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Development of Compact Dry SL for Salmonella detection

Development of Compact Dry SL for Salmonella detection. S.Mizuochi １） ,H.Teramura １） ,S.Nirazuka １） ,H.Kodaka １） ,K.Inoue ２） 、 K.Tamura ３） 1)Research Dept., Nissui Pharmaceutical, 2) Saitama Pref. Ranzango Hp., 3)National Institute of Infectious Diseases. Background.

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Development of Compact Dry SL for Salmonella detection

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  1. Development of Compact Dry SL for Salmonella detection S.Mizuochi１）,H.Teramura１）,S.Nirazuka１）,H.Kodaka１）,K.Inoue２）、K.Tamura３） 1)Research Dept., Nissui Pharmaceutical, 2) Saitama Pref. Ranzango Hp., 3)National Institute of Infectious Diseases

  2. Background • Normally, Salmonella detection from food sample is processed these following steps, Pre-culture, Selective enrichment culture, Isolation and Confirmation/Identification. There are strong customer demands to improve the test procedure for more simple and rapid one, if it could have an equivalent result. • We develop simple and easy medium for Salmonella detection (Compact Dry SL, hereinafter CDSL) that detects Salmonella by combination of biochemical reaction, specific enzyme reaction and detection of motility of Salmonella. CDSL is screening method for detecting Salmonella which inoculate pre-culture sample as a specimen. • Herewith we report CDSL evaluation results compared with conventional culture methods using meats on the market, ① growth and color forming used by pure culture strains ② recovery of Salmonella from food materials ③ comparison (sensitivity/specificity/agreement) between conventional method and CDSL

  3. Package, CDSL

  4. Comparison of operation between conventional method and CDSL Conventional CDSL Day 1 Specimen25g Specimen 25g Pre-culture Pre-culture (BPW/EEM) (BPW/EEM) ↓ ↓ Day 2Selective Enrichment CultureCDSL (TT/RV/SBG/SC) ↓ ↓ Day 3Isolation Presume, presence of Salmonella (MLCB/DHL/XLD/Rambach) Isolation ↓ ↓ Day 4Presume, presence of SalmonellaConfirmation/Identification Confirmation/Identification(Biochemical/Serological Tests) (Biochemical/Serological Tests)

  5. Operating procedure of CDSL (1) × SL Pre-culture Medium （BPW,EEM) Sample 25g Pre-incubation 20-24 hours at 35℃ Inoculate 0.1mL of Pre-culture solution on CDSL (approx. 1cm far from the edge of plate)

  6. × SL Drop 1mL of sterilized water at the opposite point where specimen dropped. ↓ Confirm water diffused all over the plate and turn over the plate capped. Incubate 20 – 24 hours at 41 - 43℃. ↓ Interpretation Operating procedure of CDSL (2)

  7. Test Principle and Features of Compact Dry SL • Base medium: DHL agar • Additives: Pigment / Magnesium chloride / Novobiocin / Other additives • Test principle: 1) Medium alkalization that changes medium color from blue-purple to yellow by Salmonella’s lysine decarboxylase ability. 2) Colony color will be greening caused by decomposition of chromogenic substrate with specific enzyme on Salmonella. 3) Salmonella produces hydrogen (not all strains) sulfide which generate black colonies. 4) Detect motility of Salmonella. • Features: 1) CDSL detects Salmonella one day earlier than conventional culture methods. 2) Isolated colonies on the plate can be fished for further identification tests. 3) Ready to use and portable plate which no need to special preparation.

