Applied and Environmental Microbiology

Enteric viruses are important contaminants of surface waters and a significant burden on public health. In aquatic ecosystems, the stability of these pathogens is differentially impacted by abiotic and biotic stressors, which can exert inactivating effects. Here, we investigated the fate of two enteroviruses, echovirus 11 (E11) and coxsackievirus B5 (CVB5), and one adenovirus, human adenovirus 2 (HAdV2), in lakewater and explored the mechanisms underlying their microbial inactivation. By combining infectivity assays, genome quantification, and capsid integrity analysis, we characterized virus-specific inactivation kinetics and mechanisms and examined the relationship between infectivity loss and capsid structural integrity. We observed rapid, intermediate, and negligible decay for HAdV2, E11, and CVB5, respectively, and confirmed that microbial proteases contribute to their inactivation. In addition, we revealed that loss of capsid structural integrity drives E11 inactivation, but not HAdV2 inactivation. Genome decay did not consistently correlate with loss of infectivity, highlighting the limitations of genome-based detection for assessing the presence of infectious viruses. These findings provide new insights into the mechanisms governing virus inactivation in aquatic environments and emphasize the importance of understanding viral fate when interpreting molecular detection data to assess virus-associated microbial risks.

Microbially-mediated mineral precipitation is important in the natural world and to a number of emerging climate, energy, and water technologies. Central to many microbial mineralization processes is the preferential accumulation of precipitates on microbial surfaces. In some systems, including ureolytic microbially-induced carbonate precipitation (MICP), precipitates may entomb cells in a process termed "encapsulation." Encapsulation is known to greatly affect microbial systems, but understanding these relationships quantitatively has proven difficult, largely because nucleic acid extraction from encapsulated cells yields inconsistent results. Here, a demineralization protocol adapted from studies of natural microbialites is shown to recover genomic DNA from carbonate-encapsulated Sporosarcina pasteurii at yields indistinguishable from unmineralized cells and of sufficient quality for quantitative PCR. This method allows the degree of encapsulation to be expressed quantitatively as the amount of precipitate formed normalized to the estimated number of cells present, and allows growing populations to be tracked with increased precision. Simultaneous growth and mineralization experiments in mixed batch reactors suggest encapsulation-induced growth suppression at calcium concentrations of 250 mM, which are commonly employed in MICP under different physicochemical conditions. Variable rates of precipitate accumulation on cells, possibly stemming from local mass transport constraints, are proposed as a mechanism of calcium concentration-dependent growth suppression. Accordingly, mass transport principles are used to derive a reaction rate scaling function, allowing the observed growth and precipitation behavior to be replicated by a numerical model. These findings lay the groundwork for a more fundamental understanding of how encapsulation shapes microbial mineralization processes.

Acinetobacter sp. strain Tol 5 is an attractive microorganism because of its ability to utilize various aromatic compounds, including toluene. However, because Tol 5 is a non-model microorganism, the utilization of carbon sources in central carbon metabolism remains unclear. In this study, the activity of the central carbon metabolism was characterized using 13C-metabolic flux analysis (13C-MFA). Tol 5 was cultured in synthetic medium using ethanol or acetate as the sole carbon source. In addition, because toluene is degraded into pyruvate and acetyl-CoA via the toluene dioxygenase pathway, this situation was mimicked by culturing with ethanol and lactate as co-substrates. The 13C-MFA confirmed flux through the glyoxylate shunt, which bypasses part of the TCA cycle when either ethanol or acetate was used as the sole carbon source. In ethanol, the flux ratio of isocitrate dehydrogenase at the isocitrate branch point decreased markedly. Unlike acetate, the conversion of ethanol to acetyl-CoA is accompanied by NADH production. Therefore, the observed change in this flux ratio was influenced by the excess NADH generated. Under ethanol-lactate conditions, Tol 5 simultaneously utilized ethanol and lactate. The ethanol consumption rate decreased to less than half of that observed when ethanol was the sole carbon source. Most of the lactate uptake was converted to pyruvate and metabolized via the TCA cycle. The glyoxylate shunt flux was lower than that in ethanol. These metabolic characteristics provide valuable insights into the use of Tol 5 as a next-generation microbial cell factory.

