Fate of Antibiotic Resistant Pseudomonas putida and Broad Host Range Plasmid in Natural Soil Microcosms. - Related Documents




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752901.0000Fate of Antibiotic Resistant Pseudomonas putida and Broad Host Range Plasmid in Natural Soil Microcosms. Plasmid conjugation is one of the dominant mechanisms of horizontal gene transfer, playing a noticeable role in the rapid spread of antibiotic resistance genes (ARGs). Broad host range plasmids are known to transfer to diverse bacteria in extracted soil bacterial communities when evaluated by filter mating incubation. However, the persistence and dissemination of broad range plasmid in natural soil has not been well studied. In this study, Pseudomonas putida with a conjugative antibiotic resistance plasmid RP4 was inoculated into a soil microcosm, the fate and persistence of P. putida and RP4 were monitored by quantitative PCR. The concentrations of P. putida and RP4 both rapidly decreased within 15-day incubation. P. putida then decayed at a significantly lower rate during subsequent incubation, however, no further decay of RP4 was observed, resulting in an elevated RP4/P. putida ratio (up to 10) after 75-day incubation, which implied potential transfer of RP4 to soil microbiota. We further sorted RP4 recipient bacteria from the soil microcosms by fluorescence-activated cell sorting. Spread of RP4 increased during 75-day microcosm operation and was estimated at around 10(-4) transconjugants per recipient at the end of incubation. Analysis of 16S rRNA gene sequences of transconjugants showed that host bacteria of RP4 were affiliated to more than 15 phyla, with increased diversity and shift in the composition of host bacteria. Proteobacteria was the most dominant phylum in the transconjugant pools. Transient transfer of RP4 to some host bacteria was observed. These results emphasize the prolonged persistence of P. putida and RP4 in natural soil microcosms, and highlight the potential risks of increased spread potential of plasmid and broader range of host bacteria in disseminating ARGs in soil.201930881351
753010.9999Enhancing control of multidrug-resistant plasmid and its host community with a prolonged thermophilic phase during composting. The plasmid-mediated horizontal transfer of antibiotic resistance genes (ARGs) among bacteria facilitates the evolution and dissemination of antibiotic resistance. Broad-host-range plasmids can be transferred to different bacterial hosts in soil, plant rhizospheres, and wastewater treatment plants. Although composting is an effective way to convert organic waste into fertilizer and reduce some ARGs, few studies have focused on its effects on the spread of ARG-carrying plasmids and their bacterial host communities during composting. In this study, a fluorescently labeled Pseudomonas putida (P. putida) harboring a broad-host-range plasmid RP4 carrying three ARGs was inoculated into a raw material microcosm and composted with different durations of the thermophilic phase. The fate of the donor and RP4 in composting was investigated. The prolonged thermophilic composting removed 95.1% of dsRed and 98.0% of gfp, and it inhibited the rebound of P. putida and RP4 during the maturation phase. The spread potential of RP4 decreased from 10(-4) to 10(-6) transconjugants per recipient after composting. In addition, we sorted and analyzed the composition of RP4 recipient bacteria using fluorescence-activated cell sorting combined with 16S rRNA gene amplicon sequencing. The recipient bacteria of RP4 belonged to eight phyla, and Firmicutes, accounting for 75.3%-90.1%, was the dominant phylum in the transconjugants. The diversity and richness of the RP4 recipient community were significantly reduced by prolonged thermophilic periods. Overall, these findings provide new insights for assessing the contribution of composting in mitigating the dissemination of plasmid-mediated ARGs, and the prolonged thermophilic phase of composting can limit the transfer of multidrug-resistant plasmids.202236060751
