Genetic Analysis of CTX Prophage and Antibiotic Resistance Determinants in Vibrio cholerae O1 Belonging to the Atypical El Tor Biotype from Kelantan, Malaysia

Epidemic and global pandemic cholera have claimed millions of lives since the first pandemic in 1817 and it continues to exact a huge annual toll, with endemics established in approximately 50 countries worldwide (Ryan, 2011). More than 200 serogroups have been reported to date (Safa et al., 2009), but only two serogroups of Vibrio cholerae, the O1 and O139 serogroups, are known to have the potential for unleashing epidemic and pandemic cholera. The O1 serogroup of V. cholerae can be further divided into the classical or El Tor biotypes based on a number of phenotypic and genotypic characteristics (Sack et al., 2004). Seven pandemics of cholera have been recorded to date, where the first six pandemics were associated with V. cholerae serogroup O1 of the classical biotype, whereas the current ongoing seventh pandemic (1961 until present day) is caused by V. cholerae serogroup O1 of the El Tor biotype (Faruque et al., 1998). In 1992, the emergence and rapid spread of V. cholerae serogroup O139 from the Indian subcontinent to neighbouring countries was viewed as a possible threat that might initiate an eighth cholera pandemic. At the height of the O139 outbreak, the Indian subcontinent saw a dramatic displacement of the V. cholerae O1 El Tor as the dominant strain. However, rather than being driven to gradual extinction like the classical biotype, an unprecedented turn of events in 1994 saw V. cholerae O1 El Tor regain its predominance over the O139 serogroup and both serogroups continue to cause disease on the Indian subcontinent (Faruque et al., 2003).

that they could not be differentiated into a specific biotype based on their phenotypic traits (Nair et al., 2002). In 2004, a second variant, designated as the Mozambique variant, was found to have typical El Tor phenotypic traits but it genetically harboured a tandem repeat of the classical CTX prophage on the small chromosome . The third, and perhaps the most significant atypical El Tor, was the altered El Tor that is uniquely recognized as carrying the classical cholera toxin while retaining almost all aspects of the prototypic seventh pandemic El Tor strain.
This altered El Tor was initially reported in Bangladesh and growing evidence suggests the wide spread of this variant around the world in recent years Okada et al., 2010;Morita et al., 2010;Sithivong et al., 2010;Ceccarelli et al., 2011). The altered El Tor was reported to have fully displaced prototypic seventh pandemic El Tor strains in several countries, including India and Bangladesh Raychoudhuri et al., 2009). Two different conjectures have been proposed for the emergence and global transmission of atypical El Tor . The emergence of altered El Tor was postulated to be the result of either clonal expansion of a single ancestral El Tor which had acquired the classical ctxB gene in a cholera endemic region or a multiclonal event occurring independently in each region from co-existing El Tor and classical strains. Transnational transmission of altered El Tor was exemplify by the recent 2010 Haiti outbreak which was thought to be introduced by human activity from South Asian countries (Chin et al., 2011) and subsequently spread to United States, Canada and Dominican Republic via importation by travellers from Haiti (CDC, 2010;Gilmour et al., 2011).
The emergence of atypical El Tor marks a significant event in the evolution of V. cholerae and the epidemiology of cholera. The 2009 cholera outbreak strain from Kelantan state on the east coast of peninsular Malaysia was characterized as belonging to the altered El Tor biotype and it carried the classical cholera toxin (ctxB) gene (Ang et al., 2010). The present study further investigated genetic aspects of the Kelantan altered El Tor strain using multiple PCR analysis to elucidate the structure of the CTX prophage and to detect the presence of class I integron and SXT element antibiotic determinants.

V. cholerae strains
A total of 20 V. cholerae isolates belonging to serogroup O1 of the altered El Tor biotype were collected during the 2009 cholera outbreak in Kelantan, Malaysia as described earlier (Ang et al., 2010). All the V. cholerae isolates were revived from glycerol stock and identification was performed using standard biochemical methods (Kay et al., 1994). Serotyping was conducted using slide agglutination tests with polyvalent O1 and monospecific Ogawa and Inaba antisera (Denka Seikan, Japan). All isolates were routinely grown on Luria-Bertani (LB) agar throughout the study.

