| Public health is seriously threatened by foodborne illnesses,hospital-acquired infections and bioterrorism caused by pathogenic bacteria.A substantial toll for health-care is exacted in the prevention and treatment of bacterial infection.Currently,the main strategy for treating bacterial infection depends on the usage of effective antibiotics.Unfortunately,with the abuse in livestock breeding and irrational usage in the hospital,antibiotics decreasingly act on pathogenic bacteria.As multi-resistant superbugs including Enterococcus faecium,Staphylococcus aureus,Klebsiella pneumoniae,Acinetobacter baumannii,Pseudomonas aeruginosa,and Enterobacter are progressively reported in the clinic,the clinical therapeutic regimens are thrown into a dilemma.Even worse,the treatment of many infectious diseases caused by pan-resistant superbug now relies on just only one or two drugs.We are in danger of stepping into a post-antibiotic era in which minor injuries and common infections will be fatal.Rapid and accurate bacterial detection is crucial to deal with pathogenic threat.After the infectious bacteria are confirmed and identified,timely administration of effective antimicrobial agents is the key to the treatment of infectious diseases.However,as the standard methods of bacteria detection and antimicrobial susceptibility testing(AST)suffer from time-consuming culture procedure,the treatments of effective anti-infective therapy are largely restricted and empiric therapies are frequently administrated.Bacteriophages are ubiquitous viruses which absolutely parasitize in the microbes such as bacteria,fungi,antinomies or Rickett’s organism.They have no machinery for producing energy and no ribosomes for translating proteins.The high specificity,long-term survivability and ability to propagating in their hosts attribute to the maintenance of the dynamic balance of bacterial species in the ecosystem.Therefore,bacteriophages and their functionalized proteins showed immense potentials to fight bacteria for establishing the platforms of bacterial detection and AST due to the specificity and robustness.In the proof-of-principle paper,Pseudomonas aeruginosa(P.aeruginosa)and Staphylococcus aureus(S.aureus)were utilized as the mode,the recognition elements specific to P.aeruginosa and S.aureus were screened and testified to establish the methods for bacterial detection and AST,as listed in detail below:1.Screening of recognition elements for pathogenic bacteria1.1 Isolation of bacteriophage PAP1 for P.aeruginosa.A virulent bacteriophage which was highly specific to P.aeruginosa was isolated from hospital sewage utilizing aλ-bacteriophage isolation protocol.The bacteriophage named PAP1 can be amplified in the Luria-Bertani broth.After NaCl desorption,PEG8000 precipitation,and chloroform purification,high purity and titer of PAP1 suspension was prepared.Subsequently,the biology characterization,optimal multiplicity of infection and single-step growth curve of PAP1 were investigated.The isolated PAP1 was a virulent bacteriophage composed of an icosahedral symmetrical head,with a size of about 70 nm and a tail with a length of about 130 nm.The capturing time and complete lysis time were approximately 20min and 60 min,respectively.1.2 Expression and purification of tail fiber protein P069.DNA sequences of putative tail fiber protein P069 was obtained from National Center for Biotechnology Information by using basic local alignment search tool.The primer was designed to contain double restriction enzyme cutting site of Nde I and Not I.The recombinant plasmid pET21a-P069 was created by cloning the DNA sequence of P069 into the plasmid pET21a for the expression of P069,followed by transforming into Escherichia coli BL21(DE3)pLysS to express and purify P069.It was found that when P069 was expressed and induced in Escherichia coli,it was only present in the inclusion body.After renaturation,purification,the molecular weight of obtained protein was 65 KD in accord with the putative P069.2.Bacterial detection and AST2.1 Highly specific bacteriophage-functionalized magnetic beads for rapid separation and sensitive detection of viable P.aeruginosa.PAP1 was used to functionalize tosyl-activated magnetic beads to establish a bacteriophage-affinity strategy for separation and detection of viable P.aeruginosa.Recognition of the target bacteria by tail fibers and baseplate of the bacteriophage led to the capture of P.aeruginosa onto the magnetic beads.After a whole replication cycle,the target bacteria were disrupted,with the intracellular adenosine triphosphate releasing.Subsequently,firefly luciferase-adenosine triphosphate bioluminescence system was used to quantitate the amount of P.aeruginosa.This bacteriophage-affinity strategy for viable P.aeruginosa detection showed a linear range of 6.0×10~2-3.0×10~5 CFU/mL,with a detection limit of 2.0×10~2 CFU/mL.The whole process for separation and detection could be completed within 2 h.Since the isolated bacteriophage recognized the target bacteria with very high specificity,the proposed strategy did not show any signal