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.

Relación clonal y detección del gen blaKPC en cepas de Klebsiella pneumoniae resistentes a carbapenémicos, en un hospital de Venezuela / Clonal relationship and detection of blaKPC gene in strains of Klebsiella pneumoniae resistant to carbapenems, at a hospital in Venezuela

In order to study the clonal relationship and blaKPC gene detection in clinical isolates of Klebsiella pneumoniae resistant to carbapenems, we analyzed 22 clinical isolates of K. pneumoniae with resistance to imipenem and/ or meropenem, isolated in the laboratory of bacteriology at the University Hospital "Antonio Patricio de Alcalá" (HUAPA) from the Cumana city, Sucre state, Venezuela, for a period of five consecutive years. Susceptibility to different antimicrobials was determined, and the presence of carbapenemases was detected by modified Hodge method, phenyl boronic acid synergy and combination discs. blaKPC gene detection was conducted by polymerase chain reaction and the clonal relationship was determined by pulsed field electrophoresis. High rates of antimicrobial resistance were found, five strains were negative, at least one phenotypic method, and all carried the blaKPC gene. Clonal spread was observed only in the intensive care unit (ICU), while in other services, polyclonality was found. We concluded that blaKPC gene is present in K. pneumoniae strains resistant to carbapenems isolated in the HUAPA and clonal spread it was only in the ICU.

Con el objetivo de estudiar la relación clonal y detección del gen blaKPC en aislados clínicos de Klebsiella pneumoniae resistentes a carbapenémicos, se analizaron 22 cepas clínicas de K. pneumoniae con resistencia a imipenem y/o meropenem, aisladas en el laboratorio de bacteriología del Hospital Universitario "Antonio patricio de Alcalá" (HUAPA) de la ciudad de cumaná, Estado Sucre, Venezuela, durante un período de cinco años continuos. Se determinó la susceptibilidad a diversos antimicrobianos, y se detectó la presencia de carbapenemasas por los métodos de Hodge modificado, sinergia con ácido fenil borónico y combinación de discos. La detección del gen blaKPC se llevó a cabo mediante la técnica de reacción de polimerasa en cadena y la determinación de la relación clonal se realizó por electroforesis de campo pulsado. Se encontraron elevados porcentajes de resistencia antimicrobiana, cinco cepas resultaron negativas, al menos, a un método fenotípico y todas portaban el gen blaKPC. Se observó diseminación de clones únicamente en la Unidad de cuidados Intensivos (UCI), mientras que, en otros servicios, se halló policlonalidad. Se concluye que el gen blaKPC se encuentra presente en cepas de K. pneumoniae resistentes a carbapenémicos aisladas en el HUAPA y que hubo diseminación clonal sólo en UCI.

[Clonal relationship and detection of blaKPC gene in strains of Klebsiella pneumoniae resistant to carbapenems, at a hospital in Venezuela]. / Relación clonal y detección del gen blaKPC en cepas de Klebsiella pneumoniae resistentes a carbapenémicos, en un hospital de Venezuela.

In order to study the clonal relationship and blaKPC gene detection in clinical isolates of Klebsiella pneumoniae resistant to carbapenems, we analyzed 22 clinical isolates of K. pneumoniae with resistance to imipenem and/ or meropenem, isolated in the laboratory of bacteriology at the University Hospital "Antonio Patricio de Alcalá" (HUAPA) from the Cumana city, Sucre state, Venezuela, for a period of five consecutive years. Susceptibility to different antimicrobials was determined, and the presence of carbapenemases was detected by modified Hodge method, phenyl boronic acid synergy and combination discs. blaKPC gene detection was conducted by polymerase chain reaction and the clonal relationship was determined by pulsed field electrophoresis. High rates of antimicrobial resistance were found, five strains were negative, at least one phenotypic method, and all carried the blaKPC gene. Clonal spread was observed only in the intensive care unit (ICU), while in other services, polyclonality was found. We concluded that blaKPC gene is present in K. pneumoniae strains resistant to carbapenems isolated in the HUAPA and clonal spread it was only in the ICU.

KPC and VIM producing Enterobacter cloacae strain from a hospital in northeastern Venezuela.

An 83-year-old male patient is admitted to the central hospital in Cumana, Venezuela with severe urinary infection, history of hospitalizaions and prolonged antimicrobial treatments. A strain of Enterobacter cloacae was isolated showing resistance to multiple types of antibiotics (only sensitive to gentamicin), with phenotype of serine- and metallo-carbapenemases. Both, bla(VIM-2) and bla(KPC) genes were detected in the isolate. This is the first report of an Enterobacteriaceae species producing both KPC carbapenemase and VIM metallo carbapenemase in Venezuela. This finding has a great clinical and epidemiological impact in the region, because of the feasibility of transferring these genes, through mobile elements to other strains of Enterobacter and to other infection-causing species of bacteria.

