nutrients

Article butyricum MIYAIRI 588 Increases the Lifespan and Multiple-Stress Resistance of Caenorhabditis elegans

Maiko Kato †, Yumi Hamazaki †, Simo Sun, Yoshikazu Nishikawa * and Eriko Kage-Nakadai *

Graduate School of Human Life Science, Osaka City University, Osaka 558-8585, Japan; [email protected] (M.K.); [email protected] (Y.H.); [email protected] (S.S.) * Correspondence: [email protected] (Y.N.); [email protected] (E.K.-N.); Tel.: +81-6-6605-2900 (Y.N.); +81-6-6605-2856 (E.K.-N.) † These authors equally contributed to this work.

 Received: 26 October 2018; Accepted: 2 December 2018; Published: 5 December 2018 

Abstract: Clostridium butyricum MIYAIRI 588 (CBM 588), one of the bacterial strains used for humans and domestic animals, has been reported to exert a variety of beneficial health effects. The effect of this probiotic on lifespan, however, is unknown. In the present study, we investigated the effect of CBM 588 on lifespan and multiple-stress resistance using Caenorhabditis elegans as a model animal. When adult C. elegans were fed a standard diet of Escherichia coli OP50 or CBM 588, the lifespan of the animals fed CBM 588 was significantly longer than that of animals fed OP50. In addition, the animals fed CBM588 exhibited higher locomotion at every age tested. Moreover, the worms fed CBM 588 were more resistant to certain stressors, including infections with pathogenic , UV irradiation, and the metal stressor Cu2+. CBM 588 failed to extend the lifespan of the daf-2/insulin-like receptor, daf-16/FOXO and skn-1/Nrf2 mutants. In conclusion, CBM 588 extends the lifespan of C. elegans probably through regulation of the insulin/IGF-1 signaling (IIS) pathway and the Nrf2 transcription factor, and CBM 588 improves resistance to several stressors in C. elegans.

Keywords: Clostridium butyricum; lifespan; stress resistance; Caenorhabditis elegans

1. Introduction are defined as live that, when administered in adequate amounts, confer health benefits to the host [1]. Clostridium butyricum, which is an anaerobic, Gram-positive, spore-forming , is one of the commercialized probiotic bacteria. C. butyricum is distributed in a variety of environments and in the gut of various animals, including humans [2]. Several studies have described the prevalence of C. butyricum; 44% of fecal samples collected from asymptomatic preterm neonates were positive for C. butyricum [3], and C. butyricum was identified in 10% to 20% of human fecal samples by microbial culture [4]. C. butyricum MIYAIRI 588 (CBM 588) is used as an anti-diarrheal medicine in Japan. In clinical practice, the administration of CBM 588 was shown to prevent antibiotic-associated diarrhea in children [5]. Numerous experimental studies have demonstrated the beneficial properties of this probiotic, including the induction of interleukin-10 (IL-10)-producing macrophages in an experimental mouse model of colitis [6], the promotion of regulatory T-cell generation in the intestine through transformation of growth factor-ß (TGF-ß) from dendritic cells [7], the improvement of high-fat diet-induced non-alcoholic fatty liver disease in rats [8], the inhibition of the cytotoxic effect of Clostridium difficile in vitro [9], and the prevention of enterohemorrhagic Escherichia coli O157:H7

Nutrients 2018, 10, 1921; doi:10.3390/nu10121921 www.mdpi.com/journal/nutrients Nutrients 2018, 10, 1921 2 of 13 infection [10] and gastric ulcers in mice [11]. The effect of C. butyricum on longevity, however, is unknown. Caenorhabditis elegans, a free-living nematode that feeds on bacteria, is a powerful experimental model due to its ease of cultivation, its short and reproducible lifespan, and the abundance of genetic tools available for studying this organism [12]. Although C. elegans was originally isolated from rich soil or compost [13], where it is mostly found in the dauer stage [14,15], feeding and reproducing stages of this species have been found in microbe-rich habitats such as rotting fruit and plant matter [16,17]. Three independent studies that provided the first description of the microbiome of C. elegans [18–20], and a meta-analysis of the data [21], showing possible functions of the naturally associated bacteria; e.g., the microbes enhance C. elegans fitness under standard and also stressful conditions, and confer resistance to infection [19]. Specific microbes correlate with the population state of C. elegans and the bacterial influence is not simply nutritional [20]. In the laboratory, C. elegans is typically maintained on agar plates and fed Escherichia coli laboratory strains (e.g., OP50) [22]. We previously showed that feeding on lactic acid bacteria, including lactobacilli and bifidobacteria, extends the lifespan of worms compared with feeding on E. coli OP50 [23]. Afterwards, several lines of investigation addressed the interactions between worms and probiotic bacteria, yielding insights into mechanisms by which probiotic bacteria enhance immune responses and increase lifespan in the C. elegans host [24,25]. Thus, C. elegans has been an accepted model for investigating dietary manipulation with probiotics [26,27]. In the present study, we examined the influence of CBM 588 on the lifespan of C. elegans. CBM 588 was shown to extend the lifespan probably through regulation of the insulin/IGF-1 signaling (IIS) pathway and the Nrf2 transcription factor and to improve resistance to several stressors in C. elegans.

2. Materials and Methods

2.1. Bacterial Strain and Culture Conditions Escherichia coli OP50, which is used as the standard feed for C. elegans cultivation, was grown on tryptone soya agar (Nissui Pharmaceutical, Tokyo, Japan) at 37 ◦C. Clostridium butyricum MIYAIRI 588 (CBM 588), which was kindly provided by Miyarisan Pharmaceutical, was cultured on GAM agar (Nissui Pharmaceutical, Japan). The Salmonella enterica serovar Enteritidis strain SE1, originally isolated from a diarrheal specimen and used as a pathogen, was grown on tryptone soya agar at 37 ◦C. Similarly, Staphylococcus aureus NBRC13276 was cultured on tryptone soya agar. The cultured bacteria were scraped and weighed. Aliquots (100 mg wet weight) of bacteria were suspended in 0.5 mL of M9 buffer (5 mM potassium phosphate, 1 mM CaCl2, and 1 mM MgSO4). 50 µL of the bacterial suspension was spread onto NGM modified to be peptone free (mNGM) plates (1.7% (w/v) agar, 50 mM NaCl, 1 mM CaCl2, 5 µg/mL cholesterol, 25 mM KH2PO4, and 1 mM MgSO4) (10 mg bacteria per plate), and used in the experimental assays.

2.2. C. elegans Strains and Culture Conditions The wild-type C. elegans strain Bristol N2 and the following derivative mutant strains were obtained from the Caenorhabditis Genetics Center: CB1370 daf-2(e1370), no outcross; CF1038 daf-16(mu86), outcrossed with N2 eleven times by the Kenyon lab.; and VC1772 skn-1(ok2315), outcrossed one time by the Moerman lab. These C. elegans strains were maintained using standard techniques [22]. The animals were cultured as follows: eggs were prepared from adult C. elegans by exposure to a sodium hypochlorite/sodium hydroxide solution. The egg suspension was incubated in M9 buffer for one day at 25 ◦C to allow for hatching and synchronization, and the resulting suspension of synchronized L1-stage worms was centrifuged at 156× g for 1 min. After the removal of the supernatant by aspiration, the remaining larvae were transferred onto mNGM plates covered with 10 mg of OP50. The transferred worms were cultured at 25 ◦C for two days (referred to as three-day-old animals). daf-2(e1370) animals were grown at 15 ◦C, the permissive temperature, until they reached the young adult stage. Nutrients 2018, 10, 1921 3 of 13

2.3. Determination of the C. elegans Lifespan Lifespan assays were performed as follows: synchronized three-day-old (young adult) animals (35 animals per plate) were placed on a 5-cm mNGM plate covered with 10 mg of OP50 alone, CBM 588 alone, or a mixture of 5 mg each of OP50 and CBM 588, and then, the plates were incubated at 25 ◦C. The animals were transferred daily to fresh plates for the first four days, and thereafter, they were transferred every other day. The number of live and dead animals was recorded every day. An animal was considered dead when it failed to respond to a gentle touch with a worm picker. Animals that crawled off the plate or died from internal hatching were considered lost and not included in the analysis. Each assay was carried out in duplicate (two plates). Two independent replicates were performed except for experiments using UV-killed bacteria and mutant analysis using daf-2(e1370). The data from the replicates were merged, and presented. Worm survival was calculated by the Kaplan-Meier method, and differences in survival were analyzed using the log-rank test. The mean lifespan (MLS) was estimated using the following formula [28]:

1 xj + xj+1 MLS = ∑ dj (1) N j 2 where dj is the number of worms that died in the age interval (xj to xj+1), and N is the total number of worms. The standard error (SE) of the estimated mean lifespan was calculated using the following equation: v u 2 1 xj + xj+1 SE = u ( − MLS) d (2) t ( − ) ∑ j N N 1 j 2

Maximum lifespan was calculated as the mean lifespan of the longest-living 15% of the worms in each group.