Genomic DNA preparation
The genomic DNA template for genetic analysis was purified using a NucleoSpin Tissue kit (Macherey-Nagel, Germany), according to the manufacturer's instructions. The purity and concentration of purified genomic DNA was determined using a Biophotometer (Eppendorf, Germany).

rstR typing
The type of rstR gene in each isolate was determined using a set of allele-specific forward primers (rstR El Tor , rstR Classical , rstR Calcutta and rstR Environment ) and a common reverse primer, rstRR (Nusrin et al., 2004;Bhattacharya et al., 2006). A list of all primers used in this study is presented in Table 1.

Genetic analysis of CTX prophage array
Genetic analysis of CTX prophage array was performed using several combinations of primer pairs, as described by Nguyen et al. (2009). The presence of a RS1 element was determined using the primer pair rstCF/rstCR. Investigations of the arrays for RS1 and CTX prophage were performed using two primer pairs: ctxBF/rstCR for the CTX prophage-RS1 array and rstCF4/rstAR for the RS1-CTX prophage array. The presence of tandem repeats of the RS1 element or CTX prophage was determined using the primer pair rstCF4/rstCR4 and ctxBF/cepR, respectively. The chromosomal localization of RS1 and CTX prophage was confirmed using the primer pairs Ch1F/rstRR and ctxBF/Ch1R for the large chromosome and Ch2F/Ch2R for the small chromosome.

rtxC PCR
Detection of the rtxC gene was performed using the primer pair rtxC-F/rtxC-R (Chow et al., 2001).

PCR detection of class I integrons
Detection of class I integrons was performed using a set of primers described by Dalsgaard et al. (2001). Briefly, the primer pairs inDS-F/inDS-B and qacE∆1-F/su1-B were used for the amplification of the 5-CS and 3-CS of the class I integron. The primer pair in-F/in-B was used to amplify gene cassettes inserted in the integron, while the primer pair in-F/aadA-B was used for the amplification of the gene cassette aad1A that encodes streptomycin resistance.

PCR detection of SXT constins
The isolates were screened for the presence of SXT constins (large conjugative elements) using the primer pair INT1/INT2. Presence of the antibiotic resistance genes floR that encodes resistance to chloramphenicol, sulII for resistance to sulfamethoxazole, strB for resistance to streptomycin, and dfrA1 for resistance to trimethoprim, were determined using the primer pairs FLOR-F/FLOR-2, Sul2-F/Sul2-B, strB-F/strB-R, and DFR1-F/DFR1-B, respectively (Hochhut et al., 2001).

Sequencing of tandem repeats of RS1 elements and upstream regions of the CTX prophage
The DNA sequence spanning tandem repeats of RS1 elements and upstream regions of the CTX prophage (~8.5 kb) was generated with the primer pairs Ch1F/rstCR, rstCF4/rstCR4, and rstCF/cepR. Amplicons from each PCR reaction were cloned into the pCR4-TOPO vector (Invitrogen, CA) and sequenced by First Base Laboratories Sdn. Bhd. (Malaysia). The sequencing data was assembled and complete nucleotide sequences of the RS1-RS1-CTX prophage arrays for isolates 03/09-KB and 27/09-KB were deposited in GenBank under the accession numbers JN545744 and JN545745.

Results
All the revived isolates were identified as V. cholerae biotype El Tor using standard biochemical tests and slide agglutination tests showed they belonged to serogroup O1 of the Ogawa serotype.

rstR typing
The rstR typing by PCR amplification of the 501 bp amplicon using the primer pair rstR El Tor /rstRR showed that all isolates possessed only the El Tor type rstR ( Fig. 1). No amplicon was produced for other allele-specific primers among all isolates analyzed, which indicated the absence of classical, Calcutta, and environmental type rstR.

Genetic analysis of the CTX prophage array
The presence of RS1 element was confirmed using the primer pair rstCF/rstCR to amplify a 197 bp region of rstC gene from all the isolates (Fig. 2a). The CTX prophage and RS1 element in each isolate was found to be arranged in the form of a RS1-CTX prophage array, as shown www.intechopen.com by the positive amplification of a 1551 bp amplicon with the primer pair rstCF4/rstAR (Fig. 2b). However, no additional RS1 element was found downstream of the CTX prophage because no amplicon was generated by the primer pair ctxBF/rstCR. The primer pair rstCF4/rstCR4 indicated the tandem arrangement of the RS1 element through the amplification of a 2629 bp amplicon, whereas no amplicon was produced for the primer pair ctxBF/cepR which indicated the presence of only a single CTX prophage. The location of the RS1-RS1-CTX prophage array on the large chromosome was verified using the primer pairs Ch1F/rstRR and ctxBF/Ch1R which amplify fragments corresponding to the upstream and downstream regions of the RS1-RS1-CTX prophage array found on the large chromosome. The absence of an RS1 element or CTX prophage on the small chromosome was confirmed through the amplification of a 910 bp amplicon using the primer pair Ch2F/Ch2R. www.intechopen.com

rtxC PCR
PCR analysis of rtxC showed that all the isolates yielded a 263 bp amplicon of the rtxC gene (Fig. 3).

