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Haemagglutinins and adherence properties to HeLa and intestine 407 cells of Klebsiella pneumoniae and Klebsiella oxytoca isolates
Zbl. Bakt. Hyg. A 263, 585-593 (1987)
Haemagglutinins and Adherence Properties to HeLa and Intestine 407 Cells of Klebsiella pneumoniae and Klebsiella oxytoca Isolates R. PODSCHUN, P. HEINEKEN, and H. G. SONNTAG Hygiene Institute, University of Heidelberg, Heidelberg, Federal Republic Germany
With 2 Figures' Received June 6, 1986 · Accepted September 23, 1986
Summary The occurrence of haemagglutination (HA) and adherence properties were examined in 50 strains of K. pneumoniae and 17 K. oxytoca strains isolated from humans. All isolates except three exhibited HA activity. Mannose-sensitive haemagglutinins (MSHA) were expressed by the majority of K. pneumoniae strains, but only by one K. oxytoca isolate. Mannose-resistant haemagglutination (MRHA) to human or guinea pig erythrocytes could not be detected; haemagglutinins of the MRiK type were found in both species with similar frequencies. Adhesive properties could be demonstrated in K. pneumoniae as often as in K. oxytoca: About half of the strains adhered to two human cell lines: HeLa and Intestine 407. The incidence of HA activity was similar in adhering and nonadhering strains. A correlation between MSHA, MRlK-HA and adherence to tissue-cultured cells could not be detected. Zusammenfassung 50 K. pneumoniae- und 17 K. oxyrocs-Stamme menschlicher Herkunft wurden auf das Vorkommen harnagglutinierender undd adharierender Eigenschaften untersucht. Mit Ausnahme dreier Isolate zeigten aile Stamme Hamagglutinationsaktivitat, Mannose-sensitive Hamagglutinine (MSHA) besaf die Mehrzahl der K. pneumoniae-Isolate, aber nur ein K. oxytoca-Stamm. Eine Mannose -resistente Hamagglutination (MRHA) gegeniiber humanen oder Meerschweinchen-Erythrozyten wurde nicht gefunden; MRlK-Hamagglutinine wur den in heiden Spezies mit gleicher Haufigkeit ermittelt. Ein Adharenzvermogen konnte bei K. pneumoniae genauso haufig wie bei K. oxytoca nachgewiesen werden: Etwa die Halfte beider Spezies adharierten an die humanen Zellinien HeLa und Intestine 407 . Adharierende und nichtadharierende Starnme zeigten ein vergleichbar haufiges Auftreten hamag glutinierender Eigenschaften. Eine Verbindung zwischen dem Besitz von MSHA, MRlK-HA und Adhasinen konnte nicht gezeigt werden. Introduction The ability of bacteria to adhere to mammalian cell surfaces, though recognized for a long time, ha s become a field of intensified research in the last decade because it is
586
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presumed to be an important pathogenicity factor. The nature of bacterial adhesins is presently under investigation, however, surface appendages called 'pili' or 'fimbriae' are reported to mediate adherence in man y gram -negati ve pathogens such as Enterobacteriaceae (1, 6, 7, 8, 11, 15, 19,20,24), Pseudomonas (29), Neisseria (26,28) and Bordetella (27). Bacterial adhesins have been classified by their haemagglutinating properties. Haemagglutination of guinea-pig erythrocytes inhibited by the presence of D-mannose (rnannose-sensitive haemagglutination, MSHA) is attributed to common or type 1 pili on bacterial surfaces (3, 4). Bacteria causing agglutination of erythrocytes without inhibition by D-mannose are expressing mannose-resistant haemagglutinins (MRHA) . Svanborg Eden and coworkers (9) reported that urinary isolates of E. coli with mannose-resistant haemagglutination of human erythrocytes adhered in high numbers to urinary tract epithelial cells, whereas bacteria inducing man nose-sensitive haemagglutination alone attached only in low numbers. Two haemagglutinins are very common in Klebsiella species: Most K. pneumoniae strains show man nose-sensitive haemagglutination of guinea-pig erythrocytes and mannose-resistant haemagglutination of tanned erythrocytes (3, 17). The latter type, found in the tribes Klebsielleae and Proteae, is associated with tye 3 pili (5, 14, 16) and designated the MR/K (mannose-resistant and Klebsiella-like) haemagglutinin. Relative ly little is known about the role of type 3 pili in adherence to mammalian cells, however, Korhonen et al. (14) demonstrated type 3 pili to mediate adhesion of Klebsiella to plant roots. In respect to the relation of type 1 pili and adherence properties, Fader et al. (6, 7) reported the piliated, MSHA-positive phase of a K. pneumoniae strain to attach to rat bladder epithelial cells, whereas the nonpiliated, MSHA-negative phase did not adhere. The present study was designed to investigate the occurrence and pattern of haernagglutination of 67 Klebsiella pneumoniae and K. oxytoca strains isolated from clinical specimens as well as their adherence activities to tissue-cultured cells. Furthermore, adherence and haemagglutination should be compared in order to demonstrate a poss ible correlation between these properties.
