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Department of Microbiology, Faculty of Veterinary Medicine, University of Yuzuncu Yil, 65080, Van, Turkey
Correspondence
Ismail Hakki Ekin
ihekin{at}yyu.edu.tr
Received 13 May 2005
Accepted 3 January 2006
Abbreviations: GBS, group B streptococci.
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INTRODUCTION |
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 TOP INTRODUCTION METHODSRESULTS AND DISCUSSIONREFERENCES |
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Traditionally, the classification and identification of streptococci from clinical and veterinary sources have been based on the serogrouping and serotyping of the carbohydrate and protein antigens of the cell walls by the method of Lancefield (Devriese, 1991). Up to now, GBS have been classified according to nine known major polysaccharide antigens (Ia, Ib, II, III, IV, V, VI, VII and VIII) and three protein antigens (Ic, R and X) (Jelinkova, 1977; Spellerberg, 2000; Shet & Ferrieri, 2004).
Bacterial adherence to host cells appears to be a multifactorial phenomenon involving specific as well as non-specific interactions. Structures that are thought to be responsible for bacterial adhesion include fimbriae and non-fimbrial adhesins, such as lipoteichoic acid and proteins. The ability of bacteria to attach to and agglutinate erythrocytes may be used in vitro as a model to study host–bacterium interaction and the mechanism of attachment (Kurl et al., 1989; Wibawan et al., 1993).
Lectins are ubiquitous, found in animals, plants and micro-organisms. A lectin usually contains two or more binding sites for carbohydrate units; some lectins contain oligomeric structures with multiple binding sites (Ottensooser et al., 1974). Because lectins are able to bind to antibodies and thereby inhibit competitively the reaction between cellular antigen and antibody, they have been used in some typing studies (Niewerth, 1987; Slifkin & Cumbie, 1987; Kellens et al., 1993; Munoz et al., 1994).
This study was performed to demonstrate the epidemiological importance of serotype distribution and the haemagglutination and lectin-agglutination activities of bovine and human GBS strains isolated in and around Van, Turkey.
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METHODS |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Reference strains. The reference strains Streptococcus agalactiae serotype Ia (090), Ib (H 36 B), Ic (A 909), II (18 RS 21), III (6313), IV (3139), V (SS 1169), VII (7271), VIII (JM9 130013), R (25/60 Compton), X (24/60 Compton) and Staphylococcus aureus Cowan I (NCTC 8530) were kindly provided by Professor Dr Christoph Lämmler, Institut für Pharmakologie und Toxikologie Fachbereich Veterinärmedizin, Justus Liebig Universität.
Serogrouping. The cultures were grown overnight on Blood Agar Base plates (Oxoid) with 5 % sheep blood. Serological grouping of isolates was performed with a commercial group B latex agglutination kit (Avipath-Strep, Omega Diagnostics) according to the manufacturer's instructions.
Serotyping. Each reference GBS serotype culture was grown overnight in 300 ml Todd–Hewitt broth (THB) and centrifuged (10 000 g, 15 min); the bacterial pellet was suspended in one-tenth of the original volume of culture medium in 0·2 M PBS (pH 7·6). The suspensions were inactivated at 60 °C for 30–60 min in a water bath. After a sterility control, the inoculum suspensions were stored at 4 °C.
Type-specific antisera were prepared in rabbits by the intravenous injection of heat-killed suspensions of reference GBS serotype Ia, Ib, Ic, II, III, IV, V, VII, VIII, R and X antigens. After immunization, polyspecific antisera were tested for homologous and heterologous antibodies. Type-specific antisera were obtained by the adsorption of the polyspecific antisera with each cross-reactive reference GBS serotype culture (Mosabi et al., 1997; Jelinkova, 1977; Ainsworth & Capley, 1986).
A staphylococcal co-agglutination test was used for serotyping the isolates. The test was performed, with minor modifications, according to the procedure described by Christensen et al. (1973).
