Reduced Glycolysis and Cytotoxicity in Staphylococcus aureus Isolates from Chronic Rhinosinusitis as Strategies for Host Adaptation.

Chronic rhinosinusitis (CRS) is a multifactorial infection of the nasal cavity and sinuses. In this study, nasal swabs from control donors (N = 128) and patients with CRS (N = 246) were analysed. Culture methods and metagenomics revealed no obvious differences in the composition of the bacterial communities between the two groups. However, at the functional level, several metabolic pathways were significantly enriched in the CRS group compared to the control group. Pathways such as carbohydrate transport metabolism, ATP synthesis, cofactors and vitamins, photosynthesis and transcription were highly enriched in CRS. In contrast, pathways related to lipid metabolism were more representative in the control microbiome. As S. aureus is one of the main species found in the nasal cavity, staphylococcal isolates from control and CRS samples were analysed by microarray and functional assays. Although no significant genetic differences were detected by microarray, S. aureus from CRS induced less cytotoxicity to lung cells and lower rates of glycolysis in host cells than control isolates. These results suggest the differential modulation of staphylococcal virulence by the environment created by other microorganisms and their interactions with host cells in control and CRS samples. These changes were reflected in the differential expression of cytokines and in the expression of Agr, the most important quorum-sensing regulator of virulence in S. aureus. In addition, the CRS isolates remained stable in their cytotoxicity, whereas the cytotoxic activity of S. aureus isolated from control subjects decreased over time during in vitro passage. These results suggest that host factors influence the virulence of S. aureus and promote its adaptation to the nasal environment during CRS.

BI 456906: Discovery and preclinical pharmacology of a novel GCGR/GLP-1R dual agonist with robust anti-obesity efficacy.

OBJECTIVE: Obesity and its associated comorbidities represent a global health challenge with a need for well-tolerated, effective, and mechanistically diverse pharmaceutical interventions. Oxyntomodulin is a gut peptide that activates the glucagon receptor (GCGR) and glucagon-like peptide-1 receptor (GLP-1R) and reduces bodyweight by increasing energy expenditure and reducing energy intake in humans. Here we describe the pharmacological profile of the novel glucagon receptor (GCGR)/GLP-1 receptor (GLP-1R) dual agonist BI 456906. METHODS: BI 456906 was characterized using cell-based in vitro assays to determine functional agonism. In vivo pharmacological studies were performed using acute and subchronic dosing regimens to demonstrate target engagement for the GCGR and GLP-1R, and weight lowering efficacy. RESULTS: BI 456906 is a potent, acylated peptide containing a C18 fatty acid as a half-life extending principle to support once-weekly dosing in humans. Pharmacological doses of BI 456906 provided greater bodyweight reductions in mice compared with maximally effective doses of the GLP-1R agonist semaglutide. BI 456906's superior efficacy is the consequence of increased energy expenditure and reduced food intake. Engagement of both receptors in vivo was demonstrated via glucose tolerance, food intake, and gastric emptying tests for the GLP-1R, and liver nicotinamide N-methyltransferase mRNA expression and circulating biomarkers (amino acids, fibroblast growth factor-21) for the GCGR. The dual activity of BI 456906 at the GLP-1R and GCGR was supported using GLP-1R knockout and transgenic reporter mice, and an ex vivo bioactivity assay. CONCLUSIONS: BI 456906 is a potent GCGR/GLP-1R dual agonist with robust anti-obesity efficacy achieved by increasing energy expenditure and decreasing food intake.

An integrative understanding of the large metabolic shifts induced by antibiotics in critical illness.

Antibiotics are commonly used in the Intensive Care Unit (ICU); however, several studies showed that the impact of antibiotics to prevent infection, multi-organ failure, and death in the ICU is less clear than their benefit on course of infection in the absence of organ dysfunction. We characterized here the compositional and metabolic changes of the gut microbiome induced by critical illness and antibiotics in a cohort of 75 individuals in conjunction with 2,180 gut microbiome samples representing 16 different diseases. We revealed an "infection-vulnerable" gut microbiome environment present only in critically ill treated with antibiotics (ICU+). Feeding of Caenorhabditis elegans with Bifidobacterium animalis and Lactobacillus crispatus, species that expanded in ICU+ patients, revealed a significant negative impact of these microbes on host viability and developmental homeostasis. These results suggest that antibiotic administration can dramatically impact essential functional activities in the gut related to immune responses more than critical illness itself, which might explain in part untoward effects of antibiotics in the critically ill.

