Bacterial pneumonia is a common infection of the lower respiratory tract that can afflict patients of all ages. Multidrug-resistant strains of Acinetobacter baumannii are increasingly responsible for causing nosocomial pneumonias, thus posing an urgent threat. Alveolar macrophages play a critical role in overcoming respiratory infections caused by this pathogen. Recently, we and others have shown that new clinical isolates of A. baumannii, but not the common lab strain ATCC 19606 (19606), can persist and replicate in macrophages within spacious vacuoles that we called Acinetobacter Containing Vacuoles (ACV). In this work, we demonstrate that the modern A. baumannii clinical isolate 398, but not the lab strain 19606, can infect alveolar macrophages and produce ACVs in vivo in a murine pneumonia model. Both strains initially interact with the alveolar macrophage endocytic pathway, as indicated by EEA1 and LAMP1 markers; however, the fate of these strains diverges at a later stage. While 19606 is eliminated in an autophagy pathway, 398 replicates in ACVs and are not degraded. We show that 398 reverts the natural acidification of the phagosome by secreting large amounts of ammonia, a by-product of amino acid catabolism. We propose that this ability to survive within macrophages may be critical for the persistence of clinical A. baumannii isolates in the lung during a respiratory infection.

Bacterial strains and culture conditions

The bacterial strains employed in this work are described in Table S1. Bacteria were seeded on lysogeny broth (LB) agar plates and incubated for 16 h at 37 °C. Thereafter, colonies were grown in LB broth under shaking conditions (200 rpm) for 16 h at 37°C. When appropriate, antibiotics were added to the cultures at the following concentrations: 50 μg/ml zeocin, 30 μg/ml kanamycin, and 15 μg/ml chloramphenicol.

Growth assays

A. baumannii strains 19606 and 398 were cultured overnight in LB broth at 37 °C under shaking conditions. Bacterial cultures were centrifugated at 6500 rpm for 5 min and the pellets were washed with Phosphate-buffered saline (PBS, Sigma, D8662). A. baumannii suspensions were diluted to OD₆₀₀ = 0.01 in 150 μl of LB buffered at pH 5 (MES 100 mM), 6 (MES 100 mM), or 7 (HEPES 100 mM) in 96-well plates and incubated 16 h at 37°C under shaking conditions. OD₆₀₀ values were measured at 30 min intervals using a BioTek microplate spectrophotometer. The experiment was performed in biological triplicate with three technical replicates per experiment for each strain and condition.

pH measurements in A. baumannii cultures

The absorbance of phenol red solutions at 560 nm is an indicator of pH, as previously described. Phenol red is a pH indicator changing color from yellow below pH 6.8 to bright pink above pH 8.2. To analyze the pH changes in A. baumannii cultures during bacterial growth, indicated strains were maintained overnight in LB broth at 37°C under shaking conditions. Bacterial cultures were washed and diluted as described for growth assays, with the following modification: non-buffered LB pH 5 with phenol red (15 µg/ml) was used. OD₆₀₀ and OD₅₆₀ values were measured every 30 min for 16 h using a BioTek microplate spectrophotometer. The OD₆₀₀ absorbance values were subtracted from the absorbance at 560 nm data to exclude the effect of bacterial density. Finally, the values obtained were used to estimate culture pH by employing a standard curve of OD₅₆₀ vs pH. The experiment was performed in triplicate with three technical replicates per each strain.

Cell culture conditions

The J774A.1 mouse macrophage cell line (ATCC TIB-67) was cultured in Dulbecco’s Modified Eagle Medium (DMEM) High Glucose (Hyclone, SH30022.01) supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS, Corning) at 37°C and 5% CO₂.

