RESUMO
BACKGROUND: The convergence of hypervirulence and carbapenem resistance in the bacterial pathogen Klebsiella pneumoniae represents a critical global health concern. Hypervirulent K. pneumoniae (hvKp) strains, frequently from sequence type 23 (ST23) and having a K1 capsule, have been associated with severe community-acquired invasive infections. Although hvKp were initially restricted to Southeast Asia and primarily antibiotic-sensitive, carbapenem-resistant hvKp infections are reported worldwide. Here, within the carbapenemase production Enterobacterales surveillance system headed by the Chilean Public Health Institute, we describe the isolation in Chile of a high-risk ST23 dual-carbapenemase-producing hvKp strain, which carbapenemase genes are encoded in a single conjugative plasmid. RESULTS: Phenotypic and molecular tests of this strain revealed an extensive resistance to at least 15 antibiotic classes and the production of KPC-2 and VIM-1 carbapenemases. Unexpectedly, this isolate lacked hypermucoviscosity, challenging this commonly used hvKp identification criteria. Complete genome sequencing and analysis confirmed the K1 capsular type, the KpVP-1 virulence plasmid, and the GIE492 and ICEKp10 genomic islands carrying virulence factors strongly associated with hvKp. Although this isolate belonged to the globally disseminated hvKp clonal group CG23-I, it is unique, as it formed a clade apart from a previously reported Chilean ST23 hvKp isolate and acquired an IncN KPC-2 plasmid highly disseminated in South America (absent in other hvKp genomes), but now including a class-I integron carrying blaVIM-1 and other resistance genes. Notably, this isolate was able to conjugate the double carbapenemase plasmid to an E. coli recipient, conferring resistance to 1st -5th generation cephalosporins (including combinations with beta-lactamase inhibitors), penicillins, monobactams, and carbapenems. CONCLUSIONS: We reported the isolation in Chile of high-risk carbapenem-resistant hvKp carrying a highly transmissible conjugative plasmid encoding KPC-2 and VIM-1 carbapenemases, conferring resistance to most beta-lactams. Furthermore, the lack of hypermucoviscosity argues against this trait as a reliable hvKp marker. These findings highlight the rapid evolution towards multi-drug resistance of hvKp in Chile and globally, as well as the importance of conjugative plasmids and other mobile genetic elements in this convergence. In this regard, genomic approaches provide valuable support to monitor and obtain essential information on these priority pathogens and mobile elements.
Assuntos
Proteínas de Bactérias , Infecções por Klebsiella , Klebsiella pneumoniae , beta-Lactamases , Humanos , Klebsiella pneumoniae/genética , Chile , Escherichia coli , Infecções por Klebsiella/epidemiologia , Infecções por Klebsiella/microbiologia , Plasmídeos , Antibacterianos/farmacologia , Carbapenêmicos/farmacologiaRESUMO
BACKGROUND: Despite representing the largest fraction of animal life, the number of insect species whose genome has been sequenced is barely in the hundreds. The order Dermaptera (the earwigs) suffers from a lack of genomic information despite its unique position as one of the basally derived insect groups and its importance in agroecosystems. As part of a national educational and outreach program in genomics, a plan was formulated to engage the participation of high school students in a genome sequencing project. Students from twelve schools across Chile were instructed to capture earwig specimens in their geographical area, to identify them and to provide material for genome sequencing to be carried out by themselves in their schools. RESULTS: The school students collected specimens from two cosmopolitan earwig species: Euborellia annulipes (Fam. Anisolabididae) and Forficula auricularia (Fam. Forficulidae). Genomic DNA was extracted and, with the help of scientific teams that traveled to the schools, was sequenced using nanopore sequencers. The sequence data obtained for both species was assembled and annotated. We obtained genome sizes of 1.18 Gb (F. auricularia) and 0.94 Gb (E. annulipes) with the number of predicted protein coding genes being 31,800 and 40,000, respectively. Our analysis showed that we were able to capture a high percentage (≥ 93%) of conserved proteins indicating genomes that are useful for comparative and functional analysis. We were also able to characterize structural elements such as repetitive sequences and non-coding RNA genes. Finally, functional categories of genes that are overrepresented in each species suggest important differences in the process underlying the formation of germ cells, and modes of reproduction between them, features that are one of the distinguishing biological properties that characterize these two distant families of Dermaptera. CONCLUSIONS: This work represents an unprecedented instance where the scientific and lay community have come together to collaborate in a genome sequencing project. The versatility and accessibility of nanopore sequencers was key to the success of the initiative. We were able to obtain full genome sequences of two important and widely distributed species of insects which had not been analyzed at this level previously. The data made available by the project should illuminate future studies on the Dermaptera.
