A membrane computing simulator of trans-hierarchical antibiotic resistance evolution dynamics in nested ecological compartments (ARES)

RESEARCH Open Access A memb ane compu ing simula o o ans-hie a chical an ibio ic esis ance e olu ion dynamics in nes ed ecological compa men s (ARES) Ma celino Campos 1,3 , Ca los Llo ens 2* , José M. Sempe e 3 , Rica do Fu ami 2 , I ene Rod iguez 1,4,5 , Pu i icación Ca asco 6 , Ra ael Capilla 2 , Ampa o La o e 5,6,7 , Te esa M. Coque 1,4,5 , And es Moya 5,6,7 and Fe nando Baque o 1,4,5* Abs ac Backg ound: An ibio ic esis ance is a majo biomedical p oblem upon which public heal h sys ems demand solu ions o cons ue he dynamics and epidemiological isk o esis an bac e ia in an h opogenically-al e ed en i onmen s. The implemen a ion o compu able models wi h ecip oci y wi hin and be ween le els o biological o ganiza ion (i.e. essen ial nes ing) is cen al o s udying an ibio ic esis ances. An ibio ic esis ance is no jus he esul o an ibio ic-d i en selec ion bu mo e p ope ly he consequence o a complex hie a chy o p ocesses shaping he ecology and e olu ion o he dis inc subcellula , cellula and sup a-cellula ehicles in ol ed in he dissemina ion o esis ance genes. Such a complex backg ound mo i a ed us o explo e he P-sys em s anda ds o memb ane compu ing an inno a i e na u al compu ing o malism ha abs ac s he no ion o mo emen ac oss memb anes o simula e an ibio ic esis ance e olu ion p ocesses ac oss nes ed le els o mic o- and mac o- en i onmen al o ganiza ion in a gi en ecosys em. Resul s: In his a icle, we in oduce ARES (An ibio ic Resis ance E olu ion Simula o ) a so wa e de ice ha simula es P-sys em model scena ios wi h i e ypes o nes ed compu ing memb anes o ien ed o emula e a hie a chy o eco-biological compa men s, i.e. a) pe iphe al ecosys em; b) local en i onmen ; c) ese oi o supplies; d) animal hos ; and e) hos ’s associa ed bac e ial o ganisms (mic obiome). Compu a ional objec s emula ing molecula en i ies such as plasmids, an ibio ic esis ance genes, an imic obials, and/o o he subs ances can be in oduced in o his amewo k and may in e ac and e ol e oge he wi h he memb anes, acco ding o a se o p e-es ablished ules and speci ica ions. ARES has been implemen ed as an online se e and o e s addi ional ools o s o age and model edi ing and downs eam analysis. (Con inued on nex page) * Co espondence: [email p o ec ed];[email p o ec ed] 2 Bio ech ana, Valencia, CEEI Building, Benjamin F anklin A . 12, Valencia Technological Pa k, 46980 Pa e na, Spain 1 Depa men o Mic obiology, Ramón y Cajal Uni e si y Hospi al, IRYCIS, Ca e e a de Colmena Viejo, km. 9,100, 28034 Mad id, Spain Full lis o au ho in o ma ion is a ailable a he end o he a icle © 2015 Campos e al. Open Access This a icle is dis ibu ed unde he e ms o he C ea i e Commons A ibu ion 4.0 In e na ional License (h p://c ea i ecommons.o g/licenses/by/4.0), which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided you gi e app op ia e c edi o he o iginal au ho (s) and he sou ce, p o ide a link o he C ea i e Commons license, and indica e i changes we e made. The C ea i e Commons Public Domain Dedica ion wai e (h p://c ea i ecommons.o g/publicdomain/ze o/1.0/) applies o he da a made a ailable in his a icle, unless o he wise s a ed. Campos e al. Biology Di ec (2015) 10:41 DOI 10.1186/s13062-015-0070-9 (Con inued om p e ious page) Conclusions: The s ochas ic na u e o he P-sys em model implemen ed in ARES explici ly links wi hin and be ween hos dynamics in o a simula ion, wi h eedback ecip oci y among he di e en uni s o selec ion in luenced by an ibio ic exposu e a a ious ecological le els. ARES o e s he possibili y o modeling p edic i e mul ile el scena ios o an ibio ic esis ance e olu ion ha can be in e oga ed, edi ed and e-simula ed i necessa y, wi h di e en pa ame e s, un il a co ec model desc ip ion o he p ocess in he eal wo ld is con incingly app oached. ARES can be accessed a h p://gydb.o g/a es. Re iewe s: This a icle was e iewed by Eugene V. Koonin, and E ic Bap es e. Keywo ds: Memb ane compu ing, P-sys em, An ibio ic esis ance, Essen ial nes ing Backg ound An ibio ic esis ance (AR) is a se ious biomedical p oblem upon which public heal h sys ems u ge o new R&D s a egies o p e en he eme gence and dissemina ion o esis an bac e ia in human inhabi ed en i onmen s [1–6]. The disco e y and clinical use o an imic obials du ing he pas 20 h cen u y has changed he cou se o medicine and he human li es yle by educing mo ali y om