Metabonomics­based analysis of Brachyspira pilosicoli's response to tiamulin reveals metabolic activity despite significant growth inhibition Article 

Accepted Version 

Creative Commons: Attribution­Noncommercial­No Derivative Works 4.0 

Le Roy, C. I., Passey, J. L., Woodward, M. J., La Ragione, R. M. and Claus, S. P. (2017) Metabonomics­based analysis of Brachyspira pilosicoli's response to tiamulin reveals metabolic activity despite significant growth inhibition. Anaerobe, 45. pp. 71­77. ISSN 1075­9964 doi: https://doi.org/10.1016/j.anaerobe.2017.03.018 Available at http://centaur.reading.ac.uk/70003/ 

It is advisable to refer to the publisher’s version if you intend to cite from the work.  See Guidance on citing  .Published version at: http://www.sciencedirect.com/science/article/pii/S1075996417300677 

To link to this article DOI: http://dx.doi.org/10.1016/j.anaerobe.2017.03.018 

Publisher: Elsevier 

All outputs in CentAUR are protected by Intellectual Property Rights law, including copyright law. Copyright and IPR is retained by the creators or other copyright holders. Terms and conditions for use of this material are defined in the End User Agreement  . 

www.reading.ac.uk/centaur   

CentAUR 

Central Archive at the University of Reading 

Reading’s research outputs online

Metabonomics-basedanalysisofBrachyspirapilosicoli’sresponseto1

tiamulinrevealsmetabolicactivitydespitesignificantgrowth2

inhibition.3

Caroline Ivanne Le Roya,b, Jade Louise Passeyc, Martin John Woodwarda, Roberto4

MarcelloLaRagionec,SandrinePauleClausa#.5

6

a Department of Food and Nutritional Sciences, University of Reading, Whiteknights,7

Reading,UK66AP,UK8

PresentaddressbDepartmentofTwinResearch&GeneticEpidemiology,King’sCollege9

London,LondonSE17EH,UK10

c FacultyofHealth andMedical Sciences, SchoolofVeterinaryMedicine,Universityof11

Surrey,Guilford,SurreyGU27XH,UK12

#Correspondingauthor:s.p.claus@reading.ac.uk,Tel+44118378871713

14

Keywords:Metabonomics,Brachyspirapilosicoli,Tiamulin,Antibiotics.15

16

Abstract17

Pathogenic anaerobes Brachyspira spp. are responsible for an increasing number of18

IntestinalSpirochaetosis(IS)casesinlivestockagainstwhichfewapprovedtreatments19

areavailable.TiamulinisusedtotreatswinedysenterycausedbyBrachyspiraspp.and20

recentlyhasbeenusedtohandleavianintestinalspirochaetosis(AIS).Thetherapeutic21

doseusedinchickensrequiresfurtherevaluationsincecasesofbacterialresistanceto22

tiamulin have been reported. In this study, we evaluated the impact of tiamulin at23

varying concentrations on the metabolism of B. pilosicoli using a 1H-NMR-based24

metabonomics approach allowing the capture of the overall bacterial metabolic25

response to antibiotic treatment. Based on growth curve studies, tiamulin impacted26

bacterialgrowthevenatvery lowconcentration(0.008µg/ml)although itsmetabolic27

activitywasbarelyaffected72hpostexposuretoantibiotictreatment.Onlythehighest28

doseof tiamulin tested(0.250µg/ml)causedamajormetabolicshift.Resultsshowed29

that below this concentration, bacteria couldmaintain a normalmetabolic trajectory30

despitesignificantgrowthinhibitionbytheantibiotic,whichmaycontributetodisease31

reemergencepostantibiotictreatment.Indeed,weconfirmedthatB.pilosicoliremained32

viableevenafterexpositiontothehighestantibioticdose.Thispaperstressestheneed33

to ensure new evaluation of bacterial viability post bacteriostatic exposure such as34

tiamulin to guarantee treatment efficacy and decrease antibiotic resistance35

development.36

37

Highlight38

• B.pilosicolimetabolismwascharacterizedusing1HNMR-basedmetabonomics39

• Tiamulin inhibited B.pilosicoli growth at very low dose (respectively < 0.01640

µg/mLand>0.032µg/mL)41

• B.pilosicoli metabolism is not inhibited for tiamulin concentration superior to42

0.032µg/mL43

• B. pilosicolimetabolism is completely repressed at 0.250 µg/mL, but remain44

viable45

1.Introduction46

Brachyspira pilosicoli is a gram-negative bacterium of the Spirochaetes family. It47

colonizesthelowerpartofthegastrointestinaltrackofalargerangeofhostsincluding48

pigs,birds,humans,monkeys,dogsandhorses[1-4].Onceintheintestinal lumen,the49

bacteriumisattractedviachemotaxistothemucinbarrier[5,6]throughwhichitswims50

mediated by its unique “corkscrew” shape and rotation of its periplasmic flagella [7]51

aidedby thesecretionofmucinedegradingenzymes [5,8].B.pilosicoli attaches to the52

enterocytesinanendonfashionandmayalsoinfectthesecells[9-12].Colonizationby53

B.pilosicoli can lead to the development of intestinal spirochaetosis (IS), the signs of54

which are diarrhea, poor overall condition, dehydration and decreased growth rate.55

