Vol. 30, No. 1
JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1992, p. 219-221
0095-1137/92/010219-03$02.00/0 Copyright © 1992, American Society for Microbiology
Occurrence of Resistance to Vibriostatic Compound 0/129 in Vibrio cholerae 01 Isolated from Clinical and Environmental Samples in Bangladesh ANWARUL
HUQ,'t MUNIRUL ALAM,' SALINA PARVEEN,1 AND R. R. COLWELL2*
International Centre for Diarrhoeal Disease Research, Bangladesh, Dhaka, Bangladesh,' and Department of Microbiology, University of Marvland, College Park, Maryland 207422 Received 8 October 1991/Accepted 10 October 1991
Fifty-one Vibrio chokrae 01 strains isolated from 734 natural water and plankton samples and 31 rectal swabs were examined. Of these strains, 32 (62.7%) were found to be resistant to vibriostatic compound 0/129. When antibiograms using the antibiotics ampicillin, tetracycline, chloramphenicol, trimethoprim-sulfamethoxazole, furoxan, and gentamicin were done, it was observed that there was a correlation of sensitivity to 0/129 with selected antibiotics. Only the Ogawa El Tor (72% of strains resistant) and Inaba classical (28% of strains resistant) biotypes of V. cholerae 01 showed resistance to 0/129. On the other hand, all Inaba El Tor and Ogawa classical strains were susceptible to 0/129. The 32 0/129-resistant and 19 0/129-sensitive isolates of V. cholerae 01 were tested for the presence of plasmid DNA. Only two strains isolated from the environment were found to carry a plasmid, and they were also found to be resistant to 0/129 and gentamicin. Thus, 0/129 resistance, although more common than previously suspected, is concluded not to be plasmid mediated in the strains tested in this study.
ical tests, including a final slide agglutination test employing specific antisera. To determine the sensitivities to 0/129 of all the isolates of V. cholerae, uniform lawns of bacteria, approximately 10' cells per ml, grown in 4-h T,Nj (1% trypticase plus 1% NaCl) broth cultures incubated at 37°C, were prepared by using a cotton-tipped swab on gelatin agar (grams per liter: peptone, 4; yeast extract, 1; gelatin, 15; NaCl, 10; and agar, 15). Impregnated discs of both 10 and 150 ,ug of the 0/129 compound were prepared and stored at 4°C for no more than 1 month before use. The discs were placed aseptically on the plates. After incubation at 37°C for 18 to 24 h, a clear zone of inhibition of growth developed around the discs, indicating that the organisms were susceptible to 0/129 at a given concentration. Organisms resistant to 0/129 developed no zone of inhibition. Resistance to 0/129 was defined as described elsewhere (4, 14). Selected antibiotic sensitivity profiles of all the strains were determined by the method of Bauer et al. (1). Commercially available antibiotic discs of ampicillin (10 ,ug), chloramphenicol (30 ,ug), trimethoprim-sulfamethoxazole (25 Rg), tetracycline (30 ,ug), gentamicin (10 j,g), and furoxan (100 pLg) (BBL, Cockeysville, Md.) were used in the tests. Discs were placed on Mueller-Hinton agar plates which had been swabbed with fresh, 4-h broth cultures of standard turbidity, containing approximately 107 cells per ml, to give a confluent lawn of growth. E. coli ATCC 25922 was used as a control strain. Sensitivity was demonstrated by the zones of inhibition produced after overnight incubation at 37°C. Tests, such as polymyxin B sensitivity (5), phage typing as described by Mukherjee et al. (12), and the chicken cell agglutination using chick erythrocytes (5), were performed to biotype the isolates of V. cholerae used in the study. Plasmids were extracted by the method described by Birnboim and Doly (2). Cells of V. cholerae 01 were grown aerobically overnight in 5 ml of Trypticase Soy Broth (Difco) in a 50-ml flask containing 0.6% yeast extract incubated in a 37°C water bath. To determine the molecular size of the
