Understanding and Evaluating

Microbiological Sampling and Testing

Udit Minocha

Microbiologist

April 2022

7/15/2022

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Today’s Presentation

• Sampling methods

• Assessing sampling plans and testing methods

• Method validations and laboratory quality assurance

• FSIS testing programs, methods and pathogen-specific issues to consider

• Industry testing activities

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USDA Food Safety and Inspection Service 

= US Offi\RTf,ENHlf AGA!CU.TLRE 

Sampling Methods

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Sampling Methods

• Destructive sampling – grab, N60, excision

o e.g., RTE, ground comminuted products, egg products, carcass excision

• Non-Destructive sampling

• Typically chosen when destructive sampling not an option

• Examples:

o Carcass rinsates or sponge

o Parts rinsate

o Environmental sponge

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What is N60?

• N60 = number of samples (n) = 60

o Multiple representative samples provides best option for detecting scattered

contamination

o Provides 95% confidence that no more than 5% of food pieces the size of each “n”

in the entire lot are contaminated

• Keys to success

o Must ensure that sampling is as representative as possible across the lot

o Large composite “N60” samples typical need a larger test portion

• Cloth rather than N60 - in-field study started on January 4, 2021 through

June 2021 – stay tuned

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Common Sampling Problems

• Small sample or sampling method may not be ideal for detection

o e.g., small swab device or environmental area sampled

• Sanitizer or residual antimicrobial chemicals might interfere with the test

o Insufficient drip time prior to carcass sample collection

o Excessive liquid carryover for parts sample collection

• Temperature abuse for the sample prior to testing

o Holding under refrigeration for long periods allows competing bacteria to grow

o Freezing can kill some pathogens (e.g., Campylobacter)

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Assessing Sampling Plans

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Sampling Methods

• All sampling plans have significant limitations

o Relative rigor of the sampling program must be evaluated

• Best sampling plans provide the opportunity but no guarantee of detection

o i.e., scattered contamination is difficult to detect

• Frequent sampling and sampling multiple sites/time points provides a better

opportunity for detection

Examples:

o Multiple samples per day vs. once per month

o N60 per lot vs. one grab sample per lot

• Does the type of sampling meet the intended need?

o Destructive vs. non-destructive sampling

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Sampling Plans

Statistical sampling plans assume:

• Uniform manufacturing conditions

• Equal probability of contamination throughout the lot (homogeneous distribution)

• Independent, random sampling (equal probability of sampling throughout the lot)

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• • • . • .. . • 

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Why are Pathogens Hard to Detect?

• They are typically not evenly distributed

• They occur at low levels

• They are often injured when found in the product

• Detection may be inhibited by material in the food product (food matrix)

o Example: high amounts of fat may inhibit PCR assays; spices, salt, acidulants can

affect isolation and detection

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E. coli O157:H7 Contamination in a N60 Sampled Lot

1 2 3 4 5 6 7 8 9 10 

a

b

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“Hot Spot”

“Sporadic/Background”

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E. coli O157:H7 Contamination in Ground Beef

% contaminated samples

40% of product contaminated

by hour 3 of production

Combo bins

<5 <5 40 30 <10

“slug”

1 3 5

100

0

Time of production, hrs 

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Assessing Testing Methods

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Key Players for Ensuring Robust Testing Methods

• The establishment that needs the testing

• The laboratory they hire

• The manufacturer of the screening test they use

• The organization validating the screening test

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Steps in Detection Methods

• Sample collection

• Sample preparation

• Enrichment for the pathogen

• Screening of the pathogen

• Confirmation of the pathogen

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Considerations for Testing Methods

• Is the method fit for the intended purpose of the analysis?

• Has the method been optimized and experimentally validated for

sensitive detection of pathogens?

• Is the laboratory complying to the validated method protocol?

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Assessing Fitness for Purpose

• Is the test portion appropriate to meet the need?

• Is the method enrichment-based with the intent to detect the

lowest possible numbers of stressed pathogen cells?

• Has the food matrix been validated for the method used?

• Are confirmation procedures appropriate for determining true

negative samples?

