Screening Of Dominant Bacteria In Bacterial Vaginitis And Preparation Of Inactivated Vaccine

Objectives In this research,pathogenic bacteria causing vaginal infection were isolated and identified,furthmore,the dominant bacteria were screened,and then inactivated vaccine for bacterial vaginitis was prepared based on above studies,so to provide experimental basis for the active immune prevention for bacterial vaginitis.Methods From October 2018 to March 2019,187 cases of vaginal secretions from patients(age of 35.01±9.96)suffered with bacterial vaginitis by clinic diagnosis in the Departments of gynecology clinic of the Affiliated Hospital of North China University of Science and Technology,as well as Tangshan Maternal and Child health hospital were collected as the study objects.Patients with menstrual period,sexual life,vaginal washing,medication,etc.were excluded from this study.Two tubes of vaginal secretions were collected with sterile cotton swabs and sent for examination within 30 minutes.1 The samples were detected by routine microscopy and vaginal quintuple test kit of vaginitis were carried out respectively,and samples were confirmed according the the detection results.In additon to that,specimen including trichomonad,mould and other types of vaginal infection than bacteria were eliminated.2 Bacterial culture and identification: Positive samples that meet the requirements were inoculated into medium and cultured,the suspicious colonies were selected for further Gram staining and pure culture,full-automatic microbial identification instrument was performed to identify the preliminary phenotype according to the biochemical reaction.After that,corresponding primers according to the preliminary identification results were decided for real-time fluorescence quantitative PCR detedtion aimed to further gene identify.3 Histogram of frequency distribution was made according to q RT-PCR results,and the dominant bacteria causing bacterial vaginitis were selected according to the frequency distribution of bacterial vaginitis.4 Preparation of inacted vaccine.The selected seed bacteria for the preparation of the inactivated vaccine were rejuvenation and pure tested.The bacterial plate counting was conducted,and the bacterial growth curve was drawn following that.The inactivation effects of seed bacteria were studied according to the designed concentrations and times of formaldehyde invovled,and the optimal inactivation concentration and time of formaldehyde were confirmed.The inactivated pure seed bacteria were mixed to prepare the inactivated vaccine,and the physicochemical properties and sterility were tested.Results 1 Among the 187 cases of bacterial vaginitis,168 cases met the requirements by jiont detection with routine microscopic examination and five joint examination kits of vaginitis.2 A total of 144 strains were isolated by bacterial culture,45 strains of Coagulase Negative Staphylococcus were identified by biochemical reaction,the isolation rate in bacterial vaginitis was 31.2%;38 strains of Escherichia coli,26.3%;27 strains of Klebsiella pneumoniae,18.8%;14 strains of Enterococcus,9.7%;8 strains of Group B Streptococcus,5.6%;7 strains of Enterobacter cloacae,4.9%;3 strains of Gardnerella,2.0%;2 strains of Corynebacterium,1.5%.PCR gene identification: 44 strains of Coagulase Negative Staphylococcus,was 30.5% in bacterial vaginitis;38 strains of Escherichia coli,26.4%;26 strains of Klebsiella pneumoniae,18.1%;12 strains of Enterococcus,8.3%;Six strains of Group B Streptococcus,4.1%;seven strains of Enterobacter cloacae,4.9%;one strain of Gardnerella,0.7%;two strains of Corynebacterium,1.4%.3 According to the results of PCR,the histograms were drawn.According to their distribution,Coagulase Negative Staphylococcus,Escherichia coli and Klebsiella pneumoniae were selected as the dominant bacteria causing bacterial vaginitis.Therefore,these three bacteria were selected as the candidate seed bacteria for the preparation of inactivated vaccine.4 According to the plate counting result of bacteria,the concentration of CNS reached the peak at 12 hours;the concentration of Escherichia coli reached the peak at 10 hours;the concentration of Klebsiella pneumoniae reached the peak at 18 hours,all of which were seed bacteria in the stable period of bacterial characteristics.At 37 ?,when the formaldehyde concentration was 0.3% and effect for 36 hours to bacteria,the inactivation effect to the bacteria was the best.After the inactivated combined vaccine was inoculated and cultured,there was no bacterial growth,and the preparations were showcased good stability.Conclusions 1 The main pathogens causing bacterial vaginitis are Coagulase Negative Staphylococcus,Escherichia coli and Klebsiella pneumoniae.Therefore,these three bacteria can be used as candidate seed bacteria for the preparation of bacterial vaginitis inactivated vaccine.2 The optimal times for harvest of Coagulase Negative Staphylococcus,Escherichia coli and Klebsiella pneumoniae were 12,10 and 18 hours respectively.The best inactivated effect of formaldehyde to bacteria under 37? were with a concentration of 0.3% and effect for 36 hours.No bacteria growth was observed after the preparations in this study was cultured,and the inactivation preparations was demonstrated good stability.Figure 6;Table 9;Reference 92.