Modulation of neutrophil chemokine receptors by Staphylococcus aureus supernate.

In a previous study, we showed that Staphylococcus aureus supernate (SaS) is a potent agonist for both neutrophils and mononuclear cells. To further investigate the immunomodulating effects of SaS, the effect on different neutrophil receptors was studied. Expression of various neutrophil receptors, before and after treatment with SaS, was quantified by flow cytometry. We found that SaS treatment of neutrophils resulted in a specific and total downregulation of the C5a and the fMLP receptor, both serpentine receptors, while other receptors were totally unaffected. Since these two receptors are both involved in chemotaxis, we tested the effect of SaS in calcium flux and chemotaxis assays. We showed that preincubation with SaS abrogated the rise in intracellular calcium concentration upon triggering with fMLP and C5a. We also showed that SaS is a potent inhibitor of neutrophil chemotaxis towards fMLP and C5a, but does not interfere with chemotaxis towards interleukin-8. These findings indicate that S. aureus produces a virulence factor extracellularly, which impairs chemotaxis towards the infected site.

Effect of clindamycin on the intracellular bactericidal capacity of human peritoneal macrophages.

Survival and growth of bacteria within peritoneal macrophages has been implicated as a cause of recurrence and relapse of Staphylococcus epidermidis peritonitis during peritoneal dialysis. We studied the effect of orally administered clindamycin (known to enter and concentrate in phagocytes) on the intracellular killing of S. epidermidis by human peritoneal macrophages. Clindamycin (300 mg qid) was taken for one day by eight CAPD patients. Peritoneal macrophages were isolated from the effluents and their capacity to phagocytose and kill S. epidermidis was measured. In effluents containing clindamycin, the macrophages showed better uptake (32 vs 17%, P less than 0.01) and intracellular killing (70 vs 42%, P less than 0.01) of S. epidermidis compared with control after 1 h incubation. After 18 h S. epidermidis within peritoneal macrophages incubated with clindamycin, showed a further decrease in viability (-0.33 decrease in log cfu/ml). In contrast, control phagocytes allowed numbers of S. epidermidis to increase over 18 h (+1.46 increase in log cfu/ml; P less than 0.01 compared to clindamycin). Antibiotics with the ability to suppress intracellular bacterial growth should be studied for treatment of CAPD-related S. epidermidis peritonitis.

Adherence of peritonitis-causing staphylococci to human peritoneal mesothelial cell monolayers.

The adherence of staphylococci to monolayers of human mesothelial cells was studied. Adherence of Staphylococcus aureus to mesothelial cell monolayers was 3.4-fold better than to plastic (P less than .01) whereas that of Staphylococcus epidermidis was 3.0-fold less than to plastic (P less than .01). Neither serum albumin nor gelatin inhibited staphylococcal binding. S. aureus adherence correlated with the amount of cell wall protein A (r = .63, P less than .05) but not with fibronectin binding; it was significantly inhibited by the addition of purified cell wall lipoteichoic acid (55% +/- 2.7%), teichoic acid (34.5% +/- 3.4%), and protein A (25.6% +/- 2.9%) but not peptidoglycan. Protein A- and teichoic acid-deficient mutants adhered less well than their parent strains, and encapsulated S. epidermidis adhere well to human monothelial cells. Staphylococcal binding may involve cell wall lipoteichoic acid, teichoic acid, and protein A.

Heterogeneity in opsonic requirements of Staphylococcus epidermidis: relative importance of surface hydrophobicity, capsules and slime.

The opsonic requirements of 65 strains of Staphylococcus epidermidis were compared in fresh and in heated normal human serum. The strains were isolated from patients with CAPD peritonitis (n = 26), neonatal septicaemia (n = 24) and nasal cultures (n = 15). A wide variation was observed in opsonic requirements between the different strains, both with fresh and with heated serum. Opsonization in heated serum proceeded less efficiently and higher concentrations (mean three-fold compared to fresh serum) were needed for adequate phagocytosis. However, a highly significant correlation was found between the minimal opsonic concentrations of fresh and of heated serum (r = 0.84, P less than 0.0005). In addition, S. epidermidis can become opsonized in agammaglobulinaemic serum. Thus, opsonization of S. epidermidis can be mediated by antibodies alone and by complement alone. Slime-producing strains and encapsulated strains did not require higher concentrations of serum to become opsonized. Opsonic requirements were highly significantly correlated with surface hydrophobicity. Enzymatic treatment rendered the strains more hydrophilic and decreased their opsonic requirements. Isolates from nasal cultures required significantly higher concentrations of both fresh and heated serum to become adequately phagocytozed, whereas isolates from CAPD peritonitis required higher concentrations of heated serum only compared to blood isolates. The uptake of S. epidermidis preopsonized in heated serum as determined in our direct phagocytosis assay did not result in a comparable chemiluminescence response.

Are intracellularly penetrating antibiotics warranted in CAPD-related peritonitis?

Survival and growth of bacteria within peritoneal macrophages has been implicated as causes of recurrences and relapses of Staphylococcus epidermidis peritonitis. We compared the effect of cephradine--known not to penetrate into peritoneal macrophages--with that of clindamycin--known to concentrate in phagocytes--on the intracellular killing of S. epidermidis by human peritoneal macrophages. Clindamycine (q.i.d. 300 mg) or cephradine (q.i.d. 250 mg) was taken orally for one day in a randomized cross-over setting by 8 stable CAPD patients. On both days peritoneal macrophages were isolated from the overnight effluents and their capacity to phagocytize and kill S. epidermidis was measured. Phagocytes isolated from and incubated in effluents containing clindamycin, showed better bacterial uptake (32 vs 17%, p less than 0.01) and killing (70 vs 42%, p less than 0.01) compared to cephradine. Moreover, clindamycin prevented S. epidermidis to multiply intracellularly (-0.33 decrease in log colony forming units (cfu)/ml after 18 h). In sharp contrast, phagocytes incubated with cephradine allowed S. epidermidis to increase over 18 h (+1.48 increase in log cfu/ml; p less than 0.01 compared to clindamycin). We conclude that antibiotics with the ability to suppress intracellular bacterial growth may provide a more optimal treatment of CAPD-related peritonitis.

Dialysis fluids and local host resistance in patients on continuous ambulatory peritoneal dialysis.

The ability of polymorphonuclear leukocytes, monocytes and peritoneal macrophages to mount a respiratory burst in continuous ambulatory peritoneal dialysis (CAPD) fluids was tested in a phorbol-myristate acetate stimulated chemiluminescence assay. Fresh CAPD fluids depressed the chemiluminescence response of all three types of phagocytes tested to less than 18% of their chemiluminescence response in control buffer. When tested in spent CAPD fluids the suppression of chemiluminescence was 30-32%. Oxygen consumption of polymorphonuclear leukocytes was depressed in fresh CAPD fluids to below 40%. Both phagocytosis of Escherichia coli by and bactericidal capacity of polymorphonuclear leukocytes and monocytes were suppressed in fresh CAPD fluids but not in spent effluents. The influence of acidic pH and hyperosmolality on phagocytic functions were studied separately by modifying the acidity or the glucose content of the control buffer. pH values below 6.0 significantly inhibited chemiluminescence but not phagocytosis. Under hypertonic conditions, both phagocytosis and chemiluminescence were inhibited. We conclude that the currently available CAPD solutions are beyond the limits of acid and osmotic tolerance of human phagocytic cells, and may thus compromise the peritoneal defenses of CAPD patients.