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The “Test Portion”

• Laboratory sample preparation => “test portion”

o “analytical unit” or “analytical portion”

o Definition: the part of the “sample” that is actually tested by

the laboratory

• The test portion determines the theoretical (i.e., best possible)

sensitivity of the test

o e.g., 1 cell/test portion

o 25-gram test portion: detecting 0.04 cells/gram is possible

o 325-gram test portion: detecting 0.003 cells/gram is possible

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Enrichment

• Test portion is incubated 8-48 hours in a culture broth

o Why?

 Contamination levels are too low for detection without

enrichment

 Must grow to high levels so very small volumes have

enough pathogen present for later detection steps

• Different pathogens require different enrichment media (broth)

o One vs. two-stage enrichment

• Primary enrichment vs. secondary enrichment

o Resuscitation vs. selective growth

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Considerations for Proper Enrichment

• Resuscitation (lag phase) can require 2-3 hours before log-phase growth begins

o Some samples support slower growth

• Has enrichment broth been tempered to warm temperature prior to incubation?

o Particularly critical for large test portions or shorter incubation periods

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Pathogen Growth During Enrichment

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Log pathogen level

(e.g., cfu, MPN/gram) 

Incubation time, hrs 2 24

lag logarithmic stationary death 

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PCR

immunoassay

Possible 

Loss of 

Sensitivity 

Prior to 

confirmatory 

retesting 

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Enrichment Period

• Different screening tests require different levels of enriched pathogen

• Shorter incubation periods (<15 hours) may warrant additional scrutiny of laboratory

compliance to the validated protocol

• Has enrichment/screening combination been validated for a larger test portion?

o Particular concern for large test portions incubated for shorter periods - e.g., 375-

gram test portion incubated for 8 hours

• Proposed incubations <8 hours may warrant OPHS review

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Confirmatory Testing

• Non-culture confirmation (e.g., PCR)

• Culture confirmation (e.g., FSIS confirmation)

o Plating the enrichment on selective and differential agar

media

o Immunomagnetic separation (IMS) necessary prior to plating

for E. coli O157:H7 and non-O157 STECs

 Suspect colonies = “presumptive positive”

• Purification and confirmatory identification tests including:

o Biochemical (e.g., identifies “E. coli”)

o Serological (e.g., identifies “O157” and “H7”)

o Genetic (e.g., identifies “stx” = Shiga toxin genes)

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Concerns for Confirmation

• Do not re-sample the lot or sample reserve!

• Non-culture confirmation

o Same considerations as the screening test

o Used under validated conditions

o Transport and storage of enrichment

• Culture confirmation- carefully assess!

o Typically expect that methods comply with a validated

procedure (e.g., MLG, FDA-BAM, ISO)

o Small changes can affect ability to recover pathogen of

interest

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Quantitative Testing

Two options:

o MPN

o Direct plating

NOTE: Quantitative testing typically cannot accommodate larger test portions and

provide the opportunity for detection that a qualitative test can provide

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Most Probable Number (MPN) Enumeration Analysis

• Traditional enrichment-based analyses are performed on three or

more dilutions, each typically in triplicate, from a single sample

homogenate (i.e., MPN = method format, not a specific method

per se)

• Advantages:

o Better sensitivity (lower LOD) than direct plating

• Disadvantages:

o Very resource intensive/expensive

• Application:

o For quantifying low levels of pathogens (e.g., Salmonella, E.

coli O157:H7, L. monocytogenes)

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Direct Plating Enumeration Methods

• Product is homogenized in diluent and small volume is directly dispensed onto agar

media (i.e., sometimes there is a 1-2 h “resuscitation” step, but enrichment is never

used prior to plating)

• Advantages:

• Allows easy inexpensive quantitative analysis

• Disadvantages:

• Accommodates only a very small test portion

• Higher LOD (i.e., often 100 CFU/g) not suitable for detecting low levels of pathogens

• Application:

• Expedient for higher level analytes (e.g., indicators, Campylobacter, S. aureus, C.

perfringens, B. cereus)

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Method Validations

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Value of Validation

• Determines performance characteristics of the method in

comparison to a gold standard (reference) method (e.g., usually

FSIS or FDA method)

• Independent evaluation provides credibility

• Rigor varies (multilab vs. single lab, # tests, etc)

• Still must consider fitness for purpose and how the method is

applied

o e.g., some AOAC-validated methods are not consistent with

FSIS goals or Compliance Guidelines

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Method Validation

• Recognized independent method validation organizations:

o Government: FSIS (MLG) and FDA (BAM)

o AOAC International (U.S.A.)

