Plasmid-mediated Transfer Research Articles

Simulated Gastric Acid Promotes the Horizontal Transfer of Multidrug Resistance Genes across Bacteria in the Gastrointestinal Tract at Elevated pH Levels.

The assessment of factors that can promote the transmission of antibiotic resistance genes (ARGs) across bacteria in the gastrointestinal tract is in great demand to understand the occurrence of infections related to antibiotic-resistant bacteria (ARB) in humans. However, whether acid-resistant enteric bacteria can promote ARG transmission in gastric fluid under high-pH conditions remains unknown. This study assessed the effects of simulated gastric fluid (SGF) at different pH levels on the RP4 plasmid-mediated conjugative transfer of ARGs. Moreover, transcriptomic analysis, measurement of reactive oxygen species (ROS) levels, assessment of cell membrane permeability, and real-time quantitative assessment of the expression of key genes were performed to identify the underlying mechanisms. The frequency of conjugative transfer was the highest in SGF at pH 4.5. Antidepressant consumption and certain dietary factors further negatively impacted this situation, with 5.66-fold and 4.26-fold increases in the conjugative transfer frequency being noted upon the addition of sertraline and 10% glucose, respectively, compared with that in the control group without any additives. The induction of ROS generation, the activation of cellular antioxidant systems, increases in cell membrane permeability, and the promotion of adhesive pilus formation were factors potentially contributing to the increased transfer frequency. These findings indicate that conjugative transfer could be enhanced under certain circumstances in SGF at elevated pH levels, thereby facilitating ARG transmission in the gastrointestinal tract. IMPORTANCE The low pH of gastric acid kills unwanted microorganisms, in turn affecting their inhabitation in the intestine. Hence, studies on the factors that influence antibiotic resistance gene (ARG) propagation in the gastrointestinal tract and on the underlying mechanisms are limited. In this study, we constructed a conjugative transfer model in the presence of simulated gastric fluid (SGF) and found that SGF could promote the dissemination of ARGs under high-pH conditions. Furthermore, antidepressant consumption and certain dietary factors could negatively impact this situation. Transcriptomic analysis and a reactive oxygen species assay revealed the overproduction of reactive oxygen species as a potential mechanism by which SGF could promote conjugative transfer. This finding can help provide a comprehensive understanding of the bloom of antibiotic-resistant bacteria in the body and create awareness regarding the risk of ARG transmission due to certain diseases or an improper diet and the subsequent decrease in gastric acid levels.

Pheromone effect of estradiol regulates the conjugative transfer of pCF10 carrying antibiotic resistance genes

Horizontal gene transfer (HGT) mediated by conjugative plasmids greatly contributes to bacteria evolution and the transmission of antibiotic resistance genes (ARGs). In addition to the selective pressure imposed by extensive antibiotic use, environmental chemical pollutants facilitate the dissemination of antibiotic resistance, consequently posing a serious threat to the ecological environment. Presently, the majority of studies focus on the effects of environmental compounds on R plasmid-mediated conjugation transfer, and pheromone-inducible conjugation has largely been neglected. In this study, we explored the pheromone effect and potential molecular mechanisms of estradiol in promoting the conjugative transfer of pCF10 plasmid in Enterococcus faecalis. Environmentally relevant concentrations of estradiol significantly increased the conjugative transfer of pCF10 with a maximum frequency of 3.2 × 10-2, up to 3.5-fold change compared to that of control. Exposure to estradiol induced the activation of pheromone signaling cascade by increasing the expression of ccfA. Furthermore, estradiol might directly bind to the pheromone receptor PrgZ and promote pCF10 induction and finally enhance the conjugative transfer of pCF10. These findings cast valuable insights on the roles of estradiol and its homolog in increasing antibiotic resistance and the potential ecological risk.

Evolution of Cytochrome P450 Enzymes and Their Redox Partners in Archaea.

