Biocontrol Science Vol. 21(2016) No. 2

　Microbial testing performed in support of pharmaceutical and biopharmaceutical production falls into three main categories: detection (qualitative), enumeration (quantitative), and characterization/identification. Traditional microbiological methods are listed in the compendia and discussed by using the conventional growth-based techniques, which are labor intensive and time consuming. In general, such tests require several days of incubation for microbial contamination (bioburden) to be detected, and therefore management seldom is able to take proactive corrective measures. In addition, microbial growth is limited by the growth medium used and incubation conditions, thus impacting testing sensitivity, accuracy, and reproducibility.
　For more than 20 years various technology platforms for rapid microbiological methods (RMM) have been developed, and many have been readily adopted by the food industry and clinical microbiology laboratories. Their use would certainly offer drug companies faster test turnaround times to accommodate the aggressive deadlines for manufacturing processes and product release. Some rapid methods also offer the possibility for real-time microbial analyses, enabling management to respond to microbial contamination events in a more timely fashion, and can provide cost savings and higher efficiencies in quality control testing laboratories. Despite the many proven business and quality benefits and the fact that the FDA's initiative to promote the use of process analytical technology (PAT) includes rapid microbial methods, pharmaceutical and biopharmaceutical industries have been somewhat slow to embrace alternative microbial methodologies for several reasons. The major reason is that the bioburden counts detected by the incubation method and rapid assay are greatly divergent.
　The use of rapid methods is a dynamic field in applied microbiology and one that has gained increased attention nationally and internationally over time. This topic has been extensively addressed at conferences and in published documents around the world. More recently, the use of alternative methods for control of the microbiological quality of pharmaceutical products and materials used in pharmaceutical production has been addressed by the compendia in an attempt to facilitate implementation of these technologies by pharmaceutical companies. The author presents some of the rapid method technologies under evaluation or in use by pharmaceutical microbiologists and the current status of the implementation of alternative microbial methods.

　The number of spoilage incidents in the food industry attributable to a species of the genus Moniliella has recently increased, but the risk of food spoilage has not yet been evaluated. The purpose of this study was to develop a method to rapidly identify high-risk species and to conduct a risk analysis study of Moniliella spp. Acetic acid resistance of M. acetoabutens and ethanol resistance of M. suaveolens were higher than for other Moniliella species. All examined strains of M. acetoabutens developed a high tolerance to acetic acid by being cultured twice in liquid media containing low concentrations of acetic acid. These findings indicate that M. acetoabutens and M. suaveolens are high-risk species for food spoilage and must be discriminated from other fungi. We developed species-specific primers to identify M. acetoabutens and M. suaveolens using the polymerase chain reaction (PCR) to amplify the D1/D2 domain of 28S rDNA. The PCR using the primer sets designed for M. acetoabutens (Mac_F1/R1) and M. suaveolens (Msu_F1/R1) was specific to the target species and did not detect other fungi involved in food spoilage or environmental contamination. This method is expected to be effective for the monitoring of raw materials and components of the food production process.

　Currently, threats caused by pathogens are serious public health problems worldwide. Protective clothing is essential when one is treating infected patients or dealing with unknown pathogens. Therefore, it is necessary to evaluate the performance of protective clothing against pathogens. In Japan, some methods for evaluating the performance of protective clothing have been established in the Japanese Industrial Standards (JIS). However, a test method against virus aerosols has not been established. Because there is a risk of infection from a live virus during the test, it is necessary to devise a safe method for the virus-aerosol-based test. Here, we propose a new method of safely measuring virus aerosols for the performance evaluation of protective clothing materials. To ensure safety, an inactivated virus was used. As a model virus, the influenza virus was selected owing to the proper small diameter of the virus particles. To quantitatively measure the particle-amount of the inactivated influenza virus, we developed an antigen-capture enzyme-linked immunosorbent assay (ELISA) targeting the M1 protein. Furthermore, we evaluated two materials using our method. Significant differences in the protection performance against the virus aerosol were observed between different sample materials, thereby confirming the applicability of our new method for performance evaluation.

