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)

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.

[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.

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.

[Molecular epidemiology of nosocomial infection by extended-spectrum beta-lactamases-producing Klebsiella pneumoniae]. / Epidemiología molecular de infección nosocomial por Klebsiella pneumoniae productora de beta-lactamasas de espectro extendido.

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.

Epidemiología molecular de infección nosocomial por Klebsiella pneumoniae productora de beta-lactamasas de espectro extendido / Molecular epidemiology of nosocomial infection by extended-spectrum beta-lactamases-producing Klebsiella pneumoniae

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