Microbial disruption in the gut promotes cerebral endothelial dysfunction.

Cerebrovascular disease is a group of conditions characterized by disorders of the cerebral vessels. Endothelial dysfunction renders the vasculature at risk of impaired blood flow and increases the potential of developing cerebrovascular disease. The gut microbiota has been recently identified as a possible risk factor of cerebrovascular disease. However, a direct link between gut microbiota and cerebral vascular function has not been established. Therefore, the aim of this study was to determine the effect of gut bacterial disruption on cerebral endothelial function. Male inbred Sprague-Dawley rats were randomly assigned to receive either drinking water with (n = 4) or without (n = 4) a cocktail of nonabsorbable broad-spectrum antibiotics (streptomycin, neomycin, bacitracin, and polymyxin B). Three weeks of antibiotic treatment resulted in a significant reduction in bacterial load and shifts within the bacterial sub-populations as assessed using flow cytometry. Using pressure myography, we found that spontaneous tone significantly increased and L-NAME-induced vasoconstriction was significantly blunted in middle cerebral arteries (MCAs) harvested from antibiotic-treated rats. ATP-mediated dilations were significantly blunted in MCAs from antibiotic-treated rats compared to their control counterparts. Immunoblotting revealed that the eNOS-P/total eNOS ratio was significantly reduced in cerebral artery lysates from antibiotic-treated rats compared to controls. Our findings suggest that disruption of the gut microbiota leads to cerebral endothelial dysfunction through reduction of eNOS activity. This study highlights the potential of the microbiota as a target to reverse endothelial dysfunction and a preventative approach to reducing risk of stroke and aneurysms.

Evaluating the Cytometric Detection and Enumeration of the Wine Bacterium, Oenococcus oeni.

Flow cytometry is a high-throughput tool for determining microbial abundance in a range of medical, environmental, and food-related samples. For wine, determining the abundance of Saccharomyces cerevisiae is well-defined and reliable. However, for the most common wine bacterium, Oenococcus oeni, using flow cytometry to determine cell concentration poses some challenges. O. oeni most often occurs in doublets or chains of varying lengths that can be greater than seven cells. This wine bacterium is also small, at 0.2-0.6 µm and may exhibit a range of morphologies including binary fission and aggregated complexes. This work demonstrates a straightforward approach to determining the suitability of flow cytometry for the chain-forming bacteria, O. oeni, and considerations when using flow cytometry for the enumeration of small microorganisms (<0.5 µm). © 2020 International Society for Advancement of Cytometry.

Changes of viral and prokaryote abundances in a high rate algal pond using flow cytometry detection.

High rate algal ponds (HRAPs) are shallow, mixed systems for wastewater treatment, which use sunlight exposure for disinfection. Little is known regarding the relationships between the bacteria and viruses within HRAP systems. Uniquely, flow cytometry permits the rapid identification of bacterial and viral populations in wastewater samples, separating populations based on genome and particle size. Treated wastewater samples were collected from an HRAP at Kingston on Murray, South Australia. Flow cytometry analysis detected bacterial populations and discriminated virus-like particles (VLP) and large VLP (LVLP). Rapid, short term, fluctuations in the abundance of all three populations were observed. Changes in the abundance of these populations was compared; wastewater composition was used as metadata for the comparisons. Linear regression determined relationships in abundances between bacteria and LVLP (R2 0.2985); LVLP and VLP (R2 0.5829) and bacteria and VLP (R2 0.5778) all with p-values of <0.001. Bacterial, LVLP and VLP abundance positively correlated with each other, indicating potential microbial interactions. Overall, the results suggest a parasitic relationship was occurring and driving the abundances of bacteria and viruses within the system.

The microbial abundance dynamics of the paediatric oral cavity before and after sleep.

