BacLight Bacterial Viability Kit Research Articles

Antibacterial efficacy of silver nanoparticles, sodium hypochlorite, chlorhexidine, and hypochlorous acid on dentinal surfaces infected with Enterococcus faecalis.

The purpose of this study was to evaluate the antibacterial effect of silver nanoparticles (AgNPs) against Enterococcus faecalis and compare it with different irrigation solutions. This study was performed using 64 dentin blocks. E. faecalis suspension was dispensed to each sample and incubated under anaerobic conditions at 37°C throughout 21 days. After the inoculation period, the following solutions were added to each group and kept for 5 min: Group 1, 5.25% sodium hypochlorite (NaOCl); Group 2, 2.5% NaOCl; Group 3, 1% NaOCl; Group 4, 2% chlorhexidine (CHX); Group 5, 200 ppm hypochlorous acid (HOCl); and Group 6, AgNPs. The samples of positive control were treated with sterile saline. Biofilm viability assay was performed using the LIVE/DEAD BacLight Bacterial Viability Kit. Samples were examined using confocal laser scanning microscopy, respectively. There was no significant difference between the 5.25% NaOCl, 2.5% NaOCl, and 1%NaOCl groups (p > .05). However, these groups showed statistically higher antibacterial activity than the 2% CHX, 200 ppm HOCl, and AgNP groups. Also, 2% CHX showed greater percentage of dead cells compared with the AgNP and HOCl groups. While AgNPs group showed lower dead cell rate than all NaOCl groups and 2% CHX, it caused higher dead cells than 200 ppm HOCl group. The 200 ppm HOCl group showed the lowest percentage of dead cells (p < .05) Although the antibacterial effect of AgNPs is not as high as NaOCl and CHX, it has considerable bactericidal activity against E. faecalis and can be improved by further studies. RESEARCH HIGHLIGHTS: New antimicrobial approaches for root canal irrigation. Antimicrobial effect of silver nanoparticles against E. faecalis. Elimination of the biofilm layer for the success of endodontic treatment.

Intracellular presence of Helicobacter pylori antigen and genes within gastric and vaginal Candida.

Helicobacter pylori infections are generally acquired during childhood and affect half of the global population, but its transmission route remains unclear. It is reported that H. pylori can be internalized into Candida, but more evidence is needed for the internalization of H. pylori in human gastrointestinal Candida and vaginal Candida. Candida was isolated from vaginal discharge and gastric mucosa biopsies. We PCR-amplified and sequenced H. pylori-specific genes from Candida genomic DNA. Using optical and immunofluorescence microscopy, we identified and observed bacteria-like bodies (BLBs) in Candida isolates and subcultures. Intracellular H. pylori antigen were detected by immunofluorescence using Fluorescein isothiocyanate (FITC)-labeled anti-H. pylori IgG antibodies. Urease activity in H. pylori internalized by Candida was detected by inoculating with urea-based Sabouraud dextrose agar, which changed the agar color from yellow to pink, indicating urease activity. A total of 59 vaginal Candida and two gastric Candida strains were isolated from vaginal discharge and gastric mucosa. Twenty-three isolates were positive for H. pylori 16S rDNA, 12 were positive for cagA and 21 were positive for ureA. The BLBs could be observed in Candida cells, which were positive for H. pylori 16S rDNA, and were viable determined by the LIVE/DEAD BacLight Bacterial Viability kit. Fluorescein isothiocyanate (FITC)-conjugated antibodies could be reacted specifically with H. pylori antigen inside Candida cells by immunofluorescence. Finally, H. pylori-positive Candida remained positive for H. pylori 16S rDNA even after ten subcultures. Urease activity of H. pylori internalized by Candida was positive. In the form of BLBs, H. pylori can internalize into gastric Candida and even vaginal Candida, which might have great significance in its transmission and pathogenicity.

The effect of the enzymes trypsin and DNase I on the antimicrobial efficiency of root canal irrigation solutions.

