Tohoku J. exp. Med., 1985, 146, 105-117

Tohoku J. exp. Med., 1985, 146, 105-117

Invasion of Haemophilus

Pseudomonas aeruginosa

induenzae and

in Humans

HIROSHI SUZUKI, SITI SOENDARI JUWONO * MATSUMOTO

Department of Internal Medicine, Institute Medicine, Nagasaki University, Nagasaki 852 Biomedical Research, National Institute of Development, Jakarta

and KEIZo

for Tropical and * Center Health and

SUZUKI, H., JUwoNO, S.S. and MATSUMOTO, K. Invasion of Haemophilus influenzae and Pseudomonas aeruginosa in Humans. Tohoku J. exp. Med., 1985, 146 (1), 105-117 Levels of serum IgG antibodies to somatic antigens of Haemophilus influenzae and Pseudomonas aeruginosa were determined in 127 healthy people, 37 patients with chronic bronchitis and 20 patients with bron-chiectasis by enzyme-linked immunosorbent assay. The logarithmic mean titers of antibodies to Haemophilus influenzae and Pseudomonas aeruginosa were significantly higher in the patients with chronic bronchitis and bronchiectasis than in healthy people. The invasion rate of Haemophilus influenzae was identical in healthy people, chronic bronchitis and bronchiectasis, while that of Pseudomonas aeruginosa was significantly lower in healthy people than in chronic bronchitis and bronchiectasis patients. These results indicate that patients with chronic bronchi-tis and bronchiectasis may often have recurrence and are easily infected because of lowered defense mechanisms in the respiratory tract. bacterial antibody ; Haemophilus influenzae infection ; Pseudomonas aeruginosa infection

Haemophilus influenzae (H. influ.) and Pseudomonas aeruginosa (P. aeru.)

are the most significant causative agents of infectious diseases of the respiratory

tract and other parts of the body (Uzuka et al. 1983). H. influ. is related to chronic respiratory infectious diseases, namely chronic bronchitis, bronchiectasis

and other chronic diseases in adults (Nordon 1978). In adults almost all H. influ. infections of the respiratory tract are caused by uncapsulated H. influ., while in

children they are caused by capsulated H. influ. (Turk and May 1967). P. aeru. is related to a severe type of respiratory infectious disease such as chronic bronchiolitis, bronchiectasis and pneumonia at the terminal stage of compromised

host (Valdivieso et al. 1977). Although the definite diagnosis of diseases caused by these bacteria has been made by isolation of the bacteria from sputum (Ma-tsumoto et al. 1978), it was not certain that these isolated bacteria are the

causative agents or not. If specific antibodies to these bacteria are disclosed in

addition to the isolation of bacteria from clinical samples, the estimation of

Received March 28, 1984; accepted for publication January 21, 1985. 105

106 H. Suzuki et al.

causative bacteria may be more definite. For this purpose investigation on the antibody to H. influ. type b was performed by enzyme-linked immunosorbent assay (ELISA) using polysachaccharide as antigen (Dahlberg 1981; Kaplan et al. 1983). As for P. aeru., hemagglutination assays and ELISA methods were used, using exotoxin A, type specific lipo-polysaccharides and cell envelope proteins as antigens (Jagger et al. 1982; Pollack et al. 1983). Furthermore, one of the active problems in H. influ. infection of the respira-tory tract is the recurrence of infection, while that in P. aeru. infection is that most antibiotics are ineffective. For the protection and cure of these infections investigations on defense agents of the host have been done as well as the study of the significance of specific antibody to these bacteria by many investigators

(Peltola et al. 1977 ; Fernandes et al. 1981). These investigations, however, have not been sufficient yet. Thus, the antibody titers to H, influ. and P, aeru. in humans were determined by the ELISA method using somatic antigens in an attempt to settle the problems.

MATERIALS AND METHODS

Subject. One hundred and twenty-seven healthy people and 57 patients with respira-tory diseases (37 with chronic bronchitis and 20 with bronchiectasis) were subjected to the

present study. The healthy people ranged in age from 20 to 78 years, with a mean of 43. The patients with chronic bronchitis ranged from 28 to 80 years (mean 61) and those with bronchiectasis from 21 to 80 (mean 55). Serum. Sera were taken from the healthy people randomly and from the patiens at the convalescent stage. They were stored at -20°C until use. Antigen. Somatic antigens of H. influ. and P. aeru. were prepared by using strains isolated from different patients suffering from bronchiectasis. The procedure of preparation of each antigen was as follows : Bacteria were cultured at 3TC for one day on agar medium. Then phosphate buffered saline (PBS), pH 7.2, 0.15 M, was added to the agar medium and the colonies were scraped so as to make suspension. The suspension was centrifuged at 6,000 rpm for 30 min at 5°C. The supernate was discarded and PBS added to the sediment. They were mixed well and then centrifuged again. These procedures were repeated three times. After the third centrifugation PBS was added to the sediment (1:50). The suspension was sonicated at 50 W, and it was then centrifuged at 6,000 rpm for 30 min at 5°C. Thereafter the supernatant was dialyzed against distilled water for two days. After that the solution was lyophilized and the powder was weighed (Burns and May 1967). Control serum. Positive sera to H. influ. and P. aeru. were obtained from infected

