Nature-based bioreactors: Tackling antibiotic resistance in urban wastewater treatment.

The overuse and misuse of antibiotics have accelerated the selection of antibiotic-resistant bacteria, significantly impacting human, animal, and environmental health. As aquatic environments are vulnerable to antibiotic resistance, suitable management practices should be adopted to tackle this phenomenon. Here we show an effective, nature-based solution for reducing antibiotic resistance from actual wastewater. We utilize a bioreactor that relies on benthic (biofilms) and planktonic microbial communities to treat secondary effluent from a small urban wastewater treatment plant (<10,000 population equivalent). This treated effluent is eventually released into the local aquatic ecosystem. We observe high removal efficiency for genes that provide resistance to commonly used antibiotic families, as well as for mobile genetic elements that could potentially aid in their spread. Importantly, we notice a buildup of sulfonamide (sul1 and sul2) and tetracycline (tet(C), tet(G), and tetR) resistance genes specifically in biofilms. This advancement marks the initial step in considering this bioreactor as a nature-based, cost-effective tertiary treatment option for small UWWTPs facing antibiotic resistance challenges.

The extent and characteristics of DNA transfer between plasmids and chromosomes.

Plasmids are extrachromosomal genetic elements that reside in prokaryotes. The acquisition of plasmids encoding beneficial traits can facilitate short-term survival in harsh environmental conditions or long-term adaptation of new ecological niches. Due to their ability to transfer between cells, plasmids are considered agents of gene transfer. Nonetheless, the frequency of DNA transfer between plasmids and chromosomes remains understudied. Using a novel approach for detection of homologous loci between genome pairs, we uncover gene sharing with the chromosome in 1,974 (66%) plasmids residing in 1,016 (78%) taxonomically diverse isolates. The majority of homologous loci correspond to mobile elements, which may be duplicated in the host chromosomes in tens of copies. Neighboring shared genes often encode similar functional categories, indicating the transfer of multigene functional units. Rare transfer events of antibiotics resistance genes are observed mainly with mobile elements. The frequent erosion of sequence similarity in homologous regions indicates that the transferred DNA is often devoid of function. DNA transfer between plasmids and chromosomes thus generates genetic variation that is akin to workings of endosymbiotic gene transfer in eukaryotic evolution. Our findings imply that plasmid contribution to gene transfer most often corresponds to transfer of the plasmid entity rather than transfer of protein-coding genes between plasmids and chromosomes.

Disinfectant polyhexamethylene guanidine triggered simultaneous efflux pump antibiotic- and metal-resistance genes propagation during sludge anaerobic digestion.

The environmental transmission of antibiotic resistance genes (ARGs) and metal resistance genes (MRGs) exerted devastating threats to global public health, and their interactions with other emerging contaminants (ECs) have raised increasing concern. This study investigated that the abundances of ARGs and MRGs with the predominant type of efflux pump were simultaneously increased (8.4-59.1%) by disinfectant polyhexamethylene guanidine (PHMG) during waste activated sludge (WAS) anaerobic digestion. The aggregation of the same microorganisms (i.e., Hymenobacter and Comamonas) and different host bacteria (i.e., Azoarcus and Thauera) were occurred upon exposure to PHMG, thereby increasing the co-selection and propagation of MRGs and ARGs by vertical gene transfer. Moreover, PHMG enhanced the process of horizontal gene transfer (HGT), facilitating their co-transmission by the same mobile genetic elements (20.2-223.7%). Additionally, PHMG up-regulated the expression of critical genes (i.e., glnB, trpG and gspM) associated with the HGT of ARGs and MRGs (i.e., two-component regulatory system and quorum sensing) and exocytosis system (i.e., bacterial secretion system). Structural equation model analysis further verified that the key driver for the simultaneous enrichment of ARGs and MRGs under PHMG stress was microbial community structure. The study gives new insights into the aggravated environmental risks and mechanisms of ECs in sludge digestion system, providing guidance for subsequent regulation and control of ECs.

Seasonal dynamics of antibiotic resistance genes and mobile genetic elements in a subtropical coastal ecosystem: Implications for environmental health risks.