The purpose of this study was to evaluate the antibacterial efficacy of using 2.5% NaOCl, 2% chlorhexidine (CHX), Irritrol, and chitosan-coated silver nanoparticles (AgCNPs) alone or in combination with deoxyribonuclease I (DNase I) and trypsin pre-enzyme applications in dentin samples contaminated with Enterococcus faecalis (E. faecalis) by CLSM. 144 dentin blocks with confirmed E. faecalis biofilm formation were divided randomly according to the irrigation protocol (n = 12): NaOCl, CHX, Irritrol, AgCNPs, trypsin before NaOCl, CHX, Irritrol, AgCNPs, and DNase I before NaOCl, CHX, Irritrol, AgCNPs. Dentin blocks were stained with the Live/Dead BacLight Bacterial Viability Kit and viewed with CLSM after irrigation applications. The percentage of dead and viable bacteria was calculated using ImageJ software on CLSM images. At a significance level of p < 0.05, the obtained data were analyzed using one-way Anova and post-hoc Tukey tests. In comparison with NaOCl, CHX had a higher percentage of dead bacteria, both when no pre-enzyme was applied and when DNase I was applied as a pre-enzyme (p < 0.05). There was no difference in the percentage of dead bacteria between the irrigation solutions when trypsin was applied as a pre-enzyme (p > 0.05). AgCNPs showed a higher percentage of dead bacteria when trypsin was applied as a pre-enzyme compared to other irrigation solutions (p < 0.05), while the pre-enzyme application did not affect the percentage of dead bacteria in NaOCl, CHX, and Irritrol (p > 0.05). No irrigation protocol tested was able to eliminate the E. faecalis biofilm. While the application of trypsin as a pre-enzyme improved the antimicrobial effect of AgCNPs, it did not make any difference over other irrigation solutions.

Antibacterial toner exhibits bactericidal effect against Cutibacterium acnes via keratin and sebum plug penetration.

Cutibacterium acnes is an opportunistic pathogen recognized as a contributing factor to acne vulgaris. The accumulation of keratin and sebum plugs in hair follicles facilitates C. acnes proliferation, leading to inflammatory acne. Although numerous antimicrobial cosmetic products for acne-prone skin are available, their efficacy is commonly evaluated against planktonic cells of C. acnes. Limited research has assessed the antimicrobial effects on microorganisms within keratin and sebum plugs. This study investigates whether an antibacterial toner can penetrate keratin and sebum plugs, exhibiting bactericidal effects against C. acnes. Scanning electron microscopy and next-generation sequencing analysis of the keratin and sebum plug suggest that C. acnes proliferate within the plug, predominantly in a biofilm-like morphology. To clarify the potential bactericidal effect of the antibacterial toner against C. acnes inside keratin and sebum plugs, we immersed the plugs in the toner, stained them with LIVE/DEAD BacLight Bacterial Viability Kit to visualize microorganism viability, and observed them using confocal laser scanning microscopy. Results indicate that most microorganisms in the plugs were killed by the antibacterial toner. To quantitatively evaluate the bactericidal efficacy of the toner against C. acnes within keratin and sebum, we immersed an artificial plug with inoculated C. acnes type strain and an isolate collected from acne-prone skin into the toner and obtained viable cell counts. The number of the type strain and the isolate inside the artificial plug decreased by over 2.2 log and 1.2 log, respectively, showing that the antibacterial toner exhibits bactericidal effects against C. acnes via keratin and sebum plug penetration.

Potential Effect of Giant Freshwater Prawn Shell Nano Chitosan in Inhibiting the Development of Streptococcus mutans and Streptococcus sanguinis Biofilm In Vitro.

