�� Vol. 1. No. 1 January–April 2010 Research Report New Biotype of Vibrio cholerae O� from Clinical Isolates in Surabaya Garry cores de Vries,1,2 Emy Koestanti sabdoningrum,2 and dadik rahardjo1,2 1 Diarrheal study group, Institute of Tropical Diseases, Airlangga University 2 Dept.of Veterinary Public Health, Faculty of Veterinary Medicine, Airlangga University abstract A surveillance of new pathogenic variants of Vibrio cholerae O1 strains was initiated to identify the emerge and spread throughout Surabaya. Findings from seven years (1994–2000) and from years 2008 until now by using a two-fold surveillance strategy was pursued involving 1) hospital-based case recognition, and 2) environment samples. Rectal swabs and environment samples were transported to ITD-UNAIR, Surabaya for culture and isolates were characterized by serotypic identification and arbitrarily primed PCR fingerprints revealed a group of strains with similar fingerprint patterns that are distinct from those of the current El Tor epidemic strain. These strains have been analyzed by in vitro technique and the group has been denominated the Surabaya-Indonesian variant of V. cholerae O1. Key words: Vibrio cholerae O1, Cholera toxin, AP-PCR finger print introduction Cholera toxin (CT) is a major virulence determinant of Vibrio cholerae. V. cholerae is indigenous to fresh and blackish water environments in worldwide especially in tropical areas primarily to developing countries with warm climates. V. cholerae causes seafood borne infection, water- borne outbreaks and epidemics in terrestrial environments (Barua, D. 1974 and, Bauer et al., 1966). Most V. cholerae isolates from the environment do not produce Cholera Toxin (CT), nor do they possess the genetic potential to produce Cholera Toxin. V. cholerae O1 and O139 are the major seroytpes associated with illness, and some V. cholerae non-O1 and non-O139 isolates produce CT. Detection of CT-producing V. cholerae using conventional culture-, biochemical- and immunological- based assays is time-consuming and laborious. A rapid, reliable and practical assay for the detection of CT- producing V. cholerae has been used as like as PCR assays which offer a more sophisticated approach to the identification of Vibrio cholerae (Brosius et al., 1981). Although PCR assays provide more rapid identification of Vibrio cholerae than conventional assays, they require the use of electrophoresis to detect amplified products, which is time-consuming and tedious. The worldwide epidemiological situation in cholera El Tor at the beginning of this century is presented; among its characteristic features are continued extensive epidemics and outbreaks in African and Asian countries with cases of import of this infection to other continents. Outbreaks caused by a new variant of the infective agent of cholera, Vibrio cholerae O139, are still registered at limited territories in the countries of South-East Asia (Oneshchenko et al., 2005). The emergence of Vibrio cholerae O139 Bengal during 1992–1993 was associated with large epidemics of cholera in India and Bangladesh and, initially, with a total displacement of the existing V. cholerae O1 strains. However, the O1 strains reemerged in 1994 and initiated a series of disappearance and reemergence of either of the two serogroups that was associated with temporal genetic and phenotypic changes sustained by the strains. Since the initial emergence of the O139 vibrios, new variants of the pathogen derived from multiple progenitors have been isolated and characterized. The clinical and epidemiological characteristics of these strains have been studied. Rapid genetic reassortment in O139 strains appears to be a response to the changing epidemiology of V. cholerae O1 and also a strategy for persistence in competition with strains of the O1 serogroup. The emergence of V. cholerae O139 has provided a unique opportunity to witness ��Vries