Assuntos
Insetos , Animais , Insetos/genética , Análise de Sequência de DNA , ChileRESUMO
The AT-hook has been defined as a DNA binding peptide motif that contains a glycine-arginine-proline (G-R-P) tripeptide core flanked by basic amino acids. Recent reports documented variations in the sequence of AT-hooks and revealed RNA binding activity of some canonical AT-hooks, suggesting a higher structural and functional variability of this protein domain than previously anticipated. Here we describe the discovery and characterization of the extended AT-hook peptide motif (eAT-hook), in which basic amino acids appear symmetrical mainly at a distance of 12-15 amino acids from the G-R-P core. We identified 80 human and 60 mouse eAT-hook proteins and biochemically characterized the eAT-hooks of Tip5/BAZ2A, PTOV1 and GPBP1. Microscale thermophoresis and electrophoretic mobility shift assays reveal the nucleic acid binding features of this peptide motif, and show that eAT-hooks bind RNA with one order of magnitude higher affinity than DNA. In addition, cellular localization studies suggest a role for the N-terminal eAT-hook of PTOV1 in nucleocytoplasmic shuttling. In summary, our findings classify the eAT-hook as a novel nucleic acid binding motif, which potentially mediates various RNA-dependent cellular processes.
Assuntos
Motivos de Aminoácidos/genética , Proteínas de Ligação a DNA/genética , Motivos de Nucleotídeos/genética , RNA/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação/genética , Ligação Competitiva , Biomarcadores Tumorais/química , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , DNA/química , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Células HCT116 , Células HeLa , Humanos , Camundongos , Microscopia de Fluorescência , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Conformação de Ácido Nucleico , Ligação Proteica , Estrutura Terciária de Proteína , RNA/química , RNA/metabolismoRESUMO
A ß-cyclodextrin (ß-CD) nanosponge (NS) was synthesized using diphenyl carbonate (DPC) as a cross-linker to encapsulate the antitumor drug cyclophosphamide (CYC), thus obtaining the NSs-CYC system. The formulation was then associated with magnetite nanoparticles (MNPs) to develop the MNPs-NSs-CYC ternary system. The formulations mentioned above were characterized to confirm the deposition of the MNPs onto the organic matrix and that the superparamagnetic nature of the MNPs was preserved upon association. The association of the MNPs with the NSs-drug complex was confirmed through field emission scanning electron microscopy, energy dispersive spectroscopy, transmission electron microscopy, X-ray photoelectron spectroscopy, dynamic light scattering, ζ-potential, atomic absorption spectroscopy, X-ray powder diffraction, selected area electron diffraction, and vibrating-sample magnetometer. The superparamagnetic properties of the ternary system allowed the release of CYC by utilizing magnetic hyperthermia upon the exposure of an alternating magnetic field (AMF). The drug release experiments were carried out at different frequencies and intensities of the magnetic field, complying with the "Atkinson-Brezovich criterion". The assays in AMF showed the feasibility of release by controlling hyperthermia of the drug, finding that the most efficient conditions were F = 280 kHz, H = 15 mT, and a concentration of MNPs of 5 mg/mL. CYC release was temperature-dependent, facilitated by local heat generation through magnetic hyperthermia. This phenomenon was confirmed by DFT calculations. Furthermore, the ternary systems outperformed the formulations without MNPs regarding the amount of released drug. The MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assays demonstrated that including CYC within the magnetic NS cavities reduced the effects on mitochondrial activity compared to those observed with the free drug. Finally, the magnetic hyperthermia assays showed that the tertiary system allows the generation of apoptosis in HeLa cells, demonstrating that the MNPs embedded maintain their properties to generate hyperthermia. These results suggest that using NSs associated with MNPs could be a potential tool for a controlled drug delivery in tumor therapy since the materials are efficient and potentially nontoxic.
