An Intact Cell Bioluminescence-Based Assay for the Simple and Rapid Diagnosis of Urinary Tract Infection.

Urinary tract infection (UTI) is one of the most common infections, accounting for a substantial portion of outpatient hospital and clinic visits. Standard diagnosis of UTI by culture and sensitivity can take at least 48 h, and improper diagnosis can lead to an increase in antibiotic resistance following therapy. To address these shortcomings, rapid bioluminescence assays were developed and evaluated for the detection of UTI using intact, viable cells of Photobacterium mandapamensis USTCMS 1132 or previously lyophilized cells of Photobacterium leiognathi ATCC 33981™. Two platform technologies-tube bioluminescence extinction technology urine (TuBETUr) and cellphone-based UTI bioluminescence extinction technology (CUBET)-were developed and standardized using artificial urine to detect four commonly isolated UTI pathogens-namely, Escherichia coli, Proteus mirabilis, Staphylococcus aureus, and Candida albicans. Besides detection, these assays could also provide information regarding pathogen concentration/level, helping guide treatment decisions. These technologies were able to detect microbes associated with UTI at less than 105 CFU/mL, which is usually the lower cut-off limit for a positive UTI diagnosis. Among the 29 positive UTI samples yielding 105-106 CFU/mL pathogen concentrations, a total of 29 urine specimens were correctly detected by TuBETUr as UTI-positive based on an 1119 s detection window. Similarly, the rapid CUBET method was able to discriminate UTIs from normal samples with high confidence (p ≤ 0.0001), using single-pot conditions and cell phone-based monitoring. These technologies could potentially address the need for point-of-care UTI detection while reducing the possibility of antibiotic resistance associated with misdiagnosed cases of urinary tract infections, especially in low-resource environments.

[The Factor Stabilizing the Bioluminescence of PVA-Immobilized Photobacteria].

Immobilization of photobacteria in the cryogel of polyvinyl alcohol (PVA) was carried out. Immobilization was found to result in increased intensity and stability of bioluminescence. The elements determining the stability of bioluminescence were investigated. Selection of the strain was found to be of the highest importance. Among immobilized cells, Photobacterium phosphoreum exhibited the most intense and prolonged light emission, while Vibrio harveyi showed the least one. The technological procedures for cryogenic immobilization of photobacteria were determined. The role of the environment of gel formation in the preservation of the bioluminescence activity was determined. In the gels formed in rich medium for submerged cultivation of photobacteria, almost 100% luminescence activity was preserved, while light emission was considerably prolonged. Bioluminescence intensity of the preparations was shown to depend significantly on pH of the incubation medium. The pH shift to acidic values during prolonged incubation of immobilized cells was shown to be one of the factors of bioluminescence quenching. The stress effects of cryogenic immobilization were found to have an insignificant effect on the temperature profile of bioluminescence. Decreased reduction rate of the luciferase flavin substrate was shown to be a possible reason for bioluminescence quenching.

Surfactants present complex joint effects on the toxicities of metal oxide nanoparticles.

The potential toxicities of nanoparticles (NPs) have been intensively discussed over the past decade. In addition to their single toxicities, NPs can interact with other environmental chemicals and thereby exert joint effects on biological systems and the environment. The present study investigated the combined toxicities of NPs and surfactants, which are among the chemicals that most likely coexist with NPs. Photobacterium phosphoreum was employed as the model organism. The results indicate that surfactants with different ion types can alter the properties of NPs (i.e., particle size and surface charge) in different ways and present complex joint effects on NP toxicities. Mixtures of different NPs and surfactants exhibited antagonistic, synergistic, and additive effects. In particular, the toxicity of ZnO was observed to result from its dissolved Zn(2+); thus, the joint effects of the ZnO NPs and surfactants can be explained by the interactions between the Zn ions and the surfactants. Our study suggests that the potential hazards caused by mixtures of NPs and surfactants are different from those caused by single NPs. Because surfactants are extensively used in the field of nanotechnology and are likely to coexist with NPs in natural waters, the ecological risk assessments of NPs should consider the impacts of surfactants.