Mortalityisoftensignificantwhenthediseaseisleftuntreated[13-15],aconsequence56

thatmakesISaseriouseconomicandwelfareprobleminfarming.57

Tiamuliniseffective in treating IS causedbyBrachyspirahyodystenteriae,B.hampsonii58

andB.pilosicoli in swine [16-18] and in poultry [19-21]. Tiamulin is a bacteriostatic59

derived from a natural pleuromutilin that binds the 50S region of the ribosome to60

inhibit protein synthesis [22]. The antibiotic blocks peptide bond formation by61

interfering with substrate binding [22-25]. Tiamulin treatment in farms generally62

resultsinclearanceofinfectionandassociatedsymptoms.However,reoccurrenceofthe63

disease canbe observedpost treatment indicating incomplete clearance andpossibly64

decreased susceptibility [26,27] in response to treatment. The reason may be an65

inappropriate dosing as there is currently there is a lack of an internationally66

recognized standardized method to determine tiamulin minimum inhibitory67

concentration (MIC) for this bacterium, which has impacts upon selection of an68

appropriate treatment dose. Furthermore, recent studies have indicated that69

Brachyspiramay acquire resistance against tiamulin and, other than blocking protein70

synthesis,nothingisyetknownofthemetabolicresponseofB.pilosicolitotiamulin.We71

argue that evaluating this using a metabonomics approach would allow a better72

understanding of the bacterial response to tiamulin and give insights into improving73

selectionofeffectivedosingregimes.74

Metabonomicsallowsnon-targetedevaluationofthemetabolicmodificationsoccurring75

in abiological system in response to a stress [28],which in this study is exposure to76

tiamulin. By providing a general overview of the metabolic response, this technique77

allows the generation of new hypotheses and to evaluate metabolic in response to78

environmental stress or genetic modification. In this study, we used an NMR-based79

metabonomicsapproachcoupledwithmultivariatestatisticstoevaluatethemetabolic80

dose-response ofB.pilosicoli to tiamulin. Bacteriawere exposed to gradual antibiotic81

doses and media were sampled over 120h in order to evaluate the evolution of its82

metaboliccompositionduringgrowth.Thisallowedtosnapshotthemetabolicresponse83

ofB.pilosicolitotiamulin.84

85

2.MaterialandMethods86

2.1.Bacterialgrowthandantibioticassay87

B.pilosicoliB2904isolatedfromchickenpresentingclinicalsignsofAISintheUK[29]88

were grown from frozen stock on agar solidified BEB plate for four days under89

anaerobic conditions (94% N2and 6% CO2) at 37°C. Colonies were transferred into90

Brachyspiraenrichmentbrothmedia(BEBsupplementedwithheartinfusion)forthree91

daysundersimilarconditions.ThebacterialconcentrationwasthenadjustedinBEBto92

1 x106CFU/ml and transferred into24well plates (2mlperwell) and incubated as93

above for 120h. Every 24 h (with a first time point at 0 h growth), the entire well94

contentwastakenandcentrifugedfor2minat2400gtoseparategrowthmediumfrom95

bacteria. The supernatant was kept at - 80°C for further analysis. This process was96

repeatedateachtimepointinsextuplettodelivertheappropriatepowerforstatistical97

analysis.98

Thesamemethodwasusedforthetiamulinassay.Bacterialcellsweregrownasabove99

and bacterial pellets were resuspended in BEB with antibiotic at six concentrations100

(0.008, 0.016, 0.031, 0.062, 0.125 and 0.250 µg/ml plus control). Bacteriawere then101

inoculated into 24 well plates as previously described and incubated for 120h. For102

metabolicanalysis,eachcondition(tiamulinconcentration)andtimepoint(every24h103

for 120 h) were also repeated in sextuplet. The medium was not changed for the104

durationoftheexperimentsothatantibioticexposurewascontinuous.105

ToevaluatetheviabilityofB.pilosicolipost-antibioticexposure,theaboveexperiment106

wasrepeatedintriplicate.Following120hincubation,B.pilosicoliwasinoculatedonto107

fastidiousanaerobicbloodagarandincubatedat370C,anaerobicallyfor48h.Following108

incubationallplateswerevisuallyinspectedforbacterialgrowth.109

B. pilosicoli growth was evaluated using the same experimental design as the one110

previously described. Bacteria were grown in a 96 well plate (0.2 ml per well) and111

bacterialgrowthwasevaluatedevery2hfor120hatanabsorbanceof600nmusinga112

Fluostar(Info).Waterwasusedasblankandbrothmediawithoutbacteriaasanegative113

control.Eachcondition(tiamulinconcentration)wasrepeatedintriplicateandresults114

arepresentedasanaverageofthelogofthebacterialconcentrationcalculatedfromthe115

absorbance observed at each tiamulin concentration per time point after correction116