et al.: New biotype of vibrio cholerae O1 genetic changes in V. cholerae that may be associated with displacement of an existing serogroup by a newly emerging one and, thus, provide new insights into the epidemiology of cholera (Faruque et al., 2003). Vibrio cholerae is causing a severe epidemic in east Java after being absent from the region for about 10 years (Tauxe et al., 1994). Vibrio cholerae typically contains a prophage that carries the genes encoding the cholera toxin, which is responsible for the major clinical symptoms of the disease (Safa et al., 2010). The taxonomy of this species has been the object of our interest, and we recently developed a method for distinguishing pathogenic groups by using arbitrarily primed PCR (APPCR) fingerprints (Coelho et al., 1993 and Coelho et al., 1995) on the basis of the general methodology of AP-PCR (Welsh et al., 1990 and Williams et al., 1990). By this technique a single oligonucleotide with an arbitrary sequence is used in a PCR with the DNA of the strain under analysis. Low- stringency conditions for hybridization are used, and the oligonucleotide can find regions of pairing, leading to the amplification of various genome fragments. Our study (6) involved four groups of pathogenic V. cholerae: classical, El Tor, Ogawa and Hikojima, all of which are distinguishable with the fingerprints. When applied to strains isolated from environment the results were similar to those from clinical isolated strains. However, when this method was used to study a group of strains from patients with diarrheal disease in the north part of Surabaya, a quite distinct fingerprint pattern emerged for some of these strains. In the work described here we further extended this observation by using other in vitro techniques to evaluate the degree of relatedness between this group and the other pathogenic strains. materials and methods isolation and identification of Vibrio cholerae serogroups o1 and o139 Enrichment in alkaline peptone water Inoculated APW with liquid stool or a rectal swab. Incubate the tube with the cap loosened at 37°C for 8 hours. Then subculture to TCBS with two loopfuls of APW from the surface of the broth. If the broth cannot be plated after 8 hours of incubation, subculture a loopful at 18 hours to a fresh tube of APW. Subculture the second tube to TCBS agar after 8 hours of incubation. inoculation of tcBs Inoculate the TCBS plate after 24 hours incubation at 37° C. Colonies suspicious for V. cholerae will appear on TCBS agar as yellow, shiny colonies, 2 to 4 mm in diameter. The yellow color is caused by the fermentation of sucrose in the medium. Sucrose-nonfermenting organisms, such as V. parahaemolyticus, produce green to blue-green colonies. isolation of suspected V. cholerae One of each type of sucrose-fermenting colony was selected from the TCBS plate to inoculate a heart infusion agar (HIA) slant. Incubate the HIA slants at 37° C for up to 24 hours; for serologic testing. Slide serology with polyvalent O1 and O139 antisera is sufficient for a presumptive identification. screening tests for suspected V. cholerae isolates oxidase test Conduct the oxidase test with fresh growth from an HIA slant medium. Place 2 to 3 drops of oxidase reagent (1% N,N,NN,NN-tetramethyl-p-phenylenediamine) on a piece of filter paper in a petri dish. In a positive reaction, the bacterial growth becomes dark purple immediately. Oxidase-negative organisms will remain colorless or will turn purple after 10 seconds. Positive and negative controls should be tested at the same time. Organisms of the Vibrio cholerae is oxidase positive. Reactions of V. cholerae in screening tests Oxidase test Positive Kligler iron agar (KIA) alkaline/acid, no gas produced (red slant/yellow butt) Triple sugar iron agar (TSI) acid/acid, no gas produced (yellow slant/yellow butt) Lysine iron agar (LIA) alkaline/alkaline, no gas produced (purple slant/purple