Bacterial antimicrobial resistance (AMR) is one of the most significant challenges to current human society. Exposing bacteria to antibiotics can activate their self-saving responses, e.g., filamentation, leading to the development of bacterial AMR. Understanding the molecular changes during the self-saving responses can understand the mechanism of AMR and reveal new inhibition methods of drug-resistant bacteria.
In this abstract, we integrated microfluidics with mass spectrometry-based omics to investigate the mechanism of bacterial AMR during bacterial filamentation when bacterial cells were treated with antibiotics. First, we used a gradient microfluidic chip to observe the morphological changes of bacteria under the impact of antibiotics. Differential proteins were identified by matrix assisted laser desorption/ionization time-of-flight mass spectrometry, and further validated by transcriptome. Combined proteomics and transcriptomic results, three potential protein biomarkers were identified, including fimbrial subunit type 3, penicillin-binding protein activator LpoB, and 30S ribosomal protein S14 from carbapenem-resistant Klebsiella pneumoniae.
We also used an online microfluidics mass spectrometry system for real-time characterization of metabolic changes of bacteria during filamentation under the stimulus of antibiotics. The proteome of bacteria in the chip was also analyzed, and significant pathways, e.g., nucleotide metabolism and coenzyme A biosynthesis, from extended-spectrum beta-lactamase-producing Escherichia coli (ESBL-E. coli) were comprehensively discovered to related to the bacterial filamentation. We further developed a method to inhibit antibiotic-resistant bacteria by combining traditional antibiotics and chemical inhibitors against the enzymes involved in the bacterial self-saving responses.
Besides, we developed single bacterial cell metabolic profiling by mass spectrometry to study bacterial AMR at single-cell level. By utilizing a microprobe controlled by a microoperation platform, single filamentous ESBL-E. coli cells stimulated by antibiotic can be extracted and spray-ionized for mass spectrometry analysis. Heterogeneous among ESBL-E. coli cells under the same antibiotic stimulus condition was observed from mass spectra as well as cell morphology. The metabolic profiles by mass spectrometry of different individual cells can be clustered into subgroups well in accordance with bacterial cell length.
In summary, we combined microfluidics with proteomics, metabolomics, and transcriptomics to comprehensively investigate the mechanism of AMR, discovery biomarkers, as well as develop new inhibition method of antibiotic-resistant bacteria.