2.4. Measurement of Body Size Three-day-old adult worms were placed on mNGM plates covered with lawns of OP50, CBM 588, or a mixture of the two strains. The plates were incubated at 25 ◦C, and the body size of the live worms was measured every 24 h until the worms reached an age of 10 days. Images of adult nematodes were taken with an M165FC stereoscopic microscope (Leica microsystems, Wetzlar, Germany) equipped with a DFC425C camera (Leica microsystems, Germany) and analyzed using ImageJ software. The area of a worm’s projection was used as an index of the body size.

2.5. Measurement of Brood Size Two L4-stage hermaphrodites were transferred to an mNGM plate covered with a lawn of OP50, CBM 588, or a mixture of the two strains. The parental animals were transferred to fresh mNGM plates every 24 h until the end of the reproductive period. The resulting eggs were left to hatch, and the number of progeny was then determined. Two independent replicates were tested to verify reproducibility, and the merged data were statistically analyzed and presented.

2.6. Measurement of Locomotion Three-day-old adult worms were placed on mNGM plates covered with lawns of OP50, CBM 588, or a mixture of the two strains. The plates were incubated at 25 ◦C, and the body size of live worms was measured every other day from days 7 to 23. The motility of the worms was then examined using the scoring method described in previous reports [25,29,30]. Briefly, worms were classified as class “A” when they exhibited spontaneous movement or vigorous locomotion in response to prodding; class “B” worms were those that did not move unless prodded or appeared to have uncoordinated movement; and class “C” worms were those that moved only their head and/or tail in response to Nutrients 2018, 10, 1921 4 of 13 prodding. Dead worms were classified as class “D”. Two independent replicates were tested, and the merged data were statistically analyzed and presented.

2.7. Stress Resistance Assays Worms were grown from an age of 3 days on mNGM plates containing OP50 or CBM 588 and then subjected to stress resistance assays. The bacterial infection was performed as previously shown [23]. Briefly, 7-day-old worms were transferred onto mNGM plate covered with 10 mg of Staphylococcus aureus or Salmonella instead of OP50 or CBM 588, cultured at 25 ◦C, and the lifespan was determined. To assess thermal tolerance, 7-day-old worms were incubated at 35 ◦C for 6 h and then scored for viability every hour. The following assays were performed as previously described [31]. 9-day-old worms were exposed to UV irradiation at 500 J/m2 using a HL-2000 HybriLinker (UVP, Upland, CA, USA), incubated at 25 ◦C, and then scored for viability every day. For the oxidative stress assay, 9-day-old worms were transferred into M9 buffer containing 0.1% cholesterol (5 mg/mL in ethanol) and 2 mM hydrogen peroxide, and then scored for viability every hour. Similarly, for the heavy metal stress assay, 9-day-old worms were transferred to a 7-mM CuCl2 solution. Survival was determined by touch-provoked movement. Worms were scored as dead when they failed to respond to repeated touching with a worm picker. The assays were performed at least twice.

2.8. RNA Sequencing Three-day-old worms were cultured for five days on mNGM plates covered with OP50 (control-fed group) or CBM 588 (CBM 588-fed group). Approximately 200 worms in each group were collected and soaked in RNAlater solution (Qiagen, Hilden, Germany). Total RNA was isolated using the RNeasy Lipid Tissue kit (Qiagen). RNA sequencing was performed by DNAFORM (Yokohama, Japan) as follows: mRNAs were purified using the Magnosphere™ UltraPure mRNA Purification Kit (Clontech, Mountain View, CA, USA). Then, libraries prepared using the SMARTer® Stranded Total RNA-Seq Kit (Clontech) were run on HiSeq as 150 bp paired-end (Illumina Inc., San Diego, CA, USA). The quality of the RNA-seq results was first assessed using FastQC (ver. 0.11.7). The raw reads were trimmed and quality-filtered using the Trim Galore! (ver. 0.4.4), Trimmomatic (ver. 0.36) [32] and cutadapt (ver. 1.16) software. Clean reads were aligned versus the N2 Caenorhabditis elegans reference genome ce11 (WBcel235.91) using STAR (ver. 2.6.1a) [33]. Mapping statistics was presented in Table S1. After the read count of gene features was performed with the featureCounts tool (ver. 1.6.1) [34], a quantitative differential expression analysis between conditions was performed using DESeq (ver. 1.30.0) [35] to compare the control-fed and CBM 588-fed groups (see Table S2). Genes with |Log2FC| ≥ 2 and p-value < 0.05 (Tables S3 and S4) were subjected to the enrichment analysis based on the Gene Ontology (GO) category for Biological Process (BP), Molecular Function (MF), and Cellular Component (CC) using clusterProfiler (ver. 3.6.0) [36] (Tables S5 and S6). The raw and processed data files were deposited at Gene Expression Omnibus (GEO) (GSE123163).

2.9. Reverse Transcription and Real-Time PCR Genomic DNA was removed and cDNA was synthesized using the QuantiTect Reverse Transcription Kit (Qiagen). Real-time PCR (quantitative PCR) was performed with a StepOnePlus Real-Time PCR system (Thermo Fisher Scientific, Waltham, MA, USA) using FastStart Universal SYBR Green Master (ROX) (Roche Diagnostics, Mannheim, Germany) with the following parameters: 95 ◦C for 10 min, and 40 cycles of 95 ◦C for 15 s and 60 ◦C for 1 min. Samples from three biological replicates were analyzed. Relative mRNA was determined with the ∆∆Ct method [37] using expression of the cyc-1 or tba-1 gene. The primers used for real-time PCR were as follows: cyc-1, (F: Forward) 5’-CGTGGTTCAAGGATCTAAACG-3’ and (R: Reverse) 5’-ACCGAGTTCTCCAAAGCGTA-3’; tba-1, (F) 5’-TCAACACTGCCATCGCCGCC-3’ and (R) 5’-TCCAAGCGAGACCAGGCTTCAG-3’; gsto-1, (F) 5’-AGCTGCTAGCGGACAGGTTA-3’ and (R) 5’-CGCTGCTTTTCATCTTTCAA-3’; and gsto-2, (F) 5’-TTGAACTTGCAAAAGCATATGATAC-3’ and (R) 5’-TGAGATAATCGACAAATCCTGGT-3’; dod-22, Nutrients 2018, 10, x FOR PEER REVIEW 5 of 13 Nutrients 2018, 10, 1921 5 of 13 5’-CGCTGCTTTTCATCTTTCAA-3’; and gsto-2, (F) 5’-TTGAACTTGCAAAAGCATATGATAC-3’ and (R) 5’-TGAGATAATCGACAAATCCTGGT-3’; dod-22, (F) 5’-CTTCTTCGTCAAACCGCAAT-3’ (F)and 5’-CTTCTTCGTCAAACCGCAAT-3’ (R) 5’-TGTGAGAGACTTCCGATGTAGG and (R) 5’-TGTGAGAGACTTCCGATGTAGG-3’;-3’; dod-23, dod-23 (F), (F) 5’-GATTCAGTGCGCTGTACCATAC-3’5’-GATTCAGTGCGCTGTACCATAC-3’ and and (R) (R) 5’-GTAGGCTTCCATCTTTGAGAGC-3’; 5’-GTAGGCTTCCATCTTTGAGAGC-3’;dod-24 dod-24, (F), 5’-CTTCTTCGTCAAACCGCAAT-3’(F) 5’-CTTCTTCGTCAAACCGCAAT and-3’ and (R) (R) 5’-TGTGAGAGACTTCCGATGTAGG-3’. 5’-TGTGAGAGACTTCCGATGTAGG-3’.