KPC and VIM producing Enterobacter cloacae strain from a Hospital in Northeastern Venezuela / Enterobacter cloacae productor de KPC y VIM en un Hospital del Noreste de Venezuela

An 83-year-old male patient is admitted to the central hospital in Cumaná, Venezuela with severe urinary infection, history of hospitalizations and prolonged antimicrobial treatments. A strain of Enterobacter cloacae was isolated showing resistance to multiple types of antibiotics (only sensitive to gentamicin), with phenotype of serine- and metallo-carbapenemases. Both, blaVIM-2 and blaKPC genes were detected in the isolate. This is the first report of an Enterobacteriaceae species producing both KPC carbapenemase and VIM metallo carbapenemase in Venezuela. This finding has a great clinical and epidemiological impact in the region, because of the feasibility of transferring these genes, through mobile elements to other strains of Enterobacter and to other infection-causing species of bacteria.

En un paciente masculino de 83 años, que ingresó al Hospital de Cumaná, Venezuela, con diagnóstico de infección urinaria severa, antecedentes de hospitalización y diferentes tratamientos antimicrobianos durante largos periodos de tiempo, se aisló una cepa de Enterobacter cloacae, la cual evidenció resistencia a múltiples tipos de antibióticos (solo sensible a gentamicina) y con fenotipo de carbapenemasas de tipo serina y metalobetalactamasa. Los genes blaVIM-2 y blaKPC fueron detectados en esta cepa. Este representa el primer reporte de una especie de Enterobacteriaceae productora simultánea de carbapenemasa KPC y metalobetalactamasa VIM en Venezuela. Esto tiene un gran impacto clínico y epidemiológico en la región por la posibilidad de transferencia de estos genes a otras cepas de Enterobacter u otras especies bacterianas causantes de infecciones, por medio de elementos móviles.

First report of metallo-ß-lactamases producing Enterobacter spp. strains from Venezuela.

Clinical strains of Enterobacter were isolated from Cumana's Central Hospital in Venezuela, and classified as E. cloacae (21), E. aerogenes (7), E. intermedium (1), E. sakazakii (1) and three unclassified. The strains showed high levels of resistance, especially to SXT (58.1%), CRO (48.8%), CAZ (46.6%), PIP (46.4%), CIP (45.2%) and ATM (43.3%). This is the first report for South America of blaVIM-2 in two E. cloacae and one Enterobacter sp., which also showed multiple mechanisms of resistance. Both E. cloacae showed blaTEM-1, but only one showed blaCTX-M-15 gene, while no blaSHV was detected.

FIRST REPORT OF METALLO-β-LACTAMASES PRODUCING Enterobacter spp. STRAINS FROM VENEZUELA / Primer reporte de cepas de Enterobacter spp productoras de metalobetalactamasas de Venezuela

Clinical strains of Enterobacter were isolated from Cumana's Central Hospital in Venezuela, and classified as E. cloacae (21), E. aerogenes (7), E. intermedium (1), E. sakazakii (1) and three unclassified. The strains showed high levels of resistance, especially to SXT (58.1%), CRO (48.8%), CAZ (46.6%), PIP (46.4%), CIP (45.2%) and ATM (43.3%). This is the first report for South America of blaVIM-2 in two E. cloacae and one Enterobacter sp., which also showed multiple mechanisms of resistance. Both E. cloacae showed blaTEM-1, but only one showed blaCTX-M-15 gene, while no blaSHV was detected.

Cepas clínicas de Enterobacter fueron aisladas del Hospital central de Cumaná en Venezuela, y se clasificaron como E. cloacae (21), E. aerogenes (7), E. intermedium (1), E. sakazakii (1) y 3 sin clasificar. Las cepas mostraron altos niveles de resistencia, especialmente a SXT (58.1%), CRO (48.8%), CAZ (46.6%), PIP (46.4%), CIP (45.2%) and ATM (43.3%). Este es el primer reporte de América del Sur de blaVIM-2 en dos cepas de E. cloacae y una de Enterobacter sp., las cuales también mostraron múltiples mecanismos de resistencia. Ambas especies de E. cloacae mostraron genes blaTEM-1, pero solo una mostro el gen blaCTX-M-15, mientras que blaSHV no fue detectado.