Cytochrome P450 monooxygenases (CYPs/P450s) and their redox partners, ferredoxins, are ubiquitous in organisms. P450s have been studied in biology for over six decades owing to their distinct catalytic activities, including their role in drug metabolism. Ferredoxins are ancient proteins involved in oxidation-reduction reactions, such as transferring electrons to P450s. The evolution and diversification of P450s in various organisms have received little attention and no information is available for archaea. This study is aimed at addressing this research gap. Genome-wide analysis revealed 1204 P450s belonging to 34 P450 families and 112 P450 subfamilies, where some families and subfamilies are expanded in archaea. We also identified 353 ferredoxins belonging to the four types 2Fe-2S, 3Fe-4S, 7Fe-4S and 2[4Fe-4S] in 40 archaeal species. We found that bacteria and archaea shared the CYP109, CYP147 and CYP197 families, as well as several ferredoxin subtypes, and that these genes are co-present on archaeal plasmids and chromosomes, implying the plasmid-mediated lateral transfer of these genes from bacteria to archaea. The absence of ferredoxins and ferredoxin reductases in the P450 operons suggests that the lateral transfer of these genes is independent. We present different scenarios for the evolution and diversification of P450s and ferredoxins in archaea. Based on the phylogenetic analysis and high affinity to diverged P450s, we propose that archaeal P450s could have diverged from CYP109, CYP147 and CYP197. Based on this study's results, we propose that all archaeal P450s are bacterial in origin and that the original archaea had no P450s.

Conjugative RP4 Plasmid-Mediated Transfer of Antibiotic Resistance Genes to Commensal and Multidrug-Resistant Enteric Bacteria In Vitro

Many antibiotic-resistant bacteria carry resistance genes on conjugative plasmids that are transferable to commensals and pathogens. We determined the ability of multiple enteric bacteria to acquire and retransfer a broad-host-range plasmid RP4. We used human-derived commensal Escherichia coli LM715-1 carrying a chromosomal red fluorescent protein gene and green fluorescent protein (GFP)-labeled broad-host-range RP4 plasmid with ampR, tetR, and kanR in in vitro matings to rifampicin-resistant recipients, including Escherichia coli MG1655, Dec5α, Vibrio cholerae, Pseudomonas putida, Pseudomonas aeruginosa, Klebsiella pneumoniae, Citrobacter rodentium, and Salmonella Typhimurium. Transconjugants were quantified on selective media and confirmed using fluorescence microscopy and PCR for the GFP gene. The plasmid was transferred from E. coli LM715-1 to all tested recipients except P. aeruginosa. Transfer frequencies differed between specific donor-recipient pairings (10-2 to 10-8). Secondary retransfer of plasmid from transconjugants to E. coli LM715-1 occurred at frequencies from 10-2 to 10-7. A serial passage plasmid persistence assay showed plasmid loss over time in the absence of antibiotics, indicating that the plasmid imposed a fitness cost to its host, although some plasmid-bearing cells persisted for at least ten transfers. Thus, the RP4 plasmid can transfer to multiple clinically relevant bacterial species without antibiotic selection pressure.

Triadimefon Promoted Plasmid-mediated Conjugative Transfer of Multiple Antibiotic Resistance Genes within Bacterial Genus

The rapid spread of antibiotic resistance genes (ARGs) has been a worldwide threat to the public health, especially via horizontal gene transfer (HGT). Conjugation is one of the major pathways of HGT. Triadimefon (TF) is a broad-spectrum antifungal compound used to control rust and powdery mildew on crops. Nevertheless, it is not known whether TF could affect the conjugation of ARGs and potential molecular mechanisms. Here, the effects of TF on conjugative transfer of RP4 plasmid within Escherichia coli were systematically investigated. The results demonstrated that TF increased the transconjugant number by 1.41–2.21 folds and the frequency of conjugative transfer by 1.49–2.22 folds at concentrations of 0.1–10 mg/L. There was no obvious change in the number of donor and recipient strains in the mating system of intra-genus with TF. The main mechanisms include increasing intracellular reactive oxygen species and membrane permeability. Our findings highlight the promotion effect of TF on ARG conjugation, providing evidence of the risk of non-antibiotic agrochemical use in ARG dissemination.

