Characterization of a Clostridioides difficile ST-293 isolate from a recurrent infection in Argentina

Resumen Clostridioides difficile es un patógeno esporulado oportunista responsable de diarrea asociada a antibióticos en humanos. C. difficile produce 2 toxinas principales: TcdAy TcdB, además de la toxina binaria (CDT), también asociada a la virulencia. Este estudio buscó caracterizar el aislamiento ALCD3, involucrado en un episodio de recurrencia de una infección nosocomial. La caracterización molecular mostró que dicho aislamiento pertenece al toxinotipo 0/v y el análisis por MLST demostró un perfil alélico adk:91, atpA:1, dxr:2, glyA: 1, recA:27, sodA: 1 y tpi:1, lo cual corresponde al ST293 (MLST clado 1). Durante el crecimiento, el aislamiento ALCD3 mostró un incremento temprano de la tasa de esporulación y valores máximos de formas termorresistentes luego de 2 días de incubación. Tanto la cinética de esporulación como la producción de formas termorresistentes fueron más rápidas en el aislamiento ALCD3 que en la cepa de referencia VPI 10463. La germinación en presencia del germinante natural taurocolato fue más rápida en el aislamiento ALCD3 que en la cepa VPI 10463, lo que indica que aquel comienza la hidrólisis del córtex antes. También, el co-germinante glicina indujo una rápida liberación de ácido dipicolínico en ALCD3. Estos hallazgos indican que el aislamiento ALCD3 es particularmente eficiente en la esporulación y en la germinación. El presente trabajo representa el primer informe de la circulación de C. difficile ST293 en Argentina. La habilidad del aislamiento ALCD3 para producir toxinas y su alta capacidad de esporulación/germinación son características claves compatibles con un alto potencial de diseminación e inducción de infecciones recurrentes.

Abstract Clostridioides difficile is an opportunistic spore-forming pathogen responsible for antibiotic-associated diarrhea in humans. C. difficile produces two main toxins: TcdA and TcdB as well as a third toxin named binary toxin (CDT) that is also involved in virulence. The present study aimed at characterizing the C. difficile isolate ALCD3 involved in a relapse episode of nosocomial infection. Molecular characterization showed that isolate ALCD3 belongs to tox-inotype 0/v and the MLST analysis demonstrated allelic profile adk:91, atpA:1, dxr:2, glyA: 1, recA:27, sodA: 1 and tpi:1 which corresponds to ST293 (MLST clade: 1). During growth, isolate ALCD3 showed an early increase in the sporulation ratio as well as maximal values of heat resis-tant forms after 2 days of incubation. Both sporulation kinetics and production of heat resistant forms were faster for isolate ALCD3 than for the reference strain VPI 10463. Germination in the presence of the natural germinant taurocholate was faster for isolate ALCD3 than for strain VPI 10463, which indicates that isolate ALCD3 starts cortex hydrolysis earlier than strain VPI 10463. Furthermore, the co-germinant glycine, induces rapid release of dipicolinic acid (DPA) in isolate ALCD3. These findings indicate that isolate ALCD3 is particularly efficient in both sporulation and germination. The present work represents the first report of the circulation of C. difficile ST293 in Argentina. The ability of isolate ALCD3 to produce toxins and its high sporulation/germination capacity are key features compatible with a microorganism with high dissemination potential and the possibility of inducing recurrent infections.

S-layer proteins as immune players: Tales from pathogenic and non-pathogenic bacteria.

In bacteria, as in other microorganisms, surface compounds interact with different pattern recognition receptors expressed by host cells, which usually triggers a variety of cellular responses that result in immunomodulation. The S-layer is a two-dimensional macromolecular crystalline structure formed by (glyco)-protein subunits that covers the surface of many species of Bacteria and almost all Archaea. In Bacteria, the presence of S-layer has been described in both pathogenic and non-pathogenic strains. As surface components, special attention deserves the role that S-layer proteins (SLPs) play in the interaction of bacterial cells with humoral and cellular components of the immune system. In this sense, some differences can be predicted between pathogenic and non-pathogenic bacteria. In the first group, the S-layer constitutes an important virulence factor, which in turn makes it a potential therapeutic target. For the other group, the growing interest to understand the mechanisms of action of commensal microbiota and probiotic strains has prompted the studies of the role of the S-layer in the interaction between the host immune cells and bacteria bearing this surface structure. In this review, we aim to summarize the main latest reports and the perspectives of bacterial SLPs as immune players, focusing on those from pathogenic and commensal/probiotic most studied species.

Clostridioides difficile: Characterization of the circulating toxinotypes in an Argentinean public hospital / Clostridioides difficile: caracterización de los toxinotipos circulantes en un hospital público de Argentina

