BioMed Cen al
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BMC Mic obiology
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Resea ch a icle
P edic i e analysis o ansmissible quinolone esis ance indica es
S eno ophomonas mal ophilia as a po en ial sou ce o a no el amily
o Qn de e minan s
Ma ía B Sánchez†1, Al a o He nández†1, José M Rod íguez-Ma ínez2,
Luis Ma ínez-Ma ínez3,4 and José L Ma ínez*1
Add ess: 1Depa amen o de Bio ecnología Mic obiana, Cen o Nacional de Bio ecnología, CSIC, Da win 3, Can oblanco, 28049-Mad id, and
CIBERESP. Spain, 2Depa amen o de Mic obiología, Uni e sidad de Se illa, A . Sanchez Pizjuan SN 41009-Se illa, Spain, 3Se ice o
Mic obiology, Uni e si y Hospi al Ma qués de Valdecilla, San ande , Spain and 4Depa men o Molecula Biology, Uni e si y o Can ab ia,
San ande , Spain
Email: Ma ía B Sánchez - [email p o ec ed]; Al a o He nández - ahd[email p o ec ed]sic.es; José M Rod íguez-Ma ínez - jm od ig[email p o ec ed];
Luis Ma ínez-Ma ínez - u10315@hum .es; José L Ma ínez* - jlm[email p o ec ed]
* Co esponding au ho †Equal con ibu o s
Abs ac
Backg ound: P edic ing an ibio ic esis ance be o e i eme ges a clinical se ings cons i u es a
no el app oach o p e en ing and igh ing esis ance o bac e ial pa hogens. To analyse he
possibili y ha no el plasmid-encoded quinolone esis ance de e minan s (Qn ) can eme ge and
dissemina e among bac e ial pa hogens, we sea ched he p esence o hose elemen s in nea ly 1000
bac e ial genomes and me agenomes.
Resul s: We ha e ound a numbe o no el po en ial qn genes in he ch omosomes o aqua ic
bac e ia and in me agenomes om ma ine o ganisms. Func ional s udies o he S eno ophomonas
mal ophilia Smqn gene show ha plasmid-encoded SmQn con e s quinolone esis ance upon i s
exp ession in a he e ologous hos .
Conclusion: Al oge he , he da a p esen ed in ou wo k suppo he no ion ha p edic i e
s udies on an ibio ic esis ance a e easible, using cu en ly a ailable in o ma ion on bac e ial
genomes and wi h he aid o bioin o ma ic and unc ional ools. Ou esul s con i m ha aqua ic
bac e ia can be he o igin o plasmid-encoded Qn , and highligh he po en ial ole o S. mal ophilia
as a sou ce o no el Qn de e minan s.
Backg ound
Quinolones a e syn he ic an ibio ics; he e o e, i was
hough ha he exis ence o ans e able quinolone
esis ance genes in na u e would be unlikely. Resis ance
mechanisms o hese d ugs we e expec ed o be only
ch omosomally encoded. I was belie ed ha only mu a-
ions in he quinolone a ge s (DNA gy ase o opoi-
some ase IV) o mu a ions ha led o a pe meabili y
dec ease and/o o e p oduc ion o mul id ug esis ance
(MDR) e lux pumps would esul in esis ance o hese
an ibio ics [1,2].
I was hus assumed ha esis ance o quinolones could
no sp ead as he consequence o ho izon al gene ans e
Published: 16 Sep embe 2008
BMC Mic obiology 2008, 8:148 doi:10.1186/1471-2180-8-148
Recei ed: 12 Ma ch 2008
Accep ed: 16 Sep embe 2008
This a icle is a ailable om: h p://www.biomedcen al.com/1471-2180/8/148
© 2008 Sánchez e al; licensee BioMed Cen al L d.
This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License (h p://c ea i ecommons.o g/licenses/by/2.0),
which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed.
BMC Mic obiology 2008, 8:148 h p://www.biomedcen al.com/1471-2180/8/148
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(HGT). La e on, he acquisi ion o quinolone esis ance
due o HGT was p oposed as a possibili y based on in i o
expe imen s [3]. This was la e con i med in 1998, wi h
he desc ip ion o a plasmid-encoded quinolone esis -
ance de e minan , ha was named Qn , in clinical isola es
[4]. Mo e ecen ly wo o he ans e able quinolone esis -
ance de e minan s ha e been desc ibed, he bi unc ional
aminoglycoside/quinolones inac i a ing aminoglycoside
ace yl ans e ase AAC(6')-Ib-c [5,6] and he quinolone
e lux de e minan , QepA [7,8].
