Effect of phage vB_EcoM_FJ1 on the reduction of ETEC O9:H9 infection in a neonatal pig cell line.

Enterotoxigenic Escherichia coli (ETEC) colonizes the intestine of young pigs causing severe diarrhoea and consequently bringing high production costs. The rise of antibiotic selective pressure together with ongoing limitations on their use, demands new strategies to tackle this pathology. The pertinence of using bacteriophages as an alternative is being explored, and in this work, the efficacy of phage vB_EcoM_FJ1 (FJ1) in reducing the load of ETEC EC43-Ph (serotype O9:H9 expressing the enterotoxin STa and two adhesins F5 and F41) was assessed. Foreseeing the oral application on piglets, FJ1 was encapsulated on calcium carbonate and alginate microparticles, thus preventing phage release under adverse conditions of the simulated gastric fluid (pH 3.0) and allowing phage availability in simulated intestinal fluid (pH 6.5). A single dose of encapsulated FJ1, provided to IPEC-1 cultured cells (from intestinal epithelium of piglets) previously infected by EC43, provided bacterial reductions of about 99.9% after 6 h. Although bacteriophage-insensitive mutants (BIMs) have emerged from treatment, the consequent fitness costs associated with this new phenotype were demonstrated, comparatively to the originating strain. The higher competence of the pig complement system to decrease BIMs' viability, the lower level of colonization of IPEC-1 cells observed with these mutants, and the increased survival rates and health index recorded in infected Galleria mellonella larvae supported this observation. Most of all, FJ1 established a proof-of-concept of the efficiency of phages to fight against ETEC in piglet intestinal cells.

The ST131 Escherichia coli H22 subclone from human intestinal microbiota: Comparison of genomic and phenotypic traits with those of the globally successful H30 subclone.

BACKGROUND: In 2006, we found healthy subjects carrying ST131 Escherichia coli in their intestinal microbiota consisting of two populations: a subdominant population of fluoroquinolone-resistant E. coli belonging to subclone H30 (H30-R or subclade C1), the current worldwide dominant ST131 subclone, and a dominant E. coli population composed of antibiotic-susceptible E. coli belonging to subclone H22 (clade B), the precursor of subclone H30. We sequenced the whole genome of fecal H22 strain S250, compared it to the genomes of ExPEC ST131 H30-Rx strain JJ1886 and commensal ST131 H41 strain SE15, sought the H22-H30 genomic differences in our fecal strains and assessed their phenotypic consequences. RESULTS: We detected 173 genes found in the Virulence Factor Database, of which 148 were shared by the three ST131 genomes, whereas some were genome-specific, notably those allowing determination of virotype (D for S250 and C for JJ1886). We found three sequences of the FimH site involved in adhesion: two in S250 and SE15 close and identical, respectively, to that previously reported to confer strong intestinal adhesion, and one in JJ1886, corresponding to that commonly present in uropathogenic E. coli. Among the genes involved in sugar metabolism, one encoding a gluconate kinase lacked in S250 and JJ1886. Although this gene was also absent in both our fecal H22 and H30-R strains, H22 strains showed a higher capacity to grow in minimal medium with gluconate. Among the genes involved in gluconate metabolism, only the ghrB gene differed between S250/H22 and JJ1886/H30-R strains, resulting in different gluconate reductases. Of the genes involved in biofilm formation, two were absent in the three genomes and one, fimB, in the JJ1886 genome. Our fecal H30-R strains lacking intact fimB displayed delayed biofilm formation relative to our fecal H22 strains. The H22 strains differed by subclade B type and plasmid content, whereas the H30-R strains were identical. CONCLUSIONS: Phenotypic analysis of our fecal strains based on observed genomic differences between S250 and JJ1886 strains suggests the presence of traits related to bacterial commensalism in our H22 strains and traits commonly found in uropathogenic E. coli in our H30-R strains.

Plasmid flux in Escherichia coli ST131 sublineages, analyzed by plasmid constellation network (PLACNET), a new method for plasmid reconstruction from whole genome sequences.