Antibiotic resistance poses a considerable global public health concern, leading to heightened rates of illness and mortality. However, the impact of seasonal variations and environmental factors on the health risks associated with antibiotic resistance genes (ARGs) and their assembly mechanisms is not fully understood. Based on metagenomic sequencing, this study investigated the antibiotic resistome, mobile genetic elements (MGEs), and microbiomes in a subtropical coastal ecosystem of the Beibu Gulf, China, over autumn and winter, and explored the factors influencing seasonal changes in ARG and MGE abundance and diversity. Results indicated that ARG abundance and diversity were higher in winter than in autumn, with beta-lactam and multidrug resistance genes being the most diverse and abundant, respectively. Similarly, MGE abundance and diversity increased in winter and were strongly correlated with ARGs. In contrast, more pronounced associations between microbial communities, especially archaea, and the antibiotic resistome were observed in autumn than in winter. The co-occurrence network identified multiple interactions between MGEs and various multidrug efflux pumps in winter, suggesting a potential for ARG dissemination. Multivariate correlation analyses and path modeling indicated that environmental factors driving microbial community changes predominantly influenced antibiotic resistome assembly in autumn, while the relative importance of MGEs increased significantly in winter. These findings suggest an elevated health risk associated with antimicrobial resistance in the Beibu Gulf during winter, attributed to the dissemination of ARGs by horizontal gene transfer. The observed seasonal variations highlight the dynamic nature of antibiotic resistance dissemination in coastal ecosystems, emphasizing the need for comprehensive surveillance and management measures to address the growing threat of antimicrobial resistance in vulnerable environments.

An overview of the occurrence, impact of process parameters, and the fate of antibiotic resistance genes during anaerobic digestion processes.

Antibiotic resistance genes (ARGs) have emerged as a significant global health threat, contributing to fatalities worldwide. Wastewater treatment plants (WWTPs) and livestock farms serve as primary reservoirs for these genes due to the limited efficacy of existing treatment methods and microbial adaptation to environmental stressors. Anaerobic digestion (AD) stands as a prevalent biological treatment for managing sewage sludge and manure in these settings. Given the agricultural utility of AD digestate as biofertilizers, understanding ARGs' fate within AD processes is essential to devise effective mitigation strategies. However, understanding the impact of various factors on ARGs occurrence, dissemination, and fate remains limited. This review article explores various AD treatment parameters and correlates to various resistance mechanisms and hotspots of ARGs in the environment. It further evaluates the dissemination and occurrence of ARGs in AD feedstocks and provides a comprehensive understanding of the fate of ARGs in AD systems. This review explores the influence of key AD parameters such as feedstock properties, pretreatments, additives, and operational strategies on ARGs. Results show that properties such as high solid content and optimum co-digestion ratios can enhance ARG removal, while the presence of heavy metals, microplastics, and antibiotics could elevate ARG abundance. Also, operational enhancements, such as employing two-stage digestion, have shown promise in improving ARG removal. However, certain pretreatment methods, like thermal hydrolysis, may exhibit a rebounding effect on ARG levels. Overall, this review systematically addresses current challenges and offers future perspectives associated with the fate of ARGs in AD systems.

Dynamics of antibiotic resistance genes and the association with bacterial community during pig manure composting with chitin and glucosamine addition.

In modern ecological systems, the overuse and misuse of antibiotics have escalated the prevalence of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs), positioning them as emerging environmental contaminants. Notably, composting serves as a sustainable method to recycle agricultural waste into nutrient-rich fertilizer while potentially reducing ARGs and MGEs. This study conducted a 47-day composting experiment using pig manure and corn straw, supplemented with chitin and N-Acetyl-D-glucosamine, to explore the impact of these additives on the dynamics of ARGs and MGEs, and to unravel the interplay between these genetic elements and microbial communities in pig manure composting. Results showed that adding 5% chitin into composting significantly postponed thermophilic phase, yet enhanced the removal efficiency of total ARGs and MGEs by over 20% compared to the control. Additionally, the addition of N-Acetyl-D-glucosamine significantly increased the abundance of tetracycline-resistant and sulfonamide-resistant genes, as well as MGEs. High-throughput sequencing revealed that N-Acetyl-D-glucosamine enhanced bacterial α-diversity, providing diverse hosts for ARGs and MGEs. Resistance mechanisms, predominantly efflux pumps and antibiotic deactivation, played a pivotal role in shaping the resistome of composting process. Co-occurrence network analysis identified the key bacterial phyla Proteobacteria, Firmicutes, Gemmatimonadota, and Myxococcota in ARGs and MGEs transformation and dissemination. Redundancy analysis indicated that physicochemical factors, particularly the carbon-to-nitrogen ratio emerged as critical variables influencing ARGs and MGEs. The findings lay a foundation for the developing microbial regulation method to reduce the risks of ARGs in animal manure composts.

