PulseNet PFGE. Kara Cooper Centers for Disease Control and Prevention NCZED/DFBMD/EDLB/PMDRL September 24, 2009

Transcription

1 PulseNet PFGE Laboratory Updates Kara Cooper Centers for Disease Control and Prevention NCZED/DFBMD/EDLB/PMDRL September 24, 2009

2 Updates Protocols Non-O157 STEC PFGE protocol Listeria protocol review Shigella flexneri update Procedure/Reagent Updates Investigation of alternative agaroses Enzyme evaluation Not so typical thiourea-dependent d t strains Alternative gels stains

3 Rapid Standardized PFGE Protocols for Subtyping Foodborne Pathogenic Bacteria Current Protocols E. coli O157:H7 Salmonella Listeria i monocytogenes Shigella sonnei Campylobacter jejuni Clostridium perfringens Vibrio cholerae Yersinia pestis Vibrio parahaemolyticus Recently Released Protocols Non-O157 STEC Clostridium botulinum Protocols Under Development Shigella flexneri (2009) Vibrio vulnificus (2010) Yersinia enterocolitica (2010) Campylobacter coli

4 Non-O157 STEC Standardized PFGE Protocol Increased number of Non-O157 STEC submissions to the National E. coli database over the last several years Non-O157 STEC PFGE patterns differ from O157 in regard to the number and distribution of bands Complicated data analysis and raised concerns about reproducibility Need for alternative electrophoresis conditions ***Additional information provided during poster session by Kara Cooper

5 Non-O157 STEC Standardized di d PFGE Protocol O157 Conditions 2.16s-54.17s Non-O157 Conditions 6.76s-35.38s The non-o157 STEC electrophoresis conditions (6.76s-35.38s) improved the ease of analysis, band distribution, and overall pattern resolution External validation of these conditions demonstrated a high degree of inter- and intra-laboratory reproducibility Incorporated into a Standardized PulseNet protocol for non-o157 STEC All laboratories ato should begin subtyping non-o157 STECs with these conditions ***Additional information provided during poster session by Kara Cooper

6 Non-O157 STEC Standardized di d PFGE Protocol Database Scripts available on MasterScripts v.5.0 (distributed 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-O157 Request certification gel and instructions from PFGE@cdc.gov Would like all laboratories analysis-certified for non- O157 by end of this year Non-O157 STEC TIFF images can be sent to PFGE@cdc.gov until laboratory becomes analysiscertified ***Additional information provided during poster session by Kara Cooper

7 Shigella flexneri PFGE Protocol Shigella flexneri is a major Shigella flexneri cause of foodborne illness 2.16s-54.17s internationally INEI - ANLIS C.G.Malbrán Buenos Aires, Argentina International Working Group The current Shigella sonnei protocol does not perform well against Shigella flexneri in regard to number and overall band distribution

8 Shigella flexneri PFGE Protocol NotI XbaI Restriction Enzymes Primary NotI Secondary XbaI Electrophoresis conditions Initial Switch Time: 5s Final Switch Time: 35s

9 External Validation Shigella flexneri PFGE Protocol Comparison of data generated by 9 laboratories against 17 strains Successfully completed Final results to be presented at the PulseNet International Meeting Currently working on the development of BioNumerics scripts and an on-line database External Validation Working Group Laboratory CDC INEI - ANLIS C.G.Malbrán NAMRU Central Public Health Laboratory Country USA ARGENTINA EGYPT OMAN Instituto t Adolfo Lutz BRASIL National Microbiology Laboratory Public Health Laboratory Centre Staten Serum Institute Instituto de Salud Pública CANADA HONK KONG DENMARK CHILE

10 Listeria monocytogenes Revised PFGE Protocol Results of implementation has varied No issues Minor optimization Unresolved issues CDC has developed an extensive troubleshooting worksheet 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 (KCooper@cdc.gov) if you would like to receive the worksheet

11 Listeria monocytogenes Examples of Lysozyme going bad AscII ApaII PFGE Protocol (Lessons Learned) Plug Preparation and Lysis issues Freezer media Lab specific optimization of cell suspension concentrations Lysozyme of poor quality or has gone bad Molecular biology grade with protein 90%

12 Listeria monocytogenes PFGE Protocol (Lessons Learned) Incomplete restriction 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 o that can lead to star activity Prolonged reaction time Suboptimal buffer or buffer concentration High (> 5% v/v) glycerol concentrations High concentration of enzyme/µg of DNA ratio Same plugs re-tested AscI AscI ApaI Overnight Restriction ApaI 2 Hour Restriction

13 Investigation of Additional Agaroses for Use in PulseNet Standardized PFGE Protocols SeaKem Gold Agarose was originally BioRad MegaBase Agarose chosen for the PusleNet protocols Stability of the plugs and gels produced Relatively short run times Previous investigations proved that the composition of PFGE quality agarose vary from different vendors Significantly more fragile Required longer run times (+2-4 hours) Electrophoretic mobility of DNA fragments differed resulting in normalization issues ***Additional information provided during poster session by Molly Freeman

