Higher prevalence of CTX-M-27-producing Escherichia coli belonging to ST131 clade C1 among residents of two long-term care facilities in Southern Spain

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ Esta es la versión aceptada del artículo publicado en: This is a accepted manuscript of a paper published in: European Journal of Clinical Microbiology & Infectious Diseases (2022) DOI: https://doi.org/10.1007/s10096-021-04380-6 Copyright: © 2025 Springer Nature El acceso a la versión publicada del artículo puede requerir la suscripción de la revista. Access to the published version may require subscription. “This version of the article has been accepted for publication, after peer review (when applicable) and is subject to Springer Nature’s AM terms of use, but is not the Version of Record and does not reflect post-acceptance improvements, or any corrections. The Version of Record is available online at: https://doi.org/10.1007/s10096-021-04380-6”. European Journal of Clinical Microbiology & Infectious Diseases Higher prevalence of CTX-M-27-producing Escherichia coli belonging to ST131 clade C1 among residents of two long-term care facilities in Southern Spain --Manuscript Draft-- Manuscript Number: EJCM-D-21-01098 Full Title: Higher prevalence of CTX-M-27-producing Escherichia coli belonging to ST131 clade C1 among residents of two long-term care facilities in Southern Spain Article Type: Original Article Keywords: CTX-M-27, ST131, long-term care facility, Corresponding Author: L. Lopez Cerero, M.D., Ph.D. University Hospital Virgen Macarena Seville, SPAIN Corresponding Author Secondary Information: Corresponding Author's Institution: University Hospital Virgen Macarena Corresponding Author's Secondary Institution: First Author: L. Lopez Cerero, M.D., Ph.D. First Author Secondary Information: Order of Authors: L. Lopez Cerero, M.D., Ph.D. Elena Salamanca, MD, PhD Mercedes Delgado-Valverde José Manuel Rodríguez-Martínez, PhD Jesús Rodríguez-Baño Álvaro Pascual Order of Authors Secondary Information: Funding Information: ministerio de economía, industria y competitividad, gobierno de españa (RD16/0016/0001) Dr. Jesús Rodríguez-Baño joint programming initiative on antimicrobial resistance (AC16/00072) Not applicable european regional development fund (Operative program Intelligent Growth 2014‐2020) Not applicable Abstract: Purpose Clone ST131, one of the prevalent clones of Escherichia coli in infections caused by both quinolone-resistant and ESBL-producing isolates, has two main clades: C1, which to date has not produced ESBLs in our environment, and C2, related to the international spread of CTX-M-15. The objective was to study the prevalence of both clades among residents of LTCFs in our region. Methods A carrier prevalence study was conducted in two LTCFs in Seville, Spain. Specific PCR was used to detect ST131 in fluoroquinolone-resistant E. coli isolates recovered from rectal swabs of 86 individuals. Genetic relatedness was studied by XbaI PFGE, and one isolate belonging to each pulsotype was subjected to massive sequencing. Resistance determinants, plasmids, fimH , virulence factors, clade and cgST were characterized for all genomes. Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation Results Twenty-eight (33%) participants were colonized with fluoroquinolone-resistant E. coli ST131 and 46% were also resistant to third-generation cephalosporins (7 CTX-M-27 producers, 3 CTX-M-15 producers, 2 CMY-2 producers and 1 both CTX-M-15 and CMY-2 producer. C1 isolates were more common than C2 in both LTCFs and also more frequently produced bla ESBL/ pAmpC genes than C2 (53% vs 33%). In half the cases, transmission and/or exposure to a common source was considered to have been recent. Genetic environment and absence of the M27PP1 region differentiated CTX-M-27 producers from the Japanese C1-M27 lineage. Conclusion This is the first time that C1-CTX-M-27 ST131 isolates have been more prevalent than C2-CTX-M-15 among LCTF residents in our country and a high transmission rate was observed. Suggested Reviewers: Marek Gniadkowski Central Laboratory of Sera and Vaccines: Narodowy Instytut Lekow [email protected] Stéphane