INFECTION AND IMMUNITY, May 1976, p. 1343-1346 Copyright © 1976 American Society for Microbiology
Vol. 13, No. 5 Printed in U.SA.
Selective Activation of Classical and Alternative Pathways of Human Complement by "Promptly Serum-Sensitive" and "Delayed Serum-Sensitive" Strains of Serratia marcescens WALTER H. TRAUB* AND INGRID KLEBER Institut fur klinische Mikrobiologie und Infektionshygiene, Universitat Erlangen-Nurnberg, 8520 Erlangen, West Germany
Received for publication 16 October 1976
Chelation of fresh human serum with 0.01 M MgCl, (Mg) plus 0.01 M ethylene glycol tetraacetic acid failed to abrogate the bactericidal activity against "delayed serum-sensitive" strains of Serratia marcescens, whereas previously "promptly serum-sensitive" strains of S. marcescens and control strain Escherichia coli C were killed after an extended period of incubation. The addition of 0.01 M ethylenediaminetetraacetate to fresh human serum neutralized bactericidal activity against S. marcescens of either serum sensitivity category. Very recently, evidence obtained in our laboratory permitted the classification of clinical isolates of Serratia marcescens, an opportunistic pathogenic microorganism, into two categories with respect to the kinetics of the bactericidal activity of either fresh human serum or fresh defibrinated human blood (12, 13). "Promptly serum-sensitive" strains of S. marcescens were killed within minutes, in a manner analogous to a serum-sensitive control strain of Escherichia coli. "Delayed serum-sensitive" isolates of S. marcescens, on the other hand, required exposure to human serum for at least several hours to allow for maximal killing. Earlier Fine et al. (4), and more recently the same group of investigators (1), had observed that chelation of fresh human serum with 0.01 M magnesium ions plus 0.01 M ethylene glycol tetraacetic acid (EGTA) blocked the classical pathway of complement activation, but left intact the alternative pathway (7-9). Furthermore, Forsgren and Quie (5) noted that, among several species of bacteria examined, the sole strain of S. marcescens used was opsonized by human serum that had been chelated with Mg + EGTA. Therefore, it was of interest to determine which activated pathway of human complement was required for serummediated killing of delayed and promptly serum-sensitive strains of S. marcescens, respectively. MATERIALS AND METHODS Bacteria. S. marcescens isolates S 86 and SM 29 (promptly serum sensitive) and S 326 n.t. and SE 142 (delayed serum sensitive) were the same as used previously (12, 13). E. coli strain C served as a control for all serum bactericidal assays.
Media. Brain heart infusion broth and agar and tryptic soy broth (TSB) were purchased from Difco, Detroit, Mich. The organisms were maintained on brain heart infusion agar slants at 4 C and in a mixture of 1 volume of brain heart infusion broth plus 1 volume of heat-inactivated bovine serum (Behringwerke, Marburg, West Germany) at -65 C. Reagents. CaCl2 and MgCl2 *6H20 were obtained from E. Merck, Darmstadt, West Germany. EDTA (disodium ethylenediaminetetraacetate) was procured from Fisher Scientific Products, Fair Lawn, N.J.; EGTA was purchased from Serva Feinbiochemica GmbH, Heidelberg, West Germany. Aqueous stock solutions (MgCl2 = 0.5 M; CaCl2, EGTA, and EDTA = 0.1 M each) were filter sterilized (0.2-pLm plain membrane filters; Nalge Sybron Corp., Rochester, N.Y. and stored at 4 C. EGTA had been dissolved according to the instructions given by Fine et al. (4). The pH of 0.1 M EGTA and 0.1 M EDTA stock solutions ranged from 7.40 to 7.45. Serum (bactericidal activity) assays. One healthy adult repeatedly served as blood donor (T-serum). Sera were processed and maintained at -65 C as before (11). Serum aliquots were heat inactivated by exposure to 56 C for 30 min in a water bath as required. All serum assays were performed in sterile, disposable tissue culture tubes (C. A. Greiner und S6hne, Nurtingen, West Germany), which were incubated stationary at 35 C. The bacterial cell inocula were exposed to 80% (vol/vol) of fresh heatinactivated and fresh T-serum that had been chelated with 0.01 M Mg + EGTA and 0.01 M EDTA, respectively. The organisms had been grown on chocolate agar at 35 C overnight (check for viability and purity). Next morning, 20 colonies of each isolate were transferred to 5 ml of TSB and incubated for 2 h, after which the turbidity of the cultures was adjusted to that of McFarland barium sulfate standard no. 0.5 and further diluted 10-I. The assay tubes (final volume = 2 ml) received 1.6 ml of fresh or heat-inactivated (56 C) T-serum plus 0.2 ml of TSB, 343
1344
INFECT. IMMUN.
