Epidemiología molecular y virulencia de cepas de Escherichia coli del grupo clonal ST131: comparación de los subclones 016:H5 H41 y 025b:H4 H30/H30-Rx

La prevalencia y la epidemiología molecular de cepas de E. coli productoras de β-lactamasas de espectro extendido (ECBLEE) está cambiando rápidamente. En los últimos años, la producción de BLEE en E. coli se ha incrementado significativamente debido especialmente a la expansión de las enzimas de tipo CTX-M. La pandemia actual es debida en gran medida a clones exitosos de alto riesgo, entre los que destaca el grupo clonal ST131 asociado a la producción de CTX-M-15. El éxito de las cepas del grupo clonal ST131 se podría explicar por la adquisición de genes de virulencia y de resistencia a antibióticos, por la enorme diversidad genética que presentan y por su capacidad de colonización a nivel intestinal. Sin embargo, se desconoce si todas las variantes/linajes/subclones de este grupo clonal emergente poseen el mismo grado de virulencia y de capacidad expansiva. La presente tesis doctoral comprende tres estudios (Dahbi G, Mora A et al. 2013 Int J Antimicrob Agents 42:347-351; Dahbi G, Mora A et al. 2014 Int J Med Microbiol 304:1247-1257; Mora A, Dahbi G et al. 2014 PLOS One 9(1):e87025) realizados con los siguientes objetivos: (A) Evaluar la prevalencia real del grupo clonal ST131 identificando todas sus variantes conocidas. (B) Caracterizar y comparar los diferentes subclones del grupo clonal ST131, determinando sus serotipos O:H, genes de virulencia, perfiles de PFGE, secuencias tipo del esquema del Instituto Pasteur, alelos fimH y sus resistencias. (C) Estudiar la virulencia in vivo de las cepas ST131 pertenecientes a los diferentes subclones empleando un modelo de sepsis murino

Serotypes, intimin variants and other virulence factors of eae positive Escherichia coli strains isolated from healthy cattle in Switzerland. Identification of a new intimin variant gene (eae-η2)

BACKGROUND: Enteropathogenic Escherichia coli (EPEC) and Shigatoxin-producing Escherichia coli (STEC) share the ability to introduce attaching-and-effacing (A/E) lesions on intestinal cells. The genetic determinants for the production of A/E lesions are located on the locus of enterocyte effacement (LEE), a pathogenicity island that also contains the genes encoding intimin (eae). This study reports information on the occurrence of eae positive E. coli carried by healthy cattle at the point of slaughter, and on serotypes, intimin variants, and further virulence factors of isolated EPEC and STEC strains. RESULTS: Of 51 eae positive bovine E. coli strains, 59% were classified as EPEC and 41% as STEC. EPEC strains belonged to 18 O:H serotypes, six strains to typical EPEC serogroups. EPEC strains harbored a variety of intimin variants with eae-β1 being most frequently found. Moreover, nine EPEC strains harbored astA (EAST1), seven bfpA (bundlin), and only one strain was positive for the EAF plasmid. We have identified a new intimin gene (η2) in three bovine bfpA and astA-positive EPEC strains of serotype ONT:H45. STEC strains belonged to seven O:H serotypes with one serotype (O103:H2) accounting for 48% of the strains. The majority of bovine STEC strains (90%) belonged to five serotypes previously reported in association with hemolytic uremic syndrom (HUS), including one O157:H7 STEC strain. STEC strains harbored four intimin variants with eae-ε1 and eae-γ1 being most frequently found. Moreover, the majority of STEC strains carried only stx1 genes (13 strains), and was positive for ehxA (18 strains) encoding for Enterohemolysin. Four STEC strains showed a virulence pattern characteristic of highly virulent human strains (stx2 and eae positive). CONCLUSION: Our data confirm that ruminants are an important source of serologically and genetically diverse intimin-harboring E. coli strains. Moreover, cattle have not only to be considered as important asymptomatic carriers of O157 STEC but can also be a reservoir of EPEC and eae positive non-O157 STEC, which are described in association with human diseases

