Genomic characterization of prevalent mcr-1, mcr-4, and mcr-5 Escherichia coli within swine enteric colibacillosis in Spain

Antimicrobial agents are crucial for the treatment of many bacterial diseases in pigs, however, the massive use of critically important antibiotics such as colistin, fluoroquinolones and 3rd–4th-generation cephalosporins often selects for co-resistance. Based on a comprehensive characterization of 35 colistin-resistant Escherichia coli from swine enteric colibacillosis, belonging to prevalent Spanish lineages, the aims of the present study were to investigate the characteristics of E. coli clones successfully spread in swine and to assess the correlation of the in vitro results with in silico predictions from WGS data. The resistome analysis showed six different mcr variants: mcr-1.1; mcr-1.10; mcr-4.1; mcr-4.2; mcr-4.5; and mcr-5.1. Additionally, blaCTX–M–14, blaCTX–M–32 and blaSHV–12 genes were present in seven genomes. PlasmidFinder revealed that mcr-1.1 genes located mainly on IncHI2 and IncX4 types, and mcr-4 on ColE10-like plasmids. Twenty-eight genomes showed a gyrA S83L substitution, and 12 of those 28 harbored double-serine mutations gyrA S83L and parC S80I, correlating with in vitro quinolone-resistances. Notably, 16 of the 35 mcr-bearing genomes showed mutations in the PmrA (S39I) and PmrB (V161G) proteins. The summative presence of mechanisms, associated with high-level of resistance to quinolones/fluoroquinolones and colistin, could be conferring adaptive advantages to prevalent pig E. coli lineages, such as the ST10-A (CH11-24), as presumed for ST131. SerotypeFinder allowed the H-antigen identification of in vitro non-mobile (HNM) isolates, revealing that 15 of the 21 HNM E. coli analyzed were H39. Since the H39 is associated with the most prevalent O antigens worldwide within swine colibacillosis, such as O108 and O157, it would be probably playing a role in porcine colibacillosis to be considered as a valuable subunit antigen in the formulation of a broadly protective Enterotoxigenic E. coli (ETEC) vaccine. Our data show common features with other European countries in relation to a prevalent clonal group (CC10), serotypes (O108:H39, O138:H10, O139:H1, O141:H4), high plasmid content within the isolates and mcr location, which would support global alternatives to the use of antibiotics in pigs. Here, we report for first time a rare finding so far, which is the co-occurrence of double colistin-resistance mechanisms in a significant number of E. coli isolatesThis study was supported by projects PI16/01477 from Plan Estatal de I+D+I 2013–2016, Instituto de Salud Carlos III (ISCIII), Subdirección General de Evaluación y Fomento de la Investigación, and FEDER; AGL2016-79343-R from the Agencia Estatal de Investigación (AEI, Spain) and FEDER; ED431C 2017/57 from the Consellería de Cultura, Educación e Ordenación Universitaria (Xunta de Galicia) and FEDER. IG-M and VG acknowledge the Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia for their pre-doctoral and post-doctoral grants (Grant Numbers ED481A-2015/149 and ED481B-2018/018, respectively). SF-S acknowledges the FPU programme from the Secretaría General de Universidades, Ministerio de Educación, Cultura y Deporte, Gobierno de España (Grant Number FPU15/02644)S

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

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 ≥ 3 of chuA, fyuA, vat, and yfcV 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 prescribingThis study was supported by the projects and funds PID2019-104439RB-C21/AEI/10.13039/501100011033 from the Agencia Estatal de Investigación (AEI, Spain), co-funded by the European Regional Development Fund of the European Union: A Way to Make Europe (ERDF); FIS PI17-00728 (Fondo de Investigación Sanitaria, Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, Spain), co-funded by ERDF; GRUPIN IDI/2022/000033 by the Regional Ministry of Science of Asturias (IDI/2022/000033). ED431C 2021/11 from the Consellería de Cultura, Educación e Ordenación Universitaria (Xunta de Galicia) and ERDF. I.G-M. and V.G. acknowledge the Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia for their post-doctoral grants (Grant Number ED481B-2021-006 and ED481-B2018/018, respectively). The research stay of I.G-M at the Hospital Universitario Central de Asturias was funded by a grant from the Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica (SEIMC). L.L. acknowledges the Ministry of Education of Spain for her predoctoral grant FPU19/01127.S

