Appendices of the Final Screening Assessment

Bacillus amyloliquefaciens 13563-0
Bacillus atrophaeus 18250-7
Bacillus licheniformis American Type Culture Collection (ATCC) 12713
Bacillus subtilis ATCC 6051A (=ATCC 6051a)
Bacillus subtilis ATCC 55405
Bacillus subtilis subspecies subtilis ATCC 6051T
Bacillus subtilis subspecies inaquosorum ATCC 55406
Bacillus species 16970-5
Bacillus species 2 18118-1
Bacillus species 4 18121-4
Bacillus species 7 18129-3

Environment Canada
Health Canada
August 2015

Appendices

Appendix 1: Colony Morphologies of Domestic Substances List (DSL) B. licheniformis/subtilis group members

Table A-1: Colony morphologies of B. amyloliquefaciens 13563-0
CharacteristicTryptone Soya Broth (TSB) agar after 7 days of growth at room temperatureaSpizizen potato agar or broth (ATCC Medium 423) at 37°C verified at 24 hours (for solid medium, add 1.5% agar) Footnote Appendix 1 Table A1 [a]
ShapeIrregularIrregular
Size (mm) diameter55
MarginUndulateSpreading, irregular edge
ElevationFlatNo data
Colour/pigmentOff-whiteWhite
TextureDullSmooth, dull
OpacityOpaqueNo data
Footnote Appendix 1 Table A1 a

Data generated by Health Canada's Healthy Environments and Consumer Safety Branch

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Table A-2: Colony morphologies of B. atrophaeus 18250-7
CharacteristicTSB agar after 24 hours of growth at room temperatureFootnote Appendix 1 Table A2 [a]TSB agar after 7 days of growth at room temperature[a]Nutrient agar at 30°C for 24 hours[a]Nutrient agar or broth (ATCC medium #3) at 30°C after 24 hours under aerobic conditionsFootnote Appendix 1 Table A2[b]Nutrient agar or broth (ATCC medium #3) at 30°C after 24 hours under aerobic conditions[b]Nutrient agar or broth (ATCC medium #3) at 30°C after 24 hours under aerobic conditions[b]
ShapeCircularIrregularCircularCircularCircularIrregular
Size (mm) diameter25-100.5No dataNo dataNo data
MarginEntireUndulateEntireEntireEntireUndulate
ElevationFlatFlatNo dataLow convexLow convexFlat
Colour/
pigmentation[a]
WhiteOff-white/beigeWhiteOrangeNo dataWhite
TextureSmooth, moistSmooth, moistNo dataGlisteningGlisteningNo data
OpacityOpaqueOpaqueNo dataNo dataOpaqueNo data
Footnote Appendix A Table A2 a

Data generated by Health Canada's Healthy Environments and Consumer Safety Branch

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Footnote Appendix A Table A2 b

ATCC description, multiple colony morphologies

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Table A-3: Colony morphologies of B. licheniformis ATCC 12713
CharacteristicTSB agar after 7 days of growth at room temperatureFootnote Appendix A Table A3 [a], Footnote Appendix A Table A3 [b]TSB agar after 7 days of growth at room temperature[a],[b]Nutrient agar at 30°C for 24 hours[a]Nutrient agar or broth (ATCC medium #3) at 30°C for 24 hours under aerobic conditionsFootnote Appendix A Table A3[c]Nutrient agar or broth (ATCC medium #3) at 30°C for 24 hours under aerobic conditions[c]
ShapeCircularIrregularIrregularNo dataIrregular
Size (mm) diameter5-75-72No dataNo data
MarginUndulateUndulate-lobateUndulate, filiformNo dataNo data
ElevationFlatUmbonateRaisedRaisedConvex
Colour/pigmentBeige/off-whiteBeige/off-whiteNo dataNo dataNo data
TextureMoist, smoothWrinkled, dryDryDry, wrinkledMucoid
OpacitySemi-translucentOpaqueOpaqueNo dataNo data
Footnote Appendix A Table A3 a

Data generated by Health Canada's Healthy Environments and Consumer Safety Branch

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Footnote Appendix A Table A3 b

Colonies stick to agar, multiple colony morphologies

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Footnote Appendix A Table A3 c

ATCC description, multiple colony morphologies

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Table A-4: Colony morphologies of B. subtilis ATCC 6051A
CharacteristicTSB agar after 7 days of growth at room temperatureFootnote Appendix A Table A4 [a]Nutrient agar at 30°C for 24 hours[a]Nutrient agar or broth (ATCC medium #3) at 30°C verified at 24 hours and up to one week under aerobic conditionsFootnote Appendix A Table A4 [b]Nutrient agar or broth (ATCC medium #3) at 30°C verified at 24 hours and up to one week under aerobic conditions[b]
ShapeCircular to irregularIrregularIrregular and spreading with ageIrregular and spreading with age
Size (mm) diameter6 to 252LargerSmaller
MarginUndulateEntireEroseEntire
ElevationFlatFlatFlatFlat
Colour/pigmentOff-whiteOff-whiteBeige/creamBeige/cream
TextureMoistDryDull, rougherDull, smoother
OpacityTranslucentSemi-translucentOpaqueOpaque
Footnote Appendix A Table A4 a

Data generated by Health Canada's Healthy Environments and Consumer Safety Branch

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Footnote Appendix A Table A4 b

ATCC description, multiple colony morphologies

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Table A-5: Colony morphologiesof B. subtilis ATCC 55405
CharacteristicTSB agar after 7 days of growth at room temperatureFootnote Appendix A Table A5 [a],Footnote Appendix A Table A5[b]TSB agar after 7 days of growth at room temperature[a],[b]TSB agar after 7 days of growth at room temperature[a],[b]Nutrient agar at 30°C for 24 hours[a]Nutrient agar or broth (ATCC medium #3) at 30°C verified at 24 hours and up to one week under aerobic conditionsFootnote Appendix A Table A5 [c]
ShapeIrregular to circularCircularIrregular to circular2Circular
Size (mm) diameter444No dataNo data
MarginEntireUndulateLobateEntireEntire
ElevationConvexFlatRaiseConvexConvex
Colour/pigmentColourless to off-whiteOff-whiteWhiteColourlessNo data
TextureGlossy, mucoidMatte, dryFlat, brittle, dryMucoidMucoid, glistening
OpacityOpaqueTranslucent to opaqueOpaqueNo dataOpaque
Footnote Appendix A Table A5 a

Data generated by Health Canada's Healthy Environments and Consumer Safety Branch

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Footnote Appendix A Table A5 b

Multiple colony morphologies

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Footnote Appendix A Table A5 c

ATCC description

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Table A-6: Colony morphologiesof B. subtilis subsp. subtilis ATCC 6051
CharacteristicTSB agar after 7 days of growth at room temperatureFootnote Appendix A Table A6 [a]Nutrient agar at 30°C for 24 hours[a]Nutrient agar or broth (ATCC medium #3) at 30°C verified at 24 hours and up to one week under aerobic conditionsFootnote Appendix A Table A6 [b]Nutrient agar or broth (ATCC medium #3) at 30°C verified at 24 hours and up to one week under aerobic conditions[b]
ShapeIrregularIrregularCircularIrregular
Size (mm) diameter20No dataNo dataNo data
MarginUndulateUndulateEntireNo data
ElevationRaisedNDLow convexFlat
Colour/pigmentOff-whiteOff-whiteNo dataNo data
TextureDryNo dataShinyRough
OpacityOpaqueOpaqueOpaqueOpaque
Footnote Appendix A Table A6 a

Data generated by Health Canada's Healthy Environments and Consumer Safety Branch

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Footnote Appendix A Table A6 b

ATCC description, multiple colony morphologies

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Table A-7: Colony morphologies of B.subtilis subsp. inaquosorum ATCC 55406
CharacteristicTSB agar after 7 days of growth at room temperatureFootnote Appendix A Table A7 [a],Footnote Appendix A Table A7[b]Nutrient agar at 30°C for 24 hours[a]Nutrient agar (ATCC medium #3) at 30°C verified at 24 hoursFootnote Appendix A Table A7 [c],Footnote Appendix A Table A7 [d]Nutrient agar (ATCC medium #3) at 30°C verified at 24 hours[c],[d]
ShapeCircularIrregularCircularCircular
Size (mm) diameter51No dataNo data
MarginUndulateNo dataEroseNo data
ElevationFlatNo dataFlatNo data
Colour/pigmentWhiteColourlessCreamWhite
TextureDullNo dataNo dataSmooth
OpacityOpaqueOpaqueOpaqueOpaque
Footnote Appendix A Table A7 a

Data generated by Health Canada's Healthy Environments and Consumer Safety Branch

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Footnote Appendix A Table A7 b

Colonies grow into agar

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Footnote Appendix A Table A7 c

ATCC description, multiple colony morphologies

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Footnote Appendix A Table A7 d

Colonies dig into agar; this organism grows better on solid media than in a broth

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Table A-8: Colony morphologies of Bacillus species 16970-5
CharacteristicTSB agar after 7 days of growth at room temperatureFootnote Appendix A Table A8 [a]Nutrient agar at 30°C for 24 hours[a]Nutrient agar or broth (ATCC medium #3) at 30°C verified at 24 hours and up to one week under aerobic conditionsFootnote Appendix A Table A8 [b]
ShapeIrregularIrregularCircular
Size (mm) diameter12 to 202No data
MarginUndulateNo dataSome with lobate margins
ElevationFlatFlatNo data
ColourOff-whiteOff-whiteNo data
TextureDullDryShiny, smooth
OpacityOpaqueOpaqueOpaque
Footnote Appendix A Table A8 a

Data generated by Health Canada's Healthy Environments and Consumer Safety Branch

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Footnote Appendix A Table A8 b

ATCC description

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Table A-9: Colony morphologies of Bacillus species 2 18118-1
CharacteristicTSB agar after 7 days of growth at room temperatureFootnote Appendix A Table A9 [a]Spizizen potato agar or broth (ATCC Medium 423) at 37°C verified at 24 hours (for solid medium, add 1.5% agar)[a]Spizizen potato agar or broth (ATCC Medium 423) at 37°C verified at 24 hours (for solid medium, add 1.5% agar)Footnote Appendix A Table A9 [b]Spizizen potato agar or broth (ATCC Medium 423) at 37°C verified at 24 hours (for solid medium, add 1.5% agar)[b]
ShapeIrregularCircularCircularNo data
Size (mm) diameter5-125No dataNo data
MarginUndulateSlightly irregular edgeSlightly irregularDull spreading irregular edge
ElevationUmbonate, raisedNo dataNo dataNo data
Colour/pigmentWhitishNo dataNo dataNo data
TextureMoist, shiny and dullNDSmooth, glisteningGlistening, smooth center
OpacityOpaqueOpaqueOpaqueNo data
Footnote Appendix A Table A9 a

Data generated by Health Canada's Healthy Environments and Consumer Safety Branch

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Footnote Appendix A Table A9 b

