Vol. 12, No. 4 Printed in U.SA.
INFECTION AND IMMUNITY, Oct. 1975, p. 779-784 Copyright C) 1975 American Society for Microbiology
Nonspecific Resistance to Escherichia coli in Mice MEHDI SHAYEGANI* AND LINDA M. PARSONS Division of Laboratories and Research, New York State Department of Health, Albany, New York 12201
Received for publication 10 June 1975
Nonspecific cell-mediated immunity to a relatively virulent strain of Escherichia coli was studied in mice infected with Staphylococcus aureus and elicited with specific antigens. The infected and elicited mice were protected against an intraperitoneal challenge by E. coli for an observation period of 7 days, whereas normal mice, given the same number of bacteria, died within 18 to 24 h. However, the amount of time elapsing between elicitation and challenge greatly affected the rate of protection. Little or no protection was observed in mice injected with S. aureus but not elicited or in mice injected with staphylococcal antigens but not infected with staphylococci.
Nonspecific cell-mediated immunity has Atlanta, Ga., as O-:K-:H14. (O or K antigens could been shown in mice infected repeatedly with not be determined by any available antisera.) E1165 Staphylococcus aureus and elicited with staphy- could not be serotyped with the 16 antisera comlococcal antigens. Peritoneal cells of such mice monly used in our laboratory, but the CDC identified 0-:K-:H49. not only demonstrated a significant increase in it as Six other strains of E. coli were also tested. Two of intracellular destruction of S. aureus (12) but these strains, also isolated from infants, were seroalso were significantly more resistant to influ- typed in our laboratory as 0127:B8 and 0126:B16. enza virus in vitro (15). Such mice were also Two strains of identical K antigen, 020a,20c: reported to be protected against challenge with K84:H3 (CDC 4918-68) and 020a,20b:K84:H26 (CDC vaccinia virus in vivo (1), and they survived 2292-55), were received from the CDC. Two strains longer than normal mice after challenge with known to be positive (339t5) and negative (Stanley) for enterotoxogenicity by the mouse intestinal loop influenza virus (15). (10) were kindly supplied by Richard A. FinEscherichia coli is an unrelated organism assay Dallas, Tex. with characteristics quite different from those kelstein, The following biochemical tests were performed of the three organisms previously used in this on these eight strains of E. coli: indole, methyl red, system (1, 12, 15). Current knowledge of anti- Voges-Proskauer, Simmons citrate, hydrogen sulbody effects on E. coli infections (6) suggests fide (TSI), urease, motility, gelatin, lysinfe and ornithat investigation should focus on the cell-me- thine decarboxylase, arginine dihydrolase, malodiated immune response. The present investiga- nate, gas from glucose, and the following carbohytion was undertaken to study the nonspecific drates: glucose, lactose, sucrose, maltose, mannitol, resistance of mice infected and elicited with the xylose, dulcitol, salicin, adonitol, inositol, sorbitol, raffinose, and rhamnose. The results staphylococcal system to a strain of E. coli viru- arabinose, were typical of E. coli according to Edwards and lent to mice. Intraperitoneal injection of a fixed (2). concentration of the live E. coli into mice was Ewing Preparation of E. coli for intraperitoneal injecselected as a test for virulence because the sur- tion. For virulence tests the E. coli strains were vival or death of the treated and untreated mice grown in Trypticase soy broth for 18 h at 37 C on a gave an unequivocal measure of the acquired roller. The bacteria were washed twice and adjusted protection. with saline to a concentration of 108 in 0.1 ml. E. coli antisera. Preparation of antisera to live E. coli E1025 was attempted in seven rabbits, using a 109/ml suspension of washed bacteria. A total of five injections was given on alternate days; the first, 0.2 ml, was subcutaneous, and the remaining four, 0.5 ml each, were intravenous. All of the rabbits were noticeably ill after the first or second injection. Five rabbits died after the fourth injection. The two survivors were seriously ill and were bled out the day after the fifth injection. E. coli E1165 antisera were prepared in rabbits in the same manner, but all animals survived with no apparent ill effect.
MATERIALS AND METHODS E. coli strains. Two strains of E. coli, E1025 and
E1165, were isolated from two infants with diarrhea. Originally E1025 gave a weak-positive reaction with anti-020:K84(B) by the slide agglutination test and a weak fluorescent reaction with 020:K84(B)-conjugated serum. After repeated subcultures the strain became nonreactive to available sera. It was later serotyped by the Center for Disease Control (CDC),
779
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SHAYEGANI AND PARSONS
These antisera contained high titers against E1025 and E1165, respectively, and were used to check the identity of the E. coli recovered from the mice. Mice. Male albino NYa:NYLAR mice (inbred in our Division's animal farm), weighing 18 to 22 g, were used throughout this study. Peritoneal recovery of E. coli from mice. A live E. coli preparation was injected into the mice. At various time intervals the mice were killed by cervical dislocation, and the peritoneal exudate was collected in 1 ml of Hanks solution containing 1% heparin. Intracellular bacteria were recovered by adding 0.5% sodium deoxycholate, which disrupts the leukocytes (3) but has no effect on the viability of the bacteria (7). The samples were diluted in saline and plated on Trypticase soy agar plates for bacterial counts. The same method was used in studies of S. aureus-infected and elicited mice. Phagocytosis of E. coli. The percentage of phagocytosis was determined by the following two methods. (i) In vivo phagocytosis. Normal mouse peritoneal cells were collected 30 min after intraperitoneal injection of the bacteria. The suspension was placed in a Leighton tube containing a cover glass, and the tube was incubated for 30 min to allow the cells to adhere to the cover glass, which was then removed, washed, and stained with Giemsa. The stained preparation was examined microscopically, and 200 to 300 peritoneal cells were counted to determine the percentage of leukocytes containing the bacteria. (ii) In vitro phagocytosis. Normal mouse peritoneal cells were harvested and washed. After 10% homologous pooled serum was added, the suspension was placed in a Leighton tube containing a cover glass. The cells were allowed to adhere to the cover glass for 30 min, and washed bacteria were then added at a ratio of 10 bacteria per peritoneal cell. After 1 h of incubation the cover glass was removed, washed, stained, and examined as above. Induction and elicitation of mice. The definition of induction and elicitation of mice and the method using S. aureus and staphylococcal antigens have been given previously (15). In the present experiment mice were infected by subcutaneous injections of 108 viable S. aureus 18Z (5) weekly for 8 weeks. One week after the last injection the hypersensitized mice were elicited by two subcutaneous 0. 1-ml doses of staphylococcal bacteriophage lysate (Staphage Lysate; Delmont Laboratories) at 48 and 24 h before challenge with E. coli. Delayed-type hypersensitivity of mice inoculated eight times with S. aureus was tested before elicitation with Staphage Lysate in some of each group of mice. Staphylococcal antigens were injected into the right hind footpad, and after 24 and 48 h the thickness was compared with that of the left hind footpad, which had been injected with saline. Normal mice were also tested this way. E. coli in serum suspensions. Mouse serum in a final concentration of 10% was added to a suspension of 106 washed E1025 E. coli per ml in Hanks solution. Several pools were prepared of normal mouse sera and of sera from mice infected with S. aureus and bled 1 day after the second elicitation. Some
INFECT. IMMUN.