Materials and Methods
Bacteria. A total of 67 Klebsiella strains, mainly isolated from the urogenital tract, respiratory secretions and wounds, were obtained from our microbiology laboratory . Sites of isolation were revealed by the laboratory records. 50 isolates could be classified as Klebsiella pneumoniae, 17 strains as K. oxytoca by API 20 E (API SystemsS. A., MontalieuVercieu, France). All isolates were kept at 4 °C on deep agar slants of brain-heart-infusion (Merck, Darmstadt) and subcultured once on blood agar plates before use. K. pneumoniae ATCC 27736 was used as reference strain. Haemagglutination tests. Citrated fresh blood samples from humans with blood group A, guinea-pigs and ox were washed three times (PBS, pH 7.2) and adjusted to 5 x 108 erythrocytes/m!. To obtain tanned ox red blood cells, one volume of washed erythrocytes (5 X 108/ ml) was treated with one volume of 0.003% tannic acid for 10 min at 3rc. Tanned erythrocytes were washed twice in PBS and adjusted to a final concentration of 5 x 108 RBC/m!. Bacteria were grown statically twice for 48 h periods at 37 °C in brain-heart-infusionbroth (BHIB). Cells were harvested by centrifugation and the bacterial pellets were resuspended in PBS giving a final concentration of 101°_1011 bacteria/m!. 50 f.t! of bacterial
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suspensions were mixed with 50 III of erythroc ytes in a porcelain tile (with or without 1% mannose ). Gently agitated, haemagglutination was observed for 3 min at room temperature and read finally after further incubation for 10 min at 4 °C. Adherence assay. HeLa and Intestine 407 cells (Flow Laboratories, Irvine, U. K.) were used to study the adherence properties of the strains . The cells were maintained in Eagle minimal essential medium (Flow Laboratories), supplemented with 10% fetal calf serum, 2 mM glutamine, 100 IU/ml penicillin, 100 ug/rnl streptomycin and 0.25 ug/ml fungizone (Gibco, Paisley, Scotland). For adhesion tests, 104 cells were seeded into each well of 10-well multitest slides (Flow) and grown for 48 h in a humidified chamber with 5 % CO 2 at 24 h before treatment with bacteria the medium was replaced by antibiotic-free medium. Bacteria were grown serially twice in tubes of BHI-broth incubated staticall y at 37 °C for 48 h period s. Cultures were washed three times with PBS, pH 7.2, passed through a 21gauge-needle to disperse aggregates and adjusted to 10 9 bacter ia/m!. Tissue cell mono layers were washed three times with PBS and 50 III bacterial suspension/ well was added. The slides were gently agitated for 30 min at 37 °C. Nonadhering bacteria were removed by washing the slides several times with PBS. The monolayers were fixed in methanol (5 min), stained in 10% Giemsa solution (10 min) and washed. Adherent bacteria on 100 individual cells were counted with the aid of a light microscope and the adhesion rate was calculated as the mean number of bacteria per cel!. Isolates with less than 1 bacterium/cell were regarded as nonadhesive. Correlation analysis. The intensities of single hemagglutination types as well as combinations were compared with the corresponding adherence ratios in the different cell lines. The probability of two features to be related were calculated by correlation coefficients, based on Kendalls tau b (10).
3rc.
Results
Haemagglutinating activity. Both species differed markedly in their ability to agglutinate guinea-pig erythrocytes: Most isolates of Klebsiella pneumoniae exhibited MS (mannose-sensitive) -haemagglutinins (72%), whereas the majority of K. oxytoca strains (94 %), despite cultural conditions allowing pellicle formation (4 ), did not agglutinate red blood cells of this species (Fig. Ia), Neither against guinea-pig nor against human A erythrocytes an y isolate showed MRHA (rnannose-resistant haemagglutination)-activity. MSHA of human erythrocytes was present in 24 % of K. pneumoniae and in 6 % of K. oxytoca strains (Fig. Ib ), With respect to MR/K-haemagglutinins, both specie s demonstrated comparable numbers of strains active against tanned ox erythrocytes. About 82 % of K. oxytoca and 90 % of K. pneumoniae isolates showed this haemagglutination type, resistant to the action of D-mannose (Fig. Ic ). Table 1. Distribution of haemagglutinins in Klebsiella strains HA rype
K. pneumoniae (n = 50)
K. oxytoca (n = 17)
only MSHA present only MRlK-HA present MS- and MRlK-HA present MS- and MRlK-HA negative
4 (8% ) 11 (22% ) 34 (68% ) 1 (2% )
1 (6% ) 13 (76%) 1 (6%) 2 (12%)
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R. Podschun, P. Heineken, and H . G. Sonntag
P e
r
C e n t II
9
e
100 90 80 70 60 50 40 30 20 10 0
+
I_
++ HA intensity
K.oxytOCll (n= 17)
o
+++
Kpneumoni lle (n=50)
Fig. 1. Haem agglutinating activity of Klebsiella isolates. Frequ ency and inten sity of MSHA to guinea pig erythrocytes (A), human A red blood cells (B) and MRlK -HA to tanned ox erythrocytes (C). Intensity of haemagglutination is graded from + to +++.
