pulsenet pfge laboratory updateslaboratory updates · eti tdc t w ki slti alternatives to etbr •...

PulseNet PFGE Laboratory UpdatesLaboratory Updates

Kara CooperCenters for Disease Control and Prevention

NCZED/DFBMD/EDLB/PMDRLSeptember 24, 2009

UpdatesUpdates• Protocols

– Non-O157 STEC PFGE protocol– Listeria protocol review

Shi ll fl i d t– Shigella flexneri update• Procedure/Reagent Updates

– Investigation of alternative agaroses– Enzyme evaluation

N t t i l thi d d t t i– Not so typical thiourea-dependent strains– Alternative gels stains

Rapid Standardized PFGE Protocols for Subtyping Foodborne

Pathogenic BacteriaCurrent Protocols Recently Released Protocols

• E. coli O157:H7• Salmonella

Li t i t

• Non-O157 STEC • Clostridium botulinum

• Listeria monocytogenes• Shigella sonnei• Campylobacter jejuni

Protocols Under Development• Shigella flexneri (2009)• Campylobacter jejuni

• Clostridium perfringens• Vibrio cholerae

Shigella flexneri (2009)• Vibrio vulnificus (2010)• Yersinia enterocolitica (2010)

• Yersinia pestis • Vibrio parahaemolyticus

• Campylobacter coli

Non-O157 STEC Standardized PFGE Protocol

Increased n mber of Non O157

Standardized PFGE Protocol

• Increased number of Non-O157 STEC submissions to the National E. coli database over the last several yearsyears

• Non-O157 STEC PFGE patterns differ from O157 in regard to the number and distribution of bandsnumber and distribution of bands

• Complicated data analysis and raised concerns about reproducibility Need for alternative electrophoresis• Need for alternative electrophoresis conditions

***Additional information provided during poster session by Kara Cooper

Non-O157 STEC St d di d PFGE P t l

O157 Conditions

2.16s-54.17s • The non-O157 STEC electrophoresis

Standardized PFGE Protocol• The non-O157 STEC electrophoresis

conditions (6.76s-35.38s) improved the ease of analysis, band distribution, and overall pattern resolutionoverall pattern resolution

• External validation of these conditions demonstrated a high degree of inter-and intra-laboratory reproducibility

Non-O157 Conditions

6.76s-35.38s

and intra laboratory reproducibility• Incorporated into a Standardized

PulseNet protocol for non-O157 STEC• All laboratories should begin abo ato es s ou d beg

subtyping non-O157 STECs with these conditions


Non-O157 STEC St d di d PFGE P t lStandardized PFGE Protocol

• Database– Scripts available on MasterScripts v.5.0 (distributed

S t b 2009)September 2009).– An online national PulseNet database is available– Re-tested historical isolates to create a unique pattern

list within the non-O157 national database • Certification

– Gel certified for E. coli – Analysis certification will be separate for O157 and

Non-O157Non O157– Request certification gel and instructions from

[email protected]– Would like all laboratories analysis-certified for non-

O157 by end of this yearO157 by end of this year– Non-O157 STEC TIFF images can be sent to

[email protected] until laboratory becomes analysis-certified


Shigella flexneri PFGE P t lPFGE Protocol

S f Shigella flexneri• Shigella flexneri is a major cause of foodborne illness internationally

Shigella flexneri

2.16s-54.17s

• INEI - ANLIS “C.G.Malbrán”Buenos Aires, Argentina

• International Working GroupInternational Working Group• The current Shigella sonnei

protocol does not perform well against Shigella flexneri inagainst Shigella flexneri in regard to number and overall band distribution


NotI

• Restriction Enzymes– Primary – NotI– Secondary – XbaI

• Electrophoresis conditions– Initial Switch Time: 5s

XbaI

– Final Switch Time: 35s


• External ValidationExternal Validation Working Group

Laboratory Country• External Validation– Comparison of data generated by

9 laboratories against 17 strains

Laboratory Country

CDC USA

INEI - ANLIS “C.G.Malbrán”

ARGENTINA

– Successfully completed– Final results to be presented at

the PulseNet International

NAMRU EGYPT

Central Public Health Laboratory

OMAN

I tit t Ad lf L t BRASILMeeting

• Currently working on the development of BioNumerics

Instituto Adolfo Lutz BRASIL

National Microbiology Laboratory

CANADA

Public Health HONK KONGdevelopment of BioNumerics scripts and an on-line database

Laboratory Centre

Staten Serum Institute DENMARK

Instituto de Salud Pública

CHILEPública

Listeria monocytogenes R i d PFGE P t lRevised PFGE Protocol

• Results of implementation has varied– No issues– Minor optimization– Unresolved issues

