High Genetic Diversity and Antimicrobial Resistance in Escherichia coli Highlight Arapaima gigas (Pisces: Arapaimidae) as a Reservoir of Quinolone-Resistant Strains in Brazilian Amazon Rivers.

The increasing prevalence of multi-drug resistant (MDR) Escherichia coli in distinct ecological niches, comprising water sources and food-producing animals, such as fish species, has been widely reported. In the present study, quinolone-resistant E. coli isolates from Arapirama gigas, a major fish species in the Brazilian Amazon rivers and fish farms, were characterized regarding their antimicrobial susceptibility, virulence, and genetic diversity. A total of forty (40) specimens of A. gigas, including 20 farmed and 20 wild fish, were included. Thirty-four quinolone-resistant E. coli isolates were phenotypically tested by broth microdilution, while resistance and virulence genes were detected by PCR. Molecular epidemiology and genetic relatedness were analyzed by MLST and PFGE typing. The majority of isolates were classified as MDR and detected harboring blaCTX-M, qnrA and qnrB genes. Enterotoxigenic E. coli pathotype (ETEC) isolates were presented in low prevalence among farmed animals. MLST and PFGE genotyping revealed a wide genetic background, including the detection of internationally spread clones. The obtained data point out A. gigas as a reservoir in Brazilian Amazon aquatic ecosystems and warns of the interference of AMR strains in wildlife and environmental matrices.

Detection of multidrug-resistant Enterobacteriaceae isolated from river waters flowing to the Guanabara Bay and from clinical samples of hospitals in Rio de Janeiro, Brazil / Detección de enterobacterias multirresistentes aisladas en aguas de los ríos que desembocan en la bahía de Guanabara y en muestras de hospitales de Río de Janeiro, Brasil

INTRODUCTION: The use of antibiotics in humans, animal husbandry and veterinary activities induces selective pressure leading to the colonization and infection by resistant strains. OBJECTIVE: We evaluated water samples collected from rivers of the Guanabara Bay, which have suffered minor and major environmental degradation, and clinical samples of hospital origin to detect evidence of the presence of resistance genes to aminoglycosides, beta-lactam antibiotics and fluoroquinolones in strains of Klebsiella pneumoniae subsp. pneumoniae, K. pneumoniae subsp. ozaenae and Escherichia coli. MATERIALS AND METHODS: For isolation of the water strains we employed culture media containing 32 µg/ml cephalotin and 8 µg/ml gentamicin. The strains from clinical materials were selected using culture media containing 8 µg/ml gentamicin. The strains were identified and subjected to antimicrobial susceptibility testing (AST), plasmid DNA extraction and polymerase chain reaction (PCR) to detect genes encoding enzymes modifying aminoglycosides (EMA), extended-spectrum beta-lactamases (ESBL) and plasmid mechanisms of quinolone resistance (PMQR). RESULTS: The AST of the isolates recovered from water samples showed multidrugresistance profiles similar to those found in isolates recovered from clinical materials. All isolates from water samples and 90% of the isolates from clinical samples showed at least one plasmid band. In the PCR assays, 7.4% of the isolates recovered from water samples and 20% of those from clinical materials showed amplification products for the three antimicrobial classes. CONCLUSION: We believe that the detection of microorganisms presenting genetic elements in environments such as water is necessary for the prevention and control of their dissemination with potential to infect humans and other animals in eventual contact with these environments.

Introducción. El uso de antibióticos en seres humanos, en la industria pecuaria y en las actividades veterinarias induce una presión selectiva que resulta en la colonización e infección con cepas resistentes. Objetivo. Determinar la presencia de genes de resistencia a aminoglucósidos, betalactámicos y fluoroquinolonas en cepas de Klebsiella pneumoniae subsp. pneumoniae, K. pneumoniae subsp. ozaenae y Escherichia coli, obtenidas de muestras de agua de los ríos que desembocan en la bahía de Guanabara y de muestras clínicas de hospitales de Río de Janeiro. Materiales y métodos. En la selección de las cepas resistentes obtenidas de las muestras de agua de los ríos, se emplearon medios de cultivo que contenían 32 µg/ml de cefalotina y 8 µg/ml de gentamicina. En el caso de las muestras de especímenes clínicos, se usaron medios de cultivo que contenían 8 µg/ml de gentamicina. Las cepas se identificaron y se sometieron a pruebas de sensibilidad antimicrobiana, extracción de ADN plasmídico y pruebas de reacción en cadena de la polimerasa (PCR) para detectar los genes que codifican aquellas enzimas que modifican los aminoglucósidos, las betalactamasas de espectro extendido (BLEE) y los mecanismos de resistencia a las quinolonas mediados por plásmidos. Resultados. Se encontraron perfiles de resistencia a los antimicrobianos similares en los dos grupos. En todas las bacterias obtenidas de las muestras de agua y en 90 % de las muestras clínicas, se evidenciaron bandas de plásmidos asociados con la transferencia de genes de resistencia. En las pruebas de PCR, se obtuvieron productos de amplificación de los genes de resistencia para las tres clases de antimicrobianos analizados, en el 7,4 % de las bacterias recuperadas de las muestras de agua y en el 20 % de aquellas recuperadas de las muestras clínicas. Conclusión. La detección de microorganismos con elementos genéticos que confieren resistencia a los antibióticos en ambientes como el agua, es una estrategia necesaria para prevenir y controlar la diseminación de estos agentes patógenos con potencial para infectar a humanos y a otros animales en dichos ambientes.

Detection and characterization of multidrug-resistant enterobacteria bearing aminoglycoside-modifying gene in a university hospital at Rio de Janeiro, Brazil, along three decades.