Since he disco e y o Qn , he p esence o di e en qn
amilies (A, B, S) in esis ance plasmids [9] has been
ound wo ldwide in di e en bac e ial pa hogens [10-15].
Mo e ecen ly, plasmid-encoded Qn de e minan s ha e
also been desc ibed in en i onmen al isola es o Ae om-
onas spp. [16]. The p esence o Qn genes in ch omo-
somes has also been shown, including Qn A in Shewanella
algae, which is likely he o igin o plasmid-encoded Qn A
de e minan s [17], and di e en membe s o he Qn am-
ily om Vib ionaceae species [18,19].
The Qn p o eins belong o he pen apep ide epea p o-
ein (PRP) amily, which is de ined by he p esence o ep-
e i ions in andem o he pa e n (A/C/S/T/V)(D/N)(L/
F)(S/T/R)(G/R) [20-22,17]. A cha ac e is ic ea u e o he
Qn p o eins is ha hey a e o med by wo domains o
pen apep ide epea s sepa a ed by a single glycine. This
s uc u e ma ches a mo i o unknown unc ion named
Clus e o O hologous G oup o P o eins (COG) 1357
h p://www.ncbi.nlm.nih.go /COG/. E en hough mem-
be s o he PRP amily ha e been iden i ied bo h in
p oka yo es and euka yo es [22], qn genes p esen ing he
abo e desc ibed COG1357 mo i ha e a much na owe
dis ibu ion, mainly in plasmids and in he ch omosomes
o some bac e ial species. The amino acid iden i y among
di e en Qn p o eins amilies a es be ween 39–60%
[23].
Some s udies ha e demons a ed ha Qn p o ec s bo h
gy ase and opoisome ase IV om he ac i i y o quinolo-
nes [15,24-26]. O he membe s o he PRP amily, ha
may play a simila ole include McbG, which p o ec s
mic ocin B17-p oducing bac e ia om he ac i i y o his
DNA eplica ion inhibi o [27] and M pA [28], a p o ein
ha mos likely binds Gy A [22] and p o ides low-le el
quinolone esis ance o Mycobac e ium ube culosis.
Al hough qn -like elemen s ha con ibu e o in insic
quinolone esis ance ha e been desc ibed in he ch omo-
some o En e ococcus aecalis [24] and mo e ecen ly in
o he G am-posi i e bac e ia [29], hei homology wi h
qn genes om G am-nega i e bac e ia is low (a ound
25%), so ha his amily o esis ance elemen s has no
been included in ou analysis.
I is impo an o no e ha plasmid-encoded quinolone
esis ance is mo e p e alen han expec ed conside ing he
da e o he i s isola ion, which sugges s ha hese plas-
mids ha e been ci cula ing o some ime be o e hey we e
i s desc ibed [30]. This highligh s he need o implemen
me hods o p edic esis ance be o e i is ecognized
wi hin clinical se ings.
A me hodology o p edic ing he possibili y o eme gence
o a new mechanism o esis ance be o e i appea s in bac-
e ial pa hogens has been ecen ly p oposed [31]. The
a ailabili y o sequenced genomes allows his ype o
analysis. This in o ma ion was used o sea ch o he p es-
ence o Qn de e minan s in a ailable sequenced bac e ial
genomes and me agenomes. A pu a i e qn gene p esen
in he ch omosome o he oppo unis ic pa hogen S eno-
ophomonas mal ophilia was chosen o pe o m unc ional
analyses. S. mal ophilia is a nosocomial pa hogen in insi-
cally esis an o se e al an imic obials [32] due o he
ac i i y o an ibio ic-inac i a ing enzymes [33-36] and
MDR e lux pumps [37-43].
The possibili y ha ch omosomally-encoded S. mal-
ophilia qn gene (he ea e named as Smqn ) could be
unc ional in a he e ologous hos has been explo ed. As
he esul o ou wo k, we ha e desc ibed new qn genes in
he ch omosomes o sequenced bac e ial genomes and
ha e ound ha he Smqn gene om S. mal ophilia
ende s a low-le el quinolone esis ance pheno ype upon
i s exp ession in Esche ichia coli. This s udy demons a es
he easibili y o u ilizing cu en ly a ailable da abases
along wi h bioin o ma ics and unc ional ools as an
app oach o p edic ing esis ance be o e i eme ges in
human pa hogens.
