Bacteriophages of Helicobacter pylori

micb-11-549084 No embe 6, 2020 Time: 13:38 # 1 MINI REVIEW published: 12 No embe 2020 doi: 10.3389/ micb.2020.549084 Edi ed by: E ic Al e mann, AgResea ch L d, New Zealand Re iewed by: Je emy J. Ba , Monash Uni e si y, Aus alia Sa ah O’Flahe y, No h Ca olina S a e Uni e si y, Uni ed S a es Junko Akada, Oi a Uni e si y, Japan *Co espondence: Angela B. Muñoz [email p o ec ed] Filipa F. Vale [email p o ec ed]; [email p o ec ed] Special y sec ion: This a icle was submi ed o E olu iona y and Genomic Mic obiology, a sec ion o he jou nal F on ie s in Mic obiology Recei ed: 04 Ap il 2020 Accep ed: 21 Oc obe 2020 Published: 12 No embe 2020 Ci a ion: Muñoz AB, S epanian J, T espalacios AA and Vale FF (2020) Bac e iophages o Helicobac e pylo i. F on . Mic obiol. 11:549084. doi: 10.3389/ micb.2020.549084 Bac e iophages o Helicobac e pylo i Angela B. Muñoz1,2*, Johanna S epanian1, Alba Alicia T espalacios1and Filipa F. Vale2* 1In ec ious Diseases Resea ch G oup, Mic obiology Depa men , Sciences Facul y, Pon i icia Uni e sidad Ja e iana, Bogo á, Colombia, 2Hos –Pa hogen In e ac ions Uni , Resea ch Ins i u e o Medicines (iMed-ULisboa), Faculdade de Fa mácia, Uni e sidade de Lisboa, Lisbon, Po ugal The bac e ium Helicobac e pylo i colonize he s omach in app oxima ely hal o he wo ld’s popula ion. In ec ion wi h his bac e ium is associa ed wi h gas i is, pep ic ulce , adenoca cinoma, and gas ic mucosa-associa ed lymphoid issue lymphoma. Besides being a pa hogen wi h wo ldwide p e alence, H. pylo i show inc easingly high an ibio ic esis ance a es, making he de elopmen o new he apeu ic s a egies agains his bac e ium challenging. Fu he mo e, H. pylo i is a gene ically di e se bac e ium, which may be in luenced by he p esence o mobile genomic elemen s, including p ophages. In his e iew, we analyze hese issues and summa ize a ious epo s and indings ela ed o phages and H. pylo i, discussing he ela ionship be ween he p esence o hese elemen s and he genomic di e si y, i ulence, and i ness o his bac e ium. We also analyze he s a e o he knowledge on he po en ial u ili y o bac e iophages as a he apeu ic s a egy o H. pylo i. Keywo ds: Helicobac e pylo i, bac e iophage, phage he apy, p ophage gene ic di e si y, phage–hos in e ac ion INTRODUCTION Helicobac e pylo i is a spi al-shaped, mic oae ophilic, G am-nega i e, pa hogenic bac e ium ha colonizes and pe sis s in he human s omach. H. pylo i in ec ion is conside ed he mos equen ch onic bac e ial in ec ion wo ldwide (Camilo e al., 2017). All indi iduals in ec ed wi h H. pylo i p esen wi h ch onic gas i is, while 20% de elop pep ic ulce and less han 1% de elop adenoca cinoma o mucosa-associa ed lymphoid issue (MALT)- ype gas ic lymphoma. Fu he , H. pylo i in ec ion is ecognized as he leading isk ac o o he de elopmen o gas ic cance (Polk and Peek, 2010). Nume ous ac o s ha e been desc ibed as esponsible o he p og ession o he in ec ion o mo e se e e diseases: al hough hese ac o s may be inhe en o he hos (Figuei edo e al., 2017;Mi ahussu u e al., 2017), se e al H. pylo i i ulence ac o s ha e also been associa ed wi h malignancy. The mos equen ly associa ed a e he Cag pa hogenici y island (cagPAI) and he VacA acuola ing cy o oxin (Denic e al., 2020). CagPAI is a gene ic locus o 40 kb, comp ising 31 genes ha encode a ype IV sec e ion sys em (T4SS). The T4SS enables he injec ion o bac e ial componen s, such as he CagA