Development of Compact Dry SL for Salmonella detection S.Mizuochi １）,H.Teramura...

Development of Compact Dry SL for Salmonella detection

S.Mizuochi １） ,H.Teramura １） ,S.Nirazuka １） ,H.Kodaka １） ,K.Inoue２）、 K.Tamura ３）

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 25gPre-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-incubation20-24 hours at 35℃

Pre-culture Medium（ BPW,EEM)

Sample 25g

Inoculate 0.1mL of Pre-culture solution on CDSL

(approx. 1cm far from the edge of plate)

SL

×

　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)

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.

Positive reactionMotility, Yellow sheet, Greening colony,

Black colonies are generated by hydrogen sulfide.

Negative reactionNo color changeRed – Red purple

Chicken (42 , 24 hours incubation)℃

CDSL reactions (1) Standard strains

Tested strains

Growth Color

1 Bacillus cereus ATCC 19637 - -2 Bacillus licheniformis ATCC 14580 - -3 Bacillus subtilis ATCC 6633 - -4 Corynebacterium minutissium ATCC 23348 - -5 Corynebacterium renale ATCC 19412 - -6 Corynebacterium xerosis ATCC 373 - -7 Enterococcus avium ATCC 14025 - -8 Enterococcus durans ATCC 19432 - -9 Enterococcus faecalis ATCC 19433 - -10 Enterococcus faecalis NIH 1001 - -11 Enterococcus faecium ATCC 19434 - -12 Enterococcus faecium NIH 2001 - -13 Lactobacillus delbrueckii subsp. Lactis ATCC 12315 - -14 Micrococcus luteus ATCC 9341 - -15 Staphylococcus aureus ATCC 6538 - -16 Staphylococcus epidermidis ATCC 14990 - -17 Streptococcus thermophilus ATCC 14485 - -18 Candida alb icans ATCC 10231 - -19 Saccharomyces cerevisiae NHL 10010 - -

CDSLTable 1: Gram Positive bacteria and Yeast (24 hours incubation at 42℃)

Tested strains Numbers Growth Color Tested strains Numbers Growth Color

(NIH, ATCC, JCM) (NIH, ATCC, JCM)

Cedecial davisae 2 - - Klebsiella pneumoniae subsp. rhinoscleromatis 2 -/+ - / purple

Cedecia lapageri 2 - - Klebsiella terrigena 2 - -Citrocacter amalonaticus 3 + - Kluyvera ascorbata 3 - -Citrobacter diversus 3(2) -/+ - / yellow Kluyvera clyocrescens 3 - -Citrobacter freundii 5 -/+ - Leclercia adecarboxylata 2 - -Cirobacter koseri 1 + - Morganella morganii 4 -/+ -Edwardiella hoshinae 2 - - Proteus mirabilis 4 + -Edwardsiella tarda 3 - - Proteus penneri 2 - -Enterobacter aerogenes 1 + - Proteus vulgaris 4(1) -/+ - / yellow

Enterobacter amnigenus 1 - - Providencia alcalifaciens 3 - -Enterobacter cloacae 3 + purple Providencia rettgeri 3(1) -/+ - / pale yellow

Enterobacter intermidius 1 + - Providencia stuartii 2 - -Enterobacter sakazakii 3 + - / purple Pseudomonas aeruginosa 6(5) + - / yellow

Escherichia coli 9 + - / purple Pseudomonas putida 1(1) + yellow

Escherichia coli Serotype O157 2 -/+ - Serratia fonticola 1 + -Escherichia fergusonii 1 + - Serratia liquefaciens 1 + -Escherichia hermannii 3 -/+ - Serratia marcescens 4 -/+ -Escherichia vulneris 2 - - Serratia rubidaea 2 -/+ -Hafnia alvei 1 - - Rehnella aquatilis 1 - -Klebsiella ornithinolytica 2 -/+ - Shigella boydii 1 - -Klebsiella oxytoca 3 + - Shigella flexneri 2 - -Klebsiella planticola 2(1) -/+ - / yellow Shigella sonnei 2 - -Klebsiella pneumoniae subsp. Ozaenae 3 + purple Yersinia enterocolitica 2 - -Klebsiella pneumoniae subsp. Pneumoniae 3 + - / purple Yokenella regensburgei 2 - -

Total number of strains tested 120(11)

Color changed to yellow around sample dropping spot (no green colonies identified)

Table 2: Gram Negative Bacteria rather than Salmonella

CDSL reactions (2) Gram Negative Bacteria rather than Salmonella

Tested strains Numbers Growth Color

(NIH, ATCC, JCM) Inoculated point Surrounding Colonies

Salmonella Agona 1 + Yellow Yellow Blue Green

Salmonella Anatum 2 + Yellow Yellow/Green Blue Green

Salmonella Blockley 2 + Yellow Yellow Blue Green

Salmonella bongori 1 + Yellow Gray Blue Green

Salmonella Cerro 1 + Yellow/Green Yellow Green

Salmonella Chester 2 + Yellow Yellow Green

Salmonella Choleraesuis 1 + Yellow Yellow Blue Green

Salmonella Cubana, H2S- 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 2 + Yellow YellowSalmonella Enteritidis 9 + Yellow/Green Yellow Green

