Ispitivanje rezistencije na antibiotike kod sojeva bakterija izolovanih od riba poreklom iz različitih sredina

Abstract

U ovom ispitivanju vršeno je uzorkovanje briseva poreklom od klinički zdravih riba koje su poticale iz različitih sredina (ribnjaci, akvarijumi, riblje pijace). Izvršena je izolacija bakterija koje su sastavni deo mikrobioma kože, škrga i creva riba i ispitivana je osetljivost ovih bakterija na određeni broj antibiotika koji se koriste u veterinarskoj i humanoj medicinskoj praksi. Precizna identifikacija ispitivanih sojeva bakterija vršena je primenom metoda PCR, sekvenciranje gena za 16S rRNK, MALDI-TOF. Primenom disk difuzionog testa i E testa ispitivano je fenotipsko ispoljavanje rezistencije na karbapeneme, ureidopeniciline sa i bez inhibitora betalaktamaza, cefalospirine III i IV generacije, aminoglikozide, tetraciklin, kolistin, flurohinolone i hloramfenikol. Prisustvo gena rezistencije, njihova lokalizacija (na hromozomu ili na mobilnim genetičkim elementima) vršena je primenom metode PCR. Kod sojeva koji su ispoljili rezistenciju na nabrojane antibiotike traženi su plazmidi i ispitivana je konjugabilnost izolovanih plazmida. Posmatrano na ukupan broj ispitanih sojeva u ovom istraživanju, bez obzira na rod i vrstu bakterija, ukupno je nađeno 55% sojeva koji su bili osetljivi na sve antibiotike, kod 22,8% sojeva nađena je rezistencija na 3 do 16 antibiotika uključujući i antibiotike koji se koriste isključivo kod ljudi (karbapenemi, ureidopenicilini, cefalosporini III i IV generacije). Ukupno 22,2% sojeva bilo je rezistentno na 1 do 2 antibiotika, mada je i među tim sojevima bilo onih koji su bili rezistentni na antibiotike registrovane samo za upotrebu kod ljudi (ceftazidim, piperacilin). Kod soja A. hydrophila izolovanom iz akvarijumske ribice gupi potvrđen je mehanizam rezistencije nalazom gena rmtB koji je bio lokalizovan na transpozonu Tn1548 smeštenom na konjugabilnom plazmidu koji je po tipu replikona bio kategorisan u grupu IncL/M. Kod sojeva Pseudomonas koji su bili rezistentni na karbapeneme, ureidopeniciline sa i bez inhibitora betalaktamaza, kao i na cefalosporine III i IV generacije, nisu nađeni geni za, karbapenemaze, MßL, ESBL, OXA i AmpC beta-laktamaze (KPC, OXA-23, OXA- 24, OXA-40, OXA-58, VIM, IMP, SPM, GIM, NDM, TEM, SHV, CTX-M-1, CTXM- 9, OXA-1, OXA-9, AmpC grupni kao i pojedinačni-MOXM, CITM, ACCM, EBCM, FOXM, DHAM).Na osnovu dobijenih rezultata primenom E testa, kod 3 soja iz roda Pseudomonas izolovanih od šarana nađena je rezistencija na kolistin sa dobijenim vrednostima MIK 4 μg/mL.

During this research, a series of microbiological smears was collected from clinically healthy fish found in different environments (aquaculture ponds, aquariums, and fish markets) has been done. Bacteria which belong to skin microbiome, gills, and fish intestines have been isolated, and their sensitivity to several antibiotics used in veterinary and human practice has been tested. Precise identification of tested strains of bacteria has been performed with PCR method, 16S rRNA gene sequencing and MALDI-TOF. Phenotypical manifestation of resistance to carbapenems, ureidopenicillins (with and without inhibitors of beta-lactamase), cephalosporins of the third and fourth generation, aminoglycosides, tetracyclines, colistin, fluoroquinolones and chloramphenicol has been tested using disc diffusion method and E test. Presence of resistant genes, their localization (on chromosome or on mobile genetic elements) has been conducted with PCR method. For strains showing resistance to the antibiotics mentioned above, plasmids have been searched and conjugation of isolated plasmids has been tested. Observing the total number of tested strains in this research, regardless of the genus and species of bacteria, 55% of examined strains were found to be sensitive to all antibiotics, and in 22.8% of strains resistance was observed to 3-16 antibiotics, including antibiotics used exclusively in human medicine (carbapenems, ureidopenicillins, cephalosporins of third and fourth generation). In additional 22.2% of strains the resistance to 1 or 2 antibiotics was recorded, including resistance to antibiotics registered for exclusive use in human medicine (ceftazidime, piperacillin). In A. hydrophila strain isolated from aquarium fish guppy that showed resistance to all 16 antibiotics, a mechanism of resistance has been confirmed by identifying gene rmtB which has been localized on transposon Tn1548 located on conjugal plasmid which belongs to group IncL/M type of replicon. In Pseudomonas strains resistant to carbapenems, ureidopenicillins (with and without inhibitors of beta-lactamase), cephalosprins of third and fourth generation, the genes for carbapenemases MßL, ESBL, OXA and AmpC beta-laktamases (KPC, OXA-23, OXA-24, OXA-40, OXA-58, VIM, IMP, SPM, GIM, NDM, TEM, SHV, CTX-M-1, CTX-M-9, OXA-1, OXA-9, group AmpC and specific MOXM, CITM, ACCM, EBCM, FOXM, DHAM) have not been found. Based on results obtained with use of E test, resistence to colistin has been found in 3 strains of Pseudomonas spp. isolated from carp with MIC values of 4 μg/mL.