Anti-Methicillin-Resistant Staphylococcus Aureus and Anti-Methicillin-Sensitive Sstaphylococcus Aureus Effects of Calligonum Comosum l'her. Methanolic Extract

Methicillin Resistant Staphylococcus aureus MRSA is a major life-threatening pathogens causing a variety of serious infections and responsible for the majority of morbidity and mortality in human. Currently, more toxic and more costly antibiotics which are used as last-line agents can only be the choice to cure MRSA infections. Therefore, search for an alternative therapy derived from natural products with effective antimicrobial activity is extensively needed. The aim of this study was to develop a combination therapy for MRSA infection by using naturally derived extract and antibiotic formulation targeting selected infective proteins. The antimicrobial activity of Calligonum comosum methanolic extract (locally known as Arta), a medicinal plant native to Saudi Arabia, was evaluated using Kirby-Bauer disc diffusion method, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), against gram positive and gram negative pathogens. The activity of the extract was determined for various clones of methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA). All MSSA and MRSA strains tested were subtyped based on multilocus sequence typing (MLST) and spa typing to differentiate the clones of these strains. Escherichia coli and Klebsiella pneumoniae were included as negative controls. The present study also verified the interactions between 17 commercial antibiotics representing 12 classes and the methanolic extract of C. comosum against MRSA and MSSA strains. The interaction was assessed using a disc-diffusion test performed on agar plates with or without dilution of C. comosum extract at sub-inhibitory concentrations, and the size of the inhibition zones (diameter) were recorded. The bioassay studies revealed that C. comosum extract showed inhibitory activity only to gram positive strains whereas gram negative organisms, Escherichia coli and Klebsiella pneumonia, showed resistant phenotypic pattern to the methanolic extract. In vitro, 10 antibiotics (penicillin, oxacillin, cefoxitin, cloxacillin, ceftriaxone, vancomycin, chloramphenicol, clindamycin, tigecycline and rifampicin) given in combination with the methanolic extract exhibited significant synergistic interaction against most of MRSA and MSSA strains (P= 0.05). Namely 4 antibiotics, rifampicin,vancomycin, cefoxitin and tigecycline (P= 0.00), presented the highest synergism rate with the extract in all MRSA and MSSA strains. These antibiotics when exposed in combination with the C. comosum extract showed greater inhibition zones when compared to the zones by single application of the antibiotics or extract. However, 6 other antibiotics had indifferent effects with the methanolic extract against most of MRSA and MSSA strains, while the extract showed antagonistic interaction with gentamicin. In addition to the bioassay experiments, antimicrobial substance activity of C. comosum extract was elucidated for effect on the changes in DNA repair gene (adaB) sequence in treated S. aureus strains through PCR and RT-PCR assay. The effect of C. comosum extract inhibitory activity on the selected gene sequence showed several nucleotide changes. The detected changes in nucleotides through substitution, insertion or deletion of the nucleotide base pairs in the adaB gene sequence led to several amino acid substitutions at different positions. Nevertheless, to find out the possibility of using the active compounds in the extract as a new alternative antibacterial agent, cytotoxic activity of C. comosum extract was determined using the MTS assay. The toxic effect of the extract was tested on skin fibroblast cells. Various concentrations were selected to evaluate the IC50 of the extract at various time intervals of 24h and 48h, respectively. As shown in vitro, inhibitory action of the extract used is much lower (250 mg/ml) than that reported for antimicrobials (100 mg/ml) derived naturally. Also, identification of crude extract compounds was carried out using gas chromatography-mass spectrometry (GC-MS). GC-MS chromatogram of the given crude sample showed 42 different peaks at various retention times ranging from 5 minutes to 28 minutes for different chemical constituents. GC–MS analysis revealed the presence of 1-((allyloxy)methyl)benzene (18.11%), acetic acid (12.57%), 2-sec-butoxybenzene-1,3-diol (10.61%), formic acid (7.76%), 5-(hydroxymethyl)-2-furancarboxaldehyde (6.67%), 2,3-Dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one (5.57%), orthomethoxyphenol (3.71%) and 1-tridecanol (1.27%) as the major components. In conclusion, C. comosum methanolic extract is justified in the present study to be a suitable alternative against MSSA and MRSA based on the inhibiting effect and the low toxicity properties. In addition, the extract in combination with several antibiotics showing synergistic activity further enhances the activity of the extract. The extract also revealed to contain potential antimicrobial chemical compounds. The C. comosum extract warrants further investigation as a drug for anti MRSA and anti MSSA

