Bordin Butr-Indr

Meticillin-resistant Staphylococcus aureus in pigs from Thailand

Livestock-associated meticillin-resistant Staphylococcus aureus (LA-MRSA) strains are increasingly isolated from pigs and humans, particularly those involved in pig farming. There is also growing evidence of transmission through the food chain. The distribution of LA-MRSA has spread throughout Europe and beyond, with the majority of strains belonging to sequence type ST398 and ST9. Here we report the emergence of LA-MRSA in pigs from Thailand. [...]

Screening of filamentous fungi for production of myrosinase

A linhagem Aspergillus sp. NR46FB, isolada de solo atraves da tecnica do agar sulfato de bario-sinigrina, foi testada quanto a producao de mirosinase. O fungo degradou completamente o glicosinolato e produziu 3,19 U.mL-1 de mirosinase, apos 48 h de cultivo. Devido a alta producao de mirosinase, esse novo isolado e um potente candidato para aplicacoes industriais.

Enhanced Production of Functional Extracellular Single Chain Variable Fragment Against HIV-1 Matrix Protein from Escherichia coli by Sequential Simplex Optimization

The optimal culture condition for extracellular recombinant single chain variable fragment anti HIV-1 p17 protein (scFv anti-p17) production in Escherichia coli HB2151 was investigated by the sequential simplex optimization (SS) method. Five variable parameters were submitted in the fermentation process. The most favorable condition obtained from 19 independent experiments was as followed: 58 µM of IPTG induction to 1.7 OD600 nm at 25.5°C for 16 h with 202 rpm agitation rate. The amount of secreted scFv anti-p17 at the optimal condition was 38% higher than under the control condition. The binding activity of soluble extracellular scFv anti-p17 protein increased 95.5% and 73.2% in comparison with the control condition and non-optimized condition respectively. The soluble scFv anti-p17 from crude HB2151 lysated was subsequently purified by immobilized metal ion affinity chromatography (IMAC) with His-tag. The purified scFv anti-p17 was intact and retained its antigen-binding affinity against HIV-1 p17. We demonstrated that the sequential simplex optimization method was a key for exertion of high yield with fewer experimental requirements for acquiring of large scale secretory protein production.

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.

A comparison of Rv0559c and Rv0560c expression in drug-resistant Mycobacterium tuberculosis in response to first-line antituberculosis drugs

Drug resistance to Mycobacterium tuberculosis is a major health problem worldwide. Mycobacterium tuberculosis can progress to be mono-drug resistant or multi-drug resistant by improper treatment. The chemical stress of M. tuberculosis was performed in this study. Rv0559c is an unknown secreted protein. Rv0560c is a putative benzoquinone methyltransferase of M. tuberculosis cell. Rv0559c gene is located downstream of Rv0560c gene. Both genes respond to salicylate stress. Drug susceptible, isoniazid resistant, rifampicin resistant and multi-drug resistant phenotypes of M. tuberculosis clinical isolates were used to determine the expression of Rv0559c and Rv0560c by qRT-PCR. In all of mycobacteria strains there was up-regulation in both genes when stressed with isoniazid. This study determined the expression of both genes, which may play important roles in the drug resistance mechanism of mycobacteria.

Multiparameter optimization method and enhanced production of secreted recombinant single-chain variable fragment against the HIV-1 P17 protein from Escherichia coli by fed-batch fermentation

ABSTRACT The single-chain fragment variable (scFv) was used to produce a completely functional antigen-binding fragment in bacterial systems. The advancements in antibody engineering have simplified the method of producing Fv fragments and made it more efficient and generally relevant. In a previous study, the scFv anti HIV-1 P17 protein was produced by a batch production system, optimized by the sequential simplex optimization method. This study continued that work in order to enhance secreted scFv production by fed-batch cultivation, which supported high volumetric productivity and provided a large amount of scFvs for diagnostic and therapeutic research. The developments in cell culture media and process parameter settings were required to realize the maximum production of cells. This study investigated the combined optimization methods, Plackett–Burman design (PBD) and sequential simplex optimization, with the aim of optimize feed medium. Fed-batch cultivation with an optimal feeding rate was determined. The result demonstrated that a 20-mL/hr feeding rate of the optimized medium can increase cell growth, total protein production, and scFv anti-p17 activity by 4.43, 1.48, and 6.5 times more than batch cultivation, respectively. The combined optimization method demonstrated novel power tools for the optimization strategy of multiparameter experiments.