Abstract
The human gut microbiota is increasingly being recognised to play an important role in maintaining health. The families Lachnospiraceae and Oscillospiraceae in particular, are often reduced in disease states but are relatively poorly represented in culture collections. Cultured representatives are required to investigate the physiology and host interactions of gut microbes. Establishing cultured isolate collections can be laborious and expensive owing to the fastidious growth requirements of these organisms and the costs associated with taxonomic classification. This study proposes a culturomics platform combining a single basal culture medium with matrix-assisted laser adsorption/ionisation coupled to time-of-flight mass spectrometry (MALDI-TOF MS) for fast and reliable isolation and identification of hundreds of novel isolates. In this study, basal YCFA medium supplemented with either glucose, apple pectin, or porcine mucin was used to cultivate a total of 724 different isolates derived from only 11 different faecal samples from healthy volunteers, of which 389 isolates belonged to the Lachnospiraceae and Oscillospiraceae families. Moreover, 27 isolates could not be assigned to known species based on their 16S rRNA gene, 17 of which may even represent novel genera. To aid MALDI-TOF MS identification of gut bacteria, the commercial database was complemented with the MaldiGut database presented here, containing a collection of 132 different Main Spectrum Profiles, including the profiles of 125 Firmicutes species, 3 Bacteroidetes species, 3 Actinobacteria species, and one Verrucomicrobia species. The culturomics platform and MaldiGut database presented here will enable further expansion of the gut culturome, especially within the understudied Lachnospiraceae and Oscillospiraceae families.
1 Introduction
Over the last two decades, there has been a growing interest in the human gut microbiota, caused by the increasing recognition that this intestinal community of viruses, fungi, and bacteria are important in both health and disease. (Gacesa et al., 2022; Gomaa, 2020; Round and Mazmanian, 2009; Tlaskalová-Hogenová et al., 2011) The rapidly expanding field of metagenomic studies has boosted the number of associations and links with health, and more predominantly with disease. In addition to gut-related diseases, such as irritable bowel syndrome and inflammatory bowel disease, autoimmune, metabolic, oncological, and neurological diseases have been linked to an altered gut microbiota compared to healthy individuals (Gacesa et al., 2022; Gomaa, 2020; Round and Mazmanian, 2009; Tlaskalová-Hogenová et al., 2011). The incidence of many of these conditions is rapidly increasing in the modernised world (GBD-2017 Inflammatory Bowel Disease Collaborators, 2020; Lerner and Matthias, 2015). These links have led to a growing interest in the design of interventions that could lead to prevention, delayed onset, or even treatment of these diseases. There is increasing evidence that certain taxa appear to be more commonly perturbed in disease states than others (Lozupone et al., 2012). For example, the numbers of butyrate-producing bacteria, and in particular, Firmicutes species such as Faecalibacterium prausnitzii, have been repeatedly shown to be decreased in inflammatory bowel disease and type one and type two diabetes, depression, rheumatoid arthritis and asthma (Gacesa et al., 2022; Harmsen and de Goffau, 2016).
The bacterial families Lachnospiraceae and Oscillospiraceae (formerly known as Ruminococcaceae) are often negatively correlated with these diseases, but are typically less represented in culture collections than other taxa (Rajilić-Stojanović and de Vos, 2014). This is possibly because many members of the Lachnospiraceae and Oscillospiraceae are extremely sensitive to oxygen, making them more difficult to culture (Stewart, 2012). This raises the pressing need for more cultured representatives that allow improved verification of the findings of metagenomic studies and will permit complementary research to provide more mechanistic insights into the interaction between the host and gut microbiota. Additionally, it has been proposed that butyrate-producing representatives of the Lachnospiraceae and Oscillospiraceae families may be utilised as novel next-generation probiotics to reverse the recurrent reduction in the prevalence of these species across various disease states (C.-J. Chang et al., 2019; Yadav et al., 2022). Therefore, microbiome research would benefit from culturomics studies that focus more on the isolation of members within the Firmicutes phylum, particularly from the Lachnospiraceae and Oscillospiraceae.
