mic oo ganisms
A icle
An Upda e on Was ewa e Mul i-Resis an Bac e ia:
Iden i ica ion o Clinical Pa hogens Such as Esche ichia coli
O25b:H4-B2-ST131-P oducing CTX-M-15 ESBL and KPC-3
Ca bapenemase-P oducing Klebsiella oxy oca
Elsa Mesqui a 1, Ri a Ribei o 1, Ca la J. C. Sil a 2, Ri a Al es 2, Ri a Bap is a 3, Síl ia Condinho 3,
Ma ia João Rosa 1, João Pe digão4, Cá ia Canei as 5,6,7 and Aida Dua e 7,8,*


Ci a ion: Mesqui a, E.; Ribei o, R.;
Sil a, C.J.C.; Al es, R.; Bap is a, R.;
Condinho, S.; Rosa, M.J.; Pe digão, J.;
Canei as, C.; Dua e, A. An Upda e
on Was ewa e Mul i-Resis an
Bac e ia: Iden i ica ion o Clinical
Pa hogens Such as Esche ichia coli
O25b:H4-B2-ST131-P oducing
CTX-M-15 ESBL and KPC-3
Ca bapenemase-P oducing Klebsiella
oxy oca.Mic oo ganisms 2021,9, 576.
h ps://doi.o g/10.3390/
mic oo ganisms9030576
Academic Edi o s: Ib ahim Bi a and
Cos as C. Papagianni sis
Recei ed: 3 Feb ua y 2021
Accep ed: 8 Ma ch 2021
Published: 11 Ma ch 2021
Publishe ’s No e: MDPI s ays neu al
wi h ega d o ju isdic ional claims in
published maps and ins i u ional a il-
ia ions.
Copy igh : © 2021 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license (h ps://
c ea i ecommons.o g/licenses/by/
4.0/).
1Wa e Quali y and T ea men Labo a o y, U ban Wa e Uni , Hyd aulics and En i onmen Depa men ,
The Labo a ó io Nacional de Engenha ia Ci il (LNEC)—Na ional Ci il Enginee ing Labo a o y,
1700-066 Lisboa, Po ugal; [email p o ec ed] (E.M.); [email p o ec ed] (R.R.); mj [email p o ec ed] (M.J.R.)
2Di eção de Ope ações de Saneamen o, ETAR de Bei olas, Rua Chen He, 1990-513 Saca ém, Po ugal;
[email p o ec ed] (C.J.C.S.); [email p o ec ed] (R.A.)
3Labo a ó io do So a en o da Águas do Alga e, Rua do Repouso, n◦10, 8000-302 Fa o, Po ugal;
[email p o ec ed] (R.B.); [email p o ec ed] (S.C.)
4Resea ch Ins i u e o Medicines (iMed.ULisboa), Facul y o Pha macy, Uni e sidade Lisboa,
A . Gama Pin o, 1649-003 Lisboa, Po ugal; jpe digao@ .ulisboa.p
5Mic obiology Resea ch Labo a o y o En i onmen al Heal h (En iHeal hMic o Lab),
Ins i u e o En i onmen al Heal h (ISAMB), Facul y o Medicine, Uni e sidade de Lisboa,
1649-028 Lisboa, Po ugal; [email p o ec ed]
6
Ins i u e o P e en i e Medicine and Public Heal h (IMP&SP), Facul y o Medicine, Uni e sidade de Lisboa,
1649-028 Lisboa, Po ugal
7Depa men o Mic obiology and Immunology, Facul y o Pha macy, A . Gama Pin o,
1649-003 Lisboa, Po ugal
8Cen o de In es igação In e disciplina Egas Moniz, Ins i u o Uni e si á io Egas Moniz,
2829-511 Mon e da Capa ica, Po ugal
*Co espondence: [email p o ec ed]; Tel.: +351-21-794-6400
Abs ac :
Was ewa e ea men plan s (WWTPs) a e signi ican ese oi s o bac e ial esis ance.
This wo k aims o iden i y he de e minan s o esis ance p oduced by G am-nega i e bac e ia in he
in luen and e luen o wo WWTPs in Po ugal. A o al o 96 was ewa e samples we e ob ained
be ween 2016 and 2019. The numbe s o o al ae obic and ecal con amina ion bac e ia we e e alua ed,
and genomic ea u es we e sea ched by polyme ase chain eac ion (PCR) and Nex -Gene a ion
Sequencing (NGS). En e obac e iaceae co esponded o 78.6% (n= 161) o he 205 isola es iden i ied by
16sRNA. The mos equen isola es we e Esche ichia spp. (57.1%, n= 117), ollowed by Ae omonas spp.
(16.1%, n= 33) and Klebsiella spp. (12.7%, n= 26). The emaining 29 isola es (14.1%) we e dis ibu ed
ac oss 10 di e en gene a. Among he 183 esis an genes de ec ed, 54 isola es p oduced ex ended
spec um
β
-lac amases (ESBL), o which bla
CTX-M-15
was p edominan (37 isola es; 68.5%). A KPC-3
ca bapenemase-p oducing K. oxy oca was iden i ied (n= 1), wi h bla
KPC-3
included in a ansposon
Tn4401 iso o m b. A highe numbe o i ulence genes (VG) (19 genes) was ound in he E. coli 5301
(O25b-ST131-B2) isola e compa ed wi h a commensal E. coli 5281 (O25b-ST410-A)
(six genes).
Bo h
sha ed i e VG [En e obac in; Ae obac in, CFA/1 (clade
α
); Type1 (clade
γ
1);
Type IV].
In conclusion,
his wo k highligh s he ole o ele an clinical bac e ia in WWTPs, such as KPC-3-p oducing
K. oxy oca, and, o he i s ime, a CTX-M-15-p oducing Och omobac um in e medium, a human
oppo unis ic pa hogen, and a SED-1-p oducing
Ci obac e a me i,
an uncommon CTX-M- ype
ex ended-spec um be a-lac amase.