TRAUB AND KLEBER
1.8 ml of fresh T-serum + 0.01 M Mg + EGTA, or 1.8 ml of fresh T-serum + 0.01 M EDTA, plus 0.2 ml of bacterial inoculum (= approximately 1.5 x 104 colony-forming units/ml at zero time). At appropriate time intervals (0, 0.3, 1, 2, 4, 6, and 22 h, unless specified otherwise), samples were withdrawn and serially diluted 10-fold in TSB; two pour plates (brain heart infusion agar) per dilution served to determine the number of viable colony-forming units per milliliter.
against E. coli C over a time span of at least 4 h; however, a diminished number of survivors was evident at 22 h after exposure. Furthermore, chelation of fresh T-serum with 0.01 M EGTA (without Mg ions) appeared to be reversible. When either 0.01 M MgCl2 or 0.01 M CaCl2 was added to EGTA-chelated T-serum 2 h later, there resulted delayed and prompt killing of E. coli C cells, respectively. Chelation of fresh Tserum with 0.01 M EDTA likewise was reversiRESULTS ble; after addition of either 0.01 M MgCl2 or 0.01 Fresh T-serum that had been chelated with M CaCl2 at 2 h after chelation, E. coli C cells 0.01 M Mg + EGTA was as bactericidally active were killed promptly. Addition of 0.01 M EDTA to heat-inactivated T-serum and TSB resulted as control fresh serum against the delayed serum-sensitive S. marcescens isolates S 326 n.t. in a delayed bactericidal and inhibitory effect (Table 1) and SE 142. These and all subsequent against E. coli C, respectively. EDTA exerted an incipient inhibitory effect at 6 h, whereas at serum assays had been performed at least three times and the kinetic data obtained were iden- 22 h after exposure there were no survivors in tical. The addition of 0.01 M EDTA completely EDTA-chelated heat-inactivated T-serum and abrogated the bactericidal activity of T-serum, a sharply reduced number of viable cells in analogous to conventional heat inactivation. In EDTA-chelated TSB (Table 4). contrast, chelation of fresh T-serum with 0.01 DISCUSSION M Mg + EGTA resulted in delayed killing of The data obtained seem to indicate that the the previously promptly serum-sensitive isolates SM 29 (Table 2) and S 86. Again, 0.01 M classical and the alternative pathways of the EDTA neutralized the bactericidal activity of human complement system are activated by T-serum. The results obtained with control promptly serum-sensitive and delayed serumstrain E. coli C were as follows: chelation of T- sensitive strains ofS. marcescens, respectively. Previously, bacterial lipopolysaccharides had serum with 0.01 M Mg + EGTA resulted in delayed killing of the cells (Table 3). However, been shown to activate either of the two pathchelation of T-serum with 0.01 M EDTA ef- ways (3), as reemphasized recently by Snyderfected incomplete neutralization of the bacteri- man and Pike (10). Furthermore, our data corcidal activity; the cells appeared to recover at 2 robrate those of Gallin et al. (6), who had differto 4 h after exposure, only to be killed upon entiated the generation of chemotactic serum prolonged incubation, in that at 22 h after expo- factors via activation of the classical or the alternative pathway of the complement system sure no survivors of E. coli C were demonstrable in EDTA-chelated serum. As previously by means of kinetic analysis; generation of noted (13), heat-inactivated T-serum exerted a chemotactic factors by the alternative pathway proceeded only after a latent period of 15 to 20 bacteriostatic effect against E. coli C. Chelation of fresh T-serum with 0.01 M min, whereas classically generated products EGTA (without Mg ions) completely neutral- were detectable within 5 min. Over 80% of all ized the bactericidal activity of the serum clinical isolates of S. marcescens examined
or
TABLE 1. Bactericidal activity of chelated human serum against delayed serum-sensitive S. marcescens isolate S 326 n.t. S. marcescens S 326 n.t.