Structure and dispersion of the conjugative mobilome in surface ocean bacterioplankton.

Mobile genetic elements (MGEs), collectively referred to as the "mobilome", can have a significant impact on the fitness of microbial communities and therefore on ecological processes. Marine MGEs have mainly been associated with wide geographical and phylogenetic dispersal of adaptative traits. However, whether the structure of this mobilome exhibits deterministic patterns in the natural community is still an open question. The aim of this study was to characterize the structure of the conjugative mobilome in the ocean surface bacterioplankton by searching the publicly available marine metagenomes from the TARA Oceans survey, together with molecular markers, such as relaxases and type IV coupling proteins of the type IV secretion system (T4SS). The T4SS machinery was retrieved in more abundance than relaxases in the surface marine bacterioplankton. Moreover, among the identified MGEs, mobilizable elements were the most abundant, outnumbering self-conjugative sequences. Detection of a high number of incomplete T4SSs provides insight into possible strategies related to trans-acting activity between MGEs, and accessory functions of the T4SS (e.g. protein secretion), allowing the host to maintain a lower metabolic burden in the highly dynamic marine system. Additionally, the results demonstrate a wide geographical dispersion of MGEs throughout oceanic regions, while the Southern Ocean appears segregated from other regions. The marine mobilome also showed a high similarity of functions present in known plasmid databases. Moreover, cargo genes were mostly related to DNA processing, but scarcely associated with antibiotic resistance. Finally, within the MGEs, integrative and conjugative elements showed wider marine geographic dispersion than plasmids.

The risk of viable but non-culturable (VBNC) enterococci and antibiotic resistance transmission during simulated municipal sludge composting.

Sludge composting is a sludge resource utilization method that can reduce pollutants, such as pathogens. Enterococci are regarded as more reliable and conservative indicators of pathogen inactivation than fecal coliforms, which are typically used as indicators of fecal pollution. Non-spore pathogenic bacteria may enter a viable but non-culturable (VBNC) state during composting, leading to residual risk. The VBNC status of bacteria is related to their survival during composting. However, the survival mechanisms of enterococci during sludge composting remain unclear. Therefore, this study aimed to investigate the VBNC state of enterococci in different phases of simulated sludge composting and the fate of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) during the composting process. This study is expected to provide a basis for subsequent exploration of possible methods to completely inactivate enterococci and reduce ARGs during sludge composting. Culturable enterococci were reduced in the thermophilic phase of sludge composting, but the proportion of VBNC subpopulation increased. It was reported for the first time that most VBNC enterococci were killed by extending the cooling phase of sludge compost, and by prolonging the cooling phase the types of ARG were reduced. However, there was a certain quantity (approximately 104/g dry weight) of culturable and VBNC enterococci in the compost products. In addition, MGEs and ARGs exist in both bacteria and compost products, leading to the risk of spreading antibiotic-resistant bacteria and antibiotic resistance when sludge compost products are used.

Pyrite from acid mine drainage promotes the removal of antibiotic resistance genes and mobile genetic elements in karst watershed with abundant calcium carbonate.

More information is needed to fully comprehend how acid mine drainage (AMD) affects the phototransformation of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in karst water and sewage-irrigated farmland soil with abundant carbonate rocks (CaCO3) due to increasing pollution of AMD formed from pyrite (FeS2). The results showed FeS2 accelerated the inactivation of ARB with an inactivation of 8.7 log. Notably, extracellular and intracellular ARGs and mobile genetic elements (MGEs) also experienced rapid degradation. Additionally, the pH of the solution buffered by CaCO3 significantly influenced the photo-inactivation of ARB. The Fe2+ in neutral solution was present in Fe(II) coordination with strong reducing potential and played a crucial role in generating •OH (7.0 µM), which caused severe damage to ARB, ARGs, and MGEs. The •OH induced by photo-Fenton of FeS2 posed pressure to ARB, promoting oxidative stress response and increasing generation of reactive oxygen species (ROS), ultimately damaging cell membranes, proteins and DNA. Moreover, FeS2 contributed to a decrease in MIC of ARB from 24 mg/L to 4 mg/L. These findings highlight the importance of AMD in influencing karst water and sewage-irrigated farmland soil ecosystems. They are also critical in advancing the utilization of FeS2 to inactivate pathogenic bacteria.

