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.

Prevalence and serotypes of Shiga toxin-producing Escherichia coli (STEC) in dairy cattle from Northern Portugal.

The prevalence of Shiga toxin (Stx)-producing Escherichia coli (STEC) was determined by evaluating its presence in faecal samples from 155 heifers, and 254 dairy cows in 21 farms at North of Portugal sampled between December 2017 and June 2019. The prevalence of STEC in heifers (45%) was significantly higher than in lactating cows (16%) (p<0.05, Fisher exact test statistic value is <0.00001). A total of 133 STEC were isolated, 24 (13.8%) carried Shiga-toxin 1 (stx1) genes, 69 (39.7%) carried Shiga-toxin 2 (stx2) genes, and 40 (23%) carried both stx1 and stx2. Intimin (eae) virulence gene was detected in 29 (21.8%) of the isolates. STEC isolates belonged to 72 different O:H serotypes, comprising 40 O serogroups and 23 H types. The most frequent serotypes were O29:H12 (15%) and O113:H21 (5.2%), found in a large number of farms. Two isolates belonged to the highly virulent serotypes associated with human disease O157:H7 and O26:H11. Many other bovine STEC serotypes founded in this work belonged to serotypes previously described as pathogenic to humans. Thus, this study highlights the need for control strategies that can reduce STEC prevalence at the farm level and, thus, prevent food and environmental contamination.

Molecular Characteristics of Extraintestinal Pathogenic E. coli (ExPEC), Uropathogenic E. coli (UPEC), and Multidrug Resistant E. coli Isolated from Healthy Dogs in Spain. Whole Genome Sequencing of Canine ST372 Isolates and Comparison with Human Isolates Causing Extraintestinal Infections.

Under a one health perspective and the worldwide antimicrobial resistance concern, we investigated extraintestinal pathogenic Escherichia coli (ExPEC), uropathogenic E. coli (UPEC), and multidrug resistant (MDR) E. coli from 197 isolates recovered from healthy dogs in Spain between 2013 and 2017. A total of 91 (46.2%) isolates were molecularly classified as ExPEC and/or UPEC, including 50 clones, among which (i) four clones were dominant (B2-CH14-180-ST127, B2-CH52-14-ST141, B2-CH103-9-ST372 and F-CH4-58-ST648) and (ii) 15 had been identified among isolates causing extraintestinal infections in Spanish and French humans in 2015 and 2016. A total of 28 (14.2%) isolates were classified as MDR, associated with B1, D, and E phylogroups, and included 24 clones, of which eight had also been identified among the human clinical isolates. We selected 23 ST372 strains, 21 from healthy dogs, and two from human clinical isolates for whole genome sequencing and built an SNP-tree with these 23 genomes and 174 genomes (128 from canine strains and 46 from human strains) obtained from public databases. These 197 genomes were segregated into six clusters. Cluster 1 comprised 74.6% of the strain genomes, mostly composed of canine strain genomes (p < 0.00001). Clusters 4 and 6 also included canine strain genomes, while clusters 2, 3, and 5 were significantly associated with human strain genomes. Finding several common clones and clone-related serotypes in dogs and humans suggests a potentially bidirectional clone transfer that argues for the one health perspective.

Clonal Structure, Virulence Factor-encoding Genes and Antibiotic Resistance of Escherichia coli, Causing Urinary Tract Infections and Other Extraintestinal Infections in Humans in Spain and France during 2016.

