Transgenic chloroplasts have become attractive systems for heterologous gene expressions because of unique advantages. Here, we report a feasibility study for producing the nontoxic B subunit of Escherichia coli heat-labile enterotoxin (LTB) via chloroplast transformation of tobacco. Stable site-specific integration of the LTB gene into chloroplast genome was confirmed by PCR and genomic Southern blot analysis in transformed plants. Immunoblot analysis indicated that plant-derived LTB protein was oligomeric, and dissociated after boiling. Pentameric LTB molecules were the dominant molecular species in LTB isolated from transgenic tobacco leaf tissues. The amount of LTB protein detected in transplastomic tobacco leaf was approximately 2.5% of the total soluble plant protein, approximately 250-fold higher than in plants generated via nuclear transformation. The GM1-ELISA binding assay indicated that chloroplast-synthesized LTB protein bound to GM1-ganglioside receptors. LTB protein with biochemical properties identical to native LTB protein in the chloroplast of edible plants opens the way for inexpensive, safe, and effective plant-based edible vaccines for humans and animals.

The cholera toxin B subunit ͑CTB͒ contains five identical polypeptides and targets glycosphingolipid receptors on eukaryotic cell surfaces. Increased expression of CTB in plants is critical for the development of edible vaccines. In this study, the coding sequence of the CTB gene was optimized, based on the modification of codon usage to that of tobacco plant genes and the removal of mRNA-destabilizing sequences. The synthetic CTB gene was cloned into a plant expression vector and expressed in tobacco plants under the control of the CaMV 35S promoter. The recombinant CTB protein constituted approximately 1.5% of the total soluble protein in transgenic tobacco leaves. This level of CTB production was approximately 15-fold higher than that in tobacco plants that were transformed with the bacterial CTB gene. The recombinant CTB produced by tobacco plants demonstrated strong affinity for GM1-ganglioside, which indicates that the sites required for binding and proper folding of the pentameric CTB structure were conserved. This is the first report on the optimization of the CTB-coding sequence to give a dramatic increase in CTB expression in plants.

. No difference was observed in the time to defervescence or length of hospital stay between the two groups (P > 0.05). In complicated scrub typhus patients (n ‫؍‬ 181), multivariate analysis showed that only APACHE II score was an independent risk factor for mortality (95% confidence interval, 1.11 to 1.56; P < 0.001). Our data suggest that outcomes of azithromycin therapy are comparable to those of doxycycline therapy in patients with complicated scrub typhus.

Escherichia coli heat-labile toxin (LT) is a potent mucosal immunogen and immunoadjuvant for coadministered antigens. We synthesized a gene encoding the B-subunit of LT (LTB) adapted to the coding sequence of tobacco plants and fused to the endoplasmic reticulum retention signal SEKDEL to enhance its level of expression in plants. The synthetic LTB gene was cloned into a plant expression vector adjacent to the CaMV 35S promoter and was introduced into tobacco by Agrobacterium-mediated transformation. The amount of LTB protein detected in transgenic tobacco leaves was 2.2% of the total soluble plant protein, which is approx 200-fold higher than in previous reports of native LTB gene expression in transgenic plants. Enzyme-linked immunosorbent assay indicated that plant-synthesized LTB protein bound specifically to GM1-ganglioside, suggesting that the LTB subunits formed active pentamers.

BackgroundMethicillin-resistant Staphylococcus aureus (MRSA) is one of the most common causes of infection in the intensive care unit (ICU). Although surveillance culture for MRSA is recommended for ICU patients, no comparative study investigating the optimal sites and frequency of culture has been performed in this population.MethodsA prospective observational cohort study was performed in an 18-bed emergency intensive care unit (EICU) in a tertiary teaching hospital. A total of 282 patients were included. Samples for MRSA detection were obtained at the time of admission, 48 h after admission, and then weekly thereafter. All subjects were routinely monitored for the development of MRSA infection during their stay in the ICU.ResultsMRSA colonization was detected in 129 (46%) patients over the course of the study. The sensitivity of MRSA surveillance culture was significantly higher in throat or tracheal aspirates (82%; 106/129) than in anterior nares (47%; 61/129) (P<0.001). The sensitivity of MRSA surveillance culture for subsequent MRSA infection and MRSA pneumonia was also higher in the throat/trachea (69 and 93%, respectively) than in the anterior nares (48 and 50%, respectively). The area under the curve for subsequent MRSA infection was higher in trachea/throat (0.675) than in the anterior nares (0.648); however, this difference was not significant (P>0.05). The area under the curve for MRSA pneumonia was significantly higher in trachea/throat (0.791; 95% CI, 0.739-0.837) than anterior nares (0.649; 95% CI, 0.590-0.705) (P = 0.044).ConclusionMRSA colonization was more common in the trachea/throat than in the anterior nares in ICU patients. Cultures from throat or tracheal aspirates were more sensitive and predictive of subsequent MRSA pneumonia than cultures from the anterior nares in this population.