Revised Sequence and Annotation of Burkholderia pseudomallei/thailandensis Bacteriophage Bp-AMP1—A Potential Agent of Natural Biocontrol of the Populations of the Melioidosis Causative Agent

Microbiology - Significant impact of Bp-AMP1-related bacteriophages on the population dynamics of Burkholderia pseudomallei in agroecosystems of the regions endemic for melioidosis has been...     

Environmental Isolates of Burkholderia pseudomallei in Ceará State,Northeastern Brazil

Melioidosis has been considered an emerging disease in Brazil since the first cases were reported to occur in the northeast region. This study investigated two municipalities in Ceará state where melioidosis cases have been confirmed to occur. Burkholderia pseudomallei was isolated in 26 (4.3%) of 600 samples in the dry and rainy seasons.Melioidosis is an endemic disease in Southeast Asia and northern Australia (2, 4) and also occurs sporadically in other parts of the world (3, 7). Human melioidosis was reported to occur in Brazil only in 2003, when a family outbreak afflicted four sisters in the rural part of the municipality of Tejuçuoca, Ceará state (14). After this episode, there was one reported case of melioidosis in 2004 in the rural area of Banabuiú, Ceará (14). And in 2005, a case of melioidosis associated with near drowning after a car accident was confirmed to occur in Aracoiaba, Ceará (11).The goal of this study was to investigate the Tejuçuoca and Banabuiú municipalities, where human cases of melioidosis have been confirmed to occur, and to gain a better understanding of the ecology of Burkholderia pseudomallei in this region.We chose as central points of the study the residences and surrounding areas of the melioidosis patients in the rural areas of Banabuiú (5°18′35″S, 38°55′14″W) and Tejuçuoca (03°59′20″S, 39°34′50′W) (Fig. ​(Fig.1).1). There are two well-defined seasons in each of these locations: one rainy (running from January to May) and one dry (from June to December). A total of 600 samples were collected at five sites in Tejuçuoca (T1, T2, T3, T4, and T5) and five in Banabuiú (B1, B2, B3, B4, and B5), distributed as follows (Fig. ​(Fig.2):2): backyards (B1 and T1), places shaded by trees (B2 and T2), water courses (B3 and T3), wet places (B4 and T4), and stock breeding areas (B5 and T5).Open in a separate windowFIG. 1.Municipalities of Banabuiú (5°18′35″S, 38°55′14″W) and Tejuçuoca (03°59′20″S, 39°34′50″W).Open in a separate windowFIG. 2.Soil sampling sites in Banabuiú and Tejuçuoca.Once a month for 12 months (a complete dry/rainy cycle), five samples were gathered at five different depths: at the surface and at 10, 20, 30 and 40 cm (Table ​(Table1).1). The samples were gathered according to the method used by Inglis et al. (9). Additionally, the sample processing and B. pseudomallei identification were carried out as previously reported (1, 8, 9).

TABLE 1.

Distribution of samples with isolates by site and soil depth

Phenotypic characterization of three clinical isolates of Burkholderia pseudomallei in Ceará, Brazil

Burkholderia pseudomallei, the causative agent of melioidosis was found in a small cluster of cases in Teju?uoca, Ceará, Brazil. Tests were carried out to determine its phenotypic characteristics: colony morphology on Ashdown agar and MacConkey agar, biochemical profile in conventional biochemical tests and API 20NE, arabinose assimilation and susceptibility testing by disk diffusion, comparing with data in the literature. This study confirms the presence of B. pseudomallei in Brazil and describes its characteristics.     

Sulfonamide inhibition profile of the γ-carbonic anhydrase identified in the genome of the pathogenic bacterium Burkholderia pseudomallei the etiological agent responsible of melioidosis

A new γ-carbonic anhydrase (CA, EC 4.1.1.1) was cloned and characterized kinetically in the genome of the bacterial pathogen Burkholderia pseudomallei, the etiological agent of melioidosis, an endemic disease of tropical and sub-tropical regions of the world. The catalytic activity of this new enzyme, BpsCAγ, is significant with a k_cat of 5.3 × 10⁵ s^?1 and k_cat/K_m of 2.5 × 10⁷ M^?1 × s^?1 for the physiologic CO₂ hydration reaction. The inhibition constant value for this enzyme for 39 sulfonamide inhibitors was obtained. Acetazolamide, benzolamide and metanilamide were the most effective (K_Is of 149–653 nM) inhibitors of BpsCAγ activity, whereas other sulfonamides/sulfamates such as ethoxzolamide, topiramate, sulpiride, indisulam, sulthiame and saccharin were active in the micromolar range (K_Is of 1.27–9.56 μM). As Burkholderia pseudomallei is resistant to many classical antibiotics, identifying compounds that interfere with crucial enzymes in the B. pseudomallei life cycle may lead to antibiotics with novel mechanisms of action.     

