Preliminary safety assessment of a new Bacteroides fragilis isolate

Food and Chemical Toxicology xxx (xxxx) xxxx

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Preliminary safety assessment of a new Bacteroides fragilis isolate Huizi Tana,b, Chen Wanga,b, Qingsong Zhanga,b, Xiaoshu Tanga,b,c, Jianxin Zhaoa,b, Hao Zhanga,b,c, Qixiao Zhaia,b,d,∗, Wei Chena,b,c,e a

State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, 214122, PR China School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, 214122, PR China National Engineering Research Center for Functional Food, Jiangnan University, Wuxi, Jiangsu, 214122, PR China d International Joint Research Laboratory for Probiotics at Jiangnan University, Wuxi, Jiangsu, 214122, China e Beijing Innovation Center of Food Nutrition and Human Health, Beijing Technology and Business University (BTBU), Beijing, 100048, PR China b c

A R T I C LE I N FO

A B S T R A C T

Keywords: Bacteroides fragilis Safety assessment Next-generation probiotics Antibiotic resistance Virulence factors Immunosuppressed mice

The novel commensal strain of Bacteroides fragilis HCK-B3 isolated from a healthy Chinese donor was discovered beneficial effects of attenuating lipopolysaccharides-induced inflammation. In order to contribute to the development of natural next-generation probiotic strains, the safety assessment was carried out with in vitro investigations of its morphology, potential virulence genes and antimicrobial resistance, and an in vivo acute toxicity study based on both healthy and immunosuppressed mice by cyclophosphamide injection. Consequently, the potential virulence genes in the genome of B. fragilis HCK-B3 have yet been identified as toxicity-associated. The absence of plasmids prevents the possibility of transferring antibiotic resistance features to other intestinal commensals. No intracorporal pathogenic properties were observed according to the body weight, hematological and liver parameters, cytokine secretions and tissue integrity. In addition, B. fragilis HCKB3 performed alleviations on part of the side effects caused by the cyclophosphamide treatment. Thus, the novel strain of B. fragilis HCK-B3 was confirmed to be non-toxigenic and did not display adverse effects in both healthy and immune-deficient mice at a routinely applicable dose.

1. Introduction The intestinal microbiota mainly comprises microorganisms from the domains of Bacteria, Archaea and Eukarya, and about 90% of which come from the phyla of Firmicutes and Bacteroidetes. These microorganisms and many of their metabolites, such as short-chain fatty acids and vitamins, facilitate the maturation of the immune system, elimination of infectious agents, maintenance of intestinal integrity, digestion of resistant carbohydrates and supplement of energies (Tan and O'Toole, 2015). The composition of the intestinal microbiota changes according to alterations in dietary habitats, health conditions and age. Disruptions in the homeostasis of intestinal micro-ecology can give rise to metabolic diseases and immune dysfunctions. Probiotics, prebiotics and antibiotics are the most relevant therapies for disorders induced by disturbed microbiota. The traditional probiotics, including Lactobacillus, Bifidobacterium, Bacillus, Weissella, Enterococcus, Escherichia coli, and Saccharomyces, are predicted to create a global turnover value of 46.55 billion US dollars by 2020 as food ingredients or supplements (O'Toole et al., 2017). With developments in

bacterial culture methodologies and sequencing techniques, Bacteroides species have been discovered to possess great potential benefits to the host. B. xylanisolvens DSM 23964 has been proved as starters in fermented milk products under Novel Food Regulation No 258/97 by the European Commission (Brodmann et al., 2017), which is the first case of Bacteroides being authorized as a food ingredient. Moreover, the nontoxigenic B. fragilis ATCC25285 is considered as promising next-generation probiotics (Troy and Kasper, 2010) due to its capabilities in attenuating pathogen-induced inflammation (Mazmanian et al., 2008) and improving autism spectrum disorders (Hsiao et al., 2013); B. fragilis ZY312 is capable of relieving Vibrio parahaemolyticus infection (Li et al., 2017) and antibiotic-induced diarrhea (Zhang et al., 2018) with no adverse effects detected in mice models (Wang et al., 2017). Nevertheless, the fact that the B. fragilis expressing fragilysin (bft) is capable of inducing diarrhea in animals and humans (Ramamurthy et al., 2013) reveals that the beneficial characteristics of Bacteroides species are strain-dependent, which raises safety concerns about individual strains. In a previous investigation, we established efficient methods for isolation and purification of relatively low-abundant Bacteroides species

∗ Corresponding author. State Key Laboratory of Food Science and Technology, Jiangnan University, No 1800 Lihu Avenue, Binhu District, Wuxi, Jiangsu, 214122, PR China. E-mail address: [email protected] (Q. Zhai).

https://doi.org/10.1016/j.fct.2019.110934 Received 11 September 2019; Received in revised form 23 October 2019; Accepted 29 October 2019 0278-6915/ © 2019 Elsevier Ltd. All rights reserved.

Please cite this article as: Huizi Tan, et al., Food and Chemical Toxicology, https://doi.org/10.1016/j.fct.2019.110934

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Potential antibiotic resistance genes and virulence genes of B. fragilis HCK-B3 were identified by using a Protein-translated nucleotide Basic Local Search Tool (tblastn) against the Comprehensive Antibiotic Resistance Database (CARD, http://arpcard.mcmaster.ca/) (McArthur et al., 2013) and the Virulence Factor Database (VFDB, http://www. mgc.ac.cn/VFs/main.htm) (Chen et al., 2012) respectively, with identity more than 30%, coverage over 70%, and e-value less than 0.01 (Salvetti et al., 2016). B. fragilis ATCC25285 (GenBank accession number NC_003228) was adopted as control for screening Bacteroidesspecific virulence genes. The genomic islands in the genome of B. fragilis HCK-B3 were searched using Island Path-DIMOB and Islander (Lu and Leong, 2016) to identify the possibilities of transferring virulence and antibiotic-resistance genetic factors. The amino acid sequences of bft, ompW, upaY, upaZ, wcfR, wcfS, cfiA, cepA and cfxA were obtained from the NCBI database (Table 1), and were blasted against the genome sequences of HCK-B3 and ATCC25285 locally by tblastn with an e-value less than 1e-5 using BioEdit v7.2.5.

