Every person involved in pharmaceutical product manufacturing has the responsibility to assure the quality of the product being produced. The aim of this paper is to validate quality assurance throughout the process of manufacturing pharmaceutical products. Additionally, within aseptic manufacturing, certain monitoring and information needs to be collected on a routine basis to continually assess the state of control of the complete operation. The basis for assessing the state of control is to have rigorous and defined information flow processes. Once the information is collected, quality assurance involves the ability to assess, evaluate, and make appropriate decisions to ensure the product has the required safety, identity, strength, quality, and purity. Quality assurance study is the process of bringing all of the information together, evaluating the information, making decisions, refining systems, and applying process knowledge. This process begins in the early stages of drug development when not a lot of specific process information about the process is known, but it is important to allow for development to progress, building knowledge about process. However, even in early development, sterility assurance requirements should be largely the same at all stages of development and routine commercial manufacturing.

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We have been investigating an advanced sterilization system that employs active oxygen species (AOS). We designed the sterilization equipment, including an evacuation system, which generates AOS from pure oxygen gas using ultraviolet irradiation, in order to study the conditions necessary for sterilization in the system’s chamber. Using Geobachillus stearothermophilus spores (10⁶ CFU) in a sterile bag as a biological indicator (BI) in the chamber of the AOS sterilization apparatus, we examined the viability of the BI as a function of exposure time, assessing the role of the decompression level in the sterilization performance. We found that the survival curves showed exponential reduction, and that the decompression level did not exert a significant influence on the survival curve. Subsequently, we investigated the sterilization effect as influenced by the spatial and environmental temperature variation throughout the chamber, and found that the sterilization effect varied with position, due to the varying environmentaltemperature in the respective areas. We confirmed that temperature is one of the most important factors influencing sterilization in the chamber, and estimated the temperature effect on the distribution of atomic oxygen concentration, using the quartz crystal microbalance (QCM) method with fluorocarbon thin film prepared by radio frequency sputtering.

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Drug susceptibility testing was carried out using 14 antibiotics in order to identify trends in the antibiotic tolerance of 142 strains of Enterobacteriaceae isolated from horsemeat commercially available for raw consumption (basashi). A comparison of the sensitivity to the 14 antibiotics using the 90% MIC (minimum inhibitory concentration) values (MIC₉₀) showed the strongest tolerance to ampicillin (ABPC) at a concentration of > 128 μg/mL, followed by that to fosfomycin (FOM) at a concentration of 128 μg/mL. When the sensitivity to these antibiotics was examined for each individual genus of tested bacteria, Hafnia spp. exhibited relative tolerance to ceftazidime (CAZ) and ceftriaxone (CTRX) at a concentration of 4 μg/mL and 2 μg/mL, respectively, which was high in comparison to that observed for the other strains. Furthermore, Raoultella spp. and Serratia spp. were found to be highly resistant to tetracycline (TC) at a concentration of 128 μg/mL and 64 μg/mL, respectively.
Of the 142 strains of test bacteria, 140 (98.6%) demonstrated resistance to ABPC, with the exception of Hafnia alvei and Klebsiella pneumonia. In addition, a total of eight strains (5.6%), seven Serratia marcescens strains and one Raoultella terrigena strain, were found to be resistant to TC. Furthermore, one strain of Citrobacter freundii exhibited resistance to nalidixic acid (NA), while another displayed resistance to ofloxacin (OFLX) (0.7% each), and one strain (0.7%) each of Enterobacter cloacae, Serratia marcescens and Citrobacter youngae demonstrated resistance to fosfomycin (FOM), streptomycin (SM) and kanamycin (KM), respectively. A single strain of C. freundii was found to be resistant to three antibiotics, ABPC, NA and OFLX. Resistance to two antibiotics was confirmed in 11 strains, including seven strains of S. marcescens and one strain of R. terrigena (a total of eight strains) resistant to ABPC and TC, and one strain each of C. youngae, S. marcescens and E. cloacae resistant to ABPC and KM, ABPC and SM, and ABPC and FOM, respectively. In addition, 128 strains were resistant to the single antibiotic of ABPC alone.
Of the 140 strains demonstrating antibiotic resistance, 137 (97.9%) retained the conjugative R-plasmid transfer factor, excluding three strains of S. marcescens. All transfer factors were ABPC and retained by a high proportion of the bacterial groups, with one strain (100%) being resistant to three antibiotics, nine (81.8%) of the 11 strains being resistant to two antibiotics, and 127 (99.2%) of the 128 strains being resistant to a single antibiotic. In addition, we examined ESBL productivity in the 140 strains of bacteria demonstrating drug tolerance; however, no strains exhibited this characteristic. Therefore, further observation is required to ascertain trends in antibiotic-tolerant bacteria.

