Shotgun Sequencing Research Articles

Screening for Pathogens and Studying Bats’ Blood and Tissue Microbiome

Bats possess unique physiological adaptations, including thermophysiological flexibility, which enable them to tolerate and harbor diverse pathogens without overt disease. Their ability to sustain powered flight and enter torpor or hibernation contributes to their immune resilience, making them important reservoirs for zoonotic pathogens such as viruses (rabies, Ebola, coronaviruses) and bacteria (Leptospira, Salmonella, Bartonella, Rickettsia). Bats' dense social roosting and migratory behaviors facilitate pathogen transmission, underscoring the need for effective surveillance. This study examined the blood microbiomes of five bat species from the Vespertilionidae and Rhinolophidae families inhabiting three caves in northern Bulgaria (Devetashka, Orlova, and Parnicite). Using pooled blood samples (n=120) and two bat hearts, DNA was extracted for microbiome analysis via shotgun metagenomic sequencing, alongside PCR-based detection of human pathogens. Bacterial phyla and genera distribution varied significantly among bat species, with prominent genera including Bacillus, Mycoplasma, Pseudomonas, and Sphingobacterium. Alpha diversity, measured by the Simpson index, revealed slightly higher microbial diversity in Miniopterus schreibersii and Myotis myotis, with no significant differences among cave populations. Principal Coordinates Analysis (PCoA) highlighted distinct microbial community compositions between bat species and cave locations. PCR results identified Mycobacterium in 26 of 34 blood samples and in 1 of 2 heart tissue samples, suggesting its prevalence in bat populations. No samples tested positive for Mycobacterium tuberculosis, Brucella spp., Coxiella burnetii, or Toxoplasma gondii, indicating the absence of these pathogens in the sampled individuals. The detection of atypical Mycobacteria and Brucella sp. in the bats' blood emphasizes its potential role as an opportunistic pathogen and the need for further investigation into its zoonotic significance. These findings highlight the diversity and zoonotic potential of bat-associated microbiomes, supporting the need for continued pathogen surveillance in bat populations. All metagenomic shotgun sequencing data were published at NCBI Sequence Read Archive under the bioproject number PRJNA1215671.

Investigation of bacterial gut microbiome in diverse Egyptian populations "pilot study".

The gut microbiota plays a huge role in human health regarding immunity, metabolism, and nutrient absorption. In this work, the gut microbiota, with its bacterial community structure, is studied using whole genome shotgun (WGS) sequencing for populations from two different geographical regions in Egypt: Cairo (urban) and Ismailia (rural). Fecal samples were obtained from six healthy individuals, three from Cairo and three from Ismailia, of ages ranging from 43 to 52 years. Alpha diversity, measured as Shannon, inverse Simpson, and OTUs, showed no significant differences between the two cities. However, beta diversity analysis by Principal Coordinate Analysis (PCoA) revealed diverse microbial compositions. Thus, only the Ismailia samples contained higher levels of butyrate-producing bacteria involved in maintaining intestinal health, such as Faecalibacterium prausnitzii and Akkermansia muciniphila. On the other hand, there was a higher prevalence in Cairo of bacteria associated with protein and fat metabolism, like Bacteroides thetaiotaomicron. Such findings explain the influence of environmental factors in shaping gut microbiota and show that to get a comprehensive understanding of regional differences, many wider-ranging studies need to be conducted.

Dataset of shotgun metagenomic evaluation of Sorghum bicolor rhizosphere microbiome in soils preceded by Glycine max.

