Bracon brevicornis is an ectoparasitoid of a wide range of larval-stage Lepidopterans, including several pests of important crops, such as the corn borer, Ostrinia nubilalis. It is also one of the earliest documented cases of complementary sex determination in Hymenoptera. Here, we present the linked-read-based genome of B. brevicornis, complete with an ab initio-derived annotation and protein comparisons with fellow braconids, Fopius arisanus and Diachasma alloeum. We demonstrate the potential of linked-read assemblies in exploring regions of heterozygosity and search for structural and homology-derived evidence of the complementary sex determiner gene (csd). Bracon brevicornis (Wesmael) is a gregarious ectoparasitoid of various Lepidoptera larvae, including many important pests, and is considered a cosmopolitan species [B. brevicornis has been classified under the genus Habrobracon [Microbracon [Habrobracon/Bracon hebetor [B. brevicornis and B. hebetor are genetically two distinct species [B. brevicornis has shown potential as a biological control agent against important pest species in stored corn stalks, such as Ostrinia nubilalis and Sesamia cretica [Opisinia arenosella [B. brevicornis attacks a wide range of larval host such as Ephesthia kuehniella, Galleria spp., and Spodoptera spp. [ species ,2. In throbracon , Microbrrobracon , or clas hebetor ; however species . In the  cretica , or agaiera spp. .B. brevicornis has included both laboratory and semi-field set-ups to determine both its efficacy as a biological control agent as well as its suitability as a study system. There are several studies on the biology of B. brevicornis, including on population growth potential [Work on otential , their hotential , interspotential , clutch otential , mate chotential ,12, dietotential .B. brevicornis falls within the subfamily Braconinae, the largest of the cyclostome-forming braconid wasps [B. brevicornis is an ectoparasitoid, and its position within a family that contains both types of parasitism lifestyles holds promise for further phylogenetic comparisons.Within a phylogenetic perspective, id wasps . The preid wasps ? Within id wasps . These sid wasps . Yet, a B. brevicornis has a haplodiploid sex determination system where males develop from unfertilized eggs and females develop from fertilized eggs [B. brevicornis belongs to a genus where sex determination and diploid male production have been widely studied . Indeeduglandis , revieweuglandis ), and rebehavior .Apis mellifera (L.) (Hymenoptera: Apidae), with the identification of the complementary sex determiner (csd) gene [Csd is a duplication of feminizer (fem), a transformer (tra) ortholog [csd initiates the female-specific splicing of fem, which then autoregulates its own female-specific splicing, ultimately resulting in female development [tra/fem have been identified in species that are presumed to have CSD [tra/fem duplications have not been analyzed for potential heterozygosity. There has been some debate on the origin of tra/fem duplications. One theory states that the duplication of fem to produce csd occurred before the divergence of ants and bees, and that the paralogs evolved in concerted evolution [fem duplications are independent events [tra/fem duplications and identify the genes underlying CSD. However, an assembled genome is usually haploid as areas of heterozygosity are collapsed in the final stages of assembly, diminishing the potential to find the gene(s) underlying CSD by its most notable feature: heterozygosity. Yet recent advances in sequencing and analysis gave us the ability to view heterozygous regions—known as “phases” in diploid assemblies—within a genome, which allow us to investigate the genes potentially involved in CSD.While straightforward to detect phenotypically through the formation of diploid males following inbreeding ,19, the sd) gene ,25. Heteortholog  that is ortholog . When heelopment ,28. Withhave CSD , but thevolution ,30, whilt events ,32. ThisB. brevicornis strain using 10X Genomics technology that relies on linked-read sequencing . Due to their long history of genetic isolation during laboratory rearing, individuals in this strain are assumed to have a high level of homozygosity, whereas a csd locus would retain its heterozygosity in the females. The 10X Genomics technology allows for generating phased data in which allelic variants can be identified after assembly. High-molecular weight DNA is partitioned into small droplets containing a unique barcode and adapter in such a way that only a few DNA molecules are present within each droplet.Here, we report on the whole-genome sequencing of a pool of females from an isolated csd candidates in the female-derived B. brevicornis genome after sequencing by studying the phased data containing the different haplotypes. Moreover, as B. brevicornis is a potential biological control agent of several pests, the availability of a full genome may provide effective ways to study and improve this species to grow it into an established biological control agent for Lepidopteran pests.Within each droplet  the DNA is used as template for random priming and elongation resulting in many short DNA fragments sharing the same unique barcode. These DNA fragments are converted into a library that can be sequenced on an Illumina sequence platform. In the assembly step, the reads originating from the same fragment are organized by barcode and put together into synthetic linked-read fragments. Importantly, it is nearly impossible that two fragments with opposing allelic variants are together in the same droplet , though B. brevicornis were taken from the laboratory colony L06. The colony was initiated in 2006 from naturally parasitized O. nubilalis larvae collected in maize fields near Leipzig, Germany. Species identification was first carried out by Matthias Schöller and Cornelis van Achterberg based on morphological characteristics [E. kuehniella [B. hebetor [Individuals of eristics . Since cehniella . The spe2O and recombined at the end of the extraction process (1.0 mL). DNA concentration was measured with an Invitrogen Qubit 2.0 fluorometer using the dsDNA HS Assay Kit  with final assessments for DNA quality, amount, and fragment size confirmed via BioAnalyzer 2100 .Immediately following emergence, 100 to 120 female wasps were flash frozen in liquid nitrogen and ground with a mortar and pestle. Genomic DNA was extracted using a protocol modified from Chang, Puryear, and Cairney . ModificB. brevicornis is relatively small for the scale of the 10X platform, there is a higher risk of overlapping fragments within single GEMs. In order to reduce this risk, genomic DNA of a larger and previously analyzed genome  (Solanaceae), commercial variety Heinz 1607) [S. lycopersicon followed the protocol of Hosmani et al. [B. brevicornis and S. lycopersicon was pooled in a 1:4 molar ratio.As the genome of nz 1607)  was usedi et al. . The DNAOne nanogram of this pooled DNA was used for 10X Genomics linked-read library preparation following the Chromium Genome Reagent Kits Version 1 User Guide (CG-00022) . Barcoded linked-read DNA fragments were recovered for final Illumina library construction . The library was used for 2 × 150 bp paired-end sequencing on one lane of an Illumina HiSeq 2500 at the business unit Bioscience of Wageningen University and Research . Sequencing data were then used for basecalling and subsequent demultiplexing using Longranger (v2.2.2) (10X Genomics) (command—mkfastq), yielding 212,910,509 paired-end reads with a read length of 150 bp.To filter sequence data from tomato carrier DNA, 23 bp (16 bp GEM + 7 bp spacer) was removed from forward reads and all reads were subsequently mapped to an in-house high quality reference assembly of the Heinz genome using BWA-MEM v0.7.17 . Using sB. brevicornis scaffolds, i.e., bacterial scaffolds from sample microbiome, was performed with BlobTools (v1.0) [−25) for taxonomical classification and BWA-MEM mapping of reads against scaffolds for coverage statistics. Reads mapping only against “Arthropoda” classified scaffolds were then extracted and used for a final k-mer analysis using jellyfish v2.1.1  [To verify whether there were no Heinz leftovers in the assembly, minimap2 v2.17-r941  was useds (v1.0) , relying000,000)  and Geno000,000)  to infercsd analysis, while the pseudohap scaffolds are now the assembly used for annotation.Assembly completeness was determined using BUSCO (v3.0.2) with the insect_odb9 ortholog set and the fly training parameter  while asFopius arisanus (Sonan) (Hymenoptera: Braconidae) and Diachasma alloeum (Muesebeck) (Hymenoptera: Braconidae). Both sets of coding sequences were retrieved from the NCBI Assembly Database, version ASM8063v1 for F. arisanus and version Dall2.0 for D. alloeum [The coding sequences of two additional braconids  were alloeum ,47,48.B. brevicornis assembly [F. arisanus were set as a query to the genome of B. brevicornis using default parameters (except minIdentity = 50) [B. brevicornis using Nasonia vitripennis   as the species parameter in Augustus (--species = nasonia –extrinsiccCfgFile = extrinsic.E.cfg).For gene prediction, Augustus (v2.5.5) was first used to predict genes from the assembly . Using BF. arisanus and D. alloeum  using Proteinortho  [B. brevicornis genes and the orthologs of F. arisanus and D. alloeum were retrieved using samtools for comparison [After prediction, the protein sequences were retrieved and compared to both = 0.001) . From thmparison . The measourceforge.net/projects/bbmap/) (ac = f) to remove all parallel pseudohaplotypes that were complete duplicates as these scaffolds were not heterozygous. The remainder of the set contained both scaffolds that previously had a duplicate, as well as solitary scaffolds that did not have a partner scaffold. These unique scaffolds were removed using the “filter by name” tool in BBTools, leaving 258 scaffolds, or 129 pairs of pseudohap2 scaffolds. Pairs were pairwise aligned in CLC Genomics Workbench 12  using default settings .The pseudohap2 files were deduplicated using the dedupe tool within BBTools (B. brevicornis assembly was performed using the Apis mellifera Feminizer protein (NP_001128300) as query in Geneious Prime 2019.1.3  with Nasonia vitripennis tra (NP_001128299) and N. vitripennis for the specific gene-finding parameters [fem duplication, a stretch of ~10 Kbp directly upstream of the annotated putative fem was again annotated using FGENESH+ (Softberry) with Nasonia vitripennis tra (NP_001128299) and N. vitripennis for the specific gene-finding parameters [A. mellifera csd (ABU68670) and fem (NP_001128300); N. vitripennis tra (XP_001604794) and B. brevicornis putative fem and B. brevicornis putative fem duplicate (fem1), using MAFFT v7.450 with the following settings: Algorithm = auto, Scoring matrix = BLOSUM62, Gap open penalty = 1.53, Offset value = 0.123 [A local tBLASTn search against the entire ous.com, ). The prous.com, ,50. Nextrameters . Only thrameters . The pre = 0.123 ,55.tra or fem in A. mellifera and N. vitripennis using a combination of the online tool SimpleSynteny [fem , tra  or the putative fem  were extracted from their respective genomes  and searched with protein sequence from the following genes: tra (GeneID: 00121203), LOC100121225, LOC100678616, LOC100680007 originating from N. vitripennis; fem (GeneID:724970), csd (GeneID:406074), LOC408733, LOC551408, LOC724886 originating from A. mellifera. The advanced settings for SimpleSynteny were as follows: BLAST E-value Threshold = 0.01, BLAST Alignment type = Gapped, Minimum Query Coverage Cutoff = 1%, Circular Genome Mode = Off. If the gene was not found within the extracted scaffold, it was searched for in the full genome assembly. For the image settings, Gene Display Mode = Project Full-Length Gene. This generated image was used together with results from the tBLASTn searches as template to draw the final figure. The final figure that we present in the Results and Discussion section depicts ~0.9 Mbp of genomic region for all three species.A microsynteny analysis was achieved by comparing the arrangement of a set of homologous genes directly upstream and downstream of eSynteny  and tBLAB. brevicornis after removal of carrier DNA and contamination, as well as the assembly, can be found in the EMBL-EBI European Nucleotide Archive (ENA) under BioProject PRJEB35412. Both the assembly file (.fasta) (https://doi.org/10.6084/m9.figshare.12674189.v2) and the complete annotation file (.gff) (https://doi.org/10.6084/m9.figshare.12073911.v2) are available in a separate repository. Contaminated pseudohap scaffolds are available for download alongside the two pseudohap2 FASTA files, more details are provided at https://doi.org/10.17026/dans-xn6-pjm8.Raw sequence data for B. brevicornis DNA was extracted, which was then reduced to 1 ng/μL for library preparation. Sequencing of the Heinz diluted library resulted in a total yield of 54 Gbp of data (corrected for 10X Genomics 23 bp segment of forward reads). Mapping against the Heinz genome assembly showed a mapping percentage of 84.8%. There was a total of 30,278,915 unmapped