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 6051
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

Table of Tables

Table of Figures

Synopsis

Pursuant to paragraph 74(b) of the Canadian Environmental Protection Act, 1999 (CEPA 1999), the Minister of the Environment and the Minister of Health have conducted a screening assessment of the following living organism strains that are listed on the DSL:

Bacillus amyloliquefaciens 13563-0
Bacillus atrophaeus 18250-7
Bacillus licheniformis ATCC^{Footnote [1]} 12713
Bacillus subtilis ATCC 6051A (also referred to as Bacillus subtilis ATCC 6051a)
Bacillus subtilis ATCC 55405
Bacillus subtilis subspecies subtilis ATCC 6051 (= type strain)
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

For the purposes of this assessment, the DSL micro-organisms listed above will collectively be referred to as the ‘DSL Bacillus licheniformis/subtilis group’ (B. licheniformis/subtilis group). The term ‘Bacillus subtilis complex’ will denote information that is not specific to these DSL strains, but relates to the broader group of species that includes the DSL strains.

Under the Masked Name Regulations pursuant to section 113 of CEPA 1999, Environment Canada assigned masked names and accession numbers to Bacillus species 16970-5, Bacillus species 2 18118-1, Bacillus species 4 18121-4 and Bacillus species 7 18129-3 in place of these organisms’ explicit biological names, which are considered confidential and must not be publicly disclosed.

Members of the broader Bacillus Subtilis (B. subtilis) complex have the ability to adapt to and thrive in many terrestrial and aquatic habitats. They may be contaminants in food and aviation fuel and transient members of the bowel microflora. Some members of the B. subtilis complex are used in the fermentation of foods. They form endospores that permit survival in sub-optimal environmental conditions. Numerous physiological variants exist in nature, indicating that members of this complex establish successfully in nearly every environment. Various characteristics of the DSL B. licheniformis/subtilis group make them suitable for use as active ingredients in commercial and consumer products.

Certain strains of Bacillus licheniformis (B. licheniformis) can cause bovine, porcine and ovine abortion as well as mastitis in cattle, but the overall impact of B. licheniformis disease in livestock is low. Members of the DSL B. licheniformis/subtilis group are susceptible to veterinary antibiotics so that in the case of livestock infection, effective treatment options are available. Negative effects in aquatic and terrestrial invertebrates exposed to strains of B. subtilis and B. licheniformis have been reported. One report implicated an isolate of B. licheniformis as the causative agent of pistachio dieback. B. subtilis complex strains also have antimicrobial properties, and can promote growth in both plants and animals.

Certain members of the B. subtilis complex are occasionally reported to cause disease in susceptible humans, including those with debilitating disease or compromised immunity, young infants and the elderly, but do so rarely in the general population. Some produce extracellular enzymes and toxins that could cause food poisoning. In laboratory analyses done by scientists at Health Canada, the DSL B. licheniformis/subtilis group strains did not produce these food poisoning toxins.

This assessment considers the aforementioned characteristics of these strains with respect to environmental and human health effects associated with product use and industrial processes subject to CEPA 1999, including releases to the environment through waste streams and incidental human exposure through environmental media. To update information about current uses, the Government launched a mandatory information-gathering survey under section 71 of CEPA 1999, as published in the Canada Gazette, Part I, on October 3, 2009 (section 71 notice). Information submitted in response to the section 71 notice indicates that the DSL B. licheniformis/subtilis group strains were used in biodegradation and bioremediation; products for surface and drain cleaning, degreasing and deodorizing; enzyme and chemical production; waste and wastewater treatment.

Considering all available lines of evidence presented in the Screening Assessment, there is low risk of harm to organisms and the broader integrity of the environment fromthe DSL B. licheniformis/subtilis group strains. It is concluded that Bacillus amyloliquefaciens 13563-0, Bacillus atrophaeus 18250-7, Bacillus licheniformis ATCC 12713, Bacillus subtilis ATCC 6051A, Bacillus subtilis ATCC 55405, Bacillus subtilis subsp. subtilis ATCC 6051, Bacillus subtilis subsp. inaquosorum ATCC 55406, Bacillus species 16970-5, Bacillus species 2 18118-1, Bacillus species 4 18121-4 or Bacillus species 7 18129-3 do not meet the criteria under paragraphs 64(a) or (b) of CEPA 1999, as they are not entering the environment in a quantity or concentration or under conditions that have or may have an immediate or long-term harmful effect on the environment or its biological diversity or that constitute or may constitute a danger to the environment on which life depends.

Also, based on the information presented in the Screening Assessment, it is concluded that Bacillus amyloliquefaciens 13563-0, Bacillus atrophaeus 18250-7, Bacillus licheniformis ATCC 12713, Bacillus subtilis ATCC 6051A, Bacillus subtilis ATCC 55405, Bacillus subtilis subsp. subtilis ATCC 6051, Bacillus subtilis subsp. inaquosorum ATCC 55406, Bacillus species 16970-5, Bacillus species 2 18118-1, Bacillus species 4 18121-4 or Bacillus species 7 18129-3 do not meet the criteria under paragraph 64(c) of CEPA 1999, as they are not entering the environment in a quantity or concentration or under conditions that constitute or may constitute a danger in Canada to human life or health.

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Introduction

Pursuant to paragraph 74(b) of the Canadian Environmental Protection Act, 1999 (CEPA 1999), the Minister of the Environment and the Minister of Health are required to conduct screening assessments of living organisms listed on the (DSL) that were in commerce between 1984 and 1986, to determine whether they present or may present a risk to the environment or human health (according to criteria as set out in section 64 of CEPA 1999).^{Footnote [2]}These strains were added to the DSL under section 10⁵(1) of CEPA 1999 because they were manufactured in or imported into Canada between January 1, 1984, and December 31, 1986 and they entered or were released into the environment without being subject to conditions under CEPA 1999 or any other federal or provincial legislation.

This screening assessment considers hazard information obtained from the public domain as well as from unpublished research data and comments from researchers in related fields. Exposure information was obtained from the public domain and from a mandatory CEPA 1999 section 71 Notice published in the Canada Gazette, Part I, on October 3, 2009. Further details on the risk assessment methodology used are available in the Risk Assessment Framework document "Framework on the Science-Based Risk Assessment of Micro-organisms under the Canadian Environmental Protection Act, 1999" (Environment Canada and Health Canada 2011).

In this report, data that are specific to the DSL Bacillus licheniformis/subtilis group strains are identified as such and includes information from the Nominators, the American Type Culture Collection (ATCC), and unpublished data generated by Environment Canada^{Footnote [3]} and Health Canada^{Footnote [4]}research scientists. Where strain-specific data were not available, surrogate information from literature searches was used. When applicable, literature searches conducted on the organism included its synonyms, and common and superseded names. Surrogate organisms are identified in each case to the taxonomic level provided by the source. Literature searches were conducted using scientific literature databases (SCOPUS, CAB Abstracts and Google Scholar), web searches, and key search terms for the identification of human health and environmental hazards of each of the DSL strains assessed in this report. Information identified as of May 2014 was considered for inclusion in this report.

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Decisions from Domestic and International Jurisdictions

Domestic

The members of the DSL B. licheniformis/subtilis group are recognized as Risk Group 1 micro-organisms by the Public Health Agency of Canada (PHAC) and by the Canadian Food Inspection Agency (CFIA).

Strains of B. subtilis have been approved in Canada for the production of enzymes used in food. Fermentation extracts from strains of B. subtilis are accepted as a feed ingredient under the Feeds Regulations, as long as they are free from antimicrobial activity and are not a source of viable microbial cells. The DSL B. licheniformis/subtilis strains have not been approved for use on the Canadian market under this Act at this time. The CFIA Fertilizer Safety Office conducted a comprehensive safety assessment of B. subtilis and exempted all strains from full safety data requirements (CFIA 2014). Strains isolated from the natural environment must be identified and distinguished the isolate to the strain level.

The Pest Management Regulatory Agency of Health Canada (PMRA-HC) has approved several other strains of the B. subtiliscomplex for use as biocontrol agents including B. subtilisvar. amyloliquefaciens strain FZB24 (2011) (PMRA-HC 2012), B. subtilis strain MBI 600 (2005) (PMRA-HC 2007a; PMRA-HC 2007c), B. subtilis strain QST 713 (2006) (PMRA-HC 2007b) and B. subtilis strain GB03 (2011) (PMRA-HC 2013). An evaluation for each microbial pest control agent and end-use product determined that they did not present an unacceptable risk to human health or the environment.

International

The United States Environmental Protection Agency (U.S. EPA) assessed several strains of B. subtilis and B. licheniformis used in the production of enzymes. It was concluded that no unreasonable risks to human health or the environment were associated with the use of these strains for the production of enzymes, antibiotics or other specialty chemicals. The Unites States Food and Drug Administration (U.S. FDA) recognizes enzymes produced by B. subtilis to be generally recognized as safe (GRAS) for use in food. The U.S. EPA approved many of the same biocontrol agents registered by PMRA-HC. In addition to the strains approved for use as biofungicides in Canada, the U.S. EPA has also approved B. amyloliquefaciens strain D747 (U.S. EPA 2011) and B. licheniformis strain SB3086 (U.S. EPA 2001) and B. a.

In Australia, modified B. amyloliquefaciens, B. licheniformis and B. subtilis were approved for use in enzyme production (ANZ 2012a; ANZ 2012b; ANZ 2013). B. subtilis PB6 has been applied in poultry to reduce clostridial isolates (C. difficile and C. perfringens) (ANZ 2011). Other strains are being considered for biocontrol purposes.

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1. Hazard Assessment

1.1 Characterisation of the DSL strains under assessment

1.1.1 Taxonomy, identification and strain history

Taxonomic designation:

Kingdom: Bacteria

Phylum: Firmicutes

Class: Bacilli

Order: Bacillales

Family: Bacillaceae

Genus: Bacillus

Species: Bacillus amyloliquefaciens13563-0

Bacillus atrophaeus 18250-7

Bacillus licheniformis ATCC 12713

Bacillus subtilis ATCC 6051A

Bacillus subtilis ATCC 55405

Bacillus subtilis subsp. subtilis ATCC 6051 (=type strain)

Bacillus subtilis subsp. inaquosorum ATCC 55406

Bacillus species 16970-5

Bacillus species 2 18118-1

Bacillus species 4 18121-4

Bacillus species 7 18129-3

Eleven strains of the 'Bacillus subtilis species complex' that are listed on the DSL are the subject of this assessment. They will be assessed collectively as the 'DSL Bacillus licheniformis/subtilis group' in this report. The term 'Bacillus subtilis complex' and the grouping of these species are supported in the literature and include the DSL strains (Sorokulova et al. 2008; De Jonghe et al. 2010). This term will be used when surrogate information is discussed. As indicated above, the names of several of the DSL B. licheniformis/subtilisgroup strains have been masked to the genus level at the request of the nominators, pursuant to the Masked Name Regulations of CEPA 1999, and may not be disclosed.

Synonyms for species of the DSL B. licheniformis/subtilis group were obtained from the 'List of Prokaryotic Names with Standing in the Nomenclature' (Euzéby 2013), the 'NCBI taxonomy browser' (Benson et al. 2009; Sayers et al. 2009) and the 'Catalogue of Life' (Shimura et al. 2013) unless otherwise indicated (Table 1-1).

**Table 1 -1: Synonyms of micro-organisms in the *B. subtilis* complex**
Current Nomenclature	Synonyms
B. amyloliquefaciens subspecies amyloliquefaciens^{Footnote Table 1-1[a]} subspecies plantarum^[a]	Bacillus amyloliquifaciens^{Footnote Table 1-1[b]} Bacillus subtilis var. amyloliquefaciens^{Footnote Table 1-1[c]} Bacillus velezensis^{Footnote Table 1-1 [d]}
B. atrophaeus	Bacillus subtilis var. niger Bacillus globigii Bacillus niger
B. licheniformis	Denitrobacillus licheniformis Clostridium licheniforme
B. subtilis subsecies inaquosorum^{Footnote Table 1-1[e]} subspecies spizizenii subspecies subtilis subspecies virginiana^{Footnote Table 1-1[f]}	Vibrio subtilis Bacillus globigii Bacillus uniflagellatus Bacillus natto

Footnote Table 1-1 a

(Borris et al. 2011)

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Footnote Table 1-1 b

(Priest et al. 1987)

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Footnote Table 1-1 c

(PMRA-HC, 2012)

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Footnote Table 1-1 d

Later heterotypic synonym of B. amyloliquefaciens (Wang et al. 2008)

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Footnote Table 1-1 e

Some strains were reclassified from B. licheniformis (Rooney et al. 2009)

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Footnote Table 1-1 f

(Zhao et al. 2011)

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1.1.1.1 Phenotypic identification and biochemical profile

Bacillus species are Gram positive but stain variably, with some species staining clearly Gram positive in young cultures only. They have rod-shaped cells with rounded or squared ends ranging from 0.5 × 1.2 to 2.5 × 10 μm in size, occurring singly or in chains, and the stability of these chains determines the form of the colony, which may vary from strain to strain (Logan and De Vos 2009; Rooney et al. 2009). While most species within the genus are aerobic some are facultatively or strictly anaerobic (Logan and De Vos 2009; Murray et al. 1995). Bacillus species are capable of forming spores that may be cylindrical, oval, round, or kidney-shaped, placed centrally, terminally or subterminally, none of which swell the sporangium (Murray et al. 1995).

Members of the B. subtilis complex can be differentiated from known human and animal pathogens of the B. cereus group (B. anthracis, B. cereus and the insect pathogen B. thuringiensis) by both morphological and biochemical means. Members of the B. subtilis complex have cell diameters which measure less than 1μm whereas members of the B. cereus group have cell diameters which are greater than 1μm (Logan and De Vos 2009). Biochemical profiles can be used to differentiate between members of the B. subtilis complex and the B. cereusgroup; select distinguishing features are provided in Table 1-2 (Santini et al. 1995).

**Table 1-2: Biochemical characteristics of *B. cereus* group species compared with *B. subtilis*complex species**
Bacillus species^{Footnote Table 1-2 [a]}	D-xylose	Mannose	Inositol	Mannitol	ONPG^{Footnote Table 1-2 [b]}
B. anthracis	-^{Footnote Table 1-2 [c]}	-	-	-	-
B. cereus	-	-	-	-	-
B. thuringiensis	-^{Footnote Table 1-2 [d]}	-^{Footnote Table 1-2 [e]}	N/A^{Footnote Table 1-2 [f]}	-^[d]	N/A
B. amyloliquefaciens	N/A^{Footnote Table 1-2 [g]}	+^{Footnote Table 1-2 [h]}	N/A	+^[g]	N/A
B. atrophaeus	+^[h]	+^[h]	N/A	+^[h]	N/A
B. licheniformis	+^{Footnote Table 1-2 [i]}	+	+	+	+
B. subtilis	+^{Footnote Table 1-2 [j]}	+	+	+	+

Footnote Table 1-2 a

(Santini et al. 1995)

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Footnote Table 1-2 b

o-nitrophenyl-β-D-galactopyranoside

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Footnote Table 1-2 c

-, less than or equal to 19% positive reactions

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Footnote Table 1-2 d

(Fakruddin et al. 2012)

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Footnote Table 1-2 e

(Swiecicka et al. 2002)

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Footnote Table 1-2 f

N/A, Not Available

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Footnote Table 1-2 g

(Borriss et al. 2011)

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Footnote Table 1-2 h

(Nakamura, 1989)

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Footnote Table 1-2 i

+, greater than or equal to 81% positive reactions

Return to footnote Table 1-2 i referrer

Footnote Table 1-2 j

20-80% positive reactions

Return to footnote Table 1-2 j referrer

1.1.1.2 Molecular identification

The genus Bacillus is large, consisting of 11 phylogenetic subclusters and over 140 species (Logan and De Vos 2009). Using 16S Ribosomal ribonucleic acid (rRNA) gene sequencing analysis, the B. subtilis complex can be differentiated from the B. cereus group due to the presence of a Hinfl restriction site between the V4 and V5 region in the B. subtilis complex (Jeyaram et al. 2011). Figure 1-1 describes the phylogenetic relationships of Bacillusspecies and closely related genera based on the alignment of 16S ribosomal RNA gene sequences generated by Health Canada scientists and publicly available sequences. This figure clearly demonstrates that species of the B. subtilis complex cluster together and apart from known pathogens of the Bacillus genus, particularly those of the B. cereus group.

The identity of the DSL B. licheniformis/subtilis group strains was independently verified by Health Canada scientists. Colony morphologies (Appendix 1) were consistent with descriptions in the literature. For example, B. atrophaeus, unlike other group members, forms a black pigment when grown on media containing tyrosine or other organic nitrogen sources (Logan and De Vos 2009; Rooney et al. 2009). The ability of B. atrophaeus strain 18250-7 to produce dark pigments was confirmed.

At Health Canada laboratories, the identification of most DSL B. licheniformis/subtilis group strains, including those that are masked at the genus level, was confirmed by 16S ribosomal RNA gene sequence, fatty acid methyl ester (FAME) analyses and total cellular content of select fatty acids to be correctly identified (Appendices 2 to 4). B. subtilis is difficult to distinguish from closely related Bacillusspecies, particularly B. amyloliquefaciens (Ash et al. 1991; Logan and De Vos 2009); however, B. amyloliquefaciens carries distinctive differences in the 16S ribosomal RNA gene sequence: the absence of two RsaI restriction sites in the V3 region that differentiates it from B. subtilis (Jeyaram et al. 2011). The lack of RsaI sites is characteristic of B. amyloliquefaciens and was observed in the ribosomal RNA gene sequence of B. subtilis ATCC 55405. Other methods used also demonstrated that B. subtilis ATCC 55405 is more similar to B. amyloliquefaciens than B. subtilis, suggesting that it may be misidentified. The Cfolrestriction site, between the V4 and V5 regions, can be used to differentiate between B. subtilis and B. licheniformis (Jeyaram et al. 2011).

Figure 1-1 : Phylogenetic relationships of Bacillaceae species based on the alignment of the 16S ribosomal RNA gene sequence coding region

Figure 1-1 (See long description below)

Long description of the figure 1-1

Figure 1-1: Phylogenetic relationships of Bacillaceae species based on the alignment of the 16S ribosomal RNA gene sequence coding region

Phylogenetic tree generated by Health Canada’s Healthy Environments and Consumer Safety Branch based on publicly available 16S ribosomal RNA gene sequences and 16S ribosomal RNA gene sequences of DSL B. licheniformis/subtilis strains generated in-house. Phylogenetic relationships of Bacillus species are based on the alignment of full length 16S rRNA gene sequences. Sequences for representative type strains were obtained from the Ribosomal Database project (RDP build 11 rdp.cme.msu.edu/) with the exception of DSL organisms whose sequences were generated in house by Health Canada’s Environmental Health Science and Research Bureau. Multiple alignments were performed using MUSCLE v3.7 and corrected for poorly aligned regions using GBLOCKS. Phylogeny was generated using PhyML 3.0 using the HKY85 substitution model and branch support calculated using an approximate likelihood-ratio Shimodaira-Hasegawa-like test (aLRT-SH) (Anisimova et al., 2006). This pipeline is available at www.phylogeny.fr/ (Depreeper et al., 2008).

B. subtilis subsp. inaquosorum and B. licheniformis both have properties that distinguish them from other B. subtilis complex members, including a lower salt tolerance, anaerobic growth and the production of toxic compounds in some strains (Salkinoja-Salonen et al. 1999). B. subtilis subsp. inaquosorum is distinguished from B. licheniformis, other subspecies of B. subtilisand other members of the B. subtilis complex by the production of a novel surfactin-like lipopeptide demonstrated by an additional major ion (mass m/z 1120.8) in its matrix-assisted laser desorption/ionization-time-of-light mass spectrometer profile, as well as differences in the total cellular content of fatty acids (Rooney et al. 2009) (Appendix 4). Recent genomic sequencing of strains of B. subtilis subsp. inaquosorum supports its taxonomic status as an independent subspecies of B. subtilis (Yi et al. 2014). For the purposes of this report, information relating to B. subtilis subsp. inaquosorum ATCC 55406 will be grouped with information on the DSL B. subtilisstrains.

1.1.1.3 Strain history

The sites of isolation of most members of the DSL B. licheniformis/subtilis group are unknown. Certain members were isolated from soil (B. subtilis ATCC 55405, B. subtilis subsp. inaquosorum ATCC 55406 and Bacillus species 16970-5) and industrial settings (Bacillus species 2 181181-1).Various strains of the DSL B. licheniformis/subtilis group that are in the American Type Culture Collection (ATCC) are also recognised under other strain designations in culture collections around the world (Table 1-3). The type strain, B. subtilis subsp. subtilis ATCC 6051, has been deposited to many culture collections and is known as the Marburg strain (Table 1-4).

**Table 1-3 : Culture collections holding DSL *B. licheniformis/subtilis* group strains and alternative recognized strain designations**
Strain	Culture Collection	Other Strain Designation
B. licheniformis ATCC 12713	Agricultural Research Service Database Culture Collection/NRRL Collection	NRRL B-1001
B. licheniformis ATCC 12713	Prairie Regional Laboratory	PRL B479
B. subtilis ATCC 6051A	Not applicable	P31K6
B. subtilis ATCC 55405	Not applicable	300
B. subtilis subsp. inaquosorum ATCC 55406	National Collection of Industrial, Food and Marine Bacteria	NCIMB 14014

**Table 1-4 : Major culture collections holding the Marburg strain (type strain), *B. subtilis* subsp. *subtilis* ATCC 6051 and alternative strain designations**
Culture Collection	Other Strain Designations
Agricultural Research Service Database Culture Collection/NRRL Collection	NRRL B-4219, NRS 1315, NRS 744
American Type Culture Collection	ATCC 6051-U
Collection Française des Bactéries Phytopathogenes and Pasteur Institute Collection (France)	CFBP 4228, CIP 52.65
Deutsche Sammlung von Mikroogansimen und Zellkulturen (Germany)	DSM 10, IMET 10758
Institute for Fermentation, Osaka (collection transferred toNBRC) (Japan)	IFO 12210, IFO 13719, IFO 16412
Japan Collection of Micro-organisms	JCM 1465, IAM 12118
National Collection (United Kingdom)	NCFB 1769, NCIB 3610, NCTC 3610
Netherlands Culture Collection of Bacteria	NCCB 32009, NCCB 53016, NCCB 70064

1.1.2 Biological and ecological properties

1.1.2.1 Natural occurrence

B. subtilis complex members can adapt to and thrive in many environments. In general, Bacillus species have been isolated from a diversity of habitats, including terrestrial (soil and vegetation) (Logan and De Vos 2009; Murray et al. 1995; Thatoi et al. 2013) and aquatic environments (Rajarajan et al. 2013; Shakir et al. 2012; Shields et al. 2013; Smitha and Bhat, 2012). Bacillus species have also been isolated from animals and as a transient part of the human bowel flora (Kramer and Gilbert, 1989; Turnbull and Kramer 1985); as contaminants of raw and prepared foods (reviewed in Fangio et al. 2010; Hosoi et al. 2000; Inatsu et al. 2006; Kramer and Gilbert 1989; Ray et al. 2000; Turnbull et al. 2001); and aviation fuels (Rauch et al. 2006). The broad range of environments exploited by the genus reflects the wide physiological variation among Bacillus species (Murray et al. 1995).

Naturally-occurring cell densities of viable B. licheniformis, B. amyloliquefaciens and B. subtilis in indoor air and settled dust of schools and daycare centres (Table 1-5) and agricultural buildings (cow shed and piggery) (Table 1-6) have been reported (Andersson et al. 1999).

