Name: nanomechanics of drug-target interactions and antibacterial resistance detection
Uploaded: 2013-10-25T09:00:00
Duration: 11 min 56 s
Description: Watch this Scientific Journal Video about Nanomechanics of Drug-target Interactions and Antibacterial Resistance Detection at JoVE.com

Joseph W. Ndieyira was supported by Engineering and Physical Sciences Research Council (EPRSC), Interdisciplinary Research Centre in Nanotechnology (IRC), the Royal Society (RS) and Biano consultancy (BNC). We thank, Alejandra Donoso Barrera, Dejian Zhou, Manuel V&ouml;gtli, Matthew Batchelor, Matthew A. Cooper, Torsten Strunz, Trevor Rayment and Gabriel Aeppli for helpful discussions.

1. Preparation of Cantilevers
<ol>
	<li>Using Teflon tweezers immerse selected number of cantilever chips (each cantilever measuring 500 &#956;m long, 100 &#956;m wide and 0.9 &#956;m thick) into a freshly prepared piranha solution (at ratio 1:1 H2SO4 and H2O2) for 20 min.</li>
	<li>After approximately 20 min remove the cantilever chips from piranha solution and rinse them thoroughly with deionized water and immediately transfer into a freshly prepared piranha solution. Repeat step 1.1&#160;above if the chips contain spots of dirt otherwise proceed to the next step.</li>
	<li>After a thorough cleaning in deionized water, rinse with pure ethanol and dry the cantilever chips on a hotplate at 75 &#176;C to remove any traces of water. Inspect them using an optical microscope to confirm their cleanliness.</li>
	<li>Transfer the cleaned cantilever chips to an evaporation chamber and pump down, targeting to achieve a vacuum pressure of 10-7 mbar.</li>
	<li>Once the required vacuum pressure is attained, coat one side of each cantilever array with a 2 nm titanium first to acts as a adhesion layer before an additional layer of 20 nm gold thick. To confirm their thickness, use quartz crystal monitor placed directly above the target source.</li>
	<li>Leave gold coated cantilever sensor chips in the chamber for 1-2 hr to cool under vacuum before opening.</li>
	<li>Transfer the freshly evaporated chips to a vacuum storage vessel filled with argon to prevent any form of contaminations.</li>
</ol>
2. Cantilever Chip Functionalization
<ol>
	<li>First, arrange micro-capillary tubes on a functionalization stage2 according to the cantilever pitch size of 250 &#956;m.</li>
	<li>Then inject 2 mM ethanolic thiol solutions of surface target molecules, i.e. bacterial mucopeptides (D-Ala and D-Lac), and an &#39;inert&#39; alkanethiol terminating in triethylene glycol (PEG) known to resist biomolecular adsorption.16 Each of the capillary tubings should contain an assorted surface capture molecules determined randomly to avoid user bias.</li>
	<li>Next incubate for 20 min the cantilevers in the microcapillaries filled with thiol solutions containing surface capturing molecules. Ensure that the solutions of the surface capture molecules are confined onto each individual cantilever sensor to avoid or minimize cross-contaminations. If this process is employed correctly, it should systematically alter the freshly prepared gold coated cantilevers into chemically active sensors formed by allowing SAMs of bacterial mucopeptide targets to form on gold coated surfaces as receptors.</li>
</ol>
3. Solution Preparations
<ol>
	<li>Dissolve 0.1 M mono- and di-basic sodium phosphate salts in ultrapure water (18.2 M&#937;&#183;cm resistivity) and mix to yield a pH value of 7.4.</li>
	<li>Add 0.002% of PS80 to the buffered solutions to minimize aggregation effects caused by nonspecific interactions of drug molecules to the glassware.</li>
	<li>Dilute drugs with freshly buffered solutions to the desired different concentrations that will enable calculation of Kd.</li>
	<li>Filter the freshly prepared drug solutions using 0.2 &#956;m filters and sonicate for 5 min at room temperature before purging with argon.</li>
	<li>Repeat the procedure with drug in buffered serum using whole serum. Gently vortex for 15 min with additional 5 min to ensure complete solubility.</li>
</ol>
4. Surface Stress Detection
<ol>
	<li>Load a functionalized cantilever sensor chip into a liquid flow cell chamber.</li>
	<li>Align the laser spot onto the free end of each sensor and confirm the alignment by heating up the liquid chamber for 1 &#176;C. All eight gold coated cantilever sensor arrays should undergo compressive downward bending because of the bimetallic effect caused by the differences in expansion rates of silicon and gold.</li>
	<li>After heating for approximately 10 min, allow the cantilevers to cool for further 10 min.</li>
	<li>Calculate the bending variation at the maximum bending signals between individual cantilever sensors and if the relative standard deviation of the bending signals is&#160;&#8804;5% then accept the alignment as desirable otherwise repeat the process.</li>
	<li>Next, measure the resonant frequencies of all eight cantilevers to calculate their spring constants. If the variation of the spring constant between each cantilever sensor within a chip is &#8804;1% then accept the chip as having consistent mechanical properties otherwise replace the cantilever chip sensor.</li>
	<li>Next, use a fluidic system (Model Genie Plus) via a six-way valve to achieve the liquid exchange within the flow cell while data acquisition should be achieved using an automated LabView software.</li>
	<li>To monitor the cantilever bending data, use the following measurement protocols: i) inject either buffer solution or serum without drug for control measurement lasting for 5-30 min to establish a baseline; ii) inject drug solution for 30-60 min; iii) inject 10 mM HCl wash for 10-60 min to dissociate bound drug complex; iv) finally inject a further washing step using buffer solution for another 5-30 min to regenerate the surface peptides and to restore the baseline signal. Always ensure that all signals are acquired under constant liquid flow rate of 30-180 &#956;l/min&#160;and at fixed temperature of 25 &#176;C in a temperature-controlled cabinet.</li>
	<li>The absolute bending signals of all eight cantilevers should be monitored using serial time multiplexed optical beam method with a single position sensitive detector.</li>
	<li>To analyze the bending signals from each concentration of drug, the resulting differential bendings are converted into a differential stress between the upper and lower sides of the cantilever using Stoney&#39;s equation.</li>
</ol>

