Povečanje učinka antibiotikov na bakterije Lactiplantibacillus plantarum z elektroporacijo

Komel, Polona (author), Klančnik, Anja (mentor), Kotnik, Tadej (comentor)

Vse večja problematika odpornosti bakterij proti antibiotikom zahteva inovativne pristope za izboljšanje njihove učinkovitosti. Uporaba elektroporacije, metode za povečanje permeabilnosti celične membrane, je eden izmed pristopov za izboljšanje delovanja antibiotikov na bakterije. V magistrski nalogi smo preučevali vpliv kombinacije elektroporacije in antibiotikov na rast po Gramu pozitivne bakterije Lactiplantibacillus plantarum, pri čemer smo se osredotočili na primerjavo učinka treh različnih antibiotikov z različnimi mehanizmi delovanja. Poleg tega smo bakterije po elektroporaciji, izvedeni pri različnih jakosti električnega polja (0, 5, 10, 15 in 20 kV/cm), inkubirali z antibiotiki eno in 24 ur, da bi ugotovili vpliv časa inkubacije na učinkovitost elektroporacije in delovanje antibiotikov. Bakterije smo elektroporirali v 250 mM saharozni raztopini z dodatkom posameznih antibiotikov: ampicilina, tetraciklina in ciprofloksacina, pri čemer smo uporabili štiri koncentracije: minimalno inhibitorno koncentracijo (MIK) in njeno tri-, deset- in tridesetkratno vrednost. Analizo učinkovitosti elektroporacije in antibiotikov na inhibicijo rasti bakterij smo izvedli z inkubacijo elektroporiranih vzorcev na ploščah z gojiščem MRS in 24-urnim spektrofotometričnim spremljanjem rasti s čitalcem mikrotitrskih plošč. Zaradi previsoke električne prevodnosti vzorcev z višjimi koncentracijami ciprofloksacina smo ta antibiotik izključili iz nadaljnjih testiranj. Rezultati so pokazali, da elektroporacija poveča učinkovitost antibiotikov, zmanjša rast bakterij za več kot eno logaritemsko enoto, pri čemer večja jakost elektroporacije in višje koncentracije antibiotikov privedejo do večje inhibicije rasti. Pri analizi učinka kombinacije elektroporacije s posameznimi antibiotiki (ampicilin in tetraciklin) smo ugotovili, da je ampicilin pri nižjih koncentracijah bolj učinkovito zaviral rast, medtem ko smo pri višjih koncentracijah zaznali večjo inhibicijo rasti pri tetraciklinu. Elektroporacija olajša prehajanje antibiotikov skozi celično steno. Ker se tarče za ampicilin nahajajo pod celično steno, je višja stopnja inhibicije vidna že pri nizkih koncentracijah. V primerjavi z ampicilinom je tarča za delovanje tetraciklina v notranjosti celice, in je zato bolj učinkovit pri višjih koncentracijah.

• Language: Slovenian
• Keywords: Lactiplantibacillus plantarum, elektroporacija, antibiotiki, mehanizmi delovanja, sinergistični učinek
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Publisher: [P. Komel]
• Year: 2024
• PID: 20.500.12556/RUL-164420
• UDC: 579.2:602.621:577.18
• COBISS.SI-ID: 213019907
• Publication date in RUL: 25.10.2024

Secondary language

• Language: English
• Title: Increased effect of antibiotics on Lactiplantibacillus plantarum with electroporation

Abstract

The increasing problem of bacterial resistance to antibiotics requires innovative approaches to improve their effectiveness. The use of electroporation, a method to increase the permeability of the cell membrane, is one such approach to improve the effect of antibiotics on bacteria. In our master's thesis, we investigated the effects of combining electroporation and antibiotics on the growth of the Gram-positive bacterium Lactiplantibacillus plantarum, focusing on comparing the effects of three different antibiotics with different mechanisms of action. We incubated the bacteria with antibiotics, with electroporation at different electric field amplitude (0, 5, 10, 15 and 20 kV/cm), for one hour and 24 hours after electroporation to determine the effects of incubation time on the efficacy of electroporation and antibiotic action. Bacteria were electroporated in a 250 mM sucrose solution containing an antibiotic (ampicillin, tetracycline, or ciprofloxacin) at four concentrations: the minimum inhibitory concentration (MIC) and its threefold, tenfold and thirtyfold values. The efficacy of electroporation and antibiotics in inhibiting bacterial growth was analyzed by incubating the electroporated samples on Petri dishes with MRS growth medium and 24-hour spectrophotometrically monitoring their growth using a microplate reader. Due to the high electric conductivity of the samples with higher concentrations of ciprofloxacin, this antibiotic was excluded from further testing. The results showed that electroporation increases the effectiveness of antibiotics and reduces bacterial growth by more than one logarithmic unit, with higher electroporation intensity and higher antibiotic concentrations leading to greater growth inhibition. When analyzing the effect of combining electroporation with an antibiotic, we found that ampicillin was more effective at lower concentrations, while tetracycline showed greater growth inhibition at higher concentrations. Electroporation eases the transport of anticotics through the cell wall. Since the targets for ampicillin are located beneath the cell wall, a higher level of inhibition is visible even at low concentrations. Compared to ampicillin, the tetracycline targets are located inside the cell, making tetracycline more effective at higher concentrations.

• Keywords: Lactiplantibacillus plantarum, electroporation, antibiotics, mode of action, synergistic effect