Transcriptional and mutational profiling of an aminoglycoside resistant Pseudomonas aeruginosa small colony variant.

1 T ansc ip ional and mu a ional p o iling o an aminoglycoside esis an Pseudomonas 1 ae uginosa small colony a ian 2 3 Monika Schniede jans,a,b Michal Koska,b Susanne Häussle ,a,b # 4 Depa men o Molecula Bac e iology, Helmhol z Cen e o In ec ion Resea ch, B aunschweig, Ge many a 5 Ins i u e o Molecula Bac e iology, TWINCORE GmbH, Cen e o Clinical and Expe imen al In ec ion 6 Resea ch, a join en u e o he Hanno e Medical School and he Helmhol z Cen e o In ec ion Resea ch, 7 Hanno e , Ge manyb 8 9 10 11 Running head: An ibio ic esis ance p o iling 12 13 # Add ess co espondence o Susanne Häussle , [email p o ec ed]. 14 15 AAC Accep ed Manusc ip Pos ed Online 5 Sep embe 2017 An imic ob. Agen s Chemo he . doi:10.1128/AAC.01178-17 Copy igh © 2017 Ame ican Socie y o Mic obiology. All Righ s Rese ed. on Sep embe 22, 2017 by Helmhol z-Zen um ue In ek ions o schung - BIBLIOTHEK-h p://aac.asm.o g/Downloaded om 2 Abs ac 16 Pseudomonas ae uginosa is a majo causa i e agen o bo h acu e and ch onic in ec ions. 17 Al hough aminoglycoside an ibio ics a e e y po en d ugs o igh such in ec ions, an ibio ic 18 ailu e is s eadily inc easing mainly due o inc easing esis ance o he bac e ia. Many 19 molecula mechanisms ha de e mine esis ance such as acquisi ion o genes encoding o 20 aminoglycoside-inac i a ing enzymes o o e exp ession o e lux pumps ha e been 21 elucida ed. Howe e , he e a e addi ional, less-well desc ibed mechanisms o 22 aminoglycoside esis ance. In his s udy we ha e p o iled a clinical ob amycin esis an P. 23 ae uginosa s ain ha exhibi ed a small colony a ian (SCV) pheno ype. Bo h, he esis ance 24 and he colony mo phology pheno ypes we e los upon passaging he isola e unde ich 25 medium condi ions. T ansc ip ional and mu a ional p o iling e ealed ha he SCV ha bo ed 26 ac i a ing mu a ions in he wo wo-componen sys ems AmgRS and Pm AB. In oduc ion o 27 hese mu a ions singula ly in o he ype s ain PA14 con e ed ob amycin and colis in 28 esis ance, espec i ely. Howe e , hei combined in oduc ion had an addi i e e ec on he 29 ob amycin esis ance pheno ype. Ac i a ion o he AmgRS sys em sligh ly educed he 30 colony size o he PA14 wild- ype, whe eas he simul aneous o e exp ession o gacA, he 31 esponse egula o o he GacSA wo componen sys em, u he educed colony size. In 32 conclusion, we unco e ed combina o ial in luences o wo-componen sys ems on clinically 33 ele an pheno ypes, such as esis ance and he exp ession o he SCV pheno ype. Ou 34 esul s clea ly demons a e ha combined ac i a ion o P. ae uginosa wo-componen 35 sys ems exhibi pleio opic e ec s wi h un o eseen consequences. 36 37 on Sep embe 22, 2017 by Helmhol z-Zen um ue In ek ions o schung - BIBLIOTHEK-h p://aac.asm.o g/Downloaded om 3 In oduc ion 38 Pseudomonas ae uginosa is one o he mos impo an oppo unis ic pa hogens and a 39 se ious conce n o public heal h. I s high po en ial o in insic and acqui ed an imic obial 40 esis ance is a majo an icipa ed p oblem in hospi als (1-3). Aminoglycosides a e highly 41 po en , b oad-spec um an ibio ics. They a e pa icula ly used o ea P. ae uginosa 42 pulmona y in ec ions in cys ic ib osis pa ien s, whe e he d ugs a e o en inhaled o each 43 high local concen a ion in he b onchial sec ions (4). In a enously adminis e ed 44 combina ion he apies mainly wi h β-lac am an ibio ics a e also common (5). Howe e , as 45 o mos an imic obial classes, highe a es o inapp op ia e empi ic an imic obial ea men 46 a e associa ed wi h he inc easing esis ance o he an imic obials in use. 47 Di e se mechanisms ha e been desc ibed o con e esis ance owa ds aminoglycosides in P. 48 ae uginosa. The mos p ominen ones include d ug inac i a ion h ough he ac i i y o 49 aminoglycoside-modi ying enzymes and dec eased d ug accumula ion inside he bac e ial 50 cell ia ac i e e lux o diminished cell wall pe meabili y. Ac i e e lux o aminoglycosides in 51 P. ae uginosa can be achie ed ia he up egula ion o he MexXY e lux sys em (6) mainly 52 caused by mu a ions in egula o y genes (7). Fu he mo e, an adap i e exp ession o he 53 MexXY e lux pump, e.g. in he p esence o ibosome- a ge ing an ibio ics has been 54 desc ibed (8). In cys ic ib osis isola es, ac i e MexXY e lux is e y common (9-11). 