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.

<ol>
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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. D., Gerry, C. J., Tan, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+Platform+for+Systematic+Quantitative+Evaluation+of+Small-Molecule+Permeability+in+Bacteria.">General Platform for Systematic Quantitative Evaluation of Small-Molecule Permeability in Bacteria.</a> ACS Chemical Biology. 9 (11), 2535-2544 (2014).</li><li>Richter, M. F., 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=Predictive+compound+accumulation+rules+yield+a+broad-spectrum+antibiotic.">Predictive compound accumulation rules yield a broad-spectrum antibiotic.</a> Nature. 545, 299 (2017).</li><li>Iyer, R., 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=Evaluating+LC-MS/MS+To+Measure+Accumulation+of+Compounds+within+Bacteria.">Evaluating LC-MS/MS To Measure Accumulation of Compounds within Bacteria.</a> ACS Infectious Diseases. 4 (9), 1336-1345 (2018).</li><li>Prochnow, H., 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=Subcellular+Quantification+of+Uptake+in+Gram-Negative+Bacteria.">Subcellular Quantification of Uptake in Gram-Negative Bacteria.</a> Analytical Chemistry. 91 (3), 1863-1872 (2019).</li><li>Dumont, E., 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=Antibiotics+and+efflux:+combined+spectrofluorimetry+and+mass+spectrometry+to+evaluate+the+involvement+of+concentration+and+efflux+activity+in+antibiotic+intracellular+accumulation.">Antibiotics and efflux: combined spectrofluorimetry and mass spectrometry to evaluate the involvement of concentration and efflux activity in antibiotic intracellular accumulation.</a> Journal of Antimicrobial Chemotherapy. 74 (1), 58-65 (2019).</li><li>Stone, M. 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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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 &#215; 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&#8482; 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&#39; 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 
			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

12.3K 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

One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. Therefore, rapid administration of adequate antibiotic therapy is of foremost importance in the treatment of bloodstream infections. The critical element in this process is timing, heavily dependent on the results of bacterial identification and antibiotic susceptibility testing. Both of these parameters are routinely obtained by culture-based testing, which is time-consuming and takes on average 24-48 hours2, 4. The aim of the study was to develop DNA-based assays for rapid identification of bloodstream infections, as well as rapid antimicrobial susceptibility testing. The first assay is a eubacterial 16S rDNA-based real-time PCR assay complemented with species- or genus-specific probes5. Using these probes, Gram-negative bacteria including Pseudomonas spp., Pseudomonas aeruginosa and Escherichia coli as well as Gram-positive bacteria including Staphylococcus spp., Staphylococcus aureus, Enterococcus spp., Streptococcus spp., and Streptococcus pneumoniae could be distinguished. Using this multiprobe assay, a first identification of the causative micro-organism was given after 2 h.
Secondly, we developed a semi-molecular assay for antibiotic susceptibility testing of S. aureus, Enterococcus spp. and (facultative) aerobe Gram-negative rods6. This assay was based on a study in which PCR was used to measure the growth of bacteria7. Bacteria harvested directly from blood cultures are incubated for 6 h with a selection of antibiotics, and following a Sybr Green-based real-time PCR assay determines inhibition of growth. The combination of these two methods could direct the choice of a suitable antibiotic therapy on the same day (Figure 1). In conclusion, molecular analysis of both identification and antibiotic susceptibility offers a faster alternative for pathogen detection and could improve the diagnosis of bloodstream infections.

The design of a straightforward one-day workflow scheme for bacterial pathogen diagnostics enables the rapid recognition of bloodstream infections. The inclusion of eight clinically relevant bacterial targets and their antibiotic resistance profiles offers the clinician an initial insight on the same day, which can lead to more adequate therapy.

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. ...

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external environment. The cell can either recover from this insult, which requires active membrane repair processes, or else die depending on the amount of toxin exposure and cell type1. In addition, these toxins induce strong inflammatory responses in infected hosts through activation of immune cells, including macrophages, which produce an array of pro-inflammatory cytokines2. Many Gram positive bacteria produce cholesterol binding toxins which have been shown to contribute to their virulence through largely uncharacterized mechanisms.
Morphologic changes in the plasma membrane of cells exposed to these toxins include their sequestration into cholesterol-enriched surface protrusions, which can be shed into the extracellular space, suggesting an intrinsic cellular defense mechanism3,4. This process occurs on all cells in the absence of metabolic activity, and can be visualized using EM after chemical fixation4. In immune cells such as macrophages that mediate inflammation in response to toxin exposure, induced membrane vesicles are suggested to contain cytokines of the IL-1 family and may be responsible both for shedding toxin and disseminating these pro-inflammatory cytokines5,6,7. A link between IL-1&beta; release and a specific type of cell death, termed pyroptosis has been suggested, as both are caspase-1 dependent processes8. To sort out the complexities of this macrophage response, which includes toxin binding, shedding of membrane vesicles, cytokine release, and potentially cell death, we have developed labeling techniques and fluorescence microscopy methods that allow for real time visualization of toxin-cell interactions, including measurements of dysfunction and death (Figure 1). Use of live cell imaging is necessary due to limitations in other techniques. Biochemical approaches cannot resolve effects occurring in individual cells, while flow cytometry does not offer high resolution, real-time visualization of individual cells. The methods described here can be applied to kinetic analysis of responses induced by other stimuli involving complex phenotypic changes in cells.

