Toxinogenic Potential of Proteus mirabilis Strains Die toxigene Wirksamkeit von Proteus mirabilis-Stämmen

Zbl. Bakt. Hyg. A 257, 83-92 (1984)

Toxinogenic Potential of Proteus mirabilis Strains Die toxigene Wirksamkeit von Proteus mirabilis-Stammen ANNA HOSTACKA, IVAN crzNAR, JOZEF MARKOVIC, and JAN KAROLCEK Research Institute of Preventive Medicine, Bratislava, Czechoslovakia (Director: Dr. Jura;

Cervenka, M. D., DrEp.) With 5 Figures· Received September 9, 1983 . Accepted December 1, 1983

Summary Live bacterial suspensions, original as well as concentrated filtrates of Proteus mirabilis cultures caused positive reaction in the rapid skin test (vascular permeability reaction) and also in the delayed test (combination of haemorrhagic reaction, dilatation of vessels and induration) as well as in the test of mice-foot edema. The test on suckling mice did not prove the presence of thermostable enterotoxin. After separating the concentrated culture filtrates the biologic activity appeared in fraction 1 (relative molecular mass over 100 000) concerning the delayed skin test and in the fraction 2 (relative molecular mass approx. 40 000) in the rapid skin test. The activity localized in the fraction 1 can be ascribed to a lipopolysaccharide. The fraction 2 with an expressed activity in the rapid skin test showed also cytotoxic and proteolytic activity. In both fractions the presence of active antigen substances with different mobility and different antigen specificity was demonstrated.

Zusammenfassung Suspensionen lebender Bakterien, und zwar urspriingliche sowie konzentrierte Filtrate von Proteus mirabilis-Kulturen, erzeugten positive Reaktionen im Hautschnelltest (vaskulare Permeabilitats-Reaktion), im verzogerten Test (Kombination aus hamorrhagischer Reaktion, GefaBerweiterung und Induration) und im MausefuBodemtest. 1m Test an Babymausen lieB sich das Vorhandensein von thermostabilem Enterotoxin nicht nachweisen. Nach Auftrennung der konzentrierten Kulturfiltrate zeigte sich die biologische Aktivitat in der Fraktion 1 (relatives Molekulargewicht iiber 100 000) im verzogerten Hauttest und in der Fraktion 2 (relatives Molekulargewicht ca. 40 000) im Hautschnelltest. Die in der Fraktion 1lokalisierte Aktivitat kann einem Lipopolysaccharid zugeschrieben werden. Die Fraktion 2 mit einer deutlichen Aktivitat im Hautschnelltest zeigte auch zytotoxische und proteolytische Aktivitat. In beiden Fraktionen wurde das Vorhandensein aktiver Antigensubstanzen mit unterschiedlicher Mobilitat und Spezifitat nachgewiesen. The Proteus mirabilis is not only one of the important pathogenic agents of the urinary tract in connection with nosocomial infections (Gould, 1968; Stamm et aI.,

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J. Markovic, and J. Karolcek

1977; Chow et aI., 1979) but it is known from references that these bacteria also play an important part in the clinical picture of enteritis (Efimov, 1979). The enterotoxin produced by Proteus mirabilis is assumed to play an important role in the pathogenesis and immunity of the infection concerned in spite of the fact that also other aspects of the virulence have to be taken into consideration (Peerboms et ai., 1982). Our work was aimed at isolation and at least partial characterization of some biologically active substances produced by strains of Proteus mirabilis.