Me hods
Bioin o ma ic ools and nomencla u e o p edic ed Qn
p o eins
Mul iple p o ein sequence alignmen s we e ca ied ou
wi h he p og am Clus alW2 h p://www.ebi.ac.uk/Tools/
clus alw2/index.h ml[56]. Simila i y sea ch was pe -
o med wi h he p og am BLAST (NCBI) h p://
www.ncbi.nlm.nih.go /blas /Blas .cgi[57] and genomic-
Blas h p://www.ncbi.nlm.nih.go /su ils/
genom_ able.cgi. Iden i ica ion o conse ed mo i s [58]
([59] was done by using Conse ed Domain Da abase
and Sea ch Se ice a h p://www.ncbi.nlm.nih.go /S uc
u e/cdd/cdd.sh ml. The Qn p o eins alignmen s and
homology ees we e ob ained using CLUSTALW2 and
Jal iew alignmen edi o wi h de aul pa ame e s [60].
Homology ee was calcula ed using he me hod "A e age
dis ance using pe cen age o iden i y" h p://
www.ebi.ac.uk/Tools/clus alw2/index.h ml.
The p edic ed Qn p o eins we e named ollowing he
ules p oposed in [61].
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Bac e ial s ains and g ow h condi ions
The bac e ial s ains and plasmids used a e shown in
Table 1. The s ains we e g own in Lu ia-Be ani (LB)
b o h [62] a 37°C unless o he wise speci ied.
Suscep ibili y an ibio ic assays
The suscep ibili y assays o he di e en an ibio ics we e
pe o med in Muelle Hin on b o h (P onadisa) plus Iso-
p opyl- hio-ß-D-galac opy anoside 0.5 mM, using he
wo old dilu ion me hod in 96-well mic o i e pla es. The
esul s we e eco ded a e 48 h o incuba ion a 37°C. To
ensu e ha he obse ed changes we e consis en , all Min-
imal Inhibi o y Concen a ions (MICs) we e de e mined
in h ee independen assays, using di e en bac e ial cul-
u es on di e en days. In all cases, a con ol s ain con-
aining he plasmid pGEM-T wi hou any inse was
included in MICs de e mina ions. In mos cases, he e
we e no in e -assay di e ences in he MIC alues. In a ew
cases, he e we e one-dilu ion di e ences. Fo he la e ,
he assay was epea ed one mo e ime o u he assu e
assay eliabili y.
The quinolones used we e cip o loxacin, enoxacin,
ga enoxacin, g epa loxacin, le o loxacin, moxi loxacin,
nalidixic acid, no loxacin, o a loxacin and spa loxacin.
DNA manipula ions
The genomic DNA was ex ac ed using he GNOME® DNA
Ki (Q-BIOgene). The PCR Mas e Mix (P omega) was
used o ampli y ull-leng h Smqn genes om he di e en
S. mal ophilia s ains wi hou hei p omo e sequences.
The eac ion con ained 100 ng o genomic DNA o each S.
mal ophilia isola e as empla e, and 1 μM o wo di e en
se s o speci ic Smqn -p ime s, Qn M+ (5'-CTTGGCAT-
GGAATCCCTGAT-3')/Qn M-(5'-TGATGCCTACGGCAC-
CAC-3') and Qn MR55+ (5'-CATGGCATGGAATCCCCGA
T-3')/Qn MR55-(5'-TGATGTCTACGGCACCAC-3'). We
used wo se s o p ime s because he egions a ound qn
a e sligh ly di e en in he sequenced S. mal ophilia s ains
K279a and R551-3. The eac ions had one dena u a ion
s ep a 94°C o 5 minu es, ollowed by 35 ampli ica ion
cycles: 94°C o 30 seconds, 61°C o 45 seconds o
annealing, and 72°C o 1 minu e o elonga ion, wi h a
inal ex ension s ep o 72°C o 5 minu es. The PCR p od-
uc s (660 bp) ob ained om he di e en S. mal ophilia
s ains (Table 1) we e elec opho esed in 1% aga ose gels
wi h TAE, pu i ied om he gel wi h GFX™ PCR DNA and
Gel Band Pu i ica ion Ki (GE Heal hca e) and cloned in
pGEM-T plasmid (P omega) gene a ing he co espond-
ing ecombinan plasmids (Table 1). The ans o ma ion
o E. coli KZM120 was made as desc ibed [63]. We chose
his pa icula E. coli s ain because i lacks he majo E.