Shigella flexneri is an enteric pathogen responsible for causing shigellosis, a type of bacillary dysentery of significant global health concern. The increasing prevalence of antibiotic resistance has driven the search for alternative treatments, including bacteriophage (phage) therapy. However, the effectiveness of orally administered phages for enteric pathogens depends on their ability to survive passage through the gastrointestinal tract. In this study, the ability of bacteriophage Sf14 to maintain infectivity was evaluated under simulated gastrointestinal conditions using a modified INFOGEST 2.0 in vitro digestion model. This model includes salivary, gastric, and intestinal fluids with physiologically relevant compositions of electrolytes, bile salts, and enzymes. Phages were incubated in each simulated phase at standard physiological temperature, then tested for infectivity using quantitative plaque assays. Results show that Sf14 remained stable during the short duration of the oral phase and for the duration of the intestinal phase. By contrast, survival in simulated gastric fluid required a pH of 2.75 or higher, with rapid and substantial loss of infectivity at pH 2.5 and below. These findings demonstrate that while Sf14 is stable in oral and intestinal environments, the gastric phase poses a critical barrier to phage survival. With in vivo fasting gastric pH ranging from 1.5 to 3, encapsulation would enable the most flexible oral phage therapy against enteric pathogens, but ingestion after eating or alongside antacids may be sufficient.

Vibrio cholerae, the facultative pathogen underlying cholera, naturally inhabits warm aquatic estuaries. Environmental persistence is enhanced by the ability of V. cholerae to colonize host reservoirs and form multicellular biofilms, causing seasonally endemic outbreaks in many tropical regions. Most toxigenic strains utilize the type IVa mannose-sensitive hemagglutinin (MSHA) pilus for host reservoir colonization and biofilm formation. Temperature and salinity can alter V. cholerae biofilm formation, yet their impact on MSHA production specifically remains largely unknown. Here, we utilized transcriptional reporters of predicted msh promoters (msh-P1/msh-P2/msh-P3) and functional assays, to determine temperature and salinity impacts on msh expression and pilus biogenesis. Under standard laboratory conditions (30{degrees}C, 1% NaCl) only msh-P1/P2 are active and inversely-coordinated with one another. Both msh-P1/P2 activity were elevated by high temperature (37{degrees}C) and low salinity (0.25%/0.5% NaCl), and reduced by low temperature (20{degrees}C/25{degrees}C) and high salinity (2%/3% NaCl). Temperature-mediated alterations in promoter activity were not immediately reflected in changes to cell-surface MSHA levels, whereas high salinity led to decreased MSHA production. Combining high temperature (37{degrees}C) and high salinity (2%/3% NaCl), attenuated the salinity-mediated reduction of msh-P1/P2 activity. Biofilm biomass levels were only substantially heightened at 25{degrees}C and 20{degrees}C, likely a result of no temperature-dependent changes in cell-surface MSHA, and additional temperature-controlled biofilm regulation previously described. We also found msh-P1/P2 promoter activity and MSHA production varies widely across toxigenic O1 and O139 serogroups despite complete sequence homology. These results shed new light on how key signals regulate MSHA pilus production to support V. cholerae persistence in aquatic environments.