Bonacorsi Assistance Publique Hopitaux de Paris: Assistance Publique - Hopitaux de Paris [email protected] Trinad Chakraborty Institute of Medical Microbiology Giessens [email protected] Yasufumi Matsumura Kyoto University Graduate School of Medicine Faculty of Medicine: Kyoto Daigaku Daigakuin Igaku Kenkyuka Igakubu [email protected] Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation 1 Title: Higher prevalence of CTX-M-27-producing Escherichia coli belonging to 1 ST131 clade C1 among residents of two long-term care facilities in Southern 2 Spain 3 4 Authors: Lorena López-Cerero1,2*, Elena Salamanca1, Mercedes Delgado-Valverde1, 5 José Manuel Rodríguez-Martínez3, Jesús Rodríguez-Baño1,3, Álvaro Pascual1,2. 6 7 Affiliation: 8 1) Unidad Clínica de Enfermedades Infecciosas y Microbiología. 9 Instituto de Biomedicina de Sevilla (IBiS). Hospital Universitario 10 Virgen Macarena, Sevilla, Spain. 11 2) Departamento de Microbiología, Universidad de Sevilla, Sevilla, Spain. 12 3) Departamento de Medicina, Universidad de Sevilla, Sevilla, Spain. 13 14 15 *Corresponding author: Lorena López- Cerero: [email protected], Phone number: 16 +34954558263 17 18 19 Manuscript Click here to access/download;Manuscript;clade CTX-M-27 borrador 20210621 REVISED JRB_MDV.docx Click here to view linked References 2 Abstract 20 Purpose: Clone ST131, one of the prevalent clones of Escherichia coli in infections 21 caused by both quinolone-resistant and ESBL-producing isolates, has two main clades: 22 C1, which to date has not produced ESBLs in our environment, and C2, related to the 23 international spread of CTX-M-15. The objective was to study the prevalence of both 24 ST131 clades among residents of LTCFs in our region. 25 Methods: A carrier prevalence study was conducted in two LTCFs in Seville, Spain. 26 Specific PCR was used to detect ST131 in fluoroquinolone-resistant E. coli isolates 27 recovered from rectal swabs of 86 individuals. Genetic relatedness was studied by XbaI 28 PFGE, and one isolate belonging to each pulsotype was subjected to massive 29 sequencing. Resistance determinants, plasmids, fimH, virulence factors, clade and cgST 30 were characterized for all genomes. 31 Results: Twenty-eight (33%) participants were colonized with fluoroquinolone-resistant 32 E. coli ST131 and 46% were also resistant to third-generation cephalosporins (7 CTX- 33 M-27 producers, 3 CTX-M-15 producers, 2 CMY-2 producers and 1 (both CTX-M-15 and 34 CMY-2 producer). C1 isolates were more common than C2 in both LTCFs and also more 35 frequently produced blaESBL/pAmpC genes than C2 (53% vs 33%). In half the cases, 36 transmission and/or exposure to a common source was considered to have been recent. 37 Genetic environment and absence of the M27PP1 region differentiated CTX-M-27 38 producers from the Japanese C1-M27 lineage. 39 Conclusion: This is the first time that C1-CTX-M-27 ST131 isolates have been more 40 prevalent than C2-CTX-M-15 among LTCF residents in our country and a high 41 transmission rate was observed. 42 Key words 43 CTX-M-27, ST131, long-term care facility 44 3 Introduction 45 Escherichia coli clonal complex ST131 is a member of the virulence-associated 46 phylogenetic group B2, which was recognized as a globally disseminated clone in 2008 47 due to the shared characteristics of CTX-M-15-producing isolates from three continents 48 (1). The study of the population structure of this complex distinguished 3 clades, each 49 associated with a particular fimH sequence: clade A with fimH41, clade B with fimH22, 50 and clade C with fimH30. Most epidemiological studies showed that clade C was 51 predominant overall and was associated with fluoroquinolone resistance (1). Using a 52 whole genome sequencing approach, two subclades were differentiated: C1 (non-ESBL 53 producer) and C2 (associated with the ESBL CTX-M-15) (2) . 