Characteristics and phylogeny of Bacillus cereus strains isolated from Maari, a traditional West African food condiment

International Journal of Food Microbiology 196 (2015) 70–78

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International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro

Characteristics and phylogeny of Bacillus cereus strains isolated from Maari, a traditional West African food condiment Line Thorsen a, Christine Kere Kando b,c, Hagrétou Sawadogo b, Nadja Larsen a, Bréhima Diawara b, Georges Anicet Ouédraogo c, Niels Bohse Hendriksen d, Lene Jespersen a,⁎ a

Department of Food Science, Faculty of Science, University of Copenhagen, Rolighedsvej 26, 1958 Frederiksberg C, Denmark Food Technology Department (DTA/IRSAT/CNRST), Ouagadougou 03 BP 7047, Burkina Faso Université Polytechnique de Bobo-Dioulasso, 01 BP 1091 Bobo-Dioulasso, Burkina Faso d Department of Environmental Science, Aarhus University, Frederiksborgvej 399, 4000 Roskilde, Denmark b c

a r t i c l e

i n f o

Article history: Received 10 July 2014 Received in revised form 10 November 2014 Accepted 24 November 2014 Available online 29 November 2014 Keywords: Maari Bacillus cereus Bacillus cereus biovar anthracis Food fermentation

a b s t r a c t Maari is a spontaneously fermented food condiment made from baobab tree seeds in West African countries. This type of product is considered to be safe, being consumed by millions of people on a daily basis. However, due to the spontaneous nature of the fermentation the human pathogen Bacillus cereus occasionally occurs in Maari. This study characterizes succession patterns and pathogenic potential of B. cereus isolated from the raw materials (ash, water from a drilled well (DW) and potash), seed mash throughout fermentation (0-96 h), after steam cooking and sun drying (final product) from two production sites of Maari. Aerobic mesophilic bacterial (AMB) counts in raw materials were of 105 cfu/ml in DW, and ranged between 6.5 × 103 and 1.2 × 104 cfu/g in potash, 109–1010 cfu/g in seed mash during fermentation and 107 – 109 after sun drying. Fifty three out of total 290 AMB isolates were identified as B. cereus sensu lato by use of ITS-PCR and grouped into 3 groups using PCR fingerprinting based on Escherichia coli phage-M13 primer (M13-PCR). As determined by panC gene sequencing, the isolates of B. cereus belonged to PanC types III and IV with potential for high cytotoxicity. Phylogenetic analysis of concatenated sequences of glpF, gmk, ilvD, pta, pur, pycA and tpi revealed that the M13-PCR group 1 isolates were related to B. cereus biovar anthracis CI, while the M13-PCR group 2 isolates were identical to cereulide (emetic toxin) producing B. cereus strains. The M13-PCR group 1 isolates harboured poly-γ-D-glutamic acid capsule biosynthesis genes capA, capB and capC showing 99-100% identity with the environmental B. cereus isolate 03BB108. Presence of cesB of the cereulide synthetase gene cluster was confirmed by PCR in M13-PCR group 2 isolates. The B. cereus harbouring the cap genes were found in potash, DW, cooking water and at 8 h fermentation. The “emetic” type B. cereus were present in DW, the seed mash at 48–72 h of fermentation and in the final product, while the remaining isolates (PanC type IV) were detected in ash, at 48–72 h fermentation and in the final product. This work sheds light on the succession and pathogenic potential of B. cereus species in traditional West African food condiment and clarifies their phylogenetic relatedness to B. cereus biovar anthracis. Future implementation of GMP and HACCP and development of starter cultures for controlled Maari fermentations will help to ensure a safe product. © 2014 Published by Elsevier B.V.

1. Introduction Maari is a spontaneously fermented alkaline food condiment produced from Baobab tree seeds (Adansonia digitata L.). Maari and similar products are produced in West African countries, especially in rural areas; they constitute an important part of the diet, as a nutritious protein rich supplement to various soups and sauces (Parkouda et al., 2009). Daily consumption of Maari in villages of Burkina Faso is commonly between 10 and 100 g per person (Kando C.

⁎ Corresponding author. Tel.: +45 3533 3230; fax: +45 3533 3513. E-mail address: [email protected] (L. Jespersen).

http://dx.doi.org/10.1016/j.ijfoodmicro.2014.11.026 0168-1605/© 2014 Published by Elsevier B.V.

K., oral communication). The microorganisms occurring in the spontaneous seeds fermentations are suggested to originate from handling, raw seeds, utensils, and containers (Odunfa, 1981). Bacillus species, notably Bacillus subtilis, are considered as the main fermenting microorganisms in these alkaline fermentations, while Staphylococcus spp. and lactic acid bacteria have been detected as well (Parkouda et al., 2009, 2010). Bacterial species belonging to Bacillus cereus sensu lato (sl) group may occur in high numbers, or even be dominant in traditional fermented African foods (Agbobatinkpo et al., 2013; Azokpota et al., 2007; Oguntoyinbo and Oni, 2004). It has been reported that B. cereus in a final Maari product comprised op to 43% of the total counts (10 log 10 cfu/g) of aerobic mesophilic bacteria (Parkouda et al., 2010).

