Biochemical and serological verification revealed that all the outbreak isolates were V. cholerae O1 serotype Ogawa, which is commonly reported in Africa. As shown in Table , only 3 of the 27 isolates were resistant to tetracycline, but all 27 strains were resistant to other antimicrobials, 13 were resistant to 3 or more of the 11 agents tested, and levofloxacin was the only tested drug to which all isolates were susceptible. We observed nalidixic acid resistance in 10 isolates, and 26 of the 27 isolates were resistant to trimethoprim/sulfamethoxazole.
Antimicrobial resistance profiles and conferring genetic elements in 27 2006 V. cholerae O1 isolates from Accra
Twenty-five of the 27 isolates produced a 0.47 kb amplicon with the dfr1a primer pair that amplifies dfrA1
cassettes, but we did not amplify cassette regions from class 1 integrons in any of the strains. The class 1 integron integrase gene was, however, detected in two strains, suggesting that these strains had very large variable regions and/or a genetic modification in the 3′ or 5′ conserved ends recognized by the cassette-region primers of Lévesque et al
Importantly, the two class 1 integron-positive strains were two of the three strains that were resistant to seven or more of the antimicrobials tested (Table ). However, although class 1 integrons were associated with multiple resistance, they could not account for most of the dfr
cassettes detected. Screening for class 2 integron-borne cassettes produced a 2.2 kb product from control strain 17-2 as well as from 22 V
isolates that produced an amplicon with the dfr1a primers. The class 2 cassette-region amplicon from strains V34 and V47 was directionally cloned into pGEMT. The resulting clones were screened for resistance to eight antimicrobials. Both were resistant to ampicillin (encoded on the vector), trimethoprim and streptomycin, but susceptible to chloramphenicol, sulphonamides, nalidixic acid and ciprofloxacin. We sequenced the cloned amplicon from the strain V34 and found that it contained three resistance gene cassettes, in the commonly recovered context that is identical to that in strain 17-2: dfrA1-sat-aadA1
. Strains that could, produced amplicons of similar size; Mbo
I and Alu
I RFLP patterns were also identical to the patterns from strains V34, V47 and EAEC strain 17-2.
Primers for the SXT integrase gene were used to screen for the SXT element, as described by Dalsgaard et al
We obtained a 0.6 kb amplicon, consistent with the expected size of 592 bp produced by strains bearing the SXT element, in 24 strains. As we did not have a positive control strain, we cloned the amplicon from the Ghanaian strain V34 into the vector pGEMT (Promega) and sequenced it. The sequence obtained was 99% identical to the SXT integrase in the GenBank database (accession number AB114188.1).
Of the 27 isolates screened, 3 carried the SXT element alone, one bore a class 2 integron with dfrA1-sat-aadA cassettes but no SXT, and 21 strains possessed both elements. All strains harbouring one or both elements showed high-level resistance to trimethoprim/sulfamethoxazole and produced an amplicon with the dfr1a primers. Of the two strains that were negative for both elements, strain V111 was susceptible to trimethoprim/sulfamethoxazole and strain V95 exhibited low-level resistance by an unknown mechanism. All the strains that had neither or only one of the two trimethoprim resistance-conferring elements were recovered from patients from Awoshie, Agona Swedru, Ga West and Tema, all of which are away from the Accra Metropolitan area (Table ). We additionally observed that although most of the strains generated an identical RAPD profile with ERIC2 primers, strains V95 and V111, both of which lacked the class 2 integron and the SXT element and were isolated from outside the Accra Metropolitan area, produced distinctly different profiles (Figure ).
Figure 1 RAPD profiles of V. cholerae isolates generated with ERIC2 primers. Lanes 2–8, V. cholerae isolates V112, V42, V47, V51, V52, V86 and V97, respectively; lanes 9–15 V. cholerae isolates V111, V84, V79, V89, V85, V78 and V90, respectively. (more ...)