Molecular Evolution And Variation Of Vibrio Cholerae Isolates Recovered In Guangdong Province,China

Background:Vibrio cholerae is the causative agent of an acute watery diarrheal disease referred to simply as cholera. So far more than 200 serogroups of V. cholerae have been identified; however, only two serogroups, O1 and O139, are capable of causing global pandemics of cholera. Seven distinct pandemics of cholera have been recorded since 1817. According to the record, cholera spread to China as early as 19 century, and remains a focused preventing intestinal infectious disease today. Located in the southern coastal area of China, Guangdong is located along the same latitude as Kolkata, India and Dhaka, Bangladesh. It is considered a cholera-epidemic area in China. In July 1961, the first outbreak caused by V. cholerae O1 E1 Tor occurred in Yangjiang city, Guangdong Province, triggering the onset of the seventh cholera pandemic in China. From 1961 to 2013, nearly 80,000 cholera cases were reported in Guangdong Province with about 2,000 deaths reported to the national cholera surveillance system. The epidemic deeply affects and harms the people’s health, production, tourism, foreign trade, transportation, and even social stability.Cholera toxin (CT) is the most important causative agent of the V. cholerae, encoding by ctxAB gene. Known as the strongest toxins causing diarrhea, Cholera toxin is the main factor that causes acute watery diarrhea of cholera. The toxigenic strains with the ctxAB gene mainly cause the cholera outbreak and pandemic, while the non-toxigenic strains without the ctxAB gene only cause sporadic diarrhea or asymptomatic status, without considering an outbreak of cholera and epidemic. The previous research had found a relatively conservative genome in the toxigenic 01 and 0139 V. Cholerae. There are only minor changes and these changes accumulate slowly over time in the toxigenic 01 ElTor V. Cholerae isolated from the seventh cholera pandemic period. However, the genome of non-toxgeinc 01 and 0139 V. Cholerae showed significant genetic diversity. All these data would engage us to apply a higher resolution molecular typing method to study the evolution and variability of the toxingenic V. Cholerae strains.Bacteria’s typing is the valuable method to inferred relationships between strains, which can be used to reconstruct evolutionary relationships, describing the structure of bacteria, can also be used for source tracking of an outbreak of infectious diseases. The original bacteria’s typing relies mainly on morphology, such as sero-typing, phage-typing, toxin-typing and antibiotic resistance spectrum. Recently, the molecular typing based on DNA sequence analysis play a more important role. Pulsed-field gel electrophoresis (PFGE) is recommended as the "golden standard" of current bacterial molecular typing techniques, with the insignificant performance in the identification of outbreak and source tracking based on the fingerprinting patterns derived from the bacterial genomic DNA digested by a specific restriction enzyme. Additionally, other molecular typing methods such as the multi locus sequence typing (MLST) and multi-locus variable-number tandem repeat analysis (MLVA), are more conducive to the rapid widespread outbreak investigation and source tracking, which greatly simplify and facilitate the exchange and sharing of data among different laboratories through the internet databases. However, only local pathogen genome genetic information was discovered, these molecular methods are limited in resolution, especially to those strains with highly clonal population or the epidemiological study with time span period. Whole-genome sequencing (WGS) bring the epidemiological analysis to the level of whole-genome sequence, which enable a comprehensive response to the pathogen’s genetic variability, greatly improve the resolution of strain evolution and source tracking analysis, quickly identify and track to the pathogen.In the 1990’s, we began to establish the molecular analysis methods such as the Ribosome typing, pulsed-field gel electrophoresis and fluorescence quantitative PCR to rapidly identify the pathogen and source tracing when cholera outbreak occurred. The comprehensive analysis of the molecular etiologic characteristics of the V.cholerae in Guangdong province remained unclear, especially in the high resolution investigation of genetic diversity and relationships of the V.cholerae strains.With the development of the