Historical trajectories of antibiotics resistance genes assessed through sedimentary DNA analysis of a subtropical eutrophic lake.

Investigating the occurrence of antibiotic-resistance genes (ARGs) in sedimentary archives provides opportunities for reconstructing the distribution and dissemination of historical (i.e., non-anthropogenic origin) ARGs. Although ARGs in freshwater environments have attracted great attention, historical variations in the diversity and abundance of ARGs over centuries to millennia remain largely unknown. In this study, we investigated the vertical change patterns of bacterial communities, ARGs and mobile genetic elements (MGEs) found in sediments of Lake Chenghai spanning the past 600 years. Within resistome preserved in sediments, 177 ARGs subtypes were found with aminoglycosides and multidrug resistance being the most abundant. The ARG abundance in the upper sediment layers (equivalent to the post-antibiotic era since the 1940s) was lower than those during the pre-antibiotic era, whereas the ARG diversity was higher during the post-antibiotic era, possibly because human-induced lake eutrophication over the recent decades facilitated the spread and proliferation of drug-resistant bacteria. Statistical analysis suggested that MGEs abundance and the bacterial community structure were significantly correlated with the abundance and diversity of ARGs, suggesting that the occurrence and distribution of ARGs may be transferred between different bacteria by MGEs. Our results provide new perspectives on the natural history of ARGs in freshwater environments and are essential for understanding the temporal dynamics and dissemination of ARGs.

Functional Roles and Genomic Impact of Miniature Inverted-Repeat Transposable Elements (MITEs) in Prokaryotes.

Prokaryotic genomes are dynamic tapestries that are strongly influenced by mobile genetic elements (MGEs), including transposons (Tn's), plasmids, and bacteriophages. Of these, miniature inverted-repeat transposable elements (MITEs) are undoubtedly the least studied MGEs in bacteria and archaea. This review explores the diversity and distribution of MITEs in prokaryotes and describes what is known about their functional roles in the host and involvement in genomic plasticity and evolution.

Combining CRISPR/Cas9 and natural excision for the precise and complete removal of mobile genetic elements in bacteria.

Horizontal gene transfer, facilitated by mobile genetic elements (MGEs), is an adaptive evolutionary process that contributes to the evolution of bacterial populations and infectious diseases. A variety of MGEs not only can integrate into the bacterial genome but also can survive or even replicate like plasmids in the cytoplasm, thus requiring precise and complete removal for studying their strategies in benefiting host cells. Existing methods for MGE removal, such as homologous recombination-based deletion and excisionase-based methods, have limitations in effectively eliminating certain MGEs. To overcome these limitations, we developed the Cas9-NE method, which combines the CRISPR/Cas9 system with the natural excision of MGEs. In this approach, a specialized single guide RNA (sgRNA) element is designed with a 20-nucleotide region that pairs with the MGE sequence. This sgRNA is expressed from a plasmid that also carries the Cas9 gene. By utilizing the Cas9-NE method, both the integrative and circular forms of MGEs can be precisely and completely eliminated through Cas9 cleavage, generating MGE-removed cells. We have successfully applied the Cas9-NE method to remove four representative MGEs, including plasmids, prophages, and genomic islands, from Vibrio strains. This new approach not only enables various investigations on MGEs but also has significant implications for the rapid generation of strains for commercial purposes.IMPORTANCEMobile genetic elements (MGEs) are of utmost importance for bacterial adaptation and pathogenicity, existing in various forms and multiple copies within bacterial cells. Integrated MGEs play dual roles in bacterial hosts, enhancing the fitness of the host by delivering cargo genes and potentially modifying the bacterial genome through the integration/excision process. This process can lead to alterations in promoters or coding sequences or even gene disruptions at integration sites, influencing the physiological functions of host bacteria. Here, we developed a new approach called Cas9-NE, allowing them to maintain the natural sequence changes associated with MGE excision. Cas9-NE allows the one-step removal of integrated and circular MGEs, addressing the challenge of eliminating various MGE forms efficiently. This approach simplifies MGE elimination in bacteria, expediting research on MGEs.

Change characteristics, bacteria host, and spread risks of bioaerosol ARGs/MGEs from different stages in sewage and sludge treatment process.