• CDC has developed an extensive troubleshooting worksheet

T id tif diff i t i t ti– To identify differences in reagent, equipment, or practices – Several laboratories have participated but numbers are still to

low to indentify contributing factors– Please send a message to Kara Cooper ([email protected]) ifPlease send a message to Kara Cooper ([email protected]) if

you would like to receive the worksheet

Listeria monocytogenes PFGE Protocol

• Plug Preparation and

PFGE Protocol(Lessons Learned)

A I A IExamples of Lysozyme going bad Plug Preparation and

Lysis issuesFreezer media

AscI ApaI

– Freezer media– Lab specific optimization

of cell suspension pconcentrations

– Lysozyme of poor quality or has gone bador has gone bad

• Molecular biology grade with protein ≥90%

Listeria monocytogenes PFGE Protocol

• Incomplete restrictionM d t h l t f

AscI ApaIPFGE Protocol(Lessons Learned)

– May need to change lots of enzyme and/or buffer

– Consider purchasing or aliquoting enzyme in smaller volumes

• Star activity - a relaxation or alteration of the specificity of a restriction enzyme

• Conditions that can lead to star A I A I

Same plugs re-tested

Overnight RestrictionCo d t o s t at ca ead to staactivity– Prolonged reaction time– Suboptimal buffer or buffer

concentration

AscI ApaI

concentration– High (> 5% v/v) glycerol

concentrations– High concentration of enzyme/µg of

DNA ratioDNA ratio

2 Hour Restriction

Investigation of Additional Agaroses for Use in PulseNet Standardized

• SeaKem Gold Agarose was originally BioRad MegaBase Agarose

PFGE Protocols

SeaKem Gold Agarose was originally chosen for the PusleNet protocols– Stability of the plugs and gels produced – Relatively short run times

BioRad MegaBase Agarose

Relatively short run times • Previous investigations proved that

the composition of PFGE quality agarose vary from different vendorsg y– Significantly more fragile– Required longer run times (+2-4 hours) – Electrophoretic mobility of DNAElectrophoretic mobility of DNA

fragments differed resulting in normalization issues

***Additional information provided during poster session

by Molly Freeman

Additional Agarose EvaluationBioRad MegaBase Amresco III

BioRad New Megabse AgaroseSeaKem Gold

g


by Molly Freeman

Additional Agarose Evaluation

• Comparison of Amresco and Comparison of XbaI PFGE patterns of Salmonella certification strains

Megabase to SeaKem Gold– Slightly more fragile, but

manageableRuns slightly slower (+0 5 1 hour)

100

80

G ACDC

CDC 98 -0 3CDC 98 -0 3

generated by 5 laboratories.

– Runs slightly slower (+0.5-1 hour)– Issues associated with normalization

were only observed on gels that run short

CDCCDCV A

V AG A

CDCCDC

CDC

CDC 98 -0 3CDC 98 -0 3CDC 98 -0 3

CDC 98 -0 3CDC 87 -0 3

CDC 87 -0 3CDC 87 -0 3

CDC 87 -0 3

• Further evaluation of these agaroses in the coming months

• We are actively recruiting 10

V A

V AG A

CDCCDC

CDCV AV A

G A

CDC 87 -0 3

CDC 87 -0 3CDC 78 -9 9

CDC 78 -9 9CDC 78 -9 9

CDC 78 -9 9CDC 78 -9 9CDC 78 -9 9

CDC 61 9 9y g

additional volunteer laboratories

G ACDC

CDCCDC

V AV A

CDC 61 -9 9CDC 61 -9 9

CDC 61 -9 9CDC 61 -9 9

CDC 61 -9 9CDC 61 -9 9


by Molly Freeman

Evaluation of Enzyme R b tRobustness

• Reason for evaluationf– Desire for time reduction

– Availability of newly engineered “rapid digest” restriction enzymes

– Changes to PFGE protocol that may enhance increased enzyme efficiency

• Enzymes EvaluatedEnzymes Evaluated– Fermentas Rapid Digest– Fermentas Conventional enzyme

