In vitro and in vivo assessment of phage therapy against Staphylococcus aureus causing bovine mastitis

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In vitro and in vivo assessment of phage therapy against Staphylococcus aureus causing bovine mastitis

C. Ngassam-Tchamba

^a

, J.N. Duprez

^a

, M. Fergestad

^b

, A. De Visscher

^c,1

, T. L ’ Abee-Lund

^b

, S. De Vliegher

^c

, Y. Wasteson

^b

, F. Touzain

^d

, Y. Blanchard

^d

, R. Lavigne

^e

, N. Chanishvili

^f

, D. Cassart

^g

, J. Mainil

^a

, D. Thiry

^a

aBacteriology,DepartmentofInfectiousandParasiticDiseases,FacultyofVeterinaryMedicineandInstituteforFundamentalandAppliedResearchinAnimals andHealth(FARAH),UniversityofLiège,QuartierVallée2,AvenueCureghem6,B-4000Liège,Belgium

bDepartmentofFoodSafetyandInfectionBiology,FacultyofVeterinaryMedicine,NorwegianUniversityofLifeSciences,P.O.Box5003,1432Ås,Norway

cM-team&MastitisandMilkQualityResearchUnit,DepartmentofReproduction,ObstetricsandHerdHealth,FacultyofVeterinaryMedicine,Ghent University,B-9820Merelbeke,Belgium

dViralGeneticsandBio-securityUnit,ANSES,Ploufragan-Plouzanélaboratory,RuedesFusillés,22440Ploufragan,France

eLaboratoryofGeneTechnology,DepartmentofBiosystems,KULeuven,3001Heverlee,Belgium

fR&DDepartment,EliavaInstituteofBacteriophages,3LevanGotuaSt,T’bilisi,Georgia

gDepartmentofMorphologyandPathology,FacultyofVeterinaryMedicineandInstituteforFundamentalandAppliedResearchinAnimalsandHealth (FARAH),UniversityofLiège,QuartierVallée2,AvenueCureghem6,B-4000Liège,Belgium

ARTICLE INFO Articlehistory:

Received11January2020

Receivedinrevisedform1April2020 Accepted4June2020

Availableonline6July2020 Keywords:

Phagetherapy Staphylococcusaureus Bovinemastitis

ABSTRACT

Objective:TheaimofthisstudywastoassesstheefﬁcacyoflyticbacteriophagesonStaphylococcusaureus causingbovinemastitis,byinvitroandinvivoassaysusingGalleriamellonellaandmurinemastitis models.

Methods:BetweenMayandDecember2016,tenS.aureus(ﬁvemethicillin-resistantandﬁvemethicillin- sensitive)isolateswereisolatedfrommilksamplesofcattlewithmastitisinBelgiumandNorway.The isolateswereassessedinvitrofortheirsusceptibilitytofourlyticbacteriophages(Romulus,Remus,ISP andDSM105264)andsubsequentlyinvivoinG.mellonellalarvaeandinmurinemastitismodel.

Results:Romulus,RemusandISPshowedalyticactivityagainsttheS.aureusisolatesinvitro.Alarvae survivalratebelow50%wasobservedat4dayspost-inoculation(DPI)inthegroupsinfectedwitha methicillin-sensitive S.aureus isolateand treated withthese three phagesin vivo. An incomplete recoveryofthemousemastitiswasobservedat48hpost-inoculation(HPI)inthegroupsinfectedand treatedwiththeISPphageinvivo.

Conclusions:Theobservationsaremuchmorepronouncedstatisticallybetweentheinfected-phosphate bufferedsaline(PBS)-treatedandinfected-phage-treatedgroupsinG.mellonellaandthemurinemastitis modeldemonstratinganeffectofthephagesagainstS.aureusassociatedwithbovinemastitis.

org/licenses/by-nc-nd/4.0/).

1.Introduction

Staphylococcusaureusis responsiblefor severalinfections in humans and animals and represents an important hazard in publichealth.Inhumans,itisresponsibleforarangeofillnesses from mild skin disorders to invasive infections and life- threateningin hospitalsettings [1].It can alsobecommunity- acquired,causingskinandsofttissueinfectionswithmoderateto

severesymptomsinhealthyandyoungerpeople[2].Inanimals,S.

aureusinfectionsimpactlivestock,companionanimalsandsome wildanimals[3,4].

Besidestheirvirulence properties,S.aureuscanalsoacquire genesconferringresistancetodifferentantimicrobials.Themost widespreadandfrequentresistantstrainsaremethicillin-resistant S.aureus(MRSA)thatareresistanttoallβ-lactams[5].Inthecaseof bovinespecies,livestock-associatedMRSAcausessubclinicaland clinicalmastitiswhich canlead tohighﬁnancial costs[6,7].To avoid the continuous selection pressure for development and disseminationofantimicrobialresistancebyantimicrobialtreat- mentprocedures,onestrategycouldbetheuseofbacteriophages ratherthantraditionalantimicrobials.