pools of each group were used fresh (within 2 h after bleeding), and some were used after having been frozen for several months. An E. coli suspension in Hanks solution was used as a control. These suspensions were incubated at 37 C on a roller, and samples were removed at various time intervals, diluted and plated on Trypticase soy agar plates for bacterial counts.
RESULTS Mouse virulence of E. coli strains by intraperitoneal injection. To determine whether the stimulated immune mechanisms of mice infected and elicited with the staphylococcal system could protect them nonspecifically against E. coli, a relatively virulent strain of this bacteria was needed. As an indicator of virulence in the mouse we used the mean lethal dose (LD50) calculated according to Reed and Muench (11). Eight isolates of E. coli were screened to select suitable strains for such a study. The results (LDso x 108) were: E1025, 0.5; CDC 4918-68, 0.59; 0126:B16, 4.4; 339t5, 5.6; E1165, 5.9; 0127:B8, 6.3; CDC 2292-55, 7.3; and Stanley, 7.7. E1025 was chosen as the virulent strain because it has the lowest LD50 and an intraperitoneal injection of 108 washed, live bacteria killed mice within 18 to 24 h. A relatively avirulent strain, E1165, was used for comparison, since the LD50 values of these strains differ by a factor of 10. Mice injected intraperitoneally with 108 E1165 survived the 7-day observation period with no apparent ill effect. For the purpose of this study, E1025 is considered virulent and E1165 avirulent. E1025 retained its virulence in mice after repeated subcultures, even though it lost its original ability to react with 020:K84(B) antiserum. Both strains could be recovered from the heart, blood, lung, liver, spleen, kidney, and feces of mice 30 min after intraperitoneal injection. The strain recovered was verified by slide agglutination tests using specific antisera. The mouse virulence of the two E. coli strains from CDC, which had an identical K antigen, was also tested by intraperitoneal injection of 108 organisms. CDC 4918-68 was virulent, killing 80% of the mice within 24 h. CDC 2292-55 was avirulent; all of the mice injected survived for 7 days. Even when passed serially through 20 mice, this strain did not become virulent. Recovery of E. coli from the peritoneal cavity. E. coli strains E1025 and E1165 were injected intraperitoneally into several groups of normal mice in order to determine the survival of these strains after exposure to peritoneal cells in vivo. Figure 1 shows the average number of E. coli recovered from the peritoneal cavities of mice. The difference is striking. The
1098 1 025 (/65
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103
1/2
2
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NONSPECIFIC RESISTANCE TO E. COLI
VOL. 12, 1975
6
,______,__,___,__,__,_ 48 72 96
24
HOURIS AFTER INJECTION
FIG. 1. Recovery c)f viable E. coli from peritoneal exudate of mice at vatrious time intervals after intraperitoneal injection o)fl1O8 of either strain E1025 (virulent) or strain E1165; (avirulent). Each point represents the average rec overy from six mice.
number of E1025 recovered increRsed during the first 6 h, and tk re mice died within 18 to 24 h. The number of aviirulent E1165 decreased steadily until, after 4 daLys, only a few hundred bacteria per ml were rebcovered. Phanoevtic acttivitv of mouse npritnnpil cells. The percentage of mouse peritoneal cells phagocytizing E. coli in vivo after intraperitoneal injection was 81% for E1025 and 85% for E1165 (average of four experiments). In the in vitro experiment 62% of the cells phagocytized E1025 and 71% phagocytized E1165 (average of four experiments). Thus both strains were phagocytized equally well by the mouse peritoneal cells. Nonspecific cell-mediated resistance to E. coli. To test the possibility that nonspecific cellmediated immunity might play a role in host defense against a virulent strain of E. coli, as has been demonstrated for other microorganisms (1, 12, 15), the following experiments were undertaken. The prime objective of each was to increase the activity of cell-mediated immunity by infection and elicitation of mice with S. aureus and its antigens. Of 100 mice, half were kept as normal controls without treatment, and the rest were infected as described in Materials and Methods. In addition, a small number of the infected mice were tested for delayed-type hypersensitiv-
ity to tnis organism by footpad injections of staphylococcal antigens. A distinct increase in the thickness of these footpads, as compared with the saline controls, was observed after 24 and 48 h in the infected mice. No reaction was observed in comparable tests with normal mice. Thirty mice were elicited 1 week after infection, as described in Materials and Methods, and 30 normal mice were injected intraperitoneally with 108, 5 x 107, and 107 live E. coli E1025 (10 mice in each group) and were observed for 7 days. To observe the effect of delayed elicitation, the 20 remaining S. aureusinfected mice were elicited 2 weeks after the last S. aureus injection and then challenged. All of the mice infected and elicited survived the challenge with 101 virulent E. coli for up to 4 days, and 90% survived for the full 7 days. All of the control mice died within 17 h. When challenged with 5 x 107 E. coli, all of the infected animals survived, compared with 10% of the controls (Table 1, experiment 1). A similar but less striking difference
the animals
were
was
observed when
elicited and challenged 2
weeks after the last injection of S.
