In vitro cytotoxic capacity against tumor cell lines and antioxidant activity of acidic polysaccharides isolated from the Andean Patagonian fungus Phylloporia boldo.

Fungal polysaccharides possess a broad biological activity, including cytotoxic and antioxidant activities. This work aimed to evaluate the cytotoxic and antioxidant activity of the acidic polysaccharides of Phylloporia boldo strain (named PBAP40). Cytotoxic activity of polysaccharide was evaluated determining the viability of three tumor cell lines by MTT assay. The effect of acidic polysaccharide on the cell cycle of HL-60 cell line was evaluated by flow cytometry, and the antioxidant activity was determined by DPPH and ABTS assays. PBAP40 showed cytotoxic effects in tumor cell lines. Results suggest that P. boldo acidic polysaccharides arrested tumor cells in the cell cycle Sub G1 phase. The acidic polysaccharides of PBAP40 strain were not cytotoxic for the non-tumor cell line. PBAP40 also showed excellent antioxidant activity. The FT-IR analysis of the acidic polysaccharides indicated the presence of glucans bearing α- and ß- type glycosidic bonds.

Selenium Nanoparticle-Enriched and Potential Probiotic, Lactiplantibacillus plantarum S14 Strain, a Diet Supplement Beneficial for Rainbow Trout.

Lactic acid bacteria (LAB), obtained from rainbow trout (Oncorhynchus mykiss) intestine, were cultured in MRS medium and probiotic candidates. Concurrently, producers of elemental selenium nanoparticles (Se0Nps) were selected. Probiotic candidates were subjected to morphological characterization and the following tests: antibacterial activity, antibiotic susceptibility, hemolytic activity, catalase, hydrophobicity, viability at low pH, and tolerance to bile salts. Two LAB strains (S4 and S14) satisfied the characteristics of potential probiotics, but only strain S14 reduced selenite to biosynthesize Se0Nps. S14 strain was identified, by 16S rDNA analysis, as Lactiplantibacillus plantarum. Electron microscopy showed Se0Nps on the surface of S14 cells. Rainbow trout diet was supplemented (108 CFU g-1 feed) with Se0Nps-enriched L. plantarum S14 (LABS14-Se0Nps) or L. plantarum S14 alone (LABS14) for 30 days. At days 0, 15, and 30, samples (blood, liver, and dorsal muscle) were obtained from both groups, plus controls lacking diet supplementation. Fish receiving LABS14-Se0Nps for 30 days improved respiratory burst and plasmatic lysozyme, (innate immune response) and glutathione peroxidase (GPX) (oxidative status) activities and productive parameters when compared to controls. The same parameters also improved when compared to fish receiving LABS14, but significant only for plasmatic and muscle GPX. Therefore, Se0Nps-enriched L. plantarum S14 may be a promising alternative for rainbow trout nutritional supplementation.

An Anaerobic Environment Drives the Harboring of Helicobacter pylori within Candida Yeast Cells.

Helicobacter pylori protects itself from stressful environments by forming biofilms, changing its morphology, or invading eukaryotic cells, including yeast cells. There is little knowledge about the environmental factors that influence the endosymbiotic relationship between bacterium and yeasts. Here, we studied if oxygen availability stimulated the growth of H. pylori within Candida and if this was a bacterial- or yeast strain-dependent relationship. Four H. pylori strains and four Candida strains were co-cultured in Brucella broth plus 5% fetal bovine serum, and incubated under microaerobic, anaerobic, or aerobic conditions. Bacteria-like bodies (BLBs) within yeast cells (Y-BLBs) were detected by microscopy. H. pylori was identified by FISH and by PCR amplification of the 16S rRNA gene of H. pylori from total DNA extracted from Y-BLBs from H. pylori and Candida co-cultures. BLBs viability was confirmed by SYTO-9 fluorescence. Higher Y-BLB percentages were obtained under anaerobic conditions and using H. pylori J99 and C. glabrata combinations. Thus, the H. pylori-Candida endosymbiotic relationship is strain dependent. The FISH and PCR results identified BLBs as intracellular H. pylori. Conclusion: Stressful conditions such as an anaerobic environment significantly increased H. pylori growth within yeast cells, where it remained viable, and the bacterium-yeast endosymbiotic relationship was bacterial strain dependent with a preference for C. glabrata.

