|Year : 2012 | Volume
| Issue : 3 | Page : 285-289
Presence of CTX gene cluster in environmental non-O1/O139 Vibrio cholerae and its potential clinical significance
B Bakhshi1, H Mohammadi-Barzelighi2, A Sharifnia3, A Dashtbani-Roozbehani3, M Rahbar4, MR Pourshafie3
1 Department of Bacteriology, Tarbiat Modares University, Tehran, Iran
2 Antimicrobial Resistance Research Center, Tehran University of Medical Sciences, Tehran, Iran
3 Department of Bacteriology, Pasteur Institute of Iran, Tehran, Iran
4 Center for Disease Control, Ministry of Health and Medical Education, Tehran, Iran
|Date of Submission||12-Jan-2012|
|Date of Acceptance||03-Apr-2012|
|Date of Web Publication||8-Aug-2012|
M R Pourshafie
Department of Bacteriology, Pasteur Institute of Iran, Tehran
Source of Support: The study was funded by Pasteur Institute of Iran grant numbers 170 and 312., Conflict of Interest: None
Purpose: The aim of this study was to understand the epidemiological linkage of clinical and environmental isolates of Vibrio cholerae and to determine their genotypes and virulence genes content. Materials and Methods: A total of 60 V. cholerae strains obtained from clinical specimens (n = 40) and surface waters (n = 20) were subjected to genotyping using PFGE and determination of their virulence-associated gene clusters. Result: PCR analysis showed the presence of chromosomally located hly and RTX genetic elements in 100% and 90% of the environmental isolates, respectively. The phage-mediated genetic elements such as CTX, TLC and VPI were detected in 5% of the environmental isolates suggesting that the environmental isolates cannot acquire certain mobile gene clusters. A total of 4 and 18 pulsotypes were obtained among the clinical and environmental V. cholerae isolates, respectively. Non-pathogenic environmentally isolated V. cholerae constituted a distinct cluster with one single non-O1, non-O139 strain (EP6) carrying the virulence genes similar to the epidemic strains. This may suggest the possible potential of conversion of non-pathogenic to a pathogenic environmental strain. Conclusions: The emergence of a single environmental isolate in our study containing the pathogenicity genes amongst the diverse non-pathogenic environmental isolates needs to be further studied in the context of V. cholerae pathogenicity sero-coversion.
Keywords: Epidemiology, genotyping, Iran, Vibrio cholerae
|How to cite this article:|
Bakhshi B, Mohammadi-Barzelighi H, Sharifnia A, Dashtbani-Roozbehani A, Rahbar M, Pourshafie M R. Presence of CTX gene cluster in environmental non-O1/O139 Vibrio cholerae and its potential clinical significance. Indian J Med Microbiol 2012;30:285-9
|How to cite this URL:|
Bakhshi B, Mohammadi-Barzelighi H, Sharifnia A, Dashtbani-Roozbehani A, Rahbar M, Pourshafie M R. Presence of CTX gene cluster in environmental non-O1/O139 Vibrio cholerae and its potential clinical significance. Indian J Med Microbiol [serial online] 2012 [cited 2019 Dec 11];30:285-9. Available from: http://www.ijmm.org/text.asp?2012/30/3/285/99487
| ~ Introduction|| |
Pathogenic O1 and O139 strains of V. cholerae contain two essential genetic elements, cholera toxin element (CTX) and the vibrio pathogenicity island (VPI) which are involved in coding cholera toxin (CTX) and toxin co-regulated pilus (TCP), respectively. , It has been reported that non-pathogenic environmental V. cholerae strains vary in their virulence gene content. , We have previously shown that different genomic organizations of CTX phage genome and resistance gene elements exist among V. cholerae isolates of clinical and environmental origin in Iran, ,, but the genetic linkage among clinical and environmental isolates is still to be explored.
It has been reported that the conversion of non-toxigenic environmental strains into epidemic strains of V. cholerae is possible if they acquire the appropriate set of virulence genes. , Toxin-linked cryptic (TLC) element and repeat in toxin (RTX) cluster are two virulence-associated clusters involved in pathogenesis of the epidemic strains. These two clusters flank the CTX integrating site on chromosome 1 in the V. cholerae genome. 
A new epidemic strain, carrying O139 rather than O1 antigen, emerged in southern Asia in 1992.  Genomes of O139 and O1 El Tor strains are closely related in most parts with difference in O-antigen genes, which may suggest the transfer of O139-specific genes from an unknown donor into a recipient El Tor strain.  This postulated mechanism of conversion emphasizes the need to monitor the environmental isolates and their virulence gene content.
Different typing methods including pulsed-field gel electrophoresis (PFGE) and PCR-based methods have been used in the epidemiological investigations in order to trace the origin and geographical distribution of V. cholerae. Data on the persistence and spread of potential epidemic strains of V. cholerae O1 and comparative analysis of clonal relationships among environmental and clinical strains collected during epidemic and inter-epidemic periods are essential in understanding of the factors that might be involved in the emergence of the pathogenic strains.
