Folia Micmbiol. 37 (5), 360-364 (1992)
Permeability Factor, Cytotoxicity and Serotyping of Pseudomonas aeruginosa Strains A. HO~TACK~ and V. MAJTAN Researct~ Institute of Preventive and Clinical Medicine, 833 01 Bratislava, CzechoslovaMa Received February 3, 1992
ABSTNAcr. We analyzed in detail the permeability and cytotoxic activity as well as the serotypes of 127 Pseudomonas aeruginosa strains. Sixty-seven strains were isolated from immunoeompromised patients (51 from patients with tumors and 16 from patients after transplantation) and 60 strains were isolated from patient's ears. Culture filtrates of strains isolated from patients after transplantation were responsible for the highest part of permeability reactions corresponding to an intermediate toxin production (68.8 %) (categories 2 and 3) and culture filtrates of strains isolated from patients with tumors caused the highest percentage of permeability reactions corresponding to a strong toxin production. Culture filtrates of strains isolated from ears of patients were responsible for the highest percentage of negative permeability reactions (15 %). With positive permeability reaction size (categories 2 - 6 ) increased also the percentage of eytotoxicity as well as the intensity of morphological changes on Vero cells after 1 and 2 d. We did not observe any relationship between a particular permeability reaction category and the most frequent serotypes (04, 06) or nontypable strains of the tested groups.
Pseudomonas aemginosa is an important Gram-negative pathogen which usually infects compromised and immunosuppressed hosts and causes relevant infections mainly in the respiratory, urinary and gastrointestinal tract (Carney et aL 1973; Rodriques and Bodey 1979; Hoiby and Rosendal 1980; H~iby et aL 1987; Bauernfeind et al. 1987). Published data indicate increasing occurrence of P. aemginosa in connection with eye and ear diseases, with wounds, etc. (Kreger 1983; Kessler and Blumberg 1987). P. aemginosa can cause severe, fife-threatening and even fatal infections (Guzzo et aL 1991). The pathogenesis of pseudomonas infections is complex and involves in vivo production of several virulence factors (Liu 1974; Nicas and Iglewski 1985; Vasil 1986). In sequel to our previous work (Majtgn et al. 1992) we tried to characterize in detail the permeability reaction caused by P. aeruginosa strains isolated from immunocompromised patients and from the ears of patients as well as to reveal the possible relationship between the permeability reaction size and cytotoxie effect on Veto cells and serotype of the studied strains.
MATERIAL AND METHODS P. aeruginosa strains were isolated from urine and sputum of patients with a tumor (n = 51), from urine of patients after kidney transplantation (n = 16) as well as from the ears of patients (n = 60). Strains were selected according to their actual occurrence in connection with the above-mentioned diagnoses. Serotyping of strains was done by the agglutination method on a slide using diagnostic agglutinating sera (Institute of Sera and Vaccines, Prague). Culture filtrates of tested strains were prepared according to Majtfin et aL (1991). 0.4 mL of bacterial suspension (A600 = 0.4) was inoculated into 30 mL of proteose peptone (Oxoid, 20 g/L) and incubated on a reciprocal shaker at 37 ~ for 24 h. By centrifugation of bacterial suspensions and Ntration of their supernatants (SartoTius, 0.22 ~m) sterile culture filtrates were obtained. Assay for vascular permeability activity was done according to Sandefur and Peterson (1976). White rabbits (2.0-2.5 kg) were injected intradermally with 0.1 mL culture filtrates. One hour after inoculation, Evans blue (2 mL/kg of a 2 % solution) was injected i.v. into each rabbit. Reactions (zone of increased vascular permeability manifested by accumulation of Evans blue) were read after another hour. Quantitative classification of permeabifity reactions was done according to Turnbull et aL (1979). Cytotoxicity on Veto ceils was tested as described previously by Hogtackfi et aL (1992). After estabfishment of monolayers in 96-weU microtiter plates (3 x 104 Vero cells per well), the growth medium was removed and the tested sample (diluted 1:10 with growth medium containing 1 % bovine
P E R M E A B I L I T Y F A C T O R O F P. aeruginosa 361
1992
serum) was applied. Morphological changes were evaluated in an optical microscope (Jenaval), • 350, after i and 2 d.
