Introduction Allogeneic hematopoietic cell transplantation (allo-HCT) is a potentially curative therapy for many hematologic diseases, but graft-versus-host-disease (GVHD) remains a significant complication. Greater intestinal microbial diversity in the 30 days post allo-HCT is linked to better survival and lower GVHD mortality. We longitudinally assessed fecal and oral microbiome diversity through 6 months post allo-HCT to better understand the microbiome's role in GVHD. Objectives To evaluate the impact of microbiome diversity on the development of GVHD after allo-HCT Methods We enrolled 27 allo-HCT patients with HLA-matched unrelated donors. All underwent myeloablative conditioning without T-cell depletion, with Tacrolimus/Methotrexate as GVHD prophylaxis and Levaquin for antibiotic prophylaxis. Fecal and oral samples were collected at baseline, neutrophil nadir, myeloid engraftment, and at 3- & 6-mon post allo-HCT. Microbiome composition was assessed by 16S rRNA sequencing. Results With each patient serving as their own control, we tracked the change in bacterial communities via Shannon diversity index (DI) over 6 months. Bacterial DI was decreased in fecal (-0.59, p<0.01) and oral (mouthwash; -0.34, p<0.01) samples at nadir. The drop in oral DI was maintained at engraftment (-0.38, p<0.01), with an apparent trend in fecal DI (-0.62, p=0.058). At 3- and 6-mon, neither fecal nor oral diversity differed from baseline.Interestingly, there was no difference in oral or fecal DI prior to allo-HCT in patients regardless of aGVHD. At count nadir, fecal microbiome DI was significantly reduced in aGVHD patients (DI 2.00 vs 3.05, p<0.01). This difference was not significant at time of engraftment, but at 3- and 6-months post allo-HCT, fecal DI remained lower in aGVHD (3-mon: DI 2.78 vs 3.29, p=0.03; 6-mon: DI 2.75 vs 3.45, p=0.028). Oral samples (buccal swab) had no significant difference in bacterial diversity related to aGVHD. However, we observed a decrease in oral bacterial DI in patients who developed oral cGVHD at 3 months (p=0.047), and in addition, we observed a lower oral bacterial DI in patients with pulmonary cGVHD both at nadir and at 6 months post allo-HCT (p=0.038). Interestingly, there was no statistical change in bacterial DI when grouping by all patients with cGVHD. Finally, there were no significant changes in infection rates based on oral or fecal diversity (p=0.13). Conclusion While studies have linked lower fecal microbiome diversity within 30 days post-transplant to poor outcomes, to our knowledge this is the first longitudinal study to link ongoing fecal microbiome diversity loss in the first 6 months post allo-HCT with higher rates of aGVHD. Furthermore, we observed a higher correlation between oral microbiome diversity and cGVHD compared to fecal microbiome. Our findings underscore the need for further research on the microbiome's impact on allo-HCT patients.

ABSTRACT:Microbial dysbiosis and metabolite changes in the gastrointestinal (GI) tract have been linked to pathogenesis and severity of many diseases, including graft-versus-host disease (GVHD), the major complication of allogeneic hematopoietic stem cell transplantation. However, published studies have only considered the microbiome and metabolome of excreted stool and do not provide insight into the variability of the microbial community and metabolite composition throughout the GI tract or the unique temporal dynamics associated with different gut locations. Because such geographical variations are known to influence disease processes, we used a multi-omics approach to characterize the microbiome and metabolite profiles of gut contents from different intestinal regions in well-characterized mouse models of GVHD. Our analysis validated analyses from excreted stool, but importantly, uncovered new biological insights from the microbial and metabolite changes between syngeneic and allogeneic hosts that varied by GI location and time after transplantation. Our integrated analysis confirmed the involvement of known metabolic pathways, including short-chain fatty acid synthesis and bile acid metabolism, and identified additional functional genes, pathways, and metabolites, such as amino acids, fatty acids, and sphingolipids, linked to GI GVHD. Finally, we validated a biological relevance for one such newly identified microbial metabolite, phenyl lactate, that heretofore had not been linked to GI GVHD. Thus, our analysis of the geographic variability in the intestinal microbiome and metabolome offers new insights into GI GVHD pathogenesis and potential for novel therapeutics.

