In Vivo Efficacy of Purified Quillaja Saponin Extracts in Protecting against Piscirickettsia salmonis Infections in Atlantic Salmon (Salmo salar).

Piscirickettsiosis, the main infectious disease affecting salmon farming in Chile, still has no efficient control measures. Piscirickettsia salmonis is a facultative intracellular bacterium that can survive and replicate within the host macrophages, evading the immune response. Triterpenic saponins obtained from the Quillaja saponaria tree have been widely studied, and have been shown to be immunomodulatory agents, suitable for feed and vaccine applications for veterinary and human uses. The impact of the oral administration of two extracts of Quillaja saponins on the infection of P. salmonis in Salmo salar and the corresponding gene expressions of immunomarkers were studied under three in vivo models. In the intraperitoneal challenge model, the group fed with Quillaja extracts showed lower mortality (29.1% treated vs. 37.5% control). Similar results were obtained in the cohabitation model trial (36.3% vs. 60.0%). In the commercial pilot trial, the results showed a significant reduction of 71.3% in mortality caused by P. salmonis (0.51% vs. 1.78%) and antibiotic use (reduction of 66.6% compared to untreated control). Also, Quillaja extracts significantly modulated the expression of IFN-II and CD8. These results represent evidence supporting the future use of purified Quillaja extracts as a natural non-pharmacological strategy for the prevention and control of P. salmonis infections in salmon.

Skeletal Muscle Atrophy Induced by Diabetes Is Mediated by Non-Selective Channels and Prevented by Boldine.

Individuals with diabetes mellitus present a skeletal muscle myopathy characterized by atrophy. However, the mechanism underlying this muscular alteration remains elusive, which makes it difficult to design a rational treatment that could avoid the negative consequences in muscles due to diabetes. In the present work, the atrophy of skeletal myofibers from streptozotocin-induced diabetic rats was prevented with boldine, suggesting that non-selective channels inhibited by this alkaloid are involved in this process, as has previously shown for other muscular pathologies. Accordingly, we found a relevant increase in sarcolemma permeability of skeletal myofibers of diabetic animals in vivo and in vitro due to de novo expression of functional connexin hemichannels (Cx HCs) containing connexins (Cxs) 39, 43, and 45. These cells also expressed P2X7 receptors, and their inhibition in vitro drastically reduced sarcolemma permeability, suggesting their participation in the activation of Cx HCs. Notably, sarcolemma permeability of skeletal myofibers was prevented by boldine treatment that blocks Cx43 and Cx45 HCs, and now we demonstrated that it also blocks P2X7 receptors. In addition, the skeletal muscle alterations described above were not observed in diabetic mice with myofibers deficient in Cx43/Cx45 expression. Moreover, murine myofibers cultured for 24 h in high glucose presented a drastic increase in sarcolemma permeability and levels of NLRP3, a molecular member of the inflammasome, a response that was also prevented by boldine, suggesting that, in addition to the systemic inflammatory response found in diabetes, high glucose can promote the expression of functional Cx HCs and activation of the inflammasome in skeletal myofibers. Therefore, Cx43 and Cx45 HCs play a critical role in myofiber degeneration, and boldine could be considered a potential therapeutic agent to treat muscular complications due to diabetes.

Antibacterial activity of a complex bacteriocin secreted by Staphylococcus epidermidis against Porphyromonas gingivalis.

OBJECTIVE: To characterize the inhibitory activity of a novel bacteriocin produced by Staphylococcus epidermidis against this periodontal pathogen. DESIGN: The bacteriocin activity was evaluated by the agar diffusion method over a lawn of P. gingivalis ATCC 33277. The bacteriocin was purified by Reverse Phase-High Performance Liquid Chromatography (RP-HPLC) and Matrix Assisted Laser Desorption Ionization -Time of Flight Mass Spectrometry (MALDI-TOF-MS). In addition, the bacteriocin host specificity, production on different media cultures and susceptibility to enzymes, pH, and heat treatment were determined. RESULTS: The bacteriocin BAC 14990 was selective to P. gingivalis, suggesting a narrow activity range. The production during the growth curve indicated that S. epidermidis had a continued production of this antimicrobial, showing the highest concentration in the stationary phase. The purification of BAC 14990 showed that bacteriocin had a molecular mass of 5795 Da. BAC 14990 was partially resistant to the treatment with proteinase K and papain, however, was fully susceptible to amylase treatment indicating the presence of sugar residues in the protein, suggesting a conjugated type of bacteriocin. Also, this diffusible inhibitory substance was heat and pH treatment resistant. CONCLUSIONS: The results indicate the isolation of a new staphylococcal complex bacteriocin that is able to eliminate a Gram-negative bacterium. These results could contribute to the development of treatments directed against pathogens in mixed communities, as is the case with oral diseases.

