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1 Department of Biology and Chemistry, City University of Hong Kong, 83 Kowloon Tong, Kowloon, Hong Kong SAR
2 International Centre for Diarrhoeal Disease Research, Bangladesh, Dhaka 1212, Bangladesh
3 Public Health Microbiology Laboratory, Forensic and Scientific Services, Cooper, Coopers Plains, Queensland, Australia
4 Department of Microbiology and Immunology, Faculty of Tropical Medicine, Mahidol University, 420/6 Rajvithi Road, Bangkok 10400, Thailand
5 Molecular Microbiology and Molecular Immunology Laboratories, Faculty of Allied Health, Thammasat University, Pratumatance 12121, Thailand
6 National Institute of Cholera and Enteric Diseases, Kolkata, India
Correspondence
Ashrafus Safa
mrpsafa{at}yahoo.com
Received June 21, 2008
Accepted September 5, 2008
Episodes of cholera stemming from indigenous Vibrio cholerae strains in Australia are mainly associated with environmental sources. In the present study, 10 V. cholerae O1 strains of Australian origin were characterized. All of the strains were serogroup O1 and their conventional phenotypic traits categorized them as belonging to the El Tor biotype. Genetic screening of 12 genomic regions that are associated with virulence in V. cholerae showed variable results. Analysis of the ctxAB gene showed that the Australian environmental reservoir contains both toxigenic and non-toxigenic V. cholerae strains. DNA sequencing revealed that all of the toxigenic V. cholerae strains examined were of ctxB genotype 2. Whole genome PFGE analysis revealed that the environmental toxigenic V. cholerae O1 strains were more diverse than the non-toxigenic environmental O1 strains, and the absence of genes that make up the Vibrio seventh pandemic island-I and -II in all of the strains indicates their pre-seventh pandemic ancestry.
Abbreviations: CCA, chicken blood cell agglutination; MSHA, mannose-sensitive haemagglutinin; VSP, Vibrio seventh pandemic island.
The GenBank/EMBL/DDBJ accession numbers for the ctxB gene sequences of V. cholerae O1 and O139 are EU828583–EU828588.
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INTRODUCTION |
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 TOP INTRODUCTION METHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSReferences |
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) is now known (Waldor & Mekalanos, 1996). Allelic variations in the different virulence-associated genes of integrated CTX and chromosomal origin in V. cholerae strains have already been reported and used as important markers to differentiate V. cholerae at the strain level (Rader & Murphy, 1988; Faast et al., 1989; Kimsey et al., 1998; Bhattacharya et al., 2006). The discovery of the self-transducing ability of the RS1 satellite phage and the exclusive presence of the rtxC gene of the RTX cluster in El Tor strains are a few of the other significant observations made in previous years (Lin et al., 1999; Faruque et al., 2002). Recently, two genomic islands, Vibrio seventh pandemic island (VSP)-I and -II, were shown to be unique to the El Tor strains of the seventh pandemic, that is, they were absent from both the pre-seventh pandemic El Tor strains and the classical biotype strains (Dziejman et al., 2002). These two islands are believed to be associated with the pandemic tendency of the seventh pandemic El Tor Vibrio strains (Faruque & Mekalanos, 2003). The first six pandemics of cholera were recorded between 1817 and 1923. The sixth and, presumably, the fifth pandemics were caused by V. cholerae O1 of the classical biotype. In 1961, the seventh pandemic, caused by the V. cholerae O1 El Tor biotype, began on the island of Sulawesi in Indonesia (Kaper et al., 1995) and has continued until today. Between the end of the sixth pandemic and the start of the seventh, there was an intervening period of 38 years during which time cholera did not spread in the form of a pandemic, but occurred intermittently or caused local outbreaks. The V. cholerae O1 strains isolated during this period are referred to as pre-seventh pandemic El Tor strains (Byun et al., 1999). The second cholera-causing serogroup, O139, was identified in late 1992 (Shimada et al., 1993). V. cholerae O139 initially spread rapidly and caused several outbreaks of cholera on the Indian subcontinent, but cholera due to O139 strains has recently declined, although they continue to coexist with strains of the O1 serogroup (Albert & Nair, 2005).
