JOURNAL OF CLINICAL MICROBIOLOGY, June 1991, p. 1262-1264
Vol. 29, No. 6
0095-1137/91/061262-03$02.00/0 Copyright © 1991, American Society for Microbiology
Incidence of Toxigenic Campylobacter Strains in South Africa HERMAN E. BOK, ANDRE S. GREEFF,* AND HEATHER H. CREWE-BROWN Department of Microbiological Pathology, Medical University of Southern Africa (MEDUNSA), P.O. MEDUNSA 0204, Republic of South Africa Received 21 November 1990/Accepted 18 March 1991
Campylobacter strains can produce a heat-labile cytotonic toxin (CTON) and various cytotoxins (CTOX). Of 22 South African Campylobacter strains tested, 86% were toxigenic (77% produced CTON, 41% produced CTOX, and 32% produced both types) and 14% were toxin negative. Campylobacterjejuni strains were 67% CTON positive and 47% CTOX positive, whereas Campylobacter coli strains were 100 and 29% positive, respectively. and to increase CTON production (22). Sodium citrate, at molar concentrations equivalent to those of the iron compound, was added to the broth phase to prevent iron precipitation. After incubation, the cell-free supematants were prepared by centrifugation of the broth phase at 10,000 x g for 30 min and membrane filtration (Millex; pore size, 0.22 ,um; Millipore) of the supernatant. Toxin assays were conducted at 37°C under 5% CO2 in a humidified CO2 incubator (Forma Scientific). Y-1 monolayers were propagated with Ham's F-10 medium with L-glutamine (M.A. Bioproducts), supplemented with 14% fetal calf serum (Delta). HeLa cells were maintained in Eagle's minimal essential medium with Earle salts and L-glutamine, supplemented with 10% fetal calf serum. The assay for CTON was done on Y-1 cells according to published procedures (14). In this assay, .50% cell rounding was considered positive for CTON (15). The CTOX assay was conducted with HeLa cells according to the dye release method of Gentry and Dalrymple (8). A .50% cell detachment was regarded as positive for CTOX. For neutralization (16), the cell-free supernatants of CTON-positive cultures were incubated for 1 h at 37°C with equal volumes of a 10-fold serial dilution of 1-,ug/ml cholera antitoxin (Swiss Serum Institute) before addition to the Y-1 monolayers in a microtiter plate. Antiserum against CTOX was not available. The results are summarized in Table 2. All cultures were confirmed as Campylobacter strains. They were said to be Campylobacter jejuni-Campylobacter coli if they showed typical darting motility in brucella broth and were suscepti-
Campylobacter spp. are recognized as a common cause of human enteritis worldwide (1, 2, 5, 9, 14, 17, 25, 30, 31). They are prevalent in children (25), particularly in developing countries (28), and rank as the third most common cause of acute diarrhea after rotavirus and enterotoxigenic Escherichia coli (5, 17, 24). Noninflammatory watery diarrhea that arises predominantly in the small bowel is the result of the action of heat-labile cytotonic toxin (CTON) (28). Klipstein et al. (17) found that all of the Campylobacter strains isolated from patients with bloody, invasive-type diarrhea elaborated cytotoxins (CTOX). Some reported incidences of toxigenic Campylobacter species from different countries are represented in Table 1. The incidence of toxin production in South African Campylobacter strains is not known and was therefore investigated among local strains by analyzing responses of Y-1 and HeLa cells to cell-free supernatants of Campylobacter stool