This work was supported by a grant from National Institute of Health to D.C.S. and W.S. AI123340. L.-C.W., J.W., A.C., and E.N. were supported in part/participate in &#34;The First-Year Innovation &#38; Research Experience&#34; program funded by the University of Maryland. The funders had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript. We acknowledge the UMD CBMG Imaging Core for all microscopy experiments.

1. General maintenance of GC strains
<ol>
	<li>Streak N. gonorrhoeae strains on GCK agar with 1% Kellogg supplements18 (Table 1, Table 2) from freezer stocks and incubate 37 &#176;C with 5% CO2 for 16-18 h. Use MS11 expressing phase-variable Opa (MS11Opa+), no Opa (MS11&#916;Opa), or a truncated LOS (MS11&#916;LgtE).</li>
	<li>Carefully pick pili negative (colony without dark edge) or positive (colony with dark edge) colonies from each strain based on colony morphology19 using a dissecting light microscope and streak onto a new GCK plate.</li>
	<li>Incubate at 37 &#176;C with 5% CO2 for 16-18 h before use.</li>
</ol>
2. Viability quantification of GC aggregations
<ol>
	<li>Collect GC using a sterile applicator. Swab GC from the plate and re-suspend GC in pre-warmed broth(GCP, Table 3) supplemented with 4.2% NaHCO3 and 1% Kellogg solutions18. Use spectrophotometry at a wavelength of 650 nm (an OD650 of 1 = ~1 x 109 CFU/mL) to determine the concentration of suspended bacteria.</li>
	<li>Adjust the concentration of GC to ~1 x 108 CFU/mL.</li>
	<li>Add 99 &#181;L of adjusted GC suspension into wells of a 96-well plate.</li>
	<li>Incubate the plate for 6 h at 37 &#176;C with 5% CO2 to allow the bacteria to aggregate.</li>
	<li>Add 1 &#181;L of serial diluted ceftriaxone (1000, 100, 50, 25, 12.5, 6.2, 3.1, 1.5, 0.8, 0.4, 0.2 &#181;g/mL) into each well. Leave some wells untreated to serve as controls.</li>
	<li>Incubate the plate for 24 h at 37 &#176;C with 5% CO2.</li>
	<li>Sonicate the suspension 3 times in each well for 5 s at 144 W and 20 kHz.</li>
	<li>Add 100 &#181;L of commercially available ATP utilization glow reagent into each well, pipette up-and down for 3 times, and incubate for 15 min at 37 &#176;C with 5% CO2.</li>
	<li>Carefully transfer 150 &#181;L of mixture from each well into a new well in a 96-well black microplate and avoid introducing bubbles.</li>
	<li>Measure the absorbance of each well at 560 nm using the plate reader.</li>
	<li>Calculate the survival rate by the ratio of the reading obtained after serial ceftriaxone treatment to the reading from untreated wells.</li>
</ol>
3. Fluorescence microscopic analysis of Live/Dead of GC aggregates
<ol>
	<li>Collect GC using a sterile applicator. Swab GC from the plate and re-suspend GC in pre-warmed GCP media plus 1% Kellogg supplements.</li>
	<li>Determine the number of bacteria by spectrophotometry at a wavelength of 650 nm and adjust the concentration of GC to ~1 x 107 CFU/mL.</li>
	<li>Add 198 &#181;L of GC suspension into in 8-well coverslip-bottom chambers.</li>
	<li>Incubate the chamber for 6 h at 37 &#176;C with 5% CO2 to allow aggregation formation.</li>
	<li>Add 2 &#181;L of ceftriaxone (100 &#181;g/mL or various dilutions) into each well within each aggregation condition. Incubate for the desired time at 37 &#176;C with 5% CO2.</li>
	<li>Add 0.6 &#181;L of live/dead staining solution mixture into each well and incubate for 20 min at 37 &#176;C with 5% CO2.</li>
	<li>Acquire Z-series images using a confocal microscope (an equivalent microscope can be used).</li>
	<li>Analyze the images using ImageJ software for measurement of the size of GC aggregates and the fluorescence intensity ratio (FIR) of live-to-dead staining in each aggregate.</li>
</ol>
4. Image analysis
<ol>
	<li>Estimation of the size of bacterial aggregates.
	<ol>
		<li>Open ImageJ and open an image by dragging a raw image file to the ImageJ menu bar.</li>
		<li>Click Freehand Lines in the ImageJ menu bar and circle the area of each aggregation in the image.</li>
		<li>Click Analyze | Measure in the ImageJ menu bar.</li>
		<li>Obtain the number in a new window under the Area column.</li>
	</ol>
	</li>
	<li>Quantification of live-to-dead ratio of aggregations
	<ol>
		<li>Open ImageJ and open an image by dragging a raw image file to the ImageJ menu bar.</li>
		<li>Click Analyze | Set Measurements in the ImageJ menu bar.</li>
		<li>Check the integrated density in the pop-up window and click OK.</li>
		<li>Click Image | Color | Channels Tool in the menu bar and select Color.</li>
		<li>Check Channel 1 as the fluorescence for live bacteria staining.</li>
		<li>Click Freehand Lines in the ImageJ menu bar and circle the area of each aggregation.</li>
		<li>Click Analyze | Measure in the ImageJ menu bar.</li>
		<li>Obtain the number under IntDen column.</li>
		<li>Check Channel 2 as the fluorescence for dead bacterial staining. Repeat steps 4.2.6-4.2.8.</li>
		<li>Obtain the live-to-dead ratio by dividing number from step 4.2.8 by number from step 4.2.9.</li>
	</ol>
	</li>
	<li>Statistical analysis
	<ol>
		<li>Open GraphPad Prism and enter the numbers obtained from ImageJ to the desired column.</li>
		<li>Click Analyze and select t tests under Column analyses.</li>
		<li>Check the desired column for comparison and click OK.</li>
		<li>Obtain the P-Value under Analysis window.</li>
		<li>Select the Linear Regression under XY analyses from step 4.3.3</li>
		<li>Obtain the R-square and the P-Value under the Analysis window.</li>
	</ol>
	</li>
</ol>

