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Emerging Microbes & Infections Dec 2023is an important human pathogen known for its resistance to carbapenem antibiotics, especially the increasing carbapenem-resistant hypervirulent variants. The carbapenem...
is an important human pathogen known for its resistance to carbapenem antibiotics, especially the increasing carbapenem-resistant hypervirulent variants. The carbapenem resistance is mainly caused by the carbapenemase gene which was commonly found on the IncFII transferable plasmids in ST11 isolates in regions of China. However, the mechanisms of the plasmid-carrying regulation by the host strain are not clear. To investigate the chromosome-encoded two-component system (TCS) that regulates the carbapenem resistance of caused by , twenty-four TCSs of a carbapenem-resistant classical ST11 clinical isolate were knocked out. The deletion mutation of the TCS regulator exhibited increased sensitivity to carbapenem, which could be restored by complementation with . Electrophoretic mobility shift, isothermal titration calorimetry and DNase I footprinting results revealed that CpxR directly bound to the promoter DNA of and the binding was abolished by disrupting the DNA-binding domain in CpxR. The subsequent assays using the reporter system and qPCR showed that CpxR upregulates the transcription of . Notably, CpxR was also found to activate the transfer of the -carrying IncFII plasmid between the hypervirulent and isolates, in which CpxR promoted the transcription of the operon via binding to its promoter region. These results provide an important insight into the regulation of the host factor CpxR in the plasmid-carrying carbapenemase gene in the classical and hypervirulent .
Topics: Humans; Anti-Bacterial Agents; Carbapenems; Klebsiella pneumoniae; Escherichia coli; Klebsiella Infections; beta-Lactamases; Plasmids; DNA
PubMed: 37672539
DOI: 10.1080/22221751.2023.2256427 -
MSystems Mar 2024Bacterial plasmids play a major role in the spread of antibiotic resistance genes. However, their characterization via DNA sequencing suffers from the low abundance of...
UNLABELLED
Bacterial plasmids play a major role in the spread of antibiotic resistance genes. However, their characterization via DNA sequencing suffers from the low abundance of plasmid DNA in those samples. Although sample preparation methods can enrich the proportion of plasmid DNA before sequencing, these methods are expensive and laborious, and they might introduce a bias by enriching only for specific plasmid DNA sequences. Nanopore adaptive sampling could overcome these issues by rejecting uninteresting DNA molecules during the sequencing process. In this study, we assess the application of adaptive sampling for the enrichment of low-abundant plasmids in known bacterial isolates using two different adaptive sampling tools. We show that a significant enrichment can be achieved even on expired flow cells. By applying adaptive sampling, we also improve the quality of plasmid assemblies and reduce the sequencing time. However, our experiments also highlight issues with adaptive sampling if target and non-target sequences span similar regions.
IMPORTANCE
Antimicrobial resistance causes millions of deaths every year. Mobile genetic elements like bacterial plasmids are key drivers for the dissemination of antimicrobial resistance genes. This makes the characterization of plasmids via DNA sequencing an important tool for clinical microbiologists. Since plasmids are often underrepresented in bacterial samples, plasmid sequencing can be challenging and laborious. To accelerate the sequencing process, we evaluate nanopore adaptive sampling as an method for the enrichment of low-abundant plasmids. Our results show the potential of this cost-efficient method for future plasmid research but also indicate issues that arise from using reference sequences.
Topics: Nanopores; Plasmids; Bacteria; DNA; Anti-Infective Agents
PubMed: 38376263
DOI: 10.1128/msystems.00945-23 -
Microbial Genomics May 2024Evidence is accumulating in the literature that the horizontal spread of antimicrobial resistance (AMR) genes mediated by bacteriophages and bacteriophage-like plasmid...
Evidence is accumulating in the literature that the horizontal spread of antimicrobial resistance (AMR) genes mediated by bacteriophages and bacteriophage-like plasmid (phage-plasmid) elements is much more common than previously envisioned. For instance, we recently identified and characterized a circular P1-like phage-plasmid harbouring a gene conferring extended-spectrum beta-lactamase (ESBL) resistance in serovar Typhi. As the prevalence and epidemiological relevance of such mechanisms has never been systematically assessed in , in this study we carried out a follow-up retrospective analysis of UK isolates previously sequenced as part of routine surveillance protocols between 2016 and 2021. Using a high-throughput bioinformatics pipeline we screened 47 784 isolates for the presence of the P1 lytic replication gene , identifying 226 positive isolates from 25 serovars and demonstrating that phage-plasmid elements are more frequent than previously thought. The affinity for phage-plasmids appears highly serovar-dependent, with several serovars being more likely hosts than others; most of the positive isolates (170/226) belonged to . Typhimurium ST34 and ST19. The phage-plasmids ranged between 85.8 and 98.2 kb in size, with an average length of 92.1 kb; detailed analysis indicated a high amount of diversity in gene content and genomic architecture. In total, 132 phage-plasmids had the p0111 plasmid replication type, and 94 the IncY type; phylogenetic analysis indicated that both horizontal and vertical gene transmission mechanisms are likely to be involved in phage-plasmid propagation. Finally, phage-plasmids were present in isolates that were resistant and non-resistant to antimicrobials. In addition to providing a first comprehensive view of the presence of phage-plasmids in , our work highlights the need for a better surveillance and understanding of phage-plasmids as AMR carriers, especially through their characterization with long-read sequencing.