  8. Chicken (42℃, 24 hours incubation) Positive reaction Motility, Yellow sheet, Greening colony, Black colonies are generated by hydrogen sulfide. Negative reaction No color change Red – Red purple

  9. CDSL reactions (1) Standard strains

  10. CDSL reactions (2) Gram Negative Bacteria rather than Salmonella

  11. CDSL reactions (3)Salmonella spp.- 1

  12. CDSL reactions (3)Salmonella spp.- 2

  13. Method Flow Chart • Food SPC • Ground Chicken Breast 5.7 x 105 CFU/mL • Dried whole Egg 6.0 x 10 CFU/mL • BPW ---- Food Sample 25g + Buffer Peptone Water 225mL Dispense 9mL each into sterile test tube Dilute S. Enteritidis (5.1 x 108CFU/mL) from 10-1 to 10-8 Dilute and inoculate 1mL each into above tube Compact Dry SL DIASALM (MERCK) Rappaport-Vassiliadis Broth Inoculation: 0.1 mL 0.1 mL 0.1 mL Incubate 24 hours at 42C° Confirmation of typical Salmonella colonies MLCB Rambach XLD Confirmation: TSI agar / LIM Medium Biochemical Tests: PYR / NPA Serological Test Recovery of Salmonella from inoculated Food samples (1)

  14. Recovery of Salmonella from inoculated Food samples (1)

  15. Method Flow Chart Chicken Breast 25g (SPC 8.3 x 103 CFU/g) Egg Yolk 25g (SPC < 10CFU/g) Inoculate 1.0mL of Pre-cultured Salmonella Typhimurium 4-207B in TSB (over night) to each food sample (Controlled 101 to 104 CFU/mL) Chicken Breast Egg Yolk EEM 225mL (Incubate 24 hours at 35C°) BPW 225mL Inoculate to below media (incubate 24 hours at 42C°) Compact Dry SL DIASALM (MERCK) Rappaport-Vassiliadis Broth Inoculation: 0.1 mL 0.1 mL 0.1 mL Confirmation of typical Salmonella colonies MLCB Rambach XLD (Incubate 24 hours at 35C°) Confirmation: TSI agar / LIM Medium Biochemical Tests: PYR / NPA Serological Test Recovery of Salmonella from inoculated Food samples (2) (Sensitivity test for pre-cultured sample)

  16. Recovery of Salmonella from inoculated Food samples (2) (Sensitivity test from pre-cultured sample)

  17. Method Flow Chart Food Specimen 25g + EEM Broth 225mL (Meat products, 60 samples) Pre-Enrichment culture <24 hours at 35C°> Compact Dry SL DIASALM (MERCK) Rappaport-Vassiliadis Broth Tetrathionate Broth Inoculation: 0.1 mL 0.1 mL 0.1 mL 1.0mL Incubate 24 hours at 42C° MLCB agar / Rambach agar / XLD agar Incubate 24 hours at 35C° Confirmation: TSI agar / LIM Medium Biochemical Tests: PYR / NPA Serological Test Comparison of Salmonella detection by methods from Selective Enrichment Media by meat products (1)

  18. Comparison of Salmonella detection by methods from Selective Enrichment Media by meat products (2)

  19. Comparison of Salmonella detection methods from Selective Enrichment Media by meat products (3) Comparison of Sensitivity, Specificity and Agreement between Compact Dry SL and Conventional method CDSL DIASALM + - + 34 0 34 - 2 24 26 36 24 60 CDSL vs DIASALM Sensitivity 34 / 34 x 100 = 100% Specificity 24 / 26 x 100 = 92.3% Agreement 58 / 60 x 100 = 96.7% CDSL R V + - + 30 0 30 - 6 24 30 36 24 60 CDSL vs RV Broth Sensitivity 30 / 30 x 100 = 100% Specificity 24 / 30 x 100 = 80% Agreement 54 / 60 x 100 = 90% CDSL T T + - + 32 0 32 - 4 24 28 36 24 60 CDSL vs Tetrathionate Broth Sensitivity 32 / 32 x 100 = 100% Specificity 24 / 28 x 100 = 85.7% Agreement 56 / 60 x 100 = 93.3%

  20. Conclusion • All Salmonella samples are being positive on Compact Dry SL. Several high concentration of Proteus and Pseudomonas strains showed “yellowed” reaction, but other Gram negatives were negative or inhibited its growth. • Sensitivity and specificity of CDSL were higher than conventional methods when detected Salmonella from meat products on the market. • Preparation of medium is not necessary for CDSL and the presumption of test result may receive one day earlier than conventional enrichment culture methods that will be useful for food manufacturers.

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