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

Summary

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.

Abstract

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.

Introduction

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 asymptomatic^2,3,4,5, 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 azithromycin^9,10, 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 aggregates^13,14,15. 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.

Protocol

1. General maintenance of GC strains

1. Streak N. gonorrhoeae strains on GCK agar with 1% Kellogg supplements¹⁸ (Table 1, Table 2) from freezer stocks and incubate 37 °C with 5% CO₂ for 16-18 h. Use MS11 expressing phase-variable Opa (MS11Opa+), no Opa (MS11ΔOpa), or a truncated LOS (MS11ΔLgtE).
2. 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.
3. Incubate at 37 °C with 5% CO₂ for 16-18 h before use.

2. Viability quantification of GC aggregations

1. 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.
2. Adjust the concentration of GC to ~1 x 10⁸ CFU/mL.
3. Add 99 µL of adjusted GC suspension into wells of a 96-well plate.
4. Incubate the plate for 6 h at 37 °C with 5% CO₂ to allow the bacteria to aggregate.
5. Add 1 µL of serial diluted ceftriaxone (1000, 100, 50, 25, 12.5, 6.2, 3.1, 1.5, 0.8, 0.4, 0.2 µg/mL) into each well. Leave some wells untreated to serve as controls.
6. Incubate the plate for 24 h at 37 °C with 5% CO₂.
7. Sonicate the suspension 3 times in each well for 5 s at 144 W and 20 kHz.
8. Add 100 µL of commercially available ATP utilization glow reagent into each well, pipette up-and down for 3 times, and incubate for 15 min at 37 °C with 5% CO₂.
9. Carefully transfer 150 µL of mixture from each well into a new well in a 96-well black microplate and avoid introducing bubbles.
10. Measure the absorbance of each well at 560 nm using the plate reader.
11. Calculate the survival rate by the ratio of the reading obtained after serial ceftriaxone treatment to the reading from untreated wells.

3. Fluorescence microscopic analysis of Live/Dead of GC aggregates

1. Collect GC using a sterile applicator. Swab GC from the plate and re-suspend GC in pre-warmed GCP media plus 1% Kellogg supplements.
2. 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.
3. Add 198 µL of GC suspension into in 8-well coverslip-bottom chambers.
4. Incubate the chamber for 6 h at 37 °C with 5% CO₂ to allow aggregation formation.
5. Add 2 µL of ceftriaxone (100 µg/mL or various dilutions) into each well within each aggregation condition. Incubate for the desired time at 37 °C with 5% CO₂.
6. Add 0.6 µL of live/dead staining solution mixture into each well and incubate for 20 min at 37 °C with 5% CO₂.
7. Acquire Z-series images using a confocal microscope (an equivalent microscope can be used).
8. 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.

4. Image analysis

1. Estimation of the size of bacterial aggregates.
   1. Open ImageJ and open an image by dragging a raw image file to the ImageJ menu bar.
   2. Click Freehand Lines in the ImageJ menu bar and circle the area of each aggregation in the image.
   3. Click Analyze | Measure in the ImageJ menu bar.
   4. Obtain the number in a new window under the Area column.
2. Quantification of live-to-dead ratio of aggregations
   1. Open ImageJ and open an image by dragging a raw image file to the ImageJ menu bar.
   2. Click Analyze | Set Measurements in the ImageJ menu bar.
   3. Check the integrated density in the pop-up window and click OK.
   4. Click Image | Color | Channels Tool in the menu bar and select Color.
   5. Check Channel 1 as the fluorescence for live bacteria staining.
   6. Click Freehand Lines in the ImageJ menu bar and circle the area of each aggregation.
   7. Click Analyze | Measure in the ImageJ menu bar.
   8. Obtain the number under IntDen column.
   9. Check Channel 2 as the fluorescence for dead bacterial staining. Repeat steps 4.2.6-4.2.8.
   10. Obtain the live-to-dead ratio by dividing number from step 4.2.8 by number from step 4.2.9.
3. Statistical analysis
   1. Open GraphPad Prism and enter the numbers obtained from ImageJ to the desired column.
   2. Click Analyze and select t tests under Column analyses.
   3. Check the desired column for comparison and click OK.
   4. Obtain the P-Value under Analysis window.
   5. Select the Linear Regression under XY analyses from step 4.3.3
   6. Obtain the R-square and the P-Value under the Analysis window.

Results

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 biofilms^20,21. Here, MS11Opa+Pil+ strain was used to examine the role of GC aggregation in antibiotic susceptibili...

Discussion

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...

Materials

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