RESUMO
This article describes the synthesis and characterization of two nanocarriers consisting of ß-cyclodextrin-based nanosponges (NSs) inclusion compounds (ICs) and gold nanorods (AuNRs) for potential near-infrared II (NIR-II) drug-delivery systems. These nanosystems sought to improve the stability of two drugs, namely melphalan (MPH) and curcumin (CUR), and to trigger their photothermal release after a laser irradiation stimulus (1064 nm). The inclusion of MPH and CUR inside each NS was confirmed by field emission scanning electron microscopy (FE-SEM), Raman spectroscopy, Fourier transform infrared spectroscopy, (FT-IR) differential scanning calorimetry (DSC), transmission electron microscopy (TEM), and proton nuclear magnetic resonance (1H-NMR). Furthermore, the association of AuNRs with both ICs was confirmed by FE-SEM, energy-dispersive spectroscopy (EDS), TEM, dynamic light scattering (DLS), ζ-potential, and UV-Vis. Moreover, the irradiation assays demonstrated the feasibility of the controlled-photothermal drug release of both MPH and CUR in the second biological window (1000-1300 nm). Finally, MTS assays depicted that the inclusion of MPH and CUR inside the cavities of NSs reduces the effects on mitochondrial activity, as compared to that observed in the free drugs. Overall, these results suggest the use of NSs associated with AuNRs as a potential technology of controlled drug delivery in tumor therapy, since they are efficient and non-toxic materials.
RESUMO
Background The convergence of hypervirulence and carbapenem resistance in the bacterial pathogen Klebsiella pneumoniae represents a critical global health concern. Hypervirulent K. pneumoniae (hvKp) strains, frequently from sequence type 23 (ST23) and having a K1 capsule, have been associated with severe community-acquired invasive infections. Although hvKp were initially restricted to Southeast Asia and primarily antibiotic-sensitive, carbapenem-resistant hvKp infections are reported worldwide. Here, within the carbapenemase production Enterobacterales surveillance system headed by the Chilean Public Health Institute, we describe the isolation in Chile of a high-risk ST23 dual-carbapenemase-producing hvKp strain, which carbapenemase genes are encoded in a single conjugative plasmid. Results Phenotypic and molecular tests of this strain revealed an extensive resistance to at least 15 antibiotic classes and the production of KPC-2 and VIM-1 carbapenemases. Unexpectedly, this isolate lacked hypermucoviscosity, challenging this commonly used hvKp identification criteria. Complete genome sequencing and analysis confirmed the K1 capsular type, the KpVP-1 virulence plasmid, and the GIE492 and ICEKp10 genomic islands carrying virulence factors strongly associated with hvKp. Although this isolate belonged to the globally disseminated hvKp clonal group CG23-I, it is unique, as it formed a clade apart from a previously reported Chilean ST23 hvKp isolate and acquired an IncN KPC-2 plasmid highly disseminated in South America (absent in other hvKp genomes), but now including a class-I integron carrying blaVIM−1 and other resistance genes. Notably, this isolate was able to conjugate the double carbapenemase plasmid to an E. coli recipient, conferring resistance to 1st-5th generation cephalosporins (including combinations with beta-lactamase inhibitors), penicillins, monobactams, and carbapenems. Conclusions We reported the isolation in Chile of high-risk carbapenem-resistant hvKp carrying a highly transmissible conjugative plasmid encoding KPC-2 and VIM-1 carbapenemases, conferring resistance to most beta-lactams. Furthermore, the lack of hypermucoviscosity argues against this trait as a reliable hvKp marker. These findings highlight the rapid evolution towards multi-drug resistance of hvKp in Chile and globally, as well as the importance of conjugative plasmids and other mobile genetic elements in this convergence. In this regard, genomic approaches provide valuable support to monitor and obtain essential information on these priority pathogens and mobile elements.
