Search Results (13)

This review attempts to summarize my three decades-long involvement in, and contribution to, the design, construction and testing of bioluminescent microbial sensor strains (bioreporters). With the understanding that such a document cannot be completely free of bias, the review focuses on studies from my own lab only, with almost no coverage of the parallel progress made by others in similar fields. This admittedly subjective approach by no way detracts from the achievements of countless excellent researchers who are not mentioned here, and whose contributions to the field are at least as important as that of my own. The review covers basic aspects of microbial sensor design, and then progresses to describe approaches to performance improvement of sensor strains aimed at the detection of either specific chemicals, groups of chemicals sharing similar characteristics, or global effects, such as toxicity and genotoxicity. The need for integration of live sensor cells into a compatible hardware platform is highlighted, as is the importance of long-term maintenance of the cells’ viability and activity. The use of multi-member sensors’ panels is presented as a means for enhancing the detection spectrum and sample “fingerprinting”, along with a list of different purposes to which such sensors have been put to use. Full article

The release of endocrine-disrupting compounds (EDCs) to the environment poses a health hazard to both humans and wildlife. EDCs can activate or inhibit endogenous endocrine functions by binding hormone receptors, leading to potentially adverse effects. Conventional analytical methods can detect EDCs at a high sensitivity and precision, but are blind to the biological activity of the detected compounds. To overcome this limitation, yeast-based bioassays have previously been developed as a pre-screening method, providing an effect-based overview of hormonal-disruptive activity within the sample prior to the application of analytical methods. These yeast biosensors express human endocrine-specific receptors, co-transfected with the relevant response element fused to the specific fluorescent protein reporter gene. We describe several molecular manipulations of the sensor/reporter circuit in a Saccharomyces cerevisiae bioreporter strain that have yielded an enhanced detection of estrogenic-like compounds. Improved responses were displayed both in liquid culture (96-well plate format) as well as in conjunction with sample separation using high-performance thin-layer chromatography (HPTLC). The latter approach allows for an assessment of the biological effect of individual sample components without the need for their chemical identification at the screening stage. Full article

The Escherichia coli Keio mutant collection has been a tool for assessing the role of specific genes and determining their role in E. coli physiology and uncovering novel functions. In this work, specific mutants in the DNA repair pathways and oxidative stress response were evaluated to identify the primary targets of silver nanoparticles (NPs) and their mechanism of action. The results presented in this work suggest that NPs mainly target DNA via double-strand breaks and base modifications since the recA, uvrC, mutL, and nfo mutants rendered the most susceptible phenotype, rather than involving the oxidative stress response. Concomitantly, during the establishment of the control conditions for each mutant, the katG and sodA mutants showed a hypersensitive phenotype to mitomycin C, an alkylating agent. Thus, we propose that KatG catalase plays a key role as a cellular chaperone, as reported previously for the filamentous fungus Neurospora crassa, a large subunit catalase. The Keio collection mutants may also be a key tool for assessing the resistance mechanism to metallic NPs by using their potential to identify novel pathways involved in the resistance to NPs. Full article

Quorum Sensing (QS) is a well-studied intercellular communication mechanism in bacteria, regulating collective behaviors such as biofilm formation, virulence, and antibiotic resistance. However, cell–cell signaling in haloarchaea remains largely unexplored. The coexistence of bacteria and archaea in various environments, coupled with the known cell–cell signaling mechanisms in both prokaryotic and eukaryotic microorganisms and the presence of cell–cell signaling mechanisms in both prokaryotic and eukaryotic microorganisms, suggests a possibility for haloarchaea to possess analogous cell–cell signaling or QS systems. Recently, N-acylhomoserine lactone (AHL)-like compounds were identified in haloarchaea; yet, their precise role—for example, persister cell formation—remains ambiguous. This study investigated the capacity of crude supernatant extract from the haloarchaeon Halorubrum saccharovorum CSM52 to stimulate bacterial AHL-dependent QS phenotypes using bioreporter strains. Our findings reveal that these crude extracts induced several AHL-dependent bioreporters and modulated pyocyanin and pyoverdine production in Pseudomonas aeruginosa. Importantly, our study suggests cross-domain communication between archaea and bacterial pathogens, providing evidence for archaea potentially influencing bacterial virulence. Using Thin Layer Chromatography overlay assays, lactonolysis, and colorimetric quantification, the bioactive compound was inferred to be a chemically modified AHL-like compound or a diketopiperazine-like molecule, potentially involved in biofilm formation in H. saccharovorum CSM52. This study offers new insights into putative QS mechanisms in haloarchaea and their potential role in interspecies communication and coordination, thereby enriching our understanding of microbial interactions in diverse environments. Full article

