BacLightTM Bacterial Viability Kit Research Articles

Assessment of biodegradation and toxicity of alternative plasticizer di(2-ethylhexyl) terephthalate: Impacts on microbial biofilms, metabolism, and reactive oxygen species-mediated stress response

Although di(2-ethylhexyl) terephthalate (DOTP) is being widely adopted as a non-phthalate plasticizer, existing research primarily focuses on human and rat toxicity. This leaves a significant gap in our understanding of their impact on microbial communities. This study assessed the biodegradation and toxicity of DOTP on microbes, focusing on its impact on biofilms and microbial metabolism using Rhodococcus ruber as a representative bacterial strain. DOTP is commonly found in mass fractions between 0.6 and 20% v/v in various soft plastic products. This study used polyvinyl chloride films (PVC) with varying DOTP concentrations (range 1–10% v/v) as a surface for analysis of biofilm growth. Cell viability and bacterial stress responses were tested using LIVE/DEAD™ BacLight™ Bacterial Viability Kit and by the detection of reactive oxygen species using CellROX™ Green Reagent, respectively. An increase in the volume of dead cells (in the plastisphere biofilm) was observed with increasing DOTP concentrations in experiments using PVC films, indicating the potential negative impact of DOTP on microbial communities. Even at a relatively low concentration of DOTP (1%), signs of stress in the microbes were noticed, while concentrations above 5% compromised their ability to survive. This research provides a new understanding of the environmental impacts of alternative plasticizers, prompting the need for additional research into their wider effects on both the environment and human health.

Distribution of Bacterial Concentration and Viability in Atmospheric Bioaerosols Under Different Weather Conditions in the Coastal Region of Qingdao

Bacteria are the most abundant microorganisms in atmospheric bioaerosols, widely distributed in the environment. Bioaerosol samples were collected using the FA-1 impact sampler from October 2013 to January 2021 in the coastal city of Qingdao, and samples stained with a BacLightTM bacterial viability kit were used to measure the concentrations of viable and non-viable bacteria with an epifluorescence microscope. The viable and non-viable bacteria in bioaerosols were characterized during different seasons, with particular attention paid to the distribution characteristics of bacteria on foggy, hazy, and dust days. The results showed that the mean concentrations of total bacteria were (1.06±0.68)×105 cells·m-3 in Qingdao during the sampling period, and those of viable and non-viable bacteria were (8.20±4.88)×103 cells·m-3and (9.74±6.72)×104 cells·m-3, respectively. The seasonal concentrations of non-viable bacteria were the highest in spring and winter and the lowest in summer, whereas that of viable bacteria was highest in spring, followed by those in summer and autumn, and the lowest in winter. Atmospheric bacterial concentrations fluctuated with by month, and total bacteria presented a similar variation pattern with that of non-viable bacteria. The monthly average concentration of non-viable bacteria showed the highest value in March during the spring and the lowest in June during the summer in 2021, whereas the highest value for viable bacteria occurred in May during the spring in 2021 and the lowest in December during the winter in 2020. Viable bacteria concentrations were significantly positively correlated with temperature and significantly negatively correlated with NO2, SO2, and CO. Non-viable bacteria were significantly positively correlated with PM. The bacteria in bioaerosol particles showed bimodal, unimodal, and skewed size distributions, varying with season and month. Under different weather conditions, the concentration of non-viable bacteria on dusty days was significantly higher than that on sunny, foggy, or hazy days, but the bacteria viability was as low as 6.85% due to long-distance transport. Anthropogenic pollution resulted in the lowest viability of bacteria at 4.10% on hazy days, whereas the highest viability in bacteria was 16.26% on foggy days due to high humidity. The size distribution of bacteria in bioaerosol particles under different pollution days showed a bimodal distribution, and the peak size depended on the weather type.

