Study On Pathogenic Factors And Drug Sensitivity Of Gardnerella Vaginalis Associated With Bacterial Vaginosis

Objective:To investigate the formation ability of Gardnerella vaginalis?GV?biofilm from gynecological secretions,antibiotic resistance,sialidase A gene carrier and clinical data of patients.To study the correlation between Gardnerella vaginalis and bacterial vaginosis?BV?.Assist clinicians in understanding the possible pathogenesis of bacterial vaginosis,guide clinical rational use of antibiotics,and ensure women's health.Methods:1.According to the inclusion and exclusion criteria established in this experiment,25 isolates of Gardnerella vaginalis were collected from gynecologic outpatients and inpatients in the First Affiliated Hospital of Dalian Medical University from January2018 to December 2018.As experimental group,25 isolates of Gardnerella vaginalis were cultured in a 5%CO2 incubator at 37?for 48-72 hours under a Gram staining microscope.Interstitial spectrum system Clin-ToF-II was identified and further confirmed by API Strep identification strip.ATCC 14018 and ATCC 14019 purchased from the US Preservation Center were used as positive control group.2.The biofilm formation ability of 25 clinical isolates of GV was detected by semi-quantitative microplate assay.Using sterile cotton swab dipped in blood plate,GV pure bacteria were mixed into BHI culture medium with 0.5 McClell turbidity,incubated in incubator at 37?and 5%CO2 for 24 hours,diluted into 96-well culture plate?200 ul/hole?with pre-arranged BHI culture medium at 1:100,incubated at 37?for 44-48 hours,abandoned bacteria solution,PBS cleaned three times,fixed with methanol for 15 minutes,dried at room temperature,dyed with 1%crystal violet for 8minutes,washed into colorless medium.The strains were dried at room temperature,decolorized with anhydrous ethanol for 5 minutes and transferred to a new 96-well plate.The OD values of each strain were measured at 570 nm wavelength by enzyme labeling instrument.The average value of OD570 measured by each strain plus 3 times standard deviation was compared with ATCC14018 to evaluate the biofilm forming ability of clinical Gardnerella strains.3.Genomic DNA of Gardnerella vaginalis was extracted and sialidase A gene of Gardnerella vaginalis was detected by qualitative and quantitative PCR.The reaction products of the qualitative PCR were electrophoretic on 2%agarose gel.The temperature,melting curve and amplification curve of quantitative PCR were analyzed and judged.The standard strain ATCC14019 was used as positive control.4.Agar dilution method was used to test the susceptibility of 25 clinical isolates of GV.Quantitative sterilized distilled water or buffer was used as the blank solution of the dilution solution in test tube.The dilution solution was prepared by 1:2,1:4 and 1:8continuous dilution method.Metronidazole was dissolved with DMSO,rifampicin was dissolved with absolute ethanol,clindamycin was dissolved in sterile water,and all drugs were diluted with sterile water.The range of the tested drugs was:rifabutin:0.0019-32 g/ml,metronidazole:0.0019-32 g/ml,clindamycin:0.0019-32 g/ml.Drug plate and drug-free control plate were incubated in anaerobic environment at 37?for42-48 hours.5.A total of 105 specimens of vaginal secretions from female patients with BV smear diagnosis and general bacterial culture were collected from a gynecological outpatient clinic and hospitalization in the First Affiliated Hospital of Dalian Medical University from January 2018 to December 2018.The age ranged from 19 to 80 years,with an average age of?33.65±8.21?years.A single colony dipped in blood plate was identified by mass spectrometry.All specimens were divided into GV group and Lactobacillus group to investigate the correlation between the distribution and age,PH value,vaginal cleanliness and BV.The relationship between the two groups of samples and age and PH value was analyzed by t test of two independent samples,P<0.05.The difference of vaginal cleanliness between GV group and Lactobacillus group was analyzed by 2 test of four-grid table,P<0.05.It was considered that there was statistical difference in vaginal cleanliness classification between GV group and Lactobacillus group.The prevalence of BV between GV group and Lactobacillus group was analyzed by 2 test of four-grid table.?Negative or positive?,P<0.05.There was statistical difference between the two groups.All clinical isolates of GV were divided into two groups:negative and positive biofilm.The differences of age,PH value,vaginal cleanliness and BV between the two groups were compared.The statistical methods used are the same as those mentioned above.Result:1.Of 25 clinical isolates of GV,11 were positive for biofilm formation,with a positive rate of 44%.2.Twenty-five clinical isolates of GV were detected by qualitative and quantitative PCR.Twenty-four of them carried sialidase A gene.The results were consistent.The positive rate of sialidase A gene was 96%in all GV strains.3.The results showed that the sensitivity of GV to rifampicin ranged from MIC_S=0.0075-0.25ug/ml,MIC₅₀=0.031ug/ml and MIC₉₀=0.25ug/ml.The sensitivity range of GV to clindamycin was MIC_S=0.031-32ug/ml,MIC₅₀=0.25ug/ml and MIC₉₀=32ug/ml.The sensitivity range of GV to metronidazole was MIC_S?16ug/ml,MIC₅₀=16ug/ml,MIC₉₀=16ug/ml.4.The age of GV group was?32.12±8.54?and that of Lactobacillus group was?34.36±9.67?.The t-test analysis of two independent samples showed that P?0.05,there was no statistical difference between the two groups.The PH value of GV group was?5.35±0.10?,and that of Lactobacillus group was?5.20±0.32?,P<0.05.There was statistical difference between the two groups.The vaginal PH value of Lactobacillus group was smaller than that of GV group.The difference of vaginal cleanliness between GV group and Lactobacillus group was analyzed by?² test of 4-grid table?P<0.05?,There was statistical difference in vaginal cleanliness classification between GV group and Lactobacillus group.The majority of vaginal cleanliness in GV group was grade III,and the vaginal cleanliness in Lactobacillus group ranged from grade II to grade III.The prevalence of BV between GV group and Lactobacillus group was analyzed by 2 test with 4 tables,?²=5.123,P<0.05.There was significant difference between the two groups.Comparing the age,vaginal PH value,vaginal cleanliness and BV distribution of GV biofilm negative and positive groups,the P value of each group was greater than 0.05,and no statistical difference was found.Pearson correlation analysis was used to evaluate the relationship between cultured GV and BV in gynecological secretions.There is a linear relationship between the two variables.According to Shapiro-Wilk test,the two variables conform to normal distribution?P?0.05?,and there is no abnormal value.There was a highly positive correlation between GV cultured from gynecological secretions and patients with BV,r=0.969,P<0.001.Conclusion1.The ability of biofilm formation of GV clinical isolates in Dalian area is similar to that of GV clinical isolates in other areas.2.The carrying rate of sialidase A gene of GV clinical isolates in Dalian area was higher than other reports,up to 96%.3.The GV isolates prevalent in Dalian area are highly sensitive to rifabutin,resistant to clindamycin to varying degrees,and highly resistant to metronidazole.4.There was no difference in age between Gardnerella vaginalis group and Lactobacillus group.There were differences in PH value and cleanliness,and there were statistical differences in BV distribution between the two groups.There is a highly positive correlation between Gardnerella vaginalis and patients with BV.The presence or absence of Gardnerella vaginalis biofilm was not correlated with the above indicators.