PCR combined with lateral flow dipstick assay (PCR-LFD) for a rapid diagnosis of melioidosis.

BACKGROUND: Melioidosis is an infectious disease caused by Burkholderia pseudomallei. Septicemic melioidosis patients have a high mortality rate within 48 hours. OBJECTIVE: To develop a polymerase chain reaction (PCR) combined with a lateral flow dipstick (LFD) assay for detection of B. pseudomallei in blood samples. METHODS: The PCR with wcbG gene primers and a PCR-LFD test were developed. The specificity and sensitivity were determined using the B. pseudomallei and other bacterial DNAs. They were evaluated using 43 B. pseudomallei positive blood samples and another 43 blood samples positive for other microbial infections. RESULTS: The detection limit of the PCR-LFD test was 50 fg of bacterial gDNA or 1.0 CFU per 200 µl of blood. All B. pseudomallei were positive while B. thailandensis and selected gram-negative bacterial strains were negative. The PCR-LFD gave all positives with all 43 B. pseudomallei culture positive patient blood samples and all negative with 43 blood samples that were culture positive for K. pneumoniae, E. gallinarum, E. faecium, E. coli, S. aureus, A. baumannii, A. hydrophila, S. haemolyticus, S. pneumoniae, P. aeruginosa, E. cloacae, S. hominis, E. aerogenes, P. mirabilis, C. neoformans, C. albicans, A. caviae, E. faecalis and K. variicola. CONCLUSION: The developed PCR-LFD assay provided 100% sensitivity and 100% specificity compared to the conventional blood culture. The technique took only 1.5 hours that is easy and quick to perform compared to the 3-7 days of culture method. The new method of PCR with LFD could facilitate the detection to be a semi-point-of-care testing (POCT).

A two-arm analysis of the immune response to heterologous boosting of inactivated SARS-CoV-2 vaccines.

Several vaccine programs were introduced during the COVID-19 pandemic, which included inactivated virus, DNA viral vectors and mRNA vaccines. Booster programs are recommended, especially for those in high-risk groups. However, many of these booster programs involve heterologous vaccines. This study enrolled volunteers who first received two full-dose CoronaVac vaccinations before receiving heterologous boosters with DNA- and/or mRNA-vaccines for an additional 2 doses (n = 40) or an additional 3 doses (n = 16). Our results showed no difference in side effects, neutralizing antibodies, or T-cell responses for any of the heterologous vaccination programs. However, the neutralizing capacity and IFN-γ responses against the Omicron variant in volunteers who received 4 or 5 doses were improved. Polarization of peripheral memory T cells after stimulation in all booster groups with Omicron peptide showed an increased trend of naïve and central memory phenotypes of both CD4+ and CD8+ T cells, suggesting that exposure to Omicron antigens will drive T cells into a lymphoid resident T cell phenotype. Our data support a continuous vaccination program to maximize the effectiveness of immunity, especially in people at high risk. Furthermore, the number of boosting doses is important for maintaining immunity.

Early Activation of iNKT Cells Increased Survival Time of BALB/c Mice in a Murine Model of Melioidosis.

Melioidosis is an infectious disease caused by Burkholderia pseudomallei. High interferon gamma (IFN-γ) levels in naive mice were reported to mediate protection against B. pseudomallei infection. Invariant natural killer T (iNKT) cells can produce and secrete several cytokines, including IFN-γ. When iNKT cell-knockout (KO) BALB/c mice were infected with B. pseudomallei, their survival time was significantly shorter than wild-type mice. Naive BALB/c mice pretreated intraperitoneally with α-galactosylceramide (α-GalCer), an iNKT cell activator, 24 h before infection demonstrated 62.5% survival at the early stage, with prolonged survival time compared to nonpretreated infected control mice (14 ± 1 days versus 6 ± 1 days, respectively). At 4 h after injection with α-GalCer, treated mice showed significantly higher levels of serum IFN-γ, interleukin-4 (IL-4), IL-10, and IL-12 than control mice. Interestingly, the IFN-γ levels in the α-GalCer-pretreated group were decreased at 4, 24, and 48 h after infection, while they were highly increased in the control group. At 24 h postinfection in the α-GalCer group, bacterial loads were significantly lower in blood (no growth and 1,780.00 ± 51.21, P < 0.0001), spleens (no growth and 34,300 ± 1,106.04, P < 0.0001), and livers (1,550 ± 68.72 and 13,400 ± 1,066.67, P < 0.0001) than in the control group, but not in the lungs (15,300 ± 761.10 and 1,320 ± 41.63, P < 0.0001), and almost all were negative at 48 h postinfection. This study for the first time shows that early activation of iNKT cells by α-GalCer helps clearance of B. pseudomallei and prolongs mouse survival.

