Contribution of Toxin-Antitoxin Systems to Adherent-Invasive E. coli Pathogenesis.

Pathobionts have been implicated in various chronic diseases, including Crohn's disease (CD), a multifactorial chronic inflammatory condition that primarily affects the gastrointestinal tract, causing inflammation and damage to the digestive system. While the exact cause of CD remains unclear, adherent-invasive Escherichia coli (AIEC) strains have emerged as key contributors to its pathogenesis. AIEC are characterized by their ability to adhere to and invade intestinal epithelial cells and survive and replicate inside macrophages. However, the mechanisms underlying the virulence and persistence of AIEC within their host remain the subject of intensive research. Toxin-antitoxin systems (TAs) play a potential role in AIEC pathogenesis and may be therapeutic targets. These systems generally consist of two components: a toxin harmful to the cell and an antitoxin that neutralizes the toxin's effects. They contribute to bacterial survival in adverse conditions and regulate bacterial growth and behavior, affecting various cellular processes in bacterial pathogens. This review focuses on the current information available to determine the roles of TAs in the pathogenicity of AIEC. Their contribution to the AIEC stress response, biofilm formation, phage inhibition, the maintenance of mobile genetic elements, and host lifestyles is discussed.

Repertoire and Diversity of Toxin - Antitoxin Systems of Crohn's Disease-Associated Adherent-Invasive Escherichia coli. New Insight of T his Emergent E. coli Pathotype.

Adherent-invasive Escherichia coli (AIEC) corresponds to an E. coli pathovar proposed as a possible agent trigger associated to Crohn's disease. It is characterized for its capacity to adhere and to invade epithelial cells, and to survive and replicate inside macrophages. Mechanisms that allow intestinal epithelium colonization, and host factors that favor AIEC persistence have been partly elucidated. However, bacterial factors involved in AIEC persistence are currently unknown. Toxin-antitoxin (TA) systems are recognized elements involved in bacterial persistence, in addition to have a role in stabilization of mobile genetic elements and stress response. The aim of this study was to elucidate the repertoire and diversity of TA systems in the reference AIEC NRG857c strain and to compare it with AIEC strains whose genomes are available at databases. In addition, toxin expression levels under in vitro stress conditions found by AIEC through the intestine and within the macrophage were measured. Our results revealed that NRG857c encodes at least 33 putative TA systems belonging to types I, II, IV, and V, distributed around all the chromosome, and some in close proximity to genomic islands. A TA toxin repertoire marker of the pathotype was not found and the repertoire of 33 TA toxin genes described here was exclusive of the reference strains, NRG857c and LF82. Most toxin genes were upregulated in the presence of bile salts and acidic pH, as well as within the macrophage. However, different transcriptional responses were detected between reference strains (NRG857c and HM605), recalling the high diversity associated to this pathotype. To our knowledge this is the first analysis of TA systems associated to AIEC and it has revealed new insight associated to this emergent E. coli pathotype.

Study of a classification algorithm for AIEC identification in geographically distinct E. coli strains.

Adherent-invasive Escherichia coli (AIEC) have been extensively implicated in Crohn's disease pathogenesis. Currently, AIEC is identified phenotypically, since no molecular marker specific for AIEC exists. An algorithm based on single nucleotide polymorphisms was previously presented as a potential molecular tool to classify AIEC/non-AIEC, with 84% accuracy on a collection of 50 strains isolated in Girona (Spain). Herein, our aim was to determine the accuracy of the tool using AIEC/non-AIEC isolates from different geographical origins and extraintestinal pathogenic E. coli (ExPEC) strains. The accuracy of the tool was significantly reduced (61%) when external AIEC/non-AIEC strains from France, Chile, Mallorca (Spain) and Australia (82 AIEC, 57 non-AIEC and 45 ExPEC strains in total) were included. However, the inclusion of only the ExPEC strains showed that the tool was fairly accurate at differentiating these two close pathotypes (84.6% sensitivity; 79% accuracy). Moreover, the accuracy was still high (81%) for those AIEC/non-AIEC strains isolated from Girona and Mallorca (N = 63); two collections obtained from independent studies but geographically close. Our findings indicate that the presented tool is not universal since it would be only applicable for strains from similar geographic origin and demonstrates the need to include strains from different origins to validate such tools.

