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1.
Biochemistry (Mosc) ; 89(4): 653-662, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38831502

RESUMO

Chromosome conformation capture techniques have revolutionized our understanding of chromatin architecture and dynamics at the genome-wide scale. In recent years, these methods have been applied to a diverse array of species, revealing fundamental principles of chromosomal organization. However, structural organization of the extrachromosomal entities, like viral genomes or plasmids, and their interactions with the host genome, remain relatively underexplored. In this work, we introduce an enhanced 4C-protocol tailored for probing plasmid DNA interactions. We design specific plasmid vector and optimize protocol to allow high detection rate of contacts between the plasmid and host DNA.


Assuntos
Plasmídeos , Plasmídeos/metabolismo , Plasmídeos/genética , DNA/química , DNA/genética , Cromatina/genética , Cromatina/metabolismo , Cromatina/química , Genoma
2.
Food Microbiol ; 122: 104568, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-38839227

RESUMO

The plasmid of emerging S. Infantis (pESI) or pESI-like plasmid in Salmonella enterica Infantis are consistently reported in poultry and humans worldwide. However, there has been limited research on these plasmids of S. Infantis isolated from eggs. Therefore, this study aimed to analyze the prevalence and characteristics of S. Infantis carrying the pESI-like plasmid from eggs in egg grading and packing plants. In this study, the pESI-like plasmid was only detected in 18 (78.3%) of 23 S. Infantis isolates, and it was absent in the other 9 Salmonella serovars. In particular, S. Infantis isolates carrying the pESI-like plasmid showed the significantly higher resistance to ß-lactams, phenicols, cephams, aminoglycosides, quinolones, sulfonamides, and tetracyclines than Salmonella isolates without the pESI-like plasmid (p < 0.05). Moreover, all S. Infantis isolates carrying the pESI-like plasmid were identified as extended-spectrum ß-lactamase (ESBL) producer, harboring the blaCTX-M-65 and blaTEM-1 genes, and carried non-ß-lactamase resistance genes (ant(3'')-Ia, aph(4)-Ia, aac(3)-IVa, aph(3')-Ic, sul1, tetA, dfrA14, and floR) against five antimicrobial classes. However, all isolates without the pESI-like plasmid only carried the blaTEM-1 gene among the ß-lactamase genes, and either had no non-ß-lactamase resistance genes or harbored non-ß-lactamase resistance genes against one or two antimicrobial classes. Furthermore, all S. Infantis isolates carrying the pESI-like plasmid carried class 1 and 2 integrons and the aadA1 gene cassette, but none of the other isolates without the pESI-like plasmid harbored integrons. In particular, D87Y substitution in the gyrA gene and IncP replicon type were observed in all the S. Infantis isolates carrying the pESI-like plasmid but not in the S. Infantis isolates without the pESI-like plasmid. The distribution of pulsotypes between pESI-positive and pESI-negative S. Infantis isolates was clearly distinguished, but all S. Infantis isolates were classified as sequence type 32, regardless of whether they carried the pESI-like plasmid. This study is the first to report the characteristics of S. Infantis carrying the pESI-like plasmid isolated from eggs and can provide valuable information for formulating strategies to control the spread of Salmonella in the egg industry worldwide.


Assuntos
Antibacterianos , Ovos , Plasmídeos , beta-Lactamases , Plasmídeos/genética , República da Coreia , Antibacterianos/farmacologia , Ovos/microbiologia , Animais , beta-Lactamases/genética , Salmonella/genética , Salmonella/isolamento & purificação , Salmonella/classificação , Salmonella/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Farmacorresistência Bacteriana Múltipla/genética , Galinhas/microbiologia , Humanos , Salmonella enterica/genética , Salmonella enterica/isolamento & purificação , Salmonella enterica/efeitos dos fármacos , Salmonella enterica/classificação
3.
Pak J Biol Sci ; 27(5): 268-275, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38840467

RESUMO

<b>Background and Objective:</b> Urinary tract infections from the use of an indwelling urinary catheter are one of the most common infections caused by <i>Proteus mirabilis</i>. Due to their biofilm-producing capacity and the increasing antimicrobial resistance in this microorganism, this study aimed to determine the prevalence, biofilm-producing capacity, antimicrobial resistance patterns, multidrug resistance and plasmid mediated resistance of the recovered isolates. <b>Materials and Methods:</b> A total of 50 urinary samples were collected from May to August, 2018 from patients on indwelling urinary catheters. Using routine microbiological and biochemical methods, 37 <i>P. mirabilis</i> were isolated. Biofilm forming capability was determined among the isolates using the tube method while antimicrobial susceptibility and plasmid curing were also performed. <b>Results:</b> All isolates were biofilm producers with 17(46%) being moderate producers while 20(54%) were strong biofilm formers. The study isolates exhibited a high resistance rate to empiric antibiotics, including ceftazidime (75.8%), cefuroxime (54.5%), ampicillin (69.7%) and amoxicillin-clavulanic acid (51.5%). Low resistance was seen in the fluoroquinolones, gentamicin and nitrofurantoin. Plasmid curing experiment revealed that most isolates lost their resistance indicating that resistance was borne on plasmids. Plasmid carriage is likely the reason for the high MDR rate of 56.8% observed. <b>Conclusion:</b> These findings necessitate the provision of infection control programs which will guide and implement policies.


