Genomic Cha ac e iza ion o Imipenem- and Imipenem-
Relebac am-Resis an Clinical Isola es o Pseudomonas
ae uginosa
Ma io López-Pé ez,
a
Jose M. Ha o-Mo eno,
a
Ca men Molina-Pa dines,
b
Ma ia Paz Ven e o,
b
Juan Ca los Rod íguez
b
a
E olu iona y Genomics G oup, Di isión de Mic obiología, Uni e sidad Miguel He nández, Alican e, Spain
b
Mic obiology Depa men , Alican e Uni e si y Gene al Hospi al, Alican e Ins i u e o Sani a y and Biomedical Resea ch (ISABIAL), Alican e, Spain
ABSTRACT Pseudomonas ae uginosa is an oppo unis ic human pa hogen and a majo
cause o nosocomial in ec ions. The global sp ead o ca bapenem- esis an s ains is g ow-
ing apidly and has become a majo public heal h challenge. Imipenem- elebac am (I/R) is a
no el ca bapenem-be a-lac amase inhibi o combina ion ha can o e come ca bapenem e-
sis ance. In his s udy, we aimed o unde s and he mechanism unde lying esis ance o imi-
penem and imipenem- elebac am. Fo his pu pose, we pe o med a genomic compa ison
o 40 new clinical P. ae uginosa s ains wi h di e en an ibio ic sensi i i y pa e ns as well as
he p esence/absence o ca bapenemases. Resul s indica ed he p esence o a educed flexi-
ble genome (15% o al) mos ly ep esen ed by phages and de ense mechanisms agains
hem, showing an impo an ole in e olu ion and pa hogenici y. We ound a high di e si y
o an ibio ic esis ance genes g ouped in small clus e s mobilized ia in eg a i e and conju-
ga i e elemen s and acili a ed by he high homologous ecombina ion de ec ed. O holog
genes we e ound in se e al pa hogenic s ains om dis an ly ela ed axa in di e en mo-
bile elemen s wi h a global dis ibu ion. The mic odi e si y ound in hose s ains wi hou
ca bapenemases did no e eal a clea pa e n ha could be associa ed wi h ca bapenem
esis ance, sugges ing mul iple mechanisms o esis ance in he co e genome. Ou esul s
p o ide new insigh in o he dynamics and high genomic plas ici y by which clinical s ains
o P. ae uginosa acqui e esis ance. This knowledge can be applied o o he mul id ug- esis -
an mic obes o c ea e p edic i e amewo ks o assessing common molecula mechanisms
o an ibio ic esis ance and in eg a ed in o new s a egies o hei p e en ion.
IMPORTANCE The g owing eme gence and sp ead o ca bapenem- esis an pa hogens
wo ldwide exace ba e he clinical challenge o ea ing hese in ec ions. Gi en he impo -
ance o ca bapenems o he ea men o in ec ions caused by Pseudomonas ae uginosa,
his s udy aimed o in es iga e he unde lying genomic p ope ies o he clinical isola es
ha exhibi ed esis ance o imipenem and imipenem- elebac am. This in o ma ion will
enhance ou abili y o o ecas ai s o esis an s ains and design eliable ea men s
agains his impo an h ea . Ou esul s p o ide new insigh in o he dynamics and high
genomic plas ici y by which clinical s ains o P. ae uginosa acqui e esis ance as well as
o e s a me hodology ha can be applied o many o he oppo unis ic pa hogens wi h
b oad an ibio ic esis ance.
KEYWORDS Pseudomonas ae uginosa, imipenem, ca bapenem esis ance, in eg a i e
and conjuga i e elemen , an ibio ic esis ance, imipenem- elebac am
Pseudomonas ae uginosa is an oppo unis ic human pa hogen ha has become a
eal conce n in hospi al-acqui ed in ec ions due o i s high a es o an ibio ic esis ance
(1). Because o he di ficul y o ea ing in ec ions caused by his pa hogen, P. ae uginosa has
been classified, acco ding o he Wo ld Heal h O ganiza ion, as “c i ical”in he global p io i y
pa hogens lis o an ibio ic- esis an bac e ia (2).
Edi o Susannah G een T inge, U.S.
