Synthetic and natural rubber associated chemicals drive functional and structural changes as well as adaptations to antibiotics in in vitro marine microbiomes.

The leaching of additives from plastics and elastomers (rubbers) has raised concerns due to their potential negative impacts on the environment and the development of antibiotic resistance. In this study, we investigated the effects of chemicals extracted from two types of rubber on microbiomes derived from a benthic sea urchin and two pelagic fish species. Additionally, we examined whether bacterial communities preconditioned with rubber-associated chemicals displayed adaptations to antibiotics. At the highest tested concentrations of chemicals, we observed reduced maximum growth rates and yields, prolonged lag phases, and increased alpha diversity. While the effects on alpha and beta diversity were not always conclusive, several bacterial genera were significantly influenced by chemicals from the two rubber sources. Subsequent exposure of sea urchin microbiomes preconditioned with rubber chemicals to the antibiotic ciprofloxacin resulted in decreased maximum growth rates. This indicates a more sensitive microbiome to ciprofloxacin when preconditioned with rubber chemicals. Although no significant interaction effects between rubber chemicals and ciprofloxacin exposure were observed in bacterial alpha and beta diversity, we observed log-fold changes in two bacterial genera in response to ciprofloxacin exposure. These findings highlight the structural and functional alterations in microbiomes originating from various marine species when exposed to rubber-associated chemicals and underscore the potential risks posed to marine life.

Archaea in boreal Swedish lakes are diverse, dominated by Woesearchaeota and follow deterministic community assembly.

Despite their key role in biogeochemical processes, particularly the methane cycle, archaea are widely underrepresented in molecular surveys because of their lower abundance compared with bacteria and eukaryotes. Here, we use parallel high-resolution small subunit rRNA gene sequencing to explore archaeal diversity in 109 Swedish lakes and correlate archaeal community assembly mechanisms to large-scale latitudinal, climatic (nemoral to arctic) and nutrient (oligotrophic to eutrophic) gradients. Sequencing with universal primers showed the contribution of archaea was on average 0.8% but increased up to 1.5% of the three domains in forest lakes. Archaea-specific sequencing revealed that freshwater archaeal diversity could be partly explained by lake variables associated with nutrient status. Combined with deterministic co-occurrence patterns this finding suggests that ecological drift is overridden by environmental sorting, as well as other deterministic processes such as biogeographic and evolutionary history, leading to lake-specific archaeal biodiversity. Acetoclastic, hydrogenotrophic and methylotrophic methanogens as well as ammonia-oxidizing archaea were frequently detected across the lakes. Archaea-specific sequencing also revealed representatives of Woesearchaeota and other phyla of the DPANN superphylum. This study adds to our understanding of the ecological range of key archaea in freshwaters and links these taxa to hypotheses about processes governing biogeochemical cycles in lakes.

Influence of pulsed and continuous substrate inputs on freshwater bacterial community composition and functioning in bioreactors.

Aquatic environments are typically not homogenous, but characterized by changing substrate concentration gradients and nutrient patches. This heterogeneity in substrate availability creates a multitude of niches allowing bacteria with different substrate utilization strategies to hypothetically coexist even when competing for the same substrate. To study the impact of heterogeneous distribution of organic substrates on bacterioplankton, bioreactors with freshwater bacterial communities were fed artificial freshwater medium with acetate supplied either continuously or in pulses. After a month-long incubation, bacterial biomass and community-level substrate uptake rates were twice as high in the pulsed treatment compared to the continuously fed reactors even if the same total amount of acetate was supplied to both treatments. The composition of the bacterial communities emerging in the two treatments differed significantly with specific taxa overrepresented in the respective treatments. The higher estimated growth yield in cultures that received pulsed substrate inputs, imply that such conditions enable bacteria to use resources more efficiently for biomass production. This finding agrees with established concepts of basal maintenance energy requirements and high energetic costs to assimilate substrates at low concentration. Our results further imply that degradation of organic matter is influenced by temporal and spatial heterogeneity in substrate availability.

Distinct Anaerobic Bacterial Consumers of Cellobiose-Derived Carbon in Boreal Fens with Different CO2/CH4 Production Ratios.

