Controlling diarrheagenic E. coli with bacteriophages: facts and challenges

Enterotoxigenic Escherichia coli (ETEC) colonizes the intestine, causing severe diarrhoea in humans and animals. The rise of antibiotic resistances and limitation on their use demands news strategies to tackle this pathology. Bacteriophages (phages), viruses specifically infecting bacteria and harmless to animals and plants, are a promising antibacterial tool. Although studies support their ability to efficiently overcome ETEC infections, they have been shown to be highly strain-specific. If this can be associated with the presence of anti-phage defense systems (APDS) in ETEC genomes, it is also true that phages can counter-evolve to escape APDS. This work aimed to define phage cocktail with broader lytic spectra, capable of overcoming APDS of ETEC, enhancing phage efficacy. We firstly sequenced 29 ETEC strains from our collection to search for the presence of APDS in their genomes. Then, we performed phage isolation and the subsequent in vitro and genomic characterization: i) evaluation of lytic spectra against ETEC collection; ii) whole-genome sequencing and phage safety evaluation (absence of undesirable genes) iii)presence of proteins responsible for escaping the main APDS. We were able to identify distinct mechanisms supporting APDS. Bacterial proteins that prevent the entry of DNA from phages (CRISPR-Cas-related proteins) or that enable the cut of phage nucleic acids (restriction-modification enzymes) were detected, however, most of them were related with the induction of abortive infection events (e.g. toxin-antitoxin systems). We also isolated 3 phages, SUS35, SUS42 and SUS65, which proved to be safe for therapy and to encode proteins enabling to escape APDS, inclusively against abortive infection. Phage-host interaction mechanisms must be considered when preparing phage- based products for therapy. This work clearly indicates that a strict selection of phages, or the construction of synthetic phages with desired traits will be a turning point in their versatility to fight against ETEC infections.info:eu-repo/semantics/publishedVersio

Estudio de las macrodestrezas linguisticas y su influencia en el desarrollo del pensamiento crítico.

Las macro destrezas lingüísticas representan el arte de comprender ideas o cualquier tipo de información para poder expresarlas de una manera crítica para mejorar el conocimiento. A lo largo de la historia el ser humano se ha interesado en adquirir el hábito de desarrollar las macro destrezas lingüísticas, ya que estos han permitido alcanzar todo tipo de conocimiento. Aprender a leer, escribir, escuchar y hablar es una tarea que empieza desde la escuela y a medida que el niño o niña avanza en la etapa estudiantil, se torna muy importante dominar estas destrezas de una forma correcta. En la Unidad Educativa Eloy Velásquez Cevallos del Cantón Milagro, evidencia el problema ya que los estudiantes tienen dificultades al momento de desarrollar estas habilidades, por esa razón se decidió realizar un estudio dirigido a los niños y niñas del Séptimo año de educación básica con la finalidad de realizar un análisis para detectar por que se dan inconvenientes para mejorar en estas destrezas. El presente trabajo de investigación se realizó en base a una encuesta, la misma que estuvo dirigida a los alumnos y padres de familia, el objetivo principal fue detectar el origen del problema, y realizar un análisis y así establecer estrategias que permitan mejorar el desarrollo de las destrezas en los alumnos y por ende su pensamiento crítico. Cabe resaltar que los beneficiados de este proyecto son los estudiantes, ya que las macro destrezas lingüísticas fundamentales en los estudios, de ella parte el proceso de aprendizaje, ya que si un alumnos tiene problemas para leer, escribir, escuchar y hablar es muy difícil que logre captar el contenido de las asignaturas que se imparten en la escuela

Estudio de las macrodestrezas linguisticas y su influencia en el desarrollo del pensamiento crítico.

Las macro destrezas lingüísticas representan el arte de comprender ideas o cualquier tipo de información para poder expresarlas de una manera crítica para mejorar el conocimiento. A lo largo de la historia el ser humano se ha interesado en adquirir el hábito de desarrollar las macro destrezas lingüísticas, ya que estos han permitido alcanzar todo tipo de conocimiento. Aprender a leer, escribir, escuchar y hablar es una tarea que empieza desde la escuela y a medida que el niño o niña avanza en la etapa estudiantil, se torna muy importante dominar estas destrezas de una forma correcta. En la Unidad Educativa Eloy Velásquez Cevallos del Cantón Milagro, evidencia el problema ya que los estudiantes tienen dificultades al momento de desarrollar estas habilidades, por esa razón se decidió realizar un estudio dirigido a los niños y niñas del Séptimo año de educación básica con la finalidad de realizar un análisis para detectar por que se dan inconvenientes para mejorar en estas destrezas. El presente trabajo de investigación se realizó en base a una encuesta, la misma que estuvo dirigida a los alumnos y padres de familia, el objetivo principal fue detectar el origen del problema, y realizar un análisis y así establecer estrategias que permitan mejorar el desarrollo de las destrezas en los alumnos y por ende su pensamiento crítico. Cabe resaltar que los beneficiados de este proyecto son los estudiantes, ya que las macro destrezas lingüísticas fundamentales en los estudios, de ella parte el proceso de aprendizaje, ya que si un alumnos tiene problemas para leer, escribir, escuchar y hablar es muy difícil que logre captar el contenido de las asignaturas que se imparten en la escuela

