Final screening assessment of Bacillus megaterium

Official title: Final screening assessment of Bacillus megaterium strain ATCC 14581

Environment and Climate Change Canada

Health Canada

February 2018

Synopsis

Pursuant to paragraph 74(b) of the Canadian Environmental Protection Act, 1999 (CEPA), the Minister of the Environment and the Minister of Health have conducted a screening assessment of Bacillus megaterium strain ATCC 14581.

B. megaterium strain ATCC 14581 is a Gram positive bacterium that has characteristics in common with other strains of this species. B. megaterium can be found in both aquatic and terrestrial environments, in association with plants, animals and humans, as a contaminant of foods and in man-made environments. Like other Bacillus species, B. megaterium is able to form thick-walled spores, which can withstand harsh conditions and nutrient depletion. It is also able to form biofilms, allowing it to persist and survive in suboptimal conditions. Various characteristics make B. megaterium suitable for applications in wastewater treatment, bioremediation and biodegradation, cleaning and deodorizing, drain and septic treatment as well as enzyme and chemical production.

B. megaterium can have both beneficial and adverse effects in terrestrial plants. In Canada, B. megaterium strain ATCC 14581 is not recognized as a plant pest and has been reported to act as a plant growth promoting rhizobacterium. Although B. megaterium or its secondary metabolites can adversely affect some invertebrate species in the context of experimental investigations into their biocontrol potential, B. megaterium strain ATCC 14581 did not cause effects in a terrestrial invertebrate. No effects in aquatic plants, invertebrates or vertebrates or terrestrial vertebrates have been reported in the scientific literature.

In spite of the widespread distribution of B. megaterium in the environment, human infection with B. megaterium is very rarely reported. Adverse human health effects have not been attributed to B. megaterium strain ATCC 14581. The Domestic Substances List (DSL) strain ATCC 14581 does not carry enterotoxin genes which have occasionally been associated with other strains of B. megaterium. Antibiotic susceptibility testing performed by Health Canada scientists demonstrated that, in the unlikely event of infection, clinically relevant antibiotics are effective against this strain.

This assessment considers the aforementioned characteristics of B. megaterium strain ATCC 14581 with respect to the environment and human health effects associated with consumer and commercial product uses and in industrial processes subject to CEPA, 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, 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 10 000 to 100 000 kg of products containing B. megaterium strain ATCC 14581 were imported into or manufactured in Canada in 2008. Reported uses include products or activities in the consumer, commercial and industrial sectors.

Based on the information available, it is concluded that B. megaterium strain ATCC 14581 does not meet the criteria under paragraph 64(a) or (b) of CEPA as it is 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. It is also concluded that B. megaterium strain ATCC 14581 does not meet the criteria under paragraph 64(c) of CEPA as it is 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.

 

Introduction

Pursuant to paragraph 74(b) of the Canadian Environmental Protection Act, 1999 (CEPA), the Minister of the Environment and the Minister of Health are required to conduct screening assessments of those living organisms added to the Domestic Substances List (DSL) by virtue of section 105 of the Act 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^{Footnote 1} . This strain was added to the DSL under subsection 25(1) of CEPA 1988 and the DSL under subsection 105(1) of CEPA because it was manufactured in or imported into Canada between January 1, 1984 and December 31, 1986.

This screening assessment considers hazard information obtained from the public domain and from unpublished research data generated by Health Canada^{Footnote 2} and Environment and Climate Change Canada^{Footnote 3}  research scientists, as well as comments from scientific peer reviewers. Exposure information was obtained from the public domain and from a mandatory CEPA 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-listed strain B. megaterium strain ATCC 14581 are identified as such. 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. Information identified up to June 2015 was considered for inclusion in this screening assessment report.

Decisions from other domestic and international jurisdictions

Domestic

B. megaterium is not considered to be a plant pest or invasive species based on the list of Pests Regulated by Canada and is included on the Canadian Food Inspection Agency (CFIA) list of “Organisms that do not Require a Plant Protection Permit to Import” under the Plant Protection Act (CFIA 2011; CFIA 2014).

B. megaterium is currently categorized as Risk Group 1 (low individual and community risk) for both humans and animals (Public Health Agency of Canada (PHAC), personal communication).

International

Germany’s Federal Institute for Occupational Safety and Health has placed B. megaterium in “Risk Group 1” (BAuA 2010; European Commission 2010) and this is considered to also apply to strain ATCC 14581.

No other mention was found regarding decisions on B. megaterium by international bodies^{Footnote 4} .

1. Hazard assessment

1.1 Characterization of Bacillus megaterium

1.1.1Taxonomic identification and strain history

Binomial name: Bacillus megaterium

Taxonomic designation:

Kingdom:  Bacteria

Phylum:    Firmicutes

Class:       Bacilli

Order:       Bacillales

Family:      Bacillaceae

Genus:      Bacillus

Species:    Bacillus megaterium de Bary 1884 (Orrell 2015; Skerman et al. 1980)

Strain:      ATCC 14581 (type strain)

Synonyms: Bacillus megatherium (Buchanan et al. 1951), Bacillus fructosus (USDA 2014a) and Bacillus megaterium de Bary 1884 (Euzéby and Tindall 2004). At one time there was consideration given to “B. carotarum” as a synonym for B. megaterium but this was rejected (Logan and Berkeley 1984).

Other equivalent type strain designations: BCRC 10608, CCM 2007, CCUG 1817, CIP 66.20, DSM 32, HAMBI 2018, IAM 13418, JCM 2506, KCTC 3007, LMG 7127, NBRC 15308, NCCB 75016, NCIMB 9376, NCTC 10342, NRIC 1710, NRRL B-14308, VKM B-512, Bacillus sp. JP44SK2, Bacillus sp. OS42, CCRC 10608, and IFO 15308 (ATCC 2014, Verslyppe et al. 2014).

Strain history

B. megaterium was named by De Bary in 1884; however, the original culture became unavailable and so the strain “Ford 19” as supplied to culture collections became the new type culture, acquiring the accession numbers ATCC 14581, NCIB 9376 and NCTC 10342 (Lapage et al. 1967; Smith et al. 1964; Sneath and Skerman 1966). It passed from Ford (strain 19) to T. Gibson (strain 1060), to R.E.Gordon and then to the ATCC (DSMZ 2014). The original source from which ATCC 14581 was isolated is unknown (Allen et al. 1983).

1.1.1.1 Phenotypic identification and biochemical profile

B. megaterium strain ATCC 14581 has rod-shaped cells that are 2 to 5 µm in length and 1.2 to 1.5 µm in diameter (Willeke et al. 1996). B. megaterium spores are between 0.5 × 1.0 µm up to 1.0 × 14.8 µm and are frequently club-shaped in appearance (Drucker and Whittaker 1971).

The colony morphology of B. megaterium strain ATCC 14581 observed by Health Canada scientists was consistent with that reported by the American Type Culture Collection (Table 1‑1).

^a Data generated by Environmental Health Science and Research Bureau, Health Canada

^b TSB, tryptic soy broth

^c Description from ATCC (ATCC 2013)

B. megaterium strain ATCC 14581 has often been grouped with members of the B. subtilis group in the scientific literature (Logan and Berkeley 1984). They are similar in terms of electrophoretic mobility of spores (White et al. 2012), position of spores, beta-galactosidase activity, lecithovitellin reaction and gelatin hydrolysis (Logan and Berkeley 1984). Additional metabolic tests were conducted by Health Canada scientists (Appendix A).

B. megaterium (including ATCC 14581) also exhibits similarities to the B. cereus group, which includes the human and animal pathogens B. cereus and B. anthracis (Beesley et al. 2010) and the insect pathogen B. thuringiensis. While most B. megaterium strains are strictly aerobic, B. megaterium strain ATCC 14581 is capable of anaerobic growth like members of the B. cereus group (Beesley et al. 2010; Xiang et al. 2011). B. megaterium, like members of the B. cereus group, have larger cells (>1.0 µm long) and can also be mistaken for B. anthracis based on antigenic similarity of the antiphagocytic capsule (Beesley et al. 2010).

When a larger number of phenotypic characteristics are compared, a pattern emerges that allows B. megaterium to be clearly differentiated from either the B. subtilis or B. cereus groups. B. megaterium is distinguishable from both species groups using the API system, which is based on biochemical methods and differences in substrate utilization (Logan and Berkeley 1984; Product sheet 2004). Based on the results of 139 observations, analysis of 600 strains of Bacillus demonstrated that B. megaterium strains formed a tight group and were distantly positioned from strains of the B. subtilis group (Logan and Berkeley 1984).

B. megaterium can be differentiated from members of the B. cereus and B. subtilis groups based on morphological, biochemical and molecular characteristics (Table 1‑2). Health Canada scientists also used fatty acid methyl ester (FAME) analysis to confirm the identity of the DSL strain (Appendix B). The guanine-cytosine content (G+C%) of B. megaterium strain ATCC 14581 is greater compared to type strains of B. anthracis and B. thuringiensis  and can be used to differentiate it from these species. (Logan and De Vos 2009).

+ indicates a positive reaction; - indicates a negative reaction

^a Data for table compiled from Logan and De Vos (2009); Slepecky and Hemphill (2006); Beesley et al. (2010); Radhika et al. (2011); Xiang et al. (2011) and Heath Canada scientists (personal communication)

^b A^-G⁺, acid negative, gas positive; A⁺G^-, acid positive, gas negative; A^-G^-, acid negative, gas negative

B. megaterium strain ATCC 14581 can be distinguished from other strains of B. megaterium by several morphological and biochemical characteristics (Table 1‑3).