Methods for purifying the cholesterol binding toxin streptolysin O from recombinant E. coli and visualization of toxin binding to live eukaryotic cells are described. Localized delivery of toxin induces rapid and complex changes in targeted cells revealing novel aspects of toxin biology.

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external ...

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify new compounds that can inhibit infection by the common drug resistant HIV-1 strains with minimal toxicity. Here we describe an efficient assay that can be used to rapidly determine the cellular cytotoxicity and efficacy of a compound against WT and mutant viral strains.
The desired target cell line is seeded in a 96-well plate and, after a 24 hr incubation, serially dilutions of the compounds to be tested are added. No further manipulations are necessary for cellular cytotoxicity assays; for anti HIV assays a predetermined amount of either a WT or drug resistant HIV-1 vector that expresses luciferase is added to the cells. Cytotoxicity is measured by using an ATP dependent luminescence assay and the impact of the compounds on infectivity is measured by determining the amount of luciferase in the presence or the absence of the putative inhibitors.
This screening assay takes 4 days to complete and multiple compounds can be screened in parallel. Compounds are screened in triplicate and the data are normalized to the infectivity/ATP levels in absence of target compounds. This technique provides a quick and accurate measurement of the efficacy and toxicity of potential anti HIV compounds.

Here we describe cellular cytotoxicity and single round infectivity assays that allow for the rapid and accurate screening of compounds to determine their cellular cytotoxicity (CC50) and IC50 values against WT and drug resistant HIV-1.

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify ...

Preparation of a Blood Culture Pellet for Rapid Bacterial Identification and Antibiotic Susceptibility Testing

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a rapid identification of the infectious agent to guide optimal antibiotic treatment. The analysis of Gram stains from positive blood culture can be rapidly conducted and already significantly impact the antibiotic regimen. However, the accurate identification of the infectious agent is still required to establish the optimal targeted treatment. We present here a simple and fast bacterial pellet preparation from a positive blood culture that can be used as a sample for several essential downstream applications such as identification by MALDI-TOF MS, antibiotic susceptibility testing (AST) by disc diffusion assay or automated AST systems and by automated PCR-based diagnostic testing. The performance of these different identification and AST systems applied directly on the blood culture bacterial pellets is very similar to the performance normally obtained from isolated colonies grown on agar plates. Compared to conventional approaches, the rapid acquisition of a bacterial pellet significantly reduces the time to report both identification and AST. Thus, following blood culture positivity, identification by MALDI-TOF can be reported within less than 1 hr whereas results of AST by automated AST systems or disc diffusion assays within 8 to 18 hr, respectively. Similarly, the results of a rapid PCR-based assay can be communicated to the clinicians less than 2 hr following the report of a bacteremia. Together, these results demonstrate that the rapid preparation of a blood culture bacterial pellet has a significant impact on the identification and AST turnaround time and thus on the successful outcome of patients suffering from bloodstream infections.

A rapid bacterial pellet preparation from a positive blood culture can be used as a sample for applications such as identification by MALDI-TOF, Gram staining, antibiotic susceptibility testing and PCR-based test. The results can be rapidly communicated to clinicians to improve the outcome of patients suffering from bloodstream infections.

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Assays for Studying the Role of Vitronectin in Bacterial Adhesion and Serum Resistance

Bacteria utilize complement regulators as a means of evading the host immune response. Here, we describe protocols for evaluating the role vitronectin acquisition at the bacterial cell surface plays in resistance to the host immune system. Flow cytometry experiments identified human plasma vitronectin as a ligand for the bacterial receptor outer membrane protein H of Haemophilus influenzae type f. An enzyme-linked immunosorbent assay was employed to characterize the protein-protein interactions between purified recombinant protein H and vitronectin, and binding affinity was assessed using bio-layer interferometry. The biological importance of the binding of vitronectin to protein H at the bacterial cell surface in evasion of the host immune response was confirmed using a serum resistance assay with normal and vitronectin-depleted human serum. The importance of vitronectin in bacterial adherence was analyzed using glass slides with and without vitronectin coating, followed by Gram staining. Finally, bacterial adhesion to human alveolar epithelial cell monolayers was investigated. The protocols described here can be easily adapted to the study of any bacterial species of interest.

