A Consortium of Fecal Microbes that Decolonizes Antibiotic-resistant Enteric Pathogens: A Potential Alternative to Fecal Microbiota Transplantation

doi:10.21203/rs.3.rs-4818769/v1

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A Consortium of Fecal Microbes that Decolonizes Antibiotic-resistant Enteric Pathogens: A Potential Alternative to Fecal Microbiota Transplantation

https://doi.org/10.21203/rs.3.rs-4818769/v1

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Fecal microbiota transplantation (FMT) has proven effective in decolonizing carbapenemase-producing Enterobacteriaceae (CPE) and vancomycin-resistant Enterococci (VRE). However, its efficacy varies among patients, and frequent implementation poses challenges. Through microbiome analysis of fecal samples from both FMT responders and non-responders, we identified a consortium of four gut commensal species, termed BM111. In an antibiotic-pretreated mouse infection model, BM111 effectively decolonized a VRE strain, demonstrating its therapeutic potential. Additionally, in a humanized mouse model with feces from CPE-infected patients transplanted into germ-free mice, BM111 significantly cleared CPE strains. In animals treated with the BM111 consortium, their microbiome diversity increased post-treatment, correlating with BM111-induced positive outcomes. Further informatic analysis of cohorts from 11 countries confirmed the high prevalence and abundance of these four species in healthy individuals, supporting their potential to restore microbiome balance in patients with infection-induced dysbiosis. The BM111 consortium presents a promising, safe, and effective alternative to FMT for combating antibiotic-resistant enteric infections, warranting further clinical research and development.

Biological sciences/Microbiology

Health sciences/Health care

Antibiotic-resistant infections

Microbiome therapeutics

FMT

consortium

Infections caused by antibiotic-resistant pathogens represent a significant global health threat, rendering common infections increasingly difficult to treat ^{1, 2}. This situation results in escalated medical expenses, prolonged hospitalizations, and higher mortality rates. Notably, infections by Vancomycin-Resistant Enterococci (VRE) and Carbapenemase-Producing Enterobacteriaceae (CPE) are rising worldwide ^{3, 4, 5}. Treating these infections is challenging due to the strains' resistance to multiple antibiotics and the limited selection of effective antibiotics. Continued antibiotic use only exacerbates antibiotic resistance and leads to recurrent infections. Therefore, there is an urgent need for antibiotic-independent strategies.

FMT was employed as an alternative treatment method for VRE and CPE infections ^{6, 7}. FMT has been shown to effectively restore the normal bacterial flora of the gut microbiome in the dysbiotic environment, leading to the rapid decolonization of infected pathogens and an increase in the diversity of the gut microbiome. Studies have also shown that implementing FMT in patients infected with VRE and CPE can downregulate the expression of antibiotic resistance genes, especially vanA and blaNDM, respectively ⁸.

FMT is generally considered safe and well-tolerated, even among high-risk patients ⁹. Nevertheless, safety concerns have arisen due to reports of severe side effects in some FMT recipients ⁹. Additionally, recent guidelines suggest that FMT cannot be recommended for gastrointestinal conditions other than recurrent Clostridium difficile infections ¹⁰. Instances of ESBL-producing E. coli bacteremia and fatalities among patients who recently underwent FMT have been documented ¹¹. Moreover, the process of choosing a healthy donor has become extremely challenging because of the introduction of stricter selection criteria ¹². Therefore, it is crucial to identify a specific set of fecal microbial strains that can be verified for safety and replicate the benefits of FMT.

In our prior FMT trials, where fecal material from a single donor was utilized, a significant number of recipients did not experience the expected efficacy ⁶. This finding implies that pre-existing conditions could impact FMT responsiveness, leading us to explore the factors influencing it. In the present study, we identified four specific strains from the donor fecal microbiome that are potentially involved in FMT responsiveness. Using animal infection models, we validated their efficacy in decolonizing both VRE and CPE pathogens. This research successfully identified well-defined strains capable of replacing FMT. Utilizing this consortium offers a promising and secure intervention against antibiotic-resistant infections.

Differential effectiveness of FMT with the same donor feces among patients. FMT proved to be successful in treating patients with recurrent Clostridium difficle infections ^{5, 13}. To assess the effectiveness of FMT on other resistant infections as well, we previously carried out FMT on 35 patients infected with VRE and/or CPE ⁶. Compared with non-FMT group, patients who received FMT showed notably better outcomes. Throughout the one-year follow-up period, only 27.1% of patients in the non-FMT group experienced spontaneous pathogen clearance (Fig. 1). After one year of FMT, 24 out of the 35 patients tested negative for the original pathogen (Fig. 1). Notably, 11 out of the 35 patients showed no response to the FMT treatment, despite receiving fecal matter from the same donor. Additionally, spontaneous decolonization was observed in the non-FMT group, necessitating a careful assessment of FMT’s actual efficacy. These findings highlight both the potential and limitations of FMT in treating antibiotic-resistant infections.

