Vibrio cholerae pathogenic clones.

We resolved the relationships between 2 pandemic clones of Vibrio cholerae. Using 26 housekeeping genes, we showed that the US Gulf clone, the Australian clone, and 3 El Tor strains isolated before the seventh pandemic were related to the seventh pandemic clone. The sixth pandemic clone was well separated from them.

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Cholera caused by Vibrio cholerae is a major disease that has caused great fear since the first recorded pandemic in 1817 because of the frequency of death and the rapidity with which it occurs (1,2). Approximately 200 O antigens have been distinguished serologically (3,4), but only O1 and O139 have been found in epidemic and pandemic cholera isolates (5,6).

The seventh pandemic (1961-present) is still widespread and has a severe impact on 3 continents. The sixth pandemic ended in 1923, but the clone persisted at least until the 1990s (7). Furthermore, several cholera outbreaks were reported after the sixth pandemic retreated but before the start of the seventh pandemic. Isolates from these outbreaks were recognized as different from those of the sixth pandemic and were allocated to the El Tor biotype, while the sixth and fifth pandemics, both of which had been studied microbiologically, were referred to as the classical biotype. These El Tor outbreaks occurred in Indonesia and the Middle East (1926-1960) (5) and are often referred to as prepandemic isolates because they were later seen as forerunners of the seventh pandemic, which was also of the E1 Tor biotype. However, now that environmental V. cholerae has been studied in some detail, major components of the El Tor phenotype are known to be present in most environmental isolates, and the classical biotype is believed to have arisen by loss of characters otherwise widely present in the species (8). Also, cases of sporadic indigenous cholera have been detected in Australia (9) and the United States (10), both of the O1 El Tor biotype. These are generally referred to as the US Gulf and Australian clones. All of the pathogenic forms discussed above had the O1 serotype, but in 1992 a variant of the seventh pandemic appeared with a new O antigen, O139; this variant is known as V. cholerae O139 Bengal (11).

The relationships of V. cholerae have been studied in several ways, but the most useful insights have come from multilocus enzyme electrophoresis (12) and more recently by multilocus sequence analyses (4,13,14). In this study, we sequenced 26 housekeeping genes from V. cholerae isolates representative of the sixth and seventh pandemic clones and other closely related toxigenic strains to determine relationships to better understand the origins of pandemic clones.

The Study

Twenty-six housekeeping genes distributed evenly on both chromosomes (online Appendix Table, available from http://www.cdc.gov/ncidod/EID/vol11no11/04-1170_ app.htm) were studied by using 5 nontoxigenic environmental isolates and 12 toxigenic V. cholerae isolates, which comprise 5 sixth and seventh pandemic isolates and 7 pathogenic isolates related to them (Table). One of the 3 seventh pandemic isolates is N16961; the sequence of its genome (15) was used in this study. All 26 housekeeping genes were successfully sequenced from the remaining 11 toxigenic isolates. However, 4 genes could not be amplified by polymerase chain reaction from 1 of the environmental isolates (sdaA for 370-94, hmpA for 905-93, glgX for 928-93, andpepN for 370-94) and were not sequenced. The GenBank accession numbers for the nucleotide sequences determined in this study are DQ020969-DQ021380.

The 5 nontoxigenic environmental isolates had different sequences for each gene. These sequences were not found in any of the 12 toxigenic isolates. The average pair-wise difference among the 5 environmental isolates in the 22 genes sequenced ranged from 0.67% to 5.29%, an indication that V. cholerae as a species has a high level of sequence variation. However 11 of the 26 housekeeping genes (glyA, gppA, pntA, icd, purM, plsX, ndh, glgX, adk, carA, and speA) were identical in all toxigenic V. cholerae isolates, confirming that they are closely related.

There were 18 mutation/recombination events among the 15 genes with sequence variation in the 12 toxigenic isolates. Three (malP, pyrC, and gyrB) of these genes had undergone 2 changes. Seven cases of single base differences, which are attributed to mutation, were clearly distinct from 11 cases of multiple base differences, which are attributed to recombination. Ten of the events attributed to recombination involve changes in 10 to 51 bases (Figure 1). The metG gene, in which the 2 sequences differ by only 4 bases, might have undergone 4 mutational events rather than a single recombination event, but we considered this to be most unlikely.

[FIGURE 1 OMITTED]

Some of the 11 genes believed to have undergone recombination may have undergone >1 recombination event. This possibility is likely because 11 of 26 genes have undergone recombination. For example, in metE the base changes are at the 2 ends and may represent 2 recombination events; the same applies, to a lesser extent, to malP.

With regard to the length of DNA involved in recombination events, segments longer than a gene are most common because only in the genes discussed above and in sdaA and gyrB are the bases that differed clustered within the gene. This is suggestive of recombination within that gene.