Escherichia coli is the main pathogen responsible for extraintestinal infections. A total of 196 clinical E. coli consecutively isolated during 2016 in Spain (100 from Lucus Augusti hospital in Lugo) and France (96 from Beaujon hospital in Clichy) were characterized. Phylogroups, clonotypes, sequence types (STs), O:H serotypes, virulence factor (VF)-encoding genes and antibiotic resistance were determined. Approximately 10% of the infections were caused by ST131 isolates in both hospitals and approximately 60% of these infections were caused by isolates belonging to only 10 STs (ST10, ST12, ST58, ST69, ST73, ST88, ST95, ST127, ST131, ST141). ST88 isolates were frequent, especially in Spain, while ST141 isolates significantly predominated in France. The 23 ST131 isolates displayed four clonotypes: CH40-30, CH40-41, CH40-22 and CH40-298. Only 13 (6.6%) isolates were carriers of extended-spectrum beta-lactamase (ESBL) enzymes. However, 37.2% of the isolates were multidrug-resistant (MDR). Approximately 40% of the MDR isolates belonged to only four of the dominant clones (B2-CH40-30-ST131, B2-CH40-41-ST131, C-CH4-39-ST88 and D-CH35-27-ST69). Among the remaining MDR isolates, two isolates belonged to B2-CH14-64-ST1193, i.e., the new global emergent MDR clone. Moreover, a hybrid extraintestinal pathogenic E.coli (ExPEC)/enteroaggregative isolate belonging to the A-CH11-54-ST10 clone was identified.

High Prevalence of ST131 Subclades C2-H30Rx and C1-M27 Among Extended-Spectrum ß-Lactamase-Producing Escherichia coli Causing Human Extraintestinal Infections in Patients From Two Hospitals of Spain and France During 2015.

The present study was carried out to evaluate the prevalence of sequence type 131 (ST131) among 188 extended-spectrum ß-lactamase-producing Escherichia coli (ESBL-EC) collected in 2015 in Lucus Augusti University hospital (Lugo, Spain) and AP-HP Beaujon hospital (Clichy, France) with regard to other STs and to characterize, the types of ESBL produced, serotypes, virulence factor (VF)-encoding genes and the ST131 clades and subclades. ST131 was detected in 33 (39.1%) and 46 (47.9%) of the isolates in Lucus Augusti and Beaujon, respectively. The 109 remaining isolates displayed 57 other STs, the following STs being displayed by at least three isolates: ST10 (8 isolates), ST23 (3), ST38 (4), ST58 (3), ST88 (5), ST95 (4), ST167 (3), ST354 (5), ST361 (3), ST410 (6), ST648 (4), ST744 (3), and ST1615 (6). ST354, ST410, and ST1615 were significantly (P < 0.05) more frequent in Lucus Augusti (5.4%, 6.5%, and 6.5%) than in Beaujon (0% for the three STs). The new globally emerging clone ST1193 among extraintestinal clinical ESBL-EC was identified in one isolate from France and one from Spain. CTX-M-15 was the commonest ESBL detected in the two hospitals (44.6% in Lucus Augusti and 50.0% in Beaujon). CTX-M-14 was significantly (P = 0.0003) more frequent in Lucus Augusti (31.5%) than in Beaujon (10.4%), whereas CTX-M-1 (20.8 vs. 7.6%; P = 0.008) and CTX-M-27 (15.6 vs. 6.5%; P = 0.0389) were more frequent in Beaujon than in Lucus Augusti. The ST131 isolates showed a higher virulence score (mean 13.367) compared with the non-ST131 isolates (mean 7.661) (P < 0.001). Among the 79 ST131 isolates, most of them (52; 65.8%) belonged to subclade C2 (also known as subclone H30Rx) followed by those belonging to subclade C1 (cluster C1-M27: 16 isolates, 20.3%; cluster non-C1-M27: 6 isolates, 7.6%) and clade A (4 isolates; 5.1%). The C2 subclade isolates showed a higher VF-encoding gene score (mean 14.250) compared with the C1-M27 cluster isolates (mean 10.875) (P < 0.001). In conclusion, this study highlights the epidemiological differences between the ESBL-EC isolated from two hospitals of France and Spain obtain in 2015 and reports, for the first time, the presence of clone ST1193 in Spain.

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.

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.

Poultry as reservoir for extraintestinal pathogenic Escherichia coli O45:K1:H7-B2-ST95 in humans.

