Identification of ISVlu1-derived translocatable units containing optrA and/or fexA genes generated by homologous or illegitimate recombination in Lactococcus garvieae of porcine origin.

The optrA gene encodes an ABC-F protein which confers cross-resistance to oxazolidinones and phenicols. Insertion sequence ISVlu1, a novel ISL3-family member, was recently reported to be involved in the transmission of optrA in Vagococcus lutrae. However, the role of ISVlu1 in mobilizing resistance genes has not yet fully explored. In this study, two complete and three truncated copies of ISVlu1 were found on plasmid pBN62-optrA from Lactococcus garvieae. Analysis of the genetic context showed that both optrA and the phenicols resistance gene fexA were flanked by the complete or truncated ISVlu1 copies. Moreover, three different-sized ISVlu1-based translocatable units (TUs) carrying optrA and/or fexA, were detected from pBN62-optrA. Sequence analysis revealed that the TU-optrA was generated by homologous recombination while TU-fexA and TU-optrA+fexA were the products of illegitimate recombinations. Importantly, conjugation assays confirmed that pBN62-optrA was able to successfully transfer into the recipient Enterococcus faecalis JH2-2. To our knowledge, this is the first report about an optrA-carrying plasmid in L. garvieae which could horizontally transfer into other species. More importantly, the ISVlu1-flanked genetic structures containing optrA and/or fexA were also observed in bacteria of different species, which underlines that ISVlu1 is highly active and plays a vital role in the transfer of some important resistance genes, such as optrA and fexA.

What role do biocontrol agents with Mg2+ play in the fate of antibiotic resistome and pathogenic bacteria in the phyllosphere?

The contamination of the plant phyllosphere with antibiotics and antibiotic resistance genes (ARGs), caused by application of antibiotics, is a significant environmental issue in agricultural management. Alternatively, biocontrol agents are environmentally friendly and have attracted a lot of interest. However, the influence of biocontrol agents on the phyllosphere resistome remains unknown. In this study, we applied biocontrol agents to control the wildfire disease in the Solanaceae crops and investigated their effects on the resistome and the pathogen in the phyllosphere by using metagenomics. A total of 250 ARGs were detected from 15 samples, which showed a variation in distribution across treatments of biocontrol agents (BA), BA with Mg2+ (T1), BA with Mn2+ (T2), and kasugamycin (T3) and nontreated (CK). The results showed that the abundance of ARGs under the treatment of BA-Mg2+ was lower than that in the CK group. The abundance of cphA3 (carbapenem resistance), PME-1 (carbapenem resistance), tcr3 (tetracycline antibiotic resistance), and AAC (3)-VIIIa (aminoglycoside antibiotic resistance) in BA-Mg2+ was significantly higher than that in BA-Mn2+ (P < 0.05). The abundance of cphA3, PME_1, and tcr3 was significantly negatively related to the abundance of the phyllosphere pathogen Pseudomonas syringae (P < 0.05). We also found that the upstream and downstream regions of cphA3 were relatively conserved, in which rpl, rpm, and rps gene families were identified in most sequences (92%). The Ka/Ks of cphA3 was 0 in all observed sequences, indicating that under the action of purifying selection, nonsynonymous substitutions are often gradually eliminated in the population. Overall, this study clarifies the effect of biocontrol agents with Mg2+ on the distribution of the phyllosphere resistome and provides evolutionary insights into the biocontrol process. IMPORTANCE: Our study applied metagenomics analysis to examine the impact of biocontrol agents (BAs) on the phyllosphere resistome and the pathogen. Irregular use of antibiotics has led to the escalating dissemination of antibiotic resistance genes (ARGs) in the environment. The majority of BA research has focused on the effect of monospecies on the plant disease control process, the role of the compound BA with nutrition elements in the phyllosphere disease, and the resistome is still unknown. We believe BAs are eco-friendly alternatives for antibiotics to combat the transfer of ARGs. Our results revealed that BA-Mg2+ had a lower relative abundance of ARGs compared to the CK group, and the phyllosphere pathogen Pseudomonas syringae was negatively related to three specific ARGs, cphA3, PME-1, and tcr3. These three genes also present different Ka/Ks. We believe that the identification of the distribution and evolution modes of ARGs further elucidates the ecological role and facilitates the development of BAs, which will attract general interest in this field.