Abundance of Live and Dead Bacteriopsammon Inhabiting Sandy Ecosystems of Recreational Marine Beaches of the Southern Baltic Sea

The study was carried out on four non-tidal sandy marine beaches located on the Polish part of the southern Baltic Sea coast. We applied a LIVE/DEAD™ BacLight™ Bacterial Viability Kit (Invitrogen™) method to determine the abundance of live and dead bacteriopsammon. Live psammon bacteria cells constituted 31–53% of the total number of bacteria inhabiting sand of the studied beaches. Abundance of live and dead psammon bacteria generally differed along the horizontal profile in all beaches. The maximum density of bacteria was noted in the dune and the middle part of the beach (dry zones) and the minimum in wet zones, i.e., under seawater surface and at the swash zone. Generally along the vertical profile, the highest numbers of two studied bacterial groups were noted in the surface sand layer, while with increasing sediment depth their numbers significantly decreased. The abundance of live and dead bacteria showed a distinct seasonal variation.

Rapid Detection of Escherichia coli Antibiotic Susceptibility Using Live/Dead Spectrometry for Lytic Agents.

Antibiotic resistance is a serious threat to public health. The empiric use of the wrong antibiotic occurs due to urgency in treatment combined with slow, culture-based diagnostic techniques. Inappropriate antibiotic choice can promote the development of antibiotic resistance. We investigated live/dead spectrometry using a fluorimeter (Optrode) as a rapid alternative to culture-based techniques through application of the LIVE/DEAD® BacLightTM Bacterial Viability Kit. Killing was detected by the Optrode in near real-time when Escherichia coli was treated with lytic antibiotics—ampicillin and polymyxin B—and stained with SYTO 9 and/or propidium iodide. Antibiotic concentration, bacterial growth phase, and treatment time used affected the efficacy of this detection method. Quantification methods of the lethal action and inhibitory action of the non-lytic antibiotics, ciprofloxacin and chloramphenicol, respectively, remain to be elucidated.

Penicillin susceptibility in biofilms formed by Streptococcus agalactiae

Background: Streptococcus agalactiae (Group B Streptococcus, GBS) is a major agent of human and animal infections. It is believed that biofilms play a major role in GBS colonization and infection despite of host type. Although GBS remains mostly susceptible to beta-lactams (including penicillin) in the planktonic form, it is still unknown how sessile forms such as biofilms respond to these antibiotics. Methods and materials: Here we evaluated the effect of penicillin (in concentration 2000 times higher than the planktonic MIC) on biofilm formation by two GBS strains (one belonging to serotype Ia/ST1002, which is commonly associated with asymptomatic colonization and another belonging to serotype III/ST17, which is the clone most associated with neonatal meningitis) that were previously characterized as strong biofilm producers and fully susceptible to penicillin by conventional testing (MIC=0.023 mg/l). Biofilms with and without penicillin were stained with LIVE, GermanyAD™ BacLight™ Bacterial Viability Kit and visualized under confocal microscopy. Images were analyzed with ImageJ and COMSTAT. Results: In the absence of penicillin, different biofilm architectures were observed for each strain: longer chains of cocci and presence of well designed channels were seen for serotype Ia/ST1002. In the presence of penicillin, overall biomasses were reduced 20% (serotype III/ST17) to 70% (serotype Ia/ST1002), and dead cells increased from nearly 2% (in the absence of penicillin) to nearly 25% in both strains. In addition, cocci chains and channels were not seen in the biofilm of strain serotype Ia/ST1002 under penicillin treatment. GBS strains were also differentiated by pilus type (PI-2a in serotype Ia/ST1002 and PI-2b in serotype III/ST17) and percentage of polysaccharides in biofilm matrix (much higher in serotype III/ST17 strain). Conclusion: Results indicate that biofilm architecture in GBS may vary according to the strain and may have implications on antimicrobial therapy of GBS infections since certain subpopulations in sessile forms can be resistant to high concentrations of penicillin even if the planktonic counterparts are fully susceptible to this drug.

Biofunctionalization of Microgroove Surfaces with Antibacterial Nanocoatings.