+ indicates a positive reaction; - indicates a negative reaction

^a QW10-11 cells were isolated from a and have adapted to a hypersaline environment (10% (w/v) NaCl)

^b Information obtained from Xiang et al. (2011)

^c Cells extend without dividing; authors attributed this morphology to physiological changes required to adapt to the hypersaline growth environment

^d Xiang et al. (2011) and Logan and De Vos (2009) report the type strain to be motile however, this characteristic was not observed by Heath Canada Scientists

1.1.1.2 Molecular identification

In spite of the above-noted phenotypic differences, molecular methods show B. megaterium to be closely related to both the B. cereus and B. subtilis groups. In one study, 51 species of Bacillus were arranged in five phylogenetically distinct clusters based on comparison of their 16S rRNA gene sequences. B. megaterium strain ATCC 14581, B. cereus, B. anthracis and B. subtilis are all grouped together (Ash et al. 1991). The close relationship between B. megaterium and species of the B. cereus and B. subtilis groups based on 16S rRNA phylogeny suggests that lateral gene transfer may be to a large extent responsible for the phenotypic differences between these species (Eppinger et al. 2011). Health Canada scientists used 16S rRNA gene sequences to analyze the phylogenetic relationship between B. megaterium strain ATCC 14581 and other Bacillus species (Figure 1‑1). The DSL strain B. megaterium strain ATCC 14581 was not observed to group with either the B. subtilis group or the pathogenic B. cereus group.

 

Figure 1‑1: phylogenetic tree derived in-house^{Footnote 5} , using 16S rRNA gene sequences of B. megaterium strain ATCC 14581 and sequences identified from literature searches

Several methods can be applied to distinguish between B. megaterium and other Bacillus species, including B. cereus and B. subtilis, such as Randomly Amplified Polymorphic DNA (RAPD) (Quingming and Zongping 1997) and Ultra Violet Raman Resonance Spectroscopy (UVRR) (Lopez-Diez and Goodacre 2004). Some B. megaterium strains, including B. megaterium strain ATCC 14581, have fully sequenced genomes which can potentially be used to determine distinguishing genotypic features between strains (Table 1‑4).

Plasmids within the genus Bacillus

Plasmids are relatively rare among Bacillus species (Yoshimura et al. 1983); however, B. megaterium strains are rich in plasmids, usually containing four or more (Slepecky and Hemphill 2006). B. megaterium strain 216 contains 10 plasmids and B. megaterium strain QM B1551 has 7 stable plasmids comprising 11% of total cellular DNA. Although the plasmids of QM B1551 carry genes encoding enzymes and proteins for heavy metal export, transport, acyl carriers, sigma factors, sterols, redox, mobilization, sporulation and germination, the plasmid-borne genes do not seem to be required for growth. In one study, a modified strain of QM B1551 with seven of its plasmids removed showed similar growth to the wild-type under laboratory conditions (Kieselburg et al. 1984; reviewed in Vary et al. 2007).

Among its plasmids, B. megaterium strain ATCC 14581 carries a 12 kb plasmid which distinguishes it from nine other strains of B. megaterium (Rosso and Vary 2005; Vary et al. 2007).

1.1.2 Biological and ecological properties

1.1.2.1 Natural occurrence

B. megaterium as a species is generally considered ubiquitous and is found in a variety of habitats, including:

• fresh and salt water (Allen et al. 1983; Rusterholtz and Mallory 1994; Kong et al. 2010) as well as soil (Smalla et al. 2001; Taupp et al. 2005; Sakurai et al. 2007);
• plant rhizosphere (Srinavasan et al. 1996; Smalla et al. 2001; Sakurai et al. 2007; Aballay et al. 2011; Xiang et al. 2011; Kavamura et al. 2013) and other plant-associated sites including ovules and seeds (Mundt and Hinkle 1976; Cottyn et al. 2001), leaves (Ercolani 1978; Purkayastha and Bhattacharyya 1982; Araújo et al. 2001; West et al. 2010;), roots (Liu and Sinclair, 1993; McInroy and Kloepper 1995; Srinavasan et al. 1996; Surette et al. 2003; West et al. 2010) and stems (McInroy and Kloepper 1995; West et al. 2010)
• in association with animals (Osborn et al. 2002; Barbosa et al. 2005; Hillesland et al. 2008; Bulushi et al. 2010) and humans (Weber et al. 1988; Rowan et al. 2001; Dib et al. 2003; Beesley et al. 2010; Subbiya and Mahalakshmi 2012); and
• compost piles (Perestelo et al. 1989), processing waste (Srinath et al. 2002; Raj et al. 2007; Priya et al. 2014), soilless growing medium (Welbaum et al. 2009), the interior of granite rock (Fajardo-Cavazos and Nicholson 2006); air ducts (Lushniak and Mattorano 1994), chewing tobacco (Rubinstein and Pedersen 2002), wood surfaces (Austin et al. 1979), paper linerboard (Namjoshi et al. 2010) and as contaminants of food (Kurtzman et al. 1971; López and Alippi 2009; Rowan et al. 2001; Yossan et al. 2006).

1.1.2.2 Growth parameters

Strains of B. megaterium, including ATCC 14581, grow over a wide range of temperatures, with minimum growth temperatures between 3°C and 10°C, an optimal growth temperature of 37°C, and a maximum growth temperature of 40 to 45°C. Although B. megaterium strain ATCC 14581 was not observed to grow at 50°C (Table 1‑2), obligate thermophilic variants are capable of growth at 55°C (Ståhl and Olsson 1977) and other variants show cardinal temperature growth maxima up to 73°C (Rilfors et al. 1978). B. megaterium is able to survive in nutrient broth for 28 days at room temperature. However, when the temperature was increased to 48°C a 1 log decrease of colony forming units (CFU) was reported (Velineni and Brahmaprakash 2011). Health Canada scientists measured the optical density of 24-hour B. megaterium strain ATCC 14581 cultures in various liquid media grown at various temperatures. B. megaterium strain ATCC 14581 was observed to grow well in TSB at 28°C but limited growth was observed in serum-containing mammalian culture media at this temperature. In contrast, as the temperature increased, growth was observed to increase slightly in most serum containing media but decreased in TSB.

B. megaterium strain ATCC 14581 grows poorly on minimal media and requires supplementation with L-threonine and L-valine whether grown at 30°C or 37°C with glucose or glycerol (White 1972); the most rapid growth occurred at 30°C with glycerol. B. megaterium strain ATCC 14581 grows well on ATCC Medium #3, with a pH of 6.8 ± 0.2 (ATCC 2013).

1.1.2.3 Survival and persistence

The persistence of B. megaterium strain ATCC 14581 in soil microcosms was investigated (Providenti et al. 2009). The results of the study indicated that an initial concentration of 1 × 10⁴ CFU/g soil would decline to the detection limit of 1 × 10² CFU/g soil within 105 days. B. megaterium would also likely be able to persist at some lower concentrations as spores.

B. megaterium may undergo sporulation in response to nutrient depletion (Brown and Hodges 1974). The formation of endospores allows Bacillus species to persist for long periods under dry conditions and to resist high temperatures, ultraviolet radiation and chemical disinfectants. Spores of Bacillus species can withstand temperatures about 45°C higher than vegetative cells (Coleman et al. 2010). Spores of B. megaterium strain ATCC 14581 are also relatively resistant to ultraviolet light compared to other Bacillus species tested (Fajardo-Cavazos and Nicholson 2006; Newcombe et al. 2005).

The conditions in which B. megaterium undergoes sporulation may affect the resistance of the spores to various conditions (Soper et al. 1976; Khoury et al. 1987). For example, spores that had undergone freezing were more susceptible to chlorine and ultraviolet light inactivation treatments compared to their unfrozen counterparts (Gao et al. 2007; Soper et al. 1976). It is also important to note that the spores of different strains of B. megaterium may behave differently than ATCC 14581 (Dr. Rebekka Biedendieck, Postdoctoral fellow, Technische Universität Braunschweig, personal communication).

B. megaterium spores demonstrated better survival in sterile river water as opposed to raw (unsterilized) river water (López et al. 1995), suggesting that predation may limit persistence of spores in this environment. However, spores may survive predation better than vegetative cells. Vegetative cells of B. megaterium do not withstand passage through the digestive system of the nematode Caenorhabditis elegans, whereas spores do (Laaberki and Dworkin 2008).

1.1.2.4 Biocontrol

B. megaterium produces antimicrobial compounds, such as the bacteriocins megacin A and megacin C, that are of potential interest for biocontrol of bacterial and fungal plant pathogens (Tersch and Carlton 1983). B. megaterium strain ATCC 14581 cytochrome P-450 (specifically CYP102A1) can oxidize (i.e., degrade) the quorum signalling molecules acyl homoserine lactones produced by other micro-organisms giving it a competitive advantage (Chowdhary et al. 2007). Some experimentally observed biocontrol activities for isolates of B. megaterium are outlined in Table 1‑5.

1.1.2.5 Biosorption of metals and biodegradation

B. megaterium is able to increase the bioavailability of metals in contaminated soils including boron, lead and cadmium (Esringu et al. 2014). A strain of B. megaterium isolated from forest soil was able to solubilize iron, manganese and copper from phosphogypsum, a waste product of the production of fertilizer from phosphate rock (Ştefănescu et al. 2011).

Some strains of B. megaterium are capable of bioaccumulating metals, including cobalt, manganese, nickel, zinc, uranium, aluminum and cadmium (Selenska-Pobell et al. 1999; Rajkumar et al. 2013). In one study, B. megaterium was reported to bioaccumulate 32.0 mg Cr/g dry weight (Srinath et al. 2002). Moderately halotolerant strains of B. megaterium have been reported to reduce selenite (SeIV) to less toxic red elemental selenium (Mishra et al. 2011).

In addition to reactions with metals, B. megaterium can degrade organic compounds, including the herbicide atrazine (Marecik et al. 2008).

1.1.2.6 Resistance to antibiotics, metals and chemical agents

Although one strain of B. megaterium isolated from a hospital environment in Nigeria was resistant to 70% of the antibiotics with which it was challenged (Atata et al. 2013), B. megaterium is generally sensitive to a greater spectrum of antibiotics than B. cereus, though not as universally susceptible as B. subtilis (Larsen et al. 2014; Reva et al. 1995; Sadiq and Ali 2013; Appendix D).

Health Canada scientists^{Footnote 6} determined the minimum inhibition concentration of antibiotics against B. megaterium strain ATCC 14581 using the TREK Sensititre broth microdilution method (Thermo Scientific) conducted in liquid medium (Table 1‑6 and Table 1‑7). Overall, B. megaterium strain ATCC 14581 was inhibited by many of the antibiotics tested.

