New therapies for COPD-related pulmonary infections unveiled by inspecting bacterial interactions

Uni e sidade do Minho Escola de Engenha ia Bea iz Maciel Fe ei a New he apies o COPD- ela ed pulmona y in ec ions un eiled by inspec ing bac e ial in e ac ions janei o de 2023 New he apies o COPD- ela ed pulmona y in ec ions un eiled by inspec ing bac e ial in e ac ions Tí ulo Tí ulo Tí ulo Tí ulo Tí ulo Tí ulo Tí ulo Bea iz Maciel Fe ei a UMinho | 2023 Uni e sidade do Minho Escola de Engenha ia Bea iz Maciel Fe ei a New he apies o COPD- ela ed pulmona y in ec ions un eiled by inspec ing bac e ial in e ac ions Disse ação de Mes ado Mes ado em Bio ecnologia T abalho e e uado sob a o ien ação da Dou o a Paula Alexand a da Sil a Jo ge janei o de 2023 I DIREITOS DE AUTOR E CONDIÇÕES DE UTILIZAÇÃO DO TRABALHO POR TERCEIROS Es e é um abalho académico que pode se u ilizado po e cei os desde que espei adas as eg as e boas p á icas in e nacionalmen e acei es, no que conce ne aos di ei os de au o e di ei os conexos. Assim, o p esen e abalho pode se u ilizado nos e mos p e is os na licença abaixo indicada. Caso o u ilizado necessi e de pe missão pa a pode aze um uso do abalho em condições não p e is as no licenciamen o indicado, de e á con ac a o au o , a a és do Reposi ó iUM da Uni e sidade do Minho. Licença concedida aos u ilizado es des e abalho A ibuição-NãoCome cial-Compa ilhaIgual CC BY-NC-SA h ps://c ea i ecommons.o g/licenses/by-nc-sa/4.0/ II Acknowledgemen s The help and suppo o se e al people we e c ucial o he accomplishmen o he disse a ion, and he e o e, I am g a e ul o all he people who con ibu ed di ec ly o indi ec ly in some way o he comple ion o my disse a ion. To my supe iso , D . Paula, o sympa hy, a ailabili y, dedica ion, pa ience, comp ehension, and coo dina ion. Fo all he knowledge ansmi ed a a heo e ical le el as well as in e ms o labo a o y echniques. Fo all he suppo , c i ical hinking, sugges ions, and cla i ica ion o all my ques ions du ing his wo k. G a e ul o e e y hing! To he MOP labo a o y g oup, o he good a mosphe e, sympa hy, a ailabili y, and cla i ica ion o all p ocedu es and ques ions h oughou he de elopmen o he labo a o y wo k. To my amily and iends in gene al, o he momen s o sha ing and happiness, companionship, dedica ion, ca e, suppo , and exchange o expe ience ha made me e ol e pe sonally and academically. To my pa en s, wo ds will ne e be enough o hank hem. Thank you o helping me in e e y hing, o gi ing me con idence, o belie ing in me and in my capaci y o con inue o accomplish my goals. Finally, o Nelson, my g ea suppo , o making my days happie , hank you o you ca e, comp ehension, a en ion, pa ience, knowledge sha ing, and encou agemen o o e come he di icul ies ha a ose h oughou his disse a ion. I'm g a e ul o e e yone. III STATEMENT OF INTEGRITY I he eby decla e ha ing conduc ed his academic wo k wi h in eg i y. I con i m ha I ha e no used plagia ism o any o m o undue use o in o ma ion o alsi ica ion o esul s along he p ocess leading o i s elabo a ion. I u he decla e ha I ha e ully acknowledged he Code o E hical Conduc o he Uni e si y o Minho. IV No as e apias pa a in ecções pulmona es elacionadas com a DPOC po inspeção de in e ações bac e ianas Resumo Anualmen e, milhões de pessoas mo em de ido à doença pulmona obs u i a c ónica (DPOC). Es a doença incu á el é ma cada po exace bações que são p incipalmen e p o ocadas po bac é ias o mado as de bio ilmes, onde NTHi e a sua in e ação com ou as bac é ias aumen am a esis ência aos agen es an imic obianos. Po an o, en ende o papel das bac é ias e das suas in e ações na DPOC é c ucial pa a o desen ol imen o de no os á macos capazes de comba e a esis ência des as aos an ibió icos. O p incipal obje i o des a disse ação consis iu no es udo das condições ó imas de c escimen o de bio ilmes de H. in luenzae e de S. au eus e das in e ações bac e ianas que se es abelecem nesses bio ilmes, bem como a a aliação da susce ibilidade das duas espécies a uma seleção de pép idos an imic obianos (AMPs). O impac o da eno ação do meio de cul u a em bio ilmes de H. in luenzae e elou inexis ência de células cul i á eis quando es e não oi eno ado às 24 h, possi elmen e de ido a me aboli os óxicos, ao esgo amen o de nu ien es, ou à exis ência de células iá eis mas não cul i á eis. Os esul ados demons a am que as di e enças no c escimen o de H. in luenzae em di e en es ma cas do meio de c escimen o (Oxoid, Lio ilchem e VWR) podem de e -se à qualidade do meio usado, dado que o meio mais ca o (Oxoid) oi o que esul ou em menos a iabilidade, an o em bio ilmes de uma ou duas espécies. Po an o, oi decidido usa es a ma ca pa a os ensaios subsequen es. Os esul ados da dinâmica populacional de bio ilmes mis os de H. in luenzae com di e en es concen ações iniciais de S. au eus suge em que ambas as espécies podem es a a bene icia o c escimen o uma da ou a. Do es udo da in luência da colonização sequencial nos bio ilmes, não se e i icou a in luência das espécies colonizado as na o mação dos bio ilmes de ou a espécie. Na a aliação do e ei o dos exop odu os p oduzidos po uma espécie na o mação de bio ilmes da ou a espécie, não se obse a am quaisque e ei os, pelo que a concen ação dos exop odu os pode e sido insu icien e pa a causa um impac o ou as células do bio ilme podem não se esponsi as aos exop odu os p oduzidos. Po im, o AMP achyplesin I demons ou melho ação bac e ios á ica e bac e icida con a ambas as es i pes em es udo, e elando esul ados p omisso es pa a abalhos u u os. V Os esul ados des e abalho pe mi i am ex ai in o mações ú eis, nomeadamen e condições ó imas de c escimen o e ipo de in e ações es abelecidas in i o , que ajuda ão o g upo de pesquisa na busca de po enciais agen es an imic obianos pa a comba e os bio ilmes o mados po es as bac é ias. Pala as-cha e: DPOC; Exace bações; Haemophilus in luenzae ; In e ações bac e ianas; S aphylococcus au eus . VI New he apies o COPD- ela ed pulmona y in ec ions un eiled by inspec ing bac e ial in e ac ions Abs ac E e y yea , millions o people die om ch onic obs uc i e pulmona y disease (COPD). This incu able disease is ma ked by exace ba ions ha a e mainly caused by bio ilm- o ming bac e ia, whe e NTHi and i s in e ac ion wi h o he bac e ia inc ease esis ance o an imic obial agen s. The e o e, unde s anding he ole o bac e ia and hei in e ac ions in COPD is c ucial o he de elopmen o new d ugs capable o comba ing hei esis ance o an ibio ics. The main objec i e o his disse a ion was o s udy he op imal condi ions o he g ow h o H. in luenzae and S. au eus bio ilms and he bac e ial in e ac ions ha a e es ablished in hese bio ilms, as well as he e alua ion o he suscep ibili y o he wo species o a selec ion o an imic obial pep ides (AMPs). The impac o enewing he cul u e medium on H. in luenzae bio ilms e ealed he absence o cul u able cells when he medium was no enewed a e 24 h, possibly due o oxic me aboli es, nu ien deple ion, o he exis ence o iable bu non-cul u able cells. The esul s showed ha he di e ences in H. in luenzae g ow h in di e en b ands o g ow h medium (Oxoid, Lio ilchem, and VWR) could be due o he quali y o he medium used, gi en ha he mos expensi e medium (Oxoid) was he one ha esul ed in less a iabili y, bo h in bio ilms o one o wo species. The e o e, i was decided o use his b and o subsequen assays. The popula ion dynamics esul s o mixed H. in luenzae bio ilms wi h di e en ini ial concen a ions o S. au eus sugges ha bo h species could be bene i ing each o he 's g ow h. F om he s udy o he in luence o sequen ial coloniza ion on bio ilms, he in luence o colonize s species on he o ma ion o bio ilms o ano he species was no e i ied. In assessing he e ec o exop oduc s p oduced by one species on bio ilm o ma ion by ano he species, no e ec s we e obse ed, so he concen a ion o exop oduc s may ha e been insu icien o cause an impac , o he bio ilm cells may no be esponsi e o he exop oduc s p oduced. Finally, AMP achyplesin I showed be e bac e ios a ic and bac e icidal ac ion agains bo h s ains unde s udy, e ealing p omising esul s o u u e wo k. The esul s o his wo k allowed ex ac ing use ul in o ma ion, namely op imal g ow h condi ions and ype o in e ac ions es ablished in i o , which will help he esea ch g oup in he sea ch o po en ial an imic obial agen s o comba he bio ilms o med by hese bac e ia. Keywo ds: Bac e ial in e ac ions; COPD; Exace ba ions; Haemophilus in luenzae ; S aphylococcus au eus . XIII Abb e ia ions: HI 2 = H. in luenzae om he second c yo ial (in use), HI 6 = H. in luenzae om he six h c yo ial (newly opened). .................................................................................................................... 35 Figu e 7 – Quan i ica ion o cul u able H. in luenzae (A) and o al biomass (B) o 48 h H. in luenzae bio ilms g own in sBHI om Oxoid and Lio ilchem wi h media eplacemen a 24 h. S anda d de ia ions a e indica ed by he e o ba s. S a is ically signi icancy: *** p<0.001 and **** p<0.0001. Abb e ia ions: HI 2 = H. in luenzae om he second c yo ial (in use), HI 6 = H. in luenzae om he six h c yo ial (newly opened). ........................................................................................................................................... 36 Figu e 8 - Quan i ica ion o cul u able H. in luenzae (A) and o al bio ilm biomass (B) om 24 h bio ilms o H. in luenzae (HI) and o H. in luenzae wi h 1% o S. au eus (1% SA) g own in sBHI om Lio ilchem and Oxoid. S a is ically signi ican di e ences: **** p < 0.0001. S anda d de ia ions a e indica ed by e o ba s. ................................................................................................................................................ 38 Figu e 9 - Quan i ica ion o cul u able H. in luenzae (A) and o al bio ilm biomass (B) om 48 h bio ilms o H. in luenzae (HI) and o H. in luenzae wi h 1% o S. au eus (1% SA) g own in sBHI om Lio ilchem and Oxoid wi hou media eplacemen a 24 h. S a is ically signi ican di e ences a e ep esen ed by *** p < 0.001 and **** p < 0.0001. S anda d de ia ions a e indica ed by e o ba s. .............................. 38 Figu e 10 - Quan i ica ion o cul u able H. in luenzae (A) and o al bio ilm biomass (B) om 48 h bio ilms o H. in luenzae (HI) and o H. in luenzae wi h 1% o S. au eus (1% SA) g own in sBHI om Lio ilchem and Oxoid wi h media eplacemen a 24 h. S a is ically signi ican di e ences a e ep esen ed by ** p < 0.01, *** p < 0.001, and **** p < 0.0001. S anda d de ia ions a e indica ed by e o ba s. .............. 40 Figu e 11 - Quan i ica ion o cul u able H. in luenzae and S. au eus (A) and o al bio ilm biomass (B) om 24 h double-species bio ilms wi h di e en ini ial concen a ions o S. au eus (1%, 10%, 25%, and 50% V/V). S a is ically signi ican di e ences a e ep esen ed by * p <0.05, ** p < 0.01, *** p < 0.001, and #### p < 0.0001. S anda d de ia ions a e indica ed by e o ba s. The symbols * and # ep esen compa isons wi h H. in luenzae and S. au eus con ol bio ilms, espec i ely (single-species con ol). .. 41 Figu e 12 - Quan i ica ion o cul u able H. in luenzae and S. au eus (A) and o al bio ilm biomass (B) om 48 h double-species bio ilms wi h di e en ini ial concen a ions o S. au eus (1%, 10%, 25%, and 50% V/V) wi hou media eplacemen a 24h. S a is ically signi ican a e ep esen ed by **** p < 0.0001, and # p < 0.05. The symbols * and # ep esen compa isons wi h H. in luenzae and S. au eus con ol bio ilms, espec i ely. ....................................................................................................................... 