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Development and testing of draft ISO/TS 6579-4: Identification of monophasic SalmonellaTyphimurium

Development and testing of draft ISO/TS 6579-4

Robin Diddens

https://creativecommons.org/licenses/by-sa/3.0/

Monophasic Salmonella Typhimurium

2 Development and testing of draft ISO/TS 6579-4

● “Salmonella Typhimurium-like” strains in food and animals

● Increasing number of cases and outbreaks had been reported in humans in Member States

● Salmonella Typhimurium (1,4,[5],12:i:1,2)

● Strains lacking expression of the phase one flagellar antigens or both not commonly been reported to be associated with significant disease in animals or humans.

Monophasic Salmonella Typhimurium


● “Salmonella Typhimurium-like” strains in food and animals

● Increasing number of cases and outbreaks have been reported in humans in Member States

● Salmonella Typhimurium (1,4,[5],12:i:1,2)

● Strains lacking expression of the phase one flagellar antigens or both not commonly been reported to be associated with significant disease in animals or humans.

● Monophasic Salmonella Typhimurium (1,4,[5],12:i:-)

Regulation (EC) No. 2073/2005● Amendment in 2011


Other serovars● Salmonella Typhimurium (1,4,[5],12:i:1,2) ● Monophasic Salmonella Typhimurium (1,4,[5],12:i:-)

● Variant of another non-target serovar– S. Lagos (1,4,[5],12:i:1,5)– S. Agama (4,12:i:1,6)– S. Farsta (4,12:i:e,n,x) – S. Tsevie (1,4,12:i:e,n,z15) – S. Gloucester (1,4,12,27:i:l,w)– S. Tumodi (1,4,12:i:z6)

● Distinction with PCR5 Development and testing of draft ISO/TS 6579-4

Draft ISO/TS 6579-4 PCR monoSTM

● 2014: decided to work on standardisation of PCR method(s) for identification of monophasic Salmonella Typhimurium

● 2015: inventory of available methods

● 2016-2017: Working Drafts (WDs) of ISO/TS 6579-4 drafted by Burkhard Malorny (NRL-Salmonella Germany) including 3 PCR protocols


PCR protocols

1. Multiplex real-time PCR assay; Primers and probes published by Maurischat et al. 2015, Int. Food Microbiol. 193:8-14.

2. Agarose gel-based multiplex PCR assay; Primers published by Tennant et al. 2010 PLoS Negl. Trop. Dis. Mar 9;4(3):e621’; and EFSA 2010, EFSA Journal, 8(10), p. 1826.

3. Agarose gel-based single target PCR assay; Primers published by Maurischat et al. 2015, Int. Food Microbiol. 193:8-14.


PCR protocols1. Multiplex real-time PCR assay2. Agarose gel-based multiplex PCR assay3. Agarose gel-based single target PCR assay

● 1, 2 and 3: Target genetic sequence to make distinction betweenSTM, and monoSTM with other Salmonella serovars

● 2: target genetic sequence – present in isolates with expressionsecond H phase antigen

● 1 and 3: – target two genetic sequences - present in isolates with

expression second H phase antigen – Internal Amplification Control (IAC)


Test PCR protocols

● Test different PCR procotols

● Call for test strains over 400 strains received

● All strains were typed at EURL-Salmonella

● Selection of 172 strains (target and non-target strains)

● Following EN ISO 16140-6:2019– Protocol for the validation of alternative (proprietary) methods for

microbiological confirmation and typing procedures


Test PCR protocols - strains

● Target strains:– Serotyped and PCR as monoSTM (1,4,[5],12:i:-)– Serotyped and PCR as (biphasic)STM (1,4,[5],12:i:1,2)– Inconsistent results with serotyping and PCR

› Serotyping monoSTM, PCR STM› Serotyping STM, PCR monoSTM

● Non-target serovars:– Including S. Lagos, S. Agama, S. Farsta, S. Gloucester

● Non-target genus strains: from family Enterobacteriaceae


Results● All 172 strains tested with the 3 PCR protocols by NRL-Salmonella

in Germany (BfR) and by EURL-Salmonella● Results were compared:

– Most strains gave similar results

– 12 strains gave different results for one or more PCR protocolsbetween two laboratories› Re-analyse by EURL-Salmonella

– 22 strains gave different results between the three PCR protocolsMajority of these strains: monoSTM with multiplex real-time PCR and gel-based single target PCR (Maurischat et al.), but (biphasic) STM with gel-based multiplex PCR (Tennant et al.)