from the fecal samples of healthy Chinese donors (Tan et al., 2018b). One of the isolates, B. fragilis HCK-B3, was verified to ameliorate lipopolysaccharides (LPS)-induced disorders in cytokine productions and the composition of the intestinal microbiota by restoring the balance of regulatory T cells (Tregs) and T helper 17 cells (Th17) (Tan et al., 2019). In order to evaluate the possibilities of further applications of this promising strain, with this study, we examined its hemolytic and motile characteristics, antibiotic resistance, genetic virulence factors, and a pilot safety assessment in vivo for determination of underlying side effects in terms of hematological and liver parameters, cytokine secretions, and tissue integrity. 2. Materials and methods 2.1. Bacterial strains and culture conditions B. fragilis HCK-B3 is maintained in-house at the Culture Collections of Food Microbiology of Jiangnan University, and has been deposited in the Guangdong Microbial Culture Center (GDMCC) with the registration number of 60342. B. fragilis ATCC25285 was purchased from the American Type Culture Collection (ATCC), USA. Salmonella enterica CMCC50335 and Escherichia coli CMCC44102 were acquired from the National Center for Medical Culture Collections, China. Both B. fragilis strains were anaerobically cultured in brain heart infusion (BHI, Hopebio, China)supplemented with hemin (Sangon Biotech, China) and vitamin K1 (BHIS) at 37 °C until early stationary phase for subsequent in vitro experiments. The bacteria solutions for animal tests were prepared with cell pellets re-suspended in phosphate buffer saline supplemented with 20% glycerol after centrifugation at 4800g for 15 min, and preserved at −80 °C. Cell viability was checked after freezing and thawing by colony-forming unit (cfu) enumeration on BHIS agar before use. S. enterica and E. coli was also cultivated in BHI medium at 37 °C anaerobically.

2.4. Antibiotic resistance analysis Fourteen antibiotics (Sangon Biotech, China) with different antimicrobial mechanisms were used to identify the antibiotic resistance breakpoint of B. fragilis HCK-B3and the type strain ATCC25285, including ampicillin, Penicillin G, vancomycin, cefoxitin, and ceftriaxone for inhibiting cell wall synthesis: clindamycin, chloromycetin, erythromycin, kanamycin, streptomycin, and tetracycline for suppressing protein synthesis; metronidazole and ciprofloxacin for restraining nucleic acid synthesis; and polymyxin B for inhibiting cytoplasmic functions. Overnight cultures (100 μl) of B. fragilis at a concentration of 107 cfu/ml were anaerobically co-cultured with serial dilutions of antibiotics from 0.125 to 1024 μg/ml in sterile 96-well plates (Wang et al., 2017). The optical density at 600 nm was checked with a microplate reader (Multiskan GO, Thermo Scientific, USA). The minimal inhibitory concentration (MIC) of each antibiotic, which refers to the lowest dose that inhibits 90% growth of the tested B. fragilis strains, was recorded (D'Aimmo et al., 2007). All of the experiments were carried out in three biological replicates.

2.2. Hemolysis and motility test The hemolytic characteristics of B. fragilis HCK-B3 were examined by inoculating 5 μl of overnight culture on Brucella agar (Hopebio, China) supplemented with hemin, vitamin K1 and 5% sheep blood (Nanjing SenBeiJia Biological Technology Co., Ltd, China) (Robertson et al., 2006). B. fragilis type strain ATCC25285 was adopted as control, and results were checked after 48-h anaerobic cultivation. The motility of B. fragilis HCK-B3 was examined by standard motility agar assays using BHIS broth supplemented with 0.5% (w/v) agar (soft agar) inoculated with 5 μl of overnight culture and incubated anaerobically for 48 h (Cousin et al., 2015). B. fragilis ATCC25285 was involved as control, as well as S.enterica CMCC50335 as the positive control and E. coli CMCC44102 as the negative control. All of the experiments were carried out in three biological replicates.

2.5. Animals Male C57 mice (7 weeks old, body weight of about 20 g, spf grade) were purchased from Shanghai Laboratory Animal Center (Shanghai, China) and were raised in the IVC rodent caging system at Jiangnan University (Wuxi, China), under a 12-h light/dark cycle, and strictly controlled temperature and humidity. Mice were caged in groups of two or three, with distilled water and standard laboratory chow provided ad libitum. Treatments were carried out at least one week after the mice arrived to allow them to acclimatize to the new environment. All procedures performed in studies involving animals were approved by the Animal Ethics Committee of Jiangnan University (JN. No. 20180415c0450730[61]), and the protocols for the care and use of the mice were based on European Community guidelines (Directive 2010/ 63/EU).

2.3. Draft genome sequencing and prediction of potential virulence genes The genomic DNA of B. fragilis HCK-B3 was extracted from cell pellets obtained from culture at the early stationary phase and sequenced with Illumina Hiseq system by Majorbio (China). Library with average insert size of 410 bp was established followed by filtration of low-quality reads. The raw data were assembled using SOAP de novo v2.04 (http://soap.genomics.org.cn/) (Li et al., 2008, 2010) with gap closure and base correction using GapCloser v1.12. A K-mer value of 23 was chosen after accuracy evaluation. Gene annotation was performed by blastp (BLAST 2.2.28+) against Nr, Swiss-prot, string and GO databases. The draft genomic sequence has been submitted to the GenBank of NCBI database (accession no: QRBX00000000), and a neighborjoining phylogenetic tree was built to indicate relationship between B. fragilis strains based on the complete genome sequences available from the NCBI database (https://www.ncbi.nlm.nih.gov/) (Fig. 1).

2.6. Acute toxicity study in normal and immunosuppressed mice Twenty-four mice were randomly allocated to one of the two categories (normal or immunosuppressed), and each category was divided into two groups (i.e., six mice per group). The normal mice included the control group (CTRL) and B. fragilis HCK-B3 group (BF), which received daily oral administration of 100 μl of PBS/glycerol solution or B. fragilis HCK-B3 solution (109 cfu), respectively, for 5 days. The immunosuppressed mice were all intraperitoneally injected with 250 mg/ kg cyclophosphamide (CTX, Sigma-Aldrich, USA) three days prior to the treatment with 100 μl of PBS/glycerol solution (CTX group) or 109 cfu B. fragilis HCK-B3 solution (CTX + BF group) by gavage every 2

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Fig. 1. Phylogenetic tree based on complete genomic sequences of B. fragilis HCK-B3, 638R, ATCC25285, BE1, BOB25, Q1F2, S14 and YCH46. Table 1 Comparison of Bacteroides-specific virulence factors in B. fragilis HCK-B3 and ATCC25285. Potential virulence factors

GenBank accession number

Size (aa)

% similarity in B. fragilis HCK-B3

% similarity in B. fragilis ATCC25285

Ref

bft ompW upaY upaZ wcfR wcfS cfiA cfxA cepA

AAF72837.1 AAL09385.1 AAK68912.1 AAK68913.1 AAK68921.1 AAK68922.1 AAK71520 CAP78899.1 AAA21538.1

63 947 172 157 407 198 111 306 300

No hit 83% (789/947) 100% (172/172) 100% (157/157) 100% (320/320) 98% (196/198) No hit 39% (107/273) 99% (297/300)

No hit 83% (790/947) 100% (172/172) 100% (157/157) 100% (407/407) 99% (197/198) No hit 39% (108/273) 100% (300/300)

Gutacker et al. (2000) Wei et al. (2001) Coyne et al. (2001)

Gutacker et al. (2002) Garcia et al. (2008) Rogers et al. (1994)

2.10. Statistical analysis

24 h for 5 days(Hirsh et al., 2004; Salva et al., 2014). The behavior and body weight of each mouse was monitored and recorded throughout the experiment. All of the mice were anesthetized with sodium pentobarbital and sacrificed by cervical dislocation. Liver, spleen and colon tissues were collected and preserved in 4% paraformaldehyde solution after sacrifice for histological analysis. Blood samples were collected for hematological measurements. Serum was obtained after centrifugation at 1200g for 10 min for determination of cytokine levels and liver parameters.

