Extraction and Characterization of Organ Components of the Malaysian Sea Cucumber Holothuria leucospilota Yielded Bioactives Exhibiting Diverse Properties.

The aim of the present study was to extract and characterize bioactive components from separate body organs of Holothuria leucospilota. Preliminary qualitative assessment of the crude extracts was positive for phenols, terpenoids, carbohydrates, flavonoids, saponins, glycosides, cardiac glycosides, steroids, phlobatannins, and tannins in all body organs evaluated. Phenolics were the most abundant group of bioactives accounting for approximately 80%. The extraction solvent mixtures that yielded most compounds evaluated were methanol/acetone (3:1, v:v) and methanol/distilled water (3:1, v:v). In other analyses, GC-MS data revealed diverse metabolic and biologically active compounds, where those in high concentrations included 2-Pentanone, 4-hydroxy-4-methyl- among the ketones; phenol- 2,4-bis(1,1-dimethylethyl)-, a phenol group; and 2-Chlorooctane, a hydrocarbon. Among FA and their methyl/ethyl esters, n-hexadecanoic acid, 5,8,11,14-eicosatetraenoic acid ethyl ester (arachidonic acid), and 5,8,11,14,17-eicosapentaenoic acid methyl ester (EPA) were among the most abundant FAMEs accounting for approximately 50% of the subgroups measured. Data from GC-FID analysis revealed methyl laurate (C12:0), methyl myristate (C14:0), methyl palmitate (C16:0), and methyl stearate (18:0) methyl esters as the most abundant saturated FA, whereas cis-9-oleic methyl ester (C18:1) and methyl linoleate (C18:2) were found as the major monounsaturated FA and PUFA FAMEs, respectively, in the body wall of the species. Taken together, the extraction and characterization of different categories of metabolically and biologically active compounds in various organ extracts of H. leucospilota suggest that the species is potentially a rich source of cholesterol-lowering, antioxidant, antimicrobial, and anticancer agents. These substances are known to benefit human health and assist in disease prevention. These findings justify the use of sea cucumbers in traditional folklore medication and the current interest and attention focused on the species to mine for bioactives in new drugs research.

Mucosal genetic immunization through microsphere-based oral carriers.

Polymeric carriers in the form of cellulose acetate phthalate (CAP) and alginate (ALG) microspheres were used for encapsulation of plasmid DNA for oral mucosal immunization. Access into the intestinal mucosa by pVAX1 eukaryotic expression plasmid vectors carrying gene-coding sequences, either for the cholera enterotoxin B subunit (ctxB) immunostimulatory antigen or the green fluorescent protein (GFP), delivered from both types of microsphere carriers were examined in orally immunized BALB/c mice. Demonstration of transgene protein expression and IgA antibody responses at local mucosal sites suggest immunological response to a potential oral DNA vaccine formulated within the microsphere carriers.

Display of the Viral Epitopes on Lactococcus lactis: A Model for Food Grade Vaccine against EV71.

In this study, we have developed a system for display of antigens of Enterovirus type 71 (EV71) on the cell surface of L. lactis. The viral capsid protein (VP1) gene from a local viral isolate was utilized as the candidate vaccine for the development of oral live vaccines against EV71 using L. lactis as a carrier. We expressed fusion proteins in E. coli and purified fusion proteins were incubated with L. lactis. We confirmed that mice orally fed with L. lactis displaying these fusion proteins on its surface were able to mount an immune response against the epitopes of EV71. This is the first example of an EV71 antigen displayed on the surface of a food grade organism and opens a new perspective for alternative vaccine strategies against the EV71. We believe that the method of protein docking utilized in this study will allow for more flexible presentations of short peptides and proteins on the surface of L. lactis to be useful as a delivery vehicle.

Genotypically different clones of Staphylococcus aureus are diverse in the antimicrobial susceptibility patterns and biofilm formations.

This study evaluated whether genotypically different clinical isolates of S. aureus have similar susceptibilities to individual antibiotics. It further aims to check the impact of biofilm on the in vitro activity of vancomycin, daptomycin, linezolid, and tigecycline against S. aureus clones. The study used a total of 60 different clinical MSSA and MRSA isolates. Susceptibilities were performed in planktonic cultures by macrobroth dilution and epsilon-test (E test) system. Biofilm production was determined using an adherent plate assay. The efficacy of antimicrobial activities against biofilms formation was checked using confocal laser scanning microscopy (CLSM). The study found that similar and different spa, MLST, and SCCmec types displayed high variation in their susceptibilities to antibiotics with tigecycline and daptomycin being the most effective. The biofilms were found resistant to high concentrations of most antibiotics tested with daptomycin being the most effective drug used in adhesive biofilms. A considerable difference exists among similar and various clone types against antibiotics tested. This variation could have contributed to the degree of virulence even within the same clonal genotype and enhanced heterogeneity in the infection potential. Thus, the development of a rapid and precise identification profile for each clone in human infections is important.

