Pathogenicity of high-dose enteral inoculation of Burkholderia pseudomallei to mice

Melioidosis is a frequently lethal tropical infection caused by the environmental saprophyte Burkholderia pseudomallei. Although transcutaneous inoculation and inhalation are considered the primary routes of infection, suggestive clinical evidence implicates ingestion as a possible alternative route. We show that in BALB/c and C57BL/6 mice, direct gastric inoculation of high doses of B. pseudomallei causes systemic infection that may be lethal or cause chronic disseminated infection. Mice may shed bacteria in the stool for weeks after infection, and high titers of B. pseudomallei-specific IgG are detectable. This report of enteric murine melioidosis supports further consideration of this route of infection.     

The cyanobacterial taxis protein HmpF regulates type IV pilus activity in response to light

Motility is ubiquitous in prokaryotic organisms including the photosynthetic cyanobacteria where surface motility powered by type 4 pili (T4P) is common and facilitates phototaxis to seek out favorable light environments. In cyanobacteria, chemotaxis-like systems are known to regulate motility and phototaxis. The characterized phototaxis systems rely on methyl-accepting chemotaxis proteins containing bilin-binding GAF domains capable of directly sensing light, and the mechanism by which they regulate the T4P is largely undefined. In this study we demonstrate that cyanobacteria possess a second, GAF-independent, means of sensing light to regulate motility and provide insight into how a chemotaxis-like system regulates the T4P motors. A combination of genetic, cytological, and protein–protein interaction analyses, along with experiments using the proton ionophore carbonyl cyanide m-chlorophenyl hydrazine, indicate that the Hmp chemotaxis-like system of the model filamentous cyanobacterium Nostoc punctiforme is capable of sensing light indirectly, possibly via alterations in proton motive force, and modulates direct interaction between the cyanobacterial taxis protein HmpF, and Hfq, PilT1, and PilT2 to regulate the T4P motors. Given that the Hmp system is widely conserved in cyanobacteria, and the finding from this study that orthologs of HmpF and T4P proteins from the distantly related model unicellular cyanobacterium Synechocystis sp. strain PCC6803 interact in a similar manner to their N. punctiforme counterparts, it is likely that this represents a ubiquitous means of regulating motility in response to light in cyanobacteria.