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B. cereus sl group includes the closely related species B. cereus sensu stricto (ss), B. thuringiensis, B. anthracis, B. weihenstephanensis (Lechner et al., 1998), B. mycoides, B. pseudomycoides (Nakamura, 1998) and B. cytotoxicus (Guinebretiere et al., 2012). B. cereus is of special concern as it may cause food poisoning, diarrhoea and emesis, through the production of enterotoxins or cereulide (Stenfors Arnesen et al., 2008), as well as various systemic and local infections (Kotiranta et al., 2000). Some B. cereus ss strains are used as human and animal probiotics, underlining the highly variable impact on human and animal health of the species (Cutting, 2011). Some B. thuringiensis strains may also cause human infections (Hernandez et al., 1998), but the species is mainly known for its production of insecticidal toxins and its use as a bio-pesticide (Aronson, 2002). B. anthracis is the cause of lethal anthrax in mammals, and has been used as a bio-weapon (Rasko et al., 2005). The ability of B. anthracis, emetic B. cereus ss, and B. thuringiensis to cause disease in mammals and insects relies on the presence of specific virulence plasmids (Ehling-Schulz et al., 2006; Rasko et al., 2005). In B. anthracis presence of two plasmids are essential for full virulence, among them, pXO1 carrying the virulence genes cya (edema factor), lef (lethal factor) and pagA (protective antigen) and pXO2 carrying genes for the poly-γ-D- glutamic acid capsule biosynthetic operon (capB, capC, capA, and capD) (Levy et al., 2014). Emetic B. cereus carries a plasmid with high similarity to pXO1 that harbours the cereulide synthetase gene cluster necessary for the production of the emetic toxin cereulide (Ehling-Schulz et al., 2006). Although currently separated into three different species, B. anthracis appears to be genetically indistinguishable from members of the B. cereus-B. thuringiensis group (Helgason et al., 2000). Recent studies using amplified fragment length polymorphism (AFLP) and multiple locus sequence typing (MLST) analysis have showed that a number of B. cereus and B. thuringiensis strains, including e.g. B. thuringiensis serovar konkukian strain 97–27, B. cereus E33L and B. cereus biovar anthracis CI, are closely related to the highly monomorphic species of B. anthracis (Han et al., 2006; Klee et al., 2006, 2010). Some strains, as B. cereus G9241 and B. cereus biovar anthracis CI, harbour plasmids that are highly similar to the B. anthracis pXO1 and pXO2 virulence plasmids, including the anthrax-causing virulence genes (Hoffmaster et al., 2004; Klee et al., 2010). It has been shown that plasmids can be transferred between the different species of B. cereus sl group (Hu et al., 2009; Van der Auwera et al., 2007). Thus, phylogeny, species delineation and pathogenic potential of B. cereus sl group is currently a subject of extensive scientific discussion (Helgason et al., 2000; Hill et al., 2004; Tourasse et al., 2006, 2011; Zwick et al., 2012; Økstad and Kolstø, 2011). Several studies have applied MLST of chromosomally encoded housekeeping genes to analyse phylogenetic relationships (Barker et al., 2005; Helgason et al., 2004; Hoffmaster et al., 2004; Klee et al., 2006; Priest et al., 2004; Sorokin et al., 2006; Tourasse et al., 2011). These analyses show that the B. cereus sl can be grouped into three main phylogenetic clusters (Økstad and Kolstø, 2011) which can be further divided into seven clusters (Guinebretiere et al., 2008; Tourasse et al., 2011). Genome analysis of sequenced B. cereus sl strains confirmed this phylogeny (Zwick et al., 2012). Guinebretiere et al. (2010) showed that B. cereus sl strains could be affiliated to these seven groups by partial sequencing of panC, and that the groups differed with regard to their growth temperature and cytoxicity. The objective of the present study was to describe the characteristics and phylogeny of B. cereus sl during traditional production of Maari at two different production sites in Burkina Faso by use of M13-PCR. DNA typing by M13-PCR previously showed to be applicable to differentiate between B. cereus strains and Bacillus species isolated from fermented Sudanese bread (Ehling-Schulz et al., 2005; Thorsen et al., 2011). Furthermore, the study analysed the phylogenetic relationship of B. cereus isolates by panC sequencing and MLST, and determined their pathogenic potential by PCR analysis for specific virulence genes.