55 Besides hese well-desc ibed s a egies o esis aminoglycoside he apy, g ow h wi hin 56 bio ilms (12), con e sion o mucoidy (13) o he eme gence o pe sis e cells (14) can 57 con ibu e o su i al in he p esence o he an ibio ic. Fu he mo e slow-g owing small 58 colony a ian (SCVs) popula ions ha e been associa ed wi h pe sis en in ec ions and 59 inc eased esis ance owa ds an ibio ics, including aminoglycosides (15). I has also been 60 shown ha aminoglycoside exposu e can induce he o ma ion o SCVs in i o and in i o 61 (16, 17). While in S aphylococcus au eus he mo pho ypic swi ch o SCVs is linked o 62 elec on- anspo de iciency in s ains ha a e auxo oph o menadione o haemin o o 63 hymidine auxo ophy (18), he unde lying mechanisms o P. ae uginosa SCV gene a ion 64 seem o be mo e he e ogeneous (19-22). In P. ae uginosa he SCV pheno ype is o en, bu 65 no exclusi ely, associa ed wi h ele a ed in acellula le els o he second messenge cyclic- 66 di-GMP (12, 19, 23-25). Al hough he an ibio ic suscep ibili y pheno ype may a y a lo (26, 67 27), many clinical SCV isola es exhibi esis ance owa ds se e al an ibio ics and exp ess 68 u he pheno ypic ea u es like hype pilia ion o inc eased bio ilm o ma ion capabili ies 69 on Sep embe 22, 2017 by Helmhol z-Zen um ue In ek ions o schung - BIBLIOTHEK-h p://aac.asm.o g/Downloaded om 4 (25, 26). Also o e p oduc ion o he exopolysaccha ides Pel and Psl (28, 29) and mo e 70 ecen ly, he in ol emen o a p ophage genomic egion (22) has been linked o SCV 71 o ma ion. 72 In his s udy we desc ibe he unde lying gene ic de e minan s o clinical P. ae uginosa 73 isola es ha p oduce no only small colonies on aga pla es bu exhibi also an 74 aminoglycoside esis an pheno ype. Th ee clonally ela ed SCVs p oduced e e an s 75 ollowing passaging unde ich medium condi ions, which exhibi ed la ge colony 76 mo phologies and aminoglycoside suscep ibili y By ansc ip ional and mu a ional p o iling 77 we unco e ed a combina o ial impac o he h ee wo-componen sys ems Pm AB, AmgRS 78 and GacSA on aminoglycoside esis ance and he SCV mo pho ype. 79 80 on Sep embe 22, 2017 by Helmhol z-Zen um ue In ek ions o schung - BIBLIOTHEK-h p://aac.asm.o g/Downloaded om 5 Resul s 81 Aminoglycoside esis ance is linked o a SCV pheno ype. We ha e p e iously iden i ied 82 h ee clonally ela ed ob amycin esis an clinical P. ae uginosa isola es (30). The esis ance 83 pheno ype o hose isola es could no be explained by he p esence o a gene encoding o 84 an aminoglycoside-modi ying enzyme, no did hey exp ess he mexXY mul id ug e lux 85 pump genes a highly ele a ed le els. (All h ee SCVs exhibi ed a below 2- old inc eased 86 mexX and mexY exp ession as compa ed o he PA14 e e ence s ain). All h ee clinical 87 isola es howe e we e small colony a ian s (SCVs) (Fig. 1). The o ma ion o SCVs has been 88 associa ed wi h pe sis en in ec ions and inc eased esis ance owa ds aminoglycoside 89 an ibio ics (15). We he e o e hypo hesized ha he e migh be a link be ween he 90 pheno ypic a ia ion and aminoglycoside esis ance in he h ee P. ae uginosa SCVs 91 MHH8607, MHH9536 and MHH9604. As shown in Fig.1, e en among he closely ela ed 92 SCVs, di e ences in colony mo phologies we e obse ed. MHH8607 seemed o exhibi an 93 e en smalle colony mo phology han he o he wo SCV isola es. Via passaging he SCVs in 94 ich medium, we gene a ed s able e e an s wi h la ge su ace colonies. Mo phological 95 di e ences we e also obse ed o he e e an s. REV_MHH8607 appea ed as shiny ci cula 96 colonies, while he o he wo exhibi ed a he la , dull colonies wi h a ough ex u e. O 97 no e, all h ee e e an s exhibi ed an inc eased suscep ibili y owa ds aminoglycosides 98 (Table 1). Howe e , only he e e an o MHH8607 (REV_MHH8607) eached a minimal 99 inhibi o y concen a ion (MIC) alue o ob amycin ha was ca ego ized as suscep ible. 100 101 T ansc ip ional p o iling e eals a