The dataset presents the microbial diversity, community structure, and functional potential of the rhizosphere microbiome associated with Sorghum bicolor in response to crop rotation involving a Glycine max precursor. Soil samples were collected from the rhizospheres of two Sorghum bicolor cultivars, Avenger and NS55, cultivated in soils previously used for Glycine max and cultivated in soils that have not previously been used for Glycine max cultivation, as follows: i) Sorghum bicolor Avenger (SA1, SA2, and SA3) cultivated in soils previously used for Glycine max, ii) Sorghum bicolor NS55 (SN1, SN2, and SN3) grown in soils that had been cultivated with Glycine max, iii) Sorghum bicolor Avenger (RA1, RA2, and RA3) cultivated in soils not previously used for Glycine max, iv) Sorghum bicolor NS55 (RN1, RN2, and RN3) grown in soils not previously cultivated with Glycine max. Thereafter, the shotgun sequencing was done to assess the microbial composition and functional genes from the extracted DNA. The effective metagenome after QC of the twelve samples include SA1 (99.72%), SA2 (99.50%), SA3 (99.68%), SN1 (99.75%), SN2 (99.76%), SN3 (99.70%), RA1 (99.72%), RA2 (99.77%), RN3 (99.72%), RN1 (99.67%), RN2 (99.68%), and RN3 (99.54%). Information from the metagenome sequences is accessible under the bioproject numbers PRJNA1166458 (SA1, SA2, and SA3), PRJNA1166463 (SN1, SN2, and SN3), PRJNA1166623 (RA1, RA2, and RA3), PRJNA1166627 (RN1, RN2 and RN3). Actinomycetota and Function unknown dominated the microbiomes across all cropping systems. The insights gained from this dataset hold promise for advancing sustainable agricultural practices, particularly through optimizing crop rotations, developing microbial bioinoculants, and enhancing soil health. Furthermore, the functional data and the function unknown from this dataset could enrich our understanding of microbial roles in nutrient cycling, plant growth promotion, and stress mitigation, which are critical for addressing challenges in food security and environmental sustainability.

Alterations in gut microbiome in age- and diet-induced colorectal cancer (CRC) progression in a preclinical model.

223 Background: CRC is the third leading cause of cancer related deaths worldwide with age and diet among the strongest risk factors. Emerging evidence suggests gut microbiome plays important role in CRC and is highly impacted by the exposome primarily the food intake. Since diet and age plays an important role in gut microbiome diversity, the present study aimed to investigate the alterations in the gut microbiome in young versus old mice harboring CRC allografts and fed with different diets. Methods: Two cohorts of C57BL/6 including young (n = 9, age = 6 weeks) and old (n = 9, age = 20-24 months) mice were implanted with 1x10 6 MSI (microsatellite instable) CRC MC38 tumor cells. Mice in both cohorts were randomized into three different diet groups: normal diet (ND; standard chow), high-fat (HF) and calorie-restricted (CR: 30% reduction in total calories). Mice were housed individually under standard laboratory conditions. Mice fecal samples were collected and subjected to DNA isolation (ZymoBIOMICS-96 MagBead DNA Kit), followed by metagenomic shotgun and whole genome sequencing (ZymoBIOMICSe and Illumina NovaSeq, respectively). P ≤ 0.05 were considered as statistically significant. Results: Fecal microbiome analysis showed that old microbiome has higher alpha diversity compared to the young mice. No statistically significant differences in were found in microbiome diversities between the diet groups. Pairwise permanova results showed statistically significant differences between microbiomes of old vs. young groups ( P : 0.001), CR vs. HF groups ( P : 0.012) and HF vs. ND groups ( P : 0.021). ANCOM-BC analysis determined the differentiating features and microbial functional pathways in the young mice compared to the old mice group and in different diet groups with relative abundance differences of larger than log 10 2. Bacteroides thetaiotaomicron ( P : 1.43E-39) and Parabacteroides goldsteinii ( P : 2.20E-23) were enriched in young and old groups, respectively. Akkermansia muciniphila ( P : 0.008) was significantly enriched in ND compared to CR group. Lactococcus lactis ( P : 9.94E-160) and Lachnospiraceae bacterium A4 ( P : 4.95E-10) were significantly enriched in HF compared to ND diet groups and vice-versa, respectively. Among the functional pathways, CMP-legionaminate biosynthesis I ( P : 2.38E-13) and L-arginine biosynthesis IV (archaebacteria) ( P : 4.77E-11) were the most significantly enriched in young and old groups, respectively. Conclusions: Our findings highlight the differential diversity and microbial features of gut microbiome in age- and diet-induced CRC progression. Collectively, alteration in diet and age of the host lead to changes in gut microbiota which has a direct impact on activation of specific signaling pathways, metabolism and local and systemic immune responses, which may in turn affect chemosensitivity and outcome in CRC

Transcriptomic analysis of key genes and signaling pathways in sepsis-associated intestinal mucosal barrier damage