Potential usefulness of viral capsid surface proteins (VP1,VP2, VP3 & VP4) for vaccination against common cold

Rhinoviruses (RVs) represent the most important etiological agents of the common cold and it is responsible for about two-thirds of acute exacerbations of chronic bronchitis, asthma and chronic obstructive pulmonary disease (COPD) in both children and adults. At present, there is no effective and approved antiviral therapies for either the prevention or treatment of diseases caused by RV infections. Furthermore, there are more than 100 types of RVs with high sequence variability hindering the progression of vaccine development. Bioinformatics tools, combined with the availability of complete genome sequence of all known RV types, provides a unique opportunity to enhance the optimal selection of potential immune targets. In vitro production or synthetic versions of these targets could be a possible alternative approach to the vaccine of choice. This study was carried out with the aim to develop a pan-serotypic vaccine that is capable of inducing the production of crossreactive antibodies that cover all or most of the RV serotypes. Firstly, a bioinformatics analysis was carried out to characterise the capsid proteins (VP1, VP2, VP3 and VP4) of all known RV serotypes and to predict potential immune motifs. In brief, complete protein sequences of each of the 100 distinct RV genomes were downloaded from the GenBank database. The sequences obtained were grouped based on their original classification [RV-A divided into two subgroups, minor LDLR(n=10) and major ICAM(n=65), and RV-B group (n=25)]. Upon grouping, sequence editing was carried out using a number of software in order to study each protein individually. The edited protein sequences were then aligned and analysed for sequence conservation, variability and to generate consensus sequences and distance matrices. This led to determining the relations between strains and identifying the ideal ones that are highly identical to others. Conserved motifs consisting at least nine-mers common across all RV-A or B serotypes (minor/major receptor) and exhibiting at least 80% representation were selected and synthesized chemically. These peptides were used alone or in combination to vaccinate groups of rabbits. On the other hand, four tagged fulllength genes coding the capsid proteins of an ideal strain (HRV-74), VP1, VP2, VP3 and VP4, whose codon uses were optimized, were constructed and cloned in vitro. Upon expression, the purified recombinant proteins adsorbed into incomplete Freund's adjuvant (IFA) as a single or combined proteins were also administered subcutaneously to other groups of rabbits. The responses and cross-reactivity of the specific immunoglobulin M (IgM) and G (IgG) to the peptides, proteins and whole viruses were measured by in-house indirect enzyme-linked immunosorbent assay (ELISA). Moreover, in vitro cross-neutralizing antibody titres against several variant strains of RV were also measured. Based on the bioinformatics analysis, 7, 8, 5 and 3 conserved regions were found among minor receptor serotypes for VP1, VP2, VP3 and VP4, respectively. The analysis of RV-A ICAM-receptor serotypes showed 3 conserved regions in each of VP1, VP2 and VP4, while 4 conserved regions were found upon alignment of VP3 sequences, respectively. The study also showed that the capsid protein of HRV-B contained at least one conserved site upon multiple sequences alignments of each protein separately. Furthermore, the analysis revealed that 72% of VP4 sequence (69 amino acids in length) as highly conserved among the RV-A major receptor group, but VP3 did not show well conserved regions. The current study also showed that VP4 sequences of the minor receptor groups (n=10) contained three highly conserved sites which accounted for 85% of its total length. RV-B VP4, in contrast,contained less conserved regions which exhibited only 25% of the protein's total length. Upon multiple sequence alignment of all RV-A, three highly conserved region were identified for each of the VP1, VP2 and VP4, while VP3 did not contain any. Based on distance matrices analysis, HRV-74 was found to be the ideal strain for vaccine development. VP1 amino acid sequence of HRV-74 was found to be identical by 80% or more of 22 serotypes, with a median identity of 75% within the RV-A group. Also, the analysis revealed HRV-74 as having the highest homology (86%) to the VP1 consensus sequence of all RV-A. A further analysis showed that HRV-74 is fully identical (100%) to the consensus sequence of RV-A VP4. Therefore, HRV-74 has been considered as the source genetic information of the recombinant proteins produced in this study. Antibodies raised to the synthetic peptides exhibited cross-reactivity against the corresponding recombinant proteins and antigenically distinct RV strains coated on plates via ELISA assay. Moreover, the specific immunoglobulin G (IgG) response to the peptides given in combination exhibited greater reactivity. Interestingly, the antipeptide antibodies obtained exhibited a cross-neutralizing activity for different RV strains in vitro. In addition, the induced antibodies against recombinant proteins also reacted successfully with relevant proteins and with whole virus particle (HRV-74) and other variant strains, as shown by ELISA. They also showed strong crossneutralizing ability against various variants of RVs. Based on the antibody cross-reactivity and neutralization towards different studied serotypes, the selected RV strain HRV-74 seemed to be the type of choice for developing RV broad protective vaccine and multiple RVs antibody based on detection assay. The findings have indicated that the peptides corresponding to the conserved region of the RV capsid proteins are potent immunogenic and suggest that their combination is crucial for extending the cross-protection against variant RVs. Such an alternative approach may raise hope for designing a novel broad-protective vaccine towards non-cultivable, hyper variable pathogen

