The bacterial community in partially purified water, which is prepared by ion exchange from tap water and is used in pharmaceutical manufacturing processes, was analyzed by denaturing gradient gel electrophoresis (DGGE). 16S ribosomal DNA fragments, including V6, -7, and -8 regions, were amplified with universal primers and analyzed by DGGE. The bacterial diversity in purified water determined by PCR-DGGE banding patterns was significantly lower than that of other aquatic environments. The bacterial populations with esterase activity sorted by flow cytometry and isolated on soybean casein digest (SCD) and R2A media were also analyzed by DGGE. The dominant bacterium in purified water possessed esterase activity but could not be detected on SCD or R2A media. DNA sequence analysis of the main bands on the DGGE gel revealed that culturable bacteria on these media were Bradyrhizobium sp., Xanthomonas sp., and Stenotrophomonas sp., while the dominant bacterium was not closely related to previously characterized bacteria. These data suggest the importance of culture-independent methods of quality control for pharmaceutical water.

Physiologically active bacteria in purified water used in the manufacturing process of pharmaceutical products were enumerated in situ. Bacteria with growth potential were enumerated using the micro‐colony technique and direct viable counting (DVC), followed by 24 h of incubation in 100‐fold diluted SCDB (Soybean Casein Digest Broth) at 30 °C. Respiring and esterase‐active bacteria were detected by fluorescent staining with 5‐cyano‐2,3‐ditolyl tetrazolium chloride (CTC) and 6‐carboxyfluorescein diacetate (6CFDA), respectively. A large number of bacteria in purified water retained physiological activity, while most could not form colonies on conventional media. The techniques applied in this study enabled bacteria to be counted within 24 h so results could be available within one working day. These rapid and convenient techniques should be useful for the systematic monitoring of bacteria in water used for pharmaceutical manufacturing.

Acriflavine resistance in the clinical meticillin-resistant Staphylococcus aureus isolate KT24 was found not to be mediated by multidrug efflux pumps encoded by qacA/B, smr, qacE, qacG, qacH, qacJ or norA. Early uptake and accumulation of ethidium bromide in MRSA KT24 was significantly lower than that in a susceptible strain, although the efflux rates were similar. Therefore, a permeability barrier in MRSA KT24 may be the conceivable mechanism of acriflavine resistance. Interestingly, it was found that MRSA KT24 had a significantly thickened cell wall, and that cellwall thickness increased gradually during bacterial growth. In contrast, cell size and surface area in MRSA KT24 were not different from those in the susceptible strain. Moreover, MRSA KT24 exposure to sub-MIC concentrations of acriflavine resulted in a thicker cell wall. These results indicate that cell-wall thickness may be responsible for acriflavine resistance in S. aureus.

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.

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