Bacterial growth inhibition by amniotic fluid

Bacterial growth inhibition IV. Studies on the nature plate-count determinations

PATRICK

SCHLIEVERT,

BRYAN

LARSEN,

WILLIAM

Iowa

city,

P.

of bacterial

inhibition

with

the use of

B.A.

M.S.

JOHNSON,

RUDOLPH

by amniotic fluid

PH.D.

GALASK,

M.D.

Iowa

Bacterial growth inhibition in amniotic fluid is associated with a compound or class of compounds which resembles antibacterial cationic peptides. The inhibitor studied in amniotic fluid is sensitive to treatment with monobasic and dibasic potassium phosphate, and the inhibitory activity of amniotic @id is lost following adsorption onto bentonite. The inhibitory component in amniotic @id interacts with bacterial cells by adsorption or is internalized by viable cells only. The inhibitory quality of amniotic @id is not destroyed by heating to IOOO C. for 15 minutes, and this heat-stable inhibitory activity is associated with the compound or compounds which are phosphate sensitive. A possible mode of antibacterial action which is consistent with the data presented is discussed.

K N o w L E D G E o F H o w the fetus is protected from infection is presently rather scant, but recent studies have shown that the amniotic fluid contains components inhibitory to bacterial growth, which may afford some measure of protection to the fetus. In preceding reports of this series, we showed not only that amniotic fluid contains an active inhibitory component’ but also that the inhibitory activity of amniotic fluid may be abrogated by interaction with phosphate ion.:: However, bacterial growth in these two studies was measured spectrophotometrically, a method which is considerably less accurate than viable plate counts. We have recently developed a From

the Departments

of

Gynecology and Microbiology,

Obstetrics The

semimicro plate-count technique which is accurate and requires a minimal amount of amniotic fluid.” Because of the availability of this new technique, the present study was undertaken to verify our previous results and to gain insight into some of the properties of the bacterial growth-inhibiting factors in amniotic fluid. Methods

Amniotic fluid for this study was obtained by amniocentesis from pregnant patients at term who were not being treated with antibiotics. These pregnancies were uncomplicated, and in each case the indication for amniocentesis was the determination of fetal maturity. Amniotic fluid containing blood was excluded from this study. All amniotic fluid was used within 21 hours from the time it was collected. Each sample of amniotic fluid was passed through a millipore filter (0.15 p pore size) to remove particulate material prior to use. Studies on bacterial growth in amniotic fluid were performed according to the semimicro plate-count technique described previously.‘< The test organism

and

University

of

lowa.

Received Revised Accepted

for publication October October

August

14, 1974.

15, 1974. 17, 1974.

Reprint requests: Dr. Rudolph P. Galask, Department of Obstetrics and Gynecology, University of Iowa Hospitals, Iowa City, Iowa

52242. 814

Volumr Number

122 7

Escherichia inoculated from culture containing was

Bacterial

growth

inhibition

by

amniotic

fluid.

IV

81.5

coli type 06. Test samples were a dilution of a log phase starter 10:’ to 10’ cells per milliliter.

Results

In order to reaffirm our previous observation that amniotic fluid contains some inhibitory component which is sensitive to phosphate ion, the following experiment was performed. Two aliquots of 0.9 ml. earh were taken from a single sample of amniotic fluid. To one of these 0.1 ml. of sterile distilled water was added, and to the other 0.1 ml. of a solution of 7.8 Gm. per liter of monopotassium dihydrogen phosphate and 23 Gm. per liter of dipotassium hydrogen phosphate was added. The amniotic fluid with or without phosphate and three controls consisting of 1 ml. of trypticase soy broth, 1 ml. of sterile distilled water, and 0.9 ml. of water with 0.1 ml. of the phosphate solution were inoculated with a diluted starter culture of Escherichia coli. The growth of the test organism in this experiment is shown in Fig. 1. This experiment shows that amniotic fluid, water, and phosphate alone failed to support bacterial growth, but, as expected, bacteria rapidly proliferated in trypticase soy broth and amniotic fluid to which phosphate had been added. A possible reason which we considered for phosphate-mediated reversal of bacterial growth inhibition by amniotic fluid was that the major inhibitory component combines with the bacterial cells and that phosphate interferes with this action by combining and inactivating the inhibitory substance. Therefore, to determine whether the inhibitor combines with bacterial cells, the following experiment was done. A single sample of amniotic fluid was divided into two aliquots. A 2 ml. aliquot of amniotic fluid was mixed with a saline-washed pellet of Escherichia coli containing in excess of lo” cells. The other aliquot served as a control. Both aliquots of amniotic fluid were placed in a 37’ C. incubator for 30 minutes to allow the inhibitor to interact with the bacterial cells. At the conclusion of the incubation period, the bacteria added to the experimental tube were removed by millipore filtration (0.45 p filter). The control sample of amniotic fluid was also passed through a millipore filter. The resulting filtrates were inoculated with Escherichia coli, and growth was measured in the usual manner. Sterile distilled water and trypticase soy broth were also inoculated and served as additional controls. Fig. 2 demonstrates that the aliquot of amniotic

