Characterization of Outer Membrane Vesicles from multiresistant bacteria

CARRERA PÁEZ LC; GONZALES MACHUCA A; KNECHT C; CARPIO DÍAZ E; VARGAS CV; ÁLVAREZ VE; PIEKAR M; DONIS, N; GAMBINO AS; QUIROGA MP; CENTRÓN D

Congreso; VII Congreso de Bioquímica y Biología Molecular de Bacterias; 2023

The external vesicles (EV) are vesicles that transport, harbor and are able to deliver its content amongst bacterial communities, including genes associated with antimicrobial resistance. Our goal was to investigate the presence of acquired antimicrobial resistance genes (ARG) in the EV of some Gram-Negative multidrug resistant clinical isolates. Strains harboring MDR plasmids from different genera were sequenced by Illumina technology: i) Serratia marcescens SM938 pDCASG6-NDM (137.269 pb, IncC) that harbors intI1 and blaNDM-1, ii) Klebsiella pneumoniae HA31kp (pDCVA3, IncFII that harbors intI1 and blaNDM-5, iii) Pseudomonas aeruginosa PAE 981 (pDCPR3, 331.929pb) that harbors intI1 and blaVIM-2 gene and iv) Escherichia coli SM5 (pDCAG-1, >112.000 pb, IncFII) that harbors blaCTX-M-15 among others. To characterize EV, we used an isolation method that was based on the International Society for Extracellular Vesicles guidelines. This procedure recommends: (i) The source of OMVs must be quantitatively defined, for that reason we adjusted the initial culture of bacteria to OD600 ~ 0.7, (ii) Total quantification of EV, in this case we quantified the proteins with the Micro BCA Protein Assay Kit, (iii) A technique that provides images of individual electric vesicles at high resolution, for this we used transmission electron microscopy (TEM), and (iv) It is needed to have evidence of individual particle analysis technique that estimate biophysical characteristics of EV, for this we did Dynamic light scattering (DSL) or nanoparticle tracking analysis (NTA). DSL and NTA results showed a size of 272nm for S. marcescens SM938, 277nm for K. pneumoniae HA31kp, 200nm for P. aeruginosa PAE 981 and 115nm for E. coli SM5. The results of DSL and NTA are correlative with the images that we obtained with TEM. Finally, by PCR assay we looked for acquired ARG. PCR assays allowed us to detect in DNA from EVs from HA31kp the intI1 and blaNDM-5 genes, from SM5 the blaCTX-M-15 gene and in EV from PAE981 the blaVIM-2 gene. The present study shows an efficient isolation method in different bacterial genera that allows the detection of genes associated with RAM by PCR in EV and highlights the production of EV-harboring ARG with the potential to disseminate to other susceptible strains within the nosocomial niche