coli MDR pump and i has been shown p e iously ha he
u iliza ion o MDR-de ec i e s ains acili a es he cha ac-
e iza ion o low-le el mechanisms o esis ance [39]. The
ans o man s we e selec ed in LB aga wi h 100 μg/ml
ca benicillin. The ecombinan plasmids we e pu i ied
using Wiza d® Plus SV Minip ep Ki (P omega) and he
inse o each plasmid was sequenced using he uni e sal
p ime s M13 o wa d and M13 e e se by Secugen S.L.
h p://www.secugen.es/ o con i m he iden i y o he
sequence and es ablish he o ien a ion o he qn gene.
The sequences o he di e en Smqn genes ha e been
deposi ed a GenBank wi h numbe s om EU681371 o
EU681385. Only hose plasmids con aining he Smqn
gene in he igh o ien a ion o allow exp ession om he
pGEM-T lac p omo e we e used in he unc ional assays.
Iden i ica ion o Smqn in di e en S. mal ophilia isola es
The po en ial p esence o Smqn in bo h clinical and en i-
onmen al S. mal ophilia s ains was e alua ed by PCR
using he p ime s qn I1 (5'-AGAAAGTGGTCGACCAG-
CAG-3')/qn I2 (5'-GCAGGTTCGACTTCTTGATG-3') and
qn I3 (5'-CAACGCCAGCTTCATGAACC-3')/qn I4 (5'-
AGTTGGCGCTGTTCCAGTCG-3'), which ampli y 312 bp
and 220 bp agmen s o wo in e nal egions in he
Smqn gene o S. mal ophilia espec i ely.
The PCR was made as desc ibed abo e, wi h he ollowing
p og am, one dena u a ion cycle a 94°C o 5 minu es,
ollowed by 35 ampli ica ion cycles: 94°C o 30 seconds,
55°C o 45 seconds o annealing, and 72°C o 30 sec-
onds o polyme iza ion, wi h a inal ex ension o 72°C
o 5 minu es. The PCR p oduc s we e analyzed in 1% aga-
ose gels wi h TAE.
Exp ession and iden i ica ion o he SmQn p o ein
The amoun o SmQn p o ein was es ima ed by SDS-
PAGE [63] and Coomassie blue s aining, using cell
ex ac s om bac e ia g own in LB a 37°C o e nigh . The
amoun o p o eins loaded in each lane was no malized o
a ound 4 × 107 cells. In all cases, he global amoun o
p o eins was equal in all lanes o a gi en gel as can be seen
a e s aining. This se es as a supplemen a y loading con-
ol [64].
To ensu e ha he p o ein wi h he p edic ed molecula
size was indeed SmQn , he co esponding band was
excised and iden i ied, as desc ibed [65,39] by Pep ide
Mass Finge p in ing using MS-MALDI TOF (P o eo ed:
h p://www.p o eo ed.o g/).
E hical conside a ions
This in es iga ion did no equi e e hical clea ance.
Resul s and Discussion
The iden i ica ion in en i onmen al mic oo ganisms o
pu a i e an ibio ic esis ance elemen s [44] ha migh
ans e in he nea u u e o pa hogenic bac e ia is an
impo an opic ha we a e jus beginning o add ess [31].
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To ha goal a combina ion o bioin o ma ic and unc-
ional ools can gi e in o ma ion o p edic esis ance
be o e i eme ges. Following his concep , we ha e
explo ed he p esence o pu a i e qn genes in a ailable
da abases o bac e ial genome sequences, because he
gene ic con ex o plasmidic qn genes suppo s he idea
ha hey ha e been acqui ed by HGT. Fo ins ance, he
plasmid-encoded qn A gene has been ound in a sul1- ype
in eg on nea an ISCR1 elemen [21,45,25]. A simila
s uc u e was ound in qn B2 [46], and qn S is adjacen o
a Tn3 ansposon s uc u e [10,11]. In con as , i has
been epo ed ha ch omosomal qn A, in Shewanella algae
[17] and se e al qn -like genes iden i ied wi hin he Vib i-
onacea amily [18] a e no linked o hese mobile gene ic
elemen s.
This sugges s ha ch omosomally-encoded qn genes
ha e no been acqui ed ecen ly by HGT because o ecen
an ibio ic selec i e p essu e and ha ye uniden i ied
ch omosomally-encoded qn genes could be a sou ce o
new ans e able quinolone esis ance genes.