ABSTRACT The Winam Gulf in the Kenyan region of Lake Victoria experiences prolific, year-round cyanobacterial harmful algal blooms (cyanoHABs) which pose threats to human, livestock, and ecosystem health. To our knowledge, there is limited molecular research on the gulf’s cyanoHABs, and thus, the strategies employed for survival and proliferation by toxigenic cyanobacteria in this region remain largely unexplored. Here, we used metagenomics to analyze the Winam Gulf’s cyanobacterial composition, function, and biosynthetic potential. Dolichospermum was the dominant bloom-forming cyanobacterium, co-occurring with Microcystis at most sites. Microcystis and Planktothrix were more abundant in shallow and turbid sites. Metagenome-assembled genomes (MAGs) of Dolichospermum harbored nitrogen fixation genes, suggesting diazotrophy as a potential mechanism supporting the proliferation of Dolichospermum in the nitrogen-limited gulf. Over 300 biosynthetic gene clusters (BGCs) putatively encoding the synthesis of toxins and other secondary metabolites were identified across the gulf, even at sites where there were no visible cyanoHAB events. Almost all BGCs identified had no known synthesis product, indicating a diverse and novel biosynthetic repertoire capable of synthesizing harmful or potentially therapeutic metabolites. Microcystis MAGs contained mcy genes encoding the synthesis of hepatotoxic microcystins which are a concern for drinking water safety. These findings illustrate the spatial variation of bloom-forming cyanobacteria in the Winam Gulf and their available strategies to dominate different ecological niches. This study underscores the need for further use of genomic techniques to elucidate the dynamics and mitigate the potentially harmful effects of cyanoHABs and their associated toxins on human, environmental, and economic health. IMPORTANCE The Winam Gulf (Kenya) is a vital resource that experiences prolific cyanobacterial harmful algal blooms (cyanoHABs). Bloom-forming cyanobacteria produce cyanotoxins, threatening human and environmental health, recreation, and fishing. However, cyanotoxin production in the gulf has not been linked to a specific type of cyanobacteria. We used DNA sequencing of whole microbial communities to track the species of cyanobacteria present across the gulf and investigate the genes responsible for synthesis of known and novel toxins. Our results reveal Dolichospermum as the main bloom-forming cyanobacteria in the gulf, often co-occurring with high abundance of toxigenic Microcystis. Over 300 unique gene clusters were found, with most predicted to encode the synthesis of uncharacterized molecules. These results provide initial insights into the diverse biosynthetic potential encoded by cyanobacteria in the Winam Gulf and underscore the need to further elucidate and investigate the effects of known and novel molecules produced in cyanoHABs in this region.

Microbiome research is becoming a mature field with a wealth of data amassed from diverse ecosystems, yet the ability to fully leverage multi-omics data for reuse remains challenging. To provide a view into researchers’ behavior and attitudes towards data reuse, we surveyed over 700 microbiome researchers to evaluate data sharing and reuse challenges. We found that many researchers are impeded by difficulties with metadata records, challenges with processing and bioinformatics, and problems with data repository submissions. We also explored the cost constraints of data reuse at each step of the data reuse process to better understand “pain points” and to provide a more quantitative perspective from sixteen active researchers. The bioinformatics and data processing step was estimated to be the most time consuming, which aligns with some of the most frequently reported challenges from the community survey. From these two approaches, we present evidence-based recommendations for how to address data sharing and reuse challenges with concrete actions for future work.

