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International Centre for Diarrhoeal Disease Research, Bangladesh, Dhaka, Bangladesh
ABSTRACT
The genomes of the recently described Matlab variants of Vibrio cholerae O1 that are hybrids between classical and El Tor biotypes were compared with those of El Tor and classical biotypes by the use of pulsed-field gel electrophoresis. Dendrograms constructed using the unweighted-pair group method using average linkages generated from NotI restriction patterns of whole-chromosomal DNA grouped these strains into two major clusters that were found to be similar but not identical to those of either of the biotypes. Strains that clustered with the classical biotype appear to have been derived from the classical strains, which are thought to be extinct.
TEXT
Vibrio cholerae is a species that has been well defined on the basis of biochemical tests and DNA homology studies (3). However, this species is not uniform with regard to its pathogenic potential (9). Cholera toxin and toxin-coregulated pilus are the two major factors that presently determine the pathogenic potential of V. cholerae (5). The genes that encode production of cholera toxin and toxin-coregulated pilus are not found in all V. cholerae isolates but are preferentially linked with the O1 and the O139 serogroups and appear to have been acquired by horizontal gene transfer (10, 13, 14). A total of 206 serogroups of V. cholerae are presently recognized on the basis of the presence of the somatic O-antigen. From an epidemiological perspective, a distinctive feature of cholera is its tendency to occur as explosive outbreaks, which have often resulted in pandemics (9). To date, seven pandemics of V. cholerae O1 have been reported, and cholera due to new serogroup O139 is believed to be the eighth pandemic (9).
The O1 serogroup of V. cholerae is further classified into two biotypes, namely, the classical and El Tor biotypes. The classical biotype was responsible for the fifth and sixth pandemics and, although there is no hard evidence, was presumably responsible for the earlier pandemics also (8). From 1961, the seventh pandemic El Tor strains gradually replaced the classical strains and continue to persist today. However, in 1982 in parts of Bangladesh, the classical biotype reemerged as the predominant epidemic strain (4, 19, 20) and coexisted with the El Tor strains, causing disease until 1993 (17). Different phenotypic traits, such as hemolysis of sheep erythrocytes, agglutination of chicken erythrocytes, and sensitivity to polymyxin B and to specific phages, and different assays such as the Voges-Proskauer test are used to differentiate the two biotypes (9). Among these traits, Voges-Proskauer reactions, agglutination of chicken erythrocytes, and hemolysis of sheep erythrocytes give negative results for classical strains but positive results for El Tor strains (9). Sensitivity to polymyxin B (50-U disk) is characteristic of classical strains, while the El Tor strains are resistant to polymyxin B. Hemolysis of sheep erythrocytes gives negative results for the classical biotype, and at present the test is of limited use, since V. cholerae El Tor strains isolated after 1960 were nonhemolytic (2).
In our previous study, we reported for the first time new variants of V. cholerae O1 that are hybrids between the classical and El Tor biotypes and were associated with sporadic acute secretory diarrhea requiring hospitalization (17). These strains were also examined using different molecular techniques such as PCR and ribotyping. All the phenotypic and genotypic traits tested failed to categorize them into the classical or the El Tor biotype classification, and these strains were found to have traits of both the biotypes, reflecting a hybrid nature. These strains were classified into three types and designated as Matlab types I, II, and III. In this study, we used pulsed-field gel electrophoresis (PFGE) to analyze the restriction patterns of the whole genomes of these hybrid strains (Matlab types I, II, and III) and the level of genomic similarity between the hybrid Matlab strains and the classical and El Tor biotypes.
Intact agarose-embedded chromosomal DNA from clinical isolates of V. cholerae was prepared, and PFGE was performed using a contour-clamped homogeneous electric field (CHEF-DRII) apparatus (Bio-Rad) by procedures described earlier (21). The conditions used for separation were as follows: 1- to 7 -s pulses for 10 h, 3- to 18-s pulses for 10 h, 3- to 28-s pulses for 10 h, and 5- to 50-s pulses for 6 h. An electric field of 6 V/cm was applied at an included field angle of 120°. Genomic DNA was digested with the NotI restriction enzyme (GIBCO-BRL, Gaithersburg, Md.) at a concentration of 20 U/μl. The restriction fragments were separated in 1% pulsed-field-certified agarose in 0.5x Tris-borate-EDTA buffer. In the post-electrophoresis-gel-treatment step, the gel was stained and destained. The DNA was visualized with a UV transilluminator, and images of the transilluminator were digitized with a 1D Gel documentation system (Bio-Rad). The images were then processed with Quantity One software (version 4.4.1; Bio-Rad).
A known strain of V. cholerae (MG-117029) was used as a molecular size marker. The test fingerprint image was normalized according to the standard, and the molecular weights of the DNA fragments were determined. The gel image analyzed included two reference lanes of classical and El Tor biotypes. The reference strain of classical V. cholerae O1 is the same as that used for our earlier report in which we described the Matlab variants (17), and the reference strain for El Tor was AR-32732, which is a standard El Tor strain that we routinely use in our laboratory for reference purposes.
