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1.
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
2.
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
3.
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
4.
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
5.
Int J Food Microbiol ; 420: 110765, 2024 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-38838541

RESUMO

Resistance to carbapenems emerged in clinical settings and has rapidly spread to other sectors, such as food and the environment, representing a One Health problem. In this regard, vegetables contaminated by critical priority pathogens have raised global concerns. Here, we have performed a whole-genome sequence-based analysis of extensively drug-resistant Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa strains isolated from cabbage, spinach, and lettuce, respectively. Genomic analysis revealed the emergence of international and high-risk clones belonging to ST340, ST155, and ST233, harboring a broad resistome to clinically important antimicrobials. In this context, K. pneumoniae, E. coli, and P. aeruginosa strains carried blaKPC-2, blaNDM-1, and blaVIM-2, respectively. The blaKPC-2 gene with a non-Tn4401 element (NTEKPC-Ic) was located on an IncX3-IncU plasmid, while the blaVIM-2 gene was associated with a Tn402-like class 1 integron, In559, on the chromosome. Curiously, the blaNDM-1 gene coexisted with the blaPER-2 gene on an IncC plasmid and the regions harboring both genes contained sequences of Tn3-like element ISKox2-like family transposase. Comparative genomic analysis showed interspecies and clonal transmission of carbapenemase-encoding genes at the human-animal-environmental interface. These findings raise a food safety alert about hospital-associated carbapenemase producers, supporting that fresh vegetables can act as a vehicle for the spread of high-risk clones.


Assuntos
Verduras , beta-Lactamases , beta-Lactamases/genética , beta-Lactamases/metabolismo , Verduras/microbiologia , Inocuidade dos Alimentos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Antibacterianos/farmacologia , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/isolamento & purificação , Pseudomonas aeruginosa/efeitos dos fármacos , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/isolamento & purificação , Klebsiella pneumoniae/enzimologia , Klebsiella pneumoniae/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Microbiologia de Alimentos , Testes de Sensibilidade Microbiana , Farmacorresistência Bacteriana Múltipla/genética , Plasmídeos/genética , Sequenciamento Completo do Genoma , Humanos
6.
Inorg Chem ; 63(24): 11450-11458, 2024 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-38823006

RESUMO

Two Ru(II) complexes, [Ru(pydppn)(bim)(py)]2+ [2; pydppn = 3-(pyrid-2'-yl)-4,5,9,16-tetraaza-dibenzo[a,c]naphthacene; bim = 2,2'-bisimidazole; py = pyridine] and [Ru(pydppn)(Me4bim)(py)]2+ [3; Me4bim = 2,2'-bis(4,5-dimethylimidazole)], were synthesized and characterized, and their photophysical properties, DNA binding, and photocleavage were evaluated and compared to [Ru(pydppn)(bpy)(py)]2+ (1; bpy = 2,2'-bipyridine). Complexes 2 and 3 exhibit broad 1MLCT (metal-to-ligand charge transfer) transitions with maxima at ∼470 nm and shoulders at ∼525 and ∼600 nm that extend to ∼800 nm. These bands are red-shifted relative to those of 1, attributed to the π-donating ability of the bim and Me4bim ligands. A strong signal at 550 nm is observed in the transient absorption spectra of 1-3, previously assigned as arising from a pydppn-centered 3ππ* state, with lifetimes of ∼19 µs for 1 and 2 and ∼270 ns for 3. A number of methods were used to characterize the mode of binding of 1-3 to DNA, including absorption titrations, thermal denaturation, relative viscosity changes, and circular dichroism, all of which point to the intercalation of the pydpppn ligand between the nucleobases. The photocleavage of plasmid pUC19 DNA was observed upon the irradiation of 1-3 with visible and red light, attributed to the sensitized generation of 1O2 by the complexes. These findings indicate that the bim ligand, together with pydppn, serves to shift the absorption of Ru(II) complexes to the photodynamic therapy window, 600-900 nm, and also extend the excited state lifetimes for the efficient production of cytotoxic singlet oxygen.