Motility is ubiquitous in prokaryotic organisms, including both swimming motility in aqueous environments and twitching or gliding motility on solid surfaces, and enables these organisms to optimize their position in response to various environmental factors. Among the photosynthetic cyanobacteria, surface motility is widespread and facilitates phototaxis to seek out favorable light environments (1, 2), and, for multicellular filamentous cyanobacteria, plays a key role in dispersal as well as the establishment of nitrogen-fixing symbioses with eukaryotes (3) and the formation of supracellular structures (3–5).Current understanding of cyanobacterial surface motility at the molecular level has been informed primarily by studies of two model organisms, the unicellular strain Synechocystis sp. strain PCC6803 (herein Synechocystis) and the filamentous strain Nostoc punctiforme ATCC29133/{"type":"entrez-protein","attrs":{"text":"PCC73102","term_id":"1245706357"}}PCC73102, where motility is exhibited only by differentiated filaments termed “hormogonia.” Motility in both organisms is powered by a type IV pilus (T4P) system where the ATPases PilB and PilT drive the extension and subsequent retraction, respectively, of pili which adhere to the substrate and pull the cells forward (for review, see ref. 6). In Synechocystis, the T4P motors are distributed throughout the entire cell, allowing a 360 ° range of motion (7), whereas in N. punctiforme they are confined to rings at the cell poles (8), resulting in movement only along the long axis of the filament. Comparative genomics implies that this mechanism of motility is widely conserved among cyanobacteria (9).Both Synechocystis and N. punctiforme employ chemotaxis-like systems to regulate motility. One of these systems, the Hmp chemotaxis-like system of N. punctiforme (3, 10), and its orthologous counterpart, the Pil chemotaxis-like system of Synechocystis (11), includes homologs to the canonical Escherichia coli chemotaxis complex (for review, see ref. 12), including the histidine kinase CheA, the adaptor protein CheW, the response regulator CheY, and the methyl-accepting chemotaxis protein MCP. These systems are essential for motility in their respective organisms and appear to regulate the T4P motors, although there are distinct differences in the phenotypes for inactivation of the components from each. In Synechocystis, null mutations either enhance or reduce the level of surface piliation (11), whereas in N. punctiforme they disrupt the coordinated polarity, but not the overall level of piliation, and affect various other aspects of hormogonium development (3, 10). In N. punctiforme, the subcellular localization of this system has been determined and has been found arrayed in static, bipolar rings similar to the T4P motors (3). However, the signals that are perceived by the MCPs and the precise mechanism by which these systems modulate T4P activity is currently undefined.Recently, an additional component of the Hmp system, HmpF, was characterized (9). HmpF is a predicted coiled-coil protein and is ubiquitous to, but confined within, the cyanobacterial lineage (9). It is essential for accumulation of surface pili and exhibits dynamic, unipolar localization to the leading poles of most cells in hormogonium filaments (9). Based on these findings, a model has been proposed where the localization of HmpF is regulated by the other components of the Hmp system, and in turn, the unipolar accumulation of HmpF leads to the activation of the T4P motors on one side of the cell to facilitate directional movement.A second chemotaxis-like system in each organism, the Ptx system of N. punctiforme (13) and the Pix system of Synechocystis (14, 15), is essential for positive phototaxis. These systems contain MCPs with cyanobacteriochrome sensory domains capable of perceiving light (for review, see ref. 16). Disruption of the Pix system results in negative phototaxis under light conditions that normally produce a positive phototactic response (14). Several other proteins containing cyanobacteriochromes, and one containing a BLUF domain, also modulate phototaxis in Synechocystis (for review, see ref. 6). In N. punctiforme, disruption of the Ptx system abolishes the phototactic response completely, resulting in uniform movement in all directions regardless of the light conditions (13), and there are currently no other proteins reported to modulate phototaxis. More recently, a motile, wild isolate of the model unicellular cyanobacterium Synechococcus elongatus sp. PCC7942 was shown to possess a chemotaxis-like system that modulates phototaxis in a manner similar to that of the N. punctiforme Ptx system (17). How these systems influence T4P activity to facilitate phototaxis is also currently unknown.There is also a substantial body of literature on motility and phototaxis in cyanobacteria, primarily based on observational studies of various filamentous strains, that predates the development of genetically tractable model organisms (for review, see ref. 18). These reports suggested that the photosystems may serve a sensory role in modulating phototaxis and that proton motive force (PMF) powers motility (19, 20), a finding that is inconsistent with the theory that cyanobacteria possess a common T4P-based gliding motor driven by ATP hydrolysis. In this study, we help reconcile this historical data with more recent molecular studies by providing evidence that the Hmp chemotaxis-like system senses light, possibly indirectly through alterations in PMF, and in turn modulates the interaction of HmpF with the T4P base to activate the motors.     

A truncated hepatitis E virus ORF2 protein expressed in tobacco plastids is immunogenic in mice