384520.9999A novel microfluidic system enables visualization and analysis of antibiotic resistance gene transfer to activated sludge bacteria in biofilm. Antibiotic resistance genes (ARGs) in environment have become a growing public concern, due to their potential to be obtained by pathogens and their duplication along cell division. Horizontal gene transfer (HGT) was reported to be responsible for ARGs dissemination in microbes, but the HGT feature in environmental biofilm was still unclear due to insufficient assay tools. To address this challenge, we applied a novel microfluidic system to cultivate thin biofilm by continuous supply of nutrients and close contact between cells. Resembling the living state of biofilm in open environment, this chip visualized the transfer of ARG-encoded plasmids RP4 and pKJK5 to the receptors, e.g., activated sludge bacteria. The average plasmid transfer frequency per receptor (T/R) from RP4-hosted Pseudomonas putida KT2440 to activated sludge bacteria was quantified to be 2.5 × 10(-3) via flow cytometry, and T/R for pKJK5-hosted Escherichia coli MG1655 was 8.9 × 10(-3), while the corresponding average frequencies per donor (T/D) were diverse for the two host strains as 4.3 × 10(-3) and 1.4 × 10(-1) respectively. The difference between T/R and T/D was explained by the plasmid transfer kinetics, implying specific purposes of the two calculations. Finally, we collected the transconjugants by fluorescent activated cell sorting and further sequenced their 16S rDNA. Bacteria from phyla Proteobacteria and Firmicutes were found more susceptible to be transconjugants than those from Bacteroidetes. Our work demonstrated that microfluidic system was advantageous in biofilm HGT study, which can provide more insights into environmental ARG control.201829909325
752830.9998Horizontal transfer of antibiotic resistance genes in a membrane bioreactor. Growing attention has been paid to the dissemination of antibiotic resistance genes (ARGs) in wastewater microbial communities. The application of membrane bioreactors (MBRs) in wastewater treatment is becoming increasingly widespread. We hypothesized that the transfer of ARGs among bacteria could occur in MBRs, which combine a high density of bacterial cells, biofilms, and antibiotic resistance bacteria or ARGs. In this study, the transfer discipline and dissemination of the RP4 plasmid in MBRs were investigated by the counting plate method, the MIDI microorganism identification system, and quantitative polymerase chain reaction (qPCR) techniques. The results showed that the average transfer frequency of the RP4 plasmid from the donor strain to cultivable bacteria in activated sludge was 2.76×10⁻⁵ per recipient, which was greater than the transfer frequency in wastewater and bacterial sludge reported previously. In addition, many bacterial species in the activated sludge had received RP4 by horizontal transfer, while the genera of Shewanella spp., Photobacterium spp., Pseudomonas spp., Proteus spp., and Vibrio spp. were more likely to acquire this plasmid. Interestingly, the abundance of the RP4 plasmid in total DNA remained at high levels and relatively stable at 10⁴ copies/mg of biosolids, suggesting that ARGs were transferred from donor strains to activated sludge bacteria in our study. Thus, the presence of ARGs in sewage sludge poses a potential health threat.201323942379
739940.9998Aquatic animals promote antibiotic resistance gene dissemination in water via conjugation: Role of different regions within the zebra fish intestinal tract, and impact on fish intestinal microbiota. The aqueous environment is one of many reservoirs of antibiotic resistance genes (ARGs). Fish, as important aquatic animals which possess ideal intestinal niches for bacteria to grow and multiply, may ingest antibiotic resistance bacteria from aqueous environment. The fish gut would be a suitable environment for conjugal gene transfer including those encoding antibiotic resistance. However, little is known in relation to the impact of ingested ARGs or antibiotic resistance bacteria (ARB) on gut microbiota. Here, we applied the cultivation method, qPCR, nuclear molecular genetic marker and 16S rDNA amplicon sequencing technologies to develop a plasmid-mediated ARG transfer model of zebrafish. Furthermore, we aimed to investigate the dissemination of ARGs in microbial communities of zebrafish guts after donors carrying self-transferring plasmids that encode ARGs were introduced in aquaria. On average, 15% of faecal bacteria obtained ARGs through RP4-mediated conjugal transfer. The hindgut was the most important intestinal region supporting ARG dissemination, with concentrations of donor and transconjugant cells almost 25 times higher than those of other intestinal segments. Furthermore, in the hindgut where conjugal transfer occurred most actively, there was remarkable upregulation of the mRNA expression of the RP4 plasmid regulatory genes, trbBp and trfAp. Exogenous bacteria seem to alter bacterial communities by increasing Escherichia and Bacteroides species, while decreasing Aeromonas compared with control groups. We identified the composition of transconjugants and abundance of both cultivable and uncultivable bacteria (the latter accounted for 90.4%-97.2% of total transconjugants). Our study suggests that aquatic animal guts contribute to the spread of ARGs in water environments.201728742284
