Genome-based characterization of two Colombian clinical Providencia rettgeri isolates co-harboring NDM-1, VIM-2, and other ß-lactamases.

BACKGROUND: Providencia rettgeri is a nosocomial pathogen associated with urinary tract infections and related to Healthcare-Associated Infection (HAI). In recent years isolates producing New Delhi Metallo-ß-lactamase (NDM) and other ß-lactamases have been reported that reduce the efficiency of clinical antimicrobial treatments. In this study, we analyzed antibiotic resistance, the presence of resistance genes and the clonal relationship of two P. rettgeri isolates obtained from male patients admitted to the same hospital in Bogotá - Colombia, 2015. RESULTS: Antibiotic susceptibility profile evaluated by the Kirby-Bauer method revealed that both isolates were resistant to third-generation carbapenems and cephalosporins. Whole-genome sequencing (Illumina HiSeq) followed by SPAdes assembling, Prokka annotation in combination with an in-house Python program and resistance gene detection by ResFinder identified the same six ß-lactamase genes in both isolates: blaNDM-1, blaVIM-2, blaCTX-M-15, blaOXA-10, blaCMY-2 and blaTEM-1. Additionally, various resistance genes associated with antibiotic target alteration (arnA, PmrE, PmrF, LpxA, LpxC, gyrB, folP, murA, rpoB, rpsL, tet34) were found and four efflux pumps (RosAB, EmrD, mdtH and cmlA). The additional resistance to gentamicin in one of the two isolates could be explained by a detected SNP in CpxA (Cys191Arg) which is involved in the stress response of the bacterial envelope. Genome BLAST comparison using CGView, the ANI value (99.99%) and the pangenome (using Roary) phylogenetic tree (same clade, small distance) showed high similarity between the isolates. The rMLST analysis indicated that both isolates were typed as rST-61,696, same as the RB151 isolate previously isolated in Bucaramanga, Colombia, 2013, and the FDAARGOS_330 isolate isolated in the USA, 2015. CONCLUSIONS: We report the coexistence of the carbapenemase genes blaNDM-1, and blaVIM-2, together with the ß-lactamase genes blaCTX-M-15, blaOXA-10, blaCMY-2 and blaTEM-1, in P. rettgeri isolates from two patients in Colombia. Whole-genome sequence analysis indicated a circulation of P. rettgeri rST-61,696 strains in America that needs to be investigated further.

Antibiotic resistance patterns of Acinetobacter calcoaceticus-A. baumannii complex species from Colombian hospitals.

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.

[Molecular characterization of an outbreak caused by CTX-M-12-producing Klebsiella pneumoniae in a Colombian hospital's neonatal intensive care unit]. / Caracterización molecular de un brote por Klebsiella pneumoniae productora de CTX-M-12 en la unidad de cuidado intensivo neonatal de un hospital Colombiano.

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.

[Distribution of extended spectrum ß-lactamases-codifying genes in Klebsiella pneumoniae isolates from hospitals of Bogota, D.C., Colombia]. / Distribución de genes codificadores de ß-lactamasas de espectro extendido en aislamientos de Klebsiella pneumoniae de hospitales de Bogotá, D.C., Colombia.

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.

Distribución de genes codificadores de beta-lactamasas de espectro extendido en aislamientos de Klebsiella pneumoniae de hospitales de Bogotá, D.C., Colombia / Distribution of extended spectrum beta-lactamases-codifying genes in Klebsiella pneumoniae isolates from hospitals of Bogota, D.C., Colombia

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.

The genomic identification of Colombian Acinetobacter baumannii clinical isolates by RFLP-PCR analysis of the 16S-23S rRNA gene spacer region / Identificación genómica de aislamientos colombianos de Acinetobacter baumannii mediante RFLP-PCR de la región intergénica espaciadora de los genes 16S y 23S rRNA

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.

Antibiotic resistance patterns of Acinetobacter calcoaceticus–A. baumannii complex species from Colombian hospitals

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.Methods16S–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 (AU)

Introducción Actualmente, los hospitales en Colombia utilizan únicamente métodos fenotípicos automatizados para la identificación de aislamientos del complejo Acinetobacter calcoaceticus - baumannii (ACB). La similitud entre estas especies no permite que se diferencien por métodos fenotípicos ya sean estos manuales o automatizados, llevando a que los aislamientos se identifiquen como A. baumannii o como pertenecientes al complejo ACB en las instituciones hospitalarias. Nuestro objetivo fue identificar a nivel de especie, 60 aislamientos de cuatro hospitales, identificados como del complejo ACB, evaluar su resistencia a antibióticos y detectar genes de resistencia. Métodos Para la identificación de especies se amplificaron la región intergénica espaciadora de los genes 16S y 23S rRNA y la secuencia parcial del gen rpoB. Estos amplificados y algunos genes de resistencia se secuenciaron y se compararon utilizando la herramienta BLAST. Se detectaron por PCR genes de resistencia a cefalosporinas, carbapenemes y aminoglicósidos. Se evaluaron posibles mutaciones en la región determinante de resistencia a quinolonas (QRDR). Se determinó por PCR la asociación de ISAba-1con los genes blaOXA y blaADC. Resultados Con las secuencias de la región ITS 16S-23S rRNA y el gen rpoB, se identificaron 51 aislamientos (..) (AU)

Caracterización molecular de un brote por Klebsiella pneumoniae productora de CTX-M-12 en la unidad de cuidado intensivo neonatal de un hospital colombiano / Molecular characterization of an outbreak caused by CTX-M-12-producing Klebsiella pneumoniae in a Colombian hospital´s neonatal intensive care unit

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.