Phenotypic and functional changes of T cell subsets after CoronaVac vaccination.

BACKGROUND: The pandemic coronavirus disease 2019 (COVID-19) is a major global public health concern and several protective vaccines, or preventive/therapeutic approaches have been developed. Sinovac-CoronaVac, an inactivated whole virus vaccine, can protect against severe COVID-19 disease and hospitalization, but less is known whether it elicits long-term T cell responses and provides prolonged protection. METHODS: This is a longitudinal surveillance study of SARS-CoV-2 receptor binding domain (RBD)-specific IgG levels, neutralizing antibody levels (NAb), T cell subsets and activation, and memory B cells of 335 participants who received two doses of CoronaVac. SARS-CoV-2 RBD-specific IgG levels were measured by enzyme-linked immunosorbent assay (ELISA), while NAb were measured against two strains of SARS-CoV-2, the Wuhan and Delta variants. Activated T cells and subsets were identified by flow cytometry. Memory B and T cells were evaluated by enzyme-linked immune absorbent spot (ELISpot). FINDINGS: Two doses of CoronaVac elicited serum anti-RBD antibody response, elevated B cells with NAb capacity and CD4+ T cell-, but not CD8+ T cell-responses. Among the CD4+ T cells, CoronaVac activated mainly Th2 (CD4+ T) cells. Serum antibody levels significantly declined three months after the second dose. INTERPRETATION: CoronaVac mainly activated B cells but T cells, especially Th1 cells, were poorly activated. Activated T cells were mainly Th2 biased, demonstrating development of effector B cells but not long-lasting memory plasma cells. Taken together, these results suggest that protection with CoronaVac is short-lived and that a third booster dose of vaccine may improve protection.

Decrease of spleen invariant natural killer T and natural killer cell numbers in Burkholderia thailandensis-infected C57BL/6 mice.

BACKGROUND: Burkholderia thailandensis is a non-pathogenic bacterium that is closely related to B. pseudomallei. Invariant natural killer T (iNKT) cells are innate lymphoid cells that play a role in bacterial infections; however the iNKT cells in B. thailandensis infections are still uncharacterized. OBJECTIVE: To study the cytokine production in α-galactosylceramide (α-GalCer)-stimulated lymphocytes from mouse organs. The numbers of spleen iNKT cells, natural killer (NK) cells, dendritic cells and macrophages in B. thailandensis- infected C57BL/6 (B6) mice were investigated. METHODS: Lymphocytes, obtained from mouse lungs, liver, and spleen, were cultured for 48 hours with α-GalCer, and their cytokine levels were determined. iNKT, dendritic, macrophage and NK cells in the spleen of B. thailandensis-infected B6 mice or iNKT knock out (KO) mice, stimulated with either phosphate-buffered saline (PBS) or α-GalCer, were analyzed by flow cytometry. This was also done in adoptive cell transfer experiments. RESULTS: Interferon gamma (IFN-γ) was predominantly produced in α-GalCer-stimulated mouse spleen and liver lymphocytes, while interleukin (IL)-13 was the main cytokine found in the lungs. B. thailandensis-infected mice had a significantly lower number of splenic iNKT, NK and dendritic cells, but not macrophages, compared to the control. Interestingly, the number of NK cells was significantly decreased in iNKT wild type and iNKT KO mice after B. thailandensis infection. The number of NK cells recovered by activation with α-GalCer or after adoptive transfer of iNKT cells into KO mice. The iNKT cell-mediated reduction of dendritic and NK cells might be related to infection by B. thailandensis. CONCLUSIONS: B. thailandensis decreased the number of iNKT and NK cells in the spleen of infected mice.