RESULTS
The results of our previous study showed the high percentage of positive permeability reactions as well as considerable cytotoxic activity on Vero cells after application of culture filtrates prepared from strains isolated from immunocompromised patients (with tumors and after kidney transplantation) as well as from the ears of patients (Majtfin et al. 1992). Fully 100 % of culture filtrates (CFs) of P. aemginosa strains isolated from patients after transplantation, 90~2 % of CFs of strains isolated from patients with tumors as well as 85 % of CFs of strains isolated from ears of patients evoked rabbit skin permeability reactions which can be grouped according to their size (TurnbuU et aL 1979) into 2 - 6 categories (Table I). Culture filtrates classified ha category 1 (15 % of CFs of strains isolated from ears of patients and 9.8 % of CFs of strains isolated from patients with tumors) were considered negative. The greatest part of permeability reactions corresponding to an intermediate (68.8 %) toxin production (categories 2 and 3) were found after application of CFs of strains isolated from patients after transplantation and the highest percentage of permeability reactions corresponding to a strong (54.9 %) toxin production (categories 4 - 6 ) were caused by CFs of strains isolated from patients with tumors (Table I).
Table L Characterization of the permeability reactions o f culture filtrates (CFs) of
P. aeruginosa strains Category VPR a
Reaction zone diameter mm
1 2 3 4 5 6
0-4.9 5 - 9.9 10-14.9 15-19.9 20-24.9 25-29.9
CFs (% of CFs) b Tu
Tr
5 (9.8) 8 (15.7) 10 (19.6) 16 (31.4) 12 (23.5) 0 (9)
0 4 7 3 2 0
(0) (25.0) (43.8) (18.7) (12.5) (0)
E 9 (15) 18 (30) 10 (16.7) 13 (21.6) 9 (15.0) I (1.7)
aVascular permeability reaction. bTu - strains isolated from patients with tumors, T r - strains isolated from patients after transplantation, E - strains isolated from patient's ears. Table IL Results of testing of culture filtrates of P. aeruginosa strains for cytotoxicity with regard to individual vascular permeability reaction categories
Vero cells, P (% of p)b Category Tu
Tr
E
V~Ra ld
1 2 3 4 5" 6
1 (20.0) 2 (25.0) 4 (40.0) 15 (93.5) 12 (100) 0 (0)
2d
2 (40.0) 4 (50.0) 6 (60.0) 15 (93.7) 12 (100) 0 (0)
ld
2d
0 (0) 0 (0) 2 (28.5) 3 (66.6) 2 (100) 0 (0)
0 (0) 1 (25.0) 5 (71.5) 3 (100) 2 (i00) 0 (0)
ld
3 (33.3) 6 (33.3) 4 (40.0) 8 (61.5) 8 (89.0) 1 (100)
2d
6 (66.6) 11 (61.2) 8 (80.0) 12 (92.3) 9 (100) 1 (100)
aVascular permeability reaction. bp _ n u m b e r of positive culture filtrates, Tu - strains isolated from patients with tumors~ T r - strains is~olated from patients after transplantation, E - strains isolated from patient's ears. T h e total n u m b e r o f culture filtrates in individual V P R categories in tester groups of strains is mentioned in Table ][.
362
A. HOS'TACK/k and V. MAJTAN
Vol. 37
Further analysis of the results showed that increasing permeability reaction size (categories 2 - 6) was accompanied by an increased percentage of cytotoxicity as well as intensity of morphological changes after 1 and 2 d (Table II). PositMty of morphological changes on Vero cells caused by CFs of strains belonging to categories 4 - 6 of permeability reactions shifted from 60 to 100 % and was mainly connected with 50-100 % of Vero cell monolayer damage (cytotoxicity degree 3 and 4) (Table II, III). We recorded positive cytotoxic reactions also in the group of negative permeability reactions (category 1) in the case of strains isolated from patients with tumors and from patient's ears. Table HL Percentage of positivity of Vero cell damage with regard to individual vascular permeability reaction categories
