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1.
Mater Sci Eng C Mater Biol Appl ; 128: 112307, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34474858

RESUMO

Gene transfer to mesenchymal stem cells (MSCs) has arisen as a powerful approach to increase the therapeutic potential of this effective cell population. Over recent years, niosomes have emerged as self-assembled carriers with promising performance for gene delivery. The aim of our work was to develop effective niosomes-based DNA delivery platforms for targeting MSCs. Niosomes based on 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA; 0, 7 or 15%) as cationic lipid, cholesterol as helper lipid, and polysorbate 60 as non-ionic surfactant, were prepared using a reverse phase evaporation technique. Niosomes dispersions (filtered or not) and their corresponding nioplexes with a lacZ plasmid were characterized in terms of size, charge, protection, and complexation abilities. DOTMA concentration had a large influence on the physicochemical properties of resulting nioplexes. Transfection efficiency and cytotoxic profiles were assessed in two immortalized cell lines of MSCs. Niosomes formulated with 15% DOTMA provided the highest values of ß-galactosidase activity, being similar to those achieved with Lipofectamine®, but showed less cytotoxicity. Filtration of niosomes dispersions before adding to the cells resulted in a loss of their biological activities. Storage of niosomes formulations (for 30 days at room temperature) caused minor modification of their physicochemical properties but also attenuated the transfection capability of the nioplexes. Differently, addition of the lysosomotropic agent sucrose into the culture medium during transfection or to the formulation itself improved the transfection performance of non-filtered niosomes. Indeed, steam heat-sterilized niosomes prepared in sucrose medium demonstrated transfection capability.


Assuntos
Lipossomos , Células-Tronco Mesenquimais , Técnicas de Transferência de Genes , Humanos , Plasmídeos/genética , Transfecção
2.
Nucleic Acids Res ; 49(15): 8732-8742, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34365511

RESUMO

CRISPR-Cas9 generates double-stranded DNA breaks (DSBs) to activate cellular DNA repair pathways for genome editing. The repair of DSBs leads to small insertions or deletions (indels) and other complex byproducts, including large deletions and chromosomal translocations. Indels are well understood to disrupt target genes, while the other deleterious byproducts remain elusive. We developed a new in silico analysis pipeline for the previously described primer-extension-mediated sequencing assay to comprehensively characterize CRISPR-Cas9-induced DSB repair outcomes in human or mouse cells. We identified tremendous deleterious DSB repair byproducts of CRISPR-Cas9 editing, including large deletions, vector integrations, and chromosomal translocations. We further elucidated the important roles of microhomology, chromosomal interaction, recurrent DSBs, and DSB repair pathways in the generation of these byproducts. Our findings provide an extra dimension for genome editing safety besides off-targets. And caution should be exercised to avoid not only off-target damages but also deleterious DSB repair byproducts during genome editing.


Assuntos
Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Reparo do DNA , Edição de Genes , Animais , Células Cultivadas , Simulação por Computador , Humanos , Camundongos , Plasmídeos/genética , Deleção de Sequência , Translocação Genética
3.
BMC Bioinformatics ; 22(1): 390, 2021 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-34332528

RESUMO

BACKGROUND: Plasmids are mobile genetic elements, key in the dissemination of antibiotic resistance, virulence determinants and other adaptive traits in bacteria. Obtaining a robust method for plasmid classification is necessary to better understand the genetics and epidemiology of many pathogens. Until now, plasmid classification systems focused on specific traits, which limited their precision and universality. The definition of plasmid taxonomic units (PTUs), based on average nucleotide identity metrics, allows the generation of a universal plasmid classification scheme, applicable to all bacterial taxa. Here we present COPLA, a software able to assign plasmids to known and novel PTUs, based on their genomic sequence. RESULTS: We implemented an automated pipeline able to assign a given plasmid DNA sequence to its cognate PTU, and assessed its performance using a sample of 1000 unclassified plasmids. Overall, 41% of the samples could be assigned to a previously defined PTU, a number that reached 63% in well-known taxa such as the Enterobacterales order. The remaining plasmids represent novel PTUs, indicating that a large fraction of plasmid backbones is still uncharacterized. CONCLUSIONS: COPLA is a bioinformatic tool for universal, species-independent, plasmid classification. Offered both as an automatable pipeline and an open web service, COPLA will help bacterial geneticists and clinical microbiologists to quickly classify plasmids.