Urinary tract infections (UTIs) are frequently caused by Escherichia coli and are increasingly associated with antimicrobial resistance, highlighting the need for alternative therapies. Although phage therapy is a promising approach, the rational selection of phages and phage cocktails remains challenging, particularly when detailed genomic information on the infecting strain is unavailable. In this study, we isolated 18 phages from environmental samples collected in Sherbrooke, Quebec, Canada. Genome sequencing identified 15 pure lytic phages representing diverse taxonomic groups and revealed prophage-derived contaminants in three isolates, underscoring the importance of genomic validation during phage discovery. Host range was determined using efficiency-of-plating (EOP) assays against UTI-associated E. coli isolates. Based on host-range overlap and cumulative EOP values, we developed a phenotype-based framework for phage cocktail design and classified combinations as redundant or complementary. Complementary cocktails composed of phages with distinct host ranges and high infection effectiveness consistently outperformed redundant combinations in time-kill assays, demonstrating that host-range diversity and lytic effectiveness are independent properties that should be considered separately during cocktail design. To evaluate cocktail performance under physiologically relevant conditions, the most effective combinations were tested in artificial urine. Both cocktails exhibited reduced activity compared with standard laboratory media, indicating that environmental conditions strongly influence phage-host interactions. Together, our results demonstrate that host-range overlap and cumulative infection effectiveness provide a practical framework for identifying effective phage combinations in the absence of detailed bacterial genomic information and may facilitate the rational development of phage therapies for UTIs.

Coastal waters often experience fecal contamination from human (e.g., septic systems, wastewater treatment catchments and outfalls) and non-human (e.g., wildlife, domesticated pets, and livestock) origins, posing human health risks through contaminated fisheries and recreational waters. Quantitative PCR (qPCR) methods can identify and quantify fecal origins for their management by targeting Bacteroides, a dominant group of gastrointestinal bacteria with high host specificity. This study identified major contributors and drivers of fecal contamination to Mobile Bay, Alabama, an urbanized estuary in the southern United States. We utilized a multi-species qPCR source tracking approach targeting total Bacteroides and human-, pig-, dog-, cow-, and fowl-associated Bacteroides. Water samples and environmental parameters were collected over one year from 13 sites that represent a range of potential fecal inputs common to coastal waters. Human-associated feces were a consistent major contributor to the system (positive in 82% of samples), with localized inputs from pig (wild or domestic) and dog. Freshwater discharge and nitrogen constituents were good predictors of Bacteroides concentrations in this system, while areas with impervious surfaces and developed land cover may be contributing elevated fecal indicator levels. The multi-species qPCR source tracking approach described in this study can be implemented in other watersheds to support the development of better detection, identification, and fecal management strategies for community health of coastal areas.

Pseudomonas aeruginosa is a metabolically versatile opportunistic pathogen capable of adapting to diverse and challenging environments, including the oxygen- and nutrient-limited conditions encountered during chronic infections. Its ability to utilize a broad range of carbon sources and to switch from aerobic to anaerobic respiration contributes to its persistence within the host and its resilience to therapeutic interventions. In particular, denitrification enables P. aeruginosa to grow under oxygen-limited conditions by using nitrate and nitrite as alternative electron acceptors, a process particularly relevant in biofilms and chronic infections such as cystic fibrosis. Ubiquinone (UQ) is essential for respiratory metabolism in P. aeruginosa, which possesses two distinct UQ biosynthetic pathways that ensure UQ production across a broad range of oxygen concentrations. Recent studies have identified the O2-independent UQ biosynthetic pathway as a key determinant of chronic infection. Because this pathway is restricted to a limited group of bacteria, we hypothesized that its selective inhibition could represent a strategy to specifically target P. aeruginosa under anaerobic conditions. To test this hypothesis, we screened analogues of 4-hydroxybenzoic acid (4-HB), the earliest precursor of the UQ biosynthetic pathway, and identified 4-aminobenzoic acid, also known as para-aminobenzoic acid (pABA), as a potential inhibitor. pABA primarily affects the anaerobic metabolism of P. aeruginosa by acting as a competing substrate for UQ biosynthetic enzymes, thereby interfering with UQ biosynthesis. This inhibitory effect positions pABA as a valuable tool for investigating, and potentially targeting, anaerobic metabolism in P. aeruginosa.

