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T ansc ip ional and mu a ional p o iling o an aminoglycoside esis an Pseudomonas
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ae uginosa small colony a ian
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Monika Schniede jans,a,b Michal Koska,b Susanne Häussle ,a,b #
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Depa men o Molecula Bac e iology, Helmhol z Cen e o In ec ion Resea ch, B aunschweig, Ge many a
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Ins i u e o Molecula Bac e iology, TWINCORE GmbH, Cen e o Clinical and Expe imen al In ec ion
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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,
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Hanno e , Ge manyb
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Running head: An ibio ic esis ance p o iling
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# Add ess co espondence o Susanne Häussle , [email p o ec ed].
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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.
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Abs ac
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Pseudomonas ae uginosa is a majo causa i e agen o bo h acu e and ch onic in ec ions.
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Al hough aminoglycoside an ibio ics a e e y po en d ugs o igh such in ec ions, an ibio ic
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ailu e is s eadily inc easing mainly due o inc easing esis ance o he bac e ia. Many
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molecula mechanisms ha de e mine esis ance such as acquisi ion o genes encoding o
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aminoglycoside-inac i a ing enzymes o o e exp ession o e lux pumps ha e been
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elucida ed. Howe e , he e a e addi ional, less-well desc ibed mechanisms o
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aminoglycoside esis ance. In his s udy we ha e p o iled a clinical ob amycin esis an P.
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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
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medium condi ions. T ansc ip ional and mu a ional p o iling e ealed ha he SCV ha bo ed
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ac i a ing mu a ions in he wo wo-componen sys ems AmgRS and Pm AB. In oduc ion o
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hese mu a ions singula ly in o he ype s ain PA14 con e ed ob amycin and colis in
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esis ance, espec i ely. Howe e , hei combined in oduc ion had an addi i e e ec on he
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ob amycin esis ance pheno ype. Ac i a ion o he AmgRS sys em sligh ly educed he
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colony size o he PA14 wild- ype, whe eas he simul aneous o e exp ession o gacA, he
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esponse egula o o he GacSA wo componen sys em, u he educed colony size. In
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conclusion, we unco e ed combina o ial in luences o wo-componen sys ems on clinically
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ele an pheno ypes, such as esis ance and he exp ession o he SCV pheno ype. Ou
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esul s clea ly demons a e ha combined ac i a ion o P. ae uginosa wo-componen
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sys ems exhibi pleio opic e ec s wi h un o eseen consequences.
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In oduc ion
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Pseudomonas ae uginosa is one o he mos impo an oppo unis ic pa hogens and a
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se ious conce n o public heal h. I s high po en ial o in insic and acqui ed an imic obial
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esis ance is a majo an icipa ed p oblem in hospi als (1-3). Aminoglycosides a e highly
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po en , b oad-spec um an ibio ics. They a e pa icula ly used o ea P. ae uginosa
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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
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high local concen a ion in he b onchial sec ions (4). In a enously adminis e ed
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combina ion he apies mainly wi h β-lac am an ibio ics a e also common (5). Howe e , as
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o mos an imic obial classes, highe a es o inapp op ia e empi ic an imic obial ea men
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a e associa ed wi h he inc easing esis ance o he an imic obials in use.
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Di e se mechanisms ha e been desc ibed o con e esis ance owa ds aminoglycosides in P.
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ae uginosa. The mos p ominen ones include d ug inac i a ion h ough he ac i i y o
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aminoglycoside-modi ying enzymes and dec eased d ug accumula ion inside he bac e ial
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cell ia ac i e e lux o diminished cell wall pe meabili y. Ac i e e lux o aminoglycosides in
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P. ae uginosa can be achie ed ia he up egula ion o he MexXY e lux sys em (6) mainly
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caused by mu a ions in egula o y genes (7). Fu he mo e, an adap i e exp ession o he
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MexXY e lux pump, e.g. in he p esence o ibosome- a ge ing an ibio ics has been
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desc ibed (8). In cys ic ib osis isola es, ac i e MexXY e lux is e y common (9-11).
