Alanine Racemase Mutants of Burkholderia pseudomallei and Burkholderia mallei and Use of Alanine Racemase as a Non-Antibiotic-Based Selectable Marker

Burkholderia pseudomallei and Burkholderia mallei are category B select agents and must be studied under BSL3 containment in the United States. They are typically resistant to multiple antibiotics, and the antibiotics used to treat B. pseudomallei or B. mallei infections may not be used as selective agents with the corresponding Burkholderia species. Here, we investigated alanine racemase deficient mutants of B. pseudomallei and B. mallei for development of non-antibiotic-based genetic selection methods and for attenuation of virulence. The genome of B. pseudomallei K96243 has two annotated alanine racemase genes (bpsl2179 and bpss0711), and B. mallei ATCC 23344 has one (bma1575). Each of these genes encodes a functional enzyme that can complement the alanine racemase deficiency of Escherichia coli strain ALA1. Herein, we show that B. pseudomallei with in-frame deletions in both bpsl2179 and bpss0711, or B. mallei with an in-frame deletion in bma1575, requires exogenous d-alanine for growth. Introduction of bpsl2179 on a multicopy plasmid into alanine racemase deficient variants of either Burkholderia species eliminated the requirement for d-alanine. During log phase growth without d-alanine, the viable counts of alanine racemase deficient mutants of B. pseudomallei and B. mallei decreased within 2 hours by about 1000-fold and 10-fold, respectively, and no viable bacteria were present at 24 hours. We constructed several genetic tools with bpsl2179 as a selectable genetic marker, and we used them without any antibiotic selection to construct an in-frame ΔflgK mutant in the alanine racemase deficient variant of B. pseudomallei K96243. In murine peritoneal macrophages, wild type B. mallei ATCC 23344 was killed much more rapidly than wild type B. pseudomallei K96243. In addition, the alanine racemase deficient mutant of B. pseudomallei K96243 exhibited attenuation versus its isogenic parental strain with respect to growth and survival in murine peritoneal macrophages

Evolution of Burkholderia pseudomallei in Recurrent Melioidosis

Burkholderia pseudomallei, the etiologic agent of human melioidosis, is capable of causing severe acute infection with overwhelming septicemia leading to death. A high rate of recurrent disease occurs in adult patients, most often due to recrudescence of the initial infecting strain. Pathogen persistence and evolution during such relapsing infections are not well understood. Bacterial cells present in the primary inoculum and in late infections may differ greatly, as has been observed in chronic disease, or they may be genetically similar. To test these alternative models, we conducted whole-genome comparisons of clonal primary and relapse B. pseudomallei isolates recovered six months to six years apart from four adult Thai patients. We found differences within each of the four pairs, and some, including a 330 Kb deletion, affected substantial portions of the genome. Many of the changes were associated with increased antibiotic resistance. We also found evidence of positive selection for deleterious mutations in a TetR family transcriptional regulator from a set of 107 additional B. pseudomallei strains. As part of the study, we sequenced to base-pair accuracy the genome of B. pseudomallei strain 1026b, the model used for genetic studies of B. pseudomallei pathogenesis and antibiotic resistance. Our findings provide new insights into pathogen evolution during long-term infections and have important implications for the development of intervention strategies to combat recurrent melioidosis

Stability of maxillary surgical movement in unilateral cleft lip and palate with preceding alveolar bone grafting

Objective: To evaluate the long-term three-dimensional stability of Le Fort I maxillary osteotomy in patients with unilateral cleft lip and palate (CLP) who had preceding alveolar bone grafting. Design: Analysis of prospectively collected data. Setting: University teaching hospital and postgraduate training center. Subjects: Thirty consecutive patients with unilateral cleft lip and palate, who underwent the procedure between 1990 and 1999, satisfied the inclusion criteria and had complete records. There were 9 males and 21 females, with an age range of 14 to 28 years (mean, 18 years), and follow-up range of 12 to 66 months (mean, 62 months). Methods: Cephalometric and study cast analyses using pre- and postoperative records (3, 6, 12, 24, and 36 months). Evaluation of surgical movement and postsurgical change at all above time intervals was carried out to determine stability of surgical maxillary movement in the horizontal and vertical planes and to identify rotational and transverse relapse. Results: Total relapse of surgical movement was 31% in the horizontal plane and 52% in the vertical plane, as well as 30% rotational. Relapse correlated with extent of surgical movement, and most relapse occurred in the first 6 months after surgery. No significant transverse relapse was documented. Conclusion: Alveolar bone grafting prior to osteotomy stabilizes the transverse dimension of the dental arch, but does not improve horizontal, vertical, or rotational relapse, which remains significant. Correlation of relapse with extent of surgical movement does suggest that planned over-correction is a reasonable option.link_to_subscribed_fulltex