pairs, comprising ~8.39 Gbp of data. This corresponds to the 4:1 ratio between Heinz and B. brevicornis DNA in the library. Further scaffold decontamination with BlobTools resulted in a separation of the assembly into B. brevicornis scaffolds and microbiome scaffolds. The final assembled genome is ~123 Mbp in size, comprised of 353 scaffolds. This is similar to the projected physical genome size of 133 Mbp  and a heterozygosity of ~0.54%. Peak coverage was 27x. The disparity in projected physical genome size and assembled genome size may be due to the decontamination process potentially removing extremely repetitive regions, as ~10.9 Mbp of the removed scaffolds returned either no hits or were classified as undefined at the taxa level. These scaffolds were clearly separated in the blobplot due to their high coverage. The potential effects are negligible, as the BUSCO analysis indicates a completeness of 98.7% .A total of 172 ng of 133 Mbp  and D. alloeum , the two closest relatives of B. brevicornis for which public data are available. Proteinortho analysis resulted in 7660 three-way orthology groups (7830 B. brevicornis genes), while 362 orthology groups contained proteins of B. brevicornis and F. arisanus (382 B. brevicornis genes), and 451 groups contained B. brevicornis and D. alloeum genes (479 B. brevicornis genes). A large number of orthology groups (2492) had no B. brevicornis genes, while 3995 predicted genes remain ungrouped.In total, 12,686 genes were predicted, with an average coding sequence length of 529.86 amino acids. The number of genes correspond well to those found in F. arisanus, the mean relative length of predicted B. brevicornis genes was 1.016, while the mean relative length for the two- and three-way orthology groups was 0.996. Similar results were obtained for comparisons to D. alloeum, where mean relative length for B. brevicornis genes was 1.011, and 0.988 for the two- and three-way orthology groups. Furthermore, the pairwise lengths of all these proteins are fairly similar  [feminizer in the genome. As feminizer  was not identified in the ab-initio annotation, we used a local tBLASTn search to find fem in the assembly. Four hits with E-value from 5.86 × 10−4 to 8.59 × 10−8 were found in scaffold 12. Searching the annotation using part of the tBLASTn result shows that it is annotated as “g7607” (locus tag = BBRV_LOCUS33129) which gave a first hit with protein O-glucosyltransferase 2  after a BLASTp search, and no fem or tra hits were found. A closer inspection showed that “g7607” is annotated as fusion protein with the N-terminal part resembling fem and the C-terminal part putatively encoding O-glucosyltransferase 2. Next, we used FGENESH+ to re-annotate the genomic region, resulting in a full-length putative B. brevicornis feminizer (Bbfem) ortholog containing seven exons  , and we en exons . We founve exons , which widentity . The codBbfem, as well as Bbfem1, are highly similar to each other and both contain all known fem/tra domains -rich region which is present only in Bbfem (124–153aa), but it otherwise appears to encode for a full-length protein. The csd-specific hypervariable domain  [We utilized the 10X Genomics linked-read approach to obtain pseudohaploid information that would allow us to search for potential t to fem  to limitgure S1) . Both gegure S1) ).B. brevicornis, A. mellifera, and N. vitripennis with the translocation of LOC408733 (A. mellifera) and the absence of LOC100121225 and LOC100678616 (N. vitripennis) in the B. brevicornis genome region. It is known that genomic regions encoding sex determination genes are dynamic in nature, showing both duplications and translocations [Tra/fem duplications have been shown in CSD systems before, most notably in A. mellifera where a fem gene duplication event resulted in it becoming a csd locus [fem with varying orientations are observed [tra duplications have been observed [fem paralogs originated due to a single duplication event and functions as csd [Bbfem paralog, Bbfem1, is a promising csd gene candidate in B. brevicornis. However, in-depth analyses are required to verify this, including in-depth sequencing of individual females, gene expression analyses, and further phylogenetic studies with other CSD Hymenopterans, all of which are now possible with an assembled, annotated, and published genome.Our synteny analysis showed low synteny between ocations . Tra/femsd locus ,28, but observed . In addiobserved ,63,64. As as csd ,30, or es as csd ,32, we s


This study was conducted to investigate the effects of hot-melt extruded ZnO nano-particles (HME-ZnO) as an alternative for P-ZnO on growth performance, nutrient digestibility, Zn bioavailability, intestinal microbiota, and intestinal morphology of weanling pigs.A total of 450 piglets (Landrace×Yorkshire×Duroc) were randomly allotted to five treatments based on initial body weight and sex. The experimental diets were fed in a meal form as phase 1 from d 0 to 14 and phase 2 from d 15 to 28. Treatments were the control diet without ZnO supplementation, the diet containing 2,500 ppm Zn as ZnO, and three diets containing 500, 1,000, or 2,500 ppm Zn as HME-ZnO.Lactobacillus spp. populations and lower Clostridium spp. (p<0.05) and Coliforms (p<0.01) populations in the ileum. Weanling pigs fed diets supplemented with increasing concentrations of HME-ZnO linearly decreased Clostridium spp. (p<0.05) and Coliforms (p<0.01) in the ileum. Lower (p<0.05) Clostridium spp. and Coliforms counts in the colon were observed in pigs fed with ZnO-supplemented diets. Weanling pigs fed diets supplemented with ZnO showed a greater (p<0.01) villus height in the duodenum.The overall result showed a higher (p<0.01) average daily gain in weanling pigs fed ZnO-supplemented diets in comparison to the control diet. There was a decrease (p<0.01) in fecal score in the ZnO-supplemented diets. Dietary supplementation of ZnO improved (p<0.05) crude protein digestibility. The weanling pigs fed the P-ZnO diet had a lower (p< 0.01) Zn digestibility in the feces than HME-ZnO supplemented treatments. Weanling pigs fed diets supplemented with ZnO had greater (p<0.05) Clostridium, villus height in duodenum and ileum. Moreover, HME-ZnO showed a higher Zn digestibility compared with P-ZnO.Dietary supplementation of HME-ZnO and P-ZnO showed a potential to improve the digestibility of protein, intestinal Coliform and  Escherchia coli (E. coli) and Clostridium perfringens as the most common infectious agents during weaning may increase the incidence of diarrhea and decrease growth of weanling pigs  agar), and Coliform (violet red bile agar) were used. The anaerobic conditions during the assay of total anaerobic bacteria and Clostridium spp. were created by using gas pack anaerobic system . The tryptic soy agar (No. 236950) and violet red bile agar (No. 216695) were purchased from Difco Laboratories (USA), and TSC agar (CM0589) were purchased from Oxoid . The bacterial concentrations were transformed to log before statistical analysis.To evaluate the microbial population, on d 28, fresh digesta samples of ileum, cecum, and colon from 2 euthanized pigs (around the average BW) in each pen were collected and kept on ice. One gram of the digesta from ileum, cecum, or colon was diluted with 9 mL of 1% peptone broth  and then homogenized. Viable counts of bacteria in the samples were then conducted by plating serial 10-fold dilutions to determine the total anaerobic bacteria (tryptic soy agar), To evaluate the small intestinal villus height and crypt depth, on d 28, intestinal samples from euthanized pigs  in each pen were collected. For each intestinal sample, three cross-sections were prepared after staining with azure A and eosin using standard paraffin embedding procedures . A totalThe apparent total tract digestibility was calculated as:diet, chromium content in the diet; nutrientfeces, nutrient content in the feces; Crfeces, chromium content in the feces; nutrientdiet, nutrient content in the diet.where, CrOrthogonal contrasts  were used to compare the possible relationship between the treatments, using the procedure PROC general linear model of SAS . In addition, orthogonal polynomial contrasts were applied to evaluate the significance of linear or quadratic models to describe the response in increasing dietary HME-ZnO levels . The pen was used as the experimental unit for the analysis of all the parameters. Probability values less than 0.05 were considered as significance.Growth performance and fecal score data are presented in The effects of dietary supplemented Zn source and concentration on nutrient digestibility are shown in IgG, IgA, and IgM levels were not affected in weanling pigs fed diets supplemented with ZnO sources in phase 1 and 2 .There was an increase (p<0.01) in the concentration of Zn in feces with ZnO supplementation in the diets in both phases . The weaLactobacillus spp. populations and lower Clostridium spp. and Coliforms populations in the ileum compared with the control. Weanling pigs fed diets supplemented with increasing concentrations of HME-ZnO decreased Clostridium spp.  and Coliforms  in the ileum. There were no differences in Lactobacillus spp., Clostridium spp., and Coliforms population in the cecum between pigs fed the ZnO-supplemented diets and the control diet, and also between HME-supplemented and P-ZnO treatments. In the cecum, Clostridium spp. population decreased  with a dietary increasing concentration of HME-ZnO. Lower (p<0.05) Clostridium spp. and Coliforms counts in colon were observed in weanling pigs fed with ZnO-supplemented diets than that of pigs fed the control diet. The counts of Clostridium spp. in cecum and colon were linearly decreased (p<0.01) in weanling pigs fed HME-ZnO diets.Effects of dietary supplemented Zn source and concentration on intestinal microbiota are shown in Effects of dietary supplemented Zn source and concentration on small intestinal morphology are shown in E. Coli [The greater overall ADG in weanling pigs fed ZnO-supplemented diets could be explained in part by the improved digestibility of nutrients. In addition, the pathogens suppression in the commensal gut microbiota in the intestine, besides a potential promotion of intestinal morphology, may further render nutrients available for weaned pigs by high dietary ZnO doses supplementation. There are two possible hypotheses that may justify the significant role of ZnO in improving the growth performance of weanling pigs. Firstly, Zn is a well-known anti-stress element and the positive effects on ADG may be explained by the antioxidative role of Zn as a main contributor to antioxidant enzymes production . EspeciaE. Coli . Un-absoE. Coli ,15. AparE. Coli –18. ThusE. Coli ,20. It wE. Coli . This agE. Coli ,19,20. HE. Coli ,5,17. ThFecal Zn concentration was greater in pigs fed ZnO-supplemented diets compared with the control. The concentration of Zn in the feces of weaning pigs fed the diets supplemented with 1,750 or 3,500 mg/kg of Zn was consistently greater than that pigs fed the control diet . It was Lactobacillus spp. in the ileum than pigs fed the control diets, which is in contrast to previous findings [Clostridium spp. in the ileum of pigs fed ZnO diets were lower than pigs fed the control diet. A 2,500 ppm ZnO/kg diet needs to be supplemented into the diet of weanling pigs to exert anti-diarrhea effects [E. coli, while Lactobacillus spp. can tolerate an acidic environment [Clostridium) populations than Lactobacillus spp. Therefore, it is assumed that ZnO has the ability to limit pathogen growth and indirectly gives an opportunity for Lactobacillus spp. to proliferate in a less competitive environment. In contrast, another study showed no additional benefits of using ZnO on ileal Lactobacillus spp. populations, but reported greater ileal total anaerobic bacteria and lower Coliforms population [Clostridium spp. population when lowering the doses of Zn to 50, 150, 250, and 1,000 ppm, most likely reflecting a decreased pathogenic bacteria in the ileum of weanling pigs receiving the high Zn level [Clostridium spp. in the ileum were linearly decreased by increasing dietary HME-ZnO supplementation. The increase of ZnO surface area by decreasing particle size can increase the antibacterial activity by efficient H2O2 generation [In the present trial, pigs fed diets supplemented with ZnO had greater findings ,17. In a effects . The ant effects . Hydrogeironment . The respulation . Anotherneration . Yamamotneration  reportedPost-weaning stress causes transient crypt hyperplasia and villus atrophy during weaning ,7. The pIn conclusion, dietary supplementation of HME-ZnO and P-ZnO improved the performance of weaned pigs, which may be associated with the regulated composition of the gut microflora, and improved digestibility of CP and intestinal villus height of piglets. In addition, dietary supplementation with sub-pharmacological dose of HME-ZnO may be an alternative to the use of P-ZnO due to the insignificant differences in performance and lower Zn excretion. However, further studies are needed to indicate what dose of HME-ZnO is the best recommendation for weanling piglets.