Microbial biofilms on macroalgae harbour diverse integron gene cassettes.

Integrons are genetic platforms that capture, rearrange and express mobile modules called gene cassettes. The best characterized gene cassettes encode antibiotic resistance, but the function of most integron gene cassettes remains unknown. Functional predictions suggest that many gene cassettes could encode proteins that facilitate interactions with other cells and with the extracellular environment. Because cell interactions are essential for biofilm stability, we sequenced gene cassettes from biofilms growing on the surface of the marine macroalgae Ulva australis and Sargassum linearifolium. Algal samples were obtained from coastal rock platforms around Sydney, Australia, using seawater as a control. We demonstrated that integrons in microbial biofilms did not sample genes randomly from the surrounding seawater, but harboured specific functions that potentially provided an adaptive advantage to both the bacterial cells in biofilm communities and their macroalgal host. Further, integron gene cassettes had a well-defined spatial distribution, suggesting that each bacterial biofilm acquired these genetic elements via sampling from a large but localized pool of gene cassettes. These findings suggest two forms of filtering: a selective acquisition of different integron-containing bacterial species into the distinct biofilms on Ulva and Sargassum surfaces, and a selective retention of unique populations of gene cassettes at each sampling location.

Antibiotics and antibiotic resistance genes removal in biological aerated filter.

Two laboratory-level biological aerated filters (BAF) were constructed to explore their treatment capacity for simulated antibiotic wastewater at high (1 - 16 mg/L) and low (0 - 0.5 mg/L) concentrations. Results showed that BAF was capable of removing both sulfonamides and tetracyclines with an efficiency of over 90 % at 16 mg/L. The main mechanism for removing antibiotics was found to be biodegradation followed by adsorption. Paenarthrobacter was identified as the key genus in sulfonamides degradation, while Hydrogenophaga played a crucial role in tetracyclines degradation. Antibiotics resistant genes such as intI1, sul1, sul2, tetA, tetW and tetX were frequently detected in the effluent, with interception rates ranging from 105 - 106 copies/mL. The dominated microorganisms obtained in the study could potentially be utilized to enhance the capacity of biological processes for treating antibiotics contaminated wastewater. These findings contribute to a better understanding of BAF treating wastewater containing antibiotics and resistant genes.

Duplicated antibiotic resistance genes reveal ongoing selection and horizontal gene transfer in bacteria.

Horizontal gene transfer (HGT) and gene duplication are often considered as separate mechanisms driving the evolution of new functions. However, the mobile genetic elements (MGEs) implicated in HGT can copy themselves, so positive selection on MGEs could drive gene duplications. Here, we use a combination of modeling and experimental evolution to examine this hypothesis and use long-read genome sequences of tens of thousands of bacterial isolates to examine its generality in nature. Modeling and experiments show that antibiotic selection can drive the evolution of duplicated antibiotic resistance genes (ARGs) through MGE transposition. A key implication is that duplicated ARGs should be enriched in environments associated with antibiotic use. To test this, we examined the distribution of duplicated ARGs in 18,938 complete bacterial genomes with ecological metadata. Duplicated ARGs are highly enriched in bacteria isolated from humans and livestock. Duplicated ARGs are further enriched in an independent set of 321 antibiotic-resistant clinical isolates. Our findings indicate that duplicated genes often encode functions undergoing positive selection and horizontal gene transfer in microbial communities.

Modeling the limits of detection for antimicrobial resistance genes in agri-food samples: a comparative analysis of bioinformatics tools.

