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Received Date : 05-May-2014 Revised Date : 28-May-2014 Accepted Date : 31-May-2014 Article type

: Original Article

Bacillus thuringiensis HD-1 Cry-: development of a safe, non-insecticidal simulant for Bacillus anthracis A.H. Bishop* and Robinson, C.V. Detection Department, Defence Science and Technology Laboratory, Porton Down, Salisbury, Wiltshire SP4 0JQ, UK.

*E-mail: [email protected]

Running headline: A new simulant for Bacillus anthracis

Keywords: Bacillus anthracis, Bacillus thuringiensis, simulant, spore, non-insecticidal, safety

Crown copyright 2014. Published with the permission of the Defence Science and Technology Laboratory on behalf of the Controller of HMSO. Abstract Aims: A representative simulant for spores of Bacillus anthracis is needed for field testing. Bacillus thuringiensis is gaining recognition as a suitable organism. A strain that does not form the insecticidal, parasporal crystals that are characteristic of this species is a more This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/jam.12560 This article is protected by copyright. All rights reserved.

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accurate physical representative of B. anthracis spores. We developed non-insecticidal derivatives of two isolates of B. thuringiensis HD-1. Methods and Results: Two plasmid-cured derivatives of B. thuringiensis HD-1, unable to make crystal toxins (‘Cry-‘), were isolated. These isolates and the existing Cry- strain, B. thuringiensis Al Hakam, were probed with PCR assays against the known insecticidal genes cry, vip and cyt. Their genomic DNA was sequenced to demonstrate a lack of insecticidal genes. This was confirmed by bioassays against a number of invertebrate species. Realtime PCR assays were developed to identify the B. thuringiensis HD-1 Cry- derivatives and an effective differential and selective medium was assessed. Conclusions: All three Cry- isolates are devoid of known insecticidal determinants. The B. thuringiensis HD-1 Cry- derivatives can easily be recovered from soil and identified by PCR with some selectivity. Significance and Impact of Study: The B. thuringiensis HD-1 Cry- derivatives represent accurate, non-genetically manipulated simulants for B. anthracis with excellent human and environmental safety records.

Keywords: Bacillus anthracis, Bacillus thuringiensis, simulant, spore, non-insecticidal, safety

Introduction For many years Bacillus atrophaeus subsp. globigii (formerly B. globigii) has been used as a spore simulant (Gibbons et al., 2011) for B. anthracis. Advances in microbiology have uncovered a number of factors that reveal B. atrophaeus to be a poor choice as a surrogate spore-forming organism. These include differences in size, morphology, hydrophobicity, heat resistance and genetics (Koshikawa et al., 1989; Carrera et al., 2007; Greenberg et al., 2010; Wood et al., 2010). The latter point is important because, with increasing molecular tools available, it is desirable to find direct orthologues of genes, found to be of interest in the

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genome of the surrogate, in that of B. anthracis and vice versa. In recent years, B. thuringiensis has become regarded by many as the best surrogate spore-former (Anon., 2003; Greenberg et al. 2010; Sinclair et al., 2012): at a genomic level B. anthracis may be regarded as the same species as B. thuringiensis (Daffonchio et al. 2000; Helgason et al., 2000). There is an overwhelming similarity in their genetic content and synteny (Keim et al., 2009). The spores of the two species are the same size and the spore integuments have the same composition and morphology (Tufts et al., 2014). The latter point is particularly important because the spores of B. atrophaeus are noticeably smaller than B. anthracis/ B. thuringiensis (Carrera et al., 2007) and they lack an exosporium which the other two species possess. The exosporium is now believed to be important in attachment to surfaces (Bozue et al., 2007; Williams et al., 2013). This and the related phenomenon of re-aerosolization are important concerns in hazard assessment (Tufts et al., 2014).

Some strains of B. thuringiensis have been used for over 60 years as biopesticides because of the characteristic, crystalline, insecticidal proteins that they accumulate in the spore mother cell (Schnepf et al., 1998). These crystals, encoded by the cry genes, may represent over 25% (Aigasse and Lereclus, 1995) of the mass of the contents of the spore mother cell and are not synthesized by B. anthracis. They represent a problem in the use of this organism as a simulant because they affect the aerodynamic properties (Tufts et al. 2014) of the spore and also interfere with decontamination studies by quenching some agents. In environmentally-sensitive areas it may also not be desirable to apply a biopesticide.

Several strains of B. thuringiensis have been used as spore simulants, these include B. thuringiensis subsp. kurstaki HD-1 (Raber and Burklund, 2010; Buckley et al., 2012; Anon. 2013;) and B. thuringiensis strain Al Hakam (Challacombe et al., 2007; Buhr et al., 2012, Omotade et al., 2013). A considerable advantage accrues to B. thuringiensis HD-1 because its use as a commercial biopesticide has generated an unrivalled safety record as an environmentally-exploited micro-organism in terms of human and environmental safety data.

