Molecular epidemiology of O139 vibrio cholerae: mutation, lateral gene transfer, and founder flush - Research

Emerging Infectious Diseases, July, 2003 by Pallavi Garg, Antonia Aydanian, David Smith, J. Glenn Morris, Jr., G. Balakrish Nair, O. Colin Stine

Vibrio cholerae in O-group 139 was first isolated in 1992 and by 1993 had been found throughout the Indian subcontinent. This epidemic expansion probably resulted from a single source after a lateral gene transfer (LGT) event that changed the serotype of an epidemic V. cholerae O1 El Tor strain to O139. However, some studies found substantial genetic diversity, perhaps caused by multiple origins. To further explore the relatedness of O139 strains, we analyzed nine sequenced loci from 96 isolates from patients at the Infectious Diseases Hospital, Calcutta, from 1992 to 2000. We found 64 novel alleles distributed among 51 sequence types. LGT events produced three times the number of nucleotide changes compared to mutation. In contrast to the traditional concept of epidemic spread of a homogeneous clone, the establishment of variant alleles generated by LGT during the rapid expansion of a clonal bacterial population may be a paradigm in infections and epidemics.

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An epidemic of cholera began in Madras, India, in 1992 and within a year had spread across the Indian subcontinent, with cases numbering in the millions (1,2). Vibrio cholerae isolates from this epidemic had a previously unidentified serotype, subsequently designated as O139 Bengal (1,2). This new serotype appears to have resulted when a lateral gene transfer (LGT) event occurred that replaced the 22 kb of the wbf region (encoding the O1 antigen) of a seventh pandemic V. cholerae O1 El Tor strain with a 37-kb region encoding the O139 polysaccharide (3-5). The epidemic spread rapidly through all age groups, as persons with previous exposure to V. cholerae O1 were not immune to O139 infection. Since 1992, O139 strains have established endemicity in this geographic region and account for a variable percentage of cholera cases every year (6).

Genetic variation observed in O139 isolates has been attributed to many causes. Variation in restriction fragment length polymorphism (RFLP) analysis of rDNA genes (7) and in recA sequence (8) has been interpreted as evidence for multiple origins. Genetic variability in RFLP of the CTX element (6) has been attributed to phage-mediated recombination. Variation in antimicrobial susceptibility (9) has been attributed to plasmid exchange in response to selective pressure from drug use. The variation in pulsed-field gel electrophoresis (PFGE) analysis of genomic restriction fragments (6,10) has been attributed to point mutations. Multilocus sequence typing (MLST), which has been used in the evaluation of a number of other bacterial species (11-14), provides an alternative method for measuring genetic relatedness and has provided data for identifying both point mutations and LGT events (14). MLST has improved discriminatory power over PFGE in some cases, e.g., Enterococcus (15) and Salmonella (16); however, in the case of Escherichia coil O157, it does not because of an absence of sequence variation in the clonally derived isolates (17). A small MLST study of O139 isolates of V. cholerae did not identify any LGT events (18).

To understand the evolutionary dynamics of V. cholerae O139, we sequenced segments from nine loci, including seven that may be classified as traditional housekeeping genes, one that carries the genes for cholera toxin, and another that is next to the insertion sequence within the O139 wbf region (3-5). Thus, the last two loci might be expected to show LGT, because they are associated with known mobile elements, but the other seven loci would not be expected to show LGT. However, we found putative LGT alleles at all nine loci in the 96 clonally related O139 isolates.

Materials and Methods

We evaluated nine loci--dnaE, lap, recA pgm, gyrB. cat, chi, rstR, and gmd--from 96 V. cholerae O139 isolated from patients seen at the Infectious Diseases Hospital, Calcutta, from 1992 to 2000 (see Appendix, online only). DNA was prepared from overnight cultures by using PrepMan Ultra (Applied Biosystems Inc., Foster City, CA) at the University of Maryland School of Medicine. Each locus was amplified by using polymerase chain reaction (PCR) with primers (Table 1) selected from a conserved region of the locus, as determined by aligning sequences from GenBank. Our primers selectively amplified the original 0139 rstR gene found in all isolates and not the additional one found in some recently inserted CTX elements (19). The presence of amplified products was confirmed on agarose gels. Purification of the products was performed by using Millipore filters. The purified PCR products were sequenced in both directions by using the same primers used for amplification and Big Dye cycle sequencing kit (ABI) in accordance with manufacturer's instructions. The fluorescently labeled products were separated and detected by using either an ABI 377 or 3700 Automatic Sequencer (ABI). The trace files were read by using Phred (20,21 ) and Phrap (22). Low-quality sequence at the ends was trimmed, and the contigs from each individual isolate were aligned by using Clustal X (23). Variable nucleotides were identified manually. Isolates with identical alleles were identified from a distance matrix obtained from PAUP (24). The alleles have been assigned GenBank accession numbers AY297845 to AY297921.