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Scientific Reports Apr 2024Incompatibility (Inc) HI2 plasmids are large (typically > 200 kb), transmissible plasmids that encode antimicrobial resistance (AMR), heavy metal resistance (HMR)...
Incompatibility (Inc) HI2 plasmids are large (typically > 200 kb), transmissible plasmids that encode antimicrobial resistance (AMR), heavy metal resistance (HMR) and disinfectants/biocide resistance (DBR). To better understand the distribution and diversity of resistance-encoding genes among IncHI2 plasmids, computational approaches were used to evaluate resistance and transfer-associated genes among the plasmids. Complete IncHI2 plasmid (N = 667) sequences were extracted from GenBank and analyzed using AMRFinderPlus, IntegronFinder and Plasmid Transfer Factor database. The most common IncHI2-carrying genera included Enterobacter (N = 209), Escherichia (N = 208), and Salmonella (N = 204). Resistance genes distribution was diverse, with plasmids from Escherichia and Salmonella showing general similarity in comparison to Enterobacter and other taxa, which grouped together. Plasmids from Enterobacter and other taxa had a higher prevalence of multiple mercury resistance genes and arsenic resistance gene, arsC, compared to Escherichia and Salmonella. For sulfonamide resistance, sul1 was more common among Enterobacter and other taxa, compared to sul2 and sul3 for Escherichia and Salmonella. Similar gene diversity trends were also observed for tetracyclines, quinolones, β-lactams, and colistin. Over 99% of plasmids carried at least 25 IncHI2-associated conjugal transfer genes. These findings highlight the diversity and dissemination potential for resistance across different enteric bacteria and value of computational-based approaches for the resistance-gene assessment.
Topics: Plasmids; Enterobacteriaceae; Drug Resistance, Bacterial; Anti-Bacterial Agents; Genotype; Enterobacter; Salmonella; Drug Resistance, Multiple, Bacterial
PubMed: 38684834
DOI: 10.1038/s41598-024-59870-2 -
Nucleic Acids Research Jul 2023PlasMapper 3.0 is a web server that allows users to generate, edit, annotate and interactively visualize publication quality plasmid maps. Plasmid maps are used to plan,...
PlasMapper 3.0 is a web server that allows users to generate, edit, annotate and interactively visualize publication quality plasmid maps. Plasmid maps are used to plan, design, share and publish critical information about gene cloning experiments. PlasMapper 3.0 is the successor to PlasMapper 2.0 and offers many features found only in commercial plasmid mapping/editing packages. PlasMapper 3.0 allows users to paste or upload plasmid sequences as input or to upload existing plasmid maps from its large database of >2000 pre-annotated plasmids (PlasMapDB). This database can be searched by plasmid names, sequence features, restriction sites, preferred host organisms, and sequence length. PlasMapper 3.0 also supports the annotation of new or never-before-seen plasmids using its own feature database that contains common promoters, terminators, regulatory sequences, replication origins, selectable markers and other features found in most cloning plasmids. PlasMapper 3.0 has several interactive sequence editors/viewers that allow users to select and view plasmid regions, insert genes, modify restriction sites or perform codon optimization. The graphics for PlasMapper 3.0 have also been substantially upgraded. It now offers an interactive, full-color plasmid viewer/editor that allows users to zoom, rotate, re-color, linearize, circularize, edit annotated features and modify plasmid images or labels to improve the esthetic qualities of their plasmid map and textual displays. All the plasmid images and textual displays are downloadable in multiple formats. PlasMapper 3.0 is available online at https://plasmapper.ca.
Topics: Software; User-Computer Interface; Plasmids; Computers; Base Sequence; Internet
PubMed: 37099365
DOI: 10.1093/nar/gkad276 -
Journal of Global Antimicrobial... Sep 2023Emergence of the plasmid-born mobile colistin resistance (mcr) gene is a growing concern in healthcare. Therefore, this study aimed to genomically characterise...
Genomic characterisation of multi-drug resistant Escherichia coli and Klebsiella pneumoniae co-harbouring mcr-1 and mcr-3 genes on a single plasmid from paediatric clinical cases.