**Table 1-5 : Naturally-occurring cell densities of viable *B. amyloliquefaciens*, *B. licheniformis* and *B. subtilis* in schools and daycare centers**
Organism	Indoor air (CFU/m³)	Settled Dust (CFU/g)
B. amyloliquefaciens	No data	10-10²
B. licheniformis	10²	10³
B. subtilis	No data	No data

**Table 1-6 : Naturally-occurring cell densities of viable *B. amyloliquefaciens*, *B. licheniformis* and *B. subtilis* in agricultural settings (cow shed and piggery)**
Organism	Indoor air (CFU/m³)	Settled Dust (CFU/g)
B. amyloliquefaciens	No data	No data
B. licheniformis	10⁴-10⁷	10⁴-10⁶
B. subtilis	10⁴-10⁷	10⁴-10⁶

1.1.2.2 Survival and persistence in the environment

Bacillus species form spores that allow them to survive inhospitable conditions and this gives them a competitive advantage over non-spore forming species in variable environments (Grossman and Losick 1988; Kramer and Gilbert 1989). Spores are more resistant to heat, chemicals, radiation and desiccation than their vegetative counterparts (Brown 2000; Logan 2012). The physiology of Bacillus thuringiensis spores is similar to those of the B. subtilis complex making it an appropriate surrogate. Spores of B. thuringiensis are reported to persist at high levels in soil for at least 13 years (Hendriksen and Hansen 2002; Hendriksen and Carstensen 2013). Nevertheless, in general, introduced microbial populations gradually decline, regardless of the source of their original isolation, due to the hostility of biotic and abiotic conditions in the soil environment (Van Veen et al. 1997). Biotic factors include predation and antagonism; abiotic factors include adverse soil pH, temperature and moisture, and nutrient scarcity (Van Veen et al. 1997). High numbers of vegetative cells are unlikely to be maintained in water or soil due to competition from other microflora (Leung et al. 1995). Plant colonization and biofilm formation may also increase the resistance of the bacteria to unfavourable conditions (Sella et al. 2012).

Three studies were identified that investigate the persistence of the B. subtilis complex in soils. In one study, long-term persistence of B. subtilis ATCC 6051 and B. subtilis ATCC 13933 in agricultural soil was investigated (Xiang et al. 2010). DeoxyriboNucleic Acid (DNA) from B. subtilis ATCC 6051 and B. subtilis ATCC 13933 could be amplified from laboratory microcosms for 8 and 127 days respectively after inoculation with cell culture suspensions containing 10⁸ to 10¹⁰ CFU/mL of the test strains (Xiang et al. 2010). Using amplified fragment length polymorphisms to develop specific DNA markers for the strains being investigated combined with quantitative real-time PCR the fate of B. subtilis ATCC 6051^T and B. subtilisATCC 13933 extracted from soil can be quantitatively tracked and can be used to estimate the concentration of cells in the soil (Figure 1-2).

Figure 1-2 : Persistence of Bacillus subtilis ATCC 6051 and Bacillus subtilis 13933 in soil, based on qPCR analyses of extractable soil DNA

Figure 1-2 (See long description below)

Long description of the figure 1-2

Figure 1-2: Persistence of Bacillus subtilis ATCC 6051T and Bacillus subtilis 13933 in soil, based on qPCR analyses of extractable soil DNA

Xiang, S., Cook, M., Saucier, S., Gillespie, P., Socha, R., Scroggins, R., and Beaudette, L.A. (2010). Development of amplified fragment length polymorphism-derived functional strain-specific markers to assess the persistence of 10 bacterial strains in soil microcosms. Appl. Environ. Microbiol. 76, 7126-7135.
Between 10e8 to 10e10 CFU/mL of B. subtilis 6051 was inoculated into soil and could be detected for up to 8 days after inoculation.
Between 10e8 to 10e10 CFU/mL of B. subtilis 13933 was inoculated into soil and could be detected for up to 127 days after inoculation.

The very different detection limits between these two strains make comparison of their persistence difficult. Sporulation of vegetative cells and less efficient recovery of DNA from spores may have played a role in the observed decline. Recovery of DNA from spores depends on the spore type, concentration of spores and the environment.

In another study, a strain of B. subtilis was inoculated into field soils and the population was observed to decline rapidly before stabilising (van Elsas et al. 1986). The populations remained low and mainly as spores over the course of 120 days.

In a third study, the persistence of B. amyloliquefaciens 13563-0, B. licheniformis ATCC 12713, B. subtilis ATCC 6051A, B. subtilis ATCC 55405 and B. subtilis subsp. inaquosorum ATCC 55406 in soil was investigated (Providenti et al. 2009). The authors suggested that if 1 × 10⁶ CFU/g soil of the vegetative cells were initially released, the detectable concentration of bacteria would likely decrease to 1 × 10² CFU/g soil or less within one to six months.

On the basis of these three studies, concentrations of the Bacillus species under assessment applied to soil are expected to decrease several fold over time, but would be likely to persist at some lower concentration as spores.

1.1.2.3 Growth parameters

Growth temperature and pH ranges vary between members of the B. subtilis complex and may vary between strains (Table 1-7) (Logan and De Vos 2009; Rooney et al. 2009).

**Table 1-7 : Growth temperature and pH ranges of membersof the *B. subtilis* complex**
Species	Growth Temperature Range (°C)	Optimal Growth Temperature (°C)	pH Range^{Footnote Table 1-7 [a]}
B. amyloliquefaciens	15-50	30-40	5.5-8.5^{Footnote Table 1-7 [b]}
B. atrophaeus	5-55	28-30	5.3-5.7^{Footnote Table 1-7 [c]}
B. licheniformis	15-55	30-40	5.7-6.8^[b]
B. subtilis subsp. subtilis	5-55	28-30	5.5-8.5^[b]
B. subtilis subsp. inaquorsorum	15-55^{Footnote Table 1-7 [d]}	28-30^[d]	5.5-5.7^[d]

Footnote Table 1-7 a

pH in Voges-Proskauer broth

Return to footnote Table 1-7 a referrer

Footnote Table 1-7 b

(Logan and De Vos 2009)

Return to footnote Table 1-7 b referrer

Footnote Table 1-7 c

(Nakamura 1989)

Return to footnote Table 1-7 c referrer

Footnote Table 1-7 d

(Rooney et al. 2009)

Return to footnote Table 1-7 d referrer

B. licheniformis and B. subtilis subsp. inaquosorum are facultative anaerobes and some strains of B. subtilis have restricted growth under anaerobic conditions (Logan and De Vos 2009). The ability to grow in both aerobic and anaerobic conditions contributes to the success of these Bacillus species in colonizing a variety of niches. In BALB/c mice inoculated orally with high concentrations of B. subtilis spores the quantity of B. subtilis (spores and vegetative cells) excreted in the feces was higher than the initial inoculation concentration (Hoa et al. 2001). This increase suggests that spores may be able to persist and germinate in the gastrointestinal tract of mice despite the anaerobic environment (Hoa et al. 2001).

1.1.2.4 Biocontrol and growth promotion^Footnote[5]

Biocontrol

B. subtilis complex strains have characteristics which make them effective biocontrol agents. As an endophytic bacterium, B. licheniformis, colonises the same sites as certain plant pathogens and may be better suited than rhizosphere bacteria to outcompete or antagonise plant pathogens (Mekete et al. 2009). B. licheniformis ATCC 14580 has chitinase and chitobiase activity which may be useful against fungal pathogens (ATCC 2012^e). B. subtilis complex members are able to produce antibiotics and extracellular chitinolytic enzymes that may inhibit plant fungal pathogens (Cordero-Ramírez et al. 2013; reviewed in Hameeda et al. 2006; Jamalizadeh et al. 2008; Pérez-García et al. 2011; Toledo et al. 2011). Bacteriocins are antagonistic peptides that may kill or inhibit the growth of other bacteria. (He et al. 2006; Tagg et al. 1976). Bacteriocins produced by B. licheniformis strains exhibit a broad range of antagonistic activity against various Gram positive and fungal pathogens but not Gram negative organisms (He et al. 2006). Antimicrobial compounds, such a bacteriocins, produced by members of the B. subtilis complex could affect microbial populations in habitats such as soils, and the microbiomes of plants, animals and humans. Recently, B. atrophaeus CAB-1 was demonstrated to have antifungal activity, making it a potential biocontrol agent (Zhang et al. 2013).

Strains of B. amyloliquefaciens and B. subtilis have been approved for use as biocontrol agents against fungal disease in terrestrial plants in Canada since 2011 and 2005, respectively (PMRA-HC 2007a; PMRA-HC 2007b; PMRA-HC 2007c; PMRA-HC 2012; PMRA-HC 2013; PMRA-HC 2014). Strains of B. amyloliquefaciens, B. licheniformis and B. subtilis have been approved for use as biocontrol agents of fungal disease in terrestrial plants in the United States since 2000, 2007 and 1992, respectively (Mendelsohn and Vaituzis 1999; U.S. EPA 2001; U.S. EPA 2006; U.S. EPA 2010; U.S. EPA 2011; U.S. EPA 2012; U.S. EPA 2013b).

Growth promotion

Members of the B. subtilis complex may promote plant growth by fixing nitrogen, producing biofertilizers and phytohormones, enhancing root nodulation, controlling plant pathogens and through their interactions with other symbiotic bacteria and fungi. These functions may be related to plant colonization and biofilm formation (Beauregard et al. 2013; Chung et al. 2010; Weng et al. 2012). B. licheniformis, B. amyloliquefaciens and B. subtilis have been isolated from the inner tissues of healthy plants and may have roles in growth promotion and plant protection (Logan, 2012). B. amyloliquefaciens, B. atrophaeus and B. licheniformis have been described within the rhizosphere of mangrove forests where they solubilize phosphate, increasing nutrient availability to the plants (Thatoi et al. 2013).

B. licheniformis and B. subtilis produce a number of enzymes (e.g. protease, lipase and amylase) that can be applied in aiding the digestion of proteins from animal feed (Ahmadnia Motlagh et al. 2012; Link and Kovác 2006). As an alternative to prophylactic antibiotic treatment, B. licheniformis has been demonstrated to protect against pathogens in aquaculture (Vinoj et al. 2013) and has been used as a probiotic for weight gain or pathogen resistance in rainbow trout (Merrifield et al. 2010a; Merrifield et al. 2010b), pigs (Link and Kovác 2006) and chickens (Rahimi and Kahsksefidi 2006). The use of some of the B. subtilis complex strains as probiotics in animals and the addition of their enzymes to feeds have been reported to result in increased weight gain and improvement of health. Other studies have investigated the immune stimulating potential of probiotic strains to enhance resistance of animal hosts against pathogens (Huang et al. 2013; reviewed in Vinoj et al. 2013).

1.1.2.5 Gene transfer

B. subtilis is naturally competent for transformation, a phenomenon that is growth-stage specific and nutrient sensitive (Dubnau and Losick 2006; Veening et al. 2008). Genetic exchange by this mechanism seems to be biased towards closely related species since the transformation frequency decreases exponentially with DNA sequence divergence (Majewski and Cohan 1998; Roberts and Cohan 1993; Zawadzki et al. 1995). This is expected to limit the possibility of B. subtilis acquiring pathogenic traits from distant species.

B. subtilis has also been implicated in the conjugal transfer of plasmids; however, most B. subtilis-like bacteria do not contain endogenous plasmid DNA (Kreft and Hughes 1982; Meijer 1995; Meijer et al. 1998; Tanaka et al. 1977). Transposable elements and prophages were reported in the genome of B. licheniformis ATCC 14580 (the type strain) (Lapidus et al. 2002), including nine identical copies of the 1,285 base pair insertion sequence IS3Bli1 and prophage sequences NZP1 and NZP3 (Rey et al. 2004).The identified prophage sequences have not been characterized. B. subtilis can also transfer transposons and integrons (Auchtung et al. 2005; Celli and Trieu-Cuot 1998; Kimura et al. 2011; K_oehler and Thorne 1987; Marra and Scott 1999; Meijer et al. 1998), and especially those of the class of integrative and conjugative elements (ICE) such as ICEBs1 (Auchtung et al. 2005), which can be transferred from B. subtilis to other Bacillus or Listeria species under conditions of host cell distress or in the presence of a high concentration of cells lacking ICEBs1. ICEs encode for proteins required for conjugal transfer, resistance to antibiotics and metabolism of alternative carbon sources (Auchtung et al. 2005).

Mobile genetic elements for some strains of the B. subtilis complex are reported in Appendix 5. Genes associated with virulence in strains of the B. subtilis complex are reported in Appendix 6. It is unknown if the DSL strains carry genes conferring virulence factors or antimicrobial resistance on mobile elements. Given their capacity for horizontal gene transfer, they could theoretically acquire such genes, but this potential is no greater for the DSL strains than for strains that are naturally present in the environment given what has been reported in the current scientific literature.

1.1.2.6 Pathogenic and Toxigenic Characteristics

Spores

The ability to form spores is integral to the etiology of Bacillus food poisoning, which has been associated with certain strains of B. licheniformis and B. subtilis. Bacillus spores survive disinfection, irradiation and cooking (Baril et al. 2012; Logan 2012). All of the DSL strains under assessment are capable of forming spores. Spores of the B. subtilis complex are highly heat resistant, with temperatures between 94.9°C and 97.7°C for B. licheniformis and between 103.2°C and 108.0°C for B. subtilis required to inactivate 90% of spores within 10 minutes (André et al. 2013). Under favourable conditions, such as when food is held at temperatures between 10°C and 50°C, the spores can germinate and proliferate (Baril et al. 2012; Brown 2000), and this permits the accumulation of sufficient cell concentrations for foodborne illness to occur.

Determinants of infectivity

In order to be an effective bacterial pathogen, a micro-organism must be able to adhere to host cell surfaces, invade host tissues and evade host defences. In one study, certain B. licheniformis and B. subtilis isolates had some ability to adhere or invade (Hep-2 and Caco-2 cell lines) while others were completely incapable of adherence or invasion (Rowan et al. 2001).

Strong hemolytic activity (as well as lecithinase activity) may indicate the presence of cytotoxic phospholipases that may facilitate invasion and are associated with virulence (Rowan et al. 2001; Sorokulova et al. 2008). Isolates of B. amyloliquefaciens, B. licheniformis and B. subtilis exhibit varying levels of hemolysis. In one study, B. amyloliquefaciens demonstrated beta-hemolysis; B. licheniformis no hemolysis; and B. subtilis alpha, beta or no hemolysis, depending on the isolate (Cordero-Ramírez et al. 2013). However, analysis by Health Canada scientists on the DSL B. licheniformis/subtilis group strains indicated no strong hemolytic activity in any strain (Appendix 7).

Catalase activity can enable a micro-organism to protect itself from reactive oxygen-induced killing from immune cells potentially making it a more effective pathogen. Catalase activity was assessed for the DSL B. licheniformis/subtilis strains by Health Canada scientists; all strains tested positive for catalase activity (Appendix 8).

Secondary Metabolites

Members of the B. subtilis complex also produce an array of secondary metabolites. Surfactin (B. subtilis) and lichenysin (B. licheniformis) are amphiphilic lipopeptides (Li et al. 2010). Both are powerful surfactants and have antimicrobial and hemolytic properties. Although they differ by only two amino acids, the hemolytic activity of lichenysin is much higher than that of surfactin (15 μmol/L vs. 200 μmol/L required to achieve 100% hemolysis, respectively) (Li et al. 2010).

Amylosin was first detected in B. amyloliquefaciens(Logan, 2012; Mikkola et al. 2007). It is an ionophore that forms K+ and Na+ channels in host cell membranes causing toxic responses including complete cell death, with extensive lysis in exposed cell lines and inhibition of motility in a boar spermatozoa assay (Mikkola et al. 2007).

Toxins

Strains of B. subtilis, B. licheniformis and B. amyloliquefaciens have been reported to produce both heat-labile and heat-stable toxins (Appendix 9) (Beattie and Williams, 1999; reviewed in De Jonghe et al. 2010; Mikkola et al. 2007; Nieminen et al. 2007). Toxins produced include some that are similar to the B. cereus emetic toxin (cereulide) (Salkinoja-Salonen et al. 1999; Taylor et al. 2005), hemolysin BL (Hbl) enterotoxin (Lindsay et al. 2000; Rowan et al. 2001) and a non-hemolytic enterotoxin (Nhe). A non-emetic heat-stable cytotoxic component has also been reported in certain strains of B. subtilis and B. amyloliquefaciens (De Jonghe et al. 2010). Isolates of B. licheniformis have been reported to produce a non-proteinaceous heat-stable toxin, which damages cell membrane integrity, depletes cellular ATP and has beta-hemolytic activity (Salkinoja-Salonen et al. 1999).

The Hbl toxin complex and BceT diarrheal toxin genes were identified in B. licheniformis and B. subtilisclinical and food isolates (Rowan et al. 2001). The growth medium was reported to affect toxin production, with more strains producing Hbl if grown in infant milk formula than in brain-heart infusion (BHI) broth. Toxin production is not related to the source of the isolate (clinical or environmental) (Beattie and Williams 1999; Madslien et al. 2012).

In testing conducted in Health Canada laboratories, strains were cultured on BHI. Three different commercial assay kits were used: all strains were tested for the HblC subunit of the Hbl enterotoxin using a commercial RPLA kit (Oxoid) and six strains^{Footnote [6]}were tested for the NheA subunit of the Nhe enterotoxin using an ELISA assay (TECRA kit). The Duopath Cereus (Millipore) kit was also used to detect both Nhe and Hbl enterotoxin production in the DSL strains. None of the DSL strains produced these toxins.

Cell-free culture supernatants of some clinical and food isolates of B. licheniformis and B. subtilis that had been implicated in food poisoning had cytotoxic activity towards both human Caco-2 and HEp-2 epithelial cell lines (Rowan et al. 2001). The growth medium affected the cytotoxic potential, and heat or trypsin treatment of the culture supernatant reduced or eliminated cytotoxic activity, indicating that it was attributable to the proteinaceous fraction (Rowan et al. 2001). In another study, food poisoning isolates of B. licheniformis and B. subtilis from street vendor food were cytotoxic to McCoy cells (Mosupye et al. 2002). In addition, whereas B. cereus isolates lost cytotoxicity following heat-treatment, some B. licheniformis and B. subtilis isolates retained their cytotoxicity (Mosupye et al. 2002). A B. licheniformis strain isolated from raw milk that was associated with food-poisoning was also cytotoxic to McCoy cells (Lindsay et al. 2000).

In testing conducted by Health Canada scientists, the cytotoxicity of the DSL B. licheniformis/subtilis strains was assessed in two cell lines, J774A.1 (macrophage cells) and HT29 (human colonic epithelial cells), with and without gentamicin. The strains did not demonstrate strong cytotoxicity towards either cell line (Appendix 10).

1.1.2.7 Antibiotic Susceptibility Profile

Information in the scientific literature on antibiotic susceptibility in B. amyloliquefaciens and B. atrophaeus is scant, presumably because these have not been implicated in cases of infection.

Variable antibiotic susceptibility profiles have been reported as part of case reports of infection with B. licheniformisand B. subtilis (Table 1-8). B. licheniformissusceptibility to the beta-lactam antibiotics ampicillin, piperacillin and ticarcillin depends on the isolate (Banerjee et al. 1988; Castagnola et al. 1997). Some isolates have an inducible beta-lactamase that may be responsible for this variable susceptibility (Filée et al. 2002; Zhu et al. 1992). Similarly, B. licheniformis ATCC 12713 is resistant to erythromycin, whereas the type strain ATCC 14580 is susceptible and variations in bacitracin synthase gene sequences are postulated to determine erythromycin resistance (Ishihara et al. 2002). A case of B. subtilis endocarditis was successfully treated with cefazolin(Tuazon et al. 1979), but in a later study, isolates were reported to be cefazolin resistant (Banerjee et al. 1988).

**Table 1-8 : Antibiotic susceptibilities of *B. licheniformis* and *B. subtilis* reported in the scientific literature**
Antibiotic	B. licheniformis	B. subtilis	Reference
Amikacin	S^{Footnote Table 1-8 [a]}	S	(Banerjee et al. 1988; Sorokulova et al. 2008)
Aminoglycosides	S	N/A^{Footnote Table 1-8 [b]}	(Ozkocaman et al. 2006)
Amoxicilin	I^{Footnote Table 1-8 [c]}	I	(Sorokulova et al. 2008)
Amoxicillin clavulanic acid	S	N/A	(Lépine et al. 2009)
Ampicillin	V^{Footnote Table 1-8 [d]}	S	(Banerjee et al. 1988; Sorokulova et al. 2008)
Azlocillin	S	S	(Banerjee et al. 1988)
Bactrim	S	S	(Sorokulova et al. 2008)
Carapenem	S	N/A	(Ozkocaman et al. 2006)
Carbenicillin	S	S	(Sorokulova et al. 2008)
Ceftazimide	R^{Footnote Table 1-8 [e]}	R	(Banerjee et al. 1988)
Cefamandol	I	S	(Sorokulova et al. 2008
Cefatolin	S	N/A	(Lépine et al. 2009)
Cefazolin	S	V	(Banerjee et al. 1988; Sorokulova et al. 2008)
Cefepime	S	N/A	(Ozkocaman et al. 2006)
Cefotaxim	R	I	(Sorokulova et al. 2008)
Cefoxitin	R	I	(Sorokulova et al. 2008)
Ceftriaxon	R	I	(Sorokulova et al. 2008)
Cephalotin	S	S	(Sorokulova et al. 2008)
Chloramphenicol	R	V	(Banerjee et al. 1988; Sorokulova et al. 2008)
Ciprofloxacin	S	S	(Castagnola et al. 1997; Sorokulova et al. 2008)
Clindamycin	R	S	(Banerjee et al. 1988; Sorokulova et al. 2008)
Cotrimoxazole	S	N/A	(Castagnola et al. 1997)
Doxycycline	S	N/A	(Lépine et al. 2009)
Enrofloxacin	S	S	(Sorokulova et al. 2008)
Erythromycin	V	S	(Ishihara et al. 2002; Sorokulova et al. 2008)
Gentamicin	S	S	(Banerjee et al. 1988; Sorokulova et al. 2008)
Imipenem	S	S	(Banerjee et al. 1988; Sorokulova et al. 2008)
Kanamycin	S	S	(Sorokulova et al. 2008)
Linezolid	S	S	(Sorokulova et al. 2008)
Meropenem	S	N/A	(Mochiduki et al. 2007)
Methicillin	R	I	(Sorokulova et al. 2008)
Mezlocillin	I	S	(Sorokulova et al. 2008)
Nafcillin	S	N/A	(Blue et al. 1995)
Neomycin	S	S	(Sorokulova et al. 2008)
Netilmicin	S	N/A	(Castagnola et al. 1997)
Nitrofurantoin	S	S	(Sorokulova et al. 2008)
Norfloxacin	S	S	(Banerjee et al. 1988; Sorokulova et al. 2008)
Ofloaxcin	S	N/A	(Lépine et al. 2009)
Oxacillin	R	R	(Castagnola et al. 1997; Sorokulova et al. 2008)
Penicillin	R	R	(Banerjee et al. 1988)
Piperacillin	V	S	(Banerjee et al. 1988)
Quinupristin + dalfopristin	N/A	N/A	(Sorokulova et al. 2008)
Rifampicin	S	S	(Sorokulova et al. 2008)
Streptomycin	S	S	(Sorokulova et al. 2008)
Telcoplanin	S	N/A	(Castagnola et al. 1997)
Tetracycline	S	S	(Sorokulova et al. 2008)
Ticarcillin	V	S	(Banerjee et al. 1988)
Tobramycin	S	S	(Castagnola et al. 1997; Sorokulova et al. 2008)
Trimethoprim	S	S	(Sorokulova et al. 2008)
Vancomycin	S	S	(Banerjee et al. 1988; Sorokulova et al. 2008)

Footnote Table 1-8 a

S, susceptible, also includes successful treatments where no other antibiotics were used

Return to footnote Table 1-8 a referrer

Footnote Table 1-8 b

N/A, not available

Return to footnote Table 1-8 b referrer

Footnote Table 1-8 c

I, intermediate

Return to footnote Table 1-8 c referrer

Footnote Table 1-8 d

V, variable (different sources gave different resistance results)

Return to footnote Table 1-8 d referrer

Footnote Table 1-8 e

R, resistant

Return to footnote Table 1-8 e referrer

Vegetative cells of the DSL B. licheniformis/subtilisgroup strains were tested for their resistance to antibiotics from a number of families by Health Canada scientists^{Footnote [7]}(Table 1-9 to Table 1-13). Interpretive categories (susceptible, intermediate, resistant or nonsusceptible) are classifications based on an in vitro response of an organism to an antimicrobial agent at levels corresponding to blood or tissue levels attainable with usually prescribed doses of that agent (CLSI, 2010). Minimum inhibitory concentration values were interpreted where possible. Interpretive criteria were not identified for some of the tested antibiotics.