Assuntos
Antibacterianos , Biofilmes , Cateteres de Demora , Testes de Sensibilidade Microbiana , Proteus mirabilis , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Proteus mirabilis/efeitos dos fármacos , Proteus mirabilis/genética , Proteus mirabilis/isolamento & purificação , Cateteres de Demora/microbiologia , Cateteres de Demora/efeitos adversos , Humanos , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Infecções Urinárias/microbiologia , Infecções Urinárias/tratamento farmacológico , Infecções Urinárias/diagnóstico , Plasmídeos/genética , Cateteres Urinários/microbiologia , Cateteres Urinários/efeitos adversos , Farmacorresistência Bacteriana , Infecções por Proteus/microbiologia , Infecções por Proteus/tratamento farmacológico , Infecções Relacionadas a Cateter/microbiologia , Infecções Relacionadas a Cateter/diagnóstico , Infecções Relacionadas a Cateter/tratamento farmacológico , Feminino , Masculino , Farmacorresistência Bacteriana Múltipla/genética
4.
BMC Genomics ; 25(1): 568, 2024 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-38840068

RESUMO

BACKGROUND: Transgenic (Tg) mice are widely used in biomedical research, and they are typically generated by injecting transgenic DNA cassettes into pronuclei of one-cell stage zygotes. Such animals often show unreliable expression of the transgenic DNA, one of the major reasons for which is random insertion of the transgenes. We previously developed a method called "pronuclear injection-based targeted transgenesis" (PITT), in which DNA constructs are directed to insert at pre-designated genomic loci. PITT was achieved by pre-installing so called landing pad sequences (such as heterotypic LoxP sites or attP sites) to create seed mice and then injecting Cre recombinase or PhiC31 integrase mRNAs along with a compatible donor plasmid into zygotes derived from the seed mice. PITT and its subsequent version, improved PITT (i-PITT), overcome disadvantages of conventional Tg mice such as lack of consistent and reliable expression of the cassettes among different Tg mouse lines, and the PITT approach is superior in terms of cost and labor. One of the limitations of PITT, particularly using Cre-mRNA, is that the approach cannot be used for insertion of conditional expression cassettes using Cre-LoxP site-specific recombination. This is because the LoxP sites in the donor plasmids intended for achieving conditional expression of the transgene will interfere with the PITT recombination reaction with LoxP sites in the landing pad. RESULTS: To enable the i-PITT method to insert a conditional expression cassette, we modified the approach by simultaneously using PhiC31o and FLPo mRNAs. We demonstrate the strategy by creating a model containing a conditional expression cassette at the Rosa26 locus with an efficiency of 13.7%. We also demonstrate that inclusion of FLPo mRNA excludes the insertion of vector backbones in the founder mice. CONCLUSIONS: Simultaneous use of PhiC31 and FLP in i-PITT approach allows insertion of donor plasmids containing Cre-loxP-based conditional expression cassettes.


Assuntos
Genoma , Integrases , Camundongos Transgênicos , Animais , Camundongos , Integrases/genética , Integrases/metabolismo , Transgenes , Marcação de Genes/métodos , Técnicas de Transferência de Genes , Plasmídeos/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Mutagênese Insercional
5.
Front Cell Infect Microbiol ; 14: 1375872, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38846355

RESUMO

Introduction: Pseudomonas aeruginosa is notorious for its multidrug resistance and its involvement in hospital-acquired infections. In this study, 20 bacterial strains isolated from soil samples near the Hindan River in Ghaziabad, India, were investigated for their biochemical and morphological characteristics, with a focus on identifying strains with exceptional drug resistance and pyocyanin production. Methods: The isolated bacterial strains were subjected to biochemical and morphological analyses to characterize their properties, with a particular emphasis on exopolysaccharide production. Strain GZB16/CEES1, exhibiting remarkable drug resistance and pyocyanin production. Biochemical and molecular analyses, including sequencing of its 16S rRNA gene (accession number LN735036.1), plasmid-curing assays, and estimation of plasmid size, were conducted to elucidate its drug resistance mechanisms and further pyocynin based target the Candida albicans Strain GZB16/CEES1 demonstrated 100% resistance to various antibiotics used in the investigation, with plasmid-curing assays, suggesting plasmid-based resistance gene transmission. The plasmid in GZB16/CEES1 was estimated to be approximately 24 kb in size. The study focused on P. aeruginosa's pyocyanin production, revealing its association with anticandidal activity. The minimum inhibitory concentration (MIC) of the bacterial extract against Candida albicans was 50 µg/ml, with a slightly lower pyocyanin-based MIC of 38.5 µg/ml. Scanning electron microscopy illustrated direct interactions between P. aeruginosa strains and Candida albicans cells, leading to the destruction of the latter. Discussion: These findings underscore the potential of P. aeruginosa in understanding microbial interactions and developing strategies to combat fungal infections. The study highlights the importance of investigating bacterial-fungal interactions and the role of pyocyanin in antimicrobial activity. Further research in this area could lead to the development of novel therapeutic approaches for combating multidrug-resistant infections.


