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At least 37 records · Page 2

Natural transformation as a tool in Acinetobacter baylyi : Evolution by amplification of gene copy number

For many years, the natural competency of Acinetobacter baylyi ADP1 facilitated studies of bacterial metabolism, biochemistry, and physiology. With the advent of synthetic biology, new opportunities arise to exploit the remarkable transformability and chromosomal plasticity of this model organism. In this chapter, we describe a recently developed method, “Evolution by Amplification and Synthetic Biology” (EASy). EASy allows the targeted amplification of chromosomal segments that give rise to new phenotypes. Increased gene dosage regulates protein expression in a rudimentary fashion by establishing a chromosomal array in which copy number adjusts via recombination between repeated DNA sequences. Selective conditions enrich for cells within the population that confer a growth advantage. Under continuous selective pressure, beneficial mutations may accumulate in any genomic region. Such mutations favor decreases in the average copy number of the target region. Thus, the genetic flexibility afforded by transient copy number variation helps accelerate the selection of engineered strains with desired traits during laboratory evolution. As a result, thanks to the extremely simple genetic manipulation of Acinetobacter baylyi ADP1, the EASy method can be readily implemented by researchers without the need for advanced instrumentation or complex cloning techniques.

59 BASIC BIOLOGICAL SCIENCES↗

Genomic insights into local adaptation and migration success in reintroduced Coho Salmon of the Wenatchee River basin

ABSTRACT Objective Reintroduction of salmonids into regions where they have been extirpated is a common conservation strategy that is often implemented through natural recolonization, translocation of natural populations, or hatchery-based programs. Locally adapting to specific environmental conditions is critical for long-term population viability, particularly for species like Coho Salmon Oncorhynchus kisutch, which face diverse selective pressures during their migration. This study focused on the mid-Columbia River Coho Salmon reintroduction program managed by Yakama Nation Fisheries, which has successfully reintroduced Coho Salmon into the Wenatchee and Methow River basins, Washington. Notably, these populations have adapted to the longer migration route than those in the founding stock, with selection favoring individuals with an earlier arrival time and that can navigate a 15-km, high-gradient canyon to reach optimal spawning grounds. The objectives of this study were to investigate whether specific genomic regions are under selection for traits associated with return location and timing in Coho Salmon. Methods Low-coverage whole-genome resequencing data were used to screen for genomic regions associated with the phenotypes of interest. Results A weak polygenic signal in female Coho Salmon was found to be associated with return group, with a subset of candidate adaptive regions occurring across eight chromosomes. Conclusions These findings provide insights into the genomic mechanisms underlying local adaptation in reintroduced salmon populations and inform broodstock selection strategies aimed at promoting natural production and long-term population sustainability.

Horn, Rebekah L.↗

Genomic Signatures of a Major Adaptive Event in the Pathogenic Fungus Melampsora larici-populina

The recent availability of genome-wide sequencing techniques has allowed systematic screening for molecular signatures of adaptation, including in nonmodel organisms. Host–pathogen interactions constitute good models due to the strong selective pressures that they entail. We focused on an adaptive event which affected the poplar rust fungus Melampsora larici-populina when it overcame a resistance gene borne by its host, cultivated poplar. Based on 76 virulent and avirulent isolates framing narrowly the estimated date of the adaptive event, we examined the molecular signatures of selection. Using an array of genome scan methods based on different features of nucleotide diversity, we detected a single locus exhibiting a consistent pattern suggestive of a selective sweep in virulent individuals (excess of differentiation between virulent and avirulent samples, linkage disequilibrium, genotype–phenotype statistical association, and long-range haplotypes). Our study pinpoints a single gene and further a single amino acid replacement which may have allowed the adaptive event. Although our samples are nearly contemporary to the selective sweep, it does not seem to have affected genome diversity further than the immediate vicinity of the causal locus, which can be explained by a soft selective sweep (where selection acts on standing variation) and by the impact of recombination in mitigating the impact of selection. Therefore, it seems that properties of the life cycle of M. larici-populina, which entails both high genetic diversity and outbreeding, has facilitated its adaptation.

