RESUMEN
This study investigated the relationship between nutrient levels, source of fecal contamination, and pathogenic Leptospira in Puerto Rico's northern coast and San Juan Bay Estuary (SJBE) aquatic ecosystems. Microbial source tracking (MST) was also used to investigate the connections between sources of feces contamination and the presence of Leptospira. Eighty-seven water samples were collected during the June (n=44) and August (n=43) in 2020. To quantify phosphorus and nitrogen concentrations, standard USEPA protocols were utilized, specifically Methods 365.4 for total and dissolved phosphorus, 351.2 for total Kjeldahl nitrogen and ammonium, and 353.2 for nitrate. Lipl32 gene-specific quantitative polymerase chain reaction (qPCR) was used to detect the presence of Leptospira. Human (HF183), canine (BacCan-UCD), and equine (HoF597) MST assays were utilized to trace the origins of fecal contamination. Forty one percent of the locations exceeded Puerto Rico's authorized total phosphorus limit of 160 g L-1, while 34% exceeded the total nitrogen limit of 1700 g L-1. Nearly half of the streams examined are affected by eutrophication. The MST analysis identified human and canine feces as the most prevalent contaminants, affecting approximately 50% of the sites. In addition, Leptospira was detected in 32% of the June samples. There was a significant correlation (r = 0.79) between the incidence of pathogenic Leptospira and the human bacterial marker (HF183). This study illuminates the central role of anthropogenic inputs in nutrient enrichment and pathogen proliferation in Puerto Rico's aquatic ecosystems.
Asunto(s)
Monitoreo del Ambiente , Leptospira , Humanos , Caballos , Animales , Perros , Monitoreo del Ambiente/métodos , Contaminación del Agua/análisis , Puerto Rico , Ecosistema , Microbiología del Agua , Heces/microbiología , Nitrógeno/análisis , Fósforo/análisisRESUMEN
Leptospirosis is an emerging zoonotic disease in the Caribbean region and the island of Puerto Rico. Information on the presence of pathogenic Leptospira in rivers and streams of Puerto Rico is currently lacking. This study aimed to evaluate seasonal shifts in the presence of pathogenic leptospires and the level of Escherichia coli from 32 coastal locations in Puerto Rico's dry and wet seasons. Physicochemical parameters (temperature, salinity, pH, and dissolved oxygen) were determined at each site. The temperature (25.8 °C) and pH (average 7.6) values were all within acceptable USEPA regulatory standards. Thirty-eight percent of the sites of the dry season and 28% of the wet season sites contained dissolved oxygen levels ≤4 mg L-1 , which is relatively low. In the dry season, 19 sites (59%) and 18 (56%) of the wet season sites had E. coli counts >410 most probable number (MPN) 100 ml-1 and would be considered unsafe for recreational use. The lipl32 gene quantitative polymerase chain reaction assay was used for the detection of pathogenic leptospires in the samples. Low concentrations of pathogenic leptospires (<60 genome copies 100 ml-1 ) at Camuy, Espíritu Santo, Río Guayanilla, Quebrada Majagual, and Río Fajardo were detected during the wet season. Pathogenic leptospires were detected (â¼40 genome copies 100 ml-1 ) at only one site, Loíza, during the dry season. There was no predictable relationship between the physicochemical parameters, concentrations of E. coli, and the presence of pathogenic leptospires in water samples.
Asunto(s)
Escherichia coli , Leptospira , Puerto Rico , Ríos , Estaciones del AñoRESUMEN
Fecal pollution in the coastal marine environments was assessed at eleven sampling locations along the Georgia coast and Trinidad, and nine sites from Puerto-Rico. Membrane filtration (EPA method 1604 and method 1600) was utilized for Escherichia coli and enterococci enumeration at each location. Quantitative polymerase chain reaction (qPCR) amplification of the 16S ribosomal RNA gene was used to determine the presence of the Helicobacter pylori in marine samples. There was no significant correlation between the levels of E. coli, enterococci and H. pylori in these water samples. H. pylori was detected at four of the 31 locations sampled; Oak Grove Island and Village Creek Landing in Georgia, Maracas river in Trinidad, and Ceiba Creek in Puerto Rico. The study confirms the potential public health risk to humans due to the widespread distribution of H. pylori in subtropical and tropical costal marine waters.
Asunto(s)
Monitoreo del Ambiente , Helicobacter pylori/crecimiento & desarrollo , Agua de Mar/microbiología , Microbiología del Agua , Escherichia coli/clasificación , Escherichia coli/crecimiento & desarrollo , Georgia , Helicobacter pylori/clasificación , Helicobacter pylori/aislamiento & purificación , Puerto Rico , Trinidad y TobagoRESUMEN
The goal of this study was to determine the potential for Enterohemorrhagic Escherichia coli O157:H7 (EHEC) contamination in tropical marine waters. Samples were collected from urban, suburban, and rural sites around the islands of Puerto Rico and The Republic of Trinidad and Tobago. Quantification of E. coli and EHEC was evaluated using MI plates and qPCR. EHEC was detected in six sites in Puerto Rico: West of La Parguera Town, Boquilla, Oro Creek, Fishers Association, Joyuda Lagoon, and Boqueron Wetland Creek and in two rural sites in Trinidad: Balandra Bay and Quinam Bay. Plate count enumeration of E. coli was not a reliable indicator for the presence of EHEC. The sites where EHEC was detected on both islands are used for recreational bathing, water sports and recreational/commercial fisheries and therefore pose a public potential health risk.