　From May 2014 to February 2015, 319 university students (male, n=173; female n=146) of 18 to 24 years of age who carried mobile phones or computer tablets were selected as subjects. Staphylococcus spp. were detected in 101 of 319 samples (31.7%). In the present study, 11 strains of S. aureus were isolated and identified, not all of which were methicillin-resistant Staphylococcus aureus (MRSA). Overall, 14 species were identified, with 11 strains (10.9%) of S. xylosus being isolated at the highest frequency. Following this were eight strains (7.9%) of S. cohnii and seven strains (6.9%) each of S. capitis and S. haemolyticus. Staphylococcus spp. isolation was performed with bacterial samples obtained from the mobile phones of 22 specific subjects (males, n=12; females, n=10). Staphylococcus spp. isolation was performed on days -1, 7 and 30 of the experiment. Staphylococcus spp. were positively detected one or more times in 12 subjects (54.5%). In one subject (8.3%), all three tests were positive. Furthermore, two tests were positive in three (25.0%). In the eight remaining subjects (66.7%) Staphylococcus spp. were detected only once. For the three abovementioned tests, we investigated the pulsed-field gel electrophoresis (PFGE) patterns of the strains derived from the mobile phone and from the fingers of three subjects in whom the same bacterial species were isolated twice. From the cases with similarities between strains derived from the fingers and the mobile phones and cases, with consistency in the strains derived from the mobile phone at different times, commonality was observed in the strains derived from the fingers and mobile phones along with chronological uniformity in the strains derived from the mobile phones. A total of 101 Staphylococcus spp. strains were isolated from mobile phones. According to drug susceptibility tests, 99 strains (98.0%) were found to have some degree of resistance to drugs (excluding one strain each of S. aureus and S. haemolyticus). Among these, the strain that showed the highest level of drug resistance was one strain (1.0%) of Staphylococcus spp., which showed resistance to nine drugs. The strain that showed the second highest level of drug resistance was one strain (1.0%) of S. caprea, which showed resistance to seven drugs. In this manner, the drug-resistant tendencies of Staphylococcus spp. isolated from mobile phones were observed.

　Bacillus thuringiensis (Bt) strains were isolated from jhum-agriculture, jhum-forest, aquatic and fallow soil samples from Mizoram by acetate selection method. Isolates were characterized for biochemical typing, cry gene and protein profiling, growth curve study and toxicity against Culex tritaeniorhynchus. Bt frequency was high in jhum-agriculture land (69.56%) whereas low in jhum-forest soils (31.57%). Bt was found to be abundant in jhum shifting cultivation soil with an index ranging between 0.010 and 0.015. Majority of the isolates from jhum soils produced oval and spherical crystals and showed eleven types of crystal proteins groups. PCR analysis revealed predominance of dipteran-active cry genes (cry4 and cry9). The variations in crystal morphology, cry genes and Cry protein (s) from the isolates of Bt revealed molecular diversity. Higher mortality, lower lethal dose, and lesser time to kill were observed in Bt isolates from jhum soils than aquatic and fallow habitats. Based on the toxicity test, SC1 and HP7 isolates containing cry 4 and cry 9 genes showed higher activity. Growth curve analysis showed significant variations among Bt isolates to reach the sporulating stage. Higher growth index and lower mean generation time were observed in SC1 and HP7 Bt isolates. Bt strains express different endotoxin genes and crystal proteins and their harvesting time also varied from strain to strain. Significant variation was found in Bt isolates from jhum habitats in relation to the cry gene composition, protein profiling and toxicity. Results from this study suggest that novel Bt entomopathogens may complement for regulating mosquito vectors.

　Our previous studies revealed that photo-irradiation of polyphenols could exert bactericidal action via reactive oxygen species (ROS). In the present study, the photo-irradiation-induced bactericidal activity of the aqueous extract from the residue of crushed grapes from winemaking was investigated in relation to ROS formation. Staphylococcus aureus suspended in the extract was irradiated with LED light at 400 nm. This solution killed the bacteria, and a 3-4 log and a >5-log reduction of the viable counts were observed within 10 and 20 min, respectively. LED light irradiation alone also killed the bacteria, but the viable counts were 2-4 log higher than those of the photo-irradiated extract. In contrast, almost no change occurred in the suspension without LED irradiation. When hydroxyl radical scavengers were added to the suspension, the bactericidal effect of the photo-irradiated extract was attenuated. Furthermore, electron spin resonance analysis demonstrated that hydroxyl radicals were generated by the photo-irradiation of the extract. The present study suggests that polyphenolic compounds in the extract exert bactericidal activity via hydroxyl radical formation upon photo-irradiation.