isolates. Campylobacter strains isolated at Ga-Rankuwa Hospital (near Pretoria) and strains donated by the Red Cross Children's Hospital in Cape Town, the South African Institute for Medical Research in Johannesburg, and private pathologists in Pretoria were used in this study (Table 2). They were all isolated from stools of patients with gastroenteritis typical of campylobacteriosis. A known CTON- and CTOX-positive strain and a CTON- and CTOX-negative strain, donated by the University of Texas Medical School, were used as controls. The identities of the cultures were confirmed by biotyping by using published procedures (6, 7, 10, 11, 19, 29). All of the Campylobacter strains were grown for 48 h at 37°C under 10% CO2 in a humidified CO2 incubator (Forma Scientific). They were stored at -70°C in glycerol-peptone medium containing 25% glycerol. When needed, they were thawed rapidly at 37°C and streaked onto tryptose blood agar (Oxoid) containing 5% lysed horse blood, Preston Campylobacter selective supplement (Oxoid), Campylobacter growth supplement (Oxoid), and 1 ,ug of amphotericin B (Fungizone) per ml. Cell-free supernatants for toxin testing were prepared by inoculation of Campylobacter strains into biphasic medium (27) containing 0.025% ferrous sulfate, sodium metabisulfite, and sodium pyruvate to maintain the characteristic morphology, motility, and viability of the Campylobacter cultures (4) *
TABLE 1. Published incidences of toxigenic
Campylobacter strains Investigators (reference)a
Johnson and Lior (14) Pang et al. (26) Ruiz-Palacios et al. (28) Mathan et al. (21)
Country
Country
Canada Malaysia Mexico South India
Incidence (%) of: C. jejuni C. coli C._jejun_C._col CTOX CTON CTOX CTON
69 48 NR NR
70 NRb 75 32
78 NR NR NR
83 NR
NR NR
a Lindblom et al. (20) reported a 32% incidence of CTON-positive Campylobacter strains in Sweden. b NR, not reported.
Corresponding author. 1262
VOL. 29, 1991
NOTES
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TABLE 2. Toxin production and biochemical characterization of Campylobacter strains Characteristica Strain no.
Identity
Identlty
Growth at:
CTON
CTOX
Hip.
Nal.
Cep.
H2S
Cat.
Ox.
250C
420C
ST167 ST152 ST116 ST111 ST65 ST52 ST23 NB11 96.86 89.86A 89.86B 87.86 85.86 71.86 207.85 206.85 192.85 AL150 435/1 L26 L28 126 IP383 136SP
C. jejuni biotype I + + + R R + + + + C. jejuni biotypelI S + R + + + C. jejuni biotype II S + + R + + + C. jejuni biotypeI S + + R + + + + C. coli S + + R + + + + C. jejuni biotype I S + + R + + + + C. jejuni biotype I S + + + R + + + + S C. coli + R + + + + C. jejuni biotype II + S + R + + + C. coli + S + R + + + C. jejuni biotype II S + + R + + C. jejuni biotype I + (+) S (+) R + + + C. coli + R + + R + + S C. jejuni biotypeI + + + + R + + C. jejuni biotype II + S + + R + + + S C. jejuni biotype I + (+) + + R + + C. coli + S + + R + + + + C. coli S (+) + R + + C. coli + S + + R + + + C. jejuni biotype II S + + + + R + + C. jejuni biotype II + + S + R + + C. jejuni biotype II S + (+) + + R + + C. coli S + + + + R C. jejuni biotypeI + + + + R R (+) a Abbreviations: CTON, CTON production; CTOX, CTOX production. Hip., hippurate hydrolysis; Nal., resistance (R) or susceptibility (S) to nalidixic acid; Cep., resistance (R) or susceptibility (S) to cephalothin; Ox., oxidase production; Cat., catalase production; H2S, H2S detected on lead acetate strips from triple sugar iron agar cultures. Symbols: +, positive; -, negative; (+), weakly positive.