Non-caloric artificial sweeteners modulate conjugative transfer of multi-drug resistance plasmid in the gut microbiota

ABSTRACT Non-caloric artificial sweeteners have been widely permitted as table sugar substitutes with high intensities of sweetness. They can pass through the intestinal tract without significant metabolization and frequently encounter the gut microbiome, which is composed of diverse bacterial species and is a pool of antibiotic resistance genes (ARGs). However, little is known about whether these sweeteners could accelerate the spread of ARGs in the gut microbiome. Here, we established an in vitro conjugation model by using Escherichia coli that carries chromosome-inserted Tn7 lacIq-pLpp-mCherry and plasmid-encoded gfpmut3b gene as the donor and murine fecal bacteria as the recipient. We found that four commonly used artificial sweeteners (saccharin, sucralose, aspartame, and acesulfame potassium) can increase reactive oxygen species (ROS) production and promote plasmid-mediated conjugative transfer to the gut microbiome. Cell sorting and 16S rRNA gene amplicon sequencing analysis of fecal samples reveal that the tested sweeteners can promote the broad-host-range plasmid permissiveness to both Gram-negative and Gram-positive gut bacteria. The increased plasmid permissiveness was also validated with a human pathogen Klebsiella pneumoniae. Collectively, our study demonstrates that non-caloric artificial sweeteners can induce oxidative stress and boost the plasmid-mediated conjugative transfer of ARGs among the gut microbiota and a human pathogen. Considering the soaring consumption of these sweeteners and the abundance of mobile ARGs in the human gut, our results highlight the necessity of performing a thorough risk assessment of antibiotic resistance associated with the usage of artificial sweeteners as food additives.

Genetic diversity and prevalence of extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae in aquatic environments receiving untreated hospital effluents.

The spread of extended-spectrum beta-lactamase (ESBL)-producing bacteria in the environment has been recognized as a challenge to public health. The aim of the present study was to assess the occurrence of ESBL-producing Escherichia coli and Klebsiella pneumoniae from selected water bodies receiving hospital effluents in Kerala, India. Nearly 69.8% of Enterobacteriaceae isolates were multi-drug resistant by the Kirby-Bauer disc diffusion method. The double disc synergy test was used to detect the ESBL production and the genes responsible for imparting resistance were detected by PCR. Conjugation experiments confirmed the mechanism of plasmid-mediated transfer of resistance. The prevalence of ESBL production in E. coli and K. pneumoniae was 49.2 and 46.8%, respectively. Among the ESBL-encoding genes, blaCTX-M was the most prevalent group followed by blaTEM, blaOXA, blaCMY, and blaSHV. The results suggest that healthcare settings are one of the key contributors to the spread of ESBL-producing bacteria, not only through cross-transmission and ingestion of antibiotics but also through the discharge of waste without a proper treatment, leading to harmful effects on the aquatic environment. The high prevalence of ESBL-producing Enterobacteriaceae with resistance genes in public water bodies even post-treatment poses a serious threat.

Bisphenols Promote the Pheromone-Responsive Plasmid-Mediated Conjugative Transfer of Antibiotic Resistance Genes in Enterococcus faecalis.

The enrichment and spread of antibiotic resistance genes (ARGs) induced by environmental chemical pollution further exacerbated the threat to human health and ecological safety. Several compounds are known to induce R plasmid-mediated conjugation through inducing reactive oxygen species (ROS), increasing cell membrane permeability, enhancing regulatory genes expression, and so forth. Up to now, there has been no substantial breakthrough in the studies of models and related mechanisms. Here, we established a new conjugation model using pheromone-responsive plasmid pCF10 and confirmed that five kinds of bisphenols (BPs) at environmentally relevant concentrations could significantly promote the conjugation of ARGs mediated by plasmid pCF10 in E. faecalis by up to 4.5-fold compared with untreated cells. Using qPCR, gene knockout and UHPLC, we explored the mechanisms behind this phenomenon using bisphenol A (BPA) as a model of BPs and demonstrated that BPA could upregulate the expression of pheromone, promote bacterial aggregation, and even directly activate conjugation as a pheromone instead of producing ROS and enhancing cell membrane permeability. Interestingly, the result of mathematical analysis showed that the pheromone effect of most BPs is more potent than that of synthetic pheromone cCF10. These findings provide new insight into the environmental behavior and biological effect of BPs and provided new method and theory to study on enrichment and spread of ARGs induced by environmental chemical pollution.

Subinhibitory antibiotic concentrations promote the horizontal transfer of plasmid-borne resistance genes from Klebsiellae pneumoniae to Escherichia coli.

