Metagenomic perspectives on antibiotic resistance genes in tap water: The environmental characteristic, potential mobility and health threat.

As an emerging environmental contaminant, antibiotic resistance genes (ARGs) in tap water have attracted great attention. Although studies have provided ARG profiles in tap water, research on their abundance levels, composition characteristics, and potential threat is still insufficient. Here, 9 household tap water samples were collected from the Guangdong-Hong Kong-Macao Greater Bay Area (GBA) in China. Additionally, 75 sets of environmental sample data (9 types) were downloaded from the public database. Metagenomics was then performed to explore the differences in the abundance and composition of ARGs. 221 ARG subtypes consisting of 17 types were detected in tap water. Although the ARG abundance in tap water was not significantly different from that found in drinking water plants and reservoirs, their composition varied. In tap water samples, the three most abundant classes of resistance genes were multidrug, fosfomycin and MLS (macrolide-lincosamide-streptogramin) ARGs, and their corresponding subtypes ompR, fosX and macB were also the most abundant ARG subtypes. Regarding the potential mobility, vanS had the highest abundance on plasmids and viruses, but the absence of key genes rendered resistance to vancomycin ineffective. Generally, the majority of ARGs present in tap water were those that have not been assessed and are currently not listed as high-threat level ARG families based on the World Health Organization Guideline. Although the current potential threat to human health posed by ARGs in tap water is limited, with persistent transfer and accumulation, especially in pathogens, the potential danger to human health posed by ARGs should not be ignored.

Canadian beach cohort study: protocol of a prospective study to assess the burden of recreational water illness.

BACKGROUND: Recreational water activities at beaches are popular among Canadians. However, these activities can increase the risk of recreational water illnesses (RWI) among beachgoers. Few studies have been conducted in Canada to determine the risk of these illnesses. This protocol describes the methodology for a study to determine the risk and burden of RWI due to exposure to fecal pollution at beaches in Canada. METHODS: This study will use a mixed-methods approach, consisting of a prospective cohort study of beachgoers with embedded qualitative research. The cohort study involves recruiting and enrolling participants at public beaches across Canada, ascertaining their water and sand contact exposure status, then following-up after seven days to determine the incidence of acute RWI outcomes. We will test beach water samples each recruitment day for culture-based E. coli, enterococci using rapid molecular methods, and microbial source tracking biomarkers. The study started in 2023 and will continue to 2025 at beaches in British Columbia, Manitoba, Ontario, and Nova Scotia. The target enrollment is 5000 beachgoers. Multilevel logistic regression models will be fitted to examine the relationships between water and sand contact and RWI among beachgoers. We will also examine differences in risks by beachgoer age, gender, and beach location and the influence of fecal indicator bacteria and other water quality parameters on these relationships. Sensitivity analyses will be conducted to examine the impact of various alternative exposure and outcome definitions on these associations. The qualitative research phase will include focus groups with beachgoers and key informant interviews to provide additional contextual insights into the study findings. The study will use an integrated knowledge translation approach. DISCUSSION: Initial implementation of the study at two Toronto, Ontario, beaches in 2023 confirmed that recruitment is feasible and that a high completion rate (80%) can be achieved for the follow-up survey. While recall bias could be a concern for the self-reported RWI outcomes, we will examine the impact of this bias in a negative control analysis. Study findings will inform future recreational water quality guidelines, policies, and risk communication strategies in Canada.

Water temperature disturbance alters the conjugate transfer of antibiotic resistance genes via affecting ROS content and intercellular aggregation.

Spread of antibiotic resistance genes (ARGs) in aquatic ecosystems poses a significant global challenge to public health. The potential effects of water temperature perturbation induced by specific water environment changes on ARGs transmission are still unclear. The conjugate transfer of plasmid-mediated ARGs under water temperature perturbation was investigated in this study. The conjugate transfer frequency (CTF) was only 7.16 × 10-7 at a constant water temperature of 5 °C, and it reached 2.18 × 10-5 at 30 °C. Interestingly, compared to the constant 5 °C, the water temperature perturbations (cooling and warming models between 5-30 °C) significantly promoted the CTF. Intracellular reactive oxygen species was a dominant factor, which not only directly affected the CTF of ARGs, but also functioned indirectly via influencing the cell membrane permeability and cell adhesion. Compared to the constant 5 °C, water temperature perturbations significantly elevated the gene expression associated with intercellular contact, cell membrane permeability, oxidative stress responses, and energy driven force for CTF. Furthermore, based on the mathematical model predictions, the stabilization times of acquiring plasmid maintenance were shortened to 184 h and 190 h under cooling and warming model, respectively, thus the water temperature perturbations promoted the ARGs transmission in natural conditions compared with the constant low temperature conditions.

