Clinical utilization of bacteriophages: a new perspective to combat the antimicrobial resistance in Brazil

w w w . e l s e v i e r . c o m / l o c a t e / b j i d

The

Brazilian

Journal

of

INFECTIOUS

DISEASES

Review

article

Clinical

utilization

of

bacteriophages:

a

new

perspective

to

combat

the

antimicrobial

resistance

in

Brazil

Willames

M.B.S.

Martins

_,

_Mark

_A.

_Toleman

_,

_Ana

_C.

_Gales

a_{UniversidadeFederaldeSãoPaulo,EscolaPaulistadeMedicina,DepartamentodeMedicinaInterna,UNIFESP,SãoPaulo,SP,Brazil} b_{CardiffUniversity,DepartmentofInfectionandImmunity,Cardiff,UnitedKingdom}

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t

i

c

l

e

i

n

f

o

Articlehistory:

Received17January2020 Accepted11April2020 Availableonline15May2020

Keywords: Virus Phagetherapy Newapproaches Infections MDR

a

b

s

t

r

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t

Duetotheemergenceofmulti-drugresistantbacteria,andtheevidentlimitationin thera-peuticoptions,alternativestocombatbacterialinfectionshavebeensought.Oneoftheseis phagetherapy,whichistheuseofbacterialvirusestokillpathogenicbacteriaresponsible fortheinfection.Thesevirusescalledbacteriophagesareveryabundantorganismsinthe worldandareharmlesstohumans.Thereareseveraladvantagesinusingphagetherapy, especiallyagainstmulti-drugresistantpathogens,whichtendtobedominatedby individ-ualstrains.Theadvantagesincludefewercollateraleffectssuchaslowerdisturbanceofgut microbiotaandlessantimicrobialsconsumption,whichitselfleadstoreducingantibiotic resistancerates.Unfortunately,fewclinicalstudieshavebeeninitiatedinBrazilandthisarea islittleexploredinourcountry.Thismanuscriptdescribesclinicalevidenceofsuccessful phageutilizationonpathogensconsideredathreatinBrazil,highlightingthebenefitsofa possiblephageutilizationasanimportanttooltocombatantimicrobialresistanceinour country.

©2020SociedadeBrasileiradeInfectologia.PublishedbyElsevierEspa ˜na,S.L.U.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/ licenses/by-nc-nd/4.0/).

AntimicrobialresistanceinBrazilhasbeenafrequenttopic ofdiscussionoverthelastfewyears.Currently,thecountry isexperiencingascenariowhereresistancetoantimicrobial agentsisemergingsoquickly,thatmanypeople are claim-ingthattheantibioticeraiscomingtoanend.Althoughnew combinationsof␤-lactamsand␤-lactamsinhibitorsarebeing introducedintoclinicaluse,multi-drugandpan-drug resis-tantisolateshavebeencharacterizedaroundtheworldcalling intoquestionhowmuchoneshouldbeheldhostagetothe

∗ _{Correspondingauthor.}

E-mailaddress:willamesbrasileiro@hotmail.com(W.M.Martins).

soleuseofantimicrobialagentsintreatinginfections.Since there are numerous review manuscripts addressing phage therapy, the purposeofthis review istoprovide ageneral overviewaboutphageutilization,highlightingtheBrazilian scenariowithincludesthemainpathogensorlineageswhich need concentrated efforts and the Brazilian investigations conducted on this topic. In this review, supportive papers willbeindicatedtoimprovetheunderstandingaboutphage utilization.

https://doi.org/10.1016/j.bjid.2020.04.010

1413-8670/©2020SociedadeBrasileiradeInfectologia.PublishedbyElsevierEspa ˜na,S.L.U.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

240

Antimicrobial

resistance

of

Gram-negative

bacilli

as

a

public

health

threat

Antimicrobialresistancehasbeenconsideredathreatto pub-lichealthin differentparts ofthe world,including middle and low-income countries such as Brazil.1 _{The increasing}

emergence of multi-drug resistant (MDR) bacteria leads to poorclinicaloutcomesfornosocomialpatientsandincreases thecostsassociatedwithhospitalization.2 _{Accordingtothe}

