Improvement Of Molecular Methods For Detection Of Pathogenic Escherichia Coli.

Diarrhea is one of the leading causes of illnesses and death among children in developing countries, where an estimated 1.3 billion episodes and 4 to 10 million deaths occur each year in children below 5 years of age. The common pathogens of diarrhea are diarrheagenic Escherichia coli (DEC), Group A rotavirus, Shigella spp, Salmonella spp, Campylobacter, and Vibrio cholerae. Microbiological insights including phenotypic and genotypic characterisation are valuable approaches with application in management and prevention of diarrheal outbreaks by E. coli. In the present study, the Random Amplified polymorphic DNA (RAPD) fingerprinting technique allowed genetic diversity assessment of 25 E. coli isolates. Six out of 20 arbitrary primers namely, OPAE 4, 9, 10, 11, 12 and 18 produced DNA fingerprinting patterns providing the discriminatory power and the display of the potential epidemiological and diagnostic markers. A highly significant finding from the DNA fingerprinting is the display of a predominant band at a size of 308 bp when arbitrar

Solid lipid nanoparticles preparation and characterization.

The presents study aimed to prepare and characterize Solid Lipid Nanoparticles (SLNs) from palm oil materials. Hydrogenated palm oil and lecithin incorporated with surfactant were mixed and formed by High Pressure Homogenization (HPH) at elevated temperature. Appropriate analytical methods are needed for the characterization of SLN. The use of several analytical techniques is a necessity such as particle size which determined using Photon Correlation Spectroscopy (PCS). The change of particle charge was studied by Zeta Potential (ZP) measurements, while the melting and recrystallization behavior characterized by Differential Scanning Calorimetry (DSC). Data showed physical stability of the formulation. In conclusion, the SLN presented here are well suited for several applications including drug delivery

Extraction of essential oil from Nigella sativa using supercritical carbon dioxide : study of antibacterial activity.

The antimicrobial activity of N. sativa essential oil obtained by supercritical fluid extraction by carbon dioxide was investigated against Gram Positive and Gram negative strains, isolated from clinical specimens. Best conditions for Black cumin oil extraction are obtained at 400 bar, 40°C and a solvent flow rate of 25 g min-1. The seed extracts were prepared by supercritical fluid extraction method. Filter paper discs impregnated with varying concentrations of N. sativa extract were tested by the disk diffusion method. Methicillin Resistant Staphylococcus Aureus (MRSA) ATCC strain (700968), E. coli ATCC strain (25922), E. coli 0157 ATCC strain (12799), Extended-Spectrum Beta-Lactamase (ESBL) Klebsiella pneumoniae ATCC strain (700603), Carbapenam Resistant acenitobacter Baumanii (CRAB) clinical strain and Vibrio cholerae 01 Ogawa and 0139 Bengal clinical strains were investigated. The inhibition zones of the Mueller Hinton agar in different extract concentrate ion showed that at 25 mg 20 μL-1, 50 mg 20 μL-1 and 100 mg 20 μL-1, the inhibition zones increased accordingly in S. aureu. However, N. sativa was found to be inactive against ESBL producers (E. coli and K. pneumoniae)

Tamoxifen drug loading solid lipid nanoparticles prepared by hot high pressure homogenization techniques.

As drug delivery systems Nanoparticulate widely investigated because of many advantages such as smaller size, controlled drug release potential, targeting ability, enhancement of therapeutic efficacy and reduction of toxicity. So, Solid Lipid Nanoparticles have recently received considerable attention as alternative drug delivery carrier. In this study Solid Lipid Nanoparticles (SLNs) containing tamoxifen, nonsteroidal antiestrogens have been loaded and to be used as breast cancer therapy, were prepared by hot High Pressure Homogenization techniques. Tamoxifen loaded SLNs seem to have dimensional properties useful for parenteral administration. Preliminary study of antiproliferative activity in vitro, carried out on MCF-7 cell line (human breast cancer cells), demonstrated that SLNs, containing tamoxifen showed an antitumoral activity comparable to free drug. Tamoxifen loaded SLNs seem to have dimensional properties useful for parenteral administration. SLN was characterized by Differential Scanning Calorimetery (DSC), Transmission Electron Microscopy (TEM), Zeta Potential and Particle Size. The results of characterization studies strongly support the potential application of Tamoxifen-loaded SLNs as a carrier system. The SLN presented here are well suited for certain drug delivery applications, particularly breast cancer therapy

Molecular characterization of horseshoe crab anti-lipopolysaccharide factor C-peptide for hybridization-based detection method of gram negative bacteria.

Recent advances in molecular techniques have revolutionized the detection of microorganism. The development of a molecular-based technique for detection of the three different targets of Enterbacteriacae was undertaken. Primer and probe were designed based on specific pepted of novel hemolymph protein of horseshoe crabs (Factor C anti-LPS) Tachypleus tridentatus that is believed to be involved in the binds to the lipopolysaccharide of Escherichia coli, Salmonella and Vibrio cholerae. The aim of our study the exploit part of cell wall polysaccharide in the development of improved detection method based on molecular approaches. In the gene detection assay, Lipopolysaccharide gene of Salmonella, V. cholera and E. coil were hybridized to anti-LPS factor gene found in the biolysate of the marine animals. The wzm and wzt genes encoding O-polysaccharide genes were amplified in these pathogens and the LPS factor C were amplified from the marine lysate. Development of a PCR-based technique for detection of the food-borne pathogens particularly Sa Salmonella, V. cholera and E. coil were achieved. Thus rapid, sensitive and reliable techniques for the detection of food-borne pathogens developed

Detection of diarrheagenic Escherichia coli isolated using molecular approaches.