Assuntos
Transferência Genética Horizontal , Genômica , Resistência Microbiana a Medicamentos , Plasmídeos/genética , Fatores de Virulência
4.
Zhonghua Yi Xue Za Zhi ; 101(31): 2478-2484, 2021 Aug 17.
Artigo em Chinês | MEDLINE | ID: mdl-34399563

RESUMO

Objective: To characterize the antibiotic resistance and virulence in a carbapenem-resistant Klebsiella pneumoniae (CRKP). Methods: A CRKP (designated K. pneumoniae C35) was isolated from a stool sample. The minimal inhibitory concentrations of antimicrobial agents were determined using the broth microdilution method. Whole-genome sequencing and genome analysis were performed to identify the antibiotic resistance and virulence genes. The genetic relationship among K. pneumoniae C35 and other CRKP isolates from our hospital was analyzed by single nucleotide polymorphism (SNP) typing of core genomes. Conjugation experiments were carried out by filter mating to evaluate the transferability and efficiency of resistance genes. The virulence phenotype was determined by Galleria mellonella infection model. Results: K. pneumoniae C35 exhibited resistance to the majority of tested antibiotics, especially carbapenems, sulbactam, and polymyxins. SNP typing showed that K. pneumoniae C35 shared a high degree of sequence homology with several CRKP isolates from different wards. This ST11 CRKP carried 13 resistance genes, including blaKPC-2, blaCTX-M-199, mcr-1, and tet(A) variant. blaKPC-2 gene was located on an IncFⅡ plasmid with>69 800 bp in size, blaCTX-M-199 and mcr-1 genes were located on an IncI2 plasmid (>64 800 bp), and tet(A) variant was located on an unknown Inc-type plasmid (83 628bp). All these three plasmids were conjugative. K. pneumoniae C35 was found to harbor rmpA, rmpA2, and iucABCD aerobactin-related genes, and was considered to be classic carbapenem-resistant hypervirulent K. pneumoniae (CR-hvKP). The virulence potential of this strain was confirmed in a Galleria mellonella infection model. The survival rate of the larvae injected with strain C35 at 48 h after infection was significantly lower than that of negative control strain (16.7% vs 80.0%). Conclusion: Multiple conjugative plasmids are identified in a faecal CR-hvKP. The IncI2 plasmid co-carrying both blaCTX-M-199 and mcr-1 genes is firstly identified in CR-hvKP. The emergence of such strain should be alerted and active surveillance is warranted.


Assuntos
Infecções por Klebsiella , Klebsiella pneumoniae , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Proteínas de Bactérias/genética , Carbapenêmicos/farmacologia , Resistência Microbiana a Medicamentos , Humanos , Infecções por Klebsiella/tratamento farmacológico , Klebsiella pneumoniae/genética , Tipagem de Sequências Multilocus , Plasmídeos/genética , Virulência/genética , beta-Lactamases
5.
Int J Mol Sci ; 22(15)2021 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-34360889

RESUMO

Despite extensive research, there is still no vaccine against the hepatitis C virus (HCV). The aim of this study was to investigate whether MSCs can exhibit adjuvant properties during DNA vaccination against hepatitis C. We used the pcNS3-NS5B plasmid encoding five nonstructural HCV proteins and MSCs derived from mice bone marrow. Five groups of DBA mice were immunized with the plasmid and/or MSCs in a different order. Group 1 was injected with the plasmid twice at intervals of 3 weeks; Group 2 with the plasmid, and after 24 h with MSCs; Group 3 with MSCs followed by the plasmid the next day; Group 4 with only MSCs; and Group 5 with saline. When the MSCs were injected prior to DNA immunization, the cell immune response to HCV proteins assessed by the level of IFN-γ synthesis was markedly increased compared to DNA alone. In contrast, MSCs injected after DNA suppressed the immune response. Apparently, the high level of proinflammatory cytokines detected after DNA injection promotes the conversion of MSCs introduced later into the immunosuppressive MSC2. The low level of cytokines in mice before MSC administration promotes the high immunostimulatory activity of MSC1 in response to a DNA vaccine. Thus, when administered before DNA, MSCs are capable of exhibiting promising adjuvant properties.


Assuntos
Genes Virais/imunologia , Hepacivirus/genética , Hepacivirus/imunologia , Hepatite C/prevenção & controle , Imunidade Celular , Células-Tronco Mesenquimais/imunologia , Vacinação/métodos , Vacinas de DNA/administração & dosagem , Proteínas não Estruturais Virais/genética , Animais , Linhagem Celular Tumoral , Citocinas/metabolismo , Feminino , Hepatite C/imunologia , Hepatite C/virologia , Humanos , Camundongos , Camundongos Endogâmicos DBA , Plasmídeos/genética , Linfócitos T/imunologia , Transfecção , Resultado do Tratamento , Vacinas de DNA/imunologia
6.
Microb Pathog ; 159: 105124, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34364978

