World Health Day: Prompting New Technologies to Explore the Mystery of Antimicrobial Resistance with M20 Genomics
In 1946, a historic gathering of 61 nations convened heralding to establish the World Health Organization (WHO), marking a pivotal moment in global health governance. Just two years later, the WHO officially came into existence on April 7th, 1948, marking a monumental leap in international efforts to combat global health challenges. Since 1950, April 7th has been recognized as World Health Day, serving as an annual reminder of our collective commitment to prioritize global health and tackling the most urgent health issues of our time. The theme for World Health Day 2024, "My Health, My Right," emphasizes the urgent need to ensure equitable access to healthcare for all individuals, irrespective of socio-economic background or geographic location. This powerful call to action urges us all to uphold and defense the right to health for every individual.
As we strive to extend healthcare access to everyone, we're faced with the daunting challenge of Antimicrobial Resistance (AMR)—one of the foremost threats to public health in the 21st century. A study published in The Lancet highlighted the devastating impact of AMR, with approximately 1.27 million deaths directly attributed to antimicrobial resistant infections in 2019, and an additional 4.95 million deaths indirectly linked to this phenomenon[1]. The WHO's predictive models suggest a worrisome scenario, forecasting that, fatalities from antibiotic-resistant bacterial infections could exceed 10 million per year (Figure 1).
Figure 1: Projected global deaths from infections with antibiotic-resistant bacteria in 2050 (Review on Antimicrobial Resistance. Antimicrobial Resistance: Tackling a Crisis for the Health and Wealth of Nations 2014)
Meanwhile, AMR also poses a direct threat to healthcare access, undermining the effectiveness of antibiotics and increasing the risk of treatment failure. Multidrug-resistant bacteria are particularly concerning, as they render conventional antibiotic treatments ineffective, leading to infections that are difficult to manage and control. This not only jeopardizes individual health outcomes but also strains healthcare systems, leading to increased healthcare costs and resource allocation challenges. Addressing AMR is essential to safeguarding healthcare access and ensuring that antibiotics remain effective for future generations.
The urgency of mitigating AMR can never be overstated. Without concerted efforts to curb AMR, we risk reverting to a pre-antibiotic era where even minor infections could prove fatal. Recognizing the gravity of the situation, the WHO took action in 2015 by initiating the Global Antimicrobial Resistance and Use Surveillance System (GLASS), aiming to monitor and report on AMR worldwide. The 2022 GLASS report showed that the number of individuals with infections of antibiotic-resistant bacteria continued to increase from 2016 to 2020 (Figure 2).
Figure 2: Trend in AMR (CTAs: countries, territories, and areas; BCIs: bacteriologically confimed infection's; AST: antimicrobial susceptibility test/testing)
The emergence of AMR renders medications ineffective, allowing infections to persist in the body and increasing the risk of transmission to others. Particularly concerning is the rapid global spread of multidrug-resistant and extensively drug-resistant bacteria (also known as "superbugs"), leading to some infections that cannot be treated with existing antimicrobial drugs such as antibiotics. Faced with this significant threat, researchers and clinicians around the world have been attempting to understand and explain bacterial heterogeneity through emerging technologies, thereby providing new insights and approaches to address this critical problem.
Tackle the Issue of Antimicrobial Resistance by Dissecting Cellular Heterogeneity in Microbes with Innovative Biotechnology
AMR poses a formidable challenge to global healthcare, driven by the heterogeneous nature of microbial populations. The development of AMR within microbial communities is a complex and dynamic process, characterized by genetic variations, transcriptional differentiation, and selective pressures exerted by antibiotics[2-3]. As such, combating AMR requires a nuanced understanding of the heterogeneity present within microbial populations, which traditional microbiological methodologies have struggled to address effectively.
Previous technologies in studies on AMR, such as microbial culture methods, MALDI-TOF-MS (Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry), FTIR (Fourier Transform Infrared Spectroscopy), and Raman spectroscopy, have limitations in effectively capturing the cellular heterogeneity in microbial populations. Furthermore, microbial culture methods suffer from low throughput and are inadequate for studying unculturable microorganisms. The latter three methods require costly equipment and the comparison of spectral signatures with established reference spectra, for which there is currently a lack of databases for effective microbial identification. These limitations significantly hinder the in-depth investigation into mechanisms of AMR, underscoring a pressing need for innovative technologies capable of effectively dissecting microbial heterogeneity.