An oral biofilm comprises a variety of bacteria including Streptococcus mutans and Streptococcus sanguinis that cause human infections, such as caries and periodontitis. Thus, biofilm management plays an important part in the prevention and treatment of oral diseases. Nano chitosan is a bioactive material that has antimicrobial activities. This in vitro study aimed to evaluate the effect of nano chitosan synthesized from giant freshwater prawn shells (PSNC) on S. mutans and S. sanguinis biofilm development. PSNC was prepared from the extracted chitosan of giant freshwater prawn (Macrobrachium rosenbergii) shells using the ionic gelation method. The effect of PSNC on S. mutans ATCC 25175 and S. sanguinis ATCC10556 biofilm formation was evaluated using the crystal violet assay. Both bacteria were inoculated in the presence of various concentrations (5, 2.5, and 1.25 mg/ml) of PSNC for 24 h and 48 h. Confocal laser scanning microscopy (CLSM) and scanning electron microscopy were performed to visualize and study the biofilm architectural features. The biofilms were stained with the BacLight Bacterial Viability Kit prior to CLSM observation to monitor the viability of the biofilm. The results showed that PSNC exposure for 24 h and 48 h inhibited the formation of S. mutans and S. sanguinis biofilms. The biofilm formation inhibition percentage increased with an increase in the PSNC concentration (p < 0.05). The highest inhibitory activity was shown at 5 mg/ml PSNC (p < 0.05). Those findings were confirmed by the subsequent findings using the CLSM and SEM analyses. The biofilm architecture was strongly disrupted upon treatment with PSNC. After exposure to 5 mg/ml PSNC, the number of bacteria significantly decreased. The remaining bacteria were seen as individual cells, showing damaged cells. In conclusion, PSNC inhibits the development of S. mutans and S. sanguinis biofilm in vitro, indicating the potential of PSNC in clinical application for oral bacterial infection, prevention, and treatment.

Estimating the optimal efflux inhibitor concentration of carvacrol as a function of the bacterial physiological state.

Our aim was to find the optimal efflux inhibitor concentration of a natural component, carvacrol, as a function of the physiological state of Escherichia coli. Using fluorescence-based measurements with two strains of E. coli, the effect of carvacrol was assessed at 17 sub-inhibitory concentrations, at which the bacterial efflux mechanism was compromised. The efficacy of carvacrol, as an efflux inhibitor, was compared to synthetic inhibitors and we found carvacrol the most efficient one. We considered the accumulation of Ethidium Bromide (EtBr) as a proxy for drugs spreading in the cell, thus measuring the efflux activity indirectly. The change in membrane integrity caused by the exposure to carvacrol was monitored using the LIVE/DEAD BacLight Bacterial Viability kit. To find the optimal inhibitory concentration of carvacrol, we used predictive microbiology methods. This optimum varied with the bacterial physiological state, as non-growing cultures were less susceptible to the effect of carvacrol than growing cultures were. Moreover, we point out, for the first time, that the efflux-mediated resistance of untreated cultures was also stronger in the non-growing than in the growing phase at population level.

Truncated analog Brevinin2-CE-N26V5K: Revelation the Augmentation of Antimicrobial Activity.

Brevinin2-CE (B2CE), a natural peptide containing 37 amino acids, was first isolated from the skin secretions of the Chinese forest frog Rana chensinensis. B2CE shows good antibacterial activity. In this study, a series of B2CE analogs with differences in cationicity, α-helicity, hydrophobicity and amphipathic properties were designed through chain-length deletion and amino acid substitution. The most potent, nontoxic analog, B2CE-N26V5K, was identified by examination of its antibacterial activity, hemolytic activity, and stability under physiological conditions. The increased cationicity, hydrophobicity and more obvious hydrophilic and hydrophobic surface of B2CE-N26-N16WA18KG23K did not improve the antibacterial activity but increased the hemolytic activity of this modified peptide. The helicity might promote antibacterial activity for brevinin-2 peptides, as the 15-aa analogs with lower helicity show decreased potency against different test bacteria (approximately 2- to 72-fold) compared to B2CE-N26V5K. Additionally, the results indicated that the "Rana box" does not affect the antimicrobial activity of brevinin-2 peptides, as B2CE, B2CE-nonDS and B2CE-C31-37S have similar strong inhibitory effects on both gram-positive and gram-negative bacteria. However, the "Rana box" does affect the hemolytic activity, as the HC50 values of the 3 peptides range from 25 ~ 130 µM. Furthermore, B2CE-N26V5K caused obvious morphological alterations of the bacterial surfaces, as shown by atomic force microscopy. Additionally, B2CE-N26V5K exhibited strong membrane-disrupting activity when examined using the LIVE/DEAD Bac Light Bacterial Viability Kit. Thus, the antibacterial effect of B2CE-N26V5K on gram-negative and gram-positive bacteria may be caused by cell membrane attack. In conclusion, the excellent candidate B2CE-N26V5K was obtained and has application prospects as a novel anti-infective agent.