down- egula ion o he Pm AB wo-componen sys em 102 in he e e an s. T ansc ip ional p o iles o he h ee SCV isola es ha e been eco ded 103 be o e (31, 32) and we e complemen ed in his s udy by RNA-sequencing o he espec i e 104 e e an s ains. Gene exp ession analysis e ealed a ange o 64 o 84 genes ha we e 105 di e en ially exp essed be ween he SCVs and hei espec i e e e an s (Fig. 2, 106 Supplemen a y Table 1). Among hose genes, we ound an o e lap o 14 genes di e en ially 107 egula ed in all h ee SCV / e e an pai s (Table 2). Mos o hem (10 genes) belonged o 108 he Pm AB wo-componen egula o y pa hway. 109 110 on Sep embe 22, 2017 by Helmhol z-Zen um ue In ek ions o schung - BIBLIOTHEK-h p://aac.asm.o g/Downloaded om 6 The senso kinase encoding gene pm B and he esponse egula o encoding gene pm A 111 we e mos s ongly down- egula ed in all e e an s. Fu he mo e, many genes o he 112 downs eam egula ed gene ope on a nBCADTEF (33) and he spe midine syn hesis gene 113 clus e speD (PA14_63110) and speE (PA14_63120) (34) we e exp essed a lowe le els in 114 he e e an s. Exp ession o he a nBCADTEF ope on is known o limi he in e ac ion and 115 sel -p omo ed up ake o polyca ionic an ibio ics (35), whe eas spe midine is a polyamine 116 and has been desc ibed be o e o p o ec cells om an ibio ic ea men and oxida i e s ess 117 (34, 36). 118 Nex o hose commonly egula ed genes, we ound in each e e an an indi idual se o 119 di e en ially exp essed genes (Supplemen a y Table 1). A close inspec ion o he isola e 120 MHH8607 (whe e he REV_MHH8607 eached aminoglycoside MIC le els in he suscep ible 121 ange) e ealed he down egula ion o a u he wo-componen sys em senso kinase 122 encoding gene, amgS (PA14_68680, anno a ed as en Z in he PA14 e e ence genome) (log2 123 old change o 3.67). The co esponding esponse egula o amgR (PA14_68700, anno a ed 124 as ompR in he PA14 e e ence genome) also appea ed o be down egula ed by a log2 old 125 change o 2.31 (al hough p- alue signi icance was no eached). The AmgRS sys em was 126 desc ibed be o e o be in ol ed in aminoglycoside esis ance, as i has been iden i ied in a 127 sc een o a ansposon mu an lib a y o genes whose inac i a ion inc eased ob amycin 128 sensi i i y (37). The AmgRS sys em is a memb ane-s ess esponsi e wo-componen sys em 129 in ol ed in he ansc ip ional egula ion o memb ane p o eases and o he memb ane 130 p o eins (38). In line wi h his, we ound a educed exp ession o he p o ease H pX 131 (encoded by PA14_27480, log2 old change o 3.99) and a memb ane p o ein o unknown 132 unc ion (encoded by PA14_72930, log2 old change o 3.97) in he REV_MHH8607 s ain. 133 In conclusion, i seems ha he down- egula ion o he Pm AB sys em in he e e an s is 134 in ol ed in he egained aminoglycoside suscep ibili y, whe eas an addi ional down- 135 egula ion o he AmgRS in he REV_MHH8607 migh accoun o he egained ull 136 suscep ibili y agains ob amycin o his pa icula s ain. 137 138 The aminoglycoside esis an SCVs exhibi inc eased pm AB and amgRS exp ession le els. 139 We nex explo ed whe he he Pm AB and AmgRS wo componen sys ems exhibi ed an 140 o e all inc eased exp ession in he SCVs and hus migh explain he aminoglycoside 141 on Sep embe 22, 2017 by Helmhol z-Zen um ue In ek ions o schung - BIBLIOTHEK-h p://aac.asm.o g/Downloaded om 7 esis ance pheno ype. We he e o e compa ed he exp ession le els o he espec i e genes 142 wi h hose o a P. ae uginosa ype s ain cul i a ed unde iden ical condi ions. Indeed all 143 h ee SCV isola es exhibi ed a high gene exp ession le el o bo h sys ems (up o 43- old 144 inc eased exp ession o pm A and up o 5- old o amgS; da a no shown) as compa ed o 145 he PA14 e e ence s ain. Also in compa ison o he median exp ession le el in 151 clinical 146 isola es ha ha e been sequenced be o e (32), we ound highe exp ession o bo h wo- 147 componen sys ems (da a no shown). 