　The antifungal activities of diglycerin monoester of fatty acids (DGCs), which have been employed as food emulsifiers, were examined against three yeasts, Saccharomyces cerevisiae, Candida albicans and Candida utilis and were compared with those of sucrose monoester of palmitic acid (SC₁₆) as another type of emulsifier and sodium benzoate (SB) as a weak acid food preservative. When the minimum growth inhibitory concentrations (MICs) of diglycerin monolaurate (DGC₁₂) against these yeasts were determined 2 d after incubation in YM broth at pH5.0, they were relatively low, being 0.01% (w/v), for both S. cerevisiae and C. utilis, whereas was high, being 4.0% (w/v), for C. albicans. On the contrary, the MICs of sucrose monopalmitate (SC₁₆) were high, being 3.0 and 4.0% (w/v), for the former two yeasts, respectively, but 0.6% (w/v) for the last yeast. In contrast to these emulsifiers, the MICs of sodium benzoate (SB) were similar independently upon the yeast strain, being in order 0.4, 0.3 and 0.5% (w/v), for the above yeasts, respectively. The anti-yeast activities of DGC₁₂ and SC₁₆ were gradually increased with a decrease in pH, in a manner similar to that of SB, except for the action of SC₁₆ on C. albicans, for which the activity was more effective at pHs 5.0 and 6.0 than at pHs 4.0 and 7.0. Among DGCs tested having different fatty acid moieties in the molecule, lauroyl ester (DGC₁₂) was more effective than myristoyl and palmitoyl esters against S. cerevisiae and C. utilis. The inhibitory effect of DGC₁₂ on the yeast growth depended upon both the cell density and the strength of aeration during the treatment. Further, DGC₁₂ was found to kill S. cerevisiae and C. utilis cells at a rather low concentration of 0.005% (w/v) in 50mM acetate buffer at pH5.0, although, against C. albicans cells, only slight fungicidal activity was demonstrated at a high concentration of 0.5% (w/v). The results obtained support the effectiveness of practical application of DGC₁₂ to acidic foods for the control of growth and survival of general yeasts such as S. cerevisiae and C. utilis.

　Sarcocystis fayeri (S. fayeri) is a newly identified causative agent of foodborne disease that is associated with the consumption of raw horse meat. The testing methods prescribed by the Ministry of Health, Labour and Welfare of Japan are time consuming and require the use of expensive equipment and a high level of technical expertise. Accordingly, these methods are not suitable for use in the routine sanitary control setting to prevent outbreaks of foodborne disease. In order to solve these problems, we have developed a new, rapid and simple testing method using LAMP, which takes only 1 hour to perform and which does not involve the use of any expensive equipment or expert techniques. For the validation of this method, an inter-laboratory study was performed among 5 institutes using 10 samples infected with various concentrations of S. fayeri. The results of the inter-laboratory study demonstrated that our LAMP method could detect S. fayeri at concentrations greater than 10⁴ copies/g. Thus, this new method could be useful in screening for S. fayeri as a routine sanitary control procedure.

　Kudoa septempunctata is the causative agent of a food-borne disease associated with the ingestion of raw olive flounder. As the current qRT-PCR method for its detection is time-consuming, a rapid and simple method is required. Recently, a new real-time loop-mediated isothermal amplification (LAMP) method and an immunochromatography method, whose sensitivities are intended to be compatible with that designated in the official analytical method (10⁵ spores/g olive flounder), have been developed. To validate these new methods, we performed an inter-laboratory study across seven laboratories. Both methods could not detect less than 10⁴ spores/g; however, these methods were able to detect more than 10⁵ spores/g in olive flounder samples. These results demonstrated that the sensitivities of these methods were compatible with the designated level in the official analytical method. We concluded that these new methods were acceptable as the screening methods for K. septempunctata.