research article impact of a complex food microbiota on...

Research ArticleImpact of a Complex Food Microbiota on Energy Metabolism inthe Model Organism Caenorhabditis elegans

Elena Zanni1 Chiara Laudenzi1 Emily Schifano1 Claudio Palleschi1 Giuditta Perozzi2

Daniela Uccelletti1 and Chiara Devirgiliis2

1Department of Biology and Biotechnology ldquoC Darwinrdquo Sapienza University of Rome Piazzale Aldo Moro 5 00185 Rome Italy2Food amp Nutrition Research Center (CRA-NUT) Agricultural Research Council Via Ardeatina 546 00178 Rome Italy

Correspondence should be addressed to Daniela Uccelletti danielauccellettiuniroma1it

Received 5 September 2014 Accepted 11 November 2014

Academic Editor Haruki Kitazawa

Copyright copy 2015 Elena Zanni et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The nematode Caenorhabditis elegans is widely used as a model system for research on aging development and host-pathogeninteractions Little is currently known about the mechanisms underlying the effects exerted by foodborne microbes We tookadvantage of C elegans to evaluate the impact of foodborne microbiota on well characterized physiological features of theworms Foodborne lactic acid bacteria (LAB) consortium was used to feed nematodes and its composition was evaluated by16S rDNA analysis and strain typing before and after colonization of the nematode gut Lactobacillus delbrueckii L fermentumand Leuconostoc lactis were identified as the main species and shown to display different worm gut colonization capacities LABsupplementation appeared to decrease nematode lifespan compared to the animals fed with the conventional Escherichia colinutrient source or a probiotic bacterial strain Reduced brood size was also observed in microbiota-fed nematodes Moreovermassive accumulation of lipid droplets was revealed by BODIPY staining Altered expression of nhr-49 pept-1 and tub-1 genesassociated with obesity phenotypes was demonstrated by RT-qPCR Since several pathways are evolutionarily conserved in Celegans our results highlight the nematode as a valuable model system to investigate the effects of a complex microbial consortiumon host energy metabolism

1 Introduction

Fermented foods result from the metabolic activity of com-plex and heterogeneous bacterial communities which prolif-erate within the food matrix using carbohydrate substrates tocarry out fermentation processes Moreover dairy fermentedproducts are frequently consumed fresh containing thereforea complex live microbial consortium mostly represented bylactic acid bacteria (LAB) which enter the human body andreach the gastrointestinal tract where they can transientlyinteract with the resident gut microflora of the host Severaldairy and meat products typical of Mediterranean countriesare obtained by traditional manufacturing procedures thatoften employ raw material relying on the natural microflorapreexisting in such ingredients whose species compositionreflects local environments [1] The food microbiota of tra-ditional fermented products is therefore extremely complex

partly specific for each food and in constant interactionwith the environmental bacterial community including thegut microbiota of animals and humans The most relevantLAB in fermented foods belong to the genera LactococcusLactobacillus Streptococcus Pediococcus and LeuconostocSeveral LAB species are also highly represented within theresident gut microbiota of healthy humans Lactobacillusspecies in particular are abundant in both food and gutmicrobiota [2] The interplay between these two microbialcommunities can greatly contribute to human health asseveral foodborne species also display probiotic properties[3] A growing body of literature suggests a link betweengutmicrobiota composition and specificmetabolic disordersincluding obesity [4 5] Alterations of intestinal microbialcomposition were identified in obese human subjects aswell as in animal obesity models and shown to affect hostmetabolism and energy storage There are different ways by

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 621709 12 pageshttpdxdoiorg1011552015621709

2 BioMed Research International

which the gut microbiota can affect host energy metabolismone is represented by an increased level of energy extractionfrom the diet involving specific bacterial phyla and classeswhile others involve a direct influence on host pathwaysby altering the expression of host genes especially thoseinvolved in fat metabolism [6] Given the capability offoodborne bacteria to transiently colonize the intestine thusinfluencing resident gut microflora composition it would beextremely important to identify the mechanisms underlyingthe effects of a complex foodborne microbial consortium onhost energy metabolism which are still poorly understoodOne of the main obstacles towards this goal is the lack ofsimple model organisms suitable for these purposes In thisworkwe tested the nematodeCaenorhabditis elegans as a sim-ple animal model to evaluate the effects of a complex food-derived microbiota on well characterized metabolic path-ways C elegans is of great value in several fields of biologicalresearch sincemany of its pathways are conserved in humansIt is widely used as a model organism for research on agingdevelopment and neurodegenerative diseases In particularfor aging studies the nematodes have the advantage of a shortand reproducible lifespan and ease of cultivationC elegans isa differentiatedmulticellular organismwith a nervous systemreproductive organs and digestive apparatus Furthermoreit has a simple structure and a short life cycle (less than 3days) and can be infected by different human pathogens thatcan replace the regular Escherichia coli food source Dietarysources such as bacteria play an important role in the controlof C elegans lifespan [7] and nematodes exhibit a decreasedlifespan when subjected to a diet of pathogens comparedto those fed with nonpathogenic laboratory microbes suchas auxotrophic strains of E coli Increasing evidences haverevealed that bacterial metabolism has a great impact on hostpathways for example in the case of folate or nitric oxidemetabolism [8 9] The nematode C elegans has been usedin several studies to identify and characterize evolutionarilypreserved traits associated with host-pathogen interactions[10] and also supplies the possibility for fast large-scale andeconomically feasible in vivo screens of new antimicrobialsalong with their mode(s) of action [11] In recent years thenematode has also been employed as a usefulmodel host for awide variety ofmicrobes relevant for humanhealth includingLAB and probiotics The majority of works demonstrate thatseveral LAB species mostly belonging to Lactobacillus genusincrease the nematode lifespan although such effects arehighly strain-dependent (reviewed in [8])On the other handsome LAB species have also been demonstrated to exertdetrimental effects on development and growth of worms[12] It was reported that different Lactobacillus speciesincluding L brevis and L plantarum isolated from fermentedvegetable foods may contribute to host defenses and prolongC elegans lifespan [13] A new functional screening methodwas recently developed to identify and characterize newpotential antioxidant probiotic bacteria revealing that astrain of L rhamnosus could protect the nematode againstoxidative stress [14] In a recent work authors provideevidence that a strain of L salivarius isolated from faecesof centenarian subjects induced longevity in nematodes bydietary restriction [15] Moreover a study investigating the

effect of Bifidobacterium infantis on C elegans longevityfound a modest dose-dependent lifespan extension when Binfantis was added to E coli conventional food source andsuch effects were also observed when nematodes were fedon cell wall or protoplast fractions of B infantis suggestingthe involvement of host protective pathways activated bybacterial cell wall components rather than the productionof bacterial metabolites or gut colonization [16] Howeverthe use of C elegans as a model organism for the studyof probiotic or foodborne bacteria has been restricted tothe analysis of single bacterial strains often deriving fromcollections while few data are available on natural uncharac-terized strains as well as on complex foodborne microbiotaas a whole Mozzarella di Bufala Campana (MBC) is anexample of traditional Italian PDO (Protected Designationof Origin) cheese that is consumed fresh within 2 weeksfrom production and contains high titer of live and complexmicroflora [17] In this work we provide evidence thatfeeding C elegans with a LAB consortium derived fromMBC influences longevity larval development fertility lipidaccumulation and gene expression related to obesity in thismodel organism as supported by transcriptional analysis ofsome genes involved in fat metabolism

2 Materials and Methods21 Cheese Microbiota Preparation Bacterial Strains andGrowth Conditions 10 g of Mozzarella di Bufala Campana(MBC) sampleswere diluted in 90mL sodiumcitrate solution(2wv) and homogenized in a BagMixer400 (InterscienceFrance) 60120583L of homogenate was inoculated in 50mL ofMRS medium (Oxoid Ltd England) and incubated at 37∘Cfor 48 h under anaerobic conditions (Anaerocult A MerckGermany) to obtain a bacterial titer of about 1times1010 CfumLcorresponding to OD

