| ObjectivesChronic endometritis(CE)is a relatively common pelvic inflammatory disease clinically,a local inflammation with unknown pathogenic mechanism,which is also an important cause of repeated implantation failure and recurrent abortion in clinical patients.Currently,there are a variety of microbial tests for chronic endometritis,including metagenomic next-generation sequencing(m NGS),16 S ribosomal RNA(16S ribosomal RNA)sequencing,PCR detection,microbial culture and other methods.In this study,metagenomic sequencing,16 S rRNA sequencing and traditional microbial culture methods were used to detect the endometrial microflora of chronic endometritis and the control group,search for possible pathogenic bacteria,and preliminarily explore the possible pathogenic factors of pathogenic bacteria,providing a new theoretical basis for accurate diagnosis and treatment of chronic endometritis and improvement of clinical pregnancy rate.Methods1.Thirty patients diagnosed with chronic endometritis in the outpatient department of Guangdong Women’s and Children’s Hospital from January 2022 to August 2022 were selected as the experimental group(CE group),and 30 patients with non-chronic endometritis during the same period were selected as the control group(NCE group).Endometrium of patients in CE group and NCE group(3 tissues from 1 patient)was collected.Metagenomic sequencing and 16 S rRNA sequencing were performed on 1 tissue,microbial culture was performed on 1 tissue,and pathological and immunological examinations were performed on the other tissue.2.Standard strain PAO1 and 8 strains of Pseudomonas aeruginosa isolated from patients with chronic endometritis were selected(ML group: ML1004-ML1011),experiments of Minimal inhibitory concentration(MIC),rhamnoolide,exercise capacity(Swarming,Twitching)and biofilm were performed.3.Two strains of Pseudomonas aeruginosa(ML1004 and ML1006)isolated from the CE group and with strong biofilm formation ability were selected for the detection of type Ⅲ secretory system related proteins.Results1.Explore the intrauterine microecological environment of chronic endometritis based on two sequencing technologies1.1 Metagenomic sequencing resultsAt the phylum classification level,Proteobacteria,actinobacteria and Firmicutes were the dominant phyla in NCE group and CE group,and there was no significant difference between groups(P > 0.05).At the genus classification level,Klebsiella,Mycobacterium,Bacillus and Lactobacillus were the dominant bacteria in NCE group and CE group,and there was no significant difference among groups(P > 0.05).1.2 16 s rRNA sequencing results1.2.1 At the phylum classification level,Firmicutes,Proteobacteria and cyanobacteria were the common dominant phyla in CE and NCE groups,and there was no significant difference between groups(P > 0.05).At the genus classification level,the relative abundance of Acinetobacter,Bacillus and Pseudomonas in CE group was higher than that in NCE group,but there was no significant difference among groups(P > 0.05).The unique bacteria of CE group were Novosphingobium,Streptococcaceae and Sphingobium.1.2.2 In this study,there were significant differences in Alpha diversity analysis and Beta diversity analysis between CE and NCE groups(P < 0.05).2.Isolation,culture and identification of intrauterine microorganisms in chronic endometritis2.1 In CE group,14 cases were positive,accounting for 47%,including 1 case of Klebsiella,2 cases of Escherichia coli,8 cases of Pseudomonas aeruginosa,2 cases of Staphylococcus aureus and 1 case of micrococcus.In the NCE group,there were 3positive cases,accounting for 10%,including 1 case of Escherichia coli,Staphylococcus aureus and Bacillus,and the difference between the groups was statistically significant(x2=9.932,P=0.002).The number of detected strains was successively named ML1001-ML1017.2.2 Microbial culture results of CE patients: Klebsiella 1 case,accounting for 7%;Escherichia coli(2 cases,14%);There were 8 cases of Pseudomonas aeruginosa(58%)and 2 cases of Staphylococcus aureus(14%).Bacillus was found in 1 case,accounting for 7%,and Pseudomonas aeruginosa was detected in the largest number of cases(named ML group,ML1004-ML1011).3.Detection of resistance and pathogenicity of Pseudomonas aeruginosa3.1 Determination of minimum inhibitory concentration(MIC)(1)The minimum inhibitory concentration of ciprofloxacin against 9 strains included in the experiment was 2ug/ml;(2)The minimum inhibitory concentration of ceftriaxone sodium on ML1004-ML1006,ML1008 and ML1009 was more than twice that of PAO1;(3)The minimum inhibitory concentration of meropenem against ML1004 was 8 times that of PAO1;(4)The minimum inhibitory concentration of levofloxacin against ML1004-ML1011 was lower than that of standard strain PAO1.