Academic Edi o : Chia a Scapoli
Recei ed: 8 Janua y 2025
Re ised: 4 Feb ua y 2025
Accep ed: 6 Feb ua y 2025
Published: 11 Feb ua y 2025
Ci a ion: He e a-Espejo, S.;
Rubio, A.; Ceballos-Rome o, L.;
Pachón, J.; Co de o, E.;
Pé ez-Pulido, A.J.;
Pachón-Ibáñez, M.E. De ec ion o
Possible Resis ance Mechanisms in
U opa hogenic Esche ichia coli S ains
Isola ed om Kidney T ansplan
Recipien s Based on Whole Genome
Sequencing. Biomolecules 2025,15, 260.
h ps://doi.o g/10.3390/
biom15020260
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Licensee MDPI, Basel, Swi ze land.
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(h ps://c ea i ecommons.o g/
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A icle
De ec ion o Possible Resis ance Mechanisms in U opa hogenic
Esche ichia coli S ains Isola ed om Kidney T ansplan
Recipien s Based on Whole Genome Sequencing
So aya He e a-Espejo 1,† , Alejand o Rubio 2,† , Lucía Ceballos-Rome o 1, Je ónimo Pachón 3,4 ,
Elisa Co de o 1,4,5,* , An onio J. Pé ez-Pulido 2,* and Ma ía Eugenia Pachón-Ibáñez 1,5
1Clinical Uni o In ec ious Diseases, Mic obiology and Pa asi ology, Ins i u e o Biomedicine o Se ille (IBiS),
Vi gen del Rocio Uni e si y Hospi al/CSIC/Uni e si y o Se ille, 41013 Se ille, Spain;
[email p o ec ed] (S.H.-E.); [email p o ec ed] (L.C.-R.); [email p o ec ed] (M.E.P.-I.)
2Andalusian Cen e o De elopmen al Biology (CABD, UPO-CSIC-JA),
Facul y o Expe imen al Sciences (Gene ics A ea), Uni e si y Pablo de Ola ide, 41013 Se ille, Spain;
[email p o ec ed]
3Ins i u e o Biomedicine o Se ille (IBiS), Vi gen del Rocio Uni e si y Hospi al/CSIC/Uni e si y o Se ille,
41013 Se ille, Spain; [email p o ec ed]
4Depa men o Medicine, School o Medicine, Uni e si y o Se ille, 41004 Se ille, Spain
5CIBER de En e medades In ecciosas (CIBERINFEC), Ins i u o de Salud Ca los III, 28029 Mad id, Spain
*Co espondence: mco de [email p o ec ed] (E.C.); ajpe [email p o ec ed] (A.J.P.-P.)
†These au ho s con ibu ed equally o his wo k.
Abs ac : Backg ound: U ina y ac in ec ions a e a global heal h conce n, wi h u opa hogenic
Esche ichia coli (UPEC) accoun ing o 80–90% o cases. Gi en he ise in an imic obial e-
sis ance, ou aim was o elucida e he gene ic mechanisms behind low-le el esis ance o
cip o loxacin and os omycin (LLCR and LLFR) in UPEC s ains, using whole-genome se-
quencing (WGS) o iden i y poin mu a ions in ch omosomal and plasmid genes. Me hods: A
coho UPEC was collec ed om kidney ansplan ecipien s a he Vi gen del Rocío Uni e -
si y Hospi al, Spain. Minimum inhibi o y concen a ions we e de e mined o cip o loxacin
and os omycin o ca ego ize s ains in o LLCR and LLFR. Twen y s ains we e selec ed
o WGS, wi h genome anno a ions. Poin mu a ions we e iden i ied and analyzed using
alignmen ools, and p o ein s abili y changes we e p edic ed. Resul s: LLCR s ains exhibi ed
mu a ions in key quinolone esis ance-de e mining egions o he gy A gene, in 83% o cases.
The qn S1 plasmid gene was ound in 17% o LLCR s ains. LLFR s ains showed mu a ions
in he glpT and cyaA genes. Mu a ions in he uhp gene amily we e linked o he os omycin-
esis an pheno ype, sugges ing a mul i-s ep esis ance e olu ion mechanism. Conclusions:
This s udy highligh s he complex in e play be ween ch omosomal and plasmid genes in
UPEC’s esis ance o cip o loxacin and os omycin. The indings con ibu e o unde s and-
ing low-le el esis ance mechanisms and may guide he de elopmen o no el he apeu ic
s a egies o comba mul id ug- esis an s ains.