aureus
(Ta-
ble 1, experiment 2). The role played by each and elicitation
procedure
in
step
of the infection
protecting the
mice
from the virulent strain of E. coli was investigated in detail. Before challenge with E. coli,
one group of mice received only staphylococcal
antigens and another group was infected with S. aureus but not elicited. Still other groups were challenged with E1025 at different times
TABLE 1. Protection ofmice against a virulent strain of E. coli by repeated infection with live S. aureus and elicitation with staphylococcal antigensa Viable
Treated mice
Normal mice
E coli
E1025
injected!
Time
mouse
Expt 1 108
Dead/ total
5 x 107
7 days 7 days
0/10 1/10 1/10 0/10
107
7 days
0/10
17 h 24 h 7 days 7 days
3/10 4/10 4/10 0/10
Expt 2 108 5 x 107
4 days 5 days
Time
17 h 17 h 7 days 7 days 17 h 24 h 17 h 7 days
Dead/ total
10/10 9/10 9/10 0/10 9/10 10/10 5/8 5/8
a Elicited 1 week (experiment 1) or 2 weeks (experiment 2) after the final injection of S. aureus.
782
INFECT. IMMUN.
SHAYEGANI AND PARSONS
after the second elicitation. In each experiment an equal number of mice served as normal controls. Table 2 shows the results of several of the experiments. The highest protection (5.8% mortality) was observed in mice challenged with 108 E1025 strain 1 day after the second elicitation. This protection decreased when the mice were challenged 2 days after the second elicitation. When the challenge was deferred until 7 days after the second elicitation, the percentage of mortality became equivalent to that of the mice infected without elicitation. Injection of staphylococcal antigens without prior infection with S. aureus did not protect the mice. These mice were as susceptible as the normal control mice (about 85% mortality). Recovery of virulent E. coli from peritoneal cavity of infected and elicited mice. One group of mice was infected with S. aureus and elicited with staphylococcal antigens and was challenged with E1025 1 day after the second elicitation. A second group was infected with S. aureus repeatedly but not elicited and was challenged 1 week after the last injection. A third group was injected with staphylococcal antigens without prior infection with S. aureus and was challenged 1 day after the second antigen injection. A fourth group was normal controls. Figure 2 displays the results ofseveral experiments using 6 to 12 mice per group for each time interval. The infected and elicited mice were able to eliminate most of the virulent E. coli from the peritoneal cavity. The other groups were susceptible to challenge, as were the normal control mice. Effect of serum from normal mice and from infected and elicited mice on viability TABLE 2. Effect of infection and/or elicitation with S. aureus and timing of challenge on protection of mice against a virulent strain of E. coli (E1025) Treatment of mice before challenge with 108 E1025 and timing of chal- Dead/total lenge
Morl-
200/265 Normal (no treatment)a 21/24 1 day after 2nd injection with antigens; no prior infection 1 week after last infection with 37/58 S. aureus; no elicitation 4/69 1 day after complete infection and elicitation 17/50 2 days after complete infection and elicitation 35/57 7 days after complete infection and elicitation
83.0 87.5
a
Total from all experiments.
ity
m
63.8
5.8 34.0 61.4
w
atr
0 W crLJ
co
IE
1/2
2
6
i
24
HOURS AFTER INJECTION
FIG. 2. Recovery of viable bacteria from peritoneal exudate of treated mice at various time intervals after intraperitoneal injection of 108 of E. coli E1025. Each point represents the average recovery from 6 to 12 mice. Symbols: 0, Infected with S. aureus and elicited with staphylococcal antigens; A, infected but not elicited; A, injected with staphylococcal antigens only; 0, normal control.
of the virulent E. coli. The possibility exists that antibodies in mice infected and elicited with staphylococci may play a role in protecting the mice against the unrelated E. coli. The pooled sera from such immunized mice were therefore tested against the virulent E. coli. The sera were from mice bled 1 day after the second elicitation, when the mice were most protected against intraperitoneal injection of virulent E. coli. Fresh and previously frozen sera from infected and elicited mice and from normal mice behaved similarly (Fig. 3). The E1025 grew in all sera but merely survived without multiplication when suspended in Hanks solution alone. DISCUSSION To study the virulence of E. coli for mice by the intraperitoneal route, Jacks and Glantz (4) tested 61 E. coli strains isolated from humans, animals, poultry, and fish (enteric, systemic, and nonenteric-nonsystemic sources). They reported LD50 values ranging from 3 x 108 to 5 x 101" (without the use of mucin adjuvant). We tested eight strains of E. coli by using the intraperitoneal route without adjuvant and selected E1025 for use because of its comparatively high virulence for mice (LD50 = 5 x 107). This high virulence in normal mice makes it a good indica-
NONSPECIFIC RESISTANCE TO E. COLJ
VOL. 12, 1975
tor of the effect of nonspecific cell-mediated immunity in modifying the response of mice to E. coli.