Several previous studies have expanded the human gut culturome (Browne et al., 2016; Forster et al., 2019; Lagier et al., 2016; Li et al., 2022; Mukhopadhya et al., 2022; Poyet et al., 2019); however, the protocols implemented in these studies are often extremely cumbersome. Most of these protocols require a broad range of media for bacterial cultivation and therefore require a substantial amount of time and practical work. Some studies have attempted to reduce this time and hands-on work by implementing matrix-assisted laser desorption/ionisation coupled with a time-of-flight mass spectrometry analyser (MALDI-TOF MS) (Asare et al., 2023; Lagier et al., 2016; Li et al., 2022). This method allows for efficient and accurate identification of bacterial isolates by replacing more time-consuming techniques, such as 16S rRNA gene Sanger sequencing or whole genome sequencing. Although MALDI-TOF MS is very accurate, the identification of bacteria depends on the availability of reference spectra in databases. The use of MALDI-TOF MS has become more established in diagnostic laboratories, and hence includes an expanding number of clinically relevant bacteria in publicly and commercially available databases (Rodrı́guez-Sánchez et al., 2019). However, most of these databases contain a limited number of non-clinically relevant bacteria, especially anaerobic gut microbial species such as those belonging to the Lachnospiraceae and Oscillospiraceae.
In this study, we established a high-throughput culturomics platform that allows a broad range of gut bacterial species to be cultivated and identified. Unlike previous studies, the focus of this study was on the isolation and identification of the phylum Firmicutes with a special interest in cultivating Lachnospiraceae and Oscillospiraceae, using a single anaerobic basal medium supplemented with a variety of single carbohydrates. The YCFA basal medium was specifically designed to support the growth of diverse anaerobic gut bacteria, especially within the Lachnospiraceae and Oscillospiraceae families, and is a convenient alternative to rumen fluid-based media (Duncan et al., 2002). Subsequent studies have shown that YCFA medium also supports the growth of other phyla and therefore allows a broad range of bacterial species to be cultivated (Browne et al., 2016; Y. Chang et al., 2019; Mukhopadhya et al., 2022). Additionally, we present a gut microbiota-specific MALDI-TOF MS reference database, named ‘MaldiGut’, that enables accurate and high-throughput identification of gut bacterial isolates.
2 Material and methods
Culturomics platform: sample collection, cultivation and MALDI-TOF MS identification
Faecal samples from 11 self-reported healthy volunteers were collected to isolate anaerobic gut bacteria after obtaining informed consent. Other requirements for ethical approval were waived by the local ethics committee of the University Medical Center Groningen (METc2014.236). The samples were used for inoculation within ten minutes after collection without prior storage to enhance the possibility of survival of the strictest anaerobic bacteria (Moore and Holdeman, 1974). In the initial inoculation round, the faecal samples were directly inoculated, without prior dilution and quantification, on YCFA (Duncan et al., 2002) agar plates supplemented with either glucose, apple pectin or porcine mucin type III (4.5 g/l) and with or without the addition of 20 μg of sulfamethoxazole, an antibiotic for which certain Lachnospiraceae and Oscillospiraceae species might have increased resistance (Bandelj et al., 2017; Foditsch et al., 2014; Khademi and Sahebkar, 2019) (Figure 1A(a)). The inoculated agar plates were incubated at 37 °C for 48 hours in an anaerobic chamber (Whitley A35 Workstation, Don Whitley Scientific Limited, Bingley, UK) with an anaerobic gas mixture (10% H2, 10% CO2, and 80% N2) (Duncan et al., 2002). After an initial incubation period that allowed the colonies to be formed on the first plate, with a possible higher diversity of bacteria due to omission of prior sample dilution, between 50-100 pure colonies per donor were selected to aim for maximal morphological diversity between colonies. These colonies were isolated by a classical pure culture technique involving at least three rounds of streaking until single colonies were obtained, of which preliminary purity was assessed using Gram staining afterwards. The identity of these novel isolates was assessed by MALDI-TOF MS (Biotyper Microflex, Bruker Daltonics GmbH, Bremen, Germany; database version 11), as described previously, and stocks of the colonies were stored at −80 °C (Figure 1A(b)) (Veloo et al., 2016). Briefly, single colonies were spotted on a stainless-steel MALDI target. Next, 1 μl of 2-Cyano-3-(4-hydroxyphenyl) acrylic acid matrix (10 mg/ml) was added to every spot and left to air-dry. Subsequently, the target was inserted into the Biotyper Microflex. The spotted isolates were identified based on spectra that were obtained by summing the measurements of 240 shots that were performed in six steps of 40 shots each. The laser was set to have a minimum power of 30% and a maximum power of 40%. (Veloo et al., 2016) According to the manufacturer’s instructions, an isolate was considered to have a reliable identification at the species level when a log score of 2 or higher was given. If the score was between 1.7 and 2, the isolate was considered to have a reliable identification to the genus level. A log score smaller than 1.7 indicated no reliable identification (Almuzara et al., 2016; Veloo et al., 2016).