RESUMO

OBJECTIVES: Pseudomonas aeruginosa is a medically important pathogen showing intrinsic low permeability to various antimicrobial agents and its potential to acquire multiple resistance mechanism. A longitudinal surveillance aimed to investigate the antimicrobial resistance and its determinants of Pseudomonas aeruginosa in Southern China. A total of 2163 P. aeruginosa isolates were obtained from patients in Southern China during 2004-2016. METHODS: The antimicrobial susceptibility of the isolates was performed by disk diffusion and Vitek 2 automated system and interpreted according to the Clinical and Laboratory Standard Institute (CLSI) 2015. RESULTS: A significant downtrend of resistant rate (>10.0%) was observed for tested antibiotic agents including ciprofloxacin (>30.0%), gentamicin (29.0%), tobramycin (24.2%) and ceftazidime (24.0%) except for aztreonam and amikacin. A total of 269 randomly selected isolates were further studied on the carriage of ß-lactam resistance genes by using 7 groups of multiplex PCRs targeting on 20 genes. ß-lactam resistance genes were rarely detected with a rate lower than 8%. Among all ß-lactam resistance genes, blaSHV acquired the highest identification rate (18/269, 6.7%), followed by blaOXA-1-like (6/269, 2.2%) and blaPER (6/269, 2.2%). In addition, 8 different plasmid replicons were amplified using 8 groups of multiplex PCRs including 18 sets of primers. Only five plasmid replicons were identified in 5 different P. aeruginosa isolates. Insignificant clonal relatedness among the positive strains identified by regular PCR were further verified by randomly amplified polymorphic DNA (RAPD)-PCR. CONCLUSION: This study has provided comprehensive knowledge on current antimicrobial resistance, ß-lactam resistance genes and plasmid replicons carriage in a large scale of clinical P. aeruginosa isolates.


Assuntos
Infecções por Pseudomonas , Pseudomonas aeruginosa , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Farmacorresistência Bacteriana , Humanos , Testes de Sensibilidade Microbiana , Plasmídeos/genética , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/genética , Técnica de Amplificação ao Acaso de DNA Polimórfico , Replicon , beta-Lactamases/genética
7.
Appl Microbiol Biotechnol ; 105(18): 6835-6852, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34448898

RESUMO

For the acetic acid bacterium (AAB) Gluconobacter oxydans only recently the first tight system for regulatable target gene expression became available based on the heterologous repressor-activator protein AraC from Escherichia coli and the target promoter ParaBAD. In this study, we tested pure repressor-based TetR- and LacI-dependent target gene expression in G. oxydans by applying the same plasmid backbone and construction principles that we have used successfully for the araC-ParaBAD system. When using a pBBR1MCS-5-based plasmid, the non-induced basal expression of the Tn10-based TetR-dependent expression system was extremely low. This allowed calculated induction ratios of up to more than 3500-fold with the fluorescence reporter protein mNeonGreen (mNG). The induction was highly homogeneous and tunable by varying the anhydrotetracycline concentration from 10 to 200 ng/mL. The already strong reporter gene expression could be doubled by inserting the ribosome binding site AGGAGA into the 3' region of the Ptet sequence upstream from mNG. Alternative plasmid constructs used as controls revealed a strong influence of transcription terminators and antibiotics resistance gene of the plasmid backbone on the resulting expression performance. In contrast to the TetR-Ptet-system, pBBR1MCS-5-based LacI-dependent expression from PlacUV5 always exhibited some non-induced basal reporter expression and was therefore tunable only up to 40-fold induction by IPTG. The leakiness of PlacUV5 when not induced was independent of potential read-through from the lacI promoter. Protein-DNA binding simulations for pH 7, 6, 5, and 4 by computational modeling of LacI, TetR, and AraC with DNA suggested a decreased DNA binding of LacI when pH is below 6, the latter possibly causing the leakiness of LacI-dependent systems hitherto tested in AAB. In summary, the expression performance of the pBBR1MCS-5-based TetR-Ptet system makes this system highly suitable for applications in G. oxydans and possibly in other AAB. KEY POINTS: • A pBBR1MCS-5-based TetR-Ptet system was tunable up to more than 3500-fold induction. • A pBBR1MCS-5-based LacI-PlacUV5 system was leaky and tunable only up to 40-fold. • Modeling of protein-DNA binding suggested decreased DNA binding of LacI at pH < 6.