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This is corroborated by biopesticide registrations by numerous bodies such as the American Environmental Protection Agency (Anon., 1986), the United States Department of Agriculture (Anon., 1992) and the World Health Organisation (Anon., 1999). B. thuringiensis strain Al Hakam is naturally lacking in the ability to form parasporal insecticidal crystal which benefits it performance as a simulant. There are, however, no publicly available data regarding the human or environmental safety that has been generated specifically for this strain.

The impetus for the work presented here was the need for a safe, spore-forming surrogate for B. anthracis which had as many favourable features as possible. To this end, acrystalliferous derivatives of two isolates of B. thuringiensis HD-1 were generated and evaluated. In parallel, confirmation was sought for the lack of insecticidal potential of B. thuringiensis Al Hakam using genetic and bioassay analysis.

Materials and methods Bacterial strains and culture conditions Two isolates of B. thuringiensis subsp. kurstaki strain HD-1, designated strains A and D, were obtained from Dr H.D. Burges (Glasshouse Crops Research Institute, UK). B. thuringiensis Al-Hakam was provided by Dr Tony Buhr, Naval Surface Warfare Center, Dahlgren, USA. B. thuringiensis subsp. galleriae HD-106 was purchased from the Bacillus Genetic Stock Center, USA. The nematicidal strain C9 was a gift from Dr Colin Berry (University of Cardiff, UK). B. thuringiensis subsp. tenebrionis and subsp. israelensis 5724 were purchased from DSM (Germany). B. thuringiensis strains 13B, 2408, HD-73 and HD-2 came from the culture collection of one of the authors (AHB). The following environmental isolates of B. thuringiensis were randomly selected from the Dstl culture collection: KpC1; KsF1; KsAc1; OpQ1; OpQ2; PO14 (Andrzejczak and Lonc, 2008); 67-S-2; 67-S-3; 86-S-7; 248-S-1; 286-S-1; 2810-S-4; 2810-S-6 (Bizzarri and Bishop, 2007); 169-S-16; 172-S-3; 65– S-47; 127-S-1; 158-S-2; 158-S-3; 216-S-2; 2810-S-3; 2810-S-8 (Bizzarri et al., 2008). Two

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B. cereus strains were used:1230-88 (donated by Prof. Per Einaar Granum, University of Oslo and 186-S-1 (Bizzarri et al., 2008). B. atrophaeus subsp. globigii came from the Dstl culture collection. Bacteria were routinely cultured at 28 oC on tryptone soy agar (TSA) or tryptone soy broth (TSB), supplemented with CCY salts (Stewart et al., 1981) to aid sporulation.

Plasmid curing Both B. thuringiensis subsp. kurstaki HD-1 isolates A and D were streaked onto TSA plates and incubated at 42 °C overnight. Colonies were re-streaked onto fresh plates at the end of vegetative growth and incubated again at 42 °C. This was repeated until colonies were found where the spore mother cells lacked the characteristic parasporal crystals (Schnepf et al., 1998), as observed using phase contrast microscopy. Such colonies were sub-cultured at 28 oC several times to check that they maintained the Cry- phenotype. One such colony from the parental strains A and D were selected and designated A- and D-, respectively.

PCR amplification of toxin genes The Cry- derivatives, A- and D-, and their respective parental strains and B. thuringiensis Al Hakam, were grown in 25 ml TSB overnight at 30 °C with shaking at 150 rev min-1. An aliquot (1ml) of each culture was centrifuged at 13,000 g in a bench-top centrifuge. The wildtype strains were re-suspended in 100 μl sterile distilled H2O by vortexing and boiled for 10 min. The boiled cells were diluted 1/1000 in H2O before use as template for PCR. Strains of B. thuringiensis used as positive controls for PCR amplification of particular toxin genes (shown in brackets) were streaked out on TSA plates and grown overnight at 28°C. A loopful of cells was added to 500 μl sterile distilled H2O, vortexed and boiled for 10 min before use as template. These were: strain B. thuringiensis HD-1 wild type (cry1); strain B. thuringiensis HD-1 wild type (cry2); subsp. tenebrionis (cry3); subsp. israelensis (cry4); strain HD-106 (cry9); subsp. israelensis (cytK) and B. thuringiensis strain HD-1 (vip3). PCR amplification was carried out using specific primers for a range of known B. thuringiensis

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genes (Table 1). PCR amplification was carried out in 25 μl reactions containing 1 ng or 50 ng DNA or 1 μl boiled cells, 1 x PCR buffer, 1 mM MgCl2, 200 μM each dNTP, 0.1 μM each primer and 5 U Taq DNA polymerase. The thermal cycle included an initial denaturing step at 94 °C for 2 min, followed by 35 cycles of 94 °C for 1 min, 50 - 54 °C (see Table 1) for 30 s, 72 °C for 45 s and a final elongation step at 72 °C for 3 min.