Keywo ds:
was ewa e ea men plan (WWTP); CTX-M-15; SED-1; ESBL; KPC-3 ca bapenemase;
Klebsiella oxy oca;Och omobac um in e medium;Ci obac e a me i; Po ugal
Mic oo ganisms 2021,9, 576. h ps://doi.o g/10.3390/mic oo ganisms9030576 h ps://www.mdpi.com/jou nal/mic oo ganisms
Mic oo ganisms 2021,9, 576 2 o 17
1. In oduc ion
Resis ance o an ibio ics is a na u al biological phenomenon due o e olu ion by
na u al selec ion. Acco ding o Sande son e al. 2016 [
1
], an ibio ic esis ance genes (ARGs)
should be conside ed a biological con aminan o eme ging conce n, conside ing ha
hey a e able o ans e , h ough gene ic p ocesses, o mul iple ypes o o ganisms and
o e long dis ances. Inc easing esis ance o an ibio ics has led o he in es iga ion o
di e en o igins, such as was ewa e ea men plan s (WWTPs), which ha e long been
conside ed o be ese oi s o bac e ial esis ance [
2
,
3
]. Al hough mul id ug- esis an
(MDR) bac e ia a e ubiqui ous in he en i onmen , WWTPs a e a key sou ce o an ibio ic
esis ance de e minan s ou side hospi als, po en ially p o iding a selec i e p essu e o
mul id ug esis ance [
4
,
5
]. WWTPs ha e dual unc ions, as hey can se e ei he as ho spo s
o gene ans e be ween bac e ia due o hei high-nu ien and high-densi y load [
6
],
o as ese oi s o ARGs, ac ing as eposi o ies o acili a ing he long- e m s o age o
an ibio ic esis ance de e minan s [
1
]. In ac , WWTPs may accele a e he e olu iona y
imeline o ARGs by inc easing he mobiliza ion o en i onmen al esis ance genes in o
pa hogenic bac e ia [7].
WWTPs gene ally ha e h ee s ages: p ima y, seconda y, and e ia y ea men .
Du ing hese p ocesses, conside able changes occu in he dis ibu ion o he bac e ial pop-
ula ion, bu he gene al obse a ion in he li e a u e is ha ea men leads o a signi ican
educ ion in bac e ial numbe s, including he o al numbe s o esis an bac e ia [
8
]. The e
a e wo main app oaches o de e mine he occu ence o MDR o ARGs in was ewa e :
cul u e o molecula based, each wi h speci ic ad an ages [
6
]. Whole-genome sequencing
(WGS) is e olu ionizing genomic medicine and public heal h, and has signi ican ly im-
p o ed ou abili y o analyze ou b eak scena ios, cha ac e ize he ansmission dynamics
o impo an pa hogens, and assess ho izon al ans e media ed by esis ance-mobilizing
gene ic elemen s [
9
]. Con en ional mic obiology, as well as molecula biology me hods,
can be complemen a y o he p esence/absence analyses o pa hogens, and hei gene ic
a ge s o an ibio ic esis ances.
The aim o his s udy was o moni o he occu ence o mul i esis an bac e ia, and
cha ac e ize he an ibio ic esis ance genes o isola es om was ewa e s om he Bei olas-
Lisboa No h and Fa o No hwes WWTPs in Po ugal, including an o e iew on he ole
o WWTPs as a ba ie agains MDR bac e ia.
2. Ma e ials and Me hods
2.1. Pheno ypic Cha ac e iza ion o Isola es om Was ewa e
2.1.1. Sample Si es and Collec ion
In luen and e luen samples om wo WWTPs, Bei olas-Lisboa No h and Fa o
No hwes in Alga e, Po ugal, we e andomly collec ed on a mon hly basis om No em-
be 2016 o Janua y 2019. A o al o 96 was ewa e samples we e collec ed in s e ile bo les
(1 L), anspo ed in e ige a ed condi ions o he Bac e iology Labo a o y o he Mic obi-
ology and Immunology Depa men , Facul y o Pha macy, Uni e si y o Lisboa, and we e
p ocessed on he same day.
2.1.2. Mic obiological Analyses
Mic obiological analyses we e pe o med using s anda dized me hods acco ding o
ISO 7704 (e alua ion o memb ane il e s used o mic obiological analyses) [
10
]; ISO 8199
(wa e quali y-gene al equi emen s o colony coun by inocula ion in a solid medium) [
11
];
and ISO 9308-1 (enume a ion o Esche ichia coli and coli o m bac e ia—Pa 1: memb ane
il a ion me hod o wa e ) [
12
]. B ie ly, 1 mL o each was ewa e sample collec ed was
aken, and se ial dilu ions we e made in s e ile pep one wa e un il a dilu ion o 10
7
was eached. The dilu ions o in luen was ewa e samples anged om 10
4
o 10
7
, and
e luen samples anged om 10
1
o 10
4
. One mL (1 mL) o each dilu ion was il e ed
h ough a 0.45
µ
m po e-size s e ile memb ane il e (Millipo e Co, PVL, Famões, Po ugal).
The memb ane il e s we e hen placed in o speci ic cul u e media: (i) COMPASS
®
cc
Mic oo ganisms 2021,9, 576 3 o 17
Aga (Bioka Diagnos ics, PVL, Famões, Po ugal) which allows he di ec enume a ion o
Esche ichia coli and coli o ms, in 24 h; (ii) COMPASS
®
En e ococcus Aga (Bioka Diagnos ics,
PVL, Famões, Po ugal), a selec i e media used o he enume a ion o en e ococci in ood
and wa e ; o (iii) Pla e Coun Aga (PCA) o enume a e ae obic bac e ia con aining glucose
and yeas ex ac (Bioka Diagnos ics, PVL, Famões, Po ugal). Memb ane il a ion is a
eliable and ecommended me hod ha allows he use o la ge sample olumes. Howe e ,
i is mo e sui able o he coun ing o speci ic bac e ia deposi ed on he il e memb ane in
selec i e media, which inhibi s he g ow h o o he bac e ia, making i easie o coun he
colonies. I is no a sui able me hod o enume a ing o al ae obic bac e ia in wa e s wi h
high bac e ial backg ound lo a, because is no easy o coun he colonies, which canno be
pe ec ly sepa a ed, and a ep oducible coun is no allowed. The e o e, in pa allel wi h a
il a ion memb ane, 0.1 mL o each dilu ion o in luen and e luen was ewa e was sp ead
on he PCA medium su ace wi h disposable L-shaped cell sp eade s (VWR In e na ional,
Amado a, Po ugal). The pla es we e incuba ed unde ae obic condi ions a 37
◦
C, and
g ow h was obse ed a e 24 h, o o al ae obic bac e ia and coli o ms, and a 44
◦
C o
24 h o E. coli and En e ococcus spp. Bac e ial coun s we e exp essed as colony- o ming
uni s pe mL (CFU/mL), and we e conside ed he o al CFUs pe memb ane o pla e wi hin
he ange o 15–300 o 1–100, espec i ely. Fo each speci ic cul u e medium, only he pla es
wi hin he ange o 1–100 ypical colonies we e conside ed o he bac e ial coun .
