Anguilla japonica) are commercially important species, harvested extensively for food. Currently, this and related species (American and European eels) are challenging to breed on a commercial basis. As a result, the wild stock is used for aquaculture. Moreover, climate change, habitat loss, water pollution, and altered ocean currents affect eel populations negatively. Accordingly, the International Union for Conservation of Nature lists Japanese eels as endangered and on its red list. Here we presented a high-quality genome assembly for Japanese eels and demonstrated that large chromosome reorganizations occurred in the events of third-round whole-genome duplications (3R-WRDs). Several chromosomal fusions and fissions have reduced the ancestral protochromosomal number of 25 to 19 in the Anguilla lineage. A phylogenetic analysis of the expanded gene families showed that the olfactory receptors (group δ and ζ genes) and voltage-gated Ca2+ channels expanded significantly. Both gene families are crucial for olfaction and neurophysiology. Additional tandem and proximal duplications occurred following 3R-WGD to acquire immune-related genes for an adaptive advantage against various pathogens. The Japanese eel assembly presented here can be used to study other Anguilla species relating to evolution and conservation.Japanese eels ( Fishes are highly diverse species living in many ecological habitats, including freshwater, estuarine, and the ocean . Over 99From the evolutionary perspective, eels are among the extant basal groups of teleost ray-finned fishes after the 3-round whole-genome duplication (3R-WGD) . The rayAnguilla japonica , was kept in a freshwater tank for a week with aeration. Blood sample was taken from the fish, snapped frozen in liquid nitrogen, and then stored at −80°C. Genomic DNA was extracted from the blood sample. DNA sequencing data were generated by different platforms, including Oxford Nanopore (ONT) long reads, PacBio continuous long reads (CLRs), Illumina short reads, Illumina mate-pair reads, 10× Chromium linked reads, DNase Hi-C (Omni-C), and Bionano optical mapping .A market-purchased female Japanese eel, RRID:SCR_006255) 3.2.10. For PacBio CLR sequencing, the SMRTbell templates were prepared using Sequel Binding Kit 1.0 and sequenced on the PacBio Sequel System . For Illumina short reads and mate-pair sequencing, the libraries were prepared using the TruSeq DNA PCRFree Kit  and Nextera Mate Pair Library Preparation Kit  (gel plus), respectively. They were sequenced with 2× 150-bp reads on an Illumina HiSeq X Ten  instrument. The library for linked reads was prepared by a 10× Genomics Chromium  system with the Chromium Genome library (v2) and sequenced with 2× 150-bp reads on an Illumina NovaSeq 6000  instrument. Dovetail Omni-C Kit  was used for Hi-C library preparation, which used NEBNext Ultra  enzyme and Illumina-compatible adapters. Biotin-containing fragments were isolated using streptavidin beads before PCR enrichment. The library was sequenced with 2× 150-bp reads on an Illumina HiSeqX platform. The Bionano optical mapping was generated by 3 enzymes, 2 from Irys (Nt.BspQI and Nb.BssSI) and 1 from Saphyr (RRID:SCR_017992) (DLE1). We stretched and captured the images of fluorescently labeled DNA molecules in Irys and Saphyr G1.2 chips. The labeling distances were extracted from the images and recorded into the raw molecule files. Molecules over 150 Kbp were assembled into consensus maps using Bionano Solve for further analysis (The library for ONT long-read sequencing was prepared using the Ligation Sequencing Kit (LSK109) and sequenced using the Nanopore PromethION P48  sequencer with the flow cells (R9.4.1) and the basecaller version Guppy  v2 [RRID:SCR_017225) v2.5 [RRID:SCR_017016) v2.71 [RRID:SCR_017642) v1.4.16 [RRID:SCR_014731) v1.23 [MitoZ software (v2.4)  was used5880) v2 , Wtdbg2 6) v2.71  separate6) v2.71  to achie1) v1.23  for 2 roRRID:SCR_021172) v1.11.08 [We applied Tigmint v1.1.2  and ARKSv1.11.08  to extende novo approaches to annotate transposable elements (TEs) in the Japanese eel genome. For the homolog-based approach, RepeatMasker v4.0.7 [RRID:SCR_021169) v21.12 database [RRID:SCR_015247) v1.06 [de novo approach, RepeatModeler (RRID:SCR_015027) v1.0.8 was used to detect the TE families and repeat boundaries by integrating 3 complementary de novo repeat finding programs. RepeatMasker collected the union of these tools' results and annotated the genome accordingly.Tandem Repeats Finder v4.09  was applr v4.0.7  and Repedatabase  to the g7) v1.06  was usedde novo, homology-based, and transcriptome-based annotations. Maker (RRID:SCR_005309) v2.31.8 [Anguilla anguilla), zebrafish (Danio rerio), Indo-Pacific tarpons , Asian arowana (Scleropages formosus), and spotted gar (Lepisosteus oculatus), based on the phylogeny of teleost fishes [Three types of methods were used to annotate the protein-coding genes in the genome, including  v2.31.8  was adopt fishes .De novo annotation was performed using Augustus (RRID:SCR_008417) v3.2.1 [RRID:SCR_002127) v1 [RRID:SCR_015530) v2.1.0 [RRID:SCR_013048) v2.10.0 [) v3.2.1  and SNAP2127) v1  by train) v2.1.0  and asseGene functional annotation was performed by aligning the predicted gene sequences to protein sequences using BLAST v2.2.31  in the sRRID:SCR_015008) v5.1.2 [RRID:SCR_011980), to compare.BUSCO  v5.1.2  was usedRRID:SCR_010835) 1.3.1 [RRID:SCR_001598) [RRID:SCR_007891) v12 [tRNAscan-SE (5) 1.3.1  was used_001598) . The mic891) v12  to the gRRID:SCR_006086) v2.2.3 [Anguilla rostrata , A. anguilla , A. japonica (Japanese eel), M. cyprinoides , S. formosus , Gadus morhua , Oryzias latipes , D. rerio ,L. oculatus , Erpetoichthys calabaricus , Latimeria chalumnae , and Callorhinchus milii . We estimated the divergence times for single-copy orthologos using mcmctree in PAML (RRID:SCR_014932) package v4.8a [RRID:SCR_021162) website: D. rerio with O. latipes (180.0–264.0 Mya), M. cyprinoides with A. anguilla (162.2–197.3 Mya), C. milii with D. rerio (442.7–515.5 Mya), and E. calabaricus with D. rerio (381.0–407.0 Mya). To estimate gene family expansion and contraction, we used CAFÉ (RRID:SCR_005983) v4.2.1 [OrthoMCL (v2.0)  was used) v2.2.3  to recon) v4.2.1  to modelRRID:SCR_011822) v2.2.26 [RRID:SCR_008493) v6.6.0 [RRID:SCR_001010) v2.2.26 to remove sequences that did not match genes already known in SwissProt and NR. InterProscan was used to determine the secondary structures of the predicted OR genes. Some genes were filtered due to lacking the 7 transmembrane domains. The maximum likelihood phylogenetic tree was reconstructed using IQ-TREE (RRID:SCR_017254) v2.2.0.3 [RRID:SCR_011811) v7.505 [We identified olfactory receptor (OR) genes using the pipeline described in GitHub , while c v2.2.26  was used) v6.6.0 . Using Ev2.2.0.3  based on) v7.505 .A. japonica, A. anguilla, A. rostrata, M. cyprinoides,and Lepisosteus oculatus. The numbers of nonsynonymous substitutions (Ka) and synonymous substitutions (Ks) were calculated using KaKs_calculator2.0 [MCscanX v1.5.1  and macrlator2.0 . In addilator2.0 , using sA. japonica (Japanese eel), M. cyprinoides (tarpon), and S. formosus . The ancestral teleosts karyotype (ATK) was constructed using zebrafish, S. formosus (arowana), and L. oculatus  [Ancestral eel/tarpon karyotype (AETK) was constructed using t-group) . This wat-group)  to obtait-group) .AB038556.2) of the NR database from NCBI  [In this study, MitoZ software was used to assemble and annotate the mitochondrial genome 16.686 Kb) of our sample to confirm the species' identity . The data matched with the Japanese eel mitochondrial genome , we identified 29,982 coding genes  loci in the 1,131 single-copy orthologs from the 12 fish species  and Cypriniformes  diverged from the Eloposteoglossocephala clade at 262.5 Mya. Above are fish groups that had undergone 3R-WGD. Compared to the out-groups, spotted gars, reed fish, coelacanths, and Australian ghost sharks underwent only 2 rounds of whole-genome duplication (2R-WGD).The orthology analysis of 12 species' coding genes identified 21,653 gene family clusters. The expansion and contraction of gene families reflect the evolution of organisms' adaptations to their environments. Ortholog analysis of genes from the 12 species  identified 21,652 gene family clusters. By removing gene families with too many (≥200) or too few (≤2) genes, we achieved 129,862 genes to evaluate the expansion and contraction of gene families Fig. . Among t2+ and K+ channel families were identified. Calcium and potassium play significant roles in neuronal excitability, muscle contraction, fertilization, and energy metabolism. Interestingly, the other expanded gene families include (i) the assembly of thick myosin filament in skeletal muscle, (ii) lipoprotein receptor–related protein (metabolic and morphogenetic pathways), and (iii) isocitrate and isopropyl malate dehydrogenases family (carbohydrate and amino acid metabolism).Comparing the