Bioin o ma ic sea ch o pu a i e qn genes
To add ess he p esence o pu a i e qn genes in he ch o-
mosomes o bac e ia ha could se e as ese oi s o his
amily o quinolone esis ance de e minan s, a bioin o -
ma ic i e a i e sea ch by sequence homology o se e al
alleles o Qn A, Qn B and Qn S was conduc ed agains
genomic o Whole Genome Sho gun Sequence (WGS)
da abases a he NCBI home page h p://
www.ncbi.nlm.nih.go /. Mos o he pu a i e qn genes
ound in his sea ch ha e been p e iously anno a ed as
hypo he ical p o eins con aining he COG1357 mo i . As
shown in Figu e 1, ch omosomally-encoded Qn p o eins
p esen a la ge deg ee o homology.
Pu a i e qn genes we e ound in 22 ou o he 960
genomes es ed. The species con aining pu a i e qn genes
belong o eigh di e en gene a (Figu e 2). Mos o hem
inhabi an aqua ic en i onmen (in some ins ances he
deep sea) sugges ing ha plasmidic qn may ha e been
o igina ed om aqua ic bac e ia [17,19,18].
Since he la ge majo i y o bac e ial species ha e no been
cul u ed so a , me agenomic s udies allow a mo e powe -
ul sea ch o ele an genes in di e en en i onmen s by
means o non-cul u e based me hodologies. Thus, besides
analysing sequenced genomes, a ailable me agenomic
sequence da abases h p://www.ebi.ac.uk/ as a33/
wgs.h ml we e also sea ched o he p esence o qn genes.
Ou s udy showed ha he me agenomes om sea-wa e
[47] con ain genes homologous o qn (he ea e named
as M gqn ), u he suppo ing he no ion ha qn genes
a e o igina ed om aqua ic mic oo ganisms. One o he
qn sequences ound in his me agenomic sea ch was
highly homologous o plasmid-encoded Qn B [15] p o-
eins (Figu e 3). I has been shown ha S. algae is likely
he o igin o plasmid-encoded qn A genes [17]. Ou anal-
yses, s ongly sugges ha plasmid-encoded qn B has also
o igina ed om a cu en ly unknown, ma ine mic oo -
ganism.
The egions su ounding he ch omosomal qn genes
ound in ou bioin o ma ics analysis we e analysed o
asce ain whe he , based in he le el o sin eny, he o igin
o hese qn de e minan s is likely monophyle ic o
polyphyle ic. As shown in Figu e 2, and in Addi ional ile
1, he egions a e e y simila o some o he analysed
species belonging o he same genus, al hough clea di e -
ences a e also obse ed o o he species.
Fo ins ance, he s uc u e su ounding qn in Vib io is e y
simila o h ee species, whe eas he s uc u e in i e
o he species and in Pho obac e ium p o undum is di e en .
The high le el o obse ed sin eny in hese egions, sug-
ges s ha qn was acqui ed by Vib io pa ahaemoly icus,
Vib io alginoly icus and Vib io ha eyi be o e hei di e -
gence, and was acqui ed by he o he Vib ionaceae a e
hei di e gence. This idea i s well wi h he ela ionship
o Vib io Qn p o eins (Figu e 4).
Sequence da a we e inspec ed o he p esence o elemen s
po en ially in ol ed in he ans e o qn genes. As shown
in Figu e 2, pu a i e ansposases o in eg ases we e ound
in he egions su ounding he qn de e minan s in some
Shewanella species, and one pu a i e ecombinase was
ound as well in Mo i ella sp. Whe he hose elemen s a e
emnan o o me gene ans e e en s o cons i u e a isk
o he ans e ence o hose qn genes h ough HGT o a
new hos , emains o be es ablished.
In any case, he analyses o a ailable sequences o bac e-
ial genomes sugges ha qn genes a e ancien elemen s
in he bac e ial ch omosomes o a speci ic subse o bac e-
ial species. No ewo hy, hose genes a e equen ly
lanked by genes coding o pu a i e e lux pumps (Figu e
2), a si ua ion ha sugges s ha Qn may ha e unc ional
oles in de oxi ica ion p ocesses in wa e -dwelling bac e-
ia. Al hough his linkage has no been so a obse ed o
plasmid-encoded qn genes, he coope a ion o wo di e -
en mechanisms o esis ance migh be an impo an ele-
men in cu en esis ance o quinolones.
