Med Microbiol Immunol (1992) 181:87-98

9 Springer-Verlag 1992

Antibacterial effect of bovine milk antibody against Escherichia coli in a mouse indigenous infection model Koji Nomoto 1, Yoshiaki Matsuoka l, Kazuhito Hayakawa 1, Makoto Ohwaki 1, Tatsuhiko Kan 1, Yasunobu Yoshikai 2, and Kikuo Nomoto 3 1Yakult Central Institute for Microbiological Research, 1796 Yaho, Kunitachi-shi, Tokyo 186, Japan 2Laboratory of Germfree life, Research Institute of Disease Mechanism and Control, Nagoya University School of Medicine, Nagoya 466, Japan 3Department of Immunology, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812, Japan Received November 4, 1991

Abstract. A skim-milk fraction and a whey-protein concentrate (WPC) fraction were prepared from the cows that had been immunized with E. coli isolated from the mouse intestine. The antibacterial effect of these fractions against E. coli was examined. They contained antibody with a high affinity for E. coli strain 48, a representative strain in the mouse intestine, which is composed of a large amount of IgG and smaller amouns of IgA and IgM. Although these fractions showed no bactericidal or bacteriostatic activity against E. coli 48 directly in vitro, they exhibited strong agglutination and opsonization activities against the bacteria in vitro. The bacteria opsonized with the WPC fraction were taken up more effectively by liver macrophages in vivo, compared with unopsonized E. coli, after an intravenous injection into mice. Oral administration of the skim-milk fraction to mice significantly reduced the susceptibility to the lethal toxicity of 5fluorouracil (5 FU). The increase in the population levels ofE. coli in the intestinal tract after administration of 5 FU was inhibited by oral administration of the skimmilk fraction. These results strongly suggest that specific antibody may be effective in the prophylaxis against the indigenous infection with gram-negative bacteria such as E. coli after a period of chemotherapy in cancer patients.

Introduction Despite recent advances in antibiotic chemotherapy, infection is still a lifethreatening problem in cancer patients, because chemotherapy itself as the main treatment for cancer accelerates the immunocompromised state of the patients (Glucksberg et al. 1981; Keating et al. 1981). There have been many reports suggesting that the infections in the immunocompromised hosts often arise from their microflora (Kreger et al. 1980; Tancr~de and Andremont 1985; Wells et al. 1987). Indigenous enteric gram-negative bacteria such as Escherichia coli have been Offprint requests to: Y. Matsuoka

88 shown to be one of the leading cause of the infections among cancer patients with severe neutropenia following chemotherapeutic periods. In recent studies, we have demonstrated that a single administration of 5fluorouracil (5 FU), one of antimetabolic chemotherapeutic agents, to specific pathogen-free (SPF) mice induced a lethal infection with indigenous E. coli. A systemic infection accompanied with a marked increase in the population levels of E. coli in the intestinal tract occurred in all mice that had received 5 FU at large doses (338mg/kg or more) without inoculation of any bacteria as pathogen (Nomoto et al. 1991). Although antibiotic chemotherapy is the main treatment for the opportunistic bacterial infections in the immunocompromised host, antibiotic administration is known to often increase the susceptibility of the host to colonization with antibiotic resistant pathogens (Pizzo 1989; Verhoef et al. 1989; Wells et al. 1984). Therefore, it is important to develop medical treatments other than antibiotics against indigenous infections. One of the desirable ways is to support the immune response of immunocompromised host against the disseminated bacteria by administration of immunoglobulins with specific antibody activity. Hilpert et al. (1977) reported that oral intake of bovine milk immunoglobulins with specific antibody activities to various serotypes of enteropathogenic E. coli was effective against infantile gastroenteritis induced by the infection with the bacteria in clinical studies. In this study, we show the results of an animal study concerning the antiinfectious effect of bovine milk containing antibody specific for endogenous E. coli. This milk was effective not only as agglutinin but also as opsonin for the phagocytosis of E. coli by mouse macrophages in vitro and exerted an inhibitory effect against the lethal toxicity of 5 FU in mice that has been shown to be due to the indigenous intestinal infection with E. coli.

