Hydrogen peroxide from L-amino acid oxidase of king cobra (Ophiophagus hannah) venom attenuates Pseudomonas biofilms.

Because of the high incidence of Pseudomonas aeruginosa biofilms-related nosocomial infections, venoms from common Thai snakes were tested. Although venoms from king cobra (Ophiophagus hannah; OH) and green pit viper (Trimeresurus albolabris) showed the broadest antibacterial spectrum, OH venom demonstrated more profound anti-biofilm activities against P. aeruginosa. Additionally, purified L-amino acid oxidase from OH venom (OH-LAAO), using a three-step chromatography and protein identification, reduced biofilm mass as indicated by the downregulation of several genes, including the genes for biofilm synthesis (algD and pslB) and biofilm regulators (algU, gacA, and siaD). Moreover, OH-LAAO disrupted Pseudomonas-preformed biofilms via upregulation of several genes for biofilm dispersion (nbdA, bdlA, and dipA) and biofilm degradation (endA and pslG), resulting in a reduction of the biofilm biomass. Due to the antimicrobial effects and anti-biofilm activities (reduced production plus increased dispersion) neutralized by catalase, a hydrogen peroxide (H2O2)-degrading enzyme, the enhanced H2O2 by OH venom might be one of the anti-biofilm mechanisms. Hence, OH-LAAO was proposed as a novel agent against Pseudomonas biofilms for either treatment or prevention. More studies are interesting.

Application of transdermal patches with new skin test reagents for detection of latent tuberculosis.

Introduction. Current intradermal tuberculin skin tests for latent tuberculosis infection (LTBI) based on purified protein derivative (PPD) have poor specificity.Aims. Developing a better skin test antigen as well as a simple skin patch test may improve and facilitate diagnostic performance.Methodology. Defined recombinant antigens that were unique to Mycobacterium tuberculosis (MTB), including two potential latency-associated antigens (ESAT-6 and Rv2653c) and five DosR-encoded latency proteins (Rv1996, Rv2031c, Rv2032, DevR and Rv3716c), were used as diagnostic skin test reagents in comparison with a standard PPD. The performance of the skin tests based on the detection of delayed-type hypersensitivity (DTH) reaction in guinea pigs sensitized to MTB and M. bovis bacille Calmette-Guérin (BCG) vaccine was evaluated.Results. The latency antigens Rv1996, Rv2031c, Rv2032 and Rv2653c and the ESAT-6 protein elicited less reactive DTH skin responses in MTB-sensitized guinea pigs than those resulting from PPD, but elicited no response in BCG-vaccinated guinea pigs. The remaining two latency antigens (DevR and Rv3716c) elicited DTH responses in both groups of animals, as did PPD. The reactivity of PPD in BCG-vaccinated guinea pigs was greater than that of any of the selected skin test reagents. Using stronger concentrations of selected skin test reagents in the patch test led to increased DTH responses that were comparable to those elicited by PPD in guinea pigs sensitized with MTB.Conclusion. Transdermal application of defined purified antigens might be a promising method for LTBI screening.

Comparative Proteomic Profiling of Mycobacterium tuberculosis and the Thai Vaccine Strain Mycobacterium bovis Bacille Calmette-Guerin Tokyo172: Diverse Biomarker Candidates for Species Differentiation.

BACKGROUND: Bacille Calmette-Guerin (BCG)-related complications can occur in vaccinated children. Comparison of the composition of cellular proteins of virulent Mycobacterium tuberculosis (MTB) H37Rv with of attenuated Mycobacterium bovis BCG Tokyo172 vaccine strain used in Thailand and identify protein candidates of value for differentiation between the two mycobacterial species may facilitate the diagnosis of etiologic agent of mycobacterial disease in vaccinated children, as most cases have been believed to have originated from BCG vaccine. MATERIALS AND METHODS: The two-dimensional electrophoresis (2DE) proteomic profiles of cellular proteins from the Thai vaccine strain M. bovis BCG Tokyo172 and MTB were compared and the matched spots in 2DE gels were submitted to mass spectrometry analysis. RESULTS: There were a number of similar protein contents with different intensity or position between MTB and M. bovis BCG Tokyo172. A higher expression of some immunogenic proteins was shown in BGG Tokyo172 when compared to MTB, while some were shown the opposite pattern. CONCLUSIONS: Proteomic approach reveals key proteins participating in different species of Mycobacteria, and may be useful for discrimination between MTB and the BCG Tokyo172 infection.

