Sorasak Intorasoot

Application of gelatin-coated magnetic particles for isolation of genomic DNA from bacterial cells

Gelatin-coated magnetic particles were implemented for bacterial genomic DNA isolation in this study. Based on structural differences in the cell wall, the standard strains Staphylococcus aureus and Escherichia coli were selected. The quantity, quality, and timing process for DNA extraction using gelatin-coated magnetite were compared to reference phenol-chloroform extraction and a commercially available kit. Approximately twice as much DNA was recovered with the use of coated magnetite, providing greater yields than other DNA extraction methods. In addition, the DNA quality was determined using 16S ribosomal DNA (rDNA) gene amplification by polymerase chain reaction (PCR). The described technique is rapid, simple, and a well-suited method to use with PCR for diagnosis of bacterial infections.

Recombinant expression of novel protegrin-1 dimer and LL-37-linker–histatin-5 hybrid peptide mediated biotin carboxyl carrier protein fusion partner

Antimicrobial peptides (AMPs) hold great promise as potential therapeutic approach for curing of infectious diseases. Prokaryotic protein expression renders high scalability with an effective purification of several heterogeneous proteins. However, it might be inappropriate for recombinant AMPs expression thereby its antimicrobial activity against the host cells. Several fusion partners demonstrated antimicrobial activity neutralization of AMPs expression and purification in Escherichia coli. In order to improve the antimicrobial effect, several hybrid AMPs have been designed and developed. As expected to increase the antimicrobial activity, a dimeric form of porcine protegrin-1 (PG-1) and human LL-37-linker-histatin-5 (LL-37-linker-Hst-5) hybrid peptide were alternatively constructed in this study. Hydroxylamine hydrochloride and thrombin cleavage sites were designed for releasing of hybrid peptide and PG-1 dimer from biotin carboxyl carrier protein (BCCP) fusion partner. The full-length AMPs gene was connected down-stream of BCCP gene using the overlap extension-PCR, cloned into pET-28a vector and expressed in E. coli BL21(DE3)pLysS. After IPTG induction, approximately 20% of BCCP-AMPs was expressed as intracytoplasmic inclusion bodies with an expected molecular weight of 24.5kDa. The mean of purified and refolded BCCP-AMPs was 1.5mg/L with 76% purity. The presence of expressed protein was subsequently determined by Western blotting analysis. Finally, radial diffusion assay supported that these peptides displayed functional antimicrobial activity against E. coli and Staphylococcus aureus standard strains. Two novel AMPs established in this study would be potentially developed as extensive intervention for treating of infectious diseases.

Secretome profile analysis of multidrug-resistant, monodrug-resistant and drug-susceptible Mycobacterium tuberculosis

The emergence of drug-resistant tuberculosis has generated great concern in the control of tuberculosis and HIV/TB patients have established severe complications that are difficult to treat. Although, the gold standard of drug-susceptibility testing is highly accurate and efficient, it is time-consuming. Diagnostic biomarkers are, therefore, necessary in discriminating between infection from drug-resistant and drug-susceptible strains. One strategy that aids to effectively control tuberculosis is understanding the function of secreting proteins that mycobacteria use to manipulate the host cellular defenses. In this study, culture filtrate proteins from Mycobacterium tuberculosis H37Rv, isoniazid-resistant, rifampicin-resistant and multidrug-resistant strains were gathered and profiled by shotgun-proteomics technique. Mass spectrometric analysis of the secreted proteome identified several proteins, of which 837, 892, 838 and 850 were found in M. tuberculosis H37Rv, isoniazid-resistant, rifampicin-resistant and multidrug-resistant strains, respectively. These proteins have been implicated in various cellular processes, including biological adhesion, biological regulation, developmental process, immune system process localization, cellular process, cellular component organization or biogenesis, metabolic process, and response to stimulus. Analysis based on STITCH database predicted the interaction of DNA topoisomerase I, 3-oxoacyl-(acyl-carrier protein) reductase, ESAT-6-like protein, putative prophage phiRv2 integrase, and 3-phosphoshikimate 1-carboxyvinyltransferase with isoniazid, rifampicin, pyrazinamide, ethambutol and streptomycin, suggesting putative roles in controlling the anti-tuberculosis ability. However, several proteins with no interaction with all first-line anti-tuberculosis drugs might be used as markers for mycobacterial identification.

