摘要 :
Binary expression systems are widely employed to analyze gene function in vivo using transgenic organisms. The tetracycline-off (Tet-Off) system, which is a binary expression system that uses a tetracycline-controlled transactivat...
展开
Binary expression systems are widely employed to analyze gene function in vivo using transgenic organisms. The tetracycline-off (Tet-Off) system, which is a binary expression system that uses a tetracycline-controlled transactivator protein (tTA) and its tetracycline operator sequence (tetO) binding site, was developed as a method for temporally controlling gene expression. To facilitate the use of the Tet-Off system in animal species other than the model organisms that are widely used for genetic analysis, we constructed two different fusion proteins containing enhanced green fluorescent protein (EGFP) as the reporter gene and tTA as the transactivator, in different configurations. We assessed the utility of these fusion proteins designated as tTA-EGFP and EGFP-tTA in transgenic fruit flies. We showed that, although EGFP of both fusion proteins was efficiently fluoresced, transcriptional activation occurred only by the tTA-EGFP fusion protein. Furthermore, tetracycline (Tc) and doxycycline (Dox) both effectively inactivated tTA-EGFP, repressing gene expression under tetO control in a concentration-dependent manner. Additionally, the removal of Tc or Dox from the diet can recover the transactivator activity of tTA-EGFP in a concentration- and time-dependent manner. The tTA-EGFP fusion protein will therefore be useful in the analysis of gene function in a wide range of animal species.
收起
摘要 :
A growing number of tet(X)-type tigecycline resistance determinants [tet(X1) to tet(X5)] constitutes an expanding family of tetracycline-inactivating enzymes, posing a potential risk to global public health. Here, we report the de...
展开
A growing number of tet(X)-type tigecycline resistance determinants [tet(X1) to tet(X5)] constitutes an expanding family of tetracycline-inactivating enzymes, posing a potential risk to global public health. Here, we report the development of an efficient multiplex PCR method to detect the family of tet(X) variants. This method is successfully applied in the screen and validation of tet(X) genes in the field and clinic bacterial samples. In addition, we found that the formerly proposed tet(X1) is a premature truncated version by the inappropriate annotation, and fixed this error. Overall, it might be the first genetic tool for the detection of different Tet(X) members.
收起
摘要 :
This study aims to demonstrate the effect of oral doxycycline on fecal microbiota of mice. Doxycycline is a common effector for control of gene expression using the tet-inducible system in transgenic mice. The effect of oral doxyc...
展开
This study aims to demonstrate the effect of oral doxycycline on fecal microbiota of mice. Doxycycline is a common effector for control of gene expression using the tet-inducible system in transgenic mice. The effect of oral doxycycline on murine gut microbiota has not been reported. We evaluated the effect of doxycycline treatment by sequencing the V4 hypervariable region of the 16S rRNA gene from fecal samples collected during a 4?week course of treatment at a dose of 2?mg/ml in the drinking water. The fecal microbiota of treated animals were distinct from control animals; the decreased richness and diversity were characterized primarily by Bacteroides sp. enrichment. These effects persisted when the treatment was temporarily discontinued for 1?week. These data suggest that doxycycline treatment can induce significant dysbiosis, and its effects should be considered when used in animal models that are or maybe sensitive to perturbation of the gut microbiota.
收起
摘要 :
The present study was conducted with the following aims: (i) To detect the multidrug-resistant Salmonella spp. isolates recovered from faeces, litter and drinking water in layers and broiler breeders’ farms and (ii) to carried ou...
展开
The present study was conducted with the following aims: (i) To detect the multidrug-resistant Salmonella spp. isolates recovered from faeces, litter and drinking water in layers and broiler breeders’ farms and (ii) to carried out the detection of the tet gene. A total of 21 Salmonella isolates were subjected to Polymerase Chain Reaction (PCR) assay to determine the presence of tet gene. Out of 21 isolates, 14 (66.7%) and 7 (33.3%) were found positive for tet(A) and tet(B), respectively. In antimicrobial susceptibility tests, the Salmonella isolates showed resistance to tetracycline, oxytetracycline, ampicillin, nalidixic acid, enrofloxacin, gentamicin and chloramphenicol. It can be concluded that the high prevalence of the tet gene indicates a high potential of Salmonella isolates for horizontal transmission of tetracycline resistance genes.
收起
摘要 :
Tetracyclines are the broad-spectrum agents used in veterinary medicine and food animal production. Known mechanisms of tetracycline resistance include ribosome protection, active efflux and enzymatic inactivation. However, the pr...