Objectives To investigate the physical properties of the modified microgroove (MG) and antibacterial nanocoated surfaces. In addition, the biological interactions of the modified surfaces with human gingival fibroblasts (HGFs) and the antibacterial activity of the surfaces against Porphyromonas gingivalis were studied. Methods The titanium nitride (TiN) and silver (Ag) coatings were deposited onto the smooth and MG surfaces using magnetron sputtering. A smooth titanium surface (Ti-S) was used as the control. The physicochemical properties including surface morphology, roughness, and hydrophilicity were characterized using scanning electron microscopy, atomic force microscopy, and an optical contact angle analyzer. The “contact guidance” morphology was assessed using confocal laser scanning microscopy. Cell proliferation was analyzed using the Cell Counting Kit-8 assay. The expression level of the main focal adhesion-related structural protein vinculin was compared using quantitative reverse transcription PCR and Western blotting. The antibacterial activity against P. gingivalis was evaluated using the LIVE/DEAD BacLight™ Bacterial Viability Kit. Results The Ag and TiN antibacterial nanocoatings were successfully deposited onto the smooth and MG surfaces using magnetron sputtering technology. TiN coating on a grooved surface (TiN-MG) resulted in less nanoroughness and greater surface hydrophilicity than Ag coating on a smooth surface (Ag-S), which was more hydrophobic. Cell proliferation and expression of vinculin were higher on the TiN-MG surface than on the Ag-coated surfaces. Ag-coated surfaces showed the strongest antibacterial activity, followed by TiN-coated surfaces. Conclusion Nano-Ag coating resulted in good antimicrobial activity; however, the biocompatibility was questionable. TiN nanocoating on an MG surface showed antibacterial properties with an optimal biocompatibility and maintained the “contact guidance” effects for HGFs.

Formation and Control of the Viable but Non-culturable State of Foodborne Pathogen Escherichia coli O157:H7.

As a common foodborne pathogen, Escherichia coli O157:H7 produces toxins causing serious diseases. However, traditional methods failed in detecting E. coli O157:H7 cells in the viable but non-culturable (VBNC) state, which poses a threat to food safety. This study aimed at investigating the formation, control, and detection of the VBNC state of E. coli O157:H7. Three factors including medium, salt, and acid concentrations were selected as a single variation. Orthogonal experiments were designed with three factors and four levels, and 16 experimental schemes were used. The formation of the VBNC state was examined by agar plate counting and LIVE/DEAD® BacLightTM bacterial viability kit with fluorescence microscopy. According to the effects of environmental conditions on the formation of the VBNC state of E. coli O157:H7, the inhibition on VBNC state formation was investigated. In addition, E. coli in the VBNC state in food samples (crystal cake) was detected by propidium monoazide–polymerase chain reaction (PMA-PCR) assays. Acetic acid concentration showed the most impact on VBNC formation of E. coli O157:H7, followed by medium and salt concentration. The addition of 1.0% acetic acid could directly kill E. coli O157:H7 and eliminate its VBNC formation. In crystal cake, 25, 50, or 100% medium with 1.0% acetic acid could inhibit VBNC state formation and kill E. coli O157:H7 within 3 days. The VBNC cell number was reduced by adding 1.0% acetic acid. PMA-PCR assay could be used to detect E. coli VBNC cells in crystal cake with detection limit at 104 CFU/ml. The understanding on the inducing and inhibitory conditions for the VBNC state of E. coli O157:H7 in a typical food system, as well as the development of an efficient VBNC cell detection method might aid in the control of VBNC E. coli O157:H7 cells in the food industry.

Antibacterial effect of chitosan and its derivative on Enterococcus faecalis associated with endodontic infection.