Serotypes, virulence genes, and PFGE patterns of enteropathogenic Escherichia coli isolated from Cuban pigs with diarrhea

Thirty-six enteropathogenic Escherichia coli strains isolated from Cuban pigs with diarrhea were serotyped and screened by PCR for the presence of virulence genes. The 36 isolates belonged to 11 O serogroups and 14 O:H serotypes, with 53% of the isolates belonging to only two serotypes: O141:H– (13 isolates) and O157:H19 (6 isolates). Genes coding for STb, STa, VT2e, and LT toxins were identified in 69, 61, 53, and 6% of the isolates, respectively. The most prevalent fimbrial adhesin was F18, detected in 22 (61%) isolates. The gene encoding F6 (P987) colonization factor was identified in three (8%) isolates. None of the 36 isolates assayed contained genes encoding F4 (K88), F5 (K99), or F41. The seropathotype O141:H–:STa/STb/VT2e/F18 (13 isolates) was the most frequently detected, followed by O157:H19:VT2e/F18 (5 isolates). A genetic diversity study, carried out by pulsed-field gel electrophoresis (PFGE) of 24 representative isolates, revealed 21 distinct restriction patterns clustered in 18 groups (I–XVIII). Isolates of the same serotype were placed together in a dendrogram, but isolates of serotype O157:H19 showed a high degree of polymorphism. The results of this study demonstrate the presence in Cuba of different clusters among one of the most prevalent serotypes isolated from pigs with diarrhea. Further experiments are needed to determine whether some of these clusters have appeared recently; if so, their evolution, as well as their possible association with pathogenicity in farms should be studied. [Int Microbiol 2006; 9(1):53-60

Tracking bacterial virulence: global modulators as indicators

The genomes of Gram-negative bacteria encode paralogues and/or orthologues of global modulators. The nucleoid-associated H-NS and Hha proteins are an example: several enterobacteria such as Escherichia coli or Salmonella harbor H-NS, Hha and their corresponding paralogues, StpA and YdgT proteins, respectively. Remarkably, the genome of the pathogenic enteroaggregative E. coli strain 042 encodes, in addition to the hha and ydgT genes, two additional hha paralogues, hha2 and hha3. We show in this report that there exists a strong correlation between the presence of these paralogues and the virulence phenotype of several E. coli strains. hha2 and hha3 predominate in some groups of intestinal pathogenic E. coli strains (enteroaggregative and shiga toxin-producing isolates), as well as in the widely distributed extraintestinal ST131 isolates. Because of the relationship between the presence of hha2/hha3 and some virulence factors, we have been able to provide evidence for Hha2/Hha3 modulating the expression of the antigen 43 pathogenic determinants. We show that tracking global modulators or their paralogues/orthologues can be a new strategy to identify bacterial pathogenic clones and propose PCR amplification of hha2 and hha3 as a virulence indicator in environmental and clinical E. coli isolatesThe authors acknowledge funding from the Spanish MICINN-FEDER (BFU2010-21836-C02-01) and Mineco (BIO2013-49148-C2-1-R and BIO2015-69085-REDC). Work in the LREC-USC-laboratory was financed by the grant CN2012/303 from Consellería de Cultura, Educación e Ordenación Universitaria (Xunta de Galicia) and The European Regional Development Fund (ERDF)S

Serotypes, virulence genes, and PFGE profiles of Escherichia coli isolated from pigs with postweaning diarrhoea in Slovakia