Northern peatlands in general have high methane (CH4) emissions, but individual peatlands show considerable variation as CH4 sources. Particularly in nutrient-poor peatlands, CH4 production can be low and exceeded by carbon dioxide (CO2) production from unresolved anaerobic processes. To clarify the role anaerobic bacterial degraders play in this variation, we compared consumers of cellobiose-derived carbon in two fens differing in nutrient status and the ratio of CO2 to CH4 produced. After [13C]cellobiose amendment, the mesotrophic fen produced equal amounts of CH4 and CO2 The oligotrophic fen had lower CH4 production but produced 3 to 59 times more CO2 than CH4 RNA stable-isotope probing revealed that in the mesotrophic fen with higher CH4 production, cellobiose-derived carbon was mainly assimilated by various recognized fermenters of Firmicutes and by Proteobacteria The oligotrophic peat with excess CO2 production revealed a wider variety of cellobiose-C consumers, including Firmicutes and Proteobacteria, but also more unconventional degraders, such as Telmatobacter-related Acidobacteria and subphylum 3 of Verrucomicrobia Prominent and potentially fermentative Planctomycetes and Chloroflexi did not appear to process cellobiose-C. Our results show that anaerobic degradation resulting in different levels of CH4 production can involve distinct sets of bacterial degraders. By distinguishing cellobiose degraders from the total community, this study contributes to defining anaerobic bacteria that process cellulose-derived carbon in peat. Several of the identified degraders, particularly fermenters and potential Fe(III) or humic substance reducers in the oligotrophic peat, represent promising candidates for resolving the origin of excess CO2 production in peatlands. IMPORTANCE: Peatlands are major sources of the greenhouse gas methane (CH4), yet in many peatlands, CO2 production from unresolved anaerobic processes exceeds CH4 production. Anaerobic degradation produces the precursors of CH4 production but also represents competing processes. We show that anaerobic degradation leading to high or low CH4 production involved distinct sets of bacteria. Well-known fermenters dominated in a peatland with high CH4 production, while novel and unconventional degraders could be identified in a site where CO2 production greatly exceeds CH4 production. Our results help identify and assign functions to uncharacterized bacteria that promote or inhibit CH4 production and reveal bacteria potentially producing the excess CO2 in acidic peat. This study contributes to understanding the microbiological basis for different levels of CH4 emission from peatlands.

Bacterial diversity along a 2600 km river continuum.

The bacterioplankton diversity in large rivers has thus far been under-sampled despite the importance of streams and rivers as components of continental landscapes. Here, we present a comprehensive dataset detailing the bacterioplankton diversity along the midstream of the Danube River and its tributaries. Using 16S rRNA-gene amplicon sequencing, our analysis revealed that bacterial richness and evenness gradually declined downriver in both the free-living and particle-associated bacterial communities. These shifts were also supported by beta diversity analysis, where the effects of tributaries were negligible in regards to the overall variation. In addition, the river was largely dominated by bacteria that are commonly observed in freshwaters. Dominated by the acI lineage, the freshwater SAR11 (LD12) and the Polynucleobacter group, typical freshwater taxa increased in proportion downriver and were accompanied by a decrease in soil and groundwater-affiliated bacteria. Based on views of the meta-community and River Continuum Concept, we interpret the observed taxonomic patterns and accompanying changes in alpha and beta diversity with the intention of laying the foundation for a unified concept for river bacterioplankton diversity.

Resistant microbial cooccurrence patterns inferred by network topology.

Although complex cooccurrence patterns have been described for microbes in natural communities, these patterns have scarcely been interpreted in the context of ecosystem functioning and stability. Here we constructed networks from species cooccurrences between pairs of microorganisms which were extracted from five individual aquatic time series, including a dystrophic and a eutrophic lake as well as an open ocean site. The resulting networks exhibited higher clustering coefficients, shorter path lengths, and higher average node degrees and levels of betweenness than those of random networks. Moreover, simulations demonstrated that taxa with a large number of cooccurrences and placement at convergence positions in the network, so-called "hubs" and "bottlenecks," confer resistance against random removal of "taxa." Accordingly, we refer to cooccurrences at convergence positions as system-relevant interdependencies, as they, like hubs and bottlenecks, determine network topology. These topology features of the cooccurrence networks point toward microbial community dynamics being resistant over time and thus could provide indicators for the state of ecosystem stability.

Auxotrophy and intrapopulation complementary in the 'interactome' of a cultivated freshwater model community.

