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Toxins and mobile antimicrobial resistance genes in Bacillus probiotics constitute a potential risk for One Health
Journal of Hazardous Materials 382 (2020) 121266
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Toxins and mobile antimicrobial resistance genes in Bacillus probiotics constitute a potential risk for One Health Cui Yifanga,1, Wang Shaolinb,1, Ding Shuangyanga, Shen Jianzhonga,b, Kui Zhua, a b
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Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China Beijing Key Laboratory of Detection Technology for Animal-Derived Food Safety and Beijing Laboratory for Food Quality and Safety, Beijing 100193, China
G R A P H I C A L A B S T R A C T
A R T I C LE I N FO
A B S T R A C T
Editor: Xiaohong Guan
Probiotic microbes conferring health benefits to the hosts have attracted great attention. However, the safety of probiotics is not guaranteed, although the increasing widespread use of probiotics with excellent overall safety records. Here, we performed a systematic evaluation of the safety of commercial Bacillus probiotics intended for usage in humans, animals, plants, aquaculture and environment in China. Nearly half of the 65 isolated Bacillus spp. strains from these commercial probiotic products were capable of producing hazardous toxins. Infections with the representative isolates could cause sepsis, intestinal inflammation and liver injury in different mouse models. Additionally, these isolates harbor multiple antimicrobial resistance genes coupled with mobile genetic elements. Collectively, the capability for producing various toxins and harboring mobile antimicrobial resistance genes in Bacillus probiotics indicates a potential risk for One Health.
Keywords: Probiotics Bacillus Toxins Antimicrobial resistance One Health
1. Introduction Probiotics are live microorganisms believed to provide health benefits on various hosts (Hill et al., 2014). It has gained worldwide attention as an alternative approach to prevent and/or treat
gastrointestinal diseases, allergies and infections in human beings (Duc et al., 2004; Strunk et al., 2015; Czaplewski et al., 2016). Probiotics have also been widely applied to crop planting, animal husbandry, aquaculture and environmental remediation for preventing infections by alleviating antimicrobial-mediated selective pressures, improving
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Corresponding author at: Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China. E-mail address: [email protected] (K. Zhu). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.jhazmat.2019.121266 Received 4 June 2019; Received in revised form 12 September 2019; Accepted 19 September 2019 Available online 20 September 2019 0304-3894/ © 2019 Elsevier B.V. All rights reserved.
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the Harvest package (Treangen et al., 2014). The tree was visualized by the online tool iTOL (Letunic and Bork, 2016).
general conditions or obtaining desired outcomes (Hill et al., 2014). Particularly, spore-forming Bacillus spp., Gram-positive, aerobic or facultative anaerobic bacteria, have advantages because their life cycles in spores are stable to heat, stomach acid and other harsh environments (Cui et al., 2019). Therefore, spores of Bacillus spp. have been widely used as probiotic preparations for different purpose (Cutting, 2011). Although the increasing widespread use of probiotics show satisfactory overall safety records, the safety of probiotics remains the top priority (Doron and Snydman, 2015). The potential risk and safety issues of probiotics arise, because evaluation and application of probiotics involves economic issues, which might be biased while ignoring the safety (Million and Raoult, 2012; Cohen, 2018). Probiotics can lead to infections in certain patient groups around the world (Bongaerts and Severijnen, 2016). For instance, consumption of Bacillus probiotics has been occasionally reported for causing septicemia in immunocompromised patients (Oggioni et al., 1998; Bottone, 2010). Additionally, bacteria in the B. cereus group are also human opportunistic pathogens responsible for foodborne poisoning with diarrhea or emetic symptoms, various human infections such as meningitis and ocular infections, as well as intoxications resulting in fulminant liver failure (Bottone, 2010; Mahler et al., 1997). On the other hand, the presence of transferrable antimicrobial resistance genes (ARGs) will endow probiotics as a reservoir with increased ability of emergence and dissemination, during their wide and inadvertent use (Berendonk et al., 2015; Cabello et al., 2016). Most probiotic Bacillus spp. will be excreted and released to the environment after enrichment in the gut of human or livestock after transient colonization (Stenfors Arnesen et al., 2008). Improperly treated animal wastes including the bedding of plant origin have been widely used as feeds in fish ponds and manure for fertilizer, creating a melting pot for these Bacillus probiotics and thereby facilitating the horizontal gene transfer of mobile ARGs (Berendonk et al., 2015; Cabello et al., 2016). Therefore, besides toxicity, the mobile ARGs in probiotic strains is a further important safety parameter. Therefore, we hypothesized that some commercial Bacillus probiotics might constitute potential risks for One Health, due to the identification of virulence factors and ARGs. Herein, we investigated the safety of Bacillus probiotics in China, with focus on the capability of toxin production and carrying mobile ARGs.
2.2. Toxicity evaluation Four main toxin genes (ces, nhe, hbl, and cytK1) were identified, and the presence of toxins were detected by immunoassays as previously described (Wehrle et al., 2009). Cereulide (an emetic toxin produced by B. cereus frequently involved in food poisoning) was confirmed by ultrahigh performance liquid chromatography tandem mass spectrometry according to the previous study (Cui et al., 2016). Cytotoxicity of the enterotoxins and heat-stable surfactin-like toxins was performed on Vero cells, while cytotoxicity of cereulide was tested on HEp-2 cells. B. cereus NVH0075/95, ATCC14579, NVH391/98 and F4810/72 were used as reference strains for non-hemolytic enterotoxin (Nhe), hemolysin BL (Hbl), cytotoxin K1 (CytK1) and cereulide, respectively (Andersson et al., 1998; Lund and Granum, 1996; Ivanova et al., 2003; Guinebretiere et al., 2013). 2.3. In vivo assessment This animal study was approved by the Beijing Association for Science and Technology (approval ID: SYXK [Beijing] 2016-0008). The entire operation followed the guidelines for the Beijing Laboratory Animal Welfare and Ethics, issued by the Beijing Administration Committee of Laboratory Animals, and the China Agricultural University Institutional Animal Care and Use Committee guidelines (ID: SKLAB-B-2010-003). Eight-week old specific pathogen-free ICR mice weighing 28–38 g (half female and half male) from the Vital River Laboratory (Beijing, China), were acclimated for 3 days prior to use. 2.3.1. Mouse sepsis model Three groups of mice (n = 6 per group, half male and half female) were injected intraperitoneally (i.p.) with 200 mg/kg cyclophosphamide (Sigma-Aldrich, St. Louis, MO, USA) 24 h prior to infection. Mice were infected with 0.3 mL of B. cereus 4 suspension (1 × 108 CFU per mouse) via intragastric administration (i.g.) (Liu et al., 2017). The placebo group were treated with 0.3 mL 0.9% NaCl solution. At one hour post-infection, one group was treated with vancomycin at single intraperitoneal doses of 10 mg/kg. Survival was observed in placebo and therapy groups for and analyzed by nonparametric logrank test (Mantel, 1966). Once the infected mice died, various organs including heart, liver, spleen, lung and kidney were removed and homogenized in sterile 0.9% NaCl solution. Surviving mice were sacrificed at 48 h postinfection, and organs were harvested. Serial dilutions of each suspension were plated on the selective Brilliance™ Bacillus cereus agar plate (Oxoid, Wesel, Germany) for enumerating bacterial colonies. The residual organs were excised and fixed with 4% paraformaldehyde solution (Sigma-Aldrich, St. Louis, Missouri, USA). Histopathological changes were stained with Hematoxylin-Eosin (H&E) then visualized through a microscope (Prophet et al., 1992).
2. Materials and methods 2.1. Sampling, isolation and taxonomy of Bacillus probiotic isolates To investigate the risk of Bacillus probiotics used in China, we collected 34 commercial probiotic products with Bacillus spp. as the main ingredient between April to July 2016 from 12 provinces in China. These products were intended for usage in humans (n = 9), animals (n = 15), plants (n = 6), aquaculture and environment (n = 4), respectively. The probiotics targeting for aquaculture and environment were mainly used for aquatic breeding and water purification by consuming excess nutrients (more details about the commercial probiotic products can be found in supplementary materials Table S1). All the Bacillus spp. strains were isolated according to the previous study, and whole genome sequencing analysis was conducted for bacterial identification (Zhu et al., 2016). Genomic DNA of all Bacillus probiotic isolates were extracted using the Wizard Genomic DNA Purification Kit (Promega, Madison, USA). Then indexed Illumina sequencing DNA libraries were constructed using KAPA Hyper Prep Kits (Kapa Biosystems, Wilmington, Massachusetts, USA) with standard protocols, and 150 base pair pairedend reads were sequenced on the Illumina HiSeq 2500 platform (Bionova, Beijing, China). The draft assemblies of the sequences were obtained using CLC Genomics Workbench 9.5.3 (CLC Bio, Aarhus, Denmark). Assembled draft genomes were aligned using muscle program, and a core-genome phylogenetic tree was generated by Parsnp in
2.3.2. Intestinal inflammation model All the mice were treated through i.g. (n = 6). Compared to the placebo group treated with 0.2 mL 0.9% NaCl solution, the group of infected mice was treated with 0.2 mL bacterial solution of B. cereus 4 (1 × 108 CFU) per mouse. At 48 h post-infection, mice were euthanized by cervical dislocation and small intestines were removed and fixed with 4% paraformaldehyde solution. Histopathological changes were analyzed by H&E stain. 2.3.3. Liver injury model In two groups (n = 6 for each group), all mice were treated through i.g. Mice in the placebo group were treated with 0.2 mL 0.9% NaCl solution. Mice for liver injury model, were infected with 0.2 mL bacterial solution of B. cereus 28-2 (1 × 108 CFU per mouse). At 48 h post2
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3.2. Toxicity evaluation
infection, all mice were euthanized by cervical dislocation. Blood of each mouse was collected for detecting serum indices based on the manufacturer’s protocols. The activity of alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) were tested with kits from Biovision (Milpitas, California, USA). The kit for alanine aminotransferase (ALT) was purchased from Cayman Chemical (Ann Arbor, Michigan, USA), and the kits for aspartate transaminase (AST) and creatinine (Cre) were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Livers were removed and fixed with 4% paraformaldehyde solution. Histopathological changes were analyzed by H&E stain.