BACKGROUND: Although the spread of antimicrobial resistance (AMR) through food and its production poses a significant concern, there is limited research on the prevalence of AMR bacteria in various agri-food products. Sequencing technologies are increasingly being used to track the spread of AMR genes (ARGs) in bacteria, and metagenomics has the potential to bypass some of the limitations of single isolate characterization by allowing simultaneous analysis of the agri-food product microbiome and associated resistome. However, metagenomics may still be hindered by methodological biases, presence of eukaryotic DNA, and difficulties in detecting low abundance targets within an attainable sequence coverage. The goal of this study was to assess whether limits of detection of ARGs in agri-food metagenomes were influenced by sample type and bioinformatic approaches. RESULTS: We simulated metagenomes containing different proportions of AMR pathogens and analysed them for taxonomic composition and ARGs using several common bioinformatic tools. Kraken2/Bracken estimates of species abundance were closest to expected values. However, analysis by both Kraken2/Bracken indicated presence of organisms not included in the synthetic metagenomes. Metaphlan3/Metaphlan4 analysis of community composition was more specific but with lower sensitivity than the Kraken2/Bracken analysis. Accurate detection of ARGs dropped drastically below 5X isolate genome coverage. However, it was sometimes possible to detect ARGs and closely related alleles at lower coverage levels if using a lower ARG-target coverage cutoff (< 80%). While KMA and CARD-RGI only predicted presence of expected ARG-targets or closely related gene-alleles, SRST2 (which allows read to map to multiple targets) falsely reported presence of distantly related ARGs at all isolate genome coverage levels. The presence of background microbiota in metagenomes influenced the accuracy of ARG detection by KMA, resulting in mcr-1 detection at 0.1X isolate coverage in the lettuce but not in the beef metagenome. CONCLUSIONS: This study demonstrates accurate detection of ARGs in synthetic metagenomes using various bioinformatic methods, provided that reads from the ARG-encoding organism exceed approximately 5X isolate coverage (i.e. 0.4% of a 40 million read metagenome). While lowering thresholds for target gene detection improved sensitivity, this led to the identification of alternative ARG-alleles, potentially confounding the identification of critical ARGs in the resistome. Further advancements in sequencing technologies providing increased coverage depth or extended read lengths may improve ARG detection in agri-food metagenomic samples, enabling use of this approach for tracking clinically important ARGs in agri-food samples.

[High-throughput qPCR and Amplicon Sequencing as Complementary Methods for Profiling Antibiotic Resistance Genes in Urban Wetland Parks].

Urban wetland parks are an important practice for urban wetland protection and utilization due to the vast ecosystem service value. As emerging contaminants, antibiotic resistance genes (ARGs) are great attractions for environmental research and public concerns. Based on high-throughput qPCR and high-throughput amplicon sequencing techniques, we investigated the occurrence, abundance, and distribution profiles of antibiotic resistance genes in the aquatic environment of Xiamen urban wetland parks (five sites). The influencing factors and driving mechanisms of antibiotic resistance genes were deciphered on the basis of microbial community structure and water quality. Diverse and abundant ARGs were observed and coexisted in urban wet parks. A total of 217 ARGs were detected in the water body of urban wetland parks, with an abundance up to 6.48×109 copies·L-1. Urban wetland parks were important hotspots and repositories of the antibiotic resistome. A total of nine bacterial genera, including Marivivens, NS5_marine_group, and Planktomarina, were identified as the potential carriers of diverse resistance genes (41 ARGs). The microbial communities could alone explain 51% of alterations in the antibiotic resistome in the aquatic environment of the urban wetland parks. Therefore, the microbial community was the key driving force for the occurrence and evolution of ARGs in urban wetland parks. Based on the results, with the presence of ARGs and antibiotic resistance bacteria, it is suggested that the water environments of urban wetland parks have potential risks of water ecological security and human health, and it is necessary to further enhance the research and control of microbial contaminants in the aquatic environment of urban wetland parks.