Depa men o Ene gy Join Genome Ins i u e
Copy igh © 2021 López-Pé ez e al. This is an
open-access a icle dis ibu ed unde he e ms
o he C ea i e Commons A ibu ion 4.0
In e na ional license.
Add ess co espondence o Ma ia Paz Ven e o,
[email p o ec ed], o Juan Ca los
Rod íguez, [email p o ec ed].
The au ho s decla e no conflic o in e es .
Recei ed 8 Oc obe 2021
Accep ed 7 No embe 2021
Published
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RESEARCH ARTICLE
24 No embe 2021
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The genome o P. ae uginosa encodes a wide a ie y o e flux pump sys ems ha
oge he wi h a low pe meabili y o he ou e memb ane make bac e ia o his species
in insically esis an o an ibio ics (1). In addi ion, mul id ug esis ance can be achie ed
by (i) ho izon al ans e o mobile gene ic elemen s (MGEs) ca ying esis ance de e mi-
nan s, (ii) mu a ion o a ge genes leading o gene dis up ion o modifica ion o gene
exp ession, and (iii) biofilm-media ed esis ance (1, 3).
Ca bapenems a e commonly used in clinical p ac ice o ea in ec ions caused by P.
ae uginosa (4). Despi e i s e ficacy, in ecen yea s, he a e o ca bapenem esis ance s ains
has inc eased wo ldwide (5, 6). The main mechanism o esis ance is he p oduc ion o ca -
bapenemases (7, 8); howe e , he e is a high pe cen age o esis an s ains ha do no p es-
en ca bapenemases. Di e en mechanisms ha e been iden ified in hese non-ca bapene-
mase-p oducing bac e ia, such as o e exp ession o e flux pumps, down egula ion o loss o
ou e memb ane po ins, and p oduc ion o ampicillin C- ype
b
-lac amases (9).
The e o e, due o he wide genomic a senal wi h which P. ae uginosa s ains a e
endowed o ace his ype o an ibio ic, i is necessa y o de elop new ea men s (10).
One o hese no el s a egies is he use o elebac am, a no el diazabicyclooc ane
be a-lac amase inhibi o , combined wi h imipenem (Im) (11). Recen ly, i s ac i i y has
been es ed in i o agains a la ge collec ion o mul id ug- esis an clinical isola es o
P. ae uginosa in se e al independen s udies, and he p e alence o suscep ible s ains
was always highe han 92% (11–13).
Gi en he impo ance o ca bapenems o he ea men o in ec ions caused by P.
ae uginosa, his s udy aimed o in es iga e he unde lying genomic p ope ies o he
clinical isola es ha exhibi ed esis ance o Im and imipenem- elebac am (I/R). Add essing
hese p ope ies will enhance ou abili y o o ecas ai s o esis an s ains and design eli-
able ea men s agains his impo an h ea .
RESULTS AND DISCUSSION
To in es iga e possible mechanisms unde lying an ibio ic esis ance in P. ae uginosa,we
isola ed 40 new s ains om di e en ypes o clinical samples (see Table S1 in he supplemen-
al ma e ial). All s ains we e iden ified using ma ix-assis ed lase deso p ion ioniza ion– ime
o fligh mass spec ome y (MALDI-TOF MS) (14) and subsequen ly sequenced o confi -
ma ion by phylogenomic analysis, as desc ibed below. The genomic ea u es o he s ains
sequenced a e shown in Table S3 in he supplemen al ma e ial. Based on he analysis o he
esis ance o be a-lac am an ibio ics as well as he p esence o ca bapenemases (see Ma e ials
Me hods) (Table S1), we we e able o clus e s ains in o 5 g oups. S ains esis an o bo h Im
and I/R wi hou ca bapenemases we e designa ed g oup 1 (G1; 6 s ains) and wi h ca bapene-
mases g oup 2 (G2; 9 s ains). G oup 3 (G3; 13 s ains) included s ains esis an o Im bu sensi-
i e o I/R wi hou ca bapenemases, and g oup 4 (G4; 2 s ains) included hose wi h ca bape-
nemases. In g oup 5 (G5; 10 s ains) we e s ains ha we e sensi i e o bo h an ibio ics.