Bacterial whole genome sequence (WGS) methods are rapidly overtaking classical sequence analysis. Many bacterial sequencing projects focus on mobilome changes, since macroevolutionary events, such as the acquisition or loss of mobile genetic elements, mainly plasmids, play essential roles in adaptive evolution. Existing WGS analysis protocols do not assort contigs between plasmids and the main chromosome, thus hampering full analysis of plasmid sequences. We developed a method (called plasmid constellation networks or PLACNET) that identifies, visualizes and analyzes plasmids in WGS projects by creating a network of contig interactions, thus allowing comprehensive plasmid analysis within WGS datasets. The workflow of the method is based on three types of data: assembly information (including scaffold links and coverage), comparison to reference sequences and plasmid-diagnostic sequence features. The resulting network is pruned by expert analysis, to eliminate confounding data, and implemented in a Cytoscape-based graphic representation. To demonstrate PLACNET sensitivity and efficacy, the plasmidome of the Escherichia coli lineage ST131 was analyzed. ST131 is a globally spread clonal group of extraintestinal pathogenic E. coli (ExPEC), comprising different sublineages with ability to acquire and spread antibiotic resistance and virulence genes via plasmids. Results show that plasmids flux in the evolution of this lineage, which is wide open for plasmid exchange. MOBF12/IncF plasmids were pervasive, adding just by themselves more than 350 protein families to the ST131 pangenome. Nearly 50% of the most frequent γ-proteobacterial plasmid groups were found to be present in our limited sample of ten analyzed ST131 genomes, which represent the main ST131 sublineages.

Phylogenetic, virulence and antibiotic resistance characteristics of commensal strain populations of Escherichia coli from community subjects in the Paris area in 2010 and evolution over 30 years.

It is important to study commensal populations of Escherichia coli because they appear to be the reservoir of both extra-intestinal pathogenic E. coli and antibiotic resistant strains of E. coli. We studied 279 dominant faecal strains of E. coli from 243 adults living in the community in the Paris area in 2010. The phylogenetic group and subgroup [sequence type complex (STc)] of the isolates and the presence of 20 virulence genes were determined by PCR assays. The O-types and resistance to 18 antibiotics were assessed phenotypically. The B2 group was the most frequently recovered (34.0 %), followed by the A group (28.7 %), and other groups were more rare. The most prevalent B2 subgroups were II (STc73), IV (STc141), IX (STc95) and I (STc131), with 22.1, 21.1, 16.8 and 13.7 %, respectively, of the B2 group strains. Virulence factors (VFs) were more common in B2 group than other strains. One or more resistances were found in 125 strains (44.8 % of the collection) but only six (2.2 % of the collection) were multiresistant; no extended-spectrum beta-lactamase-producing strain was isolated. The C phylogroup and clonal group A strains were the most resistant. No trade-off between virulence and resistance was evidenced. We compared these strains with collections of strains gathered under the same conditions 30 and 10 years ago. There has been a parallel and linked increase in the frequency of B2 group strains (from 9.4 % in 1980, to 22.7 % in 2000 and 34.0 % in 2010) and of VFs. Antibiotic resistance also increased, from 22.6 % of strains resistant to at least one antibiotic in 1980, to 31.8 % in 2000 and 44.8 % in 2010; resistance to streptomycin, however, remained stable. Commensal human E. coli populations have clearly evolved substantially over time, presumably reflecting changes in human practices, and particularly increasing antibiotic use.

Prevalence of day-care centre children (France) with faecal CTX-M-producing Escherichia coli comprising O25b:H4 and O16:H5 ST131 strains.

OBJECTIVES: Determining the prevalence of children in day-care centres (DCCs) carrying faecal extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae and molecularly characterizing those belonging to the Escherichia coli species. METHODS: Stools were collected from children's diapers (January-April 2012) in randomly chosen DCCs and plated onto ChromID ESBL. Colonies growing on this medium were identified by the Vitek 2 system and tested for antibiotic susceptibility and for ESBL production by the double-disc synergy test. ESBL genotypes were determined as well as phylogenetic groups, ERIC-2 (enterobacterial repetitive intergenic consensus) PCR profiles and sequence types (STs) for the E. coli isolates. Serotypes, virotypes, fimH alleles, ESBL-carrying plasmids and PFGE patterns were determined for the ST131 E. coli isolates. RESULTS: Among 419 children from 25 participating DCCs, 1 was colonized by CTX-M-15-producing Klebsiella pneumoniae and 27 (6.4%) by E. coli, which all produced CTX-M enzymes [CTX-M-15 (37%), CTX-M-1 (26%), CTX-M-14 (22%), CTX-M-27 (11%) and CTX-M-22 (4%)]. The 27 E. coli isolates, 55.5% belonging to group B2, displayed 20 ERIC-2 PCR profiles and 16 STs. The ST131 E. coli isolates were dominant (44%), displayed serotypes O25b:H4 and O16:H5, fimH alleles 30 and 41 and virotypes A and C. According to the PFGE patterns, one strain of E. coli ST131 producing a CTX-M-15 enzyme carried by an IncF F2:A1:B- plasmid had spread within one DCC. CONCLUSIONS: This study shows a notable prevalence (6.4%) of DCC children with faecal CTX-M-producing E. coli isolates comprising a high proportion of E. coli ST131 isolates, suggesting that these children might be a reservoir of this clone.

