Bioluminescent Bacterial Strains Research Articles

Inspired by nature: Fiber networks functionalized with tannic acid and condensed tannin-rich extracts of Norway spruce bark show antimicrobial efficacy.

This study demonstrated the antibacterial and antiviral potential of condensed tannins and tannic acid when incorporated into fiber networks tested for functional material purposes. Condensed tannins were extracted from industrial bark of Norway spruce by using pressurized hot water extraction (PHWE), followed by purification of extracts by using XADHP7 treatment to obtain sugar-free extract. The chemical composition of the extracts was analyzed by using HPLC, GC‒MS and UHPLC after thiolytic degradation. The test matrices, i.e., lignocellulosic handsheets, were produced and impregnated with tannin-rich extracts, and tannic acid was used as a commercial reference. The antibacterial and antiviral efficacy of the handsheets were analyzed by using bioluminescent bacterial strains (Staphylococcus aureus RN4220+pAT19 and Escherichia coli K12+pCGLS11) and Enterovirus coxsackievirus B3. Potential bonding of the tannin-rich extract and tannic acid within the fiber matrices was studied by using FTIR-ATR spectroscopy. The deposition characteristics (distribution and accumulation patterns) of tannin compounds and extracts within fiber networks were measured and visualized by direct chemical mapping using time-of-flight secondary ion mass spectrometry (ToF-SIMS) and digital microscopy. Our results demonstrated for the first time, how tannin-rich extracts obtained from spruce bark side streams with green chemistry possess antiviral and antibacterial properties when immobilized into fiber matrices to create substitutes for plastic hygienic products, personal protection materials such as surgical face masks, or food packaging materials to prolong the shelf life of foodstuffs and prevent the spread of infections. However, more research is needed to further develop this proof-of-concept to ensure stable chemical bonding in product prototypes with specific chemistry.

Antibacterial and Oxidative Stress-Protective Effects of Five Monoterpenes from Softwood.

Volatile organic compounds (VOC) affect the quality of indoor air. Terpenes and especially monoterpenes are the main molecules emitted from softwood material (coniferous species), which is widely used in construction. The corneal epithelium is one of the first human membranes to encounter VOCs in the air. Moreover, the industrial use of pleasant-scented monoterpenes in cosmetics, food, and detergents exposes people to monoterpenes in their daily lives. In the present study, the health effective properties of five monoterpenes from softwood were tested; cytotoxicity and oxidative stress-protective effects of α- and β-pinenes, R- and S-limonene, and 3-carene were tested in a human corneal epithelial (HCE) cell model system and with two additional in vitro antioxidant tests: oxygen radical absorbance capacity (ORAC) and hydrogen peroxide (H2O2) scavenging. Antibacterial efficacies were tested with two bioluminescent bacterial biosensor strains (Escherichia coli K12+pcGLS11 and Staphylococcus aureus RN4220+pAT19) and with minimum inhibitory concentration (MIC) test against Escherichia coli. Only very high concentrations of monoterpenes (0.3–0.5 mg/mL) demonstrated cytotoxicity against HCE cells. Contrary to the original hypothesis, monoterpenes did not exhibit strong antioxidant properties in tested concentrations. However, biosensors and MIC tests indicated clear antibacterial activities for all tested monoterpenes.

Influence of ultraviolet and gamma ray irradiation on luminescent bacteria and exploring their efficacy as biosensors.

Toxicity monitoring of harmful radiation is an indispensable issue in modern radioecology. As bioluminescent bacteria have the simplest structure to epitomize the biosphere, their bioluminescence can also act as an indicator of the conditions, therefore assay systems based on luminous bacteria can be used to monitor environmental radiotoxicity. The present investigation explored the measurement of bacterial luminescence, which can be easily computed. Bioluminescent bacterial strains were used to evaluate the effect of ultraviolet (UV) and gamma irradiation. A random amplified polymorphic DNA (RAPD) assay was carried out to observe alterations under exposure. Using a phylogenetic tree, a comparative study of the effect of UV and gamma rays was carried out. The isolated strains showed marked sensitivity towards radiation exposure present in the environment and therefore they could be used as potential biosensing elements for developing an on-site pollution monitoring biosensor.