Assuntos
Gluconobacter oxydans , Gluconobacter , Ácido Acético , Expressão Gênica , Gluconobacter oxydans/genética , Plasmídeos/genética
8.
Appl Microbiol Biotechnol ; 105(14-15): 5959-5972, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34357429

RESUMO

Production of industrially relevant compounds in microbial cell factories can employ either genomes or plasmids as an expression platform. Selection of plasmids as pathway carriers is advantageous for rapid demonstration but poses a challenge of stability. Yarrowia lipolytica has attracted great attention in the past decade for the biosynthesis of chemicals related to fatty acids at titers attractive to industry, and many genetic tools have been developed to explore its oleaginous potential. Our recent studies on the autonomously replicating sequences (ARSs) of nonconventional yeasts revealed that the ARSs from Y. lipolytica showcase a unique structure that includes a previously unannotated sequence (spacer) linking the origin of replication (ORI) and the centromeric (CEN) element and plays a critical role in modulating plasmid behavior. Maintaining a native 645-bp spacer yielded a 2.2-fold increase in gene expression and 1.7-fold higher plasmid stability compared to a more universally employed minimized ARS. Testing the modularity of the ARS sub-elements indicated that plasmid stability exhibits a pronounced cargo dependency. Instability caused both plasmid loss and intramolecular rearrangements. Altogether, our work clarifies the appropriate application of various ARSs for the scientific community and sheds light on a previously unexplored DNA element as a potential target for engineering Y. lipolytica.


Assuntos
Origem de Replicação , Yarrowia , Centrômero , Replicação do DNA , Engenharia Metabólica , Plasmídeos/genética , Yarrowia/genética
9.
Antimicrob Agents Chemother ; 65(10): e0105421, 2021 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-34339270

RESUMO

The global spread of antimicrobial-resistant bacteria has been one of the most severe threats to public health. The emergence of the mcr-1 gene has posed a considerable threat to antimicrobial medication since it deactivates one last-resort antibiotic, colistin. There have been reports regarding the mobilization of the mcr-1 gene facilitated by ISApl1-formed transposon Tn6330 and mediated rapid dispersion among Enterobacteriaceae species. Here, we developed a CRISPR/Cas9 system flanked by ISApl1 in a suicide plasmid capable of exerting sequence-specific curing against the mcr-1-bearing plasmid and killing the strain with chromosome-borne mcr-1. The constructed ISApl1-carried CRISPR/Cas9 system either restored sensitivity to colistin in strains with plasmid-borne mcr-1 or directly eradicated the bacteria harboring chromosome-borne mcr-1 by introducing an exogenous CRISPR/Cas9 targeting the mcr-1 gene. This method is highly efficient in removing the mcr-1 gene from Escherichia coli, thereby resensitizing these strains to colistin. The further results demonstrated that it conferred the recipient bacteria with immunity against the acquisition of the exogenous mcr-1 containing the plasmid. The data from the current study highlighted the potential of the transposon-associated CRISPR/Cas9 system to serve as a therapeutic approach to control the dissemination of mcr-1 resistance among clinical pathogens.


Assuntos
Proteínas de Escherichia coli , Escherichia coli , Antibacterianos/farmacologia , Sistemas CRISPR-Cas/genética , Cromossomos , Colistina/farmacologia , Farmacorresistência Bacteriana/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Humanos , Plasmídeos/genética
10.
J Environ Manage ; 298: 113541, 2021 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-34426222

RESUMO

Extracellular antibiotic resistance genes (eARG) are considered to play an important role in spread of antimicrobial resistance (AMR) in wastewater treatment and water environment. Membrane bioreactor (MBR) reportedly has better removal of ARGs in wastewater than conventional activated sludge process. However, removal of eARG is possibly limited because eARG is small to pass through microfiltration (MF) membranes. To evaluate potential removal of eARG in MBR, this study aimed to understand the initial behaviors of eARG received in MBR. The recombinant plasmid with artificial marker gene was spiked in lab-scale MBR to trace fate of eARG in MBR. Among 10 10 copies/L of the spiked gene, 2.6 × 109 copies/L was adsorbed on sludge particles at 6 h after spiking, while only 2.2 × 108-3.6 × 108 copies/L of the spiked gene was remained but constant in sludge liquid phase from 6 until 48 h. This result suggests that adsorption on sludge particles served as the main mechanism to govern the initial fate of eARG in MBR. Meanwhile, the spiked gene concentrations in membrane permeate was lower than sludge liquid phase and decreased overtime, suggesting retention of eARG in membrane filtration. Total LRV of the spiked extracellular gene were 3.4 ± 0.8 log at 48 h after spiking. LRV by adsorption corresponded to 1.7 ± 0.7 log constantly since 3 h after spiking, while LRV by membrane filtration increased from 0 to 1.7 ± 0.6 log. Linear correlation of LRV by membrane filtration with transmembrane pressure (TMP) suggested that foulant deposition on membrane governs removal of eARG by membrane filtration in MBR.