Abstract Clostridioides difficile is a spore-forming anaerobe microorganism associated to nosocomial diarrhea. Its virulence is mainly associated with TcdA and TcdB toxins, encoded by their respective tcdA and tcdB genes. These genes are part of the pathogenicity locus (PaLoc). Our aim was to characterize relevant C. difficile toxinotypes circulating in the hospital setting. The tcdA and tcdB genes were amplified and digested with different restriction enzymes: EcoRI for tcdA; HincII and AccI for tcdB. In addition, the presence of the cdtB (binary toxin) gene, TcdA and TcdB toxins by dot blot and the cytotoxic effect of culture supernatants on Vero cells, were evaluated. Altogether, these studies revealed three different circulating toxinotypes according to Rupnik's classification: 0, I and VIII, being the latter the most prevalent one. Even though more studies are certainly necessary (e.g. sequencing analysis), it is worth noting that the occurrence of toxinotype I could be related to the introduction of bacteria from different geographical origins. The multivariate analysis conducted on the laboratory values of individuals infected with the most prevalent toxinotype (VIII) showed that the isolates associated with fatal outcomes (GCD13, GCD14 and GCD22) are located in regions of the biplots related to altered laboratory values at admission. In other patients, although laboratory values at admission were not correlated, levels of urea, creatinine and white blood cells were positively correlated after the infection was diagnosed. Our study reveals the circulation of different toxinotypes of C. difficile strains in this public hospital. The variety of toxinotypes can arise from pre-existing microorganisms as well as through the introduction of bacteria from other geographical regions. The existence of microorganisms with different pathogenic potential is relevant for the control, follow-up, and treatment of the infections.

Resumen Clostridioides difficile es un anaerobio esporulado que se asocia con episodios de diarreas hospitalarias. Su virulencia se encuentra vinculada, principalmente, a las toxinas TcdA y TcdB, codificadas por sus respectivos genes, tcdA y tcdB, que son parte de un locus de patogenicidad (PaLoc). Nuestro objetivo fue caracterizar los toxinotipos de C. difficile circulantes en un hospital público. Los genes tcdA y tcdB fueron amplificados y digeridos con diferentes enzimas de restricción: EcoRI para tcdA; HincII y AccI para tcdB. Además, se evaluó la presencia de cdtB (gen de la toxina binaria B) y de las toxinas A y B (por dot blot), así como el efecto citotóxico de sobrenadantes de cultivo sobre células Vero. En conjunto, estos estudios revelaron tres toxinotipos circulantes según la clasificación de Rupnik: 0, I y VIII; el más prevalente fue el último. Aunque son necesarios más estudios (ej., secuenciación), es interesante notar que la presencia del toxinotipo I podría estar relacionada con la introducción de bacterias de diferente origen geográfico. En los pacientes infectados con el toxinotipo VIII, el análisis multivariante de los resultados de laboratorio mostró que los aislamientos asociados a decesos (GCD13, GCD14 y GCD22) estaban situados en regiones de los biplots relacionados con valores de laboratorio alterados al momento de la internación. En los otros pacientes, aunque no se observó correlación entre los valores de laboratorio al momento de la internación y la evolución clínica, los niveles de urea, creatinina y recuento de glóbulos blancos estuvieron correlacionados positivamente entre sí una vez diagnosticada la infección. Nuestro estudio revela la circulación de diferentes toxinotipos de C. difficile en un mismo hospital público. La variedad de toxinotipos puede originarse a partir de microorganismos preexistentes en la región, así como también por la introducción de bacterias provenientes de otras regiones geográficas. La existencia de microorganismos con diferente potencial patogénico es relevante para el control, el seguimiento y el tratamiento de las infecciones.

Characterization of a Clostridioides difficile ST-293 isolate from a recurrent infection in Argentina.

Clostridioides difficile is an opportunistic spore-forming pathogen responsible for antibiotic-associated diarrhea in humans. C. difficile produces two main toxins: TcdA and TcdB as well as a third toxin named binary toxin (CDT) that is also involved in virulence. The present study aimed at characterizing the C. difficile isolate ALCD3 involved in a relapse episode of nosocomial infection. Molecular characterization showed that isolate ALCD3 belongs to toxinotype 0/v and the MLST analysis demonstrated allelic profile adk:91, atpA:1, dxr:2, glyA: 1, recA:27, sodA: 1 and tpi:1 which corresponds to ST293 (MLST clade: 1). During growth, isolate ALCD3 showed an early increase in the sporulation ratio as well as maximal values of heat resistant forms after 2 days of incubation. Both sporulation kinetics and production of heat resistant forms were faster for isolate ALCD3 than for the reference strain VPI 10463. Germination in the presence of the natural germinant taurocholate was faster for isolate ALCD3 than for strain VPI 10463, which indicates that isolate ALCD3 starts cortex hydrolysis earlier than strain VPI 10463. Furthermore, the co-germinant glycine, induces rapid release of dipicolinic acid (DPA) in isolate ALCD3. These findings indicate that isolate ALCD3 is particularly efficient in both sporulation and germination. The present work represents the first report of the circulation of C. difficile ST293 in Argentina. The ability of isolate ALCD3 to produce toxins and its high sporulation/germination capacity are key features compatible with a microorganism with high dissemination potential and the possibility of inducing recurrent infections.

Clostridioides difficile: Characterization of the circulating toxinotypes in an Argentinean public hospital.

Clostridioides difficile is a spore-forming anaerobe microorganism associated to nosocomial diarrhea. Its virulence is mainly associated with TcdA and TcdB toxins, encoded by their respective tcdA and tcdB genes. These genes are part of the pathogenicity locus (PaLoc). Our aim was to characterize relevant C. difficile toxinotypes circulating in the hospital setting. The tcdA and tcdB genes were amplified and digested with different restriction enzymes: EcoRI for tcdA; HincII and AccI for tcdB. In addition, the presence of the cdtB (binary toxin) gene, TcdA and TcdB toxins by dot blot and the cytotoxic effect of culture supernatants on Vero cells, were evaluated. Altogether, these studies revealed three different circulating toxinotypes according to Rupnik's classification: 0, I and VIII, being the latter the most prevalent one. Even though more studies are certainly necessary (e.g. sequencing analysis), it is worth noting that the occurrence of toxinotype I could be related to the introduction of bacteria from different geographical origins. The multivariate analysis conducted on the laboratory values of individuals infected with the most prevalent toxinotype (VIII) showed that the isolates associated with fatal outcomes (GCD13, GCD14 and GCD22) are located in regions of the biplots related to altered laboratory values at admission. In other patients, although laboratory values at admission were not correlated, levels of urea, creatinine and white blood cells were positively correlated after the infection was diagnosed. Our study reveals the circulation of different toxinotypes of C. difficile strains in this public hospital. The variety of toxinotypes can arise from pre-existing microorganisms as well as through the introduction of bacteria from other geographical regions. The existence of microorganisms with different pathogenic potential is relevant for the control, follow-up, and treatment of the infections.