Horizontal gene transfer plays an important role in the spread of antibiotic resistance, in which plasmid-mediated conjugation transfer is the most important mechanism. While sub-minimal inhibitory concentrations (sub-MIC) of antibiotics could promote conjugation frequency, the mechanism by which sub-MIC levels of antibiotics affect conjugation frequency is not clear. Here, we used Klebsiella pneumoniae SW1780 carrying the multi-drug resistance plasmid pSW1780-KPC as the donor strain, to investigate the effects of sub-MICs of meropenem (MEM), ciprofloxacin (CIP), cefotaxime (CTX), and amikacin (AK) on conjugational transfer of pSW1780-KPC from SW1780 to Escherichia coli J53. Our results showed that the transfer frequencies increased significantly by treating SW1780 strain with sub-MIC levels of MEM, CIP, CTX and AK. Transfer frequencies at sub-MIC conditions in a Galleria mellonella were significantly higher than in vitro. To investigate gene expression and metabolic effects, RT-qPCR and LC-MS-based metabolome sequencing were performed. Transcript levels of T4SS genes virB1, virB2, virB4, virB8, and conjugation-related genes traB, traK, traE, and traL were significantly upregulated by exposure to sub-MICs of MEM, CIP, CTX, and AK. Metabolome sequencing revealed nine differentially regulated metabolites. Our findings are an early warning for a wide assessment of the roles of sub-MIC levels of antibiotics in the spread of antibiotic resistance.

Removal of antibiotic resistant bacteria, genes and inhibition of plasmid-mediated horizontal transfer by peroxymonosulfate: Efficiency and mechanisms

As the emerging contaminants, antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) have caught considerable research attention, horizontal gene transfer is one of the most primary contributors to antibiotic resistance dissemination. The application of the oxidant peroxymonosulfate (PMS) as a disinfection alternative has been systemically explored. Also, the effects of various process parameters on ARB inactivation and plasmid-mediated horizontal transfer inhibition, initial bacterial concentration, pH value and temperature, were systemically investigated. The changes in cell substances (cell membrane integrity and intracellular reactive oxygen species) were determined by flow cytometry. The results suggested that the inactivation efficiency of Escherichia coli DH5α (donor) and Pseudomonas sp. HLS-6 (recipient) by 0.8 mM PMS treatment reached 4.45 log and 4.15 log in 30 min, respectively. The conjugative transfer didn’t arise after treatment for only 1 min when the dosage of PMS exceeded 0.4 mM. PMS could work in a wide pH and ambient temperature range. It also showed significant removal efficiency on intracellular ARGs and extracellular ARGs. To identify the underlying mechanisms of restraining conjugative transfer, the mRNA expression of conjugative genes, oxidative stress genes, save our souls (SOS) response-related gene, cell outer membrane proteins genes and global regulatory genes were evaluated. The results demonstrated that the expressions of these genes were altered by 0.8 mM PMS treatment. PMS has the advantages of low cost, easy operation and technical applicability. This study illustrated that PMS treatment showed great potential for controlling the antibiotic resistance for wastewater treatment.

Dissemination and prevalence of plasmid-mediated high-level tigecycline resistance gene tet (X4).

With the large-scale use of antibiotics, antibiotic resistant bacteria (ARB) continue to rise, and antibiotic resistance genes (ARGs) are regarded as emerging environmental pollutants. The new tetracycline-class antibiotic, tigecycline is the last resort for treating multidrug-resistant (MDR) bacteria. Plasmid-mediated horizontal transfer enables the sharing of genetic information among different bacteria. The tigecycline resistance gene tet(X) threatens the efficacy of tigecycline, and the adjacent ISCR2 or IS26 are often detected upstream and downstream of the tet(X) gene, which may play a crucial driving role in the transmission of the tet(X) gene. Since the first discovery of the plasmid-mediated high-level tigecycline resistance gene tet(X4) in China in 2019, the tet(X) genes, especially tet(X4), have been reported within various reservoirs worldwide, such as ducks, geese, migratory birds, chickens, pigs, cattle, aquatic animals, agricultural field, meat, and humans. Further, our current researches also mentioned viruses as novel environmental reservoirs of antibiotic resistance, which will probably become a focus of studying the transmission of ARGs. Overall, this article mainly aims to discuss the current status of plasmid-mediated transmission of different tet(X) genes, in particular tet(X4), as environmental pollutants, which will risk to public health for the “One Health” concept.