AIM: To cost-effectively express the 23-ku pE2, the most promising subunit vaccine encoded by the E2 fragment comprising of the 3'-portion of hepatitis E virus (HEV) open reading frame 2 (ORF2) in plastids of tobacco (Nicotiana tabacum cv. SRI), to investigate the transgene expression and pE2 accumulation in plastids, and to evaluate the antigenic effect of the plastid-derived pE2 in mice. METHODS: Plastid-targeting vector pRB94-E2 containing the E2 fragment driven by rice psbA promoter was constructed. Upon delivery into tobacco plastids, this construct could initiate homologous recombination in psaB-trnfM and trnG-psbC fragments in plastid genome, and result in transgene inserted between the two fragments. The pRB94-E2 was delivered with a biolistic particle bombardment method, and the plastid-transformed plants were obtained following the regeneration of the bombarded leaf tissues on a spectinomycin-supplemented medium. Transplastomic status of the regenerated plants was confirmed by PCR and Southern blot analysis, transgene expression was investigated by Northern blot analysis, and accumulation of pE2 was measured by ELISA. Furthermore, protein extracts were used to immunize mice, and the presence of the pE2-reactive antibodies in serum samples of the immunized mice was studied by ELISA. RESULTS: Transplastomic lines confirmed by PCR and Southern blot analysis could actively transcribe the E2 mRNA. The pE2 polypeptide was accumulated to a level as high as 13.27μg/g fresh leaves. The pE2 could stimulate the immunized mice to generate pE2-specific antibodies. CONCLUSION: HEV-E2 fragment can be inserted into the plastid genome and the recombinant pE2 antigen derived is antigenic in mice. Hence, plastids may be a novel source for cost-effective production of HEV vaccines.     

Antimicrobial resistance to ceftazidime involving loss of penicillin-binding protein 3 in Burkholderia pseudomallei

Known mechanisms of resistance to β-lactam antibiotics include β-lactamase expression, altered drug target, decreased bacterial permeability, and increased drug efflux. Here, we describe a unique mechanism of β-lactam resistance in the biothreat organism Burkholderia pseudomallei (the cause of melioidosis), associated with treatment failure during prolonged ceftazidime therapy of natural infection. Detailed comparisons of the initial ceftazidime-susceptible infecting isolate and subsequent ceftazidime-resistant variants from six patients led us to identify a common, large-scale genomic loss involving a minimum of 49 genes in all six resistant strains. Mutational analysis of wild-type B. pseudomallei demonstrated that ceftazidime resistance was due to deletion of a gene encoding a penicillin-binding protein 3 (BPSS1219) present within the region of genomic loss. The clinical ceftazidime-resistant variants failed to grow using commonly used laboratory culture media, including commercial blood cultures, rendering the variants almost undetectable in the diagnostic laboratory. Melioidosis is notoriously difficult to cure and clinical treatment failure is common in patients treated with ceftazidime, the drug of first choice across most of Southeast Asia where the majority of cases are reported. The mechanism described here represents an explanation for ceftazidime treatment failure, and may be a frequent but undetected resistance event.     

Role of T cells in innate and adaptive immunity against murine Burkholderia pseudomallei infection

Antigen-specific T cells are important sources of interferon (IFN)-gamma for acquired immunity to intracellular pathogens, but they can also produce IFN- gamma directly via a "bystander" activation pathway in response to proinflammatory cytokines. We investigated the in vivo role of cytokine- versus antigen-mediated T cell activation in resistance to the pathogenic bacterium Burkholderia pseudomallei. IFN-gamma, interleukin (IL)-12, and IL-18 were essential for initial bacterial control in infected mice. B. pseudomallei infection rapidly generated a potent IFN-gamma response from natural killer (NK) cells, NK T cells, conventional T cells, and other cell types within 16 h after infection, in an IL-12- and IL-18-dependent manner. However, early T cell- and NK cell-derived IFN-gamma responses were functionally redundant in cell depletion studies, with IFN-gamma produced by other cell types, such as major histocompatibility complex class II(int) F4/80(+) macrophages being sufficient for initial resistance. In contrast, B. pseudomallei-specific CD4(+) T cells played an important role during the later stage of infection. Thus, the T cell response to primary B. pseudomallei infection is biphasic, an early cytokine-induced phase in which T cells appear to be functionally redundant for initial bacterial clearance, followed by a later antigen-induced phase in which B. pseudomallei-specific T cells, in particular CD4(+) T cells, are important for host resistance.     

Interleukin 4 is important in protective immunity to a gastrointestinal nematode infection in mice.