BACKGROUND: Postweaning diarrhoea (PWD) in pigs is usually the main infectious problem of large-scale farms and is responsible for significant losses worldwide. The disease is caused mainly by enterotoxigenic E. coli (ETEC) and Shiga-toxin producing E. coli (STEC). In this study a total of 101 E. coli isolated from pigs with PWD in Slovakia were characterized using phenotypic and genotypic methods. RESULTS: These 101 isolates belonged to 40 O:H serotypes. However, 57% of the isolates belonged to only six serotypes (O9:H51, O147:H-, O149:H10, O163:H-, ONT:H-, and ONT:H4), including two new serotypes (O163:H- and ONT:H4) not previously found among porcine ETEC and STEC isolated in other countries. Genes for EAST1, STb, STa, LT and Stx2e toxins were identified in 64%, 46%, 26%, 20%, and 5% of isolates, respectively. PCR showed that 35% of isolates carried genes for F18 colonization factor, and further analyzed by restriction endonuclease revealed that all of them were F18ac. Genes for F4 (K88), F6 (P987), F17, F5 (K99), F41, and intimin (eae gene) adhesins were detected in 19 %, 5%, 3%, 0.9%, 0.9%, and 0.9% of the isolates, respectively. The study of genetic diversity, carried out by PFGE of 46 representative ETEC and STEC isolates, revealed 36 distinct restriction profiles clustered in eight groups. Isolates of the same serotype were placed together in the dendrogram, but high degree of polymorphism among certain serotypes was detected. CONCLUSION: Seropathotype O149:H10 LT/STb/EAST1/F4 (14 isolates) was the most commonly detected followed by O163:H- EAST1/F18 (six isolates), and ONT:H4 STa/STb/Stx2e/F18 (five isolates). Interestingly, this study shows that two new serotypes (O163:H- and ONT:H4) have emerged as pig pathogens in Slovakia. Furthermore, our results show that there is a high genetic variation mainly among ETEC of O149:H10 serotype

Fecal carriage of Escherichia coli O157:H7 and carcass contamination in cattle at slaughter in northern Italy

Feedlot cattle slaughtered at a large abattoir in northern Italy during 2002 were examined for intestinal carriage and carcass contamination with Escherichia coli O157:H7. Carcass samples were taken following the excision method described in the Decision 471/2001/EC, and fecal material was taken from the colon of the calves after evisceration. Bacteria were isolated and identified according to the MFLP-80 and MFLP-90 procedures (Food Directorate’s Health Canada’s). Eighty-eight non-sorbitol-fermenting E. coli O157:H7 isolates were obtained from 12 of the 45 calves examined. In particular, E. coli O157:H7 isolates were found in 11 (24%) fecal and five (11%) carcass samples. PCR analysis showed that all 11 fecal samples and five carcass samples carried eae-γ1-positive E. coli O157:H7 isolates. In addition, genes encoding Shigatoxins were detected in O157:H7 isolates from nine and two of those 11 fecal and five carcasses, respectively. A representative group of 32 E. coli O157:H7 isolates was analyzed by phage typing and DNA macrorestriction fragment analysis (PFGE). Five phage types (PT8, PT32v, PT32, PT54, and PT not typable) and seven (I–VII) distinct restriction patterns of similarity > 85% were detected. Up to three different O157:H7 strains in an individual fecal sample and up to four from the same animal could be isolated. These findings provide evidence of the epidemiological importance of subtyping more than one isolate from the same sample. Phage typing together with PFGE proved to be very useful tools to detect cross-contamination among carcasses and should therefore be included in HACCP programs at abattoirs. The results showed that the same PFGE-phage type E. coli O157:H7 profile was detected in the fecal and carcass samples from an animal, and also in two more carcasses corresponding to two animals slaughtered the same day. [Int Microbiol 2007; 10(2):109-116

Identification of two new intimin types in atypical enteropathogenic Escherichia coli