Selenium Nanoparticles Biosynthesized by Pantoea agglomerans and Their Effects on Cellular and Physiological Parameters in the Rainbow Trout Oncorhynchus mykiss.

The applications of nanoparticles (Nps) as food additives, health enhancers, and antimicrobials in animal production are increasing. The aim of this study was to evaluate the effect of selenium (Se) nanoparticles (Se0Nps) stabilized with L-cysteine (Se0Nps/L-Cys), as a nutritional supplement, on immunological, oxidative status, and productive parameters in O. mykiss. TEM and SEM-EDS showed the accumulation of spherical Se0Nps entirely composed by elemental selenium (Se0) as intracellular and extracellular deposits in Pantoea agglomerans UC-32 strain. The in vitro antioxidant capacity of Se0Nps/L-Cys was significant more efficient ROS scavengers than Se0Nps and Na2SeO3. We also evaluate the effect of Se0Nps/L-Cys on cell viability and oxidative stress in RTgill-W1, RTS-11, or T-PHKM Oncorhynchus mykiss cell lines. Se0Nps/L-Cys showed less toxic and high antioxidant activity than Se0Nps and Na2SeO3. Finally, the dietary Se0Nps/L-Cys had a significant better effect on both plasma lysozyme and respiratory burst activity (innate immune response), on tissular Gpx activity (oxidative status), and on well-being (productive parameter) of O. mykiss when it is compared to Se0Nps and Na2SeO3. Se0Nps/L-Cys is a promising alternative for nutritional supplement for O. mykiss with better performance than Na2SeO3 and Se0Nps, ease to implementation, and reduced environmental impact.

Gastrointestinal Microbiota and Parasite-Fauna of Wild Dissostichus eleginoides Smitt, 1898 Captured at the South-Central Coast of Chile.

Dissotichus eleginoides has a discontinuous circumpolar geographic distribution restricted to mountains and platforms, mainly in Subantarctic and Antarctic waters of the southern hemisphere, including the Southeast Pacific, Atlantic and Indian oceans and in areas surrounding the peninsular platforms of subantarctic islands. The aim of this work was to determine and characterize the gastrointestinal parasitic and microbial fauna of specimens of D. eleginoides captured in waters of the south-central zone of Chile. The magnitude of parasitism in D. eleginoides captured in waters of the south-central zone of Chile is variable, and the parasite richness is different from that reported in specimens from subantarctic environments. Next-generation sequencing (NGS) of the microbial community associated to intestine showed a high diversity, where Proteobacteria, Firmicutes, and Bacteriodetes were the dominant phyla. However, both parasitic and microbial structures can vary between fish from different geographic regions.

Temperatures Outside the Optimal Range for Helicobacter pylori Increase Its Harboring within Candida Yeast Cells.

Helicobacter pylori is capable of entering into yeast, but the factors driving this endosymbiosis remain unknown. This work aimed to determine if temperatures outside the optimal range for H. pylori increase its harboring within Candida. H. pylori strains were co-cultured with Candida strains in Brucella broth supplemented with 5% fetal bovine serum and incubated at 4, 25, 37 or 40 °C. After co-culturing, yeasts containing bacteria-like bodies (Y-BLBs) were observed by optical microscopy, and the bacterium were identified as H. pylori by FISH. The H. pylori 16S rRNA gene was amplified from the total DNA of Y-BLBs. The viability of intra-yeast H. pylori cells was confirmed using a viability assay. All H. pylori strains were capable of entering into all Candida strains assayed. The higher percentages of Y-BLBs are obtained at 40 °C with any of the Candida strains. H pylori also increased its harboring within yeast in co-cultures incubated at 25 °C when compared to those incubated at 37 °C. In conclusion, although H. pylori grew significantly at 40 °C, this temperature increased its harboring within Candida. The endosymbiosis between both microorganisms is strain-dependent and permits bacterial cells to remain viable under the stressing environmental conditions assayed.