Vibrio cholerae 01 is an etiological agent of Asiatic cholera. The vibriostatic compound 0/129 (2,4-diamino-6,7,-diisopropylpteridine) is used as a primary screen in the identification of members of the family Vibrionaceae (4, 14, 19). Ninety-nine percent of V. cholerae 01 strains have been reported to be sensitive to 0/129 (5). All the isolates from the Louisiana outbreak in 1979 were sensitive to 0/129 (3). Sundaram and Murthy (17), however, described human isolates of both V. cholerae 01 and non-01 strains resistant to this compound. Kudoh et al. (8) isolated 0/129-resistant strains from Japan and other countries. Other investigators have reported that this resistance is transmissible to Escherichia coli and V. cholerae strains by conjugation and concluded that most of the 0/129 resistance associated with V. cholerae is plasmid encoded (10). Vibriostatic compound 0/129-resistant V. cholerae 01 strains from environmental samples were not reported previously. The present study was undertaken to evaluate the importance of 0/129 as a screening test for the isolation and identification of V. cholerae from clinical as well as from environmental samples. In addition to this study, a comparative study was done to determine antibiotic resistance patterns of both 0/129resistant and 0/129-sensitive strains isolated from environmental and clinical sources. A total of 734 water and plankton samples collected at 2-week intervals from eight ponds and two river sites and 31 rectal swabs from suspected cholera patients of the surveillance area of the International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B) Matlab Treatment Centre were analyzed between March 1987 and December 1988. The strains of V. cholerae 01 were isolated from these samples by using conventional culture methods described previously (5) and were confirmed by a battery of biochem* Corresponding author. t Present address: Department of Microbiology, University of Maryland, College Park, MD 20742.
219
220
J. CLIN. MICROBIOL.
NOTES
TABLE 1. Response of V. cholerae 01 to vibriostatic compound 0/129 and to commonly used antibiotics Resistance to following antibiotic" (%):
V. cholerae reaction with 0/129
No. of strains (%)
Am
Te
Ch
Sxt
Fx
Ge
Resistant Sensitive
32 (62.7) 19 (37.3)
100 0
0 0
0 0
100 0
63 11
6 0
a Am, ampicillin; Te, tetracycline; Ch, chloramphenicol; Sxt, trimethoprim-sulfamethoxazole; Fx, furoxan; Ge, gentamicin.
plasmid DNA, agarose gel electrophoresis was performed by the method described by Meyers et al. (11). Electrophoresis was carried out with a vertical lucite slab gel apparatus (model 100; Aqueboque Machine Shop, New York, N.Y.). The molecular size of the plasmid in each 0/129-resistant V. cholerae 01 strain was determined by comparing it with the standards pDK-9 (140 and 105 MDa) and Ri (62 MDa). Of 51 strains of V. cholerae 01 tested, 32 were resistant and 19 were susceptible to both 10 and 150 jig of 0/129 (Table 1). Of the 32 0/129-resistant isolates, 18 were of clinical origin and 14 were of environmental origin. Among the 19 sensitive strains, 12 and 7 were from clinical and environmental sources, respectively. When the 0/129-resistant isolates were biotyped and serotyped, 72 and 28% were found to be Ogawa El Tor and Inaba classical strains, respectively (Table 2). Inaba El Tor or Ogawa classical strains were not detected among the resistant isolates. Of 19 0/129-susceptible strains, 42% were Inaba El Tor, 42% were Ogawa El Tor, and 16% were Ogawa classical. All of the 32 0/129-resistant and 19 0/129-susceptible isolates of V. cholerae 01 were examined for plasmid DNA. Plasmid bands were not detected in the 0/129-resistant strains, except in the case of two strains which demonstrated the presence of one large plasmid and were also resistant to gentamicin. Before 1960, classical biotypes of V. cholerae 01 were associated with cholera outbreaks on the Indian subcontinent (7, 9, 17). More recently, El Tor strains replaced the classical biotype (13), and now classical and El Tor biotypes are both seen (15). Our results support this latter finding (Table 2). Serotyping of 32 0/129-resistant strains showed that 9 were Inaba classical and 22 were Ogawa El Tor; of the 19 0/129-sensitive strains, 9 were Inaba El Tor, 3 were Ogawa classical, and 8 were Ogawa El Tor (Table 2). This observation indicates that more El Tor V. cholerae 01 than classical type strains were isolated, and, with respect to percentage, the El Tor type was more frequently resistant to 0/129 as well as to the commonly used antibiotics ampicillin, trimethoprim-sulfamethoxazole, furoxan, and gentamicin. It has been reported that 0/129 resistance occurs in ca. 1% of V. cholerae strains (5). It is of great concern to find the presence of a rather large number (.62.0%) of V. cholerae 01 strains resistant to 0/129, especially strains isolated from
