Departament de Microbiologia i Parasitologia Sanitàries, Divisió de Ciències de la Salut, Facultat de Farmàcia, Universitat de Barcelona, Avda Joan XXIII s/n, 08028 Barcelona, Spain1
Author for correspondence: J. G. Lorén. Tel: +34 93 402 44 97. Fax: +34 93 402 18 96. e-mail: loren{at}farmacia.far.ub.es
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ABSTRACT |
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Keywords: MLEE, linkage disequilibrium, cholera, population genetics, electrophoretic types
Abbreviations: AFLP, amplified fragment length polymorphism; ET, electrophoretic type; MLEE, multilocus enzyme electrophoresis; RAPD, random amplified polymorphic DNA
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INTRODUCTION |
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Historically, it seems most probable that cholera emerged after the Neolithic, which began in the Middle East some 10000 years ago, when the adoption of agricultural practices by nomadic groups enabled higher densities of humans to subsist (Byun et al., 1999 ). There are references to deaths due to dehydrating diarrhoea dating back to Hippocrates and Sanskrit writings (Colwell, 1996
). Cholera has been endemic on the Indian subcontinent for centuries. The literature describes the first pandemic spread of cholera outside Asia in 1817 (Blake, 1994
). Since then seven pandemics have been recorded. The fifth and sixth were caused by the classical biotype of O1 strains, but the nature of the strains causing the first four pandemics is unknown. In contrast, in 1961 the seventh pandemic started in Indonesia and was due to the ElTor biotype. Recently, in 1992 an epidemic clone of a non-O1 strain with serogroup O139 Bengal caused a large cholera outbreak in Bangladesh and neighbouring countries (Albert et al., 1993
; Ramamurthy et al., 1993
). Several studies have shown that the O139 strain is phylogenetically and phenotypically very similar to the O1 ElTor strain, and most probably derived from an O1 seventh-pandemic clone strain by horizontal gene transfer (Bik et al., 1995
; Stroeher et al., 1997
). At the beginning of the outbreak, the O139 strain totally displaced the V. cholerae O1 strains, including both classical and ElTor biotypes, which coexisted only in Bangladesh. The subsequent emergence of a new clone of V. cholerae O1 ElTor that transiently displaced the O139 strains during 1994 and 1995, and the reemergence in 1996 of V. cholerae O139 as the main cause of cholera in Calcutta and its coexistence with the O1 ElTor strains demostrated temporal changes in the epidemiology of the cholera (Faruque et al., 1997a
, b
; Mukhopadhyay et al., 1998
). The factors that determine the emergence, disappearance or continued presence of particular clones of toxigenic V. cholerae are not clear.
The continual emergence of new toxigenic strains of V. cholerae and their selective enrichment during cholera outbreaks constitute essential mechanisms for the survival and evolution of V. cholerae and the genetic elements that mediate the transfer of virulence genes (Faruque et al., 1998 ). The molecular mechanisms of cholera pathogenesis are currently being elucidated, as exemplified by the description of the lysogenic filamentous bacteriophage (CTX
), which encodes cholera toxin (Waldor & Mekalanos, 1996
), and its receptor, the TCP (toxin-coregulated pilus), which is part of a larger genetic element, the TCP pathogenicity island (Karaolis et al., 1998
). These findings may help us to understand the possible origin of new toxigenic clones of V. cholerae, and raise the possibility that all strains of V. cholerae have the potential to become agents of epidemic cholera (Faruque et al., 1998
).
