Antibacterial efficacy of non-thermal atmospheric plasma against Streptococcus mutans biofilm grown on the surfaces of restorative resin composites

The aim of this study was to evaluate the antimicrobial efficacy of non-thermal atmospheric plasma (NTAP) against Streptococcus mutans biofilms. Resin discs were fabricated, wet-polished, UV sterilized, and immersed in water for monomer extraction (37 °C, 24 h). Biofilms of bioluminescent S. mutans strain JM10 was grown on resin discs in anaerobic conditions for (37 °C, 24 h). Discs were divided into seven groups: control (CON), 2% chlorhexidine (CHX), only argon gas 150 s (ARG) and four NTAP treatments (30 s, 90 s, 120 s, 150 s). NTAP was applied using a plasma jet device. After treatment, biofilms were analyzed through the counting of viable colonies (CFU), bioluminescence assay (BL), scanning electron microscopy (SEM), and polymerase chain reaction (PCR). All NTAP-treated biofilm yielded a significant CFU reduction when compared to ARG and CON. BL values showed that NTAP treatment for 90 s, 120 s or 150 s resulted in statistically significantly lower metabolic activity when compared to the other groups. CHX displayed the lowest means of CFU and BL. SEM showed significant morphological changes in NTAP-treated biofilm. PCR indicated damage to the DNA structure after NTAP treatment. NTAP treatment was effective in lowering the viability and metabolism of S. mutans in a time-dependent manner, suggesting its use as an intraoral surface-decontamination strategy.

The ATP bioluminescence assay: a new application and optimization for viability testing in the parasitic nematode Haemonchus contortus

The parasitic gastrointestinal nematode Haemonchus contortus causes serious economic losses to agriculture due to infection and disease in small ruminant livestock. The development of new therapies requires appropriate viability testing, with methods nowadays relying on larval motility or development using procedures that involve microscopy. None of the existing biochemical methods, however, are performed in adults, the target stage of the anthelmintic compounds. Here we present a new test for the viability of H. contortus adults and exsheathed third-stage larvae which is based on a bioluminescent assay of ATP content normalized to total protein concentration measured using bicinchoninic acid. All the procedure steps were optimized to achieve maximal sensitivity and robustness. This novel method can be used as a complementary assay for the phenotypic screening of new compounds with potential antinematode activity in exsheathed third-stage larvae and in adult males. Additionally, it might be used for the detection of drug-resistant isolates.

Photocatalytic Degradation of Rhodamine B in Heterogeneous and Homogeneous Systems

This study focuses on the photocatalytic degradation of Rhodamine B (RhB) in heterogeneous and homogeneous photo-Fenton reactions. In the heterogeneous system, iron(II) doped copper ferrite CuII(x)FeII(1-x)FeIII2O4 nanoparticles (NPs) prepared in our previous work were employed as potential catalysts. The photodegradation of RhB was carried out in a quartz cuvette located in a diode array spectrometer. The experimental conditions such as pH, NPs dosage and H2O2 dosage with regard to the photocatalytic degradation of RhB were optimized to be 7.5, 500 mg/L and 8.9x10-2 mol/L, respectively. In addition, visible light-induced photodegradation of RhB was also carried out by using \ch{H2O2} over a wide pH range in the absence of heterogeneous photocatalysts. It was observed that the reaction rate significantly increased above pH 10, resulting in a faster rate of degradation of RhB, which may be attributed to the deprotonation of hydrogen peroxide. Furthermore, the potential antibacterial property of such catalysts against the Gram-negative bacterium Vibrio fischeri in a bioluminescence assay yielded inhibition activities of more than 60% in all cases.

Evaluation of PhageDX Salmonella Assay for Salmonella Detection in Hydroponic Curly Lettuce

Lettuce is one of the most consumed leafy vegetables worldwide and has been involved in multiple foodborne outbreaks. Salmonella is one of the most prevalent etiological agents of foodborne disease (FBD) in lettuces, and its detection may take several days depending on the chosen method. This study evaluates a new rapid method that uses recombinant bacteriophages to detect Salmonella in hydroponic curly lettuce. First, the ability of the assay to detect six Salmonella serovars at three different concentrations (1, 10, and 100 CFU/well) was tested. Second, the detection of Salmonella was tested in lettuces using a cocktail of the same Salmonella serovars and concentrations after a 7 h enrichment. The results of these experiments showed that the detection limit was dependent on the serovar tested. Most serovars were detected in only 2 h when the concentration was 100 CFU/well. Salmonella was detected in 9 h (7 h enrichment + 2 h bioluminescence assay) in all lettuce samples with 10 CFU/25 g or more. Salmonella detection was not influenced by natural microbiota of lettuces. This study demonstrated that the phage assay was sensitive and faster than other detection methods, indicating that it is a better alternative for Salmonella detection on lettuces.

