MRLS is supported by an Australian Postgraduate Award (APA) and an Institute for Molecular Biosciences Research Advancement Award. Wanida Phetsang was supported by UQ International Scholarship (UQI) and IMB Postgraduate Award (IMBPA). MAC is a NHMRC principle research fellow (APP1059354) and also holds a fractional professorial research fellow appointment at the University of Queensland, with his remaining time as CEO of Inflazome Ltd, a company developing drugs to address clinical unmet needs in inflammatory disease. MATB is supported in part by Wellcome Trust Strategic Grant WT1104797/Z/14/Z and NHMRC Development grant APP1113719. Microscopy was performed at the Australian Cancer Research Foundation (ACRF)/Institute for Molecular Bioscience Cancer Biology Imaging Facility, which was established with the support of the ACRF.

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H., Dunham, S., Stover, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+liquid+chromatography/mass+spectrometry-based+drug+accumulation+assay+in+Pseudomonas+aeruginosa.">Development of a liquid chromatography/mass spectrometry-based drug accumulation assay in Pseudomonas aeruginosa.</a> Analytical Biochemistry. 385 (2), 321-325 (2009).</li><li>Bhat, J., Narayan, A., Venkatraman, J., Chatterji, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LC-MS+based+assay+to+measure+intracellular+compound+levels+in+Mycobacterium+smegmatis:+Linking+compound+levels+to+cellular+potency.">LC-MS based assay to measure intracellular compound levels in Mycobacterium smegmatis: Linking compound levels to cellular potency.</a> Journal of Microbiological Methods. 94 (2), 152-158 (2013).</li><li>Davis, T. 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R. L., Butler, M. S., Phetsang, W., Cooper, M. A., Blaskovich, M. A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+Antibiotics:+New+Research+Tools+to+Fight+Antibiotic+Resistance.">Fluorescent Antibiotics: New Research Tools to Fight Antibiotic Resistance.</a> Trends in Biotechnology. 36 (5), 523-536 (2018).</li><li>Piddock, L. J. V., Johnson, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accumulation+of+10+fluoroquinolones+by+wild-type+or+efflux+mutant+Streptococcus+pneumoniae.">Accumulation of 10 fluoroquinolones by wild-type or efflux mutant Streptococcus pneumoniae.</a> Antimicrobial Agents and Chemotherapy. 46 (3), 813-820 (2002).</li><li>Mortimer, P. G. S., Piddock, L. J. 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Z., Nikaido, H., Poole, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+MexA-MexB-OprM+in+antibiotic+efflux+in+Pseudomonas+aeruginosa.">Role of MexA-MexB-OprM in antibiotic efflux in Pseudomonas aeruginosa.</a> Antimicrobial Agents and Chemotherapy. 39 (9), 1948-1953 (1995).</li><li>Vince, R., Weiss, D., Pestka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Binding+of+N-substituted+erythromycylamines+to+ribosomes.">Binding of N-substituted erythromycylamines to ribosomes.</a> Antimicrobial Agents and Chemotherapy. 9 (1), 131-136 (1976).</li><li>Vazquez, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiotics+affecting+chloramphenicol+uptake+by+bacteria+Their+effect+on+amino+acid+incorporation+in+a+cell-free+system.">Antibiotics affecting chloramphenicol uptake by bacteria Their effect on amino acid incorporation in a cell-free system.</a> Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis. 114 (2), 289-295 (1966).</li><li>Phetsang, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+azido-oxazolidinone+antibiotic+for+live+bacterial+cell+imaging+and+generation+of+antibiotic+variants.">An azido-oxazolidinone antibiotic for live bacterial cell imaging and generation of antibiotic variants.</a> Bioorganic and Medicinal Chemistry. 22 (16), 4490-4498 (2014).</li><li>Phetsang, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+trimethoprim+conjugate+probes+to+assess+drug+accumulation+in+wild+type+and+mutant+Escherichia+coli.">Fluorescent trimethoprim conjugate probes to assess drug accumulation in wild type and mutant Escherichia coli.</a> ACS Infectious Diseases. 2 (10), 688-701 (2016).</li><li>Stone, M. 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The authors have nothing to declare.

