A total of 514 Shiga toxin-producing Escherichia coli (STEC) isolates from diarrheic and healthy cattle in Spain were characterized in this study. PCR showed that 101 (20%) isolates carried stx 1 genes, 278 (54%) possessed stx 2 genes, and 135 (26%) possessed both stx 1 and stx 2 . Enterohemolysin (ehxA) and intimin (eae) virulence genes were detected in 326 (63%) and in 151 (29%) of the isolates, respectively. STEC isolates belonged to 66 O serogroups and 113 O:H serotypes (including 23 new serotypes). However, 67% were of one of these 15 serogroups (O2, O4, O8, O20, O22, O26, O77, O91, O105, O113, O116, O157, O171, O174, and OX177) and 52% of the isolates belonged to only 10 serotypes (O4:H4, O20:H19, O22:H8, O26:H11, O77:H41, O105:H18, O113:H21, O157:H7, O171:H2, and ONT:H19). Although the 514 STEC isolates belonged to 164 different seropathotypes (associations between serotypes and virulence genes), only 12 accounted for 43% of isolates. Seropathotype O157:H7 stx 2 eae-␥1 ehxA (46 isolates) was the most common, followed by O157:H7 stx 1 stx 2 eae-␥1 ehxA (34 isolates), O113:H21 stx 2 (25 isolates), O22:H8 stx 1 stx 2 ehxA (15 isolates), O26:H11 stx 1 eae-␤1 ehxA (14 isolates), and O77:H41 stx 2 ehxA (14 isolates). Forty-one (22 of serotype O26:H11) isolates had intimin ␤1, 82 O157:H7 isolates possessed intimin ␥1, three O111:H-isolates had intimin type ␥2, one O49:Hstrain showed intimin type ␦, 13 (six of serotype O103:H2) isolates had intimin type and eight (four of serotype O156:H-) isolates had intimin . We have identified a new variant of the eae intimin gene designated (xi) in two isolates of serotype O80:H-. The majority (85%) of bovine STEC isolates belonged to serotypes previously found for human STEC organisms and 54% to serotypes associated with STEC organisms isolated from patients with hemolytic uremic syndrome. Thus, this study confirms that cattle are a major reservoir of STEC strains pathogenic for humans.

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

To ascertain whether on animal farms there reside extended-spectrum ␤-lactamase (ESBL) and plasmidic class C ␤-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.

We have analyzed the prevalence of Shiga toxin-producing Escherichia coli (STEC) in stool specimens of patients with diarrhea or other gastrointestinal alterations from the Xeral-Calde Hospital of Lugo City (Spain). STEC strains were detected in 126 (2.5%) of 5,054 cases investigated, with a progressive increase in the incidence from 0% in 1992 to 4.4% in 1999. STEC O157:H7 was isolated in 24 cases (0.5%), whereas non-O157 STEC strains were isolated from 87 patients (1.7%). STEC strains were (after Salmonella and Campylobacter strains) the third most frequently recovered enteropathogenic bacteria. A total of 126 human STEC isolates were characterized in this study. PCR showed that 43 (34%) isolates carried stx 1 genes, 45 (36%) possessed stx 2 genes and 38 (30%) carried both stx 1 and stx 2 . A total of 88 (70%) isolates carried an ehxA enterohemolysin gene, and 70 (56%) isolates possessed an eae intimin gene (27 isolates with type ␥1, 20 with type ␤1, 8 with type , 5 with type ␥2, and 3 with type ). STEC isolates belonged to 41 O serogroups and 66 O:H serotypes, including 21 serotypes associated with hemolytic uremic syndrome and 30 new serotypes not previously reported among human STEC strains in other studies. Although the 126 STEC isolates belonged to 81 different seropathotypes (associations between serotypes and virulence genes), only four accounted for 31% of isolates. Seropathotype O157:H7 stx 1 stx 2 eae-␥1 ehxA was the most common (13 isolates) followed by O157:H7 stx 2 eae-␥1 ehxA (11 isolates), O26:H11 stx 1 eae-␤1 ehxA (11 isolates), and O111:H-stx 1 stx 2 eae-␥2 ehxA (4 isolates). Our results suggest that STEC strains are a significant cause of human infections in Spain and confirm that in continental Europe, infections caused by STEC non-O157 strains are more common than those caused by O157:H7 isolates. The high prevalence of STEC strains (both O157:H7 and non-O157 strains) in human patients, and their association with serious complications, strongly supports the utilization of protocols for detection of all serotypes of STEC in Spanish clinical microbiology laboratories.

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

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This study characterized 76 atypical enteropathogenic Escherichia coli (aEPEC) strains, previously classified by the eae(+) EAF-negative stx(-) genotype, isolated from children with diarrhea in Brazil. Presence of bfpA and bfpA/perA was detected in 2 and 6 strains, respectively. The expression of bundle-forming pilus (BFP), however, was observed by immunofluorescence in 1 bfpA and 3 bfpA/perA strains, classifying them as typical EPEC (tEPEC). The remaining 72 aEPEC strains were characterized by serotyping, intimin typing, adherence patterns to HEp-2 cells, capacity to induce actin aggregation (fluorescent actin staining test), and antimicrobial resistance. Our results show that aEPEC comprise a very heterogeneous group that does not present any prevalence or association regarding the studied characteristics. It also suggest that tEPEC and aEPEC must not be classified only by the reactivity with the EAF probe, and that the search of other markers present in pEAF, as well as the BFP expression, must be considered for this matter.