E1025 was compared with a relatively avirulent strain (E1165), also administered intraperitoneally. Both strains were phagocytized equally well by peritoneal cells, but the virulent strain multiplied in the peritoneal cavity, whereas the avirulent strain was rapidly eliminated. It has been reported that local or circulating antibodies do not significantly protect the host from colonization of the intestine with certain strains of E. coli (6). The possibility of augmenting cell-mediated resistance was therefore considered as a means of protection against this virulent organism. The critical step in cell-mediated immunity involves activated macrophages. Specific induction and elicitation of laboratory animals by certain microorganisms and their antigens is known (8) to stimulate specifically sensitized thymus-modulated lymphocytes to lymphoblasts, with the release of lymphokines. The lymphokines stimulate the macrophage and in-
--a
MINUTES
FIG. 3. Viability of E. coli E1025 in a suspension containing 10% of either fresh or previously frozen, pooled normal mouse sera or sera from mice infected with S. aureus and elicited with staphylococcal antigens, as compared with a suspension without serum. Each point represents the average of two experiments for the fresh sera and five for the frozen sera. Symbols: *, Infected and elicited, fresh; 0, infected and elicited, frozen; A, normal, fresh; A, normal, frozen; 0, Hanks solution only.
783
crease its capacity to destroy intracellular pathogens of both related and nonrelated microorganisms. We reasoned that by inducing a group of mice with S. aureus and eliciting with staphylococcal antigens, we could stimulate the cellular immune system sufficiently so that the activated macrophages would nonspecifically protect these mice against the virulent E1025. The same phenomenon has been described elsewhere for other microorganisms (12, 15). S. aureus and its antigens were chosen because of the possibility of making these studies applicable to the human system. A large percentage of the human population already exhibit delayed-type hypersensitivity to staphylococcal antigens because of repeated natural exposure to the organism (9). The ability of staphylococcal bacteriophage lysate to evoke lymphoproliferative and leukocyte migration inhibition responses in human leukocytes has recently been reported (J. S. Silva, J. H. Dean, J. L. McCoy, and R. B. Herberman, Abstr. Annu. Meet. Am. Soc. Microbiol. 1975, RT30, p. 280). Lymphocytes from peripheral blood and B and T lymphocytes separated from peripheral blood exhibit lymphocyte transformation and leukocyte migration inhibition responses when cultured with this antigen. In the present study the infected and elicited mice were protected against intraperitoneal challenge of virulent E. coli. Infection without elicitation gave slight protection. Administration of the antigens alone seemed to have no effect on host resistance. Since S. aureus do not multiply within the cells (13, 14) and are therefore eventually eliminated, the presence of homologous antigens is needed for stimulation of cellular resistance. This finding confirms a previous report (12) describing the need for elicitation in which cell-mediated immunity was tested at the cellular level, using peritoneal macrophages from infected and elicited animals in order to study the intracellular destruction of S. aureus. Nonspecific cell-mediated immunity has been demonstrated in peritoneal cells harvested from infected and elicited mice and shown to be resistant to influenza virus. In vivo the mice were shown to be more resistant than normal mice to influenza virus (15) and vaccinia virus (1). In the present investigation an even more dramatic increase in host resistance to the pathogenic organism was observed when mice were similarly treated. ACKNOWLEDGMENTS The technical assistance of Robert J. Burke and Russell C. Yewdall in part of this study is gratefully acknowledged.
784
SHAYEGANI AND PARSONS LITERATURE CITED
1.
2. 3.
4.
5.
6.
7.
Allen, E. G., and S. Mudd. 1973. Protection of mice against vaccinia virus by bacterial infection and sustained stimulation with specific bacterial antigens. Infect. Immun. 7:62-67. Edwards, P. R., and W. H. Ewing. 1972. Identification of Enterobacteriaceae, 3rd ed. Burgess Publishing Co., Minneapolis. Hsu, H. S., and A. S. Radcliffe. 1968. Interactions between macrophages of guinea pigs and salmonellae. I. Fate of Salmonella typhimurium within macrophages of normal guinea pigs. J. Bacteriol. 96:191197. Jacks, T. M., and P. J. Glantz. 1967. Virulence ofEscherichia coli serotypes for mice. J. Bacteriol. 93:991995. Kapral, F. A., and M. G. Shayegani, 1959. Intracellular survival of staphylococci. J. Exp. Med. 110:123-138. Kenny, J. F., D. W. Weinert, and J. A. Gray. 1974. Enteric infection with Escherichia coli 0127 in the mouse. II. Failure of specific immunity to alter intestinal colonization of infants and adults. J. Infect. Dis. 129:10-20. Leifson, E. 1935. New culture media based on sodium desoxycholate for the isolation of intestinal pathogens and for the enumeration of colon bacilli in milk
INFECT. IMMUN. and water. J. Pathol. Bacteriol. 40:581-599. 8. Mackaness, G. B. 1964. The immunological basis of acquired cellular resistance. J. Exp. Med. 120:105120. 9. Mudd, S., J. H. Taubler, and A. G. Baker. 1970. Delayed-type hypersensitivity to Staphylococcus aureus in human subjects. J. Reticuloendothel. Soc. 8:493498. 10. Punyashthiti, K., and R. A. Finkelstein. 1971. Enteropathogenicity of Escherichia coli. I. Evaluation of mouse intestinal loops. Infect. Immun. 4:473-478. 11. Reed, L. J., and H. Muench. 1938. A simple method for estimating fifty percent end points. Am. J. Hyg. 27:493-497. 12. Shayegani, M., S. K. DeCourcy, Jr., and S. Mudd. 1973. Cell-mediated immunity in mice infected with S. aureus and elicited with specific bacterial antigens. J. Reticuloendothel. Soc. 14:44-51. 13. Shayegani, M., and F. A. Kapral. 1962. The immediate fate of staphylococci after phagocytosis. J. Gen. Microbiol. 29:625-636. 14. Shayegani, M., and F. A. Kapral. 1962. The eventual intracellular destruction of staphylococci by mononuclear cells. J. Gen. Microbiol. 29:637-644. 15. Shayegani, M., F. S. Lief, and S. Mudd. 1974. Specific and nonspecific cell-mediated resistance to influenza virus in mice. Infect. Immun. 9:991-998.