next-generation sequencing technology, the shorten time is required while the costs are reduced. The WGS can not only discover the genome evolution characteristics of pathogens, but also has great value and clear advantage in the heredity and variation of source tracking analysis.In this study,381 clinical V. cholerae O1 ElTor isolates (233 Ogawa and 148 Inaba) and 136 clinical V. cholerae O139 collected over 50 years (1961 to 2013) were characterized by sero-typing, PCR assay testing for the absence and presence of virulence genes, DNA sequence for the positive amplicons and pulsed field gel electrophoresis for genetic relatedness; isolates were obtained from cases originating different districts across Guangdong Province, either from outbreaks or sporadic cases. All these data enable us to fully understand and grasp serotype distribution and molecular characteristics of V. Cholerae in Guangdong Province. According to the PFGE fingerprinting, ctxB and tcpA genotyping results,154 typical stains were selected for whole-genome sequencing. Then, another 126 from other provinces and 105 from the international whole genome sequences were put in to perform single nucleotide polymorphism (wg-SNPs) analysis and build the maximum likelihood tree; to in-depth understand of genetic variants of V. Cholerae in Guangdong Province. We attempt to build a relationship arc of this V. Cholerae in this district along with the historical epidemiological data, and infer the evolution of the pathogens. We also expect to clarify the epidemic features of cholera in different periods and in-depth understanding the evolution and variation of V. Cholerae from the perspective of molecular genetics and molecular mechanism. We hope all this work done here will provide the experimental evidence for molecular epidemiologic surveillance, identification of outbreak and accurate traceability for cholera in future.Objectives:1. To understand and overview the characteristic of the serotype distribution, virulence gene carrying and PFGE finger type patterns of clinical strains of V.Cholerae isolated from Guangdong province from the first cholra outbreak in Guangdong province, China in 1961 to 2013.2. To describe the characteristic of genetic evolution and variability of V. Cholerae 01 and O139 in our province through the whole genome sequencing; and build a relationship arc of cholera bacteria in our province; finally to find out the molecular epidemiological characteristics and possible transmission routes of different cholera epidemic stages in our province. We expect this study will help to construct the molecular database of V. Cholera in Guangdong Province and provide the experimental basis to build a high resolution system for cholera molecular epidemiologic surveillance, identification of outbreak and traceability analysis.Methods:1. Literature research:We check out the historic surveillance data and reports to describe the overview of the cholera epidemic in Guangdong Province since 1961. Then we observed four distinct epidemic periods (1-4) based on the different dominant serotype leading its epidemic, correspond to years; or time periods from/to 1961-1969,1978-1989,1990-2000,2001-2013, respectively. A total of 381 strains of V. cholerae 01 (233 Ogawa and 148 Inaba) and 136 strains of V. cholerae 0139 were included in this study. Isolates were selected according to the different epidemic period, the severity of the symptoms, serotypes and year and city where cholera was occurring, with dates of isolation from 1961 to 2013.2. Strains recovery and identification:All freeze-dried isolates were recovered in the general nutrient broth, and then examined using the API biochemical identification test. Serotyping was determined by slide agglutination with O1-, Inaba-and Ogawa-specific antiserum.3. Virulence detection and sequencing analysis:DNA was extracted from bacterial isolates using the QIAamp DNA Mini kit according to the manufacturer’s instructions (Qiagen, Inc., Shanghai, China). Conventional PCR were carried out to determine the virulence genes. Target genes included cholera toxin B subunit (ctxB), accessory cholera enterotoxin (ace), and zonula occludens toxin (zot) of the CTX prophage, and the classical and El Tor-specific tcpA and tcpl genes of the toxin-coregulated pilus (TCP) Pathogenicity Island. Additional putative accessory virulence genes included hemolysin (hlyA), an outer membrane protein (ompU), RTX toxin (rtxC) gene and the heat-stable enterotoxin (st). The positive ctxB