Molecular characterization of high-level-cholera-toxin-producing El Tor variant Vibrio cholerae strains in the Zanzibar Archipelago of Tanzania

Abstract

Analysis of 1,180 diarrheal stool samples in Zanzibar detected 247 Vibrio cholerae O1, Ogawa strains in 2009. Phenotypic traits and PCR-based detection of rstR, rtxC, and tcpA alleles showed that they belonged to the El Tor biotype. Genetic analysis of ctxB of these strains revealed that they were classical type, and production of classical cholera toxin B (CTB) was confirmed by Western blotting. These strains produced more CT than the prototype El Tor and formed a separate cluster by pulsed-field gel electrophoresis (PFGE) analysis.

Fig 1

MAMA-PCR to detect the type of ctxB allele in representative Vibrio cholerae O1 strains isolated from the Zanzibar archipelago in Tanzania using primers (Fw-con/Rv-cla) for the classical ctxB allele (top) and Fw-con/Rv-elt for the El Tor ctxB allele (bottom). Lane 1, MCM 32; lane 2, MCM 133; lane 3, MCM 134; lane 4, MCM 146; lane 5, MCM 168; lane 6, T1; lane 7, MCF 084; lane 8, MCF 001; lane 9, WF 01; lane 10, 210200; lane 11, classical control (0395); lane 12, El Tor control (N16961).

Fig 2

Comparative nucleotide sequence analysis of the promoter region the ctxAB operon (P_ctxAB) of Zanzibar isolate MCM 133 and Kolkata isolate CRC 220. The nucleotide sequences of P_ctxAB of O395 (classical control strain) and N16961 (El Tor control strain) were obtained from GenBank. Identical residues are indicated with dots. Each solid bar indicates the missing TTTTGAT heptads. The black arrow line represents the ATG start codon of ctxA gene. The Zanzibar isolate lacks one more heptad repeat than the Kolkata isolate.

Fig 3

PCR results implicating the chromosomal organization of the CTX prophage of Vibrio cholerae O1 Ogawa isolates from Zanzibar. (A) PCR results with primers CII F and CII R showing the absence of the CTX prophage in chromosome II of Zanzibar isolates. The two black bars indicate the locations of the two primers. Lane M, 100-bp DNA ladder; lane 1, MCM 32; lane 2, MCM 133; lane 3, MCM 134; lane 4, MCM 146; lane 5, MCM 168; lane 6, T1; lane 7, MCF 084; lane 8, MCF 001. El Tor control strain N16961 and classical control strain O395 were used as positive and negative controls, respectively. (B) Agarose gel electrophoresis showing the results of PCR with primers rstC1 and rtxA1. Lane L, lambda-HindIII DNA ladder; lane 1, MCM 133; lane 2, MCM 168; lane 3, KM 282; lane 4, T1; lane 5, WM 012; lane M, 1-kb DNA ladder. (C) Predicted molecular organization of the CTX prophage of V. cholerae isolates from Zanzibar with a probable combination of rstR and ctxB in their large chromosome. The black bars indicate the locations of the two primers rstC1 and rtxA1.

Fig 4

(A) Amounts of cholera toxin production by Zanzibar variants, prototype El Tor strains, and the classical strain. Error bars show standard errors for results of tests done in triplicate. (B) Western immunoblotting results of the culture supernatant of representative Zanzibar O1 isolates. Samples of 100 ng each of the purified classical CT (lane 1) and El Tor CT (lane 2) were used as positive controls for immunoblotting with the monoclonal antibody against classical and El Tor CTB, respectively. Lane 3, CF04; lane 4, MCF147; lane 5, MCF100; lane 6, MCM79; lane 7, medium only (negative control). Numbers at left are molecular masses, in kilodaltons.

Fig 5

PFGE patterns of the NotI-digested V. cholerae strains from Zanzibar. A dendrogram analysis made using Bionumeric software (Applied Maths, Sint-Martens-Latem, Belgium) shows three distinct clusters among the Zanzibar isolates tested. Sixteen representative strains were used for the study.