Whole Genome Sequencing and Characteristics of mcr-1–Harboring Plasmids of Porcine Escherichia coli Isolates Belonging to the High-Risk Clone O25b:H4-ST131 Clade B

Porcine Escherichia coli ST131 isolates are scarcely documented. Here, whole genome sequencing and core genome (CG) and plasmidome analysis of seven isolates collected from diarrheic piglets and four from pork meat were performed. All of the 11 ST131 isolates belonged to serotype O25b:H4 and clade B and showed fimH22 allele or mutational derivatives. The 11 porcine isolates possessed virulence traits that classified the isolates as avian pathogenic, uropathogenic, and extraintestinal pathogenic E. coli–like (APEC-, UPEC-, and ExPEC-like) and constituted virotype D. The CG was performed for all porcine isolates in addition to 73 ST131 reference isolates from different origins. Within clade B, the CG showed nine subclusters, allowing us to describe five new subclades (B6, B6-like, B7, B8, and B9). There was an association between subclade B6, PST43, virotype D2, and food origin, whereas subclade B7 included PST9 isolates with virotype D5 from diarrheic piglets (p = 0.007). The distance between human and porcine isolates from subclades B6 and B7 had an average of 20 and 15 SNP/Mb, respectively. [F2:A-:B1]-IncF, ColE1-like, and IncX plasmids were the most prevalent. Besides, IncF plasmids harbored a ColV region frequent among APEC isolates. Antimicrobial resistance genes conferring resistance to penicillin, tetracycline, quinolones, and colistin were the most common. The mcr-1.1 gene was detected in 5 of 11 porcine isolates, integrated into the chromosome of one isolate and into plasmids in the remainder isolates (two MOBH11/IncHI2-ST4, one MOBP3/IncX4, and one MOBF12/IncF [F2:A-:B1] supposedly cointegrated with an IncHI2). The surrounding environments of the mcr-1 cassette showed variability. However, there were conserved structures within the same plasmid family. In conclusion, CG analysis defined five new subclades. The ST131 porcine isolates belonged to new subclades B6 and B7. Moreover, porcine and clinical human isolates were strongly related. The 11 porcine ST131 isolates harbored a wide variety of plasmids, virulence, and resistance genes. Furthermore, epidemic plasmids IncX4 and IncHI2 are responsible for the acquisition of mcr-1.1 gene. We hypothesize that the APEC-IncF plasmid acquired the mcr-1.1 gene via cointegrating an IncHI2 plasmid, which is worrying due to combination of virulence and resistance attributes in a single mobile genetic elementS-CF-S acknowledges the FPU programme for her grant (FPU15/02644) from the Secretaría General de Universidades, Spanish Ministerio de Educación, Cultura y Deporte. IG-M and VG acknowledge the Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia for his predoctoral grant (ED481A-2015/149) and her postdoctoral grant (ED481B2018/018), respectively. AM acknowledges the Ministerio de Educación, Cultura y Deporte (Spain) for the mobility grant PRX16/00023 for teachers and researchers from the Programa Estatal de Promoción del Talento y su Empleabilidad, Plan Estatal de Investigación Científica y Técnica y de Innovación 2013–2016S

Genomic characterization of escherichia coli isolates belonging to a new hybrid aepec/expec pathotype o153:H10-a-st10 eae-beta1 occurred in meat, poultry, wildlife and human diarrheagenic samples

