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Arch. Gynfik. 222, 275-278 (1977)
Gynikologie 9 J. F. Bergmann-Verlag 1977
Antibacterial Activity of Amniotic Fluid against Staphylococcus aureus, Candida albicans and Brucella abortus R. P. Jankowski*, H. E. Aikins, H. Vahrson, and K. G. Gupta* Zentrum ffir Frauenheilkunde und Geburtshilfe (Direktor: Prof. Dr. R. Kepp), and Institut ffir Bacteriologie und Immunologic (Direktor: Prof. Dr. H. Blobel) der Justus-Liebig-Universit/it Giel3en D-6300 GieBen, Federal Republic of Germany
Summary. Sixty amniotic fluid samples from sixty patients between 1 4 - 4 2 weeks gestation were studied for antibacterial activity against Staphylococcus aureus, Brucella abortus and antifungal activity against Candida albicans. Both antibacterial and antifungal activity of the amniotic fluid were observed. When antimicrobial activity was correlated with gestational age, it was found to increase with the period of gestation. Maximum antimicrobial activity of the amniotic fluid was found to be present in the samples obtained between 3 6 - 4 2 weeks of gestation. All samples before 20 weeks gestation showed week antibacterial activity against Staphylococcus aureus, but comparatively strong reaction against Candida albicans and Brucella abortus.
Antibacterial activity of amniotic fluid has been studied by various authors [1, 4, 7, 9]. Galask and Snyder [4] studied the bacteriostatic properties of sterilized amniotic fluid, when incubated with isolated strains of bacteria and concluded, that there is a definite inhibition of growth of both gram-positive and gram-negative organisms. Hartwick and associates [6] cultured the amniotic fluid from 50 patients, of which 44 were sterile and the 6 positive cultures were thought to be contaminants or secondary to vaginal examination occuring prior to the amniocentesis. This procedure is being increasingly used for various diagnostic reasons (pre-natale diagnosis, fetal maturity) in the management of the obstetric patient. The expanding use of this technique has not led to an increased incidence of amnionitis. It has been observed, that bacteria may be isolated from the amniotic fluid of a small percentage of patients, although these patients have no symptomatology indicative of an infectious process [6, 10]. This finding is suggestive, that amniotic fluid contains host-resistance factors, which prevent those organisms producing disease. * First and the last author are grateful to Alexander von Humboldt Foundation for providing them fellowships to work in Germany
276
R.P. Jankowski et al.
Several investigators have recently reported about the inhibition of growth of bacteria by amniotic fluid [1, 4, 7, 9]. Most of these studies have been made against E. eoli [8, 9, 11], which are known to be important in the infection of female genital tract and in causing abortions. Other organisms like Staphylococcus aureus, Brucella abortus and Candida albieans are also known to play some part in the infection of genitae tract of females. Therefore a preliminary study of the variability in the bacterial and fungal inhibitory capacity of the amniotic fluid of different gestational age against S. aureus, Br. abortus and C. albieans was studied. A correlation between the inhibitory capacity of amniotic fluid with gestation age has been made.
Material and Method Amniotic Fluid was obtained aseptically by transabdominal amniocentesis of during the delivery from patients visiting the Zentrum ftir Frauenheilkunde und Geburtshilfe in Giegen, between 14--42 week of gestation. Patients in this study were not ill and were not receiving any antimicrobial therapy. Amniotic fluid samples containing blood were excludes from the study. Each sample of amniotic fluid was passed though a milipore filter (0.45 V pore size) prior to use. Organisms: Staphylococcus aureus strains M-69 (human origin), H-24 (dog origin) and K-849 (cow origin), Brueella abortus (human origin) and Candida albieans were obtained from Institut ffir Bakteriologie und Immunologic in Giegen. S. aureus (on Tryptose Soya agar), Br. abortus (on Brucella agar) and C. albieans (on Sabourauds agar) were maintained at 4~ C and subcultured every month. The incubation temperature was 37~ C. Antibacterial activity: Antibacterial activity of each sample was studied by the cup plate method described by Cruickshank [3]. For quantitative measurement 100 V1of the sample was put in each cup and the zone of inhibition of bacterial growth was measured in mm.
Results and Discussion Although a modified assay procedure [11] and a spectrophotometric method [9] have been introduced for testing the antibacterial activity of amniotic fluid, in the present investigation the usual cup plate method has been used and found to be satisfactory for a preliminary investigation. On the basis of these results obtained by this procedure, the summarized data is presented in Table 1. The results showed that the antibacterial activity of amniotic fluid obtained at various gestation weeks differed. Amniotic fluid obtained between 1 4 - 2 5 gestation weeks showes a weak antistaphylococcal activity against strain M-69 (8,3--23,5%), but comparatively stronger reaction against strain H-24 (33,3-50,0%) and K-849 (25,0-37,5%). These results indicated that amniotic fluid from human is less active against human strain of S. aureus than of animal origin. Furthermore the amniotic fluid from this gestation period showed a comparatively strong reaction against C. albieans (37,5-41,6%) and Br. abortus (58,3-62,5%). It was also observed, that with increase in gestation period the antibacterial activity of amniotic fluid increased against all organism studied.