Incubation

Time

(Hours)

Fig. 1. The sensitivity of bacterial inhibition to treatment with phosphate is demonstrated. Trypticase soy broth (TSB) served as a growth control and showed normal growth of Esche&hia coli. Water served as a nutrient-free control. Amniotic fluid (AF) alone not only failed to support bacterial growth hut also caused loss of viability of the bacterial inoculum. The phosphate-treated amniotic fluid (AF + PO,) yielded good growth, while the same quantity of phosphate added to sterile distilled water did not significantly promote growth above the level of the water control. fluid which previously had been exposed to a large number of bacteria failed to inhibit bacterial growth following reinoculation, whereas the aliquot of amniotic fluid which did not have prior exposure to bacteria was inhibitory. Thus, the data from this study suggest that the bacterial inhibitor in amniotic fluid is adsorbed to the bacterial cells. To further examine interaction of the inhibitors with bacterial cells, the above experiment was

(?76 Schlievert

et ai.

TSB

4 104 v --d---A ,r--watQr

Incubation

lncubatlon Timo (Hours) Fig. 2. The effect of adsorption of amniotic fluid with 1 x 10” washed Escherichia coli cells per milliliter upon growth of Escherichia coli is demonstrated. Controls included water and trypticase soy broth (TSB). Comparison of the growth of Escherichia coli in amniotic fluid (AF) with growth in amniotic fluid-adsorbed with bacterial cells (AF-AD) reveals that factors causing bacterial growth inhibition may be removed by adsorption onto Escherichia coli cells.

repeated with heat-killed Escherichia coli, washed with normal saline. Fig. 3 depicts the results of this experiment, which show that amniotic fluid exposed to heat-killed bacterial cells does not lose its inhibitory capacity to the same degree as amniotic fluid exposed to viable cells. This suggests that either the amniotic fluid bacterial growth inhibitor binds to a bacterial structure which is substantially altered during heating or the inhibitor is taken up by the viable bacteria. However, the addition of phosphate to a third aliquot of the same amniotic fluid yielded growth to 10” cells per milliliter which demonstrated that inhibition was present and reversible but that heat-killed Escherichia coli were ineffective in reversing the inhibitor. Because of the similarity be-

Time

(HOLI~S)

Fig. 3. The failure of heat-killed Escherichia coli to effectively remove antibacterial substances from amniotic fluid is demonstrated. Water and trypticase soy broth (TSB) served as controls. Amniotic fluid (AF) failed to support bacterial growth, and amniotic fluid adsorbed with 1 x 109 heat-killed Escherichia coli per milliliter (AF-AD) did not support a significantly greater growth of bacteria than untreated amniotic fluid.

tween the inhibitory qualities of amniotic fluid and cationic peptide+ 5 demonstrated by this study, additional experiments were performed to determine if amniotic fluid possesses other properties in common with these compounds. Cationic peptide inhibitors characteristically may be removed from a solution by adsorption with bentonite.“+ Therefore, a sample of amniotic fluid was divided into two aliquots. To one of these, bentonite, 1 mg. per milliliter, was added and was allowed to react for 60 minutes at ambient temperature. Both the aliquot of amniotic fluid which contained bentonite and the untreated aliquot were centrifuged at 2,000 x g for 20 minutes followed by millipore filtration (0.45 p pore size). Bacterial growth in the bentonite-treated aliquot of amniotic fluid was compared to growth in the untreated aliquot of the amniotic fluid sample. In Fig. 4, the reversal of bacterial inhibition in amniotic fluid by bentonite extraction is evident. Because the antibacterial activity of cationic pep-

Volumr Number

122 7

Bacterial

growth

inhibition

by

amniotic

fluid.