**Table 1-9 : Minimum inhibitory concentrations (MIC) of *B. amyloliquefaciens* 13563-0**
Antibiotic	Susceptible^{Footnote Table 1-9 [a]}	Intermediate^[a]	Resistant^[a]	MIC μg/mL (interpretation)
Amoxycillin	N/A^{Footnote Table 1-9 [b]}	N/A	N/A	0.37 ± 0
Cephotaxime	less than 8	16-32	greater than or equal to 64	21.3 ± 8 (I^{Footnote Table 1-9 [c]})
Ciprofloaxcin	less than 1	2	greater than or equal to 4	0.37 ± 0 (S^{Footnote Table 1-9 [d]})
Doxycycline	N/A	N/A	N/A	0.37 ± 0
Erythromycin	less than 0.5	1-4	greater than or equal to 8 ( greater than or equal to 4^{Footnote Table 1-9[e]})	0.37 ± 0 (S)
Gentamicin	less than 4	8	greater than or equal to 16 ( greater than or equal to 4^[e])	0.52 ± 0.21 (S)
Meropenem	N/A	N/A	N/A	0.37 ± 0
Nalidixic acid	N/A	N/A	N/A	10.2 ± 8.4
Trimethoprim	less than 2	N/A	greater than or equal to 4	greater than 24 (R^{Footnote Table 1-9 [f]})
Vancomycin	less than or equal to 4	N/A	N/A ( greater than or equal to 4^[e])	0.45 ± 0.17 (S)

Footnote Table 1-9 a

Interpretive criteria (MIC μg/mL; CLSI 2010)

Return to footnote Table 1-9 a referrer

Footnote Table 1-9 b

N/A, not available

Return to footnote Table 1-9 b referrer

Footnote Table 1-9 c

I, intermediate susceptibility

Return to footnote Table 1-9 c referrer

Footnote Table 1-9 d

S, susceptible

Return to footnote Table 1-9 d referrer

Footnote Table 1-9 e

Interpretive criteria (MIC μg/mL; EFSA 2008)

Return to footnote Table 1-9 e referrer

Footnote Table 1-9 f

R, resistant

Return to footnote Table 1-9 f referrer

**Table 1-10: Minimum inhibitory concentrations (MIC) of *B. atrophaeus* 18250-7**
Antibiotic	Susceptible^{Footnote Table 1-10 [a]}	Intermediatea	Resistanta	MIC μg/mL (interpretation)
Amoxycillin	N/A^{Footnote Table 1-10 [b]}	N/A	N/A	0.75 ± 0
Cephotaxime	less than or equal to 8	16-32	greater than or equal to 64	1.5 ± 0 (S^{Footnote Table 1-10 [c]})
Ciprofloaxcin	less than or equal to 1	2	greater than or equal to 4	0.37± 0 (S)
Doxycycline	N/A	N/A	N/A	0.37± 0
Erythromycin	less than or equal to 0.5	1-4	greater than or equal to 8 ( greater than or equal to 4^{Footnote Table 1-10 [d]})	0.37 ± 0 (S)
Gentamicin	less than or equal to 4	8	greater than or equal to 16 ( greater than or equal to 4^[d])	0.37 ± 0 (S)
Meropenem	N/A	N/A	N/A	0.37 ± 0
Nalidixic acid	N/A	N/A	N/A	3 ± 0
Trimethoprim	less than or equal to 2	N/A	greater than or equal to 4	greater than 24 ± 0 (R^{Footnote Table 1-10 [e]})
Vancomycin	less than or equal to 4	N/A	N/A ( greater than or equal to 4^[d])	0.75 ± 0 (S)

Footnote Table 1-10 a

Interpretive criteria (MIC μg/mL; CLSI 2010)

Return to footnote Table 1-10 a referrer

Footnote Table 1-10 b

N/A, not available

Return to footnote Table 1-10 b referrer

Footnote Table 1-10 c

S, susceptible

Return to footnote Table 1-10 c referrer

Footnote Table 1-10 d

Interpretive criteria (MIC μg/mL;EFSA 2008)

Return to footnote Table 1-10 d referrer

Footnote Table 1-10 e

R, resistant

Return to footnote Table 1-10 e referrer

B. licheniformis ATCC 12713 appeared to be resistant to many antibiotics (most for which interpretive criteria were available; Table 1-11). This was unexpected, given that the literature on the species indicates susceptibility to a variety of antibiotic classes (Table 1-8). Resistance to vancomycin was particularly unexpected (CLSI 2010). For this reason the test results were revisited. The MIC had been strictly interpreted as the lowest concentration that completely inhibited growth of the micro-organism (CLSI 2010); however for some antibiotics, the vast majority of bacteria had been eliminated at much lower concentrations, with a small number of residual bacteria persisting through several higher concentration increments. Examination by microscopy revealed that these residual bacteria were in the form of aggregates, which is a unique behavior of this strain growing in liquid cultures. This aggregate formation may protect internal cells from contact with the antibiotic. When tests results were re-interpreted with a 95% bioreduction activity cutoff, the revised MICs were more consistent with values expected of this species. This was confirmed for vancomycin using test-strips, which showed low MIC values (1.1 ± 1.0; n=6). It was concluded that the apparent high resistance observed was an artifact of the liquid culture MIC assay.

**Table 1-11: Minimum inhibitory concentrations (MIC) of *B. licheniformis* ATCC 12713**
Antibiotic	S^{Footnote Table 1-11 [a]}	I^{Footnote Table 1-11 [b]}	R^{Footnote Table 1-11 [c]}	MIC μg/mL (interpretation)	95% bioreduction activity (interpretation)
Amikacin	less than or equal to 16	32	greater than or equal to 64	greater than 24 (not S^{Footnote Table 1-11 [d]})	greater than 24 (not S)
Amoxycillin	N/A^{Footnote Table 1-11 [e]}	N/A	N/A	greater than 24	greater than 24
Ampicillin	less than or equal to 0.25	N/A	greater than or equal to 0.5	0.37 ± 0 (I^{Footnote Table 1-11 [f]})	0.37 ± 0
Ceftazidime	less than or equal to 8	16	greater than or equal to 32	greater than 24 (R^{Footnote Table 1-11 [g]})	greater than 24 (R)
Cephotaxime	less than or equal to 8	16-32	greater than or equal to 64	12 (I)	6.0 ± 0 (S)
Chloramphenicol	less than or equal to 8	16	greater than or equal to 32 ( greater than or equal to 8^{Footnote Table 1-11 [h]})	12 (I)	12.0 ± 0 (I)
Ciprofloaxcin	less than or equal to 1	2	greater than or equal to 4	18 ± 9 (R)	0.37 ± 0 (S)
Doxycycline	N/A	N/A	N/A	24	0.56 ± 0.19
Erythromycin	less than or equal to 0.5	1-4	greater than or equal to 8 ( greater than or equal to 4^[f])	greater than 24 (R)	greater than 24 (R)
Gentamicin	less than or equal to 4	8	greater than or equal to 16 ( greater than or equal to 4^[f])	18 ± 9 (R)	2.53 ± 1.54(S)
Meropenem	N/A	N/A	N/A	24	0.37 ± 0
Nalidixic acid	N/A	N/A	N/A	greater than 24	greater than 24
Penicillin	less than or equal to 0.12	N/A	greater than or equal to 0.25	0.75 ± 0 (R)	0.75 ± 0 (R)
Rifampin	less than or equal to 1	2	greater than or equal to 4	0.5 ± 0.2 (S)	0.37 ± 0 (R)
Tetracyclin	less than or equal to 4	8	greater than or equal to 16 ( greater than or equal to 8^[f])	3 ± 0 (S)	3.0 ± 0 (S)
Trimethoprim	less than or equal to 2	N/A	greater than or equal to 4	greater than 24 (R)	0.37 ± 0 (S)
Vancomycin	less than or equal to 4	N/A	N/A ( greater than or equal to 4^[f])	18 ± 9 (non-S)	0.61 ± 0.37(S)

Footnote Table 1-11 a

Interpretive criteria (MIC μg/mL; CLSI 2010) S, susceptible

Return to footnote Table 1-11 a referrer

Footnote Table 1-11 b

Interpretive criteria (MIC μg/mL; CLSI 2010) I, intermediate susceptibility

Return to footnote Table 1-11 b referrer

Footnote Table 1-11 c

Interpretive criteria (MIC μg/mL; CLSI 2010) R, resistant

Return to footnote Table 1-11 c referrer

Footnote Table 1-11 d

S, susceptible

Return to footnote Table 1-11 d referrer

Footnote Table 1-11 e

N/A, not available

Return to footnote Table 1-11 e referrer

Footnote Table 1-11 f

I, intermediate susceptibility

Return to footnote Table 1-11 f referrer

Footnote Table 1-11 g

R, resistant

Return to footnote Table 1-11 g referrer

Footnote Table 1-11 h

Interpretive criteria (MIC μg/mL; EFSA 2008)

Return to footnote Table 1-11 h referrer

Footnote Table 1-11 i

Confirmed using test strips (1.1 ± 1.0 μg/mL, n=6)

Return to footnote Table 1-11 i referrer

Table 1-12: Minimum inhibitory concentrations (MIC, μg/mL) of DSL strains of *B. subtilis*
Antibiotic	S^{Footnote Table 1-12 [a]}	I^{Footnote Table 1-12 [b]}	R^{Footnote Table 1-12 [c]}	B. subtilis subsp. subtilis ATCC 6051	B. subtilis ATCC 6051A	B. subtilis ATCC 55405	B. subtilis subsp. inaquosorum ATCC 55406
Amoxycillin	N/A^{Footnote Table 1-12 [d]}	N/A	N/A	0.4	12.2 ± 13.6	4.3 ± 9.6	0.6 ± 0.5
Ampicillin	less than or equal to 0.25	N/A	greater than or equal to 0.5	greater than 24 (R^{Footnote Table 1-12 [e]})	greater than 24 (R)	greater than 24 (R)	No data
Aztreonam	N/A	N/A	N/A	greater than 24	greater than 24	greater than 24	No data
Cephotaxime	less than or equal to 8	16-32	greater than or equal to 64	6.1 ± 4.7 (S^{Footnote Table 1-12 [f]})	5 ± 1.7 (S)	1.3 ± 1.3 (S)	greater than 24
Ciprofloaxcin	less than or equal to 1	2	greater than or equal to 4	No data	No data	No data	greater than 24 (R)
Doxycycline	N/A	N/A	N/A	0.4	0.4	0.4	8.4 ± 3.3
Erythromycin	less than or equal to 0.5	1-4	greater than or equal to 8 ( greater than or equal to 4^{Footnote Table 1-12 [g]})	0.4 (S)	0.4 (S)	0.4 (S)	0.4 (S)
Gentamicin	less than or equal to 4	8	greater than or equal to 16 ( greater than or equal to 4^[g])	0.6 ± 0.2 (S)	0.6 ± 0.2 (S)	0.4 (S)	0.4 (S)
Meropenem	N/A	N/A	N/A	ND	No data	No data	1.2 ± 1.1
Nalidixic acid	N/A	N/A	N/A	greater than 24	8 ± 3.5	8 ± 3	9.6 ± 3.3
Trimethoprim	less than or equal to 2	N/A	greater than or equal to 4	greater than 24 (R)	greater than 24 (R)	greater than 24 (R)	greater than 24 (R)
Vancomycin	less than or equal to 4	N/A	N/A ( greater than or equal to 4^[g])	0.9 ± 0.7 (S)	0.4 (S)	0.4 (S)	0.37 (S)

Footnote Table 1-12 a

Interpretive criteria (MIC μg/mL; CLSI 2010) S, susceptible

Return to footnote Table 1-12 a referrer

Footnote Table 1-12 b

Interpretive criteria (MIC μg/mL; CLSI 2010) I, intermediate susceptibility

Return to footnote Table 1-12 b referrer

Footnote Table 1-12 c

Interpretive criteria (MIC μg/mL; CLSI 2010) R, resistant

Return to footnote Table 1-12 c referrer

Footnote Table 1-12 d

N/A, not available

Return to footnote Table 1-12 d referrer

Footnote Table 1-12 e

R, resistant

Return to footnote Table 1-12 e referrer

Footnote Table 1-12 f

S, susceptible

Return to footnote Table 1-12 f referrer

Footnote Table 1-12 g

Interpretive criteria (MIC μg/mL;EFSA 2008)

Return to footnote Table 1-12 g referrer

**Table 1-13 : Minimum inhibitory concentrations (MIC, μg/mL) of the masked *Bacillus* species on the DSL**
Antibiotic	S^{Footnote Table 1-13 [a]}	I^{Footnote Table 1-13 [b]}	R^{Footnote Table 1-13 [c]}	Bacillus species 2 18118-1	Bacillus species 4 18121-4	Bacillus species 16970-5	Bacillus species 7 18129-3
Amoxycillin	N/A^{Footnote Table 1-13 [d]}	N/A	N/A	Variable	0.37	0.9 ± 0.6	0.4
Ampicillin	less than or equal to 0.25	N/A	greater than or equal to 0.5	No data	No data	greater than 24 (R^{Footnote Table 1-13 [e]})	greater than 24 (R)
Aztreonam	N/A	N/A	N/A	greater than 24	No data	greater than 24	greater than 24
Cephotaxime	less than or equal to 8	16-32	greater than or equal to 64	1.6 ± 0.7 (S^{Footnote Table 1-13 [f]})	3 (S)	11 ± 2.4 (I^{Footnote Table 1-13 [g]})	6.1 ± 4.7 (S)
Ciprofloaxcin	less than or equal to 1	2	greater than or equal to 4	No data	0.37 (S)	No data	No data
Doxycycline	N/A	N/A	N/A	0.8 ± 0.4	0.37	0.4	0.4
Erythromycin	less than or equal to 0.5	1-4	greater than or equal to 8 ( greater than or equal to 4^{Footnote Table 1-13 [h]})	0.37 (S)	0.37 (S)	0.4 (S)	0.4 (S)
Gentamicin	less than or equal to 4	8	greater than or equal to 16 ( greater than or equal to 4^[h])	1.2 ± 0.5 (S)	1.5 (S)	0.5 ± 0.2 (S)	0.6 ± 0.2 (S)
Meropenem	N/A	N/A	N/A	No data	0.37	No data	No data
Nalidixic acid	N/A	N/A	N/A	8 ± 3	12	9.0 ± 3.3	greater than 24
Trimethoprim	less than or equal to 2	N/A	greater than or equal to 4	greater than 24 (R)	0.37 (S)	24 ± 23 (R)	greater than 24 (R)
Vancomycin	less than or equal to 4	N/A	N/A ( greater than or equal to 4^[h])	0.37 (S)	0.75 (S)	0.4 (S)	0.9 ± 0.7 (S)

Footnote Table 1-13 a

Interpretive criteria (MIC μg/mL; CLSI 2010) S, susceptible

Return to footnote Table 1-13 a referrer

Footnote Table 1-13 b

Interpretive criteria (MIC μg/mL; CLSI 2010) I, intermediate susceptibility

Return to footnote Table 1-13 b referrer

Footnote Table 1-13 c

Interpretive criteria (MIC μg/mL; CLSI 2010) R, resistant

Return to footnote Table 1-13 c referrer

Footnote Table 1-13 d

N/A, not available

Return to footnote Table 1-13 d referrer

Footnote Table 1-13 e

R, resistant

Return to footnote Table 1-13 e referrer

Footnote Table 1-13 f

S, susceptible

Return to footnote Table 1-13 f referrer

Footnote Table 1-13 g

I, intermediate susceptibility

Return to footnote Table 1-13 g referrer

Footnote Table 1-13 h

Interpretive criteria (MIC μg/mL;EFSA 2008)

Return to footnote Table 1-13 h referrer

1.1.3 Effects

1.1.3.1 Environment

B. amyloliquefaciens

B. amyloliquefaciens is widely distributed in nature in a variety of habitats. Certain strains have been released to agricultural ecosystems as biological pesticides for the control of fungal plant pathogens (PMRA-HC 2012; U.S. EPA 2011; U.S. EPA 2012); others have been released to aquatic habitats as a water treatment/conditioner (Advanced Water Technologies 2012). Despite its natural presence in and history of release into, a variety of environments, a comprehensive search of the scientific literature across a number of sources yielded no cases of infection or evidence of adverse effects in aquatic or terrestrial plants, vertebrates or invertebrates.

Studies on the effects of B. amyloliquefaciens strains FZB24 and D747 on a variety of environmental species were submitted to support their registration as biofungicides for use on terrestrial plants (Appendix 11, Table A-51 and Table A-52). Briefly, no significant pathogenicity or toxicity was observed in terrestrial vertebrates (CD and Sprague Dawley rats, Northern Bobwhite quail), aquatic vertebrates (rainbow trout), terrestrial invertebrates (honeybee adults and larvae, earthworm) or aquatic invertebrates (Daphnia magna) at the tested concentrations. Although studies on aquatic or terrestrial plants were not reported as part of the pesticide registrations, pesticides containing these strains are deliberately applied to terrestrial plants to control fungal and bacterial plant pathogens and no adverse effects on the treated plants have been reported in the scientific literature.

Murine exposure assays were conducted by Health Canada scientists. Female BALB/c mice remained asymptomatic after exposure to 10⁶ CFU of B. amyloliquefaciens 13563-0 spores or vegetative cells administered in a 25 μL volume via an endotracheal nebulizer. Aside from a transient inflammatory response, no significant changes were observed (Appendix 12, Table A-59 to Table A-63).

B. atrophaeus

B. atrophaeus is widely distributed in nature. It is used as a non-pathogenic surrogate for B. anthracis in experiments modelling airborne dispersal of spores (Carrera et al. 2007; Page et al. 2007; U.S. EPA 2013a). In spite of its natural presence in and releases into the environment, a comprehensive search of the scientific literature across a number of sources yielded no cases of infection or evidence of adverse effects in aquatic or terrestrial plants, vertebrates or invertebrates.

Murine exposure assays were conducted by Health Canada scientists. Female BALB/c mice remained asymptomatic after exposure to 10⁶ CFU of B. atrophaeus 18250-7 spores or vegetative cells administered in a 25 μL volume via an endotracheal nebulizer. Aside from a transient inflammatory response, no significant changes were observed (Appendix 12, Table A-59 to Table A-63).

B. licheniformis

Environmental isolates of B. licheniformis have the ability to form biofilms (Dat et al. 2012) which are implicated in the pathogenesis of bovine mastitis (reviewed in Contreras and Rodríguez 2011; Nieminen et al. 2007)and bovine toxemia (Murray et al. 1995). B. licheniformis has been reported to cause sporadic abortion or stillbirths in cattle as well as in buffalo, sheep, pigs and camelids (Agerholm et al. 1995; Agerholm et al. 1997; Cabell 2007; Duncanson 2012; Galiero and De Carlo 1998; Gill 1999; reviewed in Kirkbride et al. 1986; Kirkbride 1993; Madslien et al. 2012; Mitchell and Barton 1986). Other adverse effects in terrestrial vertebrates associated with B. licheniformisinclude placentitis, keraconjuntivitis, feather degradation and yolk sac infection in ostriches (Johnson et al. 1994; Gill 1999; Sheldon et al. 2002; Murray 2006; Hare et al. 2008; Rajchard 2010; Goncagul et al. 2012). B. licheniformis has been implicated in adverse effects in insects, including bed bugs, root-knot nematodes, Ecualyptus snout-beetles and moths (Reinhardt et al. 2005; Mekete et al. 2008; Molina and Santolmazza-Carbone 2010; Bilbech et al. 2012). An isolate of B. licheniformis was implicated in effects in plants as the causative agent of pistachio dieback (Baradaran and Ghasemi 2010).

A six month study attempted to determine the cause of 218 naturally-aborted bovine fetuses (Agerholm et al. 1997). The likely cause of 73 abortions was diagnosed; the most common causes were bovine diarrhea virus (13%), Neospora caninum (10%), mycosis (5%) and B. licheniformis (4%) (Agerholm et al. 1997). In another study, B. licheniformis represented 3% of bovine abortions (n=5,662) (Murray 2006). A Canadian bovine abortion update report for years 1998 to 2004 implicated B. licheniformis in 1.1 to 3.1% of abortion cases submitted to the Animal Health Laboratory (McEwen and Carman 2005). In comparison, Neospora species represented between 8.3 and 19% of cases submitted and other bacterial species represented between 6.1 and 14% for the same period of time. An etiological agent was not identified in up to 60.6% of cases between 2001 and 2002. Despite its presence at high concentrations in agricultural settings (10⁴-10⁷ CFU/m³ in indoor air and 10⁴-10⁶ CFU/g in settled dust (Andersson et al. 1999), abortion from exposure to naturally-occurring B. licheniformis populations is not common. Pathogenesis of abortion is not clear but ingestion of poor-quality/mouldy feed during gestation and subsequent hematogenous spread to the reproductive tract as well as introduction during general animal husbandry activities have been implicated (Cabell 2007; Scott 2011; Goncagul 2012). Gentamicin and ciprofloxacin were the most effective antibiotics tested against B. licheniformis isolated from the cervicovaginal mucus of repeat-breeding cows (Yadav and Kashyap 2003).

Experimental infection with B. licheniformis strain DVL 9315323 in pregnant dairy cows demonstrated placentome tropism after IV challenge doses ranging from 10⁹ to 10¹² CFU per animal (Agerholm et al. 1999). B. licheniformis bacteria were closely associated with placentome and fetal lesions, and were hypothesised to have caused abortion or premature delivery (Agerholm et al. 1999). In another mammalian study, immune depressed BALB/c mice were exposed intravenously to environmental and food isolates of B. licheniformis,including the type strain B. licheniformis ATCC 14580 at doses of less than 1 × 10⁶ to 6 × 10¹⁰ CFU per animal (Agerholm et al. 1997; Appendix 11, Table A-54). Mice were able to eliminate high numbers of the bacteria within one week however, some of the tested isolates caused pulmonary and brain lesions. Male albino Wistar rats exposed to a strain of B. licheniformis had an oral NOAEL reported to be greater than 1.1 × 10¹¹ CFU/kg body weight (Nithya et al. 2012; Appendix 11, Table A-54).

