Advances in the isolation, cultivation, and identification of gut microbes

Xu, Meng-Qi; Pan, Fei; Peng, Li-Hua; Yang, Yun-Sheng

doi:10.1186/s40779-024-00534-7

Advances in the isolation, cultivation, and identification of gut microbes

Review
Open access
Published: 03 June 2024

Volume 11, article number 34, (2024)
Cite this article

Download PDF

You have full access to this open access article

Military Medical Research Aims and scope Submit manuscript

Advances in the isolation, cultivation, and identification of gut microbes

Download PDF

Meng-Qi Xu^1,2,
Fei Pan¹,
Li-Hua Peng¹ &
…
Yun-Sheng Yang¹

772 Accesses
2 Altmetric
Explore all metrics

Abstract

The gut microbiome is closely associated with human health and the development of diseases. Isolating, characterizing, and identifying gut microbes are crucial for research on the gut microbiome and essential for advancing our understanding and utilization of it. Although culture-independent approaches have been developed, a pure culture is required for in-depth analysis of disease mechanisms and the development of biotherapy strategies. Currently, microbiome research faces the challenge of expanding the existing database of culturable gut microbiota and rapidly isolating target microorganisms. This review examines the advancements in gut microbe isolation and cultivation techniques, such as culturomics, droplet microfluidics, phenotypic and genomics selection, and membrane diffusion. Furthermore, we evaluate the progress made in technology for identifying gut microbes considering both non-targeted and targeted strategies. The focus of future research in gut microbial culturomics is expected to be on high-throughput, automation, and integration. Advancements in this field may facilitate strain-level investigation into the mechanisms underlying diseases related to gut microbiota.

Gut microbiota functions: metabolism of nutrients and other food components

Article Open access 09 April 2017

An insight into gut microbiota and its functionalities

Article 13 October 2018

E. coli as an All-Rounder: The Thin Line Between Commensalism and Pathogenicity

Background

The human gut harbors approximately 4 × 10¹³ microbes, a number comparable to the total count of human cells [1]. Recent advancements in multi-omics technologies and cost reductions in testing have significantly contributed to our understanding of the composition and function of the gut microbiome. More evidence indicates a strong correlation between the gut microbiome and human health, with conditions such as inflammatory bowel disease, colorectal cancer (CRC), metabolic syndrome, and autism spectrum disorder intricately linked to the gut health status [2,3,4,5,6,7,Full size image

Gut microbial isolation and cultivation

Traditional methods

The conventional method for isolating and culturing microbes primarily relies on plate-based techniques such as spread-plating and streak-plating procedures. Under appropriate conditions, colonies can develop on solid media and are visible to the naked eye without magnification. In practice, direct microscopic count yields a higher number of microbes in natural samples compared to plate count, which is known as the great plate count anomaly phenomenon [54]. This difference may be attributed to the fact that the media does not accurately represent the real natural environment, resulting in most microorganisms being unable to thrive. Other factors include symbiosis between cells or viable but non-colony-forming cells.

As some microbes cannot form colonies, limiting dilution in liquid media serves as another effective method for isolating individual microbial cells. Furthermore, liquid media offer a simpler experimental process and higher throughput. Goodman et al. [29] initially designed the Gut Microbiota Medium and then performed limiting dilution in 384-well microplates to achieve single-cell isolation followed by liquid anaerobic cultures. From fecal samples from a single donor, they obtained a total of 1172 isolates belonging to 48 identified species and other previously cultured species.

It is worth noting that most gut microbiota are anaerobic organisms requiring complex media with various supplements and specialized equipment to provide an anaerobic environment. The Hungate anaerobic roll-tube technique is a traditional approach for cultivating anaerobic microbes [55]. Additionally, chemically generated anaerobic systems such as AnaeroPack [56] and BBL GasPak [57] are commonly used, particularly in smaller labs. The commercialization of anaerobic chambers has also led to the development of customized systems that integrate automated colony operation workstations, albeit at a significant cost [58, 59]. These systems are complex to operate and require professional training, undoubtedly increasing the difficulty involved in culturing gut microbiota.

The renaissance of culturomics

Culturomics employs a variety of culture conditions to facilitate the growth of fastidious bacteria, specifically utilizing high-throughput culture approaches combined with advanced techniques such as 16S rRNA gene sequencing and mass-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for bacterial identification. In 2012, Lagier et al. [13] conducted a groundbreaking study on the culturomics of gut microbes. They collected fecal samples from 3 volunteers and employed 212 different culture conditions, resulting in over 30,000 isolates from 314 species, half of which were newly discovered in the human gut. Based on their findings, 70 effective culture conditions were identified, with 18 being optimal for the isolation and cultivation of microbes from fecal samples. Additionally, blood culture bottles, rumen fluid, and sheep blood were confirmed as critical nutrient substrates for microbial growth. Subsequently, Lagier et al. [14] conducted large-scale culture studies that expanded the number of known human gut microbiota to include 1525 species. Diakite et al. [30] further investigated the optimization and standardization of the culturomics workflow by confirming 16 specific culture conditions that covered 98% of isolates from previous work while simplifying the process without compromising microbial diversity. Chang et al. [31] discovered that extending pre-incubation periods, introducing fresh medium into blood culture bottles, and determining appropriate sampling frequency effectively reduced labor consumption while enhancing culture yield.

Overall, culturomics emphasizes the continuous refinement and enhancement of culture media and conditions to optimize species diversity through streamlined procedures [32, 33]. One advantage of using solid media is its ability to facilitate visual identification and selection of microbiota based on their observable morphologies. However, this method remains associated with fundamental and repetitive labor-intensive processes, including plate pouring, spread plating, and colony picking. Despite the development and commercialization of various automated equipment such as automatic media streakers and automatic microbiota selectors, their high costs and compatibility challenges with anaerobic platforms have limited their application in gut microbiota research. Therefore, there is a need for the development of novel technical solutions.

Droplet microfluidics

The utilization of droplet-based microfluidic systems enables precise manipulation of discrete fluid volumes containing immiscible phases, thereby facilitating accurate control over flow patterns and lengths for the production of water-in-oil or oil-in-water microdroplets [60]. Due to their dispersion, each microdroplet can establish an enclosed microreaction environment. The introduction of microscale droplet generation has revolutionized microbial isolation and cultivation by offering high-throughput capabilities, automation, single-cell analysis, and miniaturization features inherent in droplet microfluidics.

SlipChip

Du et al. [34] developed a SlipChip device for the generation of nanolitre-scale droplets, which were subsequently manipulated through sliding, one for microbial cultivation and the other for destructive testing. When combined with polymerase chain reaction (PCR), this approach enables the targeted isolation and cultivation of Bacteroides vulgatus, a common gut resident [35, 36]. SlipChip represents a significant endeavor in applying droplet microfluidics to gut microbes, facilitating automated as well as high-throughput isolation and cultivation of gut microbes. However, the sliding operation limits the throughput of droplet generation, manipulating only hundreds of microbial cells in a single assay. Although there has been an improvement in throughput compared to traditional methods, meeting the requirements for isolating and culturing rare species from thousands of gut microbes and conducting large-scale intervention experiments remains unmet.