Novel potential diagnostic test for Mycobacterium tuberculosis complex using combined immunomagnetic separation (IMS) and PCR‐CTPP

To exploit immunomagnetic separation combined with PCR with confronting two‐pair primers (IMS‐PCR‐CTPP) as a rapid method for detection of Mycobacterium tuberculosis complex (MTC) and identification of Mycobacterium bovis from sputum specimens.

Expression in Escherichia coli of novel recombinant hybrid antimicrobial peptide AL32-P113 with enhanced antimicrobial activity in vitro

Antibiotic-resistant pathogens have become a major public health problem worldwide. New discoveries and strategies as regards antibiotic drug development are urgently in need for curing infected patients. Antimicrobial peptides (AMPs) are short cationic peptides that play important roles in innate immune system with a broad spectrum of antimicrobial activity. Recently, hybrid AMPs have been reported to increase antimicrobial activity, stability, and in vivo half-life. In the present study, a gene encoding for AL32-P113 hybrid peptide consisting of two truncated active forms of human LL-37 and histatin-5 (Hst-5) was commercially constructed, cloned into pTXB-1 commercial plasmid, and expressed in E. coli BL21 (DE3). To increase the yield of target protein expression, IPTG concentration, time and temperature were optimized. The results indicate that AL32-P113-intein fusion protein with 33.7 kDa was expressed mostly in inclusion form and estimated to be 20% of the total protein. After chitin affinity purification, 5.7-kDa of AL32-P113 peptide was separated with an average concentration of 12.1 mg per litre of bacterial culture and over 86% purity. The minimum inhibitory concentration (MIC) was evaluated for antimicrobial activity determination of recombinant AL32-P113 compared to synthetic peptides, LL-37, Hst-5, and L31-P113. The results implied that both hybrid peptides exhibited potent antimicrobial activity against gram-negative bacteria and yeast cells whereas the L31-P113 peptide possessed approximately four times greater antimicrobial activity in gram-positive bacteria than parent LL-37. An increasing of undesired hemolysis of these hybrid peptides toward human red cells was also observed when red blood cell hemolytic assay was performed. Several factors including charge and secondary structure predicted by public software were utilized for explanation of the antimicrobial potency of both hybrid peptides. This study proved that hybrid peptides show broader and more potent antimicrobial ability against pathogens and they could be applied as a therapeutic approach for topical treatment of microbial infection in the future.

Bactericidal activity of herbal volatile oil extracts against multidrug resistant Acinetobacter baumannii

Aim: The aim of the study is to investigate the antibacterial activity of 10 volatile oils extracted from medicinal plants, including galangal (Alpinia galanga Linn.), ginger (Zingiber officinale), plai (Zingiber cassumunar Roxb.), lime (Citrus aurantifolia), kaffir lime (Citrus hystrix DC.), sweet basil (Ocimum basilicum Linn.), tree basil (Ocimum gratissimum), lemongrass (Cymbopogon citratus DC.), clove (Syzygium aromaticum), and cinnamon (Cinnamomum verum) against four standard strains of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, and 30 clinical isolates of multidrug-resistant A. baumannii (MDR-A. baumannii). Materials and Methods: Agar diffusion, minimum inhibitory concentration, and minimum bactericidal concentration (MBC) were employed for the determination of bactericidal activity of water distilled medicinal plants. Tea tree oil (Melaleuca alternifolia) was used as positive control in this study. Results: The results indicated the volatile oil extracted from cinnamon exhibited potent antibacterial activity against the most common human pathogens, S. aureus, E. coli, P. aeruginosa, and A. baumannii. Most of volatile oil extracts were less effective against non-fermentative bacteria, P. aeruginosa. In addition, volatile oil extracted from cinnamon, clove, and tree basil possessed potent bactericidal activity against MDR-A. baumannii with MBC90 of 0.5, 1, and 2 mg/mL, respectively. Conclusions: The volatile oil extracts would be useful as alternative natural product for the treatment of the most common human pathogens and MDR-A. baumannii infections.