600= 3 Bacterial counts were obtained

by serial dilution in quarter-strength Ringerrsquos solution fol-lowed by plating on MRS agar Plates were incubated at 37∘Cfor 48 h under anaerobic conditions Independent coloniesdisplaying different morphologies were isolated from theplates grown as described above and stored at minus80∘Cin 15 (vv) glycerol Lactobacillus rhamnosus GG (LGGATCC53103) was used as probiotic control where indicated

22 C elegans Strains and Growth Conditions Worms werecultured as described previously [18] and grown at 16∘Csupplemented with the Escherichia coli OP50 (originallyobtained from the Caenorhabditis Genetics Center) unlessotherwise indicated The C elegans strains used in this studyare daf-16 (mu86) mutant and Bristol N2 as standard wildtype strain provided by the Caenorhabditis Genetic Center

Worms were fed with E coli OP50 or LGG or withfoodborne microbiota LAB consortium and LGG cultureswere daily prepared as follows aliquots of frozen cheesehomogenate or frozen LGG stock were inoculated in MRSmedium and grown at 37∘C overnight under anaerobicconditions Similarly an aliquot of OP50 frozen stock wasinoculated in LBmedium and grown at 37∘C overnight underaerobic conditions Afterwards each type of bacterial lawnwas prepared by spreading 25120583L of the bacterial suspension

BioMed Research International 3

Table 1 Primers used in standard and RT-qPCR assays

Primer name Primer sequence (51015840-31015840) Target gene Organism PCR assay ReferenceP0P6

GAGAGTTTGATCCTGGCTCTACGGCTACCTTGTTAC 16S-rDNA Bacteria Standard [20]

GTG5 GTGGTGGTGGTGGTG Genomic repetitive elements Bacteria Standard [21]

pept-1 Forpept-1 Rev

GTGTTCGGAGAAGTATCTCGCAAGAGCACAGTCGTGAGTA

pept-1GenBank accession number

NM 076686C elegans Real-time This work

tub-1 Fortub-1 Rev

CCACAGCAAGTTCAAGAGTCAGCCACTACATCAGTGTTCC

tub-1GenBank accession number


nhr-49 Fornhr-49 Rev

GCTCTCAAGGCTCTGACTCGAGAGCAGAGAATCCACCT

nhr-49GenBank accession number


act-1 Foract-1 Rev

GAGCGTGGTTACTCTTTCACAGAGCTTCTCCTTGATGTC

act-1GenBank accession number


in M9 buffer corresponding to 10mg of bacterial cells onnematode growth medium (NGM) modified to be peptone-free (mNGM) in 35 cm diameter plates according to Ikedaet al (2007) [19]

23 Lifespan Assays Synchronized N2 adults were allowedto lay embryos for 2 h directly on mNGM covered with theindicated bacterial lawns and then sacrificed All lifespanassays started when the progeny became fertile (t0) Animalswere transferred to new plates spread with fresh lawns andmonitored daily They were scored as dead when they nolonger responded to gentle prodding with a platinum wireWorms that crawled off the plates were not included in theanalysis

24 Pharyngeal Pumping Assay Pharyngeal pumping wasanalyzed as described by Uccelletti et al [22] under ZeissAxiovert 25 microscope by counting the number of con-tractions (defined as backward grinder movements in theterminal bulb) on 40 animals for each treatment duringfive periods of 30 s The analysis was performed on L4 wild-type worms grown on LAB or E coli (control) starting fromembryo stage The experiment was performed after 6 daysfrom egg hatching

25 Brood Size Measurement Progeny production was eval-uated according to Zanni et al [23] with some modificationsBriefly synchronized worms obtained as above were grownonmNGMplates seeded with bacteria and then were allowedto lay embryos at 16∘C Next animals were transferred onto afresh bacteria plate every day and the number of progeny wascounted with a Zeiss Axiovert 25 microscope The procedurewas repeated for 4 days until the mother worms stoppedlaying eggs Each day the progeny production was recordedand was compared with the OP50- or LGG-fed nematodes

26 Body Size and Embryos Length Measurement Embryosand individual animals were photographed after 3 4 and 5days from egg hatching using a Leica MZ10F stereomicro-scope connected to JenoptikCCDcamera Length of embryos

or worm body was determined by using the Delta SistemiIAS software and compared to OP50- or LGG-fed worms Atleast 30 nematodes or embryos were imaged on at least threeindependent experiments

27 Lipid Droplets Visualization Approximately 100 L4nematodes grown on LAB LGG or OP50 containingmNGM plates were suspended in 1mL of M9 buffer andwashed three times Subsequently worms were incubatedwith a solution of 67120583gmLBODIPY493503 (Life technolo-gies) for 20min as indicated in the vital staining protocolreported in [24] Afterwards worms were mounted onto 3agarose pads containing 20mM sodium azide and observedwith a Zeiss Axiovert 25 microscope BODIPY images wereacquired using identical settings and exposure times to allowdirect comparisons

28 Real-Time qPCR Total RNA from LGG- or LAB-fedL4 worms was isolated with Trizol reagent (Invitrogen) andthen digested with 2U120583L DNAse I (Ambion) 800 ng ofeach sample was reverse-transcribed using oligo-dT andenhanced Avian reverse transcriptase (SIGMA Cat numberA4464) according to manufacturerrsquos instructions For real-time qPCR assay each well contained 2 120583L of cDNA usedas template SensiMix SYBR amp Fluorescein Kit purchasedfrom Bioline and the selective primers (200 nM) designedwith Primer3 software and reported in Table 1 All sampleswere run in triplicate I Cycler IQ Multicolor Real-TimeDetection System (Biorad) was used for the analysis Thereal-time qPCR conditions are described by Gorietti et al[25] Quantification was performed using a comparative CTmethod (CT = threshold cycle value) Briefly the differencesbetween the mean CT value of each sample and the CTvalue of the housekeeping gene (ama-1) were calculatedΔCTsample = CTsample minus CT119886119898119886-1 Final result was determinedas 2minusΔΔCT where ΔΔCT = ΔCTsample minus ΔCTcontrol

29 Estimation of Bacterial CFU within the Nematode GutFor each experiment 10 animals at L4 stage and at 8 days of


adulthoodwerewashed and lysed according toUccelletti et al[11] Whole worm lysates were plated onto MRS-agar platesThe number of CFU was counted after 48 h of incubation at37∘C anaerobically

210 Bacterial Species Identification and Strain Typing Totalbacterial DNA was obtained from colonies or inoculum bymicroLYSIS (Microzone Canada) according to the manu-facturersrsquo instructions and used as template for PCR ampli-fications Strain typing was performed by rep-PCR finger-printing with the GTG

5primer as described by Gevers et

al [21] (Table 1) The eubacterial P0-P6 primer pair (Table 1Invitrogen Life Technologies Italy) was used to amplify16S rRNA gene fragments [20] PCR mixtures contained200120583Meach dNTPs 1 120583Meach forward and reverse primer2mM MgCl

2 and 2U Taq DNA polymerase (Invitrogen

Italy) in the supplied buffer The PCR products were elutedfrom gels and purified by NucleoSpin Extract II Purifica-tion Kit (Macherey-Nagel Italy) and sequenced with theforward primer (Eurofins MWG Operon Sequencing Ser-vice Germany) Taxonomic identification was performedby comparing the DNA sequences of amplified 16S rDNAfragments with those reported in the Basic BLAST database[26] Representative isolates belonging to each rep groupweresubjected to 16S rDNA amplification and sequencing in orderto identify the species

211 Statistical Analysis Experimentswere performed at leastin triplicate Data are presented as mean plusmn SD and Studentrsquos119905-test or one-way ANOVA analysis coupled with a Bonfer-roni post test (GraphPad Prism 40 software) was used todetermine the statistical significance between experimentalgroups Statistical significance was defined as lowast119875 lt 005lowastlowast