Objective: Microhabitats in the oral cavity differ in microbial taxonomy. However, abundance variations of bacterial and viral communities within these microhabitats are not fully understood. Aims and Hypothesis: To assess the spatial distribution and dynamics of the microbial abundances within 6 microhabitats of the oral cavity before and after sleep. We hypothesise that the abundance distributions of these microbial communities will differ among oral sites. Methods: Using flow cytometry, bacterial and virus-like particle (VLP) abundances were enumerated for 6 oral microhabitats before and after sleep in 10 healthy paediatric sleepers. Results: Bacterial counts ranged from 7.2 ± 2.8 × 105 at the palate before sleep to 1.3 ± 0.2 × 108 at the back of the tongue after sleep, a difference of 187 times. VLPs ranged from 1.9 ± 1.0 × 106 at the palate before sleep to 9.2 ± 5.0 × 107 at the back of the tongue after sleep, a difference of 48 times. Conclusion: The oral cavity is a dynamic numerically heterogeneous environment where microbial communities can increase by a count of 100 million during sleep. Quantification of the paediatric oral microbiome complements taxonomic diversity information to show how biomass varies and shifts in space and time.

Distinct niche partitioning of marine and freshwater microbes during colonisation.

Bacteria are ubiquitous on the Earth, and many use chemotaxis to colonise favourable microenvironments. The colonisation process is continuous, where animals, plants, protists, viruses and chemical and physical factors frequently remove bacteria from wide volume ranges. Colonisation processes are poorly understood in natural communities. Here, we investigated niche partitioning during colonisation in aquatic microbial communities using bands of bacterial chemotaxis in petri dishes from mixed-species communities. The community partitioned into loiterers, primary and secondary colonisers, each having distinct abundances and taxonomy. Within marine samples, Shewanella dominated the primary colonisers, whilst Enterobacteriaceae dominated this group within the freshwater samples. Whether the success of these specific groups translates to what occurs within natural communities is uncertain, but here we show these taxa have the capacity to colonise new, unexplored environments. A strong negative association existed between the primary colonisers and viral abundance, suggesting that successful colonisers simultaneously move toward areas of heightened resources, which correlated with lower virus-like particles. Here, we show that microbial communities constantly sort themselves into distinct taxonomic groups as they move into new environments. This sorting during colonisation may be fundamental to microbial ecology, industry, technology, and disease development by setting the initial conditions that determine the winners as a community develops.

A hydrocarbon-contaminated aquifer reveals a Piggyback-the-Persistent viral strategy.

Subsurface environments hold the largest reservoir of microbes in the biosphere. They play essential roles in transforming nutrients, degrading contaminants and recycling organic matter. Here, we propose a previously unrecognised fundamental microbial process that influences aquifer bioremediation dynamics and that applies to all microbial communities. In contrast to previous models, our proposed Piggyback-the-Persistent (PtP) mechanism occurs when viruses become more dominated by those exhibiting temperate rather than lytic lifestyles driven by persistent chemicals (in our case chlorinated-hydrocarbon pollutants) that provide long-term carbon sources and that refocus the aquifer carbon cycle, thus altering the microbial community. In this ultra-oligotrophic system, the virus:microbial ratio (VMR) ranges from below the detection limit of 0.0001 to 0.6, well below the common aquatic range of 3-10. Shortest-average-path network analysis revealed VMR and trichlorethene (TCE) as nodes through which ecosystem information and biomass most efficiently pass. Novel network rearrangement revealed a hierarchy of Kill-the-Winner (KtW), Piggyback-the-Winner (PtW) and PtP nodes. We propose that KtW, PtW and PtP occur simultaneously as competing strategies, with their relative importance depending on conditions at a particular time and location with unusual nutrient sources, such as TCE, appearing to contribute to a shift in this balance between viral mechanisms.

Southern South Australian groundwater microbe diversity.

Groundwater is increasingly used globally for domestic, industrial and agricultural production. While many studies have focused on groundwater as a resource, the diverse ecosystems within are often ignored. Here, we assess 54 Southern South Australian groundwater microbial communities from the populated part of the state to assess their status and dynamics in isolated groundwater systems. We observed a strong site-to-site individuality in groundwater bacterial communities, likely due to the isolated nature of groundwater bodies leading to unique ecosystems. Rank abundance analysis indicates bacterial diversity is maintained even at low abundances and that the distribution fits classical ecological models for strong competition in resource-limited environments. Combined, our data indicates that despite overrepresentation of pollutant-associated bacterial orders in and around the Adelaide metropolitan area, microbial communities remain diverse and show little evidence of converging on a common pollutant-effected community.

