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PubMedDate not supplied

In vivo bioluminescence: a cellular reporter for research and industry.

The detection of specific bacterial pathogens, indicator microorganisms and antimicrobial substances, and the recovery of microorganisms from sub-lethal injury, are all aspects of importance to industry which are currently being targeted using in vivo bioluminescence. In all instances, a key requirement for the application of bioluminescence is the establishment of a strict correlation between in vivo bioluminescence and cell viability, as determined by colony counting on agar plates. Comparative studies for biocides (phenol, chlorhexidine diacetate, phenol thioether), for a virucide (hypochlorite) and for cellular recovery of S. typhimurium from sub-lethal injury, all indicate that such a correlation is valid. Furthermore, real-time measurements of in vivo bioluminescence reveal a major population of bacterial cells that retain functional intracellular biochemistry, but are defective in their ability to replicate post of freeze injury.

Bacteriophage lambda↗

PubMed2006

[Towards new antibacterial drugs. Interest of para-guanidinoethylcalix[4]arene].

We present here the results concerning the antibacterial properties evaluation of para-guanidinoethylcalix[4]arene, compared with its constitutive monomer, the para-guanidinoethylphenol, and hexamidine (Hexomédine), an antiseptic from the diamidine family widely used in therapeutic, chosen as a reference in this study for its resemblance in terms of functional groups. Antibacterial activities of those three compounds were evaluated by microdilution methods, in Mueller Hinton broth, onto 5 bacterial strains: Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 25923 & ATCC 29213 and Enterococcus faecalis ATCC 29212, according to CA-SFM and CLSI (formerly NCCLS) approved standards. In parallel, the effects of these three compounds on MRC-5 eukaryotic cell viability were evaluated with MTT assay. The results obtained here confirm a lack of activity for the monomer compound (MIC> or =512 mg/l) and a real antibacterial activity for the calixarene, comparable to hexamidine. This activity is expressed, both on Gram+and Gram- bacteria (MIC=4 mg/l for E. coli, 8 mg/l on both S. aureus strains) and at a lesser degree on E. faecalis and P. aeruginosa (MIC=32 mg/l). Similarly, both compounds, monomer and calixarene, slightly induce any modification on MRC-5 cells viability, and this until 168 h of treatment for concentrations reaching 10(-4) mol/L while hexamidine demonstrates a significant and increasing effect during the time of experiment and this for 100 to 1000 times lower concentrations. Thus, this study tends to confirm the significance of the organization of the para-guanidinoethylphenol monomer into its cyclic calixarenic tetramer for the gain of an antibacterial activity, similar to a widely used antiseptic one.

Anti-Bacterial Agents↗

PubMed2003

A disc-based quantitative carrier test method to assess the virucidal activity of chemical germicides.

Suspension tests for virucidal activity of chemical germicides are easier to perform, but they normally do not present the test product with a strong enough challenge. In contrast, carrier tests, where the test virus is dried on an animate or inanimate surface, offer the test formulation a higher level of challenge because it first has to penetrate successfully the inoculum to gain access to and inactivate the target organism on the carrier. Since pathogens in nature are normally found adsorbed to surfaces and/or embedded in organic or cellular debris, the results of carrier tests are more relevant to predicting the activity of chemical germicides under field situations. The method described below uses discs (1 cm in diameter) of brushed stainless steel discs as carriers. Ten micro l of the test virus in a soil load is placed on each disc and the inoculum dried under ambient conditions. The dried inoculum is then exposed to 50 micro l of the test formulation or a control solution for a defined contact time at the specified temperature. EBSS (0.95 ml) is added to each carrier holder to dilute/neutralize the germicide, the inoculum eluted and the eluates titrated in cell cultures to determine the degree of loss in virus viability. At least five test and three control carriers are used in each test. Controls are also included to test for toxicity of the test formulation to the host cells and any interference sub-cytotoxic levels of the formulation may have on the ability of the virus to infect the cells. The method has been used with several types of human and animal pathogenic viruses to test the activity of all major classes of chemical germicides against them.

Antiviral Agents↗

PubMed2002

Particulate Bioglass reduces the viability of bacterial biofilms formed on its surface in an in vitro model.