Escherichia coli strains O45:K1:H7 are implicated in severe human infections such as meningitis. Since an increasing prevalence of serogroup O45 among avian pathogenic (APEC) and human extraintestinal pathogenic (ExPEC) E. coli strains isolated in Spain have been noticed, the aims of the present study were to investigate similarities between poultry and human O45 isolates, and to investigate the evolutionary relationship of ST95 types. The genetic relatedness and virulence gene profiles of 55 O45 APEC obtained from an avian colibacillosis collection (1991-2011) and 19 human O45 ExPEC from a human septicemic/uropathogenic (UPEC) E. coli collection (1989-2010) were determined by multilocus sequence typing (MLST), pulsed-field-gel-electrophoresis (PFGE), ECOR phylogrouping, and PCR-based genotyping. Two main clonal groups were established. The most prevalent and highly pathogenic O45:K1:H7-B2-ST95 shows a successful persistence since the 90s to the present, with parallel evolution both in human and poultry, on the basis of their PFGE and virulence gene profile similarities (9 human strains and 15 avian strains showed ≥85% PFGE identity). Comparison of this group with other ST95 closely related members (O1:K1:H7 and O18:K1:H7 isolates from our collections) shows pathogenic specialization through conserved virulence genotypes. The other prevalent O45 clonal group characterized in this study, the O45:HNM/H19-D-ST371/ST2676 was only detected in APEC strains suggesting host specificity. In conclusion, poultry could be acting as a reservoir of O45:K1:H7-B2-ST95 and other pathogenic ST95 serotypes in humans. Further studies would be necessary to clarify if pathogenic mechanisms used by ST95 strains are the same in avian and human hosts.

Emergence of new variants of ST131 clonal group among extraintestinal pathogenic Escherichia coli producing extended-spectrum ß-lactamases.

Having shown that Lucus Augusti Hospital in Lugo, Spain, has been affected by Escherichia coli clone O25:H4-ST131 producing CTX-M-15, the present study was carried out to evaluate the prevalence of this clone among the extended-spectrum ß-lactamase (ESBL)-producing E. coli isolates and to identify novel variants of this clone. Of the 77 ESBL-producing E. coli isolated between January and April 2012, 47 (61%) were identified as belonging to the ST131 clonal group, comprising 38 O25b:H4-B2-ST131 (34 CTX-M-15, 2 CTX-M-14, 1 CTX-M-1 and 1 CTX-M-27), 7 O-non-typeable:H4-B2-ST131 (all CTX-M-15) and 2 O16:H5-B2-ST131 (both CTX-M-14). The 47 isolates of ST131 exhibited a significantly higher virulence score (mean of 9.1 virulence genes) compared with the 30 non-ST131 isolates (mean of 4.3 virulence genes). A new virulence profile (fimH, papG II, sat, cnf1, hlyA, iucD, kpsM II-K5, traT, malX, usp) was detected among O25b:H4-B2-ST131 isolates belonging to the new Pasteur sequence type PST621. To our knowledge, this is the first study to report the O-non-typeable:H4-B2-ST131 and O16:H5-B2-ST131 variants in Europe.

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.

Prevalence of avian pathogenic Escherichia coli (APEC) clone harboring sfa gene in Brazil.

Escherichia coli sfa+ strains isolated from poultry were serotyped and characterized by polymerase chain reaction (PCR) and amplified fragment length polymorphism (AFLP). Isolates collected from 12 Brazilian poultry farms mostly belonged to serogroup O6, followed by serogroups O2, O8, O21, O46, O78, O88, O106, O111, and O143. Virulence genes associated were: iuc 90%, fim 86% neuS 60%, hly 34%, tsh 28%, crl/csg 26%, iss 26%, pap 18%, and 14% cnf. Strains from the same farm presented more than one genotypic pattern belonging to different profiles in AFLP. AFLP showed a clonal relation between Escherichia coli sfa+ serogroup O6. The virulence genes found in these strains reveal some similarity with extraintestinal E. coli (ExPEC), thus alerting for potential zoonotic risk.

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.

Comparison of ruminant and human attaching and effacing Escherichia coli (AEEC) strains.