Streptococcus suis LuxS/AI-2 quorum sensing system regulates biofilm formation, resulting in increased pathogenicity and drug resistance, and diminished efficacy of antibiotic treatment. The remaining peony seed cake after oil extraction is rich in monoterpenoid glycosides, which can inhibit the formation of bacterial biofilm. In this study, we investigated the effect of seven major monocomponents (suffruticosol A, suffruticosol B, suffruticosol C, paeonifloin, albiflorin, trans-ε-viniferin, gnetin H) of peony seed meal on minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of S. suis. The results showed that the MICs of the seven single components were all greater than 200 μg/mL, with no significant bacteriostatic and bactericidal advantages. Crystal violet staining and scanning electron microscope observation showed that the seven single components had a certain inhibitory effect on the biofilm formation ability of S. suis at sub-MIC concentration. Among them, the ability of paeoniflorin to inhibit biofilm was significantly higher than that of the other six single components. AI-2 signaling molecules were detected by bioreporter strain Vibrio harvey BB170. The detection results of AI-2 signal molecules found that at 1/2 MIC concentration, paeoniflorin significantly inhibited the production of S. suis AI-2 signal, and the inhibitory effect was better than that of the other six single components. In addition, molecular docking analysis revealed that paeoniflorin had a significant binding activity with LuxS protein compared with the other six single components. The present study provides evidence that paeoniflorin plays a key role in the regulation of the inhibition of S. suis LuxS/AI-2 system and biofilm formation in peony seed meal. Full article

This study was conducted to propose a suitable set of methods to evaluate the efficiency of two biotreatments. For this purpose, two sets of four 7.5 L bioreactors were followed over 90 days, containing natural sediments from the Bizerte Lagoon (Tunisia) contaminated with 35 mg·kg⁻¹ benzo(a)pyrene (BaP) and 28 mg·kg⁻¹ dichlorodiphenyltrichloroethane (DDT). One set was biostimulated with N/P and bioaugmented with the indigenous Pseudomonas stutzeri, Cupriavidus metallidurans and Rhodococcus equi, and the other set was only biostimulated. In the effluent, organic carbon decreased from 42 gC·L⁻¹ to 0.2 gC·L⁻¹ for the bioaugmented treatment compared to 15 gC·L⁻¹ for biostimulation. Statistical analyses confirmed a significant difference in BaP concentration after bioaugmention from 35 mg·kg⁻¹ to 21 mg·kg⁻¹ sediment, whereas no difference was found with biostimulation. Considering DDT, biostimulation was more efficient (8.5 mg·kg⁻¹ sediment final concentration) than bioaugmentation (15 mg·kg⁻¹ final concentration). Native organotin and metals were also monitored using bioluminescent bioreporter strains. The bioaugmented treatment brought about a significant decrease in TBT content, to below 0.01 µM, whereas its concentration remained significant after biostimulation. The biostimulation did not alter As³⁺, Cu²⁺, Cd²⁺, and Hg²⁺ concentrations, whereas bioaugmentation induced a decrease of 1 to 2 log for each metal. At the end of the experimental period, toxicity decreased to 90% in the effluent of the bioaugmented reactors compared with a drop of only 48% for biostimulation, and a significant decrease in mutagenicity appeared for bioaugmention only. Interestingly, not all the strains used in the treatments were maintained, as P. stutzeri and R. equi increased up to densities of 8.3 × 10¹³ and 5.2 × 10¹² DNA·g⁻¹ sediment, respectively, while in both treatments, C. metallidurans decreased down to the detection threshold. Among the different methods used, a restricted monitoring panel of analyses appears essential to follow the change occurring over the bioremediation process: (i) organic carbon measurement reporting all biodegradation events, as well as a specific method to monitor the main compounds; (ii) dissolved N, P, O₂ and pH measurements, (iii) a qPCR method to track the degraders; and (iv) measurements of the acute toxicity and the mutagenicity. Full article

