Translocation Of Lactiplantibacillus Plantarum Wlpl04 In Pregnant Mice And Its Preventive Effect On Mastitis Induced By Staphylococcus Aureus

Mastitis is a common inflammatory disease in lactating women,with an incidence of up to 33%.At present,the main treatment method is antibiotic intervention,but antibiotics can easily lead to the production of drug-resistant bacteria and cessation of lactation,Therefore,it is necessary to develop new treatment methods.Probiotics have probiotic functions such as inhibiting pathogenic bacteria and inflammation.Breast milk is an important source of probiotics.Lactiplantibacillus plantarum WLPL04 is derived from healthy human milk and has broad-spectrum antibacterial properties,which can be colonized in the intestinal tract and transferred.Therefore,on the basis of fluorescent labeling of L.plantarum WLPL04,this study explored its migration path in the maternal body and its preventive effect on mastitis through the pregnant mouse model and the maternal mastitis model.Firstly,a WLPL04-m Cherry engineering strain labeled with red fluorescent protein was constructed.Namely,p MG36e was used as the skeleton,and the m Cherry red fluorescent protein gene driven by three promoters was used as the marker to construct the recombinant plasmids p MG36e-P_X-m Cherry,p MG36e-P₂₃-m Cherry and p MG36e-P_ldh-m Cherry,which were respectively introduced into Escherichia coli,and the recombinant was screened with erythromycin resistance,and verified by PCR,colony color observation and laser confocal microscopy observation.After successful validation,the recombinant plasmid was introduced into L.plantarum WLPL04 and validated.The results showed that the selected WLPL04 recombinants could detect specific bands of the recombinant plasmid through PCR,and there was no significant change in colony color;However,after centrifugation,it was observed that the recombinants containing p MG36e-P_X-m Cherry showed significant pink color.Laser confocal microscopy observation showed that the recombinant containing p MG36e-P_X-m Cherry had the strongest red fluorescence;The recombinant vector containing p MG36e-P₂₃-m Cherry takes the second place;The recombinants containing p MG36e-P_ldh-m Cherry exhibit weak red fluorescence.This indicates that the WLPL04engineering strain containing the recombinant plasmid has been successfully constructed(named WLPL04/p MG36e-P_X-m Cherry,WLPL04/p MG36e-P₂₃-m Cherry and WLPL04/p MG36e-P_ldh-m Cherry,respectively).The effect of the recombinant plasmid on the morphology and growth of L.plantarum was further analyzed by scanning electron microscope observation and growth curve determination.The results showed that the plasmid p MG36e-P_X-m Cherry,p MG36e-P₂₃-m Cherry and p MG36e-P_ldh-m Cherry had no significant effect on the morphology and growth of L.plantarum.In summary,this study selected WLPL04/p MG36e-P_X-m Cherry for subsequent experiments and renamed it WLPL04-m Cherry.After that,a pregnant mice model was constructed,and WLPL04-m Cherry was supplemented in the late pregnancy and lactation of the female mice.Through weight monitoring,viable bacteria count,laser confocal microscopy observation,intestinal tissue staining,and immunofluorescence analysis,the migration and possible path of WLPL04-m Cherry in pregnant rats were explored.The results showed that supplementing WLPL04-m Cherry increased the body weight of female mice,but did not affect organ index.The results of live bacterial count showed that WLPL04-m Cherry could be colonized in the intestinal tract of female mice and translocated to the mammary gland,thymus,liver,spleen,mesentery lymph nodes（MLN）,kidney,ileum,colon,cecum and brain,while the number of WLPL04-m Cherry was more in the intestinal tract and extraintestinal organs of pregnant mice.Further collection of mammary gland,placenta,umbilical cord and stomach contents of 6-day-old newborn rats（instead of maternal milk）in the third trimester of pregnancy were carried out for live count and laser confocal microscopy observation and analysis.The results showed that WLPL04-m Cherry could be detected in placenta,umbilical cord and milk and showed red fluorescence.Histological analysis of the ileum and colon of female mice showed that supplementing WLPL04-m Cherry had no significant effect on the intestinal tissue structure of female mice.The results of immunofluorescence staining showed that WLPL04-m Cherry supplementation had no significant effect on the tight junction protein in the intestine and breast of female rats.To sum up,L.plantarum WLPL04 can colonize and translocated in healthy bodies,and has no adverse effects on the intestinal tract and blood milk barrier.Finally,a female mice mastitis model induced by Staphylococcus aureus was constructed,and the preventive effect of supplementation of L.plantarum WLPL04-m Cherry on mastitis was explored through viable count,H&E staining,immunohistochemistry,immunofluorescence staining and TUNEL staining.The results showed that WLPL04-m Cherry supplementation could reduce the number of viable S.aureus in the mammary glands,MLN and liver of female mice.It can significantly reduce the histopathological score of the mammary gland of mastitis mice.It can reduce the expression and enzyme activity of MPO in the mammary gland.It can maintain the levels of tight junction proteins Claudin-1,Occludin and ZO-1.It can alleviate the apoptosis of mammary gland cells caused by S.aureus.In vitro WLPL04-m Cherry co-culture model of S.aureus showed that WLPL04-m Cherry could effectively inhibit the growth of S.aureus.To sum up,WLPL04-m Cherry supplementation can effectively prevent mastitis of female mice caused by S.aureus by enhancing the maternal blood milk barrier,inhibiting the growth of S.aureus,reducing the apoptosis of mammary gland cells.To sum up,this study successfully carried out red fluorescent labeling on L.plantarum WLPL04,derived from breast milk,and obtained the engineering strain WLPL04-m Cherry.After supplementation of WLPL04-m Cherry in the third trimester of pregnancy and lactation,WLPL04-m Cherry can be translocated from the intestinal tract to the extraintestinal organs without affecting the intestinal and mammary gland tight junction proteins,and its number is high in pregnant mice.WLPL04-m Cherry can also prevent mastitis caused by S.aureus by inhibiting the growth of S.aureus,repairing the blood milk barrier,and reducing the apoptosis of mammary gland cells.The results of this paper provide data support for the subsequent development and application of L.plantarum.