Chitosan and its derivatives have been increasingly used for bacteriostasis. To date, the effect of chitosan and N-(2-hydroxyl) propyl-3-trimethyl ammonium chitosan chloride (HTCC) on Enterococcus faecalis (E. faecalis) associated with endodontic infection has remained to be determined. Chitosan and HTCC were serially diluted with double-distilled water (DDW) or PBS at concentrations of 20-2,500 µg/ml. Various strains of E. faecalis (American Type Tissue Collection no. 29212, as well as isolated strains P25RC and P52Sa) in plankton were adjusted to an optical density at 600 nm of 0.10 and treated with chitosan or HTCC. A colony-forming unit assay was used to determine the concentration of residual bacteria after treatment. Furthermore, E. faecalis biofilms were cultured on coverslips and treated with chitosan or HTCC. The coverslips were rinsed, stained using Live/dead® BacLight™ bacterial viability kit and observed under an inverted fluorescence microscope. In addition, biofilms on dentine blocks were prepared and observed under a scanning electron microscope. MC3T3-E1 pre-osteoblasts were seeded on 96-well plates and treated with chitosan or HTCC at various concentrations. The cytotoxicity of chitosan and HTCC on MC3T3-E1 pre-osteoblasts was detected using a Cell Counting Kit-8 assay after 24, 48 and 72 h of treatment. The results revealed that the final minimum bactericidal concentrations (MBC) of chitosan and HTCC dissolved in DDW were 70 and 140 µg/ml, respectively. Chitosan and HTCC in DDW exerted a significantly greater antibacterial effect as compared with that in PBS (P<0.05). At the MBC, chitosan and HTCC in DDW, but particularly chitosan, had a significant antibacterial effect on E. faecalis biofilm. Chitosan exhibited no cytotoxicity to MC3T3-E1 pre-osteoblasts at a concentration of <625 µg/ml, while HTCC inhibited the proliferation of the cells in the concentration range of 39-10,000 µg/ml. In conclusion, chitosan and HTCC exhibited prominent antibacterial properties on E. faecalis in the planktonic state and as a biofilm via charge interaction, indicating their potential for application in root canal disinfection and fillings.

In vitro antibacterial effects of non-thermal atmospheric plasma irradiation on Staphylococcus pseudintermedius and Pseudomonas aeruginosa.

In the last decade, atmospheric plasma has been used to treating bacterial infections in humans due to its bactericidal effects; however, its efficacy in dogs is unclear. This study evaluated the in vitro bactericidal efficacy of atmospheric plasma on Staphylococcus pseudinter- medius and Pseudomonas aeruginosa, two of the most important bacterial agents isolated from canine pyodermas. Three isolates each of S. pseudintermedius and P. aeruginosa obtained from dogs with pyoderma were subjected to atmospheric plasma. The isolates from the control group were not exposed to plasma, while those from the treatment groups were exposed to plasma for 15 (7.5 J/cm2), 30 (15 J/cm2), 60 (30 J/cm2), or 90 (45 J/cm2) seconds. After each treatment, a reduction in colony formation was observed. Bacterial viability was evaluated using the LIVE/ DEAD® BacLight™ Bacterial Viability Kit. The antibacterial effects were evaluated with Image J software and significance was assessed statistically in comparison to the control group. The bactericidal effect of atmospheric plasma against both bacteria increased significantly in a time-dependent manner. These results demonstrate the bactericidal capacity of atmospheric plasma, and suggest that it could serve as an alternative treatment method for canine pyoderma. Further studies are needed to evaluate the safety and efficacy of atmospheric plasma in dogs.

Concentration, viability and size distribution of bacteria in atmospheric bioaerosols under different types of pollution

Bacteria are important components of bioaerosols with the potential to influence human health and atmospheric dynamics. However, information on the concentrations and influencing factors of viable bacteria is poorly understood. In this study, size-segregated bioaerosol samples were collected from Aug. 2017 to Feb. 2018 in the coastal region of Qingdao, China. The total microbes and viable/non-viable bacteria in the samples were measured using an epifluorescence microscope after staining with the DAPI (4′, 6-diamidino-2-phenylindole) and LIVE/DEAD® BacLight™ Bacterial Viability Kit, respectively. The concentrations of non-viable bacteria increased when the air quality index (AQI) increased from <50 to 300, with the proportion of non-viable bacteria to total microbes increasing from (11.1 ± 12.0)% at an AQI of <50 to (18.4 ± 14.7)% at an AQI of >201. However, the concentrations of viable bacteria decreased from (2.12 ± 2.04) × 104 cells·m−3 to (9.00 ± 1.72) × 103 cells·m−3 when the AQI increased from <50 to 150. The ratio of viable bacteria to total bacteria (viability) decreased from (31.0 ± 14.7)% at 0 < AQI<50 to (8.6 ± 1.0)% at 101 < AQI<150 and then increased to (9.6 ± 5.3)% at an AQI of 201–300. The results indicated that the bacterial viability decreased when air pollution occurred and increased again when pollution became severe. The mean size distribution of non-viable bacteria exhibited a bimodal distribution pattern at an AQI<50 with two peaks at 2.1–3.3 μm and >7.0 μm, while the viable bacteria had two peaks at 1.1–2.1 μm and >7 μm. When the AQI increased from 101 to 300, the size distribution of viable/non-viable bacteria varied with an increased proportion of fine particles. The multiple linear regression analysis results verified that the AQI and PM10 had important effects on the concentrations of non-viable bacteria. These results highlight impacts of air pollution on viable/non-viable bacteria and the interactions between complex environmental factors and bacteria interactions, improving our understanding of bioaerosols under air pollution conditions.