bac- e ial in ec ions. Howe e , he use o misuse o an ibio ics, medicines and d ugs may lead bac e ia o become ole an o he ac ion o an imic obials, e en ually making s and- a d ea men s ine ec i e and hus challenging key med- ical p ac ices as in ensi e ca e medicine, ansplan a ion, o he he apy o immuno-comp omised pa ien s [2, 5, 7]. AR is also a subjec o pa icula in e es o ood-chain s akeholde s who use an imic obials as g ow h-p omo ing addi i es un il ecen ly o o p e en ing c oss-in ec ion in ood p oduc ion a eas [7–12]. Unde an ibio ic exposu e, a ms ha e become signi ican ese oi s o an ibio ic e- sis an mic oo ganisms, which in u n can be ansmi ed om ood-p oducing animals and plan s o humans [7–13]. The ma e is o unde s and he isks and he unin ended consequences o he an h opogenic use and elease o an imic obial agen s in o he biosphe e, which wi hou ap- p op ia e measu es migh d i e he wo ld owa ds a pos - an ibio ic e a whe e mo ali y due o un ea able and a al in ec ions may become cus oma y, pa icula ly in unde - de eloped a eas o he wo ld [3–5, 12, 14]. Unde s anding AR e olu ion is howe e a complex issue, since i is no jus he esul o an ibio ic-d i en selec ion o mu an e- sis an bac e ial clones as i is equen ly conside ed, bu mo e p ope ly he consequence o a a ie y o ans- hie a chical in e ac ions be ween all biological ehicles in ol ed in he dissemina ion o he gene ic in o ma ion in ol ing AR [15–18]. We a e alking abou he ollowing biological en i ies: gene ic pla o ms, ansposons and/o plasmids (he e called subcellula eplica o s), bac e ial cells, gene ic exchange communi ies (GEC, communi ies in which he in e change o gene ic ma e ial occu s e- quen ly), mic obio a, hos indi iduals and hos communi- ies. In o he wo ds, no only AR genes can be ca ie s o AR gene ic in o ma ion, bu also all o he biological uni s in which AR genes can be successi ely loca ed a di e en subcellula , cellula and sup a-cellula nes ed le els o he ecosys em [19–21]. I is impo an o no e he need o un- de s anding he e ec o he whole nes ed ames associ- a ed o AR o es ablish a comp ehensi e “pa ame e space”able o desc ibe he mul idimensional e olu ion o an ibio ic esis ance (Table 1, o mo e de ails see [22]). All he a o esaid ca ie s a e uni s o selec ion ha can be simul aneously and independen ly chosen a di e en en- i onmen al le els including “in i onmen al”o mic obio- ic ecosys ems [23]. The e o e, we should expec a high complexi y in be ween-hos s demog aphical dynamics, as he hos colonized o in ec ed by esis an bac e ia usually Table 1 Key-nes ed ames associa ed o AR: a complex pa ame e space a) Densi y o colonized and colonizable hos s wi h an ibio ic esis an bac e ia b) Popula ion sizes o bac e ia pe hos du ing coloniza ion and in ec ion c) Suscep ibili y o coloniza ion o hos s, including age, gende , e hnici y, nu i ion,illness- acili a ed coloniza ion d) F equency o be ween-hos s in e ac ions i.e. ,human- o-human o animal-human in e ac ions e) Hos na u al and acqui ed immune esponse o colonizing o ganisms ) Ecological pa ame e s o colonizable a eas, including in e ac ion wi h local mic obio a and equency and ype o an ibio ic- esis an commensals g) Mig a ion and dispe sal h) An ibio ic and biocide exposu e and o e all densi y o an ibio ic use, ype o an ibio ics and mode o ac ion, dosage and du a ion o he apy, adhe ence o he apy, selec i e an ibio ic concen a ions, an ibio ic combina ions i) Mode o ansmission o esis an o ganisms om he en i onmen o hos s j) T ansmission a es be ween hos s (an ibio ic ea ed and no - ea ed, in ec ed, and no -in ec ed) k) Time o con ac be ween hos s l) Hygiene, in ec ion con ol, sani a ion m) Food, and d inking wa e con amina ion by esis an bac e ia and hos exposu e n) En i onmen al con amina ion by esis an o ganisms, including soil, sewage and wa e Campos e al. Biology Di ec (2015) 10:41 Page 2 o 13 belongs o a popula ion o in e ac ing indi iduals ha in u n belong o a communi y o in e ac ing bac e ial spe- cies. No e, o ins ance, ha lice and keds o pe s and a m animals (Melophagus o inus, Linogna hus i uli, He e o- doxus spinige ) o pes -insec s such as he house ly (Musca domes ica) and he Ge man cock oach Bla ella ge manica, which a e known o play a signi ican ole as ese oi s and ec o s o oppo unis ic bac e ial pa hogens ha a e o en esis an o an ibio ics [24–26] as hey mo e, eely and indisc imina ely, om il h and animal was e o ood, allowing esis an bac e ia o explo e new habi a s in