Occurrence and Genomic Characterization of Clone ST1193 Clonotype 14-64 in Uncomplicated Urinary Tract Infections Caused by Escherichia coli in Spain

Occurrence and Genomic Characterization of Clone ST1193 Clonotype 14-64 in Uncomplicated Urinary Tract Infections Caused by Escherichia coli in Spain Isidro García-Meniño, a , b , c Pilar Lumbreras, d , e Luz Lestón, a , b Mónica Álvarez-Álvarez, d Vanesa García, a , b Jens Andre Hammerl, c Javier Fernández, d , e , f , g Azucena Mora a , b a Laboratorio de Referencia de Escherichia coli (LREC), Dpto. de Microbioloxía e Parasitoloxía, Universidade de Santiago de Compostela (USC), Lugo, Spain b Instituto de Investigación Sanitaria de Santiago de Compostela (IDIS), Santiago, Spain c Department for Biological Safety, German Federal Institute for Risk Assessment, Berlin, Germany d Servicio de Microbiología, Hospital Universitario Central de Asturias (HUCA), Oviedo, Spain e Grupo de Microbiología Traslacional, Instituto de Investigación Sanitaria del Principado de Asturias (ISPA), Oviedo, Spain f Research & Innovation, Artificial Intelligence and Statistical Department, Pragmatech AI Solutions g CIBER de Enfermedades Respiratorias (CIBERES), Instituto de Salud Carlos III, Madrid, Spain Javier Fernández and Azucena Mora contributed equally to this work. Author order was determined based on seniority. ABSTRACT We conducted a prospective, multicenter, specific pilot study on uncomplicated urinary tract infections (uUTI). One-hundred non-duplicated uropathogenic Escherichia coli (UPEC) from uUTI occurred in 2020 in women attending 15 primary care centers of a single health region of northern Spain were characterized using a clonal diagnosis approach. Among the high genetic diversity showed by 59 different phylogroup-clonotype combinations, 11 clones accounted for 46% of the isolates: B2-ST73 (CH24-30); B2-ST73 (CH24-103); B2-ST131 (CH40-30); B2-ST141 (CH52-5); B2-ST372 (CH103-9); B2-ST404 (CH14-27); B2-ST404 (CH14-807); B2-ST1193 (CH14-64); D-ST69 (CH35-27); D-ST349 (CH36-54), and F-ST59 (CH32-41). The screening of the UPEC status found that 69% of isolates carried $3ofchuA, fyuA, vat,andyfcV genes. Multidrug resistance to at least one antibiotic of $3 antimicrobial categories were exhibited by 30% of the isolates, with the highest rates of resistance against ampicillin/amoxicillin (48%), trimethoprim (35%), norfloxacin (28%), amoxicillin-clavulanic acid (26%), and trimethoprim-sulfamethoxazole (24%). None extended-spectrum beta-lactamase/carbapenemase producer was recovered. According to our results, fosfomycin and nitrofurantoin should be considered as empirical treatment of choice for uUTI by E. coli (resistance rates 4% and 2%, respectively). We uncover the high prevalence of the pandemic fluoroquinolone-resistant ST1193 clone (6%) in uUTI, which represents the first report in Spain in this pathology. The genomic analysis showed similar key traits than those ST1193 clones disseminated worldwide. Through the SNP comparison based on the core genome, the Spanish ST1193 clustered with isolates retrieved from the Enterobase, showing high genomic similarity than the global ST1193 described in the United States, Canada and Australia. IMPORTANCE Analyzing the clonal structure and antimicrobial resistance of E. coli isolates implicated in uncomplicated urinary tract infections, one of the most frequent visits managed in primary health care, is of interest for clinicians to detect changes in the dynamics of emerging uropathogenic clones associated with the spread of fluoroquinolone resistance. It can also provide consensus concerning optimal control and antibiotic prescribing. KEYWORDS uncomplicated UTI (uUTI), antimicrobial resistance (AMR), UPEC, ST1193, ST131, fluoroquinolone resistance (FQR), Escherichia coli Editor Sadjia Bekal, Institut National de Santé Publique du Québec Copyright © 2022 García-Meniño et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Azucena Mora, [email protected]. The authors declare no conflict of interest. [This article was published on 23 May 2022 with an error in the Discussion section. The