FMT-induced changes in microbiome compositions among FMT responders. To better understand the differential responses among patients, we analyzed microbiome compositions in two groups (FMT-responders vs. FMT-nonresponders), using samples collected before and after the FMT procedure. In addition, we conducted a comparative analysis to identify similar characteristics between the responder groups: VRE-infected patients (Fig. 2A) and CPE-infected patients (Fig. 2B). Our findings revealed significant and robust changes in microbiome composition following FMT among responders in both infection groups.

Figure 2 shows microbial taxonomic units that increased or decreased significantly following FMT. The taxonomic units exhibiting significant changes are presented at the species, genus, or family levels. Three separate groups, namely the genera Bifidobacterium, Blautia, and Collinsella, exhibited the most substantial increase in their abundance after FMT (Fig. 2A, black arrows). Of note, Blautia wexlerae and Collinsella aerofaciens were identified as species belonging to the Blautia and Collinsella genera, respectively (Fig. 2A, blue arrows). Furthermore, Faecalibacterium prausnitzii, Agathobacter rectalis, and Anaerostipes hadrus were incorporated as species that displayed an elevated abundance following FMT (Fig. 2A, red arrows). Consistent with the results of the FMT-responders among VRE-infected patients, A. hadrus, C. aerofaciens, B. wexlerae, and B. catenulatum (Fig. 2B, blue arrows) showed significant increases following FMT among responders in CPE-infected patients (Fig. 2B).

Importantly, it is worth highlighting that the abundance of five taxonomic units of Enterococcus, Enterococcus faecium, Enterobacteriaceae, Escherichia, and Escherichia coli, representing the pathogens in this cohort, markedly decreased after FMT in both responder groups (Fig. 2A and 2B, dotted black arrows). Remarkably, the two species that exhibited the most substantial decrease in abundance were E. faecium and E. coli, the primary infectious agents affecting many patients who underwent the FMT procedure in our study. The successful clearance of these pathogens in the responder groups is highly correlated with the extent of induced favorable treatment responses.

FMT-induced changes in microbiome compositions among FMT non-responders. Conversely, there was a limited degree of compositional changes observed among the FMT non-responders. Notably, both E. coli and Escherichia were among the taxonomic units that exhibited the most substantial increase in population following FMT, as illustrated in Fig. 3A (red arrows). Bacteroides fragilis, a commensal microbe that can behave as an opportunistic pathogen ¹⁴, was also increased in its abundance following FMT (Fig. 3A, blue arrow). Both the genus Blautia and its specific species, Blautia wexlerae, were among the taxonomic units with increased abundance after FMT (Fig. 3A, black arrows). This outcome strongly underscores the significant capability of B. wexlerae to proficiently colonize the human intestine, consistent with the findings presented in Fig. 2.

Among FMT non-responders in VRE-infected patients, the only species exhibiting a significant decrease in abundance following FMT was Megamonas rupellensis (Fig. 3A). In the group experiencing failure in CPE decolonization, only Blautia hansenii showed a discriminative increase following FMT (Fig. 3B). Based on Fig. 3B, four taxonomic units; NFHL_s, Subdoligranulum, Megasphaera, and PAC00173_s, markedly decreased following FMT. The reason behind the decreased abundance of these groups in this case is not certain.

Contrary to the findings in Fig. 2A and 2B, the taxonomic units with significant decreases in abundance after FMT among non-responders did not include species of pathogens that infected the FMT recipients. These findings reinforce the strong correlation between FMT responsiveness and the degree of alteration in the gut microbiome after FMT. Furthermore, they suggest that individuals with greater shifts in their microbial composition are more likely to respond positively to FMT.