oncop o ein, in o hos gas ic epi helial cells (Solu i e al., 2020). VacA is a oxin sec e ed by H. pylo i ha inse s in o hos cell memb anes o o m chlo ide-sensi i e channels and dis up endolysosomal a icking, causing an accumula ion o dys unc ional lysosomes and au ophagosomes (Denic e al., 2020). Mos H. pylo i s ains isola ed ha e he acA gene. Howe e , di e ences in he alleles o he signal (s) and middle (m) egions o his gene a e esponsible o di e ing le els o cy o oxici y (Ok em-Okullu e al., 2020). F on ie s in Mic obiology | www. on ie sin.o g 1No embe 2020 | Volume 11 | A icle 549084 micb-11-549084 No embe 6, 2020 Time: 13:38 # 2 Muñoz e al. Bac e iophages o Helicobac e pylo i In addi ion o he i ulence o H. pylo i, ano he c i ical challenge o clinicians has been o ind an op imal ea men ha akes in o accoun he high le els o esis ance o H. pylo i o an ibio ics (Cama go e al., 2014;Talebi Bezmin Abadi, 2017). H. pylo i cu e a es using an ibio ic ea men ha e been epo ed o be as low as 57% (Dos San os and Ca alho, 2015). The minimum accep able a e o a i s a emp based ea men is 90% (Nije i ch e al., 2014;Jones e al., 2017). Cu en ea men s a e based on he use o an ibio ics combined wi h a p o on pump inhibi o (PPI) (Mal e heine e al., 2017). The PPI is used o inc ease in agas ic pH because, a an acidic pH, H. pylo i ans o ms in o i s an ibio ic- esis an coccoid o m (Ie a di e al., 2019). Fu he mo e, acidic pH dec eases he an imic obial ac i i y and hal -li e o an ibio ics (O e o e al., 2018). The inc easing a es o H. pylo i an ibio ic esis ance ha e necessi a ed he de elopmen o new he apeu ic s a egies (Ví o and Vale, 2011). An eme ging al e na i e ea men o an ibio ic- esis an bac e ial in ec ions is phage- he apy, which uses bac e iophages, also known as phages, o elimina e a bac e ial popula ion (Lin D. M. e al., 2017). The esu gence o his he apy has been due mainly o s udies showing ha phages a e highly speci ic and easy o isola e (Loc-Ca illo and Abedon, 2011); mo eo e , phage he apy has demons a ed e ec i eness in ea ing a ious in ec ions, e en cu ing ch onic in ec ions (Abedon, 2019). Despi e he bene i s o his he apy, i s use o he ea men o H. pylo i in ec ion is a dis an goal, conside ing ha he unde s anding o H. pylo i phage biology is s ill in i s in ancy. Phages, which a e i al pa icles ha in ec bac e ia, may be ly ic, lysogenic, o pseudo-lysogenic. Ly ic phages ecognize bac e ial su aces and injec hei nucleic acids in o he hos cell; hen, hey assemble, mul iply, and inally dis up he cell o elease phage p ogeny ha in ec new bac e ial cells (Sulak elidze, 2005). In lysogenic phages, phage DNA in eg a es in o he bac e ial genome, o ming p ophages (also known as empla e phages). P ophages p omo e bac e ial e olu ion ia ho izon al gene ans e and induce he ansduc ion o se e al genes in ol ed in he biological beha io s o he bac e ium (B üssow e al., 2004;Pa e son e al., 2010;Touchon e al., 2016). Pseudo-lysogenic phages a e p esen as episomes (i.e., he gene ic ma e ial is no in eg a ed in o he bac e ial genome) and pos pone cell lysis in nu ien - deple ed hos s (Uchiyama e al., 2013); bo h ly ic and lysogenic phages, unde ce ain condi ions, may acqui e episomal o ms (Ło´ s and We¸g zyn, 2012). Bac e iophages ep esen he absolu e majo i y o all o ganisms in he biosphe e (Ha ull and Hend ix, 2011). Among he a ious bac e iophages, he disco e y o H. pylo i p ophages has been