Characterization of the Adherence of Clostridium difficile Spores: The Integrity of the Outermost Layer Affects Adherence Properties of Spores of the Epidemic Strain R20291 to Components of the Intestinal Mucosa

Indexación: Web of Science.Clostridium difficile is the causative agent of the most frequently reported nosocomial diarrhea worldwide. The high incidence of recurrent infection is the main clinical challenge of C. difficile infections (CBI). Formation of C. difficile spores of the epidemic strain R20291 has been shown to be essential for recurrent infection and transmission of the disease in a mouse model. However, the underlying mechanisms of how these spores persist in the colonic environment remains unclear. In this work, we characterized the adherence properties of epidemic R20291 spores to components of the intestinal mucosa, and we assessed the role of the exosporium integrity in the adherence properties by using cdeC mutant spores with a defective exosporium layer. Our results showed that spores and vegetative cells of the epidemic R20291 strain adhered at high levels to monolayers of Caco-2 cells and mucin. Transmission electron micrographs of Caco-2 cells demonstrated that the hair-like projections on the surface of R20291 spores are in close proximity with the plasma membrane and microvilli of undifferentiated and differentiated monolayers of Caco-2 cells. Competitive-binding assay in differentiated Caco-2 cells suggests that spore-adherence is mediated by specific binding sites. By using spores of a cdeC mutant we demonstrated that the integrity of the exosporium layer determines the affinity of adherence of C. difficile spores to Caco-2 cells and mucin. Binding of fibronectin and vitronectin to the spore surface was concentration-dependent, and depending on the concentration, spore-adherence to Caco-2 cells was enhanced. In the presence of an aberrantly-assembled exosporium (cdeC spores), binding of fibronectin, but not vitronectin, was increased. Notably, independent of the exosporium integrity, only a fraction of the spores had fibronectin and vitronectin molecules binding to their surface. Collectively, these results demonstrate that the integrity of the exosporium layer of strain R20291 contributes to selective spore adherence to components of the intestinal mucosa.http://journal.frontiersin.org/article/10.3389/fcimb.2016.00099/ful

Molecular Epidemiology of Multidrug-Resistant Uropathogenic Escherichia coli O25b Strains Associated with Complicated Urinary Tract Infection in Children.

BACKGROUND: Uropathogenic Escherichia coli (UPEC) has increased the incidence of urinary tract infection (UTI). It is the cause of more than 80% of community-acquired cystitis cases and more than 70% of uncomplicated acute pyelonephritis cases. AIM: The present study describes the molecular epidemiology of UPEC O25b clinical strains based on their resistance profiles, virulence genes, and genetic diversity. METHODS: Resistance profiles were identified using the Kirby-Bauer method, including the phenotypic production of extended-spectrum β-lactamases (ESBLs) and metallo-β-lactamases (MBLs). The UPEC serogroups, phylogenetic groups, virulence genes, and integrons were determined via multiplex PCR. Genetic diversity was established using pulsed-field gel electrophoresis (PFGE), and sequence type (ST) was determined via multilocus sequence typing (MLST). RESULTS: UPEC strains (n = 126) from hospitalized children with complicated UTIs (cUTIs) were identified as O25b, of which 41.27% were multidrug resistant (MDR) and 15.87% were extensively drug resistant (XDR). The O25b strains harbored the fimH (95.23%), csgA (91.26%), papGII (80.95%), chuA (95.23%), iutD (88.09%), satA (84.92%), and intl1 (47.61%) genes. Moreover, 64.28% were producers of ESBLs and had high genetic diversity. ST131 (63.63%) was associated primarily with phylogenetic group B2, and ST69 (100%) was associated primarily with phylogenetic group D. CONCLUSION: UPEC O25b/ST131 harbors a wide genetic diversity of virulence and resistance genes, which contribute to cUTIs in pediatrics

Crecimiento y supervivencia del camarón Penaeus vannamei con aplicación de actinomicetos probióticos y homeopatía