The aim of this study was to analyse the epidemiological linkage of pathogenic and environmental strains by genotyping and to investigate the virulence factors in the clinical and environmental strains of V. cholerae isolates in Iran.
| ~ Materials and Methods|| |
Collection and processing of environmental samples
A total of 20 environmental isolates were analysed in this study. All of the environmental isolates were collected in a 3 month period between June and August 2006 from local surface waters in the West, East and the Central Tehran and Qom province where there were no outlets from waste water treatment plants. Sampling from each surface water source was performed three times with the interval of 30 days. Water samples were filtered using Whatman No.1 filter paper and subsequently filtered through a 0.45-μm-pore-size membrane by using vacuum pressure of 15 to 20 lb/in 2 . The membrane was cut into eight pieces and vortexed in 2 ml of 10 mM phosphate-buffered saline (PBS, pH 7.4) for 3 min. One millilitre of the suspension was added to 10 ml of alkaline peptone water (APW) containing peptone (1%, wt/vol) and NaCl (1%, wt/vol, pH 8.5), for enrichment at 37°C with shaking (100 strokes/min) for 16-18 h. Each sample was streaked on thiosulfate-citrate-bile-sucrose agar (TCBS) plates and incubated overnight at 37°C.  The resulting yellow colonies were further examined. The identity of V. cholerae strains was defined by 10 biochemical assays including: oxidase, motility, sucrose and lactose fermentation, growth in tryptone broth with 0% NaCl, arginine dehydrolase, ornithin decarboxylase, methyl red, voges-proskauer and indole test  and confirmed by PCR amplification of 16S-23S rRNA intergenic region specific for V. cholerae.  The V. cholerae strains (n 0= 20) were isolated and subjected to further analysis.
Forty clinical isolates were also obtained during summer epidemics which occurred during 2004, 2005 and 2006 in different provinces of Iran. The specimens were collected on the sterile swabs and were enriched in alkaline peptone water. The suspected samples were then placed in Carry-Blair transport and shipped to the Department of Bacteriology, Pasteur Institute of Iran, Tehran. Biochemical and PCR assays were performed to confirm the identity of V. cholerae strains. , The isolates were identified as described in the above section. A brief description of isolates is depicted in [Table 1].
Serogrouping of all isolates in this study was performed with polyvalent O1 and O139 and monospecific Inaba and Ogawa antisera using slide agglutination test (Mast Diagnostics Ltd., Bootle, Mersey side, UK).
PCR for toxin genes
The presence of ctx and zot (CTX toxin cluster), tcp (VPI pathogenic cluster), wbeT (WBE O antigen cluster), rtxA (RTX gene cluster), tlcR (TLC element) and hly (haemolysin) genes were investigated by PCR [Table 2].
DNA from isolates was obtained by a simplified method whereby one isolated colony was suspended into 200 μl of sterile distilled water and boiled for 5 min and 5 μl of supernatant was used as the template in a PCR reaction.  Each PCR reaction was performed in a 25 μl total reaction volume containing 20 μl sterile water, 2.5 μl 10x Taq polymerase buffer, 0.6 μl MgCl2 (25 mM), 0.3 μl dNTPs (10 mM), 0.5 unit Taq DNA polymerase and 25 pmol of each primer. V. cholerae ATCC 14035 was used as a positive control in each PCR assay. 
Pulsed-field gel electrophoresis
Typing of V. cholerae isolates was performed based on a PulseNet standardized protocol.  Accordingly, NotI restriction enzyme (Roche Diagnostic GmbH, Mannheim, Germany) was used for digestion of the genomic DNA of isolates and was electrophoresed in the condition consisting of: Block 1 with an initial switch time (IST) of 2 s to final switch time (FST) of 10 s and a run time of 13 h, block 2 with an IST of 20 s to a FST of 25 s with a run time of 6 h with a gradient of 6.0 V/cm and an included angle of 120 degree at 14°C. DNA molecular weight size marker for analysis of banding patterns was XbaI-digested Salmonella More Details serotype Branderup H9812. The computer-assisted clustering of isolates based on the Dice similarity coefficient using Gel compare II version 4.0 software (Applied Maths, Sint-Martens-Latem, Belgium) was performed and patterns differing by even 1 band were considered as separate pulsotypes to better compare the virulence cluster bearing types.
| ~ Results|| |
PCR analysis of the isolates revealed extensive genetic diversity. As expected, virulence-associated gene clusters were more dispersed in the isolates from surface water sources than those from clinical strains. PCR analysis of the clinical isolates showed that ctx, zot and tcp genes were present in 38 (95%) of 40 isolates. With the exception of one isolate (5%), all 20 environmental strains lacked the three toxin genes.