ObjectivesThe aim is to analyze differentially expressed genes (DEGs) in mice with sepsis-related intestinal mucosal barrier damage and to explore the diagnostic and protective mechanisms of this condition at the transcriptome level. MethodsSmall intestinal tissues from healthy male C57BL/6J mice subjected to Cecal ligation and puncture (CLP) and sham operation were collected. High-throughput sequencing was performed using the paired-end sequencing mode of the Illumina HiSeq platform. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were conducted on the differentially expressed genes (DEGs). A protein–protein interaction (PPI) network was constructed using the STRING database, and hub genes were identified with Cytoscape. These hub genes were then validated using quantitative real-time polymerase chain reaction (RT-qPCR). ResultsA total of 239 DEGs were identified, with 49 upregulated and 130 downregulated genes. KEGG enrichment analysis showed that these DEGs were primarily involved in cytokine-cytokine receptor interaction, Th1 and Th2 cell differentiation, viral protein interactions with cytokines and their receptors, and the IL-17 signaling pathway. The top 10 hub genes were selected using the cytoHubba plugin. Experimental validation confirmed that the expression levels of TBX21, CSF3, IL-6, CXCR3, and CXCL9 matched the sequencing results. ConclusionTBX21, CSF3, IL-6,CXCR3, and CXCL9 may be potential biological markers for the diagnosis and treatment the sepsis-associated intestinal mucosal barrier.

Antimicrobial-resistant Raoultella planticola isolated from preweaned dairy calf feces.

Raoultella planticola is an emerging opportunistic pathogen closely related to members of the Klebsiella genus. Genomic characterization of R. planticola isolates recovered from food animals is lacking. Here we describe the antimicrobial resistance genes, virulence factors, plasmid replicons, and phylogeny of three antimicrobial-resistant R. planticola isolates recovered from the feces of three dairy calves within 48 hours of birth. The genomes of the three R. planticola isolates were sequenced on a NextSeq 2000 platform using paired-end sequencing. The raw reads were trimmed using Trimmomatic and assembled using SPAdes v 3.15.4. Antimicrobial resistance genes, virulence factors and plasmid replicons were identified using ABRicate. Single nucleotide polymorphisms were identified among the genomes using the Harvest package and phylogenies were inferred using RAxML. In total, nine different ARGs known to confer resistance to at least five different classes of antibiotics were detected among the three R. planticola genomes. These include blaPLA, a β-lactamase found only in R. planticola, fosfomycin resistance gene fosA, and a sequence aligned with oxqAB, an efflux pump conferring resistance to antibiotics including fluoroquinolones. Each genome encoded between four and seven ARGs, with three considered genotypically to be multidrug-resistant (MDR). Two of the three genomes harbored plasmids, and all three encoded 41 to 42 virulence factors, including iron scavenging genes sitABCD. Results of this study demonstrate the presence of diverse antimicrobial-resistant R. planticola, isolated from the feces of healthy dairy calves, that had some virulence factors that may cause human disease.

Transcriptomic insight into zinc dependency of vindoline accumulation in Catharanthus roseus leaves: relevance and potential role of a CrZIP.

Foliar-applied Zn on Catharanthus roseus enhanced production of vindoline, the main impediment precursor for costly anticancer bisindoles. A leaf-abundant CrZIP was characterized for likely role in modulating vindoline metabolism. The leaf-localized Catharanthus roseus alkaloid, vindoline, is the major impediment precursor in the production of scanty and expensive anticancer bisindoles, vinblastine and vincristine. Earlier studies have hinted toward the role of micronutrients in its accumulation. Here, the effect of various micronutrients on the leaf vindoline content was analyzed. Foliar application of zinc (Zn, 100ppm) was found to be most effective for enhancing the vindoline content. Comparison of leaf samples of Zn-treated and control plants was performed using paired-end transcriptome sequencing and promising candidates among the differentially expressed genes were identified. A zinc-regulated, iron-regulated transporter-like proteins (ZIP) family member, which was found to be upregulated in Zn-treated samples, was taken up for functional characterization. Complementation of the zrt1/zrt2 yeast mutant (ZHY3) by CrZIP suggested that it encodes a functional Zn transporter. VIGS and transient overexpression of CrZIP in C. roseus showed a direct relationship between its expression and vindoline content. GFP fusion-based subcellular localization indicated chloroplast localization of CrZIP. Previously, vindoline biosynthesis has been reported to be dependent on intact chloroplasts. The chloroplast localization of CrZIP and its direct relationship with vindoline content in this study identifies the possible role of CrZIP, in modulating zinc-induced vindoline accumulation in the plant.

