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Cell Feb 2024Plasmids are extrachromosomal genetic elements that often encode fitness-enhancing features. However, many bacteria carry "cryptic" plasmids that do not confer clear...
Plasmids are extrachromosomal genetic elements that often encode fitness-enhancing features. However, many bacteria carry "cryptic" plasmids that do not confer clear beneficial functions. We identified one such cryptic plasmid, pBI143, which is ubiquitous across industrialized gut microbiomes and is 14 times as numerous as crAssphage, currently established as the most abundant extrachromosomal genetic element in the human gut. The majority of mutations in pBI143 accumulate in specific positions across thousands of metagenomes, indicating strong purifying selection. pBI143 is monoclonal in most individuals, likely due to the priority effect of the version first acquired, often from one's mother. pBI143 can transfer between Bacteroidales, and although it does not appear to impact bacterial host fitness in vivo, it can transiently acquire additional genetic content. We identified important practical applications of pBI143, including its use in identifying human fecal contamination and its potential as an alternative approach to track human colonic inflammatory states.
Topics: Humans; Bacteria; Bacteroidetes; Feces; Metagenome; Plasmids; Gastrointestinal Tract
PubMed: 38428395
DOI: 10.1016/j.cell.2024.01.039 -
The Science of the Total Environment Aug 2023Although clinical settings play a major role in the current global dissemination of antibiotic resistance, once antibiotic resistance bacteria and genes are released... (Review)
Review
Although clinical settings play a major role in the current global dissemination of antibiotic resistance, once antibiotic resistance bacteria and genes are released into the environment, their fate will be subject to complex ecological processes. One of the processes prevalent in microbial communities - horizontal gene transfer - can largely facilitate the dissemination of antibiotic resistance genes (ARGs) across phylogenetic and ecological boundaries. Especially, plasmid transfer has aroused increasing concern as it has been proved a significant role in promoting ARG dissemination. As a multi-step process, plasmid transfer can be influenced by various factors, among which those stresses caused by environmental pollutants are important elements affecting the plasmid mediated ARG transfer in the environment. In fact, diverse traditional and emerging pollutants are continuously entering the environment nowadays, as evidenced by the global occurrence of pollutants like metals and pharmaceuticals in aquatic and terrestrial systems. It is therefore imperative to understand to what extent and in which way the plasmid mediated ARG dissemination can be influenced by these stresses. Over the past decades, numerous research endeavours have been made to understand the plasmid mediated ARG transfer under various environmental relevant pressures. In this review, progress and challenges of studies on environmental stress regulating plasmid mediated ARG dissemination will be discussed, with specific focus on emerging pollutants like antibiotics and non-antibiotic pharmaceuticals, metals and their nanoparticles, disinfectants and disinfection by-products, as well as the emerging particulate matter like microplastics. Despite the previous efforts, we are still lacking insights into the in situ plasmid transfer under environmental stresses, which can be addressed by future studies considering environmental relevant pollution status and multi-species microbial communities. We believe that future development of standardized high-throughput screening platforms will assist in rapidly identifying which pollutants enhance plasmid transfer and also which ones may block such gene transfer processes.
Topics: Phylogeny; Plastics; Drug Resistance, Microbial; Anti-Bacterial Agents; Genes, Bacterial; Environmental Pollutants; Plasmids; Gene Transfer, Horizontal; Pharmaceutical Preparations
PubMed: 37149187
DOI: 10.1016/j.scitotenv.2023.163870 -
Cold Spring Harbor Protocols Aug 2023This protocol continues a series of methods for the construction of an in-frame gene deletion in strain RN4220. To this end, we describe in this protocol an...
This protocol continues a series of methods for the construction of an in-frame gene deletion in strain RN4220. To this end, we describe in this protocol an allelic-exchange procedure for We have previously described how an allelic-exchange plasmid containing a desired gene deletion (in this case, pIMAY*-Δ) can be constructed and isolated from , then introduced into electrocompetent cells by electroporation. This plasmid contains a temperature-sensitive origin of replication, a counterselectable marker (* gene) and confers chloramphenicol resistance to As a specific example, we present the construction of strain RN4220*Δ from strain RN4220 carrying the pIMAY*-Δ plasmid. The protocol can be easily adapted for the construction of other gene deletions and/or allelic-exchange plasmids.
Topics: Staphylococcus aureus; Plasmids; Sequence Deletion; Gene Deletion
PubMed: 37117017
DOI: 10.1101/pdb.prot107948 -
Nature Communications Dec 2023Antimicrobial resistance is a global health concern; Enterobacterales resistant to third-generation cephalosporins (3GCs) and carbapenems are of the highest priority....