54 During the late 2000s, blaCTX-M-15 predominated worldwide in the C2/H30-Rx group (97% 55 prevalence) and the C1 isolates were not associated with ESBL, although a new lineage 56 emerged in Japan in clade C1. When Matsumura et al (3) analyzed ST131 isolates 57 between 2001 and 2012 from Japan, they found that blaCTX-M-27 predominated in the 58 C1/H30R group (52% prevalence) and that this new lineage harbored a 11,894-bp 59 phage-specific region, named M27PP1 and more frequently, the senB enterotoxin. This 60 C1 lineage was also recently detected in Europe and some reports indicate that its 61 prevalence is increasing. In a study conducted between 2010 and 2015, Birgy et al (4) 62 found the CTX-M-27–producing E. coli ST131/fimH30 subgroup in one third of ESBL- 63 producing E. coli ST131 isolated from 1,886 children in France. In another study carried 64 out in 492 elderly patients admitted to two geriatric rehabilitation wards in Tel-Aviv (Israel) 65 between October 2008 and May 2009, Adler et al (5) found that in-hospital dissemination 66 was highest in CTX-M-27-producing ST131 compared to CTX-M-39-producing ST398, 67 CTX-M-15-producing ST131 and SHV-5-producing ST372. Meanwhile, Ghosh et al (9) 68 found blaCTX-M-27 in 15% of 953 sequenced isolates from a collection of 4,386 69 nonrepetitive ESBL-producing E. coli in Germany and its incidence increased from 0% 70 in 2009 to 45% in 2016 (6). 71 4 In several studies carried out in Spain, the C2/CTX-M-15 lineage predominated in clinical 72 isolates (7) (8). Different prevalence rates of C1/CTX-M-27-producing E. coli were found 73 in colonized patients in four European hospitals between March 2014 and February 74 2015, with C2/CTX-M-15 continuing to be the most prevalent among Spanish isolates 75 (8). Our objective was to study the current prevalence of these two subclades among 76 residents of long-term care facilities (LTCF) in our region, using the trait of 77 fluoroquinolone resistance common to both clades. 78 79 Material and methods 80 We conducted a carrier prevalence study among residents of two long-term care facilities 81 (LTCF) in the area of Seville (southern Spain). LTCF-A has capacity for 92 residents, 82 mostly (66) in double rooms with shared bathroom. LTCF-B has capacity for 50 83 residents; there are some single and double rooms and many users have individual 84 bathrooms. The study was carried out during the months of March and April 2018 and 85 was approved by the local institutional ethics committee. Informed consent was obtained 86 from participating residents, or their relatives in case of severe cognitive impairment; a 87 waiver for informed consent was obtained for those with severe cognitive impairment 88 and no family members A rectal or stool sample was taken from all participating 89 residents, and clinical and epidemiological data were collected. The samples were taken 90 either from the stool or directly from the rectum, always using FecalSwabs (Copan®). 91 Each sample was inoculated on UTI agar (Oxoid®) with 2 mg/L ciprofloxacin after an 18 92 h broth enrichment step. All morphologically different colonies of E. coli per plate were 93 selected for further analysis. ST131 screening was carried out by PCR, using specific 94 primers for O25b rfb, allele 3 of the pabB genes and phylogroup B23 typing (8). 95 Susceptibility testing was performed using Microscan WalkAway panels (Beckman 96 Coulter®). 97 5 Genetic relatedness of all O25b/pabB3/B23 positive isolates was initially performed by 98 XbaI PFGE analysis (http://www.pulsenetinternational.org/protocols/pfge.asp). 99 Dendrograms were created with BioNumerics 7.5 software (Applied Maths, BioMérieux), 100 using the Dice coefficient and the unweighted pair group method (UPGM) (position 101 tolerance 1%). Isolates exhibiting >1-band difference (corresponding to >94% Dice index 102 similarity) were assigned to different pulsotypes(9). One isolate from each pulsotype was 103 selected for further whole genome sequencing; when different susceptibility patterns 104 were found within a pulsotype, a representative of each antibiotic resistance profile was 105 also analysed. Draft genomes were generated using the Nextera Plus DNA sample 106 preparation kit and Illumina MiSeq 2000 reads and were assembled de novo with CLC 107 genomics workbench software. Genomes were sequenced at 30x average depth. 