Respiratory viruses pose a major threat to human health. Environmental virus persistence in respiratory secretions is essential for successful spread, yet studies have focused largely on a select few viruses in non-physiological matrices. Here, we compared the stability of structurally distinct human respiratory viruses, adenovirus, influenza virus, and rhinovirus, in deposited particles of pooled human saliva under indoor-relevant relative humidity (RH) conditions. Adenovirus exhibited <2.2-log10 decay over 6 h regardless of RH, while rhinovirus exhibited significantly greater degradation (>3.8-log10, at 50% and 80% RH). Influenza virus showed minimal decay at 20% and 50% RH but degraded significantly more (3-log10) at 80% RH. To distinguish aspects of human saliva that are key determinants in driving virus decay, we recapitulated saliva composition using the inorganic solute and protein concentrations determined via ion-coupled plasma mass spectrometry and proteomics, respectively. Rhinovirus was significantly more stable in simulated saliva than in human saliva at 50% RH, while influenza virus and adenovirus behaved similarly in both matrices. These findings emphasize virus-specific environmental persistence and highlight limitations of relying on a single virus and particle matrix for defining public health strategies. Ultimately, these insights will contribute to approaches for mitigating environmental transmission of distinct respiratory pathogens.

The high stability of enteric viruses in freshwater increases their risk of waterborne transmission. In aquatic environments, bacteria are known to enhance viral removal and inactivation. However, the differences in viral inactivation across freshwater ecosystems and the influence of community diversity on this process remains poorly understood. Here, we used dilution-to-extinction to generate 64 distinct freshwater communities from three lakes and three aquifers. After establishing freshwater communities in chemostat cultures, each community was challenged with two viruses, coxsackievirus B5 and human adenovirus 2. Both viruses were inactivated more rapidly in lake-derived than in groundwater-derived communities, and adenovirus was inactivated faster than coxsackievirus in both freshwater types. Community-level parameters (cell numbers, richness, evenness) were not significant predictors of viral inactivation. In contrast, differential abundance analyses identified bacterial OTUs associated with inactivation: Acidovorax sp. were associated across sources and Brevundimonas sp., Sphingopyxis sp., and Hydrogenophaga sp. showed source-specific associations. Together, these findings provide new insights into the ecological drivers of viral stability and suggest that viral inactivation may depend more strongly on specific microbial taxa and their ecological context than on overall community diversity. Identifying these associations improves our understanding of the factors governing viral persistence in freshwater environments.

Jeongeupia sacculi (HS-3) is a novel {beta}-proteobacterium with a unique multicellular life cycle. The genetic tractability and mechanisms underlying differentiation are unknown. Here, we adapt a method of homologous recombination for genetic modification in HS-3 and generate a disruption mutant of rpoS, which encodes the alternative sigma factor{sigma} S commonly associated with general stress response or major behavioral switches. We then characterize phenotypes associated with rpoS disruption. Under low-nutrient conditions, rpoS disruption reduced Congo red binding, consistent with rpoS acting as a regulator of low-nutrient response by upregulating production of biofilm-associated materials. Surprisingly, rpoS disruption did not significantly alter survival in response to other stressors in general stress response assays tested. Our work establishes a method for genetic manipulation in HS-3 and enables future investigation into the life cycle of HS-3, including whether rpoS has a role in differentiation triggered by low-nutrient response.