Optimization of culture protocols to isolate Leptospira spp. from environmental water, field investigation, and identification of factors associated with the presence of Leptospira spp. in the environment

The successful culture of&nbsp;Leptospira&nbsp;spp. from the environment is challenging. Here, we optimized the isolation of&nbsp;Leptospira&nbsp;spp. from water samples spiked with different species and initial concentrations of this organism. The time periods between water sampling and the isolation process were varied (0, 2, and 4 weeks). Bacterial cultures were observed under a microscope, and cultures were graded for cell density, weekly, for 12 weeks. Most pathogenic&nbsp;Leptospira&nbsp;spp. were difficult to culture under all conditions. All conditions of water samples spiked with novel species of&nbsp;Leptospira&nbsp;subclade P1 were culture positive within 2 weeks. For&nbsp;Leptospira&nbsp;subclade P2, storing samples for 2 weeks prior to isolation resulted in more successful isolation compared with isolation after other storage conditions. For subclade S1, all samples with initial bacterial concentrations of more than 103&nbsp;colonies/mL, under all storage conditions, were successfully cultured. These results suggest that storing contaminated water samples for 2 to 4 weeks in the dark at an ambient temperature prior to culturing can improve the isolation of&nbsp;Leptospira&nbsp;spp. from the samples. We implemented this protocol and collected water samples from natural sources accessed by both humans and animals.&nbsp;Leptospira&nbsp;spp. was identified in 32% (35/109) of water samples. The animal species using a water source influenced the likelihood of water samples being contaminated with&nbsp;Leptospira&nbsp;spp. Cultures of&nbsp;Leptospira&nbsp;spp. from environmental samples can provide useful information for understanding the complex interactions between humans, animals and the environment in the transmission of leptospirosis.</div

Chronic Wounds Healing by Innovative Herbal Medicine and Buddhist Ways to Prevent Disability in Diabetic Patients

The objectives of this research were 1) to study the properties of both scientific and traditional medicinal herbs to produce innovative herbal medicine for chronic wounds healing in diabetic patients, and 2) to analyze chronic wounds healing by innovative herbal medicine and Buddhist ways to prevent disability in 30 diabetic patients in Mahasarakham, Thailand. The results revealed that 19 medicinal herbs have properties in managing the blood, vascular, lymphatic system, the main cause of chronic wounds, reducing blood viscosity, purifying blood vessels, flushing out waste water, and healing wounds. 5 medicinal herbs were used to produce innovative herbal medicine, and Buddhist principles were used in research and for informants. Chronic wounds in diabetic patients have three characteristics: peripheral neuropathy, ischemic wounds, and infectious wounds. Herbal Oil Formula 1 and Herbal Infusion Formula 2 were used as basic medicines for healing, Herbal Capsule Formula 3 for inflamed wounds, Herbal Infusion for wounds with dark secretions and rotten blood. Wounds with a lot of secretions, foul odors, and full wounds were healed by Mangosteen Peel Powder Formula 5. Accordingly, 100% of all 3 types of chronic wounds were healed. This saved all patients from disability

Buddhism and Thai Educational System: Historical Perspectives

In Thailand, Buddhism has played an important role in terms cultivating morality and ethics for people in the society. Later, it has become an important part of the educational system of the country. This paper has its aims to clarify some backgrounds of Buddhist studies in Thai educational system from the early period to the present day. First, it discusses (1) some of the Buddha's principles of teaching first, and thereafter (2) importance of Buddhist studies in Thai society, (3) management of Buddhist Studies, and (4) History of Buddhist Studies in Thailand and (5) Buddhist Studies Curriculum will be discussed. The conclusion of this study reveals that training morality and ethics to youth is the key factor making Buddhist studies exist in the educational system of Thailand

CLM English Teaching and Learning Guidelines for Undergraduate Students in Social Studies at Phutthachinnarat Sangha College, MCU

At Phutthachinnarat Sangha College (PSC), Thailand, there is a diversity of students from monastic and secular educational backgrounds, causing learning management, particularly teaching English, more difficult compared with other institutions. To understand and improve this educational context, in this paper, we provide the guidelines for English teaching and learning by means of Cooperative Learning Management (CLM) for undergraduate students majoring in Bachelor Program in Social Studies, Mahachulalongkornrajavidyalaya University, (MCU), PSC. In so doing, the overview of English teaching and learning in the studied site will be provided and thereafter CLM together with Learning Together Technique (LTT) will be discussed