Real-time PCR assays DNA was extracted by boiling, as described above, from all of the environmental isolates of B. thuringiensis and B. cereus. B. thuringiensis strains HD-73, strain HD-1 A and D and their Cry- derivatives acted as positive controls. Real-time primers and probes (Table 1) were designed to the plasmid pHT8_2 (Embl reference CP004075). PCR amplification was carried out using a SmartCycler® PCR system (Cepheid) in 25 μl reactions containing 1 μl boiled cells, 1 x TaqMan® Fast Virus 1-Step Master Mix (Life Technologies) and 0.4 μM each primer and probe. The thermal cycle included an initial denaturing step at 95 °C for 2 min, followed by 60 °C for 30 sec then 40 cycles of 95 °C for 15 sec, 60 °C for 60 sec.

Plasmid profiles Plasmids were extracted and resolved on agarose gels using the method by ReyesRamírez and Ibarra (2008).

Cloning and sequencing of 8 Kb plasmid Small plasmids were isolated from the Cry- strains A- and D- using a plasmid isolation kit (Invitrogen), according to the manufacturer’s instructions. A plasmid of approximately 8 kB was excised from an agarose gel and digested with HindIII and BamHI. The products were cloned into pUC18 (Invitrogen) and sequenced by the Sanger method using M13 and sequence-derived primers (Beckman Coulter Cogenics, UK).

DNA sequencing and bioinformatics analysis

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DNA was extracted from B. thuringiensis Al Hakam and the Cry- isolates A- and D- using a PureGene DNA extraction kit (Qiagen). DNA was quantified using a Qubit® fluorometer (Invitrogen) and prepared for next-generation sequencing using a Nextera DNA sample prep kit (Illumina). The resulting library was sequenced on a MiSeq personal sequencer (Illumina). The quality of all sequencing reads was initially examined using the FastQC program (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and reads were subsequently truncated when the base qualities dropped below about Q30. This resulted in lengths of 250 bases for the first paired-end reads and 220 bases for the second paired-end reads, for all three organisms sequenced.

For the B. thuringiensis HD-1 A- and D- derivatives and B. thuringiensis Al Hakam, sequence analysis was carried out using the MIRA software (Chevreux et al., 1999). Initially a reference-mapping approach was used to align the sequencing data against a reference. Reference strain BMB171 (GenBank entries CP001903.1 and CP001904.1 for the genome and plasmid sequences, respectively) was used for both B. thuringiensis HD-1 derivatives, whilst the B. thuringiensis Al Hakam reference strain (GenBank entry CP000485.1) was used for the sequenced B. thuringiensis Al Hakam strain. Parameters used for mapping with MIRA included using the ‘Solexa Settings’ parameters, the highly repetitive option enabled and a paired-end insert range set as 0-750 bases. Reads which did not align to the respective references were subsequently de novo assembled into contigs with MIRA. The resulting contigs were screened for known insecticidal genes of the cry, cyt and vip toxin genes and proteins that may be found in different strains of B. thuringiensis (http://www.lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/, accessed 21st March 2013). This was performed through searching of custom databases, constructed using all known genomic and protein sequences of these toxins obtained from the NCBI GenBank and Protein databases, using the Bowtie2 (Langmead and Salzberg, 2012) and BLASTX (Altschul et al., 1990) tools respectively. These searches were performed using the default settings for both Bowtie2 and Blastx programs.

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Invertebrate bioassays Liquid cultures of the test isolates were verified to be at least 99% sporulated by microscopic examination. The cultures were centrifuged at 6,000 g for 30 min and washed in 1M NaCl. The pellets were then washed twice in sterile distilled water and enumerated by plate counts on TSA. Crystal protein content was assayed as described by Johnson et al. (1998). The negative controls contained sterile water. Preparations of either spores, for Cry- derivatives, or spores and associated crystals for wild type B. thuringiensis strains were used. For the toxic strains these were given over a range of seven concentrations to calculate the LC50 values by Probit analysis (Finney, 1974). A maximum dose of 1 x 108 CFU ml-1 of water, 1 x 108 CFU g-1 solid diet or 1 x 108 CFU cm-2 of leaf was generally used where toxicity was not apparent.