The Association of Ascorbic Acid, Deferoxamine and N-Acetylcysteine Improves Cardiac Fibroblast Viability and Cellular Function Associated with Tissue Repair Damaged by Simulated Ischemia/Reperfusion.

Acute myocardial infarction is one of the leading causes of death worldwide and thus, an extensively studied disease. Nonetheless, the effects of ischemia/reperfusion injury elicited by oxidative stress on cardiac fibroblast function associated with tissue repair are not completely understood. Ascorbic acid, deferoxamine, and N-acetylcysteine (A/D/N) are antioxidants with known cardioprotective effects, but the potential beneficial effects of combining these antioxidants in the tissue repair properties of cardiac fibroblasts remain unknown. Thus, the aim of this study was to evaluate whether the pharmacological association of these antioxidants, at low concentrations, could confer protection to cardiac fibroblasts against simulated ischemia/reperfusion injury. To test this, neonatal rat cardiac fibroblasts were subjected to simulated ischemia/reperfusion in the presence or absence of A/D/N treatment added at the beginning of simulated reperfusion. Cell viability was assessed using trypan blue staining, and intracellular reactive oxygen species (ROS) production was assessed using a 2',7'-dichlorofluorescin diacetate probe. Cell death was measured by flow cytometry using propidium iodide. Cell signaling mechanisms, differentiation into myofibroblasts and pro-collagen I production were determined by Western blot, whereas migration was evaluated using the wound healing assay. Our results show that A/D/N association using a low concentration of each antioxidant increased cardiac fibroblast viability, but that their separate administration did not provide protection. In addition, A/D/N association attenuated oxidative stress triggered by simulated ischemia/reperfusion, induced phosphorylation of pro-survival extracellular-signal-regulated kinases 1/2 (ERK1/2) and PKB (protein kinase B)/Akt, and decreased phosphorylation of the pro-apoptotic proteins p38- mitogen-activated protein kinase (p38-MAPK) and c-Jun-N-terminal kinase (JNK). Moreover, treatment with A/D/N also reduced reperfusion-induced apoptosis, evidenced by a decrease in the sub-G1 population, lower fragmentation of pro-caspases 9 and 3, as well as increased B-cell lymphomaextra large protein (Bcl-xL)/Bcl-2-associated X protein (Bax) ratio. Furthermore, simulated ischemia/reperfusion abolished serum-induced migration, TGF-ß1 (transforming growth factor beta 1)-mediated cardiac fibroblast-to-cardiac myofibroblast differentiation, and angiotensin II-induced pro-collagen I synthesis, but these effects were prevented by treatment with A/D/N. In conclusion, this is the first study where a pharmacological combination of A/D/N, at low concentrations, protected cardiac fibroblast viability and function after simulated ischemia/reperfusion, and thereby represents a novel therapeutic approach for cardioprotection.

The roles of HicBA and a novel toxin-antitoxin-like system, TsxAB, in the stability of IncX4 resistance plasmids in Escherichia coli.

OBJECTIVES: To identify toxin-antitoxin (TA)-like plasmid stability loci on IncX4 plasmids. METHODS: TA-like loci were identified bioinformatically and their contribution to stability of the IncX4 plasmid pJIE143 was tested in optimal growth conditions in vitro. The conservation of the TA-like loci identified was analysed within an updated IncX plasmid database. RESULTS: A novel TA-like locus, tsxAB, was identified on the IncX4 plasmid pJIE143, carrying the important plasmid-borne antibiotic resistance gene blaCTX-M-15. pJIE143 (the WT plasmid) and its tsxA mutant are stable for 80 bacterial generations in the absence of selective pressure but a tsxB deletion mutant of pJIE143 is relatively quickly lost without positive selection (91.1% ± 1.5% loss after 50 generations). Nine IncX subclasses were identified among 272 fully sequenced IncX plasmids, dominated by those identified as IncX3, IncX1 and IncX4 subclasses in PlasmidFinder. The novel TA-like locus, tsxAB, appears to be a feature of IncX4 plasmids, being present in 64 of 67 so identified, but only present in a single IncX1 plasmid (of 79 identified) and present in no other IncX plasmids. CONCLUSIONS: tsxAB, a novel TA-like stability locus, is highly conserved in IncX4 plasmids associated with transmission of important antibiotic resistance genes. Previous in silico analysis indicated that IncX4 encodes only HicBA among the known TA systems. Here we show that HicBA does not contribute to plasmid stability in optimal growth conditions for Escherichia coli and instead demonstrate this role for a completely novel TA-like system, TsxAB, that appears both necessary and sufficient for E. coli addiction to IncX4 resistance plasmids.