Spatial and temporal dynamics of microbes and genes in drinking water reservoirs: Distribution and potential for taste and odor generation.

Numerous reservoirs encounter challenges related to taste and odor issues, often attributed to odorous compounds such as geosmin (GSM) and 2-methylisoborneol (2-MIB). In this study, two large reservoirs located in northern and southern China were investigated. The Jinpen (JP) reservoir had 45.99 % Actinomycetes and 14.82 % Cyanobacteria, while the Xikeng (XK) reservoir contained 37.55 % Actinomycetes and 48.27 % Cyanobacteria. Most of the 2-MIB produced in surface layers of the two reservoirs in summer originated from Cyanobacteria, most of the 2-MIB produced in winter and in the bottom water originated from Actinomycetes. Mic gene abundance in the XK reservoir reached 5.42 × 104 copies/L in winter. The abundance of GSM synthase was notably high in the bottom layer and sediment of both reservoirs, while 2-MIB synthase was abundant in the surface layer of the XK reservoir, echoing the patterns observed in mic gene abundance. The abundance of odor-producing enzymes in the two reservoirs was inhibited by total nitrogen, temperature significantly influenced Actinomycetes abundance in the JP reservoir, whereas dissolved oxygen had a greater impact in the XK reservoir. Overall, this study elucidates the molecular mechanisms underlying odor compounding, providing essential guidance for water quality management strategies and the improvement of urban water reservoir quality.

Potentially pathogenic bacteria in the plastisphere from water, sediments, and commercial fish in a tropical coastal lagoon: An assessment and management proposal.

Microplastics in aquatic ecosystems harbor numerous microorganisms, including pathogenic species. The ingestion of these microplastics by commercial fish poses a threat to the ecosystem and human livelihood. Coastal lagoons are highly vulnerable to microplastic and microbiological pollution, yet limited understanding of the risks complicates management. Here, we present the main bacterial groups, including potentially pathogenic species, identified on microplastics in waters, sediments, and commercial fish from Ciénaga Grande de Santa Marta (CGSM), the largest coastal lagoon in Colombia. DNA metabarcoding allowed identifying 1760 bacterial genera on microplastics, with Aeromonas and Acinetobacter as the most frequent and present in all three matrices. The greatest bacterial richness and diversity were recorded on microplastics from sediments, followed by waters and fish. Biochemical analyses yielded 19 species of potentially pathogenic culturable bacteria on microplastics. Aeromonas caviae was the most frequent and, along with Pantoea sp., was found on microplastics in all three matrices. Enterobacter roggenkampii and Pseudomonas fluorescens were also found on microplastics from waters and fish. We propose management strategies for an Early Warning System against microbiological and microplastic pollution risks in coastal lagoons, illustrated by CGSM. This includes forming inter-institutional alliances for research and monitoring, accompanied by strengthening governance and health infrastructures.

Genetic traits and transmission of antimicrobial resistance characteristics of cephalosporin resistant Escherichia coli in tropical aquatic environments.

This study investigates the genetic traits and transmission mechanisms of cephalosporin-resistant Escherichia coli in tropical aquatic environments in Singapore. From 2016 to 2020, monthly samples were collected from wastewater treatment plants, marine niches, community sewage, beaches, reservoirs, aquaculture farms, and hospitals, yielding 557 isolates that were analyzed for antimicrobial resistance genes (ARGs) and mobile genetic elements (MGEs) using genomic methods. Findings reveal significant genotypic similarities between environmental and hospital-derived strains, particularly the pandemic E. coli ST131. Environmental strains exhibited high levels of intrinsic resistance mechanisms, including mutations in porins and efflux pumps, with key ARGs such as CMY-2 and NDM-9 predominantly carried by MGEs, which facilitate horizontal gene transfer. Notably, pathogenic EPEC and EHEC strains were detected in community sewage and aquaculture farms, posing substantial public health risks. This underscores the critical role of these environments as reservoirs for multidrug-resistant pathogens and emphasizes the interconnectedness of human activities and environmental health.