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Besides hese well-desc ibed s a egies o esis aminoglycoside he apy, g ow h wi hin
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bio ilms (12), con e sion o mucoidy (13) o he eme gence o pe sis e cells (14) can
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con ibu e o su i al in he p esence o he an ibio ic. Fu he mo e slow-g owing small
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colony a ian (SCVs) popula ions ha e been associa ed wi h pe sis en in ec ions and
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inc eased esis ance owa ds an ibio ics, including aminoglycosides (15). I has also been
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shown ha aminoglycoside exposu e can induce he o ma ion o SCVs in i o and in i o
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(16, 17). While in S aphylococcus au eus he mo pho ypic swi ch o SCVs is linked o
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elec on- anspo de iciency in s ains ha a e auxo oph o menadione o haemin o o
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hymidine auxo ophy (18), he unde lying mechanisms o P. ae uginosa SCV gene a ion
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seem o be mo e he e ogeneous (19-22). In P. ae uginosa he SCV pheno ype is o en, bu
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no exclusi ely, associa ed wi h ele a ed in acellula le els o he second messenge cyclic-
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di-GMP (12, 19, 23-25). Al hough he an ibio ic suscep ibili y pheno ype may a y a lo (26,
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27), many clinical SCV isola es exhibi esis ance owa ds se e al an ibio ics and exp ess
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u he pheno ypic ea u es like hype pilia ion o inc eased bio ilm o ma ion capabili ies
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(25, 26). Also o e p oduc ion o he exopolysaccha ides Pel and Psl (28, 29) and mo e
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ecen ly, he in ol emen o a p ophage genomic egion (22) has been linked o SCV
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o ma ion.
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In his s udy we desc ibe he unde lying gene ic de e minan s o clinical P. ae uginosa
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isola es ha p oduce no only small colonies on aga pla es bu exhibi also an
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aminoglycoside esis an pheno ype. Th ee clonally ela ed SCVs p oduced e e an s
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ollowing passaging unde ich medium condi ions, which exhibi ed la ge colony
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mo phologies and aminoglycoside suscep ibili y By ansc ip ional and mu a ional p o iling
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we unco e ed a combina o ial impac o he h ee wo-componen sys ems Pm AB, AmgRS
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and GacSA on aminoglycoside esis ance and he SCV mo pho ype.
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Resul s
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Aminoglycoside esis ance is linked o a SCV pheno ype. We ha e p e iously iden i ied
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h ee clonally ela ed ob amycin esis an clinical P. ae uginosa isola es (30). The esis ance
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pheno ype o hose isola es could no be explained by he p esence o a gene encoding o
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an aminoglycoside-modi ying enzyme, no did hey exp ess he mexXY mul id ug e lux
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pump genes a highly ele a ed le els. (All h ee SCVs exhibi ed a below 2- old inc eased
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mexX and mexY exp ession as compa ed o he PA14 e e ence s ain). All h ee clinical
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isola es howe e we e small colony a ian s (SCVs) (Fig. 1). The o ma ion o SCVs has been
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associa ed wi h pe sis en in ec ions and inc eased esis ance owa ds aminoglycoside
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an ibio ics (15). We he e o e hypo hesized ha he e migh be a link be ween he
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pheno ypic a ia ion and aminoglycoside esis ance in he h ee P. ae uginosa SCVs
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MHH8607, MHH9536 and MHH9604. As shown in Fig.1, e en among he closely ela ed
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SCVs, di e ences in colony mo phologies we e obse ed. MHH8607 seemed o exhibi an
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e en smalle colony mo phology han he o he wo SCV isola es. Via passaging he SCVs in
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ich medium, we gene a ed s able e e an s wi h la ge su ace colonies. Mo phological
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di e ences we e also obse ed o he e e an s. REV_MHH8607 appea ed as shiny ci cula
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colonies, while he o he wo exhibi ed a he la , dull colonies wi h a ough ex u e. O
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no e, all h ee e e an s exhibi ed an inc eased suscep ibili y owa ds aminoglycosides
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(Table 1). Howe e , only he e e an o MHH8607 (REV_MHH8607) eached a minimal
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inhibi o y concen a ion (MIC) alue o ob amycin ha was ca ego ized as suscep ible.
