Timely and accurate anti-infective therapy significantly benefits treatment of various life-threatening bacterial infectious diseases. Unfortunately, owing to the lack of selectivity for recognizing bacterial species, most of the reported phenotypic antimicrobial susceptibility testing protocols suffer from time-consuming bacterial isolation and identification. In this work, a highly-selective molecular recognition-based sandwich fluorimetric assay was developed to rapidly detect Staphylococcus aureus ( S. aureus ), and furtherly to assess its susceptibility to antibiotics. Immobilized porcine IgG was utilized to capture S. aureus through the selective interaction between Fc fragment of porcine IgG and protein A in the cell wall of S. aureus . Fluorescein isothiocyanate-labeled teicoplanin was adopted as the signal tracer utilizing the binding behavior between teicoplanin and d -Ala- d -Ala moieties in the peptidoglycans of Gram-positive bacterial cell wall. S. aureus could be detected within a wide linear rang of 1.0 × 10 3 to 1.0 × 10 7 CFU mL −1 . Subsequently, this protocol utilizing porcine IgG and teicoplanin as the indicator molecules was utilized to assess the antimicrobial susceptibility of S. aureus after the bacteria were treated with antibiotics. The minimum inhibitory concentrations of penicillin, cefoxitin, clindamycin, trimethoprim/sulfamethoxazole and erythromycin were estimated to be >0.25,<4, <0.25, <2/38 and <0.5 μg mL −1 , respectively. The whole process for antimicrobial susceptibility testing can be completed within 4 h since it avoided extra time-consuming isolation and identification of bacterial species.
Staphylococcus aureus is a common foodborne pathogen that can cause a suppurative infection after eating contaminated food. Detection of S. aureus plays an important role in the food industry. In this study, a strategy for the detection of S. aureus using magnetic separation (MS) technology combined with rolling circle amplification (MS-RCA) was proposed. The strategy used antibiotics to capture bacteria and employed RCA products as signal output probes. Vancomycin (Van), as a commonly used antibiotic, can recognize peptidoglycan on the cell wall of Gram-positive bacteria and can effectively identify target bacteria. Therefore, we prepared BSAylated-Van functionalized magnetic beads (Van-MBs) for the pre-enrichment of S. aureus . To ensure the selectivity of this method, we used biotin-pig IgG to bind S. aureus . In addition, to amplify the output signal of the MS-RCA strategy, we introduced streptavidin (SA) and successfully obtained the [email protected] S. aureus @biotin-pig [email protected] @biotin-RCA probe complex and used the biotin-avidin-system (BAS) by combining magnetic separation technology and RCA technology to realize the enrichment and specific detection of S. aureus . Furthermore, by optimizing the experimental conditions such as the magnetic separation time and the amount of Van-MBs, the detection performance of this method was improved. Under the optimal conditions, the detection limit of this method for S. aureus was 3.3 × 10 2 CFU/mL in fruit juice, and it was less affected by other bacteria.
Bacterial analysis and antibiotic resistance testing (ART) are of great significance in clinical diagnosis and therapy of bacterial infectious diseases. In this work, a portable antibiotic-affinity chromatographic test strip has been developed for rapid analysis of Staphylococcus aureus ( S. aureus ) and further applied for ART of this pathogen. Porcine IgG was immobilized on a nitrocellulose membrane for capturing S. aureus based on the selective binding capability of the Fc fragment of IgG toward protein A on the surface of the target bacteria. Fluorescent microspheres modified with teicoplanin (TEI) were applied as signal substances to trace S. aureus utilizing the hydrogen bond conjugation between this antibiotic and Gram-positive bacteria. S. aureus can be analyzed within the concentration range from 1.4 × 10 3 CFU mL −1 to 1.4 × 10 7 CFU mL −1 . The recovery values for spiked samples were 93.3–110.0%. The obtained results of ART 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 ART 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.
危险品化学品经营许可证（带存储）