45S5 Bioglass is a bioactive implant material which, in its particulate form, is used in the repair of periodontal defects. The surface reactions undergone by this material in an aqueous environment may exert an antibacterial effect that would be beneficial to periodontal surgical treatment. Biofilms of Streptococcus sanguis, an early plaque former, and mixed species biofilms from a salivary inoculum grown under conditions similar to those associated with periodontal implants, were grown on particulate Bioglass in a constant depth film fermenter (CDFF). Control biofilms were grown on inert glass particulates. At sample times of 3, 24 and 48 hours the viability of biofilms of S. sanguis grown on Bioglass was significantly lower than for those grown on inert glass. In the experiments with subgingivally-modelled mixed species biofilms, the total anaerobic counts were significantly lower on Bioglass after 24 and 48 hours, but not 96 or 168 hours, compared to inert glass. Thus, particulate Bioglass has the potential to reduce bacterial colonisation of its surface in vivo, a feature relevant to post-surgical periodontal wound healing.

Anti-Bacterial Agents↗

PubMed2002

A laboratory study of the effect of calcium hydroxide mixed with iodine or electrophoretically activated copper on bacterial viability in dentinal tubules.

AIM: The aim of this laboratory study was to evaluate the ability of calcium hydroxide (CH), CH/iodine-potassium iodide (IKI) and electrophoretically activated copper to kill bacteria in dentinal tubules. METHODOLOGY: In an in vitro model of dentinal tubule infection, 42 cylindrical root specimens, prepared from freshly extracted bovine teeth were used. After removal of the smear layer, intracanal dentinal tubules were infected with Enterococcus faecalis for 3 weeks. CH alone or preparations of CH with copper or IKI were placed in the root canal for 1 week. In specimens containing copper/CH, an electrophoretic current(5 mA/5 min) was applied using two electrodes follow-ing placement of the medicament in the canal. Powder dentine samples obtained from the canal wall using ISO sizes: 025, 027, 029, 031 and 033 burs were examined for the presence of viable bacteria by inoculating agar plates and counting colony forming units (cfu). RESULTS: A significant difference was found between the experimental groups and the positive control group. CH and CH/IKI significantly (P < 0.001)reduced bacterial viability in dentinal tubules to a depth of 200 microm. Specimens with CH/IKI had significantly fewer viable bacteria than CH alone in tubules between the depths of 200-500 microm. Treatment with CH/copper and electrophoresis was most effective: specimens showed no viable bacteria in dentinal tubules to a depth of 500 microm from the root-canal space. CONCLUSIONS: IKI or electrophoretically activated copper additives can significantly improve the antibacterial properties of CH in dentinal tubules.

Animals↗

PubMed1998

A chloride-inducible acid resistance mechanism in Lactococcus lactis and its regulation.

Previously, a promoter was identified in Lactococcus lactis that is specifically induced by chloride. Here, we describe the nucleotide sequence and functional analysis of two genes transcribed from this promoter, gadC and gadB. GadC is homologous to putative glutamate-gamma-aminobutyrate antiporters of Escherichia coli and Shigella flexneri and contains 12 putative membrane-spanning domains. GadB shows similarity to glutamate decarboxylases. A L. lactis gadB mutant and a strain that is unable to express both gadB and gadC was more sensitive to low pH than the wild type when NaCl and glutamate were present. Expression of gadCB in L. lactis in the presence of chloride was increased when the culture pH was allowed to decrease to low levels by omitting buffer from the medium, while glutamate also stimulated gadCB expression. Apparently, these genes encode a glutamate-dependent acid resistance mechanism of L. lactis that is optimally active under conditions in which it is needed to maintain viability. Immediately upstream of the chloride-dependent gadCB promoter Pgad, a third gene encodes a protein (GadR) that is homologous to the activator Rgg from Streptococcus gordonii. gadR expression is chloride and glutamate independent. A gadR mutant did not produce the 3kb gadCB mRNA that is found in wild-type cells in the presence of NaCl, indicating that GadR is an activator of the gadCB operon.

Amino Acid Sequence↗

PubMed2004

Antibacterial activity of hydrolyzable tannins derived from medicinal plants against Helicobacter pylori.

