ABSTRACT
Cells from all domains of life release extracellular vesicles (EVs), packages that carry a cargo of molecules that participate in communication, co-ordination of population behaviours, virulence and immune response mechanisms. Mammalian EVs play an increasingly recognised role to fight infection, yet may also be commandeered to disseminate pathogens and enhance infection. EVs released by bacterial pathogens may deliver toxins to host cells, signalling molecules and new DNA to other bacteria, and act as decoys, protecting infecting bacteria from immune killing. In this review, we explore the role of EVs in infection from the perspective of both the pathogen and host, and highlight their importance in the host/pathogen relationship. We highlight proposed strategies for EVs in therapeutics, and call attention to areas where existing knowledge and evidence is lacking.
Subject(s)
Bacteria/immunology , Bacterial Infections/immunology , Extracellular Vesicles/immunology , Signal Transduction/immunology , Animals , Bacteria/metabolism , Bacteria/pathogenicity , Bacterial Infections/metabolism , Bacterial Infections/microbiology , Drug Resistance, Microbial/immunology , Extracellular Vesicles/metabolism , Host-Pathogen Interactions/immunology , Humans , Inflammation/immunology , Inflammation/metabolism , Inflammation/microbiology , Virulence/immunologyABSTRACT
Antibiotic resistance is a serious threat to public health. The empiric use of the wrong antibiotic occurs due to urgency in treatment combined with slow, culture-based diagnostic techniques. Inappropriate antibiotic choice can promote the development of antibiotic resistance. We investigated live/dead spectrometry using a fluorimeter (Optrode) as a rapid alternative to culture-based techniques through application of the LIVE/DEAD® BacLightTM Bacterial Viability Kit. Killing was detected by the Optrode in near real-time when Escherichia coli was treated with lytic antibiotics-ampicillin and polymyxin B-and stained with SYTO 9 and/or propidium iodide. Antibiotic concentration, bacterial growth phase, and treatment time used affected the efficacy of this detection method. Quantification methods of the lethal action and inhibitory action of the non-lytic antibiotics, ciprofloxacin and chloramphenicol, respectively, remain to be elucidated.
ABSTRACT
Flow cytometry (FCM) is based on the detection of scattered light and fluorescence to identify cells with characteristics of interest. Many flow cytometers cannot precisely control the flow through its interrogation point and hence the volume and concentration of the sample cannot be immediately obtained. Here we describe the optimization and evaluation of a bead-based method for absolute cell counting applicable to basic flow cytometers without specialized counting features. Prior to the application of this method to an unknown concentration of a species of bacteria, a calibration experiment should be completed to characterize limits of detection and range of linearity with respect to the plate count method. To demonstrate the calibration process, mixtures of Escherichia coli or Staphylococcus aureus with proportions of live and dead cells ranging from 0% to 100% were prepared. These samples were stained using nucleic acid-binding dyes, and 6 µm reference beads were added (LIVE/DEAD® BacLight kit). The calibration samples were analyzed using bead-based FCM as well as the agar plate count method, and the results from both methods were compared.