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No conflicts of interest is declared

These results demonstrate that cantilever array sensors have the sensitivity to detect and quantify changes in drug-target binding interactions especially in vancomycin resistance associated with the deletion of a single H-bond from the drug&#39;s binding pocket. We show a nanomolar detection sensitivity of Van in agreement with previous Surface Plasmon Resonance (SPR) studies18,19, and reveal that the cantilever method can directly detect and quantify drug molecules in blood at clinically relevant concentrations as routinely applied in clinical practice. Our data suggest that the differential surface stress can be described by a product form equation (1), i.e. (i) a chemical term describing the specific drug-target binding events, and (ii) a geometrical term describing the mechanical connectivity between chemically reacted surface sites, implying that the local chemical interactions decouple from the global mechanical interactions of the cantilever. While the chemical term is given by the classical Langmuir adsorption isotherm, the geometrical term reveals a percolative mechanism of the drug-target induced surface stress changes. The critical threshold pc ~10% (Figure 5) was necessary to detect the differential cantilever bending, showing that surface stress is transduced collectively when a relatively large surface fraction is occupied by antibiotic molecules. For p&#8805;pc, the mechanical connectivity between chemically transformed surface sites is gradually established, and the short-range repulsive interactions such as steric interactions between the nodes of the nanomechanical network gives rise to increasing downward bending of the entire cantilever. It is speculated that our nanomechanical percolation model may play an important role in the glycopeptide antibiotic mode of action in real bacteria. These findings highlight the high sensitivity of cantilever technology for studying antibiotics&#39; mode of action and represent a novel research tool for studying drugs to improve understanding of the operations of antibiotics at the nano-scale to inform and enable discovery of powerful drugs to control the problems of superbug infections. To prepare the cantilever system for reproducible and sensitive measurements, we have addressed a set of targets in the protocol, particularly for sample loading in microfluidic cell and standard operating procedures to allow quantitative nanomechanical detections.
Significance of cantilever technology with respect to existing methods
In summary we point out that while this technology was proposed more than 25 years ago, it has not found its way into the clinic because of lack of careful and repetitive measurements on medically relevant targets. Here we demonstrate the procedures that establish the relevance of using nanomechanical cantilevers to investigate the mechanical influence of antibiotics on the bacterial cell wall targets and to detect antibacterial resistance. Conventional drug screening methods require some type of fluorescent or radioactive labeling of a reporter molecule to measure the binding of an analyte to its target, often in connection with a competitive or enzymatic binding assay and protein assays20. Labeling of biomolecules is not only time-consuming and expensive but the label can also interfere with the molecular interaction by obstructing the binding site, leading to false negatives. In addition, fluorescent compounds are often hydrophobic which can lead to background binding and false positives. Due to these limitations, there is an increasing interest in novel label-free techniques that allow virtually any molecular complex to be screened with minimal assay development. The most established label-free surface technologies at present are SPR and Quartz Crystal Microbalance (QCM). In contrast to SPR that measure the dielectric constant, a label - free cantilever technology detects the surface strain generated by a ligand-receptor interaction, which can directly measure the nanomechanical forces generated by the specific binding of ligands to surface receptors. The uniqueness of these sensors is that their sensitivity does not rely on dielectric properties caused by mass change due to the analyte binding as in SPR and QCM but rather on a minutest induced change in in-plane surface stress, making the technology uniquely suited to study the nanomechanics of drug molecules at clinically relevant antibiotic concentrations (3-27 &#956;M)17. Cantilevers are also particularly well suited to small molecule (such as DNA fragments and drugs) detection under physiological conditions including complex environments which is largely the basis of the pharmaceutical industry and will therefore serve as a complementary tool in drug discovery. Cantilever technology have successfully been applied in the fields of genomics3,5, gas sensing21, proteomics22, and drugs1. Moreover, cantilevers are fabricated using low cost silicon technology and due to their compatibility with microfabrication processes, cantilevers can be miniaturized for improved sensitivity and parallelization into large arrays of sensors for multiple drug compound screening and higher throughput information-rich screening assays. Improvements of the instrumentation and experimental design will allow a wide variety of interactions to be analyzed in real time to help advance the search for a new generation of superdrugs to tackle the problems of multidrug resistant infections.
Critical steps within the protocol
The development of robust measurement protocols is central to the applications of this technology. To achieve satisfactory quantitative drug-target measurements and to determine the lowest concentration of antibiotics that could be detected in buffer or blood serum, critical steps within the protocol were addressed. The first task involves tuning and optimizing surface capture chemistries to enhance cantilever detection specificity and sensitivity. Undeniably, surface stress transduction is a collective phenomenon, requiring a relatively large fraction of the surface to be covered to establish connectivity between chemically reactive regions. We show that by varying the density of the underlying surface peptides, a critical threshold ~p &#8805;10%, is determined where the surface stress scale as a function of peptide density and zero otherwise (Figure 5). It is important that experiments are designed to investigate the uniformity of stress along the cantilevers to ensure maximum signal deflections for sensitive measurements. In addition, efficient surface regeneration protocols need to be in place, thereby allowing multiple cycle measurements and to reduce costs for each test. While designing a receptor surface using thiolated hydrophobic end, orientation of the receptor molecule and spacing between them is vital to allow self-assembly of dense packing due to Van-der-Waals interactions between the molecules to minimize nonspecific interactions. Furthermore, the capture molecules should contain a polyethylene glycol (PEG) linker to allow some part of the sensing matrix to be hydrophilic to prevent insertion of molecules in the analyte solution from reacting directly with the uncoated gold surface. The PEG linker molecules must act as spacers to reduce steric constraints and therefore allow the solution analytes to interact specifically with the surface receptors to induce measureable surface stress change. The cantilever array sensor must be assayed with at least an in situ reference measurement and the signal displayed in real-time is a differential deflection, obtained by subtracting the absolute deflection of the reference cantilever from the sensing cantilevers. Thus a reference cantilever is essential to account for nonspecific interactions such as temperature changes, changes in refractive index or interactions on the nonfunctionalized underside of the cantilever. Optimization of the flow rate (~30-150 &#956;l/min) forms a critical step within the protocol because it ensures the efficient exchange of liquids and a sufficient steady mass transport of solution materials. The design of a liquid cell must allow optimum volume (5-80 &#956;l) for fast flow rates to enable perfect liquid exchange to overcome mass transport limitations. The flow rate is especially critical while performing kinetic measurements23. Large volume liquid chambers require uncontrollable high flow rates leading to large sample volume requirements, which unnecessarily increases the price of the assay. Previously we have used gravity flow to inject different samples into the measurement liquid chamber. Gravity flow has the advantage that it does not require any mechanical parts and therefore does not introduce additional noise into the system. However, its significant disadvantage is that it only works reliably at relatively high flow rates (~200 &#956;l/min). Obviously, a lower flow rate (&#8804;1 &#956;l/min) would require limited sample volumes per unit time but on the other hand it makes the reactions much slower and hence requires longer contact times. Moreover, gravity flow has a large variance in its flow rate as it depends on the height difference between the inlet and outlet, which decreases as the sample solutions are consumed during the experiment. To avoid gravity flow problems, a syringe pump should be used. The advantage of using a syringe pump is that it allows a constant flow rate over a long period of time allowing the experiments to be realized in a more controlled environment.