Time that tube samples were assayed
(h) 0 1 2 4 6 22 a
b
Cb
C + Mg/EGTA'
1.3 x 104 2.9 x 10:'
1.3 x 104
1.5 x 10: 1.0 x 10' 0 0
1.3 x 104 7.1 x 103 0 0 0
CFU, Colony-forming units. 80 (vol/vol) fresh T-serum. 80 (vol/vol) fresh T-serum + 0.01 M EGTA + 0.01 M MgCl2. 80 (vol/vol) fresh T-serum + 0.01 M EDTA. 80 (vol/vol) heat-inactivated (56 C, 30 min) T-serum.
(CFU/ml)Y EDTA" 1.3 x 104
C +
1.3 x 104 1.5 x 104 1.6 x 105 3.0 x 106
>108
56 C"
1.3 x 104 1.3 x 104 2.1 x 104 4.0 x 10' 3.4 x 107 >108
VOL. 13, 1976
ACTIVATION OF S. MARCESCENS COMPLEMENT PATHWAYS
1345
TABLE 2. Bactericidal activity of chelated human serum against promptly serum-sensitive S. marcescens isolate SM 29a Time that tube samples were assayed (h)
C
0 0.3 1 2 4
1.8 x 104 0 0 0 0
22
S. marcescens SM 29 (CFU/ml) C + Mg/EGTA 1.8 x 104 1.3 x 104 4.1 x 102 1.0 x 10
0
0
C + EDTA
1.8 x 1.7 x 1.7 x 1.8 x 4.0x
104 104 104 104 104
56 C 1.8 x 104 1.8 x 104
1.8 x 104 2.0 x 104 1.9X 105
>108
0
>108
See Table 1 for explanatory footnotes. TABLE 3. Bactericidal activity of chelated human serum against control strain E. coli Ca Time that tube samples were assayed (h)
0 0.3 1 2 4 22
E. coli C (CFU/ml)
C
C + Mg/EGTA
1.2 x 104 0 0 0 0 0
1.2 1.2 3.7 1.6
x x x x 0
104 104 103 102
C + EDTA
1.2 3.5 2.0 6.6 2.3
0
x x x x
104 102 102 103 x 104 0
56 C
1.2 x 104 1.4 x 104 2.3 x 104 5.1 x 104 2.8 x 105 3.6x105
See Table 1 for explanatory footnotes.
TABLE 4. Inhibitory effect of EDTA against control E. coli strain C in heat-inactivated human serum and in tryptic soy broth Time that tube samples were assayed (h) 0 1 2 4
E. coli C (CFU/ml)a 56 Cb
1.3 x 2.1 x 7.5 x 2.3 x 6 4.5 x 22 1.6 x aCFU, Colony-forming units.
b c
"
104 104 104 10-5 10'5 106
56 C + EDTA' 1.3 x 104 1.2 x 104 1.4 x 104 5.3 x 104 2.3 x 104
0
TSBd
1.3 x 104 2.0 x 104 6.9 x 104 1.8 x 10'i 2.2 x 107 >108
TSB + 0.01 M EDTA 1.3 x 104
2.1 3.0 1.6 1.0
x x x x 1.7 x
104 104 105 105 103
80% (vol/vol) heat-inactivated T-serum.