Parasitic helminths typically induce components of immediate-type hypersensitivity, including elevated serum IgE, eosinophilia, and mucosal mast cells. These responses are T-cell-dependent and associated with rapid expulsion of parasitic worms from a sensitized host; existing experimental systems have failed to define the precise role of cytokines in these responses. We report here that anti-interleukin 4 or anti-interleukin 4 receptor antibodies block the polyclonal IgE response to a parasitic nematode, Heligmosomoides polygyrus, and abrogate protective immunity to the infection. In contrast, anti-interleukin 5 antibody prevented H. polygyrus-induced eosinophilia but did not prevent protection. These data provide evidence that a specific cytokine affects the physiology and survival of a parasitic nematode in the host.     

Angiotensin II fails to induce preconditioning in vivo--the protective role of myocardial stretch due to pressure overload

BACKGROUND: Angiotensin II (Ang) has been successfully used as a preconditioning analogue in isolated rabbit hearts. It is also known that local concentrations of Ang accelerate ischemic injury in vivo, while activation of stretch receptors protects ischemic hearts. OBJECTIVES: First, to investigate further whether Ang can mimic preconditioning in vivo. Second, to test the hypothesis that there is an activation of stretch receptors, and that the larger infarct from the left atrium Ang compared with that from the intravenous Ang may be associated with the ischemic injury caused by local administration. METHODS: Male rabbits were divided into four groups - a control group, an ischemic preconditioning group with 5 min ischemia, a left atrial group and an intravenous group with 5 min Ang infusion. All animals were subjected to prolonged ischemia and reperfusion. A second series of experiments was also performed with five groups that had a 5 min mechanical obstruction of the aorta (Ao clamp), also used as a preconditioning analogue with or without the stretch receptor blocker gadolinium (Gd). RESULTS: Contrary to what was expected from the ex vivo experiments, Ang failed to mimic preconditioning (infarct size 39.6 6.1%, 13.7 4.1%, 52.2% 6.9% and 31.2 4.8%, respectively for the above groups). Interestingly, however, when Ang was infused intravenously, it produced a significantly smaller infarct compared with that observed after the same dose was infused into the left atrium (P<0.05). The infarct size was 17.0 3.7% in the Ao clamp group, which was an effect completely prevented by Gd (45.8 4.2%, P<0.01). Although Gd did not alter infarct size in the control and ischemic preconditioning groups, it increased infarct size when added to the intravenous Ang group (Gd-intravenous Ang 48.6 3.3%, P<0.05 compared with intravenous Ang). CONCLUSIONS: Ang fails to mimic preconditioning in vivo, but salvage of ischemic myocardium can be emanated from pressure overload.     

Motor-independent retraction of type IV pili is governed by an inherent property of the pilus filament

Type IV pili (T4P) are dynamic surface appendages that promote virulence, biofilm formation, horizontal gene transfer, and motility in diverse bacterial species. Pilus dynamic activity is best characterized in T4P that use distinct ATPase motors for pilus extension and retraction. Many T4P systems, however, lack a dedicated retraction motor, and the mechanism underlying this motor-independent retraction remains a mystery. Using the Vibrio cholerae competence pilus as a model system, we identify mutations in the major pilin gene that enhance motor-independent retraction. These mutants likely diminish pilin–pilin interactions within the filament to produce less-stable pili. One mutation adds a bulky residue to α1C, a universally conserved feature of T4P. We found that inserting a bulky residue into α1C of the retraction motor–dependent Acinetobacter baylyi competence T4P enhances motor-independent retraction. Conversely, removing bulky residues from α1C of the retraction motor–independent, V. cholerae toxin-coregulated T4P stabilizes the filament and diminishes pilus retraction. Furthermore, alignment of pilins from the broader type IV filament (T4F) family indicated that retraction motor–independent T4P, gram-positive Com pili, and type II secretion systems generally encode larger residues within α1C oriented toward the pilus core compared to retraction motor–dependent T4P. Together, our data demonstrate that motor-independent retraction relies, in part, on the inherent instability of the pilus filament, which may be a conserved feature of diverse T4Fs. This provides evidence for a long-standing yet previously untested model in which pili retract in the absence of a motor by spontaneous depolymerization.

