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Journal of Medical Microbiology Dec 2023. Various plasmid-mediated resistance genes have been reported in , but little is known about their global distribution features, evolution pattern and spread.. The...
. Various plasmid-mediated resistance genes have been reported in , but little is known about their global distribution features, evolution pattern and spread.. The potential mobilization mechanisms of resistance plasmids in have been poorly explored.. The aim of the study was to investigate the prevalence and diversity of plasmid-mediated resistance genes among isolates, and focus on the analysis of the features of the resistance plasmids from .. The plasmids tested were sequenced using the Illumina HiSeq platform in conjunction with PCR and inverted PCR. The susceptibility of the host strains was determined by broth microdilution. The transfer of plasmids tested was conducted by electroporation. The sequence data were compared using bioinformatics tools and the data from our laboratory and the National Center for Biotechnology Information (NCBI) database.. Nineteen plasmids were identified from our laboratory and these resistance plasmids were functional and transferable. Moreover, we clustered five types of genetic backbones of plasmids from and revealed the global distribution features of the plasmid-mediated resistance genes.. This is the first report of the coexistence of (H)-bearing type I plasmid and (C)-bearing type II plasmid in one clinical isolate. In addition, this study provides the first view of the global distribution of plasmid-mediated resistance genes and classifies the plasmids in according to their backbone regions.
Topics: Plasmids; Haemophilus parasuis; Base Sequence
PubMed: 38112519
DOI: 10.1099/jmm.0.001767 -
Bioinformatics (Oxford, England) Oct 2023Plasmids are carriers for antimicrobial resistance (AMR) genes and can exchange genetic material with other structures, contributing to the spread of AMR. There is no...
MOTIVATION
Plasmids are carriers for antimicrobial resistance (AMR) genes and can exchange genetic material with other structures, contributing to the spread of AMR. There is no reliable approach to identify the transfer of AMR genes across plasmids. This is mainly due to the absence of a method to assess the phylogenetic distance of plasmids, as they show large DNA sequence variability. Identifying and quantifying such transfer can provide novel insight into the role of small mobile elements and resistant plasmid regions in the spread of AMR.
RESULTS
We developed SHIP, a novel method to quantify plasmid similarity based on the dynamics of plasmid evolution. This allowed us to find conserved fragments containing AMR genes in structurally different and phylogenetically distant plasmids, which is evidence for lateral transfer. Our results show that regions carrying AMR genes are highly mobilizable between plasmids through transposons, integrons, and recombination events, and contribute to the spread of AMR. Identified transferred fragments include a multi-resistant complex class 1 integron in Escherichia coli and Klebsiella pneumoniae, and a region encoding tetracycline resistance transferred through recombination in Enterococcus faecalis.
AVAILABILITY AND IMPLEMENTATION
The code developed in this work is available at https://github.com/AbeelLab/plasmidHGT.
Topics: Anti-Bacterial Agents; Phylogeny; Drug Resistance, Bacterial; Plasmids; Escherichia coli; Integrons; Gene Transfer, Horizontal
PubMed: 37796811
DOI: 10.1093/bioinformatics/btad612 -
Journal of Global Antimicrobial... Dec 2023To characterize the evolution and interspecies transfer of plasmids between Klebsiella pneumoniae and Escherichia coli within a single patient.
The dynamic evolution and IS26-mediated interspecies transfer of a bla-bearing fusion plasmid leading to a hypervirulent carbapenem-resistant Klebsiella pneumoniae strain harbouring bla in a single patient.
OBJECTIVES
To characterize the evolution and interspecies transfer of plasmids between Klebsiella pneumoniae and Escherichia coli within a single patient.
METHODS
Minimum inhibitory concentrations were measured using broth microdilution assays. Conjugation assays, string tests, and Galleria mellonella infection model experiments were also conducted. Whole-genome sequencing was performed on the Illumina and Nanopore platforms. Antimicrobial resistance determinants, insertion sequences, and virulence factors were identified using ABRicate/ResFinder database, ISFinder, and virulence factor database. Wzi and capsular polysaccharide (KL) were typed using Kleborate and Kaptive. Multi-locus sequence typing (MLST), replicon typing, and single nucleotide polymorphism analyses were conducted using the BacWGSTdb server.
RESULTS
The carbapenem-resistant K. pneumoniae 2111KP was characterized as ST11, wzi64, and KL64, with a positive string test result and a relatively high virulence phenotype. Analysis of the 2111KP genome revealed that bla was located in a 268,400-bp IncFIB/IncHI1B/IncX3 conjugative plasmid (p2111KP-1), regulated by IS26, IS5, and ISKox3. p2111KP-1 was also a rmpA2-associated virulence plasmid with an iutA-iucABCD gene cluster and a IS26-mediated multidrug-resistant fusion plasmid, which contained 8-bp (AGCTGCAC or GGCCTTTG) target site duplications. Segments flanked by IS26 of p2111KP-1 were 99.99% identical to a 49,016-bp E. coli plasmid.