Different surveillance studies (2005–2015) in northwest Spain revealed the presence of eae-positive isolates of Escherichia coli O153:H10 in meat for human consumption, poultry farm, wildlife and human diarrheagenic samples. The aim of this study was to explore the genetic and genomic relatedness between human and animal/meat isolates, as well as the mechanism of its persistence. We also wanted to know whether it was a geographically restricted lineage, or whether it was also reported elsewhere. Conventional typing showed that 32 isolates were O153:H10-A-ST10 fimH54, fimAvMT78, traT and eae-beta1. Amongst these, 21 were CTX-M-32 or SHV-12 producers. The PFGE XbaI-macrorestriction comparison showed high similarity (>85%). The plasmidome analysis revealed a stable combination of IncF (F2:A-:B-), IncI1 (STunknown) and IncX1 plasmid types, together with non-conjugative Col-like plasmids. The core genome investigation based on the cgMLST scheme from EnteroBase proved close relatedness between isolates of human and animal origin. Our results demonstrate that a hybrid MDR aEPEC/ExPEC of the clonal group O153:H10-A-ST10 (CH11-54) is circulating in our region within different hosts, including wildlife. It seems implicated in human diarrhea via meat transmission, and in the spreading of ESBL genes (mainly of CTX-M-32 type). We found genomic evidence of a related hybrid aEPEC/ExPEC in at least one other countryThis study was supported by projects: AGL2013-47852-R from the Ministerio de Economía y Competitividad (MINECO, Spain) and Fondo Europeo de Desarrollo Regional (FEDER); AGL2016-79343-R from the Agencia Estatal de Investigación (AEI, Spain) and FEDER; PI16/01477 from Plan Estatal de I+D+I 2013-2016, Instituto de Salud Carlos III (ISCIII), Subdirección General de Evaluación y Fomento de la Investigación and FEDER; and ED431C2017/57 from the Consellería de Cultura, Educación e Ordenación Universitaria of Xunta de Galicia and FEDERS

High Prevalence and Diversity of Cephalosporin-Resistant Enterobacteriaceae Including Extraintestinal Pathogenic E. coli CC648 Lineage in Rural and Urban Dogs in Northwest Spain

The aim of this work was to assess the prevalence of extended spectrum-β-lactamase (ESBL)- and carbapenemase-producing Enterobacteriaceae in fecal samples recovered from rural and urban healthy dogs in Northwest Spain (Galicia) to identify potential high-risk clones and to molecularly characterize positive isolates regarding the genes coding for ESBL/pAmpC resistance and virulence. Thirty-five (19.6%) out of 179 dogs were positive for cephalosporin-resistant Enterobacteriaceae, including Escherichiacoli and Klebsiella pneumoniae (39 and three isolates, respectively). All the isolates were multidrug resistant, with high rates of resistance to different drugs, including ciprofloxacin (71.4%). A wide diversity of ESBL/pAmpC enzymes, as well as E. coli phylogroups (A, B1, C, D, E, F and clade I) were found. The eight isolates (20.5%) found to conform to the ExPEC status, belonged to clones O1:H45-clade I-ST770 (CH11-552), O18:H11-A-ST93-CC168 (CH11-neg), O23:H16-B1-ST453-CC86 (CH6-31), and O83:H42-F-ST1485-CC648 (CH231-58), with the latter also complying the uropathogenic (UPEC) status. The three K. pneumoniae recovered produced CTX-M-15 and belonged to the ST307, a clone previously reported in human clinical isolates. Our study highlights the potential role of both rural and urban dogs as a reservoir of high-risk Enterobacteriaceae clones, such as the CC648 of E. coli and antimicrobial resistance traits. Within a One-Health approach, their surveillance should be a priority in the fight against antimicrobial resistanceThis research was funded project FIS PI17-00728 (Fondo de Investigación Sanitaria, Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, Spain), cofunded by the European Regional Development Fund of the European Union: a Way to Making Europe (FEDER); Project PID2019-104439RB-C21/AEI/10.13039/501100011033 and FEDER; ED431C 2017/57 from the Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia and FEDER; and by the Strategic Researcher Cluster BioReDeS funded by the Regional Government Xunta de Galicia under the project no. ED431E 2018/09. D. Díaz-Jiménez and I. García-Meniño acknowledge the Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia for their pre-doctoral grants (ED481A-2019/022 and ED481A-2015/149, respectively). The Research stay of I. García-Meniño at the Hospital Universitario Central de Asturias was funded by a grant from the Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica (SEIMC)S

Swine enteric colibacillosis in Spain: pathogenic potential of mcr-1 ST10 and ST131 E. coli Isolates