Antimicrobial resistance is a global health concern; Enterobacterales resistant to third-generation cephalosporins (3GCs) and carbapenems are of the highest priority. Here, we conducted genome sequencing and standardized quantitative antimicrobial susceptibility testing of 4,195 isolates of Escherichia coli and Klebsiella pneumoniae resistant to 3GCs and Enterobacterales with reduced meropenem susceptibility collected across Japan. Our analyses provided a complete classification of 3GC resistance mechanisms. Analyses with complete reference plasmids revealed that among the bla extended-spectrum β-lactamase genes, bla was typically encoded in highly similar plasmids. The two major AmpC β-lactamase genes were bla and bla. Long-read sequencing of representative plasmids revealed that approximately 60% and 40% of bla and bla were encoded by such plasmids, respectively. Our analyses identified strains positive for carbapenemase genes but phenotypically susceptible to carbapenems and undetectable by standard antimicrobial susceptibility testing. Systematic long-read sequencing enabled reconstruction of 183 complete plasmid sequences encoding three major carbapenemase genes and elucidation of their geographical distribution stratified by replicon types and species carrying the plasmids and potential plasmid transfer events. Overall, we provide a blueprint for a national genomic surveillance study that integrates standardized quantitative antimicrobial susceptibility testing and characterizes resistance determinants.
Topics: Anti-Bacterial Agents; Drug Resistance, Bacterial; Bacterial Proteins; beta-Lactamases; Escherichia coli; Plasmids; Genomics; Carbapenems; Microbial Sensitivity Tests
PubMed: 38052776
DOI: 10.1038/s41467-023-43516-4 -
Science Bulletin Dec 2023The spread of hypervirulent carbapenem-resistant Klebsiella pneumoniae (Hv-CRKP) is a global health concern. Here, we report the intrahospital colonization and spread of...
The spread of hypervirulent carbapenem-resistant Klebsiella pneumoniae (Hv-CRKP) is a global health concern. Here, we report the intrahospital colonization and spread of Hv-CRKP isolates in a tertiary hospital from 2017 to 2022. Analyses of 90 nonredundant CRKP isolates from 72 patients indicated that Hv-CRKP transferability relies on the dominant ST11-K64 clone. Whole-genome sequencing of 11 representative isolates gave 31 complete plasmid sequences, including 12 KPC-2 resistance carriers and 10 RmpA virulence vehicles. Apart from the binary vehicles, we detected two types of fusion plasmids, favoring the cotransfer of RmpA virulence and KPC-2 resistance. The detection of ancestry/relic plasmids enabled us to establish genetic mechanisms by which rare fusion plasmids form. Unexpectedly, we found a total of five rmpA promoter variants (P-P) exhibiting distinct activities and varying markedly in their geographic distributions. CRISPR/Cas9 manipulation confirmed that an active P-rmpA regulator is a biomarker for the "high-risk" ST11-K64/CRKP clone. These findings suggest clonal spread and clinical evolution of the prevalent ST11-K64/Hv-CRKP clones. Apart from improved public awareness of Hv-CRKP convergence, our findings might benefit the development of surveillance (and/or intervention) strategies for the dominant ST11-K64 lineage of the Hv-CRKP population in healthcare sectors.
Topics: Humans; Klebsiella pneumoniae; Multilocus Sequence Typing; Klebsiella Infections; Plasmids; Carbapenem-Resistant Enterobacteriaceae; Carbapenems
PubMed: 37949739
DOI: 10.1016/j.scib.2023.10.038 -
Genome Medicine Dec 2023Klebsiella pneumoniae is a notorious clinical pathogen and frequently carries various plasmids, which are the main carriers of antimicrobial resistance and virulence...
BACKGROUND
Klebsiella pneumoniae is a notorious clinical pathogen and frequently carries various plasmids, which are the main carriers of antimicrobial resistance and virulence genes. In comparison to self-transmissible conjugative plasmids, mobilizable plasmids have received much less attention due to their defects in conjugative elements. However, the contribution of mobilizable plasmids to the horizontal transfer of antimicrobial resistance genes and virulence genes of K. pneumoniae remains unclear. In this study, the transfer, stability, and cargo genes of the mobilizable plasmids of K. pneumoniae were examined via genetic experiments and genomic analysis.