A pu a i e qn gene is p esen in he ch omosome o S.
mal ophilia
Once he bioin o ma ic analysis was made, we ocused
ou e o s in he s udy o he o ganism wi h highes clin-
ical ele ance. Among he o ganisms ca ying pu a i e
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Homology o p edic ed new ch omosomally-encoded Qn p o einsFigu e 1
Homology o p edic ed new ch omosomally-encoded Qn p o eins. The Figu e shows he alignmen o ch omosoma-
lly-encoded pu a i e Qn p o eins om he analysis o a ound 1000 genomes and me agenomes. The igu e includes one mem-
be o each plasmidic Qn p o ein amily (highligh ed in ed), so ha he simila i ies a e clea e . The pu a i e Qn p o ein om
he sea me agenome is also included (M gQn ). As shown in he Figu e, conse a ion is high along he p o eins, al hough he
simila i ies a e lowe a hei N- e minus. The au oma ed anno a ion o Swoqn p esen s wo s op codons. Since we igno e
whe he his is a sequence mis ake o Swoqn is a pseudogene, a manual ansla ion o i s nucleo ide sequence was pe o med
o his alignmen . S op codons a e ep esen ed wi h an X. Accession numbe s o he p o eins used o he alignmen a e:
AhQn (YP_854820.1), AsQn (YP_001143795.1), MsQn (ZP_01897167.1), Qn A1 (ACA43024), Qn B1 (ABG82188),
Qn S1 (ABU86826), SbeQn (ZP_02157732.1), S Qn (YP_750786.1), SliQn (ABO22341.1), SmQn (ZP_01643096.1),
SpaQn (ABV89003.1), Sp Qn (YP_001478290.1), SseQn (ABV35251.1), SwoQn (ZP_01539189.1), PpQn (CAG21998.1),
PsQn (EAS39797.1), ValQn (ZP_01261394.1), VanQn (ZP_01234687.1), V Qn (ZP_02136549.1), VhQn (ZP_01985396.1),
VpQn (ZP_01990287.1), VshQn (ZP_01866232.1), VspQn (ZP_00989608.1), V Qn (AAO07889.1) and M gQn
(AACY020347520).

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P edic ed qn genes in genome da abasesFigu e 2
P edic ed qn genes in genome da abases. The s uc u e o he egions su ounding p edic ed qn genes (in ed) is
shown. Elemen s ha migh con ibu e o ans e o hose genes a e highligh ed in black. The p esence o pu a i e MDR sys-
ems is also highligh ed. WGS (whole genome sho gun), CG (Comple e genome), Ch (Ch omosome). Only hose pu a i e qn
de e minan s showing mo e han 45% iden i y wi h known plasmid-encoded Qn A o Qn B de e minan s we e included in he
analysis. The abb e ia ions used in he Figu e a e shown in Addi ional ile 1.
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uncha ac e ized qn genes we chose S. mal ophilia, which
is an in insically esis an oppo unis ic pa hogen
esponsible o 4.3% o G am-nega i e in ec ions among
in ensi e ca e uni s pa ien s in he USA [48].
No ewo hy, in S. mal ophilia esis ance o luo oquinolo-
nes (pa icula ly o less ac i e ones, such as no loxacin) is
no always accompanied by high le el esis ance o nalid-
ixic acid, which is he usual and classical associa ion in
En e obac e ia. In e es ingly, esis ance o luo oquinolo-
nes and suscep ibili y o nalidixic acid in S. mal ophilia
esemble he pheno ype ecen ly obse ed by Cano ME e
al (unpublished esul s) in some En e obac e iaceae con-
aining qn genes. A pu a i e qn gene (Smqn ) was iden i-
ied in he wo a ailable ull-genome sequences o S.
mal ophilia: he clinical isola e K279a h p://
www.sange .ac.uk/P ojec s/S_mal ophilia/[49], and he
en i onmen al s ain R551-3, h p://genome.jgi-ps .o g/
d a _mic obes/s ema/s ema.home.h ml. As shown in
Figu e 2, he genomic s uc u e a ound Smqn was he
same in bo h s ains, in spi e hei di e en (clinical and
en i onmen al) sou ce o isola ion.
