State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, 100071, China.
College of Life Sciences, Wuhan University, Wuhan, 430072, China.
Sci Rep. 2017 Aug 21;7(1):8370. doi: 10.1038/s41598-017-07841-1.
Although clinical antibiotic-resistant bacteria have attracted tremendous attention in the microbiology community, the resistant bacteria that persist in natural environments have been overlooked for a longtime. We previously proposed a new species Paramesorhizobium desertii, isolated from the soil of the Taklimakan Desert in China that is highly resistant to most β-lactam antibiotics. To identify potential β-lactamase(s) in this bacteria, we first confirmed the carbapenemase activity in the freeze-thawed supernatant of a P. desertii A-3-E culture using the modified Hodge assay. We then identified a novel chromosome-encoded carbapenemase (PAD-1) in strain A-3-E, using a shotgun proteomic analysis of the supernatant and genomic information. The bioinformatics analysis indicated that PAD-1 is a class A carbapenemase. Subsequent enzyme kinetic assays with purified PAD-1 confirmed its carbapenemase activity, which is similar to that of clinically significant class A carbapenemases, including BKC-1 and KPC-2. Because the location in which A-3-E was isolated is not affected by human activity, PAD-1 is unlikely to be associated with the selection pressures exerted by modern antibiotics. This study confirmed the diversity of antibiotic-resistant determinants in the environmental resistome.
尽管临床抗生素耐药菌在微生物学界引起了极大关注，但在自然环境中持续存在的耐药菌却长期以来被忽视。我们之前提出了一个新的物种 Paramesorhizobium desertii，它是从中国塔克拉玛干沙漠的土壤中分离出来的，对大多数β-内酰胺类抗生素具有高度耐药性。为了鉴定该细菌中潜在的β-内酰胺酶，我们首先使用改良 Hodge 试验，在 P. desertii A-3-E 培养物的冻融上清液中确认了碳青霉烯酶活性。然后，我们使用上清液的鸟枪法蛋白质组分析和基因组信息，在菌株 A-3-E 中鉴定出一种新型染色体编码的碳青霉烯酶（PAD-1）。生物信息学分析表明 PAD-1 是一种 A 类碳青霉烯酶。随后用纯化的 PAD-1 进行的酶动力学测定证实了其碳青霉烯酶活性，与临床重要的 A 类碳青霉烯酶（包括 BKC-1 和 KPC-2）相似。由于 A-3-E 被分离的位置不受人类活动的影响，因此 PAD-1 不太可能与现代抗生素施加的选择压力有关。本研究证实了环境耐药组中抗生素耐药决定因素的多样性。