2.1.3. Isola ion o Resis an G am-Nega i e Bac e ia
An ibio ics we e indi idually added in o D igalsky Lac ose aga (DLA) medium o
ob ain coun s o an ibio ic esis an G am-nega i e bac e ia. The DLA cul u e medium
was used o he selec ion o esis an bac e ia, as i allows he di e en ia ion o bac e ia
acco ding o hei abili y o e men o no he lac ose, which is e ealed by di e ence o
colo (yellow o blue, espec i ely). The medium was p epa ed wi h he inco po a ion
o new an ibio ic solu ions o gi e inal concen a ions as ollows: ce o axime (
β
-lac am)
2 mg/L; gen amicin (aminoglycosides) 25 mg/L; no loxacin ( luo oquinolone) 4 mg/L;
and sul ame hoxazole 256 mg/L, acco ding o he in e na ional s anda d ISO 20776-1:2006
e ised by ISO 20776-1:2019. The selec ion o hese an ibio ics was made acco ding o he
clinical ele ance o human heal h.
A o al o 0.1 mL o each dilu ion 10
2
(in luen ) and 10
1
(e luen ) we e sp ead on he
DLA su ace, wi h and wi hou an ibio ics. Di e en dilu ions we e used o he in luen
and e luen as in luen is un ea ed was ewa e and e luen is ea ed was ewa e . Then,
he g ow h was obse ed a e 24 h o incuba ion a 37
◦
C, and he lac ose-posi i e o
-nega i e colonies we e coun ed. The esul s ob ained wi h and wi hou an ibio ic we e
compa ed, in o de o quan i y and de e mine he a e age CFU pe mL and, consequen ly,
he esis ance o isola es o hese an ibio ics. Fo pu e cul u es, colonies showing di e en
mo phologies we e selec ed and ans e ed o new DLA medium wi h selec i e an ibio ics
(p esen ed abo e). A e incuba ion, he isola es we e conse ed a
−
80
◦
C in s ock
medium (BHI b o h supplemen ed wi h 20% glyce ol) o u he pheno ype and geno ype
cha ac e iza ion.
The e ec i eness o was ewa e ea men p ocesses was measu ed using a concep
called “log emo al alues” (LRVs). The LRV is a measu e o he abili y o a ea men p o-
cess o emo e pa hogenic mic oo ganisms. LRVs a e de e mined by aking he loga i hm
o he a io o he pa hogen concen a ion in he in luen and e luen wa e o a ea men
p ocess, acco ding o he ollowing equa ion:
LRV =log10In luen Pa hogen Concen a ion
E luen Pa hogen Concen a ion
(i) an LRV o 1 is equi alen o 90% emo al o a a ge pa hogen; (ii) an LRV o 2 is
equi alen o 99% emo al; (iii) an LRV o 3 is equi alen o 99.9% emo al [13].
Mic oo ganisms 2021,9, 576 4 o 17
2.1.4. An ibio ic Suscep ibili y Tes ing
An ibio ic suscep ibili y pheno ypes we e de e mined by he aga di usion echnique
based on he Ki by–Baue me hod, acco ding o s anda d ecommenda ions o he Eu o-
pean Commi ee on An imic obial Suscep ibili y Tes ing (EUCAST, Disk Di usion Tes
Me hodology, 2017), wi h he ollowing an ibio ic disks: ce oxi in (FOX) 30
µ
g; ce o axime
(CTX) 5
µ
g; ce azidime (CAZ) 10
µ
g; imipenem (IPM) 10
µ
g; gen amicin (GM) 10
µ
g;
cip o loxacin (CIP) 5
µ
g; and ime hop im/sul ame hoxazole (SXT) 1.25
µ
g/23.75
µ
g
(Bio-Rad, Po ugal). Bac e ial suspensions, wi h u bidi y o 0.5 MacFa land S anda d we e
sp ead on Muelle Hin on aga (Bioka Diagnos ics, PVL, Famões, Po ugal) using a s e ile
co on swab. A e 24 h o incuba ion a 35
◦
C, he diame e s o an ibio ic inhibi ion o
g ow h we e measu ed, and in e p e a ion o zone diame e s was unde aken acco ding o
EUCAST b eakpoin s, 2018. An isola e is conside ed mul iple d ug esis an (MDR) i i is
esis an o h ee o mo e an ibio ic classes [14].
2.2. Genomic Cha ac e iza ion o Isola es om Was ewa e
2.2.1. DNA Ex ac ion and Sequencing
DNA was ex ac ed om pu e isola es using he NZY Mic obial gDNA Isola ion
Ki (NZYTech, Lisboa, Po ugal), ollowing he manu ac u e ’s p o ocol. Ce ain s eps
in he p o ocol we e modi ied o ob ain be e DNA yield, which included inc easing he
bead-lysis s ep o ce ain cul u es om 10 min o 20 min. Fu he mo e, he elu ion s ep
incuba ion pe iod was inc eased by an addi ional 5 min. The concen a ion and pu i y
o DNA was de e mined ia a nanopho ome e (NanoD op
®
, Isogen Li e Science, De
Mee n, The Ne he lands). The ex ac ed DNA was s o ed a
−
20
◦
C un il i was used
o polyme ase chain eac ions (PCR) and sequencing by Sange and Nex -Gene a ion
Sequencing (NGS) echnology (S ab Vida, Lisboa, Po ugal).
2.2.2. Geno ype Iden i ica ion o Isola es
The iden i ica ion o esis an isola es was comple ed by 16S RNA gene sequenc-
ing. This me hodology can iden i y bac e ia ha ha e no been iden i ied using manual
and au oma ed sys ems, and can cha ac e ize p e iously undesc ibed species. Uni e -
sal 16S RNA bac e ial p ime s 27F (5
0
-AGAGTTTGATCCTGGCTCAG-3
0
) and 1392R
(50-GGTTACCTTGTTACGACTT-30)
we e used o ampli y his gene using 10 ng o ge-
nomic DNA om each s ain. PCR p oduc s we e isualized on a 1% aga ose gel s ained
wi h Gel Red (NZYTech, Lisboa, Po ugal) unde UV ligh , o con i m he p esence o a
1350 base pai s (bp) band.
2.2.3. De ec ion o An ibio ic Resis ance Genes
Acco ding o he an ibio ype p o iles, he isola es suspec ed o p oducing
β
-lac amases
and ca bapenemases we e sc eened o bla
CTX-M-
,bla
KPC-
, and bla
OXA-
genes. The p es-
ence o Class1 in eg ons and he plasmid-media ed quinolone esis ance genes aac(6
0
)-
Ib-c ,qn A,qn B, and qn S we e cha ac e ized using p ime s and condi ions p e iously
desc ibed [15–17].
The p ime s used o ampli y he bla
OXA
- genes we e 5
0
-GTACTAATCAA
AGTGTGAA-3
0
and 5
0
-TTCCCCTAACATGAATTTGT-3
0
. S udies o he gene ic s uc u e
su ounding he bla
KPC-3
gene, in o de o iden i y he Tn3-based ansposon, Tn4401, we e
unde aken as p e iously desc ibed [3].