42 Figu e 13 – Quan i ica ion o cul u able H. in luenzae and S. au eus (A) and o al bio ilm biomass (B) om 48 h double-species bio ilms o H. in luenzae wi h di e en ini ial concen a ions o S. au eus (1%, 10%, 25%, and 50% V/V) wi h media eplacemen a 24 h. S a is ically signi ican di e ences a e XIV ep esen ed by * p < 0.05. S anda d de ia ions a e indica ed by e o ba s. No e: Due o un o eseen echnical easons, he e is no da a o he cul u abili y o H. in luenzae in H. in luenzae bio ilms wi h 50% o S. au eus. ..................................................................................................................................... 43 Figu e 14 - Quan i ica ion o cul u able S. au eus and H. in luenzae cells (A) and quan i ica ion o biomass (B) om double-species bio ilms in which H. in luenzae was he second colonize . Con ols co espond o 48 h S. au eus and 24 h H. in luenzae single-species bio ilms. The s a is ical di e ences a e ep esen ed by: * p < 0.05 and ** p < 0.01. S anda d de ia ions a e indica ed by e o ba s. ...... 45 Figu e 15 - Quan i ica ion o cul u able H. in luenzae and S. au eus cells (A) and quan i ica ion o biomass (B) om double-species bio ilms in which S. au eus was he second colonize o 24 h. Con ols co espond o 48 h H. in luenzae and 24 h S. au eus single-species bio ilms. The s a is ical di e ences a e depic ed as: * p < 0.05 and **** p < 0.0001. S anda d de ia ions a e indica ed by e o ba s. .... 46 Figu e 16 – E ec o SA CFS on HI cul u able bio ilm cells (A) and o HI CFS on SA cul u able bio ilm cells (B). The s a is ically signi ican di e ences a e ep esen ed as: **** p < 0.0001. S anda d de ia ions a e indica ed by e o ba s. ............................................................................................................... 48 XV Lis o Tables Table 1 - Func ional ca ego ies con olled by QS (G andclémen e al., 2016). ................................. 14 Table 2 - Summa y o NTHi bio ilm esis ance and ole ance mechanisms and hei espec i e model and expe imen al e ec s (adap ed om Weeks e al., 2021). ............................................................. 23 Table 3 - Calib a ion cu e equa ions o he wo bac e ial s ains p o ided by he esea ch g oup. ... 27 Table 4 – An imic obial ac i i y o empo in A, achyplesin I, palm-KGKPEG, ci opin 1.1, and cip o loxacin agains H. in luenzae DSM 4690 (ATCC 33391, NCTC 8143) and S. au eus (ATCC 25923). MIC and MBC a e exp essed in mg/L. .............................................................................................. 49 1 Chap e 1. In oduc ion This chap e p esen s he con ex ualiza ion and mo i a ion o his disse a ion, namely explaining he se e i y o COPD, cha ac e izing he pa hology, e ealing he main bac e ia esponsible o he c i ical pe iods o he disease, and no leas , he p oblem in he ea men o hese in ec ions due o an ibio ic esis ance by hese mic oo ganisms. Fu he mo e, his chap e con eys he main objec i es o his disse a ion gi en his scene y and he s uc u e o he documen . 1.1 Con ex and Mo i a ion Ch onic obs uc i e pulmona y disease (COPD) is he hi d leading cause o dea h wo ldwide ha mainly a ec s he espi a o y sys em (GOLD, 2022). Acco ding o he Wo ld Heal h O ganiza ion (WHO), in 2019, 3.23 million people died due o COPD, wi h almos 90% o hese dea hs epo ed in LMICs (WHO, 2021). COPD is a common, p e en able, and p og essi e debili a ing disease ha al hough ea able, i has no cu e and i s appea ance is mainly ela ed o smoking habi s, gene ic ac o s, and pollu ion (Awokola e al., 2022; Jiang e al., 2016; Weinbe ge e al., 2019; WHO, 2021). This disease has a high socio-economic bu den and includes wo pa hologies, namely emphysema, dema ca ed by he des uc ion o he lung pa enchyma, and ch onic b onchi is, cha ac e ized by mucus hype sec e ion, esul ing in ch onic p oduc i e cough (GOLD, 2022; Leung e al., 2017; Vogelmeie e al., 2020). COPD is cha ac e ized by equen exace ba ions ha a e deno ed by a sudden decline in lung unc ion and he consequen wo sening o symp oms (GOLD, 2022). These exace ba ions a e caused by pa hogenic b onchial coloniza ion, o which 50% a e ela ed o bac e ial in ec ions, o en esul ing in poo quali y o li e, inc eased hospi aliza ions, and highe mo ali y a es (Saxena e al., 2016; Vogelmeie e al., 2020). These bac e ial in ec ions a e he esul o bac e ial adhesion o he pulmona y epi helium and he consequen o ma ion o bio ilms (Sho e al., 2021). Bio ilms a e agg ega es o bac e ia in a sel - p oduced polyme ic ma ix ha no mally include di e en mic obial species, which in e ac wi h each o he bene i ing o ha ming one ano he (Mel on & Ande son, 2019; Welp & Bombe ge , 2020). Speci ically, in COPD exace ba ions, non- ypable Haemophilus in luenzae (NTHi) is he mos p e alen isola ed species, ollowed by Mo axella ca a halis , S ep ococcus pneumoniae , S aphylococcus au eus , Pseudomonas ae uginosa , and Klebsiella pneumoniae (Leung e al., 2017; Sho e al., 2021; Su e al., 2018). In ecen imes, he e has been a global inc easing de elopmen o an ibio ic esis ance on he pa o bac e ia (Jo ge e al., 2019). This esis ance comp omises he e ec i eness o in ec ion ea men s 2 and leads o he selec ion o esis an subpopula ions ha can make COPD exace ba ions e en mo e pe sis en and se e e (Welp & Bombe ge , 2020). I is, he e o e, c ucial o ully unde s and he in e ac ions be ween he bac e ia p esen in COPD- ela ed in ec ions in o de o y o ind new he apies adap ed o his polymic obial eali y. Thus, he ocus o his wo k was he s udy o H. in luenzae and S. au eus bio ilm in e ac ions. H. in luenzae was selec ed gi en i s high ele ance in COPD ela ed in ec ions, and S. au eus was chosen because i is he second leas s udied o he ou p e alen species ( M. ca a halis , S. pneumoniae , and P. ae uginosa ). In ac , up un il Feb ua y 2020, only nine a icles we e published ela ing S. au eus wi h H. in luenzae in COPD, mos o which only analyzed he co-occu ence o he wo species in pa ien s and none o s udied hem in he con ex o a bio ilm (Ama o, 2022). 1.2 Main Objec i es The main objec i e o his disse a ion is o unde s and and cha ac e ize he biogeog aphy o he polymic obial conso ia ha o en es ablishes in he lungs o pa ien s wi h COPD, always ocusing on NTHi. In his sense, his wo k was ocused on he in es iga ion o in e ac ions o wo species, namely H. in luenzae and S. au eus , in bio ilms, o y and iden i y he social ela ionships ha hey es ablish wi h each o he . The a ionale was o ob ain in o ma ion on he in luence o g ow h condi ions, speci ically he b and o media used on H. in luenzae and S. au eus bio ilm o ma ion, as well as he popula ion dynamics be ween he wo species wi h di e en ini ial concen a ions. Sequen ial coloniza ion was also in es iga ed o unde s and whe he he in oduc ion o a species a a la e s age induces changes o he bio ilm and, mo eo e , i he e is any bene i o damage o he o al communi y. Finally, he e ec o ex acellula p oduc s ha may e en ually b ing some bene i o ha m o he bio ilm communi y was also in es iga ed, as well as he suscep ibili y o he bac e ia o po en ial he apeu ic agen s, mo e speci ically an imic obial pep ides (AMPs). This hesis was pe o med wi hin he scope o an ESCMID Resea ch P ojec , hence he in o ma ion he e acqui ed will la e assis in he selec ion and es ing o an imic obial p oduc s o he e adica ion o hese mul ispecies agg ega es, wi h he ul ima e goal o de eloping and e alua ing new he apeu ic s a egies o ea COPD- ela ed in ec ions. 1.3 Gene al Ou line o he Thesis The s uc u e o he hesis is composed by i e chap e s. Chap e 1 add esses he con ex , scope, main objec i es, and o ganiza ion o his hesis. Chap e 2 p esen s he s a e o he a encompassing heo e ical knowledge abou COPD, he ele ance o NTHi in COPD, and he ole o bio ilms in he de elopmen o he disease. In addi ion, i add esses he inc easing esis ance o an ibio ics by bac e ia 3 and he ole o an imic obial pep ides o ea COPD in ec ions. Chap e 3 desc ibes he p ocedu es, he s ains, he g ow h media, and he main ma e ial used o ou he expe imen s. Chap e 4 e eals he esul s ob ained du ing he expe imen s pe o med and hei discussion. Finally, Chap e 5 con ains he main conclusions and sugges ed u u e wo k. 4 Chap e 2. S a e o he A Chap e 2 p esen s he de ini ion o COPD, desc ibes he wo pa hologies associa ed wi h his disease, i s o igin, and he acu e pe iods o he disease. Fu he mo e, i gi es heo ical knowledge abou he bac e ia in ol ed in he COPD ela ed in ec ions and he s a egical mechanisms ha allow hem o su i e in he hos s. This sec ion also add esses he impac o an imic obial esis ance in COPD exace ba ion ea men as well as he p omise laying in an imic obial pep ides o ea hem. 2.1 Ch onic Obs uc i e Pulmona y Disease (COPD) 2.1.1 De ini ion Ch onic Obs uc i e Pulmona y Disease (COPD) is a common p og essi e disease ha is cu en ly conside ed he mos p e alen ch onic espi a o y disease wo ldwide (F aze , 2020; Ha ipoglu, 2018). I is one o he leading causes o dea h in he wo ld, p esen ing high a es o mo ali y and mo bidi y, being an economically expensi e public heal h p oblem (B ody e al., 2020; F aze , 2020; GOLD, 2021). In his disease, s uc u al (cen al ai ways, pe iphe al ai ways, lung pa enchyma, and pulmona y ascula u e) and unc ional (gas exchange, in la ion, and ai low) changes occu in he lungs, being mainly cha ac e ized by ai way obs uc ion and pe sis en ai low limi a ion (Da idson & Bai, 2005; Papand inopoulou e al., 2012). Gene ally, hese limi a ions a e caused by excessi e mucus p oduc ion, hickening o ai way walls esul ing om edema o muscle hype ophy, and s uc u al changes in lung issue, such as loss o lung elas ici y and issue des uc ion (F aze , 2020; Papand inopoulou e al., 2012; Vogelmeie e al., 2017). COPD includes wo diso de s, namely ch onic b onchi is and emphysema. Ch onic b onchi is is caused by changes in he mucus-sec e ing sys em (Weinbe ge e al., 2019). In his case, he e is an inc ease in mucus-sec e ing glands and goble cells esponsible o b onchial sec e ions. This inc ease induces hickening o he ai way walls and excessi e p oduc ion o mucus causing blockage o he lumen (Papand inopoulou e al., 2012). In addi ion, he b onchial walls show cell in il a ion and ib osis esul ing om he in lamma o y p ocess (Weinbe ge e al., 2019). In u n, emphysema is a pa hology cha ac e ized by des uc ion o he pa enchyma (al eola walls), enla gemen o ai spaces dis al o he e minal b onchiole, loss o elas in, and abno mali ies in he o ma ion o he lung elas ic ibe (Mecham, 2018; Weinbe ge e al., 2019). In his condi ion, as he e is g adual des uc ion o he al eoli, gas exchange is comp omised, which can lead o agg a a ed ai low obs uc ion (Weinbe ge e al., 2019). 5 Al hough ai low obs uc ion has di e en o igins in hese wo pa hologies, pa ien s o en ha e he cha ac e is ics o bo h. Beyond he damage caused o he lungs, COPD may be associa ed wi h ex apulmona y dys unc ions (Jai o ich & Ba ei o, 2018). Fo example, i has been shown ha modi ica ions in b ain unc ion may be caused by COPD due o ai way obs uc ion, hypoxia, and in lamma o y media o s (M. Yin e al., 2019). Fu he mo e, changes and des uc ion o lung issue inc ease he ulne abili y o pa ien s wi h COPD o de elop hea diseases (And é e al., 2019). COPD also con ibu es o skele al muscle dys unc ion ha causes high a es o hospi aliza ions. This dys unc ion a ises om muscle a ophy and changes in ibe s, me abolism, and ana omy (Jai o ich & Ba ei o, 2018). 