The creation of a successful fluorescent antibiotic probe must begin with careful planning and consideration of the SAR of the parent drug. If the SAR is not known or fully explored, several options may need to be tested to find a site which may be selectively modified without abolishing biological activity. Once a site/s have been identified, the installation of a linker moiety is often essential in order to provide steric spacing between the biological site of action and the inactive fluorophore. Care must be taken that the reaction used to attach the linker to the antibiotic leaves a bio-stable functional group, avoiding, for example, esters that are susceptible to cleavage by esterases in vivo. Depending on the pharmacodynamic and pharmacokinetic profile of the antibiotic, a simple alkyl linker may be used, or else a less lipophilic option such as a polyethylene glycol (PEG) linker should be considered. With the linker attached, the antibacterial activity should be assessed to ensure the MICs against relevant bacteria are similar to the parent compound.
In this work, we recommend the use of Huigsen azide-alkyne [3+2] dipolar cycloaddition (click chemistry, see Figure 1) to ligate fluorophore to antibiotic, for a number of reasons. Click reactions are highly selective, meaning that protection of reactive groups on the antibiotic is not necessary, and further, the reaction leaves a stable, biocompatible triazole moiety. The azide component is introduced to the antibiotic portion in our procedures, as this is generally more easily accomplished with a variety of structural types than introduction of an alkyne. The syntheses of two alkyne-derivatized fluorophores are described here, though others could be explored if desired. NBD and DMACA were chosen due to their small size, minimizing the possibility of interfering with cell penetration and target interaction. The click reaction itself is carried out using copper catalysis, where either Cu2+ (CuSO4, with an ascorbic acid reducing agent) or Cu+ (CuI) may be used as the starting reagent. Following purification (Figure 2), the MICs should then be tested as with the azide. Even with careful consideration of fluorophore choice and site of attachment, it is possible that poor antibiotic activity will be observed. This does not, however, mean that an inactive probe is without use. As shown with the TMP probes, compounds with poor antibacterial activity may still bind to the same target as the parent drug. This can enable studies on the mode of action and examination of phenomena leading to resistance, such as efflux.
As outlined in the protocols section, it is possible to analyze bacterial labeling by the fluorescent antibiotics using either a simple spectrophotometry assay (Figure 3) or flow cytometry (Figure 4). Both methods are capable of quantifying cellular accumulation, and by lysing cells and examining fluorescence localization in lysate, it is possible to assess intracellular accumulation. In this protocol, the use of lysozyme for cell lysis is described, as this is a rapid, universal technique. Other lysis conditions, such as overnight treatment with glycine-HCl7, have also been successfully used. Using this technique, it is possible to study the impact of efflux on antibiotic cellular accumulation, which is a major mechanism of resistance. If efflux is indeed present in the bacteria, a lack of intracellular accumulation will be observed, though this can be rescued using an efflux inhibitor like CCCP.
Microscopy may also be carried out to visually inspect probe localization in different bacteria, garnering information on mode of action, and potentially also resistance (see Figure 5 for representative examples). In order to see localization within bacteria, a high resolution confocal microscope is required, equipped with capabilities such as SIM (structured illumination microscopy), SR-SIM (superresolution-SIM), Airyscan, or STED (stimulated emission depletion). Furthermore, high-performance cover slips should be used, and post-imaging analysis carried out on an appropriate software (e.g., FIJI, Zen, or Imaris). Localization of probes is compared to dyes that stain specific architectures, such as Hoechst-33342 (blue, nucleic acid), Syto-9 (green, nucleic acid), and FM4-64FX (red, membrane). The choice of dyes should be made to match the fluorescent antibiotic, so that each color used has minimal spectral overlap. In order to obtain the best possible images, optimization may be required. For example, if bacteria are too crowded on the slide, take only part of the suspended pellet, then dilute with more mounting medium. In contrast, if bacteria are too sparse on the slide, simply start with more bacteria. In this protocol, the use of a thermo reversible gel that is compatible with live cells (e.g., Cygel) is recommended for live cell imaging, as it immobilizes bacteria (including motile bacteria), but other mounting media or agarose have also been successfully used.
Overall, despite challenges that may be faced in the preparation of a biologically active fluorescent antibiotic derivatives, the simplicity of their use and their versatility make these probes attractive tools for research in AMR. Future work using fluorescent antibiotics has the potential to provide insight into mechanisms of antibiotic resistance, improve our understanding of how current antibiotics operate, and aid the development of better drugs.

Antimicrobial resistance (AMR) is a rising crisis which poses a major threat to human health around the world. Resistance to most antibiotics has been reported, and infections caused by bacteria resistant to all clinically available drugs are emerging. In order to combat the rise of AMR, we need to increase our understanding of this multifaceted phenomenon and the underlying mechanisms and interactions between antibiotics and bacteria. One aspect that has been historically poorly understood is the permeation of antibiotics into bacteria, along with the phenomena of accumulation and efflux. This knowledge is crucial in designing new drugs and understanding mechanisms of resistance. Hence, this plays a critical role in AMR research.
There are two main approaches that may be taken in order to measure antibiotic concentration: measuring the drug directly or tagging with a moiety designed to facilitate quantification. Although tagging the antibiotic improves detection, this can perturb the biological activity of the drug, such as antimicrobial activity and permeability. This is not a problem for untagged methods; however, detection can be challenging. In the past few years, technological advancements have led to a boom in research utilizing mass spectrometry (MS) to directly measure the antibiotic concentration in bacteria1,2,3,4,5,6,7. These studies have shown that it is possible to study intracellular accumulation in a variety of bacteria, with gram-negative bacteria the most widely studied. Quantification of molecule permeability has then been linked to activity and used to inform drug development2,3,4, though caution must be taken when directly conflating accumulation and target activity5. Prior to MS development, the only antibiotics whose concentration could be directly measured were those possessing intrinsic fluorescence, such as tetracycline and the quinolones8,9,10,11. Although obviously limited in scope, accumulation and efflux were examined and quantified, illustrating the usefulness of fluorescence-based quantification.
Tagged antibiotics have been used for many decades to study distributions, modes of action, and resistance, with radioactive and fluorescent tags being common. Radio-tagged probes have the advantage of being almost identical to the parent compound, hence the biological activity is unlikely to be significantly different. Isotopes such as 3H, 14C, and 15N have been frequently used due to the prominence of these elements in antibiotics, and a variety of antibiotic scaffolds have been examined1,10,12,13. While the detection of radio-probes is simple, there are a number of logistical concerns (e.g., safety, isotope half-life) that have limited the use of this approach. Another strategy is fluorescently-tagged antibiotics. These probes can be used to examine the distribution and modes of action and resistance of the parent drug, using simpler technology than MS and without the logistical problems of radiation8. The main drawback to this approach is that antibiotics are generally relatively small molecules, hence the introduction of a fluorescent moiety poses a significant chemical change. This alteration can impact physiochemical properties and antibacterial activity. Therefore, care must be taken to assess these factors in order to generate results representative of the parent antibiotic.
In this work, a method is described to synthesize, assess, and use fluorescent antibiotics, as in our previous publications14,15,16. Through previous work, a number of fluorescent antibiotics have been prepared and used for a variety of purposes (see Stone et al.8). In order to minimize the likelihood of impacting biological activity, very small fluorophores are used in this work: nitrobenzoxadiazole (NBD, green) and 7-(dimethylamino)-2-oxo-2H-chromen-4-yl (DMACA, blue). Further, the assessment of antibacterial activity using the microbroth dilution minimum inhibition concentration (MIC) assay is described, so that the effect of modifications on activity can be measured. These fluorescently-tagged probes can be used in spectrophotometric assays, flow cytometry, and microscopy. The range of possible applications is where the advantage of fluorescent antibiotics lies. Cellular accumulation can be quantified, categorized, and visualized, something not possible using MS alone. It is hoped that the knowledge gained through the use of fluorescent antibiotics will aid in our understanding of resistance, and the fight against AMR.