Microorganisms are usually studied either in highly complex natural communities or in isolation as monoclonal model populations that we manage to grow in the laboratory. Here, we uncover the biology of some of the most common and yet-uncultured bacteria in freshwater environments using a mixed culture from Lake Grosse Fuchskuhle. From a single shotgun metagenome of a freshwater mixed culture of low complexity, we recovered four high-quality metagenome-assembled genomes (MAGs) for metabolic reconstruction. This analysis revealed the metabolic interconnectedness and niche partitioning of these naturally dominant bacteria. In particular, vitamin- and amino acid biosynthetic pathways were distributed unequally with a member of Crenarchaeota most likely being the sole producer of vitamin B12 in the mixed culture. Using coverage-based partitioning of the genes recovered from a single MAG intrapopulation metabolic complementarity was revealed pointing to 'social' interactions for the common good of populations dominating freshwater plankton. As such, our MAGs highlight the power of mixed cultures to extract naturally occurring 'interactomes' and to overcome our inability to isolate and grow the microbes dominating in nature.

Productivity and salinity structuring of the microplankton revealed by comparative freshwater metagenomics.

Little is known about the diversity and structuring of freshwater microbial communities beyond the patterns revealed by tracing their distribution in the landscape with common taxonomic markers such as the ribosomal RNA. To address this gap in knowledge, metagenomes from temperate lakes were compared to selected marine metagenomes. Taxonomic analyses of rRNA genes in these freshwater metagenomes confirm the previously reported dominance of a limited subset of uncultured lineages of freshwater bacteria, whereas Archaea were rare. Diversification into marine and freshwater microbial lineages was also reflected in phylogenies of functional genes, and there were also significant differences in functional beta-diversity. The pathways and functions that accounted for these differences are involved in osmoregulation, active transport, carbohydrate and amino acid metabolism. Moreover, predicted genes orthologous to active transporters and recalcitrant organic matter degradation were more common in microbial genomes from oligotrophic versus eutrophic lakes. This comparative metagenomic analysis allowed us to formulate a general hypothesis that oceanic- compared with freshwater-dwelling microorganisms, invest more in metabolism of amino acids and that strategies of carbohydrate metabolism differ significantly between marine and freshwater microbial communities.

Disturbance induced decoupling between host genetics and composition of the associated microbiome.

BACKGROUND: Studies of oyster microbiomes have revealed that a limited number of microbes, including pathogens, can dominate microbial communities in host tissues such as gills and gut. Much of the bacterial diversity however remains underexplored and unexplained, although environmental conditions and host genetics have been implicated. We used 454 next generation 16S rRNA amplicon sequencing of individually tagged PCR reactions to explore the diversity of bacterial communities in gill tissue of the invasive Pacific oyster Crassostrea gigas stemming from genetically differentiated beds under ambient outdoor conditions and after a multifaceted disturbance treatment imposing stress on the host. RESULTS: While the gill associated microbial communities in oysters were dominated by few abundant taxa (i.e. Sphingomonas, Mycoplasma) the distribution of rare bacterial groups correlated to relatedness between the hosts under ambient conditions. Exposing the host to disturbance broke apart this relationship by removing rare phylotypes thereby reducing overall microbial diversity. Shifts in the microbiome composition in response to stress did not result in a net increase in genera known to contain potentially pathogenic strains. CONCLUSION: The decrease in microbial diversity and the disassociation between population genetic structure of the hosts and their associated microbiome suggest that disturbance (i.e. stress) may play a significant role for the assembly of the natural microbiome. Such community shifts may in turn also feed back on the course of disease and the occurrence of mass mortality events in oyster populations.

Occupational exposure to inhalable pathogenic microorganisms in waste sorting.

This study assessed microorganisms in personal inhalable work air samples aiming to identify potential human pathogens, and correlate exposure to adverse health outcomes in waste workers. Full-shift personal exposure was measured in six different waste sorting plants. Microbial concentrations in inhalable air samples were analysed using MALDI-TOF MS for cultivable, and next generation sequencing (NGS) for non-cultivable microorganisms. Concentrations of bacterial and fungal CFUs varied substantially within and between waste sorting plants, ranging from no identifiable organisms to a maximum concentration in the order of 105 CFU/m3. Bacillus and Staphylococcus were among the most abundant bacterial genera, whilst fungal genera were dominated by Aspergillus and Penicillium. Approximately 15% of all identified species were human pathogens classified in risk group 2, whereas 7% belonged to risk group 1. Furthermore, significant correlations between concentrations of fungi in risk group 1 and self-reported adverse symptoms, such as wheezing were identified in exposed workers. The combination of culture-based methods and NGS facilitated the investigation of infectious microbial species with potential pathophysiological properties as well as non-infectious biological agents in inhalable work air samples and thereby contributed to the risk assessment of occupational exposure in waste sorting.

