An impaired metabolic response to hydrostatic pressure explains Alcanivorax borkumensis recorded distribution in the deep marine water column

Alcanivorax borkumensis is an ubiquitous model organism for hydrocarbonoclastic bacteria, which dominates polluted surface waters. Its negligible presence in oil-contaminated deep waters (as observed during the Deepwater Horizon accident) raises the hypothesis that it may lack adaptive mechanisms to hydrostatic pressure (HP). The type strain SK2 was tested under 0.1, 5 and 10 MPa (corresponding to surface water, 500 and 1000 m depth, respectively). While 5 MPa essentially inactivated SK2, further increase to 10 MPa triggered some resistance mechanism, as indicated by higher total and intact cell numbers. Under 10 MPa, SK2 upregulated the synthetic pathway of the osmolyte ectoine, whose concentration increased from 0.45 to 4.71 fmoles cell(-1). Central biosynthetic pathways such as cell replication, glyoxylate and Krebs cycles, amino acids metabolism and fatty acids biosynthesis, but not beta-oxidation, were upregulated or unaffected at 10 MPa, although total cell number was remarkably lower with respect to 0.1 MPa. Concomitantly, expression of more than 50% of SK2 genes was downregulated, including genes related to ATP generation, respiration and protein translation. Thus, A. borkumensis lacks proper adaptation to HP but activates resistance mechanisms. These consist in poorly efficient biosynthetic rather than energy-yielding degradation-related pathways, and suggest that HP does represent a major driver for its distribution at deep-sea

Distribution of selected healthcare resources for influenza pandemic response in Cambodia.

INTRODUCTION: Human influenza infection poses a serious public health threat in Cambodia, a country at risk for the emergence and spread of novel influenza viruses with pandemic potential. Prior pandemics demonstrated the adverse impact of influenza on poor communities in developing countries. Investigation of healthcare resource distribution can inform decisions regarding resource mobilization and investment for pandemic mitigation. METHODS: A health facility survey performed across Cambodia obtained data on availability of healthcare resources important for pandemic influenza response. Focusing on five key resources considered most necessary for treating severe influenza (inpatient beds, doctors, nurses, oseltamivir, and ventilators), resource distributions were analyzed at the Operational District (OD) and Province levels, refining data analysis from earlier studies. Resources were stratified by respondent type (hospital vs. District Health Office [DHO]). A summary index of distribution inequality was calculated using the Gini coefficient. Indices for local spatial autocorrelation were measured at the OD level using geographical information system (GIS) analysis. Finally, a potential link between socioeconomic status and resource distribution was explored by mapping resource densities against poverty rates. RESULTS: Gini coefficient calculation revealed variable inequality in distribution of the five key resources at the Province and OD levels. A greater percentage of the population resides in areas of relative under-supply (28.5%) than over-supply (21.3%). Areas with more resources per capita showed significant clustering in central Cambodia while areas with fewer resources clustered in the northern and western provinces. Hospital-based inpatient beds, doctors, and nurses were most heavily concentrated in areas of the country with the lowest poverty rates; however, beds and nurses in Non-Hospital Medical Facilities (NHMF) showed increasing concentrations at higher levels of poverty. CONCLUSIONS: There is considerable heterogeneity in healthcare resource distribution across Cambodia. Distribution mapping at the local level can inform policy decisions on where to stockpile resources in advance of and for reallocation in the event of a pandemic. These findings will be useful in determining future health resource investment, both for pandemic preparedness and for general health system strengthening, and provide a foundation for future analyses of equity in health services provision for pandemic mitigation planning in Cambodia

Bacterial community structure and diversity along the halocline of Tyro deep-sea hypersaline anoxic basin

Purpose: Tyro is a deep hypersaline anoxic basin (DHAB) located at the seafloor of the Eastern Mediterranean sea. Tyro hosts a stratified eukaryotic microbiome moving from seawater to the brine, but no reports are available on its prokaryotic community. We provide the first snapshot of the bacterial community structure in Tyro brine, seawaterbrine interface, and the overlaying deep seawater. Methods: In this study, we combined the use of molecular analyses, i.e., DNA fingerprinting and 16S rRNA pyrosequencing for the description of the bacterial community structure and taxonomy. PiCRUST2 was used to infer information on the prokaryotes functional diversity. A culture-dependent approach was applied to enrich bacteria of interest for marine biotechnology. Results: Bacterial communities sharply clustered moving from the seawater to the Tyro brine, in agreement with the abrupt increase of salinity values. Moreover, specific taxonomic groups inhabited the seawater-brine interface compared to the overlaying seawater and their identification revealed converging taxonomy with other DHABs in the Eastern Mediterranean sea. Functional traits inferred from the prokaryote taxonomy in the upper interface and the overlaying seawater indicated metabolic pathways for the synthesis of osmoprotectants, likely involved in bacterial adaptation to the steep increasing salinity. Metabolic traits related to methane and methylated compounds and to hydrocarbon degradation were also revealed in the upper interface of Tyro. The overall capability of the Tyro microbiome for hydrocarbon metabolism was confirmed by the isolation of hydrocarbonoclastic bacteria in the sediments. Conclusions: Our results suggest that Tyro seawater-brine interface hosts a specific microbiome adapted to the polyextreme condition typical of DHABs with potential metabolic features that could be further explored for the characterization of the metabolic network connecting the brine with the deep seawater through the chemocline. Moreover, Tyro could be a reservoir of culturable microbes endowed with functionalities of interest for biotechnological applications like hydrocarbon bioremediation