Japanese eel to the other 11 species, 433 gene families increased, with a total increase of 551 genes. On the other hand, a total of 943 genes were lost from 782 gene families . It is iA. japonica, A. Anguilla, and M. cyprinoides, respectively  had many paralogous pairs after splitting from the Osteoglossomorpha lineage . The obsely Fig. . Additioely Fig. . By idenThere are 21,249 duplicated genes identified among the 29,982 coding genes in the Japanese eel genome. Based on their duplication patterns, DupGen_finder  classified the duplicated genes into 5 categories: (i) 9,890 WGDs (46.54%), (ii) 1,420 tandem duplicates , (iii) 768 proximal duplicates , (iv) 3,975 transposed duplicates , and (v) 5,196 dispersed duplicates . We then calculated the Ks and Ka/Ks values for these 5 gene categories. Ks distribution indicates that TD and PD revealed additional duplication after 3R-WGD Fig. . In addin) is 25 for tarpons, arowana, and zebrafish; 24 for medaka; and 23 for Atlantic cod. Japanese eels have a lower haploid chromosome number (n = 19). To assess the extent of interchromosomal rearrangements in Japanese eels, we reconstructed the karyotype of the common ATK and AETK  in tarpons. Comparatively, the 19 AETK chromosomes  underwent 24-fusion and 18-fission to form the 13 chromosomes  in Japanese eels (n = 19) in Japanese eels, of which Chr 1, and Chr 3–7 rearrangements are unique to Japanese eels and might play a role in speciation. Japanese eel's chromosomes  were derived from AETK's  with slight rearrangements. The patterns of chromosome rearrangements in Chr 8, 13, and 16–17 of Japanese eels were comparable with Chr 21, 6, 8, and 19 in tarpons. Without rearrangement, Japanese eel's chromosomes 10, 14, and 19 were equivalent to AETK's chromosomes 3, 6, and 22. In addition, 3 chromosomes in the Japanese eel  derived directly from AETK's chromosomes , which also correspond to tarpon's chromosomes , respectively. Figure When comparing chromosome numbers of the fishes that had all undergone 3R-WGD, the haploid chromosome number  and zeta (ζ) group genes in the freshwater eels expanded enormously, comprising about 86% of the entire gene family. Delta (δ) and ζ belong to the type I genes , which a2+ channels were the significantly expanded gene families in Japanese eels. Genome studies suggest that the cellular functions of voltage-gated ion channels emerged early in Metazoan evolution [2+ during migration between waters with great variations of calcium contents. A gene expression study in marbled eel (Anguilla marmorata) showed high expression of voltage-gated Ca2+channels in brain, skin, and osmoregulatory tissues  and its response to changes in water calcium levels [2+ homeostasis, Ca2+ signaling coordinates various physiological processes, including skeletal muscle contractions, nervous system activity, and cardiac and reproductive functions. The expanded gene families of thick myosin filament in skeletal muscle imply enhanced coordination of muscle contraction and performance [The voltage-gated Cavolution , 85 in dm levels . Besidesformance , especiaformance . Additioformance , 90. Isoformance . The dupformance . These eformance . Notablyformance  and sturformance .n) of 26 [n = 24 or 25 in extant teleost species. In this study, we reconstructed the ancestral protochromosomes AETK (n = 25) to describe the cross-species chromosome collinearity and underpin the lineage-specific genome reorganization. The chromosome number of Anguillaspecies (n = 19) was reduced as compared with M. cyprinoides (n = 25) and S. formosus (n = 25). The Anguilliformes is made up of 15 families with remarkable karyotypic diversity [n = 19 and 21. The Anguilla lineage underwent a significant structural rearrangement upon their divergence from the common ancestor of tarpons (M. cyprinoides). The fusion and fission of their chromosome structure were the primary drivers of reducing the haploid chromosome number to 19.The acquisition of evolutionary novelty by WGD duplication and the subsequent fate change of duplicated genes is necessary for phenotype alteration, environmental adaptation, and speciation . The larn) of 26 . Evolutiiversity . The hapA. japonica whole genome sequencing and assembly are publicly available on NCBI databases under the accession number PRJNA852364. The gene models are available at Zenodo [GigaScience GigaDB database [The t Zenodo . All supdatabase .Supplementary Fig. S1. The alignment plot shows our mitochondrial genomes assembled and GenBank ID AB038556.2 of Japanese eels.Supplementary Fig. S2. Circos plot for the mitochondrial genome of Japanese eel. From outer to inner circles: protein-coding genes, rRNA, and tRNA; depth of Illumina short reads; and GC content.Supplementary Fig. S3. Multiplatform Japanese eel genome assembly.Supplementary Fig. S4. Genome comparison of Japanese (A. japonica) and European (A. anguilla) eels.Supplementary Fig. S5. Venn diagram of gene annotation based on 5 databases .Supplementary Fig. S6. KEGG-based gene function classification. The numbers represent how many genes are in the particular functions.Supplementary Fig. S7. KOG-based gene function classification. The numbers represent how many genes are in the particular functions.Supplementary Fig. S8. Length distribution of messenger RNA, CDS, exon, intron, and the number of exons in Japanese eel and the other species .Supplementary Fig. S9. Ks distributions of syntenic paralogs and orthologs. Ks value distribution is used to identify genome duplication and speciation.Supplementary Fig. S10. Comparative genomic analysis of Japanese eels (A. japonica), tarpons (M. cyprinoides), and arowanas (S. formosus).Supplementary Table S1. Genome sequencing platforms for A. japonica.Supplementary Table S2. A summary of contig statistics from the ONT long-read assembly.Supplementary Table S3. A summary of contig statistics after assembly error correction.Supplementary Table S4. Scaffolding by 10× linked reads, Bionano optical mapping, and Hi-C.Supplementary Table S5. The completeness of A. japonica genome by BUSCO assessment.Supplementary Table S6. Statistical results for repeat sequences.Supplementary Table S7. A statistical analysis of the classification results for TE.Supplementary Table S8. Functional annotation of predicted genes from A. japonica.Supplementary Table S9. The completeness of A. japonica genes by BUSCO assessment.Supplementary Table S10. The average length of exons in Japanese eel and the 8 related fish species.Supplementary Table S11. GO enrichment analysis of the gene families expanded in the 3 freshwater eel genomes.Supplementary Table S12. GO enrichment analysis of the gene families expanded in the A. japonica genome.Supplementary Table S13. The karyotypes of M. cyprinoides (tarpons) and the common ancestor of eels and tarpons (AETK).Supplementary Table S14. The karyotypes of A. japonica (Japanese eel) and the common ancestor of eels and tarpons (AETK).giac120_GIGA-D-22-00177_Original_SubmissionClick here for additional data file.giac120_GIGA-D-22-00177_R1_Revision_1Click here for additional data file.giac120_GIGA-D-22-00177_Revision_2Click here for additional data file.giac120_Response_to_Reviewer_Comments_Original_SubmissionClick here for additional data file.giac120_Response_to_Reviewer_Comments_Revision_1Click here for additional data file.giac120_Reviewer_1_Report_Original_SubmissionChristiaan Henkel -- 8/9/2022 ReviewedClick here for additional data file.giac120_Reviewer_1_Report_Revision_1Christiaan Henkel -- 10/6/2022 ReviewedClick here for additional data file.giac120_Reviewer_2_Report_Original_SubmissionZhong Li -- 8/22/2022 ReviewedClick here for additional data file.giac120_Supplemental_Figures_and_TablesClick here for additional data file.AETK: ancestral eel/tarpon karyotype; ATK: ancestral teleosts karyotype; BLAST: Basic Local Alignment Search Tool; bp: base pair; BUSCO: Benchmarking Universal Single-Copy Orthologs; CLR: continuous long read; DSD: dispersed duplicate; Gb: gigabase; Kbp: kilobase pair; KEGG: Kyoto Encyclopedia of Genes and Genomes; Mb: megabase; Mbp: megabase pair; Mya: million years ago; NCBI: The National Center for Biotechnology Information; ONT: Oxford Nanopore; OR: olfactory receptor; ORF: open reading frame; PD: proximal duplicate; rRNA: ribosomal RNA; TD: tandem duplicate; TE: transposable element; TRD: transposed duplicate; tRNA: transfer RNA; WGD: whole-genome duplication; 2R-WGD: 2-round whole-genome duplication; 3R-WGD: 3-round whole-genome duplication; 4R-WGD: 4-round whole-genome duplication.The experimental plan and sequencing strategy were designed by C.K.C.W., E.L.Z., A.O.L.W., K.P.L., and T.F.C. Samples were collected by A.H.M.N. and H.T.W. Bionano optical mapping and data analysis were conducted by C.Y.L.C., E.Y.C.C., and J.Z. The sequencing data for genome assembly were analyzed by E.L.Z., H.W., B.W., J.J., E.Y.C.C., and T.F.C. The manuscript was written by C.K.C.W., H.T.W., E.L.Z., H.W., and A.O.L.W.


