-
Cell Reports Methods Feb 2024Herpesviruses are large DNA viruses and include important human and veterinary pathogens. Their genomes can be cloned as bacterial artificial chromosomes (BACs) and...
Herpesviruses are large DNA viruses and include important human and veterinary pathogens. Their genomes can be cloned as bacterial artificial chromosomes (BACs) and genetically engineered in Escherichia coli using BAC recombineering methods. While the recombineering methods are efficient, the initial BAC-cloning step remains laborious. To overcome this limitation, we have developed a simple, rapid, and efficient BAC-cloning method based on single-step transformation-associated recombination (STAR) in Saccharomyces cerevisiae. The linear viral genome is directly integrated into a vector comprising a yeast centromeric plasmid and a BAC replicon. Following transfer into E. coli, the viral genome can be modified using standard BAC recombineering techniques. We demonstrate the speed, fidelity, and broad applicability of STAR by cloning two strains of both rat cytomegalovirus (a betaherpesvirus) and Kaposi's sarcoma-associated herpesvirus (a gammaherpesvirus). STAR cloning facilitates the functional genetic analysis of herpesviruses and other large DNA viruses and their use as vaccines and therapeutic vectors.
Topics: Humans; Cloning, Molecular; Recombination, Genetic; Escherichia coli; Plasmids; Gammaherpesvirinae; Herpesvirus 8, Human
PubMed: 38266652
DOI: 10.1016/j.crmeth.2024.100696 -
Emerging Microbes & Infections Dec 2024The multi-drug resistant pathogen has gained global attention as an important clinical challenge. Owing to its ability to survive on surfaces, its capacity for...
The multi-drug resistant pathogen has gained global attention as an important clinical challenge. Owing to its ability to survive on surfaces, its capacity for horizontal gene transfer, and its resistance to front-line antibiotics, has established itself as a successful pathogen. Bacterial conjugation is a central mechanism for pathogen evolution. The epidemic multidrug-resistant ACICU harbours a plasmid encoding a Type IV Secretion System (T4SS) with homology to the F-plasmid, and plasmids with homologous gene clusters have been identified in several sequence types. However the genetic and host strain diversity, global distribution, and functional ability of this group of plasmids is not fully understood. Using systematic analysis, we show that pACICU2 belongs to a group of almost 120 T4SS-encoding plasmids within four different species of and one strain of from human and environmental origin, and globally distributed across 20 countries spanning 4 continents. Genetic diversity was observed both outside and within the T4SS-encoding cluster, and 47% of plasmids harboured resistance determinants, with two plasmids harbouring eleven. Conjugation studies with an extensively drug-resistant (XDR) strain showed that the XDR plasmid could be successfully transferred to a more divergent , and transconjugants exhibited the resistance phenotype of the plasmid. Collectively, this demonstrates that these T4SS-encoding plasmids are globally distributed and more widespread among than previously thought, and that they represent an important potential reservoir for future clinical concern.
Topics: Humans; Type IV Secretion Systems; Escherichia coli; Plasmids; Anti-Bacterial Agents; Acinetobacter baumannii; beta-Lactamases; Microbial Sensitivity Tests; Drug Resistance, Multiple, Bacterial
PubMed: 38530969
DOI: 10.1080/22221751.2024.2320929 -
Journal of Global Antimicrobial... Sep 2023Presence and dissemination of plasmid-mediated AmpC genes (pAmpCs) have made bacteria cephalosporin-resistant and assessment of their prevalence and diversity is...
OBJECTIVES
Presence and dissemination of plasmid-mediated AmpC genes (pAmpCs) have made bacteria cephalosporin-resistant and assessment of their prevalence and diversity is essential. Coexistence of pAmpCs with New Delhi metallo-β-lactamase (bla) has facilitated their spread and NDM interferes with correct pAmpC phenotypic identification.
METHODS
Assessment of pAmpCs in different species and sequence types (STs), co-transmission with bla and phenotypic detection were analysed among Klebsiella pneumoniae (n = 256) and Escherichia coli (n = 92) isolated from septicaemic neonates over 13 years.