› Re-analyse with slide agglutination


Re-analyse 12 strains● different results between one or more PCR protocols


Strain Multiplex real-time PCR Gel based multiplex PCR Gel based single target PCR

EURL BfR EURL BfR EURL BfRS030 STM STM mSTM STM STM STMS116 mSTM mSTM STM STM STM mSTMS230 STM STM mSTM STM STM STMS272 mSTM mSTM STM STM STM mSTM

S286 Other serovar STM Other

serovar STM Other serovar STM

S288 Other serovar mSTM Other

serovar mSTM Other serovar mSTM

S292 Other serovar STM Other

serovar STM Other serovar STM

S296 STM Other serovar STM Other

serovar STM Other serovar

S299 mSTM Other serovar mSTM Other

serovar mSTM Other serovar

S305 STM Other serovar STM Other

serovar STM Other serovar

S308 mSTM mSTM STM STM STM mSTMS310 mSTM mSTM STM STM STM mSTM

Re-analyse 12 strains● different results between one or more PCR protocols

● 4 strains different results with gel based single target PCREURL-Salmonella workshop September 202013

Strain Multiplex real-time PCR Gel based multiplex PCR Gel based single target PCR

EURL BfR EURL BfR EURL BfRS030 STM STM STM STM STM STMS116 mSTM mSTM STM STM STM mSTMS230 STM STM STM STM STM STMS272 mSTM mSTM STM STM STM mSTMS286 STM STM STM STM STM STMS288 mSTM mSTM mSTM mSTM mSTM mSTMS292 STM STM STM STM STM STM

S296 Other serovar

Other serovar

Other serovar

Other serovar

Other serovar

Other serovar

S299 Other serovar

Other serovar

Other serovar

Other serovar

Other serovar

Other serovar

S305 Other serovar

Other serovar

Other serovar

Other serovar

Other serovar

Other serovar

S308 mSTM mSTM STM STM STM mSTMS310 mSTM mSTM STM STM STM mSTM

Gel based single target PCR● Interpretation of weak amplification fragments

● Different final concentration hin-iroB target– 0.2 pmol/µl instead of 0.4 pmol /µl


Gel based single target PCR

EURL-Salmonella workshop September 202015

hin-iroB – final concentration: 0.4 pmol /µl

hin-iroB – final concentration: 0.2 pmol /µl

Similar interpretation of the results

Gel based multiplex PCR● Weak 1389 bp band for some strains at BfR and EURL-Salmonella

Amplification time: 1 min


Amplification time: 1.5 min1369 bp

1369 bp

1000 bp

250 bp

1000 bp

250 bp

Primer-dimers

Gel based multiplex PCR● Lower final concentration primers


Reaction setupPrimers (final concentrations)

FFLIB / RFLIA Sens-59/Antisense-83

fliA-fliB (1000 or 250 bp) fljB (1389 bp)

draft ISO-TS 6579-4 1.2 pmol/µl 0.4 pmol/µl

Tennant et al. 2010 0.4 pmol/µl 0.2 pmol/µl

EFSA 2010 0.1 pmol/µl 1.0 pmol/µl

1 0.4 pmol/µl 0.2 pmol/µl

2 0.2 pmol/µl 0.4 pmol/µl

Gel based multiplex PCR


Reaction setup 1fliA-fliB: 0.4 pmol/µl (1000 or 250 bp)fljB: 0.2 pmol/µl (1389 bp)

Reaction setup 2fliA-fliB: 0.2 pmol/µl (1000 or 250 bp)fljB: 0.4 pmol/µl (1389 bp)


● Small adjustments in PCR protocol

– Amplification time to 1.5 min

– Change in final concentrations primers


Re-analysis 22 strains with slide agglutination


Analysis sender/RIVM Multiplex Real-time PCR Gel based multiplex PCR Gel based single target

PCREURL

number Serovar EURL BfR EURL BfR EURL BfREURL-S067 STM mSTM STM_monophasic STM STM_biphasic mSTM STM_monophasicEURL-S069 STM mSTM STM_monophasic STM STM_biphasic mSTM STM_monophasicEURL-S070 STM mSTM STM_monophasic STM STM_biphasic mSTM STM_monophasicEURL-S071 STM mSTM STM_monophasic STM STM_biphasic mSTM STM_monophasicEURL-S072 STM mSTM STM_monophasic STM STM_biphasic mSTM STM_monophasicEURL-S075 STM mSTM STM_monophasic STM STM_biphasic mSTM STM_monophasicEURL-S076 STM mSTM STM_monophasic STM STM_biphasic mSTM STM_monophasicEURL-S079 STM mSTM STM_monophasic STM STM_biphasic mSTM STM_monophasicEURL-S080 STM mSTM STM_monophasic STM STM_biphasic mSTM STM_monophasicEURL-S081 STM mSTM STM_monophasic STM STM_biphasic mSTM STM_monophasicEURL-S114 STM mSTM STM_monophasic STM STM_biphasic mSTM STM_monophasicEURL-S116 STM mSTM STM_monophasic STM STM_biphasic STM STM_monophasicEURL-S197 STM mSTM STM_monophasic STM STM_biphasic mSTM STM_monophasicEURL-S198 STM mSTM STM_monophasic STM STM_biphasic mSTM STM_monophasicEURL-S206 STM mSTM STM_monophasic STM STM_biphasic mSTM STM_monophasicEURL-S226 STM mSTM STM_monophasic STM STM_biphasic mSTM STM_monophasicEURL-S266 STM mSTM STM_monophasic STM STM_biphasic mSTM STM_monophasicEURL-S267 STM STM STM_biphasic mSTM STM_monophasic STM STM_biphasicEURL-S272 STM mSTM STM_monophasic STM STM_biphasic STM STM_monophasicEURL-S308 STM mSTM STM_monophasic STM STM_biphasic STM STM_monophasicEURL-S310 STM mSTM STM_monophasic STM STM_biphasic STM STM_monophasicEURL-S313 STM STM STM_biphasic mSTM STM_monophasic STM STM_biphasic