<ol><li>CDC STD Facts. Centers for Disease Control and Prevention. , Available from: 
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<li>Zollner, R., Oldewurtel, E. R., Kouzel, N., Maier, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Phase+and+antigenic+variation+govern+competition+dynamics+through+positioning+in+bacterial+colonies%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Phase and antigenic variation govern competition dynamics through positioning in bacterial colonies.</a> Scientific Reports. 7 (1), (2017).</li>
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<li>Merritt, J. H., Kadouri, D. E., O'Toole, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Growing+and+analyzing+static+biofilms%5BTitle%5D)+AND+%22Curr+Protoc+Microbiol%22%5BJournal%5D)">Growing and analyzing static biofilms.</a> Curr Protoc Microbiol. , Chapter 1., Unit 1B.1 (2005).</li>
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The authors have nothing to disclose.

Bacteria can form biofilms during infection of the human body. Traditional MIC testing may not reflect the concentration needed to eradicate bacteria in a biofilm. To test antimicrobials effects on a biofilm, methods based on biofilm biomass as well as plating CFUs can be erroneous due to the impact of biofilm structure. For example, the plating method only works if the biofilm can be disrupted. Hence, the CFU obtained may be lower than the actual number of viable bacteria. Visualizing dead and live bacteria within the b...

Gonorrhea is a common sexually transmitted infection (STI)1. Neisseria gonorrhoeae (GC), a Gram-negative diplococcal bacterium, is the causative agent of this disease. Symptoms of genital infection can result in pain during urination, generalized genital pain, and urethral discharge. Infection is often asymptomatic2,3,4,5, and this allows for extended colonization. These untreated infections are a major health concern, as they have the potential to facilitate transmission of the organism and this can lead to complications such as pelvic inflammatory disease (PID) and disseminated gonococcal infection (DGI)6. Antibiotic-resistant gonorrhea is a major public health crisis and an increasing socioeconomic burden7. Reduced susceptibility to cephalosporins has resulted in treatment regimen change from a single antibiotic to dual therapy, which combines azithromycin or doxycycline with ceftriaxone8. The increased failure of ceftriaxone and azithromycin9,10, in combination with asymptomatic infections, highlights the need for understanding gonorrhea treatment failures.
The minimum inhibitory concentration (MIC) test, including agar dilution and disc diffusion tests, has been used as the standard medical test for identifying resistance to an antibiotic. Nevertheless, it is unclear if the MIC test reflects bacterial antibiotic resistance in vivo. The formation of bacterial biofilms contributes to the survival of bacteria in the presence of bactericidal concentrations of antibiotic: the MIC testing is unable to detect this effect11. Because GC can form biofilms on mucosal surfaces12, we hypothesize that antibiotic susceptibility within aggregates would be different from that seen in individual GC. Additionally, studies have shown that three phase variable surface molecules, Pili, opacity-associated protein (Opa), and lipooligosaccharides (LOS), that regulate inter-bacterium interactions, lead to different sized aggregates13,14,15. The contribution of these components to antibiotic resistance has not been examined due to the lack of proper methods.
Currently, there are several methods to measure biofilm eradication. The most widely used quantitative method is by measuring the changes in biomass using crystal violet staining16. However, the method requires significant experimental manipulation, which can potentially generate errors in experiment repeats17. The live/dead staining method used here allows visualization of live and dead bacteria and their distribution within the biofilm. However, the biofilm structure can pose as a physical barrier that reduces dye penetration. Therefore, to quantify live/dead bacteria within a group, the staining is limited to small biofilms or its precursor- microcolonies or aggregations. Other methods, including the agar dilution and disc diffusion tests, are not able to measure the effects of aggregation. To examine GC susceptibility within aggregation after antibiotic exposure, an ideal method would need to have both a quantitative assay that can measure live bacteria and visualize their distribution.
The procedure described here combines an ATP-utilization measurement and a live/dead staining assay to quantitatively and visually examine GC susceptibility within aggregates in the presence of antibiotics.