Haemagglutinins and Adherence Properties
589
45 P 40
e 35
r
C
30
e 25 n 20
t
II
15
9 10 e 5
o
, -
1-2
2-3 3-4 4- 5 number of bact eria/cell
K.oxytOCll (n= 17)
0
'5
Kpneumon ill e (n=50)
I
60 p
e 50
r 40 C e 30 n l 20
II
9 10 e
0
1-2
2-3 3-4 4-5 number of bacteria/cel l
'5
Fig. 2. Distribution of adherence among Klebsiella isolates to HeLa cells (A) and Intestine 407 cells (B). Adhesion is expressed as the mean number of bacterialtissue-cultured cell.
Most K. pneumoniae isolates simultaneously expressed MSHA and MR/K-HA (68%), whereas the great majority of K. oxytoca strains (76% ) exhibited only the MR/ K-type (Table 1). Twelve percent of K. oxytoca isolates, but only one K. pneumoniae strain (2%) did not show any haemagglutination type investigated. Adherence to tissue-cultured cells. Isolates were regarded as adhesive if the adhesion rate was greater/equal 1 bacterium/cell. To HeLa cells as well as to Intestine 407 cells both species exhibited only low adherence rates (1-10 bacteria/cell). On an average, the strains adhered in greater numbers and more frequently to HeLa cells. Adherence ratios are shown in Fig. 2. Reproducibility of the test results was satisfactory: Adhesion rates of a nonadhering strain (KPN 120), a low binding (KOX 135) and a moderate binding strain (KPN 47) in independent tests were determined at least four times. Only little variation in adhesiveness was found : Mean numbers of bacte ria adhering to each cell were observed to differ from 0.1-0.3 (KPN 120), 1.6-2.0 (KOX 135) and from 4.2-4.6 (KPN 47).
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R. Podschun, P. Heineken, and H. G. Sonntag
Both species showed similar frequencies of adhesive isolates: 29 (58%) of the K.
pneumoniae strains adhered to HeLa cells, 20 (40%) to Intestine 407 cells; the corresponding frequencies for K. oxytoca were 65% (11 strains) and 47% (8 strains). With regard to HeLa cell adhesion, 23 (58%) of the 40 adhering isolates showed MSHA and 36 strains (90%) exhibited MR/K-HA activity. Comparable incidences were observed in the 27 nonadhering isolates: 16 strains (59%) were MSHA-positive and in 22 strains (81%) MR/K-HA was present. Intensity and pattern of haemagglutination were compared with adherence ratios of K. pneumoniae and K. oxytoca. A correlation analysis, based on Kendalls tau b correlation coefficients, did not reveal any relation i). between possession of haemagglutinins and adherence properties or ii). between expression of a single haemagglutination type investigated and adherence to tissue-cultured cells. In each case, correlation coefficients showed values (Kendall's tau b) of less than 0.5.
Discussion Bacterial haemagglutinins are considered to be correlated to adherence properties to eucaryotic cells. Svanborg-Eden and coworkers reported the significance of MRHA in adhesion of urinary tract isolates of E. coli to uroepithelial cells (9), whereas strains with MSHA activity alone attached only in low numbers or not at all to these cells. On the other hand, Fader et al. (6, 7) demonstrated MSHA to be responsible for the adhesion of K. pneumoniae to rat bladder cells. However, his examinations involved only two strains, so that valuable conclusions about Klebsiella adherence need investigation of a greater number of isolates. Our study involved 50 K. pneumoniae and 17 K. oxytoca strains isolated from clinical specimens . Two types of haemagglutinins could be demonstrated: the mannose-sensitive haemagglutinin (MSHA) and the mannose-resistant MR/K haemagglutinino Only 3 of 67 isolates did not haemagglutinate any of the erythrocytes tested. Whereas both species showed similar proportions of strains possessing MR/K-HA activity, a striking difference of MSHA-positive isolates in both Klebsiella species was noted. MS-haemagglutinins were found in the majority of strains of K. pneumoniae but only in 2 of 17 K. oxytoca isolates. Most strains of the former species simultaneously expressed both types of haemagglutinins. The results of this study are in accordance to the report of Przondo-Hessek et al. (17), who found a similar distribution of MS- and MR/K-HA in the Klebsiella strains they investigated. Bacterial adhesion tests 'often show poor reproducibility. Especially mixed populations of cells (e.g. uro-epithelial cells), varying from donor to donor, give rise to var iable results (18, 23). To avoid heterogeneous cell populations, in our study two human cell lines were used in determining bacterial adhesion. In both Klebsiella species, only low binding rates were observed (1-10 bacteria/cell). Even if attachment of various Enterobacteriaceae to tissue-cultured cells and to cells directly obtained from human donors is reported to be comparable (21), selective adhesion should be taken into account. So P-fimbriated E. coli strains adhere in great numbers to urinary epithelial cells of P phenotypes (13) but only poorly to tissue-cultured cells (12, 25). Our findings of low binding rates of K. pneumoniae and K. oxytoca to HeLa and Intestine 407 cells, however, are in agreement with observations of Sugarman et al. (22), who found similar adhesion ratios in MSHA-positive K. pneumoniae strains to HeLa and L cells. Likewise, in E. coli strains expressing haemagglutinins other than
Haemagglutinins and Adherence Properties