Determination of a FMT-alternative consortium. Subsequently, we assessed the microbiome populations of the donor feces. Importantly, the bacterial species, found in high proportions in the post-FMT fecal samples of individuals who responded positively to FMT, corresponded to those with the most significant prevalence within the donor microbiome (Fig. 4). B. wexlerae and F. prausnitzii individually constituted more than 7% of the entire donor feces' relative abundance and these two species were the most abundant in our analysis (Fig. 4, black arrows). Anaerostipes hadrus and Collinsella aerofaciens, which exhibited robust bloom following FMT in FMT responders (Fig. 2A and 2B), constituted approximately 4.2% and 1.5% of the entire donor microbiome's relative abundance, respectively (Fig. 4, red and blue arrows). Three distinct Bifidobacterium species also exhibited substantial abundance within the donor microbiome (Fig. 4, asterisk-marked bracket), contributing notably to the heightened overall abundance of the Bifidobacterium genus in individuals who positively responded to FMT, as shown in Fig. 2. The donor feces also contained Agathobacter rectalis with a relative abundance of approximately 2% (Fig. 4, green arrow). Similarly, A. rectalis showed an increase in abundance in the post-FMT feces of FMT-responders (Fig. 2A). In the donor feces, four species of the Bacteroides genus were highly represented (Fig. 4, double asterisk-marked bracket). Nevertheless, these species did not exhibit a significant increase in their abundance following FMT (Fig. 2A and 2B).

Utilizing this dataset in combination with the availability of the candidate strains within our repository of fecal microbes, we aimed to identify a consortium of fecal microbes that could potentially collaborate to achieve outcomes, like those of FMT. Additionally, we considered the cultivability of these strains, as maintaining consistent and reproducible cultivation is vital for subsequent procedures. Notably, Agathobacter rectalis could not be isolated from the donor feces. Consequently, we decided to pursue an alternative consortium comprised of Anaerostipes hadrus, Blautia wexlerae, Collinsella aerofaciens and Bifidobacterium longum for further investigation, which we named BM111. A. hadrus and B. wexlerae fall under the Firmicutes phylum, while the other two belong to the phylum of Actinobacteria. All of them are Gram-positives.

Effects of BM111 consortium in elimination of pre-colonized vancomycin-resistant Enterococcus faecium. Next, our objective was to investigate whether the intestinal delivery of the BM111 consortium would have beneficial effects in our infection model. To establish a murine model of VRE infection, we used a Vancomycin-resistant E. faecium (VREf) strain isolated from a patient. After pretreating mice with Ampicillin (Am), we observed persistent long-term colonization of VREf within the mouse intestine. This condition created an environment to evaluate the effects of BM111 treatment. According to Fig. 5A, the mice received Am treatment for 5 days, followed by daily treatment with the BM111 mixture for an additional 5 days. The VREf infection was administered at a dose of 10⁸ CFU. In our experimental condition, the colonization of VREf was relatively well maintained for up to 9 days after infection (Fig. 5B). Long-term colonization of VREf never occurred without Am pretreatment (data not shown). Under this specific condition, BM111 robustly eliminated VREf from mouse intestine (Fig. 5B). Collectively, our results provide clear evidence that the BM111 consortium, comprising rationally selected components, effectively decolonized an antibiotic-resistant clinical strain from an infected animal host.

Persistent colonization of BM111 components in mouse intestine. We then analyzed how BM111 treatment affected overall gut microbiome structure in VREf-infected animals. First, the administration of BM111 led to a rise in the Shannon index, indicating an augmentation in microbiome diversity (Fig. 6A). The LDA effect size score clearly indicates that the taxonomic units of Enterococcus and Enterococcaceae, representing the genus- and family-level unit of E. faecium, exhibited the most significant decrease in abundance following BM111 treatment (Fig. 6B, red arrows). This result provides additional evidence of BM111's significant ability to eliminate VREf from the mouse intestine. Of particular interest, the taxonomic units of Escherichia, Enterobacteriaceae, Enterobacterales and Gamma-Proteobacteria showed a remarkable decrease following BM111 treatment (Fig. 6B, black arrows). Notably, our previous study demonstrated that Am treatment led to a significant bloom of indigenous E. coli cells of mouse origin in the mouse intestine ¹⁵. Hence, the results suggest that BM111 treatment can also effectively reduce the population of E. coli in the Am-pretreated mouse intestine.

Significantly, BM111 treatment led to a higher number of taxonomic units exhibiting an increase in abundance (Fig. 6B), suggesting a positive impact on microbiome alpha-diversity. Among these taxonomic units with increased abundance, Bifidobacterium, Coriobacteriia (a class to which Collinsella aerofaciens belongs), and Blautia were prominently featured (Fig. 6, blue arrows). This finding further supports the idea that the majority of the BM111 consortium components have the capacity to colonize the host intestine effectively. Additionally, there was a substantial increase in the abundance of Actinomycetia, the class that includes Bifidobacterium, as reflected in the composition data of responders following FTM (Fig. 6B, asterisk-marked bracket).

