Development of Compact Dry SL for Salmonella detection

Transcription

1 Development of Compact Dry SL for Salmonella detection S.Mizuochi 1),H.Teramura 1),S.Nirazuka 1),H.Kodaka 1),K.Inoue ) K.Tamura 3) 1)Research Dept., Nissui Pharmaceutical, ) 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 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 Specimen 5g Specimen 5g Pre-culture Pre-culture (BPW/EEM) (BPW/EEM) Day Selective Enrichment Culture CDSL (TT/RV/SBG/SC) Day 3 Isolation Presume, presence of Salmonella (MLCB/DHL/XLD/Rambach) Isolation Day Presume, presence of Salmonella Confirmation/Identification Confirmation/Identification (Biochemical/Serological Tests) (Biochemical/Serological Tests)

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

6 Operating procedure of CDSL () 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 hours at 1-3. Interpretation

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. ) 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. ) Detect motility of Salmonella. Features: 1) CDSL detects Salmonella one day earlier than conventional culture methods. ) 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 (, 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 Table 1: Gram Positive bacteria and Yeast ( hours incubation at ) Tested strains CDSL Growth 1 Bacillus cereus ATCC Bacillus licheniformis ATCC Bacillus subtilis ATCC Corynebacterium minutissium ATCC Corynebacterium renale ATCC Corynebacterium xerosis ATCC Enterococcus avium ATCC Enterococcus durans ATCC Enterococcus faecalis ATCC Enterococcus faecalis NIH Enterococcus faecium ATCC Enterococcus faecium NIH Lactobacillus delbrueckii subsp. Lactis ATCC Micrococcus luteus ATCC Staphylococcus aureus ATCC Staphylococcus epidermidis ATCC Streptococcus thermophilus ATCC Candida albicans ATCC Saccharomyces cerevisiae NHL Color

10 CDSL reactions () Gram Negative Bacteria rather than Salmonella Tested strains Numbers Growth Color Tested strains Numbers Growth Color (NIH, ATCC, JCM) Table : Gram Negative Bacteria rather than Salmone la (NIH, ATCC, JCM) Cedecial davisae - - Klebsiella pneumoniae subsp. rhinoscleromatis -/+ - / purple Cedecia lapageri - - Klebsiella terrigena - - Citrocacter amalonaticus Kluyvera ascorbata Citrobacter diversus 3() -/+ - / yellow Kluyvera clyocrescens Citrobacter freundii 5 -/+ - Leclercia adecarboxylata - - Cirobacter koseri Morganella morganii -/+ - Edwardiella hoshinae - - Proteus mirabilis + - Edwardsiella tarda Proteus penneri - - Enterobacter aerogenes Proteus vulgaris (1) -/+ - / yellow Enterobacter amnigenus Providencia alcalifaciens Enterobacter cloacae 3 + purple Providencia rettgeri 3(1) -/+ - / pale yellow Enterobacter intermidius Providencia stuartii - - Enterobacter sakazakii / purple Pseudomonas aeruginosa 6(5) + - / yellow Escherichia coli / purple Pseudomonas putida 1(1) + yellow Escherichia coli Serotype O157 -/+ - Serratia fonticola Escherichia fergusonii Serratia liquefaciens Escherichia hermannii 3 -/+ - Serratia marcescens -/+ - Escherichia vulneris - - Serratia rubidaea -/+ - Hafnia alvei Rehnella aquatilis Klebsiella ornithinolytica -/+ - Shigella boydii Klebsiella oxytoca Shigella flexneri - - Klebsiella planticola (1) -/+ - / yellow Shigella sonnei - - Klebsiella pneumoniae subsp. Ozaenae 3 + purple Yersinia enterocolitica - - Klebsiella pneumoniae subsp. Pneumoniae / purple Yokenella regensburgei - - Color changed to yellow around sample dropping spot (no green colonies identified) Total number of strains tested 1(11)

11 CDSL reactions (3) Salmonella spp.- 1 Table 3-1: Salmonella spp. ( hours incubation at ) Tested strains Numbers Growth Color (NIH, ATCC, JCM) Inoculated point Surrounding Colonies Salmonella Agona 1 + Yellow Yellow Blue Green Salmonella Anatum + Yellow Yellow/Green Blue Green Salmonella Blockley + Yellow Yellow Blue Green Salmonella bongori 1 + Yellow Gray Blue Green Salmonella Cerro 1 + Yellow/Green Yellow Green Salmonella Chester + Yellow Yellow Green Salmonella Choleraesuis 1 + Yellow Yellow Blue Green Salmonella Cubana, HS- 1 + Yellow Yellow Green Salmonella Derby 1 + Yellow Yellow Blue Green Salmonella enterica subsp. arizonae 1 + Yellow Yellow Blue Green Salmonella enterica subsp. diarizonae 1 + Yellow Gray small colonies Salmonella enterica subsp. houtenae + Yellow Yellow Salmonella Enteritidis 9 + Yellow/Green Yellow Green Salmonella Enteritidis, HS- 5 + Yellow Yellow Green Salmonella Fayed 1 + Yellow Yellow Green Salmonella Hadar 1 + Yellow/Green Yellow Blue Green Salmonella Havana + Yellow/Green Yellow Green Salmonella Heidelberg + Yellow Yellow Blue Green Salmonella Infantis 3 + Yellow Yellow Blue Green/Green 1

