Diversidad y presencia de bacterias resistentes a antibióticos en diferentes agroecosistemas de Costa Rica
Fecha
2024-01
Tipo
tesis doctoral
Autores
Uribe Lorío, Lorena
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ISSN de la revista
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Editor
Resumen
Este estudio empleó métodos dependientes e independientes de cultivo, secuenciación masiva
y análisis bioinformático para explorar la diversidad de comunidades bacterianas en diversos
agroecosistemas de Costa Rica, así como en el proceso de elaboración del té de vermicompost.
Además, se investigó la presencia de bacterias resistentes a antimicrobianos comúnmente utilizados
en la protección de plantas y el tratamiento veterinario de ganado bovino.
Se identificaron 116 bacterias aisladas a partir de infecciones bacterianas en 19 cultivos,
incluyendo hortalizas, frutales y ornamentales en Costa Rica. La secuenciación del gen ARN 16S y
el sistema semiautomatizado Biolog revelaron 55 especies pertenecientes a 20 géneros bacterianos,
destacando Pseudomonas, Serratia, Pantoea y Stenotrophomonas como los más abundantes. El 27%
de los aislamientos se categorizó como patogénico mediante la Reacción Hipersensible (RH),
incluyendo especies fitopatógenas. como Pseudomonas syringae, P. cichorii, Pantoea anthophila y
P. stewartii, Stenotrophomonas maltophilia, Dickeya oryzae, Erwinia billingiae, Pectobacterium
aroidearum, y Enterobacter cloacae subsp. dissolvens. Se determinó la susceptibilidad a los
antibióticos estreptomicina, tetraciclina y gentamicina de la colección mediante la prueba de difusión
en disco y a las bacterias consideradas positivas se les determinó la Concentración mínima inhibitoria
(CMI). Según las condiciones de este estudio, el 60% de las bacterias provenientes de 17 cultivos
fueron clasificadas como resistentes a alguno de los antibióticos evaluados, encontrándose CMIs
máximas en aislamientos de repollo, tomate, banano, chile dulce, apio, melón, cúrcuma, coliflor y
palmito. Dentro de los géneros con mayor abundancia se determinó una proporción de aislamientos
resistentes desde 86% (Stenotrophomonas) a 54% (Pantoea). La resistencia a estreptomicina fue la
más frecuente (35%), seguida por tetraciclina (28%) y gentamicina (9%), y la proporción de
resistencia en las bacterias con RH positiva fue mayor (74%) que en las RH negativa (54%).
Por otro lado, se analizaron mediante la secuenciación de amplicones 16S rRNA, los cambios
en la diversidad bacteriana y la presencia de resistencia a oxitetraciclina, en muestras del proceso de
vermicompostaje (excretas frescas, precompostadas y vermicompost de estas) del Módulo Lechero
de la Sede del Atlántico de la Universidad de Costa Rica, además de los tés de vermicompost con y
sin melaza, utilizados como abonos líquidos. Las comunidades de estiércol fresco y precompostado
estuvieron dominadas por bacterias de los filos Firmicutes y Bacteroidetes y compartieron una
similitud de Bray-Curtis del 71%, aunque el precompostaje provocó una disminución de la
abundancia de los géneros dominantes, incluidos coliformes. El vermicompostaje aumentó la
diversidad del sustrato y provocó un marcado cambio en la comunidad bacteriana, dominada por
bacterias fijadoras de nitrógeno y degradadoras de compuestos orgánicos complejos pertenecientes a
las Proteobacterias y Bacteroidetes. El té de vermicompost mantuvo la comunidad central “core” del
sustrato, sin embargo, la adición de melaza al té tuvo un fuerte impacto sobre la diversidad,
favoreciendo la proliferación de géneros como Acinetobacter y Aeromonas (73% de las lecturas). A
partir de todas las muestras anteriores se cultivaron bacterias en caldos suplementados con 10 μg/ml
de oxitetraciclina, donde las excretas frescas tuvieron la mayor cantidad de ASVs resistentes (32),
con géneros como Acinetobacter y Bacteroides en mayor abundancia. El período de precomposteo
disminuyó los ASVs resistentes a 10, y el vermicompostaje a 5. La preparación de los tés con o sin
suplemento de melaza aumentó los ASVs resistentes (23 y 19, respectivamente), la mayoría
compartidos con la muestra de excretas frescas. Dentro de los géneros compartidos se encuentran
Aeromonas, Acinetobacter, Escherichia, y Proteus que tienen la capacidad de diseminar
determinantes de resistencia, y, en el caso de Aeromonas, esta presente en alta abundancia en el té
suplementado con melaza.
