Within-Host Evolution of Burkholderia pseudomallei in Four Cases of Acute Melioidosis

Little is currently known about bacterial pathogen evolution and adaptation within the host during acute infection. Previous studies of Burkholderia pseudomallei, the etiologic agent of melioidosis, have shown that this opportunistic pathogen mutates rapidly both in vitro and in vivo at tandemly repeated loci, making this organism a relevant model for studying short-term evolution. In the current study, B. pseudomallei isolates cultured from multiple body sites from four Thai patients with disseminated melioidosis were subjected to fine-scale genotyping using multilocus variable-number tandem repeat analysis (MLVA). In order to understand and model the in vivo variable-number tandem repeat (VNTR) mutational process, we characterized the patterns and rates of mutations in vitro through parallel serial passage experiments of B. pseudomallei. Despite the short period of infection, substantial divergence from the putative founder genotype was observed in all four melioidosis cases. This study presents a paradigm for examining bacterial evolution over the short timescale of an acute infection. Further studies are required to determine whether the mutational process leads to phenotypic alterations that impact upon bacterial fitness in vivo. Our findings have important implications for future sampling strategies, since colonies in a single clinical sample may be genetically heterogeneous, and organisms in a culture taken late in the infective process may have undergone considerable genetic change compared with the founder inoculum.     

Landscape Changes Influence the Occurrence of the Melioidosis Bacterium Burkholderia pseudomallei in Soil in Northern Australia

Background

The soil-dwelling saprophyte bacterium Burkholderia pseudomallei is the cause of melioidosis, a severe disease of humans and animals in southeast Asia and northern Australia. Despite the detection of B. pseudomallei in various soil and water samples from endemic areas, the environmental habitat of B. pseudomallei remains unclear.

Methodology/Principal Findings

We performed a large survey in the Darwin area in tropical Australia and screened 809 soil samples for the presence of these bacteria. B. pseudomallei were detected by using a recently developed and validated protocol involving soil DNA extraction and real-time PCR targeting the B. pseudomallei–specific Type III Secretion System TTS1 gene cluster. Statistical analyses such as multivariable cluster logistic regression and principal component analysis were performed to assess the association of B. pseudomallei with environmental factors. The combination of factors describing the habitat of B. pseudomallei differed between undisturbed sites and environmentally manipulated areas. At undisturbed sites, the occurrence of B. pseudomallei was found to be significantly associated with areas rich in grasses, whereas at environmentally disturbed sites, B. pseudomallei was associated with the presence of livestock animals, lower soil pH and different combinations of soil texture and colour.

Conclusions/Significance

This study contributes to the elucidation of environmental factors influencing the occurrence of B. pseudomallei and raises concerns that B. pseudomallei may spread due to changes in land use.     

Association of the Melioidosis Agent Burkholderia pseudomallei with Water Parameters in Rural Water Supplies in Northern Australia

We analyzed water parameters and the occurrence of the melioidosis agent Burkholderia pseudomallei in 47 water bores in Northern Australia. B. pseudomallei was associated with soft, acidic bore water of low salinity but high iron levels. This finding aids in identifying water supplies at risk of contamination with this pathogenic bacterium.Melioidosis is a severe, emerging disease caused by the Gram-negative bacterium Burkholderia pseudomallei, a hydrophilic soil saprophyte that is endemic in Southeast Asia and northern Australia (3, 16, 17). Melioidosis is the most common cause of fatal community-acquired bacteremic pneumonia in northern Australia (4).Melioidosis outbreaks causing fatalities among humans and animals have been attributed to contaminated unchlorinated water supplies in northern Australia (5, 8) (B. J. Currie, personal observation). Rural water bores, of which there are 5,000 around Darwin alone, are mostly unchlorinated due to concerns of bore owners about taste, by-products, and maintenance of chlorination. We have analyzed the association of the occurrence of B. pseudomallei with environmental and physicochemical water properties in unchlorinated bore water from rural Darwin in northern Australia. This study is the first report on the habitat of B. pseudomallei in water supplies.     

Sero-Epidemiology of Lyme Disease in an Endemic Area in China

A sero-epidemiological investigation on Lyme disease was carried out in a forestry center of Hailin County, Heilongjiang Province, China. A total of 381 participants including forestry workers and their dependents completed questionnaires and had blood samples taken for detection of antibody against Borrelia burgdorferi by indirect immunofluorescent assay. Of 381 participants, 250 (65.6%) had a history of tick bites between May and July, 1987, and 379 (99.5%) at some time in the past, 56 (14.7%) developed erythema migrans at the site of tick attachment, 138 (36.2%) had late manifestations of Lyme disease, and 101 (26.2%) were seropositive. There was a significant difference in the seropositive rate between the persons with tick bites and those without tick bites in 1987 (P<0.01). The titers in the older age group (over 30) were slightly higher than those of the younger age group (under 30). No relationship between the prevalence of Lyme manifestations by tick bites and the result of serological test was observed. Further investigations are necessary to assess the actual prevalence and incidence of infections using antigen of Lyme disease borreliae isolated in China.     

