Ecotoxicological effects of ketoprofen and fluoxetine and their mixture in an aquatic microcosm.

The effects of fluoxetine (antidepressant) and ketoprofen (analgesic) on aquatic ecosystems are largely unknown, particularly as a mixture. This work aimed at determining the effect of sublethal concentrations of both compounds individually (0.050 mg/L) and their mixture (0.025 mg/L each) on aquatic communities at a microcosm scale for a period of 14 d. Several physicochemical parameters were monitored to estimate functional alterations in the ecosystem, while model organisms (Daphnia magna, Lemna sp., Raphidocelis subcapitata) and the sequencing of 16S/18S rRNA genes permitted to determine effects on specific populations and changes in community composition, respectively. Disturbances were more clearly observed after 14 d, and overall, the microcosms containing fluoxetine (alone or in combination with ketoprofen) produced larger alterations on most physicochemical and biological variables, compared to the microcosm containing only ketoprofen, which suffered less severe changes. Differences in nitrogen species suggest alterations in the N-cycle due to the presence of fluoxetine; similarly, all pharmaceutical-containing systems decreased the brood rate of D. magna, while individual compounds inhibited the growth of Lemna sp. No clear trends were observed regarding R. subcapitata, as indirectly determined by chlorophyll quantification. The structure of micro-eukaryotic communities was altered in the fluoxetine-containing systems, whereas the structure of bacterial communities was affected to a greater extent by the mixture. The disruptions to the equilibrium of the microcosm demonstrate the ecological risk these compounds pose to aquatic ecosystems.

Erwiniaceae bacteria play defensive and nutritional roles in two widespread ambrosia beetles.

Ambrosia beetles are fungal-growing insects excavating galleries deep inside the wood. Their success as invaders increased scientific interest towards them. However, most studies on their microbiota targeted their fungal associates whereas the role of bacterial associates is understudied. To explore the role of abundant microbial associates, we isolated bacteria from active galleries of two widespread ambrosia beetles, Xylosandrus crassiusculus and X. germanus. These isolates were classified within the Erwiniaceae family and through a phylogenetic analysis including isolates from other insects we showed that they clustered with isolates obtained from ambrosia and bark beetles, including Erwinia typographi. The whole genome analysis of the isolate from active galleries of X. crassiusculus suggested that this bacterium plays both a nutritional role, by providing essential amino acids and enzymes for the hydrolysis of plant biomass, and a defensive role, by producing antibiotics. This defensive role was also tested in vitro against fungi, including mutualists, common associates, and parasites. The bacteria inhibited the growth of some of the common associates and parasites but did not affect mutualists. Our study supported the hypothesis of a mutualist role of Erwiniaceae bacteria in ambrosia beetles and highlighed the importance of bacteria in maintaining the symbiosis of their host with nutritional fungi.

Wastewater-based epidemiology for surveillance of infectious diseases in healthcare settings.

PURPOSE OF REVIEW: Wastewater-based surveillance (WBS) (epidemiology) using near-source sampling (NSS) in large buildings, hospitals and care homes is reviewed covering three main areas: state-of-the-art WBS, benefits/opportunities NSS has for hospital infection control systems and new insights from hospital wastewater surveillance and policy implications. RECENT FINDINGS: Wastewater provides aggregate, anonymous sources of data where the spatial resolution can be linked to populations being served. In hospitals, clear links established between wastewater RNA-fragments signal to nosocomial COVID-19 cases/outbreaks. Detecting other targets from hospital wastewater such as antimicrobial resistance markers is considered a substantial opportunity for this technology. Other clinically relevant infections, that is influenza and monkeypox, can be perceived, and sub-variant resolution to target public health response in near real time to benefit hospital infection control. WBS can reduce hospitals' clinical testing requirements, as diagnostic costs are aggregated into fewer samples while still detecting single cases. SUMMARY: WBS using NSS can inform infectious disease monitoring earlier, faster and cheaper than conventional monitoring. Routine sampling using wastewater provides a platform for risk-based sampling and enables smarter allocation of resources. Finally, hospital wastewater can be used for the benefit of the wastewater surveillance field as a promising source to monitor emerging threats and resolve longstanding questions on faecal shedding. Hospital monitoring in low-income settings is considered a priority for future research.

Wastewater monitoring for detection of public health markers during the COVID-19 pandemic: Near-source monitoring of schools in England over an academic year.