59 BASIC BIOLOGICAL SCIENCES↗

MetaPop: a pipeline for macro- and microdiversity analyses and visualization of microbial and viral metagenome-derived populations

Abstract Background Microbes and their viruses are hidden engines driving Earth’s ecosystems from the oceans and soils to humans and bioreactors. Though gene marker approaches can now be complemented by genome-resolved studies of inter-(macrodiversity) and intra-(microdiversity) population variation, analytical tools to do so remain scattered or under-developed. Results Here, we introduce MetaPop, an open-source bioinformatic pipeline that provides a single interface to analyze and visualize microbial and viral community metagenomes at both the macro - and microdiversity levels. Macrodiversity estimates include population abundances and α- and β-diversity. Microdiversity calculations include identification of single nucleotide polymorphisms, novel codon-constrained linkage of SNPs, nucleotide diversity ( π and θ ), and selective pressures (pN/pS and Tajima’s D ) within and fixation indices ( F ST ) between populations. MetaPop will also identify genes with distinct codon usage. Following rigorous validation, we applied MetaPop to the gut viromes of autistic children that underwent fecal microbiota transfers and their neurotypical peers. The macrodiversity results confirmed our prior findings for viral populations (microbial shotgun metagenomes were not available) that diversity did not significantly differ between autistic and neurotypical children. However, by also quantifying microdiversity, MetaPop revealed lower average viral nucleotide diversity ( π ) in autistic children. Analysis of the percentage of genomes detected under positive selection was also lower among autistic children, suggesting that higher viral π in neurotypical children may be beneficial because it allows populations to better “bet hedge” in changing environments. Further, comparisons of microdiversity pre- and post-FMT in autistic children revealed that the delivery FMT method (oral versus rectal) may influence viral activity and engraftment of microdiverse viral populations, with children who received their FMT rectally having higher microdiversity post-FMT. Overall, these results show that analyses at the macro level alone can miss important biological differences. Conclusions These findings suggest that standardized population and genetic variation analyses will be invaluable for maximizing biological inference, and MetaPop provides a convenient tool package to explore the dual impact of macro - and microdiversity across microbial communities.

59 BASIC BIOLOGICAL SCIENCES↗

Divergence in heritable life history traits suggests potential for local adaptation and trade‐offs associated with a coal ash disposal site

Abstract Globally, human activities have resulted in rapid environmental changes that present unique challenges for wildlife. However, investigations of local adaptation in response to simultaneous exposure to multiple anthropogenic selection pressures are rare and often generate conflicting results. We used an in situ reciprocal transplant design within a quantitative genetic framework to examine how adaptive evolution and phenotypic plasticity contribute to the persistence of an amphibian population inhabiting an environment characterized by high levels of multiple toxic trace elements. We found evidence of phenotypic divergence that is largely consistent with local adaptation to an environment contaminated with multiple chemical stressors, tied to potential trade‐offs in the absence of contaminants. Specifically, the population derived from the contaminated environment had a reduced risk of mortality and greater larval growth and in the contaminated environment, relative to offspring from the naïve population. Further, while survival in the uncontaminated environment was not compromised in offspring from the contaminant‐exposed population, they did show delayed development and reduced growth rates over larval development, relative to the naïve population. We found no evidence of reduced additive genetic variation in the contaminant‐exposed population, suggesting long‐term selection in a novel environment has not reduced the evolutionary potential of that population. We also saw little evidence that past selection in the ASH environment had reduced trait plasticity in the resident population. Maternal effects were prominent in early development, but we did not detect any trends suggesting these effects were associated with the maternal transfer of toxic trace elements. Our results demonstrate the potential for adaptation to multiple contaminants in a wild amphibian population, which may have facilitated long‐term persistence in a heavily impacted environment.

59 BASIC BIOLOGICAL SCIENCES↗

Sporophyte Stage Genes Exhibit Stronger Selection Than Gametophyte Stage Genes in Haplodiplontic Giant Kelp