Persistent reduction of retinal microvascular vessel density in patients with moderate and severe COVID-19 disease.

OBJECTIVE: This study aims to analyse the possible recovery or worsening in retinal microvasculature after 8 months in a previously studied COVID-19 cohort. METHODS AND ANALYSIS: A cross-sectional case-control study and a prospective longitudinal cohort study. Participants were the subjects of our previous study who re-enrolled for a new examination including a fundus photograph (retinography), an optical coherence tomography (OCT) scan and an OCT angiography. COVID-19 diagnosed patients were divided into three groups: group 1: mild disease, asymptomatic/paucisymptomatic subjects who received outpatient care; group 2: moderate disease and group 3: severe disease, both of which required hospital admission because of pneumonia. Statistical analyses were performed using SPSS software (V.23.0). Cross-sectional intergroup differences were analysed by means of analysis of variance for normally distributed variables and the Kruskal-Wallis test for non-normally distributed ones. In reference to the prospective part of the study (intragroup differences, baseline with 8-month comparison), a paired t-test was used for normally distributed data and Wilcoxon signed ranks sum for non-normally distributed data. RESULTS: The fovea-centered superficial and deep vascular densities were significantly diminished in severe cases compared with mild cases (p=0.004; p=0.003, respectively, for superficial and deep) and to controls (p=0.014; p=0.010), also in moderate cases to mild group (p=0.004; p=0.003) and to controls (p=0.012; p=0.024). In the longitudinal study, no significant statistical differences were found between baseline and 8-month follow-up vessel density values. CONCLUSION: We demonstrated persistent reduction in the central vascular area over time in patients with moderate and severe COVID-19.

Atypical Sympathetic Ophthalmia Presenting with Ocular Pain Associated with Posterior Scleral Involvement.

PURPOSE: To describe an atypical case of sympathetic ophthalmia. Design: Case report. RESULTS: A 37 -year-old female presented a 3-day long acute left retroocular pain and photophobia, 1 month after having undergone evisceration of the fellow eye. Upon exploration, the patient presented conjunctival injection, macular retinal folds with peripapillary subretinal fluid, and hypocyanescent choroidal spots on indocyanine green angiography. A sympathetic ophthalmia with a reactive posterior scleritis involvement was diagnosed. The patient underwent treatment with prednisone, mycophenolate, and cyclosporine with slowly tapering, presenting a total recovery over the years. CONCLUSION: Sympathetic ophthalmia may present itself atypically as ocular pain with little vision loss secondarily to a mild panuveitis with reactive scleral involvement.

Mucosal Immunoregulatory Properties of Tsukamurella inchonensis to Reverse Experimental Food Allergy.

The intestinal mucosa is lined by epithelial cells, which are key cells to sustain gut homeostasis. Food allergy is an immune-mediated adverse reaction to food, likely due to defective regulatory circuits. Tsukamurella inchonensis is a non-pathogenic bacterium with immunomodulatory properties. We hypothesize that the anti-inflammatory effect of dead T. inchonensis on activated epithelial cells modulates milk allergy through the restoration of tolerance in a mouse model. Epithelial cells (Caco-2 and enterocytes from mouse gut) and macrophages were stimulated with T. inchonensis and induction of luciferase under the NF-κB promoter, ROS and cytokines production were studied. Balb/c mice were mucosally sensitized with cow´s milk proteins plus cholera toxin and orally challenged with the allergen to evidence hypersensitivity symptoms. After that, mice were orally administered with heat-killed T. inchonensis as treatment and then challenged with the allergen. The therapeutic efficacy was in vivo (clinical score and cutaneous test) and in vitro (serum specific antibodies and cytokines-ELISA, and cell analysis-flow cytometry) evaluated. Heat-killed T. inchonensis modulated the induction of pro-inflammatory chemokines, with an increase in anti-inflammatory cytokines by intestinal epithelial cells and by macrophages with decreased OX40L expression. In vivo, oral administration of T. inchonensis increased the frequency of lamina propria CD4+CD25+FoxP3+ T cells, and clinical signs were lower in T. inchonensis-treated mice compared with milk-sensitized animals. In vivo depletion of Tregs (anti-CD25) abrogated T. inchonensis immunomodulation. In conclusion, these bacteria suppressed the intestinal inflammatory immune response to reverse food allergy.