butt) Gram-negative curved rods serologic identification of V. cholerae o1 and o139 presumptive identification using o1 antisera Slide agglutination testing with polyvalent O1 antisera, fresh growth of suspected V. cholerae from a nonselective agar medium HIA should be used. Using growth from TCBS agar may result in false-negative reactions. Presumptive V. cholerae O1 isolates should be tested in monovalent Ogawa, Inaba antisera and Hikojima. confirmation of V. cholerae o1 using inaba and ogawa antisera The O1 serogroup of V. cholerae has been further divided into three serotypes, Inaba, Ogawa, or Hikojima. A positive reaction in either Inaba, Ogawa or both Hikojima antiserum is sufficient to confirm the identification of a V. cholerae O1 isolate. Isolates that agglutinate weakly or slowly with serogroup O1 antiserum but do not agglutinate with either Inaba or Ogawa antiserum are not considered to be serogroup O1. Agglutination reactions with Inaba and Ogawa antisera should be examined simultaneously, and the strongest and most rapid reaction should be used to identify the serotype. Strains that agglutinate very strongly and equally with both the Ogawa and Inaba antisera are suspected may be referred to as “possible serotype Hikojima.” �0 Indonesian Journal of Tropical and Infectious Disease, Vol. 1. No. 1 January–April 2010: 48-53 strains of V. cholerae. The strains used in the present study are listed in Table 1. ap-pcr. The AP-PCR mixtures consisted of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 4 mM MgCl2, 100 mM (each) deoxynucleoside triphosphate (dNTP), 30 pmol of one of the oligonucleotides, and 100 ng of DNA in a total volume of 25 ml. The mixture was overlaid with mineral oil, and 1.5 U of Taq DNA polymerase was added. The program consisted of 45 cycles, and an annealing temperature of 32o C was used (Coelho et al., 1995). Two sets of fingerprints were done, one of them with oligonucleotide 1 (5’-GGTGCGGGAA) and the other with oligonucleotide 3 (5’-CCAGATGCAC) (Coelho et al., 1995). Analysis of the amplified fragments was done on 1.4% agarose gels (GIBCO-Bethesda Research Laboratories) in Tris-borate buffer (TBE) (Sambrook et al., 1989) running at 100 V for 3 h. analysis of presence of virulence genes by pcr. The basic program for the PCRs for the specific genes included 1 min initial denaturation step at 94o C, followed by 35 three-step cycles at 94o C (45 sec), annealing 55o–60o C for (45 sec) and extension at 72o C (1 min). A final extension at 72° C for 5 min was included in all reactions. A total of 100 ng of DNA, 20 pmol of each primer, 0.25 mM dNTPs, and 1.5 U of Taq DNA polymerase were used, with a 1.5 mM MgCl2 buffer, in a total volume of 50 ml. The oligonucleotides used for ctxA1 amplification were 5’_ CGG GCA GAT TCT AGA CCT CCT G _3’ (sense) and 5’_ CGA TGA TCT TGG AGC ATT CCC AC_ 3’ (antisense), which were designed table 1. Strains of V. cholerae analyzed in the study straina placeb date serotype type VF-57 Surabaya February 1995 Ogawa El tor VF-58 Surabaya February 1995 Ogawa El tor VF-59 Surabaya February 1995 Ogawa El tor VF-60 Surabaya October 1995 Ogawa - VF-61 Surabaya February 1995 Ogawa El tor VF-62 Surabaya February 1995 Ogawa El tor VF-64 Surabaya March 1995 Ogawa Classic VF-65 Surabaya January 1995 Ogawa El tor VF-66 Surabaya March 1995 Ogawa El tor VF-67 Surabaya February 1995 Ogawa El tor VF68 Surabaya June 1995 Ogawa El tor VF 69 Surabaya June 1995 Ogawa Classic VF70 Surabaya June 1995 Ogawa El tor VF-71 Surabaya February 1995 Ogawa El tor VF-72 Surabaya February 1995 Ogawa El tor VF-73 Surabaya February 1995 Ogawa El tor VF-74 Surabaya June 1995 Ogawa El tor VF-75 Surabaya February 1995 Ogawa El tor VF-76 Surabaya February 1995 Ogawa El tor VF-77 Surabaya February 1995 Ogawa El tor VF-80 Surabaya February 1998 Ogawa - VF-81 Surabaya February 1998 Ogawa - VF-82 Surabaya March 1998 Ogawa - VF-83 Surabaya March 