Characteristics of dual carbapenemase-producing Klebsiella pneumoniae strains from an outbreak in Venezuela: a retrospective study.

OBJECTIVE: To characterize carbapenemase-producing Klebsiella pneumoniae isolated from patients treated at a hospital in Cumaná, Sucre, Venezuela. METHODS: This was a retrospective study conducted at the general hospital in Cumaná where 58 K. pneumoniae strains were analyzed for resistance to antimicrobials, specifically carbapenems, in January - June 2015. Production of metallo-ß-lactamases and serine carbapenemases was determined by the double-disc synergy test, using EDTA-sodium mercaptoacetic acid and 3-aminophenyl boronic acid discs, respectively. Multiplex-PCR was used to detect genes coding for carbapenemases. Molecular typing using ERIC-PCR determined the presence of clones. RESULTS: Four strains of K. pneumoniae resistant to carbapenems were identified. Phenotypic methods for detection of metallo-ß-lactamases and serine carbapenemases were positive, and PCR demonstrated the co-presence of bla NDM and bla KPC genes in all four strains. ERIC-PCR identified two clones circulating in the hospital. CONCLUSIONS: Infection control strategies are needed at the central hospital in Cumaná and its surrounding areas to prevent the spread of these pathogens, especially given the high levels of migration from Venezuela to other countries in South America.

Characteristics of dual carbapenemase-producing Klebsiella pneumoniae strains from an outbreak in Venezuela: a retrospective study

[ABSTRACT]. Objective. To characterize carbapenemase-producing Klebsiella pneumoniae isolated from patients treated at a hospital in Cumaná, Sucre, Venezuela. Methods. This was a retrospective study conducted at the general hospital in Cumaná where 58 K. pneumoniae strains were analyzed for resistance to antimicrobials, specifically carbapenems, in January – June 2015. Production of metallo-β-lactamases and serine carbapenemases was determined by the double-disc synergy test, using EDTA-sodium mercaptoacetic acid and 3-aminophenyl boronic acid discs, respectively. Multiplex-PCR was used to detect genes coding for carbapenemases. Molecular typing using ERIC-PCR determined the presence of clones. Results. Four strains of K. pneumoniae resistant to carbapenems were identified. Phenotypic methods for detection of metallo-β-lactamases and serine carbapenemases were positive, and PCR demonstrated the co-presence of blaNDM and blaKPC genes in all four strains. ERIC-PCR identified two clones circulating in the hospital. Conclusions. Infection control strategies are needed at the central hospital in Cumaná and its surrounding areas to prevent the spread of these pathogens, especially given the high levels of migration from Venezuela to other countries in South America.

[RESUMEN]. Objetivo. Caracterizar la Klebsiella pneumoniae productora de carbapenemasa aislada de pacientes tratados en un hospital de Cumaná (Sucre, Venezuela). Métodos. Se hizo un estudio retrospectivo en el hospital central de Cumaná, donde se analizaron 58 cepas de k. pneumoniae para estudiar la resistencia a los antimicrobianos, específicamente a los fármacos carbapenémicos, entre enero y junio del 2015. La producción de metalo-β-lactamasas y carbapenemasas de serina se determinó mediante la prueba de sinergia de doble disco, usando discos de EDTA SMA de sodio y de ácido borónico 3 aminofenil, respectivamente. Se usó la PCR múltiple para detectar la codificación de genes correspondiente a las carbapenemasas. Se determinó la presencia de clones por tipificación molecular mediante la técnica de ERIC PCR. Resultados. Se detectaron cuatro cepas de K. pneumoniae resistentes a los fármacos carbapenémicos. Los métodos fenotípicos para la detección de metalo-β-lactamasas y carbapenemasas de serina fueron positivos y se demostró mediante la PCR la copresencia de los genes blaNDM y blaKPC en las cuatro cepas. Por medio de la técnica ERIC-PCR se detectaron dos clones que circulaban en el hospital. Conclusiones. Es necesario adoptar estrategias de control de infecciones en el hospital central en Cumaná y las zonas circundantes para prevenir la propagación de estos agentes patógenos, especialmente dados los niveles altos de migración de Venezuela a otros países de América del Sur.