o pa icula impo ance in explaining his bac e ium’s emendous gene ic di e si y. This e iew aims o colla e and analyze ele an publica ions ega ding phages ela ed o H. pylo i and o unde s and how hese phages impac he di e si y and i ulence o his bac e ium. Addi ionally, we summa ize epo s on ly ic phages wi h ac i i y agains H. pylo i. H. pylo i P ophages The i s obse a ions o phage-like in acellula pa icles in p epa a ions o H. pylo i we e made sho ly a e he disco e y o his bac e ium (Ma shall e al., 1987;Goodwin e al., 1989). In he ea ly 1990s, a lysogenic s ain o H. pylo i ha p oduced phage pa icles spon aneously was desc ibed (Schmid e al., 1990). Th ee yea s la e , he ly ic cycle o his phage was ep oduced. Nega i e s ain elec on mic oscopy e ealed ha phage heads o a ound 50 o 60 nm and he DNA leng h was es ima ed o be 22,000 bp (Hein schel Von Heinegg e al., 1993). Wi h ecen ad ances in sequencing echnologies, s udies aiming a he disco e y o phages a e inc easing. Since 2011, in es iga ions ocusing on he analysis o Helicobac e spp. genomes ha e been published ha co obo a e he p esence o p ophages in he bac e ial genome and analyze hei unc ions in his con ex (Lehou s e al., 2011;Luo e al., 2012;Uchiyama e al., 2012, 2013, 2016;Vale e al., 2015, 2017;Secka e al., 2017;Vale and Lehou s, 2018) (Table 1). These s udies epo ha p ophages a e p esen in a ound 20% o H. pylo i isola es. The i s o hese s udies epo ed a p ophage sequence in H. pylo i isola ed om a pa ien wi h MALT lymphoma. This p ophage, named PhiHp33, was isola ed om H. pylo i s ain B45 and could be induced by UV ligh . Genomic sequence analysis demons a ed ha H. pylo i s ains om di e en egions ca y ull o pa ial p ophage sequences. Analysis o in eg ase and holin gene sequences allowed di e en ia ion o s ains acco ding o hei geog aphical o igin, gene a ing esul s consis en wi h classi ica ion based on mul i-locus sequence yping (MLST) (Lehou s e al., 2011;Vale e al., 2015;Secka e al., 2017). In 2012, he isola ion o a new phage (1961P) om Taiwanese H. pylo i s ains was epo ed. This phage showed cha ac e is ics compa ible wi h belonging o he amily Podo i idae. Fu he , sequence analysis iden i ied o he simila p ophages in eg a ed in o he genomes o di e en H. pylo i s ains ha had p e iously been sequenced (Luo e al., 2012). Addi ionally, in he same yea , he comple e genome sequences o wo H. pylo i bac e iophages (KHP30 and KHP40) isola ed om Japanese pa ien s we e epo ed. These p ophages we e ob ained a e spon aneous elease o i al pa icles by H. pylo i s ains (Uchiyama e al., 2012). The KHP30 phage was cha ac e ized as sphe ical, wi h a lipid en elope. I was p esen in in ec ed bac e ia as an episome, indica ing ha i likely ep esen s a new phage amily (Uchiyama e al., 2013). Subsequen ly, he possibili y o lysogeny o ac i e KHP30-like phages in 174 Japanese H. pylo i s ains was examined because hei genomes con ain a pu a i e in eg ase gene; one s ain, NY40, was ound o include a KHP30-like p ophage sequence. In his s ain, he a achmen si es, a L and a R, we e almos he same place in he genome. Fu he , hei sequences we e de ec ed p e iously by Lehou s e al., sugges ing an ac i e pa en al KHP30-like phage in eg a ed in o he ances al NY40 genome in a si e-speci ic manne (Uchiyama e al., 2016). Analysis o KHP30 has con inued, wi h esea che s in es iga ing i s e ec s on he beha io s o he hos s ain (NY43) and demons a ing