withstandardcurve.117

118

2.2.NMRspectroscopy119

ForNMRspectroscopy,0.4mlofmediawasaddedto0.2mlofNMRphosphatebuffer120

(madeinD2Ocontaining10%waterand0.05%sodium3-(tri-methylsilyl)propionate-121

2,2,3,3-d4(TSP)asa1HNMRreference)and0.5mlofthesolutionwastransferredinto122

5mmofouterdiameterNMRtubes.1H-NMRspectrawereacquiredonaBrukerAvance123

DRX700MHzNMRSpectrometer(BrukerBiopsin,Rheinstetten,Germany)operatingat124

700.19 MHz and equipped with a cryogenic probe from the same manufacturer. A125

standard 1-dimensional (1D) pulse sequence [recycle delay (RD)-90°-t1-90°-tm-90°-126

acquire free induction decay (FID)]withwater suppression applied during RD of 2s127

andamixing time (tm)of100msanda90°pulse setat10μswasapplied.Foreach128

spectrum 128 scans were recorded on a total of 32K data points. A broadening line129

function of 0.3 Hz was used to multiply all FIDs. After acquisition, all spectra were130

manually phased and baseline corrected using the software MestReNova® (version131

2.1.8-11880,MestreLab,Spain).Finally,spectrawerecalibratedtothechemicalshiftof132

TSP(δ0.00).Inordertofacilitatemetaboliteidentificationbasedonliterature,aseries133

of2Dspectraonselectedsampleswereacquiredusingcorrelationspectroscopy(COSY)134

NMRspectroscopy.135

136

3.3.Statisticalanalysis137

All spectra were scaled on unit variance and mean centered prior to analysis. To138

evaluatemetabolicvariationbetweensamples,principalcomponentanalysis(PCA)was139

used.Orthogonal projection to latent structure discriminant analysis (O-PLS-DA)was140

alsoperformed,where1H-NMRspectrawereusedasamatrixofindependentvariables141

(X)andtimeorantibioticconcentrationwereusedaspredictionvectors(Y)tocapture142

themetabolicvariations linear to timeandantibioticconcentration.O-PLSDAmodels143

were generated between each tiamulin concentration at every time point144

independently.Aheatmapwasgeneratedusingeachofthismodelstrengthinorderto145

visualizewhentiamulinimpactedbacterialmetabolismincomparisontocontrolandif146

clustersrelatedtodosecouldbeobserved.147

148

3.Results149

3.1.ModificationsofB.pilosicolimetabolismduringgrowth150

The aim of this study was to characterize B. pilosicoli metabolism under optimum151

growthconditions.Figure1presentsB.pilosicoligrowthandmetabolicactivityinbroth152

media.PC1,whichcaptured49%ofthemetabolicvariation,indicatedthatasignificant153

metabolic shiftwas recordedafter96hof incubation.Distinctionsbetween0h, 24h154

and 48 h were observed on the 3rd component, representing only 9% of the total155

variation, which suggests amodest effect on the composition of the culturemedium156

overthefirst48h.Thismetabolictrajectoryindicatesthatbacterialmetabolismmight157

changedependingonthegrowthphase(Figure1AandB).Scoresfromthesametime158

pointwereclusteredtogetherindicatinggoodreproducibilityoftheexperiment.159

In theantibiotic freeculturemedium, thegrowthofB.pilosicoliwasassociatedwitha160

decrease in glucose and an increase in amino acids (phenylalanine, alanine, tyrosine,161

lysine, valine and methionine), fermentation products (lactate, acetate, butyrate and162

isovalerate),aswellasothercompoundsinvolvedintheregulationofcellosmosissuch163

asmyo-inositolandtrimethylamine(TMA)asobservedinthePCAresultspresentedin164

Figures1AandC.165

Glucosewastheonlyreadily identifiablesubstrate thatshowedareductionover time166

(Figure1C).Decreasedconcentrationofothersubstratescouldnotbedetectedanditis167

possible that some may be below the detection limit of the NMR instrument.168

Nevertheless,itisnotunreasonabletoassumethatglucosewastheonlycarbonsource169

used for bacterial anabolism and growth. B.pilosicoli and more especially the strain170

used for this experiment (B2904) is able to use a wide range of carbohydrates and171

hexosesasprimarycarbonsources[10]butitwouldseeminthisstudythatglucosewas172

usedpreferentially.173

174

3.2.TiamulinimpactsB.pilosicoligrowthevenatverylowdoses175

Having characterized the metabolic footprint of B. pilosicoli when grown in optimal176

conditions,wethenchallengeditwithincreasingtiamulindoses.TiamulinimpactedB.177

pilosicoli’s growth at the lowest concentrations tested (0.008 and 0.016 µg/mL) as178

displayed in Figure 2. For these two doses, the bacterial count observed at the179

stationary phasewas one log lower than for the control demonstrating the ability of180

tiamulin to reduce the growth ofB.pilosicoli at low concentrations. Up to 54 hours,181

growth curves of the two lowest concentrations (0.008 and 0.016 µg/mL) were182

identicaltothecontrol(T1andT2onthegraph)buttheystoppedgrowingshortlyafter183

and entered into the stationery phase. No bacterial growth was detected for higher184

