RESUMO
Microbial biosensors that convert environmental information into real-time visual outputs are limited in their sensing abilities in complex environments, such as soil and wastewater, due to optical inaccessibility. Biosensors that could record transient exposure to analytes within a large time window for later retrieval represent a promising approach to solve the accessibility problem. Here, we test the performance of recombinase-memory biosensors that sense a sugar (arabinose) and a microbial communication molecule (3-oxo-C12-L-homoserine lactone) over 8 days (~70 generations) following analyte exposure. These biosensors sense the analyte and trigger the expression of a recombinase enzyme which flips a segment of DNA, creating a genetic memory, and initiates fluorescent protein expression. The initial designs failed over time due to unintended DNA flipping in the absence of the analyte and loss of the flipped state after exposure to the analyte. Biosensor performance was improved by decreasing recombinase expression, removing the fluorescent protein output, and using quantitative PCR to read out stored information. Application of memory biosensors in wastewater isolates achieved memory of analyte exposure in an uncharacterized Pseudomonas isolate. By returning these engineered isolates to their native environments, recombinase-memory systems are expected to enable longer duration and in situ investigation of microbial signaling, cross-feeding, community shifts, and gene transfer beyond the reach of traditional environmental biosensors.IMPORTANCEMicrobes mediate ecological processes over timescales that can far exceed the half-lives of transient metabolites and signals that drive their collective behaviors. We investigated strategies for engineering microbes to stably record their transient exposure to a chemical over many generations through DNA rearrangements. We identify genetic architectures that improve memory biosensor performance and characterize these in wastewater isolates. Memory biosensors are expected to be useful for monitoring cell-cell signals in biofilms, detecting transient exposure to chemical pollutants, and observing microbial cross-feeding through short-lived metabolites within cryptic methane, nitrogen, and sulfur cycling processes. They will also enable in situ studies of microbial responses to ephemeral environmental changes, or other ecological processes that are currently challenging to monitor non-destructively using real-time biosensors and analytical instruments.
Assuntos
Técnicas Biossensoriais , Águas Residuárias , Recombinases , DNA , Pseudomonas , CorantesRESUMO
Wastewater-based epidemiology (WBE) expanded rapidly in response to the COVID-19 pandemic. As the public health emergency has ended, researchers and practitioners are looking to shift the focus of existing wastewater surveillance programs to other targets, including bacteria. Bacterial targets may pose some unique challenges for WBE applications. To explore the current state of the field, the National Science Foundation-funded Research Coordination Network (RCN) on Wastewater Based Epidemiology for SARS-CoV-2 and Emerging Public Health Threats held a workshop in April 2023 to discuss the challenges and needs for wastewater bacterial surveillance. The targets and methods used in existing programs were diverse, with twelve different targets and nine different methods listed. Discussions during the workshop highlighted the challenges in adapting existing programs and identified research gaps in four key areas: choosing new targets, relating bacterial wastewater data to human disease incidence and prevalence, developing methods, and normalizing results. To help with these challenges and research gaps, the authors identified steps the larger community can take to improve bacteria wastewater surveillance. This includes developing data reporting standards and method optimization and validation for bacterial programs. Additionally, more work is needed to understand shedding patterns for potential bacterial targets to better relate wastewater data to human infections. Wastewater surveillance for bacteria can help provide insight into the underlying prevalence in communities, but much work is needed to establish these methods.IMPORTANCEWastewater surveillance was a useful tool to elucidate the burden and spread of SARS-CoV-2 during the pandemic. Public health officials and researchers are interested in expanding these surveillance programs to include bacterial targets, but many questions remain. The NSF-funded Research Coordination Network for Wastewater Surveillance of SARS-CoV-2 and Emerging Public Health Threats held a workshop to identify barriers and research gaps to implementing bacterial wastewater surveillance programs.