Discussion section text has been updated in the current version, posted on 26 May 2022.] Received 6 January 2022 Accepted 26 April 2022 Published 23 May 2022 May/June 2022 Volume 10 Issue 3 10.1128/spectrum.00041-22 1 RESEARCH ARTICLE Downloaded from https://journals.asm.org/journal/spectrum on 17 February 2023 by 90.164.14.193. Urinary tract infections (UTIs) are one of the most common diseases in both community and hospital settings. In the United States, UTIs were estimated in approximately 400,000 hospitalizations and $2.8 billion of costs in 2011 (1). In France, it was estimated in a mean of 70 efor one episode of a suspected UTI and 58 million from the societal perspective (2). In particular, uncomplicated urinary tract infections (uUTIs) are within the most frequent visits managed in primary health care, which implies a high impact on antibiotic prescription in ambulatory medicine (15% to 20%) (3–6). uUTI are referred as episodes of acute cystitis occurring in healthy, premenopausal, and nonpregnant women with no known structural or functional urological abnormalities of the genitourinary tract. The most frequent symptoms associated with uUTIs are strong and persistent urge to urinate, pelvic pain, burning sensation when urinating, and sign of blood in the urine (7). In Spain, a prospective study at two primary care practices in 2007 found that infectious diseases accounted for one third of the visits to a general practitioner. Among 4,353 infections observed in patients with a mean age of 44.1 years, UTIs were the third cause (452; 10.4%), after respiratory (2,196; 50.4%) and skin infections (586, 13.5%) (8). Escherichia coli is the most commonly etiological agent reported in UTIs (75% to 95%) (9). Unfortunately, there is a dramatic increase of multidrug resistance (MDR) within uropathogenic E. coli (UPEC), mainly due to a few globally disseminated clones such as ST131-H30R or ST1193-H64, which further compromises treatment (7, 10–16). While isolates causing complicated UTI have been widely analyzed and reported (17– 19), studies on uUTIs are still lacking because urine culture is rarely included in the routine diagnosis. In fact, at clinical level, the European Association of Urology and the Infectious Diseases Society of America (IDSA) recommends the treatment for uUTIs based on an empirical antimicrobial therapy guided by local susceptibility patterns without urine culture (20). Therefore, microbiological urine testing prior to treatment is only essential for patients with complicated UTIs (structural or functional abnormalities in genitourinary tracts), or when empirical therapy fails (10). In consequence, microbiological data lack for up to 70% of females with UTI symptoms. Based on a four-country primary care cohort study, Butler et al. (21) concluded that variation in management of uUTI at a country primary care network level is clinically unwarranted and highlights a lack of consensus concerning optimal symptom control and antibiotic prescribing. Based on what was stated above, we aimed the characterization of the predominant uropathogenic E. coli (UPEC) lineages associated with uUTIs to assess the importance of implementing specific surveillance programs to treat these infections. RESULTS Virulence status. The screening of virulence traits associated with a higher efficiency in the colonization of the urinary tract revealed that 69 of the 100 uUTI isolates conformed the UPEC status ($3ofspecific virulence traits chuA,fyuA,vat,andyfcV). Individually, chuA,fyuA,vat,andyfcV were present in 91%, 91%, 59%, and 67% of the isolates, respectively. Besides, the screening of iutA and kpsMII as prevailing genes within extraintestinal infections showed that 44% of the E. coli were carriers of both. Individually, they were detected in 55% and 80% of the isolates, respectively (Table S1). uUTI clonal groups. The eight E. coli phylogroups sensu stricto (A, B1, B2, C, D, E, F, and G) were represented among the 100 uUTI isolates. However, B2 and D were the most prevalent, accounting for 67% and 14% of the isolates, respectively. The remaining 19 uUTI E. coli