Australia recorded cholera just 15 years after the first pandemic began in India in 1831 (Barua, 1992), although the disease only persisted for a short time. During the seventh pandemic, apart from a few imported cases, Australia had no indigenous cases of cholera until the mid-1970s (Sutton, 1974). Following the first indigenous cholera case in south-east Queensland in 1977, Australia started to report sporadic isolation of toxigenic El Tor strains of V. cholerae O1 from patients suffering from diarrhoea (Rao & Stockwell, 1980; Rogers et al., 1980). Epidemiological investigations showed that all of these cases were directly or indirectly associated with the river waters of Australia and suggested that these water bodies may serve as a reservoir of V. cholerae (Blake, 1994). Molecular typing using techniques such as multilocus enzyme electrophoresis, ribotyping and the DNA sequencing of the ctxB gene showed that the Australian toxigenic V. cholerae strains were indigenous and likely to be clonal (Olsvik et al., 1993; Popovic et al., 1993; Wachsmuth et al., 1993).
Given the indigenous nature and genetic disparities of the Australian strains, compared with other clones of V. cholerae, we adopted a PCR-based multilocus gene profiling approach using the most current knowledge from V. cholerae research to characterize Australian V. cholerae strains of environmental and clinical origin.
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METHODS |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSReferences |
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PCR and serogrouping. All strains were confirmed as V. cholerae by ompW-specific PCR as described by Nandi et al. (2003). Serogrouping and serotyping were confirmed by serology using polyvalent O1 and monovalent Inaba- and Ogawa-specific antisera, and by using the O1 (rfbO1), O139 (rfbO139) and cholera toxin (ctxAB) gene-specific multiplex PCR assay described by Hoshino et al. (1998). PCR reagents and kits were obtained either from Perkin-Elmer or Invitrogen. PCR products were analysed by electrophoresis in 1 % agarose gels, which were stained with ethidium bromide and visualized using the Gel Doc 2000 gel documentation system (Bio-Rad).
Biotyping. All strains examined were tested for susceptibility to polymyxin B (50 U), chicken blood cell agglutination (CCA) and sensitivity to group IV (classical) and group 5 (El Tor) phages using standard procedures (WHO, 1987). To complement the biotyping tests, PCR assays targeting the tcpA (classical and El Tor variants) (Keasler & Hall, 1993) and rstR genes were performed using procedures described previously (Nusrin et al., 2004; Safa et al., 2006).
Isolation of genomic DNA. Genomic DNA was extracted from overnight cultures in Luria–Bertani (LB) broth using standard methods (Sambrook et al., 1989) with slight modifications. Briefly, cells were harvested by centrifugation and the pellets were resuspended in TES buffer (10 mM Tris/HCl, pH 8.0, 10 mM EDTA, 100 mM NaCl, 10 % SDS) and incubated at 65 °C for 10 min. Following proteinase K treatment at 50 °C for 18 h, DNA was extracted with phenol/chloroform/isoamyl alcohol (25 : 24 : 1), precipitated in ethanol and DNA dissolved in TE buffer (10 mM Tris/HCl, 1 mM EDTA, pH 8.0). RNase treatment was performed at 37 °C for 1 h, and ethanol-precipitated DNA was dissolved in TE buffer and stored at –20 °C.
Multilocus PCR analysis.
PCR-based multilocus genetic screening was performed on 12 V. cholerae O1 isolates, including classical and El Tor reference strains, by PCR amplification of internal fragments of 12 virulence-associated genes and/or gene clusters [VSP-I, VSP-II, mannose-sensitive haemagglutinin (MSHA) pilin, HlyA, VPI-1, VPI-2, pilE, RTX, RS1, CTX, toxin-linked cryptic plasmid (tlc) and class 1 integron (intl4)]. The PCR conditions and 32 pairs of primers used in this analysis were previously described by Chow et al. (2001) and O'Shea et al. (2004).
Nucleotide sequencing of ctxB. PCR amplification of ctxB was performed in a 25 µl reaction mixture in a Peltier Thermal Cycler (PTC-200; M. J. Research). The PCR primers and conditions used were as described by Mitra et al. (2001). PCR products were purified with a Microcon centrifugal filter device (Millipore) according to the manufacturer's instructions, and cycle sequencing was performed in a 20 µl reaction mixture containing 40 ng purified PCR product, 4 µl ready reaction mixture and 3.2 pmol each primer. After 25 cycles of amplification, unincorporated nucleotides were removed by ethanol precipitation. Dried template was resuspended in template suppression reagent and sequencing was performed using an automated 310 DNA sequencer (Applied Biosystems). DNA chromatograms were inspected using Chromas 2.23 (Technelysium). Nucleotide sequences of six representative test isolates (data not shown) were compared with the corresponding sequences of the N16961 El Tor and 569B classical reference strains (GenBank accesssion numbers NC_002505 and U25679, respectively) using BLAST (Altschul et al., 1997). Multiple sequence alignments were developed using CLUSTAL_X version 1.81.13 and DNA sequences were translated using GeneDoc version 2.6.002 alignment editor.