Invariant Natural Killer T (iNKT) cells response in human melioidosis.

BACKGROUND: Melioidosis is an infectious disease caused by Burkholderia pseudomallei. In infected mice, IFN-γ can provide protection against B. pseudomallei infection. Invariant Natural Killer T (iNKT) cells are a subpopulation of T lymphocytes, activated by recognition of glycolipid ligands such as α-Galactosylceramide presented by CD1d, produce and secrete several cytokines, including IFN-γ and IL-4. The response of iNKT cells in human melioidosis was then investigated. OBJECTIVE: To determine the iNKT cells response in human melioidosis. METHODS: The number of human iNKT cells and its activation states were investigated in sepsis melioidosis patients compared with healthy controls using flow cytometry. The iNKT cells activation was confirmed in vitro using heatkilled B. pseudomallei with normal peripheral blood mononuclear cells. The components induced iNKT cell were also determined using different concentration of B. pseudomallei lipopolysaccharide (LPS), heat-killed B. pseudomallei treated with or without DNase, RNase, or proteinase. RESULTS: The number of human iNKT cells was significantly lower while the percentage of activated iNKT cells was higher in sepsis melioidosis when compared to control. In addition, B. pseudomallei can stimulate human iNKT cells in vitro. Heat-killed B. pseudomallei could activate iNKT cells but not relate to nucleic acid, proteins, or LPS. CONCLUSIONS: We found for the first time that the iNKT cells were activated during B. pseudomallei infection in human. However, the roles and the mechanism of iNKT cells during early state of infection needed to be further investigated.

Evaluation of antigen-detecting and antibody-detecting diagnostic test combinations for diagnosing melioidosis.

BACKGROUND: Melioidosis, an infectious disease caused by Burkholderia pseudomallei, is endemic in many tropical developing countries and has a high mortality. Here we evaluated combinations of a lateral flow immunoassay (LFI) detecting B. pseudomallei capsular polysaccharide (CPS) and enzyme-linked immunosorbent assays (ELISA) detecting antibodies against hemolysin co-regulated protein (Hcp1) or O-polysaccharide (OPS) for diagnosing melioidosis. METHODOLOGY/PRINCIPAL FINDINGS: We conducted a cohort-based case-control study. Both cases and controls were derived from a prospective observational study of patients presenting with community-acquired infections and sepsis in northeast Thailand (Ubon-sepsis). Cases included 192 patients with a clinical specimen culture positive for B. pseudomallei. Controls included 502 patients who were blood culture positive for Staphylococcus aureus, Escherichia coli or Klebsiella pneumoniae or were polymerase chain reaction assay positive for malaria or dengue. Serum samples collected within 24 hours of admission were stored and tested using a CPS-LFI, Hcp1-ELISA and OPS-ELISA. When assessing diagnostic tests in combination, results were considered positive if either test was positive. We selected ELISA cut-offs corresponding to a specificity of 95%. Using a positive cut-off OD of 2.912 for Hcp1-ELISA, the combination of the CPS-LFI and Hcp1-ELISA had a sensitivity of 67.7% (130/192 case patients) and a specificity of 95.0% (477/502 control patients). The sensitivity of the combination (67.7%) was higher than that of the CPS-LFI alone (31.3%, p<0.001) and that of Hcp1-ELISA alone (53.6%, p<0.001). A similar phenomenon was also observed for the combination of CPS-LFI and OPS-ELISA. In case patients, positivity of the CPS-LFI was associated with a short duration of symptoms, high modified Sequential (sepsis-related) Organ Failure Assessment (SOFA) score, bacteraemia and mortality outcome, while positivity of Hcp1-ELISA was associated with a longer duration of symptoms, low modified SOFA score, non-bacteraemia and survival outcome. CONCLUSIONS/SIGNIFICANCE: A combination of antigen-antibody diagnostic tests increased the sensitivity of melioidosis diagnosis over individual tests while preserving high specificity. Point-of-care tests for melioidosis based on the use of combination assays should be further developed and evaluated.