(5)The minimum inhibitory concentration of ceftazidime against ML1004 was 8times that of PAO1;(6)The minimum inhibitory concentration of azithromycin against ML1004,ML1008 and ML1010 was twice that of PAO13.2 Rhamnoolipid secretionThe results of absorbance measurement showed that the rhamnoolipid secretion of ML group was significantly higher than that of PAO1(P < 0.05),and the rhamnoolipid secretion of ML1008,ML1009 and ML1011 was significantly higher than that of standard strain PAO1(P < 0.05).There was no significant difference between the secretion of rhamnoolipid and PAO1 in other strains(P > 0.05).3.3 Detection of motor abilityNo matter at 12 h,24h,36 h or 48 h,the Swarming ability of ML group was weakened than that of PAO1(P < 0.05),and the Swarming ability of ML1004 to ML1007 strains was significantly weakened compared with that of standard strains PAO1(P < 0.05).There was no significant difference between ML1009 to ML1011 and PAO1(P > 0.05).No matter 24 h or 48 h,the Twitching ability of ML group was stronger than that of PAO1.When the culture time was 24 h,the Twitching ability of ML1004-ML1011 was significantly increased compared with that of PAO1(P < 0.05).When the culture time was 48 h,the Twitching ability of ML1005,ML1008,ML1010 and ML1011 was significantly increased compared with PAO1(P < 0.05),while the Twitching ability of ML1004,ML1007,ML1006 and ML1009 was not significantly different compared with PAO1(P > 0.05).3.4 Biofilm productionAfter 24 h of culture,the amount of biofilm formation in ML group was higher than that in PAO1(P < 0.05),and the amount of biofilm formation in ML1006(0.79±0.37)was higher than that in standard strain PAO1(P < 0.05),but there was no significant difference between other clinical strains and PAO1(P > 0.05).At 48 h,there was no significant difference in the amount of biofilm formation between ML group and PAO1(P > 0.05),and only clinical strains ML1004 and ML1006(0.62±0.14 and 0.42±0.10,respectively)had higher biofilm formation than standard strains PAO1(P < 0.05).There was no significant difference in the amount of biofilm formation between other clinical strains and PAO1(P > 0.05).4.Protein expression levels of type Ⅲ secretory system in two strains of Pseudomonas aeruginosa with strong biofilm-forming abilityThe gene expression levels of PCRV,exs A and exo S of strain ML1004 were5.67±2.78,1.62±0.22,0.59±0.11 and 3.35±0.43,respectively,which were higher than those of PAO1(P < 0.05).There was no significant difference in exo T expression level between ML1004 strain(0.59±0.11)and PAO1 strain(P > 0.05).The expression levels of PCRV,exo A,exo T and exo S in ML1006 were 2.51±0.88,1.10±0.36,0.32±0.27 and 1.44±0.41,which showed no significant difference compared with PAO1(P > 0.05).Conclusions1.The microflora in the uterine cavity were mainly Firmicutes and Proteobacteria.2.Chronic endometritis is related to intrauterine microbial flora disorder,in which Acinetobacter,Bacillus and pseudomonas are unique microflora of chronic endometritis.3.Microbial infection may be a pathogenic factor leading to chronic endometritis,and Pseudomonas aeruginosa may be an opportunistic pathogen leading to chronic endometritis.4.Clinically,the use of ceftriaxone sodium and azithromycin in the treatment of chronic endometritis caused by Pseudomonas aeruginosa may have poor bacteriostatic effect,and antibiotics should be selected precisely according to the pathogenic characteristics of microbial flora.5.The 8 strains isolated from patients with clinical chronic endometritis showed strong virulence(rhamnoolipids,motility)and biofilm,which may be the cause of chronic endometritis caused by Pseudomonas aeruginosa.6.The type Ⅲ secretion system may be closely related to the pathogenesis of chronic endometritis caused by Pseudomonas aeruginosa infection. |
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