Genome-centric analyses of 165 metagenomes show that mobile genetic elements are crucial for the transmission of antimicrobial resistance genes to pathogens in activated sludge and wastewater.

Wastewater is considered a reservoir of antimicrobial resistance genes (ARGs), where the abundant antimicrobial-resistant bacteria and mobile genetic elements facilitate horizontal gene transfer. However, the prevalence and extent of these phenomena in different taxonomic groups that inhabit wastewater are still not fully understood. Here, we determined the presence of ARGs in metagenome-assembled genomes (MAGs) and evaluated the risks of MAG-carrying ARGs in potential human pathogens. The potential of these ARGs to be transmitted horizontally or vertically was also determined. A total of 5,916 MAGs (completeness >50%, contamination <10%) were recovered, covering 68 phyla and 279 genera. MAGs were dereplicated into 1,204 genome operational taxonomic units (gOTUs) as a proxy for species ( average nucleotide identity >0.95). The dominant ARG classes detected were bacitracin, multi-drug, macrolide-lincosamide-streptogramin (MLS), glycopeptide, and aminoglycoside, and 10.26% of them were located on plasmids. The main hosts of ARGs belonged to Escherichia, Klebsiella, Acinetobacter, Gresbergeria, Mycobacterium, and Thauera. Our data showed that 253 MAGs carried virulence factor genes (VFGs) divided into 44 gOTUs, of which 45 MAGs were carriers of ARGs, indicating that potential human pathogens carried ARGs. Alarmingly, the MAG assigned as Escherichia coli contained 159 VFGs, of which 95 were located on chromosomes and 10 on plasmids. In addition to shedding light on the prevalence of ARGs in individual genomes recovered from activated sludge and wastewater, our study demonstrates a workflow that can identify antimicrobial-resistant pathogens in complex microbial communities. IMPORTANCE: Antimicrobial resistance (AMR) threatens the health of humans, animals, and natural ecosystems. In our study, an analysis of 165 metagenomes from wastewater revealed antibiotic-targeted alteration, efflux, and inactivation as the most prevalent AMR mechanisms. We identified several genera correlated with multiple ARGs, including Klebsiella, Escherichia, Acinetobacter, Nitrospira, Ottowia, Pseudomonas, and Thauera, which could have significant implications for AMR transmission. The abundance of bacA, mexL, and aph(3")-I in the genomes calls for their urgent management in wastewater. Our approach could be applied to different ecosystems to assess the risk of potential pathogens containing ARGs. Our findings highlight the importance of managing AMR in wastewater and can help design measures to reduce the transmission and evolution of AMR in these systems.

Mechanisms of total phosphorus removal and reduction of ß-lactam antibiotic resistance genes by exogenous fungal combination activated sludge.

This study utilized Trichoderma and activated sludge to construct combined activated sludge (TAS). The metagenomic approach was employed to examine the shifts in microbial community structure and function of TAS under amoxicillin stress and investigate the mechanism underlying the reduction of ß-lactam antibiotic resistance genes (ß-ARGs). The findings demonstrated that the elevated aundance of glpa, glpd, ugpq, glpq, and glpb were primarily responsible for the reduction in total phosphorus (TP) removal by TAS. The increased abundance of Proteobacteria and Verrucomicrobia led to enhanced expression of ugpb, phnd, and phne, thereby improving the TP removal of TAS. Furthermore, antibiotic inactivation has gradually become the primary antibiotic resistance mechanism in TAS. Specifically, an increase in the abundance of OXA-309 in TAS will decrease the probability of amoxicillin accumulation in TAS. A decrease in ß-ARGs diversity confirmed this. This study presents a novel approach to reducing antibiotic and ARG accumulation in sludge.

Effects of oxytetracycline on variation in intracellular and extracellular antibiotic resistance genes during swine manure composting.