R h C ti l– Roche Conventional enzyme– New England BioLabs Conventional enzyme


by Jessica Halpin


H9812 digested with XbaI over 4 different incubation times

10m 30m 1h 2h

Fermentas 50 units

10m 30m 1h 2h

Roche 50 units

10m 30m 1h 2h

Fermentas Fast Digest 5 units

10m 30m 1h 2h

NEB 50 units


by Jessica Halpin


T ti f dditi l S l ll S

Summary of enzyme performance with 10 minute restriction

Vendor (Units/Reaction) XbaI

BlnI/AvrII/XmaJI

Fermentas Rapid

Testing of additional Salmonella Serovars with 10 minute restriction

Digest (5) Variable Variable

Fermentas Conventional Enzyme (50) Good Poor

RocheRoche Conventional Enzyme (50) Good Good

New England BioLabs

C ti lRoche XbaI, 50U, 10 min Conventional Enzyme (30) Poor Poor

Roche XbaI, 50U, 10 min


by Jessica Halpin


• Several restrictions enzymes from several vendors show the potential

• Evaluate shorter restriction times on known isolates before testing p

for time reduction • CDC PulseNet Laboratory will

continuing to evaluate this with additional enzymes and organisms

groutine isolates

• As always, laboratorians should critically review all gels and patterns for faint bandsadditional enzymes and organisms

• 10 minutes is an extreme

patterns for faint bands

RESULTS WILLRESULTS WILL VARY BETWEEN LABORATORIES


by Jessica Halpin

Typical Thoiurea-Dependent StrainsStrainsWithout Thiourea With Thiourea

Untypeable Result Typeable Resultyp yp• Addition of 50 μM thiourea in 0.5XTBE electrophoresis buffer• Include a positive control on gel

R t i t d PFGE l li f “ t bl ” t i th t “t d”– Restricted PFGE plug slice of “untypeable” strain that “typed” previously when thiourea was used in buffer.

Not So Typical Thoiurea-Dependent Strains

Typical Thoiurea-Dependent Strains without Thiourea

Dependent Strains(Salmonella Saintpaul Outbreak Strain)

St a s t out ou ea

H9812

Typical Thoiurea-Dependent Strains with ThioureaStrains with Thiourea

H9812

S SaintPaul OutbreakS. SaintPaul Outbreak Strain with Thiourea

H9812

S SaintPaul OutbreakS. SaintPaul Outbreak Strain with Thiourea

H9812

S. SaintPaul Outbreak Strain with Thiourea

H9812

Not So Typical Thoiurea-Dependent StrainsDependent Strains

• In one laboratory isolates are untypeableIn one laboratory isolates are untypeable even with the addition of Thiourea, but typeable within another laboratorytypeable within another laboratory– Observed with other Saintpaul strains as well

as other Salmonella serotypes and E colias other Salmonella serotypes and E. coli• Isolate will have to be submitted to CDC or

area laboratory for testingarea laboratory for testing

Alternative DNA StainsAlternative DNA Stains• Increased interest in

alternatives to EtBr E ti t d C t W ki S l tialternatives to EtBr• Benefits

– Reduced safety concerns– De-staining is unnecessary

StainType

StainCost

DisposalCost

TotalCost

Syber

Estimated Cost per Working Solution

De staining is unnecessary– Minimal background and

enhanced visualization of lower bands

C

SyberSafe 5.88 0 5.88

Syber Gold 11.70 0 11.70

• Concerns– Impact on appearance of

gels/patternsStability of stain over time and

Gel Red 28.50 0 28.50

EtBr 0.23 2.89 3.12

– Stability of stain over time and number of gels

– Increased cost– Which one(s)?( )

d6

d6 Don't know if due to time it would be better just to put this in the Open session or to try to power through it here.dmu0, 9/16/2009

Alternative DNA StainsAlternative DNA Stains• A 15 well gel containing H9812 was run and subsequently cut into

four parts and stained for 30 minutes with either EtBr, Gel Red, S b S f S b G ldSyber Safe, or Syber Gold.

• The gel portion stained with EtBr was de-stained before imaging while the others were imaged immediately

• Stability of the stain was tested in the same way at Day 1, 2, and 7EtBr Syber Gold Syber Safe Gel Red

Alternative DNA Stains• Observations

– All stains were stable over a 7

EtBr

day time frame– Gels stained with either of the

Syber dyes produced imagesSyber dyes produced images with limited background even with increasing integration times

– More uniform fluorescent intensity of large and small fragments

Sybr Gold

fragments

PFGE Goes Mainstream!!!See how the lab techs on CSI

Miami do PFGE!!!!Miami do PFGE!!!!Monday, October 19th

AcknowledgementsAll PulseNet participants at CDCAll PulseNet participants at CDC,

FDA, USDA, and in the State Public Health Laboratories

fThe findings and conclusions in this presentation are those of the

author and do not necessarily represent the views of the Centersrepresent the views of the Centers

for Disease Control and Prevention

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