Limitations of the protocol
The major challenge in obtaining a reproducible and specific biological detection using cantilever sensors lies in ensuring that the properties of the receptor layer are biochemically &#34;active&#34; and uniform for each and every assay. The secret to experimental success is in a careful pretreatment of the sensor chip with a standardized cleaning and immobilization protocol with linker chemistries to orient the receptor molecules in their active conformation. In our current setup, we functionalize cantilever arrays using small glass capillaries, which could be subject to some drawbacks and can be problematic in some cases. These glass capillaries are open at both ends and therefore the sample solvents can evaporate easily. For example, if the temperature of the functionalization stage is not accurately controlled, the evaporation rate can vary significantly at different times particularly when using volatile solvents like ethanol. There is also a possibility of slight variation in the incubation time from one cantilever to another given that the sensing liquids have to be loaded consecutively into the capillaries. The other limiting factor in capillary functionalization is the lack of the ability to ensure that cantilevers are always inserted into the capillaries in exactly the same way. In addition, sometimes the cantilevers have to be pulled out slightly from the capillaries in order to prevent cross-contamination as the liquid samples can flow onto the chip body. We can solve these problems by employing inkjet spotters as an alternative surface coating procedure to coat cantilevers. While it would allow the exact control of sample coating with the possibility of scaling up for large arrays, the most common drawback is that the small drops that are deposited onto the cantilevers can evaporate within seconds and requires a controlled humidity environment. Therefore, the incubation time cannot be adjusted easily, which might be desirable for some applications. The subtle interplay of the factors such as sample volume, incubation time and evaporation rate have direct impact on the exposure of the cantilevers to the functionalization sample and care must be taken to ensure optimized surface chemistries for cantilever assays as any small variation in the receptor molecular density will have a large effect on the cantilever response.
Experimental design modifications (i.e. alternative techniques or materials)
To overcome the major challenge in obtaining reproducible coating procedure for the future development of the cantilever technology, different strategies are required that would use cantilevers integrated in microfluidic channels. The idea is that special cantilever chips should be designed so that each cantilever is placed into its own channel to allow online in situ functionalization procedures where the channels are addressed individually. These experimental design modifications would allow coating process to be performed in a controlled and closed environment where the exposure to the solvent is precisely monitored by the incubation time and flow rate for automated immobilization of capture molecules on cantilever chips. The same channels could then be used for the actual binding experiments where all cantilevers could be exposed to the same analyte solution. Besides the cantilever functionalization procedure, the cantilever readout mechanism would also need improvement. The optical beam deflection method is highly sensitive and it has been used successfully in Atomic Force Microscopy (AFM) technology for many years. Nevertheless, for the free standing cantilever sensor applications the optical readout has some drawbacks, for example it does not allow measurements in opaque liquids such as blood, the alignment of an array of lasers can be time-consuming and tedious and the current configuration cannot differentiate between tilting and vertical deflections. Thus the future development of the cantilever technology will have to consider aspects of the general sensor design (e.g. cantilever geometries) and the cantilever readout for robust measurement protocols both in the fields and laboratory environments. Manalis and coworkers22 have developed novel types of hollow cantilever sensors where the cantilevers have an embedded microfluidic channel inside the beam, allowing the device to be operated in a vacuum with high-quality factors. In this mode, cantilevers act as microbalance22, and therefore it is no longer necessary to have an asymmetry between the two sides with respect to functionalization, thus the capture molecules can be physisorbed or covalently attached directly on the silicon, typically using SAMs or silanes24. The cantilevers can also be modified with a thin polymer layer which is then conjugated to antibodies25 for biochemical sensing.
Troubleshooting
A common problem associated with the cantilever measurements is the introduction of air bubbles into the microfluidic flow cell. Care must be taken to ensure that no air bubbles are introduced into the liquid cell while mounting the chip. Signal drifting is also another common problem caused by the differences in the temperature of samples. The cantilevers must be equilibrated to stabilize before measurements are undertaken. All the buffers, analytes and regeneration solutions must be stored in the same room as the cantilever instrument to allow all the solutions to have the same temperature. Although the syringe pump can provide very accurate and extremely low flow rates, it is generally associated with mechanical noise in the cantilever measurements. It is therefore important to devise a simple and effective noise reducer to avoid unnecessary noise in the deflection signals. The noise reducer consists of small liquid reservoir located between the syringe pump and the liquid cell, which absorbs the mechanical noise from the pump. Because of the sensitive nature of optical detector, the cantilever measurement system should ideally be operated in an enclosed setup to block any stray lights from interfering with the optical readout system. In addition, the quality of the sensing layers can decrease significantly if a large number of washing steps are performed on the cantilevers limiting the lifetime of the sensor chip.
Alternative or future applications after mastering this technique
Cantilevers coated with SAMs terminating in an amino- or carboxyl-group can be used as pH sensors. The functional end groups protonate or deprotonate depending on the pH of the solution and can generate a surface charge that leads the cantilever to bend24. Given that the cantilever sensors can track drug-target interactions associated with the destabilization of the cell wall of life bacteria1,6,7, they will therefore help in the search and for the development of a new generation of antibiotics to combat drug-resistant infections. In future cantilevers would be used as microbalances to measure the mass and growth rates of single cells26. The cantilever technology will prove beneficial to the study of cellular responses to different growth factors or drugs. It will also offer a novel tool for rapid detection of multiple biomarkers, which has immediate relevance in medical and point-of-care applications. Given the versatility, small size, and robustness of cantilever sensors, they will form a sensitive monitor of harmful environmental factors. They have already demonstrated their sensitivity in detecting toxic and harmful gases that can escape from the laboratory and industrial production units into the environment, such as hydrofluoric acid27 or hydrogen cyanide28.

