Application of real-time PCR array to the multiple detection of antibiotic resistant genes in glacier ice samples

Abstract

Glacier environments harbor cold-tolerant bacteria (Amato et al., 2007; Christner et al., 2003; Segawa et al., 2005). However, a variety of bacteria such as soil and enteric bacteria have been detected from these highly oligotrophic and psychrophilic environments (Segawa et al., 2005; Sheridan et al., 2003). The origin of these bacteria remains unknown. These bacteria may have been supplied at least in part from animals by means such as bird droppings (Sjolund et al., 2008). There are other plausible transmissions, such as local and global atmospheric circulation. In the case of the global atmospheric circulation, a glacier environment may receive bacteria originating from hu-man industrial activities, although most glaciers are located at a distance from such sites of human activi-ties. Emergence of antibiotic resistant bacteria has been considered as a human impact on the environment (Cabello, 2006; Chee-Sanford et al., 2001; Goni-Urriza et al., 2000). The prevalence of antibiotic resistant genes in the glacier environment can be used as an indicator of human impact on the global environment. In this experiment, the prevalence of antibiotic resis-tant genes in glacier ice samples taken from various locations in the northern hemisphere (Alaska, Central Asia) and Antarctica were evaluated by real time PCR array approach using a Taqman probe system. We collected samples of the glacial ice (1 cm in depth) or cryoconite hole samples (Table 1) using a sterilized stainless steel scoop or sterilized syringe, re-spectively. The samples were stored in 50-ml sterile plastic conical tubes (Iwaki, Tokyo). All samples were kept frozen and transported to the Tokyo Institute of Technology, Japan or National Institute of Polar Re-search, Japan. Bacterial DNA was extracted as follows within a Class 100 Clean bench (Sanyo, Tokyo, Model MCV-131BNS) at the National Institute of Polar Research (Tokyo). Ice samples were melted and a portion (1‒