Haemagglutination test. The human and bovine GBS isolates were cultivated in 10 ml THB for 18 h at 37 °C. The cultures were washed three times in 0·002 M PBS (pH 6·8) and suspended in 400 µl of the same buffer. The suspension was aliquoted into two portions. One portion was pretreated with trypsin (5 µg per 200 µl bacterial suspension) for 1 h at 37 °C, washed, and resuspended in the initial volume of PBS (Swenshon et al., 1998); the other portion was not treated with trypsin.
Haemagglutination tests were carried out with 20 µl of rabbit erythrocytes (2 %) and 20 µl of culture (trypsinized or non-trypsinized) on microscope slides. The suspensions were mixed and the slides were rotated gently, and within 30 s the haemagglutination was recorded as strong agglutination (++), agglutination (+) or no agglutination (–) (Wibawan et al., 1993; Korhonen & Finne, 1985).
Lectin-agglutination tests. The isolates were grown in 10 ml THB for 18 h at 37 °C. After centrifugation, the pellet was washed three times with 0·05 M PBS (pH 7·5) and resuspended in 400 µl PBS. The suspensions were divided into two portions; one was pretreated with a trypsin (5 µg per 200 µl bacterial suspension) for 1 h at 37 °C. The suspension was washed three times with PBS and resuspended in the initial volume of PBS (Schaefer et al., 1979; Swenshon et al., 1998).
The lectins from Arachis hypogea (Sigma L 0881), Canavalia ensiformis (Sigma L 7647), Dolichos biflorus (Sigma L 2785), Helix pomatia (Sigma L 3382) and Triticum vulgaris (Sigma L 9640) were prepared in 0·05 M PBS according to the manufacturer's instructions.
For lectin-agglutination tests, 20 µl of the lectin preparation was mixed with 20 µl of streptococcal suspension on microscope slides. The slides were rotated gently and examined for an agglutination reaction within 1 min; the reaction was recorded as strong agglutination (++), agglutination (+) or no agglutination (–) (Schaefer et al., 1979; Motlova et al., 1986; Swenshon et al., 1998).
Statistical evaluation. The chi square test was employed to test for association.
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RESULTS AND DISCUSSION |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Serotyping results
Thirty-four (44·7 %) of the bovine and 49 (68 %) of the human GBS isolates could be serotyped by the co-agglutination test. The serotype distributions of both bovine and human GBS isolates are shown in Table 1
. GBS serotype VII and III polysaccharide antigens could only be detected in bovine GBS isolates, whereas GBS serotype Ic, IV, V and R antigens were mostly found in human GBS isolates. GBS serotype VIII was detected in both bovine and human GBS isolates. Only one bovine GBS isolate had GBS serotype X antigens. The combined antigenic patterns displayed by bovine GBS isolates were II, IV; II, X; II, IV, X; and IV, R; whereas human GBS isolates showed the combined antigenic patterns III, V, VII; and VII, VIII (Table 1).
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Previous reports have shown a relative heterogeneity in the distribution of different serotypes of bovine and human isolates in diverse geographic areas (Bopp & Lämmler, 1995; Mosabi et al., 1997; Duarte et al., 2004). It has been reported that the Ia, II, III, V and X antigens are commonly observed in GBS strains isolated from bovine milk (Finch & Martin, 1984; Pasaribu et al., 1985; Kutschke, 1986; Mosabi et al., 1997; Duarte et al., 2004), whereas Ia, Ib, Ic, II, III, IV and V antigens are found in GBS from human vaginal specimens (Jelinkova, 1977; Schuctat & Wenger 1994; Berg et al., 2000; Spellerberg, 2000; Ko et al., 2001). In the present study, GBS serotype III and VII were found to be the most frequent serotypes in bovine isolates, but not in human isolates. The importance of the invasive GBS serotype III strains among human isolates is well known (Berg et al., 2000; Ko et al., 2001). GBS serotype III has been incriminated as the major colonizing strain in pregnant women (Finch & Martin, 1984; Berg et al., 2000; Moyo et al., 2002).
These findings showed that bovine milk could be regarded as a risk factor, and that it could play an important role in transmission, especially of invasive GBS serotype III strains from bovine to human.