The culturomics platform and distribution of isolates. Culturomics workflow. (A) A simplified overview of our culturomics workflow. Eleven human faecal samples were used for inoculation of three types of YCFA based agar plates with different added substrates (glucose, apple pectin and mucin) with or without sulfamethoxazole (a). Pure colonies were identified with MALDI-TOF MS (b). The pure colonies that could not be identified with MALDI-TOF MS were used for 16S rRNA gene Sanger sequencing to get an identification (c). Both species with a limited amount of reference spectra (d) and previously unidentified species with MALDI-TOF MS (e) were added to the MaldiGut database. Aliquots of the faecal samples were also used for 16S rRNA gene sequencing (f). (B) Venn diagram of all overlapping and unique identifications of isolates on YCFA supplemented with glucose (blue), mucin (red) and pectin (yellow). The numbers indicate identifications within all phyla, within Firmicutes only and within the families Lachnospiraceae and Oscillospiraceae only, in this respective order. (C) Bar plot that shows the phylum distribution of all isolates per carbohydrate supplemented medium. (D) Bar plot that shows the family distribution of all isolates within the Firmicutes per carbohydrate supplemented medium.
Citation: Beneficial Microbes 16, 1 (2025) ; 10.1163/18762891-bja00042
16S rRNA gene Sanger sequencing and analysis of unidentified isolates
Bacteria that remained unidentified by MALDI-TOF MS were clustered in a phyloproteomic dendrogram (BIOTYPER 3.0, Bruker Daltonics GmbH) and representatives of each cluster were subjected to 16S rRNA gene Sanger sequencing (Figure 1A(c)) (Sanger et al., 1977). DNA was isolated using a lysis buffer consisting of 0.05 mM NaOH and 0.25% SDS. A full 1 μl loop of bacteria from two or more single colonies, derived from a pure culture, was dissolved in 50 μl lysis buffer. Lysates were incubated for 10 min at 100 °C. Finally, 200 μl of 1× TE buffer (10 mM Tris-HCl and 1 mM EDTA, pH 8.0) was added to the lysate. PCR was performed using the universal primers 27F and 1492R or 515R, as described previously (Schuurman et al., 2004). The amplicons were sequenced on the ABI-3500XL genetic analyser with the BigDye Terminator v3.1 Cycle Sequencing kit according to the manufacturer’s instructions (Applied Biosystems, Waltham, MA, USA). The sequencing reads were manually curated using BioEdit and identified using the Ribosomal Database project (RDP release 11) as the primary database to the genus level in the RDP classifier tool and EzBioCloud as a secondary database to identify at the species level (Cole et al., 2014; Wang et al., 2007; Yoon et al., 2017). Reliable species identification was based on the closest match, and was considered valid only if the sequence identity was ≥98.5% (Stackebrandt and Ebers, 2006).
Main MALDI-TOF MS spectrum profiles database creation
To identify strictly anaerobic bacteria whose reference spectra are not currently present in the database of the manufacturer (Bruker Daltonics, GmbH; database version 11), Main Spectrum Profiles (MSPs) were created and added to our MaldiGut database (Figure 1A(d) and A(e)). Supplementary Table S1 shows all the MSPs included in the database. All isolates mentioned in Supplementary Table S2 were grown on YCFA supplemented with glucose under anaerobic conditions for 24 to 48 h unless stated otherwise. Full extraction was performed as described previously (Almuzara et al., 2016; Plomp and Harmsen, 2024; Veloo et al., 2014). Briefly, a full 1 μl loop of bacteria from one or more single colonies derived from a pure culture was used for the suspension of 300 μl ultra-pure water and 900 μl absolute ethanol. The ethanol suspensions were centrifuged twice at 13,000
DNA extraction, PCR and Illumina-based 16S rRNA gene amplicon sequencing of faecal samples
DNA was extracted from 0.25 gram of frozen aliquot of the faeces from each of the 11 samples using the repeated bead-beating method and used for 16S rRNA gene amplicon sequencing as described previously (Figure 1A(f)) (De Goffau et al., 2013). Sequencing was performed as follows, the V3-V4 region of the 16S rRNA gene was amplified by PCR using modified 314F and 806R primers (Heida et al., 2016). The reverse primer contained a 6-nucleotide barcode. PCR, DNA clean-up, and Illumina MiSeq library preparation have been described in detail previously (Heida et al., 2016). The samples were sequenced using an in-house Illumina MiSeq with a 2 × 300 bp cartridge (Illumina, Inc., San Diego, CA, USA). Reads were joined and quality controlled with a maximum error rate of 1%, and the primer sequences were trimmed off using VSEARCH (Rognes et al., 2016). Denoising was performed using a combination of USEARCH and VSEARCH (Edgar, 2010), including chimeric and singleton removal and dereplication. Amplicon sequence variants (ASVs) were assigned based on the RDP Classifier using the Ribosomal Database Project (RDP) set 18 (Cole et al., 2014; Wang et al., 2007). The relative abundances of ASVs were defined as the read count of ASVs divided by the total number of mapped reads after rarefaction to the same depth.