The molecular weight of the DNA fragments was determined by using Quantity One software (version 4.4.1; Bio-Rad). The degree of genetic diversity between the new variants and the classical and El Tor strains was determined by using Diversity Database software (version 2.2; Bio-Rad). The similarity among the strains was determined using the Dice coefficient, and the cluster analysis was carried out using the unweighted-pair group method using average linkages (UPGMA).
A set of 11 representative strains of V. cholerae hybrid strains was analyzed by PFGE. The NotI restriction digestion restricted the chromosomal genome into 17 to 22 fragments (Fig. 1). The fragments ranged from 6 to 350 kb. For cluster analysis, only fragments having a molecular size of 64 kb and above were considered. Eight pulsotypes among the 11 representative strains (Fig. 2) were revealed by using Tenover's criteria of PFGE analysis (22). Among the 8 pulsotypes, Matlab type I showed two subtypes (A1 and A2), Matlab type II showed single type (B), and Matlab type III showed 5 subtypes (C1 to C5). C4 was the most prevalent subtype among the five subtypes of Matlab type III strains. Furthermore, none of the pulsotypes produced by Matlab type I, II, and III strains were identical to each other.
The level of similarity was defined using the Dice coefficient, and the similarity matrix was converted to a dendrogram by the use of UPGMA. All the 11 strains grouped into two clusters (Fig. 2). Matlab type I clustered with the classical reference strain, while types II and III clustered with the El Tor reference strain. In a scale of similarity ranging from 0 to 1, the two major clusters showed 0.35 similarity, where 0 stands for total dissimilarity and 1 stands for being completely identical. Notably, none of the strains representing the two clusters were found to be identical when intercluster similarity was checked. A single Matlab type III hybrid strain (MH-08) showed the highest level of similarity with the reference El Tor strain, with a similarity value of 0.78. On the other hand, the representative strain of Matlab type II (MG-116226) showed 0.52 similarity with a type III Matlab strain (MG-116926).
Interbiotype differences between classical and El Tor strains of V. cholerae O1 have previously been measured according to phenotypic variations. By investigating genetic differences, we were able to detect strains of V. cholerae O1 that had traits belonging to both biotypes and were therefore described as genetic hybrids and designated as Matlab strains (17). In this study we used PFGE to analyze the genome of the hybrid strains. The strains analyzed were clinical isolates obtained between 1991 and 1994 from hospitalized patients with acute diarrhea. The analysis indicates that the Matlab type I, II, and III strains are not identical to either the classical or the El Tor biotype but seem to be derived from them. In the two major clusters, Matlab type I strains grouped with cluster A (Fig. 2), indicating they had a lineage of the classical biotype. On the other hand, Matlab type II and III grouped with cluster B, indicating that they are much closer to the El Tor biotype (Fig. 2). However, the level of similarity (less than 0.60) between the Matlab types II and III is significantly low. Previous studies have shown that the classical and El Tor strains have evolved from separate lineages (18, 11), but recent comparative genomic analysis has indicated a remarkable conservation of genomic information among V. cholerae strains of the classical and El Tor biotypes (6). Dziejman et al. (6) used microarray and DNA sequencing to show that only seven genes were absent solely from classical strains compared to the El Tor strain. It has already been proposed that El Tor and classical strains appear to be derived from environmental nontoxigenic strains that are El Tor-like (8, 6). Thus, evolution of pathogenic new variants like the Matlab variants from the existing and/or primordial source is not unexpected.
Mitra et al. (15, 16) already described phage genome involvement in the acquisition of some classical biotype specific properties by El Tor strains. However, the exact mechanism of evolution as well as the qualitative changes brought in the genome of Matlab type strains in comparison to the existing pathogenic variants is not yet known.
In 1961, with the beginning of the seventh pandemic the classical strains were gradually replaced by the El Tor biotype, and in Bangladesh the gradual replacement occurred from 1968. However, the classical biotype reappeared in 1982 (19) and caused disease until 1992 to 1993. Therefore, it is worth mentioning that these hybrid strains were isolated during a period (1991 to 1994) when the classical biotype was on the verge of extinction or already extinct (17). In our previous study, examination of the tcpA and rstR types (12), genes that are associated with the pathogenicity of V. cholerae, showed classical attributes in the Matlab type I strains. Likewise, in the present study the Matlab type I strains were clustered with the classical biotype. Therefore, even though it is thought that the classical biotype is extinct, classically derived Matlab type I strains show the presence of a reservoir of the classical genes in the Matlab environs, a rural area of Bangladesh. The previous studies of classical strains isolated between 1961 and 1992 in Bangladesh by Faruque et al. (7) also support the speculation. Matlab I-like strains have assumed added significance given that hybrids between classical and El Tor biotypes were responsible for the cholera cases that occurred during the cholera season in Beira, Mozambique, in 2004 (1). The appearance of such strains in a different continent underscores the need to institute surveillance for such strains and to see whether these types of strains represent a pandemic spread.
ACKNOWLEDGMENTS
The International Centre for Diarrhoeal Disease Research, Bangladesh, is supported by the aid agencies of the governments of Australia, Bangladesh, Belgium, Canada, Japan, the Kingdom of Saudi Arabia, The Netherlands, Sweden, Sri Lanka, Switzerland, and the United States.
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