The molecular recognition paradigm underpins large swathes of biology and medicine. In pharmacology, for example the goal is to interfere with a pathological pathway by targeting a key participant of a biochemical process such as transglycoslyation or transpeptidation that catalyzes the crosslinking of bacterial cell wall peptides. The drug design is therefore reduced to defining a suitable molecule to target a specific bacterial docking site to induce a perfect fit. A classic example emulating the success of this approach is vancomycin (Van), which targets the peptidoglycan cell wall, a conserved structural feature of a bacterium. In a nascent Gram-positive bacterium, the peptidoglycan matrix consists of peptides terminating in the sequence Lysine-D-Alanine-D-Alanine, tethered via a C55 lipid linker8-10 here termed D-Ala. These exposed peptides on the peptidoglycan precursors are fundamental for the mechanical protection of the bacterial cells against harsh environmental forces, and importantly, are not found in human cells, making them ideal targets for antibiotic drugs. Van specifically binds to the C-terminus of the bacterial cell wall peptides forming a moderately strong Van-peptide complex as shown in Figure 1. This interaction between Van and the exposed peptides blocks the actions of transpeptidases and transglycosylases, which catalyze the cross-linking of the cell walls1, effectively stopping them from crosslinking and therefore nanomechanically weakens the bacterial cell leading to its death by rupture1,6,7.
The emergence of vancomycin-resistant Enterococcus (VRE) is an increasing healthcare problem11 and because the bacterial resistance in Enterococci arises due to the subtle change of an amide linkage to an ester linkage8, this deceptively simple structural change at the surface of the bacterium deletes a single hydrogen bond from Van&#39;s docking site with subsequent modification of the binding pocket. Importantly this process alters the pentapeptide terminal alanine present in vancomycin susceptible Enterococcus (VSE) to a Lysine-D-Alanine-D-Lactate10,12, here termed D-Lac, yielding a significant reduction in Van-D-Lac binding affinity by three orders of magnitude rendering Van therapeutically ineffective against Enterococcal infections1, The alarming growth of antibiotic-resistant bacteria is therefore driving the development of innovative approaches to accelerate and restore the antibacterial activity of antibacterial agents or the discovery of more powerful drugs against multidrug resistant bacterial infections.
Our experiments using free-standing cantilevers is inspired by the systems such as cells which harness chemical energy into motion. The ability of cantilever sensors to translate chemical energy into mechanical motion makes them unique probes with considerable advantages for monitoring mechanical disruptions of bacterial cell wall targets in real time. The cantilever sensors require no reporter &#39;tags&#39; and biomolecules are detected rapidly in a single-step reaction. They can screen multiple drugs in parallel under physiological environments such as buffered solutions and whole serum. In addition, the cantilever technology uses in&#160;situ referencing for differential measurements allowing them to specifically detect drug-target interactions and, by virtue of their fabrication via standard semiconductor processing routes, they are amenable for mass production and parallelization for high-throughput screening of thousands of drugs per hour. In particular multiple cantilever sensor arrays are useful tools for studying antibiotic resistance to vancomycin - because resistance to vancomycin is essentially a mechanical problem1,6,7. The reactions between a model bacterial cell wall target and Van in solution can be detected by monitoring the changes in clinically relevant stress induced from drug-target binding interactions. The generated stress from surface reactions which manifests itself in a cantilever bending signal is analyzed optically by illuminating sensors with a laser beam. Moreover by tailoring the receptive coating of bacterial surface target molecules on top of the cantilever sensor, close to an un-limited number of analytes (Van), the specific biochemical interactions and the biomechanics of model bacterial cell wall matrix is monitored in real time. Apart from the molecular recognition event, there are several factors that can cause cantilever sensors to deflect, which include - temperature variations, unspecific binding or changes of refractive index of the solution. To account for nonspecific signals, in&#160;situ differential measurements are performed where the bending of both the measuring and reference cantilevers are continuously monitored to analyze specific interactions. Furthermore, the detection sensitivities of cantilever sensors which depend on the surface chemistries and geometry can be enhanced by tuning the surface density p (where p is defined as the ratio of the surface area occupied by capturing molecules to the entire top surface area of the cantilever covered by the total population of molecules as determined by X-ray photoelectron spectroscopy (XPS)1.
Here this protocol details how vancomycin or any other antibiotic binding to bacterial cell wall precursor analogs&#160;(mucopeptides) at clinically relevant concentrations in buffered solution and blood serum can be detected by using label-free cantilever technology. Fresh gold surfaces are used because self-assembled monolayers (SAMs) tend to easily form stable layers on clean gold13,14. SAMs typically consists of short molecules with a thiol moiety covalently attached at the gold surface while the desired capturing unit on the other end is allowed to freely interact with the analyte targets in solution. Thiols form a flexible system particularly given that many thiol compounds with different chemical end groups are commercially available or are easily synthesized in a laboratory. However, care should be taken to ensure that molecules with a thiol moiety must have the correct end groups, for example in a protein or peptide conjugation, the amino group should face inwards for the reaction with the carboxylic group of the thiol moiety tethered on the surface to occur. Here, the thiols were directly conjugated with tripeptide mimetics of bacterial lipid II from the bacterial cell wall targets synthesized by solid phase methodology using commercially available preloaded Wang-D-Ala and Wang-D-Lac resins and the standard Fmoc-protecting group chemistry15. Although this description is focused to vancomycin, clearly it can be extended to other antibiotics and indeed further studies are invited by researchers from different fields particularly in biochemistry, pharmacology, and material science to easily adopt this protocol for their own experiments.

