Evaluation of dietary single probiotic isolates and probiotic multistrain consortia in growth performance, gut histology, gut microbiota, immune regulation, and infection resistance of Nile tilapia, Oreochromis niloticus, shows superior monostrain performance.

The probiotic potential of a designed bacterial consortia isolated from a competitive exclusion culture originally obtained from the intestinal contents of tilapia juveniles were evaluated on Nile tilapia alevins. The growth performance, intestinal histology, microbiota effects, resistance to Streptococcus agalactiae challenge, and immune response were assessed. In addition, the following treatments were included in a commercial feed: A12+M4+M10 (Lactococcus lactis A12, Priestia megaterium M4, and Priestia sp. M10), M4+M10 (P. megaterium M4, and Priestia sp. M10) and the single bacteria as controls; A12 (L. lactis A12), M4 (P. megaterium M4), M10 (Priestia sp. M10), also a commercial feed without any probiotic addition was included as a control. The results showed that all probiotic treatments improved the growth performance, intestinal histology, and resistance during experimental infection with S. agalactiae in comparison to the control fish. Also, the administration of probiotics resulted in the modulation of genes associated with the innate and adaptive immune systems that were non-dependent on microbial colonization. Surprisingly, L. lactis A12 alone induced benefits in fish compared to the microbial consortia, showing the highest increase in growth rate, survival during experimental infection with S. agalactiae, increased intestinal fold length, and the number of differentially expressed genes. Lastly, we conclude that a competitive exclusion culture is a reliable source of probiotics, and monostrain L. lactis A12 has comparable or even greater probiotic potential than the bacterial consortia.

Metagenomic Analysis of Biochemical Passive Reactors During Acid Mine Drainage Bioremediation Reveals Key Co-selected Metabolic Functions.

Acid mine drainage (AMD) is the major pollutant generated by the mining industry, and it is characterized by low pH and high concentration of metals and sulfate. The use of biochemical passive reactors (BPRs) is a promising strategy for its bioremediation. To date, there are various studies describing the taxonomical composition of BPR microbial communities, generally consisting of an assemblage of sulfate-reducing organisms inside Deltaproteobacteria, and a diverse set of anaerobic (ligno)cellulolytic bacteria; however, insights about its functional metagenomic content are still scarce. In previous studies, a laboratory-scale AMD bioremediation using biochemical passive reactors was designed and performed, tracking operation parameters, chemical composition, and changes, together with taxonomic composition of the microbiomes harbored in these systems. In order to reveal the main functional content of these communities, we used shotgun metagenomics analyses to explore genes of higher relative frequencies and their inferred functions during the AMD bioremediation from three BPRs representing the main microbiome compositions detected in the system. Remarkably, genes encoding for two-component regulatory systems and ABC transporters related to metal and inorganic ions, cellulose degradation enzymes, dicarboxylic acid production, and sulfite reduction complex were all detected at increased frequency. Our results evidenced that higher taxonomic diversity of the microbiome was arising together with a functional redundancy of the specific metabolic roles, indicating its co-selection and suggesting that its enrichment on BPRs may be implicated in the cumulative efficiency of these systems.

Microbiome composition of the marine sponge Cliona varians at the neotropical southern Caribbean Sea displays a predominant core of Rhizobiales and Nitrosopumilaceae.

AIMS: This work aims to characterize the microbial diversity of the encrusting sponge Cliona varians, a pore-forming and coral reef bioeroding marine sponge of emerging spread related to ocean acidification. METHODS AND RESULTS: We analysed the microbiome composition by 16S V4 amplicon next-generation sequencing in a community of the bioeroding coral reef encrusting/excavating marine sponge Cliona varians thriving at the Southern Caribbean Sea. About 87.21% and 6.76% of the sequences retrieved were assigned to the domain Bacteria and Archaea. The most predominant operational taxonomic units were classified as members of the order Rhizobiales and family Nitrosopumilaceae, representing members of not yet characterized genera. Features found strictly conserved in the strain/genomic representatives reported in those microbial taxa are nitrogen fixation and transformation. CONCLUSION: Our results suggest, in accordance with recent results, that these microbiome members and associated functions could be contributing to the biological fitness of the sponge to be able to colonize and bioerode in environments with low access and scarce availability of nitrogen sources. SIGNIFICANCE AND IMPACT OF STUDY: Coral reefs bioresources such as sponge holobionts are intriguing and complex ecosystem units. This study contributes to the knowledge of how C. varians microbiota is composed or shaped, which is crucial to understand its ecological functions.

