US Department of Agriculture’s Food Safety and Inspection Service

Enteric Diseases Laboratory Branch

Enteric Diseases Epidemiology Branch

Outbreak Response and Prevention Branch

Epidemic Intelligence Service

Atlanta Research and Education Foundation

Acknowledgments

An Early Use of WGS for ListeriaJune 27–July 4, 2013

Day 0Minnesota Dept. of Health notes 2 PFGE-matched

isolates

Day 0PulseNet query identifies 3 more cases in other

states

Day 0All 5 patients report

soft cheeses on Listeria Initiative form

OR = 10.8 (1.8–∞)

Day 13 patients

re-interviewed; 2 name Company A cheese

Day 1 (Friday)Matching isolates found

previously from Company A plant; inspection begins

Day 7 (4th of July)Company A and FDA

announce recall

Day 6 Cheese prelim.

positive for Lm; public warning issued

Presenter

Presentation Notes

Note that this is a simplified version. Many steps took place between day 1 and day 5. Bottom line is that the outbreak was solved in record time using both epi and molecular data. It’s not typical to solve a multi-state outbreak anywhere near this fast

Pre-Packaged Salad Recall, 2014WGS Links Sporadic Case

March 13: Lettuce recall based on Listeria testing in Canada

April 21: OH inquires about PFGE pattern of lettuce isolate Finds matching case with onset

in late March 6th most common pattern in US

April 25: State learns that patient consumed multiple types of pre-packaged lettuce, including recalled brand

May 13: Canada reports clinical and lettuce isolates have 0 SNP difference among ~2.7 million base pairs examined

Presenter

Presentation Notes

On March 13th, Canada and FDA issued a recall of pre-packaged salad based on Listeria testing in Canada. On April 21, Ohio inquired about the PFGE pattern of the lettuce isolate, and by the end of the day, found a matching case with onset March 31. This PFGE pattern was, however, the 6th most common in the US, so the significance of this match was unclear. A few days later, Ohio learned that the patient consumed multiple types of pre-packaged lettuce, possibly including the recalled brand. Last month, Canada reported that the Ohio isolate and the lettuce isolate differed by zero SNPs among the about 2.7 million base pairs examined.

100

9998

CN lettuce

WGS Analysis by EDLB, CDC

66 hqSNPs [46-84]

55 hqSNPs [5-66]

24 hqSNPs [0-94]

43 hqSNPs

21.5 hqSNPs [0-33]

58 hqSNPs [46-58]

2013L-5439

2013L-5527

2013L-5545

2013L-5556

2013L-5203

2013L-5204

2013L-5308

2013L-5473

2013L-5085

2013L-5587

2013L-5333

2013L-5314

2013L-5182

2013L-5622

2013L-5623

2013L-5521

2013L-5345

2014L-6017

2013L-5548

Lettuce isolateClinical isolate

2013L-5540

2013L-5359

2013L-5297

2013L-5194

2013L-5560

2014L-6086 0.02

5 hqSNPs

wgMLSThqSNP

Presenter

Presentation Notes

WGS analysis at CDC showed similar results with a 5 SNP difference between isolates. The difference between the 2 results is minimal and results from differences in which SNPs are included in the analysis. Based on both lab and epi data, we have strong circumstantial evidence suggesting pre-packaged lettuce was the likely food vehicle in a sporadic case of listeriosis. This is the strongest link we have seen between contamination of leafy greens and a case.

An Analogy Attempt

Your OutbreakManuscript

(5,000 pages)

Your manuscript draft returns with edits…

An Analogy Attempt


Lots Your OutbreakManuscript

(5,000 pages)

An Analogy Attempt


Lots

From all 25 coauthors


(5,000 pages)

An Analogy Attempt


Lots

From all 25 coauthors (Don’t these CDC people have

anything better to do?)


(5,000 pages)

An Analogy Attempt


Lots

From all 25 coauthors (Don’t these CDC people have

anything better to do?)

No Track Changes and some of the pages are out of order


(5,000 pages)

How Can You Quantify the Changes?

Kmer Same-length confetti


(5,000 pages)


SNP Every letter

(SNP)


(5,000 pages)

CAT CAT



SNP Every letter

(SNP)

wgMLST Every section

(gene)


(5,000 pages)


listeria: how i learned to stop worrying and love the ......listeria monocytogenes was chosen for...

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