The sequences for the 26 genes were used to produce a tree with mutational and recombinational events and given equal weight, which is shown in Figure 2. By sequencing 26 genes, we have observed sufficient variation to determine the relationships of the isolates studied. The tree is unrooted because the environmental isolates share no alleles with each other or toxigenic isolates. The relatively high rate of recombination in V. cholerae means that the level of sequence similarity does not indicate relatedness of the isolates unless the sequences are very similar and differences can be attributed to mutation, as in the toxigenic strains. However, other reasons exist for believing that the root is on the long 10-event branch that includes 2 mutational changes. The 10 El Tor isolates on 1 end are, at most, 3 steps from it and the 2 classical sixth pandemic isolates are separated by 1 event. The isolates date from 1937 to 1992 for the former group and from 1921 to 1965 for the latter group. Since 1 group giving rise to the other while undergoing little divergence itself is highly improbable, we believe that the root lies somewhere on that branch. The properties that characterize the El Tor biotype are those of environmental strains, which makes it unlikely that they are derived from the sixth pandemic, and the reverse seems even less likely because the fifth pandemic was also the classical biotype. We therefore treat the tree as being rooted on the long branch, which enables us to follow the sequence of events.

[FIGURE 2 OMITTED]

Among the El Tor isolates, the Australian clone is the most closely related of the current clones to the seventh pandemic clone, with 1 and 2 events along the 2 branches separating them. The Australian clone, although not discovered until 1977, must have arisen before the seventh pandemic and spread to Australia. The prepandemic outbreak isolates are located separately among the surviving El Tor pathogenic clones, with the 1937 Indonesian 66-2 (Makassar 759) isolate located closest to the seventh pandemic clone. The US Gulf clone diverged before the Australian clone and the seventh pandemic clones diverged, with a single recombination event on the branch to the common ancestor of the Australian and the seventh pandemic clones.

The 2 sixth pandemic isolates are well separated from the other strains, differing from them at 10 loci, an average of 5 events per branch. These include recombination events affecting 8 genes. If representative, [approximately equal to] 30% of the genes have undergone recombination during divergence of the sixth pandemic clone and the El Tor group of pathogenic clones that includes the seventh pandemic clone. The extensive divergence between the sixth pandemic and other toxigenic isolates studied indicates a long period since divergence from the common ancestor, which presumably occurred well before the sixth pandemic (1899-1923). In the absence of any intermediates, we cannot allocate individual events to either branch but presume that each is equally likely to have occurred on either branch.

Conclusions

This study using sequences of 26 genes has resolved the evolutionary relationship of the 2 major pandemic clones of V. cholerae and the relationships of the seventh pandemic clone to other pathogenic El Tor clones and isolates. With the relationships established it is clear that study of the prepandemic isolates and Australian clone in particular could illuminate the events involved in the emergence of the current seventh pandemic clone from this lineage.

Acknowledgments

We thank all donors of isolates and the anonymous referees for their helpful suggestions.

This research was supported by grants from the National Health and Medical Research Council of Australia and a University of New South Wales Goldstar award.

References

(1.) Pollitzer R. Cholera. Geneva: World Health Organization; 1959. p. 147-58.

(2.) Lacey SW. Cholera: calamitous past, ominous future. Clin Infect Dis. 1995;20:1409-19.

(3.) Shimada T, Arakawa E, Itoh K, Okitsu T, Matsushima A, Asai Y, et al. Extended serotyping scheme for Vibrio cholerae. Curt Microbiol. 1994;28:175-8.

(4.) Kotetishvili M, Stine OC, Chen Y, Kreger A, Sulakvelidze A, Sozhamannan S, et al. Multilocus sequence typing has better discriminatory ability for typing Vibrio cholerae than does pulsed-field gel electrophoresis and provides a measure of phylogenetic relatedness. J Clin Microbiol. 2003;41:2191-6.

(5.) Barua D. History of cholera. In: Barua D, Greenough III WB, editors. Cholera. New York: Plenum; 1992. p. 1-36.

(6.) Albert MJ. Vibrio cholerae O139 Bengal. J Clin Microbiol. 1994;32:2345-9.

(7.) Safa A, Bhuiyan NA, Alam M, Sack DA, Nair GB. Genomic relatedness of the new Matlab variants of Vibrio cholerae O1 to the classical and El Tor biotypes as determined by pulsed-field gel electrophoresis. J Clin Microbiol. 2005;43:1401-4.

(8.) Reeves PR, Lan R. Cholera in the 1990s. Br Med Bull. 1998;54:611-23.

(9.) Desmarchelier PM, Wong FY, Mallard K. An epidemiological study of Vibrio cholerae O1 in the Australian environment based on rRNA gene polymorphisms. Epidemiol Infect. 1995;115:435-46.

(10.) Johnston JM, Martin DL, Perdue J, McFarland LM, Caraway CT, Lippy EC, et al. Cholera on a Gulf Coast oil rig. N Engl J Med. 1983;309:523-6.