What: Twenty-two scientists gathered to explore how machine learning and artificial intelligence could transform Great Lakes observations, modeling, and forecasting. Participants discussed enhancing predictive models, improving observational network design, and identifying key challenges and opportunities. They aimed to establish a framework for a Great Lakes ML/AI community of practice and to develop a strategic vision for the next decade. When: 22-23 July 2024 Where: Ann Arbor, Michigan

Despite the global expansion of cyanobacterial harmful algal blooms (cHABs), research is biased to temperate systems within the global north, such as the Laurentian Great Lakes. This lack of diversity represents a significant gap in the field and jeopardizes the health of those who reside along at-risk watersheds in the global south. The African Great Lake, Lake Victoria, is understudied despite serving as the second largest lake by surface area and demonstrating year-round cHABs. Here, we address this knowledge gap by performing a molecular survey of cHAB communities in three anthropogenically and ecologically important freshwater systems of Victoria's Kenyan watershed: Winam Gulf (Lake Victoria), Lake Simbi and Lake Naivasha. We identified a bloom of non-toxic Dolichospermum and toxic Microcystis in the Winam Gulf, with data suggesting sulfur limitation shapes competition dynamics between these two bloom-formers. Though we did not detect a bloom in Naivasha, it contained the largest diversity of cHAB genera amongst the three lakes. In turn, our results indicated methane metabolism may allow non-toxic picoplankton to outcompete cHAB genera, while suggesting Synechococcus spp. serves as a methane source and sink in this system. Lake Simbi exhibited a non-toxic Limnospira bloom at the time of sampling with very low abundances of cHAB genera present. Subsequently, these results were employed to design a cHAB screening and risk assessment framework for local stakeholders. Cumulatively, this work serves to increase cHAB research efforts on the international scale while serving as an impetus for cHAB monitoring on the local scale.

Antibiotic resistance and metal toxicity in freshwater bodies have human health impacts and carry economic implications worldwide. The presence of metal and antibiotic resistance genes within microbial communities can be informative about both issues. The Laurentian Great Lakes contain nearly 20% of the world's supply of freshwater; however, it is unclear how these genes are changing over time in this system. In this study, we characterized these genes in nearly two hundred metagenomes collected from multiple sites in western Lake Erie at a five-year time interval: 2014-2019. 11 metal resistance genes (MRGs) and nine antibiotic resistance genes (ARGs) were characterized and demonstrated significant changes in diversity and spatial distribution. Increased abundance was observed for genes like aac(3) and TEM-1B conferring resistance to aminoglycoside (gentamicin) and B-lactam antibiotics, respectively. MRGs associated with mercury, lead, and arsenic also increased in abundance over the five years. Collectively, our data point to a notable increase in both ARGs and MRGs in Lake Erie over five years, with a specific and significant increase in the abundance of genes conferring resistance to aminoglycoside and B-lactam antibiotic resistance and mercury contamination. Future integrated and systematic freshwater microbiome and public health investigations are needed to assess the potential impact on humans and environmental health from increasing microbial antibiotic and metal resistance in large freshwater reservoirs like the Great Lakes. ### Competing Interest Statement The authors have declared no competing interest.

Methylobacterium fujisawaense strain C14 was isolated from a weathered concrete cylinder. Using PacBio sequencing, we generated a complete genome for strain C14, which includes one circular chromosome (6,656,731 bp) and six putative plasmids (35,452 to 85,428 bp).

Microcystis-dominated cyanobacterial harmful algal blooms (cyanoHABs) have a global impact on freshwater environments, affecting both wildlife and human health. Microcystis diversity and function in field samples and laboratory cultures can be determined by sequencing whole genomes of cultured isolates or natural populations, but these methods remain computationally and financially expensive. Amplicon sequencing of marker genes is a lower cost and higher throughput alternative to characterize strain composition and diversity in mixed samples. However, the selection of appropriate marker gene region(s) and primers requires prior understanding of the relationship between single gene genotype, whole genome content, and phenotype. To identify phylogenetic markers of Microcystis strain diversity, we compared phylogenetic trees built from each of 2,351 individual core genes to an established phylogeny and assessed the ability of these core genes to predict whole genome content and bioactive compound genotypes. We identified single-copy core genes better able to resolve Microcystis phylogenies than previously identified marker genes. We developed primers suitable for current Illumina-based amplicon sequencing with near-complete coverage of available Microcystis genomes and demonstrate that they outperform existing options for assessing Microcystis strain composition. Results showed that genetic markers can be used to infer Microcystis gene content and phenotypes such as potential production of bioactive compounds , although marker performance varies by bioactive compound gene and sequence similarity. Finally, we demonstrate that these markers can be used to characterize the Microcystis strain composition of laboratory or field samples like those collected for surveillance and modeling of Microcystis-dominated cyanobacterial harmful algal blooms.