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2. Materials and methods 2.1. Microbial analysis Baobab seeds for Maari production were purchased at the market at Orodara and Pousghin in Burkina Faso and were brought to two different production sites (A and B) within Pousghin. The producers used the same ash and water from the same drilled well (DW) for potash production. A flow diagram for Maari production and the sampling points are shown in Fig. 1. Samples were collected from ash, DW, potash (ash mixed with DW), water after cooking (CW), fermenting seed mash throughout the first fermentation (8, 12, 24, and 48 h) and second fermentation (96 h), fermenting mash after pounding (72 h + P), after steam cooking (96 h + SC) and in the final product after sun drying (SD). Sampling was performed in two replicates collected from each production site (A and B). For all samples, except water samples, 10 g was homogenized in 90 ml sterile diluent (1% (w/v) peptone (Difco, Detroit, Michigan, USA), 0.9% (w/v) NaCl, pH 7.0) by use of a stomacher (Masticor IUL, Barcelona, Spain) for 2 min. To quantify aerobic mesophilic bacteria (AMB), 1 ml of ten-fold sample dilutions were plated into plate count agar (PCA) (Liofilchem, Roseto degli Abruzzi, Italy) and incubated at 30 °C for 72 h. For purification the isolates were successively streaked on nutrient agar (NA, Merck, Germany) (30 °C, 24 h). For long term maintenance of isolates, stock cultures were stored at − 80 °C in 20% (v/v) glycerol and 80% (v/v) nutrient broth (Merck). After purification, isolates were examined for Gram reaction and catalase production. Micro-morphology, motility and spore formation were observed by phase contrast microscopy (magnification × 1000, Olympus 40, Olympus, Japan). 2.2. Identification of B. cereus Gram positive, catalase positive and spore forming isolates were spotted on Brilliance Bacillus cereus Agar (CM1036B, supplement SR0230E, Oxoid, Denmark) and incubated at 30 °C for 24 h. Colonies with a blue/green appearance (a result of the enzymatic cleavage of 5-bromo-4-chloro-3-indolyl-ß-glucopyranoside by ß-glucosidase present in Bacillus cereus) were considered as presumptive B. cereus sl species (Fricker et al., 2008). The isolates were subjected to inter transcribed spacer polymerase chain reaction (ITS-PCR) profiling in order to verify them as B. cereus sl (Willumsen et al., 2005). The isolates were checked for the ability to produce hemolysis on TSA-sheep blood agar (Oxoid). Hemolytic activity was determined from the radii of the clarified zones around the B. cereus colonies as (++) high (r N 2 mm), (+) weak (r b 2 mm) and (-) nonhemolytic. 2.3. DNA isolation DNA was isolated from bacterial colonies by boiling in Tris-EDTA buffer as previously described (Hansen and Hendriksen, 2001). Shortly, bacteria were transferred to 200 μl of Tris-EDTA buffer and lysed by incubation at 102 °C for 10 min. Cell debris was removed by centrifugation and the DNA-containing supernatant was kept at -20 °C until use. 2.4. M13-PCR typing The B. cereus isolates were subjected to M13-PCR fingerprinting adopted from Ehling-Schulz et al. (2005). Shortly, PCR with the use of random primer PM13 (GAGGGTGGCGGCTCT) included denaturation step at 94 °C for 3 min, followed by 35 cycles of 1 min at 94 °C, 1 min at 40 °C, 8 min at 65 °C and an elongation step at 65 °C for 16 min. Cluster analysis of the obtained profiles was performed

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Raw material (Baobab tree seeds) Water from a drilled well (DW)*

Cleaning/Washing

Ash * mixed with DW*

Cooking (48 h)

Ash layer solution (Potash)*

Wringing

Dirty water, impurities

Cooking water (CW)* First Fermentation (72 h)*

Pounding (P)* Potash* Kneading/Moulding

Second Fermentation (24 h)*

Steam Cooking (SC)*

Sun drying (SD)* Maari Fig. 1. Flow diagram of a typical Maari production. The stars indicate production steps in which Bacillus cereus sl strains were isolated and characterized in this study, as follows: water from a drilled well (DW), ash, potash, cooking water (CW), fermenting seed mash at different time of fermentation, fermenting mash after pounding (P), steam cooking (SC) and sun drying (SD).

using the BioNumerics 4.5 software (Applied Maths, Sint-MartensLatem, Belgium).

2.5. Phylogenetic affiliation by panC sequencing Amplification and sequencing of the partial panC, and determination of phylogenetic affiliation into PanC groups I-VII was performed according to Guinebretiere et al., (2008; 2010). Sequencing of panC was performed for isolates selected from M13-PCR typing, as follows: 8 isolates of group 1 (2 M2, 2 M5, 2P11, 4P7, 2P14, 3 M7, 11P1 and 2 M3), 7 isolates of group 2 (23P2, 15P5, 16P6, 18P6, 13P11, 21P16 and 13P13) and 8 isolates of group 3 (17P3, 19P20, 23P1, 24P4, 6P2, 23P4, 21P and 20P10) by a commercial facility (Macrogen, The Netherlands). The sequences were corrected using the CLC main workbench vs. 5.1 (CLC, Aarhus, Denmark). Cluster analysis was performed on the panC sequences obtained in this study and the NCBI database (accession no. DQ301433, DQ301426, DQ301440, CP000903, CP000485, AE017355, CP002508, CP001907, CP000227, CP001186, CP000001, CP001746, AE017194, CP001283, CP001177, CP001407, AE017225, CP001215) with the MEGA6 software (Tamura et al., 2013) using the maximum likelihood method as described by Seiler et al., (2013).

