Evolution and progression of bacterial culture

Microbiome research has evolved significantly over the past few decades. Historically, microorganisms were obtained and identified strictly through culture-based methods and phenotypic observations, such as gram stain and carbohydrate degradation (Lagier et al. Reference Lagier, Hugon and Khelaifia2015; Russell Reference Russell1979; Salanitro et al. Reference Salanitro, Blake and Muirhead1977). These methods were expensive and time-consuming. However, until the development of more advanced molecular techniques, they were considered the “gold standard.” The development of molecular techniques, such as Sanger sequencing of the 16S rDNA gene and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) reduced the effort required to identify bacterial isolates and allowed for the easier identification of difficult-to-culture bacteria. The creation of next-generation sequencing replaced the use of conventional culture microbiology in many laboratories and has greatly increased our understanding of microbes and their communities. However, the importance of microbial culture is being re-recognized (Lagier et al. Reference Lagier, Hugon and Khelaifia2015). As our knowledge of microorganisms has increased, culture methods involving multiple technologies have emerged. Nowadays, culturomics is a high-throughput culture method involving numerous culture conditions, including but not limited to differing media compositions, pretreatments, oxygen levels, temperatures, etc., paired with techniques such as MALDI-TOF MS and full-length bacterial 16S rRNA gene sequencing to identify the isolates (Duquenoy et al. Reference Duquenoy, Ania and Boucher2020; Lagier et al. Reference Lagier, Armougom and Million2012; Wang et al. Reference Wang, Howe and Wei2021).

Culturomics workflow

MALDI-TOF mass spectrometry is a critical method to identify bacterial species in culturomics research due to its advantages in efficiency, specificity, and low cost (Bilen Reference Bilen2020). The workflow of culturomics for unknown microbes includes the following key steps (Figure 1): bacterial isolation, MALDI-TOF mass spectrometry identification, 16S rRNA sequencing, and a new species announcement. Briefly, the colony from bacterial isolates should be first analyzed through MALDI-TOF MS several times. An identification score < 1.9 suggests that the isolate may be an unknown bacterial species (Seng et al. Reference Seng, Drancourt and Gouriet2009). Then, the full-length 16S sequencing is performed. If the sequence similarity between the isolate and known species is < 98.65%, there is a high probability that the isolate is a new species (Lagier et al. Reference Lagier, Dubourg and Million2018). Finally, an article format describing the isolation process and main characteristics, including phenotypic data and taxonomic classification, is needed for the new species announcement (Fournier et al. Reference Fournier, Raoult and Drancourt2017).

Figure 1. Identification of a new species in culturomics.

Microbiome sequencing and culturomics

Since the early 2000s, the development of high-throughput sequencing technologies has continued to transform many biological fields (Goodwin et al. Reference Goodwin, McPherson and McCombie2016). Today, numerous sequencing “generations” and technologies are available (Satam et al. Reference Satam, Joshi and Mangrolia2023). These technologies have revolutionized microbial ecology and microbiome/microbiota research, allowing researchers to explore microbial communities’ diversity, structure, composition, and role in different environments (Chai et al. Reference Chai, Weiss and Beck2024; Deng et al. Reference Deng, Wang and Li2023; Wang et al. Reference Wang, Tsai and Deng2019). Additionally, the “sequencing era” has also been crucial for the development of microbe-based therapeutics (Sorbara and Pamer Reference Sorbara and Pamer2022) and bioremediation (Malla et al. Reference Malla, Dubey and Yadav2018).

However, although they have many strengths, these technologies also have shortfalls (Satam et al. Reference Satam, Joshi and Mangrolia2023). The use of these sequencing technologies found that, in the human gastrointestinal tract, approximately 80% of bacteria were unknown (Lagier et al. Reference Lagier, Dubourg and Million2018). Many of these unknown bacteria cannot be classified to lower taxonomic levels and have been described as “microbial dark matter” (Rinke et al. Reference Rinke, Schwientek and Sczyrba2013). Moreover, other weaknesses of microbiome sequencing include sequencing depth bias (Lagier et al. Reference Lagier, Dubourg and Million2018; Lynch and Neufeld Reference Lynch and Neufeld2015), lack of causality from typical microbiome association studies (Chaudhari et al. Reference Chaudhari, McCurry and Devlin2021), and differences regarding laboratory and data analysis methods (Howe et al. Reference Howe, Kegley and Powell2023). Augmenting sequencing-based approaches by including culture-dependent analyses may help researchers overcome the shortfalls of culture-independent studies, as culture-dependent analyses are integral for identifying most causal relationships and mechanisms (Zhao and Zhao Reference Zhao and Zhao2021), as illustrated by Fei et al. (Reference Fei, Bruneau and Zhang2020), Pleguezuelos-Manzano et al. (Reference Pleguezuelos-Manzano, Puschhof and Rosendahl Huber2020), and Zagato et al. (Reference Zagato, Pozzi and Bertocchi2020). Nevertheless, many microbes are not easily cultured, with some estimates being that 99% of all bacterial and archaeal species are “unculturable” using conventional methods (Jiao et al. Reference Jiao, Liu and Hua2021). This creates a problematic conundrum because increasing the number of available reference genomes is necessary to decrease microbial dark matter, which cannot be accomplished without pure cultures. These pure cultures are also required to characterize novel microbes and genes (Liu et al. Reference Liu, Moon and Zheng2022).

Culture-dependent studies allow for the analysis of low-abundance microbes, providing information regarding “rare” bacteria often “missed” by culture-independent studies (Wang et al. Reference Wang, Howe and Wei2021; Zehavi et al. Reference Zehavi, Probst and Mizrahi2018). This phenomenon has been coined the “rare biosphere,” and the potential ecological importance of these taxa is becoming recognized (Lynch and Neufeld Reference Lynch and Neufeld2015). These “rare” microbes are likely essential in their respective environments. In humans, it has been observed that members of the rare biosphere may be involved in both health and disease (Hajishengallis et al. Reference Hajishengallis, Liang and Payne2011; Jousset et al. Reference Jousset, Bienhold and Chatzinotas2017; van der Gast et al. Reference van der Gast, Walker and Stressmann2011), and in plants, members of the rare biosphere likely protect the host by producing anti-pathogen compounds (Hol et al. Reference Hol, Garbeva and Hordijk2015; Jousset et al. Reference Jousset, Bienhold and Chatzinotas2017). Clearly, studying the “rare biosphere” is highly important, and one method for this is culture. Lynch and Neufeld and Jousset et al. provide methods and considerations for studying these microbes (Jousset et al. Reference Jousset, Bienhold and Chatzinotas2017; Lynch and Neufeld Reference Lynch and Neufeld2015). Therefore, even in the sequencing age, culture-based analyses are highly important. Culturing the rare biosphere and previously uncultured members of the microbiota go hand in hand, and culturomics is a valuable tool for both.