To assess the toxin-producing potential of the Bacillus probiotics, we found that 60% of the isolates (39/65, of which 36 isolates belonging to the B. cereus group) showed hemolytic activity (Fig. 1C), which may attribute to bacterial virulence factors. To further characterize the hemolysis in these isolates, it is necessary to assess their abilities of virulence factor production. Isolates in the B. cereus group especially B. cereus and B. thuringiensis are known for the ability to produce mainly four toxins, including three enterotoxins (Hbl, Nhe and CytK1) and one peptide toxin (cereulide) (Bottone, 2010). First, almost all the B. cereus and B. thuringiensis isolates were able to produce biologically active Nhe (51%, 33/65), and most of them could also produce Hbl (40%, 26/65), except that one isolate (B. cereus 31-3) harboring the nhe gene without expression, as determined by PCR and immunoassays (Fig. 1D). Additionally, no isolate contained the cytK1 gene. Cytotoxic tests were subsequently performed to confirm the expression of intact tripartite complexes of Nhe and Hbl (Wehrle et al., 2009). Accordingly, 11 isolates (17%) were highly cytotoxic with IC50 less than 10 μg/mL (Fig. 1D; Table S2 and Fig. S1D). In such isolates, the expression levels of Nhe were similar to the reference strain NVH0075/95, which was isolated from a food poisoning case in Norway (Lund and Granum (1996)). In addition, various and cytotoxic surfactin-like toxins were found (Fig. 1E and Table S2). Therefore, it is very likely that these toxin-producing probiotics may possess a potential threat to the public health and cause or aggravate diarrhea symptoms in humans, particularly if consumed by infants or immunocompromised patients (Duc et al., 2004; Bongaerts and Severijnen, 2016). To test this notion, we challenged immunosuppressed mice with B. cereus 4 (an isolate for treating human diarrhea) to mimic immunodeficient patients or children. Infected mice without treatment all died, while infected mice treated with vancomycin all survived within 24 h (Fig. 1F). Treated mice with vancomycin had decreased bacterial counts (Fig. 1G) and alleviated pathologic changes in different organs (Fig. 1H and Fig. S2A). Lastly, we infected mice with B. cereus 4 through i.g. to simulate oral administration. Inflammatory edema on submucosa and increased numbers of goblet cells in the intestine were found (Fig. S2B). These results indicate that Bacillus probiotic isolates can produce multiple functional virulence factors. In addition, the p-value of toxicity among different targeted probiotics is 0.241, and the 95% confidence intervals are 0.233-0.249. There is no difference among these probiotics based on the sample sizes in the current study. It is noteworthy that the presence of toxin gene ces, encoding the synthesis of cereulide synthetase, was found in B. cereus 28-2 (an isolate for promoting growth in plants). The ces gene cluster located on a plasmid (Fig. 2A) (Stenfors Arnesen et al., 2008). Expression of cereulide was confirmed based on mass spectrometry analysis (Fig. 2B). Higher cytotoxicity than the reference cereulide producing strain (B. cereus F4810/72) was observed (Fig. 2C) (Cui et al., 2016). The genetic environment of cereulide synthase gene cluster in B. cereus 28-2 is consistent with the sequences in other pXO1-like plasmids (Fig. 2D and Fig. S3). Additionally, we infected mice with B. cereus 28-2 through oral administration. Apparent lesions such as vacuolization and granular degeneration in hepatocytes and local necrosis were observed in livers (Fig. 2E). Furthermore, increased serum indices were found in infected mice as well (Fig. S2C).
2.4. Antimicrobial susceptibility tests Minimum inhibitory concentrations (MICs) of Bacillus probiotic isolates were determined by classical micro-broth dilution method according to the Clinical and Laboratory Standards Institute (CLSI)’s operating directions (CLSI M100S-S26) (Clinical and Laboratory Standards Institute (CLSI), 2016). Staphylococcus aureus ATCC29213 was chosen as a standard control for antimicrobial susceptibility tests, because no data for Bacillus spp. is available according to the CLSI standards and guidelines.
2.5. Bioinformatics analysis The draft genomic sequences were searched against the ResFinder database (http://www.genomicepidemiology.org/) using the NCBI BLAST software (minimum 70% identity) (Zankari et al., 2012). The contigs carrying ARGs were extracted, counted and annotated by PARTIC annotation service (https://www.patricbrc.org/app/ Annotation) (Wattam et al., 2014). The reported mobile elements which commonly located adjacent to the detected ARGs were collected from the NCBI database (Table S3), subsequently searched against the draft genomes. The genomic sequences of a group of Bacillu strains used or potentially used in other countries were retrieved from NCBI database, and searched against the van genes reference sequences. More details can be found in Table S4.
2.6. Statistical analysis The statistical analysis in this study is performed by SPSS 23.0 (IBM, Armonk New York, USA). We performed a bivariate analysis by using Pearson’s correlation to analyze the correlation between different application targets and toxicity or antimicrobial resistance of all the isolates. We considered a 95% confidence intervals and significance level of 5% for all analyses.
3. Results 3.1. Characterization of Bacillus probiotic isolates In this study, a total of 65 isolates of Bacillus spp. were isolated from 34 products labeled as Bacillus spp. containing from 12 provinces in China (Fig. 1B). Based on colony morphology and whole genome sequencing, 37 isolates were identified to belong to the B. cereus group including B. cereus and B. thuringiensis (Fig. S1A); 22 of these isolates belonged to the B. subtilis group including B. subtilis, B. amyloliquefaciens, B. licheniformis and B. paralicheniformis (Fig. S1B). Additionally, six isolates were other Bacillus spp. including two B. oceanisediminis isolates and one each of B. flexus, B. infantis, B. meqaterium and B. safensis (Fig. S1C). All genomic assemblies of 65 isolates have been deposited in GenBank and are registered under BioProject accession number PRJNA390877, and BioSample numbers SAMN07267320SAMN07267384.
3.3. Antimicrobial resistance To understand the profiles of antimicrobial resistance in these Bacillus probiotics, all isolates were tested with 12 kinds of routinely used antimicrobials, with the resistance rates ranged from 2% to 98% (Fig. 3A and Fig. S4). The p-value of antimicrobial resistance among different targeted probiotics is 0.857, with 95% confidence intervals of 0.850-0.863. There is no difference among these probiotics based on the sample sizes in the current study. The predominant ARGs in the 3
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Fig. 1. Safety assessment of commercial Bacillus probiotics. (A) Assessment approach of toxicity and antimicrobial resistance in probiotics. (B) 65 isolates from commercial Bacillus probiotics in 12 provinces in China. Numbers in the parentheses indicate numbers of the Bacillus isolates in each province. (C) Hemolysis of Bacillus probiotic isolates on sheep blood agar plates. (D) Various enterotoxins were found in these isolates. The presence of non-hemolytic enterotoxin (Nhe), hemolysin BL (Hbl) and cytotoxin K1 (CytK1) was verified at genetic, protein and cytotoxic levels, respectively. (E) Cytotoxicity of surfactin-like toxins from Bacillus probiotics were tested. Cytotoxicity at high, medium and low levels represent IC50 < 0.5 ng/mL, 0.5 ng/ mL < IC50 ≤ 1 ng/mL, and IC50 ≥ 1 ng/mL, respectively. (F–H) Sepsis model by the isolate of B. cereus 4. Three groups of mice (n = 6 per group, half male and half female) were injected intraperitoneally with cyclophosphamide (200 mg/kg) 24 h prior to infection. Compared to the placebo group, mice were infected with 0.3 mL of bacterial suspension (1 × 108 CFU bacteria per mouse) through intraperitoneal injection. At one hour post-infection, one group was treated with vancomycin at single intraperitoneal doses of 10 mg/kg. (F) Survival curves were obtained at 48 h after infection. (G) The numbers of B. cereus in different organs were enumerated after infection. (H) Typical pathological changes in livers by Hematoxin-Eosin stain (scale bar, 50 μm). The pathological sections of other organs could be found in supplementary materials Fig. S2A.
