Characterization of Outer Membrane Vesicles from multiresistant bacteria

CARRERA PAEZ LAURA; GONZALEZ MACHUCA ADRIAN; KNECHT CAMILA; CARPIO DIAZ, EDUARDO; VARGAS, CLAUDIA; ALVAREZ, VERONICA ELIZABETH; PIEKAR MARIA; DONIS NICOLAS; GAMBINO ANAHI SAMANTA; QUIROGA MARIA PAULA; CENTRON DANIELA

Congreso; CONGRESO DE BIOQUÍMICA Y BIOLOGÍA MOLECULAR DE BACTERIAS; 2023

The external vesicles (EV) are vesicles that transport, harbor and are able to deliver itscontent amongst bacterial communities, including genes associated with antimicrobialresistance. Our goal was to investigate the presence of acquired antimicrobialresistance genes (ARG) in the EV of some Gram-Negative multidrug resistant clinicalisolates. Strains harboring MDR plasmids from different genera were sequenced byIllumina technology: i) Serratia marcescens SM938 pDCASG6-NDM (137.269 pb, IncC)that harbors intI1 and bla NDM-1 , ii) Klebsiella pneumoniae HA31kp (pDCVA3, IncFII thatharbors intI1 and bla NDM-5 , iii) Pseudomonas aeruginosa PAE 981 (pDCPR3, 331.929pb)that harbors intI1 and bla VIM-2 gene and iv) Escherichia coli SM5 (pDCAG-1, >112.000 pb,IncFII) that harbors bla CTX-M-15 among others . To characterize EV, we used an isolationmethod that was based on the International Society for Extracellular Vesicles guidelines.This procedure recommends: (i) The source of OMVs must be quantitatively defined, forthat reason we adjusted the initial culture of bacteria to OD 600 ~ 0.7, (ii) Totalquantification of EV, in this case we quantified the proteins with the Micro BCA ProteinAssay Kit, (iii) A technique that provides images of individual electric vesicles at highresolution, for this we used transmission electron microscopy (TEM), and (iv) It isneeded to have evidence of individual particle analysis technique that estimatebiophysical characteristics of EV, for this we did Dynamic light scattering (DSL) ornanoparticle tracking analysis (NTA). DSL and NTA results showed a size of 272nm forS. marcescens SM938, 277nm for K. pneumoniae HA31kp, 200nm for P. aeruginosaPAE 981 and 115nm for E. coli SM5. The results of DSL and NTA are correlative withthe images that we obtained with TEM. Finally, by PCR assay we looked for acquiredARG. PCR assays allowed us to detect in DNA from EVs from HA31kp the intI1 andbla NDM-5 genes, from SM5 the bla CTX-M-15 gene and in EV from PAE981 the bla VIM-2 gene. Thepresent study shows an efficient isolation method in different bacterial genera thatallows the detection of genes associated with RAM by PCR in EV and highlights theproduction of EV-harboring ARG with the potential to disseminate to other susceptiblestrains within the nosocomial niche.