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Abstract The role of microbial interactions and the underlying mechanisms that shape complex biofilm communities are poorly understood. Here we employ a microfluidic chip to represent porous subsurface environments and show that cooperative microbial interactions between free-living and biofilm-forming bacteria trigger active spatial segregation to promote their respective dominance in segregated microhabitats. During initial colonization, free-living and biofilm-forming microbes are segregated from the mixed planktonic inoculum to occupy the ambient fluid and grain surface. Contrary to spatial exclusion through competition, the active spatial segregation is induced by cooperative interactions which improves the fitness of both biofilm and planktonic populations. We further show that free-living Arthrobacter induces the surface colonization by scavenging the biofilm inhibitor, D-amino acids and receives benefits from the public goods secreted by the biofilm-forming strains. Collectively, our results reveal how cooperative microbial interactions may contribute to microbial coexistence in segregated microhabitats and drive subsurface biofilm community succession.

Here, Johansen et al. develop an approach, Phages from Metagenomics Binning (PHAMB), that allows the binning of thousands of viral genomes directly from bulk metagenomics data, while simultaneously enabling clustering of viral genomes into accurate taxonomic viral populations, unveiling viral-microbial host interactions in the gut.

Abstract Background Bacteria associated with plants can enhance the plants’ growth and resistance against phytopathogens. Today, growers aim to reduce the use of mineral fertilizers and pesticides. Since phytopathogens cause severe yield losses in crop production systems, biological alternatives gain more attention. Plant and also seed endophytes have the potential to influence the plant, especially seed-borne bacteria may express their beneficiary impact at initial plant developmental stages. In the current study, we assessed the endophytic seed microbiome of seven genetically diverse barley accessions by 16S rRNA gene amplicon sequencing and verified the in vitro plant beneficial potential of isolated seed endophytes. Furthermore, we investigated the impact of the barley genotype and its seed microbiome on the rhizosphere microbiome at an early growth stage by 16S rRNA gene amplicon sequencing. Results The plant genotype displayed a significant impact on the microbiota in both barley seed and rhizosphere. Consequently, the microbial alpha- and beta-diversity of the endophytic seed microbiome was highly influenced by the genotype. Interestingly, no correlation was observed between the endophytic seed microbiome and the single nucleotide polymorphisms of the seven genotypes. Unclassified members of Enterobacteriaceae were by far most dominant. Other abundant genera in the seed microbiome belonged to Curtobacterium, Paenibacillus, Pantoea, Sanguibacter and Saccharibacillus. Endophytes isolated from barley seeds were affiliated to dominant genera of the core seed microbiome, based on their 16S rRNA gene sequence. Most of these endophytic isolates produced in vitro plant beneficial secondary metabolites known to induce plant resistance. Conclusion Although barley accessions representing high genetic diversity displayed a genotype-dependent endophytic seed microbiome, a core seed microbiome with high relative abundances was identified. Endophytic isolates were affiliated to members of the core seed microbiome and many of them showed plant beneficial properties. We propose therefore that new breeding strategies should consider genotypes with high abundance of beneficial microbes.

Introduction Periprosthetic infection is one of the most severe complications following implant-based breast reconstruction affecting 5%–10% of the women. Currently, many surgeons apply antibiotics locally on the breast implant to reduce the risk of postoperative infection, but no randomised, placebo-controlled trials have tested the treatment’s efficacy.Methods and analysis The BREAST-AB trial (BREAST-AntiBiotics) is an investigator-initiated, multicentre, randomised, placebo-controlled, double-blind trial of local treatment with gentamicin, vancomycin and cefazolin on breast implants in women undergoing implant-based breast reconstruction. The trial drug consists of 80 mg gentamicin, 1 g vancomycin and 1 g cefazolin dissolved in 500 mL of isotonic saline. The placebo solution consists of 500 mL isotonic saline. The trial drug is used to wash the dissected tissue pocket and the breast implant prior to insertion. The primary outcome is all-cause explantation of the breast implant within 180 days after the breast reconstruction surgery. This excludes cases where the implant is replaced with a new permanent implant, for example, for cosmetic reasons. Key long-term outcomes include capsular contracture and quality of life. The trial started on 26 January 2021 and is currently recruiting.Ethics and dissemination The trial was approved by the Regional Ethics Committee of the Capital Region (H-20056592) on 1 January 2021 and the Danish Medicines Agency (2020070016) on 2 August 2020. The main paper will include the primary and secondary outcomes and will be submitted to an international peer-reviewed journal.Trial registration number NCT04731025.