N/A indicates information not available; S indicates susceptible; I indicates intermediate; R indicates resistant

^a Breakpoints and interpretation of MIC results according to Clinical and Laboratory Standards Institute (CLSI M45-A2, and VET01-S2) (CLSI, 2012; CLSI, 2013)

N/A indicates not available; S indicates susceptible; I indicates intermediate; R indicates resistant

^a Breakpoints and interpretation of MIC results according to Clinical and Laboratory Standards Institute (CLSI M45-A2 and VET01-S2) (CLSI, 2012; CLSI 2013)

^b (Kehrenberg and Schwarz 2006)

^c (Scott, et al. 2010)

Strains of B. megaterium are also susceptible to a number of different disinfection methods (Table 1‑8).

^a Cold atmospheric plasma is an ionized gas with decontaminating potential, that can be derived from various gases such as helium, argon, or nitrogen (Hoffmann et al. 2013)

Lytic phages have been used in the control of B. cereus group species. The lytic phage vB_BceM_Bc431v3 infects all B. cereus, B. anthracis, B. licheniformis, B. thuringiensis and B. weihenstephanensis strains, as well as B. megaterium strain ATCC 14581 (El-Arabi et al. 2013).

1.1.2.7 Pathogenic and toxigenic characteristics

Some B. megaterium strains possess characteristics of pathogenic Bacillus species that are associated with virulence, such as adherence and invasion, toxin and secondary metabolite production, biofilm formation and hemolysis.

Health Canada scientists verified the whole genome sequence of B. megaterium strain ATCC 14581 (GenBank accession JJMH00000000.1; Arya et al. 2014) using a variety of search strategies (nucleotide and protein searches) and did not find any major virulence factor genes. Another complete B. megaterium strain ATCC 14581 genome sequence that appears in GenBank (CP009920.1, CP009916.1-CP009919.1 & CP009921.1) was also investigated and yielded the same results.

1.1.2.7.1  Adherence and invasion

B. megaterium isolated from honey was reported to adhere to and invade human intestinal epithelial Caco-2 cells and the supernatant from the spent cultures resulted in detachment and necrosis of the Caco-2 cells (López et al. 2013). A strain of B. megaterium isolated from infant milk formula was determined to be non-cytotoxic, yet demonstrated adherence similar to that shown by a strain of Listeria monocytogenes as well as some invasion capability (Rowan et al. 2001). There is no evidence to indicate that the DSL strain is able to adhere to and invade mammalian cells.

1.1.2.7.2  Toxins

A clinical B. megaterium strain, associated with sepsis and pyrexia, was demonstrated to produce hemolysin BL (HBL) enterotoxin (Rowan et al. 2001). Enterotoxigenic genes cytK, bceT, those related to the HBL complex and the NHE complex have been reported in B. megaterium strains isolated from honey (López and Alippi 2010; López et al. 2013; López and Alippi 2009). Searches of the annotated genome did not identify known toxin genes or operons associated with cytK, bceT, HBL or NHE in B. megaterium strain ATCC 14581.

1.1.2.7.3  Other metabolites

In plants inoculated with pathogenic strains of B. megaterium pectolytic and cellulolytic enzymes were present, causing disintegration and collapse of invaded tissues (Abdel-Monaim et al. 2012). Relative to other Bacillus species, the activity of xylanase and cellulase in two strains of B. megaterium isolated from African soil was reported to be low (Larsen et al. 2014). There is no evidence to suggest that the DSL strain is capable of producing these enzymes.

1.1.2.7.4 Biofilm formation

Although cells of B. megaterium produce a glucose-based polysaccharide polymer that can contribute to biofilm formation (Gandhi et al. 1997; Welbaum et al. 2009), two soil isolates of B. megaterium did not form biofilms (Larsen et al. 2014). It is not known whether the DSL strain produces a biofilm.

1.1.2.7.5 Hemolysis

Hemolytic activity was demonstrated in 77% of 53 B. megaterium strains isolated from Argentinean honey (primarily of the β-hemolysis type) and 10% of these produced a discontinuous hemolytic pattern typically associated with enterotoxin activity. In addition, coagulase activity was found in 74% of the strains (López and Alippi 2009). No hemolytic activity was observed in B. megaterium strain ATCC 14581 by Health Canada scientists.

1.1.3 Effects

1.1.3.1 Environment

1.1.3.1.1  Plants

Some strains of B. megaterium are reported to be pathogenic to terrestrial plants (Shark et al. 1991) causing bacterial blight or white blotch on foliage and heads of wheat (Australian Government Department of Health and Ageing 2005; Hosford 1982; USDA 2014b). B. megaterium adversely affects the subtropical ornamental tree (Radermachera sinica) (Li et al. 2014), in which its pathogenic role was confirmed by experimental reinfection of healthy plants. B. megaterium has been implicated as one of several causative agents of wetwood wilt and dieback of certain tree species, especially elms and poplars (University of Illinois 1999). Six strains of B. megaterium were isolated from diseased lupines and re-inoculated into healthy plants, which subsequently became soft and grayish-brown, eventually collapsing (Abdel-Monaim et al. 2012). Interestingly, barley, wheat, sunflower, cocklebur, spinach and 13 other legume species were challenged with the two most pathogenic strains and no effects were reported with the exception of fava bean leaves which exhibited small necrotic spots (Abdel-Monaim et al. 2012).

Environment and Climate Change Canada scientists investigated effects in red fescue (Festuca rubra) after exposure to B. megaterium strain ATCC 14581 (Environment Canada 2005). Replicates were conducted in 1 L polypropylene vessels containing 500 ± 0.5 g wet weight of soil which were inoculated with 3.48 × 10⁸ CFU of B. megaterium strain ATCC 14581 at the start of the study. No statistically significant difference was observed between the exposed plants and the control plants as measured by mean emergence, shoot and root length, and mass after 32 days following the initial inoculation event.

B. megaterium also has beneficial effects on terrestrial plants as a plant-growth promoting rhizobacterium (Table 1‑9). B. megaterium solubilizes phosphorus (Sandeep et al. 2011; Sadiq and Ali 2013; Xiang et al. 2011), and mineralizes organic nitrogen (Sakurai et al. 2007), thereby making soil nutrients available to plants (Armada et al. 2014; Hu et al. 2013; Kieselburg et al. 1984). It also produces plant growth hormones (auxins), including indole acetic acid, and enzymes that promote growth (Armada et al. 2014; Sadiq and Ali 2013; Shaharoona et al. 2006).

An in-depth review of the scientific literature did not identify adverse effects of B. megaterium or B. megaterium strain ATCC 14581 in aquatic plant species.

1.1.3.1.2 Invertebrates

Adverse effects of B. megaterium in terrestrial invertebrates have been observed in experiments exploring its potential as a biocontrol agent. Several examples are listed below:

• 10% mortality in fifth instar moth larvae (Hylesia metabus) inoculated with 3-4 × 10⁷ CFU of B. megaterium isolated from fourth instar moth larvae (Osborn et al. 2002).
• Antagonistic effects in parasitic nematodes including Xiphinema index (Aballay et al. 2011); Meloidogyne graminicola (Padgham and Sikora 2007) and Meloidogyne incognita (Saikia et al. 2013): in M. graminicola, B. megaterium reduced nematode penetration and gall formation in rice roots and reduced migration of M. graminicola to the root zone by nearly 60% compared to non-treated roots; secondary metabolites of B. megaterium reduced nematode egg hatching by 60% compared to eggs not exposed to the bacteria.
• Antagonistic effects in weevils (Rhynchophorus ferrugineus) and moths (Dendrolimus pini (L)) (Bertone et al. 2011; Hardin and Suazo 2012).
• Secondary metabolites of B. megaterium killed mosquito larvae (Aedes aegypti) but were non-toxic to humans (Radhika et al. 2011).
• B. megaterium isolates killed aphids (Aphis pomi De Geer) (Askoy and Ozman-Sullivan 2008)

Environment and Climate Change Canada scientists investigated effects in springtails (Folsomia candida) after exposure to B. megaterium strain ATCC 14581 (Environment and Climate Change Canada 2005). Test jars were filled with 30 g wet weight of soil and inoculated with 9.28 × 10⁷ CFU of B. megaterium strain ATCC 14581 24 hours prior to the start of the study. No statistically significant difference between treatments was observed in mean adult survival or mean juvenile production after 28 days following the initial inoculation event.

An in-depth search of the scientific literature identified no reports of B. megaterium adversely affecting aquatic invertebrates with the exception of the mosquito larvae identified above; however, it has been reported to act as a growth promotant when added to shrimp feed (Olmos et al. 2011; Yuniarti et al. 2013).

1.1.3.1.3 Vertebrates

An in-depth review of the scientific literature identified no reports of B. megaterium adversely affecting aquatic or terrestrial vertebrates. However, it has been added to fish feed as a growth promotant (Parthasarathy and Ravi2011). In addition, B. megaterium strain ATCC 14581 was included with other bacteria in a patent to reduce vertebral compression syndrome in salmonid fish (Aubin et al. 2006).

1.1.3.2 Human health

In spite of its widespread presence in the environment, B. megaterium has rarely been implicated in human infections. When it has been associated with infection, it was not always clear if the clinical isolates were opportunistic pathogens or contaminants (Beesley et al. 2010). No information was identified implicating B. megaterium strain ATCC 14581 in adverse human health effects.

A strain of B. megaterium was associated with pyrexia and sepsis in the patient from which it was isolated (Rowan et al. 2001). In a study of 89 Bacillus species isolated from blood cultures associated with significant bacteremia, 13 were B. megaterium (Weber et al. 1988). B. megaterium was isolated from the infected eye in one case of lamellar keratitis 2 weeks after eye surgery (Ramos-Esteban et al. 2006). B. megaterium was also implicated in the infection of a dental crown (Subbiya and Mahalakshmi, 2012) that was thought to be related to its ability to secrete collagenase.

In clinical isolates, B. megaterium has been mistaken for B. anthracis. In one case, a non-hemolytic, non-motile Bacillus isolated from a blood culture, suspected to be B. anthracis, was in fact B. megaterium (Dib et al. 2003). In another case a B. megaterium skin infection was mistaken for cutaneous anthrax, in a young, immunocompetent patient (Duncan and Smith 2011). The infection was treated successfully with ciprofloxacin. B. megaterium cultures can be distinguished from B. anthracis based on characteristics discussed previously (1.1.1.1 Phenotypic identification and biochemical profile and 1.1.1.2 Molecular identification); however, standard clinical microbiological methods used for preliminary diagnosis may not differentiate between them (Beesley et al. 2010; Dib et al. 2003).

