Primary peritoneal macrophages were harvested from 8 to 12 week old C57/Bl6 mice (University of Louisville IACUC Protocols 16651 and 16723) injected with 3 mls of sterile Brewer's thioglycolate medium. Four days after injection, mice were humanely euthanized and peritoneal macrophages were recovered in 20 mls of Hank's Buffered Salt Solution (HBSS). Cells were isolated by centrifugation and resuspended in 10 mls of DMEM (DMEM, 100 mM glucose, sodium pyruvate; Hyclone) + 10% FBS (Biowest) as previously described (Ray and Dittel, 2010). Cells were quantified, transferred to microtiter plates or dishes, and allowed to adhere for 3 h. Non-adherent cells were then removed from adherent macrophages by washing with HBSS three times. RAW264.7 macrophages were originally obtained from ATCC and cultured in DMEM + 10% FBS. RAW264.7 cells were propagated for only up to 15 passages for these studies. Y. pestis strains used in these studies are listed in Table 1. Bacteria were propagated in Difco Brain Heart Infusion (BHI) (BD, Co.). We have previously shown that the bioluminescence generated by the Lux_PtolC bioreporter directly correlates with bacterial numbers and can be used to kinetically monitor Y. pestis intracellular survival in gentamicin protection assays (Sun et al., 2012; Connor et al., 2015).

5 × 10⁵ peritoneal macrophages in FluoroBrite DMEM (ThermoFisher), 4 mM glutamine + 10% FBS were added to a 35 mm glass bottom FluoroDish (World Precision Instruments). Macrophages were allowed to adhere to the dish for 3 h prior to infection. Y. pestis was grown overnight in BHI broth at 26°C and then diluted 1:25 in fresh BHI broth and grown for an additional 3 h until the culture reached an absorbance at 600 nm of ~1.0. The bacteria were diluted into FluoroBrite DMEM, 4 mM glutamine + 10% FBS and added to macrophages at a multiplicity of infection (MOI) of 3 bacteria per macrophage. To facilitate cell-bacteria interactions, FluoroDishes were centrifuged at 200 × g for 5 min and returned to the CO₂ incubator. Fifteen minutes later, the medium was removed, the cells were washed three times with sterile 1X PBS to remove extracellular bacteria, and fresh FluoroBrite DMEM, 4 mM glutamine + 10% FBS was added. Live imaging was performed using a Nikon A1 confocal microscope (Nikon Instruments, Inc.) with a live cell chamber equilibrated to 5% CO₂ and 37°C prior to imaging. Either a 488 nm or 561 nm laser with filter sets 525 ± 50 nm or 595 ± 50 nm were used to visualize EGFP or mCherry, respectively. Images were taken using a 40x Plan Fluro Oil objective every 20 min beginning 1 h post-infection and continuing for 24 h. The Nikon Perfect Focus System and field tiling were used to collect six fields (287 × 287 μM per field) at each time point. Bacterial numbers were estimated by calculating the area of the fluorescent signal for each field using FIJI (Schindelin et al., 2012).

1.5 × 10⁵ macrophages were aliquoted into the wells of a white 96 well microtiter plate (Greiner Bio One) and allowed to adhere for 3 or 15 h (peritoneal or RAW264.7 cells, respectively). Y. pestis was grown overnight in BHI broth at 26°C and then diluted 1:25 in fresh BHI broth and grown for an additional 3 h until the culture reached an absorbance at 600 nm of ~1.0. The bacteria were diluted into 37°C DMEM + 10% FBS and added to macrophages at a MOI of 10 bacteria per macrophage. To facilitate cell-bacteria interactions, microtiter plates were centrifuged at 200 × g for 5 min and returned to the CO₂ incubator for an additional 15 min, at which point gentamicin was added directly to the wells without washing to achieve the final desired concentrations. Concentrations of gentamicin used were not cytotoxic to macrophages. Bacterial numbers, as a function of bioluminescence produced by the Lux_PtolC bioreporter (Sun et al., 2012; Connor et al., 2015), were monitored kinetically to limit manipulation of the macrophages using a Synergy HT plate reader (0.5 s read, sensitivity of 135) (BioTek) or IVIS Spectrum camera system (5 sec with medium binning through a 500 nm emission filter) (Caliper).