ble to nalidixic acid (30-,ug disk) and resistant to cephalothin (30-,ug disk). On the basis of the hippurate hydrolysis test (11), there were 15 C. jejuni isolates and 7 C. coli isolates. Strain 85.86 cross-reacted with a DNA probe against C. jejuni-C. coli (in another experiment) and was considered to be a C. coli strain. The two hippurate-positive, nalidixic acid-resistant strains were grouped with the C. jejuni isolates (3). Strains IP383 and 136SP were the positive and negative controls, respectively. The identity of CTON was confirmed by neutralization. From Table 2, it is evident that 19 (86%) of our isolates were toxigenic: 12 (55%) produced one toxin type, and 7 (32%) produced both types. Seventeen (77%) were positive for CTON, and 9 (41%) were positive for CTOX. Johnson and Lior found concurrent production of CTON and CTOX in 53% (13) to 67% (12) of C. jejuni-C. coli isolates. Their incidence of toxin-negative isolates varied between 5% (12) and 14% (13), comparable to our 14%. Our finding that 67% of C. jejuni strains were positive for CTON is comparable to the 75% incidence of CTONpositive strains found by Ruiz-Palacios et al. (28) in Mexico and the 70% incidence reported by Johnson and Lior (14) in Canada. The South African CTON incidence figure is, however, more than double that found for CTON in South India (32%) (21). All 7 of our C. coli isolates produced CTON. Similarly, Johnson and Lior (14) also reported a high CTON incidence (83%) in 131 isolates. We found CTOX in 47% of our C. jejuni isolates and in 29% of the C. coli isolates. This is a considerably lower incidence than the 69 and 78% incidence, respectively, found by Johnson and Lior (14). In a smaller study by Klipstein et al. (17) with 20 strains, CTOX was found in cell-free culture filtrates of 6 of 6 (100%) C. jejuni strains isolated from
patients with invasive-type diarrhea. On the other hand, Moore et al. (23) found no detectable levels of CTOX in culture supernatants of 36 clinical isolates. They did, however, observe trace to low-level cell-associated cytotoxicity in 58% of their strains. The toxigenic Campylobacter incidence figures for South Africa are similar to those reported in some other countries. No direct relationship between the biotype and toxigenicity was found in this study. The role of the toxins in the pathogenesis of Campylobacter enteritis remains unclear, as some of the clinical strains were found to be negative for both toxin types. These strains may produce other toxins, such as cell-associated toxins, that were not detected in this survey. Strains from symptomatic patients can also lose their pathogenic properties (18). Other factors, such as colonization ability, invasiveness, and host resistance, may be important in the pathogenesis of infections due to Campylobacter spp. (14). We thank A. L. Lastovica (Red Cross Children's Hospital), J. Richardson (South African Institute for Medical Research), E. Gove (Niehaus and Botha, Pathologists), T. van Schalkwyk (MEDUNSA), and L. K. Pickering (University of Texas Medical School) for supplying cultures used in this study.
REFERENCES
Bopp, A. Bruce, and J. M. Hughes. 1982. Campylobacter enteritis associated with foodborne transmission. Am. J. Epidemiol. 116:886-894. 2. Blaser, M. J., J. G. Wells, R. A. Feldman, R. A. Pollard, and J. R. Allen. 1983. Campylobacter enteritis in the United States. A multicenter study. Ann. Intern. Med. 98:360-365. 3. Butzler, J. P., and M. B. Skirrow. 1979. Campylobacter enteritis. Clin. Gastroenterol. 8:737-765. 1. Blaser, M. J., P. Checko, C.
1264
NOTES
