PCR-based construction and transformation of CodY gene deletion construct in streptococcus pneumoniae

Introduction: Streptococcus pneumoniae (S. pneumoniae) is a gram-positive diplococci belonging to the genus Streptococcus and it is a well-studied pathogenic bacterium. Pneumococcal diseases such as otitis media, pneumonia, sepsis and meningitis caused by pathogenic strains of S. pneumoniae still brought significant mortality and morbidity worldwide. The pathogenicity of S. pneumoniae is exerted by various virulence factors and one of it is the enzyme hyaluronate lyase. Hyaluronate lyase plays a major role in the invasive capability of S. pneumoniae. Its mechanism of action and crystallographic structure have been determinedbut its regulatory mechanism is still poorly understood. Drawing connections between the nutritional behaviour and invasive property of S. pneumoniae, CodY regulator is hypothesized as a potential hyaluronate lyase regulator. This work was aimed to construct CodY deficient mutant of S. pneumoniae to form foundational work for the study of CodY regulatory effect on hyaluronate lyase. Materials and method: A single gene-deletion method was chosen to create CodY mutant construct containing aphA-3 gene (encoding kanamycin resistance protein) replacement cassette by employing recombinant fusion PCR method. A single band DNA product at the expected size of 2,563 bp after recombinant fusion PCR was visualized on agarose gel electrophoresis. This indicated that the linear recombinant amplicon has been successfully constructed. The recombinant amplicon was transformed into S. pneumoniae cells of Malaysian local clinical isolate by employing the competence stimulating peptide (CSP-1) to enhance and induce natural competence. Results: No viable CodY mutant was evident post-transformation. The negative transformation result was postulated to be due to the essential nature of CodY regulator. Conclusion: This work can provide basis for recombinant fusion PCR method in designing and constructing single gene deletion construct. However, further work is needed to identify the regulatory agent of hyaluronate lyas

IDENTIFICATION AND CHARACTERIZATION OF PIGMENTED BACTERIA ISOLATED FROM MALAYSIAN SEAWATER

Purpose of study: Bacteria can naturally produce pigments that can be useful for various applications as they possess antimicrobial metabolites among other numerous benefits towards the human health. This study was carried out to identify the species of marine bacterial isolates PMA, PM3C1 and PM5C1 exhibiting yellow, orange and green colors respectively. Methodology: The current study is using Polymerase Chain Reaction (PCR) amplification and sequence analysis of their 16S rRNA gene. The stability of pigments extracted from the bacterial samples was also analyzed against different temperature and light conditions. Main Findings: Sequence alignment using BLAST revealed that the yellow, orange, and green-pigmented bacteria have 84% similarity with Staphylococcus aureus, 85% similarity with Exiguobacterium profundum and 95% similarity with Pseudomonas aeruginosa respectively. The green pigment showed major changes in color following exposure to sunlight and fluorescent light, and when incubated at 24°C and 50°C. Exposure to direct sunlight also results in the reduction of color for the yellow and orange extracts, while no effect was observed for both pigments under fluorescent light. Incubation at 50°C results in the reduction of the orange color, while the yellow pigment was observed to be unaffected suggesting its stability at high temperature. Implications: Natural pigments production can provide many advantages including reduction of pollution generation, ease of disposal and other benefits to the human health

Protein produced by Bacillus subtilis ATCC21332 in the presence of Cymbopogon flexuosus essential oil