The complete chloroplast genome of Barringtonia asiatica (L.) Kurz (Lecythidaceae)

Genomic resources for Barringtonia asiatica (L.) Kurz, a pantropical beach forest species native to the Philippines, have not been published to date, despite its medicinal importance and potential for biomedical applications. In this study, we assembled and characterized the first complete chloroplast genome of B. asiatica using Illumina paired-end sequencing technology. It displayed a typical quadripartite structure with a sequence length of 158,794 bp, comprised of a large single copy of 88,196 bp, small single-copy of 18,448 bp, and a pair of inverted repeat regions of 26,075 bp each. The cp genome contained 35 tRNA genes, eight rRNA genes, and 86 protein-coding genes with an overall GC content of 36.8%. Phylogenetic analysis revealed a close evolutionary relationship between B. racemosa and B. fusicarpa, forming a monophyletic group with B. asiatica with high bootstrap support, thereby enhancing our understanding of the phylogeny, systematics, and genetics of Asian Lecythidaceae species.

P-2158. A Single Center's Utilization of Cell Free Microbial DNA Testing

Abstract Background Metagenomic next generation sequencing techniques, either through targeted (tMGS) or shotgun sequencing (sMGS), have been shown to improve the odds of identifying a causative pathogen in a variety of disease states compared to conventional microbiologic testing. However, the high cost and unfamiliarity with MGS have led to questions on when and how these should best be used. We aimed to characterize the utilization of sMGS at a large urban academic hospital system, specifically the use of the commercial Karius test, a cell free microbial DNA (cfDNA) assay. * conditions not mutually exclusive Methods We conducted a retrospective observational study through detailed chart review of the Karius tests sent from our hospital system from June 13, 2020, through April 10, 2024. Charts were reviewed for demographic, clinical and outcome information. Statistical analysis was performed using chi-square analyses.Figure 1:Pathogens with clinical additive diagnostic value (ADV) Results 110 Karius tests from 104 unique patients have been sent from our institution since 2020. Most patients were immunocompromised (72.7%); see Table 1 for demographics. Patients received on average 13.4 days of antimicrobials before cfDNA testing was sent. An average of 6.5 blood cultures were sent, and in 69 cases, invasive procedures were performed before Karius testing was sought. A pathogen was detected by Karius in 62 cases (56%). Results led to a change in antimicrobial management in 23% of cases. Karius had clinical additive diagnostic value (ADV) in 17 cases (15.6%), as defined by the detection of a causative pathogen that was not detected on conventional testing. Pathogens with clinical ADV are shown in Figure 1. There was no statistically significant difference between ADV, pathogen detection, or test concordance with conventional testing (p=0.71, p=0.64, and p=0.50 respectively) for cfDNA use in immunocompetent versus immunocompromised patients. Conclusion sMGS testing can provide clinically impactful results, although the optimal patient population and clinical scenario in which to employ these tests remains undetermined. Despite significant antimicrobial exposure prior to cfDNA testing, the Karius assay detected several fastidious pathogens that may not have been detected by conventional methods. Further research into ideal diagnostic utilization and stewardship of these tools is needed. Disclosures Wendy Szymczak, PhD, Quidel: Advisor/Consultant

P-2097. Genomic basis of Oxacillin- and Cefoxitin-Susceptible PBP2a-Positive Staphylococcus aureus

Abstract Background Methicillin-resistance Staphylococcus aureus (MRSA) is resistant to all ß-lactam antibiotics except for the fifth-generation cephalosporin drug, ceftaroline. The mecA gene, which encodes an alternative penicillin-binding protein, PBP2a, is responsible for the high-level resistance to methicillin. However, mecA positive, oxacillin- and cefoxitin susceptible MRSA has been reported, with limited understanding of the genomic basis behind this contradictory phenomenon. Here, we studied the molecular base of a “stealth” MRSA from clinical specimen. Methods A MRSA isolate was routinely isolated from a deep wound specimen, which was positive for PBP2a by lateral flow assay but negative for cefoxitin screen test and susceptible to oxacillin (MIC of 0.5 ug/ml) by VITEK 2 AST-GP75 card. It was also susceptible to cefoxitin using disk diffusion method (diameter of 26 mm). The “stealth” MRSA isolate was sequenced on illumina NovaSeq 6000 platform using 150 bp paired end sequencing. Bacteria genome was assembled and annotated using pipelines on BV-BRC (https://www.bv-brc.org/). Results Genomic sequencing revealed a point mutation (G to A) in the mecA gene resulting in a Gly599Ser substitution located in the transpeptidase domain of PBP2a, which is close to the active site of Ser403 on the 3D structure of the protein. Additionally, an insertion in the methicillin resistance regulatory sensor-transducer mecR1 gene was identified, resulting in a frameshift mutation, and predicted to cause premature termination. However, the sequence of frame shift mutation in mecR1 has been described and was not associated with reversed cefoxitin susceptibility. Therefore, the point mutation in mecA gene is most likely responsible for loss of function of PBP2a, and lead to susceptible cefoxitin and oxacillin results despite a positive PBP2a reaction. Conclusion Whole genome sequencing is valuable to understand the genomic basis of unusual phenotypic testing results. The reversion of “stealth” MRSA strain to a high-level oxacillin- and cefoxitin-resistant strain has been reported, which highlights the importance of considering tests for mecA or PBP2a in routine laboratory practice to avoid the risk of missing such strains. Disclosures All Authors: No reported disclosures