2.2.4. Phylogene ic G oup, Molecula Typing, and Sequence Analysis
Fo iden i ica ion o phylogene ic g oup associa ions in E. coli, a iplex PCR me hod
was employed using p ime s a ge ing wo genes, chuA and yjaA, and one anonymous
DNA agmen , TspE4C [
18
]. Allele-speci ic PCR was pe o med o iden i y he O25-ST131
clone o E. coli. P ime s O25pabBF (5
0
-TCCAGCAGGTGCTGGATCGT-3
0
) and O25pabB.R
(50-GCGAAATTTTTCGCCGTACTGT-3
0
) we e used o ampli y a 347 bp agmen o he
pabB gene speci ically in isola es belonging o he O25b-ST131 clone. A posi i e PCR
con ol was included in he assay o con i m ha any ampli ica ion ailu e wi h he pabB
Mic oo ganisms 2021,9, 576 5 o 17
allele-speci ic p ime s was no due o poo DNA quali y o o ailu e o he PCR i sel . This
con ol PCR a ge ed a 427 bp agmen o he pA gene, ampli ied wi h p ime s pA.F (5
0
-
GCTACGAATCTCTGTTTGCC-3
0
) and pA2.R (5
0
-GCAACGCGGCCTGGCGGAAG–3
0
) [
19
].
Mul ilocus sequence yping (MLST) yping was pe o med, in a ep esen a i e num-
be o E. coli s ains, by PCR ampli ica ion and sequencing o se en housekeeping genes
(adk, umC,gy B,icd,mdh,pu A and ecA), ollowing he p o ocols speci ied on he
E. coli
MLST websi e, as well as he p ime sequences o se en genes a ailable a h p://mls .
wa wick.ac.uk/mls /dbs/Ecoli (accessed on 15 Decembe 2020). Allele numbe s o se en
gene agmen s o each isola e we e ob ained by compa ing wi h co esponding alleles,
and he Sequence Type (ST) o each isola e was de e mined by combining se en allelic
p o iles in he MLST E. coli da abase (h p://mls .wa wick.ac.uk/mls /dbs/Ecoli, ac-
cessed on
15 Decembe 2020).
All ampli ica ion p oduc s we e pu i ied by NZYGelpu e Ki
(NZYTech, Lisboa, Po ugal), ollowing he manu ac u e ’s p o ocol. DNA agmen s o
di e en sizes we e sequenced by STAB VIDA (h ps://www.s ab ida.com/, accessed on
15 Decembe 2020).
The nucleo ide sequences we e analyzed using he NCBI Nucleo ide-
BLAST da abase (h ps://blas .ncbi.nlm.nih.go /Blas .cgi, accessed on 15 Decembe 2020),
and o gene a e alignmen s be ween h ee o mo e sequences we used he mul iple se-
quence alignmen p og am Clus al Omega (h ps://www.ebi.ac.uk/Tools/msa/clus alo/,
accessed on 15 Decembe 2020).
2.2.5. Whole-Genome Sequencing and Sequence Analysis
Two E. coli 5301 (O25b:H4-B2-ST131) and E. coli 5281 (O25b:H4-A-ST410) we e selec ed
o whole-genome sequencing. A o al o 1
µ
g DNA, p epa ed acco ding o he desc ip-
ion in Sec ion 2.1, was sen o STAB VIDA (h p://www.s ab ida.com/, accessed on
15 Decembe 2020)
o genome sequencing on he Illumina Pla o m PE150 wi h
~100–150×
o co e age. The de no o assembly app oach was used, which u ilizes an algo i hm based
on de B uijn g aphs [
20
]. A e he ini ial con ig c ea ion, he eads we e mapped back
o he con igs o assembly co ec ion. The genome assemblies we e e alua ed wi h
QUAST 5.0.2.
The assembly s a is ics and cumula i e plo s we e c ea ed [
21
]. A e ob ain-
ing he assemblies, an iden i ica ion and cha ac e iza ion o he bac e ia was pe o med
using he se ices o he Cen e o Genomic Epidemiology (CGE). The iden i ica ion o ac-
qui ed esis ance genes used ResFinde 4.0 [
22
], and i ulence genes used Vi ulenceFinde
2.0. [
23
]. Addi ionally, i ulence genes we e de ec ed by aligning hem di ec ly agains
he Vi ulence Fac o s o Pa hogenic Bac e ia Da abase (VFDB; “co e da ase ” downloaded
on
12 Decembe 2020) [24].
PlasmidFinde 2.1 [
25
] was used o de ec he incompa i-
bili y g oups o plasmids, and Se o ypeFinde 2.0 con i med he p e iously de e mined
se o ype [26].
3. Resul s
3.1. Pheno ypic Cha ac e iza ion
3.1.1. Enume a ion o Was ewa e Isola es
A o al o 96 WWTP samples we e ob ained o e he sampling pe iod be ween No em-
be 2016 and Janua y 2019, om in luen and e luen was ewa e a he Bei olas-Lisboa
(BEI) and Fa o No hwes -Alga e (FNw) WWPTs. The numbe o o al ae obic bac e ia
and aecal con amina ion bac e ia in he in luen and e luen was ewa e s we e exp essed
by CFU/mL and a e p esen ed in Table S1. Acco ding o Figu e 1, he mic obiological
analysis showed ha he alues o o al ae obic bac e ia in in luen was ewa e s o he
BEI (BEI-DSR) and FNw (FNw-AFB) WWTPs we e 5.89E+07 and 1.86E+07 CFU/mL, e-
spec i ely. In he e luen was ewa e o he BEI (BEI-EFL) and FNw (FNw-EFT) WWTPs,
he ea men p ocess p oduced a 3-log educ ion ela i e o he in luen o 7.85E+04 and
1.37E+04 CFU/mL, espec i ely. Addi ionally, in he o he pa ame e s analyzed ( o al
coli o ms, E. coli,En e ococcus spp.), he e was a 3-log educ ion a bo h WWTPs, wi h he
excep ion o he numbe o En e ococcus spp. isola es in FNw-AFB and FNw-EFT WWTP,
wi h a 2-log educ ion o 4.41E+04 and 7.93E+02 CFU/mL, espec i ely.

Mic oo ganisms 2021,9, 576 6 o 17
Mic oo ganisms 2021, 9, x 6 o 18
espec i ely. In he e luen was ewa e o he BEI (BEI-EFL) and FNw (FNw-EFT)
WWTPs, he ea men p ocess p oduced a 3-log educ ion ela i e o he in luen o
7.85E+04 and 1.37E+04 CFU/mL, espec i ely. Addi ionally, in he o he pa ame e s ana-
lyzed ( o al coli o ms, E. coli, En e ococcus spp.), he e was a 3-log educ ion a bo h
WWTPs, wi h he excep ion o he numbe o En e ococcus spp. isola es in FNw-AFB and
FNw-EFT WWTP, wi h a 2-log educ ion o 4.41E+04 and 7.93E+02 CFU/mL, espec i ely.