Keywo ds: whole-genome sequencing; an imic obial esis ance; u opa hogenic
Esche ichia coli
1. In oduc ion
U ina y ac in ec ions (UTIs) a e a common heal h p oblem ha a ec s a signi i-
can po ion o he global popula ion. E e y yea , a ound 150 million people wo ldwide
de elop UTIs, wi h subs an ial social and economic cos s [
1
,
2
]. UTIs a e mo e common
in women, wi h an es ima ed 40% de eloping a leas one UTI du ing hei li e ime, and
Biomolecules 2025,15, 260 h ps://doi.o g/10.3390/biom15020260
Biomolecules 2025,15, 260 2 o 15
11% expe iencing an episode o UTI each yea [
2
]. The mean cos o each UTI is es ima ed
a EUR 5700 and EUR 6987 in Eu ope and USA, espec i ely [
3
,
4
]. UTIs a e caused by
he p esence o bac e ia (
≥
10
5
CFU/mL) in he u ine wi h o wi hou symp oms associ-
a ed (asymp oma ic bac e iu ia). Symp oma ic UTIs a e classi ied acco ding o se e i y as
u osepsis synd ome, pyeloneph i is o uppe UTI (kidney in ec ion), and cys i is o lowe
UTI (bladde in ec ion) [
5
]. Mo eo e , UTI is a common and se ious p oblem among kidney
ansplan ecipien s (KTR), anging om 40% o 50% [
6
,
7
]. This p e alence depends on
ac o s such as p e en i e s a egies and ansplan cha ac e is ics. Se e al ac o s can
inc ease he suscep ibili y o de elop a UTI [
8
]. Among he isks ha UTI migh cause in
KTR include immunosupp ession, bladde ca he e iza ion, u e e al s en s, deceased dono
ansplan a ion, and acu e ejec ion episodes [9].
U opa hogenic Esche ichia coli (UPEC) is he p ima y cause o UTIs acqui ed in he
communi y, accoun ing o 80–90% o cases [
1
,
2
]. UPEC can be classi ied in o di e en
g oups based on he p esence o genomic pa hogenici y islands (PAIs) and i ulence
ac o s [
10
]. UPEC uses a wide a ie y o i ulence ac o s o colonize he bladde , including
lipopolysaccha ide (LPS), polysaccha ide capsule, lagella, pili, and TonB-dependen i on-
up ake ecep o s [
8
], all o which a e po en ial a ge s o he de elopmen o new d ugs
and/o accines.
A p esen , despi e he inc ease in an imic obial esis ance, he e a e nume ous op ions
o he ea men o UTIs. The Eu opean An imic obial Resis ance Repo 2021 (EARS-Ne )
in o med ha app oxima ely 50% o E. coli s ains we e esis an o a leas one g oup o
an imic obials. Cip o loxacin esis ance a es ha e inc eased up o 50% in he global sce-
na io [
11
]. Speci ically, in Spain, esis ance a es o up o 30% o cip o loxacin and 17% o
aminoglycosides ha e been epo ed [
12
]. One o he p ima y mechanisms o an ibio ic
esis ance in bac e ia is he acquisi ion o gene ic mu a ions ha con e esis ance o one o
mo e an ibio ics. Poin mu a ions, which in ol e changes in a single nucleo ide wi hin he
genome, a e a common ype o gene ic mu a ion ha can lead o an ibio ic esis ance [
13
].
Mo eo e , poin mu a ions can be classi ied as synonymous (mu a ions o al e a ions ha
do no change he amino acid sequence o he p o ein) o non-synonymous (mu a ions ha
change he amino acid sequence o he p o ein, esul ing in subs i u ions, dele ions
. . .
), and
occu spon aneously o can be induced by exposu e o he an ibio ics. These mu a ions
can lead o changes in he a ge si es o e lux pumps o an ibio ics, o al e a ions in he
bac e ial cell wall o memb ane ha educe an ibio ic up ake [
14
]. The s udy o an ibio ic
esis ance in bac e ia has adi ionally elied on pheno ypic me hods, such as es ing o
de e mine he suscep ibili y/ esis ance p o ile o a gi en s ain. Howe e , wi h he a i al
o nex -gene a ion sequencing echnologies and bioin o ma ics ools, i is now possible
o s udy he gene ic basis o an ibio ic esis ance in mo e de ail [
15
]. Cu en ly, he mos
commonly used an ibio ics a e os omycin, cip o loxacin, and amoxicillin-cla ulana e o
UTIs caused by UPEC [
16
]. Ch omosomal and plasmid-media ed mechanisms on hei
own migh con e low-le el cip o loxacin o os omycin esis ance (LLCR o LLFR), which
is clinically ansla ed as a suscep ible pheno ype. Howe e , he accumula ion o mul iple
mu a ions could lead o clinical esis ance [17]. These cumula i e an imic obial esis ance
genes could be acqui ed in na u e om o he bac e ia [18] o e en phages [19].
Thus, he aim o his s udy was o analyze ch omosomal and plasmid genes in ol ed
in low-le el esis ance o cip o loxacin and/o os omycin in UPEC s ains collec ed om a
well-cha ac e ized coho o KTR, o iden i y poin mu a ions ha migh be esponsible
o hese. By cha ac e izing hese low-le el gene ic mechanisms o esis ance, we seek
o be e unde s and he mechanisms unde lying esis ance o he men ioned an ibio ics.
Ul ima ely, he esul s may con ibu e o he de elopmen o new s a egies o comba
Biomolecules 2025,15, 260 3 o 15
in ec ions caused by hese s ains, o example, by iden i ying new he apeu ic a ge s o
by op imizing ea men .