di e ences be ween he cha ac e is ics o H. pylo i s ains in ec ed wi h p ophage and no el p ophage- ee de i a i e s ains. A ecen s udy indica ed ha he p esence o bac e iophages induces gene ic al e a ions in he hos genome, leading o he con inuous p oduc ion o p ophage- ee de i a i es ha coexis in H. pylo i mic obial communi ies F on ie s in Mic obiology | www. on ie sin.o g 2No embe 2020 | Volume 11 | A icle 549084 micb-11-549084 No embe 6, 2020 Time: 13:38 # 3 Muñoz e al. Bac e iophages o Helicobac e pylo i TABLE 1 | Main bac e iophages o H. pylo i. Bac e iophage Type Family Genome size (Kb) De ails Re e ences 1961P P ophage Podo i idae 26.8 Accession numbe : NC_019512.1 Head: 68–74 nm Tail: 23 × 13.3 nm (Luo e al., 2012) Campylobac e pilo idis s ain P ophage No da a No da a In acellula pa icles o 40 nm in diame e (Ma shall e al., 1987) Campylobac e pylo i s ain P ophage No da a No da a In acellula pa icles o 85 nm in diame e (Goodwin e al., 1989) De-M53-M P ophages No da a 28.1 P ophage popula ion: hpNEu ope Accession numbe : KX119205 (Vale e al., 2017) F -ANT170-U P ophage No da a 31.2 P ophage popula ion: hpA ica1 Accession numbe : KX119201 (Vale e al., 2017) F -B41-M P ophage No da a 29.4 P ophage popula ion: hpSWEu ope Accession numbe : KX119190 (Vale e al., 2017) F -B58-M P ophage No da a 22.6 P ophage popula ion: hpEas Asia Accession numbe : KX119193 (Vale e al., 2017) F -G12-G P ophage No da a 28.6 P ophage popula ion: hpEas Asia Accession numbe : KX119194 (Vale e al., 2017) F -GC43-A P ophage No da a 33.0 P ophage popula ion: hpEas Asia Accession numbe : KX119195 (Vale e al., 2017) F -MEG235-U P ophage No da a 31.2 P ophage popula ion: hpA ica1 Accession numbe : KX119200 (Vale e al., 2017) HP1 P ophage Sipho i idae 22 Head:50–60 nm Tail:170 ×9.5 nm (Hein schel Von Heinegg e al., 1993) KHP30 P ophage Canno be classi ied / new amily 26.2 Episome Accession numbe : NC_019928.1 Head: 67–71 nm Wi hou ail (Uchiyama e al., 2012, 2013;Takeuchi e al., 2018) KHP40 P ophage No da a 26.4 KHP30-like Accession numbe : NC_019931.1 (Uchiyama e al., 2012) PhiHp33 P ophage Sipho i idae 24.6 Inducible by UV ligh Accession numbe : NC_016568 Head: 55–70 nm Tail: 92 ×6 nm (Lehou s e al., 2011) P -1293-U P ophage No da a 30.1 P ophage popula ion: hpA ica1 Accession numbe : KX119202 (Vale e al., 2017) P -1846-U P ophage No da a 28.0 P ophage popula ion: hpA ica1 Accession numbe : KX119176 (Vale e al., 2017) P -1918-U P ophage No da a 28.7 P ophage popula ion: hpSWEu ope Accession numbe : KX119192 (Vale e al., 2017) P -212-99R-U P ophage No da a 23.0 P ophage popula ion: hpA ica1 Accession numbe : KX119193 (Vale e al., 2017) P -228_99-G P ophage No da a 30.1 P ophage popula ion: hpA ica1 Accession numbe : KX119175 (Vale e al., 2017) P -4472-G P ophage No da a 27.6 P ophage popula ion: hpSWEu ope Accession numbe : KX119190 (Vale e al., 2017) P -4481-G P ophage No da a 25.4 P ophage popula ion: hpA ica1 Accession numbe : KX119196 (Vale e al., 2017) P -4497-U P ophage No da a 29.4 P ophage popula ion: hpSWEu ope Accession numbe : KX119191 (Vale e al., 2017) P -5322-G P ophage No da a 28.3 P ophage popula ion: hpA ica1 Accession numbe : KX119198 (Vale e al., 2017) P -5771-G P ophage No da a 29.8 P ophage popula ion: hpA ica1 Accession numbe : KX119199 (Vale e al., 2017) P -B89-G P ophage No da a 27.4 P ophage popula ion: hpA ica1 Accession numbe : KX119203 (Vale e al., 2017) P -B92-G P