Dissemination of antibiotic resistance genes through soil-plant-earthworm continuum in the food production environment.

Treated municipal wastewater (TMW) can provide a reliable source of irrigation water for crops, which is especially important in arid areas where water resources are limited or prone to drought. Nonetheless, TMW may contain residual antibiotics, potentially exposing the crops to these substances. The goal of this study was to investigate the dissemination of antibiotics resistance genes (ARGs) in the soil-plant-earthworm continuum after irrigation of spinach and radish plants with TMW containing trimethoprim, sulfamethoxazole, and sulfapyridine in a greenhouse experiment, followed by feeding of earthworms with harvested plant materials. Our results showed that antibiotic resistance genes (ARGs) were enriched in the soil-plant-earthworm microbiomes irrigated with TMW and TMW spiked with higher concentrations of antibiotics. The number of ARGs and antibiotic-resistant bacteria (ARB) enrichment varied with plant type, with spinach harboring a significantly higher amount of ARGs and ARB compared to radish. Our data showed that bulk and rhizosphere soils of spinach and radish plants irrigated with MilliQ water, TMW, TMW10, or TMW100 had significant differences in bacterial community (p < 0.001), ARG (p < 0.001), and virulence factor gene (VFG) (p < 0.001) diversities. The abundance of ARGs significantly decreased from bulk soil to rhizosphere to phyllosphere and endosphere. Using metagenome assembled genomes (MAGs), we recovered many bacterial MAGs and a near complete genome (>90 %) of bacterial MAG of genus Leclercia adecarboxylata B from the fecal microbiome of earthworm that was fed harvested radish tubers and spinach leaves grown on TMW10 irrigated waters, and this bacterium has been shown to be an emerging pathogen causing infection in immunocompromised patients that may lead to health complications and death. Therefore, crops irrigated with TMW containing residual antibiotics and ARGs may lead to increased incidences of enrichment of ARB in the soil-plant-earthworm continuum.

Potential reservoirs of antimicrobial resistance in livestock waste and treated wastewater that can be disseminated to agricultural land.

Livestock manure, dairy lagoon effluent, and treated wastewater are known reservoirs of antibiotic resistance genes (ARGs), antibiotic-resistant bacteria (ARB), and virulence factor genes (VFGs), and their application to agricultural farmland could be a serious public health threat. However, their dissemination to agricultural lands and impact on important geochemical pathways such as the nitrogen (N) cycle have not been jointly explored. In this study, shotgun metagenomic sequencing and analyses were performed to examine the diversity and composition of microbial communities, ARGs, VFGs, and N cycling genes in different livestock manure/lagoon and treated wastewater collected from concentrated animal feeding operations (CAFOs) and a municipal wastewater treatment plant along the west coast of the United States. Multivariate analysis showed that diversity indices of bacterial taxa from the different microbiomes were not significantly different based on InvSimpson (P = 0.05), but differences in ARG mechanisms were observed between swine manure and other microbiome sources. Comparative resistome profiling showed that ARGs in microbiome samples belonged to four core resistance classes: aminoglycosides (40-55 %), tetracyclines (30-45 %), beta-lactam-resistance (20-35 %), macrolides (18-30 %), and >50 % of the VFGs that the 24 microbiomes harbored were phyletically affiliated with two bacteria, Bacteroidetes fragilis and Enterobacter aerogenes. Network analysis based on Spearman correlation showed co-occurrence patterns between several genes such as transporter-gene and regulator, efflux pump and involved-in-polymyxin- resistance, aminoglycoside, beta-lactam, and macrolide with VFGs and bacterial taxa such as Firmicutes, Candidatus Themoplasmatota, Actinobacteria, and Bacteroidetes. Metabolic reconstruction of metagenome-assembled genome (MAGs) analysis showed that the most prevalent drug resistance mechanisms were associated with carbapenem resistance, multidrug resistance (MDR), and efflux pump. Bacteroidales was the main taxa involved in dissimilatory nitrate reduction (DNRA) in dairy lagoon effluent. This study demonstrates that the dissemination of waste from these sources can increase the spread of ARGs, ARB, and VFGs into agricultural lands, negatively impacting both soil and human health.

Genomics discovery of giant fungal viruses from subsurface oceanic crustal fluids.