surrounded by various insertion sequences, integrases or phage proteins. Alarmingly, more than half isolates carried such mobile ARGs determinants (Fig. 3C). Furthermore, we found incomplete vancomycin resistance genes in all isolates of the B. cereus group and the isolate of B. oceanisediminis 34-1 as well (Fig. S5A). To characterize the van genes in these Bacillus isolates, the complete van gene clusters were analyzed. The van gene clusters in Bacillus isolates were categorized into four patterns (Fig. 3D). However, the absence of vanH, vanA and vanX genes was observed for all isolates. This is consistent with the fact that these
genomes of 65 isolates were cat, cfr, lsa, tet and van (Fig. 3A), in response to the resistance of choloramphenicols, choloramphenicols / lincosamides / oxazolidinones / pleuromutilins, lincosamides / pleuromutilins, tetracyclines and vancomycin, respectively. And the ARG profiles were species specific (Fig. S4). For instance, the presence of cfr gene was found in all isolates of the B. subtilis group. Nevertheless, the lsa and incomplete van genes were found in the B. cereus group. Representative flanking genetic contexts of five mobile ARGs in these isolates were shown in Fig. 3B. For example, the cat genes are
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Fig. 2. Plasmid-borne toxin cereulide in B. cereus probiotic isolate 28-2. (A) The presence of plasmid-borne ces gene. Lane 1 and 2, the extracts of plasmid (1) and genome (2) in B. cereus 28-2; Lane 3 and 4, negative controls of water (3) and B. cereus ATCC14579 (4); Lane 5 and 6, the ces genes carried by B. cereus F4810/72 (5) and B. cereus CAU45 (Cui et al., 2016) (6); M, size marker. (B) Mass spectrum of cereulide produced in B. cereus 28-2. The inset is the scheme of cereulide. (C) Cytotoxicity of cereulide secreted by B. cereus 28-2. B. cereus 28-2 produces more cereulide than the reference cereulide producing strain B. cereus F4810/72. (D) Genetic environment of the ces gene cluster in B. cereus 28-2. This sequence is identical to the ces gene clusters on plasmids of pCER270 (GenBank accession number: DQ889676.1) and pNCcld (AP007210.1), but shows high similarity (99.4%) to the reverse sequence in plasmid pH308197_258 (CP001166.1). Completed annotation can be found in supplementary materials Fig. S3. (E) Cereulide caused hepatic damages in mice by Hematoxin-Eosin stain (scale bar, 50 μm).
species of probiotic candidates accurately. Then assess the toxigenic potential on genetic, protein and cellular levels. Collectively, probiotics with the capability of virulence factor production are discouraged to use, including the identified toxins and unknown virulence factors (FAO/WHO, 2002; MEP, 2008; EFSA, 2014). It is noteworthy that the expression of cereulide was carried by one isolate (B. cereus 28-2). Cereulide is a notorious pathogenic agent responsible for fulminant liver failure in children and food poisoning with emetic symptom (Mahler et al., 1997; Stenfors Arnesen et al., 2008). The presence of plasmid-borne cereulide will endow this isolate as a reservoir with increased ability of dissemination, during its wide and inadvertent use. Taken together, such probiotic products harboring a toxigenic potential to produce multiple functional virulence factors are not recommended for use. In this study, almost all of the isolates (95%, 62/65) were resistant to lincomycin, polymyxin B or tiamulin and half of them (52%, 34/65) were resistant to ampicillin (Fig. 3A). These were mainly due to the intrinsic characteristics of Bacillus spp. (Bottone, 2010). Nevertheless, the mechanisms of lincosamides and pleuromutilins resistance in Bacillus spp. remains unclear. Additionally, more than half isolates carry the mobile ARGs. Unlike the intrinsic ARGs, the isolates with mobile ARGs have the potential to disseminate such genes to pathogenic or commensal bacteria under antimicrobial selective pressures (Berendonk et al., 2015). These mobile ARGs in probiotics would enable an accumulation or dissemination of antimicrobial resistance under a selective stress as well. For instance, due to the presence of a mobile tetracycline resistance tet(B) gene, along with insufficient data on toxicity assessment, the probiotic B. cereus strain of Esporafeed Plus® has been withdrawn in Europe (SCAN, 1999). Thus, the presence of various mobile ARGs in Bacillus probiotics indicates a potential threat to One Health. Furthermore, we found vancomycin resistance van genes in the absence of vanH, vanA and vanX genes in 38 isolates. Although none of the isolate carries the complete set of seven van genes, it still has the potential to acquire extra vanH, vanA and vanX genes through
isolates are vancomycin sensitive, because all seven genes are required for vancomycin resistance (Pootoolal et al., 2002). Additionally, the identities of van genes in the isolates used for human were significantly higher than that for animal, plant, aquaculture and environment (Fig. S5B). 4. Discussion The safety assessment of Bacillus probiotics is insufficient and should attract more attention. Because various toxins and mobile ARGs were recently reported after the long-term application of Bacillus spp. for different purposes (Cui et al., 2019). Historically, commercial Bacillus probiotic products, such as Paciflor® and Esporafeed Plus® have been withdrawn in Europe (SCAN, 1999; SCAN, 2001). In our results, more than half of the isolates showed hemolytic activity, almost half of the isolates could produce enterotoxins and various cytotoxic surfactin-like toxins. Additionally, in vivo tests confirmed that probiotic B. cereus isolates have the potential to cause sepsis (as shown in Fig. 1F to H and Fig. S2A), intestinal inflammation (Fig. S2B) and liver damage (Fig. 2E and Fig. S2C). Meanwhile, L. rhamnosus, another commonly used probiotic species, is associated with bloodstream infection in immunocompromised or defective people (Gouriet et al., 2012). Moreover, many food-poisoning cases have reported that the enterotoxins and emetic toxin produced by B. cereus sensu stricto can cause diarrhea and acute liver failure (Mahler et al., 1997; Lund and Granum, 1996), which are confirmed by our in vivo tests. In addition, comparing to the pathogenic B. cereus, such as B. cereus NVH0075/95 causing food poisoning outbreaks (Lund and Granum, 1996), many Bacillus isolates in the present study can produce higher level of toxins (Fig. 1 and Table S2). These virulent probiotic isolates and their toxins may contaminate water and foods through the food chain, and eventually ingested by consumers (Stenfors Arnesen et al., 2008). Additionally, probiotics are not all alike. The exhibition of toxicity is limited to specific strains, not every isolate of Bacillus spp. harbor all virulence genes (Cui et al., 2019). It is necessary to firstly identify strain 5
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Fig. 3. Phenotypical and genetic profiles of antimicrobial resistance in Bacillus probiotic isolates. (A) The proportion of Bacillus isolates showed the phenotypes of conferring resistance to 12 antimicrobial drugs, and the proportion of isolates carrying the corresponding antimicrobial resistance genes (ARGs). The detailed proportions data of phenotypes and genes in the different Bacillus probiotic species were showed in supplementary materials Fig. S4. (B) Schematic representation of five isolates carrying both ARGs and mobile genetic elements (MGEs). (C) Distribution of MGEs (including cat, cfr, lsa, tet and van genes) in these Bacillus isolates. (D) Four patterns of the van gene clusters in Bacillus probiotic isolates. The reference van A-type gene clusters include (EMBL accession number Y15704 (vanA), Y15705 (vanH), Y15706 (vanR), Y15707 (vanS), Y15708 (vanX) and Y17303 (vanY)) of B. circulans VR0709. (E) Comparison of the identity of van genes in reported Bacillus probiotic strains and the isolates in this study. The identities of van genes in this study were shown as average. More details about the Bacillus probiotic strains can be found in supplementary materials Table S4.
boost of pharmaceutical and biotechnological companies contributes to fast expansion of Bacillus probiotics, whereas the quality of some products barely meets the safety requirement. More importantly, the safety issues of probiotics and its potential risk to One Health is not limited to China, it becomes a global issue which attracts more attention. In conclusion, great caution should be taken when probiotics are used for people in certain groups particularly for future use in agriculture and environment, since large amounts of products consumed on a regular basis will accordingly increase probiotic related infections.
