A Pseudopaline Fluorescent Probe for the Selective Detection of <i>Pseudomonas aeruginosa</i>

Abstract

Open AccessCCS ChemistryRESEARCH ARTICLE1 Sep 2021A Pseudopaline Fluorescent Probe for the Selective Detection of Pseudomonas aeruginosa Tianhu Zhao†, Jian Zhang†, Xiaowan Han†, Jun Yang, Xin Wang, Maarten Vercruysse, Hai-Yu Hu and Xiaoguang Lei Tianhu Zhao† Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Department of Chemical Biology, College of Chemistry and Molecular Engineering, Synthetic and Functional Biomolecules Center, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871 †T. Zhao, J. Zhang, and X. Han contributed equally to this work.Google Scholar More articles by this author , Jian Zhang† Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Department of Chemical Biology, College of Chemistry and Molecular Engineering, Synthetic and Functional Biomolecules Center, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871 †T. Zhao, J. Zhang, and X. Han contributed equally to this work.Google Scholar More articles by this author , Xiaowan Han† State Key Laboratory of Bioactive Substances and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050 †T. Zhao, J. Zhang, and X. Han contributed equally to this work.Google Scholar More articles by this author , Jun Yang Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Department of Chemical Biology, College of Chemistry and Molecular Engineering, Synthetic and Functional Biomolecules Center, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871 Google Scholar More articles by this author , Xin Wang Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Department of Chemical Biology, College of Chemistry and Molecular Engineering, Synthetic and Functional Biomolecules Center, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871 Google Scholar More articles by this author , Maarten Vercruysse Roche Pharma Research & Early Development (pRED), Roche Innovation Center Basel, 4070 Basel Google Scholar More articles by this author , Hai-Yu Hu *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] State Key Laboratory of Bioactive Substances and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050 Google Scholar More articles by this author and Xiaoguang Lei *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Department of Chemical Biology, College of Chemistry and Molecular Engineering, Synthetic and Functional Biomolecules Center, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871 Google Scholar More articles by this author https://doi.org/10.31635/ccschem.020.202000517 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail The rise of resistance to all known antibiotics is a global crisis. In addition to novel treatment options, there is an urgent need to develop rapid, specific, sensitive, and reliable diagnostic methods to detect pathogenic bacteria in clinical samples and reduce the overuse and misuse antibiotics. Pseudopaline, a metallophore produced by the human pathogen Pseudomonas aeruginosa, transports divalent metal ions via a dedicated active transport system, making it an ideal carrier for a second functional moiety. In this work, we have developed a pseudopaline fluorescein-conjugated probe ( P-FL), able to specifically detect P. aeruginosa in samples (in vitro) among several bacterial species, mammalian cells, or in mouse stomach tissue sections. By replacing the fluorescein with the near-infrared fluorophore, Cyanine-7 (Cy-7) to obtain a pseudopaline-Cyanine-7 ( P-Cy7), we showed that P. aeruginosa infections could also be detected specifically in a mouse model (in vivo) using this probe. The remarkable selectivity of these pseudopaline fluorescent probes is due to pseudopaline-mediated metal transport system, exclusively specific to P. aeruginosa. Therefore, our results show that pseudopaline-based probes