2.1.2 Causes and Symp oms COPD is a complex and he e ogeneous disease ha can be caused by mul iple ac o s such as smoking, en i onmen al pollu ion, and gene ic ac o s (Weinbe ge e al., 2019). Cu en ly, smoking is a common cause o dea h by COPD (Kim e al., 2019). In 2030, 27% o dea hs om COPD will be caused by smoking (Anzue o e al., 2015). Smoking causes se ious damage o he b onchi, b onchioles, and pulmona y pa enchyma. Besides ha , he e is o e p oduc ion o mucus and de elopmen o in lamma o y p ocesses media ed by in lamma o y cells (leuko iene B4, in e leukin-8, and umo nec osis ac o -α) ha con ibu e o issue deg ada ion (Weinbe ge e al., 2019). These ac o s and he elease o eac i e oxygen species (ROS) con ibu e o he p og ession o he disease. Due o smoking habi s, al e a ions in he s uc u e o he b onchioles (small ai ways) and he appea ance o ib osis occu . Consequen ly, i se e ely hinde s he passage o ai , which in COPD pa ien s is wo isome (Weinbe ge e al., 2019). En i onmen al pollu ion has a majo impac on public heal h. I is conside ed an impo an isk ac o ha can cause o agg a a e COPD (Jiang e al., 2016). The main isk ac o s associa ed wi h COPD a e p olonged exposu e o indus ial pollu ion, a ic, and he combus ion o uels (Manisalidis e al., 2020). Exposu e o ai pollu an s may also cause exace ba ions o he disease and inc eased COPD mo bidi y and mo ali y (Manisalidis e al., 2020). In ac , ai pollu an s, such as ca bon monoxide, ni ogen oxide, sul u dioxide, g ound-le el ozone, and pa icula e ma e pollu ion, ha e ad e se e ec s on his disease (Jiang e al., 2016). Mo eo e , ola ile o ganic compounds, dioxins, and polycyclic a oma ic hyd oca bons also con ibu e o he wo sening o he disease (Manisalidis e al., 2020). Acco ding o he li e a u e, gene ic ac o s also con ibu e o he de elopmen o COPD (Weinbe ge e al., 2019). De iciency in he α1-an i ypsin glycop o ein is he mos well-known gene ic ac o ha causes COPD (Sil e man, 2020). As s a ed by Weinbe g and collabo a o s, α1-an i ypsin is p oduced in he li e and ci cula es in he blood. In no mal condi ions, α1-an i ypsin is an enzyme ha inhibi s he ac ion o 6 se ine p o eases, p o ec ing he lungs om hei ha m ul e ec s ( o example, emphysema wi h sho ness o b ea h, coughing, and wheezing) (Wise & Hopkins, 2020). This p o ein is encoded by he SERPINA1 gene. When his gene is al e ed, he e a e modi ica ions in he p o ein's s uc u e, in i s p oduc ion, and i s libe a ion. People wi h his gene ic inhe i ance ha e educed le els o α1-an i ypsin in he blood, and he p o ein may ha e an abno mal s uc u e, which induces dys unc ions. The ZZ geno ype is conside ed he mos impo an o m o α1-an i ypsin de iciency and is highly associa ed wi h p ema u e de elopmen o emphysema (Weinbe ge e al., 2019). In addi ion o he causes men ioned, COPD may also be associa ed wi h ad ancing age. Despi e no being a ully cla i ied subjec , successi e damage h oughou li e due o exposu e o agen s and he aging o he ai ways and lung pa enchyma can con ibu e o COPD. Fu he mo e, he e is a p opensi y o he de elopmen o COPD when lung in ec ions occu in childhood, and he ac o s ha a ec lung g ow h om bi h o adolescence also con ibu e o inc ease he p obabili y o de eloping his disease (GOLD, 2021). Symp oms caused by COPD ha e a majo impac on pa ien s’ li e quali y and well-being (Vogelmeie e al., 2020). Acco ding o he Global Ini ia i e o Ch onic Obs uc i e Lung Disease (GOLD), dyspnea is he symp om ha is mos e iden in COPD (GOLD, 2021). This disabling symp om is cha ac e ized by di icul y in b ea hing and is associa ed wi h anxie y (Anzue o & Mi a i lles, 2017; Vogelmeie e al., 2020). Usually, he i s symp om ha appea s in COPD is ch onic cough (GOLD, 2021). O he symp oms, such as p olonged spu um p oduc ion, a e associa ed wi h in lamma o y media o s ha a e indica i e o bac e ial exace ba ion. In si ua ions o agg a a ed illness, i may be ela ed o a igue, ano exia, and loss o weigh . Dep ession also appea s o be associa ed wi h COPD, which con ibu es o wo sening heal h s a us and g ea e suscep ibili y o exace ba ions and hospi aliza ions (GOLD, 2021; Vogelmeie e al., 2020). 2.1.3 Epidemiology in he Wo ld, Eu ope, and Po ugal Nowadays, i is es ima ed ha 328 million people su e om COPD wo ldwide. This illness is conside ed a "silen kille ” in low- and middle-income coun ies (LMICs), accoun ing o o e 90% o dea hs (Quade i & Hu s , 2018). A ise in COPD is expec ed o e he nex 40 yea s due o he con inuous exposu e o isk ac o s, inc ease in smoking in de eloping coun ies, and he aging o he wo ld popula ion. E en mo e se ious is ha , in 2060, he e may be mo e han 5.4 million annual dea hs om COPD (GOLD, 2021). In he pas , COPD was p edominan ly a disease ha a ec ed men (A yal e al., 2014). Howe e , nowadays he p e alence o COPD is equal in men and women. This change may be due o he inc ease 7 in obacco consump ion by women (GOLD, 2021). In addi ion, women's emancipa ion and cul u al changes ha e led o women being mo e exposed o isk ac o s (A yal e al., 2014). In e ms o suscep ibili y, some s udies epo ha women a e mo e suscep ible han men (GOLD, 2021). Howe e , he ela ionship be ween gen e, COPD, and suscep ibili y is complex, since biological and ho monal mechanisms can in luence he way he disease mani es s i sel (A yal e al., 2014). In Eu ope, s udies on COPD a e sca ce and he e is a lack o knowledge abou he disease (Gibson e al., 2013). A s udy ca ied ou in 19 Eu opean coun ies on people o e 40 yea s o age es ima es ha he e is a p e alence o COPD o 12.4%. Howe e , his pe cen age may no be co ec due o he absence o in o ma ion and i egula i ies in he dis ibu ion o da a ac oss Eu ope (Blanco e al., 2018). Ano he s udy es ima es ha , in Eu ope, he e is a p edominance o COPD be ween 4% and 10% (Mi a i lles e al., 2016). Acco ding o he Eu opean Lung Founda ion (ELF) and he Eu opean Respi a o y Socie y (ERS), p olonged exposu e o isk ac o s in he wo kplace is esponsible o 15% o 20% o cases. Recen s udies e eal ha , in he Eu opean Union, he p e alence o COPD in men and women was no signi ican ly di e en , which demons a es an inc ease in he p e alence o COPD in he emale popula ion. In addi ion, he e is e idence ha COPD is a c i ical heal h p oblem o women, wi h mo e and mo e cases o COPD among women (N i sos e al., 2018). Acco ding o he Po uguese Lung Socie y (Sociedade Po uguesa do Pulmão - SPP), in Po ugal, COPD is one o he main causes o dea h, a ec ing app oxima ely 800 housand people. In addi ion, i has a majo impac on public heal h and is conside ed he ch onic espi a o y disease wi h he highes mo ali y a es. I is es ima ed ha one in se en Po uguese o e 40 yea s o age has COPD. In 2016, 20.7% o dea hs om espi a o y disease we e caused by COPD (SPP, 2019). In 2017, i was esponsible o 2 627 dea hs, ep esen ing 2.4 % o o al dea hs in he coun y. The disease a ec ed mo e men han women, and abou 95% o dea hs occu ed in people o e 65 yea s (INE, 2019). In 2018, he e we e 2 834 dea hs, and he mo ali y inc eased by 7.9 % compa ed o he p e ious yea (INE, 2020). In Po ugal, COPD cases ha e been on he ise and, as his disease is unde diagnosed, he numbe o people who ha e COPD may be much highe (Munhá, 2020). Besides, he diagnosis is o en made la e and, consequen ly, when pa ien s u n o he doc o , he disease is al eady a a e y ad anced s age. Thus, i is ex emely impo an o de ec he disease in he ea ly s ages, which will allow o con ol and slow he disease p og ession. Fo his, i is necessa y o aise awa eness and in o m indi iduals abou he exis ence o COPD, i s associa ed isk ac o s, and associa ed symp oms (Simão & Ca alho, 2018). The u h is ha his ch onic espi a o y disease is no gi en due impo ance despi e i s se e i y. I is u gen ha 14 Table 1 - Func ional ca ego ies con olled by QS (G andclémen e al., 2016). Func ional ca ego ies Examples Cell main enance and p oli e a ion Exoenzymes p oduc ion, side opho es syn hesis, spo ula ion, acid esis ance Cell beha iou s Bio ilm o ma ion and dispe sal, mo ili y, adhesion Ho izon al gene ans e Plasmid conjuga ion, compe ence In e ac ions wi h hos and o he mic obes Vi ulence ac o s, exopolysaccha ide p oduc ion, bioluminescence, an ibio ics, hos coloniza ion ac o s The p ope ies o au oinduce s and he esponse hey induce in coo dina ing popula ion beha io s ensu e bac e ial su i al and p opaga ion in na u al en i onmen s whe e a a ie y o bac e ial species coexis (Fede le & Bassle , 2003; P. Smi h & Schus e , 2019). In aspecies and in e species communica ion is ca ied ou by au oinduce s (Sheela e al., 2019). In in aspecies communica ion, only he species o bac e ia ha p oduces he au oinduce can de ec and espond o i . In aspecies QS has di e ences be ween G am-posi i e and G am-nega i e bac e ia. In G am-nega i e bac e ia, he QS sys em speci ically uses he acyl-homose ine lac one (AHL) au oinduce . AHL is p oduced and eely di used in o and ou o he cell and only membe s o he same species ecognize and espond o he pep ide. In G am- posi i e bac e ia, QS is media ed by oligopep ides o au oinducing pep ides (AIPs) ha a e anspo ed o he ex acellula medium ia oligopep ide anspo e s because he bac e ial memb ane is no pe meable o AIPs. This ype o communica ion wi h bac e ia o he same species is ele an when hey li e oge he wi h se e al species o bac e ia and in an en i onmen wi h simila chemical s uc u es, as i allows hem o dis inguish hemsel es om o he species, assess hei numbe s, and coo dina e pa icula beha io s o he species. In addi ion o he species-speci ic QS communica ion sys em, all bac e ia ha e he in e species QS communica ion sys em (Fede le & Bassle , 2003). Au oinduce -2 (AI-2) is one o he main signaling molecules in he bac e ial QS p ocess wi h he abili y o con ol many p ocesses, such as he p oduc ion o i ulence ac o s, bio ilm o ma ion and mo ili y. These molecules a e p oduced by G am- posi i e and G am-nega i e bac e ia and pa icipa e in he egula ion o gene exp ession and physiological beha io s o bac e ia in in e species communica ion (A mb us e e al., 2011; Sheela e al., 2019). This ype o communica ion is e iden when some species canno p oduce hei own au oinduce s (AI-2), howe e hey ha e ecep o s o he au oinduce s o o he species (Windso , 2020). Bac e ia can also p oduce AI-2 h ough p o ein syn hesis, bu i is no de ec ed by he ecep o and can be used o egula e physiological beha io s o o he bac e ia (J. Zhao e al., 2018). QS communica ion plays a acili a ing ole in se e al unc ions, such as p o ec ion om oxins, nu ien s a a ion esponse, compe i ion wi h o he bac e ia o esou ces and limi ed space, and 15 su i al, as well as es ablishing symbiosis (Nadell e al., 2016; Sheela e al., 2019). In bac e ial coope a ion, p oduc ion by bac e ia o ex acellula p oduc s, cos ly o p oduce and sha eable o he en i e popula ion (public goods) coo dina ed by QS, can bene i neighbo ing cells in bio ilm communi ies (K. Zhao e al., 2019). Examples o public goods include ex acellula enzymes impo an o nu ien diges ion, biosu ac an s ha p omo e bac e ial mo ili y o he subs a e, and oxins ha damage hos issue o ob ain nu ien s. O he examples o public goods a e exopolysaccha ides ha p o ide s uc u e and p o ec ion o bio ilms, side opho es ha cap u e i on om he en i onmen , as well as AIs desc ibed abo e (P. Smi h & Schus e , 2019). The p oduc ion o molecules by bac e ia a e no always conside ed public goods and may es ablish compe i i e ela ionships. Fo example, bac e ial QS can be blocked by inhibi ing he p oduc ion, deli e y, o de ec ion o AIs. Blockade o QS is pe o med by enzymes, such as lac onases o acylases, which inac i a e he AI and consequen ly in e up cell- o-cell communica ion. O he compe i i e ela ionships ela e o compe i ion o nu ien s mainly o i on, p oduc ion o an ibio ics by bac e ia (bac e iocins), such as piocin, and p oduc ion o molecules o kill o in e e e wi h mic obial g ow h, as well as ans e o oxins by physical con ac (Welp & Bombe ge , 2020). 