conjugation, recombination or other mechanisms and confer resistance to vancomycin (Ligozzi et al., 1998). Moreover, compared to the Bacillus probiotic strains from other countries, such isolates from China contain more genetic elements and share high identities of van genes (Fig. 3E and Fig. S5A). High identities of van genes in our isolates, especially the isolates for human suggest that the Bacillus isolates used for human possess high ability to acquire other van genes to form an entire van gene cluster (Sanders et al., 2010). Consistent with this notion, a probiotic Lactobacillus strain has been reported to acquire vancomycin resistance in vivo (Mater et al., 2008). Here we summarize the possible health hazards of Bacillus containing commercial probiotic products in China. The verification of the absence of toxigenic potential and transferrable antimicrobial resistance gene is a prerequisite for approval of a probiotic strain. The
Declaration of Competing Interest All authors read and approved the manuscript. The authors declared no competing financial interests. 6
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Acknowledgments
Gastroenterol. Hepatol. 11, 506–514. Ivanova, N., Sorokin, A., Anderson, I., Galleron, N., Candelon, B., Kapatral, V., Bhattacharyya, A., Reznik, G., Mikhailova, N., Lapidus, A., Chu, L., Mazur, M., Goltsman, E., Larsen, N., D’Souza, M., Walunas, T., Grechkin, Y., Pusch, G., Haselkorn, R., Fonstein, M., Ehrlich, S.D., Overbeek, R., Kyrpides, N., 2003. Genome sequence of Bacillus cereus and comparative analysis with Bacillus anthracis. Nature 423, 87–91. Letunic, I., Bork, P., 2016. Interactive tree of life (iTOL) v3: an online tool for the display and annotation of phylogenetic and other trees. Nucleic Acids Res. 44, W242–245. Ligozzi, M., Lo Cascio, G., Fontana, R., 1998. vanA gene cluster in a vancomycin-resistant clinical isolate of Bacillus circulans. Antimicrob. Agents Chemother. 42, 2055–2059. Liu, Y., Ding, S., Dietrich, R., Martlbauer, E., Zhu, K., 2017. A biosurfactant-inspired heptapeptide with improved specificity to kill MRSA. Angew. Chem. Int. Ed. Engl. 56, 1486–1490. Lund, T., Granum, P.E., 1996. Characterisation of a non-haemolytic enterotoxin complex from Bacillus cereus isolated after a foodborne outbreak. FEMS Microbiol. Lett 141, 151–156. Ministry of Environmental Protection of the People’s Republic of China (MEP), 2008. Guide of safety-assessment on application of microbial blends in the environmental protection, Industry standard of the environmental protection of the People’s Republic of China HJ/T 415. MEP. http://kjs.mep.gov.cn/hjbhbz/bzwb/stzl/ 200801/W020111121498913885910.pdf. Mahler, H., Pasi, A., Kramer, J.M., Schulte, P., Scoging, A.C., Bär, W., Krähenbühl, S., 1997. Fulminant liver failure in association with the emetic toxin of Bacillus cereus. New Engl. J. Med. 336, 1142–1148. Mantel, N., 1966. Evaluation of survival data and two new rank order statistics arising in its consideration. Cancer Chemother. Rep. 50, 163–170. Mater, D.D.G., Langella, P., Corthier, G., Flores, M.J., 2008. A probiotic Lactobacillus strain can acquire vancomycin resistance during digestive transit in mice. J. Mol. Microbiol. Biotechnol. 14, 123–127. Million, M., Raoult, D., 2012. Publication biases in probiotics. Eur. J. Epidemiol. 27, 885–886. Oggioni, M.R., Pozzi, G., Valensin, P.E., Galieni, P., Bigazzi, C., 1998. Recurrent septicemia in an immunocompromised patient due to probiotic strains of Bacillus subtilis. J. Clin. Microbiol. 36, 325–326. Pootoolal, J., Neu, J., Wright, G.D., 2002. Glycopeptide antibiotic resistance. Annu. Rev. Pharmacol. Toxicol. 42, 381–408. Prophet, E.B., Mills, B., Arrington, J.B., Sobin, L.H., 1992. Laboratory Methods in Histotechnology. American Registry of Pathology, Washington D.C. Scientific Committee on Animal Nutrition (SCAN), 1999. Assessment by the Scientific Committee on Animal Nutrition (SCAN) of a Micro-organisms Product: Esporafeed Plus®, Directorate B-Scientific Health Opinions. December 3. European Commission Health & Consumer Protection Director-General. Scientific Committee on Animal Nutrition (SCAN), 2001. Assessment by the Scientific Committee on Animal Nutrition (SCAN) of the Safety of Product Paciflor® for Use As Feed Additive, Directorate C-Scientific Opinions. May 16. European Commission Health & Consumer Protection Director-General. Sanders, M.E., Akkermans, L.M., Haller, D., Hammerman, C., Heimbach, J., Hormannsperger, G., Huys, G., Levy, D.D., Lutgendorff, F., Mack, D., Phothirath, P., Solano-Aguilar, G., Vaughan, E., 2010. Safety assessment of probiotics for human use. Gut Microbes 1, 164–185. Stenfors Arnesen, L.P., Fagerlund, A., Granum, P.E., 2008. From soil to gut: Bacillus cereus and its food poisoning toxins. FEMS Microbiol. Rev. 32, 579–606. Strunk, T., Kollmann, T., Patole, S., 2015. Probiotics to prevent early-life infection. Lancet Infect. Dis. 15, 378–379. Treangen, T.J., Ondov, B.D., Koren, S., Phillippy, A.M., 2014. The Harvest suite for rapid core-genome alignment and visualization of thousands of intraspecific microbial genomes. Genome Biol. 15, 524. Wattam, A.R., Abraham, D., Dalay, O., Disz, T.L., Driscoll, T., Gabbard, J.L., Gillespie, J.J., Gough, R., Hix, D., Kenyon, R., Machi, D., Mao, C., Nordberg, E.K., Olson, R., Overbeek, R., Pusch, G.D., Shukla, M., Schulman, J., Stevens, R.L., Sullivan, D.E., Vonstein, V., Warren, A., Will, R., Wilson, M.J., Yoo, H.S., Zhang, C., Zhang, Y., Sobral, B.W., 2014. PATRIC, the bacterial bioinformatics database and analysis resource. Nucleic Acids Res. 42, D581–591. Wehrle, E., Moravek, M., Dietrich, R., Burk, C., Didier, A., Martlbauer, E., 2009. Comparison of multiplex PCR, enzyme immunoassay and cell culture methods for the detection of enterotoxinogenic Bacillus cereus. J. Microbiol. Meth. 78, 265–270. Zankari, E., Hasman, H., Cosentino, S., Vestergaard, M., Rasmussen, S., Lund, O., Aarestrup, F.M., Larsen, M.V., 2012. Identification of acquired antimicrobial resistance genes. J. Antimicrob. Chemother. 67, 2640–2644. Zhu, K., Holzel, C.S., Cui, Y., Mayer, R., Wang, Y., Dietrich, R., Didier, A., Bassitta, R., Martlbauer, E., Ding, S., 2016. Probiotic Bacillus cereus strains, a potential risk for public health in China. Front. Microbiol. 7, 718.
We thank Prof. Erwin Märtlbauer and Dr. Richard Dietrich from Ludwig-Maximilians-University Munich (LMU, Germany) for kindly providing the reference strains and monoclonal antibodies. This study is supported by the National Key Research and Development Program of China (2017YFC1600305) and the Fund of Beijing Innovation Team of Dairy Industry (BAIC06-2017). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.jhazmat.2019.121266. References Andersson, M.A., Mikkola, R., Helin, J., Andersson, M.C., Salkinoja-Salonen, M., 1998. A novel sensitive bioassay for detection of Bacillus cereus emetic toxin and related depsipeptide ionophores. Appl. Environ. Microbiol. 64, 1338–1343. Berendonk, T.U., Manaia, C.M., Merlin, C., Fatta-Kassinos, D., Cytryn, E., Walsh, F., Burgmann, H., Sorum, H., Norstrom, M., Pons, M.N., Kreuzinger, N., Huovinen, P., Stefani, S., Schwartz, T., Kisand, V., Baquero, F., Martinez, J.L., 2015. Tackling antibiotic resistance: the environmental framework. Nat. Rev. Microbiol. 13, 310–317. Bongaerts, G.P.A., Severijnen, R.S.V.M., 2016. A reassessment of the PROPATRIA study and its implications for probiotic therapy. Nat. Biotechnol. 34, 55–63. Bottone, E.J., 2010. Bacillus cereus, a volatile human pathogen. Clin. Microbiol. Rev. 23, 382–398. Cabello, F.C., Godfrey, H.P., Buschmann, A.H., Dolz, H.J., 2016. Aquaculture as yet another environmental gateway to the development and globalisation of antimicrobial resistance. Lancet Infect. Dis. 16, e127–133. Clinical and Laboratory Standards Institute (CLSI), 2016. Clinical and Laboratory Standards Institute (CLSI) Performance Standards for Antimicrobial Susceptibility Testing, CLSI Supplement M100S, 26th edition. (ISBN 1-56238-923-8 [Print]; ISBN 1-56238-924-6 [Electronic]). Cohen, P.A., 2018. Probiotic safety-no guarantees. JAMA Intern. Med. 178, 1577–1578. Cui, Y., Liu, Y., Liu, X., Xia, X., Ding, S., Zhu, K., 2016. Evaluation of the toxicity and toxicokinetics of cereulide from an emetic Bacillus cereus strain of milk origin. Toxins (Basel) 8. Cui, Y., Martlbauer, E., Dietrich, R., Luo, H., Ding, S., Zhu, K., 2019. Multifaceted toxin profile, an approach toward a better understanding of probiotic Bacillus cereus. Crit. Rev. Toxicol. 1–15. Cutting, S.M., 2011. Bacillus probiotics. Food Microbiol. 28, 214–220. Czaplewski, L., Bax, R., Clokie, M., Dawson, M., Fairhead, H., Fischetti, V.A., Foster, S., Gilmore, B.F., Hancock, R.E., Harper, D., Henderson, I.R., Hilpert, K., Jones, B.V., Kadioglu, A., Knowles, D., Olafsdottir, S., Payne, D., Projan, S., Shaunak, S., Silverman, J., Thomas, C.M., Trust, T.J., Warn, P., Rex, J.H., 2016. Alternatives to antibiotics-a pipeline portfolio review. Lancet Infect. Dis. 16, 239–251. Doron, S., Snydman, D.R., 2015. Risk and safety of probiotics. Clin. Infect. Dis. 60 (Suppl 2), S129–134. Duc, L.H., Hong, H.A., Barbosa, T.M., Henriques, A.O., Cutting, S.M., 2004. Characterization of Bacillus probiotics available for human use. Appl. Environ. Microbiol. 70, 2161–2171. European Food Safety Authority Panel on Additives and Products or Substances used in Animal Feed (EFSA), 2014. Guidance on the assessment of the toxigenic potential of Bacillus species used in animal nutrition. EFSA J. 12, 3665. Food and Agriculture Organization of the United Nations/World Health Organization (FAO/WHO), 2002. Joint FAO/WHO working group report on drafting guidelines for the evaluation of probiotics in food. FAO/WHO, London, Ontario, Canada. http:// who.int/foodsafety/fs_management/en/probiotic_guidelines.pdf. Gouriet, F., Million, M., Henri, M., Fournier, P.-E., Raoult, D., 2012. Lactobacillus rhamnosus bacteremia: an emerging clinical entity. Eur. J. Clin. Microbiol. 31, 2469–2480. Guinebretiere, 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., Sorokin, A., 2013. Bacillus cytotoxicussp. nov. is a novel thermotolerant species of theBacillus cereus Group occasionally associated with food poisoning. Int. J. Syst. Evol. Microbiol. 63, 31–40. Hill, C., Guarner, F., Reid, G., Gibson, G.R., Merenstein, D.J., Pot, B., Morelli, L., Canani, R.B., Flint, H.J., Salminen, S., Calder, P.C., Sanders, M.E., 2014. Expert consensus document. The International Scientific Association for Probiotics and Prebiotics consensus statement on the scope and appropriate use of the term probiotic. Nat. Rev.
