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1.
Nat Commun ; 12(1): 1846, 2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33758180

RESUMO

A wide repertoire of genetic switches has accelerated prokaryotic synthetic biology, while eukaryotic synthetic biology has lagged in the model organism Saccharomyces cerevisiae. Eukaryotic genetic switches are larger and more complex than prokaryotic ones, complicating the rational design and evolution of them. Here, we present a robust workflow for the creation and evolution of yeast genetic switches. The selector system was designed so that both ON- and OFF-state selection of genetic switches is completed solely by liquid handling, and it enabled parallel screen/selection of different motifs with different selection conditions. Because selection threshold of both ON- and OFF-state selection can be flexibly tuned, the desired selection conditions can be rapidly pinned down for individual directed evolution experiments without a prior knowledge either on the library population. The system's utility was demonstrated using 20 independent directed evolution experiments, yielding genetic switches with elevated inducer sensitivities, inverted switching behaviours, sensory functions, and improved signal-to-noise ratio (>100-fold induction). The resulting yeast genetic switches were readily integrated, in a plug-and-play manner, into an AND-gated carotenoid biosynthesis pathway.


Assuntos
Evolução Molecular Direcionada/métodos , Genes de Troca , Engenharia Genética/métodos , Técnicas Genéticas , Saccharomyces cerevisiae/genética , Biologia Sintética/métodos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Basidiomycota/genética , Basidiomycota/metabolismo , Citometria de Fluxo , Biblioteca Gênica , Genes Reporter , Floroglucinol/análogos & derivados , Floroglucinol/farmacologia , Regiões Promotoras Genéticas , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Razão Sinal-Ruído , Tetraciclina/farmacologia , Transativadores/química , Transativadores/genética , Transativadores/metabolismo , beta Caroteno/biossíntese , beta Caroteno/genética , beta Caroteno/metabolismo
2.
Ecotoxicol Environ Saf ; 213: 111981, 2021 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-33592372

RESUMO

Environmental pollution due to resistance genes from livestock manure has become a serious issue that needs to be resolved. However, little studies focused on the removal of resistance genes in simultaneous processing of livestock feces and urine. This study investigated the fate of antibiotic resistance genes (ARGs), metal resistance genes (MRGs), and class 1 integron-integrase gene (intI1) during thermophilic fermentation of swine manure in an ectopic fermentation system (EFS), which has been regarded as a novel system for efficiently treating both feces and urine. The abundances of MRGs and tetracycline resistance genes were 34.44-97.71% lower in the EFS. The supplementation of heavy metals significantly increased the abundance of intI1, with the enhancement effect of copper being more prominent than that of zinc. The highest abundances of resistance genes and intI1 were observed at high Cu levels (A2), indicating that Cu can increase the spreading of resistance genes through integrons. Network analysis revealed the co-occurrence of ARGs, MRGs, and intI1, and these genes potentially shared the same host bacteria. Redundancy analysis showed that the bacterial community explained most of the variations in ARGs, and environmental factors had influences on ARGs abundances by modulating the bacterial community composition. The decreased Sphingomonas, Comamonas, Acinetobacter, Lactobacillus, Bartonella, Rhizobium, and Bacteroides were mainly responsible for the reduced resistance genes. These results demonstrate that EFS can reduce resistance genes in simultaneous processing of livestock feces and urine.


Assuntos
Resistência Microbiana a Medicamentos/genética , Fermentação/fisiologia , Genes Bacterianos , Esterco/microbiologia , Metais Pesados/análise , Animais , Antibacterianos/análise , Bactérias/efeitos dos fármacos , Cobre , Integrons , Gado , Esterco/análise , Suínos , Tetraciclina/farmacologia
3.
Nucleic Acids Res ; 49(5): 2894-2915, 2021 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-33619526

RESUMO

Trans-acting regulatory RNAs have the capacity to base pair with more mRNAs than generally detected under defined conditions, raising the possibility that sRNA target specificities vary depending on the specific metabolic or environmental conditions. In Sinorhizobium meliloti, the sRNA rnTrpL is derived from a tryptophan (Trp) transcription attenuator located upstream of the Trp biosynthesis gene trpE(G). The sRNA rnTrpL contains a small ORF, trpL, encoding the 14-aa leader peptide peTrpL. If Trp is available, efficient trpL translation causes transcription termination and liberation of rnTrpL, which subsequently acts to downregulate the trpDC operon, while peTrpL is known to have a Trp-independent role in posttranscriptional regulation of antibiotic resistance mechanisms. Here, we show that tetracycline (Tc) causes rnTrpL accumulation independently of Trp availability. In the presence of Tc, rnTrpL and peTrpL act collectively to destabilize rplUrpmA mRNA encoding ribosomal proteins L21 and L27. The three molecules, rnTrpL, peTrpL, and rplUrpmA mRNA, form an antibiotic-dependent ribonucleoprotein complex (ARNP). In vitro reconstitution of this ARNP in the presence of competing trpD and rplU transcripts revealed that peTrpL and Tc cause a shift of rnTrpL specificity towards rplU, suggesting that sRNA target prioritization may be readjusted in response to changing environmental conditions.