Macrocystis pyrifera (giant kelp), a haplodiplontic brown macroalga that alternates between a macroscopic diploid (sporophyte) and a microscopic haploid (gametophyte) phase, provides an ideal system to investigate how ploidy background affects the evolutionary history of a gene. In M. pyrifera , the same genome is subjected to different selective pressures and environments as it alternates between haploid and diploid life stages. We assembled M. pyrifera gene models using available expression data and validated 8,292 genes models using the model alga Ectocarpus siliculosus . Differential expression analysis identified gene models expressed in either or both the haploid and diploid life stages while functional annotation identified processes enriched in each stage. Genes expressed preferentially or exclusively in the gametophyte stage were found to have higher nucleotide diversity (π = 2.3 × 10 –3 and 2.8 × 10 –3 , respectively) than those for sporophytes (π = 1.1 × 10 –3 and 1 × 10 –3 , respectively). While gametophyte-biased genes show faster sequence evolution, the sequence evolution exhibits less signatures of adaptations when compared to sporophyte-biased genes. Our findings contrast the standing masking hypothesis, which predicts higher standing genetic variation at the sporophyte stage, and support the strength of expression theory, which posits that genes expressed more strongly are expected to evolve slower. We argue that the sporophyte stage undergoes more stringent selection compared with the gametophyte stage, which carries a heavy genetic load associated with broadcast spawning. Furthermore, using whole-genome sequencing, we confirm the strong population structure in wild M. pyrifera populations previously established using microsatellite markers, and estimate population genetic parameters, such as pairwise genetic diversity and Tajima’s D , important for conservation and domestication of M. pyrifera .

Molano, Gary↗

Riverine bacterioplankton and phytoplankton assembly along an environmental gradient induced by urbanization

In the Anthropocene, human activities are intensifying stresses on biological communities across ecosystems. Knowledge on how environmental changes influence microbial community dynamics is crucial but currently limited. Here we unraveled ecological processes generating distribution patterns of bacterioplankton and phytoplankton communities in a river draining an urbanizing watershed in southern China. In this ecosystem, longitudinal environmental heterogeneity and directional flow potentially have counteracting effects on planktonic community assembly. Using simultaneously multiple approaches, we showed that the observed compositional patterns result from an interplay of stochastic and deterministic processes, indicating that dispersal via fluvial connectivity could not override local selective pressures. Successional shifts in the relative importance of the above processes were associated with trends in climatic factors. High diversity was observed at the intermediate levels of ecological processes alongside intense inter-species interactions maintained by a few keystone taxa. Altogether, interactions between climatic conditions, landscape and hydrology control plankton community dynamics.

54 ENVIRONMENTAL SCIENCES↗

Diverse events have transferred genes for edible seaweed digestion from marine to human gut bacteria

Humans harbor numerous species of colonic bacteria that digest fiber polysaccharides in commonly consumed terrestrial plants. More recently in history, regional populations have consumed edible macroalgae seaweeds containing unique polysaccharides. It remains unclear how extensively gut bacteria have adapted to digest these nutrients. Here, in this work, we show that the ability of gut bacteria to digest seaweed polysaccharides is more pervasive than previously appreciated. Enrichment-cultured Bacteroides harbor previously discovered genes for seaweed degradation, which have mobilized into several members of this genus. Additionally, other examples of marine bacteria-derived genes, and their mobile DNA elements, are involved in gut microbial degradation of seaweed polysaccharides, including genes in gut-resident Firmicutes. Collectively, these results uncover multiple separate events that have mobilized the genes encoding seaweed-degrading-enzymes into gut bacteria. This work further underscores the metabolic plasticity of the human gut microbiome and global exchange of genes in the context of dietary selective pressures.

42 ENGINEERING↗

Antiretroviral therapy reveals triphasic decay of intact SIV genomes and persistence of ancestral variants

The decay kinetics of HIV-1-infected cells are critical to understand virus persistence. We evaluated the frequency of simian immunodeficiency virus (SIV)-infected cells for 4 years of antiretroviral therapy (ART). The intact proviral DNA assay (IPDA) and an assay for hypermutated proviruses revealed short- and long-term infected cell dynamics in macaques starting ART ~1 year after infection. Intact SIV genomes in circulating CD4+T cells showed triphasic decay with an initial phase slower than the decay of the plasma virus, a second phase faster than the second phase decay of intact HIV-1, and a stable third phase reached after 1.6–2.9 years. Hypermutated proviruses showed bi- or mono-phasic decay, reflecting different selective pressures. Viruses replicating at ART initiation had mutations conferring antibody escape. With time on ART, viruses with fewer mutations became more prominent, reflecting decay of variants replicating at ART initiation. Collectively, these findings confirm ART efficacy and indicate that cells enter the reservoir throughout untreated infection.