The present conservation status of Juniperus forests in the South Ethiopian Endemic Bird Area

Field survey data and Landsat satellite imagery were used to evaluate the conservation status of two Juniperus forests (Mankubsa and Arero) in the south Ethiopian Endemic Bird Area. Forest cover and dense woodland decreased in both areas between 1986 and 2002, but rates of habitat change and human impact were greater at Mankubsa than at Arero. We suggest that at Mankubsa increased grazing pressure, agricultural expansion, commercial fuelwood and timber exploitation are threatening forest persistence, while most of the degradation at Arero is because of the grazing of domestic animals. Conservation efforts should focus on creating tree plantations and improving forest resource use efficiency at Mankubsa, while at Arero better results could be obtained by improving pasture quality in the habitats surrounding the forest.     

Modeling Exposure Intensity and Susceptibility-Latency Effect from Retrospective Cohort Data - Application to Lung Cancer Mortality in Blackfoot Disease Endemic Area in Taiwan

It is challenge in epidemiology to characterize the temperol aspect of exposure-disease association. The authors propose a stochastic model to deal with exposures that are time-dependent and exhibit susceptibility and latency effects. The model is applied to a retrospective cohort data on lung cancer mortality in the blackfoot disease endemic area in Taiwan. The authors compare the proposed model with the multistage model, the back-calculation model, the catalytic model, and the age-period-cohort models.     

Analysis of the Prevalence,Secretion and Function of a Cell Cycle-Inhibiting Factor in the Melioidosis Pathogen Burkholderia pseudomallei

Enteropathogenic and enterohaemorrhagic Escherichia coli express a cell cycle-inhibiting factor (Cif), that is injected into host cells via a Type III secretion system (T3SS) leading to arrest of cell division, delayed apoptosis and cytoskeletal rearrangements. A homologue of Cif has been identified in Burkholderia pseudomallei (CHBP; Cif homologue in B. pseudomallei; BPSS1385), which shares catalytic activity, but its prevalence, secretion and function are ill-defined. Among 43 available B. pseudomallei genome sequences, 33 genomes (76.7%) harbor the gene encoding CHBP. Western blot analysis using antiserum raised to a synthetic CHBP peptide detected CHBP in 46.6% (7/15) of clinical B. pseudomallei isolates from the endemic area. Secretion of CHBP into bacterial culture supernatant could not be detected under conditions where a known effector (BopE) was secreted in a manner dependent on the Bsa T3SS. In contrast, CHBP could be detected in U937 cells infected with B. pseudomallei by immunofluorescence microscopy and Western blotting in a manner dependent on bsaQ. Unlike E. coli Cif, CHBP was localized within the cytoplasm of B. pseudomallei-infected cells. A B. pseudomallei chbP insertion mutant showed a significant reduction in cytotoxicity and plaque formation compared to the wild-type strain that could be restored by plasmid-mediated trans-complementation. However, there was no defect in actin-based motility or multinucleated giant cell formation by the chbP mutant. The data suggest that the level or timing of CHBP secretion differs from a known Bsa-secreted effector and that CHBP is required for selected virulence-associated phenotypes in vitro.     

First Genome Sequence of a Burkholderia pseudomallei Isolate in China,Strain BPC006, Obtained from a Melioidosis Patient in Hainan

Melioidosis, caused by Burkholderia pseudomallei, is considered to be endemic to Northern Australia and Southeast Asia, with high mortality and relapse rates, regardless of powerful antibiotic therapy. Here we report the first genome sequence of Burkholderia pseudomallei strain BPC006, obtained from a melioidosis patient in Hainan, China. The genome sizes of the 2 chromosomes were determined to be 4,001,777 bp and 3,153,284 bp.     