1Currentaddress:FlandersResearchInstituteforAgriculture,FisheriesandFood (ILVO), Technology and Food Science, Agricultural Engineering, Burg. Van Gansberghelaan115Bus1,9820Merelbeke,Belgium.

http://dx.doi.org/10.1016/j.jgar.2020.06.020

2213-7165/©2020TheAuthor(s).PublishedbyElsevierLtdonbehalfofInternationalSocietyforAntimicrobialChemotherapy.ThisisanopenaccessarticleundertheCCBY- NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

ContentslistsavailableatScienceDirect

Journal of Global Antimicrobial Resistance

j o u r n a l h o m ep a g e: w w w . el s e v i e r . c o m / l o c at e / j g a r

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Bacteriophages are viruses able to infect and replicate within bacteria after injection of their genetic material [8]. They are functionally classiﬁed into strictly lytic and temperate phages.

Thesestrictlylyticphagesareofinterestfortherapy[9].Toassess thepotentialofphagetherapy,studiesgenerallyrelyoninvitroor invivoapproachesusingmammalianmodels.Thelackofrealistic conditions ofinvitroassay,theexpensivecostsand theethical requirementsforinvivomodelshaveledtotheuseofalternative modelssuchasG.mellonellalarvae[10].Inthisstudy,theefﬁcacyof lyticbacteriophagesonS.aureusisolatedfromcowswithmastitis inBelgiumandNorwaywasassessedinvitroandinvivowithG.

mellonellaandmurinemastitismodels.

2.Methods

2.1.Bacterialandbacteriophageselection

TenS.aureusisolatesincludingfiveMRSAandfivemethicillin sensitives(MSSA)wereisolatedinBelgiumandNorwaybetween MayandDecember2016fromquartermilksamplesofcowswith clinicalorsubclinicalmastitisandstoredat80Cwith50%(v/v) glycerol until further use (Table 1). Four well-defined lytic bacteriophages were used: Romulus, Remus, ISP [11,12] and DSM105264(phageK)[13].

2.2.Phagepreparation

ThefourphageswereampliﬁedusingtheS.aureusPS47(NCTC 8325)strainforRomulus,RemusandISPphages,andtheS.aureus DSM105272(DSMZ)strainfortheDSM105264phage,aspropaga- tion strains. Phage lysates (100

m

^L)^were ^added ^to⁵^mL ^of ^the

propagation strain culture[optical density (OD600)=0.2–0.3] in Lennoxbroth(LB;AlfaAesar,UK)[complementedwith1mMof calcium chloride (CaCl2) and magnesium sulfate (MgSO4)] and incubatedat37C,withshakingat100revolutions/min(rpm)until thesolutionbecametransparent.Then,this solutionwas added again to500mL of bacterialculture (with OD600=0.2–0.3) and incubated overnight at 37C with shaking at 100rpm. After additionof1/20ofchloroformandincubationat37Cfor10min withshakingat100rpm,thesolutionswereplacedat4Cuntilthe chloroform fraction separated. The solutions were then centri- fugedat4Cand4600rpmfor30minandthesupernatantswere ﬁlteredthroughasuperposedﬁlterof0.45

m

^m^and^0.22

m

^m.^Phage

titrationwasperformedintriplicateonabacterialoverlayofthe propagation strain (OD600 0.2–0.3) spread on an LBagar plate (Applichem, Germany). To obtain high concentrated phage solutions,apolyethyleneglycol(PEG)precipitationwasperformed [14].Aftercentrifugation(4600rpmfor40minat4C),thepellets resultingfrom50mLofphagelysatewerere-suspendedin1mLof phage buffer [10mM Tris-hydrochloride (HCl), 10mM MgSO4, 150mMNaCl,pH7.5]andstoredat4Cuntilfurtheruse.

2.3.Invitroassays

2.3.1.Hostspectrumandefﬁciencyofplating

Tenmastitis-causingS.aureusisolateswerescreenedfortheir susceptibility to the four phages (Romulus, Remus, ISP and DSM105264) on LB agar by the ‘spot-on method’ after serial dilutions[15].Lysiswererecordedasnolysis(–),semi-confluent lysis(SCL)orconfluentlysis(CL).Theefficiencyofplating(EOP) wasthencalculated(Table2)[16].

2.3.2.PhageactivityinLB

A total of 100

m

^L ^of ^the^bacterial ^suspension^at ¹⁰⁶ ^colony-

forming units (CFUs)/mL and 100

m

^L ^of ^the ^phages ^(Romulus,

Remus,ISP,mixofthethreelatterphagesorDSM105264)at10⁹ Table1 AntibioticprofileofthetenS.aureusstrains. IDOriginStrainCefoxitinAmpicillinAmoxicillin +CACiprofloxacinClindamycinColistinErythromycinFlorfenicolGentamicinLizenoidPenicillinSulfamycinSulfa- TrimTrimethoprimTetracyclineResistanceMultiresistance MRSAFlandersAni_GT_111RRSSSSSSRSRSRRR7Yes WalloniaAni_LG_017RRSRRSRSSSRSRRS8Yes Ani_LG_020RSSRRSRSSSRSRRS7Yes Ani_LG_027RRSRRSRSSSRSRRS8Yes Ani_LG_010RRSRRSRSSSRSRRS8Yes MSSAFlandersAni_Gt_110SSSSSSSSSSSSSSS0No Ani_Gt_117SSSSSSSSSSSSSSS0No WalloniaAni_Lg_028SSSSSSSSSSSSSSS0No NorwayAni_Os_001SSSSSSSSSSSSSSS0No Ani_OS_002SSSSSSSSSSSSSSS0No CA,clavulanicacid;ID,identification;MRSA,methicillinresistantS.aureus;MSSA,methicillinsensitiveS.aureus;R,resistant;S,susceptible.