3.3. ResultsResults andand DiscussionDiscussion 3.1. CBM 588 Prolonged the Longevity of C. elegans 3.1. CBM 588 Prolonged the Longevity of C. elegans To examine the effect of C. butyricum MIYAIRI 588 (CBM 588) on the C. elegans lifespan, worms To examine the effect of C. butyricum MIYAIRI 588 (CBM 588) on the C. elegans lifespan, worms were fed CBM 588 from an age of 3 days. As a result, the lifespan of the animals fed CBM 588 was were fed CBM 588 from an age of 3 days. As a result, the lifespan of the animals fed CBM 588 was E. coli significantlysignificantly longer longer than than that that of theof the animals animals fed fed E.OP50 coli OP50 (Figure (Figure1a). It is 1a). possible It is possible that the longevity that the thatlongevity resulted that from resulted the feedingfrom the of feeding CBM 588 of CBM may be588 due may to be dietary due to restriction dietary restriction (DR), which (DR), could which be considerablecould be considerable if CBM 588 if CBM contains 588 contains fewer calories fewer orcalories is too or difficult is too difficul for wormst for toworms digest. to digest. To test To this possibility,test this possibility, CBM 588 CBM was mixed588 was with mixed an equalwith an volume equal ofvolume OP50 of that OP50 was that sufficient was sufficient to avoid to DR, avoid and then,DR, and the animalsthen, the were animals fed thiswere mixture. fed this This mixture. mixture This of mixture CBM 588 of andCBM OP50 588 alsoand extendedOP50 also the extended lifespan comparedthe lifespan with comparedE. coli OP50 with E. alone coli OP50 (Figure alone1a). (Figure DR is known 1a). DR to is decrease known to body decrease weight body and weight fertility and in C.fertility elegans in[ 38C., 39elegans]. The [38,39]. body sizeThe ofbody the size animals of the fed animals CBM fed 588 CBM alone, 588 but alone, not the but mixture not the ofmixture CBM 588of andCBM OP50, 588 and was OP50, smaller was compared smaller withcompared that of with animals that fedof animals OP50 alone fed OP50 (Figure alone1b). The(Figure brood 1b). size The of thebrood worms size fedof the CBM worms 588 alone fed CBM was not588 significantlyalone was not different significantly in comparison different within comparison the size of with those the fed OP50size of alone those (Figure fed OP501c). Inalone contrast, (Figure the 1c). brood In sizecontrast, of the the mixture-fed brood size group of the was mixture larger-fed compared group was with thelarger control-fed compared group with (Figure the control1c). Taken-fed group together, (Figure these 1c). findings Taken together, indicate thatthese CBM findings 588 prolonged indicate that the longevityCBM 588 ofprolongedC. elegans theat longevity least partially of C. inelegans a DR-independent at least partially manner. in a DR-independent manner.

(a) (b) (c)

FigureFigure 1.1. ClostridiumClostridium butyricum MIYAIRI 588 (CBM 588) 588) extended extended the the lifespan lifespan of of C.C. elegans elegans. .Survival Survival curvescurves ((aa),), growthgrowth curvescurves (b) and brood size size ( (cc)) of of animals animals fed fed OP50 OP50 (control), (control), CBM CBM 588, 588, or or a amixture mixture ofof OP50OP50 and CBM 588. ( (aa)) Worms Worms were were 3 3 days days old old on on the the nominal nominal Day Day 0. OP50 0. OP50 (control) (control)-fed-fed group, group, n n= =122; 122; CBM CBM 588 588-fed-fed group, group, n n= =109; 109; mixture mixture-fed-fed group, group, n n= =112. 112. Differences Differences in in survival survival were were analyzed analyzed usingusing thethe log-ranklog-rank test.test. *** p < 0.001 vs. control control-fed-fed group. group. Detailed Detailed lifespan lifespan data data and and statistics statistics are are providedprovided in Table S7. S7. (b (b) )The The area area of ofthe the worm’s worm’s projection projection was wasmeasured. measured. n ≥ 8 forn ≥ each8 for assay. each ** assay. p < **0.01p < vs. 0.01 control vs. control-fed-fed group, group, ANOVA ANOVA followed followed by post by posthoc Tukey hoc Tukey-Kramer’s-Kramer’s test. test.(c) The (c) Thebrood brood size sizeof ofworms worms fed fed CBM CBM 588 588 alone alone was was not not significantly significantly different different in incomparison comparison with with the the size size of of those those fed fed OP50OP50 alone,alone, andand the brood size of the mixture mixture-fed-fed group group was was larger larger compared compared with with the the control control-fed-fed group.group. OP50 (control), n = 11; CBM 588, 588, nn = 12; 12; mixture, mixture, nn == 13. 13. ** ** pp << 0.01 0.01 vs. vs. control control-fed-fed group, group, StatisticalStatistical analysisanalysis waswas performed by ANOVA followed by post hoc hoc Tukey Tukey-Kramer’s-Kramer’s test. test.

3.2.3.2. UV-KilledUV-Killed CBM 588 Extended the Lifespan of of C. C. elegans elegans AlthoughAlthough probioticsprobiotics are defineddefined as “live microorganisms that, that, when when administered administered in in adequate adequate amounts,amounts, confer confer a a health health benefit benefit on on the the host” host” by the by Foodthe Food and Agriculture and Agriculture Organization Organization of the of United the NationsUnited Nations and the and World the HealthWorld Health Organization Organization [1], growing [1], growing evidence evidence has shown has shown that not that only not livingonly bacterialiving bacteria but also but dead also bacteria dead bacteria can exert can probiotic exert probiotic effects [effects40,41]. [40,41]. In addition, In addition, Hayashi Hayashi et al. showed et al. that heat-killed CBM 588 induced IL-10 production by macrophages [6]. We investigated whether

Nutrients 2018, 10, x FOR PEER REVIEW 6 of 13 Nutrients 2018, 10, x FOR PEER REVIEW 6 of 13 Nutrients 2018, 10, 1921 6 of 13 showed that heat-killed CBM 588 induced IL-10 production by macrophages [6]. We investigated showedwhether that UV heat-killed-killed CBM CBM 588 prolonged 588 induced the ILlifespan-10 production of C. elegans by. mNotably,acrophages the lifespan [6]. We of investigated worms fed whetherUV-killedUV-killed UV CBM CBM-killed 588 588 CBM prolonged was 588 increased prolonged the lifespan compared the of lifespanC. with elegans ofUV .C. Notably,-killed elegans OP50. theNotably, lifespan(Figure the of2). lifespan wormsTherefore, of fed worms UV-killednot only fed UVCBMliving-killed 588 CBM wasCBM 588 increased 588 but wasalso compared deadincreased CBM with compared 588 UV-killed exerted with longevity OP50 UV (Figure-killed effects 2OP50). in Therefore, C. (Figure elegans not . 2). onlyTherefore, living CBMnot only 588 livingbut also CBM dead 588 CBM but 588also exerteddead CBM longevity 588 exerted effects longevity in C. elegans effects. in C. elegans.