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narrow-spectrum ('man only') MRHA, comparable low binding rates to HEp2 cells are reported (25). With respect to the findings of Fader et al. (6, 7) about the importance of MShaemagglutinins for the adherence of Klebsiella, K. pneumoniae ought to show greater adherent abilities than K. oxytoca. However, tested on two human cell lines, the incidences of adhering strains were similar to those of K. oxytoca. Furthermore, adhesion ratios of both species to HeLa cells as well as to Intestine 407 cells were comparatively low (1-10 bacteria/cell) . Despite of culture conditions allowing pellicle formation, almost half of the adhering strains lacked MSHA activity; nonadhering strains exhibited identical incidences of MS-haemagglutination as adhering isolates. Two adhesive isolates neither showed MSHA nor MRlK-HA activity. Likewise, the occurence of MRlK-haernagglutinins in adhering and nonadhering strains was similar. Correlation analyses of the intensity of haemagglutination and adherence ratio did not reveal any association between adhesion and HA type or HA pattern. Concerning the strains investigated, MS- or MRlK-haemagglutinins seem to be weakly or not at all involved in the binding of Klebsiella spp. to eucaryotic cells. For E. coli, this was likewise demonstrated by Chabanon et al. (2), when they studied HA activity and adherence to Intestine 407 cells in strains isolated from the urinary tract. More than half of the adhering isolates lacked HA activity against human and guinea pig erythrocytes, and they suggested that adhesions and haemagglutinins are not clearly linked. The adhesive nature of Klebsiella seems to be a more complicated phenomenon, and other haemagglutinins than MSHA and MRlK-HA should be taken into account. Old et al. (16) recently described a new mannose-resistant haemagglutinin in Klebsiella, previously found in Proteusspp. and called MRiP-HA (rnannose-resistant and Proteuslike haemagglutinin). They reported more than half of 80 K. aerogenes strains investigated to produce this haemagglutinin. A further study is intended to clarify a possible role of the MRiP-HA for the adherence of K. pneumoniae and K. oxytoca.
Acknowledgement. We thank Mr. W. Aile for technical assistance in performing the
computer analyses.
References
1. Bosch, [. F. van den, U. Verboom-Sohmer, P. Postma, ]. de Graa((, and D . M. Mc Laren: Mannose-sensitive and mannose-resistant adherence to human uroeptihelial cells and urinary virulence of Escherichia coli. Infect. Immun. 29 (1980) 226-233 2. Chabanon, G., C. 1. Hartley, and M. H. Richmond: In vitro attachment to a human cell line of Escherichia coli strains causing urinary tract infection : occurrence of fimbriae (pili) in adhesive and non-adhesive strains. Ann. Microbiol. (Inst. Pasteur) 133 A (1982) 357-369 3. Duguid.]. P.: Fimbriae and adhesive properties in Klebsiella strains. J. gen. Microbiol. 21 (1959) 271-286 4. Duguid.], P., S. Clegg, and M. I. Wilson: The fimbrial and non-fimbrial hemagglutinins of Escherichia coli. J. Med. Microbial. 12 (1979) 213-227 5. Duguid,]. P. and D. C. Old: Adhesive properties of Enterobacteriaceae. In: E. Beachey (ed.), Bacterial Adherence, pp. 185-217. (Receptors and Recognition, Series B, Vol. 6), Chapman and Hall, London (1980)
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6. Fader, R. c., A. E. Avots-Avotins, and C. P. Davis: Evidence for pili-mediated adherence of Klebsiella pneumoniae to rat bladder epithelial cells in vitro. Infect. Immun. 25 (1979) 729-737
7. Fader, R. C. and C. P. Davis: Effect of piliation on Klebsiella pneumoniae infection in rat bladders. Infect. Immun. 30 (1980) 554-561
8. Gaastra, W. and F. K. de Graaf: Host-specific fimbrial adhesins of non invasive en-
terotoxigenic Escherichia coli strains . Microbiol. Rev. 46 (1982) 129-161 9. Hagberg, L., U. ]odal, T. K. Korhonen, G. Lidin-]anson, and C. Suanborg Eden: Adhesion, hemagglutination, and virulence of Escherichia coli causing urinary tract
infections. Infect. Immun. 31 (1981) 564-570 10. Hartung, ]. and B. Elpelt: Die Korrelationsanalyse . In: Multivariate Statistik, pp. 143-221. Olden bourg Verlag, Miinchen-Wien (1984) 11. Isaacson, R. E., P. C. Fusco, C. C. Brinton, and H. W. Moon: In vitro adhesion of Escherichia coli to porcine small intestinal epithelial cells: pili as adhesive factors. Infect. Immun. 21 (1978) 392-397 12. ]ann, K., G. Schmidt, E. Blumenstock, and K. Vosbeck : Escherichia coli adhesion to Saccharomyces cereuisiae and mammalian cells: role of piliation and surface hydrophobicity. Infect. Immun. 32 (1981) 484-489 13. Kdllenius, G., R. Mollby, H. Hultberg, S. B. Svenson, B. Cedergren, and]. Winberg: Structure of carbohydrate part of receptor on human uroepithelial cells for pyelonephritogenic Escherichia coli. Lancet 19 (1981) 604-606 14. Korhonen, T. K., E. Tarkka, H. Ranta, and K. Haahtela: Type 3 fimbriae of Klebsiella sp.: Molecular characterization and role in bacterial adhesion to plant roots. J. Bact. 155 (1983) 860-865 15. Ofek, I. and E. H. Beachey: Mannose binding and epithelial cell adherence of Escherichia coli. Infect. Immun. 22 (1978) 247-254 16. Old, D. C. and R. A. Adegbola: A new mannose-resistant hemagglutinin in Klebsiella. J. appl. Bact. 55 (1983) 165-172 17. Przondo Hessek, A. and G. Puluerer: Hemagglutinins of Klebsiella pneumoniae and Klebsiella oxytoca. Zbl. Bakt. Hyg., I. Abt. Orig. A 255 (1983) 472-478 18. Rosenstein,].]