Trajectories of freshwater microbial genomics and greenhouse gas saturation upon glacial retreat.

Due to climate warming, ice sheets around the world are losing mass, contributing to changes across terrestrial landscapes on decadal time spans. However, landscape repercussions on climate are poorly constrained mostly due to limited knowledge on microbial responses to deglaciation. Here, we reveal the genomic succession from chemolithotrophy to photo- and heterotrophy and increases in methane supersaturation in freshwater lakes upon glacial retreat. Arctic lakes at Svalbard also revealed strong microbial signatures form nutrient fertilization by birds. Although methanotrophs were present and increased along lake chronosequences, methane consumption rates were low even in supersaturated systems. Nitrous oxide oversaturation and genomic information suggest active nitrogen cycling across the entire deglaciated landscape, and in the high Arctic, increasing bird populations serve as major modulators at many sites. Our findings show diverse microbial succession patterns, and trajectories in carbon and nitrogen cycle processes representing a positive feedback loop of deglaciation on climate warming.

Bacterial bioindicators enable biological status classification along the continental Danube river.

Despite the importance of bacteria in aquatic ecosystems and their predictable diversity patterns across space and time, biomonitoring tools for status assessment relying on these organisms are widely lacking. This is partly due to insufficient data and models to identify reliable microbial predictors. Here, we show metabarcoding in combination with multivariate statistics and machine learning allows to identify bacterial bioindicators for existing biological status classification systems. Bacterial beta-diversity dynamics follow environmental gradients and the observed associations highlight potential bioindicators for ecological outcomes. Spatio-temporal links spanning the microbial communities along the river allow accurate prediction of downstream biological status from upstream information. Network analysis on amplicon sequence veariants identify as good indicators genera Fluviicola, Acinetobacter, Flavobacterium, and Rhodoluna, and reveal informational redundancy among taxa, which coincides with taxonomic relatedness. The redundancy among bacterial bioindicators reveals mutually exclusive taxa, which allow accurate biological status modeling using as few as 2-3 amplicon sequence variants. As such our models show that using a few bacterial amplicon sequence variants from globally distributed genera allows for biological status assessment along river systems.

Detecting aquatic pathogens with field-compatible dried qPCR assays.

Field-ready qPCR assays with extended shelf-life support monitoring programs for emerging aquatic pathogens and enable quick conservation and management decisions. Here, we developed, validated, and tested the shelf-life of qPCR assays targeting Gyrodactylus salaris and Aphanomyces astaci with lyophilization and air-drying.

Drivers and variability of CO2:O2 saturation along a gradient from boreal to Arctic lakes.

Lakes are significant players for the global climate since they sequester terrestrially derived dissolved organic carbon (DOC), and emit greenhouse gases like CO2 to the atmosphere. However, the differences in environmental drivers of CO2 concentrations are not well constrained along latitudinal and thus climate gradients. Our aim here is to provide a better understanding of net heterotrophy and gas balance at the catchment scale in a set of boreal, sub-Arctic and high-Arctic lakes. We assessed water chemistry and concentrations of dissolved O2 and CO2, as well as the CO2:O2 ratio in three groups of lakes separated by steps of approximately 10 degrees latitude in South-Eastern Norway (near 60° N), sub-Arctic lakes in the northernmost part of the Norwegian mainland (near 70° N) and high-Arctic lakes on Svalbard (near 80° N). Across all regions, CO2 saturation levels varied more (6-1374%) than O2 saturation levels (85-148%) and hence CO2 saturation governed the CO2:O2 ratio. The boreal lakes were generally undersaturated with O2, while the sub-Arctic and high-Arctic lakes ranged from O2 saturated to oversaturated. Regardless of location, the majority of the lakes were CO2 supersaturated. In the boreal lakes the CO2:O2 ratio was mainly related to DOC concentration, in contrast to the sub-Arctic and high-Arctic localities, where conductivity was the major statistical determinant. While the southern part is dominated by granitic and metamorphic bedrock, the sub-Arctic sites are scattered across a range of granitic to sedimentary bed rocks, and the majority of the high-Arctic lakes are situated on limestone, resulting in contrasting lake alkalinities between the regions. DOC dependency of the CO2:O2 ratio in the boreal region together with low alkalinity suggests that in-lake heterotrophic respiration was a major source of lake CO2. Contrastingly, the conductivity dependency indicates that CO2 saturation in the sub-Arctic and high-Arctic lakes was to a large part explained by DIC input from catchment respiration and carbonate weathering.