Type IV pili (T4P) are ubiquitous, hair-like appendages that dynamically extend and retract from bacterial cells (1–3). These membrane-anchored nanomachines are important for the interactions of bacteria with their environment and facilitate activities like twitching motility, surface attachment, biofilm formation, protein secretion, interbacterial interactions, and horizontal gene transfer by natural transformation (2, 4–10). The dynamic activity of T4P is central to many of these functions. T4P are also essential virulence factors in diverse pathogens (10–13). Thus, studying the regulation of these structures can uncover approaches for the development of therapeutic interventions.The surface-exposed pilus filament is a multimeric helical structure that is primarily composed of a single repeating subunit called the major pilin. This filament is built on an inner membrane platform protein commonly called PilC (14–16). Extension and retraction of the pilus is thought to occur through the interaction of cytoplasmic hexameric ATPases with the platform, whereby ATP hydrolysis facilitates coordinated conformational changes in the platform that promote polymerization or depolymerization (17, 18).There are three main categories of T4P systems among bacteria: T4aP, T4bP, and T4cP. All T4P require an extension ATPase motor for pilus polymerization (19). A detailed mechanistic understanding of the factors that promote pilus retraction, however, remains lacking in many T4P. For T4aP, pilus depolymerization is generally mediated by dedicated retraction ATPase motors commonly referred to as PilT and PilU. PilT is the dominant retraction motor, because it is independently sufficient to promote pilus retraction, while PilU is an accessory motor that facilitates retraction only in the presence of PilT (20, 21). However, even in the absence of these retraction motors, numerous T4aP retain the ability to retract (20, 22–24). Many T4bP appear to lack a dedicated retraction ATPase (25–27) yet also retract (25). Furthermore, phylogenetic analysis indicates that T4aP and T4bP most likely evolved from a common ancestor (19, 28). This suggests that diverse T4P may share a conserved mechanism for PilTU-independent retraction. Here, we investigated the mechanism of pilus retraction in the absence of dedicated retraction ATPases and find evidence to support a long-standing yet previously untested model in which pili retract via spontaneous depolymerization.     

Virulence of a Mycobacterium tuberculosis clinical isolate in mice is determined by failure to induce Th1 type immunity and is associated with induction of IFN-alpha /beta

To understand how virulent mycobacteria subvert host immunity and establish disease, we examined the differential response of mice to infection with various human outbreak Mycobacterium tuberculosis clinical isolates. One clinical isolate, HN878, was found to be hypervirulent, as demonstrated by unusually early death of infected immune-competent mice, compared with infection with other clinical isolates. The differential effect on survival required lymphocyte function because severe combined immunodeficiency (SCID) mice infected with HN878 or other clinical isolates all died at the same rate. The hypervirulence of HN878 was associated with failure to induce M. tuberculosis-specific proliferation and IFN-gamma production by spleen and lymph node cells from infected mice. In addition, 2- to 4-fold lower levels of tumor necrosis factor-alpha (TNF-alpha), IL-6, IL-12, and IFN-gamma mRNAs were observed in lungs of HN878-infected mice. IL-10, IL-4, and IL-5 mRNA levels were not significantly elevated in lungs of HN878 infected mice. In contrast, IFN-alpha mRNA levels were significantly higher in lungs of these mice. To further investigate the role of Type 1 IFNs, mice infected with HN878 were treated intranasally with purified IFN-alpha/beta. The treatment resulted in increased lung bacillary loads and even further reduced survival. These results suggest that the hypervirulence of HN878 may be due to failure of this strain to stimulate Th1 type immunity. In addition, the lack of development of Th1 immunity in response to HN878 appears to be associated with increased induction of Type 1 IFNs.     

