Bacterial population dynamics and community structure in a pharmaceutical manufacturing water supply system determined by real-time PCR and PCR-denaturing gradient gel electrophoresis

Abstract

To control bacteria in the pharmaceutical water supply system. Bacteria were enumerated by conventional culture method and fluorescent vital staining. Activated carbon treatment and storage in a tank provided favourable environments for bacterial growth. The bacterial population of the water in both the post-activated carbon treatment and the tank was analysed by denaturing gradient gel electrophoresis (DGGE) with PCR-amplified 16S rDNA fragments including V6, -7, and -8 regions. The bacterial community structure in activated carbon treated water was stable throughout the year. Several kinds of bacteria such as genus Aquaspirillum and Methylobacterium were found in the water after activated carbon treatment. The bacterial community structure was changed and other bacteria such as mycobacteria were detected after storage. Mycobacteria were quantified in water samples using real-time PCR targeting the 16S rDNA gene. Mycobacteria were also detected in tap water and their number was increased 10(3)-10(4)-fold higher after storage. These data suggest the importance of culture-independent methods for quality control of water used in pharmaceutical manufacturing. Critical steps and specified bacteria that should be controlled in the water supply system were recognized by culture-independent methods. These data will enable effective control of water used in the pharmaceutical industry.