The statistics were analyzed using Graphpad Prism v5.0 (Graph Pad Software Inc., USA). Significant differences between groups were determined by unpaired Student's t-test, with p values of less than 0.05. All of the data were presented as mean ± SD. 3. Results 3.1. Morphological characteristics

2.7. Hematological measurements and determination of liver parameters Hematological parameters were identified using automatic hematology analyzer (BC-5000,Mindray, China) within 1 h of blood sample collection. All of the associated buffers were purchased from Mindray, China. The liver parameters of the serum samples were assessed using automatic biochemical analyzer (BS-480) with corresponding kits (Mindray, China).The serum standard for quality control was purchased from Shanghai Zhicheng Biological Technology Co. Ltd, China.

B. fragilis HCK-B3 is an obligate anaerobe, and is gram-negative and rod-shaped under microscopy. The bacteria grew well at 37 °C on the Brucella Agar supplemented with laked sheep blood anaerobically, the colonies of which were circular, semi-transparent, and convex with a smooth surface. Both B. fragilis HCK-B3 and the type strain ATCC25285 were negative for characteristics of hemolysis and motility. The morphology agrees with the description of B. fragilis in Bergey's Manual (Krieg et al., 2001).

2.8. Assays of serum cytokines

3.2. Genetic characteristics and putative virulence genes

The serum concentrations of tumor necrosis factor (TNF)-alpha, interleukin-6 (IL-6), interleukin-8 (IL-8), and interleukin-10 (IL-10) were determined using corresponding mouse Elisa kits purchased from Nanjing SenBeiJia Biological Technology Co., Ltd (China) according to the manufacturer's instructions.

B. fragilis HCK-B3 was found to be most similar to B. fragilis BOB25 based on the complete genomic information (Fig. 1). HCK-B3 genome is constituted with 1 146 397 779 base pair, 75 scaffolds and 4745 predicted genes, and the GC content of which is 43.41%. 39 virulence gene homologs were identified in B. fragilis HCK-B3 according to the VFDB database (Supplementary Table 1), which are mainly related to cellular structures such as capsular polysaccharide synthesis and physiological activities such as glucosyl transferase. The predicted genes of clpV and clpB indicated the probable existence of Type VI secretion systems (T6SSs). Moreover, the typical B. fragilis enterotoxin bft was not detected in B. fragilis HCK-B3 (Table 1). The inflammatory bowel disease (IBD)-associated virulence gene encoding TonB-linked outer membrane protein (ompW) (Wei et al., 2001) was 83% identical. Four essential genes for synthesizing capsular

2.9. Histology study The histological studies of liver, spleen and colon samples were carried out by Hematoxylin-Eosin (H&E) staining after embedded in paraffin, as described (Al-Hashmi et al., 2011). Images were recorded using Pannoramic digital slide scanner (Pannoramic MIDI II, 3DHISTECH Ltd., Hungary). 3

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Table 2 Comparison of resistance profiles of B. fragilis HCK-B3 and B. fragilis ATCC25285toward antibiotics (μg/ml). Antibiotics

B. fragilis HCK-B3

B. fragilis ATCC25285

Ampicillin Penicillin G Vancomycin Cefoxitin Ceftriaxone Clindamycin Chloromycetin Erythromycin Kanamycin Streptomycin Tetracycline Metronidazole Ciprofloxacin Polymyxin B

MIC≤64 MIC≤16 MIC≤32 MIC≤8 MIC≤128 MIC≤512 MIC≤8 MIC > 1024 MIC > 1024 MIC > 1024 MIC≤32 MIC≤2 MIC≤16 MIC > 1024

MIC≤64 MIC≤32 MIC≤32 MIC≤8 MIC≤128 MIC≤0.25 MIC≤4 MIC≤1 MIC > 1024 MIC≤1024 MIC < 0.125 MIC≤1 MIC≤4 MIC≤512

polysaccharide A (PSA), including two highly reserved open reading frames (upaY and upaZ), one encoding amino sugar synthesis (wcfR) and one for undecaprenyl-phosphate galactose phosphotransferase (wcfS) (Coyne et al., 2001), had a similarity of over 98% in HCK-B3, which is very close to the PSA-producer, B. fragilis ATCC25285. Of the genes expressing β-lactamase, only cepA was confirmed in both strains.

3.3. Antibiotic resistance of B. fragilis HCK-B3 Fig. 2. Impact of B. fragilis HCK-B3 on (A) the body weight and (B) the organ indexes of normal and immunodeficient mice. The organ indexes of the liver and spleen are expressed as the percentage of the corresponding weight of organ and body, while the colon index is expressed as the total length in each animal. Data are displayed as mean ± SD, “**”: p < 0.01 vs CTX group; “#”: p < 0.05 vs CTRL group; and “###”: p < 0.001 vs CTRL group.

The potential antibiotic resistance genes predicted the high survival rate of B. fragilis HCK-B3 under the treatment of macrolide antibiotics like erythromycin, glycopeptide like vancomycin, cationic antimicrobial agents like polymyxin B, chloramphenicol and tetracycline (Supplementary Table 2). According to the classification of clinical resistance determined by an MIC of over 32 μg/ml (D'Aimmo et al., 2007), B. fragilis HCK-B3 was sensitive to penicillin G, vancomycin, cefoxitin, chloromycetin, tetracycline and antibiotics targeting nucleic acid synthesis, especially metronidazole, 2 μg/ml of which was sufficient to suppress the bacterial growth (Table 2). Erythromycin, kanamycin, streptomycin and polymyxin B had little effect on the growth of HCK-B3. However the type strain, B. fragilis ATCC25285 was found to be susceptible to two additional antibiotics, clindamycin and erythromycin.

3.5. Impact of B. fragilis HCK-B3 on body weight and organ indexes Throughout the study, no hair loss, abnormal behaviors, diarrhea or mortality occurred. The cyclophosphamide treatment led to weight reduction, but B. fragilis HCK-B3 barely impacted the body weight of normal or immunosuppressed mice (Fig. 2A). Meanwhile, HCK-B3 did not significantly influence the organ index of the liver or spleen, as determined by the ratio of organ weight to body weight, although hypertrophy of the spleen induced by drug injection was observed (p < 0.001) (Fig. 2B). The colon length of mice in the CTX + BF group was significantly higher than that in the CTX group and was similar to that of the healthy mice (p < 0.01) (Fig. 2B).

