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

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 ber protein (TFP) recombined in Escherichia coli expression system was functionalized on magnetic particles (MPs) to specically 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 specically 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 specicity of TFP, the AST can actually be conducted without the requirement of bacterial isolation and identication 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.


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 identi cation 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][10][11] , electrochemical sensor [12][13][14] , phase-shift spectroscopy detection 15 , uorescent detection 16 and micro uidic 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 timeconsuming pretreatment procedure for bacterial culture, isolation and identi cation. 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][21][22] . However, they suffer from welltrained 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 speci cally recognize their target bacteria even in harsh environment. Typical lifecycle of virulent bacteriophage involves the following steps: speci c 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 ber 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 speci city, 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 speci cally recognize P. aeruginosa without lytic activity. To investigate the application of TFP in AST detection, TFPfunctionalized magnetic particles were utilized to speci cally capture P. aeruginosa, and uorescein isothiocyanate (FITC) labeled magainin II was utilized as the uorescent tracer. A reverse assaying protocol (RAP) combined magnetic separation was developed to speci c, rapid and sensitive detection and AST of P. aeruginosa.

Instrumentations
Scanning electron micrograph (SEM) was recorded by an S-3000N scanning electron microscope (Hitachi, Japan). Fluorescence (FL) signals were obtained from an In nite M200 PRO microplate reader (TECAN, Switzerland). FL micrographs were recorded by using a NI − U FL microscope (Nikon, Japan).

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.

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.

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 magni cence 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.

Preparation procedure of TFP-functionalized MPs
TFP-functionalized MPs were obtained according to manufacturer guideline. Brie y, 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.

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.

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.

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 speci cally capture P. aeruginosa through speci c 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 uorescent 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 IIbacteria-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 de ned 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 uorescent 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.

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.

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)

Speci city
The speci city 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 speci city investigation. The speci city 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 speci city.

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.  (Fig. 5A). As shown in Fig. 5B-5D, 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 signi cantly 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. 5E). 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 signi cantly in uenced 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). 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.

Conclusion
In conclusion, a rapid, sensitive and speci c RAP using TFP and magainin II as dual recognition elements was developed to perform the detection and AST of P. aeruginosa. Since TFP can speci cally 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 identi cation, which can facilitate the decreasing frequency of irrational empiric antibiotic therapy. Based on this proof-ofprinciple 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 micro uidic system or single-cell imaging. Declarations