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

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.