both clinical and environmental sources. These results appear to cast in doubt the ability of the 0/129 test to differentiate Vibrio species from one another and from other gram-negative bacilli (8, 10, 18). Resistance of all the 0/129resistant strains to ampicillin and trimethoprim-sulfamethoxazole and the sensitivity of all 0/129-susceptible strains to ampicillin and trimethoprim-sulfamethoxazole merits further study to investigate whether this could be due to a permeability change induced when the strains become resistant, presumably to one or both of the drugs when used clinically. Plasmid-mediated antibiotic resistance in V. cholerae 01 has been reported, but this phenomenon was considered to be relatively uncommon (6). Plasmid-mediated resistance to the other antibacterial agents tested in this study, except for two gentamicin-resistant strains, was not observed for the V. cholerae strains examined. In conclusion, the usefulness of the test of 0/129 as a screen for vibrios may require further investigation; clearly, however, the resistance to 0/129 among V. cholerae 01 strains is far more frequent in both clinical and environmental strains than suspected. This research was supported by the ICDDR,B. ICDDR,B is supported by countries and agencies which share its concern about the impact of diarrheal diseases in the developing countries. Current major donors giving assistance to ICDDR,B are The Aga Khan Foundation, Arab Gulf Fund, Australia, Bangladesh, Belgium, Canadian International Development Agency (CIDA), Canadian International Development Research Centre (IDRC), Danish International Development Agency (DANIDA), France, The Ford Foundation, Japan, The Netherlands, Norwegian Agency for International Development (NORAD), SAREC (Sweden), Swiss Development Cooperation (SDC), United Kingdom, United Nations Development Programme (UNDP), United Nations Children's Fund (UNICEF), United Nations Capital Development Fund (UNCDF), United States Agency for International Development (USAID), World Health Organization (WHO), and World University Service of Canada (WUSC). The work was partially supported by National Science Foundation grant BSR-88-06509 through the University of Maryland. We acknowledge the technical help of Rezaur Rehman, Belayet Hossain, and Afsar Ali of ICDDR,B and the secretarial assistance by Mary Emad of the University of Maryland.
1. 2.
3.
4. 5.
TABLE 2. Serotypes of 0/129-resistant and -sensitive strains
of V. cholerae 01 V. cholerae
with reaction reacionwt
Resistant Sensitive
No. of
strains
32 19
Inaba
No. of subserovar strains (%) Ogawa Inaba Ogawa
classical
El Tor
classical
El Tor
9 (28) 0
0 8 (42)
0 3 (16)
23 (72) 8 (42)
6.
7.
REFERENCES Bauer, A. W., W. M. M. Kirby, J. C. Sherris, and M. Truck. 1966. Antibiotic susceptibility testing by a standardized single disc method. Am. J. Clin. Pathol. 49:983-986. Birnboim, H. C., and J. Doly. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acid Res. 7:1513-1523. Bradford, H. B., Jr., and B. Henry. 1984. An epidemiological study of V. cholerae in Louisiana, p. 59-72. In R. R. Colwell (ed.). Vibrios in the environment. John Wiley and Sons, Inc., New York. Davis, G. H. G., and R. W. A. Park. 1962. A taxonomic study of certain bacteria currently classified as Vibrio species. J. Clin. Microbiol. 27:101-119. Farmer, J. J., III, F. W. Hickman-Brenner, and M. T. Kelly. 1985. Vibrio, p. 282-301. In E. H. Lennette, A. Balows, W. J. Hausler, Jr., and H. J. Shadomy (ed.), Manual of Clinical Microbiology, 4th ed. American Society for Microbiology, Washington, D.C. Huq, M. I., R. I. M. Glass, A. R. M. A. Alim, K. Haider, and A. R. Samadi. 1984. Studies on multiply antibiotic resistant V. cholerae 01 (MARVC) biotypes El-Tor isolated from patients with gastroenteritis. Asian Med. J. 27:519-528. Janda, J. M., C. Powers, and S. L. Abbott. 1988. Current perspective on the epidemiology and pathogenesis of clinically significant Vibrio spp. Clin. Microbiol. Rev. 1:245-267.