The changing bacterial repertoires identified in humans

Due to technological limitations in earlier years, only a small minority of the microbiota could be readily cultured in vitro (Vartoukian et al. Reference Vartoukian, Palmer and Wade2010), resulting in most bacteria being considered unculturable. However, the number of bacterial species isolated from humans has been rapidly increasing and updating with the development of culturomics. Researchers have tried to build a comprehensive compilation including all human-associated prokaryotic species described. The bacterial repertoire isolated from humans has been updated from 2,172 first reported in 2015 (Hugon et al. Reference Hugon, Dufour and Colson2015), to 2,776 in 2018 (Bilen et al. Reference Bilen, Dufour and Lagier2018), and 3,253 in 2021 (Diakite et al. Reference Diakite, Dubourg and Raoult2021). Among them, the proportion of novel isolated species from 2015 to the present is up to 26% (N = 831). Bacterial species in this repertoire were mainly classified into four different phyla, including the Firmicutes (37%; N = 1200), Proteobacteria (25; N = 812), Actinobacteria (25%; N = 805), and Bacteroidetes (8%; N = 262) (Diakite et al. Reference Diakite, Dubourg and Raoult2021). Specifically, a total of 711 bacterial genera were listed in the human repertoire, mostly in Mycobacterium, Clostridium, Bacillus, Corynebacterium, and Streptococcus (Diakite et al. Reference Diakite, Dubourg and Raoult2021). It is worth noting that the number of intestinal bacterial species accounted for the largest proportion of bacteria isolated from human anatomical sites, which includes isolates from the vagina, skin, urine, respiratory tract, and so on (Diakite et al. Reference Diakite, Dubourg and Raoult2021).

Bacterial species isolated from different human body parts

In humans, bacterial species showed varied abundance in different sites of the body (Figure 2). Understanding and obtaining the gut microbiota is important for parsing the potential molecular mechanisms of human health and disease (Fan and Pedersen Reference Fan and Pedersen2021). It has been predicted that there were 10¹¹–10¹² bacteria/g of human feces (Van Houte Reference Van Houte1966), which dramatically exceeds the number identified and cultured from the gut, suggesting that a considerable portion of microbes in the gut have not been isolated using current methods (Lagier et al. Reference Lagier, Armougom and Million2012). Lagier et al. isolated 1057 bacterial species from the human gut in different geographical regions, comprising 56% Firmicutes (N = 600), 17% Actinobacteria (N = 181), 16% Proteobacteria (N = 173), and 8% Bacteroidetes (N = 88) phyla (Lagier et al. Reference Lagier, Khelaifia and Alou2016).

Figure 2. Cultured species repertoires in humans from different body sites.

The respiratory tract spans from the nostrils to the lungs, which have a large surface area of approximately 70 m² and harbor diverse microbial communities (Man et al. Reference Man, de Steenhuijsen Piters and Bogaert2017). Previous studies have shown that the microbiota in the respiratory tract could prevent pathogen colonization and maintain homeostasis (Pust et al. Reference Pust, Wiehlmann and Davenport2020; Shah et al. Reference Shah, Shah and Baloch2021). Fonkou et al. identified 756 different bacterial species totally from the respiratory tract, including the oral cavity and upper and lower respiratory tracts (Fonkou et al. Reference Fonkou, Dufour and Dubourg2018). Specifically, 355, 202, and 514 species were listed in the oral cavity, upper respiratory tract, and lower respiratory tract, respectively, with a proportion of 11.1% (N = 84) shared between all three sites. Actinobacteria (33.7%; N = 255), Proteobacteria (31.6%; N = 239), and Firmicutes (20.2%; N = 153) were the most represented phyla.

For decades, the urine of healthy individuals was usually considered to be sterile in clinical studies subjected to methodological biases (Wolfe et al. Reference Wolfe, Toh and Shibata2012). However, the ensuing evidence of detected bacteria in the urine revolutionized cognition and provided the urobiome more association with human health and disease (Hilt et al. Reference Hilt, McKinley and Pearce2014; Wolfe and Brubaker Reference Wolfe and Brubaker2019). Morand et al. reviewed a total of 562 species from human repertoires in 2018, which shared 350 species in common with the gut microbiota (Morand et al. Reference Morand, Cornu and Dufour2019). The four most represented phyla in the human urinary tract are Proteobacteria (35.5%; N = 200), Firmicutes (31.3%; N = 176), Actinobacteria (22.4%; N = 126), and Bacteroidetes (6.4%; N = 36). Importantly, most pathogenic bacteria constitute part of the microbiota composition in the human urinary tract and have the potential to lead to the occurrence of disease once the microbiota is disturbed. Dubourg et al. expanded this repertoire to 672 bacterial species by culturomics in 2020, among which 64.1% (N = 431) had been previously cultured from the human gut, hinting that a possible origin of the urine microbiota is derived from the gut (Dubourg et al. Reference Dubourg, Morand and Mekhalif2020). Strikingly, the authors also listed the 10 most prevalent bacteria from male and female urine specimens in clinical experiments, and the top 3 were Escherichia coli, Klebsiella pneumoniae, and Enterococcus faecalis.

Vaginal microbes, such as Lactobacillus iners, were reported to be involved in women’s reproductive health and disease (Bloom et al. Reference Bloom, Mafunda and Woolston2022). The imbalance of vaginal microbiota could result in bacterial vaginosis (Zhu et al. Reference Zhu, Tao and Edupuganti2022). Diop et al. inventoried a repertoire of total 571 bacteria species from the vagina in 2019 (Diop et al. Reference Diop, Dufour and Levasseur2019), comprised of 39.1% Firmicutes (N = 227), 25.8% Proteobacteria (N = 150), 17.4% Actinobacteria (N = 101), and 12.7% Bacteroidetes (N = 74), respectively. Focused on Firmicutes phylum, a predominance of Lactobacillaceae family accounted for 17.2% (N = 39). However, only 49.1% (N = 285) of this repertoire were identified through culturomics (Diop et al. Reference Diop, Dufour and Levasseur2019). The more detailed characterization of the vaginal microbiota based on culturomics anticipates further reports and analyses in the future.

Overall, these pure isolations and cultures of bacterial species fill the gaps, at least partly, in identifying bacteria through culturomics, providing a basis for a more long-term and systematic study of human–microbial interactions.

Early culture-based study on pig gut microbiota

Since the 1950s, researchers have employed culture-based methods to study the intestinal microbiota of pigs (Fewins et al. Reference Fewins, Newland and Briggs1957; Kenworthy and Crabb Reference Kenworthy and Crabb1963). One of the early studies revealed that around 30% of swine fecal bacteria could be recovered using a rumen fluid medium; and the majority (90%) of these isolated bacteria were gram-positive (Salanitro et al. Reference Salanitro, Blake and Muirhead1977), such as facultatively anaerobic Streptococcus, Eubacterium sp., Clostridium sp., and Propionibacterium acnes. Russell’s study also reported that over 90% of the bacteria he isolated were gram-positive (Russell Reference Russell1979). Furthermore, his research delved into the distribution of bacteria in different locations of the swine colon, noting a lower bacterial count in the intestinal wall tissue compared to the luminal content and surface layer. Similar results were found when bacterial populations in the pig cecum and colon were analyzed with different energy sources present in the media (Allison et al. Reference Allison, Robinson and Bucklin1979), which underscored the diversity and adaptability of microbial communities. Moreover, pig cecal content culture showed dominant Bacteroides ruminicola (35%) and Selenomonas ruminantium (21%) in the isolates (Robinson et al. Reference Robinson, Allison and Bucklin1981). As for colon microbiota, a study found that in healthy pigs, over 71% of the bacteria were gram-positive, including Streptococcus sp., Lactobacillus acidophilus, and Bifidobacterium adolescentis (Robinson et al. Reference Robinson, Whipp and Bucklin1984). In contrast, pigs with dysentery showed a higher prevalence (88%) of gram-negative bacteria. Most early studies employed strictly anaerobic techniques with several roll tube media (Hungate and Macy Reference Hungate and Macy1973) and recovered mostly gram-positive bacteria. These techniques and results laid the foundation for the future culture-dependent and -independent studies of the swine microbiome.