The oceanic igneous crust is a vast reservoir for microbial life, dominated by diverse and active bacteria, archaea, and fungi. Archaeal and bacterial viruses were previously detected in oceanic crustal fluids at the Juan de Fuca Ridge (JdFR). Here we report the discovery of two eukaryotic Nucleocytoviricota genomes from the same crustal fluids by sorting and sequencing single virions. Both genomes have a tRNATyr gene with an intron (20 bps) at the canonical position between nucleotide 37 and 38, a common feature in eukaryotic and archaeal tRNA genes with short introns (<100 bps), and fungal genes acquired through horizontal gene transfer (HGT) events. The dominance of Ascomycota fungi as the main eukaryotes in crustal fluids and the evidence for HGT point to these fungi as the putative hosts, making these the first putative fungi-Nucleocytoviricota specific association. Our study suggests active host-viral dynamics for the only eukaryotic group found in the subsurface oceanic crust and raises important questions about the impact of viral infection on the productivity and biogeochemical cycling in this ecosystem.

Viruses and Their Interactions With Bacteria and Archaea of Hypersaline Great Salt Lake.

Viruses play vital biogeochemical and ecological roles by (a) expressing auxiliary metabolic genes during infection, (b) enhancing the lateral transfer of host genes, and (c) inducing host mortality. Even in harsh and extreme environments, viruses are major players in carbon and nutrient recycling from organic matter. However, there is much that we do not yet understand about viruses and the processes mediated by them in the extreme environments such as hypersaline habitats. The Great Salt Lake (GSL) in Utah, United States is a hypersaline ecosystem where the biogeochemical role of viruses is poorly understood. This study elucidates the diversity of viruses and describes virus-host interactions in GSL sediments along a salinity gradient. The GSL sediment virosphere consisted of Haloviruses (32.07 ± 19.33%) and members of families Siphoviridae (39.12 ± 19.8%), Myoviridae (13.7 ± 6.6%), and Podoviridae (5.43 ± 0.64%). Our results demonstrate that salinity alongside the concentration of organic carbon and inorganic nutrients (nitrogen and phosphorus) governs the viral, bacteria, and archaeal diversity in this habitat. Computational host predictions for the GSL viruses revealed a wide host range with a dominance of viruses that infect Proteobacteria, Actinobacteria, and Firmicutes. Identification of auxiliary metabolic genes for photosynthesis (psbA), carbon fixation (rbcL, cbbL), formaldehyde assimilation (SHMT), and nitric oxide reduction (NorQ) shed light on the roles played by GSL viruses in biogeochemical cycles of global relevance.

Differences in chlorine and peracetic acid disinfection kinetics of Enterococcus faecalis and Escherichia fergusonii and their susceptible strains based on gene expressions and genomics.

This study was conducted to investigate mechanisms of cross-resistance to chlorine and peracetic acid (PAA) disinfectants by antibiotic-resistant bacteria. Our study evaluated chlorine and PAA based disinfection kinetics of erythromycin-resistant Enterococcus faecalis, meropenem-resistant Escherichia fergusonii, and susceptible strains of these species. Using the integrated second-order disinfectant decay model and first-order Chick-Watson's Law, it was found that the meropenem-resistant Escherichia fergusonii strain showed significantly less log inactivation compared to the susceptible E. fergusonii strain in response to both chlorine and PAA disinfection (p-value = 0.059, 3.5 × 10-6). On the other hand, the susceptible Enterococcus faecalis strain showed similar log inactivation compared to the erythromycin-resistant strain in response to either treatment (p-value = 0.075, 0.28). Meropenem-resistant E. fergusonii showed an increase in gene expression of New Delhi metallo-ß-lactamase (blaNDM-1) gene to chlorine, but there was no increase in expression to PAA. Whole genome sequencing (WGS) was then conducted to elucidate the differences in genes among both resistant and susceptible table E. fergusonii strains. The average nucleotide identity (ANI) analysis of the draft genomes (>97% similarity) suggests that meropenem-resistant E. fergusonii (S1) and meropenem-susceptible E. fergusonii (S2) are the same species but different strains. Both strains have the same genes for oxidative stress pathways, oxidative scavenger genes, and nearly 40 different antibiotic efflux pump genes. The chromosomal and plasmid draft genomes of meropenem-resistant and susceptible E. fergusonii strains each have 65 and 52 antibiotic resistance genes, respectively. Of these, the resistant E. fergusonii strain harbored the extended-spectrum beta-lactamases blaCTX-M-15 and blaTEM-1 genes located on the chromosome, and a blaTEM-1 gene on the plasmid. The overall findings of this study are significant, as they reveal that antibiotic-resistant and susceptible strains of E. fergusonii exhibit different responses towards chlorine and PAA disinfection.

In Vivo Entombment of Bacteria and Fungi during Calcium Oxalate, Brushite, and Struvite Urolithiasis.