End-to-end droplet microfluidic system

Watterson et al. [37] designed an end-to-end droplet microfluidic system integrated with an anaerobic incubator, enabling the simultaneous production of millions of picolitre single-cell droplets. Due to Poisson statistics commonly used in passive cell capture processes [61], only a fraction of the resulting droplets can effectively contain a single cell when there are background populations of empty droplets. Watterson et al. [37] also devised an image-based sorting algorithm and microfluidic control system for sorting bacterial colonies in droplets based on colony density, eliminating the need for fluorescent strains or reporters. Traditional plate-based methods favor the cultivation of fast-growing microbiota while posing limitations for slow-growing ones. Furthermore, some microbiota may be inhibited by competing interactions within their environment. The physical separation provided by droplet microfluidics effectively addresses these issues, leading to a significant increase in microbiota diversity and low-abundance microbial species. More importantly, isolation, cultivation, and sorting are tightly integrated into a standard anaerobic glove box. However, manipulation of picolitre-sized droplets poses challenges as the sorted droplets are pooled and gathered collectively rather than individually. Hence, further separation, purification, and scale-up remain necessary for subsequent studies.

Microfluidic streak plate

In a separate study, Jiang et al. [38] presented a straightforward method to directly index each droplet and achieve the addressability necessary for precise manipulation of droplets without subsequent re-separation. They introduced the microfluidic streak plate, which is based on the conventional streak plate technique. This device can generate nanolitre droplets initially and then arrange them in spiral arrays in Petri dishes pre-filled with carrier oil for individual cell cultivation purposes. Targeted recovery was achieved by imaging and aligning with the assistance of a stereoscope, followed by manual selection using sterile toothpicks. Furthermore, integrating the microfluidic streak plate platform with an anaerobic incubator to termite gut microbe culture has led to the identification of a potential new taxon [62].

Single-cell microlitre-droplet screening system

The droplets in the aforementioned studies range from picolitre to nanolitre in size, necessitating the use of a microscope for their manipulation [34,35,36,37,38, 62]. Some of these droplets require re-separated after enrichment or manual sliding or picking [34, 38]. Jian et al. [39] introduced the single-cell microlitre-droplet screening system (MISS Cell), which is capable of generating 2.0 to 2.5 µl droplets with a throughput of 10⁴ cells. MISS Cell consists of 4 interconnected modules: the sampling module, microfluidic chip module, droplet storage and culture module, and droplet detection and collection module. The system employs spectral detection and specific algorithms for accurate identification and automated sorting of droplets. It also incorporates a unique mechanical structure that enables the automatic collection of individual droplets by docking with a standardized container through droplet printing. Compared to picolitre droplets, MISS Cell utilizes a simplified optical signal detection system that accurately identifies and precisely addresses the droplets, significantly enhancing the colony picking efficiency of high-throughput applications.

The features of the current typical high-throughput droplet microfluidic culture system for gut microbes are summarised in Table 1. Droplet microfluidics is characterized by its small size (ranging from picolitres to microlitres), independent nature of the droplets, high culture throughput, low reagent consumption, and compartmentalization cultivation. This makes it a significant advancement in gut culturomics studies. It enables the discovery of rare bacteria and the reconstruction of intestinal microbial diversity due to its exceptional cultural performance. Additionally, the compatibility of droplet microfluidic equipment with anaerobic devices greatly expands its application in the field of intestinal microbiology. However, there are certain limitations associated with this technology. According to the principles of Poisson distribution, the generation of single-cell encapsulated droplets is expected to result in a significant proportion of empty droplets [61]. The fundamental challenge lies in identifying and categorizing ‘microbe growing’ droplets and empty ones. Additionally, current methods for sorting droplets lead to their aggregation; thus, making it difficult to establish a one-to-one index for subsequent analysis. This poses a challenge in achieving an optimal balance between droplet throughput and addressability. Moreover, the process of isolation impedes the proliferation of organisms that rely on other microbial or host cells [63]. Hence, the co-encapsulation of two cross-feeding auxotrophic strains within a singular droplet may prove to be a viable and efficient strategy, which can also be utilized for studying the interactions between microbiotas [64].

Table 1 Characteristics of the current typical high-throughput droplet microfluidic culture system for gut microbes

Full size table

In recent years, droplet-based microfluidic technology for single-cell isolation and sequencing has gained significant traction, enabling the cultivation of bacteria and even genome amplification within individual encapsulated cells [65]. Meng et al. [66] have employed the DREM cell platform to investigate microorganisms in the honeybee gut, facilitating strain-level analysis as well as the identification of potential novel species and specific functional strains. Additionally, the Microbe-seq technique developed by Zheng et al. [67] has been applied to analyze human fecal samples, providing species information at a strain-level resolution and facilitating in-depth functional research. Notably, this review article primarily focuses on the indexing, acquisition, and amplification of individual droplets for further causal research. These two research methods are essentially complementary.

Selection for phenotypes or genomes

Current high-throughput isolation and cultivation methods aim to enhance our understanding of the microbial ‘dark matter’. Furthermore, selectively isolating organisms with specific functional characteristics or those belonging to particular taxonomic groups is another strategy for delving deeper into gut microbiome research.

One effective method involves the use of selective media, such as the Bacteroides fragilis (B. fragilis) bile-esculin agar, which has been specifically formulated to target the B. fragilis group [68]. Oberhardt et al. [69] constructed a web-based platform that predicts suitable media based on 16S rRNA gene sequences, thereby facilitating future cultivation efforts. By employing alcohol pre-treatment on stool samples, Browne et al. [15] targeted the sporulation phenotype, and successfully isolated spore-forming bacteria, leading to the discovery of 45 potential novel species.

The technique of fluorescence-activated cell sorting (FACS) separates a heterogeneous population of cells into multiple containers based on their distinct light scattering and fluorescent properties [70]. Various strategies employing fluorescent labeling have been developed to utilize FACS for the categorization of gut microbiota with specific phenotypes or genomes. Cross et al. [40] employed single-cell genomic data to identify genes encoding membrane-associated proteins, which were then used to generate engineered antibodies. Human oral samples were incubated with these fluorescently labeled antibodies and subsequently analyzed using flow cytometry. This reverse genomics approach facilitated the isolation and cultivation of three distinct species-level lineages of human oral Saccharibacteria (TM7) and SR1 bacteria, the latter belonging to a candidate phylum without any previously cultured representatives. Moreover, Batani et al. [41] optimized the conditions affecting cell viability during fluorescence in situ hybridization (FISH), combining it with FACS to selectively isolate and cultivate specific bacterial taxa based solely on their 16S rRNA gene sequences. Although these innovative methods can target specific phenotypic or genomic traits, the sample preparation process may result in reduced cell viability, while the equipment does not provide an anaerobic environment, posing challenges in recovering obligate anaerobes. Moreover, further isolation and cultivation are still required for the sorted bacteria.