Culturomics on pig gut microbiota

The growing knowledge of swine microbiome and culture technology allows researchers to isolate and study various bacteria from pig intestines, focusing on their types, functions, and diversity. By analyzing these bacteria, scientists learn about interactions between microbes and hosts, disease development, and pigs’ overall health (Ma et al. Reference Ma, Tao and Song2024). Wang et al., using 53 different cultivation methods, demonstrated an increase in microbial diversity across different growth stages in pigs (Wang et al. Reference Wang, Howe and Wei2021). The study found that culture-dependent methods revealed higher bacterial diversity than previously known, with significant amounts of bacterial amplicon sequence variants (ASVs). It also developed reference culture maps for specific bacterial taxa, highlighting the influence of various factors like oxygen, medium, and pig age on microbiota cultivation. The findings are crucial for understanding specific bacterial roles in swine production and health, aiding in the isolation of beneficial bacteria for potential use as probiotics. One of the more recent studies identified 267 bacterial colonies, with 42 species classified into 23 genera in culturomics study of weaning pig gut microbiome (Lee et al. Reference Lee, Ryu and Kang2022). Lactobacillus species, particularly L. curvatus, L. sakei, and L. mucosae, were predominant among the cultured species. Additionally, the establishment of PiBAC, a comprehensive collection of about 1100 bacterial pure cultures from 31 different media of pig gut microbiome, marks a notable advancement in this field (Wylensek et al. Reference Wylensek, Hitch and Riedel2020). This assemblage, encompassing 117 strains that correspond to 110 species across 40 families and 9 phyla, was curated to ensure extensive species-level representation. The majority of these species are commonly found in the dominant communities within the pig gut microbiota (Figure 3). Remarkably, 38 of these strains are categorized as novel taxa, highlighting a substantial broadening in our understanding of the pig gut microbiome’s diversity. These findings are pivotal in elucidating both the functional roles and taxonomic diversity of these bacteria.

Figure 3. Most commonly cultured bacteria from swine.

Culture-based study on reproductive tract microbiome

Earlier studies on the porcine vaginal microbiota identified a dominant presence of species such as Streptococcus sp., E. coli, Staphylococcus sp., Corynebacterium sp., Micrococcus sp., and Actinobacillus sp., by cultivation from 142 isolates (Bara et al. Reference Bara, McGowan and O’boyle1993; Larsen Reference Larsen1993). In a study of large black sows, the 115 isolates from vaginal swabs revealed a broader diversity, encompassing 30 species across 16 different genera (Singh and Ebibeni Reference Singh and Ebibeni2016). In this study, Aeromonas was the most frequently isolated at 39.2%, followed by Enterococcus, Klebsiella, Escherichia, and Citrobacter. Bacterial culture and identification by MALDI-TOF MS were employed to reveal a rich population of bacterial species in the vaginal canal of sows (Poor et al. Reference Poor, Moreno and Monteiro2022), showing that healthy sows had a higher frequency of Enterococcus faecalis, Streptococcus hyovaginalis and Acinetobacter lwolffii than the purulent vulvar discharge sows (Figure 3). This suggested a complex and dynamic bacterial ecosystem within the porcine vaginal environment, with certain species potentially playing key roles in maintaining reproductive health or contributing to disease states.

Culture-based study on pig tonsil microbiome

The presence of bacteria in the tonsil of the soft palate of swine has also been well-documented (Kernaghan et al. Reference Kernaghan, Bujold and MacInnes2012), with reports up until 2012 indicating that more than 70 different bacterial species across 14 families have been cultured from this region (Figure 3). These studies have largely concentrated on identifying primary pathogens of swine and zoonotic agents. In instances involving organisms like Mycoplasma hyopneumoniae (Marois et al. Reference Marois, Le Carrou and Kobisch2007) or Salmonella enterica (Lomonaco et al. Reference Lomonaco, Decastelli and Bianchi2009), it is quite evident that these entities possess pathogenic capabilities. Conversely, bacteria such as Actinobacillus minor (Chiers et al. Reference Chiers, Haesebrouck and Mateusen2001) and Actinobacillus porcitonsillarum (Tonpitak et al. Reference Tonpitak, Rohde and Gerlach2007) were likely to be harmless commensals. This diversity in the microbial population of the swine tonsils highlighted the complexity of the microbiome in these animals, encompassing a range of organisms with varying impacts on swine health.

Culturomics in pig microbiome research is an evolving field that combines traditional culture-dependent methods with modern molecular techniques. In addition, studies have shown the importance of integrating both culture-dependent and -independent methods to gain a more coherent picture of the pig gut microbiome. One study found that using a single culture medium with selective screens identified 46 distinct bacterial species, demonstrating an effective way to increase species diversity (Fenske et al. Reference Fenske, Ghimire and Antony2020). However, these cultured species did not fully represent the most abundant taxa in the microbiome, as revealed by metagenomic analysis. This discrepancy underscored the importance of combining both approaches for a more comprehensive understanding of the pig gut microbiome. This has led to significant advancements in understanding the diversity and function of microbial communities of swine microbiome.

Rumen

The bovine rumen microbiome has been heavily researched due to its relationship with feed efficiency and methane production (Newbold and Ramos-Morales Reference Newbold and Ramos-Morales2020; O’Hara et al. Reference O’Hara, Neves and Song2020). Both the Global Rumen Census (Henderson et al. Reference Henderson, Cox and Ganesh2015) and Hungate 1000 (Creevey et al. Reference Creevey, Kelly and Henderson2014; Seshadri et al. Reference Seshadri, Leahy and Attwood2018) projects have vastly increased our knowledge of the rumen microbiota. While the Global Rumen Census described the rumen microbiota of 32 species from 35 countries, it utilized only culture-independent methods (Henderson et al. Reference Henderson, Cox and Ganesh2015), while the goal of the Hungate 1000 project is to create a reference database of genome sequences and cultured rumen microbes. A meta-analysis was conducted to identify many target organisms for the Hungate 1000 project (Creevey et al. Reference Creevey, Kelly and Henderson2014). As of 2018, the Hungate collection was comprised of 21 archaea and 480 bacterial genomes, of which 410 are from cultured microbes and 91 are from previously available reference genomes. However, this collection has been estimated to only consist of approximately 75% of taxa in the rumen at the genera level, indicating a need for additional efforts to bring the remaining 25% into cultivation. Regardless, the Hungate collection is an extremely valuable resource for rumen microbiota research (Seshadri et al. Reference Seshadri, Leahy and Attwood2018).