tiamulinconcentrations (over0.032µg/mL)confirming itsefficiency tostopbacterial185

proliferation.Interestingly,nogradualtiamulindoseresponseofbacterialgrowthwas186

observed. Indeed,growthratesweresimilar for thetwo lowestconcentrations(0.008187

and 0.016 µg/mL) while higher doses induced a complete inhibition ofB.pilosicoli’s188

growth.189

190

3.3.MetabolicresponseofB.pilosicolitotiamulin191

A clear metabolic response of B. pilosicoli to tiamulin could be observed when the192

antibiotic dose exceeded to 0.032 µg/mL. At lower doses (0.008 and 0.016 µg/mL),193

although B. pilosicoli growth was decreased by 1 log (Figure 2) compared with194

untreated control, the metabolic trajectories remained unaffected (Figure 3A,195

SupplementalFig.1and2).196

When bacteria were exposed to 0.032 µg/mL of tiamulin (Supplemental Fig. 3), a197

disruptionofthemetabolictrajectorywasobservedwhichwasduetomodificationsof198

amino acid concentration. A noticeable increase of tyrosine, methionine, valine,199

phenylalanineandlysineintothemediumfrom0to96hwasobserved.Afterthattime,200

theirconcentrationreduced,indicatingconsumptionoftheseaminoacidsuntiltheend201

of theexperiment.After120hofgrowth, themetaboliccompositionof themediawas202

comparable to control, indicating the full recovery of the metabolism following203

antibioticexposure.204

Higherdosesoftiamulin(0.062and0.125µg/mL)inducedsimilarresponsestothose205

observed at 0.032 µg/mL (Figure 3A and Supplemental Fig. 4. A and B). Amino acid206

metabolismwasaffectedinagreaterextent.207

At themaximumdosetested(0.250µg/mL)themetabolic trajectoryobservedfor the208

mediawasdrasticallymodifiedincomparisontothosedescribedpreviously(Figure3209

andSupplementalFig.5).Themetabolictrajectoryfollowedacircularshapewherethe210

scoresofthesamplescollectedafter120hofbacterialgrowthwereclusteredwiththe211

ones observed at T0, indicating metabolic similarities with the baseline time-point.212

Onceagain,aminoacidswerereleased into themediumaswellasbutyrateandmyo-213

inositol.214

215

3.4.B.pilosicolisurvivespost-antibioticexposure216

ToevaluatethesurvivingpotentialofB.pilosicoliaftertiamulinexposure,samplesfrom217

replicationofthegrowthcurveexperimentwereplatedonagarplateattheendofthe218

antibioticchallenge(120h).ForalltiamulindosesapplieditwaspossibletoobserveB.219

pilosicolicolonyformationonagarplates(SupplementTable1).Theseresultsindicate220

thatB.pilosicoli isabletorecoverfromtiamulinexposure(evenatthehighestdoseof221

0.250µg/mL)oncebackinoptimalgrowthconditions.222

223

4.Discussion224

The results obtained fromB.pilosicoli growth in a control mediumwithout tiamulin225

providenewinsightsaboutitsgeneralmetabolism.Thebacteriawereabletoproduce226

lactateandacetate fromglucose fermentationwithout secretingmethanol, suggesting227

theuseofthebifidumpathwayaccordingtothefollowingequation:glucoseà3acetate228

+ 2 lactate [30]. However, lactate was generally found in very small quantity in229

comparisontoacetate,indicatingitspotentialuseinothermetabolicreactions.Bacteria230

werealsoabletosecretebutyricacidbutnotpropionicacid.Bothoftheseshortchain231

fattyacidswerefoundtobepotentialcarbonsourcesforB.pilosicoli[10].Bacteriaalso232

released a largenumber of amino acids that couldbe caused either by synthesis and233

active secretion of these amino acids, or more likely due to exogenous protein234

degradation.ThisresultisinaccordancewiththegeneticresultspublishedbyMappley235

et al. [10] that indicated a strong proteolytic capacity of the bacterium. This specific236

strainofB.pilosicoliwasalsoshowntobeable touseaminoacidsasprimarycarbon237

source [10].However, as the bacterium favors glucose if available as primary carbon238

source, amino acidsmay here only be used for protein synthesis andmay therefore239

become in excess in the culturemediumwhere they accumulate. Finally, thebacteria240

secretedTMA.GutbacteriagenerallyproduceTMAfromdietaryL-carnitine,betaineor241

choline. Yet, it was not possible to detect a decrease in concentration of these242

compoundsindicatingthatB.pilosicolimightnotusethesemoleculesasprecursorsor243

thatthetechniqueusedwasnotsensitiveenoughtodetectsuchvariations.244

More importantly, thisworkconfirmed tiamulinability to significantly reduceatvery245

low doses (0.008 and 0.016 µg/mL) and inhibit at higher concentrationsB.pilosicoli246

growth. Decreased growth rate at such low antibiotic doses were unexpected, as247