Flow Cytometry for Rapid Enumeration and Biomass Quantification of Thraustochytrids in Coastal Seawaters.

Marine fungus-like eukaryotic unicellular protists (thraustochytrids) are considered to play an important role in the marine microbial food web. However, their abundance, distribution, and relative biomass in coastal waters have not yet been examined in detail. By using a flow cytometry method (FCM) for the rapid enumeration of thraustochytrids in nearshore and offshore stations along the Gulf of Bohai, China, we herein expanded current knowledge on their ecological significance. The FCM method allows for the rapid detection and quantification of prokaryotic and eukaryotic cells, but is rarely applied to the enumeration of small eukaryotic protists. Epifluorescence microscopy (EpiM) has been commonly used for the direct detection and enumeration of thraustochytrids; however, this method is time-consuming and inapplicable to a large-scale analysis of complex seawater samples. There is no available FCM method to track the abundance and biomass of thraustochytrids in marine habitats. The FCM enumeration of thraustochytrids in seawater samples ranged between 400 and 4,080 cells mL-1 with a biomass range of 8.15-83.96 µg C L-1. The thraustochytrid biomass contributed 10.9% to 98.1% of the total biomass of the heterotrophic microbial community comprising bacterioplankton and thraustochytrids. Their overall abundance in nearshore stations was significantly different from that in offshore stations (P<0.5). The present results provide an optimized method for the rapid detection and enumeration of thraustochytrids in seawater and facilitate large-scale studies of the ecological role of thraustochytrids in the microbial food web of coastal waters.

Microbial micropatches within microbial hotspots.

The spatial distributions of organism abundance and diversity are often heterogeneous. This includes the sub-centimetre distributions of microbes, which have 'hotspots' of high abundance, and 'coldspots' of low abundance. Previously we showed that 300 µl abundance hotspots, coldspots and background regions were distinct at all taxonomic levels. Here we build on these results by showing taxonomic micropatches within these 300 µl microscale hotspots, coldspots and background regions at the 1 µl scale. This heterogeneity among 1 µl subsamples was driven by heightened abundance of specific genera. The micropatches were most pronounced within hotspots. Micropatches were dominated by Pseudomonas, Bacteroides, Parasporobacterium and Lachnospiraceae incertae sedis, with Pseudomonas and Bacteroides being responsible for a shift in the most dominant genera in individual hotspot subsamples, representing up to 80.6% and 47.3% average abundance, respectively. The presence of these micropatches implies the ability these groups have to create, establish themselves in, or exploit heterogeneous microenvironments. These genera are often particle-associated, from which we infer that these micropatches are evidence for sub-millimetre aggregates and the aquatic polymer matrix. These findings support the emerging paradigm that the microscale distributions of planktonic microbes are numerically and taxonomically heterogeneous at scales of millimetres and less. We show that microscale microbial hotspots have internal structure within which specific local nutrient exchanges and cellular interactions might occur.

Stygofauna enhance prokaryotic transport in groundwater ecosystems.

More than 97% of the world's freshwater reserves are found in aquifers, making groundwater one of the most important resources on the planet. Prokaryotic communities in groundwater underpin the turnover of energy and matter while also maintaining groundwater purity. Thus, knowledge of microbial transport in the subsurface is crucial for maintaining groundwater health. Here, we describe for the first time the importance of stygofauna as vectors for prokaryotes. The "hitch-hiking" prokaryotes associated with stygofauna may be up to 5 orders of magnitude higher in abundance and transported up to 34× faster than bulk groundwater flow. We also demonstrate that prokaryotic diversity associated with stygofauna may be higher than that of the surrounding groundwater. Stygofauna are a newly recognized prokaryotic niche in groundwater ecosystems that have the potential to transport remediating, water purifying and pathogenic prokaryotes. Therefore, stygofauna may influence ecosystem dynamics and health at a microbial level, and at a larger scale could be a new source of prokaryotic diversity in groundwater ecosystems.

Marine and giant viruses as indicators of a marine microbial community in a riverine system.