Role of phages in the pathogenesis of Burkholderia,or ‘Where are the toxin genes in Burkholderia phages?’

Most bacteria of the genus Burkholderia are soil- and rhizosphere-associated, and rhizosphere associated, noted for their metabolic plasticity in the utilization of a wide range of organic compounds as carbon sources. Many Burkholderia species are also opportunistic human and plant pathogens, and the distinction between environmental, plant, and human pathogens is not always clear. Burkholderia phages are not uncommon and multiple cryptic prophages are identifiable in the sequenced Burkholderia genomes. Phages have played a crucial role in the transmission of virulence factors among many important pathogens; however, the data do not yet support a significant correlation between phages and pathogenicity in the Burkholderia. This may be due to the role of Burkholderia as a 'versaphile' such that selection is occurring in several niches, including as a pathogen and in the context of environmental survival.     

The molecular and cellular basis of pathogenesis in melioidosis: how does Burkholderia pseudomallei cause disease?

Melioidosis, a febrile illness with disease states ranging from acute pneumonia or septicaemia to chronic abscesses, was first documented by Whitmore & Krishnaswami (1912) . The causative agent, Burkholderia pseudomallei , was subsequently identified as a motile, gram-negative bacillus, which is principally an environmental saprophyte. Melioidosis has become an increasingly important disease in endemic areas such as northern Thailand and Australia ( Currie et al. , 2000 ). This health burden, plus the classification of B. pseudomallei as a category B biological agent ( Rotz et al. , 2002 ), has resulted in an escalation of research interest. This review focuses on the molecular and cellular basis of pathogenesis in melioidosis, with a comprehensive overview of the current knowledge on how B. pseudomallei can cause disease. The process of B. pseudomallei movement from the environmental reservoir to attachment and invasion of epithelial and macrophage cells and the subsequent intracellular survival and spread is outlined. Furthermore, the diverse assortment of virulence factors that allow B. pseudomallei to become an effective opportunistic pathogen, as well as to avoid or subvert the host immune response, is discussed. With the recent increase in genomic and molecular studies, the current understanding of the infection process of melioidosis has increased substantially, yet, much still remains to be elucidated.     

Environmental Occurrence of the Whipple’s Disease Bacterium (Tropheryma whippelii)