2.6. Multi locus sequence typing PCR amplification and sequencing of glpF (glycerol uptake facilitator protein), gmk (guanylate kinase, putative), ilvD (dihydroxy-acid dehydratase), pta (phosphate acetyltransferase), pur (phosphoribosylaminoimidazolecarboxamide), pycA (pyruvate carboxylase), and tpi (triosephosphate isomerase) was performed on selected isolates as described by Priest et al. (2004). MLST typing was performed for selected isolates of M13-PCR group 1 (2P14, 2 M5, 2 M3, 3 M7) and M13-PCR group 2 (13P11, 15P5 and 18P6). Sequencing was performed for the isolates: gmk (2P14, 2 M5, 2 M3, 3 M7, 13P11, 15P5, 18P6), glp (2 M5, 2 M3, 13P11, 15P5, 18P6), ilv (2P14, 2 M5, 2 M3, 3 M7, 15P5, 18P6), pta (2P14, 2 M5, 2 M3, 3 M7, 13P11, 15P5, 18P6), pur (2P14, 2 M5, 2 M3, 3 M7, 13P11, 15P5, 18P6), pycA (2P14, 2 M5, 2 M3, 3 M7, 15P5, 18P6) and tpi (2P14, 2 M5, 2 M3, 3 M7, 13P11, 15P5, 18P6). Concatenated sequences of 2 M5 and 15P5 were formed using the CLC main workbench. Concatenated sequences of relevant B. cereus sl isolates were obtained from the B. cereus MLST Database homepage (http://pubmlst.org/bcereus/) or by downloading gmk, ilvD, pta, pur, pucA and tpi gene sequences from the NCBI database (accession nos. CP001177, CP002508, CP001903, CP000227, CP001186, CP000001, CP001746, CP001283, CP000903, CP000485, AY729750, AY729758,

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AY729766, AY729774, AY729782, AY729790, AY729798) and concatenating them using the CLC main workbench. Cluster analysis of concatenated sequences was performed with the MEGA6 software (Tamura et al., 2013) using the maximum likelihood method as described by Seiler et al. (2013). Sequence types (ST) were identified using the MLST database referred to above.

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and after sun drying) and B (at 48 h). Isolates of M13-PCR group 3 (19 isolates) were isolated during the late fermentation stages, i.e. from 48-72 h of first fermentation and after steam cooking (after 96 h fermentation) and sun-drying from production sites A and B. A single isolate from group 3 was isolated from potash (site A). 3.3. PanC gene sequence analysis

2.7. Toxin gene amplification by PCR PCR amplification of B. anthracis virulence genes pagA, lef, cya (pXO1) and capA, capB, capC (pXO2) was performed for all isolates of M13-PCR group 1, three isolates of group 2 (18P6, 16P9 and 13P13) and three isolates of group 3 (17P3, 19P20 and 6P2) as described by Hoffmaster et al. (2006). Detection of the cesB gene of the cereulide synthetase gene cluster was conducted for selected isolates of M13-PCR group 2 (23P2, 15P5, 16P6, 18P6, 16P9, 26P2, 13P11, 21P16 and 13P13) as described by Ehling-Schulz et al. (2004). Sequencing of toxin genes was performed by Macrogen (Netherlands). 2.8. PCR amplification of pXO1 and pXO2 like replication genes Representative isolates of M13-PCR group 1 (1 M1, 2P4, 2 M5, 2P14, 3 M7, 11P1 and 4P15), group 2 (13P11, 13P3, 15P5, 16P7, 18P6), and group 3 (17P3, 19P20, 20P10, 23P3 and 26P10) were tested for the presence of pXO1 and pXO2 like replicons by PCR using the primer pairs repX-F1/R2, repX-F4/R2, repX-R1/R3, repA-F1/R2 and repA-F1/R3 as described by Hu et al. (2009). 2.9. Accession numbers The sequences obtained in the present study were submitted to GenBank. The accession numbers are KJ882849- KJ882853 and KJ939561- KJ939574. 3. Results 3.1. Identification of B. cereus group isolates Bacterial counts and fermentation conditions (pH and temperature) during Maari production are shown in Table S1 (Supplementary Data). Counts of AMB in the raw materials were 4 × 105 cfu/g in baobab seeds, 1.2 × 105 in DW and between 6.5 × 103 and 1.2 × 104 cfu/g in potash, depending on production site. The AMB counts in fermenting mash ranged between 109–1010 cfu/g during fermentation (0–96 h) and between 2.1 × 107 and 1.7 × 109 cfu/g in the sun dried Maari (Table S1). A total of 290 AMB were isolated from raw materials, fermenting mash and the final products at the Maari production sites A and B. Gram positive endospore-forming bacteria comprised 68 isolates (23.4% of AMB), while 187 isolates (64.6% of AMB) were Gram negative and 35 isolates (12% of AMB) were Gram positive non endosporeforming bacteria (results not shown). Endospore forming bacteria from the Maari productions were spotted on Brilliance Bacillus cereus agar to screen for B. cereus sl. A total of 53 isolates (18% AMB) were positive for β-glucosidase activity producing blue/green colonies, indicating their affiliation to B. cereus sl. ITS-PCR confirmed them as B. cereus sl (results not shown). 3.2. M13-PCR profiling The B. cereus sl isolates were grouped by M13-PCR profiling into 3 major groups as shown in Fig. 2. The isolates of M13-PCR group 1 (18 isolates) originated from DW, cooking water and potash from both production sites. Except for occurring after 8 h of fermentation at site A, the M13-PCR group 1 isolates were not detected at other stages of fermentation. The isolates of M13-PCR group 2 (16 isolates) were collected from DW, ash and in fermenting mash at sites A (at 72 h of fermentation