Assuntos
Antifúngicos , Candida albicans , Farmacorresistência Bacteriana Múltipla , Testes de Sensibilidade Microbiana , Plasmídeos , Pseudomonas aeruginosa , Piocianina , RNA Ribossômico 16S , Microbiologia do Solo , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Piocianina/metabolismo , Farmacorresistência Bacteriana Múltipla/genética , Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Candida albicans/genética , Candida albicans/crescimento & desenvolvimento , RNA Ribossômico 16S/genética , Índia , Plasmídeos/genética , Antibacterianos/farmacologia , Antibiose
6.
Zhonghua Yi Xue Za Zhi ; 104(22): 2066-2073, 2024 Jun 11.
Artigo em Chinês | MEDLINE | ID: mdl-38858217

RESUMO

Objective: To prepare interleukin-1ß-targeted nanoantibodies and observe their effects on apoptosis in hypoxic cardiomyocyte of mice. Methods: Using DNA recombination technology, the pET-16b and pHEN1 expression vectors were used to construct the prokaryotic expression plasmids of interleukin-1ß-targeted nanobodies (pET-16b-4G6M-VHH, pET-16b-5BVP-VHH, pET-16b-5MVZ-VHH, pHEN1-4G6M-VHH, pHEN1-5BVP-VHH and pHEN1-5MVZ-VHH, where VHH is a variable domain of heavy chain antibody, 4G6M-VHH, 5BVP-VHH, 5MVZ-VHH were three interleukin-1ß-targeted nanoantibodies respectively). The constructed plasmids were transferred into Escherichia coli Rosetta2 (DE3) for induction of expression and nickel column purification, respectively. The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting were employed to identify the expression product and purified product, and the enzyme-linked immune sorbent assay (ELISA) was performed to determine their affinity. The cardiomyocyte hypoxia model was used with the highest affinity IL-1ß-targeted nanobody (pHEN1-5MVZ-VHH), and cell survival and apoptosis rates were detected (the experiment was divided into normal control group, hypoxia model group, blank plasmid group and 12.5, 25.0, 50.0 µg/ml pHEN1-5MVZ-VHH treatment groups). Results: SDS-PAGE and Western blotting results showed that the anti-interleukin-1ß (IL-1ß) nanobodies with a relative molecular mass of about 15 000 were successfully obtained. Likewise, ELISA results found that the nanobodies expressed in pHEN1 vector group had higher affinity for IL-1ß antigen compared with pET-16b vector group (4G6M-VHH group: 3.20±0.03 vs 1.20±0.03, P<0.001; 5BVP-VHH group: 3.18±0.06 vs 1.21±0.02, P<0.001; 5MVZ-VHH group: 3.38±0.05 vs 1.62±0.04, P<0.001). Additionally, the results of cell survival assay and apoptosis assay detected that compared with the hypoxia model group, HL-1 cell activity was significantly increased in the 25.0 µg/ml and 50.0 µg/ml pHEN1-5MVZ-VHH treatment groups [(75.55±2.23)% vs (46.90±2.51)%, P<0.001; (74.36±1.96)% vs (46.90±2.51)%, P<0.001], and apoptosis rate was significantly reduced [(6.83±0.27)% vs (10.24±0.76)%, P<0.001; (6.68±0.38)% vs (10.24±0.76)%, P<0.001]. Conclusions: 4G6M-VHH, 5BVP-VHH, and 5MVZ-VHH are expressed by both pET-16b and pHEN1 expression vectors and the nanobodies produced by the pHEN1 vector display enhanced antigen affinity. Furthermore, in hypoxic cardiomyocytes, pHEN1-5MVZ-VHH treatment reduces cell apoptosis.


Assuntos
Apoptose , Interleucina-1beta , Miócitos Cardíacos , Animais , Interleucina-1beta/metabolismo , Camundongos , Miócitos Cardíacos/metabolismo , Anticorpos de Domínio Único , Plasmídeos , Escherichia coli , Hipóxia
7.
PLoS One ; 19(6): e0304599, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38829840

RESUMO

Extended-spectrum beta-lactamase (ESBL) Escherichia coli (E. coli) is an emerging pathogen of high concern given its resistance to extended-spectrum cephalosporins. Broiler chicken, which is the number one consumed meat in the United States and worldwide, can be a reservoir of ESBL E. coli. Backyard poultry ownership is on the rise in the United States, yet there is little research investigating prevalence of ESBL E. coli in this setting. This study aims to identify the prevalence and antimicrobial resistance profiles (phenotypically and genotypically) of ESBL E. coli in some backyard and commercial broiler farms in the U.S. For this study ten backyard and ten commercial farms were visited at three time-points across flock production. Fecal (n = 10), litter/compost (n = 5), soil (n = 5), and swabs of feeders and waterers (n = 6) were collected at each visit and processed for E. coli. Assessment of ESBL phenotype was determined through using disk diffusion with 3rd generation cephalosporins, cefotaxime and ceftazidime, and that with clavulanic acid. Broth microdilution and whole genome sequencing were used to investigate both phenotypic and genotypic resistance profiles, respectively. ESBL E. coli was more prevalent in backyard farms with 12.95% of samples testing positive whereas 0.77% of commercial farm samples were positive. All isolates contained a blaCTX-M gene, the dominant variant being blaCTX-M-1, and its presence was entirely due to plasmids. Our study confirms concerns of growing resistance to fourth generation cephalosporin, cefepime, as roughly half (51.4%) of all isolates were found to be susceptible dose-dependent and few were resistant. Resistance to non-beta lactams, gentamicin and ciprofloxacin, was also detected in our samples. Our study identifies prevalence of blaCTX-M type ESBL E. coli in U.S. backyard broiler farms, emphasizing the need for interventions for food and production safety.


