Understanding Antibiotic Resistances
Fluorescent Dye Facilitates the Analysis of the Survival Strategies of Disease-causing Agents
- Fig. 1: Left: chemical structure of TramTO. Right: fluorescently labeled bacteria, which were stained by TramTO and visualized by confocal live fluorescence microscopy in the red wavelength range.
- Fig. 2: Left: schematic representation of the staining of bacteria using TramTO to study the interaction with human cells, such as immune cells. Right: Microscopic image of a human phagocyte (macrophage) interacting with a TramTO-labeled bacterium (red).
The exessive of antimicrobial drugs has led to the increasing emergence of antibiotic resistant bacterial strains. This development becomes particularly dangerous when pathogenic germs that responded well to antibiotics in the past develop drug resistances and thus impede the successful treatment of patients [1,2].
In the case of pneumonia-causing agents, such as Klebsiella, resistances to all common antibiotics have emerged, rendering effective treatment of infested patients increasingly difficult. To overcome the current antibiotic crisis, the World Health Organization has given top priority to research on these bacteria . However, suitable tools to investigate the strategies of multidrug-resistant bacterial pathogens are often not available.
Interactions of Microorganisms with Cells of the Immune System
Bacterial infections are usually warded off or kept in check by the immune system. Several infectious agents however have learned to evade the immune defense and may cause long-lasting infections and associated complications. To understand the survival strategies of pathogens, it is necessary to understand how the microorganisms interact with cells of the immune system. A common approach is the fluorescent labelling of the bacteria, to make them visible by techniques such as high-resolution fluorescence microscopy or flow cytometry to study their interaction with human cells in detail. In the past, bacteria were genetically modified to produce fluorescent proteins, e.g. Green Fluorescent Protein (GFP) [4-6]. Infected human host cells interacting with the fluorescent bacteria were then visualized and enriched by fluorescence activated cell sorting (FACS) to study bacteria-host interplay in detail. For example, this FACS-based strategy has previously been used to disclose the genetic programs employed by the major bacterial pathogen Salmonella Typhimurium to establish an infection and manipulate the host immune response to the benefit of the bacterium .
While bacterial fluorescent protein labelling has unquestionably facilitated the dissection of bacterial disease-causing strategies, this approach is not applicable to all bacterial strains.
Specifically, the introduction of foreign genes such as the GFP-gene into a bacterial genome is typically a very inefficient process, so that only very few bacteria in a growth culture end up having the desired genetic modification. To enable enrichment of the few bacteria which carry the foreign gene, e.g. a GFP gene, an antibiotic resistance gene is usually coupled to it. This way, by adding antibiotics, all bacteria that have not taken up and incorporated the foreign gene into their DNA can be eliminated. Or in other words: only those bacteria survive that have taken up the foreign gene (along with the information for the antibiotics resistance). However, a major drawback of this widely used method for bacterial fluorescent labelling is that it can only work in bacterial strains which were not previously antibiotic-resistant. In addition, the procedure is too time consuming for an implementation in clinical routine analysis.
Fluorescent Labelling of Bacteria
A new approach has now been developed which allows a reliable and fast fluorescent labelling of bacteria without the need for genetic modification . A major advantage of this approach is that it also works for those bacteria that are multi-resistant against standard antibiotics. The method is based on a novel fluorescent dye, called TramTO. This dye fulfills the criteria of a straight-forward and scalable synthesis approach, low toxicity, long-lasting bacterial labelling and optimal fluorescence emission properties for FACS and microscopy analysis.
TramTO is a so-called cyanine dye whose chemical structure has been modified to confer improved properties compared to existing alternatives regarding solubility, toxicity, light emission wavelength and longevity of the bacterial labelling. The new dye emits at a red wavelength range, which allows robust detection by common microscopes and flow cytometers. The fluorescent labelling of the microorganisms takes place by simple addition of the TramTO dye to live bacterial cultures. Importantly, rapid and long-lasting fluorescent labelling of different germs such as Escherichia coli or multiresistant Klebsiella was achieved without restricting the viability of the microorganisms. These are decisive advantages over previously available dyes, which allowed either only a short-lived fluorescent labelling or were toxic to the microorganisms, so that their survival strategies could not be studied.
Exemplarily, the interaction of antibiotic-sensitive and -resistant Klebsiella with human immune cells was compared by using TramTO-labelled bacteria. First, it could be shown that uptake of both Klebsiella strains by the immune cells is significantly reduced compared to non-disease-causing E. coli bacteria. Additionally, it was observed that the antibiotic-sensitive Klebsiella interact differently with the major phagocytes of the immune system than the resistant bacteria: while the antibiotic-resistant Klebsiella were still taken up by the immune cells at a reduced rate, the antibiotics-susceptible strain was barely taken up. In summary, this suggests that Klebsiella bacteria can escape the uptake and destruction by immune cells, with antibiotic susceptible and insensitive strains taking advantage of this ability to varying degrees. Such knowledge might be used to develop tailored treatments of infected patients.
In the future, TramTO could be employed to rapidly and profoundly analyze the interaction of patient-derived bacteria with human host cells to enable tailored treatment strategies which take into account the specific pathogenic strategies of the germs. Since it could be shown that TramTO is a light-up prove, exhibiting full fluorescence emission only after binding to the DNA of organisms, further applications are conceivable, for example in the area of nucleic acid staining. Currently, the teams are still looking for suitable industrial partners to make TramTO available to a broader research community.
Leon N. Schulte, Olalla Vazquez
Institute for Lung Research
Philipps-University of Marburg, Marburg, Germany
Department of Chemistry
Philipps-University of Marburg, Marburg, Germany
 Levy, S.B. and B. Marshall, Antibacterial resistance worldwide: causes, challenges and responses. Nat Med, 2004. 10(12 Suppl): p. S122-9.
 Peleg, A.Y. and D.C. Hooper, CURRENT CONCEPTS Hospital-Acquired Infections Due to Gram-Negative Bacteria. New England Journal of Medicine, 2010. 362(19): p. 1804-1813.
 WHO, Antimicrobial Resistance: Global Report on Surveillance. 2014.
 Westermann, A.J., et al., Dual RNA-seq unveils noncoding RNA functions in host-pathogen interactions. Nature, 2016. 529(7587): p. 496-501.
 Valdivia, R.H., et al., Applications for green fluorescent protein (GFP) in the study of host-pathogen interactions. Gene, 1996. 173(1): p. 47-52.
 Schulte, L.N., et al., Analysis of the host microRNA response to Salmonella uncovers the control of major cytokines by the let-7 family. EMBO J, 2011. 30(10): p. 1977-89.
 Schulte, L.N., et al., A Far-Red Fluorescent DNA Binder for Interaction Studies of Live Multidrug-Resistant Pathogens and Host Cells. Angew Chem Int Ed Engl, 2018. 57(36): p. 11564-11568.