INFECTION AND IMMUNITY, Oct. 1975, p. 779-784 Copyright C) 1975 American Society for Microbiology
Nonspecific Resistance to Escherichia coli in Mice MEHDI SHAYEGANI* AND LINDA M. PARSONS Division of Laboratories and Research, New York State Department of Health, Albany, New York 12201
Received for publication 10 June 1975
Nonspecific cell-mediated immunity to a relatively virulent strain of Escherichia coli was studied in mice infected with Staphylococcus aureus and elicited with specific antigens. The infected and elicited mice were protected against an intraperitoneal challenge by E. coli for an observation period of 7 days, whereas normal mice, given the same number of bacteria, died within 18 to 24 h. However, the amount of time elapsing between elicitation and challenge greatly affected the rate of protection. Little or no protection was observed in mice injected with S. aureus but not elicited or in mice injected with staphylococcal antigens but not infected with staphylococci.
Nonspecific cell-mediated immunity has Atlanta, Ga., as O-:K-:H14. (O or K antigens could been shown in mice infected repeatedly with not be determined by any available antisera.) E1165 Staphylococcus aureus and elicited with staphy- could not be serotyped with the 16 antisera comlococcal antigens. Peritoneal cells of such mice monly used in our laboratory, but the CDC identified 0-:K-:H49. not only demonstrated a significant increase in it as Six other strains of E. coli were also tested. Two of intracellular destruction of S. aureus (12) but these strains, also isolated from infants, were seroalso were significantly more resistant to influ- typed in our laboratory as 0127:B8 and 0126:B16. enza virus in vitro (15). Such mice were also Two strains of identical K antigen, 020a,20c: reported to be protected against challenge with K84:H3 (CDC 4918-68) and 020a,20b:K84:H26 (CDC vaccinia virus in vivo (1), and they survived 2292-55), were received from the CDC. Two strains longer than normal mice after challenge with known to be positive (339t5) and negative (Stanley) for enterotoxogenicity by the mouse intestinal loop influenza virus (15). (10) were kindly supplied by Richard A. FinEscherichia coli is an unrelated organism assay Dallas, Tex. with characteristics quite different from those kelstein, The following biochemical tests were performed of the three organisms previously used in this on these eight strains of E. coli: indole, methyl red, system (1, 12, 15). Current knowledge of anti- Voges-Proskauer, Simmons citrate, hydrogen sulbody effects on E. coli infections (6) suggests fide (TSI), urease, motility, gelatin, lysinfe and ornithat investigation should focus on the cell-me- thine decarboxylase, arginine dihydrolase, malodiated immune response. The present investiga- nate, gas from glucose, and the following carbohytion was undertaken to study the nonspecific drates: glucose, lactose, sucrose, maltose, mannitol, resistance of mice infected and elicited with the xylose, dulcitol, salicin, adonitol, inositol, sorbitol, raffinose, and rhamnose. The results staphylococcal system to a strain of E. coli viru- arabinose, were typical of E. coli according to Edwards and lent to mice. Intraperitoneal injection of a fixed (2). concentration of the live E. coli into mice was Ewing Preparation of E. coli for intraperitoneal injecselected as a test for virulence because the sur- tion. For virulence tests the E. coli strains were vival or death of the treated and untreated mice grown in Trypticase soy broth for 18 h at 37 C on a gave an unequivocal measure of the acquired roller. The bacteria were washed twice and adjusted protection. with saline to a concentration of 108 in 0.1 ml. E. coli antisera. Preparation of antisera to live E. coli E1025 was attempted in seven rabbits, using a 109/ml suspension of washed bacteria. A total of five injections was given on alternate days; the first, 0.2 ml, was subcutaneous, and the remaining four, 0.5 ml each, were intravenous. All of the rabbits were noticeably ill after the first or second injection. Five rabbits died after the fourth injection. The two survivors were seriously ill and were bled out the day after the fifth injection. E. coli E1165 antisera were prepared in rabbits in the same manner, but all animals survived with no apparent ill effect.
MATERIALS AND METHODS E. coli strains. Two strains of E. coli, E1025 and
E1165, were isolated from two infants with diarrhea. Originally E1025 gave a weak-positive reaction with anti-020:K84(B) by the slide agglutination test and a weak fluorescent reaction with 020:K84(B)-conjugated serum. After repeated subcultures the strain became nonreactive to available sera. It was later serotyped by the Center for Disease Control (CDC),
779
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SHAYEGANI AND PARSONS
These antisera contained high titers against E1025 and E1165, respectively, and were used to check the identity of the E. coli recovered from the mice. Mice. Male albino NYa:NYLAR mice (inbred in our Division's animal farm), weighing 18 to 22 g, were used throughout this study. Peritoneal recovery of E. coli from mice. A live E. coli preparation was injected into the mice. At various time intervals the mice were killed by cervical dislocation, and the peritoneal exudate was collected in 1 ml of Hanks solution containing 1% heparin. Intracellular bacteria were recovered by adding 0.5% sodium deoxycholate, which disrupts the leukocytes (3) but has no effect on the viability of the bacteria (7). The samples were diluted in saline and plated on Trypticase soy agar plates for bacterial counts. The same method was used in studies of S. aureus-infected and elicited mice. Phagocytosis of E. coli. The percentage of phagocytosis was determined by the following two methods. (i) In vivo phagocytosis. Normal mouse peritoneal cells were collected 30 min after intraperitoneal injection of the bacteria. The suspension was placed in a Leighton tube containing a cover glass, and the tube was incubated for 30 min to allow the cells to adhere to the cover glass, which was then removed, washed, and stained with Giemsa. The stained preparation was examined microscopically, and 200 to 300 peritoneal cells were counted to determine the percentage of leukocytes containing the bacteria. (ii) In vitro phagocytosis. Normal mouse peritoneal cells were harvested and washed. After 10% homologous pooled serum was added, the suspension was placed in a Leighton tube containing a cover glass. The cells were allowed to adhere to the cover glass for 30 min, and washed bacteria were then added at a ratio of 10 bacteria per peritoneal cell. After 1 h of incubation the cover glass was removed, washed, stained, and examined as above. Induction and elicitation of mice. The definition of induction and elicitation of mice and the method using S. aureus and staphylococcal antigens have been given previously (15). In the present experiment mice were infected by subcutaneous injections of 108 viable S. aureus 18Z (5) weekly for 8 weeks. One week after the last injection the hypersensitized mice were elicited by two subcutaneous 0. 1-ml doses of staphylococcal bacteriophage lysate (Staphage Lysate; Delmont Laboratories) at 48 and 24 h before challenge with E. coli. Delayed-type hypersensitivity of mice inoculated eight times with S. aureus was tested before elicitation with Staphage Lysate in some of each group of mice. Staphylococcal antigens were injected into the right hind footpad, and after 24 and 48 h the thickness was compared with that of the left hind footpad, which had been injected with saline. Normal mice were also tested this way. E. coli in serum suspensions. Mouse serum in a final concentration of 10% was added to a suspension of 106 washed E1025 E. coli per ml in Hanks solution. Several pools were prepared of normal mouse sera and of sera from mice infected with S. aureus and bled 1 day after the second elicitation. Some
INFECT. IMMUN.