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Journal of Hazardous Materials journal homepage: www.elsevier.com/locate/jhazmat
Toxins and mobile antimicrobial resistance genes in Bacillus probiotics constitute a potential risk for One Health Cui Yifanga,1, Wang Shaolinb,1, Ding Shuangyanga, Shen Jianzhonga,b, Kui Zhua, a b
T
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Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China Beijing Key Laboratory of Detection Technology for Animal-Derived Food Safety and Beijing Laboratory for Food Quality and Safety, Beijing 100193, China
G R A P H I C A L A B S T R A C T
A R T I C LE I N FO
A B S T R A C T
Editor: Xiaohong Guan
Probiotic microbes conferring health benefits to the hosts have attracted great attention. However, the safety of probiotics is not guaranteed, although the increasing widespread use of probiotics with excellent overall safety records. Here, we performed a systematic evaluation of the safety of commercial Bacillus probiotics intended for usage in humans, animals, plants, aquaculture and environment in China. Nearly half of the 65 isolated Bacillus spp. strains from these commercial probiotic products were capable of producing hazardous toxins. Infections with the representative isolates could cause sepsis, intestinal inflammation and liver injury in different mouse models. Additionally, these isolates harbor multiple antimicrobial resistance genes coupled with mobile genetic elements. Collectively, the capability for producing various toxins and harboring mobile antimicrobial resistance genes in Bacillus probiotics indicates a potential risk for One Health.
Keywords: Probiotics Bacillus Toxins Antimicrobial resistance One Health
1. Introduction Probiotics are live microorganisms believed to provide health benefits on various hosts (Hill et al., 2014). It has gained worldwide attention as an alternative approach to prevent and/or treat
gastrointestinal diseases, allergies and infections in human beings (Duc et al., 2004; Strunk et al., 2015; Czaplewski et al., 2016). Probiotics have also been widely applied to crop planting, animal husbandry, aquaculture and environmental remediation for preventing infections by alleviating antimicrobial-mediated selective pressures, improving
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Corresponding author at: Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China. E-mail address: [email protected] (K. Zhu). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.jhazmat.2019.121266 Received 4 June 2019; Received in revised form 12 September 2019; Accepted 19 September 2019 Available online 20 September 2019 0304-3894/ © 2019 Elsevier B.V. All rights reserved.
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the Harvest package (Treangen et al., 2014). The tree was visualized by the online tool iTOL (Letunic and Bork, 2016).
general conditions or obtaining desired outcomes (Hill et al., 2014). Particularly, spore-forming Bacillus spp., Gram-positive, aerobic or facultative anaerobic bacteria, have advantages because their life cycles in spores are stable to heat, stomach acid and other harsh environments (Cui et al., 2019). Therefore, spores of Bacillus spp. have been widely used as probiotic preparations for different purpose (Cutting, 2011). Although the increasing widespread use of probiotics show satisfactory overall safety records, the safety of probiotics remains the top priority (Doron and Snydman, 2015). The potential risk and safety issues of probiotics arise, because evaluation and application of probiotics involves economic issues, which might be biased while ignoring the safety (Million and Raoult, 2012; Cohen, 2018). Probiotics can lead to infections in certain patient groups around the world (Bongaerts and Severijnen, 2016). For instance, consumption of Bacillus probiotics has been occasionally reported for causing septicemia in immunocompromised patients (Oggioni et al., 1998; Bottone, 2010). Additionally, bacteria in the B. cereus group are also human opportunistic pathogens responsible for foodborne poisoning with diarrhea or emetic symptoms, various human infections such as meningitis and ocular infections, as well as intoxications resulting in fulminant liver failure (Bottone, 2010; Mahler et al., 1997). On the other hand, the presence of transferrable antimicrobial resistance genes (ARGs) will endow probiotics as a reservoir with increased ability of emergence and dissemination, during their wide and inadvertent use (Berendonk et al., 2015; Cabello et al., 2016). Most probiotic Bacillus spp. will be excreted and released to the environment after enrichment in the gut of human or livestock after transient colonization (Stenfors Arnesen et al., 2008). Improperly treated animal wastes including the bedding of plant origin have been widely used as feeds in fish ponds and manure for fertilizer, creating a melting pot for these Bacillus probiotics and thereby facilitating the horizontal gene transfer of mobile ARGs (Berendonk et al., 2015; Cabello et al., 2016). Therefore, besides toxicity, the mobile ARGs in probiotic strains is a further important safety parameter. Therefore, we hypothesized that some commercial Bacillus probiotics might constitute potential risks for One Health, due to the identification of virulence factors and ARGs. Herein, we investigated the safety of Bacillus probiotics in China, with focus on the capability of toxin production and carrying mobile ARGs.
2.2. Toxicity evaluation Four main toxin genes (ces, nhe, hbl, and cytK1) were identified, and the presence of toxins were detected by immunoassays as previously described (Wehrle et al., 2009). Cereulide (an emetic toxin produced by B. cereus frequently involved in food poisoning) was confirmed by ultrahigh performance liquid chromatography tandem mass spectrometry according to the previous study (Cui et al., 2016). Cytotoxicity of the enterotoxins and heat-stable surfactin-like toxins was performed on Vero cells, while cytotoxicity of cereulide was tested on HEp-2 cells. B. cereus NVH0075/95, ATCC14579, NVH391/98 and F4810/72 were used as reference strains for non-hemolytic enterotoxin (Nhe), hemolysin BL (Hbl), cytotoxin K1 (CytK1) and cereulide, respectively (Andersson et al., 1998; Lund and Granum, 1996; Ivanova et al., 2003; Guinebretiere et al., 2013). 2.3. In vivo assessment This animal study was approved by the Beijing Association for Science and Technology (approval ID: SYXK [Beijing] 2016-0008). The entire operation followed the guidelines for the Beijing Laboratory Animal Welfare and Ethics, issued by the Beijing Administration Committee of Laboratory Animals, and the China Agricultural University Institutional Animal Care and Use Committee guidelines (ID: SKLAB-B-2010-003). Eight-week old specific pathogen-free ICR mice weighing 28–38 g (half female and half male) from the Vital River Laboratory (Beijing, China), were acclimated for 3 days prior to use. 2.3.1. Mouse sepsis model Three groups of mice (n = 6 per group, half male and half female) were injected intraperitoneally (i.p.) with 200 mg/kg cyclophosphamide (Sigma-Aldrich, St. Louis, MO, USA) 24 h prior to infection. Mice were infected with 0.3 mL of B. cereus 4 suspension (1 × 108 CFU per mouse) via intragastric administration (i.g.) (Liu et al., 2017). The placebo group were treated with 0.3 mL 0.9% NaCl solution. At one hour post-infection, one group was treated with vancomycin at single intraperitoneal doses of 10 mg/kg. Survival was observed in placebo and therapy groups for and analyzed by nonparametric logrank test (Mantel, 1966). Once the infected mice died, various organs including heart, liver, spleen, lung and kidney were removed and homogenized in sterile 0.9% NaCl solution. Surviving mice were sacrificed at 48 h postinfection, and organs were harvested. Serial dilutions of each suspension were plated on the selective Brilliance™ Bacillus cereus agar plate (Oxoid, Wesel, Germany) for enumerating bacterial colonies. The residual organs were excised and fixed with 4% paraformaldehyde solution (Sigma-Aldrich, St. Louis, Missouri, USA). Histopathological changes were stained with Hematoxylin-Eosin (H&E) then visualized through a microscope (Prophet et al., 1992).
2. Materials and methods 2.1. Sampling, isolation and taxonomy of Bacillus probiotic isolates To investigate the risk of Bacillus probiotics used in China, we collected 34 commercial probiotic products with Bacillus spp. as the main ingredient between April to July 2016 from 12 provinces in China. These products were intended for usage in humans (n = 9), animals (n = 15), plants (n = 6), aquaculture and environment (n = 4), respectively. The probiotics targeting for aquaculture and environment were mainly used for aquatic breeding and water purification by consuming excess nutrients (more details about the commercial probiotic products can be found in supplementary materials Table S1). All the Bacillus spp. strains were isolated according to the previous study, and whole genome sequencing analysis was conducted for bacterial identification (Zhu et al., 2016). Genomic DNA of all Bacillus probiotic isolates were extracted using the Wizard Genomic DNA Purification Kit (Promega, Madison, USA). Then indexed Illumina sequencing DNA libraries were constructed using KAPA Hyper Prep Kits (Kapa Biosystems, Wilmington, Massachusetts, USA) with standard protocols, and 150 base pair pairedend reads were sequenced on the Illumina HiSeq 2500 platform (Bionova, Beijing, China). The draft assemblies of the sequences were obtained using CLC Genomics Workbench 9.5.3 (CLC Bio, Aarhus, Denmark). Assembled draft genomes were aligned using muscle program, and a core-genome phylogenetic tree was generated by Parsnp in
2.3.2. Intestinal inflammation model All the mice were treated through i.g. (n = 6). Compared to the placebo group treated with 0.2 mL 0.9% NaCl solution, the group of infected mice was treated with 0.2 mL bacterial solution of B. cereus 4 (1 × 108 CFU) per mouse. At 48 h post-infection, mice were euthanized by cervical dislocation and small intestines were removed and fixed with 4% paraformaldehyde solution. Histopathological changes were analyzed by H&E stain. 2.3.3. Liver injury model In two groups (n = 6 for each group), all mice were treated through i.g. Mice in the placebo group were treated with 0.2 mL 0.9% NaCl solution. Mice for liver injury model, were infected with 0.2 mL bacterial solution of B. cereus 28-2 (1 × 108 CFU per mouse). At 48 h post2
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3.2. Toxicity evaluation
infection, all mice were euthanized by cervical dislocation. Blood of each mouse was collected for detecting serum indices based on the manufacturer’s protocols. The activity of alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) were tested with kits from Biovision (Milpitas, California, USA). The kit for alanine aminotransferase (ALT) was purchased from Cayman Chemical (Ann Arbor, Michigan, USA), and the kits for aspartate transaminase (AST) and creatinine (Cre) were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Livers were removed and fixed with 4% paraformaldehyde solution. Histopathological changes were analyzed by H&E stain.