Clinopodium mexicanum: potential and difficulties for the sustainable use of a Mexican medicinal plant / Clinopodium mexicanum: potencial y dificultades para el uso de una planta medicinal mexicana

Clinopodium mexicanum (Benth.) Govaerts (Lamiaceae) is a native plant of Mexico. This plant is used in traditional Mexican medicine for the treatment of cultural specific syndromes such as "susto", "nervios" or "espanto", conditions related to anxiety and depression. In addition, it has a high biocultural value for its medicinal and culinary use and for its exchange value in various indigenous areas of México. This review aims to compile updated information about the ethnobotanical, phytochemistry and commercialization aspects of Clinopodium mexicanum, and it focuses on the potential use of this species as a raw material in the phytopharmaceutical industry for the treatment of anxiety and pain. Moreover, it would be a viable productive alternative for many rural communities, which could not only produce the raw material but who could also add value to the sale of the plant.

Clinopodium mexicanum (Benth.) Govaerts, es una planta nativa de México. Es usada en la medicina tradicional mexicana para tratar síndromes de filiación cultural como "susto", "nervios" o "espanto", los cuales están asociados a la ansiedad y depresión. Además, la planta presenta un alto valor biocultural por sus usos: medicinal, culinario y por el valor de cambio que tiene en áreas indígenas de México. La revisión presentada tiene como objetivo compilar información actualizada sobre aspectos etnobotánicos, fitoquímicos y comercialización de Clinopodium mexicanum. Igualmente señalar el potencial uso de esta especie como materia prima para la elaboración de productos para la ansiedad y el dolor por parte de la industria fitofarmacéutica. Además de ser una alternativa productiva para muchas comunidades rurales, las cuales pueden venderla como materia prima o como producto derivado.

Lactobacillus strains inhibit biofilm formation of Salmonella sp. isolates from poultry.

Lactic acid bacteria (LAB) exert a strong antagonistic activity against many microorganisms including food spoilage organisms and may be used as an alternative to control biofilm formation of pathogens in food industries. The objective of this work was to investigate the ability of fifteen Salmonella strains isolated from poultry environment to form biofilms on different surfaces. In addition, the effect of Lactobacillus kefiri strains 8321 and 83113 and Lactobacillus plantarum 83114 and their surface proteins on biofilm development of Salmonella Enteritidis 115 was studied. The relationship between surface properties of bacteria (hydrophobicity, autoaggregation and coaggregation with lactobacilli) and biofilm formation was also investigated. Most of Salmonella strains were hydrophilic and five strains were moderately hydrophobic. In general, Salmonella strains showed high aggregation abilities (27-54%). S. Enteritidis 106 and S. Typhimurium 102 and 108 showed the highest percentages of autoaggregation. All Salmonella strains tested showed aggregation abilities with the three lactobacilli studied, but the percentage of coaggregation proved to be strain-specific. When comparing stainless steel, glass and polystyrene surfaces, higher levels of biofilm formation occurred on polystyrene plate than on glass surfaces or stainless steel. S. Enteritidis 115 exhibited the greatest attachment to polyestyrene surface. The preincubation or coincubation with the three lactobacilli strains significantly reduced (about 1 log CFU/ml of reduction) the ability of S. Enteritidis 115 to form biofilm compared to the control without lactobacilli. These results were confirmed by confocal microscopy. In the same way, when surface proteins extracted from lactobacilli strains were preincubated or coincubated with S. Enteritidis 115, biofilm formation of this strain was significantly decreased compared to the control. The results obtained showed that these Lactobacillus strains and their surface proteins can be used as alternatives for control of biofilm formation by Salmonella in the poultry industry.

Biofilm formation by Salmonella sp. in the poultry industry: Detection, control and eradication strategies.

Salmonella represents an important global public health problem and it is an emerging zoonotic bacterial threat in the poultry industry. Diverse registered human cases of salmonellosis shown poultry origins. Various control measures have been employed both at the farming and processing levels to address it. This review focuses on traditional and new detection techniques of biofilm formation by Salmonella spp. and different approaches that can be used to prevent and/or control biofilm formation by these bacteria. A number of methodologies based on different approximations have been recently employed to detect and evaluate bacteria attached to surfaces, including real-time polymerase chain reaction (PCR), confocal laser scanning microscopy and Optical Coherence Tomography. Due to persistence of Salmonella biofilm in food processing environments after cleaning and sanitation, control and eradication strategies in poultry industry should be constantly studied. In this sense, the use of several alternatives to control Salmonella biofilm formation, such as lactic acid bacteria, phagetherapy, extracts from aromatic plants, quorum sensing inhibitors, bacteriocins and nanomaterials, have been successfully tested and will be reviewed.

Detection of toxigenic Clostridioides --#1;Clostridium--#3; difficile: Usefulness of two commercially available enzyme immunoassays and a PCR assay on stool samples and stool isolates / Detección de Clostridioides --#1;Clostridium--#3; difficile toxigénico: utilidad de dos métodos de enzimoinmunoensayo comerciales y una PCR en las muestras de materia fecal y en los respectivos aislamientos