Figure 2. UV-killed CBM 588 prolonged the longevity of C. elegans. Survival curves of animals fed FigureFigureUV-killed 2. UVUV-killed bacteria.-killed CBM The 588 lifespan prolonged of worms the longevity fed UV-killed of C. elegans. CBM 588 Survival was significantly curves curves of of animals increased fed UVUV-killedcompared-killed bacteria. bacteria. with thos The Thee lifespanfed lifespan UV-killed of worms of wormsOP50. fed OP50, UV-killed fed UVn =- killed CBM56; CBM 588 CBM was588, 588 significantlyn = was72; UV significantly-killed increased OP50, comparedincreased n = 57; UV-killed CBM 588, n = 55. *** p < 0.001, log-rank test. Detailed lifespan data and statistics are comparedwith those with fed UV-killed those fed OP50. UV-killed OP50, OP50.n = 56; OP50, CBM 588,n = 56;n = CBM 72; UV-killed 588, n = OP50,72; UVn-=killed 57; UV-killed OP50, n CBM= 57; provided in Table S7. UV588,-killedn = 55. CBM *** p < 588, 0.001, n = log-rank 55. *** p test. < 0.001, Detailed log- lifespanrank test. data Detailed and statistics lifespan are data provided and statisticsin Table S7. are provided in Table S7. 3.3.3.3. CBM588 CBM588 Improved Improved Locomotion Locomotion duringduring AgingAging inin C.C. eleganselegans 3.3. CBM588ToTo determine determine Improved whether whether Locomotion CBM CBM 588during 588 improved improved Aging in the theC. decline elegans decline of of locomotion locomotion during during aging, aging, the the motility motility of wormsof worms was was scored. scored. The The scores scores of animals of animals fed CBMfed CBM 588 were588 were higher higher than thosethan those of animals of animals fed OP50 fed To determine whether CBM 588 improved the decline of locomotion during aging, the motility atOP50 every at age every tested age (Figure tested 3 (Figurea,b). The 3a,b). percentage The percentage of worms of in worms class “A,” in class wherein “A,” animals wherein exhibited animals of worms was scored. The scores of animals fed CBM 588 were higher than those of animals fed spontaneousexhibited spontaneous movement movement or vigorous or locomotion vigorous locomotion in response in toresponse prodding, to prodding, was significantly was significantly increased OP50 at every age tested (Figure 3a,b). The percentage of worms in class “A,” wherein animals inincreased the CBM in 588-fed the CBM group 588- (Figurefed group3c). (Figure These results3c). These support results the support hypothesis the hypothesis that CBM that 588 CBM not only 588 exhibited spontaneous movement or vigorous locomotion in response to prodding, was significantly extendsnot only the extends lifespan the of lifespanC. elegans of C.but elegans also improves but also improves health. health. increased in the CBM 588-fed group (Figure 3c). These results support the hypothesis that CBM 588 not only extends the lifespan of C. elegans but also improves health.

(a) (b) (c)

FigureFigure 3. 3. CBM588CBM588(a) improved locomotion during during aging aging(b in) in C.C. elegans elegans. (.(a,ba), bLocomotory) Locomotory activity activity(c) of C. of C.elegans elegans fedfed OP OP (a (a) )or or CBM CBM 588 588 ( (bb).). Animals Animals were were grouped grouped into into the the following following four four classes classes based based on on Figuretheirtheir locomotion: 3. locomotion: CBM588 class improved class A, robust, A, locomotion robust, coordinated coordinated during sinusoidal aging sinusoidal locomotionin C. elegans locomotion (blue. (a,b bars);) Locomotory (blue class B, bars); uncoordinated activity class of B,C. elegansand/oruncoordinated fed sluggish OP (a and/or) movementor CBM sluggish 588 (green ( bmovement). Animals bars); (green class were C, bars);grouped no forwardclass into C, nothe or forward backwardfollowing or backwardfour movement, classes movement, based but head on theirmovementsbut h locomotion:ead movements or shuddering class or A, shuddering in robust,response coordinated into prodding response to(orangesinusoidal prodding bars); locomotion (orange and class bars); D, (blue dead and bars); animalsclass D, class (black dead B, uncoordinatedbars).animals The (black frequency and/or bars). of sluggishThe each frequency class movement at the of indicatedeach (green class timebars); at the point class indicated is C, indicated. no timeforward pointn ≥ or57 is backward forindicated. each assay. movement, n ≥ (57c) Thefor butdifferenceeach head assay. movements in ( thec) The frequency difference or shuddering of “Classin the frequency A” in worms response ofwas “Class toanalyzed prodding A” worms using (orange was the analyzed log-rank bars); andusing test. class the *** plog D,< - 0.001.deadrank animalsOP50,test. ***n (black= p 61,< 0.001. CBM bars). OP50, 588, Then n =frequency =57. 61, CBM of 588, each n = class 57. at the indicated time point is indicated. n ≥ 57 for each assay. (c) The difference in the frequency of “Class A” worms was analyzed using the log-rank 3.4. Genes That Were Regulated by the CBM 588 Feeding 3.4.test. Genes *** Thatp < 0.001. Were OP50, Regulated n = 61, by CBM the CBM 588, n588 = 57. Feeding To characterize the genes that were regulated by feeding with CBM 588, RNA sequencing was To characterize the genes that were regulated by feeding with CBM 588, RNA sequencing was 3.4.performed Genes That (Tables Were S1Regulated and S2). by A the total CBM of 588 88 genesFeeding were shown to be increased in the CBM 588-fed performed (Tables S1 and S2). A total of 88 genes were shown to be increased in the CBM 588-fed group in comparison with the control-fed group with Log2FC ≥ 2 and p < 0.05 (Table S3). A total of 459 groupTo characterizein comparison the with genes the that control were-fed regulated group with by feeding Log2FC with≥ 2 and CBM p < 588, 0.05 RNA (Table sequencing S3). A total was of ≤ performedgenes were (Tables decreased S1 inand the S2). CBM A 588-fedtotal of group88 genes with were Log 2shownFC 2 to and be pincreased< 0.05 (Table in the S4). CBM Significantly 588-fed group in comparison with the control-fed group with Log2FC ≥ 2 and p < 0.05 (Table S3). A total of

Nutrients 2018, 10, x FOR PEER REVIEW 7 of 13 Nutrients 2018, 10, 1921 7 of 13

459 genes were decreased in the CBM 588-fed group with Log2FC ≤ 2 and p < 0.05 (Table S4). enrichedSignificantly Gene enriched Ontology Gene (GO) Ontology in the CBM (GO) 588-fed in the group CBM is 588 listed-fed in group Table S5 is forlisted upregulated in Table S5 genes for andupregulated in Table S6 genes for downregulated and in Table S6 genes. for downregulated Within the upregulated genes. Within genes, the top-ranked upregulated GO genes, term thefor moleculartop-ranked function GO term (MF) for was molecular “protein function heterodimerization (MF) was “protein activity” heterodimerization that includes genes activity” encoding that for histoneincludes components. genes encoding Because for histone the second-ranked components. “structural Because the constituent second-ranked of cuticle” “structural was also constituent significant inof the cuticle” downregulated was also genes,significant this GO in the term downregulated was not specifically genes, enriched this GO in the term upregulated was not specifically genes. The expressionenriched in of the genes upr encodingegulated forgenes. glucuronosyltransferases, The expression of genes endopeptidases encoding for and glucuronosyltransferases, heme-binding proteins wasendopeptidases also induced. andcpr-5 heme, an- endopeptidasebinding proteins gene was upregulated also induced. by CBM cpr 588,-5, an has endopeptidase been reported to gene be inducedupregulated by Lactobacillus by CBM 588, acidophilus has beenNCFM reported in atopmk-1 be induced/p38 MAPK-dependent by Lactobacillus acidophilus manner [24 NCFM]. Notably, in a “glutathionepmk-1/p38 MAPK transferase-dependent activity” manner was significantly [24]. Notably, enriched “glutathione in genes that transferase were upregulated activity” by CBM was 588.significantly The Omega enriched class in glutathione genes that transferase were upregulated gene gsto-1 by CBMhas been 588. reportedThe Omega to playclass key glutathione roles in counteractingtransferase gene environmental gsto-1 has been stress reported [42], andto playgsto-2 keyis roles a paralog in counter of gsto-1acting. Fold-changes environmental of stress the gsto-1 [42], and gsto-2gsto-2 geneis a wereparalog 45.5 of and gsto 5.3,-1. respectively Fold-changes (Table of the S2). gsto Using-1 and real-time gsto-2 gene PCR, were we confirmed 45.5 and that 5.3, therespectively mRNA expression (Table S2). of Usinggsto-1 realand-timegsto-2 PCR,was we increased confirmed in that the CBM-fedthe mRNA group expression compared of gsto with-1 and the control-fedgsto-2 was increased group (fold-change: in the CBM-gsto-1fed group, 39.3 compared± 7.1 (S.D.); withgsto-2 the control, 2.0 ± -1.1)fed group (Figure (fold4a and-change: Figure gsto S1).-1, Meanwhile,39.3 ± 7.1 (S.D.); the top-ranked gsto-2, 2.0 GO ± 1.1) term (Figures for biological 4a and process S1). Meanwhile, (BP) in the the downregulated top-ranked GO genes term was for “defensebiological response” process (BP) (Table in the S6). downregulated Intriguingly, dod genes (downstream was “defenseof Dresponse”AF-16) genes (Table such S6). as Intriguingly,dod-22 and dod-24dod (d,ownstream were found of in D theAF- “defense16) genes response”such as dod category-22 and dod (Table-24, were S6). Tepper found in et the al. has“defense reported response” class I (upregulated)category (Table and S6). class Tepper II (downregulated) et al. has reported DAF-16-responsible class I (upregulated) genes extracted and class from II (downregulated) a meta-analysis, andDAFdod-23-16-responsible, dod-22, and genesdod-24 extractedwere thefrom very a m topeta- 3-rankedanalysis, genesand dod in-23 class, dod II- [2243, ].and The doddod-22-24 were, dod-23 the, andvery dod-24top 3-rankedgenes weregenes allin class decreased II [43]. in The the dod CBM-22, 588-feddod-23, and group dod- in24RNA genes sequencing were all decreased (fold-change: in the 0.029,CBM 588 0.306,-fed andgroup 0.002, in RNA respectively) sequencing (Table (fold S2),-change: and also0.029, in 0.306, real-time and 0.002, PCR (0.0029respectively)± 0.0002 (Table (S.D.), S2), 0.460and also± 0.058,in real 0.0042-time PCR± 0.0012, (0.0029 respectively) ± 0.0002 (S.D.), (Figure 0.460 4±b 0.058, and Figure0.0042 S1).± 0.0012, In contrast respectively) to class (Figures II, none 4b ofand the S1) class. In contrast I genes listedto class in TableII, none 1 ofofthe theTepper’s class I genes report listed [43], in was Table found 1 of in the Table Tepper’s S3. These report results [43], suggestwas found that in the Table DAF-16-dependent S3. These results class suggest II pathway that the but DAF not-16 class-dependent I, may beclass modulated II pathway by feedingbut not withclass CBMI, may 588. be modulated by feeding with CBM 588.

