Survival of Escherichia coli DH5alpha sensitive and aminoglycoside resistant cells in conditions of osmotic and acidic stress

Otkriće antibiotika predstavlja jedno od najvažnijih medicinskih dostignuća koje je omogućilo smanjenje morbiditeta i mortaliteta. Zbog upotrebe antibiotika, dolazi do porasta rezistencije među patogenima. Aminoglikozidi su baktericidni antibiotici širokog spektra. Vežu se na A-mjesto dekodirajućeg dijela ribosomne podjedinice 30S te uzrokuju ugradnju pogrešnih aminokiselina u rastući protein. Novi mehanizam rezistencije kod patogena je metilacija 16S rRNA, tj. nukleotida G1405 i A1408 koji se nalaze u blizini ciljnog mjesta vezanja aminoglikozidnih antibiotika. 16S rRNA metil-transferaze (MT) djeluju na funkcionalno važan dio rRNA te mogu imati negativan biološki učinak na bakterijsku stanicu. Biološki učinak se može očitovati kao povećana osjetljivost bakterije pri stresnim uvjetima što može predstavljati fiziološku slabost koja bi se mogla iskoristiti u smanjenju rezistencije. Svrha rada je bila utvrditi imaju li 16S rRNA metil-transferaze biološki učinak na bakteriju. Ispitano je relativno preživljenje stanica, E. coli DH5α divljeg tipa te stanica E. coli DH5α koje eksprimiraju ispitivane metil-transferaze, u stresnim uvjetima. Stresni uvjeti su bili osmotski i kiselinski stres, dok su ispitivane metil-transferaze uključivale Sgm, RmtA, RmtB, RmtC te ArmA. Rezultati pokazuju da je biološki učinak 16S rRNA metil-transferaza relativan te ovisi o okolišu. U prisutnosti kiselinskog stresa, ekspresija ispitivanih metil-transferaza u bakteriji predstavlja prednost u usporedbi s divljim tipom bakterije, dok kod osmotskog stresa predstavlja teret. Ovaj rad pridonosi određivanju biološkog učinka 16S rRNA metil-transferaza na bakterijsku stanicu koji se može iskoristiti u pokušajima smanjenja rezistencije.The discovery of antibiotics is one of the most important medical events that helped reduce human morbidity and mortality. Due to the use of antibiotics, the frequency of resistance among pathogens has increased. Aminoglycosides are broad-spectrum bactericidal antibiotics. They bind to the A site in the decoding center of the 30S ribosomal subunit and induce misincorporation of amino acids into the growing protein. A new mechanism of resistance in pathogens is methylation of 16S rRNA, precisely nucleotides G1405 and A1408 located in close proximity of the aminoglycoside binding site. 16S rRNA methyltransferases (MTs) methylate a functionally important part of the rRNA and can be associated with a loss of fitness. This can result in hypersensitivity to stress conditions, and can pose a potential physiological weakness that may be exploitable in the reduction of resistance. The intention of this work was to determine whether acquisition of the 16S rRNA methyltransferases has a fitness cost. The survival rate of the wild type and the methyltransferase-producing E. coli strain DH5α was tested at stress conditions. The stress conditions were osmotic and acidic stress, while the tested methyltransferases included Sgm, RmtA, RmtB, RmtC and ArmA. The results show that the fitness cost of 16S rRNA methyltransferases is relative and varies in different conditions. In the presence of acidic stress, the acquisition of the tested methyltransferases by bacteria results in an advantage in comparison to the wild type E. coli, whilst it poses a burden to cells exposed to osmotic stress. This work contributes to the assessment of the fitness cost related to the acquisition of 16S rRNA methyltransferases, which can be used in the attempts to fight resistance