Assuntos
Objetivos , Pandemias , Humanos , Águas Residuárias , Vigilância Epidemiológica Baseada em Águas Residuárias , Bactérias , SARS-CoV-2RESUMO
Molecular imprinting is a promising strategy to selectively adsorb viruses, but it requires discerning and validating epitopes that serve as effective imprinting templates. In this work, glycoprotein-imprinted particles were synthesized for coronavirus capture. Adsorption was maximized at pH 6 (the glycoprotein isoelectric point) where the glycoprotein-imprinted particles outperformed non-imprinted particles, adsorbing 4.96 × 106 ± 3.33 × 103 versus 3.54 × 106 ± 1.39 × 106 median tissue culture infectious dose/mg of the target coronavirus, human coronavirus - organ culture 43, within the first 30 min (p = 0.012). During competitive adsorption, with pH adjustment (pH 6), the glycoprotein-imprinted particles adsorbed more target virus than non-target coronavirus (human coronavirus - Netherland 63) with 2.34 versus 1.94 log removal in 90 min (p < 0.01). In contrast, the non-imprinted particles showed no significant difference in target versus non-target virus removal. Electrostatic potential calculation shows that the human coronavirus - organ culture 43 glycoprotein has positively charged pockets at pH 6, which may facilitate adsorption at lower pH values. Therefore, tuning the target virus glycoprotein charge via pH adjustment enhanced adsorption by minimizing repulsive electrostatic interactions with the particles. Overall, these results highlight the effective use of glycoprotein-imprinted particles for coronavirus capture and discern the merits and limitations of glycoprotein imprinting for the capture of enveloped viruses.
Assuntos
Coronavirus , Humanos , GlicoproteínasRESUMO
Although extensive research to date has focused on enhancing removal rates of antibiotics from municipal wastewaters, the transformation products formed by anaerobic treatment processes remain understudied. The present work aims to examine the possible roles that the different microbial communities of an anaerobic membrane bioreactor (AnMBR) play in the transformation of antibiotics during wastewater treatment. As part of this work, sulfamethoxazole, erythromycin, and ampicillin were added in separate stages to the influent of the AnMBR at incremental concentrations of 10, 50, and 250 µg/L each. Antibiotic-specific transformation products detected during each stage, as identified by high resolution LC-MS, are reported herein. Results suggest that both isoxazole (sulfamethoxazole) and ß-lactam (ampicillin) ring opening could be facilitated by the AnMBR's bioprocess. Microbial community analysis results indicated that relative activity of the system's suspended biomass consistently shifted towards syntrophic groups throughout the duration of the experiment. Notable differences were also observed between the suspended biomass and the AnMBR's membrane biofilms. Membrane-attached biofilm communities showed high relative activities of several specific methanogenic (Methanothrix and Methanomethylovorans), syntrophic (Syntrophaceae), and sulfate-reducing (Desulfomonile) groups. Such groups have been previously identified as involved in the formation of the antibiotic degradation products observed in the effluent of the AnMBR. The activity of these communities within the biofilms likely confers certain advantages that aid in the biotransformation of the antibiotics tested.
Assuntos
Esgotos , Eliminação de Resíduos Líquidos , Anaerobiose , Antibacterianos , Biofilmes , Reatores Biológicos , Águas ResiduáriasRESUMO
Anaerobic membrane bioreactors (AnMBRs) can significantly reduce the release of antibiotic resistance elements to the environment. The purpose of this study was to elucidate the role of membrane fouling layers (biofilms) in mitigating the release of intracellular and extracellular antibiotic resistance genes (iARGs and eARGs) from an AnMBR. The AnMBR was equipped with three membrane modules, each exhibiting a different level of fouling. Results showed that the absolute abundance of ARGs decreased gradually in the suspended biomass during operation of the AnMBR. Normalized abundances of targeted ARGs and intI1 were found to be significantly higher in the fouling layers compared to the suspended biomass, implying adsorption or an increased potential for horizontal gene transfer of ARGs in the biofilm. Effluent ARG data revealed that the highly fouled (HF) membrane significantly reduced the absolute abundance of eARGs. However, the HF membrane effluent concomitantly had the highest absolute abundance of iARGs. Nevertheless, total ARG abundance (sum of iARG and eARG) in the effluent of the AnMBR was not impacted by the extent of fouling. These results suggest a need for a combination of different treatment technologies to effectively prevent antibiotic resistance proliferation associated with these two ARG fractions.