80% (vol/vol) heat-inactivated T-serum + 0.01 M EDTA. TSB, Tryptic soy broth (Difco).
thus far proved delayed serum sensitive (un- as yet unknown interactions between a serum published observations); thus, the alternative factor(s), certain cations, and cells of E. coli pathway of the complement system might effect strain C might have been operative in this in opsonization (5) of the majority of S. marces- vitro system. cens strains. In this context, Fearon et al. (2) ACKNOWLEDGMENT recently demonstrated that levels of the complement components Cl, C4, and C2 in patients This study was supported in part by a grant from the with gram-negative sepsis and subsequent Deutsche Forschungsgemeinschaft (Tr 136/1). shock were not significantly different from LITERATURE CITED those in patients with uncomplicated bacteremia.
Clearly, further experiments are required to
elucidate the observation that human serum, upon chelation with either EGTA or EDTA, proved bacteriostatic or even bactericidal for the control strain E. coli C after prolonged incubation, but not for isolates of S. marcescens of either serum sensitivity category. Possibly,
1. Des
Prez, R. M., C. S. Bryan, J. Hawiger, and D. G. Colley. 1975. Function of the classical and alternate pathways of human complement in serum treated with ethylene glycol tetraacetic acid and MgCl2-ethylene glycol tetraacetic acid. Infect. Immun. 11: 1235-1243. 2. Fearon, D. T., S. Ruddy, P. H. Schur, and W. R.
McCabe. 1975. Activation of the properdin pathway of complement in patients with Gram-negative bacteremia. N. Engl. J. Med. 292:937-940.
1346
TRAUB AND KLEBER
3. Fine, D. P. 1974. Activation of the classic and alternate complement pathways by endotoxin. J. Immunol. 112:763-769. 4. Fine, D. P., S. R. Marney, Jr., D. G. Colley, J. S. Sergent, and R. M. Des Prez. 1972. C3 shunt activation in human serum chelated with EGTA. J. Immunol. 109:807-809. 5. Forsgren, A., and P. G. Quie. 1974. Influence of the alternate complement pathway on opsonization of several bacterial species. Infect. Immun. 10:402404. 6. Gallin, J. I., R. A. Clark, and M. M. Frank. 1975. Kinetic analysis of chemotactic factor generation in human serum via activation of the classical and alternate complement pathways. Clin. Immunol. Immunopathol. 3:334-346. 7. Goodkofsky, I., and I. H. Lepow. 1971. Functional relationship of factor B in the properdin system to C3 proactivator of human serum. J. Immunol. 107:12001204.
INFECT. IMMUN. 8. Gotze, O., and H. J. Muller-Eberhard. 1971. The C3 activator system: an alternate pathway of complement activation. J. Exp. Med. 134(Suppl.):90-108. 9. Sandberg, A. C., and A. G. Osler. 1971. Dual pathways of complement interaction with guinea pig immunoglobulins. J. Immunol. 107:1268-1273. 10. Snyderman, R., and M. C. Pike. 1975. Interaction of complex polysaccharides with the complement system: effect of calcium depletion on terminal component consumption. Infect. Immun. 11:273-279. 11. Traub, W. H. 1969. Assay of the antibiotic activity of serum. Appl. Microbiol. 18:51-56. 12. Traub, W. H., G. Acker, and I. Kleber. 1976. Ultrastructural surface alterations of Serratia marcescens after exposure to polymyxin B and/or fresh human serum. Chemotherapy 22:104-113. 13. Traub, W. H., and I. Kleber. 1975. Studies on the additive effect of polymyxin B and the bactericidal activity of human serum against Serratia marcescens. Chemotherapy 21:189-204.