might provide a new approach to develop fast and effective diagnostics of P. aeruginosa infections. Download figure Download PowerPoint Introduction Since the discovery of penicillin in 1928, antibiotics have been widely used in the clinic, saving millions of lives each year. However, their excessive use has led to a global antibiotic resistance crisis, especially among the ESKAPE pathogens (the acronym of Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter cloacae), which are resistant to many antibiotics currently used in hospitals.1–6 To reduce the adverse effects of broad-spectrum antibiotics and slow down the resistance rate, pathogen-specific or narrow-spectrum antibiotics could be used in the future.7 The selection and effective utilization of pathogen-specific antibiotics requires the development of rapid and reliable diagnostics that identify causes of pathogen infections to guide clinicians toward narrowing antibiotics spectrum suitable for use instead of employing broad-spectrum types. Traditional methods for clinical identification of microorganisms rely on accessible sampling and pure cultivation, which usually requires 2–5 days, or even longer for slow-growing bacteria.8 Due to the challenges involved, these methods are not suitable for detecting deep tissue infections such as biomaterial-associated infections or endocarditis9,10; also several patients die even before the bacteria are identified. To address this issue, effective bacterial imaging is an emerging strategy that could diagnose bacterial infections in a matter of hours.9–11 Several types of chemical probes such as fluorescent derivatives of antibiotics or antimicrobial peptides,12–23 nitroreductase-triggered probes,24,25 glycoprotein-targeted probes,26 siderophore-based probes,27–30 maltodextrin-based probes,31 and probes with aggregation-induced emission property (AIEgen)32–36 have been reported. Near-infrared (NIR) dyes could replace the fluorescent part of these probes for live imaging to detect deep tissue infections inaccessible by the conventional sampling method.19,37–39 However, most of these probes are not species-selective, labeling multiple bacterial species simultaneously. Narrow-spectrum probes are urgently needed to enable fast diagnostics before the use of narrow-spectrum antibiotics. P. aeruginosa, one of the ESKAPE pathogens and one of the top three “critical priority pathogens” on the World Health Organization (WHO) priority list, causes severe nosocomial infections, notably in immunocompromised patients.40–43P. aeruginosa infection is often difficult to treat due to its biofilm formation, making it escape most antimicrobial therapies and host immune responses.44–47 Therefore, the development of P. aeruginosa-specific probes is urgently needed to diagnose its infection at an early stage, especially before it forms a biofilm. In this study, we targeted the P. aeruginosa metal transport machinery encoded by zrmABCD operon, which is highly expressed under zinc-limiting conditions.48–50 The metallophore, called pseudopaline, is expressed by ZrmB and ZrmC, secreted extracellularly through ZrmD to chelate metals, then imported back through the outer membrane receptor ZrmA.48–50 Moreover, the zrmABCD operon is highly upregulated in the infected host sites,51–54 suggesting that pseudopaline could serve as an ideal carrier to conjugate with a chromophore for the diagnosis of P. aeruginosa infections. Herein, based on our previous synthetic and functional studies of pseudopaline,55 we present the design, development, and evaluation of a series of pseudopaline-conjugated probes ( 1– 3, Figure 1 and Schemes 1–3) as practical tools for the selective detection of P. aeruginosa in living cells, tissue samples, and in vivo. We found that P. aeruginosa could uptake all our designed probes within 15 min. An in vitro