3 Results
Isolation of anaerobic gut bacterial species with our culturomics platform
In total, 724 pure colonies were isolated from the faecal samples and analysed by MALDI-TOF MS. Initially, only 242 out of the 724 pure colonies could be identified to the species level using the commercially available Main Spectrum Profiles (MSP) database from the manufacturer. Therefore, MALDI-TOF MS data were used to create phyloproteomic dendrograms to visualise the relatedness of the unidentified colonies. A representative isolate of each phyloproteomic cluster was selected for 16S rRNA gene Sanger sequencing, resulting in 73 representative sequences (Figure 1A(c)). Several of the representative isolates were identified to species level with more than 97% homology with references sequences. However, 30 isolates could not be identified to species level, so they were clustered with sequences of closely related bacteria. Subsequently, the isolates that were identified to species level based on 16S rRNA gene Sanger sequences were added as new MSPs in the MaldiGut database (Figure 1A(e)). This database was complemented with additional anaerobic gut bacteria that were absent or present at low frequency in the commercial database (Figure 1A(d)). These include MSPs of previously acquired anaerobic human gut bacterial strains, gut bacterial species identified during the analysis, and publicly available MSPs from type strains of anaerobic human gut bacteria (Supplementary Table S1) (URMS Data Base – IHU, 2023; Veloo et al., 2016, 2018). This MaldiGut database contains 132 MSPs with Firmicutes, our target phylum, represented by 125 different strains. Other phyla included in the database were Bacteroidetes (3 strains), Actinobacteria (3 strains), and Verrucomicrobia (1 strain) (Supplementary Table S1).
Re-analysing the final dataset of all isolated bacteria, 254 isolates should be identifiable with the Bruker database to at least genus level, an additional 348 isolates when the MaldiGut database is added, and 122 isolates are not yet identifiable to at least genus level with both MALDI-TOF MS databases.
Overall, in our culturomics, we isolated and identified 74 different species and another 160 isolates that could not be fully identified to the species level, as depicted in blue at species level in Figure 2. At the phylum level, 470 isolates were Firmicutes (64.9%), 53 were Bacteroidetes (7.3%), 96 were Actinobacteria (13.3%), 43 were Proteobacteria (5.9%), 1 was Fusobacteria (0.1%), and 61 were unidentified (8.4%).
Overview of the cultured isolates. Sankey diagram showing the overview of pure colonies isolated from the faecal samples. From left to right, the columns are volunteers (A-J), phylum, class, order, family, genus, species, different substrates added to the basal YCFA medium, SMX use, identification method.
Citation: Beneficial Microbes 16, 1 (2025) ; 10.1163/18762891-bja00042
All bacterial isolates were analysed at the family level to obtain a more comprehensive view of these results. We isolated and identified 23 different families. The most abundant identified families were Lachnospiraceae (246 isolates), Oscillospiraceae (143 isolates), Bifidobacteriaceae (57 isolates), and Bacteroidaceae (42 isolates). Since our main interest in this present study concerned two specific families within Firmicutes, we looked in more detail at the family distribution. The most frequently isolated families within this phylum were Lachnospiraceae (52.3%) and Oscillospiraceae (30.4%), followed by Clostridiaceae (4.9%), Erysipelotrichaceae (3.4%), Coprobacillaceae (2.3%), Peptostreptococcaceae (2.3%), and less frequently Streptococcaceae (0.9%), Veillonellaceae (0.9%), Eubacteriaceae (0.6%), Enterococcaceae (0.2%), and Selenomonadaceae (0.2%). Interestingly, 0.9% of the isolates were only identified at the family level, indicating that they are a particularly novel group of isolates.