Assuntos
Complexos de Coordenação , DNA , Fotoquimioterapia , Fármacos Fotossensibilizantes , Plasmídeos , Rutênio , Oxigênio Singlete , DNA/química , Complexos de Coordenação/química , Complexos de Coordenação/farmacologia , Complexos de Coordenação/síntese química , Complexos de Coordenação/efeitos da radiação , Rutênio/química , Rutênio/farmacologia , Plasmídeos/química , Oxigênio Singlete/metabolismo , Oxigênio Singlete/química , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/farmacologia , Fármacos Fotossensibilizantes/síntese química , Fármacos Fotossensibilizantes/efeitos da radiação , Estrutura Molecular , Clivagem do DNA/efeitos dos fármacos , Clivagem do DNA/efeitos da radiação
7.
Curr Microbiol ; 81(8): 225, 2024 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-38877167

RESUMO

Linezolid resistance in Enterococcus spp. is increasingly considered critically important and a public health threat which mandates the need to understand their genomic contents and dissemination patterns. Here, we used whole-genome sequencing to characterize the resistome, virulome and mobile genetic elements of nine linezolid-resistant (LZDR) enterococci (seven optrA-E. faecalis, one poxtA-E. faecium and one optrA-E. casseliflavus) previously obtained from the nares of healthy dogs, pigs, pig farmers and tracheal samples of nestling storks in Spain. Also, the relatedness of the isolates with publicly available genomes was accessed by core-genome single nucleotide polymorphism (SNP) analysis. The optrA gene of the E. faecalis and E. casseliflavus isolates was located downstream of the fexA gene. The optrA gene in the E. casseliflavus isolate was carried in a plasmid (pURX4962), while those in the seven E. faecalis isolates were chromosomally located. The OptrA proteins were mostly variants of wild type (DP-2: Y176D/T481P; RDK: I104R/Y176D/E256K; DD-3: Y176D/G393D; and EDD: K3E/Y176D/G393D), except two that were wild type (one E. faecalis and one E. casseliflavus). The poxtA gene in the E. faecium isolate was found alone within its contig. The cfrD was upstream of ermB gene in the E. casseliflavus isolate and flanked by ISNCY and IS1216. All the LZDR enterococci carried plasmid rep genes (2-3) containing tetracycline, chloramphenicol and aminoglycoside resistance genes. All isolates except E. casseliflavus carried at least one intact prophage, of which E. faecalis-ST330 (X4957) from a pig carried the highest (n = 5). Tn6260 was associated with lnuG in E. faecalis-ST330 while Tn554 was with fexA in E. feaecalis-ST59 isolates. All except E. casseliflavus (n = 0) carried at least two metal resistance genes (MRGs), of which poxtA-carrying E. faecium-ST1739 isolate contained the most (arsA, copA, fief, ziaA, znuA, zosA, zupT, and zur). SNP-based analyses identified closely related optrA-E. faecalis isolates from a pig and a pig farmer on the same farm (SNP = 4). Moreover, optrA- carrying E. faecalis-ST32, -ST59, and -ST474 isolates from pigs were related to those previously described from humans (sick and healthy) and cattle in Spain, Belgium, and Switzerland (SNP range 43-86). These findings strongly suggest the transmission of LZDR-E. faecalis between a pig and a pig farmer and potential inter-country dissemination. These highlight the need to strengthen molecular surveillance of LZDR enterococci in all ecological niches and body parts to direct appropriate control strategies.


Assuntos
Antibacterianos , Farmacorresistência Bacteriana , Enterococcus , Genoma Bacteriano , Linezolida , Filogenia , Animais , Linezolida/farmacologia , Suínos/microbiologia , Farmacorresistência Bacteriana/genética , Cães , Antibacterianos/farmacologia , Enterococcus/genética , Enterococcus/efeitos dos fármacos , Enterococcus/isolamento & purificação , Enterococcus/classificação , Infecções por Bactérias Gram-Positivas/microbiologia , Infecções por Bactérias Gram-Positivas/transmissão , Infecções por Bactérias Gram-Positivas/veterinária , Humanos , Sequenciamento Completo do Genoma , Espanha , Polimorfismo de Nucleotídeo Único , Testes de Sensibilidade Microbiana , Proteínas de Bactérias/genética , Genômica , Plasmídeos/genética
8.
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
9.
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
10.
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
11.
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
12.
Methods Mol Biol ; 2813: 65-78, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38888770

RESUMO

Reverse genetic methods to manipulate viral genomes are key tools in modern virological experimentation. They allow for the generation of reporter virus genomes to simplify the assessment of virus growth and for the analysis of the impact of specific mutations in the genome on virus phenotypes. For SARS-CoV-2, reverse genetic systems are complicated by the large size of the viral genome and the instability of certain genomic sections in bacteria requiring the use of low-copy number bacterial artificial chromosome plasmids (bacmids). However, even with the use of bacmids, faithfully amplifying SARS-CoV-2 bacmids is often challenging. In this chapter, we describe a detailed protocol to grow SARS-CoV-2 bacmids and highlight the challenges and optimal techniques to produce large quantities of SARS-CoV-2 bacmids that are free of deletions and mutations. Overall, this chapter has recapitulated an overview of the maxi-preparation procedure for large unstable bacmids like SARS-CoV-2 to facilitate downstream applications.