<table><tbody><tr><td>3-(dimethylamino)phenol</td><td>Alfa-Aesar</td><td>B23067</td><td></td></tr><tr><td>4-chloro-7-nitro-benzofuran</td><td>Sigma-Aldrich</td><td>163260-5G</td><td></td></tr><tr><td>Amicon Ultra-0.5 centrifugal filter unit with Ultracel- 10 membrane</td><td>Merck</td><td>UFC501096</td><td></td></tr><tr><td>Atlantis Prep T3 OBD (100 A, 5 uM, 10x250 mm)</td><td>Waters</td><td>186008205</td><td></td></tr><tr><td>Atlantis T3 column (100 A, 5 uM, 2.1 &amp;times; 50 mm)</td><td>Waters</td><td>186003734</td><td></td></tr><tr><td>Bruker Avance 600 MHz spectrometer</td><td>Bruker</td><td></td><td></td></tr><tr><td>Buchi Reveleris C18 12g Cartridge</td><td>Buchi</td><td>BUC145152103</td><td></td></tr><tr><td>CCCP</td><td>Sigma-Aldrich</td><td>C2759</td><td></td></tr><tr><td>Celite 545</td><td>Sigma-Aldrich</td><td>22140-5KG-F</td><td></td></tr><tr><td>Cygel</td><td>ABCAM</td><td>Ab109204</td><td></td></tr><tr><td>Elyra PS,1 SIM/STORM confocal microscope</td><td>Zeiss</td><td></td><td></td></tr><tr><td>FM4-64FX, fixable analog of FM&amp;trade; 4-64 membrane stain</td><td>Life Technologies Australia Pt</td><td>F34653</td><td></td></tr><tr><td>Gallios flow cytometer</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>Gamma 2-16 LSCplus lyophilise</td><td>CHRIST</td><td></td><td></td></tr><tr><td>Gilson HPLC 2020</td><td>Gilson</td><td></td><td></td></tr><tr><td>Hanks&#039; Balanced Salt solution, Modified, with sodium bicarbonate, without phenol red, calcium chloride and magnesium sulfate, liquid, sterile-filtered, suitable for cell culture</td><td>Sigma-Aldrich</td><td>H6648-500ML</td><td></td></tr><tr><td>Hettich Zentrifugen Rotofix 32</td><td>Hettich</td><td></td><td></td></tr><tr><td>High performance #1.5 cover slips (18 x 18 mm)</td><td>Schott/Zeiss</td><td>474030-9000-000</td><td></td></tr><tr><td>Hoechst 33342, Trihydrochloride, Trihydrate - Fluo</td><td>Life Technologies Australia Pt</td><td>H21492</td><td></td></tr><tr><td>LB</td><td>AMRESCO</td><td>J106</td><td></td></tr><tr><td>Leica STED 3X Super Resolution Microscope with White Light Laser excitation</td><td>Leica</td><td></td><td></td></tr><tr><td>Lysozyme from chicken egg white&lt;br/&gt; lyophilized powder</td><td>Sigma-Aldrich</td><td>L6876</td><td></td></tr><tr><td>Mueller Hinton II Broth Cation adjusted</td><td>Becton Dickinson</td><td>212322</td><td></td></tr><tr><td>Propargylamine</td><td>Sigma-Aldrich</td><td>P50900-5G</td><td></td></tr><tr><td>Reveleris GRACE MPLC</td><td>Buchi</td><td></td><td></td></tr><tr><td>Shimadzu LCMS-2020</td><td>Shimadzu</td><td></td><td></td></tr><tr><td>Sigma 1-15 Microcentrifuge</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Silica gel 60 (0.040-0.063 mm) for column chromatography (230-400 mesh ASTM)</td><td>Merck</td><td>1093859025</td><td></td></tr><tr><td>SYTO 9 Green Fluorescent Nucleic Acid Stain</td><td>Life Technologies Australia Pt</td><td>S34854</td><td></td></tr><tr><td>TECAN Infinite M1000 PRO</td><td>TECAN</td><td></td><td></td></tr></tbody></table>

visualization of bacterial resistance using fluorescent antibiotic probes

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant advantage over other methods. To prepare these probes, azide derivatives of antibiotics are synthesized, then coupled with alkyne-fluorophores using azide-alkyne dipolar cycloaddition by click chemistry. Following purification, the antibiotic activity of the fluorescent antibiotic is tested by minimum inhibitory concentration assessment. In order to study bacterial accumulation, either spectrophotometry or flow cytometry may be used, allowing for much simpler analysis than methods relying on radioactive antibiotic derivatives. Furthermore, confocal microscopy can be used to examine localization within the bacteria, affording valuable information about mode of action and changes that occur in resistant species. The use of fluorescent antibiotic probes in the study of antimicrobial resistance is a powerful method with much potential for future expansion.