Early pre-Hungate1000, efforts focused on increasing the number of culturable rumen microbiota organisms include (Kenters et al. Reference Kenters, Henderson and Jeyanathan2011; Nyonyo et al. Reference Nyonyo, Shinkai and Tajima2013). Kenters et al. utilized a novel culture medium with a salt composition similar to the rumen fluid and dilution method. While this study utilized sheep rumen samples, not bovine, the authors isolated 60 new isolates, 19 of which likely belonged to previously undescribed genera (Kenters et al. Reference Kenters, Henderson and Jeyanathan2011). Furthermore, Nyonyo et al. examined the inclusion of gelling agents in the culture medium and found that of the 69 unclassified isolates cultured, 24.4% were cultured on basal media (BM) supplemented with 1.8% agar (agar BM: A-BM), 56.6% were isolated on a modified basal medium (MBM) (0.1% MgCl₂ instead of KH₂PO₄) supplemented with 1.8% agar (agar MBM: A-MBM), 34.5% were isolated on the modified basal media supplemented with 0.8% Phytagel™ (Phytagel MBM: P-MBM), and 13.8% were isolated on the modified basal media modified supplemented with 1% Gelrite® (Gelrite MBM: G-MBM). The authors noted that previously uncultured bacteria were isolated from all media, but A-MBM, G-MBM, and P-MBM media increased the previously uncultured to total bacteria ratio (Nyonyo et al. Reference Nyonyo, Shinkai and Tajima2013). Although not focused on rumen samples, Ziemer observed that including cellulose and xylan–pectin in an 8-week fermentation of bovine feces resulted in the isolation of many previously uncultured microbes (Ziemer Reference Ziemer2014). Together, these studies indicate the importance of media composition in isolating fastidious microbes.

This is further emphasized by Zehavi et al., who cultured rumen samples on both defined (M10 media) and undefined (M10 + sterile rumen fluid) media with numerous sample dilutions and then sequenced the original rumen sample to estimate the percent of cultivable rumen operational taxonomic units (OTUs) (Zehavi et al. Reference Zehavi, Probst and Mizrahi2018). Overall, they observed that only 23% of the rumen microbiota detected using culture-independent methods was culturable using these two media compositions, with only 3.6% of cultured OTUs overlapping with the Hungate1000 database. After ruling out possible contamination, the authors concluded that they had confirmed the existence of a “rare rumen biosphere,” as they cultured many isolates not detected using culture-independent methods, indicating the importance of conducting culture-dependent analyses in conjunction with conventional culture-independent studies. The authors also concluded that culture repetition and sample dilution increased the microbial diversity captured by culture. Although they only utilized two culture media, Zehavi et al. clearly indicate the importance of culture media composition and dilutions in isolating rumen microbes and illustrate the clear existence of a “rare rumen biosphere” (Zehavi et al. Reference Zehavi, Probst and Mizrahi2018).

As culturomics and culture-dependent analyses have grown in popularity, and the rare biosphere has been increasingly recognized, new methods have been developed to aid in isolating lower abundance microbes. Liu et al. utilized three methods to isolate ureolytic microbes from the bovine rumen, providing a valuable methodology and workflow that can be applied to other low-abundance rumen microbes (Liu et al. Reference Liu, Yu and Zhong2023). Briefly, the authors first utilized urease gene (ureC) guided enrichment. The ureC-positive cultures were then serially diluted and embedded into agarose microspheres, which were incubated in a dialysis bag within an in situ rumen environment consisting of non-sterile rumen fluid, media, and the cattle ration. At differing time points, up to 72 hours, the microspheres were crushed to obtain the isolated microbe, and full-length 16S sequencing was performed on the isolates. Using these methods, they obtained 976 total isolates comprised of 404 unique isolates. Of these, 28 isolates belonging to 12 bacterial species contained the ureC gene, including Aliarcobacter butzleri, Citrobacter koseri, C. farmeri, C. amalonaticus, Clostridium butyricum, Corynebacterium vitaeruminis, Enterobacter hormaechei, E. cloacae, Klebsiella pneumoniae, Paraclostridium bifermentans, Pseudomonas stutzeri, and Proteus penneri (Liu et al. Reference Liu, Yu and Zhong2023).

It is clear that more wide-scale culturomics studies are needed to bring the previously uncultured microbes into cultivation and further characterize the rare rumen biosphere. While many of these studies only utilized a few different media compositions, they indicate the need for multiple media compositions, as is observed in human culturomics studies (Diakite et al. Reference Diakite, Dubourg and Dione2020). Including numerous culture conditions may result in an increased number of isolated fastidious microbes. Moreover, Zehavi et al. , Kenters et al. , and Ziemer illustrate the importance of media composition being similar to that of the niche of interest (Kenters et al. Reference Kenters, Henderson and Jeyanathan2011; Zehavi et al. Reference Zehavi, Probst and Mizrahi2018; Ziemer Reference Ziemer2014). Regardless, these provide useful media compositions for future culturomics analyses focused on isolating microbes from the rumen and a methodology for incorporating new methods, such as in situ cultivation and gene-guided enrichment, into a culturomics workflow (Liu et al. Reference Liu, Yu and Zhong2023).

Reproductive

While the uterus has been historically considered sterile, it is now well-accepted that a uterine microbiome does exist. The reproductive (vaginal and uterine) microbiome is considered low biomass, and numerous environmental factors have been observed to contribute to reproductive microbiome variation. Additionally, while uterine microbiota diversity has been linked to health, dysbiosis has been associated with metritis. It has been speculated that host-derived probiotics may be developed to maintain microbiota stability and protect against disease (Çömlekcioğlu et al. Reference Çömlekcioğlu, Jezierska and Opsomer2024).

Similarly, most culture-dependent analyses on the reproductive microbiota have focused on comparing health statuses (Kronfeld et al. Reference Kronfeld, Kemper and Hölzel2022; Paiano et al. Reference Paiano, Moreno and Gomes2022; Wagener et al. Reference Wagener, Prunner and Pothmann2015). Wagener et al. cultured 2052 isolates from the uterus of postpartum dairy calves from 76 genera. However, 24.2% and 13.2% of the isolates were from the genera Staphylococcus and Trueperella, respectively. However, only two media types (sheep blood and MacConkey) were used (Wagener et al. Reference Wagener, Prunner and Pothmann2015). Kronfeld et al. cultured bacteria from the vagina and uterus of healthy postpartum cattle and those with puerperal disorders utilizing five culture conditions consisting of aerobic, anaerobic, and microaerophilic conditions. In total, from vaginal samples, they isolated 561 bacteria from 46 genera, and from uterine samples, they isolated 409 bacteria from 42 genera. In both locations, regardless of health status, Streptococcus was the most isolated genera (Kronfeld et al. Reference Kronfeld, Kemper and Hölzel2022). Paiano et al. isolated 127 bacterial species from the uteri of healthy cattle and those with clinical and subclinical endometritis utilizing four aerobic culture conditions. In healthy cattle, 97 bacterial species were isolated, while in cattle with clinical and subclinical endometritis, 53 and 21 bacterial species were isolated, respectively. The opportunistic pathogens Bacillus cereus, Escherichia coli, and Aerococcus viridans were the most isolated bacterial species and were isolated from all health statuses. Interestingly, bacteria were unable to be cultured from 35.4%, 40%, and 18.1% of samples from healthy, subclinical, and clinical cattle, respectively (Paiano et al. Reference Paiano, Moreno and Gomes2022). However, this is likely due to the low number of culture conditions used. Wagener et al. (Reference Wagener, Prunner and Pothmann2015), Kronfeld et al. (Reference Kronfeld, Kemper and Hölzel2022), and Paiano et al. (Reference Paiano, Moreno and Gomes2022) indicate that there is increased research interest in utilizing culture-based methods to isolate both commensal microbes and opportunistic pathogens from the bovine reproductive tract and illustrate the diversity of the cultivable bovine reproductive microbiome.