Molecular epidemiology and virulence of Escherichia coli O16:H5-ST131: comparison with H30 and H30-Rx subclones of O25b:H4-ST131.

The present study was carried out to evaluate the prevalence of the clonal subgroup O16:H5-ST131 and the H30 and H30-Rx subclones among E. coli isolates causing extraintestinal infections and to know their virulence potential. The ST131 clonal group accounted for 490 (16%) of the 2995 isolates obtained from clinical samples in five Spanish hospitals during the study period (2005-2012). Among those 490 ST131 isolates, 456 belonged to serotype O25b:H4, 27 to O16:H5 and seven were O-non-typeable:H4 (ONT:H4). All 27 O16:H5 isolates showed fimH41, whereas fimH30 and fimH22 alleles were the most frequently detected among O25b:H4 isolates. The majority (381/490; 78%) of ST131 isolates belonged to H30 subclone, and 302 of 381 (79%) H30 isolates belonged to the H30-Rx subclone. Of the 27 O16:H5 isolates, 48% produced CTX-M-14; however, none produced CTX-M-15. In contrast, 46% of O25b:H4 isolates produced CTX-M-15 while only 2% produced CTX-M-14. More than a half of the O16:H5 isolates (56%) showed the ExPEC status which was significantly more prevalent within O25b:H4 isolates (81%) (P<0.01), especially among H30-Rx (97%) isolates. In the present study, a modified virotype scheme was applied within which approximately half (52%) of the O16:H5 isolates showed the C1 specific virotype. Despite their low virulence-gene score (mean of virulence genes 6.4 versus 8.5 in O25b:H4 isolates), six out of the 10 O16:H5 isolates assayed showed high virulence in the mouse model of sepsis (killed 90-100% of mice challenged). Furthermore, four O16:H5 isolates of virotypes A and C1, carrying K2 variant of group II capsule, showed lethality at 24h. Thus, certain O16:H5 fimH41 isolates show a similar in vivo virulence to that reported with the highly virulent O25b:H4 H30-Rx isolates (Mora et al., PLOS ONE 2014, e87025), supporting their potential virulence for humans.

Fluoroquinolone resistance in complicated urinary tract infections: association with the increased occurrence and diversity of Escherichia coli of clonal complex 131, together with ST1193.

Introduction: Urinary tract infections (UTIs) are one of the leading causes of multidrug-resistance (MDR) spread and infection-related deaths. Escherichia coli is by far the main causative agent. We conducted a prospective study on complicated urinary tract infections (cUTIs) i) to monitor the high-risk clones that could be compromising the therapeutic management and ii) to compare the cUTI etiology with uncomplicated infections (uUTIs) occurring in the same period and health area. Methods: 154 non-duplicated E. coli recovered from cUTIs in 2020 at the Hospital Universitario Central de Asturias (Spain) constituted the study collection. Results: Most cUTI isolates belonged to phylogroup B2 (72.1%) and met the uropathogenic (UPEC) status (69.5%) (≥3 of chuA, fyuA, vat, and yfcV genes). MDR was exhibited by 35.7% of the isolates, similarly to data observed in the uUTI collection. A significant difference observed in cUTI was the higher level of fluoroquinolone resistance (FQR) (47.4%), where the pandemic clonal groups B2-CC131 and B2-ST1193 (CH14-64) comprised 28% of the 154 E. coli, representing 52.1% of the FQR isolates. Other prevalent FQR clones were D-ST69 (CH35-27), D-ST405 (CH37-27), and B2-ST429 (CH40-20) (three isolates each). We uncovered an increased genetic and genomic diversity of the CC131: 10 different virotypes, 8 clonotypes (CH), and 2 STs. The presence of bla CTX-M-15 was determined in 12 (7.8%) isolates (all CC131), which showed 10 different core genome (cg)STs and 2 fimH types (fimH30 and fimH602) but the same set of chromosomal mutations conferring FQR (gyrA p.S83L, gyrA p.D87N, parC p.S80I, parC p.E84V, and parE p.I529L). In addition, the plasmidome analysis revealed 10 different IncF formulae in CC131 genomes. Conclusion: We proved here that non-lactose fermenting screening, together with the detection of O25b (rfbO25b), H4 (fliCH4), and H5 (fliCH5) genes, and phylogroup and clonotyping assignation, is a reasonable approach that can be easily implemented for the surveillance of emerging high-risk clones associated with FQR spread in cUTIs, such as the uncommonly reported O25b:H4-B2-ST9126-CC131 (CH1267-30). Since E. coli CC131 and ST1193 are also involved in the community uUTIs of this health area, interventions to eradicate these MDR clones, along with surveillance for other emerging ones, are essential for antibiotic use optimization programs.