Different carbohydrates aid in selection of Firmicutes and allow diverse cultivation of genera within the Lachnospiraceae and Oscillospiraceae
Using supplemented YCFA media, Firmicutes was most frequently isolated, of which Lachnospiraceae and Oscillospiraceae were the most abundant families. Thus, we focused on these two families to explore the link between supplementation with either glucose, apple pectin, or mucin and their enrichment and recovery in pure culture. (Figure 1B-D). The addition of apple pectin to the YCFA medium resulted in the highest recovered diversity of isolates; we obtained 268 isolates that belonged to 112 different species. Similarly, glucose as a carbohydrate source also resulted in high diversity, recovering 113 different species from 276 isolates. Mucin in the medium yielded the lowest number of species, but still 61 different species were obtained from 180 individual isolates. Regardless of the type of substrate added to the medium, 15 different species were retrieved from all three different media, of which six belonged to Lachnospiraceae and Oscillospiraceae (Figure 1B). This suggests that they can metabolise all three substrates or utilise other components in the medium. These overlapping species within Lachnospiraceae and Oscillospiraceae belong to the genera Anaerostipes, Agathobacter, Anaerobutyricum, Coprococcus, Dorea and Enterocloster. The other overlapping genera were Bacteroides, Bifidobacterium (two species), Collinsella, Erysipelatoclostridium, Escherichia, Hungatella, Intestinibacter, and Sutterella.
Phylogenetic tree based on the ASVs from 16S rRNA gene amplicon sequencing of 11 volunteers. The phyla were indicated by dots in different colours. The success of isolation in this study of each genus is indicated by blue (No) background and orange (Yes) background. The relative abundance of ASVs from 11 faecal samples was plotted as heatmap showing around and matching the phylogenetic tree.
Citation: Beneficial Microbes 16, 1 (2025) ; 10.1163/18762891-bja00042
Interestingly, each added substrate also resulted in the cultivation of unique species recovered solely from each individual medium type. Glucose in the medium resulted in the highest number; 83 possible unique species, of which 39 belonged to either Lachnospiraceae or Oscillospiraceae. However, within these families, apple pectin medium resulted in the highest diversity, with 42 possible unique species (Figure 1B).
To clarify the link between any nutritional preferences that might lead to enhanced enrichment and isolation of the families Lachnospiraceae and Oscillospiraceae, we determined the proportion of each phylum and families that grew on each type of enriched medium (Figure 1C and 1D, respectively). Out of the three tested media, the Firmicutes appeared to be selected best on apple pectin and glucose, accounting for 71 out of 112 isolates on apple pectin and 78 out of 113 isolates on glucose, which corresponds to a recovery of 63.4% and 69.0%, respectively. The mucin-containing YCFA appeared to be less selective for Firmicutes, with this phylum accounting for only 30 out of the 61 isolates (a recovery of 49.2%). (Figure 1C). A similar trend was observed for the recovery of Lachnospiraceae and Oscillospiraceae. Apple pectin and glucose resulted in 60 (53.6%) and 55 (48.7%) different isolates, respectively, whereas mucin yielded 20 (32.8%) different isolates within these families (Figure 1D).
Faecal profiling by 16S rRNA gene amplicon sequencing shows efficacy of the culturomics platform
To assess the efficacy of our culturomics platform, all faecal samples were subjected to 16S rRNA gene sequencing (Figure 3). According to the 16S rRNA gene profiling, by far the most amplicon sequence variants belonged to Firmicutes (82.8% to 93.5%; Figure 3, green dots), with Lachnospiraceae and Oscillospiraceae as the most abundant families in all volunteers. In total, 75 different genera were identified by 16S rRNA gene profiling. The top five identified genera with the highest relative abundances in all volunteers were Blautia, Faecalibacterium, Agathobacter, Roseburia and Coprococcus.
Efficacy of the culturomics platform compared to the consensus core microbiota. Heatmap comparing the core gut microbiota as defined by 8 different studies. Colours indicate the inclusion of the genus in the core gut microbiota per study (red: absent, blue: present). On the left is indicated whether core gut microbial genera were isolated in our culturomics study. On the right are all described genera clustered based on the presence or absence between studies, including the faecal profiling in this study (J11: Jalanka-Tuovinen et al., 2011, C09: Claesson et al., 2009, T09: Tap et al., 2009, Z14: Zhang et al., 2014, M13: Martinez et al., 2013, F16: Falony et al., 2016 and Q10: Qin et al., 2010). Asterisk indicates genus name description after 2009.
Citation: Beneficial Microbes 16, 1 (2025) ; 10.1163/18762891-bja00042
Comparison of the culturomics with 16S rRNA gene profiling showed that 94 different genera could be identified in the faecal samples with either 16S rRNA gene profiling, culturomics, or both (Figure 3). Of these genera, 37 overlapping genera (39.4%) could be retrieved by 16S rRNA gene profiling and could also be identified in this study with culturomics (Figure 3). The remaining 38 genera (40.4%) were identified exclusively by 16S rRNA gene profiling. Interestingly, 19 genera (20.2%) were only identified in our culturomics and were not retrieved by 16S rRNA gene profiling of the same samples. To assess the efficacy of our culturomics platform with the focus on only Lachnospiraceae and Oscillospiraceae, 19 out of 38 genera (50%) were identified in both the culturomics and 16S rRNA gene profiling datasets. 16S rRNA gene profiling identified 13 different genera (36.8%) exclusively, while in contrast, six genera (15.8%) were exclusively retrieved using our culturomics method.