Antibacterial Activity of Amniotic Fluid
277
Table 1. Samples of amniotic fluid of different gestational periods showing antimicrobial activity against S. aureus, C. albicans and Br. abortus Gestational week
15-20 21-25 26-30 31-35 36-40 41-42
Total no. of cases tested
No. showing antimicrobial activity against S. aureus
S. aureus
S. aureus
M-69
H-24
K-849
12 8 7 11 17 5 6O
1 1 3 7 15 4
4 4 4 9 11 3
3 3 3 4 8 3
C. albJeans
Br. abortus
5 3 3 8 12 4
7 5 6 9 15 4
Table 2. Relationship between the gestational period of amniotic fluid and the zone of inhibition Gestational period (weeks)
Variation in the zone of inhibition (mm) S. aureus strain
15-20 21-25 26-30 31-35 36-40 41-42
M-69
H-24
K-849
7 7 7-8 7-10 7-13 7-11
7-8 7-9 7-9 7-10 7-14 7-14
7-8 7 7-8 7-9 7-10 7-11
C. albicans
Br. abortus
7-9 7-9 7-9 7-11 7-13 7-11
7-10 7-11 7-12 7-12 7-19 7-14
M a x i m u m antibacterial activity was found to be present in the samples obtained between 3 6 - 4 2 weeks of gestation. W h e n the antibacterial activity of amniotic fluid of different gestation period was measurd quantitavely (Tab. 2), it was found, that late period amniotic fluid obtained between 3 1 - 4 2 weeks showed m a x i m u m activity. The zone of lysis was found to be between 7 - 1 3 m m in case of M-69, 7--11 m m in case of H-24, 7--14 m m in case of K-849, 7--13 m m in case of C. a l b i c a n s and 7 - 1 9 m m in case of B r . a b o r t u s . These results also showed, that the inhibiting capacity of amniotic fluid differed from organism to organism. B r . a b o r t u s has been found to be very sensitivy compared to S . a u r e u s and C. a l b i e a n s . Several antimicrobial factors have been demonstrated in amniotic fluid. Galask and Snyder [5] reported the presence of lysosyme, transferrin and 7S immunoglobulin. Cherry and colleagues [2] and Larsen and co-werkers [81 demonstrated the presence of peroxidase and lysozyme in amniotic fluid and correlated its levels with gestational age. They have shown, that lysozyme concentration in amniotic fluid increased up to 40 weeks and than decreased, which m a y explain our observation
278
R.P. Jankowski et al.
that (Tab. 1) from 40 weeks onword antibacterial activity of amniotic fluid against S. aureus strains and Br. abortus decreased. These results in general are in agreement with earlier observations [i, 4, 7, 9], that amniotic fluid has antibacterial activity, which could be responsible for decrease of infections in the newborn.
References 1. Bergman, U., Bercovici, B., Sacks, I.: Am. J. Obstet. Gynec. 114, 520 (1972) 2. Cherry, S. H., Filler, M., Harvey, H.: Am. J. Obstet. Gynec. 116, 639 (1973) 3. Cruickshank, R. (Ed.) Medical Microbiology, p. 894. Edenbrough and London: E. and S. Livingstone 1975 4. Galask, R. P., Snyder, I. S.: Am. J. Obstet. Gynec. 102, 949 (1968) 5. Galask, R. P., Snyder, I. S.: Am. Obstet. Gynec. 106, 59 (1970) 6. Hartwick, H. J., Luppa, J. B., Fekety, F. R.: Obstet. Gynec. 33, 256 (1969) 7. Kitzmiller, J. L., Higby, S., Lucas, W. E.: Obstet. Gynec. 41, 38 (1973) 8. Larsen, B., Galask, R. P., Snyder, I. S.: Obstet. Gynec. 44, 219 (1974) 9. Larsen, B., Snyder, I. S., Galask, R. P.: Am. J. Obstet. Gynec. 119, 492 (1974) 10. Prevedourakis, C., Papadimitrou, G., Ionnadou, A.: Am. J. Obstet. Gynec. 106, 400 (1970) 11. Schlievert, P., Larsen, B., Johnson, W., Galask, R. P.: Am. J. Obstet. Gynec. 1, 809 (1975) Received July 21, 1976
Arch. Gynfik. 222, 275-278 (1977)
Gynikologie 9 J. F. Bergmann-Verlag 1977
Antibacterial Activity of Amniotic Fluid against Staphylococcus aureus, Candida albicans and Brucella abortus R. P. Jankowski*, H. E. Aikins, H. Vahrson, and K. G. Gupta* Zentrum ffir Frauenheilkunde und Geburtshilfe (Direktor: Prof. Dr. R. Kepp), and Institut ffir Bacteriologie und Immunologic (Direktor: Prof. Dr. H. Blobel) der Justus-Liebig-Universit/it Giel3en D-6300 GieBen, Federal Republic of Germany
Summary. Sixty amniotic fluid samples from sixty patients between 1 4 - 4 2 weeks gestation were studied for antibacterial activity against Staphylococcus aureus, Brucella abortus and antifungal activity against Candida albicans. Both antibacterial and antifungal activity of the amniotic fluid were observed. When antimicrobial activity was correlated with gestational age, it was found to increase with the period of gestation. Maximum antimicrobial activity of the amniotic fluid was found to be present in the samples obtained between 3 6 - 4 2 weeks of gestation. All samples before 20 weeks gestation showed week antibacterial activity against Staphylococcus aureus, but comparatively strong reaction against Candida albicans and Brucella abortus.
