Bioluminescent detection of the total amount of viable Gram-positive bacteria isolated by vancomycin-functionalized magnetic particles

Abstract

A facile bioluminescent protocol was developed to detect the total amount of viable Gram-positive bacteria based on a novel antibiotic-affinity strategy. Vancomycin-functionalized magnetic particles were adopted to isolate Gram-positive bacteria from sample matrix, utilizing the five-point hydrogen bonds binding between vancomycin and D-Alanyl-d-Alanine moieties on the cell wall. Then the bioluminescence signal from the bacterial intracellular adenosine triphosphate was collected to quantify the captured bacteria after cells lysis. In this proof-of-principle work, the developed protocol was applied to detect four Gram-positive bacteria, including Staphylococcus aureus, Micrococcus luteus, Bacillus cereus and Streptococcus mutans. It showed a linear range of 1.0×102–1.0×107 CFU mL−1 and a detection limit of 33 CFU mL−1 for the four model bacteria. The whole assay process could be completed within 70min. Gram-negative bacteria and dead bacteria all showed negligible interference to the detection of the viable Gram-positive bacteria. The method was successfully applied to quantify the amount of viable Gram-positive bacteria in environmental, food and pharmaceutical samples with acceptable recovery values ranging from 72.0% to 120.0%. The protocol possessed numerous advantages such as high sensitivity, low cost, ideal specificity and short detection time. It could also be extended to Gram-negative bacteria detection by utilizing other antibiotics acting on Gram-negative bacteria.