Next, faecal profiling and culturomics findings were evaluated in previous studies on the core gut microbiota, which was identified mostly based on 16S rRNA gene profiling or metagenomic shotgun sequencing. As shown in Figure 4, in total 41 bacteria were designated as core gut microbiota by multiple studies, suggesting that it would be of great value to be able to culture and subsequently accurately profile this isolates from this group (Claesson et al., 2009; Falony et al., 2016; Jalanka-Tuovinen et al., 2011; Martı́nez et al., 2013; Qin et al., 2010; Tap et al., 2009; Zhang et al., 2014). Only 14 of these core members were present in the MALDI-TOF MS database of the manufacturer, whereas the MaldiGut database included 23 core members, making it easier to identify them (Supplementary Table S1). In our study, we identified 22 out of the 23 core gut microbes using 16S rRNA gene profiling and were able to culture 20 of these bacteria (Figure 4). If criteria were made stricter to define core members and bacterial genera that were found in at least half of these studies, 12 core gut microbes were identified. All of these genera, except Eubacterium, were also designated as core members in our study, and all 12 bacteria were cultured with our culturomics method and are present in the MaldiGut database, emphasizing that this culturing method is able to yield valuable bacteria, and this database is a valuable resource for subsequent identification.
Phylogenetic tree of non-identified isolates and closest neighbouring species. The phylogenetic tree of the unidentified isolates together with the closest relatives based on 771 nucleotide positions of the 16S rRNA genes starting at E. coli position 228. Isolates identified to family level are shown in purple letters and to genus level in orange letters. Branch colours indicate that the isolates are separated into Lachnospiraceae (red), Oscillospiraceae (green) and Coprobacillaceae (blue). The tree is drawn as a consensus of 10,000 bootstrap replicates based on the maximum likelihood method and Jukes-Cantor model. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test are shown next to the branches. Branches reproduced in less than 70% bootstrap replicates are collapsed.
Citation: Beneficial Microbes 16, 1 (2025) ; 10.1163/18762891-bja00042
16S rRNA gene Sanger sequencing of unidentified isolates reveals potential novel genera and species
Based on phyloproteomic clustering of the non-identified isolates with the Bruker database, we selected 73 representative isolates to further identify them using 16S rRNA Sanger sequencing; still, 30 of these isolates remained unidentified to the valid genus or species level. Therefore, the consensus sequences of these 30 isolates were classified using four different reference databases, and their closest cultured relatives and corresponding genus members were selected and used for clustering in a phylogenetic tree based on the maximum likelihood method to represent their evolutionary history (Figure 5). During the clustering analyses of the consensus sequences, three isolates were excluded from further clustering because of their inability to align with the rest. Ten of the sequenced isolates could only be identified at the genus level, and 17 isolates were identified only at the family level (Figure 5; orange and purple). This suggests that the isolated bacteria could belong to new species and genera, but more detailed genomic and biochemical analysis is needed. Phylogenetic clustering indicated that all of these possible novel isolates belonged to Firmicutes with nine Oscillospiraceae species, 17 Lachnospiraceae species, and one Coprobacillaceae (Figure 5).
The presence and relative abundances of the 27 isolates in the volunteers were determined based on faecal 16S rRNA gene profiling (Supplementary Table S3). Sixteen amplicon sequence variants (ASVs) with 100% identity at the genus level with these isolates could be retrieved from at least one volunteer up to nine different volunteers, with an average relative abundance between 0.12% (HTF-303) and 0.46% (HTF-439a). Furthermore, 51 ASVs matched with 23 different isolates at the genus level when identification was less stringent (99%). Here, the ASVs could be retrieved in one up to all volunteers with 0.12% (HTF-707) up to 0.52% (HTF-360) average relative abundance. Interestingly, the isolates HTF-235, HTF-269, HTF-840, and HTF-454a were not retrieved by faecal 16S rRNA gene profiling.
4 Discussion
Here, we provide a convenient culturomics platform suitable for the isolation of a broad spectrum of bacterial species from the gut. Importantly, our approach was successful in the isolation of the under-represented Lachnospiraceae and Oscillospiraceae families within the Firmicutes phylum, both of which have been recurrently associated with human health (Gacesa et al., 2022; Gomaa, 2020; Round and Mazmanian, 2009; Tlaskalová-Hogenová et al., 2011). This was achieved using a strategy in which a basal culture medium was combined with a limited number of carbohydrates and high-throughput identification with MALDI-TOF MS.