Phylogenomic classifica ion. Fi s , we sough o analyze he genomic di e si y o
P. ae uginosa s ains eco e ed. We pe o med a whole-genome phylogenomic ee and a -
e age nucleo ide iden i y (ANI) using genomes ob ained in his s udy oge he wi h 227 P.
ae uginosa e e ence genomes om a wide ange o geog aphical and isola ion sou ces (see
Table S2 in he supplemen al ma e ial). The esul s e ealed ha s ains clus e ed in o wo
g oups wi h ANI alues o ca. 98% (Fig. 1A, see Fig. S1 and S2 and Table S4 in he supple-
men al ma e ial), consis en wi h he cu o accep ed o designa e membe s o same species
(15). Howe e , none o ou s ains ep esen ed an independen clade, and we did no find
any significan ela ionship be ween phylogeny, an ibio ic esis ance pa e ns, he o igin o
isola ion, o i ulence (Fig. 1A, Fig. S1 and S2, and Table S2). Al hough all clinical s ains a e
capable o p oducing simila symp oma ology, hey a e all dis ibu ed in independen
clades, sugges ing a high in insic capaci y o pa hogenesis.
Pseudomonas ae uginosa pangenome. We pe o med a pangenome analysis o
e alua e he comple e genomic di e si y o he s ains. While he pe sis en genome, i.e., num-
be o gene amilies ha a e p esen in almos all he genomes (a leas 95%), apidly eached
he pla eau, he pangenome cu e had no sa u a ed, indica ing an open pangenome
(Fig. 1B). The pe sis en genome was 5,289 gene amilies which ep esen on a e age 85.2%
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o a P. ae uginosa genome. This alue also cons i u ed ca. 45% o he en i e pangenome
(11,804 gene amilies) (Fig. 1B). The numbe o genes amilies ha o med he shell genome
(a leas 15%bu less han,95% o he s ains) was 1,225 gene amilies ep esen ing 10%
o he o al pangenome. Finally, gene amilies p esen ed in less han 15% o s ains we e
classified as he cloud genome (5,290 gene amilies, comp ising ca. 45% o he pangenome).
Typically, each new isola e added ca. 380 new gene amilies.
Thegene amilieswe ecompa edagains heSEEDsubsys emsda abase(16) o unc-
ional cha ac e iza ion. The ac ion o gene amilies o he pe sis en genome ha could
be assigned o a SEED ca ego y was 58%, while o he shell and he cloud i was only 28
and 22%, espec i ely. Bo h (shell and cloud genome) o m he flexible genome, which is
ela ed o adap a ion o di e en niches, acquisi ion o di e en me abolic capabili ies, o
e en pa hogenesis, highligh ing he g ea igno ance we s ill ha e abou hese mic obes.
As migh be expec ed, he pe sis en genome was en iched in ca ego ies ela ed o
cen al me abolic p ocesses, such as amino acid biosyn hesis, ansc ip ion, ansduc ion,
and eplica ion (Fig. 1C). The shell and cloud genome sha ed se e al ca ego ies, such as he
p esence o p ophages and ansposable elemen s o esis ance o an ibio ics (be a-lac a-
mase) and o he compounds, such as me cu y, me als, and coppe , wi hin he ca ego y “ i -
ulence, disease, and de ense.”Specifically, he cloud genome was en iched in phage de ense
sys ems, such as CRISPR sys ems (“clus e ing-based subsys ems”), oxin-an i oxin sys ems
(“ egula ion and cell signaling”) o es ic ion-modifica ion sys ems (“DNA me abolism”), and
p o ec ion agains oxida i e s ess (“s ess esponse”), as well as Ton and Tol anspo sys-
ems and ype IV sec e ion sys ems wi hin he “memb ane anspo ”ca ego y (Fig. 1C).
Shell pa i ion was en iched in side opho es (“i on acquisi ion and me abolism”)(Fig.1C).
Gene amilies comp ising he flexible genome a e o en g ouped in o ho spo egions
h ough he ch omosome. Based on he pangenome g aphs, we ha e used he panRGP
me hod (17) o de e mine hese egions o genome plas ici y (RGP) using he P. ae uginosa
NCTC10332 genome as a e e ence. We ound 27 RGP ep esen ing 326 genes (Fig. 1D).
