Protozoa as Hotspots for Potential Pathogens in the Drinking Water of a Subtropical Megacity: Diversity, Treatment, and Health Risk.

Drinking water systems host a wide range of microorganisms essential for biosafety. However, one major group of waterborne pathogens, protozoa, is relatively neglected compared to bacteria and other microorganisms. Until now, little is known about the growth and fate of protozoa and their associated bacteria in drinking water systems. In this study, we aim to investigate how drinking water treatment affects the growth and fate of protozoa and their associated bacteria in a subtropical megacity. The results showed that viable protozoa were prevalent in the city's tap water, and amoebae were the major component of tap water protozoa. In addition, protozoan-associated bacteria contained many potential pathogens and were primarily enriched in amoeba hosts. Furthermore, this study showed that current drinking water disinfection methods have little effect on protozoa and their associated bacteria. Besides, ultrafiltration membranes unexpectedly served as an ideal growth surface for amoebae in drinking water systems, and they could significantly promote the growth of amoeba-associated bacteria. In conclusion, this study shows that viable protozoa and their associated bacteria are prevalent in tap water, which may present an emerging health risk in drinking water biosafety.

Metagenomic analysis of antimicrobial resistance in ducks, workers, and the environment in duck farms, southern China.

Duck farms are one of the important reservoirs of antimicrobial resistance genes (ARGs) that spread to humans and the environment. However, few studies have focused on the characteristics of antimicrobial profiles in duck farms. Here we explored the distribution characteristics and potential transmission mechanisms of ARGs in ducks, farm workers, and the environment in duck farms by a metagenomic approach. The results showed that the highest abundance and diversity of ARGs were found in duck manure. The abundance and diversity of ARGs in workers and environmental samples were higher than those in the control group. tet(X) and its variants were prevalent in duck farms, with tet(X10) being the most abundant. The genetic structure "tet(X)-like + α/ß hydrolase" was found in ducks, workers, and the environment, implying that tet(X) and its variants have been widely spread in duck farms. Network analysis indicated that ISVsa3 and IS5075 might play an important role in the coexistence of ARGs and metal resistance genes (MRGs). The Mantel tests showed that mobile genetic elements (MGEs) were significantly correlated with ARG profiles. The results suggest that duck manure may be a potential hotspot source of ARGs, including tet(X) variants that spread to the surrounding environment and workers via MGEs. These results help us optimize the antimicrobials strategy and deepen our understanding of ARG spread in duck farms.

Preliminary view of the global distribution and spread of the tet(X) family of tigecycline resistance genes.

BACKGROUND: The emergence of plasmid-mediated tet(X3)/tet(X4) genes is threatening the role of tigecycline as a last-resort antibiotic to treat clinical infections caused by XDR bacteria. Considering the possible public health threat posed by tet(X) and its variants [which we collectively call 'tet(X) genes' in this study], global monitoring and surveillance are urgently required. OBJECTIVES: Here we conducted a worldwide survey of the global distribution and spread of tet(X) genes. METHODS: We analysed a comprehensive dataset of bacterial genomes in conjunction with surveillance data from our laboratory and the NCBI database, as well as sufficient metadata to characterize the results. RESULTS: The global distribution features of tet(X) genes were revealed. We clustered three types of genetic backbones of tet(X) genes embedded or transferred in bacterial genomes. Our pan-genome analyses revealed a large genetic pool composed of tet(X)-carrying sequences. Moreover, phylogenetic trees of tet(X) genes and tet(X)-like proteins were built. CONCLUSIONS: To the best of our knowledge, our results provide the first view of the global distribution of tet(X) genes, demonstrate the features of tet(X)-carrying fragments and highlight the possible evolution of tigecycline-inactivation enzymes in diverse bacterial species and habitats.

In vitro susceptibility of four antimicrobials against Riemerella anatipestifer isolates: a comparison of minimum inhibitory concentrations and mutant prevention concentrations for ceftiofur, cefquinome, florfenicol, and tilmicosin.