The following species from the order Lepidoptera were used: Galleria mellonella (Family: Pyralidae) was purchased from Porton Aquatics and maintained on an artificial diet (Fröbius et al., 2000). Twenty, third-instar larvae were used per replicate. The positive control organism was B. thuringiensis subsp. galleriae H-106 while, for the remaining lepidopteran species, it was B. thuringiensis HD-1 wild type. Spodoptera exigua (Family: Noctuidae) larvae were provided by Dr Amit Prabhakar, University of Greenwich, UK and maintained on an artificial diet (Moar et al., 1989). Twenty, third-instar larvae were used for each replicate. Pieris brassicae (Family: Noctuidae) and Plutella xylostella (Family: Plutellidae), obtained from BBSRC Rothamsted, UK, were fed on discs of organic cabbage leaves. Ten, first-instar larvae of each species were added per disc.

Bioassays for the common mealworm (Tenebrio molitor, order Coleoptera) were conducted according to the method of Du Rand and Laing (2011). The positive control was B. thuringiensis subsp. tenebrionis. Whiteworms (Enchytraeus albidus, order Haplotaxida) were maintained on peat-free soil and ten individuals were used per replicate. There was no positive control because no activity against annelids has ever been reported for B.

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thuringiensis. The bioassays for springtails (Folsomia candida, order Collembola) were set up as for the whiteworms, above. The positive control was strain B2408 (Prabhakar and Bishop, 2011). Larvae of Musca domestica, order Diptera, were assayed using the method and positive control strain of B. thuringiensis, 13B, described by Johnson et al. (1998). Caenorhabditis elegans, order Rhabditida), obtained from Dr Keith Davies (University of Hertfordshire, UK), were incubated in tap water containing E. coli MRE162 (106 CFU ml-1). The positive control strain was B. thuringiensis C9. All of the other invertebrates, unless stated otherwise, were obtained from Dartfrog UK.

All of the assays were repeated twice with freshly prepared bacteria. The temperature was maintained at 22 oC. Any assays where mortality in the negative controls exceeded 10% were discarded. Mortality and morbidity was assessed daily for at least three days. Assay for β-exotoxin The method of Johnson et al. (1998) was used. Culture fluid was incorporated into the diet at 0, 10 and 20% (v/v). B. thuringiensis strain 13B was the positive control. There were eight replicates of ten larvae for each bacterial strain. The assay was repeated twice with fresh culture fluid.

Recovery B. thuringiensis from soil samples Grass-soil microcosms were prepared and maintained as described in Bishop et al. (2014). Spores of the Cry- derivatives A- and D- were inoculated at a level of 104 CFU g-1 dry weight of soil. After incubation for two weeks the bacteria were shaken in extraction buffer (Bishop et al., 2014). The resultant slurry was serially diluted and plated on a selective and differential medium Brilliance Bacillus cereus, Oxoid) made according to the manufacturer’s instructions except for the addition of penicillin G (Sigma) at 100 μg ml-1.

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Results Plasmid curing After several passages at elevated temperature a number of colonies from both parental strains sporulated at 28 oC to produce spore mother cells with no parasporal crystals, as seen by light microscopy. One colony each with this Cry- phenotype was selected from the wild type strains, A and D, and designated A- and D-, respectively.

Plasmid profiles It was evident that the Cry- derivatives of B. thuringiensis HD-1, A- and D-, had lost the higher molecular mass plasmids typically associated with cry genes (González et al.¸1982). It is of interest that B. thuringiensis Al Hakam, while being unable to produce parasporal δendotoxin crystals, still contains such large plasmids (Fig. 1).

Whole genome sequencing and bioinformatics analysis Sequencing reads from both Cry- derivatives of B. thuringiensis HD-1 were initially aligned against the BMB171 reference strain, while the reads from the B. thuringiensis Al Hakam strain were aligned against the B. thuringiensis Al Hakam reference. Any reads not aligned to the references were subsequently de novo assembled into contigs. This resulted in 576, 1534 and 2 contigs for the B. thuringiensis HD-1 A- and D- derivatives and B. thuringiensis Al Hakam strain, respectively. Searching these contigs against databases of known toxin genes and proteins revealed that none of the contigs from the two cry- derivatives of B. thuringiensis HD-1 or the B. thuringiensis Al Hakam strain contained sequences similar to the cry, cyt or vip (invertebrate toxicity) genes or proteins found in other B. thuringiensis strains which have invertebrate toxicity. Combined with knowledge that both B. thuringiensis BMB171 and Al Hakam references do not contain cry, cyt or vip genes, this provided evidence that none of sequenced DNA obtained from the three strains contained any parts of these genes.