Assuntos
Antibacterianos/farmacologia , Peptídeos/metabolismo , Estabilidade de RNA , RNA Mensageiro/metabolismo , Pequeno RNA não Traduzido/metabolismo , Sinorhizobium meliloti/genética , Tetraciclina/farmacologia , Pareamento de Bases , Regulação Bacteriana da Expressão Gênica , Peptídeos/química , RNA Antissenso/metabolismo , RNA Mensageiro/química , Pequeno RNA não Traduzido/química , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Sinorhizobium meliloti/efeitos dos fármacos
4.
Microbiome ; 9(1): 29, 2021 01 27.
Artigo em Inglês | MEDLINE | ID: mdl-33504360

RESUMO

BACKGROUND: Antimicrobial resistance (AMR) is a major threat to public health. Microorganisms equipped with AMR genes are suggested to have partially emerged from natural habitats; however, this hypothesis remains inconclusive so far. To understand the consequences of the introduction of exogenic antimicrobials into natural environments, we exposed lichen thalli of Peltigera polydactylon, which represent defined, highly diverse miniature ecosystems, to clinical (colistin, tetracycline), and non-clinical (glyphosate, alkylpyrazine) antimicrobials. We studied microbiome responses by analysing DNA- and RNA-based amplicon libraries and metagenomic datasets. RESULTS: The analyzed samples consisted of the thallus-forming fungus that is associated with cyanobacteria as well as other diverse and abundant bacterial communities (up to 108 16S rRNA gene copies ng-1 DNA) dominated by Alphaproteobacteria and Bacteroidetes. Moreover, the natural resistome of this meta-community encompassed 728 AMR genes spanning 30 antimicrobial classes. Following 10 days of exposure to the selected antimicrobials at four different concentrations (full therapeutic dosage and a gradient of sub-therapeutic dosages), we observed statistically significant, antimicrobial-specific shifts in the structure and function but not in bacterial abundances within the microbiota. We observed a relatively lower response after the exposure to the non-clinical compared to the clinical antimicrobial compounds. Furthermore, we observed specific bacterial responders, e.g., Pseudomonas and Burkholderia to clinical antimicrobials. Interestingly, the main positive responders naturally occur in low proportions in the lichen holobiont. Moreover, metagenomic recovery of the responders' genomes suggested that they are all naturally equipped with specific genetic repertoires that allow them to thrive and bloom when exposed to antimicrobials. Of the responders, Sphingomonas, Pseudomonas, and Methylobacterium showed the highest potential. CONCLUSIONS: Antimicrobial exposure resulted in a microbial dysbiosis due to a bloom of naturally low abundant taxa (positive responders) with specific AMR features. Overall, this study provides mechanistic insights into community-level responses of a native microbiota to antimicrobials and suggests novel strategies for AMR prediction and management. Video Abstract.


Assuntos
Anti-Infecciosos/farmacologia , Ascomicetos/efeitos dos fármacos , Ascomicetos/genética , Resistência Microbiana a Medicamentos/efeitos dos fármacos , Resistência Microbiana a Medicamentos/genética , Microbiota/efeitos dos fármacos , Microbiota/genética , Colistina/farmacologia , Glicina/análogos & derivados , Glicina/farmacologia , Pirazinas/farmacologia , RNA Ribossômico 16S/genética , Tetraciclina/farmacologia
5.
Appl Environ Microbiol ; 87(6)2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33397699

RESUMO

Little is known about the drivers of critically important antibacterial resistance in species with zoonotic potential present on farms (e.g., CTX-M ß-lactamase-positive Escherichia coli). We collected samples monthly between January 2017 and December 2018 on 53 dairy farms in South West England, along with data for 610 variables concerning antibacterial usage, management practices, and meteorological factors. We detected E. coli resistant to amoxicillin, ciprofloxacin, streptomycin, and tetracycline in 2,754/4,145 (66%), 263/4,145 (6%), 1,475/4,145 (36%), and 2,874/4,145 (69%), respectively, of samples from fecally contaminated on-farm and near-farm sites. E. coli positive for bla CTX-M were detected in 224/4,145 (5.4%) of samples. Multilevel, multivariable logistic regression showed antibacterial dry cow therapeutic choice (including use of cefquinome or framycetin) to be associated with higher odds of bla CTX-M positivity. Low average monthly ambient temperature was associated with lower odds of bla CTX-M E. coli positivity in samples and with lower odds of finding E. coli resistant to each of the four test antibacterials. This was in addition to the effect of temperature on total E. coli density. Furthermore, samples collected close to calves had higher odds of having E. coli resistant to each antibacterial, as well as E. coli positive for bla CTX-M Samples collected on pastureland had lower odds of having E. coli resistant to amoxicillin or tetracycline, as well as lower odds of being positive for bla CTX-M IMPORTANCE Antibacterial resistance poses a significant threat to human and animal health and global food security. Surveillance for resistance on farms is important for many reasons, including tracking impacts of interventions aimed at reducing the prevalence of resistance. In this longitudinal survey of dairy farm antibacterial resistance, we showed that local temperature-as it changes over the course of a year-was associated with the prevalence of antibacterial-resistant E. coli We also showed that prevalence of resistant E. coli was lower on pastureland and higher in environments inhabited by young animals. These findings have profound implications for routine surveillance and for surveys carried out for research. They provide important evidence that sampling at a single time point and/or single location on a farm is unlikely to be adequate to accurately determine the status of the farm regarding the presence of samples containing resistant E. coli.