Unlike the aforementioned studies, Webb et al. utilized both culture-dependent and -independent analyses to study the uterine and vaginal microbiota and its relationship with beef cattle fertility (Webb et al. Reference Webb, Holman and Schmidt2023). They utilized five culture conditions consisting of both aerobic and anaerobic isolation, resulting in 512 isolates from 52 genera from the vaginal samples and 221 isolates from 29 genera from the uterine samples. Streptococcus was the most isolated genera regardless of location. Although they combined both culture-dependent and -independent analyses, the authors did not comment on the percentage of the vaginal and uterine microbiota that was culturable utilizing their conditions (Webb et al. Reference Webb, Holman and Schmidt2023).

Therefore, additional culturomics studies paired with culture-independent analyses to describe the cultivability of the bovine reproductive tract, isolate key players in the bovine reproductive microbiota, and screen isolates for potential function are needed. Regardless, these studies provide valuable information such as media compositions, culture conditions, and currently isolated bacteria, which can provide researchers with a framework for designing future more in-depth culturomics studies to study the bovine reproductive tract.

Other niches of interest

Two other disease areas that could greatly benefit from increased culturomics analyses include mastitis and bovine respiratory disease (BRD). Angelopoulou et al. conducted culturomics analysis on milk samples from cattle with mastitis from five different culture conditions (Angelopoulou et al. Reference Angelopoulou, Holohan and Rea2019). They observed a large difference between the culture-independent and -dependent results. Using 16S amplicon sequencing, 36 genera were present, of which only 8 (Bacillus, Carnobacterium, Enterococcus, Escherichia/Shigella, Pseudomonas, Staphylococcus, Streptococcus, and Trueperella) were cultured. In addition, four genera (Barnesiella, Kocuria, Microbacterium, and Raoultella) were cultured but not detected using 16S amplicon sequencing. While it is possible that these bacteria are the result of contamination, they likely represent the rare biosphere discussed previously. Therefore, the results of Angelopoulou et al. indicate a need for additional culturomics studies focusing on the udder and milk microbiota (Angelopoulou et al. Reference Angelopoulou, Holohan and Rea2019).

It is well accepted that the structure, diversity, and composition of the respiratory microbiome of healthy cattle and those with BRD differs (Chai et al. Reference Chai, Capik and Kegley2022; Howe et al. Reference Howe, Kegley and Powell2023). However, no true culturomics studies and very few culture-dependent analyses focusing strictly on commensal microbes have been conducted. Holman et al. isolated commensal bacteria from the nasopharyngeal tract at feedlot entry and after 60 days within the feedlot (Holman et al. Reference Holman, Timsit and Alexander2015). They utilized three culture media (brain heart infusion (BHI), de Man, Rogosa, Sharpe (MRS), and 5% sheep blood agar) and 16S rRNA sequencing for identification paired with 454 pyrosequencing. Using these conditions, they isolated 605 isolates from 32 genera, including Moraxella, Pasteurella, Mannheimia, Corynebacterium, and Acinetobacter, most often isolated from the BHI and blood agar, and Bacillus, Staphylococcus, Lactobacillus, Aerococcus, and Streptococcus from MRS. The authors also noted differences between the culture-dependent and -independent analyses as Aerococcus, Dietzia, Proteus, Rothia, and Microccoccus were cultured but not present in the culture-independent data (Holman et al. Reference Holman, Timsit and Alexander2015). Additionally, Amat et al. isolated many commensal bacteria from the bovine nasopharyngeal tract (Amat et al. Reference Amat, Timsit and Baines2019a), focusing specifically on lactic acid-producing bacteria previously identified as potentially health-associated members of the bovine nasopharyngeal microbiota (Amat et al. Reference Amat, Holman and Timsit2019b). They isolated 300 colonies from 14 genera; however, they utilized only two culture conditions (MRS and Rogosa agar), both of which were focused on isolating lactic acid-producing bacteria. The most commonly isolated genera were Bacillus, Staphylococcus, Streptococcus, and Lactobacillus. Moreover, although they were focused on isolating lactic acid-producing bacteria, 69% of isolates were not (Amat et al. Reference Amat, Timsit and Baines2019a). These studies illustrate the need for a true culturomics study focused on the bovine respiratory microbiota.

Culture-dependent analyses, similar to culturomics, have been valuable tools in studying the bovine microbiota, especially the rumen microbiota (Kenters et al. Reference Kenters, Henderson and Jeyanathan2011; Liu et al. Reference Liu, Yu and Zhong2023; Nyonyo et al. Reference Nyonyo, Shinkai and Tajima2013; Seshadri et al. Reference Seshadri, Leahy and Attwood2018; Zehavi et al. Reference Zehavi, Probst and Mizrahi2018; Ziemer Reference Ziemer2014); however, as a whole, high-throughput culture has not been as utilized to study the bovine microbiota as it has been in humans (Bilen et al. Reference Bilen, Dufour and Lagier2018; Diakite et al. Reference Diakite, Dubourg and Dione2020; Lagier et al. Reference Lagier, Armougom and Million2012, Reference Lagier, Khelaifia and Alou2016). While it is clear that a rumen and mammary/milk rare biosphere exists (Angelopoulou et al. Reference Angelopoulou, Holohan and Rea2019; Zehavi et al. Reference Zehavi, Probst and Mizrahi2018), the true cultivability of the reproductive and respiratory tract microbiotas remains unexplored. Regardless, many microbes, both commensal and opportunistic pathogens, have been isolated from the bovine microbiota (Figure 4).

Figure 4. Most commonly cultured bacteria from bovine. * indicates an inability to differentiate due to commonly used selective agar.

While most culture-based studies have focused on the rumen, increasing the number of “culturable” rumen microbes, many members of the rumen microbiota still have not been brought into cultivation (Zehavi et al. Reference Zehavi, Probst and Mizrahi2018). Likely, future culturomics studies utilizing a wide array of culture conditions, some of which should be highly similar to the niche of interest (Kenters et al. Reference Kenters, Henderson and Jeyanathan2011; Zehavi et al. Reference Zehavi, Probst and Mizrahi2018; Ziemer Reference Ziemer2014), will increase the number of cultured microbes, providing additional insight into these microbial communities and their overall role in health and productivity. Therefore, there is a great need for large-scale bovine culturomics studies to increase the cultivability of the bovine microbiota and further evaluate the existence and role of low-abundance microbes. Doing so will aid the development of host-derived bacterial therapeutics to increase cattle health and productivity.