(11.) Bhattacharya MK, Bhattacharya SK, Garg S, Saha PK, Dutta D, Nair GB, et al. Outbreak of Vibrio cholerae non-O1 in India and Bangladesh. Lancet. 1993;341:1346-7.

(12.) Wachsmuth IK, Olsvik O, Evins GM, Popovic T. Molecular epidemiology of cholera. In: Wachsmuth IK, Blake PA, Olsvik O, editors. Vibrio cholerae and cholera, molecular to global perspectives. Washington: ASM Press; 1994. p. 357-70.

(13.) Byun R, Elbourne LDH, Lan R, Reeves PR. Evolutionary relationships of the pathogenic clones of Vibrio cholerae by sequence analysis of four housekeeping genes. Infect Immun. 1999;67:1116-24.

(14.) Farfan M, Minana-Galbis D, Fuste MC, Loren JG. Allelic diversity and population structure in Vibrio cholerae O139 Bengal based on nucleotide sequence analysis. J Bacteriol. 2002; 184:1304-13.

(15.) Heidelberg JF, Eisen JA, Nelson WC, Clayton RA, Gwinn ML, Dodson RJ, et al. DNA sequence of both chromosomes of the cholera pathogen Vibrio cholerae. Nature. 2000;406:477-83.

Anna Salim, * Ruiting Lan, ([dagger]) and Peter R. Reeves *

* University of Sydney, Sydney, New South Wales, Australia; and ([dagger]) University of New South Wales, Sydney, New South Wales, Australia

Ms Salim is a PhD student in microbiology at the University of Sydney. Her research interests include population genetics and evolution of V. cholerae.

Address for correspondence: Peter R. Reeves, School of Molecular and Microbial Biosciences, G08, University of Sydney, Sydney, New South Wales 2006, Australia; fax: [phone]; email: reeves@angis. usyd.edu.au

Table. Isolates of Vibrio cholerae tested

                       Laboratory                          Year
Original name             name        Clone/isolate      isolated

#75                       M967         6th pandemic        1921
395                      M1616         6th pandemic        1965
E506                      M794        US Gulf Coast        1974
4808                      M796        US Gulf Coast        1978
NCTC 9420                 M640       Pre-7th pandemic      1954
NCTC 5395                 M543       Pre-7th pandemic      1938
66-2 (Makassar 759)       M802       Pre-7th pandemic      1937
SIMP/77                  M2140          Australian         1977
M4287/77                 M2141          Australian         1977
2100                      M663         7th pandemic        1992
E9120                     M793         7th pandemic        1961
N16961 ([dagger])                      7th pandemic        1971
1085-93                   M549        Environmental        1993
141-94                    M553        Environmental        1994
905-93                    M555        Environmental        1993
928-93                    M557        Environmental        1993
370-94                    M563        Environmental        1994

Original name               Location          Source *    Serogroup

#75                           Japan             CDC          O1
395                           India             CVD          O1
E506                       Texas, USA           CVD          O1
4808                     Louisiana, USA         CVD          O1
NCTC 9420                 Cairo, Egypt          NCTC         O1
NCTC 5395                 Baghdad, Iraq        NICED         O1
66-2 (Makassar 759)    Sulawesi, Indonesia       IP          O1
SIMP/77                     Australia            QH          O1
M4287/77                    Australia            QH          O1
2100                     Bali, Indonesia        IMVS         O1
E9120                       Indonesia           CVD          O1
N16961 ([dagger])          Bangladesh         GenBank        O1
1085-93                      Germany            NIHJ         O37
141-94                       Germany            NIHJ         O70
905-93                      Argentina           NIHJ         O97
928-93                      Argentina           NIHJ         O6
370-94                     South Korea          NIHJ         O81

Original name           Biotype

#75                    Classical
395                    Classical
E506                     El Tor
4808                     El Tor
NCTC 9420                El Tor
NCTC 5395                El Tor
66-2 (Makassar 759)      El Tor
SIMP/77                  El Tor
M4287/77                 El Tor
2100                     El Tor
E9120                    El Tor
N16961 ([dagger])        El Tor
1085-93
141-94
905-93
928-93
370-94

* CDC, Centers for Disease Control and Prevention; CVD, Centre for
Vaccine Development (Dr James Kaper); NCTC, National Collection of Type
Cultures; NICED, National Institute for Cholera and Enteric Diseases;
IP, Institute Pasteur (Dr A. Dodin); QH, Queens land Health (Dr Denise
Murphy); IMVS, Institute of Medical and Veterinary Science, NIHJ,
National Institute of Health, Japan (Dr Tohio Shimada).

([dagger]) Sequence of the genome of isolate N16961 was used. GenBank
accession numbers for chromosomes 1 and 2 are AE003852 and AE003853,
respectively.