To assess the toxin-producing potential of the Bacillus probiotics, we found that 60% of the isolates (39/65, of which 36 isolates belonging to the B. cereus group) showed hemolytic activity (Fig. 1C), which may attribute to bacterial virulence factors. To further characterize the hemolysis in these isolates, it is necessary to assess their abilities of virulence factor production. Isolates in the B. cereus group especially B. cereus and B. thuringiensis are known for the ability to produce mainly four toxins, including three enterotoxins (Hbl, Nhe and CytK1) and one peptide toxin (cereulide) (Bottone, 2010). First, almost all the B. cereus and B. thuringiensis isolates were able to produce biologically active Nhe (51%, 33/65), and most of them could also produce Hbl (40%, 26/65), except that one isolate (B. cereus 31-3) harboring the nhe gene without expression, as determined by PCR and immunoassays (Fig. 1D). Additionally, no isolate contained the cytK1 gene. Cytotoxic tests were subsequently performed to confirm the expression of intact tripartite complexes of Nhe and Hbl (Wehrle et al., 2009). Accordingly, 11 isolates (17%) were highly cytotoxic with IC50 less than 10 μg/mL (Fig. 1D; Table S2 and Fig. S1D). In such isolates, the expression levels of Nhe were similar to the reference strain NVH0075/95, which was isolated from a food poisoning case in Norway (Lund and Granum (1996)). In addition, various and cytotoxic surfactin-like toxins were found (Fig. 1E and Table S2). Therefore, it is very likely that these toxin-producing probiotics may possess a potential threat to the public health and cause or aggravate diarrhea symptoms in humans, particularly if consumed by infants or immunocompromised patients (Duc et al., 2004; Bongaerts and Severijnen, 2016). To test this notion, we challenged immunosuppressed mice with B. cereus 4 (an isolate for treating human diarrhea) to mimic immunodeficient patients or children. Infected mice without treatment all died, while infected mice treated with vancomycin all survived within 24 h (Fig. 1F). Treated mice with vancomycin had decreased bacterial counts (Fig. 1G) and alleviated pathologic changes in different organs (Fig. 1H and Fig. S2A). Lastly, we infected mice with B. cereus 4 through i.g. to simulate oral administration. Inflammatory edema on submucosa and increased numbers of goblet cells in the intestine were found (Fig. S2B). These results indicate that Bacillus probiotic isolates can produce multiple functional virulence factors. In addition, the p-value of toxicity among different targeted probiotics is 0.241, and the 95% confidence intervals are 0.233-0.249. There is no difference among these probiotics based on the sample sizes in the current study. It is noteworthy that the presence of toxin gene ces, encoding the synthesis of cereulide synthetase, was found in B. cereus 28-2 (an isolate for promoting growth in plants). The ces gene cluster located on a plasmid (Fig. 2A) (Stenfors Arnesen et al., 2008). Expression of cereulide was confirmed based on mass spectrometry analysis (Fig. 2B). Higher cytotoxicity than the reference cereulide producing strain (B. cereus F4810/72) was observed (Fig. 2C) (Cui et al., 2016). The genetic environment of cereulide synthase gene cluster in B. cereus 28-2 is consistent with the sequences in other pXO1-like plasmids (Fig. 2D and Fig. S3). Additionally, we infected mice with B. cereus 28-2 through oral administration. Apparent lesions such as vacuolization and granular degeneration in hepatocytes and local necrosis were observed in livers (Fig. 2E). Furthermore, increased serum indices were found in infected mice as well (Fig. S2C).
2.4. Antimicrobial susceptibility tests Minimum inhibitory concentrations (MICs) of Bacillus probiotic isolates were determined by classical micro-broth dilution method according to the Clinical and Laboratory Standards Institute (CLSI)’s operating directions (CLSI M100S-S26) (Clinical and Laboratory Standards Institute (CLSI), 2016). Staphylococcus aureus ATCC29213 was chosen as a standard control for antimicrobial susceptibility tests, because no data for Bacillus spp. is available according to the CLSI standards and guidelines.
2.5. Bioinformatics analysis The draft genomic sequences were searched against the ResFinder database (http://www.genomicepidemiology.org/) using the NCBI BLAST software (minimum 70% identity) (Zankari et al., 2012). The contigs carrying ARGs were extracted, counted and annotated by PARTIC annotation service (https://www.patricbrc.org/app/ Annotation) (Wattam et al., 2014). The reported mobile elements which commonly located adjacent to the detected ARGs were collected from the NCBI database (Table S3), subsequently searched against the draft genomes. The genomic sequences of a group of Bacillu strains used or potentially used in other countries were retrieved from NCBI database, and searched against the van genes reference sequences. More details can be found in Table S4.
2.6. Statistical analysis The statistical analysis in this study is performed by SPSS 23.0 (IBM, Armonk New York, USA). We performed a bivariate analysis by using Pearson’s correlation to analyze the correlation between different application targets and toxicity or antimicrobial resistance of all the isolates. We considered a 95% confidence intervals and significance level of 5% for all analyses.
3. Results 3.1. Characterization of Bacillus probiotic isolates In this study, a total of 65 isolates of Bacillus spp. were isolated from 34 products labeled as Bacillus spp. containing from 12 provinces in China (Fig. 1B). Based on colony morphology and whole genome sequencing, 37 isolates were identified to belong to the B. cereus group including B. cereus and B. thuringiensis (Fig. S1A); 22 of these isolates belonged to the B. subtilis group including B. subtilis, B. amyloliquefaciens, B. licheniformis and B. paralicheniformis (Fig. S1B). Additionally, six isolates were other Bacillus spp. including two B. oceanisediminis isolates and one each of B. flexus, B. infantis, B. meqaterium and B. safensis (Fig. S1C). All genomic assemblies of 65 isolates have been deposited in GenBank and are registered under BioProject accession number PRJNA390877, and BioSample numbers SAMN07267320SAMN07267384.
3.3. Antimicrobial resistance To understand the profiles of antimicrobial resistance in these Bacillus probiotics, all isolates were tested with 12 kinds of routinely used antimicrobials, with the resistance rates ranged from 2% to 98% (Fig. 3A and Fig. S4). The p-value of antimicrobial resistance among different targeted probiotics is 0.857, with 95% confidence intervals of 0.850-0.863. There is no difference among these probiotics based on the sample sizes in the current study. The predominant ARGs in the 3
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Fig. 1. Safety assessment of commercial Bacillus probiotics. (A) Assessment approach of toxicity and antimicrobial resistance in probiotics. (B) 65 isolates from commercial Bacillus probiotics in 12 provinces in China. Numbers in the parentheses indicate numbers of the Bacillus isolates in each province. (C) Hemolysis of Bacillus probiotic isolates on sheep blood agar plates. (D) Various enterotoxins were found in these isolates. The presence of non-hemolytic enterotoxin (Nhe), hemolysin BL (Hbl) and cytotoxin K1 (CytK1) was verified at genetic, protein and cytotoxic levels, respectively. (E) Cytotoxicity of surfactin-like toxins from Bacillus probiotics were tested. Cytotoxicity at high, medium and low levels represent IC50 < 0.5 ng/mL, 0.5 ng/ mL < IC50 ≤ 1 ng/mL, and IC50 ≥ 1 ng/mL, respectively. (F–H) Sepsis model by the isolate of B. cereus 4. Three groups of mice (n = 6 per group, half male and half female) were injected intraperitoneally with cyclophosphamide (200 mg/kg) 24 h prior to infection. Compared to the placebo group, mice were infected with 0.3 mL of bacterial suspension (1 × 108 CFU bacteria per mouse) through intraperitoneal injection. At one hour post-infection, one group was treated with vancomycin at single intraperitoneal doses of 10 mg/kg. (F) Survival curves were obtained at 48 h after infection. (G) The numbers of B. cereus in different organs were enumerated after infection. (H) Typical pathological changes in livers by Hematoxin-Eosin stain (scale bar, 50 μm). The pathological sections of other organs could be found in supplementary materials Fig. S2A.
