A multidisciplinary approach to analyze the antimicrobial resistance in natural ecosystems.

Antimicrobial Resistance (AMR) poses a global threat to both human health and environmental well-being. Our study delved into Costa Rican wildlife reserves, uncovering a substantial human impact on these ecosystems and underscoring the imperative to pinpoint AMR hotspots. Embracing a One Health perspective, we advocated for a comprehensive landscape analysis that intricately intertwined geographic, climatic, forest, and human factors. This study illuminated the link between laboratory results and observed patterns of antimicrobial use, thereby paving the way for sustainable solutions. Our innovative methodology involved deploying open-ended questions to explore antimicrobial usage across livestock activities, contributing to establishing a comprehensive methodology. Non-invasive sampling in wildlife emerged as a critical aspect, shedding light on areas contaminated by AMR. Feline species, positioned at the apex of the food chain, acted as sentinels for environmental health due to heightened exposure to improperly disposed waste. Regarding laboratory findings, each sample revealed the presence of at least one antimicrobial resistance gene (ARG). Notably, genes encoding resistance to tetracyclines dominated (94.9%), followed by beta-lactams (75.6%), sulfonamides (53.8%), aminoglycosides (51.3%), quinolones (44.9%), phenicols (25.6%), and macrolides (20.5%). Genes encoding polymyxins were not detected. Moreover, 66% of samples carried a multi-resistant microbiome, with 15% exhibiting resistance to three antimicrobial families and 51% to four. The absence of a correlation between forest coverage and ARG presence underscored the profound human impact on wildlife reserves, surpassing previous estimations. This environmental pressure could potentially modify microbiomes and resistomes in unknown ways. As not all antimicrobial families encoding ARGs were utilized by farmers, our next step involved evaluating other human activities to identify the primary sources of contamination. This comprehensive study contributed crucial insights into the intricate dynamics of AMR in natural ecosystems, paving the way for targeted interventions and sustainable coexistence.

A case study on pharmaceutical residues and antimicrobial resistance genes in Costa Rican rivers: A possible route of contamination for feline and other species.

In this investigation, the presence of antibiotics and pharmaceuticals in Costa Rican surface waters, specifically in regions near feline habitats, was examined. The study revealed that 47% of the water samples contained detectable traces of at least one antibiotic. Ciprofloxacin and norfloxacin were the most frequently detected compounds, each with a detection rate of 27%. Other antibiotics, such as erythromycin, roxithromycin, and trimethoprim, were also found but at lower frequencies, around 14%. Notably, all antibiotic concentrations remained below 10 ng/L, with ciprofloxacin, norfloxacin, and erythromycin showing the highest concentrations. Furthermore, the investigation revealed the presence of non-antibiotic pharmaceutical residues in the water samples, typically at concentrations below 64 ng/L. Tramadol was the most frequently detected compound, present in 18% of the samples. The highest concentrations were observed for acetaminophen and tramadol, measuring 64 and 10 ng/L, respectively. Comparing these findings with studies conducted in treated wastewater and urban rivers, it became evident that the concentrations of antibiotics and pharmaceuticals were notably lower in this study. While previous research reported higher values, the limited number of studies conducted in protected areas raises concerns about the potential environmental impact on biodiversity. In summary, these results emphasize the importance of monitoring pharmaceutical residues and antimicrobial resistance genes ARGs in vulnerable ecosystems, especially those in close proximity to feline habitats in Costa Rica. Additionally, the study delved into the detection of (ARGs). All tested water samples were positive for at least one ARG, with the blaTEM gene being the most prevalent at 82%, followed by tetS at 64% and qnrB at 23%. Moreover, this research shed light on the complexity of evaluating ARGs in environmental samples, as their presence does not necessarily indicate their expression. It also highlighted the potential for co-selection and co-regulation of ARGs, showcasing the intricate behaviors of these genes in aquatic environments.

IDENTIFICATION OF ANTHROPOGENIC IMPACT ON NATURAL HABITATS BY ANTIMICROBIAL RESISTANCE QUANTIFICATION IN TWO NEOTROPICAL WILD CATS AND THEIR GEOSPATIAL ANALYSIS.

Human activities such as habitat degradation and fragmentation threaten biodiversity in Neotropical areas. This work proposes an analytical methodology to identify natural areas in Central America with anthropogenic impact, analyzing the presence of antimicrobial resistance genes (ARGs) in accordance with their theoretical relationship with human-related activities. Sixteen ARGs were quantified in feces of different individuals of 13 jaguars (Panthera onca) and 13 pumas (Puma concolor) in three conservation areas in Costa Rica by real-time PCR. At least one ARG was detected in all samples. Of the ARGs encoding tetracycline resistance, the most frequent were tetQ and tetY (85% and 69%, respectively). The sulfonamides (sulI and sulII; 69% each), phenicols (catI and catII; 19% and 54%, respectively), and quinolones (qnrS; 12%) were also detected. The presence of human settlements, livestock farms (pigs, cattle, and poultry), roads, human health centers, flood zones, and rivers were identified within each area to generate an index of human activity. We found no difference between the presence of ARG by roads, agricultural activities, and human settlements (P>0.05). However, tetW showed higher percentages with porcine and bovine farms; both tetY and tetW were more frequent in jaguars than in pumas. Of concern is that many of the most contaminated samples were taken from national parks, such as Braulio Carrillo and Tortuguero, where animals should not have direct contact with humans.