This high deg ee o sin eny, oge he wi h he lack o ele-
men s in ol ed in HGT nea Smqn sugges s ha his gene
has an ancien o igin in S. mal ophilia. Fu he con i ma-
ion o his likely monophyle ic o igin has been ob ained
om he analyses o he Smqn sequence om di e en S.
mal ophilia isola es in his s udy. The p edic ed ansla ion
o Smqn sequence is a 219 amino acid p o ein (Figu e 5),
con aining andem epe i ions o he pa e n (A/C/S/T/
V)(D/N)(L/F)(S/T/R)(G/R) ypical o he pen apep ide
amily [20,18,22], wi h wo domains o pen apep ide
epea s sepa a ed by a single glycine, ha co esponds o
a COG1357 mo i .
P e alence o he Smqn gene in S eno ophomonas
mal ophilia s ains
Since Smqn migh be in ol ed in quinolone esis ance,
we wan ed o asce ain he p e alence o his gene in S.
mal ophilia popula ions. To ha goal, and since he
sequences o he qn genes o he s ains K279a and R551-
3 we e sligh ly di e en , wo pai s o oligonucleo ides
Qn M+/- and Qn MR55+/- we e designed o ampli y
Smqn genes om di e en S. mal ophilia s ains. The se
o analysed s ains comp ised bo h clinical and en i on-
men al S. mal ophilia isola es (Table 1).
The SmQn sequences om he di e en s ains display a
high amino acid iden i y, anking om 94 o 100% (Fig-
u e 5). Howe e he amino acid iden i y o he SmQn
p o ein encoded in he ch omosome o he sequenced S.
mal ophilia s ain K279a, is low compa ed wi h o he
known Qn p o eins, sha ing 38%, 59%, 38% and 41%
wi h Qn A1 (AY070235), Qn B2 (DQ351241), Qn S2
(AB187515) and V Qn (DQ889870) espec i ely. Fo
his eason we conside ha Smqn ep esen s a possible
new sub ype o qn genes. Clus al analysis (Figu e 4) indi-
ca es ha he SmQn p o ein clus e s wi h plasmidic Qn B
p o eins, al hough o ms a sepa a e b anch. The e o e, ou
esul s sugges ha SmQn is he i s cha ac e ized ch o-
mosomally encoded Qn B-like p o ein.
We we e unable o ampli y he ull qn gene om he iso-
la es E729, F227, E301, F861 and e-p5. Since qn
sequences whe e sligh ly di e en in K279a and R551-3,
as well as in he isola es analysed in he p esen s udy, wo
new pai s o oligonucleo ides (qn I1/qn I2 and qn I3/
qn I4) we e used o ampli y an in e nal conse ed egion
o Smqn . Posi i e ampli ica ion was ob ained in all cases
(no shown) indica ing ha all S. mal ophilia isola es con-
ained he qn gene.
Alignmen o he plasmid-encoded p o ein Qn B wi h a pu a i e Qn p o ein encoded in a ma ine me agenomeFigu e 3
Alignmen o he plasmid-encoded p o ein Qn B wi h a pu a i e Qn p o ein encoded in a ma ine me agen-
ome. The pe cen age o iden i y be ween bo h p o eins was 94.9%, wi h a homology o 99.5%. Top Qn B5, bo om Qn p o-
ein om a ma ine me agenome (M gQn ). Two do s indica e iden ical amino acids, one do homologous amino acids.
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Func ional analysis o SmQn as a quinolone esis ance
de e minan in a he e ologous hos
To asce ain whe he SmQn migh con ibu e o qui-
nolone esis ance in a he e ologous hos , he di e en
alleles o he qn gene ob ained om di e en S. mal-
ophilia s ains we e cloned in o he plasmid pGEM-T.
Co esponding ecombinan plasmids, con aining he
Smqn gene, we e exp essed in E. coli, and he quinolone
suscep ibili y o s ains exp essing SmQn was compa ed
o ha o he isogenic E. coli s ain. We ound ha in ans
exp ession o SmQn om di e en ecombinan plasmid
esul s in a 2 o 32- ols dec ease in he quinolone suscep-
ibili y o E. coli (Table 2).