Topics: Plasmids; Salmonella enterica; Serogroup; Salmonella Infections; Bacteriophages; Salmonella Phages; Humans; Phylogeny; Gene Transfer, Horizontal; Retrospective Studies
PubMed: 38717818
DOI: 10.1099/mgen.0.001247 -
BMC Microbiology Oct 2023The purpose of this study is to re-sensitive bacteria to carbapenemases and reduce the transmission of the bla gene by curing the bla-harboring plasmid of...
OBJECTIVE
The purpose of this study is to re-sensitive bacteria to carbapenemases and reduce the transmission of the bla gene by curing the bla-harboring plasmid of carbapenem-resistant using the CRISPR-Cas9 system.
METHODS
The single guide RNA (sgRNA) specifically targeted to the bla gene was designed and cloned into plasmid pCas9. The recombinant plasmid pCas9-sgRNA(bla) was transformed into Escherichia coli (E.coli) carrying pET24-bla. The elimination efficiency in strains was evaluated by polymerase chain reaction (PCR) and quantitative real-time PCR (qPCR). Susceptibility testing was performed by broth microdilution assay and by E-test strips (bioMérieux, France) to detect changes in bacterial drug resistance phenotype after drug resistance plasmid clearance.
RESULTS
In the present study, we constructed a specific prokaryotic CRISPR-Cas9 system plasmid targeting cleavage of the bla gene. PCR and qPCR results indicated that prokaryotic CRISPR-Cas9 plasmid transforming drug-resistant bacteria can efficiently clear bla-harboring plasmids. In addition, the drug susceptibility test results showed that the bacterial resistance to imipenem was significantly reduced and allowed the resistant model bacteria to restore susceptibility to antibiotics after the bla-containing drug-resistant plasmid was specifically cleaved by the CRISPR-Cas system.
CONCLUSION
In conclusion, our study demonstrated that the one plasmid-mediated CRISPR-Cas9 system can be used as a novel tool to remove resistance plasmids and re-sensitize the recipient bacteria to antibiotics. This strategy provided a great potential to counteract the ever-worsening spread of the bla gene among bacterial pathogens and laid the foundation for subsequent research using the CRISPR-Cas9 system as adjuvant antibiotic therapy.
Topics: Anti-Bacterial Agents; Bacterial Proteins; beta-Lactamases; Carbapenems; CRISPR-Cas Systems; Escherichia coli; Plasmids; RNA, Guide, CRISPR-Cas Systems; Drug Resistance, Bacterial
PubMed: 37884864
DOI: 10.1186/s12866-023-03058-7 -
Microbiology Spectrum Aug 2023Escherichia coli sequence type 131 (ST131) has contributed to the spread of extended-spectrum beta-lactamase (ESBL) and has emerged as the dominant cause of hospital-...
Escherichia coli sequence type 131 (ST131) has contributed to the spread of extended-spectrum beta-lactamase (ESBL) and has emerged as the dominant cause of hospital- and community-acquired urinary tract infections. Here, we report for the first time an in-depth analysis of whole-genome sequencing (WGS) of 4 ESBL-producing E. coli ST131 isolates recovered from patients in two hospitals in Armenia using Illumina short-read sequencing for accurate base calling to determine their genotype and to infer their phylogeny and using Oxford Nanopore Technologies long-read sequencing to resolve plasmid and chromosomal genetic elements. Genotypically, the four Armenian isolates were identified as part of the 30Rx/clade C2 ( = 2) and 41/clade A ( = 2) lineages and were phylogenetically closely related to isolates from the European Nucleotide Archive (ENA) database previously recovered from patients in the United States, Australia, and New Zealand. The Armenian isolates recovered in this study had chromosomal integration of the gene in the 30Rx isolates and a high number of virulence genes found in the 41 isolates associated with the carriage of a rare genomic island (in the context of E. coli ST131) containing the S fimbrial, salmochelin siderophore, and microcin H47 virulence genes. Furthermore, our data show the evolution of the IncF[2:A2:B20] plasmid harboring both and genes, derived from the recombination of genes from an IncF[F2:A-:B-] -associated plasmid into the IncF[F1:A2:B20] -associated plasmid backbone seen in two genetically closely related 41 Armenian isolates. Combining short and long reads from whole-genome sequencing analysis provided a genetic context for uncommon genes of clinical importance to better understand transmission and evolutionary features of ESBL-producing uropathogenic E. coli (UPEC) ST131 isolates recovered in Armenia. Using hybrid genome assembly in countries lacking genomic surveillance studies can inform us about new lineages not seen in other countries with genes encoding high virulence and antibiotic resistance harbored on mobile genetic elements.