Membrane diffusion-based cultivation

In situ cultivation

Kaeberlein et al. [42] developed a diffusion chamber with 0.03 µm filter membranes on both sides, in which they placed environmental samples inoculated on an agar matrix. The equipment was placed in a natural setting to restrict microbial mobility, facilitating the natural exchange of environmental substances and eliminating the need for sophisticated medium design, thus enabling in situ microbe culture. Similarly, Gavrish et al. [43] developed a microbial trap where the agar matrix within the chamber lacked microbial inoculation. A 0.03 µm filter membrane was coated on the top of the chamber to prevent air pollutants, whereas a 0.2 mm filter membrane was placed at the bottom to allow entry of environmentally specific microbes and facilitate the natural interchange of environmental substances. Building upon this concept, Sizova et al. [44] designed a minitrap consisting of multiple micro-chambers made from a human oral prosthesis that can be worn by patients for 48 h to enable inoculation with oral anaerobic/aerobic microbes. This device revolutionized in situ isolation and cultivation of human oral microbes and offers significant advantages over conventional solid culture media in terms of microbiota quantity and species diversity.

The main concern for achieving isolation and cultivation of microorganisms lies in addressing their nutritional needs and providing optimal conditions. In the case of gut microbiota, it is both intuitive and effective to supplement with rumen fluid or sterilized fecal extract [71] and create an anaerobic environment to restore the intestinal state of the gut as closely as possible. In situ cultivation is a method that effectively mimics the natural growth conditions of microbes, thus eliminating the need for complex media design. However, its applications in studying gut microbiota are constrained by challenges such as device design and pick-and-place methodologies, highlighting the necessity for further technological advancements.

Co-culture technique

The growth of specific gut microbes depends on the metabolic support from other bacterial species. Tanaka et al. [45] designed a co-culture system using a soft agar layer composed of a basal 1.5% agar medium and two layers of 0.4% soft agar medium separated by a 0.22 µm filter membrane. This system successfully isolated and cultured 3 previously uncultured strains from fecal samples by enabling communication between microbes via the diffusion of soluble substances. Soft agar media allows for more rapid molecule diffusion compared to conventional 1.5% agar media, thereby promoting optimal growth conditions.

Gut microbial identification

The development of culture techniques is inherently intertwined with the effectiveness of identification techniques. Currently, there are two main strategies employed for gut microbial isolation and cultivation: high-throughput methods aimed at reducing the number of uncultured microbes and restoring diversity, and targeted isolation approaches designed to facilitate in-depth research on specific microbes. Non-targeted and targeted identification strategies and technologies exhibit differences.

Non-targeted identification

Classical phenoty**

The traditional method of microbial identification relies on phenoty**, which involves assessing various characteristics such as size, morphology, enzyme metabolism, carbon source utilization, organic metabolites, cellular fatty acid components, and other physiological and biochemical traits. Several commercial kits or automated systems are available for microbial phenoty**, with testing times ranging from 4 to 72 h [46]. However, classical phenoty** is not only labor-intensive but also lacks universal applicability.

16S rRNA gene

Nucleic acid sequencing of the bacterial 16S rRNA gene has been extensively used for decades to identify clinical and environmental isolates and establish phylogenetic relationships. Due to its stability throughout biological evolution, the 16S rRNA, which is commonly found in prokaryotes and exhibiting is often considered a reliable indicator of biological evolution. The 16S rRNA gene contains sequences that span highly conserved, variable, and hypervariable regions, making it well-suited for examining genetic relationships among organisms with varying evolutionary distances [47].

Initially, pure culture identification relied on PCR amplification of the full-length 16S rRNA gene followed by Sanger sequencing, whereas next-generation sequencing has predominantly been used to analyze complex microbial communities. Characterizing numerous isolates from culturomics represents a significant and costly endeavor. Zhang et al. [48] reported a high-throughput identification method based on next-generation sequencing. They introduced two-sided barcodes during library preparation targeting bacterial 16S rRNA genes to label the plates and wells containing pure DNA templates of clonal cultures in 96-well plates. These samples were subsequently pooled for Illumina sequencing. Bioinformatic analyses were performed to identify cultures and ensure the traceability of each well according to the barcodes. This method enables simultaneous identification of microbes in 48 × 96-well plates, leading to substantial cost reduction and improved throughput.

MALDI-TOF MS

MALDI-TOF MS has been widely utilized as a valuable analytical chemistry tool for detecting large and small molecules. Employing specific algorithms to compare protein spectra with reference databases, enables accurate identification of microbes. In 2009, the initial application of MALDI-TOF MS in a routine clinical microbiology laboratory successfully achieved precise identification at both genus and species levels [49]. The remarkable speed at which MALDI-TOF MS operates allows for microbial identification within minutes. Due to its rapidity, cost-effectiveness, high-throughput, and minimal training requirement, MALDI-TOF MS has emerged as a globally standardized mainstream technology [50]. This advancement in MALDI-TOF MS has sparked a renaissance in culturomics by transforming microbial isolation and cultivation into a high-throughput process.

However, the effectiveness of utilizing MALDI-TOF MS for microbial identification heavily relies on the quality and quantity of database spectra available. The success rate and accuracy of identification are directly influenced by these factors. Currently, accessible public databases are limited and expensive, highlighting an urgent need for a comprehensive, high-quality mass spectrometry library specifically tailored towards gut anaerobic microbes.

Targeted identification

Nucleic acid amplification

PCR is a widely used method for targeted identification of microbial nucleic acids, in which specific primers are designed for the target sequence and qualitative detection is achieved through exponential amplification via thermal cycling [72]. However, the integration of other equipment to achieve online monitoring becomes challenging due to the requirement of a precise temperature control module in the PCR thermal cycle reaction.

Isothermal amplification of nucleic acids is a straightforward process that rapidly and efficiently accumulates nucleic acid sequences at a constant temperature [73]. For instance, loop-mediated isothermal amplification (LAMP), an extensively studied isothermal technique, depends on primer sets with 4 to 6 specific primers for the identification of various regions in the target sequence. LAMP also provides uncomplicated qualitative detection through turbidity, colorimetry, and fluorescence [74]. Currently, commercially available Mycobacterium tuberculosis LAMP detection kits are widely used in diverse clinical diagnoses and treatments [75]. Due to its simple reaction conditions, LAMP can be well integrated with droplet microfluidic equipment and offers a wide range of applications in the identification and detection of microorganisms [51, 52].

