RESUMO
Acinetobacter baumannii es una bacteria, causante de infecciones asociadas a la atención en salud como neumonía, septicemia, meningitis e infecciones urinarias entre otras. Se caracteriza por su capacidad para desarrollar y acumular rápidamente una gran variedad de mecanismos de resistencia a antibióticos. En esta investigación se realizó el análisis genómico de una cepa de A. baumannii ABIBUN 107m que forma parte de un clon persistente en hospitales colombianos, resistente a los antibióticos carbapenémicos (imipenem y meropenem), antibióticos de elección en el tratamiento infecciones causadas por este microorganismo. El genoma de esta bacteria fue secuenciado utilizando técnicas de alto rendimiento, ensamblado y anotado, obteniéndose un genoma constituido por 3954000 pb con 56 contigs; consta de 4256 genes con un tamaño promedio de 912 pb; 3796 CDS de los cuales por anotación 2884 se asignaron a COG; 57 tRNA y un porcentaje de GC de 38,74%. A. baumannii ABIBUN 107m es resistente a β-lactámicos, aminoglicósidos, quinolonas, tetraciclina, sulfonamida y colistina. En su genoma se localizaron genes asociados con el perfil de resistencia ya que presenta serin β-lactamasas (blaADC-38, blaOXA-64, blaOXA-23, bla ampC-like, bla amp(H)-like), metalo β-lactamasa_B; proteínas de unión a penicilina de elevada masa molecular, secuencias de inserción tipo ISAba1; mutaciones en los genes de DNA girasa y topoisomerasa IV subunidad A (gyrA y parC); enzimas modificadoras de aminoglicósidos (aphA-like, aad -like); cloranfenicol aciltransferasa (cat) y dehidropteroato sintasa (sul-1). Se identificaron genes pertenecientes a cinco familias de sistemas de eflujo (RND, MATE, MSF, ATP, SMR).
Acinetobacter baumannii is a bacterium causing health care associated infections such as pneumonia, septicemia, meningitis and urinary infections amongst others. It has great capacity to quickly develop and gather a big variety of drug resistance mechanisms. In this research, the genome of strain A. baumannii ABIBUN 107m was analyzed wich forms part of a persistent clon in Colombian hospitals and its also resistant to carbapenems (imipenem and meropenem), which are the election antibiotics for treatment of infections caused by this microorganism. The genome was sequenced using high performance technology, assembled and annotated. As a result, we obtained a 3954000 bp genome, with 56 contigs; 4256 genes with average size of 912 bp; 3796 CDS; 2884 were assigned to COG; 57 tRNA and GC percentage of 38,74%. The A. baumannii strain ABIBUN 107m, is resistant to the following antibiotic groups: β-lactams, aminoglycosides, quinolones, tetracycline, sulfonamide and colistin. Genes associated with this resistance profile were found in A. baumannii ABIBUN 107m genome serino β-lactamases (blaADC-38, blaOXA-64, blaOXA-23, bla ampC-like, bla amp(H)-like), metallo β-lactamase_B; High Molecular Mass penicillin binding proteins, ISAba1 type insertion sequences, mutations of DNA gyrase and topoisomerase IV subunit A (gyrA and parC); aminoglycoside modifying enzymes (aphA-like, aadA-like); choramphenicol acyltransferase (cat) and dehydropteroate synthase (sul-1). Genes belonging to five different efflux systems were identified (RND, MATE, MSF, ATP, SMR).
RESUMO
INTRODUCTION: Only automated phenotypic methods are currently used in Colombian hospitals for identifying isolates of the Acinetobacter calcoaceticus-A. baumannii complex (ACB). The phenotypical similarities in these species mean that they cannot be differentiated by manual or automated methods, thereby leading to their identification as A. baumannii, or ACB complex in clinical settings. Our objective was to identify to the species level 60 isolates, from four hospitals, evaluate their antibiotic susceptibility, and detect resistance-related genes. METHODS: 16S-23S rRNA internal transcribed spacer (ITS) region and rpoB gene partial sequences were amplified. Resistance genes for cephalosporin, carbapenem and aminoglycoside were detected by PCR. Possible mutations in the quinolone resistance-determining region (QRDR) were evaluated. The association of ISAba-1 with blaOXA and blaADC genes was determined by PCR. Amplification products of ITS region, rpoB gene and some resistance genes were sequenced and compared using the BLAST tool. RESULTS: 16S-23S rRNA ITS region and partial rpoB gene sequence analysis allowed 51isolates to be identified as A. baumannii, 8 as A. nosocomialis, and 1 isolate as A. pitti. A. baumannii isolates were highly resistant to all antibiotics tested, while the others were susceptible to ciprofloxacin and ampicillin/sulbactam. Quinolone resistance, found only in A. baumannii, was associated with mutations in the QRDR region of gyrA and parC genes. CONCLUSION: This is the first investigation in Colombia that has identified ACB complex species using molecular methods, and determined differences in antibiotic resistance and resistance genes among the species. It is of the highest importance to identify isolates to the species level for future resistance and epidemiology studies in our region.