BACKGROUND: Mutant prevention concentration (MPC) is an alternative pharmacodynamic parameter that has been used to measure antimicrobial activity and represents the propensities of antimicrobial agents to select resistant mutants. The concentration range between minimum inhibitory concentration (MIC) and MPC is defined as mutant selection window (MSW). The MPC and MSW parameters represent the ability of antimicrobial agents to inhibit the bacterial mutants selected. This study was conducted to determine the MIC and MPC values of four antimicrobials including ceftiofur, cefquinome, florfenicol and tilmicosin against 105 Riemerella anatipestifer isolates. RESULTS: The MIC50/MIC90 values of clinical isolates tested in our study for ceftiofur, cefquinome, florfenicol and tilmicosin were 0.063/0.5、0.031/0.5、1/4、1/4 µg/mL, respectively; MPC50/ MPC90 values were 4/64、8/64、4/32、16/256 µg/mL, respectively. These results provided information on the use of these compounds in treating the R. anatipestifer infection; however, additional studies are needed to demonstrate their therapeutic efficacy. CONCLUSION: Based on the MSW theory, the hierarchy of these tested antimicrobial agents with respect to selecting resistant subpopulations was as follows: cefquinome > ceftiofur > tilmicosin > florfenicol. Cefquinome was the drug that presented the highest risk of selecting resistant mutant among the four antimicrobial agents.

Mangrove sediments are environmental hotspots for pathogenic protists.

Mangrove sediments are unique ecosystems providing habitats for diverse organisms, especially microbial communities. However, little is known about the diversity and environmental risk of a critical group of microorganisms, the protists. To address this gap, we employed metagenome sequencing technologies to provide the first comprehensive view of the protistan community in the mangrove sediment. Our results surprisingly showed that parasitic protists dominated the protistan community in mangrove sediments, with an average abundance of 59.67%, one of the highest in all ecosystems on Earth. We also found that the relative abundance of protists decreased significantly (R = -0.21, p = 0.045) with latitude but increased with depths (R = 0.7099, p < 0.001). The parasitic communities were positively influenced by microbial (bacteria, fungi, and archaea) communities, including horizontal-scale and vertical-scale. In addition, sulfate and salinity had the most significant influence on the protistan community. Our findings provide new insights into our understanding of protistan variation in mangrove sediments, including abundance, composition, and possible functions, and indicate that mangrove sediments are hotspots for environmental pathogens, posing a potential risk to human health.

Homologous recombination and gene-specific selection co-shape the vertical nucleotide diversity of mangrove sediment microbial populations.

Mangrove sediments host a diverse array of microbial populations and are characterized by high heterogeneity along their vertical depths. However, the genetic diversity within these populations is largely unknown, hindering our understanding of their adaptive evolution across the sediment depths. To elucidate their genetic diversity, we utilized metagenome sequencing to identify 16 high-frequency microbial populations comprised of two archaea and 14 bacteria from mangrove sediment cores (0-100 cm, with 10 depths) in Qi'ao Island, China. Our analysis of the genome-wide genetic variation revealed extensive nucleotide diversity in the microbial populations. The genes involved in the transport and the energy metabolism displayed a high nucleotide diversity (HND; 0.0045-0.0195; an indicator of shared minor alleles with the microbial populations). By tracking the processes of homologous recombination, we found that each microbial population was subjected to different purification selection levels at different depths (44.12% genes). This selection resulted in significant differences in synonymous/non-synonymous mutation ratio between 0-20 and 20-100 cm layers, indicating the adaptive evolutionary process of microbial populations. Furthermore, our assessment of differentiation in the allele frequencies between these two layers showed that the functional genes involved in the metabolic processes of amino acids or cofactors were highly differential in more than half of them. Together, we showed that the nucleotide diversity of microbial populations was shaped by homologous recombination and gene-specific selection, finally resulting in the stratified differentiation occurring between 0-20 and 20-100 cm. These results enhance our cognition of the microbial adaptation mechanisms to vertical environmental changes during the sedimentation process of coastal blue carbon ecosystems.

Unraveling the important role of comammox Nitrospira to nitrification in the coastal aquaculture system.

