Rapid analysis of Gram-positive bacteria in water via membrane filtration coupled with nanoprobe-based MALDI-MS.
Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) is challenging when it is directly applied to identify bacteria in water. This study demonstrates a rapid, sensitive, and selective technique for detection of Gram-positive bacteria in water. It involves a combination of membrane filtration (MF) and vancomycin-conjugated magnetite nanoparticles (VNPs) to selectively separate and concentrate Gram-positive bacteria in tap water and reservoir water, followed by rapid analysis of the isolates using whole-cell MALDI-MS. VNPs specifically recognize cells of Gram-positive bacteria, which serves as a basis for affinity capture of target Gram-positive bacteria. A two-step procedure of surface modification of bare magnetite nanoparticles was applied to synthesize VNPs. MF prior to VNP-based magnetic separation can effectively increase the enrichment factor and detection sensitivity and reduce time-consuming culture steps and the matrix effect for analysis of bacteria in MALDI-MS. The enrichment factor for the MF-VNP technique is about 6 x 10(4). A variety of bacteria, including Staphylococcus aureus, Bacillus subtilis, Bacillus cereus, and Enterococcus faecium, were successfully analyzed from aqueous solutions and their mixtures with Gram-negative bacteria. The optimal conditions of the VNP/MALDI-MS technique, including selection of the MALDI matrix, the choice of cell-washing solution, and the VNP concentration, were also investigated. The capture efficiencies of Gram-positive bacteria with VNPs were 26.7-33.3%. The mass variations of characteristic peaks of the captured bacteria were within +/-5 Da, which indicated good reproducibility of the proposed technique. The technique was applied to detect Gram-positive bacteria in tap water and reservoir water with an analysis time of around 2 h. The detection limit for Bacillus cereus, Enterococcus faecium, and Staphylococcus aureus was 5 x 10(2) cfu/ml for 2.0-l water samples.
Centre for Advanced Water Technology, Public Utilities Board, 82 Toh Guan Road East, WaterHub, #C4-03, Singapore 608576, Singapore.
Shuping
S
Li
Zhongxian
Z
Guo
Hui-Fen
HF
Wu
Ying
Y
Liu
Zhaoguang
Z
Yang
Chee Hoe
CH
Woo
D052203:Ferrosoferric Oxide; D005374:Filtration; D006094:Gram-Positive Bacteria / Q000302:isolation & purification*; D053758:Nanoparticles / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014640:Vancomycin; D014871:Water Microbiology; D014881:Water Supply
D014640:Vancomycin; D052203:Ferrosoferric Oxide