Water-Soluble Cellulose Acetate Changes the Intestinal Microbiota in Mice with Non-Alcoholic Steatohepatitis

Objectives: Non-alcoholic fatty liver disease (NAFLD) is a prevalent chronic disorder of the liver and affects many people worldwide. Intestinal bacteria are thought to be involved in the pathological progression of NAFLD; therefore, improving the intestinal microbiota may be important in controlling NAFLD. In this study, we assessed the effects of water-soluble cellulose acetate (WSCA) on the intestinal microbiota in a non-alcoholic steatohepatitis (NASH) mouse model. Methods: NASH model (STAM mice) was created by streptozotocin injection and feeding the mice a high-fat diet. The serum biochemical parameters were analyzed. Intestinal bacterial populations were analyzed using paired-end sequencing of 16S rRNA, 18S rRNA, and internal transcribed spacer gene. Results: Our findings indicated that WSCA administration tends to improve the serum alanine aminotransferase and glucose levels in STAM mice and decreased the alpha diversity and altered the beta diversity of their intestinal microbiota. Additionally, WSCA intake resulted in an increase in the abundance of Coriobacteriaceae_UCG-002 and a decrease in the abundance of Enterobacter. Conclusions: WSCA intake can alter specific microbial compositions to improve blood glucose levels and liver functions and may improve the pathogenesis of NAFLD.

High-throughput sequence-based microsatellite genotyping for the non-model Neotropical tree species Anadenanthera colubrina (Leguminosae)

Background and aims – Anadenanthera colubrina is a Neotropical native forest tree species with significant ecological importance in Seasonally Dry Tropical Forests. Developing genetic markers for this species is relevant for conservation, breeding, and evolutionary studies. Previously available genetic markers for A. colubrina consisted of a few microsatellites. Next-generation sequencing (NGS) strategies allow simple and cost-effective development of new SSR loci from low-coverage whole genome shotgun sequencing. The main aim was to develop microsatellite markers for sequence-based high-throughput genotyping (SSRseq) in the species and to characterize their information content against traditional capillary electrophoresis-based microsatellite data by estimating the amount of molecularly accessible size homoplasy of each locus. Additionally, the reliability of these markers for population genetic analysis was assessed by genotyping two age classes (reproductively mature trees and seedlings) in a typical location in Argentina. Key results – Sixty primer pairs targeting microsatellites were designed, of which 25 were validated with allelic error rates < 3% and genotype missingness < 20%. A significantly higher number of alleles per locus and heterozygosity was detected for SSRseq considering sequence polymorphisms compared to analysing the same data based on sequence size (length) only. Size homoplasy, calculated as the proportion of mismatches between datasets relative to the number of alleles differing in length, averaged 97.85% over all SSR loci. High levels of population genetic diversity were detected in adults and seedlings from Paranaense forests, exceeding those reported in previous studies of A. colubrina using traditional SSRs. The generated datasets increase the resolution of capillary-based microsatellite genotyping, allowing for more accurate inference of eco-evolutionary processes in non-model tree species.