Assuntos
Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter calcoaceticus/efeitos dos fármacos , Colômbia , Farmacorresistência Bacteriana , Hospitais , HumanosRESUMO
INTRODUCTION: Extended spectrum ß-lactamases (ESBL) are the most widely distributed enzymes in Enterobacteriaceae of Latin America and are key enzymes in resistance to antibiotics in common use. However, in Colombia, little information is available concerning the identity of genes coding for these enzymes in Klebsiella pneumoniae. OBJECTIVE: The bla genes were identified in K. pneumoniae isolated from hospitals in Bogotá D.C., Colombia. Materials and methods. One hundred seventy-seven isolates of ESBL producers were collected from 10 hospitals in Bogota between 2003 and 2005. Antibiotic susceptibility was determined by disk diffusion, and the number of ß-lactamases in each isolate was assessed by isoelectric focusing. blaCTX-M, blaSHV and blaTEM were identified by PCR and subsequent sequencing. RESULTS: Besides, the resitenance to third generation cephalosporins, 44.7 % and 49.7 % were resistant to amikacyn and thrimetoprim-sulaphametoxazole respectively. Lower resistance rates to other antibiotics were observed as well. An average of three ß-lactamases were detected by isoelectric focusing, and the genes blaCTX-M-12 (56.0%) and blaSHV-12 (33.3%) were the most prevalent. blaSHV-5 (11.8%), blaCTX-M-1-1 (4.0%), blaSHV-27 (2.8%), blaSHV-2 (2.8%), blaCTX-M-1-2 (1.7%) and blaCTX-M-1-15 (0.6%) were present in smaller percentages. In addition, three genes were identified that coded for narrow spectrum ß-lactamases. CONCLUSION: Eleven bla genes were identified, eight of which were ESBL-coding. The diversity of the bla genes suggested a continuing exposure of K. pneumoniae to strong antibiotic pressures in Bogota hospitals.
Assuntos
Farmacorresistência Bacteriana Múltipla/genética , Hospitais , Infecções por Klebsiella/enzimologia , Infecções por Klebsiella/genética , Klebsiella pneumoniae/enzimologia , Klebsiella pneumoniae/genética , beta-Lactamases/genética , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Colômbia , Genes Bacterianos , Humanos , Infecções por Klebsiella/tratamento farmacológico , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/isolamento & purificação , beta-Lactamases/farmacologia , beta-Lactamases/uso terapêuticoRESUMO
The 16S-23S rRNA gene intergenic spacer (ITS) was analysed by RFLP in this study to identify A. baumannii from 139 isolates from four hospitals (identified as A, B, C and D). One hundred and twenty of these isolates (86.3%) belonged to the A. baumannii species; those identified as being A. baumannii were found to be polyclonal (19 clone groups) when determining the genetic relationships, 16 of them being found in hospital C. Hospitals A, B and D shared two clone groups isolated during different years. This study describes a rapid and easy method for genospecies identification of Acinetobacter baumannii.
Con el objeto de identificar la genomoespecie Acinetobacter baumannii, se estudiaron 189 aislamientos pertenecientes al Complejo Acinetobacter baumannii-Acinetobacter calcoaceticus provenientes de cuatro hospitales colombianos (denominados A,B,C,D) mediante el análisis por RFLP-PCR de la región intergénica espaciador (ITS) de los genes 16S y 23S rRNA. Se encontraron 120 aislamientos (86.3%) pertenecientes a la especie A. baumannii. La estructura de la población fue policlonal, con 19 grupos clonales, 16 de los cuales se hallaron en el hospital C. En los hospitales A,B y D se encontraron 2 grupos clonales aislados durante diferentes años. En este estudio se propone un método rápido y fácil para la identificación de Acinetobacter baumannii.