Opisthorchis viverrini antigens up-regulates the expression of CD80 and MHC class II in JAWSII mouse dendritic cells and promotes IL-10 and TGF-ß secretions.

Dendritic cells (DCs) are antigen-presenting cells (APC) involved in the initiation of immune responses. Maturation of DCs is characterized by the high expression of major histocompatibility complex (MHC) class II and co-stimulatory clusters of differentiation (CD) 40, CD80, and CD86 molecules. Matured DCs are required for T cell differentiation and proliferation. However, the response of DCs to Opisthorchis viverrini antigens has not yet been understood. Therefore, this study sought to determine the expression of surface molecules of JAWSII mouse DCs stimulated by crude somatic (CS) and excretory-secretory (ES) antigens of O. viverrini. ES antigen significantly induced only mRNA expression of CD80 and MHC class II in JAWSII mouse DCs, while CS antigen promoted up-regulation of both mRNA and protein levels of CD80 and MHC class II, indicating relative maturation of JAWII mouse DCs. Moreover, the secreted cytokines from the co-cultures of O. viverrini antigens stimulated JAWSII DC with naïve CD4+ T cells was determined. Significantly increased levels of immunosuppressive cytokines interleukin (IL)-10 and transforming growth factor beta (TGF-ß) were found. The up-regulation of these cytokines may indicate the response of regulatory T cells (Treg) to CS antigen-stimulated JAWSII DC. These findings may lead to a better understanding of the role that DCs play in O. viverrini infection.

Development of an immunomagnetic separation-ELISA for the detection of Burkholderia pseudomallei in blood samples.

BACKGROUND: Septicemic melioidosis caused by Burkholderia pseudomallei is a serious cause of morbidity and mortality. An effective, rapid and simple diagnostic method is required for detection of B. pseudomallei infection. OBJECTIVE: To develop immunomagnetic beads (IMB) coupled with ELISA (IMB-ELISA) for detection of B. pseudomallei in blood samples of patients with suspected melioidosis. METHODS: For separation of B. pseudomallei from buffer, blood samples and hemoculture, 200 nm immunomagnetic beads (IMBs) coated with 4B11 monoclonal antibody (4B11-IMBs) against exopolysaccharide antigens were used. The detection was done by an ELISA based biotin-streptavidin system. The sensitivity and specificity were evaluated. RESULTS: 4B11-IMBs (100 µg) were successfully developed and used for detection of B. pseudomallei in 1 ml samples. Transmission electron microscopy (TEM) imaging demonstrated B. pseudomallei was captured by 4B11-IMBs. The IMBs showed high capture efficiency (98%) with B. pseudomallei in buffer. The IMB-ELISA assay was highly specific for B. pseudomallei. It showed no cross-reactions with other bacteria, except B. mallei. The limits of the B. pseudomallei assay detection for detecting B. pseudomallei in either buffer solution or blood was 102 CFU/ml. The IMB-ELISA detection sensitivity in blood samples was 44.5%. Although it did not give the highest sensitivity, it was useful for detection with hemoculture that was faster than conventional methods. CONCLUSION: This study suggests the IMB-ELISA assay offers a simple and highly specific method with a turnaround time of 6 h for detection of B. pseudomallei. The developed assay can be applied in hospitals for surveillance of B. pseudomallei.

D-LL-31 in combination with ceftazidime synergistically enhances bactericidal activity and biofilm destruction in Burkholderia pseudomallei.