Sensitive and Specific Molecular Detection of Burkholderia pseudomallei, the Causative Agent of Melioidosis, in the Soil of Tropical Northern Australia

Burkholderia pseudomallei, the cause of the severe disease melioidosis in humans and animals, is a gram-negative saprophyte living in soil and water of areas of endemicity such as tropical northern Australia and Southeast Asia. Infection occurs mainly by contact with wet contaminated soil. The environmental distribution of B. pseudomallei in northern Australia is still unclear. We developed and evaluated a direct soil B. pseudomallei DNA detection method based on the recently published real-time PCR targeting the B. pseudomallei type III secretion system. The method was evaluated by inoculating different soil types with B. pseudomallei dilution series and by comparing B. pseudomallei detection rate with culture-based detection rate for 104 randomly collected soil samples from the Darwin rural area in northern Australia. We found that direct soil B. pseudomallei DNA detection not only was substantially faster than culture but also proved to be more sensitive with no evident false-positive results. This assay provides a new tool to detect B. pseudomallei in soil samples in a fast and highly sensitive and specific manner and is applicable for large-scale B. pseudomallei environmental screening studies or in outbreak situations. Furthermore, analysis of the 104 collected soil samples revealed a significant association between B. pseudomallei-positive sites and the presence of animals at these locations and also with moist, reddish brown-to-reddish gray soils.     

Occurrence of Nontuberculous Mycobacterial Pulmonary Infection in an Endemic Area of Tuberculosis

The majority of investigations of the epidemiology of nontuberculous mycobacteria (NTM) have focused on highly developed nations with a low prevalence of tuberculosis. In contrast, the Para state of north Brazil represents an area of high tuberculosis prevalence and increasing NTM incidence. Toward the goal of understanding the dynamics of infection by all Mycobacterium species, we report patient characteristics and the identification of NTM strains isolated from sputum samples from patients that were residents of Para, a state in the Amazon region, Northern of Brazil, over the period January 2010 through December 2011 (2 years). The 29 NTM patients comprised 13.5% of positive mycobacterial cultures over the 2-year period. A major risk factor for NTM pulmonary disease was previous tuberculosis (76%). Further, the average age of NTM patients (52 years) was significantly higher than that of tuberculosis patients (39 years) and more were female (72.4% vs. 37.4%). Unlike other Brazilian states, NTM pulmonary patients in Para were infected with a different spectrum of mycobacteria; primarily the rapidly growing Mycobacterium massiliense and Mycobacterium simiae complex.     

Comparison of Trace Element Concentrations in Ambient Air of Industrial and Residential Areas in Tehran City

Tehran is densely populated possessing unhealthy and polluted air. There are 22 municipality districts in Tehran, and considerable numbers of industries are located within each district. Due to adverse environmental and health effects of trace elements in urban air, this study has been carried out to measure and compare concentration of trace elements including Ag, As, Br, Ce, Co, Cr, Cs, Cu, Mn, Hf, La, Sb, Sc, Sm, Th, and Zn in industrial (district 21) and residential (district 22) areas. For this purpose, total suspended particles were collected in two different time intervals in the year 2008. As a result, maximum and minimum concentrations in residential area associated with Mn (2.9 μg/m³) and Hf (2.92 × 10⁻⁴ μg/m³), respectively. Also, in industrial sites, these concentrations are higher for Mn (4.79 × 10⁻¹ μg/m³) and Hf (6.65 × 10⁻⁴ μg/m³). The concentrations of Mn (2.07 μg/m³) and Hf (7.46 × 10⁻⁴ μg/m³) in traffic zone are close to residential values. General, concentrations of Ce, Co, Cs, La, Sc, Sm, Th, Br, and Hf in industrial area were higher than in residential area. The source of these elements in Tehran's air is mainly anthropogenic. According to the achieved results, the concentration levels of As, Cr, and Zn were close together in both residential and industrial zones. Therefore, these elements could have been originated from natural sources. Also, results show that levels of Ag, Mn, Sb, and Cu in residential area were higher than in industrial area. Finally, obtained data indicated that the trends of average concentration of trace elements in industrial area (from low to high) follow as Hf<Cs<Sm<Th<La<Ce<Sc<Co<Ag<As<Sb<Cr<Br<Zn<Cu<Mn and, in residential area, are in order of Hf<Cs<La<Th<Ce<Sc<Sm<Co<As<Ag<Br<Sb<Cr<Zn<Cu<Mn.     

Paracoccidioidal Infection in HIV Patients at an Endemic Area of Paracoccidioidomycosis in Brazil

The association between paracoccidioidomycosis (PCM) and AIDS is relatively rare in contrast to the higher incidence of other systemic mycosis. The explanation may be that AIDS is still predominantly an urban disease, and the PCM is endemic in Latin American rural areas. The aim of this study was to detect the prevalence of Paracoccidioides brasiliensis infection in HIV-positive patients at an endemic area of paracoccidioidomycosis in Brazil. Skin test with purified 43 kD glycoprotein (gp43) was performed in 90 HIV/AIDS patients. The prevalence found was 12.2% and it may be even greater, considering that HIV/AIDS patients may not respond to the intradermal test, which depends on cellular immunity for its positivity.     