Studies on the effects of B. licheniformis strain SB3086 on a variety of environmental species were submitted to support its registration as a fungicide for use on terrestrial plants (Appendix 11, Table A-53). No pathogenicity or toxicity was observed in terrestrial vertebrates (rats, mallard ducks), aquatic vertebrates (rainbow trout) or terrestrial invertebrates (honeybee larvae) at the tested concentrations (U.S. EPA, 2001). Aquatic invertebrates (Daphnia magna) were exposed to the technical grade active ingredient (TGAI). The survival of daphnids exposed to 1× 10⁷ CFU/mL of the TGAI (1000 times the expected environmental concentration for pesticidal use) was 90% (two died) (PMRA-HC, personal communication). The TGAI was considered to be not toxic in terms of survival, reproduction, length and weight relative to the control. Although pathogenicity and toxicity studies on aquatic or terrestrial plants were not reported as part of the pesticide registration, the pesticide containing this strain is deliberately applied to terrestrial plants to control fungal plant pathogens. No adverse effects on the treated plants have been reported in the scientific literature or in testing performed for efficacy evaluation.

No negative effects were reported in brine shrimp, rainbow trout, pigs and chickens exposed to probiotics containing strains of B. licheniformis (Link and Kovác 2006; Merrifield et al. 2010a; Merrifield et al. 2010b; Rahimi and Kahsksefidi 2006; Vinoj et al. 2013). Increased weight gain and/or pathogen resistance were noted.

Murine exposure assays were conducted by Health Canada scientists. Female BALB/c mice remained asymptomatic after exposure to 10⁶ CFU of B. licheniformis ATCC 12713 spores or vegetative cells administered in a 25 μL volume via an endotracheal nebulizer. Aside from a transient inflammatory response, no significant changes were observed (Appendix 12, Table A-59 to Table A-63).

B. subtilis

B. subtilis occurs naturally in indoor air and settled dust of agricultural settings at elevated cell-densities (Andersson et al. 1999). Certain strains have been released to agricultural ecosystems as fungicides for use on terrestrial plants ( Mendelsohn and Vaituzis 1999; U.S. EPA 2006; PMRA-HC 2007a; PMRA-HC 2007b; PMRA-HC 2007c; U.S. EPA 2010; PMRA-HC 2013); others have been released to aquatic habitats as a water treatment/conditioner (Advanced Water Technologies 2012). Despite its natural presence in, and history of release into, a variety of environments, a comprehensive search of the scientific literature across a number of sources yielded no cases of infection or evidence of adverse effects in aquatic plants or vertebrates.

Studies on the effects of strains of B. subtilis on a variety of environmental species were submitted to support the registration of certain strains as biofungicides for use on terrestrial plants (Appendix 11, Table A-56 and Table A-57). No significant adverse effects were reported in birds, mammals, terrestrial insects, earthworms or soil micro-organisms as a result of exposure to B. subtilis strain MBI 600 (PMRA-HC 2007a). No significant adverse effects in birds, freshwater and marine fish, mammals or algae were reported as a result of exposure to B. subtilis strain QST 713 (PMRA-HC 2007b). There is some evidence of effects in aquatic and terrestrial invertebrates, but results are inconsistent. In studies reviewed by the U.S. EPA, mortalities were reported in Daphnia magna and parasitic Hymenoptera after exposure to B. subtilis QST 713 at varying concentrations (Mendelsohn and Vaituzis 1999). The cause of death and involvement of B. subtilis QST 713 in toxicity or pathogenicity could not be determined in these studies.

Murine exposure assays were conducted by Health Canada scientists. Female BALB/c mice remained asymptomatic after exposure to 10⁶ CFU of B. subtilis ATCC 6051A, B. subtilis ATCC 55405, B. subtilis subsp. subtilis ATCC 6051 and B. subtilis subsp. inaquosorum ATCC 55406 spores or vegetative cells administered in a 25 μL volume via an endotracheal nebulizer. Aside from a transient inflammatory response, no significant changes were observed (Appendix 12, Table A-59 to Table A-63).

Pathogenicity and toxicity studies were performed by Environment Canada scientists^{Footnote [8]} using Festuca rubra (red rescue), Folsomia candida (collembolan or springtail) and Eisenia andrei (earthworm) exposed to either B. subtilis ATCC 6051Aor B. subtilis ATCC 55405 in either field-collected sandy clay loam or a formulated artificial sandy loam soil (Appendix 11, Table A-58). For the red fescue, field-collected or artificial soils were inoculated with 10⁵ CFU/g soil dry weight of either B. subtilisATCC 6051Aor B. subtilis ATCC 55405. At the end of the study (day 21), a significant reduction (approximately 18%) in the mean shoot length was detected in plants exposed to B. subtilis ATCC 55405 in the field-collected soil, relative to the field-collected soil negative control.

In the springtail trials, the arthropods were exposed for 28 days to field-collected or artificial soils inoculated with either 10⁴ CFU of B. subtilis ATCC 6051Aor10³ CFU of B. subtilis ATCC 55405 per gram of dry soil. When compared with the negative control in both soils, a significant reduction (approximately 50%) in juvenile production was observed after exposure to B. subtilis ATCC 55405, while no juveniles were produced after exposure to B. subtilis ATCC 6051A. Adult survival was not affected by either of these strains.

In the earthworm trials, the invertebrate was exposed for 35 days in field-collected or artificial soils inoculated with either 10⁴ CFU of B. subtilis ATCC 6051Aor10⁵ CFU of B. subtilis ATCC 55405 per gram of dry soil. There were no adverse effects on reproduction upon exposure to either strain, regardless of soil type. A significant increase in juvenile production was observed in the field-collected soil, relative to the field-collected soil negative control, after exposure to B. subtilis ATCC 55405.

Masked DSL Bacillus Strains

Murine exposure assays were conducted by Health Canada scientists. Female BALB/c mice remained asymptomatic after exposure to 10⁶ CFU of Bacillus species 16970-5, Bacillus species 2 18118-1, Bacillus species 4 18121-4 and Bacillus species 7 18129-3 spores or vegetative cells administered in a 25 μL volume via an endotracheal nebulizer. Aside from a transient inflammatory response, no significant changes were observed (Appendix 12, Table A-59 to Table A-63).

1.1.3.2 Human Health

With the exception of B. cereus, Bacillusinfections in humans are rare. They are diverse and tend to occur in immune compromised people (Pennington et al. 1976), or in association with implanted medical devices (Banerjee et al. 1988) or recent trauma (Logan 2012). Cases of non-B. cereus food poisoning caused by Bacillus species have been reported (Kramer and Gilbert 1989; Murray et al. 1995). In recent years however, there have been no reports of food poisoning incidents or outbreaks attributed to non-B. cereus Bacillus species (Sorokulova, personal communication). As potential contaminants of tobacco products, Bacillus species have been implicated in infections, pulmonary inflammation and allergic sensitivities and plasma exudation and tissue dysfunction in the mouth (Rooney 2005; Rubinstein and Pedersen 2002).

B. amyloliquefaciens

B. amyloliquefaciens is globally distributed in a variety of ecological niches andhas a history of use in industrial fermentation and pest control. A comprehensive search of the scientific literature across all major sources yielded no reports of human infection linked to the species or of other adverse effects in humans from exposure to the organism, its metabolites or structural components.

Studies submitted to support pesticide registrations for B. amyloliquefaciens strains FZB24 and D747 included a variety of exposures in mammalian models used to predict adverse effects in humans (Appendix 11, Table A-51 and Table A-52). Oral, pulmonary and intravenous exposure studies using B. amyloliquefaciens strains FZB24 or D747 demonstrated low toxicity and no pathogenicity in CD and Sprague-Dawley rats at maximum challenge doses.

In studies conducted at Health Canada, female BALB/c mice were exposed to 10⁶ CFU of B. amyloliquefaciens13563-0 vegetative cells or spores administered in a 25 μL volume via an endotracheal nebulizer, as a model for human pulmonary exposure. The mice appeared normal and remained asymptomatic after exposures to vegetative cells and spores. All treated mice were necropsied 24 hours after exposure to vegetative cells or 1 week after exposure to spores to assess bacterial clearance, pulmonary cytokine expression and acute phase response (Appendix 12, Table A-59 to Table A-63). A statistically significant pro-inflammatory response was observed and some pulmonary cytokines were elevated 24 hours following exposure to vegetative cells. No significant changes were observed after one week following exposure to spores. Mice dosed with spores were not assessed for inflammation or cytokine expression at 24 hours, so the occurrence of transient inflammation would not have been detected. The serum amyloid A levels were slightly elevated in the acute phase response for both vegetative cells at 24 hours and spores at one week post-exposure.

No cases of hypersensitivity from glucanases or amylases produced by B. amyloliquefaciens have been reported (Caballero et al. 2007). No hypersensitivity incidents were reported during testing, production, use or handling of B. amyloliquefaciens biocontrol strains FZB24 and D747 in controlled laboratory settings during research and development (U.S. EPA 2011; U.S. EPA 2012).

B. atrophaeus

B. atrophaeus has a widespread distribution in nature and a history of environmental release as a surrogate organism for modelling airborne dispersal of pathogenic Bacillusspecies (Carrera et al. 2007; Page et al. 2007; U.S. EPA 2013a). A comprehensive search of the scientific literature across all major sources yielded no reports of human infection with B. atrophaeus or of other adverse effects in humans from exposure to the organism, its metabolites or structural components.

In studies conducted at Health Canada, female BALB/c mice were exposed to 10⁶ CFU of B. atrophaeus 18250-7 vegetative cells or spores administered in a 25 μL volume via an endotracheal nebulizer, as a model for human pulmonary exposure. The mice appeared normal and remained asymptomatic after exposures to both spores and vegetative cells. All treated mice were necropsied 24 hours after exposure to vegetative cells or 1 week after exposure to spores to assess bacterial clearance, pulmonary cytokines expression and acute phase response (Appendix 12, Table A-59 to Table A-63). A statistically significant pro-inflammatory response was observed and some pulmonary cytokines were elevated 24 hours following exposure to vegetative cells. No significant changes were observed after one week following exposure to spores. Mice dosed with spores were not assessed for inflammation or cytokine expression at 24 hours, so the occurrence of transient inflammation would not have been detected. The serum amyloid A levels were slightly elevated in the acute phase response for both vegetative cells and spores.

No cases of hypersensitivity or allergenicity as a result ofB. atrophaeus, its metabolites or structural componentshave been reported.

B. licheniformis

Although B. licheniformis is naturally present in high concentrations in a variety of environments to which humans are exposed, only 35 case reports of infection have been published in the English literature since 1966. Several were cases of bacteremia or septicemia, but a range of other infections were also reported (Blue et al. 1995; Castagnola et al. 1997; Cotton et al. 1987; Maucour et al. 1999; Murray et al. 1995; Tabbara and Tarabay 1979; Thurn and Goodman 1988). Almost all cases involved predisposing factors: immune deficiency, debilitating disease or significant breaches in natural barriers to infection.

B. licheniformis bacteremia was reported in patients with cancer (Banerjee et al. 1988; Ozkocaman et al. 2006), peritonitis (Sugar and McCloskey 1977), central venous catheters (Blue et al. 1995; Castagnola et al. 1997) and after a bronchoscopic procedure (Hong et al. 2004). It was also seen in association with foot lesions (Gayet et al. 2005) and in a pregnant woman (Peloux et al. 1976). Co-bacteremia of B. licheniformis and B. subtilis in an elderly patient with predisposing factors was also reported (La Jeon et al. 2012). In three cases of B. licheniformis septicemia, one was due to contaminated intravenous lines (Matsumoto et al. 2000), another followed arteriography (Hardy et al. 1986) and the third was in a pre-term infant (Lépine et al. 2009; Thomson et al. 1990). In two accounts, individuals deliberately injected themselves with products containing B. licheniformisspores (alone or in combination with spores of other Bacillus species), resulting in bacteremia (Galanos et al. 2009; Hannah and Ende, 1999). Bacteremia was recurrent in one case, possibly because spores, which were resistant to antibiotic treatment, remained in the tissues and germinated periodically (Hannah and Ender 1999). This kind of recurrent sepsis caused by B. licheniformis was also observed more recently, in an immune competent individual with no apparent underlying conditions (Haydushka et al. 2012).

B. licheniformis ophthalmitis or endophthalmitis (Maucour et al. 1999; Tabbara and Tarabay 1979; Thurn and Goodman 1988) and brain abscess (Jones et al. 1992) each resulting from penetrating eye trauma have been reported. A brain abscess caused by B. licheniformis was also described in a patient with acute myeloid leukemia (Mochiduki et al. 2007) and in a healthy patient which later progressed to a malignant brain tumour (Flores et al. 2001). In the last case, subsequent to being the causal organism in the formation of a brain abscess, B. licheniformis was postulated to be the oncogenic agent. Although conclusive evidence of such a causal relationship is lacking, B. licheniformis has been hypothesized to be an oncogenic bacterium along with others such as Heliobacter pylori (Wainwright and Al Talih 2003). Other infections with B. licheniformis include parotid gland abscess (Longo et al. 2003), a cutaneous infection as the result of injury (Ameur et al. 2005), prosthetic valve endocarditis (Santini et al. 1995), a pacemaker wire infection with bacteremia (Quan et al. 2000), post-operative ventriculitis where B. licheniformis was isolated from cerebrospinal fluid (Young et al. 1982) and spondylitis in association with bacteremia in a lung cancer patient (Kim et al. 2012).

The safety of B. licheniformis strain MeI (isolated from milk) was assessed for use in the food industry (Appendix 11, Table A-54). The oral no observed adverse effect level (NOAEL) was greater than 1.1 × 10¹¹ CFU/kg body weight in male albino Wistar rats (Nithya et al. 2012). Studies submitted to support pesticide registrations for B. licheniformisstrain SB3086 included a variety of exposures in standard mammalian models used to predict adverse effects in humans (Appendix 11, Table A-53). Oral, pulmonary and intravenous exposure studies using B. licheniformis strain SB3086 demonstrated low toxicity and no pathogenicity in rats at maximum challenge doses.

Artificially immune depressed mice (BALB/c mice treated intraperitoneally with cyclophosphamide at 0.2 mg/g body weight), were dosed, intravenously with less than 1 × 10⁶ to 6 × 10¹⁰ CFU per animal of clinical, environmental and food isolates of B. licheniformis, including the type strain ATCC 14580 (Agerholm et al. 1997; Appendix 11, Table A-54). Despite the immune-depressed state of the mice, they were able to eliminate high numbers of the bacteria within one week, but B. licheniformis was recovered from the liver and spleen of most mice and from the kidneys of some mice one week after exposure. Some of the tested isolates caused pulmonary and brain lesions. Signs were only observed in two mice and no deaths attributed to treatment were reported. Given the high doses, zero treatment-related mortality and the clearance of most bacteria from tissues, all tested strains of B. licheniformis were considered to be of low pathogenicity in immune depressed mice.

In studies conducted at Health Canada, female BALB/c mice were exposed to 10⁶ CFU/25 μL of B. licheniformisATCC 12713 vegetative cells or spores administered in a 25 μL volume via an endotracheal nebulizer, as a model for human pulmonary exposure. The mice appeared normal and remained asymptomatic after exposures to vegetative cells and spores. All treated mice were necropsied 24 hours after exposure to vegetative cells or 1 week after exposure to spores to assess bacterial clearance, pulmonary cytokine expression and acute phase response (Appendix 12, Table A-59 to Table A-63). A statistically significant pro-inflammatory response was observed and some pulmonary cytokines were elevated 24 hours following exposure to vegetative cells. An increase in serum amyloid A level in the acute phase response relative to the control was observed for vegetative cells of B. licheniformis ATCC 12713. No data regarding pulmonary cytokines or serum amyloid A level were available for exposure to spores of B. licheniformis ATCC 12713.

B. licheniformis has been reported in the literature as being implicated in outbreaks of food poisoning (Appendix 13). Endospore-forming bacteria, like B. licheniformis, along with heat-resistant toxic substances they produce, may survive pasteurization and other dairy processes as well as cooking temperatures (Biesta-Peters et al. 2010; Nieminen et al. 2007). For a toxic dose of enterotoxin to be produced in contaminated milk or other foods, cell counts of 10⁵ to 10⁹ CFU/g are estimated to be required(reviewed in Cosentino et al. 1997; Griffiths 1990; Logan, 2012; Lund, 1990;Rosenkvist and Hansen 1995; Salkinoja-Salonen et al. 1999). Food poisoning symptoms resulting from ingestion of B. licheniformis-contaminated food occur 5 to 12 hours after consumption (8 hour median). B. licheniformis food poisoning is similar to the diarrheal syndromes caused by Clostridium perfringens and B. cereus (reviewed in Drobniewski 1993; Kramer and Gilbert 1989). Death as a result of B. licheniformis food poisoning was reported in an infant that had consumed contaminated formula (Mikkola et al. 2000; Salkinoja-Salonen et al. 1999). Two B. licheniformis isolates obtained from the formula were reported to be toxigenic (Salkinoja-Salonen et al. 1999). B. licheniformis ATCC 14580 (the type strain) has been reported to be non-toxigenic (Pedersen et al. 2002). The DSL strain, B. licheniformis ATCC 12713, was tested at Health Canada for Hbl and Nhe toxin production and was not observed to produce these diarrheal toxins. Germination of spores and growth of Bacillus spp. in heat-treated raw milk and other foods produce "off-flavours" and poor appearance which may deter consumption and thereby prevent exposure (reviewed in Abo-Elnaga et al. 2002; Davies and Wilkinson 1973).

Glyphosate acetyltransferase from B. licheniformis used in an herbicide was evaluated for potential allergenicity and toxicity (Delaney et al. 2008). The authors concluded that at least in the context of agricultural biotechnology there are no expected adverse effects to humans and the potential for human exposure to the protein is low if expressed in transgenic plants (Delaney et al. 2008). B. licheniformis strain SB3086 has been screened for delayed contact sensitivity in guinea pigs and was determined to not be a dermal sensitizer. No reports of hypersensitivity or allergenicity implicating the DSL strain B. licheniformis ATCC 12713 have been described.

B. subtilis

B. subtilis bacteremia, septicemia and other infections have been reported (De Boer et al. 1991; reviewed in Drobniewski 1993; Ihde and Armstrong 1973; Logan 1988; Murray et al. 1995; Olszewski et al. 1999; Pennington et al. 1976; reviewed in Tuazon et al. 1979; Turnbull et al. 1979); however, B. subtilisinfections are rare, and involve predisposing conditions including immune deficiency, debilitating disease and significant breaches in normal barriers to infection. Few cases of infection and no fatalities caused by B. subtilis have been reported since 1980.

B. subtilis bacteremia has been reported in cancer patients (Banerjee et al. 1988). Nosocomial bacteremia caused by B. subtilis was reported in four of eight patients with underlying conditions (cancer, head trauma and recent surgery) who had been given a probiotic containing B. subtilis spores (10⁹ spores per tablet) (Richard et al. 1988). Septicemia caused by B. subtilis was reported in a young child(Cox et al. 1959) and in hospitalized patients who had intravenous lines (Matsumoto et al. 2000).

B. subtilis was implicated in a case of cellulitis that progressed to necrotizing fasciitis in a cancer patient (Tuazon et al. 1979). Infections where B. subtilis was implicated as the causative agent or a concomitant as the result of indwelling medical devices have been reported (Ihde and Armstrong 1973; Schoenbaum et al. 1975). Some reported B. subtilisinfections were fatal (Ihde and Armstrong 1973; Pennington et al. 1976; reviewed in Tuazon et al. 1979). In these cases, patients had serious co-morbidities and in some cases B. subtilis was thought to be a contaminant and its role as the causative agent was initially overlooked.

Studies submitted to support pesticide registrations for B. subtilis strains QST 713 and MBI 600 included a variety of exposures in standard mammalian models used to predict adverse effects in humans (Appendix 11, Table A-56 and Table A-57). Oral, pulmonary and intravenous exposure studies using B. subtilis strains QST 713 and MBI 600 demonstrated low toxicity and no pathogenicity in CD rats at maximum challenge doses.

In studies conducted at Health Canada, female BALB/c mice were exposed to 10⁶ CFU of B. subtilis ATCC 6051A, B. subtilis ATCC 55405, B. subtilis subsp. subtilis ATCC 6051 and B. subtilis subsp. inaquosorum ATCC 55406 vegetative cells and spores administered in a 25 μL volume via an endotracheal nebulizer, as a model for human pulmonary exposure. The mice appeared normal and remained asymptomatic after exposures to vegetative cells and spores. All treated mice were necropsied 24 hours after exposure to vegetative cells or 1 week after exposure to spores to assess bacterial clearance, pulmonary cytokine expression and acute phase response (Appendix 12, Table A-59 to Table A-63). Vegetative cells and spores were enumerated in the lungs, trachea and esophagus. Changes in cytokine level and serum amyloid A in the acute phase response were only reported for vegetative cells of B. subtilis subsp. inaquosorum ATCC 55406.

Endospore-forming bacteria such as Bacillus species, along with the heat-resistant toxic substances they produce, may survive pasteurization and other dairy processes (Nieminen et al. 2007). The proliferation of these micro-organisms in foods represents a potential food poisoning hazard (Beattie and Williams 1999). After consumption of food with high bacterial loads (10⁵-10⁹ CFU/g) B. subtilis food poisoning symptoms may begin 10 minutes to 14 hours (2.5 hour median) with acute onset of vomiting (Rosenkvist and Hansen 1995; Logan 2012). Foods often implicated are meat, seafood, pastry products and rice dishes. B. subtilis food poisoning has also been associated with spoiled (ropy) bread where the concentration of B. subtilis has been reported to be approximately 10⁸ CFU/g. Foodborne illness due to ropy bread is unlikely given the unattractive appearance (discoloured, sticky and soft crumb) of the affected bread as a result of the high number of cells present which breakdown starch and proteins (Rosenkvist and Hansen 1995; Logan 2012; Lund 1990). The DSL strains B. subtilis ATCC 6051A, B. subtilis ATCC 55405, B. subtilis subsp. subtilis ATCC 6051 and B. subtilis subsp. inaquosorum ATCC 55406, were tested at Health Canada for Hbl and Nhe toxin production and were not observed to produce these diarrheal toxins.

In a recent article, liver damage was reported in patients who had consumed nutritional supplements which contained B. subtilis (Logan 2012). The strain was later demonstrated to be hepatotoxic in a Hep2G cell culture assay. Several strains of B. subtilis were tested in rats and other vertebrates and no negative effects were observed.

No hypersensitivity incidents were reported during testing, production or use of B. subtilis strains QST 713 or MBI 600 (PMRA-HC 2007b; PMRA-HC 2007c). B. subtilis MBI 600 was a moderate skin sensitizer 24 to 72 hours post challenge (PMRA-HC 2007c; U.S. EPA 2012).B. subtilis produces exoenzymes that facilitate the decay of organic matter (Tjalsma et al. 2004). Subtilisins are proteolytic enzymes produced by B. subtilis that are known to elicit allergic reactions including dermatitis and respiratory allergies in humans following repeated exposure (Juniper et al. 1977; Norris et al. 1981; Schweigert et al. 2000; Thorne et al. 1986; Tripathi and Grammer 2001; Weissman and Lewis 2002). B. subtilis has been reported to produce enzymes that cause symptoms associated with allergenicity including asthma and irritation (Flindt and Hendrick 2002).

Masked DSL Bacillus Strains

In studies conducted at Health Canada, female BALB/c mice were exposed to 10⁶ CFU of Bacillus species 16970-5, Bacillus species 2 18118-1, Bacillus species 4 18121-4 and Bacillus species 7 18129-3 vegetative cells or spores administered in a 25 μL volume via an endotracheal nebulizer, as a model for human pulmonary exposure. The mice appeared normal and remained asymptomatic after exposures to vegetative cells and spores. All treated mice were necropsied 24 hours after exposure to vegetative cells or 1 week after exposure to spores to assess bacterial clearance, pulmonary cytokine expression and acute phase response (Appendix 12, Table A-59 to Table A-63). Vegetative cells and spores were enumerated in the lungs, trachea and esophagus. Changes in the cytokine levels following exposure to vegetative cells and spores of Bacillus species 16970-5, Bacillus species 2 18118-1 and Bacillus species 4 18121-4 were observed. Bacillus species 7 18129-3 was not tested. Changes in cytokine level and serum amyloid A in the acute phase response were only reported for vegetative cells of Bacillus species 16970-5, Bacillus species 2 18118-1 and Bacillusspecies 4 18121-4 and spores of Bacillus species 16970-5 and Bacillus species 2 18118-1.

