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What about s typhimurium like

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What are Salmonella Typhimurium like strains ?

What are Salmonella Typhimurium like strains ?

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  • 1. Meaning of emerging strainsSalmonella “Typhimurium-like”monophasic or non motile strainsH. Imberechts
  • 2. Salmonella Typhimurium-likeo Salmonella Typhimurium-like (STM-like) are frequently beenisolated, also in Belgiumo Questions• What kind of Salmonella are they ?• How are these reported ?• Are these really Salmonella Typhimurium, mutantserotypes, or other ?• Are they genetically homogenous ?• Are specific regulatory / methodological dispositionsrequired ?• What is known about their virulence and spread (inBelgium) ?2
  • 3. 3
  • 4. Salmonella classificationo 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 = VIo Species Salmonella bongori = Vo Classification beyound the subspecies level= SERO-typing according to Kauffmann-White-Le Minoro http://www.pasteur.fr/ip/portal/action/WebdriveActionEvent/oid/01s-000036-0894 >95% of all (human)salmonelloses, incl.typhoïd fever >1,500 serovars
  • 5. Salmonella serotypingo Technique: agglutinationo Serotype = formula• Antigen O: LPS (sugars)• Antigen H: flagella (proteins)• Two phases: H1 and H2• One phase at a time in a single bacterial cello Examples: O : H1 : H2• Enteritidis 1,9,12 g,m -• Typhimurium 1,4,[5],12 i 1,2• Virchow 6,7,14 r 1,2Conventions : x = factor encoded by a bacteriophage (lysogenic strains)[x] = factor that may or may not be present (not phage-encoded)5POS reaction NEG reaction
  • 6. Salmonella serotypingo Technique: agglutinationo Serotype = formula• Antigen O: LPS, sugars• Antigen H: flagella, proteinso 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 w6
  • 7. Technical aspectso H1 and H2 (Phase inversion medium Sven Gard, contains Ab)• "Swarming" observed after growth with Ab directedagainst one (dominant) H phaseo Difficulties• Poor Ag expression or "loss" of antigens (roughstrains, mono- or aphasic H variants)• Antiserum fails• Auto-agglutinating strainso 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 needed7
  • 8. Nomenclatureo 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 ?8
  • 9. Situation STM-like in Belgium, AHo CODA-CERVA is NRL Salmonella (Animal Health)o Number of Salmonella 4,[5],12 : i : - (= "STM-like") strainsisolated 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 isolates9
  • 10. Situation Belgium, AHo Also found are• Salmonella 4,12,[5] : - : 2• Salmonella 4,12,[5] : - : -o Found mainly in pigs but alsopoultry (layers and broilers),cattle, feed and otherso These isolates are not specifically more antibiotic resistant10Origin Nbre of strains %Pigs 216 72.0Poultry 32 10.7Cattle/Calf 14 4.7Feed 26 8.7bird 1 0.3Pigeon 1 0.3Sludge 1 0.3Turkey 1 0.3unknown 8 2.7TOTAL 300 100.0
  • 11. STM-like, geneticso 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 region11-
  • 12. Discriminative PCR test Tennant SM, Diallo S, Levy H, Livio S, Sow SO, et al. (2010) Identification by PCR of Non-typhoidalSalmonella enterica Serovars Associated with Invasive Infections among Febrile Patients in Mali. PLoSNegl Trop Dis 4(3): e621. doi:10.1371/journal.pntd.0000621 Echeita MA, Herrera S, Usera MA (2001) Atypical, fljB-negative Salmonella enterica subsp. entericastrain of serovar 4,5,12:i:- appears to be a monophasic variant of serovar Typhimurium. J ClinMicrobiol 39: 2981–298312
  • 13. Other testso Phage typing• Battery of specific bacteriophages (per Salmonella serotype)• Combination of reactions  profile or phage type (PT)• STM-like have Salmonella Typhimurium typical profileso Pulsed Field Gel Electrophoresis• STM-like have Salmonella Typhimurium typical profileso Other genetic tests• MLST• Multiple Locus Sequence Typing• MLVA/VNTR• Multiple Loci VNTR Analysis / Variable Number Tandem Repeat13
  • 14. MLVA trend of (Belgian) STM-likeo 5-marker VNTR scheme elaborated for S. Typhimurium• Multiple profiles observed• No obvious animal-specificityo Work of Cécile Boland(PhD thesis, promotor P. Wattiau)14PigsPoultryCattle
  • 15. Significance STM-Likeo Should be reported separately• Is not Salmonella Typhimurium, nor other serotypeencoding fljB flagellar genes, but not expressing them• Is related to Salmonella Typhimurium; thereforeS. Typhimurium variant, or STM-like• See: epidemiology, trend (e.g. EFSA report)• Uniform reporting in all reports needed, forcomparative purposes• Origin not clearo Virulence and antimicrobial resistance are similar to those ofSalmonella TyphimuriumIn Belgium: study is ongoing• Are 4,[5] : i : - fljB negative ?• Are these present in all food producing animals ?• Are these related to human isolates ?15
  • 16. Serotyping vs genetic testso 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)16ABCDEFGHIJ1 2 3 4 5 6 7 8 9 10 11 12Panel IPanel IIPanel III
  • 17. Genetic tests vs. (manual) serotypingo 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 systems scope: limited “geno”typeso Future: all Salmonella serotypes may be of importance forpublic health• Belgian legislation takes STM-like into account17
  • 18. Conclusionso Salmonella Typhimurium-like 4,[5] : i : - strains represent agenetic variant of Salmonella Typhimuriumo Flagellar fljB gene is absento 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 Belgiumo Are currently reported separately18
  • 19. Acknowledgementso The serotypers ..• Heidi Vander Veken• Danielle Vandergheynst• Mieke Van Hesscheo The Salmonella expert ..• Vicky Jassono The molecular scientist and Salmonella Chief ..• Pierre Wattiauo The next-generation scientist ..• Cécile BolandVisit us at: www.coda-cerva.be19