108 Assemblies were characterized using the following web-based tools: Resfinder 3.2 (10) 109 and CARD (11) to detect resistance determinants, using the 100% identity threshold for 110 beta-lactamase genes and 98% for other genes; Plasmidfinder 2.1 (12) to identify type 111 of plasmid content; MLSTFinder 2.0 (13), cgSTFinder and FimHTyper 1.0 to assign ST, 112 cgST and fimH type, respectively. VirulenceFinder (14) was used to search for virulence 113 genes, and the pMLST tool (12) for plasmid multilocus sequence typing analysis; in both 114 cases, an identification rate of 98% was considered positive for target genes. Virotypes 115 were determined using Dahby’s scheme (15) and were considered ExPEC when 2 or 116 more markers were present: papA and/or papC, afa/dra, sfa/foc, iutA, kpsMT II. The 117 genetic environment of blaESBL was studied by homology search using Blast and the 118 genetic structures classified by Matsumura et al (3). 119 Whole-genome typing was conducted with the E. coli/Shigella EnteroBase database 120 scheme (http://enterobase.warwick.ac.uk/species/ecoli) to generate GrapeTree figures 121 using the neighbour-joining (NJ) algorithm, and hierarchical clustering was developed 122 using cgMLST V1 (Hiercc), using the Silhouette index to assign the HC0 level. Strain 123 sharing was considered when 2 isolates showed an identical PFGE type, or if they 124 6 differed by at least one band with identical HC0, together with identical content in terms 125 of resistance determinants, virulence factors (9) and plasmid replicons. 126 In order to identify the clade, a whole genome single-nucleotide polymorphism (SNP)– 127 based phylogenetic analysis was conducted, comparing reads of previously clade- 128 characterized genomes from Matsumura’s study (3), including representative strains of 129 clades A, B, B0, C1, C1-M27, C2 and C0, with the 21 isolates sequenced in our study, 130 aligned against a clade A strain (Accession No. ERR161245) as reference genome with 131 the SNP tool in Enterobase. In order to confirm subclade C assignation, in silico PCR 132 was carried out, using the primers proposed by Matsumura et al. (16). A BLAST search 133 was performed for the M27PP1 region specific to clade 1 using strain KUN5781 (3) as 134 reference. Percentages were compared using Chi-square with Yates correction, and 135 p<0.05 was considered significant. 136 137 Results 138 During March and April 2018, 55 individuals in LTCF-A and 31 in LTCF-B signed 139 informed consent forms to participate. Quinolone-resistant E. coli ST131 was isolated 140 from 28 of 86 participants (33%): 19 in LTCF-A (68%) and 9 in LTCF-B (32%). All isolates 141 showed high-level fluoroquinolone resistance and double mutations in both gyrA (S83L, 142 D87N) and parC (S80I + E84V), and belonged to fimH30, consistent with the fact that 143 the isolates had been selected with a high concentration of ciprofloxacin. Thirteen of 144 them (46%) were resistant to 3rd generation cephalosporins: 7 (25%) were CTX-M-27 145 producers, 3 (11%) were CTX-M-15 producers, 2 (7%) were CMY-2 producers and 1 146 (4%) was both a CTX-M-15 and a CMY-2 producer. All isolates were ExPEC and the C2 147 virotype predominated in both centres: 14 (74%) and 6 (67%) strains in LTCF-A and 148 LTCF-B, respectively (Table 1). 149 13 Sequencing Data. J Clin Microbiol 58:1–13. 306 15. Dahbi G, Mora A, Mamani R, et al (2014). Molecular epidemiology and virulence of 307 Escherichia coli O16: H5-ST131: Comparison with H30 and H30-Rx subclones of O25b: 308 H4-ST131. Int J Med Microbiol 304:1247–1257. 309 16. Matsumura Y, Pitout JDD, Peirano G, et al (2017). Rapid identification of different 310 Escherichia coli sequence type 131 clades. Antimicrob Agents Chemother 61:1–9. 311 17. Darphorn TS, Bel K, Koenders-van Sint Anneland BB, et al (2021). Antibiotic resistance 312 plasmid composition and architecture in Escherichia coli isolates from meat. Sci Rep 313 11:1–13. 314 18. Shirakawa T, Sekizuka T, Kuroda M, et al (2020). Comparative Genomic Analysis of 315 Third-Generation- Cephalosporin-Resistant Escherichia coli Harboring the blaCMY-2- 316 Positive IncI1 Group, IncB/O/K/Z, and IncC Plasmids Isolated from Healthy Broilers in 317 Japan. Antimicrob Agents Chemother 64:1–13. 