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plaque-formingunits(PFUs)/mLwereaddedto4mLofLBbroth (complementedwith1mMofCaCl2andMgSO4)intriplicates.A positivecontrolcontaining4mLofLBbrothwith100

m

^L^of^bacteria

andnegativecontrols with4mLofLBbroth onlyor LBbroth+ 100

m

^Lôf^phage^wereâlso^tested.^The^broth^wasîncubated^for²⁴^h

at 37C with shaking at 100rpm and lysis was followed by measuringtheOD600ofthesolutionsevery3h.

2.4.Invivoassays

2.4.1.G.mellonellalarvamodel

LarvaeofG.mellonellawereobtainedfrom‘Latourterelledes bois’ (Comines-Warneton, Belgium) and stored at 4C for a maximum of 1 week. The larvae were inoculated into the haemolymphbehindthelastrightand/orleftprolegbyusinga 30-gauge (G) insulin syringe (U-100) from BD Micro-Fine^TM (FranklinLakes,NJ,USA).

2.4.1.1.Preliminaryexperiments. Basedontheinvitroassayresults, onemastitis-causingS.aureusisolate(Ani_OS001)waschosenfora ﬁrst preliminaryexperiment to assess infectivity. Analiquot of 10

m

^Lôf^the^bacterium^wasînoculatedⁱⁿ^three^groups^(A,^B,^C)ôf

tenlarvae,eachat differentbacterialtitres(A=10⁶ CFUs/10

m

^L,

B=10⁴CFUs/10

m

^L,^C⁼¹⁰²^CFUs/10

m

^L).^Another^group^(D)^of^ten

larvaewasinoculatedwith10

m

^Lôf^PBSând^servedâsâ^negative

control.Themortalityrateofthelarvaewasfolloweduntil4days post-inoculation (DPI) to choose the optimal titre of isolate Ani_OS001thatwouldkillatleast75%ofthelarvaeatDPI4.During asecondpreliminaryexperiment,fourothergroups(E,F,G,H)of tenlarvaeeach were inoculated withisolateAni_OS001 atthe optimal titre and with different concentrations of gentamicin (Sigma-Aldrich, Germany) (E=2.5

m

^g/10

m

^L, ^F⁼⁵

m

^g/10

m

^L,

G=7.5

m

^g/10

m

^L, ^H⁼¹⁰

m

^g/10

m

^L) ^to ^choose ^the concentration thatwouldgive100%oflarvasurvivalatDPI4.

2.4.1.2.Phageefﬁcacyassessment. Theﬁrstprincipalexperiment consistedoftheinoculationofisolateAni_OS001attheoptimal

titre(10⁴CFUs/10

m

^L)^with^and^without^phages^(Romulus,^Remus

and/orISP)atthemultiplicityofinfection(MOI)1000,performed intriplicate.Thegentamicin(7.5

m

^g/10

m

^L)^and^the^PBS^were^also

inoculatedaspositiveandnegativecontrols,respectively(Table3).

Bacteriaandtreatmentswereinoculated1hapart.Mortalityrate wasfollowedfor4DPIandsurvivinglarvaeweresacriﬁcedatDPI4.

In parallel, independent groups of ten larvae (inoculated as described)wereblendedatDPI2andDPI4,respectively,toassess theevolution ofthebacterialand phagetitresovertime. A10 phosphate-bufferedsaline(PBS)dilutionoftheblendedsuspen- sion of each larvae group was spotted (10

m

^L) ⁱⁿ ^triplicate ^on

Chapman agar (selective for staphylococci; VWR, Belgium) to assessthebacterialtitres.ThesamedilutionswerespottedonLB agar to estimate the phage titre after addition of 100

m

^L ^of

lysostaphin(tolysethestaphylococci;0.1mg/mL;Sigma–Aldrich, Belgium),incubationat37Cfor1h,centrifugationat46000rpm for45minandﬁltrationat0.2

m

^m.

2.4.2.Murinemastitismodel

The experimental protocol was approved by the Ethical CommitteeoftheUniversityofLiège(approvalnumber14-1719) and the experiments were conducted in a biosafety level 3 laboratoryattheFacultyofVeterinaryMedicineoftheUniversity of Liège. Female BALB/cJRj speciﬁc-pathogen-free (SPF) mice (Janvier, Saint Berthevin Cedex, France) of approximately 30g wereusedat13daysoflactation. Thepupswereremovedand euthanisedjust beforeinoculationofthemammaryglands. The fourth abdominal mammary glands pair (left and right) were inoculated usinga 30-Ginsulin syringe(U-100fromBDMicro- Fine^TM,FranklinLakes,NJ,USA).