Ov-ASP-1, the Onchocerca volvulus homologue of the activation associated secreted protein family is immunostimulatory and can induce protective anti-larval immunity

Vaccination of mice with a recombinant protein, Ov-ASP-1, the Onchocerca volvulus homologue of the activation associated secreted gene family stimulated very high titres of both IgG1 and IgG2a without adjuvant. rOv-ASP-1 was also immuno-reactive with IgG isotypes from both O. volvulus-infected (INF) and putatively immune (PI) humans, with higher IgG4 in the former group. The protein also stimulated IFN-gamma secretion by PBMC from INF and PI and IL-5 only in INF. Using a mouse diffusion chamber model, vaccination with rOv-ASP-1 resulted in partial but significant protection against challenge with infective third-stage larvae (L3) but only when formulated with Freund's complete adjuvant (FCA) or alum. Protection was Th1-dependent (highly elevated IgG2a) with FCA and contingent on a strongly Th2-skewed (IgG1) response with alum. IgE responses to rOv-ASP-1 with or without adjuvant were weak or absent. When immunization using rOv-ASP-1 in adjuvant failed to induce adequate Th1 (FCA) or Th2 (alum) responses, protection efficacy was compromised. The recombinant protein appears to stimulate a mixed Th1/Th2 response but the outcome in terms of protective immunity is the result of a subtle interplay of its intrinsic and adjuvant-augmented properties. Ov-ASP-1 is potentially secreted based on its localization in the secretory granules of L3.     

Eosinophils act as antigen-presenting cells to induce immunity to Strongyloides stercoralis in mice

The objective of the present study was to explore the ability of eosinophils to present Strongyloides stercoralis antigen in naive and immunized mice. Antigen-pulsed eosinophils were injected intraperitoneally into naive or immunized mice, and then mice were examined for antigen-specific immune responses. A single inoculation of antigen-pulsed eosinophils was sufficient to prime naive mice and to boost immunized mice for antigen-specific T helper cell type 2 (Th2) immune responses with increased interleukin (IL)-4 and IL-5 production. Mice inoculated 3 times with live eosinophils pulsed with antigen showed significant increases in parasite antigen-specific immunoglobulin (Ig) M and IgG levels in their serum. Antigen-pulsed eosinophils deficient in major histocompatibility complex class II molecules or antigen-pulsed dead eosinophils failed to induce immune responses, thereby demonstrating the requirement for direct interaction between eosinophils and T cells. These experiments demonstrate that eosinophils function as antigen-presenting cells for the induction of the primary and the expansion of the secondary Th2 immune responses to S. stercoralis in mice.     

Failure of Burkholderia pseudomallei to Grow in an Automated Blood Culture System