A global survey reveals a divergent extradiol dioxygenase clade as a widespread complementary contributor to the biodegradation of mono- and polycyclic aromatic hydrocarbons.

Extradiol dioxygenation is a key reaction in the microbial aerobic degradation of mono- and polycyclic aromatic hydrocarbon catecholic derivatives. It has been reported that many bacterial enzymes exhibiting such converging functions act on a wide range of catecholic substrates. The present study reports a new subfamily of extradiol dioxygenases (EXDOs) with broad substrate specificity, the HrbC EXDOs. The new clade belongs to the XII cluster within family 2 of the vicinal oxygen chelate superfamily (EXDO-VC2), which is typically characterized by a preference for bicyclic substrates. Coding hrbC orthologs were isolated by activity-based screening of fosmid metagenomic libraries from large DNA fragments derived from heavily PAH-contaminated soils. They occurred as solitary genes within conserved sequences encoding enzymes for amino acid metabolism and were stably maintained in the chromosomes of the Betaproteobacteria lineages harboring them. Analysis of contaminated aquifers revealed coexpression of hrbC as a polycistronic mRNA component. The predicted open reading frames were verified by cloning and heterologous expression, confirming the expected molecular mass and meta-cleavage activity of the recombinant enzymes. Evolutionary analysis of the HrbC protein sequences grouped them into a discrete cluster of 1,2-dihydroxynaphthalene dioxygenases represented by a cultured PAH degrader, Rugosibacter aromaticivorans strain Ca6. The ecological importance and relevance of the new EXDO genes were confirmed by PCR-based mapping in different biogeographical localities contaminated with a variety of mono- and polycyclic aromatic compounds. The cosmopolitan distribution of hrbC in PAH-contaminated aquifers supports our hypothesis about its auxiliary role in the degradation of toxic catecholic intermediates, contributing to the composite EXDO catabolic capacity of the world's microbiomes.

Dry and Rainy Seasons Significantly Alter the Gut Microbiome Composition and Reveal a Key Enterococcus sp. (Lactobacillales: Enterococcaceae) Core Component in Spodoptera frugiperda (Lepidoptera: Noctuidae) Corn Strain From Northwestern Colombia.

Spodoptera frugiperda is a polyphagous pest of several crops of economic importance. Nowadays, the insect is broadly distributed in America and, recently, in Africa, Asia, and Australia. The species has diverged into corn and rice strains. The role of the gut microbiota in insect physiology is relevant due to its participation in crucial functions. However, knowledge of seasonal variations that alter the gut microbiome in pests is limited. Gut microbiome composition between the dry and rainy seasons was analyzed with cultured and uncultured approaches in S. frugiperda corn strain larvae collected at Northwest Colombia, as seasonal microbiome changes might fluctuate due to environmental changes. On the basis of culture-dependent methods, results show well-defined microbiota with bacterial isolates belonging to Enterococcus, Klebsiella (Enterobacteriales: Enterobacteriaceae), Enterobacter (Enterobacterales: Enterobacteriaceae), and Bacillus (Bacillales: Bacillaceae) genera. The community composition displayed a low bacterial diversity across all samples. The core community detected with uncultured methods was composed of Enterococcus, Erysipelatoclostridium (Erysipelotrichales: Erysipelotrichaceae), Rasltonia (Burkholderiales: Burkholderiaceae), and Rhizobium (Hyphomicrobiales: Rhizobiaceae) genera, and Enterobacteriaceae family members. Significant differences in microbiome diversity were observed between the two seasons. The relative abundance of Erysipelatoclostridium was high in the dry season, while in the phylotype ZOR0006 (Erysipelotrichales: Erysipelotrichaceae) and Tyzzerella (Lachnospirales: Lachnospiraceae) genus, the relative abundance was high in the rainy season. The overall low gut bacterial diversity observed in the S. frugiperda corn strain suggests a strong presence of antagonist activity as a selection factor possibly arising from the host, the dominant bacterial types, or the material ingested. Targeting the stability and predominance of this core microbiome could be an additional alternative to pest control strategies, particularly in this moth.

Enrichment and characterization of a bilge microbial consortium with oil in water-emulsions breaking ability for oily wastewater treatment.