Assuntos
Reatores Biológicos , Membranas Artificiais , Plasmídeos/genética , Esgotos , Eliminação de Resíduos Líquidos , Águas Residuárias
11.
Int J Mol Sci ; 22(16)2021 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-34445438

RESUMO

Gram-negative bacteria release Outer Membrane Vesicles (OMVs) into the extracellular environment. Recent studies recognized these vesicles as vectors to horizontal gene transfer; however, the parameters that mediate OMVs transfer within bacterial communities remain unclear. The present study highlights for the first time the transfer of plasmids containing resistance genes via OMVs derived from Klebsiella pneumoniae (K. pneumoniae). This mechanism confers DNA protection, it is plasmid copy number dependent with a ratio of 3.6 times among high copy number plasmid (pGR) versus low copy number plasmid (PRM), and the transformation efficiency was 3.6 times greater. Therefore, the DNA amount in the vesicular lumen and the efficacy of horizontal gene transfer was strictly dependent on the identity of the plasmid. Moreover, the role of K. pneumoniae-OMVs in interspecies transfer was described. The transfer ability was not related to the phylogenetic characteristics between the donor and the recipient species. K. pneumoniae-OMVs transferred plasmid to Escherichia coli, Salmonella enterica, Pseudomonas aeruginosa and Burkholderia cepacia. These findings address the pivotal role of K. pneumoniae-OMVs as vectors for antimicrobial resistance genes spread, contributing to the development of antibiotic resistance in the microbial communities.


Assuntos
Vesículas Citoplasmáticas/genética , Transferência Genética Horizontal , Klebsiella pneumoniae/genética , Plasmídeos/genética , Antibacterianos/farmacologia , Proteínas de Bactérias , Farmacorresistência Bacteriana , Dosagem de Genes , Klebsiella pneumoniae/classificação , Klebsiella pneumoniae/efeitos dos fármacos , Filogenia
12.
Int J Mol Sci ; 22(16)2021 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-34445592

RESUMO

The Hfq protein is a bacterial RNA chaperone, involved in many molecular interactions, including control of actions of various small RNA regulatory molecules. We found that the presence of Hfq was required for survival of plasmid-containing Escherichia coli cells against high concentrations of chloramphenicol (plasmid p27cmr), tetracycline (pSC101, pBR322) and ampicillin (pBR322), as hfq+ strains were more resistant to these antibiotics than the hfq-null mutant. In striking contrast, production of Hfq resulted in low resistance to high concentrations of kanamycin when the antibiotic-resistance marker was chromosome-borne, with deletion of hfq resulting in increasing bacterial survival. These results were observed both in solid and liquid medium, suggesting that antibiotic resistance is an intrinsic feature of these strains rather than a consequence of adaptation. Despite its major role as RNA chaperone, which also affects mRNA stability, Hfq was not found to significantly affect kan and tet mRNAs turnover. Nevertheless, kan mRNA steady-state levels were higher in the hfq-null mutant compared to the hfq+ strain, suggesting that Hfq can act as a repressor of kan expression.This observation does correlate with the enhanced resistance to high levels of kanamycin observed in the hfq-null mutant. Furthermore, dependency on Hfq for resistance to high doses of tetracycline was found to depend on plasmid copy number, which was only observed when the resistance marker was expressed from a low copy plasmid (pSC101) but not from a medium copy plasmid (pBR322). This suggests that Hfq may influence survival against high doses of antibiotics through mechanisms that remain to be determined. Studies with pBR322Δrom may also suggest an interplay between Hfq and Rom in the regulation of ColE1-like plasmid replication. Results of experiments with a mutant devoid of the part of the hfq gene coding for the C-terminal region of Hfq suggested that this region, as well as the N-terminal region, may be involved in the regulation of expression of antibiotic resistance in E. coli independently.