This research aimed to investigate the alterations in extracellular (eARGs) and intracellular (iARGs) antibiotic resistance genes in response to oxytetracycline (OTC), and unravel the dissemination mechanism of ARGs during composting. The findings revealed both low (L-OTC) and high contents (H-OTC) of OTC significantly enhanced absolute abundance (AA) of iARGs (p < 0.05), compared to CK (no OTC). Composting proved to be a proficient strategy for removing eARGs, while AA of eARGs was significantly enhanced in H-OTC (p < 0.05). OTC resulted in an increase in AA of mobile genetic elements (MGEs), ATP levels, antioxidant and DNA repair enzymes in bacteria in compost product. Structural equation model further demonstrated that OTC promoted bacterial DNA repair and antioxidant enzyme activities, altered bacterial community and enhanced MGEs abundance, thereby facilitating iARGs dissemination. This study highlights OTC can increase eARGs and iARGs abundance, underscoring the need for appropriate countermeasures to mitigate potential hazards.

RNA G-quadruplexes inhibit translation of the PE/PPE transcripts in Mycobacterium tuberculosis.

The role of RNA G-quadruplexes (rG4s) in bacteria remains poorly understood. High G-quadruplex densities have been linked to organismal stress. Here we investigate rG4s in mycobacteria, which survive highly stressful conditions within the host. We show that rG4-enrichment is a unique feature exclusive to slow-growing pathogenic mycobacteria, and Mycobacterium tuberculosis (Mtb) transcripts contain an abundance of folded rG4s. Notably, the PE/PPE family of genes, unique to slow-growing pathogenic mycobacteria, contain over 50% of rG4s within Mtb transcripts. We found that RNA oligonucleotides of putative rG4s in PE/PPE genes form G-quadruplex structures in vitro, which are stabilized by the G-quadruplex ligand BRACO19. Furthermore, BRACO19 inhibits the transcription of PE/PPE genes and selectively suppresses the growth of Mtb but not Mycobacterium smegmatis or other rapidly growing bacteria. Importantly, the stabilization of rG4s inhibits the translation of Mtb PE/PPE genes (PPE56, PPE67, PPE68, PE_PGRS39, and PE_PGRS41) ectopically expressed in M. smegmatis or Escherichia coli. In addition, the rG4-mediated reduction in PE/PPE protein levels attenuates proinflammatory response upon infection of THP-1 cells. Our findings shed new light on the regulation of PE/PPE genes and highlight a pivotal role for rG4s in Mtb transcripts as regulators of post-transcriptional translational control. The rG4s in mycobacterial transcripts may represent potential drug targets for newer therapies.

Biochar induces different responses of intracellular and extracellular antibiotic resistance genes and suppresses horizontal transfer during lincomycin fermentation dregs composting.

This study aims to indicate the influence of biochar on extracellular and intracellular ARGs (e/iARGs) variation and proliferation during lincomycin fermentation dregs (LFDs) compost. Biochar addition made iARGs keep reducing but eARGs increase to the maximum at the middle thermophilic phase and reduce at the end of the compost. Compared to control 3.15-log and 5.42-log reduction of iARGs and eARGs were observed, respectively. Biochar addition, bacterial community, and MGEs were the major contributors to iARGs and eARGs removal, with the contribution percentages of 38.4%, 31.0%, 23.7%, and 27.2%, 29.1%, and 34.9%, respectively. Moreover, biochar significantly inhibited eARGs transformation and RP4 plasmid conjugative transfer among E. coli DH5α and Pseudomonas aeruginosa HLS-6. The underlying mechanism involved in broken cell membranes of bacteria, and altered expression of oxidative stress genes and save our souls (SOS) response-related genes. The results indicated that biochar addition in composting could limit the dissemination of ARGs.

Insights into the roles of biochar pores toward alleviating antibiotic resistance genes accumulation in biofiltration systems.

