Interaction Relationship Between Antimicrobial-Resistant Salmonella And Their Phages

Salmonellosis is a zoonosis which possesses great public health significance,and is one of the main problems that poultry farm should confront with.Antibiotics has always been the preference to be used to treat bacterial infections broadly.However,antibiotics abuse,the remained high-level drug resistance,the emergence of multi-resistance strains,the limited types of antibiotics and the difficulties in developing new drugs have achieved a renewed focus on phages as an antibacterial agents.Phages are highly specific and self-replicating（thus only need a small therapeutic dose）,and they could mutate with the hosts’ viriation.In this article,we have isolated the prevailing Salmonella strains in Shandong Province and screened for the targeted phages to perform the biological characteristics measurement and genome sequencing analysis;next we designed a phage-bacteria interaction transfer-culture test（subcultivation）with different combination（phages and bacteria）mode and analysed the intestinal microbiome of SPF chicken by metagenomics sequencing to demonstrate the phage-bacteria interaction networks,in order to provide relevant parameters and theoratical basis for the subsequent phage therapy of antimicrobial-resistant Salmonella infections.By the means of chromogenic media and PCR identification,we isolated 100 Salmonella strains from farms’ chicken tissues and environment,then three prevailing sequencing types（STs）was acquired by multilocus sequence typing method,in which ST 11 accounted for 96 percent.Next we carried on antimicrobial susceptibility testing with these strains,resulted in a resistance rate over 50 percent for 9 antibiotics widely used and especially,polymysin B reached up to 98 percent.We then separated phages against the strains above and 94 phages were abtained using double-plate method.Five phages whose host strains were ST11 and representative in multi-drug resistance were picked out for purification,electron microscopy observation,genome sequencing.Results showed that there are 3 Myoviridae（vB_SalM_XF,vB SalM smc,vB_SalM_LYw）and 2 Siphoviridae（vB_SalS_DZ,vB_SalS₉7）,and they all consist of an icosahedral head and a slender tail.All the five phages’ titer are over 109 pfu/ml,having an optimum growth temperature about 37℃ and a wide range of optimum pH value（that is,can grow well over pH 3）,and their optimal multiplicity of infection are less than or equal to 10-5 but they are distinct in duration of the latent and lysis phase.Besides,we designed a subculture experiment with different kinds of phage-bacteria combination like "1 phage versus 1 bacterium","1 phage（bacterium）versus multi bacteria（phages）" and "multi phages versus multi bacteria" for the simulation of phage-bacteria co-evolution and to explore the phage-bacteria interaction networks.It came out that:（1）inhibition of the host could be achieved by the co-existing of its phage and competitive strains;（2）the network formed by the interaction of multi phages and their relative hosts could balance the growth advantage of these strains and stabilize the system.Moreover,we analysed the microbiome in the intestinal tract of SPF chicken utilizing metagenome sequencing,figuring out that there are strong positive correlations exist between phages and bacteria with high abundance.Finally we treated SPF chicken with antibiotics and contrasted the abundances of both phages and bacteria before and after treated to study the influence of clinical dose antibiotics on intestinal tract microbiome and in conclusion,phage-bacteria interaction network—we named it "Stable Hysteresis Network"—could balance the growth advantage between host strains and to a certain extent buffer the fluctuations and impacts that external factors have on the intestinal systems.In the future,we will carry out animal tests to verify the conclusions above and use the phages isolated to conduct phage therapy.