Nutrient-induced blooms of the globally abundant freshwater toxic cyanobacterium Microcystis cause worldwide public and ecosystem health concerns. The response of Microcystis growth and toxin production to new and recycled nitrogen (N) inputs and the impact of heterotrophic bacteria in the Microcystis phycosphere on these processes are not well understood. Here, using microbiome transplant experiments, cyanotoxin analysis, and nanometer-scale stable isotope probing to measure N incorporation and exchange at single cell resolution, we monitored the growth, cyanotoxin production, and microbiome community structure of several Microcystis strains grown on amino acids or proteins as the sole N source. We demonstrate that the type of organic N available shaped the microbial community associated with Microcystis, and external organic N input led to decreased bacterial colonization of Microcystis colonies. Our data also suggest that certain Microcystis strains could directly uptake amino acids, but with lower rates than heterotrophic bacteria. Toxin analysis showed that biomass-specific microcystin production was not impacted by N source (i.e. nitrate, amino acids, or protein) but rather by total N availability. Single-cell isotope incorporation revealed that some bacterial communities competed with Microcystis for organic N, but other communities promoted increased N uptake by Microcystis, likely through ammonification or organic N modification. Our laboratory culture data suggest that organic N input could support Microcystis blooms and toxin production in nature, and Microcystis-associated microbial communities likely play critical roles in this process by influencing cyanobacterial succession through either decreasing (via competition) or increasing (via biotransformation) N availability, especially under inorganic N scarcity.

Here, we report on the raw and coassembled metatranscriptomes of 39 Lake Erie surface (1.0 m) water samples collected over a 2-day diel period encompassing episodic weather and bloom events. Preliminary taxonomic annotations and read mappings revealed that Microcystis spp. accounted for up to ~47% of the transcriptionally active community.

Prior work on the microbial communities of concrete focused on the surfaces of concrete structures such as sewage pipes or bridge pilings, where thick biofilms were easy to observe and sample. Because the biomass inside concrete is so low, more recent analyses of the microbial communities inside concrete used amplicon sequencing methods to describe those communities.

Extraction of high-molecular-weight DNA from Gram-positive bacterial species, with optional steps for removing surfactants. This DNA is suitable for sequencing and the protocol can be scaled up at least 5-fold. Modified from a protocol by Tina Wecke, LMU-Munich.

AbstractNutrient-induced blooms of the globally abundant freshwater toxic cyanobacteriumMicrocystisare the cause of worldwide public and ecosystem health concerns. The response ofMicrocystisgrowth and toxin production to new and recycled nitrogen (N) inputs, and the impact of heterotrophic bacteria in theMicrocystisphycosphere on these processes are not well understood. Here, using microbiome transplant experiments, cyanotoxin analysis, and stable isotope tracing to measure N incorporation and exchange at single cell resolution, we monitored the growth, cyanotoxin production, and microbiome community structure of severalMicrocystisstrains grown on amino acids and proteins as the sole N source. We demonstrate that 1) organic N availability shapes the microbiome community structure in theMicrocystisphycosphere; 2) external organic N input leads to lower bacterial colonization of the phycosphere; 3) certainMicrocystisstrains can directly uptake amino acids, but with lower rates than heterotrophic bacteria; 4) biomass-specific microcystin production is not impacted by N source (i.e., nitrate, amino acids and protein) but rather by total N availability; and 5) some bacterial communities compete withMicrocystisfor organic N, but others remineralize organic N, in the process producing bio-available N forMicrocystis. We conclude that organic N input can supportMicrocystisblooms and toxin production, andMicrocystis-associated microbial communities play critical roles by influencing cyanobacterial succession through either decreasing (via competition) or increasing (via remineralization) N availability, especially under inorganic N scarcity.