148 149 The aminoglycoside esis an SCVs exhibi gain-o - unc ion mu a ions in he wo- 150 componen sys ems. As mu a ional ac i a ion o bo h he Pm AB and he AmgRS sys ems 151 ha e been desc ibed p e iously (39-41), he gene alleles o he SCV isola es we e 152 consequen ly sc eened o sequence al e a ions, which could be gain-o - unc ion mu a ions. 153 Indeed, in all h ee clonal SCV isola es, non-synonymous sequence a ia ions in compa ison 154 o he PA14 e e ence s ain we e disco e ed in bo h genes o he pm AB ope on as well as 155 in he senso kinase amgS. The pm A sequence a ia ion caused an amino acid subs i u ion 156 in he co esponding p o ein (Leu71A g) ha is loca ed wi hin he signal ecei e domain o 157 he esponse egula o , which also con ains he phospho yla ion si e o he p o ein. Wi hin 158 pm B, wo sequence a ia ions we e de ec ed; one caused an amino acid exchange in he 159 sec e ion signal o he p o ein (Th 4Ala) while he second is loca ed in close p oximi y o he 160 his idine kinase A and he ATP binding domain (Leu323His) (40, 42). To explo e whe he 161 hese sequence a ia ions ac as ac i a ing mu a ions, we in oduced hem in o he PA14 162 e e ence s ain isola ed o in a ious combina ions and analyzed he esul ing pheno ype 163 (Table 3). 164 Aminoglycoside-suscep ibili y was sligh ly a ec ed by he mu a ion in amgS, which led o a 165 2- old inc ease o he ob amycin MIC (Table 3). The ac i a ing mu a ions wi hin he Pm AB 166 sys em did no in luence he ob amycin MIC. Howe e , he combined ac i a ion o AmgS 167 and he Pm AB sys em led o a 4- old inc ease in he ob amycin MIC. Pm AB is known o be 168 in ol ed in polymyxin esis ance by ac i a ing he a nBCADTEF ope on ha in u ns modi ies 169 he LPS s uc u e. We he e o e also es ed he gene a ed mu an s ains o changes in 170 colis in suscep ibili y. Single mu a ions in pm A o pm B led o none o only a sligh dec ease 171 in colis in suscep ibili y, while bo h pm B mu a ions in combina ion esul ed in high-le el 172 on Sep embe 22, 2017 by Helmhol z-Zen um ue In ek ions o schung - BIBLIOTHEK-h p://aac.asm.o g/Downloaded om 8 esis ance (MIC 64 µg/ml). The aminoglycoside esis ance inducing mu a ion in amgS did no 173 u he enhance polymyxin suscep ibili y. 174 175 In iguingly, he h ee in es iga ed clinical clonal SCV isola es did no exhibi colis in 176 esis ance, al hough ca ying he gain-o - unc ion mu a ions in pm AB and exhibi ing 177 o e exp ession o he sys em and he downs eam genes. The e was also no di e ence in 178 colis in suscep ibili y be ween he SCVs and hei espec i e e e an s e en hough changes 179 in a nBCADTEF exp ession occu ed. We did no iden i y any inse ions o dele ions wi hin 180 he SCV a n genes. Ne e heless, we ound o e all 19 sequence a ia ions (Supplemen a y 181 Table 2) wi hin he a n genes o he SCVs as compa ed o he PA14 WT. I canno be 182 excluded ha among hem he e is an inac i a ing mu a ion ha migh explain colis in 183 suscep ibili y despi e an gene o e exp ession. 184 Suscep ibili y owa ds Colis in in he SCVs could also no be explained by he exp ession 185 le els o he wo-componen sys ems PhoPQ-Op H ope on o Pa RS. (We ound an up o 2.3- 186 old dec eased exp ession o phoQ, phoP and op H as compa ed o he PA14 wild- ype, and 187 an up o 2.8- old inc eased exp ession o pa S, he exp ession o pa R was no signi ican ly 188 changed; da a no shown). 189 190 191 Mu a ions ha led o he swi ch o he e e an pheno ype. The Pm AB pa hway has been 192 p e iously desc ibed o con ibu e o an an ibio ic esis ance pheno ype. As his pa hway 193 was down egula ed in all h ee e e an s as compa ed o hei espec i e SCVs, we u he 194 explo ed whe he his was due o a mu a ional inac i a ion o he sys em in he e e an s. 195 We he e o e used ou RNA-sequencing da ase o iden i y mu a ions ha ha e been 196 acqui ed by he e e an s du ing he cou se o hei pheno ypic swi ch. A ange o 197 polymo phic genes o egula o y pa hways ha we e a ec ed in mo e han one e e an 198 s ain we e de ec ed (Table 4). This included he Pm AB wo-componen sys em encoding 199 genes, in which all e e an s had acqui ed a mu a ion in ei he he senso kinase o he 200 esponse egula o . Addi ionally and in line wi h he ansc ip ional da a, he wo- 201 componen sys em AmgRS ha bo ed a nonsense mu a ion in amgS in REV_MHH8607. 