Melioidosis is a severe disease caused by Burkholderia pseudomallei. The biofilm of B. pseudomallei acquires resistance to several antibiotics and may be related to relapse in melioidosis patients. Here, the killing activity of antimicrobial peptides (LL-37, LL-31) and the D-enantiomers (D-LL-37, D-LL-31) in combination with ceftazidime (CAZ) against B. pseudomallei 1026b, H777 and a biofilm mutant M10, derived from H777 grown under biofilm-stimulating conditions was observed. Using static conditions, D-LL-31 exhibited the strongest killing activity against the three isolates in a dose-dependent manner. IC50 values for D-LL-31 ranged from 1 to 6 µM, for isolates M10, H777, and 1026b, respectively. Moreover, D-LL-31 combined with CAZ synergistically decreased the IC50 values of the peptide and antibiotic and caused also disruption of biofilms of B. pseudomallei 1026b under flow conditions. Thus a combination of D-LL-31 and CAZ may enhance the efficacy of the currently used antibiotic treatments against B. pseudomallei.

Characterization and functional analysis of novel circulating NK cell sub-populations.

Natural killer (NK) cells are innate lymphoid cells having potent cytolytic function that provide host defense against microbial infections and tumors. Using our generated monoclonal antibody (mAb), named FE-1H10, new NK cell sub-populations in peripheral blood were identified. The molecules recognized by mAb FE-1H10 were expressed on a sub-population of CD3-CD56dim NK cells. The epitope recognized by mAb FE-1H10 was demonstrated to be N-glycan and proven to be different from CD57. Upon K562 stimulation, the CD56dimFE-1H10+ NK cell sub-population exhibited significantly lower cytolytic function with low ability to degranulate and release cytolytic granules compared to the CD56dimFE-1H10- NK cell sub-population. Moreover, the CD56dimFE-1H10+ NK cells produced less IFN-γ and TNF-α than the CD56dimFE-1H10- NK cells. We demonstrated here that mAb FE-1H10 could identify two sub-populations of circulating CD56dim NK cells with different functions. Our discovery of new sub-populations of NK cells improves our understanding of NK cell biology and may lead to the development of new approaches for NK cell therapy.

Burkholderia pseudomallei-absent soil bacterial community results in secondary metabolites that kill this pathogen.

Burkholderia pseudomallei is a Gram-negative bacterium found in soil and the causative agent of a severe disease in humans and animals known as melioidosis. It is intrinsically resistant to many antibiotics and has been reported resistant to the drugs of choice; ceftazidime. Microbial communities in soil in the presence and absence of B. pseudomallei were investigated using metagenomics approach. The variation in bacterial species diversity was significantly higher in soil samples without B. pseudomallei. Abundances of phyla Actinobacteria and Firmicutes were found significantly higher in B. pseudomallei-negative soils. Bacillus amyloliquefaciens KKU1 in phylum Firmicutes was discovered from negative soil and its secondary metabolites could inhibit clinical, environmental and drug resistant isolates of B. pseudomallei, together with some pathogenic Gram-negative but not Gram-positive bacteria. The antimicrobial activity from KKU 1 against B. pseudomallei was abolished when treated with proteinase K, stable in a wide range of pH and remained active after heating at 100 °C for 15 min. Precipitated proteins from KKU1 were demonstrated to cause lysis and corrugated surfaces of B. pseudomallei. The minimum inhibitory concentrations and minimum bactericidal concentrations of the precipitated proteins from KKU1 against B. pseudomallei were 0.97 µg/ml and 3.9 µg/ml. Interestingly, Native SDS-PAGE showed small active compounds of less than 6 kDa, along with other information collectively suggesting the properties of antimicrobial peptides. For the first time, culture-independent information in melioidosis endemic area could lead to a suspected source of metabolites that may help defense against B. pseudomallei and other pathogenic Gram-negative bacteria.

Hamster IFN-γ+CD4+ and IL-4+CD4+ T cell responses against leptospires are significantly higher than those of mice.

