Primates Gut Microbiome is Also Subject To Lifestyle Changes
Primates are an order of mammals belonging to tropical regions, generally as forest dwellers, surviving in a natural habitat with a different diet and lifestyle. …Read more
Microbiology took its first great step with the development of a liquid culture medium in 1860 by none other than the “father of microbiology,” Louis Pasteur. Bacterial culture techniques revolutionised the biological sciences. They allow us to isolate and investigate bacteria, yeasts and fungi, and have led to innumerable breakthroughs in diverse fields such as medicine, food technology, biotechnology and waste management. However, a clear weakness of classical microbial culture techniques is that many microorganisms cannot be cultured easily in the laboratory, or at all. This is because they are present in low amounts, are fastidious, have complex nutritional and environmental requirements, or are just problematic to identify on solid or liquid culture media.
In the meantime, molecular techniques have been developed that allow the identification of many microbes that cannot be cultured, including PCR, strain-specific gene or genome sequencing and metagenomics. These innovations have shed light onto the microbial dark matter, microbes that we knew were there but were indeterminate because we could not culture them in the laboratory. Even so, there are several disadvantages that limit the application of metagenomics. Relevant for many analyses is the depth bias, which means that microorganisms present in low concentrations are difficult to identify, even with the recent and sophisticated molecular techniques.
Culturomics is an alternative culture-based technique that uses high-throughput methods to increase the number of culture conditions applied to a sample, thus recovering, culturing and identifying a wider range of species. This method has been successfully used since it was pioneered in 2012 to identify a much more extensive assortment of bacteria present in many types of samples.
The two essential components of culturomics are (1) a wide range of different culture conditions, and (2) a high-throughput method to identify the colonies isolated. The basic procedure takes microbiota samples, which are diluted, homogenized, and aliquoted. At this stage certain pre-treatments such as pasteurisation or enrichment are applied to some. The sample aliquots are then added to a broad array of culture media and incubated under different atmospheric and nutritional conditions. Smart incubators and automated colony-picking systems to manage the large number of isolates produced provide essential analytical tools.
Culture conditions are chosen to identify microbes from particular niches, support the growth of potentially highly fastidious microbes, or to reduce the growth of ubiquitous microbes to allow less common microbes to thrive. The following treatments, among others, can be used in culturomics projects:
Researchers have found that the majority of microbes in a human microbiome sample can be identified by using about 20 different conditions, although these will likely depend on the environment from which the sample has been taken. Of note, there are microorganisms that escape detection by metagenomics and can still be cultured and vice versa.
After colonies have been isolated, an inexpensive and high-throughput method is used to identify the microbes present. Currently, the MALDI-TOF (matrix-assisted laser desorption/ionization-time of flight) mass spectrometry method is often used in culturomics projects due to its rapid turn-around-time, relatively low cost and reliable identification of microorganisms. Following minimal processing, pure colonies are mixed with a matrix solution and allowed to air dry on an ionization plate. Proteins and peptides produced by ionization travel in a vacuum tube towards a detector, which separates them on their mass-to-charge ratio. The resulting mass spectrum is compared to reference spectra within a database to reliably identify the microbes.
A current weakness in the application of MALDI-TOF MS in microbiome samples is a lack of standard spectral databases used to identify microbes. Publicly available databases tend to be narrow in focus, focusing on pathogens. Commercial and in-house databases are inaccessible to other researchers, therefore there is a need for open source spectral databases to improve the identification of microbes and the transparency of the process. Nevertheless, an intrinsic problem is that MALDI-TOF MS only recognizes known species that are already represented in the database. So during culturomics, species that are not identified by MALDI-TOF MS may be among the most interesting ones in the sample, and will need further sequencing-based characterisation.
Molecular- and culture-based techniques can work together to advance both disciplines. Microbial genomics relies on pure cultures to properly phenotype isolates and provide material for high quality genome elucidation. Culturomics essentially produces a large number of pure (single species) colonies, thus can provide crucial material needed to improve the quality of data in databases used for the omics technologies.