Assessing the genomic composition, putative ecological relevance and biotechnological potential of plasmids from sponge bacterial symbionts

Plasmid-mediated transfer of genes can have direct consequences in several biological processes within sponge microbial communities. However, very few studies have attempted genomic and functional characterization of plasmids from marine host-associated microbial communities in general and those of sponges in particular. In the present study, we used an endogenous plasmid isolation method to obtain plasmids from bacterial symbionts of the marine sponges Stylissa carteri and Paratetilla sp. and investigated the genomic composition, putative ecological relevance and biotechnological potential of these plasmids. In total, we isolated and characterized three complete plasmids, three plasmid prophages and one incomplete plasmid. Our results highlight the importance of plasmids to transfer relevant genetic traits putatively involved in microbial symbiont adaptation and host–microbe and microbe–microbe interactions. For example, putative genes involved in bacterial response to chemical stress, competition, metabolic versatility and mediation of bacterial colonization and pathogenicity were detected. Genes coding for enzymes and toxins of biotechnological potential were also detected. Most plasmid prophage coding sequences were, however, hypothetical proteins with unknown functions. Overall, this study highlights the ecological relevance of plasmids in the marine sponge microbiome and provides evidence that plasmids of sponge bacterial symbionts may represent an untapped resource of genes of biotechnological interest.

Enhancing 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.

The Spread of Antibiotic Resistance Genes In Vivo Model.

Infections caused by antibiotic-resistant bacteria are a major public health threat. The emergence and spread of antibiotic resistance genes (ARGs) in the environment or clinical setting pose a serious threat to human and animal health worldwide. Horizontal gene transfer (HGT) of ARGs is one of the main reasons for the dissemination of antibiotic resistance in vitro and in vivo environments. There is a consensus on the role of mobile genetic elements (MGEs) in the spread of bacterial resistance. Most drug resistance genes are located on plasmids, and the spread of drug resistance genes among microorganisms through plasmid-mediated conjugation transfer is the most common and effective way for the spread of multidrug resistance. Experimental studies of the processes driving the spread of antibiotic resistance have focused on simple in vitro model systems, but the current in vitro protocols might not correctly reflect the HGT of antibiotic resistance genes in realistic conditions. This calls for better models of how resistance genes transfer and disseminate in vivo. The in vivo model can better mimic the situation that occurs in patients, helping study the situation in more detail. This is crucial to develop innovative strategies to curtail the spread of antibiotic resistance genes in the future. This review aims to give an overview of the mechanisms of the spread of antibiotic resistance genes and then demonstrate the spread of antibiotic resistance genes in the in vivo model. Finally, we discuss the challenges in controlling the spread of antibiotic resistance genes and their potential solutions.

Plasmid-Mediated Transfer of Antibiotic Resistance Genes in Soil.

Due to selective pressure from the widespread use of antibiotics, antibiotic resistance genes (ARGs) are found in human hosts, plants, and animals and virtually all natural environments. Their migration and transmission in different environmental media are often more harmful than antibiotics themselves. ARGs mainly move between different microorganisms through a variety of mobile genetic elements (MGEs), such as plasmids and phages. The soil environment is regarded as the most microbially active biosphere on the Earth’s surface and is closely related to human activities. With the increase in human activity, soils are becoming increasingly contaminated with antibiotics and ARGs. Soil plasmids play an important role in this process. This paper reviews the current scenario of plasmid-mediated migration and transmission of ARGs in natural environments and under different antibiotic selection pressures, summarizes the current methods of plasmid extraction and analysis, and briefly introduces the mechanism of plasmid splice transfer using the F factor as an example. However, as the global spread of drug-resistant bacteria has increased and the knowledge of MGEs improves, the contribution of soil plasmids to resistance gene transmission needs to be further investigated. The prevalence of multidrug-resistant bacteria has also made the effective prevention of the transmission of resistance genes through the plasmid-bacteria pathway a major research priority.