119875 lt 001 and lowastlowastlowast119875 lt 0001

212 GenBank Accession Numbers Nucleotide sequences ofamplified 16S rDNA fromrepresentative isolateswere submit-ted to GenBank and the corresponding accession numbersare reported below

t0-1 KM272561t0-11 KM272562t0-12 KM272563t0-15 KM272564t0-3 KM272565t0-36 KM272566t0-51 KM272567t0-6 KM272568L4-1 KM272569L4-16 KM272570L4-22 KM272571L4-23 KM272572L4-39 KM272573L4-4 KM272574L4-6 KM272575L4-9 KM272576

3 Results

31 Effect of LAB Microbiota Supplementation on NematodeLifespan and Development Our goal was to study the effectsof a dietary source consisting of a foodborne microbialconsortium represented by the LAB component of MBCcheesemicrobiota onC elegans physiology Lifespan analysiswas performed bymonitoring LAB-fedN2wild type animalsstarting from the L1 larval stage in comparison with controlworms grown on E coli OP50 or on a claimed commercialprobiotic strain namely L rhamnosus GG (LGG) Intrigu-ingly the MBC microbiota diet induced a relevant reductionin C elegans longevity in contrast to an increased lifespanobserved in the LGG-fed worms (Figure 1(a)) Such effectsexpressed as the time at which 50 of the worm populationis dead were recorded at days 8 and 19 in LAB- and LGG-fed nematodes respectively in comparison with day 14 inE coli fed animals Notably lifespan shortening appearedto be dependent on the viability of the microbiota used asfood source Indeed dietary administration of heat killedLAB did not produce any effect on nematode lifetime (datanot shown) Microscopic observations of LAB-fed animalsresulted in a size reduction with respect to OP50-fed animalsalong all the developmental stages reaching in length 70of the control during the adulthood (Figure 1(b)) To deter-mine whether longevity and size reduction could originatefrom inefficient food uptake a pumping rate analysis wasperformed However no difference was recorded betweennematodes grown on LAB lawns and the OP50-fed worms(data not shown)

32 LAB Microbiota Diet Influences Fertility and Host EnergyMetabolism Our attention was then focused on exploringpossible F1 alterations induced in C elegans by MBC micro-biota as dietary source To this aim progeny production wasevaluated The brood size of LAB-fed worms was strikinglydecreased with a 72 reduction of progeny number com-pared with animals grown with the E coli food source Areduction was also observed for the progeny derived fromLGG-fed animals although to a lesser extent (Figure 2(a))

In addition MBC microbiota administration led to adecreased size of embryos as compared to the OP50 controlthat was not observed in the case of LGG-fed animals(Figure 2(b))

Intriguingly microscope observation after 3 days fromegg hatching and during the adulthood showed a highertransparency in LAB-fed animals with respect to OP50-fedworms (data not shown) Since this phenotype called ldquoClearrdquohas been reported in animals showing lipid homeostasisdefects [27] we used the lipophilic BODIPYdye to investigatefat storage alterations [28] LAB diet induced in the animalsan enhanced fluorescence as compared to E coli fed worms(data not shown) thus confirming an increase in lipidaccumulation However LAB-fed animals (Figures 3(d) 3(e)and 3(f)) displayed large aggregates of lipid bodies withrespect to LGG-fed nematodes (Figures 3(a) 3(b) and 3(c))

In order to identify the genes possibly responsiblefor the alterations induced by the LAB consortium withrespect to LGG real-time qPCR analysis was performed


0 5 10 15 20 250

25

50

75

100

OP50

Lifespan (days)

Surv

ival

()

LGGlowastlowastlowast

LABlowastlowastlowastlowastlowastlowast

(a)

0

200

400

600

800

1000

1200

1400

3 4 5 6Days from hatching

OP50LAB

LGG

a b ab

a

b b

a

b c

a

bb

Body

leng

th (120583

m)

(b)

Figure 1 Effect of MBC microbiota on nematode lifespan and adult body size (a) Kaplan-Meier survival plot of N2 worms fed with LABconsortium Lifespans of E coli OP50- and LGG-fed animals are reported as controls 119899 = 60 for each data point of single experiments (b)Effect of LAB feeding on C elegans body size Worms were grown in the presence of OP50 LGG (controls) or MBC-derived LAB and theirlength was measured from head to tail at the indicated time points The blue or grey asterisks indicate the 119875 values (log-rank test) againstOP50 or LGG respectively

0

100

200

300

OP50 LAB LGG

lowast

lowastlowast

lowastlowast

Embr

yos

wor

m

(a)

0

20

40

60

OP50 LAB LGG

lowastlowast

Embr

yos l

engt

h (120583

m)

(b)

Figure 2 Effect of MBC microbiota on nematode fertility (a) Average embryos production per worm of OP50- LGG- or LAB-fed animalsBars represent the mean of three independent experiments (b) Measurements of embryos length derived from worms fed with the indicatedbacteria from the L1 stage

for nhr-49 pept-1 and tub-1 genes whose involvement inlipid metabolism and obesity-related phenotypes have beendescribed [29ndash31] The results showed that the MBC micro-biota diet induced an increased transcription of all testedgenes with respect to nematodes fed with the LGG diet(Figure 4)

Since daf-16 is one of the major regulators of fatmetabolism [32] we sought to test whether the LAB dietcould alter fat metabolism by modulating this factor To thisaim we fed loss-of-function daf-16 mutant nematodes withLAB and assessed the lifespan the fertility and the fat storageThe effect of the LAB consortium on fat deposition (data not

shown) and on the lifespan of themutant animals was similarto that observed with the N2 strain (Figure 5(a)) MoreoverLAB supplementation induced a progeny reduction analo-gous to the wild type animals (Figure 5(b))

33 Colonization Capacity of Microbiota and Species Char-acterization Intestinal colonization experiments were per-formed to determine whether the microbiota was consumedby the worms To this aim worms fed LAB or LGG werelysed at different time points and the resulting whole lysatesplated on MRS and grown as described (see Section 2) wereused to evaluate the bacterial colony forming units (CFU)


100120583m

(a)

20120583m

(b)

20120583m

(c)

100120583m

(d)

20120583m

(e)

20120583m

(f)

Figure 3 Visualization of lipid droplets BODIPY staining of L4 C elegans animals grown in the presence of LGG (a b c) or LAB (d e f) asfood sources

Results reported in Figure 6 demonstrated that both dietsincreased the intestinal CFUs along the lifespanHowever theCFU number relative to MBC microbiota diet resulted to beabout 3-fold less than that relative to LGG diet at 8 days ofadulthood (Figure 6)

In order to identify the predominant bacterial speciesmost likely involved in inducing the observed phenotypesin C elegans the MBC microbiota was subjected to speciesidentification We first examined the isolates associated withdifferent colony morphology (ie large and smooth largeand rough and medium) deriving from plates obtained byserial dilution of the homogenate inoculum used to feedthe nematodes (see Section 2) Microscope observation of 10isolates representative of each colony morphology revealedthe presence of long filamentous nonmotile rods (largeand smooth colonies) short nonmotile rods (large andrough colonies) and ovoid forming cocci (medium colonies)(data not shown) 16S rDNA amplification and sequencingidentified them as Lactobacillus delbrueckii L fermentumand Leuconostoc lactis respectively

A more in-depth analysis concerned strain typing of arepresentative number of isolates ranging from 20 to 40colonies performed at time point 0 (representing the initialmicrobial LAB consortium used to feed the worms) as wellas at different nematode stages (representing the intestinalmicrobial community of C elegans fed MBC microbiota)In this latter case worms supplemented until L4 stage or8 days of adulthood with cheese microbiota were washedand lysed and the resulting lysates were plated on MRSin order to isolate bacterial colonies Strain typing wascarried out by rep fingerprinting (see Section 2) and the

Table 2 Bacterial species and related rep groups associated with thenematode gut at different time points after the initial supplementa-tion Time point 0 refers to the microbial community used to feedthe worms