(a) (b)

Figure 4.4. Relative gene expression (determined using real-timereal-time PCR) in the animals fed CBM 588 in comparison with control (OP50). ( a) The gsto-1gsto-1 and gsto-2gsto-2 expression was upregulated by the CBM 588 feeding. (b) The expression of dod-22dod-22, dod-23dod-23, dod-24dod-24 was downregulateddownregulated by CBM 588.588. Error bars indicate S.D.S.D. n = 3 biological replicates.

3.5. CBM 588 Increased Resistance to Biological Stresses in C. elegans 3.5. CBM 588 Increased Resistance to Biological Stresses in C. elegans Salmonella Staphylococcus aureus Salmonella spp., which are Gram-negative Gram-negative bacteria, bacteria, and Staphylococcus aureus, which are Gram-positive bacteria, shorten the lifespan of worms [23,44–46]. We found that probiotic Gram-positive bacteria, shorten the lifespan of worms [23,44–46]. We found that probiotic C. elegans Salmonella bifidobacteriabifidobacteria and lactobacilli could make C. elegans resilient to Salmonella infection [[23].23]. Afterward, Kim et al. reported that the L. acidophilus strain NCFM specifically enhances Gram-positive immune Kim et al. reported that the L. acidophilus strain NCFM specifically enhances Gram-positive immune L. acidophilus responses; conditioning with L. acidophilus NCFM significantlysignificantly prolonged the survival of nematodes Enterococcus faecalis Staphylococcus aureus exposed to to the the Gram-positive Gram-positive pathogens pathogens Enterococcus faecalisand and Staphylococcus aureus, but this, but strain this Pseudomonas aeruginosa Salmonella enterica hadstrain a minimalhad a minimal effect on effect Gram-negative on Gram- infectionnegative withinfection with Pseudomonas oraeruginosa or Salmonella[24]. Toenterica investigate [24]. To the investigate effect of CBM the 588effect on of the CBM biological 588 on stresses the biological caused by stresses pathogenic caused bacterial by pathogenic infection,

Nutrients 2018, 10, 1921 8 of 13 Nutrients 2018, 10, x FOR PEER REVIEW 8 of 13 bacterial7-day-old infection, worms were 7-day exposed-old worms to Salmonella were expose entericad toor SalmonellaStaphylococcus enterica aureus or .Staphylococcus Nematodes fed aureus CBM. Nematodes588 lived longer fed CBM after 588 being lived infected longer with afterSalmonella being infected enterica withor Staphylococcus Salmonella enterica aureus orcompared Staphylococcus with aureusworms compared fed OP50 with (Figure worms5a,b). fed In OP50 contrast (Figure to the 5a results,b). In contrast obtained to with the results NCFM, obtained worms fedwith CBM NCFM, 588 wormsexhibited fed increased CBM 588 resistance exhibited to increased both Gram-positive resistance andto both -negative Gram bacteria,-positiveStaphylococcus and -negative aureus bacteria,and StaphylococcusSalmonella enterica aureus. and Salmonella enterica.

(a) (b)

(c) (d)

(e) (f)

FigureFigure 5 5.. SusceptibilitySusceptibility of of CBM CBM 588 588-fed-fed worms worms to to stresses. stresses. ( (aa,b,b)) Survival Survival of of C.C. elegans elegans infectedinfected with with pathogenicpathogenic bacteria, SalmonellaSalmonella entericaenterica( a()a or) orStaphylococcus Staphylococcus aureus aureus(b). (b (a).) OP50-fed(a) OP50-fed group, group,n = 59;n = CBM 59; CBM588-fed 588 group,-fed group,n = 48. n (b )= OP50-fed 48. (b) OP50 group,-fedn = group, 59; CBM n 588-fed= 59; CBM group, 588n-=fed 50. group, (c–f) Susceptibility n = 50. (c–f of) Susceptibilityworms fed CBM of worms 588 to heatfed CBM stress 588 (c), to UV heat irradiation stress (c (),d UV), hydrogen irradiation peroxide (d), hydrogen (e) and Cu peroxide2+ (f). ( c()e) OP50, and Cun =2+ 64;(f). CBM (c) OP50, 588, n n= = 63.64; (dCBM) OP50, 588,n n= = 64; 63. CBM (d) OP50, 588, n n= = 63. 64; (e CBM) OP50, 588,n = n48; = 63. CBM (e) 588,OP50,n = n 48. = 48; (f) CBM OP50, 588,n = 63;n = CBM48. (f) 588, OP50,n = n 62. = 63; Animals CBM were588, n fed = 62. OP50 Animals (control) were or fed CBM OP50 588 from(control) an age or CBM of 3 days 588 andfrom then an ageexposed of 3 days to the and individual then exposed stresses to the at the individual ages described stresses in at the the Materials ages described and methods. in the Materials Worms wereand methods.7 days old Worms (a–c) or were 9 days 7 old days (d – oldf) on (a the–c) graph’s or 9 days nominal old (d Day–f) 0.on The the survival graph’s curves nominal were Day compared 0. The survivalwith those curves of worms were compared fed OP50. ***with Statistical those of significanceworms fed OP50. at p < *** 0.001, Statistical log-rank significance test. at p < 0.001, log-rank test.

3.6. CBM 588 Increased Resistance to UV Irradiation and Cu2+ in C. elegans

Nutrients 2018, 10, 1921 9 of 13

3.6. CBM 588 Increased Resistance to UV Irradiation and Cu2+ in C. elegans We next examined whether CBM 588 increased resistance to physical and chemical stressors. CBM 588 did not prolong the survival time of worms subjected to heat stress (Figure5c) but did increase the survival time of worms subjected to UV irradiation (Figure5d). When the animals were exposed to hydrogen peroxide, which is a known source of active oxygen, the survival rates of OP-fed and CBM 588-fed worms were not significantly different (Figure5e). In contrast, the CBM 588-fed animals lived longer compared to the OP50-fed animals in the presence of Cu2+ (Figure5f). Transgenic animals overexpressing GSTO-1 were hyperresistant to oxidative stress, and silencing the expression of GSTO-1 by RNAi resulted in worms with an increased sensitivity to oxidants and heat shock [42]. Because 2+ CBM 588 conferred resistance to UV irradiation and Cu but not to heat or H2O2 oxidative stress, upregulation of GSTO-1 is unlikely to be the underlying mechanism through which CBM 588 increases stress resistance.