Murine Model of A. baumannii pneumonia

Colonies from A. baumannii strains 398, GFP-398 and GFP-19606 were subcultured from LB agar plates and grown in LB broth 16 h at 37°C under shaking conditions. Overnight cultures obtained were diluted in sterile LB broth (1:100) and incubated for 3 h at 37 °C under shaking conditions. The bacterial inocula were prepared by centrifugation at 6500 rpm for 5 min, washed twice in PBS, and the pellet resuspended in PBS. Six- to eight-week-old female C57BL/6 mice obtained from Charles River Laboratories were intranasally inoculated with A. baumannii strains 398, GFP-398, or GFP-19606. Briefly, mice were anesthetized by inhalation of 4% isoflurane and were infected immediately with 50 μL of bacterial suspension (∼1 × 10⁸ Colony Forming Units [CFU]) that were gradually released with a micropipette into the nares. At 3 and 24 hours post-infection (hpi), bronchoalveolar lavage fluid (BALF) was collected. For BALF collection, mice were euthanized by CO₂ asphyxiation and then, the lungs and trachea were exposed by surgical dissection and polyethylene tubing (0.86x1.27mm) was inserted into the trachea. Cold PBS containing 1mM EDTA was flushed through the lungs in 1ml increments to collect a total volume of 10 ml. All pneumonia studies were performed in accordance with the guidelines of the Committee for Animal Studies at Washington University School of Medicine, and we have complied with all relevant ethical regulations.

Antibiotic protection assay

To analyze the intracellular replication of the different strains, J774A.1 cells were seeded 16 h before the experiment in 48-well plates (3x10⁵ cells/well). A. baumannii colonies from LB agar plates were inoculated in LB broth and grown overnight at 37°C under shaking conditions. Bacterial cultures were centrifugated 5 min at 6500 rpm and washed twice with PBS. Then, bacterial cultures were normalized to OD600 ≈ 1 and appropriate volumes was added to 500 µl of DMEM per well to achieve an MOI≈10. Where indicated, bafilomycin A1 (15 nM; Sigma, B1793) was added to the growth media and maintained throughout the infection. Infected cells were centrifugated 10 min at 200 x g and incubated at 37°C and 5% CO₂. After 1 h, macrophages were washed three times with PBS and treated with DMEM supplemented with 10% FBS and colistin (50 µg/mL) to eliminate the extracellular bacteria. At the time points indicated, the cells were washed three times with PBS and lysed with 500 µl of Triton X-100 (0.05%) per well. Serial dilutions of bacterial suspensions obtained were plated on LB agar and incubated overnight at 37°C, to determine number of colony forming units (CFUs).

To determine the number of total and intracellular bacteria present in BALF from A. baumannii-infected mice, two 500 µl aliquots of lavage fluid were centrifuged at 6500 rpm for 10 min. The pellets were incubated for 1 h at 37°C in PBS (total bacteria) or PBS with colistin (50 mg/mL) (intracellular bacteria). Then the cells were washed three times with PBS and lysed with 500 µl of Triton X-100 (0.05%) per aliquot. CFUs were determined by serial dilutions of the bacterial suspensions.

Ammonia production

Ammonia production by A. baumannii strains 19606 and 398 was measured using the Ammonia Assay Kit (AA0100, Sigma) according to the manufacturer’s instructions. Briefly, the strains were cultured overnight in LB broth at 37 °C under shaking conditions. Then, bacterial cultures were centrifugated and washed with PBS. A. baumannii suspensions were diluted to OD₆₀₀ = 0.01 in 150 μl of buffered LB (MES 100 mM, pH 5) in 96 well plates and incubated for 6 h at 37°C under shaking conditions. Ammonia levels were measured, at the indicated times. The experiment was performed in biological triplicate.

Intracellular ammonia production was measured using the same kit. Briefly, macrophages were infected with A. baumannii strains 19606 and 398 in duplicates. 24 hpi a set of wells was scraped in PBS to determine the number of macrophages. The remaining wells were treated with 200 µl of Triton X-100 (0.05%), centrifuged at 6500 rpm for 10 min and ammonia levels were measured in the supernatant.