人类T淋巴细胞白血病病毒流行区无偿献血人群筛查研究

人类T淋巴细胞白血病病毒(human T-lym-photropic virus,HTLV)是最早发现的人类逆转录病毒,分为I型和II型。HTLV感染后,会在人体内长期存在,潜伏期长达20年以上,并在多年后引起白血病或下肢瘫痪等致死性或致残性疾病,迄今有效的治疗药物和预防性疫苗仍未研制成功[1]。因此,唯一积极的控制办法就是及时发现HTLV感染者,并采取措施切断其传播途径。由于HTLV可经血传播,日本、美国、澳大利亚、法国及其它一些国家和地区已将其列为献血员必检项目之一,但我国各地均一直未开展此项目。自1986年以来,我国先后在多个省、市发现了HTLV感染…     

Evidence of Endemic Goiter and Iodine Deficiency in a Mountainous Area of Haiti

ObjectiveTo investigate reports of iodine-deficiency disorder in a specific area of Haiti.MethodsIn March 2008, this cross-sectional study was performed in an area 15 miles northeast of Jacmel, Haiti, within the Chaîne de la Selle Mountains. Before arrival of the study team, an announcement was made throughout local villages soliciting volunteers to meet at a central location. Of those who arrived, participants were selected in an attempt to sample individuals from all age groups, regardless of goiter status. After providing verbal informed consent, each participant was photographed and assigned a number to be used to protect privacy. An examiner performed palpation of the thyroid gland on each participant in accordance with World Health Organization criteria. Results of palpation were classified into 3 grades: grade 0, the thyroid gland was not palpable; grade 1, the thyroid gland was palpable but not visible; and grade 2, the thyroid gland was palpable and visible while the patient was in a normal position. Casual urine samples were collected from each participant and analyzed spectrophotometrically for urinary iodine concentration.ResultsEighty-eight individuals aged 2 to 72 years participated in the study. Median urinary iodine concentration was 39 μg/L. Of the 88 participants, 82 (93%) were iodine deficient (18 [20%] were severely deficient), and 45 (51%) had goiter on physical examination, including 27 with grade 1 goiters and 18 with grade 2 goiters.ConclusionsWe have documented iodine deficiency with associated endemic goiter in this previously uninvestigated Haitian population, for which world health agencies currently lack definitive data. These data have potential implications for both the local area and the country as a whole where further evaluation and treatment are needed for persons at high risk for iodine-deficiency disorder. (Endocr Pract. 2009;15:298-301)     

四川梅花鹿产仔换茸期声行为的研究

2009年4～9月在四川省若尔盖县铁布自然保护区,对四川梅花鹿Cervusnippon sichuanicus产仔换茸期的声行为进行了较系统的研究。四川梅花鹿产仔换茸期的声行为可以分为报警声、惊叫声、吼叫声、母子召唤声等20种鸣声。通过声谱分析获得了各种鸣声的语谱图及其频谱特征,并通过野外观察初步确定了这些声行为的生物学意义。在遇到危险或潜在危险时,成年雄鹿往往第一个发出报警鸣声,而雌鹿的报警声大约是雄鹿的两倍多。成年个体的主动防御鸣声频次要多于亚成体,而幼体未见有该类鸣声。对梅花鹿不同亚种和不同季节的几种声行为进行了比较。     

Environmental Biology of an Endemic Cyprinid,Varicorhinus alticorpus,in a Subtropical Mountain Stream of Taiwan

An endemic freshwater fish, Varicorhinus alticorpus, was studied from 1990 to 1993 in a fast flowing mountain stream in southern Taiwan (22°30'N, 120°30'E). The analysis of environmental conditions suggested that the fish could tolerate water temperatures between 19–24°C, a pH of 8.0–8.8, and flow velocity from 20–100cmsec^–1. The fish occupied riffles and pools. They foraged and schooled during daytime and aggregated in crevices between rocks at night. Most juveniles stayed at the shallow sandy-pebble flat by the pool where flow was slow. The species feed primarily on the periphyton growing on the rocks, and leave unique scars after grazing. One peak of juvenile recruitment was observed right after the rainy season, suggesting that reproduction of the fish has adapted to the climate. The change of abundance of adults along the habitats (at 150 to 800 m altitude) also indicated that the fish might migrate to low altitudes for spawning and disperse back to higher altitudes for exploiting new resources. The environmental biology of this fish shows an example of a cyprinid adapted to habitats in a subtropical mountain stream.     