The results showed the presence of RTX, TLC, wbe and hly elements in 100%, 95%, 95% and 97.5% of the clinical, and in 90%, 5%, 5% and 100% of environmental isolates, respectively [Table 3].
PFGE analysis of the isolates showed the presence of 4 and 18 pulsotypes among the clinical and environmental isolates, respectively. Two closely related pulsotypes were detected in all 38 clinical O1 V. cholerae isolates differing in only one single band. In general, the majority of non-O1, non-O139 V. cholerae isolates of environmental origin showed distinct pulsotype and cluster with unweighted-pair-group method using average linkages (UPGMA). The results showed the lack of close genetic interrelationship with clinical pulsotypes, even with the clinically isolated non-O1, non-O139 strains [Figure 1].
|Figure 1: UPGMA dendrogram showing PFGE patterns of NotI digested genomic DNA of clinical and environmental V. cholerae isolates. Similarity between isolates was calculated with Dice coefficient and clustering was performed by UPGMA. Each pulsotype has been shown in relation to the source and province of isolation and the presence of virulence associated gene clusters; CP: Clinical Pulsotype, EP: Environmental Pulsotype, C: Clinical, E: Environmental, 1: Tehran, 2: Qom, 3: Zahedan, 4: Golestan. All of the isolates with the same pulsotype showed uniform PCR profi le|
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| ~ Discussion|| |
The presence of chromosomally located hly and RTX genetic elements in the majority of environmental isolates (100% and 90%) in comparison with phage-mediated genetic elements of CTX, TLC and VPI (5% for each) suggests the limitations of the environmental isolates to acquire virulence gene clusters associated with phages and/or the instability of the acquired genes to be translocated within the bacterial chromosome. Chakraborty and colleagues (2000) demonstrated the presence and expression of some of the virulence genes or their homologues in diverse environmental V. cholerae, but none of their strains carried the important virulence factor genes of ctx and tcp together which would only constitute a potential reservoir of virulence genes in the environment and may play an important role in the appearance of pathogenic strains through horizontal gene transfer.  The occurrence of an environmental non-O1, non-O139 strain with a complete set of virulence determinants in our study (ctx+ , tcp+ , RTX+ , TLC+ ) is a representative of strains which may evolve through genetic recombination and/or gene exchange resulting into a new pathogenic variant.
The disability of environmental non-O1, non-O139 strains to acquire a complete or partial set of virulence genes from phages might be due to bacterial serogroup specificity. It can be postulated that since serotype variation may result in changes in V. cholerae surface structure, this could, in turn, affect the phage gene transfer. Further investigations are needed to understand whether this aptitude is restricted and related to certain V. cholerae genotypes.
The two clinical non-O1, non-O139 strains were obtained from the sporadic cases only, which propose two hypothesis: (i) pathogenic non-O1, non-O139 strains can hardly expand into epidemics and are less compatible to ecological state in Iran; (ii) the overall chromosomal nature of the clinical non-O1, non-O139 strains differ from the clinical O1 strains by more than three bands may render them unable to develop epidemics.
Our previously published data indicated the presence of identical pulsotypes among the clinical strains V. cholerae O1 in Iran and other countries including Pakistan and Hong Kong. , Accordingly, these sporadically isolated strains are unlikely to have been spread across these distant countries; rather it may have been discretely emerged from the same pulsotype from non-pathogenic environmental V. cholerae in these countries.
Among the total environmental non-O1, non-O139 serotype strains of V. cholerae, one isolate (EP6) showed a complete set of pathogenic genes like the clinical strains. As with all environmental, this EP6 isolate was obtained during non-epidemic cholera period of 2006 which, in turn, might represent a precursor of clone leading to clinical dissemination of epidemic V. cholerae. The recognition of such strains and tracking of their local and/or global prevalence may help in understanding the factors involved in the initiation of a cholera epidemic. The appearance of serogroup O139 is an example of a non-O1 V. cholerae strain which caused global warning of a pandemic strain capable of acquisition new set of genes mediating the synthesis of O139 LPS and polysaccharide capsule.  Furthermore, the Sudan strain of serogroup O37 which emerged in 1968 is another novel example of a local genotype causing a large outbreak.  This strain was later found to be closely related to the classical O1 strain with a epidemic traits acquired through horizontal exchange of genes between O1 and non-O1 strains. 
The appearance of EP6 isolate in our study with pathogenic traits amongst the diverse non-pathogenic environmental V. cholerae populations further supports the epidemiological link between the environmental reservoir and human infection in Iran. This emphasizes on the need for an effective and improved epidemiological surveillance program in Iran, where four epidemics have occurred in the past five years. ,,
| ~ Acknowledgments|| |
The study was funded by Pasteur Institute of Iran grant numbers 170 and 312. We are very grateful to Tehran Water and Sewage Company with special thanks to Mrs. Parvar and Mrs. Motallebi for kindly providing surface water samples.
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[Table 1], [Table 2], [Table 3]
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