Abstract Studying respiratory illness-specific microbial signatures and their interaction with other micro-residents could provide a better understanding of lung microbial ecology. Each respiratory illness has a specific disease etiology, however, so far no study has revealed disease—specific microbial markers. The present study was designed to determine disease-specific microbial features and their interactions with other residents in chronic obstructive pulmonary diseases (stable and exacerbated), sarcoidosis, and interstitial lung diseases. Broncho-alveolar lavage samples (n = 43) were analyzed by SSU rRNA gene sequencing to study the alveolar microbiome in these diseases. A predominance of Proteobacteria followed by Firmicutes, Bacteroidetes, Actinobacteria, and Fusobacteria was observed in all the disease subsets. Shannon diversity was significantly higher in stable COPD when compared to exacerbated chronic obstructive pulmonary disease (ECOPD) (p = 0.0061), and ILD patient samples (p = 0.037). The lung microbiome of the patients with stable COPD was more diverse in comparison to ECOPD and ILD patients (p 4). Species network analysis indicated a significant correlation (p

Mobile genetic elements have evolved anti-CRISPR (Acr) proteins to bypass the immunity provided by prokaryotic CRISPR–Cas systems. Here, the authors identify 11 type I Acrs encoded on mobile genetic elements, and show that acr loci neighborhoods can be used to discover inhibitors of other bacterial defense systems.

Abstract Background From early life, children are exposed to a multitude of environmental exposures, which may be of crucial importance for healthy development. Here, the environmental microbiota may be of particular interest as it represents the interface between environmental factors and the child. As infants in modern societies spend a considerable amount of time indoors, we hypothesize that the indoor bed dust microbiota might be an important factor for the child and for the early colonization of the airway microbiome. To explore this hypothesis, we analyzed the influence of environmental exposures on 577 dust samples from the beds of infants together with 542 airway samples from the Copenhagen Prospective Studies on Asthma in Childhood2010 cohort. Results Both bacterial and fungal community was profiled from the bed dust. Bacterial and fungal diversity in the bed dust was positively correlated with each other. Bacterial bed dust microbiota was influenced by multiple environmental factors, such as type of home (house or apartment), living environment (rural or urban), sex of siblings, and presence of pets (cat and/or dog), whereas fungal bed dust microbiota was majorly influenced by the type of home (house or apartment) and sampling season. We further observed minor correlation between bed dust and airway microbiota compositions among infants. We also analyzed the transfer of microbiota from bed dust to the airway, but we did not find evidence of transfer of individual taxa. Conclusions Current study explores the influence of environmental factors on bed dust microbiota (both bacterial and fungal) and its correlation with airway microbiota (bacterial) in early life using high-throughput sequencing. Our findings demonstrate that bed dust microbiota is influenced by multiple environmental exposures and could represent an interface between environment and child. Video Abstract

Summary Microbial conversion through enzymatic reactions has received a lot of attention as a cost‐effective and environmentally friendly way to recover amino acids and short peptides from keratin materials. However, accurate assessment of microbial keratinase activity is not straightforward, and current available methods lack sensitivity and standardization. Here, we suggest an optimized Azokeratin assay, with substrate generated directly from azo‐dyed raw keratin material. We introduced supernatant filtration in the protocol for optimal stopping of keratinase reactions instead of the widely used trichloroacetic acid (TCA), as it generated biases and impacted the sensitivity. We furthermore suggest a method for standardization of keratinase activity signals using proteinase K, a well‐known keratinase, as a reference enabling reproducibility between studies. Lastly, we evaluated our developed method with several bacterial isolates through benchmarking against a commercial assay (Keratin Azure). Under different setups, the Azokeratin method was more sensitive than commonly used Keratin Azure‐based assays (3‐fold). We argue that this method could be applied with any type of keratin substrate, enabling more robust and sensitive results which can be used for further comparison with other studies, thus representing an important progress within the field of microbial keratin degradation.