New analysis methods have permitted studies of the genetic variability of V. cholerae on a global scale (Faruque et al., 1998 ). One of them is MLEE, which analyses the electrophoretic mobility differences in multiple enzymes to study divergence of bacterial strains of the same species. Previous studies were primarily concerned with the analysis of strains belonging to the serogroup O1, collected during outbreaks, which were responsible for cholera epidemics and pandemics (Salles & Momen, 1991
; Wachsmuth et al., 1993
; Evins et al., 1995
). More recently, Beltrán et al. (1999)
applied the MLEE technique to the study of a collection of V. cholerae isolates from Mexico and Guatemala; they also included reference strains of all serogroups (toxigenic and non-toxigenic). Moreover, various molecular techniques such as PFGE, RAPD and ribotyping have also been applied in attempts to establish the genetic population structure of V. cholerae. Recent work in this field includes the comparative nucleotide sequence analysis of pathogenic isolates (Byun et al., 1999
) and the AFLP fingerprinting of clinical and environmental isolates (Jiang et al., 2000
). However, the relationship of the pathogenic clones with the environmental isolates remains unclear. In the present study, we analysed by MLEE a collection of V. cholerae strains isolated from several countries, including toxigenic (O1 and O139) and non-toxigenic serogroups from environmental and clinical sources, to determine the population structure of V. cholerae.
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METHODS |
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Electrophoresis and specific enzyme staining.
Nondenaturing vertical polyacrylamide gel electrophoresis was used for all the enzymes. The acrylamide concentration in the gels depended on the enzyme studied (10% continuous polyacrylamide gels and 10%/8% or 8%/5% discontinuous polyacrylamide gels). Tris/HCl 0·8 M (pH 8·8) buffer was used in continuous gels and Tris/HCl 0·125 M (pH 6·8) stacking buffer and Tris/HCl 0·4 M (pH 8·8) resolving buffer were used in discontinuous gels. Tris/glycine 0·19 M (pH 8·3) buffer was used for the electrode compartments. Gels were used within 24 h of preparation and run at 7 °C. A constant voltage, depending on the acrylamide concentration of the gel, was applied until the bromophenol blue band reached the bottom of the gel. All strains were run at least twice to confirm their genotype.
The staining of the gels to reveal specific enzyme activity was performed following Selander et al. (1986) , except in the case of catechol 2,3-oxygenase (Gibson, 1971
; Kataeva & Golovleva, 1990
). The following 15 enzymes were assayed: glucose-6-phosphate dehydrogenase (G6P), isocitrate dehydrogenase (IDH), alanine dehydrogenase (ALD), NAD-dependent glyceraldehyde-phosphate dehydrogenase (GP1), malate dehydrogenase (MDH), fumarase (FUM), aspartate dehydrogenase (ASD), leucine aminopeptidase (LAP), malic enzyme (ME), esterase (EST), catechol 2,3-oxygenase (C23O), nucleoside phosphorylase (NSP), xanthine dehydrogenase (XDH), phosphoglucose isomerase (PGI) and 6-phosphogluconate dehydrogenase (6PG). For each enzyme, distinct mobility variants were designated as electromorphs and numbered in order of increasing migration towards the anode. Displacement of the electromorphs was expressed in terms of relative electrophoretic mobility with respect to the bromophenol blue band. Electromorphs of an enzyme were equated with alleles at the corresponding structural gene locus. Absence of enzyme activity was attributed to a null allele, and designated as 0. Distinct combinations of alleles over the 15 loci assayed were named as electrophoretic types (ETs).
Data treatment.
Genetic diversity for a locus was calculated according to Nei (1978) . The probability that two isolates differ at the jth locus is hj=(1 -
) n/(n - 1), where pij is the frequency of allele i at locus j and n is the number of isolates. The mean genetic diversity, H, is the arithmetic mean of hj for m loci. Genotypic diversity was calculated as G= 1 -
, where gj is the frequency of the jth genotype (ET). Clustering of data obtained by MLEE was performed with the PHYLIP package (Felsenstein, 1993
) from a matrix of coefficients of distances by the unweighted pair-group method for arithmetic averages (UPGMA). Distance between pairs of ETs was calculated as the proportion of loci at which dissimilar electromorphs occurred. The cophenetic correlation coefficient was calculated using NTSYS-pc, version 1·80 (Rohlf, 1993
). Multilocus linkage disequilibrium was estimated on the basis of the distribution of allelic mismatches between pairs of bacterial isolates among all the loci examined. The ratio of the observed variance in mismatches (VO) to the expected variance at linkage equilibrium (VE) provides a measure of multilocus linkage disequilibrium that can be expressed as the index of association (IA); IA= (VO/VE) - 1 (Brown et al., 1980
; Maynard Smith et al., 1993
). For populations in linkage equilibrium, VO=VE and IA is not significantly different from zero, whereas values of IA greater than zero indicate that recombination has been rare or absent. To determine whether VO is significantly different from VE in any sample, a Montecarlo procedure was generated by randomly sampling alleles, without replacement, according to their respective frequencies at each locus (Fusté et al., 1996
). Computer programs written by T. S. Whittam (Selander et al., 1986
) and J. G. Lorén were used to calculate VO and VE and to perform the Montecarlo randomization.