Assuntos
Farmacorresistência Bacteriana , Escherichia coli/genética , beta-Lactamases/genética , Envelhecimento , Amoxicilina/farmacologia , Animais , Antibacterianos/farmacologia , Bovinos , Doenças dos Bovinos/microbiologia , Ciprofloxacino/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/microbiologia , Fazendas , Fezes/microbiologia , Estreptomicina/farmacologia , Temperatura , Tetraciclina/farmacologia
6.
Ecotoxicol Environ Saf ; 208: 111724, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33396055

RESUMO

In recent years, in order to promote animal growth and reduce the risk of disease, a variety of antibiotics are frequently added to the animal feed of livestock and poultry. However, these antibiotics can not be fully digested by animals and most of them are excreted with feces, consequently causing the enrichment of antibiotic resistance genes (ARGs) and huge environmental risks. Nowadays, composting is a better option to solve these problems. Accordingly, this study explored the effects of co-composting swine manure with different inoculants dominated by Phanerochaete chrysosporium (p), Aspergillus niger (a), and Bacillus licheniformis (b) on the simultaneous removal of multiple antibiotics and resistance genes and evolution of the bacterial community. The results showed that the highest removal extent of tetracycline and oxytetracycline occurred in pile D (p:b:a=1:5:5, biomass) reaching 89.2% and 87.8%, respectively, while the highest removal extent of doxycycline and enrofloxacin occurred in pile A (p:b:a=1:0:0, biomass) reaching 98.6% and 89%, respectively. Compared with the levels in pile B (control check), in pile D, ARGs, except those for sulfonamides, decreased by 1.059 × 10-3-6.68 × 10-2 gene copies/16S rRNA copies. Inoculation with p alone effectively reduced intI1 and intI2. Canonical correspondence analysis (CCA) that microbial community structure evolution had a greater influence on ARGs than environmental factors. In summary, this study provided a feasible way to efficiently remove the antibiotics and antibiotic resistance genes in pig manure.


Assuntos
Antibacterianos/análise , Resistência Microbiana a Medicamentos/genética , Esterco/microbiologia , Ração Animal/análise , Animais , Antibacterianos/metabolismo , Bactérias/efeitos dos fármacos , Compostagem/métodos , Genes Bacterianos , Gado , Microbiota/efeitos dos fármacos , Oxitetraciclina/farmacologia , RNA Ribossômico 16S , Suínos , Tetraciclina/farmacologia
7.
Biochem J ; 478(1): 261-279, 2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33355669

RESUMO

Herpes simplex virus 1 (HSV-1) is a human DNA virus that causes cold sores, keratitis, meningitis, and encephalitis. Ubiquitination is a post-translational protein modification essential for regulation of cellular events, such as proteasomal degradation, signal transduction, and protein trafficking. The process is also involved in events for establishing viral infection and replication. The first step in ubiquitination involves ubiquitin (Ub) binding with Ub-activating enzyme (E1, also termed UBE1) via a thioester linkage. Our results show that HSV-1 infection alters protein ubiquitination pattern in host cells, as evidenced by MS spectra and co-immunoprecipitation assays. HSV-1 induced ubiquitination of UBE1a isoform via an isopeptide bond with Lys604. Moreover, we show that ubiquitination of K604 in UBE1a enhances UBE1a activity; that is, the activity of ubiquitin-transfer to E2 enzyme. Subsequently, we investigated the functional role of UBE1a and ubiquitination of K604 in UBE1a. We found that UBE1-knockdown increased HSV-1 DNA replication and viral production. Furthermore, overexpression of UBE1a, but not a UBE1a K604A mutant, suppressed viral replication. Furthermore, we found that UBE1a and ubiquitination at K604 in UBE1a retarded expression of HSV-1 major capsid protein, ICP5. Our findings show that UBE1a functions as an antiviral factor that becomes activated upon ubiquitination at Lys604.


