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.

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.

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.

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.

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.

Clones of enterotoxigenic and Shiga toxin-producing Escherichia coli implicated in swine enteric colibacillosis in Spain and rates of antibiotic resistance.

Shiga toxin-producing E. coli (STEC) and enterotoxigenic E. coli (ETEC) are the main agents of swine colibacillosis, an infectious disease which implies important economic losses. We characterized here 186 diarrheagenic E. coli from Spanish industrial pig farms (2005-2017) to know which clones were involved in this syndrome, and the rates of antibiotic resistance. The PCR based on pathotype-associated virulence genes determined that 161 of 186 isolates (86.5 %) exhibited the ETEC pathotype, 10 (5.4 %) the STEC pathotype, and 15 (8.1 %) the hybrid ETEC/STEC pathotype. The majority of the isolates showed phylogroup A (85.5 %), clonotype CH11-24 (72 %) and belonged to the clonal complex (CC) 10, including two ETEC clones accounting for around 50 % of the 186 isolates: O157:HNM-A-ST10 (CH11-24), which exhibited mostly the fimbrial antigen F4ac; and O108:HNM-A-ST10 (CH11-24), which exhibited mainly F18. Other associations were O139:H1-E-ST1 (CH2-54) with the STEC pathotype, and both O141:H4-A-CC10 (CH11-24) and O138:HNM-E-ST42 (CH28-41) with ETEC/STEC. We found that 87.1 % of the isolates were multidrug-resistant, including 9% ESBL-producers, with the highest rates to nalidixic acid (82 %), colistin (77 %), ticarcillin (76 %) and ampicillin (76 %). Besides, more than 50 % of isolates showed non-susceptibility to gentamicin, tobramycin, doxycycline, ciprofloxacin, trimethoprim-sufamethoxazole and chloramphenicol. Additionally, 11 out of 17 ESBL-producing isolates were mcr-carriers. Results suggest that O108:HNM-A-ST10 (CH11-24) F18 is an emerging clone taking space left by other classical serogroups. Further follow-up studies on predominant clones in pig colibacillosis are essential for the update of vaccines, as alternative to the use of antibiotics.

Comprehensive Statistical Evaluation of Etest®, UMIC®, MicroScan and Disc Diffusion versus Standard Broth Microdilution: Workflow for an Accurate Detection of Colistin-Resistant and Mcr-Positive E. coli.

Four colistin susceptibility testing methods were compared with the standard broth microdilution (BMD) in a collection of 75 colistin-susceptible and 75 mcr-positive E. coli, including ST131 isolates. Taking BMD as reference, all methods showed similar categorical agreement rates (CA) of circa 90%, and a low number of very major errors (VME) (0% for the MicroScan system and Etest®, 0.7% for UMIC®), except for the disc diffusion assay (breakpoint ≤ 11 mm), which yielded false-susceptible results for 8% of isolates. Of note is the number of mcr-positive isolates (17.3%) categorized as susceptible (≤2 mg/L) by the BMD method, but as resistant by the MicroScan system. ST131 mcr-positive E. coli were identified as colistin-resistant by all MIC-based methods. Our results show that applying the current clinical cut-off (>2 mg/L), many mcr-positive E. coli remain undetected, while applying a threshold of >1 mg/L the sensitivity of detection increases significantly without loss of specificity. We propose two possible workflows, both starting with the MicroScan system, since it is automated and, importantly, it categorized all mcr-positive isolates as colistin-resistant. MicroScan should be followed by either BMD or MIC-based commercial methods for colistin resistance detection; or, alternatively, MicroScan, followed by PCR for the mcr screening.

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-ß-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 resistance.

Chicken and turkey meat: Consumer exposure to multidrug-resistant Enterobacteriaceae including mcr-carriers, uropathogenic E. coli and high-risk lineages such as ST131.