3.4. Genomic islands of B. fragilis HCK-B3 In total, only five genomic islands that are over 30 kb were discovered in the genome of B. fragilis HCK-B3 (Table 3) and were mostly identified to be metabolic-related, without integrate virulence expression or transference systems.

3.6. Impact of B. fragilis HCK-B3 on biochemical parameters No

statistically

significant

differences

were

detected

in

Table 3 Genomic islands of over 30 kb identified in B. fragilis HCK-B3. Island no.

genes in island

size (kb)

potential functional genes within island

1 2

g0167-g0219 g0938-g0967

33.3 30.3

3

g1005-g1071

44.3

4

g1023-g1087

40.6

5

g3361-g3444

68.4

tyrosine recombinase (g0167, g0204); peptidase M15 (g0179); protease (g0195) molecular chaperone DnaK (g0938); cell filamentation protein Fic (g0944); methionine sulfoxide reductase (g0946); serine/threonine protein phosphatase (g0965) hemolysin (g1012, g1013); conjugal transfer protein TraN (g1035), TraM (g1036), TraK (g1038), TraJ (g1039), TraG (g1041, g1044, g1053), TraA (g1048) conjugal transfer protein TraN (g1035), TraM (g1036), TraK (g1038), TraJ (g1039), TraG (g1041, g1044, g1053), TraA (g1048); aspartyl-tRNA amidotransferase subunit B (g1081, g1084) metallopeptidase M24 family protein (g3368); gamma carbonic anhydrase family protein (g3369); 3-deoxy-D-manno-octulosonic acid transferase (g3370); putative transmembrane anaerobic C4-dicarboxylate transporter (g3382); L-asparaginase (g3383); cobalamin adenosyltransferase (g3386); tonB-linked outer membrane, SusC/RagA family protein (g3400); histidine kinase (g3408); putative twocomponent response regulator autolysis regulator LytR (g3409); maltose O-acetyltransferase (g3416); 1-deoxy-D-xylulose-5-phosphate synthase (g3421); mannose-1-phosphate guanylyltransferase (g3442)

4

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Table 4 Summary of (A) hematological values and (B) liver parameters in both normal and immunosuppressed mice after 5-day treatment withB. fragilis HCK-B3. Data are displayed as mean ± SD, “#,”“##” and “###”indicate statistically significant differences between the CTX + BF group and the CTRL group (p < 0.05,p < 0.01 and p < 0.001, respectively). (A)

9

WBC (10 /L) Neu (109/L) Lym (109/L) Mon (109/L) Eos (109/L) Bas (109/L) Neu (%) Lym (%) Mon (%) Eos (%) Bas (%) RBC (1012/L) HGB (g/L) HCT (%) MCV (fL) MCH (pg) MCHC (g/L) RDW-CV (%) RDW-SD (fL) PLT (109/L) MPV (fL) PDW (%) PCT (%)

CTRL

BF

CTX

CTX + BF

4.36 ± 1.71 0.57 ± 0.22 3.06 ± 1.19 0.04 ± 0.02 0.02 ± 0.01 0.02 ± 0.01 13.75 ± 3.42 84.35 ± 3.90 1.03 ± 0.44 0.45 ± 0.09 0.43 ± 0.11 10.18 ± 0.18 171.75 ± 2.59 50.48 ± 0.86 49.60 ± 0.31 16.88 ± 0.11 340.25 ± 3.56 12.75 ± 0.56 28.75 ± 1.18 1267.75 ± 63.86 5.38 ± 0.04 15.48 ± 0.04 0.68 ± 0.04

3.24 ± 1.10 0.42 ± 0.14 2.99 ± 0.93 0.02 ± 0.01 0.02 ± 0.01 0.02 ± 0.02 13.00 ± 2.59 85.12 ± 2.82 0.78 ± 0.27 0.55 ± 0.35 0.55 ± 0.26 9.77 ± 0.46 167.50 ± 7.04 48.78 ± 1.83 49.97 ± 0.98 17.12 ± 0.18 343.67 ± 3.68 13.33 ± 0.96 30.15 ± 2.53 1144.17 ± 217.71 5.48 ± 0.18 15.50 ± 0.06 0.63 ± 0.11

2.57 ± 0.32 0.98 ± 0.04## 1.61 ± 0.33 0.09 ± 0.11 0.05 ± 0.02# 0.05 ± 0.02# 37.68 ± 6.95### 56.36 ± 8.94### 2.40 ± 2.21 1.72 ± 0.31### 1.84 ± 0.4### 8.54 ± 0.36### 145.40 ± 5.50### 43.04 ± 1.81### 50.40 ± 0.43# 17.04 ± 0.39 337.60 ± 7.86 13.20 ± 0.62 29.96 ± 1.69 996.33 ± 79.01## 6.20 ± 0.09### 16.04 ± 0.14### 0.56 ± 0.08#

2.12 ± 0.36 0.85 ± 0.16 1.27 ± 0.13 0.08 ± 0.01 0.06 ± 0.04 0.04 ± 0.01 37.74 ± 5.71 55.58 ± 8.57 3.38 ± 0.58 2.08 ± 0.65 1.70 ± 0.61 8.56 ± 0.11 146.71 ± 3.49 43.19 ± 0.63 50.47 ± 0.34 17.13 ± 0.30 339.71 ± 5.82 13.11 ± 0.43 29.74 ± 1.12 1125.00 ± 119.33 6.10 ± 0.12 16.00 ± 0.20 0.67 ± 0.14

(B) CTRL Glu (mmol/L) TC (mmol/L) TG (mmol/L) ALT (U/L) AST (U/L) TBIL (μmol/L) ALP (U/L) TP (g/L) ALB (g/L) CK (U/L) LDH (U/L)

5.61 ± 0.57 2.08 ± 0.17 1.33 ± 0.27 16.30 ± 5.32 126.68 ± 14.58 1.65 ± 0.09 19.00 ± 3.74 44.85 ± 2.25 28.30 ± 0.79 1293.60 ± 299.90 696.23 ± 323.24

BF

CTX

6.61 ± 0.74 1.89 ± 0.28 1.16 ± 0.32 15.45 ± 8.79 122.70 ± 13.21 1.65 ± 0.19 17.00 ± 6.63 41.35 ± 2.57 26.05 ± 1.76 1272.06 ± 387.09 669.75 ± 202.04

CTX + BF #

7.08 ± 1.10 1.62 ± 0.17## 1.60 ± 0.33 16.65 ± 1.59 135.45 ± 25.39 1.88 ± 0.29 13.20 ± 10.50 42.15 ± 1.81 26.28 ± 2.46 1025.50 ± 493.59 598.40 ± 107.53