Aedes aegypti, an important vector of several arboviral diseases, is highly adapted to human-associated environments and commonly exploits anthropogenic water-holding containers for breeding. Understanding the ecological factors governing oviposition-site selection is essential for developing sustainable vector control strategies. Previous studies have established that microbial communities in breeding waters influence mosquito oviposition-site selection through the production of volatile organic compounds. Importantly, rapid urbanization in India has increased the availability of artificial breeding habitats for Ae. aegypti, potentially facilitating the transmission and persistence of arboviral diseases. However, the composition of bacterial communities in these larval habitats and their potential role in shaping the oviposition behavior of Ae. aegypti remain poorly characterized in the Indian context. Here, we investigated the bacterial diversity and community composition of Aedes larval habitats during the 2024 Zika and chikungunya outbreaks in Pune, India. Water samples, collected from artificial urban containers and natural freshwater habitats, were subjected to 16S rRNA amplicon sequencing. Artificial breeding habitats exhibited significantly higher bacterial diversity and were enriched with Enterobacter spp. compared with natural habitats. Oviposition bioassays further demonstrated a strong preference of gravid Ae. aegypti females for Enterobacter-conditioned water. GCMS analysis of bacterial suspension identified indole as a prominent volatile metabolite associated with Enterobacter, suggesting its possible role in mediating oviposition site selection. Together, these findings provide insights into the microbial and chemical ecology of Ae. aegypti breeding habitats and establish a basis for investigating microbial volatile cues as potential targets for vector surveillance and environmentally sustainable attract-and-kill strategies.

Spontaneous vegetable fermentation is driven by the indigenous microbiota associated with the raw ingredients. The process often involves an initial heterofermentative phase followed by a predominantly homofermentative phase, both ensured by lactic acid bacteria (LAB). While microbial dynamics during fermentation are well characterized, phage communities remain poorly understood, despite evidence linking phage activity to LAB succession. We monitored viral and bacterial succession during sauerkraut and carrot fermentations using a metagenomics approach to characterize community dynamics. To further assess the impact of phage PLE001 on bacterial community dynamics, we developed a simplified carrot fermentation model comprising representative strains of Rahnella aquatilis, Leuconostoc mesenteroides and Lactiplantibacillus plantarum. Two consortia were assembled: one containing the PLE001-susceptible strain L. mesenteroides CIM3004, and the other containing also its phage-resistant derivative, CIM3004 BIM. We observed major shifts in the viral community during the first week of fermentation, coinciding with changes in bacterial structure during hetero- and homofermentative phases transition. In the simplified model, phage PLE001 reduced the population of the host during the heterofermentative phase but did not affect overall fermentation dynamics nor metabolite production. These results suggest that bacteriophages contribute to bacterial succession during fermentation, while communities remain resilient to phage infection, preserving fermentation stability.

Microbial biological control agents (mBCAs) offer a promising alternative to conventional disease management, but identifying effective strains requires screening large microbial collections against many pathogens. Here, we developed a scalable screening approach to characterize the antagonistic activity of potato-associated bacteria against multiple pathogens and investigated how bacterial origin and phylogeny relate to antagonistic phenotypes. A collection of 600 bacterial strains isolated from two potato cultivars, three plant compartments using four cultivation media was screened against six major potato pathogens: the bacteria Dickeya solani and Pectobacterium carotovorum, the fungi Alternaria solani and Rhizoctonia solani, and the oomycetes Phytophthora infestans and Pythium ultimum. A scalable confrontation method was developed for non-filamentous bacteria, while complementary lower-throughput assays were used for filamentous strains. This approach enabled successful assessment of 92% of non-filamentous and 97% of filamentous strain-pathogen combinations. Isolation cultivar, plant compartment and cultivation medium had only limited effects on the proportion of antagonistic strains. In contrast, pathogen sensitivity varied markedly: P. infestans was the most susceptible pathogen, whereas D. solani and P. carotovorum were generally resistant to bacterial antagonism. Antagonistic activity clustered phylogenetically, with Bacillus and Streptomyces containing many broad-spectrum inhibitors. In addition, several taxa showed preferential activity against the oomycetes, including P. infestans-specific antagonists from the Pseudomonas genus and P. ultimum-specific antagonists from the Frigoribacterium, Curtobacterium, Pedobacter and Phyllobacterium genera. Overall, multi-pathogen screening revealed distinct generalist and specialist antagonistic profiles and identified candidate strains and taxa for further evaluation as mBCAs.