The presence of 12 genes associated with virulence in human attaching and effacing Escherichia coli (AEEC) was studied within a collection of 20 enterohemorrhagic E. coli (EHEC) and 206 atypical enteropathogenic E. coli (EPEC) isolated from ruminants. In addition, virulence genes and the clonal relationship of 49 atypical EPEC O26 strains isolated from humans and ruminants were compared to clarify whether ruminants serve as a reservoir of atypical EPEC for humans. A great diversity in the content of virulence gene was found. Thus, the espH, espG and map genes were detected in more than 85% of ruminant AEEC strains; the tccP2, espI, efa1/lifA, ehxA and paa genes were present in 50-70% of strains; and other genes such as tccP, espP, katP and toxB were detected in <25% of strains. EHEC strains contained more virulence genes than atypical EPEC strains. Our results suggest for the first time that the efa1/lifA gene is associated with diarrhea in newborn ruminants and that the AEEC strains with the H11 flagellar antigen are potentially more virulent than the non-H11 AEEC strains. Importantly, we identified a new intimin variant gene, eaeρ, in three ruminant atypical EPEC strains. The comparison of ruminant and human EPEC O26 strains showed that some ruminant strains possess virulence gene profiles and pulse-field gel electrophoresis pulsotypes similar to those of human strains. In conclusion, our data suggest that atypical EPEC is a heterogeneous group with different pathogenic potential and that ruminants could serve as a reservoir of atypical EPEC for humans.

Emerging avian pathogenic Escherichia coli strains belonging to clonal groups O111:H4-D-ST2085 and O111:H4-D-ST117 with high virulence-gene content and zoonotic potential.

The present study characterizes, for the first time, two emerging avian pathogenic Escherichia coli (APEC) clonal groups of serogroup O111: O111:H4-D-ST117 and O111:H4-D-ST2085. The clonal group O111:H4-D-ST117 was already present in APEC strains isolated between 1991 and 2000, and was still present in strains isolated between 2004 and 2009, showing long time evolution according to the virulence-gene differences and macrorestriction profiles. Among ST117 strains, two virulence profiles could be distinguished: papG II-positive tsh-negative strains which satisfied criteria for extraintestinal pathogenic E. coli (ExPEC), and papG II-negative tsh-positive strains without ExPEC status. Interestingly, we have detected a human septicemic O111:H4-D-ST117 ExPEC strain isolated from a hemocultive in 2000 whose macrorestriction profile showed >85% similarity with four APEC strains of the study. The clonal group O111:H4-D-ST2085 was exclusively detected in 17 APEC strains isolated in 2008 and 2009, and showed short time evolution based on its homogeneity since all were nalidixic acid-resistant, all had ExPEC status, and most carried papG II and tsh genes. From the clinical point of view, O111:H4-D-ST2085 seems a successful clonal group that could be the result of the epidemiological evolution of O111:H4-D-ST117. Due to the increasing prevalence of both clonal groups among clinical APEC isolates, their high virulence-gene content, and zoonotic potential, we suggest them as possible candidates for the development of a future vaccine against avian colibacillosis.

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.

Sorogrupos e genes de virulência em Escherichia coli isoladas de psitacídeos / Serogroups and virulence genes of Escherichia coli isolated from psittacine birds

Escherichia coli isolates from 24 sick psittacine birds were serogrouped and investigated for the presence of genes encoding the following virulence factors: attaching and effacing (eae), enteropathogenic E. coli EAF plasmid (EAF), pili associated with pyelonephritis (pap), S fimbriae (sfa), afimbrial adhesin (afa), capsule K1 (neu), curli (crl, csgA), temperature-sensitive hemagglutinin (tsh), enteroaggregative heat-stable enterotoxin-1 (astA), heat-stable enterotoxin -1 heat labile (LT) and heat stable (STa and STb) enterotoxins, Shiga-like toxins (stx1 and stx2), cytotoxic necrotizing factor 1 (cnf1), haemolysin (hly), aerobactin production (iuc) and serum resistance (iss). The results showed that the isolates belonged to 12 serogroups: O7; O15; O21; O23; O54; O64; O76; O84; O88; O128; O152 and O166. The virulence genes found were: crl in all isolates, pap in 10 isolates, iss in seven isolates, csgA in five isolates, iuc and tsh in three isolates and eae in two isolates. The combination of virulence genes revealed 11 different genotypic patterns. All strains were negative for genes encoding for EAF, EAEC, K1, sfa, afa, hly, cnf, LT, STa, STb, stx1 and stx2. Our findings showed that some E. coli isolated from psittacine birds present the same virulence factors as avian pathogenic E. coli (APEC), uropathogenic E. coli (UPEC) and Enteropathogenic E. coli (EPEC) pathotypes.