The isolates were affiliated to the B. cereus phylogenetic groups I to VII with the use of panC sequencing (548 bp). The results for panC groupings are shown in Fig. 2. Isolates of M13-PCR groups 1 and 2 belonged to (panC) phylogenetic group III, while isolates of M13-PCR group 3 belonged to phylogenetic group IV. Sequence analysis of the panC (7 sequences) of M13-PCR group 1 showed 100% identity between isolates. According to a BLAST search using the GenBank database, the panC sequences of B. cereus E33L (CP000001), B. anthracis CDC684 (CP001215), and B. thuringiensis serovar konkukian 97-27 (AE017355) were the closest relatives showing 99.5%, 99.2% and 99.2% identity, respectively. Except for isolate 23P2, the panC gene sequences of M13PCR group 2 (6 sequences) were 100% identical, and showed 100% identity to cereulide producing strains, e.g. B. cereus NC7401 (AP007209.1). The panC sequence of isolate 23P2, showing a slightly different M13PCR pattern within the M13-PCR group 2 isolates, was 100% identical to Bacillus thuringiensis serovar finitimus YBT-020 (CP002508) and 99.64% identical to cereulide producing strains. Except for isolate 17P3, the panC sequences of M13-PCR group 3 (8 sequences) were also 100% identical. They were most closely related to B. thuringiensis BMB171 (CP001903.1), Bacillus cereus B4264 (CP001176.1), B. cereus ATCC 14579 (AE016877.1) and B. thuringiensis serovar kurstaki YBT1520 (CP004858.1) showing 100%, 99.8% and 98.4% identity, respectively. Isolate 17P3 showed a different M13 pattern and a nucleotide difference of 2 relative to the other group 3 isolates. This isolate was by microscopy identified as a B. thuringiensis strain (crystal inclusions, results not shown). A phylogenetic tree constructed from the panC sequences of representative isolates of M13-PCR groups 1, 2 and 3, as well as relevant members previously reported to belong to PanC groups III, IV and VI are presented in Fig. 3. 3.4. Multi locus sequence typing of M13 group 1 and 2 isolates The partial sequences of genes glpF, gmk, ilvD, pta, pur, pycA and tpi for the isolates from the M13-PCR group 1 and 2 were all identical within each of the two groups. When analysing the sequences in the B. cereus MLST database, it was revealed that isolates (exemplified by 2 M5) of M13-PCR group 1 were a new ST, with the closest match being ST 366 belonging to a human isolate B. cereus BC1 originating from Japan in 2005 (http://pubmlst.org/bcereus/). Isolate 15P5, representing M13-PCR group 2, was affiliated to ST 26, a sequence type specifically attributed to mesophilic cereulide producing B. cereus ss strains (Priest et al., 2004). To characterize the phylogenetic relationship with other B. cereus group strains, the gene sequences were concatenated for isolates 2 M5 and 15P5 and compared with corresponding concatenated sequences of relevant B. cereus group isolates (Fig. 4). Strain 2 M5 of M13-PCR group 1 clustered together with B. cereus BCI (Δ7nt), B. thuringiensis Al Hakam (Δ11nt), B. cereus 03BB102 (Δ12 nt), B. thuringiensis serovar konkukian 97-27 (Δ13nt), and B. cereus biovar anthracis CI (Δ18 nt), while strain 15P5 of M13-PCR group 2 clustered together with the cereulide producing strains B. cereus NC7401 and B. cereus AH187. 3.5. Toxin genes in isolates of M13-PCR groups 1 and 2 The isolates of M13-PCR groups were analysed for the presence of B. anthracis associated virulence genes (Table 1). Results, showed that the isolates of M13-PCR group 1 were negative for the pXO1 related virulence genes lef, cya and pagA, and positive for the pXO2 related virulence genes capA, capB and capC known to be involved in capsule

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ID Sampling Hemolysis 2M2 2P4 1M7 1M8 1M9 1M2 2P11 4P15 4P7 4M3 2M5 2P14 1M4 1M1 3M7 3M9 11P 2M3 6P16 23P2 15P5 16P6 18P6 16P2 16P9 16P7 13P3 26P2 3M4 13P11 21P16 11P2 12P2 13P13 19P14 19P10 17P3 19P20 23P1 24P3 24P4 6P2 25P19 26P10 20P8 20P7 23P4 22P10 23P7 20P9 21P 20P10 23P3

CW (B) CW (B) CW (A) CW (A) CW (A) CW (A) CW (B) Ash (A, B) Ash (A, B) Ash (A, B) CW (B) CW (B) CW (A) CW (A) DW (A, B) DW (A, B) 18h (A) CW (B) Potash (A) 96h +SC (A) 24h (A) 24h (B) 48h (B) 24h (B) 24h (B) 24h (B) 12h (A) SD (A) DW (A, B) 12h (A) 72h + P (A) 8h (A) 8h (B) 12h (A) 72h (A) 72h (A) 48h (A) 72h (A) 96h (A) SD (B) 96h + SC (B) Potash (A) 96h + SC (B) SD (A) 72h (B) 72h (B) 96h + SC (A) 72h + P (B) 96h + SC(A) 72h (B) 72h + P (A) 72h (B) 96h +SC (A)

+ + ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ + + + + + + + + + ++ + + + ++ ++ ++ ++ + ++ + ++ + + + + + + ++ + +