RESUMO
BACKGROUND: Despite representing the largest fraction of animal life, the number of insect species whose genome has been sequenced is barely in the hundreds. The order Dermaptera (the earwigs) suffers from a lack of genomic information despite its unique position as one of the basally derived insect groups and its importance in agroecosystems. As part of a national educational and outreach program in genomics, a plan was formulated to engage the participation of high school students in a genome sequencing project. Students from twelve schools across Chile were instructed to capture earwig specimens in their geographical area, to identify them and to provide material for genome sequencing to be carried out by themselves in their schools. RESULTS: The school students collected specimens from two cosmopolitan earwig species: Euborellia annulipes (Fam. Anisolabididae) and Forficula auricularia (Fam. Forficulidae). Genomic DNA was extracted and, with the help of scientific teams that traveled to the schools, was sequenced using nanopore sequencers. The sequence data obtained for both species was assembled and annotated. We obtained genome sizes of 1.18 Gb (F. auricularia) and 0.94 Gb (E. annulipes) with the number of predicted protein coding genes being 31,800 and 40,000, respectively. Our analysis showed that we were able to capture a high percentage (≥ 93%) of conserved proteins indicating genomes that are useful for comparative and functional analysis. We were also able to characterize structural elements such as repetitive sequences and non-coding RNA genes. Finally, functional categories of genes that are overrepresented in each species suggest important differences in the process underlying the formation of germ cells, and modes of reproduction between them, features that are one of the distinguishing biological properties that characterize these two distant families of Dermaptera. CONCLUSIONS: This work represents an unprecedented instance where the scientific and lay community have come together to collaborate in a genome sequencing project. The versatility and accessibility of nanopore sequencers was key to the success of the initiative. We were able to obtain full genome sequences of two important and widely distributed species of insects which had not been analyzed at this level previously. The data made available by the project should illuminate future studies on the Dermaptera.
Assuntos
Animais , Insetos/genética , Chile , Análise de Sequência de DNARESUMO
INTRODUCCIÓN: Enterobacterales productores de carbapenemasas (EPC) son una importante causa de infecciones asociadas a la atención en salud (IAAS). El principal reservorio de EPC lo constituyen pacientes infectados y colonizados, sin embargo, también se han identificado reservorios ambientales. OBJETIVO: Detectar la presencia de EPC en los sifones de lavamanos de la unidad de cuidados críticos de pacientes quemados adultos (UPC QMD) y unidad de cuidados críticos de pacientes pediátricos crónicos (UCEP). MÉTODO: Se recolectaron cuatro muestras de sifones de los lavamanos ubicados en el interior de las unidades de pacientes en UCEP y 10 de UPC QMD. A las muestras se les realizó estudio fenotípico y molecular para detección de carbapenemasas en el Instituto de Salud Pública de Chile. RESULTADOS: En los sifones estudiados de UCEP no se aislaron cepas de EPC. En UPC QMD, 50% de los sifones estudiados se aislaron cepas de EPC. CONCLUSIONES: En UPC QMD se objetivó la presencia de EPC en una alta proporción de los sifones de lavamanos testeados, lo que demuestra un reservorio ambiental de bacterias multi-resistentes.
INTRODUCTION: Carbapenemase-producing Enterobacterales (CPE) are an important cause of health care associated infections (HAI). The main reservoir is constituted by infected and colonized patients; however, environmental reservoirs have also been identified. OBJECTIVE: To detect the presence of CPE in the sink traps of the critical care unit for adult burn patients (UPC QMD) and the critical care unit for chronic pediatric patients (UCEP). MATERIAL AND METHOD: Four samples of trap were collected from the sinks located inside the patient units at PICU and 10 at UPC QMD. The samples underwent a phenotypic and molecular study for the detection of carbapenemases at the Institute of Public Health of Chile. RESULTS: In the UCEP no EPC strains were isolated. In UPC QMD, CPE was detected in 50% of the traps. CONCLUSIONS: In UPC QMD, the presence of CPE was observed in a high proportion of the tested sinks traps, which shows an environmental reservoir of multi-resistant bacteria.