pools of each group were used fresh (within 2 h after bleeding), and some were used after having been frozen for several months. An E. coli suspension in Hanks solution was used as a control. These suspensions were incubated at 37 C on a roller, and samples were removed at various time intervals, diluted and plated on Trypticase soy agar plates for bacterial counts.
RESULTS Mouse virulence of E. coli strains by intraperitoneal injection. To determine whether the stimulated immune mechanisms of mice infected and elicited with the staphylococcal system could protect them nonspecifically against E. coli, a relatively virulent strain of this bacteria was needed. As an indicator of virulence in the mouse we used the mean lethal dose (LD50) calculated according to Reed and Muench (11). Eight isolates of E. coli were screened to select suitable strains for such a study. The results (LDso x 108) were: E1025, 0.5; CDC 4918-68, 0.59; 0126:B16, 4.4; 339t5, 5.6; E1165, 5.9; 0127:B8, 6.3; CDC 2292-55, 7.3; and Stanley, 7.7. E1025 was chosen as the virulent strain because it has the lowest LD50 and an intraperitoneal injection of 108 washed, live bacteria killed mice within 18 to 24 h. A relatively avirulent strain, E1165, was used for comparison, since the LD50 values of these strains differ by a factor of 10. Mice injected intraperitoneally with 108 E1165 survived the 7-day observation period with no apparent ill effect. For the purpose of this study, E1025 is considered virulent and E1165 avirulent. E1025 retained its virulence in mice after repeated subcultures, even though it lost its original ability to react with 020:K84(B) antiserum. Both strains could be recovered from the heart, blood, lung, liver, spleen, kidney, and feces of mice 30 min after intraperitoneal injection. The strain recovered was verified by slide agglutination tests using specific antisera. The mouse virulence of the two E. coli strains from CDC, which had an identical K antigen, was also tested by intraperitoneal injection of 108 organisms. CDC 4918-68 was virulent, killing 80% of the mice within 24 h. CDC 2292-55 was avirulent; all of the mice injected survived for 7 days. Even when passed serially through 20 mice, this strain did not become virulent. Recovery of E. coli from the peritoneal cavity. E. coli strains E1025 and E1165 were injected intraperitoneally into several groups of normal mice in order to determine the survival of these strains after exposure to peritoneal cells in vivo. Figure 1 shows the average number of E. coli recovered from the peritoneal cavities of mice. The difference is striking. The
1098 1 025 (/65
E
-~
0
cr u
i
0
103
1/2
2
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NONSPECIFIC RESISTANCE TO E. COLI
VOL. 12, 1975
6
,______,__,___,__,__,_ 48 72 96
24
HOURIS AFTER INJECTION
FIG. 1. Recovery c)f viable E. coli from peritoneal exudate of mice at vatrious time intervals after intraperitoneal injection o)fl1O8 of either strain E1025 (virulent) or strain E1165; (avirulent). Each point represents the average rec overy from six mice.
number of E1025 recovered increRsed during the first 6 h, and tk re mice died within 18 to 24 h. The number of aviirulent E1165 decreased steadily until, after 4 daLys, only a few hundred bacteria per ml were rebcovered. Phanoevtic acttivitv of mouse npritnnpil cells. The percentage of mouse peritoneal cells phagocytizing E. coli in vivo after intraperitoneal injection was 81% for E1025 and 85% for E1165 (average of four experiments). In the in vitro experiment 62% of the cells phagocytized E1025 and 71% phagocytized E1165 (average of four experiments). Thus both strains were phagocytized equally well by the mouse peritoneal cells. Nonspecific cell-mediated resistance to E. coli. To test the possibility that nonspecific cellmediated immunity might play a role in host defense against a virulent strain of E. coli, as has been demonstrated for other microorganisms (1, 12, 15), the following experiments were undertaken. The prime objective of each was to increase the activity of cell-mediated immunity by infection and elicitation of mice with S. aureus and its antigens. Of 100 mice, half were kept as normal controls without treatment, and the rest were infected as described in Materials and Methods. In addition, a small number of the infected mice were tested for delayed-type hypersensitiv-
ity to tnis organism by footpad injections of staphylococcal antigens. A distinct increase in the thickness of these footpads, as compared with the saline controls, was observed after 24 and 48 h in the infected mice. No reaction was observed in comparable tests with normal mice. Thirty mice were elicited 1 week after infection, as described in Materials and Methods, and 30 normal mice were injected intraperitoneally with 108, 5 x 107, and 107 live E. coli E1025 (10 mice in each group) and were observed for 7 days. To observe the effect of delayed elicitation, the 20 remaining S. aureusinfected mice were elicited 2 weeks after the last S. aureus injection and then challenged. All of the mice infected and elicited survived the challenge with 101 virulent E. coli for up to 4 days, and 90% survived for the full 7 days. All of the control mice died within 17 h. When challenged with 5 x 107 E. coli, all of the infected animals survived, compared with 10% of the controls (Table 1, experiment 1). A similar but less striking difference
the animals
were
was
observed when
elicited and challenged 2
weeks after the last injection of S.
