a total of 58 strains of v. cholerae o1 el tor isolated in calcutta before ( march  december 1992 ; group i ) , during ( july  november 1993 ; group ii ) , and after ( march 1994june 1995 ; group iii ) the v. cholerae o139 outbreak ( 8) were included in this study . these strains , belonging to ribotypes ri , rii , and riii , were maintained in brain - heart infusion broth supplemented with 15% glycerol at 70c and grown when needed ( 8) . the 3  conserved sequence of class i integrons is characterized by antibiotic resistance gene qace1 and the sulfonamide resistance gene sul1 . to identify class i integrons , primers atcgcaatagttggcgaagt ( accession no . x12869 ) , specific for 3  cs ( 5 ) , were amplified in this region by polymerase chain reaction as described ( 5 ) . a 0.8-kb product , found in 22 of the 58 isolates , was confirmed to be the 3  cs of class i integrons by sequencing ( table ) . to identify the gene cassette in the class i integrons , primers previously used to amplify the region between the 5  cs and 3  cs ( 5 ) were used . all 22 strains produced a 1.0-kb amplicon , which on sequencing was found to contain the aada1 gene that confers resistance against aminoglycosides , streptomycin , and spectinomycin ( 4,5 ) . ( 8)  a , ampicillin ; cf , ciprofloxacin ; fz , furazolidone ; g , gentamycin ; n , neomycin ; na , nalidixic acid ; s , streptomycin ; sm , sulfamethizole ; t , tetracycline ; tr , trimethoprim . a total of 17 out of the 22 class i integron - bearing strains belonged to the before period . the remaining five , though isolated after june 1993 , probably represented carry - over strains . these five strains belonged to the same ri ribotype as the other 16 strains from the before period ( table ) and had a ctx structure identical to the other strains with ri ribotype ( 8) . resistance of v. cholerae isolates to ampicillin ( 10 g ) , ciprofloxacin ( 5 g ) , furazolidone ( 50 g ) , gentamycin ( 10 g ) , neomycin ( 30 g ) , nalidixic acid ( 30 g ) , streptomycin ( 10 g ) , sulfamethizole ( 100 g ) , tetracycline ( 30 g ) , and trimethoprim ( 25 g ) were examined by using commercial discs ( hi media , bombay , india ) as described ( 1 ) . an overall increase in the number of strains with resistance to a greater number of antibiotics was seen in the isolates of the post - o139 period ; a fourfold increase in resistance against trimethoprim , a drug often used for the treatment of cholera in children and pregnant women , was noted . almost all strains , from all isolation periods , were uniformly resistant to streptomycin and sulfamethizole , but none were found to be resistant to tetracycline , gentamycin , or ciprofloxacin . none of the strains belonging to the ribotypes rii and riii , which were all isolated during and after the o139 outbreak , carried the aada1 gene cassette ( coding for aminoglycoside resistance ) ; however , they were all resistant to streptomycin . further , all of the strains were resistant to trimethoprim . since strains of v. cholerae , which harbor the sxt element , are resistant to streptomycin and trimethoprim ( 7 ) , we sought to determine if the resistance of the post - o139 strains to streptomycin and trimethoprim could be traced to an sxt element . colony hybridization of the 58 strains with a 0.8-kb probe , specific for the sxt integrase gene ( 7 ) , showed that all trimethoprim - resistant strains from all isolation periods and all during and after streptomycin - resistant carried the sxt element ( table ) . further , five of these strains harbored both sxt and the aada1 gene cassette ( table ) . our survey thus suggested that while the streptomycin resistance of the strains isolated before the o139 outbreak was due to the aada1 gene cassette carried by the class i integrons , the sxt element was probably responsible for this phenotype in the post - o139 strains . since none of the strains harbored any plasmid and the resistance determinants present in the class i integrons and in the sxt element could account for only a few markers , other determinants of antibiotic resistance may exist . ( 4 ) found that the o1 strains isolated in vietnam in 1994 and after had the ribotype identical to that found in some of the o1 strains isolated in samutsakorn , thailand , during and after the 1993 o139 outbreak . both pre- and post - o139 isolates carried class i integrons with the aada2 gene cassette . this fact led dalsgaard et al . to conjecture that this distinct o1 strain might have been transferred between thailand and vietnam ( 5 ) . however , the possibility of its migrating from a third country could not be ruled out . the ribotype of the pre - o139 , o1 calcutta strains examined in this study was identical to that of the samutsakorn strains ( 4,5,8 ) . however , as can be seen from the data presented here , unlike the samutsakorn strains , these strains harbored aada1 gene cassette . the 3  conserved sequence of class i integrons is characterized by antibiotic resistance gene qace1 and the sulfonamide resistance gene sul1 . to identify class i integrons , primers atcgcaatagttggcgaagt ( accession no . x12869 ) , specific for 3  cs ( 5 ) , were amplified in this region by polymerase chain reaction as described ( 5 ) . a 0.8-kb product , found in 22 of the 58 isolates , was confirmed to be the 3  cs of class i integrons by sequencing ( table ) . to identify the gene cassette in the class i integrons , primers previously used to amplify the