In vivo Mouse Model of Spinal Implant Infection.

Spine implant infections portend poor outcomes as diagnosis is challenging and surgical eradication is at odds with mechanical spinal stability. The purpose of this method is to describe a novel mouse model of spinal implant infection (SII) that was created to provide an inexpensive, rapid, and accurate in vivo tool to test potential therapeutics and treatment strategies for spinal implant infections. In this method, we present a model of posterior-approach spinal surgery in which a stainless-steel k-wire is transfixed into the L4 spinous process of 12-week old C57BL/6J wild-type mice and inoculated with 1 x 103 CFU of a bioluminescent strain of Staphylococcus aureus Xen36 bacteria. Mice are then longitudinally imaged for bioluminescence in vivo on post-operative days 0, 1, 3, 5, 7, 10, 14, 18, 21, 25, 28, and 35. Bioluminescence imaging (BLI) signals from a standardized field of view are quantified to measure in vivo bacterial burden. To quantify bacteria adhering to implants and peri-implant tissue, mice are euthanized and the implant and surrounding soft tissue are harvested. Bacteria are detached from the implant by sonication, cultured overnight and then colony forming units (CFUs) are counted. The results acquired from this method include longitudinal bacterial counts as measured by in vivo S. aureus bioluminescence (mean maximum flux) and CFU counts following euthanasia. While prior animal models of instrumented spine infection have involved invasive, ex vivo tissue analysis, the mouse model of SII presented in this paper leverages noninvasive, real time in vivo optical imaging of bioluminescent bacteria to replace static tissue study. Applications of the model are broad and may include utilizing alternative bioluminescent bacterial strains, incorporating other types of genetically engineered mice to contemporaneously study host immune response, and evaluating current or investigating new diagnostic and therapeutic modalities such as antibiotics or implant coatings.

Evaluating the use of biosensors for monitoring of Penicillium digitatum infection in citrus fruit

Penicillium digitatum is a major postharvest pathogen in citrus fruit that causes losses in all citrus-growing countries. In this study, a new whole-cell-based biosensor was developed to detect the fungus’ presence in oranges. The approach was based on bacteria's luminescent responses to changes in volatile organic compounds (VOCs) following infection by a pathogenic microorganism in oranges. Differences between VOC patterns in the infected and noninfected fruit were monitored by GC–MS and then with four different genetically modified bioluminescent bacterial strains. GC–MS detected the changes in emitted VOC patterns during the first infection steps. Bioreporter strains allowed pathogen detection on the third day of infection, before the appearance of visible signs of fungal infection on the surface of the orange. Thanks to their increased sensitivity, portability and ease of use, whole-cell biosensors may serve as a new tool for monitoring rot. In the future, such technology will reduce food losses by enabling more efficient crop management throughout postharvest treatment, storage and transport.

Characterization of Bioluminescent Bacterial Strain Isolated from Nemipterus japonicus Fish of Arabian Sea

Characterization of Bioluminescent Bacterial Strain Isolated from Nemipterus japonicus Fish of Arabian Sea

Enhanced anti-microbial effect through cationization of a mono-triazatricyclodecane substituted asymmetric phthalocyanine

Antimicrobial photodynamic therapy (aPDT) is an effective way to combat infectious diseases and antibiotic resistance. Photosensitizer is a key factor of aPDT and has triggered extensive research interest. In this study, a new asymmetric Zn(II) phthalocyanine mono-substituted with a triazatricyclodecane moiety (compound 3) and its cationic N-methylated derivative (compound 4) were synthesized. Their photodynamic antimicrobial activities were evaluated using bioluminescent bacterial strains. Compound 3 showed phototoxicity only toward the Gram-positive bacteria, whereas the cationic derivative compound 4 exhibited strong anti-bacterial activity against both Gram-positive and Gram-negative strains. These bacterial species were eradicated (>4.0 logs or 99.99% killing) at appropriate concentrations of compound 4 with 12.7 J/cm2 of red light, demonstrating compound 4 as a potent aPDT agent.

