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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
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
3 Development and testing of draft ISO/TS 6579-4
● “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
4 Development and testing of draft ISO/TS 6579-4
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
6 Development and testing of draft ISO/TS 6579-4
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.
Development and testing of draft ISO/TS 6579-4
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)
Development and testing of draft ISO/TS 6579-4
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
Development and testing of draft ISO/TS 6579-49
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
Development and testing of draft ISO/TS 6579-410
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
Development and testing of draft ISO/TS 6579-411
Re-analyse 12 strains● different results between one or more PCR protocols
Development and testing of draft ISO/TS 6579-412
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
Development and testing of draft ISO/TS 6579-414
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
Development and testing of draft ISO/TS 6579-416
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
EURL-Salmonella workshop September 202017
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
Development and testing of draft ISO/TS 6579-418
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)
Gel based multiplex PCR
● Small adjustments in PCR protocol
– Amplification time to 1.5 min
– Change in final concentrations primers
EURL-Salmonella workshop September 202019
Re-analysis 22 strains with slide agglutination
Development and testing of draft ISO/TS 6579-420
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
Development and testing of draft ISO/TS 6579-421
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
Development and testing of draft ISO/TS 6579-422
Typing resultssender/
RIVM
No of strains
Multiplex real-time PCR
Gel based multiplex 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).
Development and testing of draft ISO/TS 6579-423
THANK YOU FOR LISTENING!
Special thanks:NRL-Salmonella Germany (BfR)NRLs-Salmonella who sent strains