METHODS
Carbapenem-resistant (CR) plasmid pHSKP2 and multidrug-resistant (MDR) plasmid pHSKP3 of K. pneumoniae HS11286, virulence plasmid pRJF293 of K. pneumoniae RJF293 were employed in conjugation assays to assess the transfer ability of mobilizable plasmids. Mimic mobilizable plasmids and genetically modified plasmids were constructed to confirm the cotransfer models. The plasmid morphology was evaluated through XbaI and S1 nuclease pulsed-field gel electrophoresis and/or complete genome sequencing. Mobilizable plasmid stability in transconjugants was analyzed via serial passage culture. In addition, in silico genome analysis of 3923 plasmids of 1194 completely sequenced K. pneumoniae was performed to investigate the distribution of the conjugative elements, the cargo genes, and the targets of the CRISPR-Cas system. The mobilizable MDR plasmid and virulence plasmid of K. pneumoniae were investigated, which carry oriT but lack other conjugative elements.
RESULTS
Our results showed that mobilizable MDR and virulence plasmids carrying oriT but lacking the relaxase gene were able to cotransfer with a helper conjugative CR plasmid across various Klebsiella and Escherichia coli strains. The transfer and stability of mobilizable plasmids rather than conjugative plasmids were not interfered with by the CRISPR-Cas system of recipient strains. According to the in silico analysis, the mobilizable plasmids carry about twenty percent of acquired antimicrobial resistance genes and more than seventy-five percent of virulence genes in K. pneumoniae.
CONCLUSIONS
Our work observed that a mobilizable MDR or virulence plasmid that carries oriT but lacks the relaxase genes transferred with the helper CR conjugative plasmid and mobilizable plasmids escaped from CRISPR-Cas defence and remained stable in recipients. These results highlight the threats of mobilizable plasmids as vital vehicles in the dissemination of antibiotic resistance and virulence genes in K. pneumoniae.
Topics: Humans; Klebsiella pneumoniae; Anti-Bacterial Agents; Virulence; Drug Resistance, Bacterial; Plasmids; Escherichia coli; Carbapenems; beta-Lactamases
PubMed: 38041146
DOI: 10.1186/s13073-023-01260-w -
Proceedings of the National Academy of... Dec 2023Conjugative plasmids play a key role in the dissemination of antimicrobial resistance (AMR) genes across bacterial pathogens. AMR plasmids are widespread in clinical...
Conjugative plasmids play a key role in the dissemination of antimicrobial resistance (AMR) genes across bacterial pathogens. AMR plasmids are widespread in clinical settings, but their distribution is not random, and certain associations between plasmids and bacterial clones are particularly successful. For example, the globally spread carbapenem resistance plasmid pOXA-48 can use a wide range of enterobacterial species as hosts, but it is usually associated with a small number of specific clones. These successful associations represent an important threat for hospitalized patients. However, knowledge remains limited about the factors determining AMR plasmid distribution in clinically relevant bacteria. Here, we combined in vitro and in vivo experimental approaches to analyze pOXA-48-associated AMR levels and conjugation dynamics in a collection of wild-type enterobacterial strains isolated from hospitalized patients. Our results revealed significant variability in these traits across different bacterial hosts, with spp. strains showing higher pOXA-48-mediated AMR and conjugation frequencies than strains. Using experimentally determined parameters, we developed a simple mathematical model to interrogate the contribution of AMR levels and conjugation permissiveness to plasmid distribution in bacterial communities. The simulations revealed that a small subset of clones, combining high AMR levels and conjugation permissiveness, play a critical role in stabilizing the plasmid in different polyclonal microbial communities. These results help to explain the preferential association of plasmid pOXA-48 with clones in clinical settings. More generally, our study reveals that species- and strain-specific variability in plasmid-associated phenotypes shape AMR evolution in clinically relevant bacterial communities.
Topics: Humans; Anti-Bacterial Agents; Permissiveness; Drug Resistance, Bacterial; Plasmids; Klebsiella pneumoniae; Klebsiella; Escherichia coli; Bacteria
PubMed: 38096417
DOI: 10.1073/pnas.2314135120 -
Theoretical Population Biology Dec 2023Plasmids may carry genes coding for beneficial traits and thus contribute to adaptation of bacterial populations to environmental stress. Conjugative plasmids can...