Vol. 13, No. 5 Printed in U.SA.
Selective Activation of Classical and Alternative Pathways of Human Complement by "Promptly Serum-Sensitive" and "Delayed Serum-Sensitive" Strains of Serratia marcescens WALTER H. TRAUB* AND INGRID KLEBER Institut fur klinische Mikrobiologie und Infektionshygiene, Universitat Erlangen-Nurnberg, 8520 Erlangen, West Germany
Received for publication 16 October 1976
Chelation of fresh human serum with 0.01 M MgCl, (Mg) plus 0.01 M ethylene glycol tetraacetic acid failed to abrogate the bactericidal activity against "delayed serum-sensitive" strains of Serratia marcescens, whereas previously "promptly serum-sensitive" strains of S. marcescens and control strain Escherichia coli C were killed after an extended period of incubation. The addition of 0.01 M ethylenediaminetetraacetate to fresh human serum neutralized bactericidal activity against S. marcescens of either serum sensitivity category. Very recently, evidence obtained in our laboratory permitted the classification of clinical isolates of Serratia marcescens, an opportunistic pathogenic microorganism, into two categories with respect to the kinetics of the bactericidal activity of either fresh human serum or fresh defibrinated human blood (12, 13). "Promptly serum-sensitive" strains of S. marcescens were killed within minutes, in a manner analogous to a serum-sensitive control strain of Escherichia coli. "Delayed serum-sensitive" isolates of S. marcescens, on the other hand, required exposure to human serum for at least several hours to allow for maximal killing. Earlier Fine et al. (4), and more recently the same group of investigators (1), had observed that chelation of fresh human serum with 0.01 M magnesium ions plus 0.01 M ethylene glycol tetraacetic acid (EGTA) blocked the classical pathway of complement activation, but left intact the alternative pathway (7-9). Furthermore, Forsgren and Quie (5) noted that, among several species of bacteria examined, the sole strain of S. marcescens used was opsonized by human serum that had been chelated with Mg + EGTA. Therefore, it was of interest to determine which activated pathway of human complement was required for serummediated killing of delayed and promptly serum-sensitive strains of S. marcescens, respectively. MATERIALS AND METHODS Bacteria. S. marcescens isolates S 86 and SM 29 (promptly serum sensitive) and S 326 n.t. and SE 142 (delayed serum sensitive) were the same as used previously (12, 13). E. coli strain C served as a control for all serum bactericidal assays.
Media. Brain heart infusion broth and agar and tryptic soy broth (TSB) were purchased from Difco, Detroit, Mich. The organisms were maintained on brain heart infusion agar slants at 4 C and in a mixture of 1 volume of brain heart infusion broth plus 1 volume of heat-inactivated bovine serum (Behringwerke, Marburg, West Germany) at -65 C. Reagents. CaCl2 and MgCl2 *6H20 were obtained from E. Merck, Darmstadt, West Germany. EDTA (disodium ethylenediaminetetraacetate) was procured from Fisher Scientific Products, Fair Lawn, N.J.; EGTA was purchased from Serva Feinbiochemica GmbH, Heidelberg, West Germany. Aqueous stock solutions (MgCl2 = 0.5 M; CaCl2, EGTA, and EDTA = 0.1 M each) were filter sterilized (0.2-pLm plain membrane filters; Nalge Sybron Corp., Rochester, N.Y. and stored at 4 C. EGTA had been dissolved according to the instructions given by Fine et al. (4). The pH of 0.1 M EGTA and 0.1 M EDTA stock solutions ranged from 7.40 to 7.45. Serum (bactericidal activity) assays. One healthy adult repeatedly served as blood donor (T-serum). Sera were processed and maintained at -65 C as before (11). Serum aliquots were heat inactivated by exposure to 56 C for 30 min in a water bath as required. All serum assays were performed in sterile, disposable tissue culture tubes (C. A. Greiner und S6hne, Nurtingen, West Germany), which were incubated stationary at 35 C. The bacterial cell inocula were exposed to 80% (vol/vol) of fresh heatinactivated and fresh T-serum that had been chelated with 0.01 M Mg + EGTA and 0.01 M EDTA, respectively. The organisms had been grown on chocolate agar at 35 C overnight (check for viability and purity). Next morning, 20 colonies of each isolate were transferred to 5 ml of TSB and incubated for 2 h, after which the turbidity of the cultures was adjusted to that of McFarland barium sulfate standard no. 0.5 and further diluted 10-I. The assay tubes (final volume = 2 ml) received 1.6 ml of fresh or heat-inactivated (56 C) T-serum plus 0.2 ml of TSB, 343
1344
INFECT. IMMUN.