Background: Human kidney stones form via repeated events of mineral precipitation, partial dissolution, and reprecipitation, which are directly analogous to similar processes in other natural and manmade environments, where resident microbiomes strongly influence biomineralization. High-resolution microscopy and high-fidelity metagenomic (microscopy-to-omics) analyses, applicable to all forms of biomineralization, have been applied to assemble definitive evidence of in vivo microbiome entombment during urolithiasis. Methods: Stone fragments were collected from a randomly chosen cohort of 20 patients using standard percutaneous nephrolithotomy (PCNL). Fourier transform infrared (FTIR) spectroscopy indicated that 18 of these patients were calcium oxalate (CaOx) stone formers, whereas one patient formed each formed brushite and struvite stones. This apportionment is consistent with global stone mineralogy distributions. Stone fragments from seven of these 20 patients (five CaOx, one brushite, and one struvite) were thin sectioned and analyzed using brightfield (BF), polarization (POL), confocal, super-resolution autofluorescence (SRAF), and Raman techniques. DNA from remaining fragments, grouped according to each of the 20 patients, were analyzed with amplicon sequencing of 16S rRNA gene sequences (V1-V3, V3-V5) and internal transcribed spacer (ITS1, ITS2) regions. Results: Bulk-entombed DNA was sequenced from stone fragments in 11 of the 18 patients who formed CaOx stones, and the patients who formed brushite and struvite stones. These analyses confirmed the presence of an entombed low-diversity community of bacteria and fungi, including Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria, and Aspergillus niger. Bacterial cells approximately 1 µm in diameter were also optically observed to be entombed and well preserved in amorphous hydroxyapatite spherules and fans of needle-like crystals of brushite and struvite. Conclusions: These results indicate a microbiome is entombed during in vivo CaOx stone formation. Similar processes are implied for brushite and struvite stones. This evidence lays the groundwork for future in vitro and in vivo experimentation to determine how the microbiome may actively and/or passively influence kidney stone biomineralization.

Benchmarking of single-virus genomics: a new tool for uncovering the virosphere.

Metagenomics and single-cell genomics have enabled the discovery of relevant uncultured microbes. Recently, single-virus genomics (SVG), although still in an incipient stage, has opened new avenues in viral ecology by allowing the sequencing of one single virus at a time. The investigation of methodological alternatives and optimization of existing procedures for SVG is paramount to deliver high-quality genomic data. We report a sequencing dataset of viral single-amplified genomes (vSAGs) from cultured and uncultured viruses obtained by applying different conditions in each SVG step, from viral preservation and novel whole-genome amplification (WGA) to sequencing platforms and genome assembly. Sequencing data showed that cryopreservation and mild fixation were compatible with WGA, although fresh samples delivered better genome quality data. The novel TruPrime WGA, based on primase-polymerase features, and WGA-X employing a thermostable phi29 polymerase, were proven to be with sufficient sensitivity in SVG. The Oxford Nanopore (ON) sequencing platform did not provide a significant improvement of vSAG assembly compared to Illumina alone. Finally, the SPAdes assembler performed the best. Overall, our results represent a valuable genomic dataset that will help to standardized and advance new tools in viral ecology.

Metabolic network analysis reveals microbial community interactions in anammox granules.

Microbial communities mediating anaerobic ammonium oxidation (anammox) represent one of the most energy-efficient environmental biotechnologies for nitrogen removal from wastewater. However, little is known about the functional role heterotrophic bacteria play in anammox granules. Here, we use genome-centric metagenomics to recover 17 draft genomes of anammox and heterotrophic bacteria from a laboratory-scale anammox bioreactor. We combine metabolic network reconstruction with metatranscriptomics to examine the gene expression of anammox and heterotrophic bacteria and to identify their potential interactions. We find that Chlorobi-affiliated bacteria may be highly active protein degraders, catabolizing extracellular peptides while recycling nitrate to nitrite. Other heterotrophs may also contribute to scavenging of detritus and peptides produced by anammox bacteria, and potentially use alternative electron donors, such as H2, acetate and formate. Our findings improve the understanding of metabolic activities and interactions between anammox and heterotrophic bacteria and offer the first transcriptional insights on ecosystem function in anammox granules.

Genome Sequence of Escherichia coli Tailed Phage Utah.

Escherichia coli bacteriophage Utah is a member of the chi-like tailed phage cluster in the Siphoviridae family. We report here the complete 59,024-bp sequence of the genome of phage Utah.

Insights of Phage-Host Interaction in Hypersaline Ecosystem through Metagenomics Analyses.