The best laboratory diagnostic approach to detect Clostridioides --#1;Clostridium--#3; difficile infection (CDI) is a subject of ongoing debate. With the aim of evaluating four laboratory diagnostic methods, 250 unformed stools from patients with suspected CDI submitted to nine medical center laboratories from November 2010 to December 2011, were studied using: (1) an immunochromatographic rapid assay test that combines the qualitative determination of glutamate dehydrogenase (GDH) plus toxins A and B (QAB), the CDIFF QUIK CHEK COMPLETE assay; (2) an enzyme immunoassay for qualitative determination of toxins A and B, the RIDASCREENTC. difficile Toxin A/B assay (RAB); (3) a PCR for the toxin B gene assay (PCR); and (4) the toxigenic culture (TC).C. difficile isolates from direct toxin negative stools by QAB, RAB and PCR were evaluated for toxigenicity by the same direct tests, in order to assess the contribution of the TC (QAB-TC, RAB-TC, PCR-TC). A combination of the cell culture cytotoxicity neutralization assay (CCCNA) in stools, and the same assay on isolates from direct negative samples (CCCNA-TC) was considered the reference method (CCCNA/CCCNA-TC). Of the 250 stools tested, 107 (42.8%) were positive by CCCNA/CCCNA-TC. The GDH and PCR/PCR-TC assays were the most sensitive, 91.59% and 87.62%, respectively. The QAB, RAB, QAB/QAB-TC and RAB/RAB-TC had the highest specificities, ca. 95%. A negative GDH result would rule out CDI, however, its low positive likelihood ratio (PLR) of 3.97 indicates that a positive result should always be complemented with the detection of toxins. If the RAB, QAB, and PCR assays do not detect toxins from direct feces, the toxigenic culture should be performed. In view of our results, the most accurate and reliable methods to be applied in a clinical microbiology laboratory were the QAB/QAB-TC, and RAB/RAB-TC, with PLRs >10 and negative likelihood ratios <0.30.

El mejor procedimiento para realizar el diagnóstico de laboratorio de la infección causada por Clostridioides --#1;Clostridium--#3; difficile (ICD) es aún objeto de debate. Con el fin de evaluar cuatro métodos diagnósticos de laboratorio, se estudiaron 250 muestras de heces diarreicas provenientes de pacientes con sospecha de ICD remitidas a los laboratorios de nueve centros médicos entre noviembre de 2010 y diciembre de 2011. Dichas muestras se analizaron mediante los siguientes métodos:1) un ensayo rápido inmunocromatográfico que combina la detección cualitativa de la glutamato deshidrogenasa (GDH) y de las toxinas Ay B (QAB), CDIFF QUIK CHEK COMPLETE;2) un enzimoinmunoanálisis para la determinación cualitativa de las toxinas A/B, RIDASCREENTC. difficile Toxin A/B (RAB);3) un método molecular basado en PCR para la detección del gen que codifica la toxina B (PCR) y 4) el cultivo toxigénico (TC). Como método de referencia se utilizó la combinación del ensayo de citotoxicidad sobre cultivo de células con la neutralización de toxina mediante anticuerpo específico en los filtrados de las heces (CCCNA) y el mismo método en sobrenadantes de aislamientos de C. difficile (CCCNA-TC). La toxigenicidad de las cepas aisladas de muestras directas negativas con QAB, RAB y PCR se evaluó con los mismos métodos, con el propósito de detectar la contribución del TC (QAB-TC, RAB-TC, PCR-TC). De las 250 muestras estudiadas, 107 (42,8%) fueron positivas por CCCNA/CCCNA-TC. Los métodos GDH y PCR/PCR-TC fueron los más sensibles: 91,59 y 87,62%, respectivamente. Los métodos QAB, RAB, QAB/QAB-TC y RAB/RAB-TC mostraron las mayores especificidades, del 95%, aproximadamente. Un resultado negativo para GDH excluiría la ICD, pero su baja razón de verosimilitud positiva (PLR), que fue 3,97, indica que un resultado positivo debe complementarse con la detección de toxinas. Cuando no se detectan toxinas directas por RAB, QAB ni PCR, debería realizarse el TC. De acuerdo con nuestros resultados, los métodos más precisos y confiables para ser aplicados en un laboratorio de microbiología clínica son QAB/QAB-TC y RAB/RAB-TC, con una PLR> 10 y una razón de verosimilitud negativa < 0,30.

Detection of toxigenic Clostridioides [Clostridium] difficile: Usefulness of two commercially available enzyme immunoassays and a PCR assay on stool samples and stool isolates.

The best laboratory diagnostic approach to detect Clostridioides [Clostridium] difficile infection (CDI) is a subject of ongoing debate. With the aim of evaluating four laboratory diagnostic methods, 250 unformed stools from patients with suspected CDI submitted to nine medical center laboratories from November 2010 to December 2011, were studied using: (1) an immunochromatographic rapid assay test that combines the qualitative determination of glutamate dehydrogenase (GDH) plus toxins A and B (QAB), the CDIFF QUIK CHEK COMPLETE assay; (2) an enzyme immunoassay for qualitative determination of toxins A and B, the RIDASCREEN™ C. difficile Toxin A/B assay (RAB); (3) a PCR for the toxin B gene assay (PCR); and (4) the toxigenic culture (TC). C. difficile isolates from direct toxin negative stools by QAB, RAB and PCR were evaluated for toxigenicity by the same direct tests, in order to assess the contribution of the TC (QAB-TC, RAB-TC, PCR-TC). A combination of the cell culture cytotoxicity neutralization assay (CCCNA) in stools, and the same assay on isolates from direct negative samples (CCCNA-TC) was considered the reference method (CCCNA/CCCNA-TC). Of the 250 stools tested, 107 (42.8%) were positive by CCCNA/CCCNA-TC. The GDH and PCR/PCR-TC assays were the most sensitive, 91.59% and 87.62%, respectively. The QAB, RAB, QAB/QAB-TC and RAB/RAB-TC had the highest specificities, ca. 95%. A negative GDH result would rule out CDI, however, its low positive likelihood ratio (PLR) of 3.97 indicates that a positive result should always be complemented with the detection of toxins. If the RAB, QAB, and PCR assays do not detect toxins from direct feces, the toxigenic culture should be performed. In view of our results, the most accurate and reliable methods to be applied in a clinical microbiology laboratory were the QAB/QAB-TC, and RAB/RAB-TC, with PLRs >10 and negative likelihood ratios <0.30.