2.4.2.1.Preliminaryexperiment. TheAni_OS001strainandtheISP phage(chosenbasedonitsinvitrolyticactivity)wereassessed, respectively,fortheirinfectivityandfortheirsafety.Threegroups (A⁰,B⁰,C⁰)offourmiceeachwereinoculated(50

m

^L)^with¹⁰⁵^CFUs

ofAni_OS001(inoculumtitrebasedonpreviousresults,datanot shown), 10⁸ PFU of ISP phage and PBS (negative control), Table2

HostspectrumtestandEOP.

ID Origin Strain Romulus Remus ISP DSM_105264

Lysis EOP Lysis EOP Lysis EOP Lysis EOP

MRSA Flanders Ani_GT_111 – / – / – / – /

Wallonia Ani_LG_017 – / – / CL 6.80E-01 – /

Ani_LG_020 – / – / CL 9.10E-01 – /

Ani_LG_027 – / – / CL 1.10E+00 – /

Ani_LG_010 – / – / CL 6.80E-01 – /

MSSA Flanders Ani_Gt_110 – / SCL 5.30E-01 CL 1.30E+00 – /

Ani_Gt_117 – / SCL 1.10E-01 CL 3.30E-01 – /

Wallonia Ani_Lg_028 – / SCL 7.40E-01 CL 4.50E-01 – /

Norway Ani_Os_001 CL 3.57E+00 CL 1.90E-01 CL 1.30E+00 – /

Ani_OS_002 – / – / CL 9.10E-01 – /

CL,confluentlysis;EOP,efficiencyofplating;ID,identification;MRSA,methicillinresistantS.aureus;MSSA,methicillinsensitiveS.aureus;SCL,semi-confluentlysis.

Table3

ExperimentaldesignofthephageefﬁcacyassessmentinG.mellonellalarvaeperformedintriplicate.

Groups^a S S_ROM S_REM S_ISP S_MIX S_Gent ROM REM ISP Gent PBS

Bacteria

(10⁴CFUs/10m^L)

Phage(10⁷PFUs/10m^L)

Gentamicin

(7.5m^g/10m^L)

PBS 2

aS,bacteriaandPBS;S_ROM,bacteriaandRomulus;S_REM,bacteriaandRemus;S_ISP,bacteriaandISP;S_MIX,bacteriaandthemixofthephagesRomulus,Remusand ISP;S_Gent,bacteriaandGentamycin;ROM,RomulusandPBS;REM,RemusandPBS;ISP,ISPandPBS;Gent,GentamycinandPBS;PBS,PBS2times;,10mLofinoculum.

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respectively,inoneexperiment.Onehourpost-inoculation(HPI1), allthemicewerefurtherinoculatedwith50

m

^L^of^PBS.^Two^mice

wereeuthanisedineachgroupafter24h(HPI24)and48h(HPI48) post-inoculation,respectively,theirbloodwascollectedandtheir mammary glands were dissected for histopathological analysis thenblendedwithaTissuelyserII(Qiagen,Hilden,Germany)for bacterialcounts.TheS.aureusstrain(Ani_OS001m)resultingfrom thepreliminaryexperimentwaspuriﬁedandusedasaninoculum for the phage efﬁcacy assessment. The mouse presenting an infectionwiththehighestbacterialtitrewasselectedtoisolatethe strain(Ani_OS001m)tobeusedforthemainexperimentinorder tomaximisetheinfectionsuccess.

2.4.2.2. Phage efﬁcacy assessment. The strain Ani_OS001m was used in one experiment after being checked phenotypically (antimicrobial proﬁle) and genetically (whole genome sequencing)(Fig.1).Fivegroupsoffourmicewereusedand1h wasleftbetweentwoinoculationsinthesamemammarygland.

Marboﬂoxacin(antibiotic)andthePBSwereused,respectively,as positiveand negativetreatmentcontrols.The mammaryglands weredissectedandblendedforbacterialandphagecountingatHPI 24andHPI48afterdilutionsinthePBSasdescribed.Thebloodwas alsocollectedforbacterialandphagetitrations.

2.4.2.3.Mammaryglandgrosspathologyandhistopathology. The mammaryglandswereobservedforgrosspathologyusingascore fromI(noinflammation)toV(severeinflammation) [17].Then, small samples were fixed in 4% buffered paraformaldehyde, dehydratedwithethanol, clearedwithxyleneand embeddedin paraffin for histopathological studies. Tissue sections of 4-

m

^m

thicknesswerestainedwithhaematoxylinandeosin(H&E),and examined bylight microscopyat differentmagnifications.Each samplewasevaluatedforthepresenceof necrosis,lymphocytic inflammation, inflammatory cells (polymorphonuclear neutrophils, granulocytes) and bacteria with a score from 0 (absent)to4(severe)[17].

2.5.Statisticalanalysis

Statistical analyses were performed on in vivo data with GraphPadPrism8,(GraphPadSoftware,Inc.,SanDiego,CA,USA).

Kaplan–Meier survival curves and log-rank (Mantel–Cox) tests wereperformedtoanalysetheG.mellonellalarvasurvivalcurves.

Bonferroni'smultiplecomparisonstestwasusedtocomparethe variationsofbacterialandphagetitresinsideG.mellonellalarvae andmurinemammaryglands.

2.6.WholegenomesequencingcomparisonofAni_OS001and Ani_0S001m

StaphylococcusaureusDNAwas extractedfromonecolonyof Ani_OS001and onecolonyofAni_OS001m(recoveredfromthe mousemodelpreliminaryexperiment)usingaprotocolbasedon lysostaphin followed by chloroform/isoamyl alcohol extraction.