ATCC description, multiple colony morphologies

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Table A-10: Colony morphologies of Bacillus species 4 18121-4
CharacteristicTSB agar after 7 days of growth at room temperatureFootnote Appendix A Table A10 [a]TSB agar after 7 days of growth at room temperature[a]Nutrient agar at 30°C for 24 hours[a]Nutrient agar or broth (ATCC medium #3) at 37°C for 24 hours under aerobic conditionsFootnote Appendix A Table A10[b]Nutrient agar or broth (ATCC medium #3) at 37°C for 24 hours under aerobic conditions[b]
ShapeCircularEllipticalIrregularIrregularRhizoid
Size (mm) diameter5-75-72No dataNo data
MarginUndulateUndulateUndulate, filiformUndulateFilamentous
ElevationImperfect-umbonateFlatRaisedConvexRaised
ColourBeige/off-whiteBeige/off-whiteNo dataTranslucentNo data
TextureMoistMatteDrySmooth, glisteningRough
OpacityOpaqueOpaqueOpaqueNo dataOpaque
Footnote Appendix A Table A10 a

Data generated by Health Canada's Healthy Environments and Consumer Safety Branch

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Footnote Appendix A Table A10 b

ATCC description, multiple colony morphologies

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Table A-11: Colony morphologies of Bacillus species 7 18129-3
CharacteristicTSB agar after 7 days of growth at room temperatureFootnote Appendix A Table A11 [a]Nutrient agar at 30°C for 24 hours[a]Nutrient agar or broth (ATCC medium #3) at 30°C verified at 24 hours and up to one week under aerobic conditionsFootnote Appendix A Table A11 [b]Nutrient agar or broth (ATCC medium #3) at 30°C verified at 24 hours and up to one week under aerobic conditions[b]
ShapeIrregularIrregularCircularIrregular
Size (mm) diameter20No dataNo dataNo data
MarginUndulateUndulateEntireNo data
ElevationRaisedNo dataLow convexFlat
ColourOff-whiteOff-whiteNo dataNo data
TextureDryNo dataShinyRough
OpacityOpaqueOpaqueOpaqueOpaque
Footnote Appendix A Table A11 a

Data generated by Health Canada's Healthy Environments and Consumer Safety Branch

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Footnote Appendix A Table A11 b

ATCC description, multiple colony morphologies

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Appendix 2: Characteristics of DSL B. licheniformis/subtilis group members – 16S ribosomal RNA gene sequence analysis

16S ribosomal RNA gene sequence data generated by Health Canada's Healthy Environments and Consumer Safety Branch. Restriction fragment length polymorphisms from within the V3 region and between the V4 and V5 region were identified according to Jeyaram et al. 2011. The 16S ribosomal RNA gene sequences were compared to the Ribosomal Database project release 11 (https://rdp.cme.msu.edu/) and top 10 matches are shown. The match hit format is: identification code, similarity score (if reference strain is specified), S ab score, unique common oligomers and sequence full name.

Table A-12: Results of 16S Ribosomal RNA Gene Sequence Analysis of B. amyloliquefaciens 13563Footnote Appendix 2 Table A-12 [a]
Short identificationSimilarity scoreS_ab scoreUnique common oligomersSequence full name
S0011535381.0001.0001364Bacillus sp. XI; EU779996
S0015509061.0001.0001371Bacillus subtilis; Y2; GQ148813
S0015884021.0001.0001374Bacillus amyloliquefaciens; PBT; FJ169495
S0017458991.0001.0001393Bacillus amyloliquefaciens; IMAU80205; GU125623
S0020386391.0001.0001354Bacillus amyloliquefaciens; HK1; AB279736
S0022222551.0001.0001447Bacillus amyloliquefaciens; DSM 7; DSM7; FN597644
S0022222571.0001.0001447Bacillus amyloliquefaciens; DSM 7; DSM7; FN597644
S0022222591.0001.0001447Bacillus amyloliquefaciens; DSM 7; DSM7; FN597644
S0022222611.0001.0001447Bacillus amyloliquefaciens; DSM 7; DSM7; FN597644
S0022222631.0001.0001447Bacillus amyloliquefaciens; DSM 7; DSM7; FN597644
Footnote Appendix 2 Table A12 a

The HinfI site is present and the RsaI sites are absent indicating the micro-organisms is B. amyloliquefaciens as opposed to B. subtilisto which it is closely related (Jeyaram et al. 2011).

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Table A-13: Results of 16S Ribosomal RNA Gene Sequence Analysis of B. atrophaeus 18250-7Footnote Appendix 2 Table A-13 [a]
Short identificationSimilarity scoreS_ab scoreUnique common oligomersSequence full name
S000382399Not calculated1.0001432Bacillus sp.; SSA3; AB017587
S000644416Not calculated1.0001436Bacillus atrophaeus; SCH0408; AY881241
S000980555Not calculated1.0001337Bacillus atrophaeus; K01-03; EU326483
S001872424Not calculated1.0001411Bacillus subtilis; JAM A-6-10; AB542912
S002035172Not calculated1.0001375Bacillus atrophaeus; NMTD54; GU568183
S002035195Not calculated1.0001379Bacillus atrophaeus; GBSC56; GU568206
S002166857Not calculated1.0001376Bacillus atrophaeus; RJGP16; GU969134
S002167105Not calculated1.0001372Bacillus atrophaeus; LSSC3; GU994860
S002221550Not calculated1.0001464Bacillus atrophaeus 1942; CP002207
S002221552Not calculated1.0001464Bacillus atrophaeus 1942; CP002207
Footnote Appendix 2 Table A13 a

B. atrophaeus 18250-7 ribosomal RNA gene sequence matches B. atrophaeus and Bacillus sp. sequences and the HinfI siteis present for the B. subtilis/licheniformis group(Jeyaram et al. 2011).

Return to footnote Appendix 2 Table A13 a referrer

Table A-14: Results of 16S Ribosomal RNA Gene Sequence Analysis of B. licheniformis ATCC 12713Footnote Appendix 2 Table A-14 [a]
Short identificationSimilarity scoreS_ab scoreUnique common oligomersSequence full name
S000392549Not calculated1.0001427Bacillus licheniformis; Mo1; AF372616
S000615411Not calculated1.0001409Bacillus licheniformis; ACO1; DQ228696
S000647676Not calculated1.0001421Bacillus licheniformis; K19; DQ351932
S000736754Not calculated1.0001448Bacillus licheniformis; BCRC 15413; DQ993676
S000752038Not calculated1.0001409Bacillus licheniformis; EF059752
S000824918Not calculated1.0001422Bacillus licheniformis; BCRC 12826; EF423608
S000843501Not calculated1.0001442Bacillus sp. J24; EF471917
S000901702Not calculated1.0001389Bacillus licheniformis; NBRC 12107; AB354236
S000941823Not calculated1.0001389Bacillus licheniformis; NBRC 12202; AB363734
S001153503Not calculated1.0001319Bacillus licheniformis; SVD1; EU770587
Footnote Appendix 2 Table A14 a

Bacillus licheniformis ATCC 12713 16S ribosomal RNA gene sequence matches mainly B. licheniformis ribosomal RNA gene sequences. The restriction fragment length polymorphism (RFLP) pattern (RsaI sites in V3; HinfI and CfoI site between V4 and V5) is consistent with B. licheniformis sp. (Jeyaram et al. 2011).

Return to footnote Appendix 2 Table A14 a referrer

Table A-15: Results of 16S Ribosomal RNA Gene Sequence Analysis of B. subtilis ATCC 6051AFootnote Appendix 2 Table A-15 [a]
Short identificationSimilarity scoreS_ab scoreUnique common oligomersSequence full name
S000003473Not calculated1.0001423Bacillus subtilis (T); DSM10; AJ276351
S000365537Not calculated1.0001446Bacillus sp. TUT1206; AB188212
S000383767Not calculated1.0001412Bacillus sp. CH4-4; AB055846
S000383768Not calculated1.0001412Bacillus sp. CH4-5; AB055848
S000383769Not calculated1.0001412Bacillus sp. CH15-2; AB055849
S000383770Not calculated1.0001412Bacillus sp. CH19-3; AB055850
S000383771Not calculated1.0001412Bacillus sp. CH20-1; AB055851
S000383772Not calculated1.0001412Bacillus sp. CH7-1; AB055852
S000383773Not calculated1.0001412Bacillus sp. CH10-1; AB055853
S000434646Not calculated1.0001401Bacillus subtilis; KL-073; AY030330
Footnote Appendix 2 Table A15 a

B. subtilis ATCC6051A 16S ribosomal RNA gene sequence matches Bacillus subtilis and Bacillus sp. ribosomal RNA gene sequence. The RFLP pattern (RsaI sites in V3; HinfI site between V4 and V5) is consistent for B. subtilis sp. (Jeyaram et al. 2011). However, the first putative RsaI site requires verification as it contains an ambiguous base (the dominant peak appears to correspond to A).

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Table A-16: Results of 16S Ribosomal RNA Gene Sequence Analysis of B. subtilis ATCC 55405Footnote Appendix 2 Table A-16 [a]
Short identificationSimilarity scoreS_ab scoreUnique common oligomersSequence full name
S000870716Not calculated1.0001391Bacillus amyloliquefaciens; NBRC 14141; AB325582
S001745899Not calculated1.0001393Bacillus amyloliquefaciens; IMAU80205; GU125623
S002038639Not calculated1.0001354Bacillus amyloliquefaciens; HK1; AB279736
S002222255Not calculated1.0001447Bacillus amyloliquefaciens DSM 7; DSM7; FN597644
S002222257Not calculated1.0001447Bacillus amyloliquefaciens DSM 7; DSM7; FN597644
S002222259Not calculated1.0001447Bacillus amyloliquefaciens DSM 7; DSM7; FN597644
S002228859Not calculated1.0001224Bacillus amyloliquefaciens; BAC3048; HM355639
S003280603Not calculated1.0001329Bacillus amyloliquefaciens; BSS5; JQ407053
S003285855Not calculated1.0001305Bacillus sp. SE18; JQ714100
S003313087Not calculated1.0001342Bacillus amyloliquefaciens; KU-8; JQ696827
Footnote Appendix 2 Table A16 a

B. subtilis ATCC 55405 16S ribosomal RNA gene sequence matches mainly B. amyloliquefacienssequences and the HinfI RFLP identified by for B. amyloliquefaciens is present (Jeyaram et al. 2011).

