Specific and Rapid Reverse Assaying Protocol for Detection and Antimicrobial Susceptibility Testing of Pseudomonas Aeruginosa based on Bacteriophage Tail Fiber Protein and Magainin II Recognition

Specific and Rapid Reverse Assaying Protocol for Detection and Antimicrobial Susceptibility Testing of Pseudomonas Aeruginosa based on Bacteriophage Tail Fiber Protein and Magainin II Recognition | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Specific and Rapid Reverse Assaying Protocol for Detection and Antimicrobial Susceptibility Testing of Pseudomonas Aeruginosa based on Bacteriophage Tail Fiber Protein and Magainin II Recognition Yong He, Hang Zhao, Yuanwen Liu, He Zhou This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-130554/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract The worldwide emergence and spread of antimicrobial resistance are accelerated by irrational administration and use of empiric antibiotic. A key point to the crisis is lack of rapid diagnostic protocol to antimicrobial susceptibility testing (AST) for timely and rational antibiotic prescription. Here, bacteriophage tail fiber protein (TFP) recombined in Escherichia coli expression system was functionalized on magnetic particles (MPs) to specifically capture P. aeruginosa , and FITC-labeled-magainin II was utilized as the indicator. For solving the MPs’ blocking effects, a reverse assaying protocol (RAP) based on TFP recognition was investigated the feasibility of detection and AST of P. aeruginosa . P. aeruginosa detection can be rapidly, sensitively and specifically detected within 1.5 h with a linear range of 1.0 × 10 2 to 1.0 × 10 6 CFU⋅mL − 1 and a detection limit of 3.3 × 10 CFU⋅mL − 1 . Subsequently, the results of AST which was consistent in the results of broth dilution can be obtained within 3.5 h. Due to the high specificity of TFP, the AST can actually be conducted without the requirement of bacterial isolation and identification by this RAP. Based on the proof-of-principle work, the detection and AST of other pathogens can be extended by expressing the TFP of their bacteriophages. General Biochemistry Analytical Biochemistry Antimicrobial resistance Antimicrobial susceptibility testing Bacteriophage tail fiber protein Magainin II Pseudomonas aeruginosa Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Antimicrobial resistance (AMR) is a worldwide health crisis resulting in growing economic burden and increasing mortality 1 , 2 . The most important strategies to minimize AMR are the development of rapid and accurate diagnostic protocols for antimicrobial susceptibility testing (AST), which facilitating the timing of prescription to use the effective antibiotics 3 , 4 . Therefore, numerous efforts are exerted on developing rapid, sensitive and acute detection and AST for bacterial infection. Traditional bacterial growth-based protocols are considered as the gold standard methods for bacterial detection and AST. These protocols show ideal repeatability, high standardization and good reliability. However, the isolation and identification procedure of bacterial detection usually require tedious process of 24–48 h 5 . The subsequent broth dilution or disk diffusion for AST performance require another bacterial growth cultured with given concentrations of various antibiotics, which demands almost another 24–48 h 6 , 7 . Lack of timely and accurate results of bacterial AST results in frequent empiric antibiotic therapy, causing irrational antibiotic use and the development of AMR 8 . Therefore, some other bacterial growth-based protocols are reported to aim at shortening the time of AST, such as microscopy detection 9 – 11 , electrochemical sensor 12 – 14 , phase-shift spectroscopy detection 15 , fluorescent detection 16 and microfluidic devices based on slipchip technique 17 , surface-enhanced raman scattering 18 . Nevertheless, since lack of capacity of isolating the given bacterial species, they also require time-consuming pretreatment procedure for bacterial culture, isolation and identification. Polymerase chain reaction-based protocols gradually attract attention to bacterial detection and performance of AST due to the advantages of rapidity, sensitivity and culture-free process 20 – 22 . However, they suffer from well-trained personnel, complicated molecular manipulation, prerequisite precise resistant gene information and frequent gene mutation. With the increasing global AMR, bacteriophages as the natural enemies to the bacteria have regained interest in solving the crisis caused by multi-resistant bacteria. Each strain of bacteria has one or more corresponding bacteriophages. Bacteriophages can highly specifically recognize their target bacteria even in harsh environment. Typical lifecycle of virulent bacteriophage involves the following steps: specific absorption on the bacterial cell wall, DNA injection into the bacterial cell, progenies replication and the lysis of bacteria cell for releasing progenies. Bacteriophages functional proteins (BFP) such as tail fiber protein (TFP), tailspike protein (TSP) and endolysin are the essential recognition elements which are responsible for absorption, injection and lysis, respectively 23 . Therefore, BFPs assumes the ideal molecular recognition attributes of high specificity, robustness, good anti-interference capability and universality to each bacterium 23 . Unfortunately, just like the bacteriophage entity, TSP and endolysin both have the inherent lytic activity which could be unfavorable for bacteria capture and sample manipulation. In the previous work, we had utilized to the Escherichia coli ( E. coli ) expression system to produce the TFP of Pseudomonas aeruginosa ( P. aeruginosa ) 24 . This recombinant TFP can specifically recognize P. aeruginosa without lytic activity. To investigate the application of TFP in AST detection, TFP-functionalized magnetic particles were utilized to specifically capture P. aeruginosa , and fluorescein isothiocyanate (FITC) labeled magainin II was utilized as the fluorescent tracer. A reverse assaying protocol (RAP) combined magnetic separation was developed to specific, rapid and sensitive detection and AST of P. aeruginosa . 2. Experimental 2.1. Instrumentations Scanning electron micrograph (SEM) was recorded by an S-3000N scanning electron microscope (Hitachi, Japan). Fluorescence (FL) signals were obtained from an Infinite M200 PRO microplate reader (TECAN, Switzerland). FL micrographs were recorded by using a NI − U FL microscope (Nikon, Japan). 2.2. Reagents and materials Piperacillin/Tazobactam (PIP/TAZ), ceftazidime (CAZ), tobramycin (TOB), gentamicin (GEN) and levofloxacin (LVX) were all purchased from Solarbio Life Sciences (China). Strains of P. aeruginosa , E. coli , Pseudomonas solanacearum ( P. solanacearum ), Salmonella typhimurium ( S. tyhimurium ), Staphylococcus aureus ( S. aureus ), Staphylococcus epidermidis ( S. epidermidis ), Streptococcus mutans ( S. mutans ) were purchased from Guangdong Microbiology Culture Center (China). FITC labeled magainin II were obtained from GL Biochem (Shanghai) Ltd.. (China). Tetraethyl rhodamine isothiocyanate (TRITC) and gelatin were provided by Sigma-Aldrich (USA, www.sigma-aldrich.com ). AffiAmino magnetic particles (MPs) were purchased from Lab on a Bead AB (Sweden) in which activation buffer and blocking buffer were provided. Both human urine collected from the authors and rat plasma gifted by another lab were used as the common matrices. Luria − Bertani (LB) broth consisted of 10 g⋅L − 1 NaCl, 5 g⋅L − 1 yeast extract and 10 g L − 1 tryptone. Washing buffer was composed of 10 mM PBS (pH 7.4) and 0.5% tween-20. Human beings aren’t involved in the study. 2.3. Bacterial culture and counting Strains of P. aeruginosa and other bacteria were grown in 30 mL of LB broth with continuous shaking at 80 rpm and 37 °C under aerobic condition until the value of OD 600 reached 1.0. For bacterial detection, 10 mM PBS (pH 7.4) was utilized to serially dilute the bacterial culture to reach the target concentration. The bacterial concentrations were evaluated according to standard approach of bacterial culture and counting. 