The severity of T cell-mediated gastrointestinal (GI) diseases such as graft-versus-host disease (GVHD) and inflammatory bowel diseases correlates with a decrease in the diversity of the host gut microbiome composition characterized by loss of obligate anaerobic commensals. The mechanisms underpinning these changes in the microbial structure remain unknown. Here, we show in multiple specific pathogen-free (SPF), gnotobiotic, and germ-free murine models of GI GVHD that the initiation of the intestinal damage by the pathogenic T cells altered ambient oxygen levels in the GI tract and caused dysbiosis. The change in oxygen levels contributed to the severity of intestinal pathology in a host intestinal HIF-1α- and a microbiome-dependent manner. Regulation of intestinal ambient oxygen levels with oral iron chelation mitigated dysbiosis and reduced the severity of the GI GVHD. Thus, targeting ambient intestinal oxygen levels may represent a novel, non-immunosuppressive strategy to mitigate T cell-driven intestinal diseases.

Cyanobacteria harmful algal blooms (cyanoHABs) dominated by Microcystis spp. have significant public health and economic implications in freshwater bodies around the world. These blooms are capable of producing a variety of cyanotoxins, including microcystins, that affect fishing and tourism industries, human and environmental health, and access to drinking water. In this study, we isolated and sequenced the genomes of 21 unialgal Microcystis cultures collected from western Lake Erie between 2017-2019. While some cultures isolated in different years have a high degree of genetic similarity (Average Nucleotide Identity >99%), genomic data shows that these cultures also represent much of the breadth of known Microcystis diversity in natural populations. Only 5 isolates contained all the genes required for microcystin synthesis while 2 isolates contained a previously described partial mcy operon. Microcystin production within cultures was also assessed using Enzyme-Linked Immunosorbent Assay (ELISA) and supported genomic results with high concentrations (up to 900 μg L-1) in cultures with complete mcy operons and no or low toxin detected otherwise. These xenic cultures also contained a substantial diversity of bacteria associated with Microcystis , which has become increasingly recognized as an essential component of cyanoHAB community dynamics. These results highlight the genomic diversity among Microcystis strains and associated bacteria in Lake Erie, and their potential impacts on bloom development, toxin production, and toxin degradation. This collection significantly increases the availability of environmentally relevant Microcystis strains from temperate North America, which is changing rapidly due to climate change. Highlights Twenty one xenic Microcystis cultures were isolated from western Lake Erie and capture the diversity of Microcystis strains observed in natural populations as well as their associated bacteria Microcystis strains show variability in core and accessory gene content, and genetically similar strains produce varying concentrations and congeners of microcystins This collection is a valuable resource for studying strain diversity and interactions between Microcystis and associated bacteria Our collection increases the availability of environmentally relevant strains from temperate North America, which is historically underrepresented in culture collections.

