Organizing the bacterial annotation space with amino acid sequence embeddings.

BACKGROUND: Due to the ever-expanding gap between the number of proteins being discovered and their functional characterization, protein function inference remains a fundamental challenge in computational biology. Currently, known protein annotations are organized in human-curated ontologies, however, all possible protein functions may not be organized accurately. Meanwhile, recent advancements in natural language processing and machine learning have developed models which embed amino acid sequences as vectors in n-dimensional space. So far, these embeddings have primarily been used to classify protein sequences using manually constructed protein classification schemes. RESULTS: In this work, we describe the use of amino acid sequence embeddings as a systematic framework for studying protein ontologies. Using a sequence embedding, we show that the bacterial carbohydrate metabolism class within the SEED annotation system contains 48 clusters of embedded sequences despite this class containing 29 functional labels. Furthermore, by embedding Bacillus amino acid sequences with unknown functions, we show that these unknown sequences form clusters that are likely to have similar biological roles. CONCLUSIONS: This study demonstrates that amino acid sequence embeddings may be a powerful tool for developing more robust ontologies for annotating protein sequence data. In addition, embeddings may be beneficial for clustering protein sequences with unknown functions and selecting optimal candidate proteins to characterize experimentally.

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.

QTL mapping: an innovative method for investigating the genetic determinism of yeast-bacteria interactions in wine.

The two most commonly used wine microorganisms, Saccharomyces cerevisiae yeast and Oenococcus oeni bacteria, are responsible for completion of alcoholic and malolactic fermentation (MLF), respectively. For successful co-inoculation, S. cerevisiae and O. oeni must be able to complete fermentation; however, this relies on compatibility between yeast and bacterial strains. For the first time, quantitative trait loci (QTL) analysis was used to elucidate whether S. cerevisiae genetic makeup can play a role in the ability of O. oeni to complete MLF. Assessment of 67 progeny from a hybrid S. cerevisiae strain (SBxGN), co-inoculated with a single O. oeni strain, SB3, revealed a major QTL linked to MLF completion by O. oeni. This QTL encompassed a well-known translocation, XV-t-XVI, that results in increased SSU1 expression and is functionally linked with numerous phenotypes including lag phase duration and sulphite export and production. A reciprocal hemizygosity assay was performed to elucidate the effect of the gene SSU1 in the SBxGN background. Our results revealed a strong effect of SSU1 haploinsufficiency on O. oeni's ability to complete malolactic fermentation during co-inoculation and pave the way for the implementation of QTL mapping projects for deciphering the genetic bases of microbial interactions. KEY POINTS: • For the first time, QTL analysis has been used to study yeast-bacteria interactions. • A QTL encompassing a translocation, XV-t-XVI, was linked to MLF outcomes. • S. cerevisiae SSU1 haploinsufficiency positively impacted MLF by O. oeni.

Efficacy of phage cocktail AB-SA01 therapy in diabetic mouse wound infections caused by multidrug-resistant Staphylococcus aureus.

BACKGROUND: Diabetic foot ulcer (DFU) is a serious complication of diabetes mellitus. Antibiotic-resistant Staphylococcus aureus is frequently isolated from DFU infections. Bacteriophages (phages) represent an alternative or adjunct treatment to antibiotic therapy. Here we describe the efficacy of AB-SA01, a cocktail of three S. aureus Myoviridae phages, made to current good manufacturing practice (cGMP) standards, and which has undergone two phase I clinical trials, in treatment of multidrug-resistant (MDR) S. aureus infections. RESULTS: Wounds of saline-treated mice showed no healing, but expanded and became inflamed, ulcerated, and suppurating. In contrast, AB-SA01 treatment decreased the bacterial load with efficacy similar or superior to vancomycin treatment. At the end of the treatment period, there was a significant decrease (p < 0.001) in bacterial load and wound size in infected phage- and vancomycin-treated groups compared with infected saline-treated mice. In phage-treated mice, wound healing was seen similar to vancomycin treatment. No mortality was recorded associated with infections, and post-mortem examinations did not show any evident pathological lesions other than the skin wounds. No adverse effects related to the application of phages were observed. CONCLUSION: Topical application of phage cocktail AB-SA01 is effective, as shown by bacterial load reduction and wound closure, in the treatment of diabetic wound infections caused by MDR S. aureus. Our results suggest that topical phage cocktail treatment may be effective in treating antibiotic-resistant S. aureus DFU infections.