The effect of probiotic bacteria and homeopathic medicines on growth, survival and water quality in the culture of juveniles of White Pacific shrimp Penaeus vannamei was evaluated. Four experimental groups were treated for 30 days with two probiotic strains of Streptomyces spp. RL8 (T1) and N7 (T2), homeopathic products developed in CIBNOR from pathogenic bacteria [ViP-7C + VIA-7C] (T3), homeopathic medicines for humans [PhA-7C + SIT-7C] (T4) and distilled water as a control group (T5). Shrimp treated with T2, T3 and T4 showed a significantly greater weight gain (P<0,05) compared to the T5 control. The average daily weight gain was significantly higher (P<0,05) in shrimp treated with T2 and T3, compared to T5. With respect to the survival rate, there were significant differences (P<0,05) between the treated groups (T1, T2 and T3) and the control group (T5). The density of Vibrio spp. in the culture water was significantly lower (P<0,05) in T1, T3 and T4 than in T5. With reference to T5, a significant reduction (P<0,05) of vibrios in the hepatopancreas was observed in all experimental groups. As a counterpart, the abundance of total marine heterotrophic bacteria in the hepatopancreas in all experimental groups was significantly higher (P<0.05) compared to the control. These results demonstrate that the application of probiotic actinomycetes and homeopathic medicines has potential as an alternative to the use and abuse of expensive and potentially harmful antibiotics, since in addition to maintaining good water quality they promote growth and survival of P. vannanmei, they are bio-safe and could be economically competitive for application in the commercial cultivation of the species.Se evaluó el efecto de bacterias probióticas y medicamentos homeopáticos sobre el crecimiento, la supervivencia y la calidad del agua en el cultivo de juveniles del camarón blanco del pacífico Penaeus vannamei. Cuatro grupos experimentales fueron tratados durante 30 días con dos cepas probióticas de Streptomyces spp. RL8 (T1) y N7 (T2), con productos homeopáticos desarrollados en CIBNOR a partir de bacterias patógenas [ViP-7C+VIA-7C] (T3), con medicamentos homeopáticos para humanos [PhA-7C+SIT-7C] (T4) y con agua destilada como grupo control (T5). Los camarones tratados con T2, T3 y T4 mostraron una ganancia significativamente mayor en peso (P<0.05) con respecto a T5. El peso diario ganado fue significativamente mayor (P<0,05) en los camarones tratados con T2 y T3, con respecto a T5. Se registraron diferencias significativas (P<0,05) en la tasa de supervivencia de camarones tratados con T1, T2 y T3, con respecto al control (T5). La densidad de Vibrio spp. en el agua de cultivo fue significativamente menor (P<0,05) en T1, T3 y T4, que en T5. Tomando como referencia a T5, en todos los grupos experimentales se observó una reducción significativa (P<0,05) de vibrios en el hepatopáncreas. Como contraparte, la abundancia de bacterias heterótrofas marinas totales en el hepatopáncreas en todos los grupos experimentales fue significativamente mayor (P<0,05) con respecto al control (T5). Estos resultados demuestran que la aplicación de actinomicetos probióticos y de medicamentos homeopáticos tiene potencial como alternativa al uso y abuso de antibióticos costosos y potencialmente nocivos, ya que además de mantener buena calidad del agua promueven el crecimiento y la supervivencia de P. vannanmei, son bio-seguros y podrían ser económicamente competitivos para su aplicación en el cultivo comercial de la especie

Induction of a Specific Humoral Immune Response by Nasal Delivery of Bcla2ctd of Clostridioides difficile

Clostridioides difficile, formerly known as Clostridium difficile, is a spore-forming bacterium considered as the most common cause of nosocomial infections in developed countries. The spore of C. difficile is involved in the transmission of the pathogen and in its first interaction with the host; therefore, a therapeutic approach able to control C. difficile spores would improve the clearance of the infection. The C-terminal (CTD) end of BclA2, a spore surface protein of C. difficile responsible of the interaction with the host intestinal cells, was selected as a putative mucosal antigen. The BclA2 fragment, BclA2CTD, was purified and used to nasally immunize mice both as a free protein and after adsorption to the spore of Bacillus subtilis, a well-established mucosal delivery vehicle. While the adsorption to spores increased the in vitro stability of BclA2CTD, in vivo both free and spore-adsorbed BclA2CTD were able to induce a similar, specific humoral immune response in a murine model. Although in the experimental conditions utilized the immune response was not protective, the induction of specific IgG indicates that free or spore-bound BclA2CTD could act as a putative mucosal antigen targeting C. difficile spores. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.Indexación:Scopu

Aquacultural Homoeopathy: A Focus on Marine Species

Homoeopathy is an alternative medical system proposed by Samuel Hahnemann in the eighteenth century. It uses highly diluted and agitated substances that derived from plants, minerals or animals, which have shown to be effective in human medicine, agronomy, veterinary, and as a novelty, in marine aquaculture. Aquacultural homoeopathy has developed rapidly in recent years, partially motivated by the misuse of powerful drugs (hormones, antibiotics, disinfectants) that when solving a problem generate undesirable side effects. In the last 10 years, scientific articles have been published on its application in freshwater fish native to Brazil, obtaining beneficial effects on growth, survival, hepatosomatic index, development of muscle fibres and lipid content in muscle. At Centro de Investigaciones Biológicas del Noroeste (CIBNOR, Mexico: www.cibnor.mx), we have studied the effects of homoeopathy to improve the culture of economically important marine species of molluscs, fish and shrimp. In this chapter, we show a selection of different research with preliminary or advanced results, related to the use of homoeopathy and its impact on zootechnic, biochemical, genomic and transcriptomic parameters in marine molluscs, fish and crustaceans. The results obtained suggest that homoeopathy is an eco-friendly alternative applicable in aquaculture industry to improve various productive and health aspects

