A case for the importance of following antibiotic resistant bacteria throughout the soil food web

Recei ed: 16 Augus 2023 Re ised: 17 Sep embe 2023 Accep ed: 4 Oc obe 2023 DOI: 10.1002/bies.202300153 COMMENTARY Though s & Opinion A case o he impo ance o ollowing an ibio ic esis an bac e ia h oughou he soil ood web Ca los Ga bisu1I zia Alko a2 1NEIKER −Basque Ins i u e o Ag icul u al Resea ch and De elopmen , De io, Spain 2Depa men o Biochemis y and Molecula Biology, Uni e si y o he Basque Coun y (UPV/EHU), Bilbao, Spain Co espondence I zia Alko a, Depa men o Biochemis y and Molecula Biology, Uni e si y o he Basque Coun y (UPV/EHU), P.O. Box 644, 48080 Bilbao, Spain. Email: i zi.alk[email p o ec ed] Funding in o ma ion MCIN/AEI/10.13039/501100011033, G an /Awa d Numbe : PID2020-116495RB-I00; Basque Go e nmen , G an /Awa d Numbe : IT1578-22; JRL En i onmen al An ibio ic Resis ance Abs ac I is necessa y o complemen nex -gene a ion sequencing da a on he soil esis ome wi h heo e ical knowledge p o ided by ecological s udies ega ding he sp ead o an ibio ic esis an bac e ia (ARB) in he abio ic and, especially, bio ic ac ion o he soil ecosys em. Pa icula ly, when ARB en e ag icul u al soils as a consequence o he applica ion o animal manu e as e ilize , om a mic obial ecology pe spec i e, i is impo an o know hei a e along he soil ood web, ha is, h oughou ha complex ne wo k o eeding in e ac ions among membe s o he soil bio a ha has c ucial e ec s on species ichness and ecosys em p oduc i i y and s abili y. I is c i ical o s udy how he ARB ha en e he soil h ough he applica ion o manu e can each o he axonomical g oups (e.g., ungi, p o is s, nema odes, a h opods, ea hwo ms), paying special a en ion o hei p esence in he gu mic obiomes o meso auna- mac o auna and o he possibili ies o ho izon al gene ans e o an ibio ic esis an genes. KEYWORDS An ibio ic esis ance, gu mic obiomes, ho izon al gene ans e , manu e, mic obial ecology, o ganic a ming, soil bio a COMMENTARY An ibio ic esis ance is a global heal h p oblem o he u mos impo - ance. The s udy o he eme gence, dissemina ion, and e olu ion o an ibio ic esis an bac e ia (ARB), ha bou ing an ibio ic esis ance genes (ARGs), is o en claimed o equi e a One Heal h app oach. Then, in he las decades, much esea ch has been done on he links be ween he human esis ome and he en i onmen al esis ome ( he esis ome is he collec ion o all he ARGs and hei p ecu so s p esen in pa hogenic and non-pa hogenic bac e ia). I is impo an o empha- size ha mic oo ganisms (e.g., bac e ia, ungi) ha e used an ibio ics, o compe i ion pu poses o as signalling molecules ha acili a e in a- o in e species in e ac ions wi hin mic obial communi ies,[1] o millions o yea s. The e o e, ARB and ARGs a e o en isola ed om p is ine en i onmen s, no subjec ed o an ibio ic esidues de i ed om human This is an open access a icle unde he e ms o he C ea i e Commons A ibu ion-NonComme cial-NoDe i s License, which pe mi s use and dis ibu ion in any medium, p o ided he o iginal wo k is p ope ly ci ed, he use is non-comme cial and no modi ica ions o adap a ions a e made. © 2023 The Au ho s. BioEssays published by Wiley Pe iodicals LLC. usage, indica ing ha he esis ome is an in eg al pa o he mic obial ecology o he di e en en i onmen al compa men s. Due o he use o an ibio ics in li es ock, he e a e many s udies on he po en ial consequences o e iliza ion wi h animal manu e o he in oduc ion o ARB and ARGs, he la e in many cases associa ed wi h mobile gene ic elemen s (MGEs), in ag icul u al soils, wi h concomi an isks o human heal h ia he consump ion o c ops, especially esh ho icul u al p oduce. In app oxima ely he pas decade, as a esul o he ema kable de elopmen o ad anced sequencing echnologies and bioin o ma ics ools, mos o hese s udies on he impac o e iliza ion wi h animal manu e on he soil esis ome ( he esis ance de e minan s p esen in he soil) ha e been ocused on he use o nex -gene a ion sequencing (NGS), pa icula ly 16S RNA amplicon sequencing and me agenomics ( ia, e.g., sho gun sequencing wi h Illumina, Ox o d nanopo e o PacBio BioEssays. 