Ecological Integrity Impairment and Habitat Fragmentation for Neotropical Macroinvertebrate Communities in an Agricultural Stream.

The Volcán River watershed in the south Pacific of Costa Rica comprises forests, small urban settlements, cattle fields, and intensive agriculture (mostly pineapple and sugarcane). The ecological integrity and quality of its waters was assessed from 2011-2013 and 2018-2019 by means of physical-chemical parameters (pH, conductivity, temperature, DO, DBO, nitrate, total phosphorus, and pesticide residues) and benthic macroinvertebrate (MI) sampling in eight sites (Volcán, Cañas, and Ángel Rivers, and Peje and Maura streams), resulting in high ecological integrity in all sites except the Peje stream, which is polluted with nitrates and pesticides. Only in this stream was there a marked seasonal variation in the abundance of 16 MI families including Leptohyphidae, Leptophlebiidae, Philopotamidae, Glossossomatidae, and Corydalidae, among others, whose presence was limited exclusively to the dry season (December to April), disappearing from the stream in the rainy season, with corresponding peaks in nitrate (max 20.3 mg/L) and pesticides (mainly herbicides and organophosphate insecticides). The characteristics of the watershed, with large areas of forest and excellent water quality, allow for the re-colonization of organisms into the Peje stream; however, those organisms are incapable of development and growth, providing evidence of a contaminant-driven habitat fragmentation in this stream during the rainy season.

Variación estacional y características fisicoquímicas e hidrológicas que influyen en los macroinvertebrados acuáticos, en un río tropical

Introducción: La distribución de los macroinvertebrados acuáticos en los ríos puede estar determinada por el tipo del sustrato, la forma del canal del río, el régimen hidrológico y las interacciones con factores bióticos y abióticos. En Costa Rica se desconoce en gran parte de los ríos, las variaciones en la distribución de los macroinvertebrados acuáticos producto de las transiciones y consecuentes variaciones en características fisicoquímicas e hidrológicas. Objetivos : 1) Estudiar el cambio del ensamblaje de macroinvertebrados acuáticos en dos sitios dentro del cauce del Río Naranjo, durante los cuatro periodos hidrológicos del año; 2) determinar la relación entre las variables fisicoquímicas, sustrato y el caudal con el ensamblaje. Metodología: El estudio se realizó en el Río Naranjo, Costa Rica, en dos sitios de muestreo durante un año, evaluando los cuatro periodos hidrológicos del año. En cada sitio de muestreo se obtuvieron valores de velocidad y profundidad del agua, caudal, oxígeno disuelto, pH, conductividad, sulfatos, amonio, nitrato, silicatos, fosfatos y sustrato, y se obtuvo una muestra de macroinvertebrados acuáticos por cada tipo de microhábitat determinado por la velocidad del agua. Se realizó una prueba PERMANOVA de dos vías, para determinar si existe una diferencia entre la abundancia de macroinvertebrados acuáticos, por periodo y entre sitios. Se hizo un análisis de correspondencia canónica para observar la relación entre los macroinvertebrados acuáticos y los factores ambientales. Por último, se realizó una prueba de Spearman para determinar la relación positiva o negativa de las variables con los macroinvertebrados Resultados: Hubo una mayor abundancia durante la estación seca, mientras que durante la transición de la estación seca a la lluviosa se registró la menor abundancia de organismos. El ACC indicó que el primer eje presentó una relación positiva con el sustrato limo y arena, y una relación negativa para las variables velocidad de corriente y sustrato rocoso. El segundo eje presentó una relación positiva con el caudal y los nitratos, y negativa para el sustrato canto rodado. La mayoría de los organismos mostraron una respuesta negativa al aumento del flujo y los nitratos. Discusión: Conforme aumenta el caudal, aumenta la velocidad del agua y no todos los organismos registrados en el Río Naranjo cuentan con características para sobrevivir a estas condiciones. Además, la profundidad también aumenta junto con la fuerza de arrastre, provocando el movimiento de los sustratos de menor tamaño y una remoción de organismos. El aumento de nitratos puede generar una disminución de los organismos más sensibles como Plecoptera y Ephemeroptera. A pesar de las variaciones fisicoquímicas y de caudal registradas en el Río Naranjo, éstas no fueron lo suficientemente fuertes como para variar el ensamblaje de macroinvertebrados acuáticos, por lo que la cuenca por el momento no cuenta con factores estresantes que elimine organismos durante uno o varios meses del año.

