Characteristics Of Yersinia Enterocolitica Phage VB-YenM-X1 And Its Effect Of Therapy On Enteritis In Mice

Yersinia enterocolitica is an important zoonotic pathogen,widely distributed as a food-borne pathogen worldwide,causing infections including self-limiting gastroenteritis,severe abdominal pain and life-threatening systemic infections.All ages are susceptible to Y.enterocolitica,especially children under 5 years of age,the elderly and people with low immunity have a higher risk of disease.According to surveys around the world,the pig is the main source of human foodborne infections caused by Y.enterocolitica.However,in the removal of Y.enterocolitica colonized in the intestines of farm animals,the use of antibiotics is restricted,which can cause dysbiosis in the intestinal flora of the animals,antibiotic allergies,drug resistance,and drug residues.By the end of 2020,China will comprehensively implement policies such as"forbidden resistance and restricted resistance"in livestock breeding,prompting us to seek new treatment strategies and methods to prevent and treat yersiniosis.In recent years,some successful cases of phage clinical treatment of bacterial gastrointestinal diseases have proved the feasibility and safety of phage therapy,which has great potential in replacing or supplementing antibiotics to treat bacterial diseases.To date,no cases of successful application of bacteriophage to treat Y.enterocolitica infection in vivo have been reported.In this study,a lytic bacteriophage capable of lysing Y.enterocolitica was isolated in nature.The bacteriophage exhibited broad-spectrum and highly effective bactericidal activity against Y.enterocolitica in vitro.Phage can effectively prevent the colonization of Y.enterocolitica in the gastrointestinal tract of mice,and alleviate the tissue inflammation damage caused by pathogenic bacteria.In this study,a phage named vB-YenM-X1 was was isolated and purified from domestic sewage by using Y.enterocolitica as the host bacteria.Transmission electron microscopy revealed that the bacteriophage belongs to the Myoviridae phage family,with an icosahedral head and a contracted tail.Phage whole genome sequencing results showed that the bacteriophage vB-YenM-X1 genome was 48 948 bp in length,with a G+C content of approximately 47.9%,and had 88 open reading frames.No resistance,lysogeny or bacterial virulence were found in the genome.The subsequent biological characteristics showed that bacteriophages have extremely low optimal multiplicity of infection(MOI=10^-6),shorter incubation period?15 min?and larger outbreak volume?290 PFU/Cell?,indicating the phage vB-YenM-X1 has high proliferation efficiency.Studies on the stability of bacteriophage have shown that bacteriophage vB-YenM-X1 is highly tolerant to temperature range of 4⁶0?,pH value of 4¹1 and digestive enzymes present in the gastrointestinal tract,suggesting that phage vB-YenM-X1 may be suitable for oral administration.Subsequently,serotype testing of 49 strains of Y.enterocolitica isolated from local rodents and dogs in Yunnan Province showed that the serotypes of the isolates were widely distributed,among which was mainly O:3 serotype strains which caused worldwide diarrhea predominate.The phage lysis range and activity were further determined,and it was found that it has a wide range of lytic host spectrum?27/51?and can cover multiple serotype strains with epidemiological significance,especially for the O:3 serotype strain,the highest lysis rate was 50%.In this study,a model of enteritis in mice infected with Y.enterocolitica serotype O:3 was successfully established.The phage vB-YenM-X1 was used to treat and evaluate its therapeutic effect and safety.The results showed that after 6 hours,mice challenged with bacteria?2.05×10⁸CFU/mouse?,a single dose of phage vB-YenM-X1?1.95×10⁸PFU/mouse?was administered orally.After 12 hours of treatment,the phage can significantly reduce the amount of bacterial load in the tissues,relieve the damage of Y.enterocolitica to gastrointestinal tissue,and reduce the inflammatory response in the body.The contents of proinflammatory cytokines such as IL-1?,IL-6 and TNF-?were significantly lower than those of the untreated group,and there was no significant difference from the healthy group.With the reduction of the gastrointestinal host bacterial load,48 hours after treatment,the bacteriophage vB-YenM-X1 was gradually cleared by the body,and the treated animals showed no adverse reactions.In summary,the oral bacteriophage vB-YenM-X1 can smoothly pass through the gastric acid barrier without losing the lytic activity,effectively reach the lesion to eliminate Y.enterocolitica in the intestine,and there are no adverse reactions such as drug residues.This study shows that the bacteriophage vB-YenM-X1 has great application potential in the removal of Y.enterocolitica in the environment,food and animal intestines,and can effectively reduce the potential public health and safety risks that the bacterium may bring.