hospi als, communi y se ings and ood acil- i ies [27–29]. This ype o complex scena ios, whe e bac- e ial esis an popula ions and hei gene ic pla o ms con aining AR genes mo e because hey a e selec ed, and e ol e because o hei p omiscuous mig a ion, es ablish he p econdi ion o isualize AR no only as a ma e o biological unc ion, bu also as in o ma ion low p ocess- ing. Fo hese easons, AR e olu ion is wi hou doub one o he majo biomedical challenges o esea che s in epi- demiology and sys ems biology. In e es ingly, om he epis emological in e ac ion o sys em biology, compu e science and ma hema ics, a a ie y o models ha e a isen in he las decades o con- nec pe o mances a di e en scales. These models a e known as nes ed o embedded models ( o a e iew see [30]) and ha e been used o accep ably add ess speci ic ques ions in ol ing wi hin-hos dynamics enclosed in a model o be ween-hos epidemiological scena ios. Nes ed models a e classi ied as “inessen ial”when he wi hin-hos dynamic in luences be ween-hos p ocesses bu no ice e sa,o “essen ial”when he e is a ecip ocal eedback be ween le els o o ganiza ion. In pa icula , AR modeling equi es an essen ial nes ed model; any al e a ion o he ca ie s in any speci ic esis ance ai , o in hei mecha- nisms o a ia ion and mobiliza ion (mu a ion, ecombin- a ion, ansposi ion, ho izon al gene ans e , mig a ion) may in luence he dynamics o o he uni s o highe and lowe hie a chy, ha ing logical consequences on he e- quency and dissemina ion o AR genes and, he e o e, e olu iona y and/o ecological consequences on bac e ial popula ion [31, 32]. Un il no oo long ago, he di icul y o model his ype o scena ios wi h essen ial nes ing was an impo an limi a ion o easibly s udy AR e olu ion p ocesses. Howe e , exci ing new oppo uni ies ha e e- cen ly a isen om a na u al compu ing o malism inspi ed on he s uc u e and unc ioning o biological cells, called memb ane compu ing [33–35]. Memb ane compu ing concei es any biological sys em as a hie a chical cons uc whe e he low o ma e ials can be in e p e ed as compu - ing p ocesses. In pa icula , memb ane compu ing o e s a e sa ile amewo k known as P-sys em ha consis s o a hie a chical memb ane s uc u e o nes ed compa men s whe e mul ise s o objec s a e loca ed and can mo e ac oss memb anes e ol ing acco ding o a ini e numbe o gi en ules. Memb ane compu ing ha e been p o ed o be uni e sal models o compu a ion [34] and has been success ully used o model oscilla o y sys ems [36], p o- cesses o signal ansduc ion [37, 38], gene egula ion con- ol [39], quo um sensing [40], me a-popula ions [41] and ecosys ems [42, 43] hus sugges ing ha any hing ha can be compu ed can be done so as a P-sys em. Fo mo e de- ails on he di e en app oaches eached unde memb ane compu ing, see [44], o e e o he o icial websi es o he memb ane compu ing communi y [45] and P–Lingua [46] he p og amming language used o he de elopmen o P-sys ems. In his pape we in oduce a new P-sys em model de- signed o compu ing a h ee le els o o ganiza ion (subcellula , cellula , sup a-cellula ) h ough he so wa e implemen a ion o a simula o we call An ibio ic Resis - ance E olu ion Simula o (ARES). The gene al aim o ARES is o acili a e p edic i e compu a ional models on he po en ial ans-hie a chical esponse o AR o pa - icula in e en ions in speci ic scena ios. The simula- o ´s p ojec is a wo k in p og ess, equi ing cons an e inemen s de i ed om he expe iences (“expe i- men s”) o cos ume s. The i s e sion he e in oduced, is a p o o ype ha o e s a p ede ined layou composed o i e ypes o nes ed-memb anes ha concep ually emula e an ecosys em hie a chy o biological bounda ies based on popula ion en i onmen al a eas, ese oi s, hos popula ions and bac e ial lineages o oppo unis ic pa hogens. G an ed o he implemen a ion a iendly- o- use on -end in e ace, he use is allowed o de ine a s a ing con igu a ion o elemen s (subcellula ehicles, an imic obials and o he subs ances) inhabi ing he a o esaid memb anes, speci ica ions and ules acco ding o which bo h elemen s and memb anes e ol e h ough a numbe o i e a ions. ARES is hos ed a he GyDB P ojec [47] a da abase o esea ch o mobile gene ic elemen s ( ele an ca ie s in he s udy o AR), and has been launched as an online se e accessible a h p://gydb.o g/a es. Me hods P-sys em model o simula ing ecosys ems wi h nes ed ecological bounda ies AR is a p ocess o mul ile el selec ion o nes ed uni s whe e he dis inc esis ance-ca ie s (gene ehicles) in- luence each o he o selec ion