VOL. 30, 1992
8. Kudoh, Y., S. Matsushita, S. Yamada, M. Tsuno, K. Ohta, S. Sakai, and M. Ohashi. 1981. Enterotoxin producibility and some biological features of 01 and non-01 Vibrio cholerae isolates, p. 214-224. In S. Kuwahara and Y. Zinnaka (ed.), Proceedings of Symposium on Cholera, Gifu, 1980. Toho University, Tokyo. 9. Levine, M. M., R. Black, and M. L. Clements. 1984. Pathogenesis of enteric infections caused by Vibrio, p. 109-122. In R. R. Colwell (ed.), Vibrios in the environment. John Wiley and Sons, Inc., New York. 10. Matsushita, S., Y. Kudoh, and M. Ohashi. 1984. Transferable resistance to the vibrio-static agent 2,4,-diamino-6,7-diisopropylpteridine (0/129) in Vibrio cholerae. Microbiol. Immunol. 28: 1159-1162. 11. Meyers, J. A., D. Sanchez, P. L. Elwell, and S. Falkow. 1976. Simple agarose gel electrophoretic method for the identification and characterization of plasmid deoxyribonucleic acid. J. Bacteriol. 127:1529-1537. 12. Mukherjee, S., D. K. Guha, and U. K. G. Roy. 1957. Studies on typing of cholera by bacteriophage. I. Phage typing of Vibrio cholerae from Calcutta epidemics. Ann. Biochem. Exp. Med. 17:161-176.
NOTES
221
13. Samadi, A. R., M. I. Huq, N. Shahid, M. U. Khan, A. Eusof, A. S. M. M. Rahman, M. Yunus, and A. S. G. Faruque. 1983. Classical Vibrio cholerae biotype displaces ElTor in Bangladesh. Lancet i:805-807. 14. Shewan, J. M., W. Hodgkiss, and J. Liston. 1954. A method for the rapid identification of certain non-pathogenic, asporogenous bacilli. Nature (London) 173:208-209. 15. Siddique, A. K., A. H. Baqui, A. Eursof, K. Haider, M. A. Hossain, I. Bashir, and K. Zaman. 1991. Survival of classic cholera in Bangladesh. Lancet 337:1125-1127. 16. Smith, H. L., Jr., and K. Goodner. 1958. Detection of bacterial gelatinases by gelatin-agar plate methods. J. Bacteriol. 76:662664. 17. Sundaram, S. P., and K. V. Murthy. 1983. Occurrence of 2,4,diamino-6,7,-diisopropylpteridine (0/129) resistance in human isolates of V. cholerae. FEMS Microbiol. Lett. 19:115-117. 18. Tison, D. L., and M. T. Kelly. 1984. Vibrio species of medical importance. Diagn. Microbiol. Infect. Dis. 2:263-276. 19. WHO Scientific Working Group. 1980. Cholera and other Vibrio associated diarrhoea. Bull. W.H.O. 58:353-374.
JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1992, p. 219-221
0095-1137/92/010219-03$02.00/0 Copyright © 1992, American Society for Microbiology
Occurrence of Resistance to Vibriostatic Compound 0/129 in Vibrio cholerae 01 Isolated from Clinical and Environmental Samples in Bangladesh ANWARUL
HUQ,'t MUNIRUL ALAM,' SALINA PARVEEN,1 AND R. R. COLWELL2*
International Centre for Diarrhoeal Disease Research, Bangladesh, Dhaka, Bangladesh,' and Department of Microbiology, University of Marvland, College Park, Maryland 207422 Received 8 October 1991/Accepted 10 October 1991
Fifty-one Vibrio chokrae 01 strains isolated from 734 natural water and plankton samples and 31 rectal swabs were examined. Of these strains, 32 (62.7%) were found to be resistant to vibriostatic compound 0/129. When antibiograms using the antibiotics ampicillin, tetracycline, chloramphenicol, trimethoprim-sulfamethoxazole, furoxan, and gentamicin were done, it was observed that there was a correlation of sensitivity to 0/129 with selected antibiotics. Only the Ogawa El Tor (72% of strains resistant) and Inaba classical (28% of strains resistant) biotypes of V. cholerae 01 showed resistance to 0/129. On the other hand, all Inaba El Tor and Ogawa classical strains were susceptible to 0/129. The 32 0/129-resistant and 19 0/129-sensitive isolates of V. cholerae 01 were tested for the presence of plasmid DNA. Only two strains isolated from the environment were found to carry a plasmid, and they were also found to be resistant to 0/129 and gentamicin. Thus, 0/129 resistance, although more common than previously suspected, is concluded not to be plasmid mediated in the strains tested in this study.