Synergistic effect of sulfidated nanoscale zerovalent iron in donor and recipient bacterial inactivation and gene conjugative transfer inhibition

Antibiotic resistance genes (ARGs) and antibiotic resistant bacteria (ARB) are widespread in urban wastewater treatment plants (UWTPs). In this research, a horizontal transfer model of recipient (Pseudomonas. HLS-6) and donor (Escherichia coli DH5α carries RP4 plasmid) was constructed to explore the effect of sulfidated nanoscale zerovalent iron (S-nZVI) on the efficiency of plasmid-mediated horizontal transfer. When the S/Fe was 0.1, the inactivation efficiency of 1120 mg/L S-nZVI on the donor and recipient bacteria were 2.36 ± 0.03 log and 3.50 ± 0.17 log after 30 min, respectively (initial ARB concentration ≈ 5 ×107 CFU/mL). Effects of treatment time, S/Fe molar ratio, S-nZVI dosage and initial bacterial concentration were systemically studied. S-nZVI treatment could increase the extracellular alkaline phosphatase and malondialdehyde content of the ARB, cause oxidative stress in the bacteria, destroy the cell structure and damage the intracellular DNA. This study provided evidence and insights into possible underlying mechanisms for reducing conjugative transfer, such as hindering cell membrane repair, inducing the overproduction of reactive oxygen species, inhibiting the SOS response, reducing the expression of ARGs and related transfer genes. S-nZVI could inhibit the gene conjugative transfer while inactivating the ARB. The findings provided an alternative method for controlling antibiotic resistance.

Genomic Epidemiology Insights on NDM-Producing Pathogens Revealed the Pivotal Role of Plasmids on blaNDM Transmission.

ABSTRACTIncidences of nosocomial infections mediated by New Delhi metallo-β-lactamase (NDM) enzyme-producing Enterobacterales are increasing globally, resulting in a great burden to public health. The carbapenem-resistant Enterobacterales (CRE) were collected from Henan, China during 2013–2016. The blaNDM-positive strains were characterized using PCR, antimicrobial susceptibility testing, conjugation assay, S1 nuclease pulsed-field gel electrophoresis (S1-PFGE), Southern blot, whole-genome sequencing (WGS), and bioinformatics analysis. Eighty-one NDM-producing strains were identified among 391 nonduplicate CRE strains. Among them, four strains cocarried mcr and blaNDM genes, and two carried blaIMP-4 and blaNDM genes. The coexistence of blaNDM-5 and mcr-9 in Enterobacter hormaechei was found for the first time. In total, four blaNDM subtypes were identified. Among them, blaNDM-1 and blaNDM-5 were predominant. There was an obvious increasing trend in blaNDM-5 from 2013 to 2016. Thirteen different bacterial species were found among the 81 strains, and Escherichia coli was the dominant strain. blaNDM genes were located on nine different Inc-type plasmids, most of them on the IncX3 plasmids, except for the Pr-15-2-50 strain, which was located on the chromosome. We characterized two novel plasmids: the IncHI5-like plasmid carrying blaNDM-9 found in K. pneumonia, and the IncI1 blaNDM-5-positive plasmid. These findings provide the genomic basis for the widespread transmission of blaNDM and pave the way for the formulation of more effective monitoring and control methods.IMPORTANCE To control the emergence and transmission of CRE, it is important to perform retrospective genomic investigations. It is important to evaluate the plasmid diversity, genetic environment, and evolutionary relationships of the blaNDM-positive clinical strains in the early transmission stages. This study conducted an in-depth analysis of blaNDM-positive pathogens during a 4-year period using different methods for observing the high prevalence and active transmission of blaNDM-positive CRE. Moreover, we also explored the coexistence of the blaNDM and mcr, a clinically important mobile colistin resistance gene. This study shows that the prevalence of blaNDM-positive pathogens in Henan is high and the isolation rates increase each year. Moreover, plasmid-mediated horizontal transfer plays an important role in blaNDM dissemination. The co-occurrence of multiple resistance genes highlighted a long-lasting evolutionary pathway. Therefore, we have suggested the long-term continuous surveillance of clinical pathogens carrying blaNDM to learn the future transmission trend and curb the public health risk caused by CRE.