Biofiltration systems would harbor and spread various antibiotic resistance genes (ARGs) when treating antibiotic micro-pollution, constituting a potential ecological risk. This study aimed to investigate the effects of biochar pores on ARG emergence and related microbial response mechanisms in bench-scale biofiltration systems. Results showed that biochar pores effectively reduced the absolute copies of the corresponding ARGs sul1 and sul2 by 54.1% by lowering the sorbed-SMX's bioavailability compared to non-porous anthracite. An investigation of antimicrobial resistomes revealed a considerable decrease in the abundance and diversity of ARGs and mobile gene elements. Metagenomic and metaproteomic analysis demonstrated that biochar pores induced the changeover of microbial defense strategy against SMX from blocking SMX uptake by EPS absorbing to SMX biotransformation. Microbial SOS response, antibiotic efflux pump, EPS secretion, and biofilm formation were decreased. Functions related to SMX biotransformation, such as sadABC-mediated transformation, xenobiotics degradation, and metabolism, were significantly promoted.

Long-term occurrence, resistance risk and chaotic characteristics of antibiotic resistance genes in sludge anaerobic digestion system.

The long-term occurrence, dynamics and risk of antibiotic resistance genes (ARGs) in anaerobic digestion (AD) of excess sludge (ES) are not fully understood. Therefore, 13-month metagenomic monitoring was carried out in a full-scale AD plant. The highest ARG abundance and risk scores were observed in spring. AD achieved a 35 % removal rate for the total ARG abundance, but the risk score of AD sludge was not always lower than ES samples, because of the higher proportion of Rank I ARGs in AD sludge. ARGs showed less obvious patterns under linear models compared with microbial community, implying their chaotic dynamics, which was further confirmed by nonlinearity tests. Empirical dynamic modeling performed better than the autoregressive integrated moving average model for ARG dynamics, especially for those with simple and nonlinear dynamics. This study highlighted spring for its higher ARG abundance and risk, and recommended nonlinear models for revealing the dynamics of ARGs.

Deciphering the Role of WWTPs in Cold Environments as Hotspots for the Dissemination of Antibiotic Resistance Genes.

Cold environments are the most widespread extreme habitats in the world. However, the role of wastewater treatment plants (WWTPs) in the cryosphere as hotspots in antibiotic resistance dissemination has not been well established. Hence, a snapshot of the resistomes of WWTPs in cold environments, below 5 °C, was provided to elucidate their role in disseminating antibiotic resistance genes (ARGs) to the receiving waterbodies. The resistomes of two natural environments from the cold biosphere were also determined. Quantitative PCR analysis of the aadA, aadB, ampC, blaSHV, blaTEM, dfrA1, ermB, fosA, mecA, qnrS, and tetA(A) genes indicated strong prevalences of these genetic determinants in the selected environments, except for the mecA gene, which was not found in any of the samples. Notably, high abundances of the aadA, ermB, and tetA(A) genes were found in the influents and activated sludge, highlighting that WWTPs of the cryosphere are critical hotspots for disseminating ARGs, potentially worsening the resistance of bacteria to some of the most commonly prescribed antibiotics. Besides, the samples from non-disturbed cold environments had large quantities of ARGs, although their ARG profiles were highly dissimilar. Hence, the high prevalences of ARGs lend support to the fact that antibiotic resistance is a common issue worldwide, including environmentally fragile cold ecosystems.

SOCfinder: a genomic tool for identifying social genes in bacteria.

Bacteria cooperate by working collaboratively to defend their colonies, share nutrients, and resist antibiotics. Nevertheless, our understanding of these remarkable behaviours primarily comes from studying a few well-characterized species. Consequently, there is a significant gap in our understanding of microbial social traits, particularly in natural environments. To address this gap, we can use bioinformatic tools to identify genes that control cooperative or otherwise social traits. Existing tools address this challenge through two approaches. One approach is to identify genes that encode extracellular proteins, which can provide benefits to neighbouring cells. An alternative approach is to predict gene function using annotation tools. However, these tools have several limitations. Not all extracellular proteins are cooperative, and not all cooperative behaviours are controlled by extracellular proteins. Furthermore, existing functional annotation methods frequently miss known cooperative genes. We introduce SOCfinder as a new tool to find bacterial genes that control cooperative or otherwise social traits. SOCfinder combines information from several methods, considering if a gene is likely to [1] code for an extracellular protein [2], have a cooperative functional annotation, or [3] be part of the biosynthesis of a cooperative secondary metabolite. We use data on two extensively-studied species (P. aeruginosa and B. subtilis) to show that SOCfinder is better at finding known cooperative genes than existing tools. We also use theory from population genetics to identify a signature of kin selection in SOCfinder cooperative genes, which is lacking in genes identified by existing tools. SOCfinder opens up a number of exciting directions for future research, and is available to download from https://github.com/lauriebelch/SOCfinder.