Assuntos
Antivirais/metabolismo , Herpes Simples/metabolismo , Herpesvirus Humano 1/metabolismo , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Enzimas Ativadoras de Ubiquitina/metabolismo , Animais , Sobrevivência Celular/genética , Chlorocebus aethiops , Cromatografia Líquida , Doxiciclina/farmacologia , Células HeLa , Herpes Simples/enzimologia , Herpes Simples/genética , Herpesvirus Humano 1/genética , Interações Hospedeiro-Patógeno/genética , Humanos , Ligação Proteica , Domínios Proteicos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , RNA Interferente Pequeno , Espectrometria de Massas em Tandem , Tetraciclina/farmacologia , Transfecção , Enzimas Ativadoras de Ubiquitina/genética , Ubiquitinação/efeitos dos fármacos , Células Vero , Replicação Viral/efeitos dos fármacos
8.
Appl Environ Microbiol ; 87(6)2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33361364

RESUMO

Antimicrobial-resistant pathogens display significant public health threats by causing difficulties in clinical treatment of bacterial infection. Antimicrobial resistance (AMR) is transmissible between bacteria, significantly increasing the appearance of antimicrobial-resistant pathogens and aggravating the AMR problem. In this work, the dissemination dynamics of AMR from invading multidrug-resistant (MDR) Escherichia coli to a community of pathogenic Salmonella enterica was investigated using a continuous-culture device, and the behaviors of dissemination dynamics under different levels of antibiotic stress were investigated. Three MDR E. coli invasion events were analyzed in this work: MDR E. coli-S. enterica cocolonization, MDR E. coli invasion after antibiotic treatment of S. enterica, and MDR E. coli invasion before antibiotic treatment of S. enterica It was found that both horizontal gene transfer (HGT) and vertical gene transfer (VGT) play significant roles in AMR dissemination, although different processes contribute differently under different circumstances, that environmental levels of antibiotics promote AMR dissemination by enhancing HGT rather than leading to selective advantage for resistant bacteria, and that early invasion of MDR E. coli completely and quickly sabotages the effectiveness of antibiotic treatment. These findings contribute to understanding the drivers of AMR dissemination under different antibiotic stresses, the detrimental impact of environmental tetracycline contamination, and the danger of nosocomial presence and dissemination of MDR nonpathogens.IMPORTANCE Antimicrobial resistance poses a grave threat to public health and reduces the effectiveness of antimicrobial drugs in treating bacterial infections. Antimicrobial resistance is transmissible, either by horizontal gene transfer between bacteria or by vertical gene transfer following inheritance of genetic traits. The dissemination dynamics and behaviors of this threat, however, have not been rigorously investigated. In this work, with a continuous-culture device, we studied antimicrobial resistance dissemination processes by simulating antimicrobial-resistant Escherichia coli invasion to a pathogenic Salmonella enterica community. Using this novel tool, we provide evidence on the drivers of antimicrobial resistance dissemination, on the detrimental impact of environmental antibiotic contamination, and on the danger of antimicrobial resistance in hospitals, even if what harbors the antimicrobial resistance is not a pathogen. This work furthers our understanding of antimicrobial resistance and its dissemination between bacteria and of antibiotic therapy, our most powerful tool against bacterial infection.


Assuntos
Farmacorresistência Bacteriana Múltipla/genética , Escherichia coli/genética , Salmonella enterica/genética , Antibacterianos/farmacologia , Técnicas Bacteriológicas , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Transferência Genética Horizontal , Interações Microbianas , Testes de Sensibilidade Microbiana , Filogenia , Salmonella enterica/efeitos dos fármacos , Salmonella enterica/crescimento & desenvolvimento , Tetraciclina/farmacologia
9.
Methods Mol Biol ; 2223: 115-132, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33226591

RESUMO

IL-22 is an IL-10 family cytokine that is increased in asthma and atopic dermatitis (AD). However, the specific role of IL-22 in the pathogenesis of allergic lung inflammation and AD in vivo has yet to be elucidated. We aimed to develop mouse models of allergic diseases in the lung and skin with inducible and tissue-specific expression of IL-22, using a tetracycline (Tet)-controlled system. In this chapter, we describe a series of protocols we have developed to generate a construct that contains the TRE-Tight promoter and mouse IL-22 cDNA based on this system. Furthermore, we describe how to generate TRE-Tight-IL-22 mice through pronuclear microinjection. In our approach, two Tet-on (CC10-rtTA or SPC-rtTA) and a Tet-off (K5-tTA) transgenic mouse lines are selected to crossbreed with TRE-Tight-IL-22 mice to generate inducible tissue-specific transgenic lines. The transgenic strains, CC10-rtTA/TRE-Tight-IL-22 (CC10-rtTA-IL-22) or SPC-rtTA/TRE-Tight-IL-22 (SPC-rtTA-IL-22) mice, do not produce detectable levels of IL-22 in their bronchoalveolar lavage (BAL) samples in the absence of doxycycline (Dox). However, oral Dox treatment of these mice induces IL-22 expression in the BAL, and the airway and lung epithelial cells. For K5-tTA/TRE-Tight-IL-22 (K5-tTA-IL-22) mice, to avoid potential IL-22 toxicity to mouse embryos, Dox is given starting at the time of breeding to suppress tTA and to keep the IL-22 transgene off until the K5-tTA-IL-22 mice are 6 weeks old. Experiments are then initiated by withdrawing Dox from the drinking water. In all cases, IL-22 protein can be detected by immunohistochemistry in the skin of Tg(+) animals, but not in the skin of Tg(-) animals. Utilizing transgenic technology based on the Tetracycline-controlled system, we have established inducible transgenic mouse models in which cytokine IL-22 can be expressed specifically in the lung or skin. These models are valuable for studies in vivo in a broad range of diseases involving IL-22 and will provide a new platform for research and for seeking novel therapeutics in the fields of inflammation, asthma, and allergic dermatitis.