Figure 1 Illustrates the key click chemistry reaction (A) for the preparation of the fluorescent antibiotics, and with (B) examples of structures of our published fluorescent antibiotics based on ciprofloxacin (cipro), trimethoprim (TMP), and linezolid. These probes were all synthesized from the corresponding antibiotics via an azide intermediate. They were then coupled to the NBD and DMACA fluorophores, each functionalized with an alkyne.
Figure 2 shows example LCMS traces from a ciprofloxacin-N3 and NBD-alkyne click reaction, where the azide eluted at 3.2 min and the product at 3.8 min. Comparing 1 and 2 shows how the progress of the click reaction could be followed by the disappearance of the azide peak (by UV or MS detector). Spectra 3 demonstrate the impact of purification, with erroneous peaks disappearing from the MS and UV traces. Both purity and reaction progress could be quantified by the integration of the product peak and any impurity peaks.
Figure 3 demonstrates typical results from the assessment of intracellular accumulation by fluorescence spectroscopy in the presence and absence of efflux. In this experiment, E. coli was treated with TMP-NBD with or without the addition of CCCP, which collapses the proton motive force (PMF). The intracellular fluorescence of the bacteria was significantly higher when pretreated with CCCP, indicating that efflux reduced the accumulation in these bacteria. This experiment was repeated using bacteria deficient in tolC, displaying the capacity of this assay to examine the impact of individual efflux pump components. In this case, although there was an increase in intracellular fluorescence compared to the wild type bacteria, CCCP accumulation still increased. These findings indicate that tolC takes part inTMP efflux but is not the sole PMF-drive pump involved.
Figure 4 shows the result of the same experiment as Figure 2, but with the accumulation measured by flow cytometry instead of spectroscopy. The same data trends were observed, demonstrating that either technique may be used to study the phenomenon of efflux mediated intracellular accumulation.
Figure 5 shows representative confocal microscopy images of gram-positive (S. aureus) and gram-negative bacteria (E. coli) labeled with TMP-NBD (1) and cipro-NBD (2 + 3) fluorescent probes, respectively. In both cases, the red membrane dye FM4-64FX was added in order to compare co-localization. For TMP-NBD, the blue nucleic acid dye Hoechst-33342 was also used. By overlaying these images, the localization of the antibiotic in the bacteria was visualized. Comparing panels 2 and 3 shows how the impact of efflux was examined, with the efflux inhibitor CCCP used in 2, resulting in intracellular accumulation. In panel 3, no CCCP was added. Hence, efflux is active and no probe accumulation was seen.
Figure 6 shows representative confocal microscopy images of Gram-positive (S. aureus) bacteria labeled with DMACA-labeled oxazolidinone probe Lz-NBD. The red membrane dye FM4-64FX was added in order to compare co-localization, and the green nucleic acid dye Hoechst-33342 was also used. By overlaying these images, the localization of the antibiotic in the bacteria was visualized, showing internal localization distinct from the membrane and nucleic acid.
Table 1 shows MIC values for three series of fluorescent antibiotics, ciprofloxacin, trimethoprim (TMP), and linezolid (Lz), with data presented for the parent antibiotic, NBD and DMACA derivatives of each. Representative species for each antibiotic were chosen, including both gram-positive and gram-negative. For the ciprofloxacin series, both fluorescent probes lost antibiotic activity compared to the parent drug, but retained some activity against all species. Similarly, the linezolid probes lost some activity, but remained a moderate to weak antibiotic. The TMP probes lost almost all activity against wild type bacteria, but were active against efflux deficient E. coli, indicating that the loss of antibacterial activity was due to lack of accumulation.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60743/60743fig01.jpg" /> 
Figure 1: Synthesis and structures of antibiotic-derived probes. (A) The general reaction scheme for the synthesis of fluorescent antibiotic probes from azide-antibiotics and alkyne-fluorophores. (B) The structures of our published probes based on ciprofloxacin, trimethoprim, and linezolid. <a href="https://www.jove.com/files/ftp_upload/60743/60743fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60743/60743fig02.jpg" /> 
Figure 2: Measurement of antibiotic-derived probe purity by LCMS. Analytical LCMS traces from (1) incomplete, (2) complete, and (3) HPLC purified ciprofloxacin-N3 + NBD-alkyne click reactions demonstrating the disappearance of starting material upon reaction completion, and miscellaneous peaks on purification. A = UV-Vis trace (absorbance at 250 nm), B = MS trace (positive and negative mode). <a href="https://www.jove.com/files/ftp_upload/60743/60743fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60743/60743fig03.jpg" /> 