I is no ewo hy ha 11 o he 27 RGP which ep esen 56.4% o he genes a e ela ed o
FIG 1 (A) Maximum likelihood phylogenomic ee o he P. ae uginosa genomes isola ed in his s udy. (B) Pangenome and co e genome size
accumula ion, as well as dis ibu ion o gene amilies among he 40 P. ae uginosa s ains. (C) G aphical ep esen a ion o he in e ed me abolism wi h
SEED o he di e en pa i ions o he pangenome. (D) A schema ic ep esen a ion o he egions o genome plas ici y using he P. ae uginosa NCTC10332
genome as a e e ence.
Ca bapenem-Resis an P. ae uginosa Compa ison Genomics
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phage-hos in e ac ions. We ound ou p ophages as well as i al de ense sys ems, such
as CRISPR, es ic ion-modifica ion sys ems, and glycosyla ion islands o he flagellum and
he ou e memb ane which a e a ge s uc u es used by phages (18). The e o e, his in e ac-
ion can be an impo an ac o in he popula ion dynamics and e olu ion o hese mic obes
inc easing gene ic di e si y, which could also ha e an impac on pa hogenesis. Fo his ea-
son, we iden ified p ophage sequences based on he PHASTER p edic ion (19) in all he P.
ae uginosa s ains and unc ionally anno a ed all hose genes. Th ee o hese sequences
we e ela ed o an ibio ic esis ance amilies such as class B be a-lac amases, aminoglycoside
O-nucleo idyl ans e ases, and majo acili a o supe amily e flux pumps. In e es ingly, one
o he sequences was anno a ed as a zonula occludens oxin, an en e o oxin desc ibed in
Vib io chole ae ha inc eases mucosal pe meabili y (20).
Pseudomonas ae uginosa mobilome. Gi en ha mos o he i ulence and an ibi-
o ic esis ance ac o s a e p esen in he flexible genome (cloud 1shell genome), we
decided o analyze he impac o DNA agmen ans e by analyzing ecombina ion
as well as he MGEs in ol ed in i s dispe sion.
Using he co e genome alignmen o all he isola es, we compu ed he ela i e a e o
ecombina ion o mu a ion (
g
/
m
) using mco (21), which was es ima ed o be 8.9 (s anda d
de ia ion [SD] o 0.525). These da a sugges ha ecombina ion p oduces mo e nucleo ide
eplacemen s han mu a ions. A compa ison wi h wo o he impo an G am-nega i e noso-
comial pa hogens (Klebsiella pneumoniae and Acine obac e baumannii) e ealed ha P. ae u-
ginosa has much highe ecombina ion alues. Using he same me hodology, K. pneumoniae
and A. baumannii had
g
/
m
alues o 4.2 and 1.3, espec i ely (21). Howe e , P. ae uginosa
showed lowe alues han Mycobac e ium abscessus, ano he oppo unis ic pa hogen ha
had he highes ecombina ion alues (
g
/
m
= 13) (21).
The genomic compa ison e ealed he p esence o h ee di e en ypes o in eg a-
i e and conjuga i e elemen s (ICEs) (22). We ound his ype o MGE in 90% o he
s ains (36 ou o 40), and all o hem belonged o h ee di e en ypes (Fig. 2A). The
fi s one co esponding o he pKLC102 amily was inse ed in a RNA-Gly. Ca go genes
om his egion we e ela ed o niche adap a ion, such as esis ance o di e en
FIG 2 (A) Maximum likelihood phylogenomic ee o he P. ae uginosa genomes isola ed in his s udy; he box on he igh shows he ype o ICE p esen
in each o he genomes. (B) Compa ison o he gene con en o he ICE in he s ain genomes. P edic ed open eading ames (ORFs) wi h he same colo a e
in ol ed in he same unc ion. The genomic map indica es he loca ion o he h ee ypes o ICEs in he e e ence genome (P. ae uginosa NCTC10332).