2.4. Preparation procedure of TRITC-labeled TFP One milliliter of TFP solution at the concentration of 1.0 mg⋅mL − 1 was slowly mixed with 1.0 mL of TRITC solution at 3.0 mg⋅mL − 1 dissolved in dimethyl sulfoxide, followed by 12-h reaction at 4 °C. Subsequently, NH 4 Cl solution was used to stop the reaction. Finally, the solution was dialyzed with 10 mM PBS (pH 7.4) for 48 h at room temperature. 2.5. Fluorescent microscope image of stained P. aeruginosa One milliliter of P. aeruginosa suspension at 1.0 ⊆ 10 6 CFU⋅mL − 1 was added with 200 µL of FITC labeled magainin II at 20.0 µg⋅mL − 1 and the equal volume of TRITC labeled TFP. After 1-h incubation at room temperature, the suspension was centrifuged at 2500 g and washed thrice, followed by resuspended in 10 mM PBS (pH 7.4). Subsequently, 10 µL of the stained P. aeruginosa suspension was observed by a FL microscope with the magnificence of 1000. The excitation wavelength of TRITC and FITC were 544 nm and 488 nm, respectively, and the emission wavelength of TRITC and FITC were 570 nm and 525 nm, respectively. 2.6. Preparation procedure of TFP-functionalized MPs TFP-functionalized MPs were obtained according to manufacturer guideline. Briefly, after washed by washing buffer, 100 µL of MPs was resuspended into the 1.0 mL of washing buffer. Subsequently, 50 µL of the activation buffer was mixed with the above suspension for 15 min activation. After the MPs were thoroughly washed, 1.0 mL of TFP solution at 100 µg⋅mL − 1 was added for 1-h reaction at room temperature, followed by another thorough washing. Then the residue active sites of MPs were blocked with 80 µL of the blocking buffer and 1% gelatin at room temperature for 45 min. Finally, after washed thrice the TFP-functionalized MPS were stored in 10 mM PBS (pH 7.4) at 4 °C. 2.7. RAP for P. aeruginosa One milliliter of P. aeruginosa suspension was added with 10 µL of TFP-functionalized MPs for a 45 min incubation at room temperature. After magnetic separation and thorough washing by the washing buffer thrice, the TFP-functionalized MPs was re-suspended into 100 µL of 10 mM PBS (pH 7.4). Subsequently, the suspension was mixed with 100 µL of FITC labeled magainin II at 20.0 µg⋅mL − 1 for another 45 min incubation at room temperature. Finally, after the MPs complex was separated, the supernatant solution was moved into a 96-well microplate to obtain the FL signal with the excitation wavelength and emission wavelength of 488 nm and 525 nm, respectively. 2.8. AST of P. aeruginosa The stock solution of antibiotics were prepared according to Performance Standards for Antimicrobial Susceptibility Testing of Clinical and Laboratory Standards Institute (CLSI) M100S (29th Edition). Five hundred microliters of bacterial suspensions were mixed with the same volume of serial concentrations of antibiotics solution at 37 °C for 2 h. With the above RAP for P. aeruginosa detection, the susceptibility of P. aeruginosa was assessed by the change of bacterial concentrations which was measured by the FL signal. 3. Results And Discussion 3.1. The principle of RAP for P. aeruginosa detection As shown in Fig. 1 , we developed a RAP for P. aeruginosa detection. Nonlytic recombinant TFP was functionalized on the MPs to specifically capture P. aeruginosa through specific interaction between TFP and the lipopolysaccharide on the bacterial cell wall 24 , 25 . FITC labeled magainin II anchoring on the cytoplasmic membrane of both G-positive and G-negative bacteria was utilized as the fluorescent tracer 26 . When P. aeruginosa was captured by the TFP-functionalized MPs to form bacteria-MPs complex, quantitative excess FITC labeled magainin II was added with these complex. After FITC-magainin II-bacteria-MPs complex was magnetically separated, the supernatant solution was transferred into the microplate to obtain the FL intensity. The changed values of FL intensity (ΔFL) was utilized to quantitate P. aeruginosa , where ΔFL was defined as the following equation: ΔFL = FL blank sample – FL sample . Compared to direct assaying protocol, RAP can solve the blocking effects of the MPs to the intensity of FITC since the size of MPs was much bigger than that of FITC. When the FITC-magainin II-bacteria-MPs complex was formed, only a few fraction of fluorescent tracer which was on the side of exciting light of the complex can be triggered to emit the FL signals. To testify the coinstantaneous binding capability of TFP and magainin II to P. aeruginosa , TRITC labeled TFP and FITC labeled magainin II were simultaneously mixed with P. aeruginosa to stain the cells of P. aeruginosa . As shown in Fig. 2 , red FL from TRITC and green FL from FITC can be both observed on the surface of P. aeruginosa cells. This phenomenon demonstrated that TFP and magainin II can simultaneously bind with P. aeruginosa at different sites to form the sandwich complex. 3.2. Characterization of capture of by TFP-functionalized MPs To investigate the capture capacity of TFP-functionalized MPs to P. aeruginosa , SEM was utilized to observe the capture behavior of TFP-functionalized MPs. As shown in Fig. 3 , compared to the bare MPs, P. aeruginosa cells were bound and observed on the surface of TFP-functionalized MPs. This demonstrated that after functionalized on MPs TFP remained the binding capacity to P. aeruginosa . 3.3. Condition optimization of P. aeruginosa detection To enhance the sensitivity of RAP for P. aeruginosa detection, the following parameters were evaluated including (1) the amount of TFP-functionalized MPs, (2) the incubation time for P. aeruginosa and FITC labeled magainin II, (3) the concentration of FITC labeled magainin II. As illustrated in Figure S1-S3, the values of ΔFL reached optimal when the chosen parameters were as follows: (1) 10 µL of TFP-functionalized MPs; (2) 45 min incubation time for P. aeruginosa ; (3) 45 min incubation time for FITC labeled magainin II; and (4) 20.0 µg⋅mL − 1 of FITC labeled magainin II. 3.4. Detection performance Under the optimal experimental conditions, RAP for P. aeruginosa detection showed a linear range of 1.0 × 10 2 to 1.0 × 10 6 CFU⋅mL − 1 with the detection limit of 3.3 × 10 CFU⋅mL − 1 . The regression equation was lg ΔFL (a.u.) = 1.41 + 0.404 lg C (CFU⋅mL − 1 ) with a correlation coefficient of 0.9952 (Figure S4). Here ΔFL and C represent the changed values of FL intensity and the concentration of P. aeruginosa . The relative standard deviation (RSD) values at low (1.0 × 10 2 CFU⋅mL − 1 ), medium (1.0 × 10 4 CFU⋅mL − 1 ) and high (1.0 × 10 6 CFU⋅mL − 1 ) concentrations were 6.44%, 3.67% and 2.18%, respectively. This results demonstrated RAP showed acceptable repeatability. 3.5. Specificity The specificity of RAP was investigated by selecting three Gram-negative bacteria ( E. coli , P. solanacearum and S. tyhimurium ) and three Gram-positive bacteria ( S. aureus , S. epidermidis and S. mutans ). The concentrations of these interference bacteria were all 1.0 × 10 5 CFU⋅mL − 1 for the specificity investigation. The specificity of RAP was calculated by the designed interference degree (ID) values of the above interference bacteria in the following equation. ID = ΔFL interference bacteria / ΔFL P. aeruginosa ⊆100% (1) As illustrated in Fig. 4 , the ID values of the tested interference bacteria were all below 5.26%. For the further investigation of potential interference to P. aeruginosa detection, Mixture A was composed of all the six interference bacteria and Mixture B was prepared by mixing P. aeruginosa with Mixture A. The ID value of Mixture A was 4.37%. Compared to that of P. aeruginosa , the ΔFL intensity of Mixture B showed the minor difference (3.14%). Therefore, RAP for P. aeruginosa detection showed good specificity. 