Material and Methods Strains. Six strains of Proteus mirabilis were used in the experiments (strains 35, 36, 37, 65,66, 67) isolated from patients suffering from diarrhea. Preparation of the live bacterial suspension. Brain-heart infusion (BHI, Oxoid Co) of mass concentration 37 glliter, pH 7,4 was used as cultivating media. 0.1 ml of suspension with an absorbance 0,2 at 600 nm (Spekol, Carl Zeiss lena, GDR) was inoculated into 10 ml of media in an L-test tube and statically cultivated for 48 h at 37°C. After cultivation the cells were washed with normal saline solution, centrifuged (5000 x g) and resuspended into an equivalent amount of normal saline at a temperature of 37°C. Preparation of the filtrate of culture, concentration and isolation of biologically active substances. The strains were statically cultivated for 48 h at 37°C in BHI of a mass concentration 37 gil, pH 7,4. 5 ml of inoculum with an absorbance of 0,2 was transferred into Erlenmeyer's flask (500 ml) with 100 ml of cultivating media. The bacterial culture was sterilised by filtering through a porcellain filter. The sterile filtrate of the culture after being precipitated by ammonium sulphate (55 gl100 ml) was kept at the laboratory temperature for 24 h and the centrifuged sediment dissolved in normal saline. The resulting material was intensively dialysed against distilled water and dialysed 24 h against 0,1 molll of Tris HCI + 0,15 moll1 NaC!, pH 8. The filtrate of the culture was concentrated on 1150 of the original volume and applied on the Sephadex column (Pharmacia, Sephadex G-100, 100 x 2,6 em). The calibration procedure followed the method described by Ciincir et al. (1977). Three individual fractions were obtained by the separation, these were lyophilized and dialyzed. Lipopolysaccharid (LPS) was isolated from the cell walls of Proteus mirabilis 35 killed with formaldehyde according to the method of Westphal et al. (1952). Rabbit antiserum against complete cells of Proteus mirabilis 35 (s) was prepared according to the method described by Farbakyova et al. (1974). The serum was stored at -20°C. Immunochemical assays were performed in agar according to the method described by Farbakyova et al. (1974). Immunoelectrophoresis was made on glass plates (8,5 x 8,5 em) in agar, the mass concentration was 15 gil 1 h, 120 V. Concentration of proteins was determined according to Lowry et al. (1951). Carbohydrates were determined according to the method of Trevelyan and Harrison (1952). The proteolytic activity was determined with the method according to Ryden et al. (1973). Kasein (Hammarsten, Merck) was used as substrate. The tested samples (fraction 1, 2,3) were used in a concentration of 200 ~g proteinslml. Thermostability was determined by heating the tested substances at 56°C for 30 min and at lOO°C for 10 min. Biological activity was determined by using the following tests: a) Test of increased vascular permeability (delayed skin test - DST) according to Craig (1972) and rapid skin test (RST) according to Sandefur and Peterson (1976). b) Test of the mice foot edema (Lexomboon et aI., 1971). A difference of 75 mg mass between infected and healthy foot was considered 1 biological unit (b. u.). c) Test of toxicity on mice lungs (Emody et aI., 1980).

Toxinogenic Potential of Proteus mirabilis Strains

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d) Test on suckling mice according to Dean et aI., (1972) modified for the oral application according to Jacks and Wu (1977). e) Test on ligated ileal loops of rabbit according to De and Chatterjee (1953). The fractions 1, 2, 3 were injected in an amount of 1,5 ml and with a concentration of 1 mg of proteins/ml. f) Vero test. For the determination of the cytotoxic activity a suspension of Vero cells CUSOL, Prague) gained from a continuous line of a 6-7 days old cultl}re on Roux bottles was used. MEM with bovine serum (mass concentration 100 gll- USOL, Prague) was used as cultivation media. Streptomycine-sulphate (10- 1g/l) and mycerin-sulphate (lO-lgi I) (Medexport, USSR) was added to medium. Approximately 140000 Vero cells were inoculated on a glass (25 X 25 mm) in a polystyrene Petri dish (diameter 60 mm). After obtaining a monolayer (within 24-48 h) the sample (0,4 ml) in MEM without serum was applied. After incubating for 24 h at 37°C the glass was rinsed with PBS and fixed with neutralized formaldehyde (0,3 molll) for 30 min. After further rinsing the glass was stained (May Grunwald - 10 min, Giemsa-Romanowski solution 1 : 10 - 20 min). The changes in the monolayer of Vero cells were observed in an optical microscope (Zeiss) at 100 and 200 magnification.