Genomiclibrarieswerepreparedaccordingtothemanufacturer's instructionsusingtheNexteraXTDNAlibrarypreparationkitand sequencedbytheNovaSeq6000SequencingSystem(Illumina,San Diego,CA,USA).The rawreadssequenceswereassembled into contigswiththepipelineshovill1.0.4includingtrimmomatic0.38 for the cleaning and annotated using Prokka 1.13.3 [18]. The comparisonwasperformedbyalignmentofAni_OS001mreadson Ani_OS001shovillassemblyusingbwaandVarScanvariantcalling (-varFreqoptionsetto0.6toensuremajorityvariants)[19].

Fig.1.Experimentaldesignofthephageefﬁcacyassessmentinamurinemastitismodel.HPI,hourpost-inoculation;L4/R4,left/rightfourthabdominalmammaryglandpair;

Marbo,marboﬂoxacin.

Fig.2. ActivityofthephagesonAni_OS001inLBbroth.OD600,opticaldensityat 600nm;OS001,Ani_OS001andLBbroth;OS001_ROM,Ani_OS001,RomulusandLB broth;OS001_REM,Ani_OS001,RemusandLBbroth;OS001_ISP,Ani_OS001,ISPand LBbroth;OS001_MIX,Ani_OS001,themixofthephages(Romulus,RemusandISP) andLBbroth;ROM,RomulusandLBbroth;REM,RemusandLBbroth;ISP,ISPandLB broth;MIX,mixofphagesandLBbroth;LB,LBbrothonly.

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3.Results

3.1.Invitroassay

CLandSCLwereobservedonLBagarforfourMRSAsandﬁve MSSAs with the phages Romulus, Remus and/or ISP (Table 2).

Conversely,no lysiswas observed withthephage DSM105264.

High EOP (>1) were observed for two MSSAs (Ani_OS001 and Ani_GT110)and one MRSA (Ani_LG027). TheAni_OS001 isolate thatwaslysedbythethreephageswasthereforechosenfortheG.

mellonellaexperiments.

The samethreephages, aloneor together,hadefﬁcientlytic activitiesonAni_OS001isolateinLBbrothcomparedwithpositive andnegativecontrols(Fig.2).

3.2.Invivoexperiments 3.2.1.G.mellonellamodel

AfterinoculationofthelarvaewiththeAni_OS001isolate,100%

mortalitywas observedin groupA,80% ingroupBand40% in group C at DPI 4 during the ﬁrst preliminary experiment (determinationoftheinfectiousdose).Inthesecondpreliminary experiment(determinationofthetherapeuticdoseofgentamicin) 30%ofsurvivalwasobservedingroupE,70%ingroupFand100%in groupsGandHatDPI4.Basedontheseresults,10⁴CFUs/10

m

^L

(groupB)and7.5

m

^g/10

m

^L^(group^G)^were^chosen^as^the^optimal

bacterialinfectiousdoseandoptimalgentamicintherapeuticdose, respectively,forsubsequentexperiments.

During the principal experiment, a 10% survival rate was observedinthelarvaeinoculatedwithAni_OS001isolateandPBS, whereas90%ofthelarvaeinoculatedwithAni_OS001isolateand gentamicinsurvivedatDPI4.SurvivalratesatDPI4rangedfrom 20%to35%inthegroupsinoculatedwithAni_OS001isolateand thephages(Romulus,Remus,ISPorthemixofthethreephages) (Fig.3).

HighS.aureustitres(510¹¹CFUs/mgand210¹²CFUs/mg) wereobtainedatDPI2andDPI4,respectively,fromtheblended larvaeinoculated with Ani_OS001isolateand PBS. These titres werelowerthanthelarvaeinoculatedwithAni_OS001andphage (s)(Fig.4A).ThephagetitresincreasedbetweenDPI2andDPI4in the larvae inoculated with Ani_OS001 isolate and stayed very stableinthegroupsinoculatedonlywiththephage(s)(Fig.4B).

3.2.2.Murinemastitismodel

3.2.2.1. Preliminary experiment. Mammary gland inﬂammations wereobserved ingroupA⁰ withabacterialtitreupto4.210⁴ CFUs/mgatHPI48,whereas neitherinﬂammationnorinfection

Fig.3.Kaplan–MeiersurvivalcurvesofG.mellonellalarvae.OS001_PBS,Ani_OS001 andPBS; OS001_ROM, Ani_OS001and Romulus;OS001_REM,Ani_OS001 and Remus;OS001_ISP,Ani_OS001andISP;OS001_MIX,Ani_OS001andthemixof Romulus,RemusandISP,OS001_Genta,Ani_OS001andgentamicin;DPI,dayspost- inoculation.

Fig.4.(A)BacterialtitresinblendedG.mellonella larvae.â(B)Phage titresin blended G. mellonella larvaeâ. âOS001_PBS,Ani_OS001 and PBS; OS001_ROM, Ani_OS001 and Romulus; OS001_REM, Ani_OS001 and Remus; OS001_ISP, Ani_OS001andISP;OS001_MIX,Ani_OS001andthemixofthephagesRomulus, RemusandISP;ROM,RomulusandPBS;REM,RemusandPBS;ISP,ISPandPBS;PBS, PBS2times;DPI,dayspost-inoculation;I,inoculumtitre;statisticallysignificant differencebetweenvalues(*P<0.05;**P<0.01(determinedbyBonferronimultiple comparisontest)].

