Effect of dialysate fluids on phagocytosis and killing by normal neutrophils

Abstract

Inadequate host defenses may partly explain the problem of recurrent peritonitis in patients on continuous ambulatory peritoneal dialysis. It has been suggested that these defenses may be adversely affected by the fluids used for dialysis, and so we examined the effects of unused, effluent, and infected peritoneal dialysis fluids on phagocytosis and killing by normal neutrophils. We used a clinical isolate of Staphylococcus epidermidis as the test organism, as this organism is the most commonly cultured in continuous ambulatory peritoneal dialysis peritonitis; we also used a fungal species, Candida guilliermondii. There was no phagocytosis of either organism in unused dialysate because of lack of opsonins and low pH. Phagocytosis in effluent dialysate did not occur because of inadequate opsonin levels and was variable in infected effluents, depending on quantities of immunoglobulins present. Intracellular killing of both test organisms was normal in unused dialysate in the presence of 5 % normal serum, but was reduced in effluent and infected dialysates because of factors inhibiting killing by neutrophils. These factors adversely affected the killing of S. epidermidis more than that of C. guilliermondii. These results may explain why peritonitis recurs, particularly peritonitis due to S. epidermidis, because organisms could be sequestered within the neutrophils and thus be protected from antibiotic action. Reinfection of the peritoneal cavity would then take place following neutrophil breakdown, causing a clinical relapse.

Hydrogen peroxide generation by polymorphonuclear leukocytes exposed to peritoneal dialysis effluent

“…High osmolality (15), low pH (15,16,29), and reduced availability of opsonins (24) are important in compromising PMN function. However, when these factors are corrected, PMN still fail to kill microorganisms because of the presence of a low-molecular-weight solute(s) (12,20,21). The generation of hydrogen peroxide (H 2 O 2 ) is of fundamental importance for effective PMN killing.…”

Characterization of cell envelope proteins of Staphylococcus epidermidis cultured in human peritoneal dialysate

“…The ability of coagulase-negative staphylococci to adapt and survive within a dialyzed peritoneum may be related to their capacity to (i) grow in peritoneal fluids (26,32,34), (ii) colonize intraperitoneal or plastic catheter surfaces (12,24,28), and (iii) avoid phagocytosis or phagocytic killing (17,27,28). An additional contributing factor appears to be that the staphylococcal killing capacities of peritoneal macrophages and infiltrating polymorphonuclear leukocytes are impaired in CAPD patients (11,16).…”

“…Briefly, bacteria harvested from 100 ml of growth medium were washed twice in phosphate-buffered saline (120 mM NaCl, 10 mM sodium phosphate, pH 7.4), suspended in 0.6 ml of digestion buffer (30% [wt/vol] raffinose, 1 mg of benzamidine per ml, 0.5 mg of phenylmethylsulfonyl fluoride per ml in 10 mM Tris hydrochloride, pH 7.4, containing 100 jig of lysostaphin), and incubated for 60 min at 37°C. Protoplasts were removed by centrifugation (11,600 x g for 3 min), and the supernatant containing the cell wall proteins was stored frozen at -20°C before electrophoresis. The protoplast pellet was suspended in distilled water and sonicated at 4°C for two 30-s periods.…”

Influence of carbon dioxide on growth and antibiotic susceptibility of coagulase-negative staphylococci cultured in human peritoneal dialysate

“…The variously treated pooled PUD samples were electrophoresed on SDS-12.5% PAGE gels (approximately 10 ,g of protein per lane) and then fixed and stained in acetic acidmethanol with 1% Coomassie blue (Sigma Chemical Co. Ltd., Dorset, England).…”

“…fluid represents a step closer to these conditions. The peritoneal cavities of patients undergoing continuous ambulatory peritoneal dialysis (CAPD) represent a unique growth environment for potential pathogens, and therefore in vitro studies of peritonitis-causing isolates have employed used peritoneal dialysis fluid (PUD) as a culture medium (8)(9)(10)(11)(12)(13)(14)16). In addition to differences in growth kinetics, we have shown unique phenotypic changes in staphylococcal cell surface characteristics associated with growth in PUD (17,19; S. P. Denyer, M. C. Davies, J.…”