Assuntos
COVID-19 , Cromossomos Artificiais Bacterianos , DNA Complementar , Genoma Viral , Plasmídeos , SARS-CoV-2 , SARS-CoV-2/genética , Plasmídeos/genética , Cromossomos Artificiais Bacterianos/genética , Humanos , COVID-19/virologia , DNA Complementar/genética , Genética Reversa/métodos , RNA Viral/genética
13.
Microb Genom ; 10(6)2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38896471

RESUMO

Carbapenems are last-resort antibiotics for treatment of infections caused by multidrug-resistant Enterobacterales, but carbapenem resistance is a rising global threat due to the acquisition of carbapenemase genes. Oxacillinase-48 (bla OXA-48)-type carbapenemases are increasing in abundance in Canada and elsewhere; these genes are frequently found on mobile genetic elements and are associated with specific transposons. This means that alongside clonal dissemination, bla OXA-48-type genes can spread through plasmid-mediated horizontal gene transfer. We applied whole genome sequencing to characterize 249 bla OXA-48-type-producing Enterobacterales isolates collected by the Canadian Nosocomial Infection Surveillance Program from 2010 to 2021. Using a combination of short- and long-read sequencing, we obtained 70 complete and circular bla OXA-48-type-encoding plasmids. Using MOB-suite, four major plasmids clustered were identified, and we further estimated a plasmid cluster for 91.9 % (147/160) of incomplete bla OXA-48-type-encoding contigs. We identified different patterns of carbapenemase mobilization across Canada, including horizontal transmission of bla OXA-181/IncX3 plasmids (75/249, 30.1 %) and bla OXA-48/IncL/M plasmids (47/249, 18.9 %), and both horizontal transmission and clonal transmission of bla OXA-232 for Klebsiella pneumoniae ST231 on ColE2-type/ColKP3 plasmids (25/249, 10.0 %). Our findings highlight the diversity of OXA-48-type plasmids and indicate that multiple plasmid clusters and clonal transmission have contributed to bla OXA-48-type spread and persistence in Canada.


Assuntos
Proteínas de Bactérias , Carbapenêmicos , Infecções por Enterobacteriaceae , Plasmídeos , Sequenciamento Completo do Genoma , beta-Lactamases , beta-Lactamases/genética , Plasmídeos/genética , Canadá/epidemiologia , Humanos , Carbapenêmicos/farmacologia , Proteínas de Bactérias/genética , Infecções por Enterobacteriaceae/microbiologia , Infecções por Enterobacteriaceae/epidemiologia , Enterobacteriaceae/genética , Enterobacteriaceae/efeitos dos fármacos , Enterobacteriaceae/classificação , Transferência Genética Horizontal , Antibacterianos/farmacologia , Infecção Hospitalar/microbiologia , Infecção Hospitalar/epidemiologia
14.
BMC Microbiol ; 24(1): 214, 2024 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-38886642

RESUMO

BACKGROUND: Bergeyella porcorum is a newly identified bacterium that has an ambiguous relationship with pneumonia in pigs. However, few studies have adequately characterized this species. RESULTS: In this study, we analyzed the morphological, physiological, and genomic characteristics of the newly identified B. porcorum sp. nov. strain QD2021 isolated from pigs. The complete genome sequence of the B. porcorum QD2021 strain consists of a single circular chromosome (2,271,736 bp, 38.51% G + C content), which encodes 2,578 genes. One plasmid with a size of 70,040 bp was detected. A total of 121 scattered repeat sequences, 319 tandem repeat sequences, 4 genomic islands, 5 prophages, 3 CRISPR sequences, and 51 ncRNAs were predicted. The coding genes of the B. porcorum genome were successfully annotated across eight databases (NR, GO, KEGG, COG, TCDB, Pfam, Swiss-Prot and CAZy) and four pathogenicity-related databases (PHI, CARD, VFDB and ARDB). In addition, a comparative genome analysis was performed to explore the evolutionary relationships of B. porcorum QD2021. CONCLUSIONS: To our knowledge, this is the first study to provide fundamental phenotypic and whole-genome sequences for B. porcorum. Our results extensively expand the current knowledge and could serve as a valuable genomic resource for future research on B. porcorum.