1.2 Hazard Severity

Regular exposure to members of the B. subtilis complex occurs due to their widespread distribution in the environment (Murray et al. 1995). Strains can be found on dust particles which can be inhaled (Andersson et al. 1999). Dermal contact may occur as strains are commonly found in soils and on most surfaces (Logan and De Vos 2009; Murray et al. 1995; Thatoi et al. 2013). Despite the high natural exposure to these micro-organisms there is a low rate of reported infections (Rooney, personal communication). Furthermore, B. subtilis complex members have a history of use in biocontrol, growth promotion and as probiotics, all resulting in direct exposure to humans and environmental species, and without reported adverse effects. Finally, the DSL strains are widely used in a variety of sectors in Canada (see 2.1 Sources of Exposure) and no adverse effects have been reported in association with these uses.

1.2.1 Environmental Hazard

1.2.1.1 B. amyloliquefaciens

The environmental hazard severity for B. amyloliquefaciens 13563-0 is estimated to be low because no cases of infection or adverse effects in terrestrial and aquatic vertebrates, invertebrates and plants were found in the scientific literature. Testing of B. amyloliquefaciens pesticidal strains in terrestrial and aquatic vertebrates and invertebrates indicates low pathogenic or toxic potential. Testing conducted by Health Canada scientists in murine models and cell lines indicates that B. amyloliquefaciens 13563-0 has low pathogenic potential. There is a history of safe use of B. amyloliquefaciens 13563-0 and of B. amyloliquefacienspesticidal strains

1.2.1.2 B. atrophaeus

The environmental hazard severity for B. atrophaeus18250-7 is estimated to be low because information from the scientific literature indicates that B. atrophaeus has low toxic and pathogenic potential in terrestrial and aquatic vertebrates, invertebrates and plants and no adverse effects were reported. Testing conducted by Health Canada scientists in murine models and cell lines indicates that B. atrophaeus 18250-7 has low pathogenic potential.

1.2.1.3 B. licheniformis

The environmental hazard severity for B. licheniformisATCC 12713 is estimated to be low because information from the scientific literature indicates that B. licheniformis has low pathogenic potential to terrestrial or aquatic invertebrates or plants. Though B. licheniformis abortion occurs naturally in agricultural settings it is rare and under experimental conditions, doses required to establish infection in the bovine placenta were high and resulted in higher blood concentrations of bacteria than would be expected during infection under natural conditions. In the unlikely case of infection, relevant veterinary antibiotics against B. licheniformis ATCC 12713 are available. In addition, it has been used as a probiotic in brine shrimp, rainbow trout, pigs and chickens without negative effects reported. Testing conducted by Health Canada scientists in murine models and cell lines indicates that B. licheniformis ATCC 12713 has low pathogenic potential (consistent with the Bacillus species assessed in this report).

1.2.1.4 B. subtilis

The environmental hazard severity for B. subtilis ATCC 6051A, B. subtilis ATCC 55405, B. subtilis subsp. subtilis ATCC 6051 and B. subtilis subsp. inaquosorum ATCC 55406 is estimated to be low because information from the scientific literature regarding B. subtilis indicates that it has a low toxic and pathogenic potential in terrestrial and aquatic vertebrates, invertebrates and plants. However, some adverse effects were reported following exposure to high concentrations of other strains of B. subtilis. Testing of B. subtilis pesticidal strains in terrestrial and aquatic vertebrates and invertebrates generally indicates low pathogenic or toxic potential but some effects were observed in terrestrial and aquatic invertebrates. In testing conducted by Environment Canada scientists, significant reductions in mean shoot length in terrestrial plants and in juvenile production in terrestrial arthropods were observed after exposure to B. subtilis ATCC 6051A and B. subtilis ATCC 55405. Testing conducted by Health Canada scientists in murine models and cell lines indicates that B. subtilis ATCC 6051A, B. subtilis ATCC 55405, B. subtilis subsp. subtilisATCC 6051 and B. subtilis subsp. inaquosorum ATCC 55406 have low pathogenic potential. There is a history of safe use for all the DSL B. subtilisstrains.

1.2.1.5 Masked DSL Bacillus Strains

The environmental hazard severity for Bacillus species 16970-5, Bacillus species 2 18118-1, Bacillusspecies 4 18121-4 and Bacillus species 7 18129-3 is estimated to be low because testing conducted by Health Canada scientists in murine models and cell lines indicates that these strains have low pathogenic potential. There is a history of safe use of the masked DSL Bacillus strains.

1.2.2 Human Health Hazard

1.2.2.1 B. amyloliquefaciens

The human hazard severity for B. amyloliquefaciens13563-0 is estimated to be low because information from the scientific literature indicates a low pathogenic potential and no cases of infection were reported. Testing of pesticidal strains of B. amyloliquefaciens in models of human infection indicates a low pathogenic or toxic potential. Testing conducted by Health Canada scientists in murine models and cell lines indicates that B. amyloliquefaciens 13563-0 has low pathogenic potential. Antibiotic susceptibility testing performed by Health Canada scientists demonstrated that clinically relevant antibiotics are effective against this strain. There is a history of safe use of B. amyloliquefaciens 13563-0

1.2.2.2 B. atrophaeus

The human hazard severity for B. atrophaeus 18250-7 is estimated to be low because information from the scientific literature indicates a low pathogenic potential and no cases of infection were reported. Testing conducted by Health Canada scientists in murine models and cell lines indicates that B. atrophaeus 18250-7 has low pathogenic potential.Antibiotic susceptibility testing performed by Health Canada scientists demonstrated that clinically relevant antibiotics are effective against this strain. There is a history of safe use of B. atrophaeus 18250-7.

1.2.2.3 B. licheniformis

The human hazard severity for B. licheniformis ATCC 12713 is estimated to be low because information from the scientific literature indicates that there is some pathogenic potential, however, case reports are rare, and occur mostly in individuals with compromised immunity, debilitating disease or whose normal barriers to infection are breached by implanted medical devices or wounds. In one instance, recurrent sepsis was reported in an individual with no known predisposition who made full recovery. Testing conducted by Health Canada scientists in murine models and cell lines indicates that B. licheniformis ATCC 12713 has low pathogenic potential.(consistent with the other Bacilus species assessed in this report) and no toxicity or pathogenicity was observed. B. licheniformis-associated food poisoning has been reported, however the DSL strain did not produce B. cereus-like toxins as demonstrated in testing done by Health Canada scientists. Mitigating factors such as off-flavours and appearance would likely discourage consumption of contaminated food. There is a history of safe use of B. licheniformisATCC 12713.

Antibiotic susceptibility testing performed by Health Canada scientists first indicated that B. licheniformis ATCC 12713 is resistant to many of the antibiotics it was tested against (most for which interpretive criteria were available, excepting tetracycline and rifampicin); however, after further investigation it was concluded that the apparent high resistance observed was an artefact of the liquid culture MIC assay. Reinterpreted using a 95% bioreduction activity cut-off, the susceptibility profile was consistent with values in the literature on the species, and for vancomycin, this was confirmed using a commercial test-strip method.

1.2.2.4 B. subtilis

The human hazard severity for B. subtilis ATCC 6051A, B. subtilis ATCC 55405, B. subtilis subsp. subtilis ATCC 6051 and B. subtilis subsp. inaquosorum ATCC 55406 is estimated to be low because information from the scientific literature indicates that there is some pathogenic potential in individuals with compromised immunity or whose normal barriers to infection are breached. However, the number of reports is limited, most reports pre-date 1980 and no fatalities have since been reported. Testing conducted by Health Canada scientists in murine models and cell lines indicates that B. subtilis ATCC 6051A, B. subtilis ATCC 55405, B. subtilis subsp. subtilis ATCC 6051 and B. subtilis subsp. inaquosorum ATCC 55406 have low pathogenic potential. Although B. subtilis-associated food poisoning has been reported, the DSL strains do not produce B. cereus-like toxins as demonstrated in testing done by Health Canada scientists. Mitigating factors such as off-flavours and appearance would likely discourage consumption of contaminated food. Testing of pesticidal strains of B. subtilis in models of human infection indicates a low pathogenic or toxic potential. There is a history of safe use of the DSL strains.

1.2.2.5 Masked DSL Bacillus strains

The human hazard severity for Bacillus species 16970-5, Bacillus species 2 18118-1, Bacillus species 4 18121-4 and Bacillus species 7 18129-3 is estimated to be low because Testing conducted by Health Canada scientists in murine models and cell lines indicates that these strains have low pathogenic potential. There is a history of safe use of the masked DSL Bacillus strains.

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2. Exposure Assessment

2.1 Sources of Exposure

This assessment considers exposure to the DSL B. licheniformis/subtilis group strains resulting from their addition to consumer or commercial products and their use in industrial processes in Canada.

The DSL B. licheniformis/subtilis group were nominated to the DSL for use in consumer and commercial products including products for cleaning and deodorizing, drain cleaning and degreasing, RV/septic tank treatment and in bioremediation and biodegradation, waste and wastewater treatment and water conditioning.

Responses to a voluntary questionnaire sent in 2007 to a subset of key biotechnology companies, combined with information obtained from other federal government regulatory and non-regulatory programs, indicate that DSL B. licheniformis/subtilisgroup strains were in commercial use in 2006. No information on uses of B. atrophaeus was collected at this time, as it was nominated to the DSL after the survey took place.

The Government conducted a mandatory information-gathering survey under section 71 of CEPA 1999, as published in the Canada Gazette, Part I, on October 3, 2009 (section 71 Notice). The section 71 Notice applied to any persons who, during the 2008 calendar year, manufactured or imported strains of the DSL B. licheniformis/subtilis group whether alone, in a mixture, or in a product. Commercial or consumer activity was reported for these micro-organisms in a variety of different sectors (for quantities and concentrations see Table 2-1 ). Uses reported for members of the DSL B. licheniformis/subtilisgroup include biodegradation; biological waste treatment; bioremediation; custodial cleaning and other related products; drain cleaning and degreasing; fragrance, perfume or deodorizer; enzyme and chemical production; research and development; septic tank or recreational vehicle tank additive; and waste and wastewater treatment. No information on uses of B. atrophaeus was collected through the section 71 Notice, as it was nominated to the DSL after the survey took place.

**Table 2-1 : Quantities of DSL *B. licheniformis/subtilis* group strains reported to be imported or manufactured in Canada in 2009^{Footnote Table 2-1[a]}**
Species^{Footnote Table 2-1 [b]}	Total Amount Range^{Footnote Table 2-1 [c]} (kg)	Concentration range^{Footnote Table 2-1 [d]}(CFU/mL)
Bacillus amyloliquefaciens	10,000-100,000	2.0 × 10⁸ to 1.0 × 10¹¹
Bacillus licheniformis	100,000-1,000,000	4.0× 10⁶ to 1.0 × 10¹¹
Bacillus subtilis^{Footnote Table 2-1 [e]}	100,000-1,000,000	1.0 × 10⁵ to 1.0 × 10¹¹

Footnote Table 2-1 a

No information on uses of B. atrophaeus was collected through the Notice as it was nominated to the DSL after the survey took place

Return to footnote Table 2-1 a referrer

Footnote Table 2-1 b

Includes all DSL strains of the species

Return to footnote Table 2-1 b referrer

Footnote Table 2-1 c

Combined amount of all products containing the micro-organisms manufactured in or imported to Canada

Return to footnote Table 2-1 c referrer

Footnote Table 2-1 d

Concentration range of micro-organisms reported to be imported or manufactured in Canada

Return to footnote Table 2-1 d referrer

Footnote Table 2-1 e

Including B. subtilis subsp. inaquosorum ATCC 55406

Return to footnote Table 2-1 e referrer

A search of the public domain (internet, patent databases, MSDS, etc.) suggests multiple potential uses of the B. subtiliscomplex including the DSL B. licheniformis/subtilis group strains.

B. amyloliquefaciens

As a production micro-organism of enzymes (e.g. amylase, isoprene, protease, non-structural protein 3, ribonuclease, and phytases), biosurfactants, antibiotics and detergents which have industrial and commercial applications (Madslien et al. 2012) including cleaning, degreasing, antibacterial applications (ATCC 2012c; James et al. 1995; Madslien et al. 2012; Moons et al. 2009; Pérez-García et al. 2011; Rendueles and Ghigo, 2012; Rivardo et al. 2009).
Application to surfaces to favour the formation of a B. amyloliquefaciens biofilm to displace undesirable or unknown micro-organisms (James et al. 1995; Moons et al. 2009; reviewed in Rendueles and Ghigo 2012; Rivardo et al. 2009).
Application in a mixture with other bacterial species for water and wastewater treatment to treat algal blooms, odours and sludge build-up (Advanced Water Technologies 2012; RoeTech 2014).

B. atrophaeus

Use of spores as a surrogate for weaponized B. anthracis in fine-tuning of defense monitoring equipment and as a challenge agent (Blecka et al. 2012; Carrera et al. 2007; Grinshpun et al. 2012; Page et al. 2007; U.S. EPA 2013a).
Use of spores to test the efficacy of sterilization by dry heat, ethylene oxide and steam sterilization as part of quality assurance and control in the production of pharmaceutical and personal care products (ATCC 2012d).
Pathogen transmission modelling (Gerhardts et al. 2012).

B. licheniformis

As a production organism of enzymes and biosurfactants including alpha-amylase, lichenysin, pentosanases, deoxyribonuclease (NucB), nitroreductase and levansucrase (ATCC 2013; reviewed in Komolprasert and Ofoli 1991; Moons et al. 2009; Nerurkar, 2010; reviewed in Rendueles and Ghigo 2012; Rey et al. 2004; Rivardo et al. 2009; Thatoi et al. 2013; Yakimov and Golyshin 1997).
Biosynthesis of silver nanocrystals (Kalimuthu et al. 2008) and gold nanocubes (Kalishwaralal et al. 2009).
Degradation of feather waste generated by poultry farms and processing plants (Ichida et al. 2001).
Bioremediation of heavy metals (e.g. zinc, cadmium and aluminum) (Kamika and Momba 2013).
Water and wastewater treatment to reduce algal blooms, odours and sludge build-up (Advanced Water Technologies 2012).
Bioindicator of the toxicity of sediment elutriates (Campbell et al. 1993).
Beneficial biofilm formation (James et al. 1995; Moons et al. 2009; reviewed in Rendueles and Ghigo 2012; Rivardo et al. 2009).
In probiotic products for humans and animals (Cutting 2011; Nithya et al. 2012).

B. subtilis

As a production organism of lipopeptides (biosurfactants), enzymes (e.g. amylase, protease and antibiotic compounds (e.g. aterrimin)and isoprene (ATCC 2012b; ATCC, 2012f; Moons et al. 2009; Rendueles and Ghigo 2012; Rivardo et al. 2009; reviewed in Thatoi et al. 2013).
Water and wastewater treatment to reduce algal blooms, odours, sludge build-up, septic tanks and agricultural waste pits (Advanced Water Technologies 2012; RoeTech 2014).
Fermentation of traditional foods (Inatsu et al. 2006; Leejeerajumnean 2003).
Beneficial biofilm formation (James et al. 1995; Moons et al. 2009; reviewed in Rendueles and Ghigo 2012; Rivardo et al. 2009).
Use of spores to test sterility assurance and in bacterial resistance of latex paint (ATCC 2012f).
Applications in research as a bacteriophage host (ATCC 2012f).
Diagnostic applications in blood screening for phenylketonuria (ATCC 2012f).
Application in the production of feed supplements (ATCC 2012a).
In probiotic products for humans and animals (Cutting 2011).

2.2 Exposure Characterisation

2.2.1 Environment

2.2.1.1 B. atrophaeus

Environmental exposure to B. atrophaeus 18250-7 is possible for terrestrial species, and to a lesser extent aquatic species, during its environmental release as a surrogate organism for B. anthracis in dispersal modelling and fine tuning of defense monitoring equipment. The extent of exposure will depend on the method of release, release volume, weather conditions and wind velocity. In general, exposure is expected to be low for these applications as it is a specialized activity occurring at a single, remote site in Canada. Inhalation would be the main route of exposure. Exposure as the result of dermal contact with contaminated surfaces and inadvertent ingestion through secondary contamination of food resources is expected to be low. The overall environmental exposure estimation for B. atrophaeus18250-7 is low.

2.2.1.2 B. amyloliquefaciens, B. licheniformis, B. subtilis and masked DSL Bacillus strains

Environmental exposure to the other DSL B. licheniformis/subtilis strains will be considered together, as the known and potential uses are similar.

Members of the B. subtilis complex have the ability to adapt to and thrive in many terrestrial and aquatic habitats. Numerous physiological variants exist in nature, making the complex highly successful in nearly every environment. Despite the widespread distribution of the species complex, there is evidence to demonstrate a decline in introduced populations artificially inoculated into soil microcosms and marine environments (Medina et al. 2003; Nybroe et al. 1992). High numbers of vegetative cells are unlikely to be maintained in water or soil due to competition for nutrients (Leung et al. 1995) and microbiostasis, which is an inhibitory effect of soil, resulting in the rapid decline of populations of introduced bacteria (Van Veen et al. 1997).

To estimate expected environmental concentrations from expected applications, case studies in bioremediation and wastewater treatment were explored. A mixture of Bacillus species including B. amyloliquefaciens and B. subtilis(up to 10¹¹ CFU/g) was added to treat municipal wastewater at a rate of 7.5 ppm of flow (RoeTech 2014), resulting in a concentration up to 7.5 × 10⁵ CFU/mL in the treated wastewater. In a bench scale proof of concept study, 1.5 × 10⁹ cells of a strain of B. subtilis were added to 60 g of petroleum hydrocarbon contaminated soil for a final concentration of 2.5 × 10⁷ cells/g (Wu et al. 2013). Such concentrations are unlikely to be maintained in wastewater effluent or soils as vegetative cells of the DSL B. licheniformis/subtilis strains do not have any competitive advantage over naturally-occurring populations of similar micro-organisms and would be subject to competition for nutrients with indigenous flora. Populations of vegetative cells of DSL B. licheniformis/subtilis strains introduced to soil and water will likely decrease to background levels over time. Under sub-optimal conditions, spores of the DSL B. licheniformis/subtilis strains are likely to persist and accumulate in the environment.

Exposure to the DSL strains is expected to be greatest for organisms in and around the vicinity of direct application to aquatic ecosystems for water treatment (e.g. aquaria and ponds) or to soils for bioremediation of contaminants.

Indirect exposure of environmental species resulting from the use and disposal of cleaning products is expected to be low relative to direct applications to aquatic ecosystems or soils. Growth in the market for "greener" microbial-based products may, however, increase such exposures (Spök and Klade 2009).

No relevant reports concerning the persistence of toxins produced by strains of the B. subtilis complex in the environment were found in a comprehensive search of the scientific literature over a number of sources.

The environmental exposure to the other DSL B. licheniformis/subtilis strains is expected to be medium based on the wide range of uses reported in response to the Notice.

2.2.2 Humans

2.2.2.1 B. atrophaeus

Human exposure to B. atrophaeus 18250-7 is possible for bystanders during its environmental release as a surrogate organism for B. anthracis in dispersal modelling and fine tuning of defense monitoring equipment. The extent of exposure will depend on the method of release, release volume, weather conditions, wind velocity and the proximity of bystanders to the site of application. In general, exposure is expected to be low for these applications as it is a specialized activity occurring at a single, remote site in Canada. Inhalation would be the main route of exposure. Exposure as the result of dermal contact with contaminated surfaces and inadvertent ingestion through secondary contamination of foodstuffs is expected to be low. The overall human exposure estimation forB. atrophaeus 18250-7 is low.

2.2.2.2 B. amyloliquefaciens, B. licheniformis, B. subtilis and masked DSL Bacillus strains

Human exposure to the other DSL B. licheniformis/subtilis strains will be considered together, as the known and potential uses are similar.

Human exposure is expected to be greatest through the direct use of consumer products containing spores or viable cells used for cleaning or water treatment. Handling and application of such products would be expected to result in direct exposure of the skin and inhalation of aerosolized droplets or lofted spores. Inadvertent ingestion following use on or near food preparation surfaces and contact with the eyes, are possible secondary routes of exposure.

Humans may also be exposed as bystanders during commercial application of cleaning, water treatment, agricultural or biodegradation products. The extent of bystander exposure will depend on the mode of application, the volume applied and the proximity of bystanders to the site of application. In general, exposure is expected to be low for these applications.

Indirect human exposure to the DSL B. licheniformis/subtilis strains released into the environment subsequent to their use in water treatment, agricultural applications or biodegradation is also expected to occur in the vicinity of treated sites, but is expected to be less than direct exposure from the use of these organisms in consumer products. Human exposure to bodies of water and soils treated with the DSL B. licheniformis/subtilis strains (e.g., through recreational activities), could result in exposure of the skin and eyes, as well as inadvertent ingestion; however, dilution of these products is expected to significantly reduce exposure relative to household application scenarios. Human activity on soils recently treated with the DSL B. licheniformis/subtilis strains could loft spores, which could then be inhaled and could expose the skin and eyes, but this exposure is also expected to be low relative to direct use of consumer products.

Release of the DSL B. subtilis/licheniformis strains from facilities manufacturing enzymes or biochemicals could occur, but is expected to be limited by the application of good manufacturing practices, in which measures should be taken to minimise the probability of releases of production micro-organisms.

For uses of pre- or probiotics containing spores of B. amyloliquefaciens, B. licheniformis and B. subtilis strains, direct exposure would be principally by oral ingestion. Indirect exposure could occur following disposal of probiotics or through shedding in feces into the wastewater system. In the case of feces or disposal into the sewage system, municipal wastewater treatment would be expected to reduce the microbial burden prior to the release of effluent into the environment. Human exposure to the strains through the environment is expected to be low. Disposal of unused probiotics to municipal landfills is not expected to result in significant human exposure.

In the event that spores of the DSL B. subtilis/licheniformis group enter the source waters of municipal drinking water treatment systems through release from intended and potential uses, drinking water treatment processes (e.g. coagulation, flocculation, ozonation, filtration and chlorination) are expected to effectively eliminate these micro-organisms and so limit their ingestion.

Exposure to the other DSL B. subtilis/licheniformisstrains is expected to be medium from the use of consumer products and low for indirect exposures subsequent to environmental release for biodegradation, bioremediation and water and wastewater treatment or release of effluents from facilities manufacturing enzymes and biochemicals.

Growth in the market for "greener" microbial-based products may increase direct human exposure to the DSL B. subtilis/licheniformis group which have potential applications in these products (Spök and Klade 2009).

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3. Risk Characterisation

In this assessment, risk is characterized according to a paradigm embedded in section 64 of CEPA 1999 that a hazard and exposure to that hazard are both required for there to be a risk. The risk assessment conclusion is based on the hazard, and on what is known about exposure from current uses.

The determination of risk from current uses is followed by consideration of the estimated hazard in relation to foreseeable future exposures (from new uses).

B. amyloliquefaciens

Hazard has been estimated for B. amyloliquefaciens13563-0 to be low for both the environment and human health. Environmental exposure to B. amyloliquefaciens 13563-0 is expected to be medium based on the wide range of uses reported in response to the section 71 Notice. Human exposure is expected to be medium for direct use of consumer products and low for indirect exposures subsequent to environmental release based on the wide range of uses reported in response to the section 71 Notice. The risk associated with current uses is estimated to be low for both the environment and human health.