Studies utilizing culturomics/high-throughput culture methods

Duquenoy et al. determined bacteria significantly enriched in the ceca of both low- and high-performing chicks and utilized culturomics to isolate these microbes (Duquenoy et al. Reference Duquenoy, Ania and Boucher2020). Their anaerobic culture conditions included three base media (modified Gifu Anaerobic Medium (mGAM), Luria–Bertani Miller medium, and Lactobacillus medium) supplemented with antibiotics, rumen fluid, or sodium taurocholate. Samples also underwent pretreatment with heat and ethanol to isolate spore-forming bacteria. These culture conditions allowed them to isolate 541 colonies, although only 358 could be sub-isolated. These colonies were identified utilizing MALDI-TOF MS and full-length 16S sequencing. Using the full-length 16S sequencing, the authors were able to match the isolates to the corresponding OTU, as they used the QIIME pipeline for their culture-independent analysis. Based on the 16S sequences, the authors concluded that they likely isolated a novel bacterial species from the chicken ceca. Furthermore, the isolates were then screened for anti-Campylobacter jejuni activity. In total, nine isolates were observed to have moderate to strong anti-C. jejuni activity. These isolates belonged to the following genera: Enterococcus, Lactobacillus, Bacillus, and Escherichia (Duquenoy et al. Reference Duquenoy, Ania and Boucher2020). Utilizing 16S sequencing to identify the isolated colonies alone or in conjunction with MALDI-TOF MS does increase the workload; however, it allows researchers to be confident they isolated the OTU or ASV of interest and can allow the identification of potentially novel isolates, as illustrated by Duquenoy et al. (Reference Duquenoy, Ania and Boucher2020).

Tidjani Alou et al. utilized both 16S amplicon sequencing and culturomics to study the gut microbiota of humans with kwashiorkor, also known as severe acute malnutrition (Tidjani Alou et al. Reference Tidjani Alou, Million and Traore2017). They utilized 18 culture conditions consisting of different preincubations and pretreatments, including filtration and thermic shock, as well as differing incubation temperatures and oxygen conditions, to isolate 12,000 colonies. The isolates were then identified using MALDI-TOF MS, and the ones that could not be identified using MS underwent 16S sequencing. In the kwashiorkor samples, the authors isolated nine novel bacterial species and nine novel bacterial genera. In the control samples, the authors isolated 26 new bacterial species, 8 new genera, and 1 new bacterial family. Furthermore, Tidjani Alou et al. isolated the 12 likely potential probiotics, as they were present only in the control samples, and bacteria of their classification are known to have probiotic characteristics. These potential probiotics included Alistipes indistinctus, Anerostipes caccae, Bacillus lichenformis, B. subtilis, Bacteroides salyersiae, Bifidobacterium adolescentis, Intestinimonas butyriciproducens, Lactobacillus parabuchneri, L. perolens, L. vaccinostercus, Terrisporobacter glycolicus, and Weisella confusa (Tidjani Alou et al. Reference Tidjani Alou, Million and Traore2017). However, to our knowledge, they performed no further mechanism screening confirming the isolates’ probiotic capabilities.

Li et al. utilized metagenomics and culturomics to examine the gut microbiota of centenarians (Li et al. Reference Li, Luan and Zhao2022). Based on their metagenomic sequencing data, they identified taxa that differed based on age and identified taxa associated with increased longevity. The authors then utilized 98 sample pretreatments and 23 media compositions to isolate these taxa, resulting in over 8000 isolates, which mostly belonged to 203 known bacterial species; however, novel members of the gastrointestinal tract were also isolated. Under anaerobic conditions, the authors cultured 41 “undefined” isolates that were likely novel. Under microaerobic conditions, one undefined, likely novel isolate was cultured. In addition, the authors noted that 1430 species were identified in their study. However, only 116 species were identified utilizing both culture-independent and -dependent methods, and 140 species were identified using only culturomics (Li et al. Reference Li, Luan and Zhao2022). This indicates the importance of utilizing culturomics with culture-independent methods, as it provides deeper insight into the microbial community and further emphasizes the possible existence of a rare biosphere in the human gastrointestinal tract, proving the importance of integrating culturomics with culture-independent studies when studying the microbiota. Furthermore, Li et al. cultured an increased number of bacterial species from the Enterococcus and Lactobacillus genera from centenarians, similar to their metagenomic data (Li et al. Reference Li, Luan and Zhao2022). While Li et al. did not consider these isolates potential probiotics, their methods provide a valuable example of how pairing culturomics with metagenomic sequencing can result in the isolation of taxa of interest. Future studies could then further characterize those isolates and determine if they provide health-promoting benefits within the human gastrointestinal tract (Li et al. Reference Li, Luan and Zhao2022).

To this point, Wang et al. performed culturomics on healthy human fecal samples in order to isolate potential probiotics, although they did not pair with culture-independent methods (Wang et al. Reference Wang, Howe and Wei2021). They utilized six preincubations, media types, and dilutions. Using these conditions, obtained 1100 colonies, of which 31 were isolated, identified, and screened colonies. These isolates were screened for antimicrobial resistance and both bile salt and low pH resistance to determine if they could survive the gastrointestinal tract. The isolates’ cell-free supernatant was screened for antimicrobial activity against E. coli, S. aureus, and S. typhimurium. The authors concluded that Weisella confusa isolates were potential probiotics (Wang et al. Reference Wang, Liu and Chu2020).

The studies outlined above utilized high-throughput culturomics, and in doing so, they obtained a great number of isolates, many of which were novel microbes or had never been isolated from the niche of interest before (Tidjani Alou et al. Reference Tidjani Alou, Million and Traore2017). These methods also allowed them to culture potential NGPs (Duquenoy et al. Reference Duquenoy, Ania and Boucher2020; Li et al. Reference Li, Luan and Zhao2022; Tidjani Alou et al. Reference Tidjani Alou, Million and Traore2017; Wang et al. Reference Wang, Liu and Chu2020) and potential members of the “rare biosphere” that were not detected by culture-independent analysis (Li et al. Reference Li, Luan and Zhao2022). Moreover, Wang et al. (Reference Wang, Liu and Chu2020) and Duquenoy et al. (Reference Duquenoy, Ania and Boucher2020) performed further screening of isolates deemed potential probiotics. Screening isolates to confirm their health- or production-related benefits and determine their ability to colonize their administration site is crucial. In the following section, we discuss studies where the taxa of interest were not fastidious; therefore, high-throughput culture was unnecessary. These studies employed more high-throughput screening of the isolates than the ones described previously.

Studies utilizing intensive isolate screening

Wang et al. cultured lactic acid-producing bacteria from sow milk on three media types (MRS, trypticase phytone yeast extract, and glucose yeast extract peptone), resulting in 1240 isolates from 271 taxa (Wang et al. Reference Wang, Liu and Chen2022). Additionally, 151 taxa belonged to previously identified microbes, while 120 were unidentified. Furthermore, 80 isolates belonged to the genera Pediococcus. Pediococcus pentosaceus isolates were then screened for antimicrobial activity against Salmonella typhimurium, Enterohemorrhagic Escherichia coli, Enterotoxigenic Escherichia coli, Klebsiella pneumoniae, Aeromonas punctata subsp. Caviae, Staphylococcus aureus, Listeria monocytogenes, and Clostridum perfringens. The top 10 strains based on the antimicrobial assays were then inoculated into Drosophilia melanogaster, which were then treated with paraquat. The flies that were colonized with P. pentosaceus SMM914 had an increased survival rate. Finally, P. pentosaceus SMM914 was fed to piglets before early weaning. The authors concluded that treatment with P. pentosaceus SMM914 alleviated oxidative stress and reduced liver injury (Wang et al. Reference Wang, Liu and Chen2022).