Time point Species Rep group 119873 isolates Totalisolates

0 hL delbrueckiiL fermentumLeuc lactis

VIII IXX XI XII

VII VIIa VIIb

9810

28

L4L delbrueckiiL fermentumLeuc lactis

B2 E FB B1 DA C

20146

40

8 d adults L delbrueckiiL fermentum

H H1G

287 34

results are shown in Figure 7 Isolates displaying the samefingerprinting profile were assigned to a single rep groupRep-PCR amplification identified 8 distinct rep groups outof 28 isolates analyzed at time point 0 h (Figure 7(a)) whileat L4 stage (Figure 7(b)) and at 8 d (Figure 7(c)) 8 rep groupsout of 40 isolates analyzed and 3 rep groups out of 34 wereidentified respectively Only representative rep groups areshown in Figures 7(b) and 7(c) Species assignments weredefined on the basis of sequencing results (see Section 2) orrestriction digestion (data not shown) of 16S rDNA amplifiedfrom representative isolates Correlations between the speciesand rep groups as well as their distribution in the wormgut at different stages or at time point 0 are summarizedin Table 2 Overall we observed an equal distribution of


0

1

2

LGG LAB

Relat

ive m

RNA

leve

l

lowastlowast

nhr-49

(a)

0

05

1

15

2

25

LGG LAB

Relat

ive m

RNA

leve

l

lowastlowastlowast

tub-1

(b)

0

1

2

3

LGG LAB

Relat

ive m

RNA

leve

l

lowastlowastlowastpept-1

(c)

Figure 4 RT-qPCR analysis of lipid metabolism genes Expression of (a) nhr-49 (b) tub-1 and (c) pept-1 genes in LGG- or LAB-fed animalsafter 3 days from hatching Histograms show the expression of lipid metabolism related genes detected by real-time PCR

L delbrueckii L fermentum and Leuc lactis in the initialmicrobial consortiumwhile a progressive decreasing of Leuclactis was observed in the worm gut microbial community atthe observed time points In particular only L fermentumand L delbrueckii were recovered from nematode lysates at8 days of adulthood with a prevalence of the L delbrueckiispecies (28 isolates out of 34) (Table 2)

4 Discussion

In the present work we have evaluated the impact onC elegans physiology of a complex foodborne microbialconsortium derived from a traditional fermented cheese Tothis aim nematodes were fedMBCmicrobiota fromhatchingthroughout development Longevity analysis demonstratedthat LAB-fed animals exhibited a reduced lifespan withrespect to control worms fed conventional E coli OP50 orthe LGG probiotic strain Such effect was not attributable toinefficient food uptake and was dependent on LAB viabilityAn infection-like process exerted by the consortium couldbe hypothesized however nematodes grown with MBC

microbiota did not show intestinal lumendistension (data notshown) a characteristic sign of pathogenesis [33] Moreoverlifespan analysis in the immunocompromised daf-16 mutantrevealed that LAB diet induced a reduction in the lifetimeto the same extent of that observed for C elegans wild typestrain reinforcing the idea that no infection process tookplace in LAB-fed worms

The observed effect on longevity was quite surprisingsince in the majority of previously published articles LABhave been shown to increase the nematode lifespan [13ndash16 19] However a negative effect exerted by L salivariusL reuteri and Pediococcus acidilactici on the developmentand growth of the worm was also recently reported [12]Such conflicting results could be explained by assuming thatdistinct species and strains can promote differential effects onC elegans Moreover it is important to consider that in somecases the experiments have been performed with heat killedbacteria [13 15] and that in most published studies LAB weresupplied to adult worms rather than the embryos

We consider a key aspect that bacteria must be viableto exert these effects since fermented dairy products which


0 5 10 150

20

40

60

80

100

LGG

Days of adulthood

Surv

ival

()

daf-16

LABlowastlowast

(a)

0

50

100

150

LGG LAB

Embr

yos

wor

m

lowast

(b)

Figure 5 Effect of MBC microbiota on daf-16 mutant (a) Kaplan-Meier survival plot of daf-16 worms fed with LAB consortium Lifespan ofLGG-fed animals is reported as control 119899 = 60 for each data point of single experiments (b) Average embryos production per worm of LGG-or LAB-fed daf-16 animals Bars represent the mean of three independent experimentsThe grey asterisks indicate the 119875 values (log-rank test)against LGG

L4 worm 8-day-old adult0

15

30

45

300

600

900

1200

1500

LGGLAB

CFU

wor

m

lowastlowastlowast

Figure 6 Bacterial colonization capacity Bacterial colony formingunits (CFU) recovered from nematodes were obtained by platingwhole lysates of L4 and 8-day-old adults fed LGG (grey bars) orLAB (orange bars) Bars represent the mean of three independentexperiments

are usually consumed fresh are characterized by high titersof live microflora potentially capable of colonizing thehuman gut In addition to evaluate the effects of foodbornemicrobes it would be ldquomore physiologicalrdquo to study the wholecomplex microbiota per se rather than the single speciesthat could mask the effects of the consortium Complex

bacterial environments can be composed indeed of severalunderrepresented species difficult to isolate

Analysis of MBC consortium in terms of species com-position resulted in the identification of three predominantspecies L fermentum L delbrueckii and Leuc lactis inagreement with previous findings [17] which have never beentested in C elegans so far The same species were also foundassociatedwithwormgut after ingestionwith different distri-butions relative to the time points analyzed Taken togetherthese results suggest that the overall initial cheese microbiotacomposition is maintained in the nematode intestine duringthe development while at 8 days of adulthood only Ldelbrueckii and L fermentum are still capable of survivingin the worm gut In particular prevalence of L delbrueckiiwas observed On the other hand Leuc lactis appears to beno longer able to colonize worm intestine Several factorscontribute to bacterial competition for colonization in Celegans gut such as selection by the host and strain-specificcharacteristics [34]C elegans gut colonization is still an openquestion and several works report a correlation betweenbacterial colonization and nematode lifespan as well asdefence response [35 36]

Previous findings suggest that both Enterococcus faecalisand E faecium accumulate to high titers in the nematodegut lumen during infection process although only E faecaliswas able to kill adult worms [37] Conflicting results havebeen reported by Forrester et al who demonstrated thatthe foodborne pathogen Listeria monocytogenes althoughexerting deleterious effects on C elegans did not persist inthe nematode intestine [38] suggesting that a complex setof mechanisms regulate host-bacteria interactions Similarly


M M1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16VII VII VIIa VII VII VIIb VII VII VII VII VIII X X VIII XI VIII

31 32 33 34 35 36 37 51 52 53 54 55IX IX IX IX IX IX IX XII XII XII XII XII

(a)

M 1 2 4 6 9 16 22 23 24 28 29 31 38 39A B C B1 D B E B2 E B2 E E B2 F

(b)

M 2 12 4 11G H H1 H

(c)

Figure 7 Biodiversity of nematode intestinal microbiota Rep fingerprinting profiles of bacterial colonies randomly chosen after plating MBChomogenates used to feed worms (a) or nematode intestinal lysates at L4 stage (b) and 8 days of adulthood (c) following LAB microbiotasupplementation Arabic numerals indicate each isolate while roman numerals or letters identify rep groups M 1 kb DNA ladder PromegaOnly representative rep groups are shown in panels a and c

a reduced colonization capacity has been observed in the caseof MBC microbiota with respect to LGG Further studies aretherefore necessary to unravel such mechanisms

Beyond the effects on lifespan we observed that LAB-and LGG-fed worms displayed fat accumulation as revealedby BODIPY staining In C elegans fat is mainly storedin intestinal and hypodermal lipid droplets [39 40] Inparticular LAB-fed nematodes displayed larger lipid bodieswith respect to LGG-fed worms suggesting that foodbornemicrobial consortium can have a higher impact on host fatmetabolism

