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.

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.

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.

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.

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.

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.

Recent emergence of clonal group O25b:K1:H4-B2-ST131 ibeA strains among Escherichia coli poultry isolates, including CTX-M-9-producing strains, and comparison with clinical human isolates.

To discern the possible spread of the Escherichia coli O25b:H4-ST131 clonal group in poultry and the zoonotic potential of avian strains, we made a retrospective search of our strain collection and compared the findings for those strains with the findings for current strains. Thus, we have characterized a collection of 19 avian O25b:H4-ST131 E. coli strains isolated from 1995 to 2010 which, interestingly, harbored the ibeA gene. Using this virulence gene as a criterion for selection, we compared those 19 avian strains with 33 human O25b:H4-ST131 ibeA-positive E. coli strains obtained from patients with extraintestinal infections (1993 to 2009). All 52 O25b:H4-ST131 ibeA-positive E. coli strains shared the fimH, kpsMII, malX, and usp genes but showed statistically significant differences in nine virulence factors, namely, papGIII, cdtB, sat, and kpsMII K5, which were associated with human strains, and iroN, kpsMII K1, cvaC, iss, and tsh, which were associated with strains of avian origin. The XbaI macrorestriction profiles of the 52 E. coli O25b:H4-ST131 ibeA-positive strains revealed 11 clusters (clusters I to XI) of >85% similarity, with four clusters including strains of human and avian origin. Cluster VII (90.9% similarity) grouped 10 strains (7 avian and 3 human strains) that mostly produced CTX-M-9 and that also shared the same virulence profile. Finally, we compared the macrorestriction profiles of the 12 CTX-M-9-producing O25b:H4-ST131 ibeA strains (7 avian and 5 human strains) identified among the 52 strains with those of 15 human O25b:H4-ST131 CTX-M-14-, CTX-M-15-, and CTX-M-32-producing strains that proved to be negative for ibeA and showed that they clearly differed in the level of similarity from the CTX-M-9-producing strains. In conclusion, E. coli clonal group O25b:H4-ST131 ibeA has recently emerged among avian isolates with the new acquisition of the K1 capsule antigen and includes CTX-M-9-producing strains. This clonal group represents a real zoonotic risk that has crossed the barrier between human and avian hosts.

Isolation and characterization of potentially pathogenic antimicrobial-resistant Escherichia coli strains from chicken and pig farms in Spain.

To ascertain whether on animal farms there reside extended-spectrum beta-lactamase (ESBL) and plasmidic class C beta-lactamase-producing Escherichia coli isolates potentially pathogenic for humans, phylogenetic analyses, pulsed-field gel electrophoresis (PFGE) typing, serotyping, and virulence genotyping were performed for 86 isolates from poultry (57 isolates) and pig (29 isolates) farms. E. coli isolates from poultry farms carried genes encoding enzymes of the CTX-M-9 group as well as CMY-2, whereas those from pig farms mainly carried genes encoding CTX-M-1 enzymes. Poultry and pig isolates differed significantly in their phylogenetic group assignments, with phylogroup A predominating in pig isolates and phylogroup D predominating in avian isolates. Among the 86 farm isolates, 23 (26.7%) carried two or more virulence genes typical of extraintestinal pathogenic E. coli (ExPEC). Of these, 20 were isolated from poultry farms and only 3 from pig farms. Ten of the 23 isolates belonged to the classic human ExPEC serotypes O2:H6, O2:HNM, O2:H7, O15:H1, and O25:H4. Despite the high diversity of serotypes and pulsotypes detected among the 86 farm isolates, 13 PFGE clusters were identified. Four of these clusters contained isolates with two or more virulence genes, and two clusters exhibited the classic human ExPEC serotypes O2:HNM (ST10) and O2:H6 (ST115). Although O2:HNM and O2:H6 isolates of human and animal origins differed with respect to their virulence genes and PFGE pulsotypes, the O2:HNM isolates from pigs showed the same sequence type (ST10) as those from humans. The single avian O15:H1 isolate was compared with human clinical isolates of this serotype. Although all were found to belong to phylogroup D and shared the same virulence gene profile, they differed in their sequence types (ST362-avian and ST393-human) and PFGE pulsotypes. Noteworthy was the detection, for the first time, in poultry farms of the clonal groups O25b:H4-ST131-B2, producing CTX-M-9, and O25a-ST648-D, producing CTX-M-32. The virulence genes and PFGE profiles of these two groups were very similar to those of clinical human isolates. While further studies are required to determine the true zoonotic potential of these clonal groups, our results emphasize the zoonotic risk posed especially by poultry farms, but also by pig farms, as reservoirs of ESBL- and CMY-2-encoding E. coli.

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.

Genes related to long polar fimbriae of pathogenic Escherichia coli strains as reliable markers to identify virulent isolates.

