Designing optimal experiments in metabolomics.

BACKGROUND: Metabolomics data is often complex due to the high number of metabolites, chemical diversity, and dependence on sample preparation. This makes it challenging to detect significant differences between factor levels and to obtain accurate and reliable data. To address these challenges, the use of Design of Experiments (DoE) techniques in the setup of metabolomic experiments is crucial. DoE techniques can be used to optimize the experimental design space, ensuring that the maximum amount of information is obtained from a limited sample space. AIM OF REVIEW: This review aims at providing a baseline workflow for applying DoE when generating metabolomics data. KEY SCIENTIFIC CONCEPTS OF REVIEW: The review provides insights into the theory of DoE. The review showcases the theory being put into practice by highlighting different examples DoE being applied in metabolomics throughout the literature, considering both targeted and untargeted metabolomic studies in which the data was acquired using both nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry techniques. In addition, the review presents DoE concepts not currently being applied in metabolomics, highlighting these as potential future prospects.

Roseobacter Group Probiotics Exhibit Differential Killing of Fish Pathogenic Tenacibaculum Species.

Fish-pathogenic bacteria of the Tenacibaculum genus are a serious emerging concern in modern aquaculture, causing tenacibaculosis in a broad selection of cultured finfish. Data describing their virulence mechanisms are scarce and few means, antibiotic treatment aside, are available to control their proliferation in aquaculture systems. We genome sequenced a collection of 19 putative Tenacibaculum isolates from outbreaks at two aquaculture facilities and tested their susceptibility to treatment with tropodithietic acid (TDA)-producing Roseobacter group probiotics. We found that local outbreaks of Tenacibaculum can involve heterogeneous assemblages of species and strains with the capacity to produce multiple different virulence factors related to host invasion and infection. The probiotic Phaeobacter piscinae S26 proved efficient in killing pathogenic Tenacibaculum species such as T. maritimum, T. soleae, and some T. discolor strains. However, the T. mesophilum and T. gallaicum species exhibit natural tolerance toward TDA and are hence not likely to be easily killed by TDA-producing probiotics. Tolerance toward TDA in Tenacibaculum is likely involving multiple inherent physiological features pertaining to electron and proton transport, iron sequestration, and potentially also drug efflux mechanisms, since genetic determinants encoding such features were significantly associated with TDA tolerance. Collectively, our results support the use of TDA producers to prevent tenacibaculosis; however, their efficacy is likely limited to some Tenacibaculum species. IMPORTANCE A productive and sustainable aquaculture sector is needed to meet the UN sustainable development goals and supply the growing world population with high-protein food sources. A sustainable way to prevent disease outbreaks in the industry is the application of probiotic bacteria that can antagonize fish pathogens in the aquaculture systems. TDA-producing Roseobacter group probiotics have proven efficient in killing important vibrio pathogens and protecting fish larvae against infection, and yet their efficacy against different fish pathogenic species of the Tenacibaculum genus has not been explored. Therefore, we tested the efficacy of such potential probiotics against a collection of different Tenacibaculum isolates and found the probiotic to efficiently kill a subset of relevant strains and species, supporting their use as sustainable disease control measure in aquaculture.

Chitin Degradation Machinery and Secondary Metabolite Profiles in the Marine Bacterium Pseudoalteromonas rubra S4059.

Genome mining of pigmented Pseudoalteromonas has revealed a large potential for the production of bioactive compounds and hydrolytic enzymes. The purpose of the present study was to explore this bioactivity potential in a potent antibiotic and enzyme producer, Pseudoalteromonas rubra strain S4059. Proteomic analyses (data are available via ProteomeXchange with identifier PXD023249) indicated that a highly efficient chitin degradation machinery was present in the red-pigmented P. rubra S4059 when grown on chitin. Four GH18 chitinases and two GH20 hexosaminidases were significantly upregulated under these conditions. GH19 chitinases, which are not common in bacteria, are consistently found in pigmented Pseudoalteromonas, and in S4059, GH19 was only detected when the bacterium was grown on chitin. To explore the possible role of GH19 in pigmented Pseudoalteromonas, we developed a protocol for genetic manipulation of S4059 and deleted the GH19 chitinase, and compared phenotypes of the mutant and wild type. However, none of the chitin degrading ability, secondary metabolite profile, or biofilm-forming capacity was affected by GH19 deletion. In conclusion, we developed a genetic manipulation protocol that can be used to unravel the bioactive potential of pigmented pseudoalteromonads. An efficient chitinolytic enzyme cocktail was identified in S4059, suggesting that this strain could be a candidate with industrial potential.