<table border="1">
	<tbody>
		<tr>
			<td>Items</td>
			<td>Catalog Number </td>
			<td>Company</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Scentris</td>
			<td></td>
			<td>Scentris, Veeco Instruments, Inc</td>
			<td>Not in production</td>
		</tr>
		<tr>
			<td>Six-way valve</td>
			<td></td>
			<td>MVP, Hamilton, Reno, NV</td>
			<td>Enables multiple concentration injection</td>
		</tr>
		<tr>
			<td>EB Evaporator</td>
			<td></td>
			<td>BOC Edwards Auto 500, UK</td>
			<td>Electron beam is preferable because of deformation of cantilevers caused by thermal heating problems</td>
		</tr>
		<tr>
			<td>Storage vessel</td>
			<td></td>
			<td>Agar Scientific, UK</td>
			<td>Keeps gold coated films fresh</td>
		</tr>
		<tr>
			<td>Glass capillary</td>
			<td></td>
			<td>King Precision Glass, Claremont, CA, USA</td>
			<td>For capillary functionalization of the cantilever arrays</td>
		</tr>
		<tr>
			<td>Pump</td>
			<td></td>
			<td>Model Genie Plus, Kent Scientific, Torrington, CT, USA</td>
			<td>Good for controlled flow rate of liquid samples in microfluidic channels</td>
		</tr>
		<tr>
			<td>0.2-4.5 &#956;m Filters</td>
			<td>1511940001</td>
			<td>Merck Millipore</td>
			<td>For sample filtering</td>
		</tr>
		<tr>
			<td>Cantilever chips</td>
			<td></td>
			<td>London Centre for Nanotechnology (LCN)</td>
			<td>Highly sensitive</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic</td>
			<td>10049-21-5</td>
			<td>Sigma-Aldrich, UK</td>
			<td>For making buffer solutions</td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic</td>
			<td>7558-79-4</td>
			<td>Sigma-Aldrich, UK</td>
			<td>For making buffer solutions</td>
		</tr>
		<tr>
			<td>Polysorbate 80 (PS80) or Tween 80</td>
			<td>9005-65-6</td>
			<td>Sigma-Aldrich, UK</td>
			<td>Used in buffer solutions to prevent nonspecific interactions on glassware</td>
		</tr>
		<tr>
			<td>DI water</td>
			<td></td>
			<td>Millipore Co., Billerica, MA, USA</td>
			<td>Used for making solutions</td>
		</tr>
		<tr>
			<td>Whole serum</td>
			<td>GEM-700-110-H</td>
			<td>Sera Laboratories International Ltd, UK</td>
			<td>Used in measurements that mimic physiological conditions</td>
		</tr>
		<tr>
			<td>Vancomycin (Van)</td>
			<td>1404-93-9</td>
			<td>Sigma-Aldrich, UK</td>
			<td>Drug used</td>
		</tr>
		<tr>
			<td>Sulfuric acid</td>
			<td>7664-93-9</td>
			<td>Sigma-Aldrich, UK</td>
			<td>Used for glassware cleaning procedures</td>
		</tr>
		<tr>
			<td>Hydrogen peroxide</td>
			<td>7722-84-1</td>
			<td>Sigma-Aldrich, UK</td>
			<td>Used for glassware cleaning procedures</td>
		</tr>
		<tr>
			<td>D-Ala and D-Lac</td>
			<td></td>
			<td>London Centre for Nanotechnology (LCN)</td>
			<td>Surface receptors coated on the cantilever for detecting drug-target interactions</td>
		</tr>
		<tr>
			<td>LabView</td>
			<td></td>
			<td>National Instruments Co., Austin, TX, USA</td>
			<td>Software used for instrumental interface to allow controlled measurements</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