Assuntos
Antibacterianos/farmacologia , Cromossomos Bacterianos/genética , Resistência Microbiana a Medicamentos/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Fator Proteico 1 do Hospedeiro/genética , Mutação , Plasmídeos/genética , Escherichia coli/efeitos dos fármacos
13.
Clin Lab ; 67(8)2021 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-34383410

RESUMO

BACKGROUND: To investigate the epidemics of plasmid-mediated quinolone resistance (PMQR) gene in carbapenem-resistant Enterobacteriaceae (CRE) and the resistance mechanism. METHODS: We collected CRE bacteria isolated clinically between December 2017 and December 2018 for identification and drug sensitivity testing using a VITEK2 Compact Analyzer. Furthermore, genes, including qnrA, qnrB, qnrS, qepA, and acc (6') Ib-cr, were determined through the polymerase chain reaction and sequencing. The hori-zontal transfer of PMQR gene was validated through the plasmid conjugational test. RESULTS: Drug resistance rate of carbapenem-resistant Escherichia coli against quinolones was 100%, while the rate of carbapenem-resistant Klebsiella pneumoniae ranged from 15.56% to 33.33%. The detection rate of acc (6') Ib-cr was the highest (87.72%), followed by qnrB (77.19%) and qnrS (17.54%). Additionally, there were two bacteria carrying the qnrA gene (3.51%), but qepA gene was not isolated from the samples. In total, 84.21% of these bacteria carried 2 or 3 kinds of PMQR genes. Among 8 bacteria with successful plasmid conjugation, PMQR gene transfer was detected in all of them, but with no significant change in the minimum inhibitory concentration of quinolones. CONCLUSIONS: CRE remain sensitive to quinolones in spite of the high detection rate of PMQR gene in this hospital.


Assuntos
Enterobacteriáceas Resistentes a Carbapenêmicos , Infecções por Enterobacteriaceae , Quinolonas , Antibacterianos/farmacologia , Enterobacteriáceas Resistentes a Carbapenêmicos/genética , Farmacorresistência Bacteriana/genética , Infecções por Enterobacteriaceae/diagnóstico , Infecções por Enterobacteriaceae/tratamento farmacológico , Humanos , Testes de Sensibilidade Microbiana , Plasmídeos/genética , Quinolonas/farmacologia
14.
Front Cell Infect Microbiol ; 11: 658070, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34354959

RESUMO

The emergence and prevalence of carbapenem-resistant Enterobacteriaceae (CRE) have drawn worldwide attention. Ceftazidime/avibactam (CAZ/AVI) gives us a valuable alternative strategy to treat CRE infections. Unfortunately, CAZ/AVI resistance could occur during CAZ/AVI treatment. The CAZ/AVI-resistant Carbapenem-resistant Klebsiella pneumoniae (CR-KP) (KP137060) and earlier CAZ/AVI-susceptible isolate (KP135194) from the same hospitalized patient were collected at Fujian Medical University Union Hospital between October and November 2019. In this study, CAZ/AVI MICs of CAZ/AVI-susceptible and -resistant isolates (KP135194 and KP137060) were 4 mg/L and 128 mg/L, respectively; and the two isolates had the same antibiotic resistance pattern to other carbapenems. Two strains were then submitted for whole-genome sequencing and bioinformatic analysis. ompK36 was not detected in two isolates. No mutation was observed in bla KPC-2, ompK35 and ompK37 in this study and there was no significant difference of the expression in bla KPC-2, ompK35 and ompK37 between the two isolates (p>0.05). Two isolates were sequence type 11 and harbored bla KPC-2, bla SHV-182 and bla TEM-1B. Compared with KP135194, KP137060 harbored an additional bla NDM-5 positive plasmid. bla NDM-5 gene could be successfully transferred into E. coli J53 at a conjugation frequency of 1.14×10-4. Plasmid stability testing showed that bla KPC-2- and bla NDM-5-harboring plasmids were still stably maintained in the hosts. Growth assay and growth competition experiments showed there was no significant difference in fitness cost between two CR-KP isolates. Our study described the acquisition of a bla NDM-5-harboring plasmid leading to resistance to ceftazidime/avibactam in KPC-2-producing Klebsiella pneumoniae during treatment. This phenomenon deserves further exploration.


Assuntos
Enterobacteriáceas Resistentes a Carbapenêmicos , Infecções por Klebsiella , Antibacterianos/farmacologia , Compostos Azabicíclicos , Enterobacteriáceas Resistentes a Carbapenêmicos/genética , Ceftazidima/farmacologia , Combinação de Medicamentos , Escherichia coli/genética , Humanos , Infecções por Klebsiella/tratamento farmacológico , Klebsiella pneumoniae/genética , Testes de Sensibilidade Microbiana , Plasmídeos/genética , beta-Lactamases/genética
15.
Int J Mol Sci ; 22(15)2021 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-34360567