Estimates of the genetic differentiation between subpopulations were obtained by using Neis genetic distance (Nei, 1972 ). Phylogenetic analyses were performed with the PHYLIP package by using the neighbour-joining method. The reliability of the cladograms obtained (Fig. 3
) was determined by bootstrapping as follows. Gene frequencies were randomly selected, with replacement, to produce 100 replicated data sets of the same size as the original data sets. New Neis distance matrices were then calculated and subjected to the neighbour-joining method. Comparison of the 100 bootstrapped cladograms generated was made using the Consensus program of the PHYLIP package.
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RESULTS |
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DISCUSSION |
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Bacterial population genetics is the study of the natural variability of bacterial populations and has led to the formulation of theories to account for this diversity (Maynard Smith et al., 1993 ). Classical and molecular methods have continously been used to study the population diversity of V. cholerae and have been extensively applied to characterize different serogroups and biotypes (Baumann et al., 1984
), ribotypes (Karaolis et al., 1994
), RAPD fingerprint types (Rivera et al., 1995
) and insertion sequence fingerprint types (Bik et al., 1996
). These methods have shown a considerable amount of variation in V. cholerae. MLEE constitutes the classical methodology in the study of bacterial population genetics (Selander et al., 1986
). By using MLEE data we can obtain estimates of genetic and genotypic diversity and estimates of the frequency of recombination in natural populations. These aspects of V. cholerae population biology are of great importance to molecular epidemiological studies of cholera.
Previous MLEE studies with V. cholerae have shown a limited genetic diversity among toxigenic strains of this bacterium. Pathogenic isolates from various sources all showed identical electrophoretic profiles or differed in only a few loci (Salles & Momen, 1991 ; Wachsmuth et al., 1993
; Evins et al., 1995
). Some authors indicate that the sixth-pandemic, the seventh-pandemic and the US Gulf isolates are three independent clones (Kaper et al., 1982
; Waschmuth et al., 1993
; Karaolis et al., 1994
, 1995
; Evins et al., 1995
). In a recent study, it has been postulated that all strains of the O139 serogroup belong to a unique ET; similarly the toxigenic O1 ElTor isolates cluster in only three ETs (Beltrán et al., 1999
).
Our results show that when MLEE methods were applied to a large collection of isolates of diverse origin including serogroup O1 (classical and ElTor biotypes), O139 and non-O1/non-O139 strains, the different V. cholerae populations showed a high degree of genetic variation. The number of ETs found in our work does not coincide with data previously published (Salles & Momen, 1991 ; Wachsmuth et al., 1993
; Evins et al., 1995
; Beltrán et al., 1999
). From 107 isolates analysed, 99 ETs were found, 84 containing a single strain, six containing two strains and only one containing three strains. Previous studies described a more limited diversity of V. cholerae populations, with most isolates clustering in two or three ETs with minimal differences between them, especially when toxigenic strains were included (Salles & Momen, 1991
; Wachsmuth et al., 1993
; Evins et al., 1995
). The estimate for the mean genetic diversity per locus of the total 99 ETs (H=0·50) in our study is greater than other values reported for V. cholerae (Chen et al., 1991
; Salles & Momen, 1991
; Wachsmuth et al., 1993
; Evins et al., 1995
; Beltrán et al., 1999
). Likewise, the mean number of alleles per locus (4·8), and the genotypic diversity (0·9872) were higher than those given in similar studies, with the exception of Beltrán et al. (1999)
, who reported a value of 9·5 alleles per locus as an average. Also, the work of Beltrán et al. (1999)
differed in the number of strains that fell into the same ET.