2.4 Bio ilms in COPD Ch onic espi a o y diseases, including COPD, a e o en associa ed wi h he o ma ion o polymic obial bio ilms (Sco ield & Wu, 2019; Welp & Bombe ge , 2020). Polymic obial bio ilms a e complex and dynamic mixed species communi ies ha a e cons an ly e ol ing (F. Ha ison e al., 2020; Welp & Bombe ge , 2020). Pa ien s wi h COPD ha e he ideal condi ions o bio ilm o ma ion due o impai ed cilia y clea ance sys em and in lamma o y hype plasia o mucus sec e ion. NTHi is he bac e ium ha ini ia es he p ocess o coloniza ion and a achmen o espi a o y epi helial cells. In human lungs, his mic oo ganism can su i e in he mucus- ich en i onmen and has a ailable hemin and NAD essen ial o i s su i al. I has an a senal o adhesins, such as ype T P and OMP P1, ha bind o he ICAM-1 p o ein and he CEACAM- 1 glycop o ein loca ed on he hos cell su ace (Figu e 1) ha allow he adhesion and in asion o lung cells (Figu e 3). The p oduc ion and de elopmen o EPS by agg ega es o NTHi allows he en y o new bac e ial species. The polymic obial conso ium de elops mic ocolonies wi h wa e and oxygen channels due o he de elopmen o complex in aspeci ic and in e speci ic in e ac ions, changes in me abolism and gene exp ession ha d i e he wa e and oxygen g adien . The quo um sensing sys em allows he dispe sion o he bio ilm wi h consequen dissemina ion and coloniza ion o new si es. This cycle causes 16 he in ec ion o pe sis and become ch onic, leading o ole ance and esis ance o an ibio ics and o he hos 's immune sys em (Weeks e al., 2021). Figu e 3 - Li e cycle o bio ilm in COPD. (A) Bio ilm o ma ion begins wi h he e e sible a achmen o NTHi o a sui able su ace o he ai - liquid in e ace o he acheob onchial espi a o y mucosa composed o columna epi helium p edomina ed by cilia ed epi helial cells in e spe sed wi h sec e o y cells and basal cells. (B) I e e sible ixa ion occu s due o he p esence o NTHi T P and OMP P1 adhesins ha bind o speci ic p o eins (ICAM-1, CEACAM-1, CLEC7A) p esen in espi a o y epi helial cells. NTHi also has adhesins ha bind o mucus p o eins such as mucin and lac o e in. (C) The p oduc ion o EPS by NTHi a o s he acquisi ion o seconda y colonize s. (D) Bio ilm de elops wi h he en y o seconda y colonize s. The species p esen ini ia e complex in aspeci ic and in e speci ic in e ac ions, and changes occu in gene exp ession and me abolism. As a esul , nu ien and oxygen g adien s a e o med, and bac e ial di e en ia ion akes place. The bio ilm ma u es and nu ien and wa e channels a e o med. (E) Bac e ia dispe se in plank onic o m o in bio ilm due o he quo um sensing sys em. (F) The p ocess ends wi h he coloniza ion o sui able subs a es (Weeks e al., 2021). In a polymic obial bio ilm, ecological in e ac ions, o example, syne gism, commensalism, mu ualism, compe i ion, e c., be ween bac e ial species o s ains a e ex emely impo an o he unc ioning o he bio ilm ecosys em, as well as o he exp ession o i ulen o pe sis en pheno ypes. The di e si y o he polymic obial lung mic obiome may a o in e speci ic in e ac ions be ween bac e ia. These in e speci ic in e ac ions can be indi ec due o bio ic and abio ic changes ha a o he p oli e a ion o seconda y colonize s in ai way diseases. Fo example, NTHi in ec ion inc eases in lamma ion ha can esul in mic obial changes and COPD exace ba ions. Fu he mo e, is also inc eases ai way obs uc ion and he p oduc ion o a iscous subs a e by up egula ing MUC2, which a o s he seconda y coloniza ion o bac e ia o e ime (Weeks e al., 2021). In e speci ic in e ac ions can also be mo e di ec when he e is he es ablishmen o mul i-species bio ilms ha coope a e in subs a e adhesion and s abilize he bio ilm 17 s uc u e, such as he mul i-species bio ilms o NTHi and S. pneumoniae ha oge he p oduce and sha e an EPS. NTHi and S. pneumoniae o en co-colonize he espi a o y ac o pa ien s wi h COPD, in e ac ing syne gis ically, p omo ing ini ial adhesion o he subs a e, bio ilm o ma ion, and su i al. Thus, oge he , hese bac e ia de elop an EPS ma ix made up o T P, LOS, eDNA, QS signals, p o eins, and ca bohyd a es essen ial in coope a i e adhesion and s abili y o he bio ilm s uc u e (Kyd e al., 2016; Weeks e al., 2021). In addi ion, H. in luenzae p oduces -lac amases ha p o ec S. pneumoniae om ea men wi h -lac ams. Howe e , hey also es ablish a compe i i e ela ionship because, as S. pneumoniae de elops, he pH o he bio ilm dec eases and he e is p oduc ion o a biocide, hyd ogen pe oxide, which kills NTHi (Kyd e al., 2016). Despi e killing NTHi, he p oduc ion o his biocide in he long e m s imula es he p oduc ion o neu ophils ha oge he make a selec i e p essu e o pe sis en and ROS- ole an s ains o NTHi. Fu he mo e, hyd ogen pe oxide is a sou ce o nu ien s and DNA due o bac e ial dea h and senescen hos cells ha main ain he bio ilm (Weeks e al., 2021). M. ca a halis , esponsible o 10% o exace ba ions in COPD, also a o s NTHi coloniza ion a e in ec ion due o bio ic changes. Speci ically, i s binding o epi helial cilia educes he equency o hei bea s, comp omising mucocilia y clea ance wi h consequen o ma ion o mucus plugs ha a e colonized by NTHi. In o i is media (OM) models, he highly ac i e ca alase p oduc ion by M. ca a halis p o ec s NTHi om he bac e icidal e ec o S. pneumoniae hyd ogen pe oxide (Bai & Campagna i, 2020). The p esence o M. ca a halis in he COPD lung could ha e he same e ec . As in e ac ions play an impo an ole in bio ilm s abili y, ea men o polymic obial bio ilm in ec ions should be ocused on hese in e ac ions (F. Ha ison e al., 2020). COPD p e en ion and ea men may equi e unde s anding mic obial in e ac ions ha modi y di e si y and he mic obial communi y (Welp & Bombe ge , 2020). Howe e , many o he mic obial in e ac ions in he espi a o y ac a e s ill unknown because he p esence o mixed species in ch onic in ec ions is o en con i med h ough PCR and sequencing. These echniques do no conside he spa ial o ganiza ion o he bac e ial communi y, so c ucial in o ma ion abou he composi ion o he bio ilm agg ega e, he spa ial o ganiza ion, and he possible in e ac ions be ween di e en species may be los (K ich e al., 2020). Recen ly, wo inno a i e me hods o he apid diagnosis o H. in luenzae bio ilms we e c ea ed. One o he me hods is he iden i ica ion o H. in luenzae h ough molecula imaging in eal ime due o he use o an en i onmen ally sensi i e luo opho e 7-ni obenz-2-oxa-1,3-diazole conjuga ed wi h polymyxin which luo esces in con ac wi h he lipid A componen o G am-nega i e bac e ia. The o he me hod in ol es simila sma p obes capable o iden i ying cellula me aboli es. Combined, hese wo me hods may p o ide a selec i e labeling o molecules associa ed wi h H. in luenzae bio ilms, enabling hei apid 18 diagnosis in COPD lung and e en in o he condi ions. Al hough he e a e s udies o NTHi bio ilms and hei pe sis ence du ing in ec ion, hey come om OM models and li le in o ma ion exis s on hese bio ilms in COPD (Sho e al., 2021). Mo e esea ch on NTHi bio ilms is needed in o de o cha ac e ize he bio ilm o ma ion capaci y and he bac e ia-hos in e ac ions ha will allow selec ing an i-bio ilm he apeu ic a ge s helping in he diagnosis and ea men o COPD pa ien s (Weeks e al., 2021). 2.5 An imic obial Resis ance (AMR) AMR is an eme ging p oblem wo ldwide, con ibu ing s ongly o deadly bac e ial in ec ions (Jo ge e al., 2019). In 2019, i is es ima ed ha AMR was esponsible o a leas 1.27 million dea hs wo ldwide and by 2050 he numbe is p edic ed o inc ease d ama ically o 10 million annual dea hs (O’Neill, 2016). AMR is a na u al p ocess in which mic oo ganisms (such as bac e ia, ungi, i uses and pa asi es) de elop de ense mechanisms o coun e ac he le hal e ec s o an imic obials (e.g. an ibio ics, an i ungals, an i i als, an imala ials) gi ing ise o mul id ug- esis an o ganisms (MDR) o “supe bugs” (Aslam e al., 2018; CDC, 2021; WHO, 2017). Howe e , he excessi e o inapp op ia e use o an imic obials accele a es he AMR p ocess, which leads o he dissemina ion o hese mic oo ganisms and hei esis ance mechanisms (WHO, 2017). Acco ding o he WHO, an ibio ic esis ance is, oday, one o he g ea es h ea s o public heal h, de elopmen , and ood secu i y (WHO, 2020). Al hough an ibio ics play a c ucial ole in he ea men o in ec ions, due o hei o e use and inadequa e use, i is becoming a p oblem wo ldwide (Jo ge e al., 2019). Wo yingly, i an ibio ics lose hei e ec i eness al oge he , he abili y o ea in ec ions and public heal h will be comp omised (CDC, 2020). The main causes o he eme gence o an imic obial esis ance a e: (1) he excessi e and unnecessa y use o an ibio ics in li es ock, ish a ming, and ag icul u e; (2) o e consump ion o an ibio ics due o medical o e p esc ip ion, sel -medica ion, inco ec an ibio ic use, and o e - he-coun e accessible an ibio ics; (3) lack o s anda d guidelines o an ibio ic use; (4) poo in ec ion con ol in hospi als and clinics; (5) lack o apid labo a o y es s; (6) lack o de elopmen o new an ibio ics; (7) lack o hygieniza ion and sani a ion p ac ices; (8) access o coun e ei d ugs; (9) elease o unme abolized an ibio ics o hei esidues in o he en i onmen (Aslam e al., 2018; CDC, 2019b; Jo ge e al., 2019). The e a e ou gene al mechanisms o bac e ial esis ance, which a e (1) limi ed up ake o a d ug, o example in g am-nega i e bac e ia, he limi a ion o d ug pe meabili y due o he p esence o an ou e memb ane, o by mu a ions in he numbe , ype and size o po ins p esen in he ou e memb ane, which may be es ic ed he en y o hyd ophobic an ibio ics such as β-lac ams, luo oquinolones, e acyclines, 19 and chlo amphenicol. In g am-posi i e bac e ia, such as S. au eus , he e is g ea e pep idoglycan syn hesis wi h mo e D-Ala-D-Ala esidues ha bind o he an ibio ic ancomycin, p e en ing i s binding o he a ge si e (C Reygae , 2018; Ch is aki e al., 2019; Lowy, 2003); (2) d ug a ge modi ica ion. Changes occu in he an ibio ic binding a ge s p esen in bac e ial cells, due o mu a ions, such as changes in penicillin binding p o eins (PBP) ha con e esis ance o β-lac am an ibio ics o by enzyma ic ac ion, such as 23S RNA me hyla ion ha con e s c oss- esis ance o mac olides, and lincosamides (C Reygae , 2018; Ch is aki e al., 2019); (3) d ug inac i a ion by deg ada ion. Fo example, he des uc ion o he an ibio ic by enzymes , such as β-lac amases , o by he ans e o a chemical g oup, called ans e ases (e . g ., ace yl, phospho yl, and adenyl g oups) (C Reygae , 2018); and (4) d ug e lux sys ems wi h he unc ion o pumping oxic molecules ou o he cell o egula e he in e nal bac e ial en i onmen (Ch is aki e al., 2019). In addi ion, an ibio ic esis ance can also be pe o med h ough mechanisms o ho izon al gene ans e h ough plasmids, ansposons o in eg ons o being an in eg al pa o bac e ial ch omosomal DNA (M. Kyd e al., 2011). Recen ly, he e has been an inc easing de elopmen o bac e ial esis ance o he main an ibio ics used o ea in ec ions (Aslam e al., 2018; Sho e al., 2021). This esis ance comp omises he e ec i eness o he ea men and leads o he selec ion o esis an subpopula ions ha can make COPD exace ba ions e en mo e pe sis en and se e e. Fo example, in exace ba ions, he o e use and widesp ead use o an ibio ics o he ea men o pa ien s in p ima y and seconda y heal h ca e con ibu es la gely o he esis ance o bac e ia (Beasley e al., 2012; CDC, 2019a; Sho e al., 2021). An ibio ic ea men is no necessa y in all pa ien s wi h COPD and a oiding i s unnecessa y use is impo an o limi he de elopmen o an imic obial esis ance (MacLeod e al., 2021). An ibio ics should be p esc ibed in case o mode a e o se e e exace ba ion and, acco ding o he GOLD pa ame e s, pa ien s should ha e h ee symp oms: inc eased dyspnea, spu um olume, and spu um pu ulence; ha ing wo symp oms, one o which is inc eased spu um pu ulence; o needing en ila ion (in asi e and non-in asi e) (GOLD, 2021; Nissly & P asad, 2014; Siddiqi & Se hi, 2008). The main an ibio ics used in hese si ua ions a e he ad anced mac olides (azi h omycin, cla i h omycin), ke olide ( eli h omycin), cephalospo in (ce u oxime, ce podoxime o ce dini ), doxycycline, ime hop im/sul ame hoxazole, luo oquinolone (moxi loxacin, gemi loxacin, le o loxacin), and amoxicillin/cla ulana e (Siddiqi & Se hi, 2008). A sys ema ic e iew o de e mine he p e alence, pa e ns, isk ac o s, and consequences o AMR