 AOAC Official Methods of Analysis (OMA) validations

 AOAC-RI “Performance Tested Method” validations

o AFNOR (France)

 e.g., bioMerieux Vitek biochemical confirmation tests

o Others (ISO, MicroVal, NordVal, AENOR, etc.)

• However, past validations conducted by these organizations may not be relevant to

larger test portions or other testing scenarios

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Foodborne Pathogen Test Kits Validated by

Independent Organizations

• FSIS maintains a list, updated quarterly, of methods that have

been validated by independent organizations

• https://www.fsis.usda.gov/sites/default/files/media_file/202

1-05/Validated-Test-Kit.pdf

• None of the test kits listed are implicitly approved by USDA FSIS

o A validated test kit must also be fit for purpose and

appropriate for the specific application in a food safety

program

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Process for Validating Qualitative Pathogen Methods

• Series of laboratory experiments using inoculated samples under controlled conditions

• Inoculate portions with pathogen strain at very low level where only 20-80% of

samples are positive (i.e., fractional recovery)

• Statistically compare percent of positive samples in alternative method to reference

method (FSIS MLG)

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Considerations for Validation Data

• Was method compared to an appropriate reference method (e.g.,

FSIS MLG; FDA)?

• If not performed by AOAC, AFNOR, etc., is supplemental

validation data available?

o May require additional scrutiny

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Testing Method Specifications

• Sensitivity: probability that truly positive samples are detected as

positive by analytical test

o 100 – false negative rate

• Specificity: probability that truly negative samples detected as

negative by analytical test

o 100 – false positive rate

• Level of detection (LOD): lowest level of contamination reliably

detected by analytical test

o LOD expressed as ratio of organisms to quantity tested

material (e.g., CFU per gram, MPN per mL, CFU per square-ft)

but definitions vary (e.g., LOD95, POD)

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Factors Impacting Detection and Method Specifications

• Detection as measured by sensitivity, specificity, and LOD can vary based on:

o Specific strains of pathogen

o Intrinsic factors for the sample matrix

 Levels of competing bacteria

 Fat, salt, pH and additives

• Experimental design for the validation study (e.g., cell stress, etc.)

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Complying with the Validated Protocol

• Do AOAC/AFNOR/ISO citations match the protocol in use?

o Modifications are common, and some contribute to greater potential for false

negative result

• Compare the lab procedure to the validated protocol (i.e., package insert)

• If culture confirmation is used, verify that it follows validated method as well

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Methods not Validated by Recognized Organizations

• “Supplemental” or “extension” validations

• E. coli O157:H7 and non-O157 STEC testing for 325-375g test portions

o Modifications required for AOAC validated procedures based on 25g

o Instructions for sample preparation may not be clear for the lab

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Laboratory Accreditation and Quality Assurance

• ISO 17025 = protocol for establishing and documenting a

microbiology laboratory quality program (i.e., “HACCP” for labs)

• Accrediting bodies = A2LA and others

• Accreditation implies robust quality program but does not

necessarily indicate methods meet FSIS expectations

o Laboratories are able to perform the methods they use as

expected, but methods are not “accredited” to be fit for

purpose

• Laboratories are not required to be ISO accredited, but should

have quality assurance programs that ensure results are reliable

and accurate

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FSIS Testing Programs

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FSIS Microbiological Sampling Program Objectives

• Assess effectiveness of industry process controls

• Provide critical feedback to industry

• Monitor compliance with performance standards, zero-tolerance policies

• Allow FSIS to monitor industry-wide trends

• Serve as a strong incentive to reduce the occurrence of pathogens in products

• Capture pathogen characterization information (i.e., serotype, speciation,

antimicrobial resistance, whole genome sequencing)

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FSIS Sampling Programs

• Sampling plans measure compliance with performance standards:

o Salmonella and Campylobacter verification programs (raw poultry)