Use of a Collagen Membrane to Enhance the Survival of Primary Intestinal Epithelial Cells.

Intestinal epithelial cell culture is important for biological, functional, and immunological studies. Since enterocytes have a short in vivo life span due to anoikis, we aimed to establish a novel and reproducible method to prolong the survival of mouse and human cells. Cells were isolated following a standard procedure, and cultured on ordered-cow's collagen membranes. A prolonged cell life span was achieved; cells covered the complete surface of bio-membranes and showed a classical enterocyte morphology with high expression of enzymes supporting the possibility of cryopreservation. Apoptosis was dramatically reduced and cultured enterocytes expressed cytokeratin and LGR5 (low frequency). Cells exposed to LPS or flagellin showed the induction of TLR4 and TLR5 expression and a functional phenotype upon exposure to the probiotic Bifidobacterium bifidum or the pathogenic Clostridium difficile. The secretion of the homeostatic (IL-25 and TSLP), inhibitory (IL-10 and TGF-ß), or pro-inflammatory mediators (IL-1ß and TNF) were induced. In conclusion, this novel protocol using cow's collagen-ordered membrane provides a simple and reproducible method to maintain intestinal epithelial cells functional for cell-microorganism interaction studies and stem cell expansion. J. Cell. Physiol. 232: 2489-2496, 2017. © 2016 Wiley Periodicals, Inc.

Airborne spores of Basidiomycetes in Mérida (SW Spain).

The aim of this work was to detect the presence of Basidiomycetes spores (basidiospores, teliospores, uredospores and aeciospores) in Mérida (SW Spain) and assess the influence of weather parameters. Air was sampled continuously with a volumetric seven-day Burkard spore trap for two years. Fungi spores were identified and counted at x1,000 microscope resolution. Daily and weekly meteorological data and airborne spore concentration were analysed. Twenty-three spores types were identified, including basidiospores (Amanita, Agrocybe, Cortinarius, Coprinus -2 types-, Boletus, Bovista, Calvatia, Entoloma, Ganoderma, Inocybe, Russula, Scleroderma, Telephora), teliospores (Phragmidium, Tilletia, Ustillago -4 types-), uredospores, and aeciospores (2 types), all of these types of spores included different taxa. Average concentration was of 616 spores/m(3), with maximum concentration in autumn (October), and a second concentration in spring (May-June); however, some spore types were more frequent in summer (Bovista, Ganoderma) or even in winter (Entoloma, Calvatia). The Amanita type was the most frequent (white-hyaline basidiospores); the second were teliospores of Ustilago, the third spore type was basidiospores of Coprinus (blackish basidiospores) and Agrocybe type (smoothed light to dark coloured basidiospores). Basidiospore concentration was positively correlated with temperature and negatively with relative humidity in most cases, and Ustilago teliospores concentration was positively correlated with wind speed. Differences in monthly rain were probably the origin between years. Airborne spores of Basidiomycetes may be separated into more than 20 types, and their seasonal concentration depended on meteorology as well as whether they were saprotrophic or parasitic.

Protective effect of bifidobacteria in an experimental model of Clostridium difficile associated colitis.

The aim of this study was to evaluate the ability of Bifidobacterium strains to prevent the effects associated with Clostridium difficile infection in a hamster model of enterocolitis. After clindamycin treatment (30 mg/kg), animals were infected intragastrically with C. difficile (5×108 CFU per animal). Seven days prior to antibiotic administration, probiotic treatment was started by administering bacterial suspensions of bifidobacteria in drinking water. Strains CIDCA 531, CIDCA 5310, CIDCA 5316, CIDCA 5320, CIDCA 5323 and CIDCA 5325 were used. Treatment was continued during all the experimental period. Development of diarrhoea, enterocolitis and mortality were evaluated. All the infected animals belonging to the placebo group developed enterocolitis (5/5) and only two dead (2/5) whereas in the group administered with Bifidobacterium bifidum strain CIDCA 5310 the ratio of animals with enterocolitis or dead decreased significantly (1/5 and 0/5 respectively). Biological activity of caecum contents was evaluated in vitro on Vero cells. Animals treated with strain CIDCA 5310 presented lower biological activity than those belonging to the placebo group. The present study shows the potential of selected strains of bifidobacteria to antagonise, in vivo, the virulence of C. difficile.

Ecophysiological responses to the effect of annual management on an endemic viviparous fish in central plateau of México

Studies on the biological aspects of fish typically focus on species that currently have commercial value, causing species that lack such market value to be ignored. This is the case of several freshwater fish, specifically of several members of the Goodeidae family. In the State of Querétaro there are several species of this family characterized for being viviparous and having distinctive sexual dimorphism that may have commercial potential. The subject of this study is Girardinichthys multiradiatus, a viviparous fish endemic to the upper-half of the Lerma River basin. The lack of knowledge regarding its biology and ecology has prevented the development of guidelines to manage its habitat and to preserve its population. The objective was to determine the ecophysiological responses of G. multiradiatus to its environmental management. From the sampling (24 hours every two months) population structure and dynamics were analyzed throughout a hydrological cycle using meristic data (standard length). Trophic and ecophysiological responses to fluctuations in environmental factors were also identified. Although the mexcalpique is a polytrophic species, results show that it prefers feeding on Diptera or Cladocera, while detritus is the third substance frequently found in their stomachs. Environmentally, the water regime is responsible for fluctuations in the population dynamics of the species, while temperature changes are the most influence its energy balance. These results can guide efforts to conserve this species and its habitat.