PCR detection of class I integrons
None of the isolates were positive for class I integrons, because no PCR amplicons were obtained with all the primer pairs tested.

PCR detection of SXT constins
All the isolates were shown to be positive for SXT element by the amplification of a 592 bp amplicon with the primer pair INT1/INT2 (Fig. 3). These isolates also had positive PCR results for the SulII, strB, and dfrA1 genes, because 626 bp, 470 bp, and 372 bp amplicons were generated for the respective genes. None of the isolates were positive for floR genes.

Discussion
In November 2009, Kelantan was struck by a cholera outbreak that marked the reemergence of this secretory diarrheal disease in the eastern state after years of absence. The aetiological agent was later found to be V. cholerae O1 of the altered El Tor biotype and this discovery indicated the first reported appearance of the atypical El Tor strain on Malaysian soil (Ang et al., 2010). Various genetic studies have been undertaken to gain insights into the evolution of this predominant atypical strain and research into the altered El Tor strain has primarily been directed towards the CTX prophage encoding the classical cholera toxin gene Lee et al., 2009).

www.intechopen.com
The CTX prophage found in the genome of pathogenic V. cholerae strains is actually an integrated form of a lysogenic filamentous bacteriophage known as the CTX phage (CTX). CTX is approximately 7 kb in length and it is composed of a 4.6 kb core region with a 2.4 kb RS2 region . The core region contains genes that encode for proteins involved in phage morphogenesis, specifically the core-encoded pilin (cep), pIII CTX (previously known as orfU), accessory cholera enterotoxin (ace), and zonula occludens (zot) genes. This core region also contains genes encoding for cholera toxin, so the acquisition of CTX is viewed as virulence acquisition by a host cell. The RS2 region complements the core, because it contains genes that enable the replication (rstA), integration (rstB), and regulation (rstR) of CTX. The RS1 element is another RS2-like element that is frequently found adjacent to the CTX prophage. The RS1 element is a 2.7 kb satellite phage that only differs from RS2 by an additional gene designated rstC, which encodes for a novel antirepressor to the RstR protein (Waldor et al., 1997;Heilpern & Waldor, 2003). The RS1 element provides a dual function by promoting the transcription of phage genes via an interaction between RstC and RstR, as well as enabling the replication of an adjacent CTX prophage to produce infective phage particles (Davis et al., 2002).
The rstR regulatory gene sequence found in the RS2 region also determined the type of CTX prophage carried by a V. cholerae strain. Three types of CTX prophage has been established to date, i.e., the classical CTX prophage and El Tor CTX prophage that were first detected in the V. cholerae serogroup O1 of the respective biotypes, and the Calcutta CTX prophage from the epidemic-causing serogroup O139 (Kimsey et al., 1998). A fourth type, the Mozambique CTX prophage, was proposed by Choi et al. (2010) and described based on the inclusion of other genetic features of the CTX prophage, including intergenic sequences and the rstA gene, although the CTX prophage contained a classical rstR gene. In the present study, the CTX prophage from the 2009 Kelantan cholera outbreak strain was found to be regulated by an El Tor type rstR repressor gene, but it carried a classical type ctxB gene. A CTX prophage with this combination of El Tor type rstR and classical cholera toxin gene was also designated as a hybrid CTX prophage by Grim et al. (2010). Further genetic analysis of the CTX prophage structure revealed that all isolates harboured a RS1-RS1-CTX prophage array, which was integrated on the large chromosome. As found in the prototypic seventh pandemic El Tor strains, no RS1 element or CTX prophage was integrated on the small chromosome. The RS1-RS1-CTX prophage array of the Kelantan variant represents a novel arrangement for these genetic elements among atypical El Tor strains. To the best of our knowledge, no RS1-RS1-CTX prophage array with an El Tor type rstR on the large chromosome has been demonstrated or reported elsewhere among the altered El Tor biotypes.  . Recently, the same RS1-CTX prophage array was identified in the altered El Tor isolates from Angola, Africa in 2006 (Ceccarelli et al., 2011) and from Hyderabad, India in 2009 . A study conducted by Goel et al. (2011) found that one of the altered El Tor isolates (VCH35) from Hyderabad, India harboured a tandem repeat of the CTX prophage in the small chromosome in addition to a RS1-CTX prophage array in the large chromosome. Although rstR typing of VCH35 revealed the presence of both El Tor and classical type rstR, the localization of these rstR alleles in the multiple CTX prophages was not confirmed. Various combinations of CTX prophage arrays have been documented, but the RS1-CTX prophage array appears to be the most frequently reported arrangement in altered El Tor strains associated with cholera outbreaks . Fig. 4 provides a diagrammatic comparison of various CTX prophage arrays, including the classical, prototypic seventh pandemic El Tor, and altered El Tor.