BACKGROUND: Schools are high-risk settings for infectious disease transmission. Wastewater monitoring for infectious diseases has been used to identify and mitigate outbreaks in many near-source settings during the COVID-19 pandemic, including universities and hospitals but less is known about the technology when applied for school health protection. This study aimed to implement a wastewater surveillance system to detect SARS-CoV-2 and other public health markers from wastewater in schools in England. METHODS: A total of 855 wastewater samples were collected from 16 schools (10 primary, 5 secondary and 1 post-16 and further education) over 10 months of school term time. Wastewater was analysed for SARS-CoV-2 genomic copies of N1 and E genes by RT-qPCR. A subset of wastewater samples was sent for genomic sequencing, enabling determination of the presence of SARS-CoV-2 and emergence of variant(s) contributing to COVID-19 infections within schools. In total, >280 microbial pathogens and >1200 AMR genes were screened using RT-qPCR and metagenomics to consider the utility of these additional targets to further inform on health threats within the schools. RESULTS: We report on wastewater-based surveillance for COVID-19 within English primary, secondary and further education schools over a full academic year (October 2020 to July 2021). The highest positivity rate (80.4%) was observed in the week commencing 30th November 2020 during the emergence of the Alpha variant, indicating most schools contained people who were shedding the virus. There was high SARS-CoV-2 amplicon concentration (up to 9.2x106 GC/L) detected over the summer term (8th June - 6th July 2021) during Delta variant prevalence. The summer increase of SARS-CoV-2 in school wastewater was reflected in age-specific clinical COVID-19 cases. Alpha variant and Delta variant were identified in the wastewater by sequencing of samples collected from December to March and June to July, respectively. Lead/lag analysis between SARS-CoV-2 concentrations in school and WWTP data sets show a maximum correlation between the two-time series when school data are lagged by two weeks. Furthermore, wastewater sample enrichment coupled with metagenomic sequencing and rapid informatics enabled the detection of other clinically relevant viral and bacterial pathogens and AMR. CONCLUSIONS: Passive wastewater monitoring surveillance in schools can identify cases of COVID-19. Samples can be sequenced to monitor for emerging and current variants of concern at the resolution of school catchments. Wastewater based monitoring for SARS-CoV-2 is a useful tool for SARS-CoV-2 passive surveillance and could be applied for case identification and containment, and mitigation in schools and other congregate settings with high risks of transmission. Wastewater monitoring enables public health authorities to develop targeted prevention and education programmes for hygiene measures within undertested communities across a broad range of use cases.

Dissemination of metaldehyde catabolic pathways is driven by mobile genetic elements in Proteobacteria.

Bioremediation of metaldehyde from drinking water using metaldehyde-degrading strains has recently emerged as a promising alternative. Whole-genome sequencing was used to obtain full genomes for metaldehyde degraders Acinetobacter calcoaceticus E1 and Sphingobium CMET-H. For the former, the genetic context of the metaldehyde-degrading genes had not been explored, while for the latter, none of the degrading genes themselves had been identified. In A. calcoaceticus E1, IS91 and IS6-family insertion sequences (ISs) were found surrounding the metaldehyde-degrading gene cluster located in plasmid pAME76. This cluster was located in closely-related plasmids and associated to identical ISs in most metaldehyde-degrading ß- and γ-Proteobacteria, indicating horizontal gene transfer (HGT). For Sphingobium CMET-H, sequence analysis suggested a phytanoyl-CoA family oxygenase as a metaldehyde-degrading gene candidate due to its close homology to a previously identified metaldehyde-degrading gene known as mahX. Heterologous gene expression in Escherichia coli alongside degradation tests verified its functional significance and the degrading gene homolog was henceforth called mahS. It was found that mahS is hosted within the conjugative plasmid pSM1 and its genetic context suggested a crossover between the metaldehyde and acetoin degradation pathways. Here, specific replicons and ISs responsible for maintaining and dispersing metaldehyde-degrading genes in α, ß and γ-Proteobacteria through HGT were identified and described. In addition, a homologous gene implicated in the first step of metaldehyde utilisation in an α-Proteobacteria was uncovered. Insights into specific steps of this possible degradation pathway are provided.

First insights into the prokaryotic community structure of Lake Cote, Costa Rica: Influence on nutrient cycling.