Asunto(s)
Escherichia coli Shiga-Toxigénica/crecimiento & desarrollo , Microbiología del Agua , Región del Caribe , Monitoreo del Ambiente , Agua de Mar/microbiología , Escherichia coli Shiga-Toxigénica/clasificación , Escherichia coli Shiga-Toxigénica/aislamiento & purificación , Contaminación del AguaRESUMEN
In this study, quantitative polymerase chain reaction targeting the atrazine catabolism gene, atzA, was used to detect the presence of atrazine degrading bacteria as an indicator of atrazine contamination in 11 sites in Georgia, nine coastal sites in Puerto Rico and 11 coastal sites in Trinidad. The atzA gene was detected in five stations in Georgia (Oak Grove Island entrance, Blythe Island Recreation Park, Jekyll Island., Village Creek Landing and Dunbar Creek Sea Island Rd Bridge). In Puerto Rico gene was detected in five sites (Boquilla, Oro Creek, Fishers Association, Ceiba Creek and Sabalos Creek) while seven sites in Trinidad (Carli Bay, Las Cuevas Bay, Quinam Bay, Salybia River, Salybia Bay, Maracas River and Maracas Bay) showed the presence of atzA.
Asunto(s)
Atrazina/metabolismo , Bacterias/genética , Genes Bacterianos , Herbicidas/metabolismo , Agua de Mar/microbiología , Contaminantes Químicos del Agua/metabolismo , Bacterias/metabolismo , Biodegradación Ambiental , Georgia , Puerto Rico , Agua de Mar/química , Trinidad y TobagoRESUMEN
AIMS: To assess the concentration of atrazine in Lake Oconee and develop a qPCR assay as a potential marker for the presence of atrazine-degrading bacteria indicating atrazine contamination. METHODS AND RESULTS: Water and sediment samples were collected from the Oconee Lake at four golf course sites, two residential sites, one cattle farming site and a forested site. Atrazine concentration at the study sites was determined using an ELISA kit and indicated the presence of atrazine from 0·72 ppb at the forested sites to 1·84 ppb at the golf course sites. QPCR results indicate the presence of atzA gene (atrazine chlorohydrolase) from 1·51 × 10(2) gene copies at the residential sites to 3·31 × 10(5) gene copies per 100 ml of water at the golf course regions of the lake and correlated (r = 0·64) with atrazine concentration. Sediment samples had higher atzA gene copies compared with the water samples (P < 0·05). CONCLUSIONS: Atrazine concentration and the highest quantity of atzA gene were detected in the golf course regions of the lake. Overall, atrazine concentration monitored in Lake Oconee was below the Environment Protection Agency (EPA) regulatory standards. SIGNIFICANCE AND IMPACT OF THE STUDY: Quantitative PCR is an efficient technique for assessing the presence of atrazine catabolism gene as a functional marker for atrazine-degrading bacteria and the presence of atrazine contamination.
Asunto(s)
Atrazina/análisis , Bacterias/aislamiento & purificación , Agua Dulce/química , Microbiología del Agua , Contaminantes Químicos del Agua/análisis , Agricultura , Animales , Bacterias/enzimología , Bacterias/genética , Bovinos , Recuento de Colonia Microbiana , ADN Bacteriano/genética , Agua Dulce/microbiología , Genes Bacterianos , Sedimentos Geológicos/química , Georgia , Golf , Hidrolasas/genética , Reacción en Cadena de la Polimerasa/métodos , Árboles/microbiologíaRESUMEN
Microbial community dynamics in wetlands microcosms emended with commercial products (surfactant, a biological agent, and nutrients) designed to enhance bioremediation was followed for 3 months. The effectiveness of enhanced degradation was assessed by determining residual concentrations of individual petroleum hydrocarbons by GC/MS. The size and composition of the sediment microbial community was assessed using a variety of indices, including bacterial plate counts, MPNs, and DNA hybridizations with domain- and group-specific oligonucleotide probes. The addition of inorganic nutrients was the most effective treatment for the enhancement of oil degradation, resulting in marked degradation of petroleum alkanes and a lesser extent of degradation of aromatic oil constituents. The enhanced degradation was associated with increases in the amount of extractable microbial DNA and Streptomyces in the sediment, although not with increased viable counts (plate counts, MPN). Bacteria introduced with one of the proprietary products were still detected in the microcosms after 3 months, but were not a major quantitative constituent of the community. The biological product enhanced oil degradation relative to the control, but to a lesser extent than the nutrient additions alone. In contrast, application of the surfactant to the oil-impacted sediment decreased oil degradation.