MateriaLs and methods Preparation of milk E. coli 48, a strain which had been isolated from the normal flora of an SPF BALB/c mouse kept in our animal facility was used as immunogen. E. coli 48 was cultured in trypticase soy broth (Becton Dickinson, Cockeysville, Md.) at 37~ for 18 h, harvested and then washed with distilled water. The suspension of the bacteria in distilled water was treated at 100~ for 30 rain. The lyophilized preparation of killed E. coli 48 was used as immunogen. Milk samples containing specific antibody to E. coli were prepared by Stolle Milk Biologics International (Cincinnati, Ohio). Briefly, five cows of Holstein strain were immunized intramuscularly with 2 X 10 9 cells of E. coli 48 once weekly for 4 consecutive weeks. Milk was obtained from the cows at the 7th day after the last immunization, and both skim-milk and wheyprotein concentrate (WPC) fractions were prepared from the pooled milk after pasteurization at 68 oC for 1 min. A skim-milk fraction containing natural antibody obtained from non-immunized cows and commercial milk products (New Zealand Dairy Board, Wellington, NZ) were used as controls.

Determination of antibody titer Total amounts of bovine immunoglobulin G (IgG), IgA and IgM were determined by the radial immunodiffusion kits (The binding site Ltd., Birmingham, UK). Specific antibody titers against

89

E. coli 48 were determined by enzyme-linked immunosorbent assay (ELISA). In brief, immuno plates (Nunc, Roskilde, Denmark) were coated with 100 111 of carbonate buffer (pH 9.6) with killed E. coli 48 (1 X 109 cells/ml) per well, washed, and blocked with 100 p.1 of carbonate buffer containing bovine serum albumin (BSA, 1 mg/ml). After washing, 100 ~tl of milk solution diluted from 20 to 0.032 mg/ml with phosphate-buffered saline (PBS, pH 7.0) was added, and the plates were incubated for 2 h. After washing, peroxidase-labeled goat anti-bovine IgG antibody (Kirkegaard & Perry, Gaithersburg, Md.) solution (1 ~tg/ml) in PBS containing BSA (1 mg/ml) was added. After incubation for 2 h, the plates were washed and 50 Ixl of substrate solution (0.4 mg 0-phenylene diamine and 0.2111 hydrogen peroxide in 1-ml citrate buffer, pH 5.0) was added. After incubation for 20 min, absorbance at 492 nm was read.

Agglutinin titer Killed E. coli was suspended in PBS supplemented with both Gram's safranine solution (30 ~tl/ml) and formaldehyde (5 ktl/ml). Then 50-~tl of each milk at several dilutions and 50 ~tl of the antigen solution were mixed in the wells of 96-well round-bottom microplates. The reaction was allowed to take place at room temperature for 16 h. Agglutination of the antigen-antibody complex was inspected visually and the results were expressed as the reciprocal of the highest dilution of these milks that caused agglutination.

Animals and diets SPF mice of male BALB/c strain were purchased from Charles River Japan (Narita, Japan). The mice were housed in groups of five in Clean S-CL0104 cages (Clea Japan, Tokyo, Japan) with a layer of sawdust as bedding, under controlled lighting (12-h light, 12-h dark), temperature (24 ~C), and relative humidity (55 %). They were used at 7-9 weeks of age, weighing 26 to 30 g. Food (MF diet, Oriental Yeast Co., Tokyo, Japan) was provided ad libitum.

Opsonin activity Preparation of E. coli48. Late log phase of E. coli 48 that had been incubated in trypticase soy broth at 37~ were harvested by centrifugation at 3,000 g for 15 min, and the bacteria were suspended at a concentration of 2 X 10 s colony-forming units (CFU)/ml in RPMI 1640 medium supplemented with 10% of Nu-Serum (Collaborative Research, Lexington, Mass; complete RPMI medium).

Opsonization ofE. coli 48 with milk antibody. Each milk was dissolved in complete RPMI medium at a concentration of 100 mg/ml and centrifuged at 12,000 g for 10 min to remove insoluble materials. Then, 4 ml of the supernatant was added to 2 X 108 CFU of live E. coli 48 in complete RPMI medium, and the mixture was incubated at 37~ for 15 min. After incubation, the mixture was centrifuged at 3,000 g for 10 min. After discarding of the supernatant, 0.5 ml complete RPMI medium was added to the precipitate and this was resuspended thoroughly. Preparation ofmacrophage monolayer. Peritoneal exudate macrophages were collected from the mice that had received an intraperitoneal injection of 1 ml of 10% proteose peptone 4 days previously. The peritoneal exudate cells were washed once with RPMI medium by centrifugation at 400 g for 10 min and then suspended at a concentration of 1 X 106 cells/ml in complete RPMI medium. Aliquots of the cell suspension (0.2 ml) were poured into the wells of a 96-well flatbottom microplate, and the plate was incubated at 37~ C in an atmosphere of 5 % CO2 in air for 2 h. The wells were then washed three times with warmed RPMI medium and the cells left on the bottom of the plate were used as the macrophage monolayer.