Escherichia coli strains are among the major bacterial causes of diarrheal illness. There are now seven classes of diarrheagenic E. coli (DEC), namely enteropathogenic E. coli (EPEC), enterohaemorrhagic E. coli (EHEC), enteroinvasive E. coli (EIEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), diarrhea-associated hemolytic E. coli (DHEC) and Cytolethal Distending Toxin (CDT)-producing E. coli. Due to the need for costly and labor-intensive diagnostic procedures, identification of DEC is difficult at standard laboratories. Therefore, Polymerase Chain Reaction (PCR) or dot blot has been used for genetic detection of DEC of 25 E. coli isolates from different sources. Amplification of eae (277 bp), bfp (266 bp), stx1 (154 bp), EAST (94 bp), stx2 (698 bp) and elt (450 bp) genes of a single product in separate reactions was produced. PCR showed ability to amplify and detected genes of the most common important categories of diarrheagenic E. coli isolates of different sources, it is possible implementation of this technique to diagnosis water, foodborne outbreaks related to E. coli. Dots blot and sequence analysis used to confirm the results of PCR

Antibacterial activity of honey against methicillin-resistant Staphylococcus aureus.

For centuries honey had a valued place in traditional medicine, being used in the treatment of wounds and diseases of the gut. The scientific community has now rekindled interest in the therapeutic use of honey in modern medicine and a number of published reports support its use in certain medical conditions, including burns and wounds. The aim of the present study to the effectiveness of the antimicrobial activity of honey against Methicillin-Resistant Staphylococcus aureus (MRSA) and Methicillin-Sensitive Staphylococcus aureus (MSSA) isolates collected from various Malaysian hospitals. Thirty isolated of Staphylococcus aureus were found to be resistant to routinely used higher antibiotics. Using an agar incorporation technique the sensitivity of these strains to honey was tested by the method of minimum inhibitory concentration. All the tested strains of Staphylococcus aureus showed inhibition with honey at concentrations of 25 and 30%. The present study recommended that the multidrug-resistant Staphylococcus aureus infection particularly wound and burns honey may be useful for controlling infection

Novel molecular analysis for characterization of staphylococcal cassette chromosome in a methicillin-resistant staphylococcus aureus isolated from Malaysian hospital.

Methicillin-resistant Staphylococcus aureus strains have appeared in countries worldwide and continue to be one of the most common hospital pathogens and it has become increasingly prevalent in community-acquired infections and provided strong evidence for the independent origins of health care-associated Methicillin-resistant Staphylococcus aureus and community-acquired. It has been shown that methicillin-susceptible S. aureus strains become MRSA strains by the acquisition of a staphylococcal cassette chromosome mec element carrying the mecA gene, which is responsible for methicillin resistance and has become essential for the characterization of Staphylococcus aureus clones in epidemiological studies. The objective of this study to identify the staphylococcal cassette chromosome mec types of methicillin-resistant Staphylococcus aureus isolated from different Malaysian Hospitals. PCR amplification and sequencing analysis were performed to determine the SCCmec type of MRSA. The present research successfully established molecular characteristics of local MRSA contribute as initial database of these isolates in order to fully understand the epidemiology, microbiology and pathophysiology of these infections

In silico annotation of the genes involved in biosynthesis of lipopolysaccharide for Burkholderia pseudomallei.

Burkholderia pseudomallei is the causative agent of melioidosis, a serious disease of man and animals. The high mortality of B. pseudomallei infections may cause by lipopolysaccharides, an endotoxin. The biosynthesis of LPS is complex comprising three components, lipid A, core ohgosaccharide and O-specific antigen. In the current study, by using the available B. pseudomallei genome database provided by Wellcome. The study demonstrated that the bioinformatics comparative technique was able to annotate LPS genes in Burkholderia pseudomallei. By developing a simple and easy flow chart including the using of Artemis software, total of 44 putative ORFs involved in biosynthesis of lipopolysaccharide for B. pseudomallei and the genetic mapping for the ORFs have been successfully determined using bioinformatics and laboratory approach. It is about 95.7% of success for annotation based on the 46 genes that act as references. In near future, a suitable vaccine or antimicrobial may be developed by targeting the genes encoding the various components essential in LPS biosynthesis and survival of the pathogen

Development of a PCR primer and a marker band for detection of E. coli from various sources based on arbitrary primer set.

Although, PCR methods aimed on the detection of genes associated with the pathogenicity of Escherichia coli have been reported, tests allowing the direct identification of this serotype are rare. In this study the Random Amplified Polymorphic DNA (RAPD) fingerprinting technique allowed genetic diversity assessment of 25 E. coli isolates of various sources. A highly significant finding from the DNA fingerprinting is the display of a predominant band at a size of 308 bp when arbitrary OPAE-10 primer was used. After sequencing this fragment primer called secD was designed to be used as PCR primer. secD primer pairs was highly specific to detect all isolates including E. coli O157: H7