Atlantic salmon (Salmo salar) protein hydrolysate in diets for weaning piglets ─ effect on growth performance, intestinal morphometry and microbiota composition.

Salmon protein hydrolysates (SPH) from two different rest raw materials were evaluated in diets for weaning piglets. Four experimental diets were included in the study: a diet based on plant protein with soy protein as the main protein source (Diet PP), a diet based on fishmeal in exchange for soy protein (Diet FM) and two diets in which different SPH replaced fishmeal in the FM diet. The experimental diets were fed to piglets from the day of weaning until 32 d postweaning. In addition to the record of performance data, an intestinal sampling for mucosal morphometry and microbiota 16S rRNA gene sequencing were performed at day 11 on a subset of the animals. The duodenal villi absorption area was significantly larger in piglets receiving Diets SPH compared with Diet PP (p < 0.02). A significant positive correlation between duodenal villi height and average daily gain during the first 11 d postweaning was detected. Only small differences in intestinal microbiota community and no differences in growth performance were detected between the experimental diets. To conclude, SPH seem to be an interesting novel protein source in weanling piglets.

In situ prebiotics for weaning piglets: in vitro production and fermentation of potato galacto-rhamnogalacturonan.

Postweaning diarrhea (PWD) in pigs is a leading cause of economic loss in pork production worldwide. The current practice of using antibiotics and zinc to treat PWD is unsustainable due to the potential of antibiotic resistance and ecological disturbance, and novel methods are required. In this study, an in vitro model was used to test the possibility of producing prebiotic fiber in situ in the gastrointestinal (GI) tract of the piglet and the prebiotic activity of the resulting fiber in the terminal ileum. Soluble fiber was successfully produced from potato pulp, an industrial waste product, with the minimal enzyme dose in a simulated upper GI tract model extracting 26.9% of the initial dry matter. The fiber was rich in galactose and galacturonic acid and was fermented at 2.5, 5, or 10 g/liter in a glucose-free medium inoculated with the gut contents of piglet terminal ileum. Fermentations of 5 g/liter inulin or 5 g/liter of a purified potato fiber were used as controls. The fibers showed high fermentability, evident by a dose-dependent drop in pH and an increase in the organic acid content, with lactate in particular being increased. Deep sequencing showed a significant increase in the numbers of Lactobacillus and Veillonella organisms and an insignificant increase in the numbers of Clostridium organisms as well as a decrease in the numbers of Streptococcus organisms. Multivariate analysis showed clustering of the treatment groups, with the group treated with purified potato fiber being clearly separated from the other groups, as the microbiota composition was 60% Lactobacillus and almost free of Clostridium. For animal studies, a dosage corresponding to the 5-g/liter treatment is suggested.

Characterization of the bacterial gut microbiota of piglets suffering from new neonatal porcine diarrhoea.