A suite of complementary biocontrol traits allows a native consortium of root-associated bacteria to protect their host plant from a fungal sudden-wilt disease.

The beneficial effects of plant--bacterial interactions in controlling plant pests have been extensively studied with single bacterial isolates. However, in nature, bacteria interact with plants in multitaxa consortia, systems which remain poorly understood. Previously, we demonstrated that a consortium of five native bacterial isolates protected their host plant Nicotiana attenuata from a sudden wilt disease. Here we explore the mechanisms behind the protection effect against the native pathosystem. Three members of the consortium, Pseudomonas azotoformans A70, P. frederiksbergensis A176 and Arthrobacter nitroguajacolicus E46, form biofilms when grown individually in vitro, and the amount of biofilm increased synergistically in the five-membered consortium, including two Bacillus species, B. megaterium and B. mojavensis. Fluorescence in situ hybridization and scanning electron microscopy in planta imaging techniques confirmed biofilm formation and revealed locally distinct distributions of the five bacterial strains colonizing different areas on the plant-root surface. One of the five isolates, K1 B. mojavensis produces the antifungal compound surfactin, under in vitro and in vivo conditions, clearly inhibiting fungal growth. Furthermore, isolates A70 and A176 produce siderophores under in vitro conditions. Based on these results we infer that the consortium of five bacterial isolates protects its host against fungal phytopathogens via complementary traits. The study should encourage researchers to create synthetic communities from native strains of different genera to improve bioprotection against wilting diseases.

Specificity of root microbiomes in native-grown Nicotiana attenuata and plant responses to UVB increase Deinococcus colonization.

Plants recruit microbial communities from the soil in which they germinate. Our understanding of the recruitment process and the factors affecting it is still limited for most microbial taxa. We analysed several factors potentially affecting root microbiome structure - the importance of geographic location of natural populations, the microbiome of native seeds as putative source of colonization and the effect of a plant's response to UVB exposure on root colonization of highly abundant species. The microbiome of Nicotiana attenuata seeds was determined by a culture-dependent and culture-independent approach, and the root microbiome of natural N. attenuata populations from five different locations was analysed using 454-pyrosequencing. To specifically address the influence of UVB light on root colonization by Deinococcus, a genus abundant and consistently present in N. attenuata roots, transgenic lines impaired in UVB perception (irUVR8) and response (irCHAL) were investigated in a microcosm experiment with/without UVB supplementation using a synthetic bacterial community. The seed microbiome analysis indicated that N. attenuata seeds are sterile. Alpha and beta diversities of native root bacterial communities differed significantly between soil and root, while location had only a significant effect on the fungal but not the bacterial root communities. With UVB supplementation, root colonization of Deinococcus increased in wild type, but decreased in irUVR8 and irCHAL plants compared to nontreated plants. Our results suggest that N. attenuata recruits a core root microbiome exclusively from soil, with fungal root colonization being less selective than bacterial colonization. Root colonization by Deinococcus depends on the plant's response to UVB.

In wild tobacco, Nicotiana attenuata, variation among bacterial communities of isogenic plants is mainly shaped by the local soil microbiota independently of the plants' capacity to produce jasmonic acid.

The phytohormone jasmonic acid (JA) plays a central role in defense against necrotrophic pathogens and herbivores in Nicotiana attenuata. Recently Santhanam et al.(1) showed that JA does not have a major role in shaping the root- and shoot associated bacterial communities, though a few taxa differed among control (empty vector, EV) plants and plants impaired in their capacity to produce JA (irAOC). In this addendum, we provide additional data showing that the composition of the plant bacterial communities is mainly shaped by tissue type. The qualitative data analysis revealed that at the order level, 5 bacterial OTUs formed a core community found in all tissues irrespective of genotypes, while 9 OTUs were different among roots and shoots. The heterogeneity among individual plants was high masking the potential genotype effect on bacterial communities. Using a culture-dependent approach, 3 of 18 bacterial taxa retrieved either only from one of the genotypes or from both had a growth promoting effect on EV and irAOC seedlings. The data suggest that the local soil niche in which the roots grows is a major driver of the variability in root bacterial communities recruited by different individuals, and the plant growth-promoting effects of some taxa are independent of the genotype.

Native root-associated bacteria rescue a plant from a sudden-wilt disease that emerged during continuous cropping.