One of the main issues accompanying the emergence of atypical El Tor is standardization of the nomenclature and the classification scheme used when referring to these variants. This is further complicated by the fact that the current genotypic and phenotypic diversity reported among atypical El Tor strains is only the tip of the iceberg, because there are frequent new reports. In 2009, Lee et al. proposed the classification of atypical El Tor strains into two groups based on genetic differences in their RS1 element and the CTX prophage structure on each chromosome. Group I represents atypical El Tor strains with a tandem repeat of classical CTX prophage on the small chromosome, while Group II represents those possessing the RS1 and CTX prophage with El Tor type rstR and classical ctxB on the large chromosome. Based on these criteria, the Matlab and Mozambique variants were classified into Group I, while altered El Tor, such as those described by Nguyen et al. (2009), fell into Group II. This classification system was also used by Goel et al. (2011) to categorize the VCH35 isolate from Hyderabad, India into Group I, because it carried a tandem repeat of CTX prophage on the small chromosome. The type of rstR gene determines the type of corresponding CTX prophage, so a minor discrepancy when adhering to this classification system arises when both El Tor and classical type rstR are present, as is the case with VCH35. Therefore, the exact nature of the CTX prophages in tandem arrangements needs to be elucidated to ascertain whether VCH35 truly belonged to Group I. The existence of VCH35 also questions the possible need for a subgroup within Group I, should further analysis of VCH35 reveal the presence of both an El Tor and classical type CTX prophage in a tandem arrangement. In-depth genetic analysis of VCH35 is highly warranted before any conclusions can be drawn on its classification. In contrast, only one array of the RS1-CTX prophage has been reported in Group II ). Therefore, we were able to describe a new type of array belonging to Group II based on the findings of this study. An arrangement of RS1 in tandem repeats followed by CTX prophage with an El Tor type rstR and classical ctxB was characterized in this study. This suggests that more varieties of the CTX prophage array may exist among the altered El Tor than are currently known.
The current study revealed an El Tor type rstR in the CTX prophage, but we also reported in our previous study that the 2009 Kelantan outbreak strain carried the El Tor type tcpA gene allele. In order to substantiate the El Tor lineage of this strain, we performed additional PCR analysis on the repeat in the toxin (RTX) gene cluster. The RTX gene cluster in V. cholerae was first identified and characterized in 1999 and it was found to be physically linked to the downstream region of the CTX prophage. The RTX gene cluster consists of rtxA, rtxB, rtxC, and rtxD genes, and it is responsible for the cytotoxic activity of V. cholerae in mammalian cells in vitro. However, gene deletions in the RTX gene cluster (specifically the rtxC gene and the downstream region of the rtxA gene) were noted among the classical biotype of V. cholerae O1, which resulted in defective production of cytotoxic activity (Lin et al., 1999). Based on this observation, Chow et al. (2001) developed a PCR assay targeting the rtxC gene www.intechopen.com for biotyping V. cholerae serogroup O1. We used this biotyping assay and found that all the isolates possessed the rtxC gene. This provided further evidence that the El Tor genomic backbone was preserved in the Kelantan altered El Tor strain.
The emergence of multidrug-resistant V. cholerae strains has been documented frequently in recent years (Kiiru et al., 2009;Jain et al., 2011) and this phenomenon has led to repeated calls for the more prudent use of antibiotics by the global community. Multidrug-resistant V. cholerae have been reported from Malaysia and the antibiogram profile of the Kelantan outbreak strain showed that the isolates were resistant to various antibiotics, including, tetracycline, erythromycin, sulfamethoxazole-trimethoprim, streptomycin, penicillin G, and polymyxin B. However, they were susceptible to ciprofloxacin, norfloxacin, chloramphenicol, gentamicin, and kanamycin (Ang et al., 2010). Therefore, we investigated the phenotypes of the outbreak strain by characterizing the corresponding genes encoding for antibiotic resistance. Amita et al. (2003) studied antibiotic resistance genes in V. cholerae O1 and showed that the class I integron carrying the aadA1 gene cassette was prevalent in strains isolated before 1992, whereas the SXT element was prevalent in strains isolated after 1992. Integrons are characterized by the presence of an integrase gene (intI) that mediates recombination between the attI site found on the integron and the attC site on the gene cassette. The insertion of a gene cassette into the integron results in the expression of functional proteins using a promoter found in the integron (Recchia & Hall, 1995). In agreement with Amita et al. (2003), PCR analysis of the Kelantan outbreak strain showed it was negative for the class I integron and the gene cassette aadA1 encoding resistance to streptomycin and spectinomycin.