Moving towards on-site detection of Shiga toxin-producing Escherichia coli in ready-to-eat leafy greens.

Rapid identification of Shiga toxin-producing Escherichia coli, or STEC, is of utmost importance to assure the innocuousness of the foodstuffs. STEC have been implicated in outbreaks associated with different types of foods however, among them, ready-to-eat (RTE) vegetables are particularly problematic as they are consumed raw, and are rich in compounds that inhibit DNA-based detection methods such as qPCR. In the present study a novel method based on Loop-mediated isothermal amplification (LAMP) to overcome the limitations associated with current molecular methods for the detection of STEC in RTE vegetables targeting stx1 and stx2 genes. In this sense, LAMP demonstrated to be more robust against inhibitory substances in food. In this study, a comprehensive enrichment protocol was combined with four inexpensive DNA extraction protocols. The one based on silica purification enhanced the performance of the method, therefore it was selected for its implementation in the final method. Additionally, three different detection chemistries were compared, namely real-time fluorescence detection, and two end-point colorimetric strategies, one based on the addition of SYBR Green, and the other based on a commercial colorimetric master mix. After optimization, all three chemistries demonstrated suitable for the detection of STEC in spiked RTE salad samples, as it was possible to reach a LOD50 of 0.9, 1.4, and 7.0 CFU/25 g for the real-time, SYBR and CC LAMP assays respectively. All the performance parameters reached values higher than 90 %, when compared to a reference method based on multiplex qPCR. More specifically, the analytical sensitivity was 100, 90.0 and 100 % for real-time, SYBR and CC LAMP respectively, the specificity 100 % for all three assays, and accuracy 100, 96 and 100 %. Finally, a high degree of concordance was also obtained (1, 0.92 and 1 respectively). Considering the current technological advances, the method reported, using any of the three detection strategies, demonstrated suitable for their implementation in decentralized settings, with low equipment resources.

Comparative analysis of multidrug-resistant Klebsiella pneumoniae strains of food and human origin reveals overlapping populations.

Given the increasing incidence of multidrug-resistant (MDR) Klebsiella pneumoniae infections, it is of great interest to investigate the risk of transmission associated with the prevalence of this pathogen. Some studies have described fresh raw poultry meat as a reservoir of MDR K. pneumoniae, including clinically relevant sequence types (ST) and extended-spectrum ß-lactamase (ESBL) strains, indicating possible consumer exposure. This study compared 47 MDR strains of K. pneumoniae from poultry meat and human clinical isolates to assess similarities, including analysis of antimicrobial resistance profiles and virulence factors involved in infection. In addition, several biofilm culture methods were evaluated for reproducible assessment of biofilm formation in K. pneumoniae strains. Globally, no association between strain origin and STs, hypermucoviscosity, biofilm formation or serum resistance could be found between isolates of food and clinical origin, nor an associated AMR pattern, suggesting overlapping populations. We found that LB supplemented with glucose in microaerobiosis was the best discrimination condition for biofilm formation in the active attachment biofilm cultivation model. The biofilm formation capacity was strongly dependent on culture conditions, with a strain-specific response, but only a minor increase in biofilm levels was recorded in clinical K. pneumoniae populations. Our results suggest that a similar risk of zoonosis transmission from potentially virulent foodborne strains previously observed in E. coli is also present in this high-priority pathogen. This study further confirms that foodborne isolates of K. pneumoniae pose a risk to consumers and therefore this pathogen should be included in the surveillance of foodborne pathogens with high risk of MDR infections and therapeutic failure.

Draft genome sequence of Klebsiella pneumoniae KLEB-33: a convergent biofilm hyperforming multiresistant strain belonging to the emerging ST16 lineage harboring multiple hypervirulence genes.

The emergence of convergent Klebsiella pneumoniae strains showing multiresistance, characteristic of nosocomial pathotypes and hypervirulent traits typical of community-acquired isolates, makes them important models for studying K. pneumoniae pathogenesis. Here, we describe the convergent, multidrug-resistant KLEB-33 strain harboring several hypervirulence genes and make its genome available to the scientific community.

Four main virotypes among extended-spectrum-ß-lactamase-producing isolates of Escherichia coli O25b:H4-B2-ST131: bacterial, epidemiological, and clinical characteristics.