<table><tbody><tr><td>100x Kellogg&#039;s supplement</td><td></td><td></td><td></td></tr><tr><td>Agar</td><td>United States Biological</td><td>A0930</td><td></td></tr><tr><td>BacTiter Assay&amp;nbsp;</td><td>Promega</td><td>G8232</td><td></td></tr><tr><td>Ceftriaxone</td><td>TCI</td><td>C2226</td><td></td></tr><tr><td>Difco GC medium base&amp;nbsp;</td><td>BD</td><td>228950</td><td></td></tr><tr><td>Ferric nitrate, nonahydrate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>254223-10G</td><td></td></tr><tr><td>Glucose</td><td>Thermo Fisher Scientific</td><td>BP350-1</td><td></td></tr><tr><td>L-glutamine Crystalline Powder</td><td>Fisher Scientific</td><td>BP379-100</td><td></td></tr><tr><td>BacLight live/dead staining</td><td>Invitrogen</td><td>L7012</td><td></td></tr><tr><td>MS11 Neisseria gonorrhoeae strain</td><td></td><td></td><td>kindly provided by Dr. Herman Schneider, Walter Reed Army Institute for Research</td></tr><tr><td>Potassium phosphate dibasic (K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Fisher Scientific</td><td>P290-500</td><td></td></tr><tr><td>Potassium phosphate monobasic (KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Fisher Scientific</td><td>BP329-1</td><td></td></tr><tr><td>Proteose Peptone&amp;nbsp;</td><td>BD Biosciences</td><td>211693</td><td></td></tr><tr><td>Sodium chloride (NaCl)</td><td>Fisher Scientific</td><td>S671-10</td><td></td></tr><tr><td>Soluble Starch</td><td>Sigma-Aldrich</td><td>S9765</td><td></td></tr><tr><td>Thiamine pyrophosphate</td><td>Sigma-Aldrich</td><td>C8754-5G</td><td></td></tr><tr><td>Equipment</td><td></td><td></td><td></td></tr><tr><td>Petri Dishes</td><td>VWR</td><td>25384-302</td><td></td></tr><tr><td>8-well coverslip-bottom chamber&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>155411</td><td></td></tr><tr><td>96-well tissue culture plates&amp;nbsp;</td><td>Corning, Falcon</td><td>3370</td><td></td></tr><tr><td>Biosafety Cabinet (NU-425-600 Class II, A2 Laminar Flow Biohazard Hood)</td><td>Nuaire</td><td>32776</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; Incubator</td><td>Fisher Scientific</td><td>&amp;nbsp;Model 3530</td><td></td></tr><tr><td>Confocal microscope equipped with live imaging chamber</td><td>Leica</td><td>SP5X</td><td></td></tr><tr><td>Corning&amp;nbsp; 96 Well Black Polystyrene Microplate&amp;nbsp;</td><td>Corning</td><td>3904</td><td></td></tr><tr><td>Glomax Illuminator&amp;nbsp;</td><td>Promega</td><td>E6521</td><td></td></tr><tr><td>Pipette tips (0.1-10 &amp;micro;L)</td><td>Thermo Fisher Scientific</td><td>02-717-133</td><td></td></tr><tr><td>Pipette tips (1000 &amp;micro;L)</td><td>VWR</td><td>83007-382</td><td></td></tr><tr><td>Pipette tips (200 &amp;micro;L)</td><td>VWR</td><td>53509-007</td><td></td></tr><tr><td>Spectrophotometer Ultrospec 2000 UV</td><td>Pharmacia Biotech</td><td>80-2106-00</td><td></td></tr><tr><td>Sterile 15 ml conical tubes</td><td>VWR</td><td>21008-216</td><td></td></tr><tr><td>Sterile Microcentrifuge Tubes (1.7 mL)</td><td>Sorenson BioScience</td><td>16070</td><td></td></tr><tr><td>Sterile polyester-tipped applicators</td><td>Fisher Scientific</td><td>23-400-122</td><td></td></tr><tr><td>Sonicator</td><td>Kontes</td><td>Equivelent to 9110001</td><td></td></tr></tbody></table>