Este trabajo contribuye a una mejor comprensión de la presencia de bacterias resistentes de
origen ambiental y su diversidad en diferentes agroecosistemas de nuestro país, y se espera que esta
información sirva como insumo para la toma de decisiones con respecto al control en el uso de
antibióticos en la producción agrícola y pecuaria en Costa Rica, de manera que ejerza un efecto
positivo en el ambiente productivo, ambiental y de salud pública.
This study employed both culture-dependent and independent methods, including cultivation, high-throughput sequencing, and bioinformatic analysis, to explore bacterial community diversity in various agroecosystems of Costa Rica and during the vermicomposting tea production process. The investigation also focused on identifying bacteria resistant to commonly used antimicrobials in plant protection and bovine veterinary treatment. A collection of 116 bacteria isolated from bacterial infections in 19 crops, including vegetables, fruit trees, and ornamentals in Costa Rica. The sequencing of the 16S rRNA gene and the semi-automated Biolog system revealed 55 species within 20 bacterial genera, with Pseudomonas, Serratia, Pantoea, and Stenotrophomonas being the most abundant. Approximately 27% of the isolates were categorized as pathogenic through the hypersensitivity reaction test, including phytopathogenic species like Pseudomonas syringae, P. cichorii, Pantoea anthophila, P. stewartii, Stenotrophomonas maltophilia, Dickeya oryzae, Erwinia billingiae, Pectobacterium aroidearum, and Enterobacter cloacae subsp. dissolvens. Under this study conditions, 60% of the bacteria isolated from 17 out of 19 crops were classified as resistant to at least one of the evaluated antibiotics. The highest MICs were observed in isolates from cabbage, tomato, banana, sweet pepper, celery, melon, turmeric, cauliflower, and heart of palm. Among the most abundant genera, the proportion of resistant isolates ranged from 86% (Stenotrophomonas) to 54% (Pantoea). Resistance to streptomycin was the most prevalent (35%), followed by tetracycline (28%) and gentamicin (9%). Additionally, the proportion of resistance in bacteria with positive hypersensitivity reactions was higher (74%) than in those considered non pathogenic (54%). The study further analyzed changes in bacterial diversity and the presence of oxitetracycline resistance in samples from the vermicomposting process at the Dairy Module of the Atlantic Campus of the University of Costa Rica. This included fresh and precomposted manure, vermicompost, and vermicompost teas with and without cane molasses additive. Sequencing of 16S rRNA amplicons revealed that fresh and precomposted manure communities were dominated by Firmicutes and Bacteroidetes, sharing a Bray-Curtis similarity of 71%, although precomposting led to a reduction in the abundance of dominant genera, including coliforms. Vermicomposting increased substrate diversity, shifting the bacterial community towards nitrogen-fixing and complex organic compound- degrading bacteria in Proteobacteria and Bacteroidetes. Vermicompost tea retained the substrate's core community, but the addition of molasses strongly impacted diversity, favoring genera like Acinetobacter and Aeromonas (73% of reads). Bacteria cultivated in nutrient broth supplemented with 10 μg/ml oxitetracycline revealed that fresh manure had the highest number of resistant amplicon sequence variants (ASVs) (32), with higher abundance of genera such as Acinetobacter and Bacteroides. Precomposting reduced resistant ASVs to 10, while vermicomposting reduced them to 5. Tea preparation with or without molasses increased resistant ASVs (23 and 19, respectively), mostly shared with fresh manure. Shared genera included Aeromonas, Acinetobacter, Escherichia, and Proteus, known for disseminating resistance determinants, with Aeromonas present in high abundance in molasses-supplemented tea. This research contributes to a better understanding of the presence of environmentally derived antibiotic-resistant bacteria and their diversity in different agroecosystems in our country. This information is expected to provide insights for informed decision-making regarding antibiotic control in Costa Rica's agricultural and livestock production, ultimately leading to a positive impact on productivity, the environment, and public health.