Prevalence of adhesion and regulation of biofilm-related genes in different clones of Staphylococcus aureus.

Clinical information about genotypically different clones of biofilm-producing Staphylococcus aureus is largely unknown. We examined whether different clones of methicillin-sensitive and methicillin-resistant S. aureus (MSSA and MRSA) differ with respect to staphylococcal microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) in biofilm formation. The study used 60 different types of spa and determined the phenotypes, the prevalence of the 13 MSCRAMM, and biofilm genes for each clone. The current investigation was carried out using a modified Congo red agar (MCRA), a microtiter plate assay (MPA), polymerase chain reaction (PCR), and reverse transcriptase polymerase chain reaction (RT-PCR). Clones belonging to the same spa type were found to have similar properties in adheringto the polystyrene microtiter plate surface. However, their ability to produce slime on MCRA medium was different. PCR experiments showed that 60 clones of MSSA and MRSA were positive for 5 genes (out of 9 MSCRAMM genes). icaADBC genes were found to be present in all the 60 clones tested indicating a high prevalence, and these genes were equally distributed among the clones associated with MSSA and those with MRSA. The prevalence of other MSCRAMM genes among MSSA and MRSA clones was found to be variable. MRSA and MSSA gene expression (MSCRAMM and icaADBC) was confirmed by RT-PCR.

A simplified multiplex PCR assay for fast and easy discrimination of globally distributed staphylococcal cassette chromosome mec types in meticillin-resistant Staphylococcus aureus.

A multiplex PCR assay was developed for the identification of major types and subtypes of staphylococcal cassette chromosome mec (SCCmec) in meticillin-resistant Staphylococcus aureus (MRSA) strains. The method uses a novel 9 valent multiplex PCR plus two primer pairs for S. aureus identification and detection of meticillin resistance. All 389 clinical MRSA isolates from Malaysia and 18 European isolates from the Harmony collection harbouring different SCCmec types that we tested were correctly characterized by our PCR assay. SCCmec type III and V were by far the most common types among both hospital- and community-acquired Malaysian MRSA isolates, with an apparent emergence of MRSA harbouring the IVh type.

Predominance and emergence of clones of hospital-acquired methicillin-resistant Staphylococcus aureus in Malaysia.

We define the epidemiology of predominant and sporadic methicillin-resistant Staphylococcus aureus (MRSA) strains in a central teaching and referral hospital in Kuala Lumpur, Malaysia. This is done on the basis of spa sequencing, multilocus sequence typing (MLST), staphylococcal cassette chromosome mec (SCCmec) typing, and virulence gene profiling. During the period of study, the MRSA prevalence was 44.1%, and 389 MRSA strains were included. The prevalence of MRSA was found to be significantly higher in the patients of Indian ethnicity (P < 0.001). The majority (92.5%) of the isolates belonged to ST-239, spa type t037, and possessed the type III or IIIA SCCmec. The arginine catabolic mobile element (ACME) arcA gene was detected in three (1.05%) ST-239 isolates. We report the first identification of ACME arcA gene-positive ST-239. Apart from this predominant clone, six (1.5%) isolates of ST-22, with two related spa types (t032 and t4184) and a singleton (t3213), carrying type IVh SCCmec, were detected for the first time in Asia. A limited number of community-acquired (CA) MRSA strains were also detected. These included ST-188/t189 (2.1%), ST-1/t127 (2.3%), and ST-7/t091 (1%). Panton-Valentin leukocidin (PVL) was detected in all ST-1 and ST-188 strains and in 0.7% of the ST-239 isolates. The majority of the isolates carried agr I, except that ST-1 strains were agr III positive. Virulence genes seg and sei were seen only among ST-22 isolates. In conclusion, current results revealed the predominance of ST-239-SCCmec III/IIIA and the penetration of ST-22 with different virulence gene profiles. The emergence in Malaysia of novel clones of known epidemic and pathogenic potential should be taken seriously.

Sequencing and analysis of chromosome 1 of Eimeria tenella reveals a unique segmental organization.

Eimeria tenella is an intracellular protozoan parasite that infects the intestinal tracts of domestic fowl and causes coccidiosis, a serious and sometimes lethal enteritis. Eimeria falls in the same phylum (Apicomplexa) as several human and animal parasites such as Cryptosporidium, Toxoplasma, and the malaria parasite, Plasmodium. Here we report the sequencing and analysis of the first chromosome of E. tenella, a chromosome believed to carry loci associated with drug resistance and known to differ between virulent and attenuated strains of the parasite. The chromosome--which appears to be representative of the genome--is gene-dense and rich in simple-sequence repeats, many of which appear to give rise to repetitive amino acid tracts in the predicted proteins. Most striking is the segmentation of the chromosome into repeat-rich regions peppered with transposon-like elements and telomere-like repeats, alternating with repeat-free regions. Predicted genes differ in character between the two types of segment, and the repeat-rich regions appear to be associated with strain-to-strain variation.