3.6. Practical sample detection To investigate the potential application of this RAP for P. aeruginosa detection, 5% glucose injection, rat plasma and human urine were spiked with P. aeruginosa suspension at given concentrations. As shown in Table 1, the recovery values ranged from 90.1–104.2%, with the RSD all below 5.0%. This results demonstrated the reliability of RAP for detecting P. aeruginosa in complicated matrix. Table 1 Recovery tests for P. aeruginosa detection spiked in practical samples ( n = 4). Sample Spiked (CFU mL − 1 ) Recovery (%) RSD (%) Glucose injection 1.0 × 10 6 104.2 3.2 1.0 × 10 5 95.6 3.1 1.0 × 10 4 98.7 2.8 1.0 × 10 3 93.4 4.6 Human urine 1.0 × 10 6 97.6 4.5 1.0 × 10 5 98.9 4.7 1.0 × 10 4 93.9 2.7 1.0 × 10 3 92.2 5.0 Rat plasma 1.0 × 10 6 101.6 2.1 1.0 × 10 5 93.5 3.8 1.0 × 10 4 97.2 4.6 1.0 × 10 3 90.1 3.9 The AST results of this protocol and CLSI data for P. aeruginosa (ATCC 27853). S: susceptible, R: resistant, I: intermediate. 3.7. AST of P. aeruginosa AST of P. aeruginosa was evaluated by detecting the ΔFL signals of 1.0 ⊆ 10 5 CFU⋅mL − 1 P. aeruginosa cultured with serial concentrations of antibiotics. According to the guidance of CLSI M100S, the four antibiotics of group A including PIP/TAZ, CAZ, TOB and GEN and one antibiotic of group B selected as LVX were utilized to validate the AST of P. aeruginosa to demonstrate its reliability. After P. aeruginosa was cultured with the absence (blank group, BG) and the presence (test group, TGs) of serial concentrations of antibiotics for 2 h at 37 °C, the ΔFL signals of P. aeruginosa were calculated and compared. The same amount of P. aeruginosa suspension stored at 4 °C was detected as the control group (CPs). Since P. aeruginosa at 4 °C grew extremely slowly, the concentrations of P. aeruginosa was considered as remaining almost unchanged. The results of AST were obtained through comparing the ΔFL signals of TGs with those of CGs and TGs. For the AST of P. aeruginosa to PIP/TAZ, at the concentration range from 16/4 to 128/4 µg⋅mL − 1 , the ΔFL signals of TGs were about 99.7% and 29.7% of those of CGs and BGs, respectively (Fig. 5 A). As shown in Fig. 5 B- 5 D, the similar results were also found for CAZ (8–32 µg⋅mL − 1 ), TOB (4–16 µg⋅mL − 1 ) and GEN (4–16 µg⋅mL − 1 ). This results demonstrated under the effect of these antibiotics concentration the growth of P. aeruginosa was significantly inhibited. The minimum inhibitory concentrations (MICs) of PIP/TAZ, CAZ, TOB and GEN were < 16/4, < 8, < 4 and < 4 µg⋅mL − 1 , respectively. According to the guidance of CLSI M100S (Table S1), P. aeruginosa was susceptible to these four antibiotics (Table 2 ). For the AST of P. aeruginosa to LVX, when the concentrations were 1 and 2 µg⋅mL − 1 , the ΔFL signals of TGs were about 312% and 93.2% of those of CGs and BGs, respectively (Fig. 5 E). This results demonstrated that the growth of P. aeruginosa was slightly inhibited by LVX in comparison with the normal growth of P. aeruginosa (BGs). However, the concentration of LVX reached 4 µg⋅mL − 1 , the ΔFL signals of TGs reduced to about 99.7% and 29.7% of those of CGs and BGs, respectively. It shown that the growth of P. aeruginosa was significantly influenced by LVX at the concentration of 4 µg⋅mL − 1 . Therefore, the MIC of LVX was 4 µg⋅mL − 1 and P. aeruginosa was resistant to LVX (Table 2 ). Table 2 The AST results of this protocol and CLSI data for P. aeruginosa (ATCC 27853). S: susceptible, R: resistant, I: intermediate Antibiotics PIP/TAZ CAZ TOB GEN LVX Testing results MIC (μg mL -1 ) < 16/4 < 8 < 4 < 4 4 Susceptibility S S S S R CLSI data Susceptibility S S S S I or R The AST results for all the testing antibiotics were consistent with the provided data of the document of CLSI 100S. This results demonstrated that the RAP protocol showed good reliability for the AST. 4. Conclusion In conclusion, a rapid, sensitive and specific RAP using TFP and magainin II as dual recognition elements was developed to perform the detection and AST of P. aeruginosa . Since TFP can specifically recognize the target cells of P. aeruginosa from other interference bacteria, the results of AST can actually be obtained within 4 h without the time-consuming process of bacterial isolation and identification, which can facilitate the decreasing frequency of irrational empiric antibiotic therapy. Based on this proof-of-principle work, the detection and AST of other bacteria can be facilely completed by the expression of the TFP of their bacteriophages. In the future work, we will focus on further reduce the detection time of AST based on TFP recognition through other detection technique such as microfluidic system or single-cell imaging. Declarations Acknowledgements This work was financially supported by National Natural Science Foundation of China (21964023), Science and Technology Foundation of Guizhou Province (20201Y044) and Doctoral Initiation Fund of Zunyi Medical University (No. 5). Author contributions Conceived and designed the experiments: Y.H., H.Z., Y.W.L. and H.Z. Performed the experiments: Y.H., H.Z., Y.W.L. and H.Z. Wrote the paper: Y.H. and H.Z. Conflicts of interest The authors declare no conflicts of interest. References Laxminarayan, R. et al . Antibiotic resistance—the need for global solutions. Lancet Infect. Dis. 13 , 1057-1098 (2013). Woolhouse, M. & Farrar J. An intergovernmental panel on antimicrobial resistance. Nature 509 , 555-557 (2014). Lee, J. H., Park, K. S., Karim, A. M., Lee, C. R. & Lee, S. H. How to minimize antibiotic resistance. Lancet 16 , 406-407 (2016). Tillotson, G. Antimicrobial resistance: what’s needed. Lancet Infect. Dis. 15 , 758-760 (2015). Behera, B. et al . 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Supplementary Files Supportinginformation.doc Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major revision 10 Feb, 2021 Reviews received at journal 09 Feb, 2021 Reviewers agreed at journal 30 Jan, 2021 Reviewers agreed at journal 26 Jan, 2021 Reviewers invited by journal 02 Jan, 2021 Editor assigned by journal 02 Jan, 2021 Editor invited by journal 22 Dec, 2020 Submission checks completed at journal 22 Dec, 2020 First submitted to journal 17 Dec, 2020 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-130554","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":6920130,"identity":"ecf3f46f-f45e-41cf-98d1-39657c860934","order_by":0,"name":"Yong He","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyUlEQVRIiWNgGAWjYBACAziLvbHx4QfStPAcbjaWIE2LRHqbAA8xWszZewwfF/yqiza4+bCNQYLBTk63gYAWy54zxsYz+9hyN9xObHtQwJBsbHaAkMNu5JhJ8/bwgLS0G0gwHEjcRlDL/TcgLRK5G24ebJPgIUrLDR4zaZ4fBrkbbjASq+VMWrExb0NC7swzicBANiDGL8cPb3zM86cut+/48YcPP1TYyRHUwsDAYcDA2AY3gaByEGB/wMDwhyiVo2AUjIJRMFIBAEyvRgX5x8UjAAAAAElFTkSuQmCC","orcid":"","institution":"Affiliated Hospital of Zunyi Medical College","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Yong","middleName":"","lastName":"He","suffix":""},{"id":6920131,"identity":"a155d56c-9b9f-4486-9f81-417087e95f20","order_by":1,"name":"Hang Zhao","email":"","orcid":"","institution":"Affiliated Hospital of Zunyi Medical College","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hang","middleName":"","lastName":"Zhao","suffix":""},{"id":6920132,"identity":"faf5add6-df51-488d-878e-0026736bf389","order_by":2,"name":"Yuanwen Liu","email":"","orcid":"","institution":"Affiliated Hospital of Zunyi Medical College","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yuanwen","middleName":"","lastName":"Liu","suffix":""},{"id":6920133,"identity":"709ed368-3c66-41d1-8c34-9c3e1acbea04","order_by":3,"name":"He Zhou","email":"","orcid":"","institution":"Zunyi institute for food and drug control","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"He","middleName":"","lastName":"Zhou","suffix":""}],"badges":[],"createdAt":"2020-12-17 10:14:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-130554/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-130554/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":4555354,"identity":"8878d398-a556-43a6-a4b4-e43ec74821c3","added_by":"auto","created_at":"2020-12-28 20:15:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":127568,"visible":true,"origin":"","legend":"Schematic