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en i onmen al compounds (coppe , ch oma e, a senic, and me cu y), as well as an
en ichmen in LysR ansc ip ional egula o s ha can be beneficial o he hos cell
(23) (Fig. 2B). The second ype s ood ou o ha ing es ic ion sys ems o p o ec ion
agains phages bu mainly o he p esence o wo clus e s wi h mul iple genes o e-
sis ance o an ibio ics. The fi s an ibio ic clus e was associa ed wi h a Tn3-like ans-
poson p esen in a g oup o h ee (1105-S40, 9843-S14, and 8057-S16) o ou s ains
wi h an ANI o .99% which indica es he ease o mo emen o hese elemen s among
s ains (Fig. 2B). The an ibio ic esis ance genes loca ed in his clus e we e VIM be a-lac-
amases, an aminoglycoside N-ace yl ans e ase AAC(69)-IIa, a ype B-3 chlo amphenicol O-
ace yl ans e ase, a s ep omycin 39-adenylyl ans e ase, a sul onamide- esis an dihyd op e -
oa e syn hase, and a Gcn5- ela ed N-ace yl ans e ases (Fig. 2B). On he o he hand, he sec-
ond clus e had h ee genes ha con e esis ance o chlo amphenicol, os omycin, and ami-
noglycoside an ibio ics. Fu he mo e, wi hin he 0268-S35 s ain, we also ound a de ec i e
p ophage wi h a sequence encoding he zonula occludens oxin. The hi d ype o ICE
showed only a small a iable egion among he s ains, which mos ly encodes se e al oxin-
an i oxin sys ems (Fig. 2B).
An imic obial genes. Nex , we analyzed he p esence o an imic obial esis ance
genes (ARGs) using he MEGARes 2.0 da abase (24). These p edic ions we e co ela ed wi h
hei co espondence in he di e en pangenome pa i ions. Using a 70% h eshold (BLASTP),
a o al o 154 ARGs we e de ec ed as g ouping in o 28 amilies. The mos ep esen ed amilies
we e “d ug and biocide esis ance”(21.4%), including di e en ypes o e flux pumps and eg-
ula o s ( esis ance-nodula ion-di ision [RND], ATP-binding casse e [ABC], and mul id ug and
oxic compound ex usion [MATE] anspo e s); “me cu y esis ance”(12.3%); “mul ime al e-
sis ance”(9.1%); “be a-lac ams”(8.4%); and “coppe esis ance”(5.8%). The pe sis en genome
con ains abou 54% o he ARGs and is whe e mos o he e flux pumps and egula o s a e
concen a ed. This finding highligh s he in insic capaci y o hese mic obes o an imic obial
esis ance. In he o he wo pa i ions, one-hal o he ARGs we e ela ed o esis ance o me -
cu y, be a-lac ams, and aminoglycosides in he cloud genome (ca. 29% o o al ARGs) and e-
sis ance o me cu y, coppe , and be a-lac ams in he shell genome (ca. 17% o o al ARGs).
The analysis o he genomic con ex whe e he ARGs we e loca ed in he flexible genome
e ealed ha 44% we e associa ed wi h ICEs (including associa ed ansposons), while 17%
we e loca ed in addi i e islands and only 3 sequences in plasmids. In e es ingly, an ibio ic e-
sis ance genes showed a endency o clus e in he same a ay wi hin he ICEs con e ing mul-
iple esis ance which inc eases he isk o human heal h. The es o he sequences we e
ound in con igs oo small o be able o in e whe he hey we e associa ed wi h a specificmo-
bile gene ic elemen .
Imipenem- elebac am esis ance wi h ca bapenemases. Th ough compa a i e
genomics, we e alua ed he dynamics and mechanisms ha hese s ains ha e de eloped
o acqui e esis ance o I/R. We ound six genomes belonging o he G3 and one genome
om G2, s ain 1578-S13, ha clus e ed oge he wi hin he same clonal ame (ANI , .99%)
(Fig. 1A and S1). These di e ences allowed us o s udy specific genomic di e ences ha
d i e he pheno ypic di e ences by sub ac ing he common pa o bo h pangenomes. We
ound 119 specific genes in he 1578-S13 genome. Twel e genes we e concen a ed in a
con ig o only 8 kb. Among o he s, a gene coding o an AAC(69)-Iag, an aminoglycoside
ace yl ans e ase ela ed o aminoglycoside an ibio ic esis ance, and a VIM-4 (ca bapene-
mases class B), which could pu a i ely e ade be a-lac am inhibi o s like elebac am, we e
ound. Nex o hese genes, we also ound ansposi ion genes, a TniA and TniQ module,
which a e no mally associa ed wi h a pa o class 1 in eg ons and a e de i a i es o Tn402
(also called Tn5090)(25).