Results No significant differences were observed between the tested strains as shown by the results of rapid skin test with live cells (see Table 1). A vascular permeability reaction was observed after application of live bacterial suspension of the above mentioned strains. The character of reactions at DST was qualitatively different from the results obtained at RST. Haemorrhagic reaction combined with dilatation of the vessels was prevalent at DST, accompanied by significant induration. The bacterial suspensions of tested strains caused an edema (2,5 b. u.) andlor haemorrhagic reaction within 24 h from application into the mice paw. The bacterial

Table 1. Toxic activity of Proteus mirabilis RST (cm 2)

35 36 37 65 66 67

DST (cml)

B

F

D

B

F

D

Foot edema test B.U. E/H F D B

2,5 1,9 2,4 2,3 2,0 1,7

1,7 2,0 1,5 3,0 0,5 1,1

5,3 3,9 2,5 3,4 3,4 1,6

6,3 6,5 6,3 6,3 5,3 6,6

3,6 2,4 2,5 2,3 2,7 1,8

7,0 8,2 4,4 7,5 9,0 3,9

4/3 4/2* 4/4 4/2 411 4/2

Strain

RST - Rapid skin test DST - Dalayed skin test - Bacterial suspension B F - Filtrate D - Dialysate EIH - Edema/hemorrhage B.U. - Biological unit - Death of mice *

0,3 0,4 0,8 0,6 0,4 0,4

0,9 1,2 1,0 1,3 1,4 0,8

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A.Hostacka, l.Ciznar, J.Markovic, and J.Karolcek

suspension prepared from the strain No 36 caused death to two from four mice within 48 h from application. The reactions on the mice paw caused by all tested strains were accompanied by significant suppuration and necrosis that was most intensive 96 h from application of the samples. The toxicity test on mice lungs resulted in haemorrhagic reactions on lungs, but no death of mice was observed. In the test of enterotoxicity on suckling mice a ratio of intestinal weight to remaining body weight was determined between 0,05 and 0,056 - a negative result. Further experiments were focused to the determination of the biological activity of original and concentrated sterile culture filtrates. The results are shown in Table 1. RST in the case of original culture filtrates resulted only in vascular permeability reaction. After the application of concentrated culture filtrates permeability reaction accompanied by induration was again observed. DST resulted in a combination of haemorrhagic reaction, dilatation of vessels and in the case of concentrated culture filtrates also in induration. The value of edema as shown by the mice foot test was 0,3-0,8 b. u. in the case of original filtrates and after application of concentrated culture filtrates 0,9-1,3 b. u. The enterotoxicity test on suckling mice was again negative. After the toxic properties of live bacterial suspensions have been determined, original as well as of the concentrated culture filtrates, we proceeded with the isolation of biologically active substances. As the greatest reaction at DST was caused by the 48 h concentrated static culture filtrate (Table 2) the following tests were made with such Table 2. Dependence of biological activity of dialysate on length of cultivation Proteus mirabilis 35 Dialysate DST (cm 2 )

S 3,5

24 H SH 2,0

S 7,7

48 H SH 6,1

72H S 4,5

S - Static culture SH - Shaked culture DST - Delayd skin test materials. The culture filtrates after being precipitated by ammonium sulphate and after dialysis (an approx. 50 X concentration) were separated on Sephadex G-100. The obtained profile of separation for strain Proteus mirabilis 35 was approximately the same as in the case of other concentrated filtrates of cultures tested. Three fractions, in some cases four, were obtained. Fraction 1 contained substances with relative molecular mass over 100000, fraction 2 with mass approx. 40000 and fraction 3 with a mass under 10000. Sporadically a fraction was obtained that flowed from Sephadex G-lOO in front of the blue dextrane. The biologic activity expressed in RST by vascular permeability reaction was, after separation of the concentrated culture filtrate of Proteus mirabilis 35 on the Sephadex column eluted, into fraction 2 (Table 3). Low, almost negligible activity was also noticed after application of fraction 1. The biologic activity expressed in DST was of a different character. While fraction 2 appeared quantitatively on the same level in DST