belonged to A (5%), B1 (5%), E (3%), F (3%), C (2%), and G (1%). The predictor genes associated with a higher efficiency in the colonization of the urinary tract, and therefore positive for the UPEC status was found within the isolates belonging to phylogroups B2 (65 of 67 isolates), F (3), and G (1). Both iutA and kpsMII virulence traits were present in 44 isolates of phylogroups A (2 out of 5 isolates), B2 (32 of 67), D (7 of 14), and F (3) (Table S1). By clonotyping, 59 different phylogroup-clonotype (PG-CH) combinations were revealed, and those represented by $3 isolates were further investigated by multilocus sequence typing (MLST). As a result, 46% of the 100 uUTI isolates belonged to one ST1193 in Uncomplicated UTIs Microbiology Spectrum May/June 2022 Volume 10 Issue 3 10.1128/spectrum.00041-22 2 Downloaded from https://journals.asm.org/journal/spectrum on 17 February 2023 by 90.164.14.193. of the following 11 clones: B2-ST141 (CH52-5), six isolates; B2-ST1193 (CH14-64), six isolates; B2-ST73 (CH24-103), six isolates; B2-ST73 (CH24-30), three isolates; B2-ST372 (CH103-9), four isolates; B2-ST404 (CH14-27), four isolates; B2-ST404 (CH14-807), three isolates; B2-ST131 (CH40-30), three isolates; D-ST69 (CH35-27), five isolates; D-ST349 (CH36-54), four isolates; and F-ST59 (CH32-41), three isolates. Most isolates (36 of 46) belonging to nine of the 11 prevalent clones were carriers of $3 of specific traits chuA,fyuA,vat, and yfcV associated with higher virulence, and therefore, positive for the UPEC status; and only clones D-ST349 (CH-54) and D-ST69 (CH35-27) were characteristically negative. Regarding the presence or absence of both iutA and kpsMII,we observed a positive correlation with isolates belonging to clones B2-ST73 (CH24-30), B2-ST131 (CH40-30), B2-ST404 (CH14-27), B2-ST1193 (CH14-64), and F-ST59 (CH32-41). In addition to clonotype CH40-30, determined in three isolates, the pandemic ST131 clonal group appeared also associated to CH40-22 and CH40-1196 (one isolate each) (Table 1; Table S1). Antimicrobial susceptibility testing. Antimicrobial susceptibility testing (AST) showed the highest rates of resistance to ampicillin/amoxicillin (48%), ticarcillin (45%), piperacillin (41%), trimethoprim (35%), norfloxacin (28%), amoxicillin-clavulanic acid (26%), and trimethoprim-sulfamethoxazol (24%). Furthermore, 30% of the uUTI isolates were MDR, being in vitro resistant to at least one antibiotic of $3 antimicrobial categories (Table 2). It is important to note that none of the 100 uUTI isolates exhibited phenotypic expression of ESBL enzymes. Regarding colistin, two isolates were categorized as resistant by the MicroScan System but as susceptible by the standard BMD method (both isolates displayed MICs of 0.125 mg/L). The group of uUTI isolates positive for the UPEC status exhibited lower rates of MDR (18 of 69, 26%) but without significant differences in comparison to those negative for the UPEC status (13 of 31, 42%) (P= 0.16). Particularly among the 11 prevalent uUTI clones, the global rate of MDR was 34.8% (16 out of 46 isolates): namely, B2- ST131 (CH40-30) (three isolates), B2-ST1193 (CH14-64) (four of six isolates), D- ST349 (CH36-54) (three of four isolates), B2-ST73 (CH24-30) (two of three isolates), B2-ST372 (CH103-9) (two of four isolates), B2-ST73 (CH24-103) (one of five isolates), and D-ST69 (CH35-27) (one of five isolates) (Table 1). Fluoroquinolone-resistance (FQR) was exhibited by 28% of the 100 uUTI isolates belonging to 20 different PG-CH combinations, including the six isolates of clone B2- ST1193 (CH14-64), which were further analyzed by whole genome sequencing (WGS) due to the global emergence of this FQR and virulent lineage (Table 1; Table S1). In silico characterization of B2-ST1193 (CH14-64) isolates. Table 3 summarizes the main traits of the in silico characterization. All B2-ST1193 (CH14-64) isolates were subjected to WGS. However, one isolate that had less than 20 times coverage was excluded. SerotypeFinder predicted O75:H5 for all genomes but one (LREC-270), for TABLE 1 Prevalent clones ($3 isolates) within the 100 uUTI E. coli, virulence