A total of 1,021 extended-spectrum-ß-lactamase-producing Escherichia coli (ESBLEC) isolates obtained in 2006 during a Spanish national survey conducted in 44 hospitals were analyzed for the presence of the O25b:H4-B2-ST131 (sequence type 131) clonal group. Overall, 195 (19%) O25b-ST131 isolates were detected, with prevalence rates ranging from 0% to 52% per hospital. Molecular characterization of 130 representative O25b-ST131 isolates showed that 96 (74%) were positive for CTX-M-15, 15 (12%) for CTX-M-14, 9 (7%) for SHV-12, 6 (5%) for CTX-M-9, 5 (4%) for CTX-M-32, and 1 (0.7%) each for CTX-M-3 and the new ESBL enzyme CTX-M-103. The 130 O25b-ST131 isolates exhibited relatively high virulence scores (mean, 14.4 virulence genes). Although the virulence profiles of the O25b-ST131 isolates were fairly homogeneous, they could be classified into four main virotypes based on the presence or absence of four distinctive virulence genes: virotypes A (22%) (afa FM955459 positive, iroN negative, ibeA negative, sat positive or negative), B (31%) (afa FM955459 negative, iroN positive, ibeA negative, sat positive or negative), C (32%) (afa FM955459 negative, iroN negative, ibeA negative, sat positive), and D (13%) (afa FM955459 negative, iroN positive or negative, ibeA positive, sat positive or negative). The four virotypes were also identified in other countries, with virotype C being overrepresented internationally. Correspondingly, an analysis of XbaI macrorestriction profiles revealed four major clusters, which were largely virotype specific. Certain epidemiological and clinical features corresponded with the virotype. Statistically significant virotype-specific associations included, for virotype B, older age and a lower frequency of infection (versus colonization), for virotype C, a higher frequency of infection, and for virotype D, younger age and community-acquired infections. In isolates of the O25b:H4-B2-ST131 clonal group, these findings uniquely define four main virotypes, which are internationally distributed, correspond with pulsed-field gel electrophoresis (PFGE) profiles, and exhibit distinctive clinical-epidemiological associations.

Detection of quinolone-resistant Escherichia coli isolates belonging to clonal groups O25b:H4-B2-ST131 and O25b:H4-D-ST69 in raw sewage and river water in Barcelona, Spain.

OBJECTIVES: The present study was carried out to evaluate the prevalence of clonal group O25b:H4-B2-ST131 in water environments with faecal pollution (urban sewage and river water) in the north-east of Spain and to study the virulence gene content of environmental isolates and to compare them with isolates causing human extraintestinal infections in Spain. METHODS: This study was performed with 10 sewage samples (collected in Catalonia, north-eastern Spain, in autumn 2009 from the influent raw urban sewage of a wastewater treatment plant that serves a large urban area) and 6 river water samples (collected monthly from February to April 2010 in the Llobregat river catchment area, near Barcelona, a watercourse subjected to heavy anthropogenic pressure). Escherichia coli colonies were screened by PCR for the rfbO25b gene associated with the clonal group O25b:H4-B2-ST131. Sequence types (STs), serotypes, virulence genes, PFGE profiles, antimicrobial resistance and extended-spectrum ß-lactamase (ESBL) enzymes were determined in 75 E. coli isolates positive for the O25b molecular subtype. RESULTS: Of the 75 O25b-positive isolates, 51 belonged to the O25b:H4-B2-ST131 clonal group and the remaining 24 belonged to clonal group O25b:H4-D-ST69. The majority of ST69 isolates (23 of 24) were isolated from urban sewage, whereas ST131 isolates were isolated from urban sewage (25 isolates) as well as from river water (26 isolates). ST131 and ST69 isolates carried 4-13 virulence genes, the majority (82%) being quinolone resistant. CONCLUSIONS: We showed the presence of E. coli isolates belonging to clonal groups O25b:H4-B2-ST131 and O25b:H4-D-ST69 in raw sewage and river water in Barcelona. Furthermore, we observed that the environmental O25b:H4-B2-ST131 isolates showed similar virulence and macrorestriction profiles to clinical human isolates. To our knowledge, this is the first study describing the O25b:H4-D-ST69 clonal group.

Genomics, biofilm formation and infection of bladder epithelial cells in potentially uropathogenic Escherichia coli (UPEC) from animal sources and human urinary tract infections (UTIs) further support food-borne transmission.