Differences in the serogroup, geographical origin and year of isolation could explain this diversity. Another factor that may influence the results is the methodology used; some previous studies used starch as a matrix for the gels, whose resolution could be distinct from that of polyacrylamide (Wachsmuth et al., 1993 ; Evins et al., 1995
; Beltrán et al., 1999
). Finally, the number and type of enzymes studied are different; most of them are monomorphic and only a few contribute to the genetic diversity. However, in our work all the enzymes were polymorphic with a considerable genetic diversity, thus hampering comparisons between studies.
The dendrogram obtained in the present study shows no association between isolates with regard to serogroup, biotype or geographical origin. The same serogroup is present in several lineages and does not cluster separately from the others (Fig. 1). Similar results were obtained by Beltrán et al. (1999)
, who showed that strains of the same serogroup may belong to two or more divergent ET lineages. Division II has more non-O1/non-O139 strains than other groups. On the other hand, most of the O139 serogroup strains clustered in division I (24 of 29 isolates), while other serogroups seem to be randomly distributed. With regard to the source of the strains studied, division II seems to contain most of the environmental strains, while clinical isolates predominate in the subgroup Ib. Although this clustering did not coincide with previous studies, the value of the cophenetic correlation coefficient obtained (R=0·81) falls into the range (0·740·90) of most frequently occurring cophenetic correlations reported by Sneath & Sokal (1973)
.
Unimodal allele mismatch distribution (Fig. 2) and the shape of the dendrogram (Fig. 1
) are typical of a panmictic population (Whittam, 1995
). However, evidence for clonal proliferation is provided by the multilocus linkage disequilibrium calculations, which reveal a significant level of association between alleles when the whole population sample is analysed. Values of IA among all ETs indicated that there is a nonrandom distribution of alleles, which is clear evidence of a clonal population structure, with a significant degree of linkage disequilibrium IA=1·25±0·14 (no significant differences were obtained when we considered strains or ETs). Nevertheless, when we analysed data subsets corresponding to divisions of the dendrogram, serogroups or geographical origin, some of them (divisions II, III, O1 classical and USA/Mexico strains) showed IA values less than one (0·41, 0·08, 0·64 and 0·42, respectively). The VO values of these subgroups were also within the 95% confidence limits of VE and within the maximum and minimum values of the variance obtained by the Montecarlo procedure. However, IA values corresponding to the division III and the O1 classical subgroup should be taken cautiously because of the low number of ETs in these groups (six and nine ETs, respectively). These results are partially concordant with those obtained by Beltrán et al. (1999)
, who found IA values quite similar to ours (1·248±0·083) for the whole population, and IA values close to zero when they analysed population subgroups. Therefore, our results do not rule out the possibility that the population of V. cholerae studied has a clonal structure, nor do they exclude recombination. We believe that this population has a clonal structure which has not been broken by genetic recombination. Another possibility is to consider that the population has an epidemic structure (Maynard Smith et al., 1993
), in which strains of different origin would coexist with others of identical origin corresponding to an outbreak of disease from a single person in a specific geographical area (e.g. in July, 1994, the massive outbreak of O1 ElTor V. cholerae among Rwandan refugees in Goma, Zaire: Sanchez & Taylor, 1997
).
The analysis of the genetic distances for isolates belonging to the six subpopulations that we could establish on the basis of the country of isolation (Fig. 3), showed that strains from Peru cluster in the same group as those isolated from India. This result is fully concordant with the origin of Perus strains previously determined by Wachsmuth et al. (1993)
. In the same context, we might consider the results obtained with strains isolated in USA and Mexico. USA strains are longer established and remotely related with Mexicos isolates, as Wachsmuth et al. (1993)
pointed out.
