Clarithromycin Exerts an Antibiofilm Effect against Salmonella enterica Serovar Typhimurium rdar Biofilm Formation and Transforms the Physiology towards an Apparent Oxygen-Depleted Energy and Carbon Metabolism

Abstract

Upon biofilm formation, production of extracellular matrix components and alteration in physiology and metabolism allows bacteria to build up multicellular communities which can facilitate nutrient acquisition during unfavorable conditions and provide protection towards various forms of environmental stresses to individual cells. Thus, bacterial cells become tolerant against antimicrobials and the immune system within biofilms. In the current study, we evaluated the antibiofilm activity of the macrolides clarithromycin and azithromycin. Clarithromycin showed antibiofilm activity against rdar (red, dry and rough) biofilm formation of the gastrointestinal pathogen Salmonella typhimurium ATCC14028 Nalr at 1.56 μM subinhibitory concentration in standing culture and dissolved cell aggregates at 15 μM in a microaerophilic environment suggesting that the oxygen level affects the activity of the drug. Treatment with clarithromycin significantly decreased transcription and production of the rdar biofilm activator CsgD, with biofilm genes such as csgB and adrA to be consistently downregulated. While fliA and other flagellar regulon genes were upregulated, apparent motility was downregulated. RNA sequencing showed a holistic cell response upon clarithromycin exposure, whereby not only genes involved in the biofilm-related regulatory pathways, but also genes that likely contribute to intrinsic antimicrobial resistance, and the heat shock stress response were differentially regulated. Most significantly, clarithromycin exposure shifts the cells towards an apparent oxygen- and energy- depleted status, whereby the metabolism that channels into oxidative phosphorylation is downregulated, and energy gain by degradation of propane 1,2-diol, ethanolamine and L-arginine catabolism, potentially also to prevent cytosolic, is upregulated. This analysis will allow the subsequent identification of novel intrinsic antimicrobial resistance determinants.

A reconstructed genome‐scale metabolic model of Helicobacter pylori for predicting putative drug targets in clarithromycin and rifampicin resistance conditions

“…Furthermore, our findings revealed a reduction in oxidative phosphorylation in clarithromycin‐resistant conditions. In line with our results, a study conducted on Salmonella enterica serovar Typhimurium also demonstrated that exposure to clarithromycin shifted the cellular status toward oxygen and energy depletion, leading to the downregulation of oxidative phosphorylation pathways 68 . This consistent observation underscores the impact of clarithromycin on metabolic processes across different bacterial species, highlighting its potential to induce a common response in cellular energy dynamics.…”

Gre Factors Are Required for Biofilm Formation in Salmonella enterica Serovar Typhimurium by Targeting Transcription of the csgD Gene

“…RNA was isolated from cultures grown on LB 0 plates at 28 °C for 24 h, as described previously [ 30 ]. qPCR assays were performed as described [ 7 ].…”

Innovative Strategies to Overcome Antimicrobial Resistance and Tolerance

“…While macrolides selectively affect the translation of messenger RNA into proteins by interacting with the 23S RNA of ribosomes, and prevent 50S ribosomal subunit assembly [ 126 ], transcriptional profiling of the response against subinhibitory concentrations of the semi-synthetic macrolide clarithromycin to prevent ancient rdar biofilm formation of Salmonella typhimurium , a biofilm directed by the transcriptional regulator CsgD via the expression of amyloid curli fimbriae and the exopolysaccharide cellulose, indicated selective upregulation of ribosomal subunit genes with their gene products potentially interacting with clarithromycin. Upregulation of the heat shock stress response with folding and holding chaperons is also indicative of impaired protein homeostasis [ 127 ]. Transcriptional analysis further indicated the redirection of microbial metabolism towards an oxygen- and energy-depleted status where energy is derived from L-arginine catabolism and propane-1,2-diol and ethanolamine degradation rather than by oxidative phosphorylation.…”