Prokaryotic diversity in lakes has been studied for many years mainly focusing on community structure and how the bacterial assemblages are driven by physicochemical conditions such as temperature, oxygen, and nutrients. However, little is known about how the composition and function of the prokaryotic community changes upon lake stratification. To elucidate this, we studied Lake Cote in Costa Rica determining prokaryotic diversity and community structure in conjunction with physicochemistry along vertical gradients during stratification and mixing periods. Of the parameters measured, ammonium, oxygen, and temperature, in that order, were the main determinants driving the variability in the prokaryotic community structure of the lake. Distinct stratification of Lake Cote occurred (March 2018) and the community diversity was compared to a period of complete mixing (March 2019). The microbial community analysis indicated that stratification significantly altered the bacterial composition in the epi-meta- and hypolimnion. During stratification, the Deltaproteobacteria, Chloroflexi, Bacteroidetes, Nitrospirae, and Euryarchaeota were dominant in the hypolimnion yet largely absent in surface layers. Among these taxa, strict or facultative anaerobic bacteria were likely contributing to the lake nitrogen biogeochemical cycling, consistent with measurements of inorganic nitrogen measurements and microbial functional abundance predictions. In general, during both sampling events, a higher abundance of Alphaproteobacteria, Betaproteobacteria, Actinobacteria, and Cyanobacteria was found in the oxygenated layers. Lake Cote had a unique bacterial diversity, with 80% of Amplicon Sequence Variant (ASV) recovered similar to unclassified/uncultured strains and exhibits archetypal shallow lake physicochemical but not microbial fluctuations worthy of further investigation. This study provides an example of lake hydrodynamics impacts to microbial community and their function in Central American lakes with implications for other shallow, upland, and oligotrophic lake systems.

Monitoring occurrence of SARS-CoV-2 in school populations: A wastewater-based approach.

Clinical testing of children in schools is challenging, with economic implications limiting its frequent use as a monitoring tool of the risks assumed by children and staff during the COVID-19 pandemic. Here, a wastewater-based epidemiology approach has been used to monitor 16 schools (10 primary, 5 secondary and 1 post-16 and further education) in England. A total of 296 samples over 9 weeks have been analysed for N1 and E genes using qPCR methods. Of the samples returned, 47.3% were positive for one or both genes with a detection frequency in line with the respective local community. WBE offers a low cost, non-invasive approach for supplementing clinical testing and can provide longitudinal insights that are impractical with traditional clinical testing.

Probe-based qPCR assay enables the rapid and specific detection of bacterial degrading genes for the pesticide metaldehyde in soil.

Metaldehyde, a molluscicide pesticide, has been identified as a pollutant of concern due to its repeated detection in drinking water, thereby generating numerous compliance failures for water utilities. Biological degradation potential for metaldehyde is widespread in soils, occurring at different rates, but to date, no molecular methods for its assessment have been reported. Here, three genes belonging to a shared metaldehyde-degrading gene cluster present in bacteria were used as candidates for development of a quantitative PCR (qPCR) assay for assessing the metaldehyde-degrading potential in soil. Screening of gene targets, primer pairs and optimization of reaction conditions led to the development of a sensitive and specific probe-based qPCR method for quantifying the mahY metaldehyde-degrading gene from soil. The technique was tested across 8 soils with different compositions and origins. The degrading pathway was detected in 4/8 soils, in which a higher number of gene copies correlated with periods of greater metaldehyde removal. Additionally, swift elimination of the pesticide was observed in soils with an elevated initial number of mahY gene copies. The gene cluster was not detected in other soils, even though metaldehyde removal occurred, indicating that other biological degrading pathways are also important in nature. The method described here is the first one available to estimate the microbial metaldehyde degradation potential and activity in soils, and can also be used to detect degrading microorganisms in systems such as sand filters for water purification or to monitor degrading strains in engineered processes.

Simultaneous removal of neonicotinoid insecticides by a microbial degrading consortium: Detoxification at reactor scale.

Neonicotinoid insecticides show high persistence in the environment, and standard biological approaches such as biopurification systems have shown mostly inefficient removal of such compounds. In this work, soil pre-exposed to imidacloprid was used to obtain presumptive imidacloprid-degrading consortia. Cometabolic enrichment yielded a microbial consortium composed of eight bacterial and one yeast strains, capable of degrading not only this compound, but also thiamethoxam and acetamiprid, as demonstrated in cross-degradation assays. The biological removal process was scaled-up to batch stirred tank bioreactors (STBR); this configuration was able to simultaneously remove mixtures of imidacloprid + thiamethoxam or imidacloprid + thiamethoxam + acetamiprid, reaching elimination of 95.8% and 94.4% of total neonicotinoids, respectively. Removal rates in the bioreactors followed the pattern imidacloprid > acetamiprid > thiamethoxam, including >99% elimination of imidacloprid in 6 d and 17 d (binary and ternary mixtures, respectively). A comprehensive evaluation of the detoxification in the STBR was performed using different biomarkers: seed germination (Lactuca sativa), bioluminescence inhibition (Vibrio fischeri), and acute oral tests in honeybees. Overall, ecotoxicological tests revealed partial detoxification of the matrix, with clearer detoxification patterns in the binary mixture. This biological approach represents a promising option for the removal of neonicotinoids from agricultural wastewater; however, optimization of the process should be performed before application in farms.