Oily bilge wastewater is one of the main sources of hydrocarbons pollution in marine environments due to accidental or clandestine discharges. The main technical challenge for its effective treatment is the presence of stable oil-in-water (O/W) emulsions. In this work we are reporting an enriched microbial consortium from bilge wastewater with remarkable ability to demulsify oil in water emulsions. The consortium showed emulsion-breaking ratios up to 72.6% in the exponential growth phase, while the values range from 11.9 to 8.5% in stationary phase. A positive association was observed between demulsifying ability and microbial adhesion to hydrocarbons, as well as between cell concentration and demulsifying ability. Also, an interesting ability to demulsify under different temperatures, conditions of agitation, and bilge emulsions from different vessels was observed. The Bacterial and Archaeal composition was analyzed by 16S rRNA gene amplicon lllumina sequencing analyses, revealing an assemblage composed of bacterial types highly related to well characterized bacterial isolates and also to non-yet cultured bacterial types previously detected in marine and sediment samples. Hydrocarbonoclastic microbial types such as Marinobacter, Flavobacteriaceae, Alcanivorax and Gammaproteobacteria PYR10d3 were found in high relative abundance (27.0%-11.1%) and types of marine oligotrophs and surfactant degraders such as Thallasospira, Parvibaculum, Novospirillum, Shewanella algae, and Opitutae were in a group of middle predominance (1.7-3.5%). The microbial consortium reported has promising potential for the biological demulsification of bilge wastewater and other oily wastewaters.

Enrichment and key features of a robust and consistent indigenous marine-cognate microbial consortium growing on oily bilge wastewaters.

Oily bilge wastewater (OBW) is a hazardous hydrocarbon-waste generated by ships worldwide. In this research, we enriched, characterized and study the hydrocarbon biodegradation potential of a microbial consortium from the bilges of maritime ships. The consortium cZ presented a biodegradation efficiency of 66.65% for total petroleum hydrocarbons, 72.33% for aromatics and 97.76% removal of n-alkanes. This consortium showed the ability to grow in OBWs of diverse origin and concentration. A 67-fold increase in biomass was achieved using a Sequential Batch Reactor with OBW as the only carbon and energy source. The bacterial community composition of the enriched OBW bacterial consortium at the final stable stage was characterized by 16S amplicon Illumina sequencing showing that 25 out of 915 of the emerged predominant bacterial types detected summed up for 84% of total composition. Out of the 140 taxa detected, 13 alone accumulated 94.9% of the reads and were classified as Marinobacter, Alcanivorax, Parvibaculum, Flavobacteriaceae, Gammaproteobacteria PYR10d3, Novispirillum and Xanthomonadaceae among the most predominant, followed by Thalassospira, Shewanella, Rhodospirillaceae, Gammaprotobacteria, Rhodobacteriaceae and Achromobacter. The microbial community from OBW bioreactor enrichments is intrinsically diverse with clear selection of predominant types and remarkably exhibiting consistent and efficient biodegradation achieved without any nutrient or surfactant addition. Due to there is very little information available in the OBW biodegradation field, this work contributes to the body of knowledge surrounding the treatment improvement of this toxic waste and its potential application in wastewater management.

Stable and Enriched Cenarchaeum symbiosum and Uncultured Betaproteobacteria HF1 in the Microbiome of the Mediterranean Sponge Haliclona fulva (Demospongiae: Haplosclerida).

Sponges harbor characteristic microbiomes derived from symbiotic relationships shaping their lifestyle and survival. Haliclona fulva is encrusting marine sponge species dwelling in coralligenous accretions or semidark caves of the Mediterranean Sea and the near Atlantic Ocean. In this work, we characterized the abundance and core microbial community composition found in specimens of H. fulva by means of electron microscopy and 16S amplicon Illumina sequencing. We provide evidence of its low microbial abundance (LMA) nature. We found that the H. fulva core microbiome is dominated by sequences belonging to the orders Nitrosomonadales and Cenarchaeales. Seventy percent of the reads assigned to these phylotypes grouped in a very small number of high-frequency operational taxonomic units, representing niche-specific species Cenarchaeum symbiosum and uncultured Betaproteobacteria HF1, a new eubacterial ribotype variant found in H. fulva. The microbial composition of H. fulva is quite distinct from those reported in sponge species of the same Haliclona genus. We also detected evidence of an excretion/capturing loop between these abundant microorganisms and planktonic microbes by analyzing shifts in seawater planktonic microbial content exposed to healthy sponge specimens maintained in aquaria. Our results suggest that horizontal transmission is very likely the main mechanism for symbionts' acquisition by H. fulva. So far, this is the first shallow water sponge species harboring such a specific and predominant assemblage composed of these eubacterial and archaeal ribotypes. Our data suggests that this symbiotic relationship is very stable over time, indicating that the identified core microbial symbionts may play key roles in the holobiont functioning.