CONCLUSIONS
This study provided direct evidence of plasmid fusion via IS26 between two different bacterial species within one patient and revealed the process by which genetic elements conferring carbapenem resistance and virulence were simultaneously transferred between these species. It highlights the need for strategic antibiotic use and rigorous monitoring to prevent the plasmid-mediated fusion and transmission of drug-resistance/virulence factors.
Topics: Humans; Multilocus Sequence Typing; Klebsiella pneumoniae; Escherichia coli; Klebsiella Infections; Plasmids; Carbapenems; Virulence Factors; Bacterial Outer Membrane Proteins; Escherichia coli Proteins
PubMed: 37734657
DOI: 10.1016/j.jgar.2023.08.021 -
International Microbiology : the... Nov 2023Plasmids play a fundamental role in the evolution of bacteria by allowing them to adapt to different environments and acquire, through horizontal transfer, genes that...
Plasmids play a fundamental role in the evolution of bacteria by allowing them to adapt to different environments and acquire, through horizontal transfer, genes that confer resistance to different classes of antibiotics. Using the available in vitro and in silico plasmid typing systems, we analyzed a set of isolates and public genomes of K. variicola to study its plasmid diversity. The resistome, the plasmid multilocus sequence typing (pMLST), and molecular epidemiology using the MLST system were also studied. A high frequency of IncF plasmids from human isolates but lower frequency from plant isolates were found in our strain collection. In silico detection revealed 297 incompatibility (Inc) groups, but the IncFIB (216/297) predominated in plasmids from human and environmental samples, followed by IncFII (89/297) and IncFIA/FIA(HI1) (75/297). These Inc groups were associated with clinically important ESBL (CTX-M-15), carbapenemases (KPC-2 and NDM-1), and colistin-resistant genes which were associated with major sequence types (ST): ST60, ST20, and ST10. In silico MOB typing showed 76% (311/404) of the genomes contained one or more of the six relaxase families with MOB being most abundant. We identified untypeable plasmids carrying bla, bla, and bla but for which a relaxase was found; this may suggest that novel plasmid structures could be emerging in this bacterial species. The plasmid content in K. variicola has limited diversity, predominantly composed of IncFIB plasmids dispersed in different STs. Plasmid detection using the replicon and MOB typing scheme provide a broader context of the plasmids in K. variicola. This study showed that whole-sequence-based typing provides current insights of the prevalence of plasmid types and their association with antimicrobial resistant genes in K. variicola obtained from humans and environmental niches.
Topics: Humans; Multilocus Sequence Typing; Klebsiella; Plasmids; beta-Lactamases; Anti-Bacterial Agents; Klebsiella pneumoniae; Klebsiella Infections; Microbial Sensitivity Tests
PubMed: 36971854
DOI: 10.1007/s10123-023-00346-0 -
ACS Synthetic Biology Dec 2023The soil environment adjacent to plant roots, termed the rhizosphere, is home to a wide variety of microorganisms that can significantly affect the physiology of nearby...
The soil environment adjacent to plant roots, termed the rhizosphere, is home to a wide variety of microorganisms that can significantly affect the physiology of nearby plants. Microbes in the rhizosphere can provide nutrients, secrete signaling compounds, and inhibit pathogens. These processes could be manipulated with synthetic biology to enhance the agricultural performance of crops grown for food, energy, or environmental remediation, if methods can be implemented in these nonmodel microbes. A common first step for domesticating nonmodel organisms is the development of a set of genetic engineering tools, termed a synthetic biology toolbox. A toolbox comprises transformation protocols, replicating vectors, genome engineering (e.g., CRISPR/Cas9), constitutive and inducible promoter systems, and other gene expression control elements. This work validated synthetic biology toolboxes in three nitrogen-fixing soil bacteria: , (), and a new isolate of . All three organisms were amenable to transformation and reporter protein expression, with several functional inducible systems available for each organism. and showed more reliable plasmid-based expression, resulting in successful Cas9 recombineering to create scarless deletions and insertions. Using these tools, we generated mutants with inducible nitrogenase activity and introduced heterologous genes to produce resorcinol products with relevant biological activity in the rhizosphere.
Topics: Nitrogen; Soil; Synthetic Biology; Plasmids; Genetic Engineering; CRISPR-Cas Systems
PubMed: 37988619
DOI: 10.1021/acssynbio.3c00414 -
Applied and Environmental Microbiology Apr 2024It has been nearly a century since the isolation and use of penicillin, heralding the discovery of a wide range of different antibiotics. In addition to clinical...