Kang et al. paired microbiome sequencing with culturomics analysis to analyze the gut microbiome of dogs and its relationship with aging (Kang et al. Reference Kang, Mun and Ryu2022). They observed that Lactobacillus and Enterococcus greatly decreased as dogs aged. They then utilized three culture conditions and obtained 305 total isolates. As they were primarily interested in lactic acid-producing bacteria, they selected those colonies to screen for acid and bile tolerance, mucin adhesion, antimicrobial susceptibility, and antimicrobial activity against Listeria monocytogenes, Staphylococcus aureus, Salmonella typhimurium, and Escherichia coli. Then, they utilized a Fermentation of the Intestinal Microbiota Mode model. Using this model, they cultured young and aged canine feces with mGAM media and supplemented the aged feces with Lactobacillus salivarius and Enterococcus hirae. They observed that this supplementation affected the composition and increased the diversity of the microbiota, specifically increasing the Chao1 index of the aged group back to the level of the young group (Kang et al. Reference Kang, Mun and Ryu2022).

Chuang et al. utilized culture-independent and -dependent analyses to study the gut microbiota of calves with diarrhea (Chuang et al. Reference Chuang, Chen and Hsieh2022). Using LeFse analysis, they identified health-associated microbial biomarkers, which they then utilized sample dilutions and one media composition (MRS + 0.05% L-cysteine hydrochloride monohydrate) to isolate these microbes. The isolates were identified using full-length 16S sequencing. The isolated health-associated microbes were then screened for antimicrobial activity against Bacillus cereus, Escherichia coli, Salmonella enterica, Staphylococcus aureus, and Vibrio parahaemolyticus and cytokine stimulation, specifically TNFα and IL-10) of the murine macrophage cell line RAW 264.7. Based on these results, they concluded that two strains of Bifidobacterium longum subsp. longum were NGPs for calf diarrhea (Chuang et al. Reference Chuang, Chen and Hsieh2022).

Amat et al. identified that lactic acid-producing bacteria were negatively correlated with Pasteurellaceae in the bovine nasopharyngeal tract (Amat et al. Reference Amat, Holman and Timsit2019b). To further evaluate the bovine nasopharyngeal microbiota, Amat et al. isolated 300 colonies on MRS and Rogosa agar (Amat et al. Reference Amat, Timsit and Baines2019a). These isolates were then identified using full-length 16S Sanger sequencing. The authors then screened 178 isolates for Mannheimia haemolytica, a BRD opportunistic pathogen, growth inhibition. The authors then screened 47 isolates for adherence to bovine turbinate cells in vitro. Based on adherence assay results, 15 isolates were selected for M. haemolytica competitive inhibition assay and antimicrobial susceptibility testing. From there, 10 isolates were screened for innate and adaptive immune stimulation in vitro. Based on these results, six isolates from four species of Lactobacillus were selected as the best candidates for a bacterial therapeutic cocktail (Amat et al. Reference Amat, Timsit and Baines2019a). This cocktail was then tested in vivo in two different studies (Amat et al. Reference Amat, Alexander and Holman2020, Reference Amat, Timsit and Workentine2023).

The studies outlined above clearly illustrate that there is no “tried and true” pipeline for NGP development. Duquenoy et al. (Reference Duquenoy, Ania and Boucher2020), Chuang et al. (Reference Chuang, Chen and Hsieh2022), Wang et al. (Reference Wang, Liu and Chen2022), and Amat et al. (Reference Amat, Timsit and Baines2019a, Reference Amat, Holman and Timsit2019b, Reference Amat, Alexander and Holman2020, Reference Amat, Timsit and Workentine2023) all illustrate the development of NGPs in veterinary medicine and animal agriculture. However, many of these studies did not focus on isolating fastidious bacteria and, as a result, did not utilize a wide array of culture conditions. Li et al. (Reference Li, Luan and Zhao2022) and Tidjani Alou et al. (Reference Tidjani Alou, Million and Traore2017) were true culturomics-based studies utilizing a wide array of culture conditions; however, they did not heavily screen their isolates-of-interest as was done by Wang et al. (Reference Wang, Liu and Chu2020, Reference Wang, Liu and Chen2022), Chuang et al. (Reference Chuang, Chen and Hsieh2022), Duquenoy et al. (Reference Duquenoy, Ania and Boucher2020) and Amat et al. (Reference Amat, Timsit and Baines2019a, Reference Amat, Alexander and Holman2020, Reference Amat, Timsit and Workentine2023). Such screening is necessary to confirm the NGPs’ potential beneficial health effects observed from culture-independent analyses, their ability to successfully colonize the administration site and to determine their probable function within the microbial community. Therefore, combining the methods of the studies outlined above may provide a useful framework for increasing the culture conditions in NGP development and increasing mechanistic screening of isolates in culturomics studies. Additionally, utilizing a wider variety of culture conditions may be valuable even if the microbe of interest is not fastidious, as many studies (Angelopoulou et al. Reference Angelopoulou, Holohan and Rea2019; Li et al. Reference Li, Luan and Zhao2022; Zehavi et al. Reference Zehavi, Probst and Mizrahi2018) have cultured taxa not detected by culture-independent analyses. As NGP development typically identifies taxa of interest from culture-independent studies, these microbes may have undiscovered probiotic potential. Utilizing only a few culture conditions focused only on the microbes of interest could result in missing these isolates.

Isolation methods used in culturomics

There are several effective methods for microbial isolation through culturomics (Figure 5). Traditional isolation relied on randomly picking up the colonies in the bacterial culture plate. This method is low cost; however, it comes with inevitable drawbacks such as increased time and labor and uncertainty. In addition, fast-growing bacteria may inhibit slow-growing bacteria during culture processing. The 96-well plate-based isolation could solve these problems to some extent (Zhang et al. Reference Zhang, Liu and Guo2021). In this workflow, a two-sided barcode PCR system based on Illumina technology was used to target the bacterial 16S rRNA in each well of the plate. This pipeline relieves tedious effort in picking colonies one by one but cannot avoid repeated isolation of the same dominant bacteria. Recently, an automatic machine learning-based high-throughput isolation promotes the development of culturomics (Huang et al. Reference Huang, Sheth and Zhao2023). Huang et al. developed the Culturomics by Automated Microbiome Imaging and Isolation (CAMII) platform. This machine’s workflow can be divided into four functions: (1) an imaging and machine learning algorithm component that selects colonies based on colony morphology; (2) an automated robot that isolates the selected colonies; (3) a pipeline to sequence the isolates; and (4) a bank and database containing the physical isolate and colony morphology and genomic sequence. This tool can greatly reduce the labor and time required for a culturomics project as well as increase precision colony picking. Additionally, Huang et al. have developed a public biobank database based on their results (CAMII strain biobank [microbial-culturomics.com]) with culture, morphology, and genomic data that could help researchers when developing their own culture conditions (Huang et al. Reference Huang, Sheth and Zhao2023). The approach is able to identify different colony morphology and achieve the prediction of phenotype–genotype integration in the isolation process. The authors obtained 26,997 isolates with 1,197 high-quality genomes from humans in this paper, pioneering a new and effective automated culturomics method.