Cell culture, technology: enhancing the culture of diagnosing human diseases

Cell culture involves a complex of processes of cell isolation from their natural environment (in vivo) and subsequent growth in a controlled environmental artificial condition (in vitro). Cells from specific tissues or organs are cultured as short term or established cell lines which are widely used for research and diagnosis, most specially in the aspect of viral infection, because pathogenic viral isolation depends on the availability of permissible cell cultures. Cell culture provides the required setting for the detection and identification of numerous pathogens of humans, which is achieved via virus isolation in the cell culture as the “gold standard” for virus discovery. In this review, we summarized the views of researchers on the current role of cell culture technology in the diagnosis of human diseases. The technological advancement of recent years, starting with monoclonal antibody development to molecular techniques, provides an important approach for detecting presence of viral infection. They are also used as a baseline for establishing rapid tests for newly discovered pathogens. A combination of virus isolation in cell culture and molecular methods is still critical in identifying viruses that were previously unrecognized. Therefore, cell culture should be considered as a fundamental procedure in identifying suspected infectious viral agent

Antibody and immune memory persistence post infant hepatitis B vaccination

OBJECTIVES: This study aimed to evaluate the level of hepatitis B immunity among undergraduate students 23 years after commencement of the nationwide hepatitis B childhood immunization program in Malaysia. METHODS: A total of 402 serum samples obtained from volunteer undergraduate students were screened for the presence of hepatitis B surface antibodies using qualitative ELISA. RESULTS: Results showed that 62.7% of volunteers had protective anti-hepatitis B surface antigens (≥10 IU/L), of whom 67.9% received three doses of the vaccine. The estimated post-vaccination immunity was found to be at least 20 years, indicating persistent immunity against hepatitis B and a significant association (P < 0.05) with duration of vaccination. Anamnestic response 1 month post-hepatitis B booster was 94.0% and highly significant (P < 0.01). Isolated antihepatitis B core antigen (anti-HBc) prevalence was found to be 5.0%, all having had a positive anamnestic response. CONCLUSION: Immunity after primary vaccination with hepatitis B recombinant vaccine persists for at least 20 years post-vaccination, with significant association with the number of vaccinations. Furthermore, the presence of anamnestic response to booster vaccine indicates long-lasting immunity despite decreasing antibody levels; therefore, the need for hepatitis B vaccine boosters may not be of significant benefit after complete infant vaccination