Is Cytotoxin K from Bacillus cereus a bona fide enterotoxin? Virginie Castiaux, Xiaojin Liu, Laurence Delbrassinne, Jacques Mahillon PII: DOI: Reference:
S0168-1605(15)30044-1 doi: 10.1016/j.ijfoodmicro.2015.06.020 FOOD 6960
To appear in:
International Journal of Food Microbiology
Received date: Revised date: Accepted date:
12 January 2015 20 June 2015 22 June 2015
Please cite this article as: Castiaux, Virginie, Liu, Xiaojin, Delbrassinne, Laurence, Mahillon, Jacques, Is Cytotoxin K from Bacillus cereus a bona fide enterotoxin?, International Journal of Food Microbiology (2015), doi: 10.1016/j.ijfoodmicro.2015.06.020
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ACCEPTED MANUSCRIPT Is Cytotoxin K from Bacillus cereus a bona fide enterotoxin ?
Laboratory of Food and Environmental Microbiology, Université catholique de Louvain, B-
1348 Louvain-la-Neuve, Belgium; and
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Scientific Institute of Public Health, Juliette
Wytsman street 14, B-1050 Brussels, Belgium
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Running title: CytK from B. cereus
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† These authors contributed equally to this work.
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Virginie Castiaux1,†, Xiaojin Liu1,#,†, Laurence Delbrassinne1,2,† and Jacques Mahillon1*
*Corresponding author: Jacques Mahillon
Laboratory of Food and Environmental Microbiology Croix du Sud, 2 – L7.5.12 B-1348 Louvain-la-Neuve, Belgium E-mail: [email protected] Phone: +32-10-473404 Fax: +32-10-473440
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Present address: College of Life Science, Anhui Normal University, No.1 Beijing East Road,
Wuhu, Anhui, P.R. China 1
ACCEPTED MANUSCRIPT Abstract Cytotoxin K (CytK) produced by Bacillus cereus s.l. has generally been considered to be associated with the foodborne diarrhoeal syndrome. Two distinct variants of CytK have been
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reported: CytK-1 from Bacillus cytotoxicus and CytK-2 from B. cereus. In order to determine
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whether CytK plays a significant role in the diarrhoeal disease, the occurrence of cytK genes
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was assessed among 390 B. cereus isolates with different origins including clinical and food poisoning samples and was found to be 46%. Interestingly, the cytK occurrence was slightly lower in food poisoning and clinical isolates than in environmental samples. Seventy cytK-2
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positive strains (including 28 isolates from foodborne outbreaks) were then selected in order to assess their genetic diversity. A genetic dendrogram based on the cytK-2 sequences of these 70 strains and on two cytK-1 sequences from strains NVH391-98 and 883-00 showed an
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important diversity. However, no strain clustering according to the origin or source of isolation was observed. These observations were confirmed by Multi-Locus Sequences
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Typing (MLST) based on five different loci of housekeeping genes (ccpA, recF, sucC, purF and gdpD) for which no grouping of foodborne outbreaks strains could be identified.
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Therefore, the choice of cytK as virulence factor for the diarrhoeal pathotype does not seem to be relevant per se, even though the involvement of CytK in the diarrhoeal syndrome cannot
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be fully excluded. Potential synergistic effects between CytK and other virulence factors,
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together with their potential variable expression levels should be further investigated.
Highlights:
The occurrence of cytK is not higher in food poisoning isolates than in those from the environment The cytK positive strains displayed an important genetic diversity Based on the MSLT analysis, no grouping of food poisoning isolates was observed The CytK does not seem to be a major factor in the diarrhoeal syndrome
Key words: Diarrhoeal syndrome, cytK, Bacillus cereus group, MLST
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ACCEPTED MANUSCRIPT 1. Introduction Bacillus cereus sensu lato is a complex group of closely-related Gram-positive, endospore forming, motile and facultative anaerobic bacteria. The B. cereus group contains
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seven different species that present an economical interest but pose also a threat to food safety
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and public health due to their virulence factors (Pirttijärvi et al., 2000). This group includes:
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Bacillus thuringiensis, an entomopathogen currently used as biopesticide, Bacillus anthracis, the etiologic agent of anthrax, the psychrotolerant Bacillus weihenstephanensis, the thermophilic Bacillus cytotoxicus, the rhizoid growers Bacillus mycoides and Bacillus
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pseudomycoides and finally the opportunistic pathogen, B. cereus sensu stricto. However, the concept of species to discriminate the members of B. cereus group is somewhat controversial and an alternative taxonomy has been proposed by Guinebretière and her collaborators
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(2008). This taxonomy suggests to divide the B. cereus strains into seven groups (I to VII), mainly based on their ability to grow at various temperatures and on their pathogenesis.
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B. cereus can be responsible for foodborne intoxications and causes two type of syndromes: diarrhoea and emesis (Stenfors Arnesen et al., 2008). While the emetic syndrome
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manifests as nausea and vomiting within 1-5 h after the consumption of contaminated food, the diarrhoeal syndrome includes the following symptoms: abdominal pain, profuse watery
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diarrhoea, sometime nausea and vomiting within 8-16 h. In both cases, the symptoms last generally less than 48 h. Although most cases are generally mild, more serious and even lethal cases have been reported in Europe (Dierick et al., 2005; Lund et al., 2000; Mahler et
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al., 1997; Naranjo et al., 2011).
Our current understanding of the pathogenesis of these two foodborne illnesses differs dramatically, with a simple scenario for emesis and a convoluted and still controversial situation for the diarrhoeal symptoms. It has now been established that emesis results from the consumption of food containing cereulide, a dodecadepsipeptide synthesized enzymatically via a Non-Ribosomal Peptide Synthetases (NRPS) (Magarvey et al., 2006). This toxin is believed to bind to 5-hydorxytryptamine 3 (5-HT3) receptors causing vomiting (Agata et al., 1995b). Cereulide is closely related to valinomycin which is isolated from some strains of Streptomyces spp. and acts like a potassium-specific ionophore leading to inhibition of the mitochondrial activity (Matter et al., 2009; Mikkola et al., 1999). The genetic determinants of cereulide are generally plasmid-borne (Ehling-Schulz et al., 2006; Hoton et al., 2005), although recent studies have found a chromosomal location of these determinants for several strains (Hoton et al., 2009; Mei et al., 2014). 3
ACCEPTED MANUSCRIPT Concerning the diarrhoeal syndrome, no simple and definitive hypothesis that correlates the symptoms to a unique component exists to date. Indeed, several putative enterotoxins have been reported to be potentially responsible, alone or in combination, of the diarrhoeic
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pathotypes. These include the tripartite enterotoxins as Haemolysin BL (HBL) (Beecher and
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Macmillan, 1991) and Non-haemolytic enterotoxin (Nhe) (Lund and Granum, 1996) but also the single-component toxin, Cytotoxin K (CytK) also sometimes named Haemolysin IV
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(HlyIV) (Lund et al., 2000). In addition to these three major candidates, other molecules are also cited as potential enterotoxins involved in the diarrhoeic syndrome, such as Enterotoxin FM (EntFM) (Boonchai et al., 2008), enterotoxin S (entS), enterotoxin-T (bceT) (Agata et al.,
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1995a) and pore-forming haemolysins like the Cereolysin O (CerO) (Bernheimer and Grushoff, 1967), Haemolysin II (HemII) (Coolbaugh and Williams, 1978) and Haemolysin III
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(HlyIII) (Baida and Kuzmin, 1995). Besides, other virulence factors seem to contribute to the B. cereus food-borne diseases such as phospoholipases or the Sphingomyelinase (SMase) (Doll et al., 2013; Kuppe et al., 1989).
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However, the genes coding for these putative enterotoxins are, for the most part, largely
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distributed among the B. cereus group isolates, irrespective of their diarrhoeic activities (McIntyre et al., 2008; Swiecicka et al., 2006). Moreover, with the exception of the rabbit
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ileum assay, no animal model can be used to specifically test for the diarrhoeal properties of the strains (or for purified proteins) (Beecher et al., 1995). Only classical assays on animal cell lines are readily available, but they only give information on the generic cytotoxicity of these putative enterotoxins (For a recent review, see Jeßberger et al., 2014).
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The present study aims to assess the actual contribution of Cytotoxin K (CytK) to the B. cereus diarrhoeic syndrome. CytK was initially isolated from strain NVH 391-98 of B. cereus (CytK-1, 336 aa) that was responsible for a severe food poisoning outbreak in France that killed three people (Lund et al., 2000). CytK is highly toxic towards human intestinal epithelial cells (Fagerlund et al., 2004), displays necrotic and haemolytic activities, and is able to form pores in lipid bilayers. CytK is a single-component toxin that belongs to the family of β-barrel pore-forming toxins. Two types of cytK genes have been identified and are referred as cytK-1 and cytK-2 (Guinebretière et al., 2006). Their corresponding putative proteins exhibit 89% sequence identity, compared to the > 95% identities among CytK-2 proteins themselves (Fagerlund et al., 2004). Moreover, until recently (Contzen et al., 2014) only five B. cereus strains were reported to contain cytK-1 and those strains were shown to belong to a new species named B. cytotoxicus (Guinebretière et al., 2006; 2013).
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ACCEPTED MANUSCRIPT Besides the difference in their sequences, the other important distinction between the two CytK proteins is their biological effects. CytK-2 forms pores with a lower conductance than those made by CytK-1 (Fagerlund et al., 2004; Hardy et al., 2001; Lund et al., 2000).
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Also, compared to CytK-2, CytK-1 (from NVH 391-98) displays a higher toxicity towards
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human intestinal Caco2 cells and Vero cells (Fagerlund et al., 2004). Moreover, among the cytK-1 positive strains, only NVH 391-98 and INRA AF2 were highly cytotoxic, whereas
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NVH 883/00 was shown to be non-toxic towards Vero cells (Fagerlund et al., 2007; Lund et al., 2000). Unfortunately, the cytK DNA locus of INRA AF2 has not been sequenced yet. Based on all these considerations, it is therefore difficult to conclude whether CytK-1 and/or
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CytK-2 proteins are, or participate as, causative agents of diarrhoeal syndrome caused by B. cereus.
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With the aim of assessing the potential implication of Cytotoxin K in the diarrhoeic syndrome, the genomic and genetic diversity, as well as the occurrence and the evolutionary
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ecology of cytK genes were studied in detail on a collection of B. cereus isolates.
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ACCEPTED MANUSCRIPT 2. Materials and methods 2.1. Bioinformatics
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The presence of cytK genes and their corresponding CytK putative proteins was
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screened in the NCBI database among the 229 genomes or shot-gun fragments from the B.