BACKGROUND: In recent years, new neonatal porcine diarrhoea (NNPD) of unknown aetiology has emerged in Denmark. NNPD affects piglets during the first week of life and results in impaired welfare, decreased weight gain, and in the worst-case scenario death. Commonly used preventative interventions such as vaccination or treatment with antibiotics, have a limited effect on NNPD. Previous studies have investigated the clinical manifestations, histopathology, and to some extent, microbiological findings; however, these studies were either inconclusive or suggested that Enterococci, possibly in interaction with Escherichia coli, contribute to the aetiology of NNPD. This study examined ileal and colonic luminal contents of 50 control piglets and 52 NNPD piglets by means of the qPCR-based Gut Microbiotassay and 16 samples by 454 sequencing to study the composition of the bacterial gut microbiota in relation to NNPD. RESULTS: NNPD was associated with a diminished quantity of bacteria from the phyla Actinobacteria and Firmicutes while genus Enterococcus was more than 24 times more abundant in diarrhoeic piglets. The number of bacteria from the phylum Fusobacteria was also doubled in piglets suffering from diarrhoea. With increasing age, the gut microbiota of NNPD affected piglet and control piglets became more diverse. Independent of diarrhoeic status, piglets from first parity sows (gilts) possessed significantly more bacteria from family Enterobacteriaceae and species E. coli, and fewer bacteria from phylum Firmicutes. Piglets born to gilts had 25 times higher odds of having NNPD compared with piglets born to multiparous sows. Finally, the co-occurrence of genus Enterococcus and species E. coli contributed to the risk of having NNPD. CONCLUSION: The results of this study support previous findings that points towards genus Enterococcus and species E. coli to be involved in the pathogenesis of NNPD. Moreover, the results indicate that NNPD is associated with a disturbed bacterial composition and larger variation between the diarrhoeic piglets.

Diet type dictates the gut microbiota and the immune response against Yersinia ruckeri in rainbow trout (Oncorhynchus mykiss).

This study investigated the influence of the rainbow trout (Oncorhynchus mykiss) commensal intestinal microbiota in connection to an experimental Yersina ruckeri infection, the causative agent of enteric redmouth disease. One marine and one plant diet was administered to two different groups of rainbow trout. The plant-based diet gave rise to an intestinal microbiota dominated by the genera Streptococcus, Leuconostoc and Weissella from phylum Firmicutes whereas phylum Proteobacteria/Bacteroidetes/Actinobacteria dominated the community in the marine fed fish. In connection to the Y. ruckeri bath challenge there was no effect of the diet type on the cumulative survival, but the number of Y. ruckeri positive fish as measured by plate count and the number of fish with a 'high' number of reads belonging to genus Yersinia as measured by 16S rRNA next-generation sequencing was higher for marine diet fed fish. Furthermore, the two experimental groups of fish showed a differential immune response, where Y. ruckeri challenged marine fed fish had a higher transcription of IL-1ß and MBL-2 relative to challenged plant diet fed fish. The data suggest that the plant diet gave rise to a prebiotic effect favouring the presence of bacterial taxons proving protective in connection to bath challenge by Y. ruckeri.

SecMet-FISH: labeling, visualization, and enumeration of secondary metabolite producing microorganisms.

Our understanding of the role of secondary metabolites in microbial communities is challenged by intrinsic limitations of culturing bacteria under laboratory conditions and hence cultivation independent approaches are needed. Here, we present a protocol termed Secondary Metabolite FISH (SecMet-FISH), combining advantages of gene-targeted fluorescence in situ hybridization (geneFISH) with in-solution methods (in-solution FISH) to detect and quantify cells based on their genetic capacity to produce secondary metabolites. The approach capitalizes on the conserved nature of biosynthetic gene clusters (BGCs) encoding adenylation (AD) and ketosynthase (KS) domains, and thus selectively targets the genetic basis of non-ribosomal peptide and polyketide biosynthesis. The concept relies on the generation of amplicon pools using degenerate primers broadly targeting AD and KS domains followed by fluorescent labeling, detection, and quantification. Initially, we obtained AD and KS amplicons from Pseuodoalteromonas rubra, which allowed us to successfully label and visualize BGCs within P. rubra cells, demonstrating the feasibility of SecMet-FISH. Next, we adapted the protocol and optimized it for hybridization in both Gram-negative and Gram-positive bacterial cell suspensions, enabling high-throughput single cell analysis by flow cytometry. Ultimately, we used SecMet-FISH to successfully distinguish secondary metabolite producers from non-producers in a five-member synthetic community.

RibDif2: expanding amplicon analysis to full genomes.