BrazilianHealthRegulatoryAgency(ANVISA)anincreasein carbapenemresistanceratesamongGram-negativebacillihas beenobservedinthelastfewyears,especiallyinpathogens likeAcinetobacterspp.,Klebsiellapneumoniae,andPseudomonas aeruginosa recovered from blood stream infections (BSI) in 2016(85%,46.8%,and42.9%,respectively).3_{Althoughnational}

reportsonlyfocusoncephalosporins,carbapenems,andmore recentlypolymyxin,studieshavedemonstratedthepresence ofbacterial isolateswith multi-drugresistance or extreme drugresistance(XDR)indifferentBrazilianregions.4–8

Some antibiotic resistant bacterial clones are endemic in Brazil, leading to high resistance levels at Brazilian hospitals.Amongthem,KPC-2-producing Klebsiella pneumo-niae(CC258,ST258andST11),SPM-1-producingPseudomonas aeruginosa (ST277), and carbapenem-hydrolyzing class D carbapenemase-producinginAcinetobacterbaumannnii(ST79) havegained clinicalimportanceinrecentdecades.9 _Forthe

last 5–6years,especially duethe emergence ofKPC-2 pro-ducing K. pneumoniae, polymyxin has been largely used to treatinfectionscausedbytheseBrazilianendemicclones.On theotherhand,emergenceofpolymyxinresistance,mainly among K. pneumoniae and A. baumannii isolates, has been detected,limitingevenmorethetherapeuticchoicesfor treat-ingsuchinfections.10,11

Forthisreason,researchersfrommanynationsworldwide are trying to combatinfections caused byMDR pathogens byusingalternativemethodologies.Phagetherapyisoneof thesealternatives totraditionalantibiotic therapy and has beenusedwithgreateffectivenessinsomecountries.12

Bacteriophages:

viruses

responsible

for

bacterial

biological

control

Bacteriophages,alsocalledphages,areviruseswiththeability toinfectandkillbacteriaandarethemostabundant organ-ismson Earth.13 _{Theyare foundindifferentenvironments,}

andfrequentlydetectedinwater,sewage,soil,food,clinical samples,andothers.14 _{Phagesusuallyinfectandkillonlya}

singlebacterialstrainorlineagethusnotabletoinfectalltypes ofbacteria.Thisspecificnatureisverypositiveinthe clini-calsetting,sinceonlypathogenicbacteriawouldbetargeted, infectedandkilled,preservingcommensalharmlessand per-hapsbeneficialbacteria.13,14_{Thebacteriophageinfectioncycle}

canbecomplex.15_{Afterinfectingthebacteria,thephagescan}

showalyticorlysogeniccycle.Briefly,inthelyticcycle,the mostimportantcycleforthephagetherapy,thevirusinfects thebacteriacell,multipliesitselfusingthecellmachinery,and afteraperiodoftime(typically10–20min) killsthebacteria byalytic phenomenon.Afterlysis,the countlessnewviral

particlesareabletoinfectnewbacterialcells.13,16_Incontrast,

in the lysogenic cycle, afterinfecting the cell viral genetic materialisincorporatedonthebacteriagenome,stayingthere untilexogenousstimuliactivateitslyticcycle.13,16_Itis

impor-tant tonotice thatforphagetherapy,lysogenic phagesare notusefulsincethenecessarytypeofstimulusisoftentimes unknown,orrequiresextremeconditionssuchaspH,UVlight, orlowtemperatures.17,18

When bacteriophage was first discovered, their clinical potential was quicklyappreciated. However, initial studies werefraughtwithproblemsandoftennotwell-designed yield-ing unsatisfactory results.19 _{This fact combined with the}

discoveryofantibioticsledtoadeclininginterestintheuse ofbacteriophageasausefultherapyinWesternmedicine.It isimportanttonoticethatalthoughthephagetherapywas notcogitatedduringsomeyearsinseveralpartsoftheworld, itwaslargelyusedincountriesoftheformerSovietUnion, as Georgia and Poland.20 _{However,since the expansion of}

antibioticresistanceinGramnegativesinthe90s,19_interestin

phagetherapyhasincreasedsubstantiallywithmanypapers beingpublished.Thesenewstudieswerewellconducted,and satisfactoryresultsstartedtobeobtained,stimulatingmore andmoreinterestinclinicalbacteriophageutilization.13,14_In

thelastyears,distinctphageshavebeencharacterizedwith greatpotentialtocombatseveralinfectionscausedbyMDR pathogens.