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cereus group as currently available (Table S1, Nov. 15, 2014). The cytK-1 sequence from B. cereus NVH391-98 was used to find all the homologous sequences using the Basic Local Alignment Search Tool (BLAST).
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2.2. Strain collection and growth conditions
A set of 161 strains (Table S2) coming from food poisonings (FP), food products (F),
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the environment (E) and clinical cases (C) was collected in order to investigate the presence of cytK genes. Then, a panel of cytK-2 positive strains was selected for MLST analysis (Table 1 and below). For all the experiments, the bacterial strains were grown in Luria-Bertani
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medium at 30 °C.
2.3. DNA preparation, PCR amplification and cytK sequencing
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DNA extraction was performed as described by Hu and her collaborators (Hu et al., 2009). Two sets of primers were designed on the basis of conserved flanking areas of published cytK genes, corresponding to cytK-1 and cytK-2, as shown in Table 2. For the cytK-
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1 gene amplification, the cycling program was: 95 °C for 5 min; 30 cycles of 95 °C for 30 s, 48 °C for 30 s and 72 °C for 1 min. The final extension step was 5 min at 72 °C. For cytK-2, the cycling program was: 95 °C for 5 min; 30 cycles of 95 °C for 1 min, 64 °C for 1 min and 72 °C for 1 min and a final extension step of 5 min at 72 °C. Sequencing reactions were performed by Macrogen Europe (Amsterdam, The Netherlands) and GATC (Mulhouse, France). The sequences of the new cytK-2 genes have been submitted to the NCBI database (www.ncbi.nlm.nih.gov) under the accession numbers KP409123 to KP409163.
2.4. Multiple-Locus Sequence Typing (MLST) Five loci encoding housekeeping genes with chromosomal location were chosen for MLST analysis: ccpA (catabolite control protein A), recF (DNA replication and repair protein), sucC (Succinyl CoA synthase beta-subunit) (Helgason et al., 2004), purF (Amidophosphoribosyltransferase) and gdpD (glycerophosphoryl diester phosphodiesterase) (Sorokin 6
ACCEPTED MANUSCRIPT et al., 2006). The selected primers are shown in Table 2. For amplification of ccpA, gdpD, purF, recF and sucC genes, the program was as follows: 95 °C for 5 min; 30 cycles of 95 °C for 1 min, 59 °C for 1 min and 72 °C for 1 min. The final extension step was 5 min at 72 °C.
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The genes were amplified in triplicates using High Fidelity polymerase (Fermentas). The
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same primers used for the amplification were used for DNA sequencing, performed by Macrogen Europe and GATC. The nucleotide sequences have been submitted to the MLST
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SUPERCAT database (http://mlstoslo.uio.no) and to the NCBI database under the accession numbers KP408959 to KP408999 (ccpA), KP409082 to KP409122 (sucC), KP409041 to KP409081 (purF), and KP409000 to KP409040 (gdpD).
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Forty-one strains displaying positive PCR reactions for cytK-2 (originating from China, Germany, Norway and Belgium) and 29 strains for which the cytK-2 gene was
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available in the databases, were selected for the MLST analysis (Table 1 – See also Table 4). The nucleotide sequence diversity of ccpA, recF, sucC, purF, gdpD and cytK-2 genes was analysed at two levels: by constructing a phylogenetic tree for each loci with the CLC
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Main Workbench 7 software (ClCbio, a Qiagen Company) using the Neighbour Joining (NJ)
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algorithm with Jukes Cantor as substitution rate model, and by establishing sequence types (ST) of the various strains using the non-redundant database (NRDB) for allele comparison
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(http://pubmlst.org/). In order to cluster the strains according to their ST, a BURST analysis was performed which defines a group when at least 3/5 loci were identical (http://pubmlst.org/perl/mlstanalyse/). Sequences of the five chromosomal loci were also concatenated with or without adding
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the cytK-2 gene in order to construct and compare the respective phylogeny trees generated by CLC Main Workbench 7 software using UPGMA algorithm and Kimura 80 mathematical model using. The correctness of the results was evaluated using a 100-step bootstrap test. The number of polymorphic sites was also calculated for each locus using the DnaSP software (version 5).
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ACCEPTED MANUSCRIPT 3. Results
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3.1. CytK occurrence among the B. cereus s.l. strains
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The cytK-1 sequence from B. cereus NVH 391-98 was used to collect related sequences in the NCBI database using BLAST program. Among the 229 B. cereus s.l.
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available genomes or shot-gun fragments, 94 cytK-2 genes were identified but no additional cytK-1 sequence could be found. Among these 94 strains, 61 were classified as B. cereus s.s., and 3 as B. thuringiensis. Therefore, as indicated in Table 3, the total cytK (K-1 and K-2)
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occurrence in the NCBI database was 41% (95/229).
A lab collection of 161 B. cereus s.l. strains originating from food poisoning, food,
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environmental and clinical isolates was also tested by PCR for the presence of cytK genes. Eighty-four of these strains gave a positive reaction, which indicates that at least 50% harbour the cytK-2 gene (Table 3), but no cytK-1 positive strain was found. Interestingly, the
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percentage of cytK positive strains detected in this lab collection (52%) was higher than the
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one observed for the strains reported in the databases (41%). Overall, 179 strains were identified as cytK positive, representing 46% of the 390 strains
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screened. In Table 3, the strains were sorted according to their origin: food poisoning, food, environmental, clinical isolates and strains with unknown origin. The cytK occurrence rate varied per origin from 35 to 53%, but no statistically significant differences could be established between the strains potentially more virulent (clinical and food poisoning isolates)
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and those coming from the environment (potentially less virulent). 3.2. Diversity of the cytK sequences To further explore the diversity of CytK, a panel of 70 cytK-2 positive strains was selected (41 strains from the lab collection and 29 strains whose genomes have been reported in the databases), in addition to the two cytK-1 strains: NVH391-98 and NVH 883-00. This selection was based on the origin, the year and the country of isolation and the species to obtain the most diversified panel of cyt-K positive strains. Indeed, the cytK-2 strains displayed different origins: 28 from food poisonings, 14 from food products, 15 from the environment, 8 from clinical cases and 5 with an unknown origin (Table 1). The 28 isolates associated with foodborne outbreaks included 23 strains from Belgian collective toxi-infection cases that occurred between 2006 and 2012 (TIAC strains), the NVH 0597-88 and 1230-88 strains from
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ACCEPTED MANUSCRIPT Norway (Granum et al., 1993), Bc1497 from Germany and two Canadian strains B16 and B23 (Table 1). The cytK-based dendrogram of the 72 B. cereus s.l. isolates (2 B. cytotoxicus (2.8%), 56
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B. cereus s.s (77.8%) and 14 B. thuringiensis (19.4%) was constructed using a 720-bp
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fragment and revealed two distinct groups corresponding to cytK-1 and cytK-2 (Fig. 1A). As expected, the cytK-1 group only contained the two strains NVH 391-98 and NVH 883-00.
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The cytK-2 group displayed two distinct clusters (referred to as I and II), which contained 21 and 49 strains, respectively. The division into two clusters was not linked to the geographical origin or to the type of sample. Similarly, no particular grouping of potentially toxic strains
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could be observed. For example, the 23 Belgian isolates involved in food poisonings (TIAC strains) were widely dispersed throughout the two clusters.
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Besides, the genetic diversity of cytK2 gene sequences was studied together with the diversity of housekeeping genes as illustrated in Table 4. The percentage of polymorphism among the 70 cytK-2 sequences was 15.1%.
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Concerning the CytK protein sequences, members of the CytK-2 group displayed less
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than 88% identity with those of the CytK1 group, while within each group, the proteins shared more than 96% identity.
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3.3. Genetic diversity of the cytK-2-positive strains In order to further characterize the genetic relationship and ecological diversity among
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the 70 cytK-2 positive strains of B. cereus, a multi-locus sequence typing (MLST) was performed using the following fragments of housekeeping genes: ccpA (311 bp), recF (468 bp), sucC (500 bp), purF (600 bp), gdpD (500 bp). Concatenation of the five genes resulted in a sequence of 2,379 bp, and the addition of the cytK-2 gene (760 bp) gave a 3,139 bp sequence. A global NJ tree was built with the concatenated sequences of the five loci. As shown in Fig. 1B, the 70 potentially diarrhoeal, cytK-2-positive strains of B. cereus s.s. were highly diverse and foodborne and clinical isolates were widely spread among the dendrogram. Moreover the strains could be divided in two phylogenetic groups that seem to correspond to phylogenic groups III and IV defined by Guinebretière and her collaborators (2008), based on the distribution of the strains shared between both studies. The only clustering of “diarrhoeal” strains relates to five TIAC isolates from Belgium (Fig. 1B, last five branches). However, these strains all had divergent cytK-2 sequences (Fig. 1A).
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ACCEPTED MANUSCRIPT In order to visualise the influence of cytK gene on the relatedness of the strains, a tree was also built with the sequences of the five loci concatenated with cytK-2. However, the addition of cytK-2 did not modify the layout of the dendrogram (data not shown).
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The sequence variability of each locus was also studied in details. The percentage of
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polymorphism ranged from 6.4% (ccpA) to 25.8% (gdpD) and the number of allelic profiles varied between 21 for ccpA to 37 for recF (Table 4). Based on the allelic profiles of the 5 loci,
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62 Sequence Types (ST) could be defined for all the isolates. The high number of ST is another illustration of the high diversity existing among the cytk-2-positive B. cereus s.l. strains. Of note, Table S3 displays the correspondence between the allelic profiles defined in
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this work and those reported in previous MLST schemes (Helgason et al., 2004; Sorokin et al., 2006) .
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The 70 isolates were then subjected to a BURST analysis in order to group the strains according to the similarity of their allelic profile. The isolates were grouped together when three out of the five analysed loci were identical. Based on this criterion, ten clusters were
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formed, as shown in Table 1. Table S4 displays the number of Single Locus Variants (SLV),
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Double Locus Variants (DLV) and Satellites (SAT) for each assigned sequence type (ST) and the number of strains found at each ST (FREQ). Fifty-four strains were clustered in 10 groups
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while the 16 remaining strains were considered as singletons (Table 1 and Table S4). The frequency (FREQ) showed that 5 strains were assigned to ST22, 3 strains to ST2, 2 strains to ST12 and 2 to ST36 while the other strains all display some differences in their allelic profile. Strains coming from foodborne outbreaks were spread in different groups showing that there
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was no obvious association between similar allelic profiles and geographic or source origins.