2023;45:2300153. wileyonlinelib a y.com/jou nal/bies 1o 4 h ps://doi.o g/10.1002/bies.202300153 GARBISU and ALKORTA 2o 4 echnologies), o de e mine he p esence and abundance o ARGs and MGE- ela ed genes in soils, as well as hei po en ial ela ion- ship wi h bac e ial axonomic g oups (i.e., o es ablish possible links be ween unc ional and s uc u al- axonomic-phylogene ic in o ma- ion). Fo he de ec ion and quan i ica ion o hese unc ional genes, high- h oughpu qPCR has also been used ex ensi ely. Al hough he e is no doub ha molecula echnologies and NGS in pa icula ha e b ough g ea p og ess and no el insigh s o he soil mic obial ecology ield (mos o all, he disco e y o he ex emely high ichness o soil mic obial communi ies), p ima ily hanks o he pos- sibili y o o e coming he limi a ions o cul i a ion-based echniques, la ely, i is o en poin ed ou ha , because o he cu en gene-cen ic app oach, many o he ecen soil mic obial ecology s udies (e.g., soil esis ome s udies) a e o a desc ip i e, co ela ion-based, and/o pa e n-seeking na u e, and ha e seldom con ibu ed o signi ican ad ances in knowledge and unde s anding o he ecology o soil mic oo ganisms, no ha e subs an ially inc eased ou capaci y o p e- dic soil mic obial communi y composi ion, dynamics, and esilience, no un a elled he consequences o communi y composi ion o soil p ocess a es and ecosys em unc ioning.[2]Then, he ield o soil mic obial ecology in gene al, and he opic o he impac o he applica ion o animal manu e on he soil esis ome in pa icula , need o add ess key ecological ques ions and pe o m hypo hesis-based, mechanism-based s udies ha con ibu e o he ad ance o mic obial ecology heo y and applica ions. Al hough, by de ini ion, he mic obial “ecology” ield mus inco po- a e an ecological and ecocen ic app oach, in o de o a ac a en ion o a gi en ield o knowledge, i is o en e y use ul o poin ou i s po en ial links wi h human heal h and/o well-being om an an h o- pocen ic poin o iew. In his espec , gi en he magni ude and ele ance o he an ibio ic esis ance c isis, wi hou doub i can be s a ed ha he soil esis ome and i s po en ial links wi h he human esis ome is one o he soil opics o mo e in e es om an an h o- pocen ic pe spec i e. A e all, an ibio ics, an ibio ic esidues ha emain bioac i e, ARB, and ARGs a e no only en i onmen al eme ging con aminan s o abio ic and bio ic na u e, espec i ely, bu a e pu ing a isk one o he mos impo an co ne s one o mode n medicine, ha is, an ibio ics. On he o he hand, he ood web ( he complex ne wo k o in e con- nec ing and o e lapping ood chains in an ecosys em) is a key ecological concep o pa amoun impo ance o he unde s anding o ecosys em unc ioning. Food webs ep esen complex ecological ne wo ks whose opologies ha e been widely used in ecology o illus a e he s uc u al and unc ional p ope ies o biological communi ies and ecosys ems. Explici ly, ood webs a e ep esen a ions o species and hei eed- ing ela ionships. The nodes a e o en e med “ ophic le el species o ophospecies” and he links ha e lec he di ec ion o in e ac ions “ ophic in e ac ions” (e.g., who is he p ey and who is he p eda o ). Al hough a discussion on he as complexi y o he soil ood webs and he ongoing deba es on hei s uc u e and opology is no wi hin he scope o his commen a y ( o a highly simpli ied ep esen a ion o he soil ood web, see Figu e 1), om he pi o al wo k o Hun and colleagues,[3] hey a e o en ep esen ed as webs compa men- alised in o bac e ial-based and ungal-based ene gy channels (wi h some webs domina ed by bac e ia and o he s by ungi) uni ed a highe ophic le els occupied by p o is s-p o ozoa, nema odes, a h opods, ea hwo ms, insec s, small e eb a es, and so o h. When ARB ha bou ing ARGs en e he soil ecosys