Improved Serodiagnostic Sensitivity of Strip Test for Latent Tuberculosis.

INTRODUCTION: Diagnosis of Latent Tuberculosis Infection (LTBI) is difficult due to no clinical manifestations. Cases of LTBI are mostly sputum negative. The World Health Organization recommends the Tuberculin Skin Test (TST) as the current diagnostic standard for LTBI. Our previously developed serologic strip test for LTBI detection had suboptimal sensitivity. Additional Mycobacteriumtuberculosis (MTB) latency-associated antigens may improve the detection rate of LTBI. AIM: The present study aimed to optimize sensitivity of existing strip test. MATERIALS AND METHODS: A combination of recombinant latency proteins Rv2029c, Rv2031c, Rv2032, Rv2627c, Rv3133c, and Rv3716c was used to prepare the strips and evaluate the performance with the sera of patients in four well-classified categories: LTBI, active pulmonary TB, healthy TB contacts and other non-TB diseases. RESULTS: A total of 91 serum samples from various clinical categories were screened with the strips. Among clinically diagnosed LTBI patients, strip test yielded a sensitivity of 75.0%. Among clinically diagnosed non-LTBI subjects, strip test yielded 88.1% specificity. The diagnostic positive and negative predictive values for strip test in reference to various clinical contexts were 77.4% and 86.7%, respectively. CONCLUSION: Addition of the six potential latency proteins could improve the diagnostic performance of existing strip test for LTBI. The use of suitable immunodominant antigens could maximize sensitivity in the diagnosis and differentiate MTB infection status.

Performance of a rapid strip test for the serologic diagnosis of latent tuberculosis in children.

BACKGROUND: The serodiagnostic tests for tuberculosis (TB) present a high variability in terms of sensitivity and specificity. Data on patients with latent TB infection (LTBI) and children in high prevalence settings are still limited. The present study aimed to evaluate an in-house strip test for detection of anti-M. tuberculosis antibodies in TB patients, mostly children aged under 15 y, grouped into four diagnostic categories: active TB, LTBI, healthy TB contacts, and other non-TB diseases. MATERIALS AND METHODS: The diagnostic performance of strip test was compared with the tuberculin skin test (TST) and interferon-gamma release assay (IGRA). Sensitivity and specificity were assessed for all three diagnostic tests. The detection accuracy among the tests was calculated by using a receiver operating characteristic analysis. RESULTS: TST and IGRA could diagnose the active TB cases correctly (100%). The sensitivity of strip test for active TB was 58.3% and 37.5% for LTBI, while the sensitivities of TST and IGRA for LTBI were 90.3% and 37.5%, respectively. The overall specificities of strip test and IGRA were 91.5% and 95.7%, respectively, which were superior to that of TST (68.1%). CONCLUSION: The strip test did not appear to be useful for diagnosis of active TB in comparison with the current diagnostic standard. The assay may be particularly significant in situations where TB is clinically difficult to diagnose like LTBI and could be a meaningful tool in terms of high specificity and simplicity for ruling in pediatric TB in countries with high TB infection rate. Further studies are needed to determine whether strip test can be improved in its sensitivity and should be implemented into routine clinical practice.

The fusion Vibrio campbellii luciferase as a eukaryotic gene reporter.

Bacterial luciferase from Vibrio campbellii is a thermostable enzyme with an in vitro thermal inactivation half-life of ~1020 min at 37°C. The enzyme also binds tightly to reduced FMN. In this study, a V. campbellii fusion luciferase construct in which the α and ß subunits are linked with a decapeptide was made and characterized. In general, the overall enzymatic properties of the two enzymes are similar. Expression of the enzymes in Escherichia coli demonstrated that the V. campbellii fusion luciferase emits less light than the native luciferase, but still emits a much greater amount of light than native luciferase from Vibrio harveyi and Photobacterium leiognathi TH1. The intensity of light emitted by the V. campbellii fusion luciferase was more than 80-fold greater than that from the V. harveyi native luciferase when expressed at 37°C. Biochemical characterization has shown that the V. campbellii fusion luciferase also retains a high binding affinity for reduced flavin mononucleotide and high thermostability. The levels of bioluminescence emitted by the V. campbellii fusion luciferase expressed in HEK293T cells reached ~1×10(6) Relative Light Units/mg total protein. These findings suggest that the V. campbellii fusion luciferase is a promising candidate for further development as a luciferase-based reporter for eukaryotic systems.