OBJECTIVES
Emergence of the plasmid-born mobile colistin resistance (mcr) gene is a growing concern in healthcare. Therefore, this study aimed to genomically characterise multidrug-resistant Escherichia coli and Klebsiella pneumoniae co-harbouring the mcr-1 and mcr-3 genes in young children.
METHODS
E. coli (n = 3) and K. pneumoniae (n = 2) were collected from abdominal secretions and blood, respectively. The isolates were screened using tryptone soy broth with 4 µL/mL polymyxin-B. Growing bacteria were identified using the VITEK-2 system, matrix-assisted laser desorption/ionisation time-of-flight, and 16s RNA sequencing, followed by antibiotic susceptibility testing. Metallo-β-lactamase (MBL) and extended-spectrum β-lactamase (ESBL) production was also detected. Afterwards, strains were subjected to molecular screening targeting mcr variants and ESBL/MBL-encoding genes. Conjugation, pulsed-field gel electrophoresis, Southern hybridisation, multilocus sequence typing, and phylogenic group detection were performed, along with plasmid-genome sequencing and bioinformatics analysis.
RESULTS
E. coli isolates (EC-19-322, 323, and 331) and K. pneumoniae isolates (KP-19-225 and 226) harboured both mcr-1 and mcr-3 genes. These strains were also found to be resistant to more than three classes of antibiotics. The conjugation experiment revealed the presence of mcr-1 and mcr-3 on a single plasmid, and the transmission frequency was 10 to 10. Both strains were found to be able to produce ESBLs and MBL. E. coli EC-19-322 and 323 were identified as ST131(O25a:H41); SP-19-331, as ST1577 (O16:H30); and K. pneumoniae, as ST231 (K2). All E. coli strains belonged to phylogenetic group B2, and the results of pulsed-field gel electrophoresis supported the multilocus sequence typing findings.
CONCLUSION
This study reported the co-occurrence of mcr-1 and mcr-3 genes on a single plasmid in pathogenic ESBL/MBL-producing E. coli and K. pneumoniae isolated from young children.
Topics: Humans; Child; Child, Preschool; Escherichia coli; Colistin; Klebsiella pneumoniae; Phylogeny; Plasmids; beta-Lactamases; Genomics
PubMed: 37481113
DOI: 10.1016/j.jgar.2023.07.012 -
Microbiology Spectrum Aug 2023Vibrio parahaemolyticus is a bacterial pathogen that becomes lethal to shrimps when acquiring the pVA1-type plasmid carrying the PirAB genes, causing acute...
Vibrio parahaemolyticus is a bacterial pathogen that becomes lethal to shrimps when acquiring the pVA1-type plasmid carrying the PirAB genes, causing acute hepatopancreatic necrosis disease (AHPND). This disease causes significant losses across the world, with outbreaks reported in Southeast Asia, Mexico, and South America. Virulence level and mortality differences have been reported in isolates from different locations, and whether this phenomenon is caused by plasmid-related elements or genomic-related elements from the bacteria remains unclear. Here, nine genomes of South American AHPND-causing V. parahaemolyticus (VP) isolates were assembled and analyzed using a comparative genomics approach at (i) whole-genome, (ii) secretion system, and (iii) plasmid level, and then included for a phylogenomic analysis with another 86 strains. Two main results were obtained from our analyses. First, all isolates contained pVA1-type plasmids harboring the toxin coding genes, and with high similarity with the prototypical sequence of Mexican-like origin, while phylogenomic analysis showed some level of heterogeneity with discrete clusters and wide diversity compared to other available genomes. Second, although a high genomic similarity was observed, variation in virulence genes and clusters was observed, which might be relevant in the expression of the disease. Overall, our results suggest that South American pathogenic isolates are derived from various genetic lineages which appear to have acquired the plasmid through horizontal gene transfer. Furthermore, pathogenicity seems to be a multifactorial trait where the degree of virulence could be altered by the presence or variations of several virulence factors. AHPND have caused losses of over $2.6 billion to the aquaculture industry around the world due to its high mortality rate in shrimp farming. The most common etiological agent is V. parahaemolyticus strains possessing the pVA1-type plasmid carrying the PirAB toxin. Nevertheless, complete understanding of the role of genetic elements and their impact in the virulence of this pathogen remains unclear. In this work, we analyzed nine South American AHPND-causing V. parahaemolyticus isolates at a genomic level, and assessed their evolutionary relationship with other 86 strains. We found that all our isolates were highly similar and possessed the Mexican-type plasmid, but their genomic content did not cluster with other Mexican strains, but instead were spread across all isolates. These results suggest that South American VP have different genetic backgrounds, and probably proceed from diverse geographical locations, and acquire the pVA1-type plasmid via horizontal gene transfer at different times.