60 APPLIED LIFE SCIENCES↗

Identification of over ten thousand candidate structured RNAs in viruses and phages

Structured RNAs play crucial roles in viruses, exerting influence over both viral and host gene expression. However, the extensive diversity of structured RNAs and their ability to act in cis or trans positions pose challenges for predicting and assigning their functions. While comparative genomics approaches have successfully predicted candidate structured RNAs in microbes on a large scale, similar efforts for viruses have been lacking. In this study, we screened over 5 million DNA and RNA viral sequences, resulting in the prediction of 10,006 novel candidate structured RNAs. These predictions are widely distributed across taxonomy and ecosystem. We found transcriptional evidence for 206 of these candidate structured RNAs in the human fecal microbiome. These candidate RNAs exhibited evidence of nucleotide covariation, indicative of selective pressure maintaining the predicted secondary structures. Our analysis revealed a diverse repertoire of candidate structured RNAs, encompassing a substantial number of putative tRNAs or tRNA-like structures, Rho-independent transcription terminators, and potentially cis-regulatory structures consistently positioned upstream of genes. In summary, our findings shed light on the extensive diversity of structured RNAs in viruses, offering a valuable resource for further investigations into their functional roles and implications in viral gene expression and pave the way for a deeper understanding of the intricate interplay between viruses and their hosts at the molecular level.

59 BASIC BIOLOGICAL SCIENCES↗

Permafrost microbial communities and functional genes are structured by latitudinal and soil geochemical gradients

Permafrost underlies approximately one quarter of Northern Hemisphere terrestrial surfaces and contains 25–50% of the global soil carbon (C) pool. Permafrost soils and the C stocks within are vulnerable to ongoing and future projected climate warming. The biogeography of microbial communities inhabiting permafrost has not been examined beyond a small number of sites focused on local-scale variation. Permafrost is different from other soils. Perennially frozen conditions in permafrost dictate that microbial communities do not turn over quickly, thus possibly providing strong linkages to past environments. Thus, the factors structuring the composition and function of microbial communities may differ from patterns observed in other terrestrial environments. Here, we analyzed 133 permafrost metagenomes from North America, Europe, and Asia. Permafrost biodiversity and taxonomic distribution varied in relation to pH, latitude and soil depth. The distribution of genes differed by latitude, soil depth, age, and pH. Genes that were the most highly variable across all sites were associated with energy metabolism and C-assimilation. Specifically, methanogenesis, fermentation, nitrate reduction, and replenishment of citric acid cycle intermediates. This suggests that adaptations to energy acquisition and substrate availability are among some of the strongest selective pressures shaping permafrost microbial communities. The spatial variation in metabolic potential has primed communities for specific biogeochemical processes as soils thaw due to climate change, which could cause regional- to global- scale variation in C and nitrogen processing and greenhouse gas emissions.

59 BASIC BIOLOGICAL SCIENCES↗

Structural evolution of a DNA repair self-resistance mechanism targeting genotoxic secondary metabolites

Abstract Microbes produce a broad spectrum of antibiotic natural products, including many DNA-damaging genotoxins. Among the most potent of these are DNA alkylating agents in the spirocyclopropylcyclohexadienone (SCPCHD) family, which includes the duocarmycins, CC-1065, gilvusmycin, and yatakemycin. The yatakemycin biosynthesis cluster in Streptomyces sp. TP-A0356 contains an AlkD-related DNA glycosylase, YtkR2, that serves as a self-resistance mechanism against yatakemycin toxicity. We previously reported that AlkD, which is not present in an SCPCHD producer, provides only limited resistance against yatakemycin. We now show that YtkR2 and C10R5, a previously uncharacterized homolog found in the CC-1065 biosynthetic gene cluster of Streptomyces zelensis , confer far greater resistance against their respective SCPCHD natural products. We identify a structural basis for substrate specificity across gene clusters and show a correlation between in vivo resistance and in vitro enzymatic activity indicating that reduced product affinity—not enhanced substrate recognition—is the evolutionary outcome of selective pressure to provide self-resistance against yatakemycin and CC-1065.