Motivation: As previously described, amplicon analysis of the bacterial 16S gene has several limitations owing to fundamental characteristics of both the 16S gene and technological restrictions. Previously, RibDif was introduced to help quantify these limitations by detailed analysis of a given genera and the 16S gene profile of its members, notably multiplicity and divergence of 16S alleles within genomes as well as shared alleles between species. Apart from using amplicon analysis for only the 16S gene, amplicons derived from genus-specific genes or even functional genes are increasingly being utilized. Moreover, long-read technologies are progressively being used to sequence longer amplicons, and since these inherently contain more information, they may likely alleviate the issues proposed in RibDif. Results: Taking these phenomena into account, we here propose RibDif2. RibDif2 retains the 16S-optimized functionality of the original RibDif but can now run any set of primers on any part of the genome in any set of organisms, be it prokaryote, eukaryote, or archaea. We demonstrate this new functionality by showing full species resolution of Pseudoalteromonas using complete rRNA-operon amplicons, as well as selection of optimally discriminatory primers for Staphylococcus and Pseudomonas. Moreover, we show a potential bias toward terrestrial bacteria relative to marine ones for primers amplifying biosynthetic gene clusters and lastly suggest optimal primers to differentiate the members of the insect genus Drosophila. We believe that RibDif2 will facilitate the work of all scientists using amplicon sequencing, especially in the era of long-read sequencing. Availability and implementation: Ribdif2 is freely available at https://github.com/Rob-murphys/ribdif.

Small Spatial Scale Drivers of Secondary Metabolite Biosynthetic Diversity in Environmental Microbiomes.

In the search for novel drug candidates, diverse environmental microbiomes have been surveyed for their secondary metabolite biosynthesis potential, yet little is known about the biosynthetic diversity encoded by divergent microbiomes from different ecosystems, and the environmental parameters driving this diversity. Here, we used targeted amplicon sequencing of adenylation (AD) and ketosynthase (KS) domains along with 16S sequencing to delineate the unique biosynthetic potential of microbiomes from three separate habitats (soil, water, and sediments) exhibiting unique small spatial scale physicochemical gradients. The estimated richness of AD domains was highest in marine sediments with 656 ± 58 operational biosynthetic units (OBUs), while the KS domain richness was highest in soil microbiomes with 388 ± 67 OBUs. Microbiomes with rich and diverse bacterial communities displayed the highest PK potential across all ecosystems, and on a small spatial scale, pH and salinity were significantly, positively correlated to KS domain richness in soil and aquatic systems, respectively. Integrating our findings, we were able to predict the KS domain richness with a RMSE of 31 OBUs and a R2 of 0.91, and by the use of publicly available information on bacterial richness and diversity, we identified grassland biomes as being particularly promising sites for the discovery of novel polyketides. Furthermore, a focus on acidobacterial taxa is likely to be fruitful, as these were responsible for most of the variation in biosynthetic diversity. Overall, our results highlight the importance of sampling diverse environments with high taxonomic diversity in the pursuit for novel secondary metabolites. IMPORTANCE To counteract the antibiotic resistance crisis, novel anti-infective agents need to be discovered and brought to market. Microbial secondary metabolites have been important sources of inspiration for small-molecule therapeutics. However, the isolation of novel antibiotics is difficult, and the risk of rediscovery is high. With the overarching purpose of identifying promising microbiomes for discovery of novel bioactivity, we mapped out the most significant drivers of biosynthetic diversity across divergent microbiomes. We found the biosynthetic potential to be unique to individual ecosystems, and to depend on bacterial taxonomic diversity. Within systems, and on small spatial scales, pH and salinity correlated positively to the biosynthetic richness of the microbiomes, Acidobacteria representing the taxa most highly associated with biosynthetic diversity. Ultimately, understanding the key drivers of the biosynthesis potential of environmental microbiomes will allow us to focus bioprospecting efforts and facilitate the discovery of novel therapeutics.

Comparative Genomics of Staphylococcus rostri, an Undescribed Bacterium Isolated from Dairy Mastitis.