Assuntos
Antibacterianos , Galinhas , Infecções por Escherichia coli , Escherichia coli , Plasmídeos , beta-Lactamases , Animais , beta-Lactamases/genética , Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/isolamento & purificação , Galinhas/microbiologia , Estados Unidos/epidemiologia , Plasmídeos/genética , Infecções por Escherichia coli/epidemiologia , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/veterinária , Prevalência , Antibacterianos/farmacologia , Doenças das Aves Domésticas/microbiologia , Doenças das Aves Domésticas/epidemiologia , Testes de Sensibilidade Microbiana , Fezes/microbiologia , Proteínas de Escherichia coli/genética , Fazendas
8.
Euro Surveill ; 29(23)2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38847120

RESUMO

BackgroundThe war in Ukraine led to migration of Ukrainian people. Early 2022, several European national surveillance systems detected multidrug-resistant (MDR) bacteria related to Ukrainian patients.AimTo investigate the genomic epidemiology of New Delhi metallo-ß-lactamase (NDM)-producing Providencia stuartii from Ukrainian patients among European countries.MethodsWhole-genome sequencing of 66 isolates sampled in 2022-2023 in 10 European countries enabled whole-genome multilocus sequence typing (wgMLST), identification of resistance genes, replicons, and plasmid reconstructions. Five bla NDM-1-carrying-P. stuartii isolates underwent antimicrobial susceptibility testing (AST). Transferability to Escherichia coli of a bla NDM-1-carrying plasmid from a patient strain was assessed. Epidemiological characteristics of patients with NDM-producing P. stuartii were gathered by questionnaire.ResultswgMLST of the 66 isolates revealed two genetic clusters unrelated to Ukraine and three linked to Ukrainian patients. Of these three, two comprised bla NDM-1-carrying-P. stuartii and the third bla NDM-5-carrying-P. stuartii. The bla NDM-1 clusters (PstCluster-001, n = 22 isolates; PstCluster-002, n = 8 isolates) comprised strains from seven and four countries, respectively. The bla NDM-5 cluster (PstCluster-003) included 13 isolates from six countries. PstCluster-001 and PstCluster-002 isolates carried an MDR plasmid harbouring bla NDM-1, bla OXA-10, bla CMY-16, rmtC and armA, which was transferrable in vitro and, for some Ukrainian patients, shared by other Enterobacterales. AST revealed PstCluster-001 isolates to be extensively drug-resistant (XDR), but susceptible to cefiderocol and aztreonam-avibactam. Patients with data on age (n = 41) were 19-74 years old; of 49 with information on sex, 38 were male.ConclusionXDR P. stuartii were introduced into European countries, requiring increased awareness and precautions when treating patients from conflict-affected areas.


Assuntos
Antibacterianos , Farmacorresistência Bacteriana Múltipla , Infecções por Enterobacteriaceae , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus , Plasmídeos , Providencia , Sequenciamento Completo do Genoma , beta-Lactamases , Humanos , Ucrânia/epidemiologia , beta-Lactamases/genética , Infecções por Enterobacteriaceae/microbiologia , Infecções por Enterobacteriaceae/epidemiologia , Infecções por Enterobacteriaceae/tratamento farmacológico , Farmacorresistência Bacteriana Múltipla/genética , Providencia/genética , Providencia/isolamento & purificação , Providencia/efeitos dos fármacos , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Europa (Continente)/epidemiologia , Plasmídeos/genética , Masculino , Adulto , Feminino , Pessoa de Meia-Idade , Idoso , Adulto Jovem
9.
Commun Biol ; 7(1): 695, 2024 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-38844513

RESUMO

Infection caused by KPC and NDM carbapenemases co-producing Klebsiella pneumoniae (KPC_NDM_CRKP) poses serious public health concerns. Here, we elucidate the prevalence of a hypertransmissible lncM1 plasmid, pKPC_NDM, co-carrying blaKPC-2 and blaNDM-1 genes in sequence type 1049 K_locus 5 (ST1049-KL5) KPC_NDM_CRKP isolates. Genetic and clonal relatedness analyses using pulsed-field gel electrophoresis, single nucleotide polymorphism analysis and core genome multilocus sequence typing suggested clonal dissemination of ST1049-KL5 KPC_NDM_CRKP strains in our hospital. Whole genome sequencing identified an identical 76,517 bp- blaKPC-2 and blaNDM-1 genes co-carrying IncM1 plasmid pKPC_NDM and a pLVPK-like hypervirulent plasmid in all ST1049-KL5 KPC_NDM_CRKP isolates. pKPC_NDM shared 100% identity with a previously sequenced plasmid CRKP35_unnamed4, demonstrating high transferability in conjugation assay, with conjugation frequencies reaching 10-4 and 10-5 in Escherichia coli and K. pneumoniae recipients, respectively. It also maintained favorable stability and flexible compatibility, with retention rates exceeding 80% after 10 days of continuous passage, and could be compatible with pre-existing blaKPC- or blaNDM-carrying plasmids in recipient strains. This study summarizes the characteristics of KPC_NDM_CRKP outbreaks and highlights the importance of ongoing surveillance and infection control strategies to address the challenges posed by ST1049 K. pneumoniae strains.