and tcpA amplicons were sequenced using a commercial supplier (Shenggong, Inc., Shanghai, China).4. PFGE analysis:PFGE was performed using the PulseNet standardized PFGE protocol for V. cholerae (USCDC,2006). The enzyme digestion for each plug was used NotI enzyme. PFGE patterns were analyzed using BioNumerics software (Version 6.6, Applied Maths BVBA, Sint-Martens-Latem, Belgium). Dendrograms were produced using the Dice coefficient and un-weighted pair group method with arithmetic mean algorithm (UPGMA).5. Whole genome sequencing:154 strains were then selected for further whole genome sequencing according to the PFGE fingerprinting, ctxB and tcpA genotyping results. The SOAP De novo was applied to assemble of the reads with the reference sequence of N16961. The MuMMER was applied to identify the single nucleotide polymorphism (wg-SNPs) and analyze the genetic variants of V. Cholerae in Guangdong Province. The MEGA6.0 was applied to build a maximum likelihood tree along with the historical epidemiological data, and infer the evolution of the pathogens.Results:1. Prevalence and distribution of the V. cholerae O1 and O139:Four serotypes dominated epidemic periods were observed in this time frame from the first cholra outbreak in 1961 to 2013:period 1 (1961 to 1969), isolates expressing the Ogawa serotype were responsible for cases during this cholera epidemic period. The second period (1978 to 1989) started with an Inaba strain identified in Yangjiang city in June 1978. During the third period beginning in 1990, serotype Ogawa dominated. Period 4 (the longest, starting in the year 2000) has been characterized by sporadic cases and small outbreaks with a distribution all around Guangdong Province. Serotype Inaba once again replaced Ogawa-associated isolates and became the dominant serotype in this period. From the first report of V. cholerae 0139 isolate in 1993, the V. cholerae 0139 cases have been sporadic and have not resulted in epidemic or pandemic spread in recent 20 years.2. Identification of virulence-associated genes:1) Among the 381 O1 isolates tested,367 were simultaneously positive for ctxB, ace and zot, while 374 were positive for tcpA. The amplicons sequencing indicated that 160 isolates recovered before 1992 and a single isolate recovered in 1993carried the ctxB allele of El Tor type, while the remaining 206 isolates from 1993 until 2013 harboured the ctxB of classical type. However, the entire tcpA gene was identified as El Tor type.2) Among the 136 O139 isolates tested,123 were simultaneously positive for ctxB, ace and zot. Similarly,123 isolates yielded PCR-positive results for tcpA, and all amplicons were subsequently sequenced and identified as El Tor type of tcpA. The phylogenetic tree of 123 ctxB sequences revealing multiple genotypes. The majority of isolates (n=113) belonged to ctxB genotype 3 (El Tor type), seven isolates carried genotype 1 (Classical type), and three isolates from 1999 carried genotype 5.3. PFGE patterns of the isolates:1) The 381 isolates were differentiated into 113 distinct patterns on the basis of PFGE using NotI endonuclease digestion. UPGMA analysis clustered these PFGE patterns into five clusters. Isolates from the first four clusters were associated with specific periods; cluster Ⅰ (period 1), Ⅱ (period 3), Ⅲ (period 2) and IV (period 4) respectively. Cluster V was composed of isolates from all four epidemic periods, and thus was most heterogeneous in appearance. The isolates within cluster V formed a less similar group comparatively and can be split into more than 10 different possible lineages. The Ogawa serotype was mainly associated with epidemic periods 1 and 3 (clusters 1 and 2); these isolates showed 80.3% similarity. A similar observation is apparent with serotype Inaba mainly present in clusters III and IV, which consisted of the isolates recovered from epidemic periods 2 and 4, exhibiting 81.6%similarity.2) All 136 isolates were differentiated into 75 distinct patterns by NotI endonuclease digestion and PFGE. Phylogenetic analysis using UPGMA clustered the patterns into clusters A and B. Cluster A contains seven of the O139 non-toxigenic isolates from 1998-2000 and each of the seven non-toxigenic isolates revealed a unique pattern. Cluster B contain the other group of non-toxigenic isolates (from 1998 and 2007) and all toxigenic isolates; 68 unique patterns were represented with an overall similarity of 82.4%.4. Whole genome sequencing:1) There 10396 SNPs were indentified in 387 V. cholerae