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.

Mini-review: efficacy of lytic bacteriophages on multispecies biofilms.

There is potential for phages to prevent and control bacterial biofilms, but few studies have examined the effect of phages on the multispecies biofilms that characterize most bacterial infections. This paper reviews the mechanism of action of phages, the evidence supporting the view that phage therapy will be effective against bacterial targets and the opposite viewpoint, phage application approaches, and the comparative advantage of phage therapy in multispecies biofilms. The few reports measuring the actions of lytic phages against multispecies biofilms are also reviewed. The authors are cautiously optimistic about the application of phages against their targets when in multispecies biofilms because some lysis mechanisms do not require species specificity.

Chemotaxis toward phytoplankton drives organic matter partitioning among marine bacteria.

The microenvironment surrounding individual phytoplankton cells is often rich in dissolved organic matter (DOM), which can attract bacteria by chemotaxis. These "phycospheres" may be prominent sources of resource heterogeneity in the ocean, affecting the growth of bacterial populations and the fate of DOM. However, these effects remain poorly quantified due to a lack of quantitative ecological frameworks. Here, we used video microscopy to dissect with unprecedented resolution the chemotactic accumulation of marine bacteria around individual Chaetoceros affinis diatoms undergoing lysis. The observed spatiotemporal distribution of bacteria was used in a resource utilization model to map the conditions under which competition between different bacterial groups favors chemotaxis. The model predicts that chemotactic, copiotrophic populations outcompete nonmotile, oligotrophic populations during diatom blooms and bloom collapse conditions, resulting in an increase in the ratio of motile to nonmotile cells and in the succession of populations. Partitioning of DOM between the two populations is strongly dependent on the overall concentration of bacteria and the diffusivity of different DOM substances, and within each population, the growth benefit from phycospheres is experienced by only a small fraction of cells. By informing a DOM utilization model with highly resolved behavioral data, the hybrid approach used here represents a new path toward the elusive goal of predicting the consequences of microscale interactions in the ocean.

Host-specific associations affect the microbiome of Philornis downsi, an introduced parasite to the Galápagos Islands.

The composition and diversity of bacteria forming the microbiome of parasitic organisms have implications for differential host pathogenicity and host-parasite co-evolutionary interactions. The microbiome of pathogens can therefore have consequences that are relevant for managing disease prevalence and impact on affected hosts. Here, we investigate the microbiome of an invasive parasitic fly Philornis downsi, recently introduced to the Galápagos Islands, where it poses extinction threat to Darwin's finches and other land birds. Larvae infest nests of Darwin's finches and consume blood and tissue of developing nestlings, and have severe mortality impacts. Using 16s rRNA sequencing data, we characterize the bacterial microbiota associated with P. downsi adults and larvae sourced from four finch host species, inhabiting two islands and representing two ecologically distinct groups. We show that larval and adult microbiomes are dominated by the phyla Proteobacteria and Firmicutes, which significantly differ between life stages in their distributions. Additionally, bacterial community structure significantly differed between larvae retrieved from strictly insectivorous warbler finches (Certhidea olivacea) and those parasitizing hosts with broader dietary preferences (ground and tree finches, Geospiza and Camarhynchus spp., respectively). Finally, we found no spatial effects on the larval microbiome, as larvae feeding on the same host (ground finches) harboured similar microbiomes across islands. Our results suggest that the microbiome of P. downsi changes during its development, according to dietary composition or nutritional needs, and is significantly affected by host-related factors during the larval stage. Unravelling the ecological significance of bacteria for this parasite will contribute to the development of novel, effective control strategies.

Integrating biorefinery and farm biogeochemical cycles offsets fossil energy and mitigates soil carbon losses.