TRAUB AND KLEBER
1.8 ml of fresh T-serum + 0.01 M Mg + EGTA, or 1.8 ml of fresh T-serum + 0.01 M EDTA, plus 0.2 ml of bacterial inoculum (= approximately 1.5 x 104 colony-forming units/ml at zero time). At appropriate time intervals (0, 0.3, 1, 2, 4, 6, and 22 h, unless specified otherwise), samples were withdrawn and serially diluted 10-fold in TSB; two pour plates (brain heart infusion agar) per dilution served to determine the number of viable colony-forming units per milliliter.
against E. coli C over a time span of at least 4 h; however, a diminished number of survivors was evident at 22 h after exposure. Furthermore, chelation of fresh T-serum with 0.01 M EGTA (without Mg ions) appeared to be reversible. When either 0.01 M MgCl2 or 0.01 M CaCl2 was added to EGTA-chelated T-serum 2 h later, there resulted delayed and prompt killing of E. coli C cells, respectively. Chelation of fresh Tserum with 0.01 M EDTA likewise was reversiRESULTS ble; after addition of either 0.01 M MgCl2 or 0.01 Fresh T-serum that had been chelated with M CaCl2 at 2 h after chelation, E. coli C cells 0.01 M Mg + EGTA was as bactericidally active were killed promptly. Addition of 0.01 M EDTA to heat-inactivated T-serum and TSB resulted as control fresh serum against the delayed serum-sensitive S. marcescens isolates S 326 n.t. in a delayed bactericidal and inhibitory effect (Table 1) and SE 142. These and all subsequent against E. coli C, respectively. EDTA exerted an incipient inhibitory effect at 6 h, whereas at serum assays had been performed at least three times and the kinetic data obtained were iden- 22 h after exposure there were no survivors in tical. The addition of 0.01 M EDTA completely EDTA-chelated heat-inactivated T-serum and abrogated the bactericidal activity of T-serum, a sharply reduced number of viable cells in analogous to conventional heat inactivation. In EDTA-chelated TSB (Table 4). contrast, chelation of fresh T-serum with 0.01 DISCUSSION M Mg + EGTA resulted in delayed killing of The data obtained seem to indicate that the the previously promptly serum-sensitive isolates SM 29 (Table 2) and S 86. Again, 0.01 M classical and the alternative pathways of the EDTA neutralized the bactericidal activity of human complement system are activated by T-serum. The results obtained with control promptly serum-sensitive and delayed serumstrain E. coli C were as follows: chelation of T- sensitive strains ofS. marcescens, respectively. Previously, bacterial lipopolysaccharides had serum with 0.01 M Mg + EGTA resulted in delayed killing of the cells (Table 3). However, been shown to activate either of the two pathchelation of T-serum with 0.01 M EDTA ef- ways (3), as reemphasized recently by Snyderfected incomplete neutralization of the bacteri- man and Pike (10). Furthermore, our data corcidal activity; the cells appeared to recover at 2 robrate those of Gallin et al. (6), who had differto 4 h after exposure, only to be killed upon entiated the generation of chemotactic serum prolonged incubation, in that at 22 h after expo- factors via activation of the classical or the alternative pathway of the complement system sure no survivors of E. coli C were demonstrable in EDTA-chelated serum. As previously by means of kinetic analysis; generation of noted (13), heat-inactivated T-serum exerted a chemotactic factors by the alternative pathway proceeded only after a latent period of 15 to 20 bacteriostatic effect against E. coli C. Chelation of fresh T-serum with 0.01 M min, whereas classically generated products EGTA (without Mg ions) completely neutral- were detectable within 5 min. Over 80% of all ized the bactericidal activity of the serum clinical isolates of S. marcescens examined
or
TABLE 1. Bactericidal activity of chelated human serum against delayed serum-sensitive S. marcescens isolate S 326 n.t. S. marcescens S 326 n.t.