Assuntos
Transferência Embrionária/métodos , Embrião de Mamíferos/efeitos dos fármacos , Regulação da Expressão Gênica , Interleucinas/genética , Pulmão/imunologia , Pele/imunologia , Transfecção/métodos , Células A549 , Animais , Doxiciclina/farmacologia , Embrião de Mamíferos/citologia , Embrião de Mamíferos/imunologia , Feminino , Humanos , Interleucinas/agonistas , Interleucinas/imunologia , Lipídeos/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microinjeções/métodos , Plasmídeos/química , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Tetraciclina/farmacologia , Transcrição Genética , Transgenes , Zigoto/citologia , Zigoto/crescimento & desenvolvimento , Zigoto/metabolismo
10.
PLoS One ; 15(12): e0244732, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33378396

RESUMO

On account of its stringent regulation and high rate of induction, the tetracycline regulatory system is used extensively for inducing target gene expression in eukaryotes. However, under certain circumstances, its associated background expression can be problematic, as in the expression of highly toxic proteins. We found that when using the Tet-On 3G system to drive expression of the kid toxin gene in sf9 insect cells, a higher percentage of cells were killed than when using an empty vector in the absence of the induction agent doxycycline, thereby indicating the leaky expression of this inducible expression system. Moreover, we found that the tetracycline-controlled transcriptional silencer (tTS) does not effectively reduce the background expression of the Tet-On 3G system in sf9 cells. However, Csy4, a Cas9 homologous protein in the CRISPR family with sequence-specific endonuclease activity, was found to be effective in reducing the Tet-On 3G system-associated background expression, although there was a concomitant reduction in the maximum induced expression. Nevertheless, we found that modification of the system via incorporation of TRE-controlled anti-sense csy4 in combination with a WSSVie1 (Δ23) promotor-driven sense csy4 significantly reduced the leaky expression of the Tet-On 3G system, and that the level of induction was higher than that initially obtained. This optimized Tet-On 3G system can significantly reduce cell death attributed to the background expression of Kid under uninduced conditions. Therefore, we developed a novel low-background inducible expression system for use in insect cells and potentially in other organisms including mammals based on post-transcriptional regulation using Csy4.


Assuntos
Proteínas Associadas a CRISPR/genética , Endorribonucleases/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Animais , Linhagem Celular , Vetores Genéticos/efeitos dos fármacos , Regiões Promotoras Genéticas/efeitos dos fármacos , Tetraciclina/farmacologia , Transativadores/genética
11.
BMC Genet ; 21(Suppl 2): 141, 2020 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-33339506

RESUMO

BACKGROUND: The sterile insect technique (SIT) has been successfully used in many pest management programs worldwide. Some SIT programs release both sexes due to the lack of genetic sexing strains or efficient sex separation methods but sterile females are ineffective control agents. Transgenic sexing strains (TSS) using the tetracycline-off control system have been developed in a variety of insect pests, from which females die by either of two commonly used lethal effectors: overexpression of the transcription factor tetracycline transactivator (tTA) or ectopic expression of a proapoptotic gene, such as head involution defective (hid). The lethality from tTA overexpression is thought to be due to "transcriptional squelching", while hid causes lethality by induction of apoptosis. This study aims to create and characterize a TSS of Lucilia cuprina, which is a major pest of sheep, by combining both lethal effectors in a single transgenic strain. RESULTS: Here a stable TSS of L. cuprina (DH6) that carries two lethal effectors was successfully generated, by crossing FL3#2 which carries a female-specific tTA overexpression cassette, with EF1#12 which carries a tTA-regulated LshidAla2 cassette. Females with one copy of the FL3#2 transgene are viable but up to 99.8% of homozygous females die at the pupal stage when raised on diet that lacks tetracycline. Additionally, the female lethality of FL3#2 was partially repressed by supplying tetracycline to the parental generation. With an additional LshidAla2 effector, the female lethality of DH6 is 100% dominant and cannot be repressed by maternal tetracycline. DH6 females die at the late-larval stage. Several fitness parameters important for mass rearing such as hatching rate, adult emergence and sex ratio were comparable to those of the wild type strain. CONCLUSIONS: Compared to the parental FL3#2 strain, the DH6 strain shows stronger female lethality and lethality occurs at an earlier stage of development. The combination of two tTA-dependent lethal effectors could improve strain stability under mass rearing and could reduce the risk of resistance in the field if fertile males are released. Our approach could be easily adapted for other pest species for an efficient, safe and sustainable genetic control program.