Bifidobacterium bifidum DS0908 (DS0908) and Bifidobacterium longum DS0950 (DS0950). Treatment with DS0908 and DS0950 postbiotics significantly induced the expression of the brown adipocyte-specific markers UCP1, PPARγ, PGC1α, PRDM16 and beige adipocyte-specific markers CD137, FGF21, P2RX5, and COX2 in C3H10T1/2 mesenchymal stem cells (MSCs). In mice with high-fat diet (HFD)-induced obesity, both potential probiotics and postbiotics noticeably reduced body weight and epididymal fat accumulation without affecting food intake. DS0908 and DS0950 also improved insulin sensitivity and glucose use in mice with HFD-induced obesity. In addition, DS0908 and DS0950 improved the plasma lipid profile, proved by reduced triglyceride, low-density lipoprotein, and cholesterol levels. Furthermore, DS0908 and DS0950 improved mitochondrial respiratory function, confirmed by the high expression of oxidative phosphorylation proteins, during thermogenesis induction in the visceral and epididymal fat in mice with HFD-induced obesity. Notably, the physiological and metabolic changes were more significant after treatment with potential probiotic culture-supernatants than those with the bacterial pellet. Finally, gene knockdown and co-treatment with inhibitor-mediated mechanistic analyses showed that both DS0908 and DS0950 exerted anti-obesity-related effects via the PKA/p38 MAPK signaling activation in C3H10T1/2 MSCs. Our observations suggest that DS0908 and DS0950 could potentially alleviate obesity as dietary supplements.Probiotic supplements have promising therapeutic effects on chronic diseases. In this study, we demonstrated the anti-obesity effects of two potential probiotics,  The mice were administered DS0908 or DS0950 pellets  were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 10-μm sections. Standard H&E staining was performed using standard protocols. Five random fields of each section were evaluated, and the average adipocyte diameter was measured using the ImageJ software .Tbp was an internal control. We harvested C3H10T1/2 MSCs and extracted total RNA using an RNA Extraction Kit  following the manufacturer’s guidelines. We used 1 μg RNA to synthesize cDNA using the Maxime RT PreMix Kit  on a Veriti 96-Well Thermal Cycler . qRT-PCR was performed using an iQ SYBR Green Supermix Kit  on a CFX96 Real-Time PCR Detection System (Bio-Rad). + with ImageLab (Bio-Rad). Anti-β-actin antibody was used as a loading control for each protein expression. Protein band intensities were quantified using the ImageJ software.Differentiated and treated C3H10T1/2 MSCs were prepared in RIPA lysis buffer supplemented with protease inhibitors. The total protein concentration was measured using the BCA Protein Assay Kit (Thermo Fisher Scientific). Equal amounts of protein samples were separated on a 4–20% sodium dodecyl sulfate-polyacrylamide gradient gel  and transferred onto polyvinylidene difluoride (PVDF) membranes. The PVDF membranes were then blocked and incubated with specific antibodies, as indicated in the figures. Immunoreactive protein bands were captured using the chemiluminescent ECL  assay on ChemiDoc XRSC3H10T1/2 MSCs were seeded on 6-well plates and grown to 80–90% confluence. The cells were then transfected with control siRNA , PKAα siRNA  or p38 MAPKα siRNA  oligonucleotide duplexes  using Lipofectamine 2000  according to the manufacturer’s instructions. Transfection efficiency was determined using qRT-PCR and western blotting.t-test or two-way ANOVA  were used to distinguish the statistical significance levels, where *, **, ***, and ns indicates p < 0.05, < 0.01 and < 0.001, and no significance, respectively.All values are expressed as the average ± standard error mean (SEM). The experimental determinants were confirmed by using at least triplicate biological samples. Student’s B. bifidum DS0908 (DS0908) and B. longum DS0950 (DS0950) promote brown adipocyte-, beige adipocyte-, and lipolysis-related marker expressions in 3T3-L1 pre-adipocytes. Treatment with culture supernatants of DS0908 and DS0950 reduced intracellular triglyceride (TG) accumulation during thermogenesis induction, confirmed by ORO staining and TEM analysis [Ucp1 (5.60- and 3.38-fold), Pgc1α (12.85- and 7.02-fold) and Prdm16 (4.56- and 3.59-fold) mRNA expressions and UCP1 (2.41- and 2.32-fold), PPARγ (3.12- and 5.93-fold), PGC1α (12.06- and 1.98-fold) and PRDM16 (0.70- and 0.31-fold) protein expressions in C3H10T1/2 MSCs (Fgf21 (8.94- and 4.94-fold) and P2rx5 (6.96- and 10.80-fold), as well as the brown adipocyte-specific marker Cox2 (9.76- and 7.10-fold) in C3H10T1/2 MSCs. However, we observed no significant change in the Cd137 and Tbx1 mRNA expression levels , Psat1 (1.51- and 1.36-fold), Resistin (1.43- and 1.00-fold) and Serpina3k (0.59- and 0.51-fold) in DS0908 and DS0950-treated C3H10T1/2 MSCs (Ucp1 (137.37- and 124.96-fold), Prdm16 (25.60- and 267.22-fold) and Pparγ (0.07- and 16.08-fold), as well as the beige adipocyte-specific marker Cd137 (0.85- and 5.90-fold) mRNA expressions  into seven groups as follows: NFD; (G1), HFD; (G2), HFD + BS (cell-free supernatant) of DS0908 (G3), HFD + BS (cell-free supernatant) of DS0950 (G4), HFD + BP  of DS0908 (G5), HFD + BP  of DS0950 (G6), and HFD + Rosiglitazone (G7). The HFD groups were fed a 45% HFD for four weeks to induce obesity in the animals  noticeably reduced insulin levels, suggesting improved insulin sensitivity . FollowiS133 phosphorylation , and acetyl-CoA carboxylase activity , 37, 38.ogenesis , 44. Theogenesis . We alsoB. infantis and its culture supernatants  can induce thermogenesis by promoting white adipocyte browning to beige and by improving brown adipocyte activity [EBF2 and FGF21 deletion blunt beige adipocyte formation, whereas their induction triggers thermogenesis in beige adipocytes [B. bifidum DS0908 and B. longum DS0950 increased mitochondrial UCP1 and OXPHOS protein expressions. In addition, mitochondrial biogenesis increases mitochondrial content, which is known to contribute to thermogenesis. PGC1α reportedly improves mitochondrial biogenesis and oxidative phosphorylation, induced upon B. bifidum DS0908 and B. longum DS0950 supplementation [Bifidobacterium strains considered in the current study increased the PRDM16 level.Other approaches for ameliorating obesity include non-shivering thermogenesis and lipolysis activation in mature white adipocytes. Several studies demonstrated that activity , 9. Whitipocytes , 46. PPAipocytes , 21. Botipocytes , 29. Freipocytes . In our entation , 28. PRDentation . The BifaP2), Psat1, Resistin and Serpina3k; beige and brown adipocytes, by Cd137, Tbx1 and Fgf21, as well as Cox2 and P2rx5, expressions, respectively [B. bifidum DS0908 and B. longum DS0950 supplementation [e.g., AMPK, PKA, p38 MAPK and CREB) phosphorylation, further inducing downstream thermogenic (common beige and brown) protein  expressions [B. infantis, can inhibit adiposity via AMPK and PKA/p38 MAPK signaling activation [B. longum positively regulated thermogenesis via PKA signaling activation [e.g., PKA, P38 MAPK and AMPK) [B. bifidum DS0908 and B. longum DS0950 activated PKA/p38 MAPK signaling in C3H10T1/2 MSCs during the promotion of thermogenesis. It is worth mentioning that multiple studies have investigated crude culture supernatants of probiotics as viable components to understand their role in alleviating obesity [B. bifidum DS0908 and B. longum DS0950 pellets and culture supernatants both in vitro and in vivo, these findings could be firmly bolstered by identifying key metabolites and investigating their anti-obesity effects via browning activation.Mature white adipocytes are characterized by adipocyte-binding protein 4  -51. In o obesity . AlthougB. bifidum DS0908 and B. longum DS0950 pellets and culture supernatants ameliorate obesity by activating the thermogenic program. In vivo study revealed that they improved the plasma lipid profile, insulin sensitivity, and glucose metabolism without altering food intake, suggesting that B. bifidum DS0908 and B. longum DS0950 and their culture supernatants might mitigate the clinical onset of obesity.In summary, our study provides in vitro and in vivo evidence that http://jmb.or.kr.Supplementary data for this paper are available on-line only at


Retraction Note:J Exp Clin Cancer Res38, 223 (2019)https://doi.org/10.1186/s13046-019-1210-3The Editor-in-Chief has retracted this article. After publication concerns were raised regarding irregularities present in multiple figures in this article. There is overlap in Fig. 2A with Fig. 2A of a previously published article . Figure


Klebsiella quasipneumoniae subsp. similipneumoniae strain IF3SW-P1, isolated from the International Space Station, was sequenced using Oxford Nanopore Technologies. The genome lacks a megaplasmid typical of hypervirulent and multidrug-resistant Klebsiella strains but does contain a chromosomally encoded OqxAB efflux pump associated with carbapenem resistance.The 5.2-Mb circular genome of  Klebsiella pneumoniae were described as the novel species Klebsiella quasipneumoniae , carbapenem-resistant, and hypermucoviscous strains isolated from both hospital-borne and community-acquired infections  on the ISS on 4 March 2015  using a –nano-hq and –read-error 0.03 for the Q20+ data (Escherichia coli strain Q4552 plasmid pECQ4552_IHU08 (GenBank accession number CP077071.1) (blaKPC and Inc(FII), which are known to occur in Klebsiella species (Oxford Nanopore Technologies sequencing was performed using a GridION MK1 sequencer on a R10.4 flow cell (FLO-MIN112) with a library synthesized from Q20+ EA (early access) ligation reagents (SQK-LSK112-XL). The raw reads were base called using MinKNOW v29.10.8, with a mean quality score of 16.3 and a mode of 18.03. The genome was assembled, circularized, and polished using Flye v2.9 with the parameters 20+ data . The Fly77071.1) . Notably species .K. quasipneumoniae subsp. similipneumoniae by calculating the average nucleotide identity (ANI) using the EzBioCloud calculator compared to the two subspecies’ type strains, K. quasipneumoniae subsp. quasipneumoniae 01A030T  and K. quasipneumoniae subsp. similipneumoniae 07A044T   . Strain 0T ANI, 9.63% and iutA, which encodes a ferric aerobactin receptor, although the gene encoding the associated siderophore aerobactin (iucA) is not present  and the Sequence Read Archive (SRR17974437). These data are also available at NASA GeneLab (GLDS-470).The genomic assembly and raw reads have been deposited at GenBank (accession number


The COVID-19 pandemic and lockdowns may adversely affect pregnancy outcomes due to disrupted healthcare provision and increased stress, anxiety and economic hardship. We assessed changes in perinatal outcomes in 2020 using population birth data in Europe.25 Countries in the Euro-Peristat Network implemented a federated analysis using routine national data. Countries generated anonymised aggregate data files using R scripts from individual-level data formatted to a common data model with 22 variables. We compared preterm birth, stillbirth, neonatal death and caesarean delivery rates in 2020 to 2015-2019 for 2 periods: full-year (FY) and pandemic (March-September [MS]). Data from October onward were not included in the MS period because potentially declining pandemic-related fertility may affect perinatal indicators. Country-specific relative risks (RR) for the periods, adjusted for linear trends, overall and by socio-economic (SES) group, were calculated and pooled using random effects meta-analysis.Preterm birth rates decreased slightly  0.95-0.99]; 0.98MS [0.96-1.00]) in 2020. Heterogeneity was high , with 5 countries experiencing significant declines. Neonatal mortality rates were unchanged (0.97FY [0.92-1.01]) while stillbirth rates were higher . Caesarean rates were slightly raised . Increases for stillbirth were more pronounced in the lowest (1.08FY [0.99-1.16]) versus highest SES group (1.05 FY [0.93-1.17]).In 2020, there was an unexpected decline in preterm birth in some countries, while increases in stillbirths and caesarean occurred in others. High country-level heterogeneity suggests that some government policies to mitigate the pandemic might have been more protective of pregnant women and newborns than others.