Figure 3: Plate reader measurement of antibiotic-derived probe accumulation. Fluorescence spectroscopic measurement of cellular accumulation of TMP-NBD (50 &#956;M) in wild type (1, ATCC 25922) and &#916;tolC (2, ATCC 25922) E. coli incubated (A) with and (B) without addition of CCCP (100 &#956;M). Statistical significance (**p &#8804; 0.01; ***p &#8804; 0.001) is shown between the absence or presence of CCCP and between wild type and &#916;tolCE. coli. Data reported are the mean &#177; SD for three experiments. This figure is adapted from our previous publication15, and illustrates the use of spectroscopy to elucidate the role of efflux on intracellular accumulation. <a href="https://www.jove.com/files/ftp_upload/60743/60743fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60743/60743fig04.jpg" /> 
Figure 4: Flow cytometry measurement of antibiotic-derived probe accumulation. Flow cytometry measurement of cellular accumulation using TMP-NBD in in wild type (1, ATCC 25922) and &#916;tolC (2, ATCC 25922) E. coli incubated with and without addition of CCCP (100 &#956;M). Median fluorescence activity is shown from 10,000 bacterial events, Statistical significance (***, p &#8804; 0.001; ****, p &#8804; 0.0001) is shown between the absence and presence of CCCP and between wild type and &#916;tolCE. coli. Data reported are the mean &#177; SD for three experiments. This figure is adapted from our previous publication15, and illustrates the use of flow cytometry to elucidate the role of efflux on intracellular accumulation. <a href="https://www.jove.com/files/ftp_upload/60743/60743fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60743/60743fig05.jpg" /> 
Figure 5: Confocal microscopy visualization of NBD-probe localization. Confocal microscopy images of 1) live S. aureus labeled with Hoechst-33342 (blue, nucleic acid), TMP-NBD (green), FM4-64FX (red, membrane), and overlaid; 2) live E. coli treated with CCCP (efflux inhibitor) labeled with cipro-NBD (green), FM4-64FX (red, membrane), and overlaid; 3) live E. coli labeled with cipro-NBD (green), FM4-64FX (red, membrane), and overlaid. This figure is adapted from our previous publications15,16, and illustrates the use of microscopy to examine probe localization, including the impact of efflux. <a href="https://www.jove.com/files/ftp_upload/60743/60743fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/60743/60743fig06.jpg" /> 
Figure 6: Confocal microscopy visualization of DMACA-probe localization. Confocal microscopy images of live S. aureus labeled with oxazolidinone probe Lz-DMACA (blue), Sytox green (green, nucleic acid), and FM4-64FX (red, membrane). <a href="https://www.jove.com/files/ftp_upload/60743/60743fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="3"></td>
			<td colspan="9">MIC (&#181;g/mL)</td>
		</tr>
		<tr>
			<td colspan="2">Species</td>
			<td>Strain</td>
			<td>Cipro</td>
			<td>Cipro-NBD</td>
			<td>Cipro-DMACA</td>
			<td>TMP</td>
			<td>TMP-NBD</td>
			<td>TMP-DMACA</td>
			<td>Linezolid (Lz)</td>
			<td>Lz-NBD</td>
			<td>Lz-DMACA</td>
		</tr>
		<tr>
			<td colspan="2">Staphylococcus aureus</td>
			<td>ATCC 25923</td>
			<td>0.125 - 0.5</td>
			<td>32 - &#8805;64</td>
			<td>16</td>
			<td>1</td>
			<td>16</td>
			<td>&#62;64</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2"></td>
			<td>ATCC 43300</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>1</td>
			<td>16</td>
			<td>&#62;64</td>
		</tr>
		<tr>
			<td colspan="2">Streptococcus pneumoniae</td>
			<td>ATCC 700677</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>1</td>
			<td>4</td>
			<td>64</td>
		</tr>
		<tr>
			<td colspan="2">Enterococcus faecium</td>
			<td>ATCC 35667</td>
			<td>1 - 8</td>
			<td>32</td>
			<td>32 - &#8805;64</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2"></td>
			<td>ATCC 51559</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>2</td>
			<td>16</td>
			<td>32</td>
		</tr>
		<tr>
			<td colspan="2">Klebsiella pneumoniae</td>
			<td>ATCC 13883</td>
			<td>0.015 - 0.06</td>
			<td>8 - 16</td>
			<td>8 - 32</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">Pseudomonas aeruginosa</td>
			<td>ATCC 27853</td>
			<td>0.25 - 1</td>
			<td>32 - &#8805;64</td>
			<td>32 - &#8805;64</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">Escherichia coli</td>
			<td>ATCC 25922</td>
			<td>&#8804;0.004</td>
			<td>8</td>
			<td>2</td>
			<td>0.5</td>
			<td>&#62;64</td>
			<td>&#62;64</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2"></td>
			<td>Mutant &#916;tolC</td>
			<td></td>
			<td></td>
			<td></td>
			<td>0.125</td>
			<td>0.25</td>
			<td>2</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1. Antibiotic activities of fluorescent antibiotic probes based on ciprofloxacin, trimethoprim, and linezolid against appropriate clinically relevant bacterial strains, as measured by broth microdilution MIC assays. In most cases, the probes lost some activity compared to the parent drug, but retained some measurable antibiotic potency (sufficient to be useful in further studies).