nanomechanics of drug-target interactions and antibacterial resistance detection

The cantilever sensor, which acts as a transducer of reactions between model bacterial cell wall matrix immobilized on its surface and antibiotic drugs in solution, has shown considerable potential in biochemical sensing applications with unprecedented sensitivity and specificity1-5. The drug-target interactions generate surface stress, causing the cantilever to bend, and the signal can be analyzed optically when it is illuminated by a laser. The change in surface stress measured with nano-scale precision allows disruptions of the biomechanics of model bacterial cell wall targets to be tracked in real time. Despite offering considerable advantages, multiple cantilever sensor arrays have never been applied in quantifying drug-target binding interactions.
Here, we report on the use of silicon multiple cantilever arrays coated with alkanethiol self-assembled monolayers mimicking bacterial cell wall matrix to quantitatively study antibiotic binding interactions. To understand the impact of vancomycin on the mechanics of bacterial cell wall structures1,6,7.&#160;We developed a new model1 which proposes that cantilever bending can be described by two independent factors; i) namely a chemical factor, which is given by a classical Langmuir adsorption isotherm, from which we calculate the thermodynamic equilibrium dissociation constant (Kd) and ii) a geometrical factor, essentially a measure of how bacterial peptide receptors are distributed on the cantilever surface. The surface distribution of peptide receptors (p) is used to investigate the dependence of geometry and ligand loading. It is shown that a threshold value of p ~10% is critical to sensing applications. Below which there is no detectable bending signal while above this value, the bending signal increases almost linearly,&#160;revealing that stress is a product of a local chemical binding factor and a geometrical factor combined by the mechanical connectivity of reacted regions and provides a new paradigm for design of powerful agents to combat superbug infections.