202 Fu he mo e, he GacSA sys em ha bo ed mu a ions in wo o he h ee e e an s (in 203 REV_MHH8606 in gacS and in REV_MHH9536 44 bp ups eam o gacA). Ano he mu a ional 204 on Sep embe 22, 2017 by Helmhol z-Zen um ue In ek ions o schung - BIBLIOTHEK-h p://aac.asm.o g/Downloaded om 9 ho spo was he al e na i e sigma ac o algU, whe e SNPs occu ed in close p oximi y in 205 wo e e an s ains. This la e mu a ion is likely o be he cause o he obse ed 206 mo phological di e ences among he h ee e e an s: Only REV_MHH8607 exhibi ed a 207 shiny and mo e mucoid colony su ace, while he ones wi h an acqui ed algU mu a ion and 208 hus p esumably lowe algina e p oduc ion appea ed as dull, non-mucoid colonies, as 209 desc ibed be o e (43). 210 211 212 The combined ac i a ion o he AmgRS and GacSA sys em in he ype s ain PA14 p oduces 213 an SCV pheno ype. The GacSA/RsmAZY signaling sys em is known o be in ol ed in he 214 pheno ypic swi ch be ween ch onic (pe sis ence, SCV o ma ion) and acu e in ec ious 215 li es yles. Since he e e an s o wo o he clinical SCVs ha bo ed mu a ions in he GacSA 216 pa hway, we o e exp essed he gacA gene in he PA14 wild- ype s ain as well as he PA14 217 s ain ha bo ing he gain o unc ion mu a ions in pm B and/o amgS in o de o de e mine 218 whe he we can obse e an in luence on he colony mo phology. O e exp ession o he 219 gacA gene in PA14 o in he mu an s o e exp essing Pm B did no lead o al e a ions in 220 colony mo phology o he esis ance pheno ype (da a no shown). Howe e , in he mu an 221 wi h an ac i a ion o he AmgRS wo-componen sys em, gacA o e exp ession led o an SCV 222 pheno ype. The esis ance pheno ype did no change in his s ain backg ound due o gacA 223 o e exp ession (da a no shown). 224 Ou da a hus indica e ha he combined ac i a ion o he GacSA and he AmgRS sys em 225 seem o explain he SCV pheno ype in ou clinical isola es, whe eas he combined ac i a ion 226 o he Pm AB and he AmgRS sys em con e s o enhanced aminoglycoside esis ance. 227 228 on Sep embe 22, 2017 by Helmhol z-Zen um ue In ek ions o schung - BIBLIOTHEK-h p://aac.asm.o g/Downloaded om 16 348 FIG 1: Colony mo phology o h ee clonal small-colony a ian s and hei espec i e e e an s. 349 Small-colony a ian MHH8607, MHH9536 and MHH9604 (le ) and hei espec i e e e an s wi h la ge and 350 di e se colony su aces ( igh ) a e 24 h o g ow h on Columbia blood aga pla es. 351 352 353 354 FIG 2: Di e en ially exp essed genes in he SCV isola es in compa ison o hei espec i e 355 e e an s. 356 A gene was ca ego ized as di e en ially exp essed when he absolu e alue o log2 old change was ≥ 2 and he 357 p al was ≤ 0.01. The Venn diag am was c ea ed wi h VENNY (60). 358 on Sep embe 22, 2017 by Helmhol z-Zen um ue In ek ions o schung - BIBLIOTHEK-h p://aac.asm.o g/Downloaded om 17 Re e ences 359 1. B eidens ein EB, de la Fuen e-Nunez C, Hancock RE. 2011. Pseudomonas ae uginosa: 360 all oads lead o esis ance. T ends Mic obiol 19:419-426. 361 2. Michiels JE, Van den Be gh B, Ve s ae en N, Fau a M, Michiels J. 2016. In Vi o 362 Eme gence o High Pe sis ence upon Pe iodic Aminoglycoside Challenge in he 363 ESKAPE Pa hogens. An imic ob Agen s Chemo he 60:4630-4637. 364 3. Lis e PD, Wol e DJ, Hanson ND. 2009. An ibac e ial- esis an Pseudomonas 365 ae uginosa: clinical impac and complex egula ion o ch omosomally encoded 366 esis ance mechanisms. Clin Mic obiol Re 22:582-610. 367 4. Chiappini E, Tacce i G, de Ma ino M. 2014. Bac e ial lung in ec ions in cys ic ib osis 368 pa ien s: an upda e. Pedia In ec Dis J 33:653-654. 369 5. Do ing G, Conway SP, Heije man HG, Hodson ME, Hoiby N, Smy h A, Touw DJ. 2000. 370 An ibio ic he apy agains Pseudomonas ae uginosa in cys ic ib osis: a Eu opean 371 consensus. Eu Respi J 16:749-767. 372 6. Poole K. 2001. Mul id ug esis ance in G am-nega i e bac e ia. Cu Opin Mic obiol 373 4:500-508. 374 7. Ma suo Y, Eda S, Go oh N, Yoshiha a E, Nakae T. 2004. MexZ-media ed egula ion o 375 mexXY mul id ug e lux pump exp ession in Pseudomonas ae uginosa by binding on 376 he mexZ-mexX in e genic DNA. FEMS Mic obiol Le 238:23-28. 