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The ISME Journal Oct 2023Plasmids are key disseminators of antimicrobial resistance genes and virulence factors, and it is therefore critical to predict and reduce plasmid spread within...
Plasmids are key disseminators of antimicrobial resistance genes and virulence factors, and it is therefore critical to predict and reduce plasmid spread within microbial communities. The cost of plasmid carriage is a key metric that can be used to predict plasmids' ecological fate, and it is unclear whether plasmid costs are affected by growth partners in a microbial community. We carried out competition experiments and tracked plasmid maintenance using a model system consisting of a synthetic and stable five-species community and a broad host-range plasmid, engineered to carry different payloads. We report that both the cost of plasmid carriage and its long-term maintenance in a focal strain depended on the presence of competitors, and that these interactions were species specific. Addition of growth partners increased the cost of a high-payload plasmid to a focal strain, and accordingly, plasmid loss from the focal species occurred over a shorter time frame. We propose that the destabilising effect of interspecific competition on plasmid maintenance may be leveraged in clinical and natural environments to cure plasmids from focal strains.
Topics: Microbiota; Plasmids; Ecology; Anti-Bacterial Agents
PubMed: 37558861
DOI: 10.1038/s41396-023-01487-w -
Zoological Research Sep 2023Conjugative transfer of antibiotic resistance genes (ARGs) by plasmids is an important route for ARG dissemination. An increasing number of antibiotic and nonantibiotic...
Conjugative transfer of antibiotic resistance genes (ARGs) by plasmids is an important route for ARG dissemination. An increasing number of antibiotic and nonantibiotic compounds have been reported to aid the spread of ARGs, highlighting potential challenges for controlling this type of horizontal transfer. Development of conjugation inhibitors that block or delay the transfer of ARG-bearing plasmids is a promising strategy to control the propagation of antibiotic resistance. Although such inhibitors are rare, they typically exhibit relatively high toxicity and low efficacy and their mechanisms of action are inadequately understood. Here, we studied the effects of dihydroartemisinin (DHA), an artemisinin derivative used to treat malaria, on conjugation. DHA inhibited the conjugation of the IncI2 and IncX4 plasmids carrying the mobile colistin resistance gene ( ) by more than 160-fold in , and more than two-fold (IncI2 plasmid) in a mouse model. It also suppressed the transfer of the IncX3 plasmid carrying the carbapenem resistance gene by more than two-fold . Detection of intracellular adenosine triphosphate (ATP) and proton motive force (PMF), in combination with transcriptomic and metabolomic analyses, revealed that DHA impaired the function of the electron transport chain (ETC) by inhibiting the tricarboxylic acid (TCA) cycle pathway, thereby disrupting PMF and limiting the availability of intracellular ATP for plasmid conjugative transfer. Furthermore, expression levels of genes related to conjugation and pilus generation were significantly down-regulated during DHA exposure, indicating that the transfer apparatus for conjugation may be inhibited. Our findings provide new insights into the control of antibiotic resistance and the potential use of DHA.
Topics: Mice; Animals; Escherichia coli; Escherichia coli Infections; beta-Lactamases; Anti-Bacterial Agents; Plasmids
PubMed: 37551137
DOI: 10.24272/j.issn.2095-8137.2023.084 -
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 -
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 -
Nature Jun 2024Although eukaryotic Argonautes have a pivotal role in post-transcriptional gene regulation through nucleic acid cleavage, some short prokaryotic Argonaute variants...
Although eukaryotic Argonautes have a pivotal role in post-transcriptional gene regulation through nucleic acid cleavage, some short prokaryotic Argonaute variants (pAgos) rely on auxiliary nuclease factors for efficient foreign DNA degradation. Here we reveal the activation pathway of the DNA defence module DdmDE system, which rapidly eliminates small, multicopy plasmids from the Vibrio cholerae seventh pandemic strain (7PET). Through a combination of cryo-electron microscopy, biochemistry and in vivo plasmid clearance assays, we demonstrate that DdmE is a catalytically inactive, DNA-guided, DNA-targeting pAgo with a distinctive insertion domain. We observe that the helicase-nuclease DdmD transitions from an autoinhibited, dimeric complex to a monomeric state upon loading of single-stranded DNA targets. Furthermore, the complete structure of the DdmDE-guide-target handover complex provides a comprehensive view into how DNA recognition triggers processive plasmid destruction. Our work establishes a mechanistic foundation for how pAgos utilize ancillary factors to achieve plasmid clearance, and provides insights into anti-plasmid immunity in bacteria.