Figu e 1. Mic obiological analysis o 96 WWTP samples om he Bei olas-Lisboa and Fa o No h-
wes -Alga e WWPTs o in luen was ewa e (BEI-DSR/FNw-ABF) and e luen was ewa e (BEI-
EFL/FNw-EFT), ep esen ed by he a e age numbe s ob ained o o al ae obic bac e ia, coli o ms,
Esche ichia coli, and En e ococcus spp.
3.1.2. Enume a ion o An imic obial-Resis an G am-Nega i e Bac e ia
The numbe o an ibio ic esis an bac e ia in he in luen and e luen was ewa e s
was de e mined by pla ing was ewa e samples on DLA cul u e medium, speci ically o
isola ion o G am-nega i e bac e ia, supplemen ed wi h he an ibio ics ce o axime (CTX),
gen amicin (GM), no loxacin (NF), and ime hop im-sul ame hoxazole (SXT). The DLA
cul u e medium was used wi hou an ibio ics as a con ol o bac e ial g ow h, and al-
lowed us o de e mine he ela ionship be ween he numbe o o al G am-nega i e bac-
e ia and he numbe o bac e ia ha g ow wi h an ibio ic selec ion.
Acco ding o Figu e 2, when compa ing he isola es g own on DLA cul u e medium
wi hou and wi h an ibio ics, he CFU/mL o each an ibio ic dec eased o one log10 ela-
i e o he in luen was ewa e s o BEI-DSR and FNw-AFB. Howe e , in he espec i e
e luen was ewa e s o hese WWTPs, he CFU/mL dec eased one o wo log10 alues
depending on he an ibio ic ype. Conside ing he esul s o each an ibio ic and he alue
o LRV ( he loga i hm o he a io o pa hogen concen a ion in he in luen and e luen
wa e ), in BEI-WWTP he e was a emo al e ec i eness o was ewa e ea men o 99%
(LRV o 2) o esis an isola es o he CTX an ibio ic and 90% (LRV o 1) o esis an isola es
o he GM, NF, SXT an ibio ics. Addi ionally, in FNw-WWTP, emo al e iciencies o
was ewa e ea men o 99% (LRV o 2) o esis an isola es o he GM and NF an ibio ics
and 90% (LRV o 1) o esis an isola es o he CTX and SXT an ibio ics we e achie ed.
Based on he esul s o 96 analyses, he a e age o he o al numbe o esis an isola es
was lowe in he was ewa e o Fa o FNw-AFB (3.98E+04) and FNw-EFT (1.20E+03) han
ha o he Bei olas BEI-DSR (4.32E+05) and BEI-EFL (2.79E+04) WWPTs.
Figu e 1.
Mic obiological analysis o 96 WWTP samples om he Bei olas-Lisboa and Fa o No hwes -Alga e WWPTs
o in luen was ewa e (BEI-DSR/FNw-ABF) and e luen was ewa e (BEI-EFL/FNw-EFT), ep esen ed by he a e age
numbe s ob ained o o al ae obic bac e ia, coli o ms, Esche ichia coli, and En e ococcus spp.
3.1.2. Enume a ion o An imic obial-Resis an G am-Nega i e Bac e ia
The numbe o an ibio ic esis an bac e ia in he in luen and e luen was ewa e s
was de e mined by pla ing was ewa e samples on DLA cul u e medium, speci ically o
isola ion o G am-nega i e bac e ia, supplemen ed wi h he an ibio ics ce o axime (CTX),
gen amicin (GM), no loxacin (NF), and ime hop im-sul ame hoxazole (SXT). The DLA
cul u e medium was used wi hou an ibio ics as a con ol o bac e ial g ow h, and allowed
us o de e mine he ela ionship be ween he numbe o o al G am-nega i e bac e ia and
he numbe o bac e ia ha g ow wi h an ibio ic selec ion.
Acco ding o Figu e 2, when compa ing he isola es g own on DLA cul u e medium
wi hou and wi h an ibio ics, he CFU/mL o each an ibio ic dec eased o one log10
ela i e o he in luen was ewa e s o BEI-DSR and FNw-AFB. Howe e , in he espec i e
e luen was ewa e s o hese WWTPs, he CFU/mL dec eased one o wo log10 alues
depending on he an ibio ic ype. Conside ing he esul s o each an ibio ic and he alue
o LRV ( he loga i hm o he a io o pa hogen concen a ion in he in luen and e luen
wa e ), in BEI-WWTP he e was a emo al e ec i eness o was ewa e ea men o 99%
(LRV o 2) o esis an isola es o he CTX an ibio ic and 90% (LRV o 1) o esis an isola es
o he GM, NF, SXT an ibio ics. Addi ionally, in FNw-WWTP, emo al e iciencies o
was ewa e ea men o 99% (LRV o 2) o esis an isola es o he GM and NF an ibio ics
and 90% (LRV o 1) o esis an isola es o he CTX and SXT an ibio ics we e achie ed.
Based on he esul s o 96 analyses, he a e age o he o al numbe o esis an isola es was
lowe in he was ewa e o Fa o FNw-AFB (3.98E+04) and FNw-EFT (1.20E+03) han ha
o he Bei olas BEI-DSR (4.32E+05) and BEI-EFL (2.79E+04) WWPTs.
Mic oo ganisms 2021,9, 576 7 o 17
Mic oo ganisms 2021, 9, x 7 o 18
Figu e 2. The numbe o colony- o ming uni s ha g ew on D igalsky wi hou an ibio ics (wi hou
an .) and supplemen ed wi h he an ibio ics: ce o axime (CTX), gen amicin (GM), no loxacin (NF),
and ime hop im-sul ame hoxazole (SXT). BEI-DSR: Bei olas-Lisboa in luen was ewa e ; BEI-
EFL: Bei olas-Lisboa e luen was ewa e ; FNw-AFB: Fa o No hwes -Alga e in luen
was ewa e ; FNw-EFT: Fa o No hwes -Alga e e luen was ewa e .
