Ya-Lei Chen

Remediation of PCE-contaminated aquifer by an in situ two-layer biobarrier: laboratory batch and column studies

The industrial solvent tetrachloroethylene (PCE) is among the most ubiquitous chlorinated compounds found in groundwater contamination. The objective of this study was to develop an in situ two-layer biobarrier system consisting of an organic-releasing material layer followed by an oxygen-releasing material layer. The organic-releasing material, which contained sludge cakes from a domestic wastewater treatment plant, is able to release biodegradable organics continuously. The oxygen-releasing material, which contained calcium peroxide, is able to release oxygen continuously upon contact with water. The first organic-releasing material layer was to supply organics (primary substrates) to reductively dechlorinate PCE in situ. The second oxygen-releasing material layer was to release oxygen to aerobic biodegrade or cometabolize PCE degradation byproducts from the first anaerobic layer. Batch experiments were conducted to design and identify the components of the organic and oxygen-releasing materials, and evaluate the organic substrate (presented as chemical oxygen demand (COD) equivalent) and oxygen release rates from the organic-releasing material and oxygen-releasing materials, respectively. The observed oxygen and COD release rates were approximately 0.0368 and 0.0416 mg/d/g of material, respectively. A laboratory-scale column experiment was then conducted to evaluate the feasibility of this proposed system for the bioremediation of PCE-contaminated groundwater. This system was performed using a series of continuous-flow glass columns including a soil column, an organic-releasing material column, two consecutive soil columns, and an oxygen-releasing material column, followed by two other consecutive soil columns. Anaerobic acclimated sludges were inoculated in the first four columns, and aerobic acclimated sludges were inoculated in the last three columns to provide microbial consortia for contaminant biodegradation. Simulated PCE-contaminated groundwater with a flow rate of 0.25 L/d was pumped into this system. Effluent samples from each column were analyzed for PCE and its degradation byproducts. Results show that up to 99% of PCE removal efficiency was obtained in this passive system. Thus, the biobarrier treatment scheme has the potential to be developed into an environmentally and economically acceptable remediation technology for the in situ treatment of PCE-contaminated aquifer.

Effects of soil pH, temperature and water content on the growth of Burkholderia pseudomallei.

Optimum conditions were determined for the growth ofBurkholderia pseudomallei in natural soils or waters. It grows better in paddy soil, crop-covered and fallow field than in fresh and salty water. Although the optimal temperature and pH for the growth were 37 or 42 °C, and 6.5 or 7.5 in an environmental-mimicking soil medium, this bacterium can still grow at 4 °C, which was suggested to be related with the occurrence of melioidosis in some cold areas. In soil media with water content <15.B. pseudomallei did not grow until 60 d of incubation, suggesting that water contents of soils in which it dwelled would be one important factor in determining the growth rate.

CpG-Modified Plasmid DNA Encoding Flagellin Improves Immunogenicity and Provides Protection against Burkholderia pseudomallei Infection in BALB/c Mice

ABSTRACT The plasmid DNA encoding the fliC gene of Burkholderia pseudomallei combined with CpG oligodeoxynucleotide (ODN) was injected intramuscularly into BALB/c mice, resulting in the increased production of certain humoral antibodies and flagellin-specific spleen cell clonal expansion. CpG ODN, as an immunoadjuvant, was added to the plasmid containing the fliC gene in order to obtain ongoing expression in muscle for a long period. Functional expression of flagellin from the constructed CpG-modified plasmid in transfected peritoneal exudate cells of BALB/c mice was shown by reverse transcription-PCR and Western blotting. Furthermore, BALB/c mice immunized with the modified plasmid had relatively higher resistance to B. pseudomallei infection in vivo than did mice immunized with unmodified plasmid DNA. The time course of restricted bacterial growth in spleen and liver and changes in the cytokine profiles of immunized mice suggested that the stimulated phagocytic cells would be able to kill the bacteria eventually, possibly as a consequence of the induction of Th-1-type immune polarization in vivo. Th-1-type immune polarization was detected in response to flagellin induction in mice immunized with CpG-modified plasmid DNA by the appearance of increased levels of immunoglobulin G2a antibodies and gamma interferon-secreting cells specific to flagellin. The exogenous CpG motifs added to the fliC gene would contribute to an adjuvant-like response that enhances the flagellin-specific immunogenicity and provides protection against B. pseudomallei infection. This CpG-modified plasmid DNA vaccination is an important potential strategy that should be developed to protect against melioidosis.