377 8. Mo i a Y, Sobel ML, Poole K. 2006. An ibio ic inducibili y o he MexXY mul id ug 378 e lux sys em o Pseudomonas ae uginosa: in ol emen o he an ibio ic-inducible 379 PA5471 gene p oduc . J Bac e iol 188:1847-1855. 380 9. Sobel ML, McKay GA, Poole K. 2003. Con ibu ion o he MexXY mul id ug 381 anspo e o aminoglycoside esis ance in Pseudomonas ae uginosa clinical isola es. 382 An imic ob Agen s Chemo he 47:3202-3207. 383 10. Mo i a Y, Tomida J, Kawamu a Y. 2012. MexXY mul id ug e lux sys em o 384 Pseudomonas ae uginosa. F on Mic obiol 3:408. 385 11. Islam S, Jalal S, W e lind B. 2004. Exp ession o he MexXY e lux pump in amikacin- 386 esis an isola es o Pseudomonas ae uginosa. Clin Mic obiol In ec 10:877-883. 387 12. D enka d E, Ausubel FM. 2002. Pseudomonas bio ilm o ma ion and an ibio ic 388 esis ance a e linked o pheno ypic a ia ion. Na u e 416:740-743. 389 13. Ha ch RA, Schille NL. 1998. Algina e lyase p omo es di usion o aminoglycosides 390 h ough he ex acellula polysaccha ide o mucoid Pseudomonas ae uginosa. 391 An imic ob Agen s Chemo he 42:974-977. 392 14. Moke N, Dean CR, Tao J. 2010. Pseudomonas ae uginosa inc eases o ma ion o 393 mul id ug- ole an pe sis e cells in esponse o quo um-sensing signaling molecules. 394 J Bac e iol 192:1946-1955. 395 15. Haussle S. 2004. Bio ilm o ma ion by he small colony a ian pheno ype o 396 Pseudomonas ae uginosa. En i on Mic obiol 6:546-551. 397 16. Ge be AU, Vas ola AP, B andel J, C aig WA. 1982. Selec ion o aminoglycoside- 398 esis an a ian s o Pseudomonas ae uginosa in an in i o model. J In ec Dis 399 146:691-697. 400 17. Lang o d PR, Anwa H, Gonda I, B own MR. 1989. Ou e memb ane p o eins o 401 gen amicin induced small colony a ian s o Pseudomonas ae uginosa. FEMS 402 Mic obiol Le 52:33-36. 403 18. Ga cia LG, Lemai e S, Kahl BC, Becke K, P oc o RA, Denis O, Tulkens PM, Van 404 Bambeke F. 2013. An ibio ic ac i i y agains small-colony a ian s o S aphylococcus 405 on Sep embe 22, 2017 by Helmhol z-Zen um ue In ek ions o schung - BIBLIOTHEK-h p://aac.asm.o g/Downloaded om 18 au eus: e iew o in i o, animal and clinical da a. J An imic ob Chemo he 68:1455- 406 1464. 407 19. Malone JG, Jaege T, Man edi P, Dö sch A, Blanka A, Bos R, Co nelis GR, Häussle S, 408 Jenal U. 2012. The Y iBNR signal ansduc ion mechanism e eals no el a ge s o 409 he e olu ion o pe sis en Pseudomonas ae uginosa in cys ic ib osis ai ways. PLoS 410 Pa hog 8:e1002760. 411 20. Blanka A, Du el J, Do sch A, Klinke B, Ab aham WR, Kae e V, Ri e C, Na be haus 412 F, Haussle S. 2015. Cons i u i e p oduc ion o c-di-GMP is associa ed wi h mu a ions 413 in a a ian o Pseudomonas ae uginosa wi h al e ed memb ane composi ion. Sci 414 Signal 8: a36. 415 21. Wei Q, Ta ighi S, Dö sch A, Häussle S, Müsken M, W igh VJ, Cama a M, Williams P, 416 Haenen S, Boe jan B, Bogae s A, Vie s ae e E, Ve leyen P, Schoo s L, Willae R, De 417 G oo e VN, Michiels J, Ve cammen K, C abbe A, Co nelis P. 2011. Pheno ypic and 418 genome-wide analysis o an an ibio ic- esis an small colony a ian (SCV) o 419 Pseudomonas ae uginosa. PLoS One 6:e29276. 420 22. Lim WS, Phang KK, Tan AH, Li SF, Ow DS. 2016. Small Colony Va ian s and Single 421 Nucleo ide Va ia ions in P 1 Region o PB1 Phage-Resis an Pseudomonas 422 ae uginosa. F on Mic obiol 7:282. 423 23. D'A genio DA, Cal ee MW, Rainey PB, Pesci EC. 2002. Au olysis and au oagg ega ion 424 in Pseudomonas ae uginosa colony mo phology mu an s. J Bac e iol 184:6481-6489. 425 24. Hickman JW, Ti ea DF, Ha wood CS. 2005. A chemosenso y sys em ha egula es 426 bio ilm o ma ion h ough modula ion o cyclic diguanyla e le els. P oc Na l Acad Sci 427 U S A 102:14422-14427. 428 25. Häussle S, Tümmle B, Weissb od H, Rohde M, S einme z I. 1999. Small-colony 429 a ian s o Pseudomonas ae uginosa in cys ic ib osis. Clin In ec Dis 29:621-625. 430 26. Badenoch PR, Cos e DJ. 1989. Selec ion o gen amicin- esis an a ian s o 431 Pseudomonas ae uginosa in he a co nea. J Ocul Pha macol 5:19-25. 432 27. Ki isi s MJ, P os L, S a key M, Pa sek MR. 2005. Cha ac e iza ion o colony 433 mo phology a ian s isola ed om Pseudomonas ae uginosa bio ilms. Appl En i on 434 Mic obiol 71:4809-4821. 