Vero cells, %c Category CFsb
1
2
3
4
VPR a
1
6
ld
2d
ld
2d
Tu Tr E
20.0
20.0
0
20.0
33.3
11.1
0
Tu Tr E
12.5 0 11.1
37.5 50.0 22.2
Tu Tr E
30.0 0 40.0
Tu Tr E Tu Tr E Tu Tr E
ld
2d
ld
2d
0
0
0
0
22.2
0
33.3
0
0
12.5 0 22.2
0 0 16.8
0 0 0
12.5 0 22.2
0 0 0
0 0 0
30.0 43.0 40.0
10.0 28.5 0
20.0 0 20.0
0 0 0
10.0 28.5 10.0
0 0 0
0 0 10.0
12.5 0 30.8
0 0 15.4
43.5 0 23.0
0 0 15.4
37.5 66.6 0
68.7 100 7.7
0 0 7.7
25.0 0 53.8
0 0 0
0 0 0
0 0 11.1
0 0 0
75.0 100 77.9
16.6 50.0 33.3
25.0 0 0
83.4 50.0 66.6
0
0
*
* * 0
0
0
0
100
100
aVascutar permeability reaction. bCulture filtrates: Tu - strains isolated from patients with tumors, Tr - strains isolated from patients after transplantation, E - strains isolated from patient's ears. * - no culture filtrate of competent strains was classified to this VPR group. CDegree of cytotoxicity: 1 - up to c. 30 % destruction of monolayers (formation of intercellular spaces) 2 - up to c. 50 % destruction of monolayers (formation of intercellular spaces) 3 - destruction of monolayers up to 50 - 100 % (formation of large intercellular spaces, cell shrinking, detachment) 4 - detachable cell debris
It followed from our earlier work (Majtfin et al. 1992) that the most frequent strains isolated from patients with tumors were not typing strains (NT), the most frequent serotypes of strains isolated from patients after transplantation and from patient's ears were serotype 0 4 and 06, respectively. We wanted to know how these NT strains and the most frequent serotypes were associated with the permeability reaction categories in the individual tested group. The results showed that the NT strains were connected with categories 2, 4 and 5. Serotype O4, the one most frequently isolated from patients after transplantation, was connected with all positive permeability reaction categories and the percentage of its occurrence increased with increasing permeability reaction size (Table IV). In the case of strains isolated from patient's ears with the most frequent serotype 0 6 there was a connection with categories 2 - 5 as well as with category I (negative permeability reaction).
P E R M E A B I L I T Y F A C T O R O F P. aeruginosa 363
1992
Table IV. Percentage of occurrence of serotypes (O1 - O16) of P. aeruginosa strains with regard to individual vascular permeability reaction categories
Cat.
cFs b
O1
02
03
04
05
06
07
0
20.0
0
0
0
0
0
08
09
O10
Oll
O12
0
40.0
0
0
0
O16
NrFc
PA d
0
0
40.0
0
0
0
37.5 0 5.5
12.5 0 0
0 0 0
10.0 0 0
VPR a Tu
6
Tr E
* 22.2
0
11.1
0
0
44.4
0
22.2
0
0
0
0
Tu Tr E
12.5 0 16.7
0 0 0
12.5 0 22.3
0 50.0 0
0 0 0
12.5 0 27.8
0 0 0
0 0 16.7
0 25.0 0
0 0 0
0 25.0 5.5
12.5 0 0
0 0 5.5
Tu Tr E
0 0 20.0
0 0 0
10.0 0 30.0
0 57.0 0
20.0 0 0
10.0 0 20.0
0 0 0
0 0 10.0
10.0 14.4 0
10.0 0 0
0 0 10.0
30.0 28.6 10.0
0 0 0
Tu Tr E
6.2 0 0
0 0 0
0 0 0
18.9 1~ 0
6.2 0 7.7
6.2 0 30.8
0 0 0
0 0 15.4
6.2 0 7.7
18.9 0 0
6.2 0 7.7
6.2 0 7.7
0 0 7.7
18.8 0 15.4
6.2 0 0
Tu Tr E
25.0 0 33.3
0 0 0
8.3 0 0
8.3 0 11.1
0 0 0
0 0 0
0 0 11.1
8.3 0 0
8.3 0 22.2
0 0 0
0 0 0
33.5 0 0
0 0 0
Tu Tr E
0
0
0
0
0
0
0
0
0
8.3 0 0 100 22.2 0 0
0
0
0
100
0
* *
aVascular permeability reaction. bCulture filtrates: Tu - strains isolated from patients with tumors, Tr - strains isolated from patients after transplantation, E - strains isolated from patient' ears. CNontypable strains. dpolyagglutinating strains. * - no strain isolated from a competent group of patients was classified to this V P R group. The total numb er of culture filtrates in individual V P R categories in tested groups of strains is mentioned in Table I.