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ACCEPTED MANUSCRIPT 4. Discussion Whereas the mechanisms involved in the B. cereus diarrhoeal pathogenesis are still
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largely unknown, a large panel of enterotoxins have been designated as potential causative
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agents of this syndrome. Among them, Cytotoxin K (CytK) is regularly cited as a good
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candidate because of its cytotoxic, necrotic and haemolytic activities on human intestinal cell lines (Hardy et al., 2001; Jeßberger et al., 2014). Furthermore, CytK was initially isolated from the B. cereus NVH 391-98 strain involved in a severe food poisoning outbreak that killed three people due to necrotic enteritis (Lund et al., 2000).
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In this study, 390 B. cereus strains isolated from various environments including clinical and food poisoning samples were screened in order to detect the presence of CytK genetic
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determinants. Almost half of strains contained cytK-2 while no cytK-1 strain was found. The proportion of cytK-2 positive strains varied from 35 to 55% (Table 3) but this proportion was not significantly higher for the potentially more virulent strains, namely the clinical and food
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poisoning isolates. Similar cytK occurrence was observed by Samapundo et al. (2011) among
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B. cereus strains isolated from food products (44%) and by Fagerlund et al. (2004) among foodborne or clinical B. cereus isolations (45%). However, some studies found a higher
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occurrence of cytK gene among diarrhoeal B. cereus strains (73%) by Guinebretière et al. (2002) or among B. thuringiensis strains (73%) by Swiecicka et al. (2006). Low occurrence of cytK gene was also observed by De Jonghe et al. (2010) among aerobic spore-forming
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isolates from raw milk (13%) and by Zhou et al. (2010) among B. cereus strains from ice creams (8%). Whether these more extreme values correspond to specific niches (e.g. lower in dairy products or higher in insect-related strains) or are associated with biased strain isolations remains to be determined and further investigated. The genetic diversity of cytK genes was also studied among a set of 72 strains and the constructed cytK gene-based tree did not show any clustering for the strains isolated from food poisonings. In the lab collection of 161 strains, no strain was found to contain a cytK-1 gene. It is generally recognised that the CytK-2 occurrence is higher than the one of the highly toxic CytK-1 (Fagerlund et al., 2004; Guinebretière et al., 2006). Nevertheless, Contzen and collaborators (2014) have recently isolated several strains harbouring the cytK-1 gene from various commercial potatoes products, showing that this variant could be more frequent than previously estimated. Given the high toxicity of CytK-1 on Caco-2 and Vero-cells, more 11
ACCEPTED MANUSCRIPT investigations on the occurrence of CytK-1variant in these specific products should be performed.
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4.1. Mobility of cytK
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The present study also suggests that cytK is most likely not associated to a mobile genetic element. Indeed, the MLST analysis has shown that the rate of polymorphism for
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cytK-2 was comparable to the selected housekeeping genes (Table 4) indicating that the cytK evolution fits the genetic background of the strain. Besides, the NJ tree based on six genes (five housekeeping genes and cytK-2) showed the same tendency as other published B. cereus
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s.l. phylogenetic studies (Auger et al., 2008; Barker et al., 2005; Fagerlund et al., 2007; Fricker et al., 2011; Han et al., 2006; Hu et al., 2009; Kristoffersen et al., 2011). To verify that cytK was not prone to lateral transfer, 30-kb genome fragments centred on the cytK-2
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locus (NCBI database) were also investigated. Although the genomic neighbourhood of cytK2 displayed some variability, no element potentially involved in horizontal transfer was found
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(data not shown).
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4.2. Implication of CytK in the diarrhoeal disease This study on the genetic determinants of the CytK toxin could not bring any obvious link
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between the cytK gene sequence of a strain and its virulence in the diarrhoeal pathogenesis. To assess whether CytK is or is not a significant factor in this disease, a transcriptomic study should be considered in order to take the expression of the toxin into account. Additionally,
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the potential action of CytK should be investigated in concert with other putative enterotoxins (e.g. HBL, Nhe or HlyII) and other virulence factors to evaluate the diarrhoeic potential of B. cereus strains.
To elucidate the actual involvement of these molecules in the diarrhoeal syndrome, it is necessary to find an adequate animal model. Indeed, due to their proteinaceous nature, these putative enterotoxins may be prone to a rapid inactivation in the intestinal tract, unless they would be released by the B. cereus cells in the immediate vicinity of host's intestinal epithelium being protected by the mucus layer. However this hypothesis has still to be verified. In conclusion, CytK-2 does not seem to be an essential factor in the diarrhoeal syndrome although its potential synergistic action with other virulence factors should be assessed in order to achieve a better understanding of the diarrhoeal pathogenesis. Therefore, caution should always be taken when referring to this protein as a bona fide enterotoxin. 12
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ACCEPTED MANUSCRIPT Conflict of interest
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The authors declare no conflict of interest.
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Acknowledgements
The project was supported by grants from the Federal Public Service (FOD) Health, Food Chain Safety and Environment (project RT09/2 BACEREUS). We gratefully acknowledge Pr
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P.E. Granum (The Norwegian School of Veterinary Science, Norway), Dr Guoping Zhou (Polytechnic University, Wuhan, China), Pr Marc Heyndrickx (Institute for Agricultural and
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Fisheries Research, Belgium) and Dr Paul in't Veld (Food and Consumer Product, 5600 CD Eindhoven, The Netherlands) for providing the strains. Ir Catherine Rasse (Institute for
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Multidisciplinary Research in Quantitative Modelling and Analysis, Université catholique de Louvain, Belgium) is thanked for her statistical expertise and Dr Ruiming Han for his critical
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comments on the manuscript.
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ACCEPTED MANUSCRIPT References
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Ehling-Schulz, M., Fricker, M., Grallert, H., Rieck, P., Wagner, M., Scherer, S., 2006. Cereulide synthetase gene cluster from emetic Bacillus cereus: structure and location on
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Microbiology 6, 20.
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a mega virulence plasmid related to Bacillus anthracis toxin plasmid pXO1. BMC
Fagerlund, A., Brillard, J., Fürst, R., Guinebretière, M.-H., Granum, P.E., 2007. Toxin production in a rare and genetically remote cluster of strains of the Bacillus cereus
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group. BMC Microbiology 7, 43. Fagerlund, A., Ween, O., Lund, T., Hardy, S.P., Granum, P.E., 2004. Genetic and functional analysis of the cytK family of genes in Bacillus cereus. Microbiology 150, 2689-2697. Fricker, M., Ågren, J., Segerman, B., Knutsson, R., Ehling-Schulz, M., 2001. Evaluation of Bacillus strains as model systems for the work on Bacillus anthracis spores. International Journal Food Microbiology 145, S129-136. Granum, P.E., Brynestad, S., Kramer, J.M., 1993. Analysis of enterotoxin production by Bacillus cereus from dairy products, food poisoning incidents and non-gastrointestinal infections. International Journal Food Microbiology 17, 269-279. Guinebretière, M.-H., Auger, S., Galleron, N., Contzen, M., De Sarrau, B., De Buyser, M.L., Lamberet, G., Fagerlund, A., Granum, P.E., Lereclus, D., De Vos, P., Nguyen-The, C., 16
ACCEPTED MANUSCRIPT Sorokin, A., 2013. Bacillus cytotoxicus sp. nov. is a novel thermotolerant species of the Bacillus cereus Group occasionally associated with food poisoning. International Journal of Systematic and Evolutionary Microbiology 63, 31-40.
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Guinebretière, M.-H., Thompson, F.L., Sorokin, A., Normand, P., Dawyndt, P., Ehling-
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Schulz, M., Svensson, B., Sanchis, V., Nguyen-The, C., Heyndrickx, M., De Vos, P.,
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2008. Ecological diversification in the Bacillus cereus Group. Environmental Microbiology 10, 851-865.
Guinebretière, M.-H., Fagerlund, A., Granum, P.E., Nguyen-The, C., 2006. Rapid
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discrimination of cytK-1 and cytK-2 genes in Bacillus cereus strains by a novel duplex
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PCR system. FEMS Microbiology Letters 259, 74-80. Guinebretière, M.-H., Broussolle, V., Nguyen-The, C., 2002. Enterotoxigenic profiles of food-poisoning and food-borne Bacillus cereus strains. Journal of Clinical Microbiology
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Han, C.S., Xie, G., Challacombe, J.F., Altherr, M.R., Bhotika, S.S., Brown, N., Bruce, D., Campbell, C.S., Campbell, M.L., Chen, J., Chertkov, O., Cleland, C, . Dimitrijevic, M.,
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Doggett, N.A., Fawcett, J.J., Glavina, T., Goodwin, L.A., Green, L.D., Hill, K.K., Hitchcock, P., Jackson, P.J., Keim, P., Kewalramani, A.R., Longmire, J., Lucas, S., Malfatti, S., McMurry, K., Meincke, L.J., Misra, M., Moseman, B.L., Mundt, M., Munk,
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A.C., Okinaka, R.T., Parson-Quintana, B., Reilly, L.P., Richardson, P., Robinson, D.L., Rubin, E., Saunders, E., Tapia, R., Tesmer, J.G., Thayer, N., Thompson, L.S., Tice, H., Ticknor, L.O., Wills, P.L., Brettin, T.S., Gilna, P., 2006. Pathogenomic sequence analysis of Bacillus cereus and Bacillus thuringiensis isolates closely related to Bacillus anthracis. Journal of Bacteriology 188, 3382-3390. Hardy, S.P., Lund, T., Granum, P.E., 2001. CytK toxin of Bacillus cereus forms pores in planar lipid bilayers and is cytotoxic to intestinal epithelia. FEMS Microbiology Letters 197, 47-51. Helgason, E., Tourasse, N.J., Meisal, R., Caugant, D.A., Kolsto, A.-B., 2004. Multilocus Sequence Typing Scheme for Bacteria of the Bacillus cereus Group. Applied and Environmental Microbiology 70, 191-201.
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ACCEPTED MANUSCRIPT Hoton, F.M., Fornelos, N., N’guessan, E., Hu, X., Swiecicka, I., Dierick, K., Jääskeläinen, E., Salkinoja-Salonen, M., Mahillon, J., 2009. Family portrait of Bacillus cereus and Bacillus weihenstephanensis cereulide-producing strains. Environmental Microbiology
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Reports 1, 177-183.
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Hoton, F.M., Andrup, L., Swiecicka, I., Mahillon, J., 2005. The cereulide genetic
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determinants of emetic Bacillus cereus are plasmid-borne. Microbiology 151, 21212124.