This report describes protocols for characterizing interactions between bacterial outer membrane proteins and the human complement regulator vitronectin. The protocols can be used to study the binding reactions and biological function of vitronectin in any bacterial species.

Bacteria utilize complement regulators as a means of evading the host immune response. Here, we describe protocols for evaluating the role vitronectin ...

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most colonized hosts, the commensal reservoir does serve as a repository for infectious disease, and the presence of high fungal titers in the gut is associated with inflammatory bowel disease. Here, we describe a method to visualize C. albicans cell morphology and localization in a mouse model of stable gastrointestinal colonization. Colonization is established using a single dose of C. albicans in animals that have been treated with oral antibiotics. Segments of gut tissue are fixed in a manner that preserves the architecture of luminal contents (microorganisms and mucus) as well as the host mucosa. Finally, fluorescent in situ hybridization is performed using probes against fungal rRNA to stain for C. albicans and hyphae. A key advantage of this protocol is that it allows for simultaneous observation of C. albicans cell morphology and its spatial association with host structures during gastrointestinal colonization.

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

The purpose of this protocol is to visualize Candida albicans cell shape and localization in the mammalian gastrointestinal tract.

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most ...

Antibiotic Dereplication Using the Antibiotic Resistance Platform

One of the main challenges in the search for new antibiotics from natural product extracts is the re-discovery of common compounds. To address this challenge, dereplication, which is the process of identifying known compounds, is performed on samples of interest. Methods for dereplication such as analytical separation followed by mass spectrometry are time-consuming and resource-intensive. To improve the dereplication process, we have developed the antibiotic resistance platform (ARP). The ARP is a library of approximately 100 antibiotic resistance genes that have been individually cloned into Escherichia coli. This strain collection has many applications, including a cost-effective and facile method for antibiotic dereplication. The process involves the fermentation of antibiotic-producing microbes on the surface of rectangular Petri dishes containing solid medium, thereby allowing for the secretion and diffusion of secondary metabolites through the medium. After a 6 day fermentation period, the microbial biomass is removed, and a thin agar-overlay is added to the Petri dish to create a smooth surface and enable the growth of the E. coli indicator strains. Our collection of ARP strains is then pinned onto the surface of the antibiotic-containing Petri dish. The plate is next incubated overnight to allow for E. coli growth on the surface of the overlay. Only strains containing resistance to a specific antibiotic (or class) grow on this surface enabling rapid identification of the produced compound. This method has been successfully used for the identification of producers of known antibiotics and as a means to identify those producing novel compounds.

We describe a platform that utilizes a library of isogenic antibiotic resistant Escherichia coli for the dereplication of antibiotics. The identity of an antibiotic produced by bacteria or fungi can be deduced by the growth of E. coli expressing its respective resistance gene. This platform is economically effective and time-efficient.

One of the main challenges in the search for new antibiotics from natural product extracts is the re-discovery of common compounds. To address this ...

Establishing a Porcine Ex Vivo Cornea Model for Studying Drug Treatments against Bacterial Keratitis

When developing novel antimicrobials, the success of animal trials is dependent on accurate extrapolation of antimicrobial efficacy from in vitro tests to animal infections in vivo. The existing in vitro tests typically overestimate antimicrobial efficacy as the presence of host tissue as a diffusion barrier is not accounted for. To overcome this bottleneck, we have developed an ex vivo porcine corneal model of bacterial keratitis using Pseudomonas aeruginosa as a prototypic organism. This article describes the preparation of the porcine cornea and protocol for establishment of the infection. Bespoke glass molds enable straightforward setup of the cornea for infection studies. The model mimics in vivo infection as bacterial proliferation is dependent on the ability of the bacterium to damage corneal tissue. Establishment of infection is verified as an increase in the number of colony forming units assessed via viable plate counts. The results demonstrate that infection can be established in a highly reproducible fashion in the ex vivo corneas using the method described here. The model can be extended in the future to mimic keratitis caused by microorganisms other than P. aeruginosa. The ultimate aim of the model is to investigate the effect of antimicrobial chemotherapy on the progress of bacterial infection in a scenario more representative of in vivo infections. In so doing, the model described here will reduce the use of animals for testing, improve success rates in clinical trials and ultimately enable rapid translation of novel antimicrobials to the clinic.

This article describes a step-by-step protocol to set up an ex vivo porcine model of bacterial keratitis. Pseudomonas aeruginosa is used as a prototypic organism. This innovative model mimics in vivo infection as bacterial proliferation is dependent on the ability of the bacterium to damage corneal tissue.

When developing novel antimicrobials, the success of animal trials is dependent on accurate extrapolation of antimicrobial efficacy from in vitro tests to ...

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 ...