[Environmental Monitoring in a Pharmaceutical Manufacturing Facility Using a Culture Independent Approach].

Strict microbial control is required in manufacturing facilities to ensure the quality of pharmaceuticals and foods. Environmental microbial monitoring plays a fundamental role in reducing the risk of microbial contamination. Appropriate microbial control requires an understanding of abundance and community structures of microbes in the target environment. However, most of these microbes are not culturable using conventional methods. In this study, we determined the number of microbial particles and assessed the environmental microbiome in a pharmaceutical manufacturing facility, using high-throughput sequencing of rRNA gene fragments. Our results provide fundamental data for the evaluation and control of microbes in the pharmaceutical and food industries.

Culture independent approach reveals domination of human-oriented microbes in a pharmaceutical manufacturing facility.

Strict microbial control is required in pharmaceutical manufacturing facilities, for which environmental microbial monitoring is fundamental. Appropriate microbial control is based on understanding the abundance and community structure of the microbes in the target environment, but most microbes are not culturable by conventional methods. Here, we determined the bacterial abundance and assessed the environmental microbiome in a pharmaceutical manufacturing facility using rRNA gene-targeted quantitative PCR (qPCR) and high-throughput sequencing of rRNA gene fragments. A commercially available microbial particle counter was also used for real-time measurements. In the air of the first gowning room and the passageway of the facility, the microbial particle number determined by both the particle counter and qPCR was ca. 104/m3; the number of microbial particles was about 100 times the number of culturable bacteria. Thus, the measurement of microbes using the particle counter was accurate. In the second gowning room of the facility, managed by a HEPA filter, the number of particles in the air was dependent on human movement, and was below the detection limit around 10 min after movement. Bacteria of the phyla Proteobacteria, Firmicutes, and Actinobacteria were frequently detected in samples from the facility; these bacteria are constituents of the human microbiota. Among fungi, Aspergillus and Cladosporium were detected in the air, and Malassezia was dominant on the walls. Our results provide fundamental data for the evaluation and control of microbes in pharmaceutical and food industry facilities.

Mechanisms of action of acriflavine: electron microscopic study of cell wall changes induced in Staphylococcus aureus by acriflavine.

The antimicrobial action of acriflavine, a quaternary ammonium compound, on Staphylococcus aureus was studied by electron microscopic observation. The bactericidal activity of acriflavine was dose-dependent over the 4 hr of exposure time. Scanning electron micrographs showed a wavy wrinkled cell surface following treatment with acriflavine. Transmission electron micrographs showed thickened cell walls following treatment with acriflavine. Acriflavine-induced cell wall thickness seemed to affect both the peripheral and cross walls, but was reversible after treatment removal. These findings indicate that cell wall thickness is a characteristic phenotype of S. aureus exposed to acriflavine. It is therefore believed that cell wall thickness plays an important role in the mechanism of action of acriflavine.

Cell-wall thickness: possible mechanism of acriflavine resistance in meticillin-resistant Staphylococcus aureus.

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 cell-wall 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.

16S ribosomal DNA-based analysis of bacterial diversity in purified water used in pharmaceutical manufacturing processes by PCR and denaturing gradient gel electrophoresis.

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.