Nucleic acid hybridization

Nucleic acid hybridization is a valuable tool for the identification of target genes or organisms. In FISH, fluorescently labeled oligonucleotides (typically 15 – 20 base pairs long), are specifically designed to bind to rRNAs within intact cells, thereby resulting in cellular fluorescence. Numerous adaptations of the technique have been documented, such as fixation-free FISH [76], in-solution FISH [77], and live FISH [41]. These methods can also be integrated with FACS for sorting labeled cells. However, one limitation of FISH is the need to remove unreacted probes through additional operational procedures, which restricts its potential for integrated applications.

Molecular beacons (MBs) are innovative nucleic acid probes consisting of 3 components: a loop region, a stem region, and a fluorophore/quencher. In their unbound state, MB adopts a hairpin conformation with the fluorophore and quencher nearby, resulting in fluorescence quenching. Upon binding to the complementary sequence, the conformation of MB changes, separating the fluorophore and quencher, leading to fluorescence emission that indicates the presence of the target sequence. Similarly, researchers have optimized the MBs and developed double-stranded molecular probes which are inherently compatible with liquid-phase hybridization detection without requiring probe elution. This considerably streamlines the experimental process [78]. Kaushik et al. [53] developed a DropDx platform by employing droplet microfluidics combined with fluorogenic hybridization probes for pathogen identification and antimicrobial susceptibility testing. Bacteria in urine samples are encapsulated into picolitre-sized droplets along with 16S rRNA‐specific peptide nucleic acid probes before undergoing on‐chip culture for 10 min followed by probe hybridization for 16 min. The fluorescence emitted by these droplets is then used to detect specific 16S rRNA sequences for identifying uropathogenic bacteria. Additionally, an antibiotic is introduced to perform antimicrobial susceptibility testing. The microfluidic platform integrates three modules: single-cell pathogen capture, sample preparation-free molecular probe-based detection, and nucleic acid amplification, as well as high-throughput quantitative determination through fluorescence analysis, thus enabling rapid, automated, and high-throughput clinical sample testing.

Application and future of gut culturomics

Culturomics and metagenomics are complementary to each other

The annotation of species in high-throughput sequencing necessitates the utilization of known species data through blasting [11]. The isolation, cultivation, and identification of gut microbes can expand the database of culturable gut microbes and promote genomics research. Li et al. [79] employed a combination of in-depth metagenomic sequencing and large-scale culturomics to reveal the distinctive structure of gut microbial communities within a Chinese longevity population. Only 42.17% of the isolated species were also detected using metagenomics, indicating clear complementarity between these two approaches. Certain microorganisms, despite their abundance in metagenomic analysis, may exist in a dormant state with minimal gene expression [80]. Even with ultra-sequencing depth, detecting extremely low abundance microorganisms would be challenging and result in unaffordable costs [79, 81]. We believe that integrating metagenomics and culturomics will provide a more coherent view of microbiome structure and function by encompassing the whole ecosystem structure as well as low-abundance communities.

Causal mechanism in the gut microbiome

Among human diseases associated with microbes, phenotypes are often linked to only a subset of strains within microbial clades [82]. Access to pure cultures is essential for validating the hypotheses derived from sequencing data. Investigating the causal relationship between the gut microbiome and disease phenotype using in vitro and in vivo models such as germ-free mice or organoids is important for researching, screening, and evaluating bacterial function. The relationship between Fusobacterium nucleatum and CRC serves as a notable paradigm [4, 83]. Genomic analysis detected the enrichment of F. nucleatum, a common oral anaerobic bacterium, in CRC tumor tissues before isolation for investigating the physiological characteristics and virulence factors [84, 85]. Several hypotheses were proposed and validation experiments followed suit, mounting evidence now shows that tumor-enriched F. nucleatum plays a role in multiple stages of CRC progression [85,86,87,88]. Clinical applications are currently underway with F. nucleatum potentially serving as a diagnostic biomarker, prognostic predictor, and therapeutic target in CRC [83].

Besides, the understanding of the relationship between Helicobacter pylori and gastric cancer remains incomplete. Why do only a small percentage of Helicobacter pylori-infected individuals develop gastric cancer? Do variations in pathogenicity, virulence, drug resistance, or genetic diversity among Helicobacter pylori strains play significant roles in gastric cancer progression? Could other gut microbiota also contribute? These questions are expected to be answered at the strain level through culturomics research. In some studies, metagenomics analysis may yield inconsistent results. Is it due to differences in populations and backgrounds, or the potential confusion caused by host-specific strains? Pure culture research may help clarify this uncertainty. Swift isolation of the target microorganisms could facilitate mechanistic studies in gut microbiome research.

Biotherapy of the gut microbiome

The clinical application of fecal microbiota transplantation (FMT) has been approved for treating recurrent Clostridioides difficile infections (CDI) and severe and fulminant CDI, as stated in guidelines [89]. Currently, FMT is being investigated as a potential treatment for a variety of diseases. Nevertheless, concerns regarding pathogen transmission and the challenge of fully characterizing the composition of fecal samples have restricted its broader application [90]. The exact components responsible for its efficacy or potential negative effects remain unclear, which highlights the importance of obtaining pure cultures from complex communities and fully understanding their interaction mechanism. Ideally, disease- and recipient-specific artificially designed flora should be developed to carry out standardized, and streamlined FMT procedures. The establishment of an extensive strain library through culturomics is the cornerstone of personalized medicine for the gut microbiome.

Probiotics could be a therapeutic approach to modulate the gut microbiota and enhance human health. Traditional probiotics have a well-documented history of use with a proven safety record. In contrast, next-generation probiotics (NGPs) are recently discovered strains whose safety remains unconfirmed. NGPs have been mostly identified through metagenomic analysis before isolation and even modification take place. Research focuses on NGPs such as Faecalibacterium prausnitzii, B. fragilis, and Akkermansia muciniphila [91]. An in-depth understanding of strain level is essential for probiotic research because certain Akkermansia muciniphila potentially promote colitis, while enterotoxigenic B. fragilis acts as a pathobiont [

Availability of data and materials

The data and materials used for this review are available within the manuscript.