This study characterizes 81 S. rostri isolates from bovine mastitis (of which 80 were subclinical). The isolates were first identified as S. microti by MALDI-TOF MS, but later whole genome sequencing analysis allowed reclassification as S. rostri. The isolates were derived from 52 cows and nine dairy herds in Denmark. To describe the pathogenicity of S. rostri, we used whole genome sequencing to infer the distribution of genes associated with virulence, antibiotic resistance, and mobile genetic elements. Also, we performed a core-genome phylogeny analysis to study the genetic relatedness among the isolates. All 81 isolates expressed the same virulence profile comprising two putative virulence genes, clpP and clpC. Three isolates carried a resistance gene encoding streptomycin (str) or lincomycin (lnuA) resistance. The distribution of plasmids suggested the detected antibiotic resistance genes to be plasmid-mediated. Phages were abundant among the isolates, and the single isolate from clinical mastitis acquired a phage disparate from the rest, which potentially could be involved with virulence in S. rostri. The core genome phylogeny revealed a strong genetic intra-herd conservation, which indicates the source of introduction being herd-specific and might further imply the ability of S. rostri to adapt to the bovine niche and spread from cow-to-cow in a contagious manner. With this study, we aim to acquaint clinicians and professionals with the existence of S. rostri which might have been overlooked so far.

Enhanced specificity of Bacillus metataxonomics using a tuf-targeted amplicon sequencing approach.

Bacillus species are ubiquitous in nature and have tremendous application potential in agriculture, medicine, and industry. However, the individual species of this genus vary widely in both ecological niches and functional phenotypes, which, hence, requires accurate classification of these bacteria when selecting them for specific purposes. Although analysis of the 16S rRNA gene has been widely used to disseminate the taxonomy of most bacterial species, this gene fails proper classification of Bacillus species. To circumvent this restriction, we designed novel primers and optimized them to allow exact species resolution of Bacillus species in both synthetic and natural communities using high-throughput amplicon sequencing. The primers designed for the tuf gene were not only specific for the Bacillus genus but also sufficiently discriminated species both in silico and in vitro in a mixture of 11 distinct Bacillus species. Investigating the primers using a natural soil sample, 13 dominant species were detected including Bacillus badius, Bacillus velezensis, and Bacillus mycoides as primary members, neither of which could be distinguished with 16S rRNA sequencing. In conclusion, a set of high-throughput primers were developed which allows unprecedented species-level identification of Bacillus species and aids the description of the ecological distribution of Bacilli in various natural environment.

Species and condition shape the mutational spectrum in experimentally evolved biofilms.

IMPORTANCE: Biofilm formation is a vital factor for the survival and adaptation of bacteria in diverse environmental niches. Experimental evolution combined with the advancement of whole-population genome sequencing provides us a powerful tool to understand the genomic dynamic of evolutionary adaptation to different environments, such as during biofilm development. Previous studies described the genetic and phenotypic changes of selected clones from experimentally evolved Bacillus thuringiensis and Bacillus subtilis that were adapted under abiotic and biotic biofilm conditions. However, the full understanding of the dynamic evolutionary landscapes was lacking. Furthermore, the differences and similarities of adaptive mechanisms in B. thuringiensis and B. subtilis were not identified. To overcome these limitations, we performed longitudinal whole-population genome sequencing to study the underlying genetic dynamics at high resolution. Our study provides the first comprehensive mutational landscape of two bacterial species' biofilms that is adapted to an abiotic and biotic surface.

The potential to produce tropodithietic acid by Phaeobacter inhibens affects the assembly of microbial biofilm communities in natural seawater.

Microbial secondary metabolites play important roles in biotic interactions in microbial communities and yet, we do not understand how these compounds impact the assembly and development of microbial communities. To address the implications of microbial secondary metabolite production on biotic interactions in the assembly of natural seawater microbiomes, we constructed a model system where the assembly of a natural seawater biofilm community was influenced by the addition of the marine biofilm forming Phaeobacter inhibens that can produce the antibiotic secondary metabolite tropodithietic acid (TDA), or a mutant incapable of TDA production. Because of the broad antibiotic activity of TDA, we hypothesized that the potential of P. inhibens to produce TDA would strongly affect both biofilm and planktonic community assembly patterns. We show that 1.9 % of the microbial composition variance across both environments could be attributed to the presence of WT P. inhibens, and especially genera of the Bacteriodetes were increased by the presence of the TDA producer. Moreover, network analysis with inferred putative microbial interactions revealed that P. inhibens mainly displayed strong positive associations with genera of the Flavobacteriaceae and Alteromonadaceae, and that P. inhibens acts as a keystone OTU in the biofilm exclusively due to its potential to produce TDA. Our results demonstrate the potential impact of microbial secondary metabolites on microbial interactions and assembly dynamics of complex microbial communities.