M13-PCR group

PanC group

1

III

2

III

3

IV

Fig. 2. Dendogram of M13-PCR fingerprint patterns of Bacillus cereus sensu lato sampled from Maari production sites A and B based on the Pearson correlation coefficients between the densitometric traces and the clustering method of Ward. Samples were water from drilled well (DW), ash, potash, cooking water (CW), fermenting seed mash after pounding (P), after steam cooking (SD) and sun drying (SC). Total time of seed mash fermentation is indicated as 8 h, 24 h, 48 h, 72 h (first fermentation) and 96 h (second fermentation). Affiliation to M13-PCR groups and PanC groups is indicated. Hemolytic activity of isolates was determined from the radii of clarified zones on TSA-sheep blood agar as (++) high activity (radius more than 2 mm), (+) weak activity (radius less than 2 mm) and (-) nonhemolytic.

formation. Isolates of M13-PCR groups 2 and 3 did not harbour pXO1 and pXO2 virulence genes. The analysed sequences were 100% identical between the Maari strains. The translated (partial) capA nucleotide sequence showed 99% identity to that of the capsule biosynthesis protein CapA of B. cereus 03BB108 (ZP_03115391.1), while it was only 91% identical to CapA of various B. anthracis strains and B. cereus biovar anthracis CI (CP001748.1). The translated nucleotide sequence of capB showed 100% identity to CapB of Bacillus cereus 03BB108 (ZP_03115436.1) as well as to CapB of various B. anthracis strains. The translated capC nucleotide sequence showed 98% identity to B. cereus 03BB108 (ZP_03115460.1) and Bacillus anthracis A1055 (ZP_05188102.1) CapC and 97% identity to other B. anthracis strains. The cesB gene of the cereulide synthetase gene cluster was PCR positive in all tested isolates of M13-PCR group 2 except for 23P2, indicating their ability to produce cereulide. Isolate 23P2 did not harbour the cesB gene. 3.6. Detection of pXO1 and pXO2 like replicons by PCR Selected isolates of M13-PCR groups 1, 2 and 3 were analysed for the presence of pXO1 and pXO2 like replicons (Table 1). None of the tested

isolates were PCR positive for pXO2-like replicons. M13-PCR group 2 isolates, exemplified by 13P11, 13P3, 15P5, 16P7 and 18P6, were PCR positive for the pXO1 like replicons. 3.7. Phenotypic features of M13-PCR groups 1, 2 and 3 All tested isolates of B. cereus sl were motile. Isolates of M13-PCR group 1 were strongly hemolytic and produced long filaments (Table 1, Fig. 2). Isolates of M13-PCR group 2 were non-hemolytic to weakly hemolytic and did not form filaments. The isolates of M13-PCR group 3 were characterised by different hemolytic activity and filament production. 4. Discussion Genome sequence analyses of B. cereus and B. anthracis strains revealed that these species harbour numerous genes, encoding proteolytic enzymes, peptide and amino-acid transporters, as well as genes involved in a variety of amino-acid degradation pathways, indicating an expanded capacity for amino-acid and peptide utilization (Ivanova et al., 2003).

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PanC group 85 66 91 64

B. cereus AH820 B. thuringiensis serovar konkukian 97-27 B. cereus E33L

99

B. cereus, M13-PCR group 1 (2M5) B. cereus biovar anthracis CI B. cereus 03BB102

78

99

B. anthracis CDC684 B. anthracis Sterne

97 B. cereus ATCC10987

III

B thuringiensis Al Hakam

B. thuringiensis serovar finitimus YBT-020 72

B. cereus M13-PCR group 2 (23P2) B. cereus M13-PCR group 2

97

B. cereus AH187 86

B. cereus Q1 B. weihenstephanensis WSBC10204 B. weihenstephanensis KBAB4

100

B. weihenstephanensis WSBC10206

VI

B. cereus G9842 B. thuringiensis serovar chinensis CT-43 100

B. cereus M13-PCR group 3

53 95

B. cereus M13-PCR group 3 (17P3)

IV

B. thuringiensis BMB171

0.01 Fig. 3. Phylogenetic relationship of B. cereus isolates from Maari (indicated with bold letters) and B. cereus isolates of phylogenetic groups III, IV and VI using the panC gene as a genetic marker. The phylogenetic tree was constructed with MEGA 6 using the maximum likelihood method. Bootstrap (1000 repeats) are shown next to the nodes in %.

Accordingly, the frequent occurrence of B. cereus in African spontaneously fermented food condiments based on protein rich seeds is not surprising (Parkouda et al., 2009). B. cereus in this study was isolated from Maari in high numbers (18% of AMB isolates) at two different production sites, similar to previously obtained results for Maari sampled at different geographical regions in Burkina Faso (Parkouda et al., 2010). I addition to the former study by Parkouda et al. (2009), the present study further investigates B. cereus isolates with regard to their succession pattern, genetic diversity and phylogenetic relationships. M13-PCR profiling has previously been used to describe genetic variation between the strains of B. cereus, showing that mesophilic cereulide producing isolates have almost identical patterns regardless of isolation source and geographical origin (Ehling-Schulz et al., 2005; Thorsen et al., 2010). By using M13-PCR we observed three major groups of B. cereus showing clear succession patterns associated with Maari production steps. Thus, M13-PCR group 1 isolates occurred only in samples from the DW, cooking water and ash. They were succeeded by M13-PCR group 2 isolates occurring sporadically in the DW, being dominant from an early stage of the fermentation to its termination. M13-PCR group 3 isolates were found at the latest stages of fermentation and in the final product. Though Maari was made by two different producers, they were located in the same village using the same water source (drilled well) for the cooking process and seeds purchased from the same market. Potash was produced individually at production sites A and B. The results strongly indicate that the drilled well water and cross contamination of ash were the main sources of M13-PCR groups 1 and 2 isolates, while cross contamination of potash is likely to be a source of the group 3 isolates. The panC sequence analysis affiliated M13-PCR groups 1, 2 and 3 to phylogenetic groups III, III and IV of B. cereus sl, respectively. Phylogenetic