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waspresentinthegroupsB⁰andC⁰(Table4).Nobacterialinfection wasdetectedinthemouseblood.

3.2.2.2. Main experiment. At necropsy, gross pathology scores were established for the 40 mammary glands of the 20 mice (Fig.5).ScorevaluesofIIIandVwithrednessandslimyexudateof theglandwereobservedinthemammaryglandsinoculatedwith Ani_OS001m isolate and PBS, whereas the mammary glands inoculatedwithAni_OS001misolateandtheISPphagehadscore valuesofIIandIIIwithslightlyredglands.Theotherthreegroups ofmicehadscorevaluesbetweenIandIIwithverylittlerednessof theglands.

Histopathologywasperformedonthe40mammaryglandsand anaveragescorewasestablishedforeachgroup(Fig.6).Thegroup

inoculated withAni_OS001m isolateand PBShad severetissue necrosis and high numbers of polymorphonulear neutrophils (PMN),lymphocytes and bacteria.In thegroupinoculatedwith Ani_OS001misolateandISPphage,thelesionsweremoderateand inthethreeothersgroups,theywereveryweakwithnobacteria andnotissuenecrosis.Moderateamountsofmacrophageswere alsopresentinallmice.

Ahighbacterialtitrewasobservedintheblendedmammary glandsofthegroupinoculatedwithAni_OS001misolateandPBSat HPI24andHPI48,whereasaprogressivedecreaseofthebacterial titres was observed between HPI 24 and HPI 48 in the group inoculated with Ani_OS001m isolate and ISP phage. A drastic decreasewasobservedinthegroupinoculatedwithAni_OS001m isolate and treated with marboﬂoxacin at HPI24 leading tothe absenceofbacterialdetectionatHPI48.Nobacterialgrowthwas observedintheothertwogroups(Fig.7A,Table5).Nobacteriaor phagesweredetectedinthemouseblood.

A progressive decrease of the phage titres was observed betweenHPI 24and HPI48in thetwogroups inoculatedwith phage.Thedecreaseofthephagetitrewasmorerapidanddrastic inthegroupinoculatedwithAni_OS001misolateandISPphage leading totheabsenceof phagedetection atHPI48 thanin the groupinoculated withISP phageand PBS (Fig.7B, Table5). No phagewasdetectedintheotherthreegroups.

Table4

Resultsofthepreliminaryexperiment(micemodel).^a

Groups Meanbacterialtitresoftheblendedmammaryglands(CFUs/mg) A(Ani_OS001

+PBS)

B(ISP +PBS)

C(PBS +PBS)

HPI24 2.80E+04 0.00E+00 0.00E+00

HPI48 4.20E+04 0.00E+00 0.00E+00

aHPI,hourpost-inoculation.

Fig.5.Grosspathologyofthemammaryglands.OS001_ISP,Ani_OS001mandtheISPphage;OS001_Marbo,Ani_OS001mandmarbofloxacin;OS001_PBS,Ani_OS001mand PBS;ISP_PBS,ISPphageandPBS;PBS_PBS,PBStwice,HPI,hourpost-inoculation.Clinicalscore:I,noinflammation;II,mildinflammation;III,moderateinflammation;IV, highlymoderateinflammation;V,severeinflammation.

Fig.6.Mammaryglandhistopathology.OS001_ISP,Ani_OS001mandISPphage;OS001_Marbo,Ani_OS001mandmarbofloxacin;OS001_PBS,Ani_OS001mandPBS;ISP_PBS, ISPphageandPBS;PBS_PBS,PBStwice,A,tissuenecrosis(degeneration);B,polymorphonulearneutrophils(PMNs)andlymphocytes;C,macrophages;D,bacteria;Score:0, absent,1.minimal,2,mild,3,moderate,4,severe.10,ten-foldmagnification;100,100-foldmagnification.

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3.2.3.Statisticalanalysis

A signiﬁcant difference was observed between the survivalcurvesof theinfected-gentamicin-treated,onthe one hand,andtheinfected-phage-treatedandinfected-PBS-treated G. mellonella larvae groups, on the otherhand, as determined bylog-rank(MantelCox)test(P<0.0001).Asigniﬁcantdifference wasalsodetectedbetweenthebacterialtitresoftheinfected-PBS- treated and the infected-phage-treated larva groups as determined by the Bonferroni multiple comparison test (P<0.05).

This test also showed a higher signiﬁcant difference between the bacterial titres of the infected-PBS-treated and the non- infected larva groups (P<0.01). In the murine mastitis model, the Bonferroni multiple comparison test showed a signiﬁcant difference between the bacterial titres of the infected-PBS-treated and the infected-phage-treated mammary glands (P<0.05). In both G. mellonella and mouse models, a

signiﬁcantdifference(P<0.05)wasobservedbetweenthephage titresoftheinfected-phage-treatedandthe‘non-infected’-phage- treatedgroups.

