What are Salmonella Typhimurium like strains ?

1. Meaning of emerging strains
Salmonella “Typhimurium-like”
monophasic or non motile strains
H. Imberechts

2. Salmonella Typhimurium-like
o Salmonella Typhimurium-like (STM-like) are frequently been
isolated, also in Belgium
o Questions
• What kind of Salmonella are they ?
• How are these reported ?
• Are these really Salmonella Typhimurium, mutant
serotypes, or other ?
• Are they genetically homogenous ?
• Are specific regulatory / methodological dispositions
required ?
• What is known about their virulence and spread (in
Belgium) ?
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3. 3

4. Salmonella classification
o Subspecies of Salmonella enterica
• S. enterica subsp. enterica = I
• S. enterica subsp. salamae = II
• S. enterica subsp. arizonae = IIIa
• S. enterica subsp. diarizonae = IIIb
• S. enterica subsp. houtenae = IV
• S. enterica subsp. indica = VI
o Species Salmonella bongori = V
o Classification beyound the subspecies level
= SERO-typing according to Kauffmann-White-Le Minor
o http://www.pasteur.fr/ip/portal/action/WebdriveActionEvent/
oid/01s-000036-089
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 >95% of all (human)
salmonelloses, incl.
typhoïd fever
 >1,500 serovars

5. Salmonella serotyping
o Technique: agglutination
o Serotype = formula
• Antigen O: LPS (sugars)
• Antigen H: flagella (proteins)
• Two phases: H1 and H2
• One phase at a time in a single bacterial cell
o Examples: O : H1 : H2
• Enteritidis 1,9,12 g,m -
• Typhimurium 1,4,[5],12 i 1,2
• Virchow 6,7,14 r 1,2
Conventions : x = factor encoded by a bacteriophage (lysogenic strains)
[x] = factor that may or may not be present (not phage-encoded)
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POS reaction NEG reaction

6. Salmonella serotyping
o Technique: agglutination
o Serotype = formula
• Antigen O: LPS, sugars
• Antigen H: flagella, proteins
o Examples: O : H1 : H2
• Enteritidis 9 m -
• Typhimurium 4 i 2
• Virchow 6,7 r 2
• Paratyphi B 4 b 2
• Lagos 4 i 5
• Agama 4 i 6
• Gloucester 4 i w
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7. Technical aspects
o H1 and H2 (Phase inversion medium Sven Gard, contains Ab)
• "Swarming" observed after growth with Ab directed
against one (dominant) H phase
o Difficulties
• Poor Ag expression or "loss" of antigens (rough
strains, mono- or aphasic H variants)
• Antiserum fails
• Auto-agglutinating strains
o Resolution of agglutination tests is not sufficient for
• Salmonella Enteritidis attenuated vaccines identification
• Salmonella subsp Gallinarum / Pullorum differentiation
• Salmonella Paratyphi B var Java (tartrate pos)
• Salmonella enterica subsp. II, III, IV, V, VI
• E.g. differentiation with Salmonella Indiana
• S. enterica subsp. enterica sv Indiana 4 : z : 7
• S. enterica subsp. salamae 4 : z : 7
• Additional (biochemical) tests needed
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8. Nomenclature
o If slide agglutination does not reveal enough information,
a formula is given
• Salmonella 6,7 : - : 2
• Virchow 6,7 r 2
• Salmonella 4 : i : - = STM-like ?
• Typhimurium 4 i 2
• Lagos 4 i 5
• Agama 4 i 6
• Salmonella 4 : - : 2 = STM-like ?
• Salmonella 4 : - : - = STM-like ?
o More discriminative tests are needed !
o Are flagella genes present and not expressed ?
o Are monovalent / not-motile strains mutants ?
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9. Situation STM-like in Belgium, AH
o CODA-CERVA is NRL Salmonella (Animal Health)
o Number of Salmonella 4,[5],12 : i : - (= "STM-like") strains
isolated each year in Belgian farm animals (increasing ?)
• 2005: 17 isolates
• 2006: 16 isolates
• 2007: 45 isolates
• 2008: 91 isolates
• 2009: 51 isolates
• 2010: 42 isolates
• 2011: 61 isolates
• 2012: 94 isolates
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10. Situation Belgium, AH
o Also found are
• Salmonella 4,12,[5] : - : 2
• Salmonella 4,12,[5] : - : -
o Found mainly in pigs but also
poultry (layers and broilers),
cattle, feed and others
o These isolates are not specifically more antibiotic resistant
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Origin Nbre of strains %
Pigs 216 72.0
Poultry 32 10.7
Cattle/Calf 14 4.7
Feed 26 8.7
bird 1 0.3
Pigeon 1 0.3
Sludge 1 0.3
Turkey 1 0.3
unknown 8 2.7
TOTAL 300 100.0