2. Ma e ials and Me hods
2.1. An imic obial Suscep ibili y, Whole-Genome Sequencing and Mul ilocus Sequence Typing
(MLST) o Esche ichia coli S ains
One hund ed and i een s ains we e collec ed in an obse a ional coho o adul
KTRs wi h E. coli and UTI episodes (cys i is and asymp oma ic bac e iu ia [AB]), who
signed he in o med consen o m (e hical app o al numbe s: FIS-CIP-2016-01 and FIS-
FOS-2020-01) and a ended as ou pa ien s a he Vi gen del Rocío Uni e si y Hospi al,
Se ille, Spain, om Janua y 2017 o Decembe 2019 [
20
]. Clinical s ains we e collec ed,
and he hospi al mic obiology se ice iden i ied he bac e ial s ains using a Mic oScan
WalkAway
®
Plus sys em (Beckman Coul e , Nyon, Swi ze land), and pe o med suscep ibil-
i y es ing wi h s anda d es s. Mo eo e , cip o loxacin and os omycin Minimal Inhibi o y
Concen a ions (MICs) we e de e mined in duplica e, by b o h mic odilu ion o aga di u-
sion, espec i ely [
20
]. The esul s we e in e p e ed acco ding o he Eu opean Commi ee
on An imic obial Suscep ibili y Tes ing (EUCAST) b eakpoin s o bo h an ibio ics [
21
].
An imic obials o pe o m he MIC we e pu chased as s anda d powde s (Sigma-Ald ich,
Mad id, Spain) and E. coli ATCC 25922 was used as a quali y con ol s ain. MIC b eak-
poin s used o designa e suscep ibili y and esis ance o cip o loxacin we e
≤
0.001
µ
g/mL
and >0.50
µ
g/mL and, in he case o os omycin, he b eakpoin used o designa e esis ance
was >8
µ
g/mL. MIC alues o >0.06 o 0.5 mg/L and 4 o 8 mg/L we e conside ed o be
classi ied as LLCR and LLFR, espec i ely.
A e MIC de e mina ions, 20 UPEC s ains we e selec ed o in silico s udies, 12 LLCR
and 8 LLFR. B ie ly, DNA om hese s ains was ex ac ed (QIAamp
®
DNA Mini Ki ,
Venlo, The Ne he lands). Sequencing was pe o med using he MiSeq pla o m (Illumina),
ollowing s anda d p o ocols o WGS pai ed-end, p oducing 2
×
300 bp agmen eads.
Unicycle was chosen o de no o assembly o he eads in o con igs [
22
] (BioP ojec
numbe : PRJNA1219036). P odigal 2.6.3 was used o p edic he p o ein-coding genes and
ansla e hem in o hei co esponding amino acid sequences [
23
]. The bac e ial axonomic
di ision o he UniP o 2023_05 da abase was used o he unc ional anno a ion o he
p edic ed p o eins, using Sma3s 2 [24].
The con ig iles ob ained om he assembly o each UPEC isola e we e uploaded o he
MLST web se e ( . 2.0) o he Cen e o Genomic Epidemiology (CGE) o pe o m MLST,
ollowing he schemes o Pas eu [
25
] and Ach man [
26
]. The se ings o he analysis o
hese h ee web-based p og ams we e es ablished using a h eshold o 90% iden i y and
80% o sequence co e age.
2.2. Quinolone and Fos omycin Resis an Genes Sequence Alignmen s and Analysis
Fo he s udy o cip o loxacin esis ance genes, a o al o ele en plasmid genes (aac6’-
1b-c ,qn A,qn B,qn S,qn C,qn D,qn E,qepA,oqxA,oqxB, and c pP) and eigh ch omosomal
genes (gy A,gy B,pa C,pa E,ma R,ac R,no B, and poB) associa ed wi h quinolone esis-
ance we e examined. In he case o os omycin, eigh plasmid genes ( osA3, osA4, osA5,
osB, osX, osC, omA, and omB) and nine ch omosomal genes (mu A,uhpA,uhpB,uhpC,
uhpT,glpT,p sl,cyaA, and c p) we e analyzed. To examine hese genes, he amino acid
sequences o he encoding p o eins p edic ed by P odigal we e aligned, using MAFFT
( . 7.526), o he e e ence amino acid sequences o he p o eins ob ained om The Comp e-
hensi e An ibio ic Resis ance (CARD, . 5.0.2) wi h de aul se ings [
27
]. The phea map
lib a y om he R language was used o c ea e a hea map wi h he esul s ob ained [28].
Biomolecules 2025,15, 260 4 o 15
Subsequen ly, he s uc u al s abili y o he mu a ed p o eins was in es iga ed using
he P o ein Va ia ion E ec Analyze (P o ean . 2.6.3) [
29
] and he CUPSAT [
30
] and
DDMu [
31
] algo i hms, which calcula e Gibbs ene gy o p edic changes in p o ein s abili y.
To s udy he e ec o mu a ions on he olding o he p o ein encoded by he uhpB gene, a
3D a ine c ys al s uc u e was gene a ed wi h he algo i hm AlphaFold [
32
] and isualized
using PyMol [
33
], ep esen ing he di e en mu a ions calcula ed in he p e ious s eps o
he s udy.
2.3. Iden i ica ion o Unknown Resis ance and Vi ulence Mechanisms
The da a assembled om he 20 s ains we e used o iden i y esis ance mechanisms.