Re-analysis 22 strains with slide agglutination


Analysis sender/RIVM Multiplex Real-time PCR Gel based multiplex PCR Gel based single target

PCR Results slide agglutinationEURL

number Serovar EURL BfR EURL BfR EURL BfREURL-S067 STM mSTM STM_monophasic STM STM_biphasic mSTM STM_monophasic mSTMEURL-S069 STM mSTM STM_monophasic STM STM_biphasic mSTM STM_monophasic mSTMEURL-S070 STM mSTM STM_monophasic STM STM_biphasic mSTM STM_monophasic mSTMEURL-S071 STM mSTM STM_monophasic STM STM_biphasic mSTM STM_monophasic mSTMEURL-S072 STM mSTM STM_monophasic STM STM_biphasic mSTM STM_monophasic mSTMEURL-S075 STM mSTM STM_monophasic STM STM_biphasic mSTM STM_monophasic mSTMEURL-S076 STM mSTM STM_monophasic STM STM_biphasic mSTM STM_monophasic mSTMEURL-S079 STM mSTM STM_monophasic STM STM_biphasic mSTM STM_monophasic mSTMEURL-S080 STM mSTM STM_monophasic STM STM_biphasic mSTM STM_monophasic mSTMEURL-S081 STM mSTM STM_monophasic STM STM_biphasic mSTM STM_monophasic mSTMEURL-S114 STM mSTM STM_monophasic STM STM_biphasic mSTM STM_monophasic mSTMEURL-S116 STM mSTM STM_monophasic STM STM_biphasic STM STM_monophasic STMEURL-S197 STM mSTM STM_monophasic STM STM_biphasic mSTM STM_monophasic mSTMEURL-S198 STM mSTM STM_monophasic STM STM_biphasic mSTM STM_monophasic mSTMEURL-S206 STM mSTM STM_monophasic STM STM_biphasic mSTM STM_monophasic mSTMEURL-S226 STM mSTM STM_monophasic STM STM_biphasic mSTM STM_monophasic mSTMEURL-S266 STM mSTM STM_monophasic STM STM_biphasic mSTM STM_monophasic STMEURL-S267 STM STM STM_biphasic mSTM STM_monophasic STM STM_biphasic STMEURL-S272 STM mSTM STM_monophasic STM STM_biphasic STM STM_monophasic STMEURL-S308 STM mSTM STM_monophasic STM STM_biphasic STM STM_monophasic STMEURL-S310 STM mSTM STM_monophasic STM STM_biphasic STM STM_monophasic STMEURL-S313 STM STM STM_biphasic mSTM STM_monophasic STM STM_biphasic STM

Summary results


Typing resultssender/

RIVM

No of strains

Multiplex real-time PCR


Gel based single target PCR

EURL BfR EURL BfR EURL BfR

monophasicSTM 37 37 37 37 37 37 37

biphasicSTM 65

41 (21 mSTM,

3 otherserovar)

41 (21 mSTM,

3 otherserovar)

59 (3 mSTM, 3 otherserovar)

59 (3 mSTM, 3 other serovar)

45 (17 mSTM,

3 otherserovar)

41 (21 mSTM,

3 other serovar)

otherSalmonellaserovars

43 43 43 43 43 43 43

otherEntero-

bacteriaceae27 27 27 27 27 27 27

Next steps18 May – 16 August 2020 voting for launching New Work Item Proposal (NWIP) in ISO and CEN of ‘Draft CEN ISO/TS 6579-4 ‘Microbiology of the food chain - Horizontal method for the detection, enumeration and serotyping of Salmonella - Part 4: Identification of monophasic Salmonella Typhimurium (1,4,[5],12:i:-) by polymerase chain reaction (PCR)’. ● Outcome voting in ISO and CEN: 100% approval and few

comments.● Next steps:

– Prepare document in CEN/ISO format;– Review comments;– Discuss document and next steps in ISO-WG10 (probably

November 2020).


THANK YOU FOR LISTENING!

Special thanks:NRL-Salmonella Germany (BfR)NRLs-Salmonella who sent strains

development and testing of draft iso/ts 6579-4: identification of … · 2020. 9. 23. · draft...

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