Influence of Needle Design and Irrigant Flow Rate on the Removal of Enterococcus faecalis Biofilms In Vitro.

This study aimed to evaluate the influence of needle design and irrigant flow rate on the removal of Enterococcus faecalis mature biofilms during sodium hypochlorite irrigation. Forty-eight single-rooted human teeth were instrumented (ProTaper F3), autoclaved and inoculated with Enterococcus faecalis to establish a two-week-old biofilm. E. faecalis biofilms were treated with Sodium hypochlorite that was injected in the root canals using three types of needles (NaviTip, ProRinse, IrriFlex). For the IrriFlex needle, one, two, or four bars of pressure was applied to the irrigating solution to increase flow rates. Bacteria were labeled with the LIVE/DEAD BacLight Bacterial Viability kit, and viability was assessed by flow cytometry (FCM). Results were statistically analyzed using one-way ANOVA and Tukey multiple comparison intervals (α = 0.05). Bacterial viability was significantly reduced after sodium hypochlorite passive irrigation but the number of viable bacteria retrieved from root canal specimens irrigated with the Pro-Rinse needle was significantly higher compared to NaviTip and IrriFlex needles (p < 0.05). When the irrigant flow rate was increased, the viability of bacterial biofilms was significantly reduced compared to passive irrigation using the IrriFlex needle (p < 0.05). Applying higher flow rates during irrigation using the IrriFlex needle did not further reduce bacterial viability.

Evaluation of sonic, ultrasonic, and laser irrigation activation systems to eliminate bacteria from the dentinal tubules of the root canal system.

Aiming to kill bacteria in dentin tubules of infected dental pulp cavities, we evaluated the effects of sodium hypochlorite (NaOCl) solution agitated by different irrigation protocols, i.e., conventional needle irrigation (CNI), passive ultrasonic irrigation (PUI), the EDDY tip, and the neodymium-doped yttrium aluminum perovskite (Nd:YAP) laser. The EDDY achieved good antibacterial effects as passive ultrasonic irrigation in the coronal and middle thirds. Nd:YAP laser irradiation and PUI were effective in the apical third of the root canal. To evaluate the ability of NaOCl agitated by high-frequency sonic irrigation-EDDY, PUI, and Nd:YAP laser-to kill bacteria in infected root canal walls and if the associated temperature increases at the root surface during application. Infected root canal models were established, and roots were randomly divided into six groups: negative control, positive control, CNI, PUI, sonic agitation with EDDY, and Nd:YAP laser groups. After irrigation, the teeth were split and stained using the LIVE/DEAD BacLight Bacterial Viability Kit. Dead bacteria depth was evaluated by a confocal laser scanning microscopy and the temperature at the root surface was assessed using a thermal imaging camera during the irrigation process. In the coronal and middle thirds of the root canal, PUI and EDDY had stronger antibacterial effects than CNI (p<0.05); in the apical third, the antibacterial effects of PUI and Nd:YAP laser-activated irrigation were better than CNI (p<0.05). The maximum change in temperature was significantly greater during continuous Nd:YAP laser application compared with the other methods, but intermittent irrigation helped lessening this trend. NaOCl agitated by EDDY tip and PUI exhibited a similar bacteria elimination effect in the coronal and middle root canal. Nd:YAP laser was effective in the apical third and intermittent irrigation reduced its thermal impact.