[RESUMO]. Objetivo. Caracterizar cepas de Klebsiella pneumoniae produtoras de carbapenemases isoladas de pacientes tratados em um hospital em Cumaná, Sucre, na Venezuela. Métodos. Realizamos um estudo retrospectivo no hospital geral de Cumaná, onde 58 cepas de K. pneumoniae foram analisadas para verificar a resistência a antimicrobianos, especificamente carbapenens, entre janeiro e junho de 2015. A produção de metalo-β-lactamases e serino-carbapenemases foi determinada pelo teste de sinergia de disco duplo, usando discos de EDTA sódico-ácido mercaptoacético e ácido 3-aminofenil borônico, respectivamente. Utilizamos a PCR multiplex para detectar os genes codificadores de carbapenemases. A tipagem molecular por ERIC-PCR determinou a presença de clones. Resultados. Foram identificadas quatro cepas de K. pneumoniae resistentes a carbapenens. Os métodos fenotípicos para a detecção de metalo-β-lactamases e serino-carbapenemases foram positivos, e a PCR demonstrou a co-presença dos genes blaNDM e blaKPC em todas as quatro cepas. A ERIC-PCR identificou dois clones que circulavam no hospital. Conclusões. São necessárias estratégias de controle de infecções no hospital central de Cumaná e seus arredores para prevenir a disseminação destes patógenos, especialmente devido aos altos níveis de migração da Venezuela para outros países da América do Sul.

[qnr genes in Enterobacteriaceae isolated from at a hospital in Venezuela]. / Genes qnr en Enterobacteriaceae aisladas en un hospital de Venezuela.

BACKGROUND: Enterobacteria resistant to quinolones is increasing worldwide, including Venezuela. The mechanism for this resistance could be due to genes included in the chromosome or in transmissible plasmids. AIM: To evaluate the resistance to quinolones, coded by qnr genes present in enterobacteria species, isolated in the University Hospital of Cumana, Venezuela. METHODS: Antimicrobial susceptibility tests to quinolones, beta-lactams and aminoglycosides were carried out to all the isolates. The presence of qnr genes were determined by PCR. The isolates carrying the qnr genes were used for bacterial conjugation tests to determine the presence of transferable plasmids. Antimicrobial susceptibility tests and PCR were carried out in the transconjugants to verify the transfer of the genes. RESULTS: High levels of antimicrobial resistance to quinolones and beta-lactams were found among the isolates. We found that 33.6% of the isolates carry the qnrB gene and 0.9% qnr A gene. Of the 23 transconjugants, 20 showed to have qnrB gene, but none qnrA. DISCUSSION: We concluded that the high frequency of qnr genes found in the enterobacteria isolates and their presence on transferable plasmids, complicate the use of quinolones for the treatment of bacterial infections, thus, a treatment plan should be designed with the rational use and the rotation of different types of antimicrobials, in order to avoid the selection of increasingly resistant strains.

Genes qnr en Enterobacteriaceae aisladas en un hospital de Venezuela / qnr genes in Enterobacteriaceae isolated from at a hospital in Venezuela

Resumen Introducción: La resistencia de enterobacterias a quinolonas se ha difundido por el mundo, fenómeno presente también en Venezuela. El mecanismo de esta resistencia pudiera estar mediado por genes incluidos en el cromosoma bacteriano o transmitirse en el interior de plásmidos. Objetivo: Evaluar la resistencia a quino-lonas, codificada por genes qnr, presentes en cepas de enterobacterias, aisladas en el Hospital Universitario de Cumaná, Venezuela. Métodos: A las cepas obtenidas se les realizaron pruebas de susceptibilidad antimicrobiana a quinolonas, β-lactámicos y aminoglucósidos. La presencia del gen qnr se determinó por RPC. Las enterobacterias portadoras del gen qnr fueron sometidas al proceso de conjugación bacteriana para comprobar su capacidad de transferencia. A las transconjugantes obtenidas se les realizó pruebas de susceptibilidad antimicrobiana y RPC para comprobar la transferencia de los genes. Resultados: Se encontraron elevados porcentajes de resistencia antimicrobiana a quinolonas y betalactámicos. El 33,6% de las cepas eran portadoras del gen qnrB, y 0,9% del gen qnrA. Se obtuvieron 23 cepas transconjugantes; de éstas, 20 portaban el gen qnrB, no se observó la presencia de qnrA. Discusión: En conclusión, el elevado porcentaje de genes qnr encontrado en las enterobacterias aisladas, y comprobada la presencia de éstos en plásmidos transferibles, complica la aplicación de tratamientos basados en quinolonas y fluoroquinolonas, por lo que es recomendable el uso racional de estos antimicrobianos, y proponer la rotación de la terapia antimicrobiana, a fin de evitar la selección de cepas resistentes.