Abbreviations

B. fragilis :: Bacteroides fragilis
CDI:: Clostridioides difficile infections
CRC:: Colorectal cancer
FACS:: Fluorescence-activated cell sorting
FISH:: Fluorescence in-situ hybridization
FMT:: Faecal microbiota transplantation
LAMP:: Loop-mediated isothermal amplification
MALDI-TOF MS:: Mass-assisted laser desorption/ionization time-of-flight mass spectrometry
MB:: Molecular beacon
MISS Cell:: Single-cell microlitre-droplet screening system
NGPs:: Next-generation probiotics
PCR:: Polymerase chain reaction

References

Sender R, Fuchs S, Milo R. Are we really vastly outnumbered? Revisiting the ratio of bacterial to host cells in humans. Cell. 2016;164(3):337–40.
Article CAS PubMed Google Scholar
Lee M, Chang EB. Inflammatory bowel diseases (IBD) and the microbiome-searching the crime scene for clues. Gastroenterology. 2021;160(2):524–37.
Article CAS PubMed Google Scholar
Shi N, Li N, Duan X, Niu H. Interaction between the gut microbiome and mucosal immune system. Mil Med Res. 2017;4:14.
PubMed PubMed Central Google Scholar
Wang Z, Dan W, Zhang N, Fang J, Yang Y. Colorectal cancer and gut microbiota studies in China. Gut Microbes. 2023;15(1):2236364.
Article PubMed PubMed Central Google Scholar
Wong CC, Yu J. Gut microbiota in colorectal cancer development and therapy. Nat Rev Clin Oncol. 2023;20(7):429–52.
Article PubMed Google Scholar
Michels N, Zouiouich S, Vanderbauwhede B, Vanacker J, Indave Ruiz BI, Huybrechts I. Human microbiome and metabolic health: an overview of systematic reviews. Obes Rev. 2022;23(4):e13409.
Article PubMed Google Scholar
Wan Y, Zuo T, Xu Z, Zhang F, Zhan H, Chan D, et al. Underdevelopment of the gut microbiota and bacteria species as non-invasive markers of prediction in children with autism spectrum disorder. Gut. 2022;71(5):910–8.
Article PubMed Google Scholar
** W, Gao X, Zhao H, Luo X, Li J, Tan X, et al. Depicting the composition of gut microbiota in children with tic disorders: an exploratory study. J Child Psychol Psychiatry. 2021;62(10):1246–54.
Article PubMed Google Scholar
Cui GY, Rao BC, Zeng ZH, Wang XM, Ren T, Wang HY, et al. Characterization of oral and gut microbiome and plasma metabolomics in COVID-19 patients after 1-year follow-up. Mil Med Res. 2022;9(1):32.
CAS PubMed PubMed Central Google Scholar
Li N, Wang L, Li L, Yang MZ, Wang QX, Bai XW, et al. The correlation between gut microbiome and atrial fibrillation: pathophysiology and therapeutic perspectives. Mil Med Res. 2023;10(1):51.
CAS PubMed PubMed Central Google Scholar
Almeida A, Nayfach S, Boland M, Strozzi F, Beracochea M, Shi ZJ, et al. A unified catalog of 204,938 reference genomes from the human gut microbiome. Nat Biotechnol. 2021;39(1):105–14.
Article CAS PubMed Google Scholar
Kim CY, Lee M, Yang S, Kim K, Yong D, Kim HR, et al. Human reference gut microbiome catalog including newly assembled genomes from under-represented Asian metagenomes. Genome Med. 2021;13(1):134.
Article CAS PubMed PubMed Central Google Scholar
Lagier JC, Armougom F, Million M, Hugon P, Pagnier I, Robert C, et al. Microbial culturomics: paradigm shift in the human gut microbiome study. Clin Microbiol Infect. 2012;18(12):1185–93.
Article CAS PubMed Google Scholar
Lagier JC, Khelaifia S, Alou MT, Ndongo S, Dione N, Hugon P, et al. Culture of previously uncultured members of the human gut microbiota by culturomics. Nat Microbiol. 2016;1:16203.
Article CAS PubMed Google Scholar
Browne HP, Forster SC, Anonye BO, Kumar N, Neville BA, Stares MD, et al. Culturing of ‘unculturable’ human microbiota reveals novel taxa and extensive sporulation. Nature. 2016;533(7604):543–6.
Article CAS PubMed PubMed Central Google Scholar
Forster SC, Kumar N, Anonye BO, Almeida A, Viciani E, Stares MD, et al. A human gut bacterial genome and culture collection for improved metagenomic analyses. Nat Biotechnol. 2019;37(2):186–92.
Article CAS PubMed PubMed Central Google Scholar
Poyet M, Groussin M, Gibbons SM, Avila-Pacheco J, Jiang X, Kearney SM, et al. A library of human gut bacterial isolates paired with longitudinal multiomics data enables mechanistic microbiome research. Nat Med. 2019;25(9):1442–52.
Article CAS PubMed Google Scholar
Zou Y, Xue W, Luo G, Deng Z, Qin P, Guo R, et al. 1,520 reference genomes from cultivated human gut bacteria enable functional microbiome analyses. Nat Biotechnol. 2019;37(2):179–85.
Article CAS PubMed PubMed Central Google Scholar
Lin X, Hu T, Chen J, Liang H, Zhou J, Wu Z, et al. The genomic landscape of reference genomes of cultivated human gut bacteria. Nat Commun. 2023;14(1):1663.
Article CAS PubMed PubMed Central Google Scholar
Liu C, Du MX, Abuduaini R, Yu HY, Li DH, Wang YJ, et al. Enlightening the taxonomy darkness of human gut microbiomes with a cultured biobank. Microbiome. 2021;9(1):119.
Article CAS PubMed PubMed Central Google Scholar
Zhang X, Li L, Butcher J, Stintzi A, Figeys D. Advancing functional and translational microbiome research using meta-omics approaches. Microbiome. 2019;7(1):154.
Article PubMed PubMed Central Google Scholar
Mills RH, Dulai PS, Vázquez-Baeza Y, Sauceda C, Daniel N, Gerner RR, et al. Multi-omics analyses of the ulcerative colitis gut microbiome link Bacteroides vulgatus proteases with disease severity. Nat Microbiol. 2022;7(2):262–76.
Article CAS PubMed PubMed Central Google Scholar
Li H, Liu C, Huang S, Wang X, Cao M, Gu T, et al. Multi-omics analyses demonstrate the modulating role of gut microbiota on the associations of unbalanced dietary intake with gastrointestinal symptoms in children with autism spectrum disorder. Gut Microbes. 2023;15(2):2281350.
Article PubMed PubMed Central Google Scholar
Wang WJ, Chu LX, He LY, Zhang MJ, Dang KT, Gao C, et al. Spatial transcriptomics: recent developments and insights in respiratory research. Mil Med Res. 2023;10(1):38.
PubMed PubMed Central Google Scholar
Nakayasu ES, Gritsenko MA, Kim YM, Kyle JE, Stratton KG, Nicora CD, et al. Elucidating regulatory processes of intense physical activity by multi-omics analysis. Mil Med Res. 2023;10(1):48.
CAS PubMed PubMed Central Google Scholar
Feng DC, Zhu WZ, Wang J, Li DX, Shi X, **ong Q, et al. The implications of single-cell RNA-seq analysis in prostate cancer: unraveling tumor heterogeneity, therapeutic implications and pathways towards personalized therapy. Mil Med Res. 2024;11(1):21.
PubMed PubMed Central Google Scholar
Marshall BJ, Warren JR. Unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration. Lancet. 1984;1(8390):1311–5.
Article CAS PubMed Google Scholar
Cheng AG, Ho PY, Aranda-Díaz A, Jain S, Yu FB, Meng X, et al. Design, construction, and in vivo augmentation of a complex gut microbiome. Cell. 2022;185(19):3617–36.e19.
Article CAS PubMed PubMed Central Google Scholar
Goodman AL, Kallstrom G, Faith JJ, Reyes A, Moore A, Dantas G, et al. Extensive personal human gut microbiota culture collections characterized and manipulated in gnotobiotic mice. Proc Natl Acad Sci U S A. 2011;108(15):6252–7.
Article CAS PubMed PubMed Central Google Scholar