traits, MDR, and FQR Phylogroup-ST Clonotype (CH) No. isolates (N = 46) UPEC status (N = 36) iutA +kpsMII (N = 26) MDR (N = 16) FQR (N = 10) B2-ST141 CH52-5 6 6 0 0 0 B2-ST1193 CH14-64 6 6 6 4 6 B2-ST73 CH24-103 5 5 0 1 0 CH24-30 3 3 3 2 0 B2-ST372 CH103-9 4 4 0 2 1 B2-ST404 CH14-27 4 4 4 0 0 CH14-807 3 2 2 0 0 B2-ST131 a CH40-30 3 3 3 3 0 D-ST69 CH35-27 5 0 3 1 0 D- ST349 CH36-54 4 0 2 3 2 F-ST59 CH32-41 3 3 3 0 1 a Isolates belonging to the clonal group ST131 appeared associated to three clonotypes: CH40-30, CH40-22, CH40-1196 (with three, one, and one isolates, respectively). While only the three isolates of clone B2-ST131 (CH40-30) exhibited MDR, B2-ST131 (CH40-1193) showed FQR. ST1193 in Uncomplicated UTIs Microbiology Spectrum May/June 2022 Volume 10 Issue 3 10.1128/spectrum.00041-22 3 Downloaded from https://journals.asm.org/journal/spectrum on 17 February 2023 by 90.164.14.193. which O antigen was not typeable (NT:H5). Besides, MLST (seven genes) and CHTyper tools confirmed ST1193 (CH14-64) for all genomes. The alternative MLST scheme based on the eight genes dinB,icdA,pabB,polB,putP,trpA,trpB, and uidA (22) predicted ST53 (1-7-1-9-20-20-1-6) for all genomes but LREC-270 (ST53-like; trpA, 92% identity with allele 20). cgMLSTFinder also differenciated LREC-270, which was assigned as cgST7142, while LREC-265, 269, 273, and 275 were predicted as cgST4085. All ST1193 genomes presented the same set of chromosomal mutations conferring FQR (gyrA S83L, gyrA D87N, parC S80I, and parE L416F), which correlated with the in vitro expression. Additionally, other acquired resistance traits were predicted using CGE and CARD databases (Table 3, Table S2). Using VirulenceFinder and VFDB, all genomes but LREC-270 showed the same profile, including a wide number of extraintestinal virulence genes (Table 3, Table S3). Likewise, MobileElementFinder determined the same profile of mobile genetic elements in correlation with AMR and virulence traits except for LREC-270. TheplasmidomeanalysisrevealedthepresenceofanIncF[F-:A1:B10]plasmidtypeinfour of five genomes (all but LREC-270), together with small non-conjugative Col-like plasmids in four of them (Table 3). The five ST1193 genomes were predicted as human pathogen (probability .92%) when analyzed with PathogenFinder. Finally, CRISPRCasFinder found the same types/subtypes of CRISPR-Cas system I within LREC-269, 270, and 275 (Table 3). Enterobase (https://enterobase.warwick.ac.uk/) was searched as a source of genomes belonging to ST1193 (according to the Achtman 7-gene MLST scheme), predicted as cgST4085 and cgST72142, whose assemblies were retrieved for comparative TABLE 2 Rates of antimicrobial resistance interpreted according to EUCAST 2021 breakpoints Antimicrobial categories Antimicrobial agent % Resistant isolates N = 100 Penicillins Ampicillin/amoxicillin 48 Piperacillin 41 Ticarcillin 45 Antipseudomonal penicillins 1beta-lactamase inhibitors Piperacillin-tazobactam 5 Penicillins 1beta-lactamase inhibitors Amoxicillin/clavulanic acid 26 Non-broad spectrum cephalosporins: 1st & 2nd generation Cefuroxime 3 Broad-spectrum cephalosporins: 3rd & 4th generation Cefixime 1 Cefotaxime 0 Ceftazidime 0 Cefepime 0 Broad-spectrum cephalosporins1beta-lactamase inhibitor Ceftolozane-tazobactam 0 Ceftazidime-avibactam 0 Carbapenems Imipenem 0 Ertapenem 0 Meropenem 0 Monobactams Aztreonam 1 Fluroquinolones Norfloxacin 28 Ciprofloxacin 18 Levofloxacin 17 Aminoglycosides Gentamicin 17 Tobramycin 16 Amikacin 4 Glycylcyclines Tigecycline 0 Nitrofuran derivatives Nitrofurantoin 2 Phosphonic acids Fosfomycin 4 Folate pathway inhibitors Trimethoprim 35 Trimethoprim-sulfamethoxazole 24 Polymyxins Colistin 0 MDR (resistant to $one agent in $3 antimicrobial categories) 30 ST1193 in Uncomplicated UTIs Microbiology Spectrum May/June 2022 Volume 10 Issue 3 10.1128/spectrum.00041-22 4 Downloaded from https://journals.asm.org/journal/spectrum on 17 February 2023 by 90.164.14.193. TABLE 3 In silico characterization and phenotypic AMR of the uUTI ST1193 isolates ID code a O:H antigens b ST#1/ ST#2 c cgST d CHType e Acquired resistances and point