ical tests, including a final slide agglutination test employing specific antisera. To determine the sensitivities to 0/129 of all the isolates of V. cholerae, uniform lawns of bacteria, approximately 10' cells per ml, grown in 4-h T,Nj (1% trypticase plus 1% NaCl) broth cultures incubated at 37°C, were prepared by using a cotton-tipped swab on gelatin agar (grams per liter: peptone, 4; yeast extract, 1; gelatin, 15; NaCl, 10; and agar, 15). Impregnated discs of both 10 and 150 ,ug of the 0/129 compound were prepared and stored at 4°C for no more than 1 month before use. The discs were placed aseptically on the plates. After incubation at 37°C for 18 to 24 h, a clear zone of inhibition of growth developed around the discs, indicating that the organisms were susceptible to 0/129 at a given concentration. Organisms resistant to 0/129 developed no zone of inhibition. Resistance to 0/129 was defined as described elsewhere (4, 14). Selected antibiotic sensitivity profiles of all the strains were determined by the method of Bauer et al. (1). Commercially available antibiotic discs of ampicillin (10 ,ug), chloramphenicol (30 ,ug), trimethoprim-sulfamethoxazole (25 Rg), tetracycline (30 ,ug), gentamicin (10 j,g), and furoxan (100 pLg) (BBL, Cockeysville, Md.) were used in the tests. Discs were placed on Mueller-Hinton agar plates which had been swabbed with fresh, 4-h broth cultures of standard turbidity, containing approximately 107 cells per ml, to give a confluent lawn of growth. E. coli ATCC 25922 was used as a control strain. Sensitivity was demonstrated by the zones of inhibition produced after overnight incubation at 37°C. Tests, such as polymyxin B sensitivity (5), phage typing as described by Mukherjee et al. (12), and the chicken cell agglutination using chick erythrocytes (5), were performed to biotype the isolates of V. cholerae used in the study. Plasmids were extracted by the method described by Birnboim and Doly (2). Cells of V. cholerae 01 were grown aerobically overnight in 5 ml of Trypticase Soy Broth (Difco) in a 50-ml flask containing 0.6% yeast extract incubated in a 37°C water bath. To determine the molecular size of the
Vibrio cholerae 01 is an etiological agent of Asiatic cholera. The vibriostatic compound 0/129 (2,4-diamino-6,7,-diisopropylpteridine) is used as a primary screen in the identification of members of the family Vibrionaceae (4, 14, 19). Ninety-nine percent of V. cholerae 01 strains have been reported to be sensitive to 0/129 (5). All the isolates from the Louisiana outbreak in 1979 were sensitive to 0/129 (3). Sundaram and Murthy (17), however, described human isolates of both V. cholerae 01 and non-01 strains resistant to this compound. Kudoh et al. (8) isolated 0/129-resistant strains from Japan and other countries. Other investigators have reported that this resistance is transmissible to Escherichia coli and V. cholerae strains by conjugation and concluded that most of the 0/129 resistance associated with V. cholerae is plasmid encoded (10). Vibriostatic compound 0/129-resistant V. cholerae 01 strains from environmental samples were not reported previously. The present study was undertaken to evaluate the importance of 0/129 as a screening test for the isolation and identification of V. cholerae from clinical as well as from environmental samples. In addition to this study, a comparative study was done to determine antibiotic resistance patterns of both 0/129resistant and 0/129-sensitive strains isolated from environmental and clinical sources. A total of 734 water and plankton samples collected at 2-week intervals from eight ponds and two river sites and 31 rectal swabs from suspected cholera patients of the surveillance area of the International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B) Matlab Treatment Centre were analyzed between March 1987 and December 1988. The strains of V. cholerae 01 were isolated from these samples by using conventional culture methods described previously (5) and were confirmed by a battery of biochem* Corresponding author. t Present address: Department of Microbiology, University of Maryland, College Park, MD 20742.