Assuntos
/genética , Genes Letais , Controle de Insetos , Animais , Animais Geneticamente Modificados , Desenvolvimento Embrionário , Feminino , Aptidão Genética , Ovinos/parasitologia , Tetraciclina/farmacologia
12.
Huan Jing Ke Xue ; 41(8): 3758-3764, 2020 Aug 08.
Artigo em Chinês | MEDLINE | ID: mdl-33124351

RESUMO

The tetracycline (TC) antibiotic has been widely found in different environmental matrices. The tetracycline resistant bacterium (TRB) of Shigella flexneri was screened and purified from activated sludge, and was then used to study the impact of TC stress on the gene abundances and expression levels of TC resistance genes (TC-ARGs), including tetC, tetO, and tetX, which were respectively quantified by quantitative PCR and reverse transcriptional PCR. Correlations between the TC concentration and gene abundances of TC-ARGs and their expression levels were discussed. The results showed that TC stress had an inhibiting effect on the growth of Shigella flexneri during the entire culture cycle (24 h) and that the growth rate of the bacterial concentration decreased with increasing TC concentration. However, less impact on the gene abundance of TC-ARGs was found. TC stress could promote the expression of TC-ARGs in Shigella flexneri, and the expression levels of tetC, tetO, and tetX genes first increased and then decreased. The correlation results indicated that no significant correlation was observed between the TC concentration and gene abundances of TC-ARGs and their expression levels. Nevertheless, the gene abundances of tetC and tetO were significantly correlated with their expression levels, thus indicating that they can be used to evaluate and assess expression levels to a certain extent.


Assuntos
Shigella flexneri , Resistência a Tetraciclina , Antibacterianos/farmacologia , Genes Bacterianos/genética , Shigella flexneri/genética , Tetraciclina/farmacologia , Resistência a Tetraciclina/genética
13.
Int J Food Microbiol ; 335: 108857, 2020 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-32947144

RESUMO

Cronobacter species (Cronobacter spp.) are important foodborne pathogens that can infect and cause serious life-threatening diseases in infants and immunocompromised elderly. This study aimed to acquire data on Cronobacter spp. contamination of aquatic products in China from 2011 to 2016. In total, 800 aquatic products were tested, and the overall contamination rate for Cronobacter spp. was 3.9% (31/800). The average contamination level of the positive samples was 2.05 MPN/g. Four species and nine serotypes were identified among 33 isolates, of which the C. sakazakii serogroup O1 (n = 9) was the primary serotype. The majority of Cronobacter spp. strains harbored highest resistance against cephalothin (84.8%), followed by tetracycline (6.1%), trimethoprim/sulfameth-oxazole (3.0%) and chloramphenicol (3.0%). Two isolates were resistant to three antibiotics. In total, 26 sequence types and 33 CRISPR types (including 6 new STs and 26 new CTs) were identified, which indicates the extremely high diversity of Cronobacter spp. in aquatic products. Pathogenic C. sakazakii ST4, ST1, and C. malonaticus ST7 were also observed. Overall, this large-scale study revealed the relatively low prevalence and high genetic diversity of Cronobacter spp. in aquatic products in China, and the findings provide valuable information that can guide the establishment of effective measures for the control and precaution of Cronobacter spp. in aquatic products during production processes.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Cronobacter/classificação , Cronobacter/isolamento & purificação , Farmacorresistência Bacteriana , Alimentos Marinhos/microbiologia , Antibacterianos/farmacologia , Cefalotina/farmacologia , China , Cloranfenicol/farmacologia , Cronobacter sakazakii/classificação , Cronobacter sakazakii/isolamento & purificação , Contaminação de Alimentos , Microbiologia de Alimentos , Variação Genética , Tipagem de Sequências Multilocus , Prevalência , Sorotipagem , Tetraciclina/farmacologia , Trimetoprima/farmacologia
14.
BMC Infect Dis ; 20(1): 602, 2020 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-32799799

RESUMO

BACKGROUND: The objectives of this study were to determine for the first time, in Morocco, the nasal carriage rate, antimicrobial susceptibility profiles and virulence genes of Staphylococcus. aureus isolated from animals and breeders in close contact. METHODS: From 2015 to 2016, 421 nasal swab samples were collected from 26 different livestock areas in Tangier. Antimicrobial susceptibility phenotypes were determined by disk diffusion according to EUCAST 2015. The presence of nuc, mecA, mecC, lukS/F-PV, and tst genes were determined by Polymerase Chain Reaction (PCR) for all isolates. RESULTS: The overall S. aureus nasal carriage rate was low in animals (9.97%) and high in breeders (60%) with a statistically significant difference, (OR = 13.536; 95% CI = 7.070-25.912; p < 0.001). In general, S. aureus strains were susceptible to the majority of antibiotics and the highest resistance rates were found against tetracycline (16.7% in animals and 10% in breeders). No Methicillin-Resistant S. aureus (MRSA) was detected in animals and breeders. A high rate of tst and lukS/F-PV genes has been recovered only from animals (11.9 and 16.7%, respectively). CONCLUSION: Despite the lower rate of nasal carriage of S. aureus and the absence of MRSA strains in our study, S. aureus strains harbored a higher frequency of tst and lukS/F-PV virulence genes, which is associated to an increased risk of infection dissemination in humans. This highlights the need for further larger and multi-center studies to better define the transmission of the pathogenic S. aureus between livestock, environment, and humans.