Amostras de Escherichia coli isoladas de 24 psitacídeos doentes foram sorogrupadas e investigadas para a presença de genes que codificam os seguintes fatores de virulência: attaching e effacing (eae), plasmídeo EAF (EAF), pili associado à pielonefrite (pap), fímbria S (sfa), adesina afimbrial (afa), cápsula K1 (neu), curli (crl, csgA), hemaglutinina termosensível (tsh), enterotoxina termo-estável 1 de E. coli enteroagregativa (astA), toxina termolábil (LT) e toxina termoestável (STa e STb), Shiga-like toxinas (stx1 e stx2), fator citotóxico necrotizante 1 (cnf1), hemolisina (hly), produção de aerobactina (iuc) e resistência sérica (iss). Os resultados mostraram que os isolados pertenciam a 12 sorogrupos: O7; O15; O21; O23; O54; O64; O76; O84; O88; O128; O152 e O166. Os genes de virulência encontrados foram: crl em todos os isolados, pap em 10 isolados, iss em sete isolados, csgA em cinco isolados, iuc e tsh em três isolados e eae em dois isolados. A combinação dos genes de virulência revelou 11 perfis genotípicos distintos. Todas as amostras foram negativas para os genes que codificam EAF, EAEC, K1, sfa, afa, hly, cnf, LT, STa, STb, stx1 e stx2. Estes resultados demonstraram que algumas amostras de E. coli isoladas de psitacídeos apresentam os mesmos fatores de virulência presentes nos patotipos de E. coli patogênicas para aves (APEC), uropatogênicas (UPEC) e E. coli enteropatogênicas (EPEC).

Characteristics of the Shiga-toxin-producing enteroaggregative Escherichia coli O104: H4German 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 (AU)

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Distribution of classical and nonclassical virulence genes in enterotoxigenic Escherichia coli isolates from Chilean children and tRNA gene screening for putative insertion sites for genomic islands.

Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrhea. Three adhesins (Tia, TibA, EtpA), an iron acquisition system (Irp1, Irp2, and FyuA), a GTPase (LeoA), and an autotransporter (EatA) are ETEC virulence-related proteins that, in contrast to the classical virulence factors (enterotoxins and fimbrial colonization factors) have not heretofore been targets in characterizing isolates from epidemiological studies. Here, we determined the occurrence of these nonclassical virulence genes in 103 ETEC isolates from Chilean children with diarrhea and described their association with O serogroups and classical virulence determinants. Because tia, leoA, irp2, and fyuA are harbored by pathogenicity islands inserted into the selC and asnT tRNA genes (tDNAs), we analyzed the regions flanking these loci. Ten additional tDNAs were also screened to identify hot spots for genetic insertions. Associations between the most frequent serogroups and classical colonization factor (CF)-toxin profiles included O6/LT-STh/CS1-CS3-CS21 (i.e., O6 serogroup, heat-labile [LT] and human heat-stable [STh] enterotoxins, and CFs CS1, -3 and -21), O6/LT-STh/CS2-CS3-CS21, and O104-O127/STh/CFAI-CS21. The eatA and etpA genes were detected in more than 70% of the collection, including diverse serogroups and virulence profiles. Sixteen percent of the ETEC strains were negative for classical and nonclassical adhesins, suggesting the presence of unknown determinants of adhesion. The leuX, thrW, and asnT tDNAs were disrupted in more than 65% of strains, suggesting they are hot spots for the insertion of mobile elements. Sequences similar to integrase genes were identified next to the thrW, asnT, pheV, and selC tDNAs. We propose that the eatA and etpA genes should be included in characterizations of ETEC isolates in future epidemiological studies to determine their prevalence in other geographical regions. Sequencing of tDNA-associated genetic insertions might identify new ETEC virulence determinants.

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.