14 Additional Agarose Evaluation SeaKem Gold BioRad MegaBase Amresco III BioRad New Megabse Agarose ***Additional information provided during poster session by Molly Freeman

15 Additional Agarose Evaluation Comparison of XbaI PFGE patterns of Salmonella certification strains generated by 5 laboratories GA CDC CDC CDC VA VA GA CDC CDC CDC VA VA GA CDC CDC CDC VA VA GA CDC CDC CDC VA VA CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC CDC Comparison of Amresco and Megabase to SeaKem Gold Slightly more fragile, but manageable Runs slightly slower ( hour) Issues associated with normalization were only observed on gels that run short Further evaluation of these agaroses in the coming months We are actively recruiting 10 CDC additional volunteer laboratories ***Additional information provided during poster session by Molly Freeman

16 Evaluation of Enzyme Robustness Reason for evaluation Desire for time reduction Availability of newly engineered rapid digest restriction enzymes Changes to PFGE protocol that may enhance increased enzyme efficiency Enzymes Evaluated Fermentas Rapid Digest Fermentas Conventional enzyme Roche Conventional enzyme New England BioLabs Conventional enzyme ***Additional information provided during poster session by Jessica Halpin

17 Evaluation of Enzyme Robustness H9812 digested with XbaI over 4 different incubation times Roche 50 units 10m 30m 1h 2h Fermentas 50 units 10m 30m 1h 2h NEB 50 units 10m 30m 1h 2h Fermentas Fast Digest 5 units 10m 30m 1h 2h ***Additional information provided during poster session by Jessica Halpin

18 Evaluation of Enzyme Robustness Testing of additional Salmonella ll Serovars Roche XbaI, 50U, 10 min Summary of enzyme performance with 10 minute restriction Vendor (Units/Reaction) XbaI BlnI/AvrII/ XmaJI Fermentas Rapid Digest (5) Variable Variable Fermentas Conventional Enzyme (50) Good Poor Roche Conventional Enzyme (50) Good Good New England BioLabs Conventional Enzyme (30) Poor Poor ***Additional information provided during poster session by Jessica Halpin

19 Evaluation of Enzyme Robustness Several restrictions enzymes from several vendors show the potential for time reduction CDC PulseNet Laboratory will continuing to evaluate this with additional enzymes and organisms 10 minutes is an extreme Evaluate shorter restriction times on known isolates before testing routine isolates As always, laboratorians should critically review all gels and patterns for faint bands RESULTS WILL VARY BETWEEN LABORATORIES ***Additional information provided during poster session by Jessica Halpin

20 Typical Thoiurea-Dependent Without Thiourea Strains With Thiourea Untypeable Result Typeable Result Addition of 50 μm thiourea in 0.5XTBE electrophoresis buffer Include a positive control on gel Restricted ti t dpfge plug slice of f untypeable strain that t typed previously when thiourea was used in buffer.

21 Not So Typical Thoiurea- Typical Thoiurea-Dependent Strains without Thiourea Dependent Strains (Salmonella Saintpaul Outbreak Strain) H9812 Typical Thoiurea-Dependent Strains with Thiourea H9812 S. SaintPaul Outbreak Strain with Thiourea H9812 S. SaintPaul Outbreak Strain with Thiourea H9812 S. SaintPaul Outbreak Strain with Thiourea H9812

22 Not So Typical Thoiurea- Dependent Strains In one laboratory isolates are untypeable even with the addition of Thiourea, but typeable within another laboratory Observed with other Saintpaul strains as well as other Salmonella serotypes and E. coli Isolate will have to be submitted to CDC or area laboratory for testing

23 d6 Alternative DNA Stains Increased interest in alternatives to EtBr Benefits Reduced safety concerns De-staining is unnecessary Minimal background and enhanced visualization of lower bands Concerns Impact on appearance of gels/patterns Stability of stain over time and number of gels Increased cost Which one(s)? Eti Estimated tdcost per Working Slti Solution Stain Type Stain Cost Disposal Cost Total Cost Syber Safe Syber Gold Gel Red EtBr

24 Slide 23 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

25 Alternative 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, Syber 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 7 EtBr Syber Gold Syber Safe Gel Red

26 Alternative DNA Stains EtBr Sybr Gold Observations All stains were stable over a 7 day time frame Gels stained with either of the Syber dyes produced images with limited background even with increasing integration times More uniform fluorescent intensity of large and small fragments

27 PFGE Goes Mainstream!!! See how the lab techs on CSI Miami do PFGE!!!! Miami do PFGE!!!! Monday, October 19 th

28 Acknowledgements All PulseNet participants at CDC, FDA, USDA, and in the State Public Health Laboratories The findings and conclusions in this presentation are those of the author and do not necessarily represent the views of the Centers for Disease Control and Prevention