Microbial hitchhikers harbouring antimicrobial-resistance genes in the riverine plastisphere.

BACKGROUND: The widespread nature of plastic pollution has given rise to wide scientific and social concern regarding the capacity of these materials to serve as vectors for pathogenic bacteria and reservoirs for Antimicrobial Resistance Genes (ARG). In- and ex-situ incubations were used to characterise the riverine plastisphere taxonomically and functionally in order to determine whether antibiotics within the water influenced the ARG profiles in these microbiomes and how these compared to those on natural surfaces such as wood and their planktonic counterparts. RESULTS: We show that plastics support a taxonomically distinct microbiome containing potential pathogens and ARGs. While the plastisphere was similar to those biofilms that grew on wood, they were distinct from the surrounding water microbiome. Hence, whilst potential opportunistic pathogens (i.e. Pseudomonas aeruginosa, Acinetobacter and Aeromonas) and ARG subtypes (i.e. those that confer resistance to macrolides/lincosamides, rifamycin, sulfonamides, disinfecting agents and glycopeptides) were predominant in all surface-related microbiomes, especially on weathered plastics, a completely different set of potential pathogens (i.e. Escherichia, Salmonella, Klebsiella and Streptococcus) and ARGs (i.e. aminoglycosides, tetracycline, aminocoumarin, fluoroquinolones, nitroimidazole, oxazolidinone and fosfomycin) dominated in the planktonic compartment. Our genome-centric analysis allowed the assembly of 215 Metagenome Assembled Genomes (MAGs), linking ARGs and other virulence-related genes to their host. Interestingly, a MAG belonging to Escherichia -that clearly predominated in water- harboured more ARGs and virulence factors than any other MAG, emphasising the potential virulent nature of these pathogenic-related groups. Finally, ex-situ incubations using environmentally-relevant concentrations of antibiotics increased the prevalence of their corresponding ARGs, but different riverine compartments -including plastispheres- were affected differently by each antibiotic. CONCLUSIONS: Our results provide insights into the capacity of the riverine plastisphere to harbour a distinct set of potentially pathogenic bacteria and function as a reservoir of ARGs. The environmental impact that plastics pose if they act as a reservoir for either pathogenic bacteria or ARGs is aggravated by the persistence of plastics in the environment due to their recalcitrance and buoyancy. Nevertheless, the high similarities with microbiomes growing on natural co-occurring materials and even more worrisome microbiome observed in the surrounding water highlights the urgent need to integrate the analysis of all environmental compartments when assessing risks and exposure to pathogens and ARGs in anthropogenically-impacted ecosystems. Video Abstract.

Clinically relevant antibiotic resistance genes are linked to a limited set of taxa within gut microbiome worldwide.