Current

clinical

studies

supporting

the

phages

utilization

As discussed before,phagetherapy isbased on the useof lyticphagestokillinfectiousbacteriawhilstleavinghuman cells unaffected andwith aminorimpacton human com-mensalbacteria.12_{Currently,phagesare largelyusedinthe}

foodindustry,beingconsideredbyFDAasGenerallyRegarded as Safe (GRAS).21 _{One of the biggest achievements in the}

phage field wasthe approvalof thefirst productbased on phage(ListShieldTM_{)in2006,being registeredforuseasan}

organicfoodadditiveinEurope,AustraliaandNewZealand.21

Although phage use is frequent by food companies, this reviewwillemphasizetheutilizationofthesemicroorganisms inclinical medicine.Basedonthat, manyclinicaltrials are inprogress(Fig.1)stimulated bythe goodresultsobtained in other fields. Here,we will summarize some clinical tri-alsagainstGram-negativepathogensconsideredathreatin Brazil. Itis importantto notice that this reviewwill focus onaselectionoftrialsandclinicalreportsconsideredbythe authorsasrelevanttosupportafutureclinicalutilizationof phagesinBrazil.Tobetterunderstandingthestudiesinvolving humanandanimalsrelatedtophageutilizationinemergent pathogens,readMulanietal.22

Acinetobacterbaumannii

PhagesagainstMDRA.baumanniiisolateshavemostlybeen describedinthelastcoupleofyears.Turneretal.23_performed

agenomicanalysisof37phagespreviouslydepositedin Gen-BankandrecoveredfromA.baumanniiisolates.Sofar,none of the phages described and characterizedwere recovered

Fig.1–Worlddistributionofclinicaltrialsinhumansbeingconductedornewlyconcluded.Itispossibletonotethatjust fewcountrieshaveinvestedinclinicaltrialssupportingphageutilizationinhumans.Tohavemoreinformationaboutthe clinicaltrials,seethewebsitehttps://clinicaltrials.gov/.

fromSouthAmericansamples.23_{Despite,alargenumberof}

phages that have been fully characterized, only two clini-caltrials in humans have been reported until now. In the firstone,a68-year olddiabeticpatientwasdiagnosedwith a chronic pancreaticpseudocyst aggravated byan MDR A. baumanniiinfectionduringhishospitalization.Dueto limita-tionoftherapeuticoptions(susceptiblejusttominocycline), a phagecocktail was introduced together with antibiotics. Phagetherapywasmaintainedformorethanfourmonths, withacomplete recoveryofthepatient’s condition.During phage therapy, the bacterial isolate recovered causing the infection developedresistancetosomephagesusedinthe cocktail,butinreturnshowedincreasingantibiotics suscep-tibility.Thephageresistancewascircumventedaddingnew phagestothecocktailwithactivityagainstMDRA.baumannii.

Nocollateraleffectsduetophageutilizationwereobservedin thepatient.24

ThesecondclinicaltestinvolvedA.baumanniiisolates,in a 77-year old male patient, admitted to a hospital inSan Diego, USA with a brain injury. The patient was submit-tedtocraniectomy, followed byinfection afterthesurgical procedure. Intraoperative material cultured giving growth to an MDR A. baumannii isolate only susceptible to col-istin (some cultures showed A. baumannii ColS and others ColRisolates). An attempt to curethe infection withdrug combinationsincludingnon-usualantimicrobialagentsused totreatA. baumanniiinfections,suchasrifampin+colistin, azithromycin+colistin, and chloramphenicol+colistin was unsuccessful,leavingphagetherapyasoneofthefew alterna-tivesfortreatingthepatient.Fivephagesweredetectedwith activityagainstthe isolaterecoveredfromthis patient.The phagewasadministeredintravenouslyafter12daysof hos-pitalization.Itisnotclearhowmanydifferentphageswere