What lies on macroalgal surface: diversity of polysaccharide degraders in culturable epiphytic bacteria

Macroalgal surface constitutes a peculiar ecological niche and an advantageous substratum for microorganisms able to degrade the wide diversity of algal glycans. The degrading enzymatic activities of macroalgal epiphytes are of paramount interest for the industrial by-product sector and biomass resource applications. We characterized the polysaccharide hydrolytic profle of bacterial isolates obtained from three macroalgal species: the red macroalgae Asparagopsis taxiformis and Sphaerococcus coronopifolius (Rhodophyceae) and the brown Halopteris scoparia (Phaeophyceae), sampled in South Portugal. Bacterial enrichment cultures supplemented with chlorinated aliphatic compounds, typically released by marine algae, were established using as inoculum the decaying biomass of the three macroalgae, obtaining a collection of 634 bacterial strains. Although collected from the same site and exposed to the same seawater seeding microbiota, macroalgal cultivable bacterial communities in terms of functional and phylogenetic diversity showed host specifcity. Isolates were tested for the hydrolysis of starch, pectin, alginate and agar, exhibiting a diferent hydrolytic potential according to their host: A. taxiformis showed the highest percentage of active isolates (91%), followed by S. coronopifolius (54%) and H. scoparia (46%). Only 30% of the isolates were able to degrade starch, while the other polymers were degraded by 55–58% of the isolates. Interestingly, several isolates showed promiscuous capacities to hydrolyze more than one polysaccharide. The isolate functional fngerprint was statistically correlated to bacterial phylogeny, host species and enrichment medium. In conclusion, this work depicts macroalgae as holobionts with an associated microbiota of interest for blue biotechnologies, suggesting isolation strategies and bacterial targets for polysaccharidases’ discoveryinfo:eu-repo/semantics/publishedVersio

Fluorescent-BOX-PCR for resolving bacterial genetic diversity, endemism and biogeography

<p>Abstract</p> <p>Background</p> <p>BOX-A1R-based repetitive extragenic palindromic-PCR (BOX-PCR) is one of the most used techniques in biogeography studies of microbial isolates. However the traditional separation of BOX-PCR patterns by agarose gel electrophoresis suffers many limitations. The aim of this research was to set up a fluorescent BOX-PCR (F-BOX-PCR) assay in which separation of PCR products is automated in a capillary electrophoresis system. F-BOX-PCR was compared with the traditional BOX-PCR using bacterial strains with different G+C content (<it>Bacillus cereus</it>; <it>Escherichia coli</it>; isolates of the family <it>Geodermatophilaceae</it>). Resolution, discriminatory power and reproducibility were evaluated by assaying different electrophoretic runs, PCR reactions and independent DNA extractions. BOX-PCR and F-BOX-PCR were compared for the analysis of 29 strains of <it>Modestobacter multiseptatus </it>isolated from three different microsites in an altered carbonatic wall from Cagliari, Italy, and 45 strains of <it>Streptococcus thermophilus </it>isolated from 34 samples of the hand-made, yogurt-like product Matsoni, collected in different locations in Georgia.</p> <p>Results</p> <p>Fluorophore 6-FAM proved more informative than HEX and BOX-PCR both in agarose gel electrophoresis (<it>p </it>< 0.004 and <it>p </it>< 0.00003) and in capillary electrophoresis (compared only with HEX, <it>p </it>< 2 × 10^-7). 6-FAM- and HEX-based F-BOX-PCR respectively detected up to 12.0 and 11.3 times more fragments than BOX-PCR. Replicate separations of F-BOX-PCR showed an accuracy of the size calling of ± 0.5 bp until 500 bp, constantly decreasing to ± 10 bp at 2000 bp. Cluster analysis of F-BOX-PCR profiles grouped <it>M. multiseptatus </it>strains according to the microsite of isolation and <it>S. thermophilus </it>strains according to the geographical origin of Matsoni, but resulted intermixed when a BOX-PCR dataset was used.</p> <p>Conclusion</p> <p>F-BOX-PCR represents an improved method for addressing bacterial biogeography studies both in term of sensitivity, reproducibility and data analysis.</p

Hydrolytic Profile of the Culturable Gut Bacterial Community Associated With Hermetia illucens