DISCUSSION
Our previous papers regarding the testing of biological and enzymic activity of Po aemginosa strains isolated from different clinical materials as well as from the environment showed considerable toxic potential (Majtfin et aL 1991; Hogtackfi and Majt~n 1992)o We also observed a high percentage of rabbit skin vascular permeability increase as well as considerable cytotoxic activity on Vero cells by evaluation of toxinogenicity of strains isolated from immunocompromised patients and from ears of patients (Majt~in et al. 1992). Though the percentage of permeability increase was high in each of the tested group of strains, we observed by analyzing the vascular permeability reaction differences between studied groups as to their permeability reaction sizes which reflect the presence of toxin (permeability factor). Culture filtrates (CFs) prepared from strains isolated from patients after transplantation were responsible for the highest percentage of permeability reactions corresponding to strong toxin production (54.9 %) (categories 4-6) and CFs of strains isolated from patients with tumors were responsible for the highest part of permeability reactions corresponding to an intermediate toxin production (68.8 %) (categories 2, 3). On the other hand, CFs of strains isolated from patient's ears wzre responsible for the highest percentage of negative permeability reactions (category 1) and showed very poor or no toxin production. Interesting was the fact that the percentage of cytotoxicity and intensity of morphological changes on Vero cells increased with increasing category of permeability reactions evoked by culture filtrates of all tested groups of strains. This percentage lay in the range from 60 to 100 % after application of CFs belonging to permeability reaction categories corresponding to a strong toxin production (categories 4-6). Similar results were also reached by testing the cytotoxicity of 89 B. cereus strains
364
A. HOS'TACKA and V. MAJT/~d'q
Vol. 37
isolated from different foodstuffs (Hogtack~i et aL 1992). In this case all culture filtrates connected with a strong toxin production caused a cytotoxic effect on Vero cells. It is known from published data that serotypes of strains of some bacteria correlate well with pathogenicity. A certain limited number of E. coli serotypes is for example associated with toxin production (Waehsmuth 1980)o By looking for a relationship between permeability reaction size reflecting toxin production and the most frequent serotypes or nontypable strains (the ones most frequently isolated from patients with tumors) we did not find such a correlation here. Nontypable strains were connected with permeability reaction categories 2, 4 and 5, 0 4 serotype (the one most frequently isolated from patients after transplantation) was associated with all positive permeabifity reaction categories and the 0 6 serotype (the one most frequently isolated from ears of patients) was connected with all positive permeability reaction categories ( 2 - 5) as well as with category 1 (negative permeability). The results suggest a relationship between the permeability reaction size and cytotoxic activity; then again, we did not observe any correlation between the permeability reaction size and the most frequent serotypes or nontypable strains.
REFERENCES
BAUERNFEINDA., NABER K., SAUERWEIND.: Spectrum of bacterial pathogens in uncomplicated and complicated urinary tract infections. Eur. tyrol. 13 (Suppl. 1), 9 - 1 2 (1987). CARNEY S.A., DYSTER ILE., JONES ]LJ.: The invasion of burned skin by Pseudomonas aeruginosa. Brit.J.Dermatol. 88, 539-545 (1973). G~dzzo J., DUONG F., WANDERSMANC., MURGIER M., LAZDUNSKiAo: The secretion genes of Pseudomonas aeruginosa alkaline protease are functionally related to those of Erwinia chrysanthemi proteases and Escherichia coli a-hemolysin. MoLMicrobiol. 5, 447-453 (1991). H~IBY N, ROSENDALK.." Epidemiology of Pseudomonas aeruginosa infection in patients treated at a cystic fibrosis center. Acts PathoLMicrobioLScand.Sect.B 88, 125-131 (1980). Horny N., D6R~NG G., Scmcrrz P.O.: Pathogenic mechanisms of chronic Pseudomonas aeruginosa infections in cystic fibrosis patients. Antibiot.Chemother. 39, 69- 76 (1987). Ho~rAcKA A., KOgIAROVAA., MAJTAN V., KOHOTOVAS.: Toxic properties of Bacillus cereus strains isolated from different foodstuffs. ZbLBakt. 