Beneficial bacteria in the rhizosphere are known to trigger faster and stronger plant immune responses to biotic and abiotic stressors. In the present study, we aimed to test the hypothesis that a rhizosphere microbiome transplant (RMT) may improve the immune response and reduce the disease rates of barley (Hordeum vulgare). This hypothesis was tested in a greenhouse system with the powdery mildew-causing fungus Blumeria graminis f. sp. hordei (Bgh). Detached rhizosphere microbiome from barley grown in a field soil was transplanted to barley seedlings grown in potting soil with reduced microbial diversity. Saline-treated plants served as control. At the three-leaf stage, barley was infected with Bgh. Decreased susceptibility to Bgh was observed for barley treated with the RMT as displayed by lower Bgh pustule counts in a detached leaf assay. A trend toward enhanced relative transcript abundances of the defense-related genes PR1b and PR17b was observed in leaves, 24 h after the Bgh challenge, when compared to the control. Moreover, 10 days after the Bgh challenge, the barley rhizosphere microbiome was harvested and analyzed by sequencing of 16S rRNA gene amplicons. The microbial community composition was significantly influenced by the RMT and displayed higher microbial diversity compared to the control. Furthermore, microbial beta-diversity and predicted functional profiles revealed a treatment-dependent clustering. Bacterial isolates from the RMT showed in vitro plant beneficial traits related to induced resistance. Our results showed that transplantation of a rhizosphere microbiome could be a sustainable strategy to improve the health of plants grown in potting soil with low microbial diversity under greenhouse conditions.

The toolbox available for microbiologists to study interspecies interactions is rapidly growing, and with continuously more advanced instruments, we are able to expand our knowledge on establishment and function of microbial communities. However, unravelling molecular interspecies interactions in complex biological systems remains a challenge, and interactions are therefore often studied in simplified communities. Here we perform an in-depth characterization of an observed interspecies interaction between two co-isolated bacteria, Xanthomonas retroflexus and Paenibacillus amylolyticus. Using microsensor measurements for mapping the chemical environment, we show how X. retroflexus promoted an alkalization of its local environment through degradation of amino acids and release of ammonia. When the two species were grown in proximity, the modified local environment induced a morphological change and growth of P. amylolyticus followed by sporulation. 2D spatial metabolomics enabled visualization and mapping of the degradation of oligopeptide structures by X. retroflexus and morphological changes of P. amylolyticus through e.g. the release of membrane-associated metabolites. Proteome analysis and microscopy were used to validate the shift from vegetative growth towards sporulation. In summary, we demonstrate how environmental profiling by combined application of microsensor, microscopy, metabolomics and proteomics approaches can reveal growth and sporulation promoting effects resulting from interspecies interactions.

Application of agrochemicals and mechanization enabled increasing agricultural productivity yet caused various environmental and soil health-related problems. Agricultural practices affect soil microorganisms, which are the key players of many ecosystem processes. However, less is known about whether this effect differs between time points. Therefore, soil was sampled in winter (without crop) and in summer (in the presence of maize) from a long-term field experiment (LTE) in Bernburg (Germany) managed either under cultivator tillage (CT) or moldboard plow (MP) in combination with either intensive nitrogen (N)-fertilization and pesticides (Int) or extensive reduced N-fertilization without fungicides (Ext), respectively. High-throughput sequencing of 16S rRNA gene and fungal ITS2 amplicons showed that changes in the microbial community composition were correlated to differences in soil chemical properties caused by tillage practice. Microbial communities of soils sampled in winter differed only depending on the tillage practice while, in summer, also a strong effect of the fertilization intensity was observed. A small proportion of microbial taxa was shared between soils from the two sampling times, suggesting the existence of a stable core microbiota at the LTE. In general, taxa associated with organic matter decomposition (such as Actinobacteria, Bacteroidetes, Rhizopus, and Exophiala) had a higher relative abundance under CT. Among the taxa with significant changes in relative abundances due to different long-term agricultural practices were putative pathogenic (e.g., Gibellulopsis and Gibberella) and beneficial microbial genera (e.g., Chitinophagaceae, Ferruginibacter, and Minimedusa). In summary, this study suggests that the effects of long-term agricultural management practices on the soil microbiota are influenced by the soil sampling time, and this needs to be kept in mind in future studies for the interpretation of field data.