Influence of Antibiotic Pressure on Five Plasmid-based Bioluminescent Gram-negative Bacterial Strains.

The present study aims to develop five Gram-negative bacteria expressing bacterial luciferase for use to evaluate the influence of different antibiotics on bacterial bioluminescence. The pBBR-lux plasmid was introduced into five Gram-negative bacteria; the bioluminescent signals and colony-forming unit (CFU)/ml of all the bioluminescent strains were monitored with six antibiotics at various concentrations. Dose-dependent bioluminescence signals can be used for rapid bacterial antibiotic susceptibility test (AST). All five bioluminescent bacterial strains have similar bioluminescence and CFU enhancement at sub-minimum inhibitory concentration (MIC) of six different antibiotics. The bioluminescent signals and CFU enhancement at sub-MIC antibiotic concentrations should be of value in the research of new antibiotic drugs and bioluminescent imaging.

Influence of antibiotic pressure on bacterial bioluminescence, with emphasis on Staphylococcus aureus

Bioluminescence imaging is used for longitudinal evaluation of bacteria in live animals. Clear relations exist between bacterial numbers and their bioluminescence. However, bioluminescence images of Staphylococcus aureus Xen29, S. aureus Xen36 and Escherichia coli Xen14 grown on tryptone soy agar in Etests demonstrated increased bioluminescence at sub-MICs of different antibiotics. This study aimed to further evaluate the influence of antibiotic pressure on bioluminescence in S. aureus Xen29. Bioluminescence of S. aureus Xen29, grown planktonically in tryptone soy broth, was quantified in the absence and presence of different concentrations of vancomycin, ciprofloxacin, erythromycin or chloramphenicol and was related to expression of the luxA gene under antibiotic pressure measured using real-time PCR. In the absence of antibiotics, staphylococcal bioluminescence increased over time until a maximum after ca. 6h of growth, and subsequently decreased to the detection threshold after 24h of growth owing to reduced bacterial metabolic activity. Up to MICs of the antibiotics, bioluminescence increased according to a similar pattern up to 6h of growth, but after 24h bioluminescence was higher than in the absence of antibiotics. Contrary to expectations, bioluminescence per organism (CFU) after different growth periods in the absence and at MICs of different antibiotics decreased with increasing expression of luxA. Summarising, antibiotic pressure impacts the relation between CFU and bioluminescence. Under antibiotic pressure, bioluminescence is not controlled by luxA expression but by co-factors impacting the bacterial metabolic activity. This conclusion is of utmost importance when evaluating antibiotic efficacy in live animals using bioluminescent bacterial strains.

Design of a toxicity biosensor based on Aliivibrio fischeri entrapped in a disposable card.

The degradation of the marine environment is a subject of concern for the European authorities primarily because of its contamination by hydrocarbons. The traditional methods (ISO 11348 standard) of general toxicity assessment are unsuitable in a context of in situ monitoring, such as seaports or bathing zones. Consequently, to address this issue, bacterial biosensors appear to be pertinent tools. This article presents the design of an innovative bioluminescent biosensor dedicated to in situ toxicity monitoring. This biosensor is based on the entrapment of the wild marine bioluminescent bacterial strain Aliivibrio fischeri ATCC® 49387™ in an agarose matrix within a disposable card. A pre-study was needed to select the most biological parameters. In particular, the regenerating medium's composition and the hydrogel concentration needed for the bacterial entrapment (mechanical resistance) were optimized. Based on these data, the ability of the bacterial reporter to assess the sample toxicity was demonstrated using naphthalene as a chemical model. The biosensor's results show a lower sensitivity to naphthalene (EC50 = 95 mg/L) compared with the results obtained using the reference method (EC50 = 43 mg/L). With this architecture, the biosensor is an interesting compromise among low maintenance, ease of use, appropriate sensitivity, relatively low cost and the ability to control online toxicity.

Promising Biological Indicator of Heavy Metal Pollution: Bioluminescent Bacterial Strains Isolated and Characterized from Marine Niches of Goa, India.