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Table A-17: Results of 16S Ribosomal RNA Gene Sequence Analysis of B. subtilis subsp. subtilis ATCC 6051Footnote Appendix 2 Table A-17 [a]
Short identificationSimilarity scoreS_ab scoreUnique common oligomersSequence full name
S000398967Not calculated1.0001383Bacillus subtilis; BHP6-1; AY162131
S001020073Not calculated1.0001422Bacillus subtilis; B1-33; EU435361
S001096330Not calculated1.0001435Bacillus sp. zh161; EU526087
S002038710Not calculated1.0001388Bacillus sp. PT401; AB374305
S002199724Not calculated1.0001420Uncultured Bacillus sp.; CapF3B.16; HM152583
S002199742Not calculated1.0001386Uncultured Bacillus sp.; Filt.13; HM152601
S002199744Not calculated1.0001386Uncultured Bacillus sp.; Filt.15; HM152603
S002199761Not calculated1.0001386Uncultured Bacillus sp.; Filt.32; HM152620
S002199769Not calculated1.0001385Uncultured Bacillus sp.; Filt.40; HM152628
S002199775Not calculated1.0001386Uncultured Bacillus sp.; Filt.46; HM152634
Footnote Appendix 2 Table A17 a

Bacillus species 6051 16S ribosomal RNA gene sequence matches Bacillus subtilis and Bacillus sp. ribosomal RNA gene sequence. The RFLP pattern(two RsaIsites in V3; HinfI site between V4 and V5) is consistent B. subtilis sp. (Jeyaram et al. 2011).

Return to footnote Appendix 2 Table A17 a referrer

Table A-18: Results of 16S Ribosomal RNA Gene Sequence Analysis of B. subtilis subsp. subtilis ATCC 6051Footnote Appendix 2 Table A-18 [a]
Short identificationSimilarity scoreS_ab scoreUnique common oligomersSequence full name
S001020073Not calculated0.9461422Bacillus subtilis; B1-33; EU435361
S001096330Not calculated0.9461435Bacillus sp. zh161; EU526087
S002038710Not calculated0.9461388Bacillus sp. PT401; AB374305
S002199724Not calculated0.9461420Uncultured Bacillus sp.; CapF3B.16; HM152583
S002199860Not calculated0.9461421Uncultured Bacillus sp.; Filt.132; HM152719
S002199880Not calculated0.9461421Uncultured Bacillus sp.; Filt.152; HM152739
S002410934Not calculated0.9461413Bacillus subtilis; MB5 NIOT; HQ858061
S003257857Not calculated0.9461446Bacillus subtilis subsp. inaquosorum; type strain: DSM 22148; HE582781
S003261902Not calculated0.9461388Bacillus subtilis; NBRC 3108; AB680011
S003264071Not calculated0.9461388Bacillus subtilis; NBRC 104440; AB682180
Footnote Appendix 2 Table A18 a

The 16S matches for B. subtilis subsp. inaquosorum ATCC 55406 included both Bacillus sp. and B. subtilis subsp. inaquosorum. The pattern of restriction sites within B. subtilis subsp. inaquosorum ATCC 55406 is different compared to B. licheniformis strengthening the argument that it was misnamed previously.

Return to footnote Appendix 2 Table A18 a referrer

Table A-19: Results of 16S Ribosomal RNA Gene Sequence Analysis of Bacillus species 16970-5
The results of the 16S rRNA gene sequencing analysis confirms the identity of this strain as it was purported to be when it was nominated to the Domestic Substances List. However, due to confidentiality claims the identity of Bacillus species 16970-5 and closest similarity matches using the Ribosomal Database cannot be disclosed.
Table A-20: Results of 16S Ribosomal RNA Gene Sequence Analysis of Bacillus species 2 18118-1
The results of the 16S rRNA gene sequencing analysis confirms the identity of this strain as it was purported to be when it was nominated to the Domestic Substances List. However, due to confidentiality claims the identity of Bacillus species 2 18118-1and closest similarity matches using the Ribosomal Database cannot be disclosed.
Table A-21: Results of 16S Ribosomal RNA Gene Sequence Analysis of Bacillus species 4 18121-4
The results of the 16S rRNA gene sequencing analysis confirms the identity of this strain as it was purported to be when it was nominated to the Domestic Substances List. However, due to confidentiality claims the identity of Bacillus species 4 18121-4 and closest similarity matches using the Ribosomal Database cannot be disclosed.
Table A-22: Results of 16S Ribosomal RNA Gene Sequence Analysis of Bacillus species 7 18129-3
The results of the 16S rRNA gene sequencing analysis confirms the identity of this strain as it was purported to be when it was nominated to the Domestic Substances List. However, due to confidentiality claims the identity Bacillus species 7 18129-3 and closest similarity matches using the Ribosomal Database cannot be disclosed.

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Appendix 3: Characteristics of DSL B. licheniformis/subtilis group members – Fatty acids methyl ester (FAME) analysis

Data generated by Health Canada's Healthy Environments and Consumer Safety Branch shows the best match between the sample and the environmental and clinical MIDI databases and the fatty acid profile similarity index (average of all matches) along with the number of matches (number of matches/total number of tests, parentheses). For methods and additional details, visit the Website MIDI Labs. As a general rule of thumb, samples that cluster within a Euclidian distance of 2.5, 6 and 10 represent samples derived from the same strain, subspecies and species, respectively.

Table A-23: FAME analysis of B. amyloliquefaciens 13563-0
EnvironmentalClinical
B. amyloliquefaciens 0.598 (10/15)
(B. subtilis group)
B. subtilis 0.735 (12/17)
GC subgroup A
B. subtilis 0.441 (3/15)B. subtilis 0.729 (5/17)
GC subgroup B
B. atrophaeus 0.801 (1/15) GC
subgroup A
Not applicable
Staphylococcus lutrae 0.490 (1/15)
(coag+)
Not applicable
Table A-24: FAME analysis of B. atrophaeus 18250-7
EnvironmentalClinical
B. atrophaeus 0.877 (6/10)B. atrophaeus 0.814 (5/6)
GC subgroup B
Analysis not good enough (4/10)
for library search
B. atrophaeus 0.853 (1/6)
GC subgroup A
Table A-25: FAME analysis of B. licheniformis ATCC 12713
EnvironmentalClinical
B. licheniformis 0.808 (13/14)
(B. subtilis group)
B. licheniformis 0.674 (6/17)
(B. subtilis group)
B. megaterium 0.719 (1/14) GC
subgroup A
Staphylococcus schleiferi 0.418 (6/17)
Not applicableB. pumilis-GC subgroup A 0.669 (2/17)
Not applicableB. pumilis-GC subgroup B 0.468 (2/17)
Not applicableB. subtilis 0.233 (1/17)
Table A-26: FAME analysis of B. subtilisATCC 6051A
EnvironmentalClinical
B. subtilis 0.876 (16/17)B. subtilis 0.872 (13/13)
B. amyloliquefaciens 0.798 (1/17)Not applicable
Table A-27: FAME analysis of B. subtilisATCC 55405
EnvironmentalClinical
B. subtilis 0.753 (10/15)B. subtilis 0.662 (12/12)
B. amyloliquefaciens 0.736 (5/15)
(B. subtilis group)
Not applicable
Table A-28: FAME analysis of B. subtilissubsp. subtilis ATCC 6051
EnvironmentalClinical
B. subtilis 0.911 (14/14)B. subtilis 0.760 (9/12)
Not applicableAnalysis not good enough 2/12
for library search
Not applicableMicrococcus lylae 0.292 (1/12)
GC subgroup B
Table A-29: FAME analysis of B. subtilissubsp. inaquosorum ATCC 55406
EnvironmentalClinical
B. subtilis 0.803 (10/25)B. subtilis 0.727 (12/12)
B. amyloliquefaciens 0.793 (9/25) (B. subtilis group)Not applicable
B. megaterium 0.602 (4/25) GC subgroup ANot applicable
No match 2/25Not applicable
Table A-30: FAME analysis of Bacillusspecies 16970-5
EnvironmentalClinical
The results generated from the FAME environmental database confirms the identity of this strain as it was purported to be when it was nominated to the Domestic Substances List. However, due to confidentiality claims the identity of Bacillus species 16970-5 cannot be disclosed.The results generated from the FAME environmental database confirms the identity of this strain as it was purported to be when it was nominated to the Domestic Substances List. However, due to confidentiality claims the identity of Bacillus species 16970-5 cannot be disclosed.
Table A-31: FAME analysis of Bacillusspecies 2 18118-1
EnvironmentalClinical
The results generated from the FAME environmental database confirms the identity of this strain as it was purported to be when it was nominated to the Domestic Substances List. However, due to confidentiality claims the identity of Bacillus species 2 18118-1 cannot be disclosed.The results generated from the FAME environmental database confirms the identity of this strain as it was purported to be when it was nominated to the Domestic Substances List. However, due to confidentiality claims the identity of Bacillus species 2 18118-1 cannot be disclosed.
Table A-32: FAME analysis of Bacillusspecies 4 18121-4
EnvironmentalClinical
The results generated from the FAME environmental database confirms the identity of this strain as it was purported to be when it was nominated to the Domestic Substances List. However, due to confidentiality claims the identity of Bacillus species 4 18121-4 cannot be disclosed.The results generated from the FAME clinical database confirms the identity of this strain as it was purported to be when it was nominated to the Domestic Substances List. However, due to confidentiality claims the identity of Bacillus species 4 18121-4 cannot be disclosed.
Table A-33: FAME analysis of Bacillusspecies 7 18129-3
EnvironmentalClinical
The results generated from the FAME environmental database confirms the identity of this strain as it was purported to be when it was nominated to the Domestic Substances List. However, due to confidentiality claims the identity of Bacillus species 7 18129-3 cannot be disclosed.The results generated from the FAME environmental database confirms the identity of this strain as it was purported to be when it was nominated to the Domestic Substances List. However, due to confidentiality claims the identity of Bacillus species 7 18129-3 cannot be disclosed.

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Appendix 4: Cellular content of select fatty acids

Table A-34: Cellular Content of Select Fatty Acids in DSL B. licheniformis/subtilis Group MembersFootnote Appendix 4 Table A34 [a]
DSL StrainC16:O (%)Iso-C17:1ω10c (%)
B. amyloliquefaciens 13563-03.172.4
B. atrophaeus 18250-73.071.5
B. licheniformis ATCC 127132.891.56
B. subtilis subsp. subtilis ATCC 60514.341.95
B. subtilis ATCC 6051A2.552.65
B. subtilis ATCC 554053.052.34
B. subtilis subsp. inaquosorum ATCC 554063.31.16
Bacillus species 16970-53.521.81
Bacillus species 2 18118-16.091.77
Bacillus species 4 18121-43.411.11
Bacillus species 7 18129-34.341.95
Footnote Appendix 4 Table A34 a

Unpublished data generated by Health Canada's Healthy Environments and Consumer Safety Branch

Return to footnote Appendix 4 Table A34 a referrer

Appendix 5: List of some mobile elements and associated traits identified in certain isolates of the B. subtilis complex