Antibiotic-resistant bacteria and resistance-genes in drinking water source in north Shoa zone, Amhara region, Ethiopia.

Background: The growing number of antimicrobial-resistant bacteria in a range of environments poses a serious challenge to infectious disease prevention. Good water quality is critical to human health and has a direct impact on a country's socio-economic growth. Therefore, assessing the bacteriological quality of drinking water provides benchmark data and provides insight into the development of further protection and treatment measures. Methods: A cross-sectional study was conducted from February 1, 2022, to September 31, 2023, in the diarrhea hotspot areas of North Shewa Zone (Minjar-Shenkora and Mojana-Wedera districts). Water samples were collected from drinking water sources (hand-pumps, boreholes, wells, spring water and ponds) to assess the quality following WHO guidelines. The collected water samples were processed for bacterial isolation, antimicrobial susceptibility testing, and detection of antimicrobial resistance genes. Data were entered and analyzed using the Statistical Package for the Social Sciences (SPSS) version 25. Results: A total of (49/138, 35.5%) bacteria were isolated from 138 drinking water samples, with a positive rate of (41/138, 29.7%). Among the isolates, (16/138, 11.6%) were Staphylococcus aureus while (33/138, 23.9%) were members of Enterobacteriaceae. Relatively high resistance rate among all isolates were observed for the most prescribed antibiotics in Ethiopia, including erythromycin, cotrimoxazole, doxycycline, ceftriaxone, gentamicin, and chloramphenicol. However, a low resistance was observed for early introduced antibiotics such as ciprofloxacin and recently introduced antibiotics such as cefotaxime, ceftazidime, imipenem, and meropenem. Among the 49 bacteria isolates, (32/49, 65.3%) were multidrug-resistant (MDR) pathogens while (12/49, 24.5%) were ESßL producers. Different ESßL genes were detected in most bacterial isolates. The predominant ESßL genes were blaCTX-M-gp8/25 (6/33, 18.2%), blaCTX-M-gp9 (5/33, 15.2%), and blaCTX-M-gp1 (5/33, 15.2%). Conclusion: The result of this study suggests that most water sources in the study area were contaminated by various bacterial species that are resistant to different antibiotics. Various ESßL resistance genes have also been detected. Therefore, regular sanitary inspection and bacteriological analysis should be mandatory to protect drinking water sources from contamination and the persistence of resistant bacteria.

Spatiotemporal distribution of environmental microbiota around animal farms adjacent to produce fields in central coast California.

This study aimed to identify different environmental microbiota in animal farms adjacent to produce fields and to understand their potential flow pattern. Soil and water samples were collected from 16 locations during the winter, spring, summer, and fall seasons. In addition, a high-resolution digital elevation model helped to create a stream network to understand the potential flow of the microbiome. Metagenomic analysis of the 16 S rRNA gene revealed that soil and water samples from the four seasons harbor diverse microbiome profiles. The phylogenetic relationship of operational taxonomic units (OTUs) is separated by a maximum of 0.6 Bray-Curtis distance. Similarly, the Principal Component Analysis (P = 0.001) demonstrated the soil and water microbiome clustering across different locations and seasons. The relative abundance of Proteobacteria, Bacteroidetes, and Firmicutes was higher in the water samples than in the soil samples. In contrast, the relative abundance of Actinobacteria and Chloroflexi was higher in the soil compared to the water samples. Soil samples in summer and water samples in spring had the highest abundance of Bacteroidetes and Firmicutes, respectively. A unique microbial community structure was found in water samples, with an increased abundance of Hydrogenophaga and Solirubrobacter. Genera that were significantly abundant at a 1% false discovery rate (FDR) among seasons and soil or water samples, include Nocardioides, Gemmatimonas, JG30-KF-CM45, Massilia, Gaiellales, Sphingomonas, KD4-96, Bacillus, Streptomyces, Gaiella, and Gemmatimonadaceae. The relative abundance of pathogenic genera, including Mycobacterium, Bacteroides, Nocardia, Clostridium, and Corynebacterium, were significantly (at 1% FDR) affected by seasons and environmental type. The elevation-based stream network model suggests the potential flow of microbiomes from the animal farm to the produce fields.