Crop residues are potentially significant sources of feedstock for biofuel production in the United States. However, there are concerns with maintaining the environmental functions of these residues while also serving as a feedstock for biofuel production. Maintaining soil organic carbon (SOC) along with its functional benefits is considered a greater constraint than maintaining soil erosion losses to an acceptable level. We used the biogeochemical model DayCent to evaluate the effect of residue removal, corn stover, and wheat and barley straw in three diverse locations in the USA. We evaluated residue removal with and without N replacement, along with application of a high-lignin fermentation byproduct (HLFB), the residue by-product comprised of lignin and small quantities of nutrients from cellulosic ethanol production. SOC always decreased with residue harvest, but the decrease was greater in colder climates when expressed on a life cycle basis. The effect of residue harvest on soil N2O emissions varied with N addition and climate. With N addition, N2O emissions always increased, but the increase was greater in colder climates. Without N addition, N2O emissions increased in Iowa, but decreased in Maryland and North Carolina with crop residue harvest. Although SOC was lower with residue harvest when HLFB was used for power production instead of being applied to land, the avoidance of fossil fuel emissions to the atmosphere by utilizing the cellulose and hemicellulose fractions of crop residue to produce ethanol (offsets) reduced the overall greenhouse gas (GHG) emissions because most of this residue carbon would normally be lost during microbial respiration. Losses of SOC and reduced N mineralization could both be mitigated with the application of HLFB to the land. Therefore, by returning the high-lignin fraction of crop residue to the land after production of ethanol at the biorefinery, soil carbon levels could be maintained along with the functional benefit of increased mineralized N, and more GHG emissions could be offset compared to leaving the crop residues on the land.

Microbial alignment in flow changes ocean light climate.

The growth of microbial cultures in the laboratory often is assessed informally with a quick flick of the wrist: dense suspensions of microorganisms produce translucent "swirls" when agitated. Here, we rationalize the mechanism behind this phenomenon and show that the same process may affect the propagation of light through the upper ocean. Analogous to the shaken test tubes, the ocean can be characterized by intense fluid motion and abundant microorganisms. We demonstrate that the swirl patterns arise when elongated microorganisms align preferentially in the direction of fluid flow and alter light scattering. Using a combination of experiments and mathematical modeling, we find that this phenomenon can be recurrent under typical marine conditions. Moderate shear rates (0.1 s(-1)) can increase optical backscattering of natural microbial assemblages by more than 20%, and even small shear rates (0.001 s(-1)) can increase backscattering from blooms of large phytoplankton by more than 30%. These results imply that fluid flow, currently neglected in models of marine optics, may exert an important control on light propagation, influencing rates of global carbon fixation and how we estimate these rates via remote sensing.

Lytic activity of phages against bacterial pathogens infecting diabetic foot ulcers.

Complications of diabetes, such as diabetic foot ulcers (DFUs), are common, multifactorial in origin, and costly to treat. DFUs are the cause of nearly 90% of limb amputations among persons with diabetes. In most chronic infections such as DFU, biofilms are involved. Bacteria in biofilms are 100-1000 times more resistant to antibiotics than their planktonic counterparts. Multidrug-resistant (MDR) Staphylococcus aureus and Pseudomonas aeruginosa infections in DFUs may require alternative therapeutic agents such as bacteriophages ("phages"). This study describes the lytic activity of phage cocktails AB-SA01 (3-phage cocktail) and AB-PA01 (4-phage cocktail), which target S. aureus and P. aeruginosa, respectively. The host range and lytic effect of AB-SA01 and AB-PA01 on a planktonic culture, single-species biofilm, and mixed-species biofilm were evaluated. In vitro testing showed that 88.7% of S. aureus and 92.7% of P. aeruginosa isolates were susceptible to AB-SA01 and AB-PA01, respectively, in the planktonic state. The component phages of AB-SA01 and AB-PA01 infected 66% to 94.3% of the bacterial isolates tested. Furthermore, AB-SA01 and AB-PA01 treatment significantly (p < 0.05) reduced the biofilm biomass of their hosts, regardless of the antibiotic-resistant characteristics of the isolates and the presence of a non-susceptible host. In conclusion, the strong lytic activity, broad host range, and significant biofilm biomass reduction of AB-SA01 and AB-PA01 suggest the considerable potential of phages in treating antibiotic-resistant S. aureus and P. aeruginosa infections alone or as coinfections in DFUs.

