Gentamicin Protection Assay

Introduction

The gentamycin protection assay is used to monitor the killing or growth of pathogenic bacteria by phagocytes such as macrophages and neutrophils. This assay cleanly identifies only bacteria which have successfully invaded (or been phagocytosed) by the immune cells. The assay works by "feeding" the immune cells a known number of bacteria (often opsonized) for a set period of time, after which any remaining extracellular bacteria are killed by the addition of the non-membrane permeable antibiotic gentamycin, under conditions which minimize macropinocytosis (and thus, minimizing delivery of gentamycin to phagosomes/bacterial vacuoles formed prior to the addition of the gentamycin). Once killing is complete the gentamycin is removed, and at set time points the phagocytes lysed and plated on an appropriate bacterial medium. The number of resulting colonies can then be used to enumerate the growth or killing of the pathogenic bacteria by the immune cells.

Critical Note

It is absolutely critical that the gentamycin (or other antibiotic used) be removed after extracellular bacteria are killed, as macropinocytosis by the immune cells will load phagosomes/lysosomes/bacterial vacuoles in the macrophages with solutes from the extracellular milieu. Thus, extended periods of antibiotic exposure can intoxicate bacteria within the immune cell, leading to a mis-characterization of the efficacy of bacterial killing &/or an underestimation of the measurement of bacterial growth.

Step 1 - Determining Gentamycin Dose & Killing Time

Prior to performing the experiment you must first identify a dose of gentamycin (or the antibiotic you are using) that will kill 100% of extracellular bacteria in a reasonable amount of time, under the experimental conditions (e.g. time and tissue culture media) you will be using. This process must be repeated if you change the strain or species of bacteria being used, the culture media the phagocytes will be grown in, or if you are significantly increasing the number of bacteria used in an experiment.

1. Determine the highest density of bacteria (in CFU/ml) that you expect to use. Most gentamycin protection assays use MOIs of 0.5:1 (bacteria:phagocytes) to ~100:1.
   • For example, in a standard 12-well plate sized assay, the plate will have ~250,000 phagocytes/well, in a 1 ml volume of media. Thus, if using a maximum of a 50:1 MOI, you would be adding a maximum of 12.5 million bacteria per well, in 1 ml = 12.5 x 10⁶ CFU/ml.
2. Suspend this amount of bacteria in several aliquots of 1 ml of the same serum-free medium you plan on maintaining your phagocytes in (generally DMEM or RPMI), with each tube containing an increasing concentration of gentamycin. Typical gentamycin concentrations range from 10 ug/ml to 500 ug/ml.
3. Incubate at 37C for the desired killing time (typically 60 min), wash the bacteria once in an equal volume of PBS to remove residual Gentamycin, then plate 100ul of undiluted bacteria onto an appropriate bacterial medium.
4. The lowest dose that provides 100% killing (at the shortest killing time, if possible) should be used for future experiments.

Established Antibiotic Doses

Species/strain	Dose/Antibiotic	Medium
S. aureus, USA300	Gentamycin, 100 ug/ml, 60 min	DMEM
S. pneumoniae, lab isolate	Gentamycin, 200 ug/ml, 60 min	RPMI

Step 2 - Gentamycin Protection Assay

1. Split immune cells to the desired density, typically 60% confluency, in a 12 or 6 well tissue culture plate.
2. Prepare a bacterial culture at the desired cell concentration and growth stage (lag phase versus stationary phase).
3. Wash bacterial culture and suspend in the same media as the phagocytes are cultured in, at the desired bacterial density(s).
   • If required, bacteria can be opsonized at this stage by addition of 20% pooled human serum, antibodies, etc.
4. Add the desired number of bacteria to each phagocyte-containing well in the experimental plate. Centrifuge for 1 min at ~200 g (1,000 RPM on a standard benchtop centrifuge) to force contact between the bacteria and the phagocytes.
5. Incubate for the desired period of time to allow for phagocytosis/invasion (typically 30-60 min).
6. Remove the tissue culture media, and replace with serum-free medium containing the appropriate dose of gentamycin, as determined previously.
   • Serum-free media is critical to limit macropinocytosis and thus killing/stressing of intracellular cells by the anitobiotic.
7. After completion of the killing time, replace the medium with antibiotic-free serum-containing culture medium, washing the phagocytes once with warmed serum-containing medium first
8. At the desired time points, remove the culture medium from the phagocytes and lyse the cells with 1 ml of PBS + 0.1% Triton X-100. Use a cell scraper to ensure all macrophages are recovered.
9. Vortex the phagocyte suspension briefly to complete disruption of the phagocytes and to ensure an even distribution of the bacteria.
10. Prepare dilutions of this suspension, and plate on an appropriate bacterial medium. Suspension should be diluted so that a robust (but countable) number of colonies are formed (100-300 colonies) once the sample is plated. Multiple dilutions may be required at each time point to ensure accurate enumeration and to account for variability between experiments.

Step 3 - Quantification

1. Count the number of colonies on each countable plate, and use this value to determine the CFU/ml of the phagocyte lysate.
2. Data can be expressed in several ways:
   • CFU/ml in the phagocyte culture.
   • Percent killing or percent growth.
   • Fraction killing or log-growth.