The dynamic interaction between a host and its resident microbes, including bacteria, fungi, and viruses, is a complex, dynamic, and multifaceted process that influences various aspects of host health (1) (2). This interaction is distinguished by molecular signals, biochemical communication, and a nuanced equilibrium of microbial communities, all of which have substantial implications for our well-being (3) (4). While some microorganisms are beneficial, playing crucial roles in processes such as digestion and immune system priming, others with pathogenic properties can lead to diseases and infections (5) (6). Previous studies, such as the Human Microbiome Project (HMP), have provided insights into the human microbiome in terms of human health and diseases. The first phase of this project successfully revealed the microbial communities of the human body, while the second phase, Integrative HMP (iHMP), uncovered host–microbiome interactions using the “omics” approach. The findings from these studies highlight that microbial composition and diversity vary among individuals (7).

Post-COVID-19, there has been renewed interest in whether and to what extent the presence of microbes would impact clinical severity and disease outcome after infection with a primary pathogen, like SARS-CoV-2, Dengue or Mycobacterium tuberculosis. Infectious diseases pose a persistent threat to human health because pathogens exploit host–microbe interactions to initiate infections and cause severe diseases. Understanding the complex interplay between hosts and microbes is crucial for developing effective countermeasures against these diseases. The impact of infectious diseases caused by microorganisms extends beyond posing a global burden on public health systems (8). An outstanding example is the recent outbreak caused by SARS-CoV-2, which has caused severe mortality worldwide (9). Infectious diseases, including COVID-19, dengue, and tuberculosis, are the leading causes of death worldwide, especially in low and middle-income countries (LMICs). To alleviate the burden of infectious diseases and enhance global public health, it is essential to examine each type of microbe separately to gain a better understanding of the problem (10).

Based on their immunopathology, these microbes can be classified as intracellular or extracellular. Although extracellular bacterial infections are generally easier to treat, intracellular bacteria pose significant threats to human health. Certain microbes can infiltrate host cells to harness cellular resources and conceal themselves from host defenses (11). Other microorganisms, such as Salmonella enterica and Listeria monocytogenes, can adapt their lifestyle to become intracellular for a limited period. These microorganisms use diverse endocytic mechanisms to enter and evade phagocytic and non-phagocytic cells, thereby avoiding host defense mechanisms. Identification of intracellular microbes is complex because of the limitations of traditional culture-based methods, which hinder timely and accurate identification and characterization. Despite these constraints, there is growing recognition of the significant impact of these microorganisms on host cells. Therefore, it is essential to understand the intracellular diversity of these microorganisms and their impact on health and disease. This article emphasizes the genomics approach in understanding the interaction between host and intracellular microorganisms. These microorganisms may preferentially infect specific cell types for their survival and replication, and this preference may be influenced by factors such as functional responsibilities, availability of nutrients, immune evasion mechanisms, and replication opportunities (12). These elements collectively contribute to the microorganisms’ capacity to flourish within the host cell. Various cell types can host or carry microorganisms, including epithelial cells, immune cells (13), endothelial cells, and fibroblasts (14). The diverse array of cell types contributes to differential immune responses and varying susceptibility to infection by various microorganisms (15). Several studies have indicated that HIV selectively targets CD4⁺ T cells, Mycobacterium tuberculosis and Chlamydia pneumoniae primarily affect macrophages, Epstein–Barr virus targets B cells (16), and hepatitis B virus primarily targets hepatocytes (17) (18). Although the role of immune cells in protecting the body from foreign invaders is widely acknowledged, certain microorganisms have evolved to thrive within these cells (19). Once these microbes infiltrate the host cells, they can manipulate the host’s immune response by evading phagocytosis, the complement system, and innate immune receptors (20). In addition, they hinder apoptosis, exhibit resistance to host effector mechanisms and trigger immune dysfunction, i.e., immunosuppression.

While previously viewed as potential contamination, microbial RNA inadvertently captured in host transcriptome datasets is now recognized in a limited way for its potential to elucidate diverse yet dynamic host-microbiome interactions. A study by Bost et al. allowed simultaneous profiling of infected host cells and taxonomic profiling of the responsible viruses using the Viral-Track approach (21). This method has successfully differentiated hepatitis B-infected cells in human clinical samples and identified specific host factors associated with the viral replication. The study was done on bronchoalveolar lavage (BAL) samples from the COVID-19 patients. Similarly, Lloréns-Rico et al. used an in-house pipeline for the analysis of single-cell RNA sequencing (scRNA-seq) datasets derived from the bronchoalveolar lavage (BAL) samples collected from both COVID-19 patients and non-COVID-19 pneumonia controls. Their analysis revealed a specific bacterial subset associated with various host immune cell populations, such as neutrophils, monocytes, and macrophages (22).