Toxinogenic Potential of Proteus mirabilis Strains

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Table 3. Composition of the tested samples and their biological activity Material

Filtrate Dialysate Fraction 1 Fraction 2 Fraction 3

Protein mg

Carbo- BioI. act. cm2 hydrate mg RST DST

1222 48 1.0 1.3 9.3

675 12 4.1 0.1 0. 7

600 2700 720 360 2.5 59 41 39

BioI. units (U)

RST

DST

1200 5400 720 1440 5 11 8 82 78

Spec. act. (U/mg protein) RST DST 1 15 5 63

4.4 30 118 60

RST - Rapid skin test DST - Delayed skin test BioI. unit (U) - 0.5 cm 2 of area.

as in RST, fraction 1 reacted in DST by more than 20 times greater activity. The fraction 3 was biologically without effect. Further tests were performed to determine the concentration at which fractions 1 and 2 remained effective. The results are shown in Fig. 1. It is evident that the fraction 1 in concentrations from 31 up to 250 !Lg proteins/ml remained ineffective. The concentrations 500 and 1000 !Lg proteins/ml caused in RST only a slight permeability reaction. In DST the fraction 1 remained effective in the full tested concentration range. The fraction 2 was effective in RST and DST in the concentration range 125-1000 !Lg proteins/ml. In the mice foot test (after 6 and 24 h) after application of the fraction 1 in concentration range from 31 to 1000 !Lg proteins/ml only weak edematous reaction was observed. The same concerned fraction 2 with the difference that this fraction in concentration 1000 !Lg proteins/ml 6 h after application caused a significant edematous reaction (1 b. u.).

•

o ~ST FRACTION! • DST

o RST FRACTIOtH aDST 1

RST-RAPID SKIN TEST DST-OELAYED SKIN TEST

10001"1 prolein/ ml

Fig. 1. Dose response of fraction s 1 and 2 in skin test.

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A.Hostacka, I.Ciznar, J.Markovic, and J.Karolcek

Fig. 2a. Control Vero cells.

Fig. 2b. Vero cells after exposure to fraction 2 of Proteus mirabilis 35.

Toxinogenic Potential of Proteus mirabilis Strains

89

The test on suckling mice did not prove the presence of thermostabile enterotoxin in the tested concentration range. The accumulation of fluid after application of individual fractions in the test on ligated bowel was not unambiguous. 24 h after the application of fraction 2 in concentrations 200, 100 and 50 !-tg proteins/ml significant morphological changes were observed on Vero cells. The fraction 2 in concentration 200 !-tg/ml caused a total destruction of the monolayer with extreme shrinkage of cells. In the vicinity of the nucleus some remains of cytoplasm (Fig. 2) were observed. Common for the changes caused by fraction 2 in concentrations 100 and 50 Ilg proteins/ml was the destruction of monolayer, edges on the cytoplasm, appearance of bridges and cell polymorphy. In lower concentrations (25; 12,5; 6,2; 3,1 Ilg proteins/ml) the fraction 2 remained ineffective. Fractions 1 and 3 caused no changes on Vero cells. The immunochemical test has shown (Fig. 3) that fraction 1 has formed with serum (s) one expressed diffuse line and two weaker lines. This line is identical with the line formed by the original filtrate and the dialysate. The fraction 2 formed one intensive line, that is not identical with the expressed precipitation line of the first fraction. Bya further immunochemical analysis (Fig. 4) the identity between line of fraction 1 and lipopolysaccharide prepared by phenol extraction was proved. Immunoelectrophoresis (Fig. 5) showed the separation of antigenic active components into fractions 1 and 2. They are of different migration as shown in Fig. 5. By determination of the proteolytic activity on casein substrate was shown, that this is localized in fraction 2 (Table 4). The testing of thermostability of fractions 1 and 2 (56°C/30 min and 100°C1l0 min) has shown that fraction 1 was thermo stabile and kept the biologic activity in full. Fraction 2 was partly thermo stabile, the biologic activity, however, showed some decrease according to tests as seen in Table 5. From the view point of chemical composition fraction 1 contained mostly carbohydrates, in fraction 2 and 3 the ratio of proteins to carbohydrates was 13 : 1 (Table 3).