3.2.4.GenomecomparisonofAni_OS001andAni_OS001m

ThedraftgenomeofAni_OS001andAni_OS001mareavailable from GenBank, accession numbers VXKU01000000 and VXKV01000000,respectively.TheVarScananalysisrevealedonly two single polymorphism nucleotide (SNPs) between the two strains. Oneisassociatedwitha regionwithoutanyannotation accordingtoProkka(position 25ofcontigOS001-00027C→T).

The second SNP (G→T) occurredin position60 157 of contig OS001-00001.TheProkkaannotationrevealedSdrE,agenecoding for amember of themicrobialsurface componentsrecognizing adhesive matrix family molecule (MSCRAMM). This mutation resultedinaG899Vmutationattheproteinlevel.

Fig.7.(A)Meanofthebacterialtitresinthemammarygland.â(B)Meanofthephagetitresinthemammaryglandâ.âOS001_ISP,Ani_OS001mandtheISPphage;

OS001_Marbo,Ani_OS001mandmarboﬂoxacin;OS001_PBS,Ani_OS001mandPBS;ISP_PBS,ISPphageandPBS;PBS,PBStwice.Eachbaronthegraphsrepresentsthemeanof thebacterialorphagetitresinfourmammaryglands(twomicepergroup).I,Inoculum;statisticallysigniﬁcantdifferencebetweenvalues[*P<0.05(determinedby Bonferronimultiplecomparisontest)].Errorbarsrepresentstandarderror.

Table5

Resultsofthephageefﬁcacyassessmentinmurinemastitismodel.^a

Groups OS001_ISP OS001_Marbo OS001_PBS ISP_PBS PBS_PBS

HPIn HPI24 HPI48 HPI24 HPI48 HPI24 HPI48 HPI24 HPI48 HPI24 HPI48

Mice M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11 M12 M13 M14 M15 M16 M17 M18 M19 M20

Bacterialtitres (CFU/mg)

L4 2.10E +03

1.43E +03

– – – 2,63E

+03

– – 4.30E

+06 6.25E +06

6.89E +08

3.71E +07

– – – – – – – –

R4 5.66E +05

3.28E +05

– 2.03E +04

– – – – 2.42E

+07 8.35E +07

3.24E +08

7.09E +07

– – – – – – – –

Phagetitres (PFU/mg)

L4 3.39E +02

1.17E +03

– – – – – – – – – – – 5.83E

+05 1.31E +02

1.39E +02

– – – –

R4 – 2.31E

+02

– – – – – – – – – – – – 3.39E

+03 1.93E +02

– – – –

aOS001_ISP,Ani_OS001mandtheISPphage;OS001_Marbo,Ani_OS001mandmarboﬂoxacin;OS001_PBS,Ani_OS001mandPBS;ISP_PBS,ISPphageandPBS;PBS_PBS,PBS twice;M,mouse;L4/R4=Leftandrightfourthabdominalmammaryglandpair;–,nobacterialorphagetitre;HPI,hourpost-inoculation.

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4.Discussionandconclusions

This study highlights the therapeutic potential of bacterio- phagesagainstS.aureusassociatedwithbovinemastitisbythein vitroassessmentofthephageactivitiesandtheinvivoassessment ofthephageefﬁcacyinG.mellonellaandmurinemastitismodels.

DifferentlyticactivitieswereobservedontenS.aureusisolates:

thephageRomuluswasactiveagainstonlyoneisolate,thephage RemusagainstfourisolatesandthephageISPagainstnineisolates.

Inpreviousstudies,theRomulusandRemusphageswereactive againstmoreisolatestested(70%)[11],whereasthelyticactivityof theISPphagewasobservedagainstasimilarpercentage(87%)of thetestedS.aureusisolates,includingMRSA[12,20].Conversely, noneofthetenisolatestestedwassusceptibletotheDSM105264 phage,althoughitwasdemonstratedtobeactiveagainstca.halfof thepreviouslytestedisolates(humanstrains)[13].Thedifference ofsusceptibilityobservedcouldbeexplainedbythedifferenceof theS.aureusorigins(bovinevs.human).

Thelytic activities of the three differentphages against the Ani_OS001S.aureusstraininbrothmediumconfirmtheresults observedonagarmedium.However,ahigherefficiencyhasbeen observedforthephagesISPandREMcomparedwiththoseofthe phagemix, itself moreefficient than thephageROM alone. As showed in the literature, the mix of phages usually leads to synergisticeffectinvitrointermsofabroadhostrange[21,22].

This study showed a probable lower effect of the phages mix compared with those of individual phages when using them againstthesamebacteria.