Quillaja saponaria (Molina) Extracts Inhibits In Vitro Piscirickettsia salmonis Infections.

P. salmonis infections are the cause of major bacterial disease in salmonids in Chile, and the reason for using more antibiotics compared to other salmon-producing countries. Vaccination and antibiotics have not been efficient and new approaches are needed. The safety of Quillaja saponaria extracts was measured by cytotoxicity using flow cytometry of cytopathic and death of fish cell cultures and efficacy was assessed using in vitro infection models with pathogenic P. salmonis. Cytotoxicity was low and control of in vitro infections was achieved with all products, with protection of over 90%. Minimum inhibitory concentrations were much higher than those in the infection using cell cultures. These results suggest a dual mechanism of action where less purified extracts with a combination of saponin and non-saponin components simultaneously decrease P. salmonis infection while protecting cell lines, rather than exerting a direct antimicrobial effect. Quillaja saponins controlled in vitro infections with P. salmonis and could be considered good candidates for a new, safe and sustainable method of controlling fish bacterial infectious diseases.

Blockade of Hemichannels Normalizes the Differentiation Fate of Myoblasts and Features of Skeletal Muscles from Dysferlin-Deficient Mice.

Dysferlinopathies are muscle dystrophies caused by mutations in the gene encoding dysferlin, a relevant protein for membrane repair and trafficking. These diseases are untreatable, possibly due to the poor knowledge of relevant molecular targets. Previously, we have shown that human myofibers from patient biopsies as well as myotubes derived from immortalized human myoblasts carrying a mutated form of dysferlin express connexin proteins, but their relevance in myoblasts fate and function remained unknown. In the present work, we found that numerous myoblasts bearing a mutated dysferlin when induced to acquire myogenic commitment express PPARγ, revealing adipogenic instead of myogenic commitment. These cell cultures presented many mononucleated cells with fat accumulation and within 48 h of differentiation formed fewer multinucleated cells. In contrast, dysferlin deficient myoblasts treated with boldine, a connexin hemichannels blocker, neither expressed PPARγ, nor accumulated fat and formed similar amount of multinucleated cells as wild type precursor cells. We recently demonstrated that myofibers of skeletal muscles from blAJ mice (an animal model of dysferlinopathies) express three connexins (Cx39, Cx43, and Cx45) that form functional hemichannels (HCs) in the sarcolemma. In symptomatic blAJ mice, we now show that eight-week treatment with a daily dose of boldine showed a progressive recovery of motor activity reaching normality. At the end of this treatment, skeletal muscles were comparable to those of wild type mice and presented normal CK activity in serum. Myofibers of boldine-treated blAJ mice also showed strong dysferlin-like immunoreactivity. These findings reveal that muscle dysfunction results from a pathophysiologic mechanism triggered by mutated dysferlin and downstream connexin hemichannels expressed de novo lead to a drastic reduction of myogenesis and favor muscle damage. Thus, boldine could represent a therapeutic opportunity to treat dysfernilopathies.

Myofibers deficient in connexins 43 and 45 expression protect mice from skeletal muscle and systemic dysfunction promoted by a dysferlin mutation.