Antibacterial photodynamic therapy mediated by 5-aminolevulinic acid on methicillin-resistant Staphylococcus aureus.

The emergence of drug-resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), has brought great difficulties to clinical treatment. Antibacterial photodynamic therapy (aPDT) is a new non-antibiotic treatment strategy for a variety of drug-resistant bacteria. However, there are few studies on the antimicrobial mechanism of a PDT mediated by 5-aminolevulinic acid (ALA-PDT) for MRSA. In this study, we observed the bactericidal effect of ALA-PDT on MRSA. We found that ALA-PDT had the strongest bactericidal effect when ALA was at 0.05 mM, and the bactericidal activity of aPDT increased with the increase of light dose. MRSA was almost completely eliminated at 0.05 mM and 384 Jcm-2. In addition, the bactericidal morphology was also observed under a fluorescence microscope using a LIVE/DEAD® BacLight™ Bacterial Viability Kit and an electron microscope. It was also found that proteins and DNA were damaged by ALA-PDT. Finally, the transcription level of the specific gene of MRSA, nuc, was decreased by 0.74-fold (P < 0.05) after ALA-PDT treatment by qRT-PCR analysis. The findings suggest that ALA-PDT can effectively inhibit MRSA by damaging cell membrane, cytoplasm, proteins and nucleic acid.

Optimisation of the Protocol for the LIVE/DEAD® BacLightTM Bacterial Viability Kit for Rapid Determination of Bacterial Load.

Rapid antimicrobial susceptibility testing is needed to reduce prescription of inappropriate antibiotics. A rapid alternative to standard culture-based testing is to determine reductions in cell viability using the LIVE/DEAD® BacLightTM Bacterial Viability Kit. We optimised the kit protocol for this application, focusing on simplifying the process by minimising the steps involved and on determining the optimal analytical parameters for fluorescence measurements from the dyes SYTO 9 and propidium iodide (PI). We demonstrate that for our experimental system, the intensity of emissions should be integrated from 505–515 nm for SYTO 9 and 600–610 nm for PI, and the proportion of live cells calculated from a new dye ratio formula, termed the adjusted dye ratio. We show that the pre-staining washing step is not necessary if a non-fluorescent growth media is used; however, staining must be done for each sampling as prolonged exposure to the dyes negatively impacts cell viability. The optimised methodology was able to reproducibly detect reductions in culture viability when the proportion of live cells in a sample of 1 × 108 cells/ml fell below ∼50% live in a media that supports the growth required for detecting antibiotic killing. Finally, we show that the interaction of fluorescence emission spectra from SYTO 9 and PI stained Escherichia coli cells is influenced by the proportion of dead cells in a sample. The excitation of PI by SYTO 9 was found to occur in populations containing sufficient numbers of dead cells (>25%), whereas in populations with low numbers of dead cells the dye interaction was additive in regard to red emissions, indicating that these dye interactions may offer another dimension to live/dead analysis. Fluorescence measurements from samples established according to the optimised protocol can be taken using a flow cytometer, spectrofluorometer, microplate reader, and the Optrode, a fibre-based spectroscopic system developed at the University of Auckland.