752050.9998Antibiotic Resistance Gene-Carrying Plasmid Spreads into the Plant Endophytic Bacteria using Soil Bacteria as Carriers. Applications of animal manure and treated wastewater could enrich antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in the plant microbiome. However, the mechanistic studies of the transmission of ARB and ARGs from the environment to plant endophytic bacteria were few. Herein, a genetically engineered fluorescent Escherichia coli harboring a conjugative RP4 plasmid that carries three ARGs was used to trace its spread into Arabidopsis thaliana interior in a tetracycline-amended hydroponic system in the absence or presence of a simulated soil bacterial community. Confocal microscope observation demonstrated that E. coli was internalized into plant tissues and the carried RP4 plasmid was transferred into plant endophytic bacteria. More importantly, we observed that soil bacteria inhibited the internalization of E. coli but substantially promoted RP4 plasmid spread into the plant microbiome. The altered RP4-carrying bacterial community composition in the plant microbiome and the increased core-shared RP4-carrying bacteria number between plant interior and exterior in the presence of soil bacteria collectively confirmed that soil bacteria, especially Proteobacteria, might capture RP4 from E. coli and then translocate into plant microbiome, resulting in the increased RP4 plasmid spread in the plant endophytes. Overall, our findings provided important insights into the dissemination of ARB and ARGs from the environment to the plant microbiome.202134114802
796560.9998Screening and evaluation of heavy metals facilitating antibiotic resistance gene transfer in a sludge bacterial community. Recent growing evidence suggests that heavy metals can stimulate the transfer of antibiotic resistance genes (ARGs) between bacteria. However, most previous studies focused on pure strains, the effect of heavy metals on ARG transfer in bacterial communities, especially in activated sludge, has not been clearly explored. In this study, a high-throughput method, combining computerized incubator (Bioscreen C) and flow cytometry, was developed to evaluate different concentrations of heavy metals influencing ARG transfer in sludge bacteria communities. By using Escherichia coli MG1655 as the donor of broad-host range IncP-1 plasmid pKJK5, it was found that 0.5 mmol/L Pb, 0.1 mmol/L As and 0.005 mmol/L Hg could obviously promote ARG transfer in sludge bacteria communities. Furthermore, mating assays on microfluidic chips also proved higher transfer frequencies in attached communities under the above heavy metal stresses. Transconjugants under Pb, As and Hg stresses were isolated and phylogenetically described. For As and Hg, the dominant genus was Pseudomonas, accounting for 88% and 96%, respectively. While under Pb stress, the genera Aeromonas and Enterobacter were the main transconjugants, accounting for 56% and 32% respectively. Moreover, ABC transporters and Amino acid metabolism, which were related to heavy metal transport and cellular metabolism, were dominant in the prediction of microbial metabolic function of transconjugants. This study can be helpful for risk assessment and control of ARG spreading in WWTPs.201931425984
739870.9998Spread of chloramphenicol and tetracycline resistance genes by plasmid mobilization in agricultural soil. Spread of antibiotic resistance genes (ARGs) poses a worldwide threat to public health and food safety. However, ARG spread by plasmid mobilization, a broad host range transfer system, in agricultural soil has received little attention. Here, we investigated the spread of chloramphenicol resistance gene (CRG) and tetracycline resistance gene (TRG) in agricultural soil by mobilization of pSUP106 under different conditions, including different concentrations of nutrients, temperatures, soil depths, rhizosphere soils, and soil types. The number of resistant bacteria isolated in non-sterilized soil from the experiments was approximately 10(4) to 