• Zero-tolerance policies for food pathogens

o E. coli O157:H7 and non-O157 Shiga toxin-producing E. coli (non-O157 STEC) (raw

non-intact beef or components of raw ground beef)

o Listeria monocytogenes in RTE and pasteurized egg products and on food contact

surfaces

o Salmonella in RTE and pasteurized egg products

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FSIS Methods and Pathogen-specific Issues to Consider

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Shiga Toxin-Producing E. coli (STEC) Testing

Includes:

E. coli O157:H7 and the six non-O157 Shiga toxin-producing E. coli (STEC)

(non-O157 STEC) - O26, O45, O103, O111, O121, and O145

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O157 STEC Program

• Strain must have:

o O157(+)

o stx(+) OR stx(-) and H7(+)

o biochemical(+) or Bruker MALDI Biotyper

• Currently FSIS only analyzes beef manufacturing trimmings (MT60) for non-O157

STECs

• FSIS plans to expand non-O157 STEC verification testing (85 FR 34397; June 2020):

o Ground beef (MT43), bench trim (MT65), raw ground beef components other than

trim (MT64)

o Responding to comments; final rule; grace period, etc.

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Non-O157 STEC Program

• Six non-O157 STEC = O26, O45, O103, O111, O121, O145

• Strain must have:

• stx(+) and eae(+) genes

• one of the six O-groups

• biochemical(+) or Bruker MALDI Biotyper

• Currently FSIS only analyzes beef manufacturing trimmings

(MT60) for non-O157 STECs

• Phased rollout – MT65 – MT64 – MT43

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...-------- j ---------------

- - -

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E. coli Top Seven STEC Analysis (MLG 5C.02)

Samp e Prep and Pr mary Enr chment 

42 C±1 for 15 24 hours 

Day 2 

Day 1 

Perform PCR 

A samp es that do not test PCR 

negat ve are carr ed forward for 

further ana ys s 

Day 3 

mmunomagnet c Bead Capture & 

Ra nbow Agar P at ng 

Perform 

stx/eae gene analys s 

atex agg ut nat on & genet c 

sero og ca test 

Bruker MALD B otyper 

Day 4 / 5 

Latex Agg ut nat on & Sheep B ood 

Agar P at ng 

negative or 

potential (+) – stx(+) eae(+) O group(+) 

Negative – no growth, agglutination (-) 

or agglutination(-)/rapid screen(-) 

presumptive (+) – agglutination(+) and 

rapid screen (+) or inconclusive 

Enrichment 

Screening 

Confirmation – next page 

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E. coli Top Seven STEC Analysis (MLG 5C.02) - continued

Day 4 / 5 STEC Negative – either stx(-) or eae(-) 

Perform 

stx/eae gene analys s 

atex agg ut nat on & genet c 

sero og ca test 

Bruker MALD B otyper 

stx(-) & eae(+) 

stx(+) & eae(+) O group ( ) 

STEC Negat ve 

stx(+) & eae(+) O group (+) 

Bruker MALD B otyper( ) 

perform WGS 

stx(+) & eae(+) O group (+) 

Bruker MALD B otyper(+) 

STEC Pos t ve 

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Larger E. coli O157:H7 and Non-O157 Test Portions

• Larger test portions (325-375 grams) are most important for N60 and other composite

samples containing many samples

• Less important for single “grab” samples of ground beef final product testing when:

o Trim and components have already been tested using robust sampling and 325-

375-gram test portions

o Multiple samples are collected throughout the production day

• Methods must be adapted, optimized and validated for effective use with 325-375 gram

test portions

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E. coli O157:H7 and Non-O157 STEC Testing Concerns

• Supplemental validation and special instructions for testing larger test portions

o For enrichment periods <15 hours

o 325-375g test portions typically often require longer minimum enrichment period

than 25g

• Culture-based detection and confirmation requires immunomagnetic separation (IMS)

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Listeria testing

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Includes:

L. monocytogenes testing (FSIS)

Listeria-like or Listeria spp. testing (industry)