If we consider population subsets referring to serogroup/biotype, there is also a concordance with previous references in relation to the origin of O1 and O139 serogroups (Bik et al., 1995 ; Stroeher et al., 1997
). From the analysis of the genetic distances between populations it might be deduced that V. cholerae O1 ElTor and O139 originated from a unique splitting event (Fig. 3
) and consequently, O139 and O1 ElTor might have gained a similar diversity (H=0·40 and H=0·45, respectively). Indeed, looking at Fig. 4
, we can see that O139 subpopulation strains were distributed in different ETs in the same way as those of O1 ElTor. Nonetheless, the slight differences in the genetic diversity can easily be explained if we consider the geographical origin of the two sets of strains. O139 are all Indian isolates, whereas O1 ElTor were isolated in several countries (Table 1
).
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Other population studies based on the analysis of restriction fragment length polymorphisms in genes for conserved rRNA and cholera toxin (ctxA) or in DNA sequences flanking these genes revealed four different ribotypes and four different ctx genotypes among 93 strains of V. cholerae O139 studied (Faruque et al., 1997b ). These results agree with the idea that strains belonging to the O139 serogroup may have emerged from similar serotype-specific genetic changes in more than one progenitor strain of V. cholerae. Considering this background, it is hard to believe that all the O139 strains could belong to a unique ET. Results obtained in our work are in agreement with the hypothesis of a more diverse origin of these strains, a conclusion that can also be reached from ribotyping analysis.
Data from the sequences of some housekeeping genes of V. cholerae showed a high degree of similarity when the different strains were compared, especially in clinical isolates. Karaolis et al. (1995) determined the DNA sequences of the asd (aspartate semialdehyde dehydrogenase) genes from 45 isolates of V. cholerae, which included five O139 isolates. Byun et al. (1999)
sequenced the mdh (malate dehydrogenase) and hylA (haemolysin A) genes from environmental and pathogenic strains of V. cholerae, including seven strains of the O139 serogroup. Both groups of authors found a high degree of similarity among the pathogenic isolates, suggesting that they are very closely related. These results may appear to disagree with the high diversity found in our work. This contradiction, however, is only apparent. Our results show that mdh and asd presented a low genetic diversity within the O139 isolates studied (mdh, h=0·32; asd, h=0·07). Moreover, the number of O139 strains studied by these authors is low (in our work we studied 29 O139 isolates). Recently, Jiang et al. (2000)
published a study applying the AFLP method to a collection of clinical and environmental isolates of V. cholerae, which supports the idea that pathogenic V. cholerae strains have evolved from multiple independent sources like environmental O1 or non-O1 strains. We believe that more extensive areas of the bacterial chromosome should be analysed (as in the case of MLEE) in order to reach a reasonable conclusion about the likely origin of a bacterial population.
The persistence of V. cholerae in the environment, for months and probably years, is facilitated by its ability to enter a viable nonculturable state in which its nutrient and oxygen requirements are much decreased (Ravel et al., 1995 ). It has also been described that V. cholerae can bind to chitin in crustacean shells, and colonize the surfaces of algae, phytoplankton, copepods and the roots of aquatic plants such as water hyacinth. One scenario to explain our findings is that during interepidemics V. cholerae populations might be associated with an ecological reservoir. We can considerer that all V. cholerae cells constitute a metapopulation integrated by multiple ecological populations. Previous studies have shown that bacterial recombination is too rare to prevent neutral sequence divergence between distinct ecological populations. Moreover, the episodes of periodic selection that purge diversity operate within populations and do not prevent their divergence (Young, 1989
). In their reservoir V. cholerae clones could diverge from each other. Because the genes for the major virulence factors can be acquired horizontally, we can postulate that each V. cholerae cell would have the same probability of being transformed into a toxigenic strain (Beltrán et al., 1999
). In the next epidemic episode, some unknown environmental factors might trigger an explosive multiplication of V. cholerae populations outside this reservoir (Epstein et al., 1993
; Harvell et al., 1999
). This global population would be formed by bacteria from various local populations and include a diversity of toxigenic clones that potentially could infect humans.
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ACKNOWLEDGEMENTS |
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M. Farfán is the recipient of the grant Formació en la Recerca i Docència per a alumnes de tercer cicle from the University of Barcelona. This work was supported by a grant from the Vicerectorat de Recerca of the University of Barcelona.
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Received 20 December 1999;
revised 22 May 2000;
accepted 26 June 2000.