Effects of BM111 consortium in elimination of carbapenem-resistant E. coli in a humanized mouse infection model. In our Ap-pretreated VREf infection model, we observed an endogenous bloom of inherent E. coli cells of mouse origin. Consequently, this model does not allow us to assess the effects of BM111 on eliminating externally infected E. coli. Therefore, to validate the efficacy of BM111 in inhibiting CPE infections, including those caused by E. coli, we needed an alternative infection model. To address this, we used a humanized mouse model that mimics the conditions of clinical patients infected with CPE, without requiring antibiotic-pretreated conditioning.

To construct a humanized mouse model, we transplanted a human fecal suspension into germ-free mice. In this study, we used pooled fecal samples obtained from six patients infected with CPE. These fecal samples contained very high levels of microbes belonging to the Enterobacteriaceae family, particularly E. coli, turned out to be Carbapenem-resistant (Fig. S1). We then monitored the effects of BM111 treatment on altering the titers of E. coli within the intestinal tract over an 8-day experimental period using the collected fecal samples (Fig. 7A). Two days after humanization, the E. coli cell count in fecal samples reached approximately 10⁸. BM111 treatment facilitated the decolonization of E. coli from the mouse intestine, as shown in Fig. 7B. Throughout the monitoring period, E. coli counts persistently decreased in the BM111-treated group. In contrast, titers of E. coli in the control-treated animals remained constant, albeit with a slight increase, during the same period, further indicating strong colonization capability of carbapenem-resistant E. coli of human origin inside the mouse intestine and highlights the beneficial effect of BM111 in clearing this resistant E. coli from the host intestine.

Furthermore, we investigated whether BM111 could compete with the dysbiotic microbes already present in the intestinal tract of the humanized mice. To evaluate the competitive colonization fitness of BM111 strains, we performed a quantitative analysis using qRT-PCR. This involved utilizing primer sets specific to each strain to measure their respective quantities within the intestinal tract. The BM111 strains demonstrated strong colonization within the intestinal tract of the humanized mice, despite the preexistence of dysbiotic flora derived from infected patients (Fig. S2). Although B. wexlerae did not show a statistically significant increase between the BM111-treated vs. control groups, it still exhibited a tendency to increase with BM111 treatment.

Prevalence and abundance of BM111 components in healthy human gut microbiome. Finally, we explored the prevalence of the four species as commensal organisms within the human intestine. Additionally, we sought to analyze the relative abundance of each species within individual hosts. Four species of the BM111 consortium demonstrate a prevalent presence across humans from 11 distinct countries. As an example, concerning the Korean cohort (consisting of n = 214), the presence of A. hadrus, B. longum, B. wexlerae, and C. aerofaciens are identified in 78%, 69%, 64%, and 77% of the samples, respectively (Table 1A). This strongly indicates a notable prevalence of these four species within the Korean population. Similarly, the significant prevalence of all four of these species is observed (> 50%) in the datasets from Japan, Austria, Spain, and France, as illustrated in Table 1A.

Regarding the strain abundance, each of the four species demonstrates a distinct spectrum of abundance within individual hosts, as indicated in Table 1B. For example, among healthy individuals from Mongolia, C. aerofaciens displays an average abundance of 8.71%, while the mean abundance of B. longum in healthy Koreans is 2.9%. The Austrian cohort's healthy participants display the greatest levels of abundance for all four species (Table 1B). Together, our bioinformatic analysis of the prevalence and abundance of BM111 components in the human gut microbiome reveals widespread presence and varying degrees of abundance across diverse populations, shedding light on the significance of these species within different human cohorts.

The emergence and spread of antibiotic-resistant pathogens present a

significant global health challenge ^{16, 17, 18}. In particular, the rise in infections caused by VRE and CPE has been a growing concern worldwide even before COVID-19 pandemic ^{19, 20, 21}. The limited treatment options for patients infected with these antibiotic-unresponsive strains highlight the urgent need for alternative antibiotic-independent approaches.

FMT has demonstrated potential as an effective approach to treating antibiotic-resistant infections. Despite its effectiveness, safety issues have been raised ^{11, 22, 23, 24}. Moreover, FMT carries additional risks and limitations, such as uncertainty about long-term safety ^{25, 26}, lack of standardization ^{27, 28, 29}, temporary gastrointestinal discomfort ³⁰, limited donor availability ^{31, 32}, and ethical concerns ^{33, 34}. Although FMT holds promise as a therapeutic option, it is essential to acknowledge and address the aforementioned factors to ensure its safe and responsible use in clinical practice. Additionally, the utilization of a consortium consisting of a well-defined set of fecal microbes, combined with optimized cultivation procedures, presents a potential and practical alternative to FMT.