Whipple’s disease is a systemic disorder in which a gram-positive rod-shaped bacterium is constantly present in infected tissues. After numerous unsuccessful attempts to culture this bacterium, it was eventually characterized by 16S rRNA gene analysis to be a member of the actinomycetes. The name Tropheryma whippelii was proposed. Until now, the bacterium has only been found in infected human tissues, but there is no evidence for human-to-human transmission. Here we report the detection of DNA specific for the Whipple’s disease bacterium in 25 of 38 wastewater samples from five different sewage treatment plants in the area of Heidelberg, Germany. These findings provide the first evidence that T. whippelii occurs in the environment, within a polymicrobial community. This is in accordance with the phylogenetic relationship of this bacterium as well as with known epidemiological aspects of Whipple’s disease. Our data argue for an environmental source for infection with the Whipple’s disease bacterium.Whipple’s disease, which was first described in 1907 as intestinal lipodystrophy (18), is a multisystem disorder characterized by the presence of gram-positive, rod-shaped bacteria in infected human tissues (2). Numerous attempts to culture the bacterium associated with Whipple’s disease have failed (2), and eventually its phylogenetic position within the actinomycetes was established by 16S rRNA gene analysis (11, 19). The name Tropheryma whippelii was proposed (11).The clinical presentation of Whipple’s disease is heterogeneous. Frequently, patients suffer for years from arthralgias, chronic diarrhea, and weight loss, and less often from dementia or cardiac insufficiency. If untreated, the disease is usually fatal, but with appropriate antibiotic therapy the prognosis is favorable (2). However, the pathogenesis of Whipple’s disease appears to involve more than just an infection with T. whippelii. Immunological abnormalities are presumed to play a necessary role as predisposing factors (2, 7, 8), a view which is strengthened by the detection of T. whippelii in association with AIDS (5).Two outstanding questions in the epidemiology of Whipple’s disease are the bacterium’s natural habitat and the route of infection. Until now, T. whippelii has never been found outside of infected human hosts, and although an oral route of infection has often been suspected (10), it has not been proven. There is no evidence for human-to-human transmission.A reassessment of its phylogeny revealed a close relationship of the Whipple’s disease bacterium to typical environmental bacteria, such as the cellulomonads and the rare group B peptidoglycan organisms (6). This would support the hypothesis that T. whippelii may be a soil or water inhabitant. This may also explain the difficulties involved with its culture, as an estimated 80 to 99% of bacteria occurring in such natural environments are not culturable on artificial media (14, 17) and uncultured members of the actinomycete line of descent have been found in different environments and geographical locations (12). Soil and water bacteria tend to concentrate in sewage to form communities in which a large variety of different species are found. Therefore, the search for the Whipple’s disease bacterium was concentrated at sewage treatment plants.From September 1995 to July 1996, a total of 38 effluent samples from the sedimentation ponds of five different sewage treatment plants were examined by PCR for the presence of the Whipple’s disease bacterium. The sewage treatment plants are located within a distance of 5 to 25 km from Heidelberg, Germany (Fig. ​(Fig.1).1). Effluent was sampled in 1,000-ml single-use plastic flasks and filtered as described previously (4) in portions of 25 ml through cellulose acetate prefilters (25-mm diameter, 5-μm pore size; Sartorius, Göttingen, Germany). It was then passed through polyvinylidene fluoride filters (25-mm diameter, 0.45-μm pore size; Millipore, Bedford, Mass.). The DNA from the 0.45-μm-pore-size filters was extracted with the EnviroAmp Legionella sample preparation kit (Perkin-Elmer, Norwalk, Conn.), with the addition of 0.2% bovine serum albumin to the water in the final extraction step. Distilled water for negative controls was sampled in the same flasks and treated in the same way as the sewage. Open in a separate windowFIG. 1Map of the area around Heidelberg, Germany, displaying the rivers Rhine (Rhein) and Neckar, the relevant townships (shaded areas with names in capital letters), and the five sewage treatment plants (dots) from which samples were taken. The inset in the figure shows the localization of the area within Germany.PCR amplification and detection were performed as described in detail elsewhere (15). Primers whip1 (5′-AGAGATACGCCCCCCGCAA) and whip2 (5′-ATTCGCTCCACCTTGCGA), which amplify a fragment of 267 bp from the 16S rRNA gene of the bacterium, were used. In addition to hybridization with oligonucleotide whip3 (5′-TGGTACAGAGGGTTGCAATA), a second hybridization in a separate reaction was performed with oligonucleotide whip4 (5′-GTAATGGCGGGGACTCACAG). The specificities of primers whip1 and whip2 and of probe whip3 have been tested previously (15), and testing of probe whip4 with the same set of 37 control bacteria gave the same results as those reported for whip3 (15).Of the 38 sewage samples tested, 25 were found to be positive by PCR and hybridization with both oligonucleotides (Table ​(Table1).1). Positive samples were found at each of the five sewage treatment plants. Most of the positive PCR products displayed relatively weak bands at the expected size of 267 bp. They also contained some extraneous bands, which were not of the expected size, probably due to the large amount of DNA from other organisms present in sewage. On hybridization, however, the PCR products displayed distinct bands, which were consistent between the two oligonucleotides (Fig. ​(Fig.2).2).

TABLE 1

Results of PCR for the Whipple’s disease bacterium from sewage samples

Scientists@Home: What Drives the Quantity and Quality of Online Citizen Science Participation?

Online citizen science offers a low-cost way to strengthen the infrastructure for scientific research and engage members of the public in science. As the sustainability of online citizen science projects depends on volunteers who contribute their skills, time, and energy, the objective of this study is to investigate effects of motivational factors on the quantity and quality of citizen scientists'' contribution. Building on the social movement participation model, findings from a longitudinal empirical study in three different citizen science projects reveal that quantity of contribution is determined by collective motives, norm-oriented motives, reputation, and intrinsic motives. Contribution quality, on the other hand, is positively affected only by collective motives and reputation. We discuss implications for research on the motivation for participation in technology-mediated social participation and for the practice of citizen science.     

Comparison of the amine/amino acid activation profiles of the β- and γ-carbonic anhydrases from the pathogenic bacterium Burkholderia pseudomallei

The β-class carbonic anhydrase (CA, EC 4.2.1.1) from the pathogenic bacterium Burkholderia pseudomallei, BpsCAβ, that is responsible for the tropical disease melioidosis was investigated for its activation with natural and non-natural amino acids and amines. Previously, the γ-CA from this bacterium has been investigated with the same library of 19 amines/amino acids, which show very potent activating effects on both enzymes. The most effective BpsCAβ activators were L- and D-DOPA, L- and D-Trp, L-Tyr, 4-amino-L-Phe, histamine, dopamine, serotonin, 2-pyridyl-methylamine, 1-(2-aminoethyl)-piperazine and L-adrenaline with K_As of 0.9–27?nM. Less effective activators were D-His, L- and D-Phe, D-Tyr, 2-(2-aminoethyl)pyridine and 4-(2-aminoethyl)-morpholine with K_As of 73?nM–3.42?µM. The activation of CAs from bacteria, such as BpsCAγ/β, has not been considered previously for possible biomedical applications. It would be of interest to perform studies in which bacteria are cultivated in the presence of CA activators, which may contribute to understanding processes connected with the virulence and colonization of the host by pathogenic bacteria.     

