Unveiling the Microbial Frontier: Klebsiella pneumoniae 

Klebsiella pneumoniae, a ubiquitous bacterium in human microbiomes, presents a growing paradox in modern medicine. While commonly found on skin and mucosal surfaces of healthy individuals, its multidrug-resistant strains have been declared by the World Health Organization to rank among the most urgent antimicrobial resistance threats globally. This article examines K. pneumoniae’s biological complexity, evolving epidemiology, and how single-cell technologies are revolutionizing the approach to combating resistant infections.

Biological Profile

K. pneumoniae is a rod-shaped bacterium (0.5–0.7 µm wide, 1.5–3.0 µm long) with exceptional adaptive traits. Its protective polysaccharide capsule enables immune evasion, while its ability to ferment diverse carbon sources and utilize adhesins, lipopolysaccharides, and siderophores aids in host colonization. These features allow K. pneumoniae to thrive in hostile environments. K. pneumoniae’s adaptability underlies its clinical importance, with two distinct pathotypes—classical and hypervirulent—exhibiting unique virulence traits and epidemiological impacts.

K. pneumoniae is classified into classical K. pneumoniae and hypervirulent K. pneumoniae (hvKP). Classical K. pneumoniae is globally distributed and causes 18–64% of gram-negative infections in community-acquired pneumonia and 30% in hospital-acquired pneumonia, primarily affecting males over 40 years old. hvKP, however, shows regional and racial prevalence, being more common among Asian, Pacific Islander, and Latino populations. Its heightened virulence, driven by factors like capsules, LPS, pili, and siderophores, leads to severe outcomes such as liver abscesses and brain infections. Due to all of these virulence and lethality factors, K. pneumoniae is at the heart of the ever growing bacterial antibiotic resistance threat.

Source: https://www.cdc.gov/klebsiella/about/index.html

The Antimicrobial Resistance Crisis 

Klebsiella pneumoniae poses a significant antimicrobial resistance (AMR) threat:

• Cephalosporin resistance undermines first-line treatments.
• Carbapenem-resistant pneumoniae (CRKP) has a 20% prevalence in China with a 50% mortality rate in bloodstream infections.
• Pan-drug resistance limits treatment options, including last-resort antibiotics like colistin.

The emergence of CR-hvKP strains, combining hypervirulence and carbapenem resistance, exacerbates treatment challenges. Heterogeneous resistance contributes to 30–40% of treatment failures by evading diagnostics. Research into the bacteria, however, is not progressing quickly enough to answer the resistance crisis.

Traditional research methods face significant limitations when studying bacterial antibiotic resistance. Bulk sequencing, for example, lacks effective approaches for investigating heteroresistance as it masks gene expression heterogeneity at the subpopulation level. These subpopulations can be heavily associated with resistance, leading to the loss of critical information and posing challenges for research. Single cell sequencing tools would allow researchers to investigate how mechanisms at a subpopulation level enable K. pneumoniae to withstand antibiotics across multiple classes. Thus, single-bacterium transcriptome tools would overcome the limitations of bulk sequencing and facilitates analysis of individual cells with high resolution.

The Single-Cell Revolution: VITA Technology Insights 

The VITA platform is the world’s first commercial platform capable of achieving high-throughput single-species, single-bacterium transcriptome sequencing. This innovation enables in-depth sequencing analysis at the single-bacterium level, overcoming the limitations of traditional bulk sequencing methods. The product accurately reveals gene expression patterns of individual bacteria, capturing the heterogeneity within bacterial populations.

Table 1: Summary of sequencing and analysis metrics

Demonstration data shows an example of VITA single-bacterium transcriptome sequencing data for Klebsiella pneumoniae sample (Table 1). A total of 229.3 million reads were obtained, capturing 2,625 bacteria, with a median gene count of 143 per bacterium. This sequencing library was then used to conduct unsupervised clustering analysis on the K. pneumoniae populations by their gene expression profiles and identify genetic expression profiles of the subpopulations (Figure 1).

Figure 1: 5 subpopulations were distinguished based on gene expression profiling analysis of Klebsiella pneumoniae and high expression of mrk gene family is detected in far left subpopulation coloured green and labeled Type 3 fimbriae.

Further analysis identifies five functional subgroups based on gene expression. These subpopulations underscore the inherent heterogeneity within the K. pneumoniae population, reflecting diverse functionalities as revealed by gene expression patterns. Of special interest is the far left subpopulation in the left graph of Figure 1 identified for Type 3 fimbriae and colored green. This subpopulation shows a high expression of the mrk gene family which codes for the phenotypic expression of Type 3 fimbriae, a virulence factor of K. pneumoniae.

Figure 2: Comparison of mrk gene relative expression detection between bulk level and subpopulation level sequencing for Klebsiella pneumoniae

The expression of the mrk family relative to the sample is masked at the bulk level and only evident at the subpopulation level (Figure 2). Trying to examine the mrk expression at the bulk level aggregates all of the transcriptomic data in the sample resulting in relatively low expression levels being detected. Single cell sequencing technology can identify subpopulations by their differential gene expression and highlight these expression patterns that contribute to K. pneumoniae’s virulence. This high-resolution analysis can unmask otherwise hidden heterogeneity within the bacterial population in mechanisms governing antibiotic resistance, virulence factor expression, and metabolic adaptation. This detailed gene expression profiling can also provide unprecedented insights into bacterial population dynamics at single-cell resolution, offering a powerful tool for understanding microbial adaptation and resistance mechanisms.

The VITA Platform has been used globally by hundreds of researchers to analyze thousands of bacterial samples. We firmly believe that this product will significantly advance research on clinical antibiotic resistance mechanisms in bacteria like Klebsiella pneumoniae, providing new support and perspectives for K. pneumoniae prevention and control strategies.

VITA Single-Species Single-Bacteria Transcriptome Product

Conclusion 

Klebsiella pneumoniae’s transformation from commensal to pan-resistant pathogen exemplifies the dual challenges of microbial adaptability and therapeutic innovation. As CR-hvKP strains spread globally, solutions require both biological understanding and technological breakthroughs. In accordance with the declaration by the WHO, M20 Genomics launched the Klebsiella Action Project (KAP) in September of last year. KAP is a global initiative with the aim of accelerating research on hypervirulent Klebsiella pneumoniae (hvKp) ST23 and empowering researchers to take on the threat posed by this pathogen worldwide. By decoding resistance heterogeneity and virulence dynamics at cellular resolution, we’re empowering the next generation of antimicrobial strategies. 

For more on how M20 Genomics is advancing microbiological research with cutting-edge technology, visit our website and products page.

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