surrounded by various insertion sequences, integrases or phage proteins. Alarmingly, more than half isolates carried such mobile ARGs determinants (Fig. 3C). Furthermore, we found incomplete vancomycin resistance genes in all isolates of the B. cereus group and the isolate of B. oceanisediminis 34-1 as well (Fig. S5A). To characterize the van genes in these Bacillus isolates, the complete van gene clusters were analyzed. The van gene clusters in Bacillus isolates were categorized into four patterns (Fig. 3D). However, the absence of vanH, vanA and vanX genes was observed for all isolates. This is consistent with the fact that these
genomes of 65 isolates were cat, cfr, lsa, tet and van (Fig. 3A), in response to the resistance of choloramphenicols, choloramphenicols / lincosamides / oxazolidinones / pleuromutilins, lincosamides / pleuromutilins, tetracyclines and vancomycin, respectively. And the ARG profiles were species specific (Fig. S4). For instance, the presence of cfr gene was found in all isolates of the B. subtilis group. Nevertheless, the lsa and incomplete van genes were found in the B. cereus group. Representative flanking genetic contexts of five mobile ARGs in these isolates were shown in Fig. 3B. For example, the cat genes are
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Fig. 2. Plasmid-borne toxin cereulide in B. cereus probiotic isolate 28-2. (A) The presence of plasmid-borne ces gene. Lane 1 and 2, the extracts of plasmid (1) and genome (2) in B. cereus 28-2; Lane 3 and 4, negative controls of water (3) and B. cereus ATCC14579 (4); Lane 5 and 6, the ces genes carried by B. cereus F4810/72 (5) and B. cereus CAU45 (Cui et al., 2016) (6); M, size marker. (B) Mass spectrum of cereulide produced in B. cereus 28-2. The inset is the scheme of cereulide. (C) Cytotoxicity of cereulide secreted by B. cereus 28-2. B. cereus 28-2 produces more cereulide than the reference cereulide producing strain B. cereus F4810/72. (D) Genetic environment of the ces gene cluster in B. cereus 28-2. This sequence is identical to the ces gene clusters on plasmids of pCER270 (GenBank accession number: DQ889676.1) and pNCcld (AP007210.1), but shows high similarity (99.4%) to the reverse sequence in plasmid pH308197_258 (CP001166.1). Completed annotation can be found in supplementary materials Fig. S3. (E) Cereulide caused hepatic damages in mice by Hematoxin-Eosin stain (scale bar, 50 μm).
species of probiotic candidates accurately. Then assess the toxigenic potential on genetic, protein and cellular levels. Collectively, probiotics with the capability of virulence factor production are discouraged to use, including the identified toxins and unknown virulence factors (FAO/WHO, 2002; MEP, 2008; EFSA, 2014). It is noteworthy that the expression of cereulide was carried by one isolate (B. cereus 28-2). Cereulide is a notorious pathogenic agent responsible for fulminant liver failure in children and food poisoning with emetic symptom (Mahler et al., 1997; Stenfors Arnesen et al., 2008). The presence of plasmid-borne cereulide will endow this isolate as a reservoir with increased ability of dissemination, during its wide and inadvertent use. Taken together, such probiotic products harboring a toxigenic potential to produce multiple functional virulence factors are not recommended for use. In this study, almost all of the isolates (95%, 62/65) were resistant to lincomycin, polymyxin B or tiamulin and half of them (52%, 34/65) were resistant to ampicillin (Fig. 3A). These were mainly due to the intrinsic characteristics of Bacillus spp. (Bottone, 2010). Nevertheless, the mechanisms of lincosamides and pleuromutilins resistance in Bacillus spp. remains unclear. Additionally, more than half isolates carry the mobile ARGs. Unlike the intrinsic ARGs, the isolates with mobile ARGs have the potential to disseminate such genes to pathogenic or commensal bacteria under antimicrobial selective pressures (Berendonk et al., 2015). These mobile ARGs in probiotics would enable an accumulation or dissemination of antimicrobial resistance under a selective stress as well. For instance, due to the presence of a mobile tetracycline resistance tet(B) gene, along with insufficient data on toxicity assessment, the probiotic B. cereus strain of Esporafeed Plus® has been withdrawn in Europe (SCAN, 1999). Thus, the presence of various mobile ARGs in Bacillus probiotics indicates a potential threat to One Health. Furthermore, we found vancomycin resistance van genes in the absence of vanH, vanA and vanX genes in 38 isolates. Although none of the isolate carries the complete set of seven van genes, it still has the potential to acquire extra vanH, vanA and vanX genes through
isolates are vancomycin sensitive, because all seven genes are required for vancomycin resistance (Pootoolal et al., 2002). Additionally, the identities of van genes in the isolates used for human were significantly higher than that for animal, plant, aquaculture and environment (Fig. S5B). 4. Discussion The safety assessment of Bacillus probiotics is insufficient and should attract more attention. Because various toxins and mobile ARGs were recently reported after the long-term application of Bacillus spp. for different purposes (Cui et al., 2019). Historically, commercial Bacillus probiotic products, such as Paciflor® and Esporafeed Plus® have been withdrawn in Europe (SCAN, 1999; SCAN, 2001). In our results, more than half of the isolates showed hemolytic activity, almost half of the isolates could produce enterotoxins and various cytotoxic surfactin-like toxins. Additionally, in vivo tests confirmed that probiotic B. cereus isolates have the potential to cause sepsis (as shown in Fig. 1F to H and Fig. S2A), intestinal inflammation (Fig. S2B) and liver damage (Fig. 2E and Fig. S2C). Meanwhile, L. rhamnosus, another commonly used probiotic species, is associated with bloodstream infection in immunocompromised or defective people (Gouriet et al., 2012). Moreover, many food-poisoning cases have reported that the enterotoxins and emetic toxin produced by B. cereus sensu stricto can cause diarrhea and acute liver failure (Mahler et al., 1997; Lund and Granum, 1996), which are confirmed by our in vivo tests. In addition, comparing to the pathogenic B. cereus, such as B. cereus NVH0075/95 causing food poisoning outbreaks (Lund and Granum, 1996), many Bacillus isolates in the present study can produce higher level of toxins (Fig. 1 and Table S2). These virulent probiotic isolates and their toxins may contaminate water and foods through the food chain, and eventually ingested by consumers (Stenfors Arnesen et al., 2008). Additionally, probiotics are not all alike. The exhibition of toxicity is limited to specific strains, not every isolate of Bacillus spp. harbor all virulence genes (Cui et al., 2019). It is necessary to firstly identify strain 5
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Fig. 3. Phenotypical and genetic profiles of antimicrobial resistance in Bacillus probiotic isolates. (A) The proportion of Bacillus isolates showed the phenotypes of conferring resistance to 12 antimicrobial drugs, and the proportion of isolates carrying the corresponding antimicrobial resistance genes (ARGs). The detailed proportions data of phenotypes and genes in the different Bacillus probiotic species were showed in supplementary materials Fig. S4. (B) Schematic representation of five isolates carrying both ARGs and mobile genetic elements (MGEs). (C) Distribution of MGEs (including cat, cfr, lsa, tet and van genes) in these Bacillus isolates. (D) Four patterns of the van gene clusters in Bacillus probiotic isolates. The reference van A-type gene clusters include (EMBL accession number Y15704 (vanA), Y15705 (vanH), Y15706 (vanR), Y15707 (vanS), Y15708 (vanX) and Y17303 (vanY)) of B. circulans VR0709. (E) Comparison of the identity of van genes in reported Bacillus probiotic strains and the isolates in this study. The identities of van genes in this study were shown as average. More details about the Bacillus probiotic strains can be found in supplementary materials Table S4.
boost of pharmaceutical and biotechnological companies contributes to fast expansion of Bacillus probiotics, whereas the quality of some products barely meets the safety requirement. More importantly, the safety issues of probiotics and its potential risk to One Health is not limited to China, it becomes a global issue which attracts more attention. In conclusion, great caution should be taken when probiotics are used for people in certain groups particularly for future use in agriculture and environment, since large amounts of products consumed on a regular basis will accordingly increase probiotic related infections.