However, it is of utmost importance to consider whether the presence of intracellular microbes’ influences disease severity. To address this, we need a comprehensive understanding of the dynamics of microorganisms specific to various cell types and their functional significance. Conventional methodologies are limited to only known culturable microbes because of their inherent fastidiousness and the time required to adapt according to the culture conditions. In a different approach, bulk RNA sequencing involves studying the combined genetic material of millions of cells, which provides a profile of the average gene functioning across a large group of cells. These methods restrict our ability to understand individual cells and intracellular microbes. Nevertheless, scrutiny of intracellular microorganisms that are specific to certain cell types can be achieved using other evolving methodologies that can identify intracellular microorganisms and their genomic constituents. Single-cell RNA sequencing is a classic example of such a technique (23). Single-cell RNA sequencing (scRNA-seq) allows the unbiased evaluation of cellular heterogeneity by providing comprehensive genome-wide molecular profiles across a diverse range of individual cells (24). This innovation in genomic research has made it possible to sequence intracellular microbial genomes, as a by-product of single cell whole transcriptome sequencing (WTA), thereby facilitating a deeper understanding of their interactions with specific host cells, and enabling the development of effective strategies to combat various infectious diseases (25) (26). In this perspective article, we discuss the role of intracellular microbes within specific cell types and their potential influence on the outcomes of infectious diseases. It also emphasizes emerging technologies that enhance our understanding while addressing associated challenges.

Because of our constant exposure to various pathogens, a substantial portion of the population unknowingly harbors chronic or latent infections, including those caused by viruses, bacteria, or parasites. The microbiota comprises many potential pathogenic species. Therefore, it is highly probable that any new infection will manifest as a co-infection (82). The traditional view of a single host and single pathogen interacting with one another has been challenged by numerous studies, that have introduced the concept of co-infections. Co-infection can occur either due to the reactivation of pre-existing pathogenic microorganisms in the presence of a new pathogen or because of the co-colonization of the host by new pathogenic species. Co-infection modulating the outcome of infections is now emerging as an important area in understanding multiple co-existing pathogens and the host response (83). Some examples of fungal co-infections with primary viral and bacterial infections includes-

The co-presence of microbes in a particular habitat leads to various interactions between them and the infecting primary pathogen. This co-presence results in reciprocal interactions between host-pathogen, host-microbes, and microbes-pathogen, which may result in co-infection (44). However, many of these microbes remain dormant for a long time, but possibly upon infection with any harmful pathogen changes the cellular homeostasis (dysbiosis), which may result in their reactivation. Because of the difficulties in the identification of dormant microbes, co-infection at the cellular level and its consequences in human health and infectious diseases require needs more attention.

Effective management of co-infections requires a comprehensive approach that includes early screening to identify and select specific antimicrobial treatments. Co-infections often resemble other common infections, which can complicate diagnosis and create uncertainty regarding the necessity of antibiotics. Crucial measures included ensuring environmental cleanliness, practicing thorough hand hygiene, and implementing screening and isolation protocols. Antimicrobial stewardship is vital to reduce the occurrence of drug-resistant infections. Prolonged hospital stay, illness, inadequate surveillance, and excessive antibiotic use can contribute to the development of co-infections. Following patient safety guidelines, bundles can reduce the risk of coinfection. Overall, an integrated multimodal strategy is necessary to manage co-infections (86).

Genomics involves the identification of genes and functional components in the genome of an organism. It is an invaluable tool for understanding the impact of intracellular microbes within specific cell types, including their involvement in cellular dysfunction, immune response, and disease development. Recently, Yadav et al. reported the presence of dynamic intracellular microbial species within peripheral blood mononuclear cells (PBMCs) in healthy, infected and recovered individuals from SARS-CoV-2 using scRNA-seq (12). Furthermore, unique tools such as PathogenTrack and Yeskit have been developed to identify intracellular pathogens from scRNA-seq datasets, allowing for the assessment of transcriptomic characteristics at the individual cell level. In addition, a genomics-driven approach has indicated that most of major human cancer types possess an intra-tumoral microbiota. Direct interactions between these microbial communities and immune cells were investigated using co-culture techniques.