3.1.3. An imic obial Suscep ibili y P o iles
The an imic obial suscep ibili y es s we e pe o med o isola es ha g ew on DLA
wi h an ibio ics and acco ding o colony mo phologies. Yellow colonies (en e obac e ia
lac ose posi i e) we e p e e en ially selec ed o he an imic obial suscep ibili y es s. The
isola es we e selec ed i hey we e mos ly esis an o β-lac am and any o he o he an i-
bio ics. Subsequen ly, isola es wi h high suscep ibili y we e no conside ed o molecula
cha ac e iza ion, and a o al o 315 isola es we e selec ed. Acco ding o Figu e 3, he e was
a de ia ion o he an ibio ic cip o loxacin (66.1%), ollowed by ime hop im/sul ame h-
oxazole (57.9%), gen amicin (35.1%), and imipenem (4.6%). The esis ance o he hi d gen-
e a ion cephalospo ins ce o axime (33.8%) and bo h ce azidime and ce oxi in (20.0%;
27.7%) mus be aken in o accoun , because hey a e indica o s o he p oduc ion o en-
zymes (β-lac amases) ha hyd olyze β-lac am an ibio ics, and a e s ongly associa ed
wi h bac e ia isola ed om hospi als and communi y-acqui ed in ec ions.
Figu e 3. An ibio ic suscep ibili y equency (%) o 315 isola es o ce oxi in (FOX), ce o axime
(CTX), ce azidime (CAZ), imipenem (IPM), gen amicin (GM), cip o loxacin (CIP), and ime-
hop im-sul ame hoxazole (SXT), om in luen and e luen was ewa e om he Bei olas and
Figu e 2.
The numbe o colony- o ming uni s ha g ew on D igalsky wi hou an ibio ics (wi hou
an .) and supplemen ed wi h he an ibio ics: ce o axime (CTX), gen amicin (GM), no loxacin (NF),
and ime hop im-sul ame hoxazole (SXT). BEI-DSR: Bei olas-Lisboa in luen was ewa e ; BEI-EFL:
Bei olas-Lisboa e luen was ewa e ; FNw-AFB: Fa o No hwes -Alga e in luen was ewa e ; FNw-
EFT: Fa o No hwes -Alga e e luen was ewa e .
3.1.3. An imic obial Suscep ibili y P o iles
The an imic obial suscep ibili y es s we e pe o med o isola es ha g ew on DLA
wi h an ibio ics and acco ding o colony mo phologies. Yellow colonies (en e obac e ia lac-
ose posi i e) we e p e e en ially selec ed o he an imic obial suscep ibili y es s. The iso-
la es we e selec ed i hey we e mos ly esis an o
β
-lac am and any o he o he an ibio ics.
Subsequen ly, isola es wi h high suscep ibili y we e no conside ed o molecula cha ac-
e iza ion, and a o al o 315 isola es we e selec ed. Acco ding o
Figu e 3,
he e was a de i-
a ion o he an ibio ic cip o loxacin (66.1%), ollowed by ime hop im/sul ame hoxazole
(57.9%), gen amicin (35.1%), and imipenem (4.6%). The esis ance o he hi d gene a-
ion cephalospo ins ce o axime (33.8%) and bo h ce azidime and ce oxi in (20.0%; 27.7%)
mus be aken in o accoun , because hey a e indica o s o he p oduc ion o enzymes (
β
-
lac amases) ha hyd olyze
β
-lac am an ibio ics, and a e s ongly associa ed wi h bac e ia
isola ed om hospi als and communi y-acqui ed in ec ions.
Mic oo ganisms 2021, 9, x 7 o 18
Figu e 2. The numbe o colony- o ming uni s ha g ew on D igalsky wi hou an ibio ics (wi hou
an .) and supplemen ed wi h he an ibio ics: ce o axime (CTX), gen amicin (GM), no loxacin (NF),
and ime hop im-sul ame hoxazole (SXT). BEI-DSR: Bei olas-Lisboa in luen was ewa e ; BEI-
EFL: Bei olas-Lisboa e luen was ewa e ; FNw-AFB: Fa o No hwes -Alga e in luen
was ewa e ; FNw-EFT: Fa o No hwes -Alga e e luen was ewa e .
3.1.3. An imic obial Suscep ibili y P o iles
The an imic obial suscep ibili y es s we e pe o med o isola es ha g ew on DLA
wi h an ibio ics and acco ding o colony mo phologies. Yellow colonies (en e obac e ia
lac ose posi i e) we e p e e en ially selec ed o he an imic obial suscep ibili y es s. The
isola es we e selec ed i hey we e mos ly esis an o β-lac am and any o he o he an i-
bio ics. Subsequen ly, isola es wi h high suscep ibili y we e no conside ed o molecula
cha ac e iza ion, and a o al o 315 isola es we e selec ed. Acco ding o Figu e 3, he e was
a de ia ion o he an ibio ic cip o loxacin (66.1%), ollowed by ime hop im/sul ame h-
oxazole (57.9%), gen amicin (35.1%), and imipenem (4.6%). The esis ance o he hi d gen-
e a ion cephalospo ins ce o axime (33.8%) and bo h ce azidime and ce oxi in (20.0%;
27.7%) mus be aken in o accoun , because hey a e indica o s o he p oduc ion o en-
zymes (β-lac amases) ha hyd olyze β-lac am an ibio ics, and a e s ongly associa ed
wi h bac e ia isola ed om hospi als and communi y-acqui ed in ec ions.
Figu e 3. An ibio ic suscep ibili y equency (%) o 315 isola es o ce oxi in (FOX), ce o axime
(CTX), ce azidime (CAZ), imipenem (IPM), gen amicin (GM), cip o loxacin (CIP), and ime-
hop im-sul ame hoxazole (SXT), om in luen and e luen was ewa e om he Bei olas and
Figu e 3.
An ibio ic suscep ibili y equency (%) o 315 isola es o ce oxi in (FOX), ce o axime
(CTX), ce azidime (CAZ), imipenem (IPM), gen amicin (GM), cip o loxacin (CIP), and ime hop im-
sul ame hoxazole (SXT), om in luen and e luen was ewa e om he Bei olas and Fa o No hwes
WWTPs. R- esis an ; I-suscep ible a inc eased exposu e; S-suscep ible a s anda d dose.
Mic oo ganisms 2021,9, 576 8 o 17
Conside ing he dis ibu ion o he WWTPs, he pe cen age o esis an isola es om
in luen and e luen was ewa e o bo h he Bei olas (BEI-DSR; BEI-EFL) and Fa o No h-
wes (FNw-AFB; FNw-EFT) WWTPs a e p esen ed in Figu e 4. The lowes numbe o
esis an isola es was ound in he e luen was ewa e , in acco dance wi h he esul s ob-
ained in he quan i ica ion o en e obac e ia and o he G am-nega i e bac e ia
(Figu e 2).
Howe e , o ele ance, he pe cen age o imipenem esis an bac e ia isola ed om he
FNw-AFB (in luen ) and FNw-EFT (e luen ) has inc eased om 10.6% o 22.2%.