em du ing he applica ion o animal manu e as e ilize (an ag icul u al p ac ice being encou aged, due o i s many p o en bene i s, unde a a ie y o ag icul u al sys ems such as o ganic a ming, egene a i e ag icul- u e, ecological ag icul u e, and ag oecology), hey can be iewed as “in asi e species” whose ecological success will depend on a a ie y o abio ic and bio ic ac o s (e.g., hei ecological alence unde he speci ic soil physicochemical condi ions, hei ecological i ness and compe i i eness, hei capaci y o acclima ion and adap a ion, hei dispe sal abili ies, and so on). In his espec , in a s udy on he ba - ie s ha mic obial di e si y can p o ide agains mic obial in asion by ARB, soil mic obial di e si y was ound o be nega i ely co ela ed wi h he abundance o ARGs and, hen, he au ho s concluded ha a high mic obial di e si y in soil can ac as a biological ba ie agains he sp ead o an ibio ic esis ance.[4]Likewise, clinical class 1 in e- g ons, MGEs ha ha e been widely ecognized as majo con ibu o s o he e olu ion and dissemina ion o an ibio ic esis ance, ha e been epo ed as in asi e species[5]wi h a decisi e ole in he an ibio ic esis ance c isis. These in eg ons ha e in aded many bac e ial axa, hei hos s, and, c i ically, all en i onmen s associa ed wi h human ac i i y.[5] Ano he use ul concep ual app oach o he in es iga ion o he in oduc ion o ARB in soil ia animal manu e is he s udy o commu- ni y coalescence ( he mixing o di e en biological communi ies),[6]a p ocess wi h many ami ica ions o communi y s uc u e and unc ion, shaped, among many o he ac o s, by he ypes o in e ac ions and by a ious p ocesses o communi y assembly such as selec ion, d i , dispe sal, and di e si ica ion. When en e ing ag icul u al soil, among o he possibili ies, he ARB ini ially p esen in he manu e can (i) die owing o, o ins ance, lack o adap a ion o he new en i onmen al condi ions, poo ecological com- pe i i eness, p eda ion, and so o h; (ii) su i e and hen inhabi he inanima e componen o he soil ma ix, li ing as ee li ing cells in he soil po es o , mo e o en, wi hin bio ilms a ached o he soil agg e- ga es, in ac i e o do man o ms; and (iii) su i e a e being inges ed by, o associa ed wi h, membe s o o he axonomical g oups o he soil ood web; in his espec , in many cases, hey end up inhabi ing he gu mic obiome o species belonging o he soil meso auna o mac o auna. F om all o he abo e, in his commen a y a icle, we p opose ha , ega ding he soil esis ome, i is c i ical o complemen he my iad o gene-cen ic NGS s udies wi h ecology-cen ed s udies. In pa icula , among hese ecology-cen ed s udies, we p opose ha hose deal- ing wi h he dissemina ion o he ARB ini ially p esen in he animal manu e used o ag icul u al e iliza ion should ideally ollow o e ime all possible biological-ecological ou es o p opaga ion h ough- ou he soil ood web, by means o ARB iden i ica ion and s udy in soil ungi, p o ozoa, nema odes, a h opods, ea hwo ms, insec s and so o h. O cou se, his p oposal does no exclude he s udy and moni- o ing o he a e o hose same ARB in he soil ma ix i sel , ega ding hei ans o ma ion in o bac e ia ha will li e in he po es and, mo e o en, wi hin bio ilms a ached o soil agg ega es. 15211878, 2023, 12, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1002/bies.202300153 by Uni e sidad Del Pais Vasco, Wiley Online Lib a y on [26/12/2023]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License 3o 4 GARBISU and ALKORTA FIGURE 1 Simpli ied ep esen a ion o he soil ood web. The biomass o each le el dec eases because a conside able amoun o ene gy is los (e.g., dissipa ed as hea ) wi h each ans e as en opy inc eases. I is p oposed o ollow an ibio ic esis ance bac e ia h oughou he soil ood web. Fu he mo e, we p opose o pay special a en ion o (i) he possibil- i y o ho izon al gene ans e (HGT) among he di e en axonomical g oups o he soil ood web, as his phenomenon o la e al gene ans e does