Functionality of tRNAs encoded in a mobile genetic element from an acidophilic bacterium.

The genome of the acidophilic, bioleaching bacterium Acidithiobacillus ferrooxidans, strain ATCC 23270, contains 95 predicted tRNA genes. Thirty-six of these genes (all 20 species) are clustered within an actively excising integrative-conjugative element (ICEAfe1). We speculated that these tRNA genes might have a role in adapting the bacterial tRNA pool to the codon usage of ICEAfe1 genes. To answer this question, we performed theoretical calculations of the global tRNA adaptation index to the entire A. ferrooxidans genome with and without the ICEAfe1 encoded tRNA genes. Based on these calculations, we observed that tRNAs encoded in ICEAfe1 negatively contribute to adapt the tRNA pool to the codon use in A. ferrooxidans. Although some of the tRNAs encoded in ICEAfe1 are functional in aminoacylation or protein synthesis, we found that they are expressed at low levels. These findings, along with the identification of a tRNA-like RNA encoded in the same cluster, led us to speculate that tRNA genes encoded in the mobile genetic element ICEAfe1 might have acquired mutations that would result in either inactivation or the acquisition of new functions.

Carriage of type II toxin-antitoxin systems by the growing group of IncX plasmids.

The stable maintenance of certain plasmids in bacterial populations has contributed significantly to the current worldwide antibiotic resistance (AbR) emergency. IncX plasmids, long underestimated in this regard, have achieved recent notoriety for their roles in transmission of resistance to carbapenem and colistin, the last-line antibiotics for Gram-negative infections. Toxin-antitoxin (TA) systems contribute to stable maintenance of many AbR plasmids, and a few TA systems have been previously described in the IncX plasmids. Here we present an updated overview of the IncX plasmid family and an in silico analysis of the type II TA systems carried in 153 completely sequenced IncX plasmids that are readily available in public databases at time of writing. The greatest number is in the IncX1 subgroup, followed by IncX3 and IncX4, with only a few representatives of IncX2, IncX5 and IncX6. Toxins from the RelE/ParE superfamily are abundant within IncX1 and IncX4 subgroups, and are associated with a variety of antitoxins. By contrast, the HicBA system is almost exclusively encoded by IncX4 plasmids. Toxins from the superfamily CcdB/MazF were also identified, as were less common systems such as PIN-like and GNAT toxins, and plasmids encoding more than one TA system are probably not unusual. Our results highlight the importance of the IncX plasmid group and update previous much smaller studies, and we present for the first time a detailed analysis of type II TA systems in these plasmids.

Phenolic composition and antioxidant capacity of Ugni molinae Turcz. leaves of different genotypes.

Ugni molinae Turcz. is a native shrub of Chile, known for its edible berries and its leaves, which have been the focus of recent attention, as a good source of phenolic compounds to be used in cosmetics and food products. The aim of this study was to assess the differences in the phenolic composition and antioxidant capacity of the ethanolic extracts from the leaves of 10 genotypes of U. molinae, that were cultivated under the same soil, climate and agronomical management. Antioxidant activity was assessed by complementary methods (ORAC-Fl, FRAP and DPPH assay), phenolic composition of each extract was analyzed by LC-MS. Phenolic and flavonoid total contents were determined by Folin-Ciocalteu and AlCl3 methods. Significative differences were found by these methods, and ellagitannins, gallic acid derivatives and flavonols were identified as responsible for these differences, showing the influence of the genotype on the phenolic composition of U. molinae leaves.

