Mechanisms of antibiotic resistance in bacteria mediated by silver nanoparticles.

Silver nanoparticles (AgNPs) are widely used in industry, consumer products, and medical appliances due to their efficient antimicrobial properties. However, information on environmental toxicity and bacterial impact of these particles is not completely elucidated. Results showed that AgNPs produced growth inhibition and oxidative stress in bacteria Escherichia coli (gram negative) and Staphylococcus aureus (gram positive), with half-maximal inhibitory concentrations (IC50) of 12 and 7 mg/L, respectively. Surprisingly, bacteria pre-exposed to sublethal dose of AgNPs exhibited increased resistance toward antibiotics (ampicillin and Pen-Strep) with IC50 elevated by 3-13-fold. Further, AgNP pre-exposure raised the minimal inhibitory concentration and minimal biocidal concentration by two- to eightfold when cells were challenged with antibiotics with diverse mechanisms of action (penicillin, chloramphenicol, and kanamycin). Interestingly, we found that upon exposure to ampicillin, strains pretreated with AgNPs exhibited lower levels of membrane damage and oxidative stress, together with elevated levels of intracellular ATP relative to untreated cells. Bacterial reverse mutation assay (Ames test) showed that AgNPs are highly mutagenic, consistent with further assays demonstrating abiotic reactive oxygen species (ROS) generation and intrinsic DNA cleavage activity in vitro of AgNPs. Overall, our results suggest that AgNPs enhance bacterial resistance to antibiotics by promoting stress tolerance through induction of intracellular ROS. Our data suggest potential consequences of incidental environmental exposure of bacteria to AgNPs and indicate the need to regulate use and disposal of AgNPs in industry and consumer products.

Synthesis and fluorescence properties of N-substituted 1-cyanobenz[f]isoindole chitosan polymers and nanoparticles for live cell imaging.

Highly fluorescent N-substituted 1-cyanobenz[f]isoindole chitosans (CBI-CSs) with various degrees of N-substitution (DS) were synthesized by reacting chitosan (CS) with naphthalene-2,3-dicarboxaldehyde (NDA) in the presence of cyanide under mild acidic conditions. Introduction of 1-cyanobenz[f]isoindole moieties into the CS backbone resulted in lowering of polymer thermal stability and crystallinity. The fluorescence quantum yield (Φf) of CBI-CS was found to be DS- and molecular-weight-dependent, with Φf decreasing as DS and molecular weight were increased. At similar DS values, CBI-CS exhibited 26 times higher Φf in comparison with fluorescein isothiocyanate-substituted chitosan (FITC-CS). CBI-CS/TPP nanoparticles were fabricated using an ionotropic gelation method in which pentasodium triphosphate (TPP) acted as a cross-linking agent. CS and CBI-CS exhibited low cytotoxicity to normal skin fibroblast cells over a concentration range of 0.1-1000 µg/mL, while an increased cytotoxicity level was evident in CBI-CS/TPP nanoparticles at concentrations greater than 100 µg/mL. In contrast with CBI-CS polymers, the CBI-CS/TPP nanoparticles exhibited lower fluorescence; however, confocal microscopy results showed that living normal skin fibroblast cells became fluorescent on nanoparticle uptake. These results suggest that CBI-CS and fabricated nanoparticles thereof may be promising fluorescence probes for live cell imaging.

One-tube multiplex PCR method for rapid identification of mycobacterium tuberculosis.

A rapid, inexpensive, simple, and accurate multiplex polymerase chain reaction (PCR) was developed in a single tube for identification of Mycobacterium tuberculosis. Assessment of sensitivity and specificity of simple PCR was performed with 116 strains of M. tuberculosis complex (MTC) and 144 strains of nontuberculous mycobacteria (NTM) compared with the biochemical method. Specific amplification of KS4, MTC-specific DNA fragment, was found in 98% (114/116) of MTC and not detected in 99% (143/144) of NTM. Amplification of the mtp40 gene revealed 95% sensitivity (100/105 strains of M. tuberculosis) and 77% specificity (not found in 119/155 mycobacterial strains). A multiplex PCR method based on the combination of KS4- and mtp40-derived primers was used for identification of M. tuberculosis. Crude DNA from slow growing mycobacteria with cream rough colonies that showed both 768-bp amplified product for KS4 and 396-bp for mtp40 was identified as M. tuberculosis whereas that from MTC gave only the 768-bp product.