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

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

Package, CDSL

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

Operating procedure of CDSL (1) 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) SL ×

Operating procedure of CDSL (2) × 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

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.

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

CDSL reactions (1) Standard strains

CDSL reactions (2) Gram Negative Bacteria rather than Salmonella

CDSL reactions (3) Salmonella spp.- 1

CDSL reactions (3) Salmonella spp.- 2

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

Recovery of Salmonella from inoculated Food samples (1)

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

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

Comparison of Salmonella detection by methods from Selective Enrichment Media by meat products (1) Method Flow Chart Food Specimen 25g + EEM Broth 225mL (Meat products, 60 samples) 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 Pre-Enrichment culture <24 hours at 35C°>

Comparison of Salmonella detection by methods from Selective Enrichment Media by meat products (2) Name of Product (selected unintentionally) Number of specimen Compact Dry SL Positives DIASALM Positives RV Positives TT Positives Chicken (breast/ground) 8 Chicken (dark/sliced) 4 Chicken (breast) Chicken (breast white) 6 3 Chicken (leg dark) 2 Chicken (ground) Chicken (wing) Chicken (skin) Chicken (gizzard) Chicken (steamed) Bacon Sausage Processed Ham Fried Chicken (Frozen) Japanese Style Fried Chicken (Frozen) TOTAL 60 36 34 30 32

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%

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.