Fo his, CGE’s ResFinde web se e ( . 4.1) [
34
,
35
], CGE’s Plasmid Finde web se e
( . 2.1) [
36
,
37
], and NCBI’s AMRFinde Plus ( . 3.11) [
38
] we e used o he acqui ed an imi-
c obial esis ance genes. The h esholds o he analysis in hese h ee web-based p og ams
we e es ablished as 90% iden i y and 80% sequence co e age. CGE’s Vi ulenceFinde
( . 2.0.5) [
35
,
39
,
40
] web se e was used o he iden i ica ion o genes encoding i ulence
ac o s, wi h h esholds o 90% iden i y and 80% sequence co e age.
3. Resul s
3.1. An imic obial Suscep ibili y and Mul ilocus Sequence Typing (MLST) o Esche ichia coli
S ains Selec ed o Sequencing
MICs we e de e mined o he 115 UPEC s ains om he pa ien s included in he
coho . O hese, 20 s ains p esen ed low-le el esis ance: 12 we e ca ego ized as LLCR
and 8 as LLFR (Tables 1and 2). Demog aphics and cha ac e is ics o KTRs wi h UTI
by LLCR o LLFR E. coli s ains a e desc ibed in Supplemen a y Table S1. LLCR s ains
exhibi ed a ange o MIC alues om 0.12 o 0.50
µ
g/mL and LLFR om 4 o 8
µ
g/mL. Two
cip o loxacin-suscep ible s ains (s ains 5 and 44, MIC
≤
0.015 mg/L) and wo os omycin-
suscep ible s ains (s ains 145 and 139, MIC = 1 and 0.5 mg/L, espec i ely) we e included
as suscep ibili y con ols.
Table 1. Mul ilocus sequence ype and minimum inhibi o y concen a ion o Esche ichia coli s ains
wi h low esis ance o cip o loxacin.
S ains
MLST MIC (µg/mL)
Pas eu Ach man
5 * ST509 ST625 <0.015 (S)
44 * Unknown (nea es 74) Unknown (nea es 12,985) <0.015 (S)
57 ST831 ST1136 0.12
149 ST2 ST548 0.06
14 ST14 ST68 0.25
64 ST3 ST5021 0.12
17
Unknown (nea es 132)
ST48 0.12
126 ST14 ST68 0.50
18 Unknown (nea es 35) Unknown (nea es 1439) 0.12
164 ST634 ST429 0.25
140 Unknown (nea es 24) Unknown (nea es 889) 0.50
167 ST634 ST429 0.25
26 Unknown (nea es 14) ST68 0.25
168 ST35 ST12 0.12
*: Clinical s ains 5 and 44 we e added as con ol suscep ible (S) s ains.
Biomolecules 2025,15, 260 5 o 15
Table 2. Mul i-locus sequence ype and minimum inhibi o y concen a ion o Esche ichia coli s ains
wi h low esis ance o os omycin.
S ains
MLST MIC (µg/mL)
Pas eu Ach man
145 * ST7 ST23 1 (S)
139 * ST3 ST69 0.50 (S)
44 Unknown (nea es 74) Unknown (nea es 12,985) 2
149 ST2 ST548 8
112
Unknown (nea es 966)
Unknown (nea es 4195) 2
8 ST83 ST93 2
156 ST471 ST410 2
160 ST2 ST10 2
90 ST32 ST127 2
142 Unknown (nea es 14) ST68 2
*: Clinical s ains 145 and 139 we e added as con ol suscep ible (S) s ains.
Including he suscep ibili y con ols, Ach man’s MLST scheme appea ed o be mo e
accu a e assigning ST o 11/14 (78.6%) and 8/10 (80.0%) o he s udied s ains, o
cip o loxacin and os omycin, espec i ely. Independen ly o he scheme used, he s ains
p esen ed a a ied clonali y wi hou any single clone s anding ou (Tables 1and 2). Pas eu
MLST scheme assigned a ST o 9 ou o 14 (64.3%), and 7 ou o 10 (70.0%) o he s ains o
cip o loxacin and os omycin, espec i ely.
3.2. Iden i ica ion o Poin Mu a ions in Ch omosomal o Plasmid Genes In ol ed in Cip o loxacin
o Fos omycin Resis ance
Addi ional analyses we e pe o med on poin mu a ions in he ch omosomal and
plasmid sequences o compa e low-le el esis ance e sus suscep ible s ains. Fo LLCR
s ains, 11 plasmid (aac6
′
-1b-c ,qn A,qn B,qn S,qn C,qn D,qn E,qepA,oqxA,oqxB, and
c pP) and 8 ch omosomal (gy A,gy B,pa C,pa E,ma R,ac R,sosX, and poB) genes associ-
a ed wi h quinolone esis ance we e analyzed. We ound ha mos o he poin mu a ions
we de ec ed had been p e iously desc ibed in he Comp ehensi e An ibio ic Resis ance
Da abase (CARD) as con e ing quinolone esis ance (Supplemen a y Table S2).
Rega ding ch omosomal genes, 83% (10/12) o he s ains p esen ed a poin mu a ion
in he quinolone- esis ance-de e mining egion o DNA gy ase (gy A) (Figu e 1A), a a ge
enzyme o quinolones [
41
]. Speci ically, Se 83Leu and Asp87Leu poin mu a ions we e
obse ed (Figu e 2and Supplemen a y Figu e S1). Fi y pe cen (6/12) o he s ains
ca ied a non-p e iously desc ibed subs i u ion mu a ion in pa C and pa E genes, encoding
opoisome ase implied in bac e ial eplica ion [
42
], 33% also had a subs i u ion in gy B
and ac R. While isola e 26 had a dele ion in he gy B gene, isola e 140 had a subs i u ion
plus dele ion in he gy B and pa C genes. Mo e han 91% (11/12) o he s ains p esen ed a
poin mu a ion in he ma R gene, a ep esso o he ma RAB ope on which is in ol ed in
he ac i a ion o bo h an ibio ic esis ance and oxida i e s ess genes [
43
]. In e es ingly, all
poin mu a ions ound in he al eady desc ibed genes in ol ed in cip o loxacin esis ance
we e only p esen in LLCR s ains and no poin mu a ions we e ound in con ol s ains.