This is a wide epidemiological study of 499 E. coli isolates recovered from 179 outbreaks of enteric colibacillosis from pig production farms in Spain during a period of 10 years. Most samples were of diarrheagenic cases occurred during the post-wean period (PWD) which showed to be significantly associated with ETEC (67%) followed by aEPEC (21.7%). On the contrary, aEPEC was more prevalent (60.3%) among diarrheas of suckling piglets, followed by ETEC (38.8%). STEC/ETEC or STEC were recovered in 11.3 and 0.9% of PWD and neonatal diarrhea, respectively. Detection of the F4 colonization factor was not significantly different between isolates recovered from neonatal pigs and those recovered post wean (40.5 versus 27.7%) while F18 was only present among PWD isolates (51.5% of ETEC, STEC, and STEC/ETEC isolates). We also found a high prevalence of resistance to colistin related to the presence of the mcr-1 gene (25.6% of the diarreagenic isolates). The characterization of 65 representative mcr-1 isolates showed that all were phenotypically resistant to colistin (>2 μg/ml), and most (61 of 65) multidrug-resistant (MDR). Six ETEC and one STEC mcr-1 isolates were also carriers of ESBL genes. In addition, other seven mcr-1 isolates harbored mcr-4 (three ETEC) and mcr-5 (two ETEC and two aEPEC) genes. In the phylogenetic analysis of the 65 mcr-1 diarrheagenic isolates we found that more than 50% (38 out of 65) belonged to A-ST10 Cplx and from those, 29 isolates showed the clonotype CH11-24. In this study, we also recovered 18 ST131 isolates including seven mcr-1 carriers. To the best of our knowledge, this would be the first report of ST131 mcr-1 isolation in pigs. Worryingly, the swine mcr-1 ST131 carriers also showed MDR, including to trimethoprim-sulfamethoxazole, tobramycin, gentamicin and ciprofloxacin. In the PFGE-macrorestriction comparison of clinical swine and human ST131, we found high similarities (≥85%) between two pig and two human ST131 isolates of virotype D5. Acquisition of mcr-1 by this specific clone means an increased risk due to its special feature of congregating virulence and resistance traits, together with its spread capability. Here we show a potential zoonotic swine source of ST131This study was supported by projects AGL2016-79343-R from the Agencia Estatal de Investigación (AEI, Spain) and FEDER; PI16/01477 from Plan Estatal de I C D C I 2013–2016, Instituto de Salud Carlos III (ISCIII), Subdirección General de Evaluación y Fomento de la Investigación, and FEDER; CN2012/303 andED431C 2017/57 from the Consellería de Cultura, Educación e Ordenación Universitaria (Xunta de Galicia), and FEDER IG-M acknowledges the Conselleria de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia for his grant (Ref. ED481A-2015/149) “Axudas de apoio á etapa predoutoral do Plan galego de investigación, innovación e crecemento 2011-2015S

Diversity of Multi-Drug Resistant Avian Pathogenic Escherichia coli (APEC) Causing Outbreaks of Colibacillosis in Broilers during 2012 in Spain

Avian pathogenic Escherichia coli (APEC) are the major cause of colibacillosis in poultry production. In this study, a total of 22 E. coli isolated from colibacillosis field cases and 10 avian faecal E. coli (AFEC) were analysed. All strains were characterised phenotypically by susceptibility testing and molecular typing methods such as pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). The presence of 29 virulence genes associated to APEC and human extraintestinal pathogenic E. coli (ExPEC) was also evaluated. For cephalosporin resistant isolates, cephalosporin resistance genes, plasmid location and replicon typing was assessed. Avian isolates belonged to 26 O:H serotypes and 24 sequence types. Out of 22 APEC isolates, 91% contained the virulence genes predictors of APEC; iutA, hlyF, iss, iroN and ompT. Of all strains, 34% were considered ExPEC. PFGE analysis demonstrated a high degree of genetic polymorphism. All strains were multi-resistant, including those isolated from healthy animals. Eleven strains were resistant to cephalosporins; six contained blaCTX-M-14, two blaSHV-12, two blaCMY-2 and one blaSHV-2. Two strains harboured qnrA, and two qnrA together with aac(6’)-Ib-cr. Additionally, the emergent clone O25b:H4-B2-ST131 was isolated from a healthy animal which harboured blaCMY-2 and qnrS genes. Cephalosporin resistant genes were mainly associated to the presence of IncK replicons. This study demonstrates a very diverse population of multi-drug resistant E. coli containing a high number of virulent genes. The E. coli population among broilers is a reservoir of resistance and virulence-associated genes that could be transmitted into the community through the food chain. More epidemiological studies are necessary to identify clonal groups and resistance mechanisms with potential relevance to public health.This work was partially supported by the grants AGL2011- 28836 and AGL2013-47852-R from the Ministerio de Economía y Competitividad (España) and grants CN2012/303 and EM2014/001 (Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia and the European Regional Development Fund, ERDF). Work from LMG is supported by the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) and the European Social FundS