previousevaluationofminimuminhibitoryconcentration(MIC)valuesforthisspecific248

strainwereof0.250µg/mL[31],furthermore,10-15%ofB.pilosicoliisolatespresented249

MICs>4µg/mL[31].Differences in theMICvaluescanbeexplainedbyexperimental250

conditions. Growth curveswere acquiredwhenB.pilosicoliwas grown in BEBmedia251

ratherthanonagarplatesforMICtests.TheB.pilosicolistrainB2904usedinthisstudy252

isknowntohaveanMICof0.250µg/mL[31,32]butshowedclearinhibitionofgrowth253

with concentrations below this value. Thus, our findings confirm the previously254

reported observation that lower tiamulin MIC values are generally found in broth255

comparedtoagarforB.hyodysenteriae[33].256

Despite these encouraging results regarding tiamulin efficiency to inhibit pathogen257

growth, the evaluation of B. pilosicoli metabolic viability in response to antibiotic258

treatment revealed that classic MIC calculations might not be sufficient to assess259

antibiotic efficiency. Indeed, B. pilosicoli metabolic rate reduction did not mirror260

previouslycommentedreducedgrowthrate in response to tiamulin treatment.At the261

two lowest dosesused (0.008 and0.016µg/mL),B.pilosicoli growthwas reducedby262

onelogincomparisontocontrol.However,themetabolictrajectoriesobservedbythe263

mediawere identical. Indicating that tiamulinwas able to impact growth but thatB.264

pilosicolibasicmetabolismremainedunaffected.Higherdoseswereabletoreducebut265

notsilentB.pilosicolimetabolismdespitecompletegrowthinhibition.Furthermore,the266

metabolismofB.pilosicoliappears toslightlyrecover fromall tiamulinconcentrations267

exceptfrom0.250µg/mlafter120hofgrowth.Thismightbeduetotheapparitionof268

resistance,whichisknownasbeingaslowbacterialdevelopmentprocess[34,35].269

In addition, it was demonstrated that B. pilosicoli was able to survive post-tiamulin270

exposure even at the highest antibiotic dosewhen plated on agar (after 120 h drug271

exposure). Indeed B. pilosicoli colonies were identified 48h after the end of the272

antibiotic treatment for all doses tested (0.008-0.250µg/mL).These results illustrate273

thepotentialofB.pilosicolitoenteradormancystatewhenexposedtotiamulinthatis274

reversibleoncethetreatmentperiodisover.275

Besides,resultsdemonstratedaslowresponseofthebacteriatoantibiotictreatmentas276

modification of the metabolic footprint was only observed after more than 48 h of277

growth in presence of tiamulin. From these results, it seems that metabolism was278

stressed during the exponential phase, when bacterial division is compromised.279

Metabolism modification was mainly associated with increased amino acid280

consumption(thatwereproducedinthecontrol).However,astheprovenanceofthese281

aminoacidsremainsunclear,twohypothesescanbeformulated.Firstly,inresponseto282

antibiotic stress bacteria could use amino acids as alternative energy substrates.283

Secondly, B. pilosicoli might not be able to hydrolyse proteins present in the media284

becausenewproteinsynthesis,suchassecretedproteases,isblockedattheribosome.285

Thespecificityof theaminoacidsused indicates the firstoption is themostprobable286

and that catabolism repression could be overridden to secure energy from multiple287

sources. This is an interesting hypothesis that needs confirmation by alternative288

techniquessuchastranscriptomics.289

ThefactthatB.pilosicoliremainviableandmetabolicallyactivewithoutdividingdespite290

the antibiotic treatment and is able to recover after antibiotic exposure could partly291

explaintheISrelapseobservedinfarmsaftertiamulinintervention.Indeed,itseemsto292

arisefromtheseresultsthatbacteriaremainviablebutarenotabletodivideentering293

thereforeadormancystage. It ishighlypossible that suchphenomenonoccurs in the294

intestinal lumen, where bacteria could suffer from inactivation of cell division but295

remain viable. This bacterial state might be associated with a decrease in their296

pathogenicity explaining the disappearance of associated symptoms. Nevertheless,297

bacteriamightremainviablebutata“dormancy”stateintheintestinallumenoranimal298

faecesuntil the environmentbecomes lesshostile (endof antibiotic treatment)when299

they can recover theirpathogenicproperty.However, it is impotent topoint out that300

here B. pilosicoli was recovered on agar plates that represent optimal growth301

conditions. Thismight not be the case in the intestinal lumen that is amore hostile302

environmentwhenthebacteriumalsoneedstocompetewithothercommensalbacteria303

andisconfrontedtothehost’simmunesystem.304

As increasing antibiotic resistancemechanisms developed by pathogenic bacteria are305

arising,manystudiesandreviewshavestressed theconcerns linked to inappropriate306

antibioticusage.Indeed,thisisstronglylinkedtodevelopmentofantibioticresistance,a307

burden forhealth and industry. There is therefore a surge for redefining appropriate308

antibiotic use that would help minimizing the current concern linked to decreased309

antibiotic efficiency. Thus, importance should be given to newmethods development310

aiming at better assessing antibiotic efficiency and to detect potential antibiotic311

resistance factor development. This study indicates thatmetabonomics could be and312

easy and practical way to evaluate bacterial metabolic activity and therefore assess313