Viral communities are important for ecosystem function as they are involved in critical biogeochemical cycles and controlling host abundance. This study investigates riverine viral communities around a small rural town that influences local water inputs. Myoviridae, Siphoviridae, Phycodnaviridae, Mimiviridae, Herpesviridae, and Podoviridae were the most abundant families. Viral species upstream and downstream of the town were similar, with Synechoccocus phage, salinus, Prochlorococcus phage, Mimivirus A, and Human herpes 6A virus most abundant, contributing to 4.9-38.2% of average abundance within the metagenomic profiles, with Synechococcus and Prochlorococcus present in metagenomes as the expected hosts for the phage. Overall, the majority of abundant viral species were or were most similar to those of marine origin. At over 60 km to the river mouth, the presence of marine communities provides some support for the Baas-Becking hypothesis "everything is everywhere, but, the environment selects." We conclude marine microbial species may occur more frequently in freshwater systems than previously assumed, and hence may play important roles in some freshwater ecosystems within tens to a hundred kilometers from the sea.

Enumerating Virus-Like Particles and Bacterial Populations in the Sinuses of Chronic Rhinosinusitis Patients Using Flow Cytometry.

There is increasing evidence to suggest that the sinus microbiome plays a role in the pathogenesis of chronic rhinosinusitis (CRS). However, the concentration of these microorganisms within the sinuses is still unknown. We show that flow cytometry can be used to enumerate bacteria and virus-like particles (VLPs) in sinus flush samples of CRS patients. This was achieved through trialling 5 sample preparation techniques for flow cytometry. We found high concentrations of bacteria and VLPs in these samples. Untreated samples produced the highest average bacterial and VLP counts with 3.3 ± 0.74 x 10(7) bacteria ml(-1) and 2.4 ± 1.23 x 10(9) VLP ml(-1) of sinus flush (n = 9). These counts were significantly higher than most of the treated samples (p < 0.05). Results showed 10(3) and 10(4) times inter-patient variation for bacteria and VLP concentrations. This wide variation suggests that diagnosis and treatment need to be personalised and that utilising flow cytometry is useful and efficient for this. This study is the first to enumerate bacterial and VLP populations in the maxillary sinus of CRS patients. The relevance of enumeration is that with increasing antimicrobial resistance, antibiotics are becoming less effective at treating bacterial infections of the sinuses, so alternative therapies are needed. Phage therapy has been proposed as one such alternative, but for dosing, the abundance of bacteria is required. Knowledge of whether phages are normally present in the sinuses will assist in gauging the safety of applying phage therapy to sinuses. Our finding, that large numbers of VLP are frequently present in sinuses, indicates that phage therapy may represent a minimally disruptive intervention towards the nasal microbiome. We propose that flow cytometry can be used as a tool to assess microbial biomass dynamics in sinuses and other anatomical locations where infection can cause disease.

Macroscale distribution of virioplankton and heterotrophic bacteria in the Bohai Sea.

In light of limited research into the relationship between the macroscale distribution and dynamic changes of microplankton in the shallow Bohai Sea, here we used flow cytometry to analyse samples collected from the Bohai Sea channel in winter and summer. Results showed that the average of both viral abundance (VA) and bacterial abundance (BA) were lower in winter (3.61 × 10(7) and 1.84 × 10(6) cells/mL, respectively) than in summer (7.47 × 10(7) and 5.05 × 10(6) cells/mL, respectively). At all 16 stations, VA was one order of magnitude greater than BA, with a positive relationship between one another. In the horizontal distribution, variations in VA and BA followed a similar trend, and both were obviously higher near-shore than offshore. In the vertical distribution, variations in both VA and BA did not show a clear relationship with water depth. VA and BA in summer were 2.1 and 2.7 times those in winter, respectively. Spearman correlation analysis showed that both VA and BA were correlated with the concentration of PO4-P in winter (positive) and NO3-N in summer (negative). Additionally, BA showed a negative correlation with salinity. It is clear that the macroscale distribution of these two kinds of microbes in the Bohai Sea is related to seasonal variation and nutrient availability.

Surface modification of an organic hessian substrate leads to shifts in bacterial biofilm community composition and abundance.