Increasing nitrogen (N) input to coastal ecosystems poses a serious environmental threat. It is important to understand the responses and feedback of N removal microbial communities, particularly nitrifiers including the newly recognized complete ammonia-oxidizers (comammox), to improve aquaculture sustainability. In this study, we conducted a holistic evaluation of the functional communities responsible for nitrification by quantifying and sequencing the key functional genes of comammox Nitrospira-amoA, AOA-amoA, AOB-amoA and Nitrospira-nxrB in fish ponds with different fish feeding levels and evaluated the contribution of nitrifiers in the nitrification process through experiments of mixing pure cultures. We found that higher fish feeding dramatically increased N-related concentration, affecting the nitrifying communities. Compared to AOA and AOB, comammox Nitrospira and NOB were more sensitive to environmental changes. Unexpectedly, we detected an equivalent abundance of comammox Nitrospira and AOB and observed an increase in the proportion of clade A in comammox Nitrospira with the increase in fish feeding. Furthermore, a simplified network and shift of keystone species from NOB to comammox Nitrospira were observed in higher fish-feeding ponds. Random forest analysis suggested that the comammox Nitrospira community played a critical role in the nitrification of eutrophic aquaculture ponds (40-70 µM). Through the additional experiment of mixing nitrifying pure cultures, we found that comammox Nitrospira is the primary contributor to the nitrification process at 200 µM ammonium. These results advance our understanding of nitrifying communities and highlight the importance of comammox Nitrospira in driving nitrification in eutrophic aquaculture systems.

The neonicotinoid insecticide imidacloprid has unexpected effects on the growth and development of soil amoebae.

Neonicotinoid pesticides are the most widely used insecticides worldwide and have become a global environmental issue. Previous studies have shown that imidacloprid, the most used neonicotinoid, can negatively affect a wide range of organisms, including non-target insects, fish, invertebrates, and mammals. Imidacloprid can also accumulate and persist in soils, posing threats to the terrestrial ecosystem. However, we know little about one ecologically important group of organisms, the single-celled soil protists. In this study, we used a soil amoeba, Dictyostelium discoideum, to test whether and how imidacloprid affects the growth and development of soil amoebae. We provide the first empirical evidence that environmental concentrations of imidacloprid negatively impact the fitness and development of soil amoebae. In addition, the adverse effects did not show a dose-response relationship with increased imidacloprid concentrations, where no significant difference was observed among the treatment groups. Further transcriptome analyses showed that imidacloprid affected amoeba's key DEGs related to phagocytosis, cell division, morphogenesis, and cytochrome P450. Moreover, soil amoebae show both conserved and novel transcriptional responses to imidacloprid. In conclusion, this study has expanded the non-target list of imidacloprid from animals and plants to single-celled protists, and we believe the impact of neonicotinoid pesticides on the microbiome is significantly underestimated and deserves more studies.

Antimony efflux underpins phosphorus cycling and resistance of phosphate-solubilizing bacteria in mining soils.

Microorganisms play crucial roles in phosphorus (P) turnover and P bioavailability increases in heavy metal-contaminated soils. However, microbially driven P-cycling processes and mechanisms of their resistance to heavy metal contaminants remain poorly understood. Here, we examined the possible survival strategies of P-cycling microorganisms in horizontal and vertical soil samples from the world's largest antimony (Sb) mining site, which is located in Xikuangshan, China. We found that total soil Sb and pH were the primary factors affecting bacterial community diversity, structure and P-cycling traits. Bacteria with the gcd gene, encoding an enzyme responsible for gluconic acid production, largely correlated with inorganic phosphate (Pi) solubilization and significantly enhanced soil P bioavailability. Among the 106 nearly complete bacterial metagenome-assembled genomes (MAGs) recovered, 60.4% carried the gcd gene. Pi transportation systems encoded by pit or pstSCAB were widely present in gcd-harboring bacteria, and 43.8% of the gcd-harboring bacteria also carried the acr3 gene encoding an Sb efflux pump. Phylogenetic and potential horizontal gene transfer (HGT) analyses of acr3 indicated that Sb efflux could be a dominant resistance mechanism, and two gcd-harboring MAGs appeared to acquire acr3 through HGT. The results indicated that Sb efflux could enhance P cycling and heavy metal resistance in Pi-solubilizing bacteria in mining soils. This study provides novel strategies for managing and remediating heavy metal-contaminated ecosystems.

One health genomic insights into the host-specific evolution and cross-host transmission of Staphylococcus aureus in animal farm environments, food of animal origin, and humans.