mutations (in bold) f Plasmid content Inc. group [pMLST] g Virulence genes h Mobile genetic elements (& relation to AMR and virulence traits) i CRISPR-Cas system / Cas-type/subtype j Phenotypic AMR k 51107635 LREC-265 O75:H5 1193/ 53 4085 14-64 bla TEM-1B ;aph(39')-Ib, aph(6)-Id; mdf(A), sul2; sitABCD gyrA S83L, gyrA D87N, parC S80I, parE L416F IncF [F-:A1:B10] IncI1-I [ST Unknown] IncQ1* ColBS512-like chuA, fyuA, gad, iha, irp2, iucC, iutA, kpsE, kpsMII_K1, neuC, ompT, papA_F43, sat, senB, sitA, terC, usp, vat, yfcV ISEc42 (mdf(A), ompT); MITEEc1 (mdf(A), ompT); IS30 (sitABC, sitA); ISEc31 (iha); IS629 (papA_F43); MITEEc1 (fyaA, irp2, yfcV); MITEEc1 (terC) - AMP-AM; PIP; TIC; AMC; CIP; LEV; NOR 51140663 LREC-269 O75:H5 1193/ 53 4085 14-64 bla TEM-1B ;aph(39')-Ib, aph(6)-Id; mdf(A), mph(A), sul2, dfrA17; sitABCD gyrA S83L, gyrA D87N, parC S80I, parE L416F IncF [F-:A1:B10] IncQ1* ColBS512-like ColRNAI-like* chuA, fyuA, gad, iha, irp2, iucC, iutA, kpsE, kpsMII_K1, neuC, ompT, papA_F43, sat, senB, sitA, terC, usp, vat, yfcV ISEc42 (mdf(A)); MITEEc1 (mdf(A)); MITEEc1 (terC); IS30 (sitABCD, sitA); MITEEc1 (irp2, yfcV, fyaA); ISEc31 (iha); IS629 (papA F43); IS100 (iucC, sat, iutA) Class I / CAS-Type I/Cas3-0-I; CAS-Type IIIA/Csm2-1-IIIA AMP-AM; PIP; TIC; AMC; CIP; LEV; NOR; TMP; TMP-SMX 51144630 LREC-270 NT:H5 1193/ 53-like 72142 14-64 mdf(A); sitABCD gyrA S83L, gyrA D87N, parC S80I, parE L416F IncI1-I [ST Unknown] ColBS512-like ColMG828-like cia, fyuA,gad, iha, irp2, iucC, iutA, kpsE, kpsMII_K1, senB, terC, usp ISEc31 (iha) Class I / CAS-Type I/Cas3-0-I; CAS-Type IIIA/Csm2-1-IIIA CIP; LEV; NOR 51150996 LREC-273 O75:H5 1193/ 53 4085 14-64 mdf(A); sitABCD gyrA S83L, gyrA D87N, parC S80I, parE L416F IncF [F-:A1:B10] ColBS512-like ColMG828-like* Col156-like chuA, fyuA, gad, iha, irp2, iucC, iutA, kpsE, kpsMII_K1, neuC, ompT, papA_F43, sat, senB, sitA, terC, usp, vat, yfcV ISEc42 (mdf(A)); MITEEc1 (mdf(A)); MITEEc1 (terC); IS30 (sitABCD, sitA); ISEc31 (iha); IS629 (papA_F43, iutA, sat, iucC) - CIP; LEV; NOR (Continued on next page) ST1193 in Uncomplicated UTIs Microbiology Spectrum May/June 2022 Volume 10 Issue 3 10.1128/spectrum.00041-22 5 Downloaded from https://journals.asm.org/journal/spectrum on 17 February 2023 by 90.164.14.193. TABLE 3 (Continued) ID code a O:H antigens b ST#1/ ST#2 c cgST d CHType e Acquired resistances and point mutations (in bold) f Plasmid content Inc. group [pMLST] g Virulence genes h Mobile genetic elements (& relation to AMR and virulence traits) i CRISPR-Cas system / Cas-type/subtype j Phenotypic AMR k 51152710 LREC-275 O75:H5 1193/ 53 4085 14-64 bla TEM-1B ;aph(39')-Ib, aph(6)-Id;mdf(A), sul2, dfrA14;sitABCD gyrA S83L, gyrA D87N, parC S80I, parE L416F IncF [F-:A1:B10] IncQ1 chuA, fyuA, gad, iha, irp2, iucC, iutA, kpsE, kpsMII_K1, neuC, ompT, papA_F43, sat, senB, sitA, terC, usp, vat, yfcV ISEc42 (mdf(A)); MITEEc1 (mdf(A)); MITEEc1(terC); MITEEc1(irp2; fyuA); IS30 (sitABCD, sitA); ISEc31 (iha); IS629 (papA_F43, iutA, sat, iucC) Class I / CAS-Type I/Cas3-0-I; CAS-TypeIIIA/Csm2-1-IIIA AMP-AM; PIP; TIC; CIP; LEV; NOR; TMP; TMP-SMX; FOS a Isolate and genome (LREC) identification. b O and H antigen prediction with SerotypeFinder 2.0; c Sequence types (ST#1 and ST#2) based on two different MLST schemes were applied: E. coli #1 (67) and E. coli #2 (22), respectively, and retrieved with MLST 2.0.4. d Core genome ST obtained with cgMLSTFinder1.1. software run against the Enterobase database. e Clonotypes, f acquired antimicrobial resistance genes and/or chromosomal mutations, Resistome: acquired resistance genes: beta-lactam, bla TEM-1B ; aminoglycosides, aph(39')-Ib,aph(6)-Id; macrolides, mdf(A),mph(A); sulphonamides, sul2; trimethoprim, dfrA14,dfrA17; peroxide, sitABCD (mediates transport of iron and manganese and resistance to hydrogen peroxide). Point mutations: quinolones and fluoroquinolones, gyrA S83L, TCG-TTG; gyrA D87N, GAC-AAC; parC S80I, AGC-ATC; parE L416F, CTT-TTT. g replicon/plasmid sequence types, Plasmidome: *coverage ,100%. h virulence genes, and Virulence determinants: chuA, outer membrane hemin receptor; cia, colicin Ia; fyuA, siderophore