Lpf (stands for long polar fimbriae) is one of the few adhesive factors of enterohemorrhagic Escherichia coli O157:H7 associated with colonization of the intestine. E. coli O157:H7 strains possess two lpf loci encoding highly regulated fimbrial structures. Database analysis of the genes encoding the major fimbrial subunits demonstrated that they are present in commensal as well as pathogenic (both intestinal and extraintestinal) E. coli strains and in Salmonella strains and that the lpfA1 and lpfA2 genes are highly prevalent among LEE (locus of enterocyte effacement)-positive E. coli strains associated with severe and/or epidemic disease. Further DNA sequence analysis of the lpfA1 and lpfA2 genes from different attaching-and-effacing E. coli strains has led us to the identification of several polymorphisms and the classification of the major fimbrial subunits into distinct variants. Using collections of pathogenic E. coli isolates from Europe and Latin America, we demonstrated that the different lpfA types are associated with the presence of specific intimin (eae) adhesin variants and, most importantly, that they are found in specific E. coli pathotypes. Our results showed that the use of these fimbrial genes as markers, in combination with the different intimin types, resulted in a specific test for the identification of E. coli O157:H7, distinguishing it from other pathogenic E. coli strains.

Molecular epidemiology of Escherichia coli producing extended-spectrum {beta}-lactamases in Lugo (Spain): dissemination of clone O25b:H4-ST131 producing CTX-M-15.

OBJECTIVES: Having shown that the Xeral-Calde Hospital in Lugo (Spain) has been concerned by Escherichia coli clone O25:H4-ST131 producing CTX-M-15 (Nicolas-Chanoine et al. J Antimicrob Chemother 2008; 61: 273-81), the present study was carried out to evaluate the prevalence of this clone among the extended-spectrum beta-lactamase (ESBL)-producing E. coli isolates and also to molecularly characterize the E. coli isolates producing ESBL other than CTX-M-15. METHODS: In the first part of this study, 105 ESBL-producing E. coli isolates (February 2006 to March 2007) were characterized with regard to ESBL enzymes, serotypes, virulence genes, phylogenetic groups, multilocus sequence typing (MLST) and PFGE. In the second part of this study, 249 ESBL-producing E. coli isolates (April 2007 to May 2008) were investigated only for the detection of clone O25b:H4-ST131 producing CTX-M-15 using a triplex PCR developed in this study and based on the detection of the new operon afa FM955459 and the targets rfbO25b and 3' end of the bla(CTX-M-15) gene. RESULTS: Of the 105 ESBL-producing E. coli isolates, 60 (57.1%) were positive for CTX-M-14, 23 (21.9%) for CTX-M-15, 10 (9.5%) for SHV-12 and 7 (6.7%) for CTX-M-32. Serotypes, virulence genes, phylogenetic groups and molecular typing by PFGE demonstrated high homogeneity within those producing CTX-M-15 and high diversity within E. coli producing CTX-M-14 and other ESBLs. By PFGE, CTX-M-15-producing E. coli isolates O25b:H4 belonging to the phylogenetic group B2 and MLST profile ST131 were grouped in the same cluster. The epidemic strain of clone O25b:H4-ST131 represented 23.1%, 22.5% and 20.0% of all ESBL-producing E. coli isolated in 2006, 2007 and 2008, respectively. CONCLUSIONS: CTX-M-type ESBLs, primarily CTX-M-14 and CTX-M-15, have emerged as the predominant types of ESBL produced by E. coli isolates in Lugo. In view of the reported findings, long-term care facilities for elderly people may represent a significant reservoir for E. coli clone O25b:H4-ST131 producing CTX-M-15. The triplex PCR developed in this work will be useful for rapid and simple detection of this clone.

Longitudinal study of Shiga toxin-producing Escherichia coli shedding in sheep feces: persistence of specific clones in sheep flocks.

To provide information on the persistence and maintenance of colonization with Shiga toxin-producing Escherichia coli (STEC) in sheep, pulsed-field gel electrophoresis analysis of STEC isolates (n = 145) belonging to serogroups O5, O91, and O146 from 39 healthy animals was performed in a 12-month longitudinal study carried out with four sheep flocks. At the flock level as well as the individual-animal level, the same clones were obtained on sampling occasions separated by as much as 11 months.

Extraintestinal pathogenic Escherichia coli O1:K1:H7/NM from human and avian origin: detection of clonal groups B2 ST95 and D ST59 with different host distribution.

