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

<ol>
	<li>den Heijer, C. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comprehensive+overview+of+urogenital,+anorectal+and+oropharyngeal+Neisseria+gonorrhoeae+testing+and+diagnoses+among+different+STI+care+providers:+a+cross-sectional+study.">A comprehensive overview of urogenital, anorectal and oropharyngeal Neisseria gonorrhoeae testing and diagnoses among different STI care providers: a cross-sectional study.</a> BMC Infectious Diseases. 17 (1), (2017).</li><li>Hein, K., Marks, A., Cohen, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asymptomatic+gonorrhea:+prevalence+in+a+population+of+urban+adolescents.">Asymptomatic gonorrhea: prevalence in a population of urban adolescents.</a> The Journal of Pediatrics. 90 (4), 634-635 (1977).</li><li>Hananta, I. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gonorrhea+in+Indonesia:+High+Prevalence+of+Asymptomatic+Urogenital+Gonorrhea+but+No+Circulating+Extended+Spectrum+Cephalosporins-Resistant+Neisseria+gonorrhoeae+Strains+in+Jakarta,+Yogyakarta,+and+Denpasar,+Indonesia.">Gonorrhea in Indonesia: High Prevalence of Asymptomatic Urogenital Gonorrhea but No Circulating Extended Spectrum Cephalosporins-Resistant Neisseria gonorrhoeae Strains in Jakarta, Yogyakarta, and Denpasar, Indonesia.</a> Sexually Transmitted Diseases. 43 (10), 608-616 (2016).</li><li>Chlamydia, W. H. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chlamydia+trachomatis,+Neisseria+gonorrhoeae,+syphilis+and+Trichomonas+vaginalis.+Methods+and+results+used+by+WHO+to+generate+2005+estimates.+World+Health+Organisation,+2011.+Prevalence+and+incidence+of+selected+sexually+transmitted+infections.">Chlamydia trachomatis, Neisseria gonorrhoeae, syphilis and Trichomonas vaginalis. Methods and results used by WHO to generate 2005 estimates. World Health Organisation, 2011. Prevalence and incidence of selected sexually transmitted infections.</a> World Health Organisation. , (2011).</li><li>Mayor, M. T., Roett, M. A., Uduhiri, K. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sexually+transmitted+diseases+treatment+guidelines,+2015.">Sexually transmitted diseases treatment guidelines, 2015.</a> MMWR Recommendations and Reports. 64, 1-137 (2015).</li><li>Lahra, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cooperative+Recognition+of+Internationally+Disseminated+Ceftriaxone-Resistant+Neisseria+gonorrhoeae+Strain.">Cooperative Recognition of Internationally Disseminated Ceftriaxone-Resistant Neisseria gonorrhoeae Strain.</a> Emerging Infectious Diseases. 24 (4), (2018).</li><li>Wi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+resistance+in+Neisseria+gonorrhoeae:+Global+surveillance+and+a+call+for+international+collaborative+action.">Antimicrobial resistance in Neisseria gonorrhoeae: Global surveillance and a call for international collaborative action.