illustration of RAP for P. aeruginosa detection.","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-130554/v1/a19a5bfc4fe9e6b24c66852a.png"},{"id":4555355,"identity":"1f1193e4-aea1-49d9-a858-b8ec31e9824f","added_by":"auto","created_at":"2020-12-28 20:15:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":47329,"visible":true,"origin":"","legend":"FL microscope image of the stained P. aeruginosa. (A) bright field, (B) green FL channel, (c) red FL channel.","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-130554/v1/f0f5d1e258d9c1da6889c297.png"},{"id":4555397,"identity":"cb369e4c-8143-4e5d-82cf-6efa3b219c9b","added_by":"auto","created_at":"2020-12-28 20:18:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":231685,"visible":true,"origin":"","legend":"SEM image of (A) P. aeruginosa captured on the surface of TFP-functionalized MPs and (B) bare surface of TFP-functionalized MPs. The red arrow shows the captured P. aeruginosa.","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-130554/v1/bbb6d47a2b55c358acbe1857.png"},{"id":4555358,"identity":"b01c5787-6c67-4305-9d1f-ef34e90dada7","added_by":"auto","created_at":"2020-12-28 20:15:08","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":77632,"visible":true,"origin":"","legend":"Specificity of RAP for P. aeruginosa detection. The concentrations of all the tested bacteria were 1.0 × 105 CFU⋅mL−1 (n = 4).","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-130554/v1/af82fd1f70990548f320fc9b.png"},{"id":4555357,"identity":"60748bd7-7a68-4421-a2ed-13e6e582f7d1","added_by":"auto","created_at":"2020-12-28 20:15:07","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":264465,"visible":true,"origin":"","legend":"AST of P. aeruginosa (ATCC 27853) treated by (A) PIP/TAZ, (B) CAZ, (C) TOB, (D) GEN and (E) LVX. Background signals represent the ΔFL from PBS. # signifies the P. aeruginosa suspension was kept at 4 °C before the performance of AST (n = 4).","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-130554/v1/ac921185e75634b3cbdf8cc0.png"},{"id":15671446,"identity":"833280cc-5b77-4cd7-8342-ac7b6f905aeb","added_by":"auto","created_at":"2021-11-18 14:06:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1120902,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-130554/v1/b8c811fc-15f8-4d6c-9e49-74994224cccc.pdf"},{"id":4555232,"identity":"54e717ba-41ed-4410-8120-f6367cfb6229","added_by":"auto","created_at":"2020-12-28 20:12:07","extension":"doc","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":179200,"visible":true,"origin":"","legend":"","description":"","filename":"Supportinginformation.doc","url":"https://assets-eu.researchsquare.com/files/rs-130554/v1/df9bd0d51c44ba9aca3bcad8.doc"}],"financialInterests":"","formattedTitle":"\u003cp\u003eSpecific and Rapid Reverse Assaying Protocol for Detection and Antimicrobial Susceptibility Testing of \u003cem\u003ePseudomonas Aeruginosa\u003c/em\u003e based on Bacteriophage Tail Fiber Protein and Magainin II Recognition\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":" \u003cp\u003eAntimicrobial resistance (AMR) is a worldwide health crisis resulting in growing economic burden and increasing mortality\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The most important strategies to minimize AMR are the development of rapid and accurate diagnostic protocols for antimicrobial susceptibility testing (AST), which facilitating the timing of prescription to use the effective antibiotics\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Therefore, numerous efforts are exerted on developing rapid, sensitive and acute detection and AST for bacterial infection.\u003c/p\u003e \u003cp\u003eTraditional bacterial growth-based protocols are considered as the gold standard methods for bacterial detection and AST. These protocols show ideal repeatability, high standardization and good reliability. However, the isolation and identification procedure of bacterial detection usually require tedious process of 24\u0026ndash;48 h\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. The subsequent broth dilution or disk diffusion for AST performance require another bacterial growth cultured with given concentrations of various antibiotics, which demands almost another 24\u0026ndash;48 h\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Lack of timely and accurate results of bacterial AST results in frequent empiric antibiotic therapy, causing irrational antibiotic use and the development of AMR\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Therefore, some other bacterial growth-based protocols are reported to aim at shortening the time of AST, such as microscopy detection\u003csup\u003e\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, electrochemical sensor\u003csup\u003e\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, phase-shift spectroscopy detection\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, fluorescent detection\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e and microfluidic devices based on slipchip technique\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, surface-enhanced raman scattering\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Nevertheless, since lack of capacity of isolating the given bacterial species, they also require time-consuming pretreatment procedure for bacterial culture, isolation and identification. Polymerase chain reaction-based protocols gradually attract attention to bacterial detection and performance of AST due to the advantages of rapidity, sensitivity and culture-free process\u003csup\u003e\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. However, they suffer from well-trained personnel, complicated molecular manipulation, prerequisite precise resistant gene information and frequent gene mutation.\u003c/p\u003e \u003cp\u003eWith the increasing global AMR, bacteriophages as the natural enemies to the bacteria have regained interest in solving the crisis caused by multi-resistant bacteria. Each strain of bacteria has one or more corresponding bacteriophages. Bacteriophages can highly specifically recognize their target bacteria even in harsh environment. Typical lifecycle of virulent bacteriophage involves the following steps: specific absorption on the bacterial cell wall, DNA injection into the bacterial cell, progenies replication and the lysis of bacteria cell for releasing progenies. Bacteriophages functional proteins (BFP) such as tail fiber protein (TFP), tailspike protein (TSP) and endolysin are the essential recognition elements which are responsible for absorption, injection and lysis, respectively\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Therefore, BFPs assumes the ideal molecular recognition attributes of high specificity, robustness, good anti-interference capability and universality to each bacterium\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Unfortunately, just like the bacteriophage entity, TSP and endolysin both have the inherent lytic activity which could be unfavorable for bacteria capture and sample manipulation.\u003c/p\u003e \u003cp\u003eIn the previous work, we had utilized to the \u003cem\u003eEscherichia coli\u003c/em\u003e (\u003cem\u003eE. coli\u003c/em\u003e) expression system to produce the TFP of \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e (\u003cem\u003eP. aeruginosa\u003c/em\u003e)\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. This recombinant TFP can specifically recognize \u003cem\u003eP. aeruginosa\u003c/em\u003e without lytic activity. To investigate the application of TFP in AST detection, TFP-functionalized magnetic particles were utilized to specifically capture \u003cem\u003eP. aeruginosa\u003c/em\u003e, and fluorescein isothiocyanate (FITC) labeled magainin II was utilized as the fluorescent tracer. A reverse assaying protocol (RAP) combined magnetic separation was developed to specific, rapid and sensitive detection and AST of \u003cem\u003eP. aeruginosa\u003c/em\u003e.