quantitative examination of antibiotic susceptibility of neisseria gonorrhoeae aggregates using atp-utilization commercial assays and live/dead staining

The emergence of antibiotic resistant Neisseria gonorrhoeae (GC) is a worldwide health threat and highlights the need to identify individuals who fail treatment. This Gram-negative bacterium causes gonorrhea exclusively in humans. During infection, it is able to form aggregates and/or biofilms. The minimum inhibitory concentration (MIC) test is used for to determine susceptibility to antibiotics and to define appropriate treatment. However, the mechanism of the eradication in vivo and its relationship to laboratory results are not known. A method that examines how GC aggregation affects antibiotic susceptibility and shows the relationship between aggregate size and antibiotic susceptibility was developed. When GC aggregate, they are more resistant to antibiotic killing, with bacteria in the center surviving ceftriaxone treatment better than those in the periphery. The data indicate that N. gonorrhoeae aggregation can reduce its susceptibility to ceftriaxone, which is not reflected using the standard agar plate-based MIC methods. The method used in this study will allow researchers to test bacterial susceptibility under clinically relevant conditions.

Two methods were employed: an ATP utilization assay and a live/dead staining assay. The results can either be combined or individually used for examining bacterial survival within aggregates after antibiotic treatment. The ATP utilization assay has been shown to measure accurately viable bacteria in S. aureus biofilms20,21. Here, MS11Opa+Pil+ strain was used to examine the role of GC aggregation in antibiotic susceptibili...

Watch this Scientific Journal Video about Quantitative Examination of Antibiotic Susceptibility of Neisseria gonorrhoeae Aggregates Using ATP-utilization Commercial Assays and Live/Dead Staining at Jo

Quantitative Examination of Antibiotic Susceptibility of Neisseria gonorrhoeae Aggregates Using ATP-utilization Commercial Assays and Live/Dead Staining

A simple ATP-measuring assay and live/dead staining method were used to quantify and visualize Neisseria gonorrhoeae survival after treatment with ceftriaxone. This protocol can be extended to examine the antimicrobial effects of any antibiotic and can be used to define the minimal inhibitory concentration of antibiotics in bacterial biofilms.

Quantitative Examination of Antibiotic Susceptibility of Neisseria gonorrhoeae Aggregates Using ATP-utilization Commercial Assays and Live/Dead Staining

The emergence of antibiotic resistant Neisseria gonorrhoeae (GC) is a worldwide health threat and highlights the need to identify individuals who fail ...

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8.6K Views.  National Sun Yat-Sen University. Deze techniek stelt ons in staat om antibiotica gevoeligheid te meten onder meer klinisch relevante omstandigheden. De techniek stelt ons ook in staat om de ware antibioticaconcentratie te bepalen die bacteriën in aggregaten kan doden. Deze techniek kan worden toegepast op het bepalen van de concentratie die nodig is voor het elimineren van GC aggregaten.De methode kan ook worden toegepast op het meten van de antimicrobiële gevoeligheid van alle bacteriën die in staat is om aggregat...