Shining a light on antibiotic selection: optimised live/dead fluorescence spectrometry for rapid antimicrobial susceptibility testing

Antibiotic resistance is a serious threat to public health. The empiric use of the wrong antibiotic occurs due to urgency in treatment combined with slow, culture-based diagnostic techniques. Inappropriate antibiotic choice can promote the development of antibiotic resistance. We propose to use live/dead spectrometry as a rapid alternative to culture-based techniques through application of the LIVE/DEAD® BacLightTM Bacterial Viability Kit. We have developed a spectroscopic device (Optrode) to measure fluorescence from SYTO 9 and propidium iodide stained cells that can be used to enumerate the bacterial load. We propose a procedure using the Optrode that will take bacteria in a clinical sample, challenge with a panel of antibiotics, and measure live/dead ratios to determine the best bactericidal choice. Using calibration data we optimised the live/dead spectrometry protocol outlined in the kit instructions, improving upon media selection for growth and staining, and analytical parameters. We applied the optimised methodology to detect live and dead Escherichia coli in populations challenged with ampicillin. Killing was detected by the Optrode in near real-time when E. coli was treated with ampicillin and stained with SYTO 9 and/or PI. Following on from the promising results generated with ampicillin, live/dead spectrometry of ampicillin challenged cells was characterised in terms of antibiotic concentration, growth phase, and susceptibility to treatment for each treatment time. The generated data demonstrated that reliable detection of E. coli knockdown by ampicillin using live/dead spectrometry requires log phase cells challenged with a suitable concentration for a particular treatment time.

Antibacterial effects of bio-inspired nanostructured materials

ABSTRACTSeveral properties of bio-inspired surfaces like chemical composition, surface topography, surface hydrophilicity and even surface charge could influence bacterial adhesion to implant materials. Therefore, a nanostructured surface is being investigated to avoid bacterial colonization by their physico-mechanical and chemical aspects.Both smooth and rough-surfaced titanium (PT, SLA) and zirconia (M and ZLA) surfaces were used as controls. Titanium SLA was modified by two-step-etching to create nanostructured surface. Antibacterial properties of the materials were tested by adhesion of Porphyromonas gingivalis (ATCC 33277). The vitality of bacteria was assessed by Live/Dead BacLight™ Bacterial Viability Kit or by conventional culturing on Columbia blood agar.Conventional culturing revealed reduction of bacteria on nanostructured titanium (5.27±0.8 x 104 CFU/mm2) in comparison to rough-surfaced control materials (ZLA 6.16±4.86 x 104 and SLA 1.53±0.75 x 105 CFU/mm2). However, smooth-surfaced control materials (M 2.25±0.84 x 104 and PT 6.63±5.77 x 103 CFU/mm2) showed similar results to the nanostructured material. Live/dead staining demonstrated the antimicrobial efficacy of the nanostructured material revealing reduction of vital bacteria population up to 70%. This effect was not observed on the control materials (bacterial vitality ≥95%).In conclusion, nanostructured titanium surface shows a reduction of vital bacteria. Therefore, bio-inspired nanostructures can modify the bacteria–titanium interaction.

Stress response of Escherichia coli induced by surface streamer discharge in humid air

Inactivation of Escherichia coli by means of surface streamer discharge has been investigated to obtain new insights into the key mechanisms involved, with a particular emphasis placed on the microbial response to plasma-induced stress. The surface streamer discharge was produced in coplanar dielectric barrier discharge electrode geometry, and was driven by an amplitude-modulated ac high voltage in humid synthetic air at atmospheric pressure. The response to plasma-induced stress was evaluated by using conventional cultivation, sublethal injury and resazurin assay and the LIVE/DEAD® BacLight™ Bacterial Viability kit. Compared to conventional cultivation, the LIVE/DEAD® test labels bacteria with damaged membranes, while resazurin assay tracks their metabolic activity. Our results clearly demonstrate that the treated bacteria partly lost their ability to grow properly, i.e. they became injured and culturable, or even viable but nonculturable (VBNC). The ability to develop colonies could have been lost due to damage of the bacterial membrane. Damage of the membranes was mainly caused by the lipid peroxidation, evidencing the key role of oxygen reactive species, in particular ozone. We conclude that the conventional cultivation method overestimates the decontamination efficiency of various plasma sources, and must therefore be complemented by alternative techniques capable of resolving viable but nonculturable bacteria.