Bacillus species, including strains of B. megaterium, isolated from tobacco products have been implicated in infections, pulmonary inflammation and allergic sensitivities, and plasma exudation and tissue dysfunction in the mouth (Rooney et al. 2005; Rubinstein and Pedersen 2002).

1.2 Hazard severity

1.2.1 Environment

The environmental hazard potential of B. megaterium strain ATCC 14581 is assessed to be low because in spite of the ubiquity of B. megaterium, no adverse effects have been reported at the population level in the environment. B. megaterium can have both beneficial and adverse effects in terrestrial plants. In Canada, B. megaterium strain ATCC 14581, is not recognized as a plant pest and has been reported to act as a plant growth promoting rhizobacterium. No negative effects were reported in plants exposed to B. megaterium strain ATCC 14581 as observed in studies conducted by Environment and Climate Change Canada scientists. Although B. megaterium or its secondary metabolites can adversely affect some invertebrate species under biocontrol conditions, the DSL strain, B. megaterium strain ATCC 14581 did not cause effects when tested on the terrestrial invertebrate Folsomia candida. No adverse effects in aquatic plants, invertebrates or vertebrates or terrestrial vertebrates have been reported. Growth promotion was observed in aquatic invertebrates (shrimp) and in aquatic vertebrates (fish) fed B. megaterium as a feed supplement. Although B. megaterium strain ATCC 14581 has been used in Canada for several decades, there are no reports in the literature implicating it in adverse environmental effects.

1.2.2 Human health

The human hazard potential of B. megaterium strain ATCC 14581 is assessed to be low because in spite of its widespread distribution in the environment, human infection with B. megaterium is very rarely reported. Adverse human health effects have not been attributed to B. megaterium strain ATCC 14581 and it has not been shown to carry enterotoxin genes which have occasionally been associated with other strains of B. megaterium. Antibiotic susceptibility testing performed by Health Canada scientists demonstrated that, in the unlikely event of infection, clinically relevant antibiotics are effective against this strain.

Hazards related to micro-organisms used in the workplace should be classified under the Workplace Hazardous Materials Information System (WHMIS)^{Footnote 7} .

2. Exposure assessment

2.1 Sources of exposure

This assessment considers exposure to B. megaterium strain ATCC 14581 resulting from its deliberate addition to consumer or commercial products and its use in industrial processes in Canada.

B. megaterium strain ATCC 14581 was nominated to the DSL for use in consumer or commercial products.

Responses to a voluntary questionnaire sent in 2007 to a subset of biotechnology companies, combined with information obtained from other federal government regulatory and non-regulatory programs, indicate that B. megaterium strain ATCC 14581 was in commercial use in 2006.

The Government conducted a mandatory information-gathering survey under section 71 of CEPA, 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 B. megaterium strain ATCC 14581, whether alone, in a mixture or in a product. Between 10,000 and 100,000 kg of products containing B. megaterium strain ATCC 14581 were imported into or manufactured in Canada in 2008 for a variety of uses in consumer, commercial and industrial applications.

B. megaterium strain ATCC 14581 is available for purchase from the ATCC. As it is on the DSL, and can be used in Canada without prior notification under CEPA, it could be an attractive choice for further commercialization. A search of the public domain (internet, patent databases, MSDS, etc.) revealed the following consumer, commercial and industrial applications of other strains of B. megaterium. These represent possible uses of the DSL strain, as B. megaterium strain ATCC 14581 is likely to share characteristics (modes of action) with other commercialized B. megaterium strains. Growth in the market for “greener” microbial-based products for both commercial and consumer applications may increase direct human exposure to B. megaterium strain ATCC 14581, which has potential applications in these products (Spök and Klade 2009).

• as a production or modifying organism for a variety of biochemicals and biopolymers (Kieselburg et al. 1984; Honorat et al. 1990; Shark et al. 1991; reviewed in Vary 1994; Xu et al. 1997; Schallmey et al. 2004; Biedendieck et al. 2007; Shimizu et al. 2007; Obruca et al. 2009; Muffler et al. 2011; Hastings et al. 2013; Mohammed et al. 2014);
• in biocontrol (Branly and Atkins 2001; Product sheet 2004; Chowdhary et al. 2007; Drahos and Petersen 2010);
• in biodegradation (Saxena et al. 1987; Quinn et al. 1989; Bianchi et al. 2008; Marecik et al. 2008; Singh et al. 2013);
• in bioremediation (Esringu et al. 2014);
• in wastewater treatment (Bianchi et al. 2007);
• in commercial cleaning and deodorizing products for use in drain and septic maintenance (Calfarme 2010; Vandini et al. 2014);
• in calcite precipitation to provide improved strength of fly ash-amended concrete (Achal and Pan 2011; Dhami et al. 2013; Soon et al. 2014);
• in bioleaching or “biohydrometallurgy” (Vasanthakumar et al. 2013);
• in the rapid biosynthesis of silver nanoparticles (Saravanan et al. 2011);
• as a bioindicator (Lillehoj and Ciegler 1970; Garvey et al. 2013; Verma et al. 2013);
• as an aquaculture feed additive (Aubin et al. 2006) and probiotic (Barbosa et al. 2005; Olmos et al. 2011; Parthasarathy and Ravi 2011; Yuniarti et al. 2013);
• in the production of food flavouring agents (Taupp et al. 2005); and
• as an adjuvant modulator of the immune system in mice (Ruiz-Bravo et al. 1981).

2.2 Exposure characterization

2.2.1 Environment

The environmental exposure to B. megaterium strain ATCC 14581 is expected to be medium based on the wide range of uses reported in response to the section 71 notice.

The magnitude of environmental exposure to B. megaterium strain ATCC 14581 will depend on the nature, quantity, duration and frequency of use and on its persistence and survival in the environment to which it is released. The concentration of vegetative cells of B. megaterium strain ATCC 14581 introduced into soil was demonstrated to decrease by two orders of magnitude within 105 days (Providenti et al. 2009). 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 that results in the rapid decline of populations of introduced bacteria (van Veen et al. 1997). Spores of B. megaterium strain ATCC 14581 are more resistant to harsh conditions compared to their vegetative counterparts. It is unlikely that populations of B. megaterium strain ATCC 14581 will be maintained as vegetative cells in soil and water since they have little competitive advantage over naturally-occurring populations and would be subject to predation and competition for nutrients with indigenous flora. Spores of B. megaterium strain ATCC 14581 are likely to persist and could accumulate in the environment.

Aquatic exposure to B. megaterium strain ATCC 14581 is expected to be greatest for organisms in the vicinity of direct application to aquatic ecosystems for water treatment. Aquatic species may also be exposed to B. megaterium strain ATCC 14581, from its introduction into the wastewater through use in consumer or commercial products and as a result of runoff from terrestrial applications.

Terrestrial exposure to B. megaterium strain ATCC 14581 is expected through agricultural applications, biodegradation and bioremediation and bioleaching, in the vicinity of treated sites.

Aquatic or terrestrial exposure to B. megaterium strain ATCC 14581 as a result of its release from facilities manufacturing enzymes or biochemicals is expected to be limited by the application of good manufacturing processes (for example, to be in conformity with municipal and provincial waste water regulations).

2.2.2 Human health

Human exposure to B. megaterium strain ATCC 14581 is expected to be medium based on the wide range of uses reported in the section 71 survey. Human exposure to B. megaterium strain ATCC 14581 is expected to be greatest through the direct use of consumer products containing spores or viable cells. 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. Growth in the market for “greener” microbial-based products may increase direct human exposure to B. megaterium strain ATCC 14581, which has potential applications in these products (Spök and Klade 2009).

Humans may also be exposed to B. megaterium strain ATCC 14581 as bystanders during the 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 B. megaterium strain ATCC 14581 released into the environment subsequent to its 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 treated with B. megaterium strain ATCC 14581 (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 the use of consumer products. Human activity on soils recently treated with B. megaterium strain ATCC 14581 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.

In the event that spores of B. megaterium strain ATCC 14581 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.

3. Risk characterization

In this assessment, risk is characterized according to a paradigm whereby 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.

Hazard has been estimated for B. megaterium strain ATCC 14581 to be low for the environment and low for human health. Environmental and human exposure to B. megaterium strain ATCC 14581 is estimated to be medium based on the wide range of uses reported for this strain, so the risk associated with current uses is estimated to be low for both the environment and human health.

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. megaterium strain ATCC 14581 has properties that make it suitable for use in a range of products, and there is reason to expect new uses of B. megaterium strain ATCC 14581 will emerge. In particular, there is growth in the market for “greener” microbial-based cleaning products, that may increase human exposure to B. megaterium strain ATCC 14581 because of its potential application in these products (Spök and Klade 2009; Vandini et al. 2014). Nevertheless, the risk from foreseeable future uses is also expected to be low, given the low hazard associated with B. megaterium strain ATCC 14581.

4. Conclusion

Based on the information presented in this screening assessment, it is concluded that B. megaterium strain ATCC 14581 is 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 concluded that this substance does not meet the criteria as set out in section 64 of the CEPA.

References

Aballay, E., Martensen, A., and Persson, P. (2011). Screening of rhizosphere bacteria from grapevine for their suppressive effect on Xiphinema index Thorne & Allen on in vitro grape plants. Plant Soil 347, 313-325.

Abdel-Monaim, M.K., Gabr, M.R., El-Gantiry, S.M., Shaat, M.N., and El-Bana, A.A. (2012). Bacillus megaterium, a new pathogen on lupine plants in Egypt. J Bacteriol Res 4, 24-32.

Achal, V., and Pan, X. (2011). Characterization of urease and carbonic anhydrase producing bacteria and their role in calcite precipitation. Curr Microbiol 62, 894-902.

Allen, D., Austin, B., and Colwell, R. (1983). Numerical taxonomy of bacterial isolates associated with a freshwater fishery. J Gen Microbiol 129, 2043-2062.