Distribution of bacterial concentration and viability in atmospheric aerosols under various weather conditions in the coastal region of China

Airborne bacteria have an important role in atmospheric processes and human health. However, there is still little information on the transmission and distribution of bacteria via the airborne route. To characterize the impact of foggy, haze, haze-fog (HF) and dust days on the concentration and viability of bacteria in atmospheric aerosols, size-segregated bioaerosol samples were collected in the Qingdao coastal region from March 2018 to February 2019. The total airborne microbes and viable/non-viable bacteria in the bioaerosol samples were measured using an epifluorescence microscope after staining with DAPI (4′, 6-diamidino-2-phenylindole) and a LIVE/DEAD® BacLight Bacterial Viability Kit. The average concentrations of total airborne microbes on haze and dust days were 6.75 × 105 and 1.03 × 106 cells/m3, respectively, which increased by a factor of 1.3 and 2.5 (on average), respectively, relative to those on sunny days. The concentrations of non-viable bacteria on haze and dust days increased by a factor of 1.2 and 3.6 (on average), respectively, relative to those on sunny days. In contrast, the concentrations of viable bacteria on foggy and HF days were 7.13 × 103 and 5.74 × 103 cells/m3, decreases of 38% and 50%, respectively, compared with those on sunny days. Foggy, haze, dust and HF days had a significant effect on the trend of the seasonal variation in the total airborne microbes and non-viable bacteria. Bacterial viability was 20.8% on sunny days and significantly higher than the 14.1% on foggy days, 11.2% on haze days, 8.6% during the HF phenomenon and 6.1% on dust days, indicating that special weather is harmful to some bacterial species. Correlation analysis showed that the factors that influenced the bacterial concentration and viability depended on different weather conditions. The main influential factors were temperature, NO2 and SO2 concentrations on haze days, and temperature, particulate matter (PM2.5) and NO2 concentrations on foggy days. The median size of particles containing viable bacteria was 1.94 μm on sunny days and decreased to 1.88 μm and 1.74 μm on foggy and haze days, respectively, but increased to 2.18 μm and 2.37 μm on dust and HF days, respectively.

Antibacterial efficacy of copper-added chitosan nanoparticles: a confocal laser scanning microscopy analysis.

The aim of this study was to evaluate the antibacterial efficacy of copper added chitosan nanoparticles (CU-CNPs) as an irrigation solution with different irrigants in terms of eliminating Enterococcus Faecalis (E. faecalis) from the root canals. Fifty mandibular premolar teeth were prepared and infected with E. faecalis for 21days. After the incubation period, samples were randomly divided into a control group irrigated with distilled water and 4 experimental groups (n = 10) irrigated with as follows, %6 NaOCl, %6 NaOCl + %9 editronate (HEBP), Chitosan nanoparticles (CNPs), and CU-CNPs. To calculate the proportion of dead E. faecalis cell volume, stained using LIVE/DEAD BacLight Bacterial Viability Kit and were scanned using confocal laser scanning microscope (CLSM). All the irrigation solutions significantly (P < .05) killed the bacteria in the canal, except for the control group. CU-CNPs solution killed the highest (P < .05) number of bacteria compared with the other experimental groups. No significant difference was found between CNPs, NaOCl + HEBP, and NaOCl in terms of antibacterial activity. CU-CNPs solution was exhibited higher antibacterial efficacy against E. faecalis.

Nutrient Deficiency Promotes the Entry of Helicobacter pylori Cells into Candida Yeast Cells.