In addi ion, in plasmid mu a ions analysis, wo s ains (17%) ca ied a subs i u ion in he
qn S1 gene (Supplemen a y Table S2).
Biomolecules 2025,15, 260 6 o 15
Biomolecules 2025, 15, x FOR PEER REVIEW 6 o 15
While none o he LLFR s ains ca ied poin mu a ions in he plasmidic genes es ed,
75% (6/8) o he LLFR s ains ca ied a poin mu a ion in cyaA, glpT, and p sI genes. Con-
e sely o ha obse ed in LLCR s ains, hese genes we e mu a ed in one o he con ol
suscep ible s ains es ed (Figu e 1B). Howe e , he s ains used as con ols, since hey
p esen a suscep ible pheno ype in he an ibio ics analyzed, a e clinical s ains isola ed
om UTI in KTR pa ien s, and i is expec ed o ind poin mu a ions in hem. In addi ion,
50% (4/8) o he s ains ca ied subs i u ions in he uhp genes amily, speci ically 25% (2/8)
o he LLFR s ains ca ied a subs i u ion affec ing he coding egion o hexose-phospha e
anspo e (UhpT) gene in ol ed in he os omycin anspo a ion in o he bac e ia [44].
Also, subs i u ions in he uhp genes amily we e obse ed in con ol suscep ible s ains,
and only 3/9 (33%) ch omosomic genes analyzed showed no mu a ions in con ol s ains.
Speci ically, o he uhpB gene, some o he amino acidic subs i u ions ca ied by LLFR
s ains we e ound among posi ions om 311 o 499, he his idine kinase domain (Figu e
3).
Finally, isola e 160 ca ied a poin mu a ion in he mu A and c p genes. Addi ionally,
no mu a ions we e obse ed in he analysis o plasmid mu a ions (Figu e 1B and Supple-
men a y Table S3).
Figu e 1. Hea map o he accumula ion o poin mu a ions in he low-le el cip o loxacin esis ance
(LLCR) and low-le el os omycin esis ance (LLFR) s ains. Abundance o poin mu a ion in genes
(axis Y) in ol ed in (A) low-le el quinolone esis ance (LLCR, axis X) s ains and (B) low-le el
os omycin esis ance (LLFR, axis X) s ains. Blue: Suscep ible s ains; Salmon: LLCR s ains; G ay:
LLFR s ains; Whi e: LLCR o LLFR s ains wi h any poin mu a ion in he s udied genes in ol ed
in esis ance. Range o colo s om b own o blue: LLCR o LLFR s ains wi h an accumula ion om
1 o 8 poin mu a ions in he s udied genes in ol ed in esis ance.
Figu e 1. Hea map o he accumula ion o poin mu a ions in he low-le el cip o loxacin esis ance
(LLCR) and low-le el os omycin esis ance (LLFR) s ains. Abundance o poin mu a ion in genes
(axis Y) in ol ed in (A) low-le el quinolone esis ance (LLCR, axis X) s ains and (B) low-le el
os omycin esis ance (LLFR, axis X) s ains. Blue: Suscep ible s ains; Salmon: LLCR s ains; G ay:
LLFR s ains; Whi e: LLCR o LLFR s ains wi h any poin mu a ion in he s udied genes in ol ed in
esis ance. Range o colo s om b own o blue: LLCR o LLFR s ains wi h an accumula ion om
1 o 8 poin mu a ions in he s udied genes in ol ed in esis ance.
Biomolecules 2025, 15, x FOR PEER REVIEW 6 o 15
While none o he LLFR s ains ca ied poin mu a ions in he plasmidic genes es ed,
75% (6/8) o he LLFR s ains ca ied a poin mu a ion in cyaA, glpT, and p sI genes. Con-
e sely o ha obse ed in LLCR s ains, hese genes we e mu a ed in one o he con ol
suscep ible s ains es ed (Figu e 1B). Howe e , he s ains used as con ols, since hey
p esen a suscep ible pheno ype in he an ibio ics analyzed, a e clinical s ains isola ed
om UTI in KTR pa ien s, and i is expec ed o ind poin mu a ions in hem. In addi ion,
50% (4/8) o he s ains ca ied subs i u ions in he uhp genes amily, speci ically 25% (2/8)
o he LLFR s ains ca ied a subs i u ion affec ing he coding egion o hexose-phospha e
anspo e (UhpT) gene in ol ed in he os omycin anspo a ion in o he bac e ia [44].
Also, subs i u ions in he uhp genes amily we e obse ed in con ol suscep ible s ains,
and only 3/9 (33%) ch omosomic genes analyzed showed no mu a ions in con ol s ains.