IntheG.mellonellamodel,usingonlytheAni_OS001strain,no significantdifferencewas observedbetweenthesurvivalrateof theinfected-phage-treatedandinfected-PBS-treatedlarvaegroup, whereas this difference was significant in comparison to the infected-gentamicin-treated group. The higher bacterial titre observedatDPI4intheblendedlarvae(groupOS001_ISP)with theISPphagecomparedwiththoseobservedwithRomulusand Remus,althoughitshigherlyticactivityinvitrocanbeexplained byitsinitialadsorptionandbiofilm-degradationthatareslower thanthoseofRomulusandRemusphages[11].Anotherexplana- tioncouldbetheoccurrenceofamutationinthebacterialgenome duringthephagetreatmentmakingapartoftheAni_OS001isolate populationresistanttotheISPphage[23,24].NoSPFlarvaeexist, and naturalcarriage ofS. aureuscouldaffect theresultsof the blendedlarvaebacterialtitres.Theveryweakimpactofnatural carriage on our results has been confirmed by the Bonferroni multiplecomparisontestshowingahighersignificantdifference (P<0.01)betweenthebacterialtitresoflarvagroupswithnatural carriage(REM,ISP,PBS)comparedwiththoseoftheinfected-PBS- treated larva groups. Moreover, a decrease in phage titre was observedfortheblendedlarvaeatDPI2andDPI4.Thiscouldbe explained by the single dose of phages inoculated and their possible inactivation as demonstrated in recent studies where bacteriophageshavebeenshown tobeefficientinG. mellonella larvaeaftermultipleinoculationsinthepresenceofthebacteria anddependingontheMOI[25,26].Thedecreaseofthephagetitre observed in this study is slower in the infected-phage-treated groups compared with the ‘non-infected’-phage-treated groups meaningthatasimultaneousreplicationofthephageandbacteria occurredinsidethe larvae.Thisstudyreports forthefirst time resultsonlyticbacteriophagesassessmentonS.aureusintheG.

mellonella model, whereas many other studies were already performed,but onotherpathogenbacteria[10,25,27].However, theresultsobservedinthismodelneedtobeconﬁrmedinother infectious modelsin mammals, suchas in themurine mastitis model.

Anincreaseofthevirulencegeneexpressionafterapreviousin vivo passage is often observed as reviewed by Angelichio and

Camilli[28].Inthemurinemastitismodel,Ani_OS001mshoweda moreefﬁcientinfectionthanAni_OS001.TheWGSofbothstrains revealed a mutation in the SdrE gene of Ani_OS001m. SdrE is presented as a key factor for S. aureus being invasive and has already been described in an S. aureus strain showing higher virulenceinmastitiscases[29].

A reductionof the inﬂammationinmicemammaryglands was obtained in the infected-phage-treated group compared withthoseobservedintheinfected-PBS-treatedgroupwiththe strainAni_OS001m.Theseobservationshavealsobeendescribed inrecentstudiesonthetherapeuticassessmentofphageagainst S.aureus inamurinemastitismodel[17,30].Anincreaseofthe bacterialtitre(from710⁴to210⁸CFUs/mg) observedinthe infected-PBS-treatedgroupand a decreaseofthebacterialtitre (from710⁴to510³CFUs/mg)observedintheinfected-phage- treated grouphighlighttheefﬁcacyof thephageagainst theS.

aureusstraininthemicemammaryglands,asalreadyshownwith otherphagesinpreviousstudies[30,31].Nevertheless,thisphage efﬁcacy was less important than the one obtained with antimicrobials in themurine model[17].Moreover, adecrease of the phage titre was observed in the ‘non-infected’-phage- treated group (from 1.310⁸ to 910² PFUs/mg) and more drasticallyintheinfected-phage-treatedgroup(from1.310⁸to 0 PFUs/mg),suggestingan eliminationor a degradationof the phage in the mouse mammary gland. Studies show that the stimulation of the immune response (macrophages, serum complement system) is more important when bacteria are inoculated with phages in the mice mammary glands, which canexplainthedrasticdecreaseofthephagetitreintheinfected- phage-treatedgroups[17,23,32].Theintra-mammaryroutecan be favourable to thephage inactivation by some proteinsand lipidspresentsinthemilkleadingtoadecreaseofthephagetitre in themammary gland[23,33]. Conversely,nodecreaseof the phagetitrewasobservedinthemicemammaryglandsshowing moderateinﬂammationasdescribedbyBreyneet al.[17],who assessed a phage cocktail effect onS. aureus at MOI 10⁵. This contradictionwithourstudycouldbeexplainedbytheuseofa singlephageinoculationatalowerMOI(10³),whichwouldhave beenquicklyeliminated.

Thisstudyhighlightsthelyticactivityofthreebacteriophages (Romulus,RemusandISP)againstS.aureusassociatedwithbovine mastitisinvitroandtheirefﬁcacyinvivomaterialisedbyalarvae survivalratebelow50%atDPI4inthegroupsinfectedandtreated (withthethreephages)andanincompleterecoveryofthemouse mastitisatHPI48inthegroupsinfectedandtreated(withtheISP phage).Moreover,signiﬁcantdifferenceswereobservedbetween infected-PBS-treated and infected-phage-treated groups in G.

mellonella and murine mastitis model demonstrating an effect of the three phages against S. aureus associated with bovine mastitis.Theweakefﬁcacyofphageobservedinvivo(lowlarvae survival rate, incomplete mammary gland recovery) could be relatedtotheuniquebacteriophagedoseused,theratiophage/

bacteria(MOI),theroleoftheimmune systemandtherouteof inoculation as described in previous studies [32,34]. Further studiesarerequiredtoassesstheseparametersinordertoconﬁrm the phage therapy efﬁcacy against S. aureus causing bovine mastitis.

Funding None.

Competinginterests None.