Background: Enterobacteria resistant to quinolones is increasing worldwide, including Venezuela. The mechanism for this resistance could be due to genes included in the chromosome or in transmissible plasmids. Aim: To evaluate the resistance to quinolones, coded by qnr genes present in enterobacteria species, isolated in the University Hospital of Cumana, Venezuela. Methods: Antimicrobial susceptibility tests to quinolones, beta-lactams and aminoglycosides were carried out to all the isolates. The presence of qnr genes were determined by PCR. The isolates carrying the qnr genes were used for bacterial conjugation tests to determine the presence of transferable plasmids. Antimicrobial susceptibility tests and PCR were carried out in the transconjugants to verify the transfer of the genes. Results: High levels of antimicrobial resistance to quinolones and beta-lactams were found among the isolates. We found that 33.6% of the isolates carry the qnrB gene and 0.9% qnr A gene. Of the 23 transconjugants, 20 showed to have qnrB gene, but none qnrA. Discussion: We concluded that the high frequency of qnr genes found in the enterobacteria isolates and their presence on transferable plasmids, complicate the use of quinolones for the treatment of bacterial infections, thus, a treatment plan should be designed with the rational use and the rotation of different types of antimicrobials, in order to avoid the selection of increasingly resistant strains.

Determinación del gen que codifica la enzima APH-(3´)-VIa en aislamientos de Acinetobacter 13TU:RUH1139 de origen hospitalario / Determination of the gene which codifies the APH-(3’)-VIa enzyme in Acinetobacter 13TU:RUH1139 isolates of nosocomial origin

El objetivo de este trabajo fue evaluar el fenotipo de resistencia y la presencia del gen aminoglucósido-O-fosfotransferasa-3´-VIa (aph-(3´)-VIa) en 23 aislamientos identificados como Acinetobacter 13TU:RUH 1139, provenientes de neonatos y soluciones parenterales, administradas a los mismos, en la Unidad de Alto Riesgo Neonatal (UARN) del Instituto Autónomo Hospital Universitario de Los Andes (IAHULA). Para la determinación de la susceptibilidad antimicrobiana se probaron seis aminoglucósidos mediante la prueba de difusión en agar (amikacina, gentamicina, tobramicina, netilmicina, dibekacina y neomicina), así como kanamicina y estreptomicina por dilución en agar. Todos los aislamientos resultaron resistentes a estreptomicina, más del 90% de los mismos mostraron resistencia ante amikacina, gentamicina y tobramicina, y el 82,6% fue resistente a netilmicina y dibekacina. Se detectó el gen aph-(3´)-VIa en 15 aislamientos de los neonatos y en 2 provenientes de las soluciones parenterales. Debido al elevado porcentaje de aislamientos resistentes a los aminoglucósidos, así como la presencia del gen aph-(3´)-VIa en Acinetobacter 13TU:RUH 1139, el uso de estos antimicrobianos como monoterapia debe ser restringido en el IAHULA y es necesario vigilar continuamente la diseminación genética de dicha resistencia.

The purpose of this work was to evaluate the resistance phenotype and the presence of aminoglucoside-0-phosphotransferase-3’-VIa (aph-(3’)-VIa) in 23 isolates identified as Acinetobacter 12TU:RUH 1139, obtained from neonates and parenteral solutions administered to them, at the Neonatal High Risk Unit (NHRU) of the Instituto Autónomo Hospital Universitario de Los Andes (IAHULA). Six aminoglucosides were examined for determination of antimicrobial susceptibility by the agar-diffusion test (amikacin, gentamycin, tobramycin, netilmycin, dibekacin, and neomycin), as well kanamycin and streptomycin by the agar-dilution test. All the isolates were streptomycin resistant, and over 90% of them showed resistance to amikacin, gentamycin, and tobramycin, and 82.6% was resistant to netilmycin and dibekacin. Gene aph-(3`)-VIa was detected in 15 isolates from neonates and in 2 from the parenteral solutions. Due to the high percentage of aminoglucoside-resistant isolates, as well as the presence of the gene aph-(3’)-VIa in Acinetobacter 13TU:RUH 1139, the use of these antimicrobials as monotherapy should be restricted at the AUHLA, and it is necessary to constantly survey the genetic dissemination of this resistance.