B. atrophaeus

Hazard has been estimated for B. atrophaeus 18250-7 to be low for both the environment and human health. Environmental exposure to B. atrophaeus 18250-7 is expected to be medium and human exposure is expected to be low based on the known uses. The risk associated with current uses is estimated to be low for both the environment and human health.

The risk from foreseeable future uses is also expected to be low, given the low hazard associated with B. atrophaeus18250-7.

B. licheniformis

Hazard has been estimated for B. licheniformis ATCC 12713 to be low for both the environment and human health because the scientific literature and laboratory results specific to the DSL strain indicate a low pathogenic potential (consistent with the other strains under assessment), and there is a history of safe use of the DSL strain. B. licheniformis has been associated with livestock abortion. Routes of exposure leading to B. licheniformis abortion in livestock are thought to includeingestion of poor-quality, mouldy feed during gestation and subsequent hematogenous spread to the reproductive tract as well as introduction during general animal husbandry activities (e.g. natural breeding, artificial insemination, parturition and during examination) (Cabell, 2007; Scott, 2011; Goncagul, 2012). Current applications of the DSL strain are not expected to significantly increase exposure of livestock by these routes. Environmental exposure to B. licheniformis ATCC 12713 is expected to be medium based on the wide range of uses reported in response to the section 71 Notice. Human exposure is expected to be medium for direct use of consumer products and low for indirect exposures subsequent to environmental releases based on the wide range of uses reported in response to the section 71 Notice. The risk associated with current uses is estimated to be low for both the environment and human health.

Growth in the market for "greener" microbial-based products may increase human exposure to the DSL B. subtilis/licheniformis group which have potential applications in these products (Spök and Klade, 2009), however, the risk from foreseeable future uses is expected remain low for both humans and the environment given the low hazard associated with B. licheniformis ATCC 12713.

It is proposed to conclude that B. licheniformis ATCC 12713 does not meet any of the criteria set out in section 64 of CEPA 1999.

B. subtilis

Hazard has been estimated for B. subtilis ATCC 6051A, B. subtilis ATCC 55405, B. subtilis subsp. subtilis ATCC 6051 and B. subtilis subsp. inaquosorum ATCC 55406 to be low for both the environment and human health. Environmental exposure to B. subtilisATCC 6051A, B. subtilis ATCC 55405, B. subtilissubsp. subtilis ATCC 6051 and B. subtilis subsp. inaquosorum ATCC 55406 is expected to be medium based on the wide range of uses reported in response to the section 71 Notice. Human exposure is expected to be medium for direct use of consumer products and low for indirect exposures subsequent to environmental release based on the wide range of uses reported in response to the section 71 Notice. The risk associated with current uses is estimated to be low for both the environment and human health.

Growth in the market for "greener" microbial-based products may increase human exposure to the DSL B. subtilis/licheniformis group which have potential applications in these products (Spök and Klade, 2009), however, the risk from foreseeable future uses is also expected to be low, given the low hazard associated withB. subtilis ATCC 6051A, B. subtilis ATCC 55405, B. subtilis subsp. subtilis ATCC 6051^T and B. subtilissubsp. inaquosorum ATCC 55406 associated with both human and environmental health.

Masked DSL Bacillus Strains

Hazard has been estimated for Bacillus species 16970-5, Bacillus species 2 18118-1, Bacillus species 4 18121-4 and Bacillus species 7 18129-3 to be low for both the environment and human health based on laboratory results specific to the masked DSL strains and a history of safe use. Environmental exposure to Bacillus species 16970-5, Bacillus species 2 18118-1, Bacillus species 4 18121-4 and Bacillus species 7 18129-3 is expected to be medium based on the wide range of uses reported in response to the section 71 Notice. Human exposure is expected to be medium for direct use of consumer products and low for indirect exposures subsequent to environmental release based on the wide range of uses reported in response to the section 71 Notice. The risk associated with current uses is estimated to be low for both the environment and human health

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4. Conclusions

Based on information presented in this Screening Assessment, it is concluded that Bacillus amyloliquefaciens 13563-0, Bacillus atrophaeus 18250-7, Bacillus licheniformis ATCC 12713, Bacillus subtilis ATCC 6051A, Bacillus subtilis ATCC 55405, Bacillus subtilis subsp. subtilis ATCC 6051, Bacillus subtilis subsp. inaquosorum ATCC 55406, Bacillus species 16970-5, Bacillus species 2 18118-1, Bacillus species 4 18121-4, Bacillusspecies 7 18129-3 are not entering the environment in a quantity or concentration or under conditions that:

have or may have an immediate or long-term harmful effect in the environment or its biological diversity;
constitute or may constitute a danger to the environment on which life depends; or
constitute or may constitute a danger in Canada to human life or health.

Therefore, it is proposed that this substance does not meet the criteria as set out in section 64 of CEPA 1999.

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References

Abo-Elnaga, H.I., Hegazi, F.Z., and Abo-Elnaga, I.G. (2002). Spore-forming rods surviving boiling the raw milk and implicated in later spoilage of the product. Arch. Lebensmittelhyg. 53,86-89.

Abriouel, H., Franz, C.M.A.P., Omar, N.B., and Galvez, A. (2011). Diversity and applications of Bacillus bacteriocins. FEMS Microbiol. Rev. 35, 201-232.

Advanced Water Technologies. (2012). Advanced Water Technologies: Freshwater and Saltwater Liquid and Powder Based Bacterial Products. MSDS 1-2.

Agerholm, J.S., Jensen, N.E., Dantzer, V., Jensen, H.E., and Aarestrup, F.M. (1999). Experimental Infection of Pregnant Cows with Bacillus licheniformis Bacteria. Vet Pathol 36, 191-201.

Agerholm, J.S., Jensen, N.E., Giese, S.B., and Jensen, H.E. (1997). A preliminary study on the pathogenicity of Bacillus licheniformis bacteria in immunodepressed mice. APMIS 10⁵, 48-54.

Agerholm, J.S., Krogh, H.V., and Jensen, H.E. (1995). A Retrospective Study of Bovine Abortions Associated with Bacillus licheniformis. J. Vet. Med. B. 42,225-234.

Agerholm, J.S., Willadsen, C.M., Nielsen, T.K., Giese, S.B., Holm, E., Jensen, L., and Agger, J.F. (1997). Diagnostic Studies of Abortion in Danish Dairy Herds. J Vet Med A 44,551-558.

Agerholm, J.S., Jensen, H.E., and Jensen, N.E. (1995). Experimental infection in mice with Bacillus licheniformis. Zentralblatt Veterinarmedizin Reihe B 42, 247-256.

Ahmadnia Motlagh, H.R., Farhangi, M., Rafiee, G., and Noori, F. (2012). Modulating gut microbiota and digestive enzyme activities of Artemia urmiana by administration of different levels of Bacillus subtilis and Bacillus licheniformis. Aquacult. Int. 20, 693-705.

Ameur, M.A., Dubrous, P., and Koeck, J.L. (2005). Bacillus licheniformis: An unusual cause of erysipelosis. Med. Mal. Infect. 35, 417-418.

Andersson, A.M., Weiss, N., Rainey, F., and Salkinoja-Salonen, M.S. (1999). Dust-borne bacteria in animal sheds, schools and children's day care centres. J. Appl. Microbiol. 86,622-634.

André, S., Zuber, F., and Remize, F. (2013). Thermophilic spore-forming bacteria isolated from spoiled canned food and their resistance. Results of a French ten-year survey. Int J Food Microbiol 165, 134-143.

Anisimova M., Gascuel O. (2006). Approximate likelihood ratio test for branches: A fast, accurate and powerful alternative. Syst. Biol. 55, 539-552.

ANZ. (2013). Application A1044 - Pullanase from Bacillus subtilis as a Processing Aid (Enzyme). ANZ Food Standards.

ANZ. (2012a). Application A1050 - Acyltransferase from Bacillus licheniformis for use as an emulsifier. ANZ Food Standards.

ANZ. (2012b). Application A1061 - Amylomaltase from Bacillus amyloliquefaciens as a Processing Aid (Enzyme). ANZ Food Standards.

ANZ. (2011). Application summary for Application No 50240: Registration of a 2 × 10 exp 11 CFU/kg Bacillus subtilis prowder product for application in poultry. Aust Pest Vet Med Authority 1-3.

Ash, C., Farrow, J.A.E., Wallbanks, S., and Collins, M.D. (1991). Phylogenetic heterogeneity of the genus Bacillus revealed by comparative analysis of small-subunit-ribosomal RNA sequences. Lett. Appl. Microbiol. 13, 202-206.

ATCC. (2013). Product sheet: Bacillus licheniformis ATCC 12713.

ATCC. (2012a). Product sheet: Bacillus subtilis (Ehrenberg) Cohn (ATCC® 13933).

ATCC. (2012b). Product sheet: Bacillus subtilis(Ehrenberg) Cohn (ATCC® 6051a).

ATCC. (2012c). Product sheet: Bacillus amyloliquefaciens ATCC 23842.

ATCC. (2012d). Product sheet: of Bacillus atrophaeusATCC 9372. ATCC 2013,

ATCC. (2012e). Product sheet: Bacillus licheniformis ATCC14580.

ATCC. (2012f). Product sheet: Bacillus subtilis subsp. subtilis (Ehrenberg) Cohn (ATCC® 6051).

Auchtung, J.M., Lee, C.A., Monson, R.E., Lehman, A.P., and Grossman, A.D. (2005). Regulation of a Bacillus subtilis mobile genetic element by intercellular signaling and the global DNA damage response. Proc. Natl. Acad. Sci. U. S. A. 10², 12554-12559.

Banerjee, C., Bustamante, C.I., Wharton, R., Talley, E., and Wade, J.C. (1988). Bacillus infections in patients with cancer. Arch. Intern. Med. 148, 1769-1774.

Baradaran, G.R., and Ghasemi, A. (2010). Introducing Bacillus licheniformis as the causal agent of pistachio dieback in Iran.43, 597-601.

Baril, E., Coroller, L., Couvert, O., El Jabri, M., Leguerinel, I., Postollec, F., Boulais, C., Carlin, F., and Mafart, P. (2012). Sporulation boundaries and spore formation kinetics of Bacillus spp. as a function of temperature, pH and aw. Food Microbiol. 32, 79-86.

Beattie, S.H., and Williams, A.G. (1999). Detection of toxigenic strains of Bacillus cereus and other Bacillus spp. with an improved cytotoxicity assay. Lett. Appl. Microbiol. 28,221-225.

Beauregard, P.B., Chai, Y., Vlamakis, H., Losick, R., and Kolter, R. (2013). Bacillus subtilis biofilm induction by plant polysaccharides. Proc. Natl. Acad. Sci. U. S. A. 110,E1621-E1630.

Begley, M., Cotter, P.D., Hill, C., and Ross, R.P. (2009). Identification of a novel two-peptide lantibiotic, lichenicidin, following rational genome mining for LanM proteins. Appl. Environ. Microbiol. 75, 5451-5460.

Benson, D.A., Karsch-Mizrachi, I., Lipman, D.J., Ostell, J., and Sayers, E.W. (2009). GenBank. Nuc Acids Res 37,D26-31.

Biesta-Peters, E.G., Reij, M.W., Blaauw, R.H., In't Veld, P.H., Rajkovic, A., Ehling-Schulz, M., and Abee, T. (2010). Quantification of the emetic toxin cereulide in food products by liquid chromatography-mass spectrometry using synthetic cereulide as a standard. Appl. Environ. Microbiol. 76,7466-7472.

Blecka, M.I., Rataj, M., and Szymanski, G. (2012). Passive Detection of Biological Aerosols in the Atmosphere with a Fourier Transform Instrument (FTIR)-the Results of the Measurements in the Laboratory and in the Field.42, 101-111.

Blibech, I., Ksantini, M., Chaieb, I., Jlassi, B., Rhouma, A., Jaoua, S., and Aifa, S. (2012). Isolation of entomopathogenic Bacillus from a biodynamic olive farm and their pathogenicity to lepidopteran and coleopteran insect pests.31, 72-77.

Blue, S.R., Singh, V.R., and Saubolle, M.A. (1995). Bacillus licheniformis bacteremia: Five cases associated with indwelling central venous catheters. Clin. Infect. Dis. 20,629-633.

Borriss, R., Chen, X.H., Rueckert, C., Blom, J., Becker, A., Baumgarth, B., Fan, B., Pukall, R., Schumann, P., Spröer, C., Junge, H., Vater, J., Pühler, A., Klenk, H.P.(2011). Relationship of Bacillus amyloliquefaciens clades associated with strains DSM7^T and FZB42^T: a proposal for Bacillus amyloliquefaciens subsp. amyloliquefaciens subsp. nov. and Bacillus amyloliquefaciens subsp. plantarum subsp. nov. based on complete genome sequence comparisons. Int. J. Syst. Evol. Microbiol. 61, 1786-1801.

Brown, K.L. (2000). Control of bacterial spores. Br. Med. Bull. 56, 158-171.

Caballero, M.l., Gomez, M., Gonzalez-Muiioz, M., Reinoso, l., Rodriguez-Perez, R., Alday, E., and Moneo, I. (2007). Occupational sensitization to fungal enzymes used in animal feed undustry. Int Arch Allergy Immunol 144, 231-472.

Cabell, E. (2007). Bovine abortion: aetiology and investigations. In Pract. 29, 455-463.

Campbell, M., Bitton, G., and Koopman, B. (1993). Toxicity testing of sediment elutriates based on inhibition of alpha- glucosidase biosynthesis in Bacillus licheniformis. Arch. Environ. Contam. Toxicol. 24, 469-472.

Carrera, M., Zandomeni, R.O., Fitzgibbon, J., and Sagripanti, J. (2007). Difference between the spore sizes of Bacillus anthracis and other Bacillus species. J. Appl. Microbiol. 10², 303-312.

Castagnola, E., Conte, M., Venzano, P., Garaventa, A., Viscoli, C., Barretta, M.A., Pescetto, L., Tasso, L., Nantron, M., Milanaccio, C., and Giacchino, R. (1997). Broviac catheter-related bacteraemias due to unusual pathogens in children with cancer: Case reports with literature review. J. Infect. 34,215-218.

CDC. (1995). Foodborne Disease Outbreak Line Listing. CDC 1-11.

Celli, J., and Trieu-Cuot, P. (1998). Circularization of Tn916 is required for expression of the transposon-encoded transfer functions: Characterization of long tetracycline-inducible transcripts reading through the attachment site. Mol Microbiol 28, 10³-117.

CFIA. (2014). Identification and taxonomic classification of microorganism(s) represented for use as supplements under the Fertilizers Act. http://www.inspection.gc.ca/plants/fertilizers/registration-requirements/taxonomic-cassification/eng/1346524491267/1346527009874

Chung, S., Lim, J., and Kim, S. (2010). Powder formulation using heat resistant endospores of two multi-functional plant growth promoting rhizobacteria Bacillus strains having phytophtora blight suppression and growth promoting functions. J. Appl. Biol. Chem. 53, 485-492.

CLSI. (2010). Methods for Antimicrobial Dilution and Disk Susceptibility Testing of Infrequently Isolated or Fastidious Bacteria; Approved Guideline - Second Edition. CLSI document M45-A2. Wayne, PA, Clinical and Laboratory Standard Institute.

Contreras, G.A., and Rodríguez, J.M. (2011). Mastitis: Comparative etiology and epidemiology. J. Mammary Gland Biol. Neoplasia 16, 339-356.

Cordero-Ramírez, J.D., López-Rivera, R., Figueroa-Lopez, A.M., Mancera-López, M.E., Martínez-Álvarez, J.C., Apodaca-Sánchez, M.A., and Maldonado-Mendoza, I.E. (2013). Native soil bacteria isolates in Mexico exhibit a promising antagonistic effect against Fusarium oxysporum f. sp. radicis-lycopersici. J. Basic Microbiol.

Cosentino, S., Mulargia, A.F., Pisano, B., Tuveri, P., and Palmas, F. (1997). Incidence and biochemical characteristics of Bacillus flora in Sardinian dairy products. Int. J. Food Microbiol. 38, 235-238.

Cotton, D.J., Gill, V.J., Marshall, D.J., Gress, J., Thaler, M., and Pizzo, P.A. (1987). Clinical features and therapeutic interventions in 17 cases of Bacillus bacteremia in an immunosuppressed patient population. J. Clin. Microbiol. 25, 672-674.

Cox, R., Sockwell, G., and Landers, B. (1959). Bacillus subtilis septicemia: report of a case and review of the literature. N. Engl. J. Med. 261, 894-896.

Cutting, S.M. (2011). Review: Bacillus Probiotics. Food. Microbiol. 28, 214-220.

Dat, N.M., Hamanaka, D., Tanaka, F., and Uchino, T. (2012). Control of milk pH reduces biofilm formation of Bacillus licheniformis and Lactobacillus paracasei on stainless steel.23, 215-220.

Davies, F.L., and Wilkinson, G. (1973). Bacillus cereus in milk and dairy products. In The Microbiological Safety of Food, Hobbs, B. C., and Christian, J. H. B. eds., (London: Academic Press) pp. 3-126.

De Boer, W., Gunnewiek, P.J.A.K., Veenhuis, M., Bock, E., and Laanbroek, H.J. (1991). Nitrification at low pH by aggregated chemolithotrophic bacteria. Appl. Environ. Microbiol. 57,3600-3604.

De Jonghe, V., Coorevits, A., De Block, J., Van Coillie, E., Grijspeerdt, K., Herman, L., De Vos, P., and Heyndrickx, M. (2010). Toxinogenic and spoilage potential of aerobic spore-formers isolated from raw milk. Int. J. Food Microbiol. 136,318-325.

Delaney, B., Zhang, J., Carlson, G., Schmidt, J., Stagg, B., Comstock, B., Babb, A., Finlay, C., Cressman, R.F., Ladics, G., et al. (2008). A gene-shuffled glyphosate acetyltransferase protein from Bacillus licheniformis (GAT4601) shows no evidence of allergenicity or toxicity. Toxicol. Sci. 10²,425-432.

Dereeper A, Guignon V, Blanc G, Audic S, Buffet S, Chevenet F, Dufayard JF, Guindon S, Lefort V, Lescot M, Claverie JM, Gascuel O. (2008). Dedicated to reconstructing and analysing phylogenetic relationships between molecular sequences.Nuc. Acid. Res. 36, W465-9

Dischinger, J., Josten, M., Szekat, C., Sahl, H., and Bierbaum, G. (2009). Production of the novel two-peptide lantibiotic lichenicidin by Bacillus licheniformis DSM 13. PLoS ONE 4,

Drobniewski, F.A. (1993). Bacillus cereus and related species. Clin. Microbiol. Rev. 6, 324-338.

Dubnau, D., and Losick, R. (2006). Bistability in bacteria. Mol Microbiol 16, 269-275.

Duncanson, G.R. Nutrition and metabolic diseases in. Veterinary tremtnet of llamas and alpacas (Wallingford: CABI), 13-21.

Echeverri-Molina, D., and Santolamazza-Carbone, S. (2010). Toxicity of synthetic and biological insecticides against adults of the Eucalyptus snout-beetle Gonipterus scutellatus Gyllenhal (Coleoptera: Curculionidae). J. Pest Sci. 83, 297-305.

EFSA. (2008). Technical guidance: Update of the criteria used in the assessment of bacterial resistance to antibiotics of human or veterinatry importance. EFSA J. 732, 1-15.

Environment Canada and Health Canada. (2011). Framework on the Science-Based Risk Assessment of Micro-organisms under the Canadian Environmental Protection Act, 1999. http://www.ec.gc.ca/subsnouvelles-newsubs/default.asp?lang=En&n=120842D5-1.

Euzéby, J.P. (2013). List of Prokaryotic Names with Standing in Nomenclature - Genus Bacillus. LPSN 2013.

Fakruddin, M., Sarker, N., Ahmed, M.M., and Noor, R. (2012). Protein Profiling of Bacillus thuringiensis Isolated from Agro-forest Soil in Bangladesh. AsPac J Mol Biol Biotechnol 20, 139-145.

Fangio, M.F., Roura, S.I., and Fritz, R. (2010). Isolation and identification of Bacillus spp. and related genera from different starchy foods. J. Food Sci. 75, M218-M221.

Filée, P., Benlafya, K., Delmarcelle, M., Moutzourelis, G., Frère, J., Brans, A., and Joris, B. (2002). The fate of the BlaI repressor during the induction of the Bacillus licheniformis BlaP ß-lactamase. Mol. Microbiol. 44, 685-694.

Flindt, M.L., Hendrick, D.J. (2002). Papers that have changed the practice of occupational medicine: Pulmonary disease due to inhalation of derivatives of Bacillus subtilis containing proteolytic enzyme by M.L.H. Flindt. 1969. Occup Med (Lond). 52, 57–63.

Flores, C.M., Ravishankar, J., Ajiboye, P., and Kramer, M. (2001). Bacillus licheniformis causing brain abscess in a normal host. Clin Infect Dis 33, 146.

From, C., Pukall, R., Schumann, P., Hormazábal, V., and Granum, P.E. (2005). Toxin-producing ability among Bacillus spp. outside the Bacillus cereus group. Appl. Environ. Microbiol. 71,1178-1183.

Galanos, J., Perera, S., Smith, H., O'Neal, D., Sheorey, H., and Waters, M.J. (2009). Bacteremia Due to Three BacillusSpecies in a Case of Muchausen's Syndrome. J Clin Microbiol 41, 2247-2248.

Galiero, G., and De Carlo, E. (1998). Abortion in water buffalo (Bubalus bubalis) associated with Bacillus licheniformis. Vet. Rec. 143, 640.

Galvez, A., Valdivia, E., Gonzalez-Segura, A., Lebbadi, M., Martinez-Bueno, M., and Maqueda, M. (1993). Purification, characterization, and lytic activity against Naegleria fowleri of two amoebicins produced by Bacillus licheniformis A12. Appl. Environ. Microbiol. 59, 1480-1486.

Gayet, S., Garcin, O., Mazodier, K., Kaplanski, G., Bernit, E., Schleinitz, N., Veit, V., and Harlé, J.R. (2005). Bacillus licheniformis bacteremia in an immunocompetent man [2]. Rev. Med. Interne 26, 250-251.

Gerhardts, A., Hammer, T.R., Balluff, C., Mucha, H., and Hoefer, D. (2012). A model of the transmission of micro-organisms in a public setting and its correlation to pathogen infection risks. J. Appl. Microbiol. 112, 614-621.

Gill, J.M. (1999). Bacillus species - an occasional cause of sheep abortion.39-40.

Goncagul, G., SeyrekIntas, K., Kumru, I.H., Ozakin, C., Ozdemir, S.E.S., Weiss, R., and PrengerBerninghoff, E. (2012). Bacterial infertility and ascending uterine infections with respect to pneumovagina and urovagina in cows.2, 583-586.

Griffiths, M.W. (1990). Toxin production by psychotropic Bacillus spp. present in milk. J Food Prot 53,790-792.

Grinshpun, S.A., Adhikari, A., Yermakov, M., Reponen, T., Dreizin, E., Schoenitz, M., Hoffmann, V., and Zhang, S. (2012). Inactivation of aerosolized Bacillus atrophaeus (BG) endospores and MS2 viruses by combustion of reactive materials.46,7334-7341.

Grossman, A.D., and Losick, R. (1988). Extracellular Control of Spore Formation in Bacillus subtilis. Proc. Natl. Acad. Sci. U. S. A. 85, 4369-4373.

Hameeda, B., Rupela, O.P., and Reddy, G. (2006). Antagonistic activity of bacteria inhabiting composts against soil-borne plant pathogenic fungi. Indian J. Microbiol. 46, 389-396.