435 28. Malone JG, Jaege T, Spangle C, Ri z D, Spang A, A ieume lou C, Kae e V, 436 Landmann R, Jenal U. 2010. Y iBNR media es cyclic di-GMP dependen small colony 437 a ian o ma ion and pe sis ence in Pseudomonas ae uginosa. PLoS Pa hog 438 6:e1000804. 439 29. S a key M, Hickman JH, Ma L, Zhang N, De Long S, Hinz A, Palacios S, Manoil C, Ki isi s 440 MJ, S a ne TD, Wozniak DJ, Ha wood CS, Pa sek MR. 2009. Pseudomonas ae uginosa 441 ugose small-colony a ian s ha e adap a ions ha likely p omo e pe sis ence in he 442 cys ic ib osis lung. J Bac e iol 191:3492-3503. 443 30. Khaledi A, Schniede jans M, Pohl S, Raine R, Bodenho e U, Xia B, Klawonn F, 444 B uchmann S, P eusse M, Eckweile D, Do sch A, Haussle S. 2016. T ansc ip ome 445 P o iling o An imic obial Resis ance in Pseudomonas ae uginosa. An imic ob Agen s 446 Chemo he 60:4722-4733. 447 31. Do sch A, Schniede jans M, Khaledi A, Ho nische K, Schulz S, Bielecka A, Eckweile D, 448 Pohl S, Haussle S. 2015. The Pseudomonas ae uginosa T ansc ip ional Landscape Is 449 Shaped by En i onmen al He e ogenei y and Gene ic Va ia ion. MBio 6:e00749. 450 32. Pohl S, Klockge he J, Eckweile D, Khaledi A, Schniede jans M, Chou a ine P, 451 Tummle B, Haussle S. 2014. The ex ensi e se o accesso y Pseudomonas 452 ae uginosa genomic componen s. FEMS Mic obiol Le 356:235-241. 453 on Sep embe 22, 2017 by Helmhol z-Zen um ue In ek ions o schung - BIBLIOTHEK-h p://aac.asm.o g/Downloaded om 19 33. McPhee JB, Lewenza S, Hancock RE. 2003. Ca ionic an imic obial pep ides ac i a e a 454 wo-componen egula o y sys em, Pm A-Pm B, ha egula es esis ance o 455 polymyxin B and ca ionic an imic obial pep ides in Pseudomonas ae uginosa. Mol 456 Mic obiol 50:205-217. 457 34. Johnson L, Mulcahy H, Kane e s U, Shi Y, Lewenza S. 2012. Su ace-localized 458 spe midine p o ec s he Pseudomonas ae uginosa ou e memb ane om an ibio ic 459 ea men and oxida i e s ess. J Bac e iol 194:813-826. 460 35. Fe nandez L, Goode ham WJ, Bains M, McPhee JB, Wiegand I, Hancock RE. 2010. 461 Adap i e esis ance o he "las hope" an ibio ics polymyxin B and colis in in 462 Pseudomonas ae uginosa is media ed by he no el wo-componen egula o y 463 sys em Pa R-Pa S. An imic ob Agen s Chemo he 54:3372-3382. 464 36. Kwon DH, Lu CD. 2006. Polyamines induce esis ance o ca ionic pep ide, 465 aminoglycoside, and quinolone an ibio ics in Pseudomonas ae uginosa PAO1. 466 An imic ob Agen s Chemo he 50:1615-1622. 467 37. Lee S, Hinz A, Baue le E, Ange meye A, Juhaszo a K, Kaneko Y, Singh PK, Manoil C. 468 2009. Ta ge ing a bac e ial s ess esponse o enhance an ibio ic ac ion. P oc Na l 469 Acad Sci U S A 106:14570-14575. 470 38. Hinz A, Lee S, Jacoby K, Manoil C. 2011. Memb ane p o eases and aminoglycoside 471 an ibio ic esis ance. J Bac e iol 193:4790-4797. 472 39. Lau CH, F aud S, Jones M, Pe e son SN, Poole K. 2013. Mu a ional ac i a ion o he 473 AmgRS wo-componen sys em in aminoglycoside- esis an Pseudomonas 474 ae uginosa. An imic ob Agen s Chemo he 57:2243-2251. 475 40. Moskowi z SM, B annon MK, Dasgup a N, Pie M, Sgamba i N, Mille AK, Selg ade SE, 476 Mille SI, Den on M, Conway SP, Johansen HK, Hoiby N. 2012. Pm B mu a ions 477 p omo e polymyxin esis ance o Pseudomonas ae uginosa isola ed om colis in- 478 ea ed cys ic ib osis pa ien s. An imic ob Agen s Chemo he 56:1019-1030. 479 41. Moskowi z SM, E ns RK, Mille SI. 2004. Pm AB, a wo-componen egula o y sys em 480 o Pseudomonas ae uginosa ha modula es esis ance o ca ionic an imic obial 481 pep ides and addi ion o aminoa abinose o lipid A. J Bac e iol 186:575-579. 482 42. Olai an AO, Mo and S, Rolain JM. 2014. Mechanisms o polymyxin esis ance: 483 acqui ed and in insic esis ance in bac e ia. F on Mic obiol 5:643. 484 43. Yu H, Schu MJ, De e ic V. 1995. Func ional equi alence o Esche ichia coli sigma E 485 and Pseudomonas ae uginosa AlgU: E. coli poE es o es mucoidy and educes 486 sensi i i y o eac i e oxygen in e media es in algU mu an s o P. ae uginosa. J 487 Bac e iol 177:3259-3268. 488 44. Adams MD, Nickel GC, Bajaksouzian S, La ende H, Mu hy AR, Jacobs MR, Bonomo 489 RA. 2009. Resis ance o colis in in Acine obac e baumannii associa ed wi h 490 mu a ions in he Pm AB wo-componen sys em. An imic ob Agen s Chemo he 491 53:3628-3634. 