RESULTS
pAmpCs were present in 9% (30/348) of strains, 5% in K. pneumoniae and 18% in E. coli. pAmpC genes (bla and bla) were detected, bla and bla variants being predominant. Strains were resistant to most antimicrobials tested. bla and bla were dominant among E. coli (14/17) and K. pneumoniae (9/13), respectively. pAmpC-bearing strains belonged to diverse STs, including epidemic K. pneumoniae ST11 and ST147. Some strains co-harboured carbapenemase genes, bla (17/30) and bla (5/30). In 40% (12/30) of strains, pAmpC genes were transferred by conjugation, of which 8/12 exhibited co-transfer with bla. pAmpCs were frequently found in replicons as follows: bla with IncHIB-M, bla with IncA/C, bla with IncA/C, and bla with IncFII. The combination disk-diffusion test correctly detected pAmpC in 77% (23/30) of pAmpC-bearing strains. However, correct detection of pAmpC was higher in strains that did not harbour bla vs. those with bla (85% vs. 71%).
CONCLUSION
Presence of pAmpCs along with carbapenemases, linkage with multiple STs, and replicon types indicated their potential for spread. pAmpCs can go undetected in the presence of bla; hence, regular surveillance is required.
Topics: Infant, Newborn; Humans; Escherichia coli; Klebsiella pneumoniae; Anti-Bacterial Agents; Plasmids; Escherichia coli Infections
PubMed: 37328061
DOI: 10.1016/j.jgar.2023.05.012 -
The ISME Journal Jan 2024Large cointegrate plasmids recruit genetic features of their parental plasmids and serve as important vectors in the spread of antibiotic resistance. They are now...
Large cointegrate plasmids recruit genetic features of their parental plasmids and serve as important vectors in the spread of antibiotic resistance. They are now frequently found in clinical settings, raising the issue of how to limit their further transmission. Here, we conducted evolutionary research of a large blaNDM-positive cointegrate within Escherichia coli C600, and discovered that adaptive evolution of chromosome and plasmid jointly improved bacterial fitness, which was manifested as enhanced survival ability for in vivo and in vitro pairwise competition, biofilm formation, and gut colonization ability. From the plasmid aspect, large-scale DNA fragment loss is observed in an evolved clone. Although the evolved plasmid imposes a negligible fitness cost on host bacteria, its conjugation frequency is greatly reduced, and the deficiency of anti-SOS gene psiB is found responsible for the impaired horizontal transferability rather than the reduced fitness cost. These findings unveil an evolutionary strategy in which the plasmid horizontal transferability and fitness cost are balanced. From the chromosome perspective, all evolved clones exhibit parallel mutations in the transcriptional regulatory stringent starvation Protein A gene sspA. Through a sspA knockout mutant, transcriptome analysis, in vitro transcriptional activity assay, RT-qPCR, motility test, and scanning electron microscopy techniques, we demonstrated that the mutation in sspA reduces its transcriptional inhibitory capacity, thereby improving bacterial fitness, biofilm formation ability, and gut colonization ability by promoting bacterial flagella synthesis. These findings expand our knowledge of how cointegrate plasmids adapt to new bacterial hosts.
Topics: Escherichia coli; Plasmids; Bacteria; Drug Resistance, Microbial; Chromosomes; Anti-Bacterial Agents
PubMed: 38438143
DOI: 10.1093/ismejo/wrae037 -
Emerging Microbes & Infections Dec 2024To investigate the epidemiology of ST20 carbapenem-resistant (CRKP) in China, and further explore the genomic characteristics of and coharboring isolates and plasmid...
To investigate the epidemiology of ST20 carbapenem-resistant (CRKP) in China, and further explore the genomic characteristics of and coharboring isolates and plasmid contributions to resistance and fitness. Seven ST20 CRKP isolates were collected nationwide, and antimicrobial susceptibility testing was performed. Antimicrobial resistance genes, virulence genes, and plasmid replicons were identified via whole-genome sequencing, and clonality assessed via core-genome multilocus sequence typing. Furthermore, we found four dual-metallo-β-lactamases (MBL)-harbouring isolates, the gene location was detected by Southern blotting, and plasmid location analysis showed that was located on a separate plasmid, a self-conjugative fusion plasmid, or the bacterial chromosome. These isolates were subjected to long-read sequencing, the presence of in different locations was identified by genomic comparison, and transposon units were detected via inverse PCR. We subsequently found that on the fusion plasmid and bacterial chromosome was formed via intact plasmid recombination by the IS and , respectively, and the circular transposon unit was related to cointegration, however, in different locations did not affect the gene stability. The -harbouring plasmid contributed to the increased resistance to β-lactams and shortened survival lag time which was revealed in plasmid cured isolates. In summary, the ST20 clone is a high-risk resistant clone. With the use of ceftazidime/avibactam, MBL-positive isolates, especially dual-MBL-harbouring isolates, should be given additional attention.