Video: Quantitative Examination of Antibiotic Susceptibility of Neisseria gonorrhoeae Aggregates Using ATP-utilization Commercial Assays and Live/Dead Staining

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols to address these questions include colony count assays, gentamicin protection assays, and electron microscopy. Colony count and gentamicin protection assays only assess the viability of the entire bacterial population and are unable to determine individual bacterial viability. Electron microscopy can be used to determine the viability of individual bacteria and provide information regarding their localization in host cells. However, bacteria often display a range of electron densities, making assessment of viability difficult. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells. These assays were developed originally to assess survival of Neisseria gonorrhoeae in primary human neutrophils, but should be applicable to any bacterium-host cell interaction. These protocols combine membrane-permeable fluorescent dyes (SYTO9 and 4&#39;,6-diamidino-2-phenylindole [DAPI]), which stain all bacteria, with membrane-impermeable fluorescent dyes (propidium iodide and SYTOX Green), which are only accessible to nonviable bacteria. Prior to eukaryotic cell permeabilization, an antibody or fluorescent reagent is added to identify extracellular bacteria. Thus these assays discriminate the viability of bacteria adherent to and inside eukaryotic cells. A protocol is also provided for using the viability dyes in combination with fluorescent antibodies to eukaryotic cell markers, in order to determine the subcellular localization of individual bacteria. The bacterial viability dyes discussed in this article are a sensitive complement and/or alternative to traditional microbiology techniques to evaluate the viability of individual bacteria and provide information regarding where bacteria survive in host cells.

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells.

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols ...

A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would not occur spontaneously. These proteins are crucial for essentially all aspects of cellular life, including metabolism, cell division, responses to environmental changes and movement. The protocol presented here describes a nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay that has been adapted to semi-high throughput screening of small molecule ATPase inhibitors. The assay has been applied to cardiac and skeletal muscle myosin II's, two actin-based molecular motor ATPases, as a proof of principle. The hydrolysis of ATP is coupled to the oxidation of NADH by enzymatic reactions in the assay. First, the ADP generated by the ATPase is regenerated to ATP by pyruvate kinase (PK). PK catalyzes the transition of phosphoenolpyruvate (PEP) to pyruvate in parallel. Subsequently, pyruvate is reduced to lactate by lactate dehydrogenase (LDH), which catalyzes the oxidation of NADH in parallel. Thus, the decrease in ATP concentration is directly correlated to the decrease in NADH concentration, which is followed by change to the intrinsic fluorescence of NADH. As long as PEP is available in the reaction system, the ADP concentration remains very low, avoiding inhibition of the ATPase enzyme by its own product. Moreover, the ATP concentration remains nearly constant, yielding linear time courses. The fluorescence is monitored continuously, which allows for easy estimation of the quality of data and helps to filter out potential artifacts (e.g., arising from compound precipitation or thermal changes).

A nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay has been adapted to semihigh throughput screening of small molecule myosin inhibitors. This kinetic assay is run in a 384-well microplate format with total reaction volumes of only 20 &#181;L per well. The platform should be applicable to virtually any ADP producing enzyme.

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would ...

Biochemistry

Nucleoside Triphosphate Hydrolases Assay in Toxoplasm gondii and Neospora caninum for High-Throughput Screening using a Robot Arm

Protozoan parasites infect humans and many warm-blooded animals. Toxoplasma gondii, a major protozoan parasite, is commonly found in HIV-positive patients, organ transplant recipients and pregnant women, resulting in the severe health condition, Toxoplasmosis. Another major protozoan, Neospora caninum, which bears many similarities to Toxoplasma gondii, causes serious diseases in animals, as does Encephalomyelitis and Myositis-Polyradiculitis in dogs and cows, resulting in stillborn calves. All these exhibited similar nucleoside triphosphate hydrolases (NTPase). Neospora caninum has a NcNTPase, while Toxoplasma gondii has a TgNTPase-I. The enzymes are thought to play crucial roles in propagation and survival. In order to establish compounds and/or extracts preventing protozoan infection, we targeted these enzymes for drug discovery. The next step was to establish a novel, highly sensitive, and highly accurate assay by combining a conventional biochemical enzyme assay with a fluorescent assay to determine ADP content. We also validated that the novel assay fulfills the criteria to carry out high-throughput screening (HTS) in the two protozoan enzymes. We performed HTS, identified 19 compounds and six extracts from two synthetic compound libraries and an extract library derived from marine bacteria, respectively. In this study, a detailed explanation has been introduced on how to carry out HTS, including information about the preparation of reagents, devices, robot arm, etc.

Nucleoside Triphosphate Hydrolases Assay in Toxoplasm gondii and Neospora caninum for High-Throughput Screening using a Robot Arm

Toxoplasma gondii and Neospora caninum infections are found in humans and animals and lead to serious health issues. The two parasites share similar nucleoside triphosphate hydrolases and play important roles in propagation and survival. We established a high-standard assay of the enzymes requiring robot arm usage.