RESUMO

Resistance to antimicrobials is a growing problem of worldwide concern. Plasmids are thought to be major drivers of antibiotic resistance spread. The present work reports a simple way to recover replicative plasmids conferring antibiotic resistance from the bacteria in cheese. Purified plasmid DNA from colonies grown in the presence of tetracycline and erythromycin was introduced into plasmid-free strains of Lactococcus lactis, Lactiplantibacillus plantarum and Lacticaseibacillus casei. Following antibiotic selection, the plasmids from resistant transformants were isolated, analyzed by restriction enzyme digestion, and sequenced. Seven patterns were obtained for the tetracycline-resistant colonies, five from L. lactis, and one each from the lactobacilli strains, as well as a single digestion profile for the erythromycin-resistant transformants obtained in L. lactis. Sequence analysis respectively identified tet(S) and ermB in the tetracycline- and erythromycin-resistance plasmids from L. lactis. No dedicated resistance genes were detected in plasmids conferring tetracycline resistance to L. casei and L. plantarum. The present results highlight the usefulness of the proposed methodology for isolating functional plasmids that confer antibiotic resistance to LAB species, widen our knowledge of antibiotic resistance in the bacteria that inhabit cheese, and emphasize the leading role of plasmids in the spread of resistance genes via the food chain.


Assuntos
Antibacterianos/farmacologia , Queijo/microbiologia , Resistência Microbiana a Medicamentos , Eritromicina/farmacologia , Lactobacillales/crescimento & desenvolvimento , Plasmídeos/genética , Animais , Lactobacillales/efeitos dos fármacos , Lactobacillales/isolamento & purificação
16.
Front Cell Infect Microbiol ; 11: 681588, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34327151

RESUMO

In this study, multidrug-resistant (MDR) Escherichia coli isolates from retail food and humans assigned into similar Multilocus Sequence Types (MLST) were analyzed using whole genome sequencing (WGS). In silico analysis of assembled sequences revealed the existence of multiple resistance genes among the examined E. coli isolates. Of the six CTX-M-producing isolates from retail food, bla CTX-M-14 was the prevalent variant identified (83.3%, 5/6). Two plasmid-mediated fosfomycin resistance genes, fosA3, and fosA4, were detected from retail food isolates (one each from chicken and beef), where fosA4 was identified in the chicken isolate 82CH that also carried the colistin resistance gene mcr-1. The bla CTX-M-14 and fosA genes in retail food isolates were located adjacent to insertion sequences ISEcp1 and IS26, respectively. Sequence analysis of the reconstructed mcr-1 plasmid (p82CH) showed 96-97% identity to mcr-1-carrying IncI2 plasmids previously identified in human and food E. coli isolates from Egypt. Hierarchical clustering of core genome MLST (HierCC) revealed clustering of chicken isolate 82CH, co-harboring mcr-1 and fosA4 genes, with a chicken E. coli isolate from China at the HC200 level (≤200 core genome allelic differences). As E. coli co-harboring mcr-1 and fosA4 genes has only been recently reported, this study shows rapid spread of this genotype that shares similar genetic structures with regional and international E. coli lineages originating from both humans and food animals. Adopting WGS-based surveillance system is warranted to facilitate monitoring the international spread of MDR pathogens.


Assuntos
Escherichia coli , Contaminação de Alimentos , Carne/microbiologia , Animais , Antibacterianos/farmacologia , Galinhas , China , Farmacorresistência Bacteriana Múltipla , Egito , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Proteínas de Escherichia coli/genética , Humanos , Tipagem de Sequências Multilocus , Plasmídeos/genética , beta-Lactamases/genética
17.
Acta Biochim Biophys Sin (Shanghai) ; 53(7): 933-942, 2021 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-34223883

RESUMO

The transgenic glyphosate-tolerant soybean MON87712 event was developed by the agrochemical and agricultural biotechnology company Monsanto (USA) and commercialized in 2013. Due to the absence of matrix-based and genomic DNA-positive reference material for MON87712, it is very difficult to detect and monitor this event. In this study, we developed a recombinant 760-bp linearized plasmid, including 150 bp of the soybean endogenous lectin gene and 610 bp of the exogenous BBX32 gene plus its 3' flanking sequence of MON87712 by In-Fusion cloning technology. In addition, a duplex real-time polymerase chain reaction for the detection of MON87712 and the soybean endogenous lectin gene was established. By using this method, we achieved specific and quantitative detection of MON87712 in 45 other kinds of crops, with a detection limit of 10 copies/µl. This method provides a new technical means for the accurate detection of transgenic soybean MON87712, as well as technical support for the supervision of agricultural transgenic organisms.