A BLAST sea ch o he VIM-4 gene sequence agains he non edundan (n ) NCBI da abase
showed ha his gene was ound in a ple ho a o MGEs, such as in eg ons, plasmids, o ICEs,
in clinical s ains isola ed wo ldwide wi hin membe s o Pseudomonadales,Bu kholde iales,
and En e obac e ales o de s (see Fig. S3 in he supplemen al ma e ial). The e o e, he p es-
ence o o hologs o his gene in di e en mobile elemen s o di e en axonomic anges
mul iplies he isk o inc easing esis ance o he I/R combina ion in a sho pe iod o ime.
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Imipenem- elebac am esis ance wi hou ca bapenemases. To elucida e gene ic
ac o s associa ed wi h he esis ance o I/R, we compa ed genomes om G1 agains hose
om G3 (Fig. 1). We sub ac ed he common pa o he pangenomes om bo h g oups o an-
alyze hose gene amilies unique o G3 and ob ained a o al o 1,174 genes amilies. Howe e ,
an analysis o he p e alence o hese amilies among he G3 genomes showed ha none o
hem was ound in mo e han one genome. This esul could sugges ha (i) he e is a wide
di e si y o mechanisms in ol ed in esis ance and (ii) he esis ance mechanism could be
loca ed in he pe sis en genome. In he la e case, he mu a ion o some gene o noncoding
egion could lead o he modifica ion o i s exp ession. The e o e, we decided o analyze he
mic odi e si y be ween esis an and sensi i e s ains o de ec possible a ge genes.
Imipenem- elebac am esis ance (mic odi e si y). We nex sough o de e mine
whe he he esis ance o ca bapenems is due o he p esence o single-nucleo ide polymo -
phisms (SNPs). The e o e, we g ouped 7 isola es suscep ible o Im and compa ed hem o 11
and 6 isola es esis an o Im and I/R, espec i ely. Only isola es wi h clea e idence o no ha -
bo ca bapenemases we e conside ed. Fo a gi en posi ion, we conside ed all he possible nu-
cleo ides p esen in he suscep ible g oup as nonsignifican mu a ions h ough a window o
100 nucleo ides and significan hose SNPs p esen in he esis an g oups and di e en om
he suscep ible one (see Ma e ials and Me hods). In he end, we could e ie e se e al signals
o high nucleo ide a ia ions o Im and I/R on which mo e han 40% o he s ains in hese
g oups ha e SNPs (Fig. 3). Due o high ecombina ion de ec ed, we ound SNPs in common
p o eins, such as in eg ases, ansposases, me hyl ans e ases, and es ic ion-modifica ion
FIG 3 Dis ibu ion o single-nucleo ide polymo phisms (SNPs) along he genome o 2696_S6. SNP equency conside ing se e al s ains esis an o
imipenem- elebac am (5 s ains) (A) and imipenem (11 s ains) (B). Numbe ed peaks a e anno a ed on he bo om. In bold, p o eins wi h a leas one
memb ane domain. Red do s indica ed sha ed p o eins be ween ea men s.
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sys ems, and along he ICE. Rema kably, se en p o eins ca ying memb ane domains, mos ly
hypo he ical, had also a high p opo ion o SNPs. We ound among hese p o eins he ou e
memb ane po in Op D in bo h an ibio ics. This p o ein pa icipa es in he passi e up ake o
basic amino acids ac oss he ou e memb ane, bu i is also pe meable o ca bapenems (26).
By eal- ime PCR and p o ein de ec ion, p e ious s udies demons a ed ha he low- o-absen
exp ession o he op D gene is equen ly no ed in ca bapenem- esis an isola es wi hou ca -
bapenemase ac i i y (27). We pe o med he alignmen o he Op D p o ein among suscep i-
ble and esis an isola es o iden i y hose possible mu a ions ha would con e esis ance o
Im (see Fig. S4 in he supplemen al ma e ial). In he end, a o al o 103 and 32 synonymous
and nonsynonymous mu a ions, espec i ely, we e de ec ed along his gene. I is impo an o
highligh ha 81% o he esis an s ains had mu a ions ha p oduced a p ema u e s op
codon, ega dless o he p esence o elebac am, as expec ed gi en ha he compound ac s
as a be a-lac amase inhibi o (28). On he o he hand, h ee esis an isola es ca ied he ull
p o ein. Howe e , due o he amino acid a iabili y wi hin he suscep ible g oup, we could no
de e mine any significan mu a ion ha migh co ela e wi h hei esis ance o Im; hus, o he
molecula mechanisms may ake place. Ou esul s ag ee wi h p e ious epo s ha indica ed
ha unca ed Op D p o eins a e esponsible o he esis ance o Im (29).