Plants maintain microbial associations whose functions remain largely unknown. For the past 15 y, we have planted the annual postfire tobacco Nicotiana attenuata into an experimental field plot in the plant's native habitat, and for the last 8 y the number of plants dying from a sudden wilt disease has increased, leading to crop failure. Inadvertently we had recapitulated the common agricultural dilemma of pathogen buildup associated with continuous cropping for this native plant. Plants suffered sudden tissue collapse and black roots, symptoms similar to a Fusarium-Alternaria disease complex, recently characterized in a nearby native population and developed into an in vitro pathosystem for N. attenuata. With this in vitro disease system, different protection strategies (fungicide and inoculations with native root-associated bacterial and fungal isolates), together with a biochar soil amendment, were tested further in the field. A field trial with more than 900 plants in two field plots revealed that inoculation with a mixture of native bacterial isolates significantly reduced disease incidence and mortality in the infected field plot without influencing growth, herbivore resistance, or 32 defense and signaling metabolites known to mediate resistance against native herbivores. Tests in a subsequent year revealed that a core consortium of five bacteria was essential for disease reduction. This consortium, but not individual members of the root-associated bacteria community which this plant normally recruits during germination from native seed banks, provides enduring resistance against fungal diseases, demonstrating that native plants develop opportunistic mutualisms with prokaryotes that solve context-dependent ecological problems.

Pyridine-type alkaloid composition affects bacterial community composition of floral nectar.

Pyridine-type alkaloids are most common in Nicotiana species. To study the effect of alkaloid composition on bacterial community composition in floral nectar, we compared the nicotine-rich wild type (WT) N. attenuata, the nicotine biosynthesis-silenced N. attenuata that was rich in anatabine and the anabasine-rich WT N. glauca plants. We found that the composition of these secondary metabolites in the floral nectar drastically affected the bacterial community richness, diversity and composition. Significant differences were found between the bacterial community compositions in the nectar of the three plants with a much greater species richness and diversity in the nectar from the transgenic plant. The highest community composition similarity index was detected between the two wild type plants. The different microbiome composition and diversity, caused by the different pyridine-type alkaloid composition, could modify the nutritional content of the nectar and consequently, may contribute to the change in the nectar consumption and visitation. These may indirectly have an effect on plant fitness.

Silencing a key gene of the common symbiosis pathway in Nicotiana attenuata specifically impairs arbuscular mycorrhizal infection without influencing the root-associated microbiome or plant growth.

While the biochemical function of calcium and calmodulin-dependent protein kinase (CCaMK) is well studied, and plants impaired in the expression of CCaMK are known not to be infected by arbuscular mycorrhizal fungi (AMF) in glasshouse studies, the whole-plant and ecological consequences of CCaMK silencing are not well understood. Here we show that three independently transformed lines of Nicotiana attenuata plants silenced in CCaMK (irCCaMK) are neither infected by Rhizophagus irregularis in the glasshouse nor by native fungal inoculum in the field. The overall fungal community of field-grown roots did not differ significantly among empty vector (EV) and the transgenic lines, and the bacterial communities only showed minor differences, as revealed by the alpha-diversity parameters of bacterial OTUs, which were higher in EV plants compared with two of the three transformed lines, while beta-diversity parameters did not differ. Furthermore, growth and fitness parameters were similar in the glasshouse and field. Herbivory-inducible and basal levels of salicylic acid, jasmonic acid and abscisic acid did not differ among the genotypes, suggesting that activation of the classical defence pathways are not affected by CCaMK silencing. Based on these results, we conclude that silencing of CCaMK has few, if any, non-target effects.

Analysis of plant-bacteria interactions in their native habitat: bacterial communities associated with wild tobacco are independent of endogenous jasmonic acid levels and developmental stages.

Jasmonic acid (JA) mediates defense responses against herbivores and necrotrophic pathogens but does it influence the recruitment of bacterial communities in the field? We conducted field and laboratory experiments with transformed Nicotiana attenuata plants deficient in jasmonate biosynthesis (irAOC) and empty vector controls (EV) to answer this question. Using both culture-dependent and independent techniques, we characterized root and leaf-associated bacterial communities over five developmental stages, from rosette through flowering of plants grown in their natural habitat. Based on the pyrosequencing results, alpha and beta diversity did not differ among EV and irAOC plants or over ontogeny, but some genera were more abundant in one of the genotypes. Furthermore, bacterial communities were significantly different among leaves and roots. Taxa isolated only from one or both plant genotypes and hence classified as 'specialists' and 'generalists' were used in laboratory tests to further evaluate the patterns observed from the field. The putative specialist taxa did not preferentially colonize the jasmonate-deficient genotype, or alter the plant's elicited phytohormone signaling. We conclude that in N. attenuata, JA signaling does not have a major effect on structuring the bacterial communities and infer that colonization of plant tissues is mainly shaped by the local soil community in which the plant grows.