PFGE. Intact agarose-embedded chromosomal DNA was prepared from V. cholerae O1 isolates and PFGE was performed using a contour-clamped homogeneous electric field (CHEF-Mapper) apparatus (Bio-Rad) according to the PulseNet V. cholerae subtyping protocol (Cooper et al., 2006). Genomic DNA was digested with NotI (10 U µl–1 stock; Invitrogen) and DNA fragments were separated in 1 % SeaKem Gold agarose in 0.5x Tris/borate/EDTA buffer. Genomic DNA of Salmonella braenderup H9812 digested with 40 U XbaI (Invitrogen) was used as a size marker. Following electrophoresis, gels were stained with ethidium bromide (50 µg ml–1) for 30 min and destained with reagent grade water. Images were captured using the Gel Doc 2000 and Gel Doc XR systems (Bio-Rad).
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RESULTS AND DISCUSSION |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSReferences |
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. Except for the clinical isolate, Cis77, all of the strains were resistant to polymyxin B (50 U) and agglutinated with chicken blood cells, which are typical traits of the El Tor biotype. Eight of the 10 strains were resistant to classical phage IV but sensitive to El Tor phage 5, and the remaining two were resistant to both phages (Table 1). The PCR-based multilocus genetic analysis of the 12 regions, namely VSP-I, VSP-II, MSHA pilin, hlyA, VPI-1, VPI-2, pilE, RTX, RS1, CTX, tlc and intl4, accounted for approximately 165 kb of the V. cholerae genome. In the VPI-1 gene cluster, the major virulence-associated genes, tcpA, toxT and acfB, were present in seven isolates but absent in three (Tables 1 and 2). These genes are mainly associated with the colonization that occurs prior to infection. Most of the pathogenic strains of V. cholerae that were isolated during the sixth, seventh, and pre-seventh pandemics were shown to possess these genes (O'Shea et al., 2004). The MSHA cluster encodes a type-IV pilus and is expressed by strains of the El Tor biotype, but rarely by those of the classical biotype (Jonson et al., 1991). All of the genes of the MSHA gene cluster were present in all of the Australian strains (data not shown). The stn gene of the VPI-2 gene cluster encodes a heat-stable enterotoxin in V. cholerae strains, which is believed to be present only in non-O1 strains of V. cholerae. Later study showed that stn can also be present in toxigenic V. cholerae O1 strains, although this occurs less frequently in O1 strains than it does in non-O1 strains (Takeda et al., 1991). Interestingly, most of the ctxAB-positive V. cholerae O1 strains were positive for stn, except for 6732/80 and Cis77. The RTX gene cluster encodes one kind of cytotoxin, and toxin activity is regulated by the rtxC gene (Lin et al., 1999). It was demonstrated that some regions of the RTX gene cluster, including rtxC, are absent in the genome of the classical strains, whereas the cluster is intact in strains of the El Tor biotype (Lin et al., 1999; Dziejman et al., 2002). We tested two genes of this cluster, rtxC and rtxA, and found that both were present in all of the strains, including the VPI-1-negative strains (Table 2). RS1 usually flanks the entire CTX genome, i.e. the core and the RS2 region. RS2 and RS1 are almost the same; the major difference between them is that the latter possesses an additional gene, rstC. This gene is unique to strains of the El Tor biotype; it is absent in classical strains (Waldor et al., 1997). In this study, only four strains were positive for rstC. The remaining six, including the clinical strain, were negative (Table 2). The rstC gene provides antirepressor activity to the phage replication process, but the impact of its absence on the cellular process is not currently well-understood (Davis & Waldor, 2003). Overall observations indicate that these strains are virulent and have similarities to the V. cholerae strains of the El Tor biotype.