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Sequencing of 8 Kb plasmid Sequence analysis showed that an 8 Kb plasmid, present in both B. thuringiensis Cryisolates A- and D- was identical to that of pHT8_2 in B. thuringiensis strain HD-73 (Embl reference CP004075). A real-time PCR set was designed to an 84 bp section of this plasmid (Table 1) as a means to identify these strains.

Real time PCR of Cry- derivatives Only two (OpQ2 and 286-S-2) of the 24 environmental isolates of B. cereus and B. thuringiensis produced positive signals using the real-time PCR primers based upon the pHT8_2 sequence (Table 1). As expected, B. thuringiensis strain HD-73 and B. thuringiensis HD-1 A- and D- produced strong positive responses.

Bioassays Spore/crystal preparations of both the A and D parental strains of B. thuringiensis HD-1 produced very similar mortality in the lepidopteran species and so are presented as one strain (Table 2). No toxicity was observed with these strains with the fourth lepidopteran species, G. mellonella, nor with any of the other invertebrate species. When added at 1 x 108 CFU g-1 of diet the positive control strain, B. thuringiensis strain HD-106, killed all of the G. mellonella larvae. At a similar dosage the positive control organism, strain B2408, only caused an average of 27% mortality in the springtails (F. candida). Similarly, the positive control, strain C9, resulted in an average of 17% mortality to the nematode C. elegans. The spore preparations of the Cry- derivatives, B. thuringiensis HD-1 A- and D- and B. thuringiensis strain Al Hakam exhibited no toxicity to any of the invertebrate species tested, even at the 1 x 108 CFU dose rate (Table 2).

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Assay for β-exotoxin There was no mortality in D. hydei at even 20% incorporation rate of autoclaved supernatant into the diet for any of the Cry- derivatives or B. thuringiensis HD-1 wild types. All of the larvae pupated successfully and there were no deformities in the emerging adults. When the supernatant from the positive control organism, 13B, comprised 10% of the diet there was 100% larval mortality within 96 h.

Recovery of B. thuringiensis from soil samples Both B. thuringiensis HD-1 A- and D- produced bright blue-green colonies with beige haloes when grown on the supplemented, selective and differential medium (Fig. 2). The appearance of contaminating colonies did not appear until two days after the B. thuringiensis colonies were clearly visible. No attempt was made to screen the other environmental isolates of B. cereus or B. thuringiensis for their ability to grow on the penicillinsupplemented medium.

Discussion The finished sequence of the genome of B. thuringiensis Al Hakam has already been published (Challacombe et al., 2007). The sequence data generated here showed that the isolate held at Dstl only differs from it at two sites. These differences could be explained in sequencing errors. The bioinformatic analysis of the sequences generated for this strain and for B. thuringiensis HD-1 A- and D- showed that there were no fragment sequences of any known cry, vip or cyt genes. Hence, the likelihood of toxin gene fragments begin recombined or transferred to generate new insecticidal strains is non-existent. This corroborates the PCR assays directed against regions of a number of cry, cyt and vip genes (Table 1). The Cryisolates exhibited no invertebrate toxicity, confirming that the lost plasmids carried all of the invertebrate toxin genes. In particular, there was no effect when the spore preparations were fed to larvae of the four species of Lepidoptera (Table 2). Three of these (P. xylostella, P.

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brassicae and S. exigua) were susceptible to the parental strains of B. thuringiensis HD-1. The LC50 value for S. exigua was thirteen times lower than the challenge given with the acrystalliferous strains. In the cases of P. xylostella and P. brassicae no toxicity was observed when over ten thousand times the LC50 values for the parental strains was administered. The bioassay data also showed a lack of toxicity to any of the other invertebrate species tested. For the non-insect invertebrates, where feeding avoidance at the higher spore levels was not apparent, doses of 1 x 109 CFU g-1 were non-toxic for any of the strains.

The B. thuringiensis HD-1 wild type strains were not expected to produce β-exotoxin, in keeping with their biopesticide usage (Johnson et al., 1998). Neither their Cry- derivatives nor B. thuringiensis Al Hakam were found to do so either, even at high doses. The βexotoxins are non-ribosomally synthesized neurotoxins (Espinasse et al., 2004) whose basis of production is uncertain so bioassays or other analyses (Levinson, 1990; Johnson et al., 1998) are necessary to verify their absence. This is the first published demonstration that B. thuringiensis Al Hakam has no invertebrate toxicity, at least to the species tested.