Y. pestis was grown overnight in BHI broth at 26°C and then diluted 1:25 in fresh BHI broth and grown for an additional 3 h until the culture reached an absorbance at 600 nm of ~1.0. The bacteria were diluted into 37°C DMEM + 10% FBS and aliquoted at 1.5 × 10⁶ CFU per well in a 96 well plate. Gentamicin was diluted in 37°C DMEM + 10% FBS and added to the bacteria to achieve the final desired concentration (0–128 μg/ml). One h after treatment, bacterial numbers were determined by conventional enumeration using serial dilution and plating on BHI agar (Sun et al., 2012).

For short incubations with the antibiotic, bacterial numbers were directly enumerated by serial dilution to allow for separate calculations of extracellular and intracellular bacterial numbers after gentamicin treatment. Macrophages were infected with Y. pestis as described above and treated with gentamicin for 1 h. After treatment with gentamicin, the culture medium was collected into a separate tube, the cells were washed three times with sterile 1X PBS, which was also collected and combined with the collected medium, and the combined medium + 1x PBS washes was serial diluted and plated on BHI agar plates to enumerate the extracellular bacteria. For the intracellular bacteria, washed macrophages were lysed with 0.1% Triton X-100 and serial dilutions were plated on BHI agar plates for enumeration (Sun et al., 2012).

All studies were repeated three times to ensure reproducibility. When needed, mean values from individual treatment groups were compared to the 0 μg/ml gentamicin group using the ANOVA with the Dunnett's post-test or across all samples with the Tukey post-test. A p-value <0.5 was consider to be statistically significant.

To establish the fate of Y. pestis during infection of macrophages in the absence of gentamicin treatment, primary peritoneal macrophages were infected with a Y. pestis strain expressing mCherry fluorescent protein (YPA165). Twenty minutes post-infection, macrophages were washed to remove extracellular bacteria and imaged every 20 min by laser confocal microscopy for 24 h (Supplemental Movie 1). Changes in bacterial number, as a function of the total area of mCherry signal, were calculated at each time point (Figure 1). Over the first ~12 h, most bacteria appeared to be intracellular and we observed a steady increase in the area of mCherry over time, indicating intracellular survival and growth (Figure 1A). However, a small number of infected macrophages began to lyse ~12 h post-infection and release Y. pestis into the culture medium, though cell death was not synchronous and most infected cells were still intact through the first 18 h (Figure 1C). We continued to observe increases in the area of mCherry through 24 h of observation (Figure 1B), and while bacteria appeared to continue to grow intracellularly, extracellular bacteria released from dead macrophages were also replicating in the medium and represented a large portion of the mCherry signal at later time points (Figures 1B,C, (Supplemental Movie 1). By 24 h post-infection, almost all of the infected macrophages had lysed and the majority of the mCherry signal was from extracellular bacteria (Figure 1C, (Supplemental Movie 1).

To determine if the Y. pestis Ysc type 3 secretion system (T3SS) influenced the intracellular fate of the bacterium, macrophages were infected with a Y. pestis strain lacking the pCD1 virulence plasmid encoding the Ysc T3SS and expressing EGFP (YPA081) and imaged by laser confocal microscopy as described above (Supplemental Movie 2). As observed for the T3SS positive strain, total area of EGFP increased over the entire course of the experiment (Figures 1A,B). However, unlike the Y. pestis pCD1⁽⁺⁾ strain, we did not observe macrophage lysis, bacterial release, or extracellular proliferation over the 24 h time frame of the experiment, indicating that the increased area of EGFP was a function of only intracellular growth. Together these data: (1) confirm data from previous in vitro assays that Y. pestis survives intracellularly in primary macrophages (Straley and Harmon, 1984a,b); (2) confirm that Y. pestis lacking the T3SS can survive in macrophages (Straley and Harmon, 1984a,b; Pujol and Bliska, 2003); (3) demonstrate that macrophages infected with Y. pestis with a functional T3SS lyse more rapidly than macrophages infected with Y. pestis lacking the T3SS; (4) macrophage cell death releases intracellular bacteria into the environment; and (5) bacteria released from macrophages are viable and can replicate in the cell culture medium. While intracellular vs. extracellular bacteria can be differentiated during live imaging, in vitro assays that do not use microscopy are not able to differentiate between the two, thus requiring the use of an antibiotic protection assay. However, as a wide variety of concentrations of gentamicin have been reported in the Yersinia literature (Pujol and Bliska, 2003; Benedek et al., 2004; Leigh et al., 2005; Ponnusamy et al., 2011; Sha et al., 2013; Spinner et al., 2014; Tiner et al., 2015; van Lier et al., 2015), we next wanted to determine if extended incubation with gentamicin could influence intracellular proliferation of Y. pestis.