Flow Cytometry

BALF samples were centrifugated at 300 x g for 5 min and incubated in 500 µl Pharm Lyse Buffer (BD Biosciences 555899) for 3 min at room temperature to lyse red blood cells. The cells were then washed with flow cytometry buffer [PBS + 0.5% Bovine Serum Albumin (BSA) (Fisher BioReagents, BP9706100) + 2 mM EDTA] and incubated at 4°C with Fc Block (BD Biosciences, 553142) for 10 min. Samples were then stained for 30 min with CD45-BV510 (Biolegend, 103138) and Ly6G-BV421 (Biolegend,127628), CD11b-Alexa700 (BD Biosciences, 557960), CD11c-APC (BD Biosciences, 550261), and Siglec-F-BV786 (BD Biosciences, 740956). Finally, cells were washed and fixed in 2% paraformaldehyde until acquisition on a Benton Dickinson (BD) LSR II Fortessa cytometer. GFP signal was acquired in the FITC channel. Total cell counts per mouse were calculated using Precision Count Beads (Biolegend, 424902) according to the manufacturer’s instructions.

Cytospin of BALF cells

BALF samples were centrifugated at 300 x g for 5 min and the pellets were resuspended in 500 µl Pharm Lyse Buffer (BD Biosciences, 555899) for 3 min at room temperature to lyse red blood cells. The cells were resuspended in PBS and total cells and viability was determined using Trypan Blue solution (Sigma, T8154) and counted using the TC20 automated cell counter (BioRad). Samples were centrifugated at 300 x g for 5 min onto CytoPro Poly-L-Lysine Coated Microscope Slides (ELITechGroup, SS-118). The slides were air-dried overnight at 4 °C and fixed in 4% paraformaldehyde. Samples were incubated with permeabilizing and blocking solution [PBS + 0.1% saponin + 0.5% BSA (Fisher BioReagents, BP9706100) + 10% FBS (Corning)]. Cells were stained with Alexa Fluor 647 Rat Anti-Mouse Siglec-F antibody (BD, 562680) and nuclei with 4',6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI) solution (Invitrogen, D1306) 1 h at 37°C. After staining, the samples were rinsed with washing solution [PBS + 0.1% saponin + 0.5% BSA (Fisher BioReagents, BP9706100)], then rinsed with water, and mounted with a coverslip in Invitrogen ProLong Gold Antifade Mountant (Invitrogen, P36930). Finally, the cells were analyzed by confocal microscopy.

Immunofluorescence staining

1.3x10⁵ J774A.1 cells were plated onto glass coverslips in 24 well plates and incubated for 16 to 18 h at 37 °C and 5% CO₂. Inocula of the indicated A. baumannii strains were prepared by centrifugation of overnight cultures at 6500 rpm for 5 min, washing twice in PBS, and resuspension of the pellet in PBS. Bacterial suspensions were normalized to OD≈1 and an appropriate volume was used to infect the cells (MOI≈10). Afterward, the plates were centrifugated for 10 min at 200 x g to enhance bacterial contact with the host cells and incubated for 1 h at 37 °C and 5% CO₂. Cells were washed three times with PBS, and extracellular bacteria were killed by treatment of the cells with colistin (50 µg/mL) for 1 h. When necessary, cells were incubated with DQ Green BSA (10 µg/ml; Invitrogen, D12050) 1 h prior to the end of the infection. At the indicated time points, samples were fixed with 4% paraformaldehyde for 15 min at 37°C and then stored in permeabilizing and blocking solution [PBS + 0.1% saponin + 0.5% BSA (Fisher BioReagents, BP9706100) + 10% FBS (Corning)]. The glass coverslips were incubated with the indicated primary antibodies produced in rabbit: anti-LC3 (Sigma, L7543), anti-EEA1 (Invitrogen, PA1-063A), anti-LAMP1 (Abcam, ab24170) or anti-A. baumannii at a 1:100 dilution for 1 h at 37 °C. The cells were then washed 3 times with washing solution and incubated with the indicated secondary antibody goat anti-rabbit: Alexa Fluor 647 (Invitrogen, A-21244) at a 1:250 dilution, Alexa Fluor 555 phalloidin (0.33 µM; CST, #8953) and DAPI for 1 h at 37°C. Afterwards, samples were washed with PBS, rinsed with water, and mounted with a coverslip in Invitrogen ProLong Gold Antifade Mountant (Invitrogen, P36930). Stained samples were analyzed by confocal microscopy. 