usedinthecocktail,butanimprovementinhishealth con-ditionwasnoted.Twelvedaysthereafterthepatientwasonly treatedwithphagesbecausecolistinwassuspendedonday13 duetokidneyinjury.Thepatientreceivedeightdaysofphage therapy.However,beforeadministrationofthesecondphage cocktail,hisfamilyoptedtowithdrawtreatmentandphage therapywasdiscontinuedonday19,andthepatientdiedon day20.Nevertheless,itisinterestingtonotethatwhilethe phageswerebeingadministeredthepatient’shealthcondition wasmaintainedwithsomeclinicalimprovement.25

Pseudomonasaeruginosa

AlthoughP.aeruginosainfectionsweretreatedwith bacterio-phagesmorethan 50yearsago,26_{this therapyhasrecently}

reemergedasanattractivealternativetreatment.27_P.

aerugi-nosaisanopportunistpathogen,historicallyassociatedwith severe nosocomial infections and with a strong link with cysticfibrosisandburninfections.ItoftendisplaysanMDR phenotype andnumerous studies involvingphagesagainst

P.aeruginosahavebeenreported.27,28 _{Besidesthecapacityto}

accumulatedistinctresistancemechanisms,this microorgan-ismisalsoabletoproduceahighlevelofbiofilm,especially oninanimatesurfaces.Therefore,phagesactiveagainstthe biofilm and the planktoniccultures are also importantfor treatingP.aeruginosainfections.28

Tenyearsago,aclinicaltrialinvolvingbacteriophage ther-apyforP.aeruginosainfectionswaspublisheddemonstrating that bacteriophage therapy againstP. aeruginosa infections was safeandclinicallyeffective. Twenty-fourpatientswith external otitiscaused by P. aeruginosastrains were divided intotwo(n=12each)treatmentgroups.Group1wastreated withacocktailcontainingsixphageswithactivityagainstP.

242

aeruginosa(105_{PFUofeachphagedilutedinasolution)while}

thegroup2wastreatedwithplacebo.Tobepartofthestudy, thepatients’bacteriaweresubmittedtomicrobiologicaltests tocheckiftheyweresusceptibletoatleasttoonephage con-tained onthe cocktail. Thephagetherapy waseffective in combatingtheinfectionandimprovingtheearhealthofthe patients.Inaddition,itwaspossibletodetecthowlongthe phagesremainedintheregionwherethesolutionhadbeen applied.Accordingtotheauthors,whenthetargetbacterial isreduced or completely eliminated,the phagereplication ceasesleadingtophageelimination.Nosignaloftoxicityor collateraleffectswerereportedinthisstudy,highlightingthe safetyofthisphagetherapy.29

InEurope, aplatform named PhageBurnrepresents one of the biggest research studies in the phage field against

P. aeruginosa and Escherichia coli from burn infections, and involvedcountriessuchasBelgium,France,andSwitzerland (http://www.phagoburn.eu).Recently,adouble-blind random-ized controlledtrial waspublished using phagesagainstP. aeruginosaresponsibleforburninfections.Inthisstudy two groupsofpatientseitherreceivedaphagecocktail(n=12/13) containing12phagesactiveagainstP.aeruginosaisolates,or atraditionaltreatment (n=13) usingastandard ofcarefor patientswith burninfections (1% sulfadiazinesilver emul-sioncream).Thephagetherapywasgiventopatientsthrough atopicalcreamcontainingthephages.Unfortunately,some unexpectedeventscompromisedthestudy,andthetrialwas stopped oneyear and a half afterits beginning. Afterthe creamwasmanufactured,thephageconcentrationdecreased from1×106_PFUto1×102_{PFU,(lessthaninitiallyexpected).}