In present study, several marine water samples collected from the North Goa Beaches, India for isolation of luminescent bacterial species. Isolates obtained labelled as DP1-5 and AB1-6. Molecular characterization including identification of a microbial culture using 16S rRNA gene based molecular technique and phylogenetic analysis confirmed that DP3 & AB1 isolates were Vibrio harveyi. All of the isolates demonstrated multiple metal resistances in terms of growth, with altered luminescence with variable metal concentration. Present investigations were an attempt towards exploring and reporting an updated diversity of bioluminescent bacterial species from various sites around the Goa, India which would be explored in future for constructing luminescence based biosensor for efficiently monitoring the level of hazardous metals in the environment.

Tn5/7-lux: a versatile tool for the identification and capture of promoters in gram-negative bacteria.

BackgroundThe combination of imaging technologies and luciferase-based bioluminescent bacterial reporter strains provide a sensitive and simple non-invasive detection method (photonic bioimaging) for the study of diverse biological processes, as well as efficacy of therapeutic interventions, in live animal models of disease. The engineering of bioluminescent bacteria required for photonic bioimaging is frequently hampered by lack of promoters suitable for strong, yet stable luciferase gene expression.ResultsWe devised a novel method for identification of constitutive native promoters in Gram-negative bacteria. The method is based on a Tn5/7 transposon that exploits the unique features of Tn5 (random transposition) and Tn7 (site-specific transposition). The transposons are designed such that Tn5 transposition will allow insertion of a promoter-less bacterial luxCDABE operon downstream of a bacterial gene promoter. Cloning of DNA fragments from luminescent isolates results in a plasmid that replicates in pir+ hosts. Sequencing of the lux-chromosomal DNA junctions on the plasmid reveals transposon insertion sites within genes or operons. The plasmid is also a mini-Tn7-lux delivery vector that can be used to introduce the promoter-lux operon fusion into other derivatives of the bacterium of interest in an isogenic fashion. Alternatively, promoter-containing sequences can be PCR-amplified from plasmid or chromosomal DNA and cloned into a series of accompanying mini-Tn7-lux vectors. The mini-Tn5/7-lux and mini-Tn7-lux vectors are equipped with diverse selection markers and thus applicable in numerous Gram-negative bacteria. Various mini-Tn5/7-lux vectors were successfully tested for transposition and promoter identification by imaging in Acinetobacter baumannii, Escherichia coli, and Burkholderia pseudomallei. Strong promoters were captured for lux expression in E. coli and A. baumannii. Some mini-Tn7-lux vectors are also equipped with attB sites for swapping of the lux operon with other reporter genes using Gateway technology.ConclusionsAlthough mini-Tn5-lux and mini-Tn7-lux elements have previously been developed and used for bacterial promoter identification and chromosomal insertion of promoter-lux gene fusions, respectively, the newly developed mini-Tn5/7-lux and accompanying accessory plasmids streamline and accelerate the promoter discovery and bioluminescent strain engineering processes. Availability of vectors with diverse selection markers greatly extend the host-range of promoter probe and lux gene fusion vectors.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0354-3) contains supplementary material, which is available to authorized users.

Using bacterial bioluminescence to evaluate the impact of biofilm on porous media hydraulic properties

Biofilm formation in natural and engineered porous systems can significantly impact hydrodynamics by reducing porosity and permeability. To better understand and characterize how biofilms influence hydrodynamic properties in porous systems, the genetically engineered bioluminescent bacterial strain Pseudomonas fluorescens HK44 was used to quantify microbial population characteristics and biofilm properties in a translucent porous medium. Power law relationships were found to exist between bacterial bioluminescence and cell density, fraction of void space occupied by biofilm (i.e. biofilm saturation), and hydraulic conductivity. The simultaneous evaluation of biofilm saturation and porous medium hydraulic conductivity in real time using a non-destructive approach enabled the construction of relative hydraulic conductivity curves. Such information can facilitate simulation studies related to biological activity in porous structures, and support the development of new models to describe the dynamic behavior of biofilm and fluid flow in porous media. The bioluminescence based approach described here will allow for improved understanding and control of industrially relevant processes such as biofiltration and bioremediation.