Diakite A, Dubourg G, Dione N, Afouda P, Bellali S, Ngom II, et al. Optimization and standardization of the culturomics technique for human microbiome exploration. Sci Rep. 2020;10(1):9674.
Article CAS PubMed PubMed Central Google Scholar
Chang Y, Hou F, Pan Z, Huang Z, Han N, Bin L, et al. Optimization of culturomics strategy in human fecal samples. Front Microbiol. 2019;10:2891.
Article PubMed PubMed Central Google Scholar
Hirano R, Nishita I, Nakai R, Bito A, Sasabe R, Kurihara S. Development of culture methods capable of culturing a wide range of predominant species of intestinal bacteria. Front Cell Infect Microbiol. 2023;13:1056866.
Article CAS PubMed PubMed Central Google Scholar
Tao X, Huang W, Pan L, Sheng L, Qin Y, Chen L, et al. Optimizing ex vivo culture conditions to study human gut microbiome. ISME Commun. 2023;3(1):38.
Article PubMed PubMed Central Google Scholar
Du W, Li L, Nichols KP, Ismagilov RF. SlipChip. Lab Chip. 2009;9(16):2286–92.
Article CAS PubMed PubMed Central Google Scholar
Ma L, Datta SS, Karymov MA, Pan Q, Begolo S, Ismagilov RF. Individually addressable arrays of replica microbial cultures enabled by splitting SlipChips. Integr Biol (Camb). 2014;6(8):796–805.
Article CAS PubMed Google Scholar
Ma L, Kim J, Hatzenpichler R, Karymov MA, Hubert N, Hanan IM, et al. Gene-targeted microfluidic cultivation validated by isolation of a gut bacterium listed in Human Microbiome Project’s Most Wanted taxa. Proc Natl Acad Sci U S A. 2014;111(27):9768–73.
Article CAS PubMed PubMed Central Google Scholar
Watterson WJ, Tanyeri M, Watson AR, Cham CM, Shan Y, Chang EB, et al. Droplet-based high-throughput cultivation for accurate screening of antibiotic resistant gut microbes. Elife. 2020;9:e56998.
Article CAS PubMed PubMed Central Google Scholar
Jiang CY, Dong L, Zhao JK, Hu X, Shen C, Qiao Y, et al. High-throughput single-cell cultivation on microfluidic streak plates. Appl Environ Microbiol. 2016;82(7):2210–8.
Article CAS PubMed PubMed Central Google Scholar
Jian X, Guo X, Cai Z, Wei L, Wang L, **ng XH, et al. Single-cell microliter-droplet screening system (MISS Cell): an integrated platform for automated high-throughput microbial monoclonal cultivation and picking. Biotechnol Bioeng. 2023;120(3):778–92.
Article CAS PubMed Google Scholar
Cross KL, Campbell JH, Balachandran M, Campbell AG, Cooper CJ, Griffen A, et al. Targeted isolation and cultivation of uncultivated bacteria by reverse genomics. Nat Biotechnol. 2019;37(11):1314–21.
Article CAS PubMed PubMed Central Google Scholar
Batani G, Bayer K, Böge J, Hentschel U, Thomas T. Fluorescence in situ hybridization (FISH) and cell sorting of living bacteria. Sci Rep. 2019;9(1):18618.
Article CAS PubMed PubMed Central Google Scholar
Kaeberlein T, Lewis K, Epstein SS. Isolating “uncultivable” microorganisms in pure culture in a simulated natural environment. Science. 2002;296(5570):1127–9.
Article CAS PubMed Google Scholar
Gavrish E, Bollmann A, Epstein S, Lewis K. A trap for in situ cultivation of filamentous actinobacteria. J Microbiol Methods. 2008;72(3):257–62.
Article CAS PubMed PubMed Central Google Scholar
Sizova MV, Hohmann T, Hazen A, Paster BJ, Halem SR, Murphy CM, et al. New approaches for isolation of previously uncultivated oral bacteria. Appl Environ Microbiol. 2012;78(1):194–203.
Article CAS PubMed PubMed Central Google Scholar
Tanaka Y, Benno Y. Application of a single-colony coculture technique to the isolation of hitherto unculturable gut bacteria. Microbiol Immunol. 2015;59(2):63–70.
Article CAS PubMed Google Scholar
Blazevic DJ, MacKay DL, Warwood NM. Comparison of Micro-ID and API 20E systems for identification of Enterobacteriaceae. J Clin Microbiol. 1979;9(5):605–8.
Article CAS PubMed PubMed Central Google Scholar
Church DL, Cerutti L, Gürtler A, Griener T, Zelazny A, Emler S. Performance and application of 16S rRNA gene cycle sequencing for routine identification of bacteria in the clinical microbiology laboratory. Clin Microbiol Rev. 2020;33(4):e00053–e119.
Article CAS PubMed PubMed Central Google Scholar
Zhang J, Liu Y-X, Guo X, Qin Y, Garrido-Oter R, Schulze-Lefert P, et al. High-throughput cultivation and identification of bacteria from the plant root microbiota. Nat Protoc. 2021;16(2):988–1012.
Article CAS PubMed Google Scholar
Seng P, Drancourt M, Gouriet F, La Scola B, Fournier PE, Rolain JM, et al. Ongoing revolution in bacteriology: routine identification of bacteria by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Clin Infect Dis. 2009;49(4):543–51.
Article CAS PubMed Google Scholar
Elbehiry A, Aldubaib M, Abalkhail A, Marzouk E, Alqarni A, Moussa I, et al. How MALDI-TOF mass spectrometry technology contributes to microbial infection control in healthcare settings. Vaccines (Basel). 2022;10(11):1881.
Article CAS PubMed PubMed Central Google Scholar
Azizi M, Zaferani M, Cheong SH, Abbaspourrad A. Pathogenic bacteria detection using RNA-based loop-mediated isothermal-amplification-assisted nucleic acid amplification via droplet microfluidics. ACS Sens. 2019;4(4):841–8.
Article CAS PubMed Google Scholar
Wu H, Cao X, Meng Y, Richards D, Wu J, Ye Z, et al. DropCRISPR: a LAMP-Cas12a based digital method for ultrasensitive detection of nucleic acid. Biosens Bioelectron. 2022;211:114377.
Article CAS PubMed Google Scholar
Kaushik AM, Hsieh K, Mach KE, Lewis S, Puleo CM, Carroll KC, et al. Droplet-based single-cell measurements of 16S rRNA enable integrated bacteria identification and pheno-molecular antimicrobial susceptibility testing from clinical samples in 30 min. Adv Sci (Weinh). 2021;8(6):2003419.
Article CAS PubMed Google Scholar
Staley JT, Konopka A. Measurement of in situ activities of nonphotosynthetic microorganisms in aquatic and terrestrial habitats. Annu Rev Microbiol. 1985;39:321–46.
Article CAS PubMed Google Scholar
Holdeman LV, Moore WE. Roll-tube techniques for anaerobic bacteria. Am J Clin Nutr. 1972;25(12):1314–7.
Article CAS PubMed Google Scholar
Delaney ML, Onderdonk AB. Evaluation of the AnaeroPack system for growth of clinically significant anaerobes. J Clin Microbiol. 1997;35(3):558–62.
Article CAS PubMed PubMed Central Google Scholar
Collee JG, Watt B, Fowler EB, Brown R. An evaluation of the Gaspak system in the culture of anaerobic bacteria. J Appl Bacteriol. 1972;35(1):71–82.
Article CAS PubMed Google Scholar
Aranki A, Freter R. Use of anaerobic glove boxes for the cultivation of strictly anaerobic bacteria. Am J Clin Nutr. 1972;25(12):1329–34.
Article CAS PubMed Google Scholar
Cox ME, Mangels JI. Improved chamber for the isolation of anaerobic microorganisms. J Clin Microbiol. 1976;4(1):40–5.
Article CAS PubMed PubMed Central Google Scholar
Amirifar L, Besanjideh M, Nasiri R, Shamloo A, Nasrollahi F, de Barros NR, et al. Droplet-based microfluidics in biomedical applications. Biofabrication. 2022;14(2):022001.
Collins DJ, Neild A, deMello A, Liu AQ, Ai Y. The Poisson distribution and beyond: methods for microfluidic droplet production and single cell encapsulation. Lab Chip. 2015;15(17):3439–59.
Article CAS PubMed Google Scholar
Zhou N, Sun YT, Chen DW, Du W, Yang H, Liu SJ. Harnessing microfluidic streak plate technique to investigate the gut microbiome of Reticulitermes chinensis. Microbiologyopen. 2019;8(3):e00654.