Empirical parameterisation and dynamical analysis of the allometric Rosenzweig-MacArthur equations.

Allometric settings of population dynamics models are appealing due to their parsimonious nature and broad utility when studying system level effects. Here, we parameterise the size-scaled Rosenzweig-MacArthur differential equations to eliminate prey-mass dependency, facilitating an in depth analytic study of the equations which incorporates scaling parameters' contributions to coexistence. We define the functional response term to match empirical findings, and examine situations where metabolic theory derivations and observation diverge. The dynamical properties of the Rosenzweig-MacArthur system, encompassing the distribution of size-abundance equilibria, the scaling of period and amplitude of population cycling, and relationships between predator and prey abundances, are consistent with empirical observation. Our parameterisation is an accurate minimal model across 15+ orders of mass magnitude.

Metagenomic comparison of microbial communities inhabiting confined and unconfined aquifer ecosystems.

A metagenomic analysis of two aquifer systems located under a dairy farming region was performed to examine to what extent the composition and function of microbial communities varies between confined and surface-influenced unconfined groundwater ecosystems. A fundamental shift in taxa was seen with an overrepresentation of Rhodospirillales, Rhodocyclales, Chlorobia and Circovirus in the unconfined aquifer, while Deltaproteobacteria and Clostridiales were overrepresented in the confined aquifer. A relative overrepresentation of metabolic processes including antibiotic resistance (ß-lactamase genes), lactose and glucose utilization and DNA replication were observed in the unconfined aquifer, while flagella production, phosphate metabolism and starch uptake pathways were all overrepresented in the confined aquifer. These differences were likely driven by differences in the nutrient status and extent of exposure to contaminants of the two groundwater systems. However, when compared with freshwater, ocean, sediment and animal gut metagenomes, the unconfined and confined aquifers were taxonomically and metabolically more similar to each other than to any other environment. This suggests that intrinsic features of groundwater ecosystems, including low oxygen levels and a lack of sunlight, have provided specific niches for evolution to create unique microbial communities. Obtaining a broader understanding of the structure and function of microbial communities inhabiting different groundwater systems is particularly important given the increased need for managing groundwater reserves of potable water.

Shifts in evolutionary balance of phenotypes under environmental changes.

Environments shape communities by driving individual interactions and the evolutionary outcome of competition. In static, homogeneous environments a robust, evolutionary stable, outcome is sometimes reachable. However, inherently stochastic, this evolutionary process need not stabilize, resulting in a dynamic ecological state, often observed in microbial communities. We use evolutionary games to study the evolution of phenotypic competition in dynamic environments. Under the assumption that phenotypic expression depends on the environmental shifts, existing periodic relationships may break or result in formation of new periodicity in phenotypic interactions. The exact outcome depends on the environmental shift itself, indicating the importance of understanding how environments influence affected systems. Under periodic environmental fluctuations, a stable state preserving dominant phenotypes may exist. However, rapid environmental shifts can lead to critical shifts in the phenotypic evolutionary balance. This might lead to environmentally favoured phenotypes dominating making the system vulnerable. We suggest that understanding of the robustness of the system's current state is necessary to anticipate when it will shift to a new equilibrium via understanding what level of perturbations the system can take before its equilibrium changes. Our results provide insights in how microbial communities can be steered to states where they are dominated by desired phenotypes.

Potential for bacteriophage therapy for Staphylococcus aureus pneumonia with influenza A coinfection.

The ability of influenza A virus to evolve, coupled with increasing antimicrobial resistance, could trigger an influenza pandemic with great morbidity and mortality. Much of the 1918 influenza pandemic mortality was likely due to bacterial coinfection, including Staphylococcus aureus pneumonia. S. aureus resists many antibiotics. The lack of new antibiotics suggests alternative antimicrobials, such as bacteriophages, are needed. Potential delivery routes for bacteriophage therapy (BT) include inhalation and intravenous injection. BT has recently been used successfully in compassionate access pulmonary infection cases. Phage lysins, enzymes that hydrolyze bacterial cell walls and which are bactericidal, are efficacious in animal pneumonia models. Clinical trials will be needed to determine whether BT can ameliorate disease in influenza and S. aureus coinfection.

