Department of Biology, Lakehead University, 955 Oliver Road, Thunder Bay, Ontario, Canada P7B 5E1.
J Microbiol Methods. 2010 Jul;82(1):19-27. doi: 10.1016/j.mimet.2010.03.012. Epub 2010 Mar 30.
Sphingomonas species can be found ubiquitously in the environment and can be frequently found in surface biofilms. Some Sphingomonas strains are well known for metabolizing complex organic pollutants but some are opportunistic human pathogens. Despite the importance of the Sphingomonas species, a reliable system to isolate this group of bacteria from the environment has not been developed. In this study, a combined streptomycin-piperacillin selective growth medium/polymerase chain reaction (PCR) detection approach is developed to isolate and identify the Sphingomonas bacteria. A total of 72 known Sphingomonas strains (including 21 different Sphingomonas species type strains) and 14 non-Sphingomonas species were tested using a new Sphingomonas-specific growth medium containing 100 and 50 microg/ml streptomycin and piperacillin, respectively. All the Sphingomonas strains showed positive growth on the selective medium and no growth was shown by the non-Sphingomonas species. In addition, two sets of PCR primers targeting the serine palmitoyltransferase gene (spt), a crucial sphingolipid biosynthesis gene, were developed. With the exception of the Sphingomonas subarctica type strain, 71 of the 72 known Sphingomonas samples were amplified positively by either one or both of the spt-specific primers. None of the non-Sphingomonas bacteria were amplified by the spt primers. To verify the effectiveness of this novel approach for use in environmental screening applications the Sphingomonas selective medium was used to isolate 165 potential Sphingomonas isolates, including 101 yellow, 4 orange and 58 unpigmented isolates, from the influent water and biofilm samples of a pulp and paper mill in Northwestern Ontario. Screening of these isolates with the two Sphingomonas spt-PCR primer sets showed that 98% of the yellow isolates and 100% of the orange isolates were positive to the spt-PCR test. None of the unpigmented isolates was positive to the spt-PCR assay. The 16S rDNA of 17% of the spt+ve and -ve isolates were sequenced and analyzed. All of the yellow and orange pigmented isolates were Sphingomonas while none of the unpigmented isolates were Sphingomonas. REP-PCR was performed on 79 Sphingomonas samples randomly selected from the paper mill and hospital isolates and showed that a diverse group of Sphingomonas can be grown or isolated by our Sphingomonas selective growth medium. Therefore, by using the streptomycin-piperacillin selective growth medium in combination with the colour pigmentation and the positive spt-PCR reactions of the isolates, a diverse population of Sphingomonas strains can be isolated and identified from complex microbial communities with high accuracy.
鞘氨醇单胞菌属的物种在环境中无处不在，经常可以在表面生物膜中发现。一些鞘氨醇单胞菌菌株以代谢复杂的有机污染物而闻名，但有些是机会性人类病原体。尽管鞘氨醇单胞菌属很重要，但尚未开发出一种从环境中分离该细菌群的可靠系统。在这项研究中，开发了一种组合链霉素-哌拉西林选择性生长培养基/聚合酶链反应 (PCR) 检测方法，用于分离和鉴定鞘氨醇单胞菌。使用含有 100 和 50 μg/ml 链霉素和哌拉西林的新鞘氨醇单胞菌特异性生长培养基测试了总共 72 种已知的鞘氨醇单胞菌菌株（包括 21 种不同的鞘氨醇单胞菌属模式菌株）和 14 种非鞘氨醇单胞菌属菌株。所有鞘氨醇单胞菌菌株在选择性培养基上均显示出阳性生长，而非鞘氨醇单胞菌属菌株则未显示生长。此外，还开发了两组针对丝氨酸棕榈酰转移酶基因 (spt) 的 PCR 引物，spt 是一种关键的鞘脂生物合成基因。除了鞘氨醇单胞菌 subarctica 模式菌株外，72 种已知的鞘氨醇单胞菌样品中的 71 种均被一种或两种 spt 特异性引物扩增为阳性。非鞘氨醇单胞菌属菌株均未被 spt 引物扩增。为了验证这种新方法在环境筛选应用中的有效性，将鞘氨醇选择性培养基用于从安大略省西北部的纸浆和造纸厂的进水和生物膜样品中分离 165 种潜在的鞘氨醇分离株，包括 101 种黄色、4 种橙色和 58 种非色素分离株。用两种鞘氨醇 spt-PCR 引物对这些分离株进行筛选，结果表明，98%的黄色分离株和 100%的橙色分离株对 spt-PCR 检测呈阳性。非色素分离株均未对 spt-PCR 检测呈阳性。对 17%的 spt+ve 和 -ve 分离株的 16S rDNA 进行测序和分析。所有黄色和橙色色素分离株均为鞘氨醇单胞菌，而非色素分离株均不是鞘氨醇单胞菌。从纸厂和医院分离株中随机选择 79 种鞘氨醇单胞菌样本进行 REP-PCR，结果表明，我们的鞘氨醇选择性生长培养基可培养或分离出多种鞘氨醇单胞菌。因此，通过使用链霉素-哌拉西林选择性生长培养基结合分离物的颜色色素沉着和阳性 spt-PCR 反应，可以从复杂的微生物群落中以高精度分离和鉴定出多种鞘氨醇单胞菌菌株。