., D. Grady,]. M. T. Hamilton-Miller, and W. Brumfitt: Relationship between adhesion of Escherichia coli to uro-epithelial cells and the pathogenesis of urinary infection: problems in methodology and analysis. J. Med. Microbiol. 20 (1985) 335-344 . 19. Savoia, D., P. Martinetto, A. Achino, and A. Pugliese: Adhesion of Proteus species to various cell types. Europ. ]. Clin. Microbiol. 2 (1983) 571-576 20. Siluerblatt, F. ]. and I. Ofek: Effects of pili on susceptibility of Proteus mirabilis to phagocytosis and on adherence to bladder cells. In: Kass, E. H. and W. Brumfitt (eds.), Infections of the Urinary Tract, pp. 49-59. University of Chicago Press, Chicago (1978) 21. Sugarman, B. and S. T. Donta: Specificity of attachment of certain Enterobacteriaceae to mammalian cells. ]. gen. Microbiol. 115 (1979) 509-512 22. Sugarman, B., L. R. Epps, and W. A. Stenback: Antibiotic and non antibiotic ionophores can alter bacterial adherence to mammalian cells. Proc. Soc. expo BioI. Med. 173 (1983) 588-597 23. Svanborg Eden, B. Eriksson, and L. A. Hansson: Adhesion of Escherichia coli to human uroepithelial cells in vitro. Infect. Immun. 18 (1977) 767-774 24. Suanborg Eden, L. Hagberg, L. A. Hansson, T. Korhonen, H. Leffler, and S. Olleng: Adhesion of Escherichia coli in urinary tract infection. In: Adhesion and Microorganism Pathogenicity, pp. 161-187. Ciba Foundation Symposium 80. Pitman Medical, London (1981) 25. Tauendale, A. and D. C. Old: Hemagglutinins and adhesion of Escherichia coli to HEp2 epithelial cells. J. Med. Microbiol. 20 (1985) 345-353 26. Tramont, E. C;, Adhesion of Neisseria gonorrhoea and disease. In: Adhesion and Microorganism Pathogenicity. Ciba Foundation Symposium 80. Pitman Medical, London (1981)
c., c.,
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27. Tuomanen, E. j ., ]. Nedelman,]. O. Hendley, and E. L. Hewlett: Species specificity of Bordetella adherence to hu man and anima l ciliated respirato ry epithelial cells. Infect. Immun. 42 (1983) 692- 695 28. Virji, M. and ]. E. Heckels: The role of common and type-specific pilus ant igenic domains in adhe sion and viru lence of gonococ ci for hum an epithelial cells. ]. gen. Mi crobi al. 130 (1984) 1089-1095 . 29. Woods, E. D., D. C. Straus, W. G. j ohanson, V. K. Berry, and ]. A. Bass: Role of pili in adherence of Pseudomonas aeruginosa to mammalian buccal epithel ial cells. Infect. Immun . 29 (1980) 1146-11 51 Prof. Dr. med. H. G. Sonntag, Hygiene-Institut der Universitat Heidelberg, 1m Neuenheimer Feld 324, D-6900 Heidelber g
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Zbl. Bakt. Hyg. A 263/4
Haemagglutinins and Adherence Properties to HeLa and Intestine 407 Cells of Klebsiella pneumoniae and Klebsiella oxytoca Isolates R. PODSCHUN, P. HEINEKEN, and H. G. SONNTAG Hygiene Institute, University of Heidelberg, Heidelberg, Federal Republic Germany
With 2 Figures' Received June 6, 1986 · Accepted September 23, 1986
Summary The occurrence of haemagglutination (HA) and adherence properties were examined in 50 strains of K. pneumoniae and 17 K. oxytoca strains isolated from humans. All isolates except three exhibited HA activity. Mannose-sensitive haemagglutinins (MSHA) were expressed by the majority of K. pneumoniae strains, but only by one K. oxytoca isolate. Mannose-resistant haemagglutination (MRHA) to human or guinea pig erythrocytes could not be detected; haemagglutinins of the MRiK type were found in both species with similar frequencies. Adhesive properties could be demonstrated in K. pneumoniae as often as in K. oxytoca: About half of the strains adhered to two human cell lines: HeLa and Intestine 407. The incidence of HA activity was similar in adhering and nonadhering strains. A correlation between MSHA, MRlK-HA and adherence to tissue-cultured cells could not be detected. Zusammenfassung 50 K. pneumoniae- und 17 K. oxyrocs-Stamme menschlicher Herkunft wurden auf das Vorkommen harnagglutinierender undd adharierender Eigenschaften untersucht. Mit Ausnahme dreier Isolate zeigten aile Stamme Hamagglutinationsaktivitat, Mannose-sensitive Hamagglutinine (MSHA) besaf die Mehrzahl der K. pneumoniae-Isolate, aber nur ein K. oxytoca-Stamm. Eine Mannose -resistente Hamagglutination (MRHA) gegeniiber humanen oder Meerschweinchen-Erythrozyten wurde nicht gefunden; MRlK-Hamagglutinine wur den in heiden Spezies mit gleicher Haufigkeit ermittelt. Ein Adharenzvermogen konnte bei K. pneumoniae genauso haufig wie bei K. oxytoca nachgewiesen werden: Etwa die Halfte beider Spezies adharierten an die humanen Zellinien HeLa und Intestine 407 . Adharierende und nichtadharierende Starnme zeigten ein vergleichbar haufiges Auftreten hamag glutinierender Eigenschaften. Eine Verbindung zwischen dem Besitz von MSHA, MRlK-HA und Adhasinen konnte nicht gezeigt werden. Introduction The ability of bacteria to adhere to mammalian cell surfaces, though recognized for a long time, ha s become a field of intensified research in the last decade because it is
586
R. Podschun, P. Heineken, and H. G. Sonntag