Nutrient Deficiency Promotes the Entry of Helicobacter pylori Cells into Candida Yeast Cells.

Helicobacter pylori, a Gram-negative bacterium, has as a natural niche the human gastric epithelium. This pathogen has been reported to enter into Candida yeast cells; however, factors triggering this endosymbiotic relationship remain unknown. The aim of this work was to evaluate in vitro if variations in nutrient concentration in the cultured medium trigger the internalization of H. pylori within Candida cells. We used H. pylori-Candida co-cultures in Brucella broth supplemented with 1%, 5% or 20% fetal bovine serum or in saline solution. Intra-yeast bacteria-like bodies (BLBs) were observed using optical microscopy, while intra-yeast BLBs were identified as H. pylori using FISH and PCR techniques. Intra-yeast H. pylori (BLBs) viability was confirmed using the LIVE/DEAD BacLight Bacterial Viability kit. Intra-yeast H. pylori was present in all combinations of bacteria-yeast strains co-cultured. However, the percentages of yeast cells harboring bacteria (Y-BLBs) varied according to nutrient concentrations and also were strain-dependent. In conclusion, reduced nutrients stresses H. pylori, promoting its entry into Candida cells. The starvation of both H. pylori and Candida strains reduced the percentages of Y-BLBs, suggesting that starving yeast cells may be less capable of harboring stressed H. pylori cells. Moreover, the endosymbiotic relationship between H. pylori and Candida is dependent on the strains co-cultured.

Antibiotics as a Stressing Factor Triggering the Harboring of Helicobacter pylori J99 within Candida albicans ATCC10231.

First-line treatment for Helicobacter pylori includes amoxicillin and clarithromycin or metronidazole plus a proton pump inhibitor. Treatment failure is associated with antibiotic resistance and possibly also with internalization of H. pylori into eukaryotic cells, such as yeasts. Factors triggering the entry of H. pylori into yeast are poorly understood. Therefore, the aim of this study was to evaluate whether clarithromycin or amoxicillin trigger the entry of H. pylori into C. albicans cells. METHODS: H. pylori J99 and C. albicans ATCC 10231 were co-cultured in the presence of subinhibitory concentrations of amoxicillin and clarithromycin as stressors. Bacterial-bearing yeasts were observed by fresh examination. The viability of bacteria within yeasts was evaluated, confirming the entry of bacteria into Candida, amplifying, by PCR, the H. pylori16S rRNA gene in total yeast DNA. RESULTS: Amoxicillin significantly increased the entry of H. pylori into C. albicans compared to the control. CONCLUSION: the internalization of H. pylori into C. albicans in the presence of antibiotics is dependent on the type of antibiotic used, and it suggests that a therapy including amoxicillin may stimulate the entry of the bacterium into Candida, thus negatively affecting the success of the treatment.

Characterization of Arsenite-Oxidizing Bacteria Isolated from Arsenic-Rich Sediments, Atacama Desert, Chile.

Arsenic (As), a semimetal toxic for humans, is commonly associated with serious health problems. The most common form of massive and chronic exposure to As is through consumption of contaminated drinking water. This study aimed to isolate an As resistant bacterial strain to characterize its ability to oxidize As (III) when immobilized in an activated carbon batch bioreactor and to evaluate its potential to be used in biological treatments to remediate As contaminated waters. The diversity of bacterial communities from sediments of the As-rich Camarones River, Atacama Desert, Chile, was evaluated by Illumina sequencing. Dominant taxonomic groups (>1%) isolated were affiliated with Proteobacteria and Firmicutes. A high As-resistant bacterium was selected (Pseudomonas migulae VC-19 strain) and the presence of aio gene in it was investigated. Arsenite detoxification activity by this bacterial strain was determined by HPLC/HG/AAS. Particularly when immobilized on activated carbon, P. migulae VC-19 showed high rates of As(III) conversion (100% oxidized after 36 h of incubation). To the best of our knowledge, this is the first report of a P. migulae arsenite oxidizing strain that is promising for biotechnological application in the treatment of arsenic contaminated waters.