Assuntos
Antibacterianos , Águas Residuárias , Anaerobiose , Antibacterianos/farmacologia , Reatores Biológicos , Resistência Microbiana a Medicamentos/genética , Genes Bacterianos , Membranas ArtificiaisRESUMO
Aeration accounts for the largest energy demand in conventional activated sludge wastewater treatment. Emerging aeration control strategies for energy conservation have significantly reduced operational bulk liquid dissolved oxygen (DO) from above 2 mg/L to at or below 0.5 mg/L. As we move toward low DO treatment processes, there is a need to understand how low DO impacts the kinetics of micropollutant biotransformation. The objective of this study was to characterize the impact of DO concentration on pharmaceutical biotransformation rates via two approaches: (1) Determine oxygen half saturation constants that describe the community-wide impact of DO on biotransformation rates. (2) Evaluate shifts in the microbial community 16S rRNA pool due to DO concentration. Batch experiments were performed at several DO concentrations using biomass from a full-scale wastewater treatment plant. Results reveal that substantial reductions in bulk liquid DO concentrations to 0.5 mg-O2/L are possible without compromising pharmaceutical biotransformation rates. Sequencing of cDNA generated from community rRNA revealed that diverse, low abundance community members may play important roles in pharmaceutical transformation. The results of this work advance our ability to predict and model the impact of DO on pharmaceutical biotransformations during wastewater treatment and identify taxonomic groups associated with those biotransformations.
Assuntos
Microbiota , Esgotos , Reatores Biológicos , Oxigênio , RNA Ribossômico 16S , Eliminação de Resíduos Líquidos , Águas ResiduáriasRESUMO
Anaerobic membrane bioreactors (AnMBRs) are an emerging technology with potential to improve energy efficiency and effluent reuse in mainstream wastewater treatment. However, their contribution to the proliferation of contaminants of emerging concern, such as antibiotic resistance genes (ARGs), remains largely unknown. The purpose of this study was to determine the effect of select influent antibiotics at varying concentrations on the presence and abundance of ARGs in an AnMBR system and its effluent. Quantification of targeted ARGs revealed distinct profiles in biomass and effluent, with genes conferring resistance to different antibiotic classes dominating in biomass (macrolides) and effluent (sulfonamides). Effluent sul1 gene abundance was strongly correlated with abundance of intl1, signifying the potential importance of mobile genetic elements in ARG release from AnMBR systems. The addition of specific antibiotics also affected normalized abundances of their related ARGs, exemplifying the potential impact of selective pressures at both low (10 µg/L) and high (250 µg/L) influent antibiotic concentrations.
Assuntos
Antibacterianos , Eliminação de Resíduos Líquidos , Anaerobiose , Reatores Biológicos , Resistência Microbiana a Medicamentos , Genes Bacterianos , Águas ResiduáriasRESUMO
The objective of this study was to evaluate emerging anaerobic membrane bioreactor (AnMBR) technology in comparison with conventional wastewater energy recovery technologies. Wastewater treatment process modeling and systems analyses were combined to evaluate the conditions under which AnMBR may produce more net energy and have lower life cycle environmental emissions than high rate activated sludge with anaerobic digestion (HRAS+AD), conventional activated sludge with anaerobic digestion (CAS+AD), and an aerobic membrane bioreactor with anaerobic digestion (AeMBR+AD). For medium strength domestic wastewater treatment under baseline assumptions at 15 °C, AnMBR recovered 49% more energy as biogas than HRAS+AD, the most energy positive conventional technology considered, but had significantly higher energy demands and environmental emissions. Global warming impacts associated with AnMBR were largely due to emissions of effluent dissolved methane. For high strength domestic wastewater treatment, AnMBR recovered 15% more net energy than HRAS+AD, and the environmental emissions gap between the two systems was reduced. Future developments of AnMBR technology in low energy fouling control, increased flux, and management of effluent methane emissions would make AnMBR competitive with HRAS+AD. Rapid advancements in AnMBR technology must continue to achieve its full economic and environmental potential as an energy recovery strategy for domestic wastewater.
Assuntos
Reatores Biológicos , Conservação de Recursos Energéticos/métodos , Membranas Artificiais , Águas Residuárias , Purificação da Água/instrumentação , Purificação da Água/métodos , Anaerobiose , Conservação de Recursos Energéticos/economia , Custos e Análise de Custo , Aquecimento Global , Modelos Teóricos , Esgotos/química , Incerteza , Purificação da Água/economiaRESUMO
Wastewater surveillance for SARS-CoV-2 provides early warnings of emerging variants of concerns and can be used to screen for novel cryptic linked-read mutations, which are co-occurring single nucleotide mutations that are rare, or entirely missing, in existing SARS-CoV-2 databases. While previous approaches have focused on specific regions of the SARS-CoV-2 genome, there is a need for computational tools capable of efficiently tracking cryptic mutations across the entire genome and investigating their potential origin. We present Crykey, a tool for rapidly identifying rare linked-read mutations across the genome of SARS-CoV-2. We evaluated the utility of Crykey on over 3,000 wastewater and over 22,000 clinical samples; our findings are three-fold: i) we identify hundreds of cryptic mutations that cover the entire SARS-CoV-2 genome, ii) we track the presence of these cryptic mutations across multiple wastewater treatment plants and over three years of sampling in Houston, and iii) we find a handful of cryptic mutations in wastewater mirror cryptic mutations in clinical samples and investigate their potential to represent real cryptic lineages. In summary, Crykey enables large-scale detection of cryptic mutations in wastewater that represent potential circulating cryptic lineages, serving as a new computational tool for wastewater surveillance of SARS-CoV-2.