Recent advancements in single-cell transcriptome technologies for microbes have revolutionized our ability to study the heterogeneity in microbial populations and their response to antimicrobial substances. These cutting-edge tools offer unprecedented insights into the molecular dynamics underlying AMR, providing researchers with a deeper understanding of the mechanisms driving resistance. For instance, the development of Micro-SPLiT in 2021 enabled researchers to investigate heterogeneous stress responses across distinct subpopulations of Bacillus subtilis[4]. In the meantime, mDrop-Seq facilitated single-cell transcriptome analysis of Candida albicans and its adaptive mechanisms in response to fluconazole, a common antifungal agent[5].
Furthermore, the introduction of smRandom-seq in 2023 has expanded our ability to study microbial heterogeneity in various bacterial species, including Escherichia coli, Acinetobacter baumannii, Streptococcus pneumoniae, Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus aureus at the single-cell level. This technology has enabled researchers to delve into the single-cell transcriptome mechanisms underlying antibiotic resistance development in Escherichia coli, further enhancing our understanding of AMR at the molecular level[6].
Figure 3: Overview of smRandom-seq
These technological advancements underscore the transformative potential of single-cell transcriptomics in microbes to unravel the complexities of AMR and inform future therapeutic interventions.
Unveiling the Mysteries of Antimicrobial Resistance: Advancing Insights with M20 Genomics
The introduction of our VITA products in 2022 marked a significant breakthrough in the realm of single-cell trancriptomics in microbes. Offering a uniquely precise profiling of transcriptomics in bacteria at the single-cell level, our VITA products empower researchers to delve into the complexities of antimicrobial resistance mechanisms with unprecdented precision. Surpassing the constraints of conventional bulk RNA sequencing methods, our VITA products provide detailed insights into resistant bacterial subpopulations, thus serving as a potent tool for understanding characteristics of the heterogeneity in bacterial populations and investigating antimicrobial resistance mechanisms and pathogen characteristics.
Figure 4: VITA products for microbial single-cell full-length transcriptome sequencing by M20 Genomics
The Global Antimicrobial Resistance and Use Surveillance System (GLASS), with its focus on monitoring eight groups of highly resistant bacterial pathogens, including Acinetobacter species, Escherichia coli, Klebsiella pneumoniae, Salmonella species, Staphylococcus aureus, Streptococcus pneumoniae, Shigella species, and Neisseria gonorrhoeae, highlights the critical importance of analyzing these pathogenic species. In this context, we present the outcomes of VITA products's analysis of Klebsiella pneumoniae, mixed culture of Klebsiella pneumoniae and Escherichia coli, and a human gut microbiota sample, showcasing unprecedented precision. These findings underscore VITA products's efficacy in clinically relevant samples, further validating its significance in advancing our understanding of AMR and enhancing strategies for combating this global health threat.
Single-Bacterium Transcriptome Analysis in Axenic Bacterial Cultures
With its alarming propensity for developing resistance to multiple antibiotics, Klebsiella pneumoniae poses a significant threat to public health worldwide. Utilizing the cutting-edge VITA products, we conducted comprehensive transcriptome profiling on this clinically relevant species. These high-resolution insights into transcriptomic profiles have the potential to facilitate a deeper understanding of resistance mechanisms and profiles.
The sequencing resulted in 69.8 G of high-quality raw data, capturing an estimated number of 2,625 bacteria. A sequencing saturation rate of 70.5% was achieved, reflecting robust data acquisition. Additionally, the median gene count stood at 121 per cell, while the median UMI count reached 293 per cell (Table 1). These results underscore the effectiveness of the VITA products in delivering high-quality data output.
Table 1: Data derived from a Klebsiella pneumoniae sample with VITA products and Illumina Novaseq 6000 sequencer
To further elucidate the functional roles of bacterial subgroups, we conducted an analysis of gene expression across six subpopulations (Figure 5).
Based on the pathways and metabolic processes associated with these differentially expressed genes, we annotated each bacterial subpopulation with their major function. Subpopulations 1 to 5 were found to participate in stress response, ethanolamine utilization, translation, inositol catabolism, and PST sugar transporter, respectively (Figure 6).
Figure 6: UMAP clustering and functional annotation of 6 subpopulations within a Klebsiella pneumoniae sample
Leveraging the capabilities of the VITA products, this comprehensive annotation sheds light on the heterogeneous functional phenotypes within the bacterial population and allows to gain a deeper understanding of molecular mechanisms underlying the cellular heterogeneity within the bacterial population.
