Drug Resistance Analysis Of Proteus Mirabilis Isolated From Swine In Guangxi And Isolation,Identification And Preliminary Application Of A Phage Of Proteus Mirabilis

Proteus mirabilis(P.mirabilis)is a common zoonotic opportunistic pathogen,which can cause gastrointestinal tract,respiratory tract and urinary tract infections in animals and humans.In recent years,it has been found that P.mirabilis has the characteristics of carrying and transmitting drug-resistant genes.The emergence of multi-drug resistant P.mirabilis has brought great challenges to clinical treatment and animal production,which has aroused people's attention and vigilance.Phages have specific bactericidal properties,which make them an ideal alternative to antibiotics.In this study,the virulence and drug resistance of P.mirabilis isolated from swine in Guangxi province was investigated.Meanwhile,in order to explore the application prospect of phage in killing P.mirabilis,a phage that could lyse multi-drug resistant P.mirabilis was screened.The biological characteristics and whole genome were analyzed,and the effect of its application in food safety and clinical treatment was evaluated through bactericidal experiments.The drug resistance and virulence of 21 strains P.mirabilis isolated from the 98 samples collected from swine farms in Guangxi were investigated.The results showed that the drug resistance rate of 21 strains P.mirabilis to tetracycline,doxycycline,chloramphenicol,ampicillin and sulfamethoxazole reached 100%,and the drug resistance rate of cefalexin,cefuroxime,cefotaxime,imipenem and kanamycin was above 57.1%,and all the strains were multi-drug resistant bacteria.The proportion of strains carrying ESBLs or Amp C genes was 57.1%(12/21),14.3%(3/21),and the proportion of strains carrying both ESBLs and Amp C genes was 14.3%(3/21).The gene detection rates of qnr A,qnr B,qnr S and aac(6')-Ib-cr were 9.5%,0,4.8%and 80.1%,respectively.The class?integron positive rate was 61.9%,nine different gene cassette arrays were detected(aad A2-lin F;est X;dfr A32-ere A-aad A2;drf A5;drf A12-orf F-aad A2;arr3-aac(6')-Ib-cr;aac(6')-Ib-cr-bla_OXA-1-cat B3-aar3;drf A1-orf C;aad A2);the class?integron positive rate 76.2%,only one gene cassette array was detected(drf A1-sat1-aad A1).The positive rate of virulence gene atf A,hpm A,ire A,mrp A,pmf A,pta,ure C,and zap A of 21 strains P.mirabilis were 100%,and the positive rate of uca A was 95.2%.A lytic phage,named v B?Pmi P?p Pm05,was isolated using multi-drug resistant P.mirabilis as host bacterium.The biological characteristics and whole genome analysis were analyzed.The results showed that the plaque of v B?Pmi P?p Pm05 was transparent and clear without halo,and the best multiplicity of infection(MOI)of its host was 0.001.It has temperature stability and can maintain high activity at 50?.In addition,it has acid and alkali-resistant stability and can maintain high titer at p H 4.0-9.0.The incubation period was 10 minutes and the explosive quantity was 44 PFU/cell.The full length of the phage genome was 90486 bp and the GC content was34.44%.It contained 131 open reading frames(ORF),that did not carry drug resistance and virulence genes.The genome shared 98.51%identity with Proteus phage v B?Pmi P-P59,so it should be classified as a member of the order Caudovirales,family Podoviridae.The bactericidal effect of phage v B?Pmi P?p Pm05 on liquid culture medium,milk and pork was evaluated.The results showed that phage v B?Pmi P?p Pm05 had great bactericidal effect on liquid culture medium,milk and pork at 4?,and the bactericidal effect on culture medium and milk was better than that on pork.The safety and therapeutic effect of phage v B?Pmi P?p Pm05 was evaluated.The results showed that there was no abnormal behavior,no death and no tissue lesion in the mice 2 weeks after the injected of phage v B?Pmi P?p Pm05,which showed good safety.The treatment of P.mirabilis infected mice helped to relieve bacteremia and significantly reduce the bacterial load in the spleen.In conclusion,P.mirabilis strains isolated from swine in Guangxi have strong virulence and serious drug resistance,which should be vigilant.Phage v B?Pmi P?p Pm05 has potential in controlling P.mirabilis contamination in food and in clinical treatment of P.mirabilis infection.It can be developed into phage preparations and use in the field of food safety and clinical treatment in the future.