Topics: Humans; Escherichia coli; Plasmids; Escherichia coli Infections; Escherichia coli Proteins; beta-Lactamases; Anti-Bacterial Agents
PubMed: 37466446
DOI: 10.1128/spectrum.00356-23 -
Communications Biology Oct 2023Salmonella enterica serotype 1,4,[5],12:i:- (Typhimurium monophasic variant) of sequence type (ST) 34 has emerged as the predominant pandemic genotype in recent decades....
Salmonella enterica serotype 1,4,[5],12:i:- (Typhimurium monophasic variant) of sequence type (ST) 34 has emerged as the predominant pandemic genotype in recent decades. Despite increasing reports of resistance to antimicrobials in Southeast Asia, Salmonella ST34 population structure and evolution remained understudied in the region. Here we performed detailed genomic investigations on 454 ST34 genomes collected from Vietnam and diverse geographical sources to elucidate the pathogen's epidemiology, evolution and antimicrobial resistance. We showed that ST34 has been introduced into Vietnam in at least nine occasions since 2000, forming five co-circulating major clones responsible for paediatric diarrhoea and bloodstream infection. Most expansion events were associated with acquisitions of large multidrug resistance plasmids of IncHI2 or IncA/C2. Particularly, the self-conjugative IncA/C2 pST34VN2 (co-transferring bla, mcr-3.1, and qnrS1) underlies local expansion and intercontinental spread in two separate ST34 clones. At the global scale, Southeast Asia was identified as a potential hub for the emergence and dissemination of multidrug resistant Salmonella ST34, and mutation analysis suggests of selection in antimicrobial responses and key virulence factors.
Topics: Humans; Child; Salmonella enterica; Serogroup; Drug Resistance, Multiple, Bacterial; Plasmids; Salmonella; Asia, Southeastern; Anti-Infective Agents
PubMed: 37789208
DOI: 10.1038/s42003-023-05365-1 -
ACS Synthetic Biology Dec 2023There is a growing need for applications capable of handling large synthesis biology experiments. At the core of synthetic biology is the process of cloning and...
There is a growing need for applications capable of handling large synthesis biology experiments. At the core of synthetic biology is the process of cloning and manipulating DNA as plasmids. Here, we report the development of an application named DNAda capable of writing automation instructions for any given DNA construct design generated by the J5 DNA assembly program. We also describe the automation pipeline and several useful features. The pipeline is particularly useful for the construction of combinatorial DNA assemblies. Furthermore, we demonstrate the platform by constructing a library of polyketide synthase parts, which includes 120 plasmids ranging in size from 7 to 14 kb from 4 to 7 DNA fragments.
Topics: Plasmids; DNA; Gene Library; Automation; Synthetic Biology; Cloning, Molecular
PubMed: 37948662
DOI: 10.1021/acssynbio.3c00292 -
Sheng Wu Gong Cheng Xue Bao = Chinese... Nov 2023Plasmids are the most commonly used gene carriers in the field of gene synthesis and sequencing. However, the main problems faced by traditional plasmid DNA extraction...
Plasmids are the most commonly used gene carriers in the field of gene synthesis and sequencing. However, the main problems faced by traditional plasmid DNA extraction technology are low extraction throughput and high production cost, so they cannot meet the growing demand. In this study, a double-magnetic-bead method (DMBM) for plasmid extraction was developed based on the principle of plasmid extraction. The effects of the input of magnetic beads, the size of plasmid DNA fragments, and the volume of bacterial on plasmid DNA extraction were explored. In addition, the quality, throughput, and cost of plasmid DNA extraction were also compared between this technique and the commercial plasmid DNA extraction kits. The results showed that the DMBM can meet the needs of extracting plasmid DNA with different cell densities and fragment lengths. Moreover, the sensitivity and quality of plasmid extraction by the DMBM method were both superior to those of the centrifugal adsorption column method. In addition, this technique could be applied on a 96-channel automated nucleic acid extractor, resulting in higher purity of the extracted plasmid DNA, 80% reduction in extraction time, and 57.1% reduction in cost. It also reduces manual operations, achieving high-throughput and low-cost plasmid DNA extraction, thus may facilitate gene synthesis and sequencing.