Antibacterial activity of amniotic fluid has been studied by various authors [1, 4, 7, 9]. Galask and Snyder [4] studied the bacteriostatic properties of sterilized amniotic fluid, when incubated with isolated strains of bacteria and concluded, that there is a definite inhibition of growth of both gram-positive and gram-negative organisms. Hartwick and associates [6] cultured the amniotic fluid from 50 patients, of which 44 were sterile and the 6 positive cultures were thought to be contaminants or secondary to vaginal examination occuring prior to the amniocentesis. This procedure is being increasingly used for various diagnostic reasons (pre-natale diagnosis, fetal maturity) in the management of the obstetric patient. The expanding use of this technique has not led to an increased incidence of amnionitis. It has been observed, that bacteria may be isolated from the amniotic fluid of a small percentage of patients, although these patients have no symptomatology indicative of an infectious process [6, 10]. This finding is suggestive, that amniotic fluid contains host-resistance factors, which prevent those organisms producing disease. * First and the last author are grateful to Alexander von Humboldt Foundation for providing them fellowships to work in Germany
276
R.P. Jankowski et al.
Several investigators have recently reported about the inhibition of growth of bacteria by amniotic fluid [1, 4, 7, 9]. Most of these studies have been made against E. eoli [8, 9, 11], which are known to be important in the infection of female genital tract and in causing abortions. Other organisms like Staphylococcus aureus, Brucella abortus and Candida albieans are also known to play some part in the infection of genitae tract of females. Therefore a preliminary study of the variability in the bacterial and fungal inhibitory capacity of the amniotic fluid of different gestational age against S. aureus, Br. abortus and C. albieans was studied. A correlation between the inhibitory capacity of amniotic fluid with gestation age has been made.
Material and Method Amniotic Fluid was obtained aseptically by transabdominal amniocentesis of during the delivery from patients visiting the Zentrum ftir Frauenheilkunde und Geburtshilfe in Giegen, between 14--42 week of gestation. Patients in this study were not ill and were not receiving any antimicrobial therapy. Amniotic fluid samples containing blood were excludes from the study. Each sample of amniotic fluid was passed though a milipore filter (0.45 V pore size) prior to use. Organisms: Staphylococcus aureus strains M-69 (human origin), H-24 (dog origin) and K-849 (cow origin), Brueella abortus (human origin) and Candida albieans were obtained from Institut ffir Bakteriologie und Immunologic in Giegen. S. aureus (on Tryptose Soya agar), Br. abortus (on Brucella agar) and C. albieans (on Sabourauds agar) were maintained at 4~ C and subcultured every month. The incubation temperature was 37~ C. Antibacterial activity: Antibacterial activity of each sample was studied by the cup plate method described by Cruickshank [3]. For quantitative measurement 100 V1of the sample was put in each cup and the zone of inhibition of bacterial growth was measured in mm.
Results and Discussion Although a modified assay procedure [11] and a spectrophotometric method [9] have been introduced for testing the antibacterial activity of amniotic fluid, in the present investigation the usual cup plate method has been used and found to be satisfactory for a preliminary investigation. On the basis of these results obtained by this procedure, the summarized data is presented in Table 1. The results showed that the antibacterial activity of amniotic fluid obtained at various gestation weeks differed. Amniotic fluid obtained between 1 4 - 2 5 gestation weeks showes a weak antistaphylococcal activity against strain M-69 (8,3--23,5%), but comparatively stronger reaction against strain H-24 (33,3-50,0%) and K-849 (25,0-37,5%). These results indicated that amniotic fluid from human is less active against human strain of S. aureus than of animal origin. Furthermore the amniotic fluid from this gestation period showed a comparatively strong reaction against C. albieans (37,5-41,6%) and Br. abortus (58,3-62,5%). It was also observed, that with increase in gestation period the antibacterial activity of amniotic fluid increased against all organism studied.