Single-Bacterium Transcriptome Analysis of a Mixed Bacterial Culture
The single-bacterium analysis of a mixed culture of Klebsiella pneumoniae and Escherichia coli with VITA products yielded a total of 35.9 G of raw data. In this sample, an estimated number of 3,150 bacterial cells were detected, resulting in the identification of 5,464 total genes. The median gene count was 215, and the median UMI count 611 per cell. The sequencing saturation rate was 66.0%, indicating a high level of data acquisition.
Table 2: Data derived from a mixed culture of Klebsiella pneumoniae and Escherichia coli with VITA products and Illumina Novaseq 6000 sequencer
We conducted dimensionality reduction and clustering on the obtained dataset. The results unveiled two distinct clusters in the sample, representing Klebsiella pneumoniae and Escherichia coli, respectively.
Figure 7: UMAP showing distinct clusters identifying different bacterial species within the mixed culture of Klebsiella pneumoniae and Escherichia coli in the VITA data
Single-Bacterium Transcriptome Analysis in the Human Gut Microbiome
The analysis of a human gut microbiota sample with VITA products yielded a total of 65.4 G of raw data. In this sample, an estimated number of 5,726 cells were captured. Additionally, the median gene count stood at 148 per cell, while the median UMI count reached 467 per cell (Table 3) showcasing the excellent performance of VITA products.
Table 3: Data derived from a human gut microbiome sample with VITA products and Illumina Novaseq 6000 sequencer
We conducted dimensionality reduction and clustering on a pathogenic bacterial species identified within the human microbiota sample, revealing the presence of five distinct subpopulations. Notably, the analysis uncovered an enrichment of genes associated with vancomycin resistance within subpopulation 1, while subpopulation 3 exhibited an enrichment of genes associated to bacterial infection.
Figure 8: Pathway enrichment analysis for subpopulations of a pathogenic bacterial species in a human gut microbiome sample
Unraveling the Complexity of Antimicrobial Resistance with VITA Products
In the ever-evolving landscape of AMR, a comprehensive understanding of its intricate mechanisms is paramount for safeguarding human health. To date, the VITA products have been used on over 5,000 samples, ranging from single-species culture to complex gut microbiota. Such extensive explorations have encompassed a diverse array of clinically relevant species, including Escherichia coli, Acinetobacter baumannii, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Lactobacillus, and Clostridium difficile. By extending the scope of single-cell transcriptomics to bacteria, the VITA products lay the groundwork to gain essential insights into complex resistance mechanisms.
Romain Rolland once said, "Wisdom and friendship are the only lights that illuminate our dark night." This sentiment deeply resonated with the founding team of M20 Genomics, shaping our mission from the very beginning. In the face of the looming threat of Antimicrobial Resistance to global human health and economic development, we recognize the urgent need for global collective action. Leveraging the power of new technologies, we are dedicated to advance global collaboration to tackle significant challenges and ensure the health and safety of all humanity. This steadfast commitment remains our guiding principle
* For further inquiries or to learn more about our VITA products, please feel free to contact us at info@m20genomics.com. We welcome the opportunity to collaborate and contribute to the collective efforts in microbiological research and the fight against AMR
References
[1] Antimicrobial Resistance Collaborators. Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. Lancet. 2022 12; 399(10325):629-655. Erratum in: Lancet. 2022; 400(10358):1102.
[2] Stojowska-Swędrzyńska K, et al. Antibiotic Heteroresistance in Klebsiella pneumoniae. International Journal of Molecular Sciences. 2022; 23(1): 449.
[3] Andersson D I, et al. Mechanisms and clinical relevance of bacterial heteroresistance. Nat Rev Microbiol. 2019; 17: 479–496.
[4] Kuchina A. et al. Microbial single-cell RNA sequencing by split-pool barcoding. Science. 2021; 371: eaba5257.
[5] Dohn R, et al. mDrop-Seq: Massively Parallel Single-Cell RNA-Seq of Saccharomyces cerevisiae and Candida albicans. Vaccines (Basel). 2021; 10(1): 30.
[6] Xu Z, et al. Droplet-based high-throughput single microbe RNA sequencing by smRandom-seq. Nat Commun. 2023; 14(1): 5130.