Listeria monocytogenes (MLG 8.13)

possible(+) 

confirm (-) – both must be negative 

Samp e Prep and Pr mary Enr chment 

S omach 25g samp e + 225 m UVM 

ncuba e 30 C or 20 26 hrs 

P a ng Secondary Enr chment 

ncubate 35 C MOX 24 28 hrs 

ncubate 35 C MOPS BLEB 18 24 hrs Day 2 

Day 1 

S reak p a es o next day 

3M Mo ecu ar De ec on Assay 2 

Horse b ood and MOX p ates 

Day 4 

Day 3 

Res reak or hemo ys s 

ncuba e 35 C var ab e t me 

Day Bruker MALD B o ype and restreak 5 

Day 6 Fur her charac e za on morpho og ca and 

a yp ca so ate analys s 

presumptive(+) – hemolysis 

presumptive (+) – hemolytic 

Confirmed (+) – on Bruker MALDI Biotyper 

Enrichment 

Screening 

Confirmation presumptive (+) from previous day are 

Confirmed (+) – by Bruker MALDI Biotyper 

Perform WGS 

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Expectations for Listeria Environmental Testing Equivalence

• Compliance Guidelines – Controlling Lm in Post-lethality

Exposed RTE Meat and Poultry Products Jan 2014

• For optimal sensitivity of detection, method for food contact

surface testing must:

o Validated by a recognized body (e.g., AOAC, AFNOR)

o Be enrichment-based

o Enrich the entire sponge/swab sample

 e.g., an aliquot from sponge/swab does not provide

opportunity to detect bacteria trapped in the sponge

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Analytes for Industry Food Contact or Environmental 

Surface Testing

Establishment laboratories test for one of the following:

• Listeria monocytogenes

• Use internationally recognized enrichment-based method that

biochemically confirms culture as L. monocytogenes

• Listeria spp.

• Use internationally recognized enrichment-based method that uses

ELISA, PCR or other screening technology to provide more rapid but less

specific Listeria spp. result

• “Listeria-like” indicator bacteria

• Use the first part of an internationally recognized enrichment-based

method to find suspect Listeria colonies (e.g., darkened colonies on MOX

using the FSIS method)

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Salmonella Testing

• Raw products

o Meat and turkey carcass sponge samples

o Chicken carcass/parts rinsates

o Raw meat and comminuted poultry

• Processed products

• RTE (325g portion)

• Pasteurized egg

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Salmonella (MLG Ch. 4.11)

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Samp e Prep and Pr mary Enr chment S omach samp e + BPW 

ncuba e 350C or 18 24 hrs RTE 20 24 hrs pou ry 

Day 1 

Perform PCR A samp es ha do no es PCR nega ve a e 

ca ed orward o RV and TT bro h 

ncuba e 42 C or 22 24 h s 

Day 3 

Day 2 

S reak RV and TT on BGS 

and DML A p ates 

ncuba e 35 C or 18 24 hrs 

P ck suspec co ony rom P ate med um to 

TS and L A s ants 

ncuba e 350C for 22 26 hrs Day 4 

Day 5 S reak on SBA or b ochem ca test ng 

ncuba e 18 24 h s at 350C 

Day Bruker MALD B o ype 6 

confirm (-) 

(+) - 2° enrichment 

confirm (-/+) 

presumptive (+) 

confirm (-)

Enrichment 

Screening 

Confirmation 

Campylobacter Testing

Qualitative

• Enrichment-based (as opposed to direct plating) since Aug

27, 2018 - exception: “other raw chicken parts”

(EXP_CPT_OT01 and LO_CPT_OT01)

Targets

• C. jejuni, C. lari or C. coli

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Campylobacter (MLG 41.06) - Qualitative

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Samp e Prep and Pr mary Enr chment or P ate 

Samp e + BF BEB or p a e Campy Ce ex 

ncuba e 42 ± 10C or 48 ± 2 hrs 

Day 1-2 Enrich or plate 

PCR Screen & P at ng/ so at on 

Campy Ce ex 

ncuba e 42 ± 10C or 48 ± 2 hrs 

Day 3 

Day 5 Bruker MALD B o ype Ana ys s 

La ex agg u na on confirm (-/+) 

Plating/isolation 

Confirmation 

confirm (-) 

Confirmed

Negative

Confirmed

Positive

Inconclusive

Confirmed

Positive isolates

WGS performed on

confirmed positive isolates &

inconclusive isolates

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Issues for Campylobacter Testing

• Campylobacter is highly vulnerable to freezing

o Do not freeze samples

• Can be a challenging test (inconsistent results across labs)

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Industry Testing Programs

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Establishment Documentation for Testing Methods

• Does the establishment have the necessary documentation?

o Can the establishment provide the method used for microbial

detection?

o Can the establishment provide evidence that the method used

was properly validated by an independent body?

o Can the establishment explain why the method fits the need?