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genome showed that the major toxin-producing gene, ctxAB, is present in the seven strains and thereby classified as toxigenic. Other CTX-encoded genes were also present in these seven toxigenic strains (Table 2
), which suggests that there is an intact core region. In contrast, most of the genes, including ctxAB, were absent in three strains, 12129(1), 220386, and P358, representing a truncated phage genome. These non-toxigenic strains possess only the zot gene, which is known to play an important role in phage assembly. Such non-toxigenic strains are sporadically isolated from tourists with gastrointestinal illness, which indicates that they may have the potential to initiate gastrointestinal symptoms but not cholera (Kaper et al., 1995). The diversity of CTX among the biotypes is mainly due to variations in the repeat sequence elements, particularly in the rstR gene region. This gene encodes a regulatory aspect of phage replication, and four variants have been reported to date, rstRclassical, rstREl Tor, rstRCalcutta and rstRenvironmental. These variants were named after the biotype, source or location of the strains in which they were first identified. An assortment of different alleles of the rstR gene in a single V. cholerae strain was first reported in 1998 (Kimsey et al., 1998). In this study, all seven toxigenic strains were positive for the rstRclassical and/or rstRenvironmental type, and the remaining two types of the rstR gene were not found in any of the strains tested (Table 2). Since the beginning of the seventh pandemic, classical strains have gradually been replaced by the El Tor biotype, and none has been reported recently. It is thus interesting to find classical-type rstR genes in El Tor-like strains. These may have been horizontally transferred from the classical strains either before their extinction or from an unknown gene pool. The presence of rstRenvironmental in the V. cholerae strains of environmental origin may be considered to be further confirmation of their source of isolation. Finally, we made an effort to analyse these strains using the two most recently identified gene clusters, VSP-I and -II (Dziejman et al., 2002), which are unique to the seventh pandemic El Tor strains but absent in pre-seventh pandemic El Tor strains. PCR analysis revealed that both of the clusters were absent in most of the test strains, including the classical reference strain. They were present only in the N16961 El Tor reference strain (Table 2). The absence of VSP-I and -II in most of the strains indicates their similarity to the pre-seventh pandemic strains that prevailed after the sixth pandemic.
To understand the clonal relationship, all 10 of the V. cholerae O1 strains isolated from Australia were analysed by PFGE. NotI digestion restricted the chromosomal genome to 17–22 fragments, and these fragments ranged from 6 to 350 kb. Based on the banding pattern, five major pulsotypes, designated A, B, C, D and E, were observed among the 10 strains (Fig. 1). Pulsotype A was further divided into four subtypes, designated A1–A4, based on minor banding differences (Fig. 1). A1 was the most prevalent subtype of the four, whereas only the clinical isolate Cis77 showed subtype A3. PFGE analysis revealed that the environmental toxigenic V. cholerae O1 strains were less diverse than the non-toxigenic environmental O1 strains (Fig. 1). The clinical strain, Cis77, showed significant similarity to those of environmental origin, which is an indication of possible genetic relatedness between strains from two different sources. Heterogeneity within the ctxB gene and translated protein was first reported in the early 1990s and based on this, three ctxB genotypes of the V. cholerae O1 strains have been identified (Olsvik et al., 1993). Based on amino acid residue substitution at positions 39, 46 and 68, all of the classical and US Gulf Coast El Tor strains have been categorized as genotype 1, the Australian El Tor strains as genotype 2 and the El Tor strains of the seventh pandemic and Latin American epidemic as genotype 3. The ctxB sequencing data in this study are consistent with previous findings (Olsvik et al., 1993) that Australian V. cholerae strains are local inhabitants of the Australian environment and display genotype 2, which is different from genotypes 1 and 3.
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ACKNOWLEDGEMENTS |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSReferences |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSReferences |
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. Infect Immun 70, 163–170. and evolution of new pathogenic Vibrio cholerae. Lancet 352, 457–458.[Medline] array and its position in the genome. Microbiology 149, 89–97. are encoded by region RS2. Mol Microbiol 24, 917–926.[CrossRef][Medline]This article has been cited by other articles:
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  J. Chun, C. J. Grim, N. A. Hasan, J. H. Lee, S. Y. Choi, B. J. Haley, E. Taviani, Y.-S. Jeon, D. W. Kim, J.-H. Lee, et al. Comparative genomics reveals mechanism for short-term and long-term clonal transitions in pandemic Vibrio cholerae PNAS, September 8, 2009; 106(36): 15442 - 15447. [Abstract] [Full Text] [PDF]
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