Characterization Of Microflora In Granulomatous Lobular Mastitis By 16S RDNA High-throughput Sequencing

Objective: To improve the method of bacterial detection in granulomatous lobular mastitis lesions,evaluate and compare the differences in the distribution of bacteria in granulomas and paralesional tissues in granulomatous lobular mastitis,and further study the relationship between granulomatous lobular mastitis and bacterial infection.Methods: The clinical data of 46 patients with pathologically confirmed granulomatous lobular mastitis who were initially treated in Hunan Provincial People's Hospital from June 2018 to April 2020 were collected.Fourteen patients who did not receive anti-infective treatment 2 weeks before operation were selected as the study subjects.One granulomatous lesion,one paralesional tissue(tissue between normal tissue and lesion,and at least 1 ~ 2 cm away from the lesion)and one pus sample were collected from each patient under aseptic conditions in the operating room.A total of 36 samples(including 9 pus samples,14 granuloma samples and 13 paralesional tissue samples)were obtained.The collected 9 pus samples were timely inoculated in the culture medium for microbial culture and identification.Another 14 granuloma tissue samples collected were set as RY group and 13 paralesional tissue samples were set as ZF group.DNA was extracted from the two groups of samples.High-throughput sequencing technology was used to sequence bacteria in RY and ZF group.QIIME and other software were used to compare the sequencing results with 16 S database for species annotation.Species classification and abundance analysis,Alpha diversity analysis and Beta diversity analysis were performed according to species abundance information.The correlation analysis was further performed for the types,composition,distribution and characteristics of flora in the lesions and perilesional tissues of patients with granulomatous lobular mastitis.Results:1.Only one of 9 pus samples was positive after culture,and no anaerobic growth was observed,with a positive rate of 11.1%(1/9),which was identified as Xanthomonas after isolation and purification.2.The bacterial community of a total of 27 samples from granulomas and paralesional tissues was sequenced and the data of low-quality bases were screened and filtered.The unqualified sequences were removed,resulting in a total of 1935,765 valid sequences with an average fragment length of 409 bp,which was consistent with the sequence length of the V3-V4 region of the 16 S rRNA gene.By alignment with the database Silva132,species annotation was performed,and statistics at different taxonomic levels revealed that there were a total of 1937 OTUs,of which 1899(98.04%)OTUs could be annotated to the database.All samples could be annotated to the library.The proportion of their annotation to the kingdom level was 98.04%,the proportion to the phylum level was 78.06%,the proportion to the class level was 75.22%,the proportion to the order level was 69.95%,the proportion to the family level was 64.02%,the proportion to the genus level was 44.91%,and the proportion to the species level was 15.07%.3.At the phylum level,we found that the dominant species included mainly Proteobacteria,Firmicutes,Bacteroidetes.At the genus level,the dominant species were Stenotrophomonas,Photobacterium,Brevundimonas.Corynebacterium was detected in low relative abundance(< 0.001)in three samples from two patients,of which two samples from the same patient were finally identified as Corynebacterium kroppenstedtii.4.The rarefaction curve and species cumulative box diagram showed that the sampling results were sufficient and data analysis could be performed.According to the species annotation,the complexity of sample bacteria in the two groups was further analyzed.The ? diversity indices such as Chao1,ACE,Shannon,Simpson and Good's coverage of the two groups of samples were calculated.The results showed that there was no significant difference in the total number of species and bacterial diversity measured by the two groups of samples(P > 0.05).5.We also performed ? diversity analysis.Pco A was used to analyze the flora structure of the samples.The results showed that the two groups of samples were relatively clustered.Similarity analysis(Anosim)showed statistic R value =-0.04006 < 0,indicating that the difference within the group was greater than the difference(P = 0.765 > 0.05),and there was no significant difference in the bacterial structure in each sample.Conclusion:1.It was preliminarily determined that bacterial infections were associated with the occurrence of granulomatous lobular mastitis,mainly mixed infections.Possible pathogens included Stenotrophomonas,Photobacterium,and Brevundimonas.2.The distribution of flora in necrotic granulation tissue and paralesional tissue was approximately the same in patients with granulomatous lobular mastitis.3.Among the etiological detection methods for granulomatous lobular mastitis,although 16 S rDNA-based high-throughput sequencing technology has the advantages of high throughput,high sensitivity,and specificity,its clinical application value remains to be further explored.