7.02 ± 0.75 1.86 ± 0.20 1.25 ± 0.24 17.33 ± 2.95 120.36 ± 12.80 2.07 ± 0.25 12.86 ± 1.36 44.06 ± 3.23 27.60 ± 1.94 988.33 ± 244.23 528.78 ± 23.80

Abbreviations: WBC, white blood cells; Neu, neutrophils; Lym, lymphocytes; Mon, monocytes; Eos, eosinophils; Bas, basophils; RBC, red blood cells; HGB, hemoglobin concentration; HCT, hematocrit value; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; RDW, red cell distribution width; CV, coefficient of variation; SD, standard deviation; PLT, platelet; MPV, mean platelet volume; PDW, platelet distribution width; PCT, thrombocytocrit. Abbreviations: Glu, glucose; TC, total cholesterol; TG, triglyceride; ALT, alanine aminotransferase; AST, aspartate aminotransferase; TBIL, total bilirubin; ALP, alkaline phosphatase; TP, total protein; ALB, albumin; CK, creatine kinase; LDH, lactic dehydrogenase.

hematological measurements (Table 4A) or liver parameters (Table 4B) during B. fragilis treatment of both normal and immunosuppressed mice. Notably, the cyclophosphamide injection remarkably changed the concentration of the hematological parameters, and significantly downregulated total cholesterol in serum, which were, to some extent, restored by oral administration of HCK-B3. 3.7. Impact of B. fragilis HCK-B3 on cytokine production As shown in Fig. 3, the 5-day treatment of B. fragilis HCK-B3 did not notably influence the cytokine production in normal mice. The disturbed cytokine levels induced by cyclophosphamide administration were restored by B. fragilis HCK-B3 treatment, especially the secretion of IL-10, which was significantly upregulated (p < 0.05).

Fig. 3. Impact of B. fragilis HCK-B3 on cytokine productions in normal and immunodeficient mice. Data are displayed as mean ± SD, “*”: p < 0.05 vs CTX group.

B3 (Fig. 4). Other than liver and colon, the histological structure of spleen was severely destroyed by CTX injection. The boundaries of red and white pulp in the spleen of mice from CTX group were blurry. And the splenic cells were irregularly aligned, with fibrosis and hemorrhage.

3.8. Impact of B. fragilis HCK-B3 on tissue histology No obvious histopathological damage was observed in the liver, spleen or colon tissues of the healthy mice treated with B. fragilis HCK5

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Fig. 4. Impact of B. fragilis HCK-B3 on the tissue histology of normal and immunodeficient mice.

of bacteria, as well as inhibition of the proliferation of lymphocytes and the production of lymphokines and immunoglobulins (Hillman et al., 1993). Fortunately, neither of these pathogenic factors were found in the novel strain HCK-B3. Bacteroides species are regarded as opportunistic pathogens due to the contributions to intestinal inflammation via producing toxins such as bft, a metalloprotease-type toxin (Wick and Sears, 2010), and ompW, one of the bacterial pANCA antigens isolated from IBD patients (Wei et al., 2001). HCK-B3 was confirmed to be bft-free, but possessed genes with high similarity to ompW. However, apart from homology with RagA from Porphyromonas gingivalis (Wei et al., 2001), which is correlated with tissue damage, the Ton-B complex is also an essential receptor for the utilization of complex polysaccharides via the starchutilization system C (SusC), which is considered fundamental to the exclusively powerful carbohydrates-utilization characteristics of Bacteroides species (Sonnenburg et al., 2006). Therefore, the pathogenesis of the ompW-like genes in HCK-B3 requires further determination. Although the T6SS is considered as toxin delivery system in Proteobacteria species, it acts as prevalent antagonistic system in Bacteroidales under limited conditions when sensitive bacterial cells contact during occupation of the same niche (Chatzidaki-Livanis et al., 2016). Moreover, apart from clpV and clpB, multiple core proteins such as VgrG and Hcp which are crucial to the T6SS (Coyne et al., 2016) are absent in B. fragilis HCK-B3. Compared with the non-enterotoxigenic B. fragilis ATCC25285, the conserved genes for synthesizing PSA were confirmed in HCK-B3, which forecasted the existence of the capsular polysaccharides. It is manifested that the PSA-producing B. fragilis would facilitate inflammation and abscess formation after entering the abdominal cavity to initiate IL-10 producing T cells and avoid excessive inflammatory responses and prevent diseases (Cohen-Poradosu et al., 2011; Mazmanian et al., 2008), which may explain the significantly upregulated NF-κB signals in the colon and intestinal permeability during HCK-B3 administration (Tan et al., 2019). B. fragilis HCK-B3 is resistant to penicillin G and cefoxitin, which is consensus with the facts that B. fragilis is more resistant to β-lactam antibiotics compared to other Bacteroides species(Sutter and Finegold, 1976). Moreover, the three genes encoding β-lactamase in B. fragilis, including cfxA for class A cephalosporinase, cfiA for class B metallo-βlactamase and cepA for endogenous cephalosporinase (Garcia et al., 2008), were partially identified in HCK-B3; especially the low similarity of cfxA, which is associated with the conjugative transposon Tn4555 and responsible for transference, indicates a low likelihood of

However, the oral administration of B. fragilis HCK-B3 did not perform aggravating effects to the CTX-treated mice (Fig. 4). 4. Discussion Commercialized probiotics, mainly corresponding to lactic acid bacteria, are generally isolated from traditional fermented foods with a long history of consumption and thereby rarely create safety concerns. However, safety assessment is crucial for authorized intake of beneficial intestinal candidates with a wider variety and greater efficiency of modulatory effects. Yet, no specific regulations for the applications of these next-generation probiotics have been officially released. Based on our previous findings that B. fragilis HCK-B3 exhibited little effects on the production of secretory immunoglobulin A (sIgA) and chemokine (C-X-C motif) ligand 2 (CXCL2) and the balance of Tregs/Th-17 cells, and even promoted the diversity of the intestinal microbiota (Tan et al., 2019), in this study, a pilot and systematic safety evaluation of B. fragilis HCK-B3 was carried out according to the guidelines for developing traditional probiotics suggested by FAO/WHO which emphasize the importance of strain preservation, acquisition of the integrated bacterial characterizations including original source, cultivated history, phenotype, genotype, antibiotic resistance, and virulence factors, and three-step clinical trials, including safety assessment and functional characterization in vitro and animal models; double blind, randomized, placebo-controlled human studies; and efficiency comparisons with standard treatments, as well as safety studies of traditional and nextgeneration probiotics, some of which have already been authorized as food ingredients (Fernandez-Murga and Sanz, 2016; Jia et al., 2011; Tan et al., 2018a; Ulsemer et al., 2012a, 2012b; Wang et al., 2017; Yakabe et al., 2009). The search-and-locate mechanism (motility and chemotaxis) gives motile bacterial cells competitive advantages over non-motile cells in the intestinal ecosystem, in terms of nutrient acquisition, niche colonization (Lane et al., 2007; Neville et al., 2012), biofilm formation (Houry et al., 2010) and virulence protein secretion by pathogens (Konkel et al., 2004). Furthermore, flagellin, a well-characterized microbial-associated molecular pattern, secreted by motile bacteria has been confirmed to be pro-inflammatory as it stimulates IL-8 production from human intestinal epithelial cells by activating the NF-κB pathway via Toll-like receptor 5 (Neville et al., 2012). Hemolysins produced by bacteria contribute to the direct lysis of a variety of cell types and damages in vascular permeability, which lead to intercellular propagation 6