Thisresultedininefficacy ofPP1131 (aphagecocktail) due totheextensivetimetakentodecreasethebacterialdensity onthe woundinfection.However,it isimportanttonotice thatalthoughalowerphageconcentrationwasused,the bac-terialburdeninburnwoundswasdecreased, showingthat phageswereinfactactive,butinferiortothestandardofcare treatment.30

Klebsiellapneumoniae

Severalstudieshavefocusedorsuggestedalternativestothe treatmentof infections caused byK. pneumoniae.12,31 _Thus,

severaldifferenttypesofphageshavebeendiscoveredinthe lastfewyears.Althoughitrepresentsanimportantadvance inthefield,thenumberofclinicaltrialsusingphagesagainst

K.pneumoniae clonesare insufficienttodeterminewhether phagetherapyisaviableoptionfortreatingKlebsiella infec-tionsinhumans.32–34

Recently,Corbellino etal.35_{demonstrated theutilization}

of a custom-made bacteriophage lytic preparation to gut decolonizationofapatientcolonizedbyKPC-3-producingK. pneumoniae. A 57-year old patient diagnosed with Crohn’s disease had multiple sites of colonization by an MDR K. pneumoniae belonging to ST307. The isolate was suscepti-ble toceftazidime-avibactam, which was successfully with reversionofallinfectionsigns,butwithlaterremission. Prob-ably, the repeated casesof infections (UTI and BSI) bythe samepathogen couldbeattributedtothepatient coloniza-tion,evidencedbyswabcultures.Fiveisolatesrecoveredfrom

the patientweresent toGeorgiEliava Instituteof Bacterio-phagesinTbilisi, Georgiaforcustomofphagepreparation. Interestingly, after three weeks of phage use by oral and intra-rectalroutesandnoadverseeffects,thepatientdidhad nourine,blood,orswabpositivecultureswith carbapenem-resistance.Almost oneyearafterthephageutilization,the patient had other cases of BSI and UTI by K. pneumo-niae;however, therecovered isolatesbelongedtoother STs (ST1109andST247),susceptibletomostantimicrobialagents, and notrelated tothe KPC-3-producing K.pneumoniaeonly susceptible to ceftazidime-avibactam frequently detected before.35

Kumari et al.36 _{comparedphagetherapyand its}

respec-tive protection in a wound burn model. For that study, three groups of mice were created: the first consisted of untreated animals, the second were treated with a hydrogelcontainingthephagesagainstaK.pneumoniae iso-late, while the third group was treated with gentamicin plus silver nitrate. The results demonstrated that a sin-gle dose of phages resulted in decreased mortality of the group treated withphageswhen comparedwith the other groups.

Role

of

phages

in

infection

control:

a

good

way

to

combat

MDR

pathogens

Phages are recognizedasuseful toolstocombat infections caused by many typesof microorganisms, including MDR-pathogens.16 _{Somestudies havealsoused phageson hand}

sanitizersolutionaimingtoimprovehandhygieneandavoid thespreadofMDRpathogens.37_{Otherauthorshave}

demon-stratedthecapacityofdecolonizationofspecificisolatesby phage action.38,39 _{Chhiber et al.}39 _{have demonstrated that}

phagesinassociation withmuropirocinwereable to effec-tively eradicate the colonizing MRSA population from the nares ofBALB/c mice.More importantly, administrationof phage MR-10 combined withmuropirocin reduced the fre-quencyofemergenceofspontaneousmuropirocinresistant isolates atnegligible levels. In addition, phages also could be used for effective removal of hospital pathogens from hard surfacesathospitalsettings,especially whenusedin combination with detergents.40 _{In atwo-phaseprospective}

interventionstudyperformedinTaiwan,theuseofanaerosol formulation containing bacteriophage inthe ICU led toan important reductionof new acquisition carbapenem resis-tantA.baumaniiinfectionfrom8.57per1000patients-dayto 5.11per1000patients-day(p=0.0029).Moreover,adecreasein carbapenem-resistancewasobserved(87.76%to46.07%)inthe ICUs.41_{Theseactionscouldreducethenumberofinfections}

and, indirectly, reduce antibiotic usage for treating severe infections.41

Itiswellknownthattheuseoflargeamountsof antimi-crobial agentsisresponsible fortheselectionofresistance clones,contributingtohighratesofantimicrobialresistance thatarefrequentlyreportedinBrazil.3_{Anotheradvantagethat}

supportsphageutilizationindevelopingcountriesisthelow cost associated withphage isolation and administration.14