BACKGROUND: Extraintestinal pathogenic Escherichia coli (ExPEC) strains of serotype O1:K1:H7/NM are frequently implicated in neonatal meningitis, urinary tract infections and septicemia in humans. They are also commonly isolated from colibacillosis in poultry. Studies to determine the similarities of ExPEC from different origins have indicated that avian strains potentially have zoonotic properties. RESULTS: A total of 59 ExPEC O1:K1:H7/NM isolates (21 from avian colibacillosis, 15 from human meningitis, and 23 from human urinary tract infection and septicemia) originated from four countries were characterized by phylogenetic PCR grouping, Multilocus Sequence Typing (MLST), Pulsed Field Gel Electrophoresis (PFGE) and genotyping based on several genes known for their association with ExPEC or avian pathogenic Escherichia coli (APEC) virulence.APEC and human ExPEC isolates differed significantly in their assignments to phylogenetic groups, being phylogroup B2 more prevalent among APEC than among human ExPEC (95% vs. 53%, P = 0.001), whereas phylogroup D was almost exclusively associated with human ExPEC (47% vs. 5%, P = 0.0000). Seven virulence genes showed significant differences, being fimAvMT78 and sat genes linked to human isolates, while papGII, tsh, iron, cvaC and iss were significantly associated to APEC. By MLST, 39 of 40 ExPEC belonging to phylogroup B2, and 17 of 19 belonging to phylogroup D exhibited the Sequence Types (STs) ST95 and ST59, respectively. Additionally, two novel STs (ST1013 and ST1006) were established. Considering strains sharing the same ST, phylogenetic group, virulence genotype and PFGE cluster to belong to the same subclone, five subclones were detected; one of those grouped six strains of human and animal origin from two countries. CONCLUSION: Present results reveal that the clonal group B2 O1:K1:H7/NM ST95, detected in strains of animal and human origin, recovered from different dates and geographic sources, provides evidence that some APEC isolates may act as potential pathogens for humans and, consequently, poultry as a foodborne source, suggesting no host specificity for this type of isolates. A novel and important finding has been the detection of the clonal group D O1:K1:H7/NM ST59 almost exclusively in humans, carrying pathogenic genes linked to the phylogenetic group D. This finding would suggest D O1:K1:H7/NM ST59 as a host specific pathotype for humans.

Biofilm formation as a novel phenotypic feature of adherent-invasive Escherichia coli (AIEC).

BACKGROUND: Crohn's disease (CD) is a high morbidity chronic inflammatory disorder of unknown aetiology. Adherent-invasive Escherichia coli (AIEC) has been recently implicated in the origin and perpetuation of CD. Because bacterial biofilms in the gut mucosa are suspected to play a role in CD and biofilm formation is a feature of certain pathogenic E. coli strains, we compared the biofilm formation capacity of 27 AIEC and 38 non-AIEC strains isolated from the intestinal mucosa. Biofilm formation capacity was then contrasted with the AIEC phenotype, the serotype, the phylotype, and the presence of virulence genes. RESULTS: Specific biofilm formation (SBF) indices were higher amongst AIEC than non-AIEC strains (P = 0.012). In addition, 65.4% of moderate to strong biofilms producers were AIEC, whereas 74.4% of weak biofilm producers were non-AIEC (P = 0.002). These data indicate that AIEC strains were more efficient biofilm producers than non-AIEC strains. Moreover, adhesion (P = 0.009) and invasion (P = 0.003) indices correlated positively with higher SBF indices. Additionally, motility (100%, P < 0.001), H1 type flagellin (53.8%, P < 0.001), serogroups O83 (19.2%, P = 0.008) and O22 (26.9%, P = 0.001), the presence of virulence genes such as sfa/focDE (38.5%, P = 0.003) and ibeA (26.9%, P = 0.017), and B2 phylotype (80.8%, P < 0.001) were frequent characteristics amongst biofilm producers. CONCLUSION: The principal contribution of the present work is the finding that biofilm formation capacity is a novel, complementary pathogenic feature of the recently described AIEC pathovar. Characterization of AIEC specific genetic determinants, and the regulatory pathways, involved in biofilm formation will likely bring new insights into AIEC pathogenesis.

Invasiveness as a putative additional virulence mechanism of some atypical Enteropathogenic Escherichia coli strains with different uncommon intimin types.

BACKGROUND: Enteropathogenic Escherichia coli (EPEC) produce attaching/effacing (A/E) lesions on eukaryotic cells mediated by the outer membrane adhesin intimin. EPEC are sub-grouped into typical (tEPEC) and atypical (aEPEC). We have recently demonstrated that aEPEC strain 1551-2 (serotype O non-typable, non-motile) invades HeLa cells by a process dependent on the expression of intimin sub-type omicron. In this study, we evaluated whether aEPEC strains expressing other intimin sub-types are also invasive using the quantitative gentamicin protection assay. We also evaluated whether aEPEC invade differentiated intestinal T84 cells. RESULTS: Five of six strains invaded HeLa and T84 cells in a range of 13.3%-20.9% and 5.8%-17.8%, respectively, of the total cell-associated bacteria. The strains studied were significantly more invasive than prototype tEPEC strain E2348/69 (1.4% and 0.5% in HeLa and T84 cells, respectively). Invasiveness was confirmed by transmission electron microscopy. We also showed that invasion of HeLa cells by aEPEC 1551-2 depended on actin filaments, but not on microtubules. In addition, disruption of tight junctions enhanced its invasion efficiency in T84 cells, suggesting preferential invasion via a non-differentiated surface. CONCLUSION: Some aEPEC strains may invade intestinal cells in vitro with varying efficiencies and independently of the intimin sub-type.