90

Phagocytosis and killing of E. coli 48 by macrophages. The opsonized E. coli 48 (50 gl) at a concentration of 4 X 108 CFU/ml, prepared as described above, were added to the macrophage monolayers in the microplate, and the macrophages were incubated on ice for l0 rain for attachment of the bacteria to macrophages without killing of the bacteria. Then, the wells were washed three times with warmed RPMI medium to remove the extracellular free bacteria. The macrophages were incubated at 37~ for 0, 30 and 60 min, and 200 gl of sterilized water containing 0.3% Triton X-100 was added to the wells. The microplate was sonicated for 1 min to disrupt macrophages completely. The numbers of live E. coli 48 in the wells were determined by culture on MacConkey agar plates (Nissui Seiyaku, Tokyo, Japan). Assays were conducted in quadruplicate.

Clearance of opsonized E. cob 48 in vivo. Opsonized E. coli 48 cells (108) were inoculated intravenously into both untreated mice and the mice that had received an intravenous injection of heat-killed Lactobacillus casei YIT 9018 (LC 9018, Yakult Honsha, Tokyo, Japan), a nonspecific macrophage-activating agent, at a dose of 20 mg/kg 13 days previously. The numbers of viable bacteria both in the liver and peripheral blood were determined at various intervals after the injection ofE. coli 48. Effect of skim milk containing specific antibody on the population levels of E. coli in the cecum of mice after administration of 5 FU Three independent experiments were done to examine the effect of skim milk containing specific antibody to E. coli on the population levels ofE. coli in the cecum of mice after administration of 5 F U (Kyowa Hakko Kogyo Co., Tokyo, Japan). In experiments 1 and 2, skim milk, which had been sterilized by y irradiation at 10 kGy, was fed to mice ad libitum at a concentration of 30 mg/ml in sterile distilled water throughout the experiment. Usually, five mice drank 25-30 ml or 10-15 ml of the milk solution per day before or after administration of 5 FU, respectively. There was no significant difference of the amounts among the different groups. In experiment 3, skim milk at a dose of 70 rag/mouse was administered orally twice a day. In every experiment, 5 F U at a dose of 400mg/kg was administered on the 7th day after the beginning of administration of milks. Eight mice from each group were killed at different days before and after administration of 5 FU under ether anesthesia, and the population levels of E. co# in the cecum were examined. Briefly, the cecum was homogenized in 5 ml of sterile saline. The homogenates were diluted and the numbers of E. coli were counted by culture on MacConkey agar plates. Results were expressed as the geometric mean _+SD of the number of E. coli per entire cecum. The average weights of the cecum were 0.79g on day --7 and --1, 0.53 g on day 1 and 0.44 g on day 3 respectively (administration of 5 FU day 0). There was no significant difference of the average weights of the cecum between the different groups.

Protective effect of skim milk containing specific antibody against the lethal toxicity of 5 FU in mice Skim milks at a dose of 20 mg/mouse were administered orally once a day for 5 days from day 1 to day 5 (experiment A), or at a dose of 70 mg/mouse orally twice a day for 13 consecutive days from day --6 to day + 6 (experiment B). 5 F U at a dose of 400 mg/kg (experiment A) or 450 mg/kg (experiment B) was administered to 20 mice in four cages per group on day 0. The mice were observed for survival for 30 days after the administration of 5 FU.

Statistical analysis The numbers of bacteria were expressed as means _+SD (in macrophages and in liver) or as geometric means + SD (in cecum). These data were analyzed by ANOVA followed by Tukey's test

91 Table 1. Composition of milks Commercial skim milk (control)

Skimmilk containing natural antibody (control)

Skimmilk containing specific antibody

Commercial WPC (control)

WPC containing specific antibody

Total energy (kJ/100 g) Protein (g/100 g) Carbohydrate (g/100 g) Lipid (g/100 g) Ash (g/100 g) Ca (g/100 g) K (g/100 g) Na (g/100 g) P (g/100 g) Fe (mg/100 g) Retinol (~tg/100 g) Thiamine (mg/100 g) Riboflavin (mg/100 g) Ascorbic acid (rag/100 g) Water g/100 g)

1484 34.8 52.7 0.5 8.3 1.9 0.9 0.4 1.0 0.7 3.6 0.2 2.0 1 3.7

1509 39.9 46.8 1.3 8.4 1.9 0.9 0.5 0.9 1.0 11.7 0.3 3.8 5 3.6

1505 34.0 54.8 0.5 6.9 1.1 1.0 0.3 1.0 0.7 2.7 0.2 1.8 ND 3.8

1760 64.4 17.7 8.8 3.3 0.7 0.2 0.1 0.4 2.5 6.6 0.1 0.5 ND 5.8

1622 68.2 20.1 2.5 3.8 0.7 0.5 0.2 0.4 1.9 13.8 0.1 1.3 2 5.4

IgM (mg/100 g) IgA (mg/100 g) IgG (mg/100 g)

ND ND ND

80 60 480

40 30 450

ND ND 90

300 210 3240

WPC, whey-protein concentrate; ND, not detected for multiple pairwise comparisons. The difference in host survival were determined with the generalized Wilcoxon test. Statistical analysis was performed using the computer package Statistical Library II (Yukms Co., Tokyo, Japan).