Helicobacter pylori is a major etiological agent in gastroduodenal disorders. In this study, we isolated 36 polyphenols and 4 terpenoids from medicinal plants, and investigated their antibacterial activity against H. pylori in vitro. All hydrolyzable tannins tested demonstrated promising antibacterial activity against H. pylori. Monomeric hydrolyzable tannins revealed especially strong activity. Other compounds demonstrated minimal antibacterial activity with a few exceptions. A monomeric hydrolyzable tannin, Tellimagrandin I demonstrated time- and dose-dependent bactericidal activity against H. pylori in vitro. On the other hand, hydrolyzable tannins did not affect the viability of MKN-28 cells derived from human gastric epithelium. Hydrolyzable tannins, therefore, have potential as new and safe therapeutic regimens against H. pylori infection. Furthermore, we investigated effects of hydrolyzable tannins on lipid bilayer membranes. All the hydrolyzable tannins tested demonstrated dose-dependent membrane-damaging activity. However, it remains to be elucidated whether their membrane-damaging activity directly contributes to their antibacterial action.

Acids↗

PubMed2007

Detection of Vibrio cholerae by real-time nucleic acid sequence-based amplification.

A multitarget molecular beacon-based real-time nucleic acid sequence-based amplification (NASBA) assay for the specific detection of Vibrio cholerae has been developed. The genes encoding the cholera toxin (ctxA), the toxin-coregulated pilus (tcpA; colonization factor), the ctxA toxin regulator (toxR), hemolysin (hlyA), and the 60-kDa chaperonin product (groEL) were selected as target sequences for detection. The beacons for the five different genetic targets were evaluated by serial dilution of RNA from V. cholerae cells. RNase treatment of the nucleic acids eliminated all NASBA, whereas DNase treatment had no effect, showing that RNA and not DNA was amplified. The specificity of the assay was investigated by testing several isolates of V. cholerae, other Vibrio species, and Bacillus cereus, Salmonella enterica, and Escherichia coli strains. The toxR, groEL, and hlyA beacons identified all V. cholerae isolates, whereas the ctxA and tcpA beacons identified the O1 toxigenic clinical isolates. The NASBA assay detected V. cholerae at 50 CFU/ml by using the general marker groEL and tcpA that specifically indicates toxigenic strains. A correlation between cell viability and NASBA was demonstrated for the ctxA, toxR, and hlyA targets. RNA isolated from different environmental water samples spiked with V. cholerae was specifically detected by NASBA. These results indicate that NASBA can be used in the rapid detection of V. cholerae from various environmental water samples. This method has a strong potential for detecting toxigenic strains by using the tcpA and ctxA markers. The entire assay including RNA extraction and NASBA was completed within 3 h.

Animals↗

PubMed1999

Suppression of replication of multidrug-resistant HIV type 1 variants by combinations of thymidylate synthase inhibitors with zidovudine or stavudine.

The replication of recombinant multidrug-resistant HIV-1 clones modeled on clinically derived resistant HIV-1 strains from patients receiving long-term combination therapy with zidovudine (AZT) plus 2',3'-dideoxycytidine was found to regain sensitivity to AZT and stavudine (D4T) as a consequence of a pharmacologically induced decrease in de novo dTMP synthesis. The host-cell system used was phytohemagglutinin-stimulated peripheral blood mononuclear cells; dTMP and dTTP depletion were induced by single exposures to a low level of the thymidylate synthase inhibitor 5-fluorouracil (5-FU) or its deoxynucleoside, 2'-deoxy-5-fluorouridine. The host-cell response to the latter was biphasic: a very rapid decrease in the rate of de novo dTMP formation and, consequently, in intracellular dTTP pools, followed by slower recovery in both indices over 3 to 24 h. With the additional presence of AZT or D4T, however, replication of the multidrug-resistant HIV-1 strains remained inhibited, indicating dependence of HIV DNA chain termination by AZT-5'-monophosphate or 2',3'-didehydro-2', 3'-dideoxythymidine-5'-monophosphate in these resistant strains on simultaneous inhibition of host-cell de novo synthesis of thymidine nucleotides. No effect on viability of control (uninfected) phytohemagglutinin-stimulated/peripheral blood mononuclear cells was noted on 6-day exposures to 5-FU or 2'-deoxy-5-fluorouridine alone or in combination with AZT or D4T, even at drug levels severalfold higher than those used in the viral inhibition studies. These studies may provide useful information for the potential clinical use of AZT/5-FU or D4T/5-FU combinations for the prevention or reversal of multidrug resistance associated with long-term dideoxynucleoside combination therapy.

