Mice with a humanized immune system are resilient to transplantation of human microbiota

Human gut microbiota has co-evolved with human, and plays important roles in regulating the development and functioning of the host immune system. To study the human-specific microbiome-immunune interaction in an animal model is challenging as the animal model needs to capture both the human-specific immune functions and the human-specific microbiome composition. Here we combined two widely-used humanization procedures to generate a humanized mouse model (HMA-huCD34) with functional human leukocytes developed from engrafted huCD34+ cells and human fecal microbes introduced through fecal microbiota transplantation, and investigated how the two introduced human components interact. We found that the engrafted human leukocytes are resilient to the transplanted human microbes, while reciprocally the transplanted microbial community in the huCD34 mice was significantly different from mice without a humanized immune system. By tracking the colonization of human fecal Bacteroides strains in the mouse gut, we found that the composition of the strain population changes over time, the trajectory of which depends upon the type of mouse. On the other hand, different from Bacteroides, Akkermansia muciniphila exhibited consistent and rapid fixation of a single donor strain in all tested mice, suggesting strong purifying selection common to all mouse types. Our prospect study illustrated the complex interactions between the transplanted microbiome and different host factors, and suggested that the humanized mouse model may not faithfully reproduce the human-specific microbiome-immune interaction.

Drivers and Determinants of Strain Dynamics Following Faecal Microbiota Transplantation

Faecal microbiota transplantation (FMT) is an efficacious therapeutic intervention, but its clinical mode of action and underlying microbiome dynamics remain poorly understood. Here, we analysed the metagenomes associated with 142 FMTs, in a time series-based meta-study across five disease indications. We quantified strain-level dynamics of 1,089 microbial species based on their pangenome, complemented with 47,548 newly constructed metagenome-assembled genomes. Using subsets of procedural-, host- and microbiome-based variables, LASSO-regularised regression models accurately predicted the colonisation and resilience of donor and recipient microbes, as well as turnover of individual species. Linking this to putative ecological mechanisms, we found these sets of variables to be informative of the underlying processes that shape the post-FMT gut microbiome. Recipient factors and complementarity of donor and recipient microbiomes, encompassing entire communities to individual strains, were the main determinants of individual strain population dynamics, and mostly independent of clinical outcomes. Recipient community state and the degree of residual strain depletion provided a neutral baseline for donor strain colonisation success, in addition to inhibitive priority effects between species and conspecific strains, as well as putatively adaptive processes. Our results suggest promising tunable parameters to enhance donor flora colonisation or recipient flora displacement in clinical practice, towards the development of more targeted and personalised therapies.

Functional characterization of a gene cluster responsible for inositol catabolism associated with hospital-adapted isolates of Enterococcus faecium

Enterococcus faecium is a nosocomial, multidrug-resistant pathogen. Whole genome sequence studies revealed that hospital-associated E. faecium isolates are clustered in a separate clade A1. Here, we investigated the distribution, integration site and function of a putative iol gene cluster that encodes for myo-inositol (MI) catabolism. This iol gene cluster was found as part of an ~20 kbp genetic element (iol element), integrated in ICEEfm1 close to its integrase gene in E. faecium isolate E1679. Among 1644 E. faecium isolates, ICEEfm1 was found in 789/1227 (64.3 %) clade A1 and 3/417 (0.7 %) non-clade A1 isolates. The iol element was present at a similar integration site in 180/792 (22.7 %) ICEEfm1-containing isolates. Examination of the phylogenetic tree revealed genetically closely related isolates that differed in presence/absence of ICEEfm1 and/or iol element, suggesting either independent acquisition or loss of both elements. E. faecium iol gene cluster containing isolates E1679 and E1504 were able to grow in minimal medium with only myo-inositol as carbon source, while the iolD-deficient mutant in E1504 (E1504∆iolD) lost this ability and an iol gene cluster negative recipient strain gained this ability after acquisition of ICEEfm1 by conjugation from donor strain E1679. Gene expression profiling revealed that the iol gene cluster is only expressed in the absence of other carbon sources. In an intestinal colonization mouse model the colonization ability of E1504∆iolD mutant was not affected relative to the wild-type E1504 strain. In conclusion, we describe and functionally characterise a gene cluster involved in MI catabolism that is associated with the ICEEfm1 island in hospital-associated E. faecium isolates. We were unable to show that this gene cluster provides a competitive advantage during gut colonisation in a mouse model. Therefore, to what extent this gene cluster contributes to the spread and ecological specialisation of ICEEfm1-carrying hospital-associated isolates remains to be investigated.