The presence of a microbial genome can be a consequence of the following, i) the intracellular presence of dormant or inactive forms of microbes, which may remain within host cells without actively replicating or causing harm, ii) microbial remnants persist even after successful elimination by the host’s defense mechanisms, including their genetic material (DNA or RNA), which then undergoes lysis within the cells (87), iii) uptake of microbial products, (RNA, proteins, and metabolites) from the microbes residing in the extracellular environment by certain cell types, iv) uptake of apoptotic bodies generated from infected cells, and v) transport of exosomes or vesicles containing microbial RNA, from infected cells to neighboring cells, which may unhouse microbes.

Recent research has greatly enhanced our understanding of the human microbiota including, skin, oral, gut and organ-specific microbes. Nevertheless, many unknowns and challenges remain in disentangling intracellular microbes and differential infection outcomes.

The innate immune system comprises a range of defense mechanisms within the host, including physical and chemical barriers. It uses a wide array of mechanisms to combat a broad spectrum of microbial threats and remove harmful substances, including toxins and allergens. Although the innate and adaptive immune systems are often depicted as separate, they typically work in tandem. The innate immune system relies on several components, including complement proteins, phagocytic cells (such as monocytes, macrophages, and neutrophils), and natural killer (NK) cells, to provide immediate defense against various threats. These host defense mechanisms are inaccessible to intracellular microbes. The components of the innate immune system, such as antigen-presenting cells, further activate the adaptive immune system, which employs a more powerful immune response to eliminate infectious agents. In addition, certain microbes have developed the capability to persist within the host despite the presence of both innate and adaptive immune responses. For example, pathogens such as M. tuberculosis, S. enterica, and Neisseria can evade clearance by the immune system even after continuous immune surveillance (90) (Figure 2).

The interaction between intracellular microbes and the host follows a cascade of immune reactions in which the host activates various microbicidal mechanisms at different stages of infection, while the microbes employ different evasion strategies. In particular, for intracellular microbes, both phagocytic and non-phagocytic cells are involved in microbial uptake and target intracellular destruction. Understanding the immune response involved in intracellular killing can deepen our understanding of cell-type-specific mechanisms and the pathogenesis of infection. The first counteraction against intracellular microbes is provided by the components of the innate immune system. The products released by intracellular bacteria are recognized by Toll-like receptors (TLRs) and Nod-like receptors (NLRs), and activates the effector phagocytic cells (46). Phagocytic cells generally recognize microbes based on unique molecular patterns (PAMPs) in the microorganisms, irrespective of their pathogenicity (11). TLRs are responsible for initiating a response that prompts macrophages to produce proteins and peptides with antimicrobial properties. They also activate the transcription of genes involved in the production of reactive oxygen species (ROS), which permits lysosome fusion (91). Some pathogenic microbes, such as Leishmania, Staphylococci, Coxiella and Salmonella, are well organized to survive even the acidic environment and continue to replicate. For example, PhoP/PhoQ regulation in Salmonella helps its intracellular survival (34). Together, these pathogen recognition receptors (PRRs) result in the production of antimicrobial peptides and induction of the inflammatory response mediated by interferons and pro-inflammatory cytokines (TNF-α). Macrophages can also be activated via natural killer cells through interferon (IFN-γ) production, which plays a major role in the killing of intracellular pathogens (92).

Despite decades of research, we still have limited insights into the factors that regulate the severity of infection between individuals. Studying intracellular microbes, especially through culture-based methods, fails to detect scrupulous or metabolically active but unculturable bacteria. One of the challenges in accurately describing the current state of microbial diversity is the difficulties associated with environmental circumstances (pH, osmotic pressure, nutritional components) (95). The major limitations of traditional techniques include the following: i) these methods are time-consuming, as they require the isolation and cultivation of microorganisms in the laboratory; ii) the diversity and complexity of microbial communities may not be accurately known, and iii) not all microorganisms can be cultured, and challenges such as environmental conditions, stress, or acclimatization can hinder their recovery (Figure 3).

Microscopy allows the direct visualization of intracellular microbes, providing valuable morphological information. It is limited by the resolution and size of the microbes that can be visualized, whereas NGS can detect and identify microbes at the molecular level, even if they are too small to be seen under a microscope. Microscopy allows studying a small number of microbes at a time, making it difficult to capture the full diversity and complexity of microbial communities. Conventional microscopy-based methods for identifying intracellular bacteria also have drawbacks, such as resolution limitations that make distinguishing between different species difficult. Precisely localizing internal microorganisms may require additional labelling techniques.