Proteins levels produced by bacteria may be increased in stressful surroundings, such as in the presence of antibiotics. It appears that many antimicrobial agents or antibiotics, when used at low concentrations, have in common the ability to activate or repress gene transcription, which is distinct from their inhibitory effect. There have been comparatively few studies on the potential of antibiotics or natural compounds in nature as a specific chemical signal that can trigger a variety of biological functions. Therefore, this study was focusing on the effect of essential oil from Cymbopogon flexuosus in regulating proteins production by Bacillus subtilis ATCC21332. The Minimum Inhibition Concentration (MIC) of the C. flexuosus essential oil on B. subtilis was determined by using microdilution assay, resulting 1.76mg/ml. The bacteria cells were further exposed to the C. flexuosus essential oil at concentration of 0.01 MIC for 72 h. The proteins were then isolated and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins profile showed that a band with approximate size of 30 kDa was appeared for the treated bacteria with C. flexuosus essential oil. Thus, B. subtilis ATCC21332 in stressful condition with the presence of C. flexuosus essential oils at low concentration could induce the protein production. The isolated protein also showed antimicrobial activity against selected Gram-positive and Gram-negative bacteria

Thirdhand Tobacco Smoke: Emerging Evidence and Arguments for a Multidisciplinary Research Agenda

There is broad consensus regarding the health impact of tobacco use and secondhand smoke exposure, yet considerable ambiguity exists about the nature and consequences of thirdhand smoke (THS). We introduce definitions of THS and THS exposure and review recent findings about constituents, indoor sorption-desorption dynamics, and transformations of THS; distribution and persistence of THS in residential settings; implications for pathways of exposure; potential clinical significance and health effects; and behavioral and policy issues that affect and are affected by THS. Physical and chemical transformations of tobacco smoke pollutants take place over time scales ranging from seconds to months and include the creation of secondary pollutants that in some cases are more toxic (e.g., tobacco-specific nitrosamines). THS persists in real-world residential settings in the air, dust, and surfaces and is associated with elevated levels of nicotine on hands and cotinine in urine of nonsmokers residing in homes previously occupied by smokers. Much still needs to be learned about the chemistry, exposure, toxicology, health risks, and policy implications of THS. The existing evidence on THS provides strong support for pursuing a programmatic research agenda to close gaps in our current understanding of the chemistry, exposure, toxicology, and health effects of THS, as well as its behavioral, economic, and sociocultural considerations and consequences. Such a research agenda is necessary to illuminate the role of THS in existing and future tobacco control efforts to decrease smoking initiation and smoking levels, to increase cessation attempts and sustained cessation, and to reduce the cumulative effects of tobacco use on morbidity and mortality

Antimicrobial properties of local green seaweed (Caulerpa Racemosa) in Langkawi

The antimicrobial activity is widespread among marine seaweeds. However, antimicrobial defenses of marine micro algae are largely uncharacterized for locally available seaweed species. The aim of this study was to evaluate the antimicrobial activity of Caulerpa racemosa (‘Latok Lada’), an edible green seaweed species available in Langkawi Island, Kedah. Methanol extracts of the dried samples of C. racemosa was tested against Staphylococcus aureus, Escherichia coli, Pseudomonas aureginosa, Bacillus subtilis and MRSA (Methilcillin-resistant Staphylococcus aureus). Phosphate Buffer Saline (PBS) extracts of fresh sample of C. racemosa was tested against four different fungi; Candida albicans, Candida krusei, Candida tropicalis and Cryptococcus neoformans. C. racemosa showed good antibacterial activity against Gram-positive and Gram-negative bacteria tested, with a significant antimicrobial effect against B. subtilis (ZI=3-9mm), S. aureus (ZI=3-9mm), P. aureginosa (ZI=1-3mm) and MRSA (ZI=3-6mm). C. racemosa did not demonstrate any antifungal properties against any of the four fungi tested. Further studies needs to be performed on other types of bacteria and fungi as well as utilizing different extraction methods to further investigate the antimicrobial properties of C. racemosa. These preliminary results showed promising trend of antimicrobial properties in local seaweeds species collected in Langkawi coastline

Empirical Modeling of the Viscosity of Supercritical Carbon Dioxide Foam Fracturing Fluid under Different Downhole Conditions