10(7) per gram of soil, belonging to 5-10 species from four genera, including nonpathogen, opportunistic pathogen, pathogen bacteria, and gram-positive and gram-negative bacteria, depending on the experiment conditions. In sterilized soil, higher levels of nutrients and higher temperatures promoted plasmid mobilization and ARG expression. Topsoil and deep soil might not support the spread of antibiotic resistance, while ARG dissemination by plasmid mobilization was better supported by maize rhizosphere and loam soils. All these factors might change bacterial growth and the activity of bacteria and lead to the above influence. Introduction of only the donor and helper, or the donor alone also resulted in the transfer of ARGs and large numbers of antibiotic resistant bacteria (ARB), indicating that some indigenous bacteria contain the elements necessary for plasmid mobilization. Our results showed that plasmid mobilization facilitated dissemination of ARGs and ARB in soil, which led to the disturbance of indigenous bacterial communities. It is important to clear ARG dissemination routes and inhibit the spread of ARGs.202031991360
384480.9998Effects of Nutrient Level and Growth Rate on the Conjugation Process That Transfers Mobile Antibiotic Resistance Genes in Continuous Cultures. Bacteria in the effluent of wastewater treatment plants (WWTPs) can transfer antibiotic resistance genes (ARGs) to the bacteria in receiving water through conjugation; however, there is a lack of quantitative assessment of this phenomenon in continuous cultures. Our objective was to determine the effects of background nutrient levels in river water column and growth rates of bacteria on the conjugation frequency of ARGs from effluent bacteria to river bacteria, as well as on the resulting resistance level (i.e., MICs) of the river bacteria. Chemostats were employed to simulate the discharge points of WWTPs into rivers, where effluent bacteria (donor cells) meet river bacteria (recipient cells). Both donor and recipient cells were Escherichia coli cells, and the donor cells were constructed by filter mating with bacteria in the effluent of a local WWTP. Results showed that higher bacterial growth rate (0.45 h(-1) versus 0.15 h(-1)) led to higher conjugation frequencies (10(-4) versus 10(-6) transconjugant per recipient). The nutrient level also significantly affected the conjugation frequency, albeit to a lesser extent than the growth rate. The MIC against tetracycline increased from 2 mg/L in the recipient to 64 to 128 mg/L in transconjugants. In comparison, the MIC only increased to as high as 8 mg/L in mutants. Whole-genome sequencing showed that the tet-containing plasmid in both the donor and the transconjugant cells also occur in other fecal bacterial genera. The quantitative information obtained from this study can inform hazard identification related to the proliferation of wastewater-associated ARGs in surface water. IMPORTANCE WWTPs have been regarded as an important hot spot of ARGs. The discharge point of WWTP effluent, where ARGs may be horizontally transferred from bacteria of treated wastewater to bacteria of receiving water, is an important interface between the human-dominated ecosystem and the natural environment. The use of batch cultures in previous studies cannot adequately simulate the nutrient conditions and growth rates in receiving water. In this study, chemostats were employed to simulate the continuous growth of bacteria in receiving water. Furthermore, the experimental setup allowed for separate investigations on the effects of nutrient levels (i.e., simulating background nutrients in river water) and bacterial growth rates on conjugation frequencies and resulting resistance levels. The study generates statistically sound ecological data that can be used to estimate the risk of wastewater-originated ARGs as part of the One Health framework.202236094214