The spread of antibiotic resistance genes (ARGs) in the atmospheric environment has seriously threatened human health. Wastewater treatment plants (WWTPs) are an important source of aerosol ARGs. A large WWTP, including sewage treatment process (SWP) and sludge treatment process (SDP), was selected in North China for sampling in this study. The content of ARGs, mobile genetic elements (MGEs), and bacterial genera in sewage/sludge and aerosols from different process stages was detected. The possible correlation between ARGs/ MGEs and bacteria was analyzed. The risk of antibiotic-resistant bacteria was evaluated and the diffusion of ARGs/MGEs was simulated. The results showed that the concentration of ARGs/MGEs varied as the process progressed, and which in the aeration tank was relatively high. The ARGs/MGEs content in SWP aerosol (8.35-163.27 copies/m3) was higher than that in SDP (5.52-16.36 copies/m3). The main ARGs/MGEs detected in SWP aerosol were tnpA-05, tnpA-04, and ermF, while the main ARGs/MGEs detected in SDP aerosol were sul1, ermF, and blaPAO. ARGs were positively correlated with most bacteria and Escherichia coli with ARGs carries higher cytotoxicity. ARGs/MGEs mainly diffused towards the southeast, which may cause harm to urban residents with the diffusion of aerosols. This study provides clues and theoretical basis for preventing the hazards of ARGs from WWTP sources.

Airborne ARGs/MGEs from two sewage types during the COVID-21: Population, microbe interactions, cytotoxicity, formation mechanism, and dispersion.

During the COVID-2021 epidemic, a large number of antibiotics were used for clinical treatment in hospitals or daily prevention. Sewage from hospital sewage treatment centers (HSTC) and wastewater treatment plants (WWTP) produced a lot of antibiotic-resistance genes/mobile genetic elements (ARGs/MGEs). In this study, the sewage and bioaerosol in the biochemical tank (BT) of an HSTC and a WWTP were sampled throughout the year. The results showed that the average absolute abundance of sewage in BT of WWTP (BTW-W) was higher than sewage in BT of HSTC (BTW-H). Sewage was an important source of microorganisms and ARGs/MGEs in the air of BT. Microorganisms and MGEs were the factors affecting the differences in ARGs/MGEs. Cytotoxicity experiment proved that the cytotoxicity changed from Grade III to Grade IV with the increase in drug-resistant Escherichia coli concentration. According to the formation mechanism formula, the average generation rate of ARGs/MGEs in BT of HSTC was lower than that in WWTP. The diffusion range of ARGs/MGEs of HSTC was larger than that of WWTP. According to the above results, this study found that when people were far away from BT, the health risk of HSTC caused by the diffusion of bioaerosol was higher than WWTP; When people were close to BT, the health risk of WWTP was higher than HSTC due to the aeration of BT. This study provided a basis for public protection of ARGs. In the future, the elimination of airborne ARGs and crowd protection can be further studied in detail.

Analysis of extracellular and intracellular antibiotic resistance genes in commercial organic fertilizers reveals a non-negligible risk posed by extracellular genes.

Livestock manure is known to be a significant reservoir of antibiotic resistance genes (ARGs), posing a major threat to human health and animal safety. ARGs are found in both intracellular and extracellular DNA fractions. However, there has been no comprehensive analysis of these fractions in commercial organic fertilizers (COFs). The present study conducted a systematic survey of the profiles of intracellular ARGs (iARGs) and extracellular ARGs (eARGs) and their contributing factor in COFs in Northern China. Results showed that the ARG diversity in COFs (i.e., 57 iARGs and 53 eARGs) was significantly lower than that in cow dung (i.e., 68 iARGs and 69 eARGs). The total abundance of iARGs and eARGs decreased by 85.7% and 75.8%, respectively, after compost processing, and there were no significant differences between iARGs and eARGs in COFs (P > 0.05). Notably, the relative abundance of Campilobacterota decreased significantly (99.1-100.0%) after composting, while that of Actinobacteriota and Firmicutes increased by 21.1% and 29.7%, respectively, becoming the dominant bacteria in COFs. Co-occurrence analysis showed that microorganisms and mobile genetic elements (MGEs) were more closely related to eARGs than iARGs in COFs. And structural equation models (SEMs) further verified that microbial community was an essential factor regulating iARGs and eARGs variation in COFs, with a direct influence (λ = 0.74 and 0.62, P < 0.01), following by similar effects of MGEs (λ = 0.59 and 0.43, P < 0.05). These findings indicate the need to separate eARGs and iARGs when assessing the risk of dissemination and during removal management in the environment.