A series of 25 new benzothiazole–urea–quinoline hybrid compounds were synthesized successfully via a three-step synthetic sequence involving an amidation coupling reaction as a critical step. The structures of the synthesized compounds were confirmed by routine spectroscopic tools (¹H and ¹³C NMR and IR) and by mass spectrometry (HRMS). In vitro evaluation of these hybrid compounds for their antitubercular inhibitory activity against the Mycobacterium tuberculosis H₃₇Rv pMSp12::GPF bioreporter strain was undertaken. Of the 25 tested compounds, 17 exhibited promising anti-TB activities of less than 62.5 µM (MIC₉₀). Specifically, 13 compounds (6b, 6g, 6i–j, 6l, 6o–p, 6r–t, and 6x–y) showed promising activity with MIC₉₀ values in the range of 1–10 µM, while compound 6u, being the most active, exhibited sub-micromolar activity (0.968 µM) in the CAS assay. In addition, minimal cytotoxicity against the HepG2 cell line (cell viability above 75%) in 11 of the 17 compounds, at their respective MIC₉₀ concentrations, was observed, with 6u exhibiting 100% cell viability. The hybridization of the quinoline, urea, and benzothiazole scaffolds demonstrated a synergistic relationship because the activities of resultant hybrids were vastly improved compared to the individual entities. In silico ADME predictions showed that the majority of these compounds have drug-like properties and are less likely to potentially cause cardiotoxicity (QPlogHERG > −5). The results obtained in this study indicate that the majority of the synthesized compounds could serve as valuable starting points for future optimizations as new antimycobacterial agents. Full article

This study presents an inexpensive, eco-friendly, and simple green synthesis of ZnO nanoparticles using Origanum vulgare extract. These nanoparticles are non-hazardous, environmentally friendly, and cheaper than other methods of biosynthesis. Ongoing research determines the role of phytochemicals in the fabrication and biosynthesis of ZnO NPs and their role in antibacterial activity and biomedical applications. Characterizations by fourier transform infrared spectroscopy (FTIR), diffuse reflectance UV-visible spectroscopy, X-ray diffraction (XRD), scanning electron microscopy (SEM), and transmission electron microscopy (TEM) determine the successful biosynthesis of ZnO NPs. Meanwhile, TEM and X-ray diffraction studies approximated the spherical morphology and crystalline nature of biosynthesized ZnO NPs of nano size in the range of 20–30 nm. The global increase in drug resistance necessitates the search for new drugs with different mechanisms of action. Quorum sensing (QS), a cell-to-cell communication, has gained attention as an emerging drug target. It controls numerous biochemical processes in bacteria, which are essential for their survival and pathogenicity. The potential of nanomedicines has also been tested to synthesize new antibiotics to tackle drug resistance. ZnO NPs were explored for their antibacterial, antiquorum sensing, and antibiofilm activities with a bioreporter strain of Chromobacterium violaceum. Susceptibility testing results indicated the potential antibacterial activity of ZnO NPs with a minimum inhibitory concentration (MIC) of 4 µg/mL and minimum bactericidal concentration (MBC) of 16 µg/mL. Antiquorum-sensing assays revealed that these nanoparticles inhibit quorum sensing with minimum antiquorum sensing activity (MQSIC) of 1 µg/mL, without causing any bacterial growth inhibition. In addition, ZnO NPs inhibit biofilm formation at inhibitory and higher concentrations. RT-qPCR results supported the downregulation of the quorum sensing genes when C. violaceum was treated with ZnO NPs. The outcomes of this study are promising with regard to the biofilm and quorum sensing, emphasizing the potential applications of ZnO NPs against bacterial communication and biofilm formation. Full article

Three new cyanobactins, trikoramides B (1)–D (3), have been isolated from the marine cyanobacterium, Symploca hydnoides, following a preliminary bioassay-guided isolation of the two most active polar fractions based on the brine shrimp toxicity assay. These new cyanobactins are new analogues of the previously reported cytotoxic trikoramide A (4) with differences mainly in the C-prenylated cyclotryptophan unit. Their planar structures were elucidated from their 1D and 2D NMR spectral data in combination with the HRMS/MS data. Marfey’s method, 2D NOESY NMR spectroscopic and ECD spectra analyses were used to determine the absolute stereochemistry of trikoramides B (1)–D (3). Trikoramides B (1) and D (3) exhibited cytotoxicity against MOLT-4 acute lymphoblastic leukemia cell line with IC₅₀ values of 5.2 µM and 4.7 µM, respectively. Compounds 1 and 3 were also evaluated for their quorum-sensing inhibitory assay based on the Pseudomonas aeruginosa PAO1 lasB-gfp and rhlA-gfp bioreporter strains. Although trikoramide B (1) exhibited weak quorum-sensing inhibitory activity, the Br-containing trikoramide D (3) exhibited moderate to significant dose-dependent quorum-sensing inhibitory activities against PAO1 lasB-gpf and rhlA-gfp bioreporter strains with IC₅₀ values of 19.6 µM and 7.3 µM, respectively. Full article