Next generation SARM1 knockout and epitope tagged CRISPR-Cas9-generated isogenic mice reveal that SARM1 does not participate in regulating nuclear transcription, despite confirmation of protein expression in macrophages

ABSTRACTSARM1 is an ancient and highly conserved TIR-domain containing protein, with a diverse range of proposed roles in both innate immunity and neuronal death and degeneration. Murine SARM1 has been reported to regulate the transcription of specific chemokines in both neurons and macrophages, however the extent and mechanism by which SARM1 contributes to transcription regulation remains to be fully understood. Here, using RNA sequencing we identify differential gene expression in bone marrow-derived macrophages (BMDM) from C57BL/6 congenic 129 ES cell-derived Sarm1-/- mice compared to wild type (WT). However, we show that passenger genes which are derived from the 129 donor strain of mice flank the Sarm1 locus, confounding interpretation of results, since many of the identified differentially regulated genes come from the region containing passenger genes. To re-examine the transcriptional role of SARM1 in the absence of such passenger genes, we generated three different Sarm1-/- mice using CRISPR/Cas9 technology. Vincristine treatment of ex vivo cultured post-natal neurons from these mice confirmed SARM1’s previously identified key function as an executor of axon degeneration. However, using these mice, we show that the absence of SARM1 has no impact on transcription of genes previously shown to be altered in macrophages or in the brainstem. To gain further insight into SARM1 function, we generated and characterized a mouse expressing epitope-tagged SARM1, as it has been difficult to date to confirm which cells and tissues express SARM1 protein. In these mice we see high SARM1 protein expression in the brain and brainstem, and lower but detectable levels in macrophages. Overall, the generation of these next generation SARM1 knockout and epitope-tagged mice has clarified that SARM1 is expressed in mouse macrophages but has no general role in transcriptional regulation in these cells, and has provided important new animal models to further explore SARM1 function.

Intraspecies strain exclusion, antibiotic pretreatment, and donor selection control microbiota engraftment after fecal transplantation

Fecal microbiota transplantation (FMT) is both a promising therapeutic approach to treat microbiota-associated pathologies and an experimental tool to establish a causal role of microbiome dysbiosis in human pathologies. Although clearly efficacious in resolving recurrent Clostridioides difficile infection (rCDI), the therapeutic value of FMT in other pathologies is not yet established, and our mechanistic and ecological understanding of how FMT alters the microbiome in patients is incomplete. Here, we assembled the most comprehensive FMT trial microbiota dataset to date, including new and previously generated fecal metagenomes from FMT trials in rCDI, inflammatory bowel disease (IBD), metabolic syndrome (MetS), drug-resistant pathogen colonization (MDR), and resistance to immune checkpoint inhibitor anti-tumor therapy (ICI). We characterized post-FMT microbiota assembly in the recipients by establishing the origin of the detected strains, and we identified the clinical and ecological factors that determine the engraftment of donor strains. Our findings showed little coexistence of donor and recipient strains and linked the magnitude of donor strain engraftment to dysbiosis of the recipient microbiome. Dysbiosis and strain engraftment were low in pathologies other than rCDI but could be enhanced through pretreatment with antibiotics and lavage. Using generalized linear mixed-effects models, we demonstrate that both ecological (low recipient and high donor ɑ-diversity and relative species abundance) and clinical (antibiotic pretreatment, bowel lavage, multiple rounds of FMT) variables are associated with increased donor microbiota engraftment, and that donor strain engraftment events are predictable for individual patients and strains. Overall donor strain engraftment was not linked to FMT outcome in IBD patients but was higher in ICI patients that responded to immunotherapy after FMT. Our findings provide an ecological framework for post-FMT microbiota assembly that can predict donor strain engraftment and determine its importance for clinical outcomes, informing more targeted and personalized approaches to increase the therapeutic benefits of FMTs.

Horizontal transfer of the n-TASE gene cluster in Burkholderia seminalis TC3.4.2R3 inferred from phylogenetic and molecular methods

This study describes the n-TASE cluster in Burkholderia seminalis TC3.4.2R3, which was present in B. contaminans (CP046609.1), but absent in other related Burkholderia species. Phylogeny, comparative genomics and molecular analysis indicated it is not common to B. seminalis species, presenting similarity with homologous genes presents Aquamicrobium sp. SK-2 and B. contaminans LMG23361, probably acquired by an HGT (Horizontal Gene Transfer) event. It was not possible to determine which was the most likely donor strain of the n-TASE cluster. The HGT event did not occur in all strains of the Bcc group, nor in the B. seminalis, but it did occur punctually in the strain B. seminalis TC34.2R3. It has a correlation in biotechnological applications related processes. Aiming at understanding the involvement of the n-TASE cluster in the interaction of this bacterium in the environment, genes in this cluster will be inactivated, next.