Topics: Humans; Vibrio parahaemolyticus; Plasmids; Toxins, Biological; Genomics; Aquaculture; Necrosis
PubMed: 37272817
DOI: 10.1128/spectrum.04851-22 -
Porin deficiency or plasmid copy number increase mediated carbapenem-resistant resistance evolution.Emerging Microbes & Infections Dec 2024This study investigated resistance evolution mechanisms of conjugated plasmids and bacterial hosts under different concentrations of antibiotic pressure. Ancestral...
This study investigated resistance evolution mechanisms of conjugated plasmids and bacterial hosts under different concentrations of antibiotic pressure. Ancestral strain ECNX52 was constructed by introducing the -carrying IncX3 plasmid into C600, and was subjected to laboratory evolution under different concentrations of meropenem pressure. Minimal inhibitory concentrations and conjugation frequency were determined. Fitness of these strains was assessed. Whole genome sequencing and transcriptional changes were performed. Ancestral host or plasmids were recombined with evolved hosts or plasmids to verify plasmid or host factors in resistance evolution. Role of the mutation on plasmid copy number was determined. Two out of the four clones (EM2N1 and EM2N3) exhibited four-fold increase in MIC when exposed to a continuous pressure of 2 μg/mL MEM (1/32 MIC), by down regulating expression of outer membrane protein . Besides, all four clones displayed four-fold increase in MIC and higher conjugation frequency when subjected to a continuous pressure of 4 μg/mL MEM (1/16 MIC), attributing to increasing plasmid copy number generated by D140Y (GAT→TAT) mutation. Bacterial hosts and conjugative plasmids can undergo resistance evolution under certain concentrations of antimicrobial pressure by reducing the expression of outer membrane proteins or increasing plasmid copy numbers.
Topics: Escherichia coli; Plasmids; Microbial Sensitivity Tests; Anti-Bacterial Agents; Porins; Escherichia coli Proteins; Carbapenems; Meropenem; Mutation; Evolution, Molecular; Conjugation, Genetic; Carbapenem-Resistant Enterobacteriaceae; Whole Genome Sequencing; Gene Dosage; beta-Lactamases
PubMed: 38712634
DOI: 10.1080/22221751.2024.2352432 -
Frontiers in Cellular and Infection... 2023Antibiotic resistance represents one of the greatest threats to global health. The spread of antibiotic resistance genes among bacteria occurs mostly through horizontal...
Antibiotic resistance represents one of the greatest threats to global health. The spread of antibiotic resistance genes among bacteria occurs mostly through horizontal gene transfer via conjugation mediated by plasmids. This process implies a direct contact between a donor and a recipient bacterium which acquires the antibiotic resistance genes encoded by the plasmid and, concomitantly, the capacity to transfer the acquired plasmid to a new recipient. Classical assays for the measurement of plasmid transfer frequency (i.e., conjugation frequency) are often characterized by a high variability and, hence, they require many biological and technical replicates to reduce such variability and the accompanying uncertainty. In addition, classical conjugation assays are commonly tedious and time-consuming because they typically involve counting colonies on a large number of plates for the quantification of donors, recipients, and transconjugants (i.e., the bacteria that have received the genetic material by conjugation). Due to the magnitude of the antibiotic resistance problem, it is critical to develop reliable and rapid methods for the quantification of plasmid transfer frequency that allow the simultaneous analysis of many samples. Here, we present the development of a high-throughput, reliable, quick, easy, and cost-effective method to simultaneously accomplish and measure multiple conjugation events in 96-well plates, in which the quantification of donors, recipients, and transconjugants is estimated from the time required to reach a specific threshold value (OD value) in the bacterial growth curves. Our method successfully discriminates different plasmid transfer frequencies, yielding results that are equivalent to those obtained by a classical conjugation assay.