\u003c/p\u003e "},{"header":"2. Experimental","content":" \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Instrumentations\u003c/h2\u003e \u003cp\u003eScanning electron micrograph (SEM) was recorded by an S-3000N scanning electron microscope (Hitachi, Japan). Fluorescence (FL) signals were obtained from an Infinite M200 PRO microplate reader (TECAN, Switzerland). FL micrographs were recorded by using a NI\u0026thinsp;\u0026minus;\u0026thinsp;U FL microscope (Nikon, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Reagents and materials\u003c/h2\u003e \u003cp\u003ePiperacillin/Tazobactam (PIP/TAZ), ceftazidime (CAZ), tobramycin (TOB), gentamicin (GEN) and levofloxacin (LVX) were all purchased from Solarbio Life Sciences (China). Strains of \u003cem\u003eP. aeruginosa\u003c/em\u003e, \u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003ePseudomonas solanacearum\u003c/em\u003e (\u003cem\u003eP. solanacearum\u003c/em\u003e), \u003cem\u003eSalmonella typhimurium\u003c/em\u003e (\u003cem\u003eS. tyhimurium\u003c/em\u003e), \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (\u003cem\u003eS. aureus\u003c/em\u003e), \u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e (\u003cem\u003eS. epidermidis\u003c/em\u003e), \u003cem\u003eStreptococcus mutans\u003c/em\u003e (\u003cem\u003eS. mutans\u003c/em\u003e) were purchased from Guangdong Microbiology Culture Center (China). FITC labeled magainin II were obtained from GL Biochem (Shanghai) Ltd.. (China). Tetraethyl rhodamine isothiocyanate (TRITC) and gelatin were provided by Sigma-Aldrich (USA, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"http://www.sigma-aldrich.com\" target=\"_blank\"\u003ewww.sigma-aldrich.com\u003c/a\u003e\u003c/span\u003e\u003c/span\u003e). AffiAmino magnetic particles (MPs) were purchased from Lab on a Bead AB (Sweden) in which activation buffer and blocking buffer were provided. Both human urine collected from the authors and rat plasma gifted by another lab were used as the common matrices. Luria\u0026thinsp;\u0026minus;\u0026thinsp;Bertani (LB) broth consisted of 10 g\u0026sdot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NaCl, 5 g\u0026sdot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e yeast extract and 10\u0026nbsp;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e tryptone. Washing buffer was composed of 10\u0026nbsp;mM PBS (pH 7.4) and 0.5% tween-20. Human beings aren\u0026rsquo;t involved in the study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Bacterial culture and counting\u003c/h2\u003e \u003cp\u003eStrains of \u003cem\u003eP. aeruginosa\u003c/em\u003e and other bacteria were grown in 30\u0026nbsp;mL of LB broth with continuous shaking at 80\u0026nbsp;rpm and 37\u0026nbsp;\u0026deg;C under aerobic condition until the value of OD\u003csub\u003e600\u003c/sub\u003e reached 1.0. For bacterial detection, 10\u0026nbsp;mM PBS (pH 7.4) was utilized to serially dilute the bacterial culture to reach the target concentration. The bacterial concentrations were evaluated according to standard approach of bacterial culture and counting.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Preparation procedure of TRITC-labeled TFP\u003c/h2\u003e \u003cp\u003eOne milliliter of TFP solution at the concentration of 1.0 mg\u0026sdot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was slowly mixed with 1.0\u0026nbsp;mL of TRITC solution at 3.0 mg\u0026sdot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e dissolved in dimethyl sulfoxide, followed by 12-h reaction at 4\u0026nbsp;\u0026deg;C. Subsequently, NH\u003csub\u003e4\u003c/sub\u003eCl solution was used to stop the reaction. Finally, the solution was dialyzed with 10\u0026nbsp;mM PBS (pH 7.4) for 48\u0026nbsp;h at room temperature.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Fluorescent microscope image of stained P. aeruginosa\u003c/h2\u003e \u003cp\u003eOne milliliter of \u003cem\u003eP. aeruginosa\u003c/em\u003e suspension at 1.0 \u0026sube; 10\u003csup\u003e6\u003c/sup\u003e CFU\u0026sdot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was added with 200 \u0026micro;L of FITC labeled magainin II at 20.0 \u0026micro;g\u0026sdot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and the equal volume of TRITC labeled TFP. After 1-h incubation at room temperature, the suspension was centrifuged at 2500\u0026nbsp;\u003cem\u003eg\u003c/em\u003e and washed thrice, followed by resuspended in 10\u0026nbsp;mM PBS (pH 7.4). Subsequently, 10 \u0026micro;L of the stained \u003cem\u003eP. aeruginosa\u003c/em\u003e suspension was observed by a FL microscope with the magnificence of 1000. The excitation wavelength of TRITC and FITC were 544\u0026nbsp;nm and 488\u0026nbsp;nm, respectively, and the emission wavelength of TRITC and FITC were 570\u0026nbsp;nm and 525\u0026nbsp;nm, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Preparation procedure of TFP-functionalized MPs\u003c/h2\u003e \u003cp\u003eTFP-functionalized MPs were obtained according to manufacturer guideline. Briefly, after washed by washing buffer, 100 \u0026micro;L of MPs was resuspended into the 1.0\u0026nbsp;mL of washing buffer. Subsequently, 50 \u0026micro;L of the activation buffer was mixed with the above suspension for 15\u0026nbsp;min activation. After the MPs were thoroughly washed, 1.0\u0026nbsp;mL of TFP solution at 100 \u0026micro;g\u0026sdot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was added for 1-h reaction at room temperature, followed by another thorough washing. Then the residue active sites of MPs were blocked with 80 \u0026micro;L of the blocking buffer and 1% gelatin at room temperature for 45\u0026nbsp;min. Finally, after washed thrice the TFP-functionalized MPS were stored in 10\u0026nbsp;mM PBS (pH 7.4) at 4\u0026nbsp;\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. RAP for P. aeruginosa\u003c/h2\u003e \u003cp\u003eOne milliliter of \u003cem\u003eP. aeruginosa\u003c/em\u003e suspension was added with 10 \u0026micro;L of TFP-functionalized MPs for a 45\u0026nbsp;min incubation at room temperature. After magnetic separation and thorough washing by the washing buffer thrice, the TFP-functionalized MPs was re-suspended into 100 \u0026micro;L of 10\u0026nbsp;mM PBS (pH 7.4). Subsequently, the suspension was mixed with 100 \u0026micro;L of FITC labeled magainin II at 20.0 \u0026micro;g\u0026sdot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for another 45\u0026nbsp;min incubation at room temperature. Finally, after the MPs complex was separated, the supernatant solution was moved into a 96-well microplate to obtain the FL signal with the excitation wavelength and emission wavelength of 488\u0026nbsp;nm and 525\u0026nbsp;nm, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. AST of P. aeruginosa\u003c/h2\u003e \u003cp\u003eThe stock solution of antibiotics were prepared according to Performance Standards for Antimicrobial Susceptibility Testing of Clinical and Laboratory Standards Institute (CLSI) M100S (29th Edition). Five hundred microliters of bacterial suspensions were mixed with the same volume of serial concentrations of antibiotics solution at 37\u0026nbsp;\u0026deg;C for 2\u0026nbsp;h. With the above RAP for \u003cem\u003eP. aeruginosa\u003c/em\u003e detection, the susceptibility of \u003cem\u003eP. aeruginosa\u003c/em\u003e was assessed by the change of bacterial concentrations which was measured by the FL signal.