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Ultraviolet-A (UV-A) can damage microbes by generating reactive oxygen species (ROS), singlet oxygen, superoxides, hydrogen peroxide and hydroxyl radicals. These species readily react with lipids, proteins, DNA and other constituents of cells, leading to oxidative deterioration and the eventual death of the microbe. However, the oxidative ability of these reactive species also harms the viability of mammalian cells such as fibroblasts and keratinocytes, as they cause both acute and chronic damage, photo-aging, and photo-carcinogenesis. This study describes a UV-A treatment that does not affect the viability or growth of human neonate dermal fibroblasts, as determined by examining the post-irradiation cell density after the addition of polyphenols as antioxidants. The results demonstrate the possible wide applicability of UV-A sterilization. The potency of polyphenols for attenuating UV-A-induced ROS generation in cells was tested using (+)-catechin hydrate, (-)- epigallocatechin gallate hydrate, morin hydrate, quercetin hydrate and resveratrol. The lowest concentration of polyphenols required to reduce ROS by 50% in cells upon exposure to a dose of 15 J cm^-2 was determined and defined as its IC₅₀. Pre-treatment with morin hydrate at its IC₅₀ allowed cells irradiated with 5.0 J cm^-2 UV-A to recover to the level of the specific growth rate of cells incubated without UV-A irradiation. However, the growth rate of cells exposed to 15 J cm^-2 UV-A irradiation was scarcely influenced by co-incubation with morin hydrate; this dose of UV-A also suppressed cell growthcompletely in the absence of morin hydrate, although co-incubation resulted in no decrease in cell viability. This study demonstrates the potential of polyphenols for protecting both the viability of cells and their ability to proliferate from damage caused by UV-A-irradiation.

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Bisphenol A (BPA, 2,2’-Bis (4-hydroxyphenyl) propane) is an artificial pollutant that is easily detected in soil and water environments. BPA decomposition and removal from the environment is relatively difficult due to its stability. This study evaluated the BPA decomposition and removal activities of the microbial community existing in the soil with or without Sphingomonas bisphenolicum AO1, and revealed the toxic effects of BPA towards the microbial community. The microbial community in soil was able to degrade BPA at 1.0 mg·g^-1 soil or lower, although its degradation was slow. On the other hand, BPA at more than 10 mg·g^-1 soil was not only degraded by the microbial community but also decreased its diversity, suggesting that BPA is harmful to many microorganisms. PCR-TTGE analysis and the cloned 16S rRNA gene sequence analysis indicated that Sphingomonadales, Xanthomonadales, Burkholderiales and Pseudomonadales in the microbial community might independently or cooperatively degrade BPA. On the other hand, supplementation with strain AO1 was able to significantly improve the BPA decomposition activity of the microbial community in soil even at 10 mg BPA·g^-1 soil, although BPA at 100 mg·g^-1 soil overwhelmed the BPA decomposition activity of strain AO1. Furthermore, it was also concluded that strain AO1 could not inhabit BPA purified soil after decomposition of BPA by strain AO1 and the soil microbial community, suggesting that the application of strain AO1 could be a low-burden method for the decomposition and removal of BPA from the natural environment.

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The antimicrobial activity of weakly acidified chlorous acid water (WACAW) against Staphylococcus aureus, non-pathogenic Escherichia coli, enterohemorrhagic E. coli (EHEC O157:H7), Candida albicans, and spore-forming Bacillus and Paenibacillus species was evaluated in vitro. The antiviral activity was also examined using feline calicivirus (FCV). Diluted WACAW (>100 ppm) effectively reduced the number of non-spore-forming bacteria (>4 log₁₀ CFU reductions) within 5 min. Treatment with this sanitizer at 400 ppm for 30 min achieved>5 log₁₀ CFU reductions in spore-forming Bacillus and Paenibacillus species while an equivalent concentration of sodium hypochlorite (NaClO) resulted in only a 0.98 and 2.72 log₁₀ CFU reduction, respectively. The effect of this sanitizer against FCV was equivalent to that of NaClO. Immersion in WACAW (400 ppm) achieved >4 and 2.26 log₁₀ CFU reductions in Campylobacter jejuni and EHEC, respectively, on artificially contaminated broiler carcass pieces. Finally, theantimicrobial activity of this sanitizer was shown to be maintained for at least 28 d when in contact with nonwoven fabric (100% cotton). This study showed that pH control of chlorous acid is expected to modify its antimicrobial activity and stability. WACAW is expected to have applications in various settings such as the food processing and healthcare industries.

