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

Aims: To control bacteria in the pharmaceutical water supply system. Methods and Results: 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. Conclusion: These data suggest the importance of culture-independent methods for quality control of water used in pharmaceutical manufacturing. Significance and Impact of the Study: 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.

16S Ribosomal RNA Gene-Based Phylogenetic Analysis of Abundant Bacteria in River, Canal and Potable Water in Bangkok, Thailand

“…Thus, this simple method is suitable for analyzing many samples simultaneously. TaqGold is often used for PCR amplification of eubacterial 16S rRNA genes from freshwater samples 10,11,14,15) ; however, this DNA polymerase was not very effective in our study.…”

“…In particular, denaturing gradient gel electrophoresis (DGGE) 9) has been used to determine the genetic diversity of natural microbial communities and to identify the phylogenetic affiliation of community members. [10][11][12] Polymerase chain reaction (PCR)-amplified DNA fragments of the eubacterial 16S ribosomal RNA (rRNA) gene that have the same length but different sequences can be separated by DGGE. However, PCR-DGGE analysis is often difficult to implement with freshwater samples from eutrophic environments that contain PCR-inhibitors, such as humic acid.…”

Bacterial Population Dynamics in a Reverse-Osmosis Water Purification System Determined by Fluorescent Staining and PCR-Denaturing Gradient Gel Electrophoresis

“…For DGGE of the PCR product, a 40 bp GC-rich sequence (GCclamp; 5'-CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGC-ACGGGGGG-3') was attached to the 5' end of the primer EUB f933. PCR was performed with Ampli Taq Gold (Applied Biosystem, Foster City, CA, USA) according to the method used by Kawai et al (8,14).…”

“…Culturebased techniques, however, have limitations in environmental microbiology, and currently fluorescence microscopy is recognized as a superior method for the enumeration of bacteria in their natural environment (2,10). Application of culture-independent methods such as fluorescent vital staining (1,11,24) has clarified the abundance and activity of bacteria in pharmaceutical water (7)(8)(9), dialysis fluid (22) and platelet concentrate (16): areas where bacterial detection by culture-dependent methods alone was difficult. These new methods provide sufficient information to assure, rapidly and accurately, the microbiological quality of the water produced and medical products.…”

“…The processed water, reverse-osmosis water (RO water), is a critical component of food, drink and pharmaceutical products. Quality control of RO water, particularly microbiological quality control, is especially important when RO water will be used to create an ingestible product (7)(8)(9). Microorganisms which survive the water purification process and proliferate can become a source of microbial contamination, while water must meet strict quality standards which should be maintained until use.…”