presumed to be an important pathogenicity factor. The nature of bacterial adhesins is presently under investigation, however, surface appendages called 'pili' or 'fimbriae' are reported to mediate adherence in man y gram -negati ve pathogens such as Enterobacteriaceae (1, 6, 7, 8, 11, 15, 19,20,24), Pseudomonas (29), Neisseria (26,28) and Bordetella (27). Bacterial adhesins have been classified by their haemagglutinating properties. Haemagglutination of guinea-pig erythrocytes inhibited by the presence of D-mannose (rnannose-sensitive haemagglutination, MSHA) is attributed to common or type 1 pili on bacterial surfaces (3, 4). Bacteria causing agglutination of erythrocytes without inhibition by D-mannose are expressing mannose-resistant haemagglutinins (MRHA) . Svanborg Eden and coworkers (9) reported that urinary isolates of E. coli with mannose-resistant haemagglutination of human erythrocytes adhered in high numbers to urinary tract epithelial cells, whereas bacteria inducing man nose-sensitive haemagglutination alone attached only in low numbers. Two haemagglutinins are very common in Klebsiella species: Most K. pneumoniae strains show man nose-sensitive haemagglutination of guinea-pig erythrocytes and mannose-resistant haemagglutination of tanned erythrocytes (3, 17). The latter type, found in the tribes Klebsielleae and Proteae, is associated with tye 3 pili (5, 14, 16) and designated the MR/K (mannose-resistant and Klebsiella-like) haemagglutinin. Relative ly little is known about the role of type 3 pili in adherence to mammalian cells, however, Korhonen et al. (14) demonstrated type 3 pili to mediate adhesion of Klebsiella to plant roots. In respect to the relation of type 1 pili and adherence properties, Fader et al. (6, 7) reported the piliated, MSHA-positive phase of a K. pneumoniae strain to attach to rat bladder epithelial cells, whereas the nonpiliated, MSHA-negative phase did not adhere. The present study was designed to investigate the occurrence and pattern of haernagglutination of 67 Klebsiella pneumoniae and K. oxytoca strains isolated from clinical specimens as well as their adherence activities to tissue-cultured cells. Furthermore, adherence and haemagglutination should be compared in order to demonstrate a poss ible correlation between these properties.
Materials and Methods
Bacteria. A total of 67 Klebsiella strains, mainly isolated from the urogenital tract, respiratory secretions and wounds, were obtained from our microbiology laboratory . Sites of isolation were revealed by the laboratory records. 50 isolates could be classified as Klebsiella pneumoniae, 17 strains as K. oxytoca by API 20 E (API SystemsS. A., MontalieuVercieu, France). All isolates were kept at 4 °C on deep agar slants of brain-heart-infusion (Merck, Darmstadt) and subcultured once on blood agar plates before use. K. pneumoniae ATCC 27736 was used as reference strain. Haemagglutination tests. Citrated fresh blood samples from humans with blood group A, guinea-pigs and ox were washed three times (PBS, pH 7.2) and adjusted to 5 x 108 erythrocytes/m!. To obtain tanned ox red blood cells, one volume of washed erythrocytes (5 X 108/ ml) was treated with one volume of 0.003% tannic acid for 10 min at 3rc. Tanned erythrocytes were washed twice in PBS and adjusted to a final concentration of 5 x 108 RBC/m!. Bacteria were grown statically twice for 48 h periods at 37 °C in brain-heart-infusionbroth (BHIB). Cells were harvested by centrifugation and the bacterial pellets were resuspended in PBS giving a final concentration of 101°_1011 bacteria/m!. 50 f.t! of bacterial
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suspensions were mixed with 50 III of erythroc ytes in a porcelain tile (with or without 1% mannose ). Gently agitated, haemagglutination was observed for 3 min at room temperature and read finally after further incubation for 10 min at 4 °C. Adherence assay. HeLa and Intestine 407 cells (Flow Laboratories, Irvine, U. K.) were used to study the adherence properties of the strains . The cells were maintained in Eagle minimal essential medium (Flow Laboratories), supplemented with 10% fetal calf serum, 2 mM glutamine, 100 IU/ml penicillin, 100 ug/rnl streptomycin and 0.25 ug/ml fungizone (Gibco, Paisley, Scotland). For adhesion tests, 104 cells were seeded into each well of 10-well multitest slides (Flow) and grown for 48 h in a humidified chamber with 5 % CO 2 at 24 h before treatment with bacteria the medium was replaced by antibiotic-free medium. Bacteria were grown serially twice in tubes of BHI-broth incubated staticall y at 37 °C for 48 h period s. Cultures were washed three times with PBS, pH 7.2, passed through a 21gauge-needle to disperse aggregates and adjusted to 10 9 bacter ia/m!. Tissue cell mono layers were washed three times with PBS and 50 III bacterial suspension/ well was added. The slides were gently agitated for 30 min at 37 °C. Nonadhering bacteria were removed by washing the slides several times with PBS. The monolayers were fixed in methanol (5 min), stained in 10% Giemsa solution (10 min) and washed. Adherent bacteria on 100 individual cells were counted with the aid of a light microscope and the adhesion rate was calculated as the mean number of bacteria per cel!. Isolates with less than 1 bacterium/cell were regarded as nonadhesive. Correlation analysis. The intensities of single hemagglutination types as well as combinations were compared with the corresponding adherence ratios in the different cell lines. The probability of two features to be related were calculated by correlation coefficients, based on Kendalls tau b (10).