Our study began with the observation that FMT treatment for patients infected with VRE and/or CPE yielded varying degrees of effectiveness. While some individuals achieved complete pathogen clearance, others showed no response despite receiving FMT using the same donor feces. This prompted us to investigate the underlying factors contributing to these differential responses. Microbiome analysis revealed significant changes in the gut microbiome compositions of FMT responders. Notably, there was an increase in the abundance of certain taxonomic units, including four species found in the BM111 consortium. This increase suggests that specific microbial changes are closely linked to the successful clearance of VRE and/or CPE. In contrast, FMT non-responders exhibited minimal compositional changes in their gut microbiome. These contrasting outcomes highlight the strong association between the degree of microbiome alteration and the effectiveness of FMT. Based on these findings, we hypothesized that administering the BM111 consortium repeatedly to FMT non-responders could modify their microbiome environment and potentially lead to pathogen clearance over time. This approach may help overcome the challenges observed in FMT non-responders, providing a more effective treatment strategy for antibiotic-resistant infections.

Four strains that make up the BM111 consortium have been proposed as potential treatments for improving human health and addressing disturbances in the gut microbiome. B. wexlerae brings about metabolic shifts in amino acids and carbohydrates, leading to alterations in the composition of gut bacteria and triggering anti-inflammatory effects in a mouse model of obesity induced by a high-fat diet ^{35, 36}. Additionally, depletion of B. wexlerae is linked to metabolic inflammation that promotes insulin resistance in overweight children ³⁷. A. hadrus holds a dominant position among the beneficial microbes in the human gut and serves as a significant producer of butyrate ³⁸. This component plays a crucial role in preserving gut balance and mitigating disorders associated with the gastrointestinal tract. Lactate and acetate, produced by B. longum, serve as substrates for the subsequent utilization by butyrate-producing bacteria ³⁹. Despite C. aerofaciens not traditionally being viewed as a beneficial microbe, its impact on reducing Treg cells stimulates immune responses and ultimately fosters anti-tumor immunity ⁴⁰.

Intestinal tracts compromised in immune function might serve as reservoirs for the proliferation of VRE and CRE ^{41, 42}. Therefore, the re-establishment of intestinal barriers becomes a crucial approach for eliminating the harmful effects triggered by VRE and CRE infections ⁴³. Utilizing commensals, which efficiently restore gut microbial balance, holds promise as a treatment for inhibiting the colonization and growth of pathogenic bacteria in the intestinal tract ^{42, 44}. Caballero and colleagues also documented that a specific consortium of commensal bacteria reinstates colonization resistance against VRE through their collaborative inter-species activities ⁴⁵. In comparison with what was presented in Caballero’s study, however, our findings stand out because four species of the BM111 are derived from comprehensive microbiome analysis from human FMT clinical trials.

After selecting the four species, we conducted a series of in vivo efficacy experiments. Further research is required to elucidate the precise mechanisms by which these four species collaborate within the host's intestine. Key questions to address include: (i) Can the robust intestinal colonization of BM111 components alone effectively displace pre-existing pathogens? (ii) What additional changes are prompted by BM111 transplantation, especially at the metabolomic level? (iii) How does the administration of BM111 impact intestinal immune status?

Recent studies provide relevant insights into the last two questions. For instance, Lactobacillus stimulates the growth of Clostridiales, creating an environment unfavorable for the proliferation of multidrug-resistant Enterobacteriaceae by increasing butyrate levels and reducing nutrient availability ⁴⁶. Additionally, the commensal microbe Bacteroides influences immune responses via IL-37 signaling, hindering Klebsiella pneumoniae colonization and transmission while acting as a sentinel through its polysaccharide utilization capability ⁴¹. Exploring these questions can provide valuable direction in understanding the potential impact and mechanisms of BM111 in addressing antibiotic-resistant infections within the gut.

In our animal experiments, BM111 treatment led to the decolonization of antibiotic-resistant bacteria and restoration of the gut microbiome ecosystem, which is often disrupted in patients infected with antibiotic-resistant pathogens. Numerous diseases arise from dysbiosis in the gut microbiome ^{47, 48, 49, 50, 51}. Therefore, BM111 not only serves as a therapeutic option for antibiotic-resistant infections but also holds the potential to treat various diseases associated with gut microbiome dysbiosis.