Disturbance-based management of ecosystem services and disservices in partial nitritation-anammox biofilms.

The resistance and resilience provided by functional redundancy, a common feature of microbial communities, is not always advantageous. An example is nitrite oxidation in partial nitritation-anammox (PNA) reactors designed for nitrogen removal in wastewater treatment, where suppression of nitrite oxidizers like Nitrospira is sought. In these ecosystems, biofilms provide microhabitats with oxygen gradients, allowing the coexistence of aerobic and anaerobic bacteria. We designed a disturbance experiment where PNA biofilms, treating water from a high-rate activated sludge process, were constantly or intermittently exposed to anaerobic sidestream wastewater, which has been proposed to inhibit nitrite oxidizers. With increasing sidestream exposure we observed decreased abundance, alpha-diversity, functional versatility, and hence functional redundancy, among Nitrospira in the PNA biofilms, while the opposite patterns were observed for anammox bacteria within Brocadia. At the same time, species turnover was observed for aerobic ammonia-oxidizing Nitrosomonas populations. The different exposure regimens were associated with metagenomic assembled genomes of Nitrosomonas, Nitrospira, and Brocadia, encoding genes related to N-cycling, substrate usage, and osmotic stress response, possibly explaining the three different patterns by niche differentiation. These findings imply that disturbances can be used to manage the functional redundancy of biofilm microbiomes in a desirable direction, which should be considered when designing operational strategies for wastewater treatment.

Two different approaches of microbial community structure characterization in riverine epilithic biofilms under multiple stressors conditions: Developing molecular indicators.

Microbial communities are major players in the biogeochemical processes and ecosystem functioning of river networks. Despite their importance in the ecosystem, biomonitoring tools relying on prokaryotes are still lacking. Only a few studies have employed both metabarcoding and quantitative techniques such as catalysed reported deposition fluorescence in situ hybridization (CARD-FISH) to analyse prokaryotic communities of epilithic biofilms in river ecosystems. We intended to investigate the efficacy of both techniques in detecting changes in microbial community structure associated with environmental drivers. We report a significant correlation between the prokaryotic community composition and pH in rivers from two different geographical areas in Norway. Both CARD-FISH and metabarcoding data were following the pattern of the environmental variables, but the main feature distinguishing the community composition was the regional difference itself. Beta-dispersion analyses on both CARD-FISH abundance and metabarcoding data revealed higher accuracy of metabarcoding to differentiate regions and river systems. The CARD-FISH results showed high variability, even for samples within the same river, probably due to some unmeasured microscale ecological variability which we could not resolve. We also present a statistical method, which uses variation coefficient and overall prevalence of taxonomic groups, to detect possible biological indicators among prokaryotes using metabarcoding data. The development of new prokaryotic bioindicators would benefit from both techniques used in this study, but metabarcoding seems to be faster and more reliable than CARD-FISH for large scale bio-assessment.

Multiple thresholds and trajectories of microbial biodiversity predicted across browning gradients by neural networks and decision tree learning.

Ecological association studies often assume monotonicity such as between biodiversity and environmental properties although there is growing evidence that nonmonotonic relations dominate in nature. Here, we apply machine-learning algorithms to reveal the nonmonotonic association between microbial diversity and an anthropogenic-induced large-scale change, the browning of freshwaters, along a longitudinal gradient covering 70 boreal lakes in Scandinavia. Measures of bacterial richness and evenness (alpha-diversity) showed nonmonotonic trends in relation to environmental gradients, peaking at intermediate levels of browning. Depending on the statistical methods, variables indicative for browning could explain 5% of the variance in bacterial community composition (beta-diversity) when applying standard methods assuming monotonic relations and up to 45% with machine-learning methods taking non-monotonicity into account. This non-monotonicity observed at the community level was explained by the complex interchangeable nature of individual taxa responses as shown by a high degree of nonmonotonic responses of individual bacterial sequence variants to browning. Furthermore, the nonmonotonic models provide the position of thresholds and predict alternative bacterial diversity trajectories in boreal freshwater as a result of ongoing climate and land-use changes, which in turn will affect entire ecosystem metabolism and likely greenhouse gas production.