3.7. CBM 588 Could Extend the Lifespan of Worms through Regulation of the IIS Pathway and the Nrf2 Transcription Factor The insulin/IGF-1-like signaling (IIS) pathway plays a central role in stress resistance and longevity. In C. elegans, IIS is initiated by the binding of insulin-like peptides to the receptor DAF-2, which eventually leads to the activation of downstream IIS kinases, which phosphorylate and inhibit the FOXO transcription factor DAF-16 [47–50]. Moreover, transcriptional profiling using RNA sequencing implicated the involvement of the DAF-16-dependent class II pathway. To investigate whether the IIS pathway is involved in the prolongevity effects of CBM 588, the lifespans of the C. elegans loss-of-function mutants daf-2 and daf-16 were measured. CBM 588 failed to prolong the lifespan of strains harboring individual mutations in daf-2 or daf-16 (Figure6). SKN-1 (an ortholog of mammalian Nrf2) is one of the key factors that regulates the stress responses and longevity of C. elegans [51,52]. Notably, CBM 588 failed to extend the lifespan of the skn-1 mutants (Figure6). These results suggest that both DAF-16 and SKN-1 may be required for the CBM 588-mediated lifespan extension. Because SKN-1 has been shown to be directly suppressed by DAF-2 [53], DAF-2 signaling via SKN-1 activity may also regulate the lifespan of worms fed CBM 588. The p38 MAPK pathway activates SKN-1 in several contexts, including pathogen infection and stress responses [54–56], raising the possibility that SKN-1 activation via the p38 MAPK pathway contributes to the prolongevity of animals fed CBM 588. However, we could not determine whether pmk-1/p38 MAPK is required for the prolonged lifespan caused by CBM 588 because the pmk-1 mutation caused severe egg-laying defects when these mutants were fed CBM 588 (data not shown). Further analyses will be necessary to elucidate the role of the p38 MAPK pathway in the increased lifespan resulting from CBM 588. The long-lived mutants daf-2 and age-1 exhibited increased resistance to heat [57], oxidants [58,59], UV [60], and metal stress [61], indicating that longevity and stress resistance are closely related. Based on our results from the CBM 588-mediated lifespan extension, suppression of DAF-2 may partially mediate the increased resistance of animals fed CBM 588. However, the possibility that the impact of CBM 588 on longevity was insufficient to further extend the lifespan of the long-lived daf-2 mutants, cannot be ruled out. In addition, the experiments using mutants that were not outcrossed with our own N2 strain, require caution in the interpretation of these results, because it has been reported that the impact of simply a different N2 strain on lifespan can be huge [62]. Nutrients 2018, 10, 1921 10 of 13 Nutrients 2018, 10, x FOR PEER REVIEW 10 of 13

(a) (b)

(c) (d)

Figure 6.6. Effects of CBM 588 on the lifespan of C. elegans loss-of-functionloss-of-function mutants. Survival curves of the wild wild-type-type animals (a) and the daf-2daf-2 (b), daf-16daf-16 (c), and skn-1skn-1 mutants (d). CBM 588 failed to extend the lifespan lifespan of of these these mutants. mutants. The The survival survival curves curves were were compared compared with with those those of worms of worms fed fedOP50. OP50. *** ***Statistical Statistical significance significance at atp

4. Conclusions 4. Conclusions In the present study, we demonstrated for the first time that CBM 588 extends the lifespan of In the present study, we demonstrated for the first time that CBM 588 extends the lifespan of C. C. elegans. The involved mechanisms seemed to depend on the DAF-16/FOXO and the SKN-1/Nrf2 elegans. The involved mechanisms seemed to depend on the DAF-16/FOXO and the SKN-1/Nrf2 transcription factors. Our transcriptional profiling comparing CBM 588-fed and control-fed animals transcription factors. Our transcriptional profiling comparing CBM 588-fed and control-fed animals suggested that DAF-16-dependent class II genes were regulated by CBM 588. Taken together with suggested that DAF-16-dependent class II genes were regulated by CBM 588. Taken together with the fact that reduced IIS extends the lifespan of C. elegans by upregulating stress response (class I) the fact that reduced IIS extends the lifespan of C. elegans by upregulating stress response (class I) and downregulating other (class II) genes in a DAF-16-dependent manner [43], CBM 588 may exert and downregulating other (class II) genes in a DAF-16-dependent manner [43], CBM 588 may exert its prolongevity through modulating of the DAF-16 class II pathway. Because DAF-16/FOXO is its prolongevity through modulating of the DAF-16 class II pathway. Because DAF-16/FOXO is well-conserved transcription factor that regulate longevity among animal species including humans, it well-conserved transcription factor that regulate longevity among animal species including humans, would be worth studying the DAF-16-dependent mechanisms that govern the effects of the probiotic it would be worth studying the DAF-16-dependent mechanisms that govern the effects of the CBM 588. probiotic CBM 588. Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6643/10/12/ 1921/s1Supplementary, Figure S1: Materials: Gene expression The following (real-time are PCR) available in the online animals at fed www.mdpi.com/xxx/s1, OP50 or CBM 588. Table Figure S1: MappingS1: Gene expressionstatistics. Table (real S2:-time Differential PCR) in expression the animals analysis fed OP50 using or DESeq CBM (CBM 588. Table 588 vs. S1: OP50 Mapping (control)). statistics. Table S3: Table Genes S2: Differentialupregulated expression in CBM-fed analysis animals. using Table DESeq S4: Genes (CBM downregulated 588 vs. OP50 in (control)). CBM-fed animals. Table S3: Table Genes S5: upregulated Gene ontology in (GO)CBM-fed enrichment animals. for Table genes S4: upregulated Genes downregulated by CBM 588. in Table CBM S6:-fed Gene animals. ontology Table (GO) S5: enrichment Gene ontology for genes (GO) downregulated by CBM 588. Table S7: Summary of lifespan experiments. enrichment for genes upregulated by CBM 588. Table S6: Gene ontology (GO) enrichment for genes Authordownregulated Contributions: by CBM 588.Investigation, Table S7: Summary M.K., Y.H., of lifespan S.S.; writing—original experiments. draft preparation, E.K.-N., Y.N.; supervision, E.K.-N., Y.N.; project administration, E.K.-N., Y.N. Author Contributions: Investigation, M.K., Y.H. S.S.; writing—original draft preparation, E.K.-N., Y.N.; Funding: This research received no external funding. supervision, E.K.-N., Y.N.; project administration, E.K.-N., Y.N. Acknowledgments: We thank the Caenorhabditis Genetics Center (University of Minnesota, Minneapolis, MN; Funding:supported This by the research National received Institutes no ofexternal Health-National funding. Center for Research Resources) for providing the C. elegans strains, and Miyarisan Pharmaceutical for providing C. butyricum MIYAIRI 588. Acknowledgments: We thank the Caenorhabditis Genetics Center (University of Minnesota, Minneapolis, MN; supported by the National Institutes of Health-National Center for Research Resources) for providing the C. elegans strains, and Miyarisan Pharmaceutical for providing C. butyricum MIYAIRI 588.

Nutrients 2018, 10, 1921 11 of 13

Conflicts of Interest: The authors declare no conflict of interest.