Topics: Klebsiella pneumoniae; Anti-Bacterial Agents; Carbapenems; Drug Resistance, Multiple, Bacterial; Plasmids; beta-Lactamases; Carbapenem-Resistant Enterobacteriaceae; Multilocus Sequence Typing; Microbial Sensitivity Tests
PubMed: 38584569
DOI: 10.1080/22221751.2024.2339942 -
Cell Host & Microbe Jul 2023Antibiotic resistance plasmids can be disseminated between different Enterobacteriaceae in the gut. Here, we investigate how closely related Enterobacteriaceae...
Antibiotic resistance plasmids can be disseminated between different Enterobacteriaceae in the gut. Here, we investigate how closely related Enterobacteriaceae populations with similar nutrient needs can co-bloom in the same gut and thereby facilitate plasmid transfer. Using different strains of Salmonella Typhimurium (S.Tm SL1344 and ATCC14028) and mouse models of Salmonellosis, we show that the bloom of one strain (i.e., recipient) from very low numbers in a gut pre-occupied by the other strain (i.e., donor) depends on strain-specific utilization of a distinct carbon source, galactitol or arabinose. Galactitol-dependent growth of the recipient S.Tm strain promotes plasmid transfer between non-isogenic strains and between E. coli and S.Tm. In mice stably colonized by a defined microbiota (OligoMM), galactitol supplementation similarly facilitates co-existence of two S.Tm strains and promotes plasmid transfer. Our work reveals a metabolic strategy used by Enterobacteriaceae to expand in a pre-occupied gut and provides promising therapeutic targets for resistance plasmids spread.
Topics: Animals; Mice; Escherichia coli; Plasmids; Salmonella typhimurium; Salmonella Infections; Galactitol; Anti-Bacterial Agents
PubMed: 37348498
DOI: 10.1016/j.chom.2023.05.029 -
Emerging Microbes & Infections Dec 2023Epidemiological characteristics and molecular features of carbapenem-resistant (CR-) species remain unclear in China. In this study, we performed a genomic study on 92...
Epidemiological characteristics and molecular features of carbapenem-resistant (CR-) species remain unclear in China. In this study, we performed a genomic study on 92 isolates from -caused infections from a multicenter study in China. Whole genome sequencing (WGS) was used to determine the genome sequence of 92 non-duplicated CR- strains collected from multiple tertiary health centres. The precise species of strains were identified by average nucleotide identity (ANI) and in silico DNA-DNA hybridization (isDDH). Molecular features of high-risk CR- sequence type (ST) lineages and carbapenemase-encoding plasmids were determined. The result revealed that the most common human-source CR- species in China was (66/92, 71.93%), and the proportion of carbapenemase-producing (CP-) in CR- was high (72/92, 78.26%) in comparison to other global regions. Furthermore, ST171 and ST116 were the major lineages of CP- strains, and ST171 was more likely to cause infections in older patients. Genomic analysis also highlighted the likelihood of intra-hospital/inter-hospital clonal transmission of ST171 and ST116 . In addition, the -harbouring IncX3-type plasmid was identified as the prevalent carbapenemase-encoding plasmid carried by CR- strains, and was experimentally confirmed to be able to self-transfer with high frequency. This study detailed the genomic and clinical characteristics of CR- in China in the form of multicenter for the first time. The high risk of carbapenemase-producing ST171 and ST116 , and the -harbouring IncX3-type plasmid were detected and emphasized.
Topics: Aged; Humans; Bacterial Proteins; beta-Lactamases; Carbapenem-Resistant Enterobacteriaceae; China; Enterobacter; Enterobacteriaceae Infections; Genomics; Microbial Sensitivity Tests; Plasmids
PubMed: 36382635
DOI: 10.1080/22221751.2022.2148562 -
Nature Communications Aug 2023Infections caused by metallo-beta-lactamase-producing organisms (MBLs) are a global health threat. Our understanding of transmission dynamics and how MBLs establish...