Long-read MinION™ sequencing of 16S and 16S-ITS-23S rRNA genes provides species-level resolution of Lactobacillaceae in mixed communities.

The Lactobacillaceae are lactic acid bacteria harnessed to deliver important outcomes across numerous industries, and their unambiguous, species-level identification from mixed community environments is an important endeavor. Amplicon-based metataxonomics using short-read sequencing of partial 16S rRNA gene regions is widely used to support this, however, the high genetic similarity among Lactobacillaceae species restricts our ability to confidently describe these communities even at genus level. Long-read sequencing (LRS) of the whole 16S rRNA gene or the near complete rRNA operon (16S-ITS-23S) has the potential to improve this. We explored species ambiguity amongst Lactobacillaceae using in-silico tool RibDif2, which identified allele overlap when various partial and complete 16S rRNA gene and 16S-ITS-23S rRNA regions were amplified. We subsequently implemented LRS by MinION™ to compare the capacity of V3-V4, 16S and 16S-ITS-23S rRNA amplicons to accurately describe the diversity of a 20-species Lactobacillaceae mock community in practice. In-silico analysis identified more instances of allele/species overlap with V3-V4 amplicons (n = 43) compared to the 16S rRNA gene (n = 11) and partial (n = up to 15) or complete (n = 0) 16S-ITS-23S rRNA amplicons. With subsequent LRS of a DNA mock community, 80% of target species were identified using V3-V4 amplicons whilst the 16S rRNA gene and 16S-ITS-23S rRNA region amplicons resulted in 95 and 100% of target species being identified. A considerable reduction in false-positive identifications was also seen with 16S rRNA gene (n = 3) and 16S-ITS-23S rRNA region (n = 9) amplicons compared with V3-V4 amplicons (n = 43). Whilst the target species affected by allele overlap in V3-V4 and 16S rRNA gene sequenced mock communities were predicted by RibDif2, unpredicted species ambiguity was observed in 16S-ITS-23S rRNA sequenced communities. Considering the average nucleotide identity (ANI) between ambiguous species (~97%) and the basecall accuracy of our MinION™ sequencing protocol (96.4%), the misassignment of reads between closely related taxa is to be expected. With basecall accuracy exceeding 99% for recent MinION™ releases, the increased species-level differentiating power promised by longer amplicons like the 16S-ITS-23S rRNA region, may soon be fully realized.

Tropodithietic Acid, a Multifunctional Antimicrobial, Facilitates Adaption and Colonization of the Producer, Phaeobacter piscinae.

In the marine environment, surface-associated bacteria often produce an array of antimicrobial secondary metabolites, which have predominantly been perceived as competition molecules. However, they may also affect other hallmarks of surface-associated living, such as motility and biofilm formation. Here, we investigate the ecological significance of an antibiotic secondary metabolite, tropodithietic acid (TDA), in the producing bacterium, Phaeobacter piscinae S26. We constructed a markerless in-frame deletion mutant deficient in TDA biosynthesis, S26ΔtdaB. Molecular networking demonstrated that other chemical sulfur-containing features, likely related to TDA, were also altered in the secondary metabolome. We found several changes in the physiology of the TDA-deficient mutant, ΔtdaB, compared to the wild type. Growth of the two strains was similar; however, ΔtdaB cells were shorter and more motile. Transcriptome and proteome profiling revealed an increase in gene expression and protein abundance related to a type IV secretion system, and to a prophage, and a gene transfer agent in ΔtdaB. All these systems may contribute to horizontal gene transfer (HGT), which may facilitate adaptation to novel niches. We speculate that once a TDA-producing population has been established in a new niche, the accumulation of TDA acts as a signal of successful colonization, prompting a switch to a sessile lifestyle. This would lead to a decrease in motility and the rate of HGT, while filamentous cells could form the base of a biofilm. In addition, the antibiotic properties of TDA may inhibit invading competing microorganisms. This points to a role of TDA in coordinating colonization and adaptation. IMPORTANCE Despite the broad clinical usage of microbial secondary metabolites with antibiotic activity, little is known about their role in natural microbiomes. Here, we studied the effect of production of the antibiotic tropodithietic acid (TDA) on the producing strain, Phaeobacter piscinae S26, a member of the Roseobacter group. We show that TDA affects several phenotypes of the producing strain, including motility, cell morphology, metal metabolism, and three horizontal gene transfer systems: a prophage, a type IV secretion system, and a gene transfer agent. Together, this indicates that TDA participates in coordinating the colonization process of the producer. TDA is thus an example of a multifunctional secondary metabolite that can mediate complex interactions in microbial communities. This work broadens our understanding of the ecological role that secondary metabolites have in microbial community dynamics.