Finally, he same me hodology was applied o he compa ison be ween Im e sus
I/R esis an s ains. Conside ing he same h eshold o 40% o he s ains ha ing a SNP in a
gi en posi ion, we de ec ed 54 ho spo s o SNP accumula ion. Nine peaks co esponded o
in e genic space s, whe eas he emaining 46 peaks we e ound in coding sequences, such
as he flagella biosyn hesis ope on, ype VI sec e ion p o eins, non ibosomal pep ide-syn-
he ase (NRPS) sys ems, and a ew memb ane p o eins, which we e mos ly anspo e s
ela ed o i on (signals 1, 27, and 30), cyana e (9), and lipop o ein (38) (see Fig. S5 in he sup-
plemen al ma e ial). Besides, se en signals we e ound in p o eins, and hei unc ions ha e
no been cha ac e ized ye . The high di e gence be ween s ains leads o a la ge accumula-
ion o mu a ions which makes i di ficul o de e mine an exac a ge associa ed wi h esis -
ance. Howe e , he analysis e ealed se e al anspo e s o po ins ha should be s udied in
de ail by knockou . In addi ion, he de elopmen o in i o esis an mu an s would be a
good app oach o complemen he esul s ob ained.
The da a sugges ha bo h he flexible and pe sis en (o co e) genome a e closely
ela ed o I/R esis ance. This esul , oge he wi h he high me abolic flexibili y due o he
la ge numbe o ansc ip ional egula o s ha con ol he exp ession o he sec e ion o
quo um sensing sys ems, make i possible o hese mic obes o adap apidly o en i on-
men al s esses, such as an ibio ics (30). Howe e , he analysis o mu a ions o p esence/ab-
sence o genes did no yield a clea esul , which could sugges ha he p ocesses leading
o I/R esis ance a e mul i ac o ial o ha egula ion occu s a he ansc ip ional le el exe -
ing i s e ec on gene exp ession. Thus, de ec ion by adi ional PCR-based means would no
be easible. Fu u e s udies should be di ec ed o ansc ip ional analysis be ween sensi i e
and esis an s ains.
The high ecombina ion and he concen a ion o mul iple esis ances in small clus e s o
genes easily ansmi ed mainly by ICEs make he he apeu ic op ions in he figh agains an-
ibio ic esis ance inc easingly limi ed. Due o he u gency and se iousness o he p oblem,
we mus an icipa e he eme gence o h ea s ha may a ise, which is why he implemen a-
ion o genomics in he hospi al en i onmen is a c ucial poin in he figh agains esis ance.
Analyzing genomic di e si y as well as acking new genes esponsible o esis ance is c ucial
o designing mo e accu a e a ian de ec ion and moni o ing s a egies. O e all, his s udy
no only ad ances he knowledge o gene ic di e si y in hese s ains bu also o e s a me h-
odology ha can apply o many o he oppo unis ic pa hogens wi h b oad an ibio ic esis -
ance, such as A. baumannii o K. pneumoniae.
MATERIALS AND METHODS
P. ae uginosa isola ion and sequencing. S ains we e collec ed du ing 2020 om clinical isola es o
pa ien s hospi alized a Hospi al Gene al Uni e si a io de Alican e (Spain). An imic obial suscep ibili y
es was ca ied ou wi h he Mic oScan WalkAway sys em (Beckman). The o igin o he isola es and hei
an ibio ic esis ance pa e ns a e de ailed in Table S1. MALDI-TOF mass spec ome y was used o species
Ca bapenem-Resis an P. ae uginosa Compa ison Genomics
No embe /Decembe 2021 Volume 6 Issue 6 e00836-21 msphe e.asm.o g 7
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iden ifica ion. The p esence o ca bapenemases was de e mined using he Gene Xpe sys em (Cepheid).
DNA was ex ac ed using Chelex 100 Resin (Bio-Rad) and checked o quali y on a 1% aga ose gel. Sequencing
was pe o med using he Illumina HiSeq 2000 (100-bp pai ed-end eads) pla o m.