276, 303-312 (1992). HOS~ACKA A., MA.rrANV.: Bfological and enzymic activity of Pseudomonas aeruginosa strains. (In Slovak) Biol6gia, in press (1992). KESSLER E.~ BLUMBERGS.: Specific inhibitors of Pseudomonas aeruginosa as potential drugs for the treatment of Pseudomonas keratitis. Antibiot.Chemother. 39, 103-112 (1987). KREGER A.S.: Pathogenesis of Pseudomonas aeruginosa ocular diseases. Rev.Infect.Dis. 5 (Suppl. 5), $931- $935 (1983). LIu P.V.: Extracellular toxins of Pseudomonas aeruginosa. J.Infect.Dis. 130 (Suppl.), $94- $99 (1974). MArrAN V., Ho~rACKA A., KOglAROVX A., MArrANovA E., KOHOTOVA S.: On the toxinogenity of Pseudomonas aeruginosa strains. ZHyg.EpidemioLMicrobiol.Immunol. 35, 217-224 (1991). MArrAN V , HORrACKAA~, ~TKOVI~ B., KOvAcs P.: Toxinogenic potential of Pseudomonas aeruginosa strains isolated from immunocompromised patients. (In Slovak) Biol6gia, in press (1992). NICAS T.L, IGLEWSKI B.H.: The contribution of exoproducts to virulence of Pseudomonas aeruginosa. Can.ZMicrobioL 31, 387- 392 (1985). ROOmQUES V., BOoEY G.P.: Epidemiology, clinical manifestations and treatment in cancer patients, pp. 368-404 in Pseudomonas aeruginosa Clinical Manifestations of Infection and Current Therapy (R.G. Dogyett, Ed). Academic Press, New York 1979. SANDEFUR P.D., PErEP.SOIq J.W.: Isolation of skin permeability factors from culture filtrates of Salmonella lyphimurium. Infect.Immun. 14, 671-679 (1976). TURNBULL P.C.B., KRAMERJ.M., J~RGENSEN K., GILBERT P,.J., MELLINGJ.: Properties and production characteristics of vomiting, diarrheal and necrotizing toxins of Bacillus cereus. Mm.J.Clin.Nutr. 32, 219 - 228 (1979). VAS]L M.L.: Pseudomonas aeruginosa: biology, mechanisms of virulence, epidemiology. ZPediatr.Res. 108, 800 -805 (1986). WACHSMOTH I.IC: Esclmrichia coli typing and pathogenicity. Clin.Microbiol.Newsletter 2, 4 - 7 (1980).
Permeability Factor, Cytotoxicity and Serotyping of Pseudomonas aeruginosa Strains A. HO~TACK~ and V. MAJTAN Researct~ Institute of Preventive and Clinical Medicine, 833 01 Bratislava, CzechoslovaMa Received February 3, 1992
ABSTNAcr. We analyzed in detail the permeability and cytotoxic activity as well as the serotypes of 127 Pseudomonas aeruginosa strains. Sixty-seven strains were isolated from immunoeompromised patients (51 from patients with tumors and 16 from patients after transplantation) and 60 strains were isolated from patient's ears. Culture filtrates of strains isolated from patients after transplantation were responsible for the highest part of permeability reactions corresponding to an intermediate toxin production (68.8 %) (categories 2 and 3) and culture filtrates of strains isolated from patients with tumors caused the highest percentage of permeability reactions corresponding to a strong toxin production. Culture filtrates of strains isolated from ears of patients were responsible for the highest percentage of negative permeability reactions (15 %). With positive permeability reaction size (categories 2 - 6 ) increased also the percentage of eytotoxicity as well as the intensity of morphological changes on Vero cells after 1 and 2 d. We did not observe any relationship between a particular permeability reaction category and the most frequent serotypes (04, 06) or nontypable strains of the tested groups.
Pseudomonas aemginosa is an important Gram-negative pathogen which usually infects compromised and immunosuppressed hosts and causes relevant infections mainly in the respiratory, urinary and gastrointestinal tract (Carney et aL 1973; Rodriques and Bodey 1979; Hoiby and Rosendal 1980; H~iby et aL 1987; Bauernfeind et al. 1987). Published data indicate increasing occurrence of P. aemginosa in connection with eye and ear diseases, with wounds, etc. (Kreger 1983; Kessler and Blumberg 1987). P. aemginosa can cause severe, fife-threatening and even fatal infections (Guzzo et aL 1991). The pathogenesis of pseudomonas infections is complex and involves in vivo production of several virulence factors (Liu 1974; Nicas and Iglewski 1985; Vasil 1986). In sequel to our previous work (Majtgn et al. 1992) we tried to characterize in detail the permeability reaction caused by P. aeruginosa strains isolated from immunocompromised patients and from the ears of patients as well as to reveal the possible relationship between the permeability reaction size and cytotoxie effect on Veto cells and serotype of the studied strains.