Time that tube samples were assayed
(h) 0 1 2 4 6 22 a
b
Cb
C + Mg/EGTA'
1.3 x 104 2.9 x 10:'
1.3 x 104
1.5 x 10: 1.0 x 10' 0 0
1.3 x 104 7.1 x 103 0 0 0
CFU, Colony-forming units. 80 (vol/vol) fresh T-serum. 80 (vol/vol) fresh T-serum + 0.01 M EGTA + 0.01 M MgCl2. 80 (vol/vol) fresh T-serum + 0.01 M EDTA. 80 (vol/vol) heat-inactivated (56 C, 30 min) T-serum.
(CFU/ml)Y EDTA" 1.3 x 104
C +
1.3 x 104 1.5 x 104 1.6 x 105 3.0 x 106
>108
56 C"
1.3 x 104 1.3 x 104 2.1 x 104 4.0 x 10' 3.4 x 107 >108
VOL. 13, 1976
ACTIVATION OF S. MARCESCENS COMPLEMENT PATHWAYS
1345
TABLE 2. Bactericidal activity of chelated human serum against promptly serum-sensitive S. marcescens isolate SM 29a Time that tube samples were assayed (h)
C
0 0.3 1 2 4
1.8 x 104 0 0 0 0
22
S. marcescens SM 29 (CFU/ml) C + Mg/EGTA 1.8 x 104 1.3 x 104 4.1 x 102 1.0 x 10
0
0
C + EDTA
1.8 x 1.7 x 1.7 x 1.8 x 4.0x
104 104 104 104 104
56 C 1.8 x 104 1.8 x 104
1.8 x 104 2.0 x 104 1.9X 105
>108
0
>108
See Table 1 for explanatory footnotes. TABLE 3. Bactericidal activity of chelated human serum against control strain E. coli Ca Time that tube samples were assayed (h)
0 0.3 1 2 4 22
E. coli C (CFU/ml)
C
C + Mg/EGTA
1.2 x 104 0 0 0 0 0
1.2 1.2 3.7 1.6
x x x x 0
104 104 103 102
C + EDTA
1.2 3.5 2.0 6.6 2.3
0
x x x x
104 102 102 103 x 104 0
56 C
1.2 x 104 1.4 x 104 2.3 x 104 5.1 x 104 2.8 x 105 3.6x105
See Table 1 for explanatory footnotes.
TABLE 4. Inhibitory effect of EDTA against control E. coli strain C in heat-inactivated human serum and in tryptic soy broth Time that tube samples were assayed (h) 0 1 2 4
E. coli C (CFU/ml)a 56 Cb
1.3 x 2.1 x 7.5 x 2.3 x 6 4.5 x 22 1.6 x aCFU, Colony-forming units.
b c
"
104 104 104 10-5 10'5 106
56 C + EDTA' 1.3 x 104 1.2 x 104 1.4 x 104 5.3 x 104 2.3 x 104
0
TSBd
1.3 x 104 2.0 x 104 6.9 x 104 1.8 x 10'i 2.2 x 107 >108
TSB + 0.01 M EDTA 1.3 x 104
2.1 3.0 1.6 1.0
x x x x 1.7 x
104 104 105 105 103
80% (vol/vol) heat-inactivated T-serum.