A machine learning approach for rapid early detection of Campylobacter spp. using absorbance spectra collected from enrichment cultures.

Enumeration of Campylobacter from environmental waters can be difficult due to its low concentrations, which can still pose a significant health risk. Spectrophotometry is an approach commonly used for fast detection of water-borne pollutants in water samples, but it has not been used for pathogen detection, which is commonly done through a laborious and time-consuming culture or qPCR Most Probable Number enumeration methods (i.e., MPN-PCR approaches). In this study, we proposed a new method, MPN-Spectro-ML, that can provide rapid evidence of Campylobacter detection and, hence, water concentrations. After an initial incubation, the samples were analysed using a spectrophotometer, and the spectrum data were used to train three machine learning (ML) models (i.e., supported vector machine - SVM, logistic regression-LR, and random forest-RF). The trained models were used to predict the presence of Campylobacter in the enriched water samples and estimate the most probable number (MPN). Over 100 stormwater, river, and creek samples (including both fresh and brackish water) from rural and urban catchments were collected to test the accuracy of the MPN-Spectro-ML method under various scenarios and compared to a previously standardised MPN-PCR method. Differences in the spectrum were found between positive and negative control samples, with two distinctive absorbance peaks between 540-542nm and 575-576nm for positive samples. Further, the three ML models had similar performance irrespective of the scenario tested with average prediction accuracy (ACC) and false negative rates at 0.763 and 13.8%, respectively. However, the predicted MPN of Campylobacter from the new method varied from the traditional MPN-PCR method, with a maximum Nash-Sutcliffe coefficient of 0.44 for the urban catchment dataset. Nevertheless, the MPN values based on these two methods were still comparable, considering the confidence intervals and large uncertainties associated with MPN estimation. The study reveals the potential of this novel approach for providing interim evidence of the presence and levels of Campylobacter within environmental water bodies. This, in turn, decreases the time from risk detection to management for the benefit of public health.

Analysis of 16S rRNA amplicon data illuminates the major role of environment in determining the marine plastisphere microbial communities.

Microplastics (MPs) are a potential threat to the marine environment and its associated ecosystem functions. Earlier investigations revealed that the microbiome plays a crucial role in deciding the fate of MPs in the environment. Further studies also highlighted the influences of environment and polymer types on the plastisphere microbiome. Nevertheless, the major factor that determines the plastisphere microbiome remains elusive. Thus, we examined the publicly available marine plastisphere data generated from polyethylene (PE), polypropylene (PP), and polystyrene (PS), collected from three different locations to identify the importance of environment and/or polymer types in shaping the microbiome. The beta diversity analyses showed a clear distinction between samples collected from different locations. The PERMANOVA results illustrated a significant influence of environment and sample type (control/PE/PP/PS) on the microbial communities. However, the influence of sample type on microbial diversity was not significant (P-value > 0.05) when the control samples were removed from the dataset but the environment remained a significant factor (P-value < 0.05). Further, the differential abundance analyses explicitly showed the abundance of many bacterial taxa to be significantly influenced (adjusted P-value < 0.05) by the locations rather than the polymer types. The validation analysis also supports the findings. Thus, this study suggests that both the surrounding environment and polymer types determine the microbial communities on marine MPs, but the role of the environment in shaping the microbial composition is greater than that of polymer types.

The piranha gut microbiome provides a selective lens into river water biodiversity.

Advances in omics technologies have enabled the in-depth study of microbial communities and their metabolic profiles from all environments. Here metagenomes were sampled from piranha (Serrasalmus rhombeus) and from river water from the Rio São Benedito (Amazon Basin). Shotgun metagenome sequencing was used to explore diversity and to test whether fish microbiomes are a good proxy for river microbiome studies. The results showed that the fish microbiomes were not significantly different from the river water microbiomes at higher taxonomic ranks. However, at the genus level, fish microbiome alpha diversity decreased, and beta diversity increased. This result repeated for functional gene abundances associated with specific metabolic categories (SEED level 3). A clear delineation between water and fish was seen for beta diversity. The piranha microbiome provides a good and representative subset of its river water microbiome. Variations seen in beta biodiversity were expected and can be explained by temporal variations in the fish microbiome in response to stronger selective forces on its biodiversity. Metagenome assembled genomes construction was better from the fish samples. This study has revealed that the microbiome of a piranha tells us a lot about its river water microbiome and function.