Asunto(s)
Biodegradación Ambiental , Sedimentos Geológicos/microbiología , Petróleo/microbiología , Microbiología del Suelo , Contaminantes del Suelo , Recuento de Colonia Microbiana , Sondas de ADN/química , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , Hibridación de Ácido Nucleico , Petróleo/efectos adversos , Petróleo/metabolismo , Hidrocarburos Policíclicos Aromáticos/metabolismo , ARN Ribosómico 16S/genética , Análisis de Regresión , Contaminantes del Suelo/efectos adversos , Contaminantes del Suelo/metabolismo , Tensoactivos/farmacologíaRESUMEN
DNA extracts from sediment and water samples are often contaminated with coextracted humic-like impurities. Estuarine humic substances and vascular plant extract were used to evaluate the effect of the presence of such impurities on DNA hybridization and quantification. The presence of humic substances and vascular plant extract interfered with the fluorometric measurement of DNA concentration using Hoechst dye H33258 and PicoGreen reagent. Quantification of DNA amended with humic substances (20-80 ng/microl) using the Hoechst dye assay was more reliable than with PicoGreen reagent. A simple procedure was developed to improve the accuracy for determining the DNA concentration in the presence of humic substances. In samples containing up to 80 ng/microl of humic acids, the fluorescence of the samples were measured twice: first without Hoechst dye to ascertain any fluorescence from impurities in the DNA sample, followed with Hoechst dye addition to obtain the total sample fluorescence. The fluorescence of the Hoechst dye-DNA complex was calculated by subtracting the fluorescence of the impurities from the fluorescence of the sample. Vascular plant extract and humic substances reduced the binding of DNA onto the nylon membrane. Low amounts (<2.0 microg) of humic substances derived from estuarine waters did not affect the binding of 100 ng of target DNA to nylon membranes. DNA samples containing 1.0 microg of humic substances performed well in DNA hybridizations with DIG-labeled oliogonucleotide and chromosomal probes. Therefore, we suggest that DNA samples containing low concentrations of humic substances (<20 ng/microl) could be used in quantitative membrane hybridization without further purification.
Asunto(s)
ADN Bacteriano/análisis , ADN Bacteriano/efectos de los fármacos , Escherichia coli/genética , Sustancias Húmicas/farmacología , Hibridación de Ácido Nucleico/efectos de los fármacos , Bisbenzimidazol/metabolismo , Colorantes Fluorescentes/metabolismo , Fluorometría/métodos , Compuestos Orgánicos , Extractos Vegetales/farmacología , Espectrofotometría/métodos , ÁrbolesRESUMEN
The effectiveness of maintaining prokaryotic RNA in Synechococcus and Pseudomonas cells, fixed in 96% ethanol, 4% paraformaldehyde, or suspended in RNAlater, and held in cold storage for 3 months was compared. Fluorometric determination of the RNA extracted from Synechococcus and Pseudomonas cells indicated that the cell storage treatments tested were equally effective at maintaining their total RNA content. There was not any detectable decrease in the quantity of RNA isolated from the preserved samples during storage. Intact mRNA transcripts of the RuBisCO (rbcL) and nir genes were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) from preserved bacterial cells throughout 3 months of storage. In contrast, RT-PCR failed to amplify the mRNA of the rbcL and nitrite reductase genes in unfixed and/or unpreserved bacterial samples, suggesting that bacterial mRNA can be well maintained during a prolonged storage when cells are preserved properly. In addition, RNAlater is a useful reagent for the storage and maintenance of high quality RNA in unfrozen samples.
Asunto(s)
Bacterias/genética , Preservación Biológica , ARN Mensajero/análisis , ARN Mensajero/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Bacterias/aislamiento & purificación , ADN Bacteriano/análisis , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , ARN Bacteriano/análisis , ARN Bacteriano/genética , ARN Bacteriano/aislamiento & purificación , ARN Mensajero/genéticaRESUMEN
The effect of oil amendment in salt marsh sediment microcosms was examined by most probable number (MPN), DNA-hybridization with domain-specific oligonucleotide probes and whole community 16S rDNA-hybridizations. Gas chromatography (GC/MS) analysis of oil residues in sediments from microcosms after 3 months of operation showed that the quantity of petroleum hydrocarbons was lower in microcosms amended with oil compared to microcosms amended with oil+plant detritus. Bacterial numbers (total-MPN) increased in all experimental microcosms (amended with plant detritus, oil, and oil+plant detritus). In comparison to the intact sediment, the proportions of oil-degrading bacteria increased >100-fold in the oil amended microcosm and >10-fold in the plant detritus and the oil+plant detritus amended microcosms. DNA-hybridizations with Bacteria, Archaea and Eukarya oligonucleotide probes indicated few changes in the petroleum contaminated sediment community profile. In contrast, rDNA-hybridizations indicated that the bacterial community profile of the oil-impacted sediments, after 1 month of exposure, was significantly different from the control sediment.