High hydrostatic pressure effects on bacterial bioluminescence

The mechanism that leads to microbial inactivation by high hydrostatic pressure remains elusive. In this study, a high-pressure system interfaced with a photomultiplier tube (PMT) was developed to monitor cellular metabolism in situ using bioluminescent bacterial strains. Preliminary characterization of the system was performed using Pseudomonas fluorescens 5RL expressing lux proteins from Vibrio fischeri. Stepwise increases in pressure at 34 MPa and above resulted in decreased bioluminescence. Square wave exposure to pressures of 69, 103 and 138 MPa showed bioluminescence reductions greater than 95%, but when cells were returned to ambient pressure bioluminescence returned to 51, 38, and 4% of initial bioluminescence values, respectively. An Escherichia coli strain expressing lux proteins from V. fischeri was constructed to determine whether this reversible effect could be observed in another bacterial genus. Square wave perturbations of 69, 103 and 138 MPa resulted in bioluminescence reductions of about 94% at the highest pressure treatment. Upon decompression, bioluminescence returned to 74, 58 and 30% of the initial bioluminescence values for cells treated at 69, 103 and 138 MPa, respectively. These results suggest that square wave exposure to pressure up to 138 MPa induces reversible cell damage in P. fluorescens 5RL and E. coli VF lux.

Antimicrobial screening of natural product extracts using a bioluminescent assay

We have developed a high throughput screening method for the discovery of new antimicrobial compounds, using a novel approach with recombinant bioluminescent bacterial strain Escherichia coli K-12/pTetLux. This recombinant strain can particularly be used to indicate compounds that affect microbial transcriptional and translational events [1]. The developed method improves the feasibility of using natural product extracts as a screening resource as the assay is less prone to interference from coloured or turbid samples. Compared to conventional antimicrobial testing, this biosensor-based method offers other important improvements such as simultaneous data on antimicrobial activity and mode of action and reduction of assay time from 24h to 2–3h. Results reported here include method validation and miniaturisation into 384-well plate format as well as implementation on 22 plant extracts from our in-house collection Based on the results, fractions of seven promising extracts that had been prepared by HPLC into 96 well-plates were further tested. The results show that the developed bioluminescent technology is well suitable for natural product screening and is sensitive enough to identify bioactive fractions.

Improvement of the Identification of Four Heavy Metals in Environmental Samples by Using Predictive Decision Tree Models Coupled with a Set of Five Bioluminescent Bacteria

A primary statistical model based on the crossings between the different detection ranges of a set of five bioluminescent bacterial strains was developed to identify and quantify four metals which were at several concentrations in different mixtures: cadmium, arsenic III, mercury, and copper. Four specific decision trees based on the CHAID algorithm (CHi-squared Automatic Interaction Detector type) which compose this model were designed from a database of 576 experiments (192 different mixture conditions). A specific software, 'Metalsoft', helped us choose the best decision tree and a user-friendly way to identify the metal. To validate this innovative approach, 18 environmental samples containing a mixture of these metals were submitted to a bioassay and to standardized chemical methods. The results show on average a high correlation of 98.6% for the qualitative metal identification and 94.2% for the quantification. The results are particularly encouraging, and our model is able to provide semiquantitative information after only 60 min without pretreatments of samples.

Non invasive real-time monitoring of bacterial infection & therapeutic effect of anti-microbials in five mouse models

Background & objectives:In vivo imaging system has contributed significantly to the understanding of bacterial infection and efficacy of drugs in animal model. We report five rapid, reproducible, and non invasive murine pulmonary infection, skin and soft tissue infection, sepsis, and meningitis models using Xenogen bioluminescent strains and specialized in vivo imaging system (IVIS).Methods:The progression of bacterial infection in different target organs was evaluated by the photon intensity and target organ bacterial counts. Genetically engineered bioluminescent bacterial strains viz. Staphylococcus aureus Xen 8.1, 29 and 31; Streptococcus pneumoniae Xen 9 and 10 and Pseudomonas aeruginosa Xen-5 were used to induce different target organs infection and were validated with commercially available antibiotics.Results:The lower limit of detection of colony forming unit (cfu) was 1.7-log10 whereas the lower limit of detection of relative light unit (RLU) was 4.2-log10. Recovery of live bacteria from different target organs showed that the bioluminescent signal correlated to the live bacterial count.Interpretation & conclusions:This study demonstrated the real time monitoring and non-invasive analysis of progression of infection and pharmacological efficacy of drugs. These models may be useful for pre-clinical discovery of new antibiotics.