group III contains B. anthracis and the emetic B. cereus, thus, isolates from this group are generally considered cytotoxic and many of them highly cytotoxic. Group IV contains a number of B. cereus ss and B. thuringiensis isolates, among which some are associated with intestinal infections, therefore, isolates from this group are generally considered cytotoxic and some highly cytotoxic (Guinebretiere et al., 2008, 2010). So it is very likely that Maari can harbour B. cereus with a pathogenic potential, similar to other studies in which traditional fermented African foods have been shown to harbour B. cereus sl with a pathogenic potential (Agbobatinkpo et al., 2013; Oguntoyinbo and Sanni, 2007; Oguntoyinbo and Oni, 2004; Ouoba et al., 2008; Padonou et al., 2009; Thorsen et al., 2010). BLAST analysis of the panC sequences allowed identification of unusual B. cereus isolates phylogenetically resembling B. cereus biovar anthracis as well as isolates that have an emetic potential. While cereulide producers previously have been isolated from African alkaline fermented food condiments (Agbobatinkpo et al., 2013; Thorsen et al., 2010), to the best of our knowledge, this study is the first to describe B. cereus biovar anthracis-like B. cereus sl isolates in a food production environment based on panC genotyping. The MLST analyses of concatenated sequences of M13-PCR group 1 isolates, exemplified by 2 M5, confirmed the results for phylogenetic affiliation to B. cereus biovar anthracis isolates obtained by the panC sequence analysis. Most of the previously reported B. cereus sl strains, including B. cereus biovar anthracis, are of clinical rather than environmental origin and, consequently, considered highly virulent (Han et al., 2006; Hoffmaster et al., 2004, 2006; Klee et al., 2006). In the present study the B. cereus isolates that were phylogenetically related to B. cereus biovar anthracis were isolated from a food production environment, including water in a drilled well used for every day purposes, and seemingly without affecting humans. This information reveals that

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B thuringiensis Al Hakam B. cereus 03BB102

41

B. cereus biovar anthracis CI

99

B. thuringiensis serovar konkukian 97-27

41

B. cereus 2M5, M13-PCR group 1

73

B. cereus BCI

83

67

B. cereus E33L B. cereus Q1

60

B. cereus AH820 B. anthracis ATCC14186

49

45

B. anthracis Sterne 34 F2

100

B. anthracis K2478/A0102 B. cereus G9241 B. thuringiensis serovar finitimus YBT-020

54

100

B. cereus ATCC10987

46

B. cereus AH187

74 100

B. cereus NC7401 B. cereus 15P5, M13-PCR group 2

B. anthracis 17003-14 B. cereus G9842 B. thuringiensis BMB171

100

B. thuringiensis serovar kurstakii T03a001

100

B. weihenstephanensis DSM11821 B. weihenstephanensis WSBC10202

100

B. weihenstephanensis KBAB4

98

B. weihenstephanensis 189

0.01 Fig. 4. Phylogenetic relationship of B. cereus sensu stricto inferred from concatenated housekeeping gene sequences (glpF, gmk, ilvD, pta, pur, pycA and tpi). The tree was constructed with MEGA 6 using the maximum likelihood method. Bootstrap (1000 repeats) is shown next to the nodes in %. B. cereus isolates from Maari are indicated in bold letters.

a full understanding of the pathogenic potentials of B. cereus sl strains with resemblances to B. cereus biovar anthracis is not yet obtained. The reason why such bacteria have not been isolated from food environments previously might be due to the differences in sampling procedures, methods for sample characterization, the hygienic standards as well as regional differences.

The M13-PCR group 1 isolates did not seem to harbour pXO1 or pXO2-like plasmids, as there was no detection of the related replicons (repX and repA) by PCR. Absence of pXO1 or pXO2-like plasmids is similar to B. cereus E33L and B. thuringiensis serovar konkukian 97-27 (Han et al., 2006), but not to B. cereus G9241 which harbours an almost complete pXO1 named pBCXO1 and a plasmid pBC218 containing the

Table 1 Characterization of B. cereus sensu lato strains isolated from Maari production. Phylogenetic relations

Phenotypic features

Origin

M13-PCR group

PanC groupa

MLSTa,b

Motility

Hemolysisd

Filaments

Ash, drill water and cooking water sites, (8 h ferm at site A) Ash, 8 to 48 h of fermentation (site B) and 8 h fermentation to final product (site A) Late fermentation and after sun drying sites A and B