The stress change measured using nano-scale precision with unprecedented sensitivity to a single H-bond deletion, is exploited to track the disruptions of model bacterial cell wall biomechanics in real time (Figures 2a-d). The limit of drug detection sensitivity for Van was investigated by serially diluting its concentrations in steps from 1,000 &#956;M to &#8804;10 nM, revealing 10 nM (~15 ng/ml) as the lowest detectable concentration giving rise to an average of ~ -9&#177;2 nm as the differential bending signals (Figure 2c). The ability to detect antibiotics under physiological environment was further investigated in serum at a clinically relevant concentration range of 3-27 &#956;M17. Upon injection of 7 &#956;M Van in serum (90% fetal calf serum plus 10% sodium phosphate buffer pH 7.4) across all cantilevers under identical conditions, the differential signal for DAla in serum was 105&#177;4 nm while for DLac coated cantilevers, no bending was observed (Figure 3). The considerable bending signals observed for DAla coated (vancomycin susceptible) cantilevers are caused by the strong drug-target binding interactions. However for DLac coated (vancomycin resistant) cantilevers, the presence of ground-state repulsion of the oxygen lone pair and the reduced NH bond in the vancomycin binding pocket10,12 contributes to the weakening of the drug-target binding interactions which results in less or no cantilever bending, particularly for lower vancomycin concentrations (Figure 3). However, for high vancomycin concentration, we observe measureable bending signals for DLac. Furthermore, our experimental design is important for in&#160;situ referencing where all cantilevers coated with both DAla and DLac are simultaneously exposed to the same analyte in real time.
To understand the precise role of chemistry and geometry, we designed a model1 describing the correlation between solvent interactions and surface mechanics. This produces the surface stress:
<img alt="Equation 1" src="/files/ftp_upload/50719/50719eq1.jpg" /> for p &#62; pc and zero otherwise (1)
The first term in equation (1) is the Langmuir Adsorption Isotherm, accounting for drug-target binding events and the second term is the power law form describing the large-scale mechanical consequences of stressed network formation. The constant a corresponds to maximum surface stress when all the accessible binding sites are occupied and Kd is the surface equilibrium dissociation constant on the cantilever. The analysis as shown in Figure 4 is the outcome of the global fit of equation (1) superimposed onto measured differential stress signals revealing; a&#160; ~29.7&#177;1.0 or 14.1&#177;3.0 mN/m and Kd ~1.0&#177;0.3 or 800&#177;310 &#956;M for D-Ala or DLac peptides, where a corresponds to a measure of surface stress when all the accessible binding sites are occupied. The enhanced sensitivity of cantilever was tuned by systematically varying the peptide densities p while monitoring the stress data as a function of p while antibiotic concentrations fixed at 10, 100, and 250 &#956;M, respectively (Figure 5). The process was repeated with stress as a function of Van concentrations while the peptide densities fixed at p ~100%.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/50719/50719fig1.jpg" /> 
Figure 1. Cantilever detection of antibiotic surface binding interactions. a) Schematic representation of a cantilever sensor array and the mode by which the drug-target interactions are detected nanomechanically. b) Chemical binding interaction between a drug molecule (Van) and the bacterial mucopeptide analog&#160;(D-Ala or D-Lac). c) The mechanism by which a mutated vancomycin susceptible bacteria (VSE) acquires resistance to vancomycin by deleting a single H-bond in the binding pocket. <a href="https://www.jove.com/files/ftp_upload/50719/50719fig1large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/50719/50719fig2.jpg" /> 
Figure 2. Tracking the disruptions of the model bacterial cell wall biomechanics in real time. a) Absolute bending signals of D-Ala , D-Lac and PEG coated cantilevers in phosphate buffer and 250 &#956;M vancomycin. The differential PEG reference signals are shown in black lines. b) Differential bending signals of D-Ala and D-Lac coated cantilevers in phosphate buffer and 250 &#956;M vancomycin. c) The differential cantilever deflection of a dose dependent signal as a function of time in the presence of vancomycin concentrations in the order of 10 nM (yellow line), 100 nM (red line) and 1,000 nM (dark red line) respectively. d) The differential cantilever deflection signals for three D-Ala coated cantilever sensors as a function of time in the presence of 10 nM vancomycin. <a href="https://www.jove.com/files/ftp_upload/50719/50719fig2large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 3" fo:content-width="4in" src="/files/ftp_upload/50719/50719fig3.jpg" /> 
Figure 3. Tracking of the disruptions of the model bacterial cell wall biomechanics in real time for a D-Ala and D-Lac coated cantilevers in the presence of 7 &#956;M vancomycin in calf fetal serum.
<img alt="Figure 4" fo:content-width="4in" src="/files/ftp_upload/50719/50719fig4.jpg" /> 
Figure 4. Investigating the nanomechanics of drug-target interactions. Plot showing the measured differential surface stress response for D-Ala (black circles) and D-Lac (red circles) coated cantilevers as a function of vancomycin concentration in solution [Van]. The data are described by equation (1) (solid lines).1
<img alt="Figure 5" fo:content-width="4in" src="/files/ftp_upload/50719/50719fig5.jpg" /> 
Figure 5. Optimization of nanomechanical detection sensitivity of receptor-ligand interactions by investigating the dependence of receptor loading and geometry via mixed monolayers. Measured differential surface stress responses of cantilever sensors as a function of D-Ala surface coverage, p in the presence of fixed vancomycin concentrations in solution at 10 &#956;M (black line), 100 &#956;M (red line), and 250 &#956;M (green line).