\u003c/p\u003e \u003c/div\u003e "},{"header":"3. Results And Discussion","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n\u003ch2\u003e3.1. The principle of RAP for P. aeruginosa detection\u003c/h2\u003e\n\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, we developed a RAP for \u003cem\u003eP. aeruginosa\u003c/em\u003e detection. Nonlytic recombinant TFP was functionalized on the MPs to specifically capture \u003cem\u003eP. aeruginosa\u003c/em\u003e through specific interaction between TFP and the lipopolysaccharide on the bacterial cell wall\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. FITC labeled magainin II anchoring on the cytoplasmic membrane of both G-positive and G-negative bacteria was utilized as the fluorescent tracer\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. When \u003cem\u003eP. aeruginosa\u003c/em\u003e was captured by the TFP-functionalized MPs to form bacteria-MPs complex, quantitative excess FITC labeled magainin II was added with these complex. After FITC-magainin II-bacteria-MPs complex was magnetically separated, the supernatant solution was transferred into the microplate to obtain the FL intensity. The changed values of FL intensity (\u0026Delta;FL) was utilized to quantitate \u003cem\u003eP. aeruginosa\u003c/em\u003e, where \u0026Delta;FL was defined as the following equation: \u0026Delta;FL\u0026thinsp;=\u0026thinsp;FL\u003csub\u003eblank sample\u003c/sub\u003e \u0026ndash; FL\u003csub\u003esample\u003c/sub\u003e. Compared to direct assaying protocol, RAP can solve the blocking effects of the MPs to the intensity of FITC since the size of MPs was much bigger than that of FITC. When the FITC-magainin II-bacteria-MPs complex was formed, only a few fraction of fluorescent tracer which was on the side of exciting light of the complex can be triggered to emit the FL signals.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo testify the coinstantaneous binding capability of TFP and magainin II to \u003cem\u003eP. aeruginosa\u003c/em\u003e, TRITC labeled TFP and FITC labeled magainin II were simultaneously mixed with \u003cem\u003eP. aeruginosa\u003c/em\u003e to stain the cells of \u003cem\u003eP. aeruginosa\u003c/em\u003e. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, red FL from TRITC and green FL from FITC can be both observed on the surface of \u003cem\u003eP. aeruginosa\u003c/em\u003e cells. This phenomenon demonstrated that TFP and magainin II can simultaneously bind with \u003cem\u003eP. aeruginosa\u003c/em\u003e at different sites to form the sandwich complex.\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n\u003ch2\u003e3.2. Characterization of capture of by TFP-functionalized MPs\u003c/h2\u003e\n\u003cp\u003eTo investigate the capture capacity of TFP-functionalized MPs to \u003cem\u003eP. aeruginosa\u003c/em\u003e, SEM was utilized to observe the capture behavior of TFP-functionalized MPs. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, compared to the bare MPs, \u003cem\u003eP. aeruginosa\u003c/em\u003e cells were bound and observed on the surface of TFP-functionalized MPs. This demonstrated that after functionalized on MPs TFP remained the binding capacity to \u003cem\u003eP. aeruginosa\u003c/em\u003e.\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n\u003ch2\u003e3.3. Condition optimization of P. aeruginosa detection\u003c/h2\u003e\n\u003cp\u003eTo enhance the sensitivity of RAP for \u003cem\u003eP. aeruginosa\u003c/em\u003e detection, the following parameters were evaluated including (1) the amount of TFP-functionalized MPs, (2) the incubation time for \u003cem\u003eP. aeruginosa\u003c/em\u003e and FITC labeled magainin II, (3) the concentration of FITC labeled magainin II. As illustrated in Figure S1-S3, the values of \u0026Delta;FL reached optimal when the chosen parameters were as follows: (1) 10 \u0026micro;L of TFP-functionalized MPs; (2) 45\u0026nbsp;min incubation time for \u003cem\u003eP. aeruginosa\u003c/em\u003e; (3) 45\u0026nbsp;min incubation time for FITC labeled magainin II; and (4) 20.0 \u0026micro;g\u0026sdot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of FITC labeled magainin II.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n\u003ch2\u003e3.4. Detection performance\u003c/h2\u003e\n\u003cp\u003eUnder the optimal experimental conditions, RAP for \u003cem\u003eP. aeruginosa\u003c/em\u003e detection showed a linear range of 1.0\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e2\u003c/sup\u003e to 1.0\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e6\u003c/sup\u003e CFU\u0026sdot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e with the detection limit of 3.3\u0026thinsp;\u0026times;\u0026thinsp;10 CFU\u0026sdot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The regression equation was lg \u003cem\u003e\u0026Delta;FL\u003c/em\u003e (a.u.)\u0026thinsp;=\u0026thinsp;1.41\u0026thinsp;+\u0026thinsp;0.404 lg \u003cem\u003eC\u003c/em\u003e (CFU\u0026sdot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) with a correlation coefficient of 0.9952 (Figure S4). Here \u003cem\u003e\u0026Delta;FL\u003c/em\u003e and \u003cem\u003eC\u003c/em\u003e represent the changed values of FL intensity and the concentration of \u003cem\u003eP. aeruginosa\u003c/em\u003e. The relative standard deviation (RSD) values at low (1.0\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e2\u003c/sup\u003e CFU\u0026sdot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), medium (1.0\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e4\u003c/sup\u003e CFU\u0026sdot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and high (1.0\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e6\u003c/sup\u003e CFU\u0026sdot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) concentrations were 6.44%, 3.67% and 2.18%, respectively. This results demonstrated RAP showed acceptable repeatability.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n\u003ch2\u003e3.5. Specificity\u003c/h2\u003e\n\u003cp\u003eThe specificity of RAP was investigated by selecting three Gram-negative bacteria (\u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003eP. solanacearum\u003c/em\u003e and \u003cem\u003eS. tyhimurium\u003c/em\u003e) and three Gram-positive bacteria (\u003cem\u003eS. aureus\u003c/em\u003e, \u003cem\u003eS. epidermidis\u003c/em\u003e and \u003cem\u003eS. mutans\u003c/em\u003e). The concentrations of these interference bacteria were all 1.0\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e5\u003c/sup\u003e CFU\u0026sdot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for the specificity investigation. The specificity of RAP was calculated by the designed interference degree (ID) values of the above interference bacteria in the following equation.\u003c/p\u003e\n\u003cp\u003eID\u0026thinsp;=\u0026thinsp;\u003cem\u003e\u0026Delta;FL\u003c/em\u003e\u003csub\u003einterference bacteria\u003c/sub\u003e/\u003cem\u003e\u0026Delta;FL\u003c/em\u003e\u003csub\u003e\u003cem\u003eP. aeruginosa\u003c/em\u003e\u003c/sub\u003e\u0026sube;100% (1)\u003c/p\u003e\n\u003cp\u003eAs illustrated in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e, the ID values of the tested interference bacteria were all below 5.26%. For the further investigation of potential interference to \u003cem\u003eP. aeruginosa\u003c/em\u003e detection, Mixture A was composed of all the six interference bacteria and Mixture B was prepared by mixing \u003cem\u003eP. aeruginosa\u003c/em\u003e with Mixture A. The ID value of Mixture A was 4.37%. Compared to that of \u003cem\u003eP. aeruginosa\u003c/em\u003e, the \u0026Delta;FL intensity of Mixture B showed the minor difference (3.14%). Therefore, RAP for \u003cem\u003eP. aeruginosa\u003c/em\u003e detection showed good specificity.\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n\u003ch2\u003e3.6. Practical sample detection\u003c/h2\u003e\n\u003cp\u003eTo investigate the potential application of this RAP for \u003cem\u003eP. aeruginosa\u003c/em\u003e detection, 5% glucose injection, rat plasma and human urine were spiked with \u003cem\u003eP. aeruginosa\u003c/em\u003e suspension at given concentrations. As shown in Table\u0026nbsp;1, the recovery values ranged from 90.1\u0026ndash;104.2%, with the RSD all below 5.0%. This results demonstrated the reliability of RAP for detecting \u003cem\u003eP. aeruginosa\u003c/em\u003e in complicated matrix.\u003c/p\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u0026nbsp;\u003c/div\u003e\n\u003cdiv class=\"CaptionNumber\"\u003e\n\u003cp\u003eRecovery tests for \u003cem\u003eP. aeruginosa\u003c/em\u003e detection spiked in practical samples (\u003cem\u003en \u003c/em\u003e= 4).