antibiotic efficiency to totally inactivate pathogens and therefore avoid infection314

relapse.315

316

5.Conclusion317

ThisworkgaveaclearerunderstandingofB.pilosicolimetabolisminoptimumgrowth318

conditions, including indication regarding favored fermentation pathways and amino319

acids metabolism. It supports the fact that tiamulin can inhibit efficiently bacterial320

growthatlowconcentrations.However,itwassurprisingtoobservethattiamulincould321

impactB.pilosicoligrowthwithoutinfluencingitsbasicmetabolism.Italsorevealsthat322

thebacteriumtrytomaintainmetabolichomeostasisdespiteanobviousstressvisible323

on the growth curve, demonstrating that in response to xenobiotic stress, bacterial324

division is the first mechanism to be suspended. This indicates that tiamulin might325

presentagoodsolutionagainstAISoutbreaks,asitisabletosignificantlyreduceorstop326

bacterial growth, provided that the efficient dose is achieved in the gut of every327

individual.Evenso,thetreatmentmaynotbesufficienttoavoidrelapseofthedisease.328

Suchfindingssuggestthatmeasurementofbacterialactivitymightbeneededinorder329

to assess antibiotic efficiency against potential reoccurrence of the disease. In that330

prospect, metabonomics appeared as a potential solution to evaluate if antibiotic331

treatmentcaninactivatemicrobialmetabolicactivity.332

333

References334

[1] Stanton,T.B.&Hampson,D.J.PhysiologyofRuminalandintestinalspirochaetes.335CABInt.7–45(1997).336

[2] Duhamel,G.E.,Stryker,C.J.,Lu,G.,Wong,V.J.&Tarara,R.P.Colonicspirochetosis337ofcolony-raisedrhesusmacaquesassociatedwithBrachyspiraandHelicobacter.338Anaerobe.9,45–55(2003).339

[3] Hidalgo,A.,Rubio,P.,Osorio,J.&Carvajal,A.PrevalenceofBrachyspirapilosicoli340and ‘Brachyspira canis’ in dogs and their association with diarrhoea. Vet.341Microbiol.146,356–60(2010).342

[4] Trott,D.J.,Stanton,T.B.,Jensen,N.S.,Duhamel,G.E.,Johnson,J.L.,&Hampson,D.343J.Serpulinapilosicoli sp., theagentofporcine intestinal spirochetosis. Int.J.Syst.344Bacteriol.46,206–15(1996).345

[5] Naresh, R. & Hampson, D. J. Attraction of Brachyspira pilosicoli to mucin.346Microbiology.156,191–7(2010).347

[6] Hopwood,D.,Pethick,D.&Hampson,D.Increasingtheviscosityoftheintestinal348

contents stimulates proliferation of enterotoxigenic Escherichia coli and349Brachyspirapilosicoliinweanerpigs.Br.J.Nurition.88,523–532(2007).350

[7] Prapasarakul, N., Lugsomya, K., Disatian, S., Lekdumrongsak, T., Banlunara,W.,351Chetanachan,P.,&Hampson,D. J. Faecal excretionof intestinal spirochaetesby352urbandogs,andtheirpathogenicityinachickmodelofintestinalspirochaetosis.353Res.Vet.Sci.91,e38–43(2011).354

[8] Li,C.,Motaleb,A.,Sal,M.,Goldstein,S.F.&Charon,N.W.Spirocheteperiplasmic355flagellaandmotility.J.Mol.Microbiol.Biotechnol.2,345–54(2000).356

[9] Dworkin,M.Theprokaryotes:Vol.7:Proteobacteria:DeltaandEpsilonSubclasses.357S.Falkow,E.Rosenberg,K.H.Schleifer,&E.Stackebrandt(Eds.).SpringerScience358&BusinessMedia.2006.359

[10] Mappley,L.J.,Black,M.L.,AbuOun,M.,Darby,A.C.,Woodward,M.J.,Parkhill, J.,360Turner,A.K.,Bellgard,M.I.,La,T.,Philips,N.D.,LaRagione,R.M.&Hampson,D.361J. Comparative genomics of Brachyspira pilosicoli strains: genome362rearrangements, reductions and correlation of genetic compliment with363phenotypicdiversity.BMCGenomics.13,454(2012).364

[11] Dassanayake,R.P.Biochemicalpropertiesofmembrane-associatedproteasesof365Brachyspira pilosicoli isolated from humans with intestinal disorders. J. Med.366Microbiol.53,319–323(2004).367

[12] Nakamura, S., Adachi, Y., Goto, T. & Magariyama, Y. Improvement in Motion368Efficiency of the Spirochete Brachyspira pilosicoli. Biophys. J. 90, 3019–3029369(2006).370

[13] Fellström, C. & Gunnarsson, A. Phenotypical characterisation of intestinal371spirochaetesisolatedfrompigs.Res.Vet.Sci.59,1–4(1995).372

[14] Duhamel, G. E., Hunsaker, B. D., Mathiesen, M. R., & Moxley, R. A. Intestinal373spirochaetosisandgiardasisinabeaglepupwithdiarrhoea.Vet.Pathol.33,360-374362(1996)375