Intracellular Presence of Helicobacter pylori and Its Virulence-Associated Genotypes within the Vaginal Yeast of Term Pregnant Women.

BACKGROUND: Helicobacter pylori transmission routes are not entirely elucidated. Since yeasts are postulated to transmit this pathogen, this study aimed to detect and genotype intracellular H. pylori harbored within vaginal yeast cells. METHODS: A questionnaire was used to determine risk factors of H. pylori infection. Samples were seeded on Sabouraud Dextrose Agar and horse blood-supplemented Columbia agar. Isolated yeasts were identified using and observed by optical microscopy searching for intra-yeast H. pylori. Total yeast DNA, from one random sample, was extracted to search for H. pylori virulence genes by PCR and bacterial identification by sequencing. RESULTS: 43% of samples contained yeasts, mainly Candida albicans (91%). Microscopy detected bacteria such as bodies and anti-H. pylori antibodies binding particles in 50% of the isolated yeasts. Total DNA extracted showed that 50% of the isolated yeasts were positive for H. pylori 16S rDNA and the sequence showed 99.8% similarity with H. pylori. In total, 32% of H. pylori DNA positive samples were cagA+ vacAs1a vacAm1 dupA-. No relationship was observed between possible H. pylori infection risk factors and vaginal yeasts harboring this bacterium. CONCLUSION: H. pylori having virulent genotypes were detected within vaginal yeasts constituting a risk for vertical transmission of this pathogen.

The Influence of pCO2-Driven Ocean Acidification on Open Ocean Bacterial Communities during A Short-Term Microcosm Experiment in the Eastern Tropical South Pacific (ETSP) off Northern Chile.

Due to the increasing anthropogenic CO2 emissions, Ocean Acidification (OA) is progressing rapidly around the world. Despite the major role that microorganisms play on the marine biogeochemical cycling and ecosystem functioning, the response of bacterial communities upon OA scenarios is still not well understood. Here, we have conducted a detailed characterization of the composition and relative abundance of bacterial communities in the water column of an open-ocean station in the Eastern Tropical South Pacific (ETSP) off northern Chile and their interactions with environmental factors. In addition, through a short-term microcosm experiment, we have assessed the effect of low pH/high pCO2 conditions over the abundance and genetic diversity of bacterial communities. Our results evidence a clear partitioning of community composition that could be attributed mostly to dissolved oxygen. However, our experimental approach demonstrated that low pH/high pCO2 conditions might modify the structure of the bacterial community, evidencing that small changes in pH may impact significantly the abundance and diversity of key microorganisms. This study constitutes a first step aiming to provide insight about the influence of changing carbonate chemistry conditions on natural bacterial communities and to shed light on the potential impact of OA in biogeochemical cycles on the ETSP region.

Characterization of the Bacterial Biofilm Communities Present in Reverse-Osmosis Water Systems for Haemodialysis.