318 19. Flament-Simon SC, García V, Duprilot M, et al (2020). High Prevalence of ST131 319 Subclades C2-H30Rx and C1-M27 Among Extended-Spectrum β-Lactamase-Producing 320 Escherichia coli Causing Human Extraintestinal Infections in Patients From Two 321 Hospitals of Spain and France During 2015. Front Cell Infect Microbiol 10:1–9. 322 20. Merino I, Hernández-García M, Turrientes MC, et al (2018). Emergence of ESBL- 323 producing Escherichia coli ST131-C1-M27 clade colonizing patients in Europe. J 324 Antimicrob Chemother 73:2973–2980. 325 21. Nobrega D, Peirano G, Lynch T, et al (2021). Spatial distribution of Escherichia coli 326 ST131 C subclades in a centralized Canadian urban region . J Antimicrob Chemother 327 76:1135-1139. 328 22. Overdevest I, Haverkate M, Veenemans J, et al (2016). Prolonged colonisation with 329 Escherichia coli O25:ST131 versus other extended-spectrum beta-lactamase-producing 330 Escherichia coli in a long-term care facility with high endemic level of rectal colonisation, 331 the Netherlands, 2013 to 2014. Eurosurveillance 21:30376. 332 14 23. Madigan T, Johnson JR, Clabots C, et al (2015). Extensive household outbreak of 333 urinary tract infection and intestinal colonization due to extended-spectrum β-lactamase- 334 producing Escherichia coli sequence type 131. Clin Infect Dis 61:e5–e12. 335 24. Reeves PR, Liu B, Zhou Z, et al (2011). Rates of mutation and host transmission for an 336 Escherichia coli clone over 3 years. PLoS One 6:e26907. 337 338 339 15 Table 1. Distribution of virotypes and resistance determinants of the E. coli ST131 lineages in two LTCFs Centre clade cgST1HC02 Resistance pattern No. Isolates ESBL/pAmpC bla OXA-1 bla TEM-1 aac(6´)-Ib-cr Other determinants IncF RST Other plasmids Virotype3 LTCF-A C1 11088 131873 5 bla CTX-M-27 aadA5, aph(3´´)-I, aph(6)-Id, su1, sul2, dfr, tetA F1:A2:B20 (2), F2:A2:B20 (3) col C2 131868 2 Negative 1 None F1:A2:B20 IncX4 C2 131877 1Negative 1 None F49:A-:B- C2 C1 131561 2Negative None F4:A2:B20 IncI1, Col C2 131562 R1 1Negative qnrS1, sul1, dfr, aadA5 F4:A2:B20 IncX4, IncB/O/K/Z, Col C2 R2 1Negative 1 aac(3´)-II F4:A2:B20 IncX4, IncB/O/K/Z, IncN, IncI1, Col C2 R3 1Negative 1 aadA5, aph(3´´)-I, aph(6)-Id, aac(3´)_II, sul, dfr, tetA F4:A2:B20 IncX4, IncB/O/K/Z, IncN, IncI1, Col C2 131560 1Negative 1 aadA5, aph(3´´)-I, aph(6)-Ic, aac(3´)-II, sul1, sul2, dfr, tetA F4:A2:B20 IncI1, IncN C2 C2 10134 131564 1 bla CTX-M-15 1 1 aadA5, sul1, dfr, tetA F2:A1:B- D3 C2 115041 131563 2 bla CTX-M-15 1 1 aadA5, sul1, dfr F2:A1:B- D3 131876 1Negative 1 1 qnrS1, dfr F2:A1:B- IncN D3 131872 1Negative None F2:A1:B- D3 LTCF-B C1 8116 131905 1 bla CTX-M-27 aadA5, aph(3´´)-I, aph(6)-Id, su1, sul2, tetA F1:A2:B10 C2 C1 116883 131906 1 bla CTX-M-27 aadA5, aph(3´´)-I, aph(6)-Id, su1, sul2, dfr, tetA F-:A2:B20 C2 C1 131879 132351 1 bla CTX-M-15, bla CMY-2 1 1 aac(3)-II F1:A2:B20 IncB/O/K/Z, Col C3 C1 33578 131879 2 bla CMY-2 None F1:A2:B20 IncB/O/K/Z C3 C2 7808 131904 3Negative 3 3 aadA5, aph(3´´)-II, aac(3)-II, sul1, sul2, dfr, tetA F1:A1:B16 C2 131907 1Negative tetA F1:A1:B16 C2 1 according to cgSTFinder 2 according to hierarchical clustering HierCC0 of cgMLST data of Enterobase scheme 3 according to scheme proposed by Dhaby et al. 40943 16 Figure 1. Whole-genome based grape trees of E. coli ST131 from the two LTCFs using the wgMLST scheme of Enterobase. Node colour indicates the relevant third-generation cephalosporin resistance determinants. The numbers on the branches of the tree express the number of alleles of difference. Circles with a dashed line encompass genomes with the same cgST according to cgSTFinder. Circles with a solid line encompass genomes with the same cgST according to the HierCC0 hierarchy of the Enterobase scheme. Numbers in red indicate the number of isolates with an identical PFGE profile corresponding to that genome. The clades are indicated at the beginning of the branch. 17 Figure S1. Single-nucleotide polymorphism (SNP)-based phylogenetic tree of Escherichia coli ST131 isolates. The reference genome was a clade A isolate (Accession No. ERR161245). Node colour indicates country of isolation and red stars indicate the sequenced isolates in this study. A dashed blue line separates clade C1 from C2.