[15] Taylor,D.J.,Simmons,J.R.&Laird,H.M.Productionofdirrhoeaanddysenteryin376pigs by feeding pure cultures of spirochaete differing from Treponemu377hyodysenteriae.Vet.106,326–332(1980).378

[16] Johnston,W.T.,Dewey,C.E.,Friendship,R.M.,Smart,N.,McEwen,B. J.,Stalker,379M.,&deLange,C.F.Aninvestigationoftheetiologyofamilddiarrheaobservedin380agroupofgrower/finisherpigs.Can.Vet.J.42,33–37(2001).381

[17] Wilberts, B. L., Arruda, P.H.,Warneke,H. L., Erlandson, K. R., Hammer, J.M.,&382Burrough, E. R. Cessation of clinical disease and spirochete shedding after383tiamulin treatment inpigsexperimentally infectedwith ‘Brachyspirahampsonii’.384

Res.Vet.Sci.97,341–347(2014).385

[18] Burch,D.TiamulinactivityagainstBrachyspirahyodysenteriae.Vet.Rec.163,698386(2008).387

[19] Burch, D. & Klein, U. Treatment ofBrachyspira species with highMICs against388Tiamulininlayers.6thInt.Conf.AvianIntest.SpyrochaetosisInfect.Anim.Humans.38951(2013).390

[20] Stephens, C. P. & Hampson, D. J. Evaluation of tiamulin and lincomycin for the391treatment of broiler breeders experimentally infected with the intestinal392spirochaeteBrachyspirapilosicoli.AvianPathol.31,299–304(2002).393

[21] Burch, D. G. S., Harding, C., Alvarez, R. & Valks,M. Treatment of a field case of394avian intestinal spirochaetosis caused by Brachyspira pilosicoli with tiamulin.395AvianPathol.35,211–6(2006).396

[22] Schlünzen,F.,Pyetan,E.,Fucini,P.,Yonath,A.&Harms,J.M.Inhibitionofpeptide397bond formation by pleuromutilins: the structure of the 50S ribosomal subunit398fromDeinococcusradioduransincomplexwithtiamulin.Mol.Microbiol.54,1287–3991294(2004).400

[23] Poulsen,S.M.,Karlsson,M.,Johansson,L.B.,&Vester,B.Thepleuromutilindrugs401tiamulinandvalnemulinbindtotheRNAatthepeptidyltransferasecentreonthe402ribosome.Mol.Microbiol.41,1091–1099(2001).403

[24] Long, K. S., Hansen, L. H., Jakobsen, L. & Vester, B. Interaction of pleuromutilin404derivatives with the ribosomal peptidyl transferase center. Antimicrob. Agents405Chemother.50,1458–1462(2006).406

[25] Forschungsinstitut, S. TheModeof actionof pleuromutilinderivatives effect on407cell-freepolypeptidesynthesis.Europ.J.Biochem.533,527–533(1974).408

[26] Stephens, C. P. & Hampson, D. J. Evaluation of tiamulin and lincomycin for the409treatment of broiler breeders experimentally infected with the intestinal410spirochaeteBrachyspirapilosicoli.AvianPathol.31,299–304(2002).411

[27] Burch, D. G. S., Harding, C., Alvarez, R. & Valks,M. Treatment of a field case of412avian intestinal spirochaetosis caused by Brachyspira pilosicoli with tiamulin.413AvianPathol.35,211–6(2006).414

[28] Sperling, D., Smola, J. & Cízek, A. Characterisation ofmultiresistantBrachyspira415hyodysenteriaeisolatesfromCzechpigfarms.Vet.Rec.168,215(2011).416

[29] Karlsson, M., Aspan, A., Landén, A., & Franklin, A. Further characterization of417porcine Brachyspira hyodysenteriae isolates with decreased susceptibility to418tiamulin.J.Med.Microbiol.53,281–285(2004).419

[30] Nicholson, J. K., Lindon, J. C. & Holmes, E. ‘Metabonomics’: understanding the420

metabolic responses of living systems to pathophysiological stimuli via421multivariatestatisticalanalysisofbiologicalNMRspectroscopicdata.Xenobiotica.42229,1181–9(1999).423

[31] Mappley,L.J.,Tchórzewska,M.a,Cooley,W.a,Woodward,M.J.&LaRagione,R.424M. Lactobacilli antagonize the growth, motility, and adherence of Brachyspira425pilosicoli: a potential intervention against avian intestinal spirochetosis. Appl.426Environ.Microbiol.77,5402–5411(2011).427

[32] Gottschalk,G.Bacterialmetabolism.SpringerScience&BusinessMedia.(2012).428

[33] Pringle, M., Landén, A., Unnerstad, H. E., Molander, B. & Bengtsson, B.429Antimicrobial susceptibility of porcine Brachyspira hyodysenteriae and430BrachyspirapilosicoliisolatedinSwedenbetween1990and2010.ActaVet.Scand.43154,54(2012).432

