The Antagonistic Interactions Between A Polyvalent Phage SaP7 And β-lactam Antibiotics On Combined Therapies

Salmonella is a common zoonotic strain that can cause invasive diseases,including acute diarrhea,gastroenteritis,and meningitis.In recent years,the rise of bacterial resistance has put more and more pressure on the traditional antibiotic treatment of bacterial infections,which has brought great pressure to microbial control through antibiotics,and posed a serious challenge to the treatment of multi-drug resistance（MDR）Salmonella infection.In order to find an alternative approach to antibiotic treatment,we isolated a phage,and evaluated the efficacy of phage therapy and the combination of phage and antibiotics in the treatment of multidrug-resistant Salmonella infection through in vivo experiments.In this study,a Salmonella strain named S7 was isolated from piglets with diarrhea,and its virulence and drug resistance were investigated.The results showed that Salmonella S7 carried both inv A and fim A virulence genes.Meanwhile,S7 showed resistance to amoxicillin,amoxicillin clavulanate potassium,aztrconam,gentamicin,doxycycline,levofloxacin and florfenicol.In addition,a polyvalent phage named Sa P7 was isolated from the sewage of a pig farm in Nanning using MDR Salmonella S7 as host.The biological characteristics and full genome of Sa P7 were also analyzed.The results showed that the plaque of phage Sa P7 was clear and bright without halo at the edge,and it can also lyse another 6 strains of Escherichia coli from different sources besides the host Salmonella.The optimal multiplicity of infection（MOI）was 10^-4,the incubation period was 20 min,the outbreak lasted 50 min,and the explosive quantity was21 PFU/Cell.In addition,it has good stability and can maintain high activity at 60°C or p H 5-7.Transmission electron microscopic observation showed that Sa P7 belonged to the order Caudovirales.Phage Sa P7 contains 254 open reading frames（ORF）with a total genome length of 240 613 bp,without any genes related to antibiotic resistance or virulence.The genome has 96.51%homology with Salmonella phage SPN3US,which belongs to the family Myoviridae,genus Seoulvirus.Bactericidal efficacy of phage Sa P7 combined withβ-lactam antibiotics was evaluated in piglets and BALB/c mice.The results showed that we discovered the antagonism efficacy of Sa P7 combined amoxicillin/potassium clavulanate（AMC）in counteracting Salmonella S7in piglet-models by measuring the bacterial level,tissue section or survival rate.The consistent result as above between Sa P7 and amoxicillin（AMX）was further verified in BALB/c mice-models.Furthermore,in vitro,plaque assay and minimum inhibitory concentration（MIC）determinations showed that AMX or AMC or cefepime（FEP）inhibited Sa P7 plaque formation respectively and Sa P7decreased bacterial susceptibility of Salmonella S7 to AMX,AMC and FEP,suggesting phage Sa P7 existed antagonism with severalβ-lactam antibiotics.Phage-antibiotic interactions in vitro were evaluated in liquid medium using microtitration plates.And the negative interference of Sa P7 with the bacteriostasis to Salmonella S7 of these threeβ-lactam antibiotics was observed in planktonic cultures via microtiter plates,but could not prevent the bacteriostasis of high titer of phage or high concentration of antibiotics.In conclusion,our study indicates that polyvalent phage Sa P7 has antagonistic effects combined with threeβ-lactam antibiotics and explains to some extent the mechanism by which phage resistance can reduce bacterial susceptibility to antibiotics.It is therefore crucial to fully and cautiously evaluate phage/antibiotic interactions and probable outcomes to avoid antagonistic impacts and failure of antibiotic and phage combination therapy.