A number of bacteria have been used as simulants for B. anthracis, particularly for the spore form (Sagripanti et al., 2007; Raber and Burklund, 2010; Gibbons et al., 2011, Buhr et al., 2012; Murphy et al., 2012). A bar-coded derivative of B. thuringiensis subsp. kurstaki has been developed which allows a unique PCR tag to be inserted into the chromosome (Buckley et al., 2013). This strain would probably not be permitted for use in some countries because it would be regarded as genetically modified. It also suffers the drawbacks, outlined above, relating to the parasporal, insecticidal crystals that are still produced.

B. thuringiensis subsp. kurstaki HD-1 is not the only strain approved for biopesticide use and which, therefore, possesses a long safety record. Curing a bioinsecticide strain of B.

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thuringiensis subsp. israelensis, for example, was considered at the outset of the work. This was discounted because the bacteria in this subspecies may be orientated to survival in wetland or aquatic conditions (Guidi et al., 2011); members of sub-species kurstaki, being isolated from terrestrial environments might have survival characteristics in soil more similar to those of B. anthracis.

It may be important to track the distribution and persistence of a bacterial simulant after release. Indeed, one of the early attractions to the use of B. atrophaeus as a simulant was that it can produce distinctively coloured colonies on solid media (Gibbons et al., 2011). The selective and differential medium reported here facilitates recovery of the simulant derivatives even from a large background of contaminating organisms. The real-time PCR assay, targeting a section of the pHT8_2 plasmid present in both of the Cry- derivatives, produced a positive response in only 8% (2/ 24) of the randomly selected, environmental isolates. This would provide a useful level of discrimination to differentiate the applied simulant from most other environmental strains of B. cereus or B. thuringiensis.

No organism can be a completely accurate simulant for B. anthracis in all respects but isolates A- and D- are superior to the parental B. thuringiensis HD-1 strains as simulants because they lack the insecticidal crystal proteins. Furthermore, these Cry- derivatives were not produced by genetic modification and so may be acceptable for use in many countries. The Cry- derivatives of B. thuringiensis strain HD-1 described here benefit from the highly documented human and environmental safety record of their parent strains. This is a very important advantage over B. thuringiensis Al Hakam which has no reported environmental or human safety record. This is particularly a concern where the environmental release of aerosols is being considered.

Future work will characterise the behaviour of the B. thuringiensis HD-1 Cry- simulants in aerosols, their attachment to surfaces, propensity for re-aerosolisation and environmental

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survival. Their response to physical and chemical decontamination will be compared to that of B. anthracis. Preliminary results indicate that spores of isolate D- respond more similarly to those of B. anthracis when exposed to decontaminants than do spores of isolate A(Bishop and Robinson, unpublished data).

Acknowledgements This work was funded by the Ministry of Defence, UK. The authors are grateful to Dr Tom Piggot, Dstl, for bioinformatic analysis. Conflict of Interest No conflict of interest declared. References Agaisse, H. and Lereclus, D. (1995). How does Bacillus thuringiensis produce so much insecticidal crystal protein? J Bacteriol 17, 6027–6032. Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. (1990) Basic local alignment search tool. J. Mol. Biol. 215, 403-410. Andrzejczak, S. and Lonc, E. (2008) Selective isolation of Bacillus thuringiensis from soil by use of L-serine as minimal medium supplement. Pol J Microbiol 57, 333-335. Anon. (1986) U.S. Environmental Protection Agency. Pesticide fact sheet for Bacillus thuringiensis. Fact sheet no. 93. Office of Pesticide Programs. Washington, DC. Anon. (1999) Environmental Health Criteria 217. Microbial Pest Control Agent Bacillus thuringiensis. World Health Organisation, Geneva. Ben-Dov, E., Zaritsky, A., Dahan, E., Barak, Z., Sinai, R., Manasherob, R., Khamaraev, A., Troitskaya, E., Dubitsky, A., Berezina, N., Margalith, Y. (1997) Extended screening by PCR for seven cry group genes from field-collected strains of Bacillus thuringiensis. Appl Environ Microbiol 63, 4883–4890. Bishop, A.H., Rachwal, A.H. and Vaid, A. (2014) Identification of genes required by Bacillus thuringiensis for survival in soil by transposon-directed insertion site sequencing. Curr Microbiol 68, 477-485.