IV

817

Bentonite

Adsorbed-AF 10’

106 r

&2ntOnitQ+Water

E \ ln =

105

s

I/

AF

lncubatlon

lo3r Wa

TlmQ (Hours)

Fig. 4. The loss of bacterial inhibition from amniotic fluid following treatment with bentonite is shown. As in the previous experiments, water and amniotic fluid failed to support bacterial growth. Trypticase soy broth (TSB) showed good growth. Significant growth was obtained in amniotic fluid which had been treated with bentonite (Bentonite Adsorbed-AF). An additional control, water treated with bentonite, failed to show growth.

tides depends upon their positive charge rather than molecular conformation, their antibacterial properties are relatively resistant to denaturation by heat.!’ To determine whether the amniotic fluid bacterial growth inhibitor was thermostable, an aliquot of a sample of amniotic fluid was placed in a boiling water bath for 15 minutes. A second aliquot from the same sample of amniotic fluid remained untreated. Following this procedure, both aiquots were passed through a millipore filter (0.45 p) and inoculated with the test organism. Bacterial growth was measured in the usual manner. The results of this experiment are presented in Fig. 5. It is evident that heating does not destroy bacterial inhibition in amniotic fluid but appears to enhance its inhibitory nature. Although no loss of inhibition by heating was a consistent finding in the fluids studied, the increase in inhibitory activity was not always seen. To ensure that the heat-stable inhibition was not caused by a substance distinct from the phosphatesensitive

inhibition,

done. A single sample

the

following

of amniotic

experiment

was

fluid was divided

Boiled

Incubation

Time

AF

(Hours)

Fig. 5. The effect of boiling upon bacterial growth inhibition in human amniotic fluid is shown. Neither amniotic fluid (AF) nor water supported bacterial growth. Escherichia coli grew normally in trypticase soy broth (TSB). Amniotic fluid which was placed in a 100” C. water bath for 15 minutes (Boiled AF) showed an enhanced inhibition of bacterial growth.

in half. One aliquot was heated to 100’ C. for 15 minutes, and the other sample was untreated. Each aliquot was again divided, and bacterial growth studies were performed on the boiled amniotic fluid, the boiled amniotic fluid with phosphate added, the untreated amniotic fluid, and the untreated amniotic fluid with phosphate added. Fig. 6 shows that the heat-stable inhibition may be reversed by the addition

of phosphates. Comment

The amniotic

purposes fluid

of this contains

study an

were inhibitory

to verify component

that

818

Schlievert

et al.

109

Bolled

108 AF.

/

/

Incubation

BAF+P04

PO?

Tlme~Hours)

Fig. 6. The

phosphate sensitivity of the heat-stable bacterial inhibitor is demonstrated. Growth of bacteria in control media (first graph) is the same as in previous studies. The second graph shows that the unboiled aliquot of amniotic fluid did not support bacterial growth (AF) but supported bacterial growth when phosphate was added (AF + PO,). The third graph demonstrates that boiled amniotic fluid (BAF) did not support bacterial growth, whereas phosphatetreated boiled amniotic fluid (BAF +PO,) supported bacterial growth.

which may be rendered ineffective by the addition of phosphate compounds and to further investigate properties of the bacterial growth inhibition which may suggest either the nature of the inhibitory substance or its mode of action. For two reasons we believed it was necessary to use the plate-count technique to re-examine bacterial inhibition and interference by phosphate. First, because of the low sensitivity of the spectrophotometric technique for detecting bacterial growth, it is important that earlier studies be re-evaluated. Second, because sensitivity of the amniotic fluid bacterial growth inhibitor to phosphate relates to the basic mode of inhibitory action, it was imperative that phosphate sensitivity be verified by the most accurate means available. As anticipated, amniotic fluid exhibited bacterial growth-inhibiting properties which were readily reversed by the addition of phosphate. In considering the possible reasons for phosphate sensitivity of the bacterial growth inhibitor in amniotic fluid, it was evident that the observed interactions could be explained if the inhibitor is or possesses the qualities of an antibacterial cationic peptide. Cationic peptides represent a class of compounds which are able to associate with the normally anionic bacterial cell surface. This interaction has been shown by others to be related to a loss in