Hu, X., Swiecicka, I., Timmery, S., Mahillon, J., 2009. Sympatric soil communities of
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Bacillus cereus sensu lato: population structure and potential plasmid dynamics of
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pXO1- and pXO2-like elements. FEMS Microbiology Letters 70, 344-355. Jeßberger, N., Dietrich, R., Bock, S., Didier, A., Märtlbauer, E., 2014. Bacillus cereus enterotoxins act as major virulence factors and exhibit distinct cytotoxicity to different
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human cell lines. Toxicon 77, 49-57.
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Kristoffersen, S.M., Tourasse, N.J., Kolstø, A.B., Økstad, O.A., 2011. Interspersed DNA repeats bcr1-bcr18 of Bacillus cereus group bacteria form three distinct groups with
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different evolutionary and functional patterns. Molecular Biology and Evolution 28, 963-
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Kuppe, A., Evans, L.M., McMillen, D.A., Griffith, O.H., 1989. Phosphatidylinositol-specific phospholipase C of Bacillus cereus: cloning, sequencing, and relationship to other phospholipases. Journal of Bacteriology 171, 6077-6083. Lund, T., De Buyser, M.-L., Granum, P.E., 2000. A new cytotoxin from Bacillus cereus that may cause necrotic enteritis. Molecular Microbiology 38, 254-261. Lund, T., Granum, P.E., 1996. Characterisation of a non-haemolytic enterotoxin complex from Bacillus cereus isolated after a foodborne outbreak. FEMS Microbiology Letters 141, 151-156. Magarvey, N.A., Ehling-Schulz, M., Walsh, C.T., 2006. Characterization of the cereulide NRPS alpha-hydroxy acid specifying modules: activation of alpha-keto acids and chiral
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ACCEPTED MANUSCRIPT reduction on the assembly line. Journal of the American Chemical Society 128, 1069810699. Mahler, H., Pasi, A., Kramer, J.M., Schulte, P., Scoging, A.C., Bär, W., Krähenbühl, S., 1997.
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Fulminant liver failure in association with the emetic toxin of Bacillus cereus. The New
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Matter, A.M., Hoot, S.B., Anderson, P.D., Neves, S.S., Cheng, Y.-Q., 2009. Valinomycin biosynthetic gene cluster in Streptomyces: conservation, ecology and evolution. PloS
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One 4, e7194.
McIntyre, L., Bernard, K., Beniac, D., Isaac-Renton, J.L., Naseby, D.C., 2008. Identification
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of Bacillus cereus group species associated with food poisoning outbreaks in British Columbia, Canada. Applied and Environmental Microbiology 74, 7451-7453.
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Mei, X., Xu, K., Yang, L., Yuan, Z., Mahillon, J., Hu, X., 2014. The genetic diversity of cereulide biosynthesis gene cluster indicates a composite transposon Tnces in emetic
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Bacillus weihenstephanensis. BMC Microbiology 14, 149.
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Mikkola, R., Saris, N.-E.L., Grigoriev, P.A., Andersson, M.A., Salkinoja-Salonen, M.S., 1999. Ionophoretic properties and mitochondrial effects of cereulide. The emetic toxin of B. cereus. European Journal of Biochemistry 263, 112-117.
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Naranjo, M., Denayer, S., Botteldoorn, N., Delbrassinne, L., Veys, J., Waegenaere, J., Sirtaine, N., Driesen, R.B., Sipido, K.R., Mahillon, J., Dierick, K., 2011. Sudden death of a young adult associated with Bacillus cereus food poisoning. Journal of Clinical Microbiology 49, 4379-4381. Pirttijärvi, T.S.M., Andersson, M.A., Salkinoja-Salonen, M.S., 2000. Properties of Bacillus cereus and other bacilli contaminating biomaterial-based industrial processes. International Journal of Food Microbiology 60, 231-239. Samapundo, S., Heyndrickx, M., Xhaferi, R., Devlieghere, F., 2011. Incidence, diversity and toxin gene characteristics of Bacillus cereus group strains isolated from food products marketed in Belgium. International Journal Food Microbiology 150, 34-41.
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ACCEPTED MANUSCRIPT Sorokin, A., Candelon, B., Guilloux, K., Galleron, N., Wackerow-Kouzova, N., Ehrlich, S.D., Bourguet, D., Sanchis, V., 2006. Multiple-locus sequence typing analysis of Bacillus cereus and Bacillus thuringiensis reveals separate clustering and a distinct population
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structure of psychrotrophic strains. Applied and Environmental Microbiology 72, 1569-
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1578.
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Stenfors Arnesen, L.P., Fagerlund, A., Granum, P.E., 2008. From soil to gut: Bacillus cereus and its food poisoning toxins. FEMS Microbiology Reviews 32, 579-606. Swiecicka, I., Van der Auwera, G.A., Mahillon, J., 2006. Hemolytic and nonhemolytic
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enterotoxin genes are broadly distributed among Bacillus thuringiensis isolated from
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wild mammals. Microbial Ecology 52, 544-551.
Zhou, G., Zheng, D., Dou, L., Cai, Q., Yuan, Z., 2010. Occurrence of psychrotolerant Bacillus cereus group strains in ice creams. International Journal Food Microbiology 137, 143-
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ACCEPTED MANUSCRIPT Legends to Figures
Fig. 1. Relationship dendrograms. A. Dendrogram of the cytK gene sequences from 72 B.
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cereus s.l. isolates. A cytK gene-based UPGMA tree of 72 B. cereus s.l. isolates. The cytK-1
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group contains two strains while all the other cytK-2 sequences are organized into two clusters
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(I and II). B. Relationship dendrogram of the 70 B. cereus strains containing cytK-2, based on the concatenated sequences of five housekeeping loci. All these strains belong to group III or IV as defined by Guinebretière et al., 2008. In both parts, the origin of the strains is indicated within brackets: food poisoning (FP), clinical isolates (C), food (F), environment (E) and
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unknown (U).
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Fig. 1A
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Fig. 1B
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ACCEPTED MANUSCRIPT Table 1: Origin, typing and BURST-grouping data of B. cereus s.l. strains.
Bc_AH1134* Bc_ATCC10876* Bc_F65185* Bc_MHI2385 Bc_MHI1687 Bc_B16 Bc_B23 Bc_I11-1 Bt_T13001* Bc_I8-5 TIAC468 TIAC70 TIAC1120 TIAC299 Bc_95_8201* Bc_AH820* Bt_BGSC4AJ1* Bc_NVH0597-99* Bt_97-27* Bt_BGSC4BA1* Bt_ATCC35646* Bt_T01001* TIAC949 Bc_NVH1230-88
USA
Belgium USA USA France Belgium Belgium USA Belgium Belgium Germany Norway Germany
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Norway
U
USA
4
5 6
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3
Germany Germany Canada Canada China Pakistan China Belgium Belgium Belgium Belgium UK Norway Mexico Norway Yugoslavia India Israel Canada Belgium Norway
gdpD
purF
recF
sucC
6 7 8 9 12 12 13 16 17 18 50 58 59 4 19 20 21 26 27 28 29 5 22 22 22 22 22 23 24 25 30 31 35 36 36 37 39 40 51 52 53 1
3 3 3 3 3 3 3 3 3 4 13 19 19 2 5 5 5 5 5 5 5 2 5 5 5 5 5 5 5 5 5 5 8 8 8 8 8 8 14 14 14 1
3 3 3 3 13 13 13 34 34 3 3 3 3 2 2 5 5 10 10 11 16 6 6 6 6 6 6 6 6 6 27 31 9 9 9 9 19 21 20 20 36 1
3 3 25 26 3 3 21 21 30 4 3 24 25 2 2 6 6 6 6 10 2 5 5 5 5 5 5 5 26 29 3 27 9 9 9 16 16 9 18 18 31 1
3 8 3 3 3 3 26 8 8 3 3 8 8 2 5 6 10 5 14 10 5 7 5 5 5 5 5 20 32 5 5 5 12 15 15 21 12 12 24 24 24 1
3 3 3 3 3 3 3 3 3 3 3 18 3 2 2 2 2 2 8 2 2 4 4 4 4 4 4 4 4 22 4 4 7 7 7 7 7 7 13 16 16 1
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Germany
ccpA
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China
Belgium
STb
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Bt_BMB171*
Germany
Type of samplea U F FP FP E U E FP FP F E FP FP FP F F F E C U C U F FP FP F E F FP FP FP FP C C E FP C E E E FP FP
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Norway
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Bc_15 Bc_MHI32 TIAC179 TIAC180 Bc_ATCC14579* Bc_BDRD-Cer4* Bt_Bt407* TIAC75 TIAC77 Bc_MHI1520 Bc_Rock1-15* TIAC1112 TIAC176 Bc_MHI1497 Bc_45 Bc_MHI1694 Bc_67
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Country
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Group Strain
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Germany China Belgium Belgium Belgium Belgium
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Belgium Belgium Norway Norway USA Belgium Belgium Belgium Canada
Namibia USA
Pakistan Japan U
Belgium
1 1 1 1 12 15 4 4 28 1 26 29 3 23 7 8 17 23 21 6 11 13 14 19 20 22 3 30
23 23 23 30 16 3 4 19 6 6 33 14 29 31 9 11 22 28 6 13 13 17 18 12 25 27 8 34
12 12 12 12 3 3 3 3 20 20 2 2 19 21 5 6 7 9 23 9 9 10 11 7 14 15 17 24
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Belgium
1 1 1 30 14 14 4 4 6 29 32 32 28 31 7 8 18 26 33 37 12 15 17 22 23 24 25 35
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Brazil
1 1 1 1 10 10 3 3 8 8 20 20 3 3 6 7 8 8 8 8 9 11 12 15 16 17 18 21
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U
2 2 2 3 46 47 10 11 34 42 60 61 14 15 32 33 38 41 43 44 45 48 49 54 55 56 57 62
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Belgium
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Singletons
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France
E C E FP U F F F FP FP FP FP FP FP F F E FP FP FP F E C E E C FP FP
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Israel
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Bt_ATCC10792* Bt_IBL200* Bt_T03a001* TIAC294 Bc_B4264* Bc_m1550* Bc_MHI1544 Bc_I38-8 TIAC59 TIAC219 TIAC73 TIAC71 TIAC126 TIAC297 Bc_NVH390-88 Bc_NVH762-00 Bc_Rock3-42 * TIAC1108 TIAC74 TIAC959 Bc_ATCC10987* Bc_E33L* BC_G9241* Bt_BGSC4CC1* Bt_BGSC4Y1* Bt_IBL4222* TIAC1107 TIAC896
Detailed allelic profiles for the five housekeeping genes (ccpA, gdpD, purF, sucC and recF) are given for the ST (Sequence Types (detailed in Table S3). The sequences of strains with an asterisk (29 strains) are from NCBI databases whereas the data of the 41 other strains are from this study. Numbers were arbitrary assigned to allele fragment for each locus. The STs were grouped by BURST analysis: fifty-four strains were divided into ten groups based on the number of differences in the allelic profiles (Table S42). The remaining 16 strains could not be grouped and were considered as singletons. Note that the genetic diversity associated with these genes, expressed as the percentage of polymorphism, is displayed in Table 4. a
: Abbreviations are as follows: C: clinical isolates, E: environmental isolates, F: food isolates,
FP: food poisoning isolates and U: strains with unknown origin. b
: ST stands for Sequence Type and corresponds to the specific allelic profile.