Dysferlinopathy is a genetic human disease caused by mutations in the gene that encodes the dysferlin protein (DYSF). Dysferlin is believed to play a relevant role in cell membrane repair. However, in dysferlin-deficient (blAJ) mice (a model of dysferlinopathies) the recovery of the membrane resealing function by means of the expression of a mini-dysferlin does not arrest progressive muscular damage, suggesting the participation of other unknown pathogenic mechanisms. Here, we show that proteins called connexins 39, 43 and 45 (Cx39, Cx43 and Cx45, respectively) are expressed by blAJ myofibers and form functional hemichannels (Cx HCs) in the sarcolemma. At rest, Cx HCs increased the sarcolemma permeability to small molecules and the intracellular Ca2+ signal. In addition, skeletal muscles of blAJ mice showed lipid accumulation and lack of dysferlin immunoreactivity. As sign of extensive damage and atrophy, muscles of blAJ mice presented elevated numbers of myofibers with internal nuclei, increased number of myofibers with reduced cross-sectional area and elevated creatine kinase activity in serum. In agreement with the extense muscle damage, mice also showed significantly low motor performance. We generated blAJ mice with myofibers deficient in Cx43 and Cx45 expression and found that all above muscle and systemic alterations were absent, indicating that these two Cxs play a critical role in a novel pathogenic mechanism of dysfernolophaties, which is discussed herein. Therefore, Cx HCs could constitute an attractive target for pharmacologic treatment of dyferlinopathies.

High genetic diversity of species A rotaviruses detected in swine farms in Chile.

Rotavirus A is one of the main causative agents of diarrhoea in lactating and weaned pigs worldwide. Its impact in the swine industry is well documented. However, in Chile, the current epidemiological status of rotavirus on porcine farms is unknown. This study evaluated the current epidemiologic status of rotavirus A infection in Chile using on-farm detection techniques, electrophoretic confirmation, genotyping and phylogenetic clustering by analysis of partial sequences of VP4 and VP7 genes. Rotavirus A was detected in four out of five farms with an overall prevalence of 17.7 % in diarrhoeic pigs. The average age of diarrhoea onset was at 32±6.2 days, corresponding to weaning pigs, and rotavirus was not detected in lactating piglets. Molecular characterization indicated that genotypes G5, G3, P[7] and P[13] are currently the most widely represented on these pigs farms. The phylogenetic analysis showed that farms shared similar G types (VP7), which might denote a common origin. Meanwhile, [P] types (VP4) showed considerable genetic diversity, and this might represent a high rate of reassortment of this genetic segment in rotavirus circulating in the researched area. These findings demonstrate the importance of considering both the geographical and production factors to accurately determine rotavirus prevalence status at the national level, and have relevant implications in determining effective strategies for rotavirus infection control on porcine farms.

Salmonella enterica: una revisión de la trilogía agente, hospedero y ambiente, y su trascendencia en Chile / Salmonella enterica: a review or the trilogy agent, host and environment and its importance in Chile

Salmonella enterica is a major foodborne pathogen worldwide, being the main cause of outbreaks by food consumption in Chile. Despite all efforts deployed for control and prevention, the high incidence in people still persists, with several factors that could be influencing the epidemiological behavior of this infection. The objective of this review is to identify these factors belonging to the biological agent, the human host and the environment, which probably have a greater importance in Chile. Thus, priority areas for research of S. enterica are inferred, which hopefully will help to understand its spread in nature and its success as a wide host range pathogen. In the future, increased understanding of these determinants will facilitate the implementation of biosecurity and surveillance strategies for the prevention of disease in people and animals.

Salmonella enterica es uno de los principales patógenos transmitidos por los alimentos en el mundo, siendo la primera causa de brotes de intoxicación alimentaria en Chile. A pesar de todos los esfuerzos de control y prevención desplegados, la incidencia en las personas se ha mantenido alta, por lo que diversos factores podrían estar influenciando el comportamiento epidemiológico de esta infección. El objetivo de esta revisión es describir factores referidos tanto al agente biológico, al hospedero humano y al medio ambiente, que podrían tener mayor trascendencia en Chile. De esta forma, se infieren ámbitos prioritarios para la investigación de S. enterica, que permitan entender su dispersión en la naturaleza y su éxito como patógeno de un amplio rango de hospederos. A futuro, el mayor conocimiento de estos determinantes facilitará la implementación de estrategias de bioseguridad y vigilancia para la prevención de la enfermedad en las personas y en los animales.

[Salmonella enterica: a review or the trilogy agent, host and environment and its importance in Chile]. / Salmonella enterica: una revisión de la trilogía agente, hospedero y ambiente, y su trascendencia en Chile.