Assuntos
Acinetobacter baumannii/isolamento & purificação , Acinetobacter baumannii/enzimologia , Acinetobacter baumannii/fisiologia , Acinetobacter baumannii/genética , Acinetobacter baumannii/imunologia , Acinetobacter baumannii/metabolismo , Acinetobacter baumannii/patogenicidade , Acinetobacter baumannii/químicaRESUMO
Introducción. Las beta-lactamasas de espectro extendido son las enzimas de Enterobacteriaceae de mayor distribución en Latinoamérica. No obstante, en Colombia existe poca información sobre la identidad de los genes codificadores de estas enzimas en Klebsiella pneumoniae. Objetivo. Identificar genes bla presentes en K. pneumoniae procedentes de hospitales de Bogotá, D.C., Colombia. Materiales y métodos. Se recolectaron 177 aislamientos productores de beta-lactamasas de espectro extendido de 10 hospitales de Bogotá, entre 2003 y 2005. Se determinó su sensibilidad antibiótica por difusión en disco y el número de beta-lactamasas presentes por isoelectroenfoque. Se identificaron los genes blaCTX-M, blaSHV y blaTEM, mediante PCR y posterior secuenciación. Resultados. Además de la resistencia a las cefalosporinas de tercera generación, el 44,7 % y el 49,7 % fueron resistentes a la gentamicina y al trimetoprim-sulfametoxazol, respectivamente. Se observaron porcentaje de resistencia más bajos con otros antibióticos. En promedio, se detectaron por aislamiento tres beta-lactamasas, y los genes blaCTX-M-12 (56%) y blaSHV-12 (33,3%) fueron los más prevalentes. Además, se identificaron blaSHV-5 (11,8%), blaCTX-M-1 (4%), blaSHV-27 (2,8%), blaSHV-2 (2,8%), blaCTX-M-2 (1,7%) y blaCTX-M-15 (0,6%). Además, en nuestros aislamientos se identificaron tres genes codificadores de beta-lactamasas de espectro ampliado. Conclusión. Se identificaron once genes bla, de los cuales, ocho eran codificadores de beta-lactamasas de espectro extendido. La diversidad encontrada de los genes bla sugiere la continua exposición de K. pneumoniae a fuertes presiones antibióticas, como las observadas en nuestros hospitales.
Introduction. Extended spectrum beta-lactamases (ESBL) are the most widely distributed enzymes in Enterobacteriaceae of Latin America and are key enzymes in resistance to antibiotics in common use. However, in Colombia, little information is available concerning the identity of genes coding for these enzymes in Klebsiella pneumoniae. Objective. The bla genes were identified in K. pneumoniae isolated from hospitals in Bogotá D.C., Colombia. Materials and methods. One hundred seventy-seven isolates of ESBL producers were collected from 10 hospitals in Bogota between 2003 and 2005. Antibiotic susceptibility was determined by disk diffusion, and the number of beta-lactamases in each isolate was assessed by isoelectric focusing. blaCTX-M, blaSHV and blaTEM were identified by PCR and subsequent sequencing. Results. Besides, the resitenance to third generation cephalosporins, 44.7 % and 49.7 % were resistant to amikacyn and thrimetoprim-sulaphametoxazole respectively. Lower resistance rates to other antibiotics were observed as well. An average of three beta-lactamases were detected by isoelectric focusing, and the genes blaCTX-M-12 (56.0%) and blaSHV-12 (33.3%) were the most prevalent. blaSHV-5 (11.8%), blaCTX-M-1 (4.0%), blaSHV-27 (2.8%), blaSHV-2 (2.8%), blaCTX-M-2 (1.7%) and blaCTX-M-15 (0.6%) were present in smaller percentages. In addition, three genes were identified that coded for narrow spectrum beta-lactamases. Conclusion. Eleven bla genes were identified, eight of which were ESBL-coding. The diversity of the bla genes suggested a continuing exposure of K. pneumoniae to strong antibiotic pressures in Bogota hospitals.