Araújo, W.L., Jr, W.M., Aguilar-Vildoso, C.I., Barroso, P.A., Saridakis, H.O., and Azevedo, J.L. (2001). Variability and interactions between endophytic bacteria and fungi isolated from leaf tissues of citrus rootstocks. Can J Microbiol 47, 229-236.

Armada, E., Portela, G., Roldán, A., and Azcón, R. (2014). Combined use of beneficial soil microorganism and agrowaste residue to cope with plant water limitation under semiarid conditions. Geoderma 232-234, 640-648.

Arya, G., Petronella, N., Crosthwait, J., Carrillo, C.D., and Shwed, P.S. (2014). Draft Genome Sequence of Bacillus megaterium Type Strain ATCC 14581. Genome Announc 2, e01124-14.

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.

Askoy, H.M. and Ozman-Sullivan, S.K. (2008) Isolation of Bacillus Megaterium from Aphis pomi (Homoptera: Aphididae) and Assessment of Its Pathogenicity. J Plant Pathol 90, 449-452.

Atata, R.F., Ibrahim, Y.K.E., Giwa, A., and Akanbi II, A.A.A. (2013). Antibiotics resistance profile of bacterial isolates from surgical site and hospital environment in a university teaching hospital in Nigeria. JMMS 4, 181-187.

ATCC. (2013). Product sheet: Bacillus megaterium (ATCC® 14581™). ATCC 1-2.

ATCC. (2014). American Type Culture Collection: Bacillus megaterium de Bary (ATCC^® 14581™).

Aubin, J., Labbe, L., Gatesoupe, J.F., and Lebrun, L. (2006). United States Patent Application Publication: Use of bacilli bacteria in order to produce a composition for the prevention of vertebral compression syndrome in salmonids. US Patents 10/562,204, 1-11.

Austin, B., Allen, D., Zachary, A., Belas, M., and Colwell, R. (1979). Ecology and taxonomy of bacteria attaching to wood surfaces in a tropical harbor. Can J Microbiol 25, 447-461.

Australian Government Department of Health and Ageing. (2005). The Biology and Ecology of Bread Wheat (Triticum aestivum L. em Thell.) in Australia.

Barbosa, T.M., Serra, C.R., Ragione, R.M.L., Woodward, M.J., and Henriques, A.O. (2005). Screening for Bacillus isolates in the broiler gastrointestinal tract. Appl Environ Microbiol 71, 968-978.

BAuA. (2010). Technical Rules for Biological Agents: Classification of Prokaryotes (Bacteria and Archaea) into Risk Groups. Fed Inst Occupational Safety Health TRBA 466

Bédard, J., and Lefebvre, G.M. (1989). L-alanine and inosine enhancement of glucose triggering in Bacillus megaterium spores. Can J Microbiol 35, 760-763.

Beesley, C.A., Vanner, C.L., Helsel, L.O., Gee, J.E., and Hoffmaster, A.R. (2010). Identification and characterization of clinical Bacillus spp. isolates phenotypically similar to Bacillus anthracis. FEMS Microbiol Lett 313, 47-53.

Berkeley, R.C.W., Logan, N.A., Shute, L.A., and Capey, A.G. (1984). Identification of Bacillus Species. Methods Microbiol 16, 291-328.

Bertone, C., Michalak, P., and Roda, A. (2011). New Pest Response Guidelines . Red Palm Weevil Rynchophorus ferrugineus.

Bianchi, G., Benedusi, M., and Altheimer, L. (2007). Waste water treatment. CIPO CA 2629217.

Bianchi, G., Benedusi, M., and Altheimer, L. (2008). Bag for the collection of organic waste and method for its manufacturing. WIPO Patent Application WO/2008/135845 Kind Code: A1

Biedendieck, R., Yang, Y., Deckwer, W.D., Malten, M., and Jahn, D. (2007). Plasmid system for the intracellular production and purification of affinity-tagged proteins in Bacillus megaterium. Biotechnol Bioeng 96, 525-537.

Branly, K., and Atkins, R. (2001). Agricultural compositions containing bacteria.

Broadwater, W.T., Hoehn, R.C., and King, P.H. (1973). Sensitivity of three selected bacterial species to ozone. Appl Microbiol 26, 391-393.

Brown, M.R.W., and Hodges, N.A. (1974). Growth and sporulation characterisitics of Bacillus megaterium under different conditions of nutrient limitation. J Pharm Pharmac 26, 217-227.

Buchanan, R. E.; Breed, R. S.; St. John-Brooks, R. (1951). "Opinion 1. The Correct Spelling of the Specific Epithet in the Species Name Bacillus Megaterium De Bary 1884: Approved by the Judicial Commission of the International Committee on Bacteriological Nomenclature". Int B Bact Nomencl T 1, 35–36.

Bulushi, I.M.A., Poole, S.E., Barlow, R., Deeth, H.C., and Dykes, G.A. (2010). Speciation of Gram-positive bacteria in fresh and ambient-stored sub-tropical marine fish. Int. J Food Microbiol 138, 32-38.

Calfarme. (2010). Calfarme Waterless Urinal Cleaner & Deodorizer/Drain & Septic System Maintainer.

CBSG. (2013). Canadian Biosafety Standards and Guidelines (CBSG) for Facilities Handling Human and Terrestrial Animal Pathogens, Prions, and Biological Toxins: First Edition.

CFIA. (2014). Pests Regulated By Canada.

CFIA. (2011). Organisms that do not require a Plant Protection Permit to Import.

Chowdhary, P.K., Keshavan, N., Nguyen, H.Q., Peterson, J.A., Gonzàlez, J.E., and Haines, D.C. (2007). Bacillus megaterium CYP102A1 oxidation of acyl homoserine lactones and acyl homoserines. Biochemistry (N. Y.) 46, 14429-14437.

CLSI (Clinical and Laboratory Standards Institute). (2012). Method for Antimicrobial Dilution and Disk Susceptibility Testing of Infrequently Isolated or Fastidious Bacteria; Approved Guideline- Second Edition M45-A2:30 no.18. 30

CLSI (Clinical and Laboratory Standards Institute). (2013). Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals; second informational supplement, VET01-S2. CLSI Document VET01-S2.

Coleman, W.H., Zhang, P., Li, Y.Q., and Setlow, P. (2010). Mechanism of killing of spores of Bacillus cereus and Bacillus megaterium by wet heat. Lett Appl Microbiol 50, 507-514.

Cottyn, B., Regalado, E., Lanoot, B., Cleene, M.D., Mew, T.W., and Swings, J. (2001). Bacterial populations associated with rice seed in the tropical environment. Phytopathology 91, 282-292.

Daligault, H.E., Davenport, K.W., Minogue, T.D., Bishop-Lilly, K.A., Broomall, S.M., Bruce, D.C., Chain, P.S., Coyne, S.R., Frey, K.G., Gibbons, H.S., et al. (2014). Twenty Whole-Genome Bacillus sp. Assemblies. Genome Announc 2, e00958.

de Bary, A. (1884). Vergleichende Morphologie und Biologie der Pilze, Mycetozoen und Bacterien. Wilhelm Engelmann, Leipzig, 500.

Deutsche Sammlung von Microoorganismen und Zellkulturen (DSMZ). (2014). Bacillus megaterium de Bary 1884.

Dhami, N.K., Reddy, M.S., and Mukherjee, A. (2013). Biomineralization of calcium carbonate polymorphs by the bacterial strains isolated from calcareous sites. J Microbiol Biotechn 23, 707-714.

Dib, E.G., Dib, S.A., Korkmaz, D.A., Mobarakai, N.K., and Glaser, J.B. (2003). Nonhemolytic, nonmotile gram-positive rods indicative of Bacillus anthracis. Emerg Infect Dis 9, 1013-1015.

Drahos, D., and Petersen, S. (2010). Methods, compositions and systems for controlling fouling of a membrane. US Patents 12/942,610, 1-27.

Drucker, D.B., and Whittaker, D.K. (1971). Microstructure of colonies of rod-shaped bacteria. J Bacteriol 108, 515-525.

Duncan, K.O., and Smith, T.L. (2011). Primary cutaneous infection with Bacillus megaterium mimicking cutaneous anthrax. J Am Acad Dermatol 65, e60-1.

Dursun, A., Ekinci, M., and Donmez, M.F. (2010). Effects of Foliar Application of Plant Growth Promoting Bacterium on Chemical Contents, Yield and Growth of Tomato (Lycopersicon esculentum L.) and Cucumber (Cucumis sativus L.). Pak J Bot 42, 3349-3356.

El-Arabi, T.F., Griffiths, M.W., She, Y.M., Villegas, A., Lingohr, E.J., and Kropinski, A.M. (2013). Genome sequence and analysis of a broad-host range lytic bacteriophage that infects the Bacillus cereus group. Virol J 10, 48-422-10-48.

Enomoto, A., Nakamura, K., Hakoda, M., and Amaya, N. (1997). Lethal effect of high-pressure carbon dioxide on a bacterial spore. J Ferment Bioeng 83, 305-307.

Environment and Climate Change Canada. (2005). An Assessment of the Pathogenicity and/or Toxicity of Bacillus licheniformis, B. megaterium, B. amyloliquefaciens, B. polymyxa, Paenibacillus polymyxa, B. circulans, and B. subtilis on Terrestrial Organisms in Soil. Environment and Climate Change Canada 1-30.

Eppinger, M., Bunk, B., Johns, M.A., Edirisinghe, J.N., Kutumbaka, K.K., Koenig, S.S., Creasy, H.H., Rosovitz, M.J., Riley, D.R., Daugherty, S., et al. (2011). Genome sequences of the biotechnologically important Bacillus megaterium strains QM B1551 and DSM319. J Bacteriol 193, 4199-4213.

Ercolani, G. (1978). Pseudomonas savastanoi and other bacteria colonizing the surface of olive leaves in the field. J Gen Microbiol 109, 245-257.

Esringu, A., Turan, M., Gunes, A., and Rustu, M. (2014). Roles of Bacillus megaterium in Remediation of Boron, Lead, and Cadmium from Contaminated Soil. Commun Soil Sci Plant Anal 45, 1741-1759.

