Search

Introduction: Recurrent urinary tract infections (RUTIs) caused by uropathogenic Escherichia coli are costly public health problems impacting patients' quality of life., Aim: In this work, a comparative genomics analysis of three clinical RUTI strains isolated from bladder biopsy specimens was performed., Materials and Methods: One hundred seventy-two whole genomes of urinary tract E. coli strains were selected from the NCBI database. The search for virulence factors, fitness genes, regions of interest, and genetic elements associated with resistance was manually carried out. The phenotypic characterization of antibiotic resistance, haemolysis, motility, and biofilm formation was performed. Moreover, adherence and invasion assays with human bladder HTB-5 cells, and transmission electron microscopy (TEM) were performed., Results: The UTI-1&#95;774U and UTI-3&#95;455U/ST1193 strains were associated with the extraintestinal pathotypes, and the UTI-2&#95;245U/ST295 strain was associated with the intestinal pathotype, according to a phylogenetic analysis of 172 E. coli urinary strains. The three RUTI strains were of clinical, epidemiological, and zoonotic relevance. Several resistance genes were found within the plasmids of these strains, and a multidrug resistance phenotype was revealed. Other virulence genes associated with CFT073 were not identified in the three RUTI strains (genes for type 1 and P fimbriae, haemolysin hlyA, and sat toxin). Quantitative adherence analysis showed that UTI-1&#95;774U was significantly ( p  < 0.0001) more adherent to human bladder HTB-5 cells. Quantitative invasion analysis showed that UTI-2&#95;245U was significantly more invasive than the control strains. No haemolysis or biofilm activity was detected in the three RUTI strains. The TEM micrographs showed the presence of short and thin fimbriae only in the UTI-2&#95;245U strain., Conclusion: The high variability and genetic diversity of the RUTI strains indicate that are a mosaic of virulence, resistance, and fitness genes that could promote recurrence in susceptible patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Flores-Oropeza, Ochoa, Cruz-Córdova, Chavez-Tepecano, Martínez-Peñafiel, Rembao-Bojórquez, Zavala-Vega, Hernández-Castro, Flores-Encarnacion, Arellano-Galindo, Vélez-Sánchez and Xicohtencatl-Cortes.)

The coliphage mEp021 belongs to a phage group with a unique immunity repressor, and its life cycle requires the host factor Nus. mEp021 has been classified as non-lambdoid based on its specific characteristics. The mEp021 genome carries a gene encoding an N λ -like antiterminator protein, termed Gp17, and three nut sites (nutL, nutR1, and nutR2). Analysis of plasmid constructs containing these nut sites, a transcription terminator, and a GFP reporter gene showed high levels of fluorescence when Gp17 was expressed, but not in its absence. Like lambdoid N proteins, Gp17 has an arginine-rich motif (ARM), and mutations in its arginine codons inhibit its function. In infection assays using the mutant phage mEp021ΔGp17::Kan (where gp17 has been deleted), gene transcripts located downstream of transcription terminators were obtained only when Gp17 was expressed. In contrast to phage lambda, mEp021 virus particle production was partially restored (>1/3 relative to wild type) when nus mutants (nusA1, nusB5, nusC60, and nusE71) were infected with mEp021 and Gp17 was overexpressed. Our results suggest that RNA polymerase reads through the third nut site (nutR2), which is more than 7.9 kbp downstream of nutR1., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Thyroid cancer is the most frequent endocrine malignancy, and its incidence and prevalence are increasing worldwide. Despite its generally good prognosis, the observed mortality rates are higher in the less-developed regions. This indicates that timely diagnosis and appropriate initial management of this disease are important to achieve a positive outcome. We performed an observational study in order to describe the frequency of the BRAF 1799T>A mutation in Mexican mestizo patients with thyroid nodules, a scarcely studied ethnic group with large populations. Competitive allele-specific Taqman PCR was performed in 147 samples of thyroid tissue DNA obtained from patients histologically diagnosed with papillary thyroid cancer (PTC), colloid goiters, and follicular adenomas. The BRAF 1799T>A mutation frequency was 61.1% in PTC samples ( p = 4.99 × 10 -11 ). Potential diagnostic values were as follows: sensitivity, 61.1%; specificity, 96%; PPV, 94.2%; NPV, 69.5%; accuracy, 77.9%. Taking into account the fact that this mutation is not frequently found in cytologically indeterminate nodules, we suggest that the BRAF mutational analysis should be implemented in the clinical setting along with other diagnostic criteria such as USG, in order to contribute to diagnosis and to surgical decision-making during the initial management of thyroid nodules in Mexican public hospitals.

In order to identify host components involved in the infective process of bacteriophages, we developed a wide-range strategy to obtain cell envelope mutants, using Escherichia coli W3110 and its specific phage mEp213. The strategy consisted in four steps: (1) random mutagenesis using transposon miniTn10Km(r); (2) selection of phage-resistant mutants by replica-plating; (3) electroporation of the phage-resistant mutants with mEp213 genome, followed by selection of those allowing phage development; and (4) sequencing of the transposon-disrupted genes. This strategy allowed us to distinguish the host factors related to phage development or multiplication within the cell, from those involved in phage infection at the level of the cell envelope.

