An update on wastewater multi-resistant bacteria : identification of clinical pathogens such as Escherichia coli O25b:H4-B2-ST131-producing CTX-M-15 ESBL and KPC-3 Carbapenemase-Producing Klebsiella oxytoca

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 12. ISO. Wa e Quali y-Enume a ion o Esche ichia coli and Coli o m Bac e ia—Pa 1: Memb ane Fil a ion Me hod o Wa e s wi h Low Bac e ial Backg ound Flo a. In In e na ional S anda d ISO 9308-1; In e na ional O ganiza ion o S anda diza ion: Gene a, Swi ze land, 2014. 13. Wa e Resea ch Aus alia Wa e o he wellbeing o all Aus alians. Log Remo al Values in Was ewa e T ea men . 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