1

III

ST 366c

+

+/++

+

a

Cereulide synthetase cesBa

B. anthracis related virulence genesa

pXO1 and pXO2 like plasmidsa

pXO1 lef cya pagA

pXO2 capA, capB, capC

pXO1 repX

pXO2 repA

nd

-

+

-

-

18

e

-

-

+

-

16

-

-

-

-

19

2

III

ST 26

+

+/−

-

+

3

IV

nd

+

−/+/++

+/−

nd

# of isolates

PCR and sequencing were performed for selected isolates only. The MLST scheme was derived from the seven alleles: glpF, gmk, ilvD, pta, purn pycA, and tpi. M13-PCR group 1 isolates (based on 2 M5) represents a new ST, however ST366 was the closest match, see Results Section 3.4. d Hemolytic activity was determined from the radii of clarified zones on TSA-sheep blood agar as high (++) – radius more than 2 mm, weak (+) – radius less than 2 mm, and nonhemolytic (-). e All tested isolates were positive for the cesB gene except for 23P2 and16P9; nd = not determined. b c

L. Thorsen et al. / International Journal of Food Microbiology 196 (2015) 70–78

capsule encoding genes (Hoffmaster et al., 2004), or to B. cereus biovar anthracis CI which harbours as well pCI-XO1 as pCI-XO2 (Klee et al., 2010). Isolates of M13-PCR group 2 positive for the cereulide synthetase gene cesB were also positive for the pXO1-like replicon repX. This result is supported by a previous study showing that the cereulide synthetase gene cluster is located on a plasmid with high similarity to pXO1 (Ehling-Schulz et al., 2006). Results obtained for panC gene sequencing and MLST analysis are in agreement with previous observations that mesophilic emetic B. cereus strains belong to a single evolutionary lineage of B. cereus which are genetically highly homogenous regardless of isolation source and geographical origin (Ehling-Schulz et al., 2005). Strains of M13-PCR group 1 did not harbour any of the pXO1 related virulence genes pagA (protective antigen), lef (lethal factor) or cya (edema factor) contrary to the results obtained for the clinical B. cereus isolates 03BB102, 03BB87 (Hoffmaster et al., 2006) and B. cereus biovar anthracis CI (Klee et al., 2006, 2010), but similar to the environmental isolate 03BB108 (Hoffmaster et al., 2006). In the M13PCR group 1 we did detect the pXO2 related virulence genes capA, capB and capC involved in capsule biosynthesis. The translated capA, capB and capC gene sequences of the M13-PCR group 1 showed the highest homology to an environmental B. cereus strain 03BB108 isolated from a welder’s worksite (Hoffmaster et al., 2006). It has been reported that isolate 03BB108 was not able to produce the capsule under in vitro conditions which normally induce capsule formation in B. anthracis (Hoffmaster et al., 2006). Supporting these observations, we did not detect the presence of a capsule for the M13-PCR group 1 isolates (results not shown). The translated gene sequences, except for capB, showed higher similarity to B. cereus 03BB108 than to B. anthracis, confirming a closer relationship to B. cereus, as also evidenced by the MLST analysis. In B. anthracis, depending on the infection model and strain used, the strains showed variable virulence or even loss of virulence when cured for the pXO1 or pXO2 virulence plasmids (Levy et al., 2014). Further studies are needed to evaluate the virulence potential of M13PCR group 1 isolates harbouring the cap genes, to estimate the risk of disease. Isolates of M13-PCR group 1 were hemolytic and motile contrary to non-motile and non-hemolytic B. anthracis (Hoffmaster et al., 2006). The obtained results for hemolysis and motility are similar to the results previously reported for B. cereus related to B. anthracis, among them B. cereus strains G9241, 03BB102, 03BB108, 03BB87 (Hoffmaster et al., 2006) and B. thuringiensis serovar konkukian strain 97-27 (Hernandez et al., 1998). The diarrheal toxin encoding genes cytK-2, hbl(A,B,C and D) and nhe (A,B,C) are widely distributed among the species of B. cereus group, whereas the cesB gene is mainly detected in a monomorphic group of B. cereus ss strains (Ehling-Schulz et al., 2005; Guinebretiere et al., 2010; Hansen and Hendriksen, 2001; Padonou et al., 2009; Thorsen et al., 2011). In the present study potentially cereulide producing isolates (M13-PCR group 2) were detected at both production sites. Cereulide producers have been isolated from African alkaline fermented foods as afitin, iru and sonru made from African locust beans (Thorsen et al., 2010) and from some additives yanyanku and ikpiru used for the production of iru and sonru (Agbobatinkpo et al., 2013). These findings indicate that alkaline types of fermentations may favour the presence of cereulide producing B. cereus. In conclusion, three phylogenetically different distinct groups of B. cereus sl were isolated from Maari. These groups included (i) isolates that are phylogenetically related to B. cereus biovar anthracis and that possess the pXO2 associated virulence genes capA, capB and capC involved in the synthesis of poly-γ-D- glutamic acid capsule (M13-PCR group 1, PanC group III), (ii) isolates with potential to produce cereulide, the emetic toxin (M13-PCR group 2, PanC group III) and (iii) isolates of PanC type IV (M13-PCR group 3). The presence of these bacteria in Maari rises food safety concern, however no incidences of human disease has been reported in relation to the consumption of Maari or other traditional alkaline fermented African food products.

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Still, the results of this study highlight the importance of developing starter cultures, upgrading production facilities, as well as introducing the use of GMP and HACCP to manage the occurrence of B. cereus and other pathogens. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.ijfoodmicro.2014.11.026.

Acknowledgements This work was financially supported by DANIDA through the research project “Value added processing of underutilized savannah tree seeds for improved food security and income generation in West Africa (SeedFood)”.

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