59 BASIC BIOLOGICAL SCIENCES↗

Conserved features of TERT promoter duplications reveal an activation mechanism that mimics hotspot mutations in cancer

Mutations in the TERT promoter represent the genetic underpinnings of tumor cell immortality. Beyond the two most common point mutations, which selectively recruit the ETS factor GABP to activate TERT, the significance of other variants is unknown. In seven cancer types, we identify duplications of wildtype sequence within the core promoter region of TERT that have strikingly similar features including an ETS motif, the duplication length and insertion site. The duplications recruit a GABP tetramer by virtue of the native ETS motif and its precisely spaced duplicated counterpart, activate the promoter and are clonal in a TERT expressing multifocal glioblastoma. We conclude that recurrent TERT promoter duplications are functionally and mechanistically equivalent to the hotspot mutations that confer tumor cell immortality. The shared mechanism of these divergent somatic genetic alterations suggests a strong selective pressure for recruitment of the GABP tetramer to activate TERT.

60 APPLIED LIFE SCIENCES↗

Novel adaptive immune systems in pristine Antarctic soils

Antarctic environments are dominated by microorganisms, which are vulnerable to viral infection. Although several studies have investigated the phylogenetic repertoire of bacteria and viruses in these poly-extreme environments with freezing temperatures, high ultra violet irradiation levels, low moisture availability and hyper-oligotrophy, the evolutionary mechanisms governing microbial immunity remain poorly understood. Using genome-resolved metagenomics, we test the hypothesis that Antarctic poly-extreme high-latitude microbiomes harbour diverse adaptive immune systems. Our analysis reveals the prevalence of prophages in bacterial genomes (Bacteroidota and Verrucomicrobiota), suggesting the significance of lysogenic infection strategies in Antarctic soils. Furthermore, we demonstrate the presence of diverse CRISPR-Cas arrays, including Class 1 arrays (Types I-B, I-C, and I-E), alongside systems exhibiting novel gene architecture among their effector cas genes. Notably, a Class 2 system featuring type V variants lacks CRISPR arrays, encodes Cas1 and Cas2 adaptation module genes. Phylogenetic analysis of Cas12 effector proteins hints at divergent evolutionary histories compared to classified type V effectors and indicates that TnpB is likely the ancestor of Cas12 nucleases. Our findings suggest substantial novelty in Antarctic cas sequences, likely driven by strong selective pressures. These results underscore the role of viral infection as a key evolutionary driver shaping polar microbiomes.

59 BASIC BIOLOGICAL SCIENCES↗

Climate change drives convergent evolution of root traits on Sky Island climate relicts

Roots are essential to the strategies plants use to survive in variable environments, yet we know little of how they vary within species. Experimental conditions demonstrate that intraspecific plant root traits respond strongly to variation in the environment; however, it is unclear when these responses can be characterized as evolution in response to selective pressures of climate change over many generations. Sky Islands are model, natural climate relict ecosystems to examine climate-change driven evolution. Utilizing a common garden with replicate genotypes of Populus angustifolia (Narrowleaf cottonwood) from six Sky Island (SI) populations and nine adjacent Mountain Chain (MC) populations across three genetic provenances, we hypothesized that SI root traits have diverged due to historical isolation in warmer, drier climates. When grown in common conditions, populations originating on SI’s showed convergent evolution across three distinct genetic provenances, which was characterized by 44.16% decreased total root length, 42.64% decreased average root volume, 43.31% decreased root surface area, and significantly less root trait variation, relative to adjacent mountain chains. Convergent evolution of root traits from trees originating on SI’s is correlated with changes in mean annual precipitation and potential evapotranspiration in the field over the past ~ 125 years. These results demonstrate a consistent pattern in root trait evolution at the landscape scale and the role of climate on the evolution of root traits in a genetic and geographic context relevant to climate change.

Convergent evolution↗

Evolving dual-trait EPSP synthase variants using a synthetic yeast selection system

The enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) functions in the shikimate pathway which is responsible for the production of aromatic amino acids and precursors of other essential secondary metabolites in all plant species. EPSPS is also the molecular target of the herbicide glyphosate. While some plant EPSPS variants have been characterized with reduced glyphosate sensitivity and have been used in biotechnology, the glyphosate insensitivity typically comes with a cost to catalytic efficiency. Thus, there exists a need to generate additional EPSPS variants that maintain both high catalytic efficiency and high glyphosate tolerance. Here, we create a synthetic yeast system to rapidly study and evolve heterologous EPSP synthases for these dual traits. Using known EPSPS variants, we first validate that our synthetic yeast system is capable of recapitulating growth characteristics observed in plants grown in varying levels of glyphosate. Next, we demonstrate that variants from mutagenesis libraries with distinct phenotypic traits can be isolated depending on the selection criteria applied. By applying strong dual-trait selection pressure, we identify a notable EPSPS mutant after just a single round of evolution that displays robust glyphosate tolerance (K i of nearly 1 mM) and improved enzymatic efficiency over the starting point (~2.5 fold). Finally, we show the crystal structure of corn EPSPS and the top resulting mutants and demonstrate that certain mutants have the potential to outperform previously reported glyphosate-resistant EPSPS mutants, such as T102I and P106S (denoted as TIPS), in whole-plant testing. Altogether, this platform helps explore the trade-off between glyphosate resistance and enzymatic efficiency.

Science & Technology - Other Topics↗

Transport of metformin metabolites by guanidinium exporters of the small multidrug resistance family

Proteins from the small multidrug resistance (SMR) family are frequently associated with horizontally transferred multidrug resistance gene arrays found in bacteria from wastewater and the human-adjacent biosphere. Recent studies suggest that a subset of SMR transporters might participate in the metabolism of the common pharmaceutical metformin by bacterial consortia. Here, we show that both genomic and plasmid-associated transporters of the SMRGdx functional subtype export byproducts of microbial metformin metabolism, with particularly high export efficiency for guanylurea. We use solid-supported membrane electrophysiology to evaluate the transport kinetics for guanylurea and native substrate guanidinium by four representative SMRGdx homologs. Using an internal reference to normalize independent electrophysiology experiments, we show that transport rates are comparable for genomic and plasmid-associated SMRGdx homologs, and using a proteoliposome-based transport assay, we show that 2 proton:1 substrate transport stoichiometry is maintained. Additional characterization of guanidinium and guanylurea export properties focuses on the structurally characterized homolog, Gdx-Clo, for which we examined the pH dependence and thermodynamics of substrate binding and solved an x-ray crystal structure with guanylurea bound. Together, these experiments contribute in two main ways. By providing the first detailed kinetic examination of the structurally characterized SMRGdx homolog Gdx-Clo, they provide a functional framework that will inform future mechanistic studies of this model transport protein. Second, this study casts light on a potential role for SMRGdx transporters in microbial handling of metformin and its microbial metabolic byproducts, providing insight into how native transport physiologies are co-opted to contend with new selective pressures.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Treatment of antibiotic-manufacturing wastewater enriches for Aeromonas veronii, a zoonotic antibiotic-resistant emerging pathogen

Antibiotic-manufacturing wastewater treatment plants primarily target chemical pollutants, but their processes may select for antibiotic-resistant pathogens and antibiotic resistance genes. Leveraging the combined strengths of deep metagenomic sequencing, 16S rRNA gene sequencing, quantitative polymerase chain reaction, and bacterial culturing, we investigated bacterial communities and antibiotic resistomes across eleven treatment units in a full-scale antibiotic-manufacturing wastewater treatment plant processing wastewater from a β-lactam manufacturing facility. Both bacterial communities and antibiotic resistance gene compositions varied across the treatment units, but were associated. Certain antibiotic resistance gene persisted through treatment, either carried by identical bacterial species, or linked to mobile genetic elements in different species. Despite the satisfactory performance in chemical removal, this plant continuously enriched zoonotic antibiotic-resistant Aeromonas veronii (an emerging pathogen responsible for substantial economic losses in aquaculture and human health) from influent to effluent, probably due to prolonged β-lactam selection pressure and aquatic nature of A. veronii. This enrichment resulted in a significantly higher abundance of A. veronii than other aquatic samples worldwide. Furthermore, the closest evolutionary relative to the retrieved A. veronii was an isolate obtained from the stool of a local diarrhea patient. These findings highlighted a substantial public health risk posed by antibiotic-manufacturing wastewater treatment, underlining its potential role in enriching and disseminating zoonotic antibiotic-resistant pathogens. Beyond chemical monitoring, enhanced surveillance of antibiotic-resistant pathogens and antibiotic resistance genes is needed in effluent discharge standard for antibiotic-manufacturing wastewater treatment plants.

Wang, Xingshuo↗