OBJECTIVES: Staphylococcus aureus is an opportunistic pathogen that is considered a high priority for research. However, comparative studies of S. aureus strains from different environments and hosts are still lacking. METHODS: Here, we performed a high-resolution bioinformatics analysis of 576 S. aureus genomes isolated from livestock, farm environments, farm workers, animal-origin food, and humans. RESULTS: The S. aureus isolates showed high diversity in genetic lineages and demonstrated host specialization and multi-host range in the population phylogeny. Recent transmission events, historical divergences, and frequent host switching in specific sequence types (STs) and through the food chain and animal farm mediums were observed. Frequent gene transfer may quickly give rise to new fitness to colonize their host or switch to other hosts, even in isolates with the closest vertical evolutionary history. The large multi-host-shared antibiotic resistance gene (ARG) pool was the major factor shaping antibiotic resistance in S. aureus isolates. We revealed the genetic backgrounds of mec, cfr, and optrA, which could be spread among isolates from different species, hosts, and environments. CONCLUSION: Overall, our findings provide One Health genomic insights into the evolution, transmission, gene content characteristics, and antibiotic resistance profiles of S. aureus from different hosts, suggesting that, despite well-formed host specificity during the evolution of S. aureus, the ever-expanding host range and the cross-hosts/niches transmission (at both the strain and genetic level) may be facilitated by diverse bacterial vehicles (e.g., food chain, farm environments, and workers), which will lead to emerging antibiotic resistance consequences and threaten public health and food safety.

The vertically-stratified resistomes in mangrove sediments was driven by the bacterial diversity.

Early evidence has elucidated that the spread of antibiotic (ARGs) and metal resistance genes (MRGs) are mainly attributed to the selection pressure in human-influenced environments. However, whether and how biotic and abiotic factors mediate the distribution of ARGs and MRGs in mangrove sediments under natural sedimentation is largely unclear. Here, we profiled the abundance and diversity of ARGs and MRGs and their relationships with sedimental microbiomes in 0-100 cm mangrove sediments. Our results identified multidrug-resistance and multimetal-resistance as the most abundant ARG and MRG classes, and their abundances generally decreased with the sediment depth. Instead of abiotic factors such as nutrients and antibiotics, the bacterial diversity was significantly negatively correlated with the abundance and diversity of resistomes. Also, the majority of resistance classes (e.g., multidrug and arsenic) were carried by more diverse bacterial hosts in deep layers with low abundances of resistance genes. Together, our results indicated that bacterial diversity was the most important biotic factor driving the vertical profile of ARGs and MRGs in the mangrove sediment. Given that there is a foreseeable increasing human impact on natural environments, this study emphasizes the important role of biodiversity in driving the abundance and diversity of ARGs and MRGs.

PCycDB: a comprehensive and accurate database for fast analysis of phosphorus cycling genes.

BACKGROUND: Phosphorus (P) is one of the most essential macronutrients on the planet, and microorganisms (including bacteria and archaea) play a key role in P cycling in all living things and ecosystems. However, our comprehensive understanding of key P cycling genes (PCGs) and microorganisms (PCMs) as well as their ecological functions remains elusive even with the rapid advancement of metagenome sequencing technologies. One of major challenges is a lack of a comprehensive and accurately annotated P cycling functional gene database. RESULTS: In this study, we constructed a well-curated P cycling database (PCycDB) covering 139 gene families and 10 P metabolic processes, including several previously ignored PCGs such as pafA encoding phosphate-insensitive phosphatase, ptxABCD (phosphite-related genes), and novel aepXVWPS genes for 2-aminoethylphosphonate transporters. We achieved an annotation accuracy, positive predictive value (PPV), sensitivity, specificity, and negative predictive value (NPV) of 99.8%, 96.1%, 99.9%, 99.8%, and 99.9%, respectively, for simulated gene datasets. Compared to other orthology databases, PCycDB is more accurate, more comprehensive, and faster to profile the PCGs. We used PCycDB to analyze P cycling microbial communities from representative natural and engineered environments and showed that PCycDB could apply to different environments. CONCLUSIONS: We demonstrate that PCycDB is a powerful tool for advancing our understanding of microbially driven P cycling in the environment with high coverage, high accuracy, and rapid analysis of metagenome sequencing data. The PCycDB is available at https://github.com/ZengJiaxiong/Phosphorus-cycling-database . Video Abstract.

