Antimicrobial resistance (AMR) is a growing global concern, and carbapenem-resistant Enterobacterales (CRE) are one of the most urgent threats due to limited antibiotic therapies. CRE, specifically carbapenemase-producing (CP) strains, are found in clinical, environmental, and food samples worldwide, causing many hospitalizations and deaths. Early detection is crucial in minimizing and controlling their detrimental impact. However, many detection techniques usually require pure culture after retrieving the pathogens from matrices, which takes several days. Thus, rapid detection of the causative bacteria directly from samples is needed for outbreak prevention. In this study, two nanoparticles, glycan-coated magnetic nanoparticles (gMNPs) and dextrin-coated gold nanoparticles (dGNPs), were used to extract and detect, respectively, CP E. coli, particularly those harboring Klebsiella pneumoniae carbapenemase (KPC), from spiked tap water and food samples. Experiments for magnetic extraction and plasmonic detection were first conducted using pure cultures of resistant E. coli (R) isolates and susceptible E. coli (S) acting as reference, followed by applying them in E. coli-contaminated matrices.The gMNPs offer rapid and cost-effective extraction and concentration of bacteria. A confocal laser scanning microscopy and transmission electron microscopy (TEM) initially confirmed the binding of gMNPs to bacterial cells in a buffer solution. The gMNP-cell binding capacity was expressed as concentration factor (CF), quantified through the standard plating method. Results showed that the CF of all E. coli (R) isolates was lower than that of E. coli (S). This study further illustrated that the lower CF could be the effect of cell surface characteristics of E. coli (R) isolates, where they displayed heterogeneous cell shape (rod and round cells) and lower negative zeta potential (cell surface charge). In addition, bacterial load and solution pH on gMNP-cell interaction were evaluated. Results showed that the higher bacterial load and pH environment resulted in lower CF of both E. coli (R) and E. coli (S) isolates. Further, the effectiveness of gMNPs in large-volume water and food samples was tested. The gMNPs successfully extracted E. coli (R) and E. coli (S) isolates from buffer solution, tap water, and food samples (raw chicken breast, ground beef, and romaine lettuce), as confirmed by TEM and the selective plating method. The CF of both E. coli (R) and E. coli (S) in buffer solution and tap water was higher than in food samples. The variable CF in food samples could be the effect of food microparticles and natural microflora, where non-selective gMNPs may bind to plant and animal tissues and natural microflora. Whereas the gMNPs successfully extracted bacterial cells from foods and water, their specific and rapid detection is also significant. To achieve this, a plasmonic biosensor was designed for feasibility. Gold nanoparticle-based plasmonic biosensors have recently drawn attention due to their unique surface plasmon resonance (SPR) properties. GNPs change color upon aggregation or agglomeration, allowing for simple use without expensive and complex equipment and data analysis. This work utilized highly stable dextrin-coated GNPs to detect CP bacteria, specifically Klebsiella pneumoniae carbapenemase (KPC)-producing bacteria, targeting the blaKPC gene. The assay only requires dGNPs, an oligonucleotide probe specific to blaKPC, and DNA samples to detect the target DNA within 30 min without PCR amplification. The stability of dGNPs under an acidic environment indicates the presence of target DNA due to probe-binding and dGNPs’ protection, maintaining their red appearance. The absence of target DNA was indicated by the aggregation of dGNPs, corresponding to a color change from red to blue or purple. The stability and aggregation of dGNPs were further confirmed by TEM and quantified by a shift in absorbance spectra. The plasmonic biosensor was tested in 47 bacterial isolates: 14 KPC-producing target bacteria and 33 non-target bacteria. The biosensor successfully detected and differentiated blaKPC-positive bacteria, regardless of bacterial type. The diagnostic sensitivity and specificity were 79% and 97%, respectively, along with a detection limit of 2.5 ng/μL. For specific bacterial (E. coli) detection, an earlier developed plasmonic biosensor with a uidA probe was further used. Finally, the two biosensor platforms were implemented to detect KPC-producing E. coli from water and food samples. Magnetically extracted bacteria from the artificially contaminated tap water, romaine lettuce, ground beef, and chicken breast samples were followed by short enrichment and DNA extraction. The plasmonic biosensors successfully detected KPC-producing E. coli from each sample by parallel detection targeting of uidA and blaKPC genes, with no false positives for the samples contaminated with non-target bacteria. The biosensor successfully detected the target organism, where the original concentration prior to magnetic extraction was 103 CFU/mL. The biosensor results were further verified with the standard PCR test. As proof-of-concept, these findings indicate promising applications of the integrated platform for cost-effective and rapid bacterial detection from complex matrices within <7 h. The gained insights may facilitate future application of this platform; recommendations for further optimizations and studies have also been identified.
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