MATERIAL AND METHODS P. aeruginosa strains were isolated from urine and sputum of patients with a tumor (n = 51), from urine of patients after kidney transplantation (n = 16) as well as from the ears of patients (n = 60). Strains were selected according to their actual occurrence in connection with the above-mentioned diagnoses. Serotyping of strains was done by the agglutination method on a slide using diagnostic agglutinating sera (Institute of Sera and Vaccines, Prague). Culture filtrates of tested strains were prepared according to Majtfin et aL (1991). 0.4 mL of bacterial suspension (A600 = 0.4) was inoculated into 30 mL of proteose peptone (Oxoid, 20 g/L) and incubated on a reciprocal shaker at 37 ~ for 24 h. By centrifugation of bacterial suspensions and Ntration of their supernatants (SartoTius, 0.22 ~m) sterile culture filtrates were obtained. Assay for vascular permeability activity was done according to Sandefur and Peterson (1976). White rabbits (2.0-2.5 kg) were injected intradermally with 0.1 mL culture filtrates. One hour after inoculation, Evans blue (2 mL/kg of a 2 % solution) was injected i.v. into each rabbit. Reactions (zone of increased vascular permeability manifested by accumulation of Evans blue) were read after another hour. Quantitative classification of permeabifity reactions was done according to Turnbull et aL (1979). Cytotoxicity on Veto ceils was tested as described previously by Hogtackfi et aL (1992). After estabfishment of monolayers in 96-weU microtiter plates (3 x 104 Vero cells per well), the growth medium was removed and the tested sample (diluted 1:10 with growth medium containing 1 % bovine
P E R M E A B I L I T Y F A C T O R O F P. aeruginosa 361
1992
serum) was applied. Morphological changes were evaluated in an optical microscope (Jenaval), • 350, after i and 2 d.
RESULTS
The results of our previous study showed the high percentage of positive permeability reactions as well as considerable cytotoxic activity on Vero cells after application of culture filtrates prepared from strains isolated from immunocompromised patients (with tumors and after kidney transplantation) as well as from the ears of patients (Majtfin et al. 1992). Fully 100 % of culture filtrates (CFs) of P. aemginosa strains isolated from patients after transplantation, 90~2 % of CFs of strains isolated from patients with tumors as well as 85 % of CFs of strains isolated from ears of patients evoked rabbit skin permeability reactions which can be grouped according to their size (TurnbuU et aL 1979) into 2 - 6 categories (Table I). Culture filtrates classified ha category 1 (15 % of CFs of strains isolated from ears of patients and 9.8 % of CFs of strains isolated from patients with tumors) were considered negative. The greatest part of permeability reactions corresponding to an intermediate (68.8 %) toxin production (categories 2 and 3) were found after application of CFs of strains isolated from patients after transplantation and the highest percentage of permeability reactions corresponding to a strong (54.9 %) toxin production (categories 4 - 6 ) were caused by CFs of strains isolated from patients with tumors (Table I).
Table L Characterization of the permeability reactions o f culture filtrates (CFs) of
P. aeruginosa strains Category VPR a
Reaction zone diameter mm
1 2 3 4 5 6
0-4.9 5 - 9.9 10-14.9 15-19.9 20-24.9 25-29.9
CFs (% of CFs) b Tu
Tr
5 (9.8) 8 (15.7) 10 (19.6) 16 (31.4) 12 (23.5) 0 (9)
0 4 7 3 2 0
(0) (25.0) (43.8) (18.7) (12.5) (0)
E 9 (15) 18 (30) 10 (16.7) 13 (21.6) 9 (15.0) I (1.7)
aVascular permeability reaction. bTu - strains isolated from patients with tumors, T r - strains isolated from patients after transplantation, E - strains isolated from patient's ears. Table IL Results of testing of culture filtrates of P. aeruginosa strains for cytotoxicity with regard to individual vascular permeability reaction categories
Vero cells, P (% of p)b Category Tu
Tr
E
V~Ra ld
1 2 3 4 5" 6
1 (20.0) 2 (25.0) 4 (40.0) 15 (93.5) 12 (100) 0 (0)
2d
2 (40.0) 4 (50.0) 6 (60.0) 15 (93.7) 12 (100) 0 (0)
ld
2d
0 (0) 0 (0) 2 (28.5) 3 (66.6) 2 (100) 0 (0)
0 (0) 1 (25.0) 5 (71.5) 3 (100) 2 (i00) 0 (0)
ld
3 (33.3) 6 (33.3) 4 (40.0) 8 (61.5) 8 (89.0) 1 (100)
2d
6 (66.6) 11 (61.2) 8 (80.0) 12 (92.3) 9 (100) 1 (100)
aVascular permeability reaction. bp _ n u m b e r of positive culture filtrates, Tu - strains isolated from patients with tumors~ T r - strains is~olated from patients after transplantation, E - strains isolated from patient's ears. T h e total n u m b e r o f culture filtrates in individual V P R categories in tester groups of strains is mentioned in Table ][.