Foodborne microbes are introduced in the human bodythrough the food chain and those capable of surviving theharsh conditions of the upper GI tract can reach the colonwhere they can interact and modify the autochthonous gut

microbiota However the contribution of foodborne bacteriato the modulation of host energy metabolism is still unclearOne of the mechanisms by which the gut microbes can leadto metabolic disorders is the alteration of expression of hostgenes involved in energy expenditure and conservation [41]We found that genes previously described as responsiblefor obesity phenotypes in C elegans that is pept-1 nhr-49 and tub-1 are induced in MBC microbiota-fed wormsdemonstrating for the first time the capability of foodbornemicrobes to alter lipid metabolism and accumulation

It has been reported that different E coli strains sig-nificantly affect fat storage levels through the involvementof the intestinal peptide transporter Pept-1 [42] The factthat LAB-fed worms showed higher level of pept-1 transcriptwith respect to LGG-fed animals suggests that the uptake


of di- and tripeptides derived from foodborne microbiotacould be not enough to guarantee the building blocks forprotein synthesis However we can not exclude that reducedfunctionality of the transporter could be caused by an alteredintestinal cell pH originating from LAB metabolism insidethe worm gut that leads to an increased mRNA level as acompensatory mechanism This hypothesis is in agreementwith the fact that pept-1 mutants showed phenotypes similarto those observed in LAB-fed nematodes [43]

Foodborne microbiota induced in C elegans alsoincreased transcription of the nuclear hormone receptorgene nhr-49 although to a lesser extent compared to theother genes tested namely pept-1 and tub-1 This geneencodes a transcription factor localized within nematodebody wall muscle cells [44] and involved in the controlof pathways that regulate fat consumption and maintainnormal balance of fatty acids saturation by modulating theexpression of genes involved in fatty acid beta-oxidation [29]It could be speculated that the increased nhr-49 transcriptlevels in LAB-fed animals represent an attempt to decreasethe fat storages by increasing the beta-oxidation process

Lipid accumulation has been connected inC eleganswiththe sensory cilia implicated in sensing the chemical andorphysical extracellular environments Tub-1 the transcriptionfactor homologous to the mouse protein Tubby results to belocalized to the axons dendrites and cilia [45] and influencesfat metabolism [46] Increased levels of tub-1 transcript werealso found in LAB-fed nematodes suggesting a link betweenthe sensory neuron and the diet that will deserve furtherinvestigations

Taken together these results suggest that supplementa-tion of nematodes with a foodborne microbiota influenceshost energy metabolism Both microbiota- and LGG-fedanimals showed reduced progeny this can be in agreementwith the fact that worms spend part of their energy onreproduction Embryos use yolk transferred from the intes-tine to the gonadal arms as a primary food source [47] wetherefore can hypothesize that LAB and LGG diet may alterthe reproductive behaviour indirectly through modificationsof fat stores

Intriguingly the predominant species identified in LABconsortium used to feed the nematodes and triggeringobesity-like phenotypes belong to Firmicutes phylum Bothdiet- and genetic-induced obesity are associated with animbalance between Bacteroidetes and Firmicutes the twomajor phyla of gut bacteria with Firmicutes prevailing inobese subjects [48]

Studies aimed at investigating the role of gut bacteriain metabolic disease progression have greatly profited fromgnotobiotic animal models [6] However the availability ofsimpler model organisms would be extremely valuable interms of ease of cultivation reduced costs and ethic issuesWe have provided evidence that foodborne microbes cancolonize C elegans gut and modulate host gene expressionOverall our results could represent a first step towards theidentification ofmetabolic pathways influenced by foodbornebacteria

5 Conclusions

C elegans has been extensively used in biology research andmost recently some studies have taken advantage of thismodel to study host-microbiota interactions using isolatedfoodborne bacteria or probiotic strains

Herein we found that supplementation of nematodeswith a foodborne microbiota derived from a traditionalfermented dairy product influences longevity and fertilityas well as lipid accumulation and gene expression relatedto obesity as supported by transcriptional analysis of somegenes involved in fat metabolism We provide evidence thatin foodborne microbiota-fed worms expression of genesencoding transcriptional factors involved in fatty acid beta-oxidation or nutrient sensing and influencing fat metabolismand storage is induced demonstrating the capability offoodborne microbes to alter host lipid metabolism and accu-mulation Moreover we found that the main bacterial species(L fermentum L delbrueckii and Leuc lactis) characterizingthe consortium are able to colonize worm gut

Overall our work extends the applicability of such modelin the field of host-microbiota interaction by evaluating theinfluence exerted by a cheese derived LAB consortium ondifferent physiological aspects of the nematodes

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Theauthors acknowledge theCaenorhabditisGenetics Center(University of Minnesota Minneapolis) for the nematodeand E coliOP50 strainsThis work was financially supportedby Grants NUME (DM 3688730308) and MEDITO (DM12487730311) from the ItalianMinistry of Agriculture Foodamp Forestry (MiPAAF) Elena Zanni was partially supportedby Pasteur Institute-Cenci Bolognetti Foundation

References

[1] M Morea F Baruzzi and P S Cocconcelli ldquoMolecular andphysiological characterization of dominant bacterial popula-tions in traditional Mozzarella cheese processingrdquo Journal ofApplied Microbiology vol 87 no 4 pp 574ndash582 1999

[2] S-Q Liu ldquoPractical implications of lactate and pyruvatemetabolismby lactic acid bacteria in food and beverage fermen-tationsrdquo International Journal of Food Microbiology vol 83 no2 pp 115ndash131 2003

[3] C Devirgiliis S Barile and G Perozzi ldquoAntibiotic resistancedeterminants in the interplay between food and gut micro-biotardquo Genes amp Nutrition vol 6 no 3 pp 275ndash284 2011

[4] V Tremaroli and F Backhed ldquoFunctional interactions betweenthe gut microbiota and host metabolismrdquo Nature vol 489 no7415 pp 242ndash249 2012

[5] E Angelakis F Armougom M Million and D RaoultldquoThe relationship between gut microbiota and weight gain inhumansrdquo Future Microbiology vol 7 no 1 pp 91ndash109 2012


[6] F Backhed ldquoHost responses to the human microbiomerdquo Nutri-tion Reviews vol 70 supplement 1 pp S14ndashS17 2012

[7] S So T Tokumaru K Miyahara and Y Ohshima ldquoControl oflifespan by food bacteria nutrient limitation and pathogenicityof food in C elegansrdquo Mechanisms of Ageing and Developmentvol 132 no 4 pp 210ndash212 2011

[8] L C Clark and J Hodgkin ldquoCommensals probiotics andpathogens in the Caenorhabditis elegans modelrdquo CellularMicrobiology vol 16 no 1 pp 27ndash38 2014

[9] T P Nguyen and C F Clarke ldquoFolate status of gut microbiomeaffects Caenorhabditis elegans lifespanrdquo BMC Biology vol 10article 66 2012

[10] CD Sifri J Begun and FMAusubel ldquoThewormhas turnedmdashmicrobial virulence modeled in Caenorhabditis elegansrdquo Trendsin Microbiology vol 13 no 3 pp 119ndash127 2005

[11] D Uccelletti E Zanni L Marcellini C Palleschi D Barraand M L Mangoni ldquoAnti-Pseudomonas activity of frog skinantimicrobial peptides in a Caenorhabditis elegans infectionmodel a plausible mode of action in vitro and in vivordquoAntimicrobial Agents andChemotherapy vol 54 no 9 pp 3853ndash3860 2010

[12] M K Fasseas C Fasseas K C Mountzouris and P SyntichakildquoEffects of Lactobacillus salivarius Lactobacillus reuteri andPediococcus acidilactici on the nematodeCaenorhabditis elegansinclude possible antitumor activityrdquo Applied Microbiology andBiotechnology vol 97 no 5 pp 2109ndash2118 2013

[13] J Lee H S Yun K W Cho et al ldquoEvaluation of probioticcharacteristics of newly isolated Lactobacillus spp immunemodulation and longevityrdquo International Journal of FoodMicro-biology vol 148 no 2 pp 80ndash86 2011