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ACCEPTED MANUSCRIPT Table 2. Primers used in the study.
Amplicon
Gene
Primers
Sequences (5’-3’)
purF
purF_F
YGAAGAATGTGGCGTTTTYGGA
purF_R
GAAATACTAGAGTCTGGTACAC
gdpD_F
GGTGGTTATGCAAATGCGACRAATG
gdpD_R
YGCCATAATATAAAATGGYGTCACG
recF_F
GCGATGGCGAAATCTCATAG
recF_R
CAAATCCATTGATTCTGATACATC
sucC_F
GGCGGAACAGAAATTGAAGA
sucC_R
TCACACTTCATAATGCCACCA
ccpA_F
GTTTAGGATACCGCCCAAATG
ccpA_R
TGTAACTTCTTCGCGCTTCC
L2-2
GAGTTTTGRAVGATARMGAT
R2
AGGCATCTCTTCACCTTTAT
CytK_F1
ACAGATATCGGKCAAAATGC
CytK_R1
TCCAACCCAGTTWSCAGTTC
Sorokin et al., 2006
574
Sorokin et al., 2006
560
Helgason et al., 2004
590
Helgason et al., 2004
460
Helgason et al., 2004
1,263
This study
760
This study
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cytK-2
790
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cytK-1
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ccpA
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sucC
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recF
Reference
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gdpD
size (bp)
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The nucleotide codes are as follows: Y for C or T, R for A or G, V for G or A or C, M for C
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or A, K for G or T, W for A or T, and S for G or C.
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ACCEPTED MANUSCRIPT Table 3: CytK gene occurrence among the B. cereus s.l. strains.
Origins
Database (22941 strains)
Lab collection (161 strains)
Total (390402 strains)
cytK+
cytK+ %
cytK–
cytK+
cytK+ %
cytK+ ratio
cytK+ %
Clinical isolates
12
119
483
3
1
25
120/275
440
Food poisoning
7
2
22
325
27
464
29/6871
431
Food
86
45
3345
401
5148
564
553/1030
53
Environment
8575
6354
4639
21
3
6075
6657/143
4640
Unknown
3241
1520
323
01
21
10050
1721/4963
353
Mean
13451
950
4137
7781
840
520
1790/390402
462
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cytK–
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ACCEPTED MANUSCRIPT Table 4: Genetic diversity of the five housekeeping gene loci and cytK-2.
Fragment length (bp)
No. of polymorphic sites*
No. of alleles
ccpA
311
20 (6.4)
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gdpD
500
129 (25.8)
purF
600
91 (15.2)
recF
468
92 (19.7)
sucC
500
56 (11.2)
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cytK2
760
115 (15.1)
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Locus
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37 31 34
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*In brackets: the percentage of polymorphism (the number of polymorphic sites divided by
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the length of the fragment).
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ACCEPTED MANUSCRIPT Highlights: The occurrence of cytK is not higher in food poisoning isolates than in those from the
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environment
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The cytK positive strains displayed an important genetic diversity
Based on the MSLT analysis, no grouping of food poisoning isolates was observed
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The CytK does not seem to be a major factor in the diarrhoeal syndrome
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S0168-1605(15)30044-1 doi: 10.1016/j.ijfoodmicro.2015.06.020 FOOD 6960
To appear in:
International Journal of Food Microbiology
Received date: Revised date: Accepted date:
12 January 2015 20 June 2015 22 June 2015
Please cite this article as: Castiaux, Virginie, Liu, Xiaojin, Delbrassinne, Laurence, Mahillon, Jacques, Is Cytotoxin K from Bacillus cereus a bona fide enterotoxin?, International Journal of Food Microbiology (2015), doi: 10.1016/j.ijfoodmicro.2015.06.020
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Is Cytotoxin K from Bacillus cereus a bona fide enterotoxin ?
Laboratory of Food and Environmental Microbiology, Université catholique de Louvain, B-
1348 Louvain-la-Neuve, Belgium; and
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Scientific Institute of Public Health, Juliette
Wytsman street 14, B-1050 Brussels, Belgium
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Running title: CytK from B. cereus
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† These authors contributed equally to this work.
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Virginie Castiaux1,†, Xiaojin Liu1,#,†, Laurence Delbrassinne1,2,† and Jacques Mahillon1*
*Corresponding author: Jacques Mahillon
Laboratory of Food and Environmental Microbiology Croix du Sud, 2 – L7.5.12 B-1348 Louvain-la-Neuve, Belgium E-mail: [email protected] Phone: +32-10-473404 Fax: +32-10-473440
#
Present address: College of Life Science, Anhui Normal University, No.1 Beijing East Road,
Wuhu, Anhui, P.R. China 1
ACCEPTED MANUSCRIPT Abstract Cytotoxin K (CytK) produced by Bacillus cereus s.l. has generally been considered to be associated with the foodborne diarrhoeal syndrome. Two distinct variants of CytK have been
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reported: CytK-1 from Bacillus cytotoxicus and CytK-2 from B. cereus. In order to determine
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whether CytK plays a significant role in the diarrhoeal disease, the occurrence of cytK genes
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was assessed among 390 B. cereus isolates with different origins including clinical and food poisoning samples and was found to be 46%. Interestingly, the cytK occurrence was slightly lower in food poisoning and clinical isolates than in environmental samples. Seventy cytK-2
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positive strains (including 28 isolates from foodborne outbreaks) were then selected in order to assess their genetic diversity. A genetic dendrogram based on the cytK-2 sequences of these 70 strains and on two cytK-1 sequences from strains NVH391-98 and 883-00 showed an
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important diversity. However, no strain clustering according to the origin or source of isolation was observed. These observations were confirmed by Multi-Locus Sequences
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Typing (MLST) based on five different loci of housekeeping genes (ccpA, recF, sucC, purF and gdpD) for which no grouping of foodborne outbreaks strains could be identified.
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Therefore, the choice of cytK as virulence factor for the diarrhoeal pathotype does not seem to be relevant per se, even though the involvement of CytK in the diarrhoeal syndrome cannot
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be fully excluded. Potential synergistic effects between CytK and other virulence factors,
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together with their potential variable expression levels should be further investigated.
Highlights:
The occurrence of cytK is not higher in food poisoning isolates than in those from the environment The cytK positive strains displayed an important genetic diversity Based on the MSLT analysis, no grouping of food poisoning isolates was observed The CytK does not seem to be a major factor in the diarrhoeal syndrome
Key words: Diarrhoeal syndrome, cytK, Bacillus cereus group, MLST
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ACCEPTED MANUSCRIPT 1. Introduction Bacillus cereus sensu lato is a complex group of closely-related Gram-positive, endospore forming, motile and facultative anaerobic bacteria. The B. cereus group contains
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seven different species that present an economical interest but pose also a threat to food safety
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and public health due to their virulence factors (Pirttijärvi et al., 2000). This group includes:
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Bacillus thuringiensis, an entomopathogen currently used as biopesticide, Bacillus anthracis, the etiologic agent of anthrax, the psychrotolerant Bacillus weihenstephanensis, the thermophilic Bacillus cytotoxicus, the rhizoid growers Bacillus mycoides and Bacillus
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pseudomycoides and finally the opportunistic pathogen, B. cereus sensu stricto. However, the concept of species to discriminate the members of B. cereus group is somewhat controversial and an alternative taxonomy has been proposed by Guinebretière and her collaborators
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(2008). This taxonomy suggests to divide the B. cereus strains into seven groups (I to VII), mainly based on their ability to grow at various temperatures and on their pathogenesis.
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B. cereus can be responsible for foodborne intoxications and causes two type of syndromes: diarrhoea and emesis (Stenfors Arnesen et al., 2008). While the emetic syndrome
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manifests as nausea and vomiting within 1-5 h after the consumption of contaminated food, the diarrhoeal syndrome includes the following symptoms: abdominal pain, profuse watery
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diarrhoea, sometime nausea and vomiting within 8-16 h. In both cases, the symptoms last generally less than 48 h. Although most cases are generally mild, more serious and even lethal cases have been reported in Europe (Dierick et al., 2005; Lund et al., 2000; Mahler et
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al., 1997; Naranjo et al., 2011).
Our current understanding of the pathogenesis of these two foodborne illnesses differs dramatically, with a simple scenario for emesis and a convoluted and still controversial situation for the diarrhoeal symptoms. It has now been established that emesis results from the consumption of food containing cereulide, a dodecadepsipeptide synthesized enzymatically via a Non-Ribosomal Peptide Synthetases (NRPS) (Magarvey et al., 2006). This toxin is believed to bind to 5-hydorxytryptamine 3 (5-HT3) receptors causing vomiting (Agata et al., 1995b). Cereulide is closely related to valinomycin which is isolated from some strains of Streptomyces spp. and acts like a potassium-specific ionophore leading to inhibition of the mitochondrial activity (Matter et al., 2009; Mikkola et al., 1999). The genetic determinants of cereulide are generally plasmid-borne (Ehling-Schulz et al., 2006; Hoton et al., 2005), although recent studies have found a chromosomal location of these determinants for several strains (Hoton et al., 2009; Mei et al., 2014). 3
ACCEPTED MANUSCRIPT Concerning the diarrhoeal syndrome, no simple and definitive hypothesis that correlates the symptoms to a unique component exists to date. Indeed, several putative enterotoxins have been reported to be potentially responsible, alone or in combination, of the diarrhoeic
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pathotypes. These include the tripartite enterotoxins as Haemolysin BL (HBL) (Beecher and
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Macmillan, 1991) and Non-haemolytic enterotoxin (Nhe) (Lund and Granum, 1996) but also the single-component toxin, Cytotoxin K (CytK) also sometimes named Haemolysin IV
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(HlyIV) (Lund et al., 2000). In addition to these three major candidates, other molecules are also cited as potential enterotoxins involved in the diarrhoeic syndrome, such as Enterotoxin FM (EntFM) (Boonchai et al., 2008), enterotoxin S (entS), enterotoxin-T (bceT) (Agata et al.,
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1995a) and pore-forming haemolysins like the Cereolysin O (CerO) (Bernheimer and Grushoff, 1967), Haemolysin II (HemII) (Coolbaugh and Williams, 1978) and Haemolysin III
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(HlyIII) (Baida and Kuzmin, 1995). Besides, other virulence factors seem to contribute to the B. cereus food-borne diseases such as phospoholipases or the Sphingomyelinase (SMase) (Doll et al., 2013; Kuppe et al., 1989).
