Exploring the antagonistic effect of Lacticaseibacillus rhamnosus CRL2244 against ESKAPE Pathogens

CECILIA RODRÍGUEZ; DEMA RAMLAOUI; BRIEA GASCA; AZIZ, ADIBA; LOPEZ, CHRISTINA; SOPHIA GOGEK; CAMILA LEAL; NICHOLAS SALZAMEDA; BONOMO ROBERT A; RAYA RAUL RICARDO; RAMIREZ MARIA SOLEDAD

Conferencia; Gordon research Conference: New Antibacterial Discovery and Development; 2024

In recent years, there has been a marked increase in infections caused by multidrug resistant (MDR) bacteria with high antibiotic resistance, a situation that generates a considerable increase in healthcare costs. Both, the World Health Organization (WHO) and Center for Diseases Control and Prevention (CDC) recognized the urgent need to develop new strategies to combat MDR pathogens1. The aim of this project is to determine if lactic acid bacteria (LAB), generally regarded as safe or GRAS, can be used to treat MDR pathogens belonging to the ESKAPE group of organisms2,3. LAB are part of our normal healthy microbiota, and have been shown to provide many essential health benefits. Previous experiments from Dr. Ramirez’s lab have tested the inhibitory activity of ten LAB strains against MDR pathogens showing promising results. Considering, the previous observation, the specific objectives of this research were to determine the if the LAB Lacticaseibacillus rhamnosus CRL 2244 can be used to inhibit the growth of MDR pathogens and to determine the transcriptional, and morphological effects of LAB bacteria on MDR pathogens. In addition, the initial characterization of the Lcb. rhamnosus CRL 2244 active component was assessed.The Cell-free conditioned media “CFCM” of the CRL 2244 strain was prepared and used to determine inhibitory activity against 30 different MDR strains. In addition, a transwell migration assay was used to determine if cell to cell contact between the MDR pathogens and the LAB strains was required. To determine if the LAB strains could affect pathogen gene expression, RNA extraction and RT-qPCR were used. Scanning electron microscopy was used to observation changes at the morphological level when both bacteria were cultured together. Lastly, CFCM 3k filtration and the study of the physical-chemical characteristics (pH, solubility, temperature and enzyme treatment) were performed to start with the characterization of the active component of CRL 2244. Based on our results, it was determined that Lcb. rhamnosus CRL 2244 has inhibitory activity against MDR pathogens and that cell-to-cell contact was not required for inhibition. Our results also show that bacterial biofilm and outer membrane vesicle gene expression is increased in the presence of CRL 2244, and iron transport gene expression is decreased. Lastly, the 3k filtration showed that the active component is a small water-soluble metabolite that can resist different pH treatments. Temperatures and different enzymes also do not affect its antimicrobial activity. These results are significant because they demonstrate that Lcb. rhamnosus CRL 2244 produces and secretes an active metabolite with inhibitory activity against a wide range of MDR pathogens. With further research, the active component could be isolated and potentially used as an adjuvant to antibiotic therapies to improve the health of people suffering from MDR infections.