Genome compa ison and phylogeny. The gene a ed eads we e immed and assembled using
T immoma ic 0.36 (31) and SPAdes 3.11.1 (32), espec i ely. The esul ing genes on he assembled
con igs we e p edic ed using P odigal 2.6 (33). RNA genes we e p edic ed using RNAscan-SE 1.4 (34)
and ssu-align 0.1.1 (35) along wi h me a- na (36) o RNA genes. P edic ed p o ein sequences we e
compa ed agains he NCBI n da abase using DIAMOND (37) and agains COG (38) and TIGRFAMs (39)
using HMMscan 3.1b2 (40) o axonomic and unc ional anno a ion. AMRs we e de ec ed in ou sam-
ples using he MEGARes 2.0 da abase (24). Assembled p o eins we e aligned o he e e ence da abase
using DIAMOND ($50% iden i y, $50% alignmen leng h, E alue o ,10
25
). A e age nucleo ide iden i y
(ANI) and co e age be ween pai s o genomes we e calcula ed using he PYANI so wa e (41). We applied he
so wa e mco (21) o in e he pa ame e s o homologous ecombina ion, i.e., he a e o ecombina ion o
mu a ion (
g
/
m
). To classi y he s ains phylogenomically, genomes we e analyzed using TIGRFAMs o iden i y
and conca ena e all he conse ed p o eins. The conca ena ed p o eins we e aligned using Kalign (42), and a
maximum likelihood ee was made using Fas T ee (43) using a JTT 1CAT model and a gamma app oxima-
ion. As a e e ence, all a ailable genomes belonging o he species Pseudomonas ae uginosa we e down-
loaded om he Pseudomonas Genome Da abase (h ps://www.pseudomonas.com/)(TableS2).
Pangenome analysis. Pangenomes we e gene a ed using PPanGGOLiN so wa e, and gene amilies
we e di ided in o pe sis en /shell/cloud pa i ions (44). Then, he SEED subsys em da abase was used o de e -
mine he unc ional anno a ion o genes ha cons i u ed each pa i ion (16). The compa ison was made using
DIAMOND (37), keeping all ma ches wi h an E alue o ,0.001 and alignmen leng h o .0.5 o bo h subjec
and que y. The panRGP me hod (17) was used o p edic egions o genome plas ici y (RGPs) using pange-
nome g aphs made o all a ailable genomes ob ained om PPanGGOLiN analysis.
Iden ifica ion o single-nucleo ide polymo phisms (SNPs). SNPs we e de e mined by aligning
immed Illumina eads o he e e ence genomes using Bow ie 2 (45). Only alignmen s wi h an e o a e o
,0.1% we e conside ed. Va ian s we e hen de ec ed using Va scan (46), conside ing ha , o a gi en polymo -
phism, i had o be p esen in a leas 80% o he aligned eads. Las ly, SnpE (47) was used o disc imina e
be ween synonymous, nonsynonymous, and in e genic mu a ions. We called a mu a ion only i he SNP was di -
e en om hose de ec ed in he suscep ible g oup (e.g., A and G a ia ions in he suscep ible g oup, only T and
C a e called mu a ions in he esis an s ain). Only isola es wi h clea e idence o no ha bo ca bapenemases
we e analyzed. Finally, he amino acid alignmen o Op D p o ein sequences was pe o med wi h MUSCLE (48).
Da a a ailabili y. The genomes ha e been deposi ed unde BioP ojec PRJNA754264.
SUPPLEMENTAL MATERIAL
Supplemen al ma e ial is a ailable online only.
FIG S1, PDF file, 0.04 MB.
FIG S2, PDF file, 0.2 MB.
FIG S3, PDF file, 0.04 MB.
FIG S4, PDF file, 0.1 MB.
FIG S5, PDF file, 0.1 MB.
TABLE S1, XLSX file, 0.01 MB.
TABLE S2, XLSX file, 0.03 MB.
TABLE S3, XLSX file, 0.01 MB.
TABLE S4, XLSX file, 0.8 MB.
ACKNOWLEDGMENTS
This wo k was suppo ed by g an IISP 57739 om Me ck & Co., Inc. o Juan Ca los
Rod íguez.
We decla e ha we ha e no compe ing in e es s.
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