Here, the authors present the results of a mother–child cohort randomized clinical trial of n-3 LCPUFA and vitamin D maternal supplementation, finding an association between supplement-induced microbiota changes in infant airways at age 1-month but not the infant fecal or maternal vaginal microbiome.

Here, Thorsen et al. examine the microbiota during the first three months of life in a cohort of 700 children and find that microbial diversity and the relative abundances of Veillonella and Prevotella in the airways at one month of age are associated with topical immune mediators and asthma by age 6 years.

Plasmids carrying metal resistance genes (MRGs) have been suggested to be key ecological players in the adaptation of metal-impacted microbial communities, making them promising drivers of bio-remediation processes. However, the impact of metals on plasmid-mediated spread of MRGs through selection, plasmid loss, and transfer is far from being fully understood. In the present study, we used two-member bacterial communities to test the impact of lead on the dispersal of the IncP plasmid pKJK5 from a Pseudomonas putida KT2440 plasmid donor and two distinct recipients, Variovorax paradoxus B4 or Delftia acidovorans SPH-1 after 4 and 10 days of mating. Two versions of the plasmid were used, carrying or not carrying the lead resistance pbrTRABCD operon, to assess the importance of fitness benefit and conjugative potential for the dispersal of the plasmid. The spread dynamics of metal resistance conveyed by the conjugative plasmid were dependent on the recipient and the lead concentration: For V. paradoxus, the pbr operon did not facilitate neither lead resistance nor variation in plasmid spread. The growth gain brought by the pbr operon to D. acidovorans SPH-1 and P. putida KT2440 at 1 mM Pb enhanced the spread of the plasmid. At 1.5 mM Pb after 4 days, the proteomics results revealed an oxidative stress response and an increased abundance of pKJK5-encoded conjugation and partitioning proteins, which most likely increased the transfer of the control plasmid to D. acidovorans SPH-1 and ensured plasmid maintenance. As a consequence, we observed an increased spread of pKJK5-gfp. Conversely, the pbr operon reduced the oxidative stress response and impeded the rise of conjugation- and partitioning-associated proteins, which slowed down the spread of the pbr carrying plasmid. Ultimately, when a fitness gain was recorded in the recipient strain, the spread of MRG-carrying plasmids was facilitated through positive selection at an intermediate metal concentration, while a high lead concentration induced oxidative stress with positive impacts on proteins encoding plasmid conjugation and partitioning.

This cohort study aimed to characterize the oral microbiome of children with CLP, from two different age groups, and evaluate the effect of supervised or unsupervised toothbrushing on the microbiome of the cleft over time. Swab samples were collected from the cleft area at three different time points (A; no brushing, B; after 15 days and C; after 30 days) and were analyzed using next-generation sequencing to determine the microbial composition and diversity in these time points. Overall, brushing significantly decreased the abundance of the genera Alloprevotella and Leptotrichia in the two age groups examined, and for Alloprevotella this decrease was more evident for children (2–6 years old). In the preteen group (7–12 years old), a significant relative increase of the genus Rothia was observed after brushing. In this study, the systematic brushing over a period of thirty days also resulted in differences at the intra-individual bacterial richness.

Abstract Objective To describe the prevalence of lifetime psychiatric hospital diagnoses among men registered in an outpatient alcohol clinic and compare the prevalence with matched controls. To assess temporality of alcohol use disorder (AUD) diagnoses and another psychiatric hospital diagnosis and examine the prevalence of lifetime psychiatric hospital diagnoses according to this temporal order. Methods The study included 8,412 Danish men registered in an outpatient alcohol clinic, and 8,412 unregistered controls from the Danish Conscription Database matched on birth date, lifespan, intelligence and draft board district. Information on first outpatient AUD treatment was retrieved from the Copenhagen Alcohol Cohort. Information on lifetime psychiatric hospital diagnoses was retrieved from national Danish psychiatric registers and based on the International Classification of Diseases the 8th and 10th Revisions. Prevalence estimates of lifetime psychiatric hospital diagnoses were compared with odds ratios (OR) between men registered in an outpatient alcohol clinic and the control population. Results Among men registered in an outpatient alcohol clinic, 66.6% had a lifetime psychiatric hospital diagnosis. In total, 8.6% had neuroses and anxiety disorders, while 25.3% had personality disorders. The OR of a lifetime psychiatric hospital diagnosis was 9.77 (95%CI: 8.87–10.75) when comparing men registered in an outpatient alcohol clinic with the control population. Among men with a lifetime psychiatric hospital diagnosis, 42.8% was registered with another psychiatric hospital diagnosis before registration with an AUD diagnosis. Conclusion Among men with a lifetime psychiatric hospital diagnosis, AUD is rarely diagnosed without psychiatric comorbidity at first‐time admissions to psychiatric hospital departments.