Results As shown in Table 1, there was no significant difference in the chemical composition between skim milk containing specific antibody and control milks of this type. Milk of WPC type contained about twice the amount of protein and much less carbohydrate than the skimmed-milk type. However, the chemical composition was much the same between WPC containing specific antibody and control milk of this type. Table 1 also shows the concentrations of three classes of immunoglobulins. The major type was IgG in both skim milk and WPC, although the concentrations of immunoglobulins were different between these groups. There was no immunoglobulin in commercial skim milk and no difference was found in the concentrations of immunoglobulin between skim milks obtained from immunized and nonimmunized cows. We tested the agglutinin activities of these milks to E. coli 48, which had been used as the immunogen for preparation of these milks. Both types of milk fractions prepared from immunized cows showed higher agglutinin titer as compared with their relevant controls (Table 2). The results of E L I S A correlated well with those of agglutinin titration (Fig. 1). We also examined the antibody

92 Table 2. Agglutinin titers a of milks against E. coli 48 Commercial skim milk

Skim milk containing natural antibody

Skim milk containing specific antibody

Commercial WPC

WPC containing specific antibody

ND

8

32

2

256

JResults are expressed as the reciprocal of the highest value of serial doubling dilution of milk solution (100 mg/ml) which caused agglutination against E. coli 48 WPC, whey-protein concentrate; ND, not detected

1.0 eft 0.8 9~

0.6 0.4

.o 0.2

20

4

0.8

0.16

0.032

Concentration of milk (mg/ml)

Fig. l. Antibody activity of milks against E. coli 48 measured by enzyme-linked immunosorbent assay. Values are expressed as means of the absorbance values at 492 nm in quadruplicate wells. O, Commercial skim milk; ~, skim milk containing natural antibody; 0, skim milk containing specific antibody; [3, commercial whey-protein concentrate (WPC); n, w P c containing specific antibody titer of these milk fractions to another several E. coli strains isolated from the intestines of BALB/c mice by ELISA and the results were much the same as the result shown in Fig. 1 for all E. coli strains examined (data not shown). We tested the role of the specific antibody in these milks in phagocytosis and killing of E. coli by murine macrophages. As shown in Fig. 2, milk fractions containing specific antibody significantly augmented the engulfment of the bacteria by macrophages. The activity of skimmed-milk type and that of WPC type were about 250% and 500% greater than their relevant controls, respectively. The bacteria opsonized with the milk fractions containing specific antibody were killed 200%-300% greater than that opsonized with control milks by the macrophages during 60-min incubation (Fig. 3). The differences in the opsonin activities of the two types of milks containing specific antibody may reflect the difference in the antibody concentrations. The opsonin activity disappeared when the specific antibodies were preabsorbed by E. coli 48, although not by other bacteria such as Aerobacteraerogenes (data not shown). There was no difference in the opsonin activity of commercial skim milk and skim milk containing natural antibody.

93

4 \

3

o X

1441

z C

Skkw-C Skim-NA Skim-SA WPC~C WPC-SA

Fig. 2, Opsonin activity of milks against E. eoli 48 in vitro. E. coil 48 (2 • 107 CFU) that had been opsonized with commercial skim milk (Skim-c), skim milk containing natural antibody (SkimNA), skim milk containing specific antibody (Skim-SA), commercial WPC (WPC-C) or WPC containing specific antibody (WPC-SA) were added onto the macrophage monolayers, and the macrophages were incubated on ice for 10 rain. The numbers of bacteria engulfed in the macrophages were then determined. Values are expressed as means _+ SD of the numbers of bacteria in quadruplicate wells. C, Untreated c?ntrol. Asterisks indicate a significant difference from the respective control, P < 0.01 (ANOVA)

4

J

ii

iiii

iiii

iiii

lilt

III

I

> 3 o uJr o

z

0

30

6-0-

Incubation time (min)