Coexistence of Antibiotic Resistance Genes and Virulence Factors Deciphered by Large-Scale Complete Genome Analysis.

Widespread use of antibiotics has enhanced the evolution of highly resilient pathogens and poses a severe risk to human health via coselection of antibiotic resistance genes (ARGs) and virulence factors (VFs). In this study, we rigorously evaluate the abundance relationship and physical linkage between ARGs and VFs by performing a comprehensive analysis of 9,070 bacterial genomes isolated from multiple species and hosts. The coexistence of ARGs and VFs was observed in bacteria across distinct phyla, pathogenicities, and habitats, especially among human-associated pathogens. The coexistence patterns of gene elements in different habitats and pathogenicity groups were similar, presumably due to frequent gene transfer. A shorter intergenic distance between mobile genetic elements and ARGs/VFs was detected in human/animal-associated bacteria, indicating a higher transfer potential. Increased accumulation of exogenous ARGs/VFs in human pathogens highlights the importance of gene acquisition in the evolution of human commensal bacteria. Overall, the findings provide insights into the genic features of combinations of ARG-VF and expand our understanding of ARG-VF coexistence in bacteria.IMPORTANCE Antibiotic resistance has become a serious global health concern. Despite numerous case studies, a comprehensive analysis of ARG and VF coexistence in bacteria is lacking. In this study, we explore the coexistence profiles of ARGs and VFs in diverse categories of bacteria by using a high-resolution bioinformatics approach. We also provide compelling evidence of unique ARG-VF gene pairs coexisting in specific bacterial genomes and reveal the potential risk associated with the coexistence of ARGs and VFs in organisms in both clinical settings and environments.

Metagenomic insights into the distribution of antibiotic resistome between the gut-associated environments and the pristine environments.

Antibiotic resistance genes (ARGs) in the environment are promoted by anthropogenic activities, which cause potential risks to human health. However, large-scale quantitative data on antibiotic resistome from the pristine and anthropogenic environments remains largely unexplored. Here, we used metagenome-wide analysis to investigate the share and divergence in ARG profiles and their potential bacterial hosts between the pristine and gut-associated environments. We found that the abundance of total ARGs in gut-associated environments was significantly higher than the pristine environments (P < 0.001). The mcr-1 and tetX, the genes resistant to the last resort antibiotics (colistin and tigecycline, respectively), were in high abundance (4.57 copies/Gb and 3.39 copies/Gb, respectively) in gut-associated environments, suggesting the ARG pollution caused by anthropogenic antibiotics. Metagenomic assembly-based host-tracking analysis identified Escherichia, Bacteroides, and Clostridium as the predominant bacterial hosts of ARGs in gut-associated environments, while Alteromonas, Vibrio, and Proteobacteria as the predominant bacterial hosts of ARGs in pristine environments. We first described the broad diversity of ARG hosts in different environments using metagenome-wide analysis. Our results revealed the heterogeneous distribution of ARGs and their hosts among different microbial niches in gut-associated environments and the pristine environments.

Metagenomic Insights Into the Contribution of Phages to Antibiotic Resistance in Water Samples Related to Swine Feedlot Wastewater Treatment.

In this study, we examined the types of antibiotic resistance genes (ARGs) possessed by bacteria and bacteriophages in swine feedlot wastewater before and after treatment using a metagenomics approach. We found that the relative abundance of ARGs in bacterial DNA in all water samples was significantly higher than that in phages DNA (>10.6-fold), and wastewater treatment did not significantly change the relative abundance of bacterial- or phage-associated ARGs. We further detected the distribution and diversity of the different types of ARGs according to the class of antibiotics to which they confer resistance, the tetracycline resistance genes were the most abundant resistance genes and phages were more likely to harbor ATP-binding cassette transporter family and ribosomal protection genes. Moreover, the colistin resistance gene mcr-1 was also detected in the phage population. When assessing the contribution of phages in spreading different groups of ARGs, ß-lactamase resistance genes had a relatively high spreading ability even though the abundance was low. These findings possibly indicated that phages not only could serve as important reservoir of ARG but also carry particular ARGs in swine feedlot wastewater, and this phenomenon is independent of the environment.