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eSample\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eSpiked (CFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eRecovery (%)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eRSD (%)\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGlucose injection\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026times;\"\u003e\n\u003cp\u003e1.0\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e104.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e3.2\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026times;\"\u003e\n\u003cp\u003e1.0\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e95.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e3.1\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026times;\"\u003e\n\u003cp\u003e1.0\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e98.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e2.8\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026times;\"\u003e\n\u003cp\u003e1.0\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e93.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e4.6\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHuman urine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026times;\"\u003e\n\u003cp\u003e1.0\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e97.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e4.5\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026times;\"\u003e\n\u003cp\u003e1.0\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e98.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e4.7\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026times;\"\u003e\n\u003cp\u003e1.0\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e93.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e2.7\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026times;\"\u003e\n\u003cp\u003e1.0\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e92.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e5.0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eRat plasma\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026times;\"\u003e\n\u003cp\u003e1.0\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e101.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e2.1\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026times;\"\u003e\n\u003cp\u003e1.0\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e93.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e3.8\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026times;\"\u003e\n\u003cp\u003e1.0\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e97.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e4.6\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026times;\"\u003e\n\u003cp\u003e1.0\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e90.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e3.9\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003ctfoot\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"4\"\u003eThe AST results of this protocol and CLSI data for \u003cem\u003eP. aeruginosa\u003c/em\u003e (ATCC 27853). S: susceptible, R: resistant, I: intermediate.\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tfoot\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n\u003ch2\u003e3.7. AST of P. aeruginosa\u003c/h2\u003e\n\u003cp\u003eAST of \u003cem\u003eP. aeruginosa\u003c/em\u003e was evaluated by detecting the \u0026Delta;FL signals of 1.0 \u0026sube; 10\u003csup\u003e5\u003c/sup\u003e CFU\u0026sdot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e \u003cem\u003eP. aeruginosa\u003c/em\u003e cultured with serial concentrations of antibiotics. According to the guidance of CLSI M100S, the four antibiotics of group A including PIP/TAZ, CAZ, TOB and GEN and one antibiotic of group B selected as LVX were utilized to validate the AST of \u003cem\u003eP. aeruginosa\u003c/em\u003e to demonstrate its reliability. After \u003cem\u003eP. aeruginosa\u003c/em\u003e was cultured with the absence (blank group, BG) and the presence (test group, TGs) of serial concentrations of antibiotics for 2\u0026nbsp;h at 37\u0026nbsp;\u0026deg;C, the \u0026Delta;FL signals of \u003cem\u003eP. aeruginosa\u003c/em\u003e were calculated and compared. The same amount of \u003cem\u003eP. aeruginosa\u003c/em\u003e suspension stored at 4\u0026nbsp;\u0026deg;C was detected as the control group (CPs). Since \u003cem\u003eP. aeruginosa\u003c/em\u003e at 4\u0026nbsp;\u0026deg;C grew extremely slowly, the concentrations of \u003cem\u003eP. aeruginosa\u003c/em\u003e was considered as remaining almost unchanged. The results of AST were obtained through comparing the \u0026Delta;FL signals of TGs with those of CGs and TGs.\u003c/p\u003e\n\u003cp\u003eFor the AST of \u003cem\u003eP. aeruginosa\u003c/em\u003e to PIP/TAZ, at the concentration range from 16/4 to 128/4 \u0026micro;g\u0026sdot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, the \u0026Delta;FL signals of TGs were about 99.7% and 29.7% of those of CGs and BGs, respectively (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA). As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB-\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD, the similar results were also found for CAZ (8\u0026ndash;32 \u0026micro;g\u0026sdot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), TOB (4\u0026ndash;16 \u0026micro;g\u0026sdot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and GEN (4\u0026ndash;16 \u0026micro;g\u0026sdot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). This results demonstrated under the effect of these antibiotics concentration the growth of \u003cem\u003eP. aeruginosa\u003c/em\u003e was significantly inhibited. The minimum inhibitory concentrations (MICs) of PIP/TAZ, CAZ, TOB and GEN were \u0026lt;\u0026thinsp;16/4, \u0026lt; 8, \u0026lt; 4 and \u0026lt;\u0026thinsp;4 \u0026micro;g\u0026sdot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively. According to the guidance of CLSI M100S (Table S1), \u003cem\u003eP. aeruginosa\u003c/em\u003e was susceptible to these four antibiotics (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). For the AST of \u003cem\u003eP. aeruginosa\u003c/em\u003e to LVX, when the concentrations were 1 and 2 \u0026micro;g\u0026sdot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, the \u0026Delta;FL signals of TGs were about 312% and 93.2% of those of CGs and BGs, respectively (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eE). This results demonstrated that the growth of \u003cem\u003eP. aeruginosa\u003c/em\u003e was slightly inhibited by LVX in comparison with the normal growth of \u003cem\u003eP. aeruginosa\u003c/em\u003e (BGs). However, the concentration of LVX reached 4 \u0026micro;g\u0026sdot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, the \u0026Delta;FL signals of TGs reduced to about 99.7% and 29.7% of those of CGs and BGs, respectively. It shown that the growth of \u003cem\u003eP. aeruginosa\u003c/em\u003e was significantly influenced by LVX at the concentration of 4 \u0026micro;g\u0026sdot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Therefore, the MIC of LVX was 4 \u0026micro;g\u0026sdot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and \u003cem\u003eP. aeruginosa\u003c/em\u003e was resistant to LVX (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eThe AST results of this protocol and CLSI data for \u003cem\u003eP. aeruginosa \u003c/em\u003e(ATCC 27853). S: susceptible, R: resistant, I: intermediate\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"2\" width=\"253\"\u003e\n\u003cp\u003eAntibiotics\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003ePIP/TAZ\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003eCAZ\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003eTOB\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003eGEN\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003eLVX\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" width=\"132\"\u003e\n\u003cp\u003eTesting