Simple SummaryHelicobacter pylori is a pathogenic bacterium which causes several gastric and extra-gastric pathologies in humans. This pathogen is capable of entering eukaryotic cells of humans or of other species, including yeasts of the genus Candida. These yeasts are resistant to stressing environmental conditions (such as pH changes or scarce nutrients) which threaten the viability of H. pylori. Therefore, Candida yeasts may harbor this bacterium when subjected to stressing conditions or become transmission vehicles for it. Further research is required to establish the stressing environmental factors triggering the harboring of H. pylori within yeasts. The present work evaluated if deficiency or absence of the necessary nutrients favors the endosymbiotic relationship between these two microorganisms, facilitating the viability of the bacterium. In fact, a deficiency of nutrients increased the harboring of viable H. pylori cells within the yeast cells. On the contrary, in the complete absence of nutrients, the presence of intra-yeast bacteria was reduced. Therefore, yeast cells may contribute to the subsistence of this pathogenic bacterium when subjected to nutrient deficiency until it may infect an appropriate host, such as humans. The present work may also contribute, with further studies, to elucidate the transmission routes used by the pathogen H. pylori to infect its hosts.Helicobacter pylori, a Gram-negative bacterium, has as a natural niche the human gastric epithelium. This pathogen has been reported to enter into Candida yeast cells; however, factors triggering this endosymbiotic relationship remain unknown. The aim of this work was to evaluate in vitro if variations in nutrient concentration in the cultured medium trigger the internalization of H. pylori within Candida cells. We used H. pylori–Candida co-cultures in Brucella broth supplemented with 1%, 5% or 20% fetal bovine serum or in saline solution. Intra-yeast bacteria-like bodies (BLBs) were observed using optical microscopy, while intra-yeast BLBs were identified as H. pylori using FISH and PCR techniques. Intra-yeast H. pylori (BLBs) viability was confirmed using the LIVE/DEAD BacLight Bacterial Viability kit. Intra-yeast H. pylori was present in all combinations of bacteria–yeast strains co-cultured. However, the percentages of yeast cells harboring bacteria (Y-BLBs) varied according to nutrient concentrations and also were strain-dependent. In conclusion, reduced nutrients stresses H. pylori, promoting its entry into Candida cells. The starvation of both H. pylori and Candida strains reduced the percentages of Y-BLBs, suggesting that starving yeast cells may be less capable of harboring stressed H. pylori cells. Moreover, the endosymbiotic relationship between H. pylori and Candida is dependent on the strains co-cultured.

Bactericidal Effect of a Novel Alkaline EDTA Root Canal Cleaning Solution.

Objectives In this study, we aimed to evaluate the bactericidal effect and cytotoxicity of an ethylenediaminetetra-acetic acid (EDTA)-based root canal irrigant solution capable of efficiently removing both the organic matter and the smear layer. We prepared a strong alkaline EDTA (AE) solution with an acid buffer capacity similar to that of sodium hypochlorite. Materials and Methods AE was used at concentrations of 1%, 2%, and 3%. The bactericidal effect of AE on Enterococcus faecalis was evaluated by determining the colony number and biofilm removal rate. Biofilms were visualized using a Live/Dead BacLight bacterial viability kit. Viability of AE-treated cells were determined using a CCK-8 cell counting assay. Statistical Analysis One-way analysis of variance followed by a Dunnett’s multiple comparison test were used for comparisons among groups. Results Significant reduction in cell viability and biofilm formation were observed in case of 3% and 2% AE. AE exerted bactericidal effects in a concentration-dependent manner. Damage of normal human fibroblasts was not observed at any of the AE concentrations. Conclusions Our results suggest that the AE solution could be used as an effective canal irrigant for the removal of bacterial biofilm.

Nanometric Deposition of Fluoride Ions on Titanium Alloys and its Influence on In Vitro Bacterial Adhesion and Viability

Bacterial colonization plays a key role on the pathogenesis of peri-implantitis and may be influenced by titanium surface topography. The aim of this in vitro study was to evaluate the influence of titanium topography after fluoride ions deposition in the bacterial colonization. Machined (M), double acid-etched (DE) and double acid-etched surface with fluoride ions deposition (Nano F-) were analyzed by scanning electron microscopy, contact angle and roughness (Ra). Streptococcus mutans viability was quantified by Live/Dead Baclight bacterial viability kit. The mean Ra/contact angle values were 0.20 μm/69.13°, 0.53 μm /92.82° and 0.56 μm/94.33° for M, DE and Nano F-, respectively. M surface presented significantly lower live bacterial counts when compared to the Nano F- surface (p=0.007). The dead bacteria count was higher on the Nano F- surface (p=0.001) than on the M and DE surfaces. Crystalline deposition of fluoride ions (Nano F-) promoted an increase in dead bacteria on the tested titanium surface.