Prevalence and epidemiological distribution of indicators of pathogenic bacteria in households drinking water in Ethiopia: a systematic review and meta-analysis.

BACKGROUND: Ensuring the availability of safe drinking water remains a critical challenge in developing countries, including Ethiopia. Therefore, this paper aimed to investigate the prevalence of fecal coliform and E. coli bacteria and, geographical, children availability, and seasonal exposure assessment through a meta-analysis. METHODS: Two independent review groups extensively searched internet databases for English-language research articles published between 2013 and 2023. This systematic review and meta-analysis followed PRISMA guidelines. The methodological quality of each included study was evaluated using the STROBE guidelines. Publication bias was assessed by visual inspection of a funnel plot and then tested by the Egger regression test, and meta-analysis was performed using DerSimonian and Laird random-effects models with inverse variance weighting. Subgroup analyses were also conducted to explore heterogeneity. RESULTS: Out of 48 potentially relevant studies, only 21 fulfilled the inclusion criteria and were considered for meta-analysis. The pooled prevalence of fecal coliform and E. coli was 64% (95% CI: 56.0-71.0%, I2 = 95.8%) and 54% (95% CI: 45.7-62.3%, I2 = 94.2%), respectively. Subgroup analysis revealed that the prevalence of fecal coliform bacteria increased during the wet season (70%) compared to the dry season (60%), particularly in households with under-five children (74%) compared to all households (61%), in rural (68%) versus urban (66%) areas, and in regions with high prevalence such as Amhara (71%), Gambela (71%), and Oromia (70%). Similarly, the prevalence of E. coli was higher in households with under-five children (66%) than in all households (46%). CONCLUSIONS: The analysis highlights the higher prevalence of fecal coliform and E. coli within households drinking water, indicating that these bacteria are a significant public health concern. Moreover, these findings emphasize the critical need for targeted interventions aimed at improving drinking water quality to reduce the risk of fecal contamination and enhance public health outcomes for susceptible groups, including households with under-five children, in particular geographical areas such as the Amhara, Gambela, and Oromia regions, as well as rural areas, at point-of-use, and during the rainy season. REGISTRATION: This review was registered on PROSPERO (registration ID - CRD42023448812).

Extended-spectrum beta-lactamase (ESBL)- and non-ESBL producing Escherichia coli surveillance in surface water sources in Edo State, Nigeria: a public health concern.

This research explores the antimicrobial resistance (AMR) profiles and prevalence of extended-spectrum beta-lactamase (ESBL) and non-ESBL-producing Escherichia coli in Ojerame Dam and Ovokoto Spring, Edo State, Nigeria. Over 12 months, water was systematically sampled to accommodate seasonal variations and analyzed by employing an ESBL-selective medium for bacterial species. Additionally, bacterial isolates underwent identification and characterization using polymerase chain reaction (PCR) and disk diffusion methods to evaluate their susceptibility to antimicrobials. Results indicated significant prevalence of ESBL-producing E. coli, which exhibited complete resistance to common antimicrobials like ceftriaxone, ceftazidime, cefotaxime, and ampicillin while demonstrating 100% sensitivity to ertapenem, imipenem, meropenem, and nitrofurantoin. Non-ESBL-producing E. coli were resistant to ampicillin but sensitive to other antimicrobials mentioned earlier. Furthermore, both ESBL and non-ESBL-producing E. coli displayed multidrug resistance to varying degrees. Specific ESBL genes, including blaTEM, blaCTX-M-1, and blaCTX-M-15, were identified, alongside resistance genes like tetA, tetM, sul1, sul2, sul3, qnrA, qnrB, and qnrS in E. coli. This study pioneers the documentation of ESBL-producing E. coli in surface water in the region. This signals impending health risks associated with water being a reservoir of resistant genes while emphasizing the urgency for further research and public awareness concerning the quality of surface water.