796690.9998How heavy metal stress promotes dissemination of antibiotic resistance genes in the activated sludge process. Heavy metals have been recently revealed as promoters to antibiotic resistance gene (ARG) dissemination in water environment, but their influence on ARG transfer in the activated sludge process has not been clear. In this study, a set of sequencing batch reactors (SBRs) and micro-scale microfluidic chips were established to quantify the impacts of heavy metals (0.5 mM of Pb, 0.1 mM of As, and 0.005 mM of Hg) on the ARG spreading in the activated sludge consortium. Under heavy metal stress, transfer frequencies were 1.7-3.6 folds increase compared to the control. Gram-negative bacteria increased significantly after heavy metal added, which were more prone to receiving resistant plasmid from donors. Meanwhile, the relative expression of genes related to conjugation changed in activated sludge, especially the expression of outer membrane protein and oxidative stress regulatory genes increased by 2.9-7.4 folds and 7.8-13.1 folds, respectively. Furthermore, using microfluidic chips, the dynamics of ARG transfer was observed at single cell level under heavy metal pressure. Heavy metals firstly promoted conjugation and then vertical gene transfer played an important part for ARG spreading. The results provided in-depth understanding of the influence of heavy metals on ARG behavior in the environment.202235724617
7201100.9998Evolution of microbial community and drug resistance during enrichment of tetracycline-degrading bacteria. Recently, the microbial degradation of tetracycline has been widely reported. However, its potential risks in treating wastewater containing high concentrations of tetracycline have not been fully evaluated. In this study, the evolution of the microbial community and drug resistance was traced during the enrichment of tetracycline-degrading bacteria. The results showed that some minor compositions such as Shewanella, Bacillus, and Pseudomonas in the seed sludge became the predominant genera in the enrichment cultures when continuously using tetracycline as the sole carbon source, especially some possible pathogenic bacteria increased significantly in this process. The abundances of most TRGs/16S rDNA were increased after enrichment, although the relative abundance of tetA and tetL genes decreased to some extent. From the enrichment culture, 7 predominant tetracycline-degrading strains were isolated, of which TD-1 (Bacillus) and TD-5 (Shewanella) presented high degradation efficiencies (6-day degradation rate > 95%, half-life period of tetracycline ≈ 24 h). In addition, multiple TRGs, mobile genetic elements (MGEs) and even gene cassettes were found in each tetracycline-degrading isolate. The findings suggested that some risks such as the pathogenicity of isolates and the spread of ARGs should be considered when the biodegradation method is used to treat wastewater polluted with high concentrations of tetracycline.201930660087
7400110.9998Investigating the effects of municipal and hospital wastewaters on horizontal gene transfer. Horizontal gene transfer (HGT) plays an important role in the dissemination of antibiotic resistance genes. In sewer systems, human-associated and environmental bacteria are mixed together and exposed to many substances known to increase HGT, including various antibacterial compounds. In wastewaters, those substances are most often detected below concentrations known to induce HGT individually. Still, it is possible that such wastewaters induce HGT, for example via mixture effects. Here, a panel of antibiotics, biocides and other pharmaceuticals was measured in filter-sterilized municipal and hospital wastewater samples from Gothenburg, Sweden. The effects on HGT of the chemical mixtures in these samples were investigated by exposing a complex bacterial donor community together with a GFP-tagged E. coli recipient strain. Recipients that captured sulfonamide resistance-conferring mobile genetic elements (MGEs) from the bacterial community were enumerated and characterized by replicon typing, antibiotic susceptibility testing and long read sequencing. While exposure to municipal wastewater did not result in any detectable change in HGT rates, exposure to hospital wastewater was associated with an increase in the proportion of recipients that acquired sulfonamide resistance but also a drastic decrease in the total number of recipients. Although, concentrations were generally higher in hospital than municipal wastewater, none of the measured substances could individually explain the observed effects of hospital wastewater. The great majority of the MGEs captured were IncN plasmids, and resistance to several antibiotics was co-transferred in most cases. Taken together, the data show no evidence that chemicals present in the studied municipal wastewater induce HGT. Still, the increased relative abundance of transconjugants after exposure to hospital wastewater could have implications for the risks of both emergence and transmission of resistant bacteria.202133631686