80% (vol/vol) heat-inactivated T-serum + 0.01 M EDTA. TSB, Tryptic soy broth (Difco).
thus far proved delayed serum sensitive (un- as yet unknown interactions between a serum published observations); thus, the alternative factor(s), certain cations, and cells of E. coli pathway of the complement system might effect strain C might have been operative in this in opsonization (5) of the majority of S. marces- vitro system. cens strains. In this context, Fearon et al. (2) ACKNOWLEDGMENT recently demonstrated that levels of the complement components Cl, C4, and C2 in patients This study was supported in part by a grant from the with gram-negative sepsis and subsequent Deutsche Forschungsgemeinschaft (Tr 136/1). shock were not significantly different from LITERATURE CITED those in patients with uncomplicated bacteremia.
Clearly, further experiments are required to
elucidate the observation that human serum, upon chelation with either EGTA or EDTA, proved bacteriostatic or even bactericidal for the control strain E. coli C after prolonged incubation, but not for isolates of S. marcescens of either serum sensitivity category. Possibly,
1. Des
Prez, R. M., C. S. Bryan, J. Hawiger, and D. G. Colley. 1975. Function of the classical and alternate pathways of human complement in serum treated with ethylene glycol tetraacetic acid and MgCl2-ethylene glycol tetraacetic acid. Infect. Immun. 11: 1235-1243. 2. Fearon, D. T., S. Ruddy, P. H. Schur, and W. R.
McCabe. 1975. Activation of the properdin pathway of complement in patients with Gram-negative bacteremia. N. Engl. J. Med. 292:937-940.
1346
TRAUB AND KLEBER
3. Fine, D. P. 1974. Activation of the classic and alternate complement pathways by endotoxin. J. Immunol. 112:763-769. 4. Fine, D. P., S. R. Marney, Jr., D. G. Colley, J. S. Sergent, and R. M. Des Prez. 1972. C3 shunt activation in human serum chelated with EGTA. J. Immunol. 109:807-809. 5. Forsgren, A., and P. G. Quie. 1974. Influence of the alternate complement pathway on opsonization of several bacterial species. Infect. Immun. 10:402404. 6. Gallin, J. I., R. A. Clark, and M. M. Frank. 1975. Kinetic analysis of chemotactic factor generation in human serum via activation of the classical and alternate complement pathways. Clin. Immunol. Immunopathol. 3:334-346. 7. Goodkofsky, I., and I. H. Lepow. 1971. Functional relationship of factor B in the properdin system to C3 proactivator of human serum. J. Immunol. 107:12001204.
INFECT. IMMUN. 8. Gotze, O., and H. J. Muller-Eberhard. 1971. The C3 activator system: an alternate pathway of complement activation. J. Exp. Med. 134(Suppl.):90-108. 9. Sandberg, A. C., and A. G. Osler. 1971. Dual pathways of complement interaction with guinea pig immunoglobulins. J. Immunol. 107:1268-1273. 10. Snyderman, R., and M. C. Pike. 1975. Interaction of complex polysaccharides with the complement system: effect of calcium depletion on terminal component consumption. Infect. Immun. 11:273-279. 11. Traub, W. H. 1969. Assay of the antibiotic activity of serum. Appl. Microbiol. 18:51-56. 12. Traub, W. H., G. Acker, and I. Kleber. 1976. Ultrastructural surface alterations of Serratia marcescens after exposure to polymyxin B and/or fresh human serum. Chemotherapy 22:104-113. 13. Traub, W. H., and I. Kleber. 1975. Studies on the additive effect of polymyxin B and the bactericidal activity of human serum against Serratia marcescens. Chemotherapy 21:189-204.