P-1371. Identifying Occurrences of Plasmid-Borne Antimicrobial Resistance Gene Transfer between Diverse Bacterial Species within Environmental Biofilms Using Proximity-Ligation Sequencing

Abstract Background Antimicrobial resistance is a critical global concern. Pathogens may become resistant through acquisition of mobile genetic elements harboring antimicrobial resistance genes (ARGs). Environmental biofilms provide an excellent venue for ARG transfer via mobile elements. Mobile ARG dissemination pathways in the environment require a better understanding. Here we use proximity-ligation (Hi-C) and metagenomic sequencing to pair plasmids and ARGs to their bacterial hosts and present evidence of plasmid sharing between bacterial species. Methods Eight environmental biofilm swabs collected from water sources in Sindh Province, Pakistan were equally aliquoted for preparation by 1) DNA cross-linking for proximity ligation and Hi-C metagenomic sequencing, and 2) traditional shotgun metagenomic sequencing. Metagenomic Hi-C was used to analyze the proximity-ligation and shotgun sequencing data to construct metagenomic-assembled genomes, determine if ARGs were chromosomally- or plasmid-located, and associate mobile genetic elements and ARGs with their host cells. Results Actinomycetia, Gammaproteobacteria, and Alphaproteobacteria were the most frequently detected taxa in most samples, with Bacteroidia also being at higher numbers than other taxa in most samples. Multiple classes of ARGs in the sample were identified as being located on plasmids, including aminoglycoside ARGs (e.g. aph(3'), aph(6)), beta-lactam RGs (blaCTX-M-15, blaEC, blaTEM-1, blaOKP-A), macrolide RGs (ere(A), erm(X), mph(A), mef(C), mph(G)), quinolone RG qnrS1, sulfonamide RG sul1, and several tetracycline and metal ARGs. TetC, blaA2, and aac(3)I, were only found on bacterial chromosomes. Analysis of unique assembled ARG-bearing plasmids and taxonomic clusters revealed plasmid sharing between bacterial species, some of which were not closely related taxa. Conclusion Mobile ARGs are common in environmental biofilms. These ARGs have the potential to spread within biofilms between species, as evidenced by shared plasmid between different bacterial species. The environment should be considered an important source for mobile ARG dissemination between bacterial species. Disclosures All Authors: No reported disclosures

P-1850. Acute Babesia microti Infection in Humans Is Associated With Marked Changes In The Expression Of Peripheral Blood Coding And Noncoding RNA

Abstract Background Babesiosis is a potentially life-threatening disease transmitted by ticks. However, host and parasite determinants of human babesiosis are completely unknown. In the current study we evaluated the impact of B. microti infection, the primary cause of human babesiosis in the US, on the peripheral blood transcriptome. Figure 1. PCA of top 500 differentially expressed genes. PCA distinguishes Babesia patients at presentation from healthy controls. Methods Patients with confirmed B. microti acute infection were enrolled into a 1-year follow up cohort study at Stony Brook University Hospital, NY. Total RNA was isolated from blood using PaxGene RNA tubes and library preparation performed using Illumina's Stranded Total RNAPrep with Ribo-zero plus kit to remove human ribosomal RNA and globin RNA. 100 M RNA paired end reads were sequenced (2 x 151 bps). Low expression RNAs with less than 10 reads in 17 samples were filtered out prior to identification of differentially expressed genes (DEGs) (p< 0.05).Figure 2.Volcano Plot comparing differential gene and noncoding RNA expression between healthy controls and patients with babesiosis at presentation.Red circles represent genes whose expression are increased or decreased in patients with babesiosis at presentation compared to healthy controls. Blue circles represent genes whose expression are not significantly different. Results The peripheral blood transcriptome from 20 healthy controls was compared to 42 patients at presentation for B. microti infection and 1, 3, 6, 9 and 12 months after treatment for patients who completed the longitudinal protocol after the first visit. A total of 1860 genes or noncoding RNAs were differentially expressed in patients with babesiosis at presentation (V0) compared to healthy controls; 370 were decreased in patients with babesiosis and 1490 were increased (adj p < 0.01; expression fold change >+/- 1.) PCA analysis (Fig 1), and the Volcano Plot (Fig. 2) demonstrate that patients with babesiosis have a distinct peripheral blood gene expression profile compared to healthy control individuals. The heat map of DEGs in Fig. 3 shows the peripheral blood transcriptome in patients with babesiosis largely returned to baseline levels by 1-month post-treatment. The Qiagen IPA Graphical Summary based on DEGs is shown in Figure 4 and highlights activation of diverse immune-mediators, including TNF and interferon families, as well as growth factors, and vascularization pathways in response to B. microti infection.Figure 3.Heat map of differentially expressed genes of patients with babesiosis at presentation (0) and 1, 3, 6, 9 and 12 months post-treatment compared to healthy control individuals.Gene expression in patients with babesiosis is markedly different at presentation but is similar to levels for healthy controls by 1 month post-treatment. Conclusion Infection with B. microti results in marked changes in expression of genes and noncoding RNA in peripheral blood that largely returns to baseline by 1-month post-treatment.Figure 4:Qiagen IPA Graphical Summary of pathways likely increased (orange) or decreased (blue) in patients presenting with babesiosis. Disclosures Paul Arnaboldi, PhD, Biopeptides, Corp: Patent Holder|Biopeptides, Corp: Employee|DiaSorin: Advisor/Consultant