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Bozue, J., Moody, K.L., Cote, C.K., Stiles, B.G., Friedlander, A.M., Welkos, S.L. and Hale, M.L. (2007). Bacillus anthracis spores of the bclA mutant exhibit increased adherence to epithelial, fibroblast, and endothelial cells but not macrophages. Infect Immun 75, 4498–4505. Buckley, P., Rivers, B., Katoski, S., Kim, M.H., Kragl, F.J., Broomall, S., Krepps, M., Skowronski, E. W., Rosenzweig, C. N., Paikoff, S., Emanuel, P. and Gibbons, H. S. (2012) Genetic barcodes for improved environmental tracking of an anthrax simulant. Appl Environ Microbiol 78, 8272–8280. Buhr, T.L., Young, A. A., Minter, Z.A., Wells, C.M., McPherson, D.C., Hooban, C.L., Johnson, C.A., Prokop, E.J., and Crigler, J.R. (2012) Test method development to evaluate hot, humid air decontamination of materials contaminated with Bacillus anthracis Delta Sterne and B. thuringiensis Al Hakam spores. J Appl Microbiol 113,1037-1051. Carrera, M., Zandomeni, R.O., Fitzgibbon, J. and Sagripanti, J.L. (2007) Difference between the spore sizes of Bacillus anthracis and other Bacillus species. J Appl Microbiol 102, 303-12. Challacombe, J.F., Altherr, M.R., Xie, G., Bhotika, S.S., Brown, N., Bruce, D., Campbell, C. S., Campbell, M. L., Chen, J., Chertkov, O., Cleland, C., Dimitrijevic, M., Doggett, N. A., Fawcett, J.J., Glavina, T., Goodwin, L.A., Green, L.D., Han, C.S., Hill, K.K., Hitchcock, P., Jackson, P.J., Keim, P., Kewalramani, A.R., Longmire, J., Lucas, S., Malfatti, S., Martinez, D., McMurry, K., Meincke, L. J., Misra, M., Moseman, B.L., Mundt, M., Munk, A.C., Okinaka, R.T., Parson-Quintana, B., Reilly, L.P., Richardson, P., Robinson, D. L., Saunders, E., Tapia, R., Tesmer, J. G., Thayer, N., Thompson, L. S., Tice, H., Ticknor, L. O., Wills, P. L., Gilna, P. and Brettin, T. S. (2007) The complete genome sequence of Bacillus thuringiensis Al Hakam. J Bacteriol 189, 3680-1.

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Johnson, C., Bishop, A.H. and Turner, C.L. (1998) Isolation and activity of strains of Bacillus thuringiensis toxic to larvae of the housefly (Diptera: Muscidae) and tropical blowflies (Diptera: Calliphoridae). J Inv Pathol 77:138-144. Koshikawa T., Yamazaki M., Yoshimi M., Ogawa S., Yamada A., Watabe K., Torii M. (1989) Surface hydrophobicity of spores of Bacillus spp. J Gen Microbiol 135, 2717-2722. Langmead B, Salzberg S. (2012) Fast gapped-read alignment with Bowtie 2. Nature Meth. 2012, 9:357-359. Lund, T., de Buyser, M.L. and Granum, P.E. (2000) A new cytotoxin from Bacillus cereus that may cause necrotic enteritis. Mol Microbiol 38, 254–261. Moar, W.T., Trumble, J.T. and Federici, B. A. (1989) Comparative toxicities of spores and crystals from NRD-12 and HD-1 strains of Bacillus thuringiensis to neonate beet army worm (Lepidoptera: Noctuidae). J Econ Entomol 82, 1593-1603. Murphy, S.B., Holmes, M.D. and Wright, S.M. (2012) Bacillus pumilus: possible model for the bioweapon Bacillus anthracis. Adv Microbiol 2, 382-387. Omotade, T.O., Heffron, J.D., Klimko, C.P., Marchand, C.L., Miller, L.L., Halasahoris, S.A., Bozue, J.A., Welkos S.L. and Cote C.K. (2013) D-cycloserine or similar physiochemical compounds may be uniquely suited for use in Bacillus anthracis spore decontamination strategies. J Appl Microbiol 68, 1343-1356.

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surfaces have similar sensitivity to chemical decontaminants. J Appl Microbiol 102, 11-21. Sauka, D.G., Basurto-Rios, R.E., Ibarra, J.E. and Benintende, G.B. (2010) Characterization of an Argentine isolate of Bacillus thuringiensis similar to the HD-1 strain. Neotrop Entomol 39, 767-773. Stewart, G.S.A.B., Johnstone, K., Hagelberg, E. and Ellar, D.J. (1981) Commitment of bacterial spores to germinate. Biochem J 198, 101-106. Tufts, J.A.M., Calfee, M.W., Lee, S.D. (2014) Bacillus thuringiensis as a surrogate for Bacillus anthracis in aerosol research. World J Microbiol Biotechnol 30, 1453-1461. Williams, G., Linley, E., Nicholas, R. and Baillie L. (2013) The role of the exosporium in the environmental distribution of anthrax. J Appl Microbiol 114, 396-403. Wood, J.P., Lemieux, P., Betancourt, D., Kariher, P. and Gatchalian, N. G. (2010) Dry thermal resistance of Bacillus anthracis (Sterne) spores and spores of other Bacillus species: implications for biological agent destruction via waste incineration. J Appl Microbiol 109, 99-106.