Premature birth, especially if born before week 32 of gestation, is associated with increased risk of neonatal morbidity and mortality. Prophylactic use of probiotics has been suggested to protect preterm infants via supporting a healthy gut microbiota (GM) development, but the suggested strains and doses vary between studies. In this study, we profiled the GM of 5, 10 and 30-day fecal samples from two cohorts of preterm neonates (born

In the barren semiarid landscape individual plant species improve soil structure, reducing the erosion risks, whereby microaggregates form the most fundamental soil structural components. Extracellular polymeric substances (EPS) are considered an important glue determining aggregation in addition to inorganic binding agents such as carbonates. However, the role of the prokaryotic community in EPS formation and consequently for microaggregation in natural environments is not substantially clarified yet. EPS should be particularly important under semiarid conditions as it forms a protection mechanism of the prokaryotes against desiccation. Therefore, we examined the influence of the prokaryotic community on soil EPS content and subsequently on soil microaggregation in semiarid grasslands, depending on the parent material, common plant species and the distance to the plant. Soil samples were taken over a distance gradient from two major semiarid grassland plant species in Southern Spain, the legume shrub Anthyllis cytisoides and the grass tussock Macrochloa tenacissima, to the surrounding bare soil at two sites, rich and poor in carbonates. Total community DNA and EPS were extracted, followed by quantification of EPS-saccharide, bacterial abundance and examination of the prokaryotic community composition. Further, the particle size distribution of the microaggregate fraction was determined as an indication of microaggregation. We found that the overall prokaryotic community composition differed between the two sites, but not between plant species. Nonetheless, a link between the community composition and EPS content was established, whereby soil organic matter (OM) seems to be a regulating factor as increasing soil OM contents resulted in more EPS-saccharide. Furthermore, microaggregation was enhanced by the canopy, especially at the edge of Macrochloa tussocks. Contrary to the expectation that increasing inorganic C contents would diminish importance of EPS, the parent material richest in inorganic C resulted in a significant effect of EPS-saccharide contents on microaggregation according to the structural equation model. For the inorganic C poor site, EPS-saccharide had no observed direct effect on microaggregation. Based on our results we conclude that the availability of decomposable OM influences the prokaryotic community composition and thereby triggers EPS production whereas large contents of polyvalent cations promote the stabilizing effect of EPS on microaggregates.

Abstract Background It remains unclear whether adaptation and changes in diversity associated to a long-term perturbation are sufficient to ensure functional resilience of soil microbial communities. We used RNA-based approaches (16S rRNA gene transcript amplicon coupled to shotgun mRNA sequencing) to study the legacy effects of a century-long soil copper (Cu) pollution on microbial activity and composition, as well as its effect on the capacity of the microbial community to react to temporal fluctuations. Results Despite evidence of microbial adaptation (e.g., iron homeostasis and avoidance/resistance strategies), increased heterogeneity and richness loss in transcribed gene pools were observed with increasing soil Cu, together with an unexpected predominance of phage mRNA signatures. Apparently, phage activation was either triggered directly by Cu, or indirectly via enhanced expression of DNA repair/SOS response systems in Cu-exposed bacteria. Even though total soil carbon and nitrogen had accumulated with increasing Cu, a reduction in temporally induced mRNA functions was observed. Microbial temporal response groups (TRGs, groups of microbes with a specific temporal response) were heavily affected by Cu, both in abundance and phylogenetic composition. Conclusion Altogether, results point toward a Cu-mediated “decoupling” between environmental fluctuations and microbial activity, where Cu-exposed microbes stopped fulfilling their expected contributions to soil functioning relative to the control. Nevertheless, some functions remained active in February despite Cu, concomitant with an increase in phage mRNA signatures, highlighting that somehow, microbial activity is still happening under these adverse conditions.