A multi-channel bioluminescent bacterial biosensor for the on-line detection of metals and toxicity. Part I: design and optimization of bioluminescent bacterial strains

This study describes the construction of inducible bioluminescent strains via genetic engineering along with their characterization and optimization in the detection of heavy metals. Firstly, a preliminary comparative study enabled us to select a suitable carbon substrate from pyruvate, glucose, citrate, diluted Luria-Bertani, and acetate. The latter carbon source provided the best induction ratios for comparison. Results showed that the three constructed inducible strains, Escherichia coli DH1 pBzntlux, pBarslux, and pBcoplux, were usable when conducting a bioassay after a 14-h overnight culture at 30 °C. Utilizing these sensors gave a range of 12 detected heavy metals including several cross-detections. Detection limits for each metal were often close to and sometimes lower than the European standards for water pollution. Finally, in order to maintain sensitive bacteria within the future biosensor-measuring cell, the agarose immobilization matrix was compared to polyvinyl alcohol (PVA). Agarose was selected because the detection limits of the bioluminescent strains were not affected, in contrast to PVA. Specific detection and cross-detection ranges determined in this study will form the basis of a multiple metals detection system by the new multi-channel Lumisens3 biosensor.

Randomly distributed arrays of optically coded functional microbeads for toxicity screening and monitoring

We have successfully developed optically coded functional microbeads by co-encapsulating both bioluminescent reporter bacterial cells and fluorescent microspheres within a common alginate microbead. These microbeads harboring an individual self-identification code using fluorescent microspheres could be randomly scattered on any multi-well chip plate as long as the size of the microbeads are made to fit on it with the result that, since cell types are identified on the basis of fluorescent color, microbead arrays were fabricated without pre-designation of an individual well. As an example of this method, five different stress specific bioluminescent bacterial strains, each with a different optical code, were successfully implemented to make five different types of optically coded functional microbeads, with a speed of about 30 microbeads/min. Each functional microbead has a specific stress-specific bacterial strain and, as an identification optical code, one of five optical codes generated from fluorescence microspheres such as yellow, green, red, yellow + green, or no fluorescence. This final randomly scattered functional microbeads array biochip, with a fast fabrication of each chip at every 2 min, successfully demonstrated its ability in toxicity screening and monitoring for samples with a few examples for five different stress chemicals. This simple and fast, but not tedious and complicated procedure should be widely and practically used in making cell array chips for the monitoring of environmental toxicity, new-borne chemicals, pharmaceutical drugs and cosmic rays in the space station or spaceships in future.

Lead Bioavailability in Soil and Soil Components

The bioavailability of Pb in polluted soil as well as in Pb-spiked individual soil components (carbonates, Fe-oxides, clays, organic matter, and quartz) was determined using a genetically engineered bioluminescent bacterial reporter strain. Calculated bioavailability was compared to total concentrations of the metal in the samples and to its fraction leached into the aqueous assay medium. Our data show that adsorbed Pb is unavailable to the bacterial reporter, which responds only to dissolved Pb. We also found that leaching with aqueous solutions do not desorb any measurable Pb (<0.1 ppm) from contaminated soils, but does so from most of the freshly spiked soil components, at different efficiencies (10%, 25–60%, 25–55%, 0%, and 0–3% for carbonate, quartz, clay, Fe-oxide, and organic matter, respectively). This tendency to retain or release Pb, and hence determine its bioavailability, corresponds to the affinity of the various soil fractions to Pb as reported in the literature. For example, Fe-oxides which have the highest affinity for Pb retain all its Pb, making it unavailable to microorganisms in the soil. Nonetheless, even in the soil fractions with the lowest affinity for Pb (quartz), only 55% of the desorbed Pb is bioavailable.