no only occu among bac e ia o be ween bac e ia and a chaea, bu also be ween p oka yo es and euka yo es; and (ii) he p esence o ARB in he gu mic obiomes o he soil meso auna and mac o auna. Fo example, HGT s udies can be pe o med in i o, using a ailable cul i a ion-dependen classical and/o high- h oughpu conjuga ion assays, wi h he isola ed en i onmen al ARB as dono and ecipien bac e ia (en i onmen al pe spec i e), o al e na i ely wi h he isola ed ARB as dono s and po en ial human pa hogens (e.g., Esche ichia coli)as ecipien s (an h opocen ic pe spec i e). Unlike many s udies ha a e ocused on he p esence o ARB and, abo e all, ARGs in he gu mic obiomes o speci ic axa o he soil meso- auna o mac o auna a a gi en poin in ime, we p opose o s udy o e ime he p opaga ion o ARB in all o , ailing ha , in as many as possi- ble axa o he soil ood web in he same soil, in an a emp o achie e a comp ehensi e, empo al, and dynamic ep esen a ion o he di e - en soil biological-ecological ou es ha ARB can ollow once hey ha e been in oduced in o ag icul u al soil. We a e ce ainly awa e o he ac ha his p oposal can p o e a somewha daun ing ask bu , a he same ime, we do eckon ha his ecological-ecocen ic app oach, as compa ed o he gene-cen ic app oach, has he po en ial o gene a e much needed mic obial ecol- ogy knowledge, as well as o b ing ou impo an scien i ic ques ions and hypo heses o he soil mic obial ecology ield in ela ion o (i) he links be ween he soil esis ome and he human esis ome, a opic o much alue and in e es om a One Heal h pe spec i e; and (ii) he su - i al and ecological implica ions o he in oduc ion and p opaga ion o ARB in ag icul u al soils, a subjec o much ele ance o he biodi e - si y, unc ioning, esis ance, and esilience o he soil ecosys em, whose heal h is c i ical o he p o ision o essen ial se ices and, in gene al, o he unc ioning o e es ial ecosys ems. On he posi i e side, i mus be s a ed ha new de elop- men s in molecula p obes, luo escen p obes, low cy ome y, ad anced mic oscopy (e.g., Raman mic o-spec oscopy, FT-IR mic o- spec oscopy), and o he analy ical echniques a e acili a ing he acking and moni o ing o bac e ia in soil. On he nega i e side, i mus be men ioned ha soil ood webs a e ex emely complex, o en wi h i e ophic le els, and no well unde s ood. P eda o s in soil ood webs appea o be gene alis s (e en wi h omni o ous eeding habi s), hus being able o inges o ganisms om a ious ophic le els, which means ha hey can acqui e bac e ia om o ganisms belonging o di e en ophic le els. Finally, a ew s udies ela ed o he opic unde conside a ion he e a e b ie ly p esen ed. Since bac e ia and ungi a e he dominan g oups o soil bio a in e ms o bo h abundance and di e si y, i is no su - p ising ha hey cons an ly in e ac , physically and me abolically, wi h each o he . Fungal hyphae p o ide a habi a o bac e ial communi- ies ha can o m bio ilms on hem (he e, i mus be emembe ed ha wi hin bio ilms, he HGT o ARGs is o en in ensi ied), bu , mos impo an ly, ungal hyphae se e as ec o s o bac e ia (e.g., ARB) o a el ac oss he soil, he so-called hyphal highways along which bac e ia can swim in he wa e ilm ha coa s he hyphae. Al e na- i ely, bac e ia can a el passi ely by se ling a he ip o he g owing hyphae.[7] 15211878, 2023, 12, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1002/bies.202300153 by Uni e sidad Del Pais Vasco, Wiley Online Lib a y on [26/12/2023]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License GARBISU and ALKORTA 4o 4 Pe aining o p o is s, al hough soil p o ozoa a e known o eed on bac e ia, many bac e ia engul ed in he esicles o p o ozoa ha e he capaci y o a oid being diges ed, hus opening up oppo uni ies o cell- o-cell con ac among bac e ia p esen in he esicles, esul - ing, o ins ance, in some p o ozoa enhancing he equency o ARG conjuga ion among hem.