Division of Microbiology, Osaka Institute of Public Health, Japan.
Department of Bacteriology, Toyama Institute of Health, Japan.
Jpn J Infect Dis. 2021 Nov 22;74(6):592-599. doi: 10.7883/yoken.JJID.2020.926. Epub 2021 Mar 31.
Genes conferring carbapenem resistance have spread worldwide among gram-negative bacteria. Subtyping of these genes has epidemiological value due to the global cross-border movement of people. Subtyping of bla genes that frequently detected in Japan appears to be important in public health settings; however, there are few useful tools for this purpose. We developed a subtyping screening tool based on PCR direct sequencing, which targets the internal sequences of almost all bla genes. The tool used bipartite multiplex primers with M13 universal sequences at the 5'-end. According to in silico analysis, among the 78 known IMP-type genes, except for bla, 77 detected genes were estimated to be differentiated. In vitro evaluation indicated that sequences of amplicons of IMP-1, IMP-6, IMP-7, and IMP-20 templates were identical to their respective subtypes. Even if the amplicons were small or undetectable through the first PCR, sufficient amplicons for DNA sequencing were obtained through a second PCR using the M13 universal primers. In conclusion, our tool can be possibly used for subtype screening of bla, which is useful for the surveillance of bacteria with bla in clinical and public health settings or environmental fields.
具有碳青霉烯耐药性的基因已在全球范围内的革兰氏阴性细菌中传播。由于人员在全球范围内的跨境流动，对这些基因进行亚型分析具有流行病学价值。在日本经常检测到的 bla 基因的亚型分析似乎在公共卫生环境中很重要；然而，为此目的提供了很少有用的工具。我们开发了一种基于 PCR 直接测序的分型筛选工具，该工具针对几乎所有 bla 基因的内部序列。该工具使用带有 M13 通用序列的双份多重引物在 5'-端。根据计算机分析，在 78 种已知的 IMP 型基因中，除了 bla 基因之外，估计有 77 种检测到的基因具有差异性。体外评估表明，IMP-1、IMP-6、IMP-7 和 IMP-20 模板的扩增子序列与其各自的亚型相同。即使通过第一轮 PCR 无法检测到或扩增子较小，通过使用 M13 通用引物进行第二轮 PCR 也可以获得足够用于 DNA 测序的扩增子。总之，我们的工具可用于 bla 的亚型筛选，这对于临床和公共卫生环境或环境领域中 bla 细菌的监测非常有用。