Advantages and limitations of microtiter biofilm assays in the model of antibiofilm activity of Klebsiella phage KP34 and its depolymerase

One of the potential antibiofilm strategies is to use lytic phages and phage-derived polysaccharide depolymerases. The idea is to uncover bacteria embedded in the biofilm matrix making them accessible and vulnerable to antibacterials and the immune system. Here we present the antibiofilm efficiency of lytic phage KP34 equipped with virion-associated capsule degrading enzyme (depolymerase) and its recombinant depolymerase KP34p57, depolymerase-non-bearing phage KP15, and ciprofloxacin, separately and in combination, using a multidrug-resistant K. pneumoniae biofilm model. The most effective antibiofilm agents were (1) phage KP34 alone or in combination with ciprofloxacin/phage KP15, and (2) depolymerase KP34p57 with phage KP15 and ciprofloxacin. Secondly, applying the commonly used biofilm microtiter assays: (1) colony count, (2) LIVE/DEAD BacLight Bacterial Viability Kit, and (3) crystal violet (CV) biofilm staining, we unravelled the main advantages and limitations of the above methods in antibiofilm testing. The diverse mode of action of selected antimicrobials strongly influenced obtained results, including a false positive enlargement of biofilm mass (CV staining) while applying polysaccharide degrading agents. We suggest that to get a proper picture of antimicrobials’ effectiveness, multiple examination methods should be used and the results must be read considering the principle of each technique and the antibacterial mechanism.

The Effect of Pulpotomy Base Material on Bacterial Penetration and Proliferation for Pulpotomized Primary Molar Teeth: A Confocal Laser Scanning Microscopy Study.

Introduction: the study aimed to evaluate Enterococcus Faecalis colonization in the pulp chamber in pulpotomized extracted human teeth filled by different pulpotomy base materials (PBMs), using confocal laser scanning microscopy (CLSM). Study design: Cavity preparations were made in 70 extracted primary molars. The pulp chambers were filled using either Intermediate restorative material (IRM), Mineral Trioxide Aggregate (MTA) or Glass ionomer (GI). Twenty-five teeth served controls. The specimens were sterilized, and coronally filled with bacterial suspension for 21 days. The specimens were cut through the furcation area, stained using LIVE/DEAD BacLight Bacterial Viability Kit and evaluated using CLSM. Results: The extent of fluorescent staining was larger in the GI group, compared to the IRM and MTA groups, and larger in the IRM group compared to the MTA group (P<0.05). The minimal and maximal bacterial penetration depths into the dentinal tubules were 55 and 695&mu;m, respectively (mean 310&mu;m), without differences between the materials (GI, IRM, MTA, p>0.05). The ratio of live bacteria to dead bacteria within the evaluated areas was higher in the GI group compared to the IRM and the MTA groups, and higher in the IRM group compared to the MTA group (P<0.05). There were no differences between the mesial, distal and apical parts in any of the evaluations (p>0.05). Conclusions: bacteria colonize the interface between the PBM and dentin and penetrate deeply into the dentinal tubules. The extent and the vitality of the colonized bacteria may be affected by the type of PBM.

Antimicrobial photodynamic therapy against multidrug-resistant Acinetobacter baumannii clinical isolates mediated by aloe-emodin: An in vitro study.

Antimicrobial photodynamic therapy (aPDT) has received considerable attention as an emerging and promising approach for treating superficial infections. The aim of this study was to investigate aPDT mediated by aloe emodin (AE), a natural compound isolated from Aloe vera and Rheum palmatum, against multidrug-resistant (MDR) Acinetobacter baumannii clinical isolates in vitro. The photodynamic inactivation (PDI) efficacies of AE on three MDR A. baumannii isolates were assessed by colony forming units (CFU) assay. The aPDT effects mediated by AE on the genomic DNA, membrane integrity, and cellular structure of MDR A. baumannii were also investigated. AE showed no obvious dark toxicity, but inactivated the MDR A. baumannii isolates in an AE concentration and light energy dose-dependent manner. Agarose gel electrophoresis and LIVE/DEAD BacLight Bacterial Viability kit assay indicated that the genomic DNA and membrane integrity of MDR A. baumannii were damaged after AE-mediated aPDT treatment. Transmission electron microscopy (TEM) images demonstrated that AE-mediated aPDT could induce rupture of bacterial cell wall and membrane, and condensation of ribosomes in the cytoplasm. The results obtained in this study suggested that AE could serve as a potential antibacterial photosensitizer in the treatment of superficial infections caused by MDR A. baumannii.