whole genome by MuMMER.3351 SNPs were special for the pre-seventh-pandemic isolate M66 before 1961, which was used as an outgroup strain to root the tree. Another 5534 SNPs were used to distinguish the 387 genomes from the current seventh cholera pandemic.2) Genetic recombination was rare in the 01 El Tor V.cholarae isolated from Guangdong province. There only 1511 SNPs were induced by the genetic recombination, mostly occurred in the O-Antigen gene cluster and Vibrio Seventh Pandemic Island II. All the other SNPs were induced by mutation.3) The 01 El Tor V.cholarae isolated from the early pandemic period I in Guangdong province show more genetic proximity with the strains from Indonesia in the same period and more closer to the root of the Maximun-likelihood tree than the strains from the other province in China. Similarity, some strains from the early pandemic period Ⅱ in Guangdong province formed a cluster and showed closer to the root of the Maximun-likelihood tree than the strains from the other province in China.4) At least two O1 E1 Tor V.cholarae isolated from Guangdong province were the most recently common ancestor, located in two branch roots of the Maximun-likelihood tree, followed with the strains form the South Asia in the same period, and then strains from the other province in China.5) In the 0139 V.cholarae genetic branch, two strains isolated from Guangdong province in 1993 were located in the root of the branch and showed close genetic distance with the O1 El Tor Vcholerae. Different from the strains after 1998, most of the 0139 strains before 1998 were located in the genetic branch root.Conclusion:1. This study indicated that from 1961 to 2013, among the cholera epidemic periods in Guangdong Province, there were four distinct periods, dominated by serotypes shifting of Ogawa (1961-1964) to Inaba (1978-1989) to Ogawa (1990-2000) to Inaba (2001-2013) in each epidemic period. This serotype shifting is consistent with what has been observed in other provinces in China, as well as in most countries in Asia.2. In this study, sequencing revealed that all of the ctxB alleles in isolates before 1992 were ctxBEl Tor, however, after 1992, the genotype switched to ctxBclassical, similar to serotype shifting at national level in China, as well as other countries of Asia, Africa and Latin America.3. The previous study show that the 0139 isolates collected in China were entirely El Tor type (genotype 3) of ctxB. In this study, we detected genotype 1 (classical type) and genotype 5 type of ctxB carried isolates from Guangdong province, which to the best of our knowledge, is the first report of variant types (but not El Tor type) of ctxB from V. cholerae 0139 isolates in China. Our study also indicated that the ctxB variants are not limited to the O1 serogroup in China.4. PFGE clustered the isolates into five main clusters, highly related isolates, as determined by PFGE banding patterns were also from the same time epidemic period. These results suggest that each epidemic period began with the input of a genetic variant that was immunologically divergent enough to cause disease in a previously exposed community.5. Our study indicated that, toxigenic (cholera toxin produced) 0139 isolates maintained a relatively high clonality, although isolates collected from widely different geographic locations, nontoxigenic isolates, however, exhibited greater diversity and multiple clonal lineages.6. The wg-SNPs analysis indicated a high genetic singlet in the 01/0139 V.cholerae in Guangdong province and the genetic difference were mostly caused by mutation.7. Some of the stains in Guangdong province formed clusters in the O1 El Tor V.cholerae genetic branch, which reflected the regional relation. The results may be caused by the limited transmission ability, which prevented these strains to spread out of Guangdong province. It may also be caused by the sampling bias in this study.8. The genetic evolution characteristics of the O1/O139 V.cholerae changed obviously with the cholera spread and epidemic pattern. In the early periods of O139 epidemic, its spread characteristics was similar with O1 E1 Tor V.cholerae, while the food-borne turned out to be the most common way of cholera after 1998.9. The genetic evolution characteristics of the O1/O139 V.cholerae in Guangdong province were distinctly to the domestic and international strains, which may be caused by the special geographical location and frequent trade exchanges between Guangdong and South Asia.