Topics: Plasmids; DNA; Nucleic Acids; Genetic Techniques; Magnetic Phenomena
PubMed: 38013194
DOI: 10.13345/j.cjb.230277 -
Frontiers in Cellular and Infection... 2023Today, the emergence of with the gene cassette in patients has presented a significant clinical challenge.
OBJECTIVE
Today, the emergence of with the gene cassette in patients has presented a significant clinical challenge.
METHODS
To present the detailed genetic features of the gene cassette of strain F4_plasmid pA, the whole bacterial genome was sequenced by Illumina and nanopore platforms, and mobile genetic elements related to antibiotic resistance genes were analyzed with a series of bioinformatics methods.
RESULTS
strain F4 was determined to be a class A+C beta-lactamase, and was resistant to routinely used antibiotics, especially tigecycline, because of the gene localized on the F4_chromosome and on F4_plasmid A. After plasmid transfer assays, the F4_plasmid pA or F4_plasmid pB could be recovered with an average conjugation frequencies of 3.42×10 or 4.19×10. F4_plasmid pA carried and accompanied by genetic intermixing of Tn, Tn, Tn, and In641, while F4_plasmid pB, bearing , had structural overlap of Tn and In641.
CONCLUSIONS
We suggested that plasmids carrying might be strongly related to IS-integrated loop intermediates. This study showed that due to the structural evolution of F4 and related strains, their resistances were so strong that effective antibiotics were virtually unavailable, therefore their spread and prevalence should be strictly controlled.
Topics: Humans; Klebsiella pneumoniae; Klebsiella Infections; Plasmids; beta-Lactamases; Anti-Bacterial Agents; Microbial Sensitivity Tests
PubMed: 37900313
DOI: 10.3389/fcimb.2023.1260066 -
Applied Microbiology and Biotechnology May 2024The emergence and quick spread of the plasmid-mediated tigecycline resistance gene tet(X4) and colistin resistance gene mcr-1 have posed a great threat to public health...
The emergence and quick spread of the plasmid-mediated tigecycline resistance gene tet(X4) and colistin resistance gene mcr-1 have posed a great threat to public health and raised global concerns. It is imperative to develop rapid and accurate detection systems for the onsite surveillance of mcr-1 and tet(X4). In this study, we developed one-tube recombinase polymerase amplification (RPA) and CRISPR-Cas12b integrated mcr-1 and tet(X4) detection systems. We identified mcr-1- and tet(X4)-conserved and -specific protospacers through a comprehensive BLAST search based on the NCBI nt database and used them for assembling the detection systems. Our developed one-tube RPA-CRISPR-Cas12b-based detection systems enabled the specific detection of mcr-1 and tet(X4) with a sensitivity of 6.25 and 9 copies within a detection time of ~ 55 and ~ 40 min, respectively. The detection results using pork and associated environmental samples collected from retail markets demonstrated that our developed mcr-1 and tet(X4) detection systems could successfully monitor mcr-1 and tet(X4), respectively. Notably, mcr-1- and tet(X4)-positive strains were isolated from the positive samples, as revealed using the developed detection systems. Whole-genome sequencing of representative strains identified an mcr-1-carrying IncI2 plasmid and a tet(X4)-carrying IncFII plasmid, which are known as important vectors for mcr-1 and tet(X4) transmission, respectively. Taken together, our developed one-tube RPA-CRISPR-Cas12b-based mcr-1 and tet(X4) detection systems show promising potential for the onsite detection of mcr-1 and tet(X4). KEY POINTS: • One-tube RPA-CRISPR-Cas12b-based mcr-1 and tet(X4) detection systems were developed based on identified novel protospacers. • Both detection systems exhibited high sensitivity and specification with a sample-to-answer time of less than 1 h. • The detection systems show promising potential for onsite detection of mcr-1 and tet(X4).
Topics: CRISPR-Cas Systems; Escherichia coli Proteins; Escherichia coli; Plasmids; Drug Resistance, Bacterial; Swine; Animals; Colistin; Sensitivity and Specificity; Nucleic Acid Amplification Techniques; Anti-Bacterial Agents
PubMed: 38801527
DOI: 10.1007/s00253-024-13191-6