Stochasticity Highlights the Development of Both the Gastrointestinal and Upper-Respiratory-Tract Microbiomes of Neonatal Dairy Calves in Early Life

The microbiome of dairy calves undergoes extensive change due to various forces during the first weeks of life. Importantly, diseases such as bovine respiratory disease (BRD) and calf diarrhea can have profound impacts on the early-life microbiome. Therefore, a longitudinal, repeated-measures pilot study was designed to characterize the establishment of nasal and fecal microbiomes of dairy calves, assess the governing forces of microbial assembly, and evaluate how disease states impact these microbial ecologies. Dairy calves (n = 19) were clinically evaluated for gastrointestinal and respiratory disease across three weeks beginning at age ≤ seven days old. Fecal (n = 57) and nasal (n = 57) microbial samples were taken for paired-end 16S rRNA gene amplicon sequencing. Taxonomy and diversity analyses were used to characterize early-life nasal and fecal microbiomes. Stochasticity and determinism were measured using normalized stochasticity testing (NST) and Dirichlet multinomial model (DMM). All analyses were tested for statistical significance. Clinical diarrhea was observed in 11 of the 19 calves. Clinical BRD was not independently observed among the cohort; however, two calves presented clinical signs of both BRD and diarrhea. Taxonomic analysis revealed that fecal samples were highlighted by Bacteroidaceae (40%; relative abundance), Ruminococcaceae (13%), and Lachnospiraceae (10%), with changes in diversity (Kruskal–Wallis; p < 0.05) and composition (PERMANOVA; p < 0.05). Clinical diarrhea reduced diversity in the fecal microbiome but did not impact composition. Nasal samples featured Moraxellaceae (49%), Mycoplasmataceae (16%), and Pasteurellaceae (3%). While no diversity changes were seen in nasal samples, compositional changes were observed (p < 0.05). NST metrics (Kruskal–Wallis; p > 0.01) and DMM (PERMANOVA; p < 0.01) revealed that stochastic, neutral theory-based assembly dynamics govern early-life microbial composition and that distinct microbial populations drive community composition in healthy and diarrheic calves.

Transcriptomic Response of White Lupin Roots to Short-Term Sucrose Treatment

Abstract: White lupin (Lupinus albus) has become a model plant for understanding plant adaptations to phosphorus (P) and iron (Fe) deficiency, two major limiting factors for plant productivity. In response to both nutrient deficiencies, white lupin forms cluster roots, bottle-brush-like root structures that aid in P and Fe acquisition from soil. While the cluster root function is well-studied, not much is known about the signaling pathways involved in sensing and responding to a P and Fe deficiency. Sucrose has been identified as a long-distance signal sent in increased concentrations from shoot to root in response to both a P and Fe deficiency. Thus, sucrose plays a dual role both as a signal and as a major source of energy for the root. To unravel the responses to sucrose as a signal, we performed an Illumina paired-end cDNA sequencing of white lupin roots treated with sucrose for 20, 40 or 80 min, compared to untreated controls (0 min). We identified 634 up-regulated and 956 down-regulated genes in response to sucrose. Twenty minutes of sucrose treatment showed the most responses, with the ethylene-activated signaling pathway as the most enriched Gene Ontology (GO) category. The number of up-regulated genes decreased at 40 min and 80 min, and protein dephosphorylation became the most enriched category. Taken together, our findings indicate active responses to sucrose as a signal at 20 min after a sucrose addition, but fewer responses and a potential resetting of signal transduction pathways by the dephosphorylation of proteins at 40 and 80 min.