in COPD e ealed ha a leas one o he h ee bac e ia H. in luenzae , S. pneumoniae , and M. ca a halis p esen in COPD pa ien s exhibi s high le els o esis ance o a leas one o he an ibio ics 20 amoxicillin, doxycycline, and cla i h omycin, which may con ibu e o ea men ailu e and eme gence o esis ance (D. Smi h e al., 2022). Acco ding o GOLD, 2022, ea men o azi h omycin and cla i h omycin o one yea in pa ien s p one o exace ba ions educes he isk o exace ba ions compa ed wi h usual ca e (GOLD, 2022). Howe e , long- e m ea men wi h mac olides, namely azi h omycin and cla i h omycin, can inc ease bac e ial esis ance o his ype o an ibio ics (Y. Cui e al., 2018). Mac olide esis ance can be caused by modi ica ion o mac olide a ge sequences, as well as mac olide e lux pump sys ems (Djamin e al., 2020). In addi ion, ke olides can also be inhibi ed by he Ac AB e lux pump p esen in H. in luenzae , and he o e p oduc ion o e lux pumps by P. ae uginosa in COPD con ibu es o esis ance mechanisms ac ing on he inhibi ion o luo oquinolones, -lac ams, aminoglycosides, and polymyxin B (M. Kyd e al., 2011). Acquisi ion o exp ession o β-lac amases is a common esis ance mechanism ha causes β- lac amic ing des uc ion o β-lac am an ibio ics. H. in luenzae , M. ca a halis , and P. ae uginosa , p oduce his enzyme and, he e o e, he use o hese an ibio ics, such as amoxicillin, o ea COPD ela ed in ec ions is no ecommended. In H. in luenzae , inhibi ion o β-lac ams occu s ei he h ough he p oduc ion o β-lac amases o ia he al e a ion o PBPs. Mos s ains only ha e one mechanism, bu many s ains ha e bo h and a e known o be β-lac amase-posi i e amoxicillin cla ulana e- esis an . Also, he al e a ions in po ins, men ioned abo e, may be in ol ed in he esis ance mechanisms in COPD, whe e, o example, he al e a ion in po in 2 p esen in H. in luenzae pa ially con ibu es o he esis ance o ampicillin, penicillin, cephalo hin, and chlo amphenicol (M. Kyd e al., 2011). In iew o he esis ance mechanisms exp essed by bac e ia, namely he p oduc ion o β- lac amases, al e a ion o memb ane pe meabili y, e lux pump sys ems, and mu a ions in mic obial a ge s, he selec ion o an imic obial he apy o exace ba ions is a challenge since i is necessa y o ob ain mic obial clea ance and a oidance o mic obial s imula ion (M. Kyd e al., 2011; Sho e al., 2021). To ci cum en his ad e si y, mucoly ic agen s a e cu en ly being p esc ibed on a la ge scale by clinicians as hey dec ease mucus iscosi y and inc ease i s luidi y, helping wi h mic obial clea ance (Papi e al., 2020; Weeks e al., 2021). Speci ically, e dos eine can be used as an adjunc o he apy in COPD as i has a signi ican po en ia ing e ec o an ibio ics agains ch onic espi a o y in ec ions (Papi e al., 2020). 2.6 The Role o H. in luenzae Bio ilms in AMR The o ma ion o bio ilms is a o m o AMR and is conside ed a ecu ing p oblem due o hei pe sis ence in in ec ions (Jo ge e al., 2019; Welp & Bombe ge , 2020). Bio ilms p o ec bac e ia and 21 make hem ole an o an ibio ics, hus hampe ing he clinical ea men o a bio ilm ela ed in ec ion (Mel on & Ande son, 2019). In bio ilms, due o poo g ow h condi ions, i is common o ind ole ance in esiden bac e ia. Tole ance can be de ined as he abili y o he mic oo ganism o su i e a b ie exposu e o high an ibac e ial concen a ions. The e o e, he an ibio ic akes longe o kill he bac e ia. This happens because bio ilms ha e a g adien o oxygen and nu ien s, which makes he bac e ia ha a e in he lowe laye s ha e less access o hem. This phenomenon induces hese bac e ia o educe hei me abolic ac i i y and en e a slow g ow h o s a e o do mancy, gi ing ise o he pe sis e cell pheno ype. When a bac e ial subpopula ion has a pe sis en pheno ype, al hough he an imic obial can elimina e he o he cells, he subpopula ion su i es he ea men and can g ow again, causing he in ec ion o elapse. This pe sis ence pheno ype is one o he main causes o he ine iciency o some an imic obials (Jo ge e al., 2019). In ac , a p o eomics s udy indica ed ha H. in luenzae bio ilms in a semi-do man s a e ha e educed cellula me abolism and p o ein syn hesis (Pos e al., 2014). This al e ed cellula ac i i y enhances he limi ed di usion o d ugs h ough he bio ilm ECM componen s, and he dec eased p o ein syn hesis minimizes he ac i i y o p o ein syn hesis inhibi o an ibio ics, such as mac olides (Sho e al., 2021). In bio ilms, he ma ix ha su ounds hem also con ibu es o hei high esis ance/ ole ance o ex e nal s esses, namely he ac ion o an imic obials and he immune sys em (Jo ge e al., 2019). The p oduc ion o a hick ECM by H. in luenzae con ibu es o esis ance o neu ophils and NETs, blocks mac ophage access o bio ilm cells, hus p e en ing phagocy osis, and aids in clea age o immunoglobulin (IgA), which is conside ed a majo componen o he immune sys em and a i s -line de ense mechanism o he mucosal su ace, ia IgA p o eases. The eDNA belonging o he ECM is also an impo an componen o an ibio ic esis ance, because i s imula es bio ilm o ma ion and binds o human β-de ensins p esen in g ea e amoun s in COPD compa ed o heal hy indi iduals, educing hei an imic obial p ope ies (Sho e al., 2021). H. in luenzae is ole an o imipenem and, in a bio ilm communi y, his ole ance can be s eng hened due o he di e en ial beha io o bio ilm cells, such as luc ua ing gene exp ession and cell me abolism ha can lead o he de elopmen o he e o esis an cell pheno ypes and pe sis ence (Sho e al., 2021). In e es ingly, β-lac am an ibio ics, p obably due o a s ess esponse, induce he o ma ion o H. in luenzae bio ilms. Fu he mo e, in polymic obial bio ilms, M. ca a halis has he abili y o sec e e β-lac amases, which con e p o ec ion o H. in luenzae om ampicillin ea men , and he signaling molecule AI-2 om H. in luenzae induces esis ance o M. ca a halis o cla i h omycin and ime hop im- sul ame hoxazole (Jo ge e al., 2019). The syne ge ic in e ac ion o he in i o polymic obial bio ilm o 22 bo h H. in luenzae and S. pneumoniae can also p omo e p o ec ion om he e ec s o an ibio ics, since he p oduc ion o β-lac amases by H. in luenzae and he p oduc ion o ECM by bo h bac e ia we e able o p o ec S. pneumoniae bio ilms om amoxicillin ea men in chinchillas’ middle ea in OM models (Sho e al., 2021). NTHi bio ilms in COPD lung a e cu en ly being s udied, bu mos a ailable in o ma ion on he bio ilm li e o hese bac e ia is gi en h ough OM s udies o in i o bac e ial monocul u e models (Weeks e al., 2021). NTHi bio ilms exhibi a se ies o mechanisms o esis ance and ole ance o se e al commonly used an ibio ics (Table 2). Fo example, in OM, he an ibio ics cip o loxacin, azi h omycin, and amoxicillin, which a e clinically e ec i e o plank onic s a es o NTHi, a e ine ec i e o NTHi bio ilms. In addi ion, he e ec o he an ibio ics chlo hexidine, glucu ona e, ampicillin, and cip o loxacin is also ine ec i e due o he ich eDNA ma ix (Weeks e al., 2021). A ecen s udy wi h a COPD model o e e lung exposed o ciga e e smoke showed ha NTHi bac e ia ha e he abili y o agg ega e and exp ess genes ela ed o g ow h, QS, and en i onmen al and oxida i e s ess ole ance, which induces o ma ion and pe sis ence o NTHi bio ilms (Hun e al., 2020). Thus, wi h he inc ease in AMR, i is necessa y o ind new ways o comba hese e sa ile and adap able mic oo ganisms. The e o e, i is essen ial o s udy and explo e he mechanisms o bac e ial esis ance, cha ac e ize he bio ilm o ma ion capaci y o NTHi, iden i y he hos -pa hogen in e ac ions in COPD, as well as an i-bio ilm he apeu ic a ge s. These s a egies can ha e a posi i e impac on diagnosis and ea men , no only in COPD, bu also in e ms o o he diseases, wi h he ul ima e goal o minimizing his g ea es sou ce o mo ali y and mo bidi y (C Reygae , 2018; Weeks e al., 2021). 23 Table 2 - Summa y o NTHi bio ilm esis ance and ole ance mechanisms and hei espec i e model and expe imen al e ec s (adap ed om Weeks e al., 2021). Mechanism and D ug Model/Expe imen Expe imen al e ec s Ci a ion Gen amycin ole ance Clinical CF1 NTHi isola es adhe ed o human ai way epi helia Bio ilms su i ed ea men wi h high gen amycin concen a ion. (S a ne e al., 2006) β-lac am an ibio ics & ca bohyd a e me abolism NTHi bio ilms g own on ai way epi helia Inc ease in ca bohyd a e me abolism gene exp ession in esponse o sub-MIC ampicillin and amoxicillin. (Wu e al., 2014) eDNA- ich EPS ma ix p o ec s agains an imic obials 8 clinical NTHi isola es s a ic bio ilms in i o Biocide esis ance media ed by he cohesi e and p o ec i e p ope ies o he bio ilm ma ix. (Izano e al., 2009) Low me abolic ac i i y p o ec s bio ilms agains β- lac am an ibio ics Me abolomic and p o eomic analysis o 814 p o eins ac oss bio ilm and plank onic s ains. 127 di e en ially exp essed p oduc s sugges ed ha NTHi bio ilms su i e β- lac am an ibio ics in a do man s a e wi h dec eased ene gy me abolism and p o ein syn hesis. (Pos e al., 2014) 2.7 AMPs agains bac e ia Bac e ial esis ance o an ibio ics, which a e one o he main ways o ea ing in ec ions, is a majo conce n in mode n medicine. Cu en ly, he slow pace o disco e y and de elopmen o new an ibio ics in he ace o he apid sp ead o esis ance by hese mic oo ganisms o hese d ugs causes se ious impac s on he economy and public heal h (Aslam e al., 2018; He nández-A is izábal & Ocampo-Ibáñez, 2021). The e o e, i is u gen o ind new an imic obial d ugs agains bac e ia. AMPs a e mul i unc ional molecules o he inna e immune sys em o p oka yo ic and euka yo ic o ganisms ha ac agains in ec ions caused by bac e ia, ungi, i uses, and some p o ozoa (He nández- A is izábal & Ocampo-Ibáñez, 2021; Wu e al., 2018). Gene ally, in na u e, he di e en ypes o AMPs a e cons i u ed by ou , six, o eigh cys eines in e connec ed by disul ide b idges ha con e s abili y, ha e be ween 12 and 50 amino acids, α-helical s uc u e, a e mos ly ca ionic, and show amphiphilici y (Dena di e al., 2022; He nández-A is izábal & Ocampo-Ibáñez, 2021; H. Wang e al., 2022). The α- 1 CF – Cys ic Fib osis 30 ANOVA and wo-way ANOVA using Bon e oni's mul icompa ison es . The signi ican di e ences be ween he g oups a e ep esen ed in he g aphs by he as e isk (*) o he ca dinal (#) symbols, whe e */# equals p < 0.05, **/## equals p < 0.001, ***/### equals p < 0.001, and ****/#### equals p < 0.0001. 31 Chap e 4. Resul s and Discussion Chap e 4 is composed o he desc ip ion and depic ion o he esul s ob ained, hei c i ical analysis as well as hei discussion. 4.1 Impac o media enewal on he g ow h o H. in luenzae bio ilm To s udy he impac o g ow h media enewal in he bio ilm o ma ion o H. in luenzae , 48 h bio ilms we e o med, wi h and wi hou media eplacemen a 24 h. Based on he analysis o Figu e 4, i is possible o e i y ha , when he e is no eplacemen o media a 24 h, he e a e no cul u able bac e ia a 48 h. On he o he hand, when he medium is eplenished a 24 h, he e a e cul u able bac e ia a 48 h. The esul s sugges ha sBHI medium eplacemen a ec s he amoun o cul u able bac e ia p esen in he bio ilm, since he e a e s a is ically signi ican di e ences be ween H. in luenzae coun s wi hou sBHI medium eplacemen compa ed o H. in luenzae coun s wi h sBHI medium eplacemen (p < 0.0001) (Figu e 4). These esul s a e in ag eemen wi h hose p e iously ob ained by ou esea ch g oup (da a no published). Figu e 4 - Quan i ica ion o cul u able H. in luenzae in 48 h bio ilms g own in sBHI (Lio ilchem), wi h and wi hou media eplacemen a 24 h. On he g aph, s anda d de ia ions a e indica ed by e o ba s. S a is ically signi ican di e ences: **** p<0.0001. The de elopmen o bio ilms and hei bac e ial and inal chemical composi ion a e a ec ed by he amoun , a ailabili y, and ype o nu ien s and by hei pe usion o he cells inside o he bio ilm (Bowden & Li, 1997; Dunne, 2002). Hence, he absence o cul u able cells obse ed could be explained by bac e ial un iabili y caused by consump ion and consequen deple ion o nu ien s by he bac e ia o e ime, such as he g ow h ac o s hemin and NAD. H. in luenzae cells a e no well adap ed o su i al unde non-op imal condi ions, equi ing an en iched cul u e medium, namely BHI, wi h adequa e supplemen a ion o hemin and NAD (Poje & Red ield, 2003). 