Salmonella enterica is a major foodborne pathogen worldwide, being the main cause of outbreaks by food consumption in Chile. Despite all efforts deployed for control and prevention, the high incidence in people still persists, with several factors that could be influencing the epidemiological behavior of this infection. The objective of this review is to identify these factors belonging to the biological agent, the human host and the environment, which probably have a greater importance in Chile. Thus, priority areas for research of S. enterica are inferred, which hopefully will help to understand its spread in nature and its success as a wide host range pathogen. In the future, increased understanding of these determinants will facilitate the implementation of biosecurity and surveillance strategies for the prevention of disease in people and animals.

Isolation of a novel Aggregatibacter actinomycetemcomitans serotype b bacteriophage capable of lysing bacteria within a biofilm.

A bacteriophage specific for Aggregatibacter actinomycetemcomitans serotype b, able to kill the bacterium within a biofilm, was isolated. Random mutagenesis of this phage rendered a bacteriophage able to kill 99% of the bacteria within a biofilm. This is the first report of a biocontrol experiment against A. actinomycetemcomitans.

Modified intracellular-associated phenotypes in a recombinant Salmonella Typhi expressing S. Typhimurium SPI-3 sequences.

A bioinformatics comparison of Salmonella Pathogenicity Island 3 sequences from S. Typhi and S. Typhimurium serovars showed that ten genes are highly conserved. However three of them are pseudogenes in S. Typhi. Our aim was to understand what functions are lost in S. Typhi due to pseudogenes by constructing a S. Typhi genetic hybrid carrying the SPI-3 region of S. Typhimurium instead of its own SPI-3. We observed that under stressful conditions the hybrid strain showed a clear impairment in resistance to hydrogen peroxide and decreased survival within U937 culture monocytes. We hypothesized that the marT-fidL operon, encoded in SPI-3, was responsible for the new phenotypes because marT is a pseudogen in S. Typhi and has a demonstrated role as a transcriptional regulator in S. Typhimurium. Therefore we cloned and transferred the S. Typhimurium marT-fidL operon into S. Typhi and confirmed that invasion of monocytes was dramatically decreased. Finally, our findings suggest that the genomic and functional differences between SPI-3 sequences have implications in the host specificity of Typhi and Typhimurium serovars.

Characterization of MgtC, a virulence factor of Salmonella enterica Serovar Typhi.

The MgtC is a virulence factor in Salmonella Typhimurium that is required for growth at low-Mg2+ concentrations and intramacrophage survival. This gene is codified in a conserved region of the Salmonella pathogenicity island 3 (SPI-3), and is also present in the chromosome of other Salmonella serovars. In this study we characterized the MgtC factor in S. Typhi, a human specific pathogen, by using mgtC and SPI-3 mutant strains. We found that MgtC is the most important factor codified in the SPI-3 of S. Typhi for growth in low-Mg2+ media and survival within human cells. In addition, by using reporter genes we determined that the low-Mg2+ concentration, acidic media and PhoP regulator induce mgtC expression in S. Typhi. We suggest that MgtC is the most important virulence factor codified in the SPI-3 of S. Typhi.

The Salmonella Typhi hlyE gene plays a role in invasion of cultured epithelial cells and its functional transfer to S. Typhimurium promotes deep organ infection in mice.

Comparison of genome sequences of Salmonella enterica serovars Typhi and Typhimurium reveals that S. Typhi has a small 2.3kb genomic island missing in S. Typhimurium, designated Salmonella pathogenicity island 18 (SPI-18), which includes two potential genes. One of these, hlyE, encodes a hemolysin related to the Escherichia coli K12 HlyE hemolysin. PCR assays show that SPI-18 is present in S. Typhi and in many other, but not all, serovars of S. enterica subsp. enterica belonging to the SARB collection. HlyE activity cannot be detected in S. Typhi by means of standard plate assays. Nevertheless, we were able to reveal this activity upon lysis of bacterial cells with phages, in the presence of ampicillin, and in a ompA genetic background, conditions that compromise the integrity of the bacterial envelope. Almost all serovars of the SARB collection shown to cause systemic infections in humans have SPI-18 and hlyE and express an active hemolysin revealed upon bacterial envelope destabilization. S. Typhi hlyE mutants are impaired in invasion of human epithelial cells in vitro, and its heterologous expression in S. Typhimurium improves the colonization of deep organs in mice, demonstrating that the HlyE hemolysin is a new virulence determinant.