Hannah, W.N., and Ender, P.T. (1999). Persistent Bacillus licheniformis bacteremia associated with an intentional injection of organic drain cleaner. Clin. Infect. Dis. 29,659-661.

Hardy, A., Christmann, D., Feger, J.M., Pasquali, J.L., and Storck, D. (1986). État septique après artériographie. Du rôle d'un germe réputé non pathogène : Bacillus licheniformis.16, 37-38.

Hare, W.R., Hoyt, P.G., Hohn, C., and Higgins, J.A. (2008). Ribosomal RNA-based analysis of the bacterial flora from the conjunctivae of cattle with bovine keratoconjunctivitis (BKC). Vet. Microbiol. 131, 358-368.

Haydushka, I.A., Markova, N., Kirina, V., and Atanassova, M. (2012). Recurrent sepsis due to Bacillus licheniformis. J. Global Infect. Dis. 4, 82-83.

He, L., Chen, W.L., and Liu, Y. (2006). Production and partial characterization of bacteriocin-like pepitdes by Bacillus licheniformis ZJU12. Microbiol. Res. 161, 321-326.

Hendriksen, N.B., and Carstensen, J. (2013). Long-term survival of Bacillus thuringiensis subsp. kurstaki in a field trial. Can. J. Microbiol. 59, 34-38.

Hendriksen, N.B., and Hansen, B.M. (2002). Long-term survival and germination of Bacillus thuringiensis car. kurstaki in a field trial. Can J Microbiol 48,256-261.

Hoa, T.T., Duc, L.H., Isticato, R., Baccigalupi, L., Ricca, E., Van, P.H., and Cutting, S.M. (2001). Fate and Dissemination of Bacillus subtilis Spores in a Murine Model. Appl. Environ. Microbiol. 67, 3819-3823.

Hosoi, T., Ametani, A.Kiuchi, K., Kaminogawa. (2000). Improved growth and viability of lactobacilli in the presence of Bacillus subtilis (natto), catalase, or subtilisin." Can. J. Microbiol. 46, 892-897.

Hong, T.W., Kim, H.Y., Jee, M.G., Choi, J.W., Youg, S.J., Shin, K.C., and Lee, W.Y. (2004). A case of Bacillus licheniformis bacteremia associated with bronchoscopy. Tuberc. Respir. Dis. 57, 553-556.

Huang, J.-M., La Ragione, R. M., Nunez, A., Cutting, S.M. (2008). Immunostimulatory activity of Bacillus spores. FEMS Immunol. Med. Microbiol. 53, 195-203.

Huang, Q., Xu, X., Mao, Y., Huang, Y., Rajput, I.R., Li, W. (2013). Effects of Bacillus subtilis B10 spores on viability and biological functions of murine macrophages. Animal Sci. J. 84,247-252.

Ichida, J.M., Krizova, L., LeFevre, C.A., Keener, H.M., Elwell, D.L., and Burtt E.H., J. (2001). Bacterial inoculum enhances keratin degradation and biofilm formation in poultry compost. J. Microbiol. Methods 47, 199-208.

Ihde, D.C., and Armstrong, D. (1973). Clinical spectrum of infection due to Bacillus species. Am. J. Med. 55,839-845.

Inatsu, Y., Nakamura, N., Yuriko, Y., Fushimi, T., Watanasiritum, L., and Kawamoto, S. (2006). Characterization of Bacillus subtilis strains in Thya nao, a traditional fermented soybean food in northern Thailand. Lett Appl Microbiol 43, 237-242.

Ishihara, H., Takoh, M., Nishibayashi, R., and Sato, A. (2002). Distribution and variation of bacitracin synthetase gene sequences in laboratory stock strains of Bacillus licheniformis. Curr. Microbiol. 45, 18-23.

Jamalizadeh, M., Etebarian, H.R., Alizadeh, A., and Aminian, H. (2008). Biological control of gray mold on apple fruits by Bacillus licheniformis (EN74-1). Phytoparasitica 36, 23-29.

James, G.A., Beaudette, L., and Costerton, J.W. (1995). Interspecies bacterial interactions in biofilms. J Ind Microbiol 15, 257-262.

Jephcott, A.E., Barton, B.W., Gilbert, R.J., and Shearer, C.W. (1977). An unusual outbreak of food-poisoning associated with meals-on-wheels. Lancet 129-130.

Jeyaram, K., Romi, W., Singh, T.A., Adewumi, G.A., Basanti, K., and Oguntoyinbo, F.A. (2011). Distinct differentiation of closely related species of Bacillus subtilis group with industrial importance. J Microbiol Methods 87, 161-164.

Johnson, C.T., Lupson, G.R., and Lawrence, K.E. (1994). The bovine placentome in bacterial and mycotic abortions. Vet. Rec. 134, 263-266.

Jones, B.L., Hanson, M.F., and Logan, N.A. (1992). Isolation of Bacillus licheniformis from a brain abscess following a penetrating orbital injury. J. Infect. 24,10³-10⁴.

Juniper, C.P., How, M.J., Goodwin, B.F.J., and Kinshott, A.K. (1977). Bacillus subtilis enzymes: a 7 year clinical, epidemiological and immunological study of an industrial allergen. J. Soc. Occup. Med. 27, 3-12.

Kalimuthu, K., Suresh Babu, R., Venkataraman, D., Bilal, M., and Gurunathan, S. (2008). Biosynthesis of silver nanocrystals by Bacillus licheniformis. Colloids Surf. B Biointerfaces 65,150-153.

Kalishwaralal, K., Deepak, V., Ram Kumar Pandian, S., and Gurunathan, S. (2009). Biological synthesis of gold nanocubes from Bacillus licheniformis. Bioresour. Technol. 100,5356-5358.

Kamika, I., and Momba, M.N.B. (2013). Assessing the resistance and bioremediation ability of selected bacterial and protozoan species to heavy metals in metal-rich industrial wastewater. BMC Microbiol. 13,

Katz, E., and Demain, A.L. (1977). The Peptide Antibiotics of Bacillus: Chemistry, Biogensis, and Possible Functions. Bacteriol Rev 41, 449-474.

Kim, H., Lee, E., Bae, E., Kim, M., Hur, J., Cho, O., Kang, D.H., Kim, S., Jun, J., and Bae, I. (2012). A case of Bacillus licheniformis spondylitis and bacteremia in a patient with lung cancer. Infect. Chemother. 44, 512-515.

Kimura, K., Tran, L.P., and Funane, K. (2011). Loss of poly-γ-glutamic acid synthesis of Bacillus subtilis (natto) due to IS4Bsu1 translocation to swrA gene. Food Sci. Technol. Res. 17, 447-451.

Kirkbride, C.A. (1993). Bacterial agents detected in a 10-year study of bovine abortions and stillbirths.5, 64-68.

Kirkbride, C.A., Collins, J.E., and Gates, C.E. (1986). Porcine abortion caused by Bacillus sp. J. Am. Vet. Med. Assoc. 188, 10⁶0-10⁶1.

Koehler, T.M., and Thorne, C.B. (1987). Bacillus subtilis (natto) plasmid pLS20 mediates interspecies plasmid transfer. J Bacteriol 169, 5271-5278.

Komolprasert, V., and Ofoli, R.Y. (1991). A dispersion model for predicting the extent of starch liquefaction by Bacillus licheniformis α-amylase during reactive extrusion. Biotechnol. Bioeng. 37, 681-690.

Kramer, J.M., and Gilbert, R.J. (1989). In Bacillus cereus and other Bacillus species; Foodborne Bacterial Pathogens, Doyle, M. P. ed., (New York: Marcel Dekker Inc) pp. 21-70.

Kreft, J., and Hughes, C. (1982). Cloning vectors derived from plasmids and phage of Bacillus. Curr Top Microbiol Immunol 96, 1-17.

La Jeon, Y., Yang, J.J., Kim, M.J., Lim, G., Cho, S.Y., Park, T.S., Suh, J., Park, Y.H., Lee, M.S., Kim, S.C., and Lee, H.J. (2012). Combined Bacillus licheniformis and Bacillus subtilis infection in a patient with oesophageal perforation. J. Med. Microbiol. 61, 1766-1769.

Lapidus, A., Galleron, N., Andersen, J.T., Jorgensen, P.L., Ehrlich, S.D., and Sorokin, A. (2002). Co-linear scaffold of the Bacillus licheniformis and Bacillus subtilis genomes and its use to compare their competence genes. FEMS Microbiol. Lett. 209,23-30.

Lebbadi, M., Galvez, A., Valdivia, E., Martinez-Bueno, M., and Maqueda, M. (1994). Biological activity of amoebicin m4-A from Bacillus licheniformis M-4. Antimicrob. Agents Chemother. 38, 1820-1823.

Leejeerajumnean, A. (2003). Thus nao: Alkali Fermented Soybean from Bacillus subtilis. Silpakorn University Int J. 3, 277-292.

Lépine, A., Michel, F., Nicaise, C., Imbert, G., Vialet, R., Thomachot, L., Di Marco, J., Lagier, P., and Martin, C. (2009). Bacillus licheniformis septicemia in a very-low-birth-weight neonate: A case report. Infection 37, 156-158.

Leung, K., Trevors, J.T., and Lee, H. (1995). Survival of and lacZ expression recombinant Pseudomonas strains introduced into river water microcosms. Can. J. Microbiol. 41,461-469.

Li, Y., Yang, S., and Mu, B. (2010). The surfactin and lichenysin isoforms produced by Bacillus licheniformis HSN 221. Anal Lett 43, 929-940.

Lindsay, D., Mosupye, F.M., Brözel, V.S., and Von Holy, A. (2000). Cytotoxicity of alkaline-tolerant dairy-associated Bacillus spp. Lett. Appl. Microbiol. 30, 364-369.

Link, R., and Kovác, G. (2006). The effect of probiotic BioPlus 2B on feed efficiency and metabolic parameters in swine. Biologia 61, 783-787.

Logan, N.A. (1988). Bacillus species of medical and veterinary importance. J Med Microbiol 25, 157-165.

Logan, N.A. (2012). Bacillus and relatives in foodborne illness. J. Appl. Microbiol. 112, 417-429.

Logan, N.A., and De Vos, P. (2009). In Genus I. Bacillus; Bergey's Manual of Systematic Bacteriology. Volume 3: The Firmicutes, De Vos, P., Garrity, G., Jones, D., Krieg, N. R., Ludwig, W., Rainey, F. A., Schleifer, K. -H and Whitman, W. B. Eds eds., (New York: Springer) pp. 21-127.

Longo, F., Pavone, E., and Califano, L. (2003). About a case of parotid gland abscess by Bacillus Licheniformis [2]. Br. J. Plast. Surg. 56, 424-425.

Lund, B.M. (1990). Foodborne disease due to Bacillus and Clostridium species. Lancet 336, 982-986.

Madslien, E.H., Olsen, J.S., Granum, P.E., and Blatny, J.M. (2012). Genotyping of B. licheniformis based on a novel multi-locus sequence typing (MLST) scheme.BMC Microbiol.12,230-237.

Majewski, J., and Cohan, F.M. (1998). The effect of mismatch repair and heteroduplex formation on sexual isolation in Bacillus. Genetics 148, 13.

Mandal, M.D., Mandal, S., and Pal, N.K. (2005). Plasmid-mediated dimethoate degradation by Bacillus licheniformis isolated from a fresh water fish Lobeo rohita. J. Biomed. Biotechnol. 2005, 280-286.

Marra, D., and Scott, J.R. (1999). Regulation of excision of the conjugative transposon Tn916. Mol. Microbiol. 31,609-621.

Matros, L., Wheeler, T., Microbiology Team, IVS Sacramento. (2001). Microbiology Guide to Interpreting MIC (Minimum Inhibitory Concentration). http://the-vet.net/DVMWiz/Vetlibrary/Lab-%20Microbiology%20Guide%20to%20Interpreting%20MIC.htm

Matsumoto, S., Suenaga, H., Naito, K., Sawazaki, M., Hiramatsu, T., and Agata, N. (2000). Management of suspected nosocomial infection: An audit of 19 hospitalized patients with septicemia caused by Bacillus species. Jpn. J. Infect. Dis. 53,196-202.

Maucour, M.F., Brugniart, C., Ducasse, A., Brasme, L., and Bajolet, O. (1999). [Bacillary endophthalmitis. Four case reports]. J. Fr. Ophtalmol. 22, 371-376.

McEwen, B., Carman, S. (2005). Animal Health Laboraotry Reports - Cattle: Bovine abortion update, 1998-2004. Can. Vet. J. 46, 46

Medina, A., Probanza, A., Gutierrez Mañero, F.J., and Azcón, R. (2003). Interactions of arbuscular-mycorrhizal fungi and Bacillus strains and their effects on plant growth, microbial rhizosphere activity (thymidine and leucine incorporation) and fungal biomass (ergosterol and chitin). Appl. Soil Ecol. 22, 15-28.

Meijer, W.J.J. (1995). Replication and maintenance of plasmids in Bacillus subtilis. State Univerity of Groningen, the Netherlands

Meijer, W.J.J., Wisman, G.B.A., Terpstra, P., Thorsted, P.B., Thomas, C.M., Holsappel, S., Venema, G., and Bron, S. (1998). Rolling-circle plasmids from Bacillus subtilis: Complete nucleolide sequences and analyses of genes of pTA1015, pTA1040, pTA1050 and pTA1060, and comparisons with related plasmids from Gram-positive bacteria. FEMS Microbiol. Rev. 21, 337-368.

Mekete, T., Hallmann, J., Kiewnick, S., and Sikora, R. (2009). Endophytic bacteria from ethiopian coffee plants and their potential to antagonise meloidogyne incognita. Nematology 11, 117-127.

Mendelsohn, M., and Vaituzis, Z. (1999). Non-target Organisms Registration Data Requirements Risk Assessment for AgraQuest, Inc.'s Product Containing the Microbial Active Ingredient Bacillus subtilis train QST-731. U.S. EPA 1-44.

Merrifield, D.L., Bradley, G., Baker, R.T.M., and Davies, S.J. (2010a). Probiotic applications for rainbow trout (Oncorhynchus mykiss Walbaum) II. Effects on growth performance, feed utilization, intestinal microbiota and related health criteria postantibiotic treatment. Aquacult. Nutr. 16, 496-503.

Merrifield, D.L., Dimitroglou, A., Bradley, G., Baker, R.T.M., and Davies, S.J. (2010b). Probiotic applications for rainbow trout (Oncorhynchus mykiss Walbaum) I. Effects on growth performance, feed utilization, intestinal microbiota and related health criteria. Aquacult. Nutr. 16, 504-510.

Mikkola, R., Andersson, M.A., Teplova, V., Grigoriev, P., Kuehn, T., Loss, S., Tsitko, I., Apetroaie, C., Saris, N.L., Veijalainen, P., and Salkinoja-Salonen, M.S. (2007). Amylosin from Bacillus amyloliquefaciens, a K+ and Na+ channel-forming toxic peptide containing a polyene structure. Toxicon 49, 1158-1171.

Mikkola, R., Kolari, M., Andersson, M.A., Helin, J., and Salkinoja-Salonen, M.S. (2000). Toxic lactonic lipopeptide from food poisoning isolates of Bacillus licheniformis. Eur. J. Biochem. 267, 4068-4074.

Mitchell, G., and Barton, M.G. (1986). Bovine abortion associated with Bacillus licheniformis. Aust Vet J 63, 160-161.

Mochiduki, Y., Amemiya, T., and Yabe, M. (2007). Brain abscess induced by Bacillus licheniformis complications in acute myeloid leukemia (AML). Kansenshogaku Zasshi 81, 592-596.

Moons, P., Michiels, C.W., and Aertsen, A. (2009). Bacterial interactions in biofilms. Crit Rev Microbiol 35,157-168.

Mosupye, F.M., Lindsay, D., Damelin, L.H., and Von Holy, A. (2002). Cytotoxicity assessment of Bacillus strains isolated from street-vended foods in Johannesburg, South Africa. J. Food Saf. 22, 95-10⁵.

Mullany, P., Wilks, M., and Tabaqchali, S. (1995). Transfer of macrolide-lincosamide-streptogramin B (MLS) resistance in Clostridium difficile is linked to a gene homologous with toxin A and is mediated by a conjugative transposon, Tn5398. J. Antimicrob. Chemother. 35, 305-315.

Murray, P.R., Baron, E.J., Jorgensen, J.H., Landry, M.L., and Pfaller, M.A. (1995). Bacillus. In Manual of Clinical Microbiology. ASM 8-349-356.

Murray, R.D. (2006). Practical approach to infectious bovine abortion diagnosis. 24^th World Buiatrics Congress, Nice, France. World Association of Buiatrics: Paris. 260-268.

Nakamura, L.K. (1989). Taxonomic relationship of black-pigmented Bacillus subtilis strains and a proposal for Bacillus atrophaeus sp. nov. Int. J. Syst. Bacteriol. 39, 295-300.

Nerurkar, A.S. (2010). Structural and molecular characteristics of lichenysin and its relationship with surface activity. Adv Exp Med Biol 672, 304-315.

Nieminen, T., Rintaluoma, N., Andersson, M., Taimisto, A., Ali-Vehmas, T., Seppälä, A., Priha, O., and Salkinoja-Salonen, M. (2007). Toxinogenic Bacillus pumilus and Bacillus licheniformis from mastitic milk. Vet. Microbiol. 124, 329-339.

Nithya, V., Muthukumar, S.P., and Halami, P.M. (2012). Safety assessment of Bacillus licheniformis Me1 isolated from milk for probiotic application. Int. J. Toxicol. 31, 228-237.

Norris, J.R., Berkeley, R.C.W., Logan, N.A., and O'Donnell, A.G. (1981). The genera Bacillus and Sporolactobacillus; The Prokaryotes: A handbook on habitats, isolation, and identification of bacteria (Berlin: Springer-Verlag).

Nybroe, O., Christoffersen, K., and Riemann, B. (1992). Survival of Bacillus licheniformis in seawater model ecosystems. Appl. Environ. Microbiol. 58, 252-259.

Oguntoyinbo, F.A., and Sanni, A.I. (2007). Determination of toxigenic potentials of Bacillus strains isolated from Okpehe, a Nigerian fermented condiment. World J Microbiol Biotechnol 23, 65-70.

Olszewski, W., Jamal, S., Manokaran, G., Pani, S., Kumaraswami, V., and Kubicka, U. (1999). Bacteriological studies of blood, tissue fluid, lymph and lymph nodes in patients with acute dermatolymphangioadenitis (DLA) in course of filarial lymphedema. Acta Tropica 73, 217-224.

Ozkocaman, V., Ozcelik, T., Ali, R., Ozkalemkas, F., Ozkan, A., Ozakin, C., Akalin, H., Ursavas, A., Coskun, F., Ener, B., and Tunali, A. (2006). Bacillus spp. among hospitalized patients with haematological malignancies: clinical features, epidemics and outcomes. J. Hosp. Infect. 64, 169-176.

Page, W.F., Young, H.A., and Crawford, H.M. (2007). Long-term health effects of participation in project SHAD (500 Fifth Street, NW, Washington, DC: The National Academies Press).

Pavic, S., Brett, M., Petric, I., Laštre, D., Smoljanovic, M., Atkinson, M., Kovacic, A., Cetinic, E., and Ropac, D. (2005). An outbreak of food poisoning in a kindergarten caused by milk powder containing toxigenic Bacillus subtilis and Bacillus licheniformis. Arch. Lebensmittelhyg. 56, 20-22.

Pedersen, P.B., Bjørnvad, M.E., Rasmussen, M.D., and Petersen, J.N. (2002). Cytotoxic potential of industrial strains of Bacillus sp. Regul. Toxicol. Pharmacol. 36, 155-161.

Peloux, Y., Charrel Taranger, C., and Gouin, F. (1976). New opportunist infection due to a bacillus. A case of bacteremia with Bacillus licheniformis. PATH. BIOL. 24, 97-98.

Pennington, J.E., Gibbons, N.D., Strobeck, J.E., Simpson, G.L., and Myerowitz, R.L. (1976). Bacillus species infection in patients with hematologic neoplasia. J Am Med Assoc 235,1473-1474.

Pérez-García, A., Romero, D., and de Vicente, A. (2011). Plant protection and growth stimulation by microorganisms: Biotechnological applications of Bacilli in agriculture. Curr. Opin. Biotechnol. 22, 187-193.

Peypoux, F., Bonmatin, J.M., and Wallach, J. (1999). Recent trends in the biochemistry of surfactin. Appl. Microbiol. Biotechnol. 51, 553-563.

Phelps, R.J., and McKillip, J.L. (2002). Enterotoxin Production in Natural Isolates of Bacillaceae outside of the Bacillus cereus Group. Appl Environ Microbiol 68,3147-3151.

PMRA-HC. (2014). Pesticides and Pest Management - Protecting Your Health and the Environment - Public Registry - Product Information - By Active.

PMRA-HC. (2013). Proposed Registration Decision PRD2013-14, Bacillus subtilis strain GB03.

PMRA-HC. (2012). Proposed Decision Decision PRD2012-32: Bacillus subtilis var. amyloliquefaciens strain FZB24.

PMRA-HC. (2007a). Bacillus subtilis strain MBI 600. Proposed Registration Decision.

PMRA-HC. (2007b). Evaluation Report for Bacillus subtilis strain QST 713. ERC2007-06.

PMRA-HC. (2007c). Proposed Registration Decision for Bacillus subtilis strain MBI 600. PDR2007 -05.

Pragai, Z., Tran, S.L.P., Nagy, T., Fulop, L., Holczinger, A., and Sik, T. (1994). Transposon Tn917PF1 mutagenesis in Bacillus licheniformis. Microbiol. 140, 3091-3097.

Priest, F.G., Goodfellow, M., Shute, L.A., and Berkeley, R.C.W. (1987). Bacillus amyloliquefaciens sp. nov., nom. rev. Int. J. Syst. Bacteriol. 37, 69-71.

Providenti, M.A., Begin, M., Hynes, S., Lamarche, C., Chitty, D., Hahn, J., Beaudette, L.A., Scroggins, R., and Smith, M.L. (2009). Identification and application of AFLP-derived genetic markers for quantitative PCR-based tracking of Bacillus and Paenibacillus spp. released in soil. Can. J. Microbiol. 55, 1166-1175.

Quan, J., Darer, J.D., Sewell, D.L., and Strausbaugh, L.J. (2000). Pacemaker wire infection caused by Bacillus licheniformis. Clin. Microbiol. Newsl. 22, 110-111.

Rahimi, S., and Kahsksefidi, A. (2006). A comparison between the effects of a probiotic (Bioplus 2B) and an antibiotic (virginiamycin) on the performance of broiler chickens under heat stress conditions. Iran. J. Vet. Res 7, 23-28.

Rajarajan, N., Ward, A.C., Burgess, J.G., and Glassey, J. (2013). Use of physiological information and process optimisation enhances production of extracellular nuclease by a marine strain of Bacillus licheniformis. Bioresour. Technol. 130,552-558.

Rajchard, J. (2010). Biologically active substances of bird skin: a review. Vet. Med. 55, 413-421.

Rauch, M.E., Graef, H.W., Rozenzhak, S.M., Jones, S.E., Bleckmann, C.A., Kruger, R.L., Naik, R.R., and Stone, M.O. (2006). Characterization of microbial contamination in United States Air Force aviation fuel tanks. J. Ind. Microbiol. Biotechnol. 33, 29-36.

Ray, P., Sanchez, C., O'Sullivan, D.J., McKay, L.L. (2000). Classification of a bacterial isolate, from pozol, exhibiting antimicrobial activity against several gram-positive and gram-negative bacteria, yeasts, and molds." J. Food Prot. 63, 1123-1132.