4. Chou, S. P., R. Dular, and S. Kasatiya. 1983. Effect of ferrous sulphate, sodium metabisulfite, and sodium pyruvate on survival of Campylobacterjejuni. J. Clin. Microbiol. 18:986-987. 5. Crewe-Brown, H. H., A. S. Greeff, P. J. Fripp, M. T. Bothma, A. D. Steele, H. E. Bok, G. C. Clay, T. V. van Schalkwyk, A. Geyer, and S. N. Kalian. 1989. Aetiology of summer diarrhoea at Ga-Rankuwa Hospital, p. 232-233. In H. J. Koornhof (eds.), Proceedings of the Symposium on Infections in Developing Countries. South African Medical Research Council, Parow, South Africa. 6. Edmonds, P., C. M. Patton, T. J. Barrett, G. K. Morris, A. G. Steigerwalt, and D. J. Brenner. 1985. Biochemical and genetic characteristics of atypical Campylobacter fetus subsp. fetus strains isolated from humans in the United States. J. Clin. Microbiol. 21:936-940. 7. Fox, J. G., K. 0. Maxwell, N. S. Taylor, C. D. Runsick, P. Edmonds, and D. J. Brenner. 1989. "Campylobacter upsaliensis" isolated from cats as identified by DNA relatedness and biochemical features. J. Clin. Microbiol. 27:2376-2378. 8. Gentry, M. K., and J. M. Dalrymple. 1980. Quantitative microtiter cytotoxicity assay for Shigella toxin. J. Clin. Microbiol. 12:361-366. 9. Guerrant, R. L., C. A. Wanke, R. A. Pennie, L. J. Barrett, A. A. M. Lima, and A. D. O'Brien. 1987. Production of a unique cytotoxin by Campylobacter jejuni. Infect. Immun. 55:25262530. 10. Hebert, G. A., D. G. Hollis, R. E. Weaver, M. A. Lambert, M. J. Blaser, and C. W. Moss. 1982. 30 years of campylobacters: biochemical characteristics and a biotyping proposal for Campylobacterjejuni. J. Clin. Microbiol. 15:1065-1073. 11. Hwang, M., and G. M. Ederer. 1975. Rapid hippurate hydrolysis method for presumptive identification of group B streptococci. J. Clin. Microbiol. 1:114-115. 12. Johnson, W. M., and H. Lior. 1984. Toxins produced by Campylobacter jejuni and Campylobacter coli. Lancet ii:229230. 13. Johnson, W. M., and H. Lior. 1985. Toxigenicity studies of Campylobacter species, p. 153. In A. D. Pearson, M. B. Skirrow, H. Lior, and B. Rowe (ed.), Campylobacter III. Proceedings of the Third International Workshop on Campylobacter Infections, Ottawa (Canada), 7 to 10 July 1985. Public Health Laboratory Service, London. 14. Johnson, W. M., and H. Lior. 1986. Cytotoxic and cytotonic factors produced by Campylobacterjejuni, Campylobacter coli, and Campylobacter laridis. J. Clin. Microbiol. 24:275-281. 15. Keusch, G. T., and S. T. Donta. 1975. Classification of enterotoxins on the basis of activity in cell culture. J. Infect. Dis. 131:58-63. 16. Klipstein, F. A., and R. F. Engert. 1984. Properties of crude Campylobacter jejuni heat-labile enterotoxin. Infect. Immun. 45:314-319. 17. Klipstein, F. A., R. F. Engert, H. Short, and E. A. Schenk. 1985. Pathogenic properties of Campylobacter jejuni: assay and cor-
J. CLIN. MICROBIOL.
relation with clinical manifestations. Infect. Immun. 50:43-49. 18. Klipstein, F. A., R. F. Engert, and H. B. Short. 1986. Enzymelinked immunosorbent assays for virulence properties of Campylobacterjejuni clinical isolates. J. Clin. Microbiol. 23:10391043. 19. Leaper, S., and R. J. Owen. 1981. Identification of catalaseproducing Campylobacter species based on biochemical characteristics and on cellular fatty acid composition. Curr. Microbiol. 6:31-35. 20. Lindblom, G. B., B. Kaijser, and E. Sjogren. 1989. Enterotoxin production and serogroups of Campylobacter jejuni and Campylobacter coli from patients with diarrhea and from healthy laying hens. J. Clin. Microbiol. 27:1272-1276. 21. Mathan, V. I., D. P. Rajan, F. A. Klipstein, and R. F. Engert. 1984. Enterotoxigenic Campylobacter jejuni among children in South India. Lancet ii:981. 22. McCardell, B. A., J. M. Madden, and J. T. Stanfield. 1986. Effect of iron concentration on toxin production in Campylobacter jejuni and Campylobacter coli. Can. J. Microbiol. 32: 395-401. 23. Moore, M. A., M. J. Blaser, G. I. Perez-Perez, and A. D. O'Brien. 1988. Production of a Shiga-like cytotoxin by Campylobacter. Microb. Pathog. 4:455-462. 24. Olusanya, O., J. 0. Adebayo, and B. Williams. 1983. Campylobacterjejuni as a bacterial cause of diarrhoea in Ile-Ife, Nigeria. J. Hyg. 91:77-80. 25. Pai, G. H., S. Sorger, K. Lackman, R. E. Sinai, and M. I. Marks. 1979. Campylobacter gastroenteritis in children. J. Pediatr. 94:589-591. 26. Pang, T., C. L. Koh, N. Puthucheary, N. Parasakthi, P. Y. Wong, M. Dharan, K. W. Chang, and K. Sihotang. 