Assuntos
Nariz/microbiologia , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/veterinária , Staphylococcus aureus/efeitos dos fármacos , Animais , Animais Domésticos/microbiologia , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Portador Sadio , Farmacorresistência Bacteriana/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Humanos , Leucocidinas/genética , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Testes de Sensibilidade Microbiana , Nuclease do Micrococo/genética , Marrocos/epidemiologia , Proteínas de Ligação às Penicilinas/genética , Infecções Estafilocócicas/epidemiologia , Staphylococcus aureus/genética , Staphylococcus aureus/isolamento & purificação , Staphylococcus aureus/patogenicidade , Tetraciclina/farmacologia , Virulência/genética
15.
Water Res ; 185: 116241, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32777596

RESUMO

The transmission of antibiotic resistance in surface water has attracted much attention due to its increasing threat to human health. The role of sunlight irradiation and the effect of dissolved organic matter (DOM) on the transmission of antibiotic resistance are still unclear. In this study, photo-inactivation of antibiotic resistant bacteria (ARB) was investigated using antibiotic resistant E. coli (AR E. coli) that contained the tetracycline resistance gene (Tc-ARG) as a representative. The results showed that AR E. coli underwent significant photo-inactivation due to the membrane damage induced by direct irradiation and by the generated reactive oxygen species. Simulated sunlight irradiation specifically suppressed the expression of tetracycline resistance, which is attributed to the destruction of tetracycline-specific efflux pump. Tetracycline inhibited the photo-inactivation of AR E. coli due to its selective pressure on tetracycline resistant E. coli and competitive light absorption effect. Suwannee River fulvic acid (SRFA), a representative DOM, promoted the inactivation of AR E. coli and further inhibited the expression of tetracycline resistance gene due to the generation of its excited triplet state, singlet oxygen, and hydroxyl radical. The extracellular Tc-ARG also underwent fast photodegradation under light irradiation and in the presence of SRFA, which leads to the decrease of its transformation efficiency. This study provided insight into the sunlight-induced inactivation of ARB, which is of significance for understanding the transmission of tetracycline resistance in surface water.


Assuntos
Luz Solar , Poluentes Químicos da Água , Antibacterianos/farmacologia , Bactérias , Escherichia coli/genética , Fotólise , Tetraciclina/farmacologia , Poluentes Químicos da Água/análise
16.
Nat Commun ; 11(1): 3834, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32737309

RESUMO

The transcriptional inducer anhydrotetracycline (aTc) and the bacteriostatic antibiotic tetracycline (Tc) are commonly used in all fields of biology for control of transcription or translation. A drawback of these and other small molecule inducers is the difficulty of their removal from cell cultures, limiting their application for dynamic control. Here, we describe a simple method to overcome this limitation, and show that the natural photosensitivity of aTc/Tc can be exploited to turn them into highly predictable optogenetic transcriptional- and growth-regulators. This new optogenetic class uniquely features both dynamic and setpoint control which act via population-memory adjustable through opto-chemical modulation. We demonstrate this method by applying it for dynamic gene expression control and for enhancing the performance of an existing optogenetic system. We then expand the utility of the aTc system by constructing a new chemical bandpass filter that increases its aTc response range. The simplicity of our method enables scientists and biotechnologists to use their existing systems employing aTc/Tc for dynamic optogenetic experiments without genetic modification.


Assuntos
Escherichia coli/efeitos dos fármacos , Optogenética/métodos , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Tetraciclina/farmacologia , Tetraciclinas/farmacologia , Transcrição Genética/efeitos dos fármacos , Clonagem Molecular , Relação Dose-Resposta a Droga , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Genes Reporter , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Fotólise , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Raios Ultravioleta
17.
Environ Sci Pollut Res Int ; 27(33): 42150-42157, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32851521

RESUMO

Residues of antibiotics in the soil can have adverse effects on crop plants related closely to humans. However, there is a lack of knowledge on the phytotoxicity of antibiotics to sensitive species. The aim of our study was mainly to reveal tetracycline stress on the mobilization of seed storage proteins, lipids, and minerals of Chinese cabbage (Brassica rapa L.) during radicle elongation after germination. The Chinese cabbage seed was incubated for 48 h in tetracycline solutions at 1 and 5 µM. For the cotyledons of the seed, the result of TEM showed that tetracycline significantly hindered the mobilization of protein bodies, which was in line with the result of FTIR spectroscopy. However, the mobilization of oil bodies and mineral bodies in cotyledons was not significantly disturbed by the stress of tetracycline, as well as the energy supply in different organs including the cotyledons, hypocotyl, and radicle of the seed. To our knowledge, this is the first study to demonstrate the disturbance on mobilization of protein bodies in seed storage reserves caused by the stress of tetracycline at low levels during radicle elongation after germination.