References

1. Sanders, M.E. Probiotics: Definition, sources, selection, and uses. Clin. Infect. Dis. 2008, 46 (Suppl. 2), S58–S61, discussion S144–S151. [CrossRef][PubMed] 2. Cassir, N.; Benamar, S.; La Scola, B. Clostridium butyricum: From beneficial to a new emerging pathogen. Clin. Microbiol. Infect. 2016, 22, 37–45. [CrossRef][PubMed] 3. Ferraris, L.; Butel, M.J.; Aires, J. Antimicrobial susceptibility and resistance determinants of Clostridium butyricum isolates from preterm infants. Int. J. Antimicrob. Agents 2010, 36, 420–423. [CrossRef][PubMed] 4. Sato, R.; Tanaka, M. Intestinal distribution and intraluminal localization of orally administered Clostridium butyricum in rats. Microbiol. Immunol. 1997, 41, 665–671. [CrossRef][PubMed] 5. Seki, H.; Shiohara, M.; Matsumura, T.; Miyagawa, N.; Tanaka, M.; Komiyama, A.; Kurata, S. Prevention of antibiotic-associated diarrhea in children by Clostridium butyricum MIYAIRI. Pediatr. Int. 2003, 45, 86–90. [CrossRef][PubMed] 6. Hayashi, A.; Sato, T.; Kamada, N.; Mikami, Y.; Matsuoka, K.; Hisamatsu, T.; Hibi, T.; Roers, A.; Yagita, H.; Ohteki, T.; et al. A single strain of Clostridium butyricum induces intestinal IL-10-producing macrophages to suppress acute experimental colitis in mice. Cell Host Microbe 2013, 13, 711–722. [CrossRef][PubMed] 7. Kashiwagi, I.; Morita, R.; Schichita, T.; Komai, K.; Saeki, K.; Matsumoto, M.; Takeda, K.; Nomura, M.; Hayashi, A.; Kanai, T.; et al. Smad2 and Smad3 Inversely Regulate TGF-β Autoinduction in Clostridium butyricum-Activated Dendritic Cells. Immunity 2015, 43, 65–79. [CrossRef] 8. Seo, M.; Inoue, I.; Tanaka, M.; Matsuda, N.; Nakano, T.; Awata, T.; Katayama, S.; Alpers, D.H.; Komoda, T. Clostridium butyricum MIYAIRI 588 improves high-fat diet-induced non-alcoholic fatty liver disease in rats. Dig. Dis. Sci. 2013, 58, 3534–3544. [CrossRef] 9. Woo, T.D.; Oka, K.; Takahashi, M.; Hojo, F.; Osaki, T.; Hanawa, T.; Kurata, S.; Yonezawa, H.; Kamiya, S. Inhibition of the cytotoxic effect of Clostridium difficile in vitro by Clostridium butyricum MIYAIRI 588 strain. J. Med. Microbiol. 2011, 60, 1617–1625. [CrossRef] 10. Takahashi, M.; Taguchi, H.; Yamaguchi, H.; Osaki, T.; Komatsu, A.; Kamiya, S. The effect of probiotic treatment with Clostridium butyricum on enterohemorrhagic Escherichia coli O157:H7 infection in mice. FEMS Immunol. Med. Microbiol. 2004, 41, 219–226. [CrossRef] 11. Wang, F.Y.; Liu, J.M.; Luo, H.H.; Liu, A.H.; Jiang, Y. Potential protective effects of Clostridium butyricum on experimental gastric ulcers in mice. World J. Gastroenterol. 2015, 21, 8340–8351. [CrossRef][PubMed] 12. Finch, C.E.; Ruvkun, G. The genetics of aging. Annu. Rev. Genom. Hum. Genet. 2001, 2, 435–462. [CrossRef] 13. Hodgkin, J.; Doniach, T. Natural variation and copulatory plug formation in Caenorhabditis elegans. Genetics 1997, 146, 149–164. [PubMed] 14. Barrière, A.; Félix, M.A. High local genetic diversity and low outcrossing rate in Caenorhabditis elegans natural populations. Curr. Biol. 2005, 15, 1176–1184. [CrossRef][PubMed] 15. Barrière, A.; Félix, M.A. Temporal dynamics and linkage disequilibrium in natural Caenorhabditis elegans populations. Genetics 2007, 176, 999–1011. [CrossRef][PubMed] 16.F élix, M.A.; Duveau, F. Population dynamics and habitat sharing of natural populations of Caenorhabditis elegans and C. briggsae. BMC Biol. 2012, 10, 59. [CrossRef][PubMed] 17. Frézal, L.; Félix, M.A. C. elegans outside the Petri dish. eLife 2015, 4.[CrossRef][PubMed] 18. Berg, M.; Stenuit, B.; Ho, J.; Wang, A.; Parke, C.; Knight, M.; Alvarez-Cohen, L.; Shapira, M. Assembly of the Caenorhabditis elegans gut microbiota from diverse soil microbial environments. ISME J. 2016, 10, 1998–2009. [CrossRef] 19. Dirksen, P.; Marsh, S.A.; Braker, I.; Heitland, N.; Wagner, S.; Nakad, R.; Mader, S.; Petersen, C.; Kowallik, V.; Rosenstiel, P.; et al. The native microbiome of the nematode Caenorhabditis elegans: Gateway to a new host-microbiome model. BMC Biol. 2016, 14, 38. [CrossRef] 20. Samuel, B.S.; Rowedder, H.; Braendle, C.; Félix, M.A.; Ruvkun, G. Caenorhabditis elegans responses to bacteria from its natural habitats. Proc. Natl. Acad. Sci. USA 2016, 113, E3941–E3949. [CrossRef] 21. Zhang, F.; Berg, M.; Dierking, K.; Félix, M.A.; Shapira, M.; Samuel, B.S.; Schulenburg, H. Caenorhabditis elegans as a Model for Microbiome Research. Front. Microbiol. 2017, 8, 485. [CrossRef][PubMed] 22. Brenner, S. The genetics of Caenorhabditis elegans. Genetics 1974, 77, 71–94. [PubMed] Nutrients 2018, 10, 1921 12 of 13