This study employed both culture-dependent and independent methods, including cultivation, high-throughput sequencing, and bioinformatic analysis, to explore bacterial community diversity in various agroecosystems of Costa Rica and during the vermicomposting tea production process. The investigation also focused on identifying bacteria resistant to commonly used antimicrobials in plant protection and bovine veterinary treatment. A collection of 116 bacteria isolated from bacterial infections in 19 crops, including vegetables, fruit trees, and ornamentals in Costa Rica. The sequencing of the 16S rRNA gene and the semi-automated Biolog system revealed 55 species within 20 bacterial genera, with Pseudomonas, Serratia, Pantoea, and Stenotrophomonas being the most abundant. Approximately 27% of the isolates were categorized as pathogenic through the hypersensitivity reaction test, including phytopathogenic species like Pseudomonas syringae, P. cichorii, Pantoea anthophila, P. stewartii, Stenotrophomonas maltophilia, Dickeya oryzae, Erwinia billingiae, Pectobacterium aroidearum, and Enterobacter cloacae subsp. dissolvens. Under this study conditions, 60% of the bacteria isolated from 17 out of 19 crops were classified as resistant to at least one of the evaluated antibiotics. The highest MICs were observed in isolates from cabbage, tomato, banana, sweet pepper, celery, melon, turmeric, cauliflower, and heart of palm. Among the most abundant genera, the proportion of resistant isolates ranged from 86% (Stenotrophomonas) to 54% (Pantoea). Resistance to streptomycin was the most prevalent (35%), followed by tetracycline (28%) and gentamicin (9%). Additionally, the proportion of resistance in bacteria with positive hypersensitivity reactions was higher (74%) than in those considered non pathogenic (54%). The study further analyzed changes in bacterial diversity and the presence of oxitetracycline resistance in samples from the vermicomposting process at the Dairy Module of the Atlantic Campus of the University of Costa Rica. This included fresh and precomposted manure, vermicompost, and vermicompost teas with and without cane molasses additive. Sequencing of 16S rRNA amplicons revealed that fresh and precomposted manure communities were dominated by Firmicutes and Bacteroidetes, sharing a Bray-Curtis similarity of 71%, although precomposting led to a reduction in the abundance of dominant genera, including coliforms. Vermicomposting increased substrate diversity, shifting the bacterial community towards nitrogen-fixing and complex organic compound- degrading bacteria in Proteobacteria and Bacteroidetes. Vermicompost tea retained the substrate's core community, but the addition of molasses strongly impacted diversity, favoring genera like Acinetobacter and Aeromonas (73% of reads). Bacteria cultivated in nutrient broth supplemented with 10 μg/ml oxitetracycline revealed that fresh manure had the highest number of resistant amplicon sequence variants (ASVs) (32), with higher abundance of genera such as Acinetobacter and Bacteroides. Precomposting reduced resistant ASVs to 10, while vermicomposting reduced them to 5. Tea preparation with or without molasses increased resistant ASVs (23 and 19, respectively), mostly shared with fresh manure. Shared genera included Aeromonas, Acinetobacter, Escherichia, and Proteus, known for disseminating resistance determinants, with Aeromonas present in high abundance in molasses-supplemented tea. This research contributes to a better understanding of the presence of environmentally derived antibiotic-resistant bacteria and their diversity in different agroecosystems in our country. This information is expected to provide insights for informed decision-making regarding antibiotic control in Costa Rica's agricultural and livestock production, ultimately leading to a positive impact on productivity, the environment, and public health.
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Palabras clave
BACTERIA, GENÉTICA, MICROBIOLOGÍA, AGRICULTURA, SUELO