Sequence types, clonotypes, serotypes, and virotypes of extended-spectrum beta-lactamase-producing Escherichia coli causing bacteraemia in a Spanish hospital over a 12-year period (2000 to 2011)

The aim of the present study was to examine the prevalence and determine the molecular characteristics of extended-spectrum β-lactamase-producing Escherichia coli (ESBL-EC) causing bacteraemia in a Spanish Hospital over a 12-year period (2000 to 2011). As far as we know, this is the first study which has investigated and compared the serotypes, phylogroups, clonotypes, virotypes, and PFGE profiles of ST131 and non-ST131 clones of bacteraemia ESBL-EC isolates. Of the 2,427 E. coli bloodstream isolates, 96 (4.0%) were positive for ESBL production: 40 for CTX-M-15, 36 for CTX-M-14, eight for CTX-M-1, four for CTX-M-9, CTX-M-32, and SHV-12. The number of ESBL-EC increased from 1.0% during 2000 to 2005 to 5.5% during 2006–2011 (P < 0.001). The 96 ESBL-EC isolates belonged to 36 different STs. The commonest was ST131 (41 isolates), followed by ST58, ST354, ST393 and ST405 (four isolates each). Most CTX-M-15 isolates (87.5%, 35/40) were ST131, whereas the 36 CTX-M-14 isolates belonged to 23 different STs and only 3 (8.3%) of them were ST131. The 35 ST131 CTX-M-15-producing isolates belonged to the H30Rx subclone and 29 of them showed the virotype A. A drastic change in ST131 virotypes happened in 2011 due to the emergence of the virotypes E (sat, papGII, cnf1, hlyA, and kpsMII-K5) and F (sat, papGII, and kpsMII-K5) which displaced virotype A (afa/draBC, afa operon FM955459, sat, and kpsMII-K2). Although the 96 ESBL-EC isolates showed 21 O serogroups and 17 H flagellar antigens, 39 belonged to serotype O25b:H4 (ST131 isolates). The second most prevalent serotype (O15:H1) was found to be associated with another important high-risk clone (ST393). In conclusion, the ST131 was the most frequent sequence type, being the H30Rx subclone responsible for the significant increase of ESBL-EC isolates since 2006. Here, we report two new virotypes (E and F) of the H30Rx subclone emerged in 2011. Future molecular studies are needed to understand the dynamics of expansion of this successful high-risk subclone in order to prevent its spread and establish the importance of the two new virotypesThis study was supported by the following projects: PI16/01477 from the Plan Estatal de I+D+I 2013-2016, Instituto de Salud Carlos III (ISCIII), Subdirección General de Evaluación y Fomento de la Investigación, and Fondo Europeo de Desarrollo Regional (FEDER); AGL2013-47852-R from the Spanish Ministerio de Economía y Competitividad (MINECO) and FEDER; AGL2016-79343-R from the Spanish Agencia Estatal de Investigación (AEI) and FEDER; and CN2012/303 and ED431C2017/57 from the Consellería de Cultura, Educación e Ordenación Universitaria, (Xunta de Galicia) and FEDERS

Ciclo audiovisual y herramienta de visionado para las asignaturas de primer y segundo ciclo del área de Escultura de la Facultad de Bellas Artes de la Universidad de Salamanca

Memoria ID2022-191 Ayudas de la Universidad de Salamanca para la innovación docente, curso 2022-2023