Quantitative DNA Analysis for the Effect of Anti-Bacterial Household Products on Multiple Antibiotic Resistance Using Nanoparticles

Magnetic/luminescent nanoparticles (NPs) were used for quantitative monitoring of antibiotic resistance genes and gene-expression in environmental samples. We investigated the impact of antibacterial chemicals (i.e., triclosan (TCS) and triclocarban (TCC)), commonly used in personal care products, on antibiotic resistance of microbial populations. The potential risk of increased bacterial antibiotic resistance via multiple antibiotic resistance was assessed by quantitative monitoring of tetracycline (tetQ) and MLSB (ermC) resistance by a newly developed NPs-DNA assay in microcosm incubations. Fe3O4/Eu:Gd2O3 NPs were synthesized by spray pyrolysis and biofunctionalized by neutravidin. After immobilization of biotinylated probe DNA on particle surfaces, target (gDNA or cDNA) and signaling probe DNA were hybridized in a 96- well platform. Linear standard curves (R2 = 0.99) of target tetQ and ermC genes were determined from the normalized fluorescence (Cy3/Eu NPs) of DNA-NP hybrids. NPs provide a magnetic property and stable fluorescence as an internal calibration in the assay. The abundance of tet Q and ermC genes after a 4 week-incubation showed 1) a significant increase of tetQ gene copies with the addition of tetracycline, TCS, or TCC and 2) an increase of ermC gene copies with the addition of tetracycline only but not with the other chemicals. Gene expression analysis elucidated by complementary DNA (cDNA) quantification also indicated a significant expression of tetQ or ermC gene in response to TCS or TCC, and demonstrated the possibility of a multiple antibiotic effect resulting from other co-existing antibacterial reagents. A real-time qPCR assay was used to evaluate the NPs assay sensitivity and range of quantification. The tetQ gene copy numbers in microcosms determined by NPs-DNA hybridization were positively correlated with numbers measured by real-time qPCR assay (R2 = 0.93). This high-throughput, non-PCR based NPs-DNA assay has potential for application in monitoring of antibiotic resistance and multiple resistance effects in the environment.