3rc.
Results
Haemagglutinating activity. Both species differed markedly in their ability to agglutinate guinea-pig erythrocytes: Most isolates of Klebsiella pneumoniae exhibited MS (mannose-sensitive) -haemagglutinins (72%), whereas the majority of K. oxytoca strains (94 %), despite cultural conditions allowing pellicle formation (4 ), did not agglutinate red blood cells of this species (Fig. Ia), Neither against guinea-pig nor against human A erythrocytes an y isolate showed MRHA (rnannose-resistant haemagglutination)-activity. MSHA of human erythrocytes was present in 24 % of K. pneumoniae and in 6 % of K. oxytoca strains (Fig. Ib ), With respect to MR/K-haemagglutinins, both specie s demonstrated comparable numbers of strains active against tanned ox erythrocytes. About 82 % of K. oxytoca and 90 % of K. pneumoniae isolates showed this haemagglutination type, resistant to the action of D-mannose (Fig. Ic ). Table 1. Distribution of haemagglutinins in Klebsiella strains HA rype
K. pneumoniae (n = 50)
K. oxytoca (n = 17)
only MSHA present only MRlK-HA present MS- and MRlK-HA present MS- and MRlK-HA negative
4 (8% ) 11 (22% ) 34 (68% ) 1 (2% )
1 (6% ) 13 (76%) 1 (6%) 2 (12%)
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R. Podschun, P. Heineken, and H . G. Sonntag
P e
r
C e n t II
9
e
100 90 80 70 60 50 40 30 20 10 0
+
I_
++ HA intensity
K.oxytOCll (n= 17)
o
+++
Kpneumoni lle (n=50)
Fig. 1. Haem agglutinating activity of Klebsiella isolates. Frequ ency and inten sity of MSHA to guinea pig erythrocytes (A), human A red blood cells (B) and MRlK -HA to tanned ox erythrocytes (C). Intensity of haemagglutination is graded from + to +++.
Haemagglutinins and Adherence Properties
589
45 P 40
e 35
r
C
30
e 25 n 20
t
II
15
9 10 e 5
o
, -
1-2
2-3 3-4 4- 5 number of bact eria/cell
K.oxytOCll (n= 17)
0
'5
Kpneumon ill e (n=50)
I
60 p
e 50
r 40 C e 30 n l 20
II
9 10 e
0
1-2
2-3 3-4 4-5 number of bacteria/cel l
'5
Fig. 2. Distribution of adherence among Klebsiella isolates to HeLa cells (A) and Intestine 407 cells (B). Adhesion is expressed as the mean number of bacterialtissue-cultured cell.
Most K. pneumoniae isolates simultaneously expressed MSHA and MR/K-HA (68%), whereas the great majority of K. oxytoca strains (76% ) exhibited only the MR/ K-type (Table 1). Twelve percent of K. oxytoca isolates, but only one K. pneumoniae strain (2%) did not show any haemagglutination type investigated. Adherence to tissue-cultured cells. Isolates were regarded as adhesive if the adhesion rate was greater/equal 1 bacterium/cell. To HeLa cells as well as to Intestine 407 cells both species exhibited only low adherence rates (1-10 bacteria/cell). On an average, the strains adhered in greater numbers and more frequently to HeLa cells. Adherence ratios are shown in Fig. 2. Reproducibility of the test results was satisfactory: Adhesion rates of a nonadhering strain (KPN 120), a low binding (KOX 135) and a moderate binding strain (KPN 47) in independent tests were determined at least four times. Only little variation in adhesiveness was found : Mean numbers of bacte ria adhering to each cell were observed to differ from 0.1-0.3 (KPN 120), 1.6-2.0 (KOX 135) and from 4.2-4.6 (KPN 47).