Assuntos
Composição de Bases , Genoma Bacteriano , Filogenia , Sequenciamento Completo do Genoma , Animais , China , Genoma Bacteriano/genética , Suínos , Flavobacteriaceae/genética , Flavobacteriaceae/isolamento & purificação , Flavobacteriaceae/classificação , Doenças dos Suínos/microbiologia , DNA Bacteriano/genética , Ilhas Genômicas , Plasmídeos/genética , Infecções por Flavobacteriaceae/microbiologia , Infecções por Flavobacteriaceae/veterinária , Análise de Sequência de DNA , Anotação de Sequência Molecular
15.
Antimicrob Resist Infect Control ; 13(1): 66, 2024 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-38886812

RESUMO

BACKGROUND: Carbapenem-resistant E. coli (CREco) pose a significant public health threat due to their multidrug resistance. Colistin is often a last-resort treatment against CREco; however, the emergence of colistin resistance gene mcr-1 complicates treatment options. METHODS: Two E. coli strains (ECO20 and ECO21), recovered from hospitalized patients in distinct wards, exhibited resistance to carbapenems and colistin. Whole-genome sequencing and phenotypic characterization were employed to study resistance patterns, plasmid profiles, transferability of resistance and virulence genes, and siderophore production capabilities. Comparative genome analysis was used to investigate the genetic environment of mcr-1, blaNDM-7, and virulence clusters. RESULTS: Both E. coli strains exhibited thr presence of both mcr-1 and blaNDM-7 genes, showing high resistance to multiple antibiotics. Genomic analysis revealed the clonal transmission of these strains, possessing identical plasmid profiles (pMCR, pNDM, and pVir) associated with colistin resistance, carbapenem resistance, and virulence factors. Conjugation experiments confirmed the transferability of these plasmids, indicating their potential to disseminate resistance and virulence traits to other strains. Comparative genomic analyses unveiled the distribution of mcr-1 (IncX4-type) and blaNDM (IncX3-type) plasmids across diverse bacterial species, emphasizing their adaptability and threat. The novelty of pVir indicates its potential role in driving the evolution of highly adaptable and pathogenic strains. CONCLUSIONS: Our findings underscore the co-occurrence of mcr-1, blaNDM-7, and siderophore-producing plasmids in E. coli, which poses a significant concern for global health. This research is crucial to unravel the complex mechanisms governing plasmid transfer and recombination and to devise robust strategies to control their spread in healthcare settings.


Assuntos
Antibacterianos , Farmacorresistência Bacteriana Múltipla , Infecções por Escherichia coli , Proteínas de Escherichia coli , Escherichia coli , Plasmídeos , Sideróforos , Plasmídeos/genética , Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , Proteínas de Escherichia coli/genética , Humanos , Infecções por Escherichia coli/microbiologia , Antibacterianos/farmacologia , China , Farmacorresistência Bacteriana Múltipla/genética , Sequenciamento Completo do Genoma , Colistina/farmacologia , Testes de Sensibilidade Microbiana , beta-Lactamases/genética , Hospitais , Carbapenêmicos/farmacologia , Fatores de Virulência/genética
16.
Biotechnol J ; 19(6): e2300685, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38900035

RESUMO

Recombinant adeno-associated virus (rAAV) is the leading vector for the delivery of gene therapies. However, low viral genome (VG) titers are common and the proportion of "full" capsids containing the therapeutic gene payload can be highly variable. The coordinated molecular design of plasmids encoding viral components and Helper functions remains a major challenge for rAAV manufacturing. Here we present the design of improved Rep/Cap and Helper plasmids for rAAV2/8 production, (i) a Rep/Cap expression vector harboring independently controllable rep and cap genes and (ii) an improved Helper plasmid harboring E4 gene deletion variants. First, an optimized Rep/Cap vector utilized a truncated p5 promoter, a p5 cis-regulatory element at the 3' end in combination with a heterologous promoter to drive Cap expression and an additional copy of the rep52/40 gene to overexpress short Rep proteins. We demonstrate that Rep78 is essential for efficient rAAV2/8 production in HEK293 cells, and a higher ratio of short Rep to long Rep proteins enhances genome packaging. Second, we identified regulators and open reading frames within the Helper plasmid that contribute to increased rAAV2/8 production. While L4-33k/22k is integral to optimal production, the use of E4orf6-6/7 subset significantly enhanced VG titer. Together, an optimal combination of engineered Rep/Cap and Helper plasmid variants increased VG titer by 3.1-fold. This study demonstrates that configuring and controlling the expression of the different AAV genetic elements contributes toward high rAAV production and product quality (full/empty capsid ratio).