Comparative study of anti-inflammatory activity and qualitative-quantitative composition of triterpenoids from ten genotypes of Ugni molinae / Estudio comparativo de la actividad antiinflamatoria y composición química cualitativa y cuantitativa de triterpenoides de diez genotipos de Ugni molinae

The aim of this study was to assess the differences in qualitative-quantitative composition of triterpenoids and total phenolic contents, together with anti-inflammatory activity of Ugni molinae leaves obtained from ten genotypes. The ethyl acetate (EAE) and ethanol extracts (ETE) were obtained and analyzed. The plant genotypes were grown under same soil and climate conditions and under same agronomic management; the leaves were also harvested under the same conditions. Anti-inflammatory activity was evaluated by mice ear edema induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) at a single dose of 200 mg/kg BW of each extract. Composition of triterpenoids and total phenolic contents was determined by HPLC-DAD and Folin-Ciocalteu method, respectively. Ugni molinae leaves of different plant genotypes exhibited significant differences in regard to their anti-inflammatory activity, as well as in qualitative-quantitative composition of triterpenoids and total phenolic content.

El objetivo de este estudio fue establecer las diferencias en la composición cualitativa y cuantitativa de triterpenoides y en los contenidos totales de fenoles, junto con la actividad antiinflamatoria de las hojas de Ugni molinae provenientes de diez genotipos. Los extractos de acetato de etilo (EAE) y etanólicos (ETE) fueron obtenidos y analizados. Los genotipos fueron cultivados bajo las mismas condiciones edafo-climáticas y con el mismo manejo agronómico; las hojas fueron cosechadas bajo las mismas condiciones. La actividad antiinflamatoria fue evaluada en ratones a los que se les indujo un edema en la oreja mediante la aplicación del 12-O-tetradecanoilforbol-13 acetato (TPA) y los extractos fueron evaluados a una dosis única de 200 mg/kg de peso corporal. La composición en triterpenoides y los contenidos de fenoles totales fueron determinados por CLAE-DAD y por el método de Folin-Ciocalteu, respectivamente. Las hojas provenientes de los diferentes genotipos de U. molinae, exhibieron significativas diferencias en sus actividades antiinflamatorias, así como, en el contenido cualitativo y cuantitativo de triterpenoides y en el contenido de fenoles totales.

Evaluación de la actividad antioxidante y determinación del contenido de fenoles totales y flavonoides de hojas de diferentes genotipos de Ugni molinae Turcz / Evaluation of the antioxidant activity and determination of the content of total phenols and flavonoids of leaves of different genotypes of Ugni molinae Turcz

Ugni molinae, Myrtaceae, es un arbusto silvestre que crece en el sur de Chile, especialmente en la cordillera de la Costa y parte de la precordillera andina. La planta produce frutos globosos y pequeños, de agradable sabor y aroma, y las infusiones de sus hojas han sido utilizadas por la medicina folclórica para el tratamiento de distintos tipos de dolor e inflamación. Las propiedades de la murtilla se deben, en parte, a la presencia de diferentes compuestos fenólicos en sus hojas. Debido a que estos compuestos pueden variar por diversos factores, como el genotipo, en este estudio se realizó un análisis comparativo respecto a la capacidad antioxidante (ensayo FRAP), el contenido fenólico total (CFT) (ensayo de Folin-Ciocalteu) y el contenido de flavonoides (ensayo colorimétrico con AlCl3) de extractos etanólicos seriados (EETs) de hojas de 10 genotipos de murtilla (8-2, 14-4, 19-1, 19-1ha, 22-1, 23-2, 27-1, 31-1 y ZF-18), los cuales fueron cultivados bajos las mismas condiciones. De acuerdo a los resultados obtenidos, los EETs de los 10 genotipos presentaron diferencias significativas entre sí (p<0,05), siendo el EET ZF-18 el que obtuvo el mayor CFT, (260,6 ± 3,7 mg EAG/g ES) y el genotipo 31-1 el que obtuvo la mayor cantidad de flavonoides (53,5 ± 0,8 mg quercetina/g ES). En relación a la actividad antioxidante, los EETs de los genotipos ZF-18 y 27-1 fueron los que obtuvieron los mayores valores FRAP en todos los distintos tiempos de medición, con un máximo de 5,40 ± 0,12 y 4,93 ± 0,05 mmol Fe2+/g ES a los 60 minutos, respectivamente. La información de esta memoria permitió aportar a los estudios realizados con hojas murtilla en el laboratorio de Productos Naturales de la Universidad de Chile, principalmente en relación a la influencia del genotipo sobre su composición fenólica y actividad antioxidante.