High-quality supercritical CO2 (sCO2) foam as a fracturing fluid is considered ideal for fracturing shale gas reservoirs. The apparent viscosity of the fracturing fluid holds an important role and governs the efficiency of the fracturing process. In this study, the viscosity of sCO2 foam and its empirical correlations are presented as a function of temperature, pressure, and shear rate. A series of experiments were performed to investigate the effect of temperature, pressure, and shear rate on the apparent viscosity of sCO2 foam generated by a widely used mixed surfactant system. An advanced high pressure, high temperature (HPHT) foam rheometer was used to measure the apparent viscosity of the foam over a wide range of reservoir temperatures (40–120 °C), pressures (1000–2500 psi), and shear rates (10–500 s−1). A well-known power law model was modified to accommodate the individual and combined effect of temperature, pressure, and shear rate on the apparent viscosity of the foam. Flow indices of the power law were found to be a function of temperature, pressure, and shear rate. Nonlinear regression was also performed on the foam apparent viscosity data to develop these correlations. The newly developed correlations provide an accurate prediction of the foam’s apparent viscosity under different fracturing conditions. These correlations can be helpful for evaluating foam-fracturing efficiency by incorporating them into a fracturing simulator

Towards multifunctional cellulosic fabric: UV photo-reduction and in-situ synthesis of silver nanoparticles into cellulose fabrics

Herein, the highly multifunctional cotton fabric surfaces were designed with excellent coloration, UV-protection function, and antimicrobial activity. These multifunctional functions were developed by in-situ synthesis of silver nanoparticles (Ag NPs) into the cotton fabric surface using a simple green one-pot “UV-reduction” method. Cotton fabrics were pretreated with non-anionic detergent, immersed into alcoholic silver nitrate solution (concentration ranging from 100 to 500 ppm), squeezed to remove excess solution and then exposed to UV-irradiation (range 320–400 nm) for 1 h. The influence UV-irradiation on the thermal, chemical, optical and biological properties of the cotton fabric surface was discussed in details. The UV-irradiation promotes reducing of Ag+ ions and the cotton fabrics act as seed medium for Ag NPs formation by “heterogeneous nucleation”. Increasing Ag+ concentration (from 100 to 500 ppm) results in Ag NPs of particle size (distribution) of 50–100 nm. Interestingly, the Ag NPs exhibited different localized surface Plasmon resonance properties causing a coloration of the cotton fabrics with different color shades ranging from bright to dark brown with excellent color fastness properties. The treated cotton fabrics also show high protecting functions against UV-transmission (reduction of 65%) and Escherichia coli growth (99%). The side-effects of the UV-reduction process are further investigated

Influence of Sand Fines Transport Velocity on Erosion-Corrosion Phenomena of Carbon Steel 90-Degree Elbow

Erosion-corrosion is an ineluctable flow assurance problem confronted in hydrocarbon transportation and production systems. In this work, the effect of sand fines velocity on the erosion-corrosion behavior of AISI 1018 carbon steel long radius 90° elbows was experimentally and numerically investigated for liquid-solid flow conditions. Experiments were effectuated for sand fines of mean diameter 50 µm circulated in a flow loop with three different velocities (0.5, 1 and 2 m/s). To elucidate the erosion-corrosion mechanism and degradation rate, the material loss analysis, multilayer paint modeling (MPM) and microscopic imaging technique were employed, with computational fluid dynamics (CFD) and discrete phase modeling (DPM) also capacitating to evaluate the erosion distribution. It was perceived that increasing slurry velocity significantly changes the particle-wall impaction mechanism, leading to an increase in material degradation in the elbow bottom section up to 2 times in comparison to the low transport velocity. The erosion scars and pits development at the elbows internal surface was found to govern the wear mechanism in the carbon steel and made downstream section susceptible to erosion and corrosion. The material removal mechanisms were ascertained to change from cutting to pitting and plastic deformation with an increase of sand fines transportation velocity from 0.5 m/s to 2 m/s