Effects of disinfectant type and dosage on biofilm's activity, viability, microbiome and antibiotic resistome in bench-scale drinking water distribution systems.

Drinking water distribution systems (DWDSs) are important for supplying high-quality water to consumers and disinfectant is widely used to control microbial regrowth in DWDSs. However, the disinfectant's influences on microbial community and antibiotic resistome in DWDS biofilms and the underlying mechanisms driving their dynamics remain elusive. The study investigated the effects of chlorine and chloramine disinfection on the microbiome and antibiotic resistome of biofilms in bench-scale DWDSs using metagenomics assembly. Additionally, the biofilm activity and viability were monitored based on adenosine triphosphate (ATP) and flow cytometer (FCM) staining. The results showed that both chlorine and chloramine disinfectants decreased biofilm ATP, although chloramine at a lower dosage (1 mg/L) could increase it. Chloramine caused a greater decrease in living cells than chlorine. Furthermore, the disinfectants significantly lowered the microbial community diversity and altered microbial community structure. Certain bacterial taxa were enriched, such as Mycobacterium, Sphingomonas, Sphingopyxis, Azospira, and Dechloromonas. Pseudomonas aeruginosa exhibited high resistance towards disinfectants. The disinfectants also decreased the complexity of microbial community networks. Some functional taxa (e.g., Nitrospira, Nitrobacter, Nitrosomonas) were identified as keystones in chloramine-treated DWDS microbial ecological networks. Stochasticity drove biofilm microbial community assembly, and disinfectants increased the contributions of stochastic processes. Chlorine had greater promotion effects on antibiotic resistance genes (ARGs), mobile genetic elements (MGEs) and ARG hosts than chloramine. The disinfectants also selected pathogens, such as Acinetobacter baumannii and Klebsiella pneumonia, and these pathogens also harbored ARGs and MGEs. Overall, this study provides new insights into the effects of disinfectants on biofilm microbiome and antibiotic resistome, highlighting the importance of monitoring and managing disinfection practices in DWDSs.

Metagenome sequencing to unveil the occurrence and distribution of antibiotic resistome and in a wastewater treatment plant.

The emergence and persistence of antibiotic resistance genes (ARGs) in wastewater treatment plants (WWTPs) has aroused growing public concern for its risk to human health and ecological safety. Moreover, heavy metals concentrated in sewage and sludge could potentially favour co-selection of ARGs and heavy metal resistance genes (HMRGs). In this study, the profile and abundance of antibiotic and metal resistance genes in influent, sludge and effluent were characterized based on the Structured ARG Datebase (SARG) and Antibacterial Biocide and Metal Resistance Gene Datebase (BacMet) by metagenomic analysis. Sequences were aligning against the INTEGRALL, ISFinder, ICEberg and NCBI RefSeq databases to obtain the diversity and abundance of mobile genetic elements (MGEs, e.g.plasmid and transposon). Among them, 20 types of ARGs and 16 types of HMRG were detected in all samples, the influent metagenomes contained many more resistance genes (both ARGs and HMRGs) than the sludge and the influent sample, large reductions in the relatively abundance and diversity of ARG were achieved by biological treatment. ARGs and HMRGs cannot be completely eliminated during the oxidation ditch. A total of 32 species of the potential pathogens were detected, relative abundances of pathogens had no obvious changes. It is suggested that more specific treatments are required to limit their proliferation in the environment. This study can be helpful for further understanding the removal of antibiotic resistance genes in the sewage treatment process via metagenomic sequencing.

Antibiotic resistance gene distribution in Shine Muscat grapes and health risk assessment of streptomycin residues in mice.