[14] G Grompone P Martorell S Llopis et al ldquoAnti-inflammatoryLactobacillus rhamnosus CNCM I-3690 strain protects againstoxidative stress and increases lifespan in Caenorhabditis ele-gansrdquo PLoS ONE vol 7 no 12 Article ID e52493 2012

[15] Y Zhao L Zhao X Zheng T Fu H Guo and F RenldquoLactobacillus salivarius strain FDB89 induced longevity inCaenorhabditis elegans by dietary restrictionrdquo Journal of Micro-biology vol 51 no 2 pp 183ndash188 2013

[16] T Komura T Ikeda C Yasui S Saeki and Y NishikawaldquoMechanismunderlying prolongevity induced by bifidobacteriain Caenorhabditis elegansrdquo Biogerontology vol 14 no 1 pp 73ndash87 2013

[17] C Devirgiliis A Caravelli D Coppola S Barile and GPerozzi ldquoAntibiotic resistance andmicrobial composition alongthe manufacturing process of Mozzarella di Bufala CampanardquoInternational Journal of Food Microbiology vol 128 no 2 pp378ndash384 2008

[18] S Brenner ldquoThe genetics of Caenorhabditis elegansrdquo Geneticsvol 77 no 1 pp 71ndash94 1974

[19] T Ikeda C Yasui K Hoshino K Arikawa and Y NishikawaldquoInfluence of lactic acid bacteria on longevity of Caenorhabditiselegans and host defense against Salmonella enterica serovarEnteritidisrdquo Applied and Environmental Microbiology vol 73no 20 pp 6404ndash6409 2007

[20] F Di Cello and R Fani ldquoA molecular strategy for the studyof natural bacterial communities by PCR-based techniquesrdquoMinerva Biotecnologica vol 8 no 3 pp 126ndash134 1996

[21] D Gevers G Huys and J Swings ldquoApplicability of rep-PCRfingerprinting for identification of Lactobacillus speciesrdquo FEMSMicrobiology Letters vol 205 no 1 pp 31ndash36 2001

[22] D Uccelletti A Pascoli F Farina et al ldquoAPY-1 a novelCaenorhabditis elegans apyrase involved in unfolded proteinresponse signalling and stress responsesrdquo Molecular Biology ofthe Cell vol 19 no 4 pp 1337ndash1345 2008

[23] E Zanni G de Bellis M P Bracciale et al ldquoGraphitenanoplatelets and Caenorhabditis elegans insights from an invivomodelrdquo Nano Letters vol 12 no 6 pp 2740ndash2744 2012

[24] M KlapperM Ehmke D Palgunow et al ldquoFluorescence-basedfixative and vital staining of lipid droplets in Caenorhabditiselegans reveal fat stores using microscopy and flow cytometryapproachesrdquo The Journal of Lipid Research vol 52 no 6 pp1281ndash1293 2011

[25] D Gorietti E Zanni C Palleschi et al ldquoDepletion of caseinkinase I leads to a NAD(P)+NAD(P)H balance-dependentmetabolic adaptation as determined by NMR spectroscopy-metabolomic profile in Kluyveromyces lactisrdquo Biochimica etBiophysica ActamdashGeneral Subjects vol 1840 no 1 pp 556ndash5642014

[26] S F Altschul T L Madden A A Schaffer et al ldquoGappedBLAST and PSI-BLAST a new generation of protein databasesearch programsrdquo Nucleic Acids Research vol 25 no 17 pp3389ndash3402 1997

[27] T Nomura M Horikawa S Shimamura T Hashimoto andK Sakamoto ldquoFat accumulation in Caenorhabditis elegans ismediated by SREBP homolog SBP-1rdquo Genes and Nutrition vol5 no 1 pp 17ndash27 2010

[28] E Bonilla and A Prelle ldquoApplication of nile blue and nile redtwo fluorescent probes for detection of lipid droplets in humanskeletal musclerdquo Journal of Histochemistry ampCytochemistry vol35 no 5 pp 619ndash621 1987

[29] M R vanGilst H Hadjivassiliou A Jolly and K R YamamotoldquoNuclear hormone receptor NHR-49 controls fat consumptionand fatty acid composition in C elegansrdquo PLoS Biology vol 3no 2 article e53 2005

[30] A Mukhopadhyay B Deplancke A J M Walhout and H ATissenbaum ldquoC elegans tubby regulates life span and fat storageby two independent mechanismsrdquo Cell Metabolism vol 2 no 1pp 35ndash42 2005

[31] B Spanier K Lasch S Marsch et al ldquoHow the intestinalpeptide transporter PEPT-1 contributes to an obesity phenotypein Caenorhabditits elegansrdquo PLoS ONE vol 4 no 7 Article IDe6279 2009

[32] S S Lee S Kennedy A C Tolonen and G Ruvkun ldquoDAF-16target genes that control C elegans Life-span and metabolismrdquoScience vol 300 no 5619 pp 644ndash647 2003

[33] J E Irazoqui E R Troemel R L Feinbaum L G LuhachackB O Cezairliyan and FMAusubel ldquoDistinct pathogenesis andhost responses during infection of C elegans by P aeruginosaand S aureusrdquoPLoS Pathogens vol 6 no 7 Article ID e1000982pp 1ndash24 2010

[34] C Portal-Celhay and M J Blaser ldquoCompetition and resiliencebetween founder and introduced bacteria in theCaenorhabditiselegans gutrdquo Infection and Immunity vol 80 no 3 pp 1288ndash1299 2012

[35] F Gomez G C Monsalve V Tse et al ldquoDelayed accumulationof intestinal coliform bacteria enhances life span and stressresistance in Caenorhabditis elegansfed respiratory deficient Ecolirdquo BMCMicrobiology vol 12 article 300 2012

[36] Y Kim and EMylonakis ldquoCaenorhabditis elegans immune con-ditioningwith the probiotic bacterium Lactobacillus acidophilusstrain ncfm enhances gram-positive immune responsesrdquo Infec-tion and Immunity vol 80 no 7 pp 2500ndash2508 2012


[37] D A Garsin C D Sifri E Mylonakis et al ldquoA simplemodel host for identifying Gram-positive virulence factorsrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 98 no 19 pp 10892ndash10897 2001

[38] S Forrester S R Milillo W A Hoose M Wiedmann andU Schwab ldquoEvaluation of the pathogenicity of Listeria spp inCaenorhabditis elegansrdquo Foodborne Pathogens and Disease vol4 no 1 pp 67ndash73 2007

[39] B C Mullaney R D Blind G A Lemieux et al ldquoRegulationof C elegans fat uptake and storage by acyl-CoA synthase-3 isdependent on NR5A family nuclear hormone receptor nhr-25rdquoCell Metabolism vol 12 no 4 pp 398ndash410 2010

[40] S O Zhang A C Box N Xu et al ldquoGenetic and dietaryregulation of lipid droplet expansion inCaenorhabditis elegansrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 107 no 10 pp 4640ndash4645 2010

[41] D M Tsukumo B M Carvalho M A Carvalho-Filho andJ A S Mario ldquoTranslational research into gut microbiotanew horizons in obesity treatmentrdquo Arquivos Brasileiros deEndocrinologia e Metabologia vol 53 no 2 pp 139ndash144 2009

[42] K K Brooks B Liang and J L Watts ldquoThe influence ofbacterial diet on fat storage in C elegansrdquo PLoS ONE vol 4 no10 Article ID e7545 2009

[43] B Meissner M Boll H Daniel and R Baumeister ldquoDeletionof the intestinal peptide transporter affects insulin and TORsignaling in Caenorhabditis elegansrdquo The Journal of BiologicalChemistry vol 279 no 35 pp 36739ndash36745 2004

[44] B Meissner T Rogalski R Viveiros et al ldquoDeterminingthe sub-cellular localization of proteins within Caenorhabditiselegans body wall musclerdquo PLoS ONE vol 6 no 5 Article IDe19937 2011