probe in the series, pseudopaline fluorescein-conjugated probe 1 ( P-FL), was utilized for selective labeling of P. aeruginosa in the presence of other bacterial species, mammalian cells, and tissue sections. Most importantly, the pseudopaline-derived probe 2 ( P-Cy7) containing a NIR carbocyanine fluorophore displayed a high selectivity detection of P. aeruginosa infection in a mouse model. Figure 1 | Chemical structures of P-FL (1), P-Cy7 (2), and P-Cy5 (3). Download figure Download PowerPoint Experimental Methods General information Proton nuclear magnetic resonance (1H NMR) spectra were recorded on a Bruker 400 or 600 MHz spectrometer (Bruker, Shanghai, China) at ambient temperature with D2O or D2O/30% CD3CN as the solvent. 13C NMR spectra were recorded on a Bruker 151 or 239 MHz spectrometer with complete proton decoupling at ambient temperature. Chemical shifts are reported in parts per million relative to deuterium oxide (1H, δ 4.79 ppm). Data for 1H NMR is reported as follow: chemical shift, integration, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet) and coupling constants. High-resolution mass spectra were obtained at Peking University Mass Spectrometry Laboratory using a Bruker Fourier Transform Ion Cyclotron Resonance Mass Spectrometer Solarix XR. The samples were analyzed by high-performance liquid chromatography (HPLC)/mass spectrometry (MS) on a Waters Auto Purification LC/MS system (3100 Mass Detector, 2545 Binary Gradient Module, 2767 Sample Manager, and 2998 Photodiode Array (PDA) Detector; Waters, Shanghai, China). The system was equipped with Waters C18 5 μm SunFire separation column (150 × 4.6 mm) equilibrated with HPLC grade water with 0.1% HCOOH (solvent A) and HPLC grade CH3CN (solvent B) with a flow rate of 0.3 mL/min at room temperature. Preparative HPLC/MS on a Waters AutoPurification LC/MS system (3100 Mass Detector, 2545 Binary Gradient Module, 515 HPLC pump, 2767 Sample Manager 2998 PDA Detector). The system was equipped with Waters C18 5 μm X-bridge separation column (150 × 19 mm). The yields refer to chromatographically and spectroscopically pure materials. All reactions were carried out in oven-dried glassware under an argon atmosphere unless otherwise stated. Sulfo-Cy7-acid was purchased from Psaitong Co. (Beijing, China), and Sulfo-Cy7-Succinimidyl Ester ( S-3) was purchased from MedChemExpress (Shanghai, China). Scheme 2 | Synthesis of the pseudopaline-Cyanine-7 probe. Download figure Download PowerPoint Synthesis of compound 1 ( P-FL) To a solution of S-1 (5.6 mg, 12.3 μmol, 1 equiv) in water (1 mL) was added Et3N (8.5 μL, 61.5 μmol, 5 equiv) and S-2 (4.8 mg, 12.3 μmol, 1 equiv) at room temperature. After stirring overnight at room temperature, the reaction mixture was subjected directly to prep-HPLC (20% acetonitrile in 0.02% HCl in H2O for 3 min then a gradient of 20–90% acetonitrile in 0.02% HCl in H2O for 10 min) to afford the pure product 1 (2.3 mg, 2.71 μmol, 22%) and the recovered S-1 (2 mg). Scheme 3 | Synthesis of the pseudopaline-Cyanine-5 probe. Download figure Download PowerPoint 1H NMR (400 MHz, D2O, δ): 8.27 (s, 1H), 7.97 (s, 1H), 7.78 (d, J = 8.4 Hz, 1H), 7.62 (d, J = 9.8 Hz, 2H), 7.41 (d, J = 8.1 Hz, 1H), 7.35 (d, J = 2.0 Hz, 2H), 7.17 (dd, J = 9.2, 2.0 Hz, 2H), 4.48 (t, J = 5.6 Hz, 2H), 4.36 (d, J = 5.7 Hz, 1H), 4.12 (t, J = 6.4 Hz, 1H), 4.08 (t, J = 6.4 Hz, 1H), 3.64–3.56 (m, 2H), 3.50–3.38 (m, 4H), 2.72–2.57 (m, 2H), 2.48–2.33 (m, 2H), 2.29–2.16 (m, 2H), 2.03–1.93 (m, 2H), 1.65–1.56 (m, 2H). 