E. coli isolates from suckling piglets with colibacillosis.Colibacillosis is a frequent enteric disease in the pig industry that causes significant economic losses. The objective of this study was to investigate the molecular characteristics of fluoroquinolone (FQ)-resistant E. coli isolates were tested in this study and all isolates showed multi-drug resistance (MDR) and mutations in quinolone resistance determining regions (gyrA or parC). Especially, FQ-resistant E. coli isolates with double mutations in both gyrA and parC were shown a high FQs minimum inhibitory concentration . Among 43 FQ-resistant E. coli isolates, 12 (27.9%) were showed plasmid-mediated quinolone resistance (PMQR) positive E. coli. Prevalence of PMQR gene, aac(6’)-Ib-cr, qnrS, and qepA, were identified in 7, 3, and 2 E. coli isolates, respectively. We identified the following in PMQR-positive E. coli isolates: the tetracycline resistance genes tetD , tetE , tetA , and tetB ; β-lactamases–encoding blaCMY-2 , blaTEM-1 , blaOXA-1 , blaSHV-1 , and blaAAC-2 ; and the chloramphenicol resistance genes ; the sulfonamide resistance genes sul1  and sul2 ; the aminoglycoside modifying enzyme gene aac(3)-II . The F4 , LT:STb:EAST1 , and paa  were most common fimbrial antigen, combinations of toxin genes, and non-fimbrial adhesins genes, respectively. All PMQR-positive E. coli carried class I integrons but only 4 isolates carried the gene cassette. The most prevalent plasmid replicon was FIB , followed by FIC, HI1, and N , respectively.A total of 43 FQ-resistant E. coli can serve as a reservoir of FQ resistant genetic determinants that can be transferred to pathogenic bacteria in humans or pigs, this represents a public health hazard.Because FQ-resistant The online version contains supplementary material available at 10.1186/s12866-022-02632-9. Escherichia coli (E. coli) in pigs is the most frequent enteric disease and an important cause of death in suckling piglets [Colibacillosis caused by  piglets . This di piglets . The useE. coli or other gram-negative bacteria. The World Health Organization (WHO) has classified FQs as ‘‘critically important antimicrobials’’ because of their clinical importance in both human and animal medicine [E. coli have emerged in Korea after enrofloxacin was licensed to be used for veterinary purposes [FQs are broad-spectrum antimicrobials agents and have been used for the treatment of various infections caused by medicine . Becausepurposes .qnr-mediated inhibition of quinolone binding to DNA [qepA encoded efflux pump [aac(6’)-Ib-cr mediated FQ acetylation [E. coli isolated from suckling piglets with diarrhea. Therefore, the purpose of this study was to investigate the molecular characteristics of FQ-resistant E. coli isolates from suckling piglets with colibacillosis.Multiple mechanisms are involved in resistance to FQ in Enterobacteriaceae. The major mechanisms of resistance to FQ involve mutations of chromosomal genes encoding DNA gyrase and/or topoisomerase IV , 6. In ag to DNA , 8, qepAlux pump , and thetylation , 11. Thetylation . Althougtylation –15, therE. coli isolates collected from each colibacillosis clinical case in suckling piglets from 2007–2018 were tested. The farms consisted of 42 different pig herds (50 to 100 sows per each herd). The aseptically collected intestinal contents and feces were inoculated on MacConkey agar containing 4 mg/mL of ciprofloxacin . Subsequently, suspected E. coli colonies were identified by VITEK II system . Thus, a total of 43 CIP-resistant E. coli were tested in this study.E. coli ATCC 25,922 was included as a quality control. Multidrug-resistance (MDR) was defined as acquired resistance to at least one agent in three or more antimicrobial classes [The disk diffusion method was performed according to the Clinical and Laboratory Standards Institute (CLSI) guidelines . The 19  classes .E. coli Reference Laboratory, Quebec, Canada).O-serogroup typing was performed using rabbit antisera purchased from SSI  with the slide agglutination technique of the Animal and Plant Quarantine Agency . A standard strain was obtained from Dr. J.M. Fairbrother  to identify mutations in 43 FQ-resistant E. coli isolates using primers and conditions described previously [http://www.ncbi.nlm.nih.gov/BLAST). PMQR genes -Ib-cr, and qepA) were detected by PCR amplification and sequencing analysis, as described in Table SPCR was carried out to amplify quinolone resistance determining regions (QRDRs) of the target genes -II, and ant(2’’)-I), β-lactam antimicrobials , chloramphenicols (cmlA and catA1), sulfonamides (sul1 and sul2), and tetracyclines . The virulence factor genes associated with the toxins , fimbriae , and non-fimbrial adhesins  were also confirmed by PCR as previously described [For detection of antimicrobial resistance genes and virulence genes, PCR was performed using DNA extracted from PMQR-positive escribed .E. coli isolates. The primers used in this study targeted 18 different replicons [For plasmid replicon typing and detection of integrons and gene cassettes, PCR was performed using DNA extracted from PMQR-positive eplicons  and clasE. coli J53 used as the recipient as previously described [To determine the transferability of PMQR and other genes, conjugation assays were performed using the broth mating method, with escribed . TranscoE. coli isolates from colibacillosis are shown in Table E. coli, all isolates showed MDR against 6 to 11 classes of antimicrobial agents. The rates of resistance to the various antimicrobial classes were as follows: aminoglycosides , penicillins , quinolones , tetracyclines , phenicols , β-lactam/β-lactamase inhibitor combinations , folate pathway inhibitors , cephems , monobactams , and polypeptides . The rate of resistance to 9 antimicrobial classes was the highest at 39.5%, and one (2.3%) FQ-resistant E. coli isolate was identified resistance to 11 antimicrobial classes.MDR patterns of FQ-resistant E. coli isolates are shown in Table gyrA amino acid substitutions were S83L (43 isolates), D87N (22 isolates), D87G (5 isolates), and D87E (2 isolates), and the parC mutations were S80I (23 isolates), S80R (9 isolates), E84A (5 isolates), S80K (3 isolates), S80N (1 isolates), S80W (1 isolates), E84G (1 isolates), A56C (1 isolates), and S57Q (1 isolates). The MIC ranges for CIP, ENR, and NOR were 4–256 mg/mL, 8–512 mg/ml, and 8–512 mg/mL, respectively, and isolates with double mutations in gyrA were relatively higher than those of other isolates with single mutations in gyrA. In particular, FQ-resistant E. coli isolates with a high level of MICs range  were shown to carry double mutations in gyrA in combination with double mutations in parC. PMQR genes were detected in 12 (27.9%) of the 43 FQ-resistant E. coli isolates. The aac(6’)-Ib-cr, qnrS, and qepA genes were identified in seven, three, and two FQ-resistant E. coli isolates, respectively. Among 12 PMQR-positive E. coli isolates, one isolate that showed the highest MICs for CIP (256 mg/mL), ENR (512 mg/mL), and NOR (512 mg/mL), also carried the PMQR gene aac(6’)-Ib-cr.The molecular characteristics of 43 FQ-resistant E. coli isolates carried the following β-lactamase encoding genes: blaCMY-2 , blaTEM-1 , blaOXA-1 , blaSHV-1 , and blaAAC-2 . Tetracycline-resistance genes were detected in all PMQR-positive E. coli isolates as follows: tetD , tetE , tetA , and tetB . Two types of aminoglycoside-modifying enzyme genes were examined, but aac(3)-II was found only in 2 (16.7%) PMQR-positive E. coli isolates. The sul1 and sul2 sulfonamide-resistance genes were detected in 9 isolates (75.0%) and 10 isolates (83.3%), respectively. The cmlA chloramphenicol-resistance gene was found in 10 isolates (83.3%). Distributions of the virotypes are shown in Table E. coli isolates, all isolates were found to have class 1 integrons. Class 1 integrons contained four types of gene cassette arrangements, aadA1-dfrA1 (2 isolates), aadA1-aadA2- aadB (1 isolate), and aadA1-aadA2- aadB-cmlA6 (1 isolate). Eight isolates did not carry any of the gene cassettes. A total of 10 plasmid replicon types were identified in all 12 PMQR-positive E. coli isolates. The most common plasmid replicon was FIB , followed by FIC, HI1, and N , respectively. Transferability was only identified in 7 isolates among 12 PMQR-positive FQ-resistant E. coli isolates.The prevalence of antimicrobial resistance genes is shown in Table E. coli worldwide [E. coli isolates showed were MDR  with high levels of resistance to several antimicrobials: aminoglycosides (100.0%), penicillins (100.0%), tetracyclines (97.7%), and phenicols (90.7%). In particular, five isolates showed resistance to more than 10 classes. These results were consistent with previous studies showing co-association of resistance to other classes of antimicrobials and high MDR rates among FQ-resistant E. coli [Suckling piglets are vulnerable to colibacillosis for many reasons such as changes in environmental conditions, a decline in maternal antibody titers, and various stresses. Antimicrobials are used in intensive pig production systems to control infectious diseases. In particular, FQs are highly effective antimicrobial class with many advantages including high oral absorption, large volume of distribution, and broad-spectrum antimicrobial activity . Howeverorldwide ; thus, torldwide . In this E. coli , 27. Thi E. coli .E. coli and variety of O-serogroups has been associated with diarrhea [gyrA mutations, and 29 isolates (67.4%) were double amino acid substitutions (S83L plus substitution in aspartic acid 87). These results were consistent with previous studies showing that DNA gyrase is the primary target of FQ in gram-negative bacteria, and gyrA mutations are dominant mutations in E. coli [E. coli isolates had mutations at codon 80 in parC in the QRDRs, and the most common type of amino-acid substitution was S80I in parC. Previous studies reported that the substitution S80I was most frequently observed among substitutions in the QRDR of parC [E. coli isolates from humans [gyrA were relatively higher than those of other isolates with single mutations in gyrA. Notably, FQ-resistant E. coli isolates carrying double mutations in gyrA in combination with double mutations in parC were identified at high levels of MICs . These results are consistent with those of recent studies showing that the total number of point mutations in QRDR was positively correlated with the increased MIC.The O-serogroup is considered one of the major virulence factors of diarrhea –30. In tdiarrhea , 32. In  