219
220
J. CLIN. MICROBIOL.
NOTES
TABLE 1. Response of V. cholerae 01 to vibriostatic compound 0/129 and to commonly used antibiotics Resistance to following antibiotic" (%):
V. cholerae reaction with 0/129
No. of strains (%)
Am
Te
Ch
Sxt
Fx
Ge
Resistant Sensitive
32 (62.7) 19 (37.3)
100 0
0 0
0 0
100 0
63 11
6 0
a Am, ampicillin; Te, tetracycline; Ch, chloramphenicol; Sxt, trimethoprim-sulfamethoxazole; Fx, furoxan; Ge, gentamicin.
plasmid DNA, agarose gel electrophoresis was performed by the method described by Meyers et al. (11). Electrophoresis was carried out with a vertical lucite slab gel apparatus (model 100; Aqueboque Machine Shop, New York, N.Y.). The molecular size of the plasmid in each 0/129-resistant V. cholerae 01 strain was determined by comparing it with the standards pDK-9 (140 and 105 MDa) and Ri (62 MDa). Of 51 strains of V. cholerae 01 tested, 32 were resistant and 19 were susceptible to both 10 and 150 jig of 0/129 (Table 1). Of the 32 0/129-resistant isolates, 18 were of clinical origin and 14 were of environmental origin. Among the 19 sensitive strains, 12 and 7 were from clinical and environmental sources, respectively. When the 0/129-resistant isolates were biotyped and serotyped, 72 and 28% were found to be Ogawa El Tor and Inaba classical strains, respectively (Table 2). Inaba El Tor or Ogawa classical strains were not detected among the resistant isolates. Of 19 0/129-susceptible strains, 42% were Inaba El Tor, 42% were Ogawa El Tor, and 16% were Ogawa classical. All of the 32 0/129-resistant and 19 0/129-susceptible isolates of V. cholerae 01 were examined for plasmid DNA. Plasmid bands were not detected in the 0/129-resistant strains, except in the case of two strains which demonstrated the presence of one large plasmid and were also resistant to gentamicin. Before 1960, classical biotypes of V. cholerae 01 were associated with cholera outbreaks on the Indian subcontinent (7, 9, 17). More recently, El Tor strains replaced the classical biotype (13), and now classical and El Tor biotypes are both seen (15). Our results support this latter finding (Table 2). Serotyping of 32 0/129-resistant strains showed that 9 were Inaba classical and 22 were Ogawa El Tor; of the 19 0/129-sensitive strains, 9 were Inaba El Tor, 3 were Ogawa classical, and 8 were Ogawa El Tor (Table 2). This observation indicates that more El Tor V. cholerae 01 than classical type strains were isolated, and, with respect to percentage, the El Tor type was more frequently resistant to 0/129 as well as to the commonly used antibiotics ampicillin, trimethoprim-sulfamethoxazole, furoxan, and gentamicin. It has been reported that 0/129 resistance occurs in ca. 1% of V. cholerae strains (5). It is of great concern to find the presence of a rather large number (.62.0%) of V. cholerae 01 strains resistant to 0/129, especially strains isolated from
both clinical and environmental sources. These results appear to cast in doubt the ability of the 0/129 test to differentiate Vibrio species from one another and from other gram-negative bacilli (8, 10, 18). Resistance of all the 0/129resistant strains to ampicillin and trimethoprim-sulfamethoxazole and the sensitivity of all 0/129-susceptible strains to ampicillin and trimethoprim-sulfamethoxazole merits further study to investigate whether this could be due to a permeability change induced when the strains become resistant, presumably to one or both of the drugs when used clinically. Plasmid-mediated antibiotic resistance in V. cholerae 01 has been reported, but this phenomenon was considered to be relatively uncommon (6). Plasmid-mediated resistance to the other antibacterial agents tested in this study, except for two gentamicin-resistant strains, was not observed for the V. cholerae strains examined. In conclusion, the usefulness of the test of 0/129 as a screen for vibrios may require further investigation; clearly, however, the resistance to 0/129 among V. cholerae 01 strains is far more frequent in both clinical and environmental strains than suspected. This research was supported by the ICDDR,B. ICDDR,B is supported by countries and agencies which share its concern about the impact of diarrheal diseases in the developing countries. Current major donors giving assistance to ICDDR,B are The Aga Khan Foundation, Arab Gulf Fund, Australia, Bangladesh, Belgium, Canadian International Development Agency (CIDA), Canadian International Development Research Centre (IDRC), Danish International Development Agency (DANIDA), France, The Ford Foundation, Japan, The Netherlands, Norwegian Agency for International Development (NORAD), SAREC (Sweden), Swiss Development Cooperation (SDC), United Kingdom, United Nations Development Programme (UNDP), United Nations Children's Fund (UNICEF), United Nations Capital Development Fund (UNCDF), United States Agency for International Development (USAID), World Health Organization (WHO), and World University Service of Canada (WUSC). The work was partially supported by National Science Foundation grant BSR-88-06509 through the University of Maryland. We acknowledge the technical help of Rezaur Rehman, Belayet Hossain, and Afsar Ali of ICDDR,B and the secretarial assistance by Mary Emad of the University of Maryland.