A New Ultrasensitive Bioluminescence-Based Method for Assaying Monoacylglycerol Lipase

A novel bioluminescent Monoacylglycerol lipase (MAGL) substrate 6-O-arachidonoylluciferin, a D-luciferin derivative, was synthesized, physico-chemically characterized, and used as highly sensitive substrate for MAGL in an assay developed for this purpose. We present here a new method based on the enzymatic cleavage of arachidonic acid with luciferin release using human Monoacylglycerol lipase (hMAGL) followed by its reaction with a chimeric luciferase, PLG2, to produce bioluminescence. Enzymatic cleavage of the new substrate by MAGL was demonstrated, and kinetic constants Km and Vmax were determined. 6-O-arachidonoylluciferin has proved to be a highly sensitive substrate for MAGL. The bioluminescence assay (LOD 90 pM, LOQ 300 pM) is much more sensitive and should suffer fewer biological interferences in cells lysate applications than typical fluorometric methods. The assay was validated for the identification and characterization of MAGL modulators using the well-known MAGL inhibitor JZL184. The use of PLG2 displaying distinct bioluminescence color and kinetics may offer a highly desirable opportunity to extend the range of applications to cell-based assays.

ATP Bioluminescence for Rapid and Selective Detection of Bacteria and Yeasts in Wine

Microbial contamination may represent a loss of money for wine producers as several defects can arise due to a microorganism’s growth during storage. The aim of this study was to implement a bioluminescence assay protocol to rapidly and simultaneously detect bacteria and yeasts in wines. Different wines samples were deliberately contaminated with bacteria and yeasts at different concentrations and filtered through two serial filters with decreasing mesh to separate bacteria and yeasts. These were resuscitated over 24 h on selective liquid media and analyzed by bioluminescence assay. ATP measurements discriminated the presence of yeasts and bacteria in artificially contaminated wine samples down to 50 CFU/L of yeasts and 1000 CFU/L of bacteria. The developed protocol allowed to detect, rapidly (24 h) and simultaneously, bacteria and yeasts in different types of wines. This would be of great interest for industries, for which an early detection and discrimination of microbial contaminants would help in the decision-making process.

Study of Stem Cells Influence on Cardiac Cells Cultured with a Cyanide-P-Trifluoromethoxyphenylhydrazone in Organ-on-a-Chip System

Regenerative medicine and stem cells could prove to be an effective solution to the problem of treating heart failure caused by ischemic heart disease. However, further studies on the understanding of the processes which occur during the regeneration of damaged tissue are needed. Microfluidic systems, which provide conditions similar to in vivo, could be useful tools for the development of new therapies using stem cells. We investigated how mesenchymal stem cells (MSCs) affect the metabolic activity of cardiac cells (rat cardiomyoblasts and human cardiomyocytes) incubated with a potent uncoupler of mitochondrial oxidative phosphorylation under microfluidic conditions. A cyanide p-trifluoromethoxyphenylhydrazone (FCCP) was used to mimic disfunctions of mitochondria of cardiac cells. The study was performed in a microfluidic system integrated with nanofiber mats made of poly-l-lactid acid (PLLA) or polyurethane (PU). The microsystem geometry allows four different cell cultures to be conducted under different conditions (which we called: normal, abnormal—as both a mono- and co-culture). Metabolic activity of the cells, based on the bioluminescence assay, was assessed in the culture’s performed in the microsystem. It was proved that stem cells increased metabolic activity of cardiac cells maintained with FCCP.

ATP Bioluminescence for Assessing the Efficacy of the Manual Cleaning Procedure during the Reprocessing of Reusable Surgical Instruments

Achieving sterilization by adopting proper practices is essential to ensure that surgical instruments do not transmit microorganisms to patients. As the effectiveness of sterilization mandates effective cleaning, it is necessary to verify the success of cleaning procedures. In this study, we used the adenosine triphosphate (ATP) bioluminescence method for assessing the efficacy of the manual cleaning procedure during the reprocessing of reusable surgical instruments. The ATP bioluminescence assay was performed on 140 surgical instruments of 12 different types, both before being cleaned (baseline) and after each of the cleaning procedures (i.e., decontamination, manual washing, drying, and visual inspection). For each instrument, two swabs were used as follows: one to sample the entire surface (test point 1) and the other to sample the most difficult part of the surface to clean (test point 2). Overall, for each type of instrument, there was a decrease in contamination ranging from 99.6 to >99.9% (log reduction from 2.40 to 3.76). Thus, in order to standardize the assessment of cleanliness, it may be useful to introduce the bioluminescence method into the daily routine or, at least, at regular time intervals as a complementary check combined with visual inspection. This would allow real-time verification of the achievement of an adequate level of cleanliness.