Los estudios sobre aspectos biológicos de los peces se centran, generalmente, en especies que actualmente tienen interés comercial, lo que ocasiona que las especies que carecen de tal valor en el mercado estén prácticamente olvidadas; tal es el caso de varios peces de agua dulce y más específicamente de algunos integrantes de la familia Godeidae. En el estado de Querétaro se encuentran varias especies pertenecientes a esta familia que se caracterizan por ser vivíparas y presentar un marcado dimorfismo sexual, aspectos que pudieran definir un potencial comercial. El pez objeto de este estudio es Girardinichthys multiradiatus, especie endémica de la parte alta-media de la cuenca del río Lerma; los lugares donde habita presentan procesos de degradación, fragmentación del hábitat y extracción de agua, que ponen en riesgo su existencia. Además, la falta de conocimiento sobre su biología y ecología, no permiten que se elaboren pautas de gestión de sus poblaciones o hábitats con fines de conservación y preservación de la especie o de los procesos ecológicos que mantienen la estabilidad del ecosistema que ocupa. En el presente trabajo se estudió la población de G. multiradiatus localizada en el bordo de San Martín, Amealco. El objetivo del presente trabajo fue determinar las respuestas ecofisológicas de G. multiradiatus debido al manejo de su ambiente. Se hicieron ciclos de 24 horas en muestreos bimensuales a lo largo de un ciclo hidrológico en el que se analizaron la estructura y dinámica de la población; asimismo, se determinaron las respuestas tróficas y ecofisiológicas de la población ante las fluctuaciones de los factores ambientales de su entorno. Los resultados muestran que aunque el mexcalpique es polítrofo, prefiere dípteros, cladóceros y detritus, habiendo diferencias alimentarias entre las clases de edades. G. multiradiatus presenta 12 clases de talla que van de 8 a 48 mm de longitud patrón. En el medio ambiente, el régimen de agua es responsable de las fluctuaciones en la dinámica poblacional de las especies, mientras que el cambio de temperatura es el factor de mayor influencia sobre su balance energético. Estos resultados pueden guiar los esfuerzos para conservar esta especie y su hábitat.

Bifidobacterium CECT 7765 improves metabolic and immunological alterations associated with obesity in high-fat diet-fed mice.

OBJECTIVES: To evaluate the effects of administration of Bifidobacterium pseudocatenulatum CECT 7765 on metabolic and immune alterations in obese mice. DESIGN AND METHODS: Adult male wild-type C57BL-6 mice were fed a standard diet or high-fat diet (HFD), supplemented or not with B. pseudocatenulatum CECT 7765 for 7 weeks. The assessments included biochemical and immunological parameters, insulin resistance, glucose tolerance, histology of liver, white-adipose and intestinal tissues, immunocompetent cell functions, and microbiota-related features. RESULTS: B. pseudocatenulatum CECT 7765 reduced serum cholesterol, triglyceride, and glucose levels and decreased insulin resistance and improved glucose tolerance in obese mice. This strain reduced serum levels of leptin, interleukin (IL)-6 and monocyte chemotactic protein-1, while increased those of IL-4 in HFD-fed mice. B. pseudocatenulatum CECT7765 reduced liver steatosis and the number of larger adipocytes and number of fat micelles in enterocytes of obese mice. The strain also improved the function of macrophages and dendritic cells in relation to phagocytosis, cytokine production, and induction of T-lymphocyte proliferation. The strain administration increased bifidobacteria and reduced enterobacteria and the inflammatory properties of the gut content in HFD-fed mice. CONCLUSION: B. pseudocatenulatum CECT 7765 was shown to ameliorate both metabolic and immunological dysfunctions related to obesity in HFD-fed mice.

El afrontamiento determinó las prácticas de autocuidado de una pareja viviendo con VIH/SIDA / Coping determined the self-care practices of a couple living with HIV / AIDS / Coping determinou as práticas de autocuidado de um casal vivendo com HIV / aids

El ser humano está capacitado para adaptarse a casi cualquier situación, así sea la más adversa, la enfermedad es un ejemplo de ello, sin embargo existen enfermedades que han sido etiquetadas por la sociedad, ejemplos de ello son el cáncer y recientemente el VIH/SIDA, recibir la noticia de padecer alguna de ellas, representa para el enfermo como recibir una sentencia de muerte. No obstante la persona atraviesa por una serie de etapas que lo llevan a adaptarse a ella y vivir con la enfermedad. El presente estudio, pretende investigar, cuales son las reacciones delas personas que tienen VIH/SDA, así como de interrogarlos sobre sus sentimientos al saber la noticia, para de esta manera determinar cómo vivieron el proceso de afrontamiento, todo ello desde la óptica de la metodología cualitativa y utilizando la fenomenología como herramienta, para entender sus vivencias de convivir con la enfermedad. Del mismo modo indagar sobre sus prácticas de autocuidado antes y después de saberse infectados, para ello recurrió a la historia de vida, además de realizar dos entrevistas a profundidad, cabe señalar que se realzo un estudio de caso Dell matrimonio de una pareja viviendo co VIH/SIDA. Todo complementado por observación y notas de campo, que permitieron visualizar un amplio panorama de la dinámica familiar, y así planear los resultados que se obtuvieron. Se detectaron 7 categorías que se dividieron en 16 subcategorías y se pudo observar que según como afrontaron la enfermedad, determino las prácticas de autocuidado de la pareja en estudio y algo que no esperaba encontrar en este estudio, fue el surgimiento de una categoría que nombre como anclaje, que es motivo muy especial que las personas se fijan para lograr un objetivo en este caso lo fueron los hijos de ambos, para lograr la adaptación al VIH/SIDA y vivir con la enfermedad.