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However, the genes coding for these putative enterotoxins are, for the most part, largely
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distributed among the B. cereus group isolates, irrespective of their diarrhoeic activities (McIntyre et al., 2008; Swiecicka et al., 2006). Moreover, with the exception of the rabbit
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ileum assay, no animal model can be used to specifically test for the diarrhoeal properties of the strains (or for purified proteins) (Beecher et al., 1995). Only classical assays on animal cell lines are readily available, but they only give information on the generic cytotoxicity of these putative enterotoxins (For a recent review, see Jeßberger et al., 2014).
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The present study aims to assess the actual contribution of Cytotoxin K (CytK) to the B. cereus diarrhoeic syndrome. CytK was initially isolated from strain NVH 391-98 of B. cereus (CytK-1, 336 aa) that was responsible for a severe food poisoning outbreak in France that killed three people (Lund et al., 2000). CytK is highly toxic towards human intestinal epithelial cells (Fagerlund et al., 2004), displays necrotic and haemolytic activities, and is able to form pores in lipid bilayers. CytK is a single-component toxin that belongs to the family of β-barrel pore-forming toxins. Two types of cytK genes have been identified and are referred as cytK-1 and cytK-2 (Guinebretière et al., 2006). Their corresponding putative proteins exhibit 89% sequence identity, compared to the > 95% identities among CytK-2 proteins themselves (Fagerlund et al., 2004). Moreover, until recently (Contzen et al., 2014) only five B. cereus strains were reported to contain cytK-1 and those strains were shown to belong to a new species named B. cytotoxicus (Guinebretière et al., 2006; 2013).
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ACCEPTED MANUSCRIPT Besides the difference in their sequences, the other important distinction between the two CytK proteins is their biological effects. CytK-2 forms pores with a lower conductance than those made by CytK-1 (Fagerlund et al., 2004; Hardy et al., 2001; Lund et al., 2000).
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Also, compared to CytK-2, CytK-1 (from NVH 391-98) displays a higher toxicity towards
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human intestinal Caco2 cells and Vero cells (Fagerlund et al., 2004). Moreover, among the cytK-1 positive strains, only NVH 391-98 and INRA AF2 were highly cytotoxic, whereas
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NVH 883/00 was shown to be non-toxic towards Vero cells (Fagerlund et al., 2007; Lund et al., 2000). Unfortunately, the cytK DNA locus of INRA AF2 has not been sequenced yet. Based on all these considerations, it is therefore difficult to conclude whether CytK-1 and/or
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CytK-2 proteins are, or participate as, causative agents of diarrhoeal syndrome caused by B. cereus.
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With the aim of assessing the potential implication of Cytotoxin K in the diarrhoeic syndrome, the genomic and genetic diversity, as well as the occurrence and the evolutionary
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ecology of cytK genes were studied in detail on a collection of B. cereus isolates.
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ACCEPTED MANUSCRIPT 2. Materials and methods 2.1. Bioinformatics
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The presence of cytK genes and their corresponding CytK putative proteins was
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screened in the NCBI database among the 229 genomes or shot-gun fragments from the B.
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cereus group as currently available (Table S1, Nov. 15, 2014). The cytK-1 sequence from B. cereus NVH391-98 was used to find all the homologous sequences using the Basic Local Alignment Search Tool (BLAST).
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2.2. Strain collection and growth conditions
A set of 161 strains (Table S2) coming from food poisonings (FP), food products (F),
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the environment (E) and clinical cases (C) was collected in order to investigate the presence of cytK genes. Then, a panel of cytK-2 positive strains was selected for MLST analysis (Table 1 and below). For all the experiments, the bacterial strains were grown in Luria-Bertani
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medium at 30 °C.
2.3. DNA preparation, PCR amplification and cytK sequencing
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DNA extraction was performed as described by Hu and her collaborators (Hu et al., 2009). Two sets of primers were designed on the basis of conserved flanking areas of published cytK genes, corresponding to cytK-1 and cytK-2, as shown in Table 2. For the cytK-
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1 gene amplification, the cycling program was: 95 °C for 5 min; 30 cycles of 95 °C for 30 s, 48 °C for 30 s and 72 °C for 1 min. The final extension step was 5 min at 72 °C. For cytK-2, the cycling program was: 95 °C for 5 min; 30 cycles of 95 °C for 1 min, 64 °C for 1 min and 72 °C for 1 min and a final extension step of 5 min at 72 °C. Sequencing reactions were performed by Macrogen Europe (Amsterdam, The Netherlands) and GATC (Mulhouse, France). The sequences of the new cytK-2 genes have been submitted to the NCBI database (www.ncbi.nlm.nih.gov) under the accession numbers KP409123 to KP409163.
2.4. Multiple-Locus Sequence Typing (MLST) Five loci encoding housekeeping genes with chromosomal location were chosen for MLST analysis: ccpA (catabolite control protein A), recF (DNA replication and repair protein), sucC (Succinyl CoA synthase beta-subunit) (Helgason et al., 2004), purF (Amidophosphoribosyltransferase) and gdpD (glycerophosphoryl diester phosphodiesterase) (Sorokin 6
ACCEPTED MANUSCRIPT et al., 2006). The selected primers are shown in Table 2. For amplification of ccpA, gdpD, purF, recF and sucC genes, the program was as follows: 95 °C for 5 min; 30 cycles of 95 °C for 1 min, 59 °C for 1 min and 72 °C for 1 min. The final extension step was 5 min at 72 °C.
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The genes were amplified in triplicates using High Fidelity polymerase (Fermentas). The
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same primers used for the amplification were used for DNA sequencing, performed by Macrogen Europe and GATC. The nucleotide sequences have been submitted to the MLST
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SUPERCAT database (http://mlstoslo.uio.no) and to the NCBI database under the accession numbers KP408959 to KP408999 (ccpA), KP409082 to KP409122 (sucC), KP409041 to KP409081 (purF), and KP409000 to KP409040 (gdpD).
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Forty-one strains displaying positive PCR reactions for cytK-2 (originating from China, Germany, Norway and Belgium) and 29 strains for which the cytK-2 gene was
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available in the databases, were selected for the MLST analysis (Table 1 – See also Table 4). The nucleotide sequence diversity of ccpA, recF, sucC, purF, gdpD and cytK-2 genes was analysed at two levels: by constructing a phylogenetic tree for each loci with the CLC
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Main Workbench 7 software (ClCbio, a Qiagen Company) using the Neighbour Joining (NJ)
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algorithm with Jukes Cantor as substitution rate model, and by establishing sequence types (ST) of the various strains using the non-redundant database (NRDB) for allele comparison
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(http://pubmlst.org/). In order to cluster the strains according to their ST, a BURST analysis was performed which defines a group when at least 3/5 loci were identical (http://pubmlst.org/perl/mlstanalyse/). Sequences of the five chromosomal loci were also concatenated with or without adding
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the cytK-2 gene in order to construct and compare the respective phylogeny trees generated by CLC Main Workbench 7 software using UPGMA algorithm and Kimura 80 mathematical model using. The correctness of the results was evaluated using a 100-step bootstrap test. The number of polymorphic sites was also calculated for each locus using the DnaSP software (version 5).
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ACCEPTED MANUSCRIPT 3. Results
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3.1. CytK occurrence among the B. cereus s.l. strains
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The cytK-1 sequence from B. cereus NVH 391-98 was used to collect related sequences in the NCBI database using BLAST program. Among the 229 B. cereus s.l.
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available genomes or shot-gun fragments, 94 cytK-2 genes were identified but no additional cytK-1 sequence could be found. Among these 94 strains, 61 were classified as B. cereus s.s., and 3 as B. thuringiensis. Therefore, as indicated in Table 3, the total cytK (K-1 and K-2)
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occurrence in the NCBI database was 41% (95/229).
A lab collection of 161 B. cereus s.l. strains originating from food poisoning, food,
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environmental and clinical isolates was also tested by PCR for the presence of cytK genes. Eighty-four of these strains gave a positive reaction, which indicates that at least 50% harbour the cytK-2 gene (Table 3), but no cytK-1 positive strain was found. Interestingly, the
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percentage of cytK positive strains detected in this lab collection (52%) was higher than the
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one observed for the strains reported in the databases (41%). Overall, 179 strains were identified as cytK positive, representing 46% of the 390 strains
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screened. In Table 3, the strains were sorted according to their origin: food poisoning, food, environmental, clinical isolates and strains with unknown origin. The cytK occurrence rate varied per origin from 35 to 53%, but no statistically significant differences could be established between the strains potentially more virulent (clinical and food poisoning isolates)
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and those coming from the environment (potentially less virulent). 3.2. Diversity of the cytK sequences To further explore the diversity of CytK, a panel of 70 cytK-2 positive strains was selected (41 strains from the lab collection and 29 strains whose genomes have been reported in the databases), in addition to the two cytK-1 strains: NVH391-98 and NVH 883-00. This selection was based on the origin, the year and the country of isolation and the species to obtain the most diversified panel of cyt-K positive strains. Indeed, the cytK-2 strains displayed different origins: 28 from food poisonings, 14 from food products, 15 from the environment, 8 from clinical cases and 5 with an unknown origin (Table 1). The 28 isolates associated with foodborne outbreaks included 23 strains from Belgian collective toxi-infection cases that occurred between 2006 and 2012 (TIAC strains), the NVH 0597-88 and 1230-88 strains from
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ACCEPTED MANUSCRIPT Norway (Granum et al., 1993), Bc1497 from Germany and two Canadian strains B16 and B23 (Table 1). The cytK-based dendrogram of the 72 B. cereus s.l. isolates (2 B. cytotoxicus (2.8%), 56
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B. cereus s.s (77.8%) and 14 B. thuringiensis (19.4%) was constructed using a 720-bp
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fragment and revealed two distinct groups corresponding to cytK-1 and cytK-2 (Fig. 1A). As expected, the cytK-1 group only contained the two strains NVH 391-98 and NVH 883-00.
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The cytK-2 group displayed two distinct clusters (referred to as I and II), which contained 21 and 49 strains, respectively. The division into two clusters was not linked to the geographical origin or to the type of sample. Similarly, no particular grouping of potentially toxic strains
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could be observed. For example, the 23 Belgian isolates involved in food poisonings (TIAC strains) were widely dispersed throughout the two clusters.