Introduction: The severity of T cell-mediated gastrointestinal graft-versus-host disease (GI-GVHD) correlates with a decrease in the gut microbiome diversity, loss of obligate anaerobic bacteria and alterations in levels of salutary metabolites like short chain fatty acids (SCFAs). However, it is unknown whether the intestinal anatomic geography of the microbiome and its metabolites impacts GI GVHD severity. Herein, we investigate the pattern of the gut microbiome and metabolites from four distinct anatomical sites of intestines after allogeneic stem cell transplant (allo-SCT) via shotgun metagenomics and untargeted mass-spectrometry. Methods: To determine the composition of the gut microbiome and gut metabolites of mice after allo-SCT, we utilized contemporaneous analyses of shotgun metagenomic sequencing and untargeted mass spectrometry on the gut contents of mice 7 or 21 days after major histocompatibility complex (MHC)-disparate BALB/c→C57BL/6 (B6) allogeneic SCT. We investigated the microbial community composition and gut metabolites from four distinct gut locations (terminal ileum, cecum, transverse colon, descending colon) and performed integrative analysis to assess for novel pathways that may regulate GVHD biology. For shotgun metagenomics analyses, reads were depth normalized with BBnorm v.38.96 and assembled per-sample with MEGAHIT v.1.2.9. Prodigal v.2.6.3 was used to find genes in each bin. KofamScan v.1.3.0 was used to assign KEGG ortholog IDs (KO) to translated genes predicted by Prodigal. Community composition profiles were produced with Kraken2 v.2.1.2 and Bracken v.2.6.1. Functional profiles for each sample were determined with the Kraken community composition profiles and Humann3 v.3.0.0, with a GTDB database built with Struo2. Results: Consistent with previous reports from direct stool analyses, our data demonstrated that after allo-SCT, gut microbial diversity is reduced, relative abundance of obligate anaerobes decreases, and relative abundance of facultative anaerobes increases at all the intestinal anatomic sites, but the difference was maximal in ileo-cecal contents. Furthermore, metabolomic analyses reveal a decrease in previously described metabolites such as, SCFAs, bile acids and indoles at these sites. The distinct patterns of change between control and allogeneic samples depended not only on the gut location probed but also correlated with timepoint after transplant (7 days or 21 days) highlighting the importance of probing different gut locations and timepoints. The largest driver of differences between sample groups was (1) gut location (2) time after transplant (3) transplant type. Integrative analyses demonstrated loss of microbial functional genes related to butyrate processing following allo-SCT complementing previous studies that demonstrated reduction in butyrate levels. Further bioinformatic analyses identified novel microbial functional genes and metabolites that may have biological implications. Analyses revealed that after allo-SCT, phenyllactic acid (PLA) levels increased in the transverse colon and distal colon (fig.1). PLA is an anti-microbial compound and has been shown to have immune modulatory properties. Administration of PLA in drinking water of mice after allo-SCT worsened GVHD severity and increased mortality indicating PLA may play a role in inducing dysbiosis and worsening GVHD severity. Conclusion: Contemporaneous analyses of shotgun metagenomic and untargeted mass spectrometry of gut contents after allo-SCT reveal changes in microbial composition and metabolites consistent with previous data from analyses from stool. However, our analysis demonstrates distinct patterns of key metabolites and microbes in between syngeneic and allogeneic depending on gut location and time after transplant. Furthermore, our analyses have identified novel functional genes and metabolites that are implicated in GVHD severity.

Nutrient-induced blooms of the globally abundant freshwater toxic cyanobacterium Microcystis are the cause of worldwide public and ecosystem health concerns. The response of Microcystis growth and toxin production to new and recycled nitrogen (N) inputs, and the impact of heterotrophic bacteria in the Microcystis phycosphere on these processes are not well understood. Here, using microbiome transplant experiments, cyanotoxin analysis, and stable isotope tracing to measure N incorporation and exchange at single cell resolution, we monitored the growth, cyanotoxin production, and microbiome community structure of several Microcystis strains grown on amino acids and proteins as the sole N source. We demonstrate that 1) organic N availability shapes the microbiome community structure in the Microcystis phycosphere; 2) external organic N input leads to lower bacterial colonization of the phycosphere; 3) certain Microcystis strains can directly uptake amino acids, but with lower rates than heterotrophic bacteria; 4) biomass-specific microcystin production is not impacted by N source (i.e., nitrate, amino acids and protein) but rather by total N availability; and 5) some bacterial communities compete with Microcystis for organic N, but others remineralize organic N, in the process producing bio-available N for Microcystis . We conclude that organic N input can support Microcystis blooms and toxin production, and Microcystis -associated microbial communities play critical roles by influencing cyanobacterial succession through either decreasing (via competition) or increasing (via remineralization) N availability, especially under inorganic N scarcity.### Competing Interest StatementThe authors have declared no competing interest.