Culture-independent methods, such as metagenomics, metatranscriptomics, metaproteomics, and metabolomics, provide insights into the functional potential and activities of microbial communities in their natural environment (96). Metagenomics involves studying the genomes of host and microbial organisms. Metatranscriptomics provides information on total gene expression in a specific environment. Metaproteomics and metabolomics are other omics branches based on protein expression and metabolite identification and quantification, which help us to understand the active microbial pathways (97) (98).

One might wonder what motivates the in-depth investigation of these microorganisms at the single-cell level. It is important to determine the functional role of intracellular microbes inside specific cells and whether they can modulate the severity of diseases. The answer lies in the fact that various tissues, each responsible for unique physiological functions, exhibit diversity and specialized functions attributed to variations in gene expression and unique characteristics among their constituent cells. Individual cells within tissues show heterogeneity in gene expression and function. A high-throughput cutting-edge alternative emerged, single-cell technology, which can address these limitations, as it enables the sequencing of individual cells and provides different information, including proteomic, epigenomic, transcriptomic, and metagenomic, thereby offering a more comprehensive view of cellular diversity and function.

Bulk RNA sequencing revealed microbes in different samples, including blood (99). However, various cell types, make it challenging to study the specific interactions between microbes and individual cell types such as neutrophils (100). Because of their limited lifespan and inability to be cryopreserved, they are difficult to study. Our understanding of distinct cell and tissue types is compromised because heterogeneity among these cells remains masked. Furthermore, there is still a significant gap in our understanding of which microbes are associated with specific immune cells. This knowledge is particularly vital in the context of immune cells, which play a pivotal role in combating infections and pathogens. It is essential to understand the composition of pre-existing microbes and how they evolve throughout the progression of diseases (101). The key to our knowledge of human infection biology is how the immune system combines signals and coordinated responses from many cell types and how inter-individual diversity in these cell types translates to variations in infection outcomes.

Single-cell RNA-seq (scRNA-seq) technology has made recent advancements that enable the disintegration of complex tissues and host compartments into cell types and their importance in health and diseases. A scRNA-seq study by Hoffman et al. revealed the diverse outcomes of bacterial infection by macrophage subsets. Some cells may allow the replication of microbes, some may inhibit the growth and eliminate the microbe, while some may probably not respond to either side (49). The primary steps involved in scRNA-seq techniques are isolation and capture of single cells, cell lysis, reverse transcription, cDNA amplification, and library preparation. However, these processes vary in procedure depending on the sequencing platform. Among these steps, the isolation and sorting of individual cells are critical yet labor-intensive processes within the single-cell RNA-seq (Figure 4). Cell separation techniques encompass various methods, such as fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), microfluidics (10x Genomics), and microwell-based systems (BD Rhapsody), which are geared toward high-throughput applications. The design of the microwell system is particularly noteworthy because it allows the accommodation of individual cells coupled with a single bead. Subsequently, the process involves cell lysis and mRNA hybridization to the beads, followed by reverse transcription and amplification for sequencing, which serves as a universal PCR priming site. It is equipped with a combination of elements, including a distinctive cell label, a unique molecular index, and an mRNA capture sequence composed of deoxythymidine/oligo (dT). To date, single cell RNA-seq data have mainly been used to investigate host response by analyzing mRNA expression. Accordingly, analyzing either host or microbe alone may limit the understanding of host-microbe interactions. Although, simultaneous study of host and microbe genomes increases the complexity, scRNA-seq can elucidate the influence of diverse intracellular microbial species on the composition and host transcriptional profiles of diverse cells. The “dual RNA-seq” studies allow us to investigate both host and pathogen genomes in parallel (11) (102). New state-of-the-art tools have been developed to analyze the single cell transcriptomic datasets to identify intracellular pathogens at the single-cell level (50, 51). Hence, it can capture all transcripts, including microbial/bacterial transcripts having poly A (not necessarily more than 50 A) (103). Undoubtedly, not all microbes exhibit poly-A tails in their transcripts; nevertheless, a significant majority of microbes manifest this trait, thereby accentuating the value of this approach in elucidating bacterial diversity.