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T ansc ip ional p o iling e eals a down- egula ion o he Pm AB wo-componen sys em
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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
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be o e (31, 32) and we e complemen ed in his s udy by RNA-sequencing o he espec i e
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e e an s ains. Gene exp ession analysis e ealed a ange o 64 o 84 genes ha we e
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di e en ially exp essed be ween he SCVs and hei espec i e e e an s (Fig. 2,
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Supplemen a y Table 1). Among hose genes, we ound an o e lap o 14 genes di e en ially
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egula ed in all h ee SCV / e e an pai s (Table 2). Mos o hem (10 genes) belonged o
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he Pm AB wo-componen egula o y pa hway.
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The senso kinase encoding gene pm B and he esponse egula o encoding gene pm A
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we e mos s ongly down- egula ed in all e e an s. Fu he mo e, many genes o he
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downs eam egula ed gene ope on a nBCADTEF (33) and he spe midine syn hesis gene
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clus e speD (PA14_63110) and speE (PA14_63120) (34) we e exp essed a lowe le els in
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he e e an s. Exp ession o he a nBCADTEF ope on is known o limi he in e ac ion and
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sel -p omo ed up ake o polyca ionic an ibio ics (35), whe eas spe midine is a polyamine
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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
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(34, 36).
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Nex o hose commonly egula ed genes, we ound in each e e an an indi idual se o
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di e en ially exp essed genes (Supplemen a y Table 1). A close inspec ion o he isola e
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MHH8607 (whe e he REV_MHH8607 eached aminoglycoside MIC le els in he suscep ible
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ange) e ealed he down egula ion o a u he wo-componen sys em senso kinase
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encoding gene, amgS (PA14_68680, anno a ed as en Z in he PA14 e e ence genome) (log2
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old change o 3.67). The co esponding esponse egula o amgR (PA14_68700, anno a ed
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as ompR in he PA14 e e ence genome) also appea ed o be down egula ed by a log2 old
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change o 2.31 (al hough p- alue signi icance was no eached). The AmgRS sys em was
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desc ibed be o e o be in ol ed in aminoglycoside esis ance, as i has been iden i ied in a
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sc een o a ansposon mu an lib a y o genes whose inac i a ion inc eased ob amycin
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sensi i i y (37). The AmgRS sys em is a memb ane-s ess esponsi e wo-componen sys em
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in ol ed in he ansc ip ional egula ion o memb ane p o eases and o he memb ane
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p o eins (38). In line wi h his, we ound a educed exp ession o he p o ease H pX
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(encoded by PA14_27480, log2 old change o 3.99) and a memb ane p o ein o unknown
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unc ion (encoded by PA14_72930, log2 old change o 3.97) in he REV_MHH8607 s ain.
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In conclusion, i seems ha he down- egula ion o he Pm AB sys em in he e e an s is
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in ol ed in he egained aminoglycoside suscep ibili y, whe eas an addi ional down-
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egula ion o he AmgRS in he REV_MHH8607 migh accoun o he egained ull
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suscep ibili y agains ob amycin o his pa icula s ain.
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The aminoglycoside esis an SCVs exhibi inc eased pm AB and amgRS exp ession le els.
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We nex explo ed whe he he Pm AB and AmgRS wo componen sys ems exhibi ed an
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o e all inc eased exp ession in he SCVs and hus migh explain he aminoglycoside
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esis ance pheno ype. We he e o e compa ed he exp ession le els o he espec i e genes
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wi h hose o a P. ae uginosa ype s ain cul i a ed unde iden ical condi ions. Indeed all
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h ee SCV isola es exhibi ed a high gene exp ession le el o bo h sys ems (up o 43- old
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inc eased exp ession o pm A and up o 5- old o amgS; da a no shown) as compa ed o
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he PA14 e e ence s ain. Also in compa ison o he median exp ession le el in 151 clinical
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isola es ha ha e been sequenced be o e (32), we ound highe exp ession o bo h wo-
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componen sys ems (da a no shown).
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The aminoglycoside esis an SCVs exhibi gain-o - unc ion mu a ions in he wo-
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componen sys ems. As mu a ional ac i a ion o bo h he Pm AB and he AmgRS sys ems
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ha e been desc ibed p e iously (39-41), he gene alleles o he SCV isola es we e
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consequen ly sc eened o sequence al e a ions, which could be gain-o - unc ion mu a ions.