Assuntos
Dependovirus , Vetores Genéticos , Dependovirus/genética , Células HEK293 , Humanos , Vetores Genéticos/genética , Plasmídeos/genética , Regiões Promotoras Genéticas/genética , Genoma Viral/genética , Proteínas Virais/genética
17.
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
18.
Biotechnol J ; 19(6): e2400140, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38896410

RESUMO

Artificial Intelligence (AI) technology is spearheading a new industrial revolution, which provides ample opportunities for the transformational development of traditional fermentation processes. During plasmid fermentation, traditional subjective process control leads to highly unstable plasmid yields. In this study, a multi-parameter correlation analysis was first performed to discover a dynamic metabolic balance among the oxygen uptake rate, temperature, and plasmid yield, whilst revealing the heating rate and timing as the most important optimization factor for balanced cell growth and plasmid production. Then, based on the acquired on-line parameters as well as outputs of kinetic models constructed for describing process dynamics of biomass concentration, plasmid yield, and substrate concentration, a machine learning (ML) model with Random Forest (RF) as the best machine learning algorithm was established to predict the optimal heating strategy. Finally, the highest plasmid yield and specific productivity of 1167.74 mg L-1 and 8.87 mg L-1/OD600 were achieved with the optimal heating strategy predicted by the RF model in the 50 L bioreactor, respectively, which was 71% and 21% higher than those obtained in the control cultures where a traditional one-step temperature upshift strategy was applied. In addition, this study transformed empirical fermentation process optimization into a more efficient and rational self-optimization method. The methodology employed in this study is equally applicable to predict the regulation of process dynamics for other products, thereby facilitating the potential for furthering the intelligent automation of fermentation processes.


Assuntos
Reatores Biológicos , Escherichia coli , Fermentação , Aprendizado de Máquina , Plasmídeos , Plasmídeos/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/crescimento & desenvolvimento , Reatores Biológicos/microbiologia , Técnicas de Cultura Celular por Lotes/métodos , Biomassa
19.
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
20.
ACS Synth Biol ; 13(6): 1831-1841, 2024 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-38863339

RESUMO

Antimicrobial resistance poses a significant global challenge, demanding innovative approaches, such as the CRISPR-Cas-mediated resistance plasmid or gene-curing system, to effectively combat this urgent crisis. To enable successful curing of antimicrobial genes or plasmids through CRISPR-Cas technology, the development of an efficient broad-host-range delivery system is paramount. In this study, we have successfully designed and constructed a novel functional gene delivery plasmid, pQ-mini, utilizing the backbone of a broad-host-range Inc.Q plasmid. Moreover, we have integrated the CRISPR-Cas12f system into the pQ-mini plasmid to enable gene-curing in broad-host of bacteria. Our findings demonstrate that pQ-mini facilitates the highly efficient transfer of genetic elements to diverse bacteria, particularly in various species in the order of Enterobacterales, exhibiting a broader host range and superior conjugation efficiency compared to the commonly used pMB1-like plasmid. Notably, pQ-mini effectively delivers the CRISPR-Cas12f system to antimicrobial-resistant strains, resulting in remarkable curing efficiencies for plasmid-borne mcr-1 or blaKPC genes that are comparable to those achieved by the previously reported pCasCure system. In conclusion, our study successfully establishes and optimizes pQ-mini as a broad-host-range functional gene delivery vector. Furthermore, in combination with the CRISPR-Cas system, pQ-mini demonstrates its potential for broad-host delivery, highlighting its promising role as a novel antimicrobial tool against the growing threat of antimicrobial resistance.


Assuntos
Antibacterianos , Sistemas CRISPR-Cas , Bactérias Gram-Negativas , Plasmídeos , Sistemas CRISPR-Cas/genética , Plasmídeos/genética , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/efeitos dos fármacos , Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , Técnicas de Transferência de Genes , Edição de Genes/métodos
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