It has been nearly a century since the isolation and use of penicillin, heralding the discovery of a wide range of different antibiotics. In addition to clinical applications, such antibiotics have been essential laboratory tools, allowing for selection and maintenance of laboratory plasmids that encode cognate resistance genes. However, antibiotic resistance mechanisms can additionally function as public goods. For example, extracellular beta-lactamases produced by resistant cells that subsequently degrade penicillin and related antibiotics allow neighboring plasmid-free susceptible bacteria to survive antibiotic treatment. How such cooperative mechanisms impact selection of plasmids during experiments in laboratory conditions is poorly understood. Here, we show in multiple bacterial species that the use of plasmid-encoded beta-lactamases leads to significant curing of plasmids in surface-grown bacteria. Furthermore, such curing was also evident for aminoglycoside phosphotransferase and tetracycline antiporter resistance mechanisms. Alternatively, antibiotic selection in liquid growth led to more robust plasmid maintenance, although plasmid loss was still observed. The net outcome of such plasmid loss is the generation of a heterogenous population of plasmid-containing and plasmid-free cells, leading to experimental confounds that are not widely appreciated.IMPORTANCEPlasmids are routinely used in microbiology as readouts of cell biology or tools to manipulate cell function. Central to these studies is the assumption that all cells in an experiment contain the plasmid. Plasmid maintenance in a host cell typically depends on a plasmid-encoded antibiotic resistance marker, which provides a selective advantage when the plasmid-containing cell is grown in the presence of antibiotic. Here, we find that growth of plasmid-containing bacteria on a surface and to a lesser extent in liquid culture in the presence of three distinct antibiotic families leads to the evolution of a significant number of plasmid-free cells, which rely on the resistance mechanisms of the plasmid-containing cells. This process generates a heterogenous population of plasmid-free and plasmid-containing bacteria, an outcome which could confound further experimentation.
Topics: Humans; Plasmids; Anti-Bacterial Agents; Bacteria; beta-Lactamases; Penicillins
PubMed: 38446071
DOI: 10.1128/aem.02311-23 -
Soft Matter Aug 2023Bacteria have numerous large dsDNA molecules that freely interact within the cell, including multiple plasmids, primary and secondary chromosomes. The cell membrane...
Bacteria have numerous large dsDNA molecules that freely interact within the cell, including multiple plasmids, primary and secondary chromosomes. The cell membrane maintains a micron-scale confinement, ensuring that the dsDNA species are proximal at all times and interact strongly in a manner influenced by the cell morphology ( whether cell geometry is spherical or anisotropic). These interactions lead to non-uniform spatial organization and complex dynamics, including segregation of plasmid DNA to polar and membrane proximal regions. However, exactly how this organization arises, how it depends on cell morphology and number of interacting dsDNA species are under debate. Here, using an nanofluidic model, featuring a cavity that can be opened and closed , we address how plasmid copy number and confinement geometry alter plasmid spatial distribution and dynamics. We find that increasing the plasmid number alters the plasmid spatial distribution and shortens the plasmid polar dwell time; sharper cavity end curvature leads to longer plasmid dwell times.
Topics: DNA; Plasmids; Anisotropy; Cell Membrane
PubMed: 37599597
DOI: 10.1039/d3sm00491k -
Microbiology Spectrum Aug 2023Plasmids contribute to microbial diversity and adaptation, providing microorganisms with the ability to thrive in a wide range of conditions in extreme environments....
Plasmids contribute to microbial diversity and adaptation, providing microorganisms with the ability to thrive in a wide range of conditions in extreme environments. However, while the number of marine microbiome studies is constantly increasing, very little is known about marine plasmids, and they are very poorly represented in public databases. To extend the repertoire of environmental marine plasmids, we established a pipeline for the assembly of plasmids in the marine environment by analyzing available microbiome metagenomic sequencing data. By applying the pipeline to data from the Red Sea, we identified 362 plasmid candidates. We showed that the distribution of plasmids corresponds to environmental conditions, particularly, depth, temperature, and physical location. At least 7 of the 362 candidates are most probably real plasmids, based on a functional analysis of their open reading frames (ORFs). Only one of the seven has been described previously. Three plasmids were identified in other public marine metagenomic data from different locations all over the world; these plasmids contained different cassettes of functional genes at each location. Analysis of antibiotic and metal resistance genes revealed that the same positions that were enriched with genes encoding resistance to antibiotics were also enriched with resistance to metals, suggesting that plasmids contribute site-dependent phenotypic modules to their ecological niches. Finally, half of the ORFs (50.8%) could not be assigned to a function, emphasizing the untapped potential of the unique marine plasmids to provide proteins with multiple novel functions. Marine plasmids are understudied and hence underrepresented in databases. Plasmid functional annotation and characterization is complicated but, if successful, may provide a pool of novel genes and unknown functions. Newly discovered plasmids and their functional repertoire are potentially valuable tools for predicting the dissemination of antimicrobial resistance, providing vectors for molecular cloning and an understanding of plasmid-bacterial interactions in various environments.