Mic oo ganisms 2021, 9, x 8 o 18
Fa o No hwes WWTPs. R- esis an ; I-suscep ible a inc eased exposu e; S-suscep ible a s anda d
dose.
Conside ing he dis ibu ion o he WWTPs, he pe cen age o esis an isola es om
in luen and e luen was ewa e o bo h he Bei olas (BEI-DSR; BEI-EFL) and Fa o No h-
wes (FNw-AFB; FNw-EFT) WWTPs a e p esen ed in Figu e 4. The lowes numbe o e-
sis an isola es was ound in he e luen was ewa e , in acco dance wi h he esul s ob-
ained in he quan i ica ion o en e obac e ia and o he G am-nega i e bac e ia (Figu e
2.). Howe e , o ele ance, he pe cen age o imipenem esis an bac e ia isola ed om
he FNw-AFB (in luen ) and FNw-EFT (e luen ) has inc eased om 10.6% o 22.2%.
Figu e 4. Pe cen age o esis an isola es o in luen and e luen was ewa e om he Bei olas and
Fa o No hwes WWTPs. An ibio ics es ed: ce oxi in (FOX), ce o axime (CTX), ce azidime (CAZ),
imipenem (IPM), gen amicin (GM), cip o loxacin (CIP), and ime hop im-sul ame hoxazole
(SXT). BEI-DSR: Bei olas-Lisboa in luen was ewa e ; BEI-EFL: Bei olas-Lisboa e luen
was ewa e ; FNw-AFB: Fa o No hwes -Alga e in luen was ewa e ; FNw-EFT: Fa o No hwes -
Alga e e luen was ewa e .
3.2. Genomic Cha ac e iza ion
3.2.1. Iden i ica ion o Isola es by 16S RNA
Two hund ed and i e isola es (n = 205) esis an o wo o mo e an ibio ics, wi h
emphasis on β-lac ams, luo oquinolones, and ime hop im-sul ame hoxazole, we e se-
lec ed o iden i ica ion and molecula cha ac e iza ion. A e sequencing, he p oduc s o
ampli ica ion ob ained by 16S RNA we e success ully iden i ied wi h simila i y alues
highe han 98% wi h he ype s ain o an accu a ely named species, and we e he e o e
conside ed membe s o ha genus. Mos o he was ewa e ’s 117 isola es (57.1%) we e
iden i ied in genus Esche ichia, namely E. coli (n = 95; 46.3%), E. e gusoni (n = 18; 8.7%) and
E. ma mo ae (n = 4; 1.9%). Twen y-six isola es (12.7%) we e iden i ied as belonging o he
genus Klebsiella, namely K. pneumoniae (n = 16; 7.8%), K. michiganensis (n = 7; 3.4%), K. oxy-
oca, K. quasipneumoniae, and K. quasi a iicola (n = 1; 0.5% each). Nine di e en species o
he genus Ae omonas (n = 33; 16.1%) we e iden i ied, namely: A. ca iae (n = 16; 7.8%), A.
media (n = 5; 2.4%), A. hyd ophila (n = 3; 1.5%), A. aiwanensis, A. e oni and A. dhakensis (n =
2; 1.0% each) and A. en e opelogenes, A. i ipollensis and A. sana ellii (n = 1; 0.5% each). The
emaining 29 isola es (14.1%) we e dis ibu ed ac oss 10 gene a (Table S2). Rela i e o he
dis ibu ion o he 205 isola es o each WWTP, and acco ding o Figu e 5, he was ewa e
isola es we e g ouped by axonomic iden i y: Esche ichia spp., o he En e obac e iaceae,
non- e men e bac e ia, and Ae omonas spp.
Figu e 4.
Pe cen age o esis an isola es o in luen and e luen was ewa e om he Bei olas
and Fa o No hwes WWTPs. An ibio ics es ed: ce oxi in (FOX), ce o axime (CTX), ce azidime
(CAZ), imipenem (IPM), gen amicin (GM), cip o loxacin (CIP), and ime hop im-sul ame hoxazole
(SXT). BEI-DSR: Bei olas-Lisboa in luen was ewa e ; BEI-EFL: Bei olas-Lisboa e luen was ewa-
e ; FNw-AFB: Fa o No hwes -Alga e in luen was ewa e ; FNw-EFT: Fa o No hwes -Alga e
e luen was ewa e .
3.2. Genomic Cha ac e iza ion
3.2.1. Iden i ica ion o Isola es by 16S RNA
Two hund ed and i e isola es (n= 205) esis an o wo o mo e an ibio ics, wi h
emphasis on
β
-lac ams, luo oquinolones, and ime hop im-sul ame hoxazole, we e se-
lec ed o iden i ica ion and molecula cha ac e iza ion. A e sequencing, he p oduc s o
ampli ica ion ob ained by 16S RNA we e success ully iden i ied wi h simila i y alues
highe han 98% wi h he ype s ain o an accu a ely named species, and we e he e o e
conside ed membe s o ha genus. Mos o he was ewa e ’s 117 isola es (57.1%) we e
iden i ied in genus Esche ichia, namely E. coli (n= 95; 46.3%), E. e gusoni (n= 18; 8.7%)
and E. ma mo ae (n= 4; 1.9%). Twen y-six isola es (12.7%) we e iden i ied as belonging
o he genus Klebsiella, namely K. pneumoniae (n= 16; 7.8%), K. michiganensis (n= 7; 3.4%),
K. oxy oca,
K. quasipneumoniae, and K. quasi a iicola (n= 1; 0.5% each). Nine di e en species
o he genus Ae omonas (n= 33; 16.1%) we e iden i ied, namely: A. ca iae (n= 16; 7.8%),
A. media
(n= 5; 2.4%), A. hyd ophila (n= 3; 1.5%), A. aiwanensis,A. e oni and A. dhakensis
(n= 2;
1.0% each) and A. en e opelogenes,A. i ipollensis and A. sana ellii (n= 1; 0.5% each).
The emaining 29 isola es (14.1%) we e dis ibu ed ac oss 10 gene a (Table S2). Rela i e o
he dis ibu ion o he 205 isola es o each WWTP, and acco ding o Figu e 5, he was ewa-
e isola es we e g ouped by axonomic iden i y: Esche ichia spp., o he En e obac e iaceae,
non- e men e bac e ia, and Ae omonas spp.
Mic oo ganisms 2021,9, 576 9 o 17
Mic oo ganisms 2021, 9, x 9 o 18
Figu e 5. F equency (%) o he 205 was ewa e isola es by axonomic iden i y: Esche ichia spp.,
o he En e obac e iaceae, non- e men e bac e ia, and Ae omonas spp., om he Bei olas (BEI-
DSR/EFL) and Fa o (FNw-AFB/EFT) WWTPs. BEI-DSR: Bei olas-Lisboa in luen was ewa e ; BEI-
EFL: Bei olas-Lisboa e luen was ewa e ; FNw-AFB: Fa o No hwes -Alga e in luen
was ewa e ; FNw-EFT: Fa o No hwes -Alga e e luen was ewa e .