Assuntos
beta-Lactamases , Farmacorresistência Bacteriana , Klebsiella pneumoniae , Análise de Sequência de DNA , Colômbia , Epidemiologia MolecularRESUMO
INTRODUCTION: Molecular characterisation of Klebsiella pneumoniae strains is a tool that assits in the reduction of the disemination of drug resistance and the control of nosocomial infections that are caused by this pathogen. Objective. Molecular description of an outbreak of nosocomial infection caused by Klebsiella pneumoniae in a neonatal intensive care unit in a tertiary level hospital in Bogotá. METHODS: Eleven Klebsiella pneumoniae isolates were analysed. Production of Extended Spectrum Beta-Lactamases was verified by agar diffusion tests. Isoelectric points of the enzymes were determined by isoelectric focusing. The bla(CTX-M-12) gene was detected by PCR and pulsed field gel electrophoresis genotyping was done. RESULTS: All the isolates were Extended Spectrum Beta-Lactamase producers. Pulsed field gel electrophoresis and BOX-PCR genotyping grouped two isolates from hospital objects and eight infection-causing isolates into a single epidemic clone. The isolate from a thermometer was not grouped into the epidemic clone and showed a different resistance pattern. Isoelectric focusing revealed simultaneous beta-lactamase production having different isoelectric points. PCR amplification revealed the presence of the bla(CTX-M-12) gene in the 11 isolates studied. CONCLUSION: This is the first report of a molecularly characterised outbreak of CTX-M-12-producing Klebsiella pneumoniae from Colombia. The results of this study provide additional evidence of the global dissemination of CTX-M ESBL and the need for epidemiological follow-up in our hospitals.
Assuntos
Infecção Hospitalar/epidemiologia , Infecção Hospitalar/microbiologia , Surtos de Doenças , Unidades de Terapia Intensiva Neonatal , Infecções por Klebsiella/epidemiologia , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/isolamento & purificação , Klebsiella pneumoniae/metabolismo , beta-Lactamases/biossíntese , Colômbia/epidemiologia , Feminino , Humanos , Recém-Nascido , Masculino , beta-Lactamases/análiseRESUMO
Introducción. La caracterización molecular de cepas de Klebsiella pneumoniae es una herramienta que contribuye a disminuir la diseminación de la resistencia y al control de las infecciones nosocomiales causadas por este patógeno. Objetivo. Describir molecularmente un brote de infección nosocomial por Klebsiella pneumoniae en la Unidad de Cuidado Intensivo Neonatal de un hospital de tercer nivel de Bogotá. Materiales y métodos. Se analizaron once aislamientos. Se verificó la producción de betalactamasas de espectro extendido mediante pruebas de difusión en agar. Se determinaron los puntos isoeléctricos de las betalactamasas mediante isoelectroenfoque. Se detectó el gen blaCTX-M-12 por PCR y se realizó genotipificación mediante BOX- PCR y electroforesis en gel con campos pulsados (PFGE). Resultados. Los aislamientos fueron productores de beta-lactamasas de espectro extendido. La genotipificación por PFGE y por BOX-PCR, agrupó a dos aislamientos provenientes de objetos hospitalarios y a los ocho aislamientos causantes de infección en un grupo clonal epidémico. El aislamiento proveniente de un termómetro no fue agrupado en el grupo clonal epidémico y mostró un patrón de resistencia diferente. Se observó la producción simultánea de beta-lactamasas con diferentes puntos isoeléctricos. La PCR reveló el gen blaCTX-M-12 en los 11 aislamientos estudiados. Conclusión: Este es el primer informe en Colombia de un brote por Klebsiella pneumoniae productora de CTX-M-12, caracterizado molecularmente. Este estudio da evidencia adicional de la diseminación global de BLEE de tipo CTX-M y alerta sobre la necesidad de actividades especificas de prevención para cortar la cadena de transmisión y del seguimiento de tipo epidemiológico en nuestros centros hospitalarios
Introduction. Molecular characterisation of Klebsiella pneumoniae strains is a tool that assits in the reduction of the disemination of drug resistance and the control of nosocomial infections that are caused by this pathogen. Objective. Molecular description of an outbreak of nosocomial infection caused by Klebsiella pneumoniae in a neonatal intensive care unit in a tertiary level hospital in Bogotá. Methods: Eleven Klebsiella pneumoniae isolates were analysed. Production of Extended Spectrum Beta-Lactamases was verified by agar diffusion tests. Isoelectric points of the enzymes were determined by isoelectric focusing. The blaCTX-M-12 gene was detected by PCR and pulsed field gel electrophoresis genotyping was done. Results. All the isolates were Extended Spectrum Beta-Lactamase producers. Pulsed field gel electrophoresis and BOX-PCR genotyping grouped two isolates from hospital objects and eight infection-causing isolates into a single epidemic clone. The isolate from a thermometer was not grouped into the epidemic clone and showed a different resistance pattern. Isoelectric focusing revealed simultaneous beta-lactamase production having different isoelectric points. PCR amplification revealed the presence of the blaCTX-M-12 gene in the 11 isolates studied. Conclusion. This is the first report of a molecularly characterised outbreak of CTX-M-12-producing Klebsiella pneumoniae from Colombia. The results of this study provide additional evidence of the global dissemination of CTX-M ESBL and the need for epidemiological follow-up in our hospitals.