1998 Ogawa - VF-84 Surabaya March 1998 Ogawa - VF-85 Surabaya March 1998 Ogawa - VF-86 Surabaya March 1998 Ogawa - VF-87 Surabaya March 1998 Ogawa - VF-88 Surabaya September 1998 Ogawa - VF-89 Surabaya September 1999 Ogawa - VF-90 Surabaya September 1998 Ogawa - VF-91 Surabaya September 1998 Ogawa - VF-92 Surabaya April 1997 Ogawa El tor VF-94 Surabaya March 1998 Ogawa - VF-95 Surabaya June 1998 Ogawa - VF-96 Surabaya September 2008 Ogawa - VF-192 Surabaya June 2009 Hikojima VF-193 Surabaya July 2009 Ogawa - VF-194 Surabaya July 2009 Hikojima ��Vries et al.: New biotype of vibrio cholerae O1 for classical strains. The oligonucleotides used for ctxA2 amplification were 5’_ ACA GAG TGA GTA CTT TGA CC_ 3’ (sense) and 5’_ ATA CCA TCC ATA TAT TTG GGA G_ 3’ (antisense), which were designed for classical strains (Lipp et al., 2003). Biochemical identification. The biochemical characterization of Surabaya- Indonesian strains was done by a battery of standard tests (Farmer et al., 1985.) including oxidase, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, requirement for Na+ (growth in nutrient broth with 0, 1, and 3% NaCl), motility, indole, gas from glucose, susceptibility to O/129 (150-mg discs), and acid production from D-glucose, L-arabinose, cellobiose, lactose, maltose, D-mannitol, salicin, and sucrose. Identification of biotypes was performed by detection of acetylmethylcarbinol (Voges-Proskauer test) and determination of susceptibility to polymyxin B (Oxoid) by spot inoculation onto Mueller- Hinton agar (Difco) containing 15mg of polymyxin B per ml (Roy et al., 1965), hemolysis of sheep erythrocytes, and hemagglutination activity for human (O group) and chicken erythrocytes. o1 somatic antigen characterization. Expression of O1 antigen by Ogawa-El Tor strains was further evaluated by tube agglutination tests against polyvalent O1 antisera prepared by immunizing rabbits with heat-killed cells of Inaba or Ogawa and then absorption of the antisera with the heat-killed cells of the heterologous serotype. Polyvalent O1 antiserum was obtained by mixing the two monospecific antisera. Tests were performed with live cultures grown for 3 h (Barua, 1974.). antimicrobial susceptibility tests. Antimicrobial susceptibility testing was carried out on Mueller-Hinton agar (Difco) by the disc diffusion method (Clinical and Laboratory Standards Institute, 2008) for levofloxacin, nalidixic acid, ampicillin, chloramphenicol, imipenem and ceftriaxone. figure 1. Pathogenic V. cholerae O1 fingerprints obtained by AP-PCR with oligonucleotide ctxA1 and ctxA2. Lanes 1 to 5, Surabaya-Indonesian variant strains (biotype Ogawa-El Tor VF-69, VF-59, VF-76, VF-77 and VF-92) respectively; lanes 6 to 9, biotype Hikojima strains VF192, VF-200, biotype Ogawa VF-193 and VF-196, respectively; lane 10 1-kb ladder size marker (GIBCO Bethesda Research Laboratories). results and discussion We studied 43 V. cholerae O1 strains (Table 1) isolated in 1995, 1997, 1998, 2008 and 2009 in east and south Surabaya and, in particular, from the villages adjacent region north of Surabaya. Most of them came from patients with diarrhea, and their co-cultures did not show other enteropathogenic bacteria. A screening was done with these strains by using biochemical identification and AP-PCR fingerprints. Two oligonucleotides, oligonucleotides ctxA1 and ctxA2, were used in separate reactions. Each of the oligonucleotides showed that there were two markedly different groups of strains in the sample. One of these groups yielded the fingerprints found with other El Tor strains (Coelho et al., 1995 and Lipp et al., 2003), and the other group, comprising five strains Hokojima and denominated in Surabaya- Indonesian variant in 2009–2010, produced fingerprints different from those of the other pathogenic groups studied straina placeb date serotype type VF-195 Surabaya September 2009 Hikojima VF-196 Surabaya September 