Quorum sensing (QS) plays an essential role in the production of virulence factors, in biofilm formation and antimicrobial resistance. Consequently, inhibiting QS is being considered a promising target for antipathogenic/anti-virulence therapies. This study aims to screen 2-nitrovinylfuran derivatives structurally related to Furvina (a broad-spectrum antibiotic already used for therapeutic purposes) for their effects on QS and in biofilm prevention/control. Furvina and four 2-nitrovinylfuran derivatives (compounds 1–4) were tested to assess the ability to interfere with QS of Staphylococcus aureus using bioreporter strains (S. aureus ALC1742 and ALC1743). The activity of Furvina and the most promising quorum-sensing inhibitor (QSI) was evaluated in biofilm prevention and in biofilm control (combined with fusidic acid). The biofilms were further characterized in terms of biofilm mass, viability and membrane integrity. Compound 2 caused the most significant QS inhibition with reductions between 60% and 80%. Molecular docking simulations indicate that this compound interacts preferentially with the protein hydrophobic cleft in the LytTR domain of AgrA pocket. Metabolic inactivations of 40% for S. aureus ALC1742 and 20% for S. aureus ALC1743 were reached. A 24 h-old biofilm formed in the presence of the QSI increased the metabolic inactivation by fusidic acid to 80%, for both strains. The overall results highlight the effects of compound 2 as well as the potential of combining QSI with in-use antibiotics for the management of skin and soft tissues infections. Full article

The persistence of endocrine disrupting compounds (EDCs) throughout wastewater treatment processes poses a significant health threat to humans and to the environment. The analysis of EDCs in wastewater remains a challenge for several reasons, including (a) the multitude of bioactive but partially unknown compounds, (b) the complexity of the wastewater matrix, and (c) the required analytical sensitivity. By coupling biological assays with high-performance thin-layer chromatography (HPTLC), different samples can be screened simultaneously, highlighting their active components; these may then be identified by chemical analysis. To allow the multiparallel detection of diverse endocrine disruption activities, we have constructed Saccharomyces cerevisiae-based bioreporter strains, responding to compounds with either estrogenic or androgenic activity, by the expression of green (EGFP), red (mRuby), or blue (mTagBFP2) fluorescent proteins. We demonstrate the analytical potential inherent in combining chromatographic compound separation with a direct fluorescent signal detection of EDC activities. The applicability of the system is further demonstrated by separating influent samples of wastewater treatment plants, and simultaneously quantifying estrogenic and androgenic activities of their components. The combination of a chemical separation technique with an optical yeast-based bioassay presents a potentially valuable addition to our arsenal of environmental pollution monitoring tools. Full article

This paper investigated the effects of binary mixtures of bioluminescence inducers (toluene, xylene isomers, m-toluate) and of metals (Cu, Cd, As(III), As(V), and Cr) on bioluminescence activity of recombinant (P_m-lux) strain KG1206. Different responses and sensitivities were observed depending on the types and concentrations of mixtures of inducers or metals. In the case of inducer mixtures, antagonistic and synergistic modes of action were observed, whereas metal mixtures showed all three modes of action. Antagonistic mode of action was most common for mixtures of indirect inducers, which showed bioluminescence ranging from 29% to 62% of theoretically expected effects (P(E)). On the other hand, synergistic mode of action was observed for mixtures of direct and indirect inducers, which showed bioluminescence between 141% and 243% of P(E). In the case of binary metal mixtures, bioluminescence activities were ranged from 62% to 75% and 113% to 164% of P(E) for antagonistic and synergistic modes of action, respectively (p-values 0.0001–0.038). Therefore, mixture effects could not be generalized since they were dependent on both the types and concentrations of chemicals, suggesting that biomonitoring may constitute a better strategy by investigating types and concentrations of mixture pollutants at contaminated sites. Full article

Initially described in 1990, Pseudomonas fluorescens HK44 served as the first whole-cell bioreporter genetically endowed with a bioluminescent (luxCDABE) phenotype directly linked to a catabolic (naphthalene degradative) pathway. HK44 was the first genetically engineered microorganism to be released in the field to monitor bioremediation potential. Subsequent to that release, strain HK44 had been introduced into other solids (soils, sands), liquid (water, wastewater), and volatile environments. In these matrices, it has functioned as one of the best characterized chemically-responsive environmental bioreporters and as a model organism for understanding bacterial colonization and transport, cell immobilization strategies, and the kinetics of cellular bioluminescent emission. This review summarizes the characteristics of P. fluorescens HK44 and the extensive range of its applications with special focus on the monitoring of bioremediation processes and biosensing of environmental pollution. Full article