European Commission. (2010). Directive 2000/54/EC of the European parliament and of the council of 18 September 2000 on the protection of workers from risks related to exposure to biological agents at work (seventh individual directive within the meaning of article 16(1) of directive. EC 54.

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

Ewing, D. (1971). Streptomycin resistance in Bacillus megaterium. Mutat Res 12, 315-319.

Euzéby, J.P. and Tindall, B.J. (2004) Valid publication of new names or new combinations: making use of the Validation Lists. ASM News. 70, 258-259.

Fajardo-Cavazos, P., and Nicholson, W. (2006). Bacillus endospores isolated from granite: close molecular relationships to globally distributed Bacillus spp. from endolithic and extreme environments. Appl Environ Microbiol 72, 2856-2863.

Gandhi, H.P., Ray, R.M., and Patel, R.M. (1997). Exopolymer production by Bacillus species. Carbohydr Polym 34, 323-327.

Gao, W., Smith, D., and Li, Y. (2007). Effects of Freezing on the Survival of Escherichia coli and Bacillus and Response to UV and Chlorine After Freezing. Water Environ Res 79, 507-513.

Gao, Y., and Xu, G. (2014). Development of an Effective Nonchemical Method against Plasmodiophora brassicae on Chinese Cabbage. Int J Agron 2014, 1-5.

Garvey, M., Clifford, E., O'Reilly, E., and Rowan, N.J. (2013). Efficacy of Using Harmless Bacillus Endospores to Estimate the Inactivation of Cryptosporidium parvum Oocysts in Water. J Parasitol 99, 448-452.

Ghosh, S., and Setlow, P. (2009). Isolation and characterization of superdormant spores of Bacillus species. J Bacteriol 191, 1787-1797.

Gordon, R., Haynes, W., and Pang, C. (1973). The genus bacillus. US Department of Agriculture Handbook 109-126.

Government of Canada. (2009). Human Pathogens and Toxins Act. (S.C. 2009, c. 24).

Hardin, J., and Suazo, A. (2012). New Pest Response Guidelines. Dendrolimus Pine Moths. USDA, 1-200.

Hastings, W.J., Ritter, M.A., Chamakura, K.R., and Everett, G.F.K. (2013). Complete Genome of Bacillus megaterium Siphophage Staley. Genome Announcements 1, 10.1128/genomeA.00864-13.

Hillesland, H., Read, A., Subhadra, B., Hurwitz, I., McKelvey, R., Ghosh, K., Das, P., and Durvasula, R. (2008). Identification of aerobic gut bacteria from the kala azar vector, Phlebotomus argentipes: a platform for potential paratransgenic manipulation of sand flies. Am J Trop Med Hyg 79, 881-886.

Hoffmann, C., Berganza, C., and Zhang, J. (2013). Cold Atmospheric Plasma: methods of production and application in dentistry and oncology. Med GasRes 3, 21-9912-3-21.

Honorat, A., Monot, F., and Ballerini, D. (1990). Synthesis of L-alanine and L-valine by enzyme systems from Bacillus megaterium. Enzyme Microb Technol 12, 515-520.

Hosford, R.M. (1982). White blotch incited in wheat by Bacillus megaterium pv. cerealis. Phytopathology 72, 1453-1459.

Hu, X., Roberts, D.P., Xie, L., Maul, J.E., Yu, C., Li, Y., Zhang, S., and Liao, X. (2013). Development of a biologically based fertilizer, incorporating Bacillus megaterium A6, for improved phosphorus nutrition of oilseed rape. Can J Microbiol 59, 231-236.

Kavamura, V.N., Santos, S.N., Silva, J.L., Parma, M.M., Avila, L.A., Visconti, A., Zucchi, T.D., Taketani, R.G., Andreote, F.D., and Melo, I.S. (2013). Screening of Brazilian cacti rhizobacteria for plant growth promotion under drought. Microbiol Res 168, 183-191.

Kehrenberg C, Schwarz S. 2006. Distribution of florfenicol resistance genes fexA and cfr among chloramphenicol-resistant Staphylococcus isolates. Antimicrob Agents Chemother 50:1156–1163

Khoury, P.H., Lombardi, S.J., and Slepecky, R.A. (1987). Perturbation of the Heat Resistance of Bacterial Spores by Sporulation Temperature and Ethanol. Curr Microbiol 15, 15-19.

Kieselburg, M.K., Weickert, M., and Vary, P.S. (1984). Analysis of Resident and Transformant Plasmids in Bacillus megaterium. Nat Biotechnol 2, 254-259.

Kong, Q., Shan, S., Liu, Q., Wang, X., and Yu, F. (2010). Biocontrol of Aspergillus flavus on peanut kernels by use of a strain of marine Bacillus megaterium. Int J Food Microbiol 139, 31-35.

Kumar, A., Maurya, B.R., and Raghuwanshi, R. (2014). Isolation and characterization of PGPR and their effect on growth, yield and nutrient content in wheat (Triticum aestivum L.). Biocatal Agri Biotechnol 3, 121-128.

Kurtzman, C.P., Rogers, R., and Hesseltine, C.W. (1971). Microbiological spoilage of mayonnaise and salad dressings. Appl Microbiol 21, 870-874.

Laaberki, M., and Dworkin, J. (2008). Death and survival of spore-forming bacteria in the Caenorhabditis elegans intestine. Symbiosis 46, 95-100.

Lapage, S.P., Hill, L.R., Midgley, J., and Shelton, J.E. (1967). Annotations from the NCTC on the List of Type and Reference Strains of Bacteria, Sneath and Skerman 1966. Int J Syst Bacteriol 17, 93-103.

Larsen, N., Thorsen, L., Kpikpi, E.N., Stuer-Lauridsen, B., Cantor, M.D., Nielsen, B., Brockmann, E., Derkx, P.M., and Jespersen, L. (2014). Characterization of Bacillus spp. strains for use as probiotic additives in pig feed. Appl Microbiol Biotechnol 98, 1105-1118.

Li, Y.L., Zhou, Z., and Yuan, Y.C. (2014). First Report of a Leaf Spot of Radermachera sinica in China Caused by Bacillus megaterium. Plant Dis 98, 1425.

Lillehoj, E.B., and Ciegler, A. (1970). Aflatoxin B1 induction of lysogenic bacteria. Appl Microbiol 20, 782-785.

Liu, L., Li, Y., Zhang, J., Zou, W., Zhou, Z., Liu, J., Li, X., Wang, L., and Chen, J. (2011). Complete genome sequence of the industrial strain Bacillus megaterium WSH-002. J Bacteriol 193, 6389-6390.

Liu, Z., and Sinclair, J.B. (1993). Colonization of soybean roots by Bacillus megaterium B 153-2-2. Soil Biol Biochem 25, 849-855.

Logan, N.A., and Berkeley, R.C.W. (1984). Identification of Bacillus strains using the API system. J Gen Microbiol 130, 1871-1882.

Logan, N.A., and De Vos, P. (2009) Genus I. Bacillus. In Bergey’s Manual of Systemic Bacteriology, Second Edition, Volume Three: The Firmicutes, P. De Vos, G.M. Garrity, D. Jones, N.R. Krieg, W. Ludwig, F.A. Rainey, K.-H. Shleifer, and W.B. Whitman, eds. (Springer Dordrecht Heidelberg London New York), pp. 21-127.

López, N.I., Floccari, M.E., Steinbüchel, A., García, A.F., and Mébdez, B.S. (1995). Effect of poly(3-hydroxybutyrate) (PHB) content on the starvation-survival of bacteria in natural waters. FEMS Microbiol Ecol 16, 95-102.

López, A.C., and Alippi, A.M. (2010). Enterotoxigenic gene profiles of Bacillus cereus and Bacillus megaterium isolates recovered from honey. Rev Argent Microbiol 42, 216-225.

López, A.C., Minnaard, J., Perez, P.F., and Alippi, A.M. (2013). In vitro interaction between Bacillus megaterium strains and Caco-2 cells. Int Microbiol 16, 27-33.

López, A.C., and Alippi, A.M. (2009). Diversity of Bacillus megaterium isolates cultured from honeys. LWT-Food Sci. Technol. 42, 212-219.

Lopez-Diez, E.C., and Goodacre, R. (2004). Characterization of microorganisms using UV resonance Raman spectroscopy and chemometrics. Anal Chem 76, 585-591.

Lushniak, B., and Mattorano, D. (1994). Health Hazard Evaluation Report. No. 93-0928.,

Marecik, R., Króliczak, P., Czaczyk, K., Białas, W., Olejnik, A., and Cyplik, P. (2008). Atrazine degradation by aerobic microorganisms isolated from the rhizosphere of sweet flag (Acorus calamus L.). Biodegradation 19, 293-301.

McInroy, J.A., and Kloepper, J.W. (1995). Survey of indigenous bacterial endophytes from cotton and sweet corn. Plant Soil 173, 337-342.

Mishra, R.R., Prajapati, S., Das, J., Dangar, T.K., Das, N., and Thatoi, H. (2011). Reduction of selenite to red elemental selenium by moderately halotolerant Bacillus megaterium strains isolated from Bhitarkanika mangrove soil and characterization of reduced product. Chemosphere 84, 1231-1237.

Mohammed, Y., Lee, B., Kang, Z., and Du, G. (2014). Development of a two-step cultivation strategy for the production of vitamin B12 by Bacillus megaterium. Microb Cell Fact 13, 102-112.

Muffler, K., Leipold, D., Scheller, M., Haas, C., Steingroewer, J., Bley, T., Neuhaus, H.E., Mirata, M.A., Schrader, J., and Ulber, R. (2011). Biotransformation of triterpenes. Process Biochem 46, 1-15.

Mundt, J.O., and Hinkle, N.F. (1976). Bacteria within ovules and seeds. Appl Environ Microbiol 32, 694-698.

Namjoshi, K., Johnson, S., Montello, P., and Pullman, G.S. (2010). Survey of bacterial populations present in US-produced linerboard with high recycle content. J Appl Microbiol 108, 416-427.

Newcombe, D.A., Schuerger, A.C., Benardini, J.N., Dickinson, D., Tanner, R., and Venkateswaran, K. (2005). Survival of spacecraft-associated microorganisms under simulated martian UV irradiation. Appl Environ Microbiol 71, 8147-8156.

