Development of Compact Dry SLfor 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 materials③ 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/IdentificationConfirmation/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 pointwhere 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 strainsGrowth Color

1 Bacillus cereus ATCC 19637 - -2 Bacillus licheniformis ATCC 14580 - -3 Bacillus sub tilis 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 delb rueckii 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: G ram 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 - - Kleb siella 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 mirab ilis 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) + - / yellowEscherichia 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 rub idaea 2 -/+ -Hafnia alvei 1 - - Rehnella aquatilis 1 - -Klebsiella ornithinolytica 2 -/+ - Shigella b oydii 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 regensb urgei 2 - -

Total number of strains tested 120(11)

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

Table 2: Gram Negative Bacteria rather than Salm onella

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 GreenSalmonella Anatum 2 + Yellow Yellow/Green Blue GreenSalmonella Blockley 2 + Yellow Yellow Blue GreenSalmonella bongori 1 + Yellow Gray Blue GreenSalmonella Cerro 1 + Yellow/Green Yellow GreenSalmonella Chester 2 + Yellow Yellow GreenSalmonella Choleraesuis 1 + Yellow Yellow Blue GreenSalmonella Cubana, H2S- 1 + Yellow Yellow GreenSalmonella Derby 1 + Yellow Yellow Blue GreenSalmonella enterica subsp. arizonae 1 + Yellow Yellow Blue GreenSalmonella enterica subsp. diarizonae 1 + Yellow Gray small coloniesSalmonella enterica subsp. houtenae 2 + Yellow YellowSalmonella Enteritidis 9 + Yellow/Green Yellow GreenSalmonella Enteritidis, H2S- 5 + Yellow Yellow GreenSalmonella Fayed 1 + Yellow Yellow GreenSalmonella Hadar 1 + Yellow/Green Yellow Blue GreenSalmonella Havana 2 + Yellow/Green Yellow GreenSalmonella Heidelberg 4 + Yellow Yellow Blue GreenSalmonella 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 GreenSalmonella Livingstone 1 + Yellow Yellow GreenSalmonella Manhattan 1 + Yellow Yellow Blue GreenSalmonella Mbandaka 1 + Yellow Yellow Blue GreenSalmonella Montevideo 1 + Yellow/Green Yellow Blue GreenSalmonella Muenchen 1 + Yellow Yellow GreenSalmonella Muenster 1 + Yellow Yellow BlackSalmonella Newport 1 + Yellow Yellow Blue GreenSalmonella Paratyphi B 1 + Yellow Yellow Blue GreenSalmonella Schleissheim 1 + Yellow Yellow Blue GreenSalmonella Schwarzengrund 1 + Yellow Yellow Blue GreenSalmonella Senftenberg 1 + Yellow Yellow Blue GreenSalmonella Solna 2 + Yellow/Green Yellow Blue GreenSalmonella Thompson 3 + Yellow Yellow Blue Green/GreenSalmonella Tshiongwe 1 + Yellow Yellow GreenSalmonella Typhi 1 + Yellow YellowSalmonella Typhimurium 17 + Yellow/Green Yellow Blue Green/GreenSalmonella 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 (Nissui) 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 SPC1. Ground Chicken Breast 5.7 x 105 CFU/mL2. Dried whole Egg 6.0 x 10 CFU/mL3. 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 coloniesMLCB 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 NoneCDSL ++ ++ ++ ++ ++ ++ + + -

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 NoneCDSL ++ ++ ++ ++ ++ ++ + 1 -

DIASALM + + + + + + - - -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 NoneCDSL ++ ++ ++ ++ ++ ++ + - -

DIASALM + + + + + + - - -RV + + + + + + + - -

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

Table 4: Recovery of Salm onella from inoculated Food sam ple (G round C hicken Breast M eat: 5.7 x 105 C FU/m L)

Inoculated amount of Salmonella (CFU/mL)

Table 5: Recovery of Salm onella from inoculated Food sam ple (Dried W hole Egg : 6.0 x 10 C FU/m L)

Inoculated amount of Salmonella (CFU/mL)

Table 6: Recovery of Salm onella from inoculated Food sam ple (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 (Nissui) DIASALM (MERCK) Rappaport-Vassiliadis Broth

Inoculation: 0.1 mL 0.1 mL 0.1 mL

Confirmation: TSI agar / LIM MediumBiochemical 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 coloniesMLCB 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 (Nissui) DIASALM (MERCK) Rappaport-Vassiliadis Broth Tetrathionate BrothInoculation: 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 MediumBiochemical Tests: PYR / NPA

Serological Test

Pre-Enrichment culture <24 hours at 35C°>

Comparison of Salmonella detection by methods fromSelective Enrichment Media by meat products (2)

3230343660TOTAL

00002Japanese Style Fried Chicken (Frozen)

00004Fried Chicken (Frozen)

00002Processed Ham

00006Sausage

00004Bacon

00222Chicken (steamed)

00222Chicken (gizzard)

22222Chicken (skin)

22224Chicken (wing)

44444Chicken (ground)

42346Chicken (leg dark)

44346Chicken (breast white)

44444Chicken (breast)

44444Chicken (dark/sliced)

88888Chicken (breast/ground)

TT PositivesRV PositivesDIASALM PositivesCompact Dry SL Positives

Number of specimen

Name of Product(selected unintentionally)

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

CDSLR V + -

+ 30 0 30 - 6 24 30

36 24 60

CDSLT 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 Salmonellafrom 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.