Mechanisms of colistin resistance in clinical isolates of Klebsiella pneumoniae

Klebsiella pneumoniae, gram-negativna bakterija iz porodice Enterobacterales, ljudski je patogen koji uzrokuje širok spektar infekcija. Pojava rezistencije na karbapeneme, visoko djelotvornu skupinu antibiotika, u enterobakterija uvjetovala je sve češću upotrebu kolistina kao antibiotika zadnje linije obrane. Upravo je zato pojava sojeva K. pneumoniae rezistentnih na kolistin od velikog kliničkog značaja. Cilj ovog istraživanja bio je otkriti mehanizme rezistencije na kolistin u kliničkih izolata K. pneumoniae prikupljenih u Zagrebu. Metodom mikrodilucije u Mueller-Hinton bujonu ispitana je osjetljivost izolata K. pneumoniae na kolistin i utvrđeno je kako je svih 12 izolata rezistentno na taj antibiotik. Metodama lančane reakcije polimerazom i gel elektroforezom ispitana je prisutnost plazmidnih gena mcr i kromosomskih gena pmrA, pmrB, mgrB, phoP i phoQ te je utvrđeno kako je rezistencija na kolistin u ispitivanih izolata posljedica mutacija u genu mgrB. Sekvenciranjem gena mgrB otkriveno je kako je rezistencija uzrokovana dvjema vrstama mutacija: insercijom elementa IS1 unutar regulatorne regije gena te točkastom mutacijom u samom genu. Metodom ERIC-PCR ustanovljeno je da svi ispitivani izolati pripadaju dvama bakterijskim sojevima, što je u skladu s očekivanjima budući da se u svih dvanaest pacijenata radilo o bolničkoj infekciji.Klebsiella pneumoniae, a gram-negative bacterium of the family Enterobacterales, is a human pathogen which causes a broad spectrum of infections. Occurrence of resistance to carbapenems, a highly-effective group of antibiotics, in enterobacteria led to an increase in usage of colistin as a last-resort antibiotic. That is why occurrence of colistin-resistant strains of K. pneumoniae is of great clinical significance. The aim of this experiment was to determine the mechanisms of resistance to colistin in clinical isolates of K. pneumoniae collected in Zagreb. Sensitivity of the isolates to colistin was determined by microdilution in Mueller-Hinton broth, which showed all 12 of the isolates were resistant to the antibiotic. PCR and gel electrophoresis were used to test the presence of plasmid mcr genes and chromosomal pmrA, pmrB, mgrB, phoP and phoQ genes and showed the resistance to colistin was the result of mutations in mgrB gene. Sequencing of mgrB gene showed the resistance was caused by two types of mutations: insertion of IS1 element in the regulatory region of the gene and point mutation within the gene. ERIC-PCR analysis showed all the isolates belong to two bacterial strains, which makes sense since all patients acquired the infection in a hospital