Cholera in Vietnam: changes in genotypes and emergence of class I integrons containing aminoglycoside resistance gene cassettes in vibrio cholerae O1 strains isolated from 1979 to 1996

Abstract

The number of cholera cases and the mortality rates reported from different regions of Vietnam varied considerably in the period from 1979 to 1996, with between 2,500 and 6,000 cases reported annually from 1992 to 1995. Annual mortality rates ranged from 2.0 to 9.6% from 1979 to 1983 to less than 1.8% after 1983. Major cholera outbreaks were reported from the High Plateau region for the first time in 1994 and 1995; this is an area with limited access to health services and safe drinking-water supplies. All cases were associated with Vibrio cholerae O1. Using ribotyping, cholera toxin (CT) genotyping, and characterization of antibiotic susceptibility patterns and antibiotic resistance genes by PCR, we show that strains isolated after 1990 were clearly different from strains isolated before 1991. In contrast to strains isolated before 1991, 94% of 104 strains isolated after 1990 showed an identical ribotype R1, were resistant to sulfamethoxazole and streptomycin, and showed a different CT genotype. Furthermore, PCR analysis revealed that sulfamethoxazole-resistant strains harbored class I integrons containing a gene cassette ant(3")-1a encoding resistance to streptomycin and spectinomycin. This is, to our knowledge, the first report of class I integrons in V. cholerae. The development of cholera and the changes in the phenotypic and genotypic properties of V. cholerae O1 shown in the present study highlight the importance of monitoring V. cholerae O1 in Vietnam as in other parts of the world. In particular, the emergence of the new ribotype R1 strain containing class I integrons should be further studied.

FIG. 1

Association between integron structure and PCR products modified from Sandvang et al. (36). The lines below the integron structure represent amplicons. The numbers above the PCR products refer to the primers used on V. cholerae O1 (see Table 3). 5′-CS and 3′-CS represent the 5′ and 3′ conserved segments of the integron; qacE and sul1 encode resistance to disinfectant and sulfonamide, respectively. Numbers correspond to the sequence positions in GenBank accession number D43625 (20).

FIG. 2

Examples of BglI ribotypes of V. cholerae O1 strains associated with cholera outbreaks in Vietnam from 1979 to 1996, of clinical strains from Thailand and India, and of strains used in an oral vaccine. Unless otherwise stated, explanations for the contents of the lanes indicate the strain designation, the place and date of isolation, and the ribotype designation. Lanes: A, 1-kb molecular mass standard; B, Gia Lai, 1995, type R2; C, Ho Chi Minh City, June 1994, type R1; D, 33/97, Bangkok 1997, type R1; E, 697/38, Samut Sakorn 1994, type R1; F, VC20, Calcutta, 1992, type R1; G, Dong Thap, March 1995, type R8; H, Thuan Hai, December 1979, type R6; I, Minh Hai, November 1994, type R3; J, Ham Tan Thuan Hai, August 1985, type R5; K, Hue, June 1983, type R4; L, 1076/25, Samut Sakorn, Thailand, 1982, type R4; M, Hue, May 1995, type R7; N, 4260B, V. cholerae O139 vaccine strain; O, Phil 6973, vaccine strain, type R4; P, Cairo 48, vaccine strain; Q, Cairo 50, vaccine strain; R, 569B, vaccine strain; and S, 1-kb molecular mass standard.

FIG. 3

Examples of PCR for the presence of class I integrons among V. cholerae O1 strains isolated in Vietnam by using the primers qacEΔ1 F and sul1 B. Unless indicated otherwise, the following explanations for the contents of the lanes indicate province and year of isolation, antibiogram, and ribotype. Lanes: A, 100-bp molecular mass standard; B, Khanh Hoa, 1979, Cl;Sm_I, type R6; C, Ham Tan-Thuan Hai, 1985, Cl;Sm_I, type R5; D, Hue, 1990, Cl;Sm_I, type R4; E, Ben Tre, 1994, Cl;Sm;Su, type R1; F, Dak Lak, 1994, Cl;Sm,Su type R1; G, Hue, 1995, Cl;Sm;Su, type R1; H, Gia Lai, 1995, Cl;Sm_I, type R1; I, Dong Thap, 1995, Cl;Sm;Su, type R8; J, Dak Lak, 1995, Cl;Sm;Su, type R2; K, Hai Hung, 1996, Cl;Sm;Su, type R1; L, PCR mixture, negative control, M, R4-96, negative control; N, 9720921, positive control; and O, 100-bp molecular mass standard.

FIG. 4

Examples of PCR products of V. cholerae O1 strains isolated in Vietnam by using the primers int1 F and int1 B (lanes B to G) and int1 F and ant(3")-Ia B (lanes K to P). Unless indicated otherwise, the following explanations for the contents of the lanes indicate the province and year of isolation, antibiogram, and ribotype. Lanes: A, 100-bp molecular mass standard; B, Ben Tre, 1994, Cl;Sm;Su, type R1; C, Dak Lak, 1994, Cl;Sm,Su, type R1; D, Hue, 1995, Cl;Sm;Su, type R1; E, Dong Thap, 1995, Cl;Sm;Su, type R8; F, Dak Lak, 1995, Cl;Sm;Su, type R2; G, Hai Hung, 1996, Cl;Sm;Su, type R1; H, R4-96, negative control; I, 9720921, positive control; J, PCR mixture, negative control; K, Ben Tre, 1994, Cl;Sm;Su, type R1; L, Dak Lak, 1994, Cl;Sm,Su, type R1; M, Hue, 1995, Cl;Sm;Su, type R1; N, Dong Thap, 1995, Cl;Sm;Su, type R8; O, Dak Lak, 1995, Cl;Sm;Su, type R2; P, Hai Hung, 1996, Cl;Sm;Su, type R1; Q, 9720921, positive control; R, R4-96, negative control; and S, 100-bp molecular mass standard.

FIG. 5

Southern hybridization analysis of genomic DNA from 15 V. cholerae strains isolated from patients in Vietnam and 5 vaccine strains digested with BglI and probed with a cholera toxin probe. Unless stated otherwise, explanations of the contents of the lanes indicate the strain designation, the province/region of isolation, and the ribotype. Lanes: A, HindIII digest of phage lambda as molecular size markers; B, VC-351, Khanh Hoa/Center ribotype R1; C, VC-320, Hue/Center, type R1; D, VC-233, Dong Thap/South, type R1; E, VC-288, Gia Lai/High Plateau, type R1; F, VC-378, Dak Lak/High Plateau, type R1; G, VC-281, Dong Thap/South, type R8; H, VC-181, Dak Lak/High Plateau, type R2; I, VC-182, Gia Lai/High Plateau, type R2; J, VC-203, Minh Hai/South, type R3; K, VC-367, Khanh Hoa/Center, type R4; L, VC-362, Ham Tan-Huan Hai/Center, type R5; M, VC-364, Da nang/Center, type R5; N, VC-336, Hue/Center, type R6; O, VC-346, Khanh Hoa/Center, type R6; P, VC-302, Hue/Center, type R7; Q, Cairo 48, vaccine strain; R, Cairo 50, vaccine strain; S, 569B, vaccine strain; T, 4260 B, vaccine strain; U, Phil 6973, vaccine strain; and V, HindIII digest of phage lambda as molecular size markers. Since the approximately 6.7-kb fragment appeared weakly (lanes J to N) in repeated testing and was difficult to visualize, the position of the fragment is indicated by arrowheads.