Clinical efficacy and increased donor strain engraftment after antibiotic pretreatment in a randomized trial of ulcerative colitis patients receiving fecal microbiota transplant

Fecal microbiota transplant is a promising therapy for ulcerative colitis. Parameters maximizing effectiveness and tolerability are not yet clear, and it is not known to what degree the transmission of donor microbes to patients is important. Here (clinicaltrails.gov: NCT03006809) we have tested the effects of antibiotic pretreatment (neomycin, vancomycin, and metronidazole, 500 mg twice a day for 5 days) and compared two modes of maintenance dose delivery, capsules versus enema, in a randomized, pilot, open-label, 2x2 factorial design with 22 patients analyzed (30 patients randomized) with mild to moderate UC (total Mayo score 4-9). Clinically, the treatment was well-tolerated with favorable safety profile. Patients receiving antibiotic pretreatment trended toward greater remission after six weeks of treatment (55% vs. 18%, p=0.18), and no significant differences were found between maintenance dosing via capsules versus enema. In exploratory analyses, microbiome turnover at both the species and strain levels was extensive and significantly more pronounced in the pretreated patients. Associations were also revealed between taxonomic turnover and changes in the composition of primary and secondary bile acids. Together these findings suggest that antibiotic pretreatment contributes to microbiome engraftment and possibly clinical effectiveness, and validate longitudinal strain tracking as a powerful way to monitor the dynamics and impact of microbiota transfer.

Copy-out-paste-in transposition of a Tn6283-like integrative element assists interspecies antimicrobial resistance gene transfer from Vibrio alfacsensis

The exchange of antimicrobial resistance (AMR) genes between aquaculture and terrestrial microbial populations has emerged as a serious public health concern. However, the nature of the mobile genetic elements in marine bacteria is poorly documented. To gain insight into the genetic mechanisms underlying AMR gene transfer from marine bacteria, we mated a multi-drug resistant Vibrio alfacsensis strain with an Escherichia coli strain, and then determined the complete genome sequences of the donor strain and multidrug-resistant transconjugants. Sequence analysis revealed a conjugative plasmid of the MOBH family in the donor strain, which was integrated into the chromosome of the recipient. The plasmid backbone in the transconjugant chromosome was flanked by two copies of a 7.1 kb integrative element, designated Tn 6945, harboring a beta-lactamase gene that conferred ampicillin resistance to the host cell. Use of a recA mutant E. coli strain as the recipient yielded a transconjugant showing ampicillin resistance but not multidrug resistance, suggesting the involvement of homologous recombination in plasmid integration into the chromosome. Polymerase chain reaction experiments revealed that Tn 6945 generates a circular copy without generating an empty donor site, suggesting that it moves via a copy-out-paste-in mode, as previously reported for Tn 6283. Transposition of the integrative element into multiple loci in the recipient chromosome increased the resistance level of the transconjugants. Overall, these results suggest that Tn 6283-like copy-out integrative elements and conjugative plasmids additively spread AMR genes among marine bacteria and contribute to the emergence of isolates with high-level resistance through amplification of AMR genes.

Kin discrimination promotes horizontal gene transfer between unrelated strains in Bacillus subtilis

Bacillus subtilis is a soil bacterium that is competent for natural transformation. Genetically distinct B. subtilis swarms form a boundary upon encounter, resulting in killing of one of the strains. This process is mediated by a fast-evolving kin discrimination (KD) system consisting of cellular attack and defence mechanisms. Here, we show that these swarm antagonisms promote transformation-mediated horizontal gene transfer between strains of low relatedness. Gene transfer between interacting non-kin strains is largely unidirectional, from killed cells of the donor strain to surviving cells of the recipient strain. It is associated with activation of a stress response mediated by sigma factor SigW in the donor cells, and induction of competence in the recipient strain. More closely related strains, which in theory would experience more efficient recombination due to increased sequence homology, do not upregulate transformation upon encounter. This result indicates that social interactions can override mechanistic barriers to horizontal gene transfer. We hypothesize that KD-mediated competence in response to the encounter of distinct neighbouring strains could maximize the probability of efficient incorporation of novel alleles and genes that have proved to function in a genomically and ecologically similar context.

Evaluation Of The Transfer Of The TOL Plasmid Pseudomonas Putida To Groundwater-Derived Biofilms In A Model Rock-Fracture Aquifer

Non-aqueous phase liquids (NAPLs) can become entrapped in subsurface rock fractures and become a long-term groundwater contaminant source. While remediation technologies exist, they can be expensive. Subsurface microorganisms can also degrade NAPLs trapped in the subsurface; however, this is a slow process. The possibility of enhancing microbial degradation of NAPLs via a plasmid transfer mechanism in a model rock fracture aquifer was explored. There was no indication that introduction of donor strain Pseudomonas putida SM1443::gfp2x-pWW0::dsRed into the model system led to transfer of the degradative TOL plasmid pWWO, or led to increased degradation of model NPL toluene. Plate matings with the donor strain and a groundwater-derived microbial consortium indicated that few potential recipients existed in the community. Nutrient concentration was ruled out as a limiting factor of plasmid transfer.