Prevalencia del síndrome de dolor y disfunción temporomandibular y factores de riesgo en estudiantes de odontología / Prevalence of temporomandibular dysfunction and pain syndrome and associated factors in dental students

Introducción: El síndrome de dolor y disfunción temporomandibular (SDDTM) se ha identificado como una de las princi - pales causas de dolor no dental en la región orofacial. Objetivo: Describir la prevalencia del SDDTM y factores asociados en los estu- diantes de la Facultad de Odontología de la Universidad Cooperativa de Colombia (UCC), sede Envigado. Materiales y métodos: estudio observacio - nal, descriptivo y transversal. Se evaluaron 98 estudiantes seleccionados aleatoriamen - te. Se realizó una encuesta, examen clíni - co, índice de Helkimo, test emocional de ansiedad y angustia. Se realizó un análisis univariado y bivariado (Chi 2 , p <0,05). Resultados: El 66% fueron mujeres. La edad promedio fue 21 años (DE±3). El 26,5% presentó hábito de apretamiento dental, 26,5% onicofagia y 18,4% bruxis- mo. El 31,6% refirió ruido articular, 30,6% cefalea-dolor de cuello, 18,4% dolor en articulación temporomandibular (ATM) y 12,2% rigidez mandibular. Clínicamente 44,5% presentó masticación unilateral. El 62,2% desviación durante la apertura y al cierre el 81,6%. El 42,9% presentó tras - torno de ansiedad y en menor proporción trastornos de angustia. El 74,7% presentó SDDTM leve. No se encontró diferen - cia con significación estadística entre la prevalencia del SDDTM por sexo (Chi 2 , p =0,255), ni asociación con los trastornos de ansiedad (Chi 2, p=0,060), ni angustia. Conclusión: El SDDTM leve fue el más frecuente entre la población estudiantil, se identificaron hábitos de apretamiento, onicofagia y masticación unilateral. No se encontró diferencia por sexo, ni asociación con trastorno de ansiedad ni de angustia. Llama la atención la alta prevalencia de masticación unilateral y del trastorno de ansiedad, son importantes estudiar

Background: Temporomandibular joint dysfunction and pain syndrome (TMJD) has been identified as one of the principle causes of non-dental pain in the orofacial region. Objective: To describe the prevalence of TMJD and associated factors in the students of the faculty of odontology of the Uni - versity Cooperativa de Colombia (UCC), Envigado campus Materials and methods: Observational, descriptive and cross-sectional study. 98 randomly selected students were evaluated. A survey, clinical examination, Helkimo index, emotional distress and anxiety test was performed. Univariate and bivariate analysis was performed (Chi 2 , p <0,05). Results: 66% were women. The average age was 21 years (SD±3). 26,5% had dental clenching habit, 26,5% and 18,4% onicofagia bruxism. 31,6% reported joint click, 30,6% headache or neck pain, 18,4% pain in the temporomandibular joint (TMJ) and 12,2% mandibular stiffness. Clinically, 44,5% presented unilateral chewing. 62,2% showed deviation on opening and 81,6% on closing. 42,9% had anxiety disorder and to a lesser extent panic disorder. 74,7% had mild TMJD. No difference was found with statistical significance between the preva - lence of sex TMJD. (Chi 2 , p =0,255) or any association with anxiety disorders (Chi 2 , p =0,060), and distress. Conclusions: Mild TMJD was most frequent among the student population. Clenching habits, nail biting and unilateral chewing were identified. No difference was found by gender or any association with anxiety disorder or anxiety. Particularly striking is the high prevalence of unilateral chewing and anxiety disorder, which are important to study

Toxin-antitoxin systems in the mobile genome of Acidithiobacillus ferrooxidans.