Impact of oxytetracycline and bacterial bioaugmentation on the efficiency and microbial community structure of a pesticide-degrading biomixture.

An experimental study evaluating the effect of bioaugmentation and antibiotic (oxytetracycline) application on pesticide degradation and microbial community structure of a biomixture used in a biopurification system (BPR) was conducted. The bioaugmentation employed a carbofuran-degrading bacterial consortium. The non-bioaugmented biomixture showed excellent performance for removal of atrazine (t1/2: 9.9 days), carbendazim (t1/2: 3.0 days), carbofuran (t1/2: 2.8 days), and metalaxyl (t1/2: 2.7 days). Neither the addition of oxytetracycline nor bioaugmentation affected the efficiency of pesticide removal or microbial community (bacterial and fungal) structure, as determined by DGGE analysis. Instead, biomixture aging was mainly responsible for microbial population shifts. Even though the bioaugmentation did not enhance the biomixtures' performance, this matrix showed a high capability to sustain initial stresses related to antibiotic addition; therefore, simultaneous elimination of this particular mixture of pesticides together with oxytetracycline residues is not discouraged.

Expanding the application scope of on-farm biopurification systems: Effect and removal of oxytetracycline in a biomixture.

Antibiotic-containing wastewaters produced in agricultural activities may depress the pesticide-degrading capacity of biomixtures contained in biopurification systems. This work aimed to assay the effect of oxytetracycline (OTC) on the removal of carbofuran (CFN) in an optimized biomixture, and to determine the capacity of the system to dissipate OTC. During co-application of CFN+OTC, CFN removal and its accelerated degradation were not negatively affected. Similarly, different doses of OTC (10-500mgkg-1) did not significantly affect CFN mineralization, and the process even exhibited a hormetic-like effect. Moreover, the biomixture was able to remove OTC with a half-life of 34.0 d. DGGE-cluster analyses indicated that fungal and bacterial communities remained relatively stable during OTC application and CFN+OTC co-application, with similarities of over 70% (bacteria) and 80% (fungi). Overall, these findings support the potential use of this matrix to discard OTC-containing wastewater in this system originally intended for CFN removal.

Elimination of fungicides in biopurification systems: Effect of fungal bioaugmentation on removal performance and microbial community structure.

Bioaugmentation with ligninolytic fungi represents a potential way to improve the performance of biomixtures used in biopurification systems for the treatment of pesticide-containing agricultural wastewater. The fungus Trametes versicolor was employed in the bioaugmentation of a biomixture to be used in the simultaneous removal of seven fungicides. Liquid cultures of the fungus were able to remove tebuconazole, while no evidence of carbendazim, metalaxyl and triadimenol depletion was found. When applied in the biomixture, the bioaugmented matrix failed to remove all the triazole fungicides (including tebuconazole) under the assayed conditions, but was efficient to eliminate carbendazim, edifenphos and metalaxyl (the latter only after a second pesticide application). The re-addition of pesticides markedly increased the elimination of carbendazim and metalaxyl; nonetheless, no clear enhancement of the biomixture performance could be ascribed to fungal bioaugmentation, not even after the re-inoculation of fungal biomass. Detoxification efficiently took place in the biomixture (9 d after pesticide applications) according to acute tests on Daphnia magna. DGGE-analysis revealed only moderate time-divergence in bacterial and fungal communities, and a weak establishment of T. versicolor in the matrix. Data suggest that the non-bioaugmented biomixture is useful for the treatment of fungicides other than triazoles.

Antibiotics do not affect the degradation of fungicides and enhance the mineralization of chlorpyrifos in biomixtures.