The acquisition of antimicrobial resistance (AR) genes has rendered important pathogens nearly or fully unresponsive to antibiotics. It has been suggested that pathogens acquire AR traits from the gut microbiota, which collectively serve as a global reservoir for AR genes conferring resistance to all classes of antibiotics. However, only a subset of AR genes confers resistance to clinically relevant antibiotics, and, although these AR gene profiles are well-characterized for common pathogens, less is known about their taxonomic associations and transfer potential within diverse members of the gut microbiota. We examined a collection of 14,850 human metagenomes and 1666 environmental metagenomes from 33 countries, in addition to nearly 600,000 isolate genomes, to gain insight into the global prevalence and taxonomic range of clinically relevant AR genes. We find that several of the most concerning AR genes, such as those encoding the cephalosporinase CTX-M and carbapenemases KPC, IMP, NDM, and VIM, remain taxonomically restricted to Proteobacteria. Even cfiA, the most common carbapenemase gene within the human gut microbiome, remains tightly restricted to Bacteroides, despite being found on a mobilizable plasmid. We confirmed these findings in gut microbiome samples from India, Honduras, Pakistan, and Vietnam, using a high-sensitivity single-cell fusion PCR approach. Focusing on a set of genes encoding carbapenemases and cephalosporinases, thus far restricted to Bacteroides species, we find that few mutations are required for efficacy in a different phylum, raising the question of why these genes have not spread more widely. Overall, these data suggest that globally prevalent, clinically relevant AR genes have not yet established themselves across diverse commensal gut microbiota.

VBCG: 20 validated bacterial core genes for phylogenomic analysis with high fidelity and resolution.

BACKGROUND: Phylogenomic analysis has become an inseparable part of studies of bacterial diversity and evolution, and many different bacterial core genes have been collated and used for phylogenomic tree reconstruction. However, these genes have been selected based on their presence and single-copy ratio in all bacterial genomes, leaving out the gene's 'phylogenetic fidelity' unexamined. RESULTS: From 30,522 complete genomes covering 11,262 species, we examined 148 bacterial core genes that have been previously used for phylogenomic analysis. In addition to the gene presence and single-copy rations, we evaluated the gene's phylogenetic fidelity by comparing each gene's phylogeny with its corresponding 16S rRNA gene tree. Out of the 148 bacterial genes, 20 validated bacterial core genes (VBCG) were selected as the core gene set with the highest bacterial phylogenetic fidelity. Compared to the larger gene set, the 20-gene core set resulted in more species having all genes present and fewer species with missing data, thereby enhancing the accuracy of phylogenomic analysis. Using Escherichia coli strains as examples of prominent bacterial foodborne pathogens, we demonstrated that the 20 VBCG produced phylogenies with higher fidelity and resolution at species and strain levels while 16S rRNA gene tree alone could not. CONCLUSION: The 20 validated core gene set improves the fidelity and speed of phylogenomic analysis. Among other uses, this tool improves our ability to explore the evolution, typing and tracking of bacterial strains, such as human pathogens. We have developed a Python pipeline and a desktop graphic app (available on GitHub) for users to perform phylogenomic analysis with high fidelity and resolution. Video Abstract.

Pet cats may shape the antibiotic resistome of their owner's gut and living environment.

BACKGROUND: Companion animals can contribute to the physical and mental health of people and often live in very close association with their owners. However, the antibiotic resistome carried by companion animals and the impact they have on their owners and living environment remain unclear. In this study, we compared the ARG profiles of cats, humans, and their living environments using metagenomic analysis to identify the core ARGs in the cat and human gut and explore the potential impact of cats on ARGs in the human gut through the environment. RESULTS: Results showed that the abundance of ARGs in the cat gut was significantly higher than that in the human gut (P < 0.0001), with aminoglycoside and tetracycline resistance genes being the dominant ARGs in the cat gut. There was no significant difference in the abundance of total ARGs in the guts of cat owners and non-owners (P > 0.05). However, the abundance of aminoglycoside resistance genes including APH(2'')-IIa and AAC(6')-Im was significantly higher in cat owners than that in non-cat owners (P < 0.001). Also, ARG abundance was positively correlated with the frequency of cat activity in the living environment. Enterobacteriaceae was the dominant ARG host co-occurring in the cat gut, human gut, and living environment. CONCLUSIONS: Our results show that cats may shape the living environment resistome and thus the composition of some ARGs in the human gut, highlighting the importance of companion animal environment health. Video Abstract.