ophage No da a 30.5 P ophage popula ion: hpA ica1 Accession numbe : KX119197 (Vale e al., 2017) Sw-A626-G P ophage No da a 31 P ophage popula ion: hpNEu ope Accession numbe : KX119177 (Vale e al., 2017) Sw-577-G P ophage No da a 26.9 P ophage popula ion: hpNEu ope Accession numbe : KX119204 (Vale e al., 2017) UK-EN31-U P ophage No da a 30.5 P ophage popula ion: hpNEu ope Accession numbe : KX119174 (Vale e al., 2017) UK-EN32-U P ophages No da a 29.9 P ophage popula ion: hpNEu ope Accession numbe : KX119206 (Vale e al., 2017) φHPE1 Ly ic Podo i idae No da a Ti e : 109PFU/ml Head: 62 nm Sho noncon ac ile ail: 12 ×6 nm (Abdel-Haliem and Asko a, 2013) φHPE2 Ly ic Sipho i idae No da a Ti e : 1010 PFU/ml Head: 95 nm Tail: 180 ×15 nm (Abdel-Haliem and Asko a, 2013) Hp ϕLy ic No da a No da a Used in syne gy wi h lac o e icin and hyd oxyapa i e. Adso p ion a e: 1.89 ×109 mL/min La en pe iod: 45 min Bu s size: 80 PFU (Cuomo e al., 2020) H. pylo i s ain BAPOUI Ly ic No da a No da a Ob ained om human eces Head: 100 nm Wi hou ail (Vale e al., 2008) and con e di e si y in mo phology, mo ili y, iabili y, and pa hogenici y. Fu he , his s udy epo ed ha KHP30 p ophage could speci ically supp ess CagA exp ession (Takeuchi e al., 2018). Ano he s udy conduc ed using an H. pylo i s ain isola ed om an e hnic mino i y pa ien in China e ealed he p esence o a 32.5 kb p ophage in eg a ed in o he genome and demons a ed ha mos o i s genes (30/33) we e he same as hose o KHP30; howe e , in his case, he p ophage was inse ed be ween wo pu a i e i ulence genes, oipA and homB, which encode adhe ence ac o s ha media e in e ac ions be ween H. pylo i and he hos mic oen i onmen . The au ho s sugges ha he p esence o a p ophage inse ed in his egion indica es ha HomB and OipA may ac as ecep o s o he phage (You e al., 2015). F on ie s in Mic obiology | www. on ie sin.o g 3No embe 2020 | Volume 11 | A icle 549084 micb-11-549084 No embe 6, 2020 Time: 13:38 # 4 Muñoz e al. Bac e iophages o Helicobac e pylo i In addi ion o he cha ac e iza ion o H. pylo i p ophages, he ela ionship be ween he p esence o p ophages and bac e ial gene ic di e si y has been widely discussed. H. pylo i is a gene ically di e se species. This di e si y has been associa ed wi h he geog aphic o igin o popula ions (Ma a e al., 2017) and likely in luenced by he p esence o mobile genomic elemen s, such as p ophages, among o he ac o s (Vale and Lehou s, 2018). Since 2015, Vale e al. ha e epo ed ha H. pylo i p ophages may con ibu e o bac e ial gene ic di e si y. Mos obse a ions indica ed a phylogeog aphic ag eemen be ween phage and bac e ial genes, sugges ing a co-e olu ion model be ween he i us and i s bac e ial hos . None heless, some p ophages we e assigned o popula ions dis inc om hei hos , while o he s exhibi ed ecombina ion signs among popula ions (Yaha a e al., 2019). The phylogeog aphic dis ibu ion o H. pylo i p ophages can be cha ac e ized by Bayesian clus e ing analysis, based on a yping scheme ha includes he sequences o wo p ophage genes: in eg ase (which is esponsible o he in eg a ion o he phage genome in o he bac e ial ch omosome) and holin (which is in ol ed in cell lysis du ing a ly ic cycle). Fou p ophage popula ions ha e been desc ibed: hpA ica1, hpEas Asia, hpNEu ope, and hpSWEu ope, no ably subdi iding he Eu opean popula ion (Vale e al., 2015). Recen ly, based on he whole-genome analysis o H. pylo i, wo subpopula ions o hpEu