The g oup Esche ichia spp. was he majo i y in was ewa e isola es o bo h WWTPs,
con ibu ing o 57.1% o he o al o isola es s udied. Al hough he in luen om BEI-DSR
had a lowe numbe o Esche ichia spp. isola es (51.8%), he numbe o isola es iden i ied
in his in luen was highe (39.5%) han in he e luen BEI-EFL (25.9%), as well as om
he in luen FNw-AFB (27.8%) and e luen FNw-EFT (6.8%). I should be emphasized ha
Ae omonas spp. was ound in 35.7% o isola es om e luen was ewa e (FNw-EFT), and
was highe han in e luen was ewa e BEI-EFL, wi h 15.1% o isola es. A Fa o WWTP,
he las s age o e luen (FNw-EFT) was disin ec ion by ul a iole adia ion, which could
explain he p esence o Ae omonas spp. [27]. Among non- e men e isola es, we should
men ion some species ha we e ubiqui ous, no speci ically pa hogenic o humans, bu
possibly esponsible o in ec ions in he hospi al and in he communi y en i onmen ,
such as Acine obac e spp. (A. ge ne i, A. pi ii and A. nosocomialis), Comamonas spp., Och o-
mobac um spp. and U ubu uella suis, a no el species wi hin he Neisse iaceae amily.
3.2.2. De ec ion and Sequencing o An ibio ic Resis ance Genes
Acco ding o he suscep ibili y esul s, 205 was ewa e isola es we e selec ed o
sc eening o esis ance de e minan s, which was pe o med by PCR wi h speci ic p ime s,
and a e sequencing, 183 esis an genes we e iden i ied in 97 isola es, as p esen ed in
Table 1. The cha ac e iza ion o suscep ibili y o an ibio ics and esis an genes ound by
isola e is p esen ed in Table S3 (n = 97). Among he genes ha exp essed enzymes espon-
sible o esis ance o β-lac ams, he gene blaCTX-M-15 was de ec ed in 37 isola es, ollowed
by blaCTX-M-14 (n = 8) and blaCTX-M-27 (n = 4), while he genes blaCTX-M-1, blaCTX-M-2, blaCTX-M-32, and
blaCTX-M-174 we e de ec ed in one isola e each. The gene blaSED-1 was iden i ied in Ci obac e
a me i and he gene blaCTX-M-15 in Och omobac um in e medium. These genes exp ess en-
zymes denomina ing he ex ended spec um o β-lac amases (ESBLs), con e ing e-
sis ance o hi d gene a ion cephalospo ins, and a e p e alen among En e obac e iaceae.
Figu e 5.
F equency (%) o he 205 was ewa e isola es by axonomic iden i y: Esche ichia spp., o he
En e obac e iaceae, non- e men e bac e ia, and Ae omonas spp., om he Bei olas (BEI-DSR/EFL) and
Fa o (FNw-AFB/EFT) WWTPs. BEI-DSR: Bei olas-Lisboa in luen was ewa e ; BEI-EFL: Bei olas-
Lisboa e luen was ewa e ; FNw-AFB: Fa o No hwes -Alga e in luen was ewa e ; FNw-EFT: Fa o
No hwes -Alga e e luen was ewa e .
The g oup Esche ichia spp. was he majo i y in was ewa e isola es o bo h WWTPs,
con ibu ing o 57.1% o he o al o isola es s udied. Al hough he in luen om BEI-DSR
had a lowe numbe o Esche ichia spp. isola es (51.8%), he numbe o isola es iden i ied
in his in luen was highe (39.5%) han in he e luen BEI-EFL (25.9%), as well as om
he in luen FNw-AFB (27.8%) and e luen FNw-EFT (6.8%). I should be emphasized
ha Ae omonas spp. was ound in 35.7% o isola es om e luen was ewa e (FNw-EFT),
and was highe han in e luen was ewa e BEI-EFL, wi h 15.1% o isola es. A Fa o
WWTP, he las s age o e luen (FNw-EFT) was disin ec ion by ul a iole adia ion,
which could explain he p esence o Ae omonas spp. [
27
]. Among non- e men e isola es,
we should men ion some species ha we e ubiqui ous, no speci ically pa hogenic o
humans, bu possibly esponsible o in ec ions in he hospi al and in he communi y en i-
onmen , such as
Acine obac e spp.
(A. ge ne i,A. pi ii and A. nosocomialis), Comamonas spp.,
Och omobac um spp. and U ubu uella suis, a no el species wi hin he Neisse iaceae amily.
3.2.2. De ec ion and Sequencing o An ibio ic Resis ance Genes
Acco ding o he suscep ibili y esul s, 205 was ewa e isola es we e selec ed o
sc eening o esis ance de e minan s, which was pe o med by PCR wi h speci ic p ime s,
and a e sequencing, 183 esis an genes we e iden i ied in 97 isola es, as p esen ed in
Table 1.
The cha ac e iza ion o suscep ibili y o an ibio ics and esis an genes ound by
isola e is p esen ed in Table S3 (n= 97). Among he genes ha exp essed enzymes esponsi-
ble o esis ance o
β
-lac ams, he gene bla
CTX-M-15
was de ec ed in 37 isola es, ollowed by
bla
CTX-M-14
(n= 8) and bla
CTX-M-27
(n= 4), while he genes bla
CTX-M-1
,bla
CTX-M-2
,bla
CTX-M-32
,
and bla
CTX-M-174
we e de ec ed in one isola e each. The gene bla
SED-1
was iden i ied in
Ci obac e a me i and he gene bla
CTX-M-15
in Och omobac um in e medium. These genes
exp ess enzymes denomina ing he ex ended spec um o
β
-lac amases (ESBLs), con e ing
esis ance o hi d gene a ion cephalospo ins, and a e p e alen among En e obac e iaceae.
Addi ionally, in he
β
-lac amases amily, ca bapenemases ha con e esis ance o
ca bapenems can be ound. In his s udy, he KPC-3 enzyme in one K. oxy oca wi h he
bla
KPC-3
gene was iden i ied, which included a ansposon Tn4401 iso o m b. Fu he , his
bac e ium co-p oduced a ch omosomal
β
-lac amase OXY-2. This enzyme belongs o he
g oup o na ow-spec um
β
-lac amases, as well he OXA-2 p oduced by U ubu uella suis.
Mic oo ganisms 2021,9, 576 16 o 17
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