Induction and Recovery of the Viable but Nonculturable State of Hop-Resistance Lactobacillus brevis.

Lactobacillus brevis is a major hop-resistance bacterium which poses significant challenge for the brewing industry, mainly due to the difficulty or incapability in detection by routine culturing methodology and its beer spoilage ability.This study aimed at investigating the VBNC state of a hop-resistance strain, L. brevis BM-LB13908. The culturable, total and viable numbers of L. brevis cells were calculated by MRS agar plate counting, acridine orange direct count (AODC) method and Live/Dead BacLight bacterial viability kit with fluorescence microscope. VBNC formation was induced by 189 ± 5.7 days under low-temperature storage or 27 ± 1.2 subcultures by continuous passage in beer, and VBNC cells induced by both strategies were recovered by adding catalase. In addition, insignificant difference in beer-spoilage ability was found in 3 states of L. brevis, including logarithmic growing, VBNC and recovered cells. This is the first study on the formation of VBNC state for L. brevis and beer-spoilage ability of both VBNC and recovered cells, which indicate L. brevis strain could cause beer spoilage without being detected by routine methodologies. The results derived from this study may support further study on L. brevis and other hop-resistance bacteria, and guidance on beer spoilage prevention and control, such as improvement for brewers on the microbiological quality control by using the improved culture method with catalase supplementation.

Susceptibility of Streptococcus suis to liamocins from Aureobasidium pullulans

Abstract Streptococcus suis is a major pathogen of swine that is also considered an emerging zoonotic pathogen of humans. Because of increasing antibiotic resistance in S. suis, new antimicrobial agents are being sought. Liamocins are unique polyol lipids produced by certain strains of the fungus Aureobasidium pullulans. Liamocins recently have been found to have antibacterial activity with specificity for Streptococcus species. However, liamocins have not previously been tested for inhibition of S. suis. Ten strains of S. suis isolated from domestic pigs were tested for susceptibility to liamocins by the broth microdilution method. All 10 were susceptible, with MICs ranging from 39 to 78 μg/mL. Viability assays using the LIVE/DEAD BacLight Bacterial Viability Kit indicated that liamocins are bactericidal to S. suis. Cell-leakage assays showed that cells treated with liamocins leaked small molecular mass nucleic acids into the extracellular matrix, suggesting that liamocins act via disruption of the cell membrane. Liamocins thus show potential for development as a specific antibacterial to reduce colonization of S. suis in swine.

Investigation of live and dead status of airborne bacteria using UVAPS with LIVE/DEAD® BacLight Kit

Although the amount of allergens and toxins being originated from viable bacteria is higher than that from dead bacteria, monitoring concentrations of dead bacteria as well as live bacteria is an important issue related to human health. The ultraviolet aerodynamic particle sizer (UVAPS) is an instrument to measure the size distribution of only viable bioaerosols. Therefore, the main objective of this study is to detect percentages of dead as well as live airborne bacteria by using a LIVE/DEAD BacLight Bacterial Viability Kit (L7012, Molecular probes, USA) together with UVAPS. Two different bacteria species, Escherichia coli (E.coli) and Micrococcus luteus (M. luteus), were used for tests. Percentages of fluorescence emitted from these bacteria were compared with those from standard PSL and dust particles. The increased percentages for dead E. coli and dead M. luteus were 41% and 75%, respectively, while this kit did not show any effective influence on fluorescence percentages of polystyrene latex and dust particles. Field test was carried out in various indoor environments to investigate percentages of live and dead airborne bacteria.