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Objectives and Methods: Whipple's disease is a rare multisystem chronic infection, involving the intestinal tract as well as various other organs. Tropheryma whipplei is a slow-growing facultative intracellular bacterium that remains poorly understood. In vitro antibiotic susceptibility testing has previously been assessed in cells using a real-time quantitative PCR assay. In this study, we have evaluated the antibiotic susceptibility of three strains of T. whipplei grown in axenic medium using the same assay., Results: The active compounds in axenic medium were doxycycline, macrolide compounds, penicillin G, streptomycin, rifampicin, chloramphenicol, thiamphenicol, teicoplanin, vancomycin, amoxicillin, gentamicin, aztreonam, levofloxacin and ceftriaxone, with MICs in the range 0.06-1 mg/L. Cefalothin was less active, with MICs in the range 2-4 mg/L. We found that co-trimoxazole was active with MICs in the range 0.5-1 mg/L, and sulfamethoxazole alone was active with MICs in the range 0.5-1 mg/L. MICs of trimethoprim varied from 64-128 mg/L., Conclusions: Co-trimoxazole was effective in vitro, but this activity was due to sulfamethoxazole alone. These results were in accordance with the fact that T. whipplei does not contain the encoding gene for dihydrofolate reductase, the target for trimethoprim.

Non-typeable Haemophilus influenzae, which is a cause of disease in the upper and lower respiratory tract, can survive intracellularly in human epithelial cells and macrophages. We studied the in vitro activity of five antibiotics against intracellular non-typeable H. influenzae in human type II alveolar epithelial cells. The eukaryotic cells were loaded with bacteria, and extracellular bacteria were killed by gentamicin. After the cells were washed, antibiotics were added at concentrations of 0.12-64 mg/L for 18 h before the numbers of viable intracellular bacteria were determined. Of the antibiotics tested, ciprofloxacin and quinupristin/dalfopristin were the most potent agents, followed by clarithromycin and telithromycin. Ampicillin was not active against intracellularly localized, non-typeable H. influenzae.

The purpose of this study was to compare the extra- and intracellular activity of antifolates on Listeria monocytogenes. The fortuitous discovery of elongated bacteria in response to trimethoprim revealed a novel effect on the morphology of Listeria in cell culture medium and after phagocytosis. This phenomenon permitted the quantification of trimethoprim activity, revealing comparable activity intra- and extracellularly. Subinhibitory concentrations of trimethoprim resulted in bacterial elongation, which was reversed after removal of trimethoprim. We attribute this effect of trimethoprim to an inhibition of cell wall synthesis and/or cell separation of Listeria.

Intracellular persistence assays were performed with small-colony variants (SCVs) derived from a patient with Darier's disease from whom different phenotypes and genotypes of Staphylococcus aureus were isolated over a 28-month period; the assays revealed that >100-fold more SCV cells persisted intracellularly relative to the normal phenotype. The presence of intracellular S. aureus SCVs may protect against host defenses and antibiotic therapy and thus may have contributed to this patient's very prolonged skin infection.

Helicobacter pylori colonizes the gastric epithelium of humans and plays a causative role in peptic ulcer disease and perhaps gastric cancer. H. pylori proliferates in the mucus layer over the epithelium and is not cleared by the host immune response. Although the mucus layer is the major reservoir of H. pylori in vivo, a growing body of evidence suggests that H. pylori can persist in multiple intracellular locales. Clinical isolates of H. pylori invade epithelial monolayers at least as well as Shigella. The intracellular organisms are cytotoxic, and bacterial microcolonies form on the exposed basement membrane. Both mononuclear phagocytes and neutrophils phagocytose unopsonized H. pylori. However, the internalized organisms are not killed efficiently and our recent data suggest that H. pylori disrupts phagosome maturation. Collectively, the data support the hypothesis that intracellular H. pylori represent a reservoir of organisms that contributes to bacterial persistence, host tissue damage, and treatment failure.

Intracellular growth patterns of Legionella pneumophila were examined in monocytes obtained from carriers of human T-lymphotropic virus type I (HTLV-1) and controls who were HTLV-1 seronegative. All subjects were seronegative for antibodies against L. pneumophila. Bacterial growth was determined 0, 1, 2, and/or 3 days after infecting peripheral blood mononuclear cells (PBMCs) with the bacteria. The intracellular growth of L. pneumophila was markedly inhibited in HTLV-1 carriers compared with normal controls. When the lymphocytes were depleted from the HTLV-1 carrier PBMC cultures before infection, this inhibition was abolished. Inhibition reappeared, however, when the 72-h culture supernatants of PBMCs from HTLV-1 carriers were added to the lymphocyte-depleted cultures. Culture supernatants of infected and uninfected PBMCs from HTLV-1 carriers exhibited markedly increased levels of interferon-gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) compared with the HTLV-1 seronegative controls. In the HTLV-1 carriers, IFN-gamma was produced by the CD4+ lymphocytes, whereas TNF-alpha was secreted by the monocytes. Addition of anti-IFN-gamma or anti-TNF-alpha antibodies to the HTLV-1 carrier PBMC cultures diminished the inhibition of intracellular growth of L. pneumophila. Results suggest that the monocytes are activated in HTLV-1 carriers. These findings may explain why an opportunistic infection by certain intracellular pathogens is rarely seen among HTLV-1 carriers.