Variations in Microbial Diversity and Metabolite Profiles of the Tropical Marine Sponge Xestospongia muta with Season and Depth.

Xestospongia muta is among the most emblematic sponge species inhabiting coral reefs of the Caribbean Sea. Besides being the largest sponge species growing in the Caribbean, it is also known to produce secondary metabolites. This study aimed to assess the effect of depth and season on the symbiotic bacterial dynamics and major metabolite profiles of specimens of X. muta thriving in a tropical marine biome (Portobelo Bay, Panamá), which allow us to determine whether variability patterns are similar to those reported for subtropical latitudes. The bacterial assemblages were characterized using Illumina deep-sequencing and metabolomic profiles using UHPLC-DAD-ELSD from five depths (ranging 9-28 m) across two seasons (spring and autumn). Diverse symbiotic communities, representing 24 phyla with a predominance of Proteobacteria and Chloroflexi, were found. Although several thousands of OTUs were determined, most of them belong to the rare biosphere and only 23 to a core community. There was a significant difference between the structure of the microbial communities in respect to season (autumn to spring), with a further significant difference between depths only in autumn. This was partially mirrored in the metabolome profile, where the overall metabolite composition did not differ between seasons, but a significant depth gradient was observed in autumn. At the phyla level, Cyanobacteria, Firmicutes, Actinobacteria, and Spirochaete showed a mild-moderate correlation with the metabolome profile. The metabolomic profiles were mainly characterized by known brominated polyunsaturated fatty acids. This work presents findings about the composition and dynamics of the microbial assemblages of X. muta expanding and confirming current knowledge about its remarkable diversity and geographic variability as observed in this tropical marine biome.

A composite taxonomical and functional framework of microbiomes under acid mine drainage bioremediation systems.

Mining-industry is one of the most important activities in the economic development of many countries and produces highly significant alterations on the environment, mainly due to the release of a strong acidic metal-rich wastewater called acid mine drainage (AMD). Consequently, the establishment of multiple wastewater treatment strategies remains as a fundamental challenge in AMD research. Bioremediation, as a constantly-evolving multidisciplinary endeavor had been complemented during the last decades by novel tools of increasingly higher resolution such as those based on omics approaches, which are providing detailed insights into the ecology, evolution and mechanisms of microbial communities acting in bioremediation processes. This review specifically addresses, reanalyzes and reexamines in a composite comparative manner, the available sequence information and associated metadata available in public databases about AMD impacted microbial communities; summarizing our understanding of its composition and functions, and proposing potential genetic enhancements for improved bioremediation strategies. 16 S rRNA gene-targeted sequencing data from 9 studies previously published including AMD systems reported and studied around the world, were collected and reanalyzed to compare and identify the core and most abundant genera in four distinct AMD ecosystems: surface biofilm, water, impacted soils/sediments and bioreactor microbiomes. We determined that the microbial communities of bioreactors were the most diverse in bacterial types detected. The metabolic pathways predicted strongly suggest the key role of syntrophic communities with denitrification, methanogenesis, manganese, sulfate and iron reduction. The perspectives to explore the dynamics of engineering systems by high-throughput sequencing and biochemical techniques are discussed and foreseen application of synthetic biology and omics exploration on improved AMD biotransformation are proposed.

Delftia sp. LCW, a strain isolated from a constructed wetland shows novel properties for dimethylphenol isomers degradation.

