Use of five probiotic strains to determine sensitivity in vitro on pathogenic bacteria growth isolated from sick fishes

ABSTRACT Ornamental aquaculture is an activity in clear economic growth, both globally and in Mexico where the development is particularly relevant to freshwater species. Infectious diseases produced by fungus, bacteria and virus are considered one of the principal limitations during the productive process. Between implemented strategies for reduction of antibiotic use, which are "living microorganisms that confer a health benefit to the host if they are given in adequate quantities"; lactic acid bacteria and yeast are among the most common used microorganism in aquaculture. This investigation, prove the effect of isolated probiotic bacteria from the digestive tract of healthy fish, belonging to specie: Bacillus sp., Bacillus laterosporus, Bacillus subtilis, Lactobacillus sp, and Lactococcus lactis, at different dilutions (10 9 ,10 8 , 10 7 ,10 6 , 10 5 and 10 4 ) in vitro growth of pathogenic bacteria: Citrobacter freundii, Enterobacter sakasakii, Klebsiella oxytoca, Proteus vulgaris, and Vibrio fluvialis, isolated from kidney of sick fish, cultured and purified through successive inoculations and identificated by the amplification of gene 16S of rRNA (PCR) using universal primers 8 for. (5'-AGACTTTGATCATGGCTCAG-3') and 1492 rev. (5'-TACGGCTACCTTGTTACGACTT-3') and comparison with GENEBANK sequences base. Probiotic strains were previously isolated from the digestive tract of different healthy fish in the laboratory. In order to perform in vitro challenge tests, pathogenic strains were inoculated three times each in BHI agar boxes at a concentration of 1x10 7 CFU mL -1 and subsequently using the well diffusion method, 70 µL from a suspension with each of the probiotic strains were added. Agar boxes were incubated 24 h at 30ºC to observe the formation of inhibition halos. Obtained values from inhibition halos were transformed to qualitative data with the following premise: halo diameter < 2.0 mm negative effect; halo diameter > 2.0 mm positive effect. In this study, it was determined that probiotic strains B. subtilis was the one that gave better results to inhibit the growth of pathogenic bacteria P. vulgaris, E. sakazakii, V. fluvialis, K. oxytoca and C. freundii in most of used dilutions. Making it a strain with high potential in aquaculture. Key words: Growth, halos, probiotics, bacteria, sensibility. RESUMEN La acuicultura de especies ornamentales es una actividad económica en franco crecimiento, tanto a nivel mundial como en México, en donde tiene particular desarrollo lo correspondiente a especies dulceacuícolas. Las enfermedades infeccionas producidas por hongos, bacterias y virus, están consideradas una de las limitantes principales durante el proceso productivo. Entre las estrategias implementadas para disminuir el uso de antibióticos para el control de patógenos, se encuentra el control biológico mediante el uso de organismos probióticos, los cuales son "microorganismos vivos los cuales, administrados en cantidades adecuadas, confieren un beneficio en la salud del hospedador"; entre los de uso más común en acuicultura se encuentran las lactobacterias y las levaduras. En el presente trabajo, se probó el efecto de bacterias probióticas aisladas del tracto digestivo de peces sanos, pertenecientes a las especies: Bacillus sp., Bacillus laterosporus, Bacillus subtilis, Lactobacillus sp, y Lactococcus lactis, a diferentes diluciones (10 9 ,10 8 , 10 7 ,10 6 , 10 5 y 10 4 ) en el crecimiento in vitro de las bacterias patógenas: Citrobacter freundii, Enterobacter sakasakii, Klebsiella oxytoca, Proteus vulgaris y Vibrio fluvialis, aisladas del riñón de peces enfermos, cultivadas y purificadas a través de resiembras sucesivas e identificadas mediante la amplificación del gen 16S del ARNr (PCR) utilizando los primers universales 8 for. 24 AGACTTTGATCATGGCTCAG-3') y 1492 rev. (5'-TACGGCTACCTTGTTACGACTT-3') y su comparación con la base de secuencias GENEBANK. Las cepas probióticos fueron aisladas previamente del tracto intestinal de diversos peces sanos en el laboratorio. Para llevar a cabo las pruebas de desafío in vitro, las cepas patógenas se sembraron por triplicado en cajas de agar BHI a una concentración de 1x10 7 UFC mL -1 y posteriormente, utilizando el método de difusión en pozos, se adicionaran 70 µL de una suspensión con cada una de las cepas probióticas Las placas se incubaron durante 24 h a 30ºC para observar la formación de halos de inhibición. Los valores obtenidos de los halos de inhibición fueron transformados a datos cualitativos con la siguiente premisa: diámetro halo < 2.0 mm efecto negativo; diámetro de halo > 2.0 mm efecto positivo. En este estudio, se determinó que la cepas probiótica B. subtilis fue la que dio mejores resultados al inhibir el crecimiento de las bacterias patógenas P. vulgaris, E. sakazakii, V. fluvialis, K. oxytoca y C. freundii en la mayoría de las diluciones utilizadas. Por lo que es una cepa con alto potencial en acuicultura