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

Plasmid flux in Escherichia coli ST131 sublineages, analyzed by plasmid constellation network (PLACNET), a new method for plasmid reconstruction from whole genome sequences

This is an open-access article distributed under the terms of the Creative Commons Attribution License.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.Work was financed by the Spanish Ministry of Economy and Competitivity (BFU2011-26608 to FdlC, FIS-PI09/01273 and AGL2013-47852-R to JB and FIS-PI12-01581 and CB06/02/0053 to TMC), by the European Seventh Framework Program (612146/FP7-ICT-2013-10 to FdlC and 282004/FP7-HEALTH-2011-2.3.1-2 to FdlC and TMC); by Red Española de Investigación en Patología­ Infecciosa (REIPI RD06/0008/1018-1016) to JB, by Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia and European Regional Development Fund, ERDF (CN2012/303 and EM2014/001) to JB and by the regional government of Madrid (PROMPT-S2010/BMD2414) to TMC. We are also grateful to the Spanish Network for the Study of Plasmids and Extrachromosomal Elements (REDEEX) for funding cooperation among Spanish microbiologists working on the biology of MGEs (Spanish Ministry of Science and Innovation BFU2011-14145-E).Peer Reviewe

Studies on the interaction of three lytic bacteriophages with a wide collection of Escherichia coli strains implicated in swine enteric colibacillosis

bioRxiv - the Preprint Server for BiologyThe misuse of antibiotics in the swine industry and their on-going restriction requires alternatives to control enterotoxigenic and shiga toxin-producing Escherichia coli (ETEC and STEC, respectively). This study evaluates the potential of three coliphages, vB_EcoM_FJ1, vB_EcoM_FN and vB_EcoM_SP1 against 104 ETEC, STEC and ETEC/STEC strains isolated from pig colibacillosis in Portuguese (2018-2020) and Spanish farms (2006-2016), encompassing 71.2% mcr-positive strains (33.7% with mcr-1, 1.9% mcr-2, 35.6% mcr-4 and 2.9% mcr-5) and 18.3% positive strains for TEM (1%), SHV (6.7%), and CTX-M (11.5%) extended-spectrum beta-lactamase-encoding genes. In general, all bacteriophages presented a narrow lytic spectrum (up to 2.9%) against the 104 ETEC, STEC and ETEC/STEC. Bacteriophages shared >80% overall nucleotide identity with E. coli phage T4 (Tevenvirinae subfamily), but a particular look at the distal part of the long tail fiber (gp38) revealed no homology. All bacteriophages recognize lipopolysaccharides as receptors, and additionally, FN binds to an outer membrane protein A. Bacteriophage-insensitive mutants of vB_EcoM_FJ1 (90%) and vB_EcoM_FN (100%) were shown to be more susceptible to pig serum inactivation comparatively to the parental strain and furthermore, their adhesion capacity to porcine intestinal cells was diminished by, approximately, 90%. Contrariwise, vB_EcoM_SP1 insensitive variants did not display phenotypic differences comparing to the wild-type strain. This study demonstrates that besides being T4-like, these bacteriophages revealed a narrow lytic spectrum against diarrhoeagenic E. coli strains and that the acquisition of novel bacteriophage-encoded adhesins (gp38) seems to be determinant for such results.info:eu-repo/semantics/publishedVersio

Necrotoxigenic Escherichia coli from sheep and goats produce a new type of cytotoxic necrotizing factor (CNF3) associated with the eae and ehxA genes

Fecal samples from sheep and goats were screened by tissue-culture assays and PCR for the presence of necrotoxigenic Escherichia coli (NTEC) producing cytotoxic necrotizing factors (CNFs). Of the 18 NTEC strains assayed, four were positive for the cnf1 gene while 14 strains were negative for the cnf1 and cnf2 genes. All of the NTEC strains had the eae gene and most of them also carried the ehxA gene. Moreover, all the cnf1– cnf2– NTEC strains were negative for several virulence markers associated with CNF1+ or CNF2+ strains. The cnf gene present in one of these strains was sequenced and analysis of the gene product revealed a new type of CNF, which was named CNF3 (and the coding gene cnf3). Oligonucleotide primers were designed to PCR-amplify a fragment of cnf3. The results showed that all strains examined in this study, except one cnf1+strain, were cnf3+. The association of cnf3 with eae and ehxA suggests that cnf3+ NTEC strains might be pathogenic for humans. [Int Microbiol 2007; 10(1):47-55

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