Confocal microscopy

Infected cells were analyzed with a Zeiss LSM880 laser scanning confocal microscope (Carl Zeiss Inc.) equipped with 405nm diode, 488nm Argon, 543nm HeNe, and 633nm HeNe lasers. A Plan-Apochromat 63X (NA 1.4) DIC objective and ZEN black 2.1 SP3 software were used for image acquisition. Live images were acquired with a Zeiss spinning disk confocal microscope (Carl Zeiss Inc.) equipped with 488nm and 560nm lasers. A Plan-Apochromat 63X/1.3 Oil Ph 3 (UV) VIS-IR M27 objective and ZEN black 2.1 SP3 software were employed for image acquisition. Images were analyzed using ImageJ software (NIH, USA).

Live imaging

5x10⁵ J774A.1 macrophages were plated in a 10 mm Glass Bottom Culture 35 mm petri dish (MATEK corporation, P35G-0-14-C) and incubated 12-16 h at 37°C and 5% CO₂. The next day, cells were infected with mCherry-398 at an MOI≈10 as described above. 15 minutes prior to the indicated infection time points, cells were washed twice with PBS and incubated in DMEM with LysoSensor Green DND-189 (1µM; Molecular Probes, Invitrogen, L7535). The cells were then rinsed twice in PBS and were immediately analyzed by confocal microscopy. During image acquisition, the cells were maintained at 37°C and 5% CO₂ in a temperature-controlled CO₂ chamber on the microscope.

Transmission electron microscopy

Cells from BALF were centrifuged at 300 x g for 5 minutes and incubated in 500µl Pharm Lyse Buffer (BD Biosciences 555899) for 3 minutes at room temperature to lyse red blood cells. The cells were centrifugated and resuspended in PBS. Total cell number and viability were measured using Trypan Blue solution (Sigma T8154) and counted with TC20 automated cell counter (BioRad). At least 1x106 cells were fixed in 2% paraformaldehyde/2.5% glutaraldehyde (Polysciences Inc., Warrington, PA) in 100 mM sodium cacodylate buffer pH 7.2 for 1 h at room temperature and subsequently incubated to 4˚C overnight. Samples were washed in sodium cacodylate buffer at room temperature and postfixed in 1% osmium tetroxide (Polysciences Inc.) for 1 h. The cells were then rinsed in distilled water, and bloc-stained for 1 h with 1% aqueous uranyl acetate (Ted Pella Inc., Redding, CA). Subsequently, the samples were rinsed in distilled water several times, dehydrated in a graded series of ethanol, and finally embedded in Eponate 12 resin (Ted Pella Inc.). Sections of 95 nm were cut with a Leica Ultracut UCT ultramicrotome (Leica Microsystems Inc., Bannockburn, IL), stained with uranyl acetate and lead citrate, and viewed on a JEOL 1200 EX transmission electron microscope (JEOL USA Inc., Peabody, MA) equipped with an AMT 8-megapixel digital camera and AMT Image Capture Engine V602 software (Advanced Microscopy Techniques, Woburn, MA). Images were processed using ImageJ software.

Statistical analysis

Statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software Inc., La Jolla, CA). Datasets were analyzed by a Mann–Whitney test, Welch's t-test, one-way ANOVA-test or two-way ANOVA-test, as indicated.