Frankiaceae are bacterial endosymbionts that are also found free-living in soil. Here, we present the genome sequences of two novel bacterial members of the order Frankiales, class Actinobacteria, isolated from temperate terrestrial forest soils. The genomes for MT45 and GAS493 indicate a genetic capacity for carbohydrate degradation but not nitrogen fixation. Frankiaceae are bacterial endosymbionts that are also found free-living in soil. Here, we present the genome sequences of two novel bacterial members of the order Frankiales, class Actinobacteria, isolated from temperate terrestrial forest soils. The genomes for MT45 and GAS493 indicate a genetic capacity for carbohydrate degradation but not nitrogen fixation. Frankiaceae are best known as spore-forming, nitrogen-fixing actinobacteria  (Genomic DNA was extracted using the Qiagen Genomic-tip 500/G kit. SMRTbell libraries were constructed and sequenced on the PacBio RS platform at the U.S. Department of Energy (DOE) Joint Genome Institute (JGI) , generatGenes were identified using Prodigal , manuallGeodermatophilus species, which had D values of 0.241 and 0.244, respectively. MT45 has two rRNA operons; the two 16S rRNA genes are identical in sequence and bear 99% identity to those of Jatrophihabitans sp. GAS493 and less than 97% to any other cultivated bacteria.Genomes were compared by average nucleotide identity (ANI) and using Mash  in KBaseFrankiaceae (Frankia spp. can fix nitrogen based on the acetylene reduction assay without NifH homologs detected by PCR amplification (Carbohydrate utilization is observed among only some nkiaceae , and botnkiaceae . Both lankiaceae . Both genkiaceae . Althougfication , the mecTogether, these genomes suggest multiple mechanisms for growth in soils where nutrients can be poor and sparsely available.SRX2158410 for Frankineae sp. MT45 and accession no. SRX3048269 for Jatrophihabitans sp. GAS493. Genome assemblies have been deposited in GenBank under the accession no. LT629697 for Frankineae sp. MT45 and accession no. LT907982 for Jatrophihabitans sp. GAS493.Raw reads are available for download in the Sequence Read Archive under the accession no.


Porphyromonas gingivalis, a prominent component of oral microorganism communities, is the main pathogen that causes periodontitis. As a result of the extensive analysis of this organism, the evidence of its connection to systemic diseases has become more apparent over the last decade. A significant amount of research has explored the role of Porphyromonas gingivalis in atherosclerosis, Alzheimer’s disease, rheumatoid arthritis, diabetes, and adverse pregnancy outcomes, while relatively few studies have examined its contribution to respiratory diseases, nonalcoholic fatty liver disease, and depression. Here, we provide an overview of the current state of knowledge about Porphyromonas gingivalis and its systemic impact in an aim to inform readers of the existing epidemiological evidence and the most recent preclinical studies. Additionally, the possible mechanisms by which Porphyromonas gingivalis is involved in the onset or exacerbation of diseases, together with its effects on systemic health, are covered. Although a few results remain controversial, it is now evident that Porphyromonas gingivalis should be regarded as a modifiable factor for several diseases.The relationship between periodontitis and systemic diseases, notably including atherosclerosis and diabetes, has been studied for several years.  Porphyromonas gingivalis  has proved to be closely correlated with the occurrence and development of many systemic diseases, such as atherosclerosis, cancer, and Alzheimer’s disease [P. gingivalis in systemic diseases have been discussed for several years, and several reviews have summarized the effects and the inner mechanisms [P. gingivalis and one specific disease or one class of diseases. A global understanding of the effect of P. gingivalis on overall health is lacking. In this review, we systematically and comprehensively provide an update and summary of the literature on P. gingivalis-related systemic diseases that affect the whole body, as well as the internal mechanisms, to provide a more comprehensive understanding of P. gingivalis and its relationship with systemic diseases.Chronic periodontitis, a multifactorial chronic inflammatory disease resulting from dysbacteriosis, is characterized by the destruction of connective tissue and alveolar bone, and it has become the primary reason for tooth loss in adults. It affects nearly 50% of the population worldwide, representing one of the most common inflammatory diseases in humans . Over th disease ,3,4. Thechanisms ,7,8,9,10P. gingivalis, one of over 700 bacterial species in the oral cavity, is a Gram-negative, anaerobic, rod-shaped bacteria that forms black colonies on blood agar and requires the presence of heme or hemin and vitamin K in its growth milieu. It is a successful colonizer of the oral epithelium and an important component of subgingival microbiomes [P. gingivalis is responsible for the chronic form of periodontitis, as it can remodel the commensal bacterial community to promote a state of dysbiosis [P. gingivalis largely depend on its diverse virulence factors, including its own structural components  and secretory components (gingipains and outer membrane vesicles).robiomes . P. gingysbiosis . Throughysbiosis . The surP. gingivalis to specifically bind to eukaryotic cells and other species of bacteria to enhance bacterial motility, biofilm formation, and bacterial invasion of the cells [P. gingivalis lipopolysaccharide (LPS) can trigger the innate immune response via activation of Toll-like receptors (TLRs) [P. gingivalis LPS exhibits two predominant variations of acylation that are attributed to different strains and microenvironmental conditions: the penta-acylated LPS activates TLR4, while tetra-acylated LPS acts as a TLR4 antagonist and TLR2 agonist [P. gingivalis is remarkably immunogenic and plays a critical role in P. gingivalis-induced autoimmune diseases [P. gingivalis are enriched in major virulence mediators, such as gingipains, LPS, and the capsule, and participate in biofilm development, host interaction, colonization, and immune defense evasion [P. gingivalis-associated systemic disorders [Fimbriae are crucial for enabling he cells . It can he cells . P. gings (TLRs) . The lip agonist . The headiseases . Gingipadiseases . Outer m evasion . Moreoveisorders .P. gingivalis in local periodontal tissue can enter the vasculature through ulcerated epithelium [P. gingivalis can survive in other organs besides the oral cavity. Viable P. gingivalis has been detected in human atherosclerotic plaque tissues [P. gingivalis in some cells. For example, live P. gingivalis has been isolated from human aortic endothelial cells [ithelium  and lympithelium  shortly  tissues  and mous tissues  through al cells , human pal cells , and humal cells . All of P. gingivalis has been as high as 100% [P. gingivalis and CVDs has been deeply studied in recent decades.Cardiovascular diseases (CVDs) are a cluster of conditions that involve the heart and blood vessels, and they remain the most common cause of death throughout the world. Considerable epidemiologic evidence has suggested that periodontal disease is a risk factor for CVDs . For exa as 100% . The relP. gingivalis in clinical samples at a detection rate of 82.61% by fluorescent in situ hybridization assay [P. gingivalis on AS. Infection with P. gingivalis exacerbated atherogenesis in apolipoprotein E (ApoE)-deficient mice [P. gingivalis-inoculated mice was 2-fold larger than that in control mice [P. gingivalis may enter the lesion area and promote the development of AS.ACVDs, including coronary artery disease and stroke, are a form of CVDs with high morbidity and mortality rates. Atherosclerosis (AS), resulting from the progressive accumulation of lipids, calcium, macrophages, and other components in the artery wall, is the pathological basis of ACVDs. Much evidence has linked periodontitis to an increased risk of AS . Periodoon assay . Subsequent mice , and theP. gingivalis can promote AS by affecting the function of all of these cells , which might affect the inflammatory response and fibrinolysis [P. gingivalis and its virulence components  are involved in each phase of monocyte activity during the formation of AS by supporting monocyte migration to the endothelial surface, intimal infiltration, and differentiation into pro-inflammatory macrophages and eventually foam cells [P. gingivalis can play a distinct role in foam cell formation, which is a critical step in the atherosclerotic process. P. gingivalis LPS promotes the accumulation of lipids in macrophages and the formation of macrophage-derived foam cells by upregulating CD36 (a scavenger receptor for low-density lipoprotein and oxidized low-density lipoprotein) as a result of c-Jun/AP-1 pathway activation and by downregulating ATP-binding cassette transporter A1 (cholesterol efflux moderator) due to increased calpain activity [P. gingivalis promoted lipid uptake in macrophages by inducing the expression of fatty acid-binding protein 4, which may be dependent on the JNK pathway [P. gingivalis enhanced the TLR2-CD36/SR-B2-dependent systemic release of IL-1β, leading to a subsequent increase in lipid uptake by macrophages and foam cell formation as a result of encountering IL-1β in the vessel wall [P. gingivalis and AS progression. P. gingivalis infection may also contribute to AS by modulating lipid metabolism and homeostasis. Fifth, vascular calcification is a prominent feature of AS. P. gingivalis LPS can stimulate the proliferation and calcification of SMCs, resulting in vascular calcification [P. gingivalis OMVs were also reported to promote vascular SMC calcification through ERK1/2-RUNX2 [P. gingivalis could contact or invade the SMC layer in blood vessels after endothelial cell injury and induce the expression of S100A9 , which triggers the transformation of SMCs from a contractile to a proliferative phenotype, stimulating further cell growth and contributing to aortic intimal hyperplasia [Most scholars regard AS as an excessive inflammatory response after arterial wall endothelial dysfunction resulting from many damage factors. The pathogenesis mechanisms of AS are highly complex and include the activation of endothelial cells and platelets, recruitment of leukocytes (mainly monocytes and macrophages), migration and proliferation of smooth muscle cells (SMCs), and formation of a lipid core, along with thrombosis and plaque instability. Studies have shown that se cells . First, poptosis . Throughpoptosis ,33. Thison of AS . Furthertability . Second,regation . A receninolysis . Third, am cells ,38. Four pathway . In addisel wall . Furthersel wall . Evidencfication . P. ging/2-RUNX2 . AdditioP. gingivalis to AS. Endothelial cells express HSPs under some conditions, such as exposure to bacteria or CKs. P. gingivalis carries homologs to human HSPs, and P. gingivalis HSP60 antibodies cross-react with human HSPs [P. gingivalis and human HSPs have been found in the peripheral blood of CVD patients and in AS plaques [P. gingivalis may cause cross-reactivity, resulting in endothelial damage and contributing to the pathogenesis of AS.In addition to the mechanisms described above, molecular mimicry is also a hub that links man HSPs . In addi plaques . TherefoP. gingivalis infection, especially clones with type II fimbriae, has been found to decrease the population of regulatory T cells (Tregs) in AS patients [P. gingivalis could influence T-cell differentiation and promote a pro-inflammatory Th17 response, suggesting that Th17/Treg imbalance plays a role during the process of AS [P. gingivalis to manipulate components of the complement system, especially C5a, and antimicrobial peptides in remote locations may be implicated in AS development [In the last two decades, accumulating evidence has supported the role of T cells as critical drivers and modifiers during AS progression. patients . Additioss of AS . This wass of AS . Moreoveelopment .P. gingivalis was the most frequently detected species, with an average prevalence of 40% [P. gingivalis bacteremia induced myocarditis and/or MI in mice [MI is the irreversible necrosis of cardiac cells and scar formation resulting from insufficient blood supply to the heart. Many studies have reported the promoting role of periodontitis in the development of MI. A large multicenter study showed that the risk of a first MI was markedly increased among periodontitis patients: The odds ratio was 1.28 after multi-variable adjustment . Furthere of 40% . Further in mice .P. gingivalis and MI. Firstly, P. gingivalis participates in the progression of MI through direct action with cardiomyocytes. It promotes cardiomyocyte apoptosis by activating Bax and increases matrix metalloproteinase-9 (MMP-9) activity by enhancing oxidative stress, thereby exerting a strongly detrimental effect on the healing process of the infarcted myocardium and subsequently leading to cardiac rupture after MI [P. gingivalis may aggravate myocardial injury after MI by elevating the expression of high mobility group box 1, a nuclear protein from necrotic cells that can induce an inflammatory response [P. gingivalis-associated production of IL-17A might play an essential role in the pathology of MI, as IL-17A was found to exacerbate ventricular remodeling after acute MI in mice [Researchers have attempted to explore the underlying biological mechanisms that link after MI . Additioresponse . Lastly, in mice .P. gingivalis was 85%, the highest among all detected periodontal pathogens [P. gingivalis resulted in a significant increase in the abdominal aortic diameter or intraluminal thrombus (ILT) expansion [AAA is a permanent localized widening of the abdominal aorta. A prospective study in 2004 first detected periodontal bacteria by PCR in aneurysmal specimens from AAA patients, and the detection rate of athogens . Subsequxpansion ,55.P. gingivalis mainly promotes AAA progression via three key processes: inflammation, proteolysis, and SMC apoptosis. Firstly, recognition of P. gingivalis by TLR-2 induces an inflammatory response and accelerates the development of experimental AAAs [P. gingivalis may also promote neutrophil recruitment and activation in AAA thrombus, along with the formation of neutrophil extracellular traps (NETs) in ILT, ultimately stimulating AAA growth [P. gingivalis, which suggests more active proteolytic activities and neutrophil chemotaxis in the luminal layer of ILT [P. gingivalis infection resulted in a significant elevation of myeloperoxidase–DNA complexes in aneurysm samples and plasma [tal AAAs . P. gingA growth . Secondlr of ILT . Lastly,d plasma , therebyd plasma .P. gingivalis exacerbated the hypertensive response to angiotensin II in mice [P. gingivalis on hypertension.Hypertension, currently defined as high systolic blood pressure (BP) (≥140 mmHg) and/or high diastolic BP (≥90 mmHg), is a growing public health problem worldwide. In recent years, a positive link between periodontitis and hypertension has been reported by several epidemiological studies. The results show that patients with diagnosed periodontitis (both moderate-severe and severe) have an increased likelihood of developing hypertension . Moreove in mice , suggestP. gingivalis has been reported to affect these processes and thereby promote hypertension. It was shown that repeated exposure of live P. gingivalis or bacteria LPS induced the release of pro-inflammatory CKs and angiotensin II in human coronary artery endothelial cells, together with P. gingivalis-associated mediators of systemic inflammation , contributing to both endothelial dysfunction and the development of arterial hypertension [P. gingivalis antigens is response for elevated BP [P. gingivalis can induce high expression of endothelial cell adhesion molecules, platelet aggregation, and SMC proliferation, thus impairing vasomotor function [Hypertension is mainly caused by functional changes in blood vessels, such as increased vasoconstrictor responses and endothelial dysfunction. Its etiology is complicated, with no simple mechanism that entirely explains it, and inflammation and the immune response are both implicated. rtension . Recentlvated BP . Moreovefunction ,34,42.P. gingivalis and digestive system cancer have been reported. Intriguingly, increased mortality from P. gingivalis-related digestive system cancer was also exhibited in otherwise healthy individuals, providing evidence for a direct role that is independent of periodontal disease. Additionally, the authors identified P. gingivalis as a valuable microbial marker of mortality risk in digestive system cancer [As the major cause of death across the world, cancer is characterized by uncontrolled cell growth due to aberrant cell cycle progression. Periodontal disease patients were found to have a greater risk of cancer, with a small but significant correlation . With fum cancer .P. gingivalis was shown to increase the chance of OSCC, and its colonization in tumor tissues has reduced patient survival [P. gingivalis by immunohistochemical staining was more than 33% higher in gingival carcinoma tissues than in normal gingival tissues [P. gingivalis in OSCC. It was found that P. gingivalis accelerated OSCC progression in an immune microenvironment through the secretion of C-C motif chemokine 2, chemokine (C-X-C motif) ligand 2, IL-6, and IL-8 from infected oral dysplastic keratinocytes to recruit myeloid-derived suppressor cells [P. gingivalis promoted 4-nitroquinoline-1-oxide-induced tongue tumorigenesis and aggravated the disturbance of fatty acid metabolism during oral carcinoma progression [Oral squamous cell carcinoma (OSCC) often occurs in the tongue, floor of the mouth, buccal mucosa, and gingiva. A meta-analysis revealed that periodontitis increased the risk of OSCC by nearly 2-fold . Furthersurvival . The det tissues . Afterwaor cells . In addiP. gingivalis in OSCC, including epithelial–mesenchymal transition (EMT) of oral epithelial cells, the inhibition of epithelial cell apoptosis, the promotion of immune evasion, the proliferation and invasion of tumor cells, and so on , which are transcription factors that regulate EMT through GSK-3β [P. gingivalis-induced EMT of OSCC cells and human primary oral epithelial cells [P. gingivalis modulates epithelial cell apoptosis via multiple anti-apoptotic/survival pathways, including the activation of the PI3K/Akt and JAK/Stat pathways, release of survivin, upregulation of anti-apoptotic Bad and Bcl-2, downregulation of pro-apoptotic Bax, and inhibition of cytochrome c release and caspase-9 and caspase-3 activation [P. gingivalis infection upregulates miR-203, which directly inhibits suppressor of cytokine signaling 3 and leads to increased Stat3 activation [P. gingivalis, confers epithelial cells with an anti-apoptotic phenotype by binding to and phosphorylating HSP27, which inhibits cytochrome c release and caspase-9 activation [P. gingivalis can also scavenge ATP and inhibit P2X7-mediated host-cell apoptosis [P. gingivalis infection promotes a prosurvival phenotype in human primary oral epithelial cells by regulating cyclins and p53 [P. gingivalis-induced reactive oxygen species (ROS) activates the multipurpose transcriptional regulator FOXO1 via JNK signaling and then initiates an anti-apoptotic program in epithelial cells [P. gingivalis upregulates the expression of B7-H1 and B7-DC receptors on oral cancer cells via a receptor-interacting serine/threonine-protein kinase 2-dependent mechanism, contributing to the escape of tumor cells from immunosurveillance [P. gingivalis has antagonist effects on ATP activation of P2X7 receptors, leading to reduced IL-1β production in epithelial cells, thereby promoting the immune evasion of tumor cells [P. gingivalis promotes the proliferation of OSCC cells by regulating the expression of cyclin D1 through the miR-21/PDCD4/AP-1 negative signaling pathway [P. gingivalis-infected OSCC cells can increase invasiveness through EMT-like changes [P. gingivalis may have the ability to induce proMMP9 expression via ERK1/2-Ets1, p38/HSP27, and PAR2/NF-kB pathways, which subsequently activate MMP9, promoting OSCC cell invasion [P. gingivalis exposure also increases the invasive ability of oral cancer cells via the upregulation of MMPs in an IL-8-dependent fashion, including MMP-1, MMP-2, and MMP-10 [P. gingivalis could induce cell proliferation, mutagenesis, oncogene activation, angiogenesis, and immunosuppression, thus facilitating the development of OSCC [Large studies have explored the mechanisms of nd so on . First, h GSK-3β  and β-cah GSK-3β . A revieal cells . Second,tivation ,71. P. gtivation , which mtivation . NDK fropoptosis . Moreove and p53 . P. gingal cells . Third, eillance . Furtheror cells . Fourth, pathway . Fifth,  changes . In addiinvasion . P. gingd MMP-10 ,82. Beyo of OSCC .P. gingivalis and higher esophageal squamous cell carcinoma (ESCC) risk. The expression of whole P. gingivalis antigen and unique Kgp, along with 16S rDNA, was much higher in cancerous tissues compared with adjacent and normal tissues from ESCC patients [P. gingivalis was inversely related to the ESCC survival rate [P. gingivalis IgA or IgG tended to have a worse prognosis [P. gingivalis in ESCC.Esophageal cancer is one of the most aggressive cancers in the world. A positive correlation was found between patients . Moreoveval rate . Patientrognosis , indicatP. gingivalis dehydrogenates ethanol to its carcinogenic derivative, acetaldehyde, at levels that are capable of inducing DNA damage, mutagenesis, and excessive proliferation of the epithelium. Carcinogen metabolism by P. gingivalis may therefore be a possible mechanism [P. gingivalis to evade immune surveillance, and they determined that the blockade of immune-checkpoint B7-H4 and lysine demethylase 5b in ESCC conferred resistance against P. gingivalis infection and tumor challenge, proposing them as potential therapeutic targets for controlling P. gingivalis infection and its associated neoplasia [P. gingivalis can enhance ESCC cell proliferation and metastasis by triggering the NF-κB signaling pathway [In order to investigate the underlying mechanisms, a series of in vitro experiments has been carried out. echanism . Furthereoplasia . Further pathway  or via t pathway .P. gingivalis antibodies were associated with a two-fold increased risk of pancreatic cancer [P. gingivalis had a 59% greater risk of developing pancreatic cancer compared with those in whom the species was undetected [P. gingivalis and pancreatic cancer.Pancreatic cancer, one of the most rapidly fatal diseases, represents one of the primary causes of cancer-related mortality. People with a history of periodontal disease showed a higher risk of developing pancreatic cancer, which was up to 1.64 times higher relative to those without periodontal disease . Subsequc cancer . In anotdetected . This evP. gingivalis infection could induce differential expression of TLR pathway-associated genes [P. gingivalis invasion leads to p53 activation, a tumor suppressor gene, whose mutation rate is higher in pancreatic cancer and whose abnormal expression plays an important role in pancreatic tumorigenesis [P. gingivalis and pancreatic cancer development. Although partly clarified, further observations are needed to elucidate how P. gingivalis participates in the progression of pancreatic cancer.Some possible mechanisms have been proposed. ed genes , and TLRed genes . Additioigenesis . Thus, mP. gingivalis infection significantly elevate the risk of neuropsychiatric disorders, such as Alzheimer’s disease [Mental and neurological disorders, involving alterations in brain or nervous system