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Issues for Industry Labs

• On-site vs. off-site labs

o Shipment of samples/handling during shipment

• Overarching concerns for on-site labs

o Is testing effective?

o Is testing safe in that facility?

 Enrichment of pathogens in an establishments

• Evaluate the following:

o Are personnel qualified?

o Does the lab have proper equipment and materials for testing

and disposal of contaminated media?

o Do they follow the validated testing protocol?

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Establishment Responsibilities for Laboratory Testing

• The establishment is ultimately responsible for the testing they

request from private laboratories

• Has the establishment properly conveyed testing needs?

o e.g., test portion equivalent to FSIS as opposed to the default

25g in protocols

• Is the laboratory aware of FSIS expectations?

o Directives, Notices and guidance

• Establishment should provide documented detailed methodology

and validation information for FSIS review

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FSIS Verification of Establishment Sampling and

Testing Programs

Effectiveness verified by FSIS

• Reviews/observations of EIAOs during FSA

• Establishment provides supporting documentation

• Technical and policy support provided through askFSIS

• Establishment, not laboratory, is responsible for implementing effective program

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FSIS Verification of Establishment Sampling and

Testing Programs

Focus of FSIS’ evaluation

• Is the method fit for the intended purpose?

• Does the method support the hazard analysis decisions?

• Is the method comparable to the appropriate FSIS method (or is there justification

for an alternative)?

• Is a comparable or appropriate test portion used?

• Is the method validated and used under validated conditions?

• Does the laboratory assure the quality of the results?

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Helpful Guidance

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Existing Agency Guidance – Compliance Guides

Ready-to-Eat

• FSIS-GD-2021-0014 - Appendix A – “FSIS Salmonella Compliance Guidelines for

Small and Very Small Meat and Poultry Establishments that Produce Ready-to-Eat

(RTE) Products and Revised Appendix A” (December 2021) – Being updated

• FSIS-GD-2021-0013 - Appendix B – “FSIS Compliance Guideline for Stabilization

(Cooling and Hot-Holding) of Fully and Partially Heat-Treated RTE and NRTE Meat

and Poultry Products Produced by Small and Very Small Establishments and

Revised Appendix B” (December 2021) – Being updated

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Existing Agency Guidance – Compliance Guides

Shiga Toxin-producing E. coli (STEC)

• FSIS-GD-2021-0007 – FSIS Industry Guideline for Minimizing the Risk of Shiga

Toxin-Producing Escherichia coli (STEC) in Beef (including Veal) Processing

Operations (July 2021)

• FSIS-GD-2021-0008 - FSIS Industry Guideline for Minimizing the Risk of Shiga

Toxin-Producing Escherichia coli (STEC) in Beef (including Veal) Slaughter

Operations (July 2021)

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Existing Agency Guidance – Compliance Guides

HACCP

• FSIS-GD-2018-0005 – “Meat and Poultry Hazards and Controls Guide”

(March 2018)

• FSIS-GD-2015-0011 – “FSIS Compliance Guideline: HACCP Systems Validation”

(April 2015)

WGS

• News & Events (under Full Menu; right side) – Events & Meetings (left side under

the picture) – search for WGS in the advanced search

• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6653787/

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Existing Agency Guidance – Compliance Guides

Microbiological Test Methods and Laboratories

• FSIS-GD-2013-009 -Establishment Guidance for the Selection of a Commercial

or Private Microbiological Testing Laboratory” (June 2013)

go to: Policy-FSIS Guidelines – search for “selection of a commercial lab”

• FSIS-GD-2010-0004 – “FSIS Guidance for Test Kit Manufacturers, Laboratories:

Evaluating the Performance of Pathogen Test Kit Methods” (October 2010)

• FSIS-GD-2019-0008 – “Foodborne Pathogen Test Kits Validated by Independent

Organizations” (February 2020)

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Whole Genome Sequencing (WGS)

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Whole Genome Sequencing – A Collaborative Approach

• FSIS worked with the Food and Drug Administration, the Centers

for Disease Control and Prevention (CDC), with PulseNet partners

on:

o How to perform WGS – methodology (aligned methods)

o Analyze WGS data

o Interpret WGS data

• FSIS began performing WGS for Listeria monocytogenes (Lm) in

FY13 (along side PFGE) and for all pathogens starting in early FY16.