cellular viability.4, ’ Therefore, to determine if the bacterial inhibitor functions in a manner similar to that of the cationic peptides, it was necessary that an association of the amniotic fluid-inhibitory factor or factors with bacterial cells be demonstrated. Studies which showed that inhibition is lost following exposure of amniotic fluid to living bacteria strongly suggest that binding of the inhibitor by bacterial cells does occur. However, the results of the experiment in which amniotic fluid was exposed to heat-killed bacteria prior to inoculation suggest that the inhibitor is internalized by viable bacteria. Alternatively, the interaction of the inhibitor with living cells could occur by means of a heat-labile structure such as the bacterial cytoplasmic membrane. Notably, Spitznagel’, B has suggested that the lethal interaction of cationic proteins with bacteria may be by a detergent effect upon the bacterial membrane. Additional experiments have demonstrated that other similarities exist in the properties associated with amniotic fluid inhibition of bacterial growth and characteristics of the cationic peptide inhibitors. This investigation demonstrated that the inhibitor in amniotic fluid was extractable by bentonite, a characteristic common to the cationic peptides. Rentonite is an earth which may be used to adsorb

Volume

Number

122 7

Bacterial

cationic peptides and has been used extensively in the past for the preparation of the cationic protein lysozyme. The additional characteristic of heat stability is shared by the phosphate-sensitive amniotic fluid inhibitor and cationic antibacterial peptides. The reason that a moderately inhibitory sample of amniotic fluid becomes more hostile to bacteria following boiling is presently unclear. Perhaps certain growthpromoting materials are destroyed by heat, superimposing nutrient deficiency upon active bacterial inhibition and resulting in accelerated bacterial destruction. Alternatively, activation of an inhibitory compound and destruction of amniotic fluid components which may be chemically similar to the inhibitor and compete for binding sites on the bacterial cells are possible reasons for the greater inhibition following heat treatment. In any case, the data presented in this study showing the heat stability of the inhibitor should negate the presence of complement interaction. Collectively, the data obtained in this study indicate that bacterial growth inhibition by amniotic fluid may be in part caused by an unknown substance or substances which have several characteristics in common with the cationic antibacterial peptides described elsewhere.a, ’ Although it is not possible to conclude that the inhibitor in amniotic

growth

inhibition

by

amniotic

fluid.

IV

819

fluid is a cationic protein, it is likely that it is a cationic substance which becomes associated with a heat-labile structure on the bacterial cell surface. Because bacterial cells can be protected against the action of cationic peptides by allowing them to first react with anionic substances such as deoxyribonucleic acid, ribonucleic acid, or hog gastric mucin, it is possible that phosphate may function in a similar manner.4. ‘, lo This hypothesis is consistent with the experimental data obtained in this investigation. Although this study did not show that the phosphate-sensitive, heat-stable inhibitor present in amniotic fluid is the only agent responsible for bacterial growth inhibition, it is nevertheless responsible for much of the inhibitory capacity. This concept is clearly evident when bacterial growth in amniotic fluid is compared with growth in amniotic fluid to which phosphate was added. The extent to which the inhibitory component described by this paper and other potentially antibacterial factors, which were discussed in the preceding paper of this series, complement each other to produce the antibacterial effect seen in amniotic fluid is unknown. As our studies on the isolation and chemical characterization of this phosphate-sensitive, heat-stable inhibitor progress, we will be better equipped to determine more accurately its role within the fetal environment.

REFERENCES

1. Larsen, B., Snyder, I. S., and Galask, R. P.: AM. J. OBSTET. GYNECOL. 119:492, 1974. 2. Larsen, B., Snyder, I. S., and Galask, R. P.: AM. J. OBSTET.GYNECOL. 119:497. 1974. 3. Schlievert, P., Larsen, B., Johnson, W., and Galask, R. P.: AM. J. OBSTET. GYNECOL. 122: 809, 1975. 4. Spitznagel, J. K.: J. Exp. Med. 114: 1063, 1961. 5. Spitznagel, J. K.: J. Exp. Med. 114: 1079, 1961.

Note

to

authors:

Change

in

reference

6. Lundblad, G., and H&in, E.: Stand. J. Clin. Lab. Invest. 18: 201, 1966. 7. Pandy, S. R., and Schmid, K.: Biochem. Biophy. Res. Commun. 43: 1112, 1971. 8. Wardlow, A.: J. Exp. Med. 115: 1231, 1967. 9. Skarnes, R. C., and Watson, D. W.: Bacterial. Rev. 21: 273, 1957. 10. Olitzki, L.: Bacterial. Rev. 12: 149, 1948.

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