conjugation, recombination or other mechanisms and confer resistance to vancomycin (Ligozzi et al., 1998). Moreover, compared to the Bacillus probiotic strains from other countries, such isolates from China contain more genetic elements and share high identities of van genes (Fig. 3E and Fig. S5A). High identities of van genes in our isolates, especially the isolates for human suggest that the Bacillus isolates used for human possess high ability to acquire other van genes to form an entire van gene cluster (Sanders et al., 2010). Consistent with this notion, a probiotic Lactobacillus strain has been reported to acquire vancomycin resistance in vivo (Mater et al., 2008). Here we summarize the possible health hazards of Bacillus containing commercial probiotic products in China. The verification of the absence of toxigenic potential and transferrable antimicrobial resistance gene is a prerequisite for approval of a probiotic strain. The
Declaration of Competing Interest All authors read and approved the manuscript. The authors declared no competing financial interests. 6
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Acknowledgments
Gastroenterol. Hepatol. 11, 506–514. Ivanova, N., Sorokin, A., Anderson, I., Galleron, N., Candelon, B., Kapatral, V., Bhattacharyya, A., Reznik, G., Mikhailova, N., Lapidus, A., Chu, L., Mazur, M., Goltsman, E., Larsen, N., D’Souza, M., Walunas, T., Grechkin, Y., Pusch, G., Haselkorn, R., Fonstein, M., Ehrlich, S.D., Overbeek, R., Kyrpides, N., 2003. Genome sequence of Bacillus cereus and comparative analysis with Bacillus anthracis. Nature 423, 87–91. Letunic, I., Bork, P., 2016. Interactive tree of life (iTOL) v3: an online tool for the display and annotation of phylogenetic and other trees. Nucleic Acids Res. 44, W242–245. Ligozzi, M., Lo Cascio, G., Fontana, R., 1998. vanA gene cluster in a vancomycin-resistant clinical isolate of Bacillus circulans. Antimicrob. Agents Chemother. 42, 2055–2059. Liu, Y., Ding, S., Dietrich, R., Martlbauer, E., Zhu, K., 2017. A biosurfactant-inspired heptapeptide with improved specificity to kill MRSA. Angew. Chem. Int. Ed. Engl. 56, 1486–1490. Lund, T., Granum, P.E., 1996. Characterisation of a non-haemolytic enterotoxin complex from Bacillus cereus isolated after a foodborne outbreak. FEMS Microbiol. Lett 141, 151–156. Ministry of Environmental Protection of the People’s Republic of China (MEP), 2008. Guide of safety-assessment on application of microbial blends in the environmental protection, Industry standard of the environmental protection of the People’s Republic of China HJ/T 415. MEP. http://kjs.mep.gov.cn/hjbhbz/bzwb/stzl/ 200801/W020111121498913885910.pdf. Mahler, H., Pasi, A., Kramer, J.M., Schulte, P., Scoging, A.C., Bär, W., Krähenbühl, S., 1997. Fulminant liver failure in association with the emetic toxin of Bacillus cereus. New Engl. J. Med. 336, 1142–1148. Mantel, N., 1966. Evaluation of survival data and two new rank order statistics arising in its consideration. Cancer Chemother. Rep. 50, 163–170. Mater, D.D.G., Langella, P., Corthier, G., Flores, M.J., 2008. A probiotic Lactobacillus strain can acquire vancomycin resistance during digestive transit in mice. J. Mol. Microbiol. Biotechnol. 14, 123–127. Million, M., Raoult, D., 2012. Publication biases in probiotics. Eur. J. Epidemiol. 27, 885–886. Oggioni, M.R., Pozzi, G., Valensin, P.E., Galieni, P., Bigazzi, C., 1998. Recurrent septicemia in an immunocompromised patient due to probiotic strains of Bacillus subtilis. J. Clin. Microbiol. 36, 325–326. Pootoolal, J., Neu, J., Wright, G.D., 2002. Glycopeptide antibiotic resistance. Annu. Rev. Pharmacol. Toxicol. 42, 381–408. Prophet, E.B., Mills, B., Arrington, J.B., Sobin, L.H., 1992. Laboratory Methods in Histotechnology. American Registry of Pathology, Washington D.C. Scientific Committee on Animal Nutrition (SCAN), 1999. Assessment by the Scientific Committee on Animal Nutrition (SCAN) of a Micro-organisms Product: Esporafeed Plus®, Directorate B-Scientific Health Opinions. December 3. European Commission Health & Consumer Protection Director-General. Scientific Committee on Animal Nutrition (SCAN), 2001. Assessment by the Scientific Committee on Animal Nutrition (SCAN) of the Safety of Product Paciflor® for Use As Feed Additive, Directorate C-Scientific Opinions. May 16. European Commission Health & Consumer Protection Director-General. Sanders, M.E., Akkermans, L.M., Haller, D., Hammerman, C., Heimbach, J., Hormannsperger, G., Huys, G., Levy, D.D., Lutgendorff, F., Mack, D., Phothirath, P., Solano-Aguilar, G., Vaughan, E., 2010. Safety assessment of probiotics for human use. Gut Microbes 1, 164–185. Stenfors Arnesen, L.P., Fagerlund, A., Granum, P.E., 2008. From soil to gut: Bacillus cereus and its food poisoning toxins. FEMS Microbiol. Rev. 32, 579–606. Strunk, T., Kollmann, T., Patole, S., 2015. Probiotics to prevent early-life infection. Lancet Infect. Dis. 15, 378–379. Treangen, T.J., Ondov, B.D., Koren, S., Phillippy, A.M., 2014. The Harvest suite for rapid core-genome alignment and visualization of thousands of intraspecific microbial genomes. Genome Biol. 15, 524. Wattam, A.R., Abraham, D., Dalay, O., Disz, T.L., Driscoll, T., Gabbard, J.L., Gillespie, J.J., Gough, R., Hix, D., Kenyon, R., Machi, D., Mao, C., Nordberg, E.K., Olson, R., Overbeek, R., Pusch, G.D., Shukla, M., Schulman, J., Stevens, R.L., Sullivan, D.E., Vonstein, V., Warren, A., Will, R., Wilson, M.J., Yoo, H.S., Zhang, C., Zhang, Y., Sobral, B.W., 2014. PATRIC, the bacterial bioinformatics database and analysis resource. Nucleic Acids Res. 42, D581–591. Wehrle, E., Moravek, M., Dietrich, R., Burk, C., Didier, A., Martlbauer, E., 2009. Comparison of multiplex PCR, enzyme immunoassay and cell culture methods for the detection of enterotoxinogenic Bacillus cereus. J. Microbiol. Meth. 78, 265–270. Zankari, E., Hasman, H., Cosentino, S., Vestergaard, M., Rasmussen, S., Lund, O., Aarestrup, F.M., Larsen, M.V., 2012. Identification of acquired antimicrobial resistance genes. J. Antimicrob. Chemother. 67, 2640–2644. Zhu, K., Holzel, C.S., Cui, Y., Mayer, R., Wang, Y., Dietrich, R., Didier, A., Bassitta, R., Martlbauer, E., Ding, S., 2016. Probiotic Bacillus cereus strains, a potential risk for public health in China. Front. Microbiol. 7, 718.
We thank Prof. Erwin Märtlbauer and Dr. Richard Dietrich from Ludwig-Maximilians-University Munich (LMU, Germany) for kindly providing the reference strains and monoclonal antibodies. This study is supported by the National Key Research and Development Program of China (2017YFC1600305) and the Fund of Beijing Innovation Team of Dairy Industry (BAIC06-2017). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.jhazmat.2019.121266. References Andersson, M.A., Mikkola, R., Helin, J., Andersson, M.C., Salkinoja-Salonen, M., 1998. A novel sensitive bioassay for detection of Bacillus cereus emetic toxin and related depsipeptide ionophores. Appl. Environ. Microbiol. 64, 1338–1343. Berendonk, T.U., Manaia, C.M., Merlin, C., Fatta-Kassinos, D., Cytryn, E., Walsh, F., Burgmann, H., Sorum, H., Norstrom, M., Pons, M.N., Kreuzinger, N., Huovinen, P., Stefani, S., Schwartz, T., Kisand, V., Baquero, F., Martinez, J.L., 2015. Tackling antibiotic resistance: the environmental framework. Nat. Rev. Microbiol. 13, 310–317. Bongaerts, G.P.A., Severijnen, R.S.V.M., 2016. A reassessment of the PROPATRIA study and its implications for probiotic therapy. Nat. Biotechnol. 34, 55–63. Bottone, E.J., 2010. Bacillus cereus, a volatile human pathogen. Clin. Microbiol. Rev. 23, 382–398. Cabello, F.C., Godfrey, H.P., Buschmann, A.H., Dolz, H.J., 2016. Aquaculture as yet another environmental gateway to the development and globalisation of antimicrobial resistance. Lancet Infect. Dis. 16, e127–133. Clinical and Laboratory Standards Institute (CLSI), 2016. Clinical and Laboratory Standards Institute (CLSI) Performance Standards for Antimicrobial Susceptibility Testing, CLSI Supplement M100S, 26th edition. (ISBN 1-56238-923-8 [Print]; ISBN 1-56238-924-6 [Electronic]). Cohen, P.A., 2018. Probiotic safety-no guarantees. JAMA Intern. Med. 178, 1577–1578. Cui, Y., Liu, Y., Liu, X., Xia, X., Ding, S., Zhu, K., 2016. Evaluation of the toxicity and toxicokinetics of cereulide from an emetic Bacillus cereus strain of milk origin. Toxins (Basel) 8. Cui, Y., Martlbauer, E., Dietrich, R., Luo, H., Ding, S., Zhu, K., 2019. Multifaceted toxin profile, an approach toward a better understanding of probiotic Bacillus cereus. Crit. Rev. Toxicol. 1–15. Cutting, S.M., 2011. Bacillus probiotics. Food Microbiol. 28, 214–220. Czaplewski, L., Bax, R., Clokie, M., Dawson, M., Fairhead, H., Fischetti, V.A., Foster, S., Gilmore, B.F., Hancock, R.E., Harper, D., Henderson, I.R., Hilpert, K., Jones, B.V., Kadioglu, A., Knowles, D., Olafsdottir, S., Payne, D., Projan, S., Shaunak, S., Silverman, J., Thomas, C.M., Trust, T.J., Warn, P., Rex, J.H., 2016. Alternatives to antibiotics-a pipeline portfolio review. Lancet Infect. Dis. 16, 239–251. Doron, S., Snydman, D.R., 2015. Risk and safety of probiotics. Clin. Infect. Dis. 60 (Suppl 2), S129–134. Duc, L.H., Hong, H.A., Barbosa, T.M., Henriques, A.O., Cutting, S.M., 2004. Characterization of Bacillus probiotics available for human use. Appl. Environ. Microbiol. 70, 2161–2171. European Food Safety Authority Panel on Additives and Products or Substances used in Animal Feed (EFSA), 2014. Guidance on the assessment of the toxigenic potential of Bacillus species used in animal nutrition. EFSA J. 12, 3665. Food and Agriculture Organization of the United Nations/World Health Organization (FAO/WHO), 2002. Joint FAO/WHO working group report on drafting guidelines for the evaluation of probiotics in food. FAO/WHO, London, Ontario, Canada. http:// who.int/foodsafety/fs_management/en/probiotic_guidelines.pdf. Gouriet, F., Million, M., Henri, M., Fournier, P.-E., Raoult, D., 2012. Lactobacillus rhamnosus bacteremia: an emerging clinical entity. Eur. J. Clin. Microbiol. 31, 2469–2480. Guinebretiere, 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., Sorokin, A., 2013. Bacillus cytotoxicussp. nov. is a novel thermotolerant species of theBacillus cereus Group occasionally associated with food poisoning. Int. J. Syst. Evol. Microbiol. 63, 31–40. Hill, C., Guarner, F., Reid, G., Gibson, G.R., Merenstein, D.J., Pot, B., Morelli, L., Canani, R.B., Flint, H.J., Salminen, S., Calder, P.C., Sanders, M.E., 2014. Expert consensus document. The International Scientific Association for Probiotics and Prebiotics consensus statement on the scope and appropriate use of the term probiotic. Nat. Rev.
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