aureus
(Ta-
ble 1, experiment 2). The role played by each and elicitation
procedure
in
step
of the infection
protecting the
mice
from the virulent strain of E. coli was investigated in detail. Before challenge with E. coli,
one group of mice received only staphylococcal
antigens and another group was infected with S. aureus but not elicited. Still other groups were challenged with E1025 at different times
TABLE 1. Protection ofmice against a virulent strain of E. coli by repeated infection with live S. aureus and elicitation with staphylococcal antigensa Viable
Treated mice
Normal mice
E coli
E1025
injected!
Time
mouse
Expt 1 108
Dead/ total
5 x 107
7 days 7 days
0/10 1/10 1/10 0/10
107
7 days
0/10
17 h 24 h 7 days 7 days
3/10 4/10 4/10 0/10
Expt 2 108 5 x 107
4 days 5 days
Time
17 h 17 h 7 days 7 days 17 h 24 h 17 h 7 days
Dead/ total
10/10 9/10 9/10 0/10 9/10 10/10 5/8 5/8
a Elicited 1 week (experiment 1) or 2 weeks (experiment 2) after the final injection of S. aureus.
782
INFECT. IMMUN.
SHAYEGANI AND PARSONS
after the second elicitation. In each experiment an equal number of mice served as normal controls. Table 2 shows the results of several of the experiments. The highest protection (5.8% mortality) was observed in mice challenged with 108 E1025 strain 1 day after the second elicitation. This protection decreased when the mice were challenged 2 days after the second elicitation. When the challenge was deferred until 7 days after the second elicitation, the percentage of mortality became equivalent to that of the mice infected without elicitation. Injection of staphylococcal antigens without prior infection with S. aureus did not protect the mice. These mice were as susceptible as the normal control mice (about 85% mortality). Recovery of virulent E. coli from peritoneal cavity of infected and elicited mice. One group of mice was infected with S. aureus and elicited with staphylococcal antigens and was challenged with E1025 1 day after the second elicitation. A second group was infected with S. aureus repeatedly but not elicited and was challenged 1 week after the last injection. A third group was injected with staphylococcal antigens without prior infection with S. aureus and was challenged 1 day after the second antigen injection. A fourth group was normal controls. Figure 2 displays the results ofseveral experiments using 6 to 12 mice per group for each time interval. The infected and elicited mice were able to eliminate most of the virulent E. coli from the peritoneal cavity. The other groups were susceptible to challenge, as were the normal control mice. Effect of serum from normal mice and from infected and elicited mice on viability TABLE 2. Effect of infection and/or elicitation with S. aureus and timing of challenge on protection of mice against a virulent strain of E. coli (E1025) Treatment of mice before challenge with 108 E1025 and timing of chal- Dead/total lenge
Morl-
200/265 Normal (no treatment)a 21/24 1 day after 2nd injection with antigens; no prior infection 1 week after last infection with 37/58 S. aureus; no elicitation 4/69 1 day after complete infection and elicitation 17/50 2 days after complete infection and elicitation 35/57 7 days after complete infection and elicitation
83.0 87.5
a
Total from all experiments.
ity
m
63.8
5.8 34.0 61.4
w
atr
0 W crLJ
co
IE
1/2
2
6
i
24
HOURS AFTER INJECTION
FIG. 2. Recovery of viable bacteria from peritoneal exudate of treated mice at various time intervals after intraperitoneal injection of 108 of E. coli E1025. Each point represents the average recovery from 6 to 12 mice. Symbols: 0, Infected with S. aureus and elicited with staphylococcal antigens; A, infected but not elicited; A, injected with staphylococcal antigens only; 0, normal control.
of the virulent E. coli. The possibility exists that antibodies in mice infected and elicited with staphylococci may play a role in protecting the mice against the unrelated E. coli. The pooled sera from such immunized mice were therefore tested against the virulent E. coli. The sera were from mice bled 1 day after the second elicitation, when the mice were most protected against intraperitoneal injection of virulent E. coli. Fresh and previously frozen sera from infected and elicited mice and from normal mice behaved similarly (Fig. 3). The E1025 grew in all sera but merely survived without multiplication when suspended in Hanks solution alone. DISCUSSION To study the virulence of E. coli for mice by the intraperitoneal route, Jacks and Glantz (4) tested 61 E. coli strains isolated from humans, animals, poultry, and fish (enteric, systemic, and nonenteric-nonsystemic sources). They reported LD50 values ranging from 3 x 108 to 5 x 101" (without the use of mucin adjuvant). We tested eight strains of E. coli by using the intraperitoneal route without adjuvant and selected E1025 for use because of its comparatively high virulence for mice (LD50 = 5 x 107). This high virulence in normal mice makes it a good indica-
NONSPECIFIC RESISTANCE TO E. COLJ
VOL. 12, 1975
tor of the effect of nonspecific cell-mediated immunity in modifying the response of mice to E. coli.