We compared the organisms isolated from 30,210 pairs of blood culture bottles by using BacT/Alert system and the conventional system. Overall, 2,575 (8.5%) specimens were culture positive for pathogenic organisms. The sensitivity for detection of pathogenic organisms with the BACT/Alert system (85.6%, 2,203 of 2,575) was significantly higher than that with the conventional method (74.1%, 1,908 of 2,575; P < 0.0001). However, Burkholderia pseudomallei was isolated less often with the BacT/ALERT system (73.5%, 328 of 446) than with the conventional system (90.3%, 403 of 446; P < 0.0001). This finding suggests that use of the conventional culture method in conjunction with the BacT/Alert system may improve the isolation rate for B. pseudomallei in melioidosis-endemic areas.The Gram-negative bacillus Burkholderia pseudomallei is a Tier 1 select agent and the cause of melioidosis.¹ The disease accounts for 20% of all community-acquired septicemias in northeastern Thailand,² where melioidosis is the third most frequent cause of death from infectious diseases.³ Melioidosis is notoriously difficult to cure despite appropriate antimicrobial therapy and has a case-fatality rate of up to 43%.³ More than half of all melioidosis patients are bacteremic, and positive blood cultures for B. pseudomallei obtained at hospital admission and/or during hospitalization are strong prognostic markers for death.⁴Although the automated blood culture system (BacT/Alert) is convenient and currently used in many laboratories in provincial hospitals in Thailand, it is unclear whether its sensitivity for the detection of pathogens is similar to that obtained using a conventional low tech system still commonly used in small hospitals in resource-limited settings.In a retrospective study conducted during January 1, 2009–July 31, 2011 as part of routine patient care at Sappasithiprasong Hospital, a 1,000-bed tertiary-care hospital in northeastern Thailand, we compared the organisms isolated from more than 30,000 pairs of blood culture bottles by using the BacT/Alert system and the conventional system.In conventional system, the culture medium is made in-house, blood culture bottles are incubated in a conventional incubator, and bacterial detection is made by direct visualization with or without regular sub-culture. During the study period, two 5-mL blood samples were regularly obtained from each patient 10–15 minutes apart. The first 5 mL of blood was inoculated into a 40 mL culture media BacT/Alert SA bottle (catalog no. 259789; bioMérieux, Durham, NC). The second 5 mL of blood was inoculated into an in-house bottle containing 40 mL of broth, which consisted of 37 g of brain heart infusion medium broth (catalogue no, 211059; Becton Dickinson, Franklin Lakes, NJ) and 0.25 g of sodium polyanatholesulfonate (catalog no.1000907362; Sigma, St. Louis, MO), in 1 liter of purified water. BacT/Alert bottles were incubated in the BacT/Alert automated blood culture system (bioMérieux) at 35°C for 7 days, and in-house bottles were incubated aerobically in a normal incubator at 35°C for 7 days. Examination of BacT/Alert bottles was done according to the directions provided by the manufacturer, and positive cultures were indicated on the computer screen accompanied by a beeping sound. Positive cultures in the in-house bottles were detected by direct visualization of cloudy broth.Positive bottles from both systems were sub-cultured by using airway needles (bioMérieux) to place approximately 15–20 μL of the culture on chocolate agar, blood agar, and eosin-methylene blue agar. In addition, all in-house bottles were routinely sub-cultured onto blood agar on day 2 of incubation, and all in-house and BacT/Alert bottles were routinely sub-cultured onto blood agar on day 7 of incubation. Blood agar and eosin-methylene blue agar plates were incubated aerobically at 35°C and inspected at 24 hours. Chocolate agar plates were incubated in an atmosphere of 5% CO₂ at 35°C and were inspected at 48 hours. Bacterial or fungal colonies that grew on culture plates were identified by using standard biochemical tests and colonies of presumptive B. pseudomallei were identified by typical colony morphology on Ashdown agar, resistance to gentamicin and colistin, and a positive result for a highly specific latex agglutination test, as described.^5,6A total of 30,210 pairs of blood culture bottles were collected during the study period (10,208, 12,574 and 7,428 in 2009, 2010 and 2011, respectively). Overall, 2,575 (8.5%) specimens were culture positive for pathogenic organisms. A total of 1,536 (59.7%) grew with both methods, 667 (25.9%) grew only with the BacT/Alert system, and 372 (14.4%) grew only with the conventional method.The pathogenic organisms isolated were gram-negative bacteria (68.1%), gram-positive bacteria (20.0%), fungi (9.7%) and polymicrobial organisms (2.2%). The most common pathogens were Escherichia coli (18.9%, 486 of 2,575), B. pseudomallei (17.3%, 446 of 2,575), Klebsiella species (10.3%, 266 of 2,575), Staphylococcus aureus (8.5%, 219 of 2,575), and Pseudomonas spp. (8.0%, 207 of 2,575) (

Development of antibodies to Burkholderia pseudomallei during childhood in melioidosis-endemic northeast Thailand

A cross-sectional serological survey of 2,214 children living in northeast Thailand was conducted to define the antibody response to Burkholderia pseudomallei from birth to 14 years. There was a sharp rise in detectable antibodies from birth to 4 years followed by reactivity in approximately 60-70% of children thereafter.     