1. 2.
3.
4. 5.
TABLE 2. Serotypes of 0/129-resistant and -sensitive strains
of V. cholerae 01 V. cholerae
with reaction reacionwt
Resistant Sensitive
No. of
strains
32 19
Inaba
No. of subserovar strains (%) Ogawa Inaba Ogawa
classical
El Tor
classical
El Tor
9 (28) 0
0 8 (42)
0 3 (16)
23 (72) 8 (42)
6.
7.
REFERENCES Bauer, A. W., W. M. M. Kirby, J. C. Sherris, and M. Truck. 1966. Antibiotic susceptibility testing by a standardized single disc method. Am. J. Clin. Pathol. 49:983-986. Birnboim, H. C., and J. Doly. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acid Res. 7:1513-1523. Bradford, H. B., Jr., and B. Henry. 1984. An epidemiological study of V. cholerae in Louisiana, p. 59-72. In R. R. Colwell (ed.). Vibrios in the environment. John Wiley and Sons, Inc., New York. Davis, G. H. G., and R. W. A. Park. 1962. A taxonomic study of certain bacteria currently classified as Vibrio species. J. Clin. Microbiol. 27:101-119. Farmer, J. J., III, F. W. Hickman-Brenner, and M. T. Kelly. 1985. Vibrio, p. 282-301. In E. H. Lennette, A. Balows, W. J. Hausler, Jr., and H. J. Shadomy (ed.), Manual of Clinical Microbiology, 4th ed. American Society for Microbiology, Washington, D.C. Huq, M. I., R. I. M. Glass, A. R. M. A. Alim, K. Haider, and A. R. Samadi. 1984. Studies on multiply antibiotic resistant V. cholerae 01 (MARVC) biotypes El-Tor isolated from patients with gastroenteritis. Asian Med. J. 27:519-528. Janda, J. M., C. Powers, and S. L. Abbott. 1988. Current perspective on the epidemiology and pathogenesis of clinically significant Vibrio spp. Clin. Microbiol. Rev. 1:245-267.
VOL. 30, 1992
8. Kudoh, Y., S. Matsushita, S. Yamada, M. Tsuno, K. Ohta, S. Sakai, and M. Ohashi. 1981. Enterotoxin producibility and some biological features of 01 and non-01 Vibrio cholerae isolates, p. 214-224. In S. Kuwahara and Y. Zinnaka (ed.), Proceedings of Symposium on Cholera, Gifu, 1980. Toho University, Tokyo. 9. Levine, M. M., R. Black, and M. L. Clements. 1984. Pathogenesis of enteric infections caused by Vibrio, p. 109-122. In R. R. Colwell (ed.), Vibrios in the environment. John Wiley and Sons, Inc., New York. 10. Matsushita, S., Y. Kudoh, and M. Ohashi. 1984. Transferable resistance to the vibrio-static agent 2,4,-diamino-6,7-diisopropylpteridine (0/129) in Vibrio cholerae. Microbiol. Immunol. 28: 1159-1162. 11. Meyers, J. A., D. Sanchez, P. L. Elwell, and S. Falkow. 1976. Simple agarose gel electrophoretic method for the identification and characterization of plasmid deoxyribonucleic acid. J. Bacteriol. 127:1529-1537. 12. Mukherjee, S., D. K. Guha, and U. K. G. Roy. 1957. Studies on typing of cholera by bacteriophage. I. Phage typing of Vibrio cholerae from Calcutta epidemics. Ann. Biochem. Exp. Med. 17:161-176.
NOTES
221
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