E1025 was compared with a relatively avirulent strain (E1165), also administered intraperitoneally. Both strains were phagocytized equally well by peritoneal cells, but the virulent strain multiplied in the peritoneal cavity, whereas the avirulent strain was rapidly eliminated. It has been reported that local or circulating antibodies do not significantly protect the host from colonization of the intestine with certain strains of E. coli (6). The possibility of augmenting cell-mediated resistance was therefore considered as a means of protection against this virulent organism. The critical step in cell-mediated immunity involves activated macrophages. Specific induction and elicitation of laboratory animals by certain microorganisms and their antigens is known (8) to stimulate specifically sensitized thymus-modulated lymphocytes to lymphoblasts, with the release of lymphokines. The lymphokines stimulate the macrophage and in-
--a
MINUTES
FIG. 3. Viability of E. coli E1025 in a suspension containing 10% of either fresh or previously frozen, pooled normal mouse sera or sera from mice infected with S. aureus and elicited with staphylococcal antigens, as compared with a suspension without serum. Each point represents the average of two experiments for the fresh sera and five for the frozen sera. Symbols: *, Infected and elicited, fresh; 0, infected and elicited, frozen; A, normal, fresh; A, normal, frozen; 0, Hanks solution only.
783
crease its capacity to destroy intracellular pathogens of both related and nonrelated microorganisms. We reasoned that by inducing a group of mice with S. aureus and eliciting with staphylococcal antigens, we could stimulate the cellular immune system sufficiently so that the activated macrophages would nonspecifically protect these mice against the virulent E1025. The same phenomenon has been described elsewhere for other microorganisms (12, 15). S. aureus and its antigens were chosen because of the possibility of making these studies applicable to the human system. A large percentage of the human population already exhibit delayed-type hypersensitivity to staphylococcal antigens because of repeated natural exposure to the organism (9). The ability of staphylococcal bacteriophage lysate to evoke lymphoproliferative and leukocyte migration inhibition responses in human leukocytes has recently been reported (J. S. Silva, J. H. Dean, J. L. McCoy, and R. B. Herberman, Abstr. Annu. Meet. Am. Soc. Microbiol. 1975, RT30, p. 280). Lymphocytes from peripheral blood and B and T lymphocytes separated from peripheral blood exhibit lymphocyte transformation and leukocyte migration inhibition responses when cultured with this antigen. In the present study the infected and elicited mice were protected against intraperitoneal challenge of virulent E. coli. Infection without elicitation gave slight protection. Administration of the antigens alone seemed to have no effect on host resistance. Since S. aureus do not multiply within the cells (13, 14) and are therefore eventually eliminated, the presence of homologous antigens is needed for stimulation of cellular resistance. This finding confirms a previous report (12) describing the need for elicitation in which cell-mediated immunity was tested at the cellular level, using peritoneal macrophages from infected and elicited animals in order to study the intracellular destruction of S. aureus. Nonspecific cell-mediated immunity has been demonstrated in peritoneal cells harvested from infected and elicited mice and shown to be resistant to influenza virus. In vivo the mice were shown to be more resistant than normal mice to influenza virus (15) and vaccinia virus (1). In the present investigation an even more dramatic increase in host resistance to the pathogenic organism was observed when mice were similarly treated. ACKNOWLEDGMENTS The technical assistance of Robert J. Burke and Russell C. Yewdall in part of this study is gratefully acknowledged.
784
SHAYEGANI AND PARSONS LITERATURE CITED
1.
2. 3.
4.
5.
6.
7.
Allen, E. G., and S. Mudd. 1973. Protection of mice against vaccinia virus by bacterial infection and sustained stimulation with specific bacterial antigens. Infect. Immun. 7:62-67. Edwards, P. R., and W. H. Ewing. 1972. Identification of Enterobacteriaceae, 3rd ed. Burgess Publishing Co., Minneapolis. Hsu, H. S., and A. S. Radcliffe. 1968. Interactions between macrophages of guinea pigs and salmonellae. I. Fate of Salmonella typhimurium within macrophages of normal guinea pigs. J. Bacteriol. 96:191197. Jacks, T. M., and P. J. Glantz. 1967. Virulence ofEscherichia coli serotypes for mice. J. Bacteriol. 93:991995. Kapral, F. A., and M. G. Shayegani, 1959. Intracellular survival of staphylococci. J. Exp. Med. 110:123-138. Kenny, J. F., D. W. Weinert, and J. A. Gray. 1974. Enteric infection with Escherichia coli 0127 in the mouse. II. Failure of specific immunity to alter intestinal colonization of infants and adults. J. Infect. Dis. 129:10-20. Leifson, E. 1935. New culture media based on sodium desoxycholate for the isolation of intestinal pathogens and for the enumeration of colon bacilli in milk
INFECT. IMMUN. and water. J. Pathol. Bacteriol. 40:581-599. 8. Mackaness, G. B. 1964. The immunological basis of acquired cellular resistance. J. Exp. Med. 120:105120. 9. Mudd, S., J. H. Taubler, and A. G. Baker. 1970. Delayed-type hypersensitivity to Staphylococcus aureus in human subjects. J. Reticuloendothel. Soc. 8:493498. 10. Punyashthiti, K., and R. A. Finkelstein. 1971. Enteropathogenicity of Escherichia coli. I. Evaluation of mouse intestinal loops. Infect. Immun. 4:473-478. 11. Reed, L. J., and H. Muench. 1938. A simple method for estimating fifty percent end points. Am. J. Hyg. 27:493-497. 12. Shayegani, M., S. K. DeCourcy, Jr., and S. Mudd. 1973. Cell-mediated immunity in mice infected with S. aureus and elicited with specific bacterial antigens. J. Reticuloendothel. Soc. 14:44-51. 13. Shayegani, M., and F. A. Kapral. 1962. The immediate fate of staphylococci after phagocytosis. J. Gen. Microbiol. 29:625-636. 14. Shayegani, M., and F. A. Kapral. 1962. The eventual intracellular destruction of staphylococci by mononuclear cells. J. Gen. Microbiol. 29:637-644. 15. Shayegani, M., F. S. Lief, and S. Mudd. 1974. Specific and nonspecific cell-mediated resistance to influenza virus in mice. Infect. Immun. 9:991-998.