6767120.9998Effects of iron mineral adhesion on bacterial conjugation: Interfering the transmission of antibiotic resistance genes through an interfacial process. Bacterial conjugation is one of the most prominent ways for antibiotic resistance genes (ARGs) transmission in the environment. Interfacial interactions between natural colloidal minerals and bacteria can alter the effective contact of bacteria, thereby affecting ARGs conjugation. Understanding the impact of iron minerals, a core component of colloidal minerals, on ARGs conjugation can help assess and intervene in the risk of ARGs transmission. With three selected iron minerals perturbation experiments, it was found that the conjugative transfer of plasmid that carried kanamycin resistance gene was 1.35 - 3.91-fold promoted by low concentrations of iron minerals (i.e., 5 - 100 mg L(-1)), but inhibited at high concentrations (i.e., 1000 - 2000 mg L(-1)) as 0.10 - 0.22-fold. Conjugation occurrence was highly relevant to the number of bacteria adhering per unit mass of mineral, thus switch in the adhesion modes of mineral-bacterial determined whether the conjugate transfer of ARGs was facilitated or inhibited. In addition, a unified model was formularized upon the physicochemical and physiological effects of adhesion on conjugation, and it can be used in estimating the critical inhibitory concentration of different iron minerals on conjugation. Our findings indicate natural colloidal minerals have great potential for applications in preventing the environmental propagation of ARGs through interfacial interactions.202235472548
7193130.9998Plasmid-mediated transfer of antibiotic resistance genes and biofilm formation in a simulated drinking water distribution system under chlorine pressure. The effects of disinfectants and plasmid-based antibiotic resistance genes (ARGs) on the growth of microorganisms and the plasmid-mediated transfer of ARGs in the water and biofilm of the drinking water distribution system under simulated conditions were explored. The heterotrophic plate count of the water in reactors with 0.1 mg/L NaClO and NH(2)Cl was higher than in the control groups. There was no similar phenomenon in biofilm. In the water of reactors containing NaClO, the aphA and bla genes were lower than in the antibiotic resistant bacteria group, while both genes were higher in the water of reactors with NH(2)Cl than in the control group. Chloramine may promote the transfer of ARGs in the water phase. Both genes in the biofilm of the reactors containing chlorine were lower than the control group. Correlation analysis between ARGs and water quality parameters revealed that the copy numbers of the aphA gene were significantly positively correlated with the copy numbers of the bla gene in water and significantly negatively correlated in biofilm (p < 0.05). The results of the sequencing assay showed that bacteria in the biofilm, in the presence of disinfectant, were primarily Gram-negative. 1.0 mg/L chlorine decreased the diversity of the community in the biofilm. The relative abundance of some bacteria that may undergo transfer increased in the biofilm of the reactor containing 0.1 mg/L chlorine.202539617560
7630140.9997Coexistence of silver ion and tetracycline at environmentally relevant concentrations greatly enhanced antibiotic resistance gene development in activated sludge bioreactor. Antibiotic resistance has become a global public health problem. Recently, various environmental pollutants have been reported to induce the proliferation of antibiotic resistance. However, the impact of multiple pollutants (e.g., heavy metals and antibiotics), which more frequently occur in practical environments, is poorly understood. Herein, one widely distributed heavy metal (Ag(+)) and one frequently detected antibiotic (tetracycline) were chosen to investigate their coexisting effect on the proliferation of antibiotic resistance in the activated sludge system. Results show that the co-occurrence of Ag(+) and tetracycline at environmentally relevant concentrations exhibited no distinct inhibition in reactor performances. However, they inhibited the respiratory activity by 42%, destroyed the membrane structure by 218%, and increased membrane permeability by 29% compared with the blank control bioreactor. Moreover, the relative abundances of target antibiotic resistance genes (ARGs) (e.g., tetA, bla(TEM-1), and sulII) in effluent after exposure of coexisting Ag(+) and tetracycline were increased by 92-1983% compared with those in control reactor, which were 1.1-4.3 folds higher than the sum of the sole ones. These were possibly attributed to the enrichments of antibiotic-resistant bacteria. The results would illumine the coexisting effect of heavy metals and antibiotics on the dissemination of ARGs in activated sludge system.202234482077