362
A. HOS'TACK/k and V. MAJTAN
Vol. 37
Further analysis of the results showed that increasing permeability reaction size (categories 2 - 6) was accompanied by an increased percentage of cytotoxicity as well as intensity of morphological changes after 1 and 2 d (Table II). PositMty of morphological changes on Vero cells caused by CFs of strains belonging to categories 4 - 6 of permeability reactions shifted from 60 to 100 % and was mainly connected with 50-100 % of Vero cell monolayer damage (cytotoxicity degree 3 and 4) (Table II, III). We recorded positive cytotoxic reactions also in the group of negative permeability reactions (category 1) in the case of strains isolated from patients with tumors and from patient's ears. Table HL Percentage of positivity of Vero cell damage with regard to individual vascular permeability reaction categories
Vero cells, %c Category CFsb
1
2
3
4
VPR a
1
6
ld
2d
ld
2d
Tu Tr E
20.0
20.0
0
20.0
33.3
11.1
0
Tu Tr E
12.5 0 11.1
37.5 50.0 22.2
Tu Tr E
30.0 0 40.0
Tu Tr E Tu Tr E Tu Tr E
ld
2d
ld
2d
0
0
0
0
22.2
0
33.3
0
0
12.5 0 22.2
0 0 16.8
0 0 0
12.5 0 22.2
0 0 0
0 0 0
30.0 43.0 40.0
10.0 28.5 0
20.0 0 20.0
0 0 0
10.0 28.5 10.0
0 0 0
0 0 10.0
12.5 0 30.8
0 0 15.4
43.5 0 23.0
0 0 15.4
37.5 66.6 0
68.7 100 7.7
0 0 7.7
25.0 0 53.8
0 0 0
0 0 0
0 0 11.1
0 0 0
75.0 100 77.9
16.6 50.0 33.3
25.0 0 0
83.4 50.0 66.6
0
0
*
* * 0
0
0
0
100
100
aVascutar permeability reaction. bCulture filtrates: Tu - strains isolated from patients with tumors, Tr - strains isolated from patients after transplantation, E - strains isolated from patient's ears. * - no culture filtrate of competent strains was classified to this VPR group. CDegree of cytotoxicity: 1 - up to c. 30 % destruction of monolayers (formation of intercellular spaces) 2 - up to c. 50 % destruction of monolayers (formation of intercellular spaces) 3 - destruction of monolayers up to 50 - 100 % (formation of large intercellular spaces, cell shrinking, detachment) 4 - detachable cell debris
It followed from our earlier work (Majtfin et al. 1992) that the most frequent strains isolated from patients with tumors were not typing strains (NT), the most frequent serotypes of strains isolated from patients after transplantation and from patient's ears were serotype 0 4 and 06, respectively. We wanted to know how these NT strains and the most frequent serotypes were associated with the permeability reaction categories in the individual tested group. The results showed that the NT strains were connected with categories 2, 4 and 5. Serotype O4, the one most frequently isolated from patients after transplantation, was connected with all positive permeability reaction categories and the percentage of its occurrence increased with increasing permeability reaction size (Table IV). In the case of strains isolated from patient's ears with the most frequent serotype 0 6 there was a connection with categories 2 - 5 as well as with category I (negative permeability reaction).