1988. Cytotoxin and plasmid studies with Malaysian isolates of Campylobacterjejuni, p. 235-236. In B. Kaijser and E. Falsen (ed.), Campylobacter IV. Proceedings of the 4th International Workshop on Campylobacter Infections, Goteborg (Sweden), 16 to 18 June, 1988. University of Goteborg, Sweden. 27. Roilings, D. M., J. C. Coolbaugh, R. I. Walker, and E. Weiss. 1983. Biphasic culture system for rapid Campylobacter cultivation. Appl. Environ. Microbiol. 45:284-289. 28. Ruiz-Palacios, G. M., J. Torres, N. I. Torres, E. Escamilla, B. R. Ruiz-Palacios, and J. Tamayo. 1983. Cholera-like enterotoxin produced by Campylobacter jejuni. Characterization and clinical significance. Lancet ii:250-253. 29. Steele, T. W., and R. J. Owen. 1988. Campylobacter jejuni subsp. doylei subsp. nov., a subspecies of nitrate-negative campylobacters isolated from human clinical specimens. Int. J. Syst. Bacteriol. 38:316-318. 30. Svedhem, A., and B. Kaijser. 1980. Campylobacter fetus subspecies jejuni: a common cause of diarrhoea in Sweden. J. Infect. Dis. 142:353-359. 31. Walder, M. 1982. Epidemiology of Campylobacter enteritis. Scand. J. Infect. Dis. 14:27-33.
Vol. 29, No. 6
0095-1137/91/061262-03$02.00/0 Copyright © 1991, American Society for Microbiology
Incidence of Toxigenic Campylobacter Strains in South Africa HERMAN E. BOK, ANDRE S. GREEFF,* AND HEATHER H. CREWE-BROWN Department of Microbiological Pathology, Medical University of Southern Africa (MEDUNSA), P.O. MEDUNSA 0204, Republic of South Africa Received 21 November 1990/Accepted 18 March 1991
Campylobacter strains can produce a heat-labile cytotonic toxin (CTON) and various cytotoxins (CTOX). Of 22 South African Campylobacter strains tested, 86% were toxigenic (77% produced CTON, 41% produced CTOX, and 32% produced both types) and 14% were toxin negative. Campylobacterjejuni strains were 67% CTON positive and 47% CTOX positive, whereas Campylobacter coli strains were 100 and 29% positive, respectively. and to increase CTON production (22). Sodium citrate, at molar concentrations equivalent to those of the iron compound, was added to the broth phase to prevent iron precipitation. After incubation, the cell-free supematants were prepared by centrifugation of the broth phase at 10,000 x g for 30 min and membrane filtration (Millex; pore size, 0.22 ,um; Millipore) of the supernatant. Toxin assays were conducted at 37°C under 5% CO2 in a humidified CO2 incubator (Forma Scientific). Y-1 monolayers were propagated with Ham's F-10 medium with L-glutamine (M.A. Bioproducts), supplemented with 14% fetal calf serum (Delta). HeLa cells were maintained in Eagle's minimal essential medium with Earle salts and L-glutamine, supplemented with 10% fetal calf serum. The assay for CTON was done on Y-1 cells according to published procedures (14). In this assay, .50% cell rounding was considered positive for CTON (15). The CTOX assay was conducted with HeLa cells according to the dye release method of Gentry and Dalrymple (8). A .50% cell detachment was regarded as positive for CTOX. For neutralization (16), the cell-free supernatants of CTON-positive cultures were incubated for 1 h at 37°C with equal volumes of a 10-fold serial dilution of 1-,ug/ml cholera antitoxin (Swiss Serum Institute) before addition to the Y-1 monolayers in a microtiter plate. Antiserum against CTOX was not available. The results are summarized in Table 2. All cultures were confirmed as Campylobacter strains. They were said to be Campylobacter jejuni-Campylobacter coli if they showed typical darting motility in brucella broth and were suscepti-