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R. Podschun, P. Heineken, and H. G. Sonntag
Both species showed similar frequencies of adhesive isolates: 29 (58%) of the K.
pneumoniae strains adhered to HeLa cells, 20 (40%) to Intestine 407 cells; the corresponding frequencies for K. oxytoca were 65% (11 strains) and 47% (8 strains). With regard to HeLa cell adhesion, 23 (58%) of the 40 adhering isolates showed MSHA and 36 strains (90%) exhibited MR/K-HA activity. Comparable incidences were observed in the 27 nonadhering isolates: 16 strains (59%) were MSHA-positive and in 22 strains (81%) MR/K-HA was present. Intensity and pattern of haemagglutination were compared with adherence ratios of K. pneumoniae and K. oxytoca. A correlation analysis, based on Kendalls tau b correlation coefficients, did not reveal any relation i). between possession of haemagglutinins and adherence properties or ii). between expression of a single haemagglutination type investigated and adherence to tissue-cultured cells. In each case, correlation coefficients showed values (Kendall's tau b) of less than 0.5.
Discussion Bacterial haemagglutinins are considered to be correlated to adherence properties to eucaryotic cells. Svanborg-Eden and coworkers reported the significance of MRHA in adhesion of urinary tract isolates of E. coli to uroepithelial cells (9), whereas strains with MSHA activity alone attached only in low numbers or not at all to these cells. On the other hand, Fader et al. (6, 7) demonstrated MSHA to be responsible for the adhesion of K. pneumoniae to rat bladder cells. However, his examinations involved only two strains, so that valuable conclusions about Klebsiella adherence need investigation of a greater number of isolates. Our study involved 50 K. pneumoniae and 17 K. oxytoca strains isolated from clinical specimens . Two types of haemagglutinins could be demonstrated: the mannose-sensitive haemagglutinin (MSHA) and the mannose-resistant MR/K haemagglutinino Only 3 of 67 isolates did not haemagglutinate any of the erythrocytes tested. Whereas both species showed similar proportions of strains possessing MR/K-HA activity, a striking difference of MSHA-positive isolates in both Klebsiella species was noted. MS-haemagglutinins were found in the majority of strains of K. pneumoniae but only in 2 of 17 K. oxytoca isolates. Most strains of the former species simultaneously expressed both types of haemagglutinins. The results of this study are in accordance to the report of Przondo-Hessek et al. (17), who found a similar distribution of MS- and MR/K-HA in the Klebsiella strains they investigated. Bacterial adhesion tests 'often show poor reproducibility. Especially mixed populations of cells (e.g. uro-epithelial cells), varying from donor to donor, give rise to var iable results (18, 23). To avoid heterogeneous cell populations, in our study two human cell lines were used in determining bacterial adhesion. In both Klebsiella species, only low binding rates were observed (1-10 bacteria/cell). Even if attachment of various Enterobacteriaceae to tissue-cultured cells and to cells directly obtained from human donors is reported to be comparable (21), selective adhesion should be taken into account. So P-fimbriated E. coli strains adhere in great numbers to urinary epithelial cells of P phenotypes (13) but only poorly to tissue-cultured cells (12, 25). Our findings of low binding rates of K. pneumoniae and K. oxytoca to HeLa and Intestine 407 cells, however, are in agreement with observations of Sugarman et al. (22), who found similar adhesion ratios in MSHA-positive K. pneumoniae strains to HeLa and L cells. Likewise, in E. coli strains expressing haemagglutinins other than
Haemagglutinins and Adherence Properties
591
narrow-spectrum ('man only') MRHA, comparable low binding rates to HEp2 cells are reported (25). With respect to the findings of Fader et al. (6, 7) about the importance of MShaemagglutinins for the adherence of Klebsiella, K. pneumoniae ought to show greater adherent abilities than K. oxytoca. However, tested on two human cell lines, the incidences of adhering strains were similar to those of K. oxytoca. Furthermore, adhesion ratios of both species to HeLa cells as well as to Intestine 407 cells were comparatively low (1-10 bacteria/cell) . Despite of culture conditions allowing pellicle formation, almost half of the adhering strains lacked MSHA activity; nonadhering strains exhibited identical incidences of MS-haemagglutination as adhering isolates. Two adhesive isolates neither showed MSHA nor MRlK-HA activity. Likewise, the occurence of MRlK-haernagglutinins in adhering and nonadhering strains was similar. Correlation analyses of the intensity of haemagglutination and adherence ratio did not reveal any association between adhesion and HA type or HA pattern. Concerning the strains investigated, MS- or MRlK-haemagglutinins seem to be weakly or not at all involved in the binding of Klebsiella spp. to eucaryotic cells. For E. coli, this was likewise demonstrated by Chabanon et al. (2), when they studied HA activity and adherence to Intestine 407 cells in strains isolated from the urinary tract. More than half of the adhering isolates lacked HA activity against human and guinea pig erythrocytes, and they suggested that adhesions and haemagglutinins are not clearly linked. The adhesive nature of Klebsiella seems to be a more complicated phenomenon, and other haemagglutinins than MSHA and MRlK-HA should be taken into account. Old et al. (16) recently described a new mannose-resistant haemagglutinin in Klebsiella, previously found in Proteusspp. and called MRiP-HA (rnannose-resistant and Proteuslike haemagglutinin). They reported more than half of 80 K. aerogenes strains investigated to produce this haemagglutinin. A further study is intended to clarify a possible role of the MRiP-HA for the adherence of K. pneumoniae and K. oxytoca.
Acknowledgement. We thank Mr. W. Aile for technical assistance in performing the
computer analyses.
References
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