BACKGROUND: Dimethylphenols (DMP) are toxic compounds with high environmental mobility in water and one of the main constituents of effluents from petro- and carbochemical industry. Over the last few decades, the use of constructed wetlands (CW) has been extended from domestic to industrial wastewater treatments, including petro-carbochemical effluents. In these systems, the main role during the transformation and mineralization of organic pollutants is played by microorganisms. Therefore, understanding the bacterial degradation processes of isolated strains from CWs is an important approach to further improvements of biodegradation processes in these treatment systems. RESULTS: In this study, bacterial isolation from a pilot scale constructed wetland fed with phenols led to the identification of Delftia sp. LCW as a DMP degrading strain. The strain was able to use the o-xylenols 3,4-DMP and 2,3-DMP as sole carbon and energy sources. In addition, 3,4-DMP provided as a co-substrate had an effect on the transformation of other four DMP isomers. Based on the detection of the genes, proteins, and the inferred phylogenetic relationships of the detected genes with other reported functional proteins, we found that the phenol hydroxylase of Delftia sp. LCW is induced by 3,4-DMP and it is responsible for the first oxidation of the aromatic ring of 3,4-, 2,3-, 2,4-, 2,5- and 3,5-DMP. The enzyme may also catalyze both monooxygenation reactions during the degradation of benzene. Proteome data led to the identification of catechol meta cleavage pathway enzymes during the growth on ortho DMP, and validated that cleavage of the aromatic rings of 2,5- and 3,5-DMPs does not result in mineralization. In addition, the tolerance of the strain to high concentrations of DMP, especially to 3,4-DMP was higher than that of other reported microorganisms from activated sludge treating phenols. CONCLUSIONS: LCW strain was able to degraded complex aromatics compounds. DMPs and benzene are reported for the first time to be degraded by a member of Delftia genus. In addition, LCW degraded DMPs with a first oxidation of the aromatic rings by a phenol hydroxylase, followed by a further meta cleavage pathway. The higher resistance to DMP toxicity, the ability to degrade and transform DMP isomers and the origin as a rhizosphere bacterium from wastewater systems, make LCW a suitable candidate to be used in bioremediation of complex DMP mixtures in CWs systems.

Comparative Assessment of Mediterranean Gorgonian-Associated Microbial Communities Reveals Conserved Core and Locally Variant Bacteria.

Gorgonians are key habitat-forming species of Mediterranean benthic communities, but their populations have suffered from mass mortality events linked to high summer seawater temperatures and microbial disease. However, our knowledge on the diversity, dynamics and function of gorgonian-associated microbial communities is limited. Here, we analysed the spatial variability of the microbiomes of five sympatric gorgonian species (Eunicella singularis, Eunicella cavolini, Eunicella verrucosa, Leptogorgia sarmentosa and Paramuricea clavata), collected from the Mediterranean Sea over a scale of ∼1100 km, using next-generation amplicon sequencing of the 16S rRNA gene. The microbiomes of all gorgonian species were generally dominated by members of the genus Endozoicomonas, which were at very low abundance in the surrounding seawater. Although the composition of the core microbiome (operational taxonomic units consistently present in a species) was found to be unique for each host species, significant overlap was observed. These spatially consistent associations between gorgonians and their core bacteria suggest intricate symbiotic relationships and regulation of the microbiome composition by the host. At the same time, local variations in microbiome composition were observed. Functional predictive profiling indicated that these differences could be attributed to seawater pollution. Taken together, our data indicate that gorgonian-associated microbiomes are composed of spatially conserved bacteria (core microbiome members) and locally variant members, and that local pollution may influence these local associations, potentially impacting gorgonian health.

Development of a genetic tool for functional screening of anti-malarial bioactive extracts in metagenomic libraries.

BACKGROUND: The chemical treatment of Plasmodium falciparum for human infections is losing efficacy each year due to the rise of resistance. One possible strategy to find novel anti-malarial drugs is to access the largest reservoir of genomic biodiversity source on earth present in metagenomes of environmental microbial communities. METHODS: A bioluminescent P. falciparum parasite was used to quickly detect shifts in viability of microcultures grown in 96-well plates. A synthetic gene encoding the Dermaseptin 4 peptide was designed and cloned under tight transcriptional control in a large metagenomic insert context (30 kb) to serve as proof-of-principle for the screening platform. RESULTS: Decrease in parasite viability consistently correlated with bioluminescence emitted from parasite microcultures, after their exposure to bacterial extracts containing a plasmid or fosmid engineered to encode the Dermaseptin 4 anti-malarial peptide. CONCLUSIONS: Here, a new technical platform to access the anti-malarial potential in microbial environmental metagenomes has been developed.

Interaction and effects of temperature preference under a controlled environment on the diversity and abundance of the microbiome in Lutzomyia longipalpis (Diptera: Psychodidae).

