Salmonella isolation, identification and Serotyping from Food and Animals the practical angle

Transcription

1 Salmonella isolation, identification and Serotyping from Food and Animals the practical angle

2 Why use ISO-6579: 2002 standard The Strong side: It s well known and recommended by the WHO. It s standardised and revised in It s very sensitive. It diminish the risk of obtaining false negative results. The down side: May not propagate Salmonella Typhi and Salmonella Paratyphi. It s laborious

3 Isolation Pre-enrichment in non-selective medium (buffered peptone water). Selective enrichment in Tetrathionate broth (Müller-Kauffmann) and Rappaport Vassiliadis soy peptone (RVS) broth. Subcultivation on Xylose Lysine Desoxycholate (XLD) agar and on Brilliant Green agar (BGA) (or another selective agar media).

4 The two times two media procedure. Sample of 25g to 225ml buffered peptone water (1:9) Incubate at 36 C/18-24h Transfer 1ml to 10ml of Tetrathionate broth Incubate at 36 C/18-24h Transfer 0.1ml to 10ml of Rappaport Vassiliadis Soy Peptone broth Incubate at 41.5 C/18-24h Inoculate XLD agar plates by loop Incubate at 36 C/18-24h Inoculate BGA agar plates or other by Incubate at 36 C/18-24h

5 Typical colonies on the selective agar XLD Agar: Slightly transparent zone of reddish colour Black center, pink-red zone may be seen BG Agar: Gray-reddish / pink slightly convex

6 Or use a commercial available identification kit e.g. API systems. Revised in The identification step. Test Positive or negative reaction Percentage of Salmonella inoculations showing the reaction TSI glucose (acid formation) TSI glucose (gas formation) TSI lactose TSI sucrose TSI hydrogen sulphide Urea splitting Lysine decarboxylation β-galactosidase reaction Voges-Proskauer reaction Indole reaction ) ) ) )

7 Biochemical confirmation with the use of quality control strains. Triple Sugar Iron Agar (TSI) Acid production if the butt is yellow, and the slope is red, acid production is only from glucose. On the contrary if acid production is from lactose and/or sucrose Only the surface will be Yellow. Gas production Air bubbles in butt. H 2 S production. Reduction of thiosulphate into hydrogen sulphide cause the ferro-ions to precipitate as black ferro-sulphide Salmonella Negative

8 Biochemical confirmation with the use of quality control strains. Lysine decarboxylase test Decarboxylase convert amino acids to alkalic amines as lysine converts into kadaverin. Initial glucose are fermented and cause a drop in ph (colour change from brownish to yellow). Secondly, when the amino acid are decarboxylased into amines the ph will raise even higher and change the colour of the media to purple. Negative Salmonella

9 Biochemical confirmation with the use of quality control strains. Indole test. The amino acid tryptofan (indolyl-alanin) can split alanin during production of indol. Indole extracts into the top layer of amyl alcohol and forms a red dye with p-di-methylamino-benzaldehyd. Often combined with motility and ornithine (Salm = pos.) Salmonella Negative

10 Biochemical confirmation with the use of quality control strains. Urea broth Urea can be split into carbon dioxide and ammonia, which react with the phenol-red indicator in the media which change the colour of the media into rose pink deep cerise Positive Salmonella

11 Biochemical confirmation with the use of quality control strains. Simmons Citrate agar Organisms growing on Simmons Citrate Agar are capable of using citrate as the sole carbon source and they can metabolize the ammonium salt in the medium. Use of citrate increases the ph of the medium. The increase in ph then causes color change in the bromothymol blue indicator, turning it blue. Under acidic condition it changes to yellow colour. Salmonella Negative

12 Biochemical confirmation with the use of quality control strains.

13 Salmonella serotyping The best bacterial phenotyping method ever made Established in 1929 (White) then Kauffmann Phenotypic characterization of strains based on the immunologic reactivity of two surface structures: Lipopolysaccharide (O antigen) Flagellin protein (H antigen) +/- Vi antigen

14 Schematic Representation of Salmonella Serotype Antigens O-antisera O-antigens (LPS) Vi-antigens (Capsule) Vi-antisera H-antisera H antigens (flagella) Salmonella cell H-antisera

15 Genus Salmonella Two species S. enterica S. bongori Six subspecies 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) Biochemical tests Serotyping Numerous serotypes

16 Vi antigen Typhi +++, Paratyphi C, Dublin Determination by slide agglutination with a single polyclonal antisera Can hide O antigens (destroy Vi antigen by boiling before performing O agglutination)