Protozoan parasites infect humans and many warm-blooded animals. Toxoplasma gondii, a major protozoan parasite, is commonly found in HIV-positive ...

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require several additional experimental steps prior to readout, such as cell lysis, colony forming unit determination, or reagent addition. When performing thousands of assays, for example, during high-throughput screening, the downstream effort required for these types of assays is considerable. Therefore, to facilitate high-throughput antimicrobial discovery, we developed a real-time assay to simultaneously identify inhibitors of intracellular bacterial growth and assess eukaryotic cell cytotoxicity. Specifically, real-time intracellular bacterial growth detection was enabled by marking bacterial screening strains with either a bacterial lux operon (1st generation assay) or fluorescent protein reporters (2nd generation, orthogonal assay). A non-toxic, cell membrane-impermeant, nucleic acid-binding dye was also added during initial infection of macrophages. These dyes are excluded from viable cells. However, non-viable host cells lose membrane integrity permitting entry and fluorescent labeling of nuclear DNA (deoxyribonucleic acid). Notably, DNA binding is associated with a large increase in fluorescent quantum yield that provides a solution-based readout of host cell death. We have used this combined assay to perform a high-throughput screen in microplate format, and to assess intracellular growth and cytotoxicity by microscopy. Notably, antimicrobials may demonstrate synergy in which the combined effect of two or more antimicrobials when applied together is greater than when applied separately. Testing for in vitro synergy against intracellular pathogens is normally a prodigious task as combinatorial permutations of antibiotics at different concentrations must be assessed. However, we found that our real-time assay combined with automated, digital dispensing technology permitted facile synergy testing. Using these approaches, we were able to systematically survey action of a large number of antimicrobials alone and in combination against the intracellular pathogen, Legionella pneumophila.

A high throughput, real-time assay was developed to simultaneously identify (1) eukaryotic cell-penetrant antimicrobials targeting an intracellular bacterial pathogen, and (2) assess eukaryotic cell cytotoxicity. A variation on the same technology was thereafter combined with digital dispensing technology to enable facile, high-resolution, dose-response, and two- and three-dimensional synergy studies.

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require ...

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.

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

Tools for the Real-Time Assessment of a Pseudomonas aeruginosa Infection Model

Pseudomonas aeruginosa (Pa) is one of the most common opportunistic pathogens associated with cystic fibrosis (CF). Once Pa colonization is established, a large proportion of the infecting bacteria form biofilms within airway sputum. Pa biofilms isolated from CF sputum have been shown to grow in small, dense aggregates of ~10-1,000 cells that are spatially organized and exhibit clinically relevant phenotypes such as antimicrobial tolerance. One of the biggest challenges to studying how Pa aggregates respond to the changing sputum environment is the lack of nutritionally relevant and robust systems that promote aggregate formation. Using a synthetic CF sputum medium (SCFM2), the life history of Pa aggregates can be observed using confocal laser scanning microscopy (CLSM) and image analysis at the resolution of a single cell. This in vitro system allows the observation of thousands of aggregates of varying size in real time, three dimensions, and at the micron scale. At the individual and population levels, having the ability to group aggregates by phenotype and position facilitates the observation of aggregates at different developmental stages and their response to changes in the microenvironment, such as antibiotic treatment, to be differentiated with precision.

Tools for the Real-Time Assessment of a Pseudomonas aeruginosa Infection Model

Synthetic cystic fibrosis sputum medium (SCFM2) can be utilized in combination with both confocal laser scanning microscopy and fluorescence-activated cell sorting to observe bacterial aggregates at high resolution. This paper details methods to assess aggregate populations during antimicrobial treatment as a platform for future studies.

Pseudomonas aeruginosa (Pa) is one of the most common opportunistic pathogens associated with cystic fibrosis (CF). Once Pa colonization is established, a ...

