Isolation,Identification And Its Safety Evaluation Of The Lactobacillus Gasseri Strain HMV18 Which Inhibits The Growth Of Pathogenic Bacteria

Objectives:To improve the Oxford Cup antibacterial test method.To isolate and identify the Lactobacillus strains which had antibacterial activity,we collected samples of vaginal secretions from women,then.Combined with the traditional phenotypic test and genomics method,we evaluated the safety of the strain with good antibacterial activity,in order to provide new ideas and targets for clinical diagnosis and treatment of infectious diseases.Methods:1. Research objects and samples collection100 female vaginal swabs were collected in Shijiazhuang Great Wall Integrated Traditional Chinese and Western Medicine Hospital in 2018.We excluded the patients who had taken antibiotics in the past month or had diseases such as cancer,hepatitis B,hepatitis C,syphilis,AIDS,or diabetes.The collected swabs were inoculated into MRS liquid culture medium to increase the concentration of lactobacilli;then the cultured samples were separated into supernatants and precipitates,and stored frozen below-20°C.2.Oxford Cup antibacterial testThis study is the improvement of the Oxford Cup antibacterial test method.We improved the results by comparing a various concentration of sensitive bacteria,the concentration of agarose supplemented in the solid culture medium and the volume poured into the petri dish,when and where to put the Oxford cups,the volume of the antibacterial active substance to be added in the cup/well,and the incubation time.The method strives for more accurate experimental results and better repeatability,which lays a good foundation for subsequent experiments.3.Isolation,identification,safety analysis of Lactobacillus and active substances detectionAfter the Lactobacillus supernatants with good antibacterial activity was selected,we inoculated the bacteria on MRS agar by streaking for separation.After incubated anaerobically at 37°C for 72 hours,and picked white smooth suspicious colonies,transferred to MRS liquid medium,and incubated at 37°C for anaerobic culture.The Suspicious colony was smeared,dried,fixed,and applied to Gram Staining,and then observed under an oil microscope.Then the new isolate was analysed by Catalase test,Lactobacillus biochemical identification and the 16S r DNA sequencing.The whole genome sequencing was also performed for further characterization.Hemolysis test was performed to evaluate its pathogenesis.The crude extracts of the cell-free supernatants was sent for protein sequencing.The bacteriocin containing supernatants was applied for the Oxford cup test to evaluate its antibacterial activities.Both of the experiments suggested that it was bacteriocin which was active to inhibit the pathogens and it was secreted into the supernatants.Results:1. Optimize the method of the Oxford Cup antibacterial testThe details were as follows:35ml culture medium with 1.5%agarose was poured into each petri dish.The sensitive pathogenic bacteria were diluted to10~5CFU/ml and spread on the surface of the plate.After all the liquid were absorbed,the Oxford Cups were placed on the surface of each plate.The Oxford Cups should distribute evenly and be placed no more than 5 cups on each plate.There were 230μl tested liquid at maximal to be added in to the cups.Then the plates were placed at 4°C for 24h for the absorbance of the tested liquid,and then transferred into the incubator at 37°C for 24h for the bacteria growth.2. Select the clinical sample which showed pathogen-inhibition activities by the Oxford cup testMRS enriched cultures of 100 female vaginal swabs,was separated by centrifugation.The supernatants were applied for the Oxford cup test against the three pathogens:E.coli、K.oxytaca and S.aureus.The results showed that19 samples showed antibacterial activities,of which the No.47 showed antibacterial activities against all the three pathogens.3. Isolation and identification of Lactobacillus which showed good antibacterial activitiesThe 47th sample was streaked on a plate for separation and identification the most effective isolate.The bacterial cells of specimen No.47 in the second part were streaked and separated on MRS agar,incubated at 37°C for 72hours,and picked white smooth suspicious colonies,transferred to MRS liquid medium,and incubated at 37°C for anaerobic culture.The Suspicious colony was smeared,dried,fixed,and applied to Gram Staining,and then observed under an oil microscope.The results showed that the new isolate was Catalase negative.The results of monosaccaride fermentation showed that it could degraded cellobiose,lactose,inulin,raffinose,sucrose,salicin,and maltose to produce acids.Lactobacillus biochemical results and the 16S r DNA sequencing results showed that the suspicious colony was a new isolate of Lactobacillus gasseri,which was named HMV18.It is related to 29 strains of Lactobacillus gasseri already existing in Genebank.4.Safety evaluation of L gasseri HMV18 and the detection of its active componentsL.gasseri HMV18 was sequenced by whole genome and showed that its genome includes a 1.95Mb chromosome,a 4.6kb small plasmid,a 32kb large plasmid and a 3.9kb phage.The gene prediction results showed that the bacteria had no genes encoding antibiotic resistance on the plasmid or phage,indicating that the bacteria did not have the threat of horizontal transfer of the antibiotic resistance.Whole genome sequencing results showed that the gat AX genes were located on its chromosome,encoding Gassericin T(Gat AX).The culture supernatant and its crude extract of L.gasseri HMV18 were respectively subjected to SDS-PAGE,and their molecular weights were identified after Commassie Blue staining.The crude protein strip was cut off and sent for sequencing.The supernatant of Gassericin T culture was tested for bacteriostatic activity,and it was confirmed that the bacteriostatic active substance was present in the culture supernatant of L.gasseri HMV18.The results of the hemolysis test showed that there wasα-hemolysis around the colony of L.gasseri HMV18.Conclusion:1.The improved Oxford cup bacteriostatic test process can ensure the accuracy and repeatability of the bacteriostatic test,which is suitable for a large number of clinical samples screening.2.In this study,a new strain HMV18 was purified from vaginal secretions of women of childbearing age,which can inhibit the growth and reproduction of conditioned pathogens or pathogenic bacteria E.coli,K.oxytaca,S.aureus.3.The traditional phenotypic test and genomic results of the new L.gasseri strain HMV18 confirmed that the strain had no virulent gene,did not have the threat of horizontal transfer resistance,and could produce the bacteriocin Gassericin T,which had an antagonistic effect on the vagina pathogenic bacteria.4. L.gasseri HMV18 produceα-hemolysis,which may related to the vaginal self-cleaning.