patients and the titers were preliminarily determined by using the method of counter immunoelectrophoresis (Ingram et al. 1972). Each negative serum to H. influ. and P. aeru. was prepared by the absorption method. The period of incubation was 1 hr at 37°C, followed by 24 hr at 5°C. It was then centrifuged at 6,000 rpm for 30 min at 5°C. The supernatant was filtered through a membrane filter, 0.45 dam in pore size, and was stored at - 20C C until use.

ELISA system. Preparation of the antigen coating plate was carried out as follows : Antigen was diluted with coating buffer solution. After 100,1 of diluted antigen was

poured into the well of a microplate "Flat Bottom Microelisa Plates, Immulon of Dynatech Microelisa Systems", the microplate was incubated at 5°C over night. The plate was washed with PBS-Tween for 3 min. This process was repeated three times. After the plate was dried up, it was kept at -20°C until use. 100 pl of the test serum diluted in PBS-Tween

H. influenzae and P. aeruginosa Infection 107

was added into the wells and the microplate was incubated at 37°C for 1 hr in a humid chamber. After the incubation the microplate was washed with PBS-Tween three times. On the following step, 100,1 of antihuman IgG immunoglobulin alkaline phosphatase conjugate, Tago Inc., Surlingame, Ca., USA, which was diluted 1:1,000 with PBS-Tween, was added into each well and the microplate was incubated again for 1 hr at 37°C in a humid chamber. After the incubation the washing procedure with PBS-Tween was repeated as described above. 100,ul of substrate which was prepared by adding 1 mg of p-nitrophenylphosphate to 1 ml of diethanolamine buffer, pH 9.8, was added into each well and the microplate was incubated for 30 min at room temperature (Yolken et al. 1978). After this step the absorbance of each well was read in a "Microelisa Auto Reader" (Dynatech Instruments, Inc., Santa Monica, Ca., USA). The result of the ELISA assay was expressed either as end point titration or as a positive/negative ratio. The end point of titration of serum was the highest dilution of serum yielded by an optical density (O.D.) which was three times higher than that of negative serum on the plate. Furthermore, a method of curve fitting was used for the estimation of the endpoint (Rodbard et al. 1969; Yolken et al. 1978). It was decided that less than 1:10 of serum dilution was negative. Concentration of antigen. Appropriate concentrations of antigens of H. influ. and P. aeru. were determined by using positive and negative sera and by the method of checker-board titration at different incubation time. O.D. of the test was read at 30 min after adding the substrate into wells of the microplate.

RESULTS

Concentration of antigen

As shown in Table 1 the highest titer of antibody to H. influ. was observed at 60 min and 120 min of incubation of the test serum. The concentration of

antigen in a well at 60 min was 1 X 10-s mg and that at 120 min was 1 X 10-4 mg. The highest titer of antibody to P. aeru. was observed at 60 min and 120 min of

incubation as shown in Table 2. The concentration of antigen in a well was 1 X 10 mg at 60 and 120 min. From these results it was denoted that the appropri-ate concentrations of antigens of H. influ. and P. aeru. were 1 X 10 mg and 1 X

10 mg in a well at 60 min of incubation.

Antibody distribution to H. influenzae

The distribution of antibody to H, influ. as determined with 1 X 10 J3 mg/well of antigen of H. influ. at 60 min of incubation in the ELISA system is shown in

Table 3 and Fig. 1. As shown in Fig. l the antibody distribution of healthy

people was lower than that of chronic bronchitis and bronchiectasis patients. Distribution of antibody to H. influ. of bronchiectasis was the highest.

Level of antibody to H. influenzae

At the level of 50% of all examined cases antibody to H. influ. in healthy

people was 1:119, it was 1: 223 in chronic bronchitis patients and it was 1: 640 in bronchiectasis cases in serum dilution as shown in Fig. 1. The level of antibody was lowest in healthy people, and highest in bronchiectasis cases. For the

comparison of levels of antibody each antibody titer was expressed in logarithm and the results are shown in Table 4. The comparison was carried out among


mean levels for individual subjects. There differences between mean levels of antibody chiectasis cases and healthy people at the level

were statistically significant

of chronic bronchitis or bron-

of five percent and one percent,

TABLE 1. Checkerboad titration between antigen and antibody for H. influenzae

Fig. 1. Cumulative curve of antibody to H. influenzae.

