Abstract: Objective To study the inhibitory effect of Huidu Yinhua powder from the Orthodox Manual of External Medicine on methicillin-resistant Staphylococcus aureus ( MRSA) , virulence factor α-hemolysin ( Hla) activity,and biofilm formation, and to explore the optimal ratios of Huidu Yinhua powder and provide experimental support for its use. Methods The inhibitory effects of Huidu Yinhua powder and the herbs in the formula on USA300 were analyzed by the minimum inhibitory concentration (MIC) , minimum bactericidal concentration (MBC) , and disk diffusion assay (K-B method ) . Hemolysis, neutralization, oligomerization, and Western blot assays were used to verify in which form the drug inhibits the activity of virulence factor α-hemolysin ( Hla) . A biofilm assay was performed to evaluate the inhibitory effect of Huidu Yinhua powder on biofilm. Orthogonal experiments were performed to explore the optimal ratio of Huidu Yinhua powder. Results Huidu Yinhua powder inhibited the MRSA strain with a MIC90 of 64 mg / mL and an MBC of 256 mg / mL with antibacterial circle diameter of ( 7. 50 ± 0. 50) mm. Huidu Yinhua powder inhibited Hla activity by inhibiting Hla secretion. The minimum effective concentration (MEC) was 16 mg / mL, and the MEC of biofilm was 8 mg / mL. In HuiduYinhua powder, honeysuckle and astragalus only affected the hemolytic activity of MRSA and biofilm formation without inhibiting bacterial growth. The hemolytic activity and biofilm of MEC were both 32 mg / mL. Glycyrrhiza had a strong bacterial inhibitory capacity with a MIC90 of 8 mg / mL and biofilm MEC of 1 mg / mL without showing inhibitory hemolytic activity at subinhibitory concentrations. The orthogonal experiment showed that, at a ratio of honeysuckle, astragalus, and glycyrrhiza in Huidu Yinhua powder of 1 ∶ 2 ∶ 4, the MIC90 was 16 mg / mL, MEC of hemolytic activity was 8 mg / mL and that of biofilm was 4 mg / mL, both of which were the lowest among the nine groups. Conclusions Huidu Yinhua powder affects the hemolytic activity and biofilm formation of MRSA at subinhibitory concentrations with the optimal ratio of honeysuckle, astragalus, and glycyrrhiza being 1 ∶ 2 ∶ 4.

Abstract:Objective To establish a model of acute and chronic oviduct inflammatory infertility in rats infected with Staphylococcus aureus. Methods A 4 × 10 9 / mL S. aureus suspension was prepared. A total of 108 rats were randomly divided into a normal group, a model group, and a control group, with 36 rats in each. The model group was injected in the fallopian tube with the S. aureus suspension, and the control group was injected with saline in the fallopian tube. The morphology of the fallopian tube was observed on the 7th, 14th, 21st, and 28th days after surgery, and S. aureus culture and pathological examination were also performed. Results After the injection of S. aureus into the fallopian tube, acute oviduct inflammation emerged on postoperative days 7 and 21 to form chronic tubal inflammatory models, and on day 28 to form an oviduct inflammatory model of infertility. Conclusions The injection of 4 ×10 9 / mL S. aureus into rat oviduct successfully established an acute salpingitis and chronic tubal phlogistic sex infertility rat model.

Abstract:Objective Aiming at detecting Staphylococcus aureus、Pseudomonas aeruginosa and Klebsiella pneumoniae in laboratory animals,the paper provides a rapid,sensitive and simple test method.Methods According to Staphylococcus aureus nuc gene,Pseudomonas aeruginosa LasI gene,Klebsiella pneumonia PhoE geneand general 16S rRNA gene,designed specific primers; Through the optimization of multiplex PCR primer concentrations and annealing temperature, the specificity and sensitivity of detection, establishing multiplex PCR system.Application of the PCR system test specimens of artificial infections and experiment animal feces is compared with traditional test method.Results Multiplex PCR amplification of Staphylococcus aureus (153 bp), Pseudomonas aeruginosa (600 bp) with Klebsiella pneumoniae (368 bp) and general (520 bp).The multiplex sensitivity for the purpose of 10pg, specificity of detection was not detected from other pathogens.Application of establishing multiplex PCR system to detect the artificial positive samples, and detect 1 Pseudomonas aeruginosa positive case in 76 fecals. Conclusions This paper established the multiplex PCR method which has the advantages of specific,sensitive,simple and rapid, and provides a reliable way for rapid test in laboratory animals microbiology.

Abstract:Objective To compare the efficiency of bacteria culture and PCR assays for detection of Staphylococcus aureus (S. aureus), Pseudomonas aeruginosa (P. aeruginosa) and Klebsiella pneumoniae (K. pneumoniae) in laboratory rats and mice. Methods Bacteria culture combined with biochemical identification and PCR assay were used to detect 78 SPF rats and 422 SPF mice and the results of the two methods were compared. Results All the 78 rats were negative. Of the 422 mice, the positive rate by culture was 7.11% (30/422), of which, 10 were S. aureus, 22 were P. aeruginosa, and 2 were K. pneumoniae. The positive rate by PCR was 7.58% (32/422), of which, 10 were S. aureus, 25 were P. aeruginosa, and 2 were K. pneumoniae. Conclusions The high sensitivity, rapid procedure and easy to operate of PCR assay makes it valuable for rapid bacteria diagnosis and large-scale screening in laboratory animals.

Abstract:ObjectiveTo establish a loop-mediated isothermal amplification (LAMP) technique for detecting Staphylococcus aureus in laboratory animals. MethodsFour primers were designed and screened specifically to recognize the nuc gene of Staphylococcus aureus. Optimal reaction conditions were screened. Results2 cfu extracted crude DNA and no cross reaction with other commonly seen pathogenic bacteria was found when the conditions were 60℃ for 40 min at least, Mg2+at a concentration of 8.0 mmol/L, dNTP 2.0 mmol/L and betaine 0.8mmol/L. The result of reaction can be directly observed with naked eye by adding a fluorescent SYBR green I dye. ConclusionsThis LAMP technique for detectiong Staphylococcus aureus is sensitive, rapid and simple to perform, and can satisfy the requirements of grassroots lab, mass samples and quick detection.

Abstract:Objective To establish a sensitive and specific molecular biological method for detecting Staphylococcus aureus (S. aureus, SA) in laboratory animals. Methods A DNA fragment of 676 bps of the nuc gene was applified from SA genome DNA by PCR and labeled with biotin as the probe. The purified genomic DNAs of SA1800, SAm0109 and Staphylococcus epidermidis, Streptococcus pyogenes, Escherichia coli were blotted onto the Hybond N nylon membrane, fixed using UV irradiation, hybridized under high stringency conditions, and detected using the Enhanced Chemiluminesence kit (ECL). Results The probe reacted positively with the SA genomic DNAs, but not with that of the non-SA with a sensitivity of I pg. 322 clinical samples taken randomly from specific pathogen free (SPF) mice were submitted to detection by the dot blotting and previously established PCR and culture with a positive detection rate of 3.1% (10/322) and an agreement of 100 %. Conclusion The dot-hybridization assay was a new rapid, sensitive and specific method and could be applied to detect SA in laboratory animals.