Factors Affecting Infestation by Triatoma infestans in a Rural Area of the Humid Chaco in Argentina: A Multi-Model Inference Approach

Vector-borne transmission of Chagas disease remains a major public health problem in parts of Latin America. Triatoma infestans is the main vector in the countries located in the South American Cone, particularly in the Gran Chaco ecoregion where residual insecticide control has achieved only a moderate, irregular impact. To contribute to improved control strategies, we analyzed the factors associated with the presence and abundance of T. infestans in 327 inhabited houses in a well-defined rural area with no recent vector control interventions in the humid Argentine Chaco. Bugs were found mainly in domiciles, kitchens, storerooms, and chicken coops and nests, particularly where adequate refuge and animal hosts (humans, dogs, cats or poultry) were available. Domiciles constructed from mud were the most often infested, but brick-and-cement domiciles, even in good conditions, were also found infested. Availability of refuge and hosts for T. infestans are key targets for vector control. Ten-fold variations in domestic infestation observed across neighboring villages, and differences in the relevant factors for T. infestans presence with respect to other areas of the Gran Chaco region suggest that host management, building techniques and insecticide use need to be tailored to the local environment, socio-economic characteristics, and climatic conditions

Species-Specific Effects of Epigeic Earthworms on Microbial Community Structure during First Stages of Decomposition of Organic Matter

Background: Epigeic earthworms are key organisms in organic matter decomposition because of the interactions they establish with microorganisms. The earthworm species and the quality and/or substrate availability are expected to be major factors influencing the outcome of these interactions. Here we tested whether and to what extent the epigeic earthworms Eisenia andrei, Eisenia fetida and Perionyx excavatus, widely used in vermicomposting, are capable of altering the microbiological properties of fresh organic matter in the short-term. We also questioned if the earthworm-induced modifications to the microbial communities are dependent on the type of substrate ingested. Methodology/Principal Findings: To address these questions we determined the microbial community structure (phospholipid fatty acid profiles) and microbial activity (basal respiration and microbial growth rates) of three types of animal manure (cow, horse and rabbit) that differed in microbial composition, after being processed by each species of earthworm for one month. No differences were found between earthworm-worked samples with regards to microbial community structure, irrespective of type of manure, which suggests the existence of a bottleneck effect of worm digestion on microbial populations of the original material consumed. Moreover, in mesocosms containing cow manure the presence of E. andrei resulted not only in a decrease in bacterial and fungal biomass, but also in a reduced bacterial growth rate and total microbial activity, while no such reduction was found with E. fetida and P. excavatus

Comparing genomic variant identification protocols for Candida auris.

Genomic analyses are widely applied to epidemiological, population genetic and experimental studies of pathogenic fungi. A wide range of methods are employed to carry out these analyses, typically without including controls that gauge the accuracy of variant prediction. The importance of tracking outbreaks at a global scale has raised the urgency of establishing high-accuracy pipelines that generate consistent results between research groups. To evaluate currently employed methods for whole-genome variant detection and elaborate best practices for fungal pathogens, we compared how 14 independent variant calling pipelines performed across 35 Candida auris isolates from 4 distinct clades and evaluated the performance of variant calling, single-nucleotide polymorphism (SNP) counts and phylogenetic inference results. Although these pipelines used different variant callers and filtering criteria, we found high overall agreement of SNPs from each pipeline. This concordance correlated with site quality, as SNPs discovered by a few pipelines tended to show lower mapping quality scores and depth of coverage than those recovered by all pipelines. We observed that the major differences between pipelines were due to variation in read trimming strategies, SNP calling methods and parameters, and downstream filtration criteria. We calculated specificity and sensitivity for each pipeline by aligning three isolates with chromosomal level assemblies and found that the GATK-based pipelines were well balanced between these metrics. Selection of trimming methods had a greater impact on SAMtools-based pipelines than those using GATK. Phylogenetic trees inferred by each pipeline showed high consistency at the clade level, but there was more variability between isolates from a single outbreak, with pipelines that used more stringent cutoffs having lower resolution. This project generated two truth datasets useful for routine benchmarking of C. auris variant calling, a consensus VCF of genotypes discovered by 10 or more pipelines across these 35 diverse isolates and variants for 2 samples identified from whole-genome alignments. This study provides a foundation for evaluating SNP calling pipelines and developing best practices for future fungal genomic studies