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Annealin g Prime r

Sequence

Un1(d )

CATGATTCATGCGGCA GATAAAC

Un1(r)

TTGTGACACTTCTGCTT CCCATT

Un2(d )

GTTATTCTTAATGCAGA TGAA TGGG

Un2(r)

CGGATAAAATAATCTGG GAAATAGT

Un3(d )

CGTTATCGCAGAGAGA TGACATTAAC

Un3(r)

CATCTGTTGTTTCTGGA GGCAAT

Un4(d )

GCATATGATGTAGCGA AACAAGCC

Un4(r)

GCGTGACATACCCATTT CCAGGTCC

cytK(f)

CGACGTCACAAGTTGT AACA

cytK(r)

CGTGTGTAAATACCCC AGTT

VIP3A -F

ACATCCTCCCTACACTT TCTAATA

VIP3A -R

TCTTCTATGGACCCGTT CTCTAC

9GP

CGGCAAATTTAGTGTC RTGCTTATC

temperat ure

Forwa rd Rever se Probe

Reference strain

Universal cry1

B. thuringiensis subsp. kurstaki HD-1

54

54

Universal cry2

B. thuringiensis subsp. kurstaki HD-1

Ben-Dov et al.,

Ben-Dov et al., 1997

Universal cry3

B. thuringiensis subsp. tenebrionis

Ben-Dov et al., 1997

54

Universal cry4

54

Cytotoxin K

B. thuringiensis subsp. israelensis

Vegetativ e insecticid al protein

B. thuringiensis subsp. kurstaki HD-1

cry9

B. thuringiensis subsp. galleriae HD-106

50

Reference

1997

54

50 9GN

Target

B. thuringiensis subsp. israelensis

Ben-Dov et al., 1997

AATTCAAGATTTCCTAR CGTCGC

Lund et al., 1997 Bizzarri and Bishop, 2008 Sauka et al., 2010

GCCAATTTCATAACTTG CTTTGC

TGCGAGCATTTCTACAACT GAAG 6(FAM)ATCCACTCAATAA AACCCCTTCAAAATCACCC A(BHQ1)

pHT8_2 60

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B. thuringiensis strain HD-73

This study

Accepted Article

Table 1. Oligonucleotides used for PCR amplification

Invertebrate species G. mellonella P. xylostella

P. brassicae

S. exigua

T. molitor E. albidus F. candida M. domestica C. elegans

B. thuringiensis strain/ isolate HD-1 wild type HD-1 Cry (A ) Al Hakam 1 Cry (D ) NTD NTD NTD NTD NTD 1.14 x 104 CFU g1 diet 3 (5.70 x 10 – 1.90 4 x 10 ) 3 NTD NTD 2.50 x 10 CFU -2 cm diet (2.27 x 103 – 2.77 x 103) NTD NTD 7.65 x 106 CFU g1 diet 6 (5.35 x 10 – 1.06 x 107) NTD NTD NTD NTD NTD NTD NTD NTD NTD NTD NTD NTD NTD NTD NTD

HDNTD NTD

NTD

NTD

NTD NTD NTD NTD NTD

Table 2. LD50 concentrations for spore/crystal preparations (for the B. thuringiensis HD-1 wild type strain) or spore preparations (for the Cry- organisms) against invertebrate organisms. NTD, no toxicity detected at a challenge of 1 x 108 CFU. The data are given as CFU cm -2 of diet for P. brassicae and T. molitor; CFU ml-1 for C. elegans and CFU g-1 of diet for the other species. The 95% confidence limits are given in parentheses.

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1

2

3

4

5

Plasmids typically carrying crystal protein genes

Chromosomal DNA

Figure 1. Plasmid profiles of B. thuringiensis strains. Lane 1, Al Hakam; Lane 2, HD-1 strain D, Cry-; Lane 3, HD-1 strain D, wild type; Lane 4, HD-1 strain A Cry-; Lane 5, HD-1 strain A, wild type. The large molecular mass plasmids, often associated with carrying cry genes, have disappeared in the Cry- strains, but not in Al Hakam.

Figure 2. Appearance of colonies of B. thuringiensis HD-1 strain D- on the supplemented, selective and differential medium when recovered from a plant/ soil microcosm.

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Bacillus thuringiensis HD-1 Cry- : development of a safe, non-insecticidal simulant for Bacillus anthracis.

A representative simulant for spores of Bacillus anthracis is needed for field testing. Bacillus thuringiensis is gaining recognition as a suitable or...
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