</a> PLOS Medicine. 14 (7), 1002344 (2017).</li><li>Singh, S., Singh, S. 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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+Opacity+Proteins+Interferes+with+the+Transmigration+of+Neisseria+gonorrhoeae.+across+Polarized+Epithelial+Cells.">Expression of Opacity Proteins Interferes with the Transmigration of Neisseria gonorrhoeae. across Polarized Epithelial Cells.</a> PLoS One. 10 (8), 0134342 (2015).</li><li>LeVan, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+and+characterization+of+a+derivative+of+Neisseria+gonorrhoeae+strain+MS11+devoid+of+all+opa+genes.">Construction and characterization of a derivative of Neisseria gonorrhoeae strain MS11 devoid of all opa genes.</a> Journal of Bacteriology. 194 (23), 6468-6478 (2012).</li><li>Merritt, J. H., Kadouri, D. E., O'Toole, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growing+and+analyzing+static+biofilms.">Growing and analyzing static biofilms.</a> Curr Protoc Microbiol. , (2005).</li><li>Peeters, E., Nelis, H. J., Coenye, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+multiple+methods+for+quantification+of+microbial+biofilms+grown+in+microtiter+plates.">Comparison of multiple methods for quantification of microbial biofilms grown in microtiter plates.</a> Journal of Microbiological Methods. 72 (2), 157-165 (2008).</li><li>White, L. A., Kellogg, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neisseria+Gonorrhoeae+Identification+in+Direct+Smears+by+a+Fluorescent+Antibody-Counterstain+Method.">Neisseria Gonorrhoeae Identification in Direct Smears by a Fluorescent Antibody-Counterstain Method.</a> Journal of Applied Microbiology. 13, 171-174 (1965).</li><li>Swanson, J., Kraus, S. J., Gotschlich, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+gonococcus+infection.+I.+Pili+and+zones+of+adhesion:+their+relation+to+gonococcal+growth+patterns.">Studies on gonococcus infection. I. Pili and zones of adhesion: their relation to gonococcal growth patterns.</a> Journal of Experimental Medicine. 134 (4), 886-906 (1971).</li><li>Herten, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+in+Vitro+Quantification+of+S.+aureus+Biofilms+on+Vascular+Graft+Surfaces.">Rapid in Vitro Quantification of S. aureus Biofilms on Vascular Graft Surfaces.</a> Frontiers in Microbiology. 8, 2333 (2017).</li><li>Gracia, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+development+of+Staphylococcus+aureus+biofilms+using+slime-producing+variants+and+ATP-bioluminescence+for+automated+bacterial+quantification.">In vitro development of Staphylococcus aureus biofilms using slime-producing variants and ATP-bioluminescence for automated bacterial quantification.</a> Luminescence. 14 (1), 23-31 (1999).</li><li>Webb, J. 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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.