In treacherous waters: detection of colistin-resistant bacteria in water and plastic litter from a recreational estuary.

Colistin resistance poses a major therapeutic challenge and resistant strains have now been reported worldwide. However, the occurrence of such bacteria in aquatic environments is considerably less understood. This study aimed to isolate and characterize colistin-resistant strains from water and plastic litter collected in an urban recreational estuary. Altogether, 64 strains with acquired colistin resistance were identified, mainly Acinetobacter spp. and Enterobacter spp. From these, 40.6% were positive for at least one mcr variant (1-9), 26.5% harbored, extended-spectrum beta-lactamases, 23.4% harbored, sulfonamide resistance genes, and 9.3% harbored, quinolone resistance genes. merA, encoding mercury resistance, was detected in 10.5% of these strains, most of which were also strong biofilm producers. The minimum inhibitory concentration toward colistin was determined for the mcr-positive strains and ranged from 2 to ≥512 µg ml-1. Our findings suggest that Gram-negative bacteria highly resistant to a last-resort antimicrobial can be found in recreational waters and plastic litter, thereby evidencing the urgency of the One Health approach to mitigate the antimicrobial resistance crisis.

Extreme trophic tales: deciphering bacterial diversity and potential functions in oligotrophic and hypereutrophic lakes.

BACKGROUND: Oligotrophy and hypereutrophy represent the two extremes of lake trophic states, and understanding the distribution of bacterial communities across these contrasting conditions is crucial for advancing aquatic microbial research. Despite the significance of these extreme trophic states, bacterial community characteristics and co-occurrence patterns in such environments have been scarcely interpreted. To bridge this knowledge gap, we collected 60 water samples from Lake Fuxian (oligotrophic) and Lake Xingyun (hypereutrophic) during different hydrological periods. RESULTS: Employing 16S rRNA gene sequencing, our findings revealed distinct community structures and metabolic potentials in bacterial communities of hypereutrophic and oligotrophic lake ecosystems. The hypereutrophic ecosystem exhibited higher bacterial α- and ß-diversity compared to the oligotrophic ecosystem. Actinobacteria dominated the oligotrophic Lake Fuxian, while Cyanobacteria, Proteobacteria, and Bacteroidetes were more prevalent in the hypereutrophic Lake Xingyun. Functions associated with methanol oxidation, methylotrophy, fermentation, aromatic compound degradation, nitrogen/nitrate respiration, and nitrogen/nitrate denitrification were enriched in the oligotrophic lake, underscoring the vital role of bacteria in carbon and nitrogen cycling. In contrast, functions related to ureolysis, human pathogens, animal parasites or symbionts, and phototrophy were enriched in the hypereutrophic lake, highlighting human activity-related disturbances and potential pathogenic risks. Co-occurrence network analysis unveiled a more complex and stable bacterial network in the hypereutrophic lake compared to the oligotrophic lake. CONCLUSION: Our study provides insights into the intricate relationships between trophic states and bacterial community structure, emphasizing significant differences in diversity, community composition, and network characteristics between extreme states of oligotrophy and hypereutrophy. Additionally, it explores the nuanced responses of bacterial communities to environmental conditions in these two contrasting trophic states.

Carbapenemase and extended-spectrum beta-lactamase-producing bacteria in waters originating from a single landfill in Slovenia.

Groundwater, rainwater, and leachate associated with a single landfill were analysed to detect extended-spectrum beta-lactamase (ESBL)-producing and carbapenemase (CP)-producing bacteria. After cultivation on three commercial selective-differential media, 240 bacterial isolates were obtained and identified by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). Isolates from clinically relevant species were further genotyped by enterobacterial repetitive intergenic consensus polymerase chain reaction, and tested for antibiotic susceptibility and presence of CPs and ESBL enzymes. Two ESBL-producing isolates and two isolates producing CPs were detected in rainwater, groundwater, and leachate: Klebsiella oxytoca complex with the gene for the ESBL enzyme CTX-M-1 and the gene for the CP OXA-48, Serratia fonticola with the gene for the ESBL enzyme FONA-2, and Pseudomonas aeruginosa with the gene coding Verona integron-encoded Metallo-beta-lactamases (VIM) metallo-beta-lactamase. Our study indicates that bacteria with ESBL and CP genes can be present in landfill-associated waters.