Assuntos
Terapia Intensiva Neonatal , Infecção Hospitalar/prevenção & controle , Klebsiella pneumoniae/isolamento & purificação , CefalosporinasRESUMO
Molecular epidemiology applied to the study of nosocomial infection has been fundamental in formulating and evaluating control methods. From patients in a level 3 Bogota hospital, Klebsiella pneumoniae samples were isolated that produced extended-spectrum beta-lactamases (ESBL). Each of 15 isolates was characterized microbiologically and by molecular characters realized by pulsed field gel electrophoresis (PFGE) and by repetitive-DNA sequences amplification (REP-PCR). Antimicrobial susceptibility and ESBL production was determined in accordance with NCCLS guidelines. The beta-lactamases were evaluated by isoelectric-focusing and PCR. Twelve (80%) of the isolates were associated with nosocomial infection; 11 of them were from intensive care units. The antibiotic susceptibility displayed 13 resistance patterns--87% presented co-resistance to amikacin, 53% to gentamicin, 33% to ciprofloxacin, 40% to cefepime, 67% to piperacillin/tazobactam, 60% to trimethoprim/sulfamethoxazole and 47% to chloranphenicol. All were sensitive to imipenem. Production of TEM and SHV beta-lactamases was detected simultaneously in most isolates by isoelectric focusing and 93.3% produced a ceftazidimase of pl 8.2 of the SHV-5 type. The 15 isolates were grouped into 11 and 12 electrophoretic patterns by PFGE and REP-PCR, respectively. The degree of genetic variability indicated an endogenous origin of the nosocomial infections.
Assuntos
DNA Bacteriano/genética , Infecções por Klebsiella/epidemiologia , Infecções por Klebsiella/genética , Klebsiella pneumoniae/genética , beta-Lactamases/genética , Adolescente , Idoso , Antibacterianos/uso terapêutico , Pré-Escolar , Colômbia/epidemiologia , Infecção Hospitalar/epidemiologia , Infecção Hospitalar/genética , Eletroforese em Gel de Campo Pulsado , Feminino , Humanos , Lactente , Recém-Nascido , Infecções por Klebsiella/tratamento farmacológico , Klebsiella pneumoniae/isolamento & purificação , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Epidemiologia Molecular , Resistência beta-Lactâmica/genéticaRESUMO
La epidemiología molecular aplicada al estudio de las infecciones nosocomiales ha sido fundamental para la formulación y la evaluación de las medidas de control; con este fin, se caracterizaron microbiológica y molecularmente aislamientos de Klebsiella pneumoniae productores de beta-lactamasas de espectro extendido (BLEE) obtenidos de pacientes en un hospital de tercer nivel de Bogotá, D.C., Colombia. Se tipificaron quince aislamientos por electroforesis en gel de campo pulsado (PFGE) y por amplificación de secuencias de AND repetidas (REP-PCR). La susceptibilidad antimicrobiana y la producción de BLEE se determinaron de acuerdo con las normas de NCCLS. Las beta-lactamasas se evaluaron por isoelectroenfoque y PCR. El 80 por ciento de estos aislamientos se asociaron con infección nosocomial y de éstos, el 91,7 por ciento provenía de unidades de cuidado intensivo. La susceptibilidad antibiótica mostró 13 patrones de resistencia; 87 por ciento de los aislamientos presentó corresistencia a amikacina, 53 por ciento a gentamicina, 33,3 por ciento a ciprofloxacina, 40 por ciento a cefepime, 66,7 por ciento a piperacilina/tazobactam, 60 por ciento trimetoprim/sulfametoxazol y 46,7por ciento a cloranfenicol. Todos fueron sensibles a imipenem. En la mayoría de los aislamientos se detectó producción simultánea de beta-lactamasas del tipo TEM y SHV y el 93,3 por ciento produjo ceftazidimasa de pI 8.2 del tipo SHV-5. Los 15 aislamientos fueron agrupados por PFGE y REP-PCR en 11 y 12 patrones electroforéticos, respectivamente. Esta variabilidad genética está relacionada con infecciones nosocomiales de origen endógeno más que por infecciones cruzadas