Antibacterial Characteristics And Safety Evaluation Of Enterococcus Faecium LS170308 Strain

Enterococcus faecium is a common intestinal normal bacterium.It is generally considered that Enterococcus faecium is weak in toxicity,and it can inhibit the growth of pathogenic bacteria and promote the digestion and absorption of nutrients.Therefore,it has been widely used as microecological preparation.In the aquaculture industry,but Enterococcus faecium has obvious strain differences when used as a probiotic,so a lot of work is needed for the screening of probiotics.In this study,a strain of Enterococcus faecium with inhibitory pathogen growth was isolated from animal-derived fecal samples,and its antibacterial effect was evaluated.The safety of the strain was evaluated by means of genomics and other methods theoretical support.1.Enterococcus faecium selection medium formula screeningSince the Enterococcus faecium selection medium did not appear on the market,only the Enterococcus selection medium,the Enterococcus faecalis selection medium and the Enterococcus faecium color identification medium were included,and the target strain Enterococcus faecium could not be easily obtained by the above method.Therefore,this study explored the culture characteristics of Enterococcus faecium,selected bile salts,sodium azide and cefepime hydrochloride to screen non-Enterococcus bacteria,and screened Enterococcus faecium by adjusting the culture temperature.The study used single factor study and orthogonal test to determine the medium formula.The results showed that the basal medium was added with 0.04%sodium azide,1.5%bile salt,250?g/mL cefepime and 5%calf serum,and the culture temperature was adjusted to 50°C,only Enterococcus faecium grew on the medium,which was in line with the expected test results.The culture method has better screening effect,simple operation,no special equipment,low cost and certain market application value.2.Screening of probiotic strains of Enterococcus faecium and its probiotic mechanismIn this study,a number of strains of Enterococcus faecium were screened from the faeces samples of the forest carp by self-made selection medium,and contacted with Acinetobacter solid medium on the contact medium,and finally a strain was obtained to inhibit the growth of Acinetobacter.The Enterococcus faecium strain,named Enterococcus faecium LS170308,was tested by experiments to inhibit the growth of Escherichia coli,Acinetobacter,Yersinia,Pseudomonas,Staphylococcus aureus,etc.The inhibition was most obvious when it was cultured for 4 to 16 hours,but the strain had a poor growth inhibition effect on K.pneumoniae.Further studies showed that the main antibacterial substances Enterococcus faecium supernatant is greater than 50 kDa protein,the culture supernatant was analyzed by tandem mass spectrometry,the active protein is predicted glycan peptidases hydrolyzing sagA.The protein was purified and expressed to verify the antibacterial function,and it was confirmed that the peptidoglycan hydrolase sagA has the effect of inhibiting the growth of other bacteria.The experiment confirmed that the strain has the application value and can effectively inhibit the growth of other bacteria,but it also needs to be verified in vivo by the strain.3.Safety evaluation of Enterococcus faecium strainsIn order to determine the safety of the strain application,the strain was evaluated from bacterial pathogenicity and drug resistance,and the application safety of the strain was determined by comprehensive investigation of in vitro safety and in vivo safety.The whole gene sequencing method was used to determine the virulence genes,drug resistance genes,and amine-related genes carried by the strains.At the same time,the bacterial susceptibility test and the pathogenicity test in mice and rabbits were carried out.The experimental results confirmed that the strain carried efmA,AAC(6')-Ie-APH(2'')-Ia,ErmA,dfrG,AAC(6')-Ii,lias,lnuB,ANT(6)-Ia,lsaE,ANT(9)-Ia,cat,tetM,tet(L),ErmB and other drug resistance genes,the drug sensitivity test also confirmed that the strain against amoxicillin,ticarcillin,cephalosporin,doxycycline,kanamycin,tobramycin,cotrimoxazole,norfloxacin,and trimethoprim are completely resistant.It is worth noting that this strain is sensitive to penicillin and streptomycin.For antibiotics such as ciprofloxacin,amikacin,clindamycin,and erythromycin;sensitive to chloramphenicol,florfenicol,and nitrofurantoin antibiotics.The genome showed that the strain did not carry the virulence genes Cyl,gelE,asal,efaA and Hylefm related to the pathogenicity of Enterococcus faecium.The pathogenicity of the strain was extremely weak by animal experiments,and the concentration of the inoculum reached 2.38×10~111 CFU/mL,the disease did not occur in the animal and the pathological diagnosis confirmed that the cell barrier was not passed through,but the strain was metabolized to produce tyramine.Therefore,the safety of the strain still needs further verification.In this study,a strain of Enterococcus faecium with significant inhibition of the growth of other bacteria was successfully isolated from animal feces samples.The strain has potential production and application value.The safety evaluation of the strain found that the strain is extremely weak,but The strain carries a variety of drug resistance genes on chromosomes and plasmids,and has a similar drug resistance island structure,indicating that the drug resistance genes may be transmitted between species,but relevant experimental studies are still needed to confirm the spread of drug resistance genes.At the same time,the strain has not been tested for inhibition in vivo,and relevant experiments are needed to further confirm its production and application value.