Survival in Macrophages . ... Table 1. Bacterial strains. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....S. aureus strains Relevant genotype/markers Relevant properties Source/reference . ... ATCC 25923 clinical isolate ATCC .... Newman wild type laboratory strain T.J. Foster ... COL wild type laboratory strain S. Foster . ... 8325-4 NCTC 8325 cured of prophages S. Foster ... . SH1000 rsbU+ 8325-4 derivative S. Foster .... Newman (sarA2) sarA::EmR sarA2; isogenic mutant of Newman T.J. Foster ... Newman (agr2) agr::TcR agr2; isogenic mutant of Newman T.J. Foster ... . Newman (sigB2) sigB::TcR sigB2; isogenic mutant of Newman T.J. Foster .... Newman (hla2) hla::EmR hla2; isogenic mutant of Newman T.J. Foster ... Newman (aur2) aur::EmR aur2; isogenic mutant of Newman T.J. Foster . ... Newman (sspA2) sspA::EmR sspA2; isogenic mutant of Newman T.J. Foster .... Newman (srtA2) srtA::EmR srtA2; isogenic mutant of Newman O. Schneewind ... Newman (spA2) unmarked spA gene deleted T.J. Foster . . . .. doi:10.1371/journal.pone.0001409.t001 In summary, it is evident that the mechanism for survival of S. aureus inside macrophages differs considerably from its interactions with other cells types. S. aureus preserves the integrity of the macrophage, using it as an intracellular niche, rather than destroying it. Nothing is yet known about how macrophages affect transcriptional regulation of intracellular S. aureus or why bacteria do not quickly escape from phagosomes as they do from other cells. However from the data presented here, it is clear that intracellular S. aureus can survive within human macrophages for several days until a point where bacteria escaped the intracellular confinement, proliferated in the conditioned medium and killed cells. In comparison to the more stealthy strategy of conversion to small colony variants, which allows for survival for months or even years [82], it would appear that hiding within macrophages is a short-term solution. Nevertheless, it may provide S. aureus with an advantage since, as is the case for L. monocytogenes and M. tuberculosis, infected mobile macrophages can serve as vehicles for the dissemination of infection [83–85]. This would explain the ability of S. aureus to invade the circulation from localized sites of infection and to disseminate systemically. MATERIALS AND METHODS Reagents Acridine orange, propidium iodide, ethidium bromide, Bodipy 493/503 were from Molecular Probes (Invitrogen). FCS (Fetal Calf Serum), gentamycin, erythromycin, tetracycline, chloram- phenicol, sodium cacodylate trihydrate, RNase A, proteinase K, bovine serum albumin (BSA), saponin, recombinant human interferon c (IFNc), tryptic soy agar (TSA), tryptic soy broth (TSB), lysostaphin and agarose were obtained from Sigma. RPMI 1640 and CMF-PBS (without Ca2+ and Mg2+) and Ficoll-Paque were obtained from Gibco and Amersham Bioscience, respective- ly. LDH Kit was obtained from Promega. Genomic DNA Prep Plus Kit was purchased from A&A Biotechnology and Taq recombinant polymerase was obtained from MBI Fermentas. Cell culture hMDMs were obtained from peripheral blood mononuclear cells (PBMCs). Briefly, PBMCs were isolated from human blood using a Ficoll-Paque density gradient and plated at 36106/well in 24-well plates (Sarstedt) in RPMI1640 supplemented with 2 mM L- glutamine, 50 mg ml21 gentamycin (Sigma), and 10% autological human serum. After 24 h, non-adherent PBMCs were removed by washing with complete medium, and adherent cells were routinely cultured in this medium for 7 days with fresh medium changed every 2 days [86]. For some experiments hMDMs were cultured for 14 days. Bacterial strains, storage and growth conditions The laboratory S. aureus strains used in this study are listed in Table 1. The strains were stored in TSB medium containing glycerol (50% v/v) at 280uC. Cultures were inoculated from stocks into 10 ml media. S. aureus strains were grown overnight under constant rotation (180 rpm) to stationary growth phase at 37uC. If required, media were supplemented with antibiotics at the following concentration: erythromycin at 5 mg ml21 or 10 mg ml21; tetracycline at 2 mg ml21. The bacterial cells were collected by centrifugation (5,0006g, 8 min), washed with phosphate- buffered saline (PBS) and resuspended in PBS to the desired OD600nm. For some experiments, staphylococci were opsonized by incubation at 37uC for 30 min in heat inactivated FCS, washed and resuspended in PBS. Accuracy of preparation of bacterial samples for phagocytosis assay was routinely verified by plating dilutions on agar plates and counting colonies to determine CFU per ml. Occasionally, viability of staphylococci used to incubate with hMDMs was evaluated using a LIVE/DEADH BacLightTM bacterial viability kit from Molecular Probes and found .90% viable. Phagocytosis assay and samples preparations A suspension of S. aureus in PBS was added to 36105 hMDMs cultured in RPMI1640 medium containing L-glutamine and 10% heat inactivated human autologous serum in the 24-well culture plate to the final amount of 1.56106 CFU, 36106 CFU, 7.56106 CFU, 1.56107 CFU, and 36107 CFU (the ratio bacteria : hMDM = from 5:1 to 100:1, multiplicity of infection, MOI = 5, 10, 25, 50 and 10, respectively). hMDMs and staphylococci were co- cultured for 2h in a humidified atmosphere containing 5% CO2. Phagocytosis was stopped by putting the plates on ice and washing the hMDMs cultures twice with ice-cold PBS to remove non- phagocytosed bacteria. Any remaining extracellular bacteria were killed by overnight culturing hMDMs in RPMI1640 medium PLoS ONE | www.plosone.org 12 January 2008 | Issue 1 | e1409