Table A-35: List of Some Mobile Elements and Associated Traits Identified in some strains of B. licheniformis
Element NameAssociated TraitsReferences
Plasmid (unknown name)Dimethoate resistance and additional genes for antibiotic and heavy metal resistance (Na, Er, Ch, Cz, Cf, Ba2+ and Zn2+)(Mandal et al. 2005)
Plasmid (pBL1, pBL10, pBL2)Not specified(Zawadzki et al. 1996)
Insertion element (IS3Bli1)Encodes two predicted overlapping protein coding sequences, designated orfA and orfB in relative translational reading frames of 0 and 1. Eight of these elements lie in intergenic regions and one interrupts the comPgene(Rey et al. 2004)
Prophase sequences (NZP1 and NZP3)Codes for the large subunit of terminase, a signature protein that is highly conserved among prophages(Rey et al. 2004)
Table A-36: List of Some Mobile Elements and Associated Traits Identified in some strains of B. subtilis
Element NameAssociated TraitsReferences
Transposon (Tn917)Used in transposition mutagenesis (rapid cloning and construction of transcriptional gene fusions and the characterization of genes which are over-expressed)(Pragai et al. 1994)
Plasmid (pLS20)Promotes transfer of tetracycline resistance plasmid pBC16 from B. subtilis (natto) to the Bacillus species B. anthracis, B. cereus, B. licheniformis, B. megaterium, B. pumilus, B. subtilis and B. thuringiensis.(Koehler and Thorne, 1987)
Transposon (Tn5)Neomycin phosphotransferase gene(Sprengel et al. 1985)
Rolling circle replication (RCR) plasmid pTA family (pTA1015, pTA1040, pTA1050 and pTA1060)Contains genes encoding a type I signal peptidase and genes encoding proteins belonging to the family of response regulator aspartate phosphatases likely to be involved in the regulation of post-exponential phase processes(Meijer et al. 1998)
Plasmid (pBS4, pBS12, pBS7, pBS8, pBS6)Not specified(Zawadzki et al. 1996)
Transposon (integrative and conjugative elements class: ICEBs1)
  • Integrated into the trnS-leu2 gene is regulated by the SOS response and the RapI-PhrI cell-cell peptide signaling system
  • When DNA damage occurs or high concentrations of potential mating partners that lack the element, ICEBs1 is excised from the chromosome and transferred to recipients.
(Auchtung et al. 2005)
Tn916 (transposon)
  • Implicated in the horizontal transfer of antibiotic resistance genes in many species of Gram positive bacteria
  • Transfer of this element may increase the presence of tetracycline
(Celli and Trieu-Cuot, 1998; Marra and Scott, 1999)
Tn5397 (transposon)
  • Originates from Clostridium difficile; element transfers to and from B. subtilis
  • Encodes a conjugation system that is similar to that of Tn916
  • Contains a group II intron
(Roberts et al. 2001; Wang and Mullany, 2000)
Tn5398 (transposon)
  • Originates from C. difficile
  • Facilitates the transfer ofan MLS resistance gene (ermBZ)
(Mullany et al. 1995)
IS4Bsu1 (mobile element)Spontaneously translocates to the swrA gene in B. subtilis natto; causes a defect in poly-gamma- glutamic acid (gamma-PGA) synthesis(Kimura et al. 2011)

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Appendix 6: Virulence genes

Table A-37: List of Some Virulence Genes Identified in Certain Isolates of the B. subtilisComplex
SpeciesVirulence Gene(s)Associated TraitsReferences
B. amyloliquefaciensHblC, HblD, HblA, NheB, NheAEnterotoxin production and discontinuous beta-hemolysis(Phelps and McKillip, 2002)
B. licheniformiscesACereulide synthase(Nieminen et al. 2007)
B. licheniformislchAA, lchAB, lchACLichenysin synthase(Nieminen et al. 2007)
B. licheniformisbceT, hblC, hblA, hblDHbl enterotoxin, Bacillus hemolytic enterotoxin(Oguntoyinbo and Sanni, 2007; Rowan et al. 2001)
B. licheniformislchAA, lchAB and lchAC (lichenysin synthase genes)
  • Surfactant lichenysin
  • Heat-stable cyclic lipopeptide toxins; immobilizes boar sperm
  • Structurally similar to cereulide, but the toxic activity appears to be different; it has the potential to form ion channels in host cell membranes and has a surfactant effect
(Logan, 2012; Mikkola et al. 2000; Nieminen et al. 2007; Peypoux et al. 1999)
B. licheniformisbacA, bacB, bacC (bacitracin synthetases genes)
  • Cyclic polipeptides; interferes with cell wall and peptidoglycan synthesis of Gram positive and negative bacteria Possibly/indirectly related to erythromycin resistance
(Ishihara et al. 2002)
B. licheniformis and B. subtilisbceT, hblC, hblA, hblDDiarrhoeagenic enterotoxin production(Rowan et al. 2001)

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Appendix 7: Virulence and Pathogenicity Testing of DSL B. licheniformis/subtilis Strains: Hemolytic Activity

Data generated by Health Canada's Healthy Environments and Consumer Safety Branch. Strains were streaked onto 5% sheep blood agar and incubated for 37°C for 24 hours. Hemolysis was judged by clearing zones around colonies which indicate lysis of red blood cells.

Table A-38: Hemolytic activity of DSL B. licheniformis/subtilis strains
DSL StrainHemolytic Activity
B. amyloliquefaciens 13563-0+Footnote Appendix 7 Table A38 [a],Footnote Appendix 7 Table A38 [b]
B. atrophaeus 18250-7-Footnote Appendix 7 Table A38 [c]
B. licheniformis ATCC 12713+Footnote Appendix 7 Table A38 [d]
B. subtilis ATCC 6051AwFootnote Appendix 7 Table A38 [e]
B. subtilis ATCC 55405w
B. subtilis subsp. subtilis ATCC 6051w
B. subtilis subsp. inaquosorum ATCC 55406+
Bacillus species 16970-5w
Bacillus species 2 18118-1w
Bacillus species 4 18121-4-
Bacillus species 7 18129-3w
Footnote Appendix 7 Table A38 a

+, hemolytic activity

Return to footnote Appendix 7 Table A38 a referrer

Footnote Appendix 7 Table A38 b

Hemolysis was seen in 5 to 10% of colonies

Return to footnote Appendix 7 Table A38 b referrer

Footnote Appendix 7 Table A38 c

-, no hemolytic activity

Return to footnote Appendix 7 Table A38 c referrer

Footnote Appendix 7 Table A38 d

Hemolysis was seen in 70 to 80% of colonies

Return to footnote Appendix 7 Table A38 d referrer

Footnote Appendix 7 Table A38 e

w, weak hemolytic activity – clearing zones do not extend past colony margin

Return to footnote Appendix 7 Table A38 e referrer

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Appendix 8: Virulence and pathogenicity testing of DSL B. licheniformis/subtilis strains: Catalase production

Data generated by Health Canada's Healthy Environments and Consumer Safety Branch. Bacteria were propagated on TSB agar at 28°C for 48 hours. Hydrogen peroxide dropped on to colony to determine conversion to water and hydrogen. Catalase positive reaction indicated that a given bacteria has the capacity to protect itself from reactive oxygen-induced killing from immune cells.

Table A-39: Catalase production of DSL B. licheniformis/subtilis strains
DSL StrainCatalase Production
B. amyloliquefaciens 13563-0+ (positive)
B. atrophaeus 18250-7+
B. licheniformis ATCC 12713+
B. subtilis ATCC 6051A+
B. subtilis ATCC 55405+
B. subtilis subsp. subtilis ATCC 6051+
B. subtilis subsp. inaquosorum ATCC 55406+
Bacillus species 16970-5+
Bacillus species 2 18118-1+
Bacillus species 4 18121-4+
Bacillus species 7 18129-3+

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Appendix 9: Antimicrobial Compound, Other Metabolites and Toxins Produced by certain isolates of the B. subtilis Complex

Table A-40: Antimicrobial compounds produced in some strains of B. amyloliquefaciens
Substance NameActivityReferences
Bacteriocin-like peptidesBroad antibacterial spectrum with activity against Gram positive bacteria(Reviewed in Abriouel et al. 2011; Smitha and Bhat, 2012)
BLISFootnote Appendix 9 Table A40 [a]5940, BLIS RC-2, BLIS 5006Antibacterial and antifungal activity(Reviewed in Abriouel et al. 2011)
Surfactin, iturin, bacillomycine, azalomycin F, acivicin, arthrobactin, rhodutorola acid, valinomycin, stenothricin, enterochelin, nocardaminAntibacterial and antifungal activity, inhibition of growth(Wulff et al. 2002)
Subtilosin A
  • Bacteriocin with bactericidal activity against Gram negative bacteria
  • Spermicidal activity against boar, bovine, horse, rat and human spermatozoa
(Reviewed in Abriouel et al. 2011)
Footnote Appendix 9 Table A40 a