We utilized two distinct mouse infection models to validate the potential efficacy of the BM111 consortium in treating VRE and CPE infections. Employing appropriate animal models is crucial for demonstrating in vivo efficacy, and we successfully conducted this validation. Through these models, we confirmed that the BM111 consortium could effectively combat both VRE and CPE infections. This strategic use of infection models underscores the robustness of our approach and provides a strong foundation for potential clinical applications of BM111 in managing persistent bacterial infections.

In conclusion, the study identified and characterized a consortium of fecal microbes, BM111, comprising Anaerostipes hadrus, Blautia wexlerae, Collinsella aerofaciens, and Bifidobacterium longum. The findings demonstrate the BM111 consortium's potential as a promising treatment approach, independent of antibiotics, for tackling recalcitrant infections, particularly those caused by CPE and VRE. Further investigations and clinical trials are warranted to validate the safety and efficacy of BM111 as a therapeutic intervention to address the growing challenges of antibiotic resistance in clinical settings. The BM111 consortium may offer a transformative and valuable alternative to FMT, enhancing the management of antibiotic-resistant infections and improving patient outcomes.

Microbiome composition analysis. Fecal microbial DNA was extracted using a DNeasy PowerSoil Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The extracted DNA was quantified using Quant-IT PicoGreen (Invitrogen). Paired-end (2×300 bp) sequencing was performed by Macrogen on the MiSeq platform (Illumina, San Diego, CA, USA). Sequencing libraries were prepared according to the Illumina 16S Metagenomic Sequencing Library protocols to amplify the V3 and V4 regions. The universal primer pair sequences with an Illumina adapter overhang used for the first amplification were as follows: V3-F: 5′-TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG CCT ACG GGN GGC WGC AG -3′, V4-R: 5′- GTC TCG TGG CTC GGA GAT GTG TAT AAG AGA CAG GAC TAC HVG GGT ATC TAA TCC − 3′. The 1st PCR product was purified using AMPure beads (Agencourt Biosciences). Following purification, 2 ml of the 1st PCR product was PCR-amplified for final library construction containing the index using the NexteraXT Indexed Primer. The PCR products were purified using AMPure beads. The final purified product was quantified using qPCR according to the qPCR Quantification Protocol Guide (KAPA Library Quantification kits for Illumina Sequencing platforms) and qualified using a TapeStation D1000 ScreenTape (Agilent Technologies, Waldbronn, Germany).

Illumina MiSeq raw data were processed by classifying the raw reads using index sequences to generate paired-end FASTQ files. Sequencing adapter and primer sequences were removed using Cutadapt (v3.2), trimming forward and reverse reads to 250 bp and 200 bp, respectively. The DADA2 (v1.18.0) package in R (v4.0.3) was used to correct errors, excluding reads with expected errors over 2. A batch-specific error model was applied to remove noise, and error-corrected sequences were assembled into single sequences. Chimera sequences were removed with DADA2's Consensus method, forming ASVs, with those shorter than 350bp excluded. Reads were normalized using QIIME (v1.9) based on the sample with the lowest read count. Each ASV sequence was matched against the NCBI 16S Microbial DB using BLAST+ (v2.9.0) for taxonomy assignment, excluding matches below 85% coverage or identity. Multiple alignments were performed with mafft (v7.475) and phylogenetic trees generated with FastTreeMP (v2.1.10). QIIME was used for comparative microbial community analysis, assessing species diversity and evenness with Shannon and Inverse Simpson indices, alpha diversity with Rarefaction curves and Chao1 values, and beta diversity with UniFrac distances, visualized through PCoA and UPGMA trees.

Statistical Analysis of Taxonomic Abundance Differences. The study utilized the Linear Discriminant Analysis Effect Size (LEfSe) method for high-dimensional class comparisons to identify biologically meaningful features ⁵². To detect significant differences in feature abundance between the BM111-treated and control groups across various taxonomic units, the Kruskal-Wallis sum-rank test was applied. To further validate the biological relevance and consistency of these findings, pairwise tests among subclasses were conducted using the Wilcoxon rank-sum test, with a significance threshold set at α ≤ 0.05.

Animal husbandry and mouse models. We used 7–9 week-old female C57BL/6 mice purchased from Orient Bio (Sungnam, Korea). The mice were acclimatized for 1 week before the experiments. All experiments involving mice were conducted according to the guidelines of the Department of Animal Resources, Yonsei Avison Biomedical Research Institute. The Committee on the Ethics of Animal Experiments at Yonsei University College of Medicine approved this study (permit number, 2020 − 0228).