The use of antibiotics in agriculture produces residues in wastewaters. The disposal of such wastewaters in biopurification systems (BPS) employed for the treatment of pesticides could result in the inhibition of the degrading capacity of the biomixtures used in the BPS. We assayed the effect of two commercial formulations of antibiotics used in agriculture, one containing kasugamycin (KSG) and the other oxytetracycline plus gentamicin (OTC+GTM), on the biomixture performance. Doses from 0.1mgkg-1 to 1000mgkg-1 of KSG increased the respiration of the biomixture, and low doses enhanced the mineralization rate of the insecticide 14C-chlorpyrifos. On the contrary, OTC+GTM depressed the respiration of the biomixture and the initial mineralization rate of 14C-chlorpyrifos; nonetheless, the antibiotics did not decrease overall mineralization values. The application of both formulations in the biomixture at a relevant concentration did not harm the removal of the fungicides carbendazim and metalaxyl, or their enhanced degradation; on the other hand, the biomixture was unable to dissipate tebuconazol or triadimenol, a result that was unchanged during the addition of the antibiotic formulations. These findings reveal that wastewater containing these antibiotics do not affect the performance of BPS. However, such a response may vary depending on the type of pesticide and microbial consortium in the biomixture.

Aging of biomixtures: Effects on carbofuran removal and microbial community structure.

The aim of this work was to determine the efficiency of a straw/compost/soil biomixture for pesticide depuration during its aging and continuous use, for a period of over a year, based on its capacity to remove carbofuran (CFN), while simultaneously monitoring the variations in microbial community structure. Successive CFN spikings were applied in the biomixture at 6-week intervals, and the removal efficiency was determined 48 h post-application. Initially, only a discrete degradation performance was observed (9.9%), but one CFN application was sufficient to induce efficient elimination (>88.5%) of the pesticide at subsequent influxes for a period of over 6 months. A statistically significant reduction on CFN removal efficiency after this time was detected, reaching levels similar to the fresh-prepared biomixture (14.8%) at the end of the experiment. Simultaneous DGGE analyses showed only modest changes on microbial community patterns through time for both, bacteria and fungi. The clustering of genetic fingerprints in chronological groups corresponding to significantly different CFN degradation efficiencies indicates that biomixture aging changes not only the composition of microbial communities, but also their suitability to engage in pesticide degradation. Periodic substitution of straw/compost/soil biomixture in biopurification systems or regular provision of easily-degradable organic substrates should be considered to maintain an adequate depuration capacity on this system.

Accelerated biodegradation of selected nematicides in tropical crop soils from Costa Rica.

Degradation and mineralization behavior of selected nematicides was studied in soil samples from fields cultivated with banana, potato, and coffee. Degradation assays in most of the studied soils revealed shorter half-lives for carbofuran (CBF) and ethoprophos (ETP) in samples with a history of treatment with these compounds, which may have been caused by enhanced biodegradation. A short half-life value for CBF degradation was also observed in a banana field with no previous exposure to this pesticide, but with a recent application of the carbamate insecticide oxamyl, which supports the hypothesis that preexposure to oxamyl may cause microbial adaptation towards degradation of CBF, an observation of a phenomenon not yet tested according to the literature reviewed. Mineralization assays for CBF and terbufos (TBF) revealed that history of treatment with these nematicides did not cause higher mineralization rates in preexposed soils when compared to unexposed ones, except in the case of soils from coffee fields. Mineralization half-lives for soils unexposed to these pesticides were significantly shorter than most reports in the literature in the same conditions. Mineralization rates for soils with a previous exposure to these pesticides were also obtained, adding to the very few reports found. This paper contributes valuable data to the low number of reports dealing with pesticide fate in soils from tropical origin.

On-farm biopurification systems: role of white rot fungi in depuration of pesticide-containing wastewaters.

Environmental contamination with pesticides is an undesired consequence of agricultural activities. Biopurification systems (BPS) comprise a novel strategy to degrade pesticides from contaminated wastewaters, consisting of a highly active biological mixture confined in a container or excavation. The design of BPS promotes microbial activity, in particular by white rot fungi (WRF). Due to their physiological features, specifically the production of highly unspecific ligninolytic enzymes and some intracellular enzymatic complexes, WRF show the ability to transform a wide range of organic pollutants. This minireview summarizes the potential participation of WRF in BPS. The first part presents the potential use of WRF in biodegradation of pollutants, particularly pesticides, and includes a brief description of the enzymatic systems involved in their oxidation. The second part presents an outline of BPS, focusing on the elements that influence the participation of WRF in their operation, and includes a summary of the studies regarding the fungal-mediated degradation of pesticides in BPS biomixtures and other solid-phase systems that mimic BPS.