functions, together with perception and response to the environment, have afflicted the global human population. The relationship between periodontitis and neuropsychiatric disorders has recently captured the interest of scientists. Epidemiological evidence from several studies has indicated that chronic periodontitis and  disease  and depr disease .P. gingivalis DNA, LPS [P. gingivalis in AD pathogenesis. P. gingivalis exposure induced AD-like phenotypes in mice, which presented as microglia-mediated neuroinflammation, β-amyloid (Aβ) accumulation in neurons, impaired cognitive function, and a reduction in learning and memory [AD is a neurodegenerative disease. It is the most common reason for dementia and is becoming a major health problem in aging societies worldwide. A historical cohort study found that chronic periodontitis patients had an elevated risk of AD , and P. DNA, LPS , and ginDNA, LPS  have beed memory ,99.P. gingivalis may be involved in the progression of AD through these processes  of tooth loss, poor oral hygiene, and halitosis . It has pression . More stRespiratory diseases are among the most common pathologies in humans across the world. Many published epidemiological studies have reported a positive link between periodontitis and respiratory diseases  through P. gingivalis developed severe bronchopneumonia and lung abscess [Lower respiratory tract infections, including pneumonia, were the fourth highest cause of death worldwide in 2016 . A recen abscess ,110.P. gingivalis and pneumonia, and some key findings have been reported. Evidence from in vivo experiments suggests that gingipains are essential factors in mediating aspiration pneumonia due to P. gingivalis infection, and the authors recommended it as a useful adjunct treatment site [P. gingivalis [P. gingivalis might affect pneumonia progression through interactions with other respiratory pathogens. Co-infection of P. gingivalis and H1N1 in lung epithelial cells promoted the production of inflammatory CKs and NO, resulting in increased levels of apoptosis via the Bcl-2/Bax/caspase-3 pathway [P. gingivalis can modulate Pseudomonas aeruginosa-induced respiratory epithelial cell apoptosis via the STAT3 signaling pathway by causing transient inhibition and, ultimately, exerting promoting effects [Studies have investigated the correlation between ent site . Interesngivalis . Further pathway . P. ging effects .P. gingivalis was found in tracheal aspirates from patients with severe acute exacerbation of COPD [P. gingivalis-associated enzymes in saliva (such as proteases) may alter mucosal surface adhesion receptors, destroy salivary pellicles on pathogenic bacteria, or degrade the salivary pellicles on mucosal layer, all of which promote the colonization of respiratory pathogens in the respiratory epithelium [P. gingivalis plays a role in COPD progression by modulating the colonization of respiratory pathogens.COPDs are a group of diseases characterized by airflow limitation in the airway and include chronic bronchitis and emphysema. A positive relationship between periodontitis and COPD was reported , and per of COPD , suggestithelium . These fP. gingivalis to several metabolic diseases, especially diabetes and nonalcoholic fatty liver disease.Metabolic diseases refer to a class of disorders caused by the abnormal metabolism of glucose, proteins, or lipids. Their prevalence has markedly increased across the world in the last few decades. Many studies have linked P. gingivalis or its components can induce insulin resistance in mice [Diabetes, a series of chronic metabolic disorders characterized by hyperglycemia, is the result of defects in insulin secretion, insulin action, or both. Considerable epidemiologic evidence has shown a two-way interrelationship between periodontitis and diabetes, and many researchers regard periodontal infection as a risk factor for diabetes progression. Diabetic individuals with periodontitis were found to have a significantly higher prevalence of diabetes-related complications . Further in mice ,120,121,P. gingivalis-related mechanisms involved in the course of diabetes, insulin resistance is considered to be the most important. P. gingivalis infection increased systemic inflammation, especially in adipose tissue, through the induction of endotoxemia [P. gingivalis may promote insulin resistance. P. gingivalis LPS induced pro-inflammatory adipokine secretion and oxidative stress in adipocytes through the regulation of TLR-mediated pathways and redox enzymes and contributed to obesity-associated insulin resistance [P. gingivalis aggravated high-fat diet (HFD)-induced insulin resistance in mice via its biosynthesis of branched-chain amino acids, which can activate the mammalian target of rapamycin and downstream genes, which leads to the dephosphorylation of insulin receptor substrate 1 [P. gingivalis OMVs were loaded with gingipains and translocated to the liver in mice, leading to the attenuation of insulin sensitivity and inhibition of hepatic glycogen synthesis, partly by attenuating the Akt and GSK-3β pathway [P. gingivalis are reported to be linked to higher prediabetes prevalence among diabetes-free adults [P. gingivalis LPS stimulated insulin secretion by pancreatic β-cells and had significant implications in the progression of β-cell compensation in prediabetes in subjects with periodontitis [P. gingivalis induced the translocation of P. gingivalis/gingipains to the pancreas in mice, leading to significant changes in islet architecture and β-cell apoptosis, which may be involved in the development of prediabetes [P. gingivalis attenuated the phosphorylation and translocation of FOXO1, which is regulated by insulin in HepG2 cells, thereby increasing hepatic gluconeogenesis [Among the potential otoxemia , alteratotoxemia , or an iotoxemia , ultimatotoxemia . Thus, isistance . A recenstrate 1 . Further pathway . Importae adults . P. gingdontitis . Moreovediabetes . Lastly,ogenesis  and poteP. gingivalis and NAFLD has also been shown. The detection frequency of P. gingivalis DNA (46.7%) was markedly higher in NAFLD patients compared with control subjects [P. gingivalis was also immunohistochemically detected (52.5%) in the liver of nonalcoholic steatohepatitis (NASH) patients, and NASH patients with P. gingivalis infection had higher fibrosis scores than those who did not [P. gingivalis with type IV fimbriae tended to increase with fibrosis progression among NASH patients [P. gingivalis infection can exacerbate HFD-induced NAFLD/NASH progression [NAFLD is a hepatic manifestation of metabolic syndrome characterized by abnormal accumulation of fat. Epidemiological evidence in the last decade has supported a positive correlation between periodontitis and high prevalence odds of NAFLD. In NAFLD patients, liver function parameters (ALT and AST) were improved after they underwent periodontal therapy . A potensubjects . P. ging did not . Similarpatients . Moreovegression ,131,132.P. gingivalis to NAFLD have been explored. The involvement of P. gingivalis in the pathogenesis of NAFLD in mice may occur through the activation of the P. gingivalis LPS-associated TLR2 pathway and inflammasomes [P. gingivalis injection aggravated NAFLD in mice, along with impaired glucose tolerance and insulin resistance [P. gingivalis related to insulin resistance that may also have effects on NAFLD. This was also mentioned in related review [P. gingivalis and hepatic stellate cells (HSCs), which act as effector cells of hepatic fibrosis. The presented data suggested that P. gingivalis odontogenic infection could play a role in aggravated fibrosis in a NASH mouse model through HSCs activation caused by the production of TGF-β1 and galectin-3 in HSCs and hepatocytes [P. gingivalis, along with the production of CKs and ROS triggered by P. gingivalis [Over the past decade, potential underlying mechanisms that connect mmasomes , the sevmmasomes . Moreovesistance . We prevd review . A recenatocytes . Other pngivalis . DespiteP. gingivalis DNA and antigens have been found in the placenta, umbilical cord [P. gingivalis within the placenta or umbilical cord of pregnant women and many pregnancy complications, including preterm birth (PB), preeclampsia, and pregnancy-related hypertensive disorders [P. gingivalis in APOs through direct invasion and damage to utero-placental tissues. Subsequently, various rodent models confirmed that P. gingivalis infection can induce diverse utero-placental pathologies  and cause a diverse array of APOs, including fetal growth restriction (FGR), low birth weight (LBW), and PB [Adverse pregnancy outcomes (APOs) refer to any maternal, fetal, and/or neonatal complications that occur during pregnancy, labor, and/or the postpartum period. Epidemiologic evidence has consistently suggested a positive correlation between periodontitis and APOs . Furthercal cord , and amncal cord . A positisorders ,135,136., and PB ,138,139.P. gingivalis to the placenta and its pathogenicity mechanisms have been proposed in many animal experiments. It has been reported that P. gingivalis could translocate to local placental tissues in rats and enhance the expression of Fas, FasL, and TLR2, leading to PB and LBW [P. gingivalis infection in pregnant mice induced a fetus-specific placental immune response and increased the placental Th1/Th2 cytokine ratio , which may be related to the occurrence of FGR [P. gingivalis induced a maternal immune response and enhanced FGR in mice, with an elevation in the maternal serum TNF-α and a decrease in IL-10 [P. gingivalis LPS directly or indirectly upregulated inflammatory factors such as TNF-α, IL-8, and cyclooxygenase 2 via the production of galectin-3 in the placenta, inducing PB [P. gingivalis on trophoblast cells. P. gingivalis can invade human extravillous trophoblasts and induce G1 arrest and cell apoptosis, which may involve ERK1/2 and DNA damage response pathways, thereby disrupting the maintenance of pregnancy [P. gingivalis’ persistence and survival within utero-placental tissues via sophisticated invasive strategies and immune response evasion, development of polymicrobial dysbiosis and synergy with other commensal bacteria to increase the overall microbial burden at the maternal–fetal interface, increased expression of acute-phase proteins such as CRP and pentraxin3, excessive generation of ROS, increased production of fetal adrenal cortisone, and fetal stress [The direct dissemination of  and LBW . P. ginge of FGR . The samin IL-10 . Furtherucing PB . Researcregnancy . The actregnancy . Other ml stress .P. gingivalis, were detected in synovial fluid from patients with RA at the DNA level (42.1%) [P. gingivalis increased the incidence and progression of RA [As a chronic autoimmune disease, rheumatoid arthritis (RA) is characterized by persistent inflammatory synovitis and progressive degeneration of cartilage and bone. A meta-analysis found a higher RA prevalence in periodontitis patients compared with controls , and non (42.1%) . Subsequon of RA ,147,148.P. gingivalis is the only microorganism known to express the PAD enzyme. A previous review investigated and proposed that P. gingivalis PAD may set in motion a chain of events that break immunotolerance to citrullinated proteins, leading to the development of RA [P. gingivalis [P. gingivalis prior to RA activated the immune system by inducing a TLR2- and IL-1-driven Th17 cell response, which suggests that exposure to P. gingivalis may accelerate RA progression predominantly through Th17-related pathways [P. gingivalis, which alters the gut immune system and gut microbiota composition [n-3 DPA, which weakens the intestinal barrier function and facilitates a switch in P. gingivalis behavior to the pathogenic tendency, thus further promoting gut bacterial translocation and increasing joint inflammation [The etiology of RA is very complicated. Autoantibodies against citrullinated