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 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

Abstract 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 multidrug-resistance 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.

Resumen 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.

The combination of different carbon sources enhances bacterial growth efficiency in aquatic ecosystems.

The dissolved organic carbon (DOC) pool is composed of several organic carbon compounds from different carbon sources. Each of these sources may support different bacterial growth rates, but few studies have specifically analyzed the effects of the combination of different carbon sources on bacterial metabolism. In this study, we evaluated the response of several metabolic parameters, including bacterial biomass production (BP), bacterial respiration (BR), bacterial growth efficiency (BGE), and bacterial community structure, on the presence of three DOC sources alone and in combination. We hypothesized that the mixture of different DOC sources would increase the efficiency of carbon use by bacteria (BGE). We established a full-factorial substitutive design (seven treatments) in which the effects of the number and identity of DOC sources on bacterial metabolism were evaluated. We calculated the expected metabolic rates of the combined DOC treatments based on the single-DOC treatments and observed a positive interaction on BP, a negative interaction on BR, and, consequently, a positive interaction on BGE for the combinations. The bacterial community composition appeared to have a minor impact on differences in bacterial metabolism among the treatments. Our data indicate that mixtures of DOC sources result in a more efficient biological use of carbon. This study provides strong evidence that the mixture of different DOC sources is a key factor affecting the role of bacteria in the carbon flux of aquatic ecosystems.

Tropical freshwater ecosystems have lower bacterial growth efficiency than temperate ones.

Current models and observations indicate that bacterial respiration should increase and growth efficiency (BGE) should decrease with increasing temperatures. However, these models and observations are mostly derived from data collected in temperate regions, and the tropics are under-represented. The aim of this work was to compare bacterial metabolism, namely bacterial production (BP) and respiration (BR), bacterial growth efficiency (BGE) and bacterial carbon demand (BCD) between tropical and temperate ecosystems via a literature review and using unpublished data. We hypothesized that (1) tropical ecosystems have higher metabolism than temperate ones and, (2) that BGE is lower in tropical relative to temperate ecosystems. We collected a total of 498 coupled BP and BR observations (N total = 498; N temperate = 301; N tropical = 197), calculated BGE (BP/(BP+BR)) and BCD (BP+BR) for each case and examined patterns using a model II regression analysis and compared each parameter between the two regions using non-parametric Mann-Whitney U test. We observed a significant positive linear regression between BR and BP for the whole dataset, and also for tropical and temperate data separately. We found that BP, BR and BCD were higher in the tropics, but BGE was lower compared to temperate regions. Also, BR rates per BP unit were at least two fold higher in the tropics than in temperate ecosystems. We argue that higher temperature, nutrient limitation, and light exposure all contribute to lower BGE in the tropics, mediated through effects on thermodynamics, substrate stoichiometry, nutrient availability and interactions with photochemically produced compounds. More efforts are needed in this study area in the tropics, but our work indicates that bottom-up (nutrient availability and resource stoichiometry) and top-down (grazer pressure) processes, coupled with thermodynamic constraints, might contribute to the lower BGE in the tropics relative to temperate regions.

DOC removal paradigms in highly humic aquatic ecosystems.

BACKGROUND, AIM, AND SCOPE: Dissolved humic substances (HS) usually comprise 50-80% of the dissolved organic carbon (DOC) in aquatic ecosystems. From a trophic and biogeochemical perspective, HS has been considered to be highly refractory and is supposed to accumulate in the water. The upsurge of the microbial loop paradigm and the studies on HS photo-degradation into labile DOC gave rise to the belief that microbial processing of DOC should sustain aquatic food webs in humic waters. However, this has not been extensively supported by the literature, since most HS and their photo-products are often oxidized by microbes through respiration in most nutrient-poor humic waters. Here, we review basic concepts, classical studies, and recent data on bacterial and photo-degradation of DOC, comparing the rates of these processes in highly humic ecosystems and other aquatic ecosystems. MATERIALS AND METHODS: We based our review on classical and recent findings from the fields of biogeochemistry and microbial ecology, highlighting some odd results from highly humic Brazilian tropical lagoons, which can reach up to 160 mg C L(-1). RESULTS AND DISCUSSION: Highly humic tropical lagoons showed proportionally lower bacterial production rates and higher bacterial respiration rates (i.e., lower bacterial growth efficiency) than other lakes. Zooplankton showed similar delta(13)C to microalgae but not to humic DOC in these highly humic lagoons. Thus, the data reviewed here do not support the microbial loop as an efficient matter transfer pathway in highly humic ecosystems, where it is supposed to play its major role. In addition, we found that some tropical humic ecosystems presented the highest potential DOC photo-chemical mineralization (PM) rates reported in the literature, exceeding up to threefold the rates reported for temperate humic ecosystems. We propose that these atypically high PM rates are the result of a joint effect of the seasonal dynamics of allochthonous humic DOC input to these ecosystems and the high sunlight incidence throughout the year. The sunlight action on DOC is positive to microbial consumption in these highly humic lagoons, but little support is given to the enhancement of bacterial growth efficiency, since the labile photo-chemical products are mostly respired by microbes in the nutrient-poor humic waters. CONCLUSIONS: HS may be an important source of energy for aquatic bacteria in humic waters, but it is probably not as important as a substrate to bacterial growth and to aquatic food webs, since HS consumption is mostly channeled through microbial respiration. This especially seems to be the case of humic-rich, nutrient-poor ecosystems, where the microbial loop was supposed to play its major role. Highly humic ecosystems also present the highest PM rates reported in the literature. Finally, light and bacteria can cooperate in order to enhance total carbon degradation in highly humic aquatic ecosystems but with limited effects on aquatic food webs. RECOMMENDATIONS AND PERSPECTIVES: More detailed studies using C- and N-stable isotope techniques and modeling approaches are needed to better understand the actual importance of HS to carbon cycling in highly humic waters.

Ecological aspects of the antimicrobial resistance in bacteria of importance to human infections

In view of the intimate relationship of humans with coastal lagoons (used for recreation, tourism, water supply, etc.), the discharge of domestic effluents may lead to the establishment of routes of dissemination of pathogenic microorganisms, including microorganisms carrying genes for resistance to antimicrobials, through the surrounding human communities. The objective of the present investigation was to relate the presence of antimicrobial-resistant bacteria to the environmental characteristics of three coastal lagoons, comparing the results with those from hospital sewage. Of the lagoons evaluated, two (Geriba and Imboassica) receive domestic sewage discharge, and the other (Cabiunas) is still in a natural state. We isolated in a culture medium containing 32 1/4ìg/ml of Cephalothin, fecal coliforms (E. coli), non-fecal coliforms (Klebsiella, Enterobacter, Serratia, and Citrobacter), non-glucose-fermenting Gram-negative bacilli, and Aeromonas sp. In cultures from the hospital drain we found strains showing numerous markers for resistance to most of the 11 antimicrobials tested. On the other hand, in cultures from Cabiunas and Imboassica lagoons, we found strains showing resistance only to antibiotics frequently observed in non-selective situations (considered as "common" markers). The capacity for dilution in the ecosystem, and salinity appeared related with the occurrence of multi-resistant bacterial strains. The intensity of recent fecal contamination was not shown to be associated with the numbers and types of markers found.