The human being is able to adapt to almost any situation, even if it is the most adverse, the disease is an example of this, however there are diseases that have been labeled by society, examples of this are cancer and recently HIV / AIDS , receive the news of suffering some of them, represents for the patient how to receive a death sentence. However, the person goes through a series of stages that lead him to adapt to it and live with the disease. The present study intends to investigate, what are the reactions of people who have HIV / SDA, as well as to question them about their feelings when they know the news, in order to determine how they lived the coping process, all from the perspective of the qualitative methodology and using phenomenology as a tool, to understand their experiences of coexisting with the disease. In the same way inquire about their self-care practices before and after knowing they were infected, for that reason they resorted to the life story, in addition to conducting two in-depth interviews, it should be noted that a case study was carried out. The marriage of a couple living with HIV /AIDS. All complemented by observation and field notes, which allowed to visualize a broad panorama of family dynamics, and thus plan the results that were obtained. Seven categories were detected that were divided into 16 subcategories and it was observed that depending on how they faced the disease, I determined the self-care practices of the couple under study and something that I did not expect to find in this study, was the emergence of a category that I named as anchoring, which is a very special reason that people are set to achieve an objective in this case were the children of both, to achieve adaptation to HIV / AIDS and live with the disease.

O ser humano é capaz de se adaptar a qualquer situação, se o mais adverso, a doença é um exemplo, mas não são as doenças que foram marcadas pela sociedade, exemplos incluem o cancro e, recentemente, o HIV / SIDA , receber a notícia de sofrer alguns deles, representa para o paciente como receber uma sentença de morte. No entanto, a pessoa passa por uma série de passos que o levam a se adaptar a ele e viver com a doença. O presente estudo tem como objetivo investigar, quais são as reações delas pessoas que vivem com HIV / SDA e interrogá-los sobre seus sentimentos com a notícia, para determinar assim como viveram o processo de enfrentamento, tudo a partir da perspectiva de metodologia qualitativa e usando a fenomenologia como uma ferramenta para compreender as suas experiências de vida com a doença. Assim como perguntar sobre suas práticas de auto-cuidado antes e depois de conhecer infectado, por isso virou-se para a história da vida, além de duas entrevistas em profundidade, deve-se notar que um estudo de caso Dell casamento de um casal vida foi reforçada co HIV / AIDS. Tudo é complementado por notas de observação e de campo, o que permitiu-nos visualizar uma visão ampla da dinâmica familiar e, assim, planejar os resultados obtidos. 7 categorias foram divididas em 16 subcategorias e observou-se que, de acordo como eles enfrentaram a doença foi detectada, determinada práticas de auto-atendimento do casal em estúdio e algo inesperada descoberta neste estudo foi o surgimento de um nome de categoria como âncora, o que é motivo muito especial que as pessoas são definidas para alcançar um objetivo, neste caso, foram os filhos de ambos, para alcançar a adaptação ao HIV / SIDA e vivem com a doença.

Surface proteins from Lactobacillus kefir antagonize in vitro cytotoxic effect of Clostridium difficile toxins.

In this work, the ability of S-layer proteins from kefir-isolated Lactobacillus kefir strains to antagonize the cytophatic effects of toxins from Clostridium difficile (TcdA and TcdB) on eukaryotic cells in vitro was tested by cell detachment assay. S-layer proteins from eight different L. kefir strains were able to inhibit the damage induced by C. difficile spent culture supernatant to Vero cells. Besides, same protective effect was observed by F-actin network staining. S-layer proteins from aggregating L. kefir strains (CIDCA 83115, 8321, 8345 and 8348) showed a higher inhibitory ability than those belonging to non-aggregating ones (CIDCA 83111, 83113, JCM 5818 and ATCC 8007), suggesting that differences in the structure could be related to the ability to antagonize the effect of clostridial toxins. Similar results were obtained using purified TcdA and TcdB. Protective effect was not affected by proteases inhibitors or heat treatment, thus indicating that proteolytic activity is not involved. Only preincubation with specific anti-S-layer antibodies significantly reduced the inhibitory effect of S-layer proteins, suggesting that this could be attributed to a direct interaction between clostridial toxins and L. kefir S-layer protein. Interestingly, the interaction of toxins with S-layer carrying bacteria was observed by dot blot and fluorescence microscopy with specific anti-TcdA or anti-TcdB antibodies, although L. kefir cells did not show protective effects. We hypothesize that the interaction between clostridial toxins and soluble S-layer molecules is different from the interaction with S-layer on the surface of the bacteria thus leading a different ability to antagonize cytotoxic effect. This is the first report showing the ability of S-layer proteins from kefir lactobacilli to antagonize biological effects of bacterial toxins. These results encourage further research on the role of bacterial surface molecules to the probiotic properties of L. kefir and could contribute to strain selection with potential therapeutic or prophylactic benefits towards CDAD.