The process ofdiscovery and productionofa new antimi-crobial agentis expensivein terms oftimeand resources.

Incontrast,phagescouldbeeasilyidentifiedinwater envi-ronments,especiallyinsewage,throughasimpleprocessof isolationandpurification.14_{InBelgium,forinstance,phages}

canbeusedasamagistralformula,manipulatedbya phar-macist, following a series of criteria which determine the phagesafety.42,43 _{Regarding administration, whilerepeated}

dosesofantibioticsneedtobeadministratedperiodicallyto achievethepharmacokineticandpharmacodynamic param-eters,small doses ofphagesare enoughsincethe number of viral particles will expand after the cell infection.20 _In

countrieswithhighsocialinequalitiesandlimitedresources to invest in public health, low cost and effective alterna-tivescould beessential totackle the high rates of deaths causedeveryyearbyMDR-pathogensinnosocomial environ-ments.

In addition, phages could be used incombination with antimicrobial agents increasing therapeutic efficacy and decreasing antimicrobial resistance development during therapy.44,45 _{Another importantadvantagerelated tophage}

utilizationisthecapacitytorestoreantimicrobialsensitivity.46

A study performed with a phage named OMKO1 revealed the ability of this phage to interact with the outer mem-brane proteins of MexAB and MexXY efflux systems in P. aeruginosaisolates.Inastrategytosurviveviralattack, the cellseemstodevelopamechanismbywhichseveral muta-tions in the outer membrane proteins are selected during bacteriapassage,decreasingvirus-bacteriabinding.However, thismodificationontheouter membraneproteinsequence decreasedthecapacityofbacteriatoextrudetheantibiotics, causingadropintheMICs.46

Brazilian

studies

using

phages

Unfortunately,fewphagestudieshavebeenreportedinBrazil. We strongly believe that research in this field is currently under development in Brazil. However, to the best of our knowledge,noclinicalstudieshavebeenpublished. Interest-ingly,arecentlypublishedhistoricalreviewpointedBrazilas onethepioneersofclinicalphageutilizationagainstS.aureus

anddiarrhealbacillieveninthe1920decade.47_{However,after}

theintroductionofantimicrobialagents,andmainlydueto discontinuationofresearchinthisfield,investigationswith bacteriophages were practically abandoned inBrazil.47 _Old

reportsreiteratetheBraziliancapacitytoinvestin bacterio-phagetechnology,aimingatcombatingbacterialinfections. The resumption ofstudies related tophagesin Brazil was startedinthelastdecadeandtheseinvestigationsarerelated todifferentfieldssuchasenvironmentalresearch,veterinary application,humanhealth,andothers,thoughleggingbehind intermsoftypeofphagesstudyconductedworldwide(Fig.2). Althoughmorerelatedwithcontaminationofaquatic envi-ronment,awell-conductedstudy about somaticcoliphages was carriedout inthecoastal regionsofSãoPaulo.In this study,it waspossibletoidentifyand characterizedifferent typesofphagesagainstE.coliisolates.Interestingly,the pres-enceofsomaticcoliphagesisawell-establishedindicationof fecalcontaminationoftheaquaticenvironment.48

Aimingtoperformthe decontaminationofchickenskin bySalmonellaenteritidis,apanelofphageswasusedwith suc-cessbyresearchersofEmbrapa inSanta Catarina,Brazil.49