Toxin-antitoxin (TA) systems are genetic modules composed of a pair of genes encoding a stable toxin and an unstable antitoxin that inhibits toxin activity. They are widespread among plasmids and chromosomes of bacteria and archaea. TA systems are known to be involved in the stabilization of plasmids but there is no consensus about the function of chromosomal TA systems. To shed light on the role of chromosomally encoded TA systems we analyzed the distribution and functionality of type II TA systems in the chromosome of two strains from Acidithiobacillus ferrooxidans (ATCC 23270 and 53993), a Gram-negative, acidophilic, environmental bacterium that participates in the bioleaching of minerals. As in other environmental microorganisms, A. ferrooxidans has a high content of TA systems (28-29) and in twenty of them the toxin is a putative ribonuclease. According to the genetic context, some of these systems are encoded near or within mobile genetic elements. Although most TA systems are shared by both strains, four of them, which are encoded in the active mobile element ICEAfe1, are exclusive to the type strain ATCC 23270. We demonstrated that two TA systems from ICEAfe1 are functional in E. coli cells, since the toxins inhibit growth and the antitoxins counteract the effect of their cognate toxins. All the toxins from ICEAfe1, including a novel toxin, are RNases with different ion requirements. The data indicate that some of the chromosomally encoded TA systems are actually part of the A. ferrooxidans mobile genome and we propose that could be involved in the maintenance of these integrated mobile genetic elements.

ICE Afe 1, an actively excising genetic element from the biomining bacterium Acidithiobacillus ferrooxidans.

Integrative conjugative elements (ICEs) are self-transferred mobile genetic elements that contribute to horizontal gene transfer. An ICE (ICEAfe1) was identified in the genome of Acidithiobacillus ferrooxidans ATCC 23270. Excision of the element and expression of relevant genes under normal and DNA-damaging growth conditions was analyzed. Bioinformatic tools and DNA amplification methods were used to identify and to assess the excision and expression of genes related to the mobility of the element. Both basal and mitomycin C-inducible excision as well as expression and induction of the genes for integration/excision are demonstrated, suggesting that ICEAfe1 is an actively excising SOS-regulated mobile genetic element. The presence of a complete set of genes encoding self-transfer functions that are induced in response to DNA damage caused by mitomycin C additionally suggests that this element is capable of conjugative transfer to suitable recipient strains. Transfer of ICEAfe1 may provide selective advantages to other acidophiles in this ecological niche through dissemination of gene clusters expressing transfer RNAs, CRISPRs, and exopolysaccharide biosynthesis enzymes, probably by modification of translation efficiency, resistance to bacteriophage infection and biofilm formation, respectively. These data open novel avenues of research on conjugative transformation of biotechnologically relevant microorganisms recalcitrant to genetic manipulation.

Aislamiento de los microorganismos cultivables de la rizosfera de Ornithogalum umbellatum y evaluación del posible efecto biocontrolador en dos patógenos del suelo / Isolating cultured Ornithogalum umbellatum rhizosphere microorganisms and assessment of possible biocontrolling effect on two ground pathogens

Los microorganismos presentes en el suelo ejercen una acción específica cuando se relacionan con una especie vegetal. Ornithogalum umbellatum es una planta silvestre usada en el país como ornamental pero reconocida en otras partes del mundo por los componentes químicos del bulbo y la aplicación de estas sustancias en el tratamiento alternativo de diferentes enfermedades. Esta característica permitió plantear la hipótesis de que la microflora existente en la rizosfera podría tener posibles efectos biocontroladores en fitopatógenos del suelo y a realizar una evaluación del efecto antagónico in vitro con Botrytis cinerea y Sclerotinia sclerotiorum. El estudio se realizó tomando 12 muestras de rizosfera y sembrándolas por duplicado en medios sólidos, agar papa dextrosa, agar Rosa de Bengala (con y sin antibiótico), agar yeast mold y agar Luria Bertani. Se aislaron 8 morfotipos de levaduras, 5 morfotipos de bacterias y 9 morfotipos de mohos; la mayoría de ellos demostraron antagonismo in vitro con los fitopatógenos seleccionados. Este estudio permite concluir que en la rizósfera de la planta Ornithogalum umbellatum se encuentran microorganismos que demostraron antagonismo in vitro y que podrían ser utilizados como biocontroladores de Botrytis cinerea y Sclerotinia sclerotiorum. Además, se observó que algunos de los morfotipos microbianos aislados presentaban un antagonismo muy discreto pero ocasionaban cambios en la estructura y color de las hifas de los hongos fitopatógenos utilizados; posiblemente la concentración influye sobre los mecanismos de biocontrol.