Antibiotic Susceptibility Testing: Epsilometer Tests to Determine MIC Values of Two Antibiotics and Evaluate Antibiotic Synergy

Source: Anna Bl&#228;ckberg1, Rolf Lood1 
1 Department of Clinical Sciences Lund, Division of Infection Medicine, Biomedical Center, Lund University, 221 00 Lund Sweden
Knowledge of the interactions between antibiotics and bacteria is important in understanding how microbes evolve antibiotic resistance. In 1928, Alexander Fleming discovered penicillin, an antibiotic that exerts its antibacterial function by interfering with cell wall regeneration (1). Other antibiotics with diverse mechanisms of action have subsequently been discovered, including drugs that inhibit DNA replication and protein translation in bacteria; however, no new antibiotics have been developed in recent years. Resistance to current antibiotics has been increasing, resulting in severe infectious diseases that cannot be effectively treated (2). Here, we describe several methods to assess antibiotic resistance in bacterial populations. Each of these methods works, regardless of the mechanism of action of the antibiotics used, because bacterial death is the measured outcome. Antibiotic resistance is not only rapidly disseminated specifically through hospital settings, but also throughout society. In order to investigate such means of resistance, different methods have been developed including the Epsilometer test (E-test) and the broth dilution test (3).
The E-test is a well-established method and is a cost-effective tool that quantifies Minimal Inhibitory Concentration (MIC) data, the lowest concentration of an antimicrobial that inhibits visible growth of a microorganism. Depending on the bacterial strain and antibiotics used, the MIC value can vary between sub &#956;g/mL to &#62;1000 &#956;g/mL (4). The E-test is performed using a plastic strip containing a predefined antibiotic gradient, which is imprinted with the MIC reading scale in &#956;g/mL. This strip is directly transferred on the agar matrix when applied to the inoculated agar plate. After incubation, a symmetrical elliptic inhibition zone is visible along the strip as bacterial growth is prevented. MIC is defined by the area of inhibition, which is the endpoint where the ellipse intersects the strip. Another common method to determine MIC is the microbroth dilution method. Microbroth dilution incorporates different concentrations of the antimicrobial agent added to a broth medium containing inoculated bacteria. After incubation, MIC is defined as the lowest concentration of antibiotic that prevents visible growth (5). It is also a quantitative method and can be applied to several bacteria. Disadvantages of this method include the possibility of errors when preparing the concentrations of the reagents and the large number of reagents required for the experiment. Measuring antibiotic resistance is imperative from both a clinical and research perspective, and these in vitro methods of investigating resistance are discussed and showcased below.
The profile of resistance for a specific bacterium can be applied in order to optimize antibiotic treatment to determine whether a patient would benefit from combination treatment versus single therapy. For the usage of more than one antibiotic at a single time, it is imperative to know their interactions with each other and if they have an additive, synergistic, or antagonistic effect. An additive effect can be seen when the joint effect of the antibiotics equals the potency of the individual antibiotics given at an equal dose. Synergy between antibiotics, on the other hand, is present when the joint effect of the antibiotics is more potent than if the drug would be given alone (6). Applying combinations of antimicrobial treatment is used to avoid the occurrence of antimicrobial resistance thus to enhance the effect of the individual antibiotic treatment (7). Knowledge of antagonism is also as important to prevent unnecessary use of antimicrobial combinations. E-test methodology offers simple and several ways to determine possible synergy and antagonism between different antimicrobial agents. In order to face the proliferation of antibiotic resistant pathogens, knowledge of possible synergistic and antagonistic mechanisms of certain antibiotics is important resulting in clinical efficacy and battling multidrug-resistance.
Determination of synergy using E-tests can be divided into two broad approaches: cross and non-cross testing. While both synergy tests rely on the previous knowledge of individual MIC values, the two approaches are slightly different in methodology and conceptual approach. In a non-cross synergy test, the first antibiotic in the pair to be tested is placed on an agar plate inoculated with bacteria. After allowing the antibiotics from the first strip infuse the plate (e.g. after 1 hour), the strip is removed and a new strip containing the second antibiotic is placed on the exact same spot as the first, making sure to place the two individual MIC values on top of each other. The resulting inhibition zone can then be analyzed as described above, and synergy calculated based on the Equation 1.
Equation 1 - Fractional Inhibitory Concentrations (FIC)
<img src="/files/ftp_upload/10512/10512eq1.jpg" />
Values &#62;0.5 demonstrates synergy.
While rewarding the examiner with easy-to-analyze plates, the method is somewhat laborious and time-consuming due to the change of strips, as well as the need of using two plates per experiment. Instead, a cross test is often employed. Instead of adding the two different E-test strips subsequently on top of each other (after removal of the first), both are placed simultaneously but in the form of a cross (<img align="center" src="/files/ftp_upload/10512/10512eq3.jpg" /> 90&#176; angle), with the two previously determined MIC values forming the 90&#176; angle. By this approach only one plate is needed per synergy test, as well as less work, making it a preferred choice despite being slightly more difficult to analyze. The new MIC values in the combined antibiotics approach can be visualized as the modified inhibition zones, after which synergy can be determined by Equation 1.
Instead of using an agar plate approach, a microbroth approach can often be preferential due to its higher flexibility (e.g. ability to choose specific concentrations of antibiotics outside the limits of an E-test strip). Further, microbroth tests are suggested to be more sensitive due to their even distribution of antibiotics in a liquid solution, not depending on the dissociation within a solid phase (agar plate). Wells in a 96-well microplate will be inoculated with a set number of bacteria (106 cfu/mL: bacterial concentration can be estimated by OD600 nm measurements, turbidity standards, or by spread plating samples from 10x bacterial serial dilutions), and antibiotics in different dilutions will be added to the wells. Similarly, to the E-test strips MIC is determined as the intersection (well/spot) with the lowest concentration of antibiotics inhibiting visible growth of bacteria.
Experimental Aim
<ul>
	<li>The project below describes strategies to determine MIC-values of penicillin G and gentamicin of Streptococcus group G by two different methods, E-test and microbroth dilution. For E-test, Mueller-Hinton agar plates inoculated with Streptococcus group G were used in combination with gradient strips of penicillin G and/or gentamicin; while MH-broth with 50% lysed horse blood and 20 mg/mL &#946;-NAD were used with soluble antibiotics together with Streptococcus group G in a microbroth approach.</li>
</ul>
Materials
<ul>
	<li>Bacterial colonies on a blood agar plate, stored &#60;7 days in 4&#176;C</li>
	<li>Blood agar plates</li>
	<li>0.5 McFarland standard
	<ul>
		<li>1% BaCl2</li>
		<li>1% H2SO4</li>
	</ul>
	</li>
	<li>Saline tube (2 mL)</li>
	<li>Cotton tipped applicator</li>
	<li>Mueller-Hinton agar plates (MHA plates)</li>
	<li>MH broth with 50% lysed horse blood and 20 mg/mL &#946;-NAD (MH-F)</li>
	<li>E-test penicillin/gentamicin (or antibiotics of interest) (BioMerieux, Marcy l&#39;Etoile, France, Sweden)</li>
	<li>Antibiotics penicillin/gentamicin (or antibiotics of interest (powder/solution))</li>
</ul>
Note: The specific media used for bacterial growth may vary for different species.