2009 Ogawa - VF-200 Surabaya December 2009 Hikojima VF-217 Surabaya January 2010 ? a The 36 first strains correspond to the original group of strains analyzed. The seven El Tor strains included in this group were used in the tests described in the text. The last four Hikojima strains were identified later. All isolates came from patients; isolates VF-96 were obtained from environment. b All locations are in Surabaya and adjacent region. �� Indonesian Journal of Tropical and Infectious Disease, Vol. 1. No. 1 January–April 2010: 48-53 previously. The fingerprints of the strains within the group were identical. The results with oligonucleotide ctxA1 (Fig. 1) showed a completely different pattern for the Surabaya- Indonesian strains, in which a 1.3-kb band seemed to be the only band common to the bands for the El Tor strains. In the case of oligonucleotide ctxA2, a prominent 0.55-kb El Tor band was absent from the fingerprints of the Surabaya- Indonesian strains; the other bands were the same (data not shown). These same oligonucleotides have been used against representative El Tor, classical and Inaba strains (Coelho et al., 1995). A serotype difference is not detected with these oligonucleotides. All of the Surabaya-Indonesian strains tested belonged to the Ogawa serotype, but other Ogawa strains in the sample were normal El Tor isolates, producing their characteristic AP-PCR fingerprints. The Surabaya-Indonesian variant strains came from the small town (villages) bordering of Surabaya¸ and a few other villages 50 km adjacent Surabaya. A further search of our collection revealed five other Surabaya-Indonesian strains among the Ogawa strains from the same region. The AP-PCR fingerprints used in the present study are tuned at a less discriminative level that groups together the El Tor strains (except the environment strains that form a separate group). This grouping occurs because of the choice of oligonucleotides, which were selected to distinguish between broad pathogenic groups and not strains within a group (Coelho et al., 1995). A more conspicuous distinction between the Surabaya-Indonesian variant and the El Tor strains was produced with AP-PCR. The Surabaya-Indonesian strains behaved in the biochemical tests as typical representatives of V. cholerae. Biological markers such as O-antigen specificity and antimicrobial susceptibility were also evaluated. It was shown that all Surabaya-Indonesian strains but one exhibited O1 agglutination titers of 1,024 (five strains) or 2,048 (three strains). The homologous titer for the classical Ogawa strain was 4,096. Surabaya-Indonesian strain VF-217 was autoagglutinable, preventing its testing. Further testing with monospecific antiserum showed that Surabaya-Indonesian strains reacted only with Ogawa antiserum. Antimicrobial susceptibility tests showed that the Surabaya-Indonesian and El Tor strains isolated from the same geographical area were equally susceptible to all antimicrobial agents tested. Biotyping showed that these strains were Voges- Proskauer test positive and susceptible to polymyxin. Taken together these results support the previous findings of AP-PCR analyses that Surabaya-Indonesian strains are a separate group distinct from the El Tor (polymyxin resistant, Voges Proskauer test positive) and the classical (polymyxin susceptible, Voges-Proskauer test negative) biotypes of V. cholerae O1. The presence of ctxA gene was investigated in various ways in the Surabaya-Indonesian strains. PCR amplifications were done with positive results for all Hikojima strain and 2 Ogawa El Tor strains. The restriction fragment length polymorphisms of the ctx genes were tested. A cholera toxin DNA fragment of 982 bp was used as a probe. This DNA fragment was produced by PCR amplification from an El Tor strain, and it includes most of the ctxA1 and ctxA2 genes. This indicates that the toxin genes, if present, have a very divergent