Reinhardt, K., Naylor, R.A., and SivaJothy, M.T. (2005). Potential sexual transmission of environmental microbes in a traumatically inseminating insect. Ecol. Entomol. 30,607-611.

Rendueles, O., and Ghigo, J. (2012). Multi-species biofilms: How to avoid unfriendly neighbors. FEMS Microbiol. Rev. 36,972-989.

Rey, M.W., Ramaiya, P., Nelson, B.A., Brody-Karpin, S.D., Zaretsky, E.J., Tang, M., Lopez de Leon, A., Xiang, H., Gusti, V., Clausen, I.G., et al. (2004). Complete genome sequence of the industrial bacterium Bacillus licheniformis and comparisons with closely related Bacillus species. Genome Biol. 5.

Richard, V., Van Der Auwera, P., Snoeck, R., Daneau, D., and Meunier, F. (1988). Nosocomial bacteremia caused by Bacillus species.7, 783-785.

Rivardo, F., Turner, R.J., Allegrone, G., Ceri, H., and Martinotti, M.G. (2009). Anti-adhesion activity of two biosurfactants produced by Bacillus spp. prevents biofilm formation of human bacterial pathogens. Appl. Microbiol. Biotechnol. 83, 541-553.

Roberts, M., and Cohan, F. (1993). The effect of DNA sequence divergence on sexual isolation in Bacillus. Genetics 134, 401.

Roberts, A.P., Braun, V., Von Eichel-Streiber, C., and Mullany, P. (2001). Demonstration that the group II intron from the clostridial conjugative transposon Tn5397 undergoes splicing in vivo. J. Bacteriol. 183, 1296-1299.

RoeTech. (2014). CASE STUDY: Treatment of Municipal Wastewater Treatment Plant for Hydrogen Sulfide Control with RoeTech 10⁶ Bacteria Culture.

Rooney, A.P., Sewzey, J.L., Wicklow, D.T., McAtee, M.J. (2005). Bacterial Species Diversity in Cigarettes Linked to an Investigation of Severe Pneumonitis in U.S. Military Personnel Deployed in Operation Iraqi Freedom. Curr. Microbiol. 51, 46-52.

Rooney, A.P., Price, N.P.J., Ehrhardt, C., Sewzey, J.L., and Bannan, J.D. (2009). Phylogeny and molecular taxonomy of the Bacillus subtilis species complex and description of Bacillus subtilis subsp. inaquosorum subsp. nov. Int. J. Syst. Evol. Microbiol. 59, 2429-2436.

Rosenkvist, H., and Hansen, Å. (1995). Contamination profiles and characterisation of Bacillus species in wheat bread and raw materials for bread production. Int. J. Food Microbiol. 26, 353-363.

Rowan, N.J., Deans, K., Anderson, J.G., Gemmell, C.G., Hunter, I.S., and Chaithong, T. (2001). Putative virulence factor expression by clinical and food isolates of Bacillus spp. after growth in reconstituted infant milk formulae. Appl. Environ. Microbiol. 67, 3873-3881.

Rubinstein, I., Pedersen, G.W. (2002). Bacillus Species Are Present in Chewing Tobacco Sold in the United States and Evoke Plasma Exudation from the Oral Mucosa. Clin. Diag. Lab. Immunol. 9, 10⁵7-10⁶0.

Ruckert, C., Blom, J., Chen, X., Reva, O., and Borriss, R. (2011). Genome sequence of B. amyloliquefaciens type strain DSM7^T reveals differences to plant-associated B. amyloliquefaciens FZB42. J Biotechnol 155, 78-85.

Salkinoja-Salonen, M.S., Vuorio, R., Andersson, M.A., Kämpfer, P., Andersson, M.C., Honkanen-Buzalski, T., and Scoging, A.C. (1999). Toxigenic strains of Bacillus licheniformis related to food poisoning. Appl. Environ. Microbiol. 65, 4637-4645.

Santini, F., Borghetti, V., Amalfitano, G., and Mazzucco, A. (1995). Bacillus licheniformis prosthetic aortic valve endocarditis. J. Clin. Microbiol. 33, 3070-3073.

Sayers, E.W., Barrett, T., Benson, D.A., Bryant, S.H., Canese, K., Chetvernin, V., Church, D.M., DiCuccio, M., Edgar, R., Federhen, S., et al. (2009). Database resources of the National Centre for Biotechnology Information. Nuc Acids Res 37, D5-15.

Schoenbaum, S.C., Gardner, P., and Shillito, J. (1975). Infections of cerebrospinal fluid shunts: epidemiology, clinical manifestations, and therapy. J. Infect. Dis. 131,543-552.

Schweigert, M.K., Mackenzie, D.P., and Sarlo, K. (2000). Occupational asthma and allergy associated with the use of enzymes in the detergent industry - A review of the epidemiology, toxicology and methods of prevention.30, 1511-1518.

Scott, P. (2011). Cattle self-assessment. Livestock. 16, 27-28.

Seligy, V.L., Beggs, R.W., Rancourt, J.M., and Tayabali, A.F. (1997). Quantitative bioreduction assays for calibrating spore content and viability of commercial Bacillus thuringiensis insecticides. J Ind Microbiol Biotechnol 18, 370-378.

Sella, S.R.B.R., Guizelini, B.P., Gouvea, P.M., Figueiredo, L.F.M., Ribeiro, C.A.O., Vandenberghe, L.P.S., Minozzo, J.C., and Soccol, C.R. (2012). Relations between phenotypic changes of spores and biofilm production by Bacillus atrophaeus ATCC 9372 growing in solid-state fermentation. Arch. Microbiol. 194,815-825.

Shakir, A., Elbadawey, M.R., Shields, R.C., Jakubovics, N.S., and Burgess, J.G. (2012). Removal of biofilms from tracheoesophageal speech valves using a novel marine microbial deoxyribonuclease.147, 509-514.

Sheldon, I.M., Noakes, D.E., Rycroft, A.N., Pfeiffer, D.U., and Dobson, H. (2002). Influence of uterine bacterial contamination after parturition on ovarian dominant follicle selection and follicle growth and function in cattle.123, 837-845.

Shields, R.C., Mokhtar, N., Ford, M., Hall, M.J., Burgess, J.G., ElBadawey, M.R., and Jakubovics, N.S. (2013). Efficacy of a Marine Bacterial Nuclease against Biofilm Forming Microorganisms Isolated from Chronic Rhinosinusitis. PLoS ONE 8.

Shimura, J., Hiraki, K., and Garrity, G.M. (2013). BIOS: Bacteriology Insight Orienting System. In: Species 2000 & ITIS Catalogue of Life, 2013 Annual Checklist. Roskov, Y., Kunze, T., Paglinawan, L., Orrell, T., Nicolson, D., Culham, A., Bailly, N., Kirk, P., Bourgoin, T., Baillargeon, G., et al. eds., (Reading, UK: Species 2000)

Smitha, S., and Bhat, S.G. (2012). Thermostable Bacteriocin BL8 from Bacillus licheniformis isolated from marine sediment. J. Appl. Microbiol. 114, 688-694.

Sorokulova, I.B., Pinchuk, I.V., Denayrolles, M., Osipova, I.G., Huang, J.M., Cutting, S.M., and Urdaci, M.C. (2008). The safety of two Bacillus probiotic strains for human use. Dig. Dis. Sci. 53, 954-963.

Spök, A., and Klade, M. (2009). Environmental, Health and Legal Aspects of Cleaners Containing Living Microbes as Active Ingredients. IFZ 1-17.

Sprengel, R., Reiss, B., and Schaller, H. (1985). Translationally coupled initiation of protein synthesis in Bacillus subtilis. Nucleic Acids Res. 13, 893-909.

Stein, T. (2005). MicroReview: Bacillus subtilisantibiotic: structures, syntheses and specific functions. Mol. Microbiol. 56, 845-857.

Sugar, A.M., and McCloskey, R.V. (1977). Bacillus licheniformis sepsis. JAMA 238, 1180-1181.

Swiecicka, I., Fiedoruk, K., and Bednarz, G. (2002). The occurrence and properties of Bacillus thuringiensis isolated from free-living animals. Lett Appl Microbiol 34, 194-198.

Tabbara, K.F., and Tarabay, N. (1979). Bacillus licheniformis corneal ulcer. Am. J. Ophthalmol. 87, 717-719.

Tagg, J.R., Dajani, A.S., and Wannamaker, L.W. (1976). Bacteriocins of gram positive bacteria. Bacteriol. Rev. 40, 722-756.

Tanaka, T., Kuroda, M., and Sakaguchi, K. (1977). Isolation and characterization of four plasmids from Bacillus subtilis. J Bacteriol 129, 1487-1494.

Tareq, F.S., Kim, J.H., Lee, M.A., Lee, H., Lee, Y., Lee, J.S., and Shin, H.J. (2012). Ieodoglucomides A and B from a marine-derived bacterium Bacillus licheniformis. Org. Lett. 14, 1464-1467.

Taylor, J.M., Sutherland, A.D., Aidoo, K.E., and Logan, N.A. (2005). Heat-stable toxin production by strains of Bacillus cereus, Bacillus firmus, Bacillus megaterium, Bacillus simplex and Bacillus licheniformis. FEMS Microbiol. Lett. 242, 313-317.

Thatoi, H., Behera, B.C., Mishra, R.R., and Dutta, S.K. (2013). Biodiversity and biotechnological potential of microorganisms from mangrove ecosystems: A review.63, 1-19.

Thomson, A.P.J., Damjanovic, V., Hart, C.A., and Cooke, R.W.I. (1990). Bacillus licheniformis infection in a preterm neonate. PEDIATR. REV. COMMUN. 4, 147-149.

Thorne, P.S., Hillebrand, J., Magreni, C., Riley, E.J., and Karol, M.H. (1986). Experimental sensitization to subtilisin. I. Production of immediate- and late-onset pulmonary reactions. Toxicol Appl Pharmacol 86, 112-123.

Thurn, J.R., and Goodman, J.L. (1988). Post-traumatic ophthalmitis due to Bacillus licheniformis. Am. J. Med. 85, 708-710.

Tjalsma, H., Antelmann, H., Jongbloed, J.D.H., Braun, P.G., Darmon, E., Dorenbos, R., and et al. (2004). Proteomics of protein secretion by Bacillus subtilis: Separating the "secrets" of the secretome. Microbiol Mol Biol Rev 68, 207-233.

Toledo, A.V., Alippi, A.M., and De Remes Lenicov, A.M.M. (2011). Growth inhibition of Beauveria bassiana by bacteria isolated from the cuticular surface of the corn leafhopper, Dalbulus maidis and the planthopper, Delphacodes kuscheli, two important vectors of maize pathogens. J. Insect Sci. 11,

Tong, J.L., Engle, H.M., Cullyford, J.S., Shimp, D.J., and Love, C.E. (1962). Investigation of an Outbreak of Food Poisoning Traced to Turkey Meat. Am. J. Publ. Health 52, 976-990.

Tripathi, A., Grammer, L.C. (2001). Extrinsic allergic alveolitis from a proteolytic enzyme. Ann Allergy Asthma Immunol. 86, 425–427.

Tuazon, C.U., Murray, H.W., Levy, C., Solny, M.N., Curtin, J.A., and Sheagren, J.N. (1979). Serious Infections From Bacillus sp. JAMA 241, 1137-1140.

Turnbull, P.C.B., and Kramer, J.M. (1985). Intestinal Carriage of Bacillus cereus: Faecal Isolation Studies in Three Population Groups. J Hyg 95, 629-638.

Turnbull, G.A., Ousley, M., Walker, A., Shaw, E., and Morgan, J.A.W. (2001). Degradation of Substituted Phenylurea Herbicides by Arthrobacter globiformis strain D47 and Characterization of a Plasmid-Associated Hydrolase Gene, puhA. Appl. Environ. Microbiol. 67, 2270-2275.

Turnbull, P.C., Jorgensen, K., Kramer, J.M., Gilbert, R.J., and Parry, J.M. (1979). Severe clinical conditions associated with Bacillus cereus and the apparent involvement of exotoxins. J. Clin. Pathol. 32, 289-293.

U.S. EPA. (2013a). Bio-response Operational Testing and Evaluation (BOTE) Project. EPA/600/R-13/168 i-799.

U.S. EPA. (2013b). Registration Review: Microbial Cases Schedule: 2012-2015.

U.S. EPA. (2012). Bacillus subtilis var. amyloliquefaciens strain FZB24 (006480) Biopesticide Registration Action Document.

U.S. EPA. (2011). Bacillus amyloliquefaciens strain D747. Biopesiticides Registration Action Document Docket Number EPA-HQ-OPP-2011-0853-0011.

U.S. EPA. (2010). Bacillus subtilis Final Registration Review Decision Case 6012. Docket Number EPA-HQ-OPP-2007-10²6..

U.S. EPA. (2006). Biopesticide Registration Action Document Bacillus subtilis Strain QST 713, PC Code 006479. BRAD PC Code 006479, 1-41.

U.S. EPA. (2001). Biopesticide Registration Action Document: Bacillus licheniformis strain SB3086. BRAD PC Code 006492, 1-49.

U.S. EPA. (1993). Ecological Effects Review of a 154A-16 Tier I Avian oral study of GUS Concentrate Biological Fungicide with Bacillus stubtilis as Active Ingredient. MRID No. 425783-01 1-7.

van Elsas, J.D., Dijkstra, A.F., Govaert, J.M., and van Veen, J.A. (1986). Survival of Pseudomonas fluorescens and Bacillus subtilis introduced into two soils of different texture in field microplots. FEMS Microbiol. Lett. 38, 151-160.

Van Veen, J.A., Van Overbeek, L.S., and Van Elsas, J.D. (1997). Fate and activity of microorganisms introduced into soil. Microbiol. Mol. Biol. Rev. 61, 121-135.

Veening, J.W., Smits, W.K., and Kuipers, O.P. (2008). Bistability, epigenetics, and bet-hedging in bacteria. Annu Rev Microbiol 62, 193-210.

Vinoj, G., Vaseeharan, B., DavidJayaseelan, B., Rajakumaran, P., and Ravi, C. (2013). Inhibitory effects of Bacillus licheniformis (DAB1) and Pseudomonas aeruginosa (DAP1) against Vibrio parahaemolyticus isolated from Fenneropenaeus indicus. Aquacult. Int. 21, 1121-1135.

Wainwright, M., and Al Talih, A. (2003). Is this the historical 'cancer germ'? Med Hypotheses 60, 290-292.

Wang, H., and Mullany, P. (2000). The large resolvase TndX is required and sufficient for integration and excision of derivatives of the novel conjugative transposon Tn5397. J. Bacteriol. 182, 6577-6583.

Wang, L.T., Lee, F.L., Tai, C.J., Kasai, H. (2007). Comparison of gyrB gene sequences, 16S rRNA gene sequences and DNA–DNA hybridization in the Bacillus subtilis group. Int. J. Syst. Evol. Microbiol. 57, 1846-1850.

Wang, L.T., Lee, F.L., Tai, C.J., Kuo, H.P. (2008). Bacillus velezensis is a later heterotypic synonym of Bacillus amyloliquefaciens. Int. J. Syst. Evol. Microbiol. 58, 671-675.

Weissman, D.N., and Lewis, D.M. (2002). Allergic and latex-specific sensitization: Route, frequency, and amount of exposure that are required to initiate IgE production. J. Allergy Clin. Immunol. 110, S57-S63.

Weng, J., Wang, Y., Li, J., Shen, Q., and Zhang, R. (2012). Enhanced root colonization and biocontrol activity of Bacillus amyloliquefaciens SQR9 by abrB gene disruption. Appl. Microbiol. Biotechnol. 1-8.

Wu, G., Coulon, F., Yang, Y., Li, H., and Sui, H. (2013). Combining Solvent Extraction and Bioremediation for Removing Weathered Petroleum from Contaminated Soil.23,455-463.

Wulff, E.G., Mguni, C.M., Mansfeld-Giese, K., Fels, J., Lübeck, M., and Hockenhull, J. (2002). Biochemical and molecular characterization of Bacillus amyloliquefaciens, B. subtilis and B. pumilus isolates with distinct antagonistic potential against Xanthomonas campestris pv. campestris. Plant Pathol. 51,574-584.

Xiang, S., Cook, M., Saucier, S., Gillespie, P., Socha, R., Scroggins, R., and Beaudette, L.A. (2010). Development of amplified fragment length polymorphism-derived functional strain-specific markers to assess the persistence of 10 bacterial strains in soil microcosms. Appl. Environ. Microbiol. 76, 7126-7135.

Yadav, G., and Kashyap, S.K. (2003). Efficacy of Different Antibitoics Against Bacterial Isolated from Cervicovaginal Mucus of Repeat Breeding Holstein friesian Crossbred Cows. Indian J. Comp. Microbiol. Immunol. Infect. Dis. 24,10⁷-10⁸.

Yakimov, M.M., Fredrickson, H.L., and Timmis, K.N. (1996). Effect of heterogeneity of hydrophobic moieties on surface activity of lichenysin A, a lipopeptide biosurfactant from Bacillus licheniformis BASSO. Biotechnol Appl Biochem 23,13-18.

Yakimov, M.M., and Golyshin, P.N. (1997). Com-A-dependent transcriptional activation of lichenysin A synthetase promoter in Bacillus subtilis cells. Biotechnol Prog 13, 757-761.

Yi, H., Chun, J., Cha, C.-J. (2014). Genomic insights into the taxonomic status of the three subspecies of Bacillus subtilis. Syst. Appl. Microbiol. 37, 95-99

Young, R.F., Yoshimori, R.N., Murray, D.L., and Chou, P.J. (1982). Postoperative neurosurgical infections due to bacillus species. Surg. Neurol. 18, 271-273.

Zawadzki, P., Roberts, M.S., and Cohan, F.M. (1995). The log-linear relationship between sexual isolation and sequence divergence in Bacillus transformation is robust. Genetics 140, 917.

Zawadzki, P., Riley, M.A., and Cohan, F.M. (1996). Homology among nearly all plasmids infecting three Bacillus species. J. Bacteriol. 178, 191-198.

Zhang, X., Li, B., Wang, Y., Guo, Q., Lu, X., Li, S., and Ma, P. (2013). Lipopeptides, a novel protein, and volatile compounds contribute to the antifungal activity of the biocontrol agent Bacillus atrophaeus CAB-1. Appl. Microbiol. Biotechnol. 97, 9525-9534.

Zhao, K., Liu, J., Li, Z., Chang, Z., Shi, P., Ping, W., Zhou, D. (2011). Bacillus subtilis subspecies virginiana, a new subspecies of antitermitic compound-producing endophytic bacteria isolated from Juniperus virginiana. J Econ Entomol. 10⁴, 1502-1508.

Zhu, Y., Englebert, S., Joris, B., Ghuysen, J., Kobayashi, T., and Lampen, J.O. (1992). Structure, function, and fate of the BlaR signal transducer involved in induction of ß-

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Appendices

Footnotes

Footnote 1

American Type Culture Collection.

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Footnote 2

A determination of whether one or more of the criteria of section 64 of CEPA 1999 are met is based upon an assessment of potential risks to the environment and/or to human health associated with exposure in the general environment. For humans, this includes, but is not limited to, exposure from air, water and the use of products containing the substances. A conclusion under CEPA 1999, on DSL Bacillus licheniformis/subtilis group strains, is not relevant to, nor does it preclude, an assessment against the hazard criteria for Workplace Hazardous Materials Information System (WHMIS) that are specified in the Controlled Products Regulations for products intended for workplace use.

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Footnote 3

Testing conducted by Environment Canada's Biological Methods Division

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Footnote 4

Testing conducted by Health Canada's Environmental Health Science and Research Bureau

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Footnote 5

Biocontrol or growth promotions activities are not within the scope of this assessment.

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Footnote 6

B. licheniformis ATCC 12713, B. subtilis ATCC 6051A, B. subtilis subsp. subtilis ATCC 6051,B. subtilis subsp. inaquosorum ATCC 55406, Bacillus species 2 18118-1 and Bacillus species 7 18129-3.

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Footnote 7

Data generated by Health Canada's Healthy Environments and Consumer Safety Branch. Work conducted using TSB-MTT liquid assay method to determine the MIC values for bacteria based on replicate experiments (Seligy et al. 1997). Values correspond to the minimal inhibitory concentration (µg/mL) for select Bacillus species grown in the presence of antibiotic for 24 hours at 37°C.

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Footnote 8

Tests done according to Environment Canada's "Guidance Document for Testing the Pathogenicity and Toxicity of New Microbial Substances to Aquatic and Terrestrial Organisms (EPS 1/RM/44, March 2004)".

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Date modified:: 2015-07-31

Final Screening Assessment

Environment CanadaHealth CanadaAugust 2015

Table of Contents

Table of Tables

Table of Figures

Synopsis

Introduction

Decisions from Domestic and International Jurisdictions

Domestic

International

1. Hazard Assessment

1.1 Characterisation of the DSL strains under assessment

1.1.1 Taxonomy, identification and strain history

Taxonomic designation:

1.1.1.1 Phenotypic identification and biochemical profile

1.1.1.2 Molecular identification

Figure 1-1 : Phylogenetic relationships of Bacillaceae species based on the alignment of the 16S ribosomal RNA gene sequence coding region

1.1.1.3 Strain history

1.1.2 Biological and ecological properties

1.1.2.1 Natural occurrence

1.1.2.2 Survival and persistence in the environment

Figure 1-2 : Persistence of Bacillus subtilis ATCC 6051 and Bacillus subtilis 13933 in soil, based on qPCR analyses of extractable soil DNA

1.1.2.3 Growth parameters

1.1.2.4 Biocontrol and growth promotionFootnote[5]

Biocontrol

Growth promotion

1.1.2.5 Gene transfer

1.1.2.6 Pathogenic and Toxigenic Characteristics

Spores

Determinants of infectivity

Secondary Metabolites

Toxins

1.1.2.7 Antibiotic Susceptibility Profile

1.1.3 Effects

1.1.3.1 Environment

B. amyloliquefaciens

B. atrophaeus

B. licheniformis

B. subtilis

Masked DSL Bacillus Strains

1.1.3.2 Human Health

B. amyloliquefaciens

B. atrophaeus

B. licheniformis

B. subtilis

Masked DSL Bacillus Strains

1.2 Hazard Severity

1.2.1 Environmental Hazard

1.2.1.1 B. amyloliquefaciens

1.2.1.2 B. atrophaeus

1.2.1.3 B. licheniformis

1.2.1.4 B. subtilis

1.2.1.5 Masked DSL Bacillus Strains

1.2.2 Human Health Hazard

1.2.2.1 B. amyloliquefaciens

1.2.2.2 B. atrophaeus

1.2.2.3 B. licheniformis

1.2.2.4 B. subtilis

1.2.2.5 Masked DSL Bacillus strains

2. Exposure Assessment

2.1 Sources of Exposure

B. amyloliquefaciens

B. atrophaeus

B. licheniformis

B. subtilis

2.2 Exposure Characterisation

2.2.1 Environment

2.2.1.1 B. atrophaeus

2.2.1.2 B. amyloliquefaciens, B. licheniformis, B. subtilis and masked DSL Bacillus strains

2.2.2 Humans

2.2.2.1 B. atrophaeus

2.2.2.2 B. amyloliquefaciens, B. licheniformis, B. subtilis and masked DSL Bacillus strains

3. Risk Characterisation

B. amyloliquefaciens

B. atrophaeus

B. licheniformis

B. subtilis

Masked DSL Bacillus Strains

4. Conclusions

References

Environment Canada
Health Canada
August 2015

1.1.2.4 Biocontrol and growth promotion^Footnote[5]