Streptomyces erringtonii sp. nov. and Streptomyces kaempferi sp. nov., isolated from a hay meadow soil.

Two filamentous actinomycetes isolated from a hay meadow soil were provisionally assigned to the genus Streptomyces based on morphological features. The isolates were found to have chemical and morphological properties typical of the genus Streptomyces and formed distinct phyletic lines in the 16S rRNA gene tree. Isolate I36(T) was most closely related to Streptomyces glauciniger NBRC 100913(T) and isolate I37(T) to Streptomyces mirabilis NBRC 13450(T). Low DNA:DNA relatedness values were recorded between each of the isolates and their closest phylogenetic neighbour. The isolates were also distinguished from their nearest phylogenetic neighbour, and from one another, using a combination of phenotypic properties. These data indicate that the isolates should be recognised as new species in the genus Streptomyces. The names proposed for these new taxa are Streptomyces erringtonii sp. nov. and Streptomyces kaempferi sp. nov. with isolate I36(T) (=CGMCC 4.7016(T) = KACC 15424(T)) and isolate I37(T) (=CGMCC 4.7020(T) = KACC 15428(T)) as the respective type strains.

Streptomyces bullii sp. nov., isolated from a hyper-arid Atacama Desert soil.

A Streptomyces strain isolated from a hyper-arid Atacama Desert soil was characterised using a polyphasic taxonomic approach. The strain, designated C2(T), had chemical and morphological properties typical of the genus Streptomyces. The isolate formed a branch in the Streptomyces 16S rRNA gene tree together with the type strain of Streptomyces chromofuscus and was also loosely related to Streptomyces fragilis NRRL 2424(T). DNA:DNA relatedness values between the isolate and its two phylogenetic neighbours showed that it formed a distinct genomic species. The strain was readily distinguished from these organisms using a combination of morphological and phenotypic data. Based on the genotypic and phenotypic results, isolate C2(T) represents a novel species in the genus Streptomyces, for which the name Streptomyces bullii sp. nov. is proposed. The type strain is C2(T) (=CGMCC 4.7019(T) = KACC 15426(T)).

Streptomyces deserti sp. nov., isolated from hyper-arid Atacama Desert soil.

The taxonomic position of a Streptomyces strain isolated from a hyper-arid desert soil was established using a polyphasic approach. The organism had chemical and morphological properties typical of the genus Streptomyces and formed a phyletic line at the periphery of the Streptomyces coeruleorubidus subcluster in the 16S rRNA gene tree. DNA:DNA relatedness values between the isolate and its nearest phylogenetic neighbours, Streptomyces lomondensis NRRL 3252(T) and Streptomyces lusitanus NRRL B-12501(T) were 42.5 (±0.48)% and 25.0 (±1.78)%, respectively. The isolate was readily distinguished from these organisms using a combination of morphological and phenotypic properties. On the basis of these results, it is proposed that isolate C63(T) (CGMCC 4.6997(T), = KACC 15425(T)) be classified as the type strain of Streptomyces deserti sp. nov.

Streptomyces atacamensis sp. nov., isolated from an extreme hyper-arid soil of the Atacama Desert, Chile.

The taxonomic position of a Streptomyces strain isolated from an extreme hyper-arid soil sample collected from the Atacama Desert was determined using a polyphasic approach. The strain, isolate C60(T), had chemical and morphological features typical of members of the genus Streptomyces and formed a distinct phyletic line in the Streptomyces 16S rRNA gene tree, together with the type strain of Streptomyces radiopugnans. The two strains were distinguished readily using a combination of phenotypic properties and by a DNA-DNA relatedness value of 23.17 (± 0.95)%. On the basis of these genotypic and phenotypic data, it is proposed that isolate C60(T) (=CGMCC 4.7018(T)=KACC 15492(T)) be classified in the genus Streptomyces as Streptomyces atacamensis sp. nov.