Antibiotic residues and antibiotic resistance genes (ARGs) in fruits and vegetables pose public health risks via the food chain, attracting increased attention. Antibiotics such as streptomycin, used directly on seedless grapes or introduced into vineyard soil through organic fertilizers. However, extensive data supporting the risk assessment of antibiotic residues and resistance in these produce remains lacking. Utilizing metagenomic sequencing, we characterized Shine Muscat grape antibiotic resistome and mobile genetic elements (MGEs). Abundant MGEs and ARGs were found in grapes, with 174 ARGs on the grape surface and 32 in the fruit. Furthermore, our data indicated that soil is not the primary source of these MGEs and ARGs. Escherichia was identified as an essential carrier and potential transmitter of ARGs. In our previous study, streptomycin residue was identified in grapes. Further short-term exposure experiments in mice revealed no severe physiological or histological damage at several environment-related concentrations. However, with increased exposure, some ARGs levels in mouse gut microbes increased, indicating a potential threat to animal health. Overall, this study provides comprehensive insights into the resistance genome and potential hosts in grapes, supporting the risk assessment of antibiotic resistance in fruits and vegetables.

Distribution and major driving elements of antibiotic resistance genes in the soil-vegetable system under microplastic stress.

Microplastics (MPs) and antibiotic resistance genes (ARGs) are both enriched in soil-vegetable systems as a consequence of the prolonged use of agricultural mulches. MPs can form unique bacterial communities and provide potential hosts for ARGs. Therefore, MPs stress may promote the spread of ARGs from soil to crops. Increasing ARGs pollution in soil-vegetable system. In our research, we investigated the distribution and major driving elements of antibiotic resistance genes in the soil-vegetable system under microplastic stress. The results showed that MPs treatment decreased the relative abundance of ARGs in non-rhizosphere soil. High concentrations of MPs promoted the enrichment of tetracycline antibiotic resistance genes in rhizosphere soil. MPs treatment promoted the enrichment of ARGs and mobile genetic elements (MGEs) in lettuce tissues, and the overall abundance of ARGs in root after 0.5 %, 1 %, and 2 % (w/w, dry weight) polyethylene (PE) administration was considerably higher compared to that in the untreated group (p < 0.05). At the same time, high PE concentrations promoted the spread of sulfa ARGs from root to leaf. MPs also impacted the bacterial communities in the soil-plant system, and the changes in ARGs as well as MGEs in each part of the soil-vegetable system were significantly correlated with the bacterial diversity index (p < 0.05). Correlation analysis and network analysis showed that bacterial communities and MGEs were the main drivers of ARGs variation in soil-lettuce systems.

The mobilome of Lactobacillus crispatus M247 includes two novel genetic elements: Tn7088 coding for a putative bacteriocin and the siphovirus prophage ΦM247.

Lactobacillus crispatus is a member of the vaginal and gastrointestinal human microbiota. Here we determined the complete genome sequence of the probiotic strain M247 combining Nanopore and Illumina technologies. The M247 genome is organized in one circular chromosome of 2 336 109 bp, with a GC content of 37.04 % and 2303 ORFs, of which 1962 could be annotated. Analysis of the M247 mobilome, which accounts for 14 % of the whole genome, revealed the presence of: (i) Tn7088, a novel 14 105 bp long integrative and mobilizable element (IME) containing 16 ORFs; (ii) ΦM247, a novel 42 510 bp long siphovirus prophage containing 52 ORFs; (iii) three clustered regularly interspaced short palindromic repeats (CRISPRs); and (iv) 226 insertion sequences (ISs) belonging to 14 different families. Tn7088 has a modular organization including a mobilization module encoding FtsK homologous proteins and a relaxase, an integration/excision module coding for an integrase and an excisionase, and an adaptation module coding for a class I bacteriocin and homologous to the listeriolysin S (lls) locus of Listeria monocytogenes. Genome-wide homology search analysis showed the presence of Tn7088-like elements in 12 out of 23 L. crispatus complete public genomes. Mobilization and integration/excision modules are essentially conserved, while the adaptation module is variable since it is the target site for the integration of different ISs. Prophage ΦM247 contains genes for phage structural proteins, DNA replication and packaging, lysogenic and lytic cycles. ΦM247-like prophages are present in seven L. crispatus complete genomes, with sequence variability mainly due to the integration of ISs. PCR and sequencing showed that the Tn7088 IME excises from the M247 chromosome producing a circular form at a concentration of 4.32×10-5 copies per chromosome, and reconstitution of the Tn7088 chromosomal target site occurred at 6.65×10-4 copies per chromosome. The ΦM247 prophage produces an excised form and a reconstituted target site at a level of 3.90×10-5 and 2.48×10-5 copies per chromosome, respectively. This study identified two novel genetic elements in L. crispatus. Tn7088 represents the first example of an IME carrying a biosynthetic gene cluster for a class I bacteriocin in L. crispatus.