and in og essi e c oss- ing o esis ance o an ibio ics a di e en en i onmen al le els (subcellula , cellula and sup acellula ) [18, 20, 48]. This is an eco-biological model ha can be o mally gen- e alized acco ding o he ollowing uple: Y¼V;μ;w1;w2;…;wn;R1;ρ1 ðÞ;…Rn;ρn  Campos e al. Biology Di ec (2015) 10:41 Page 3 o 13 whe e Vis a wo king alphabe o objec s; μis a memb ane s uc u e consis ing o nmemb anes labeled 1, 2, . . . , n ep esen s a oo ed ee; and w 1 ,...,w n a e s ings o e V ha ep esen mul ise s o objec s ini ially placed in he s uc u e o nmemb anes, which, om ha poin on will be e e ed as ecological bounda ies (EBs) h ough he es o his a icle. In he Valphabe , ou model akes in o accoun he ollowing en i ies o be ea ed as objec s: Bac e ial esis ance genes compu ed ei he as an independen uni o cons i u ing a combina ion oge he wi h a pa icula subcellula eplica o ( o ins ance a plamid). He e, we use he se o symbols AR i o desc ibe genes encoding AR, whe e ideno es he objec ’s iden i y. Should an AR gene is designa ed in i s single o m (AR-like) i will be conside ed as a genomic gene (i.e. p esen as a locus wi hin he bac e ial hos genome) by he model, bu i i is a ached o a pa icula subcellula eplica o hen i will be conside ed o be pa o he subcellula eplica o (i.e. ca ied by he subcellula eplica o ). Subcellula eplica o s inside bac e ial cells such as plasmids, in eg a i e-conjuga i e elemen s (ICE), ansposons, o any o he gene ic elemen wi h sel - eplica ion abili y. Fo he sake o simplici y, in his i s e sion we only conside plasmid-like objec s, which ou model compu es wi h he se o symbols PL i . As p e iously indica ed abo e, he model pe mi s simula ion o plasmids ca ying AR genes by in oducing a egula exp ession ha de ine complex objec s as ollows: le us o conside kdi e en plasmids and jdi e en AR genes, hen a complex objec o he combina ion o plasmids wi h genes belongs o he ollowing exp ession (wi h λbeing an emp y s ing in he absence o a name): (PL i +…+ PL k )(AR i +λ)…(AR j +λ). Fo example he s ings “PL 1 ”,“PL 1 -AR 1 ”,“PL 1 -AR 2 ”, and “PL 1 -AR 1 -AR 2 ” espec i ely co espond o objec s ep esen ing ou di e en o ms o he same plasmid –“no ca ying AR genes”,“ca ying gene AR 1 ”,“ca ying gene AR 2 ” and “ca ying bo h AR genes”. Fo compu a ional sake, he cu en e sion o ou P-sys em model pe mi s only simula ion o plasmids ca ying up o wo di e en AR genes. Ex e nal chemicals and/o bioma e ials eleased in o he en i onmen , including any kind o molecule inhibi ing bac e ial g ow h such as an ibio ics and biocides. We use he se o symbols A i o desc ibe hese objec s. Managemen Clocks a e objec s labeled wi h G i symbols used o pe iodically add objec s o speci ic memb anes acco ding o ecosys em in luences expec ed o be cyclic. In he memb ane s uc u e μ he model implemen s i e hie a chical EBs labeled as ECO,P i ,RS i ,H i and B i ECO is he skin EB ep esen ing he pe iphe al ecosys em o ul ima e con aine o all popula ions and en i onmen s. P i is he second EB ype in he memb ane amewo k le el ha designa es simula ion o pa icula en i onmen al a eas o he ecosys em o he sp ead o hos s and esis an bac e ia and e en ually o he bac e ial pa hogens (inhabi able spaces, a eas o ood acquisi ion, and o he e en ual in as uc u es). H i is a ype o EB on he hi d le el o nes ing wi hin he memb ane amewo k hie a chy. We use H-like EBs o de ine hos indi iduals ca ying mic obio a (hos -speci ic assemblies o bac e ial mic oo ganisms, de ined by i s mic obio a composi ion). RS i is ano he ype o EB, also on he hi d amewo k le el, ha he model uses in o de o abs ac (when sui able) he compu a ion o h ee di e en ypes o physical o concep ual ese oi s. Two o hese a e called Food and Wa e supplies and a e used du ing he simula ion as ese oi -EBs o hese esou ces. I is wo h o no e ha o compu a ional sake ood and wa e a e no ea ed as objec s bu as quan i a i e in e nal esou ces o he P-sys em being necessa y o hos s´ li e ha mus he e o e be pe iodically gene a ed by managemen clocks and consumed by he simula ed hos s acco ding o he a es s a ed by he use s in he con igu a ion o ules. Once he hos popula ion g ow h ou paces he a ailabili y o ood and wa e he model ac i a es an in e nal mal husian ule ha andomly kills (elimina es om he simula ion) a numbe o hos s equi alen o he popula ion su plus. The hi d RS-like EB is called “Sewage”and i e e s o any body o wa e con eying all wa e - ca ied was e (ei he na u al o an h opogenic) being emo ed om