The Lancet | New Treatment Strategies for Multidrug-Resistant Gram-Negative Bacteria

Introduction

In January 2025, The Lancet published a comprehensive review titled "Multidrug-Resistant Gram-Negative Bacterial Infections." This article details the worldwide impact of multidrug-resistant Gram-negative bacteria (MDR-GNB) infections, how they resist treatment, ways to diagnose them, and novel ways to combat their growing threat.

MDR-GNB infections are a major global public health threat. Data from 2021 show that approximately 4.71 million deaths were associated with bacterial resistance, and these pathogens have become the main causative agents of both community-acquired and hospital-acquired infections. However, the development of new therapies for MDR-GNB has been slow, and clinical treatment faces severe challenges.

Main Categories and Resistance Mechanisms of MDR-GNB

The evolution of resistance in MDR-GNB has rendered many traditional antibiotics ineffective, greatly limiting treatment options and increasing clinical complexity. Common Gram-negative pathogens and their mechanisms include:

Enterobacterales

• Examples include Escherichia coli, Klebsiella pneumoniae, Proteus species, Enterobacter species, and Serratia marcescens. These bacteria are usually found in the human gut and can cause infections in the urinary tract, abdomen, and bloodstream. Their resistance mechanisms include:
  • Hydrolysis of third-generation cephalosporins via extended-spectrum β-lactamases (ESBLs) and AmpC β-lactamases.
  • Production of carbapenemases (such as KPC, NDM), which hydrolyze carbapenem antibiotics and render them ineffective.

Pseudomonas aeruginosa and Acinetobacter baumannii

• Both are typical hospital-acquired pathogens with relatively complex resistance mechanisms:
  • Pseudomonas aeruginosa can actively expel various antibiotics through efflux pumps (such as MexAB-OprM) or produce multiple β-lactamases, including carbapenemases (such as VIM, IMP), leading to resistance.
  • Acinetobacter baumannii mediates resistance through porin mutations (such as CarO) and production of OXA-type carbapenemases (such as OXA-23) or metallo-β-lactamases (such as NDM, VIM, IMP).

Other Gram-Negative Bacteria

• For example, Stenotrophomonas maltophilia possesses intrinsic resistance to many antibiotics and often causes infections in immunocompromised patients.

Figure 1. Key mechanisms of action of antibiotics for treating MDR-GNB infections

Advancement in Novel Antibiotics

To combat multidrug-resistant Gram-negative bacteria (MDR-GNB), novel β-lactam/β-lactamase inhibitor combinations (e.g., ceftazidime-avibactam, ceftolozane-tazobactam) have transformed treatment paradigms, targeting carbapenem-resistant Enterobacterales (CRE) and pathogens producing ESBLs, AmpC, and OXA-48 enzymes. Cefiderocol, a siderophore cephalosporin, exploits bacterial iron transport to overcome porin/efflux pump resistance, demonstrating broad activity against CRE, carbapenem-resistant P. aeruginosa, and A. baumannii. Eravacycline, a modified tetracycline, circumvents common resistance mechanisms but remains ineffective against P. aeruginosa. Despite these advances, emerging resistance highlights the need for vigilant antimicrobial stewardship and ongoing clinical surveillance

Advancements in antibiotic development are increasingly driven by breakthroughs in high-resolution technologies. One such innovation, single-bacterium transcriptomics, has emerged as a powerful tool for unraveling antibiotic persistence/resistance mechanisms and host-pathogen interactions. In a study published in Nature Communications in 2023, researchers utilized the smRandom-seq single-microbe transcriptome method—now available as the VITA Single-Bacterium Transcriptome Platform—to profile gene expression alterations in E. coli exposed to antibiotics at the single-bacterial cell level. This high-throughput technique enabled the investigation of gene expression heterogeneity in E. coli under antibiotic pressure, especially the gene expression profiles of the persister subpopulations. For instance, E. coli treated with ciprofloxacin (Fig. 2A) exhibited increased resistance through reduced expression of outer membrane proteins (Fig. 2D) and activation of the SOS response (Fig. 2E). On the other hand, ampicillin exposure led to upregulation of DNA damage response genes and downregulation of metabolic pathways (data not shown here; please refer to the original article for details), potentially contributing to the emergence of persistent subpopulations. The study highlights how smRandom-seq offers high sensitivity and single-bacterium resolution of transcriptional heterogeneity. Such dynamic, molecular-level insights may facilitate the discovery of novel antibiotic targets and the design of drug optimization strategies.