In one of the most remarkable events in the recorded history of cholera, a novel serogroup of V. cholerae emerged in 1992 that was designated O139 and it replaced the O1 El Tor biotype in Bangladesh and the Indian subcontinent where it became the dominant strain, although its reign was short-lived (Faruque et al., 2003). The V. cholerae serogroup O139 differed from serogroup O1 in having a different somatic antigen and it was also uniquely characterized by its antibiotic resistance to sulfamethoxazole, trimethoprim, chloramphenicol, and streptomycin. Interestingly, a distinctive pattern of antibiotic resistance was found after the re-emergence of serogroup O1 El Tor in 1994 where all the El Tor strains were found to be resistant to these four antibiotics, which strikingly resembled the profile of serogroup O139 . The corresponding antibiotic resistance genes in serogroup O1 El Tor were collectively referred to as ICEVchInd1 (previously known as SXT ET ) and they were genetically closely related to the SXT MO10 , which encodes for antibiotic resistance to the same four antibiotics mentioned above by serogroup O139. Based on a comparative DNA analysis, both the ICEVchInd1 and the SXT MO10 elements were considered to be derived from a common precursor (Hochhut et al., 2001;Burrus et al., 2006). The results of PCR conducted on the SXT constin in this study showed that all the Kelantan outbreak strains contained the SXT element. Genes conferring resistance to sulfamethoxazole (SulII), trimethoprim (dfrA1), and streptomycin (strB) were detected in all isolates, with the exception of the chloramphenicol resistance gene (floR). The detection of the antibiotic genes was consistent with the findings in the phenotypic antibiotic susceptibility testing and the SXT constin detected in the present outbreak strain appeared to have a deletion of the floR gene, when compared with the STX ET reported from elsewhere (Hochhut et al., 2001). The SXT constin without the floR gene represented a variant of the SXT ET constin and, other than this study, the SXT variant has also been found among altered El Tor strains from India (Goel & Jiang, 2010). It was recently reported that an altered El Tor strain carrying both an integron and an SXT element had been identified among outbreak strains from Solapur, India . These findings are important, because the management of cholera patients usually entails fluid replacement therapy to replace the electrolytes lost during profuse diarrheal bouts. However, antibiotic therapy serves as an adjunct to fluid replacement therapy to reduce the duration of the disease and the excretion of the bacterium (Lindenbaum et al., 1967). Thus, continuous monitoring of changes in antibiotic resistance patterns is highly recommended, because the SXT constin harbouring various antibiotic resistance genes can be acquired easily via lateral gene transfer (Iwanaga et al., 2004).

Conclusion
Genetic analysis performed on the Kelantan altered El Tor strain isolated during a cholera outbreak in 2009 revealed a novel CTX prophage array where a tandem repeat of the RS1 element was found upstream of the CTX prophage on the large chromosome. This is the first report of a RS1-RS1-CTX prophage array among altered El Tor strains that fit into Group II according to the classification system of Lee et al. (2009). All isolates carried the SXT constin and we identified genes conferring resistance to sulfamethoxazole, trimethoprim and chloramphenicol, which correlated with their phenotypic expression in the antibiogram profile.
The emergence of the altered El Tor is viewed as an evolutionary optimization of V. cholerae strains in the development of a successor to the current cholera pandemic. The altered El Tor strains are poised to become epidemiologically dominant and they might hold the key to sustaining the current seventh pandemic. Equipped with the unique characteristics of the classical and El Tor biotypes, the altered El Tor has already been associated with more severe cases of cholera (Siddique et al., 2010) and it appears to be widely disseminated around the globe. The research community should actively unravel the wealth of knowledge that lies within these atypical El Tor strains and gain a better understanding that can be translated into measures to combat and conquer cholera.