Widespread occurrence of fecal indicator bacteria in oligotrophic tropical streams. Are common culture-based coliform tests appropriate?

Monitoring of stream water quality is a key element of water resource management worldwide, but methods that are commonly used in temperate habitats may not be appropriate in humid tropical systems. We assessed the influence of four land uses on microbial water quality in 21 streams in the Panama Canal Watershed over a one-year period, using a common culture-based fecal indicator test and 16S rDNA metabarcoding. Each stream was located within one of four land uses: mature forest, secondary forest, silvopasture, and traditional cattle pasture. Culturing detected total coliforms and Escherichia coli across all sites but found no significant differences in concentrations between land uses. However, 16S rDNA metabarcoding revealed variability in the abundance of coliforms across land uses and several genera that can cause false positives in culture-based tests. Our results indicate that culture-based fecal indicator bacteria tests targeting coliforms may be poor indicators of fecal contamination in Neotropical oligotrophic streams and suggest that tests targeting members of the Bacteroidales would provide a more reliable indication of fecal contamination.

Successful Isolation of Diverse Verrucomicrobiota Strains through the Duckweed-Microbes Co-cultivation Method.

The "duckweed-microbes co-cultivation method" is a microbial isolation technique that effectively recovers diverse microbes, including rarely cultivated bacterial phyla, from environmental samples. In this method, aseptic duckweed and microbes collected from an environmental sample are co-cultivated for several days, and duckweed-associated microbes are then isolated from its roots using a conventional agar plate-based cultivation method. We herein propose several improvements to the method in order to specifically obtain members of the rarely cultivated bacterial phylum, Verrucomicrobiota. In systems using river water as the inoculum, the marked enrichment of Verrucomicrobiota was observed after 10 days of co-cultivation, particularly in the roots and co-cultivated media. We also successfully isolated 44 strains belonging to subdivisions 1, 3, and 4 of the phylum Verrucomicrobiota from these systems. This was achieved by changing the concentration of nitrogen in the co-cultivation medium, which is known to affect duckweed growth and/or metabolism, and by subjecting the fronds and co-cultivated media as well as the roots after co-cultivation to microbial isolation.

Quantitative fecal pollution assessment with bacterial, viral, and molecular methods in small stream tributaries.

Stream water quality can be impacted by a myriad of fecal pollution sources and waste management practices. Identifying origins of fecal contamination can be challenging, especially in high order streams where water samples are influenced by pollution from large drainage areas. Strategic monitoring of tributaries can be an effective strategy to identify conditions that influence local water quality. Water quality is assessed using fecal indicator bacteria (FIB); however, FIB cannot differentiate sources of fecal contamination nor indicate the presence of disease-causing viruses. Under different land use scenarios, three small stream catchments were investigated under 'wet' and 'dry' conditions (Scenario 1: heavy residential; Scenario 2: rural residential; and Scenario 3: undeveloped/agricultural). To identify fecal pollution trends, host-associated genetic targets HF183/BacR287 (human), Rum2Bac (ruminant), GFD (avian), and DG3 (canine) were analyzed along with FIB (Escherichia coli and enterococci), viral indicators (somatic and F+ coliphage), six general water quality parameters, and local rainfall. Levels of E. coli exceeded single sample maximum limits (235 CFU/100 mL) in 70.7 % of samples, enterococci (70 CFU/100 mL) in 100 % of samples, and somatic coliphage exceeded advisory thresholds (600 PFU/L) in 34.1 % of samples. The detection frequency for the human-associated genetic marker was highest in Scenario 3 (50 % of samples) followed by Scenario 2 (46 %), while the ruminant-associated marker was most prevalent in Scenario 1 (64 %). Due to the high proportion of qPCR-based measurements below the limit of quantification, a Bayesian data analysis approach was applied to investigate links between host-associated genetic marker occurrence with that of rainfall and fecal indicator levels. Multiple trends associated with small stream monitoring were revealed, emphasizing the role of rainfall, the utility of fecal source information to improve water quality management. And furthermore, water quality monitoring with bacterial or viral methodologies can alter the interpretation of fecal pollution sources in impaired waters.