Article PubMed Google Scholar
He X, McLean JS, Edlund A, Yooseph S, Hall AP, Liu SY, et al. Cultivation of a human-associated TM7 phylotype reveals a reduced genome and epibiotic parasitic lifestyle. Proc Natl Acad Sci U S A. 2015;112(1):244–9.
Article CAS PubMed Google Scholar
Terekhov SS, Smirnov IV, Malakhova MV, Samoilov AE, Manolov AI, Nazarov AS, et al. Ultrahigh-throughput functional profiling of microbiota communities. Proc Natl Acad Sci U S A. 2018;115(38):9551–6.
Article CAS PubMed PubMed Central Google Scholar
Lloréns-Rico V, Simcock JA, Huys GRB, Raes J. Single-cell approaches in human microbiome research. Cell. 2022;185(15):2725–38.
Article PubMed Google Scholar
Meng Y, Li S, Zhang C, Zheng H. Strain-level profiling with picodroplet microfluidic cultivation reveals host-specific adaption of honeybee gut symbionts. Microbiome. 2022;10(1):140.
Article CAS PubMed PubMed Central Google Scholar
Zheng W, Zhao S, Yin Y, Zhang H, Needham DM, Evans ED, et al. High-throughput, single-microbe genomics with strain resolution, applied to a human gut microbiome. Science. 2022;376(6597):eabm1483.
Article CAS PubMed Google Scholar
Livingston SJ, Kominos SD, Yee RB. New medium for selection and presumptive identification of the Bacteroides fragilis group. J Clin Microbiol. 1978;7(5):448–53.
Article CAS PubMed PubMed Central Google Scholar
Oberhardt MA, Zarecki R, Gronow S, Lang E, Klenk HP, Gophna U, et al. Harnessing the landscape of microbial culture media to predict new organism-media pairings. Nat Commun. 2015;6:8493.
Article CAS PubMed Google Scholar
Bacon K, Lavoie A, Rao BM, Daniele M, Menegatti S. Past, present, and future of affinity-based cell separation technologies. Acta Biomater. 2020;112:29–51.
Article CAS PubMed PubMed Central Google Scholar
Ahn Y, Sung K, Rafii F, Cerniglia CE. Effect of sterilized human fecal extract on the sensitivity of Escherichia coli ATCC 25922 to enrofloxacin. J Antibiot (Tokyo). 2012;65(4):179–84.
Article CAS PubMed Google Scholar
Canene-Adams K, General PCR. Methods Enzymol. 2013;529:291–8.
Article CAS PubMed Google Scholar
Zhao Y, Chen F, Li Q, Wang L, Fan C. Isothermal amplification of nucleic acids. Chem Rev. 2015;115(22):12491–545.
Article CAS PubMed Google Scholar
Park JW. Principles and applications of loop-mediated isothermal amplification to point-of-care tests. Biosensors (Basel). 2022;12(10):857.
Article CAS PubMed Google Scholar
Pham TH, Peter J, Mello FCQ, Parraga T, Lan NTN, Nabeta P, et al. Performance of the TB-LAMP diagnostic assay in reference laboratories: results from a multicentre study. Int J Infect Dis. 2018;68:44–9.
Article PubMed PubMed Central Google Scholar
Yilmaz S, Haroon MF, Rabkin BA, Tyson GW, Hugenholtz P. Fixation-free fluorescence in situ hybridization for targeted enrichment of microbial populations. ISME J. 2010;4(10):1352–6.
Article PubMed Google Scholar
Haroon MF, Skennerton CT, Steen JA, Lachner N, Hugenholtz P, Tyson GW. In-solution fluorescence in situ hybridization and fluorescence-activated cell sorting for single cell and population genome recovery. Methods Enzymol. 2013;531:3–19.
Article CAS PubMed Google Scholar
Meserve D, Wang Z, Zhang DD, Wong PK. A double-stranded molecular probe for homogeneous nucleic acid analysis. Analyst. 2008;133(8):1013–9.
Article CAS PubMed Google Scholar
Li C, Luan Z, Zhao Y, Chen J, Yang Y, Wang C, et al. Deep insights into the gut microbial community of extreme longevity in south Chinese centenarians by ultra-deep metagenomics and large-scale culturomics. NPJ Biofilms Microbiomes. 2022;8(1):28.
Article CAS PubMed PubMed Central Google Scholar
Schirmer M, Franzosa EA, Lloyd-Price J, McIver LJ, Schwager R, Poon TW, et al. Dynamics of metatranscription in the inflammatory bowel disease gut microbiome. Nat Microbiol. 2018;3(3):337–46.
Article CAS PubMed PubMed Central Google Scholar
** H, You L, Zhao F, Li S, Ma T, Kwok LY, et al. Hybrid, ultra-deep metagenomic sequencing enables genomic and functional characterization of low-abundance species in the human gut microbiome. Gut Microbes. 2022;14(1):2021790.
Article PubMed PubMed Central Google Scholar
Liwinski T, Leshem A, Elinav E. Breakthroughs and bottlenecks in microbiome research. Trends Mol Med. 2021;27(4):298–301.
Article PubMed Google Scholar
Wang N, Fang JY. Fusobacterium nucleatum, a key pathogenic factor and microbial biomarker for colorectal cancer. Trends Microbiol. 2023;31(2):159–72.
Article CAS PubMed Google Scholar
Castellarin M, Warren RL, Freeman JD, Dreolini L, Krzywinski M, Strauss J, et al. Fusobacterium nucleatum infection is prevalent in human colorectal carcinoma. Genome Res. 2012;22(2):299–306.
Article CAS PubMed PubMed Central Google Scholar
Kostic AD, Chun E, Robertson L, Glickman JN, Gallini CA, Michaud M, et al. Fusobacterium nucleatum potentiates intestinal tumorigenesis and modulates the tumor-immune microenvironment. Cell Host Microbe. 2013;14(2):207–15.
Article CAS PubMed PubMed Central Google Scholar
Yang Y, Weng W, Peng J, Hong L, Yang L, Toiyama Y, et al. Fusobacterium nucleatum increases proliferation of colorectal cancer cells and tumor development in mice by activating Toll-like receptor 4 signaling to nuclear factor-κB, and up-regulating expression of microRNA-21. Gastroenterology. 2017;152(4):851–66.e24.
Article CAS PubMed Google Scholar
Casasanta MA, Yoo CC, Udayasuryan B, Sanders BE, Umaña A, Zhang Y, et al. Fusobacterium nucleatum host-cell binding and invasion induces IL-8 and CXCL1 secretion that drives colorectal cancer cell migration. Sci Signal. 2020;13(641):eaba9157.
Article CAS PubMed PubMed Central Google Scholar
Ternes D, Tsenkova M, Pozdeev VI, Meyers M, Koncina E, Atatri S, et al. The gut microbial metabolite formate exacerbates colorectal cancer progression. Nat Metab. 2022;4(4):458–75.
Article CAS PubMed PubMed Central Google Scholar
Kelly CR, Fischer M, Allegretti JR, LaPlante K, Stewart DB, Limketkai BN, et al. ACG clinical guidelines: prevention, diagnosis, and treatment of Clostridioides difficile infections. Am J Gastroenterol. 2021;116(6):1124–47.
Article CAS PubMed Google Scholar
Sorbara MT, Pamer EG. Microbiome-based therapeutics. Nat Rev Microbiol. 2022;20(6):365–80.
Article CAS PubMed Google Scholar
Kaźmierczak-Siedlecka K, Skonieczna-Żydecka K, Hupp T, Duchnowska R, Marek-Trzonkowska N, Połom K. Next-generation probiotics - do they open new therapeutic strategies for cancer patients?. Gut Microbes. 2022;14(1):2035659.
Article PubMed PubMed Central Google Scholar
Wang F, Cai K, **ao Q, He L, **e L, Liu Z. Akkermansia muciniphila administration exacerbated the development of colitis-associated colorectal cancer in mice. J Cancer. 2022;13(1):124–33.
Article CAS PubMed PubMed Central Google Scholar
Chung L, Thiele Orberg E, Geis AL, Chan JL, Fu K, DeStefano Shields CE, et al. Bacteroides fragilis toxin coordinates a pro-carcinogenic inflammatory cascade via targeting of colonic epithelial cells. Cell Host Microbe. 2018;23(2):203–14.e5.
Article CAS PubMed PubMed Central Google Scholar