proteins are confirmed to be one of its pathological bases. Protein citrullination is carried out by peptidyl-arginine deiminases (PAD), and, so far, nt of RA . Treatmengivalis . Additiopathways . Furtherpathways , and oraposition , may alsammation . P. gingivalis and several systemic diseases, many researchers naturally want to explore whether controlling P. gingivalis can be regarded as a feasible disease management strategy. Proposed approaches include broad-spectrum antibiotics and specific inhibitors that target P. gingivalis. Firstly, administration of doxycycline or metronidazole resulted in a significant reduction in the percentage of atherosclerotic lesions in P. gingivalis-inoculated mice [P. gingivalis load and Aβ production in the mouse brain, suggesting that it could be valuable for treating P. gingivalis brain colonization and neurodegeneration in AD [P. gingivalis-mediated aspiration pneumonia [P. gingivalis HSP60 or nasal immunization with OMVs prior to P. gingivalis injection also significantly reduced AS lesion formation in ApoE-deficient mice, and it may be an effective strategy for preventing naturally occurring or P. gingivalis-accelerated AS [P. gingivalis (such as gingipains) by pre-immunization or, if possible, small-molecule inhibitors may reduce the ability of the bacteria to translocate to remote tissues and instigate and/or exacerbate pathology in some chronic inflammatory diseases [Given the inseparable link between ted mice ,152. Addted mice . Gingipaon in AD . More reon in AD . Pretreaneumonia . Besidesrated AS ,155. Tardiseases . DespiteP. gingivalis and certain systemic diseases, especially AD and AS, and an increasing amount of evidence has been accumulating. P. gingivalis invades the human body and influences general health in four main ways  and play pivotal roles in the progression of local diseases [P. gingivalis [The latest studies have tended to focus on exploring possible direct and indirect links between ain ways . (i) Bacnstrated . Transieocedures , which e and IFN . A low-ldiseases ,120.  to emerging areas . Some of the clearer and more direct mechanisms are discussed here. However, many of the exact mechanisms are not fully clear yet, and a few conclusions are controversial. Current evidence emphasizes that promoting oral health should be encouraged as an indispensable part of a healthy lifestyle to reduce the burden of global chronic noncommunicable and communicable diseases [P. gingivalis, and should be regarded as a modifiable factor for diseases. Reducing the load of P. gingivalis through periodontal intervention has potential benefits for oral health and a direct or indirect positive impact on overall health, even if it prevents the possibility of such a connection. We should be aware of the risk and address it as soon as possible to avoid threatening our health. Moreover, interrelations between P. gingivalis and other oral pathogens (such as Actinobacillus actinomycetemcomitans and Fusobacterium nucleatum), together with gut microbiota, are another subject of intensive study.In this article, we comprehensively summarize the adverse effects of diseases , which w


Gene fusions can generate immunogenic neoantigens that mediate anticancer immune responses. However, their computational prediction from RNA sequencing (RNA-seq) data requires deep bioinformatics expertise to assembly a computational workflow covering the prediction of: fusion transcripts, their translated proteins and peptides, Human Leukocyte Antigen (HLA) types, and peptide-HLA binding affinity. Here, we present NeoFuse, a computational pipeline for the prediction of fusion neoantigens from tumor RNA-seq data. NeoFuse can be applied to cancer patients’ RNA-seq data to identify fusion neoantigens that might expand the repertoire of suitable targets for immunotherapy.https://icbi.i-med.ac.at/NeoFuse/.NeoFuse source code and documentation are available under GPLv3 license at Bioinformatics online. Besides being major determinants of response to immune checkpoint blockers, neoantigens are at the basis of other cancer immunotherapies like personalized cancer vaccines and adoptive T cell therapy  data have been proposed recently (https://sylabs.io) and Docker (https://www.docker.com) images to simplify installation and analysis.Here, we present NeoFuse, a user-friendly pipeline for the prediction of fusion neoantigens from tumor RNA-seq data. NeoFuse is available as Singularity (https://github.com/suhrig/arriba), considering both fusion junctions and 3’ out-of-frame sequences. We chose Arriba because it outperformed competitor prediction methods in the DREAM Somatic Mutation Calling–RNA Challenge (https://www.synapse.org/SMC_RNA). Moreover, it computes a confidence score reflecting the likelihood that a fusion is caused by a tumor-specific genomic rearrangement and is not due to technical artifacts. The third module uses MHCflurry . Although not distributed as part of NeoFuse, netMHCpan  [project n. T 974-B30 to F.F.] and by the European Research Council [advanced grant agreement n. 786295 to Z.T.].Conflict of Interest: none declared. btz879_Supplementary_DataClick here for additional data file.


Nosema ceranae is a gut intracellular parasite of honey bees which destroys epithelial cells and gut tissue integrity. Studies have shown protective impacts of honey bee gut microbiota towards N. ceranae infection. However, the impacts of N. ceranae on the relative abundance of honey bee gut microbiota remains unclear, and has been confounded during prior infection assays which resulted in the co-inoculation of bacteria during Nosema challenges. We used a novel method, the suppression of N. ceranae with specific siRNAs, to measure the impacts of Nosema on the gut microbiome.Gut microbial communities can contribute positively and negatively to host health. So far, eight core bacterial taxonomic clusters have been reported in honey bees. These bacteria are involved in host metabolism and defenses. N. ceranae led to significant positive effects on microbial abundance. Nevertheless, 15 bacterial taxa, including three core taxa, were negatively correlated with N. ceranae levels. In particular, one co-regulated group of 7 bacteria was significantly negatively correlated with N. ceranae levels.Suppressing N. ceranae are negatively correlated with the abundance of 15 identified bacteria. Our results provide insights into interactions between gut microbes and N. ceranae during infection. Animals evolve with their associated microorganisms as a unit and symbiotic microbes can facilitate the survival of hosts toward diverse stresses. When the host encounters pathogens, the microbial community responds and the dynamics can change dramatically . As micrNosema ceranae is a unicellular fungal parasite which infects honey bee mid-gut epithelial cells. Infection starts from ingestion of N. ceranae spores. The proliferation cycle of N. ceranae is approximately 4 days, at which point a large number of offspring spores are released from infected cells [N. ceranae infection negatively affects honey bee physiology, behavior and immune responses [N. ceranae has spread globally and this agent is one of the factors implicated in honey bee colony collapses [N. ceranae proliferation [ed cells . N. ceraesponses . N. ceraollapses , 12. A fferation –15.N. ceranae proliferation, this parasite inevitably encounters the gut microbial community. Within the honey bee gut, bacteria are dominated by eight species/clusters. Three bacteria are within the Gram-negative phylum Proteobacteria, including Gilliamella apicola, Frischella perrara and Snodgrassella alvi. Two bacteria are within the Gram-positive phylum Firmicutes, including Lactobacillus mellis, Lactobacillus kunkeei and Lactobacillus Firm5 . One Bifidobacterium cluster within the phylum Actinomycetes, including B. asteroids, B. actinocoloniiforme and B. bohemicum. Two other species clusters are within phylum Alphaproteobacteria of Bartonellaceae and Acetobacteraceae [. Some of these taxa are likely to be involved in honey and pollen digestion, along with many low-frequency opportunistic microbes [N. ceranae infection [N. ceranae infection by regulating gut microbes, providing a long-lasting strategy to improve overall bee health. Quantifying the responses of microbes to N. ceranae infection is a critical step in understanding these interactions. This is challenging, because inoculating honey bees with N. ceranae spores generally leads to co-inoculation with associated gut bacteria. Even after several rounds of Percoll purification, a fraction of high-throughput sequence reads from purified spores can still be aligned to bacteria [N. ceranae proliferation using bees challenged with N. ceranae and control bees can lead to artefacts.During teraceae . Some ofmicrobes , 7, 17. nfection . Consequbacteria , 20. So,Dicer is a key component of the RNA induced silencing complex, which is involved in developmental and physiological gene regulation [Dicer using siRNA interrupted N. ceranae proliferation [N. ceranae infection and gut bacteria loads.The gene gulation , 22. We feration , 24. We N. ceranae RNA polymerase II small subunit  was lower in the siRNA-Dicer group compared with the infection and siRNA-scramble groups at 3 dpi  and significantly negatively correlated with N. ceranae RPB1 . Additionally, marker genes for Serratia sp. G5_1_1BCO2, Serratia marcescens, Serratia nematodiphila and Snodgrassella alvi were co-expressed (co-regulation group 2 of 4 bacteria), which was also negatively correlated with N. ceranae levels, even though this correlation was not statistically significant . Bartonella apis, Propionibacterium sp. B4, Lactobacillus apis and Gilliamella apicola did not cluster into any co-regulation group. None of these studied 15 microbes were statistically significantly differentially expressed at each day post-infection . However, the co-regulated groups (P < 0.0001) and siRNA treatment (P < 0.0001) showed significant effects on specific microbe counts during the entire experimental period within the generalized linear model. Additionally, 6 bacteria in co-regulation group 1 , 3 bacteria in co-regulation group 2  and G. apicola were significantly enhanced towards siRNA treatment during the entire experimental period (P < 0.001).As expected, sequence reads which aligned to the bee genome decreased while reads aligned to microbes increased gradually over the experimental period Fig.  