Watch this Scientific Journal Video about Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes at JoVE.com

Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes

Fluorescently tagged antibiotics are powerful tools that can be used to study multiple aspects of antimicrobial resistance. This article describes the preparation of fluorescently tagged antibiotics and their application to studying antibiotic resistance in bacteria. Probes can be used to study mechanisms of bacterial resistance (e.g., efflux) by spectrophotometry, flow cytometry, and microscopy.

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant ...

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Research

JoVE Journal

Immunology and Infection

12.5K Views.  The University of Queensland. Fluorescently tagged antibiotics are powerful tools that can be used to study multiple aspects of antimicrobial resistance. This article describes the preparation of fluorescently tagged antibiotics and their application to studying antibiotic resistance in bacteria. Probes can be used to study mechanisms of bacterial resistance (e.g., efflux) by spectrophotometry, flow cytometry, and microscopy.

Video: Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes

Monitoring Spatial Segregation in Surface Colonizing Microbial Populations

Microbes provide an intriguing system to study social interaction among individuals within a population. The short generation times and relatively simple genetic modification procedures of microbes facilitate the development of the sociomicrobiology field. To assess the fitness of certain microbial species, selected strains or their genetically modified derivatives within one population, can be fluorescently labelled and tracked using microscopy adapted with appropriate fluorescence filters. Expanding colonies of diverse microbial species on agar media can be used to monitor the spatial distribution of cells producing distinctive fluorescent proteins.
Here, we present a detailed protocol for the use of green- and red-fluorescent protein producing bacterial strains to follow spatial arrangement during surface colonization, including flagellum-driven community movement (swarming), exopolysaccharide- and hydrophobin-dependent growth mediated spreading (sliding), and complex colony biofilm formation. Non-domesticated isolates of the Gram-positive bacterium, Bacillus subtilis can be utilized to scrutinize certain surface spreading traits and their effect on two-dimensional distribution on the agar-solidified medium. By altering the number of cells used to initiate colony biofilms, the assortment levels can be varied on a continuous scale. Time-lapse fluorescent microscopy can be used to witness the interaction between different phenotypes and genotypes at a certain assortment level and to determine the relative success of either.

The role of assortment (spatial segregation) in evolutionary scenarios can be examined using simple microbial systems in the laboratory that allow controlled adjustment of spatial distribution. By modifying the founder cell density, various assortment levels can be visualized using fluorescently labelled bacterial strains in the colony biofilms of Bacillus subtilis.

Microbes provide an intriguing system to study social interaction among individuals within a population. The short generation times and relatively simple ...

Genetics

A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some factors act directly on virulence genes; others control pathogenesis by adjusting the expression of downstream regulators or the accumulation of signals that affect regulator activity. While regulation has been studied extensively during in vitro growth, relatively little is known about how gene expression is adjusted during infection. Such information is important when a particular gene product is a candidate for therapeutic intervention. Transcriptional approaches like quantitative, real-time RT-PCR and RNA-Seq are powerful ways to examine gene expression on a global level but suffer from many technical challenges including low abundance of bacterial RNA compared to host RNA, and sample degradation by RNases. Evaluating regulation using fluorescent reporters is relatively easy and can be multiplexed with fluorescent proteins with unique spectral properties. The method allows for single-cell, spatiotemporal analysis of gene expression in tissues that exhibit complex three-dimensional architecture and physiochemical gradients that affect bacterial regulatory networks. Such information is lost when data are averaged over the bulk population. Herein, we describe a method for quantifying gene expression in bacterial pathogens in situ. The method is based on simple tissue processing and direct observation of fluorescence from reporter proteins. We demonstrate the utility of this system by examining the expression of Staphylococcus aureus thermonuclease (nuc), whose gene product is required for immune evasion and full virulence ex vivo and in vivo. We show that nuc-gfp is strongly expressed in renal abscesses and reveal heterogeneous gene expression due in part to apparent spatial regulation of nuc promoter activity in abscesses fully engaged with the immune response. The method can be applied to any bacterium with a manipulatable genetic system and any infection model, providing valuable information for preclinical studies and drug development.

Described here is a method for analyzing bacterial gene expression in animal tissues at a cellular level. This method provides a resource for studying the phenotypic diversity occurring within a bacterial population in response to the tissue environment during an infection.

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some ...

Stress-induced Antibiotic Susceptibility Testing on a Chip

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria survive these stressful conditions. The hypothesis behind this method is new: stress activation of biochemical pathways, which are targets of antibiotics, can accelerate antibiotic susceptibility testing. As compared to standard antibiotic susceptibility testing methods, the rate-limiting step -&#160;bacterial growth -&#160;is omitted during antibiotic application. The technical implementation of the method is in a combination of standard techniques and innovative approaches. The standard parts of the method include bacterial culture protocols, defining microfluidic channels in polydimethylsiloxane&#160;(PDMS), cell viability monitoring with fluorescence, and batch image processing for bacteria counting. Innovative parts of the method are in the use of culture media flow for mechanical stress application, use of enzymes to damage but not kill the bacteria, and use of microarray substrates for bacterial attachment. The developed platform can be used in antibiotic and nonantibiotic related drug development and testing. As compared to the standard bacterial suspension experiments, the effect of the drug can be turned on and off repeatedly over controlled time periods. Repetitive observation of the same bacterial population is possible over the course of the same experiment.

We have developed a microfluidic platform for rapid antibiotic susceptibility testing. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria can survive these stressful conditions.

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over ...

Bioengineering

Quantification of Plasmid-Mediated Antibiotic Resistance in an Experimental Evolution Approach

Plasmids play a major role in microbial ecology and evolution as vehicles of lateral gene transfer and reservoirs of accessory gene functions in microbial populations. This is especially the case under rapidly changing environments such as fluctuating antibiotics exposure. We recently showed that plasmids maintain antibiotic resistance genes in Escherichia coli without positive selection for the plasmid presence. Here we describe an experimental system that allows following both the plasmid genotype and phenotype in long-term evolution experiments. We use molecular techniques to design a model plasmid that is subsequently introduced to an experimental evolution batch system approach in an E. coli host. We follow the plasmid frequency over time by applying replica plating of the E. coli populations while quantifying the antibiotic resistance persistence. In addition, we monitor the conformation of plasmids in host cells by analyzing the extent of plasmid multimer formation by plasmid nicking and agarose gel electrophoresis. Such an approach allows us to visualize not only the genome size of evolving plasmids but also their topological conformation-a factor highly important for plasmid inheritance. Our system combines molecular strategies with traditional microbiology approaches and provides a set-up to follow plasmids in bacterial populations over a long time. The presented approach can be applied to study a wide range of mobile genetic elements in the future.