Pyrogenic black carbon, such as biochar and wildfire char, possesses a reversible electron storage capacity (ESC), allowing it to serve as an electron donor or acceptor for microorganisms. However, not all the ESC is bioavailable, and the proportion available varies by culture. We hypothesized microbes capable of producing soluble redox mediators could access the ESC in biochar interior and thus utilize a higher percentage of the ESC than organisms without a mediator. To test this hypothesis, we grew wild-type Shewanella oneidensis (MR-1) and {Delta}bfe MR-1, a mutant incapable of exporting flavins, on air-oxidized wood biochar. Cell counts and redox titration show that both strains of MR-1 grew to similar cell densities and accessed comparable amounts of biochar ESC (~19%). The addition of exogenous flavin mononucleotide (FMN) yielded no measurable increase in final cell density, due to the strong adsorption of FMN to biochar. SEM images show attachment of MR-1 and {Delta}bfe to biochar surfaces and the presence of nanowires. This study demonstrates MR-1 can utilize biochar ESC for growth, through direct physical contact without the involvement of flavins. Understanding how microbes metabolize biochar provides insight into the impacts and biogeochemical roles of pyrogenic carbon.

Bacterial genomes are dynamic and harbour accessory regions that can be gained, lost, or rearranged in response to selective pressures. Members of the genus Komagataeibacter are used for bacterial cellulose production and researched as platforms for engineered living materials. However, their genomes are rich in mobile genetic elements (MGEs), including insertion sequences (ISs), genomic islands and prophage remnants, which drive genome rearrangements under agitated cultivation conditions. Whether this genome architecture contributes to genomic changes during routine strain maintenance and distribution remains poorly understood. Comparative genome sequencing of independent Komagataeibacter rhaeticus iGEM stocks revealed recurrent loss of a 210 kbp chromosomal region flanked by homologous IS5 sequences. This deletion was identified in a stock received as an agar stab and reproduced from an intact-genome population subjected to simulated agar-stab handling. Phylogenomic analysis of the affected locus revealed gene-tree/species-tree discordance, heterogeneous GC content and multiple ISs, supporting its characterization as a dynamic accessory region. Populations carrying the larger genome reduction showed improved growth and cellulose production relative to the intact genome reference, whereas populations carrying a smaller nested deletion showed reduced performance. These findings demonstrate that routine strain handling is not necessarily genomically neutral and can expose structural genomic variabilities that generate population-level genomic heterogeneity with phenotypic consequences. For bacteria with MGE-rich genomes, handling history may represent an underappreciated source of genomic and phenotypic variation between laboratories. More broadly, identifying unstable accessory regions may highlight genomic content that is dispensable under laboratory conditions, providing candidates for genome streamlining and strain improvement.

The anaerobic biotransformation of lindane ({gamma}-hexachlorocyclohexane), a persistent legacy pesticide and challenging subsurface contaminant was modelled using an integrated Monod biokinetics framework. Calibrated against batch experimental data, this model accounted for the dissolution of solid phase lindane and the thermodynamics of reactions involved. The study focused on a methanogenic, lindane-degrading enrichment culture (GT2) that used ethanol as the electron donor, where ethanol fermentation supplied hydrogen to support reductive dechlorination of lindane. A Dehalobacter strain was identified as the lindane-degrading organism, while a member of bacterial order Geobacterales carried out ethanol fermentation. First-order decay constants for lindane-degrading Dehalobacter were estimated from experimental data and showed variation across different growth conditions. The biokinetics model captured interactions among key microbial processes, namely lindane dechlorination, ethanol fermentation, hydrogenotrophic methanogenesis, and acetoclastic methanogenesis. Thirteen biokinetics parameters including maximum substrate utilization rates (k), half-velocity constants (Ks), and decay constants for these microbial processes were estimated through a Bayesian optimization approach. Global sensitivity analysis showed that eight parameters, primarily k and Ks, had significant linear impact on the model output, whereas responses to decay constants were more variable and potentially non-linear. This study proposed an effective modelling approach for characterizing syntrophic microbial growth within enrichment cultures and provides valuable biokinetics parameters estimates for real-world applications of anaerobic lindane bioremediation.