BLIS: bacteriocin-like inhibitory substances

Return to footnote Appendix 9 Table A40 a referrer

Table A-41: Antimicrobial compounds produced in some strains of B. atrophaeus
Substance NameActivityReferences
Subtilosin A
  • Bacteriocin with bactericidal activity against Gram negative bacteria
  • Spermicidal activity against boar, bovine, horse, rat and human spermatozoa
(Reviewed in Abriouel et al. 2011)
Table A-42: Antimicrobial compounds produced in some strains of B. licheniformis
Substance NameActivityReferences
Amoebicins (A12-A and A12-B)
  • Amoebolytic activity against Naegkriafowkri
  • Antibiotic activity against yeasts (Saccharomyces heterogenicus and Cryptococcus neoformans) and several fungal species
(Galvez et al. 1993)
Amoebicin (m4-A)Bactericidal and bacteriolytic activity on Bacilus megaterium GR10(Lebbadi et al. 1994)
Antibiotics (bacitracin, licheniformin, proticin)Antibiotics which are secreted and may inhibit competing organisms including Gram positive and Gram negative bacteria, yeasts and molds(Reviewed in Katz and Demain, 1977)
Bacillocin, BLIS P40, BLIS ZJU12, BLIS MKU3, peptide A-12 CAntibacterial and antifungal activity(Reviewed in Abriouel et al. 2011)
Bacteriocin BL8
  • Thermostable, broad pH range
  • Antibacterial against Gram positive bacteria
(Smitha and Bhat, 2012)
Bacteriocin-like peptideBroad spectrum antagonistic activity activities against fungal pathogens and Gram positive bacteria but not most Gram negative bacteria(He et al. 2006)
Ieodoglucomides A and B
  • Cytotoxic activity against lung and stomach cancer cells lines
  • Moderate antimicrobial activity
(reviewed in Tareq et al. 2012)
Lichenicidin (α, β)Antibacterial activity associated with the cell surface(Reviewed in Abriouel et al. 2011; Begley et al. 2009; Dischinger et al. 2009)
LicheninBacteriocin produced under anaerobic conditions(Reviewed in Abriouel et al. 2011)
LichenysinLipopeptide that acts as an anionic biosurfactin as well as an antimicrobial(Li et al. 2010; Nerurkar, 2010; Nieminen et al. 2007)
Surfactin, iturin, acivicin, arthrobactin, rhodutorola acid, valinomycinAntibacterial and antifungal activity, inhibition of growth(Wulff et al. 2002)
Table A-43: Antimicrobial compounds produced in some strains of B. subtilis
Substance NameActivityReference
Anti-bacterials (ericin S and A, sublancin 168, subtilin B, subtilosin A and A1, mersacidin, betacin, MJP1, Bac 14B, LFB112)Bacterial activity against bacterial pathogens(Reviewed in Abriouel et al. 2011)
Antibiotics (mycobacillin, subtilin, bacilysin, bacillomycin, fungistatin, bulbiformin, bacillin, subsporin, bacillocin, mycosubtilin, fungocin, iturin, neocidin, eumycin)Antibiotics which may inhibit competing organisms including Gram positive and negative bacteria as well as yeasts and molds(reviewed in Katz and Demain, 1977)
Antibiotics (subtilin, ericin, mersacidin, sublancin 168, subtilosin, surfactin, iturin, bacillomycin, mycosubtilin, fengycin plipastatin, corneybactin, bacilysin, bacilysocin, amicoumacin, mycobacillin, TL-119, rhizocticin, difficidin, 3,3'-neotrehalos-adiamine 168)Anti-microbial activity, biofilm and swarming development, pheromones in quorum sensing and 'killing factor'.(reviewed in Stein, 2005)
Heat-stable, protease resistant antimicrobial substanceInhibits growth of many bacteria(reviewed in Sorokulova et al. 2008)
Subtilin
  • Antimicrobial peptide
  • Affects pore formation in the cytoplasmic membrane
  • Produced in higher amounts under starvation conditions to eliminate competing species and increase available nutrients
(Reviewed in Abriouel et al. 2011)
Surfactin
  • Lipopeptide that acts as a biosurfactin and a potent antimicrobial
(Li et al. 2010)
Table A-44: Toxic metabolites produced by some strains of B. amyloliquefaciens
Substance NameActivityReferences
Amylosin
  • Inhibits boar sperm motility Cytotoxic to feline lung cells
(Mikkola et al. 2007)
Table A-45: Toxic metabolites produced by some strains of B. licheniformis
Substance NameActivityReferences
Non-emetic heat stable cytotoxic componentCytotoxicity activity(De Jonghe et al. 2010)
Heat labile cytotoxic substanceCytotoxicity activity(De Jonghe et al. 2010)
B. cereus-like protein toxin
  • Reduction in cellular metabolic activity
  • Cytotoxic activity
(Beattie and Williams, 1999)
Emetic toxin
  • Induces vomiting if ingested
  • Ionophoric uptake of K+ resulting in the dissipation of the transmembrane potential, stimulating swelling and respiration in mitochondria which leads to their inactivation
(Biesta-Peters et al. 2010; Reviewed in From et al. 2005)
Hemolysin BL (Hbl) enterotoxinCauses diarrhea(Rowan et al. 2001)
Heat labile B. cereus diarrheal-like toxin
  • Cytotoxic to McCoy cells causing leaky membranes, disrupts cell surfaces and decreases metabolic activity
(Lindsay et al. 2000)
Lichenysin
  • Inhibits sperm motility
  • Synthesized in both aerobic and anaerobic condition during growth
  • Species specific variations (A, B, C, D, G and surfactant BL86)
(Li et al. 2010; Nerurkar, 2010; Nieminen et al. 2007)
Lichenysin A
  • Causes loss of motility, damage to plasma membrane and acrosome, loss of cellular NADH and ATP in boar spermatozoa
  • Toxic towards natural (non-malignant) mammalian cells
  • May be produce aerobically and anaerobically
  • More powerful compared to surfactin and lichenysin B
(Mikkola et al. 2000; Yakimov et al. 1996)
Non-proteinaceous, heat-stable, sperm toxic agent
  • Inhibits sperm motility and swells acrosome
  • Damages cell membrane integrity
  • Depletes cellular ATP
  • Beta-hemolytic activity
(Salkinoja-Salonen et al. 1999)
NucB
  • Degrades extracellular DNA that is an essential building block of both single species and mixed biofilms
  • Nontoxic deoxyribonuclease
  • Sporulation-specific enzyme
(Rajarajan et al. 2013; Shakir et al. 2012)
SurfactinInhibits phytopathogenic fungi(Nerurkar, 2010)
Table A-46: Toxic metabolites produced by some strains of B. subtilis
Substance NameActivityReferences
Hemolysin BL (Hbl) enterotoxinCauses diarrhea(Rowan et al. 2001)
Non-emetic heat stable cytotoxic component and a heat labile cytotoxic substanceCytotoxicity activity(De Jonghe et al. 2010)
Protolytic and lipolytic substancesLysis of proteins and lipids(De Jonghe et al. 2010)
Putative emetic toxinCauses nausea and vomiting(From et al. 2005)

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Appendix 10: Virulence and pathogenicity testing of DSL B. licheniformis/subtilis strains: Cytotoxicity

Data generated by Health Canada's Healthy Environments and Consumer Safety Branch. The MTT Assay was used to determine the cytotoxic potential of the strains towards HT29 (colonic epithelial cells) and J774A.1 (macrophage cells). MTT is a yellow, soluble bromide salt which is reduced to a purple, insoluble formazan crystal by dehydrogenase enzymes of living cells (indicating mitochondrial activity). In the crystal state after reduction, it is trapped inside the cell. DMSO or another solvent such as isopropanol or mineral oil can be used to solubilize the formazan, which can then exit the cell, turning the solvent a purple colour that is detectable with a spectrophotometer. This assay is suitable for animal cells that are adherent. Metabolically active bacterial cells can also reduce MTT also. Since most animal cells are not adherent bacteria and their formazan contribution can be rinsed away with PBS prior to solubilisation.

HT29 and J774A.1 were incubated at 37°C in the presence of 5% carbon dioxide. Mammalian cells were dosed with 106CFU/well of vegetative bacteria for 2, 4 and 24 hours. Dose cells were washed twice with PBS before adding MTT.

Loss in bioreduction activity of the cell lines toward MTT was measured to determine the cytotoxic potential of the DSL B. licheniformis/subtilis group strains. Cytotoxicity is related to increased losses in bioreduction activity of the cell lines.

Table A-47: Cyototoxic potential of DSL B. licheniformis/subtilis strains towards HT29 cells with gentamicin at 2, 4 and 24 hours
DSL StrainFootnote Appendix 10 Table A47[a]2 hours4 hours24 hours
B. amyloliquefaciens 13563-0wFootnote Appendix 10 Table A47 [b]ww
B. atrophaeus 18250-7wFootnote Appendix 10 Table A47 [c]ww
B. licheniformis ATCC 12713www
B. subtilis subsp. inaquosorum ATCC 55406w--
Bacillus species 16970-5w-w
Bacillus species 2 18118-1---
Bacillus species 4 18121-4www
Footnote Appendix 10 Table A47 a

No data available for B. subtilis ATCC 6051A, B. subtilis ATCC 55405, B. subtilis subsp. subtilis ATCC 6051T and Bacillusspecies 7 18129-3

Return to footnote Appendix 10 Table A47 a referrer

Footnote Appendix 10 Table A47 b

w, weak cytotoxic activity (5-50% bioreduction loss)

Return to footnote Appendix 10 Table A47 b referrer

Footnote Appendix 10 Table A47 c

Related to structural components

Return to footnote Appendix 10 Table A47 c referrer

Table A-48: Cyototoxic potential of DSL B. licheniformis/subtilis strains towards HT29 cells without gentamicin at 2, 4 and 24 hours
DSL Strain2 hours4 hours24 hours
B. amyloliquefaciens 13563-0wFootnote Appendix 10 Table A48 [a]ww
B. atrophaeus 18250-7wFootnote Appendix 10 Table A48 [b]ww
B. licheniformis ATCC 12713ww+Footnote Appendix 10 Table A48 [c],Footnote Appendix 10 Table A48 [d]
B. subtilis ATCC 6051ANDFootnote Appendix 10 Table A48[e]ww
B. subtilis ATCC 55405ND-Footnote Appendix 10 Table A48 [f]-
B. subtilis subsp. subtilis ATCC 6051TNDww
B. subtilis subsp. inaquosorum ATCC 55406ww+
Bacillus species 16970-5ww+
Bacillus species 2 18118-1ww+[c]
Bacillus species 4 18121-4www
Bacillus species 7 18129-3NDww
Footnote Appendix 10 Table A48 a

w, weak cytotoxic activity (5-50% bioreduction loss)

Return to footnote Appendix 10 Table A48 a referrer

Footnote Appendix 10 Table A48 b

Related to structural components

Return to footnote Appendix 10 Table A48 b referrer

Footnote Appendix 10 Table A48 c

+, cytotoxic activity ( greater than 50% bioreduction loss)

Return to footnote Appendix 10 Table A48 c referrer

Footnote Appendix 10 Table A48 d

Growth-related

Return to footnote Appendix 10 Table A48 d referrer

Footnote Appendix 10 Table A48 e

ND, no data

Return to footnote Appendix 10 Table A48 e referrer

Footnote Appendix 10 Table A48 f

-, no cytotoxic activity ( less than 5% bioreduction loss)

Return to footnote Appendix 10 Table A48 f referrer

Table A-49: Cyototoxic potential of DSL B. licheniformis/subtilis strains towards J774A.1 cells with gentamicin at 2, 4 and 24 hours
DSL StrainFootnote Appendix 10 Table A49[a]2 hours4 hours24 hours
B. amyloliquefaciens 13563-0-Footnote Appendix 10 Table A49 [b]--
B. atrophaeus 18250-7wFootnote Appendix 10 Table A49 [c],Footnote Appendix 10 Table A49 [d]ww
B. licheniformis ATCC 12713--w
B. subtilis subsp. inaquosorum ATCC 55406www
Bacillus species 16970-5www
Bacillus species 2 18118-1---
Bacillus species 4 18121-4-ww
Footnote Appendix 10 Table A49 a

No data available for B. subtilis ATCC 6051A, B. subtilis ATCC 55405, B. subtilis subsp. subtilis ATCC 6051T and Bacillusspecies 7 18129-3

Return to footnote Appendix 10 Table A49 a referrer

Footnote Appendix 10 Table A49 b

-, no cytotoxic activity ( less than 5% bioreduction loss)

Return to footnote Appendix 10 Table A49 b referrer

Footnote Appendix 10 Table A49 c

w, weak cytotoxic activity (5-50% bioreduction loss)

Return to footnote Appendix 10 Table A49 c referrer

Footnote Appendix 10 Table A49 d

Related to structural components

Return to footnote Appendix 10 Table A49 d referrer

Table A-50: Cyototoxic potential of DSL B. licheniformis/subtilis strains towards J774A.1 cells without gentamicin at 2, 4 and 24 hours
DSL Strain2 hours4 hours24 hours
B. amyloliquefaciens 13563-0-Footnote Appendix 10 Table A50 [a]--
B. atrophaeus 18250-7wFootnote Appendix 10 Table A50 [b],Footnote Appendix 10 Table A50 [c]ww
B. licheniformis ATCC 12713w-w
B. subtilis ATCC 6051ANDFootnote Appendix 10 Table A50[d]ww
B. subtilis ATCC 55405ND-w
B. subtilis subsp. subtilis ATCC 6051TND-w
B. subtilis subsp. inaquosorum ATCC 55406--+Footnote Appendix 10 Table A50 [e]
Bacillus species 16970-5--w
Bacillus species 2 18118-1--+Footnote Appendix 10 Table A50 [f]
Bacillus species 4 18121-4--w
Bacillus species 7 18129-3ND-w
Footnote Appendix 10 Table A50 a