Chamaecyparis obtusa Suppresses Virulence Genes in Streptococcus mutans

Chamaecyparis obtusa (C. obtusa) is known to have antimicrobial effects and has been used as a medicinal plant and in forest bathing. This study aimed to evaluate the anticariogenic activity of essential oil of C. obtusa on Streptococcus mutans, which is one of the most important bacterial causes of dental caries and dental biofilm formation. Essential oil from C. obtusa was extracted, and its effect on bacterial growth, acid production, and biofilm formation was evaluated. C. obtusa essential oil exhibited concentration-dependent inhibition of bacterial growth over 0.025 mg/mL, with 99% inhibition at a concentration of 0.2 mg/mL. The bacterial biofilm formation and acid production were also significantly inhibited at the concentration greater than 0.025 mg/mL. The result of LIVE/DEAD® BacLight™ Bacterial Viability Kit showed a concentration-dependent bactericidal effect on S. mutans and almost all bacteria were dead over 0.8 mg/mL. Real-time PCR analysis showed that gene expression of some virulence factors such as brpA, gbpB, gtfC, and gtfD was also inhibited. In GC and GC-MS analysis, the major components were found to be α-terpinene (40.60%), bornyl acetate (12.45%), α-pinene (11.38%), β-pinene (7.22%), β-phellandrene (3.45%), and α-terpinolene (3.40%). These results show that C. obtusa essential oil has anticariogenic effect on S. mutans.

Testing a dual-fluorescence assay to monitor the viability of filamentous cyanobacteria

Filamentous cyanobacteria are currently being engineered to produce long-chain organic compounds, including 3rd generation biofuels. Because of their filamentous morphology, standard methods to quantify viability (e.g., plate counts) are not possible. This study investigated a dual-fluorescence assay based upon the LIVE/DEAD® BacLight™ Bacterial Viability Kit to quantify the percent viability of filamentous cyanobacteria using a microplate reader in a high throughput 96-well plate format. The manufacturer's protocol calls for an optical density normalization step to equalize the numbers of viable and non-viable cells used to generate calibration curves. Unfortunately, the isopropanol treatment used to generate non-viable cells released a blue pigment that altered absorbance readings of the non-viable cell solution, resulting in an inaccurate calibration curve. Thus we omitted this optical density normalization step, and carefully divided cell cultures into two equal fractions before the isopropanol treatment. While the resulting calibration curves had relatively high correlation coefficients, their use in various experiments resulted in viability estimates ranging from below 0% to far above 100%. We traced this to the apparent inaccuracy of the propidium iodide (PI) dye that was to stain only non-viable cells. Through further analysis via microplate reader, as well as confocal and wide-field epi-fluorescence microscopy, we observed non-specific binding of PI in viable filamentous cyanobacteria. While PI will not work for filamentous cyanobacteria, it is possible that other fluorochrome dyes could be used to selectively stain non-viable cells. This will be essential in future studies for screening mutants and optimizing photobioreactor system performance for filamentous cyanobacteria.

Multifunctional cationic polymer decorated and drug intercalated layered silicate (NLS) for early gastric cancer prevention

