The Biological Characteristics Of Aerococcus Viridans Phage AVP And Its Endolysin And Their Experimental Treatment Of Mouse Mastitis Model

Aerococcus viridans are Gram-positive cocci,which are an important zoonotic pathogen.They are widely distributed in the environment and can cause widespread infection and even death in humans and animals.In veterinary clinics,Aerococcus viridans can cause meningitis,pneumonia,urinary tract infections and arthritis in pigs.In addition,it is a pathogenic bacteria in bovine clinical and subclinical mastitis.However,due to the similar morphology and biochemical characteristics of enterococcus and streptococcus,the former Aerococcus viridans were mostly misidentified as these two bacteria.In recent years,due to the improvement of identification methods,more and more Aerococcus viridans have been isolated and identified,and the emergence of drug-resistant strains due to the long-term irregular use of antibiotics,which makes the treatment of infection caused by the bacteria increasingly difficult.Phage can infect and kill bacteria specifically,so it can be used as a candidate drug in the treatment of bacterial infection.Phage therapy is a promising candidate therapy that can assist or even replace antibiotics.At present,antibiotics are still the main way to control the infection of Aerococcus viridans,but there are no reports about its phage and its application in infection treatment.This study first isolated and identified 14 strains of Aerococcus viridans from clinically affected swine farms and bovine farms.The first Aerococcus viridans bacteriophage was isolated from sewage with Aerococcus viridans AV-X1 as the host bacteria,and it was named vB_AviM_AVP(AVP)according to the standard nomenclature.Its basic biological characteristics were further studied.The bacteriophage AVP can form small and translucent plaques on the BHI solid double-layer plate.The isometric,icosahedral heads and contractile tails under transmission electron microscopy indicate that the bacteriophage belongs to the Myoviridae family.The one-step growth curve showed that the eclipse period of the phage AVP was 10 min,the latent period was 15 min,and the burst size was about 139 PFU / cell.Temperature and pH stability tests also show that the phage has better temperature and pH tolerance.In addition,the lysis profile measured by the spot method showed that the phage AVP could lyse 8 of the 14 existing Aerococcus viridans.Next,the whole genome of the phage was analyzed.The genetic material of the phage AVP was double-stranded DNA,the full-length genome was 133 806 bp,and the G + C content was 34.51%.A total of 165 putative open reading frames,without any known lysogen,virulence or antibiotic resistancee-related genes,provided the possibility of phage AVP as an antibacterial agent.By comparison and analysis with known sequences in the database,ORF96 was determined to be a lysin gene encoding the phage AVP,and the endolysin AVPL was prokaryotically expressed and purified.The in vitro activity measurement showed that AVPL had better bactericidal activity in pasteurized milk and was not inhibited by the components in milk.Furthermore,its 3D structure and key action sites were predicted.The Amidase domain of LysGH15 was used as a template to predict the 3D structure of AVPL catalytic domain with 100% confidence.According to the predicted structure,site-directed mutagenesis of 99 E,159H,and 160 V at its possible key action sites was performed.After the latter two sites were mutated,the activity of AVPL was completely lost,while the activity of 99 E did not affect its activity.This indicates that the 159 H and 160 V sites are the key sites for the catalytic activity of the lyase AVPL.This article successfully established a mouse mastitis model caused by Aerococcus viridans infection,and evaluated the in vivo therapeutic effect of the lysin AVPL and phage AVP.The results showed that after 4 hours of intra-mammary challenge,a single dose of 5 × 10~6 PFU of phage was given to each mammary gland to achieve an effective therapeutic effect.The number of colonies in the mammary gland was reduced,the pathological changes in mammary gland were reduced,and the inflammation was reduced.The levels of cytokines TNF-?,IL-1? and IL-6 and myeloperoxidase(MPO)activity were also significantly lower than those in the PBS-treated group.At the same time,there was no obvious pathological change or inflammatory reaction in breast tissue 24 hours after single dose of 5 × 10~7 PFU phage,which indicated that the phage AVP had good safety in vivo application.Endolysin treatment only reduced the bacterial load in the mammary gland,but did not reduce the inflammatory response.In conclusion,this study isolates and systematically identifies the first strain of Aerococcus viridans at home and abroad,recombinantly expressed its lysin and used it for experimental treatment of a mouse mastitis model,providing a new way for the prevention and control of drug-resistant Aerococcus viridans.