Circulating cell-free HPV DNA is a strong marker for disease severity in cervical cancer.

For cervical cancer (CC), circulating cell-free HPV DNA (ccfHPV) may establish disease severity. Furthermore, HPV integration has been correlated to viral load and survival. In this study, pre-treatment plasma from 139 CC cases (50 primary surgery patients, 22 primary surgery + adjuvant oncological therapy patients, and 67 primary oncological therapy patients) was collected (2018-2020). Furthermore, plasma from 25 cervical intraepithelial neoplasia grade 3 patients and 15 healthy women (negative controls) were collected. Two next-generation sequencing (NGS) panels were used to establish ccfHPV presence and human papillomavirus type 16 (HPV16) integration status. ccfHPV was detected in four primary surgery (8.0%), eight primary surgery + adjuvant oncology (36.4%), and 54 primary oncology (80.6%) patients. For primary oncology patients with HPV16-related cancer (n = 37), more ccfHPVneg than ccfHPVpos patients had HPV16 integration (P = 0.04), and in patients with HPV16 integration (n = 13), ccfHPVpos patients had higher disease stages than ccfHPVneg patients (P = 0.05). In summary, ccfHPV presence is related to disease severity and may add to the debated Sedlis criteria used for identifying patients for adjuvant oncological therapy. However, ccfHPV detection is influenced by HPV integration status and disease stage, and these factors need to be considered in ccfHPVneg patients.

Soil conditions and the plant microbiome boost the accumulation of monoterpenes in the fruit of Citrus reticulata 'Chachi'.

BACKGROUND: The medicinal material quality of Citrus reticulata 'Chachi' differs depending on the bioactive components influenced by the planting area. Environmental factors, such as soil nutrients, the plant-associated microbiome and climatic conditions, play important roles in the accumulation of bioactive components in citrus. However, how these environmental factors mediate the production of bioactive components of medicinal plants remains understudied. RESULTS: Here, a multi-omics approach was used to clarify the role of environmental factors such as soil nutrients and the root-associated microbiome on the accumulation of monoterpenes in the peel of C. reticulata 'Chachi' procured from core (geo-authentic product region) and non-core (non-geo-authentic product region) geographical regions. The soil environment (high salinity, Mg, Mn and K) enhanced the monoterpene content by promoting the expression of salt stress-responsive genes and terpene backbone synthase in the host plants from the core region. The microbial effects on the monoterpene accumulation of citrus from the core region were further verified by synthetic community (SynCom) experiments. Rhizosphere microorganisms activated terpene synthesis and promoted monoterpene accumulation through interactions with the host immune system. Endophyte microorganisms derived from soil with the potential for terpene synthesis might enhance monoterpene accumulation in citrus by providing precursors of monoterpenes. CONCLUSIONS: Overall, this study demonstrated that both soil properties and the soil microbiome impacted monoterpene production in citrus peel, thus providing an essential basis for increasing fruit quality via reasonable fertilization and precision microbiota management. Video Abstract.