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transmission of β-lactamase between bacteria. Only one fifth of B. fragilis strains have been identified to be resistant to clindamycin (Nakano and Avila-Campos, 2004), whereas HCK-B3 and ATCC25285 displayed opposite responses to this antibiotic. Genomic islands containing virulence-related genes are considered as pathogenicity island (PAI), which are generally over 30 kb, commonly existed in pathogenic species, and would be easily exchanged among bacteria (Hacker et al., 1997; Hacker and Kaper, 2000). Although there are hemolysin-associated genes found in one of the genomic islands of HCK-B3, the previous experiment had confirmed the negative of hemolysis, which indicate that this potential PAI is unlikely to be expressed. Meanwhile, no plasmid was detected in the genome of HCK-B3. which prevents the possibility of transferring the any potential characteristics of virulence or antibiotic resistance to other intestinal commensals. The acute toxicity of HCK-B3 was assessed by administering a moderate daily dose of 109 cfu of bacterial cells to both normal and CTX-induced immune-deficient mice. The hematological and liver parameters, serum cytokine concentrations and the histological analysis revealed that HCK-B3 had no side effects on healthy animals, suggesting that 3.5 × 1012 cfu of live HCK-B3 cells would be safe for a 70 kg healthy human adult. Previous researches have suggested that the best concentration for applications of microorganisms after lyophilization and rehydration which guarantees both the viability and integrity of cells is109 cfu/g (Miyamoto-Shinohara et al., 2000).The bacterial dose used in both the previous characterization study and the current safety assessment is therefore of feasibility for industrial applications. Cyclophosphamide is one of the most frequently used chemotherapeutic treatments for leukemia, multiple myeloma, lymphoma and autoimmune diseases, and pretreatments of bone marrow transplantation (Salva et al., 2014). The effect of CTX is dose dependent, which high dose (> 100 mg/kg) leading to immunosuppression can be applied in myeloablative therapy and low dose or in combination facilitates antitumor immuno-augmentation therapies (Ehrke, 2003). Correspondingly, hepatotoxic effects of CTX were observed according to the biochemical parameters, of which total cholesterol (p < 0.01) and glucose (p < 0.05) were deviant to that of the control group; abnormal indexes of spleen, serious injuries in the histological structure of the spleen, reductions in lymphocytes (p < 0.001), hemoglobins (p < 0.001) and platelets (p < 0.01), and decreases in body weight, revealing remarkable dysfunctions in the immune system of the mice, and the increase of spleen cellularity was considered as the consequence of myeloid cell expansion during recovery (Sefc et al., 2003). Furthermore, throughout the 5-day administration to the immunosuppressed animals, B. fragilis HCK-B3 did not display adverse effects in liver which is the first target organ detoxifying xenobiotics (Singh et al., 2018), spleen which is the most important immune organ (Zong et al., 2018) and intestinal mucosa which is the essential barrier for potential pathogenic microorganisms and metabolites (Munoz et al., 2011). Rather, it improved their health conditions, as shown by remarkable recoveries of the colon length and immune system, via modulating cytokine productions to normal levels and repairing the CTX-induced damage in biochemical parameters, although to a nonsignificant extent.

conditions of bacterial invasion (Ulsemer et al., 2012b). Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 31871773 and 31470161, and Key Program No. 31530056), the Natural Science Foundation of Jiangsu Province (BK20160175), the National First-Class Discipline Program of Food Science and Technology (JUFSTR20180102), and the Collaborative Innovation Center of Food Safety and Quality Control in Jiangsu Province. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.fct.2019.110934. References Al-Hashmi, S., Hassan, Z., Sadeghi, B., Rozell, B., Hassan, M., 2011. Dynamics of early histopathological changes in GVHD after busulphan/cyclophosphamide conditioning regimen. Int. J. Clin. Exp. Pathol. 4, 596–605. Brodmann, T., Endo, A., Gueimonde, M., Vinderola, G., Kneifel, W., de Vos, W.M., Salminen, S., Gomez-Gallego, C., 2017. Safety of novel microbes for human consumption: practical examples of assessment in the European Union. Front. Microbiol. 8, 15. Chatzidaki-Livanis, M., Geva-Zatorsky, N., Comstock, L.E., 2016. Bacteroides fragilis type VI secretion systems use novel effector and immunity proteins to antagonize human gut Bacteroidales species. Proc. Natl. Acad. Sci. U.S.A. 113, 3627–3632. Chen, L.H., Xiong, Z.H., Sun, L.L., Yang, J., Jin, Q., 2012. VFDB 2012 update: toward the genetic diversity and molecular evolution of bacterial virulence factors. Nucleic Acids Res. 40, D641–D645. Cohen-Poradosu, R., McLoughlin, R.M., Lee, J.C., Kasper, D.L., 2011. Bacteroides fragilisstimulated interleukin-10 contains expanding disease. J. Infect. Dis. 204, 363–371. Cousin, F.J., Lynch, S.M., Harris, H.M.B., McCann, A., Lynch, D.B., Neville, B.A., Irisawa, T., Okada, S., Endo, A., O'Toole, P.W., 2015. Detection and genomic characterization of motility in Lactobacillus curvatus: confirmation of motility in a species outside the Lactobacillus salivarius clade. Appl. Environ. Microbiol. 81, 1297–1308. Coyne, M.J., Roelofs, K.G., Comstock, L.E., 2016. Type VI secretion systems of human gut Bacteroidales segregate into three genetic architectures, two of which are contained on mobile genetic elements. BMC Genomics 17, 21. Coyne, M.J., Tzianabos, A.O., Mallory, B.C., Carey, V.J., Kasper, D.L., Comstock, L.E., 2001. Polysaccharide biosynthesis locus required for virulence of Bacteroides fragilis. Infect. Immun. 69, 4342–4350. D'Aimmo, M.R., Modesto, M., Biavati, B., 2007. Antibiotic resistance of lactic acid bacteria and Bifidobacterium spp. isolated from dairy and pharmaceutical products. Int. J. Food Microbiol. 115, 35–42. Ehrke, M.J., 2003. Immunomodulation in cancer therapeutics. Int. Immunopharmacol. 3, 1105–1119. Fernandez-Murga, M.L., Sanz, Y., 2016. Safety assessment of Bacteroides uniformis CECT 7771 isolated from stools of healthy breast-fed infants. PLoS One 11, 21. Garcia, N., Gutierrez, G., Lorenzo, M., Garcia, J.E., Piriz, S., Quesada, A., 2008. Genetic determinants for cfxA expression in Bacteroides strains isolated from human infections. J. Antimicrob. Chemother. 62, 942–947. Gutacker, M., Valsangiacomo, C., Bernasconi, M.V., Piffaretti, J.C., 2002. recA and glnA sequences separate the Bacteroides fragilis population into two genetic divisions associated with the antibiotic resistance genotypes cepA and cfiA. J. Med. Microbiol. 51, 123–130. Gutacker, M., Valsangiacomo, C., Piffaretti, J.C., 2000. Identification of two genetic groups in Bacteroides fragilis by multilocus enzyme electrophoresis: distribution of antibiotic resistance (cfiA, cepA) and enterotoxin (bft) encoding genes. MicrobiologyUk 146, 1241–1254. Hacker, J., BlumOehler, G., Muhldorfer, I., Tschape, H., 1997. Pathogenicity islands of virulent bacteria: structure, function and impact on microbial evolution. Mol. Microbiol. 23, 1089–1097. Hacker, J., Kaper, J.B., 2000. Pathogenicity islands and the evolution of microbes. Annu. Rev. Microbiol. 54, 641–679. Hillman, J.D., Maiden, M.F.J., Pfaller, S.P., Martin, L., Duncan, M.J., Socransky, S.S., 1993. Characterization of hemolytic bacteria in subgingival plaque. J. Periodontal. Res. 28, 173–179. Hirsh, M., Carmel, J., Kaplan, V., Livne, E., Krausz, M.M., 2004. Activity of lung neutrophils and matrix metalloproteinases in cyclophosphamide-treated mice with