ope we e also obse ed (Tho ell e al., 2017). A ailable in o ma ion on H. pylo i p ophage di e si y indica es ha p ophages and bac e ia sha e a complex e olu iona y his o y (Vale e al., 2017) and ha he bac e ial genome has been widely modi ied, in di e se egions, ia ho izon al gene ans e (Takeuchi e al., 2018). A ew s udies ha e in es iga ed he p esence o phages in o he H. pylo i s ains; o example, a s udy om 2016 epo ed he de ec ion o a 31.7 kb p ophage in an H. pylo i s ain isola ed om a Mexican pa ien wi h gas ic cance (Muci o-Va ela e al., 2016). Fu he , Kuma e al. (2017) analyzed H. pylo i s ains om Kuwai and epo ed a p ophage in a s ain isola ed om a pa ien wi h ac i e ch onic gas i is and ocal in es inal me aplasia. In silico analyses ha e e ealed p ophage o hologous genes in he genomes o bac e ial s ains wi h i ulence genes such as cagA o acA (Ky illos e al., 2016). Repo s sugges ha p ophages inse ed in he H. pylo i genome ep esen essen ial elemen s o he adap a ion o his bac e ium o hos ile en i onmen s, because me abolic ad an ages o ole ance mechanisms ha can be in luenced by p ophages may imp o e H. pylo i compe i i eness. Ne e heless, no speci ic p ophage unc ions ha e, as ye , been ecognized in H. pylo i, and he oles o p ophage in disease ha e a ely been eco ded (Vale e al., 2017). Long- e m coloniza ion o he human s omach by H. pylo i has allowed i o e ol e oge he wi h i s human hos o mo e han 100,000 yea s (Moodley e al., 2012). Fu he mo e, because o equen mu a ion and ecombina ion e en s and ho izon al gene ans e e en s, H. pylo i is an ex emely di e se bac e ium. Speci ically, H. pylo i is na u ally compe en o ans o ma ion and highly compe en in ecombina ion, making he exchange o ch omosomal DNA agmen s be ween s ains equen and highly e icien (Fische e al., 2020). Phages ha e gene ally been conside ed as ac i ely in ol ed in he p ocesses o ecombina ion and ho izon al gene ic exchange. In his con ex , a ecen s udy p esen ed a quan i a i e analysis o he desc ibed ecombina ion e en s o H. pylo i phage genes, e ealing equen ecombina ion among phage co e genes p e iously epo ed as less p one o ecombina ion. This obse a ion appea s o e lec he co-e olu iona y ela ionships o hese phages wi h hei hos s, e ealing he e olu iona y a ms ace ha exis s, ei he o con ibu e o phage escape om bac e ial immuni y o o p o ec hos s ha p oduce de ec i e phages. The high ecombina ion a e in H. pylo i phages sugges s ha hey a e among he mos ecombinogenic phages on ea h (Yaha a e al., 2019). To u he ad ance knowledge o his opic, i is impo an o in es iga e he biological and genomic cha ac e is ics o H. pylo i phages in de ail. Likewise, aluable in o ma ion may eme ge om he con inued sea ch o phage in s ains associa ed wi h di e en pa hologies and om a ious geog aphical loca ions. Fu he mo e, impo an insigh s may be ob ained h ough in es iga ion o phage inse ion si es and hei oles in he bac e ial genome, conside ing ha p ophages can ac as gene ese oi s, which may bene i pa hogens in ways ha a e jus beginning o be de e mined. Ly ic Bac e iophages o H. pylo i The ise o an ibio ic esis ance inc eased he in e es in s udying bac e iophages, pa icula ly he ly ic ones. Indeed, phage he apy has a ious po en ial ad an ages han an ibio ic use because phages and phage lysins, a e highly speci ic, a ec ing he a ge s ain bu no he mic obiome (B üssow, 2017). Fu he , phages only eplica e a he in