Anti-HIV Agents↗

PubMed2026

Biogenic Silver Nanoparticles from the Cell-Free Supernatant of Mychonastes sp. B1: Antibacterial and Antibiofilm Effects, and Wound Healing Activity Supported by Gene and Protein Expression Analysis.

The biogenic synthesis of silver nanoparticles (AgNPs) using microalgae provides a sustainable alternative to conventional physicochemical methods. In this study, AgNPs were synthesized from the cell-free supernatant of the freshwater microalga Mychonastes sp. B1 and characterized by ultraviolet-visible spectroscopy (UV-Vis), transmission electron microscopy (TEM), dynamic light scattering (DLS), Fourier transform infrared spectroscopy (FTIR), and field-emission scanning electron microscopy with energy-dispersive X-ray spectroscopy (FE-SEM/EDS). The nanoparticles were predominantly spherical (15-55 nm), highly stable (ζ =  - 42.8 mV), and appeared to be capped by extracellular polymeric substances. The biogenic AgNPs (GS-AgNPs) exhibited potent antibacterial activity, with minimum inhibitory concentrations (MICs) of 2.0 µg/mL against Staphylococcus aureus and 2.5 µg/mL against Pseudomonas aeruginosa, and significantly (p < 0.05) inhibited biofilm formation. Fibroblast viability remained at or above 80% at AgNP concentrations up to 1.5 µg/mL, which promoted cell migration and increased wound closure by 8.1% at 24 h (p < 0.05). Exposure to 1.5 µg/mL AgNPs significantly upregulated extracellular matrix markers (Col1a1 2.3-fold, Fn1 3.3-fold at mRNA level; COL1A1 2.1-fold, FN1 2.7-fold at the protein level). These findings indicate that GS-AgNPs possess antimicrobial and wound healing properties, highlighting their potential as biocompatible nanomaterials for biomedical applications.

Silver↗

PubMed1994

Killing of cariogenic bacteria by light from a gallium aluminium arsenide diode laser.

Suspensions of Streptococcus mutans, S. sobrinus, Lactobacillus casei and Actinomyces viscosus were exposed to light from a gallium aluminium arsenide laser in the presence of aluminium disulphonated phthalocyanine and the numbers of survivors determined. Exposure to the laser light in the absence of the dye, or the dye in the absence of the laser light, had no significant effect on the viability of the organisms. However, a light-dose-related decrease in the viable count of all four target organisms was found on exposure to the laser light in the presence of the dye. The kills attributable to lethal photosensitization amounted to approximately 10(6) CFU in the case of each organisms. As appreciable kills were achieved within clinically convenient exposure times (30-90 s), these results imply that lethal photosensitization may be a useful technique for eliminating bacteria from carious lesions prior to restoration.

Actinomyces viscosus↗

PubMed2006

Effects of subinhibitory concentrations of clindamycin on the morphological, biochemical and genetic characteristics of Bacteroides fragilis.

The effects of subinhibitory concentrations of clindamycin on the morphological, biochemical and genetic characteristics of species of the Bacteroides fragilis group isolated from children with diarrhea were determined. The minimal inhibitory and subinhibitory concentrations for clindamycin were determined. Minimal inhibitory concentration values ranging from 0.25 to 512 microg mL(-1) were observed. Cultures grown with clindamycin were used to determine the macroscopic morphological characteristics, cellular viability, ultrastructural characteristics and DNA integrity. Clindamycin did not alter colonial morphology, but after 6 h elongated cells were observed. Also, extracellular vesicles and electron-lucent areas inside the cytoplasm were observed. Bacteria treated with clindamycin also showed fragmentation of DNA as determined by electrophoresis. The alterations produced by clindamycin might be indicative of a possible modification of the structures involved in bacterial pathogenesis.