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			<td height="21" style="height:21px;width:323px;">100x Kellogg&#39;s supplement</td>
			<td style="width:229px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Agar</td>
			<td style="width:229px;">United States Biological</td>
			<td style="width:119px;">A0930</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">BacTiter Assay&#160;</td>
			<td style="width:229px;">Promega</td>
			<td style="width:119px;">G8232</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Ceftriaxone</td>
			<td style="width:229px;">TCI</td>
			<td style="width:119px;">C2226</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Difco GC medium base&#160;</td>
			<td style="width:229px;">BD</td>
			<td style="width:119px;">228950</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ferric nitrate, nonahydrate&#160;</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td style="width:119px;">254223-10G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glucose</td>
			<td style="width:229px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">BP350-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-glutamine Crystalline Powder</td>
			<td style="width:229px;">Fisher Scientific</td>
			<td style="width:119px;">BP379-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BacLight live/dead staining</td>
			<td style="width:229px;">Invitrogen</td>
			<td style="width:119px;">L7012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MS11 Neisseria gonorrhoeae strain</td>
			<td style="width:229px;"></td>
			<td style="width:119px;"></td>
			<td>kindly provided by Dr. Herman Schneider, Walter Reed Army Institute for Research</td>
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			<td height="26" style="height:26px;width:323px;">Potassium phosphate dibasic (K2HPO4)</td>
			<td style="width:229px;">Fisher Scientific</td>
			<td style="width:119px;">P290-500</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">Potassium phosphate monobasic (KH2PO4)</td>
			<td style="width:229px;">Fisher Scientific</td>
			<td style="width:119px;">BP329-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Proteose Peptone&#160;</td>
			<td style="width:229px;">BD Biosciences</td>
			<td align="right" style="width:119px;">211693</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Sodium chloride (NaCl)</td>
			<td style="width:229px;">Fisher Scientific</td>
			<td style="width:119px;">S671-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Soluble Starch</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td style="width:119px;">S9765</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thiamine pyrophosphate</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td style="width:119px;">C8754-5G</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Equipment</td>
			<td style="width:229px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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			<td height="21" style="height:21px;width:323px;">Petri Dishes</td>
			<td style="width:229px;">VWR</td>
			<td style="width:119px;">25384-302</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">8-well coverslip-bottom chamber&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:119px;">155411</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;">96-well tissue culture plates&#160;</td>
			<td>Corning, Falcon</td>
			<td style="width:119px;">3370</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:323px;">Biosafety Cabinet (NU-425-600 Class II, A2 Laminar Flow Biohazard Hood)</td>
			<td style="width:229px;">Nuaire</td>
			<td style="width:119px;">32776</td>
			<td></td>
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			<td height="26" style="height:26px;width:323px;">CO2 Incubator</td>
			<td style="width:229px;">Fisher Scientific</td>
			<td style="width:119px;">&#160;Model 3530</td>
			<td></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:323px;">Confocal microscope equipped with live imaging chamber</td>
			<td style="width:229px;">Leica</td>
			<td style="width:119px;">SP5X</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Corning&#160; 96 Well Black Polystyrene Microplate&#160;</td>
			<td style="width:229px;">Corning</td>
			<td style="width:119px;">3904</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Glomax Illuminator&#160;</td>
			<td style="width:229px;">Promega</td>
			<td style="width:119px;">E6521</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Pipette tips (0.1-10 &#181;L)</td>
			<td style="width:229px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">02-717-133</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pipette tips (1000 &#181;L)</td>
			<td>VWR</td>
			<td style="width:119px;">83007-382</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Pipette tips (200 &#181;L)</td>
			<td style="width:229px;">VWR</td>
			<td style="width:119px;">53509-007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spectrophotometer Ultrospec 2000 UV</td>
			<td style="width:229px;">Pharmacia Biotech</td>
			<td style="width:119px;">80-2106-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Sterile 15 ml conical tubes</td>
			<td style="width:229px;">VWR</td>
			<td style="width:119px;">21008-216</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Sterile Microcentrifuge Tubes (1.7 mL)</td>
			<td style="width:229px;">Sorenson BioScience</td>
			<td style="width:119px;">16070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile polyester-tipped applicators</td>
			<td>Fisher Scientific</td>
			<td>23-400-122</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:323px;">Sonicator</td>
			<td style="width:229px;">Kontes</td>
			<td style="width:119px;">Equivelent to 9110001</td>
			<td></td>
		</tr>
	</tbody>
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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...