Ethanol administration generates oxidative stress in the pancreas and liver,but fails to induce heat-shock proteins in rats

BACKGROUND: Heat-shock proteins (HSP) play an essential role in the sequestration and reparation of denatured cellular proteins. Because ethanol treatment can result in oxidative stress-induced protein damage, it is possible that expression of HSP is altered after ethanol consumption. Dose-response and time-course studies were performed to investigate whether acute and chronic intragastric ethanol administration can induce tissue damage, oxidative stress and expression of the heat-shock proteins HSP60 and HSP72 in the pancreas and liver of male Wistar rats. METHODS: Laboratory and morphological analysis of pancreatic and liver damage were investigated. The degree of oxidative stress was assessed by measurement of the reduced glutathione content, lipid peroxidation and protein oxidation. The levels of HSP were examined by western blot analysis. RESULTS: Ethanol administration dose- and time-dependently elevated the serum ethanol concentration and hepatic enzyme activities. Chronic ethanol treatment also resulted in morphological damage of the liver. We observed that acute and chronic ethanol consumption had markedly different effects on the oxidative parameters in the pancreas and liver. Acute ethanol administration caused oxidative stress in the liver, whereas there was no such effect in the pancreas. In contrast, chronic ethanol feeding resulted in oxidative stress in both the pancreas and the liver. Furthermore, neither acute nor chronic ethanol intake induced the synthesis of HSP, a major defense system against cellular damage in the examined organs. CONCLUSION: Ethanol administration generates oxidative stress in the pancreas and liver, but fails to induce HSP in rats.     

A successful dietary treatment fails to normalize plasma triglyceride postprandial response in type IV patients.

The role of plasma triglycerides as a risk factor for cardiovascular disease is still under scrutiny. While recent studies have shown that postprandial triglyceridemia is an independent risk factor, normalization of fasting plasma triglycerides through modification of nutritional habits remains the primary approach in the treatment of hypertriglyceridemia. To address the issue of whether a satisfactory dietary regimen results in the control of postprandial lipemia, 53 type IV hypertriglyceridemic patients underwent an hypolipidemic diet for 3 months. All patients had a reduction of fasting lipid parameters (average TG: from 516+/-208 to 229+/-99 mg/dl; total cholesterol (Chol): from 261+/-42 to 213+/-40 mg/dl and HDL Chol: from 33+/-9 to 38+/-8 mg/dl). Taking plasma TG < or =200 mg/dl as the target for dietary intervention 26 patients were classified as 'responders' while the remaining 27 were 'non responders'. Even if fasting total TG, total Chol, HDL and LDL Chol were normal, both responders and non responders (P<0.0001) showed an exaggerated postprandial response to an oral fat load as compared to controls (20 normolipidemic subjects). Also when 10 responders and 10 controls, all male, were matched for plasma TG (129+/-43 versus 121+/-41 mg/dl) and other lipid parameters, a statistically significant difference between the two groups was observed at the time of each of the postprandial tests (P<0.0001) and for the area under the curve. The fact that the post prandial response is poorly modified by a dietary regimen, that effectively reduces plasma fasting TG, suggests that commonly used dietary regimens fail to restore a normal postprandial metabolism. Whether the cardiovascular risk for these patients is reduced after diet remains, therefore, to be addressed.     

VAR2CSA domains expressed in Escherichia coli induce cross-reactive antibodies to native protein

The variant surface antigen VAR2CSA is a pregnancy malaria vaccine candidate, but its size and polymorphism are obstacles to development. We expressed 3D7-type VAR2CSA domains in Escherichia coli as insoluble His-tagged proteins (Duffy binding-like [DBL] domains DBL1, DBL3, DBL4, and DBL5) that were denatured and refolded or as soluble glutathione S-transferase-tagged protein (DBL6). Anti-DBL5 antiserum cross-reacted with surface proteins of chondroitin sulfate A (CSA)-binding laboratory strains (3D7-CSA and FCR3-CSA) and a clinical pregnancy malaria isolate, whereas anti-DBL6 antiserum reacted only to 3D7 surface protein. This is the first report that E. coli-expressed VAR2CSA domains induce antibody to native VAR2CSA.