results\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"121\"\u003e\n\u003cp\u003eMIC (\u0026mu;g mL\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e\u0026lt; 16/4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003e\u0026lt; 8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003e\u0026lt; 4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003e\u0026lt; 4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e4\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"121\"\u003e\n\u003cp\u003eSusceptibility\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eS\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003eS\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003eS\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003eS\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003eR\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"132\"\u003e\n\u003cp\u003eCLSI data\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"121\"\u003e\n\u003cp\u003eSusceptibility\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eS\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003eS\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003eS\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003eS\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003eI or R\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eThe AST results for all the testing antibiotics were consistent with the provided data of the document of CLSI 100S. This results demonstrated that the RAP protocol showed good reliability for the AST.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e"},{"header":"4. Conclusion","content":" \u003cp\u003eIn conclusion, a rapid, sensitive and specific RAP using TFP and magainin II as dual recognition elements was developed to perform the detection and AST of \u003cem\u003eP. aeruginosa\u003c/em\u003e. Since TFP can specifically recognize the target cells of \u003cem\u003eP. aeruginosa\u003c/em\u003e from other interference bacteria, the results of AST can actually be obtained within 4\u0026nbsp;h without the time-consuming process of bacterial isolation and identification, which can facilitate the decreasing frequency of irrational empiric antibiotic therapy. Based on this proof-of-principle work, the detection and AST of other bacteria can be facilely completed by the expression of the TFP of their bacteriophages. In the future work, we will focus on further reduce the detection time of AST based on TFP recognition through other detection technique such as microfluidic system or single-cell imaging.\u003c/p\u003e "},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eThis work was financially supported by National Natural Science Foundation of China (21964023), Science and Technology Foundation of Guizhou Province (20201Y044) and Doctoral Initiation Fund of Zunyi Medical University (No. 5).\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eAuthor contributions\u003c/h2\u003e\n\u003cp\u003eConceived and designed the experiments: Y.H., H.Z., Y.W.L. and H.Z. Performed the experiments: Y.H., H.Z., Y.W.L. and H.Z. Wrote the paper: Y.H. and H.Z.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eConflicts of interest\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLaxminarayan, R. \u003cem\u003eet al\u003c/em\u003e. Antibiotic resistance\u0026mdash;the need for global solutions. \u003cem\u003eLancet Infect. Dis.\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 1057-1098 (2013).\u003c/li\u003e\n\u003cli\u003eWoolhouse, M. \u0026amp; Farrar J. An intergovernmental panel on antimicrobial resistance. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e509\u003c/strong\u003e, 555-557 (2014).\u003c/li\u003e\n\u003cli\u003eLee, J. H., Park, K. S., Karim, A. M., Lee, C. R. \u0026amp; Lee, S. H. How to minimize antibiotic resistance. \u003cem\u003eLancet\u003c/em\u003e \u003cstrong\u003e16\u003c/strong\u003e, 406-407 (2016).\u003c/li\u003e\n\u003cli\u003eTillotson, G. 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W. \u0026amp; Schneider, G. Designing antimicrobial peptides: form follows function. \u003cem\u003eNat. Rev. Drug. Discov.\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 37-51 (2012).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Antimicrobial resistance, Antimicrobial susceptibility testing, Bacteriophage tail fiber protein, Magainin II, Pseudomonas aeruginosa ","lastPublishedDoi":"10.21203/rs.3.rs-130554/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-130554/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe worldwide emergence and spread of antimicrobial resistance are accelerated by irrational administration and use of empiric antibiotic. A key point to the crisis is lack of rapid diagnostic protocol to antimicrobial susceptibility testing (AST) for timely and rational antibiotic prescription. Here, bacteriophage tail fiber protein (TFP) recombined in \u003cem\u003eEscherichia coli\u003c/em\u003e expression system was functionalized on magnetic particles (MPs) to specifically capture \u003cem\u003eP. aeruginosa\u003c/em\u003e, and FITC-labeled-magainin II was utilized as the indicator. For solving the MPs\u0026rsquo; blocking effects, a reverse assaying protocol (RAP) based on TFP recognition was investigated the feasibility of detection and AST of \u003cem\u003eP. aeruginosa\u003c/em\u003e. \u003cem\u003eP. aeruginosa\u003c/em\u003e detection can be rapidly, sensitively and specifically detected within 1.5\u0026nbsp;h with a linear range of 1.0\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e2\u003c/sup\u003e to 1.0\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e6\u003c/sup\u003e CFU\u0026sdot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and a detection limit of 3.3\u0026thinsp;\u0026times;\u0026thinsp;10 CFU\u0026sdot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Subsequently, the results of AST which was consistent in the results of broth dilution can be obtained within 3.5\u0026nbsp;h. Due to the high specificity of TFP, the AST can actually be conducted without the requirement of bacterial isolation and identification by this RAP. Based on the proof-of-principle work, the detection and AST of other pathogens can be extended by expressing the TFP of their bacteriophages.\u003c/p\u003e","manuscriptTitle":"Specific and Rapid Reverse Assaying Protocol for Detection and Antimicrobial Susceptibility Testing of Pseudomonas Aeruginosa based on Bacteriophage Tail Fiber Protein and Magainin II Recognition","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-12-28 20:12:05","doi":"10.21203/rs.3.rs-130554/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2021-02-10T13:13:11+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2021-02-09T15:57:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"cb20c62e-310a-462a-8565-122d9a4cada5","date":"2021-01-30T14:33:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"0a754651-1c7f-43ca-9b72-fea448293c15","date":"2021-01-26T11:39:20+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2021-01-02T20:03:29+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2021-01-02T19:47:38+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2020-12-22T11:54:57+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2020-12-22T10:16:16+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2020-12-17T10:07:17+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a99fcd1d-9985-406e-8a6f-4ce259c0b220","owner":[],"postedDate":"December 28th, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":1645455,"name":"General Biochemistry"},{"id":1645456,"name":"Analytical Biochemistry"}],"tags":[],"updatedAt":"2021-05-11T05:29:11+00:00","versionOfRecord":[],"versionCreatedAt":"2020-12-28 20:12:05","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-130554","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-130554","identity":"rs-130554","version":["v1"]},"buildId":"-HB7Z8yhvgn0wM9Nzuekk","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}