Speci ically, o he uhpB gene, some o he amino acidic subs i u ions ca ied by LLFR
s ains we e ound among posi ions om 311 o 499, he his idine kinase domain (Figu e
3).
Finally, isola e 160 ca ied a poin mu a ion in he mu A and c p genes. Addi ionally,
no mu a ions we e obse ed in he analysis o plasmid mu a ions (Figu e 1B and Supple-
men a y Table S3).
Figu e 1. Hea map o he accumula ion o poin mu a ions in he low-le el cip o loxacin esis ance
(LLCR) and low-le el os omycin esis ance (LLFR) s ains. Abundance o poin mu a ion in genes
(axis Y) in ol ed in (A) low-le el quinolone esis ance (LLCR, axis X) s ains and (B) low-le el
os omycin esis ance (LLFR, axis X) s ains. Blue: Suscep ible s ains; Salmon: LLCR s ains; G ay:
LLFR s ains; Whi e: LLCR o LLFR s ains wi h any poin mu a ion in he s udied genes in ol ed
in esis ance. Range o colo s om b own o blue: LLCR o LLFR s ains wi h an accumula ion om
1 o 8 poin mu a ions in he s udied genes in ol ed in esis ance.
Figu e 2. Logo o he amino acid sequence encoded by he gy A gene in low-le el cip o loxacin
esis ance (LLCR) s ains. Sequence conse a ion o amino acids encoding Gy A c ea ed om he
aligned sequences o he 12 LLCR E. coli s ains. Comple e logo sequence is shown in Supplemen a y
Figu e S1. Le e s depic he consensus amino acid o each posi ion; Blue: Amino acids wi h side
chain cha ge posi i ely; Red: Amino acids wi h side chain cha ge nega i ely; G ay: No-pola amino
acids; G een: Pola amino acids wi hou changes; Blue s a depic di e ences; The ggseqlogo lib a y
was used o c ea e logo g aph om he mul iple sequence alignmen .
On he o he hand, in LLFR s ains, eigh plasmid ( osA3, osA4, osA5, osB, osX, osC,
omA, and omB) and nine ch omosomal (mu A,uhpA,uhpB,uhpC,uhpT,glpT,p sl,cyaA, and
c p) genes we e analyzed. Mos o he poin mu a ions ound we e p e iously desc ibed in
he cu a ed CARD da abase ha con e s esis ance o os omycin (Supplemen a y Table S3).
While none o he LLFR s ains ca ied poin mu a ions in he plasmidic genes es ed,
75% (6/8) o he LLFR s ains ca ied a poin mu a ion in cyaA,glpT, and p sI genes. Con-
e sely o ha obse ed in LLCR s ains, hese genes we e mu a ed in one o he con ol
suscep ible s ains es ed (Figu e 1B). Howe e , he s ains used as con ols, since hey
p esen a suscep ible pheno ype in he an ibio ics analyzed, a e clinical s ains isola ed
om UTI in KTR pa ien s, and i is expec ed o ind poin mu a ions in hem. In addi-
ion, 50% (4/8) o he s ains ca ied subs i u ions in he uhp genes amily, speci ically
Biomolecules 2025,15, 260 7 o 15
25% (2/8) o he LLFR s ains ca ied a subs i u ion a ec ing he coding egion o hexose-
phospha e anspo e (UhpT) gene in ol ed in he os omycin anspo a ion in o he
bac e ia [
44
]. Also, subs i u ions in he uhp genes amily we e obse ed in con ol sus-
cep ible s ains, and only 3/9 (33%) ch omosomic genes analyzed showed no mu a ions
in con ol s ains. Speci ically, o he uhpB gene, some o he amino acidic subs i u ions
ca ied by LLFR s ains we e ound among posi ions om 311 o 499, he his idine kinase
domain (Figu e 3).
Biomolecules 2025, 15, x FOR PEER REVIEW 7 o 15
Figu e 2. Logo o he amino acid sequence encoded by he gy A gene in low-le el cip o loxacin
esis ance (LLCR) s ains. Sequence conse a ion o amino acids encoding Gy A c ea ed om he
aligned sequences o he 12 LLCR E. coli s ains. Comple e logo sequence is shown in Supplemen a y
Figu e S1. Le e s depic he consensus amino acid o each posi ion; Blue: Amino acids wi h side
chain cha ge posi i ely; Red: Amino acids wi h side chain cha ge nega i ely; G ay: No-pola amino
acids; G een: Pola amino acids wi hou changes; Blue s a depic diffe ences; The ggseqlogo lib a y
was used o c ea e logo g aph om he mul iple sequence alignmen .
Figu e 3. P esence o poin mu a ion in UhpB p o ein. (A) Rende ed s uc u e and poin mu a ion
ou side/wi hin he unc ional domain and (B) abundance o poin mu a ion in uhpB gene in he low-
le el os omycin esis ance (LLFR) s ains. In (A), g een mu a ions: ca ied by LLFR s ains ou side
o he unc ional domain ( ed squa e (IPR005467—His idine kinase domain); blue mu a ions: ca -
ied by con ol s ains ou side o inside o he unc ional domain ( ed squa e); ed mu a ion: p o ein
due o his single amino acid a ia ion is no s able; pu ple mu a ion: ca ied by LLFR s ains ou side
o he unc ional domain ( ed squa e). In (B), o ange do s: p esence/absence o he mu a ion.