12 CDSL reactions (3) Salmonella spp.- Table 3-: Salmonella spp. ( hours incubation at ) Tested strains Numbers Growth Color (NIH, ATCC, JCM) Inoculated point Surrounding Colonies Salmonella Lexington 1 + Yellow Yellow Green Salmonella Livingstone 1 + Yellow Yellow Green Salmonella Manhattan 1 + Yellow Yellow Blue Green Salmonella Mbandaka 1 + Yellow Yellow Blue Green Salmonella Montevideo 1 + Yellow/Green Yellow Blue Green Salmonella Muenchen 1 + Yellow Yellow Green Salmonella Muenster 1 + Yellow Yellow Black Salmonella Newport 1 + Yellow Yellow Blue Green Salmonella Paratyphi B 1 + Yellow Yellow Blue Green Salmonella Schleissheim 1 + Yellow Yellow Blue Green Salmonella Schwarzengrund 1 + Yellow Yellow Blue Green Salmonella Senftenberg 1 + Yellow Yellow Blue Green Salmonella Solna + Yellow/Green Yellow Blue Green Salmonella Thompson 3 + Yellow Yellow Blue Green/Green Salmonella Tshiongwe 1 + Yellow Yellow Green Salmonella Typhi 1 + Yellow Yellow Salmonella Typhimurium 17 + Yellow/Green Yellow Blue Green/Green Salmonella Virchow 1 + Yellow Yellow Green 37

13 Recovery of Salmonella from inoculated Food samples (1) Method Flow Chart Food Sample 5g + Buffer Peptone Water 5mL Food SPC 1. Ground Chicken Breast 5.7 x 1 5 CFU/mL. Dried whole Egg 6. x 1 CFU/mL 3. BPW ---- Dispense 9mL each into sterile test tube Dilute S. Enteritidis (5.1 x 1 8 CFU/mL) from 1-1 to 1-8 Dilute and inoculate 1mL each into above tube Compact Dry SL (Nissui) DIASALM (MERCK) Rappaport-Vassiliadis Broth Inoculation:.1 ml.1 ml.1 ml Incubate hours at C Confirmation of typical Salmonella colonies MLCB Rambach XLD Confirmation: TSI agar / LIM Medium Biochemical Tests: PYR / NPA Serological Test

14 Recovery of Salmonella from inoculated Food samples (1) Table : Recovery of Salmonella from inoculated Food sam ple (Ground Chicken Breast Meat: 5.7 x 1 5 CFU/m L) Inoculated amount of Salmonella (CFU/mL) 5.1 x x x x x x x None CDSL DIASALM W - RV : Potitive ++ : Whole Yellowed Positive +W : Weak Postive Table 5: Recovery of Salmonella from inoculated Food sam ple (Dried Whole Egg : 6. x 1 CFU/m L) Inoculated amount of Salmonella (CFU/mL) 5.1 x x x x x x x None CDSL DIASALM RV : Potitive ++ : Whole Yellowed Positive +W : Weak Postive Table 6: Recovery of Salmonella from inoculated Food sam ple (BPW) Inoculated amount of Salmonella (CFU/mL) 5.1 x x x x x x x None CDSL DIASALM RV : Potitive ++ : Whole Yellowed Positive +W : Weak Postive

15 Recovery of Salmonella from inoculated Food samples () (Sensitivity test for pre-cultured sample) Method Flow Chart Chicken Breast 5g (SPC 8.3 x 1 3 CFU/g) Egg Yolk 5g (SPC < 1CFU/g) Inoculate 1.mL of Pre-cultured Salmonella Typhimurium -7B in TSB (over night) to each food sample (Controlled 1 1 to 1 CFU/mL) Chicken Breast Egg Yolk EEM 5mL (Incubate hours at 35C ) BPW 5mL Inoculate to below media (incubate hours at C ) Compact Dry SL (Nissui) DIASALM (MERCK) Rappaport-Vassiliadis Broth Inoculation:.1 ml.1 ml.1 ml Confirmation of typical Salmonella colonies MLCB Rambach XLD (Incubate hours at 35C ) Confirmation: TSI agar / LIM Medium Biochemical Tests: PYR / NPA Serological Test

16 Recovery of Salmonella from inoculated Food samples () (Sensitivity test from pre-cultured sample) Table 7: Comparison of Salmonella detection from each medium (Chicken Breast meat) Inoculate CDSL DIASALM TT RV (CFU/5g) : Whole reaction are confirmed + : Partial reaction are confirmed Inoculate Table 8: Comparison of Salmonella detection from each medium (Egg Yolk) CDSL DIASALM TT RV (CFU/5g) : Whole reaction are confirmed + : Partial reaction are confirmed

17 Comparison of Salmonella detection by methods from Selective Enrichment Media by meat products (1) Method Flow Chart Food Specimen 5g + EEM Broth 5mL (Meat products, 6 samples) Pre-Enrichment culture < hours at 35C > Compact Dry SL (Nissui) DIASALM (MERCK) Rappaport-Vassiliadis Broth Tetrathionate Broth Inoculation:.1 ml.1 ml.1 ml 1.mL Incubate hours at C MLCB agar / Rambach agar / XLD agar Incubate hours at 35C Confirmation: TSI agar / LIM Medium Biochemical Tests: PYR / NPA Serological Test

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

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 CDSL vs DIASALM Sensitivity 3 / 3 x 1 = 1% Specificity / 6 x 1 = 9.3% Agreement 58 / 6 x 1 = 96.7% CDSL R V CDSL vs RV Broth Sensitivity 3 / 3 x 1 = 1% Specificity / 3 x 1 = 8% Agreement 5 / 6 x 1 = 9% CDSL T T CDSL vs Tetrathionate Broth Sensitivity 3 / 3 x 1 = 1% Specificity / 8 x 1 = 85.7% Agreement 56 / 6 x 1 = 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.