Our experimental approach provides a strategy to follow plasmid abundance and antibiotic resistance over time in bacterial populations.

Plasmids play a major role in microbial ecology and evolution as vehicles of lateral gene transfer and reservoirs of accessory gene functions in microbial ...

Live-Cell Fluorescence Microscopy to Investigate Subcellular Protein Localization and Cell Morphology Changes in Bacteria

Investigations of factors influencing cell division and cell shape in bacteria are commonly performed in conjunction with high-resolution fluorescence microscopy as observations made at a population level may not truly reflect what occurs at a single cell level. Live-cell timelapse microscopy allows investigators to monitor the changes in cell division or cell morphology which provide valuable insights regarding subcellular localization of proteins and timing of gene expression, as it happens, to potentially aid in answering important biological questions. Here, we describe our protocol to monitor phenotypic changes in Bacillus subtilis and Staphylococcus aureus using a high-resolution deconvolution microscope. The objective of this report is to provide a simple and clear protocol that can be adopted by other investigators interested in conducting fluorescence microscopy experiments to study different biological processes in bacteria as well as other organisms.

This article provides a step-by-step guide to investigate protein subcellular localization dynamics and to monitor morphological changes using high-resolution fluorescence microscopy in Bacillus subtilis and Staphylococcus aureus.

Investigations of factors influencing cell division and cell shape in bacteria are commonly performed in conjunction with high-resolution fluorescence ...

Kinetic Visualization of Single-Cell Interspecies Bacterial Interactions

Polymicrobial communities are ubiquitous in nature, yet studying their interactions at the single-cell level is difficult. Thus, a microscopy-based method has been developed for observing interspecies interactions between two bacterial pathogens. The use of this method to interrogate interactions between a motile Gram-negative pathogen, Pseudomonas aeruginosa and a non-motile Gram-positive pathogen, Staphylococcus aureus is demonstrated here. This protocol consists of co-inoculating each species between a coverslip and an agarose pad, which maintains the cells in a single plane and allows for visualization of bacterial behaviors in both space and time.
Furthermore, the time-lapse microscopy demonstrated here is ideal for visualizing the early interactions that take place between two or more bacterial species, including changes in bacterial species motility in monoculture and in coculture with other species. Due to the nature of the limited sample space in the microscopy setup, this protocol is less applicable for studying later interactions between bacterial species once cell populations are too high. However, there are several different applications of the protocol which include the use of staining for imaging live and dead bacterial cells, quantification of gene or protein expression through fluorescent reporters, and tracking bacterial cell movement in both single species and multispecies experiments.

This live-bacterial cell imaging protocol allows for visualization of interactions between multiple bacterial species at the single-cell level over time. Time-lapse imaging allows for observation of each bacterial species in monoculture or coculture to interrogate interspecies interactions in multispecies bacterial communities, including individual cell motility and viability.

Polymicrobial communities are ubiquitous in nature, yet studying their interactions at the single-cell level is difficult. Thus, a microscopy-based method ...

Monitoring Intraspecies Competition in a Bacterial Cell Population by Cocultivation of Fluorescently Labelled Strains

Many microorganisms such as bacteria proliferate extremely fast and the populations may reach high cell densities. Small fractions of cells in a population always have accumulated mutations that are either detrimental or beneficial for the cell. If the fitness effect of a mutation provides the subpopulation with a strong selective growth advantage, the individuals of this subpopulation may rapidly outcompete and even completely eliminate their immediate fellows. Thus, small genetic changes and selection-driven accumulation of cells that have acquired beneficial mutations may lead to a complete shift of the genotype of a cell population. Here we present a procedure to monitor the rapid clonal expansion and elimination of beneficial and detrimental mutations, respectively, in a bacterial cell population over time by cocultivation of fluorescently labeled individuals of the Gram-positive model bacterium Bacillus subtilis. The method is easy to perform and very illustrative to display intraspecies competition among the individuals in a bacterial cell population.

Bacteria may accumulate either detrimental or beneficial mutations during their lifetime. In a population of cells individuals that have accumulated beneficial mutations may rapidly outcompete their fellows. Here we present a simple procedure to visualize intraspecies competition in a bacterial cell population over time using fluorescently labeled individuals.&#160;

Many microorganisms such as bacteria proliferate extremely fast and the populations may reach high cell densities. Small fractions of cells in a ...

Biology

Live Cell Fluorescence Microscopy to Observe Essential Processes During Microbial Cell Growth

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of proteins which are essential for bacterial survival. A series of target-specific dyes can be used as probes to better understand these processes. Staining with lipophilic dyes enables the observation of membrane structure, visualization of lipid microdomains, and detection of membrane blebs. Use of fluorescent-d-amino acids (FDAAs) to probe the sites of peptidoglycan biosynthesis can indicate potential defects in cell wall biogenesis or cell growth patterning. Finally, nucleic acid stains can indicate possible defects in DNA replication or chromosome segregation. Cyanine DNA stains label living cells and are suitable for time-lapse microscopy enabling real-time observations of nucleoid morphology during cell growth. Protocols for cell labeling can be applied to protein depletion mutants to identify defects in membrane structure, cell wall biogenesis, or chromosome segregation. Furthermore, time-lapse microscopy can be used to monitor morphological changes as an essential protein is removed and can provide additional insights into protein function. For example, the depletion of essential cell division proteins results in filamentation or branching, whereas the depletion of cell growth proteins may cause cells to become shorter or rounder. Here, protocols for cell growth, target-specific labeling, and time-lapse microscopy are provided for the bacterial plant pathogen Agrobacterium tumefaciens. Together, target-specific dyes and time-lapse microscopy enable characterization of essential processes in A. tumefaciens. Finally, the protocols provided can be readily modified to probe essential processes in other bacteria.