Nguyen, M.T., Ranamukhaarachchi, S.L., and Hannaway, D.B. (2011). Efficacy of Antagonist Strains of Bacillus megaterium, Enterobacter cloacae, Pichia guilliermondii and Candida ethanolica against Bacterial Wilt Disease of Tomato. J Phytopathol 3, 1-10.

Obruca, S., Marova, I., Melusova, S., and Ondruska, V. (2009). Production of polyester-based bioplastics by Bacillus megaterium grown on waste cheese whey substrate under exogenous stress. New Biotechnol 25S, S257.

Olmos, J., Ochoa, L., Paniagua-Michel, J., and Contreras, R. (2011). Functional feed assessment on Litopenaeus vannamei using 100% fish meal replacement by soybean meal, high levels of complex carbohydrates and Bacillus probiotic strains. Mar Drugs 9, 1119-1132.

Orrell T. (2015). ITIS Global: The Integrated Taxonomic Information System (version Mar 2015). In: Species 2000 & ITIS Catalogue of Life, 23rd June 2015 (Roskov Y., Abucay L., Orrell T., Nicolson D., Kunze T., Culham A., Bailly N., Kirk P., Bourgoin T., DeWalt R.E., Decock W., De Wever A., eds). Species 2000: Naturalis, Leiden, the Netherlands

Osborn, F., Berlioz, L., Vitelli-Flores, J., Monsalve, W., Dorta, B., and Lemoine, V.R. (2002). Pathogenic effects of bacteria isolated from larvae of Hylesia metabus Crammer (Lepidoptera: Saturniidae). J Invertebr Pathol 80, 7-12.

Padgham, J.L., and Sikora, R.A. (2007). Biological control potential and modes of action of Bacillus megaterium against Meloidogyne graminicola on rice. Crop Prot 26, 971-977.

Parthasarathy, R., and Ravi, D. (2011). Probiotic bacteria as growth promoter and biocontrol agent against Aeromonas hydrophila in Catla catla (Hamilton, 1922). Indian J Fish 58, 87-93.

Patil, H.S.R., Naik, T.V., Avin, B.R.V., and Sayeswara, H.A. (2013). Isolation and molecular characterization of Bacillus megaterium isolated from various agro climatic zones of Karnataka and its effect on medicinal plant Ruta gladiolus. Curr Res Microbiol Biotechnol 1, 173-182.

Perestelo, F., Falcón, M.A., Pérez, M.L., Roig, E.C., and de la Fuente Martin, G. (1989). Bioalteration of kraft pine lignin by Bacillus megaterium isolated from compost piles. J Ferment Bioeng 68, 151-153.

PHAC. (2014). Pathogen Safety Data Sheets and Risk Assessment. Public Health Agency of Canada. Poleatewich, A.M., Ngugi, H.K., and Backman, P.A. (2012). Assessment of Application Timing of Bacillus spp. to Suppress Pre- and Postharvest Diseases of Apple. Plant Dis 96, 211-220.

Porcel, R., Zamarreno, A.M., Garcia-Mina, J.M., and Aroca, R. (2014). Involvement of plant endogenous ABA in Bacillus megaterium PGPR activity in tomato plants. BMC Plant Biology 14, 36-2229-14-36.

Priya, J.D., Divakar, K., Prabha, M.S., Selvam, G.P., and Gautam, P. (2014). Isolation, purification and characterisation of an organic solvent-tolerant Ca2+-dependent protease from Bacillus megaterium AU02. Appl Biochem Biotechnol 172, 910-932.

Product sheet. (2004). Turf Pro USA - Liquid Products, Class A, Non-pathogenic, Bacillus spores Added.

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.

Purkayastha, R., and Bhattacharyya, B. (1982). Antagonism of micro-organisms from jute phyllosphere towards Colletotrichum corchori. T Brit Mycol Soc 78, 509-513.

Quingming, Y., and Zongping, X. (1997). Rapid Classification of Bacillus Isolates Using RAPD Technique. Wuhan Univ. J Nat Sci 2, 105-109.

Quinn, J.P., Peden, J.M.M., and Dick, R.E. (1989). Carbon-phosphorus bond cleavage by Gram-positive and Gram-negative soil bacteria. Appl Microbiol Biotechnol 31, 283-287.

Radhika, D., Ramathilaga, A., Prabu, C.S., and Murugesan, A. (2011). Evaluation of larvicidal activity of soil microbial isolates (Bacillus and Acinetobactor Sp.) against Aedes aegypti (Diptera: Culicidae) - the vector of Chikungunya and Dengue. Proc Int Acad Ecol Environ Sci 1, 169-178.

Raj, A., Reddy, M.K., Chandra, R., Purohit, H.J., and Kapley, A. (2007). Biodegradation of kraft-lignin by Bacillus sp. isolated from sludge of pulp and paper mill. Biodegradation 18, 783-792.

Rajkumar, M., Ma, Y., and Freitas, H. (2013). Improvement of Ni phytostabilization by inoculation of Ni resistant Bacillus megaterium SR28C. J Environ Manage 128, 973-980.

Ramos-Esteban, J.C., Servat, J.J., Tauber, S., and Bia, F. (2006). Bacillus megaterium delayed onset lamellar keratitis after LASIK. J Refract Surg 22, 309-312.

Reva, O.N., Vyunitskaya, V.A., Reznik, S.R., Kozachko, I.A., and Smirnov, V.V. (1995). Antibiotic susceptibility as a taxonomic characteristic of the genus Bacillus. Int J Syst Bacteriol 45, 409-411.

Rilfors, L., Wieslander, A., and Stahl, S. (1978). Lipid and protein composition of membranes of Bacillus megaterium variants in the temperature range 5 to 70 degrees C. J Bacteriol 135, 1043-1052.

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.

Rosso, M.L., and Vary, P.S. (2005). Distribution of Bacillus megaterium QM B1551 plasmids among other B. megaterium strains and Bacillus species. Plasmid 53, 205-217.

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., and 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 Diagn Lab Immunol 9, 1057-1060.

Ruiz-Bravo, A., Kouwatli, K., Cienfuegos, G.A.d., and Ramos-Cormenzana, A. (1981). Immunomodulation in mice by Bacillus megaterium and its dependence on culture conditions. Immunol Lett 3, 39-43.

Rusterholtz, K., and Mallory, L. (1994). Density, activity, and diversity of bacteria indigenous to a karstic aquifer. Microb Ecol 28, 79-99.

Sadiq, A., and Ali, B. (2013). Growth and yield enhancement of Triticum aestivum L. by rhizobacteria isolated from agronomic plants. Aust J Crop Sci 7, 1544-1550.

Saikia, S.K., Tiwari, S., and Pandey, R. (2013). Rhizospheric biological weapons for growth enhancement and Meloidogyne incognita management in Withania somnifera cv. Poshita. Biol Control 65, 225-234.

Sakurai, M., Suzuki, K., Onodera, M., Shinano, T., and Osaki, M. (2007). Analysis of bacterial communities in soil by PCR-DGGE targeting protease genes. Soil Biol Biochem 39, 2777-2784.

Salih, F. (2001). Prediction of growth of Bacillus megaterium spores as affected by gamma radiation dose and spore load. J Appl Microbiol 91, 176-181.

Sandeep, C., Raman, R.V., Radhika, M., Thejas, M.S., Patra, S., Gowda, T., Suresh, C.K., and Mulla, S.R. (2011). Effect of inoculation of Bacillus megaterium isolates on growth, biomass and nutrient content of Peppermint. J Phytol 3, 19-24.

Saravanan, R., Dhachinamnorthi, D., Renuga, G., and Senthilkumar, K. (2011). Production of L-asparaginase from Pectobacterium carrotovorum (MTCC 14288) and Bacillus eirculanc (MTCC 490). Res J Pharm Technol 4, 1323-1327.

Saxena, A., Zhang, R.W., and Bollag, J.M. (1987). Microorganisms capable of metabolizing the herbicide metolachlor. Appl Environ Microbiol 53, 390-396.

Schallmey, M., Singh, A., and Ward, O.P. (2004). Developments in the use of Bacillus species for industrial production. Can J Microbiol 50, 1-17.

Scott BA, Mortensen JE, McKeever TM, Logas DB, McKeever PJ. 2010. Efficacy of tylosin tartrate on canine Staphylococcus intermedius isolates in vitro. Vet Ther 11:E1-7

Selenska-Pobell, S., Panak, P., Miteva, V., Boudakov, I., Bernhard, G., and Nitsche, H. (1999). Selective accumulation of heavy metals by three indigenous Bacillus strains, B. cereus, B. megaterium and B. sphaericus, from drain waters of a uranium waste pile. FEMS Microbiol Ecol 29, 59-67.

Shaharoona, B., Bibi, R., Arshad, M., Zahir, Z.A., and Zia-Ul-Hassan. (2006). 1-Aminocylopropane-1-carboxylate (ACC)-deaminase rhizobacteria extenuates ACC-induced classical triple response in etiolated pea seedlings. Pak J Bot 38, 1491-1499.

Shark, K.B., Smith, F.D., Harpending, P.R., Rasmussen, J.L., and Sanford, J.C. (1991). Biolistic transformation of a procaryote, Bacillus megaterium. Appl Environ Microbiol 57, 480-485.

Shimizu, K., Nakamura, H., and Ashiuchi, M. (2007). Salt-inducible bionylon polymer from Bacillus megaterium. Appl Environ Microbiol 73, 2378-2379.

Shimizu, S., Barczyk, S., Rettberg, P., Shimizu, T., Klaempfl, T., Zimmermann, J.L., Hoeschen, T., Linsmeier, C., Weber, P., Morfill, G.E., and Thomas, H.M. (2014). Cold atmospheric plasma - A new technology for spacecraft component decontamination. Planet Space Sci 90, 60-71.

Skerman, V.B.D., McGowan, V., Sneath, P.H.A. (1980). Approved list of bacterial names. Int J Syst Bacterial 30 225-420.

Slepecky, R.A., and Hemphill, H.E. (2006). Dworkin, M., Falkow, S., Rosenberg, E., Schleifer, K.-H., Stackebrandt, E. eds., (Berlin: Springer) 530-562.

