Molecular characterization of a new ribotype of Vibrio cholerae O139 Bengal associated with an outbreak of cholera in Bangladesh

Abstract

Vibrio cholerae O139 Bengal initially appeared in the southern coastal region of Bangladesh and spread northward, causing explosive epidemics during 1992 and 1993. The resurgence of V. cholerae O139 during 1995 after its transient displacement by a new clone of El Tor vibrios demonstrated rapid changes in the epidemiology of cholera in Bangladesh. A recent outbreak of cholera in two north-central districts of Bangladesh caused by V. cholerae O139 led us to analyze strains collected from the outbreak and compare them with V. cholerae O139 strains isolated from other regions of Bangladesh and neighboring India to investigate their origins. Analysis of restriction fragment length polymorphisms in genes for conserved rRNA (ribotype) revealed that the recently isolated V. cholerae O139 strains belonged to a new ribotype which was distinct from previously described ribotypes of toxigenic V. cholerae O139. All strains carried the genes for toxin-coregulated pili (tcpA and tcpI) and accessory colonization factor (acfB), the regulatory gene toxR, and multiple copies of the lysogenic phage genome encoding cholera toxin (CTXPhi) and belonged to a previously described ctxA genotype. Comparative analysis of the rfb gene cluster by PCR revealed the absence of a large region of the O1-specific rfb operon downstream of the rfaD gene and the presence of an O139-specific genomic region in all O139 strains. Southern hybridization analysis of the O139-specific genomic region also produced identical restriction patterns in strains belonging to the new ribotype and those of previously described ribotypes. These results suggested that the new ribotype of Bengal vibrios possibly originated from an existing strain of V. cholerae O139 by genetic changes in the rRNA operons. In contrast to previously isolated O139 strains which mostly had resistance to trimethoprim, sulfamethoxazole, and streptomycin encoded by a transposon (SXT element), 68.6% of the toxigenic strains analyzed in the present study, including all strains belonging to the new ribotype, were susceptible to these antibiotics. Molecular analysis of the SXT element revealed possible deletion of a 3.6-kb region of the SXT element in strains which were susceptible to the antibiotics. Thus, V. cholerae O139 strains in Bangladesh are also undergoing considerable reassortments in genetic elements encoding antimicrobial resistance.

FIG. 1

Southern hybridization analysis of rRNA genes in V. cholerae O139 strains isolated between 1995 and 1998 in Bangladesh, India, and Thailand. Genomic DNA was digested with BglI and probed with a 7.5-kb BamHI fragment of the E. coli rRNA clone pKK3535. Restriction pattern I, produced by two strains belonging to designated ribotype B-I isolated from Bangladesh and Thailand, respectively, are shown in lanes 1 and 2; restriction pattern II, produced by three strains belonging to ribotype B-II isolated in Bangladesh, India, and Thailand, respectively, are shown in lanes 3 through 5; and lanes 6 through 8 show restriction pattern III, produced by the new ribotype of V. cholerae O139 designated B-III, isolated from a recent outbreak in two north-central districts of Bangladesh. The pattern produced by the nontoxigenic O139 strain isolated in Argentina is shown in lane 9. Numbers indicating molecular sizes of bands correspond to a 1-kb DNA ladder (BRL) used as a molecular size marker.

FIG. 2

Southern hybridization analysis of ctxA genes in V. cholerae O139 strains isolated from the epidemic in the north-central region of Bangladesh (lanes 6 through 9) in 1997 and from other districts between 1995 and 1998 (lanes 1 through 5). Genomic DNA was digested with BglI and probed with a 0.5-kb fragment of the ctxA gene. Restriction patterns corresponding to ctxA genotype A are shown in lanes 1 through 4, whereas those representing ctxA genotype B are shown in lanes 5 through 9. Numbers indicating molecular sizes of bands correspond to a 1-kb DNA ladder (BRL) used as a molecular size marker.

FIG. 3

Analysis of the SXT element in V. cholerae O139 strains isolated between 1995 and 1998 in Bangladesh. Genomic DNA was digested with BglI and probed with the SXT gene probe. Lanes 1 through 5 show restriction patterns corresponding to SXT genotype 1, whereas lanes 6 through 10 represent SXT genotype 2. Lane numbers, place, and year of isolation of strains are as follows: 1 and 2, Matlab, 1995; 3 and 4, Dhaka, 1995; 5, Sunamganj, 1996; 6 and 7, Mymensingh, 1997; 8 and 9, Kishoreganj, 1997; 10, Bakerganj, 1998. Numbers indicating molecular sizes of bands correspond to a 1-kb DNA ladder (BRL).