The simplest antibiotic protection assay would be to add gentamicin to the infected cells and maintain that concentration in the medium throughout the entire course of the study. However, extended incubations have been shown to influence intracellular proliferation of other bacteria (Utili et al., 1991; Drevets et al., 1994; Menashe et al., 2008). To determine if extended incubations with gentamicin impacted survival of intracellular Y. pestis, peritoneal macrophages were infected with a Y. pestis strain containing the Lux_PtolC bioreporter (YPA050), which we have previously shown can be used to accurately monitor intracellular Y. pestis as a function of bioluminescence (Sun et al., 2012; Connor et al., 2015). Twenty minutes post-infection, serial dilutions of gentamicin were added to each well to achieve final concentrations of 128 to 1 μg/ml and beginning 2 h after infection bacterial numbers, as a function of bioluminescence, were determined every 3 h for 20 h using a plate reader. We observed that at the lowest concentrations tested, 1 and 2 μg/ml, bacterial bioluminescence increased over time, although at 2 μg/ml, bioluminescence was approximately 2–3-fold lower than at 1 μg/ml (Figures 2A,B). At concentrations greater than 2 μg/ml, bioluminescence no longer increased, indicating that bacteria were no longer able to proliferate. At 4 μg/ml, we observed an initial decrease in bioluminescence between 2 and 5 h, at which time bioluminescence remained constant over the remainder of the assay (Figure 2C). Gentamicin concentrations greater than 4 μg/ml resulted in a steady decrease in bacterial bioluminescence over the entire course of the assay (Figures 2D–H). Compared to our live imaging experiments without gentamicin, these data demonstrate that extended incubation with even low concentrations of gentamicin can have a dramatic influence on Y. pestis intracellular survival, and can lead to intracellular killing of the bacterium.

As pinocytosis is suggested to be the main mechanism for gentamicin uptake by macrophages, sensitivity of intracellular bacteria to gentamicin could vary depending on cell type used. RAW264.7 cells are an immortal cell line originally derived from mouse macrophages and are commonly used as a macrophage model (Raschke et al., 1978). To determine if the sensitivity of intracellular Y. pestis to gentamicin could vary depending on cell type, RAW264.7 cells were infected with Y. pestis YPA050. Twenty minutes later, serial dilutions of gentamicin were added to each well to achieve final concentrations of 128 to 1 μg/ml and cells were incubated for 20 h. Bacterial numbers were monitored as a function of bioluminescence, normalized to T = 2 h, and compared to similarly treated primary peritoneal macrophages (Figure 3). Similar to peritoneal cells, bacterial bioluminescence increased in RAW264.7 macrophages when treated with 1 μg/ml, indicating intracellular proliferation. However, unlike bacteria in peritoneal macrophages, Y. pestis bioluminescence continued to increase in RAW264.7 cells until gentamicin concentrations reached 16 μg/ml. After 16 μg/ml, bioluminescence began to decrease overtime, indicating bacterial killing, and approached levels similar to that seen for peritoneal macrophages at 128 μg/ml. These data highlight that gentamicin inhibition of intracellular Y. pestis can vary between cell types.

Since extended incubations with gentamicin dramatically inhibited the proliferation of intracellular Y. pestis, we next sought to identify the minimal concentration of gentamicin needed to inhibit Y. pestis growth in tissue culture medium in a 1 h period. Toward this end, Y. pestis YPA050 was diluted in DMEM + 10% FBS with increasing concentrations of gentamicin. One h after addition of gentamicin, bacteria were serially diluted and plated on BHI agar to determine remaining viable bacteria (Figure 4A). All doses of gentamicin tested resulted in a significant decrease in the number of recovered bacteria compared to untreated (p ≤ 0.0001) and the degree of inhibition was dependent on the concentration of the antibiotic. While bacteria were reduced ~1,000-fold within 1 h with 4 μg/ml gentamicin, no detectable viable bacteria were recovered after incubation with ≥8 μg/ml. Similar inhibition was observed for the Medivalis biovar KIM D19 (Supplemental Figure 1).