Download references

Acknowledgements

Not applicable.

Funding

Not applicable.

Author information

Authors and Affiliations

Department of Gastroenterology and Hepatology, the First Medical Center of Chinese, PLA General Hospital, Bei**g, 100853, China
Meng-Qi Xu, Fei Pan, Li-Hua Peng & Yun-Sheng Yang
Medical School of Chinese PLA, Bei**g, 100853, China
Meng-Qi Xu

Authors

Meng-Qi Xu
View author publications
You can also search for this author in PubMed Google Scholar
Fei Pan
View author publications
You can also search for this author in PubMed Google Scholar
Li-Hua Peng
View author publications
You can also search for this author in PubMed Google Scholar
Yun-Sheng Yang
View author publications
You can also search for this author in PubMed Google Scholar

Contributions

YSY conceived this review. MQX and YSY performed the review and wrote the manuscript. FP and LHP helped with the review and writing of the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Yun-Sheng Yang.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that there are no conflicts of interest in this study.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Cite this article

Xu, MQ., Pan, F., Peng, LH. et al. Advances in the isolation, cultivation, and identification of gut microbes. Military Med Res 11, 34 (2024). https://doi.org/10.1186/s40779-024-00534-7

Download citation

Received: 27 September 2023
Accepted: 17 April 2024
Published: 03 June 2024
DOI: https://doi.org/10.1186/s40779-024-00534-7

Advances in the isolation, cultivation, and identification of gut microbes

Abstract

Similar content being viewed by others

Gut microbiota functions: metabolism of nutrients and other food components

An insight into gut microbiota and its functionalities

E. coli as an All-Rounder: The Thin Line Between Commensalism and Pathogenicity

Background

Gut microbial isolation and cultivation

Traditional methods

The renaissance of culturomics

Droplet microfluidics

SlipChip

End-to-end droplet microfluidic system

Microfluidic streak plate

Single-cell microlitre-droplet screening system

Selection for phenotypes or genomes

Membrane diffusion-based cultivation

In situ cultivation

Co-culture technique

Gut microbial identification

Non-targeted identification

Classical phenoty**

16S rRNA gene

MALDI-TOF MS

Targeted identification

Nucleic acid amplification

Nucleic acid hybridization

Application and future of gut culturomics

Culturomics and metagenomics are complementary to each other

Causal mechanism in the gut microbiome

Biotherapy of the gut microbiome

Availability of data and materials

Abbreviations

References

Acknowledgements

Funding

Author information

Authors and Affiliations

Contributions

Corresponding author

Ethics declarations

Ethics approval and consent to participate

Consent for publication

Competing interests

Rights and permissions

About this article

Cite this article

Share this article

Keywords

Search

Navigation