Single-cell transcriptomics has ushered in formidable methodologies for determining microbial diversity. Pioneering-modified techniques, such as Fluidigm C1, Prokaryotic single-cell RNA sequencing (PETRI-seq), microbial split-pool ligation transcriptomics (microSPLiT), multiple annealing and dC-tailing-based quantitative single-cell RNA-seq (MATQ-seq), and Bacterial droplet-based single-cell RNA-seq (BacDrop), have been developed to effectively circumvent the limitations of 16S rDNA-based microbiota profiling (104). Techniques such as Smart‐seq, Smart‐seq2, MATQ‐seq (multiple annealing and dC‐tailing‐based quantitative single‐cell RNA‐seq), MARS‐seq (massively parallel single‐cell RNA‐sequencing), and CEL‐seq (Cell Expression by Linear amplification and sequencing) use fluorescence-activated cell sorting (FACS) for single-cell isolation (105–107). MARS‐seq and CEL‐seq amplify cDNA using an in vitro transcription method wherein the 5′ end of cDNA is connected to poly(A)/poly(C) to build common adaptors in the PCR reaction. Fluidigm C1, Smart‐seq2, MATQ‐seq, Drop-seq, and 10x Genomics use Moloney Murine Leukemia Virus (MMLV) reverse transcriptase to incorporate template‐switching oligos as adaptors for further PCR amplification. These methods also use unique molecular identifiers (UMI) to eliminate PCR amplification bias. Fluidigm C1 has two configurations: Fluidigm C1 96 scRNA-seq and Fluidigm C1 HT scRNA-seq, each tailored for different throughput requirements. This method captures and processes cells on an integrated microfluidic chip (IFC) with multiple capture sites, each capable of capturing a single cell. For library preparation, cDNAs were fragmented, barcoded, and amplified using the protocols recommended by Fluidigm. The libraries were then quantified and sequenced using high-throughput sequencing platforms such as Illumina HiSeq, NovaSeq and recently Nanopore has been used for the same (52, 108, 109). Despite encountering obstacles such as the diversity of cell walls, which are difficult to lyse, high abundance of rRNA, which makes it difficult to isolate non-polyadenylated mRNA (limited mRNA abundance), and mRNA instability, recent discoveries illustrate that single-cell techniques can capture bacterial sequences to a certain extent.

Well, understood and characterized intracellular survival strategies are limited to bacteria and a few parasites (at the moment) and are almost devoid for the fungal species. To understand how fungi manipulate their intracellular environment to ensure their persistence in mammalian hosts, it is necessary to investigate the specific mechanisms employed by these organisms. Fungi possess a rigid cell wall, which makes them less likely to be captured compared to bacteria and viruses (110). Additionally, they contain only 1-3 pg of total RNA per cell, which is 10 times less than that in mammalian cells, necessitating a specific RNA extraction procedure. Some methods are designed for single-cell sequencing of yeast organisms, such as Smart-seq, which uses zymolyase for efficient lysis of yeast cells within the droplets during single-cell droplet formation in the 10x Genomics Chromium Single Cell 3’ protocol (111). The authors validated these results using single-cell time-lapse microscopy. Yeast single-cell RNA sequencing (YscRNA-seq) is capable of detecting the expression of low-abundance noncoding RNAs and nearly half of the protein-coding genome in each cell. The cells were sorted using fluorescence-activated flow cytometry (FACS) in the 96-well plates, and full-length cDNA libraries were generated from biotinylated oligo(dT) and tagged libraries that were captured using streptavidin beads. In contrast, single-cell RNA barcoding and sequencing (SCRB-seq) specifically enriched 3’-end transcripts at the tagmentation stage after FACS sorting (112). However, these techniques have not yet been applied to human host cells; therefore, their diversity remains unknown. Several studies have demonstrated the diverse nature of the host response to antifungal infections through RNA-seq analysis of peripheral blood mononuclear cells (PBMCs) infected with various fungal species. For example, pathogens such as Yersinia pestis and Candida albicans invade and survive within macrophages as part of their life cycle (113, 114). These microorganisms cause a shift from pro-inflammatory to anti-inflammatory states and upregulate genes involved in the inflammasome activation, resulting in bimodality, and influencing infection outcomes in the healthy individuals. Moreover, fungal pathogens like Cryptococcus neoformans, Histoplasma capsulatum, Candida glabrata, Candida albicans, and Aspergillus fumigatus inhibit lysosomal fusion by altering intra-phagosomal pH, manipulating cytokine secretion, and inducing programmed cell death pathways such as pyroptosis and apoptosis. For instance, Candida albicans has been shown to trigger increased arginase activity upon sensing chitin, which leaves nitric oxide synthase (NOS) without a substrate and impairs macrophages’ ability to eliminate the fungus (115). This revelation not only broadens our understanding of microbial interactions within host-associated tissues but also presents a promising prospect for future therapeutic and disease applications. The advancement of integrated host-microbiome single-cell genomics and transcriptomics techniques has established a technological basis that may pave the way for personalized treatment strategies. By understanding complex host-microbiota interactions at the cellular level, these methods may provide insights into disease mechanisms and microbial pathogenesis, potentially resulting in targeted therapies and interventions.