Recent studies have reported that Staphylococcus aureus small colony variants (SCVs) can cause highly persistent infections in humans and in cultured endothelial cells. To understand the process by which SCVs of S. aureus appear in subjects who have not received antibiotic treatment, bovine endothelial cells were coincubated with a wild S. aureus strain for 72 h in the presence of lysostaphin. Intracellular bacteria were harvested and screened for stable SCVs. Intracellular bacteria developed the SCV phenotype at a greater rate than control bacteria not exposed to endothelial cells: The intracellular induction rate was approximately 10(-3) versus a spontaneous rate of <10(-7). This observation suggest that SCVs are induced by the intracellular milieu and suggest a possible mechanism for the intriguing pathophysiology of tissue persistence of staphylococci.

We studied the effect in vitro of azithromycin on the clinical strain Helicobacter pylori H:72 growing intracellularly in monolayers of HEp-2 epithelial cells. After using gentamicin to eradicate extracellular bacteria, different concentrations of azithromycin were added to the infected cells and samples were taken after 0, 4, 8 and 24 h. Infected cells not exposed to antibiotic were included as controls. The MIC of azithromycin to the H. pylori was 0.25 mg/L and the MBC 0.5 mg/L in a broth dilution plate count method. A bactericidal effect was observed on intracellular H. pylori, with inhibition increasing with increasing azithromycin concentrations. However, extracellular concentrations of 200 x MBC were necessary to achieve intracellular killing. Our results show that azithromycin is active against intracellular H. pylori suggesting that it might be possible to exploit this activity when treating infections due to the organism.

The agent 1,25-dihydroxyvitamin D3 (D3) induces the differentiation of HL-60 human leukemia cells into functional monocyte-like cells that can support the intracellular multiplication of Legionella pneumophila. 22-Oxacalcitriol (OCT), a synthetic analogue of D3, exhibits greater differentiation-inducing activity than D3 in WEHI-3 mouse leukemia cells and has been suggested to be clinically more useful because of its lower hypercalcemic activity. The abilities of OCT and D3 to induce the functional differentiation of human leukemia HL-60 cells have now been investigated. OCT induced the differentiation of HL-60 cells into monocyte-like cells to a similar extent as D3. Thus, both OCT and D3 increased (1) the surface expression of CD11b, CD11c, CD14, and CD35; (2) nonspecific esterase staining; and (3) phagocytic activity toward fluorescent beads. HL-60 cells differentiated in response to OCT also supported the intracellular multiplication of L. pneumophila. Activation of both OCT- and D3-treated HL-60 cells with human recombinant interferon-gamma (IFN-gamma) for 24 h before infection markedly inhibited L. pneumophila multiplication. IFN-gamma activation enhanced superoxide anion generation by D3-treated HL-60 cells but not by OCT-treated HL-60 cells, suggesting that the inhibition of L. pneumophila multiplication in IFN-gamma-activated cells is independent of superoxide generation. Finally, D3, but not OCT, markedly stimulated the formation of osteoclast-like multinucleated cells from mouse bone marrow cells, consistent with the lower hypercalcemic activity of OCT.

We examined leukemic cells, HL-60, an acute promyelocytic leukemia cell line, after differentiation induced by 1,25-dihydroxyvitamin D3 (D3) and retinoic acid (A) for infection of Legionella pneumophila, the etiologic agent of Legionnaires' disease. We investigated the effect of interferon gamma (IFN-gamma) on the differentiated cells and on the intracellular growth of the bacteria. An examination of morphological and antigenic changes in the cells was also included in the study. After 4-day incubation with 10(-6)M D3 or A, the HL-60 cells differentiated into monocyte-like (D3-HL-60) or mature granulocyte-like (A-HL-60) cells, respectively. They were then infected with L. pneumophila. Intracellular multiplication of the bacteria was evident in D3-HL-60 cells but not in HL-60 or A-HL-60 cells. D3-HL-60 cells required a 24-h infection time for the intracellular growth of L. pneumophila. D3-HL-60 cells activated with human recombinant IFN-gamma for 1-24 h (gamma-IFN-D3-HL-60 cells) before infection markedly inhibited L. pneumophila multiplication, the effect of IFN-gamma being dose dependent. Surface marker analysis was carried out in HL-60, D3-HL-60, and gamma-IFN-D3-HL-60 cells. On D3-HL-60 cells, CD11b, CD11c, CD14, and CD35 antigen increased, whereas CD71 and HLA-DR antigen decreased. This finding suggested that HL-60 cells differentiated into monocyte-like cells; the acquisition of the complement receptors, CD11b(CR3) and CD35(CR1), seemed to be important for phagocytosis and for the subsequent intracellular multiplication of L. pneumophila. The gamma-IFN-D3-HL-60 cells showed an increase of CD16, CD36, CD71, and HLA-DR antigen, suggesting that they were in an activated state. Our study indicated, first, that D3 can induce human leukemic cells to differentiate into functional monocyte-macrophage-like cells that can support the intracellular multiplication of L. pneumophila and, second, that these differentiated leukemic cells can be activated by IFN-gamma to markedly inhibit bacterial growth.