Development of a bioluminescent bacteria sheet for the measurement of oxygen concentration.

The aim of this study was the preparation and characterization of a bioluminescence bacteria sheet in view of the fabrication of an oxygen marker inside a biofilm. Photobacterium kishitanii sheets were prepared using sodium alginate, and a biofilm of Pseudomonas aeruginosa was dropped over it. The relationship between the dissolved oxygen (DO) concentration in the biofilm and luminescence from the sheet was examined. As a conclusion, the alginate-gel film method was effective in fabricating a DO-monitoring thin film.

Effects of Americium-241 and humic substances on Photobacterium phosphoreum: bioluminescence and diffuse reflectance FTIR spectroscopic studies.

The integral bioluminescence (BL) intensity of live Photobacterium phosphoreum cells (strain 1883 IBSO), sampled at the stationary growth stage (20 h), was monitored for further 300 h in the absence (control) and presence of (241)Am (an α-emitting radionuclide of a high specific activity) in the growth medium. The activity concentration of (241)Am was 2 kBq l(-1); [(241)Am]=6.5×10(-11) M. Parallel experiments were also performed with water-soluble humic substances (HS, 2.5 mg l(-1); containing over 70% potassium humate) added to the culture medium as a possible detoxifying agent. The BL spectra of all the bacterial samples were very similar (λ(max)=481±3 nm; FWHM=83±3 nm) showing that (241)Am (also with HS) influenced the bacterial BL system at stages prior to the formation of electronically excited states. The HS added per se virtually did not influence the integral BL intensity. In the presence of (241)Am, BL was initially activated but inhibited after 180 h, while the system (241)Am+HS showed an effective activation of BL up to 300 h which slowly decreased with time. Diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy, applied to dry cell biomass sampled at the stationary growth phase, was used to control possible metabolic responses of the bacteria to the α-radioactivity stress (observed earlier for other bacteria under other stresses). The DRIFT spectra were all very similar showing a low content of intracellular poly-3-hydroxybutyrate (at the level of a few percent of dry biomass) and no or negligible spectroscopic changes in the presence of (241)Am and/or HS. This assumes the α-radioactivity effect to be transmitted by live cells mainly to the bacterial BL enzyme system, with negligible structural or compositional changes in cellular macrocomponents at the stationary growth phase.

Expression, purification, and characterization of the recombinant putative periplasmic hemin-binding protein (HutB) of Photobacterium damselae subsp. piscicida.

HutB, the periplasmic hemin binding protein of Photobacterium damselae subsp. piscicida, was produced as a recombinant protein. UV-Vis spectrophotometrical analysis showed absorption spectral changes in hemin upon mixing it with the recombinant protein, indicating complex formation. Spectrophotometric titration of HutB with hemin showed saturation at a heme/HutB ratio of 1:1 and a binding affinity (K(d)) of 10 microM.

[Research of a bioluminent bacterial-based optical fiber sensor to detecting acute effects of pollutants in water].

Field detection of general toxicity is urgent demand in these years, therefore rapid, sensitive, convenient biosensor was studied to detect cute toxicity of water pollutant, which is a novel optical fiber sensor immobilized bioluminent bacterial, Photobacterium phosphorem T3. This bioluminence light was measured by light detection system after it coupling to optical fiber. For the activity and bioluminence of bacterial was seriously affected by immobilizing conditions, optimization of immobilization was studied, including the concentration of Sodium alga gel and CaCl2, immobilizing time, pH, and the preservation time. When the bacteria was immobilized with 3% sodium alga in 3% CaCl2 solution for 15 minutes, the bacteria had the best activity, and the immobilized bacteria would maintain its activity for the longest time in the 3% NaCl solution. The dose-response curves of Zn2+, NH3, nitrobenzenne and cresol are detected, and the EC50 are also calculated, which are 5.1, 10.2, 70.4, 77.0 mg x L(-1). The EC50 are quite coherent to the results of standard bioluminescent bacteria method. The optical fiber biosensor could be disposable, small sized, convenient operation for field application.