BACKGROUND: Leptospirosis is a bacterial disease caused by the Leptospira interrogans. The hamster is considered a susceptible host while the mouse is resistant. The knowledge of hamster T cell immunity is limited compared to the mouse. The reason why the hamster and the mouse give different responses to leptospires remains unclear. OBJECTIVE: To determine the differential responses of CD4+ T cells between hamsters and mice using Leptospira interrogans as an infectious model. METHODS: The CD4+ T-cell reactivity and their intracellular cytokine responses after infection with live L.interrogans serovar Autumnalis or leptospiral antigens, or injection with recombinant LipL32 protein (rLipL32) were elucidated. For secondary immune responses, mononuclear cells were re-stimulated with leptospiral crude antigens (LAg) or rLipL32. Intracellular cytokines and CD4+ T cells were determined using flow cytometry. RESULTS: There were no significant differences between the percentages of hamster and mouse CD4+ and CD25+CD4+ T cell responses to live bacteria. Mouse CD4+ (24.50±1.98%) and CD25+CD4+ T cells (3.83±0.88) responded significantly higher than those of hamster (15.07±2.82% and 2.00±0.37%) when infected and re-stimulated with LAg. The numbers of IFN-γ and IL-4 producing cells in hamsters at 1.76±0.10% and 0.82±0.25% for IFN-γ+CD4+ and IL-4+CD4+ T cells were significantly higher than those in resistant mice at 0.10±0.02% and 0.23±0.03% for IFN-γ+CD4+ and IL-4+CD4+ T cells. CONCLUSION: Hamsters responded significantly higher in secondary stimulation especially in the levels of the IFN-γ+ and IL-4+CD4+ T cells. The mechanisms of this dissimilarity remain to be elucidated.

Ocular involvement in melioidosis: a 23-year retrospective review.

BACKGROUND: Ocular involvement in melioidosis is rare and has devastating outcomes. Although there have been few reports on the condition, Khon Kaen, a city in northeast Thailand, has been called the "capital of melioidosis" due to the high prevalence of the condition in the region. We retrospectively reviewed all admitted cases of melioidosis with ocular involvement from the two largest hospitals in Khon Kaen. We reviewed cases from Srinagarind Hospital (a university hospital) of patients admitted between 1993 and 2016 and from Khon Kaen Hospital (a provincial hospital) of patients who presented from 2012 to 2016. RESULTS: We identified 16 cases of ocular involvement. Eight of these cases were proven from positive culture, and the remaining eight were implied from high melioidosis titer. The prevalence was estimated as being from 0.49 to 1.02%. Most patients had underlying diseases (14, 88%), of which diabetes mellitus was the most prevalent (12, 75%). Nine cases (56%) were part of disseminated septicemia. Patients suffered from blindness in 11 (73%) of the 15 cases in which visual acuity was recorded. Orbital cellulitis was the most common manifestation (7, 44%) followed by endophthalmitis (4, 25%). Interestingly, all patients with necrotizing fasciitis (100%) developed septic shock as a consequence. In most of the cases, patients underwent surgery (13, 81%) including incision and drainage, debridement, and pars plana vitrectomy. Despite appropriate management, the visual outcomes were disappointing (9, 64%). CONCLUSION: To summarize, ocular melioidosis is a highly destructive disease. Early detection and prompt surgical management may reduce morbidity and mortality from septic shock.

BURKHOLDERIA PSEUDOMALLEI BIOFILM PLAYS A KEY ROLE IN CHRONIC INFLAMMATION IN C57BL/6 MICE.