展开
Tetracyclines are the broad-spectrum agents used in veterinary medicine and food animal production. Known mechanisms of tetracycline resistance include ribosome protection, active efflux and enzymatic inactivation. However, the presence of two different tet genes conferring different resistance mechanisms on the same plasmid has rarely been reported. In this study, we identified the tandem tetracycline resistance genes tet(61)-tet(58) on the novel plasmid pT4303. These tet genes were identified for the first time in Aerococcus urinaeequi. Reduced susceptibility to doxycycline was observed in S. aureus RN4220 harboring tet(61) when an extra tet(58) was expressed. Plasmid pT4303 was electrotransformed into S. aureus RN4220, E. faecalis JH2- 2, S. suis BAA and E. coli DH5 alpha and conferred tetracycline resistance (MIC >= 16) in both Gram-positive and Gram-negative bacteria, assuming that it might serve as a vehicle for the dissemination of the tetracycline resistance genes tet(61) and tet (58).
收起
摘要 :
Background Recent emergence of high-level tigecycline resistance is mediated by tet(X) genes in Gram-negative bacteria, which undoubtedly constitutes a serious threat for public health worldwide. This study aims to identify tigecy...
展开
Background Recent emergence of high-level tigecycline resistance is mediated by tet(X) genes in Gram-negative bacteria, which undoubtedly constitutes a serious threat for public health worldwide. This study aims to identify tigecycline non-susceptible isolates and detect the presence of genes that are responsible for tigecycline resistance among local isolates in Iraq for the first time. Methods Thirteen clinical isolates of Klebsiella pneumonia, Acinetobacter baumannii and Pseudomonas aeruginosa tigecycline non-susceptible were investigated from blood, sputum and burns specimens. The susceptibility of different antibiotics was tested by the VITEK-2 system. To detect tigecycline resistance genes, PCR was employed. Results Strains studied in this work were extremely drug-resistant and they were resistant to most antibiotic classes that were studied. The plasmid-encoded tet(X), tet(X1), tet(X2), tet(X3), tet(X4), tet(X5), tet(M) and tet(O) genes were not detected in the 13 isolates. The results showed that there is a clear presence of tet(A) and tet(B) genes in tigecycline non-susceptible isolates. All 13 (100%) tigecycline non-susceptible K. pneumoniae, A. baumannii and P. aeruginosa isolates harbored the tet(B) gene. In contrast, 4 (30.77%) tigecycline non-susceptible P. aeruginosa isolates harbored the tet(A) gene and there was no tigecycline non-susceptible A. baumannii isolate harboring the tet(A) gene (0%), but one (7.69%) tigecycline non-susceptible K. pneumoniae isolate harbored the tet(A) gene. A phylogenetic tree, which is based on the nucleotide sequences of the tet(A) gene, showed that the sequence of the local isolate was 87% similar to the nucleotide sequences for all the isolates used for comparison from GenBank and the local isolate displayed genetic diversity. Conclusions According to this study, tet(B) and tet(A) play an important role in the appearance of tigecycline non-susceptible Gram-negative isolates.
收起
摘要 :
The use of antimicrobials has increased the number of resistant bacteria to these drugs; however, the organic production has restricted the use of these compounds. The objectives of this work were to assess counts of tetracycline-...
展开
The use of antimicrobials has increased the number of resistant bacteria to these drugs; however, the organic production has restricted the use of these compounds. The objectives of this work were to assess counts of tetracycline-resistant bacteria using conventional microbiology, to compare these results with those obtained for tet(A) and tet(B) genes by qPCR and to investigate both genes in conventional and organic meat. Counts of mesophilic aerobic bacteria were higher in organic beef, while chicken meat obtained higher counts for Enterobacteriaceae. Only tet(B) was higher in conventional pork and chicken meat than in their organic counterparts. The tet(A) gene was found in almost 100% of samples and tet(B) gene changed according to the type of meat. The presence of tet genes suggests that they are widely distributed, especially tet(A), in food of animal origin, even in organic meat samples obtained from animals in which the use of antimicrobials is restricted. El uso de los antimicrobianos ha incrementado sustancialmente el numero de bacterias resistentes a estos farmacos sin embargo, la produccion ecologica, ha limitado el uso de estos medicamentos. Los objetivos del trabajo fueron evaluar los recuentos obtenidos de bacterias resistentes a tetraciclina mediante microbiologia convencional, obtener recuentos de bacterias con los genes tet(A) y tet(B)mediante qPCR e investigar la distribucion de ambos genes en carne convencional y ecologica. Los recuentos de bacterias aerobias mesofilas fueron significativamente mayores en carne ecologica de ternera, mientras que los recuentos de Enterobacteriaceae fueron superiores en carne convencional de pollo. Solo el gen tet(B) fue significativamente mayor en carne convencional de cerdo y de pollo que en sus homologas ecologicas. El gen tet(A) se encontro en casi todas las muestras mientras que el tet(B) vario segun la especie. La presencia de los genes tet sugiere que estan ampliamente distribuidos, especialmente tet(A), en alimentos de origen animal, incluso en aquellos derivados de animales en los que el uso de antimicrobianos esta seriamente restringido.