Colonization of commensal bacteria is thought to impact immune development, especially in the earliest years of life. Here, the authors show, by analyzing the development of the gut microbiome of 690 children, that microbial composition at the age of 1 year is associated with asthma diagnosed in the first 5 years of life.

Abstract Microbial biofilms are omnipresent in nature and relevant to a broad spectrum of industries ranging from bioremediation and food production to biomedical applications. To date little is understood about how multi-species biofilm communities develop and function on a molecular level, due to the complexity of these biological systems. Here we apply a meta-proteomics approach to investigate the mechanisms influencing biofilm formation in a model consortium of four bacterial soil isolates; Stenotrophomonas rhizophila, Xanthomonas retroflexus, Microbacterium oxydans and Paenibacillus amylolyticus. Protein abundances in community and single species biofilms were compared to describe occurring inter-species interactions and the resulting changes in active metabolic pathways. To obtain full taxonomic resolution between closely related species and empower correct protein quantification, we developed a novel pipeline for generating reduced reference proteomes for spectral database searches. Meta-proteomics profiling indicated that community development is dependent on cooperative interactions between community members facilitating cross-feeding on specific amino acids. Opposite regulation patterns of fermentation and nitrogen pathways in Paenibacillus amylolyticus and Xanthomonas retroflexus may, however, indicate that competition for limited resources also affects community development. Overall our results demonstrate the multitude of pathways involved in biofilm formation in mixed communities.

Although organic matter may accumulate sometimes (e.g. lignocellulose in peat bog), most natural biodegradation processes are completed until full mineralization. Such transformations are often achieved by the concerted action of communities of interacting microbes, involving different species each performing specific tasks. These interactions can give rise to novel "community-intrinsic" properties, through e.g. activation of so-called "silent genetic pathways" or synergistic interplay between microbial activities and functions. Here we studied the microbial community-based degradation of keratin, a recalcitrant biological material, by four soil isolates, which have previously been shown to display synergistic interactions during biofilm formation; Stenotrophomonas rhizophila, Xanthomonas retroflexus, Microbacterium oxydans and Paenibacillus amylolyticus. We observed enhanced keratin weight loss in cultures with X. retroflexus, both in dual and four-species co-cultures, as compared to expected keratin degradation by X. retroflexus alone. Additional community intrinsic properties included accelerated keratin degradation rates and increased biofilm formation on keratin particles. Comparison of secretome profiles of X. retroflexus mono-cultures to co-cultures revealed that certain proteases (e.g. serine protease S08) were significantly more abundant in mono-cultures, whereas co-cultures had an increased abundance of proteins related to maintaining the redox environment, e.g. glutathione peroxidase. Hence, one of the mechanisms related to the community intrinsic properties, leading to enhanced degradation from co-cultures, might be related to a switch from sulfitolytic to proteolytic functions between mono- and co-cultures, respectively.

The capacity of microbes to degrade recalcitrant materials has been extensively explored for environmental remediation and industrial production. Significant achievements have been made with single strains, but focus is now going toward the use of microbial consortia owning to their functional stability and efficiency. However, assembly of simplified microbial consortia (SMC) from complex environmental communities is still far from trivial due to large diversity and the effect of biotic interactions. Here we propose a strategy, based on enrichment and dilution-to-extinction cultures, to construct SMC with reduced diversity for degradation of keratinous materials. Serial dilutions were performed on a keratinolytic microbial consortium pre-enriched from a soil sample, monitoring the dilution effect on community growth and enzymatic activities. An appropriate dilution regime (10–9) was selected to construct a SMC library from the enriched microbial consortium. Further sequencing analysis and keratinolytic activity assays demonstrated that obtained SMC displayed actual reduced microbial diversity, together with various taxonomic composition, and biodegradation capabilities. More importantly, several SMC possessed equivalent levels of keratinolytic efficiency compared to the initial consortium, showing that simplification can be achieved without loss of function and efficiency. This methodology is also applicable to other types of recalcitrant material degradation involving microbial consortia, thus considerably broadening its application scope.

