2.1 Strains and drugs
Staphylococcus aureus [CMCC (B) 26003], Pseudomonas aeruginosa [CMCC (B) 10104], Escherichia coli [CMCC (B) 44102], Candida albicans [CMCC (F) 98001], and Aspergillus niger [CMCC(F) 98003] were provided by the China Medical Bacterial Collection Management Center (CMCC). The strains for the preparation of bacterial suspensions were all third generation.
Betastatin besylate nasal spray (Shanghai Modern Pharmaceutical Preparation Engineering Research Center Co., Ltd. China; specification, 100 μL per spray) containing 2.5 mg of betastatin besylate (7.5 mL per bottle) was used in this study. The nasal spray batch numbers 2015071401, 2015071402, and 2015071403 contained 0.02%, 0.0125%, and 0.005% benzalkonium chloride, respectively.
Trypticase soy agar (TSA) medium (batch No. 3303005), Sabouraud dextrose agar (SDA) medium (batch No. 20160901), trypticase soy meal broth (TSB) (batch No. 3302035), and Sabouraud dextrose broth (SDB) (batch No. 3302126) were purchased from Guangdong Huan Kai Microbiology Technology Co., Ltd., China. The applicability of the media was in compliance with the Chinese Pharmacopoeia 2015 edition. The media were prepared according to the manufacturer’s instructions.
2.3 Microbial Suspension Preparation
The microbial suspensions were prepared to appropriate concentrations according to the method described in the Chinese Pharmacopoeia 2015 edition Four General Rules 1121 "Antibacterial efficacy test method."
2.4 Method Suitability Study
In order to meet the requirements of Chinese Pharmacopoeia 2015 edition of the four general rules 1105 "microbial limit test for non-sterile products: microbiological counting method", the recovery rate of betastatin besylate nasal spray with high antibacterial content (batch No. 2015071401, containing benzalkonium chloride 0.02%) should not be less than 70%.
2.5 Antimicrobial Efficacy Assay
2.5.1 Microbial Inoculation
Sterile bottles (5 bottles for each batch) containing betastatin besylate nasal spray with 0.02%, 0.0125%, and 0.005% benzalkonium chloride, respectively, were inoculated with 50 µL of S. aureus, P. aeruginosa, E. coli, C. albicans, and A. niger at a concentration of about 105–106 colony forming units per mL (CFU/mL). Then, the suspension was mixed well and stored at 23°C in dark.
2.5.2 Quantification of Viable Microbial Cells
The viable microbial cells were quantified according to the product type and Chinese Pharmacopoeia 2015 edition General Rules 1121 "Antibacterial Effectiveness Check Method" Table 2-2. In brief, 1 mL of the test sample was collected on day 2, 7, 14, and 28, respectively, inoculated onto a TSA plate (for bacteria) and SDA plate (for fungi), and the number of viable cells was determined by counting and verified by the method suitability test. Finally, the survival numbers of each test bacterium at each interval were calculated and converted into lg value.
2.5.3 Results Assessment
The criteria for assessing the antimicrobial effectiveness of the nasal preparation are shown in Table 1. The "reduced lg value" in the table refers to the difference between the lg value of the number of microbial cells measured at each interval and the lg value of the number of microbial cells inoculated in 1 mL of the test sample. Standard “A” indicates the antimicrobial efficacy standard that should be achieved. In special cases, if the antimicrobial agent increased the risk of adverse reactions, standard “B” antimicrobial efficacy was used in the test.