E. coli . Moreove of parC , 35. Them humans , 37. AlsE. coli isolates carried three types of PMQR genes, aac(6’)-Ib-cr (7 isolates), qnrS (3 isolates), and qepA (2 isolates). These PMQR variants have been previously detected in E. coli from livestock, including in healthy animals and retail meats in the Czech Republic [gyrA and harbored qnrS in its plasmid. This result showed that PMQR genes play a role in FQ resistance [In this study, 12 FQ-resistant Republic , China [Republic , and theRepublic , as wellRepublic . Also, tRepublic . Howeversistance .E. coli isolates carried a variety of antimicrobial resistance genes such as blaCMY, blaTEM, blaOXA, blaSHV, blaAAC, tetA, tetB, tetD, tetE, aac(3)-II, sul1, sul2, and cmlA and harbored mobile elements such as integrons and gene cassettes at the same time. The bla genes hydrolyze the characteristic β-lactam ring and confer resistance to most β-lactam antimicrobials, including cephalosporins [bla positive-E. coli were detected at high levels [E. coli isolates harbored class 1 integrons and four isolates also contained gene cassettes aadA or dfrA or both genes. These genes are frequently detected in class 1 integrons isolated from animals and humans in Korea [E. coli isolates from suckling piglets may have acquired the genetic determinants of drug resistance, which could become a concern.The rise of antimicrobial resistance is thought to be closely associated with the widespread transfer of resistance genes between bacterial species. In this study, all 12 PMQR-positive osporins . Previouh levels . The preh levels . Also, ain Korea . TherefoE. coli isolates. The most common plasmid replicon was IncF plasmids including FIB, and FIC. IncF plasmids were associated with the important role in the spread of virulence and resistance to important classes of antimicrobials including quinolones, β-lactams, TEs, sulfonamides, chloramphenicol, and aminoglycosides among Enterobacteriaceae [Plasmids are small DNA molecules that are distinct from chromosomes and can provide beneficial effects to bacteria such as antibiotic resistance through horizontal gene transfer . In thiseriaceae .E. coli virulence factors is important for diagnosing and establishing preventative measures for colibacillosis [E. coli isolates, respectively. These LT, STa, and STb genes damage the vessel and cause edema and a high mortality in pigs [E. coli isolates were identified as having the paa gene and coexisting with the F4 gene. Although the specific role of the paa gene in the development of pathogenic E. coli has not yet been clearly defined, various virotypes may also appear as a result of horizontal gene transferability of the paa gene [The detection of cillosis . In this in pigs . Also, t in pigs –52. Prev in pigs , 53. In paa gene .E. coli isolated from suckling piglets with colibacillosis. All FQ-resistant E. coli isolates showed an MDR phenotype, and the most prevalent of the mutations were double point mutations in gyrA and a single mutation in parC. Also, FQ-resistant E. coli isolates with PMQR genes carried various antimicrobial genes and harbored mobile elements and plasmid replicons. Antimicrobial resistance may become a serious problem because many drugs are probably ineffective for the treatment of colibacillosis and resistance elements can be horizontally transferred on pig farms. Also, this represents a public health hazard because FQ-resistant E. coli can serve as a reservoir of FQ resistant genetic determinants that can be transferred to pathogenic bacteria in humans or pigs. These data support the critical need for comprehensive surveillance of antimicrobial resistance on pig farms.This study investigated the molecular characteristics of FQ-resistant Additional file 1:Figure S1-1. LC716475 (SSC-1) blast alignment. Figure S1-2. LC716476 (SSC-2) blast alignment. Figure S1-3. LC716477 (SSC-3) blast alignment. Figure S1-4. LC716478 (SSC-4) blast alignment. Figure S1-5. LC716479 (SSC-7) blast alignment. Figure S1-6. LC716480 (SSC-8) blast alignment. Figure S1-7. LC716481 (SSC-10) blast alignment. Figure S1-8. LC716482 (SSC-11) blast alignment. Figure S1-9. LC716483 (SSC-12) blast alignment. Figure S1-10. LC716484 (SSC-13) blast alignment. Figure S1-11. LC716485 (SSC-14) blast alignment. Figure S1-12. LC716486 (SSC-15) blast alignment. Figure S1-13. LC716487 (SSC-16) blast alignment. Figure S1-14. LC716488 (SSC-17) blast alignment. Figure S1-15. LC716489 (SSC-19) blast alignment. Figure S1-16. LC716490 (SSC-20) blast alignment. Figure S1-17. LC716491 (SSC-21) blast alignment. Figure S1-18. LC716492 (SSC-22) blast alignment. Figure S1-19. LC716493 (SSC-23) blast alignment. Figure S1-20. LC716494 (SSC-26) blast alignment. Figure S1-21. LC716495 (SSC-27) blast alignment. Figure S1-22. LC716496 (SSC-28) blast alignment. Figure S1-23. LC716497 (SSC-29) blast alignment. Figure S1-24. LC716498 (SSC-30) blast alignment. Figure S1-25. LC716499 (SSC-31) blast alignment. Figure S1-26. LC716500 (SSC-32) blast alignment. Figure S1-27. LC716501 (SSC-33) blast alignment. Figure S1-28. LC716502 (SSC-34) blast alignment. Figure S1-29. LC716503 (SSC-35) blast alignment. Figure S1-30. LC716504 (SSC-36) blast alignment. Figure S1-31. LC716505 (SSC-37) blast alignment. Figure S1-32. LC716506 (SSC-38) blast alignment. Figure S1-33. LC716507 (SSC-39) blast alignment. Figure S1-34. LC716508 (SSC-40) blast alignment. Figure S1-35. LC716509 (SSC-41) blast alignment. Figure S1-36. LC716510 (SSC-42) blast alignment. Figure S1-37. LC716511 (SSC-43) blast alignment. Figure S1-38. LC716512 (SSC-44) blast alignment. Figure S1-39. LC716513 (SSC-45) blast alignment. Figure S1-40. LC716514 (SSC-46) blast alignment. Figure S1-41. LC716515 (SSC-47) blast alignment. Figure S1-42. LC716516 (SSC-48) blast alignment. Figure S1-43. LC716517 (SSC-49) blast alignment. Additional file 2:Table S1. Primers used for PCR and DNA sequencing.Additional file 3:Table S2. History of samples and isolates separated from each sample.


To date, 10 mcr variants, mcr-1 to mcr-10, have been described. mcr-8 was first identified in a carbapenem-resistant Klebsiella pneumoniae isolate  pathogens infections . Liu et  isolate . Unlike eumoniae , 5. In rfections . CurrentE. cloacae clinical isolate SD21 was recovered from the sputum sample of a male patient suffering from chronic obstructive pulmonary disease at a tertiary hospital in Shandong, China. The isolate was initially identified using mass spectrometry and confirmed by next-generation sequencing. MICs of 15 antimicrobial agents were determined by the broth microdilution method. The MIC of tigecycline was interpreted according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. And the interpretation of MICs of the remaining antimicrobials was based on the Clinical and Laboratory Standards Institute (CLSI) guidelines. This isolate exhibited a multidrug-resistant phenotype, with resistance to polymyxin B (>8 μg/mL), cefmetazole, ceftazidime, cefotaxime, cefepime, tigecycline, ciprofloxacin, amikacin, and aztreonam (Table S1). Conjugation assays were performed using the Escherichia coli J53 as the recipient strain  and long-read nanopore MinION (CP093914) and four plasmids, namely pSD21_mcr8 (CP093916), pSD21_266kb (CP093915), pSD21_54kb (CP093917), and pSD21_4kb (CP093918). According to multilocus sequence typing, SD21 was assigned as ST1718. Notably, a mcr-8 variant exhibiting 100% identity to mcr-8.2 was identified in pSD21_mcr8 (100 852 bp). The plasmid pSD21_mcr8, belonging to IncFIA(HI1)/IncFII(K), harbored mcr-8.2 within the genetic context IS903B-orf-ISRor7-orf-dgkA-baeS-copR-ISEcl1-orf-mcr-8.2-orf-ISKpn26  from K. pneumoniae , showing 99.91% identity and 100% coverage to p2019036D-mcr8-345kb. Besides, the lack of an intact tra region may be one of the aspects limiting the mobilization of pSD21_mcr8. However, the lack of tra operon did not prevent the mobilization of mcr-8-carrying plasmids between K. pneumoniae and E. cloacae. There could be other underlying mobilization mechanisms that mediate the dissemination of mcr-8-carrying plasmids in different species.To investigate the genetic structure of -ISKpn26 . The mcrblaCTX-M-55, blaTEM-1B), fosfomycin (fosA3), aminoglycosides (aac(3)-IId, aph(3′)-Ia, aph(6)-Id, rmtB, aadA22), macrolides (mph[A]), quinolones (qnrS1), sulfonamides (sul3), trimethoprim (dfrA14), tetracyclines (tet[A]), rifamycin (ARR-2), amphenicols (floR), and lincosamides (lnu[F]). Therefore, this plasmid enabled SD21 to exhibit resistance to multiple antimicrobial agents. The pSD21_266kb shared 99.99% identity with p16-6773.1 (CP039861.1) from Salmonella sp. . The other two plasmids in SD21 did not harbor accessory resistance genes.In addition to pSD21_mcr8, SD21 was found to harbor another resistance plasmid, designated pSD21_266kb. The pSD21_266kb with IncHI2/IncHI2A replicon type contained genes encoding resistance to β-lactams (mcr-8 in E. cloacae, identification of an E. cloacae isolate with mcr-8.2 indicates the further dissemination of the mcr-8 variant in different species. Furthermore, the mcr-8.2-bearing plasmid coexisted with a multidrug-resistant plasmid, which increased the difficulty of clinical treatment. In conclusion, we first reported an MDR E. cloacae strain with mcr-8.2 isolated from a patient in China. We hypothesize that the mcr-8.2-located transferable genetic elements may be the genetic basis for the transmission of mcr-8.2 among different species. Further research on epidemiology and transmission mechanism should be conducted to better understand the potential dissemination of mcr-8.Given that no reports have described the existence of CP093914, CP093915, CP093916, CP093917, and CP093918 under the BioProject PRJNA816615.The complete sequences of SD21 were deposited in the NCBI database with accession numbers


Retraction Note:J Exp Clin Cancer Res37, 202 (2018)https://doi.org/10.1186/s13046-018-0875-3The Editor-in-Chief has retracted this article because data presented in Figs. 2, 3 and 4 were duplicated from previously published articles. Specifically, a set of staining images in Fig. 2I had previously been published as Fig. 3E in . The act