Phylogeographic pattern and extensive mitochondrial DNA divergence disclose a species complex within the Chagas disease vector Triatoma dimidiata.

ABSTARCT: Previous studies have shown that "bioequivalent" generic products of vancomycin are less effective in vivo against Staphylococcus aureus than the innovator compound. Considering that suboptimal bactericidal effect has been associated with emergence of resistance, we aimed to assess in vivo the impact of exposure to innovator and generic products of vancomycin on S. aureus susceptibility. A clinical methicillin-resistant S. aureus (MRSA) strain from a liver transplant patient with persistent bacteremia was used for which MIC, minimum bactericidal concentration (MBC), and autolytic properties were determined. Susceptibility was also assessed by determining a population analysis profile (PAP) with vancomycin concentrations from 0 to 5 mg/liter. ICR neutropenic mice were inoculated in each thigh with ∼7.0 log(10) CFU. Treatment with the different vancomycin products (innovator and three generics; 1,200 mg/kg of body weight/day every 3 h) started 2 h later while the control group received sterile saline. After 24 h, mice were euthanized, and the thigh homogenates were plated. Recovered colonies were reinoculated to new groups of animals, and the exposure-recovery process was repeated until 12 cycles were completed. The evolution of resistance was assessed by PAP after cycles 5, 10, 11, and 12. The initial isolate displayed reduced autolysis and higher resistance frequencies than S. aureus ATCC 29213 but without vancomycin-intermediate S. aureus (VISA) subpopulations. After 12 cycles, innovator vancomycin had significantly reduced resistant subpopulations at 1, 2, and 3 mg/liter, while the generic products had enriched them progressively by orders of magnitude. The great capacity of generic vancomycin to select for less susceptible organisms raises concerns about the role of therapeutic inequivalence of any antimicrobial on the epidemiology of resistance worldwide

Heightened resistance to host type 1 interferons characterizes HIV-1 at transmission and after antiretroviral therapy interruption

Type 1 interferons (IFN-I) are potent innate antiviral effectors that constrain HIV-1 transmission. However, harnessing these cytokines for HIV-1 cure strategies has been hampered by an incomplete understanding of their antiviral activities at later stages of infection. Here, we characterized the IFN-I sensitivity of 500 clonally derived HIV-1 isolates from the plasma and CD4+ T cells of 26 individuals sampled longitudinally after transmission or after antiretroviral therapy (ART) and analytical treatment interruption. We determined the concentration of IFNα2 and IFNβ that reduced viral replication in vitro by 50% (IC50) and found consistent changes in the sensitivity of HIV-1 to IFN-I inhibition both across individuals and over time. Resistance of HIV-1 isolates to IFN-I was uniformly high during acute infection, decreased in all individuals in the first year after infection, was reacquired concomitant with CD4+ T cell loss, and remained elevated in individuals with accelerated disease. HIV-1 isolates obtained by viral outgrowth during suppressive ART were relatively IFN-I sensitive, resembling viruses circulating just before ART initiation. However, viruses that rebounded after treatment interruption displayed the highest degree of IFNα2 and IFNβ resistance observed at any time during the infection course. These findings indicate a dynamic interplay between host innate responses and the evolving HIV-1 quasispecies, with the relative contribution of IFN-I to HIV-1 control affected by both ART and analytical treatment interruption. Although elevated at transmission, host innate pressures are the highest during viral rebound, limiting the viruses that successfully become reactivated from latency to those that are IFN-I resistant

Mangrove trees affect the community structure and distribution of anammox bacteria at an anthropogenic-polluted mangrove in the Pearl River Delta reflected by 16S rRNA and hydrazine oxidoreductase (HZO) encoding gene analyses