Burkholderia pseudomallei is a causative agent of melioidosis. Clinical signs of melioidosis vary from acute septicemia to chronic inflammation or subclinical infection. This study investigated the role of B. pseudomallei biofilm in chronic inflammation in lungs of infected C57BL/6 mice. Low doses of B. pseudomallei H777 and its biofilm defective M10 mutant were fed intra-gastrically to C57BL/6 mice and inflammatory responses were investigated by histopathological techniques. Two hundred colony forming units (CFUs) of B. pseudomallei H777 induced chronic inflammatory responses in mice on day 20 post-infection, with discrete interstitial infiltration by mononuclear inflammatory cells. On day 40 postinfection, there were marked thickening of alveolar septa and congested capillaries, which increased in severity by day 60. On the other hand, mice infected with B. pseudomallei M10 showed less mononuclear infiltration. The results indicate that B. pseudomallei defective in biofilm production gave rise to less severe pathology, resulting a higher rate of survival in infected mice; and pulmonary melioidosis could be developed in C57BL/6 mice by intra-gastric feeding makes it a possible animal model of chronic human melioidosis.

Burkholderia pseudomallei resistance to antibiotics in biofilm-induced conditions is related to efflux pumps.

Burkholderia pseudomallei, the causative agent of melioidosis, has been found to increase its resistance to antibiotics when growing as a biofilm. The resistance is related to several mechanisms. One of the possible mechanisms is the efflux pump. Using bioinformatics analysis, it was found that BPSL1661, BPSL1664 and BPSL1665 were orthologous genes of the efflux transporter encoding genes for biofilm-related antibiotic resistance, PA1874-PA1877 genes in Pseudomonas aeruginosa strain PAO1. Expression of selected encoding genes for the efflux transporter system during biofilm formation were investigated. Real-time reverse transcriptase PCR expression of amrB, cytoplasmic membrane protein of AmrAB-OprA efflux transporter encoding gene, was slightly increased, while BPSL1665 was significantly increased during growth of bacteria in biofilm formation. Minimum biofilm inhibition concentration and minimum biofilm eradication concentration (MBEC) of ceftazidime (CTZ), doxycycline (DOX) and imipenem were found to be 2- to 1024-times increased when compared to their MICs for of planktonic cells. Inhibition of the efflux transporter by adding phenylalanine arginine ß-napthylamide (PAßN), a universal efflux inhibitor, decreased 2 to 16 times as much as MBEC in B. pseudomallei biofilms with CTZ and DOX. When the intracellular accumulation of antibiotics was tested to reveal the pump inhibition, only the concentrations of CTZ and DOX increased in PAßN treated biofilm. Taken together, these results indicated that BPSL1665, a putative precursor of the efflux pump gene, might be related to the adaptation of B. pseudomallei in biofilm conditions. Inhibition of efflux pumps may lead to a decrease of resistance to CTZ and DOX in biofilm cells.

CpG oligodeoxynucleotides with crude parasite antigens reduce worm recovery in Opisthorchis viverrini infected hamsters.

Opisthorchis viverrini, a human liver fluke, is still an endemic parasitic infection in Thailand and nearly all countries in Southeast Asia. O. viverrini induces a chronic stage of infection in hamsters. During the first 2 weeks of infection, Th1 inducing cytokine, IL-12, increased but was down regulated in chronic infection. In this study it was found that unmethylated-CpG ODN (oligodeoxynucleotides) 1826 increased hamster mononuclear cell proliferation and stimulated IFN-γ production in vitro. The IFN-γ levels in hamster sera were significantly increased in hamsters injected with CpG ODN 1826 alone or plus crude somatic antigens (CSAg). Further investigation using the flow cytometer found that CD4+T cells and IFN-γ+ CD4+T cells (Th1-like cells) in the hamster blood were significantly increased. The role of these cells in the protective responses in hamsters was evaluated by challenging with 25 metacercaria and observation for 3 months. The number of worms recovered was significantly reduced in the hamsters injected with CpG ODN 1826 with CSAg, but not in CpG ODN 1826 alone groups when compared to PBS control. The percent of reduction in hamsters against this parasite were 32.95% and 21.49% in the CpG ODN 1826 with CSAg and CpG ODN 1826 alone. This study indicates that CpG ODN 1826 plus parasite antigens elicit a Th1-like response that leads to the enhancement of worm reduction.

Relapsed Melioidosis Model in C57BL/6 Mice.