-, no cytotoxic activity ( less than 5% bioreduction loss)

Return to footnote Appendix 10 Table A50 a referrer

Footnote Appendix 10 Table A50 b

w, weak cytotoxic activity (5-50% bioreduction loss)

Return to footnote Appendix 10 Table A50 b referrer

Footnote Appendix 10 Table A50 c

Cytotoxic activity related to structural components

Return to footnote Appendix 10 Table A50 c referrer

Footnote Appendix 10 Table A50 d

ND, No data

Return to footnote Appendix 10 Table A50 d referrer

Footnote Appendix 10 Table A50 e

+, cytotoxic activity ( greater than 50% bioreduction loss)

Return to footnote Appendix 10 Table A50 e referrer

Footnote Appendix 10 Table A50 f

Cytotoxic activity related to growth

Return to footnote Appendix 10 Table A50 f referrer

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Appendix 11: Pathogenicity, toxicity and irritation testing results for strains of the B. subtilis complex on terrestrial and aquatic vertebrates, invertebrates and plants

Table A-51: Pathogenicity, toxicity and irritation testing results for B. amyloliquefaciens strain FZB24Footnote Appendix 11 Table A51 [a]
Study TypeTarget organismDose concentrationOutcome
Acute oral toxicity and infectivityCD rats1.3 × 108 CFU/animalLow toxicity, not pathogenic
LD50 greater than 1.3 × 108 CFU/animal
Acute pulmonary toxicity and infectivityCD rats0.1 mL of at least 1.4 × 108 viable CFU/animalLow toxicity, not pathogenic
LD50 greater than 1.4 × 108 CFU/animal
Acute pulmonary toxicity and infectivityRats1.3 × 108 CFUNot toxic/
Not pathogenic
Acute intravenous infectivityCD rats0.5 mL of at least 1.0 × 107 CFU/animalNot pathogenic
LD50 greater than 1.0 × 107 CFU/animal
Acute intravenous infectivityRats1.7 × 108 CFUNot toxic/
Not pathogenic
Acute dermal toxicityRabbits2000 mg/kg BWFootnote Appendix 11 Table A51 [b](1.5 to 2 × 1012 CFU/animal)Low toxicity but severely irritating
LD50 greater than 2000 mg/kg
Primary dermal irritationRabbits0.5 g granular test substance (7.0 × 1010 CFU/g) and 0.5 mL of 1.5% w.v suspensionNo irritation observed
Primary eye irritationRabbit3.6 × 1010 CFUEye irritant
Acute dermal irritationNew Zealand white rabbit2000 mg/kg/BW (1.5 to 2 × 1012 CFU/animal)Low toxicity and severely irritating
LD50 greater than 2000 mg/kg/BW
Avian oral toxicityNorthern bobwhite (Colinus virginianus)10 mL/kg BW or 1.0 × 109 CFU/g BWNOELFootnote Appendix 11 Table A51 [c]: 1.0 × 109 CFU/animal
Terrestrial arthropod toxicityAdult bees (Apis meelifera)105 CFU/mLNo signs of toxicity or pathogenicity
LC50 greater than 1.0 × 106 CFU/mL
Terrestrial arthropod toxicityLarva (Apis meelifera)6.0 × 103CFU/larvaLC50 greater than 6.0 ×103CFU/larva
Terrestrial non-arthropod invertebratesWorm (Eisenia fetida)6.0 × 1011 CFU/kg soilNOECFootnote Appendix 11 Table A51 [d]: 6.0 × 1011 CFU/kg soil
Freshwater fish toxicity/pathogenicityRainbow trout (Oncorhynchus mykiss)1.85 × 109 CFU/kg
to 1.85 × 1010 CFU/L (active ingredient)
LC50 greater than 1.85 × 1010CFU/L
NOEC: 1.85 × 1010 CFU/L
Freshwater invertebrate toxicity/pathogenicityDaphnia magnaUp to 1.85 × 1010 CFU/LLC50 greater than 1.85 × 1010CFU/L
NOEC: 1.85 × 1010 CFU/L
Footnote Appendix 11 Table A51 a

Studies done with the technical grade active ingredient and not the end-use product containing the micro-organism, (PMRA-HC, 2012; U.S. EPA, 2012)

Return to footnote Appendix 11 Table A51 a referrer

Footnote Appendix 11 Table A51 b

BW, body weight

Return to footnote Appendix 11 Table A51 b referrer

Footnote Appendix 11 Table A51 c

NOEL, no observed effect level is the highest dose of the test substance in the test substrate at which no statistically significant effect on the test organism was observed, relative to the control

Return to footnote Appendix 11 Table A51 c referrer

Footnote Appendix 11 Table A51 d

NOEC, no observed effect concentration is the highest concentration of the test substance in the test substrate at which no statistically significant effect on the test organism was observed, relative to the control

Return to footnote Appendix 11 Table A51 d referrer

Table A-52 : Pathogenicity, toxicity and irritation testing results for B. amyloliquefaciens strain D747Footnote Appendix 11 Table A52 [a]
Study TypeTarget organismDose concentrationOutcome
Acute oral toxicitySprague-Dawley rats (5 week old, male and female)108 CFU/animalNot toxic, infective or pathogenic
LD50 greater than 5000 mg/kg
Acute pulmonary toxicitySprague-Dawley rats (5 week old, male and female)107 CFU/animalNot toxic or pathogenic
LC50 greater than 2.18 mg/L
Acute injectionSprague-Dawley rats (5 week old, male and female)107 CFU/animalNot toxic, infective, or pathogenic
LD50 greater than 5050 mg/kg
Acute eye irritationNew Zealand white rabbits0.1 mL of the end-use productEye irritant
Primary dermal irritationNew Zealand white rabbits500 mg of the end-use productNo evidence of irritation
Avian oral toxicityNorthern bobwhite quail (Colinus virginianus)8.9 × 109 spores/birdNot toxic
LD50 greater than 4.5 × 1011 spores/kg BW or greater than 8.0 × 1010 spores/bird
Freshwater fish toxicity/pathogenicityRainbow trout (Oncorhynchus mykiss)1.7 × 108 CFU/LLC50: 8.1 × 1010 CFU/L
NOEC: 1.44 × 1010 CFU/L
Freshwater invertebrate toxicity/pathogenicityDaphnia magna1.7 × 108 CFU/LEC50: 3.7 × 1010 CFU/L
NOEC: 2.84 × 108 CFU/L
Footnote Appendix 11 Table A52 a

Studies done with the technical grade active ingredient and not the end-use product containing the micro-organism, (U.S. EPA, 2011)

Return to footnote Appendix 11 Table A52 a referrer

Table A-53: Pathogenicity, toxicity and irritation testing results for B. licheniformis strain SB3086Footnote Appendix 11 Table A53 [a]
Study TypeTarget organismDose concentrationOutcome
Acute oral toxicity/pathogenicityRats1.0 × 108 CFU/animalFootnote Appendix 11 Table A53 [b]Not toxic, infective, or pathogenic
LD50 greater than 5000 mg/kg
Acute pulmonary toxicity/pathogenicityRats1.1 × 108CFU/animal[b]Not toxic, infective, or pathogenic
Acute intravenous toxicity/pathogenicityRats1.0 × 107CFU/animal[b]Not toxic, infective, or pathogenic
Acute dermal toxicityNew Zealand white rabbitsNDFootnote Appendix 11 Table A53[c],Footnote Appendix 11 Table A53[d]LD50 greater than 5050 mg/kg
Primary eye irritationNew Zealand white rabbits0.1 mL/animal (concentration not provided)dNon-irritating
Delayed contact hypersensitivityGuinea PigsND[d]Not a dermal sensitizer
Avian oral toxicity/pathogenicityYoung mallards (Anas platyrhynchos)4.5 × 1010 CFU/kg of BW[b]No signs of illness or abnormal behaviour observed
Fresh water fish toxicity/pathogenicityRainbow trout (Oncorhynchus mykiss)LC50 (of Formula 710-132) greater than 1.1 × 106 CFU/mL[b]No effects as the result of the active microbial agent observed
Fresh water aquatic invertebrate toxicity/pathogenicity (21-day renewal life-cycle)Daphnia magna1 × 104 CFU/mL[b]LC50: 1.8 × 106 CFU/mL
NOAEC[d]:1.2 × 106CFU/mL[b],Footnote Appendix 11 Table A53 [e]
2 daphnids died at the end of the test at 1 × 107CFU/mL (1000 times the expected environmental concentration)
Invertebrate toxicity/pathogenicityHoneybee larvae (Apis mellifera L.)1.6 × 106 CFU/mL (of Formula 710-132)Footnote Appendix 11 Table A53 [f]No statistically significant effects on larvae survival, adverse behaviour or developmental abnormalities observed
Footnote Appendix 11 Table A53 a

(U.S. EPA, 2001)

Return to footnote Appendix 11 Table A53 a referrer

Footnote Appendix 11 Table A53 b

TGAI, technical grade active ingredient

Return to footnote Appendix 11 Table A53 b referrer

Footnote Appendix 11 Table A53 c

ND, no data

Return to footnote Appendix 11 Table A53 c referrer

Footnote Appendix 11 Table A53 d

NOAEC, no observed adverse effect concentration

Return to footnote Appendix 11 Table A53 d referrer

Footnote Appendix 11 Table A53 e

Formulation ingredients are known aquatic toxicants at high concentrations

Return to footnote Appendix 11 Table A53 e referrer

Footnote Appendix 11 Table A53 f

EP, end product

Return to footnote Appendix 11 Table A53 f referrer

Table A-54: Pathogenicity, toxicity and irritation testing results for several strains of B. licheniformisand B. subtilis
Study TypeTarget organismDose concentrationOutcome
Experimental infection (intravenous inoculation)Footnote Appendix 11 Table A54 [a]Immune depressed BALB/c mice4 × 107 CFUNumerous and larger lesions in many organs and more severe infection with lesions occurring in more organs compared to healthy/normal mice used in the study
Experimental infection (intravenous inoculation)[a]Normal mice4 × 107 CFULesions observed in liver and kidneys
Experimental infection (intravenous inoculation)Footnote Appendix 11 Table A54 [b]Immune depressed BALB/c miceIntravenous doses of less than 106 to 1010 CFUOnly brain and pulmonic lesions could be definitely attributed to B. licheniformis. Mice were able to clear high numbers of bacteria within 1 week
Experimental infection (intravenous inoculation)Footnote Appendix 11 Table A54 [c]Pregnant crossbred Red Danish Dairy X American Brown Swiss cows (6-8 months of gestation, n=8) and their calvesIntravenous doses of less than 109 to 1012 CFU (once or on 4 consecutive days)Demonstration of the abortifacient potential of B. licheniformis and the tropism for the bovine placenta. Lesions in the fetal membranes, the fetal side of the placentomes, necrosis in the fetal compartment of the placenta and inflammation in some calves. Two abortions were observed
Cytotoxic ActivityFootnote Appendix 11 Table A54[d]Boar sperm motility inhibition1-10 μg/mLEC50: 20-30 μg/mL
Acute eye irritation studyFootnote Appendix 11 Table A54 [e]Male albino rabbits0.1 g of 1.1 × 1011 CFU/kg BWNo irritation or negative symptoms in the cornea or iris
Acute skin irritation study[e]Male albino rabbits0.5 g of 1.1 × 1013 CFU/kg BWNo clinical signs of erythema or oedema
Acute oral toxicity (14-day) [e]Adult male albino Wistar rats1.1 × 1011 CFU/kg BWNo treatment-related changes
Subchronic oral toxicity (13-week)[e]Male and female Wistar rats1.1 × 1011 CFU/kg BW and 1.1 × 1011CFU/kg BWNOAELFootnote Appendix 11 Table A54 [f]greater than 1.1 × 1011 CFU/kg BW
Micronucleus assay (2 days at 24 hour intervals)[e]Adult male and female Swiss albino mice (CFT strain)1.1 × 1010 CFU/kg BW and 1.1 × 1011CFU/kg BWNo signs of bone marrow cytotoxicity and no observed genocyctotoxicity
Oral Pathogenicity and Toxicity StudyFootnote Appendix 11 Table A54 [g]Bobwhite quails (Colinus virginianus)3,333 kg/mg daily for 5 daysLD50 greater than 2,000 mg/kg
Footnote Appendix 11 Table A54 a