ec ion si e, and no seconda y e ec s ha e been desc ibed (Ma suzaki e al., 2005). Al hough hese indings indica e he po en ial o use phages o e adica e H. pylo i, epo s in his ield a e sca ce. A he ime o his e iew, he e we e only h ee s udies on ly ic bac e iophages (Table 1): he i s was published by Vale e al. (2008), who isola ed one ly ic phage om human eces. No many cha ac e is ics o his phage a e known. I was only epo ed ha i is a phage wi hou a ail and an app oxima e size o 100 nm. The second s udy was published in 2013 by Abdel- Haliem and Asko a, who isola ed and cha ac e ized wo ly ic phages (8HPE1 and 8HPE2) om was ewa e (Abdel-Haliem and Asko a, 2013). These phages we e classi ied, by elec onic mic oscopy, as belonging o he Podo i idae and Sipho i idae amilies, espec i ely. The i e s o 8HPE1 and 8HPE2 we e 109PFU/ml and 1010 PFU/ml, espec i ely. These s udies did no o e da a on he la en pe iod, bu s size, o an imic obial po en ial o hese phages. A hi d ecen s udy by Cuomo e al. (2020) desc ibes a new he apeu ic op ion based on using a ly ic phage o H. pylo i (Hp ϕ), isola ed om gas ic biopsies. The phage was es ed alone and combined wi h lac o e in (LA) and adso bed on hyd oxyapa i e (HA) nanopa icles. The cha ac e iza ion o Hp ϕ e ealed ha he adso p ion a e was 1.89 ×109mL/min, he la en pe iod was 45 min, and he bu s size was only 80 PFU. These analyses showed ha he use o he complex (Hp ϕ+ LF-HA) enhances he ac i i y o Hp ϕup o 4 imes. The use o HA as a ehicle can imp o e he na u al p ope ies o bo h Hp ϕand LF because i p o ec s he gas ic acid en i onmen . F on ie s in Mic obiology | www. on ie sin.o g 4No embe 2020 | Volume 11 | A icle 549084 micb-11-549084 No embe 6, 2020 Time: 13:38 # 5 Muñoz e al. Bac e iophages o Helicobac e pylo i An impo an inding, conside ing ha phages a e o en sensi i e o ex eme pH alues such as hose associa ed wi h s omach acidi y (Da¸b owska and Abedon, 2019). The s udies a ailable o da e ha e no add essed ce ain aspec s impo an o de ining phage- he apy. The genome sequences o he phages emain una ailable. Genomic analysis could de e mine whe he he phage genome comp ises genes in ol ed in lysogenic e en s, encode oxins, o ep esen de e minan s o an imic obial esis ance, o whe he hey code o ly ic cycle ep esso s, o si e-speci ic in eg ases o ecombinases (Fe nández e al., 2019). Likewise, conside ing he speci ic de ense mechanisms moun ed by bac e ia agains bac e iophage in ec ions, i is essen ial o add ess he possible esis ance ha H. pylo i may de elop agains hese phages (Hyman and Abedon, 2010). The s udy by Cuomo e al. (2020) does no de ine he minimum e ec i e combined doses o Hp ϕand LF-HA. Addi ional o he use o ly ic phages, he use o phage lysins and ela ed bac e ioly ic enzymes a e also being conside ed in phage he apy (Fische i, 2018). Many phages ha e polysaccha ide depolyme ases and lysins ha ecognize, bind, and deg ade he polysaccha ide compounds (La ka e al., 2017). The enzyme holin could also be used as a he apeu ic al e na i e. This enzyme is in ol ed in he holin-endolysin pa hway esponsible o he i s s eps o bac e ial lysis, which begins when he holin o ms mic ome e -scale holes in he bac e ial inne memb ane, eleasing ac i e endolysin in o he pe iplasm o deg ade pep idoglycan (Cahill and Young, 2019). S udies ha e e alua ed he e ec o enzymes o he ea men o bac e ial in ec ions mainly caused by g am-nega i e bac e ia, and