For the first time, this study evaluates consumer exposure via poultry meat to Enterobacteriaceae with capacity to develop severe extraintestinal infections by either bacterial virulence and/or antibiotic resistance traits. The characterization of 256 isolates and the assessment of five parameters, showed that 96 of 100 poultry meat samples from supermarkets of northwest Spain posed ≥ one potential risk: i) 96% carried Enterobacteriaceae resistant to antimicrobials of categories A (64% to monobactams) or B (95% to cephalosporins 3rd and 4rd- generation, quinolones and/or polymixins) of the new categorization of EMA. ii) More than one extended-spectrum-ß-lactamase (ESBL)-producing Enterobacteriaceae species were recovered from 28% of poultry meat. iii) High-risk lineages of E. coli, including multidrug-resistant ST131-H22, were present in 62% of samples. iv) E. coli recovered from 25% of samples conformed the ExPEC status. v) E. coli from 17% of samples satisfied the UPEC status. Of note, the recovery from different samples of two E. coli CC10-A (CH11-54) carrying mcr-1.1-bearing IncX4 plasmids, and four E. coli CC10-A (eae-beta1) of the hybrid pathotype aEPEC/ExPEC. (ESBL)-producing K. pneumoniae were isolated from 27% of samples. In summary, poultry meat microbiota is a source of genetically diverse Enterobacteriaceae, resistant to relevant antimicrobials and potentially pathogenic for consumers.

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 country.

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 MOB H 11/IncHI2-ST4, one MOB P 3/IncX4, and one MOB F 12/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 element.

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, bla CTX-M- 14, bla CTX-M- 32 and bla SHV- 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 isolates.

Sequence Types, Clonotypes, Serotypes, and Virotypes of Extended-Spectrum ß-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 ß-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 virotypes.

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 ST131.

Bilayer polymeric nanocapsules: A formulation approach for a thermostable and adjuvanted E. coli antigen vaccine.

One of the strategies used to improve the immunogenicity of purified protein antigens has relied on their association with synthetic nanocarriers, which, in general, have functioned as simple antigen containers. Here, we present a more advanced strategy based on the design of an antigen nanocarrier at the molecular level. The nanocarrier is composed of a vitamin E oily core, surrounded by two layers: a first layer of chitosan and a second of dextran sulphate. The selected antigen, IutA protein from Escherichia coli, was harboured between the two polymeric layers. The final bilayer nanocapsules had a nanometric size (≈ 200 nm), a negative zeta potential (< -40 mV) and a good antigen association efficiency (≈ 70%). The bilayer architecture led to an improvement on the formulation stability and the controlled release of the associated antigen. Remarkably, after being administered to mice, bilayer nanocapsules elicited higher IgG levels than those obtained with antigen precipitated with Alum. Moreover, freeze-dried nanocapsules were stable at room temperature for, at least, 3 months. These promising data, in addition to their contribution to the development of an uropathogenic E. coli vaccine, has allowed us to validate these novel bilayer nanocapsules as adequate platforms for the delivery of protein antigens.

Co-occurrence of mcr-1, mcr-4 and mcr-5 genes in multidrug-resistant ST10 Enterotoxigenic and Shiga toxin-producing Escherichia coli in Spain (2006-2017).

Colistin is an antimicrobial polypeptide commonly employed for controlling and treating neonatal and post-weaning diarrhoea (PWD) diseases caused by Enterotoxigenic and Shiga toxin-producing Escherichia coli (ETEC and STEC). The plasmid-mediated colistin resistance gene, mcr-1 was first described in late 2015 and, since then, multiple studies have reported its global distribution. In addition, five different mcr genes have been identified. The aim of this study was to characterise the colistin-resistant E. coli clonal groups implicated in PWD in farms of intensive pig production. Of 186 ETEC and STEC isolated in Spain from 2006 to 2017, 76.9% showed resistance to colistin. Of those, 102 were mcr-4 carriers, 37 mcr-1 and 5 mcr-5, with co-occurrence of mcr-1/mcr-4, mcr-1/mcr-5 and mcr-4/mcr-5 in five isolates. Three different mcr-4 variants were detected, including the new mcr-4.4 and mcr-4.5 described here. Interestingly, the clonal group ST10-A (CH11-24) appears to be primarily responsible for the spread of mcr-4. In summary, our results show that the pig industry is an important reservoir of colistin-resistant E. coli, carriers of other additional risk genes, such as blaESBL. These food-producing animals might be spreading a cocktail of multiple resistances, posing a worrisome threat to human health.