Bacteriological Study On Granulomatous Lobular Mastitis In South China

Granulomatous lobular mastitis(GLM)is a chronic inflammation of the breast that occurs in the mammary gland,which is characterized by granuloma formation.Clinically,GLM may be present as a firm,red,tender lesion that suggests the presence of an abscess,or as a hard mass closely resembling a malignancy,especially inflammatory breast cancer.Therefore,differential diagnosis and symptomatic treatment of GLM are challenges in the prevention and treatment of this breast disease.At present,the pathogenesis of GLM and the lack of understanding of the development process of the disease,and the unknown of etiology and pathogenesis of the disease,lead to the blindness in clinical treatment of GLM.It is not uncommon that misdiagnosis of medical records were in the hospital,which impacts the patient’s physical and mental health.In order to discriminate the potential pathogens due to GLM,we studied the pus and tissue specimens of GLM in South China.Moreover,the similarities and differences between the potential bacteria and GLM in different areas would be analyzed by other study.The potential pathogenic bacteria involved GLM in South China were identified by bacterial culture,Gram stain and bacterial 16 S rDNA sequencing.The phylogenetic tree was constructed by NJ method to determine the classification status of the bacteria.This study would provide new theories on the etiology and pathogenesis of GLM.Methods:In this study,32 patients of granulomatous lobular mastitis in South China who were outpatient treatment and hospitalization in Sun Yat-sen Memorial Hospital of Zhongshan University from June 2014 to June 2016 were checked.14 pus specimens and 18 tissue specimens constituted the 32 samples.All specimens were obtained in the operating room under the conditions of sterile conditions.The collection of 14 cases of pus specimens were inoculated in blood agar plate,incubated at 37℃ constant temperature,in air supplemented with 5% CO2 for more than 48 hours.The Gram stain was carried out when the culture was positive.The bacterial genomic DNA was extracted from the positive bacteria culture and the 18 tissue samples.The bacterial 16 S rDNA sequences were amplified by PCR using the bacteria specific primers.The amplicon were sequenced after cloning.The sequencing results were compared with NCBI Gene database.To determine the classification of the bacteria status,the phylogenetic analysis was carried out by MEGA software.Results:In the fourteen pus specimens,only 4 specimens were found to grow colonies on the medium after two days later.The detection rate of bacteria in the pus was 28.57%.The bacterial clonies were small and smooth,and then the bacteria were stained with Gram stain.Under the oil microscope,the bacteria were observed in a rod shape,and purple.It can be initially identified as Gram-positive bacilli.The sequencing of the bacterial 16 S rDNA sequences were compared with the NCBI gene database,of which two cases have 99.9% similarity to the C.kroppenstedtii and the other two cases also owned 100% similarity to M.abscessus and P.avidum respectively.The phylogenetic analysis was carried out by using Neighbor-Joining(NJ)and phylogenetic tree was constructed to determine the classification of these four bacteria.Eighteen cases of tissue specimens were stained with HE and found that there were inflammatory ductus arteriosus,secretions,lymphocytes and plasma cell infiltration around GLM.The total DNA was extracted,PCR and clone.Only two samples were able to amplify the 16 S rDNA sequences successfully.One of the sequences is related to the T.aquatica available in GenBank and the similarity reaches 99.9%.The other sequences have the similarity of 99.9% to the C.amycolatum.The phylogenetic tree with these two sequences was constructed by NJ method with DNAClub and MEGA software.Conclusion:Based on the specimens of our study,we concluded that there was a close relationship between GLM and C.kroppenstedtii,M.abscessus,P.avidum,C.amycolatum and T.aquatic in South China.The bacteria can be identified by microbiology analysis including pure culture and independent of the culture in South China.The total bacterial detection rate reached 18.75%.The non-specific infection of these bacteria may cause GLM to occur.