No ewo hy, di e en ecombinan s ains p esen ed
sligh , bu consis en , di e ences in hei suscep ibili y o
se e al quinolones (Table 2). I has been p e iously s a ed
ha ansconjugan s o plasmids con aining he qn gene
p esen dispa a e le els o quinolone esis ance, likely due
o di e en le els o exp ession o he Qn p o ein in he
Clus al analysis o Qn p o einsFigu e 4
Clus al analysis o Qn p o eins. Cha ac e ized and p edic ed Qn p o eins we e clus e ed. The ee was calcula ed using
he a e age pe cen age o iden i y. O he wo membe s o he pen apep ide epea amily wi h p o en esis ance o luo oqui-
nolones, M pA om Mycobac e ium ube culosis and McbG om Esche ichia coli, we e included in he analysis, bu a e no shown
because hey plo ed ou o he Qn ee. Ch omosomally-encoded Qn p o eins a e highligh ed in bold. The posi ion o he
SmQn p o ein om S. mal ophilia as a membe o he Qn B clus e is highligh ed wi h a squa e. No el Qn p o eins a e
named acco ding o he species whe e hey a e o igina ed: AhQn (Ae omonas hyd ophila), AsQn (Ae omonas salmonicida),
MsQn (Mo i ella sp), Sp Qn (Se a ia p o eamaculans), SbeQn (Shewanella ben hica), S Qn (Shewanella igidima ina), SliQn
(Shewanella loihica), SpaQn (Shewanella pealeana), SseQn (Shewanella sediminis), SwoQn (Shewanella woodyi), SmQn (S eno o-
phomonas mal ophilia), PsQn (Psych omonas), ValQn (Vib io alginoly icus), VanQn (Vib io angus um), V Qn (Vib io ische i),
VhQn (Vib io ha eyi), VshQn (Vib io shilonii), VspQn (Vib io splendidus), V Qn (Vib io ulni icus).
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Alignmen o deduced amino acid sequences o di e en SmQn p o einsFigu e 5
Alignmen o deduced amino acid sequences o di e en SmQn p o eins. The amino acids ha changed in he di -
e en SmQn alleles a e highligh ed in a g ey squa e. The glycine esidue (G) sepa a ing he wo domains o pen ape ide
epea s, is ma ked wi h a box.
di e en ansconjugan s [50]. To asce ain whe he his
could be he si ua ion wi h he di e en SmQn -exp ess-
ing plasmids, he exp ession o he SmQn p o ein was
es ima ed in he di e en E. coli ans o man s. As shown
in Figu e 6A, ans o man s con aining ei he pBS3.25 o
pBS3.13 exp essed highe le els o SmQn han he o he
ans o man s.
Du ing ou wo k, we de ec ed ha some Qn -exp essing
plasmids we e los a e subcul u ing e en in he p esence
o ampicillin, al hough hey could be main ained in he
p esence o ca benicillin. Plasmid loss a e he bac e ia
has deg aded he selec i e an ibio ic is an indica ion o
physiological bu den [51]. Ou esul s sugges ha high-
le el exp ession o SmQn is likely ha m ul o E. coli. I is
possible ha , as desc ibed o o he sys ems [52], a as
adap a ion be ween he hos cell and he plasmid
occu ed ha esul s in educed le els o SmQn and ha
his allowed some o he clones o o e come he pu a i e
SmQn oxici y. To add ess his possibili y, E. coli
KZM120 was e ans o med wi h he plasmids pBS3.8,
pBS3.25 and pBS3.13. Two di e en clones om each
e ans o ma ion we e chosen and hei suscep ibili y o
quinolones es ed. As shown in Table 3, an o e all educ-
ion in MICs as well as in he le el o SmQn exp ession
(Figu e 6B) was obse ed o he new ans o man s con-
aining he plasmids pBS3.8, pBS3.25 and pBS3.13. How-
e e , he pheno ype o educed suscep ibili y o
quinolones was main ained. Inc eased esis ance due o
highe exp ession o an ibio ic esis ance genes has been
desc ibed mainly associa ed wi h plasmidic be a-lac ama-
ses [53-55]. Ou esul s oge he wi h al eady published
da a indica e ha gene-dosage migh be ele an o qn -
media ed quinolone esis ance.
Conclusion
By using a combina ion o bioin o ma ics and unc ional
ools, we ound a numbe o no el pu a i e qn genes in
he ch omosomes o aqua ic bac e ia and in me agen-
omes om ma ine o ganisms. This u he suppo s he
no ion ha he o igins o qn de e minan s a e in wa e -
dwelling bac e ia. I has been ecen ly shown ha Ae om-
onas spp. ob ained om wa e samples con ain plasmid-
encoded qn genes [16]. These esul s ha e been in e -
p e ed as a consequence o he p esence o quinolones in
i e s ha d i e he e olu ion o aqua ic bac e ia owa ds
quinolone esis ance. This explana ion is likely ue o
plasmid-encoded qn genes. Howe e , he p esence o qn -