In our antibiotic-pretreated infection model, mice received ampicillin-supplemented drinking water at a concentration of 0.5 g/L daily for 5 days before being infected with VRE. Two days after the VRE infection, the drinking water was replaced with antibiotic-free water. The mice were then randomly divided into two groups: control and BM111, with 8 mice in each group. Germ-free (GF) mice of C57BL/6 were manipulated in the same facility, and their sterile state was confirmed before each study, as previously reported ⁵³.

Preparation of BM111 consortium, VRE isolate, and CPE-infected fecal suspension. A. hadrus, C. aerofaciens, B. wexlerae and B. longum isolated from healthy humans were either supplied by YSFlora^®, BioMe Inc's proprietary human microbiome repository ⁵⁴. All anaerobic culture media were deoxygenated for 48 hrs before use and bacteria were cultured at 37°C in an anaerobic chamber with mixed anaerobic gas (5% carbon dioxide, 5% hydrogen, 90% nitrogen). Tryptic soy broth (TSB, BD, USA) supplemented with menadione (Menadione, Sigma, MO, USA), hemin (Hemin, Sigma, MO, USA), vitamin K1 (Vitamin K1, Sigma, MO, USA), and L-cysteine (L-Cysteine hydrochloride monohydrate, Sigma, MO, USA) was used to cultivate BM111 strains. After incubation, each BM111 strain was centrifuged at 8,000 rpm for 20 min to remove the supernatant and suspended in sterile 1× phosphate-buffered saline (PBS, Sigma, MO, USA) for washing. After the 1× PBS wash, the four strains were mixed, each 1:1:1:1, dissolved in 12.5% glycerol (Glycerol, Supelco, USA), and stored at -80°C until use. Mice were treated with BM111 by oral gavage at 1×10⁹ for 5 days.

Vancomycin-resistant Enterococcus faecium strain (VREf) was isolated from a patient at Severance Hospital and generously provided by Professor Dong Eun Yong, Department of Laboratory Medicine, Yonsei University College of Medicine. The VREf strain was streaked onto Enterococcocel Agar (EC, BD, USA) supplemented with vancomycin (64 µg/ml) (Vancomycin Hydrochloride, Duchefa, Netherlands) to selectively culture VREf from a VRE stock and incubated in 37°C for 24 hrs. Single colonies were then picked and aerobically incubated in Brain Heart Infusion Broth (BHI, BD, USA) at 37°C for 24 hrs. Mice were infected with 10⁸ CFU of VREf suspended in sterile 1× PBS by oral gavage.

For our humanized mouse infection model, we selected six different patient fecal samples with a high prevalence of Carbapenem-resistant E. coli. The viable cell counts for E. coli in the pooled fecal sample suspension were 2.0×10⁵ (data not shown).

Viable cell counting of VRE and CPE. Fresh stools were obtained from mice at every time point and then suspended in 1 mL of 1×PBS and spun down for 5–10 seconds using a mini centrifuge, and the homogenate of the supernatant was serially diluted. To verify the quantity of VRE cells in mouse fecal contents, the dilutions were then spotted at 10 µl onto Enterococcosel agar (EC, BD, USA) plates supplemented with vancomycin 64 ug/ml and incubated at 37°C for 24 hrs, and colonies were counted. Meanwhile, CHROM mSuperCARBA Agar plates (ASAN Pharm. Co., Ltd.) were used for E. coli counting in fecal matter and bacterial cultures.

Quantitative analysis of BM111. Total bacterial DNA was extracted using the Quick-DNA Fecal/Soil Microbe Kit (Zymo Research, USA) from fresh stool samples collected from mice at various time points. Specific primer sets were designed using the whole genome sequences of each strain to amplify bacterial DNA at the strain-specific level from the fecal DNA. The accuracies and sensitivities of the designed primer sets were evaluated through multiple assessments. The target genes for each ultimately selected primer set are as follows: Galactitol 1-phosphate 5-dehydrogenase for A. hardus, Deoxyribose-phosphate aldolase 1 for C. aerofaciens, Melibiose operon regulatory protein for B. wexlerae, and α-maltose-1-phosphate synthase for B. longum. Primer sequences are presented in Fig. S2.