P E R M E A B I L I T Y F A C T O R O F P. aeruginosa 363
1992
Table IV. Percentage of occurrence of serotypes (O1 - O16) of P. aeruginosa strains with regard to individual vascular permeability reaction categories
Cat.
cFs b
O1
02
03
04
05
06
07
0
20.0
0
0
0
0
0
08
09
O10
Oll
O12
0
40.0
0
0
0
O16
NrFc
PA d
0
0
40.0
0
0
0
37.5 0 5.5
12.5 0 0
0 0 0
10.0 0 0
VPR a Tu
6
Tr E
* 22.2
0
11.1
0
0
44.4
0
22.2
0
0
0
0
Tu Tr E
12.5 0 16.7
0 0 0
12.5 0 22.3
0 50.0 0
0 0 0
12.5 0 27.8
0 0 0
0 0 16.7
0 25.0 0
0 0 0
0 25.0 5.5
12.5 0 0
0 0 5.5
Tu Tr E
0 0 20.0
0 0 0
10.0 0 30.0
0 57.0 0
20.0 0 0
10.0 0 20.0
0 0 0
0 0 10.0
10.0 14.4 0
10.0 0 0
0 0 10.0
30.0 28.6 10.0
0 0 0
Tu Tr E
6.2 0 0
0 0 0
0 0 0
18.9 1~ 0
6.2 0 7.7
6.2 0 30.8
0 0 0
0 0 15.4
6.2 0 7.7
18.9 0 0
6.2 0 7.7
6.2 0 7.7
0 0 7.7
18.8 0 15.4
6.2 0 0
Tu Tr E
25.0 0 33.3
0 0 0
8.3 0 0
8.3 0 11.1
0 0 0
0 0 0
0 0 11.1
8.3 0 0
8.3 0 22.2
0 0 0
0 0 0
33.5 0 0
0 0 0
Tu Tr E
0
0
0
0
0
0
0
0
0
8.3 0 0 100 22.2 0 0
0
0
0
100
0
* *
aVascular permeability reaction. bCulture filtrates: Tu - strains isolated from patients with tumors, Tr - strains isolated from patients after transplantation, E - strains isolated from patient' ears. CNontypable strains. dpolyagglutinating strains. * - no strain isolated from a competent group of patients was classified to this V P R group. The total numb er of culture filtrates in individual V P R categories in tested groups of strains is mentioned in Table I.
DISCUSSION
Our previous papers regarding the testing of biological and enzymic activity of Po aemginosa strains isolated from different clinical materials as well as from the environment showed considerable toxic potential (Majtfin et aL 1991; Hogtackfi and Majt~n 1992)o We also observed a high percentage of rabbit skin vascular permeability increase as well as considerable cytotoxic activity on Vero cells by evaluation of toxinogenicity of strains isolated from immunocompromised patients and from ears of patients (Majt~in et al. 1992). Though the percentage of permeability increase was high in each of the tested group of strains, we observed by analyzing the vascular permeability reaction differences between studied groups as to their permeability reaction sizes which reflect the presence of toxin (permeability factor). Culture filtrates (CFs) prepared from strains isolated from patients after transplantation were responsible for the highest percentage of permeability reactions corresponding to strong toxin production (54.9 %) (categories 4-6) and CFs of strains isolated from patients with tumors were responsible for the highest part of permeability reactions corresponding to an intermediate toxin production (68.8 %) (categories 2, 3). On the other hand, CFs of strains isolated from patient's ears wzre responsible for the highest percentage of negative permeability reactions (category 1) and showed very poor or no toxin production. Interesting was the fact that the percentage of cytotoxicity and intensity of morphological changes on Vero cells increased with increasing category of permeability reactions evoked by culture filtrates of all tested groups of strains. This percentage lay in the range from 60 to 100 % after application of CFs belonging to permeability reaction categories corresponding to a strong toxin production (categories 4-6). Similar results were also reached by testing the cytotoxicity of 89 B. cereus strains
364
A. HOS'TACKA and V. MAJT/~d'q
Vol. 37
isolated from different foodstuffs (Hogtack~i et aL 1992). In this case all culture filtrates connected with a strong toxin production caused a cytotoxic effect on Vero cells. It is known from published data that serotypes of strains of some bacteria correlate well with pathogenicity. A certain limited number of E. coli serotypes is for example associated with toxin production (Waehsmuth 1980)o By looking for a relationship between permeability reaction size reflecting toxin production and the most frequent serotypes or nontypable strains (the ones most frequently isolated from patients with tumors) we did not find such a correlation here. Nontypable strains were connected with permeability reaction categories 2, 4 and 5, 0 4 serotype (the one most frequently isolated from patients after transplantation) was associated with all positive permeabifity reaction categories and the 0 6 serotype (the one most frequently isolated from ears of patients) was connected with all positive permeability reaction categories ( 2 - 5) as well as with category 1 (negative permeability). The results suggest a relationship between the permeability reaction size and cytotoxic activity; then again, we did not observe any correlation between the permeability reaction size and the most frequent serotypes or nontypable strains.
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