32 The oxic me aboli es p oduced by he bac e ia hemsel es a e also ano he ac o ha could explain he absence o cul u able bac e ia. Dunne desc ibes ha , in bac e ial bio ilms, bac e ial g ow h is limi ed by he exp ession o quo um-sensing molecules eleased in esponse o nu ien limi a ion and he accumula ion o oxic by-p oduc s (Dunne, 2002). H. in luenzae is subjec o oxida i e s ess esul ing om i s own me abolism du ing ae obic g ow h (Ha ison e al., 2012). Oxida i e s ess can cause damage o all ypes o bac e ial cell componen s including p o eins, lipids, and DNA, leading o bac e ial dea h (Ez a y e al., 2017; Fasnach & Polacek, 2021). A hi d hypo hesis o explain he absence o cul u able cells is ha H. in luenzae could be iable bu no cul u able (VBNC). In he VBNC s a e, he bio ilm bac e ia a e ali e bu do no g ow o di ide, and hei p esence is no de ec able by con en ional me hods ( hey do no o m colonies on solid media and do no change he appea ance o he b o h) (Năşcuţiu, 2010). The VBNC s a e is a unique su i al s a egy in he ace o ad e se en i onmen al condi ions such as an ibio ic p essu e, high o low empe a u e, s a a ion, chlo ina ion, pH change, and oxygen s ess (Ramamu hy e al., 2014). I is known ha VBNC has been associa ed wi h longe pe iods o bio ilm o ma ion and, u he mo e, he cen al a eas o he same end o be hypoxic, wi h limi ed esou ces, and acidic due o he deposi s o me abolic esidues ha consequen ly induce he VBNC s a e (Ay ape yan e al., 2018; Cas o e al., 2022). The VBNC s a e can be e e sible unde a o able g ow h condi ions wi h an ideal ene gy sou ce and s oichiome ic a io o ca bon o ino ganic elemen s (Ramamu hy e al., 2014). 4.2 In luence o G ow h Media B and in H. in luenzae Bio ilm Fo ma ion P e ious assays wi hin he esea ch g oup ound beha io al a ia ions in he g ow h o H. in luenzae . The assays we e highly consis en o abou 5 mon hs, bu a iabili y s a ed o be mo e wo isome a e wa ds (da a no published). Based on Chap e 3 o he book " Haemophilus in luenzae p o ocols", g owing H. in luenzae on cheape g ow h media can cause hese g ow h p oblems as well as pla ing p oblems (Poje & Red ield, 2003). As such, he in luence o media b and on H. in luenzae bio ilm g ow h was es ed. 4.2.1 H. in luenzae C yo ial Va iabili y in Di e en B ands o G ow h Media Su p isingly, he a iabili y de ec ed in he g ow h o H. in luenzae by he esea ch g oup was suspec ed o co ela e wi h di e en c yo ials o he same s ain. Al hough all he ials we e p epa ed and s ocked a he same ime, epea ed use o some could be comp omising he cells. Fo his eason, he di e en ial g ow h be ween wo c yo ials was es ed, one o which was al eady in use and he o he was opened o he i s ime. In addi ion, i was also in es iga ed whe he he a iabili y in he g ow h o hese 33 c yo ials was dependen o no on he b and o g ow h media used, namely, Oxoid (mos expensi e), VWR (a e age p ice), and Lio ilchem (cheape and used in all p e ious s udies wi hin he g oup). Fo his s udy, 24 h and 48 h bio ilms o H. in luenzae (wi h and wi hou media eplacemen ) we e o med in sBHI o di e en b ands. The sBHI om VWR was disca ded a he beginning o he assay because HI 2 ( H. in luenzae om c yo ial 2, in use) did no g ow in i , hence no compa ison was pe o med o his b and. Howe e , his was in i sel an in e es ing esul , since i was possible o see ha he e is in ac di e ences in bac e ial g ow h dependen on he media b and and he c yo ial used. 4.2.1.1 24 h Bio ilms Figu e 5 illus a es he esul s o cul u able cells and o al biomass ob ained in he cul i a ion o 24 h H. in luenzae bio ilms. I is possible o obse e ha , in sBHI om Oxoid, he numbe o cul u able cells (Figu e 5-A) as well as he amoun o o al biomass o he bio ilm (Figu e 5-B) is simila o he wo c yo ials. In con as , wi h he sBHI om Lio ilchem, HI 2 has ewe cul u able cells (1 log) (p < 0.01) (Figu e 5-A) and d as ically lowe biomass (p < 0.0001) (Figu e 5-B) compa ed o HI 6 ( H. in luenzae om c yo ial 6, newly opened). In addi ion, i is obse ed ha he Oxoid b and eached a highe numbe o cul u able cells compa ed o he Lio ilchem b and (Figu e 5-A) and, in e ms o biomass (Figu e 5-B), sBHI om Oxoid pe o med equal o HI 6 and be e o HI 2. Figu e 5 - Quan i ica ion o cul u able H. in luenzae cells (A) and o al biomass (B) o 24 h H. in luenzae bio ilms g own in sBHI om Oxoid and Lio ilchem. S anda d de ia ions a e indica ed by e o ba s. S a is ically signi icancy: ** p < 0.001 and **** p < 0.0001. Abb e ia ions: HI 2 = H. in luenzae om he second c yo ial (in use), HI 6 = H. in luenzae om he six h c yo ial (newly opened). The g ow h di e ences obse ed sugges ha he p oli e a ion o H. in luenzae may be dependen on he b and o sBHI used, which could be ela ed o i s highe o lowe quali y. The esul s also showed ha he amoun o biomass (Figu e 5-B) does no di ec ly co ela e wi h he amoun o cul u able cells, since HI 6 p oduced a simila amoun o biomass in bo h b ands bu had a lowe amoun o cul u able 34 cells when g own in sBHI om Lio ilchem (Figu e 5-A). This simila i y in he amoun o biomass in bo h b ands can be explained by he CV me hod which, in addi ion o quan i ying iable cells, also quan i ies dead cells and ex acellula ma ix (Xu e al., 2016). In e es ingly, he amoun o biomass di e ed signi ican ly o he Lio ilchem b and. This phenomenon can be explained by he g ea e p oduc ion o ex acellula ma ix o HI 6 compa ed o HI 2, e en hough hey a e o he same s ain. This could indica e ha HI 2 su e ed modi ica ions de i ed om i s use ha caused cells o lowe hei abili y o p oduce ma ix. Addi ionally, i was e i ied ha bio ilms wi h 24 h ha e cul u able cells o H. in luenzae . Thus, he p oblems men ioned in he p e ious sec ion (4.1), such as lack o nu ien s and p oduc ion o oxic me aboli es by H. in luenzae ha can a ec he cul u abili y o he bio ilm, a e no ye p esen in bio ilms cul u ed o 24 h. 4.2.1.2 48 h Bio ilms wi hou Media Renewal Figu e 6 shows he same compa ison as be o e bu o 48 h bio ilms wi hou media enewal a 24 h. Compa ed o 24 h H. in luenzae bio ilms, he amoun o cul u able cells (Figu e 5-A) educed o all condi ions, excep o HI 2 wi h he sBHI om Lio ilchem (Figu e 6-A). This could indica e ha he cell g ow h o ial HI 2 was slowe han ha o ial HI 6 in Lio ilchem and, he e o e, hey managed o g ow a li le mo e om 24 h o 48 h, as hey could s ill be a he end o he exponen ial phase o in he s a iona y phase. In he o he h ee condi ions, as he cells could ha e g own as e , hey could ha e al eady eached he dead phase o in he bio ilm de achmen s age, esul ing in lowe cell cul u abili y a 48 h. Fo sBHI om Oxoid, he amoun o cul u able HI 2 cells was 2 logs lowe compa ed o HI 6 (p < 0.05) (Figu e 6-A) bu he amoun o biomass was iden ical o bo h c yo ials (Figu e 6-B). Fu he mo e, Figu e 6-A shows ha he e is a signi ican di e ence (p < 0.01) in he numbe o cul u able cells be ween HI 2 and HI 6 o sBHI om Lio ilchem. This can be explained by he same eason desc ibed abo e. In e ms o biomass, he e is a signi ican educ ion (p < 0.0001) be ween he wo c yo ials (Figu e 6-B) ha was also obse ed in he 24 h H. in luenzae bio ilms (Figu e 5-B) and discussed abo e. I is no ewo hy ha he amoun o cul u able cells and biomass di e s be ween b ands and c yo ials, excep o he amoun o biomass in c yo ials o sBHI om Oxoid (Figu e 6-B). 35 Figu e 6 - Quan i ica ion o cul u able H. in luenzae (A) and o al biomass (B) o 48 h H. in luenzae bio ilms g own in sBHI om Oxoid and Lio ilchem wi hou media eplacemen a 24h. S anda d de ia ions a e indica ed by e o ba s. S a is ically signi icancy: * p < 0.05 ** p < 0.01, and **** p < 0.0001. Abb e ia ions: HI 2 = H. in luenzae om he second c yo ial (in use), HI 6 = H. in luenzae om he six h c yo ial (newly opened). In e es ingly, cul u able cells o hese 48 h bio ilms wi hou media enewal we e obse ed o bo h b ands (Figu e 6-A), con a y o he ini ial expe ience (Figu e 4), which was pe o med wi h sBHI om Lio ilchem. This con i med he a iabili y p e iously obse ed by he esea ch g oup and elimina ed he ope a o as he possible causing ac o . This e en can be ela ed o he uni e sal s ess p o ein (UspA). Al hough lack o sBHI eplacemen is associa ed wi h loss o cul u abili y p obably due o de iciency o nu ien s and p oduc ion o oxic me aboli es, as discussed p e iously, i is known ha H. in luenzae can su i e due o inc eased exp ession o UspA. Acco ding o he li e a u e, when bac e ia a e exposed o s ess ul en i onmen al condi ions ha comp omise cell iabili y, UspA exp ession is inc eased, enhancing he bac e ial su i al a e (Sousa & McKay, 2001). In his case, he p obable s ess caused by nu ien deple ion and p oduc ion o eac i e oxygen species esul ing om i s own me abolism, which could comp omise cell iabili y, may ha e induced an inc ease in UspA exp ession. 4.2.1.3 48 h Bio ilms wi h Media Renewal Finally, Figu e 7 shows he same compa isons as be o e bu o 48 h bio ilms wi h media enewal a 24 h. I is possible o obse e ha he amoun o cul u able H. in luenzae cells o sBHI om Oxoid was signi ican ly highe (p < 0.001) (Figu e 7-A) in ial HI 2 han in ial HI 6. Rega ding o al biomass, he e was simila p oduc ion o bo h ials (Figu e 7-B). The amoun o cul u able H. in luenzae cells o sBHI om Lio ilchem was also highe (p < 0.001) (Figu e 7-A) in HI 2 han in HI 6. In con as , he amoun o biomass p oduced was signi ican ly lowe (p < 0.0001) (Figu e 7-B) in HI 2 compa ed o HI 6. This di e ence in he quan i ica ion o biomass be ween c yo ials o sBHI om Lio ilchem (p < 0.0001) (Figu e 7-B) has also been obse ed p e iously (Figu e 5-B and 6-B). 36 Figu e 7 – Quan i ica ion o cul u able H. in luenzae (A) and o al biomass (B) o 48 h H. in luenzae bio ilms g own in sBHI om Oxoid and Lio ilchem wi h media eplacemen a 24 h. S anda d de ia ions a e indica ed by he e o ba s. S a is ically signi icancy: *** p<0.001 and **** p<0.0001. Abb e ia ions: HI 2 = H. in luenzae om he second c yo ial (in use), HI 6 = H. in luenzae om he six h c yo ial (newly opened). Compa ing he esul s o 48 h bio ilms wi hou (Figu e 6) and wi h media enewal (Figu e 7), i is possible o e i y an inc ease in he numbe o cul u able cells o bo h c yo ials (Figu e 7-A) and o al biomass (Figu e 7-B) o sBHI om Oxoid. As e i ied in he ini ial expe imen (Figu e 4), he eplacemen o sBHI a 24 h showed again ha he es i u ion o nu ien s and he possible emo al o oxic me aboli es could be wha is he e in luencing cell cul u abili y. In conclusion, i was ound ha he e we e beha io al di e ences be ween HI 2 and HI 6 c yo ials, e en hough hey we e om he same s ain and es ablished a he same ime. Thus, i was seen ha ial HI 2, which had al eady been opened and consequen ly subjec ed o mo e empe a u e a ia ions, due o i s eezing and hawing, had a slowe bac e ial g ow h compa ed o he ial HI 6, which was opened o he i s ime o be used in his expe imen . Fu he mo e, H. in luenzae g ow h di e ences we e dependen on he b and o g ow h medium used, and a iabili y and poo e /slowe g ow h and biomass o ma ion we e mos no iceable o sBHI om Lio ilchem. The esul s o sBHI om Oxoid we e mo e consis en among he c yo ials es ed. Thus, he Oxoid b and was chosen o ca y on he emainde assays. 