Bioinformatic analysis of prevalence and abundance of BM111 component species. To investigate the abundance and prevalence of four species (A. hadrus, B. longum, B. wexlerae, C. aerofaciens) in healthy individuals, a total of 1,543 fecal samples from 11 countries were used: South Korea ^{55, 56, 57, 58}, Japan ^{59, 60}, China ^{61, 62, 63}, Mongolia ⁶⁴, India ^{65, 66, 67}, USA ^{68, 69, 70, 71}, UK ⁷², Spain ⁷³, France ⁷⁴, Austria ⁷⁵, and Republic of Congo ⁷⁶. All raw shotgun sequences were processed consistently. Low-quality reads were removed using Sickle ⁷⁷ with the condition of Phred quality scores > 20 and read length options > 90 bp (pe -q 20 -t sanger -l 90), and reads containing ‘N’ were removed. The retained reads were mapped to the human genome using Bowtie2 ⁷⁸ to remove mapped reads for host contamination. Following preprocessing, the sequencing reads were analyzed using MetaPhlAn (version 3.0) ⁷⁹ to profile microbial composition with default settings. Only species with an abundance greater than 0.1% in each individual sample were retained for further analysis.

Whole genome sequencing of BM111 strains. BM111 strains were cultured in TSB supplemented with menadione, hemin, vitamin K1 and L-cysteine, and the cultures were harvested and centrifuged to obtain pellets. Genomic DNA was extracted and purified from the bacteria using the MGTM Genomic DNA Purification Kit. Sequencing libraries were prepared according to the manufacturer's instructions for the TruSeq Nano DNA High Throughput Library Prep Kit (Illumina). Briefly, 100 ng of genomic DNA was sheared using adaptive focused acoustic technology (Covaris) and the fragmented DNA was end-repaired to generate 5'-phosphorylated, blunt-ended dsDNA molecules. After end-repair, the DNA was sized using a bead-based method. These DNA fragments undergo single 'A' base addition and ligation to TruSeq DNA UD Indexing Adapters. The products are then purified and enriched by PCR to generate the final DNA library. The libraries were quantified by qPCR according to the qPCR Quantification Protocol Guide (KAPA Library Quantification kits for Illumina sequencing platforms) and qualified using the Agilent Technologies 4200 TapeStation D1000 Screen Tape (Agilent Technologies). And then we sequenced using the HiSeq (Illumina).

Statistical analysis

Data are expressed as means ± standard deviation (SD). All data calculations and statistical analyses were performed using GraphPad Prism ver. 9.4.1 (GraphPad Software Inc., La Jolla, CA, USA). Differences between groups were analyzed using multiple t-tests or the Mann-Whitney U test, and p values < 0.05 were considered statistically significant.

Competing Interests

The authors declare no competing interests

Funding

This work was supported by grants from the National Research Foundation of Korea, funded by the Korean Government (2022M3A9F301750613, 2022R1A2C1009903 and 2019R1A6A1A03032869). This work was also supported by a grant from the Tech Incubator Program for Startups (TIPS) funded by the Ministry of SMEs and Startups, Republic of Korea (S3287890). This work was also supported by a grant from the Korea National Institute of Infectious Diseases, Korea National Institute of Health (grant number 2023-ER2107-00).

Author Contribution

MYY and SSY designed the study. MYY, SSY, and JYC obtained funds and managed the study. MYY, UJJ, and SYC performed experiments. MYY, HYJ, MH, EHL, and JHK performed sequencing analyses and bioinformatic analyses. EK and MR performed bioinformatic cohort analyses. SSL collected patient stools. YWC and JHR provided germ-free mice. MYY, UJJ, and SSY drafted the manuscript. All authors contributed to the article and approved the submitted version.

Acknowledgement

Data Availability

The metagenome amplicon sequencing data can be accessed at the following URL: https://dataview.ncbi.nlm.nih.gov/object/PRJNA1139362.

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Table 1 is available in the Supplementary Files section.

No competing interests reported.

Figures240731Tab.1.pdf
Prevalence and abundance of the BM111 strains in gut microbial composition of healthy individuals across different countries. a Species prevalence (%) and b Species abundance (Mean±SD) of the BM111 strains in gut microbial composition were evaluated following the methodologies outlined in the Materials and Methods section. The analysis encompassed a total of 1,543 datasets showcasing bacterial compositions derived from stool samples of healthy individuals across 11 diverse countries.
Figures240731Fig.S1.pdf
Figures240731Fig.S2.pdf
Supplementaryinformation0731Final.docx

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A Consortium of Fecal Microbes that Decolonizes Antibiotic-resistant Enteric Pathogens: A Potential Alternative to Fecal Microbiota Transplantation

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