Figure 2. smRandom-seq analysis of E. coli under different antibiotic stresses showed decreased expression of outer membrane protein genes (ompF, tsx, lamB) and increased expression of genes related to the SOS response and ROS degradation.

Leveraging the core advantages of smRandom-seq, M20 Genomics has advanced it into the VITA Single-Bacterium Transcriptome Platform, significantly expanding its accessibility and range of applications. Experimental data show that this platform demonstrates high sensitivity and stability in the single-bacterium analysis of not only E. coli but also other Gram-negative pathogens, precisely capturing transcriptional heterogeneity at the single-bacterium level.

In a pure culture sample of Pseudomonas aeruginosa, the VITA platform captured 2,626 bacterial cells with a median gene count of 115 (Table 1). Dimensionality reduction and clustering based on gene expression profiles divided the bacterial cells into five distinct functional subpopulations (Fig. 3). Such high-resolution analytical capabilities of the VITA platform allow precise dissection of transcriptional heterogeneity in persistent/resistant subpopulations, offering valuable insights into the mechanisms of MDR-GNB infections, potentially accelerating the discovery of new antibiotic targets and improving drug design efficiency.

Table 1. Quality Control Parameters of a Pseudomonas aeruginosa Samples

Figure 3. Five functional subpopulations were identified in the Pseudomonas aeruginosa sample with the VITA platform.

Novel Non-Antibiotic Therapies: Phage Therapy

In addition to the development of new antibiotics, alternative and adjunctive non-antibiotic therapies, such as phage therapy, are gaining increasing attention for combating MDR-GNB. Phage therapy achieves precise treatment by specifically lysing pathogenic bacteria, such as ESBL-E, carbapenem-resistant Enterobacteriaceae, and multidrug-resistant P. aeruginosa.  This targeted approach preserves the host’s commensal microbiota and may help curb the spread of antimicrobial resistance. Multiple clinical trials are currently assessing the efficacy of phage therapy against these difficult-to-treat infections.

Despite this promise, phage therapy faces significant challenges. These include the complexity of host-phage interactions and the lack of efficient methods for identifying optimal bacteria-phage pairs. Addressing these bottlenecks requires high-resolution data and advanced analytical tools. In a study published in Protein & Cell, researchers used the VITA platform combined with the MIC-Phage analysis tool to identify, for the first time at the single-bacterium level, transcriptional activity associations between bacterial hosts and phages in the human gut microbiome. They identified 373 reliable host-phage pairs, about 325 of which were previously unreported. The remaining 48 matched predictions from the Gut Phage Database (GPD). By providing high-resolution single-bacterium transcriptomics data, the VITA platform aids in identifying host-phage specificity, revealing functional heterogeneity, and optimizing phage selection. This technical breakthrough is a key advancement for phage therapy development and helps to overcome the challenges of treating resistant infections.

Figure 4. The VITA platform identified specific bacteria-phage associations in a human gut microbiome sample.

Conclusion

The VITA Single-Bacterium Transcriptome Platform (Fig. 5) stands at the forefront of microbiology research. To date, it has been used to analyze the single-bacterium transcriptome of over 7,000 microbial samples and has contributed to research published in leading journals such as ,Protein & Cell, Angewandte Chemie, Nature Communications, and Nature Microbiology. With its high-resolution analytical capabilities, the VITA platform accurately captures the heterogeneity of resistant subpopulations and tracks host-phage interactions. Its precise single-cell transcriptome profiling helps accelerate the understanding of resistance mechanisms, the development of new antibiotics, and the clinical translation of phage therapy, providing essential technical support to address the challenges of MDR-GNB.

Figure 5. VITA Single-Bacterium Transcriptomics Platform

References

1. Macesic, Nenad et al. “Multidrug-resistant Gram-negative bacterial infections.” Lancet (London, England) vol. 405,10474 (2025): 257-272. 

2.  Xu, Ziye et al. “Droplet-based high-throughput single microbe RNA sequencing by smRandom-seq.” Nature communications vol. 14,1 5130. 23 Aug. 2023

3.  Shen, Yifei et al. “High-throughput single-microbe RNA sequencing reveals adaptive state heterogeneity and host-phage activity associations in human gut microbiome.” Protein & cell vol. 16,3 (2025): 211-226. doi:10.1093/procel/pwae027