The Impact of Phytochemicals on Antibiotic-Resistant Microbiota and Advanced Biotyping on Multidrug-Resistant Bacteria

Abstract

Baggrund: Udviklingen af multi-resistente bakterier er blevet en af de største problematikker for den offentlige sundhedssektor. Bakterier udvikler resistens mekanismer, som udvidet spektrum β-laktamase, der forhindrer virkningen af antibiotika og derved begrænser behandlingsmulighederne. Af denne ˚arsag er det vigtigt at finde løsninger, der kan forhindre yderligere eskalering. Her er faktorer som, antiobiotisk stewardship programmer, nye antibakterielle midler og hurtigere identifisering af antibiotisk resistens vigtige nølgepunkter i kampen mod disse mikroogranismer. Metode: I dette studie, testede vi antibakterielle egenskaber af fytokemikalier fra Salicornia Ramosissima, udtaget enten vandligt eller organisk. Den baktericide og bakteriostatiske virkning blev testet ved hjælp af agar brønd diffundering og en optisk-tæthed test. Ydermere, analyserede vi potentialet i at analysere bakterielle resistens proteiner til at identificere resistens mekanismer ved brug af LC-MS/MS. Dette involverede udtrækning af bakterielle proteiner ved hjælp af S-trap baseret in-situ proteolyse og hurtig analyse ved hjælp af hurtig LC-MS/MS analysis tilkoblet ion mobilitet tandem masse spektrometri. Resultater: Vi observerede ingen baktercid eller bakteriostatisk effekt fra Salicornia Ramosissima ekstraktet med den mulige koncentration af bioaktive stoffer. Dog i forhold til den advancerede biotyping, identificerede vi 30 resistens proteiner ved brug af LC-MS/MS, hvoraf tre (TEM-55, SHV-65 og CTX-M-82) er relaterede til ESBL. Konklusion: Vi kan i dette studie konkludere at koncentrationen af fytokemikalier skal være højere for at f˚a en antibakteriel effekt. Dog vidste LC-MS/MS lovende resultater i forbindelse med identificering af resistens proteiner og denne metodes potentiale i AST bør derfor yderligere analyseres.

Background: The occurrence of multidrug-resistant bacteria has led to one of the greatest challenges in public health care. Bacteria are continuously evolving resistance mechanisms, such as extended spectrum β-lactamases, which alters the effect of antibiotics and thereby limits treatment options. It is therefore crucial to find possible solutions in order to prevent further escalation. Factors such as antibiotic stewardship programs, new antibacterial agents, and faster antibiotic susceptibility testing are keynotes in the combat against these microorganisms. Methodology: In this project, we evaluated the antibacterial capabilities of phytochemicals extracted from Salicornia Ramosissima obtained by aqueous or organic solvent based extraction. Bactericidal and bacteriostatic tests were performed using the agar well diffusion method and an optical density test. In addition, the potential of determining bacterial resistance proteins to identify resistance mechanisms using fast LC-MS/MS for novel antibiotic susceptibility testing by advanced biotyping was also assessed. This involved extraction of bacterial proteins with S-trap based in-situ proteolysis and fast analysis through the Fast LC-MS/MS analysis coupled to ion-mobility tandem mass spectrometry. Results: We did not observe any bactericidal or bacteriostatic effect from the phytochemicals extracted from Salicornia Ramosissima at the available concentrations of bioactive compounds. For the advanced biotyping, we were, however, able to identify 30 resistance proteins using LC-MS/MS, three of which (TEM-55, SHV-65, and CTX-M-82) are related to ESBL. Conclusion: In this study, we can conclude that the concentration of phytochemicals needs to be higher before an antibacterial effect is possible. However, LC-MS/MS demonstrated promising results in determining resistance proteins and the potential of incorporating this system in AST should further analysed.

A master thesis from Aalborg University

The Impact of Phytochemicals on Antibiotic-Resistant Microbiota and Advanced Biotyping on Multidrug-Resistant Bacteria