Characterization of the temperature effects on the abundance and richness of the microbiota of Lutzomyia longipalpis, insect vector of Leishmania infantum in America, is an aspect of pivotal importance to understand the interactions between temperature, bacteria, and Leishmania infection. We developed and used a customized device with a temperature gradient (21-34 °C) to assess the temperature preferences of wild females of Lu. longipalpis collected in a rural area (Ricaurte, Cundinamarca, Colombia). Each replicate consisted of 50 females exposed to the gradient for an hour. At the end of the exposure time, insects were collected and separated by the temperature ranges selected varying from 21 °C to 34 °C. They were organized in 17 pools from which total DNA extracts were obtained, and samples were subjected to 16S rRNA amplicon sequencing analyzes. The most abundant phyla across the different temperature ranges were Proteobacteria (17.22-90.73 %), Firmicutes (5.99-77.21 %) and Actinobacteria (1.56-59.85 %). Results also showed an abundance (30 % to 57.36 %) of Pseudomonas (mainly at temperatures of 21-29 °C and 34 °C) that decreased to 6.55 %-13.20 % at temperatures of 31-33 °C, while Bacillus increase its abundance to 67.24 % at 29-33 °C. Serratia also had a greater representation (49.79 %), specifically in sand flies recovered at 25-27 °C. No significant differences were found at α-diversity level when comparing richness using the Shannon-Wiener, Simpson, and Chao1 indices, while ß-diversity differences were found using the Bray-Curtis index (F-value of 3.5073, p-value < 0.013, R-squared of 0,4889), especially in the groups of Lu. longipalpis associated at higher temperatures (29-33 °C). It was also possible to detect the presence of endosymbionts such as Spiroplasma and Arsenophonus in the range of 29-33 °C. Rickettsia was only detected in Lu. longipalpis sand flies recovered between 25-27 °C. It was possible to characterize Lu. longipalpis microbiota in response to intraspecific temperature preferences and observe changes in bacterial communities and endosymbionts at different ranges of said environmental variable, which may be important in its vector competence and environmental plasticity to adapt to new climate change scenarios.

Isocyanides inhibit bacterial pathogens by covalent targeting of essential metabolic enzymes.

Isonitrile natural products, also known as isocyanides, demonstrate potent antimicrobial activities, yet our understanding of their molecular targets remains limited. Here, we focus on the so far neglected group of monoisonitriles to gain further insights into their antimicrobial mode of action (MoA). Screening a focused monoisonitrile library revealed a potent S. aureus growth inhibitor with a different MoA compared to previously described isonitrile antibiotics. Chemical proteomics via competitive cysteine reactivity profiling, uncovered covalent modifications of two essential metabolic enzymes involved in the fatty acid biosynthetic process (FabF) and the hexosamine pathway (GlmS) at their active site cysteines. In-depth studies with the recombinant enzymes demonstrated concentration-dependent labeling, covalent binding to the catalytic site and corresponding functional inhibition by the isocyanide. Thermal proteome profiling and full proteome studies of compound-treated S. aureus further highlighted the destabilization and dysregulation of proteins related to the targeted pathways. Cytotoxicity and the inhibition of cytochrome P450 enzymes require optimization of the hit molecule prior to therapeutic application. The here described novel, covalent isocyanide MoA highlights the versatility of the functional group, making it a useful tool and out-of-the-box starting point for the development of innovative antibiotics.

Screening Privileged Alkyl Guanidinium Motifs under Host-Mimicking Conditions Reveals a Novel Antibiotic with an Unconventional Mode of Action.

Screening large molecule libraries against pathogenic bacteria is often challenged by a low hit rate due to limited uptake, underrepresentation of antibiotic structural motifs, and assays that do not resemble the infection conditions. To address these limitations, we present a screen of a focused library of alkyl guanidinium compounds, a structural motif associated with antibiotic activity and enhanced uptake, under host-mimicking infection conditions against a panel of disease-associated bacteria. Several hit molecules were identified with activities against Gram-positive and Gram-negative bacteria, highlighting the fidelity of the general concept. We selected one compound (L15) for in-depth mode of action studies that exhibited bactericidal activity against methicillin-resistant Staphylococcus aureus USA300 with a minimum inhibitory concentration of 1.5 µM. Structure-activity relationship studies confirmed the necessity of the guanidinium motif for antibiotic activity. The mode of action was investigated using affinity-based protein profiling with an L15 probe and identified the signal peptidase IB (SpsB) as the most promising hit. Validation by activity assays, binding site identification, docking, and molecular dynamics simulations demonstrated SpsB activation by L15, a recently described mechanism leading to the dysregulation of protein secretion and cell death. Overall, this study highlights the need for unconventional screening strategies to identify novel antibiotics.