Expression and Immunogenicity of Mycoplasma hyopneumoniae Heat Shock Protein Antigen P42 by DNA Vaccination

ABSTRACT Mycoplasma hyopneumoniae is the etiological agent of swine enzootic pneumonia, a chronic nonfatal disease affecting pigs of all ages. The goal of this study was to design DNA vaccines by constructing plasmid pcDNA3/P42, carrying the heat shock protein gene P42 of M. hyopneumoniae, and to evaluate the immune responses elicited in BALB/c mice. The expression of P42 was first examined in transfected NIH 3T3 cells by reverse transcription-PCR to ensure that the construct was functional. The humoral and cell-mediated immune responses induced by the plasmid were further evaluated in BALB/c mice through intramuscular injection. Both immunoglobulin G1 (IgG1) and IgG2a levels were 64 times those of the control groups during the first 8 weeks. The levels of interleukin-2 (IL-2), IL-4, and gamma interferon mRNAs in the immunized animals were elevated, and the proliferation of spleen cells was also enhanced in the immunized animals. The results indicate that pcDNA3/P42 DNA immunization induces both Th1 and Th2 immune responses. In addition, antiserum from the immunized animals was found to inhibit the growth of M. hyopneumoniae. The present study reveals that DNA vaccination could be a new strategy against infection by M. hyopneumoniae and may have potential for developing vaccines for other infectious diseases as well.

Prevalence of Melioidosis in the Er-Ren River Basin, Taiwan: Implications for Transmission

ABSTRACT An increase in melioidosis cases compared to other areas in Taiwan was observed in the Er-Ren River Basin, southwestern Taiwan, from November 2001 to August 2006. The objective of this study was to determine the association between the level of exposure to Burkholderia pseudomallei and the incidence rate of melioidosis and to survey the transmission modes of B. pseudomallei in the Er-Ren River Basin. The serosurveillance of melioidosis gave seropositivity rates of 36.6%, 21.6%, and 10.9%, respectively, for residents in regions A, B, and C within the Er-Ren Basin area. Culture and PCR-based detection of B. pseudomallei from soil demonstrated that the geographical distribution of this bacterium was confined to a particular site in region B. The distribution of seropositive titers was significantly associated with the incidence rate of melioidosis (120, 68, or 36 incidence cases per 100,000 population in region A, B, or C in 2005), whereas it did not correlate with the geographical distribution of B. pseudomallei within the soil. A survey of transmission modes showed that residents with seropositivity were linked to factors such as having confronted flooding and having walked barefoot on soil, which are potential risk factors associated with exposure to B. pseudomallei. Our findings indicated that the Er-Ren River Basin in Taiwan has the potential to become a high-prevalence area for melioidosis. This is the first report that documents a high prevalence of melioidosis in an area north of latitude 20°N.

Distribution of Melioidosis Cases and Viable Burkholderia pseudomallei in Soil: Evidence for Emerging Melioidosis in Taiwan

ABSTRACT A survey for the prevalence if Burkholderia pseudomallei in soil in Taiwan found that its incidence is comparable to that in other regions of the world where melioidosis is endemic. The presence of identical genetic patterns among the clinical and environmental isolates evaluated suggested a link between the pathogens present in contaminated soil and the emergence of indigenous melioidosis.