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Besides, the genetic diversity of cytK2 gene sequences was studied together with the diversity of housekeeping genes as illustrated in Table 4. The percentage of polymorphism among the 70 cytK-2 sequences was 15.1%.
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Concerning the CytK protein sequences, members of the CytK-2 group displayed less
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than 88% identity with those of the CytK1 group, while within each group, the proteins shared more than 96% identity.
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3.3. Genetic diversity of the cytK-2-positive strains In order to further characterize the genetic relationship and ecological diversity among
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the 70 cytK-2 positive strains of B. cereus, a multi-locus sequence typing (MLST) was performed using the following fragments of housekeeping genes: ccpA (311 bp), recF (468 bp), sucC (500 bp), purF (600 bp), gdpD (500 bp). Concatenation of the five genes resulted in a sequence of 2,379 bp, and the addition of the cytK-2 gene (760 bp) gave a 3,139 bp sequence. A global NJ tree was built with the concatenated sequences of the five loci. As shown in Fig. 1B, the 70 potentially diarrhoeal, cytK-2-positive strains of B. cereus s.s. were highly diverse and foodborne and clinical isolates were widely spread among the dendrogram. Moreover the strains could be divided in two phylogenetic groups that seem to correspond to phylogenic groups III and IV defined by Guinebretière and her collaborators (2008), based on the distribution of the strains shared between both studies. The only clustering of “diarrhoeal” strains relates to five TIAC isolates from Belgium (Fig. 1B, last five branches). However, these strains all had divergent cytK-2 sequences (Fig. 1A).
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The sequence variability of each locus was also studied in details. The percentage of
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polymorphism ranged from 6.4% (ccpA) to 25.8% (gdpD) and the number of allelic profiles varied between 21 for ccpA to 37 for recF (Table 4). Based on the allelic profiles of the 5 loci,
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62 Sequence Types (ST) could be defined for all the isolates. The high number of ST is another illustration of the high diversity existing among the cytk-2-positive B. cereus s.l. strains. Of note, Table S3 displays the correspondence between the allelic profiles defined in
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this work and those reported in previous MLST schemes (Helgason et al., 2004; Sorokin et al., 2006) .
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The 70 isolates were then subjected to a BURST analysis in order to group the strains according to the similarity of their allelic profile. The isolates were grouped together when three out of the five analysed loci were identical. Based on this criterion, ten clusters were
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formed, as shown in Table 1. Table S4 displays the number of Single Locus Variants (SLV),
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Double Locus Variants (DLV) and Satellites (SAT) for each assigned sequence type (ST) and the number of strains found at each ST (FREQ). Fifty-four strains were clustered in 10 groups
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while the 16 remaining strains were considered as singletons (Table 1 and Table S4). The frequency (FREQ) showed that 5 strains were assigned to ST22, 3 strains to ST2, 2 strains to ST12 and 2 to ST36 while the other strains all display some differences in their allelic profile. Strains coming from foodborne outbreaks were spread in different groups showing that there
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was no obvious association between similar allelic profiles and geographic or source origins.
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ACCEPTED MANUSCRIPT 4. Discussion Whereas the mechanisms involved in the B. cereus diarrhoeal pathogenesis are still
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largely unknown, a large panel of enterotoxins have been designated as potential causative
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agents of this syndrome. Among them, Cytotoxin K (CytK) is regularly cited as a good
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candidate because of its cytotoxic, necrotic and haemolytic activities on human intestinal cell lines (Hardy et al., 2001; Jeßberger et al., 2014). Furthermore, CytK was initially isolated from the B. cereus NVH 391-98 strain involved in a severe food poisoning outbreak that killed three people due to necrotic enteritis (Lund et al., 2000).
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In this study, 390 B. cereus strains isolated from various environments including clinical and food poisoning samples were screened in order to detect the presence of CytK genetic
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determinants. Almost half of strains contained cytK-2 while no cytK-1 strain was found. The proportion of cytK-2 positive strains varied from 35 to 55% (Table 3) but this proportion was not significantly higher for the potentially more virulent strains, namely the clinical and food
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poisoning isolates. Similar cytK occurrence was observed by Samapundo et al. (2011) among
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B. cereus strains isolated from food products (44%) and by Fagerlund et al. (2004) among foodborne or clinical B. cereus isolations (45%). However, some studies found a higher
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occurrence of cytK gene among diarrhoeal B. cereus strains (73%) by Guinebretière et al. (2002) or among B. thuringiensis strains (73%) by Swiecicka et al. (2006). Low occurrence of cytK gene was also observed by De Jonghe et al. (2010) among aerobic spore-forming
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isolates from raw milk (13%) and by Zhou et al. (2010) among B. cereus strains from ice creams (8%). Whether these more extreme values correspond to specific niches (e.g. lower in dairy products or higher in insect-related strains) or are associated with biased strain isolations remains to be determined and further investigated. The genetic diversity of cytK genes was also studied among a set of 72 strains and the constructed cytK gene-based tree did not show any clustering for the strains isolated from food poisonings. In the lab collection of 161 strains, no strain was found to contain a cytK-1 gene. It is generally recognised that the CytK-2 occurrence is higher than the one of the highly toxic CytK-1 (Fagerlund et al., 2004; Guinebretière et al., 2006). Nevertheless, Contzen and collaborators (2014) have recently isolated several strains harbouring the cytK-1 gene from various commercial potatoes products, showing that this variant could be more frequent than previously estimated. Given the high toxicity of CytK-1 on Caco-2 and Vero-cells, more 11
ACCEPTED MANUSCRIPT investigations on the occurrence of CytK-1variant in these specific products should be performed.
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4.1. Mobility of cytK
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The present study also suggests that cytK is most likely not associated to a mobile genetic element. Indeed, the MLST analysis has shown that the rate of polymorphism for
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cytK-2 was comparable to the selected housekeeping genes (Table 4) indicating that the cytK evolution fits the genetic background of the strain. Besides, the NJ tree based on six genes (five housekeeping genes and cytK-2) showed the same tendency as other published B. cereus
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s.l. phylogenetic studies (Auger et al., 2008; Barker et al., 2005; Fagerlund et al., 2007; Fricker et al., 2011; Han et al., 2006; Hu et al., 2009; Kristoffersen et al., 2011). To verify that cytK was not prone to lateral transfer, 30-kb genome fragments centred on the cytK-2
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locus (NCBI database) were also investigated. Although the genomic neighbourhood of cytK2 displayed some variability, no element potentially involved in horizontal transfer was found
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(data not shown).
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4.2. Implication of CytK in the diarrhoeal disease This study on the genetic determinants of the CytK toxin could not bring any obvious link
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between the cytK gene sequence of a strain and its virulence in the diarrhoeal pathogenesis. To assess whether CytK is or is not a significant factor in this disease, a transcriptomic study should be considered in order to take the expression of the toxin into account. Additionally,
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the potential action of CytK should be investigated in concert with other putative enterotoxins (e.g. HBL, Nhe or HlyII) and other virulence factors to evaluate the diarrhoeic potential of B. cereus strains.
To elucidate the actual involvement of these molecules in the diarrhoeal syndrome, it is necessary to find an adequate animal model. Indeed, due to their proteinaceous nature, these putative enterotoxins may be prone to a rapid inactivation in the intestinal tract, unless they would be released by the B. cereus cells in the immediate vicinity of host's intestinal epithelium being protected by the mucus layer. However this hypothesis has still to be verified. In conclusion, CytK-2 does not seem to be an essential factor in the diarrhoeal syndrome although its potential synergistic action with other virulence factors should be assessed in order to achieve a better understanding of the diarrhoeal pathogenesis. Therefore, caution should always be taken when referring to this protein as a bona fide enterotoxin. 12
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ACCEPTED MANUSCRIPT Conflict of interest
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The authors declare no conflict of interest.
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Acknowledgements
The project was supported by grants from the Federal Public Service (FOD) Health, Food Chain Safety and Environment (project RT09/2 BACEREUS). We gratefully acknowledge Pr
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P.E. Granum (The Norwegian School of Veterinary Science, Norway), Dr Guoping Zhou (Polytechnic University, Wuhan, China), Pr Marc Heyndrickx (Institute for Agricultural and
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Fisheries Research, Belgium) and Dr Paul in't Veld (Food and Consumer Product, 5600 CD Eindhoven, The Netherlands) for providing the strains. Ir Catherine Rasse (Institute for
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Multidisciplinary Research in Quantitative Modelling and Analysis, Université catholique de Louvain, Belgium) is thanked for her statistical expertise and Dr Ruiming Han for his critical
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comments on the manuscript.
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England Journal of Medicine 336, 1142-1148.
Matter, A.M., Hoot, S.B., Anderson, P.D., Neves, S.S., Cheng, Y.-Q., 2009. Valinomycin biosynthetic gene cluster in Streptomyces: conservation, ecology and evolution. PloS
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One 4, e7194.
McIntyre, L., Bernard, K., Beniac, D., Isaac-Renton, J.L., Naseby, D.C., 2008. Identification
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of Bacillus cereus group species associated with food poisoning outbreaks in British Columbia, Canada. Applied and Environmental Microbiology 74, 7451-7453.
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Mei, X., Xu, K., Yang, L., Yuan, Z., Mahillon, J., Hu, X., 2014. The genetic diversity of cereulide biosynthesis gene cluster indicates a composite transposon Tnces in emetic
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Bacillus weihenstephanensis. BMC Microbiology 14, 149.
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Mikkola, R., Saris, N.-E.L., Grigoriev, P.A., Andersson, M.A., Salkinoja-Salonen, M.S., 1999. Ionophoretic properties and mitochondrial effects of cereulide. The emetic toxin of B. cereus. European Journal of Biochemistry 263, 112-117.
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Naranjo, M., Denayer, S., Botteldoorn, N., Delbrassinne, L., Veys, J., Waegenaere, J., Sirtaine, N., Driesen, R.B., Sipido, K.R., Mahillon, J., Dierick, K., 2011. Sudden death of a young adult associated with Bacillus cereus food poisoning. Journal of Clinical Microbiology 49, 4379-4381. Pirttijärvi, T.S.M., Andersson, M.A., Salkinoja-Salonen, M.S., 2000. Properties of Bacillus cereus and other bacilli contaminating biomaterial-based industrial processes. International Journal of Food Microbiology 60, 231-239. Samapundo, S., Heyndrickx, M., Xhaferi, R., Devlieghere, F., 2011. Incidence, diversity and toxin gene characteristics of Bacillus cereus group strains isolated from food products marketed in Belgium. International Journal Food Microbiology 150, 34-41.