• FSIS suspended PFGE analysis for Lm and started using WGS data

Jan 15, 2018.

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Whole Genome Sequencing – Benefits

• WGS benefits FSIS and its mission to protect public health:

o Detects harborage and cross-contamination of pathogens in

FSIS-regulated facilities,

o Traceback from human illness outbreak data to regulated food

products, and

o Identification of unique genes related to virulence,

pathogenicity, survival, adaptation, and resistance to biocides

(sanitizers, metal, etc.) and antimicrobials.

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0 

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Whole Genome Sequencing – Analysis

• FSIS uses different tools to analyze WGS information including:

o Public Sequence Typing

o Multi-locus Sequence Typing (MLST)

o Core genome analysis (~1800 genes for Lm)

o Phylogenetic analysis

o High-quality Single Nucleotide Polymorphisms (hqSNP)

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Whole Genome Sequencing – Single Nucleotide

Polymorphism (SNP)

Single Nucleotide Polymorphism (SNP)

ATGTTCCTC isolate A

ATGTTGCTC isolate B

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Whole Genome Sequencing – Sequence Typing

Multi-locus Sequence Typing (MLST)

• MLST can generate a pattern name or designation based on differences in a pre-defined

set of genes.

• MLST Results will be Provided by FSIS as Follows:

o Public Sequence Type (“MLST ST”, “ST”, or “pubST”)

 small number of genes (i.e., 6-12)

 named using the publicly available database developed by Jolley & Maiden (2010)

(e.g., publicST09)

• Allele Code

o compares ~1,800 genes for Lm

o named by using CDC PulseNet numerical code

(e.g., LMO1.1-5.1.1.2.5.1) 75

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Whole Genome Sequencing – Single Nucleotide

Polymorphism (SNP)

ST09

LMO 1.1- 37.3.2.33

LMO 1.1- 37.3.2.36.1 LMO 1.1- 37.3.2.11.7.13

Public Sequence 

Type 

Allele Code is more specific than Public Sequence Type;

one Public Sequence Type can be inclusive of many Allele

Codes. 76

Whole Genome Sequencing – Single Nucleotide

Polymorphism (SNP)

a “field”

If the first four fields

between two isolates

match, the isolates may

be closely related

LMO – L.

monocytogenes

Version 1.1

Allele codes are a

nomenclature scheme

created by CDC

Like PFGE patterns,

allele codes simplify

how we communicate

about pathogen

strains

Example: LMO1.1 - 5.1.1.2.5.1

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Background: What does allele code tell you?

LM1.0 – 1.5.4.4.2.5

LM1.0 – 1.5.4.4.5.9

LM1.0 – 1.5.4.6.7.2

Last digit same or different

differ by 0 – 19 alleles

“closely related”

3rd to last digit different

differ by > 19 alleles

“not closely related”

LM1.0 – 1.5.4.4.2.3

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26
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

-

1--1,''-_--'--_'-....... --'\+1 ---'=--+--=---'---+---"-'-+----'----i+--

--------,..--·- ·

--·--·- ·-------------·'"""'----""--- ... -­ ~ .. ---·- ----...------~- ---~------,,_ ..... ---~ ___ ------------ .,._w:-,_ 

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Establishment-specific Datasets

Allele codes for Lm have been reported since 2019

Fields were created for Salmonella and STEC allele codes (Campylobacter in development)

Date Stamp format (allele code (space) date mm/dd/yyyy

LM1.0-23.5.6.0 04/05/2022 Retrieval of the allele code from PulseNet

Allele codes may change over time, a date-stamp supports use of the data in static reports

Whole Genome Sequencing – Allele Codes

• Allele codes are a nomenclature scheme created by CDC.

• Like PFGE patterns, allele codes simplify how we communicate about pathogen strains.

• Allele codes can be used for trend analysis and to interpret relatedness.