Escherichia coli is the main cause of urinary tract infections (UTI). While genomic comparison of specific clones recovered from animals, and human extraintestinal infections show high identity, studies demonstrating the uropathogenicity are lacking. In this study, comparative genomics combined with bladder-cell and biofilm formation assays, were performed for 31 E. coli of different origins: 7 from meat (poultry, beef, and pork); 2 from avian-farm environment; 12 from human uncomplicated UTI, uUTI; and 10 from human complicated UTI, cUTI. These isolates were selected based on their genetic uropathogenic (UPEC) status and phylogenetic background. In silico analysis revealed similar virulence-gene profiles, with flagella, type 1 and curli fimbriae, outer-membrane proteins (agn43, ompT, iha), and iron-uptake (iutA, entA, and fyuA) associated-traits as the most prevalent (>65%). In bladder-cell assays, moderate to strong values of association (83%, 60%, 77.8%) and invasion (0%, 70%, 55.5%) were exhibited by uUTI, cUTI, and animal-derived isolates, respectively. Of interest, uUTI isolates exhibited a significantly lower invasive capacity than cUTI isolates (p < 0.05). All isolates but one produced measurable biofilm. Notably, 1 turkey meat isolate O11:H6-F-ST457, and 2 cUTI isolates of the pandemic lineages O83:H42-F-ST1485-CC648 and O25b:H4-B2-ST131, showed strong association, invasion and biofilm formation. These isolates showed common carriage of type 1 fimbriae and csg operons, toxins (hlyF, tsh), iron uptake systems (iutA, entA, iroN), colicins, protectins (cvaC, iss, kpsM, traT), ompT, and malX. In summary, the similar in vitro behaviour found here for certain E. coli clones of animal origin would further reinforce the role of food-producing animals as a potential source of UPEC. Bladder-cell infection assays, combined with genomics, might be an alternative to in vivo virulence models to assess uropathogenicity.

Diagnostic strategy for identifying avian pathogenic Escherichia coli based on four patterns of virulence genes.

In order to improve the identification of avian pathogenic Escherichia coli (APEC) strains, an extensive characterization of 1,491 E. coli isolates was conducted, based on serotyping, virulence genotyping, and experimental pathogenicity for chickens. The isolates originated from lesions of avian colibacillosis (n = 1,307) or from the intestines of healthy animals (n = 184) from France, Spain, and Belgium. A subset (460 isolates) of this collection was defined according to their virulence for chicks. Six serogroups (O1, O2, O5, O8, O18, and O78) accounted for 56.5% of the APEC isolates and 22.5% of the nonpathogenic isolates. Thirteen virulence genes were more frequently present in APEC isolates than in nonpathogenic isolates but, individually, none of them could allow the identification of an isolate as an APEC strain. In order to take into account the diversity of APEC strains, a statistical analysis based on a tree-modeling method was therefore conducted on the sample of 460 pathogenic and nonpathogenic isolates. This resulted in the identification of four different associations of virulence genes that enables the identification of 70.2% of the pathogenic strains. Pathogenic strains were identified with an error margin of 4.3%. The reliability of the link between these four virulence patterns and pathogenicity for chickens was validated on a sample of 395 E. coli isolates from the collection. The genotyping method described here allowed the identification of more APEC isolates with greater reliability than the classical serotyping methods currently used in veterinary laboratories.

Seropathotypes, Phylogroups, Stx subtypes, and intimin types of wildlife-carried, shiga toxin-producing escherichia coli strains with the same characteristics as human-pathogenic isolates.

The objectives of this study were to investigate the presence of Shiga toxin-producing Escherichia coli (STEC) strains in wildlife that have spread in Europe, living near human settlements; to analyze their epidemiological role in maintenance and transmission to domestic livestock; and to assess the potential health risk of wildlife-carried strains. STEC strains were recovered from 53% of roe deer, 8.4% of wild boars, and 1.9% of foxes sampled in the northwest of Spain (Galicia). Of the 40 serotypes identified, 21 were classified as seropathotypes associated with human disease, accounting for 81.5% of the wildlife-carried STEC strains, including the enterohemorrhagic serotypes O157:H7-D-eae-γ1, O26:[H11]-B1-eae-ß1, O121:H19-B1-eae-ε1, and O145:[H28]-D-eae-γ1. None of the wildlife-carried strains belonged to the highly pathogenic serotype O104:H4-B1 from the recent Germany outbreak. Forty percent of wildlife-carried STEC strains shared serotypes, phylogroups, intimin types, and Stx profiles with isolates from human patients from the same geographic area. Furthermore, wildlife-carried strains belonging to serotypes O5:HNM-A, O26:[H11]-B1, O76:H19-B1, O145:[H28]-D, O146:H21-B1, and O157:H7-D showed pulsed-field gel electrophoresis (PFGE) profiles with >85% similarity to human-pathogenic STEC strains. We also found a high level of similarity among STEC strains of serotypes O5:HNM-A, O26:[H11]-B1, and O145:HNM-D of bovine (feces and beef) and wildlife origins. Interestingly, O146:H21-B1, the second most frequently detected serotype in this study, is commonly associated with human diarrhea and isolated from beef and vegetables sold in Galicia. Importantly, at least 3 STEC isolates from foxes (O5:HNM-A-eae-ß1, O98:[H21]-B1-eae-ζ1, and O146:[H21]-B1) showed characteristics similar to those of human STEC strains. In conclusion, roe deer, wild boar, and fox in Galicia are confirmed to be carriers of STEC strains potentially pathogenic for humans and seem to play an important role in the maintenance of STEC.