13C NMR (239 MHz, D2O/30%CD3CN, δ): 180.8, 176.2, 171.3, 170.8, 170.2, 168.7, 166.0, 156.7, 141.0, 140.7, 131.8, 129.8, 129.6, 128.5, 124.9, 117.3, 114.4, 102.9, 100.0, 62.8, 60.3, 60.0, 59.0, 50.3, 44.0, 29.9, 27.1, 26.6, 25.5, 25.2, 25.1. High-resolution mass spectrometry (HRMS) [electrospray ionization (ESI)]: [M + H]+ calcd for C39H40N7O13S, 846.2410; found, 846.2440. Scheme 1 | Synthesis of the pseudopaline-fluorescein probe. brsm, based on recovered starting materials. Download figure Download PowerPoint Synthesis of compound 2 ( P-Cy7) To a solution of S-1 (3.5 mg, 6.4 μmol, 1 equiv) and NaHCO3 (2.7 mg, 32 μmol, 5 equiv) in water (1 mL) were added acetonitrile (ACN) (1 mL) and S-3 (6.0 mg, 7.7 μmol, 1.2 equiv) at room temperature. After stirring for 2 h at room temperature, ACN was removed in vacuo and then purified by gel column to afford 2 (3.8 mg, 3.0 μmol, 47%) as a green solid. 1H NMR (600 MHz, D2O/30% CD3CN, δ): 8.66 (s, 1H), 8.21–8.13 (m, 2H), 8.11–8.04 (m, 5H), 7.88–7.77 (s, 1H), 7.55 (d, J = 30.5 Hz, 2H), 6.89–6.73 (m, 2H), 6.53 (dd, J = 37.9, 12.1 Hz, 2H), 4.60 (br s, 2H), 4.33 (br s, 2H), 4.27 (br s, 2H), 4.18 (br s, 1H), 3.96–3.82 (m, 2H), 3.54 (br s, 4 H), 3.33 (br s, 2H), 2.72 (br s, 2H), 2.41 (br s, 2H), 2.08 (br s, 2H), 2.03 (br s, 3 H), 1.93 (s, 12H), 1.84 (br s, 3H), 1.67–1.50 (m, 9H). 13C NMR (151 MHz, D2O/30% CD3CN, δ): 175.9, 172.8, 172.7, 172.3, 172.2, 156.7, 152.6, 151.8, 144.6, 144.0, 142.0, 141.6, 141.5, 140.2, 139.9, 139.8, 126.9, 126.8, 126.7, 126.6, 124.7, 120.0, 120.0, 110.9, 104.6, 104.4, 63.0, 62.8, 50.1, 49.8, 49.3, 49.2, 49.1, 49.0, 44.0, 39.5, 39.0, 38.6, 35.7, 27.1, 26.9, 26.8, 26.8, 26.6, 26.5, 25.9, 25.7, 25.3, 23.9, 11.8. HRMS [ESI]: [M − 2H]2−/2 calcd for C53H68N8O15S2, 560.2128; found, 560.2130. Synthesis of compound 3 ( P-Cy5) To a solution of S-1 (5.5 mg, 10 μmol, 1 equiv) and NaHCO3 (5.0 mg, 60 μmol, 5 equiv) in water (1 mL) were added ACN (1 mL) and S-4 (7.0 mg, 12 μmol, 1.2 equiv) at room temperature. After stirring for 2 h at room temperature, the reaction mixture was subjected directly to prep-HPLC (10% acetonitrile in 0.05% HCOOH in H2O for 1 min then a gradient of 10–95% acetonitrile in 0.05% HCOOH in H2O for 8.5 min) to afford the pure product 3 (6.3 mg, 8.1 μmol, 81%) as a blue solid. 1H NMR (400 MHz, D2O/30% CD3CN, δ): 8.51 (t, J = 13.1 Hz, 2H), 8.24 (s, 1H), 7.94 (dd, J = 6.9, 3.3 Hz, 2H), 7.91–7.84 (m, 2H), 7.77–7.68 (m, 4H), 6.99 (t, J = 12.4 Hz, 1H), 6.68 (dd, J = 13.7, 9.9 Hz, 2H), 4.52 (dd, J = 14.5, 7.3 Hz, 2H), 4.47 (t, J = 7.3 Hz, 2H), 4.36 (t, J = 5.6 Hz, 1H), 4.07 (dd, J = 13.2, 6.7 Hz, 2H), 3.77–3.69 (m, 4H), 3.55 (t, J = 6.9 Hz, 2H), 3.09–2.93 (m, 2H), 2.78–2.68 (m, 2H), 2.64–2.55 (m, 4H), 2.32–2.26 (m, 2H), 2.25–2.18 (m, 2H), 2.11 (s, 12H), 2.08–2.02 (m, 2H), 1.88–1.81 (m, 4H), 1.78 (t, J = 7.7 Hz, 3 H). 13C NMR (126 MHz, D2O/30% CD3CN, δ): 177.1, 175.6, 173.6, 173.4, 166.2, 154.1, 154.0, 142.6, 142.2, 141.9, 141.7, 141.7, 129.1, 129.1, 125.6, 125.6, 124.8, 124.7, 122.7, 122.7, 111.5, 111.3, 103.3, 103.1, 67.0, 62.8, 62.0, 60.6, 50.3, 49.6, 49.5, 44.3, 44.1, 39.4, 38.8, 36.0, 30.7, 27.3, 27.3, 27.1, 26.3, 26.0, 25.9, 25.6, 20.0, 12.1. HRMS (ESI): [M]+ calcd for C51H69N8O9, 937.5182; found, 937.5171. Absorption and fluorescence spectra of probes The probe in dimethyl sulfoxide (DMSO) stock solution was diluted to 10 μM in phosphate-buffered saline (PBS). The spectra of P-FL, P-Cy5, and P-Cy7 were recorded on a Spark 10M Multimode Microplate Reader (TECAN, Männedorf, Switzerland), and shown in Supporting Information Figure S14. Bacteria and cell culture P. aeruginosa PA14,56P. aeruginosa (American Type Culture Collection (ATCC) 27853), A. baumannii (ATCC 19606), K. pneumoniae (clinically isolated, unpublished), Escherichia coli (ATCC 80739), P. putida KT-2440, and Pseudomonas fluorescens (ATCC 17400) were typically grown in Luria-Bertani (LB) medium, Enterobacter cloacae (ATCC 13047) in Nutrient Broth (NB) medium, S. aureus (ATCC 25923) in Tryptic Soy Broth (TSB) medium, and E. faecium (CICC 10840) in De Man, Rogosa, and Sharpe (MRS) Broth medium. P. putida KT-2440 and P. fluorescens (ATCC 17400) were grown overnight at 30 °C with shaking at 200 rpm, while all