Larvae of the black soldier fly (BSF) Hermetia illucens (L.) convert organic waste into high valuable insect biomass that can be used as alternative protein source for animal nutrition or as feedstock for biodiesel production. Since insect biology and physiology are influenced by the gut microbiome, knowledge about the functional role of BSF-associated microorganisms could be exploited to enhance the insect performance and growth. Although an increasing number of culture-independent studies are unveiling the microbiota structure and composition of the BSF gut microbiota, a knowledge gap remains on the experimental validation of the contribution of the microorganisms to the insect growth and development. We aimed at assessing if BSF gut-associated bacteria potentially involved in the breakdown of diet components are able to improve host nutrition. A total of 193 bacterial strains were obtained from guts of BSF larvae reared on a nutritious diet using selective and enrichment media. Most of the bacterial isolates are typically found in the insect gut, with major representatives belonging to the Gammaproteobacteria and Bacilli classes. The hydrolytic profile of the bacterial collection was assessed on compounds typically present in the diet. Finally, we tested the hypothesis that the addition to a nutritionally poor diet of the two isolates Bacillus licheniformis HI169 and Stenotrophomonas maltophilia HI121, selected for their complementary metabolic activities, could enhance BSF growth. B. licheniformis HI169 positively influenced the larval final weight and growth rate when compared to the control. Conversely, the addition of S. maltophilia HI121 to the nutritionally poor diet did not result in a growth enhancement in terms of larval weight and pupal weight and length in comparison to the control, whereas the combination of the two strains positively affected the larval final weight and the pupal weight and length. In conclusion, we isolated BSF-associated bacterial strains with potential positive properties for the host nutrition and we showed that selected isolates may enhance BSF growth, suggesting the importance to evaluate the effect of the bacterial administration on the insect performance

Destabilization of the Bacterial Interactome Identifies Nutrient Restriction-Induced Dysbiosis in Insect Guts

Stress-associated dysbiosis of microbiome can have several configurations that, under an energy landscape conceptual framework, can change from one configuration to another due to different alternating selective forces. It has been proposed—according to the Anna Karenina Principle—that in stressed individuals the microbiome are more dispersed (i.e., with a higher within-beta diversity), evidencing the grade of dispersion as indicator of microbiome dysbiosis. We hypothesize that although dysbiosis leads to different microbial communities in terms of beta diversity, these are not necessarily differently dispersed (within-beta diversity), but they form disrupted networks that make them less resilient to stress. To test our hypothesis, we select nutrient restriction (NR) stress that impairs host fitness but does not introduce overt microbiome selectors, such as toxic compounds and pathogens. We fed the polyphagous black soldier fly, Hermetia illucens, with two NR diets and a control full-nutrient (FN) diet. NR diets were dysbiotic because they strongly affected insect growth and development, inducing significant microscale changes in physiochemical conditions of the gut compartments. NR diets established new configurations of the gut microbiome compared to FN-fed guts but with similar dispersion. However, these new configurations driven by the deterministic changes induced by NR diets were reflected in rarefied, less structured, and less connected bacterial interactomes. These results suggested that while the dispersion cannot be considered a consistent indicator of the unhealthy state of dysbiotic microbiomes, the capacity of the community members to maintain network connections and stability can be an indicator of the microbial dysbiotic conditions and their incapacity to sustain the holobiont resilience and host homeostasis

Cell phenotype changes and oxidative stress response in Vibrio spp. induced into viable but non-culturable (VBNC) state

Purpose: Aquatic bacteria of the genus Vibrio include animal and human pathogens. The occurrence of Vibrio-related diseases has been associated with the current climate change-driven increase of sea surface temperature. Vibrio spp. can enter into the viable but non-culturable (VBNC) state, as a consequence of starvation in seawater at low temperatures. In such physiological state, Vibrio cells are no longer culturable on standard media agar plates but can resuscitate if incubated at 30 °C prior to plating, retaining virulence. Since limited information is available on regards to this topic, in this work, we characterized the phenotypic changes of four Vibrio spp. strains (one laboratory strain and three environmental isolates) in cold seawater microcosms, investigating the relationship between resuscitation and a hydrogen peroxide-induced oxidative stress. Methods: Cell phenotypic changes and the effect of hydrogen peroxide and/or catalase addition to the medium were studied on VBNC and resuscitated cells by flow cytometry in microcosm experiments, paralleled by culturability experiments by plating. Results: The cells of all the Vibrio strains changed their phenotype upon the induction of the VBNC state resulting in cell dwarfing and decrease in DNA quantity, losing the ability to grow on solid media. These features were partially or totally reverted when the cells were treated for resuscitation. Hydrogen peroxide at concentrations as low as 0.007 mM prevented resuscitation and a prolonged exposure to hydrogen peroxide at concentrations far under those inhibiting the growth of log-phase cells permanently damaged VBNC cells, which could not be resuscitated. However, the potential of culturability of VBNC cells could be preserved, at least for a part of the population, by plating the cells in the presence of catalase. The study also showed that during the resuscitation process, the cells gradually increased their resistance to hydrogen peroxide. Conclusions: The timing and mode of induction of the VBNC state, as well as cell resuscitation and response to hydrogen peroxide, differed among Vibrio strains, indicating that induction and resuscitation from dormancy could vary in the context of species belonging to a single genus