Mobile colistin resistance (MCR), extended-spectrum beta-lactamase (ESBL) and multidrug resistance monitoring in Escherichia coli (commensal and pathogenic) in pig farming: need of harmonized guidelines and clinical breakpoints.

Current data on antimicrobial resistance in pig production is essential for the follow-up strategic programs to eventually preserve the effectiveness of last-resort antibiotics for humans. Here, we characterized 106 Escherichia coli recovered in routine diagnosis (2020-2022) from fecal sample pigs, belonging to 74 Spanish industrial farms, affected by diarrhea. The analysis of virulence-gene targets associated with pathotypes of E. coli, determined 64 as pathogenic and 42 as commensal. Antimicrobial susceptibility testing (AST) performed by minimal inhibitory concentration (MIC) assay, was interpreted by applying breakpoints/cut-off values from the different standards EUCAST/TECOFF 2022, CLSI VET ED5:2020, and CASFM VET2020. Comparisons taking EUCAST as reference exhibited moderate to high correlation except for enrofloxacin, neomycin, and florfenicol. Of note, is the lack of clinical breakpoints for antibiotics of common use in veterinary medicine such as cefquinome, marbofloxacin, or florfenicol. AST results determined multidrug resistance (MDR) to ≥3 antimicrobial categories for 78.3% of the collection, without significant differences in commensal vs pathogenic isolates. Plasmid-mediated mobile colistin resistance gene (mcr) was present in 11.3% of 106 isolates, all of them pathogenic. This means a significant decrease compared to our previous data. Furthermore, 21.7% of the 106 E. coli were ESBL-producers, without differences between commensal and pathogenic isolates, and mcr/ESBL genes co-occurred in 3 isolates. Phylogenetic characterization showed a similar population structure (A, B1, C, D, and E), in both commensal and pathogenic E. coli, but with significant differences for B1, C, and E (38.1 vs 20.3%; 19 vs 1.6%; and 7.1 vs 25%, respectively). Additionally, we identified one B2 isolate of clone O4:H5-B2-ST12 (CH13-223), positive for the uropathogenic (UPEC) status, and in silico predicted as human pathogen. We suggest that a diagnosis workflow based on AST, detection of mcr and ESBL genes, and phylogenetic characterization, would be a useful monitoring tool under a "One-Health" perspective.

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 prescribing.

Spread of Escherichia coli O25b:H4-B2-ST131 producing CTX-M-15 and SHV-12 with high virulence gene content in Barcelona (Spain).

OBJECTIVES: The present study was carried out to evaluate the current prevalence of the clonal group O25b:H4-B2-ST131 among extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli (ESBLEC) collected in the Hospital Vall d'Hebron in Barcelona (Spain) with regard to other clonal groups and to characterize their genetic background. METHODS: Ninety-four consecutive non-duplicate ESBLEC isolates collected from May to December 2008 were studied. ESBL enzymes, phylogenetic groups, serotypes, virulence genes, sequence types (STs) and PFGE profiles were determined. Results The most prevalent ESBLs were CTX-M-14 (47%), CTX-M-15 (26%) and SHV-12 (19%). Thirty (32%) of the 94 ESBLEC isolates belonged to the clonal group O25b:H4-B2-ST131 of which 19 (63%) carried the bla(CTX-M-15) gene and eight (27%) the bla(SHV-12) gene. Moreover, five additional clonal groups (O15/O25a:H1/HNM-D-ST393, O78:HNM-A-ST369, ONT:H21,42/HNM-B1-ST101, O9:H4-A-ST410 and O8:H19-B1-ST162) were detected among 16 isolates producing CTX-M-14 and SHV-12. The 30 ST131 isolates exhibited a significantly higher virulence score (mean number of virulence genes 9.60 versus 5.84) compared with the 64 non-ST131 isolates. In particular, the SHV-12-producing ST131 isolates showed the highest virulence score (range 8-13, mean score 11.75). RESULTS: also revealed that the 30 ST131 isolates were distributed in five different groups according to their virulence, XbaI macrorestriction and resistance patterns. CONCLUSIONS: We report for the first time the clonal spread of SHV-12-producing O25b:H4-B2-ST131 isolates characterized by high virulence gene content. Moreover, we describe the distribution of the ST131 isolates within different virulence groups.

