Molecular responses during bacterial filamentation reveal inhibition methods of drug- resistant bacteria

Bacterial antimicrobial resistance (AMR) is among 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 reveal new inhibition methods of drug-resistant bacteria. Herein, we used an online microfluidics mass spectrometry system for real -time characterization of metabolic changes of bacte-ria during filamentation under the stimulus of antibiotics. Significant pathways, e.g., nucleotide metabolism and coenzyme A biosynthesis, correlated to the filamentation of extended-spectrum beta-lactamase-producing Escherichia coli (ESBL-E. coli) were identified. A cyclic dinucleotide, c- di- GMP, which is derived from nucleotide metab-olism and reported closely related to bacterial resistance and tolerance, was observed significantly up-regulated during the bacterial filamentation. By using a chemical inhibitor, ebselen, to inhibit diguanylate cyclases which catalyzes the synthesis of c- di- GMP, the minimum inhibitory concentration of ceftriaxone against ESBL-E. coli was significantly decreased. This inhibitory effect was also verified with other ESBL-E. coli strains and other beta-lactam antibiotics, i.e., ampicillin. A mutant strain of ESBL-E. coli by knocking out the dgcMgene was used to demonstrate that the inhi-bition of the antibiotic resistance to beta-lactams by ebselen was mediated through the inhibition of the diguanylate cyclase DgcM and the modulation of c- di- GMP levels. Our study uncovers the molecular changes during bacterial filamentation and proposes a method to inhibit antibiotic-resistant bacteria by combining traditional antibiotics and chemical inhibitors against the enzymes involved in bacterial self-saving responses.

Automated summary

Bacterial antimicrobial resistance (AMR) is a major challenge to current society, and understanding the molecular changes during bacterial self-saving responses can lead to new inhibition methods. This study used microfluidics mass spectrometry to identify metabolic changes in extended-spectrum beta-lactamase-producing Escherichia coli (ESBL-E. coli) during filamentation. The up-regulation of cyclic dinucleotide c-di-GMP, which is related to bacterial resistance and tolerance, was observed. By inhibiting the synthesis of c-di-GMP through a chemical inhibitor, the minimum inhibitory concentration of ceftriaxone against ESBL-E. coli was significantly decreased. This study uncovers molecular changes during bacterial filamentation and proposes a method to inhibit antibiotic-resistant bacteria.