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To evaluate the removal of airborne microbes by air cleaners, a technique for generating airborne fungal spores in the dry state in a test chamber (dry dispersion) become necessary. The Society of Indoor Environment Japan (SIEJ) published SIEJ Standard Method No. 20110001 (SIEJ standard),in which an aerial ultrasonic oscillator was used as the device for dry dispersion. However, a more versatile apparatus is also necessary from a practical point of view. Therefore, we developed a new device using glass beads for the dispersion. Glass beads and a fungal sheet containing spores of Wallemia sebi were set in a midget impinger, which was connected to a compressor and a compact test chamber (1 m³). Air was blown into the impinger from the compressor. The spores on the fungal sheet were released by impingement of the glass beads when the beads were induced to float by the air blown into the impinger, and the spores were introduced to the chamber by the airflow. This newly developed technique can be used in a compact chamber system and could be applicable as an improved method for generating airborne fungal spores in the dry state in the SIEJ standard.

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Acanthamoeba castellanii, a ubiquitous organism in water environments, is pathogenic toward humans and also is a host for bacteria of the genus Legionella, a causative agent of legionellosis. Oakmoss, a natural fragrance ingredient, and its components are antibacterial agents specifically against the genus Legionella. In the present study, oakmoss and its components were investigated for their amoebicidal activity against A. castellanii ATCC 30234 and the inhibitory effect on the uptake of L. pneumophila JCM 7571 (ATCC 33152) into A. castellanii. The oakmoss and its components 3-hydroxy-5-methylphenyl 2,4-dihydroxy-6-methylbenzoate(5), and 6,8-dihydroxy-3-pentyl-1H-isochromen-1-one (12) exhibited high amoebicidal activity (IC₅₀ values; 10.5 ± 2.3, 16.3 ± 4.0 and 17.5 ± 2.8 μg/mL, respectively) after 48 h of treatment, which were equivalent to that of the reference compound, chlorhexidine gluconate. Pretreatment of L. pneumophila with sub-minimal inhibitory concentration of oakmoss, compound 5, 3-hydroxy-5-methylphenyl 2-hydroxy-4-methoxy-6-methylbenzoate (10) and 8-(2,4-dihydroxy-6-pentylphenoxy)-6-hydroxy-3-pentyl-1H-isochromen-1-one (14) obviously reduced the uptake of L. pneumophila into A.castellanii (p < 0.05).The inhibitory effect of compound 5 on the uptake of L. pneumophila was almost equivalent to that of ampicillin used as a reference. Thus, the oakmoss and its components were considered to be good candidates for disinfectants against not only genus Legionella but also A. castellanii.

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To determine whether the DNA gyrase (gyrB) and 16S ribosomal RNA (16S rRNA) genes can be used as indicators of the biological activities of Legionella pneumophila, the expression levels were estimated. The ratio of mRNA/DNA in gyrB was 0.7 in mid log phase and decreased drastically after the log phase. For 16S rRNA, the ratio was highest in mid log phase (7.0×10³), and the value that was about 10% of that in the log phase was maintained for six days. The rRNA may be vital in the resting or active but nonculturable cells that are not growing but physiologically active. The expression levels of gyrB mRNA and 16S rRNA can be used as indicators of the growth activity and the physiological activity of L. pneumophila, respectively. Therefore, by measurement of these indicators, we can evaluate the activities of Legionella cells in various environments.

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We detected Legionella species in 111 bath water samples and 95 cooling tower water samples by using a combination of conventional plate culture, quantitative polymerase chain reaction (qPCR) and qPCR combined with ethidium monoazide treatment (EMA-qPCR) methods. In the case of bath water samples, Legionella spp. were detected in 30 samples by plate culture, in 85 samples by qPCR, and in 49 samples by EMA-qPCR. Of 81 samples determined to be Legionella-negative by plate culture, 56 and 23 samples were positive by qPCR and EMA-qPCR, respectively. Therefore, EMA treatment decreased the number of Legionella-positive bath water samples detected by qPCR. In contrast, EMA treatment had no effect on cooling tower water samples. We therefore expect that EMA-qPCR is a useful method for the rapid detection of viable Legionella spp. from bath water samples.

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