Phosphorus Availability Promotes Bacterial DOC-Mineralization, but Not Cumulative CO2-Production.

The current trend of increasing input of terrestrially derived dissolved organic carbon (DOC) to boreal freshwater systems is causing increased levels of carbon dioxide (CO2) supersaturation and degassing. Phosphorus (P) is often the most limiting nutrient for bacterial growth and would thus be expected to increase overall mineralization rates and CO2 production. However, high carbon (C) to P ratios of terrestrially derived DOC could also cause elevated cell-specific respiration of the excess C in heterotrophic bacteria. Using data from a survey of 75 Scandinavian lakes along an ecosystem gradient of DOC, we estimated in situ CO2 production rates. These rates showed a unimodal response with DOC-specific CO2 production negatively related to DOC:total phosphorus (TP) ratio, and a turning point at 5 mg C L-1, indicating higher DOC turnover rates in productive than in unproductive lakes. To further assess the dependency of bacterial respiration (BR) on DOC and P, we monitored CO2 production in incubations of water with a gradient of DOC crossed with two levels of inorganic P. Finally, we crossed DOC and P with a temperature gradient to test the temperature dependency of respiration rates [as oxygen (O2) consumption]. While total CO2 production seemed to be unaffected by P additions, respiration rates, and growth yields, as estimated by ribosomal gene copy numbers, suggest increased bacterial growth and decreased cell-specific respiration under non-limited P conditions. Respiration rates showed a sigmoid response to increasing DOC availability reaching a plateau at about 20 mg C L-1 of initial DOC concentrations. In addition to these P and DOC level effects, respiration rates responded in a non-monotonic fashion to temperature with an increase in respiration rates by a factor of 2.6 (±0.2) from 15 to 25°C and a decrease above 30°C. The combined results from the survey and experiments highlight DOC as the major determinant of CO2 production in boreal lakes, with P and temperature as significant modulators of respiration kinetics.

Diazotroph Genomes and Their Seasonal Dynamics in a Stratified Humic Bog Lake.

Aquatic N-fixation is generally associated with the growth and mass development of Cyanobacteria in nitrogen-deprived photic zones. However, sequenced genomes and environmental surveys suggest active aquatic N-fixation also by many non-cyanobacterial groups. Here, we revealed the seasonal variation and genomic diversity of potential N-fixers in a humic bog lake using metagenomic data and nif gene clusters analysis. Groups with diazotrophic operons were functionally divergent and included Cholorobi, Geobacter, Desulfobacterales, Methylococcales, and Acidobacteria. In addition to nifH (a gene that encodes the dinitrogenase reductase component of the molybdenum nitrogenase), we also identified sequences corresponding to vanadium and iron-only nitrogenase genes. Within the Chlorobi population, the nitrogenase (nifH) cluster was included in a well-structured retrotransposon. Furthermore, the presence of light-harvesting photosynthesis genes implies that anoxygenic photosynthesis may fuel nitrogen fixation under the prevailing low-irradiance conditions. The presence of rnf genes (related to the expression of H+/Na+-translocating ferredoxin: NAD+ oxidoreductase) in Methylococcales and Desulfobacterales suggests that other energy-generating processes may drive the costly N-fixation in the absence of photosynthesis. The highly reducing environment of the anoxic bottom layer of Trout Bog Lake may thus also provide a suitable niche for active N-fixers and primary producers. While future studies on the activity of these potential N-fixers are needed to clarify their role in freshwater nitrogen cycling, the metagenomic data presented here enabled an initial characterization of previously overlooked diazotrophs in freshwater biomes.

Corrigendum: Phosphorus Availability Promotes Bacterial DOC-Mineralization, but Not Cumulative CO2-Production.

[This corrects the article DOI: 10.3389/fmicb.2020.569879.].