Campylobacter spp. are recognized as a common cause of human enteritis worldwide (1, 2, 5, 9, 14, 17, 25, 30, 31). They are prevalent in children (25), particularly in developing countries (28), and rank as the third most common cause of acute diarrhea after rotavirus and enterotoxigenic Escherichia coli (5, 17, 24). Noninflammatory watery diarrhea that arises predominantly in the small bowel is the result of the action of heat-labile cytotonic toxin (CTON) (28). Klipstein et al. (17) found that all of the Campylobacter strains isolated from patients with bloody, invasive-type diarrhea elaborated cytotoxins (CTOX). Some reported incidences of toxigenic Campylobacter species from different countries are represented in Table 1. The incidence of toxin production in South African Campylobacter strains is not known and was therefore investigated among local strains by analyzing responses of Y-1 and HeLa cells to cell-free supernatants of Campylobacter stool isolates. Campylobacter strains isolated at Ga-Rankuwa Hospital (near Pretoria) and strains donated by the Red Cross Children's Hospital in Cape Town, the South African Institute for Medical Research in Johannesburg, and private pathologists in Pretoria were used in this study (Table 2). They were all isolated from stools of patients with gastroenteritis typical of campylobacteriosis. A known CTON- and CTOX-positive strain and a CTON- and CTOX-negative strain, donated by the University of Texas Medical School, were used as controls. The identities of the cultures were confirmed by biotyping by using published procedures (6, 7, 10, 11, 19, 29). All of the Campylobacter strains were grown for 48 h at 37°C under 10% CO2 in a humidified CO2 incubator (Forma Scientific). They were stored at -70°C in glycerol-peptone medium containing 25% glycerol. When needed, they were thawed rapidly at 37°C and streaked onto tryptose blood agar (Oxoid) containing 5% lysed horse blood, Preston Campylobacter selective supplement (Oxoid), Campylobacter growth supplement (Oxoid), and 1 ,ug of amphotericin B (Fungizone) per ml. Cell-free supernatants for toxin testing were prepared by inoculation of Campylobacter strains into biphasic medium (27) containing 0.025% ferrous sulfate, sodium metabisulfite, and sodium pyruvate to maintain the characteristic morphology, motility, and viability of the Campylobacter cultures (4) *
TABLE 1. Published incidences of toxigenic
Campylobacter strains Investigators (reference)a
Johnson and Lior (14) Pang et al. (26) Ruiz-Palacios et al. (28) Mathan et al. (21)
Country
Country
Canada Malaysia Mexico South India
Incidence (%) of: C. jejuni C. coli C._jejun_C._col CTOX CTON CTOX CTON
69 48 NR NR
70 NRb 75 32
78 NR NR NR
83 NR
NR NR
a Lindblom et al. (20) reported a 32% incidence of CTON-positive Campylobacter strains in Sweden. b NR, not reported.
Corresponding author. 1262
VOL. 29, 1991
NOTES
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TABLE 2. Toxin production and biochemical characterization of Campylobacter strains Characteristica Strain no.
Identity
Identlty
Growth at:
CTON
CTOX
Hip.
Nal.
Cep.
H2S
Cat.
Ox.
250C
420C
ST167 ST152 ST116 ST111 ST65 ST52 ST23 NB11 96.86 89.86A 89.86B 87.86 85.86 71.86 207.85 206.85 192.85 AL150 435/1 L26 L28 126 IP383 136SP
C. jejuni biotype I + + + R R + + + + C. jejuni biotypelI S + R + + + C. jejuni biotype II S + + R + + + C. jejuni biotypeI S + + R + + + + C. coli S + + R + + + + C. jejuni biotype I S + + R + + + + C. jejuni biotype I S + + + R + + + + S C. coli + R + + + + C. jejuni biotype II + S + R + + + C. coli + S + R + + + C. jejuni biotype II S + + R + + C. jejuni biotype I + (+) S (+) R + + + C. coli + R + + R + + S C. jejuni biotypeI + + + + R + + C. jejuni biotype II + S + + R + + + S C. jejuni biotype I + (+) + + R + + C. coli + S + + R + + + + C. coli S (+) + R + + C. coli + S + + R + + + C. jejuni biotype II S + + + + R + + C. jejuni biotype II + + S + R + + C. jejuni biotype II S + (+) + + R + + C. coli S + + + + R C. jejuni biotypeI + + + + R R (+) a Abbreviations: CTON, CTON production; CTOX, CTOX production. Hip., hippurate hydrolysis; Nal., resistance (R) or susceptibility (S) to nalidixic acid; Cep., resistance (R) or susceptibility (S) to cephalothin; Ox., oxidase production; Cat., catalase production; H2S, H2S detected on lead acetate strips from triple sugar iron agar cultures. Symbols: +, positive; -, negative; (+), weakly positive.