Next we sought to determine whether the presence of macrophages impacted the minimal concentration of gentamicin required to inhibit Y. pestis growth in cell culture medium. Peritoneal macrophages were infected with Y. pestis YPA050 for 20 min and serial dilutions of gentamicin were added to each well to achieve final concentrations of 128 to 1 μg/ml. One h after addition, the gentamicin containing medium was removed and combined with washes and viable extracellular bacteria were determined by serial dilution and conventional enumeration (Figure 4B). Extracellular bacterial numbers were significantly lower in all the samples that received greater than 1 μg/ml of gentamicin as compared to absence of gentamicin (p ≤ 0.0001). However, no significant differences were observed in bacterial numbers between concentrations of 4–32 μg/ml. Furthermore, while we observed no significant difference in bacterial numbers between the 64 and 128 μg/ml concentrations, we observed ~2.5-fold lower numbers of extracellular bacteria at 64 and 128 μg/ml samples (average 180 CFU) compared to the 4–32 μg/ml samples (average 458 CFU) that was statistically significant (p ≤ 0.05).

Finally, we enumerated the number of intracellular bacteria in macrophages after 1 h treatment with gentamicin at both 1 and 24 h post-treatment to determine if gentamicin concentration during short incubations could impact intracellular survival of Y. pestis (Figure 5). At 1 h post-treatment, statistically significant differences in the number of intracellular bacteria recovered were not observed until gentamicin concentrations were greater than 8 μg/ml (Figure 5A). In concentrations >8 μg/ml, a dose dependent decrease in viable intracellular bacteria was observed. However, even at the highest doses, intracellular numbers only varied by ~1.3-fold as compared to macrophages receiving lower doses of antibiotic. To determine if long term intracellular survival of Y. pestis was affected by short exposures to gentamicin, a separate group of infected cells were washed 1 h after gentamicin treatment to remove residual gentamicin and fresh medium without gentamicin was added. Twenty three hour later, the medium was removed, the cells were washed, lysed, and intracellular bacterial numbers were enumerated (Figure 5B). We observed bacterial proliferation in all samples containing <64 μg/ml gentamicin, and there were no significant differences in intracellular numbers in samples containing 0–32 μg/ml gentamicin. However, bacteria did not proliferate in macrophages treated with 64 μg/ml of the antibiotic, and at the highest dose of 128 μg/ml, intracellular bacterial numbers decreased over time. Together these data indicate that at least 4 μg/ml of gentamicin is required to achieve optimal killing of extracellular Y. pestis in 1 h during macrophage infection assays. However, as gentamicin concentrations increase, even short incubations with the antibiotic can impact proliferation of intracellular Y. pestis, especially at higher doses of the antibiotic.

Together these data suggest that an optimized gentamicin assay for Y. pestis should include two doses of gentamicin. The first should be an initial dose to efficiently kill the majority of the extracellular bacteria in a short incubation period (e.g., 1 h) without inhibiting intracellular survival. The data from short term incubations with gentamicin (Figure 5) suggest that this concentration should not exceed 8 μg/ml. After the 1 h incubation, the medium should be removed as continued incubation in 8 μg/ml results in inhibition of intracellular bacteria (Figure 2). The medium should then be replaced with a lower dose that inhibits extracellular bacterial growth but not intracellular survival of the bacteria during extended incubations. The data from extended incubations with gentamicin (Figure 2) suggest that this maintenance dose should not exceed 2 μg/ml. To test these empirically derived gentamicin concentrations, peritoneal or RAW264.7 macrophages were infected with YP043, a fully virulent strain of Y. pestis CO-92 expressing the Lux_PtolC bioluminescent bioreporter (Sun et al., 2012). Twenty minutes later, gentamicin was added at a final concentration of 8 μg/ml. Cells were incubated for 1 h, washed with 1X PBS, and the medium was replaced with medium containing 2 μg/ml gentamicin. Intracellular bacterial survival was monitored by bacterial bioluminescence every 2 h for 20 h using an IVIS optical imager (Figure 6). In peritoneal macrophages, bioluminescence slightly decreased over the first ~8 h of infection, indicating an initial inhibition of bacterial survival by the macrophages, but then at ~12 h, bacterial bioluminescence began to increase, reaching a steady state level at 16 h that was maintained for the remainder of the infection (Figure 6A). Based on our live imaging experiments, this steady state level at later time points likely represents a balance between intracellular replication and gentamicin killing of extracellular bacteria from lysed macrophages (Figure 1). In RAW264.7 macrophages we did not observe an initial decrease in bacterial bioluminescence, supporting that this cell type is more permissive to intracellular bacterial infection (Figure 6B). Instead, we observed a ~2-fold increase in bioluminescence over the first 4 h, which plateaued at this level for the first 10 h of the infection. Then, similar to the peritoneal macrophages, at ~12 h post-infection, bacterial bioluminescence began to increase, indicating further intracellular bacterial proliferation.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.