H. influenzae an d P, aeruginosa Infection 109

respectively. The difference was, however, not significant between chronic bron-

chitis and bronchiectasis cases.

Antibody distribution to P. aeruginosa

In Table 5 and Fig. 2 the distribution of antibody to P. aeru. is shown. Fig.

2 shows that the lineal trends of healthy people and chronic bronchitis cases are

similar, but that they are different from that of bronchiectasis cases.

Level of antibody to P. aeruginosa

As shown in Fig. 2 antibody levels to P. aeru. were compared at the level of 50 ̀ percent in each subject. The levels of healthy people, chronic bronchitis and bronchiectasis patients were 1:12,1: 25 and 1: 68 in serum dilution, respectively.

In healthy people the distribution level was lowest. Antibody titers were convert-

ed into logarithm and their means were compared. As shown in Table 6 the means of antibody titers of chronic bronchitis and bronchiectasis cases were 1:1.44

and 1.93. They were significantly higher than that of healthy people, which was

0.89, at the level of one percent. But the difference in mean of the antibody titer

TABLE 2. Checkerboad titration between antigen and antibody for P. aerugznosa


H, influenzae an d P. aeruginosa Infection 111




between chronic bronchitis and bronchiectasis cases was not significant.

Invasion rate

As shown in Fig. 3, invasion rates of H. influ. and P. aeru.were compared in different groups of subjects. The invasion rate of P. aeru. was significantly higher in chronic bronchitis and bronchiectasis cases than in healthy people at the level of one percent, while there was no difference among these subjects in invasion rate of H, influ. Furthermore, the invasion rates of H. infdu. and P. aeru. were compared in each group. In healthy people the invasion rate of H. influ. was significantly higher than that of P. aeru. at the level of one percent. The difference between invasion rates of H. influ. and P. aeru. in chronic bronchitis or bronchiectasis cases was however not significant.

Fig. 2. Cumulative curve of antibody to P. aerugznosa.

Fig . 3. Comparison of invasion rate in humans. N, negative rate of infection ; P, positive rate of infection ; H.P., healthy

people ; C.B., chronic bronchitis cases ; B.E., bronchiectasis cases. * Significant at p <0 .01. Statistical evaluation was carried out by f-test.

H. inf iuenzae an d P. aeruginosa Infection 115

Correlation of antibody level

The correlation between antibody levels of H. influ. and P. aeru. is shown in Fig. 4. The distribution of antibody levels of H. influ. was similar in the three

groups of subjects, while the distribution of antibody levels of P. aeru. was lower in healthy people, higher in bronchiectasis cases, and slightly higher in chronic bronchitis cases than in healthy people, but lower than in bronchiectasis cases.

DISCUSSION

This study examined the antibody level and the invasion rate of H. influ. and P. aeru. in healthy people, chronic bronchitis and bronchiectasis patients by the method of ELISA using somatic antigens. Because somatic antigen of H. influ. is common to the uncapsulated strain and all encapsulated types (Tunevall 1953), it was used for the present investigation. Somatic antigen of P. aeru., on the other hand, has some type specificities to different strains, although exotoxin A is

Fig. 4.

H

Correlation of antibody to influenzae and P. aeruginosa.


produced by a majority of P. aeru. strains (Pollack et al. 1977, 1983). As a common antigen of P. aeru. exotoxin A is superior to the somatic antigen. The somatic antigen of P. aeru. was however utilized in this investigation. The invasion rate of H. influ. was detected to be the same in healthy people,

chronic bronchitis and bronchiectasis cases, whereas antibodies to H. influ. were

found to be higher in chronic bronchitis and bronchiectasis patients than in healthy people. There was no significant difference in H.influ. antibody level

between chronic bronchitis and bronchiectasis patients. It has been already

pointed out, however, that hemagglutinating activity against H. influ. was detectable in about 60 percent of normal human adults (Anderson et al. 1972). On the other hand, the invasion rate of P. aeru. was higher in chronic bronchitis

and bronchiectasis than in healthy people. Futhermore, P. aeru. antibody titers in chronic bronchitis and bronchiectasis patients were higher than in healthy

people. Antibody titers to H. influ, were also higher in chronic bronchitis and bronchiectasis patients than in healthy people, but the antibodies had no preven-tive effect. Other investigators also pointed out that antibodies to the somatic antigens were not effective for prevention (Anderson et al. 1972). In addition, it

has been shown that there is cross-reaction caused by antigens from different bacteria which will elevate non-specific antibodies (Schneerson and Robbins

1975). This may account for the fact that, in spite of high antibody titers as revealed by our study, the invasion rate is high. Structural changes of the

respiratory mucosa and low secretion of IgA may also be responsible for the high invasion rate (Pryjma 1982).

References

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Tohoku J. exp. Med., 1985, 146, 105-117