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Title: Molecular epidemiology and evolution of atypical strains of Vibrio cholerae 01
Other Titles: Huo luan hu jun fei dian xing jun zhu 01 de fen zi liu xing bing xue yu jin hua
霍亂弧菌非典型菌株 01 的分子流行病學與進化
Authors: Safa, Ashrafus
Department: Department of Biology and Chemistry
Degree: Doctor of Philosophy
Issue Date: 2010
Publisher: City University of Hong Kong
Subjects: Vibrio cholerae -- Molecular aspects.
Molecular epidemiology.
Notes: CityU Call Number: QR201.C5 S23 2010
xxiv, 200 leaves : ill. (some col.) 30 cm.
Thesis (Ph.D.)--City University of Hong Kong, 2010.
Includes bibliographical references (leaves 149-178)
Type: thesis
Abstract: The gamma-proteobacterium, V. cholerae, is an autochthonous inhabitant of the aquatic ecosystem. Several pathogenic forms of V. cholerae are responsible for the gastrointestinal disease called ‘cholera’, which is characterized by a severe form of acute secretory diarrhoea in humans. To date, more than 206 serogroups of V. cholerae have been identified based on variations in O-antigen structure. Serogroups O1 and O139 are responsible for all of the major cholera epidemics and pandemics on record. Based on phenotypic differences, V. cholerae O1 strains have been classified into two biotypes: 'classical' or 'El Tor'. The sixth pandemic and presumably the fifth pandemic were caused by V. cholerae of the classical biotype. The ongoing seventh pandemic started in 1961 by strains of the El Tor biotype. In 1992, a new pathogenic serogroup, O139, caused cholera epidemics in India and Bangladesh, which subsequently spread to other countries. In recent years, the emergence of a new group of V. cholerae O1 variants has been reported in Bangladesh and Mozambique, which display a mixture of phenotypic and genotypic traits typical of the classical and El Tor biotypes, suggesting that they may be genetic hybrids. The new variants are now known as ‘atypical’ strains of V. cholerae O1. This study aims to study the molecular epidemiology and evolution of these atypical V. cholerae O1 strains. Additionally, this study aims to investigate the genetic and pathogenic differences between typical and atypical El Tor strains of V. cholerae O1 using the nematode, Caenorhabditis elagans, as a model host. We examined 41 clinical strains of V. cholerae O1 from Asia and Africa (isolated between 1986 and 2004), which were confirmed as serogroup O1 by rfbO1-specific PCR that targeted a specific region of the O-antigen biosynthesis gene cluster. Biotyping was performed using standard procedures, and all strains were confirmed as El Tor. All strains were also examined for the biotype-specific tcpA allele and for the rtxC gene, which is present only in strains of the El Tor biotype. PCR results revealed that all of the strains examined harbour the tcpA gene of the El Tor type and absence of the rtxC gene. The findings were in agreement with the biotyping results and confirmed that all of the 41 test strains were of the El Tor biotype. PCR amplification of the ctxA and ctxB genes (that encode for the CtxA and CtxB subunits, respectively, of cholera toxin - a key virulence factor directly responsible for the major clinical symptoms of the disease) and detection of cholera toxin (CT) production by ELISA revealed that all 41 V. cholerae O1 strains are toxigenic. The DNA and deduced amino acid sequence of the ctxB gene is known to differ among toxigenic V. cholerae strains. DNA sequencing of the ctxB gene of all 41 strains showed that the deduced CtxB amino acid sequences of 30 strains were of the classical CT type (histidine at position 39, phenylalanine at position 46, and threonine at position 68), while those of the remaining 11 strains were of the El Tor CT type (with tyrosine at position 39, phenylalanine at position 46, and isoleucine at position 68). The overall analysis showed that all 41 test strains are of El Tor biotype but 30 of these harbour the classical ctxB gene. These 30 variants were classified as atypical El Tor strains of V. cholerae O1. The major finding from this study therefore revealed that atypical El Tor V. cholerae O1 strains that harbor the classical CT gene are not limited only to the Bangladesh and Mozambique, but have now spread to several other countries in Asia and Africa. These findings provided compelling evidence that the classical CT has reappeared but that for these cases its carrier has been El Tor. Based on the fact that V. cholerae O1 classical strains (the major reservoir/carrier of the classical CT gene) are now extinct, it is hypothesized that there are alternative sources of the classical CT gene in the environment that has yet to be discovered. To better understand the virulence and pathogenic potential of the newly described atypical El Tor V. cholerae O1 strains from different Asian and African countries, various genoptypic traits that are associated with the pathogenicity of V. cholerae strains and those that are used to differentiate clinical strains of the classical and El Tor biotypes were used to examine a total of 41 strains (that consisted of both typical and atypical El Tor strains of V. cholerae O1). In this study, various chromosome-encoded virulence genes - hlyA, nanH, stn, and ORFs VC1449, VCA0316 and VCA0728-VCA0730 - were analysed using a variety of molecular techniques. It is widely known that V. cholerae O1 El Tor harbours a functional hlyA gene while the hlyA in V. cholerae classical is non-functional due to an 11-bp deletion in the gene. Southern hybridization analysis revealed that 22 out of 30 of the atypical El Tor V. cholerae O1 strains analysed harbour a functional hlyA gene indicating that the atypical strains are highly similar in the hlyA gene locus to the El Tor biotype. Several virulence-associated open reading frames (ORFs) such as VC1449, VCA0316 and VCA0728-VCA0730 are believed to be present only in the El Tor biotype; and PCR analysis demonstrated that these ORFs are present in the genomes of all atypical El Tor V. cholerae O1 and typical El Tor strains examined in this study. Moreover, PCR analyses revealed that the nanH (neuraminidase) gene is present while the stn (heat-stable enterotoxin) gene is absent in all typical and atypical V. cholerae O1 El Tor strains examined in this study. Examination by PCR of the virulence genes, cep, ace and zot, which are carried on the cholera toxin prophage (CTXØ), also confirmed the presence of all these genes in atypical El Tor strains. Using rstR allele-specific PCR, this study also revealed that 21 out of the 30 atypical V. cholerae O1 strains analysed harbour the rstR allele of the classical type. Differences in the VSP-I and -II (Vibrio seventh pandemic island I and II), and RTX (repeat in toxin) gene clusters have been previously reported whereby VSP-I and -II were found to be unique to El Tor strains, while RTX (which comprises four genes, rtxA, rtxB, rtxC and rtxD) is intact in El Tor strains but is truncated in classical strains (which lacks rtxC). Another gene cluster, named MSHA (mannose-sensitive haemagglutinin), is known to be present in typical El Tor strains but absent in most classical strains. Long-range PCR analysis demonstrated that all of the atypical El Tor strains analysed harbour intact forms of these four pathogenicity islands. Overall, molecular analysis of various virulence genetic loci revealed that all atypical El Tor strains possess the key pathogenicity genetic traits necessary to initiate a pandemic spread. Importantly, the findings indicated that the atypical El Tor strains are more similar to the typical El Tor than the classical biotype, which strongly suggest that atypical El Tor highly likely originated from the typical El Tor biotype. To study the evolutionary relatedness of the atypical El Tor V. cholerae O1 strains to the prototypical classical and El Tor biotype strains, Multilocus Sequence Typing (MLST), Pulsed-field Gel Electrophoresis (PFGE) and Randomly Amplified Polymorphic DNA (RAPD) experiments were carried out. MLST analysis of the three house-keeping genes, pyrC, mdh, and recA, revealed that atypical El Tor strains are more similar to the El Tor biotype strains, and PFGE analysis strongly indicated that the genomes of atypical El Tor strains are highly similar to and possibly evolved from El Tor biotype strains although the genomes of atypical El Tor may have undergone a significant level of independent genetic rearrangements. By contrast, RAPD analysis indicated that the genomes of atypical El Tor strains are a mélange of typical classical and El Tor strains. Comparative genomic hybridization (CGH) analysis was carried out to investigate the level of genomic similarity between selected strains of atypical El Tor and typical El Tor V. cholerae O1. A total of 3811 ORFs of a typical V. cholerae O1 El Tor were examined in three representative atypical El Tor strains. Approximately 30 ORFs were found to be absent in total from chromosomes 1 and 2 of the three atypical El Tor strains examined, most of which are known to encode for hypothetical proteins. V. cholerae-Caenorhabditis elegans interaction studies were conducted in an attempt to better understand the differences in pathogenicity between atypical El Tor and typical El Tor strains. The killing efficiencies of C. elegans by typical and atypical El Tor strains were examined, and the atypical El Tor V. cholerae O1 strains consistently showed a faster killing rate on C. elegans than the typical El Tor strains. Several Pathogenicity Islands (PAIs) - VSP-I, MSHA and RTX - were correspondingly subcloned from both typical and atypical El Tor strains into E. coli hosts using a cosmid vector, and the killing effects of the E. coli recombinants on C. elegans were examined. No significant difference in the killing rate of C. elegans was observed between PAIs subcloned from atypical and typical El Tor strains. Although there is ongoing speculation regarding the origins of the atypical El Tor strains, this study revealed that these strains have spread to many countries around the world, and currently co-exist with the prototype El Tor clone. This study also indicated that atypical El Tor strains have likely evolved through lateral gene transfers of various virulence genetic traits between strains of the classical and El Tor biotypes that facilitated either better survival and/or epidemic potential of the atypical El Tor variants.
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