Topics: Plasmids; Anti-Bacterial Agents; Drug Resistance, Microbial; Conjugation, Genetic; Gene Transfer, Horizontal
PubMed: 37886666
DOI: 10.3389/fcimb.2023.1269732 -
Journal of Global Antimicrobial... Mar 2024This study aimed to delineate the ability of a plasmid, pS130-4, which harboured both hypervirulence and multidrug resistance genes, to disseminate within Klebsiella...
OBJECTIVES
This study aimed to delineate the ability of a plasmid, pS130-4, which harboured both hypervirulence and multidrug resistance genes, to disseminate within Klebsiella pneumoniae, as well as its potential formation mechanism.
METHODS
We employed whole-genome sequencing to decipher the genetic architecture of pS130-4. Its capability to conjugate and transfer was assessed through a series of experiments, including plasmid stability, competitive growth, and growth curve analysis. Its expression stability was further evaluated using drug sensitivity, larval survival, and biofilm formation tests.
RESULTS
pS130-4 contained four intact modules typical of self-transmissible plasmids. BLAST analysis revealed a sequence identity exceeding 90% with other plasmids from a variety of hosts, suggesting its broad prevalence. Our findings indicated the plasmid's formation resulted from IS26-mediated recombination, leading us to propose a model detailing the creation of this conjugative fusion plasmid housing both bla and hypervirulence genes. Our conjugation experiments established that pS130-4, when present in the clinical strain S130, was self-transmissible with an estimated efficiency between 10 and 10. Remarkably, pS130-4 showcased a 90% retention rate and did not impede the growth of host bacteria. Galleria mellonella larval infection assay demonstrated that S130 had pronounced toxicity when juxtaposed with high-virulence control strain NTUH-K2044 and low-toxicity control strain ATCC700603. Furthermore, pS130-4's virulence remained intact postconjugation.
CONCLUSION
A fusion plasmid, encompassing both hypervirulence and multidrug resistance genes, was viable within K. pneumoniae ST11-KL64 and incurred minimal fitness costs. These insights underscored the criticality of rigorous monitoring to pre-empt the escalation and distribution of this formidable super-plasmid.
Topics: Animals; Genes, MDR; Klebsiella pneumoniae; Larva; Plasmids
PubMed: 38307249
DOI: 10.1016/j.jgar.2024.01.010 -
Antimicrobial Agents and Chemotherapy Jul 2023The emergence of carbapenem-resistant, hypervirulent Klebsiella pneumoniae is a new threat to health care. We studied the molecular epidemiology of carbapenem-resistant...
The emergence of carbapenem-resistant, hypervirulent Klebsiella pneumoniae is a new threat to health care. We studied the molecular epidemiology of carbapenem-resistant Klebsiella pneumoniae isolates in Qatar using whole-genome sequence data. We also characterized the prevalence and genetic basis of hypervirulent phenotypes and established the virulence potential using a Galleria mellonella model. Of 100 Klebsiella isolates studied, NDM and OXA-48 were the most common carbapenemases. Core genome single-nucleotide polymorphism (SNP) analysis indicated the presence of diverse sequence types and clonal lineages; isolates belonging to Klebsiella quasipneumoniae subsp. sequence type 196 (ST196) and ST1416 may be disseminated among several health care centers. Ten K. pneumoniae isolates carried and/or truncated , and 2 isolates belonged to KL2, indicating low prevalence of classical hypervirulent isolates. Isolates carrying both carbapenem resistance and hypervirulence genes were confined mainly to ST231 and ST383 isolates. One ST383 isolate was further investigated by MinION sequencing, and the assembled genome indicated that was located on an IncHI1B-type plasmid (pFQ61_ST383_NDM-5) which coharbored several virulence factors, including the regulator of the mucoid phenotype (), the regulator of mucoid phenotype 2 (), and aerobactin ( and ), likely resulting from recombination events. Comparative genomics indicated that this hybrid plasmid may be present in two additional Qatari ST383 isolates. Carbapenem-resistant, hypervirulent K. pneumoniae ST383 isolates pose an emerging threat to global health due to their simultaneous hypervirulence and multidrug resistance.