sequence. Only seven strains (VF-92, VF-192, VF-193, VF-194, VF-195, Vf-196 and VF-200) produced cholera toxin. The presence of other V. cholerae virulence genes was investigated by PCR. Oligonucleotides specific for ctxB. All of these amplifications gave negative results. Positive controls with an El Tor strain were included in all of the experiments. Preliminary phenetic analysis places the Surabaya- Indonesian clone at a considerable distance from other pathogenic O1 clones (ElTor, Classical, and Hikojima). The Surabaya-Indonesian variant seems to be restricted, for the time being, to a small area of the Surabaya and adjacent region and is probably unable to compete with the invading El Tor strains. A parallel may be traced with the early isolates of El Tor from the hospitals. Their epidemiological relevance is, at present, negligible, but as in the latter case, future developments of cholera in Indonesia may outline its importance. references 1. Barua, D. 1974. Laboratory diagnosis of cholera, p. 85–126. In D. Barua and W. Burrows (ed.), Cholera. The W. B. Saunders Co., Philadelphia. 2. Bauer, A. W., W. M. M. Kirby, J. C. Sherris, and M. Turck. 1966. Antibiotic susceptibility testing by a standardized single disk method. Am. J. Clin. Pathol. 45:493–496. 3. Coelho, A., A. Vicente, H. Momen, and C. Salles. 1993. Typing of Vibrio cholerae using arbitrary primers, p. 211. Volume of abstracts. Seventeenth International Congress of Genetics, Birmingham, United Kingdom. The Quadgraphics Partnership, Thatcham, United Kingdom. 4. Coelho, A., A. C. P. Vicente, M. A. S. Baptista, H. Momen, F. R. W. dos Santos, and C. A. Salles. 1995. The distinction of pathogenic Vibrio cholerae groups using arbitrarily primed PCR fingerprints. The Quadgraphics Partnership, Thatcham, United Kingdom. 5. Coelho, A., O. R. Joa., C. Andrade., A. C. P. Vicente, and C. S. A. Salles. 1995. New Variant of Vibrio cholerae O1 from Clinical Isolates in Amazonia. Journal Of Clinical Microbiology. 33:114– 118. 6. Clinical and Laboratory Standards Institute. 2008. Performance Standards for Antimicrobial Susceptibility Testing; Eighteenth Informational Supplement. M100-S18. 28:74–76. 7. DiRita, V., C. Parsot, G. Jander, and J. J. Mekalanos. 1991. Regulatory cascade controls virulence in Vibrio cholerae. Proc. Natl. Acad. Sci. USA 88: 5403–5407. 8. Faruque, S.M., D.A Sack, R.B Sack, R.R Colwell, Y. Takeda and G.B Nair. 2003. Emergence and evolution of Vibrio cholerae O139. Proceedings of the National Academy of Sciences of the United States of America. Volume 100, Issue 3. pp 1304–1309. 9. Hayashi S, M. Okura, R. Osawa. 2006. Soft-agar-coated filter method for early detection of viable and thermostable direct hemolysin (TDH)- or TDH-related hemolysin-producing Vibrio parahaemolyticus in seafood. Appl Environ Microbiol, 72:4576–4582. 10. Higgins, D. E., E. Nazareno, and V. J. DiRita. 1992. The virulence gene activator ToxT from Vibrio cholerae is a member of the AraC family of transcriptional activators. J. Bacteriol. 174:6974–6980. 11. Iwanaga, M., and K. Yamamoto. 1985. New medium for the production of cholera toxin by Vibrio cholerae O1 biotype El Tor. J. Clin. Microbiol. 22:405–408. ��Vries et al.: New biotype of vibrio cholerae O1 12. Jonson, G., J. Holmgren, and A.-M. Svennerholm. 1991. Identification of a mannose-binding pilus on Vibrio cholerae El Tor. Microb. Pathog. 11:433–441. 13. Jonson, G., J. Sanchez, and A.-M. Svennerholm. 1989. Expression and detection of different biotype-associated cell-bound haemoagglutinins of Vibrio cholerae O1. J. Gen. Microbiol. 135:111–120. 14. Keasler, S. P., and R. H. Hall. 1993. Detecting and biotyping Vibrio cholerae O1 with multiplex polymerase chain reaction. Lancet 341:1661. 15. Koblavi, S., F. Grimont, and P. A. D. Grimont. 1990. Clonal diversity of Vibrio cholerae O1 evidenced by rRNA gene restriction patterns. Res. Microbiol. 141:645–657. 