Assuntos
Infecções por Klebsiella , Klebsiella pneumoniae , Plasmídeos , beta-Lactamases , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/isolamento & purificação , beta-Lactamases/genética , beta-Lactamases/metabolismo , Plasmídeos/genética , Infecções por Klebsiella/epidemiologia , Infecções por Klebsiella/microbiologia , Humanos , Prevalência , Enterobacteriáceas Resistentes a Carbapenêmicos/genética , Enterobacteriáceas Resistentes a Carbapenêmicos/isolamento & purificação , Antibacterianos/farmacologia , Carbapenêmicos/farmacologia , Sequenciamento Completo do Genoma , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Testes de Sensibilidade Microbiana
10.
Nat Commun ; 15(1): 4731, 2024 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-38830889

RESUMO

Major antibiotic groups are losing effectiveness due to the uncontrollable spread of antimicrobial resistance (AMR) genes. Among these, ß-lactam resistance genes -encoding ß-lactamases- stand as the most common resistance mechanism in Enterobacterales due to their frequent association with mobile genetic elements. In this context, novel approaches that counter mobile AMR are urgently needed. Collateral sensitivity (CS) occurs when the acquisition of resistance to one antibiotic increases susceptibility to another antibiotic and can be exploited to eliminate AMR selectively. However, most CS networks described so far emerge as a consequence of chromosomal mutations and cannot be leveraged to tackle mobile AMR. Here, we dissect the CS response elicited by the acquisition of a prevalent antibiotic resistance plasmid to reveal that the expression of the ß-lactamase gene blaOXA-48 induces CS to colistin and azithromycin. We next show that other clinically relevant mobile ß-lactamases produce similar CS responses in multiple, phylogenetically unrelated E. coli strains. Finally, by combining experiments with surveillance data comprising thousands of antibiotic susceptibility tests, we show that ß-lactamase-induced CS is pervasive within Enterobacterales. These results highlight that the physiological side-effects of ß-lactamases can be leveraged therapeutically, paving the way for the rational design of specific therapies to block mobile AMR or at least counteract their effects.


Assuntos
Antibacterianos , Escherichia coli , Testes de Sensibilidade Microbiana , beta-Lactamases , beta-Lactamases/genética , beta-Lactamases/metabolismo , Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , Antibacterianos/farmacologia , Sensibilidade Colateral a Medicamentos/genética , Plasmídeos/genética , Azitromicina/farmacologia , Colistina/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Resistência beta-Lactâmica/genética
11.
J Vis Exp ; (207)2024 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-38829124

RESUMO

Functional genomics screening offers a powerful approach to probe gene function and relies on the construction of genome-wide plasmid libraries. Conventional approaches for plasmid library construction are time-consuming and laborious. Therefore, we recently developed a simple and efficient method, CRISPR-based modular assembly (CRISPRmass), for high-throughput construction of a genome-wide upstream activating sequence-complementary DNA/open reading frame (UAS-cDNA/ORF) plasmid library. Here, we present a protocol for CRISPRmass, taking as an example the construction of a GAL4/UAS-based UAS-cDNA/ORF plasmid library. The protocol includes massively parallel two-step test tube reactions followed by bacterial transformation. The first step is to linearize the existing complementary DNA (cDNA) or open reading frame (ORF) cDNA or ORF library plasmids by cutting the shared upstream vector sequences adjacent to the 5' end of cDNAs or ORFs using CRISPR/Cas9 together with single guide RNA (sgRNA), and the second step is to insert a UAS module into the linearized cDNA or ORF plasmids using a single step reaction. CRISPRmass allows the simple, fast, efficient, and cost-effective construction of various plasmid libraries. The UAS-cDNA/ORF plasmid library can be utilized for gain-of-function screening in cultured cells and for constructing a genome-wide transgenic UAS-cDNA/ORF library in Drosophila.


Assuntos
Sistemas CRISPR-Cas , Biblioteca Gênica , Fases de Leitura Aberta , Plasmídeos , Plasmídeos/genética , Animais , Sistemas CRISPR-Cas/genética , Fases de Leitura Aberta/genética , DNA Complementar/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Drosophila melanogaster/genética
12.
Antonie Van Leeuwenhoek ; 117(1): 86, 2024 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-38829455

RESUMO

Yersinia is an important genus comprising foodborne, zoonotic and pathogenic bacteria. On the other hand, species of the so-called group Yersinia enterocolitica-like are understudied and mostly characterized as non-pathogenic, despite of some reports of human infections. The present study aimed to provide genomic insights of Yersinia frederiksenii (YF), Yersinia intermedia (YI) and Yersinia kristensenii (YK) isolated worldwide. A total of 22 YF, 20 YI and 14 YK genomes were searched for antimicrobial resistance genes, plasmids, prophages, and virulence factors. Their phylogenomic relatedness was analyzed by Gegenees and core-genome multi-locus sequence typing. Beta-lactam resistance gene blaTEM-116 and five plasmids replicons (pYE854, ColRNAI, ColE10, Col(pHAD28) and IncN3) were detected in less than five genomes. A total of 59 prophages, 106 virulence markers of the Yersinia genus, associated to adherence, antiphagocytosis, exoenzymes, invasion, iron uptake, proteases, secretion systems and the O-antigen, and virulence factors associated to other 20 bacterial genera were detected. Phylogenomic analysis revealed high inter-species distinction and four highly diverse YF clusters. In conclusion, the results obtained through the analyses of YF, YI and YK genomes suggest the virulence potential of these strains due to the broad diversity and high frequency of prophages and virulence factors found. Phylogenetic analyses were able to correctly distinguish these closely related species and show the presence of different genetic subgroups. These data contributed for a better understanding of YF, YI and YK virulence-associated features and global genetic diversity, and reinforced the need for better characterization of these Y. enterocolitica-like species considered non-pathogenic.