The mechanisms underlying diverse infection outcomes due to host-microbe interactions in the presence of pathogens remain poorly understood. Infection outcomes are not solely defined by the primary pathogen infection abilities. Rather, pre-existing microbiota and host cellular factors play an important role. However, the existing tools for the identification of these intracellular microbes are still limited to obtain a better understanding of their existence and role inside definitive cell types. Hence, the development of new tools and computational pipelines is already forthcoming Several noteworthy research gaps have been identified. These lacunae serve as valuable points for future scientific inquiries. Foremost among these gaps is the pressing need to advance our holistic understanding of the intracellular microbiome. Presently, microbiome research predominantly focuses on the bacteriome, with an emphasis on anatomical regions such as the skin, oral cavity, nasal passages, and gastrointestinal tract. In stark contrast, there is a conspicuous dearth of attention paid to the intracellular diversity of microbiota. Profiling intracellular microbes, which encompass a diverse array of entities including bacteria, archaea, fungi, and viruses, presents serious challenges. These challenges stem from the scarcity of requisite technical and analytical resources, encompassing methodological approaches and computational pipelines. Moreover, genomic-based approaches and novel molecular-based technologies, including single-cell genomics, are becoming accessible, enabling the characterization of the transcriptome at the granular level. This greatly enhances our capacity to capture microbiome diversity and heterogeneity in a seemingly uniform cell population. However, thorough attempts to advance technologies used to analyze pathogens (such as bacteria, fungi, and viruses) accurately and efficiently at the single-cell level are critical. These limitations represent formidable barriers to researchers seeking to delve into the intricacies of the intracellular microbiome. scRNA-seq is an emerging technique to profile host and microbial transcriptomes. Unlike traditional approaches, scRNA-seq can simultaneously capture host and microbial transcriptomes, providing direct insight into host–microbe interactions. However, this part of scRNA-seq is still in its infancy, and it is only used to understand the transcript of either host or microbe. In light of these considerations, addressing these knowledge gaps and developing cutting-edge tools with respect to both experimental facets and computational pipelines to facilitate a comprehensive evaluation of the intracellular microbiome should assume a central position in future scientific investigations.

The interactions between the intracellular microbiota and the host immune system are significant, as they play a critical role in shaping immune development and function. Additionally, they help maintain the symbiosis and integrity of the immune system. While techniques like microscopy, culturing, and DNA sequencing have been successful in environmental and human-associated microbiomes, identifying intracellular microbiomes remains challenging due to diverse cell wall structures and low mRNA abundances. However, single-cell genomics shows promise in capturing microbial sequences along with the host transcriptome, paving the way for joint host-microbiome transcriptome analysis. These advancements have revealed individual variability within the cell-type specific microbial communities, addressing their role in persistent infections and determining differential disease severity. Harnessing genomics strategies enables one to investigate the specific prevalence and function of microbes within the intricate environment of the host immune system. Intracellular microbes significantly impact disease outcomes by modulating cellular functions, yet our understanding of their role in disease severity is limited due to their unculturable nature. The implications of cell-to-cell variability (specifically immune cell types) towards internalizing specific microbes are unclear, yet significant in the background of the COVID-19 pandemic. Our recent lab work (DOI: 10.1016/j.isci.2023.108357) highlighted the non-canonical usage of Single Cell Transcriptomics to understand the microbial dynamics with immune cell types. This review highlights the intricate interactions of the intracellular microbiome, offering insights into predicting disease severity. This approach provides insight into the interactions, defense evasion mechanisms, and possible impact on varying disease outcomes. More innovations in the non-canonical genomic applications needs greater participation, integration, sharing and dissemination for public health benefits.

RP: Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Writing – review & editing. JS: Data curation, Formal analysis, Investigation, Resources, Visualization, Writing – original draft.