Synthesis of vancomycin fluorescent probes that retain antimicrobial activity, identify Gram-positive bacteria, and detect Gram-negative outer membrane damage.

Antimicrobial resistance is an urgent threat to human health, and new antibacterial drugs are desperately needed, as are research tools to aid in their discovery and development. Vancomycin is a glycopeptide antibiotic that is widely used for the treatment of Gram-positive infections, such as life-threatening systemic diseases caused by methicillin-resistant Staphylococcus aureus (MRSA). Here we demonstrate that modification of vancomycin by introduction of an azide substituent provides a versatile intermediate that can undergo copper-catalysed azide-alkyne cycloaddition (CuAAC) reaction with various alkynes to readily prepare vancomycin fluorescent probes. We describe the facile synthesis of three probes that retain similar antibacterial profiles to the parent vancomycin antibiotic. We demonstrate the versatility of these probes for the detection and visualisation of Gram-positive bacteria by a range of methods, including plate reader quantification, flow cytometry analysis, high-resolution microscopy imaging, and single cell microfluidics analysis. In parallel, we demonstrate their utility in measuring outer-membrane permeabilisation of Gram-negative bacteria. The probes are useful tools that may facilitate detection of infections and development of new antibiotics.

Current and Emerging Technologies for the Detection of Norovirus from Shellfish.

Reports of norovirus infections associated with the consumption of contaminated bivalve molluscan shellfish negatively impact both consumers and commercial shellfish operators. Current virus recovery and PCR detection methods can be expensive and time consuming. Due to the lack of rapid, user-friendly and onsite/infield methods, it has been difficult to establish an effective virus monitoring regime that is able to identify contamination points across the production line (i.e., farm-to-plate) to ensure shellfish quality. The focus of this review is to evaluate current norovirus detection methods and discuss emerging approaches. Recent advances in omics-based detection approaches have the potential to identify novel biomarkers that can be incorporated into rapid detection kits for onsite use. Furthermore, some omics techniques have the potential to simultaneously detect multiple enteric viruses that cause human disease. Other emerging technologies discussed include microfluidic, aptamer and biosensor-based detection methods developed to detect norovirus with high sensitivity from a simple matrix. Many of these approaches have the potential to be developed as user-friendly onsite detection kits with minimal costs. However, more collaborative efforts on research and development will be required to commercialize such products. Once developed, these emerging technologies could provide a way forward that minimizes public health risks associated with shellfish consumption.

Fluorescent Trimethoprim Conjugate Probes To Assess Drug Accumulation in Wild Type and Mutant Escherichia coli.

Reduced susceptibility to antimicrobials in Gram-negative bacteria may result from multiple resistance mechanisms, including increased efflux pump activity or reduced porin protein expression. Up-regulation of the efflux pump system is closely associated with multidrug resistance (MDR). To help investigate the role of efflux pumps on compound accumulation, a fluorescence-based assay was developed using fluorescent derivatives of trimethoprim (TMP), a broad-spectrum synthetic antibiotic that inhibits an intracellular target, dihydrofolate reductase (DHFR). Novel fluorescent TMP probes inhibited eDHFR activity with comparable potency to TMP, but did not kill or inhibit growth of wild type Escherichia coli. However, bactericidal activity was observed against an efflux pump deficient E. coli mutant strain (ΔtolC). A simple and quick fluorescence assay was developed to measure cellular accumulation of the TMP probe using either fluorescence spectroscopy or flow cytometry, with validation by LC-MS/MS. This fluorescence assay may provide a simple method to assess efflux pump activity with standard laboratory equipment.