other bacteria were grown at 37 °C, accordingly. The lung adenocarcinoma cells (A549) were cultured in McCoy’s 5A (modified) medium, and human embryonic kidney cells (HEK293T), leukemic monocyte-macrophage cells (Raw 264.7), and cervical cancer cell line (Hela cells) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) under a humidified atmosphere of 5% CO2 at 37 °C. Confocal imaging of P. aeruginosa treated with P-FL/Zn, P-FL, and FL (5-Carboxyfluorescein) An overnight culture of P. aeruginosa PA14 was collected, washed twice with PBS, resuspended in PBS, diluted 100-fold in metal-limited chemically defined medium (CDM),57 and grown at 37 °C for another 12 h with shaking at 200 rpm. The P-FL was preincubated with ZnSO4 at a molar ratio of 1∶1 for 15 min at 37 °C to get P-FL/Zn. Then P. aeruginosa culture was collected, resuspended in PBS (OD600 = 1.0), and treated with 10 μM P-FL/Zn, P-FL, or FL (5-Carboxyfluorescein) for 15 min at 37 °C. cells were washed three with PBS, resuspended in PBS, and fluorescent were on with at Fluorescent labeling of and toward bacteria of P. aeruginosa A. baumannii, and S. aureus were collected, washed twice with PBS, resuspended in PBS, diluted 100-fold in and grown at 37 °C for another 12 h with shaking at 200 rpm. The P-Cy7 and P-Cy5 were preincubated with ZnSO4 at a molar ratio of 1∶1 for 15 min at 37 °C, All bacterial were collected, resuspended in PBS (OD600 = 1.0), and treated with 10 μM or 10 μM for 15 min at 37 °C. cells were washed three with PBS, resuspended in PBS, and the bacterial fluorescence was detected using Spark 10M Multimode Microplate Reader at for and at for Fluorescent labeling of toward P. aeruginosa under the An overnight culture of P. aeruginosa PA14 was collected, washed twice with PBS, resuspended in PBS, diluted 100-fold in + 5 μM or + 5 μM and grown at 37 °C for another 12 h with shaking at 200 rpm. The P-FL was preincubated with ZnSO4 at a molar ratio of 1∶1 for 15 min at 37 °C. Then P. aeruginosa culture was collected, resuspended in PBS (OD600 = 1.0), and treated with 10 μM for 15 min at 37 °C. cells were washed three with PBS, resuspended in PBS, and the fluorescence of the bacteria was detected using an Reader Fluorescent labeling of toward P. aeruginosa in the presence of pseudopaline An overnight culture of P. aeruginosa PA14 was collected, washed twice with PBS, resuspended in PBS, diluted 100-fold in and grown at 37 °C for another 12 h with shaking at 200 rpm. The P-FL was preincubated with ZnSO4 at a molar ratio of 1∶1 for 15 min at 37 °C, and pseudopaline was added Then P. aeruginosa culture was collected, resuspended in PBS (OD600 = 1.0), and treated with 10 μM containing pseudopaline of or for 15 min at 37 °C. cells were washed three with PBS, resuspended in PBS, and the bacterial fluorescence was detected using an Reader Confocal imaging of bacteria treated with The fluorescent labeling of bacteria was using probe and detected to with of P. aeruginosa P. aeruginosa (ATCC 27853), A. baumannii, K. pneumoniae, E. E. faecium, E. S. aureus, P. and P. fluorescens were collected, washed twice with PBS, resuspended in PBS, diluted 100-fold in and grown at 37 °C for P. putida and P. at 30 for another 12 h with shaking at 200 rpm. The P-FL was preincubated with ZnSO4 at a molar ratio of 1∶1 for 15 min at 37 °C before to the bacterial All bacterial were collected, resuspended in PBS (OD600 = 1.0), and the of the were added to obtain a 10 μM Then the were at 37 °C for 15 min for P. putida and P. fluorescens at 30 cells were washed three with PBS, resuspended in PBS, and fluorescent were on the or Confocal with at Confocal imaging of mammalian cells treated with The fluorescent labeling of the P-FL probe toward mammalian cells was carried out as 200 of the or or or cells (1 × was an with a at the and cultured for h in McCoy’s 5A (modified) medium cells) or with 10% and cells) under a humidified atmosphere of 5% CO2 at 37 °C. the medium was and the cells were washed twice with The P-FL was preincubated with ZnSO4 at a molar ratio of 1∶1 for 15 min at 37 °C, diluted in PBS to obtain a 10 μM and then added to the cells per The cells were with for 30 min at 37 °C. The were and the cells were washed three with 200 PBS was added to each by using the with Shanghai, China) at Selective labeling of toward P. aeruginosa in the bacterial culture of P. aeruginosa A. baumannii, K. pneumoniae, E. E. faecium, E. S. aureus, P. and P. fluorescens were collected, washed twice with PBS, resuspended in PBS, diluted 100-fold in and grown at 37 °C for P. putida and P. fluorescens at 30 for another 12 h with All bacterial were and resuspended in PBS (OD600 = The P. aeruginosa culture was with an of another bacterial and resuspended in The P-FL was preincubated with ZnSO4 at a molar ratio of 1∶1 for 15 min at 37 °C and added to the bacterial culture to obtain a 10 μM The bacterial culture with was at 37 °C for 15 min P. putida and P. fluorescens at 30 cells were washed three with PBS, resuspended in PBS, and fluorescent were on the or Confocal with at E. which expressed the fluorescent the were at Selective labeling of in the of P. aeruginosa and mammalian cells 200 of mammalian cells (1 × was an with a at the and cultured for h in McCoy’s 5A (modified) medium cells) or with 10% and cells) under a humidified atmosphere of 5% CO2 at 37 °C. An overnight culture of P. aeruginosa was collected, washed twice with PBS, resuspended in PBS, diluted 100-fold in and grown at 37 °C for another 12 Then the bacterial cells were and resuspended in The P-FL was preincubated with ZnSO4 at a molar ratio of 1∶1 for 15 min at 37 °C and added to the P. aeruginosa PBS culture to obtain 10 μM After the medium for mammalian cells was by 200 P. aeruginosa PBS culture (OD600 = containing and for 30 min at 37 °C. The were and the cells were washed three with 200 of PBS was added to each were on the with at Selective labeling of toward P. aeruginosa in of mouse stomach stomach tissue were from and and of the All to were in with the on of the Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College (Beijing, China). The stomach was and in tissue medium temperature and for μm by Beijing & (Beijing, China) for imaging An overnight culture of P. aeruginosa was collected, washed twice with PBS, resuspended in PBS, diluted 100-fold in and grown at 37 °C for another 12 Then the bacterial cells were and resuspended in The P-FL was preincubated with ZnSO4 at a molar ratio of 1∶1 for 15 min at 37 °C and added to the P. aeruginosa PBS culture to obtain a 10 μM The were treated with 400 P. aeruginosa PBS culture (OD600 = containing at 37 °C for 1 After the were washed three with PBS and for 5 5 medium containing was on the before with a were using the with at for and 30 for The ratio was using J ( In of P. aeruginosa using with an of at the of were used this of P. aeruginosa and A. baumannii were collected, washed twice with PBS, resuspended in PBS, diluted 100-fold in and grown at 37 °C for another 12 Then the bacterial cells were and resuspended in PBS and the bacterial culture to at The of the were infected with P. aeruginosa μL, = while the of the were infected with A. baumannii μL, = Then the was through the infection or The with an of and infected of were used as The were h and The and were at were on the imaging system using the of × emission was used for to detect the of P-FL and P-Cy7 The and cells were on a containing 1 × cells per in and in a humidified 5% CO2 at 37 °C for h before the probe. After with of P-FL or P-Cy7 at 37 °C for solution was added to each and to for another 3 The was at using a Spark 10M Multimode Microplate Reader was as × − − of the of the of the