Assuntos
Genoma Bacteriano , Filogenia , Fatores de Virulência , Yersinia , Yersinia/genética , Yersinia/classificação , Yersinia/patogenicidade , Yersinia/isolamento & purificação , Fatores de Virulência/genética , Brasil , Yersiniose/microbiologia , Yersiniose/veterinária , Humanos , Genômica , Prófagos/genética , Plasmídeos/genética , Tipagem de Sequências Multilocus , Virulência/genética
13.
J Nanobiotechnology ; 22(1): 325, 2024 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-38858695

RESUMO

BACKGROUND: Osteoarthritis (OA) is an aging-related degenerative joint disorder marked by joint discomfort and rigidity. Senescent chondrocytes release pro-inflammatory cytokines and extracellular matrix-degrading proteins, creating an inflammatory microenvironment that hinders chondrogenesis and accelerates matrix degradation. Targeting of senescent chondrocytes may be a promising approach for the treatment of OA. Herein, we describe the engineering of an injectable peptide-hydrogel conjugating a stem cell-homing peptide PFSSTKT for carrying plasmid DNA-laden nanoparticles and Tanshinon IIA (pPNP + TIIA@PFS) that was designed to attenuate OA progression by improving the senescent microenvironment and fostering cartilage regeneration. RESULTS: Specifically, pPNP + TIIA@PFS elevates the concentration of the anti-aging protein Klotho and blocks the transmission of senescence signals to adjacent healthy chondrocytes, significantly mitigating chondrocyte senescence and enhancing cartilage integrity. Additionally, pPNP + TIIA@PFS recruit bone mesenchymal stem cells and directs their subsequent differentiation into chondrocytes, achieving satisfactory chondrogenesis. In surgically induced OA model rats, the application of pPNP + TIIA@PFS results in reduced osteophyte formation and attenuation of articular cartilage degeneration. CONCLUSIONS: Overall, this study introduces a novel approach for the alleviation of OA progression, offering a foundation for potential clinical translation in OA therapy.


Assuntos
Condrócitos , Condrogênese , Glucuronidase , Hidrogéis , Proteínas Klotho , Células-Tronco Mesenquimais , Osteoartrite , Plasmídeos , Ratos Sprague-Dawley , Animais , Osteoartrite/terapia , Osteoartrite/tratamento farmacológico , Hidrogéis/química , Ratos , Condrócitos/metabolismo , Condrócitos/efeitos dos fármacos , Glucuronidase/metabolismo , Glucuronidase/farmacologia , Condrogênese/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Masculino , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/metabolismo , Progressão da Doença , Nanopartículas/química , Humanos , DNA , Senescência Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos
14.
Nat Commun ; 15(1): 5011, 2024 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-38866742

RESUMO

Site-directed insertion is a powerful approach for generating mutant alleles, but low efficiency and the need for customisation for each target has limited its application. To overcome this, we developed a highly efficient targeted insertional mutagenesis system, CRIMP, and an associated plasmid toolkit, CRIMPkit, that disrupts native gene expression by inducing complete transcriptional termination, generating null mutant alleles without inducing genetic compensation. The protocol results in a high frequency of integration events and can generate very early targeted insertions, during the first cell division, producing embryos with expression in one or both halves of the body plan. Fluorescent readout of integration events facilitates selection of successfully mutagenized fish and, subsequently, visual identification of heterozygous and mutant animals. Together, these advances greatly improve the efficacy of generating and studying mutant lines. The CRIMPkit contains 24 ready-to-use plasmid vectors to allow easy and complete mutagenesis of any gene in any reading frame without requiring custom sequences, modification, or subcloning.


Assuntos
Sistemas CRISPR-Cas , Mutagênese Insercional , Plasmídeos , Peixe-Zebra , Mutagênese Insercional/métodos , Animais , Plasmídeos/genética , Peixe-Zebra/genética , Vetores Genéticos/genética , Edição de Genes/métodos , Alelos
15.
PLoS One ; 19(6): e0305431, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38865304

RESUMO

BACKGROUND: The incidence of antimicrobial resistance is alarmingly high because it occurs in humans, environment, and animal sectors from a "One Health" viewpoint. The emergence of plasmid-carried mobile colistin-resistance (MCR) genes limits the efficacy of colistin, which is the last-line treatment for multidrug resistance (MDR) against gram-negative infections. OBJECTIVES: The current study aimed to investigate emergence of colistin-resistance (MCR 1-5) genes in E. coli isolated from patients with urinary tract infections (UTIs) in Jordan. METHODS: E. coli (n = 132) were collected from urine specimens. The E. coli isolated from human UTI patients were examined the resistance to colistin based on the presence of MCR (1-5). All isolates were tested against 20 antimicrobials using the standard disk diffusion method. The broth microdilution technique was used to analyze colistin resistance. In addition, the MCR (1-5) genes were detected using multiplex PCR. RESULTS: Out of the 132 isolates, 1 isolate was colistin-resistant, having a minimum inhibitory concentration of 8 µg/mL and possessing MCR-1. All the E. coli isolates showed high resistance to penicillin (100%), amoxicillin (79.55%), cephalexin (75.76%), nalidixic acid (62.88%), tetracycline (58.33%), or cefepime (53.79). CONCLUSION: To our knowledge, this is the first report on the presence of plasmid-coded MCR-1 in E. coli from a patient with UTIs in Jordan. This is a problematic finding because colistin is the last-line drug for the treatment of infections caused by MDR gram-negative bacteria. There is a crucial need to robustly utilize antibiotics to control and prevent the emergence and prevalence of colistin-resistance genes.


