Browsing by Subject "bioluminescence"
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(2015)Susceptibility to antibiotics is constantly developing in bacteria due to selection pressure caused by use of antibiotics. For this reason, finding new antimicrobial substances is imperative. High-throughput screening (HTS) is an important tool to find new active substances. The need to analyse as many substances in as small time as possible is emphasised in modern drug development. Robust methods, suitable for fast throughput of substances, miniaturisation and automation, are particularly useful. In the context of antimicrobial screening, methods utilising bioluminescence can correspond this need, and genetic engineering can help in developing bacterial strains with beneficial features for screening. In this work, two screening methods were developed and optimised using genetically engineered Escherichia coli strains. The screening methods make use of the bioluminescent properties of the strains, and the methods can be used to screen compound libraries for antimicrobials rapidly enough to approach HTS. The strain E. coli WZM120/pCGLS 11 is constitutively luminescent, so weakening of luminescence means the cell viability weakens. The strain E. coli K12/pCSS305, where luminescence is produced by a heat-inducible runaway plasmid, can be used to especially detect compounds inhibiting DNA replication. In developing the method, workflow was optimised and conditions were validated so as to enable possible HTS campaigns. The target was to create as simple, fast and reproducible a method as possible. The Z' values calculated in assessing the performance are excellent for a cell-based method. The signal is readily distinguishable, the bacterial strains are in a stable manner, and the method is well reproducible. It is possible to continue assay development from 96-well format to 384-well format.
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(2022)The increasing use of antimicrobials causes a heavy pollution load on the environment and can enhance antimicrobial resistance of pathogenic bacteria, thus having a negative impact on human and animal health. Antibiotic concentrations in the environment are constantly monitored, however traditional chemical analyses fail to provide data on the bioavailability of antimicrobials. Whole-cell bacterial bioreporters have been developed to detect a wide variety of environmental pollutants including antimicrobials. These living, genetically engineered organisms can also be used for the measurement of the bioavailable fraction in a sample and thus bioreporters could give insights on the role of antimicrobial pollution in the dissemination of antimicrobial resistance. The aim of this study was to design and construct an improved bioluminescent bioreporter for detection of macrolide antibiotics. The mrx gene and the mph(A)R repressor gene were coupled with the mph(A) promotor of the macrolide resistance operon mph(A) and the reporter genes of the luciferase operon luxCDABE. The main objective was to determine whether Mrx, the hydrophobic and putative transmembrane transport protein of the macrolide resistance operon mph(A), would improve the sensitivity and reduce the induction time. Another aim was to optimize the use of three existing bioreporters with other macrolides than erythromycin, which was used earlier in testing their performance. The mrx-mph(A)R fragment was cloned into the pmph(A)luxCDABE vector, and the bioreporter plasmid was introduced to Escherichia coli strain DH10B. After verification of the construct pmph(A)luxCDABE-mrx-mph(A)R, the usability of the new whole-cell biosensor was compared against the three existing macrolide bioreporters with three different macrolide and lincosamide antibiotics, erythromycin, tylosin and clindamycin. The cloning of the new bioluminescent bioreporter for macrolides was performed successfully. However, the addition of erythromycin, tylosin or clindamycin to a suspension of E. coli DH10B(pmph(A)luxCDABE-mrx-mph(A)R) did not stimulate the expression of the lux genes. High concentrations of all three antibiotics triggered a light response with the existing bioreporters although the response was slow. The results indicated that further studies on the mrx gene and its encoded Mrx protein are still needed. The response of the mph(A) operon to other macrolide and lincosamide antibiotics than erythromycin was a positive and encouraging finding, since this enables detection of other synthetic macrolides than erythromycin as well as lincosamides with the existing bioreporters after optimization.
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(2022)The increasing use of antimicrobials causes a heavy pollution load on the environment and can enhance antimicrobial resistance of pathogenic bacteria, thus having a negative impact on human and animal health. Antibiotic concentrations in the environment are constantly monitored, however traditional chemical analyses fail to provide data on the bioavailability of antimicrobials. Whole-cell bacterial bioreporters have been developed to detect a wide variety of environmental pollutants including antimicrobials. These living, genetically engineered organisms can also be used for the measurement of the bioavailable fraction in a sample and thus bioreporters could give insights on the role of antimicrobial pollution in the dissemination of antimicrobial resistance. The aim of this study was to design and construct an improved bioluminescent bioreporter for detection of macrolide antibiotics. The mrx gene and the mph(A)R repressor gene were coupled with the mph(A) promotor of the macrolide resistance operon mph(A) and the reporter genes of the luciferase operon luxCDABE. The main objective was to determine whether Mrx, the hydrophobic and putative transmembrane transport protein of the macrolide resistance operon mph(A), would improve the sensitivity and reduce the induction time. Another aim was to optimize the use of three existing bioreporters with other macrolides than erythromycin, which was used earlier in testing their performance. The mrx-mph(A)R fragment was cloned into the pmph(A)luxCDABE vector, and the bioreporter plasmid was introduced to Escherichia coli strain DH10B. After verification of the construct pmph(A)luxCDABE-mrx-mph(A)R, the usability of the new whole-cell biosensor was compared against the three existing macrolide bioreporters with three different macrolide and lincosamide antibiotics, erythromycin, tylosin and clindamycin. The cloning of the new bioluminescent bioreporter for macrolides was performed successfully. However, the addition of erythromycin, tylosin or clindamycin to a suspension of E. coli DH10B(pmph(A)luxCDABE-mrx-mph(A)R) did not stimulate the expression of the lux genes. High concentrations of all three antibiotics triggered a light response with the existing bioreporters although the response was slow. The results indicated that further studies on the mrx gene and its encoded Mrx protein are still needed. The response of the mph(A) operon to other macrolide and lincosamide antibiotics than erythromycin was a positive and encouraging finding, since this enables detection of other synthetic macrolides than erythromycin as well as lincosamides with the existing bioreporters after optimization.
Now showing items 1-3 of 3