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.

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

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

Virulence patterns in a murine sepsis model of ST131 Escherichia coli clinical isolates belonging to serotypes O25b:H4 and O16:H5 are associated to specific virotypes.

Escherichia coli sequence type (ST)131 is an emerging disseminated public health threat implicated in multidrug-resistant extraintestinal infections worldwide. Although the majority of ST131 isolates belong to O25b:H4 serotype, new variants with different serotypes, STs using the discriminative multilocus sequence typing scheme of Pasteur Institute, and virulence-gene profiles (virotypes) have been reported with unknown implications on the pattern of spread, persistence and virulence. The aim of the present study was to compare virulence in a mouse subcutaneous sepsis model of representative ST131 clinical isolates belonging to 2 serotypes (O25b:H4, O16:H5) and nine virotypes and subtypes (A, B, C, D1, D2, D3, D4, D5 and E). Fourteen out of the 23 ST131 isolates tested (61%) killed 90 to 100% of mice challenged, and 18 of 23 (78%) at least 50%. Interestingly, different virulence patterns in association with virotypes were observed, from highly rapid lethality (death in less than 24 h) to low final lethality (death at 7 days) but with presence of an acute inflammation. This is the first study to assess virulence of ST131 isolates belonging to serotype O16:H5, which exhibited virotype C. In spite of their low virulence-gene score, O16:H5 isolates did not show significant differences in final lethality compared with highly virulent O25b:H4 isolates of virotypes A, B and C, but killed mice less rapidly. Significant differences were found, however, between virotypes A, B, C (final lethality ≥80% of mice challenged) and virotypes D, E. Particularly unexpected was the low lethality of the newly assigned virotype E taking into account that it exhibited high virulence-gene score, and the same clonotype H30 as highly virulent O25b:H4 isolates of virotypes A, B and C. In vivo virulence diversity reported in this study would reflect the genetic variability within ST131 clonal group evidenced by molecular typing.

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.

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.

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

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

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

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

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.

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.

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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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.

Enterotoxins, colonization factors, serotypes and antimicrobial resistance of enterotoxigenic Escherichia coli (ETEC) strains isolated from hospitalized children with diarrhea in Bolivia.

UNLABELLED: Enterotoxigenic Escherichia coli (ETEC) is recognized as the main cause of bacterial diarrhoea among children in Asia, Africa and Latin America but less investigated in Bolivia. OBJECTIVE: To determine the relation between enterotoxins, CFs and serotypes as well as the antimicrobial resistance patterns in a set of ETEC isolates collected from hospitalized children with acute diarrhea. In the present study we characterized 43 ETEC strains isolated from 2002 to 2006 from hospitalized children (0-5 years) with acute diarrhea in Bolivia. The strains were analyzed for heat-labile (LT) and heat-stable (ST) enterotoxins and colonization factor (CF) profiles, as well as for serogroups and antimicrobial resistance using phenotypic (ELISA, dot blot, slide agglutination and disc diffusion) and genotypic (Multiplex PCR) methods. Among the ETEC isolates tested, 30 were positive for LT, 3 for STh and 10 for LT/STh. Sixty-five percent (28/43) of the strains expressed one or more CF. The most common CFs were CS17 (n = 8) and CFA/I (n = 8). The phenotypical and genotypical results for toxins and CFs were congruent except for CS21 that was amplified in 10 of the strains by multiplex PCR, but CS21 pili was only detected phenotypically in four of these strains. The ETEC strains had diverse O and H antigens and the most common types were O8:H9 LT CS17 (n = 6; 14%) and O78:HNM LT-ST CFA/I (n = 4; 9%). The analysis of antibiotic resistance showed that 67% (n = 29/43) of the strains were resistant to one or several of the antimicrobial agents tested. Presence of CFs was associated with antibiotic resistance. CONCLUSION: The most common toxin profile was LT 70%, LT/STh 23% and STh 7%. High antimicrobial resistance to ampicillin among serogroups O6, O8 and O78 were the most common.