[45] H Y Mak L S Nelson M Basson C D Johnson andG Ruvkun ldquoPolygenic control of Caenorhabditis elegans fatstoragerdquo Nature Genetics vol 38 no 3 pp 363ndash368 2006

[46] K Ashrafi F Y Chang J L Watts et al ldquoGenome-wide RNAianalysis of Caenorhabditis elegans fat regulatory genesrdquo Naturevol 421 no 6920 pp 268ndash272 2003

[47] H M Kubagawa J L Watts C Corrigan et al ldquoOocytesignals derived from polyunsaturated fatty acids control spermrecruitment in vivordquoNature Cell Biology vol 8 no 10 pp 1143ndash1148 2006

[48] L M Cox and M J Blaser ldquoPathways in microbe-inducedobesityrdquo Cell Metabolism vol 17 no 6 pp 883ndash894 2013

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


which the gut microbiota can affect host energy metabolismone is represented by an increased level of energy extractionfrom the diet involving specific bacterial phyla and classeswhile others involve a direct influence on host pathwaysby altering the expression of host genes especially thoseinvolved in fat metabolism [6] Given the capability offoodborne bacteria to transiently colonize the intestine thusinfluencing resident gut microflora composition it would beextremely important to identify the mechanisms underlyingthe effects of a complex foodborne microbial consortium onhost energy metabolism which are still poorly understoodOne of the main obstacles towards this goal is the lack ofsimple model organisms suitable for these purposes In thisworkwe tested the nematodeCaenorhabditis elegans as a sim-ple animal model to evaluate the effects of a complex food-derived microbiota on well characterized metabolic path-ways C elegans is of great value in several fields of biologicalresearch sincemany of its pathways are conserved in humansIt is widely used as a model organism for research on agingdevelopment and neurodegenerative diseases In particularfor aging studies the nematodes have the advantage of a shortand reproducible lifespan and ease of cultivationC elegans isa differentiatedmulticellular organismwith a nervous systemreproductive organs and digestive apparatus Furthermoreit has a simple structure and a short life cycle (less than 3days) and can be infected by different human pathogens thatcan replace the regular Escherichia coli food source Dietarysources such as bacteria play an important role in the controlof C elegans lifespan [7] and nematodes exhibit a decreasedlifespan when subjected to a diet of pathogens comparedto those fed with nonpathogenic laboratory microbes suchas auxotrophic strains of E coli Increasing evidences haverevealed that bacterial metabolism has a great impact on hostpathways for example in the case of folate or nitric oxidemetabolism [8 9] The nematode C elegans has been usedin several studies to identify and characterize evolutionarilypreserved traits associated with host-pathogen interactions[10] and also supplies the possibility for fast large-scale andeconomically feasible in vivo screens of new antimicrobialsalong with their mode(s) of action [11] In recent years thenematode has also been employed as a usefulmodel host for awide variety ofmicrobes relevant for humanhealth includingLAB and probiotics The majority of works demonstrate thatseveral LAB species mostly belonging to Lactobacillus genusincrease the nematode lifespan although such effects arehighly strain-dependent (reviewed in [8])On the other handsome LAB species have also been demonstrated to exertdetrimental effects on development and growth of worms[12] It was reported that different Lactobacillus speciesincluding L brevis and L plantarum isolated from fermentedvegetable foods may contribute to host defenses and prolongC elegans lifespan [13] A new functional screening methodwas recently developed to identify and characterize newpotential antioxidant probiotic bacteria revealing that astrain of L rhamnosus could protect the nematode againstoxidative stress [14] In a recent work authors provideevidence that a strain of L salivarius isolated from faecesof centenarian subjects induced longevity in nematodes bydietary restriction [15] Moreover a study investigating the

effect of Bifidobacterium infantis on C elegans longevityfound a modest dose-dependent lifespan extension when Binfantis was added to E coli conventional food source andsuch effects were also observed when nematodes were fedon cell wall or protoplast fractions of B infantis suggestingthe involvement of host protective pathways activated bybacterial cell wall components rather than the productionof bacterial metabolites or gut colonization [16] Howeverthe use of C elegans as a model organism for the studyof probiotic or foodborne bacteria has been restricted tothe analysis of single bacterial strains often deriving fromcollections while few data are available on natural uncharac-terized strains as well as on complex foodborne microbiotaas a whole Mozzarella di Bufala Campana (MBC) is anexample of traditional Italian PDO (Protected Designationof Origin) cheese that is consumed fresh within 2 weeksfrom production and contains high titer of live and complexmicroflora [17] In this work we provide evidence thatfeeding C elegans with a LAB consortium derived fromMBC influences longevity larval development fertility lipidaccumulation and gene expression related to obesity in thismodel organism as supported by transcriptional analysis ofsome genes involved in fat metabolism

2 Materials and Methods21 Cheese Microbiota Preparation Bacterial Strains andGrowth Conditions 10 g of Mozzarella di Bufala Campana(MBC) sampleswere diluted in 90mL sodiumcitrate solution(2wv) and homogenized in a BagMixer400 (InterscienceFrance) 60120583L of homogenate was inoculated in 50mL ofMRS medium (Oxoid Ltd England) and incubated at 37∘Cfor 48 h under anaerobic conditions (Anaerocult A MerckGermany) to obtain a bacterial titer of about 1times1010 CfumLcorresponding to OD

600= 3 Bacterial counts were obtained

by serial dilution in quarter-strength Ringerrsquos solution fol-lowed by plating on MRS agar Plates were incubated at 37∘Cfor 48 h under anaerobic conditions Independent coloniesdisplaying different morphologies were isolated from theplates grown as described above and stored at minus80∘Cin 15 (vv) glycerol Lactobacillus rhamnosus GG (LGGATCC53103) was used as probiotic control where indicated

22 C elegans Strains and Growth Conditions Worms werecultured as described previously [18] and grown at 16∘Csupplemented with the Escherichia coli OP50 (originallyobtained from the Caenorhabditis Genetics Center) unlessotherwise indicated The C elegans strains used in this studyare daf-16 (mu86) mutant and Bristol N2 as standard wildtype strain provided by the Caenorhabditis Genetic Center

Worms were fed with E coli OP50 or LGG or withfoodborne microbiota LAB consortium and LGG cultureswere daily prepared as follows aliquots of frozen cheesehomogenate or frozen LGG stock were inoculated in MRSmedium and grown at 37∘C overnight under anaerobicconditions Similarly an aliquot of OP50 frozen stock wasinoculated in LBmedium and grown at 37∘C overnight underaerobic conditions Afterwards each type of bacterial lawnwas prepared by spreading 25120583L of the bacterial suspension










tub-1 Fortub-1 Rev








act-1 Foract-1 Rev
























3 Results







0 5 10 15 20 250

25

50

75

100

OP50

Lifespan (days)

Surv

ival

()



(a)

0

200

400

600

800

1000

1200

1400


OP50LAB

LGG

a b ab

a

b b

a

b c

a

bb

Body

leng

th (120583

m)

(b)


0

100

200

300

OP50 LAB LGG

lowast

lowastlowast

lowastlowast

Embr

yos

wor

m

(a)

0

20

40

60

OP50 LAB LGG

lowastlowast

Embr

yos l

engt

h (120583

m)

(b)







100120583m

(a)

20120583m

(b)

20120583m

(c)

100120583m

(d)

20120583m

(e)

20120583m

(f)








VIII IXX XI XII

VII VIIa VIIb

9810

28


B2 E FB B1 DA C

20146

40


H H1G

287 34



0

1

2

LGG LAB

Relat

ive m

RNA

leve

l

lowastlowast

nhr-49

(a)

0

05

1

15

2

25

LGG LAB

Relat

ive m

RNA

leve

l

lowastlowastlowast

tub-1

(b)

0

1

2

3

LGG LAB

Relat

ive m

RNA

leve

l


(c)