17 O Polyvalent Antisera OMA = 2 (A) - 4 (B) - 9 (D 1 ) - 9,46 (D 2 ) - 3,10 (E 1 ) - 1,3,19 (E 4 ) -21 (L) OMB = 7 (C 1 ) - 8 (C 1 -C 2 ) - 11 (F) - 13 (G) - 6,14 (H). OMC = OMD = OME = OMF = OMG =

18 H Polyvalent Antisera HMA = a + b + c + d + i + z 10 + z 29 HMB = E -complex + G -complex HMC = k + y + z + L -complet + Z 4 -complex + r HMD = z 35 + z 36 + z 38 + z 39 + z 41 + z 42 + z 44 + z 60 HME = z 52 + z 53 + z 54 + z 54 + z 55 + z 57 + z 61

19 Confirmation of Salm. Inoculate the strain onto a none selective agar e.g. Nutrient agar and Swarm agar Incubate o.n at 37ºC

20 Confirmation of Salm. Dispense a drop of antisera onto a slide Culture from the Nutrient agar or the swarm agar are mixed with the antiserum Rock the slide gently for about 30 sec.

21 Confirmation of Salm. Positive reaction visible clumping = agglutination Negative reaction homogenous milky turbidity Controls : Saline and negative reaction in another antisera Negative Positive

22 Slide agglutination - O-Antigen O: 10 O : 46 O : 9 O : 34 O : 20 Conc. H : z 10 O : 5 O :27 O : 14 Conc. H : i O : 4 O : 19 O : 8 Conc. H : d Conc. H : r O : 2 O : 15 O : 7 Conc. H : c Conc. H : L Conc. H : z29 O : 1 O : 12 O : 6 O: Vi Conc. H : b Conc. H : G Conc. H : z 6 OMA OMB Conc. H : a Conc. H : E Conc. H : 1 complex - Try Positive the factor reaction sera = represented O: 6,7 in the positive pool until you find a positive reaction. - After having completed the O-typing, continue with H-typing. Test the isolate (Nutrient agar) against the polyvalent O- antisera

23 Slide agglutination - H-Antigen H : z 10 H : f H : 7 H : i H : G complex H: p H : z 6 H : d H : z15 H : t H : 6 H : c H : x H : s H : r H : 5 H : b H : h H : q H : w H : 2 H : a H : E complex H : m H : L complex H : 1 complex H : z29 HMA HMB HMC HMD HME Test the isolate (Swarm agar) against the polyvalent H- antisera. Try - Positive the factor reaction sera = represented H : r in the positive pool, until you find a positive reaction.

24 Previous results: I:6.7 : r :? Next step - phase inversion.

25 Phase inversion preparation Add 10µl of concentrated antisera in a small petri dish corresponding to the phase whish has been identified Ex. H:r Pour approximately 10ml of melted swarm agar Leave to solidify

26 Inoculation - phase inversion. Inoculate the plate in the centre with af loop Incubate o.n at 37ºC

27 Detection - the 2. phase of the H Use culture from the edge of the growth zone for slide agglutination Select the relevant H antisera, by using the Kauffmann - White Scheme

28 Using Kauffmann-White Scheme

29 Final Results: Antigenic formula: Salmonella Virchow - 6,7 : r : 1,2

30 Species and subspecies Some have the same antigenic formula Should be seperated by biochemical tests Look in the Kauffmann-White Scheme at page 13

31 Species and subspecies Salmonella are divided into two species S. enterica and S. bongori

32 Species and subspecies - S. enterica: divided into 6 subspecies - S.enterica subspecies enterica have all a name in the KW - Others: do not have a name but a number e.g. I, II.

33 Species and subspecies Seperate them by biochemical tests Choose one to five tests, depending on which one you have to distinguish between

34 Species and subspecies Here you have to distinguish between S.enterica subsp enterica (S.Indiana) and S.enterica subsp salamae Consult your KW scheme

35 Species and subspecies - Ex Malonat. - Malonat : S. Indiana - Malonat + : S. enterica subsp. Salamae - S.II 4.12: z : 1,7

36 Limitations of serotyping Availability of antisera Costs Of the about 200 antisera (needed for serotyping of all known serotypes), 140 are not commercialized. Production is time-consuming (inoculation of rabbits, numerous absorptions to prevent cross-reactions) Distribution of serotypes Often 2 serotypes are predominant (Enteritidis and Typhimurium) so weak discriminatory power for outbreak investigations (need for subtyping methods) Untypeable isolates : rough, monophasic or non-motile

37 Thank you for your attention Rene S. Hendriksen, PhD Research group of Antimicrobial Resistance and Molecular Epidemiology WHO Collaborating Centre for Antimicrobial Resistance in Food borne Pathogens European Union Reference Laboratory for Antimicrobial Resistance National Food Institute Denmark