To facilitate the use of fast and reliable bacterial identification by the scientific community, we presented here our MALDI-TOF MS reference database called ‘MaldiGut’ that focuses on members of the gut microbiota, especially within the Firmicutes phylum. This reference database was created out of necessity, because non-clinical bacteria, especially intestinal Lachnospiraceae and Oscillospiraceae species, are either absent or underrepresented in most reference spectra databases (Asare et al., 2023). The identification of bacteria using MALDI-TOF MS relies heavily on the availability of these reference spectra. The urgency for the culturomics field to fill these gaps in the databases and thereby improve the identification of these bacteria is also highlighted by the fact that recent efforts have already been made to expand the reference spectra for bacteria within this phylum (Asare et al., 2023). The name MaldiGut was proposed, since the overall goal of our database originally was to increase the identification rate of gut bacteria. Moreover, all reference isolates in the database originate from the human gut. The new MaldiGut database, containing 125 MSPs of Firmicutes bacteria, has remarkably increased the identification rate of faecal isolates in this culturomics pipeline. Indeed, the improved reference database allowed rapid and reliable identification of an additional 348 isolates out of the total number of 724 isolates, representing a 2.4-fold increase in classification capacity compared to the use of the manufacturer database alone. Therefore, this database will aid future culturomics studies by enabling more accurate and extensive identification of isolates. This will allow the scientific community and commercial enterprises to feasibly identify cultured Firmicutes species.
In contrast to this study, previous culturomics protocols have used a considerable number of culture conditions (C.-J. Chang et al., 2019; Lagier et al., 2016; Li et al., 2022; Poyet et al., 2019) or were less effective at isolating Lachnospiraceae and Oscillospiraceae. (Y. Chang et al., 2019; Groussin et al., 2021; Lagier et al., 2016; Mukhopadhya et al., 2022). An important difference between our studies and previous studies is that we employed direct faecal inoculations, without prior serial dilutions, under anaerobic conditions within just 10 minutes after defaecation to ensure maximum bacterial viability of anaerobic species. In addition, we reasoned that the omission of sample dilutions during the first inoculation round keeps the bacterial community as intact as possible. This allows interactions among the bacterial community, normally present in vivo, to take place during the first incubation, as was shown to be effective before between Faecalibacterium and Desulfovibrio (Khan et al., 2023). Thus, metabolites in the sample, both human and bacterial, and specific bacterial interactions, such as quorum sensing, may enable adaptation to our conditions and subsequent growth of species that were previously labelled as hard to culture (Almeida et al., 2019). Other studies have applied methods to improve cultured diversity, for instance by using liquid precultures. Still, these precultures were serial diluted for downstream isolation (Naud et al., 2020). The culture conditions in our study were limited by solely exchanging the carbohydrates in the YCFA culture medium. Here, three different carbohydrates were investigated. Glucose was used, because it is a common sugar that is widely used by bacteria and is therefore a suitable carbohydrate to capture a high cultured diversity for comparison with other carbohydrates. The other two carbohydrates, apple pectin and mucin, are degraded by certain bacteria in the gut and their enzymatic capability is distributed among the most dominant phyla, but mostly between Bacteroidetes and Firmicutes (Chung et al., 2016; Crost et al., 2016; Martens et al., 2008; Png et al., 2010; Raba et al., 2021; Wexler and Goodman, 2017; Zhernakova et al., 2024). However, the media in our study were slightly acidic, which seems to favour the degradation of these carbohydrates by species within the Firmicutes and seems to inhibit growth of certain Bacteroidetes species, especially species within Bacteroides, which are well-known mucin and pectin degraders (Chung et al., 2016; Martens et al., 2008; Raba et al., 2021; Wexler and Goodman, 2017). Interestingly, mostly species belonging to the families Lachnospiraceae and Oscillospiraceae are described by these studies to degrade these carbohydrates and are especially favoured by the slightly acidic conditions that were used in our media (Chung et al., 2016; Lopez-Siles et al., 2012; Png et al., 2010; Raba et al., 2021; Zhernakova et al., 2024), which is indeed in line with the findings in this study. Compared to previous culturomics attempts that have reached yields of Firmicutes between 23% and 67%, the current approach yielded a higher percentage of this phylum (69%) to the highest described yield in these studies (Groussin et al., 2021; Lagier et al., 2016; Li et al., 2022; Mukhopadhya et al., 2022). Moreover, when the number of culture conditions was considered, the present approach with only six different culture conditions performed notably better compared to other protocols that required much more effort as a result of using a greater number of culture conditions (Groussin et al., 2021; Lagier et al., 2016; Li et al., 2022; Mukhopadhya et al., 2022). The aforementioned culture approaches included 16 to 70 different culture conditions and analysed between 525 and more than 900,000 isolates to reach this yield of cultured bacteria within the Firmicutes. One of the culture conditions used here was the addition of sulfamethoxazole to the culture medium. Although this addition resulted in specific genera that were not isolated without this antibiotic, at the phylum level, this did not make a significant difference. Therefore, the culture approach could be limited to three different conditions if the focus is on the total yield of anaerobic intestinal Firmicutes.