Topics: Humans; Klebsiella pneumoniae; Carbapenems; Qatar; Klebsiella Infections; Klebsiella; beta-Lactamases; Plasmids; Genomics; Anti-Bacterial Agents
PubMed: 37310284
DOI: 10.1128/aac.00030-23 -
ACS Synthetic Biology Oct 2023, a genus of Gram-positive bacteria, is known as nature's medicine maker, producing a plethora of natural products that have huge benefits for human health, agriculture,...
, a genus of Gram-positive bacteria, is known as nature's medicine maker, producing a plethora of natural products that have huge benefits for human health, agriculture, and biotechnology. To take full advantage of this treasure trove of bioactive molecules, better genetic tools are required for the genetic engineering and synthetic biology of . We therefore developed CUBIC, a novel CUmate-Based Inducible CRISPR interference (CRISPRi) system that allows highly efficient and inducible gene knockdown in . Its broad application is shown by the specific and nondisruptive knockdown of genes involved in growth, development and antibiotic production in various species. To facilitate hyper-efficient plasmid construction, we adapted the Golden Gate assembly to achieve 100% cloning efficiency of the protospacers. We expect that the versatile plug-and-play CUBIC system will create new opportunities for research and innovation in the field of .
Topics: Humans; Streptomyces; Promoter Regions, Genetic; Clustered Regularly Interspaced Short Palindromic Repeats; Genetic Engineering; Plasmids
PubMed: 37801665
DOI: 10.1021/acssynbio.3c00464 -
Microbiology Spectrum Aug 2023Plasmid-mediated quinolone resistance (PMQR) determinants, such as genes, have been widely reported in spp. while other types of PMQR genes were rarely reported in...
Plasmid-mediated quinolone resistance (PMQR) determinants, such as genes, have been widely reported in spp. while other types of PMQR genes were rarely reported in these bacteria. This study characterized the phenotypic and genotypic features of foodborne spp. carrying , a key PMQR gene in . Among a total of 1,811 foodborne isolates tested, 34 (1.88%) were found to harbor the gene. The allele was the most prevalent, but coexistence with other alleles was common. Missense mutations in the quinolone resistance-determining region (QRDR) of the and genes were only found in 11 of the 34 -bearing isolates. Antimicrobial susceptibility tests showed that all 34 -bearing isolates were resistant to ampicillin and that a high percentage also exhibited resistance to cefotaxime, ceftriaxone, and trimethoprim-sulfamethoxazole. Genetic analysis showed that these phenotypes were attributed to a diverse range of resistance elements that the -bearing isolates harbored. The gene could be found in both the chromosome and plasmids; the plasmid-borne genes could be found on both conjugative and nonconjugative plasmids. pAQU-type -bearing conjugative plasmids were able to mediate expression of phenotypic resistance to both ciprofloxacin and cephalosporins. Transmission of this plasmid among spp. would speed up the emergence of multidrug-resistant (MDR) pathogens that are resistant to the most important antibiotics used in treatment of infections, suggesting that close monitoring of emergence and dissemination of MDR spp. in both food samples and clinical settings is necessary. spp. used to be very susceptible to antibiotics. However, resistance to clinically important antibiotics, such as cephalosporins and fluoroquinolones, among clinically isolated strains is increasingly common. In this study, we found that plasmid-mediated quinolone resistance (PMQR) genes, such as , that have not been previously reported in spp. can now be detected in food isolates. The gene alone could mediate expression of ciprofloxacin resistance in spp.; importantly, this gene could be found in both the chromosome and plasmids. The plasmids that harbor the gene could be both conjugative and nonconjugative, among which the pAQU-type -bearing conjugative plasmids were able to mediate expression of resistance to both ciprofloxacin and cephalosporins. Transmission of this plasmid among spp. would accelerate the emergence of multidrug-resistant pathogens.
Topics: Ciprofloxacin; Cephalosporins; Drug Resistance, Bacterial; Anti-Bacterial Agents; Quinolones; Plasmids; Monobactams; Vibrio; Microbial Sensitivity Tests
PubMed: 37395663
DOI: 10.1128/spectrum.01032-23