B. licheniformis ATCC 14580, (Agerholm et al. 1995)

Return to footnote Appendix 11 Table A54 a referrer

Footnote Appendix 11 Table A54 b

13 strains of B. licheniformis, (Agerholm et al. 1997)

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Footnote Appendix 11 Table A54 c

B. licheniformis strain DVL 9315323, (Agerholm et al. 1999)

Return to footnote Appendix 11 Table A54 c referrer

Footnote Appendix 11 Table A54 d

B. licheniformis strains NR 5160 and NR 6768 (toxic heat-stable non-protein substance), (Nieminen et al. 2007)

Return to footnote Appendix 11 Table A54 d referrer

Footnote Appendix 11 Table A54 e

B. licheniformis strain Me I, The concentration used in the study corresponds to 77 × 1011 CFUs for an average 70 kg human being and thus the concentration used can be considered to be 2566 to 77000 times safe for human consumption (suggested human dose range: 1 × 108 to 3 × 109 CFUs) (Nithya et al. 2012)

Return to footnote Appendix 11 Table A54 e referrer

Footnote Appendix 11 Table A54 f

NOAEL, no observed adverse effect level

Return to footnote Appendix 11 Table A54 f referrer

Footnote Appendix 11 Table A54 g

B. subtilis strain GB03, (U.S. EPA, 1993)

Return to footnote Appendix 11 Table A54 g referrer

Table A-55: Pathogenicity, toxicity and irritation testing results for a mixture containing two strains of B. licheniformis and B. subtilisFootnote Appendix 11 Table A55 [a]
Study TypeTarget organismDose concentrationOutcome
Acute toxicityBALB/c mice5 × 107 to 2 × 1011 CFU/mouse (oral administration)No changes in tissues, organs or weight
Acute toxicityBALB/c mice5 × 107 to 5 × 109 CFU/mouse (IPFootnote Appendix 11 Table A55 [b] or IVFootnote Appendix 11 Table A55 [c] administration)No changes in tissues, organs or weight
Chronic toxicityMice1 × 106 CFU/dayNo effect on health status
Chronic toxicityRabbits1 × 109 CFU/dayNo effect on health status
Chronic toxicityPiglets1 × 109 CFU/dayNo effect on health status
Footnote Appendix 11 Table A55 a

B. licheniformis strain VKPM B2336 and B. subtilis strain VKPM B2335, (Sorokulova et al. 2008)

Return to footnote Appendix 11 Table A55 a referrer

Footnote Appendix 11 Table A55 b

IP, intraperitoneal

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Footnote Appendix 11 Table A55 c

IV, intravenous

Return to footnote Appendix 11 Table A55 c referrer

Table A-56: Pathogenicity, toxicity and irritation testing results for B. subtilis strain QST 713Footnote Appendix 11 Table A56 [a]
Study TypeTarget organismDose concentrationOutcome
Acute oral toxicity and infectivityCD rats1.13 × 108 CFU/animalNon-toxic and not infective
LD50 greater than 1.13 × 108 CFU/animal
Acute pulmonary toxicity and infectivityCD rats1.2 × 108 CFU/animalNon-toxic and not infective
LD50 greater than 1.2 × 108 CFU/animal
Intravenous InfectivityCD rats9.4 × 108 CFU/animalNon-infective
Acute dermal toxicityCD rats2 g/kg BW (2.3 × 1011 to 2.73 × 1011CFU/animalLow toxicity
LD50 greater than 2g/kg BW
Eye irritationRabbits0.1 ml (4.8 × 109 CFU/animal)Minimally irritating
Dermal irritationRabbits500 mg (2.4 × 1010 CFU/animal)Slightly irritating
Avian oral toxicityNorthern bobwhite quail (Colinus virginianus)1 × 108 CFU/g BW/day (5000 mg/kg BW/day)LD50 greater than 5000 mg/kg BW
Freshwater fishRainbow trout (Oncorhynchus mykiss)Max dose: 1.4 × 107 CFU/mLLC50: 1.4 × 107 CFU/mL
Freshwater aquatic invertebrate (48-hour)Daphnia magna5 × 105 CFU/mL, 1 × 106 CFU/mL, 2 × 106 CFU/mL and 4 × 106 CFU/mLMortality of 15, 15, 45
and 85% respectively and lethargy of surviving daphnids in the 100mg/L treatment
Freshwater aquatic invertebrate (21-day)Daphnia magna5 × 105 CFU/mL,
1 × 106 CFU/mL,
2 × 106 CFU/mL and 4 × 106 CFU/mL
LC50 ~ 3 × 105 CFU/mL
NOEC: 7.5 × 103CFU/mL
Freshwater aquatic invertebrate (21-day)Daphnia magna7.9 × 105 CFU/mL, 1.8 × 106 CFU/mL, 3.4 × 106 CFU/mL, 7.3 × 106 CFU/mL and 2.0 × 107 CFU/mLLC50 ~ 1.6 × 106 CFU/mL
NOEC: 7.9 × 105 CFU/mL.
Freshwater aquatic invertebrateGrass shrimp (Palaemonetes pugio)4.0 × 106 CFU/gLC50 greater than 4.0 × 106 CFU/mL
Aqueous plantSingle cell green alga (Scenedesmus subspicatus)Max dose: 5.1 × 105 CFU/mLNOEC greater than 100 mg/L
LOEC greater than 100 mg/L
Non-target insect study (oral/dietary)Honey Bee -Apil mellifera L.Max dose: 100,000 ppmLD50 greater than 100,000 ppm
Dietary toxicity/ pathogenicityHoney Bee -Apil mellifera L.600, 6,000 and 60,000 ppmLC50: 5663 ppm
Non-target insect study (oral/dietary)Green lacewing (Chrisoperla carnea)Max dose: 60000 ppmLC50 greater than 60,000 ppm
Non-target insect study (oral/dietary)Ladybird beetle -Hippodamia convergensMax dose: 1.2 × 109 CFU/mL (60000 ppm)LC50 greater than 60,000 ppm
NOEC: 60,000 ppm (1.2 × 109 CFU/g)
Toxicity and pathogenicity test (30 days)Parasitic Hymenoptera - Nasonia vitripenis600, 6,000 and 60,000 ppmLC50: 28,000 ppm (15 days)
Non-target insect study (oral/dietary)Parasitic Hymenoptera - Nasonia vitripenisMax dose: 3.2 × 109 CFU/mL (60000 ppm)LC50 ~ 24,739 ppm
Footnote Appendix 11 Table A56 a

Studies done with the technical grade active ingredient (Mendelsohn and Vaituzis, 1999; U.S. EPA, 2006; U.S. EPA, 2010; PMRA-HC, 2007b).

Return to footnote Appendix 11 Table A56 a referrer

Table A-57: Pathogenicity, toxicity and irritation testing results for B. subtilis strain MBI 600Footnote Appendix 11 Table A57 [a]
Study TypeTarget organismDose concentrationOutcome
Acute oral toxicity and infectivityCD Rats2.0 × 108 sporesLow toxicity, not infective
LD50 greater than 2 × 108 CFU
Acute pulmonary toxicity and infectivityCD Rats3.3 × 108 to 3.7 × 108 sporesToxic, not infective
LD50 greater than 3.5 × 108 CFU
IV infectivityCD Rats107 sporesNo significant signs of toxicity
Acute dermal toxicityNew Zealand White Rabbits2mL/kg body weightLow toxicity (slight oedema)
LD50 greater than 2mL/kg body weight
Eye irritationNew Zealand White Rabbits1.0 × 109 CFUMinimally irritating
Dermal irritationNew Zealand White Rabbits2.0 × 1010 CFUMinimally irritating
Acute Avian Oral Toxicity and PathogenicityBobwhite quails (Colinus virginianus)GUS 378 Concentrate: 4000 mg/kg BW/dayLow toxicity, not pathogenic
Acute Avian Oral Toxicity and PathogenicityBobwhite quails (Colinus virginianus)Water-soluble metabolites: 240 mg/kg BW/dayLow toxicity, not pathogenic
Acute Avian Oral Toxicity and PathogenicityBobwhite quails (Colinus virginianus)Washed spores: 3680 mg/kg
BW/day
Low toxicity, not pathogenic
Plant Toxicity and PathogenicitySoybean seeds105 to 107 viable spores/seedNot pathogenic
Freshwater Fish Toxicity and InfectivityCarp2.0 × 106 CFU/mL, 2.0 × 107 CFU/mL, 2.0 × 108 CFU/mLNo treatment-related toxicity or pathogenicity
Footnote Appendix 11 Table A57 a

Studies done with the technical grade active ingredient and not the end-use product containing the micro-organism, (PMRA-HC, 2007a)

Return to footnote Appendix 11 Table A57 a referrer

Table A-58: Pathogenicity and toxicity testing results for B. subtilis ATCC 6051A and ATCC 55405Footnote Appendix 11 Table A58 [a]
Study TypeTarget organismDose concentrationOutcome
Pathogenicity/toxicity testingRed fescue(Festuca rubra)105 CFU/g soil dry weightShoot length significantly affected (p = 0.03)Footnote Appendix 11 Table A58 [b]
Pathogenicity/toxicity testingSpringtail(Folsomia candida)103 CFUFootnote Appendix 11 Table A58[c]/g soil dry weight ;
104 CFUFootnote Appendix 11 Table A58[d]/g soil dry weight
  • Significant reduction (p less than 0.01) in juvenile production[c]
  • No juvenile production (statistical analysis could not be performed)[d]
  • Adult survival not affected by either strain
Pathogenicity/toxicity testingEarth worm(Eisenia andrei)105 CFU[c]/g soil dry weight;
104 CFU[d]/g soil dry weight
No adverse effects reported
Footnote Appendix 11 Table A58 a

Data generated by Environment Canada's Biological Methods Division

Return to footnote Appendix 11 Table A58 a referrer

Footnote Appendix 11 Table A58 b

The survival, growth and reproduction of test organisms were significantly inhibited in the field-collect soil relative to the artificial soil

Return to footnote Appendix 11 Table A58 b referrer

Footnote Appendix 11 Table A58 c

B. subtilis ATCC 55405

Return to footnote Appendix 11 Table A58 c referrer

Footnote Appendix 11 Table A58 d

B. subtilis ATCC 6051A

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Appendix 12: Virulence and pathogenicity testing of the DSL B. licheniformis/subtilis strains

Murine exposure data generated by Health Canada's Healthy Environments and Consumer Safety Branch. Female BALB/c mice were exposed to 106 CFU/25μL of bacteria (vegetative cells or spores) via an endotracheal nebulizer for pulmonary exposure. Animals were necropsied at 24 hours and 1 week for vegetative cells and spores exposures respectively.