红喉雉鹑繁殖季的夜栖行为与夜栖地选择

红喉雉鹑（Tetraophasisobscurus）是中国特有种,国家一级重点保护野生动物,研究其繁殖季的夜栖地选择,能丰富该物种的基础资料,理解其生存策略,为该物种的保护和深入研究提供科学依据。2021年和2022年的3～7月,采用跟踪观察和系统搜索法,对四川王朗国家级自然保护区繁殖期红喉雉鹑的夜栖行为和夜栖地选择进行了调查,共记录夜栖地样方34个。采用卡方检验、配对样本T检验、Mann-WhitneyU检验和逻辑斯蒂回归模型方法对数据进行分析。红喉雉鹑主要夜栖于方枝柏（Sabina saltuaria）等针叶树,常见1只单独夜栖或2只共同夜栖,未见有集群夜栖;夜栖树通常位于上坡位,距离林缘和最近乔木较近,具有较大的胸径和郁闭度,栖枝距离地面高度6.6 m左右,栖枝长度约1.66m;夜栖地具有较大的坡度和落叶盖度,而灌木盖度较小。安全因素（夜栖树胸径、坡度）和食物因素（落叶盖度）是红喉雉鹑选择夜栖地的最关键因素。     

Degradation of Chlorobenzenes at Nanomolar Concentrations by Burkholderia sp. Strain PS14 in Liquid Cultures and in Soil

The utilization of 1,2,4,5-tetrachloro-, 1,2,4-trichloro-, the three isomeric dichlorobenzenes and fructose as the sole carbon and energy sources at nanomolar concentrations was studied in batch experiments with Burkholderia sp. strain PS14. In liquid culture, all chlorobenzenes were metabolized within 1 h from their initial concentration of 500 nM to below their detection limits of 0.5 nM for 1,2,4,5-tetrachloro- and 1,2,4-trichlorobenzene and 7.5 nM for the three dichlorobenzene isomers, with 63% mineralization of the tetra- and trichloroisomers. Fructose at the same initial concentration was, in contrast, metabolized over a 4-h incubation period down to a residual concentration of approximately 125 nM with 38% mineralization during this time. In soil microcosms, Burkholderia sp. strain PS14 metabolized tetrachlorobenzene present at 64.8 ppb and trichlorobenzene present at 54.4 ppb over a 72-h incubation period to below the detection limits of 0.108 and 0.09 ppb, respectively, with approximately 80% mineralization. A high sorptive capacity of Burkholderia sp. strain PS14 for 1,2,4,5-tetrachlorobenzene was found at very low cell density. The results demonstrate that Burkholderia sp. strain PS14 exhibits a very high affinity for chlorobenzenes at nanomolar concentrations.     

Continuous Cultivation and Drug Susceptibility Testing of Plasmodium falciparum in a Malaria Endemic Area

ABSTRACT Isolates (UCH-23 and OM) and cloned strains of Plasmodium falciparum (Clones W-2 and D-6) were maintained in continuous culture for 28 to 150 days using culture media supplemented with 10% (v/v) heat inactivated semi-immune human plasma. Microscopic appearance and growth rates (R) of the parasites in media supplemented with semi-immune human plasma [R = 1.13 (W-2), 0.92 (D-6), 0.75 (OM) and 0.84 (UCH-23)] were comparable to those of parallel cultures maintained in media supplemented with 10% (v/v) heat inactivated non-immune human plasma [R = 1.42 (W-2), 0.83 (D-6), 0.66 (OM) and 0.89 (UCH-23)]. In addition, IC50 for chloroquine and mefloquine against the two cloned strains of P. falciparum maintained in culture media supplemented with either non-immune human plasma or semi-immune human plasma were identical. Although growth rates of new isolates (UCH-23 and OM) fluctuated over time, they stabilized between the 12th and 19th day of adaptation to culture. This fluctuation in growth rates of the new isolates underscores the influence of population dynamics during adaptation of P. falciparum to continuous culture. Sixty-eight percent of the primary isolates (170 of 250) obtained from patients in Ibadan were successfully adapted and maintained in continuous culture using semi-immune human plasma. The results of these studies indicate that semi-immune human plasma is a suitable supplement for continuous cultivation and drug susceptibility testing of P. falciparum. This finding will have practical implications in malaria endemic areas where difficulties in obtaining non-immune human plasma or serum limits establishment of continuous culture of P. falciparum and its application in studies on malaria.