Topics: Argonaute Proteins; Bacterial Proteins; Cryoelectron Microscopy; Deoxyribonucleases; DNA Helicases; DNA, Single-Stranded; Models, Molecular; Plasmids; Protein Domains; Protein Multimerization; Vibrio cholerae
PubMed: 38740055
DOI: 10.1038/s41586-024-07515-9 -
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 -
Bulletin of Mathematical Biology Apr 2024In this study, we present a mathematical model for plasmid spread in a growing biofilm, formulated as a nonlocal system of partial differential equations in a 1-D free...
In this study, we present a mathematical model for plasmid spread in a growing biofilm, formulated as a nonlocal system of partial differential equations in a 1-D free boundary domain. Plasmids are mobile genetic elements able to transfer to different phylotypes, posing a global health problem when they carry antibiotic resistance factors. We model gene transfer regulation influenced by nearby potential receptors to account for recipient-sensing. We also introduce a promotion function to account for trace metal effects on conjugation, based on literature data. The model qualitatively matches experimental results, showing that contaminants like toxic metals and antibiotics promote plasmid persistence by favoring plasmid carriers and stimulating conjugation. Even at higher contaminant concentrations inhibiting conjugation, plasmid spread persists by strongly inhibiting plasmid-free cells. The model also replicates higher plasmid density in biofilm's most active regions.
Topics: Biofilms; Gene Transfer, Horizontal; Plasmids; Mathematical Concepts; Models, Biological; Models, Genetic; Conjugation, Genetic; Anti-Bacterial Agents
PubMed: 38664322
DOI: 10.1007/s11538-024-01289-x -
Methods in Molecular Biology (Clifton,... 2024Several mammarenaviruses cause hemorrhagic fever (HF) disease in humans and pose a significant public health problem in their endemic regions. The Old World (OW)...
Several mammarenaviruses cause hemorrhagic fever (HF) disease in humans and pose a significant public health problem in their endemic regions. The Old World (OW) mammarenavirus Lassa virus (LASV) is estimated to infect several hundred thousand people yearly in West Africa, resulting in high numbers of Lassa fever (LF) cases, a disease associated with high morbidity and mortality. No licensed vaccines are available to combat LASV infection, and anti-LASV drug therapy is limited to the off-label use of ribavirin whose efficacy remains controversial. The development of reverse genetics approaches has provided investigators with a powerful approach for the investigation of the molecular, cell biology and pathogenesis of mammarenaviruses. The use of cell-based minigenome systems has allowed examining the cis- and trans-acting factors involved in viral genome replication and gene transcription, assembly, and budding, which has facilitated the identification of several anti-mammarenavirus candidate drugs. Likewise, it is possible now to rescue infectious recombinant mammarenaviruses from cloned cDNAs containing predetermined mutations in their genomes to investigate virus-host interactions and mechanisms of viral pathogenesis. Reverse genetics have also allowed the generation of mammarenaviruses expressing foreign genes to facilitate virus detection, to identify antiviral drugs, and to generate live-attenuated vaccine (LAV) candidates. Likewise, reverse genetics techniques have allowed the generation of single-cycle infectious, reporter-expressing mammarenaviruses to study some aspects of the biology of HF-causing human mammarenavirus without the need of high security biocontainment laboratories. In this chapter, we describe the experimental procedures to generate recombinant (r)LASV using state-of-the-art plasmid-based reverse genetics.
Topics: Humans; Lassa virus; Reverse Genetics; Arenaviridae; Lassa Fever; Hemorrhagic Fevers, Viral; Plasmids
PubMed: 38064030
DOI: 10.1007/978-1-0716-3533-9_8