RESUMEN
Two metallo-ß-lactamase-producing Klebsiella pneumoniae (HA30 and HA31) were isolated in a hospital in Argentina during 2018. K. pneumoniae HA30 was isolated from a rectal swab during the epidemiological surveillance for carbapenemase-producing strains, while K. pneumoniae HA31 was collected from the same patient 4 days after hospitalization. The aim of the present study was to identify the clonal relationships and resistome of these two NDM-producing K. pneumoniae strains isolated from a patient with a fatal outcome. Whole-genome sequencing (WGS) was performed using Illumina MiSeq-I, and subsequent analysis involved genome assembly, annotation, antibiotic resistance gene identification, multilocus sequence typing (MLST), and plasmid characterization using bioinformatics tools. Conjugation assays to E. coli J53 was conducted as previously described. K. pneumoniae HA30 exhibited extensively drug-resistant phenotype, while HA31 was multidrug-resistant as defined by Magiorakos et al., including both resistance to carbapenems, aminoglycosides and ciprofloxacin with blaNDM-5, blaCTX-M-15 and rmtB genes found in both strains. MLST analysis showed that both strains belonged to ST11, differing by only 4 cgSNPs, indicating that K. pneumoniae HA30 and HA31 were the same strain. Conjugation assays revealed that K. pneumoniae HA31 strain possessed a transferable plasmid to E. coli J53. Bioinformatics studies identified that the same strain colonizing an inpatient during hospital admission subsequently caused the infection leading to a fatal outcome, being the first report of blaNDM-5, rmtB and blaCTX-M-15 genes in a K. pneumoniae ST11 strain from Latin America. Our results also highlighted the importance of focusing on epidemiological surveillance programs.
Asunto(s)
Escherichia coli , Klebsiella pneumoniae , Humanos , Klebsiella pneumoniae/genética , Tipificación de Secuencias Multilocus , Genómica , Antibacterianos/farmacología , beta-Lactamasas/genéticaRESUMEN
OBJECTIVES: The worldwide dissemination of carbapenemase-producing Escherichia coli lineages belonging to high-risk clones poses a challenging public health menace. The aim of this work was to investigate genomic features of a colonizing multidrug-resistant strain of Klebsiella pneumoniae carbapenemase (KPC)-producing E. coli from our institution. METHODS: Whole-genome sequencing was done by Illumina MiSeq-I, and de novo assembly was achieved using SPAdes. Resistome, mobilome, plasmids, virulome, and integrons were analysed using ResFinder, AMRFinder, ISFinder, PlasmidFinder, MOB-suite, VirulenceFinder, and IntegronFinder. Sequence types (STs) were identified with pubMLST and BIGSdb databases. Conjugation assays were also performed. RESULTS: Escherichia coli HA25pEc was isolated from a rectal swab sample taken within the framework of the hospital epidemiological surveillance protocol for detection of carbapenemase-producing Enterobacterales. Escherichia coli HA25pEc corresponded to the first report of ST648 co-harbouring blaKPC-2 and blaCTX-M-15 in Latin America from a colonized patient. It had 19 antibiotic resistance genes (ARGs), including blaKPC-2, located on a Tn4401a isoform. Conjugation assays revealed that blaKPC-2 was not transferred by conjugation to E. coli J53 under our experimental conditions. CONCLUSION: Escherichia coli ST648 has been detected previously in companion and farm animals as well as in hospital- and community-acquired infections worldwide. Although scarcely reported as KPC-producers, our finding in a culture surveillance with several acquired ARGs, including blaCTX-M-15, alerts the potential of this clone for worldwide unnoticed spreading of extreme drug resistance to ß-lactams. These data reinforce the importance of carrying out molecular surveillance to identify reservoirs and warn about the dissemination of new international clones in carbapenemase-bearing patients.
Asunto(s)
Farmacorresistencia Bacteriana Múltiple , Escherichia coli , Escherichia coli/genética , Farmacorresistencia Bacteriana Múltiple/genética , beta-Lactamasas/genética , Antibacterianos/farmacología , Klebsiella pneumoniae , Genómica , HospitalesRESUMEN
Objetivo. Estandarizar el cultivo de células HeLa en diferentes condiciones, con el fin de utilizarlo en protocolos de infección con Chlamydia trachomatis serovar L2. Materiales y métodos. Este estudio se llevó a cabo en cuatro fases principales que son: 1.Viabilidad celular por la técnica de azul de tripán y su posterior observación, 2. Estandarización del cultivo de células HeLa, 3. Coloración de Giemsa, 4. Cultivo de células HEp-2.Resultados. Se determinó que la línea celular HeLa debe ser cultivada en medio DMEM, 0,1% de L- glutamina, 10% de SFB. Así mismo, que la coloración de Giemsa es mejor realizarla en un tiempo de 40 minutos por que se evidencia una clara definición de núcleo y citoplasma. Frente a la comparación de las dos líneas celulares se obtuvo que la línea HeLa desde el primer día muestra un crecimiento adecuado y alcanza rápidamente la confluencia esperada, en contraposición la línea HEp-2 presenta un crecimiento más lento pero alcanzando la confluencia deseada al último día.
Objective. The goal of this study was to standardize the cultivation of HeLa cells under different conditions, to be used in Chlamydia trachomatis serovar L2 infection's protocols. Materials and Methods. This study was conducted in four phases that are: 1.Cell Viabilidad by trypan blue technique and his subsequent remark, 2. Standardization HeLa cell culture, 3. Giemsa, 4. Cultivation of Hep-2 cells. Results. As a result of standardization it is determined that the HeLa cell line should be cultured in DMEM, 0.1% L-glutamine, 10% FBS.It was determined that the Giemsa perform better over time of 40 minutes that a clear definition of nucleus and cytoplasm is evident. Comparing against both cell lines HeLa was obtained that the line from day growing well and quickly reaches the expected confluence, the opposed line HEp-2 has a slower growth but achieving the desired confluence the last day.