File S11. The geGilliamella apicola and Snodgrassella alvi support food digestion and pathogen defenses, even though sequence variance may lead to functional variation [Frischella perrara triggers the honey bee melanization response [When a parasite infects a host, infection success depends on host immune defenses, microbial community responses, and even competition from other parasites . In turnariation , 28. In response . Honey bN. ceranae proliferation, suggesting N. ceranae reduces bacterial abundance, at least for the studied 15 bacteria. In the gut microbiome, diet shapes bacterial diversity and responses towards N. ceranae infection, where a negative correlation between S. alvi and N. ceranae was found, which is consistent with our data [N. ceranae and other opportunistic pathogens [Lactobacillus strains as food additives showed suppressive effects on N. ceranae spores [As two co-regulation groups were identified, the relative abundance of bacteria may not be independent of each other in the mid-gut tissue. Additionally, all of the studied bacteria were negatively correlated with our data . When thathogens , 31. Adme spores .S. alvi [F. perrara and S. alvi were significantly enhanced with the reduction of N. ceranae. It remains unclear why N. ceranae negatively affected gut bacteria. Even though sugar was the only food resource in our studied bees, the chance is low that N. ceranae directly competes for this food resource with bacteria, as N. ceranae proliferates within epithelia cells and bacteria live in the lumen. Tissue destruction caused by infection might lead to the deterioration of the gut lumen, thus impacting bacteria.Competition for limited food resources may lead to the decrease/exclusion of other competitors, as suggested by the competitive exclusion principle . TherefoS. alvi , 36. FroN. ceranae and the studied microbes was correlative. However, the siRNA Dicer treatment and the reduced N. ceranae level was causative. We provided novel insights into interactions between gut parasites and other microbes over the entire life cycle of the parasite. Our study is limited to mid-gut tissues of newly-emerged workers. It will be interesting to screen N. ceranae and bacterial interactions in older forager bees, and in bees collected during different seasons.Although microbial treatments provide a promising chemical-free strategy to control pathogen infection , cautionN. ceranae are negatively correlated with levels of gut bacteria. Gut bacteria were not independent with each other and were co-regulated. The relative abundance between the gut bacteria and N. ceranae was negatively correlated.Levels of Apis mellifera mellifera), which is neither an endangered nor protected species.Three apiaries  for bee sample collection were established at the USDA-ARS Bee Research Laboratory, Beltsville, Maryland, USA. No specific permits were required for the described studies. Studies involved the European honey bee  and centrifuged 5 min at 3000 rpm. Spores were further purified using a Percoll gradient procedure [5 N. ceranae spores without siRNA treatment, as the infection group; (2) with 105 N. ceranae spores and 1.5 μg siRNA (targeting N. ceranae gene Dicer) as the siRNA-Dicer group; (3) with 105 N. ceranae spores and 1.5 μg of a non-matching (scrambled) siRNA, as the siRNA-scramble group. The sequences of the designed siRNA were provided in a previous study [2, the mid-gut tissue was dissected and the RNA was immediately extracted with TRizol [rocedure . Sealed us study . Forty hh TRizol . Sequenchttps://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and SEQTK (https://github.com/lh3/seqtk). Sequencing reads were aligned to the honey bee genome assembly (Amel_HAv3.1) with the hisat2 package under default settings [N. ceranae and other bee-associated microbes. The Holobee Bar database is a curated resource for microbes associated with honey bees . In order to remove false-positive microbial classifications and restrict reads to the more prevalent bacteria responding towards the parasite infection, microbes found in fewer than 22 libraries of the sequencing libraries were removed. The counts were normalized using the weighted trimmed mean of M-values (TMM). Two replicates of each treatment for each post-infection day were used to calculate the variance in order to identity significantly regulated bacteria with edgeR [P values were corrected for multiple comparisons using false discovery rate (FDR). Significantly differentially expressed taxa met three criteria: (1) the relative abundance of the bacterium is significantly different between the infection group and the siRNA-Dicer group; (2) the relative abundance of the bacterium is significantly different between the siRNA-Dicer group and the siRNA-scramble group; and (3) the relative abundance of the bacterium is not significantly different between the siRNA-scramble group and the infection group.On average, 58 million reads (114 nucleotides for each paired-read member) were generated from each library, after trimming and quality control using FastQC . In order to analyze bacterial patterns over the infection period, co-regulation between N. ceranae and bacteria clusters was analyzed using the WGCNA package [N. ceranae were treated as phenotype. To further test the effect of siRNA treatment on the microbe enrichment, a generalized linear model was performed using R (version 3.5.1). Within the linear model (counts ~ treatment + day + cage + group + microbe), counts indicate normalized microbe counts. Treatment indicates siRNA-Dicer group, siRNA-scramble group and infection group. Day indicates number of days post infection. Cage indicates each of the two cage replicates. Group indicates co-expression groups obtained from WGCNA analysis. Microbe indicates each of 15 microbe species. The code is provided in supplementary file SSignificantly differentially expressed taxa were tested using t-tests at each time point and the  package . The norAdditional file 1 File S1 sequencing alignment statistics and normalized microbe counts.Additional file 2 File S2 code used for the statistics.


Aeromonas hydrophila are ubiquitous in the environment and are highly distributed in aquatic habitats. They have long been known as fish pathogens but are opportunistic human pathogens. Aeromonas spp. have persisted through food-processing safeguards and have been isolated from fresh grocery vegetables, dairy, beef, pork, poultry products and packaged ready-to-eat meats, thus providing an avenue to foodborne illness. A beta-hemolytic, putative Escherichia coli strain collected from diarrheic neonatal pigs in Oklahoma was subsequently identified as A. hydrophila, and designated CVM861. Here we report the whole-genome sequence of A. hydrophila CVM861, SRA accession number, SRR12574563; BioSample number, SAMN1590692; Genbank accession number SRX9061579. The sequence data for CVM861 revealed four Aeromonas-specific virulence genes: lipase (lip), hemolysin (hlyA), cytonic enterotoxin (ast) and phospholipid-cholesterolacyltransferase (GCAT). There were no alignments to any virulence genes in VirulenceFinder. CVM861 contained an E. coli resistance plasmid identified as IncQ1_1__M28829. There were five aminoglycoside, three beta-lactam, and one each of macrolide, phenicol, sulfonamide, tetracycline and trimethoprim resistance genes, all with over 95% identity to genes in the ResFinder database. Additionally, there were 36 alignments to mobile genetic elements using MobileElementFinder. This shows that an aquatic pathogen, rarely considered in human disease, contributes to the resistome reservoir and may be capable of transferring resistance and virulence genes to other more prevalent foodborne strains such as E. coli or Salmonella in swine or other food production systems. Aeromonas spp. comprise numerous species that are widely distributed in aquatic environments ranging from sewage, marine and freshwater to water treatment plants [Aeromonas spp. have persisted through food-processing safeguards and have been isolated from fresh grocery vegetables, dairy, beef, pork, poultry products, and packaged ready-to-eat meats [Aeromonas spp. are opportunistic pathogens and have been identified as the etiologic agent in a variety of infections in immunocompetent and immunocompromised humans [A. hydrophila may encode a number of virulence factors including hemolysins, aerolysins, adhesins, enterotoxins phospholipase, and lipase [t plants ,2,3. Thet plants ,5. Aeromat meats ,7. Some at meats . Aeromond humans ,9. Severd humans ,10,11,12d lipase .Aeromonas spp. are Gram-negative bacilli that are oxidase and catalase positive. They are distinguishable based on temperature growth traits [A. hydrophila and displays optimal growth between 35 °C and 37 °C. Another group is psychrophilic, non-motile, and is associated with fish diseases. This group is typified by A. salmonicida and displays optimal growth between 22 °C and 25 °C [Aeromonas spp. as transboundary pathogens. h traits . One groAquaculture increasingly provides a sustainable, safe alternative to more traditional livestock and poultry production as a food source. The amount of fish consumption coming from aquaculture compared to capture has increased over the years. For the time periods 1986–1995, 1996–2005, 2006–2015, and 2016–2018 the average contributions of fish production from aquaculture were 14.6%, 27.2%, 39.9%, and 46.0%, respectively [Traditionally, clinical and agriculture settings have been the primary the focus of public health officials for monitoring antimicrobial-resistant microorganisms. Currently, a wider scope includes the environmental microbiome, mobilome, and resistome. A collaborative effort by many countries to address antimicrobial resistance has been termed “One Health”. One Health is an effort by health professionals to minimize antimicrobial resistance in human and veterinary medicine, agriculture, aquaculture, environmental niches and molecular genetic reservoirs ,18. To aWhole-genome sequencing and metagenomic next-generation sequencing have provided insight into the complex antimicrobial resistance reservoirs that exist. A horizontal flow of antimicrobial resistance and virulence genes occurs due to mobile genetic elements such as plasmids, transposons, integrons and integrative and conjugative elements (ICEs) that can acquire resistance and virulence genes as they move between bacteria. A. hydrophila CVM861 was chosen for WGS to obtain the complete genotype including virulence genes that A. hydrophila intrinsically possesses. This potentially elucidates the possible fluidity via recombination between chromosomal and plasmid locations of virulence and resistance genes. It also provides information on the resistome of food production animals and the larger global resistome.Aeromonas hydrophila CVM861 was previously identified and found to possess the macrolide inactivation gene cluster mphA-mrx-mphR. The EMBL GenBank accession sequence for the previously characterized sequence of 9948bp is AY522923. The GenBank BioSample accession number for the WGS of CVM861 is SAMN15950692. The BioProject number is PRJNA660480.® automated antimicrobial susceptibility system was used according to the manufacturer’s instructions  using the National Antibiotic Resistance Monitoring System (NARMS) panel CMV3AGNF (Trek Diagnostic Systems). The following antimicrobials were tested: amoxicillin/clavulanic acid, ampicillin, cefoxitin, ceftiofur, ceftriaxone, chloramphenicol, ciprofloxacin, gentamicin, nalidixic acid, streptomycin, sulfisoxazole, tetracycline, and trimethoprim/sulfamethoxazole. E. coli ATCC 25922, E. coli ATCC 35218, and Enterococcus faecalis ATCC 29212 were used as quality control organisms. Data were interpreted using CLSI break points unless unavailable, and then break points from the NARMS 2010 annual report were used [Susceptibility testing was performed by broth microdilution according to Clinical Laboratory Standards Institute (CLSI) methods  on CVM86® sequencer (150-bp paired-end reads). Sequences were uploaded to PATRIC for assembly [https://cge.cbs.dtu.dk/services/MobileElementFinder/ were downloaded for sequence alignments. The alignments were performed with MagicBlast [A. hydrophila CVM861 contigs were also aligned with Aeromonas-specific virulence genes in Genbank using Magic-Blast [DNA purification employed the QIAamp 96 DNA QIAcube HT kit . DNA quality and quantity were assessed by absorbance and fluorescence using a FLUOstar Omega plate reader . DNA was sent  for WGS on an Illumina MiSeqassembly . Assemblassembly , Virulenassembly  PlasmidFgicBlast . A. hydric-Blast .The sequences generated from the WGS were submitted to Genbank as reads. The Genbank accession information was: SRA accession number, SRR12574563; BioSample number, SAMN1590692; BioProject number PRJNA660480; Genbank accession number SRX9061579.Assembly with the Unicycler option produced 92 contigs with a genome of 4800554 bp, a GC content of 61.45% and N50 of 132950. The assembly using the Metaspades option produced 261 contigs with a total length of 4933241 bp, and GC content of 61.42% and an N50 of 130230. Aeromonas-specific virulence gene sequences from Genbank were aligned (Aeromonas-specific virulence genes were identified in the CVM861 genome: lipase (lip), hemolysin A (hlyA), cytonic