Watch this Scientific Journal Video about Nanomechanics of Drug-target Interactions and Antibacterial Resistance Detection at JoVE.com

Nanomechanics of Drug-target Interactions and Antibacterial Resistance Detection

Acquired resistance to antibiotics is a major public healthcare problem and is presently ranked by the WHO as one of the greatest threats to human life. Here we describe the use of cantilever technology to quantify antibacterial resistance, critical to the discovery of novel and powerful agents against multidrug resistant bacteria.

The cantilever sensor, which acts as a transducer of reactions between model bacterial cell wall matrix immobilized on its surface and antibiotic drugs in ...

nanomechanics-drug-target-interactions-antibacterial-resistance

Research

JoVE Journal

Immunology and Infection

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below 50-75 copies/ml) is necessary to gain insight to viral dynamics and virus host interactions in patients who naturally control the infection and those who are on combination antiretroviral therapy (cART). 
Here we describe how to amplify viral genomes by single genome sequencing (the SGS protocol) 13, 19 and how to accurately quantify HIV-1 RNA in patients with low viral loads (the single-copy assay (SCA) protocol) 12, 20. 
The single-copy assay is a real-time PCR assay with sensitivity depending on the volume of plasma being assayed. If a single virus genome is detected in 7 ml of plasma, then the RNA copy number is reported to be 0.3 copies/ml. The assay has an internal control testing for the efficiency of RNA extraction, and controls for possible amplification from DNA or contamination. Patient samples are measured in triplicate.
The single-genome sequencing assay (SGS), now widely used and considered to be non-labor intensive 3, 7, 12, 14, 15,is a limiting dilution assay, in which endpoint diluted cDNA product is spread over a 96-well plate. According to a Poisson distribution, when less than 1/3 of the wells give product, there is an 80% chance of the PCR product being resultant of amplification from a single cDNA molecule. SGS has the advantage over cloning of not being subjected to resampling and not being biased by PCR-introduced recombination 19. However, the amplification success of SCA and SGS depend on primer design. Both assays were developed for HIV-1 subtype B, but can be adapted for other subtypes and other regions of the genome by changing primers, probes, and standards.

Quantifying levels of HIV-1 RNA in plasma and sequencing single HIV-1 genomes from individuals with viral loads below the limit of detection (50-75 copies/ml) is difficult. Here we describe how to extract and quantify plasma viral RNA using a real time PCR assay that reliably measures HIV-1 RNA down to 0.3 copies/ml and how to amplify viral genomes by single genome sequencing, from samples with very low viral loads.

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below ...

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. ...

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable method of analysis by flow cytometry to study nanoparticle interaction with immune cells. Primary immune cells can be easily purified from human or mouse tissues by antibody-mediated magnetic isolation. In the first instance, the different cell populations running in a flow cytometer can be distinguished by the forward-scattered light (FSC), which is proportional to cell size, and the side-scattered light (SSC), related to cell internal complexity. Furthermore, fluorescently labeled antibodies against specific cell surface receptors permit the identification of several subpopulations within the same sample. Often, all these features vary when cells are boosted by external stimuli that change their physiological and morphological state. Here, 50 nm FITC-SiO2 nanoparticles are used as a model to identify the internalization of nanostructured materials in human blood immune cells. The cell fluorescence and side-scattered light increase after incubation with nanoparticles allowed us to define time and concentration dependence of nanoparticle-cell interaction. Moreover, such protocol can be extended to investigate Rhodamine-SiO2 nanoparticle interaction with primary microglia, the central nervous system resident immune cells, isolated from mutant mice that specifically express the Green Fluorescent Protein (GFP) in the monocyte/macrophage lineage. Finally, flow cytometry data related to nanoparticle internalization into the cells have been confirmed by confocal microscopy.

Analysis of nanoparticle interaction with defined subpopulations of immune cells by flow cytometry.

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

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 ...

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or A. baumannii

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, preferably in models which mimic the intended clinical indication. A method for inducing robust lung infections in immunocompetent rats and mice is described which allows for the assessment of treatments in a model of serious pneumonia caused by S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae or A. baumannii. Animals are anesthetized, and an agar-based inoculum is deposited deep into the lung via nonsurgical intratracheal intubation. The resulting infection is consistent, reproducible, and stable for at least 48 h and up to 96 h for most isolates. Studies with marketed antibacterials have demonstrated good correlation between in vivo efficacy and in vitro susceptibility, and concordance between pharmacokinetic/pharmacodynamic targets determined in this model and clinically accepted targets has been observed. Although there is an initial time investment when learning the technique, it can be performed quickly and efficiently once proficiency is achieved. Benefits of the model include elimination of the neutropenic requirement, increased robustness and reproducibility, ability to study more pathogens and isolates, improved flexibility in study design and establishment of a challenging infection in an immunocompetent host.

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or  A. baumannii

A method for establishing lung infections in immunocompetent rodents is described. With proficiency, this method can be performed quickly and easily to induce stable infection with many isolates of S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae and A. baumannii.

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

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 ...

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.

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.