To investigate clinical characteristics, management, and prognosis of Epstein-Barr virus (EBV)-positive lymphoma-associated hemophagocytic syndrome (LAHS) patients in real-world practice.This was a retrospective, single-center cohort study. EBV-positive LAHS patients diagnosed from January 2010 to December 2021 in our center were enrolled. Clinical characteristics, treatment, overall response rate (ORR), and overall survival (OS) were investigated. Univariate and multivariate analysis of potential factors were conducted.P=0.033, P=0.000, and P=0.004, respectively). Combined treatment of anti-hemophagocytic lymphohistiocytosis (HLH) and anti-lymphoma treatment was conducted in 24 patients; anti-HLH treatment was conducted in 18 patients; anti-lymphoma treatment was conducted in three patients; glucocorticoid treatment was conducted in one patient. ORR was 47.8%, and the median OS was 61  days for overall patients. Patients who received anti-HLH treatment and turned to anti-lymphoma treatment early displayed higher ORR and OS than those of anti-HLH patients . Elevated alanine aminotransferase level was the independent risk factor of EBV-positive LAHS prognosis.Of the 51 patients, 44 were T/NK cell lymphoma; five were B cell lymphoma; two were Hodgkin lymphoma. EBV-positive T/NK cell LAHS patients were significantly younger and showed lower fibrinogen levels and C-reactive protein levels than EBV-positive B cell LAHS patients (Prognosis of EBV-positive LAHS patients was poor. Anti-lymphoma treatment should be initiated as soon as HLH was rapidly controlled. EBV maind PD-L2 , 5. The nd PD-L2 . Howeveragenesis , 8.Hemophagocytic syndrome, also named hemophagocytic lymphohistiocytosis (HLH), is a rare but lethal hyperinflammatory syndrome. Aberrant activation of lymphocytes, monocytes, and macrophages leads to substantial release of inflammatory cytokines. HLH typically manifests itself as fever, hepatosplenomegaly, and multiple organ dysfunction syndrome (MODS). HLH can be categorized into primary HLH and secondary HLH. Primary HLH is characterized by early onset and cytotoxicity-related gene mutations. Secondary HLH mainly attacks adult patients secondary to malignancy, infection, autoimmune disease, pregnancy, and other triggers, with the incidence of 0.125 per 100,000 per year . Lymphomin situ hybridization. Besides antiviral treatment, induction treatment was divided into anti-HLH treatment, anti-lymphoma treatment, and the combination of both. Anti-HLH treatment consisted of HLH-1994 regimen, HLH-2004 regimen, and GED regimen (gemcitabine-etoposide-dexamethasone). Anti-lymphoma treatment was chemotherapy depending on different subtypes of lymphoma. Treatment response was evaluated according to the following conditions. Complete response (CR) required disappearance of all HLH-related symptoms, and HLH-related lab findings returning to normal range, including ferritin, soluble CD25 (sCD25), complete blood count, triglyceride, alanine aminotransferase (ALT), etc. Consciousness should recover to normal if central nervous system was involved. Partial response (PR) required normal body temperature and improvement by over 25% in at least two symptoms or lab findings, including sCD25 decrease by at least one third, ferritin and triglyceride decrease by at least 25%, ALT decrease by at least 50% if formerly over 400U/L, and neutrophil increase by 100% without transfusion. If neutrophil counts were formerly less than 0.5×109/L, they should exceed 0.5×109/L after treatment. If neutrophil counts were formerly over 0.5×109/L but less than 2×109/L, they should exceed 2×109/L after treatment. No response (NR) was defined as being unable to reach CR or PR. Overall response rate (ORR) was calculated as (CR+PR)/total patients × 100%. OS was defined as the interval between HLH diagnosis and all-cause death. The patients were followed up until December, 2021. This study was conducted with the approval of the West China Hospital ethics committee, according to Declaration of Helsinki.Patients consecutively diagnosed as EBV-positive LAHS from January, 2010 to December, 2021 at West China Hospital, Sichuan University were enrolled , 17. EBVt test and U test, respectively. Enumeration data were evaluated with χ² test. Survival analysis was assessed with Kaplan-Meier methods, and compared with Log rank test. Univariate and multivariate analysis of prognostic predictors was conducted with Cox proportional hazard model. It was regarded as statistically significant with P<0.05.Statistical analysis was conducted with SPSS 23.0 and GraphPad Prism 7.0 software. Measurable data with normal distribution and skewed distribution were evaluated with Fifty-one EBV-positive LAHS patients were enrolled in this study, including 35 (68.6%) male patients and 16 (31.4%) female patients. Median age at HLH diagnosis was 37 (13-64) years. Three (5.9%) patients were newly diagnosed as lymphoma, while 48 (94.1%) patients were relapsed/refractory lymphoma cases. Clinical characteristics of these patients at baseline were listed in P=0.033). Fibrinogen level and C-reactive protein (CRP) level were significantly lower in T/NK cell LAHS patients than B cell LAHS patients . Plasma EBV DNA level tended to be higher in B cell LAHS patients than in T/NK cell LAHS patients, but the difference was not significant. There was no significant difference in sex, IPI score, ECOG score, clinical manifestations, complete blood count, liver and kidney function, triglyceride, ferritin, sCD25, hemophagocytosis phenomenon, and the occurrence order of lymphoma and HLH between EBV-positive T/NK cell LAHS patients and EBV-positive B cell LAHS patients . EBV-positive LAHS patients displayed significantly higher hemoglobin and triglyceride levels at baseline than EBV-negative LAHS patients . T/NK-cell lymphoma tended to account for a higher proportion of EBV-positive LAHS patients (86.3%) than EBV-negative LAHS patients (69.8%), but the difference was insignificant  were significantly younger at HLH diagnosis than EBV-negative LAHS patients  patients who only received anti-HLH treatment, the median weeks of induction treatment was 4 (1-8). The majority of  patients received HLH-1994 regimen. HLH-2004 regimen  and GED regimen  were conducted in three patients each. Three (6.5%) patients only received anti-lymphoma chemotherapy. One ENKL patient and one ANKL patient received GLIDE regimen, and one DLBCL patient received chidamide-RCHOP regimen. Median number of chemotherapy cycles was 3 (2-8). One (2.2%) patient only received glucocorticoid treatment.For patients who were at higher risk or failed to achieve CR with a poor control of EBV infection, consolidation treatment was conducted, which included PD-1 monoclonal antibody treatment, chidamide treatment, and autologous or allogenic stem cell transplantation. For patients whose biopsy revealed positive PD-L1 staining of tumor cells, especially HL patients and DLBCL patients, PD-1 monoclonal antibody treatment was conducted. For patients who displayed aberrant epigenetic alterations, especially PTCL patients and double expression DLBCL patients, chidamide treatment was conducted. For young, fit, and high-risk patients with sufficient stem cells collected who were willing to receive transplantation, autologous or allogenic stem cell transplantation was conducted. Overall, seven (15.2%) patients underwent consolidation therapy. Three (6.5%) patients  received PD-1 monoclonal antibody treatment, of whom the DLBCL patient also received autologous stem cell transplantation after PD-1 monoclonal antibody treatment. Two (4.3%) patients  received autologous stem cell transplantation. One (2.2%) ANKL patient received allogenic stem cell transplantation. Two (4.3%) AITL patients received chidamide treatment.P=0.103). There was no significant difference in ORR among different treatment groups  patients, and PR was achieved in 18 (39.1%) patients. Twenty-four (52.2%) patients remained NR. ORR was 47.8%. In the combined treatment group, CR was reached in two (8.3%) patients, and PR was reached in 12 (50%) patients. NR was present in 10 (41.7%) patients. ORR was 58.3%. For patients who first received anti-HLH treatment and turned to anti-lymphoma chemotherapy subsequently, CR was achieved in two (10.5%) patients, and PR was achieved in 10 (52.6%) patients. Seven (36.8%) patients remained NR. ORR was 63.2%. For those who first received anti-lymphoma chemotherapy and turned to anti-HLH treatment subsequently, PR was achieved in two (40%) patients, and three (60%) patients remained NR. ORR was 40%. In anti-HLH treatment group, six (33.3%) patients achieved PR and twelve (66.7%) patients remained NR. ORR was 33.3% for the total anti-HLH treatment group, the HLH-1994 regimen subgroup, the HLH-2004 regimen subgroup, and the GED regimen subgroup. In anti-lymphoma treatment group, CR was achieved in two (66.7%) patients while one (33.3%) patient remained NR. ORR was 66.7%. One patient only received glucocorticoid treatment but remained NR. Patients who first received anti-HLH treatment and turned to anti-lymphoma treatment subsequently tended to show a higher ORR than those who received anti-HLH treatment only, but the difference was not significant . In terms of treatment group, median plasma EBV-DNA of patients after treatment in combined treatment group and anti-HLH group was 272.5 copies/mL (range 0-64800 copies/mL), and 3130 copies/mL (range 0-33800 copies/mL), respectively. For the three patients in anti-lymphoma group, median plasma EBV-DNA of patients after treatment was 0 copies/mL (range 0-50 copies/mL). For the only patient in glucocorticoid group, his plasma EBV-DNA level decreased from 158000 copies/mL to 88800 copies/mL after treatment. There was no significant difference in plasma EBV-DNA level after treatment among treatment groups.At baseline, plasma EBV-DNA of all patients was positive . After induction treatment, plasma EBV-DNA decreased significantly in all patients but one, who was a ANKL patient only receiving anti-HLH treatment but remaining NR . Plasma EBV-DNA of ten patients after treatment was negative. Another three patients had a plasma EBV-DNA level lower than 50 copies/mL after treatment. There was no significant difference in plasma EBV-DNA after treatment between T/NK-cell LAHS  and B-cell LAHS patients  patients received combined treatment. Sixteen (40%) patients first underwent anti-HLH treatment of 3.5 (1-9) weeks, and then turned to anti-lymphoma chemotherapy. Median number of chemotherapy cycles was 1 (1-8). Another five (12.5%) patients first received anti-lymphoma chemotherapy and turned to HLH-1994 regimen subsequently. The median number of chemotherapy cycles was 1 (1-3). The median weeks of HLH-1994 treatment was 4 (2-4). In the 16 (40%) patients who only received anti-HLH treatment, the median weeks of induction treatment was 4 (1-8). Two (5%) patients only received anti-lymphoma