Biofilm in reverse osmosis (RO) membranes is a common problem in water treatment at haemodialysis facilities. Bacteria adhere and proliferate on RO membranes, forming biofilms, obstructing and damaging the membranes and allowing the transfer of bacteria and/or cellular components potentially harmful to the health of haemodialysis patients. Our aim was to characterize the bacterial community associated to biofilm of RO membranes and to identify potentially pathogenic bacteria present in the haemodialysis systems of two dialysis centres in Chile. The diversity of the bacterial communities present on RO membranes and potable and osmosed water samples was evaluated using Illumina sequencing. Additionally, bacteria from potable water, osmosed water and RO membrane samples were isolated, characterized and identified by Sanger's sequencing. The molecular analyses of metagenomics showed that the phyla having a greater relative abundance in both dialysis centres were Proteobacteria and Planctomycetes. Pseudomonas, Stenotrophomonas, Agrobacterium, Pigmentiphaga, Ralstonia, Arthrobacter, Bacteroides and Staphylococcus were bacterial genera isolated from the different samples obtained at both haemodialysis centres. Pseudomonas spp. was a bacterial genus with greater frequency in all samples. Pseudomonas and Staphylococcus showed higher levels of resistance to the antibiotics tested. Results demonstrated the presence of potentially pathogenic bacteria, showing resistance to antimicrobials on RO membranes and in osmosed water in both dialysis centres studied.

In Vitro Incorporation of Helicobacter pylori into Candida albicans Caused by Acidic pH Stress.

Yeasts can adapt to a wide range of pH fluctuations (2 to 10), while Helicobacter pylori, a facultative intracellular bacterium, can adapt to a range from pH 6 to 8. This work analyzed if H. pylori J99 can protect itself from acidic pH by entering into Candida albicans ATCC 90028. Growth curves were determined for H. pylori and C. albicans at pH 3, 4, and 7. Both microorganisms were co-incubated at the same pH values, and the presence of intra-yeast bacteria was evaluated. Intra-yeast bacteria-like bodies were detected using wet mounting, and intra-yeast binding of anti-H. pylori antibodies was detected using immunofluorescence. The presence of the H. pylori rDNA 16S gene in total DNA from yeasts was demonstrated after PCR amplification. H. pylori showed larger death percentages at pH 3 and 4 than at pH 7. On the contrary, the viability of the yeast was not affected by any of the pHs evaluated. H. pylori entered into C. albicans at all the pH values assayed but to a greater extent at unfavorable pH values (pH 3 or 4, p = 0.014 and p = 0.001, respectively). In conclusion, it is possible to suggest that H. pylori can shelter itself within C. albicans under unfavorable pH conditions.

Effect of the natural arsenic gradient on the diversity and arsenic resistance of bacterial communities of the sediments of Camarones River (Atacama Desert, Chile).

Arsenic (As), a highly toxic metalloid, naturally present in Camarones River (Atacama Desert, Chile) is a great health concern for the local population and authorities. In this study, the taxonomic and functional characterization of bacterial communities associated to metal-rich sediments from three sites of the river (sites M1, M2 and M3), showing different arsenic concentrations, were evaluated using a combination of approaches. Diversity of bacterial communities was evaluated by Illumina sequencing. Strains resistant to arsenic concentrations varying from 0.5 to 100 mM arsenite or arsenate were isolated and the presence of genes coding for enzymes involved in arsenic oxidation (aio) or reduction (arsC) investigated. Bacterial communities showed a moderate diversity which increased as arsenic concentrations decreased along the river. Sequences of the dominant taxonomic groups (abundances ≥1%) present in all three sites were affiliated to Proteobacteria (range 40.3-47.2%), Firmicutes (8.4-24.8%), Acidobacteria (10.4-17.1%), Actinobacteria (5.4-8.1%), Chloroflexi (3.9-7.5%), Planctomycetes (1.2-5.3%), Gemmatimonadetes (1.2-1.5%), and Nitrospirae (1.1-1.2%). Bacterial communities from sites M2 and M3 showed no significant differences in diversity between each other (p = 0.9753) but they were significantly more diverse than M1 (p<0.001 and p<0.001, respectively). Sequences affiliated with Proteobacteria, Firmicutes, Acidobacteria, Chloroflexi and Actinobacteria at M1 accounted for more than 89% of the total classified bacterial sequences present but these phyla were present in lesser proportions in M2 and M3 sites. Strains isolated from the sediment of sample M1, having the greatest arsenic concentration (498 mg kg-1), showed the largest percentages of arsenic oxidation and reduction. Genes aio were more frequently detected in isolates from M1 (54%), whereas arsC genes were present in almost all isolates from all three sediments, suggesting that bacterial communities play an important role in the arsenic biogeochemical cycle and detoxification of arsenical compounds. Overall, results provide further knowledge on the microbial diversity of arsenic contaminated fresh-water sediments.