Infections caused by metallo-beta-lactamase-producing organisms (MBLs) are a global health threat. Our understanding of transmission dynamics and how MBLs establish endemicity remains limited. We analysed two decades of bla evolution in a hospital using sequence data from 270 clinical and environmental isolates (including 169 completed genomes) and identified the bla gene across 7 Gram-negative genera, 68 bacterial strains and 7 distinct plasmid types. We showed how an initial multi-species outbreak of conserved IncC plasmids (95 genomes across 37 strains) allowed endemicity to be established through the ability of bla to disseminate in successful strain-genetic setting pairs we termed propagators, in particular Serratia marcescens and Enterobacter hormaechei. From this reservoir, bla persisted through diversification of genetic settings that resulted from transfer of bla plasmids between bacterial hosts and of the integron carrying bla between plasmids. Our findings provide a framework for understanding endemicity and spread of MBLs and may have broader applicability to other carbapenemase-producing organisms.
Topics: Integrons; beta-Lactamases; Bacterial Proteins; Plasmids; Serratia marcescens; Carbapenems; Genomics; Microbial Sensitivity Tests; Anti-Bacterial Agents
PubMed: 37553339
DOI: 10.1038/s41467-023-39915-2 -
Bioinformatics (Oxford, England) Jul 2023With recent advances in sequencing technologies, it is now possible to obtain near-perfect complete bacterial chromosome assemblies cheaply and efficiently by combining...
SUMMARY
With recent advances in sequencing technologies, it is now possible to obtain near-perfect complete bacterial chromosome assemblies cheaply and efficiently by combining a long-read-first assembly approach with short-read polishing. However, existing methods for assembling bacterial plasmids from long-read-first assemblies often misassemble or even miss bacterial plasmids entirely and accordingly require manual curation. Plassembler was developed to provide a tool that automatically assembles and outputs bacterial plasmids using a hybrid assembly approach. It achieves increased accuracy and computational efficiency compared to the existing gold standard tool Unicycler by removing chromosomal reads from the input read sets using a mapping approach.
AVAILABILITY AND IMPLEMENTATION
Plassembler is implemented in Python and is installable as a bioconda package using 'conda install -c bioconda plassembler'. The source code is available on GitHub at https://github.com/gbouras13/plassembler. The full benchmarking pipeline can be found at https://github.com/gbouras13/plassembler_simulation_benchmarking, while the benchmarking input FASTQ and output files can be found at https://doi.org/10.5281/zenodo.7996690.
Topics: Sequence Analysis, DNA; High-Throughput Nucleotide Sequencing; Software; Plasmids; Benchmarking
PubMed: 37369026
DOI: 10.1093/bioinformatics/btad409 -
Cell Reports Methods Dec 2023We created a generalizable pipeline for antibiotic-resistance-gene-free plasmid (ARGFP)-based cloning using a dual auxotrophic- and essential-gene-based selection...
We created a generalizable pipeline for antibiotic-resistance-gene-free plasmid (ARGFP)-based cloning using a dual auxotrophic- and essential-gene-based selection strategy. We use auxotrophic selection to construct plasmids in engineered E. coli DH10B cloning strains and both auxotrophic- and essential-gene-based selection to (1) select for recombinant strains and (2) maintain a plasmid in E. coli Nissle 1917, a common chassis for engineered probiotic applications, and E. coli MG1655, the laboratory "wild-type" E. coli strain. We show that our approach has comparable efficiency to that of antibiotic-resistance-gene-based cloning. We also show that the double-knockout Nissle and MG1655 strains are simple to transform with plasmids of interest. Notably, we show that the engineered Nissle strains are amenable to long-term plasmid maintenance in repeated culturing as well as in the mouse gut, demonstrating the potential for broad applications while minimizing the risk of antibiotic resistance spread via horizontal gene transfer.
Topics: Animals; Mice; Anti-Bacterial Agents; Escherichia coli; Plasmids; Drug Resistance, Microbial; Cloning, Molecular
PubMed: 38086386
DOI: 10.1016/j.crmeth.2023.100669