Introduction: The distribution of aquatic macroinvertebrates in rivers may be determined by the type of substrate, the shape of the river channel, the hydrological regime, and interactions with biotic and abiotic factors. Variations in the distribution of aquatic macroinvertebrates due to transitions and variations in physicochemical and hydrological characteristics are unknown in most rivers. Objectives: 1) to study the change in the aquatic macroinvertebrates assemblages in two sites in Naranjo River, during the four hydrological seasons; 2) to determine the relationship between the physicochemical variables, substrate and flow with the assemblages. Methodology: The study was conducted in the Naranjo River, Costa Rica, at two sampling sites during one year, evaluating the four seasons of the year. In each station we measure, values of water velocity and depth, flow rate, dissolved oxygen, pH, conductivity, sulfates, ammonium, nitrate, silicates, phosphates, and substrate were obtained, and a sample of aquatic macroinvertebrates was obtained for each type of microhabitat determined by water velocity. Two-way PERMANOVA was made to determine if there is a difference among aquatic macroinvertebrate abundance, per period and between sites. Canonical correspondence analysis was used to observe the relationship between aquatic macroinvertebrates and environmental factors. Finally, a Spearman's rank correlation coefficient was performed to determine the positive or negative relationship of variables with macroinvertebrates. Results: There was a greater abundance during the dry season, while during the transition from dry to the rainy season, the lowest abundance of organisms was recorded. The CCA indicates that the first axis presented a positive relationship with the silt and sand substrate and a negative relationship for the current velocity and rock substrate variables. The second axis showed a positive relationship with flow and nitrates and negative for the pebble substrate. Most organisms showed a negative response to increased flow and nitrates. Discussion: As flow increases, current velocity increases, and not all organisms recorded in the Naranjo River have the characteristics to survive these conditions. In addition, the depth also increases along with the drag force, causing movement of smaller substrates and removal of organisms. Increased nitrates can lead to a decrease in more sensitive organisms, such as Plecoptera and Ephemeroptera. In spite of the physicochemical and flow variations registered in the Naranjo River, these were not strong enough to vary the assemblages of aquatic macroinvertebrates, so the basin does not have stressful factors that eliminate organisms during one or several months of the year.

Colonización de macroinvertebrados acuáticos en tres tipos de sustratos artificiales, en un río tropical

Introducción: Existen diferentes metodologías para la recolecta de macroinvertebrados acuáticos (p.e., la red D y Red Surber), sin embargo, algunas de estas no son efectivas en todos los cuerpos de agua o representa un riesgo para la persona que adquiere la muestra. Los sustratos artificiales pueden emplearse en ríos profundos, contaminados o con presencia de cocodrilos. En Costa Rica hay estudios donde se utilizó los sustratos artificiales, para determinar la riqueza y abundancia en humedales tipo lago y lagunas, no obstante se desconoce la efectividad de un tipo de sustrato sobre otro y en sistemas lóticos. El objetivo del estudio fue demostrar la efectividad de tres tipos de sustratos artificiales. Métodos: las muestras se obtuvieron de manera bimensual entre los años 2013 y 2017, en el Río Parismina, Costa Rica, en cuatro sitios a lo largo del río y se evaluó los sustratos piedra, madera y hojas. Se aplicó una prueba de Kruskal-Wallis para determinar las diferencias entre la abundancia y el tipo de sustrato, como también para determinar la diferencia entre la abundancia y el sitio de muestreo. Además, para determinar si existe una diferencia entre los sustratos por sitio, se aplicó una prueba Permanova de dos vías. Un análisis de correspondencia múltiple para determinar el grado de asociación de la comunidad con respecto al tipo de sustrato. Resultados: Un total de 1159 organismos fueron recolectados, siendo Diptera y Ephemeroptera los órdenes más abundantes. La madera y hojas fueron los sustratos con mayor biodiversidad, como también los sitios con mayor vegetación. Conclusiones: El uso en conjunto de los tres tipos de sustratos artificiales, es efectivo para obtener una mayor riqueza de organismos, ya que se observó afinidad por un sustrato según el género de macroinvertebrado acuático.

Introduction: Different methodologies exist for sampling aquatic macroinvertebrates, however, some of these (e.g., kick-net, Surber) are not well suited for deep, contaminated rivers or rivers with presence of crocodiles, which represent a risk to the person who must take the sample. One alternative is to use artificial substrates. The use of artificial substrates to evaluate the aquatic macroinvertebrate biodiversity in wetlands has been studied previously in Costa Rica, but no research has been conducted to determine which types of substrates are most effective. The present study aimed to evaluate the effectiveness of three artificial substrates (i.e., stones, leaves and wood). Methods: Samples of the three substrates were taken bi-monthly between 2013 and 2017 in four sites of the Parismina River. A Kruskal-Wallis test was applied to determine differences between the macroinvertebrate abundance among substrate type. Beside to determine if there is a difference between substrates per site, a two-way PERMANOVA test was applied and a multiple correspondence analysis was used to correlate the substrate type with the macroinvertebrate community. Results: A total of 1 159 organisms were collected, with Diptera and Ephemeroptera being the most abundant. Wood and leaves were the substrates with greatest biodiversity and abundance of organisms; in addition the two sites with greatest vegetation were the most diverse. Conclusions: Most of the organisms were registered on any of the three types of artificial substrates, thus we recommend the joint use of the three types of substrates for biomonitoring or taxa richness studies.