Anaerobic ammonium oxidizing (anammox) bacterial community structures were investigated in surface (1–2 cm) and lower (20–21 cm) layers of mangrove sediments at sites located immediately to the mangrove trees (S0), 10 m (S1) and 1000 m (S2) away from mangrove trees in a polluted area of the Pearl River Delta. At S0, both 16S rRNA and hydrazine oxidoreductase (HZO) encoding genes of anammox bacteria showed high diversity in lower layer sediments, but they were not detectable in lower layer sediments in mangrove forest. S1 and S2 shared similar anammox bacteria communities in both surface and lower layers, which were quite different from that of S0. At all three locations, higher richness of anammox bacteria was detected in the surface layer than the lower layer; 16S rRNA genes revealed anammox bacteria were composed by four phylogenetic clusters affiliated with the “Scalindua” genus, and one group related to the potential anammox bacteria; while the hzo genes showed that in addition to sequences related to the “Scalindua”, sequences affiliated with genera of “Kuenenia”, “Brocadia”, and “Jettenia” were also detected in mangrove sediments. Furthermore, hzo gene abundances decreased from 36.5 × 104 to 11.0 × 104 copies/gram dry sediment in lower layer sediments while increased from below detection limit to 31.5 × 104 copies/gram dry sediment in lower layer sediments from S0 to S2. The results indicated that anammox bacteria communities might be strongly influenced by mangrove trees. In addition, the correlation analysis showed the redox potential and the molar ratio of ammonium to nitrite in sediments might be important factors affecting the diversity and distribution of anammox bacteria in mangrove sediments

Eco-bio-social determinants for house infestation by non-domiciliated Triatoma dimidiata in the Yucatan peninsula, Mexico

Background Chagas disease is a vector-borne disease of major importance in the Americas. Disease prevention is mostly limited to vector control. Integrated interventions targeting ecological, biological and social determinants of vector-borne diseases are increasingly used for improved control. Methodology/principal findings We investigated key factors associated with transient house infestation by T. dimidiata in rural villages in Yucatan, Mexico, using a mixed modeling approach based on initial null-hypothesis testing followed by multimodel inference and averaging on data from 308 houses from three villages. We found that the presence of dogs, chickens and potential refuges, such as rock piles, in the peridomicile as well as the proximity of houses to vegetation at the periphery of the village and to public light sources are major risk factors for infestation. These factors explain most of the intra-village variations in infestation. Conclusions/significance These results underline a process of infestation distinct from that of domiciliated triatomines and may be used for risk stratification of houses for both vector surveillance and control. Combined integrated vector interventions, informed by an Ecohealth perspective, should aim at targeting several of these factors to effectively reduce infestation and provide sustainable vector control

Development of Sensory, Motor and Behavioral Deficits in the Murine Model of Sanfilippo Syndrome Type B

BACKGROUND: Mucopolysaccharidosis (MPS) IIIB (Sanfilippo Syndrome type B) is caused by a deficiency in the lysosomal enzyme N-acetyl-glucosaminidase (Naglu). Children with MPS IIIB develop disturbances of sleep, activity levels, coordination, vision, hearing, and mental functioning culminating in early death. The murine model of MPS IIIB demonstrates lysosomal distention in multiple tissues, a shortened life span, and behavioral changes. PRINCIPAL FINDINGS: To more thoroughly assess MPS IIIB in mice, alterations in circadian rhythm, activity level, motor function, vision, and hearing were tested. The suprachiasmatic nucleus (SCN) developed pathologic changes and locomotor analysis showed that MPS IIIB mice start their daily activity later and have a lower proportion of activity during the night than wild-type controls. Rotarod assessment of motor function revealed a progressive inability to coordinate movement in a rocking paradigm. Purkinje cell counts were significantly reduced in the MPS IIIB animals compared to age matched controls. By electroretinography (ERG), MPS IIIB mice had a progressive decrease in the amplitude of the dark-adapted b-wave response. Corresponding pathology revealed shortening of the outer segments, thinning of the outer nuclear layer, and inclusions in the retinal pigmented epithelium. Auditory-evoked brainstem responses (ABR) demonstrated progressive hearing deficits consistent with the observed loss of hair cells in the inner ear and histologic abnormalities in the middle ear. CONCLUSIONS/SIGNIFICANCE: The mouse model of MPS IIIB has several quantifiable phenotypic alterations and is similar to the human disease. These physiologic and histologic changes provide insights into the progression of this disease and will serve as important parameters when evaluating various therapies