[34] Woodward,M.J.,Mappley,L.,LeRoy,C.,Claus,S.P.,Davies,P.,Thompson,G.,&La433Ragione,R.M.DrinkingwaterapplicationofDenagard®Tiamulin forcontrolof434Brachyspirapilosicoliinfectionoflayingpoultry.Res.Vet.Sci.103,87–95(2015).435

[35] Rohde, J., Kessler, M., Baums, C. G. & Amtsberg, G. Comparison of methods for436antimicrobial susceptibility testing and MIC values for pleuromutilin drugs for437Brachyspira hyodysenteriae isolated in Germany. Vet. Microbiol. 102, 25–32438(2004).439

[36] Bock,A.,Turnowsky,F.,Hogenauer,G.,Universitat,M.D.&Germany,W.Tiamulin440resistancemutationsinEscherichiacoli.J.Bacteriol.151,1253–1260(1982).441

[37] Karlsson, M., Gunnarsson, A. & Franklin, A. Susceptibility to pleuromutilins in442Brachyspira(Serpulina)hyodysenteriae.Anim.Heal.Res.Rev.2,59–66(2001).443

444

Figuresandtables445

446

Figure 1: B. pilosicoli consumed glucose and released amino acids and fermentation447

products in its environment. (A) PCA scores plot. (B) B. pilosicoli growth curve in448

Brachyspiraenrichmentbrothmediaunderanaerobicconditionsmadeintriplicate.(C)449

Associatedloadingsofthefirstcomponent.Themetabolictrajectoriesdescribedbythe450

arrowsweredeterminedbythepositionofthecentroidscalculatedateachtimepoint451

usingthecoordinateoftheassociatedscoresontheprincipalcomponents.452

453

Figure 2: Impact of tiamulin onB.pilosicoli growth inBrachyspira enrichment broth454

mediaunderanaerobicconditions.Growthwasmeasuredintriplicateevery2hfor120455

h.456

457

Figure3:(A)MetabolictrajectoriesderivedfromthePCAanalysisperformedusingall458

thesamplepopulation(N=252)ofthestudy(i.e.controlplus6tiamulindilution)onPC1459

and PC3 displaying the centroids for each time points of the control and three460

concentrations of tiamulin. (B) Heatmap representing the O-PLS DAmodel strength461

existing between each tiamulin concentration at each time point, based on R2Y462

(goodness of fit of themodel) and Q2Y (goodness of prediction of themodel) values463

usingthefollowingformula:𝑀𝑜𝑑𝑒𝑙𝑠𝑡𝑟𝑒𝑛𝑔𝑡ℎ = ./0×2/0./032/0

.464

465

Supplementalmaterial466

467

Supplement Fig. 1:Metabolic trajectories ofB.pilosicoli footprints in brothmedia for468

120hatatiamulinconcentrationof:0.008μg/mL(A)and0.016μg/mL(B).Thearrows469

indicate themetabolic trajectory. Themetabolic trajectories described by the arrows470

weredeterminedbythepositionofthecentroidscalculatedateachtimepointusingthe471

coordinateoftheassociatedscoresonthePCs.472

473

Supplement Fig. 2: Metabolic variation related to PCA scores plots presented in474

SupplementFig.1.(A)loadingsoftheprincipalcomponent1ofthemodelpresentedin475

Fig A.1.A. (B) loadings of the principal component 3 of the model presented in476

SupplementFig.1.A.(C)loadingsoftheprincipalcomponent1ofthemodelpresented477

in Supplement Fig. 1.B. (D) loadings of the principal component 4 of the model478

presentedinSupplementFig.1.B.479

480

SupplementFig.3:MetabolictrajectoriesofB.pilosicolifootprintinbrothmediafor120481

hatatiamulinconcentrationof0.032μg.mlonprincipalcomponent1and3.(A)PCA482

scoreplot.(B)AssociatedloadingplotforPC1.Themetabolictrajectoriesdescribedby483

the arrowswere determined by the position of the centroids calculated at each time484

pointusingthecoordinateoftheassociatedscoresonthePCs.485

486

SupplementFig.4:MetabolictrajectoriesofB.pilosicolifootprintinbrothmediafor120487

h at a tiamulin concentration of: 0.062 μg/ml (A) and 0.125 μg/ml (B). The arrows488

indicate themetabolic trajectory. Themetabolic trajectories described by the arrows489

weredeterminedbythepositionofthecentroidscalculatedateachtimepointusingthe490

coordinateoftheassociatedscoresonthePCs.491

492

SupplementFig.5:MetabolictrajectoriesofB.pilosicolifootprintinbrothmediafor120493

hatatiamulinconcentrationof0.250μg/ml.(A)PCAscoreplot.(B)Associatedloading494

plot of PC1. The arrows indicate themetabolic trajectory. Themetabolic trajectories495

describedbythearrowsweredeterminedbythepositionofthecentroidscalculatedat496

eachtimepointusingthecoordinateontheassociatedscoresonthePCs.497

498

SupplementTab.1:GrowthscoresofB.pilosicolionagarplatepost-antibioticexposure.499

DetectionofB.pilosicoli’sgrowth48hafterplatingareindicatedbyasign‘+’andby‘-‘if500

nogrowthwasobserved.501