Effects of squamous cell carcinoma and smoking status on oropharyngeal and laryngeal microbial communities.

BACKGROUND: Still, little is known about microbial dysbiosis in oropharyngeal and laryngeal tissue as risk factor for development of local squamous cell carcinoma. The site-specific microbiota at these regions in healthy and cancer tissue and their modulation by environmental factors need to be defined. METHODS: The local microbiota of cancer tissue and healthy controls was profiled by 16S rRNA gene amplicon sequencing and statistical analysis using 111 oropharyngeal and 72 laryngeal intraoperative swabs. RESULTS: Oropharynx and larynx harbor distinct microbial communities. Clear effects of both smoking and cancer were seen in the oropharynx whereas effects in the larynx were minor. CONCLUSION: The distinct microbial communities at larynx and oropharynx partially explain why the effects of cancer and smoking were distinct at those sites. Thus, the use of microbiota supposed to mirror community changes in another target location should be avoided and more studies on the actual cancerous environment are necessary.

Analysis of the microbial gene landscape and transcriptome for aromatic pollutants and alkane degradation using a novel internally calibrated microarray system.

Despite various efforts to develop tools to detect and compare the catabolic potential and activity for pollutant degradation in environmental samples, there is still a need for an open-source, curated and reliable array method. We developed a custom array system including a novel normalization strategy that can be applied to any microarray design, allowing the calculation of the reliability of signals and make cross-experimental comparisons. Array probes, which are fully available to the scientific community, were designed from knowledge-based curated databases for key aromatic catabolic gene families and key alkane degradation genes. This design assigns signals to the respective protein subfamilies, thus directly inferring function and substrate specificity. Experimental procedures were optimized using DNA of four genome sequenced biodegradation strains and reliability of signals assessed through a novel normalization procedure, where a plasmid containing four artificial targets in increased copy numbers and co-amplified with the environmental DNA served as an internal calibration curve. The array system was applied to assess the catabolic gene landscape and transcriptome of aromatic contaminated environmental samples, confirming the abundance of catabolic gene subfamilies previously detected by functional metagenomics but also revealing the presence of previously undetected catabolic groups and specifically their expression under pollutant stress.

Microbiome analysis of Spodoptera frugiperda (Lepidoptera, Noctuidae) larvae exposed to Bacillus thuringiensis (Bt) endotoxins.

Background: Spodoptera frugiperda (or fall armyworm, FAW) is a polyphagous pest native to Western Hemisphere and recently discovered in the Eastern Hemisphere. In Colombia, S. frugiperda is recognized as a pest of economic importance in corn. The species has genetically differentiated into two host populations named "corn" and "rice" strains. In 2012, a study made in central Colombia demonstrated that the corn strain is less susceptible to Bacillus thuringiensis (Bt) endotoxins (Cry1Ac and Cry 1Ab) than the rice strain. In this country, Bt transgenic corn has been extensively produced over the last 15 years. Since gut microbiota plays a role in the physiology and immunity of insects, and has been implicated in promoting the insecticidal activity of Bt, in this study an analysis of the interaction between Bt endotoxins and FAW gut microbiota was made. Also, the detection of endosymbionts was performed here, as they might have important implications in the biological control of a pest. Methods: The composition and diversity of microbiomes associated with larval specimens of S. frugiperda(corn strain) was investigated in a bioassay based on six treatments in the presence/absence of Bt toxins and antibiotics (Ab) through bacterial isolate analyses and by high throughput sequencing of the bacterial 16S rRNA gene. Additionally, species specific primers were used, to detect endosymbionts from gonads in S. frugiperda corn strain. Results: Firmicutes, Proteobacteria and Bacteroidota were the most dominant bacterial phyla found in S. frugiperda corn strain. No significant differences in bacteria species diversity and richness among the six treatments were found. Two species of Enterococcus spp., E. mundtii and E. casseliflavus were detected in treatments with Bt and antibiotics, suggesting that they are less susceptible to both of them. Additionally, the endosymbiont Arsenophonus was also identified on treatments in presence of Bt and antibiotics. The results obtained here are important since little knowledge exists about the gut microbiota on this pest and its interaction with Bt endotoxins. Previous studies made in Lepidoptera suggest that alteration of gut microbiota can be used to improve the management of pest populations, demonstrating the relevance of the results obtained in this work.