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Indeed, in all h ee clonal SCV isola es, non-synonymous sequence a ia ions in compa ison
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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
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in he senso kinase amgS. The pm A sequence a ia ion caused an amino acid subs i u ion
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in he co esponding p o ein (Leu71A g) ha is loca ed wi hin he signal ecei e domain o
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he esponse egula o , which also con ains he phospho yla ion si e o he p o ein. Wi hin
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pm B, wo sequence a ia ions we e de ec ed; one caused an amino acid exchange in he
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sec e ion signal o he p o ein (Th 4Ala) while he second is loca ed in close p oximi y o he
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his idine kinase A and he ATP binding domain (Leu323His) (40, 42). To explo e whe he
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hese sequence a ia ions ac as ac i a ing mu a ions, we in oduced hem in o he PA14
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e e ence s ain isola ed o in a ious combina ions and analyzed he esul ing pheno ype
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(Table 3).
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Aminoglycoside-suscep ibili y was sligh ly a ec ed by he mu a ion in amgS, which led o a
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2- old inc ease o he ob amycin MIC (Table 3). The ac i a ing mu a ions wi hin he Pm AB
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sys em did no in luence he ob amycin MIC. Howe e , he combined ac i a ion o AmgS
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and he Pm AB sys em led o a 4- old inc ease in he ob amycin MIC. Pm AB is known o be
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in ol ed in polymyxin esis ance by ac i a ing he a nBCADTEF ope on ha in u ns modi ies
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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
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colis in suscep ibili y. Single mu a ions in pm A o pm B led o none o only a sligh dec ease
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in colis in suscep ibili y, while bo h pm B mu a ions in combina ion esul ed in high-le el
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esis ance (MIC 64 µg/ml). The aminoglycoside esis ance inducing mu a ion in amgS did no
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u he enhance polymyxin suscep ibili y.
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In iguingly, he h ee in es iga ed clinical clonal SCV isola es did no exhibi colis in
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esis ance, al hough ca ying he gain-o - unc ion mu a ions in pm AB and exhibi ing
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o e exp ession o he sys em and he downs eam genes. The e was also no di e ence in
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colis in suscep ibili y be ween he SCVs and hei espec i e e e an s e en hough changes
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in a nBCADTEF exp ession occu ed. We did no iden i y any inse ions o dele ions wi hin
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he SCV a n genes. Ne e heless, we ound o e all 19 sequence a ia ions (Supplemen a y
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Table 2) wi hin he a n genes o he SCVs as compa ed o he PA14 WT. I canno be
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excluded ha among hem he e is an inac i a ing mu a ion ha migh explain colis in
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suscep ibili y despi e an gene o e exp ession.
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Suscep ibili y owa ds Colis in in he SCVs could also no be explained by he exp ession
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le els o he wo-componen sys ems PhoPQ-Op H ope on o Pa RS. (We ound an up o 2.3-
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old dec eased exp ession o phoQ, phoP and op H as compa ed o he PA14 wild- ype, and
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an up o 2.8- old inc eased exp ession o pa S, he exp ession o pa R was no signi ican ly
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changed; da a no shown).
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Mu a ions ha led o he swi ch o he e e an pheno ype. The Pm AB pa hway has been
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p e iously desc ibed o con ibu e o an an ibio ic esis ance pheno ype. As his pa hway
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was down egula ed in all h ee e e an s as compa ed o hei espec i e SCVs, we u he
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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.
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We he e o e used ou RNA-sequencing da ase o iden i y mu a ions ha ha e been
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acqui ed by he e e an s du ing he cou se o hei pheno ypic swi ch. A ange o
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polymo phic genes o egula o y pa hways ha we e a ec ed in mo e han one e e an
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s ain we e de ec ed (Table 4). This included he Pm AB wo-componen sys em encoding
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genes, in which all e e an s had acqui ed a mu a ion in ei he he senso kinase o he
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esponse egula o . Addi ionally and in line wi h he ansc ip ional da a, he wo-
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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
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REV_MHH8606 in gacS and in REV_MHH9536 44 bp ups eam o gacA). Ano he mu a ional
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ho spo was he al e na i e sigma ac o algU, whe e SNPs occu ed in close p oximi y in
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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).
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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
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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
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FIG 2: Di e en ially exp essed genes in he SCV isola es in compa ison o hei espec i e
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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).
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