Figure 5. Different isolation methods for culturomics.

Culturomics strategies and conditions

The National Institute of Health and the Human Microbiome Project have developed a Most Wanted List describing the taxa they deem the highest priority to bring into cultivation (https://www.hmpdacc.org/most_wanted/) (Fodor et al. Reference Fodor, DeSantis and Wylie2012). In this section, we provide a framework for readers when designing culturomics studies and discuss some new methods that can be utilized to culture particularly fastidious microbes of interest.

To the authors’ knowledge, the first culturomics study utilized 212 unique culture conditions, including various pretreatments, nutrient and antibiotic supplementation, oxygen conditions, and filtering steps, to culture microbes from the human gastrointestinal tract. The authors noted that all isolated bacteria could be cultured utilizing only 70 conditions (Lagier et al. Reference Lagier, Armougom and Million2012). Consequently, additional studies have focused on reducing these conditions to a more manageable number and expanding them to culture a wider variety of microbes. Utilizing the previous conditions and additional ones to cultivate a wider variety of microbes, Lagier et al. isolated 1057 unique microbial species. These included 197 novel species, 187 bacterial species, and 1 archaeal species that had never been isolated from humans, and 146 species that had never been isolated from the human gastrointestinal tract (Lagier et al. Reference Lagier, Khelaifia and Alou2016). Furthermore, Diakite et al. reduced the culture conditions even further (from 58 to 25) and noted that 98% of cultured bacteria could be isolated using only 16 conditions (Diakite et al. Reference Diakite, Dubourg and Dione2020). These studies illustrate that pretreating samples with alcohol allow for isolating spore-forming bacteria (Afouda et al. Reference Afouda, Hocquart and Pham2020; Diakite et al. Reference Diakite, Dubourg and Dione2020) and that filtering and antibiotic supplementation allow for isolating low-abundant microbes (Lagier et al. Reference Lagier, Armougom and Million2012). Although these studies list the media compositions utilized, the specific culture conditions used should mimic the natural environment of the sample being cultured as closely as possible (Kaeberlein et al. Reference Kaeberlein, Lewis and Epstein2002). This can include ensuring the salt composition of the media is similar to that of the sample (Kenters et al. Reference Kenters, Henderson and Jeyanathan2011) or by providing undescribed growth factors from the environment, such as a sterilized sample (Zehavi et al. Reference Zehavi, Probst and Mizrahi2018; Ziemer Reference Ziemer2014). It is possible that the microbe of interest may require an unknown growth factor produced by another microbe within the community. In this instance, coculture methods can be performed to isolate the microbe (Strandwitz et al. Reference Strandwitz, Kim and Terekhova2019).

Coculture can also allow for microbes that require intensive conditions for cultivation to be cultured more easily, as illustrated by Khelaifia et al., who cocultured Bacteroides thetaiotaomicron with Methanobrevibacter smithii. Using this method, M. smithii was able to be cultured aerobically (Khelaifia et al. Reference Khelaifia, Lagier and Nkamga2016). Similarly, Wang et al. illustrated that many bacteria considered strict anaerobes, such as Dorea, Clostridium, Megasphaera, Blautia, Mogibacterium, Prevotella, and Bacteroides could be cultured under aerobic conditions on specific media compositions (Wang et al. Reference Wang, Howe and Wei2021). Both the methods and conditions employed by Khelaifia et al. (Reference Khelaifia, Lagier and Nkamga2016) and Wang et al. (Reference Wang, Howe and Wei2021) can be used to investigate growth conditions that can be used to more easily culture other strict anaerobes aerobically. In addition, Khan et al. developed an oxygen adaptation protocol that increased Faecalibacterium prausnitzii’s oxygen tolerance (Khan et al. Reference Khan, Dwibedi and Sundh2023). Similar methodologies may be utilized for other anaerobic species once cultured. Moreover, as metabolites and growth factors produced by host cells can affect the microbiota (Jensen et al. Reference Jensen, Young and Mathes2020), it is possible that a host cell from the niche of interest may be producing a metabolite required for growth. Jalili-Firoozinezhad et al. developed an “intestine-on-a-chip” model to culture and analyze community interactions between both anaerobic and aerobic microbes and the human intestinal epithelium. While they utilized this method to analyze the microbiota and its interactions (Jalili-Firoozinezhad et al. Reference Jalili-Firoozinezhad, Gazzaniga and Calamari2019), we speculate that, in the future, this model, or ones like it, may be tweaked to isolate fastidious microbes requiring unknown growth factors produced by host cells or multiple microbes.

As previously discussed, antibiotic supplementation and filtering are commonly used to isolate low abundant microbes (Lagier et al. Reference Lagier, Armougom and Million2012), dilutions (Zehavi et al. Reference Zehavi, Probst and Mizrahi2018), as well as single-cell isolation, can also be utilized to selectively culture microbes present in a sample at lower abundances that may be outcompeted by more abundant, less fastidious microbes. Bellais et al. (have illustrated that flow cytometry cell sorting can isolate fastidious microbes anaerobically. Briefly, authors generated antibodies for ATCC strains of Faecalibacterium prausnitzii and DSM strain of Christensenella minuta. These antibodies were then used with flow cytometry to sort F. prausnitzii and C. minuta from fecal samples. They observed that Live/Dead staining and antibody labeling did not affect the cultivability of the bacteria and that the antibody labeling and sorting enriched the microbe of interest (Bellais et al. Reference Bellais, Nehlich and Ania2022).

Culture-independent sequencing data can also be utilized to design culture conditions to isolate fastidious microbes. Metagenomic data can guide cultivation through gene-targeted isolation and stable-isotope probing guided Raman-activated microbial cell sorting (Liu et al. Reference Liu, Moon and Zheng2022). Liu et al. (Reference Liu, Yu and Zhong2023) provide a detailed methodology for utilizing gene-guided isolation methods to isolate ureolytic bacteria from the rumen (Liu et al. Reference Liu, Yu and Zhong2023). Jing et al. illustrates the use and methodology of stable-isotope probing guided Raman-activated microbial cell sorting combined with culturing and sequencing (scRACS-Seq/Culture) to isolate phosphate solubilizing microbes from sewage (Jing et al. Reference Jing, Gong and Pan2022). Additionally, the bioinformatics software Traitar can be used to analyze bacterial genomes and metagenomic assemblies, providing information on 67 traits related to oxygen requirements, carbon and energy sources, and antibiotic susceptibility, providing data valuable for formulating media compositions and developing culture conditions (Weimann et al. Reference Weimann, Mooren and Frank2016). While this review has focused mostly on culturomics as it applies to bacteria, culturomics has also been utilized to study archaea and fungi, and the review by Tidjani Alou et al. discusses considerations for utilizing culturomics for archaea and fungi, as well as bacteria (Tidjani Alou et al. Reference Tidjani Alou, Naud and Khelaifia2020). To aid researchers, we have compiled a table of basal media used in the studies cited in this review (Supplementary Table S1). For brevity, we did not include the preincubations, nutritive additives, or antimicrobial additives used by these studies. If a specific culture condition is needed, researchers can see the corresponding citation. Additionally, the review by Tidjani Alou et al. also contains a table of media compositions utilized by their citations as well (Tidjani Alou et al. Reference Tidjani Alou, Naud and Khelaifia2020).