receptor; gad, glutamate decarboxylase; iha, adherence protein; irp2, high molecular weight protein 2 nonribosomal peptide synthetase; iucC, aerobactin synthetase; iutA, ferric aerobactin receptor; kpsE, capsule polysaccharide export inner-membrane protein; kpsMII_K1, polysialic acid transport protein group 2 capsule; neuC, polysialic acid capsule biosynthesis protein; ompT, outer membrane protease (protein protease 7); papA_F43, major pilin subunit F43; sat, secreted autotransporter toxin; senB, plasmid-encoded enterotoxin; sitA, iron transport protein; terC, tellurium ion resistance protein; usp, uropathogenic specific protein; vat, vacuolating autotransporter toxin; yfcV,fimbrial protein. i the mobile genetic elements associated with AMR and virulence traits were also predicted using: CHtyper 1.0, ResFinder 4.1, PlasmidFinder 2.1, pMLST 2.0, VirulenceFinder 2.0 and MobileElementFinder 1.03 online tools at the Center of Genomic Epidemiology (http://www.genomicepidemiology.org/services/), respectively. j CRISPRCasFinder software (https://crisprcas.i2bc.paris-saclay.fr/) was used to identify and type CRISPR and Cas systems within the genomes. k Phenotypic resistances were interpreted according to EUCAST 2021 breakpoints. Antimicrobial abbreviation: PIP, piperacillin; TIC, ticarcillin; AMC, amoxicillin-clavulanic acid; AMP-AM, ampicillin-amoxicillin; CIP, ciprofloxacin; LEV, levofloxacin; NOR, norfloxacin; TMP, trimethoprim; TMP-SMX, trimethoprim-sulfamethoxazole; FOS, fosfomycin. ST1193 in Uncomplicated UTIs Microbiology Spectrum May/June 2022 Volume 10 Issue 3 10.1128/spectrum.00041-22 6 Downloaded from https://journals.asm.org/journal/spectrum on 17 February 2023 by 90.164.14.193. purposes. We found one cgST4085 genome (assembly code ESC_FA9684AA_AS) as part of BioProject PRJEB10018 registered in 2016 by the Sanger Institute. We also retrieved two of the three cgST72142 informed as isolates of human origin, Australia, 2007 (ESC_GB6748AA_AS) and 2008 (ESC_TA3850AA_AS) as part of BioProjects PRJNA487890 (submitted by University of Minnesota) and PRJNA493523 (submitted by the University of Queensland), respectively. The in silico analysis of these three genomes are summarized in Table S4, and show quite conserved traits regarding acquired resistances, the same set of chromosomal mutations (gyrA S83L, gyrA D87N, parC S80I, parE L416F), and a constant in the profile of virulence (chuA,fyuA,gad,iha, irp2,iucC,iutA,kpsE,kpsMII K1,neuC,ompT,papA_F43,sat,senB,sitA,terC,tratT,usp, vat,yfcV). Besides, PlasmidFinder confirmed the presence of an IncF [F-:A1:B10] plasmid type in the three genomes. The three ST1193 genomes retrieved from Enterobase were also predicted as human pathogen (probability .92%) when analyzed with PathogenFinder (Table S4). The genomic similarity of the cgST4085 and cgST72142 assemblies (five from the present study and three from Enterobase) were further investigated through the SNP comparison based on the core genome sequence (82.69% of the reference genome LREC-269) using CSI phylogeny 1.4 (Fig. 1). The phylogenetic dendrogram showed that seven of the eight ST1193 genomes clustered with only 15 to 103 SNP differences. The LREC-270 exhibited the maximum distance, ranging between 140 (in comparison with ESC_TA3850AA) to 204 SNPs (in comparison with LREC-273). DISCUSSION Despite the public health burden implications and derived costs, uUTIs are not systematically monitored, which also means a lack of data regarding the clonal diversity of the E. coli involved. Within scientific references, most studies do not differentiate complicated UTI from uUTI, or even from other extraintestinal infections. Likewise, it is not always possible to discriminate the origin of the isolate (hospital or primary health care centers), giving rise to misinterpretations. To avoid this possible bias, we addressed the characterization, using a clonal diagnosis approach, of nonduplicated E. coli isolates causing uUTI in women attending primary health care centers associated to the HUCA hospital under a specific pilot study on uUTI named URIPROAS. It is important to