a communi y. Sewage can also be used o simula e he s ool emains (o ecal d oppings) pe iodically eleased by H-like indi iduals o he en i onmen . The h ee RS-like ypes o EBs a e ep esen ed only once wi hin each (P-like) en i onmen and al hough hey a e p o ide s o wa e and ood hey may also con ain subs ances and mic obial con amina ion eleased h ough he d oppings o animal hos s and he con e sion o dead animals in o ood. In o he wo ds, he use o ese oi s allows he use o simula e supplies o ood and wa e bu also ecycling o mic obio a eleased in o he ecosys em by animal hos s (H-like memb anes) du ing he inal ac o diges ion o o Campos e al. Biology Di ec (2015) 10:41 Page 4 o 13 u n in o ood any o ganism ha dies o is p eda ed by o he o ganisms. B ij ± is he las EB le el o he hie a chy con empla ed in ou model and i is used o simula e bac e ial cells. Each cell has se e al a ibu es he e de ined as ollows: he supe sc ip “plus/minus”is used o indica e i he cell is g am-nega i e o g am posi i e; he subsc ip iis used o ep esen cell popula ions as lineages e m he e used o highligh ha he use can design he simula ion o a mic obiome acco ding o a common his o ical o sp ing o cells a any axonomical le el (a lineage can he e o e e e o a domain, phylum, class, o de , amily, genus, species and clones, depending on he expe imen ); jis used o assign wo o mo e cells o a pa icula o unique communi y o cells o GEC. Fo ins ance, wi hin a mic obiome, he use can de ine a subse o cells o belong o GEC 1 and ano he subse o belong o GEC 2 using he subsc ip di e en ia ion. The dynamics, speci ici y and beha io o he dis inc memb anes and objec s du ing he P-sys em simula ion a e adminis a ed by a ini e se o ules (R) ixed o each memb ane ha can be anked by o de o p io - i ies (ρ). Pa icula ly, ou P-sys em model conside s ules o he p ocesses o ansi ion, in e ac ions and so- cial beha io , bi h, dea h, inac i a ion, and e olu ion o he dis inc objec s and memb anes. The model also conside s ules called speci ica ions o ecosys em e- sou ce limi a ions in space and ime (as limi s in space, o li e expec ancy). Following a e some examples o ules desc ibed in he o mal de ini ion ollowed by he memb ane compu ing communi y, and whe e o he sake o simplici y, we omi p io i ies and s ochas ic pa ame e s. Example 1: ansi ion ules wi h mo emen o hie - a chically adjacen egions: “Subs ance Ai en e s bac e ial cells”o “Subs ance Ai lea es bac e ial cells”. ½ui½ jk→½ui0 ½ jkinsideðÞ ½ui ½ jk→½ui0½ jkou sideðÞ Example 2: ansi ion ule wi h mo emen o win adjacen egions: “A bac e ial cell ansmi s a plasmid o o he membe s o he same GEC”. ui ½ j½ i→½ jui0 ½ i Example 3: ac i e memb ane ules wi h mo emen o win adjacen egions: “An indi idual hos ansmi s bac e ia o ano he indi idual”. ½½  kj½ j→½ j½½ ki He e, egion kis mo ed om egion j o egion i, ha is a he same le el. Then, egions inside egion kcan be mo ed acco ding o hei co esponding ules. Example 4: ac i e memb ane ules o memb ane di ision: “G ow h o bac e ial cells j in egion i.” ½½  ji→½½ j½ ji He e, he con en o egion jis copied oge he wi h i s ules and all he memb anes i con ains in a hie a ch- ical manne . In summa y, ou P-sys em consis s o a memb ane s uc u e composed o i e ypes o EBs and a wo king alphabe Vo objec s whose in e ac i e eedback is de- e mined by he se o ules assigned a e e y EB. This amewo k can be g aphically ep esen ed as a Venn dia- g am (Fig. 1). As shown in he igu e, he con aine dia- g am (ECO) ep esen s he skin EB. Wi hin ECO, he wo nex diag ams designa ed as –P i and P j – ep esen wo P-like en i onmen al EBs ( he use can howe e de- sign as many P-like EBs as equi ed). P-like EBs a e allowed o con ain RS-like, H-like and B-like EBs ( ep e- sen ed as diag ams o smalle size). RS-like EBs (desig- na ed as i, j, k) ep esen ood, wa e and sewage ese oi s and a e allowed o con ain B-like memb anes (bu no H-like memb anes). H-like EBs can be dis in- guished in sub ypes (social classes, species, e c.) using subsc ip assigna ions. Fo example, in he igu e we con empla e 3 popula ions (i, j, k) ha may be espec - i ely composed o a numbe o indi iduals ( o ins ance 100, 50 and 150, e c.). Each H-like EB is allowed o con- ain a numbe o in e nal B-like EBs (bu no RS-like EBs) de ining i s in insic mic obio a. B-like EBs can be placed no only wi hin RS-like and H-like EBs bu also in P-like EBs and can be di e en ia ed in lineages o which g am and GEC s a us can be assigned using sub- and supe sc ip s. The