response to all of the tested interfering bacteria including Pseudomonas solanacearum and Pseudomonas putida.The proposed strategy had been applied for detection of P.aeruginosa in pharmaceutical and biological samples such as glucose injection,human urine,and rat plasma.In the further work,this facile bacteriophage-affinity strategy could be extended for detection of other pathogens by utilizing virulent bacteriophage specific to other targets.2.2 AST by using virulent phages to evaluate bacterial viability.Since phages have no their own metabolic machineries,the progenies replication and host cell lysis absolutely depend on the host metabolic machineries such as DNA topoisomerase and ribosome.Many categories of antibiotics such as aminoglycosides,macrolides,lincosamides and rifamycin target on the ribosome,while quinolones work against DNA topoisomerase.Therefore,the life cycle of virulent bacteriophage can be inhibited by effective antibiotics.If bacteria were sensitive to antibiotics,there was no viable bacterial cells and thus no difference between the bioluminescence signals of phage group and solution group.Conversely,in case that bacteria were resistant to antibiotics,there still existed some viable bacterial cells,therefore there was difference between the bioluminescence signals of phage group and solution group.The MICs of tobramycin,gentamicin,ceftazidime,piperacillin and levofloxacin were evaluated to be<4μg/mL,8μg/mL,>32μg/mL,>128μg/mL and>8μg/mL within 3 h.It has immense potentials to reduce the frequency of empirical antibiotic therapy and alleviate antimicrobial resistance in clinical therapy of infectious diseases.It can be extended to AST of other pathogens by isolation of their virulent phages.2.3 Specific and non-lytic phage tail fiber protein as promising biorecognition element for bacterial detection.TFP showed lack of the lytic activity,and might only be responsible for host recognition.P069 could specifically distinguish the difference of the species of P.aeruginosa within 15 min.Subsequently,one-site recognition based BL method and two-site recognition based sandwich fluorescence method were developed to detect P.aeruginosa,with the linear range of 2.0×10~3 CFU/mL-2.0×10~6 CFU/mL and 4.0×10~2 CFU/mL-4.0×10~6 CFU/mL,respectively.The detection limit of BL method and fluorescence method were 6.7×10~2 CFU/mL and 1.3×10~2 CFU/mL,respectively.P.aeruginosa solutions spiked into human urine,glucose injection and rat serum were successfully detected by using one-site recognition based BL method with the acceptable recovery.2.4 Molecular recognition strategy for detection and AST of S.aureus by utilizing teicoplanin and porcine IgG as sensing agents.A highly-selective molecular recognition-based sandwich fluorimetry was developed to rapidly detect Staphylococcus aureus(S.aureus),and furtherly to assess its susceptibility to antibiotics.Immobilized porcine IgG was utilized to capture S.aureus through the selective interaction between Fc fragment of porcine IgG and protein A in the cell wall of S.aureus.Fluorescein isothiocyanate-labeled teicoplanin was adopted as the signal tracer utilizing the binding behavior between teicoplanin and D-Ala-D-Ala moieties in the peptidoglycans of Gram-positive bacterial cell wall.S.aureus could be detected within a wide linear rang of 1.0×10~3 to 1.0×10~7 CFU/mL and detection limit of 3.3×10~2 CFU/mL.Subsequently,this protocol utilizing porcine IgG and teicoplanin as the sensing agents was utilized to assess the antimicrobial susceptibility of S.aureus after the bacteria were treated with antibiotics.The minimum inhibitory concentrations of penicillin,cefoxitin,clindamycin,trimethoprim/sulfamethoxazole and erythromycin were estimated to be>0.25,<4,<0.25,<2/38 and<0.5μg/mL,respectively.Additionally,this methodology platform also can be utilized to AST to teicoplanin as the resistance to teicoplanin can hinder the binding between teicoplanin and S.aureus.The whole process for AST can be completed within 4 h since it avoided extra time-consuming isolation and identification of bacterial species.This fluorimetry shows the promise of providing rapid and accurate AST data for infectious diseases.In summary,since porcine IgG,bacteriophage and tail fiber protein can specifically bind with the targets on the surface of bacteria,the targets bacteria were separated and concentrated from the complicated matrix.Combined with different luminescence analysis method,pathogenic bacteria can be rapidly,accurately and specifically detected.Furthermore,AST was successfully performed with evaluation of bacterial viability by virulent phage or molecular recognition-affinity strategy,which can identify the bacterial species without extra time-consuming culture process.It showed immense potentials to alleviate antimicrobial resistance in clinical therapy of infectious diseases.Additionally,we successful cloned and expressed physiologically active tail fiber protein,which was expected to play an important role in pathogenic bacterial detection and AST. |
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