A multifunctional compound that can prevent early gastric cancer is produced by intercalating 3.20% and 1.64% of 5-FU into the interlayer of montmorillonite (MMT) and attapulgite (At), respectively. A low molecular weight cationic polymer, polyethylenimine (PEI1200), is incorporated into the surface of the 5-FU-MMT and 5-FU-At to form the multifunctional layered silicate (NLS). The chemical structure and surface morphology of the NLS are characterized and the model drug of 5-FU is intercalated into the MMT and At. The cell viability determined by the MTT assay on the BGC-823 cell lines show that over 80% of the cells are live under the experimental conditions. The PEI-5-FU-MMT and PEI-5-FU-At can carry the report gene to the BGC-823 and COS-7 cell lines efficiently. Western blotting assay shows that the pTrail protein of the BGC-823 cell lines treated with PEI-5-FU-MMT/pTrail and PEI-5-FU-At/pTrail is up-regulated, whereas the cFLIP protein is down-regulated at 48 h compared to free 5-FU, PEI1200, MMT, and At, providing evidence that the NLS can increase the sensitivity of pTrail gene and improve the effects of pTrail gene therapy. Moreover, the Helicobacter pylori (HP) bacteria are adsorbed and immobilized efficiently on the surface of the NLS according to the LIVE/DEAD® BacLight™ Bacterial Viability Kit in the confocal fluorescence analysis. The histochemical analyses provide evidence that NLS/pTrail can prevent early gastric mucosa effectively.

Antimicrobial/anti-biofilm activity of expired blood platelets and their released products

Although platelets are not part of the classical immune system, they have many features that indicate their role in the anti-infective host defense. They come into interactions with microorganisms, which results in co-aggregation and co-adhesion or destruction of the microbes due to the action of antimicrobial peptides released from platelets.The aim of this study was to evaluate the killing effect of platelets against planktonic and biofilm cultures of Staphylococcus aureus and to test their synergy with antibiotics. S. aureus ATCC 29213; platelet rich plasma (1-3 days post shelf life). Evaluation of bactericidal activity of platelets or their lysates against planktonic cultures of S. aureus--CFU calculation after 4- and 24-hour co-incubation. Assessment of S. aureus biofilm viability under the influence of platelets--Live/Dead® BacLight™ Bacterial Viability Kit. Determination of minimum inhibitory concentrations (MICs) (oxacillin, vancomycin, linezolid) and estimation of the synergistic action of antibiotics and platelet lysates--a gradient-diffusion test strip. Microbicidal activity of "expired" platelets and their lysates has been shown as a significant reduction in the population of staphylococci in their planktonic cultures by 56-87% and a decrease in metabolic activity of biofilm formation by 7-38%. These activities were enhanced after activation with ADP. Platelet lysates showed a synergistic effect with β-lactam antibiotic (oxacillin) and glycopeptide (vancomycin) but not with oxazolidinone (linezolid). In summary, platelets even after the medical expiry date are still a good source of antimicrobial low molecular weight proteins (PMPs). Testing of bacterial resistance to PMPs may be advisable as a predictive indicator of susceptibility to treatment of infections such as infective endocarditis and other local infections of biofilm nature.

Applicability of LIVE/DEAD BacLight Stain with Glutaraldehyde Fixation for the Measurement of Bacterial Cell Concentration and Viability in the Air

ABSTRACTData on the concentration and viability of microorganisms in the atmosphere provide crucial information with regard to their dispersion, roles in environmental changes, and effects on ecosystems. In this study, we investigated the application of the LIVE/DEAD® BacLight™ Bacterial Viability Kit (BacLight stain) with regard to the enumeration of viable and non-viable bacterial cells in the air, with the 4',6-diamidino-2-phenylindole (DAPI) stain used as the control of total cell counts. Two cultured bacterial strains isolated from an air sample, Bacillus subtilis and Micrococcus sp., were used in laboratory experiments. The results of BacLight staining agreed well with those of DAPI staining in detecting total cell counts, and the detection efficiency of Bacillus subtilis was 76–112%. Bacterial viability (number ratio of viable cells to total cells) showed consistency among repeated experiments with the same sample replicates, indicating the high confidence of counting viable cells, as well as total cells with the stain. Fixation of samples with glutaraldehyde prevented fluorescence bleaching at the exposure to fluorescence and increased detection accuracy. Application of the BacLight stain with and without fixation to air samples that were collected with a bio-sampler at an urban site proved the effectiveness of this approach in determining the cell concentration and viability of airborne bacteria at a 1-hour time resolution. A combination of BacLight staining and glutaraldehyde fixation treatment, in parallel with experiments without this treatment in field measurements, is proposed to ensure the detection accuracy. The method described here is able to measure the concentration and viability of bacterial cells in the air, and their variation with weather conditions