4 Discussion






0 5 10 150

20

40

60

80

100

LGG

Days of adulthood

Surv

ival

()

daf-16

LABlowastlowast

(a)

0

50

100

150

LGG LAB

Embr

yos

wor

m

lowast

(b)



15

30

45

300

600

900

1200

1500

LGGLAB

CFU

wor

m

lowastlowastlowast









(a)


(b)

M 2 12 4 11G H H1 H

(c)














5 Conclusions






Acknowledgments


References


























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology










tub-1 Fortub-1 Rev








act-1 Foract-1 Rev
























3 Results







0 5 10 15 20 250

25

50

75

100

OP50

Lifespan (days)

Surv

ival

()



(a)

0

200

400

600

800

1000

1200

1400


OP50LAB

LGG

a b ab

a

b b

a

b c

a

bb

Body

leng

th (120583

m)

(b)


0

100

200

300

OP50 LAB LGG

lowast

lowastlowast

lowastlowast

Embr

yos

wor

m

(a)

0

20

40

60

OP50 LAB LGG

lowastlowast

Embr

yos l

engt

h (120583

m)

(b)







100120583m

(a)

20120583m

(b)

20120583m

(c)

100120583m

(d)

20120583m

(e)

20120583m

(f)








VIII IXX XI XII

VII VIIa VIIb

9810

28


B2 E FB B1 DA C

20146

40


H H1G

287 34



0

1

2

LGG LAB

Relat

ive m

RNA

leve

l

lowastlowast

nhr-49

(a)

0

05

1

15

2

25

LGG LAB

Relat

ive m

RNA

leve

l

lowastlowastlowast

tub-1

(b)

0

1

2

3

LGG LAB

Relat

ive m

RNA

leve

l


(c)



4 Discussion






0 5 10 150

20

40

60

80

100

LGG

Days of adulthood

Surv

ival

()

daf-16

LABlowastlowast

(a)

0

50

100

150

LGG LAB

Embr

yos

wor

m

lowast

(b)



15

30

45

300

600

900

1200

1500

LGGLAB

CFU

wor

m

lowastlowastlowast









(a)


(b)

M 2 12 4 11G H H1 H

(c)














5 Conclusions






Acknowledgments


References


























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology












3 Results







0 5 10 15 20 250

25

50

75

100

OP50

Lifespan (days)

Surv

ival

()



(a)

0

200

400

600

800

1000

1200

1400


OP50LAB

LGG

a b ab

a

b b

a

b c

a

bb

Body

leng

th (120583

m)

(b)


0

100

200

300

OP50 LAB LGG

lowast

lowastlowast

lowastlowast

Embr

yos

wor

m

(a)

0

20

40

60

OP50 LAB LGG

lowastlowast

Embr

yos l

engt

h (120583

m)

(b)







100120583m

(a)

20120583m

(b)

20120583m

(c)

100120583m

(d)

20120583m

(e)

20120583m

(f)








VIII IXX XI XII

VII VIIa VIIb

9810

28


B2 E FB B1 DA C

20146

40


H H1G

287 34



0

1

2

LGG LAB

Relat

ive m

RNA

leve

l

lowastlowast

nhr-49

(a)

0

05

1

15

2

25

LGG LAB

Relat

ive m

RNA

leve

l

lowastlowastlowast

tub-1

(b)

0

1

2

3

LGG LAB

Relat

ive m

RNA

leve

l


(c)



4 Discussion






0 5 10 150

20

40

60

80

100

LGG

Days of adulthood

Surv

ival

()

daf-16

LABlowastlowast

(a)

0

50

100

150

LGG LAB

Embr

yos

wor

m

lowast

(b)



15

30

45

300

600

900

1200

1500

LGGLAB

CFU

wor

m

lowastlowastlowast









(a)


(b)

M 2 12 4 11G H H1 H

(c)














5 Conclusions






Acknowledgments


References


























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0 5 10 15 20 250

25

50

75

100

OP50

Lifespan (days)

Surv

ival

()



(a)

0

200

400

600

800

1000

1200

1400


OP50LAB

LGG

a b ab

a

b b

a

b c

a

bb

Body

leng

th (120583

m)

(b)


0

100

200

300

OP50 LAB LGG

lowast

lowastlowast

lowastlowast

Embr

yos

wor

m

(a)

0

20

40

60

OP50 LAB LGG

lowastlowast

Embr

yos l

engt

h (120583

m)

(b)







100120583m

(a)

20120583m

(b)

20120583m

(c)

100120583m

(d)

20120583m

(e)

20120583m

(f)








VIII IXX XI XII

VII VIIa VIIb

9810

28


B2 E FB B1 DA C

20146

40


H H1G

287 34



0

1

2

LGG LAB

Relat

ive m

RNA

leve

l

lowastlowast

nhr-49

(a)

0

05

1

15

2

25

LGG LAB

Relat

ive m

RNA

leve

l

lowastlowastlowast

tub-1

(b)

0

1

2

3

LGG LAB

Relat

ive m

RNA

leve

l


(c)



4 Discussion






0 5 10 150

20

40

60

80

100

LGG

Days of adulthood

Surv

ival

()

daf-16

LABlowastlowast

(a)

0

50

100

150

LGG LAB

Embr

yos

wor

m

lowast

(b)



15

30

45

300

600

900

1200

1500

LGGLAB

CFU

wor

m

lowastlowastlowast









(a)


(b)

M 2 12 4 11G H H1 H

(c)














5 Conclusions






Acknowledgments


References


























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


100120583m

(a)

20120583m

(b)

20120583m

(c)

100120583m

(d)

20120583m

(e)

20120583m

(f)








VIII IXX XI XII

VII VIIa VIIb

9810

28


B2 E FB B1 DA C

20146

40


H H1G

287 34



0

1

2

LGG LAB

Relat

ive m

RNA

leve

l

lowastlowast

nhr-49

(a)

0

05

1

15

2

25

LGG LAB

Relat

ive m

RNA

leve

l

lowastlowastlowast

tub-1

(b)

0

1

2

3

LGG LAB

Relat

ive m

RNA

leve

l


(c)



4 Discussion






0 5 10 150

20

40

60

80

100

LGG

Days of adulthood

Surv

ival

()

daf-16

LABlowastlowast

(a)

0

50

100

150

LGG LAB

Embr

yos

wor

m

lowast

(b)



15

30

45

300

600

900

1200

1500

LGGLAB

CFU

wor

m

lowastlowastlowast









(a)


(b)

M 2 12 4 11G H H1 H

(c)














5 Conclusions






Acknowledgments


References


























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

1

2

LGG LAB

Relat

ive m

RNA

leve

l

lowastlowast

nhr-49

(a)

0

05

1

15

2

25

LGG LAB

Relat

ive m

RNA

leve

l

lowastlowastlowast

tub-1

(b)

0

1

2

3

LGG LAB

Relat

ive m

RNA

leve

l


(c)



4 Discussion






0 5 10 150

20

40

60

80

100

LGG

Days of adulthood

Surv

ival

()

daf-16

LABlowastlowast

(a)

0

50

100

150

LGG LAB

Embr

yos

wor

m

lowast

(b)



15

30

45

300

600

900

1200

1500

LGGLAB

CFU

wor

m

lowastlowastlowast









(a)


(b)

M 2 12 4 11G H H1 H

(c)














5 Conclusions






Acknowledgments


References


























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0 5 10 150

20

40

60

80

100

LGG

Days of adulthood

Surv

ival

()

daf-16

LABlowastlowast

(a)

0

50

100

150

LGG LAB

Embr

yos

wor

m

lowast

(b)



15

30

45

300

600

900

1200

1500

LGGLAB

CFU

wor

m

lowastlowastlowast









(a)


(b)

M 2 12 4 11G H H1 H

(c)














5 Conclusions






Acknowledgments


References


























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




(a)


(b)

M 2 12 4 11G H H1 H

(c)














5 Conclusions






Acknowledgments


References


























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








5 Conclusions






Acknowledgments


References


























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

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