Antifouling strategies to limit biofilms on submerged surfaces in the marine environment are of particular interest due to the economic and environmental impacts in industries such as shipping and aquaculture. Here, we investigate the influence of chemically modified hessian bag surfaces on the bacterial abundance and community composition of biofilm formation using flow cytometry and 16S rRNA pyrosequencing. Hessian bags were coated with 5% and 10% Propyl(trimethoxy)silane (PTMS) and half of the bags had their lignin and hemicellulose removed via NaOH mercerisation. Significantly lower bacterial abundance was observed on mercerised bags treated with 5% PTMS (p<0.01). Significant shifts in bacterial taxa were also observed (p=0.0004), whereby unmercerised bags exhibited higher relative abundances of the anaerobic family Desulfovibrionaceae (4.5±1.7%), while mercerised bags displayed higher relative abundances of the aerobic family Phyllobacteriaceae (3.6±1.7%). This suggests that the mercerisation process may lower colonization rates and subsequently produce a thinner biofilm. This hypothesis is strengthened by the lower abundance of bacteria on mercerised bags, particularly on the 5% PTMS coating. Our results show that modifying a hessian surface via non-toxic coating and mercerisation reduces biofilm formation and also shifts the dominant taxa, increasing our understanding of antifouling strategies in the marine environment.

Distributions of Virus-Like Particles and Prokaryotes within Microenvironments.

Microbial interactions are important for ecosystem function, but occur at the microscale and so are difficult to observe. Previous studies in marine systems have shown significant shifts in microbial community abundance and composition over scales of micrometres to centimetres. This study investigates the microscale abundance distributions of virus-like particles (VLPs) and prokaryotes in the lower reaches of a river to determine the extent to which microscale microbial patchiness exists in freshwater systems. Here we report local hotspots surrounded by gradients that reach a maximum 80 and 107 fold change in abundance over 0.9 cm for prokaryotic and VLP subpopulations. Changes in prokaryotic and VLP hotspots were tightly coupled. There were no gradients at tens of centimetres across the boundary layers, which is consistent with strong mixing and turbulence-driven aggregation found in river systems. Quantification of the patchiness shows a marked asymmetry with patches 10 times greater than background common, but depletions being rare or absent in most samples. This consistent asymmetry suggests that coldspots depleted by grazing and lysis are rapidly mixed to background concentrations, while the prevalence of hotspots indicates persistence against disruption. The hotspot to coldspot relative abundance may be useful for understanding microbial river dynamics. The patchiness indicates that the mean- field approach of bulk phase sampling misses the microbially relevant community variation and may underestimate the concentrations of these important microbial groups.

Putative Effect of Aquifer Recharge on the Abundance and Taxonomic Composition of Endemic Microbial Communities.

Drought events and the overexploitation of freshwater resources have led to the increased need to manage groundwater reserves. Aquifer storage and recovery (ASR), whereby artificial water is injected into aquifers for storage, is one of the proposed methods by which freshwater supplies can be increased. Microbial clogging following injection, however, is a major issue. Here, during laboratory simulations of ASR, we used flow cytometry and bar-coded pyrosequencing to investigate changes in microbial abundance and community dynamics. Bacterial abundance ranged from 5.0 × 104 to 1.4 × 107 cells ml-1 before the addition of synthetic wastewater. Following wastewater addition, a 25-fold decrease in abundance was observed, coinciding with a 12-fold increase in viral abundance. Taxa shifted from an overrepresentation of Sphingomonadales, Sphingobacteriales, Rhodospirillales, Caulobacterales, Legionellales, Bacillales, Fusobacteriales and Verrucomicrobiales prior to the addition of synthetic wastewater to Burkholderiales, Actinomycetales, Pseudomonadales, Xanthomonadales, Rhodobacterales, Thizobiales and Thiotrichales following the addition of synthetic wastewater. Furthermore, a significant difference in overall taxonomic composition between the groundwater samples before and after the addition of synthetic wastewater was observed, with water samples exhibiting more similarity to sediment samples after wastewater was added. Collectively, these results suggest that ASR may alter the taxonomic composition of endemic microbial communities and that complete profiles of groundwater properties, including microbial community abundance and composition need to be taken into consideration when selecting aquifers for ASR practices.