Simultaneous removal of C and N from fish effluents in filter reactors: Effect of recirculation ratio on the axial distribution of microbial communities.

We simultaneously removed carbon (C) and nitrogen (N) from fish effluents in compact filter reactors operating at different recirculation ratios (RRs) (2, 10 and without recirculation) to demonstrate microbial coexistence and determine the effect of the RR on the axial bacterial stratification. We also examined the global performance of anoxic, anaerobic and aerobic processes. Microbial communities (bacteria and archaea) were analyzed using 16s rRNA amplification followed by DGGE analyses. Their banding profiles were analyzed using ecological parameters and the most representative bands were sequenced. TOC removal was larger than 98% in the three reactors. The total N removal was 48% for the RR-2 reactor, whereas in the RR-10 reactor, there was no N removal due to the absence of nitrification in the final aerobic step. Coexistence and stratification of microorganisms were observed. The microbial communities were correlated with distinct biochemical processes in each reactor fraction. The RR had a large effect on the distribution of the microbial communities. When the RR increased from 2 to 10, the stratification decreased from 60 to 30%, suggesting a close relationship between reactor performance and the presence of nitrifiers. In the RR-10 reactor, the nitrifier concentration was only 4%. Thus, in combined processes, filter reactors should operate with a moderate RR to favor bacterial stratification and improve performance.

Arsenite oxidation by Pseudomonas arsenicoxydans immobilized on zeolite and its potential biotechnological application.

Pseudomonas arsenicoxydans has been recently described as a new arsenite oxidizing bacterial species. Arsenite detoxification activity by this species was determined by HPLC/HG/AAS. P. arsenicoxydans showed a high rate of As(III) conversion, particularly when immobilized (it oxidizes 100 % of 500 µg arsenite present in the medium after 48 of incubation). Arsenite oxidizing activity, mediated by a constitutive periplasmic enzyme, was determined following the transfer of reducing equivalents from arsenite to 2,4-dichlorophenolindophenol (DCIP) showing that approximately 75 % (0.173 µmol DCIP min(-1) mg(-1)) of the total activity (0.231 µmol DCIP min(-1) mg(-1)) was detected in the periplasmic fraction. Using PCR with primers specific for arsenite oxidase gene showed the presence of a gene encoding for arsenite oxidase in P. arsenicoxydans. Results show the potential biotechnological application of P. arsenicoxydans as a candidate for detoxification of As(III).

Pseudomonas arsenicoxydans sp nov., an arsenite-oxidizing strain isolated from the Atacama desert.

A Gram-negative, arsenite-oxidizing bacterial strain, designated VC-1, was isolated from sediment samples from the Camarones Valley in the Atacama Desert, Chile. Strain VC-1 was strictly aerobic, oxidase and catalase positive, rod shaped, of about 5.5 microm in length and 0.5-1.0 microm in diameter. It was motile by means of multiple polar flagella. The phylogenetic reconstruction of the 16S rRNA gene sequence, an MLSA study by concatenating six genes, and DDH studies indicated that the strain differed genotypically from its closest relatives and was therefore recognized as a new species within the genus Pseudomonas. Phenotypic analysis combining metabolic tests, fatty acid profiles and MALDI-TOF profiles of total cell extracts supported the classification of the new species for which we propose the designation Pseudomonas arsenicoxydans sp. nov. The type strain is accessible under the culture collection numbers CCUG 58201(T) and CECT 7543(T).