Photobacterium lutimaris sp. nov., isolated from a tidal flat sediment in Korea.

A Gram-negative, motile, pale-yellow-pigmented, oval-shaped bacterial strain, DF-42T, was isolated from a tidal flat sediment in Korea. Strain DF-42T grew optimally at 25-30 degrees C and in the presence of 2-3 % (w/v) NaCl. It contained Q-8 as the predominant ubiquinone and C(16 : 0), C(18 : 1)omega7c and summed feature 3 (C(16 : 1)omega7c and/or iso-C(15 : 0) 2-OH) as the major fatty acids. The DNA G+C content was 48.3 mol%. Phylogenetic trees based on 16S rRNA gene sequences showed that strain DF-42T falls within the evolutionary radiation enclosed by the genus Photobacterium. Strain DF-42T exhibited 16S rRNA gene sequence similarity values of 93.8-97.9 % to the type strains of Photobacterium species with validly published names. DNA-DNA relatedness data and differential phenotypic properties made it possible to categorize strain DF-42T as representing a species that is separate from previously described Photobacterium species. The name Photobacterium lutimaris sp. nov. is proposed, with strain DF-42T (=KCTC 12723T=JCM 13586T) as the type strain.

Photobacterium halotolerans sp. nov., isolated from Lake Martel in Spain.

A halotolerant bacterium was isolated from a saline lake located in Mallorca, Spain. Cells of the strain, designated MACL01T, were Gram-negative, rod-shaped and motile by means of polar flagella. Colonies of strain MACL01T were white to cream in TSA medium, turning brown after 7 days of incubation; they were blue in thiosulphate/citrate/bile salts/sucrose agar medium. A neighbour-joining phylogenetic analysis based on 16S rRNA gene sequences showed that strain MACL01T belongs to the genus Photobacterium, in which it forms a distinct lineage together with Photobacterium rosenbergii and Photobacterium ganghwense (showing 96.9 and 96.2 % similarity, respectively). The most closely related taxon according to phylogenetic analysis of the rpoA gene is also P. rosenbergii (90 % similarity). The recA gene also showed low similarity (83.7, 83.4 and 82.4 %, respectively) with respect to those of Vibrio proteolyticus LMG 3772T, Photobacterium leiognathii LMG 4228T and P. rosenbergii LMG 22223T. Neighbour-joining phylogenetic analysis of the rpoA and recA genes confirms that strain MACL01T belongs to the genus Photobacterium, forming a branch together with P. rosenbergii. Strain MACL01T was able to grow in 0-8 % NaCl. Growth occurred between 4 and 37 degrees C (optimum, 28 degrees C) and at pH 5-8.5. Luminescence was negative on marine agar. Strain MACL01T was found to be sensitive to the vibriostatic agent O/129. It reduced nitrate to nitrite, produced beta-galactosidase and hydrolysed gelatin, but did not produce arginine dihydrolase, indole or acetoin. Strain MACL01T used several carbohydrates and fermented glucose, L-arabinose and sucrose. The most abundant fatty acids were summed feature 3 (32.6 %; comprising C16 : 1omega7c and/or C15 : 0 iso 2-OH), C16 : 0 (21.2 %) and C18 : 1omega7c (19.9 %). The G+C content of the genomic DNA was 49.8 mol%. On the basis of genotypic, phenotypic, chemotaxonomic and phylogenetic results, strain MACL01T (=LMG 22194T=CECT 5860T) should be classified as the type strain of a novel species of the genus Photobacterium, for which the name Photobacterium halotolerans sp. nov. is proposed.

Analysis of synchronous photon emissions from the bacterium Photobacterium phosphoreum during colony formation from a single cell.