Fig. 3. Effect of milk antibody as opsonin on in vitro bactericidal activity of macrophages. E. coli 48 was treated with commercial skim milk (O), skim milk containing natural antibody (@), skim milk containing specific antibody (e), commerical WPC (El) or WPC containing specific antibody (11), and then incubated with macrophages under the same condition as given in the legend to Fig. 2, and the macrophages were incubated again at 37~ for 0, 30 or 60 rain. The number of live bacteria in the macrophages was determined. Values are expressed as the mean • SD of the number of bacteria in quadruplicate wells. ~, untrated control

To assess the opsonin activity o f the milk fraction containing specific antibody in vivo, E. coli pretreated with W P C containing specific antibody or commercial W P C was inoculated intravenously into mice. Trapping of the bacteria in the liver was enhanced by opsonization of the bacteria with W P C containing specific antibody and the bacteria were cleared more rapidly from the organ (Fig. 4A). To confirm the p h e n o m e n a , we tested the opsonin activity of W P C containing specific

94 10 *

A

8

6 ,-,4

X I

I

LMI

B

,,.6 0

6 z4

10

60 Time after injection (min)

180

Fig. 4A, B. Effect of opsonization ofE. coli with WPC milks on the clearance of the bacteria by liver macrophages of mice. E. coli (1X 10s CFU) that had been opsonized with commercial WPC (vq) or WPC containing specific antibody (11) were injected intravenously to untreated BALB/mice (A), or mice that had received an i.v. injection of LC9018 at a dose of 20 mg/kg 13 days previously (B). The number of bacteria in their livers were determined 10, 60 and 180 min after injection of the bacteria. Values are expressed as means _+SD of the numbers of bacteria per entire liver of five mice. A, Untreated control Asterisk indicates a significant difference from the respective controls at P < 0.01 (ANOVA)

antibody in vivo using mice pretreated with LC9018, a potent macrophageactivating agent (Miake et al. 1985; N o m o t o et al. 1985). The liver macrophages stimulated by LC 9018 exerted more rapid killing of E. coli, and opsonization of the bacteria with WPC containing specific antibody resulted in more rapid clearance of the bacteria both in the liver (Fig. 4B) and peripheral blood (data not shown). The lethal toxicity of 5 FU at doses of approximately 400 m g / k g in SPF mice is due to the indigenous infection with E. coli following a dramatic increase in the population levels ofE. coli in the intestinal tract. Antibiotic treatment of the mice after administration of such doses of 5 FU protected the mice completely from the lethality of the drug and inhibited the overgrowth of the bacteria in the intestine (Nomoto et al. 1991). Thus, we tested the effect of skim milk containing specific antibody against the population levels ofE. coli in the cecum after administration of 5 FU (Table 3). In the control mice, a dramatic increase in the population levels of E. coli was observed after administration of 5 F U , and skim milk containing specific antibody inhibited the increase at an early period after administration of the drug. Next, we assessed the effect of skim milk containing specific antibody on the lethal toxicity of 5 FU. All 20 mice of non-treated control group died during the 10 to 18 days after administration of 5 FU at a dose of 400 mg/kg. Of the 20 mice given skim milk containing specific antibody 9 survived the lethal toxicity of 5 FU for 30 days, while control skim milk showed little protective effect (Fig. 5A). In another experiment (Fig. 5B), every mouse died during the 7 to 15 days after

95 Effect of milks on the population levels of E. col• in the cecum of mice after administration of 5 FU

Table3.

Logl0 no. of E. col• in cecum a

Days before or after administration of 5 FU

Untreated control

Commercial skim milk

Skim milk containing natural antibody

Skim milk containing specific antibody

Expt. 1b 7 -- 1 § 1

3.51 +0.74 3.68 • 0.57 8.19 + 0.57

3.51 _+0.74 3.34 _+0.55 8.00 + 0.57

NT NT NT

3.51 +0.74 3.85 + 0.81 6.71 + 1.88 * *

Expt. 2b + 3 + 7 + 10

8.09 + 0.24 8.72 + 0.11 8.08 _+0.23

NT NT NT

NT NT NT

6.39 + 1.04" 8.38 _+0.28" 8.01 + 0.27

NT NT NT

3.00 _+0.55 3.33 + 0.70 8.01 • 0.74

3.00 + 0.55 3.00 + 0.68 7.78 + 0.78

3.00 + 0.55 2.46 _+0.46" 7.49 + 0.60

Expt 3 c 7 --

1

+ 3

Values are expressed as means _+ SD, n = 8 mice; NT, not tested. P values of the difference by ANOVA were; * P

Antibacterial effect of bovine milk antibody against Escherichia coli in a mouse indigenous infection model.

A skim-milk fraction and a whey-protein concentrate (WPC) fraction were prepared from the cows that had been immunized with E. coli isolated from the ...
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