Salmonella Enteritidis, H2S- 5 + Yellow Yellow Green

Salmonella Fayed 1 + Yellow Yellow Green

Salmonella Hadar 1 + Yellow/Green Yellow Blue Green

Salmonella Havana 2 + Yellow/Green Yellow Green

Salmonella Heidelberg 4 + Yellow Yellow Blue Green

Salmonella Infantis 3 + Yellow Yellow Blue Green/Green

41

Table 3- 1: Salmonella spp. (24 hours incubation at 42℃)

CDSL reactions (3) Salmonella spp.- 1

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 2 + Yellow/Green Yellow Blue Green

Salmonella Thompson 3 + Yellow Yellow Blue Green/Green

Salmonella Tshiongwe 1 + Yellow Yellow Green

Salmonella Typhi 1 + Yellow YellowSalmonella Typhimurium 17 + Yellow/Green Yellow Blue Green/Green

Salmonella Virchow 1 + Yellow Yellow Green

37

Table 3- 2: Salmonella spp. (24 hours incubation at 42℃)

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 MediumBiochemical Tests: PYR / NPA

Serological Test

Dispense 9mL each into sterile test tube

Food SPC

1. Ground Chicken Breast 5.7 x 105 CFU/mL

2. Dried whole Egg 6.0 x 10 CFU/mL

3. 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

5.1 x 107 5.1 x 106 5.1 x 105 5.1 x 104 5.1 x 103 5.1 x 102 5.1 x 101 5.1 None

CDSL ++ ++ ++ ++ ++ ++ + + -DIASALM + + + + + + + +W -

RV + + + + + + + - -+ : Potitive ++ : Whole Yellowed Positiv e +W : Weak Postive

5.1 x 107 5.1 x 106 5.1 x 105 5.1 x 104 5.1 x 103 5.1 x 102 5.1 x 101 5.1 None

CDSL ++ ++ ++ ++ ++ ++ + 1 -DIASALM + + + + + + - - -


5.1 x 107 5.1 x 106 5.1 x 105 5.1 x 104 5.1 x 103 5.1 x 102 5.1 x 101 5.1 None

CDSL ++ ++ ++ ++ ++ ++ + - -DIASALM + + + + + + - - -


Table 4: Recovery of Salmonella from inoculated Food sample (Ground Chicken Breast Meat: 5.7 x 105 CFU/ mL)Inoculated amount of Salmonella (CFU/mL)

Table 5: Recovery of Salmonella from inoculated Food sample (Dried Whole Egg : 6.0 x 10 CFU/ mL)Inoculated amount of Salmonella (CFU/mL)

Table 6: Recovery of Salmonella from inoculated Food sample (BPW)Inoculated amount of Salmonella (CFU/mL)

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


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

Inoculate

(CFU/25g) 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5

1 92000 ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ + + + + + + + + + +

2 9200 ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ + + + + + + + + + +

3 920 ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ + + + + + + + + + +

4 92 ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ + + + + + + + + + +

5 7 ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ + + + + + + + + + +

6 - - - - - - - - - - - - - - - - - - - - -++ : Whole reaction are confirmed

+ : Partial reaction are confirmed

Inoculate

(CFU/25g) 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5

1 410 ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ + + + + + + + + + +

2 41 ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ + + + + + + + + + +

3 - - - - - - - - - - - - - - - - - - - - -

++ : Whole reaction are confirmed

+ : Partial reaction are confirmed

Table 7: Comparison of Salmonella detection from each medium (Chicken Breast meat)

Table 8: Comparison of Salmonella detection from each medium (Egg Yolk)

CDSL DIASALM TT RV

CDSL DIASALM TT RV

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 BrothInoculation: 0.1 mL 0.1 mL 0.1 mL 1.0mL


MLCB agar / Rambach agar / XLD agar



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 8 8 8 8

Chicken (dark/sliced) 4 4 4 4 4

Chicken (breast) 4 4 4 4 4

Chicken (breast white) 6 4 3 4 4

Chicken (leg dark) 6 4 3 2 4

Chicken (ground) 4 4 4 4 4

Chicken (wing) 4 2 2 2 2

Chicken (skin) 2 2 2 2 2

Chicken (gizzard) 2 2 2 0 0

Chicken (steamed) 2 2 2 0 0

Bacon 4 0 0 0 0

Sausage 6 0 0 0 0

Processed Ham 2 0 0 0 0

Fried Chicken (Frozen) 4 0 0 0 0

Japanese Style Fried Chicken (Frozen)

2 0 0 0 0

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

CDSLDIASALM + - + 34 0 34 - 2 24 26 36 24 60

CDSL R V + - + 30 0 30 - 6 24 30 36 24 60

CDSL T T + - + 32 0 32 - 4 24 28 36 24 60

CDSL vs DIASALMSensitivity 34 / 34 x 100 = 100%Specificity 24 / 26 x 100 = 92.3%Agreement 58 / 60 x 100 = 96.7%

CDSL vs RV BrothSensitivity 30 / 30 x 100 = 100%Specificity 24 / 30 x 100 = 80%Agreement 54 / 60 x 100 = 90%

CDSL vs Tetrathionate BrothSensitivity 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.

Development of Compact Dry SL for Salmonella detection S.Mizuochi １）,H.Teramura...

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