Plasmids may carry genes coding for beneficial traits and thus contribute to adaptation of bacterial populations to environmental stress. Conjugative plasmids can horizontally transfer between cells, which a priori facilitates the spread of adaptive alleles. However, if the potential recipient cell is already colonized by another incompatible plasmid, successful transfer may be prevented. Competition between plasmids can thus limit horizontal transfer. Previous modeling has indeed shown that evolutionary rescue by a conjugative plasmid is hampered by incompatible resident plasmids in the population. If the rescue plasmid is a mutant variant of the resident plasmid, both plasmids transfer at the same rates. A high conjugation rate then has two, potentially opposing, effects - a direct positive effect on spread of the rescue plasmid and an increase in the fraction of resident plasmid cells. This raises the question whether a high conjugation rate always benefits evolutionary rescue. In this article, we systematically analyze three models of increasing complexity to disentangle the benefits and limits of increasing horizontal gene transfer in the presence of plasmid competition and plasmid costs. We find that the net effect can be positive or negative and that the optimal transfer rate is thus not always the highest one. These results can contribute to our understanding of the many facets of plasmid-driven adaptation and the wide range of transfer rates observed in nature.
Topics: Conjugation, Genetic; Plasmids; Biological Evolution; Gene Transfer, Horizontal; Bacteria
PubMed: 37923145
DOI: 10.1016/j.tpb.2023.10.001 -
Molecular Pharmaceutics Feb 2024Over the past decades, significant progress has been made in utilizing nucleic acids, including DNA and RNA molecules, for therapeutic purposes. For DNA molecules,... (Review)
Review
Over the past decades, significant progress has been made in utilizing nucleic acids, including DNA and RNA molecules, for therapeutic purposes. For DNA molecules, although various DNA delivery systems have been established, viral vector systems are the go-to choice for large-scale commercial applications. However, viral systems have certain disadvantages such as immune response, limited payload capacity, insertional mutagenesis and pre-existing immunity. In contrast, nonviral systems are less immunogenic, not size limited, safer, and easier for manufacturing compared with viral systems. What's more, nonviral DNA vectors have demonstrated their capacity to mediate specific protein expression for diverse therapeutic objectives containing a wide range of diseases such as cancer, rare diseases, neurodegenerative diseases, and infectious diseases, yielding promising therapeutic outcomes. However, exogenous plasmid DNA is prone to degrade and has poor immunogenicity . Thus, various strategies have been developed: (i) designing novel plasmids with special structures, (ii) optimizing plasmid sequences for higher expression, and (iii) developing more efficient nonviral DNA delivery systems. Based on these strategies, many interesting clinical results have been reported. This Review discusses the development of DNA-based nonviral gene therapy, including novel plasmids, nonviral delivery systems, clinical advances, and prospects. These developments hold great potential for enhancing the efficacy and safety of nonviral gene therapy and expanding its applications in the treatment of various diseases.
Topics: Gene Transfer Techniques; Genetic Vectors; Plasmids; Genetic Therapy; DNA
PubMed: 38198640
DOI: 10.1021/acs.molpharmaceut.3c00907 -
Molecular Systems Biology Apr 2024Antimicrobial resistance (AMR) in bacteria is a major public health threat and conjugative plasmids play a key role in the dissemination of AMR genes among bacterial... (Review)
Review
Antimicrobial resistance (AMR) in bacteria is a major public health threat and conjugative plasmids play a key role in the dissemination of AMR genes among bacterial pathogens. Interestingly, the association between AMR plasmids and pathogens is not random and certain associations spread successfully at a global scale. The burst of genome sequencing has increased the resolution of epidemiological programs, broadening our understanding of plasmid distribution in bacterial populations. Despite the immense value of these studies, our ability to predict future plasmid-bacteria associations remains limited. Numerous empirical studies have recently reported systematic patterns in genetic interactions that enable predictability, in a phenomenon known as global epistasis. In this perspective, we argue that global epistasis patterns hold the potential to predict interactions between plasmids and bacterial genomes, thereby facilitating the prediction of future successful associations. To assess the validity of this idea, we use previously published data to identify global epistasis patterns in clinically relevant plasmid-bacteria associations. Furthermore, using simple mechanistic models of antibiotic resistance, we illustrate how global epistasis patterns may allow us to generate new hypotheses on the mechanisms associated with successful plasmid-bacteria associations. Collectively, we aim at illustrating the relevance of exploring global epistasis in the context of plasmid biology.
Topics: Anti-Bacterial Agents; Drug Resistance, Bacterial; Epistasis, Genetic; Plasmids; Genome, Bacterial; Bacteria
PubMed: 38409539
DOI: 10.1038/s44320-024-00012-1