4.3 Popula ion Dynamics in Dual-species Bio ilms In bio ilms, he in e ac ion o bac e ia wi h each o he is ine i able. These in e ac ions can be coope a i e o compe i i e, and can in luence he g ow h and su i al o species, ha ing a spa ial and empo al impac on he o ma ion o a highly o ganized communi y (Giaou is e al., 2015). Th oughou he de elopmen o he bio ilm, mic oo ganisms a e dis ibu ed in a non- andom manne depending on 37 social in e ac ions and nu ien a ailabili y (W. Liu e al., 2018; Paula e al., 2020). In a mul ispecies bio ilm, he e a e in e ac ions ha bene i he whole g oup, such as he a ailabili y o syn hesized compounds and he escape o hos de enses, bu he e a e also ha m ul in e ac ions, such as species elimina ion due o compe i ion om di e en coexis ing species (Reigada e al., 2021). In es iga ion o popula ion dynamics is essen ial o unde s anding in e ac ions be ween species, making possible he selec ion o d ugs ha can inhibi in e ac ions be ween species and hei abili y o o m mul ispecies bio ilms (Tikhomi o a & Kidd, 2013a). 4.3.1 Popula ion Dynamics o Dual-Species Bio ilms in G ow h Media o Di e en B ands Gi en he a iabili y obse ed p e iously ega ding he use o di e en media b ands in he g ow h o H. in luenzae bio ilms, a p elimina y assay was pe o med o check i he media b and had he same e ec s when S. au eus was mixed wi h H. in luenzae . In his way, 24 h and 48 h H. in luenzae bio ilms (single-species con ol) and H. in luenzae bio ilms wi h 1% o S. au eus (s a ing inoculum %) we e o med in sBHI om Lio ilchem and om Oxoid. 4.3.1.1 24 h Bio ilms The esul s o 24 h H. in luenzae bio ilms g own in sBHI om Lio ilchem show s a is ically signi ican ly lowe cul u able cells when compa ed o Oxoid (p < 0.0001) (Figu e 8-A), in con as o he amoun o biomass, which is simila ) (Figu e 8-B). This ac occu s o he same easons explained in he p e ious s udy, namely he g ea e p obabili y o sBHI om Lio ilchem o cause g ow h p oblems. In e ms o o al biomass, al hough equal alues we e ob ained, bio ilm composi ion may be di e en , as i may con ain a g ea e amoun o dead cells o sBHI om Lio ilchem due o he lowe cul u able cells coun s. The esul s o H. in luenzae bio ilms g own wi h 1% S. au eus showed no signi ican di e ence in he numbe o cul u able cells (Figu e 8-A) o biomass (Figu e 8-B) be ween he wo b ands. In e es ingly, he amoun o cul u able H. in luenzae cells was highe in he double-species bio ilm when compa ed wi h he single-species con ol o sBHI om Lio ilchem (Figu e 8-A). Thus, S. au eus may ha e a posi i e in luence on H. in luenzae g ow h, possibly by p o iding he necessa y nu ien s o main ain he cul u abili y o H. in luenzae cells. In ac , H. in luenzae and S. au eus we e al eady epo ed o es ablish a coope a i e ela ionship in polymic obial in ec ions, in which H. in luenzae eached a g ea e numbe o colonies when S. au eus was he esiden colonize due o nu ien s ha he la e p o ided, such as hemin and NAD (Nai e al., 2014). In his case, he mechanism o ob aining nu ien s by H. in luenzae was di e en since sBHI does no ha e e y h ocy es o he hemolysins p oduced by S. au eus o cause 38 hei lysis and elease hemin and NAD. In e ms o o al biomass, his was main ained o all bio ilms in he di e en b ands. Howe e , i s composi ion may a y because, o he easons explained p e iously. Figu e 8 - Quan i ica ion o cul u able H. in luenzae (A) and o al bio ilm biomass (B) om 24 h bio ilms o H. in luenzae (HI) and o H. in luenzae wi h 1% o S. au eus (1% SA) g own in sBHI om Lio ilchem and Oxoid. S a is ically signi ican di e ences: **** p < 0.0001. S anda d de ia ions a e indica ed by e o ba s. 4.3.1.2 48 h Bio ilms wi hou Media Renewal In he 48 h H. in luenzae bio ilms wi hou sBHI eplacemen , a g ea e numbe o cul u able cells we e ob ained o sBHI om Oxoid (p < 0.0001) (Figu e 9-A). As o he quan i ica ion o he o al biomass, he H. in luenzae bio ilms cul i a ed in sBHI om Lio ilchem eached he highes amoun o biomass (p < 0.0001) (Figu e 9-B), bu hey we e he ones ha ob ained he lowes numbe o cul u able cells (Figu e 9-A), he e o e i can be in e ed ha he amoun o biomass comes mainly om dead cells and/o ma ix. In bio ilms wi h wo species, he numbe o cul u able H. in luenzae cells was lowe o sBHI om Lio ilchem (p < 0.001) (Figu e 9-A), as well as he o al biomass (p < 0.0001) (Figu e 9-B). Figu e 9 - Quan i ica ion o cul u able H. in luenzae (A) and o al bio ilm biomass (B) om 48 h bio ilms o H. in luenzae (HI) and o H. in luenzae wi h 1% o S. au eus (1% SA) g own in sBHI om Lio ilchem and Oxoid wi hou media eplacemen a 24 h. S a is ically signi ican di e ences a e ep esen ed by *** p < 0.001 and **** p < 0.0001. S anda d de ia ions a e indica ed by e o ba s. 39 In his case, i is again no o ious ha he p esence o S. au eus esul ed in a g ea e amoun o cul u able H. in luenzae cells. As desc ibed abo e, S. au eus can p omo e he cul u abili y o H. in luenzae due o nu ien supply. I is also impo an o no e ha when S. au eus is p esen , he amoun o biomass is lowe in sBHI om Lio ilchem compa ed o he single-species con ol. This may be due o S. au eus making H. in luenzae p oduce less ma ix, o example. Compa ing H. in luenzae bio ilms a 24 h (Figu e 8-A) and a 48 h wi hou media enewal, i was obse ed ha he e was a signi ican dec ease in cell cul u abili y, namely o sBHI om Lio ilchem (app oxima ely 3 logs) as well as om Oxoid (app oxima ely 1.5 logs) (Figu e 9-A). Fu he mo e, while in he 24 h double-species bio ilms (Figu e 8-A), he numbe o cul u able cells was simila o he di e en b ands, in his case, he numbe o cul u able cells was lowe (app oxima ely 1 log) o Lio ilchem (Figu e 9-A). Thus, i appea s ha he lack o sBHI enewal impai ed he cul u abili y o H. in luenzae . In he quan i ica ion o bio ilm biomass (Figu e 9-B), i was shown ha , o e he 48 h wi hou sBHI eplacemen , he amoun o biomass p oduced was lowe o bo h bio ilms in compa ison o he 24 h bio ilms (Figu e 8-B). Fu he mo e, he amoun o biomass showed mo e a iabili y be ween he Lio ilchem and Oxoid b ands in he 48 h bio ilms (Figu e 9-B), which did no happen in he 24 h bio ilms (Figu e 8-B). Resul s once again show ha he quali y o he g ow h media coupled wi h he lack o sBHI eplacemen can in luence H. in luenzae g ow h. This is mainly shown when he 24 h and 48 h bio ilms a e compa ed. Fo example, when H. in luenzae bio ilms a e cul i a ed in sBHI om Lio ilchem, hey o en each a lowe numbe o cul u able cells in ela ion o sBHI om Oxoid and, when hey a e cul i a ed o a pe iod o 48 h wi hou media eplacemen , hey end o dec ease cell cul u abili y. In e es ingly, S. au eus posi i ely in luenced he amoun o cul u able H. in luenzae cells, which leads o he in e ence ha S. au eus may play an impo an ole in p o iding nu ien s necessa y o he de elopmen o H. in luenzae . 4.3.1.3 48 h Bio ilms wi h Media Renewal In he 48 h H. in luenzae bio ilms wi h media enewal a 24 h, he numbe o cul u able cells was highe o sBHI om Oxoid (p < 0.01) (Figu e 10-A). Rega ding he amoun o biomass, he e was a highe p oduc ion o sBHI om Lio ilchem (p < 0.0001) (Figu e 10-B) and, as p e iously explained, i may come om dead cells and/o ma ix, since he e was less cells cul u ed. Fo double-species bio ilms, he numbe o cul u able H. in luenzae cells (p < 0.01) (Figu e 10-A) and he amoun o biomass (p < 0.001) (Figu e 10-B) was g ea e o sBHI om Oxoid. Fu he mo e, i was obse ed ha he H. in luenzae bio ilm had a highe amoun o biomass han he double-species bio ilm (Figu e 10-B). 46 he double-species bio ilm had a highe biomass p oduc ion compa ed o he 24 h single-species con ol (p < 0.0001) (Figu e 15-B). Figu e 15 - Quan i ica ion o cul u able H. in luenzae and S. au eus cells (A) and quan i ica ion o biomass (B) om double-species bio ilms in which S. au eus was he second colonize o 24 h. Con ols co espond o 48 h H. in luenzae and 24 h S. au eus single-species bio ilms. The s a is ical di e ences a e depic ed as: * p < 0.05 and **** p < 0.0001. S anda d de ia ions a e indica ed by e o ba s. The inse ion o S. au eus in o he p e-es ablished H. in luenzae bio ilm a e 24 hou s had no in luence on adhesion o bio ilm o ma ion because he numbe o cul u able cells ob ained was simila o he con ols and he biomass was simila o he i s colonize . Gi en ha S. au eus is known o be able o co-colonize wi h o he bac e ia, namely wi h H. in luenzae , when i was in oduced in o he p e- es ablished bio ilm, i may ha e de eloped a coope a i e ela ionship wi h H. in luenzae (Nai e al., 2014). Al hough coloniza ion o he seconda y species had no signi ican e ec s on he exis ing bio ilm o he o he species, Esin and colleagues ound ha p e-coloniza ion by H. in luenzae p omo ed adhesion and bio ilm o ma ion o S. au eus in ympanos omy ubes (TTs), sugges ing ha he e is a p ocess ac i e and syne gis ic in which H. in luenzae p omo es he a achmen o S. au eus , such as cell- o-cell signaling, which is a c i ical ea ly s ep in bio ilm o ma ion (Esin e al., 2015). In conclusion, al hough he li e a u e desc ibes ha he p e-es ablishmen o he bio ilm by one species can p omo e he adhesion, bio ilm o ma ion, and g ow h o o he bac e ia, in his s udy, i was seen ha he e was no in luence o he colonize species on he adhesion and bio ilm o ma ion o ano he species. 47 4.5 In e species In luence o Exop oduc s on Bio ilm Fo ma ion Bio ilm- o ming bac e ia can elease EPS such as polysaccha ides, small amoun s o p o eins, DNA, and o he molecules in o he su ounding en i onmen ha can help o ha m o he bac e ia p esen in he bio ilm, a ec ing hei e olu iona y dynamics (Nadell e al., 2008). In addi ion, h ough he QS cell- cell communica ion sys em, he e is he p oduc ion and elease o AIs ha p o ide a pla o m o in a- and in e species c oss alk and he con ol o di e se bac e ial ac i i ies, such as he o ma ion and de elopmen o he bio ilm, sec e ion o i ulence ac o s, and su i al in a cons an ly changing en i onmen (Sah een e al., 2022). CFS is he liquid esul ing om he emo al o bac e ial cells and con ains me aboli es om bac e ial g ow h (some lis ed abo e) and esidual nu ien s om he medium used and has been s udied mainly due o i s an imic obial po en ial agains bac e ia. E en hough he e is no li e a u e abou he e ec o S. au eus and H. in luenzae CFS on H. in luenzae and S. au eus bio ilms, espec i ely, some expe imen s wi h CFS agains hese s ains ha e been done. Fo example, in a s udy by Coleman and colleagues, on he in i o abili y o alpha haemoly ic s ep ococcus (AHS) and lac obacilli (LBs) om indigenous Aus alian child en o inhibi he g ow h o espi a o y pa hogens, i was shown ha LBs- ee cells we e e ec i e in inhibi ing H. in luenzae , educing he ini ial concen a ion by 3 logs (Coleman e al., 2022). Ano he s udy on he use o ex acellula ex ac s o lac ic acid bac e ia (LAB) and bi idobac e ia o he inhibi ion o S. au eus demons a ed ha CFS, mainly p oduced by LAB, d as ically inhibi ed he g ow h o he pa hogen S. au eus (Ho & Liong, 2014). On he o he hand, exop oduc s can also bene i he bio ilm. A s udy by Hou e al. ound ha he bio ilm o an EPS-p oducing S. au eus ATCC 12600 exhibi s a highe esis ance o mechanical p essu e han ha o a non-EPS-p oducing S. au eus 5298, due o an immedia e inc ease in polysaccha ide con en (Hou e al., 2018). Ano he example includes he educ ion o pene a ion by some an ibio ics in o he S. au eus bio ilm due o exop oduc s (Yin e al., 2019). Addi ionally, he EPS ma ix o med by H. in luenzae p o ides a p o ec i e ba ie ha allows hem o su i e he s ess caused by an imic obial agen s and he hos 's immune esponse (Tikhomi o a & Kidd, 2013b). In o de o s udy he in luence o exop oduc s om one species on bio ilms o ano he species, CFS om H. in luenzae was added o 48 h S. au eus bio ilms a 24 h and ice e sa. The esul s show ha he amoun o cul u able cells om HI bio ilms wi h SA CFS and om SA bio ilms wi h HI CFS was sligh ly highe (p < 0.0001) (Figu e 16) when compa ed o he espec i e con ols. Howe e , hese di e ences a e no biologically signi ican , gi en he alue p oximi y. 48 Figu e 16 – E ec o SA CFS on HI cul u able bio ilm cells (A) and o HI CFS on SA cul u able bio ilm cells (B). The s a is ically signi ican di e ences a e ep esen ed as: **** p < 0.0001. S anda d de ia ions a e indica ed by e o ba s. The bac e ial iabili y o he 48 h bio ilms may be ela ed o he addi ion o CFS.This could be due o he educ ion in he numbe o cells compe ing o nu ien s, as a esul o emo ing plank onic cells and adding CFS om 24 h bio ilms ha s ill ha e nu ien s and EPS o he species (such as polysaccha ides, p o eins, eDNA, and lipids) ha enhance he o e all s uc u e o he bio ilm (Di Ma ino, 2018; Mani-López e al., 2022). Compa ed o he con ols, he exop oduc s o S. au eus and H. in luenzae did no ha e a nega i e o posi i e in luence on H. in luenzae and S. au eus bio ilms, espec i ely (Figu e 16). This may ha e occu ed because exop oduc s p oduced by bio ilms du ing 24 h may no be a a high enough concen a ion o ha e an e ec on he amoun o cul u able cells. Ano he ac o is ha S. au eus and H. in luenzae bio ilms may simply no be suscep ible o he exop oduc s p oduced by he o he species. 