Fig. 3. Double diffusion test of filtrate (F), dialysate (D) and fractions 1,2,3 (1,2,3) with rabbit antiserum against Proteus mirabilis 35 (s). Fig. 4. Double diffusion test of fraction 1 (1) and LPS with rabbit antiserum against Proteus mirabi/is 35 (s).

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A. HostacH, I. Ciznar, J. Markovic, and J. Karolcek

Table 4. Proteolytic activity Fractions

U/ml

2

3

124

10

1

o

U/ml of proteolytic activity is defined as the amount of enzyme giving an increase of 0.01 optical density unit per hour at 280 nm.

Fig. 5. Immunodiffusion assay of filtrate (F), dialysate (D) and fractions 1, 2, 3 (1,2,3) with rabbit antiserum against Proteus mirabilis 35 (s).

Table 5. Biological activity of the fraction 2 after incubation at 56 °C/30 min and 100 °CI 10 min Material Fraction 2 Fraction 2 56 °C/30 min Fraction 2 100 °CIl 0 min

RST (cm 2 )

%

DST (cm 2)

%

4.9

100

4.2

100

3.2

65

1.7

41

0.6

12

0.2

5

RST - Rapid skin test DST - Delayed skin test

Yero cells destruction partial destruction without change

Toxinogenic Potential of Proteus mirabilis Strains

91

Discussion and Conclusions There are no many data in literature on the production of exoproducts by Proteus mirabilis. We aimed, therefore, at a more detailed characterization of some toxic properties of biologically active substances obtained from culture filtrates of these strains. The greatest biological activity after 48 h of static cultivation was determined in DST. The live bacterial suspensions, original as well as concentrated culture filtrates caused a vascular permeability reaction in RST. The result of DST was haemorrhagic reaction, dilatation of vessels and induration. A similar character of reactions appeared also at testing strains of Proteus morganii (Cizndr, Hostackd, 1982). The tests on mice foot were positive. The test on suckling mice did not prove the presence of thermostabiIe enterotoxin. The separation profile of concentrated culture filtrate on the column of Sephadex G100 was similar to the profile obtained while separating the concentrated culture filtrates of Vibrio cholerae non-01 (Cizndr et aI., 1977) and Aeromonas hydrophila (Hostackd et aI., 1982). The tests showed that the biological activity in RST was after separation mostly localized in fraction 2. In DST the fraction 1 appeared significantly. Differently as in case of the first fraction, the proteolytic and cytotoxic activity was localized in fraction 2. In both fractions the presence of antigenic substances was determined, but having different migration and different antigenic specificity. Further experiments have shown that fraction 1 contains most of the carbohydrates and the immunochemical assays proved the presence of LPS. It should be noticed that fractions 1 and 2 were differently behaving, considering the reactivity in RST and DST. While fraction 1 with contents of LPS gave a significant positive reaction in DST, the fraction 2 with protein characteristic gave a positive reaction in RST. It seems that the differences in RST and DST could express the separation, presence or absence of LPS in the fraction concerned. The presence of thermostabile enterotoxin in culture filtrates of Proteus mirabilis has not been proved by suckling mouse model test. A similar enterotoxic activity as in case of culture filtrates of Escherichia coli, vibrios or other enteric bacteria has in case of exoproducts of Proteus mirabilis not been unambiguously proved. We have been able, however, to prove a significant biological activity of at least two components with different molecular masses. The component having a greater molecular mass is active and this activity can be ascribed to lipopolysaccharide. The second component showing significant activity in RST had also cytotoxic and proteolytic activity. It is difficult to say to what extent the proteolytic activity contributes to the observed toxic effects. The results show the complexity of pathogenetic factors in strains of Proteus mirabilis, requinng further research. We thank Dr. G. Bartkavd for the supply of antiserum used in this study and Mrs. A. Korenackava for skilled technical assistance.

References

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