chemotherapy. Median number of chemotherapy cycles was 2.5 (2-3). One (2.5%) patient only received glucocorticoid treatment.Of the 40 evaluable EBV-positive T/NK cell LAHS patients, CR was achieved in three (7.5%) patients, and PR was achieved in 14 (35%) patients. Twenty-three (57.5%) patients remained NR. ORR was 42.5%. In the combined treatment group, CR was reached in two (9.5%) patients, and PR was reached in nine (42.9%) patients. NR was present in 10 (47.6%) patients. ORR was 52.4%. For patients who first received anti-HLH treatment and turned to anti-lymphoma chemotherapy subsequently, CR was achieved in two (12.5%) patients, and PR was achieved in seven (43.75%) patients. Seven (43.75%) patients remained NR. ORR was 56.25%. For those who first received anti-lymphoma chemotherapy and turned to anti-HLH treatment subsequently, PR was achieved in two (40%) patients, and three (60%) patients remained NR. ORR was 40%. In anti-HLH treatment group, five (31.25%) patients achieved PR and 11 (68.75%) patients remained NR. ORR was 31.25%. In anti-lymphoma treatment group, CR was achieved in one (50%) patient while one (50%) patient remained NR. ORR was 50%. One patient only received glucocorticoid treatment but remained NR. There was no significant difference in ORR among different treatment groups.P=0.033, We also compared treatment and response of EBV-positive LAHS patients and EBV-negative LAHS patients in our department. Treatment choice for these patients was significantly different, with a higher proportion of EBV-positive LAHS patients (52.2%) receiving combined treatment than EBV-negative LAHS patients  tended to display worse OS compared to patients whose plasma EBV DNA was no more than 105 copies/mL , but the difference was not significant (P=0.159) (P=0.060) (P=0.041) (P=0.003) (P=0.003) (P=0.006)  days, the median OS of the total cohort was 61 (95% CI 47.9-74.1) days. The median OS of EBV-positive NK/T cell LAHS patients was 65 (95% CI 52.7-77.3) days, while that of EBV-positive B cell LAHS or EBV-positive HL LAHS was not evaluable due to limited sample size. Patients whose baseline plasma EBV DNA exceeded 10P=0.001, P=0.004, and P=0.009, respectively). ENKL or ANKL tended to associate with worse OS (P=0.069). There was no significant association between plasma EBV-DNA level after treatment and OS. There was no significant relationship between other baseline characteristics and OS. When combining baseline ALT level, combined treatment of first anti-HLH regimen and subsequent anti-lymphoma regimen, treatment response, and ENKL or ANKL subtype in multivariate analysis, elevated baseline ALT level was the independent risk factor of OS (P=0.025). Combined treatment of first anti-HLH regimen and subsequent anti-lymphoma regimen and achieving CR or PR were independent protective factors of OS   Table 4.P=0.001, P=0.016, P=0.044, P=0.017, and P=0.013, respectively). When combining these factors in multivariate analysis, lower ALT level, combined treatment of first anti-HLH regimen and subsequent anti-lymphoma regimen, and achieving CR or PR were independent protective factors of OS .Regarding EBV-positive T/NK cell LAHS patients, univariate analysis revealed lower ALT level, lower IL6 level, higher PT level, combined treatment of first anti-HLH regimen and subsequent anti-lymphoma regimen, achieving CR or PR were significantly related to improved OS , and achieving CR or PR were significantly related to improved OS . When combining both factors in multivariate analysis, only achieving CR or PR remained significantly related to improved OS , serving as protective factor of prognosis.We also compared prognosis of EBV-positive LAHS patients and EBV-negative LAHS patients in our department. OS of EBV-positive LAHS patients was similar to that of EBV-negative LAHS patients , Achievement Transformation Project (No. CGZH21001), 1.3.5 Project for Disciplines of Excellence, West China Hospital, Sichuan University (No. ZYJC21007), Translational Research Grant of NCRCH (No. 2021WWB03), and China Postdoctoral Science Foundation (No. 2021M692310).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Irritable bowel syndrome (IBS) includes diarrhea-predominant (IBS-D) and constipation-predominant (IBS-C) subtypes. We combined breath testing and stool microbiome sequencing to identify potential microbial drivers of IBS subtypes.2), methane (CH4), and hydrogen sulfide (H2S) levels were measured by gas chromatography, and baseline stool microbiome composition was analyzed by 16S rRNA sequencing. Microbial metabolic pathways were analyzed using Kyoto Encyclopedia of Genes and Genomes collection databases.IBS-C and IBS-D subjects from 2 randomized controlled trials (NCT03763175 and NCT04557215) were included. Baseline breath carbon dioxide, hydrogen  of stool methanogens, predominantly Methanobrevibacter, as well as higher absolute abundance of Methanobrevibacter smithii in stool. IBS-D subjects had higher breath H2 that correlated with lower microbial diversity and higher breath H2S that correlated with higher RA of H2S-producing bacteria, including Fusobacterium and Desulfovibrio spp. The predominant H2 producers were different in these distinct microtypes, with higher RA of Ruminococcaceae and Christensenellaceae in IBS-C/CH4+ (which correlated with Methanobacteriaceae RA) and higher Enterobacteriaceae RA in IBS-D. Finally, microbial metabolic pathway analysis revealed enrichment of Kyoto Encyclopedia of Genes and Genomes modules associated with methanogenesis and biosynthesis of methanogenesis cofactor F420 in IBS-C/CH4+ subjects, whereas modules associated with H2S production, including sulfate reduction pathways, were enriched in IBS-D.IBS-C subjects had higher breath CHM. smithii and H2S producers such as Fusobacterium and Desulfovibrio spp, respectively.Our findings identify distinct gut microtypes linked to breath gas patterns in IBS-C and IBS-D subjects, driven by methanogens such as  Irritable bowel syndrome (IBS) is estimated to affect 1 in 10 people globally, with a prevalence of 11.8%–14.0% in North America . Based o2) on the breath test (2S), may also be involved in diarrheal conditions. H2S is a gasotransmitter  and is involved in numerous functions throughout the body, including inflammation and mucosal repair in the gastrointestinal (GI) tract (2S) have been linked to colorectal cancer and ulcerative colitis  tract . However colitis ,8, which colitis . More reth IBS-D ,11, alth2 generated by syntrophic bacterial species for the generation of methane (4) is directly linked to slowing of intestinal transit in an animal model and an increased motility index in methane-producing IBS subjects  . Methano methane . Interessubjects  and may subjects ,16.2, CH4, and H2S) breath testing and stool microbiome sequencing to identify potential microbial drivers of clinical phenotypes in IBS.These findings demonstrate that changes in the gut microbiome are not uniform in IBS as a whole. Rather, different microbial compositions (microtypes) may account for the differing phenotypes of IBS. Identifying these microtypes may more clearly define possible microbial pathomechanisms in IBS in general. In this study, we combine 3-gas . IBS-C was diagnosed based on Rome IV criteria  based on Rome IV criteria . Subject2), H2, CH4, and H2S . Interpretation of breath test results was based on the North American Consensus for breath testing  lactulose breath testing using a system that allows the measurement of carbon dioxide  and stored at room temperature before DNA extraction. Stool form was classified according to the Bristol Stool Form Scale  stool samples were self-collected, immediately refrigerated, and then transported to the laboratory. For the IBS-C trial, only CHrm Scale .DNA extraction was performed using the MagAttract PowerSoil DNA KF Kit (Qiagen) with some modifications as described previously . ExtractMethanobrevibacter smithii and Methanosphaera stadtmanae, in stool from IBS-C and IBS-D subjects were determined by quantitative polymerase chain reaction (qPCR) using primers and probes targeting the beta subunit of RNA polymerase (rpoB) gene of each species (M. smithii stock culture and from M. stadtmanae DSM 3091 from the Leibniz Institute Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ)  were extracted using the same protocol, and standard curves with ten-fold serial dilutions was prepared for use as qPCR standards.Levels of 2 methanogenic archaeal species,  species . Assays http://links.lww.com/AJG/C678).Details of 16S sequencing and analysis protocols are provided in the Supplementary Digital Content , SAS 9.4 , RStudio , and GraphPad Prism 9 . Graph construction was performed using GraphPad Prism 9.02 (GraphPad Software). Significance was set at P < 0.05.The descriptive analysis is presented as mean ± SD. Categorical variables were compared with χhttp://links.lww.com/AJG/C678). Among IBS-C subjects, 58 (47%) were CH4 negative (IBS-C/CH4−) and 66 (53%) were CH4 positive (IBS-C/CH4+) and considered to have IMO (4 positive. IBS-C/CH4+ subjects were significantly older than IBS-D (P = 0.037) and IBS-C/CH4− (P = 0.029) subjects. No differences in sex distribution or body mass index were identified between groups .A total of 171 IBS subjects were included (47 with IBS-D and 124 with IBS-C). Subjects' demographics and clinical characteristics are shown in the Supplementary Digital Content  (P < 0.0001).Stool form for all baseline stool samples was assessed using the Bristol Stool Form Scale. IBS-D subjects had a significantly higher average score (4.7 ± 1.27), indicating looser, more watery stools, compared with IBS-C/CH2 was higher in IBS-D compared with all IBS-C subjects . Within IBS-C, 35.09% of IBS-C/CH4− subjects were positive for H2 SIBO, compared with 25.76% of IBS-C/CH4+ subjects  for HFigure 2 , as weres Figure b. Signifhttp://links.lww.com/AJG/C678). Breath H2 dynamics were markedly different between IBS-D subjects and IBS-C subcategories . H2 delta values (120 minutes after lactulose ingestion vs preingestion levels) were significantly higher in IBS-D subjects vs IBS-C/CH4− (P = 0.038) and IBS-C/CH4+ . By 15 minutes after lactulose ingestion, H2 levels were already higher in IBS-D vs IBS-C/CH4+ subjects  and remained significantly higher at all time points during the breath test , 15 (P < 0.0001), 30 (P = 0.011), and 75 (P = 0.037) minutes . CH4 dynamics were also different between IBS-C and IBS-D subjects , with higher CH4 levels in IBS-C/CH4+ vs IBS-D subjects at all time points during the breath