The inclusion of an increased number of volunteers could also boost the diversity of isolated species and may result in an even higher yield of Lachnospiraceae and Oscillospiraceae in addition to other families or phyla. Indeed, this trend has already been observed in other culturomics and is already a well-established phenomenon in other fields, such as genomics (Li et al., 2022; Mukhopadhya et al., 2022; Saary et al., 2017) and environmental ecology (Chao and Shen, 2004). The inclusion of more samples will be more feasible with a convenient culturomics protocol. This study is a step forward to achieve such protocols and therefore will aid in the cultivation of more cultured representatives, especially within the Lachnospiraceae and Oscillospiraceae families.
Another approach to making culturomics more convenient is the implementation of robotics in combination with artificial intelligence. In this way, inoculation, colony picking, and streaking are automated and require little intervention from humans. Recent efforts were made by Huang et al. to create such a high-throughput platform, where they combined robotic strain isolation with machine learning to isolate bacteria (Huang et al., 2023). Although this is an exciting step forward in the field, the implementation of such techniques is still very technical and relatively expensive compared to the protocol proposed here. Although automation allows for higher numbers of isolates with less effort, optimisation of these kinds of protocols, such as omission of sample dilutions, could lead to higher diversity of species and isolation of gut anaerobes that are difficult to culture, especially within the Lachnospiraceae and Oscillospiraceae. Furthermore, the implementation of MALDI-TOF MS with a dedicated database, such as that presented in this study, instead of sequencing, will reduce turnover time and costs.
A natural progression of this research is to test other single or combinations of carbon sources or other medium components, such as different electron acceptors to suit different redox potentials, to generate selective pressure towards the growth of Lachnospiraceae and Oscillospiraceae (Sikora et al., 2017). More importantly, continuing efforts to expand the number of reference spectra within MALDI-TOF MS databases will enable the accurate identification of even more isolates and make identifications more reliable in the future. Besides culturing samples from healthy volunteers, incorporating culture-based studies of patient samples may provide increased insights into strain-specific features of the gut microbiome associated with disease. Approaches such as those adopted in our study will enable expansion of the human gut culturome and ensure an enhanced array of cultured gut bacterial representatives for further research.
Corresponding author; e-mail: h.j.m.harmsen@umcg.nl
Supplementary material
Supplementary material is available online at: https://doi.org/10.6084/m9.figshare.27087943
Acknowledgements
N.P. was supported by a grant from the Graduate School of Medical Sciences of the University of Groningen, the Netherlands. L.L. was supported by a joint fellowship from the University Medical Center Groningen and the China Scholarship Council (CSC) (grant number CSC201908320432). A.W.W. and the Rowett Institute receive core funding support from the Scottish Government’s Rural and Environmental Science and Analytical Services (RESAS) division. The funders played no role in study design, data collection, analysis and interpretation of data, or the writing of this manuscript.
Authors’ contribution
N.P. analysed and interpreted all the data, created MSPs, constructed the ‘MaldiGut’ database and was the major contributor in writing the manuscript. L.L. analysed the 16S rRNA gene profiling data and visualisation. E.T. performed the bacterial isolation. A.C.M.V helped in creating MSPs. All authors read and approved the final manuscript.
Conflict of interest
E.T. and P.O.S were financially supported by a research grant from Chr Hansen A/S, Copenhagen, Denmark, obtained by H.M.J.H and A.W.W. All other authors report that there are no competing interests to declare.
Data availability
The 16S rRNA amplicon sequence data were submitted to the NCBI Sequence Read Archive (SRA) under SRA numbers SRR26203441 to SRR26203451. The 16S rRNA gene Sanger sequencing data is submitted under the NCBI GenBank numbers OR610160 to OR610186. The MALDI-TOF MS database MALDIGUT can be downloaded from https://github.com/NPlmp/MaldiGut.
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