The effects of antibiotic use on the dynamics of the microbiome and resistome in pigs.

Antibiotics are widely used in pig farming across the world which has led to concerns about the potential impact on human health through the selection of antibiotic resistant pathogenic bacteria. This worry has resulted in the development of a production scheme known as pigs Raised Without Antibiotics (RWA), in which pigs are produced in commercial farms, but are ear-tagged as RWA until slaughter unless they receive treatment, thus allowing the farmer to sell the pigs either as premium priced RWA or as conventional meat. Development of antibiotic resistance in pig farming has been studied in national surveys of antibiotic usage and resistance, as well as in experimental studies of groups of pigs, but not in individual pigs followed longitudinally in a commercial pig farm. In this study, a cohort of RWA designated pigs were sampled at 10 time points from birth until slaughter along with pen-mates treated with antibiotics at the same farm. From these samples, the microbiome, determined using 16S sequencing, and the resistome, as determined using qPCR for 82 resistance genes, was investigated, allowing us to examine the difference between RWA pigs and antibiotic treated pigs. We furthermore included 176 additional pigs from six different RWA farms which were sampled at the slaughterhouse as an endpoint to substantiate the cohort as well as for evaluation of intra-farm variability. The results showed a clear effect of age in both the microbiome and resistome composition from early life up until slaughter. As a function of antibiotic treatment, however, we observed a small but significant divergence between treated and untreated animals in their microbiome composition immediately following treatment, which disappeared before 8 weeks of age. The effect on the resistome was evident and an effect of treatment could still be detected at week 8. In animals sampled at the slaughterhouse, we observed no difference in the microbiome or the resistome as a result of treatment status but did see a strong effect of farm origin. Network analysis of co-occurrence of microbiome and resistome data suggested that some resistance genes may be transferred through mobile genetic elements, so we used Hi-C metagenomics on a subset of samples to investigate this. We conclude that antibiotic treatment has a differential effect on the microbiome vs. the resistome and that although resistance gene load is increased by antibiotic treatment load, this effect disappears before slaughter. More studies are needed to elucidate the optimal way to rear pigs without antibiotics.

The Gut Microbiota of Healthy and Flavobacterium psychrophilum-Infected Rainbow Trout Fry Is Shaped by Antibiotics and Phage Therapies.

In the aquaculture sector, there is an increased interest in developing environmentally friendly alternatives to antibiotics in the treatment and prevention of bacterial infections. This requires an understanding of the effects of different treatments on the fish microbiota as a measure for improving the fish health status. In this study, we focused on the freshwater pathogen Flavobacterium psychrophilum and investigated the effects of antibiotics (florfenicol) and phage therapies on the gut microbiota of healthy and infected rainbow trout fry (1-2 g). Florfenicol-coated feed was administered for 10 days, starting two days after the infection procedure. A two-component mix of phage targeting F. psychrophilum (FpV4 and FPSV-D22) was continuously delivered by feed with a prophylactic period of 12 days. Samples of the distal intestine were collected over time (day -1 and 1, 8, and 33 days post-infection) and analyzed by community analysis targeting the 16S rRNA gene (V3-V4 region). Results showed the dysbiosis effect caused both by the infection and by florfenicol administration. Shifts in the overall composition were detected by ß-diversity analysis, and changes in specific populations were observed during taxonomic mapping. Measures of α-diversity were only affected in infected fish (large variation observed 1 and 8 dpi). These community alterations disappeared again when fish recovered from the infection and the antibiotic treatment was terminated (33 dpi). Interestingly, phage addition altered the microbiota of the fish independently of the presence of their target bacterium. The overall gut bacterial community in fish fed phage-treated feed was different from the controls at each time point as revealed by ß-diversity analysis. However, it was not possible to identify specific bacterial populations responsible for these changes except for an increase of lactic acid bacteria 33 dpi. Overall, the results indicate that the administered phages might affect the complex network of phage-bacteria interactions in the fish gut. Nevertheless, we did not observe negative effects on fish health or growth, and further studies should be directed in understanding if these changes are beneficial or not for the fish health with an additional focus on the host immune response.