Background: Cancer development involves an "injury" to the respiratory machinery (Warburg effect) due to decreased or impaired mitochondrial function. This circumstance results in a down regulation of some of the ATPase subunits of the malignant tissue. The objective of this work was to assess and compare the relative expression of mRNA of mitochondrial ATPase subunits between samples of thyroid cancer and benign nodules., Material and Methods: Samples from 31 patients who had an operation for PTC at the General Hospital of Mexico were snap-frozen and stored at -70°C. Thirty-five patients who had an operation for benign tumors were also included in the study. mRNA expression levels of alpha, beta, gamma, and epsilon subunits of F1 and "c12" of subunit Fo were determined by real-time RT-PCR (by duplicate), in order to determine if abnormal expression of these genes could partially explain the Warburg effect in papillary thyroid cancer (PTC)., Results: ATP5E transcript alteration (down-expression) was highly associated to PTC diagnosis OR=11.76 (95% confidence interval, 1.245-237.98; p=0.04)., Conclusions: Relative down-expression of ATP5E transcript was highly associated with PTC diagnosis. This transcript alteration may be used as a tumoral marker in papillary thyroid cancer.

This work was aimed to develop a multiplex PCR assay to detect infectious agents such as Clavibacter michiganensis subsp. michiganensis, Fusarium sp, Leveillula taurica, and begomoviruses in tomato (Solanum lycopersicum) plants. Specific primer sets of each pathogen were designed based on intergenic ribosomal RNA sequences for the first three, whereas for begomoviruses, primers were designed based on conserved regions. The design also considered that the length (200-800 bp) of the PCR products was resolvable by electrophoresis; thus 296, 380, 457, and 731 bp fragments for Clavibacter, Fusarium, Leveillula, and begomoviruses, respectively, were considered. PCR conditions were optimized to amplify all the products in a single tube from genomic DNA and circumvent PCR inhibitors from infected plants. Finally, when the multiplex PCR assay was tested with tomato plants infected with any of the four pathogens, specific PCR products confirmed the presence of the pathogens. Optimized PCR multiplex allowed for the accurate and simultaneous detection of Clavibacter, Fusarium, Leveillula, and begomoviruses in infected plants or seeds from tomato.

We have developed a direct and efficient strategy, based on a three-step method, to select bacterial cell-envelope mutants resistant to bacteriophage infection. Escherichia coli K-12 strain W3110 underwent classical transposon mutagenesis followed by replica plating and selection for mutants resistant to infection by coliphage mEp213. To verify that phage resistance was due to mutations in the cell envelope, we transformed host cells with the viral genome using electroporation and selected those in which virions were subsequently detected in the supernatant. Among the nine mutants resistant to coliphage infection that we selected, six were in the fhuA gene, two were mutated in the waaC gene, and one was mutated in the gmhD gene. The latter two gene products are involved in the synthesis of lipopolysaccharide (LPS). The efficiency of plating and adsorption of phage mEp213 was affected in these mutants. We verified that LPS is required for the efficient infection of phage λ as well. We propose that this mutation-and-selection strategy can be used to find host factors involved in the initial steps of phage infection for any cognate pair of phage and bacteria.

Lysogenic Escherichia coli K-12 harbouring the prophage mEp021 displays haemolytic activity. From a genomic library of mEp021, we identified an open reading frame (ORF 4) that was responsible for the haemolytic activity. However, the ORF 4 sequence contains four initiation codons in the same frame: ORF 4.1-ORF 4.4, coding for 83-a.a., 82-a.a., 77-a.a. and 72-a.a. products, respectively. The expression of the cloned ORF 4.3, or inducer of pleiotropic effects (ipe), reproduced the haemolytic phenotype in a native strain carrying the gene hlyE(+), but not in the mutant hlyE(-) strain. The overexpression of Ipe induced several pleiotropic effects, such as the inhibition of cell growth and the deregulation of cell division, which resulted in a mixture of normal and desiccated-like cells: normal-filamentous, desiccated-like-filamentous bacilli, minicells etc. Other effects included abnormalities of the cell membrane, the production of vesicles containing HlyE, and finally, cell death. These events were analysed at the molecular level by microarray assays. The global transcription profile of E. coli K-12 strain MC4100, which expressed Ipe after 4 h, revealed differential expression of various genes, most of which were related either to cell membrane and murein biosynthesis or to cell division. The up-regulation of some of these transcripts was confirmed by qRT-PCR. Additional research is needed to determine whether these effects are directly related to Ipe activity or are consequences of the cellular responses to putative structural damage induced by Ipe., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

A group of previously isolated heterogeneous mEp lambdoid phages (43) from 19 different immunity groups for phage infection was further characterized to gain insight into some phenotypic traits and to assess their relationship with phage lambda. Interestingly, the FhuA host receptor was required by the majority of mEp phages (37 out of 43; approximately 85%). The cor gene, which has been reported to be involved in FhuA-dependent exclusion of lambdoid phages, was also found in most of the FhuA-dependent phages. Accordingly, no cor amplification by PCR was obtained among the six FhuA-independent mEp lambdoid phages. In contrast, it was found that around 25% of the population (10 out of 43 phages) required the specific and essential lambda N antitermination function, and the lambda site-specific DNA recombination function was observed only in two members (4.6%). Thus, a larger proportion of phages require the FhuA receptor for infection, and this is frequently correlated with the cor gene.