4.6 Suscep ibili y H. in luenzae and S. au eus o AMPs The suscep ibili y o H. in luenzae and S. au eus o ou syn he ic AMPs, namely empo in A, achyplesin I, palm-KGK-PEG, and ci opin 1.1, was assessed by he MIC and MBC (Table 4). Resul s show ha he wo species ha e di e en pa e ns o suscep ibili y o he chosen AMPs. Tachyplesin I showed he bes bac e ios a ic and bac e icidal ac i i y agains bo h species. In H. in luenzae , he second mos ac i e AMP was palm-KGK-PEG, ollowed by ci opin 1.1 and, las ly, empo in A. In S. au eus , a e achyplesin I, empo in A was he second bes , ollowed by palm-KGK-PEG, and inally ci opin 1.1. To ensu e ha he assays we e well pe o med, cip o loxacin, which ac s agains bo h species, was used as a con ol. 49 Table 4 – An imic obial ac i i y o empo in A, achyplesin I, palm-KGKPEG, ci opin 1.1, and cip o loxacin agains H. in luenzae DSM 4690 (ATCC 33391, NCTC 8143) and S. au eus (ATCC 25923). MIC and MBC a e exp essed in mg/L. H. in luenzae S. au eus AMPs/An ibio ic MIC MBC MIC MBC Tempo in A 64 1024 16 128 - 256 Tachyplesin I 32 128 16 - 32 16 - 32 Palm-KGK-PEG 64 128 - 256 32 - 64 64 - 128 Ci opin 1.1 64 - 256 256 - 512 64 - 128 128 Cip o loxacin < 0.015 0.06 - 0.125 0.001 > 0.002 Acco ding o EUCAST abula ed b eakpoin alues, he MIC alues o cip o loxacin indica e ha H. in luenzae and S. au eus a e no esis an o his an ibio ic (EUCAST, 2022b). This is in acco dance o wha is desc ibed o hese s ains , hence he assays we e conside ed well execu ed (Chan e al., 2017; Kashe e al., 2020). All AMPs unde s udy ha e hei mechanism o ac ion a ge ing he bac e ial memb ane (Al es e al., 2016; Boland & Sepa o ic, 2006; C. Liu e al., 2018; Mangoni & Shai, 2009). Tachyplesin I was he AMP ha showed he g ea es e icacy agains H. in luenzae and S. au eus , which migh be explained by he ac ha , in addi ion o a ge ing he bac e ial memb ane, i also ac s a he in acellula le el, a ge ing DNA (C. Liu e al., 2018; Yonezawa e al., 1992; Yu e al., 2020). Fo achyplesin I, he MIC alues we e high (32 mg/L and 16 – 32 mg/L o H. in luenzae and S. au eus , espec i ely) (Table 4) compa ed o MIC alues ound in he li e a u e (0.8 - 12.5 mg/L) o G am-nega i e ( H. in luenzae ) and G am-posi i e ( S. au eus ) bac e ia (Xue e al., 2018). Repea ed AMP eezing and de os ing cycles o make aliquo s and i s p e ious use can ha e made he AMP suscep ible o deg ada ion, con ibu ing o lowe an imic obial ac i i y and consequen ly highe MIC alues (GenSc ip , 2022). On he o he hand, i is possible ha hese s ains may be esis an o achyplesin I. Howe e , his hypo hesis would ha e o be con i med h ough o he assays. Tempo in A showed g ea e an imic obial ac i i y agains S. au eus compa ed o H. in luenzae . This esul is consis en wi h he li e a u e, since empo ins a e mainly e ec i e and ac quickly agains G am-posi i e bac e ia. The ac i i y agains S. au eus p esen s MIC alues (16 mg/L) wi hin hose men ioned in he li e a u e, since gene ally he MIC o empo ins anges om 2.5 o 20 mg/L o G am- 50 posi i e bac e ia (Mangoni & Shai, 2009). Fo H. in luenzae , he MIC alue was subs an ially lowe han wha is published in he li e a u e o G am-nega i e bac e ia (100-400 mg/L) (Rosen eld e al., 2006). Ci opin 1.1 is conside ed o ha e a b oad spec um o ac ion agains bo h G am-posi i e and G am- nega i e bac e ia (Boland & Sepa o ic, 2006). In his s udy, ci opin 1.1 demons a ed g ea e an ibac e ial ac i i y agains he G am-posi i e bac e ia S. au eus . In suscep ibili y esea ches wi h S. au eus (ATCC 25923), ci opin 1.1 showed g ea e an imic obial ac i i y wi h lowe MIC and MBC alues (16 mg/L) (Jo ge e al., 2017; Neubaue e al., 2017) han he alues ob ained he e (MIC o 64 - 128 mg/L and MBC o 128 mg/L). The p oblems ha may be associa ed wi h he highe MIC and MBC alues ob ained a e he same as hose p e iously epo ed o he AMP achyplesin I. Palm-KGK-PEG is a pegyla ed lipopep ide ha demons a ed be e an ibac e ial ac i i y agains S. au eus compa ed o H. in luenzae . Acco ding o he li e a u e, Palm-KGK-NH2 (wi hou PEG) has a po en an ibac e ial ac i i y and is an excellen al e na i e o an ibio ics since i has a low p opensi y o inducing mic obial esis ance (Al es e al., 2016; A ahami & Shai, 2004). In addi ion, he associa ion o AMP wi h PEG has he ad an age o inc easing wa e solubili y, p o ec ing he pep ide cons i uen s om p o ease deg ada ion, p oducing la ge conjuga es ha a oid apid enal il a ion o p olong ci cula ion in he bloods eam, and u he dec easing he po en ial e ec s o cy o oxici y agains mammalian cells (Z. Cui e al., 2021). Simila ac i i y o his lipopep ide wi hou PEG agains S. au eus was ound in p e ious s udies whe e, mo eo e , he ac i i y agains G am-posi i e bac e ia was also s onge compa ed o G am- nega i e bac e ia (Al es e al., 2016; Paduszynska e al., 2019). Gi en ha achyplesin I demons a ed he bes bac e icidal and bac e ios a ic ac i i y agains H. in luenzae and S. au eus , i was conside ed he bes AMP o he ou AMPs es ed. 51 Chap e 5. Conclusions and Fu u e Wo k COPD, a ch onic, p og essi e, and incu able disease, is he hi d leading cause o mo ali y wo ldwide. The main causes o his disease a e smoking, pollu ion and gene ic de iciency o alpha-1 an i ypsin. Exace ba ions, which a e cha ac e ized by a wo sening o COPD symp oms, occu ecu en ly and a e mainly caused by bac e ial in ec ions. These in ec ions a e dange ous because hey encompass he o ma ion o polymic obial bio ilms, usually composed o NTHi wi h o he bac e ia, ha o en esis he adminis e ed an ibio ics. The ine ec i eness o hese d ugs leads o a educ ion in he quali y o li e o pa ien s, como bidi ies, and high mo ali y a es. As he p esence o se e al species can agg a a e bac e ial esis ance and in ec ion esolu ion, knowledge on he bac e ial in e ac ions aking place is u gen o design be e ea men s. This s udy examined some o he g ow h condi ions o H. in luenzae and S. au eus bio ilms, bo h isola ed and combined, including he e ec o enewal he g ow h media and he in luence o he di e en b ands o g ow h media on he o ma ion o bio ilms. I also in es iga ed he dynamics o bac e ial in e ac ions ha occu wi hin he double-species bio ilms namely he impac o sequen ial coloniza ion and o exop oduc s, and he suscep ibili y o bo h species o AMPs. The esul s showed ha he lowe numbe o cul u able H. in luenzae cells in 48 h bio ilms wi hou sBHI enewal a 24 h was p obably due o nu ien deple ion, gi en ha when media is eplaced a 24 h, he numbe o cul u able cells is highe , bu oxic me aboli es p oduced by he bac e ia i sel o he exis ence o VBNC could also be in ol ed. The esul s also e ealed di e ences in he g ow h o H. in luenzae when di e en media b ands we e used, which may be due o he quali y o he medium, and ha he epea ed use o HI c yo ials may impac bac e ial quali y and cause lowe g ow h a es. I was also ound ha he g ow h o H. in luenzae , bo h in single and in double-species bio ilms, had less a iabili y when i was g own in sBHI om Oxoid, making his he chosen b and o he ollowing assays. Rega ding he dynamics o double-species bio ilms wi h di e en ini ial concen a ions o S. au eus , i was highligh ed ha H. in luenzae and S. au eus appea o imp o e each o he ’s cul u abili y, bu mos di e ences encoun e ed we e small. Al hough i is epo ed in he li e a u e ha he densi y o H. in luenzae inc eases when S. au eus is he p e ious colonize , in his case, he colonize species had no in luence on he o ma ion o he bio ilm o he o he species. Addi ionally, he esul s also showed ha he exop oduc s o one species had no in luence on he o ma ion o he bio ilms o he o he species, which could be due o he concen a ion o he exop oduc no being enough o igge an e ec , o he cells o he bio ilms no be suscep ible o he exop oduc s p oduced by he o he species. Finally, o he ou AMPs es ed, achyplesin I showed he bes bac e ios a ic and bac e icidal ac ion agains H. 52 in luenzae and S. au eus , hus being mo e p omising o u he de elopmen o ea men s agains he double-species conso ia. Hope ully, he in es iga ion o he g ow h condi ions and o he in e ac ions be ween hese wo species conduc ed in his wo k will p o ide he esea ch eam wi h aluable insigh s o he de elopmen o an imic obial agen s o ea in ec ions caused by hese bac e ia. To enhance he esea ch conduc ed in his hesis, u u e wo k should include he disc imina ion o he mic obial popula ion o bio ilms, namely iable, non- iable, and VBNC cells, o example h ough low cy ome y. Fu he in es iga ions may be ca ied ou , such as he iden i ica ion o ex acellula me aboli es p oduced by bio ilms when cul u ed wi h and wi hou sBHI enewal, by mass spec ome y (MS). Ano he undamen al poin would be he analysis o he spa ial a angemen o he di e en species in he bio ilm, whe e he luo escence in si u hyb idiza ion (FISH) echnique could be used. Fo a mo e ealis ic s udy, bio ilms could be g own in a i icial mucus ha mimics he mucus in he ai ways. I would be in e es ing o compa e all he cha ac e iza ions indica ed abo e be ween he di e en g ow h media (sBHI and a i icial mucus). Since, in ch onic in ec ions, mos bio ilms o m small agg ega es o cells no a ached o a su ace bu embedded in hos ma e ial, he bio ilms, in addi ion o being o med on mic o i e pla es, could also be o med on algina e beads (Sønde holm e al., 2017). Algina e beads ep esen a simple and lexible model o in i o bio ilms. This would allow us o unde s and bio ilm o ma ion in di e en s uc u es: mic o i e pla es (adhe ed bio ilm) s algina e g anules (agg ega ed bio ilm). Finally, o he de elopmen o a possible d ug, i would be essen ial o p edic esis ance o gene a ions o H. in luenzae and S. au eus o achyplesin I. Resis ance is es ed o bac e ial gene a ions because a bac e ial cell ha is exposed o an an imic obial agen can de elop esis ance and ansmi his esis ance o he descendan s h ough he gene ic ma e ial. By es ing esis ance in se e al gene a ions o bac e ia, i will be possible o de e mine how easily esis ance is ansmi ed and how quickly i sp eads wi hin a popula ion. In his way, he suscep ibili y es o bac e ial gene a ions could be pe o med. Gi en he signi icance o bio ilms in his con ex , i would also be impo an o s udy he suscep ibili y o hese bac e ia o achyplesin I when hey a e in single and double bio ilms in o de o gain a comp ehensi e unde s anding o how hey a e a ec ed by his p omising AMP. 53 Re e ences Ahea n, C. P., Gallo, M. C., & Mu phy, T. F. (2017). Insigh s on pe sis en ai way in ec ion by non- ypeable Haemophilus in luenzae in ch onic obs uc i e pulmona y disease. In Pa hogens and disease (Vol. 75, Issue 4, p. 42). Ox o d Academic. h ps://doi.o g/10.1093/ emspd/ x042 Al es, D., Magalhães, A., G zywacz, D., Neubaue , D., Kamysz, W., & Pe ei a, M. O. (2016). Co- immobiliza ion o Palm and DNase I o he de elopmen o an e ec i e an i-in ec i e coa ing o ca he e su aces. 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