Analysis o plasmid esis ance genes ound ha all s ains, bo h LLCR and LLFR,
ca ied a leas one plasmid (Supplemen a y Tables S2 and S3).
3.3. Iden i ica ion o Unknown Resis ance Ch omosomal o Plasmid Genes and Vi ulence Fac o s
in LLCR and LLFR S ains
Bo h he LLCR and LLFR s ains showed a leas one gene in ol ed in An imic obial
Resis ance (AMR). Rega ding he analysis o he p esence o an imic obial esis ance
genes in he selec ed s ains exhibi ing low-le el esis ance, 83% (10/12) o he LLCR
s ains ca ied mu a ions in gy A, and 13% (1/8) o he LLFR s ains ca ied osA. This gene
encodes me alloenzymes which hyd olyze os omycin [41]. Fu he mo e, mos o hese
s ains ca ied a wide a ie y o genes associa ed wi h esis ance o aminoglycosides, β-
lac ams, sul onamides, and e acyclines (Tables 3 and 4). Howe e , no clea associa ion
was de ec ed among he p esence o hese AMR genes and he s ainsʹ MIC pheno ype.
Only wo LLCR s ains p esen ed he aminoglycoside modi ying enzymes (AMEs),
aac(3′)-IIa (encoding ob amycin and gen amycin esis ance) [45]. Simila ly, AME was
a ely p esen in LLFR s ains, wi h only one s ain p esen ing an aac(3′)-Ib in ol ed in
s ep omycin esis ance [46]. Wi h espec o β-lac am esis ance genes, bo h genes encod-
ing class A and D β-lac amases we e iden i ied in all LLCR s ains, especially, he bla
TEM-1
gene, esponsible o ampicillin, pipe acillin, amoxicillin, and ica cillin esis ance [47].
Mo eo e , 62% (5/8) o he LLFR s ains also ca ied he bla
TEM-1
gene. Howe e , 75% (9/12)
and 37% (3/8) o he LLCR and LLFR s ains, espec i ely, ca ied genes in ol ed in ola e
Figu e 3. P esence o poin mu a ion in UhpB p o ein. (A) Rende ed s uc u e and poin mu a ion
ou side/wi hin he unc ional domain and (B) abundance o poin mu a ion in uhpB gene in he low-
le el os omycin esis ance (LLFR) s ains. In (A), g een mu a ions: ca ied by LLFR s ains ou side o
he unc ional domain ( ed squa e (IPR005467—His idine kinase domain); blue mu a ions: ca ied by
con ol s ains ou side o inside o he unc ional domain ( ed squa e); ed mu a ion: p o ein due o
his single amino acid a ia ion is no s able; pu ple mu a ion: ca ied by LLFR s ains ou side o he
unc ional domain ( ed squa e). In (B), o ange do s: p esence/absence o he mu a ion.
Finally, isola e 160 ca ied a poin mu a ion in he mu A and c p genes. Addi ion-
ally, no mu a ions we e obse ed in he analysis o plasmid mu a ions (Figu e 1B and
Supplemen a y Table S3).
Analysis o plasmid esis ance genes ound ha all s ains, bo h LLCR and LLFR,
ca ied a leas one plasmid (Supplemen a y Tables S2 and S3).
3.3. Iden i ica ion o Unknown Resis ance Ch omosomal o Plasmid Genes and Vi ulence Fac o s in
LLCR and LLFR S ains
Bo h he LLCR and LLFR s ains showed a leas one gene in ol ed in An imic obial
Resis ance (AMR). Rega ding he analysis o he p esence o an imic obial esis ance genes
in he selec ed s ains exhibi ing low-le el esis ance, 83% (10/12) o he LLCR s ains
ca ied mu a ions in gy A, and 13% (1/8) o he LLFR s ains ca ied osA. This gene
encodes me alloenzymes which hyd olyze os omycin [
41
]. Fu he mo e, mos o hese
s ains ca ied a wide a ie y o genes associa ed wi h esis ance o aminoglycosides,
β-lac ams
, sul onamides, and e acyclines (Tables 3and 4). Howe e , no clea associa ion
was de ec ed among he p esence o hese AMR genes and he s ains’ MIC pheno ype. Only
wo LLCR s ains p esen ed he aminoglycoside modi ying enzymes (AMEs), aac(3
′
)-IIa
(encoding ob amycin and gen amycin esis ance) [
45
]. Simila ly, AME was a ely p esen
in LLFR s ains, wi h only one s ain p esen ing an aac(3
′
)-Ib in ol ed in s ep omycin
esis ance [
46
]. Wi h espec o
β
-lac am esis ance genes, bo h genes encoding class A and
D
β
-lac amases we e iden i ied in all LLCR s ains, especially, he bla
TEM-1
gene, esponsible
o ampicillin, pipe acillin, amoxicillin, and ica cillin esis ance [
47
]. Mo eo e , 62% (5/8)
Biomolecules 2025,15, 260 8 o 15
o he LLFR s ains also ca ied he bla
TEM-1
gene. Howe e , 75% (9/12) and 37% (3/8) o he
LLCR and LLFR s ains, espec i ely, ca ied genes in ol ed in ola e pa hway an agonis s,
con e ing esis ance o ime hop im [
48
]. Fu he mo e, and ega ding he sul onamide
esis ance, genes encoding sul1 and sul2, a ge enzyme dihyd op e oa e syn hase [
49
],
we e ound in 75% (9/12) and 50% (4/8) o LLCR and LLFR s ains, espec i ely. Finally,
83% (10/12) o LLCR and 62% (5/8) o LLFR s ains ca ied e A o e B genes, which
encodes an e lux p o ein o he memb ane ha con e s esis ance o an ibio ics o he
e acycline amily, such as e acycline, minocycline, and doxycycline [50].