INTRODUCTION: Multidrug-resistant Enterobacteriaceae, particularly those resistant to gentamicin, have become one of the most important causes of nosocomial infections. OBJECTIVE: We sought to investigate the presence of genes conferring resistance to aminoglycosides, specially to gentamicin, in Klebsiella pneumoniae and Escherichia coli multidrug-resistant strains isolated from different clinical materials among patients hospitalized in a university hospital in Rio de Janeiro, Brazil. MATERIALS AND METHODS: Ten colonization strains and 20 infection strains were evaluated during three decades (1980 to 2010) using selective media containing 8 µg/ml of gentamicin. Thirty strains were tested for antimicrobial susceptibility. Twenty two strains were subjected to plasmid DNA extraction and 12 to hybridization assays using as probe a 1.9 kb plasmid DNA fragment from one of the K. pneumoniae strains isolated from faecal samples. This fragment was sequenced and assigned to the GQ422439 GenBank record. PCR was also performed using oligonucleotides designed for aminoglycoside-modifying enzymes. RESULTS: An accC2 acetylase, besides transposons and insertion sequences, were evidenced. Twenty-four (80%) of the isolates were positive for the aacC2 gene in agreement with antibiotic susceptibility testing profiles, indicating the persistent presence of this gene throughout the three decades. We detected high molecular weight plasmids in 54,5% of the strains. Of the tested strains, 91% showed positive signal in the hybridization assays. CONCLUSION: A gene codifying for one specific aminoglycoside-modifying enzyme was detected all throughout the three decades. Our data back the adoption of preventive measures, such as a more conscious use of antimicrobial agents in hospital environments, which can contribute to control the dissemination of microorganisms harboring resistance gene plasmids.

Detection and characterization of multidrug-resistant enterobacteria bearing aminoglycoside-modifying gene in a university hospital at Rio de Janeiro, Brazil, along three decades / Detección y caracterización de enterobacterias resistentes a múltiples fármacos, portadoras del gen modificador de aminoglucósidos en un hospital universitario de Río de Janeiro, Brasil, durante tres décadas

Introduction: Multidrug-resistant Enterobacteriaceae, particularly those resistant to gentamicin, have become one of the most important causes of nosocomial infections. Objective: We sought to investigate the presence of genes conferring resistance to aminoglycosides, specially to gentamicin, in Klebsiella pneumoniae and Escherichia coli multidrug-resistant strains isolated from different clinical materials among patients hospitalized in a university hospital in Rio de Janeiro, Brazil. Materials and methods: Ten colonization strains and 20 infection strains were evaluated during three decades (1980 to 2010) using selective media containing 8 µg/ml of gentamicin. Thirty strains were tested for antimicrobial susceptibility. Twenty two strains were subjected to plasmid DNA extraction and 12 to hybridization assays using as probe a 1.9 kb plasmid DNA fragment from one of the K. pneumoniae strains isolated from faecal samples. This fragment was sequenced and assigned to the GQ422439 GenBank record. PCR was also performed using oligonucleotides designed for aminoglycoside-modifying enzymes. Results: An accC2 acetylase, besides transposons and insertion sequences, were evidenced. Twenty-four (80%) of the isolates were positive for the aacC2 gene in agreement with antibiotic susceptibility testing profiles, indicating the persistent presence of this gene throughout the three decades. We detected high molecular weight plasmids in 54,5% of the strains. Of the tested strains, 91% showed positive signal in the hybridization assays. Conclusion: A gene codifying for one specific aminoglycoside-modifying enzyme was detected all throughout the three decades. Our data back the adoption of preventive measures, such as a more conscious use of antimicrobial agents in hospital environments, which can contribute to control the dissemination of microorganisms harboring resistance gene plasmids.

Introducción. Las enterobacterias resistentes a la gentamicina se asocian frecuentemente a infecciones hospitalarias. Objetivo. Verificar la presencia de los genes que confieren resistencia a los aminoglucósidos, específicamente a la gentamicina, en cepas de Klebsiella pneumoniae y Escherichia coli multirresistentes, obtenidas de pacientes internados en un hospital universitario de Río de Janeiro. Materiales y métodos. Se recolectaron y evaluaron 10 cepas de colonización y 20 de infección entre 1980 y 2010, utilizando medios selectivos enriquecidos con gentamicina (8 µg/ml). Se obtuvieron 30 cepas en las que se determinó la resistencia a los antibióticos por medios fenotípicos. Veintidós muestras se sometieron a extracción de ADN plasmídico y se hicieron ensayos de hibridización en 12 de ellas, usando como sonda un fragmento de ADN plasmídico de 1,9 kb obtenido de una cepa de K. pneumoniae aislada de muestra fecal. Este fragmento fue secuenciado y correspondió al registro GQ422439 del GenBank. Se verificó la presencia de genes de enzimas modificadoras de aminoglucósidos mediante reacción en cadena de la polimerasa. Resultados. En las cepas analizadas se evidenció la presencia de la acetilasa accC2, además de transposones y secuencias de inserción. Veinticuatro aislamientos (80 %) fueron positivos para el gen aacC2 en concordancia con los perfiles de sensibilidad a los antibióticos, lo que indicó su persistencia a lo largo de las tres décadas. Se detectaron plásmidos de alto peso molecular en 54,5 % de las cepas. El 91 % de las cepas analizadas mostró signos positivos en las pruebas de hibridación. Conclusión. Se detectó la persistencia de un gen codificador de una enzima modificadora de aminoglucósidos a lo largo de las tres décadas. Los resultados indican que las medidas de prevención, tales como un uso más responsable de los agentes antimicrobianos en el ambiente hospitalario, pueden contribuir al control de la diseminación de microorganismos que albergan plásmidos de genes de resistencia.