The effects of ethyleneglycoltetra-acetic acid on bactericidal activity of human serum against Escherichia coli

Abstract

SUMMARY.The effects of ethyleneglycoltetra-acetic acid (EGTA) and EGTA+magnesium (MgEGTA) on the viable counts of 10 strains of Escherichia coli 0 6 have been studied in normal human serum (NHS), heat-inactivated serum (HIS) and in culture media with and without the addition of a fl-lactam antibiotic. The addition of EGTA to NHS largely prevented bactericidal activity against serum-sensitive strains while, in contrast, it reduced the growth of a serum-resistant strain. These apparently paradoxical effects are due to the lower growth rate permitted by the reduced amount of available magnesium in the presence of EGTA.Experiments with equimolar concentrations of EGTA and magnesium indicated that whilst MgEGTA is a reagent allowing alternative complement-pathway activity, such activity must be determined by comparison with results in HIS+MgEGTA rather than in HIS alone, classical-pathway activity being taken as the difference between the results in NHS and in NHS + MgEGTA.By these criteria, prompt killing by serum was found to occur via the classical pathway while delayed serum bactericidal activity occurred by the alternative pathway in some strains and by the classical pathway in others.

The relative importance of the classical and alternative complement pathways in serum bactericidal activity against Escherichia coli

“…In our previous study of the effect of MgEGTA on the bactericidal activity of human serum (Roberts and Phillips, 1981) we found that promptly serum-sensitive strains of E. coli serogroup 0 6 were killed rapidly by the classical pathway and that, when this pathway was eliminated by the chelator, delayed killing occurred by the alternative pathway; this was comparable with the findings of Traub and Kleber (1976) for strains of Serratia marcescens. In the present study of other 0-serogroups common amongst urinary isolates of E. coli, most strains again responded in a similar way but further patterns of response to serum have emerged; the 0 1 isolate, no.…”

“…These differences could be a reflection of nutritional requirements or of the effectiveness of a non-complement-dependent bacteriostatic mechanism (Traub and Kleber, 1976). The generally reduced growth when MgEGTA is added to HIS has been noted before for E. coli 0 6 strains (Roberts and Phillips, 1981) and probably indicates that MgEGTA chelates other divalent cations important for growth.…”

“…The preparation of serum and the assay of bactericidal activity have been described previously (Roberts and Phillips, 1981). Briefly, to 1.6 ml of fresh human serum was added 0-2 ml of normal saline or MgEGTA and 0-2 ml of a bacterial suspension containing c. 2 x lo5 actively-dividing bacteria in normal saline.…”

“…We have shown recently that both the classical and the alternative complement pathways are involved in serum bactericidal activity against most urinary isolates of Escherichia coli of serogroup 0 6 (Roberts and Phillips, 1981). It was considered important to establish whether this is also true for other 0-serogroups of E. coli commonly associated with urinary-tract infections.…”

Alternative pathway amplification and infections

“…The extremely labile nature of nascent C3b (half‐life of approximately 60 μs 48 ) and low activation rates because of the bias toward inhibition of the pathway by FH and FI makes the “tickover” mechanism alone highly unlikely to effectively surveil pathogens. Therefore, it is no surprise that killing of most pathogens is either abrogated or considerably delayed when only the AP is active 49–58 …”