Microorganisms inhabiting geothermal springs and volcanic systems experience fluctuating temperatures that can periodically exceed their upper thermal limits, but the demographic consequences of such exposure remain poorly understood. Here, we investigated demographic responses of the thermophilic archaeon Sulfolobus acidocaldarius to an extreme temperature (94.1{degrees}C) under two regimes: sustained exposure varying in duration, and episodic exposure interspersed with recovery at a permissive temperature (75{degrees}C). Under sustained exposure, populations showed no detectable loss of viability after 15 min but declined thereafter, decreasing by approximately five orders of magnitude after 120 min. Under episodic exposure, populations remained viable across nine exposure-recovery cycles but declined in density with successive cycles. Similar responses were observed for three strains, including a DNA mismatch repair knockout ({Delta}nucS), indicating that mismatch repair deficiency did not affect viability or recovery. Together, these results demonstrate that S. acidocaldarius can withstand brief and repeated exposure to near-boiling temperatures, with mortality determined primarily by cumulative exposure duration rather than a fixed thermal threshold.

Biological filtration in newly established aquaria depends on the establishment of nitrifying microorganisms that prevent accumulation of toxic ammonia and nitrite. Many aquarists use commercial nitrifying supplements to accelerate biofilter establishment and reduce ammonia and nitrite accumulation. However, the microbial composition and in situ efficacy of these products remain poorly characterized. We evaluated the ability of enrichment cultures containing comammox Nitrospira (CX) and the ammonia-oxidizing archaeon Ca. Nitrosotenuis aquarius (AQ) to serve as aquarium nitrifying supplements, tested alongside a conventional supplement (CS) containing ammonia-oxidizing bacteria. Inoculation of new aquaria containing zebrafish (Danio rerio) with nitrifying supplements resulted in distinct nitrifier profiles within biofilters of each treatment group corresponding with significant reductions in peak ammonia and nitrite concentrations, which were most evident when applied at the maximum tested supplement dose. Treating aquaria with CX, AQ, or CS maintained ammonia and nitrite at low concentrations, and CX effectively prevented ammonia and nitrite accumulation through all stages of aquarium operation and biofilter establishment. After removing all fish from the aquaria, ammonia "spike-in" experiments demonstrated biofilters treated with CX and AQ conferred long-term protection against nitrite accumulation following acute ammonia loading events. Whereas 16S rRNA gene sequences associated with CS were rarely detected within biofilter profiles, those associated with CX and AQ established early and persisted temporally. Overall, results demonstrate how nitrifiers might be differentially adapted to home aquarium conditions and the potential of AOA and comammox Nitrospira to improve biofilter establishment and maintaining safe water quality in newly established freshwater aquaria.

Wastewater sequencing using probe-capture enrichment of public health-relevant viruses is a powerful, non-intrusive approach to monitor infectious diseases, but performance depends on sample type and processing approach. We compared two processing pipelines from wastewater influent samples collected at three timepoints across three wastewater treatment facilities. Each sample was split for parallel processing, with viruses concentrated (1) directly from whole influent using nano-sized affinity particles and (2) by settling and dewatering the solids fraction. Combined DNA/RNA extracts were prepared into libraries using a pre-commercial Illumina protocol, then probe-capture enrichment was performed with Illumina Viral Surveillance Panel (VSP) v1 (66 targets) and v2 (242 targets). Sequencing depth was increased for VSP v2 to accommodate the expanded target set. On average, 160 +/- 146 viral species were detected per sample, with wastewater fraction (sample type) explaining 19% of the variance in viral abundances. Solids contained a higher abundance of DNA viruses compared to RNA viruses, while influent exhibited the inverse. Metagenomic analysis of the VSP v2 sequencing data detected more viral species in influent (median 358) than in solids (median 184), but probe-capture enrichment produced more comparable target-virus readouts across fractions, supporting the use of either fraction for wastewater-based epidemiology.