Iron defecation by sperm whales stimulates carbon export in the Southern Ocean.

The iron-limited Southern Ocean plays an important role in regulating atmospheric CO(2) levels. Marine mammal respiration has been proposed to decrease the efficiency of the Southern Ocean biological pump by returning photosynthetically fixed carbon to the atmosphere. Here, we show that by consuming prey at depth and defecating iron-rich liquid faeces into the photic zone, sperm whales (Physeter macrocephalus) instead stimulate new primary production and carbon export to the deep ocean. We estimate that Southern Ocean sperm whales defecate 50 tonnes of iron into the photic zone each year. Molar ratios of C(export):Fe(added) determined during natural ocean fertilization events are used to estimate the amount of carbon exported to the deep ocean in response to the iron defecated by sperm whales. We find that Southern Ocean sperm whales stimulate the export of 4 × 10(5) tonnes of carbon per year to the deep ocean and respire only 2 × 10(5) tonnes of carbon per year. By enhancing new primary production, the populations of 12 000 sperm whales in the Southern Ocean act as a carbon sink, removing 2 × 10(5) tonnes more carbon from the atmosphere than they add during respiration. The ability of the Southern Ocean to act as a carbon sink may have been diminished by large-scale removal of sperm whales during industrial whaling.

Efficacy of Lytic Phage Cocktails on Staphylococcus aureus and Pseudomonas aeruginosa in Mixed-Species Planktonic Cultures and Biofilms.

The efficacy of phages in multispecies infections has been poorly examined. The in vitro lytic efficacies of phage cocktails AB-SA01, AB-PA01, which target Staphylococcus aureus and Pseudomonas aeruginosa, respectively, and their combination against their hosts were evaluated in S. aureus and P. aeruginosa mixed-species planktonic and biofilm cultures. Green fluorescent protein (GFP)-labelled P. aeruginosa PAO1 and mCherry-labelled S. aureus KUB7 laboratory strains and clinical isolates were used as target bacteria. During real-time monitoring using fluorescence spectrophotometry, the density of mCherry S. aureus KUB7 and GFP P. aeruginosa PAO1 significantly decreased when treated by their respective phage cocktail, a mixture of phage cocktails, and gentamicin. The decrease in bacterial density measured by relative fluorescence strongly associated with the decline in bacterial cell counts. This microplate-based mixed-species culture treatment monitoring through spectrophotometry combine reproducibility, rapidity, and ease of management. It is amenable to high-throughput screening for phage cocktail efficacy evaluation. Each phage cocktail, the combination of the two phage cocktails, and tetracycline produced significant biofilm biomass reduction in mixed-species biofilms. This study result shows that these phage cocktails lyse their hosts in the presence of non-susceptible bacteria. These data support the use of phage cocktails therapy in infections with multiple bacterial species.

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.

Heavy metal toxicity of kidney and bone tissues in South Australian adult bottlenose dolphins (Tursiops aduncus).

Metallothioneins (MT) concentration, renal damage, and bone malformations were investigated in 38 adult Tursiops aduncus carcasses to determine any associations with cadmium, copper, zinc, mercury, lead and selenium. Significantly higher concentrations of cadmium, copper, and zinc in the liver were observed in dolphins showing evidence of more advanced renal damage. No significant differences in metal or selenium concentrations in the liver were observed between groups differing in level of bone malformations. Some dolphins displayed evidence of toxicity and knowledge of metal toxicity pathways were used to elucidate the cause of these abnormalities. Two dolphins had high metal burdens, high MT concentrations, renal damage, and evidence of bone malformations, indicating possible severe and prolonged metal toxicity. One dolphin showed evidence of renal damage, but the lack of any other symptoms suggests that this was unlikely to be caused by metal toxicity. We recommend examining a range of metal toxicity symptoms simultaneously to aid in distinguishing metal toxicity from unrelated aetiologies.