enterotoxin (ast) and phospholipid-cholesterolacyltransferase (GCAT). Lipase had a 94% identity. The other three had a >95% identity.There were no alignments to 2538 virulence gene sequences provided by CGE. Therefore, ten  aligned . Four AeCVM861 aligned to 13 sequences in the ResFinder database (3100 gene sequences), all of the alignments had a >95% identity . CorrespEscherichia coli plasmid RSF1010 sequence that was in the Enterobacteriaceae database. Comparison of CVM861 to the PlasmidFinder database (469 gene sequences) resulted in the alignment to a single plasmid, IncQ1 (identified as IncQ1_1__M28829 in the database). The CVM861 sequence fragment displayed a 100% alignment with an IncQ Salmonella genomic island 1 sequences (SGI1), four each: insertion sequences (IS), and Intergrative and conjugative elements (ICEs). Overall, there were alignments to 34 different potential mobile elements.CVM861 was aligned to the MobileElementFinder database version v1.0.2 (9 June 2020) containing 4452 elements . When boAeromonas infections can be difficult to control for many reasons. They have inducible chromosomal β-lactamase resistance, primarily to ampicillin [picillin ,26. Chropicillin ,28,29,30picillin ,32. WholAeromonas spp. [Aeromonas spp. isolated from fish or the environment. However, they are of concern because they are broad-range plasmids that are often found in clinical or animal environments [Aeromonas sp. isolated from the Ter river in Spain was fully sequenced in 2012 and found to carry IncU plasmid (pP2G1) [Bacterial cells can possess multiple plasmids, of varying size, without substantially affecting their growth . In thisnas spp. . All of ronments . An Aero (pP2G1) . The genmphA-mrx-mphR) was first found in E. coli [E. coli isolates for mphA-mrx-mphR in 2005. It was discovered in CVM861 which was then determined to be Aeromonas hydrophila and not E. coli [aadA2, sul1 and dfrA. The aph(3′)-Ia gene was located downstream of the MphA gene cluster. All were downstream of a Tn21 transposon [Macrolide phosphotransferase (MphA) is an enzyme that phosphorylates erythromycin and other macrolide antibiotics; thereby, inactivating their antibiotic properties. Phosphorylation confers high-level resistance to macrolides. An MphA gene cluster ( E. coli . Our lab E. coli . The Mph E. coli . At thatansposon . E. coli or Aeromonas spp., due to efflux pumps that transport macrolide antimicrobials out of the cell. However, it demonstrates how dissemination of resistance genes occurs when they are physically linked on mobile genetic elements that can be transferred to other genera or strains of microorganisms [High-level resistance to macrolides is not necessary for Gram-negative bacteria, such as rganisms ,37.E. coli. The plasmid characterized, pRSB225 was an IncF plasmid that displayed resistance to the same categories of antimicrobials as CVM861 including high-level resistance to macrolide antimicrobials [An unidentified bacterial spp. was collected from a municipal sewage treatment plant in Germany . Conjugacrobials . Resistacrobials . catB3 gene identified in CVM861 encodes chloramphenicol acetyltransferase that catalyzes the acetylation of the 3′-OH of chloramphenicol and provides one mechanism for resistance to phenicol antimicrobials [Overall, CVM861 produced genomic alignments that were anticipated for a multidrug-resistant strain. Initially, alignment to seven antimicrobial classes from ResFinder corresponded with the phenotypic susceptibility profile with the exception of chloramphenicol. The crobials . Resistacrobials ,41. HoweThe lack of virulence genes in CVM861 to those in Enterobacteriaceae suggests that none had been translocated to the IncQ plasmid. Aeromonas is a rarely considered a human pathogen, it still contributes to the resistome reservoir and may be capable of transferring resistance and virulence genes to other more prevalent foodborne strains such as E. coli or Salmonella in swine or other food production systems. The ability to provide comprehensive knowledge of the entire genome including mobile genetic elements allows elucidation into the global dissemination of these elements. It is important to continue studies of this type for both health and food safety management.Although


Autochthonous hydatidosis in France and western Europa are uncommon since the beginning of the 21st century. We report here an authentic indigenous cystic echinococcosis case in a French shepherd. The risk of remerging pathology should not be neglected and measures to interrupt parasite transmission are still relevant. Echinococcus granulosus is a cestode of the Tæniidæ family which mainly infects domestic dogs as a permanent host. It is transmitted to a wide range of intermediate-host domestic ungulates  which harbor the hydatid cysts [id cysts . Cystic id cysts . CE is uid cysts , and lesid cysts . NeverthHydatidosis has a worldwide distribution from tropical to Artic areas . It typiIn June 2017, a 78-year-old Frenchman, was hospitalized for episodic pain in the right hypochondrium for one month. His medical history included prostate local resection in 2013, inguinal hernia and daily both alcohol drinking and tobacco smoking. The patient was a traditional shepherd who never traveled outside the South-East of France. He had daily contact with sheep, sheepdogs, donkeys, horses and cats. Physical examination revealed a painful mass in the right hypochondrium. Computed tomography and magnetic resonance imaging scan showed three liver cysts, located in segments IV, V and VI , that weEchinococcus granulosus sensu stricto genotype G1 (99 % on gene bank: HF947568.1). ABZ treatment was continued up to May 2018. At 2 months of follow-up, the patient had no pain and CT-scan images showed only the 4 cm calcified cyst in segment IV.Based on CT-scan images, a diagnosis of liver hydatid cyst was retained. The patient received albendazole (ABZ) 400 mg twice per day for six months. ABZ was stopped temporarily during a course of 2 months because of lymphopenia. The follow-up showed a suspected pre-rupture of the largest cyst: the serology switched to positive, thus precipitating the decision of surgical treatment by resection of the protruding dome in March 2018. The postoperative biological examination of the puncture fluid revealed scolex and typical hooklets . MoleculE. granulosus  at slaughterhouses in southern France. Despite this fact, only one human case [In western Europe, helminthiasis are usually diagnosed in migrants or travelers  as indigThe national survey of Umhang et al.  demonstrE. granulosus stricto senso (recovered from sheep) are endemic in south of France. In line with these data, the genotype G1 was involved in this patient’s cyst. The standardized classification of CE proposed by the WHO-Informal Working Group on Echinococcosis (WHO-IWGE) [i. e. surgery, percutaneous treatment such as PAIR , albendazole chemotherapy or ‘watch & wait’ (WW). Our patient presented with 3 cysts, two classified CE5 and one CE3b. Conventionally, WW is appropriate for uncomplicated inactive CE5 cyst [i.e. alopecia, liver toxicity, and agranulocytosis) and cyst leakage.The genotypes G1, G2 and G3 of HO-IWGE)  defined CE5 cyst , whereasCE5 cyst  and thatCE5 cyst . TherefoThis patient’s case, associated with the other reported indigenous cases in western Europe, highlight that CE risk persists in these rural areas. Therefore, some recommended measures to interrupt parasite transmission are still relevant, including: controlling livestock slaughtering, proper disposal of offal particularly viscera, regular deworming of dogs with praziquantel and vaccination of intermediate hosts . AutochtFlorent Darriet: Writing - original draft. Nadim Cassir: Resources, Visualization. David J. Birnbaum: Resources. Jérôme Soussan: Resources. Estelle Menu: Resources. Stéphane Ranque: Supervision. Coralie L’Ollivier: Writing - review & editing.All authors declare any conflicts of interest or sources of funding


Escherichia coli and Staphylococcus aureus, we designed an ecological experiment to monoculture and co-culture E. coli and S. aureus. We co-cultured 45 strains of E. coli and S. aureus, as well as each species individually to measure growth over 36 h. We implemented a genome wide association study (GWAS) based on growth parameters  to identify significant single nucleotide polymorphisms (SNPs) of the bacteria. Three commonly used growth regression equations, Logistic, Gompertz, and Richards, were used to fit the bacteria growth data of each strain. Then each equation’s Akaike’s information criterion (AIC) value was calculated as a commonly used information criterion. We used the optimal growth equation to estimate the four parameters above for strains in co-culture. By plotting the estimates for each parameter across two strains, we can visualize how growth parameters respond ecologically to environment stimuli. We verified that different genotypes of bacteria had different growth trajectories, although they were the same species. We reported 85 and 52 significant SNPs that were associated with interaction in E. coli and S. aureus, respectively. Many significant genes might play key roles in interaction, such as yjjW, dnaK, aceE, tatD, ftsA, rclR, ftsK, fepA in E. coli, and scdA, trpD, sdrD, SAOUHSC_01219 in S. aureus. Our study illustrated that there were multiple genes working together to affect bacterial interaction, and laid a solid foundation for the later study of more complex inter-bacterial interaction mechanisms.To accurately explore the interaction mechanism between  There are extremely complex interactions between organisms and their environment, which play a crucial role in nature, particularly in microbial environments  with longitudinal phenotypes provides a novel platform to identify genetic variant associations and how they change over time   using g(t) denote the growth of a trait at time t. These equations are expressed asBacterial growth follows a standard s-shaped curve. A typical growth curve spans three continuously connected phases: the lag phase , the exponential phase , and the stationary phase (growth rate continues to decelerate to zero and bacteria reached a stable state). Many mathematical equations have been derived to reflect the features of a growth curve, including Gompertz, logistic, and Richards equations. Let λ (lag time), R (maximum specific growth rate) and A (asymptotic growth) to describe three continuous phases of bacterial growth curves, respectively. Moreover, these three key parameters were used as phenotypic data in GWAS analyses. In the Richards equation s is a shape parameter that describes the curvature of a growth curve  of each association, from which Manhattan plots were derived.We analyzed the associations between SNPs and microbial abundances measured for bacterial populations reared in monocultures and co-cultures. We calculated the logIn co-cultures, we used binary correlation coefficients to treat the three parameters as three different periods of phenotypic data combined with genotype data for comparative analyses. We constructed two-dimensional Manhattan plots, thereby obtaining significant SNPs of interaction between the two strains under the same culture conditions.E. coli and 45 S. aureus strains  parameter and 12 under the R parameter. Thirty-four significant SNPs were under the A parameter and shape (s) parameters, respectively  parameter, 13 under the R parameter, and seventeen under the shape (s) parameter. A parameter SNPs were the most diverse, with 19 significant SNPs  was predicted for the encoded cell wall hydrolase   and S. aureus (b) in co-culture.Additional file 4: Table S3. Gene annotation of E. coli and S. aureus in monoculture.Additional file 5: Table S4. Fitting R2 of growth curves in E. coli and S. aureus in co-culture.Additional file 6: Table S5. Gene annotation of E. coli and S. aureus in co-culture.