Bacteriophages, as the most abundant biological entities on Earth, place significant predation pressure on their hosts. This pressure plays a critical role in the evolution, diversity, and abundance of bacteria. In addition, phages modulate the genetic diversity of prokaryotic communities through the transfer of auxiliary metabolic genes. Various studies have been conducted in diverse ecosystems to understand phage-host interactions and their effects on prokaryote metabolism and community composition. However, hypersaline environments remain among the least studied ecosystems and the interaction between the phages and prokaryotes in these habitats is poorly understood. This study begins to fill this knowledge gap by analyzing bacteriophage-host interactions in the Great Salt Lake, the largest prehistoric hypersaline lake in the Western Hemisphere. Our metagenomics analyses allowed us to comprehensively identify the bacterial and phage communities with Proteobacteria, Firmicutes, and Bacteroidetes as the most dominant bacterial species and Siphoviridae, Myoviridae, and Podoviridae as the most dominant viral families found in the metagenomic sequences. We also characterized interactions between the phage and prokaryotic communities of Great Salt Lake and determined how these interactions possibly influence the community diversity, structure, and biogeochemical cycles. In addition, presence of prophages and their interaction with the prokaryotic host was studied and showed the possibility of prophage induction and subsequent infection of prokaryotic community present in the Great Salt Lake environment under different environmental stress factors. We found that carbon cycle was the most susceptible nutrient cycling pathways to prophage induction in the presence of environmental stresses. This study gives an enhanced snapshot of phage and prokaryote abundance and diversity as well as their interactions in a hypersaline complex ecosystem, which can pave the way for further research studies.

Whole-Community Metagenomics in Two Different Anammox Configurations: Process Performance and Community Structure.

Anaerobic ammonia oxidation (anammox) combined with partial nitritation (PN) is an innovative treatment process for energy-efficient nitrogen removal from wastewater. In this study, we used genome-based metagenomics to investigate the overall community structure and anammox species enriched in suspended growth (SGR) and attached growth packed-bed (AGR) anammox reactors after 220 days of operation. Both reactors removed more than 85% of the total inorganic nitrogen. Metagenomic binning and phylogenetic analysis revealed that two anammox population genomes, affiliated with the genus Candidatus Brocadia, were differentially abundant between the SGR and AGR. Both of the genomes shared an average nucleotide identify of 83%, suggesting the presence of two different species enriched in both of the reactors. Metabolic reconstruction of both population genomes revealed key aspects of their metabolism in comparison to known anammox species. The community composition of both the reactors was also investigated to identify the presence of flanking community members. Metagenomics and 16S rRNA gene amplicon sequencing revealed the dominant flanking community members in both reactors were affiliated with the phyla Anaerolinea, Ignavibacteria, and Proteobacteria. Findings from this research adds two new species, Ca. Brocadia sp. 1 and Ca. Brocadia sp. 2, to the genus Ca. Brocadia and sheds light on their metabolism in engineered ecosystems.

Effect of short term external perturbations on bacterial ecology and activities in a partial nitritation and anammox reactor.

This research investigated the short term effects of temperature changes (lasting 2-4weeks each) from 35±2°C to 21±2°C and 13±2°C and sulfide toxicity on partial nitrification-anammox (PN/A) system. Temperatures below 20°C and sulfide content as low as 5mgSL(-1) affected both aerobic and anaerobic catabolic activities of ammonia oxidation and the expression of related functional gene markers. The activity of AOB was inversely correlated with ammonium monooxygenase (amoA) gene expression. In contrast, the activity of AMX bacteria was positively correlated with the expression of their hydrazine synthase (hzsA) gene. Although the overall activities of AMX bacteria decreased at lower temperatures, the AMX bacteria were still active at the low temperatures. The inverse correlation between amoA gene expressions and the corresponding AOB activities was surprising. 16S rDNA based high throughput amplicon sequencing revealed the dominance of Chloroflexi, Planctomycetes and Proteobacteria phyla the distribution of which changed with temperature changes.

Microbiological study of bacteriophage induction in the presence of chemical stress factors in enhanced biological phosphorus removal (EBPR).

Polyphosphate accumulating organisms (PAOs) are responsible for carrying the enhanced biological phosphorus removal (EBPR). Although the EBPR process is well studied, the failure of EBPR performance at both laboratory and full-scale plants has revealed a lack of knowledge about the ecological and microbiological aspects of EBPR processes. Bacteriophages are viruses that infect bacteria as their sole host. Bacteriophage infection of polyphosphate accumulating organisms (PAOs) has not been considered as a main contributor to biological phosphorus removal upsets. This study examined the effects of different stress factors on the dynamics of bacteriophages and the corresponding effects on the phosphorus removal performance in a lab-scale EBPR system. The results showed that copper (heavy metal), cyanide (toxic chemical), and ciprofloxacin (antibiotic), as three different anthropogenic stress factors, can induce phages integrated onto bacterial genomes (i.e. prophages) in an enriched EBPR sequencing batch reactor, resulting in a decrease in the polyphosphate kinase gene ppk1 clades copy number, phosphorus accumulation capacity, and phosphorus removal performance. This study opens opportunities for further research on the effects of bacteriophages in nutrient cycles both in controlled systems such as wastewater treatment plants and natural ecosystems.