Climate,Livestock, and Vegetation: What Drives Fire Increase in the Arid Ecosystems of Southern Russia?

Fire is an important natural disturbance process in arid grasslands but current fire regimes are largely the result of both human and natural processes and their interactions. The collapse of the Soviet Union in 1991 spurred substantial socioeconomic changes and was ultimately followed by a rapid increase in burned area in southern Russia. What is unclear is whether this increase in burned area was caused by decreasing livestock numbers, vegetation changes, climate change, or interactions of these factors. Our research goal was to identify the driving forces behind the increase in burned area in the arid grasslands of southern Russia. Our study area encompassed 19,000 km² in the Republic of Kalmykia in southern Russia. We analyzed annual burned area from 1986 to 2006 as a function of livestock population, NDVI, precipitation, temperature, and broad-scale oscillation indices using best subset regressions and structural equation modeling. Our results supported the hypothesis that vegetation recovered within 5–6 years after the livestock declined in the beginning of the 1990s, to a point at which large fires could be sustained. Climate was an important explanatory factor for burning, but mainly after 1996 when lower livestock numbers allowed fuels to accumulate. Ultimately, our results highlight the complexity of coupled human-natural systems, and provide an example of how abrupt socioeconomic change may affect fire regimes.     

What lies in the future of high-pressure bioscience?

Without being comprehensive in this mini-review, I will address perspectives, some speculative, for the development and use of high pressure to explore biochemical phenomena. This will be illustrated with several examples.     

What is new in the treatment of hypospadias?

Hypospadias is a congenital anomaly characterized by a ventrally placed urethral meatus in a more proximal position on the midline than its normal position in the glanular part of the penis. In 1961, C. E. Horton and C. J. Devine, Jr., developed single-stage modern surgical techniques, namely, local skin flaps and free skin grafts, for urethra reconstruction in hypospadias repair, which may be applied to almost any case with different localizations of the meatus. Later, two new methods, advancement of the urethra and preputial island flap techniques, were added to the surgical algorithm. Because acceptable results were observed, the authors have insisted on using these four techniques for all hypospadias cases since 1972. Complication rates (mainly fistula formation) were quite high (50 percent) in their early series of adults as a result of erection and hematoma formation. The complication rate of their patient population, which is now mainly composed of preschool children, has decreased to 7 to 8 percent, primarily as a result of careful selection of appropriate techniques for each individual case, the development of better surgical materials and equipment, and taking necessary precautions for postoperative care. A brief summary of modern hypospadias repair techniques is presented in four major classes. The results of the authors' 30-year experience and the precautions necessary to avoid postoperative complications are evaluated. The authors conclude that the four modern techniques and their modifications should be performed meticulously for successful hypospadias repair.     

Motive for Killing: What Drives Prey Choice in Wild Predators?

Carnivorous animals are assumed to consume prey to optimise energy intake. Recently, however, studies using Nutritional Geometry (NG) have demonstrated that specific blends of macronutrients (e.g. protein, fat and in some cases carbohydrates), rather than energy per se, drive the food selection and intake of some vertebrate and invertebrate predators in the laboratory. A vital next step is to examine the role of nutrients in the foraging decisions of predators in the wild, but extending NG studies of carnivores from the laboratory to the field presents several challenges. Biologging technology offers a solution for collecting relevant data which when combined with NG will yield new insights into wild predator nutritional ecology.     

What is the role of the helical domain of Gsalpha in the GTPase reaction?

Structural analysis of Gsalpha shows that it is composed of two domains: the ras-like domain (RD) that is conserved in all members of the GTPase superfamily and is homologous to the monomeric G-proteins (e.g., p21ras) and an alpha-helical domain (HD) that is unique to heterotrimeric G-proteins. Little is known about the function of the HD. Recent experiments by Bourne and co-workers, who expressed both the RD and the HD of Gsalpha separately and found that GTP hydrolysis is very slow if only recombinant RD is present but is accelerated when the HD is added, suggest that the HD serves as an intrinsic GTPase-activating protein (GAP). In this work, the GTP hydrolysis in Gsalpha was studied. The results obtained by calculating catalytic effects with and without the HD provide evidence for the role of the HD as a GAP. It is demonstrated that a major part of the catalysis is obtained because of an allosteric influence of the HD on the RD. Structural as well as energetic considerations suggest that the HD confines the RD to a more compact conformation, pushing the phosphate into an orientation where it is further stabilized, thus lowering the overall reaction barrier. The resemblance between the behavior of rasGAP and the HD suggests that the conclusion may be a general conclusion, applicable for all of the G-protein members.