underlined that no set of genes has been found to unequivocally characterize ExPEC and the different categories within a group defined by its isolation from infections outside the intestinal tract. Many cases of bloodstream infections are preceded by an episode of UTI, and therefore UPEC are considered as a subgroup of ExPEC, which also comprise avian pathogenic E. coli (APEC) and neonatal meningitis E. coli (NMEC) (23). Nevertheless, certain virulence traits have been statistically associated with the pathogenic potential of the isolates, which can be used in a predictive way (24, 25) together with the identification of the so-called global extraintestinal clonal groups of E. coli such as ST131 (26). In this study, we used two concepts, namely, the pathogenic categorization and the status of the isolates. Thus, the 100 E. coli analyzed here, and clinically recovered from urine as the only pathogen involved in uUTI cases, are UPEC, defined as a subcategory of the ExPEC group. The second concept applied here is the status (UPEC and ExPEC) to differentiate, based on statistical criteria, isolates with higher pathogenic potential. Spurbeck et al. (25) found that E. coli isolates carrying vat,fyuA,chuA,and yfcV efficiently colonize the urinary tract. Their results indicated that a predictor gene (PG) score of 3 or 4 of those is indicative of status UPEC, and isolates with a PG score of 4 may be highly virulent. In addition, iutA and kpsM II genes are the most prevalent traits of the status ExPEC scheme (24) for positive isolates, and they are also prevailing in isolates causing extraintestinal infections (18, 27). The IDSA guidelines recommend trimethoprim-sulfamethoxazole if the local resistance prevalence in E. coli is #20%, and the use of nitrofurantoin and fosfomycin as next-choice antibiotics for uUTI. For pyelonephritis, empirical fluoroquinolone (FQ) treatment is suggested if local resistance prevalence in E. coli is #10% (7). In our study, ST1193 in Uncomplicated UTIs Microbiology Spectrum May/June 2022 Volume 10 Issue 3 10.1128/spectrum.00041-22 7 Downloaded from https://journals.asm.org/journal/spectrum on 17 February 2023 by 90.164.14.193. conducted in 2020, the resistance rates observed for fosfomycin (4%) and nitrofurantoin (2%) were similar to those reported for Spain by the ECOSENS European multicenter study on the antimicrobial resistance of uUTI-associated E. coli recovered between 1999 and 2000 (11), and by the ARESC (Antimicrobial Resistance Epidemiological FIG 1 Phylogenetic dendrogram based on the SNP counts per substitution within the core genome of the ST1193 isolates (five recovered in our study and three retrieved from Enterobase: ESC_FA9684AA_AS, ESC_TA3850AA_AS, ESC_GB6748AA_AS). The comparison of the WGS data sets resulting in a core genome represented by 82.69% of the reference genome (LREC-269). CSI phylogeny version 1.4 (CGE, https://cge.cbs.dtu .dk/services/CSIPhylogeny/; parameters used for phylogenetic analysis: min. depth at SNP positions 10 x; min. relative depth at SNP positions: 10 x; min. distance between SNPs (prune): 10 bp; min. SNP quality: 30; min. read mapping quality: 25, a min. Z-score of 1.96 and by ignoring heterozygous SNPs). ST1193 in Uncomplicated UTIs Microbiology Spectrum May/June 2022 Volume 10 Issue 3 10.1128/spectrum.00041-22 8 Downloaded from https://journals.asm.org/journal/spectrum on 17 February 2023 by 90.164.14.193. Survey on Cystitis) in a study conducted between 2003 and 2006 in nine European countries (28). We retrospectively checked resistance rates against the same antibiotics among 241 E. coli isolates recovered from women aged 18 to 45 years attending the same primary care centers from August to December of 2020. As a result, we found similar data to those reported for the 100 isolates prospectively studied, which further support the suggestion of fosfomycin and nitrofurantoin as empirical treatment of choice for uUTI by E. coli (data not shown). Furthermore, our results corroborate the stable resistance rates (;,4%) reported for both antibiotics in specific health areas of Spain such as Galicia, Navarra