Assuntos
COVID-19 , Genoma Viral , Mutação , SARS-CoV-2 , Águas Residuárias , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Águas Residuárias/virologia , COVID-19/virologia , COVID-19/epidemiologia , COVID-19/diagnóstico , Humanos , Genoma Viral/genética , Biologia Computacional/métodosRESUMO
Wastewater surveillance is a powerful tool to assess the risks associated with antibiotic resistance in communities. One challenge is selecting which analytical tool to deploy to measure risk indicators, such as antibiotic resistance genes (ARGs) and their respective bacterial hosts. Although metagenomics is frequently used for analyzing ARGs, few studies have compared the performance of long-read and short-read metagenomics in identifying which bacteria harbor ARGs in wastewater. Furthermore, for ARG host detection, untargeted metagenomics has not been compared to targeted methods such as epicPCR. Here, we 1) evaluated long-read and short-read metagenomics as well as epicPCR for detecting ARG hosts in wastewater, and 2) investigated the host range of ARGs across the wastewater treatment plant (WWTP) to evaluate host proliferation. Results highlighted long-read revealed a wider range of ARG hosts compared to short-read metagenomics. Nonetheless, the ARG host range detected by long-read metagenomics only represented a subset of the hosts detected by epicPCR. The ARG-host linkages across the influent and effluent of the WWTP were characterized. Results showed the ARG-host phylum linkages were relatively consistent across the WWTP, whereas new ARG-host species linkages appeared in the WWTP effluent. The ARG-host linkages of several clinically relevant species found in the effluent were identified.
Assuntos
Antibacterianos , Águas Residuárias , Antibacterianos/farmacologia , Genes Bacterianos , Vigilância Epidemiológica Baseada em Águas Residuárias , Bactérias/genética , Farmacorresistência Bacteriana/genética , Metagenômica/métodosRESUMO
Wastewater monitoring is an efficient and effective way to surveil for various pathogens in communities. This is especially beneficial in areas of high transmission, such as preK-12 schools, where infections may otherwise go unreported. In this work, we apply wastewater disease surveillance using school and community wastewater from across Houston, Texas to monitor three major enteric viruses: astrovirus, sapovirus genogroup GI, and group A rotavirus. We present the results of a 10-week study that included the analysis of 164 wastewater samples for astrovirus, rotavirus, and sapovirus in 10 preK-12 schools, 6 wastewater treatment plants, and 2 lift stations using newly designed RT-ddPCR assays. We show that the RT-ddPCR assays were able to detect astrovirus, rotavirus, and sapovirus in school, lift station, and wastewater treatment plant (WWTP) wastewater, and that a positive detection of a virus in a school sample was paired with a positive detection of the same virus at a downstream lift station or wastewater treatment plant over 97 % of the time. Additionally, we show how wastewater detections of rotavirus in schools and WWTPs were significantly associated with citywide viral intestinal infections. School wastewater can play a role in the monitoring of enteric viruses and in the detection of outbreaks, potentially allowing public health officials to quickly implement mitigation strategies to prevent viral spread into surrounding communities.
Assuntos
Rotavirus , Sapovirus , Instituições Acadêmicas , Águas Residuárias , Águas Residuárias/virologia , Sapovirus/isolamento & purificação , Rotavirus/isolamento & purificação , Texas , Monitoramento Ambiental/métodos , Humanos , Mamastrovirus/isolamento & purificaçãoRESUMO
Wastewater surveillance has proven a cost-effective key public health tool to understand a wide range of community health diseases and has been a strong source of information on community levels and spread for health departments throughout the SARS- CoV-2 pandemic. Studies spanning the globe demonstrate the strong association between virus levels observed in wastewater and quality clinical case information of the population served by the sewershed. Few of these studies incorporate the temporal dependence present in sampling over time, which can lead to estimation issues which in turn impact conclusions. We contribute to the literature for this important public health science by putting forward time series methods coupled with statistical process control that (1) capture the evolving trend of a disease in the population; (2) separate the uncertainty in the population disease trend from the uncertainty due to sampling and measurement; and (3) support comparison of sub-sewershed population disease dynamics with those of the population represented by the larger downstream treatment plant. Our statistical methods incorporate the fact that measurements are over time, ensuring correct statistical conclusions. We provide a retrospective example of how sub-sewersheds virus levels compare to the upstream wastewater treatment plant virus levels. An on-line algorithm supports real-time statistical assessment of deviations of virus level in a population represented by a sub-sewershed to the virus level in the corresponding larger downstream wastewater treatment plant. This information supports public health decisions by spotlighting segments of the population where outbreaks may be occurring.