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ACCEPTED MANUSCRIPT Sorokin, A., Candelon, B., Guilloux, K., Galleron, N., Wackerow-Kouzova, N., Ehrlich, S.D., Bourguet, D., Sanchis, V., 2006. Multiple-locus sequence typing analysis of Bacillus cereus and Bacillus thuringiensis reveals separate clustering and a distinct population
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structure of psychrotrophic strains. Applied and Environmental Microbiology 72, 1569-
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1578.
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Stenfors Arnesen, L.P., Fagerlund, A., Granum, P.E., 2008. From soil to gut: Bacillus cereus and its food poisoning toxins. FEMS Microbiology Reviews 32, 579-606. Swiecicka, I., Van der Auwera, G.A., Mahillon, J., 2006. Hemolytic and nonhemolytic
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enterotoxin genes are broadly distributed among Bacillus thuringiensis isolated from
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wild mammals. Microbial Ecology 52, 544-551.
Zhou, G., Zheng, D., Dou, L., Cai, Q., Yuan, Z., 2010. Occurrence of psychrotolerant Bacillus cereus group strains in ice creams. International Journal Food Microbiology 137, 143-
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ACCEPTED MANUSCRIPT Legends to Figures
Fig. 1. Relationship dendrograms. A. Dendrogram of the cytK gene sequences from 72 B.
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cereus s.l. isolates. A cytK gene-based UPGMA tree of 72 B. cereus s.l. isolates. The cytK-1
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group contains two strains while all the other cytK-2 sequences are organized into two clusters
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(I and II). B. Relationship dendrogram of the 70 B. cereus strains containing cytK-2, based on the concatenated sequences of five housekeeping loci. All these strains belong to group III or IV as defined by Guinebretière et al., 2008. In both parts, the origin of the strains is indicated within brackets: food poisoning (FP), clinical isolates (C), food (F), environment (E) and
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unknown (U).
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Fig. 1A
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Fig. 1B
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ACCEPTED MANUSCRIPT Table 1: Origin, typing and BURST-grouping data of B. cereus s.l. strains.
Bc_AH1134* Bc_ATCC10876* Bc_F65185* Bc_MHI2385 Bc_MHI1687 Bc_B16 Bc_B23 Bc_I11-1 Bt_T13001* Bc_I8-5 TIAC468 TIAC70 TIAC1120 TIAC299 Bc_95_8201* Bc_AH820* Bt_BGSC4AJ1* Bc_NVH0597-99* Bt_97-27* Bt_BGSC4BA1* Bt_ATCC35646* Bt_T01001* TIAC949 Bc_NVH1230-88
USA
Belgium USA USA France Belgium Belgium USA Belgium Belgium Germany Norway Germany
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Norway
U
USA
4
5 6
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3
Germany Germany Canada Canada China Pakistan China Belgium Belgium Belgium Belgium UK Norway Mexico Norway Yugoslavia India Israel Canada Belgium Norway
gdpD
purF
recF
sucC
6 7 8 9 12 12 13 16 17 18 50 58 59 4 19 20 21 26 27 28 29 5 22 22 22 22 22 23 24 25 30 31 35 36 36 37 39 40 51 52 53 1
3 3 3 3 3 3 3 3 3 4 13 19 19 2 5 5 5 5 5 5 5 2 5 5 5 5 5 5 5 5 5 5 8 8 8 8 8 8 14 14 14 1
3 3 3 3 13 13 13 34 34 3 3 3 3 2 2 5 5 10 10 11 16 6 6 6 6 6 6 6 6 6 27 31 9 9 9 9 19 21 20 20 36 1
3 3 25 26 3 3 21 21 30 4 3 24 25 2 2 6 6 6 6 10 2 5 5 5 5 5 5 5 26 29 3 27 9 9 9 16 16 9 18 18 31 1
3 8 3 3 3 3 26 8 8 3 3 8 8 2 5 6 10 5 14 10 5 7 5 5 5 5 5 20 32 5 5 5 12 15 15 21 12 12 24 24 24 1
3 3 3 3 3 3 3 3 3 3 3 18 3 2 2 2 2 2 8 2 2 4 4 4 4 4 4 4 4 22 4 4 7 7 7 7 7 7 13 16 16 1
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Germany
ccpA
T
China
Belgium
STb
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Bt_BMB171*
Germany
Type of samplea U F FP FP E U E FP FP F E FP FP FP F F F E C U C U F FP FP F E F FP FP FP FP C C E FP C E E E FP FP
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Norway
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Bc_15 Bc_MHI32 TIAC179 TIAC180 Bc_ATCC14579* Bc_BDRD-Cer4* Bt_Bt407* TIAC75 TIAC77 Bc_MHI1520 Bc_Rock1-15* TIAC1112 TIAC176 Bc_MHI1497 Bc_45 Bc_MHI1694 Bc_67
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Country
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Group Strain
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Germany China Belgium Belgium Belgium Belgium
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Belgium Belgium Norway Norway USA Belgium Belgium Belgium Canada
Namibia USA
Pakistan Japan U
Belgium
1 1 1 1 12 15 4 4 28 1 26 29 3 23 7 8 17 23 21 6 11 13 14 19 20 22 3 30
23 23 23 30 16 3 4 19 6 6 33 14 29 31 9 11 22 28 6 13 13 17 18 12 25 27 8 34
12 12 12 12 3 3 3 3 20 20 2 2 19 21 5 6 7 9 23 9 9 10 11 7 14 15 17 24
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Belgium
1 1 1 30 14 14 4 4 6 29 32 32 28 31 7 8 18 26 33 37 12 15 17 22 23 24 25 35
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Brazil
1 1 1 1 10 10 3 3 8 8 20 20 3 3 6 7 8 8 8 8 9 11 12 15 16 17 18 21
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2 2 2 3 46 47 10 11 34 42 60 61 14 15 32 33 38 41 43 44 45 48 49 54 55 56 57 62
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Belgium
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Singletons
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France
E C E FP U F F F FP FP FP FP FP FP F F E FP FP FP F E C E E C FP FP
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Israel
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Bt_ATCC10792* Bt_IBL200* Bt_T03a001* TIAC294 Bc_B4264* Bc_m1550* Bc_MHI1544 Bc_I38-8 TIAC59 TIAC219 TIAC73 TIAC71 TIAC126 TIAC297 Bc_NVH390-88 Bc_NVH762-00 Bc_Rock3-42 * TIAC1108 TIAC74 TIAC959 Bc_ATCC10987* Bc_E33L* BC_G9241* Bt_BGSC4CC1* Bt_BGSC4Y1* Bt_IBL4222* TIAC1107 TIAC896
Detailed allelic profiles for the five housekeeping genes (ccpA, gdpD, purF, sucC and recF) are given for the ST (Sequence Types (detailed in Table S3). The sequences of strains with an asterisk (29 strains) are from NCBI databases whereas the data of the 41 other strains are from this study. Numbers were arbitrary assigned to allele fragment for each locus. The STs were grouped by BURST analysis: fifty-four strains were divided into ten groups based on the number of differences in the allelic profiles (Table S42). The remaining 16 strains could not be grouped and were considered as singletons. Note that the genetic diversity associated with these genes, expressed as the percentage of polymorphism, is displayed in Table 4. a
: Abbreviations are as follows: C: clinical isolates, E: environmental isolates, F: food isolates,
FP: food poisoning isolates and U: strains with unknown origin. b
: ST stands for Sequence Type and corresponds to the specific allelic profile.
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ACCEPTED MANUSCRIPT Table 2. Primers used in the study.
Amplicon
Gene
Primers
Sequences (5’-3’)
purF
purF_F
YGAAGAATGTGGCGTTTTYGGA
purF_R
GAAATACTAGAGTCTGGTACAC
gdpD_F
GGTGGTTATGCAAATGCGACRAATG
gdpD_R
YGCCATAATATAAAATGGYGTCACG
recF_F
GCGATGGCGAAATCTCATAG
recF_R
CAAATCCATTGATTCTGATACATC
sucC_F
GGCGGAACAGAAATTGAAGA
sucC_R
TCACACTTCATAATGCCACCA
ccpA_F
GTTTAGGATACCGCCCAAATG
ccpA_R
TGTAACTTCTTCGCGCTTCC
L2-2
GAGTTTTGRAVGATARMGAT
R2
AGGCATCTCTTCACCTTTAT
CytK_F1
ACAGATATCGGKCAAAATGC
CytK_R1
TCCAACCCAGTTWSCAGTTC
Sorokin et al., 2006
574
Sorokin et al., 2006
560
Helgason et al., 2004
590
Helgason et al., 2004
460
Helgason et al., 2004
1,263
This study
760
This study
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cytK-2
790
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cytK-1
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ccpA
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sucC
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recF
Reference
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gdpD
size (bp)
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The nucleotide codes are as follows: Y for C or T, R for A or G, V for G or A or C, M for C
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or A, K for G or T, W for A or T, and S for G or C.
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ACCEPTED MANUSCRIPT Table 3: CytK gene occurrence among the B. cereus s.l. strains.
Origins
Database (22941 strains)
Lab collection (161 strains)
Total (390402 strains)
cytK+
cytK+ %
cytK–
cytK+
cytK+ %
cytK+ ratio
cytK+ %
Clinical isolates
12
119
483
3
1
25
120/275
440
Food poisoning
7
2
22
325
27
464
29/6871
431
Food
86
45
3345
401
5148
564
553/1030
53
Environment
8575
6354
4639
21
3
6075
6657/143
4640
Unknown
3241
1520
323
01
21
10050
1721/4963
353
Mean
13451
950
4137
7781
840
520
1790/390402
462
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cytK–
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ACCEPTED MANUSCRIPT Table 4: Genetic diversity of the five housekeeping gene loci and cytK-2.
Fragment length (bp)
No. of polymorphic sites*
No. of alleles
ccpA
311
20 (6.4)
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gdpD
500
129 (25.8)
purF
600
91 (15.2)
recF
468
92 (19.7)
sucC
500
56 (11.2)
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cytK2
760
115 (15.1)
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Locus
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*In brackets: the percentage of polymorphism (the number of polymorphic sites divided by
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the length of the fragment).
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ACCEPTED MANUSCRIPT Highlights: The occurrence of cytK is not higher in food poisoning isolates than in those from the
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environment
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The cytK positive strains displayed an important genetic diversity
Based on the MSLT analysis, no grouping of food poisoning isolates was observed
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The CytK does not seem to be a major factor in the diarrhoeal syndrome
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