Understanding the function of essential processes in bacteria is challenging. Fluorescence microscopy with target-specific dyes can provide key insights into microbial cell growth and cell cycle progression. Here, Agrobacterium tumefaciens is used as a model bacterium to highlight methods for live cell imaging for characterization of essential processes.

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of ...

Population and Single-Cell Analysis of Antibiotic Persistence in Escherichia coli

Antibiotic persistence refers to the capacity of small bacterial subpopulations to transiently tolerate high doses of bactericidal antibiotics. Upon bactericidal antibiotic treatment, the bulk of the bacterial population is rapidly killed. This first rapid phase of killing is followed by a substantial decrease in the rate of killing as the persister cells remain viable. Classically, persistence is determined at the population level by time/kill assays performed with high doses of antibiotics and for defined exposure times. While this method provides information about the level of persister cells and the killing kinetics, it fails to reflect the intrinsic cell-to-cell heterogeneity underlying the persistence phenomenon. The protocol described here combines classical time/kill assays with single-cell analysis using real-time fluorescence microscopy. By using appropriate fluorescent reporters, the microscopy imaging of live cells can provide information regarding the effects of the antibiotic on cellular processes, such as chromosome replication and segregation, cell elongation, and cell division. Combining population and single-cell analysis allows for the molecular and cellular characterization of the persistence phenotype.

Population and Single-Cell Analysis of Antibiotic Persistence in Escherichia coli

Antibiotic persistence describes the ability of small subpopulations within a sensitive isogenic population to transiently tolerate high doses of bactericidal antibiotics. The present protocol combines approaches to characterize the antibiotic persistence phenotype at the molecular and cellular levels after exposing Escherichia coli to lethal doses of ofloxacin.

Antibiotic persistence refers to the capacity of small bacterial subpopulations to transiently tolerate high doses of bactericidal antibiotics. Upon ...

Visualizing Antibiotic Persistence in Bacterial Pathogens

Time-Lapse Epifluorescence Microscopy Imaging of Pseudomonas aeruginosa and Staphylococcus aureus Heterogeneous Phenotypes

Antibiotic persistence is a phenomenon in which a small number of bacterial cells in a genetically susceptible population survive antibiotic treatment that kills the other genetically identical cells. Bacterial persisters can resume replication once antibiotic treatment ends and are commonly thought to&#160;underlie clinical treatment failure.&#160;Recent work harnessing the power of time-lapse fluorescence microscopy, in which bacteria are labeled with fluorescent transcriptional reporters, translational reporters, and/or dyes for a variety of cellular features, has advanced our understanding of Escherichia coli persisters beyond what could be learned from population-level antibiotic survival assays. Such single-cell approaches, rather than bulk population assays, are essential for delineating the mechanisms of persister formation, damage response, and survival. However, methods for studying persisters in other important pathogenic species at this level of detail remain limited.
This study provides an adaptable approach for time-lapse imaging of Pseudomonas aeruginosa (a gram-negative rod) and Staphylococcus aureus (a gram-positive coccus)&#160;during antibiotic treatment and recovery. We discuss molecular genetic approaches to introduce fluorescent reporters into these bacteria. Using these reporters, as well as dyes, we can track the phenotypic changes, morphological features, and fates of individual cells in response to antibiotic treatment. Additionally, we are able to observe the phenotypes of individual persisters as they resuscitate following treatment. In all, this work serves as a resource for those interested in tracking the survival and gene expression of individual antibiotic-treated cells, including persisters, both during and after treatment, in clinically important pathogens.

In this manuscript, we provide a comprehensive protocol for assessing antibiotic survival for Pseudomonas aeruginosa and Staphylococcus aureus, transforming plasmids into P. aeruginosa and S. aureus to create reporter strains and visualize&#160;phenotypic variants, such as persisters, by time-lapse epifluorescence&#160;microscopy.

Antibiotic persistence is a phenomenon in which a small number of bacterial cells in a genetically susceptible population survive antibiotic treatment ...

Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes

Summary

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Chapters in this video

Monitoring Spatial Segregation in Surface Colonizing Microbial Populations

A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues

Stress-induced Antibiotic Susceptibility Testing on a Chip

Quantification of Plasmid-Mediated Antibiotic Resistance in an Experimental Evolution Approach

Live-Cell Fluorescence Microscopy to Investigate Subcellular Protein Localization and Cell Morphology Changes in Bacteria

Kinetic Visualization of Single-Cell Interspecies Bacterial Interactions

Monitoring Intraspecies Competition in a Bacterial Cell Population by Cocultivation of Fluorescently Labelled Strains

Live Cell Fluorescence Microscopy to Observe Essential Processes During Microbial Cell Growth

Population and Single-Cell Analysis of Antibiotic Persistence in Escherichia coli

Time-Lapse Epifluorescence Microscopy Imaging of Pseudomonas aeruginosa and Staphylococcus aureus Heterogeneous Phenotypes