Assuntos
Germinação , Sementes , Antibacterianos/farmacologia , Cotilédone , Tetraciclina/farmacologia
18.
Chemosphere ; 255: 127011, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32679630

RESUMO

The environmental toxicity of tetracycline antibiotics to aquatic organisms has attracted increasing attention. The adverse impacts of tetracycline antibiotics on ciliates should be detailed considering the significant roles of protozoa in the microfood web in the soils and other eco-systems. This study for the first time investigated the toxicity of two typical tetracycline antibiotics, tetracycline (TC) and tetracycline hydrochloride (HTC) on two primary model ciliates, Stentor coeruleus and Stylonychia lemnae. The concentrations for 50% of maximal effect (24h‒EC50) of TC and HTC to Stentor coeruleus were 94.4 mg/L and 8.39 mg/L, respectively. Correspondingly, the 24h‒EC50 values of TC and HTC to Stylonychia lemnae were 40.1 mg/L and 14.0 mg/L, respectively. The TC and HTC inhibited the growth rates, reduced the activities of antioxidant enzymes, and damaged the ultra-structures of the tested ciliate cells, with the latter having larger impacts than the former. Based on the experimental works reported herein, the two model protozoan species were proposed to be the toxicity indicators for tetracycline antibiotics, which could work as supplements with the other existing protocols, such as Brochydanio rerio (zebrafish), Limnodrilus (a worm), Chlorogonium elongatum (a green alga) also studied herein.


Assuntos
Antibacterianos/toxicidade , Cilióforos/efeitos dos fármacos , Tetraciclina/toxicidade , Antioxidantes/farmacologia , Tetraciclina/farmacologia , Testes de Toxicidade
19.
Artigo em Inglês | MEDLINE | ID: mdl-32539543

RESUMO

Wild animals like pheasant seem to be a good source of information about human activities. Therefore, the wild pheasants and relative stable appendix microcenosis were selected for antibiotic resistance testing. Penicillin resistance by MALDI-TOF Mass Spectrometry and tetracyclines resistance by genetic methods using specific primers were tested. Differences between tetracycline and penicillin resistance were detected. Results showed high prevalence of resistant Escherichia coli isolated from wild pheasant appendix. E. coli isolated from wild pheasant appendix carried plasmids for penicillins and tetracyclines resistance where they were responsible for enzymatic degradation of penicillin and carried genes for regulating efflux pumps for tetracyclines. Results showed that tetracyclines and penicillins resistance is widespread between wild pheasants with a carrier as Escherichia coli isolated from relative stable microcenosis of appendix.


Assuntos
Monitoramento Ambiental/métodos , Escherichia coli/genética , Galliformes/microbiologia , Resistência às Penicilinas/genética , Resistência a Tetraciclina/genética , Ampicilina/farmacologia , Animais , Animais Selvagens , Apêndice/microbiologia , Escherichia coli/efeitos dos fármacos , Humanos , Eslováquia , Tetraciclina/farmacologia
20.
Int J Infect Dis ; 97: 102-107, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32474200

RESUMO

OBJECTIVES: Bhutan suffers from a high prevalence of Helicobacter pylori (H. pylori) infection and gastric cancer-related mortality. In preparation for a countrywide H. pylori eradication program, the antibiotic resistance patterns of H. pylori infection were surveyed in different geographical regions. METHODS: Dyspeptic patients in 6 districts including Thimphu, Punakha, Wangdue, Trongsa, Bumthang, and Haa underwent upper gastrointestinal endoscopy during GASTROCAMP and were enrolled between December 2010 and April 2015. Gastric biopsies were obtained for rapid urease test, histopathology, and H. pylori culture. Antimicrobial susceptibility testing was later performed if the culture was positive. RESULTS: A total of 1178 patients were surveyed. The overall H. pylori infection in Bhutan was 66.2%. Punakha had the highest prevalence of H. pylori infection (85.6%). Thimphu and Punakha (city areas) had higher prevalence of H. pylori infection than rural districts (73.5% vs 63.3%, OR=1.61, 95% CI 1.22-2.13, p=0.0008). There were 357 patients (30.3%) with positive H. pylori culture completed antimicrobial susceptibility testing. The mean age was 40.5 years with female predominance (57.1%). No amoxicillin resistant strains were found. Metronidazole resistance was 81% followed by levofloxacin resistance (8.1%). Clarithromycin (2%) and tetracycline (0.6%) resistance was rare except in Thimphu, the capital city (10%) vs 0% in rural areas, p<0.001. The metronidazole resistance rate remained stable at approximately 80% during the past 5 years of study. Levofloxacin-resistant strains gradually rose from 5.3% in 2010 to 9.9% in 2015. CONCLUSIONS: Bhutan had a high prevalence of H. pylori infection. Metronidazole resistance was extremely high, whereas clarithromycin resistance was quite low in this specific area. Antibiotic resistance pattern could be good evidence for guiding a proper treatment regimen for H. pylori infection in Bhutan.


Assuntos
Farmacorresistência Bacteriana , Infecções por Helicobacter/epidemiologia , Helicobacter pylori/efeitos dos fármacos , Adulto , Amoxicilina/farmacologia , Antibacterianos/farmacologia , Butão/epidemiologia , Claritromicina/farmacologia , Feminino , Infecções por Helicobacter/microbiologia , Helicobacter pylori/isolamento & purificação , Humanos , Levofloxacino/farmacologia , Masculino , Metronidazol/farmacologia , Pessoa de Meia-Idade , Prevalência , Tetraciclina/farmacologia
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