BACKGROUND: Burkholderia pseudomallei are the causative agents of melioidosis, a disease that has a high relapse rate in endemic areas. The mechanism of relapse is unclear OBJECTIVE: This study aimed to establish relapsed melioidosis in C57BL/6 mice by induction with B. pseudomallei. MATERIAL AND METHOD: Low doses of B. pseudomallei H777 and its biofilm defective mutant (M10) were intra-gastric fed to C57BL/6 mice. All the infected mice had suppressed immune status by intra-peritoneal injection of hydrocortisone at 2.5 mg per mouse at day 60 post-infection. Inflammatory response to the infection was investigated by histo-pathological studies and monitoring bacterial counts in the blood and organs. RESULTS: All the infected mice were found to have a high infiltration of mononuclear cells at day 60 post-infection. The results showed high bacterial counts in the blood in both strains post-suppressed immune status after two days. The biofilm mutant and wild type strains produced relapse in C57BL/6 mice but the latter was responsible for significantly more severe inflammation than the biofilm mutant. CONCLUSION: Low immune status may cause relapsed melioidosis in hosts with chronic inflammation.

A PCR-BASED DETECTION OF BURKHOLDERIA PSEUDOMALLEI DIVERSITY USING MYOVIRIDAE PROPHAGE TYPING.

PCR-based detection of Myoviridae lysogenic phages in Burkholderia pseudomallei was developed using primers targeting K96243 prophage GI2, phiE12-2 and phi52237/phiX216. Investigation of 50 clinical and 50 environmental (soil) isolates revealed that K96243 prophage GI2 was the most common (48%) among the isolates, followed by phiE12-2 (38%) and phi52237/phiX216 (35%), with K96243 prophage GI2 being significantly more frequent in soil (64%) than clinical (32%) samples. Twenty-four percent of soil isolates contained all three prophage types, while clinical isolates harbored no more than two types. Although B. pseudomallei isolates from soil were found to be more diverse based on prophage typing, all isolates were equally susceptible to a battery of lytic phages (although to different extents), suggesting the possibility of using lytic phages to control environmental B. pseudomallei.

THE CONTRIBUTION OF SOIL PHYSICOCHEMICAL PROPERTIES TO THE PRESENCE AND GENETIC DIVERSITY OF BURKHOLDERIA PSEUDOMALLEI.

Burkholderia pseudomallei (Bp), the causative agent of melioidosis, is unevenly distributed in the complex soil environment. Physicochemical factors in the soil have been reported to affect microbial communities in the soil. The effect of physicochemical factors on the number and diversity of organisms in the soil has not been reported. Twenty-five each B. pseudomallei-positive and -negative soil samples were collected from a melioidosis-endemic area. The amount of Bp in each soil sample was measured by culture and quantitative PCR (qPCR). The following physicochemical properties from each soil sample were measured: pH, total organic carbon (TOC), total nitrogen (TN), carbon to nitrogen ratio (C:N ratio), exchangeable calcium (EC) and extractable iron (EI). All the physico- chemical properties measured were significantly different between the Bp-positive and -negative soil samples. The Bp-positive soil samples had lower C:N ratios and lower EC and a higher EI (p < 0.05) than the Bp-negative samples. The average pH was lower (3.7-5.0) in the Bp-negative samples. Among the Bp-positive soil samples, the EC was negatively correlated with the PCR copy number. The amount of bacteria detected with the qPCR method was higher than with the culture method, suggesting the presence of unculturable forms of bacteria that might re-grow when the environmental conditions was suitable. A total of 117 Bp isolates obtained from the soil samples were classified into 25 groups using BOX-PCR. The genetic diversity of Bp, did not correlate with the physicochemical factors investigated. A suitable pH range and C:N ratio may be important for the presence of Bp. The EI supports the needs and EC probably alters the growth of Bp. The genetic diversity of the bacteria was not influenced by the soil factors investigated in this study. This information shows the environment conducive to the growth of Bp. This gives us information about how to potentially control or decrease Bp in the soil in the future.