Assuntos
Antibacterianos , Colistina , Infecções por Escherichia coli , Escherichia coli , Testes de Sensibilidade Microbiana , Infecções Urinárias , Humanos , Colistina/farmacologia , Infecções Urinárias/microbiologia , Infecções Urinárias/tratamento farmacológico , Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/isolamento & purificação , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Feminino , Masculino , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/tratamento farmacológico , Adulto , Pessoa de Meia-Idade , Proteínas de Escherichia coli/genética , Farmacorresistência Bacteriana/genética , Idoso , Jordânia , Adolescente , Adulto Jovem , Farmacorresistência Bacteriana Múltipla/genética , Plasmídeos/genética , Criança
16.
Nat Commun ; 15(1): 4956, 2024 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-38858376

RESUMO

A crucial step in life processes is the transfer of accurate and correct genetic material to offspring. During the construction of autonomous artificial cells, a very important step is the inheritance of genetic information in divided artificial cells. The ParMRC system, as one of the most representative systems for DNA segregation in bacteria, can be purified and reconstituted into GUVs to form artificial cells. In this study, we demonstrate that the eGFP gene is segregated into two poles by a ParM filament with ParR as the intermediate linker to bind ParM and parC-eGFP DNA in artificial cells. After the ParM filament splits, the cells are externally induced to divide into two daughter cells that contain parC-eGFP DNA by osmotic pressure and laser irradiation. Using a PURE system, we translate eGFP DNA into enhanced green fluorescent proteins in daughter cells, and bacterial plasmid segregation and inheritance are successfully mimicked in artificial cells. Our results could lead to the construction of more sophisticated artificial cells that can reproduce with genetic information.


Assuntos
Células Artificiais , Proteínas de Fluorescência Verde , Plasmídeos , Plasmídeos/genética , Plasmídeos/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Fluorescência Verde/genética , Células Artificiais/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Segregação de Cromossomos , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo
17.
Sheng Wu Gong Cheng Xue Bao ; 40(5): 1559-1570, 2024 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-38783816

RESUMO

To develop an accurate and efficient protocol for multi-fragment assembly and multi-site mutagenesis, we integrated and optimized the common multi-fragment assembly methods and validated the established method by using fructose-1,6-diphosphatase 1 (FBP1) with 4 mutant sites. The fragments containing mutations were assembled by introducing mutant sites and Bsa I recognition sequences. After digestion/ligation, the ligated fragment was amplified with the primers containing overlap region to the linearized vector. The amplified fragment was ligated to the linearized vector and the ligation product was transformed into Escherichia coli. After screening and sequencing, the recombinant plasmid with 4 mutant sites was obtained. This protocol overcame the major defects of Gibson assembly and Golden Gate assembly, serving as an efficient solution for multi-fragment assembly and multi-site mutagenesis.


Assuntos
Escherichia coli , Frutose-Bifosfatase , Recombinação Homóloga , Escherichia coli/genética , Escherichia coli/metabolismo , Frutose-Bifosfatase/genética , Frutose-Bifosfatase/metabolismo , Plasmídeos/genética , Vetores Genéticos/metabolismo , DNA/genética , Mutação , Mutagênese Sítio-Dirigida , Clonagem Molecular
18.
PLoS One ; 19(5): e0303999, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38781126

RESUMO

Serine integrases (Ints) are a family of site-specific recombinases (SSRs) encoded by some bacteriophages to integrate their genetic material into the genome of a host. Their ability to rearrange DNA sequences in different ways including inversion, excision, or insertion with no help from endogenous molecular machinery, confers important biotechnological value as genetic editing tools with high host plasticity. Despite advances in their use in prokaryotic cells, only a few Ints are currently used as gene editors in eukaryotes, partly due to the functional loss and cytotoxicity presented by some candidates in more complex organisms. To help expand the number of Ints available for the assembly of more complex multifunctional circuits in eukaryotic cells, this protocol describes a platform for the assembly and functional screening of serine-integrase-based genetic switches designed to control gene expression by directional inversions of DNA sequence orientation. The system consists of two sets of plasmids, an effector module and a reporter module, both sets assembled with regulatory components (as promoter and terminator regions) appropriate for expression in mammals, including humans, and plants. The complete method involves plasmid design, DNA delivery, testing and both molecular and phenotypical assessment of results. This platform presents a suitable workflow for the identification and functional validation of new tools for the genetic regulation and reprogramming of organisms with importance in different fields, from medical applications to crop enhancement, as shown by the initial results obtained. This protocol can be completed in 4 weeks for mammalian cells or up to 8 weeks for plant cells, considering cell culture or plant growth time.


Assuntos
Células Eucarióticas , Integrases , Integrases/metabolismo , Integrases/genética , Humanos , Células Eucarióticas/metabolismo , Plasmídeos/genética , Serina/metabolismo , Edição de Genes/métodos
19.
West Afr J Med ; 41(3): 301-310, 2024 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-38788127