Clearance following endotracheal exposure

Table A-59: Enumeration of vegetative cells (CFU/mg) of DSL B. licheniformis/subtilis group strains following endotracheal exposure
StrainFootnote Appendix 12 Table A59[a]LungTracheaEsophagus
B. amyloliquefaciens 13563-0468.5124.116.1
B. atrophaeus 18250-7270.796.645.0
B. licheniformis ATCC 12713323.131.414.6
B. subtilis subsp. inaquosorum ATCC 55406228.41.54.8
Bacillus species 16970-5249.217.630.2
Bacillus species 2 18118-1235.051.817.8
Bacillus species 4 18121-4310.747.344.3
Footnote Appendix 12 Table A59 a

No data available for B. subtilis ATCC 6051A, B. subtilis ATCC 55405, B. subtilis subsp. subtilis ATCC 6051T and Bacillusspecies 7 18129-3

Return to footnote Appendix 12 Table A59 a referrer

Figure A-1: Enumeration of vegetative cells (CFU/mg) of DSL B. licheniformis/subtilis group strains following endotracheal exposure

Figure A-1 (See long description below)

Long description of the figure A-1

Figure A-1: Enumeration of vegetative cells (CFU/mg) of DSL B. licheniformis/subtilis group strains following endotracheal exposure

Table A-60: Enumeration of spores (CFU/mg) of DSL B. licheniformis/subtilis group strains following endotracheal exposure
StrainFootnote Appendix 12 Table A60[a]LungTracheaEsophagus
B. amyloliquefaciens 13563-0132.229.520.7
B. atrophaeus 18250-754.77.02.6
B. licheniformis ATCC 127136.91.31.6
B. subtilis ATCC 6051A0.00.00.0
B. subtilis ATCC 5540552.185.120.0
B. subtilis subsp. subtilis ATCC 6051T7.00.00.0
B. subtilis subsp. inaquosorum ATCC 5540610.61.40.3
Bacillus species 2 18118-154.88.50.4
Bacillus species 4 18121-428.415.04.1
Bacillus species 7 18129-37.00.00.0
Footnote Appendix 12 Table A60 a

No data available for Bacillus species 16970-5

Return to footnote Appendix 12 Table A60 a referrer

Figure A-2: Enumeration of spores (CFU/mg) of DSL B. licheniformis/subtilis group strains following endotracheal exposure

Figure A-2 (See long description below)

Long description of the figure A-2

Figure A-2: Enumeration of spores (CFU/mg) of DSL B. licheniformis/subtilis group strains following endotracheal exposure

Pulmonary Cytokines

Table A-61: Pulmonary cytokine expression (pg/mL) from vegetative cell exposures
DSL StrainFootnote Appendix 12 Table A61[a]IL-1 betaIL-6MCP-1IL-12 (p70)KCTNF-alpha
Control (saline)73.58 ± 13.262.02 ± 0.611501.94 ± 288.1918.07 ± 2.267.32 ± 1.16728.57 ± 107.77
B. amyloliquefaciens 13563-0141.39 ± 60.311.46 ± 1.111597.70 ± 177.8713.05 ± 5.7762.46 ± 16.28575.77 ± 58.43
Bacillus atrophaeus 18250-71619.34 ± 564.474.20 ± 0.734446.72 ± 1536.1516.49 ±9.96452.83 ± 160.76718.82 ± 135.08
B. licheniformis ATCC 127131818.28 ± 573.735.80 ± 1.946032.81 ± 2094.6519.08 ± 7.44387.79 ± 146.88705.84 ± 228.92
B. subtilis subsp. inaquosorum ATCC 55406262.22 ± 44.631.10 ± 0.491624.45 ± 242.7812.23 ± 4.6534.61 ± 19.19591.58 ± 87.85
Bacillus species 16970-5101.13 ± 5.941.10 ± 0.531651.50 ± 319.1116.38 ± 5.7826.29 ± 18.31667.01 ± 133.49
Bacillus species 2 18118-11444.876 ± 778.684.53 ± 2.245554.10 ± 2162.6413.95 ± 2.00660.90 ± 74.56647.20 ± 205.74
Bacillus species 4 18121-47545.602 ± 1988.0153.43 ± 21.7020278.06 ± 7401.5424.40 ± 7.212082.50 ± 501.70983.90 ± 172.32
Footnote Appendix 12 Table A61 a

No data available for B. subtilis ATCC 6051A, B. subtilis ATCC 55405, B. subtilis subsp. subtilis ATCC 6051and Bacillus species 7 18129-3

Return to footnote Appendix 12 Table A61 a referrer

Table A-62: Pulmonary cytokine expression (pg/mL) from spore exposures
DSL StrainFootnote Appendix 12 Table A62[a]IL-betaIL-6MCP-1IL-12 (p70)KCTNF-alpha
Control (saline)90.86 ± 13.652.29 ± 1.27837.12 ± 147.2012.62 ± 8.059.13 ± 2.46484.79 ± 160.58
B. amyloliquefaciens 13563-098.07 ± 11.212.87 ± 1.75 921.78 ± 187.969.82 ± 7.6211.89 ± 4.39476.05 ± 167.15
Bacillus atrophaeus 18250-7120.36 ± 52.912.53 ± 1.60750.55 ± 146.43 14.23 ± 8.829.48 ± 2.70521.90 ± 110.22
Bacillus species 2 18118-1104.37 ± 9.542.13 ± 1.78843.20 ± 101.7113.86 ± 6.7310.0 ± 3.74526.06 ± 115.86
Bacillus species 4 18121-490.31 ± 19.543.06 ± 1.47884.30 ± 105.0517.10 ± 6.727.17 ± 0.74374.76 ± 91.07
Footnote Appendix 12 Table A62 a

No data available for B. licheniformisATCC 12713, B. subtilis ATCC 6051A, B. subtilisATCC 55405, B. subtilis subsp. inaquosorum ATCC 55406, Bacillus species 16970-5 and Bacillusspecies 7 18129-3

Return to footnote Appendix 12 Table A62 a referrer

Acute phase response

Table A-63: Serum Amyloid A (SAA)Footnote Appendix 12 Table A63 [a] Levels (µg/mL) in serum samples obtained from BALB/c mice treated with vegetative cells or spores of DSL strains
StrainFootnote Appendix 12 Table A63[b]Vegetative cellsSpores
Control (saline)13.80 ± 3.5214.08 ± 0.63
B. amyloliquefaciens 13563-017.87 ± 2.7316.60 ± 2.09
Bacillus atrophaeus 18250-720.73 ± 1.2121.56 ± 14.0
B. licheniformis ATCC 1271339.54 ± 28.54No data
B. subtilis subsp. inaquosorum ATCC 5540617.76 ± 4.34No data
Bacillus species 16970-513.72 ± 2.6613.88 ± 3.10
Bacillus species 2 18118-123.35 ± 3.2916.04 ± 2.38
Bacillus species 4 18121-417.68 ± 7.89No data
Footnote Appendix 12 Table A63 a

Serum amyloid A, an indicator of systemic effects, was measured using ELISA

Return to footnote Appendix 12 Table A63 a referrer

Footnote Appendix 12 Table A63 b

No data available for B. subtilis ATCC 6051A, B. subtilis ATCC 55405, B. subtilis subsp. subtilis ATCC 6051 and Bacillus species 7 18129-3

Return to footnote Appendix 12 Table A63 b referrer

Figure A-3: Serum Amyloid A (SAA) Levels (µg/mL) in serum samples obtained from BALB/c mice treated with vegetative cells of DSL strains

Figure a-3 (See long description below)

Long description of the figure A-3

Figure A-3: Serum Amyloid A (SAA) Levels (µg/mL) in serum samples obtained from BALB/c mice treated with vegetative cells of DSL strains

Figure A-4: Serum Amyloid A (SAA) Levels (µg/mL) in serum samples obtained from BALB/c mice treated with spores of DSL strains

Figure A-4 (See long description below)

Long description of the figure A-4

Figure A-4: Serum Amyloid A (SAA) Levels (µg/mL) in serum samples obtained from BALB/c mice treated with spores of DSL strains

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Appendix 13: Food poisoning outbreaks

Table A-64: Food poisoning outbreaks involving B. licheniformis
PlaceYear# of CasesFootnote Appendix 13 Table A64[a]SymptomsCauseDeath(s)
Veterans Administration Hospital, Denver, COFootnote Appendix 13 Table A64 [b]1959161Gastroenteritis including abdominal cramps, diarrhoea and vomitingCooked turkey meat that was held at room temperature overnight1
AustraliaFootnote Appendix 13 Table A64[c]197649Abdominal pain, diarrhoea and vomitingMeals-on-wheels co-contaminated with Clostridium perfringens and B. cereus1
Prison in Ohio, USAFootnote Appendix 13 Table A64[d]1995165No dataTurkey and gravy were implicatedNo data
Kindergarten in Split, CroatiaFootnote Appendix 13 Table A64 [e]200012Nausea, headache and vomitingContaminated milk powder that was prepared two hours prior to consumption and not boiled. Co-contamination with B. subtilis0
Footnote Appendix 13 Table A64 a

Case refers to an individual person diagnosed with food poisoning

Return to footnote Appendix 13 Table A64 a referrer

Footnote Appendix 13 Table A64 b

(Tong et al. 1962), though the authors implicate B. subtilis as the causative agent, the food poisoning was likely caused by B. licheniformis as the onset and symptoms are more in line with the description by Lund (1990) and biochemical testing results appear to be closer to B. licheniformis (e.g. growth in salt and anaerobic growth).

Return to footnote Appendix 13 Table A64 b referrer

Footnote Appendix 13 Table A64 c

(Jephcott et al. 1977)

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Footnote Appendix 13 Table A64 d

(CDC 1995)

Return to footnote Appendix 13 Table A64 d referrer

Footnote Appendix 13 Table A64 e

(Pavic et al. 2005), contamination of food as the result of toxin-producing isolated of B. licheniformisand B. subtilis was proven via vacuolation assay and MTT cell culture test.

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