Smalla, K., Wieland, G., Buchner, A., Zock, A., Parzy, J., Kaiser, S., Roskot, N., Heuer, H., and Berg, G. (2001). Bulk and rhizosphere soil bacterial communities studied by denaturing gradient gel electrophoresis: plant-dependent enrichment and seasonal shifts revealed. Appl Environ Microbiol 67, 4742-4751.

Smith, N., Gibson, T., Gordon, R.E., and Sneath, P. (1964). Type cultures and proposed neotype cultures of some species in the genus Bacillus. J Gen Microbiol 34, 269-272.

Sneath, P.H.A., and Skerman, V.B.D. (1966). A List of Type and Reference Strains of Bacteria. Int J Syst Bacteriol 16, 1-133.

Soon, N.W., Lee, L.M., Khun, T.C., and Ling, H.S. (2014). Factors Affecting Improvement in Engineering Properties of Residual Soil through Microbial-Induced Calcite Precipitation. J Geotech Geoenviron Eng 140.

Soper, C.J., Whistler, J.M., and Davies, D.J. (1976). The response of bacterial spores to vacuum treatments. II. Germination and viability studies. Cryobiology 13, 71-79.

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

Srinath, T., Verma, T., Ramteke, P.W., and Garg, S.K. (2002). Chromium (VI) biosorption and bioaccumulation by chromate resistant bacteria. (Science for Environmental Technology). Chemosphere 48, 427-435.

Srinavasan, M., Holt, F.B., and Petersen, D.J. (1996). Influence of indoleacetic-acid-producing Bacillus isolates on the nodulation of Phaseolus vulgaris by Rhizobium etli under gnotobiotic conditions. Can J Microbiol 42, 1006-1014.

Ståhl, S., and Olsson, O. (1977). Temperature range variants of Bacillus megaterium. Arch Microbiol 113, 221-229.

Ştefănescu, I.A., Gavrilă‚ L., and Mocanu, R. (2011). Evaluation of the solubilization ability of two strains of Bacillus megaterium for heavy metals from residual phosphogypsum. Rom Biotech Lett 16, 6513-6522.

Subbiya, A., and Mahalakshmi, K. (2012). Bordetella avium and Bacillus megaterium in Endodontic Infection. Indian J Multidiscip Dent. 2, 411-414.

Surette, M.A., Sturz, A.V., Lada, R.R., and Nowak, J. (2003). Bacterial endophytes in processing carrots (Daucus carota L. var. sativus): their localization, population density, biodiversity and their effects on plant growth. Plant Soil 253, 381-390.

Suzuki, Y., and Rode, L.J. (1969). Effect of lysozyme on resting spores of Bacillus megaterium. J Bacteriol 98, 238-245.

Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S. (2013). MEGA6: Molecular evolutionary genetic analysis version 6.0. Mol. Biol. Evol. 30, 2725-2729.

Taupp, M., Harmsen, D., Heckel, F., and Schreier, P. (2005). Production of natural methyl anthranilate by microbial N-demethylation of N-methyl methyl anthranilate by the topsoil-isolated bacterium Bacillus megaterium. J Agric Food Chem 53, 9586-9589.

Tersch, M.A.V., and Carlton, B.C. (1983). Megacinogenic plasmids of Bacillus megaterium. J Bacteriol 155, 872-877.

Thomas, P. (2012). Long-term survival of Bacillus spores in alcohol and identification of 90% ethanol as relatively more spori/bactericidal. Curr Microbiol 64, 130-139.

Tiwari, R.P., Dham, C.K., Bhalla, T.C., Saini, S.S., and Vadehra, D.V. (1985). Mechanism of action of aflatoxin B1 in Bacillus megaterium. Appl Environ Microbiol 49, 904-907.

University of Illinois. (1999). Bacterial Wetwood and Slime Flux of Landscape Trees. Integrated Pest Management RPD No. 656.

USDA. (2014a). Thesaurus: Bacillus megaterium.  

USDA. (2014b). GrainGenes Species Report: Bacillus megaterium pv. cerealis.USEPA. (2014). Substance Details - Bacillus megaterium.

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

Vandini, A., Temmerman, R., Frabetti, A., Caselli, E., Antonioli, P., Balboni, P.G., Platano, D., Branchini, A., and Mazzacane, S. (2014). Hard Surface Biocontrol in Hospitals Using Microbial-Based Cleaning Products. PLOS One 9, 1-13.

Vary, P.S. (1994). Prime time for Bacillus megaterium. Microbiology 140, 1001-1013.

Vary, P.S., Biedendieck, R., Fuerch, T., Meinhardt, F., Rohde, M., Deckwer, W.D., and Jahn, D. (2007). Bacillus megaterium--from simple soil bacterium to industrial protein production host. Appl Microbiol Biotechnol 76, 957-967.

Vasanthakumar, B., Ravishankar, H., and Subramanian, S. (2013). Microbially induced selective flotation of sphalerite from galena using mineral-adapted strains of Bacillus megaterium. Colloid Surface B. 112, 279-286.

Velineni, S., and Brahmaprakash, G. (2011). Survival and Phosphate Solubilizing Ability of Bacillus megaterium in Liquid Inoculants under High Temperature and Desiccation Stress. J Agric Sci Technol 13, 795-802.

Verma, N., Singh, N.A., Kumar, N., Singh, V.K., and Raghu, H.V. (2013). Development of Field Level Chromogenic Assay for Aflatoxin M21 Detection in Milk. Adv Dairy Res 1, 1-8.

Verslyppe B., De Smet W., De Baets B., De Vos P., Dawyndt P. (2014). StrainInfo introduces electronic passports for microorganisms. Syst Appl Microbiol 37, 42-50.

Weber, D.J., Saviteer, S.M., Rutala, W.A., and Thomann, C.A. (1988). In vitro susceptibility of Bacillus spp. to selected antimicrobial agents. Antimicrob Agents Chemother 32, 642-645.

Welbaum, G.E., Shen, Z., Watkinson, J.I., Wang, C., and Nowak, J. (2009). Priming soilless growing medium with disaccharides stimulated microbial biofilm formation, and increased particle aggregation and moisture retention during muskmelon transplant production. J Am Soc Hort Sci 134, 387-395.

West, E.R., Cother, E.J., Steel, C.C., and Ash, G.J. (2010). The characterization and diversity of bacterial endophytes of grapevine. Can J Microbiol 56, 209-216.

White, C.P., Popovici, J., Lytle, D.A., Adcock, N.J., and Rice, E.W. (2012). Effect of pH on the electrophoretic mobility of spores of Bacillus anthracis and its surrogates in aqueous solutions. Appl Environ Microbiol 78, 8470-8473.

White, P.J. (1972). The nutrition of Bacillus megaterium and Bacillus cereus. J Gen Microbiol 71, 505-514.

Willeke, K., Qian, Y., Donnelly, J., Grinshpun, S., and Ulevicius, V. (1996). Penetration of airborne microorganisms through a surgical mask and a dust/mist respirator. Am Ind Hyg Assoc J 57, 348-355.

Xiang, W., Liang, H., Liu, S., Luo, F., Tang, J., Li, M., and Che, Z. (2011). Isolation and performance evaluation of halotolerant phosphate solubilizing bacteria from the rhizospheric soils of historic Dagong Brine Well in China. World J Microb Biot 27, 2629-2637.

Xu, G., Mitchell, K.W., and Monticello, D.J. (1997). Process for demetalizing a fossil fuel.

Yoshimura, K., Yamamoto, O., Seki, T., and Oshima, Y. (1983). Corrected Version Distribution of Heterogeneous and Homologous Plasmids in Bacillus spp. Appl Environ Microbiol 46, 1268-1275.

Yossan, S., Reungsang, A., and Yasuda, M. (2006). Purification and Characterization of Alkaline Protease from Bacillus megaterium isolated from Thai Fish Sauce Fermentation Process. ScienceAsia 32, 377-383.

Yuniarti, A., Guntoro, D., Maftuch, and Hariati, A. (2013). Response of Indigenous Bacillus megaterium Supplementation on the Growth of Litopenaeus vannamei (Boone), a New Target Species for Shrimp Culture in East Java of Indonesia. J Basic Appl Sci 3, 747-754.

Appendices

Appendix A: Metabolism by B. megaterium strain ATCC 14581

Data generated by Environmental Health Science and Research Bureau, Health Canada

^a Cells were motile in fetal bovine serum at 37^°C; however, the bacterium was non-motile within minutes of removal from the incubator

Appendix B: Characteristics of B. megaterium – Fatty acid methyl ester (FAME) analysis

Data generated by Environmental Health Science and Research Bureau, Health Canada shows the best match between the sample and the environmental and clinical MIDI databases and the fatty acid profile similarity index (average of all matches) along with the number of matches (number of matches/total number of tests, parentheses). For methods and additional details, see MIDI labs.

Appendix C: Growth characteristics of B. megaterium strain ATCC 14581

Data generated by Environmental Health Science and Research Bureau, Health Canada

^a TSB, tryptic soy broth

^b Long chains observed by microscopy

^c FBS, fetal bovine serum

^d Substantial bacilli observed by microscopy

^e Spores/debris observed by microscopy

^f Spores observed by microscopy

^g Lysed bacilli observed by microscopy

^h DMEM, Dulbecco’s Modified Eagle’s Medium

ⁱ Appears to be fewer than time=0 min (by microscopy)

Appendix D: Antibiotic susceptibility profiles of B. megaterium strains reported in the literature

^a Breakpoints for susceptibility cannot be determined using the disk diffusion method because limited data currently exist for this genus (CLSI 2013)

^b Results of 10 strains of B. megaterium, 10 strains of B. cereus and 30 strains of B. subtilis (Reva et al. 1995)

^S indicates susceptible; I indicates intermediate; R indicates resistant

^a Data and interpretation of antibiotic susceptibility from Sadiq and Ali (2013)

^S indicates susceptible; I indicates intermediate susceptibility; R indicates resistant: NR indicates, not required

^a (Larsen et al. 2014)

^b Strains with MIC values (mg/L) higher than the breakpoint are considered to be resistant (EFSA 2008)