s a us o G am posi i e o G am- nega i e o ganisms is assigned using a supe sc ip wi h wo s a es (minus and plus). In he igu e we obse e ou sub ypes (i _ ,j,k,l) acco ding o he le subsc ip . Those labeled wi h he le subsc ip jbelong he GEC-j and hose labeled wi h he subsc ip kbelong o GEC-k. Those ha ing he supe sc ip plus a e conside ed o be g am-posi i e cells, and hose assigned he supe sc ip minus a e g am-nega i e. Logically, popula ion size can also be assigned o each lineage ( o example 10 9 cells pe bac e ial lineage). The wo king alphabe is composed o ou ypes o objec s (also di e en ia ed in sub ypes) summa ized below he Venn diag am. In pa icula he Campos e al. Biology Di ec (2015) 10:41 Page 5 o 13 igu e shows wo AR-like objec s (i, j) de ining wo di - e en AR genes; ou A-like objec s (i, j, k, l) de ining ou dis inc subs ances ( o ins ance wo an ibio ics and wo insec icides wi h di e en p ope ies); eigh PL-like objec s ep esen ing wo plasmids (i, j) each one wi h ou possible s a es (wi hou AR genes, ca ying an AR i gene, an AR j gene o ca ying bo h AR genes); and ou G-like objec s (i, j, k, l) ep esen ing managemen clocks. AR-like and PL-like objec s a e es ic ed o B-like EBs bu hey can mo e om a B-like EB in o ano he ( o emula e ho izon al ans e e en s). No e, howe e , ha AR objec s can be only ans e ed when ca ied by a Fig. 1 P-sys em model o AR e olu ion in complex ecosys ems. Venn diag am ep esen a ion showing o he amewo k o memb anes and ocabula y o objec s, on which ou P-sys em model is based; memb anes a e illus a ed as nes ed diag ams labeled a bo om acco ding o he model´s code o symbols we use o e e ing memb anes; objec s a e also ep esen ed using symbols summa ized below he igu e; and ules assigned o each memb ane a ea a e, o simplici y´s sake, indica ed as ex indica ions colo ed g een Campos e al. Biology Di ec (2015) 10:41 Page 6 o 13 plasmid objec . A-like and G-like objec s a e allowed in all EBs (excep ing ECO which is he ul ima e con aine ) as hey ei he de ine subs ances expec ed o sp ead ac oss all en i onmen s o pe iodical ac ions (in he case o G-like managemen clocks) s a ed by he use . Finally, e e y EB is assigned a se o speci ic ules (R) designed and uned wi h he aim o go e n he dynamic o in e ac ions and e olu- iona y e en s wi hin each EB acco ding o gi en p io i ies (ρ), pa ame e s and condi ions indica ed by he use . Resul s and discussion In oducing ARES: simula o de ice co e and se e implemen a ion The P-sys em model p e iously de ined in Me hods was implemen ed as a simula o so wa e, which was p o- g ammed using he Ja a objec -o ien ed compu e p o- g amming language [49] and P-lingua ounda ions [50]. In pa icula , he simula o eads an xml ile ha con- ains he s a ing con igu a ion o a P-sys em and un simula ion o he P-sys em case s udy con igu ed du ing a use -de ined numbe o in e ac ions con empla ing ou s eps pe i e a ion; 1) s ep o e olu ion whe e all ules o e olu ion and in e ac ion a e applied; 2) s ep o mo emen whe e all ules o mo emen apply; 3) s ep o g ow h whe e all memb anes allowed o di ide do so. A e each s ep, an upda ing p e-s ep p epa es he model o he nex one. The ules applying o each egion ac simul aneously, in a pa allel way, o all he objec s and egions, and he s ochas ic beha io o he sys em is achie ed by applying he ules acco ding o hei s o- chas ic pa ame e s in a nai e p obabilis ic manne . Rules apply i : a) he objec s in need o he ule applica ion a e in he egion and b) he e is no ule wi h highe p io i y ha uses common objec s. The ansi ion om one con- igu a ion o he nex is ca ied ou by applying all he ules a e e y egion in a non-de e minis ic maximal pa allel mode; he sys em is always unning om one con igu a ion o he nex and only hal s i no ule can be applied. Hence, he hal ing con igu a ion con ains he ou pu o he sys em and he inal con igu a ion. In addi ion, s ochas ic pa ame e s o model he popula ion dynamics o he sys em a e also in oduced in he ules. The simula o has been ins alled wi hin an engine co e wi hin a 4× 6 Co e Se e wi h Linux OS and 128 GB o RAM, and has been coupled wi h he ollowing sub- sys ems; 1) a MySQL Managemen Sys em o s o age o P -sys em con igu a ions; 2) an engine o ou pu con e - sion o CSV o ma ; 3) he ou pu ´s a chi e, which is an eposi o y o ou pu olde s; 4) a se e sec ion o upload aining u o ials; 5) a collec ion o sc ip s o s a is ical analysis de eloped using he R p og amming language [51]; 6) A on -end in e