Molecular Evidence of Cholera Outbreak Caused by a Toxigenic Vibrio cholerae O1 El Tor Variant Strain in Kelantan,Malaysia

A total of 20 Vibrio cholerae isolates were recovered for investigation from a cholera outbreak in Kelantan, Malaysia, that occurred between November and December 2009. All isolates were biochemically characterized as V. cholerae serogroup O1 Ogawa of the El Tor biotype. They were found to be resistant to multiple antibiotics, including tetracycline, erythromycin, sulfamethoxazole-trimethoprim, streptomycin, penicillin G, and polymyxin B, with 35% of the isolates being resistant to ampicillin. All isolates were sensitive to ciprofloxacin, norfloxacin, chloramphenicol, gentamicin, and kanamycin. Multiplex PCR analysis confirmed the biochemical identification and revealed the presence of virulence genes, viz., ace, zot, and ctxA, in all of the isolates. Interestingly, the sequencing of the ctxB gene showed that the outbreak strain harbored the classical cholera toxin gene and therefore belongs to the newly assigned El Tor variant biotype. Clonal analysis by pulsed-field gel electrophoresis demonstrated that a single clone of a V. cholerae strain was responsible for this outbreak. Thus, we present the first molecular evidence that the toxigenic V. cholerae O1 El Tor variant has invaded Malaysia, highlighting the need for continuous monitoring to facilitate early interventions against any potential epidemic by this biotype.Vibrio cholerae strains belonging to the O1 and O139 serogroups are agents of endemic and pandemic cholera, a potentially life-threatening diarrheal disease. As a water- and food-borne disease, cholera infection is linked to poverty and poor sanitation in many developing countries (13). From an epidemiological point of view, cholera tends to occur in explosive outbreaks throughout several regions simultaneously; likewise, extensive pandemics have followed a progressive pattern, affecting many countries across the continents and extending over many years (17). Cholera pandemics long have been believed to be exclusively associated with the toxigenic V. cholerae O1 serogroup, which consists of two biotypes: classical and El Tor. The classical biotype was responsible for the world''s first six pandemics, but the seventh pandemic was caused by the O1 El Tor biotype and was exceedingly more extensive in geographic spread and duration (13).The first non-O1 strain recognized as having caused an explosive cholera epidemic was discovered in 1992, but it did not belong to any of the 138 serogroups previously described. Thus, the new epidemic strain was designated serogroup O139 and subsequently has been linked to extensive outbreaks in various regions of Bangladesh and India (14). The discovery of its ability to cause large outbreaks and rapid spread to neighboring countries suggested the possibility that the new serogroup will facilitate the eighth pandemic of cholera if outbreaks continue to occur and more countries are affected (42). The strains of V. cholerae O1 El Tor in the seventh pandemic experienced an initial displacement by O139 strains in the Indian subcontinent (1) but recovered their prominence within a year and established a coexistence with O139 strains, causing subsequent cholera outbreaks throughout India and Bangladesh (12, 27).In Malaysia, cholera caused by V. cholerae O1 El Tor is endemic and often has been associated with sporadic outbreaks (24, 33, 46). El Tor is one of the most established biotypes within the V. cholerae O1 serogroup and is differentiated from the classical biotype based on a number of phenotypic traits, such as polymyxin B susceptibility, chicken cell agglutination, Voges-proskauer (VP) test positivity, and phage susceptibility (17). In addition, comparative genomic analysis has revealed the presence of genes that are unique to the El Tor biotype (11). Some of these genes, including the major toxin-coregulated pilus (TCP) tcpA (19) and rtxC of the repeat in toxin (RTX) cluster, have since been used as genetic markers for the identification of El Tor strains (8). The classical and El Tor biotypes also differ in the infection pattern of disease they elicit; the El Tor strains have a higher ratio of asymptomatic carriers (37), survive better in the environment and human host, and are more efficiently transmitted from host to host (http://www.ncbi.nlm.nih.gov/bookshelf/br.fegi?book=mmed&part=A1358).The public health significance of a V. cholerae isolate is routinely assessed by two critical properties: the production of cholera toxin (CT) and the possession of either the O1 or O139 antigen, which acts as a marker of epidemic potential (17). So far, agents of endemic and pandemic cholera have been represented exclusively by CT-producing V. cholerae strains. CT has been shown to be the key virulence factor responsible for the manifestation of massive, dehydrating diarrhea (21). Although CTs from classical and El Tor strains are structurally and functionally similar, differences in immunological and genetic attributes have enabled several methods to differentiate between the two biotypes. Specifically, the molecular techniques include the direct sequencing of the ctxB gene (30), a ganglioside GM₁ enzyme-linked immunosorbent assay (GM₁-ELISA) with specific monoclonal antibody to classical or El Tor CT (29), and a mismatch amplification mutation assay (MAMA) PCR to detect polymorphism at nucleotide position 203 in the ctxB genes (26).In the study presented here, we performed the genetic analysis and molecular typing of V. cholerae O1 isolated from patients during a cholera outbreak in Kelantan, Malaysia, that occurred between November and December 2009. The existence of an outbreak was established based on the region''s previous incidence rate of cholera, which was reported to be 0.0 per 100,000 for four consecutive years (2005 to 2008) (http://statistics.gov.my). The duration of the outbreak was only 21 days, and 33 cholera cases were confirmed. Among those 33, 21 victims were local Kelantanese and the remaining 12 were migrant Thai workers. The patient demographic data revealed that six different districts were involved, and the patients ranged widely in age. Isolates recovered from symptomatic patients were characterized for their antimicrobial susceptibility patterns, the presence of various toxigenic and pathogenic genes, the production of cholera toxin, the nucleotide sequence of cholera toxin B subunits, and the demonstration of the clonal relationships between these isolates. The ultimate findings from this research have provided important insights into the causative agent of the recent cholera outbreak in peninsular Malaysia.(This study was presented in part at the 2nd National Conference on Environment and Health, Kota Bharu, Kelantan, Malaysia, 17 to 18 March 2010.)