Name: visualization of bacterial resistance using fluorescent antibiotic probes
Uploaded: 2020-03-02T13:00:00
Duration: 8 min 23 s
Description: Watch this Scientific Journal Video about Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes at JoVE.com

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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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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