Assuntos
Plantas Geneticamente Modificadas/genética , Plasmídeos/genética , Soja/genética
18.
Nano Lett ; 21(13): 5697-5705, 2021 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-34228937

RESUMO

Polyelectrolyte complex particles assembled from plasmid DNA (pDNA) and poly(ethylenimine) (PEI) have been widely used to produce lentiviral vectors (LVVs) for gene therapy. The current batch-mode preparation for pDNA/PEI particles presents limited reproducibility in large-scale LVV manufacturing processes, leading to challenges in tightly controlling particle stability, transfection outcomes, and LVV production yield. Here we identified the size of pDNA/PEI particles as a key determinant for a high transfection efficiency with an optimal size of 400-500 nm, due to a cellular-uptake-related mechanism. We developed a kinetics-based approach to assemble size-controlled and shelf-stable particles using preassembled nanoparticles as building blocks and demonstrated production scalability on a scale of at least 100 mL. The preservation of colloidal stability and transfection efficiency was benchmarked against particles generated using an industry standard protocol. This particle manufacturing method effectively streamlines the viral manufacturing process and improves the production quality and consistency.


Assuntos
DNA , Polietilenoimina , DNA/genética , Tamanho da Partícula , Plasmídeos/genética , Reprodutibilidade dos Testes , Transfecção
19.
Viruses ; 13(6)2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-34205958

RESUMO

Picornaviruses are non-enveloped, single-stranded RNA viruses that cause highly contagious diseases, such as polio and hand, foot-and-mouth disease (HFMD) in human, and foot-and-mouth disease (FMD) in animals. Reverse genetics and minigenome of picornaviruses mainly depend on in vitro transcription and RNA transfection; however, this approach is inefficient due to the rapid degradation of RNA template. Although DNA-based reverse genetics systems driven by mammalian RNA polymerase I and/or II promoters display the advantage of rescuing the engineered FMDV, the enzymatic functions are restricted in the nuclear compartment. To overcome these limitations, we successfully established a novel DNA-based vector, namely pKLS3, an FMDV minigenome containing the minimum cis-acting elements of FMDV essential for intracytoplasmic transcription and translation of a foreign gene. A combination of pKLS3 minigenome and the helper plasmids yielded the efficient production of uncapped-green florescent protein (GFP) mRNA visualized in the transfected cells. We have demonstrated the application of the pKLS3 for cell-based antiviral drug screening. Not only is the DNA-based FMDV minigenome system useful for the FMDV research and development but it could be implemented for generating other picornavirus minigenomes. Additionally, the prospective applications of this viral minigenome system as a vector for DNA and mRNA vaccines are also discussed.


Assuntos
Vírus da Febre Aftosa/genética , Febre Aftosa/virologia , Regulação Viral da Expressão Gênica , Genoma Viral , Plasmídeos/genética , RNA Mensageiro/genética , Animais , Antivirais/química , Antivirais/farmacologia , Linhagem Celular , Febre Aftosa/tratamento farmacológico , Vírus da Febre Aftosa/efeitos dos fármacos , Ordem dos Genes , Humanos , Modelos Moleculares , Estrutura Molecular , RNA Mensageiro/química , Relação Estrutura-Atividade , Transfecção , Replicação Viral/efeitos dos fármacos
20.
Int J Med Microbiol ; 311(6): 151519, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34280738

RESUMO

Rhodococcus equi is a saprophytic soil bacterium and intracellular pathogen that causes refractory suppurative pneumonia in foals and has emerged as a pathogenic cause of zoonotic disease. Several studies have reported human infections caused by R. equi harboring a recently described third type of virulence plasmid, the ruminant-associated pVAPN, which carries the vapN virulence determinant. Herein, we analyzed pathogenicity and genomic features of nine vapN-harboring R. equi isolated from human patients with and without HIV/AIDS. Four of these strains showed significant VapN production and proliferation in cultured macrophages. These strains were lethally pathogenic after inoculation with 1.0 × 108 CFU in mice and reproduced a necrotizing granulomatous inflammation in the liver and spleen similar to that observed in humans. Additionally, we determined entire genome sequences of all nine strains. Lengths of sequences were 5.0-5.3 Mbp, and GC contents were 68.7 %-68.8 %. All strains harbored a 120- or 125-kbp linear plasmid carrying vapN (Type I or Type II pVAPN) classified on the basis of differences in the distal sequences on the 3' side. Interestingly, VapN production differed significantly among strains harboring nearly identical types of pVAPN with variation limited to several SNPs and short base pair indels. The pVAPN sequences possessed by the VapN-producing strains did not retain any common genetic characteristics, and more detailed analyses, including chromosomal genes, are needed to further elucidate the VapN expression mechanism.


Assuntos
Infecções por Actinomycetales , Rhodococcus equi , Rhodococcus , Infecções por Actinomycetales/veterinária , Animais , Genômica , Cavalos , Humanos , Camundongos , Plasmídeos/genética , Rhodococcus equi/genética , Virulência
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