ace layou o manage all o he sub-sys ems, p og ammed in a PHP amewo k on La a el 4 ollowing he Model–View–Con olle pa e n an a chi ec u al model whe e se e in e aces a e in e - changeable [52]. This In as uc u e is wha we call ARES. Managing ARES Use s can simula e dynamics o AR e olu ion using ARES o design a P-sys em model scena io adap ed o he case s udy speci ied by he use and hen un simula- ion o his scena io as many imes as necessa y co ec - ing pa ame e s un il a ealis ic desc ip ion o he AR p ocess is app oxima ed o alida ed i eal wo ld obse - a ions a e a ailable. Use o ARES is ee, bu i s acces- sion is passwo d p o ec ed in o de o allow he use s o open and main ain a use accoun ha will needed o s o e and un model p ojec s in p i a e session (a simu- la ion may las hou s o e en days depending on he complexi y o a P-sys em scena io). ARES is managed ia an easy- o-use in e ace ha implemen s a cen al- ized menu (Fig. 2a) o accessing he sys em o o ms he use need o sequen ially comple e in o de o in oduce he s a ing con igu a ion o a P-sys em scena io, un a simula ion, and access he esul s. All menu- o ms access- ible wi h his menu can be na iga ed back and o h o edi ing he P-sys em con igu a ion, change o add EBs, objec s and ules whe e o when necessa y. A scheme o he whole ARES in as uc u e and he wo k low o con igu a ion and simula ion o P-sys em scena ios is depic ed on Fig. 2b. The usual p ocedu e can be syn he- ized in he ollowing s eps. The o m designa ed as “ECO”mus be i s accessed ( ia menu) and comple ed o c ea e he P basal skin EB; hen “ENVIRONMENTS”has o be accessed o con igu e as many P-like (en i onmen- al) EBs as needed wi hin ECO; nex , “RESERVOIRS”and “HOSTS”mus be illed o con igu e RS-like ( ese oi s) and H-like (hos s) EBs wi hin he p e iously c ea ed P EBs; a e his, “MICROBIOMES”mus be used o con ig- u e a se ies o B-like (bac e ial) EBs ha can be ei he placed wi hin he p e iously c ea ed P-, RS- and H-like EBs; hen “OBJECTS”mus be used o c ea e as many as PL- (plasmids), AR- (AR genes), A-like (an ibio ics and/ o o he subs ances), and G-like (clocks) objec s as e- qui ed wi hin he p e iously c ea ed EBs (excep o ECO, since i is he skin memb ane); inally, he o ms “SPECIFICATIONS”and “RULES”mus be comple ed o s a e he ules assigned o each EB by selec ing hem om a lis o p e-designed ules p o ided in an unde - s andable and gene alized way allowing he use o choose and une ules wi h he alues and he pa ame e s needed o app oxima e he equencies, beha io s, condi- ions and p io i ies ha go e n he dynamic o in e ac- ions among he di e en memb anes and objec s o he P-sys em model o be simula ed. Once he P-sys em s a ing con igu a ion has been de- ined, i is au oma ically w i en o an xml ile (which is he inpu in o he simula o ), which is s o ed in ARES. Campos e al. Biology Di ec (2015) 10:41 Page 7 o 13 Fig. 2 ARES in e ace and se e o ganiza ion. aSc eensho o he ARES in e ace. The in e ace implemen s a menu ha gi es access o he dis inc se e o ms ha apply o con igu a ion, s o age and simula ion o P-sys em model scena ios. A he bo om o he in e ace he use can access o he suppo sec ions o managing ARES o o s a is ical in e oga ion o he ou pu gene a ed by he simula o de ice. bARES se e scheme and wo k low o c ea ion, edi ion and simula ion o P-sys em model scena ios Campos e al. Biology Di ec (2015) 10:41 Page 8 o 13 The o m “RUN”is an in e ace ha p o ides accession o he simula o engine co e accompanied by a lis o all xml iles eady o simula ion. The use only needs o se- lec an xml ile om such a lis , hen de e mine he numbe o i e a ions he simula ion will las (each i e - a ion is se o be co espond o one day) and un he simula ion. I is wo h o no e ha xml iles can be expo ed o impo ed om he use ’s PC as con enience, using he “RUN”in e ace. Once he simula ion s a s, ARES au oma ically c ea es a olde (labeled wi h he name gi en o he P-sys em du ing he con igu a ion) in he ou pu s’a chi e assigned o his pa icula simula ion and hen gene a es he xml inpu ile ha is placed wi hin his oupu olde . When he simula ion inishes he sys em i s deli e s a aw ou pu , which is a plain ile con aining he sampling coun s pe i - e a ion o all simula ed objec s and EBs. The aw ou pu is di icul o manage because o he de aul o ma i en- closes. To o e come his di icul y, ARES implemen s an ou pu con e e engine ha p ocesses and spli s he aw ou pu in o a se o 5 o ma ed cs iles appoin ed as ECO-like.cs , P-like.cs , RS-like.cs , H-like.cs , B-like.cs . These iles cons i u e oge he he ou pu ha ARES de- li e s a e simula ion o he use in o he ou pu ’s a ch- i e ( he ou pu ’s a chi e can be accessed using he “OUTPUTS” ab in he ARES menu). Each cs con ains he coun s o each EB and objec sampled a he P-sys em EB le el e e ed in he ile name. Fo ins ance, ECO-like.cs has he coun s o all EB and objec s sampled a he ecosys em le el, H-like.cs has he coun s o all objec s and EBs sampled in he H-like EBs bu no in hose o highe le els (i.e. ECO and P-like); and B-like.cs ile has he coun s o all objec s sampled in B-like EBs. In each cs , i e a ions co espond wi h he ows and a e o ganized in ascending o de (being he s a ing con igu a ions he i s coun in he ile) and memb anes and objec s co espond wi h he columns. ARES also o e s o he sec ions o use suppo , which can be accessed ia he submenu a ailable a he oo e o he ARES in e ace. 3 o hese sec ions called “R-TOOLS”,“TUTORIALS”,and “AVAILABLE RULES” a e o pa icula in e es .The i s sec ion (R-TOOLS) gi es access o an in e ace (also managed ia menu) o e ing he use s di e en sc ip s mainly bu no exclu- si ely de eloped in “R”, o downs eam in e oga ion o cs ou pu s. No e howe e ha he use o R-TOOLS is no a manda o y ask as cs s a e open plain iles ha can be p ocessed using any o he ool o s a is ical pack- ages such as Excel, Gnuplo , Ma lab and Ma hema ica, e c. The second sec ion (“TUTORIALS”) is a eposi o y whe e use s can upload and download u o ials o P- sys em con igu a ion and managemen o ARES (see also he sec ion below, “Tu o ials and aining ma e ial”). Finally he hi d sec ion (“AVAILABLE RULES”)isa sec ion whe e we summa ize all p e-designed ules o da e a ailable o each ype o EB in o de o le he use o make p elimina y e alua ions o he ules o ake o a pa - icula simula ion be o e c ea ing he s a ing con igu a ion o he P-sys em. This sec ion also includes a o m o use s o make i necessa y any speci ic sugges ion o he implemen a ion o new ules no ye a ailable. Tu o ials and aining ma e ial Al hough managemen o ARES is qui e in ui i e, he design and p epa a ion o he s a ing con igu a ion o a P-sys em model scena io can be an a duous ask o e- sea che s no amilia ized wi h memb ane compu ing (con igu a ion o i ems, assigna ion o ules, e c.). Taking his in o p ima y conside a ion, we ha e p epa ed wo u o ials aimed o gi e he eade some aining ma e ial ha can be downloaded om he “TUTORIALS”sec ion o ARES unde he labels “Nosocomial Scena io”and “Two cock oach a ms” espec i ely. The i s u o ial con empla es a simpli ied nosocomial scena io p o ided wi h he sole objec i e o allowing he use o ake he i s s eps in lea ning how o con igu e an exempla y P- sys em c ea ing wo memb ane en i onmen s (a commu- ni y and a hospi al en i onmen ), hos EBs ( o ins ance, pa ien s), mic obial communi ies wi hin hos s, composed o dis inc bac e ial EBs and plasmids, ca ying AR genes objec s wi hin he bac e ial EBs. The u o ial also exempli- ies how o c ea e clocks o in oduce o he objec s such as an ibio ics in he simula ion o how o con igu e and une a basic package o ules. The second u o ial con em- pla es a mo e elabo a ed scena io ha can be add essed a e comple ing he i s u o ial. This u o ial speci ically ocuses on he simula ion o wo popula ions o B. ge ma- nica (a model insec o ganism able o implan an in es- inal mic obio a simila o ha o humans [53–56]) espec i ely emplaced in wo sepa a e cages wi h he pos- sibili y o mig a ing om one o he o he . These wo boxes concep ually ep esen en i onmen al EBs o hos- pi al and u ban-communi y indi iduals, he e designa ed as P 1 and P 2 . Cock oaches o bo h a ms a e hos s (H-like EBs) ca ying he same in es inal mic obio a, which ac- co ding o Ca asco e al. [56] is p edominan ly composed o eigh bac e ial lineages (B-like EBs). Fou o hese cellu- la lineages will be simula ed as G am-nega i e, while he ou o he will be G am-posi i e. All bac e ial cells o all lineages a e allowed o ca y h ee dis inc ypes o in a- cellula plasmids (PL 1 ,PL 2 and PL 3 ) capable o ho izon al ans e . One o hese (PL 1 ) is ca ie o an AR gene (AR 1 ) con e ing esis ance o a g am-nega i e speci ic an ibio ic designa ed as “A 1 ”while ano he plasmid ype (PL 2 ) ca ies an AR gene (AR 2 ) o e ing esis ance o a g am-posi i e speci ic an ibio ic labeled as A 2 . In he s a ing con igu a ion, he hi d plasmid ype (PL 3 ) does no ca y AR genes bu du ing he cou se o he Campos e al. Biology Di ec (2015) 10:41 Page 9 o 13