Assuntos
COVID-19 , Águas Residuárias , Humanos , Fatores de Tempo , RNA Viral/genética , SARS-CoV-2/genética , Estudos Retrospectivos , COVID-19/epidemiologia , Vigilância Epidemiológica Baseada em Águas ResiduáriasRESUMO
Tiled amplicon sequencing has served as an essential tool for tracking the spread and evolution of pathogens. Over 15 million complete SARS-CoV-2 genomes are now publicly available, most sequenced and assembled via tiled amplicon sequencing. While computational tools for tiled amplicon design exist, they require downstream manual optimization both computationally and experimentally, which is slow and costly. Here we present Olivar, a first step towards a fully automated, variant-aware design of tiled amplicons for pathogen genomes. Olivar converts each nucleotide of the target genome into a numeric risk score, capturing undesired sequence features that should be avoided. In a direct comparison with PrimalScheme, we show that Olivar has fewer mismatches overlapping with primers and predicted PCR byproducts. We also compare Olivar head-to-head with ARTIC v4.1, the most widely used primer set for SARS-CoV-2 sequencing, and show Olivar yields similar read mapping rates (~90%) and better coverage to the manually designed ARTIC v4.1 amplicons. We also evaluate Olivar on real wastewater samples and found that Olivar has up to 3-fold higher mapping rates while retaining similar coverage. In summary, Olivar automates and accelerates the generation of tiled amplicons, even in situations of high mutation frequency and/or density. Olivar is available online as a web application at https://olivar.rice.edu and can be installed locally as a command line tool with Bioconda. Source code, installation guide, and usage are available at https://github.com/treangenlab/Olivar .
Assuntos
COVID-19 , Primers do DNA , Genoma Viral , SARS-CoV-2 , SARS-CoV-2/genética , COVID-19/virologia , COVID-19/diagnóstico , Humanos , Genoma Viral/genética , Primers do DNA/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Águas Residuárias/virologia , Reação em Cadeia da Polimerase Multiplex/métodos , SoftwareRESUMO
Public health surveillance systems for COVID-19 are multifaceted and include multiple indicators reflective of different aspects of the burden and spread of the disease in a community. With the emergence of wastewater disease surveillance as a powerful tool to track infection dynamics of SARS-CoV-2, there is a need to integrate and validate wastewater information with existing disease surveillance systems and demonstrate how it can be used as a routine surveillance tool. A first step toward integration is showing how it relates to other disease surveillance indicators and outcomes, such as case positivity rates, syndromic surveillance data, and hospital bed use rates. Here, we present an 86-week long surveillance study that covers three major COVID-19 surges. City-wide SARS-CoV-2 RNA viral loads in wastewater were measured across 39 wastewater treatment plants and compared to other disease metrics for the city of Houston, TX. We show that wastewater levels are strongly correlated with positivity rate, syndromic surveillance rates of COVID-19 visits, and COVID-19-related general bed use rates at hospitals. We show that the relative timing of wastewater relative to each indicator shifted across the pandemic, likely due to a multitude of factors including testing availability, health-seeking behavior, and changes in viral variants. Next, we show that individual WWTPs led city-wide changes in SARS-CoV-2 viral loads, indicating a distributed monitoring system could be used to enhance the early-warning capability of a wastewater monitoring system. Finally, we describe how the results were used in real-time to inform public health response and resource allocation.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , COVID-19/epidemiologia , Águas Residuárias , RNA Viral , PandemiasRESUMO
As clinical testing declines, wastewater monitoring can provide crucial surveillance on the emergence of SARS-CoV-2 variant of concerns (VoCs) in communities. In this paper we present QuaID, a novel bioinformatics tool for VoC detection based on quasi-unique mutations. The benefits of QuaID are three-fold: (i) provides up to 3-week earlier VoC detection, (ii) accurate VoC detection (>95% precision on simulated benchmarks), and (iii) leverages all mutational signatures (including insertions & deletions).