Caracterización de la vía endocítica de esporas de Clostridium difficile en células epiteliales intestinales y su implicancia en infecciones recurrentes de Clostridium difficile en un modelo murino

Tesis (Magíster en Biotecnología)Clostridium difficile es un patógeno Gram-positivo, anaerobio estricto y formador de esporas. Este patógeno es el responsable de la mayoría de muertes causadas por diarreas asociadas a antibióticos. Las infecciones por C. difficile (ICD) tienen una elevada tasa de recurrencia, que varía entre el 20% después del primer episodio, a un 40 y 60% después del segundo y tercero, respectivamente. Se ha demostrado que las esporas son el morfotipo de transmisión y persistencia en C. difficile para generar infecciones por C. difficile recurrentes (ICD-R). Sin embargo, poco es conocido sobre los mecanismos de persistencia de las esporas en el hospedero. En este sentido, datos no publicados de nuestro laboratorio demuestran que las esperos de C. difficile son capaces de entrar en células epiteliales intestinales (CEIs) en un proceso dependiente de la polimerización actina, el cual podría ser uno de los mecanismos de persistencia. El objetivo de esta tesis es identificar las vías de endocitosis involucradas en la entrada de las esporas de la cepa R20291 de C. difficile y evaluar si el uso de inhibidores de la endocitosis de esporas reducen los casos de ICD-R en un modelo murino. En este trabajo encontramos que; i) mediante análisis de micrografías electrónicas de transmisión, microscopía confocal de fluorescencia e inhibidores farmacológicos, las esporas de C. difficile se asocian y requiren vesículas de clatrina, caveolina y macropinosomas para ingresar a las células; ii) el uso de un inhibidor de endocitosis en ratones infectados por C. difficile, redujo la incidencia de diarrea durante la ICD-R en un 67% y un 80% al ser administrados de forma intraperitoneal y oral respectivamente, lo cual sugiere que la endocitosis de esporas es el mecanismo de persistencia de C. difficile en el hospedero para generar ICD-R. En consecuencia, este trabajo amplía las aristas para generar terapias farmacológicas combinadas para tratar infecciones recurrentes por C. difficile

Clostridium difficile exosporium cysteine-rich proteins are essential for the morphogenesis of the exosporium layer, spore resistance, and affect C. difficile pathogenesis.

Clostridium difficile is a Gram-positive spore-former bacterium and the leading cause of nosocomial antibiotic-associated diarrhea that can culminate in fatal colitis. During the infection, C. difficile produces metabolically dormant spores, which persist in the host and can cause recurrence of the infection. The surface of C. difficile spores seems to be the key in spore-host interactions and persistence. The proteome of the outermost exosporium layer of C. difficile spores has been determined, identifying two cysteine-rich exosporium proteins, CdeC and CdeM. In this work, we explore the contribution of both cysteine-rich proteins in exosporium integrity, spore biology and pathogenesis. Using targeted mutagenesis coupled with transmission electron microscopy we demonstrate that both cysteine rich proteins, CdeC and CdeM, are morphogenetic factors of the exosporium layer of C. difficile spores. Notably, cdeC, but not cdeM spores, exhibited defective spore coat, and were more sensitive to ethanol, heat and phagocytic cells. In a healthy colonic mucosa (mouse ileal loop assay), cdeC and cdeM spore adherence was lower than that of wild-type spores; while in a mouse model of recurrence of the disease, cdeC mutant exhibited an increased infection and persistence during recurrence. In a competitive infection mouse model, cdeC mutant had increased fitness over wild-type. Through complementation analysis with FLAG fusion of known exosporium and coat proteins, we demonstrate that CdeC and CdeM are required for the recruitment of several exosporium proteins to the surface of C. difficile spores. CdeC appears to be conserved exclusively in related Peptostreptococcaeace family members, while CdeM is unique to C. difficile. Our results sheds light on how CdeC and CdeM affect the biology of C. difficile spores and the assembly of the exosporium layer and, demonstrate that CdeC affect C. difficile pathogenesis