6768150.9997Biofilm formation mechanisms of mixed antibiotic-resistant bacteria in water: Bacterial interactions and horizontal transfer of antibiotic-resistant plasmids. Over 95 % of bacteria on water supply pipeline surfaces exist in biofilms, which are hotspots for antibiotic resistance gene (ARG) transmission. This study established mixed biofilm culture systems on a metal iron substrate using Escherichia coli: antibiotic-sensitive bacteria (ASB) and antibiotic-resistant bacteria (ARB). The growth rate and extracellular polymeric substances (EPS) content of mixed biofilm surpassed single-species biofilms due to synergistic interactions among different bacteria. However, the composition of mixed biofilms formed by ASB and ARB became unstable after 72 h, linked to reduced polysaccharide proportions in EPS and inter-bacterial competition. The bacterial composition and conjugative transfer frequency of ARGs in mixed biofilms indicate that biofilm formation significantly enhances horizontal transfer of ARGs. Notably, the conjugative transfer frequency of the mixed biofilm formed by two ARB increased 100-fold within five days. In contrast, the conjugative transfer frequency in the mixed biofilm formed by ASB and ARB was unstable; inter-bacterial competition led to plasmid loss associated with horizontal transfer of ARGs, ultimately resulting in biofilm shedding. Furthermore, genes associated with ARG transfer and biofilm growth up-regulated by 1.5 - 6 and 2 - 7 times, respectively, in mixed biofilm. These findings highlight a mutually reinforcing relationship between biofilm formation and horizontal ARG transmission, with significant environmental implications.202539566460
3843160.9997Bacterivorous Ciliate Tetrahymena pyriformis Facilitates vanA Antibiotic Resistance Gene Transfer in Enterococcus faecalis. Background: Wastewater treatment plants (WWTPs) are hotspots for the emergence and spread of antibiotic resistance genes (ARGs). In activated sludge treatment systems, bacterivorous protozoa play a crucial role in biological processes, yet their impact on the horizontal gene transfer in Gram-positive enteric bacteria remains largely unexplored. This study investigated whether the ciliate Tetrahymena pyriformis facilitates the transfer of antibiotic resistance genes between Enterococcus faecalis strains. Methods: Conjugation assays were conducted under laboratory conditions using a vanA-carrying donor and a rifampicin-resistant recipient at an initial bacterial concentration of 10(9) CFU/mL and ciliate density of 10(5) N/mL. Results: Transconjugant numbers peaked at 2 h when experiments started with recipient bacteria harvested in the exponential growth phase, and at 24 h when bacteria were in the stationary phase. In both cases, vanA gene transfer frequency was highest at 24 h (10(-4)-10(-5) CFU/mL), and the presence of energy sources increased gene transfer frequency by one order of magnitude. Conclusions: These findings suggest that ciliate grazing may contribute to vanA gene transfer in WWTP effluents, potentially facilitating its dissemination among permissive bacteria. Given the ecological and public health risks associated with vanA gene persistence in wastewater systems, understanding protozoan-mediated gene transfer is crucial for mitigating the spread of antibiotic resistance in aquatic environments.202540426515
7190170.9997Dynamics of microbial community and tetracycline resistance genes in biological nutrient removal process. The occurrence of antibiotics in wastewater has become a serious concern due to the possible development of antibiotic resistant bacteria in wastewater treatment process. In order to understand the dynamics of microbial community and tetracycline resistance genes in biological nutrient removal (BNR) process, three lab-scale sequencing batch reactors (SBRs) were operated under the stress of tetracycline. Results indicated that microbial community structure was altered, and tetracycline efflux pump genes were enhanced over 150-day operation in the presence of trace tetracycline of 20 and 50 μg L(-1), respectively. Furthermore, when the initial tetracycline concentrations were increased to 2 and 