Recombinant Truncated Flagellin of Burkholderia pseudomallei as a Molecular Probe for Diagnosis of Melioidosis

ABSTRACT Current serological tests for melioidosis, using impure or uncharacterized cell antigens from Burkholderia pseudomallei, have problems in detection sensitivity and specificity. Therefore, we designed and expressed the recombinant flagellin (truncated at both the N- and C-terminal ends), and used the antigen to develop an indirect enzyme-linked immunosorbent assay (ELISA) to diagnose melioidosis. Comparison of the immunoreactivities of the full-length and truncated flagellins reveals that the truncated flagellin performed much better in detection specificity and sensitivity. Only the full-length flagellin was recognized by other bacterial causing septicemia and gave a false-positive result in Western analysis, indicating that the cross-reactive epitopes were located on the more highly conserved N- and C-terminal regions of flagellin. The indirect ELISA using recombinant truncated flagellin as the antigen achieved 93.8% sensitivity and 96.3% specificity and offered a more efficient serodiagnosis of melioidosis.

Presence of the exoU Gene of Pseudomonas aeruginosa Is Correlated with Cytotoxicity in MDCK Cells but Not with Colonization in BALB/c Mice

ABSTRACT A total of 141 independent strains of Pseudomonas aeruginosa with different heterogeneities in the exo gene (exoS, exoT, exoU, and exoY) background were examined for their pathogenic roles. Results indicated that the exoU gene is the major contributor to cytotoxicity in Madin-Darby canine kidney cells but is not related to bacterial colonization in mice.

The Use of Nanoscale Visible Light-Responsive Photocatalyst TiO2-Pt for the Elimination of Soil-Borne Pathogens

Exposure to the soil-borne pathogens Burkholderia pseudomallei and Burkholderia cenocepacia can lead to severe infections and even mortality. These pathogens exhibit a high resistance to antibiotic treatments. In addition, no licensed vaccine is currently available. A nanoscale platinum-containing titania photocatalyst (TiO2-Pt) has been shown to have a superior visible light-responsive photocatalytic ability to degrade chemical contaminants like nitrogen oxides. The antibacterial activity of the catalyst and its potential use in soil pathogen control were evaluated. Using the plating method, we found that TiO2-Pt exerts superior antibacterial performance against Escherichia coli compared to other commercially available and laboratory prepared ultraviolet/visible light-responsive titania photocatalysts. TiO2-Pt-mediated photocatalysis also affectively eliminates the soil-borne bacteria B. pseudomallei and B. cenocepacia. An air pouch infection mouse model further revealed that TiO2-Pt-mediated photocatalysis could reduce the pathogenicity of both strains of bacteria. Unexpectedly, water containing up to 10% w/v dissolved soil particles did not reduce the antibacterial potency of TiO2-Pt, suggesting that the TiO2-Pt photocatalyst is suitable for use in soil-contaminated environments. The TiO2-Pt photocatalyst exerted superior antibacterial activity against a broad spectrum of human pathogens, including B. pseudomallei and B. cenocepacia. Soil particles (<10% w/v) did not significantly reduce the antibacterial activity of TiO2-Pt in water. These findings suggest that the TiO2-Pt photocatalyst may have potential applications in the development of bactericides for soil-borne pathogens.

Burkholderia multivorans acts as an antagonist against the growth of Burkholderia pseudomallei in soil

In this study, it was demonstrated, by using agar diffusion tests and a Transwell system, that Burkholderia multivorans NKI379 has an antagonistic effect against the growth of B. pseudomallei. Bacterial representatives were isolated from agricultural crop soil and mixed to construct a partial bacterial community structure that was based on the results of reproducible patterns following PCR‐denaturing gradient gel electrophoresis analysis of total soil chromosomes. The antagonistic effect of B. multivorans on B. pseudomallei was observed in this imitate community. In a field study of agricultural crop soil, the presence of B. pseudomallei was inversely related to the presence of the antagonistic strains B. multivorans or B. cenocepacia. B. multivorans NKI379 can survive in a broader range of pH, temperatures and salt concentrations than B. pseudomallei, suggesting that B. multivorans can adapt to extreme environmental changes and therefore predominates over B. pseudomallei in natural environments.