ha e shown e icacy in educing bac e ial i ulence (D’and ea e al., 2017;Lin H. e al., 2017;Majkowska-Sk obek e al., 2018; Solo ie a e al., 2018). Conside ing he p esence o p ophages in H. pylo i and he backg ound in o he bac e ial models, he in es iga ion o hese kinds o enzymes in H. pylo i is easible. CONCLUSION The indings e iewed he e show ha H. pylo i gene ic di e si y is in luenced by p ophages, which a e equen ly ound in s ains o his bac e ium, ep esen a s uc u ed popula ion, and a e highly ecombinogenic. The addi ional gene ic di e si y ha phages may p o ide o H. pylo i con e s ad an ages o his bac e ium in e ms o pe sis ence and coloniza ion o di e en human popula ions. The s udy o H. pylo i phages has gained impo ance conside ing he subs an ial challenges posed by in ec ion wi h his bac e ium, including low e adica ion a es and an ibio ic esis ance. Phages appea o be a p omising app oach o he design o u u e he apies. Ad ances in esea ch o imp o e unde s anding o phage and H. pylo i in e ac ions a e equi ed, and s udies in es iga ing whe he p ophage genes belonging o he lysis casse e a e use ul o phage- he apy a e wa an ed. Fu he , iden i ica ion o mo e ly ic phages o H. pylo i would allow conside ing he phage- he apy as an al e na i e app oach o e adica ing H. pylo i, which would undoub edly ma k a miles one in he his o y o applica ions o H. pylo i phages. AUTHOR CONTRIBUTIONS AM, JS, AT, and FV concei ed he e iew. AM and JS collec ed he in o ma ion. AM and FV w o e he manusc ip . All au ho s con ibu ed o he manusc ip e ision, ead, and app o ed he submi ed e sion. FUNDING This wo k was suppo ed by he ollowing sou ces: AM is a ecipien o a schola ship om he Cen o de Es udios In e disciplina ios Básicos y Aplicados (CEIBA Founda ion), Colombia. AM, JS, and AT a e ecipien s o a p ojec g an (120380763025/2018) om he Depa amen o Adminis a i o de Ciencia, Tecnología e Inno ación de Colombia (Colciencias) and a p ojec g an (PPTA_7676) om Resea ch Vice-Rec o y, Pon i icia Uni e sidad Ja e iana. FV is he ecipien o a p ojec g an (PTDC/BTM-SAL/28978/2017) om he Fundação pa a a Ciência e a Tecnologia (FCT). ACKNOWLEDGMENTS We hank he en i ies ha inancially suppo ed he de elopmen o his wo k. REFERENCES Abdel-Haliem, M. E. F., and Asko a, A. (2013). Isola ion and cha ac e iza ion o bac e iophages o Helicobac e pylo i isola ed om Egyp . Fu u e Vi ol. 8, 821–826. doi: 10.2217/ l.13.58 Abedon, S. T. (2019). 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Compa a i e genomics o aHelicobac e pylo i isola e om a Chinese Yunnan Naxi e hnic abo igine sugges s high gene ic di e gence and phage inse ion. PLoS One 10:e0120659. doi: 10.1371/jou nal.pone.0120659 Con lic o In e es : The au ho s decla e ha he esea ch was conduc ed in he absence o any comme cial o inancial ela ionships ha could be cons ued as a po en ial con lic o in e es . Copy igh © 2020 Muñoz, S epanian, T espalacios and Vale. This is an open-access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License (CC BY). The use, dis ibu ion o ep oduc ion in o he o ums is pe mi ed, p o ided he o iginal au ho (s) and he copy igh owne (s) a e c edi ed and ha he o iginal publica ion in his jou nal is ci ed, in acco dance wi h accep ed academic p ac ice. No use, dis ibu ion o ep oduc ion is pe mi ed which does no comply wi h hese e ms. F on ie s in Mic obiology | www. on ie sin.o g 7No embe 2020 | Volume 11 | A icle 549084