Antibiotic Affinity Strategy For Fluorescent Detection Of Pathogenic Bacteria Based On A Dual-Site Recognition Mode

Pathogenic bacteria are distributed almost everywhere,which pose great threats to human health by causing infectious diseases.According to a report in Lancet Infectious Diseases,infections led by antibiotic-resistant bacteria were estimated to account for more than 30,000 deaths in the EU and the European Economic Area in2015.The number was close to the total number of deaths led by influenza,tuberculosis and HIV.The emergences of various multiresistant bacteria are bringing huge challenge to the antimicrobial treatment of bacterial infectious diseases.Inappropriate empirical usage of antibiotics is a major cause of aggravated drug resistance and poor treatment effect.Rapid bacterial analysis and antibiotic resistance testing are two key approaches for alleviating the crisis.Infection caused by Staphylococcus aureus?S.aureus?and Pseudomonas aeruginosa?P.aeruginosa?is very common in clinic,and shows very high morbidity and mortality.Therefore,it is of great significance to develop timely and accurate methodological platform for detecting them and testing their antibiotic resistance.In our study,two novel methods were developed for fluorescent analysis of the two pathogenic bacteria utilizing the affinity between antibiotic and bacterial whole cells based on a dual-site recognition mode.?1?Antibiotic-affinity chromatographic test strip for quantitative analysis and antibiotic resistance testing of S.aureus.Fc fragment of immunoglobulin G?IgG?can selectively recognize protein A widely distributed in the cellular wall of S.aureus.As a glycopeptide antibiotic,teicoplanin can form five hydrogen bonds with the D-alanyl-D-alanine terminals of peptidoglycan in the cellular wall of Gram-positive bacteria to inhibit the synthesis of cellular wall.Therefore,IgG was immobilized on a nitrocellulose membrane for capturing S.aureus.Fluorescent microspheres modified with teicoplanin were applied as signal probes to trace S.aureus.With the two reagents,a portable antibiotic-affinity chromatographic test strip was developed for rapid analysis of S.aureus and further applied for antibiotic resistance testing of this pathogen.S.aureus can be analyzed within the concentration range from 1.4×10³ CFU m L^-1 to 1.4×10⁷ CFU mL^-1.The recovery values for spiked samples were 93.3%-110.0%.The obtained results of antibiotic resistance testing for penicillin,daptomycin,gentamicin,cefoxitin and clindamycin against S.aureus showed agreement with those of traditional broth dilution method.The procedures for bacterial analysis and antibiotic resistance testing can be accomplished within 20 and 110 min,respectively.The antibiotic-affinity chromatographic test strip showed great promise in point-of-care testing because of its ideal portability and rapidity.?2?A fluorescent protocol for the detection of P.aeruginosa using polymyxin B and bacteriophage tail fiber protein P069 as the dual-siterecognition reagents.Polymyxin B?PMB?is a wide-spectrum antibiotic toward many Gram-negative bacteria,which can bind with lipid A on the outer membrane of bacteria through electrostatic and hydrophobic interactions.Bacteriophages can selectively recognize the host bacteria through their tail fiber protein.In this study,a novel fluorescent protocol was proposed for rapid detection of P.aeruginosa based on a dual-site recognition of P.aeruginosa by PMB and bacteriophage tail fiber protein P069.Tail fiber protein P069 was immobilized on 96 microwell plate to capture P.aeruginosa as the first recognition reagent.PMB modified with fluorescein isothiocyanate?FITC?was applied as second recognition agent and signal probes to trace P.aeruginosa.Thus a dual-site recognition mode was developed for fluorescent detection of P.aeruginosa.The target bacteria can be analyzed within the concentration range from2.0×10³ CFU m L^-1 to 2.0×10⁷ CFU m L^-1.The proposed protocol can exclude the interference from other Gram-negative bacteria and all Gram-positive bacteria.It was successfully applied for the detection of P.aeruginosa in lake water,physiological saline injection,human urine and milk with acceptable recovery values ranging from83.3%to 126.7%,showing great prospect for practical application.