Stool specimens of patients with diarrhea or other gastrointestinal alterations who were admitted to Xeral-Calde Hospital (Lugo, Spain) were analyzed for the prevalence of typical and atypical enteropathogenic Escherichia coli (EPEC). Atypical EPEC strains (eae+ bfp–) were detected in 105 (5.2%) of 2015 patients, whereas typical EPEC strains (eae+ bfp+) were identified in only five (0.2%) patients. Atypical EPEC strains were (after Salmonella) the second most frequently recovered enteropathogenic bacteria. In this study, 110 EPEC strains were characterized. The strains belonged to 43 O serogroups and 69 O:H serotypes, including 44 new serotypes not previously reported among human EPEC. However, 29% were of one of three serogroups (O26, O51, and O145) and 33% belonged to eight serotypes (O10:H–, O26:H11, O26:H–, O51:H49, O123:H19, O128:H2, O145:H28, and O145:H–). Only 14 (13%) could be assigned to classical EPEC serotypes. Fifteen intimin types, namely, α1 (6 strains), α2 (4 strains), β1 (34 strains), ξR/β2 (6 strains), γ1 (13 strains), γ2/θ (16 strains), δ/k (5 strains), ε1 (9 strains), νR/ε2 (5 strains), ζ (6 strains), ι1 (1 strain), μR/ι2 (1 strain), νB (1 strain), ξB (1 strain), and ο (2 strains), were detected among the 110 EPEC strains, but none of the strains was positive for intimin types μ1, μ2, λ, or μB. In addition, in atypical EPEC strains of serotypes O10:H–, O84:H–, and O129:H–, two new intimin genes (eae-νB and eae-ο) were identified. These genes showed less than 95% nucleotide sequence identity with existing intimin types. Phylogenetic analysis revealed six groups of closely related intimin genes: (i) α1, α2, ζ, νB, and ο; (ii) ι1 and μR/ι2; (iii) β1, ξR/β2B, δ/β2O, and κ; (iv) ε1, ξB, η1,η2, and νR/ε2; (v) γ1, μB, γ2, and θ; and (vi) λ. These results indicate that atypical EPEC strains belonging to large number of serotypes and with different intimin types might be frequently isolated from human clinical stool samples in Spain. [Int Microbiol 2006; 9(2):103-110

Tracking bacterial virulence: global modulators as indicators

The genomes of Gram-negative bacteria encode paralogues and/or orthologues of global modulators. The nucleoid-associated H-NS and Hha proteins are an example: several enterobacteria such as Escherichia coli or Salmonella harbor H-NS, Hha and their corresponding paralogues, StpA and YdgT proteins, respectively. Remarkably, the genome of the pathogenic enteroaggregative E. coli strain 042 encodes, in addition to the hha and ydgT genes, two additional hha paralogues, hha2 and hha3. We show in this report that there exists a strong correlation between the presence of these paralogues and the virulence phenotype of several E. coli strains. hha2 and hha3 predominate in some groups of intestinal pathogenic E. coli strains (enteroaggregative and shiga toxin-producing isolates), as well as in the widely distributed extraintestinal ST131 isolates. Because of the relationship between the presence of hha2/hha3 and some virulence factors, we have been able to provide evidence for Hha2/Hha3 modulating the expression of the antigen 43 pathogenic determinants. We show that tracking global modulators or their paralogues/orthologues can be a new strategy to identify bacterial pathogenic clones and propose PCR amplification of hha2 and hha3 as a virulence indicator in environmental and clinical E. coli isolates

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

<p>Abstract</p> <p>Background</p> <p>Enteropathogenic <it>Escherichia coli </it>(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.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>Some aEPEC strains may invade intestinal cells <it>in vitro </it>with varying efficiencies and independently of the intimin sub-type.</p

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&nbsp;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&nbsp;reports of serotype O104:H4, which is very rare in humans and has never been detected in animals or food. Several research&nbsp;groups obtained the complete genome sequence of isolates of the German outbreak strain as well as the genome sequences of&nbsp;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 theoutbreak in Germany. Epidemiologic investigations supported a linkage between the outbreaks in Germany and France and&nbsp;traced their origin to fenugreek seeds imported from Africa. However, there has been no isolation of the causative strain&nbsp;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&nbsp;June and July 2011, 200 vegetable samples from different origins were analyzed. All were negative for the virulent serotype&nbsp;O104:H4 and only one lettuce sample (0.6%) was positive for a STEC strain of serotype O146:H21 (stx1, stx2), considered&nbsp;of low virulence. Despite the single positive case, the hygienic and sanitary quality of Spanish vegetables proved to be quite&nbsp;good. In 195 of the 200 samples (98%), &lt;10 colony-forming units (cfu) of E. coli per gram were detected, and the microbiological levels of all samples were satisfactory (&lt;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 serotypeO157:H7 and other serotypes have been isolated from beef, milk, cheese, and domestic (cattle, sheep, goats) and wild (deer,&nbsp;boar, fox) animals in Spain. Nevertheless, STEC outbreaks in Spain are rare