5. Conclusions The novel strain of B. fragilis HCK-B3, which was selected for its beneficial potentials to alleviate LPS infection-induced dysfunctions in vivo, was confirmed to be non-toxigenic and did not display adverse effects in both healthy and immune-deficient mice at a routinely applicable dose. We acknowledged the pilot aspects of this study, and further investigations featuring variable doses in both animal models and human volunteers should be performed before safe applications can be suggested. Moreover, intraperitoneal injection of live cells should be considered to simulate extremely uncomfortable and impossible 7

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leads to high-level beta-lactamase expression in Bacteroides fragilis clinical isolates. J. Bacteriol. 176, 4376–4384. Salva, S., Marranzino, G., Villena, J., Aguero, G., Alvarez, S., 2014. Probiotic Lactobacillus strains protect against myelosuppression and immunosuppression in cyclophosphamide-treated mice. Int. Immunopharmacol. 22, 209–221. Salvetti, E., Orru, L., Capozzi, V., Martina, A., Lamontanara, A., Keller, D., Cash, H., Felis, G.E., Cattivelli, L., Torriani, S., Spano, G., 2016. Integrate genome-based assessment of safety for probiotic strains: Bacillus coagulans GBI-30, 6086 as a case study. Appl. Microbiol. Biotechnol. 100, 4595–4605. Sefc, L., Psenak, O., Sykora, V., Sulc, K., Necas, E., 2003. Response of hematopoiesis to cyclophosphamide follows highly specific patterns in bone marrow and spleen. J. Hematother. Stem Cell Res. 12, 47–61. Singh, C., Prakash, C., Tiwari, K.N., Mishra, S.K., Kumar, V., 2018. Premna integrifolia ameliorates cyclophosphamide-induced hepatotoxicity by modulation of oxidative stress and apoptosis. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie 107, 634–643. Sonnenburg, E.D., Sonnenburg, J.L., Manchester, J.K., Hansen, E.E., Chiang, H.C., Gordon, J.I., 2006. A hybrid two-component system protein of a prominent human gut symbiont couples glycan sensing in vivo to carbohydrate metabolism. Proc. Natl. Acad. Sci. U.S.A. 103, 8834–8839. Sutter, V.L., Finegold, S.M., 1976. Susceptibility of anaerobic bacteria to 23 antimicrobial agents. Antimicrob. Agents Chemother. 10, 736–752. Tan, H.Z., O'Toole, P.W., 2015. Impact of diet on the human intestinal microbiota. Curr. Opin. Food Sci. 2, 71–77. Tan, H.Z., Yu, Z.M., Wang, C., Zhang, Q.S., Zhao, J.X., Zhang, H., Zhai, Q.X., Chen, W., 2018a. Pilot safety evaluation of a novel strain of Bacteroides ovatus. Front. Genet. 9, 12. Tan, H.Z., Zhao, J.X., Zhang, H., Zhai, Q.X., Chen, W., 2018b. Isolation of low-abundant Bacteroidales in the human intestine and the analysis of their differential utilization based on plant-derived polysaccharides. Front. Microbiol. 9, 10. Tan, H.Z., Zhao, J.X., Zhang, H., Zhai, Q.X., Chen, W., 2019. Novel strains of Bacteroides fragilis and Bacteroides ovatus alleviate the LPS-induced inflammation in mice. Appl. Microbiol. Biotechnol. 103, 2353–2365. Troy, E.B., Kasper, D.L., 2010. Beneficial effects of Bacteroides fragilis polysaccharides on the immune system. Front. Biosci. 15, 25–34. Ulsemer, P., Toutounian, K., Schmidt, J., Karsten, U., Goletz, S., 2012a. Preliminary safety evaluation of a new Bacteroides xylanisolvens isolate. Appl. Environ. Microbiol. 78, 528–535. Ulsemer, P., Toutounian, K., Schmidt, J., Leuschner, J., Karsten, U., Goletz, S., 2012b. Safety assessment of the commensal strain Bacteroides xylanisolvens DSM 23964. Regul. Toxicol. Pharmacol. 62, 336–346. Wang, Y., Deng, H.M., Li, Z.C., Tan, Y.F., Han, Y.P., Wang, X.Y., Du, Z.M., Liu, Y.Y., Yang, R.F., Bai, Y., Bi, Y.J., Zhi, F.C., 2017. Safety evaluation of a novel strain of Bacteroides fragilis. Front. Microbiol. 8, 10. Wei, B., Dalwadi, H., Gordon, L.K., Landers, C., Bruckner, D., Targan, S.R., Braun, J., 2001. Molecular cloning of a Bacteroides caccae TonB-linked outer membrane protein identified by an inflammatory bowel disease marker antibody. Infect. Immun. 69, 6044–6054. Wick, E.C., Sears, C.L., 2010. Bacteroides spp. and diarrhea. Curr. Opin. Infect. Dis. 23, 470–474. Yakabe, T., Moore, E.L., Yokota, S., Sui, H., Nobuta, Y., Fukao, M., Palmer, H., Yajima, N., 2009. Safety assessment of Lactobacillus brevis KB290 as a probiotic strain. Food Chem. Toxicol. 47, 2450–2453. Zhang, W.D., Zhu, B., Xu, J.H., Liu, Y.Y., Qiu, E.Q., Li, Z.J., Li, Z.C., He, Y., Zhou, H.W., Bai, Y., Zhi, F.C., 2018. Bacteroides fragilis protects against antibiotic-associated diarrhea in rats by modulating intestinal defenses. Front. Immunol. 9, 15. Zong, S., Li, J.L., Yang, L., Huang, Q.L., Ye, Z.Y., Hou, G.H., Ye, M., 2018. Synergistic antitumor effect of polysaccharide from Lachnum sp in combination with cyclophosphamide in hepatocellular carcinoma. Carbohydr. Polym. 196, 33–46.

