PDF(Pubmed)
Abstract：
The soil bacterium Burkholderia pseudomallei causes melioidosis, a potentially fatal and greatly underdiagnosed tropical disease. Detection of B. pseudomallei in the environment is important to trace the source of infections, define risk areas for melioidosis and increase the clinical awareness. Although B. pseudomallei polymerase chain reaction (PCR)-based environmental detection provides important information, the culture of the pathogen remains essential but is still a methodological challenge. B. pseudomallei can catabolize erythritol, a metabolic pathway, which is otherwise rarely encountered among bacteria. We recently demonstrated that replacing threonine with erythritol as a single carbon source in the pH-neutral threonine-basal salt solution (TBSS-C50) historically used improved the isolation of B. pseudomallei from rice paddy soils. However, further culture medium parameters for an optimized recovery of B. pseudomallei strains from soils are still ill-defined. We, therefore, aimed to design a new erythritol-based medium by systematically optimizing parameters such as pH, buffer capacity, salt and nutrient composition. A key finding of our study is the enhanced erythritol-based growth of B. pseudomallei under acidic medium conditions. Our experiments with B. pseudomallei strains from different geographical origin led to the development of a phosphate-buffered acidic erythritol (ACER) medium with a pH of 6.3, higher erythritol concentration of 1.2%, supplemented vitamins and nitrate. This highly selective medium composition shortened the lag phase of B. pseudomallei cultures and greatly increased growth densities compared to TBSS-C50 and TBSS-C50-based erythritol medium. The ACER medium led to the highest enrichments of B. pseudomallei as determined from culture supernatants by quantitative PCR in a comparative validation with soil samples from the central part of Vietnam. Consequently, the median recovery of B. pseudomallei colony forming units on Ashdown\'s agar from ACER subcultures was 5.4 times higher compared to TBSS-C50-based erythritol medium (p = 0.005) and 30.7 times higher than TBSS-C50 (p < 0.001). In conclusion, our newly developed ACER medium significantly improves the isolation of viable B. pseudomallei from soils and, thereby, has the potential to reduce the rate of false-negative environmental cultures in melioidosis risk areas.
摘要：
土壤细菌Burkholderiapseudomallei导致类石病，一种潜在致命且严重未被诊断的热带病。检测环境中的假单胞菌对追踪感染源很重要，确定类lioidosis的风险区域并提高临床意识。尽管基于聚合酶链反应（PCR）的环境检测提供了重要信息，病原体的培养仍然是必不可少的，但仍然是方法上的挑战。假单胞菌可以分解代谢赤藓糖醇，一种代谢途径，否则在细菌中很少遇到。我们最近证明，在历史上使用的pH中性苏氨酸基础盐溶液（TBSS-C50）中，用赤藓糖醇代替苏氨酸作为单一碳源可以改善从稻田土壤中分离假单胞菌。然而,从土壤中优化回收假单胞菌菌株的进一步培养基参数仍然不确定。我们,因此,旨在通过系统地优化pH等参数来设计一种新的赤藓糖醇基培养基，缓冲能力,盐和营养成分。我们研究的一个关键发现是在酸性培养基条件下假单胞菌的基于赤藓糖醇的生长增强。我们使用来自不同地理来源的假单胞菌菌株进行的实验导致开发了pH为6.3，赤藓糖醇浓度为1.2％的磷酸盐缓冲酸性赤藓糖醇（ACER）培养基，补充维生素和硝酸盐。与基于TBSS-C50和TBSS-C50的赤藓糖醇培养基相比，这种高度选择性的培养基组合物缩短了假单胞菌培养物的滞后期，并大大提高了生长密度。在与来自越南中部的土壤样品的比较验证中，通过定量PCR从培养上清液中确定，ACER培养基导致最高的假单胞菌富集。因此,ACER继代培养的Ashdown's琼脂上的假单胞菌菌落形成单位的平均回收率是基于TBSS-C50的赤藓糖醇培养基的5.4倍(p=0.005)和TBSS-C50的30.7倍(p<0.001)。总之,我们新开发的ACER培养基显着改善了从土壤中分离活的假单胞菌，因此，有可能降低类霉菌病风险地区环境培养的假阴性率。