Topics: Indian Ocean; Plasmids; Anti-Bacterial Agents; Metagenomics; Metagenome
PubMed: 37395658
DOI: 10.1128/spectrum.00400-23 -
Microbiology Spectrum Aug 2023Colistin is still commonly used and misused in animal husbandry driving the evolution and dissemination of transmissible plasmid-mediated colistin resistance (). is a...
Colistin is still commonly used and misused in animal husbandry driving the evolution and dissemination of transmissible plasmid-mediated colistin resistance (). is a rare variant and, so far, has only been detected in Escherichia coli obtained from a hospitalized patient in Germany in 2018. Recently, it was also notified in fecal samples from a pigeon in Lebanon. We report on the presence of 16 colistin-resistant, -carrying extended-spectrum beta-lactamase (ESBL)-producing and commensal E. coli isolated from poultry samples in Germany, of which retail meat was the most common source. Short- and long-read genome sequencing and bioinformatic analyses revealed the location of exclusively on IncX4 plasmids. was identified on two different IncX4 plasmid types of 33 and 38 kb and was associated with an IS-like element. Based on the genetic diversity of E. coli isolates, transmission of the resistance determinant is mediated by horizontal transfer of IncX4 plasmids, as confirmed by conjugation experiments. Notably, the 33-kb plasmid is highly similar to the plasmid reported for the human sample. Furthermore, we identified the acquisition of an additional beta-lactam resistance linked to a Tn transposon on the IncX4 plasmids of three isolates, indicating progressive plasmid evolution. Overall, all described -carrying plasmids contain a highly conserved core genome necessary for colistin resistance development, transmission, replication, and maintenance. Variations in the plasmid sequences are mainly caused by the acquisition of insertion sequences and alteration in intergenic sequences or genes of unknown function. Evolutionary events causing the emergence of new resistances/variants are usually rare and challenging to predict. Conversely, common transmission events of widespread resistance determinants are quantifiable and predictable. One such example is the transmissible plasmid-mediated colistin resistance. The main determinant, , has been notified in 2016 but has successfully established itself in multiple plasmid backbones in diverse bacterial species across all One Health sectors. So far, 34 variants of are described, of which some can be used for epidemiological tracing-back analysis to identify the origin and transmission dynamics of these genes. Here, we report the presence of the rare gene in E. coli isolated from poultry since 2014. Based on the temporal occurrence and high similarity of the plasmids between poultry and human isolates, our study provides first indications for poultry husbandry as the primary source of and its transmission between different niches.
Topics: Animals; Humans; Colistin; Escherichia coli; Anti-Bacterial Agents; Poultry; Escherichia coli Proteins; Plasmids; Genomics; Drug Resistance, Bacterial; Microbial Sensitivity Tests
PubMed: 37358464
DOI: 10.1128/spectrum.01015-23 -
Nucleic Acids Research Jul 2023Many novel traits such as antibiotic resistance are spread by plasmids between species. Yet plasmids have different host ranges. Restriction-modification systems (R-M...
Many novel traits such as antibiotic resistance are spread by plasmids between species. Yet plasmids have different host ranges. Restriction-modification systems (R-M systems) are by far the most abundant bacterial defense system and therefore represent one of the key barriers to plasmid spread. However, their effect on plasmid evolution and host range has been neglected. Here we analyse the avoidance of targets of the most abundant R-M systems (Type II) for complete genomes and plasmids across bacterial diversity. For the most common target length (6 bp) we show that target avoidance is strongly correlated with the taxonomic distribution of R-M systems and is greater in plasmid genes than core genes. We find stronger avoidance of R-M targets in plasmids which are smaller and have a broader host range. Our results suggest two different evolutionary strategies for plasmids: small plasmids primarily adapt to R-M systems by tuning their sequence composition, and large plasmids primarily adapt through the carriage of additional genes protecting from restriction. Our work provides systematic evidence that R-M systems are important barriers to plasmid transfer and have left their mark on plasmids over long evolutionary time.
Topics: DNA Restriction-Modification Enzymes; Plasmids; Bacteria; Adaptation, Physiological; Drug Resistance, Microbial
PubMed: 37254807
DOI: 10.1093/nar/gkad452