23. Ikeda, T.; Yasui, C.; Hoshino, K.; Arikawa, K.; Nishikawa, Y. Influence of lactic acid bacteria on longevity of Caenorhabditis elegans and host defense against salmonella enterica serovar enteritidis. Appl. Environ. Microbiol. 2007, 73, 6404–6409. [CrossRef][PubMed] 24. Kim, Y.; Mylonakis, E. Caenorhabditis elegans immune conditioning with the probiotic bacterium Lactobacillus acidophilus strain NCFM enhances gram-positive immune responses. Infect. Immun. 2012, 80, 2500–2508. [CrossRef][PubMed] 25. Komura, T.; Ikeda, T.; Yasui, C.; Saeki, S.; Nishikawa, Y. Mechanism underlying prolongevity induced by bifidobacteria in Caenorhabditis elegans. Biogerontology 2013, 14, 73–87. [CrossRef][PubMed] 26. Cabreiro, F.; Gems, D. Worms need microbes too: Microbiota, health and aging in Caenorhabditis elegans. EMBO Mol. Med. 2013, 5, 1300–1310. [CrossRef][PubMed] 27. Clark, L.C.; Hodgkin, J. Commensals, probiotics and pathogens in the Caenorhabditis elegans model. Cell. Microbiol. 2014, 16, 27–38. [CrossRef][PubMed] 28. Wu, D.; Rea, S.L.; Yashin, A.I.; Johnson, T.E. Visualizing hidden heterogeneity in isogenic populations of C. elegans. Exp. Gerontol. 2006, 41, 261–270. [CrossRef][PubMed] 29. Gruber, J.; Ng, L.F.; Fong, S.; Wong, Y.T.; Koh, S.A.; Chen, C.B.; Shui, G.; Cheong, W.F.; Schaffer, S.; Wenk, M.R.; et al. Mitochondrial changes in ageing Caenorhabditis elegans—What do we learn from superoxide dismutase knockouts? PLoS ONE 2011, 6, e19444. [CrossRef] 30. Hosono, R.; Sato, Y.; Aizawa, S.I.; Mitsui, Y. Age-dependent changes in mobility and separation of the nematode Caenorhabditis elegans. Exp. Gerontol. 1980, 15, 285–289. [CrossRef] 31. Yaguchi, Y.; Komura, T.; Kashima, N.; Tamura, M.; Kage-Nakadai, E.; Saeki, S.; Terao, K.; Nishikawa, Y. Influence of oral supplementation with sesamin on longevity of Caenorhabditis elegans and the host defense. Eur. J. Nutr. 2014, 53, 1659–1668. [CrossRef][PubMed] 32. Bolger, A.M.; Lohse, M.; Usadel, B. Trimmomatic: A flexible trimmer for Illumina sequence data. Bioinformatics 2014, 30, 2114–2120. [CrossRef][PubMed] 33. Dobin, A.; Davis, C.A.; Schlesinger, F.; Drenkow, J.; Zaleski, C.; Jha, S.; Batut, P.; Chaisson, M.; Gingeras, T.R. STAR: Ultrafast universal RNA-seq aligner. Bioinformatics 2013, 29, 15–21. [CrossRef][PubMed] 34. Liao, Y.; Smyth, G.K.; Shi, W. featureCounts: An efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 2014, 30, 923–930. [CrossRef] 35. Anders, S.; Huber, W. Differential expression analysis for sequence count data. Genome Biol. 2010, 11, R106. [CrossRef] 36. Yu, G.; Wang, L.G.; Han, Y.; He, Q.Y. clusterProfiler: An R package for comparing biological themes among gene clusters. OMICS 2012, 16, 284–287. [CrossRef] 37. Livak, K.J.; Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001, 25, 402–408. [CrossRef] 38. Klass, M.R. Aging in the nematode Caenorhabditis elegans: Major biological and environmental factors influencing life span. Mech. Ageing Dev. 1977, 6, 413–429. [CrossRef] 39. Tain, L.S.; Lozano, E.; Sáez, A.G.; Leroi, A.M. Dietary regulation of hypodermal polyploidization in C. elegans. BMC Dev. Biol. 2008, 8, 28. [CrossRef] 40. Tanaka, A.; Seki, M.; Yamahira, S.; Noguchi, H.; Kosai, K.; Toba, M.; Morinaga, Y.; Miyazaki, T.; Izumikawa, K.; Kakeya, H.; et al. Lactobacillus pentosus strain b240 suppresses pneumonia induced by Streptococcus pneumoniae in mice. Lett. Appl. Microbiol. 2011, 53, 35–43. [CrossRef] 41. Chen, M.F.; Weng, K.F.; Huang, S.Y.; Liu, Y.C.; Tseng, S.N.; Ojcius, D.M.; Shih, S.R. Pretreatment with a heat-killed probiotic modulates monocyte chemoattractant protein-1 and reduces the pathogenicity of influenza and enterovirus 71 infections. Mucosal Immunol. 2017, 10, 215–227. [CrossRef][PubMed] 42. Burmeister, C.; Lüersen, K.; Heinick, A.; Hussein, A.; Domagalski, M.; Walter, R.D.; Liebau, E. Oxidative stress in Caenorhabditis elegans: Protective effects of the Omega class glutathione transferase (GSTO-1). FASEB J. 2008, 22, 343–354. [CrossRef][PubMed] 43. Tepper, R.G.; Ashraf, J.; Kaletsky, R.; Kleemann, G.; Murphy, C.T.; Bussemaker, H.J. PQM-1 complements DAF-16 as a key transcriptional regulator of DAF-2-mediated development and longevity. Cell 2013, 154, 676–690. [CrossRef][PubMed] 44. Aballay, A.; Yorgey, P.; Ausubel, F.M. Salmonella typhimurium proliferates and establishes a persistent infection in the intestine of Caenorhabditis elegans. Curr. Biol. 2000, 10, 1539–1542. [CrossRef] Nutrients 2018, 10, 1921 13 of 13

45. Labrousse, A.; Chauvet, S.; Couillault, C.; Kurz, C.L.; Ewbank, J.J. Caenorhabditis elegans is a model host for Salmonella typhimurium. Curr. Biol. 2000, 10, 1543–1545. [CrossRef] 46. Sifri, C.D.; Begun, J.; Ausubel, F.M.; Calderwood, S.B. Caenorhabditis elegans as a model host for Staphylococcus aureus pathogenesis. Infect. Immun. 2003, 71, 2208–2217. [CrossRef][PubMed] 47. Murphy, C.T.; McCarroll, S.A.; Bargmann, C.I.; Fraser, A.; Kamath, R.S.; Ahringer, J.; Li, H.; Kenyon, C. Genes that act downstream of DAF-16 to influence the lifespan of Caenorhabditis elegans. Nature 2003, 424, 277–283. [CrossRef][PubMed] 48. Berman, J.R.; Kenyon, C. Germ-cell loss extends C. elegans life span through regulation of DAF-16 by kri-1 and lipophilic-hormone signaling. Cell 2006, 124, 1055–1068. [CrossRef] 49. Mukhopadhyay, A.; Oh, S.W.; Tissenbaum, H.A. Worming pathways to and from DAF-16/FOXO. Exp. Gerontol. 2006, 41, 928–934. [CrossRef] 50. Samuelson, A.V.; Carr, C.E.; Ruvkun, G. Gene activities that mediate increased life span of C. elegans insulin-like signaling mutants. Genes Dev. 2007, 21, 2976–2994. [CrossRef] 51. Park, S.K.; Tedesco, P.M.; Johnson, T.E. Oxidative stress and longevity in Caenorhabditis elegans as mediated by SKN-1. Aging Cell 2009, 8, 258–269. [CrossRef][PubMed] 52. Blackwell, T.K.; Steinbaugh, M.J.; Hourihan, J.M.; Ewald, C.Y.; Isik, M. SKN-1/Nrf, stress responses, and aging in Caenorhabditis elegans. Free Radic. Biol. Med. 2015, 88, 290–301. [CrossRef][PubMed] 53. Tullet, J.M.; Hertweck, M.; An, J.H.; Baker, J.; Hwang, J.Y.; Liu, S.; Oliveira, R.P.; Baumeister, R.; Blackwell, T.K. Direct inhibition of the longevity-promoting factor SKN-1 by insulin-like signaling in C. elegans. Cell 2008, 132, 1025–1038. [CrossRef][PubMed] 54. Inoue, H.; Hisamoto, N.; An, J.H.; Oliveira, R.P.; Nishida, E.; Blackwell, T.K.; Matsumoto, K. The C. elegans p38 MAPK pathway regulates nuclear localization of the transcription factor SKN-1 in oxidative stress response. Genes Dev. 2005, 19, 2278–2283. [CrossRef][PubMed] 55. Hoeven, R.; McCallum, K.C.; Cruz, M.R.; Garsin, D.A. Ce-Duox1/BLI-3 generated reactive oxygen species trigger protective SKN-1 activity via p38 MAPK signaling during infection in C. elegans. PLoS Pathog. 2011, 7, e1002453. [CrossRef] 56. Papp, D.; Csermely, P.; S˝oti, C. A role for SKN-1/Nrf in pathogen resistance and immunosenescence in Caenorhabditis elegans. PLoS Pathog. 2012, 8, e1002673. [CrossRef][PubMed] 57. MacColl, G.S.; Quinton, R.; Bülow, H.E. Biology of KAL1 and its orthologs: Implications for X-linked Kallmann syndrome and the search for novel candidate genes. Front. Horm. Res. 2010, 39, 62–77. [CrossRef] [PubMed] 58. Larsen, P.L. Aging and resistance to oxidative damage in Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA 1993, 90, 8905–8909. [CrossRef] 59. Honda, Y.; Honda, S. The daf-2 gene network for longevity regulates oxidative stress resistance and Mn-superoxide dismutase gene expression in Caenorhabditis elegans. FASEB J. 1999, 13, 1385–1393. [CrossRef] 60. Murakami, S.; Johnson, T.E. A genetic pathway conferring life extension and resistance to UV stress in Caenorhabditis elegans. Genetics 1996, 143, 1207–1218. 61. Barsyte, D.; Lovejoy, D.A.; Lithgow, G.J. Longevity and heavy metal resistance in daf-2 and age-1 long-lived mutants of Caenorhabditis elegans. FASEB J. 2001, 15, 627–634. [CrossRef][PubMed] 62. Zhao, Y.; Long, L.; Xu, W.; Campbell, R.F.; Large, E.E.; Greene, J.S.; McGrath, P.T. Changes to social feeding behaviors are not sufficient for fitness gains of the. eLife 2018, 7.[CrossRef][PubMed]

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).