Harrison et al. (1995) have reported that the isolation rate of GBS serotype V has increased from 2·6 to 20 % in human samples. In the present study, serotype V was frequently found in the human isolates.
These results could be considered as the basis of an epidemiological marker for use in Turkey. The distribution of GBS serotypes can vary in different geographical regions, and this may be an important characteristic for the development of vaccine and treatment strategies.
Haemagglutination test results
Because seven (9·2 %) of 76 bovine and eight (11·1 %) of 72 human GBS strains showed auto-agglutination, these strains could not be used in both haemagglutination and lectin-agglutination tests. Of the 69 bovine GBS strains, eight (11·6 %) agglutinated rabbit erythrocytes, but 23 (33·3 %) gave a positive reaction after trypsin treatment. On the other hand, none of the human GBS strains agglutinated rabbit erythrocytes, although 15 (23·4 %) human GBS strains were positive after trypsin treatment (Table 2). The difference between bovine and human GBS strains with respect to haemagglutination properties without trypsin treatment was found to be statistically significant (P<0·05). In addition, comparable differences were observed between the typable and non-typable human GBS strains after treatment with trypsin.
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, the haemagglutinating properties also appeared not to be directly related to the occurrence of the type antigens, because haemagglutination was observed among both typable and non-typable GBS strains. In contrast to our results, Kurl et al. (1989) have reported that one of 130 human GBS isolates shows haemagglutination activity, and that haemagglutination properties do not increase, even if the cultures are treated with trypsin. Although no report is available about this mechanism, changes in the adhesion activities of GBS cultures treated with trypsin may occur much less under in vitro conditions than in the infectious process in vivo (Kurl et al., 1989; Wibawan et al., 1993).
Lectin-agglutination test results
Lectins are glycoproteins, which combine specifically with defined sugar structures on the cell wall. This has often been applied to the identification and characterization of micro-organisms, exploiting the specificity of binding to cell surface sugars (Ottensooser et al., 1974; Slifkin & Cumbie, 1987; Kellens et al., 1993). In the present study, none of the GBS strains agglutinated with the lectins used. However, bovine and human GBS strains did agglutinate with lectins when the bacteria were treated with trypsin, as shown in Table 3. In agreement with our findings, Kellens et al. (1993) have reported that of 95 untreated ß-haemolytic streptococci, none gave a visible reaction with any of the lectins tested, whereas 42 strains agglutinated with one or more lectin when the cells were treated with trypsin. The results suggest that certain adhesins are exposed after the bacteria have been treated with trypsin.
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As reported elsewhere (Slifkin & Gil, 1983; Niewerth, 1987), the Dolichos biflorus lectin-agglutination reaction is especially recommended for rapid identification of group C streptococci. In the present study, comparable results were also observed with bovine GBS strains. This relationship could be based on the cross-reactive components of C-polysaccharide antigens of both GBS and group C streptococci. Although the results of the present study indicated that the pattern of lectin-agglutination reactions might be characteristic of bovine and human GBS, it could not be safely used for rapid identification of GBS or differentiation of streptococci.
In this first report, the serotype distribution of GBS isolates in Turkey was found to be similar to those of GBS isolated in various regions of the world. The results of the study indicated also that the treatment of GBS with trypsin is important for the demonstration of the haemagglutination and lectin-agglutination properties of GBS. The lectin-agglutination tests used in the study could be useful for differentiating bovine and human GBS strains, but not for identification of GBS strains. Further surveillance of GBS strains is important to provide data on serotype distribution, the formulation of a possible GBS vaccine, antibiotic prophylaxis and treatment recommendations for Turkey.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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kel. Amsterdam: Elsevier.This article has been cited by other articles:
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  K. M. Puopolo and L. C. Madoff Type IV Neonatal Early-Onset Group B Streptococcal Disease in a United States Hospital J. Clin. Microbiol., April 1, 2007; 45(4): 1360 - 1362. [Abstract] [Full Text] [PDF]
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