Assuntos
Humanos , Criança , Aparelho Sanitário/microbiologia , Infecções por Enterobacteriaceae , Enterobacteriáceas Resistentes a Carbapenêmicos/isolamento & purificação , Proteínas de Bactérias , beta-Lactamases , Reservatórios de Doenças , Acessórios Sanitários , Cuidados Críticos , Higiene das MãosRESUMO
INTRODUCCIÓN: La aparición y diseminación de Enterobacterales resistentes a carbapenémicos ha generado un gran impacto en las infecciones asociadas a la atención de salud en el mundo. Recientemente, en Chile se detectó un brote por Klebsiella pneumoniae productora de carbapenemasas tipo oxacilinasas (OXA) de la subfamilia tipo OXA-48, reportándose los primeros casos en pacientes hospitalizados mayoritariamente en la zona norte del país. OBJETIVO: Determinar los perfiles fenotípicos, genotípicos y de susceptibilidad antimicrobiana de 16 cepas referidas durante mayo del año 2021 desde las regiones de Antofagasta y Metropolitana al Laboratorio de Referencia del Instituto de Salud Pública. METODOLOGÍA: Las cepas provenientes de muestras clínicas fueron analizadas mediante técnicas tradicionales (Kirby-Bauer y epsilometría) y automatizadas, además de técnicas colorimétricas, inmunocromatográficas y moleculares (RPC y PFGE). Resultados: Se detectó la presencia de los genes blaoxa-48 y blaoxa-232 con una resistencia inusual, tanto a carbapenémicos (ertapenem, imipenem y meropenem) como a cefalosporinas (cefepime, cefotaxima y ceftazidima), además de piperacilina/tazobactam y temocilina. Se detectaron dos subtipos por PFGE, siendo predominante el clon CL-Kpn-Spe-329 (93,8%) con dos mecanismos de resistencia identificados: carbapenemasa y β-lactamasa de espectro extendido (BLEE). CONCLUSIÓN: Ante esta alerta epidemiológica es necesario unificar criterios existentes en la red asistencial nacional para la oportuna detección, vigilancia y control de posibles brotes de cepas productores de oxacilinasa tipo OXA-48.
BACKGROUND: The appearance and spread of carbapenems-resistant Enterobacterales have generated a major impact on health care-associated infections worldwide. Recently, a Klebsiella pneumoniae outbreak expressing OXA-48 like-carbapenemases was detected in Chile, the first reported cases corresponded to hospitalized patients mainly from northern Chile. AIM: To characterize the phenotypic and genotypic profiles of antimicrobial susceptibility of 16 clinical isolates referred during May 2021 from Antofagasta and Metropolitan regions to the Reference Laboratory of Instituto de Salud Publica. METHODS: Antimicrobial susceptibility of all strains was analyzed using traditional (Kirby-Bauer and epsilometry) and automated methods, and complemented with colorimetric, immunochromatographic and molecular (PCR and PFGE) techniques. RESULTS: As a result of the genetic characterization, blaoxa-48 and blaoxa-232 genes were detected, showing the isolates an unusual resistance profile to both carbapenems (ertapenem, imipenem, and meropenem) and cephalosporins (cefepime, cefotaxime, and ceftazidine), as well as piperacillin/ tazobactam and temocillin. Two subtypes were detected by PFGE, with a predominant clone CL-Kpn-Spe-329 (93.8%), with two resistance mechanisms identified: carbapenemase and extended-spectrum β-lactamase (ESBL). CONCLUSION: Due to this epidemiological alert, it is essential the establishment of national guidelines for early detection, surveillance, and control of future outbreaks of OXA-48 like carbapenemases isolates.
Assuntos
Humanos , Klebsiella pneumoniae/isolamento & purificação , Klebsiella pneumoniae/genética , Fenótipo , beta-Lactamases , Testes de Sensibilidade Microbiana , Chile , Genótipo , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/enzimologia , Antibacterianos/farmacologiaRESUMO
The aim of this work was to determine the genetic environment and transferability of blaKPC as well as the pulsotypes of KPC-producing Klebsiella pneumoniae strains isolated from clinical samples in Chilean hospitals. Seventeen strains, principally isolated in Santiago (the capital of Chile) during the years 2012 and 2013, were included. The genetic environment of blaKPC was elucidated by PCR mapping and sequencing. Molecular typing was performed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Curing and conjugation experiments were performed with six strains of different sequence types (STs) and pulsotypes. Thirteen pulsotypes and six STs, mainly belonging to clonal complex 258, were found. In addition, seven strains belonged to a new ST assigned ST1161. The blaKPC sequence indicated that 16 strains had the KPC-2 variant; in only one strain (UC331) an amino acid change (R6P) was detected, corresponding to a new KPC variant designated KPC-24. Molecular characterisation of the blaKPC genetic environment revealed two distinct platforms, namely variant 1a and the Tn4401a isoform, with the first being the most common (11/17 strains). Mating experiments failed to produce transconjugants; however, loss of blaKPC was achieved by plasmid curing in all assayed strains. In conclusion, in Chilean strains of K. pneumoniae, blaKPC is primarily found associated with the variant 1a and is located in non-transferable plasmids. In addition, this study highlights the description of the new ST1161 and the new KPC-24 variant.