Light emission from Photobacterium phosphoreum was analysed during cell growth on an agar plate from a single cell to colony formation. Temporal analysis of image intensified light was set so that a quadratic window covered a single cell. Intensity of light emission from a single cell through colony formation showed an initial decrease, a prolonged lag phase, and then a rapid increase. These responses on an agar plate were similar to those from liquid cultures. The image analysis showed repeated bursts of light emission in the phases when light was increasing and decreasing. Statistical analysis of light emission also emphasized the presence of bursts of light emission, suggesting the metabolic synchronism of luciferase reactions in either a single cell or suggesting the metabolic synchronism of luciferase reactions in either a single cell or synchronously divided cells. The repetitive bursts of light occurred in a single cell and continued during the growth phase in which the cell population and the light emission was increasing. In a single cell, however, periodicity of light emission was not defined directly from fast Fourier transformation, although it was indicated on oscillation of mean level of fluctuated light emission, at initial phase of culture on agar plate.

Bioluminescence and cell growth of Photobacterium phosphoreum.

The bioluminescence activity of Photobacterium phosphoreum was compared at different times after cell division by the methods of density gradient centrifugation and synchronous culture. The bioluminescence intensity per cell mass increased linearly at a rate of 1.5 times per doubling time. The luciferase system in the cell is continuously activated during growth, independent of cell division.

Properties and kinetics of salt activation of a membrane-bound NADH dehydrogenase from a marine bacterium Photobacterium phosphoreum.

A membrane-bound NADH dehydrogenase, solubilized and partially purified from a marine bacterium Photobacterium phosphoreum, contains FAD as the prosthetic group, and is specific for NADH. Ferricyanide, various other redox dyes and cytochrome c can act as electron acceptors. The enzymatic activity when assayed with electron acceptors other than cytochrome c, is activated by monovalent cations (Na+ and K+) and deactivated by high concentrations of monovalent anions (SCN-, NO3-, and Cl-) but not by phosphate ions. The enzymatic reaction follows a ping-pong mechanism and kinetic analysis of the enzyme showed that the activation by monovalent cations is due to increase of affinity of the enzyme for substrates; Vm was not affected. The increase of affinity was 62- and 46-fold for NADH and 57- and 31-fold for 2,6-dichlorophenol indophenol in the presence of Na+ and K+, respectively. On the other hand, NADH-cytochrome c reductase activity of the enzyme was strongly inhibited by these cations.

Structure and arrangement of flagella in species of the genus Beneckea and Photobacterium fischeri.

Species of marine bacteria belonging to the genus Beneckea and strains of Photobacterium fischeri were negatively stained and examined by means of the electron microscope to determine the structure and arrangement of their flagella. All of the species of the genus Beneckea had single, polar, sheathed flagella when grown in liquid medium. When grown on solid medium, most strains of B. campbellii and B. neptuna and all strains of B. alginolytica and B. parahaemolytica had unsheathed, peritrichous flagella in addition to the single, sheathed, polar flagellum. The remaining species, B. nereida, B. pelagia, and B. natriegens, had a single, polar, sheathed flagellum when grown on solid medium. Strains of P. fischeri had sheathed flagella arranged in polar tufts. Only one group (B-2) of marine bacteria included in this study was found to have polar, unsheathed flagella.

Presence of rhapidosomes in various species of bacteria and their morphological characteristics.

Rod-shaped structures have been observed in cells of Pseudomonas, Photobacterium, Proteus, and Saprospira by use of the negative-contrast stain. These structures, referred to as rhapidosomes, appear to be normal components of these cells. Other bacteria including Escherichia, Salmonella, Shigella, Klebsiella, Micrococcus, Bacillus, Mycobacterium, Rhodospirillum, and Hydrogenomonas genera failed to reveal these structures. The rhapidosomes of Saprospira were found to consist of two rods, one encasing a narrower, longer structure. In contrast, the rhapidosomes of Pseudomonas, Proteus, and Photobacterium were without the rigid inner structure, but were occasionally seen filled with a homogeneous material as observed by the negative stain. Ultrathin sections of Pseudomonas cells indicate that these rhapidosomes are embedded within or are in close association with the nucleoplasm.