[8] Mo eo e , ARGs ha e been ound o be di e se and abundan in nema ode gu mic obiomes and, mos impo an ly, o ha e he capaci y o be ans e ed along belowg ound ood chains.[9] In ela ion o a h opods, i has been epo ed ha an ibio ic expo- su e can al e he gu mic obiome o he common soil collembolan Folsomia candida and signi ican ly inc ease he di e si y and abundance o ARGs in he collembolan gu .[10] In e es ingly, an ibio ic-p oducing S ep omyces (a genus o soil ac i- nobac e ia well-known o hei capaci y o p oduce an ibio ics and o he seconda y me aboli es used in human medicine) and o he ac i- nobac e ia can li e as symbion s o soil in e eb a es, whe e hey play a p o ec i e ole by p oducing an ibio ics ha a e used o de end he hos s agains bac e ial in ec ions.[11]These symbioses in which he hos eeds and p o ec s he ac inobac e ia and, in e u n, he ac i- nobac e ia p oduce an ibio ics o p o ec he hos om pa hogens a e mos in e es ing o he s udy o he sp ead and e olu ion o ARB, as S ep omyces cells ca y esis ance o hei own an ibio ics and, unde selec i e p essu e, hei ARGs can sp ead o o he bac e ia ia HGT.[11] Al hough he ole o ea hwo ms in he sp ead o an ibio ic esis- ance is s ill con o e sial, di e en s udies ha e shown ha manu e e iliza ion can inc ease he abundance o ARGs in ea hwo m gu mic obiomes,[12]a ac which mus in e p e ed aking in o close con- side a ion he ole o ea hwo ms as ecosys em enginee s ha can in luence he mo emen o o he soil o ganisms, including bac e ia. To inish, we do hope his commen a y p omo es he de elopmen o mo e ecologically ele an s udies on soil mic obial ecology and, in pa icula on he opic o an ibio ic esis ance in soil. AUTHOR CONTRIBUTIONS I zia Alko a: Concep ualiza ion, w i ing – o iginal d a , w i ing – e iew and edi ing; Ca los Ga bisu: Concep ualiza ion, w i ing – o ig- inal d a , w i ing – e iew and edi ing. ACKNOWLEDGEMENTS This wo k was suppo ed by MCIN/AEI/10.13039/501100011033 (PID2020-116495RB-I00), Basque Go e nmen (IT1578-22), and Euskampus – JRL En i onmen al An ibio ic Resis ance. CONFLICT OF INTEREST STATEMENT The au ho s decla e no con lic o in e es . DATA AVAILABILITY STATEMENT Da a sha ing is no applicable o his a icle as no da ase s we e gene a ed o analysed du ing he cu en s udy. ORCID Ca los Ga bisu h ps://o cid.o g/0000-0002-5577-6151 I zia Alko a h ps://o cid.o g/0000-0001-6978-389X REFERENCES 1. Rome o, D., T axle , M. F., López, D., & Kol e , R. (2011). An ibio ics as signal molecules. 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Fa es o an ibio ic esis ance genes in he gu mic obiome om di e en soil auna unde long- e m e iliza ion. En i onmen al Science and Technology,55, 423–432. h ps://doi.o g/10. 1021/acs.es .0c03893 10. Zhu, D., An, X.-L., Chen, Q.-L., Yang, X.-R., Ch is ie, P., Ke, X., Wu, L.-H., & Zhu, Y.-G. (2018). An ibio ics dis u b he mic obiome and inc ease he incidence o esis ance genes in he gu o a common soil collembolan. En i onmen al Science and Technology,52, 3081–3090. h ps://doi.o g/ 10.1021/acs.es .7b04292 11. Seipke, R. F., Kal enpo h, M., & Hu chings, M. I. (2012). S ep omyces as symbion s: An eme ging and widesp ead heme? FEMS Mic obiol- ogy Re iews,36, 862–876. h ps://doi.o g/10.1111/j.1574-6976.2011. 00313.x 12. Zhou, S., Zhu, D., Giles, M., Daniell, T., Neilson, R., & Yang, X. (2020). Does educed usage o an ibio ics in li es ock p oduc ion mi iga e he sp ead o an ibio ic esis ance in soil, ea hwo m gu s, and he phyl- losphe e? En i onmen al In e na ional,136, 105359. h ps://doi.o g/10. 1016/j.en in .2019.105359 How o ci e his a icle: Ga bisu, C., & Alko a, I. (2023). A case o he impo ance o ollowing an ibio ic esis an bac e ia h oughou he soil ood web. BioEssays,45, e2300153. h ps://doi.o g/10.1002/bies.202300153 15211878, 2023, 12, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1002/bies.202300153 by Uni e sidad Del Pais Vasco, Wiley Online Lib a y on [26/12/2023]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License