Integrated Resistome and Quantitative Proteomics Reveal Coordinated Resistance Architecture in MDR and XDR Gram-Negative ICU Pathogens

Objectives

Antimicrobial resistance (AMR) in Gram-negative pathogens is driven by complex and coordinated molecular mechanisms that remain incompletely characterized. This study integrated phenotypic, genomic, and quantitative proteomic analyses to characterize multidrug-resistant (MDR) and extensively drug-resistant (XDR) Gram-negative bacteria circulating in an intensive care unit (ICU) in Northeastern Brazil.

Methods

A total of 259 Gram-negative isolates collected between 2019 and 2021 underwent species identification, antimicrobial susceptibility testing, and targeted qPCR for resistance genes. Klebsiella pneumoniae , Acinetobacter baumannii , and Pseudomonas aeruginosa representing susceptible, MDR, and XDR phenotypes were selected for whole-genome sequencing and label-free quantitative proteomics. Differential protein abundance was assessed using Limma with |log2FC| > 1 and p < 0.05.

Results

K. pneumoniae (47%), A. baumannii (24%), and P. aeruginosa (21%) predominated. Carbapenem resistance reached 44%, 93%, and 61%, respectively, and MDR/XDR phenotypes occurred in >30% of isolates. Genomic analyses revealed dense resistomes with coexisting β-lactamases (blaKPC, blaNDM, blaCTX-M, OXA) and widespread efflux systems. Proteomic profiling demonstrated phenotype-associated differences in outer membrane proteins, transport systems, regulatory proteins, and metabolic pathways. XDR isolates showed additional enrichment of envelope remodeling proteins, stress response mechanisms, and proteostasis-associated factors.

Conclusions

MDR and XDR Gram-negative ICU pathogens exhibit coordinated resistance architecture characterized by accumulation of resistance genes and adaptive proteomic remodeling. Integrated multi-omics approaches provide mechanistic insight into antimicrobial resistance and support improved surveillance and therapeutic strategies.

What is known?

• Antimicrobial resistance is a priority and a serious problem in global health, resulting in high rates of morbidity and mortality.

• Klebsiella pneumoniae , Acinetobacter baumannii , and Pseudomonas aeruginosa are on the World Health Organization’s (WHO) priority list as major causes of morbidity and mortality worldwide.

• Classical characterization of susceptibility and resistance phenotypes does not capture the complexity of antimicrobial resistance and hampers effective control measures and actions to minimize the evolutionary dynamics of resistance in these bacteria.

What is new?

• The study characterizes the phenotypic pattern of antimicrobial susceptibility, the presence and sequencing of the resistome and virulome, and analyzes the label-free quantitative proteome of susceptible, MDR, and XDR phenotypes in strains of K. pneumoniae , A. baumannii , and P. aeruginosa circulating in hospital ICUs in Brazil.

• MDR and XDR gram-negative phenotypes are associated with a dense resistome, with widespread dissemination of beta-lactamase genes ( bla_KPC , bla_NDM , bla_CTX-M , and OXA ) and RND-type ( MEX s) and acrAB-tolC efflux pumps, without changes in virulence genes.

• Proteomic analysis demonstrated increased production of beta-lactamases, components of efflux pump systems, outer membrane protein synthesis, protection for oxidative stress mechanisms, proteins for iron acquisition, and systemic regulators. XDR strains additionally showed enhanced remodeling of the cell envelope, activation of proteostasis, and metabolic adaptation.