Fig.2–Timelineofthemaineventsrelatedtophage’sresearchduringthelastdecades.Atthetopofthefigure,world eventsrelatedtophageresearch,whileinthebottom,themainresearchesthatwereconductedinBrazil.Theinvestigations involvingphagesinBrazilwereobtainedthroughresearchinthePubMed,usingtheindicators“BacteriophagesANDBrazil” or“PhagesANDBrazil”.Forabetterunderstandingortorequestmoreinformationabouteventsshownonthetopofthe figure,wesuggestreadingSalmondandFineran.14_{Additionally,formoreinformationabouttheearlystudiescarriedoutin}

244

Similarresultswereobservedalmost10yearslaterby Hun-garoetal.,50_{whoshowedtheefficacyofphageutilizationin}

reducingSalmonellaspp.isolatesfromchickenskin.Recently, useofphagesagainstP.aeruginosainthepoultryindustrywas alsoevidencedinMinasGeraisState.51

Astudyinvolvingclinicalisolates,conductedinSãoPaulo, detectedthepresenceofphagesagainstclinicalisolatesofP. aeruginosa.Surprisingly,thephageswereisolatedfroma col-lectionofP.aeruginosaisolatesrecoveredfromclinicalsamples ofdifferenthospitals.SomephagesshowedactivityagainstP. aeruginosaMDRandmucoidisolates.Althoughaclinical rela-tionhadbeenobserved,nodataabouttheclinicalutilization ofthesephageshavebeenreported.52

Recently,aphagewithactivity againstP. aeruginosawas detectedinthesewagefromTietêriverinSãoPaulocity.This phage,namedBrSP1,wasmicrobiologicallyandgenomically characterizedandhaditsactivitytestedagainstP.aeruginosa

isolatesfromdomesticanimals.BrSP1phagebelongedtothe Caudoviralesorder,agenomewith66,189bp,andhadactivity against51.4%oftestedisolates.Thisfacthighlightsthe possi-bilityofphageutilizationinhumanandveterinarymedicine.53

Limitation

of

bacteriophages

use

in

human

health

and

the

environment

Likealltreatmentoptions,phageutilizationalsopresents lim-itationsthat should be considered beforeclinical use. The absenceoflargerandomizedclinicaltrials,safetyevaluation ofphagetherapy,andthenumerousstepsnecessarybefore clinicalutilizationofanidentifiedphageareoftenreported asthemainlimitationsofthistypeoftherapy.Furthermore, littleisknownabouttheroleofthesebacteriophagesinthe environmentandtheircapacitytocarryresistancegenes.To abetterunderstandingofthelimitationsofphagetreatment, werecommendreadingPrincipietal.54

Conclusions

Wehaveprovidedevidencethatuseofphagetherapycouldbe anewwaytocombatMDR-pathogensandthatthisapproach wouldbeusefulinBrazilwheretheratesofantibiotic resis-tance areon the rise.Currently,Brazil isgoingthrough an economiccrisis,amomentthatalwaysrequiresinexpensive alternatives to tackle different national challenges. In this case,lyticphageutilizationcouldbeoneofthesealternatives, sincetheseagentsaresimple,inexpensiveandabundantin differentenvironments.Inaddition,morestudiesneedtobe supportedinthisfield,aimingtoreducealluncertainties con-cerningthistypeoftherapy,togainanewtoolinthefight againstantimicrobialresistanceinBrazil.

Funding

W.M.B.S.M.waspartially supportedbyCoordinationforthe ImprovementoftheHigherEducationPersonnel(CAPES) [Pro-cess number 88887.32142/2019-00] and fully supported by São Paulo Research Foundation (FAPESP) [Process number 2018/24431-4].

Conflicts

of

interest

A.C.G.hasrecentlyreceivedresearchfundingand/or consul-tationfeesfromBayer,Eurofarma,MSD,Pfizer,andZambon. Otherauthorshavenothingtodeclare.

Acknowledgments

WewouldliketothankstheCoordinationfortheImprovement oftheHigherEducationPersonnel(CAPES)andtheSãoPaulo ResearchFoundation(FAPESP)forprovidingfundingstothis researcher.

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