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Whole Genome Sequencing – Analysis - Microbial 

Characterization Branch (MCB) - Eastern Lab, Athens, GA

LIMS ID

Form ID

FSIS Identifier

Allele Code

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 .____I _ __JI 

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Harborage and Cross-contamination

• Harborage or persistent contamination of the post-lethality environment, is

suggested if WGS analysis indicates closely related Lm isolates are found in

product, food contact, or non-food contact environmental samples that were

collected over multiple days, weeks, months, or years.

• Cross-contamination is suggested when closely related Lm isolates are found

in product, food contact, and environmental (non-food contact) samples

collected during the same sampling event.

If Lm is isolated from a post-lethality exposed product sample and from a

food contact surface sample, the food contact surface is more likely to be the

source, unless under-processing of RTE product is suspected.

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Harborage and Cross-contamination

Harborage (likely)

different collection

LM-1 LM-1

LM-1

LM-1

dates

SAL-1

SAL-2

SAL 3

SAL 3

Source

Cross-contamination (possible)

same collection date Recommend corrections to food safety controls 83

PulseNet Cluster Search

• EIAOs assigned to perform PHRE at Cat 3 Est request search through AskFSIS

(Directive 10,250.2)

Search strategy:

• Obtain all Salmonella WGS from all raw poultry sampling projects

obtained in past 52-weeks from the establishment.

• Determine if any such sequences are closely related to a recent

clinical isolate associated with a PulseNet cluster.

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PulseNet Cluster Search

• EIAOs assigned to perform PHRE at Cat 3 Est request search through

askFSIS (Directive 10,250.2)

Search strategy:

• Obtain all Salmonella WGS from all raw poultry sampling projects

obtained in past 52-weeks from the establishment.

• Determine if any such sequences are closely related to a recent

clinical isolate associated with a PulseNet cluster.

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PulseNet Cluster Search

Sample collection dates (FSIS

projects)

Sample collection

dates (PulseNet

human cases)

FSIS sample matches

PulseNet cluster (yes/no)

FSIS

sample

matches

recent

PulseNet

clinical

isolate 86

Asking for More Information

• When performing a PHRE in establishments with more than one positive RTE sample,

EIAOs are to:

o Use the Form ID to Request WGS analysis of previous matches from

the OPHS - Microbial Characterization Branch (OPHS-MCB) from

Outbreaks_WGS@fsis.usda.gov

o The WGS analysis will indicate if there is a history of harborage or crosscontamination in the establishment.

• After an IVT/RLm positive, EIAOs are to make a request through the

Outbreaks_WGS@fsis.usda.gov Outlook mailbox for WGS analyses.

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I Q Q C '--------------1 

I I I 

I l~C] 

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Whole Genome Sequencing – The Future

• FSIS continually works with FDA, CDC PulseNet, local & state health departments to

harmonize interpretation and reporting.

• Future plans -- pathogens that will be reported by allele code:

o STEC and Campylobacter jejuni allele codes were released in early 2021

o Salmonella is still being finalized by PulseNet

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Whole Genome Sequencing – The Future

Abbreviations

• FDA – Food and Drug Administration

• WGS – Whole genome sequencing

• Lm – Listeria monocytogenes

• NCBI – National Center for

Biotechnology Information

• PFGE – Pulsed field gel electrophoresis

• NARMS – National Antimicrobial

Resistance Monitoring System

• STEC – Shiga toxin-producing E. coli

FY14

FY15 FY16

FY17

FY13 FY18

FY19

FY20

1- FSIS began WGS

on STEC and Lm

2- Uploaded first

Lm sequences to

NCBI

OPHS Eastern Laboratory

runs first sequences for

outbreak investigations

FDA ran WGS

on Lm for FSIS

FSIS began WGS on

Salmonella and

Campylobacter

for select cecal

isolates

WGS on all

Salmonella and

Campylobacter

(product and cecal)

1- PFGE stopped

for Lm

2- Began WGS on

NARMS cecal

indicator

organisms

PFGE stopped on

Campylobacter,

Salmonella,

STEC

FSIS begins to

obtain Salmonella

serotype from WGS

FSIS hosts WGS

Public Meeting

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fsis.usda.gov

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