National survey of Escherichia coli causing extraintestinal infections reveals the spread of drug-resistant clonal groups O25b:H4-B2-ST131, O15:H1-D-ST393 and CGA-D-ST69 with high virulence gene content in Spain.

OBJECTIVES: To evaluate the current prevalence of the three clonal groups O25b:H4-B2-ST131, O15:H1-D-ST393 and CGA-D-ST69 (where ST stands for sequence type) among Escherichia coli isolates causing extraintestinal infections in Spain and to characterize their virulence background, 500 consecutive non-duplicate E. coli isolates causing extraintestinal infections were analysed. METHODS: The 500 isolates were collected during February 2009 from five hospitals in different Spanish regions. Phylogenetic groups, STs, serotypes, virulence genes, PFGE profiles, antimicrobial resistance and extended-spectrum ß-lactamase (ESBL) enzymes were determined. RESULTS: The three clonal groups accounted for 19% of the 500 isolates. Furthermore, they accounted for 37% of the isolates exhibiting trimethoprim/sulfamethoxazole plus ciprofloxacin resistance, 34% of aminoglycoside-resistant isolates and 30% of multidrug-resistant isolates. Clonal group ST131 was the most prevalent, and accounted for 12% of isolates overall and for 23% of multidrug-resistant isolates. The ST131 isolates exhibited a significantly higher virulence score (mean of virulence genes 8.1) compared with the ST393 (6.0) and ST69 (5.4) isolates. The prevalence of ESBL-producing isolates was 7%. Six (10%) of the 59 ST131 isolates were positive for CTX-M-15 and one (6%) of the 16 ST393 isolates was positive for CTX-M-14, whereas none of the 22 ST69 isolates produced ESBL enzymes. CONCLUSIONS: The three clonal groups investigated accounted for 30% of the multidrug-resistant isolates, which gives evidence of an important clonal component in the emergence of resistances among extraintestinal pathogenic E. coli. Notably, a single high virulence clonal group (O25b:H4-B2-ST131) causes approximately 1 in every 10 extraintestinal infections in Spain, representing an important public health threat. A new variant of the ST131 clonal group, which is non-ESBL-producing but trimethoprim/sulfamethoxazole resistant and with high virulence content, is reported.

Characteristics of the Shiga-toxin-producing enteroaggregative Escherichia coli O104:H4 German outbreak strain and of STEC strains isolated in Spain.

A Shiga-toxin-producing Escherichia coli (STEC) strain belonging to serotype O104:H4, phylogenetic group B1 and sequence type ST678, with virulence features common to the enteroaggregative E. coli (EAEC) pathotype, was reported as the cause of the recent 2011 outbreak in Germany. The outbreak strain was determined to carry several virulence factors of extraintestinal pathogenic E. coli (ExPEC) and to be resistant to a wide range of antibiotics. There are only a few reports of serotype O104:H4, which is very rare in humans and has never been detected in animals or food. Several research groups obtained the complete genome sequence of isolates of the German outbreak strain as well as the genome sequences of EAEC of serotype O104:H4 strains from Africa. Those findings suggested that horizontal genetic transfer allowed the emergence of the highly virulent Shiga-toxin-producing enteroaggregative E. coli (STEAEC) O104:H4 strain responsible for the outbreak in Germany. Epidemiologic investigations supported a linkage between the outbreaks in Germany and France and traced their origin to fenugreek seeds imported from Africa. However, there has been no isolation of the causative strain O104:H4 from any of the samples of fenugreek seeds analyzed. Following the German outbreak, we conducted a large sampling to analyze the presence of STEC, EAEC, and other types of diarrheagenic E. coli strains in Spanish vegetables. During June and July 2011, 200 vegetable samples from different origins were analyzed. All were negative for the virulent serotype O104:H4 and only one lettuce sample (0.6%) was positive for a STEC strain of serotype O146:H21 (stx1, stx2), considered of low virulence. Despite the single positive case, the hygienic and sanitary quality of Spanish vegetables proved to be quite good. In 195 of the 200 samples (98%), <10 colony-forming units (cfu) of E. coli per gram were detected, and the microbiological levels of all samples were satisfactory (<100 cfu/g). The samples were also negative for other pathotypes of diarrheagenic E. coli (EAEC, ETEC, tEPEC, and EIEC). Consistent with data from other countries, STEC belonging to serotype O157:H7 and other serotypes have been isolated from beef, milk, cheese, and domestic (cattle, sheep, goats) and wild (deer, boar, fox) animals in Spain. Nevertheless, STEC outbreaks in Spain are rare.