test .In IBS-D and IBS-C/CH4+ subjects and 40 IBS-D subjects provided baseline stool samples. Of IBS-C/CH4+ subjects, 88.09% had detectable levels of the methanogen M. smithii compared with 17.94% of IBS-D subjects (P < 0.0001). M. stadtmanae was less abundant in stool and was only detectable in 10% of IBS-C/CH4+ and 7.69% of IBS-D subjects (P = 1). Absolute M. smithii abundance correlated positively with breath CH4 levels, regardless of time point during the breath test, but a higher correlation coefficient was obtained using the maximum CH4 level reached during the breath test . Absolute M. smithii abundance also correlated negatively with breath H2 levels at 105 minutes  and 120 minutes  .A total of 42 IBS-C/CH4+ and 40 IBS-D subjects) were also used for 16S rRNA sequencing. After denoising and removal of low-quality reads, a total of 3,780,543 reads were retained for taxonomic analysis . Microbial alpha diversity analysis was performed using 3 different indices, Chao1, Simpson index, and Shannon index. Chao1 is an estimator based on abundance, Simpson index gives more weight to common or dominant species , while Shannon index assumes all species (including rare species) are represented in a sample    . Interestingly, higher breath CH4 AUC correlated with higher stool microbial alpha diversity  . This al4+ and IBS-D subjects were evident even at higher taxonomic levels. The relative abundance (RA) of the archaeal phylum Euryarchaeota was higher in the stool microbiome of IBS-C/CH4+ subjects compared with IBS-D . Regarding bacterial taxa, the RA of phylum Firmicutes was 1.27-fold higher in IBS-C/CH4+ vs IBS-D (FDR P = 0.04), and the RA of phyla Tenericutes, Lentisphaerae, and Synergistetes were also higher in IBS-C/CH4+ vs IBS-D  . By contrast, the RA of phyla Bacteroidetes , Fusobacteria , Proteobacteria , Epsilonbacteraeota , and Spirochetes  were higher in IBS-D subjects vs IBS-C/CH4+ .Differences in microbial profiles between IBS-C/CH4+ subjects was characterized by higher RA of methanogenic archaea from families Methanobacteriaceae  and Methanomassiliicoccaceae  when compared with IBS-D subjects. The RA of genus Methanobrevibacter was higher in IBS-C/CH4+ subjects vs IBS-D , confirming the qPCR results. Bacterial families with higher RA in IBS-C/CH4+ subjects vs IBS-D included Anaeroplasmataceae , Flavobacteriaceae , Christensenellaceae , Enterococcaceae , and Ruminococcaceae , among others . Notably, the RA of family Methanobacteriaceae correlated positively with RA of these bacterial families in IBS-C/CH4+ subjects, indicating possible syntropic relationships between these microbes , Spirochaetaceae , Fusobacteriaceae , and Bacteroidaceae  . Most of these bacterial families negatively affected stool microbial diversity  and of Spirochaetaceae correlated with high breath H2S AUC  and an unknown genus from family Desulfovibrionaceae  . There was no significant difference in RA of genus Desulfovibrio in IBS-D vs IBS-C/CH4+ subjects after P value correction , but the RA of several Desulfovibrio OTUs was higher in IBS-D subjects vs IBS-C/CH4+ .At the genus level, 35 known and unknown genera had higher RA in the stool microbiome of IBS-D subjects when compared to IBS-C/CH4+ and IBS-D subjects , microorganisms encoding enzymes necessary for H2S and CH4 production correlated with breath H2S and CH4 levels in IBS subjects. RA of Fusobacterium and an unknown Desulfovibrio species correlated positively with AUC for H2S . RA of genus Methanobrevibacter correlated positively with breath CH4 levels at all time points (P < 0.0001) and with AUC for CH4 , consistent with the findings for stool M. smithii levels by PCR. Moreover, Methanobrevibacter RA correlated negatively with H2 levels at 105 minutes  and 120 minutes .Although RA of numerous microbial taxa was different between IBS-C/CH4+ subjects and included enrichment of Kyoto Encyclopedia of Genes and Genomes (KEGG) modules associated with methane production from CO2, methanol, and methylamine . The KEGG module predicting biosynthesis of F420, a cofactor used during methanogenesis (4+ subjects (P < 0.0001), and this module correlated with breath CH4 levels at all time points  and with CH4 AUC .Microbial metabolic pathway analysis further supported these associations. A signature associated with biomethanation was characteristic of the stool microbiome in IBS-C/CHogenesis , was als2S production, including dissimilatory and assimilatory sulfate reduction pathways . Although there were no direct associations between these pathways and breath H2S levels, the assimilatory sulfate reduction pathway correlated with H2 levels .A biochemical signature associated with sulfur metabolism was characteristic of the stool microbiome in IBS-D subjects due to enrichment of KEGG modules associated with H4 breath tests, breath CH4 levels were linked to higher stool levels of the methanogenic archaeon M. smithii, confirmed by both qPCR and sequencing. IBS-C/CH4+ subjects had a distinct gut microtype when compared to IBS-D, characterized by higher RA of the archaeal family Methanobacteriaceae (which includes M. smithii) that correlated with higher RA of specific H2-producing bacterial families, Ruminococcaceae and Christensenellaceae, which include known syntrophs of M. smithii. By contrast, IBS-D subjects were characterized by elevated breath levels of H2 and H2S. Breath H2S levels correlated with RA of gut bacterial H2S producers in IBS-D subjects, including genus Fusobacterium and an unknown species from genus Desulfovibrio, and the RA of family Fusobacteriaceae correlated with the RA of the H2-producing family Enterobacteriaceae. In addition, predicted microbial metabolic pathway analysis indicated enrichment of KEGG modules associated with methane production in IBS-C/CH4+ subjects and enrichment of KEGG modules associated with H2S production in IBS-D subjects. These findings suggest that increases in M. smithii and in bacterial H2S producers including Fusobacterium and Desulfovibrio species may contribute to the predominant constipation and diarrheal subtypes in IBS subjects, respectively.In this study, we identify breath gas profiles and associated gut microtypes characteristic of different IBS phenotypes. Specifically, in IBS-C subjects with positive CH2 nor CH4 is produced by human cells  correlated with the presence of SIBO  and breath CH4 in IBS-C/CH4+ subjects. Interestingly, there was also an inverse relationship between CH4 and H2 levels, which may be consistent with the syntropic relationship between fermenting bacteria and hydrogenotrophic methanogens  and a constipation phenotype. By contrast, IBS-D subjects are characterized by higher breath H2 and by higher breath H2S linked to increased prevalence of H2S-producing bacteria (predominantly Fusobacterium and Desulfovibrio spp) and a diarrhea phenotype. Furthermore, the predominant H2 producers were different in these distinct microtypes, with higher RA of Ruminococcaceae and Christensenellaceae in IBS-C/CH4+ and higher Enterobacteriaceae RA in IBS-D. Identification of these distinct microtypes may facilitate a better understanding of the relationship between the gut microbiome and the heterogeneous phenotypes of IBS and allow us greater precision in the development of targeted microbiome-based therapies.In conclusion, our data identify distinct gut microtypes linked to breath gas patterns in subjects with IBS-C and IBS-D. IBS-C subjects are characterized by detectable breath CHGuarantor of the article: Mark Pimentel, MD.Specific author contributions: Conceptualization: M.P., R.M. Formal analysis: G.L., M.J.V.M., J.W., A.R., M.P. Methodology: M.J.V.M., G.L., W.M., S.W., M.P. Investigation: M.J.V.M., G.L., G.P., M.L.P., G.M.B., M.S., S.A., D.C., S.W., C.C., M.R., A.H., A.F., B.C., N.P., A.R., M.P. Visualization: M.J.V.M., G.L. Funding acquisition: G.B., R.M., M.P. Project administration: R.M., M.P. Supervision: W.M., S.W., C.C., M.R., R.M., M.P. Writing–original draft: M.J.V.M., G.L., G.B., W.M., J.W., M.P., Writing–review & editing: M.J.V.M., G.L., G.B., W.M., J.W., A.R., R.M., M.P.Financial support: This study was supported in part by funds from The Monica Lester Charitable Trust (R.M.), The Elias, Genevieve, and Georgianna Atol Charitable Trust (R.M.), Synthetic Biologics, Inc (A.R.), Bausch Health (M.P.) and The National Philanthropic Trust (M.P.).Potential competing interests: M.P. is a consultant for Bausch Health, Ferring Pharmaceuticals Inc., and Vivante Health Inc. M.P. has received grant support from Bausch Health and Synthetic Biologics. R.M. has received grant support from Valeant Pharmaceuticals. A.R. is a consultant/speaker for and has received grant support from Bausch Health. In addition, Cedars-Sinai Medical Center has licensing agreements with Bausch Health and Gemelli Biotech. A.R., M.P., and R.M. have equity in Gemelli Biotech and M.P. has equity in Synthetic Biologics. All other authors report no conflicts of interest.Data availability: The data sets generated during the current study are available in the National Center for Biotechnology Information (NCBI) BioProject Repository https://www.ncbi.nlm.nih.gov/bioproject under BioProject PRJNA804225.✓ Irritable bowel syndrome (IBS) includes diarrhea-predominant (IBS-D) and constipation-predominant (IBS-C) subtypes.✓ The gut microbiome is associated with IBS, but the roles of specific gut microbial populations are poorly understood.2), hydrogen sulfide (H2S), and methane (CH4) are produced by gut microbes.✓ The gases hydrogen (H4 on the breath test is associated with IBS-C and correlates with increased predominance of methanogens, including Methanobrevibacter smithii.✓ Increased CH2 levels do not directly correlate with diarrhea, but H2S has recently been linked to a diarrhea phenotype.✓ H✓ Distinct gut microtypes are linked to breath gas patterns in IBS-C and IBS-D subjects.4+ IBS-C subjects, increased breath CH4 correlated with increased gut microbial diversity.✓ In CH2 correlated with lower microbial diversity and increased breath H2S correlated with increased predominance of H2S producers, including Fusobacterium and Desulfovibrio species.✓ In IBS-D subjects, increased breath H2 producers in IBS-C subjects were Ruminococcaceae and Christensenellaceae, which include known bacterial syntrophs for methanogens.✓ The predominant H4+ subjects and enrichment of pathways associated with H2S production in IBS-D subjects.✓ Predicted microbial metabolic pathway analysis indicated enrichment of pathways associated with methanogenesis in IBS-C/CH