Assuntos
COVID-19 , Humanos , COVID-19/diagnóstico , SARS-CoV-2/genética , Águas Residuárias , BenchmarkingRESUMO
We present Crykey, a computational tool for rapidly identifying cryptic mutations of SARS-CoV-2. Specifically, we identify co-occurring single nucleotide mutations on the same sequencing read, called linked-read mutations, that are rare or entirely missing in existing databases, and have the potential to represent novel cryptic lineages found in wastewater. While previous approaches exist for identifying cryptic linked-read mutations from specific regions of the SARS-CoV-2 genome, there is a need for computational tools capable of efficiently tracking cryptic mutations across the entire genome and for tens of thousands of samples and with increased scrutiny, given their potential to represent either artifacts or hidden SARS-CoV-2 lineages. Crykey fills this gap by identifying rare linked-read mutations that pass stringent computational filters to limit the potential for artifacts. We evaluate the utility of Crykey on >3,000 wastewater and >22,000 clinical samples; our findings are three-fold: i) we identify hundreds of cryptic mutations that cover the entire SARS-CoV-2 genome, ii) we track the presence of these cryptic mutations across multiple wastewater treatment plants and over a three years of sampling in Houston, and iii) we find a handful of cryptic mutations in wastewater mirror cryptic mutations in clinical samples and investigate their potential to represent real cryptic lineages. In summary, Crykey enables large-scale detection of cryptic mutations representing potential cryptic lineages in wastewater.
RESUMO
Wastewater surveillance is a passive and efficient way to monitor the spread of infectious diseases in large populations and high transmission areas such as preK-12 schools. Infections caused by respiratory viruses in school-aged children are likely underreported, particularly because many children may be asymptomatic or mildly symptomatic. Wastewater monitoring of SARS-CoV-2 has been studied extensively and primarily by sampling at centralized wastewater treatment plants, and there are limited studies on SARS-CoV-2 in preK-12 school wastewater. Similarly, wastewater detections of influenza have only been reported in wastewater treatment plant and university manhole samples. Here, we present the results of a 17-month wastewater monitoring program for SARS-CoV-2 (n = 2176 samples) and influenza A and B (n = 1217 samples) in 51 preK-12 schools. We show that school wastewater concentrations of SARS-CoV-2 RNA were strongly associated with COVID-19 cases in schools and community positivity rates, and that influenza detections in school wastewater were significantly associated with citywide influenza diagnosis rates. Results were communicated back to schools and local communities to enable mitigation strategies to stop the spread, and direct resources such as testing and vaccination clinics. This study demonstrates that school wastewater surveillance is reflective of local infections at several population levels and plays a crucial role in the detection and mitigation of outbreaks.
Assuntos
COVID-19 , Influenza Humana , Criança , Humanos , Influenza Humana/epidemiologia , SARS-CoV-2 , Águas Residuárias , COVID-19/epidemiologia , RNA Viral , Vigilância Epidemiológica Baseada em Águas ResiduáriasRESUMO
Tiled amplicon sequencing has served as an essential tool for tracking the spread and evolution of pathogens. Over 2 million complete SARS-CoV-2 genomes are now publicly available, most sequenced and assembled via tiled amplicon sequencing. While computational tools for tiled amplicon design exist, they require downstream manual optimization both computationally and experimentally, which is slow and costly. Here we present Olivar, a first step towards a fully automated, variant-aware design of tiled amplicons for pathogen genomes. Olivar converts each nucleotide of the target genome into a numeric risk score, capturing undesired sequence features that should be avoided. In a direct comparison with PrimalScheme, we show that Olivar has fewer SNPs overlapping with primers and predicted PCR byproducts. We also compared Olivar head-to-head with ARTIC v4.1, the most widely used primer set for SARS-CoV-2 sequencing, and show Olivar yields similar read mapping rates (~90%) and better coverage to the manually designed ARTIC v4.1 amplicons. We also evaluated Olivar on real wastewater samples and found that Olivar had up to 3-fold higher mapping rates while retaining similar coverage. In summary, Olivar automates and accelerates the generation of tiled amplicons, even in situations of high mutation frequency and/or density. Olivar is available as a web application at https://olivar.rice.edu. Olivar can also be installed locally as a command line tool with Bioconda. Source code, installation guide and usage are available at https://github.com/treangenlab/Olivar.