In addition, molecular techniques can predict nutrient requirements of difficult-to-culture microbes. Functional analyses can be used to determine likely growth conditions. Some software programs based on artificial intelligence or machine learning algorithms are able to match microbes to existing media that they are likely to grow either well or poorly on based on the genes or functional pathways present. Therefore, genomics can greatly assist identifying hard-to-culture microorganisms by providing additional information on how to grow them in the laboratory more readily.
Culturomics offers potential to cultivate medically important microbes that grow poorly in the laboratory, and to isolate them from specimens at lower cell concentrations normally required by molecular biology. Culturomics can also be used to confirm the presence of viable microbes in a sample of a particular type, and the technique lends itself to identifying the presence of fast-growing yet low concentration microbes in a clinical setting. Even so, the applications of the technique have not yet been developed to the stage that they can reliably be used to diagnose infectious disease. Culturomics essentially is an exploratory tool aimed at the discovery of new species. Once identified, dedicated molecular tests can be easily developed.
An article by Vaca (2022) outlines the difficulties in diagnosing five different fastidious microorganisms. Their highly specific growth conditions arise from their adaptation to their human host, which is difficult to replicate adequately in the laboratory. The Rickettsias in particular are intracellular parasites that require the support of eukaryotic cells for growth. Culturomics offers a means to identify better growth conditions in the laboratory for these microbes, although there are still considerable hurdles to be overcome before culturomics can become an integral part of the diagnosis of infectious diseases.
In addition to medical microbiology, human microbiota projects can expand their scope with the addition of culturomics. Estimates of biodiversity on Earth indicate that we vastly underestimate the number of species present, and these can be found in many types of samples. The Human Microbiome Project has been extremely important for scientists to obtain a thorough understanding of the depth of the human microbiota. However, we know that species are being missed. Minority populations, and bacteria that are difficult to culture, are still not reliably identified.
Culturomics has emerged as a tool to identify an even greater proportion of microorganisms within a sample from a particular ecological niche. The creation of pure cultures via this method is also essential to further characterise the microbial world. For example, culturomics has been shown to increase the number of species recovered from a microbiome sample by 30% compared to pyrosequencing (Lagier, 2012).
Microbes are important players in the biotechnology industry and they are used to make many different products especially food, biofuels and medicines. Large screening programs in industry are used to find microorganisms with useful characteristics for biotech applications. However, there is a bias in how microbes are selected: the majority of microbes from a given environment are difficult to isolate and grow in the laboratory. This means that current screening programs only use a very small proportion of the microorganisms found in any given environment, thus a great deal of potential is missed.
Culturomics offers a way to expand the number of culturable microbes found in a given sample. By using information about growth requirements derived by the interpretation of genomic data, the range of culture conditions used to isolate new microbial strains for biotechnology can be tailored towards isolating strains of interest in the sample. In particular, screening extremophiles discovered via culturomics could identify a wide range of useful enzymes that are particularly suited to high efficiency biotech processes.
The animal microbiome has not yet been widely investigated with culturomics. Yet, the technique could be extremely useful in identifying causal relationships between the gut microbiome and health in a wide range of livestock. In one example, the culturomics technique was performed under anaerobic conditions, and this resulted in a much greater microbial richness compared to culture-independent methods, particularly for low-abundance taxa (Wang, 2021).The technique was able to identify a wide range of strains that differ by biochemical or physiological requirements at the species level that are otherwise difficult to pick up via classical culture or genomic techniques. Novel probiotic strains for animal health applications could be identified. There is also the potential to identify new genes in the sample, or to gain a more thorough understanding of the animal gut microbiome.
Researchers are continuing to improve culturomics to render the technique faster, more efficient and more reliable. Miniaturisation, robotics and advanced sample/isolate handling are important for high throughput microbial culture. Faster cultures that make use of shortened enrichment and incubation times are being developed to aid in the use of culturomics in the clinical setting. Critical analysis of culture conditions can help pare down the number of conditions tested whilst retaining the ability to identify a wide range of microorganisms.
Culturomics has broadened our understanding of the microbial world, and there is promise for its application in myriad settings. However, considerable development work is needed to bring the technology from explorative studies to wider use in many different applications.