RESUMO

INTRODUCTION: According to the World Health Organization, antimicrobial resistance (AMR) is a silent global pandemic that plagues everyone. It makes therapy of infectious diseases more difficult and eventually increases morbidity and mortality. AIM: The purpose of this work is to examine existing data on plasmid-mediated quinolone resistance (PMQR), to assess the prevalence of PMQR genes in Enterobacterales, and to determine any knowledge gaps from sub-Saharan Africa. METHODOLOGY: The Preferred Reporting Items of Systematic Reviews and Meta-analyses (PRISMA) standard was followed when conducting this systematic review. The main internet databases examined for pertinent publications were PubMed, Google Scholar, and Ajol. A set of qualifying criteria were used to evaluate the qualified articles. Using the eligibility criteria, 56 full-text articles were chosen for screening. RESULT: Thirty-two (32) articles with the majority originating from West and North Africa and only one article reporting a study carried out in Central Africa were selected for this review. Escherichia coli and Ciprofloxacin were the most reported Enterobacterales and Quinolone respectively. The PMQR genes include qnr (qnrA,qnrB, qnrC, qnrD, and qnrS), aac (6') Ib, aac (6') Ib-cr, oqxAB and qepA gene. The most prevalent PMQR gene is the aac (6') Ib-cr gene (32%) followed by qnrS (26%). CONCLUSION: This study highlighted the requirement for an efficient antimicrobial resistance surveillance system in the continent and revealed a significant incidence of PMQR genes.


INTRODUCTION: Selon l'Organisation mondiale de la santé, la résistance aux antimicrobiens (RAM) est une pandémie mondiale silencieuse qui touche tout le monde. Elle rend le traitement des maladies infectieuses plus difficile et finit par augmenter la morbidité et la mortalité. OBJECTIF: L'objectif de ce travail est d'examiner les données existantes sur la résistance plasmidique aux quinolones (PMQR), d'évaluer la prévalence des gènes PMQR chez les Enterobacterales et de déterminer d'éventuelles lacunes de connaissances en Afrique subsaharienne. MÉTHODOLOGIE: La norme Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) a été suivie lors de la réalisation de cette revue systématique. Les principales bases de données Internet examinées pour des publications pertinentes étaient PubMed, Google Scholar et Ajol. Un ensemble de critères d'admissibilité a été utilisé pour évaluer les articles qualifiés. En utilisant les critères d'éligibilité, 56 articles en texte intégral ont été choisis pour le dépistage. RÉSULTAT: Trente-deux (32) articles, dont la majorité provient d'Afrique de l'Ouest et du Nord, et un seul article rapportant une étude menée en Afrique centrale, ont été sélectionnés pour cette revue. Escherichia coli et la ciprofloxacine étaient les Enterobacterales et les quinolones les plus signalées respectivement. Les gènes PMQR comprennent les gènes qnr (qnrA, qnrB, qnrC, qnrD et qnrS), aac (6 ') Ib, aac (6 ') Ib-cr, oqxAB et qepA. Le gène PMQR le plus prévalent est le gène aac (6 ') Ib-cr (32 %), suivi de qnrS (26 %). CONCLUSION: Cette étude a souligné la nécessité d'un système efficace de surveillance de la résistance aux antimicrobiens sur le continen`t et a révélé une incidence significative des gènes PMQR. MOTS-CLÉS: Enterobacterales, Escherichia coli, Quinolone, Ciprofloxacine, PMQR, "aac(6')-Ib", "aac(6')-Ib-cr", "qnr", "qepA", "oqxAB", "résistance aux antibiotiques".


Assuntos
Antibacterianos , Farmacorresistência Bacteriana , Infecções por Enterobacteriaceae , Enterobacteriaceae , Fluoroquinolonas , Plasmídeos , Humanos , Fluoroquinolonas/farmacologia , Antibacterianos/farmacologia , Plasmídeos/genética , Farmacorresistência Bacteriana/genética , Enterobacteriaceae/efeitos dos fármacos , Enterobacteriaceae/genética , Infecções por Enterobacteriaceae/epidemiologia , Infecções por Enterobacteriaceae/tratamento farmacológico , Infecções por Enterobacteriaceae/microbiologia , África/epidemiologia
20.
Environ Sci Pollut Res Int ; 31(23): 34295-34308, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38700770

RESUMO

Fertilization can change the composition of antibiotic resistance genes(ARGs) and their host bacteria in agricultural fields, while complex microbial activities help ARGs into crops and transmit them to humans through agricultural products.Therefore, this study constructed a farmland food chain with soil-lettuce-snail as a typical structure, added genetically engineered Pseudomonas fluorescens containing multidrug-resistant plasmid RP4 to track its spread in the farmland food chain, and used different fertilization methods to explore its influence on the spread and diffusion of ARGs and intl1 in the farmland food chain. It was found that exogenous Pseudomonas can enter plants from soil and pass into snails' intestines, and there is horizontal gene transfer phenomenon of RP4 plasmid in bacteria. At different interfaces of the constructed food chain, the addition of exogenous drug-resistant bacteria had different effects on the total abundance of ARGs and intl1. Fertilization, especially manure, not only promoted the spread of Pseudomonas aeruginosa and the transfer of RP4 plasmid levels, but also significantly increased the total abundance of ARGs and intl1 at all interfaces of the constructed food chain. The main ARGs host bacteria in the constructed food chain include Proteobacteria, Bacteroides, and Firmicutes, while Flavobacterium of Bacteroides is the unique potential host bacteria of RP4 plasmid. In conclusion, this study provides a reference for the risk assessment of ARGs transmitted to the human body through the food chain, and has important practical significance to reduce the antibiotic resistance contamination of agricultural products and ensure the safety of vegetable basket.


Assuntos
Resistência Microbiana a Medicamentos , Cadeia Alimentar , Plasmídeos , Microbiologia do Solo , Plasmídeos/genética , Resistência Microbiana a Medicamentos/genética , Animais , Caramujos , Solo/química , Transferência Genética Horizontal , Antibacterianos/farmacologia
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