Ralstonia solanacearum (biovar2, race3) is the causal agent of bacterial wilt and this quarantine phytopathogen is responsible for massive losses in several commercially important crops. Biological control of this pathogen might become a suitable plant protection measure in areas where R. solanacearum is endemic. Two bacterial strains, Bacillus velezensis (B63) and Pseudomonas fluorescens (P142) with in vitro antagonistic activity toward R. solanacearum (B3B) were tested for rhizosphere competence, efficient biological control of wilt symptoms on greenhouse-grown tomato, and effects on the indigenous rhizosphere prokaryotic communities. The population densities of B3B and the antagonists were estimated in rhizosphere community DNA by selective plating, real-time quantitative PCR, and R. solanacearum-specific fliC PCR-Southern blot hybridization. Moreover, we investigated how the pathogen and/or the antagonists altered the composition of the tomato rhizosphere prokaryotic community by 16S rRNA gene amplicon sequencing. B. velezensis (B63) and P. fluorescens (P142)-inoculated plants showed drastically reduced wilt disease symptoms, accompanied by significantly lower abundance of the B3B population compared to the non-inoculated pathogen control. Pronounced shifts in prokaryotic community compositions were observed in response to the inoculation of B63 or P142 in the presence or absence of the pathogen B3B and numerous dynamic taxa were identified. Confocal laser scanning microscopy (CLSM) visualization of the gfp-tagged antagonist P142 revealed heterogeneous colonization patterns and P142 was detected in lateral roots, root hairs, epidermal cells, and within xylem vessels. Although competitive niche exclusion cannot be excluded, it is more likely that the inoculation of P142 or B63 and the corresponding microbiome shifts primed the plant defense against the pathogen B3B. Both inoculants are promising biological agents for efficient control of R. solanacearum under field conditions.

Purpose: To compare three region-of-interest (ROI) settings in the assessment of ADC in a clinical trial, and to evaluate the effectiveness of ADC in assessing therapy-induced changes and predicting clinical outcomes. Methods: In a 52-week clinical trial involving patients with axial spondyloarthritis, mean sacroiliac joint (SIJ) ADC measurements using structured, lesion-based, and index-lesion ROI-settings were assessed at baseline and weeks 4, 16, and 52. Variation among the three ROI-settings, correlations with Spondyloarthritis Research Consortium of Canada (SPARCC)-bone marrow edema (BME) SIJ inflammation indices, standardized response means (SRMs), and effectiveness in predicting clinical outcomes were analyzed. Results: Forty of the 53 patients had at least one assessable SIJ lesion on ADC at baseline. The mean of the structured ROI ADC (ADCstruc) was 230 μmm2/s (standard deviation [SD] = 120). This was significantly lower (p 0.92) and ADCindex (SRM > 0.87) while moderate for ADCstruc (SRM:0.54-0.67). Baseline ADC and changes in ADC did not predict clinical outcomes. Conclusions: Lesion-based and index-lesion ROI ADC could both be used to evaluate the effectiveness of tumor necrosis factor inhibitor therapy. None of the methods could predict clinical outcomes.