Table 3. Analysis o he p esence o an imic obial esis ance genes in he selec ed s ains exhibi ing
low-le el cip o loxacin esis ance.
An ibio ic Family A ec ed Gene Abundance in LLCR (%) Speci ic An imic obials A ec ed
Aminoglycoside
aac(3)-II 25% Tob amycin, sisomicin, ap amycin,
dibekacin, ne ilmicin
aph(3′′)-I 75% S ep omycin
aph(6)-I 75% S ep omycin
aadA1 42% S ep omycin, spec inomycin
aadA5 25% S ep omycin, spec inomycin
Amphenicol ca A1 33% Chlo amphenicol
cmlA1 8% Chlo amphenicol
loR 33% Flo enicol, chlo amphenicol
β-lac am
ampC 8%
Ce oxi in, ampicillin,
ampicillin/cla ulanic acid,
ce o axime, ce azidime
blaCARB-2 8% Ampicillin, pipe acillin, amoxicillin
blaEC 100% Class C
blaOXA-1 8%
Ampicillin, amoxicillin/cla ulanic
acid, pipe acillin, amoxicillin,
ce epime, pipe acillin/ azobac am
blaOXA-10 8% Ampicillin, pipe acillin/ azobac am,
amoxicillin, pipe acillin, az eonam
blaTEM-1A 83% Ampicillin, cephalo hin, pipe acillin,
amoxicillin, ica cillin
blaTEM-1B 83% Ampicillin, cephalo hin, pipe acillin,
amoxicillin, ica cillin
Fola e pa hway an agonis
d A1 42% T ime hop im
d A8 8% T ime hop im
d A14 8% T ime hop im
d A17 25% T ime hop im
d A36 8% T ime hop im
sul1 58% Sul ame hoxazole
sul2 67% Sul ame hoxazole
sul3 8% Sul ame hoxazole
Fos omycin cyaA 8% Fos omycin
uhpT 42% Fos omycin
Mac olide mph(A) 42% E y h omycin, eli h omycin,
azi h omycin, spi amycin
Pe oxide si ABCD 58% (hyd ogen pe oxide)
Biomolecules 2025,15, 260 9 o 15
Table 3. Con .
An ibio ic Family A ec ed Gene Abundance in LLCR (%) Speci ic An imic obials A ec ed
Qua e na y
ammonium compound
qacE 58%
Ce ylpy idinium chlo ide, e hidium
b omide, benzylkonium
chlo ide, chlo hexidine
qacL 8%
Ce ylpy idinium chlo ide, e hidium
b omide, benzylkonium
chlo ide, chlo hexidine
Quinolone
ac 33% Cip o loxacin
gy A 83% Cip o loxacin and nalidixic acid
gy B 33% Cip o loxacin and nalidixic acid
pa C 50% Cip o loxacin and nalidixic acid
pa E 50% Cip o loxacin and nalidixic acid
qn S1 17% Cip o loxacin
Ri amycin ARR-2 8% Ri ampicin
Te acycline e (A) 58% Doxycycline, e acycline
e (B) 17%
Te acycline, doxycycline, minocycline
Va ious e m(B) 8%
S ep og amin B, lincosamide
(clindamycin, lincomycin), and
mac olide (e y h omycin)
Table 4. Analysis o he p esence o an imic obial esis ance genes in he selec ed s ains exhibi ing
low-le el os omycin esis ance.
An ibio ic Family A ec ed Gene Abundance in LLFR (%) Speci ic An imic obials A ec ed
Aminoglycoside aph(3′′)-I 50% S ep omycin
aph(6)-I 50% S ep omycin
aadA1 38% S ep omycin, spec inomycin
Amphenicol ca A1 13% Chlo amphenicol
oqxB 13% Flo enicol, chlo amphenicol
β-lac am
blaEC 88% Class C
blaMIR-6 13%
Ampicillin, amoxicillin/cla ulanic
acid, ica cillin/cla ulanic acid,
ce oxi in, pipe acillin, amoxicillin,
pipe acillin/ azobac am, ce azidime,
ica cillin, ce o axime,
ampicillin/cla ulanic acid
blaTEM-1B 50% Ampicillin, cephalo hin, pipe acillin,
amoxicillin, ica cillin
Fola e pa hway an agonis
d A1 42% T ime hop im
d A5 8% T ime hop im
sul1 25% Sul ame hoxazole
sul2 38% Sul ame hoxazole
Fos omycin cyaA 13% Fos omycin
osA 13% Fos omycin
uhpT 25% Fos omycin
Pe oxide si ABCD 38% Hyd ogen pe oxide
Qua e na y
ammonium compound qacE 25%
Ce ylpy idinium chlo ide, e hidium
b omide, benzylkonium
chlo ide, chlo hexidine