The difference of oropharyngeal microbiome during acute respiratory viral infections in infants and children

Acute respiratory infections (ARI) with multiple types of viruses are common in infants and children. This study was conducted to assess the difference of oropharyngeal microbiome during acute respiratory viral infection using whole-genome shotgun metagenomic sequencing. The overall taxonomic alpha diversity did not differ by the types of infected virus. The beta diversity differed by disease severity, disease-related symptoms, and types of infected virus. Nine species had significantly higher abundance in outpatients than in inpatients, with five of them in the genus Achromobacter. Three microbial community types were identified. The prevalence of community type (CT) 1 was higher among patients with influenza virus, enterovirus, and human adenvirus; CT2 was higher among patients with human metapneumovirus; and CT3 was higher among patients with respiratory syncytial virus and human adenvirus infections. Our results suggest that the oropharyngeal microbiome is associated with ARI disease severity, disease-related symptoms, and the types of infected virus.

Microbiome and Colorectal Cancer in Humans: A Review of Recent Studies

The tumor microenvironment has recently been well-studied in various gastrointestinal cancers, including colorectal cancer (CRC). The gut microbiota, a collection of microorganisms in the human gastrointestinal tract, is one of the microenvironments associated with colon carcinogenesis. It has been challenging to elucidate the mechanisms by which gut microbiota contributes to carcinogenesis and cancer progression due to complex interactions with the host, including its metabolites and immune and inflammatory responses. Various studies described the influence of diet on reported changes in the composition and microbiota of gut bacteria and its association with CRC. In recent years, metagenomic techniques such as shotgun sequencing and genome-wide association studies focused on understanding the role of the microbiota and the metabolome on early CRCs and colon carcinogenesis to determine if there are modifiable or intervenable targets for CRC. In this review, we will attempt to provide an overview of gut microbiota related to CRC, with particular attention to the findings of recent studies.

Fecal microbiota transplantation to prevent acute graft-versus-host disease: pre-planned interim analysis of donor effect

Gut microbiota disruptions after allogeneic hematopoietic cell transplantation (alloHCT) are associated with increased risk of acute graft-versus-host disease (aGVHD). We designed a randomized, double-blind placebo-controlled trial to test whether healthy-donor fecal microbiota transplantation (FMT) early after alloHCT reduces the incidence of severe aGVHD. Here, we report the results from the single-arm run-in phase which identified the best of 3 stool donors for the randomized phase. The primary and key secondary endpoints were microbiota engraftment and severe aGVHD, respectively. Three cohorts of patients (20 total) received FMT, each from a different donor. FMT was safe and effective in restoring microbiota diversity and commensal species. Microbiota engraftment, determined from shotgun sequencing data, correlated with larger microbiota compositional shifts toward donor and better clinical outcomes. Donor 3 yielded a median engraftment rate of 66%, higher than donors 1 (P = 0.02) and 2 (P = 0.03) in multivariable analysis. Three patients developed severe aGVHD; all 3 had received FMT from donor 1. Donor 3 was selected as the sole donor for the randomized phase. Our findings suggest a clinically relevant donor effect and demonstrate feasibility of evidence-based donor selection. FMT is a holistic microbiota restoration approach that can be performed as a precision therapeutic. ClinicalTrials.gov identifier NCT06026371

Longitudinal phage–bacteria dynamics in the early life gut microbiome

Microbial colonization of the human gut occurs soon after birth, proceeds through well-studied phases and is affected by lifestyle and other factors. Less is known about phage community dynamics during infant gut colonization due to small study sizes, an inability to leverage large databases and a lack of appropriate bioinformatics tools. Here we reanalysed whole microbial community shotgun sequencing data of 12,262 longitudinal samples from 887 children from four countries across four years of life as part of the The Environmental Determinants of Diabetes in the Young (TEDDY) study. We developed an extensive metagenome-assembled genome catalogue using the Marker-MAGu pipeline, which comprised 49,111 phage taxa from existing human microbiome datasets. This was used to identify phage marker genes and their integration into the MetaPhlAn 4 bacterial marker gene database enabled simultaneous assessment of phage and bacterial dynamics. We found that individual children are colonized by hundreds of different phages, which are more transitory than bacteria, accumulating a more diverse phage community over time. Type 1 diabetes correlated with a decreased rate of change in bacterial and viral communities in children aged one and two. The addition of phage data improved the ability of machine learning models to discriminate samples by country. Finally, although phage populations were specific to individuals, we observed trends of phage ecological succession that correlated well with putative host bacteria. This resource improves our understanding of phage–bacteria interactions in the developing early life microbiome.