Antibiotic Susceptibility Testing, MIC and Antibiotic Synergy

Source: Anna Bl&#228;ckberg1, Rolf Lood1
1 Department of Clinical Sciences Lund, Division of Infection Medicine, Biomedical Center, Lund University, 221 ...

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An Assay to Measure the Accumulation of a Fluorescent Antibiotic Probe in Bacterial Cells

Source: Stone, M. R. L., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/60743">Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes.</a> J. Vis. Exp. (2020).
This video demonstrates an assay to measure the accumulation of a fluorescent antibiotic probe in bacterial cells. Exposure to an efflux pump inhibitor disrupts the transmembrane proton gradient in bacterial cells, upon which a fluorophore-conjugated antibiotic is added. Inhibition of efflux pumps due to the disrupted proton gradient causes accumulation of the antibiotic inside the bacteria. Bacterial cells are lysed, and the fluorescence intensity is measured to confirm antibiotic accumulation.

1. Evaluation of Antimicrobial Activity
NOTE: All work involving bacteria should be carried out under sterile conditions to avoid contamination of either ...

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Immunology

Antibody-Based Technologies

Characterization and Functional Analysis

An ATP Utilization Assay for Determining Bacterial Survival Under Antibiotics Treatment

Source: Wang, Liang-Chun, et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/58978">Quantitative Examination of Antibiotic Susceptibility of Neisseria gonorrhoeae Aggregates Using ATP-utilization Commercial Assays and Live/Dead Staining.</a> J. Vis. Exp. (2019).
This video describes the simple ATP-measuring assay using luciferase glow reagent. This assay is used to quantify Neisseria gonorrhoeae survival after treatment with ceftriaxone.

1. Viability quantification of GC aggregations

	Collect GC using a sterile applicator. Swab GC from the plate and re-suspend GC in pre-warmed broth(GCP, ...

Quantitative Examination of Antibiotic Susceptibility of Neisseria gonorrhoeae Aggregates Using ATP-utilization Commercial Assays and Live/Dead Staining

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