Enterotoxins, colonization factors, serotypes and antimicrobial resistance of enterotoxigenic Escherichia coli (ETEC) strains isolated from hospitalized children with diarrhea in Bolivia

Enterotoxigenic Escherichia coli (ETEC) is recognized as the main cause of bacterial diarrhoea among children in Asia, Africa and Latin America but less investigated in Bolivia. OBJECTIVE: To determine the relation between enterotoxins, CFs and serotypes as well as the antimicrobial resistance patterns in a set of ETEC isolates collected from hospitalized children with acute diarrhea. In the present study we characterized 43 ETEC strains isolated from 2002 to 2006 from hospitalized children (0-5 years) with acute diarrhea in Bolivia. The strains were analyzed for heat-labile (LT) and heat-stable (ST) enterotoxins and colonization factor (CF) profiles, as well as for serogroups and antimicrobial resistance using phenotypic (ELISA, dot blot, slide agglutination and disc diffusion) and genotypic (Multiplex PCR) methods. Among the ETEC isolates tested, 30 were positive for LT, 3 for STh and 10 for LT/STh. Sixty-five percent (28/43) of the strains expressed one or more CF. The most common CFs were CS17 (n = 8) and CFA/I (n = 8). The phenotypical and genotypical results for toxins and CFs were congruent except for CS21 that was amplified in 10 of the strains by multiplex PCR, but CS21 pili was only detected phenotypically in four of these strains. The ETEC strains had diverse O and H antigens and the most common types were O8:H9 LT CS17 (n = 6; 14 percent) and O78:HNM LT-ST CFA/I (n = 4; 9 percent). The analysis of antibiotic resistance showed that 67 percent (n = 29/43) of the strains were resistant to one or several of the antimicrobial agents tested. Presence of CFs was associated with antibiotic resistance. CONCLUSION: The most common toxin profile was LT 70 percent, LT/STh 23 percent and STh 7 percent. High antimicrobial resistance to ampicillin among serogroups O6, O8 and O78 were the most common.

Emergence of clonal groups O1:HNM-D-ST59, O15:H1-D-ST393, O20:H34/HNM-D-ST354, O25b:H4-B2-ST131 and ONT:H21,42-B1-ST101 among CTX-M-14-producing Escherichia coli clinical isolates in Galicia, northwest Spain.

CTX-M enzymes, mainly CTX-M-14 and CTX-M-15, have emerged as the most prevalent extended-spectrum ß-lactamase (ESBL) type produced by Escherichia coli in Spain, with successful dissemination of clonal group O25b:H4-B2-ST131 producing CTX-M-15 within the hospital and community settings. However, until now CTX-M-14-producing E. coli in Spain had been shown to belong to a wide variety of serotypes with no predominance of a certain clonal group. In the present study, 654 E. coli strains positive for ESBL production obtained between 2005 and 2008 from inpatients and outpatients of four hospitals in Galicia, northwest Spain, were analysed. The strains were characterised with regard to ESBL enzymes, serotype, virulence genes, phylogenetic group, multilocus sequence type, and pulsed-field gel electrophoresis of XbaI-digested DNA. As a result, the emergence of certain clonal groups of extraintestinal pathogenic E. coli producing CTX-M-14 has been detected in this geographic area, including O1:HNM-D-ST59, O15:H1-D-ST393/ST1394, O20:H34/HNM-D-ST354, O25b:H4-B2-ST131 and ONT:H21,42-B1-ST101. These five clonal groups showed a high virulence potential as they harboured more than eight virulence factors, which could explain their successful dissemination.

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.

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.

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.

Distribution of espM and espT among enteropathogenic and enterohaemorrhagic Escherichia coli.

Enterohaemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) translocate dozens of type III secretion system effectors, including the WxxxE effectors Map, EspM and EspT that activate Rho GTPases. While map, which is carried on the LEE pathogenicity island, is absolutely conserved among EPEC and EHEC strains, the prevalence of espM and espT is not known. Here we report the results of a large screen aimed at determining the prevalence of espM and espT among clinical EPEC and EHEC isolates. The results suggest that espM, detected in 51 % of the tested strains, is more commonly found in EPEC and EHEC serogroups that are linked to severe human infections. In contrast, espT was absent from all the EHEC isolates and was found in only 1.8 % of the tested EPEC strains. Further characterization of the virulence gene repertoire of the espT-positive strains led to the identification of a new zeta2 intimin variant. All the espT-positive strains but two contained the tccP gene. espT was first found in Citrobacter rodentium and later in silico in EPEC E110019, which is of particular interest as this strain was responsible for a particularly severe diarrhoeal outbreak in Finland in 1987 that affected 650 individuals in a school complex and an additional 137 associated household members. Comparing the protein sequences of EspT to that of E110019 showed a high level of conservation, with only three strains encoding EspT that differed in 6 amino acids. At present, it is not clear why espT is so rare, and what impact EspM and EspT have on EPEC and EHEC infection.

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.