Enrofloxacin Promotes Plasmid-Mediated Conjugation Transfer of Fluoroquinolone-Resistance Gene qnrS

This study aimed to determine the effect of enrofloxacin (ENR) on the transfer of the plasmid-mediated quinolone resistance (PMQR) gene qnrS from opportunistic pathogen Escherichia coli (E2) to Salmonella Enteritidis (SE211) and to analyze the resistance characteristics of SE211-qnrS isolates. The plasmid carrying qnrS gene of E2 was sequenced by Oxford Nanopore technology. The plasmid carrying qnrS gene belonged to incompatibility group IncY. In vitro, the transfer experiment of IncY plasmid was performed by the liquid medium conjugation method. The conjugation transfer frequency of the IncY plasmid was 0.008 ± 0.0006 in the absence of ENR, 0.012 ± 0.003 in 1/32 MICENR, 0.01 ± 0.008 in 1/8 MICENR, and 0.03 ± 0.015 (Mean±SD) in 1/2 MICENR, respectively. After inoculation of E. coli E2 and SE211, chickens were treated with different doses of ENR (3.03, 10, and 50 mg/kg b.w.) for 7 days consecutively. To screen the SE211-qnrS strains from intestinal tract of chickens, the resistance genes and susceptibility of isolates were identified. The amount of E. coli E2 and the copy number of qnrS gene in the chicken intestinal tract were determined by colony counting and qPCR, respectively. In vivo, more SE211-qnrS strains were isolated from the treated group compared with the untreated group. SE211-qnrS strains not only obtained IncY plasmid, but also showed similar resistance phenotype as E2. In conclusion, ENR treatment can promote the spread of a IncY-resistance plasmid carrying the qnrS fluoroquinolone-resistance gene in Escherichia coli and the development of drug-resistant bacteria.

Low-concentration of trichloromethane and dichloroacetonitrile promote the plasmid-mediated horizontal transfer of antibiotic resistance genes

Disinfection by-products (DBPs) are one of the unintended consequences of water disinfection that are commonly detected in various water environments. Although DBPs are known to induce antimicrobial resistance via stimulation of chromosomal mutations, it remains unclear whether low-concentration of DBPs could stimulate the conjugative transfer of antibiotic resistance genes (ARGs). The present study aimed to investigate the effect of two typical DBPs, namely trichloromethane (TCM) and dichloroacetonitrile (DCAN), on the conjugative transfer of RP4 plasmid in Escherichia coli genera. The results of the study demonstrated that exposure to low concentrations of TCM and DCAN significantly stimulated conjugative transfer of ARGs, wherein application of 25 μg/L of TCM and 10 μg/L of DCAN resulted in maximum fold change of ~5.5– and ~6.0–fold, respectively, at 16 h of exposure. Further, assessment of underlying mechanisms revealed the involvement of intracellular reactive oxygen species generation, SOS response, increase in cell membrane permeability, upregulation of expression of genes and proteins related to pilus generation, ATP synthesis, and RP4 gene expression. Our findings provided a better understanding of the hidden biological effects and the ecological risks of DBPs in the water environment, especially concerning their effect on the spread of antibiotic resistance.

Melatonin prevents conjugative transfer of plasmid-mediated antibiotic resistance genes by disrupting proton motive force

The widespread dissemination of antibiotic resistance genes (ARGs) is a serious problem and constitutes a threat for public health. Plasmid-mediated conjugative transfer of ARGs is recognized as one of the most important pathways accounting for this global crisis. Inhibiting the conjugative transfer of resistant gene-bearing plasmids provides a feasible strategy to prevent the spread of antibiotic resistance. Here we found that melatonin, a neurohormone secreted from pineal gland, substantially inhibited the horizontal transfer of RP4–7 plasmid in a dose-dependent manner. Furthermore, melatonin could also suppress the conjugal frequency of different types of clinical plasmids that carrying colistin resistance gene mcr-1 rather than blaNDM or tet(X) genes. Next, we investigated the mechanisms underlying the inhibitory effect of melatonin on conjugation. As a result, we showed that the addition of melatonin markedly reduced bacterial membrane permeability and inhibited the oxidative stress. In line with these observations, the conjugative transfer-related genes were regulated accordingly. Most importantly, we uncovered that melatonin disrupted bacterial proton motive force (PMF), which is an essential bacterial energy metabolism substance and is important for conjugative process. Collectively, these results provide implications that some non-antibiotics such as melatonin are effective inhibitors of transmission of ARGs and raise a promising strategy to confront the increasing resistant infections.