ABSTRACT Nitrification, the oxidative process converting ammonia to nitrite and nitrate, is driven by microbes and plays a central role in the global nitrogen cycle. Our earlier investigations based on 16S rRNA and amoA amplicon analysis, amoA quantitative PCR and metagenomics of groundwater-fed biofilters indicated a consistently high abundance of comammox Nitrospira. Here, we hypothesized that these nonclassical nitrifiers drive ammonia-N oxidation. Hence, we used DNA and RNA stable isotope probing (SIP) coupled with 16S rRNA amplicon sequencing to identify the active members in the biofilter community when subjected to a continuous supply of NH4+ or NO2− in the presence of 13C-HCO3− (labeled) or 12C-HCO3− (unlabeled). Allylthiourea (ATU) and sodium chlorate were added to inhibit autotrophic ammonia- and nitrite-oxidizing bacteria, respectively. Our results confirmed that lineage II Nitrospira dominated ammonia oxidation in the biofilter community. A total of 78 (8 by RNA-SIP and 70 by DNA-SIP) and 96 (25 by RNA-SIP and 71 by DNA-SIP) Nitrospira phylotypes (at 99% 16S rRNA sequence similarity) were identified as complete ammonia- and nitrite-oxidizing, respectively. We also detected significant HCO3− uptake by Acidobacteria subgroup10, Pedomicrobium, Rhizobacter, and Acidovorax under conditions that favored ammonia oxidation. Canonical Nitrospira alone drove nitrite oxidation in the biofilter community, and activity of archaeal ammonia-oxidizing taxa was not detected in the SIP fractions. This study provides the first in situ evidence of ammonia oxidation by comammox Nitrospira in an ecologically relevant complex microbiome. IMPORTANCE With this study we provide the first in situ evidence of ecologically relevant ammonia oxidation by comammox Nitrospira in a complex microbiome and document an unexpectedly high H13CO3− uptake and growth of proteobacterial and acidobacterial taxa under ammonia selectivity. This finding raises the question of whether comammox Nitrospira is an equally important ammonia oxidizer in other environments.

Microbial cells in bioprocesses are usually described with averaged parameters. But in fact, single cells within populations vary greatly in characteristics such as stress resistance, especially in response to carbon source gradients. Our aim was to introduce tools to quantify population heterogeneity in bioprocesses using a combination of reporter strains, flow cytometry, and easily comprehensible parameters. We calculated mean, mode, peak width, and coefficient of variance to describe distribution characteristics and temporal shifts in fluorescence intensity. The skewness and the slope of cumulative distribution function plots illustrated differences in distribution shape. These parameters are person-independent and precise. We demonstrated this by quantifying growth-related population heterogeneity of Saccharomyces cerevisiae and Escherichia coli reporter strains in steady-state of aerobic glucose-limited chemostat cultures at different dilution rates and in response to glucose pulses. Generally, slow-growing cells showed stronger responses to glucose excess than fast-growing cells. Cell robustness, measured as membrane integrity after exposure to freeze-thaw treatment, of fast-growing cells was strongly affected in subpopulations of low membrane robustness. Glucose pulses protected subpopulations of fast-growing but not slower-growing yeast cells against membrane damage. Our parameters could successfully describe population heterogeneity, thereby revealing physiological characteristics that might have been overlooked during traditional averaged analysis.

Manure application to agricultural soil introduces antibiotic residues and increases the abundance of antibiotic-resistant bacteria (ARB) carrying antibiotic resistance genes (ARGs), often located on mobile genetic elements (MGEs). The rhizosphere is regarded as a hotspot of microbial activity and gene transfer, which can alter and prolong the effects of organic fertilizers containing antibiotics. However, not much is known about the influence of plants on the effects of doxycycline applied to soil via manure. In this study, the effects of manure spiked with or without doxycycline on the prokaryotic community composition as well as on the relative abundance of ARGs and MGEs in lettuce rhizosphere and bulk soil were investigated by means of a polyphasic cultivation-independent approach. Samples were taken 42 days after manure application, and total community DNA was extracted. Besides a pronounced manure effect, doxycycline spiking caused an additional enrichment of ARGs and MGEs. High-throughput quantitative PCR revealed an increase in tetracycline, aminoglycoside, and macrolide–lincosamide–streptogramin B (MLSB) resistance genes associated with the application of manure spiked with doxycycline. This effect was unexpectedly lower in the rhizosphere than in bulk soil, suggesting a faster dissipation of the antibiotic and a more resilient prokaryotic community in the rhizosphere. Interestingly, the tetracycline resistance gene tetA(P) was highly enriched in manure-treated bulk soil and rhizosphere, with highest values observed in doxycycline-treated bulk soil, concurring with an enrichment of Clostridia. Thus, the gene tetA(P) might be a suitable marker of soil contamination by ARB, ARGs, and antibiotics of manure origin. These findings illustrate that the effects of manure and doxycycline on ARGs and MGEs differ between rhizosphere and bulk soil, which needs to be considered when assessing risks for human health connected to the spread of ARGs in the environment.