5 mg L(-1), substantial enhancement of tetracycline resistance was observed, accompanied with a sharp shift in microbial community structure. In this study, horizontal gene transfer was found to be the main mechanism for the development of tetracycline resistance genes under the long-terms stress of trace tetracycline. About 90.34% of the observed variations in tetracycline resistance genes could be explained by the dynamics of potential hosts of tetracycline resistance genes and class 1 integron. It should be noticed that the functional bacteria (e.g. Nitrospira, Dechloromonas, Rhodobacter and Candidatus_Accumulibacter) responsible for nutrient removal were positively correlated with tetracycline resistance, which might promote the prevalence of tetracycline resistance during biological wastewater treatment. Consequently, this study provided in-depth insights into the occurrence and prevalence of tetracycline resistance genes and their microbial hosts in BNR process.201930849601
7383180.9997Spread of plasmids carrying antibiotic resistance genes in soil-lettuce-snail food chain. Fertilization can change the composition of antibiotic resistance genes(ARGs) and their host bacteria in agricultural fields, while complex microbial activities help ARGs into crops and transmit them to humans through agricultural products.Therefore, this study constructed a farmland food chain with soil-lettuce-snail as a typical structure, added genetically engineered Pseudomonas fluorescens containing multidrug-resistant plasmid RP4 to track its spread in the farmland food chain, and used different fertilization methods to explore its influence on the spread and diffusion of ARGs and intl1 in the farmland food chain. It was found that exogenous Pseudomonas can enter plants from soil and pass into snails' intestines, and there is horizontal gene transfer phenomenon of RP4 plasmid in bacteria. At different interfaces of the constructed food chain, the addition of exogenous drug-resistant bacteria had different effects on the total abundance of ARGs and intl1. Fertilization, especially manure, not only promoted the spread of Pseudomonas aeruginosa and the transfer of RP4 plasmid levels, but also significantly increased the total abundance of ARGs and intl1 at all interfaces of the constructed food chain. The main ARGs host bacteria in the constructed food chain include Proteobacteria, Bacteroides, and Firmicutes, while Flavobacterium of Bacteroides is the unique potential host bacteria of RP4 plasmid. In conclusion, this study provides a reference for the risk assessment of ARGs transmitted to the human body through the food chain, and has important practical significance to reduce the antibiotic resistance contamination of agricultural products and ensure the safety of vegetable basket.202438700770
6892190.9997Metagenomic deciphers the mobility and bacterial hosts of antibiotic resistance genes under antibiotics and heavy metals co-selection pressures in constructed wetlands. Both antibiotics and heavy metals exert significant selection pressures on antibiotic-resistance genes (ARGs). This study aimed to investigate the co-selection effects of doxycycline (DC) and cadmium (Cd) on ARGs in constructed wetlands (CWs). The results demonstrated that under antibiotic and heavy metal co-selection pressures, single high concentration DC/Cd or double high, relative abundances of metagenomics assembled genomes all reached 55.1%; meanwhile, the average ratio of ARG-containing contigs located on chromosomes was 61.8% (ranging from 50.4% to 70.6%) suggesting a more stable inheritance of ARGs. Antibiotic and heavy metal co-selection in single high concentration DC/Cd or double high groups stimulate the enrichment of ARG host bacteria, which exhibited complex multiple-resistant patterns accompanied by a host-specific pattern. Additionally, the potential transfer abilities of ARGs mediated by plasmids and integrative and conjugative elements (ICEs) were enhanced under single high-concentration DC/Cd or double high stresses. Together, antibiotic and heavy metal co-selection pressures increased occurrence frequencies of ARGs, MGEs, and their combinations and altered structural communities of ARG host bacteria, increasing the risk of the spread of ARGs. This study was helpful in understanding the dissemination of ARGs and simultaneously preventing the spread of heavy metal-resistant bacteria and ARGs under antibiotic and heavy metal co-selection in small- and micro-wetlands.202539848523