Antibacterial Activity of Amniotic Fluid
277
Table 1. Samples of amniotic fluid of different gestational periods showing antimicrobial activity against S. aureus, C. albicans and Br. abortus Gestational week
15-20 21-25 26-30 31-35 36-40 41-42
Total no. of cases tested
No. showing antimicrobial activity against S. aureus
S. aureus
S. aureus
M-69
H-24
K-849
12 8 7 11 17 5 6O
1 1 3 7 15 4
4 4 4 9 11 3
3 3 3 4 8 3
C. albJeans
Br. abortus
5 3 3 8 12 4
7 5 6 9 15 4
Table 2. Relationship between the gestational period of amniotic fluid and the zone of inhibition Gestational period (weeks)
Variation in the zone of inhibition (mm) S. aureus strain
15-20 21-25 26-30 31-35 36-40 41-42
M-69
H-24
K-849
7 7 7-8 7-10 7-13 7-11
7-8 7-9 7-9 7-10 7-14 7-14
7-8 7 7-8 7-9 7-10 7-11
C. albicans
Br. abortus
7-9 7-9 7-9 7-11 7-13 7-11
7-10 7-11 7-12 7-12 7-19 7-14
M a x i m u m antibacterial activity was found to be present in the samples obtained between 3 6 - 4 2 weeks of gestation. W h e n the antibacterial activity of amniotic fluid of different gestation period was measurd quantitavely (Tab. 2), it was found, that late period amniotic fluid obtained between 3 1 - 4 2 weeks showed m a x i m u m activity. The zone of lysis was found to be between 7 - 1 3 m m in case of M-69, 7--11 m m in case of H-24, 7--14 m m in case of K-849, 7--13 m m in case of C. a l b i c a n s and 7 - 1 9 m m in case of B r . a b o r t u s . These results also showed, that the inhibiting capacity of amniotic fluid differed from organism to organism. B r . a b o r t u s has been found to be very sensitivy compared to S . a u r e u s and C. a l b i e a n s . Several antimicrobial factors have been demonstrated in amniotic fluid. Galask and Snyder [5] reported the presence of lysosyme, transferrin and 7S immunoglobulin. Cherry and colleagues [2] and Larsen and co-werkers [81 demonstrated the presence of peroxidase and lysozyme in amniotic fluid and correlated its levels with gestational age. They have shown, that lysozyme concentration in amniotic fluid increased up to 40 weeks and than decreased, which m a y explain our observation
278
R.P. Jankowski et al.
that (Tab. 1) from 40 weeks onword antibacterial activity of amniotic fluid against S. aureus strains and Br. abortus decreased. These results in general are in agreement with earlier observations [i, 4, 7, 9], that amniotic fluid has antibacterial activity, which could be responsible for decrease of infections in the newborn.
References 1. Bergman, U., Bercovici, B., Sacks, I.: Am. J. Obstet. Gynec. 114, 520 (1972) 2. Cherry, S. H., Filler, M., Harvey, H.: Am. J. Obstet. Gynec. 116, 639 (1973) 3. Cruickshank, R. (Ed.) Medical Microbiology, p. 894. Edenbrough and London: E. and S. Livingstone 1975 4. Galask, R. P., Snyder, I. S.: Am. J. Obstet. Gynec. 102, 949 (1968) 5. Galask, R. P., Snyder, I. S.: Am. Obstet. Gynec. 106, 59 (1970) 6. Hartwick, H. J., Luppa, J. B., Fekety, F. R.: Obstet. Gynec. 33, 256 (1969) 7. Kitzmiller, J. L., Higby, S., Lucas, W. E.: Obstet. Gynec. 41, 38 (1973) 8. Larsen, B., Galask, R. P., Snyder, I. S.: Obstet. Gynec. 44, 219 (1974) 9. Larsen, B., Snyder, I. S., Galask, R. P.: Am. J. Obstet. Gynec. 119, 492 (1974) 10. Prevedourakis, C., Papadimitrou, G., Ionnadou, A.: Am. J. Obstet. Gynec. 106, 400 (1970) 11. Schlievert, P., Larsen, B., Johnson, W., Galask, R. P.: Am. J. Obstet. Gynec. 1, 809 (1975) Received July 21, 1976
![[Antibacterial activity of the amniotic fluid against Staphylococcus aureus, Candida albicans, and Brucella abortus (proceedings)].](/assets/img/hold.png)