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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 ...

This technique allows us to measure antibiotic susceptibility under more clinically relevant conditions. The technique also allows us to determine the true antibiotic concentration that can kill bacteria in aggregates. This technique can be applied to determining the concentration needed for eliminating GC aggregates.The method can also be applied to measuring the antimicrobial susceptibility of any bacteria that is able to form aggregates. Pay careful attention to the lysis steps to ensure the lysis is complete. Otherwise the ATP measuring assay will underestimate the number of viable bacteria.Visualization of this method will allow other researchers to follow and replicate this experiment with consistency. Begin by streaking Neisseria gonorrhoeae, or GC strains, on GCK agar supplemented with 1%Kellogg for a 16 to 18-hour incubation at 37 degrees Celsius and 5%carbon dioxide. The next morning, use a light microscope to carefully select pili-negative colonies without dark edges or pili-positive colonies with dark edges from each plate, and streak the picked colonies onto new GCK plates for a 16 to 18-hour culture at 37 degrees Celsius and 5%carbon dioxide.The next morning, use a sterile applicator to collect GC colonies from each plate and resuspend each swab in warm broth supplemented with 4.2%sodium bicarbonate and 1%Kellogg solution. Measure the optical density at 650 nanometers, or OD650, to determine the concentration of the suspended bacteria and adjust the GC concentration to about one times 10 to the eight colony forming units per milliliter. Next, add 99 microliters of the GC suspension into individual wells of a 96-well plate and allow the bacteria to aggregate at 37 degrees Celsius and 5%carbon dioxide for six hours.At the end of the incubation, add one microliter of serially diluted ceftriaxone to each well leaving some wells untreated to serve as controls and return the plate to the incubator for another 24 hours. The next day, sonicate the suspension in each well three times at 144 watts and 20 kilohertz for five seconds per sonication and add 100 microliters of commercially available ATP utilization glow reagent to each well. Carefully transfer 150 microliters of mixture from each well into individual wells of a new 96-well black microplate and measure the absorbance of each well at 560 nanometers on a plate reader.Then, calculate the survival rate by the ratio of the reading obtained after serial antibiotic treatment to the reading from the untreated wells. For visualization of the live/dead GC aggregates, use a sterile applicator to collect GC from the overnight cultured plates and resuspend the GC in prewarmed GCP medium supplemented with 1%Kellogg. Measure the OD650 by spectrophotometry and adjust the concentration to approximately one times 10 to the seven colony-forming units per milliliter.Next, add 198 microliters of GC suspension into eight-well coverslip-bottom chambers and incubate the chambers at 37 degrees Celsius and 5%carbon dioxide for six hours. At the end of the incubation, treat the bacteria with two microliters of ceftriaxone per aggregation condition and return the chambers to the incubator for the appropriate experimental incubation period. At the end of the incubation, add 0.6 microliters of live/dead staining solution into each well and return the chambers to the incubator for an additional 20 minutes.Then, use a confocal microscope to obtain z-series images. For measurement of the size of the GC aggregates, open the first image in ImageJ and select freehand lines to circle the area of each aggregation in the image. Click analyze and measure.The size of the aggregates will be indicated under the area column. For quantification of the fluorescence intensity ratio of the live to dead staining in each aggregate, click analyze and set measurements and check the integrated density in the popup window. Click OK and click image, color and channels tool.Select color and channel one as the fluorescence for dead bacteria staining. Select freehand lines to circle the area of each aggregation and click analyze and measure to obtain the fluorescence intensity ratio for each aggregate in the integrated density column. Select channel two for the fluorescence for the live bacterial staining and measure the fluorescence intensity in each aggregate as just demonstrated.Then divide the live fluorescence intensity data by the dead fluorescence intensity data to obtain the live to dead fluorescence intensity ratio. In this representative experiment, nonaggregated pili positive, aggregated pili positive or aggregated and disrupted pili positive bacteria were treated with serial dilutions of ceftriaxone before measurement of their ATP levels. Preaggregated GC demonstrated a significantly higher survival than non-aggregated or aggregation disrupted GC when treated with equal concentrations of ceftriaxone or higher.Pili-positive strains that form larger aggregates exhibited the highest ATP levels after ceftriaxone treatment compared to the mutant strains. Regardless of strain tested, live/dead staining before antibiotic treatment revealed dead bacteria largely located at the outer layers of the culture, while live GC were identified mainly within the core of ceftriaxone-treated aggregates. As expected, pili-positive bacteria formed the largest aggregates, while pili-negative cultures formed the smallest.Notably, pili-positive aggregates were still alive in their core layers after antibiotic treatment, whereas GC in the small, loose mutant aggregates were dead. Based on the size and survival of the GC, a correlation graph can be plotted to examine the relationship between the aggregation size and antibiotic survival of the bacteria. Sonication is important for making sure that ATP from each individual bacterium is released within the tightly bound aggregates.Otherwise, the measurements will not be consistent. It is important to plate the bacteria after they aggregate and are treated with antibiotics to measure both the viable and the replication-capable bacteria. The technique measures the bacteria aggregation influence on antibiotic susceptibility allowing researchers to develop better drugs for treating diseases.

Research

JoVE Journal

Immunology and Infection

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