Anti-Bacterial Agents↗

PubMed1998

A rapid method for simultaneous detection of phenotypic resistance to inhibitors of protease and reverse transcriptase in recombinant human immunodeficiency virus type 1 isolates from patients treated with antiretroviral drugs.

Combination therapy with protease (PR) and reverse transcriptase (RT) inhibitors can efficiently suppress human immunodeficiency virus (HIV) replication, but the emergence of drug-resistant variants correlates strongly with therapeutic failure. Here we describe a new method for high-throughput analysis of clinical samples that permits the simultaneous detection of HIV type 1 (HIV-1) phenotypic resistance to both RT and PR inhibitors by means of recombinant virus assay technology. HIV-1 RNA is extracted from plasma samples, and a 2.2-kb fragment containing the entire HIV-1 PR- and RT-coding sequence is amplified by nested reverse transcription-PCR. The pool of PR-RT-coding sequences is then cotransfected into CD4+ T lymphocytes (MT4) with the pGEMT3deltaPRT plasmid from which most of the PR (codons 10 to 99) and RT (codons 1 to 482) sequences are deleted. Homologous recombination leads to the generation of chimeric viruses containing PR- and RT-coding sequences derived from HIV-1 RNA in plasma. The susceptibilities of the chimeric viruses to all currently available RT and/or PR inhibitors is determined by an MT4 cell-3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-based cell viability assay in an automated system that allows high sample throughput. The profile of resistance to all RT and PR inhibitors is displayed graphically in a single PR-RT-Antivirogram. This assay system facilitates the rapid large-scale phenotypic resistance determinations for all RT and PR inhibitors in one standardized assay.

Acquired Immunodeficiency Syndrome↗

PubMed2003

Attenuation of itraconazole fungicidal activity following preexposure of Aspergillus fumigatus to fluconazole.

Fluconazole (FLC), a triazole with limited activity against Aspergillus species, is frequently used as prophylaxis in leukemia patients and bone marrow transplant recipients. Prior FLC use has been associated with an increasing incidence of invasive aspergillosis in these patients. We hypothesized that prior exposure of Aspergillus fumigatus to FLC could result in altered in vitro susceptibility of this fungus to other, more active triazoles. Thus, we performed serial passages of conidia of 10 clinical isolates of A. fumigatus (all itraconazole [ITC] susceptible) on FLC-containing yeast agar glucose plates. The MICs and minimal fungicidal concentrations (MFCs) of amphotericin B, FLC, ITC, and voriconazole (VRC) for A. fumigatus conidia were measured following four passages on FLC-containing medium according to the National Committee for Clinical Laboratory Standards microdilution method. Serial passages on FLC-containing plates resulted in a fourfold increase in the MFCs (but not the MICs) of ITC for nine isolates. The attenuated ITC fungicidal activity against A. fumigatus following FLC preexposure was medium independent and was also observed against FLC-preexposed A. fumigatus hyphae with the viability staining FUN-1 dye. Moreover, FLC preexposure of A. fumigatus conidia resulted in an analogous increase in the MFCs (but not the MICs) of VRC. Our findings suggest that preexposure of A. fumigatus to FLC attenuates the in vitro fungicidal activity of subsequent ITC use against it. This phenotypic adaptation is not captured by a routine MIC determination but requires MFC measurement. The in vivo significance of this in vitro phenomenon requires further investigation.

Antifungal Agents↗

PubMed2004

Colorimetric method for identifying plant essential oil components that affect biofilm formation and structure.

The specific biofilm formation (SBF) assay, a technique based on crystal violet staining, was developed to locate plant essential oils and their components that affect biofilm formation. SBF analysis determined that cinnamon, cassia, and citronella oils differentially affected growth-normalized biofilm formation by Escherichia coli. Examination of the corresponding essential oil principal components by the SBF assay revealed that cinnamaldehyde decreased biofilm formation compared to biofilms grown in Luria-Bertani broth, eugenol did not result in a change, and citronellol increased the SBF. To evaluate these results, two microscopy-based assays were employed. First, confocal laser scanning microscopy (CLSM) was used to examine E. coli biofilms cultivated in flow cells, which were quantitatively analyzed by COMSTAT, an image analysis program. The overall trend for five parameters that characterize biofilm development corroborated the findings of the SBF assay. Second, the results of an assay measuring growth-normalized adhesion by direct microscopy concurred with the results of the SBF assay and CLSM imaging. Viability staining indicated that there was reduced toxicity of the essential oil components to cells in biofilms compared to the toxicity to planktonic cells but revealed morphological damage to E. coli after cinnamaldehyde exposure. Cinnamaldehyde also inhibited the swimming motility of E. coli. SBF analysis of three Pseudomonas species exposed to cinnamaldehyde, eugenol, or citronellol revealed diverse responses. The SBF assay could be useful as an initial step for finding plant essential oils and their components that affect biofilm formation and structure.