experimental sepsis. Int. J. Exp. Pathol. 85, 147–157. Houry, A., Briandet, R., Aymerich, S., Gohar, M., 2010. Involvement of motility and flagella in Bacillus cereus biofilm formation. Microbiology-(UK) 156, 1009–1018. Hsiao, E.Y., McBride, S.W., Hsien, S., Sharon, G., Hyde, E.R., McCue, T., Codelli, J.A., Chow, J., Reisman, S.E., Petrosino, J.F., Patterson, P.H., Mazmanian, S.K., 2013. Microbiota modulate behavioral and physiological abnormalities associated with neurodevelopmental disorders. Cell 155, 1451–1463. Jia, X., Wang, W., Song, Y., Li, N., 2011. A 90-day oral toxicity study on a new strain of Lactobacillus paracasei in rats. Food Chem. Toxicol. 49, 1148–1151. Konkel, M.E., Klena, J.D., Rivera-Amill, V., Monteville, M.R., Biswas, D., Raphael, B., Mickelson, J., 2004. Secretion of virulence proteins from Campylobacter jejuni is dependent on a functional flagellar export apparatus. J. Bacteriol. 186, 3296–3303. Krieg, N.R., Ludwig, W., Euzeby, J., Whitman, W.B., 2001. Bergey's Manual of Systematic Bacteriology, second ed. Springer, New York. Lane, M.C., Alteri, C.J., Smith, S.N., Mobley, H.L.T., 2007. Expression of flagella is coincident with uropathogenic Escherichia coli ascension to the upper urinary tract. Proc. Natl. Acad. Sci. U.S.A. 104, 16669–16674. Li, R.Q., Li, Y.R., Kristiansen, K., Wang, J., 2008. SOAP: short oligonucleotide alignment program. Bioinformatics 24, 713–714. Li, R.Q., Zhu, H.M., Ruan, J., Qian, W.B., Fang, X.D., Shi, Z.B., Li, Y.R., Li, S.T., Shan, G., Kristiansen, K., Li, S.G., Yang, H.M., Wang, J., Wang, J., 2010. De novo assembly of human genomes with massively parallel short read sequencing. Genome Res. 20, 265–272. Li, Z.C., Deng, H.M., Zhou, Y.Z., Tan, Y.F., Wang, X.Y., Han, Y.P., Liu, Y.Y., Wang, Y., Yang, R.F., Bi, Y.J., Zhi, F.C., 2017. Bioluminescence imaging to track Bacteroides fragilis inhibition of Vibrio parahaemolyticus infection in mice. Front. Cell. Infect. Microbiol. 7, 9. Lu, B.X., Leong, H.W., 2016. Computational methods for predicting genomic islands in microbial genomes. Comput. Struct. Biotechnol. J. 14, 200–206. Mazmanian, S.K., Round, J.L., Kasper, D.L., 2008. A microbial symbiosis factor prevents intestinal inflammatory disease. Nature 453, 620–625. McArthur, A.G., Waglechner, N., Nizam, F., Yan, A., Azad, M.A., Baylay, A.J., Bhullar, K., Canova, M.J., De Pascale, G., Ejim, L., Kalan, L., King, A.M., Koteva, K., Morar, M., Mulvey, M.R., O'Brien, J.S., Pawlowski, A.C., Piddock, L.J.V., Spanogiannopoulos, P., Sutherland, A.D., Tang, I., Taylor, P.L., Thaker, M., Wang, W.L., Yan, M., Yu, T., Wright, G.D., 2013. The comprehensive antibiotic resistance database. Antimicrob. Agents Chemother. 57, 3348–3357. Miyamoto-Shinohara, Y., Imaizumi, T., Sukenobe, J., Murakami, Y., Kawamura, S., Komatsu, Y., 2000. Survival rate of microbes after freeze-drying and long-term storage. Cryobiology 41, 251–255. Munoz, J.A.M., Chenoll, E., Casinos, B., Bataller, E., Ramon, D., Genoves, S., Montava, R., Ribes, J.M., Buesa, J., Fabrega, J., Rivero, M., 2011. Novel probiotic Bifidobacterium longum subsp infantis CECT 7210 strain active against rotavirus infections. Appl. Environ. Microbiol. 77, 8775–8783. Nakano, V., Avila-Campos, M.J., 2004. Virulence markers and antimicrobial susceptibility of bacteria of the Bacteroides fragilis group isolated from stool of children with diarrhea in Sao Paulo, Brazil. Mem. Inst. Oswaldo Cruz 99, 307–312. Neville, B.A., Forde, B.M., Claesson, M.J., Darby, T., Coghlan, A., Nally, K., Ross, R.P., O'Toole, P.W., 2012. Characterization of pro-inflammatory flagellin proteins produced by Lactobacillus ruminis and related motile Lactobacilli. PLoS One 7, 12. O'Toole, P.W., Marchesi, J.R., Hill, C., 2017. Next-generation probiotics: the spectrum from probiotics to live biotherapeutics. Nat. Microbiol 2, 6. Ramamurthy, D., Pazhani, G.P., Sarkar, A., Nandy, R.K., Rajendran, K., Sur, D., Manna, B., Ramamurthy, T., 2013. Case-control study on the role of enterotoxigenic Bacteroides fragilis as a cause of diarrhea among children in Kolkata, India. PLoS One 8, 7. Robertson, K.P., Smith, C.J., Gough, A.M., Rocha, E.R., 2006. Characterization of Bacteroides fragilis hemolysins and regulation and synergistic interactions of HlyA and HlyB. Infect. Immun. 74, 2304–2316. Rogers, M.B., Bennett, T.K., Payne, C.M., Smith, C.J., 1994. Insertional activation of cepA

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