收起
摘要 :
The administration and the mass use of antimicrobial therapy without the appropriate dose or exposure time, enhance the emergence of antimicrobial resistance leading to increasing the difficulty to control the infectious bacterial...
展开
The administration and the mass use of antimicrobial therapy without the appropriate dose or exposure time, enhance the emergence of antimicrobial resistance leading to increasing the difficulty to control the infectious bacterial agents in dairy farms and hence impose negative economic impact on dairy industry. Tetracycline resistance conferred with different genes but in this study, two genes were found tet (k) and tet (38). The tet (38) gene had high prevalence than the tet (k) gene and one of the interesting features found in this work that the tet (k) was found only in most of MRSA isolates (mecA gene harboring) while the tet (38) gene was found in all S. aureus isolates (MRSA and MSSA). High level of similarities were found between these genes isolated from different stains and their corresponding reference sequences retrieved from the GeneBank by applying the multiple sequence alignment and the phylogenetic analysis. Further analysis would be done to determine other genes of resistance to explore the whole picture of antibiotic resistance but on the genetic level.
收起
摘要 :
To determine whether the tetracycline resistance genes tet(34), tet(M), and tet(S) can be transferred among bacteria, we used a filter mating experiment allowing intimate cell-cell contact between donor and recipient. The let (34)...
展开
To determine whether the tetracycline resistance genes tet(34), tet(M), and tet(S) can be transferred among bacteria, we used a filter mating experiment allowing intimate cell-cell contact between donor and recipient. The let (34) gene, conveyed on achromosome of Vibrio species (No. 6 and SW-42) was not transferred to Escherichia coli JM109, suggesting that it is not transferred among bacterial species. The tet(M) gene was transferred from three Vibrio strains (4-E, SW-18, and SW-38) to E. coli at frequencies of 8.5x10~(-5) to 2.1x10~(-6). The tet(S) gene was transferred from Lactococcus garvieae KHS98032 to E. coli at a frequency of 1.8x10~(-6). Transconjugated recipients showed increased minimum inhibitory concentrations against oxytetracycline.Although the donors possess the Tn976-Tn1545 transposons, they were not detected in transformed recipients, suggesting that the transfer of tet(M) and tet(S) is mediated by elements or mechanisms. Two ribosomal protect protein genes were also transmissible from marine bacteria to E. coli, suggesting gene hopping among marine, terrestrial, and human environments.
收起
摘要 :
In Aspergillus, controlled gene expression is often achieved using the reverse tetracycline-controlled transactivator (rtTA) dependent Tet-on system, whereby transcription is activated in a titratable manner by addition of the tet...
展开
In Aspergillus, controlled gene expression is often achieved using the reverse tetracycline-controlled transactivator (rtTA) dependent Tet-on system, whereby transcription is activated in a titratable manner by addition of the tetracycline derivative doxycycline. The complementary Tet-off system utilises the tetracycline-controlled transactivator (tTA) component to quantitatively reduce gene expression. In this study, we utilised a synthetic biological approach to engineer highly optimised Tet-off conditional expression systems in Aspergillus niger and Aspergillus fumigatus. Steps for delivery of these tools include utilising codon optimised cassette components, testing several promoters for improved genetic stability and validating two modified luciferase reporters for highly accurate measurements of gene expression. The Tet-off cassettes developed in this study enable facile and quantitative functional analysis, as validated by Tet-off analysis of genes involved in chitin synthesis and cell wall polarity in A. niger, and para-aminobenzoic acid synthesis in A. fumigatus. We also used a racA(G18V) dominant allele to demonstrate that Tet-off in A. niger enables gene over-expression and downregulation in a single isolate. Additionally, we used the improved luciferase reporters to show that the Tet-off cassette in A. niger enables quantification of gene oscillations. In order to demonstrate that synthetic biological approaches developed here are broadly applicable to engineering transcriptional circuits in filamentous fungi, we used our strategy for improving cassette stability by promoter replacement in the A. niger Tet-on system, which resulted in a modified Tet-on cassette with higher stability in recipient genomes. (C) 2015 Elsevier Inc. All rights reserved.
收起
原文获取