Bacterial Adhesion↗

PubMed1984

Adenosine triphosphate content of Mycobacterium leprae from leprosy patients.

Mycobacterium leprae obtained from randomly selected lepromatous leprosy patients were used to evaluate the ATP assay technique for detecting viability of these cells. The findings were further confirmed by the standard mouse foot pad technique. While the latter takes about 8-12 months to obtain any valid information on the status of M. leprae, the ATP data can be generated within hours and at much lower cost. It is hoped that the ATP data could also instantaneously identify viable bacilli from patients taking dapsone and thereby identify dapsone-resistant patients so that alternative treatment could be given. The advantages of this method over other currently available methods are discussed.

Adenosine Triphosphate↗

PubMed2003

Cold and carbon dioxide used as multi-hurdle preservation do not induce appearance of viable but non-culturable Listeria monocytogenes.

AIMS: To study whether the exposure to cold (4 degrees C) and carbon dioxide which results in the elongation of Listeria cells, induces a viable but nonculturable (VBNC) state. METHODS AND RESULTS: When cold and CO2 stressed L. monocytogenes were observed under a fluorescence microscope, using the LIVE/DEAD BacLight bacteria viability kit (Molecular Probes, Eugene, OR, USA), the healthy, mildly injured, and the putative VBNC cells accounted for 31.0% of the stressed cell population. By using the selective plate count, 31.4% of the same stressed cell population was found to be healthy and mildly injured (putative VBNC cells not included). If there were VBNC state cells present, we should have observed a significant difference between the above two numbers. In fact, there was no significant difference between the results obtained from those two methods. CONCLUSIONS: There were no VBNC state cells observed in the stressed cell population. We conclude that cold and CO2 do not induce L. monocytogenes to enter a VBNC state. SIGNIFICANCE AND IMPACT OF THE STUDY: Cold and modified atmospheres are widely used in fresh muscle food and fruit preservation. Whether they would induce L. monocytogenes into a VBNC state is of a great concern for microbial food safety.

Adenosine Triphosphate↗

PubMed2003

Comparative survival of Cryptosporidium, coxsackievirus A9 and Escherichia coli in stream, brackish and sea waters.

Discharge of inadequately treated wastewater into streams may result in the dissemination of pathogens and the contamination of surface water sources. Determining the die-off rate of pathogenic microorganisms in stream and sea waters may serve as the basis for evaluating the health risks posed by the presence of pathogens in seawater. This study was conducted to determine the effect of microbial load, temperature, salinity and turbidity on the die-off of oocysts of Cryptosporidium as compared to that of coxsackie A9 virus (Cox A9) and E. coli. The test microorganisms were seeded into stream, outfall or sea waters and incubated at either 30 degrees C (summer) or 15 degrees C (winter). At 30 degrees C, the fastest die-off was observed for Cox A9 where < 5-log was reduced regardless of the water quality. At 1 degrees C Cox A9 persistence was similar to that of Cryptosporidium where no change was detected in the concentration of either throughout the study period. E. coli die-off reached 5 orders of magnitude within 10d then its concentration remained unchanged. The die-off of E. coli was faster than observed for Cox A9 at 15 degrees C regardless of the water quality. No decrease was observed in the viability of Cryptosporidium under all tested conditions throughout the study period indicating the unsuitability of E. coli to serve as an indicator for the presence of parasites and viruses in stream and marine waters. The prolonged persistence of pathogenic microorganisms in marine waters suggested that discharge of contamination into streams may present a serious environmental health risk.

Animals↗