region between the 5  cs and 3  cs ( 5 ) were used . all 22 strains produced a 1.0-kb amplicon , which on sequencing was found to contain the aada1 gene that confers resistance against aminoglycosides , streptomycin , and spectinomycin ( 4,5 ) . sharma et al . ( 8)  a , ampicillin ; cf , ciprofloxacin ; fz , furazolidone ; g , gentamycin ; n , neomycin ; na , nalidixic acid ; s , streptomycin ; sm , sulfamethizole ; t , tetracycline ; tr , trimethoprim . a total of 17 out of the 22 class i integron - bearing strains belonged to the before period . the remaining five , though isolated after june 1993 , probably represented carry - over strains . these five strains belonged to the same ri ribotype as the other 16 strains from the before period ( table ) and had a ctx structure identical to the other strains with ri ribotype ( 8) . resistance of v. cholerae isolates to ampicillin ( 10 g ) , ciprofloxacin ( 5 g ) , furazolidone ( 50 g ) , gentamycin ( 10 g ) , neomycin ( 30 g ) , nalidixic acid ( 30 g ) , streptomycin ( 10 g ) , sulfamethizole ( 100 g ) , tetracycline ( 30 g ) , and trimethoprim ( 25 g ) were examined by using commercial discs ( hi media , bombay , india ) as described ( 1 ) . an overall increase in the number of strains with resistance to a greater number of antibiotics was seen in the isolates of the post - o139 period ; a fourfold increase in resistance against trimethoprim , a drug often used for the treatment of cholera in children and pregnant women , was noted . almost all strains , from all isolation periods , were uniformly resistant to streptomycin and sulfamethizole , but none were found to be resistant to tetracycline , gentamycin , or ciprofloxacin . none of the strains belonging to the ribotypes rii and riii , which were all isolated during and after the o139 outbreak , carried the aada1 gene cassette ( coding for aminoglycoside resistance ) ; however , they were all resistant to streptomycin . further , all of the strains were resistant to trimethoprim . since strains of v. cholerae , which harbor the sxt element , are resistant to streptomycin and trimethoprim ( 7 ) , we sought to determine if the resistance of the post - o139 strains to streptomycin and trimethoprim could be traced to an sxt element . colony hybridization of the 58 strains with a 0.8-kb probe , specific for the sxt integrase gene ( 7 ) , showed that all trimethoprim - resistant strains from all isolation periods and all during and after streptomycin - resistant carried the sxt element ( table ) . further , five of these strains harbored both sxt and the aada1 gene cassette ( table ) . our survey thus suggested that while the streptomycin resistance of the strains isolated before the o139 outbreak was due to the aada1 gene cassette carried by the class i integrons , the sxt element was probably responsible for this phenotype in the post - o139 strains . since none of the strains harbored any plasmid and the resistance determinants present in the class i integrons and in the sxt element could account for only a few markers , other determinants of antibiotic resistance may exist . ( 4 ) found that the o1 strains isolated in vietnam in 1994 and after had the ribotype identical to that found in some of the o1 strains isolated in samutsakorn , thailand , during and after the 1993 o139 outbreak . both pre- and post - o139 isolates carried class i integrons with the aada2 gene cassette . this fact led dalsgaard et al . to conjecture that this distinct o1 strain might have been transferred between thailand and vietnam ( 5 ) . however , the possibility of its migrating from a third country could not be ruled out . the ribotype of the pre - o139 , o1 calcutta strains examined in this study was identical to that of the samutsakorn strains ( 4,5,8 ) . however , as can be seen from the data presented here , unlike the samutsakorn strains , these strains harbored aada1 gene cassette . we had shown previously that the post - o139 , o1 strains isolated in calcutta could have migrated to guinea - bissau in africa ( 9 ) . further evidence has shown that this strain , which caused an outbreak in 19941995 , could have subsequently acquired class i integrons bearing the [ ant(3)-1a ] gene cassette by a 150-kb plasmid from an unknown source ( 10 ) . while the class i integron with aada1 gene cassette was widely distributed among the pre - o139 o1 strains isolated in calcutta , it was mostly absent in the post - o139 o1 strains ( figure ) . this finding is in contrast to other studies involving the sxt element ; 80% of all pre - o139 strains were devoid of sxt , whereas all post - o139 strains ( with the exception of a few carry - over strains ) had it ( figure ) . when the data presented in this paper are considered together with the information presented by others ( 4,5,8,10 ) , it appears that both pre- and post - o139 , o1 strains , isolated in calcutta , probably could have moved to other countries and became established there . distribution of class i integrons and sxt elements in vibrio cholerae el tor strains isolated before , during , and after the o139 outbreak .
we examined the distribution of class i integrons and sxt elements in vibrio cholerae o1 el tor strains , isolated in calcutta , india , before and after the v. cholerae o139 outbreak in 1992 . 
 class i integrons , with aada1 gene cassette , were detected primarily in the pre - o139 strains ; the sxt element was found mainly in the post - o139 strains .