11. STM-like, genetics
o Do STM-like strains contain the flagellar genes ?
• PCR of second-phase fljB, coding for structural flagel gene
• STM-like do not have fljB
• PCR of fliB-fliA region; Salmonella Typhimurium harbour IS200 sequence
• STM-like do have a large, S. Tyhimurium-specific fljB-fljA region
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12. Discriminative PCR test
 Tennant SM, Diallo S, Levy H, Livio S, Sow SO, et al. (2010) Identification by PCR of Non-typhoidal
Salmonella enterica Serovars Associated with Invasive Infections among Febrile Patients in Mali. PLoS
Negl Trop Dis 4(3): e621. doi:10.1371/journal.pntd.0000621
 Echeita MA, Herrera S, Usera MA (2001) Atypical, fljB-negative Salmonella enterica subsp. enterica
strain of serovar 4,5,12:i:- appears to be a monophasic variant of serovar Typhimurium. J Clin
Microbiol 39: 2981–2983
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13. Other tests
o Phage typing
• Battery of specific bacteriophages (per Salmonella serotype)
• Combination of reactions  profile or phage type (PT)
• STM-like have Salmonella Typhimurium typical profiles
o Pulsed Field Gel Electrophoresis
• STM-like have Salmonella Typhimurium typical profiles
o Other genetic tests
• MLST
• Multiple Locus Sequence Typing
• MLVA/VNTR
• Multiple Loci VNTR Analysis / Variable Number Tandem Repeat
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14. MLVA trend of (Belgian) STM-like
o 5-marker VNTR scheme elaborated for S. Typhimurium
• Multiple profiles observed
• No obvious animal-specificity
o Work of Cécile Boland
(PhD thesis, promotor P. Wattiau)
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Pigs
Poultry
Cattle

15. Significance STM-Like
o Should be reported separately
• Is not Salmonella Typhimurium, nor other serotype
encoding fljB flagellar genes, but not expressing them
• Is related to Salmonella Typhimurium; therefore
S. Typhimurium variant, or STM-like
• See: epidemiology, trend (e.g. EFSA report)
• Uniform reporting in all reports needed, for
comparative purposes
• Origin not clear
o Virulence and antimicrobial resistance are similar to those of
Salmonella Typhimurium
In Belgium: study is ongoing
• Are 4,[5] : i : - fljB negative ?
• Are these present in all food producing animals ?
• Are these related to human isolates ?
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16. Serotyping vs genetic tests
o Based on the detection (PCR, array) of
• Genes coding for somatic and flagellar genes
• Marker genes representative for certain serotypes
• E.g. Premi-Test® Salmonella, by Check-Points
(Wageningen, NL)
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A
B
C
D
E
F
G
H
I
J
1 2 3 4 5 6 7 8 9 10 11 12
Panel I
Panel II
Panel III

17. Genetic tests vs. (manual) serotyping
o Advantages
• Fast and firm reporting delay (1.5 working day)
• Highly reproducible and traceable results
• Not dependent on antigen expression of isolates
• Easily traceable and measurable (accreditation!)
o Disadvantages
• Definition of serotype; legal aspects
• Depending on system's scope: limited “geno”types
o Future: all Salmonella serotypes may be of importance for
public health
• Belgian legislation takes STM-like into account
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18. Conclusions
o Salmonella Typhimurium-like 4,[5] : i : - strains represent a
genetic variant of Salmonella Typhimurium
o Flagellar fljB gene is absent
o STM-like show similarities with S. Typhimurium
• IS200 sequences in fliB-fliA region
• Phage typing
• PFGE profiles
• Other genetic tests (MLVA, MLST)
o Are of epidemiological significance, also in Belgium
o Are currently reported separately
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19. Acknowledgements
o The serotypers ..
• Heidi Vander Veken
• Danielle Vandergheynst
• Mieke Van Hessche
o The Salmonella expert ..
• Vicky Jasson
o The molecular scientist and Salmonella Chief ..
• Pierre Wattiau
o The next-generation scientist ..
• Cécile Boland
Visit us at: www.coda-cerva.be
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