Bactericidal Properties Of Stenotrophomonas Maltophilia Phages And Study On The Activity Of Phage DNA Polymerase

Stenotrophomonas maltophilia(S.maltophilia)is an important global opportunistic nosocomial pathogen that causes considerable morbidity in immunocompromised patients.In addition,S.maltophilia is intrinsically resistant to many antibiotics.With the irrational use of antibiotics,the drugresistant species of S.maltophilia is increasing,leading to fewer and fewer drugs that can effectively treat S.maltophilia infections.Therefore,there is an urgent need to seek new therapeutic approaches.Bacteriophages(phages),viruses that infect bacteria,are the most abundant and diverse biological entities on the planet and are considered an alternative to antibiotics for treating drug-resistant bacterial infections.Phages have had a tremendous impact on molecular biology and applied biology since their discovery.However,there is still a huge gap in our understanding of phage diversity and protein function.Screening of new phages and studies on the characterization of their life activities and protein functions will enrich the understanding of phages and contribute to the development of phage applications.Therefore,we took the application of phages as a starting point for phage screening and bactericidal assay against clinically drug-resistant S.maltophilia,as well as study phage DNA polymerase for nucleic acid detection.First,we isolated two phages with lysis ability against drug-resistant S.maltophilia from hospital sewage,named BUCT555 and BUCT603,respectively.Transmission electron microscopy analysis showed that BUCT555 had a short tail,while BUCT603 had a long bendable tail.BUCT555 infected the host with an incubation period of 30 min and a lysis period of 70 min,whereas BUCT603 infected the host with an incubation period of 20 min and a lysis period of 60 min.Host range tests showed that BUCT555 lysed 13.3% of the test bacteria,while BUCT603 lysed 66.7%.Genome sequencing analysis showed that the genome of BUCT555 was composed of 39,440 bp with 57 open reading frames(ORFs),but contained genes related to lysogeny;the genome of BUCT603 was composed of 44,912?bp with 64 ORFs,and no lysogeny or virulence-related genes were identified.BLASTn analysis showed that BUCT555 and BUCT603 had less than 2% homology with other phages,suggesting that they are two novel phages.A multifaceted comparison showed that BUCT603 was more suitable for phage therapy,not only inhibiting the growth of drug-resistant bacteria within 1 h in vitro,but also significantly improving the survival rate of lung-infected mice.However,BUCT603 alone did not completely kill resistant bacteria due to the development of phage resistance in vitro testing.Subsequently,we used evolutionary studies of bacteria–phage co-cultures in vitro to elucidate the mechanism of resistance development between BUCT603 and SMA118,and to seek for ways to improve the therapeutic efficacy of the phage.Our results revealed that SMA118 resisted phage infection through mutations in the cell membrane proteins,while BUCT603 reinfected the resistant strain primarily through mutations in structure-related proteins.Compared with the wildtype strain SMA118,the evolved phageresistant strain R118-2 showed reduced virulence,weakened biofilm-formation ability,and reduced resistance to aminoglycosides.In addition,the evolved phage BUCT603B1 in combination with kanamycin could inhibit the development of phage-resistant in SMA118 in vitro,thereby killing SMA118 completely.Altogether,these results suggest that in vitro coevolutionary phage training leads to greater bacterial suppression.In addition to being used to lyse drug-resistant bacteria,phages’ diverse genomes also provide a valuable resource for mining proteins for specific functions.In order to obtain novel,faithful and efficient DNA polymerases for nucleic acid detection,we expressed and purified four phage DNA polymerases in vitro and functionally characterized them.By comparing the activities of the four DNA polymerases,we found that IME199 DNA polymerase showed better activity.IME199 DNA polymerase had 3′-5′ exonuclease activity that removes mismatched bases from DNA replication to ensure replication fidelity.In addition,IME199 DNA polymerase had d NTPs-dependent 5′-3′ polymerase activity and could amplify DNA at 15-35 °C and a p H range of 5.5-9.5.What’s more,IME199 DNA polymerase had powerful strand displacement and processive synthesis capabilities,and could perform rolling circle amplification and multiple displacement amplification with very low error rates.Meanwhile,IME199 DNA polymerase showed better amplification efficiency compared with the widely used phage phi29 DNA polymerase,which also indicated that IME199 DNA polymerase had a good application prospect.Finally,based on the properties of IME199 DNA polymerase,it could be used to develop a nucleic acid assay for DNA amplification at room temperature.Nucleic acid detection is one of the indispensable tools in life sciences,which is mainly based on PCR.However,PCR requires complex and expensive thermal cycling instruments that cannot perform point of care testing.Although isothermal amplification techniques were developed to eliminate thermal cycling equipment,they still require simple heating and detection equipment.To break through this limitation,we developed a visual nucleic acid assay based on rolling circle amplification and dependent on p H changes using IME199 DNA polymerase.By optimizing the reaction conditions of this method,accurate detection of target nucleic acids could be achieved by color change at room temperature(20-30°C)for 30 min without any equipment.In summary,this study enriched the understanding of the phage diversity and bactericidal capacity of S.maltophilia,and provided experimental basis for the use of phage to control infections with drug-resistant S.maltophilia.Additionally,we obtained a phage DNA polymerase with efficient amplification capability and developed the room temperature visual nucleic acid detection method,which has important potential application value.