16. Lan, R., and P. R. Reeves. 2002. Pandemic Spread of Cholera: Genetic Diversity and Relationships within the Seventh Pandemic Clone of Vibrio cholerae Determined by Amplified Fragment Length Polymorphism. JCM. 40:172-181. 17. Lipp,E. K., I. N. G. Rivera, A. I. Gil, E. M. Espeland, .N. Choopun, V. R. Louis, E. R. Cohen, A. Huq, and R. R. Colwell. 2003. Direct Detection of Vibrio cholerae and ctxA in Peruvian Coastal Water and Plankton by PCR. Applied and Environmental Microbiology. 69: 3676–3680. 18. Louis, V. R., E. R. Cohen, N. Choopun, I. N. G. Rivera, B. Gangle, S. C. Jiang, A. Rubin, J. A. Patz, A. Huq, and R. R. Colwell. 2003. Predictability of Vibrio cholerae in Chesapeake Bay. Applied and Environmental Microbiology. 69:2773–2785. 19. Ogawa, A., J.-I. Kato, H. Watanabe, B. G. Nair, and T. Takeda. 1990. Cloning and nucleotide sequence of a heat-stable enterotoxin gene from Vibrio cholerae non-O1 isolated from a patient with traveler’s diarrhea. Infect. Immun. 58:3325–3329. 20. Oneshchenko G. G, I. M. Lomov, E. A. Moskvitina, I. M. Fedorov, L.S. Podosinnikova, A. V. Gorobets. 2005. Cholera at the beginning of the XXI century. Prognosis. Zhurnal Mikrobiologii Epidemiologii i Immunobiologii. pp. 44-48. 21. Popovic, T., C. Bopp, Ø. Olsvik, and K. Wachsmuth. 1993. Epidemiologic application of a standardized ribotype scheme for Vibrio cholerae O1. J. Clin. Microbiol. 31:2474–2482. 22. Roy, C., K. Mridha, and S. Mukerjee. 1965. Action of polymyxin of cholera vibrios: techniques of determination of polymyxin-sensitivity. Proc. Soc. Exp. Biol. Med. 119:893–896. 23. Salles, C. A., H. Momen, A. Coelho, E. F. de Oliveira, A. C. P. Vicente, and G. B. Nair. 1994. Bengal: El Tor cholera Vibrio in a new robe. Mem. Inst. Oswaldo Cruz 89:115–116. 24. Salles, C. A., H. Momen, A. C. P. Vicente, and A. Coelho. 1993. Vibrio cholerae in South America: polymerase chain reaction and zymovar analysis. Trans. R. Soc. Trop. Med. Hyg. 87:272. 25. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd. ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 26. Safa A, Nair G.B, Kong R.Y.C. 2010. Trends in Microbiology. 18:46–54. 27. Silhavy, T. J., M. L. Berman, and L. W. Enquist. 1994. Experiments with gene fusions, pp. 137–139. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 28. Simanjuntak, C. H., W. Larasati, S. Arjoso, Maidy Putri, M. Lesmana, B. A. Oyofo, N. Sukri, D. Nurdin, R. P. Kusumaningrum, N. H. Punjabi, D. Subekti, S. Djelantik, Sukarma, Sriwati, Muzahar, A. Lubis, H. Siregar, B. Mas’ud, M. Abdi, A. Sumardiati, S. Wibisana, Hendarwanto, B. Setiawan, W. Santoso, Eka Putra, S. Sarumpaet, H. Ma’ani, C. Lebron, S. A. Soeparmanto, J. R. Campbell, and A. L. Corwin. 2001. Cholera In Indonesia In 1993–1999. Am. J. Trop. Med. Hyg., 65: 788–797. 29. Tauxe, R., L. Seminario, R. Tapia, and M. Libel. 1994. The Latin American epidemic, p. 321–344. In I. K. Wachsmuth, P. A. Blake, and Ø. Olsvik (ed.), Vibrio cholerae and cholera. American Society for Microbiology, Washington, D.C. 30. Welsh, J., and M. McClelland. 1990. Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Res. 18:7213–7218. 31. Williams, J. G. K., A. R. Kubelik, K. J. Livak, J. A. Rafalski, and S. V. Tingey. 1990. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res. 18:6531–6535. 32. Yamamoto, K., Y. Takeda, T. Miwatani, and J. P. Craig. 1983. Purification and some properties of a non-O1 Vibrio cholerae enterotoxin that is identical to cholera enterotoxin. Infect. Immun. 39:1128–1135. 33. Yamazaki, W., K. Seto, M. Taguchi, M. Ishibashi and K. Inoue. 2008. Sensitive and rapid detection of cholera toxin-producing Vibrio cholerae using a loop-mediated isothermal amplification. BMC Microbiology, 8:94. IJTID vol 1 no 1 Jan-Apr 2010.50.pdf IJTID vol 1 no 1 Jan-Apr 2010.51.pdf IJTID vol 1 no 1 Jan-Apr 2010.52.pdf IJTID vol 1 no 1 Jan-Apr 2010.53.pdf IJTID vol 1 no 1 Jan-Apr 2010.54.pdf IJTID vol 1 no 1 Jan-Apr 2010.55.pdf