ble to nalidixic acid (30-,ug disk) and resistant to cephalothin (30-,ug disk). On the basis of the hippurate hydrolysis test (11), there were 15 C. jejuni isolates and 7 C. coli isolates. Strain 85.86 cross-reacted with a DNA probe against C. jejuni-C. coli (in another experiment) and was considered to be a C. coli strain. The two hippurate-positive, nalidixic acid-resistant strains were grouped with the C. jejuni isolates (3). Strains IP383 and 136SP were the positive and negative controls, respectively. The identity of CTON was confirmed by neutralization. From Table 2, it is evident that 19 (86%) of our isolates were toxigenic: 12 (55%) produced one toxin type, and 7 (32%) produced both types. Seventeen (77%) were positive for CTON, and 9 (41%) were positive for CTOX. Johnson and Lior found concurrent production of CTON and CTOX in 53% (13) to 67% (12) of C. jejuni-C. coli isolates. Their incidence of toxin-negative isolates varied between 5% (12) and 14% (13), comparable to our 14%. Our finding that 67% of C. jejuni strains were positive for CTON is comparable to the 75% incidence of CTONpositive strains found by Ruiz-Palacios et al. (28) in Mexico and the 70% incidence reported by Johnson and Lior (14) in Canada. The South African CTON incidence figure is, however, more than double that found for CTON in South India (32%) (21). All 7 of our C. coli isolates produced CTON. Similarly, Johnson and Lior (14) also reported a high CTON incidence (83%) in 131 isolates. We found CTOX in 47% of our C. jejuni isolates and in 29% of the C. coli isolates. This is a considerably lower incidence than the 69 and 78% incidence, respectively, found by Johnson and Lior (14). In a smaller study by Klipstein et al. (17) with 20 strains, CTOX was found in cell-free culture filtrates of 6 of 6 (100%) C. jejuni strains isolated from
patients with invasive-type diarrhea. On the other hand, Moore et al. (23) found no detectable levels of CTOX in culture supernatants of 36 clinical isolates. They did, however, observe trace to low-level cell-associated cytotoxicity in 58% of their strains. The toxigenic Campylobacter incidence figures for South Africa are similar to those reported in some other countries. No direct relationship between the biotype and toxigenicity was found in this study. The role of the toxins in the pathogenesis of Campylobacter enteritis remains unclear, as some of the clinical strains were found to be negative for both toxin types. These strains may produce other toxins, such as cell-associated toxins, that were not detected in this survey. Strains from symptomatic patients can also lose their pathogenic properties (18). Other factors, such as colonization ability, invasiveness, and host resistance, may be important in the pathogenesis of infections due to Campylobacter spp. (14). We thank A. L. Lastovica (Red Cross Children's Hospital), J. Richardson (South African Institute for Medical Research), E. Gove (Niehaus and Botha, Pathologists), T. van Schalkwyk (MEDUNSA), and L. K. Pickering (University of Texas Medical School) for supplying cultures used in this study.
REFERENCES
Bopp, A. Bruce, and J. M. Hughes. 1982. Campylobacter enteritis associated with foodborne transmission. Am. J. Epidemiol. 116:886-894. 2. Blaser, M. J., J. G. Wells, R. A. Feldman, R. A. Pollard, and J. R. Allen. 1983. Campylobacter enteritis in the United States. A multicenter study. Ann. Intern. Med. 98:360-365. 3. Butzler, J. P., and M. B. Skirrow. 1979. Campylobacter enteritis. Clin. Gastroenterol. 8:737-765. 1. Blaser, M. J., P. Checko, C.
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