and Madrid (1999 to 2016) in E. coli implicated in UTI (29–31), which further support that these antibiotics should be considered as empirical treatment of choice by the community-acquired uUTI by E. coli. In contrast, high percentages of resistance to FQ and trimethoprim-sulfamethoxazole were described for Spain in those same reports (29–31). The variability of non-susceptible isolates to FQ ranged between 10.7% in 2003 to 2006 (28) and 36.6% in 2014 to 2016 (29). For trimethoprim-sulfamethoxazole, resistance ranged between 33.8% of the Spanish isolates within the multicenter European study (2003 to 2006) (28) and 25% of the E. coli analyzed in 2016 in Madrid (30). Within our uUTI collection, the resistance rates of trimethoprim-sulfamethoxazole (24%) and FQ (ciprofloxacin: 18%; levofloxacin: 17%; and norfloxacin: 28%) also exceed the limit recommended by the therapeutic guides for the use of these antibiotics as empirical treatments (7). Globally, similar circumstances have been described in different countries for UTIs caused by E. coli (uncomplicated or not), where the FQ resistance exceeds even 20%, complicating empirical treatment (18, 32, 33). Clonotype-level antibiograms can provide a more accurate guidance for empirical treatment than the standard based on species-level antibiograms, as previously showed by Tchesnokova et al. (34, 35) Furthermore, clonal antibiograms could be usable over the time and places due to their stability. But before a clinical implementation, the clonotyping test would benefit from the development of clonal reference databases for the follow up of the dynamics of predominant UTI E. coli lineages (32). In the present study, we defined the clonal structure of our collection by means of phylogrouping and clonotyping, followed by MLST for the most prevalent PG-CH combinations. In addition, we performed the screening of specific traits associated with the virulence status of isolates due to the strong correlation found previously between virulence-gene profiles and specificlineages of E. coli (18, 27, 36). From the hands-on time and cost point of view, this workflow seems a reasonable approach for the surveillance of emerging clones. As reported in different studies, the phylogenetic group B2 is the predominant within UTI isolates, even though other seven E. coli phylogroups sensu stricto appear represented to a greater or lesser extent (18, 33, 37, 38). Among the 59 different PG-CH combinations determined in our study, 11 E. coli clones comprised 46% of the 100 uUTI isolates. These 11 clones include major pandemic clonal lineages of ExPEC, such as ST73, ST141, ST131, ST69, or ST1193, which are globally responsible for up to 50% of all ExPEC-associated extraintestinal infections worldwide (in particular, UTIs, bloodstream infections, and health care-associated infections) (23, 26, 37, 39). Our findings can be compared with those resulting from the clonal characterization of 196 consecutively isolated E. coli causing UTIs and other extraintestinal infections during 2016 in Spain (100 from Lucus Augusti hospital, HULA, in Lugo) and France (96 from Beaujon hospital in Clichy) (18). Nine out of the 11 uUTI prevalent clones recovered in 2020 in our study were also present within the study collection of Flament-Simon et al. (18): B2-ST73 (CH24-103); B2-ST73 (CH24-30); B2-ST131 (CH40-30); B2-ST141 (CH52-5); B2- ST372 (CH103-9); B2-ST404 (CH14-27); B2-ST1193 (CH14-64); D-ST69 (CH35-27); and F-ST59 (CH32-41). A significant difference found between both studies is the prevalence rate of ST131, which accounted for 12% and 11.5% of the isolates in the Spanish (HULA) and French hospitals, respectively, versus 5% of the present study (health care centers associated to HUCA). In contrast, the MDR rates are quite similar (30% here versus 37.2% for the Spanish and French collection), with individual resistant prevalence .20%inbothstudies ST1193 in Uncomplicated UTIs Microbiology Spectrum May/June 2022 Volume 10 Issue 3 10.1128/spectrum.00041-22 9 Downloaded from https://journals.asm.org/journal/spectrum on 17 February 2023 by 90.164.14.193.