492 45. Yeaman MR, Youn NY. 2003. Mechanisms o an imic obial pep ide ac ion and 493 esis ance. Pha macol Re 55:27-55. 494 46. Lewenza S. 2013. Ex acellula DNA-induced an imic obial pep ide esis ance 495 mechanisms in Pseudomonas ae uginosa. F on Mic obiol 4:21. 496 47. Lee JY, Ko KS. 2014. Mu a ions and exp ession o Pm AB and PhoPQ ela ed wi h 497 colis in esis ance in Pseudomonas ae uginosa clinical isola es. Diagn Mic obiol In ec 498 Dis 78:271-276. 499 on Sep embe 22, 2017 by Helmhol z-Zen um ue In ek ions o schung - BIBLIOTHEK-h p://aac.asm.o g/Downloaded om 20 48. Jochumsen N, Ma ig RL, Damkiae S, Jensen RL, Paulande W, Molin S, Jelsbak L, 500 Folkesson A. 2016. The e olu ion o an imic obial pep ide esis ance in Pseudomonas 501 ae uginosa is shaped by s ong epis a ic in e ac ions. Na Commun 7:13002. 502 49. Pou na as S, Poulou A, Da opoulou K, Chabane YN, K is o I, Mak is D, Ha douin J, 503 Cose e P, Tsak is A, De E. 2014. G ow h e a da ion, educed in asi eness, and 504 impai ed colis in-media ed cell dea h associa ed wi h colis in esis ance de elopmen 505 in Acine obac e baumannii. An imic ob Agen s Chemo he 58:828-832. 506 50. Sun S, Neg ea A, Rhen M, Ande sson DI. 2009. Gene ic analysis o colis in esis ance 507 in Salmonella en e ica se o a Typhimu ium. An imic ob Agen s Chemo he 53:2298- 508 2305. 509 51. H aiech S, Roch A, Lepidi H, A ieh T, Audoly G, Rolain JM, Raoul D, B unel JM, 510 Papazian L, B egeon F. 2013. Impai ed i ulence and i ness o a colis in- esis an 511 clinical isola e o Acine obac e baumannii in a a model o pneumonia. An imic ob 512 Agen s Chemo he 57:5120-5121. 513 52. Sni kin ES, Zelazny AM, Gup a J, P og am NCS, Palmo e TN, Mu ay PR, Seg e JA. 514 2013. Genomic insigh s in o he a e o colis in esis ance and Acine obac e 515 baumannii du ing pa ien ea men . Genome Res 23:1155-1162. 516 53. Lopez-Rojas R, Dominguez-He e a J, McConnell MJ, Docobo-Pe ez F, Smani Y, 517 Fe nandez-Reyes M, Ri as L, Pachon J. 2011. Impai ed i ulence and in i o i ness o 518 colis in- esis an Acine obac e baumannii. J In ec Dis 203:545-548. 519 54. Da ies JA, Ha ison JJ, Ma ques LL, Foglia GR, S emick CA, S o ey DG, Tu ne RJ, 520 Olson ME, Ce i H. 2007. The GacS senso kinase con ols pheno ypic e e sion o 521 small colony a ian s isola ed om bio ilms o Pseudomonas ae uginosa PA14. FEMS 522 Mic obiol Ecol 59:32-46. 523 55. Malone JG. 2015. Role o small colony a ian s in pe sis ence o Pseudomonas 524 ae uginosa in ec ions in cys ic ib osis lungs. In ec D ug Resis 8:237-247. 525 56. Libe a i NT, U bach JM, Miya a S, Lee DG, D enka d E, Wu G, Villanue a J, Wei T, 526 Ausubel FM. 2006. An o de ed, non edundan lib a y o Pseudomonas ae uginosa 527 s ain PA14 ansposon inse ion mu an s. P oc Na l Acad Sci U S A 103:2833-2838. 528 57. Wes SE, Schweize HP, Dall C, Sample AK, Runyen-Janecky LJ. 1994. Cons uc ion o 529 imp o ed Esche ichia-Pseudomonas shu le ec o s de i ed om pUC18/19 and 530 sequence o he egion equi ed o hei eplica ion in Pseudomonas ae uginosa. 531 Gene 148:81-86. 532 58. Wiegand I, Hilpe K, Hancock RE. 2008. Aga and b o h dilu ion me hods o 533 de e mine he minimal inhibi o y concen a ion (MIC) o an imic obial subs ances. 534 Na P o oc 3:163-175. 535 59. Hoang TT, Ka kho -Schweize RR, Ku chma AJ, Schweize HP. 1998. A b oad-hos - 536 ange Flp-FRT ecombina ion sys em o si e-speci ic excision o ch omosomally- 537 loca ed DNA sequences: applica ion o isola ion o unma ked Pseudomonas 538 ae uginosa mu an s. Gene 212:77-86. 539 60. Oli e os JC. 2007. VENNY. An in e ac i e ool o compa ing lis s wi h Venn Diag ams. 540 h p://bioin ogp.cnb.csic.es/ ools/ enny/index.h ml 541 542 on Sep embe 22, 2017 by Helmhol z-Zen um ue In ek ions o schung - BIBLIOTHEK-h p://aac.asm.o g/Downloaded om on Sep embe 22, 2017 by Helmhol z-Zen um ue In ek ions o schung - BIBLIOTHEK-h p://aac.asm.o g/Downloaded om on Sep embe 22, 2017 by Helmhol z-Zen um ue In ek ions o schung - BIBLIOTHEK-h p://aac.asm.o g/Downloaded om