Unique organization of the CTX genetic element in Vibrio cholerae O139 strains which reemerged in Calcutta, India, in September 1996

Abstract

We studied the restriction fragment length polymorphism of the rRNA gene and CTX genetic element in Vibrio cholerae O139 Bengal, which resurged in Calcutta in September 1996 after a gap of 32 months. While the strains from this resurgence were indistinguishable from the earlier strains by ribotyping, the structure of the CTX genetic element present in the current O139 strains was found to be unconventional. Vibrio cholerae O139, the second etiological serogroup of cholera, came into being dramatically in September 1992 in Southern India and thereafter spread explosively to all areas of cholera endemicity in India (11) and to neighboring countries (12). Inexplicably, towards the end of 1993 the incidence of V. cholerae O139 declined in Calcutta, India (10), and in other parts of the subcontinent (3), and the O139 serogroup was replaced by a new clone of V. cholerae O1 biotype El Tor (4, 18, 21). However, in September 1996, V. cholerae O139 staged a resurgence. Phenotypically, the reemerged V. cholerae O139 strains were different from those that appeared in late 1992 and 1993 in that the current O139 strains were sensitive to cotrimoxazole (9). However, like the O139 strains isolated in 1992 to 1993 (16), the O139 strains that resurged were resistant to ampicillin, furazolidone, neomycin, and streptomycin (9). Toxigenic strains of V. cholerae contain a compound transposon-like structure known as the CTX genetic element, which comprises a 4.5-kb central core region that contains the ctxAB, zot, ace, orfU, and cep genes and has one or more copies of a 2.7-kb repetitive sequence (designated RS1) flanking it (14, 19). The number and arrangement of the CTX element are known to vary in different strains of toxigenic V. cholerae (20) and have formed a useful basis for the study of the clonality of strains. We embarked on a study to characterize the ribotype, organization, and chromosomal location of the CTX genetic element of the current O139 strains to assess the extent of genotypic variations in these compared to the strains isolated in 1992 to 1993. A total of 10 strains of V. cholerae O139, namely, AS207, AS209, AS210, AS212, AS213, AS231, AS233, AS258, AS259, and AS260, isolated from hospitalized patients with cholera from the Infectious Diseases Hospital, Calcutta, India, between 21 August and 20 September 1996 were selected randomly by blinding the date of isolation from the person who selected the strains. MO1, a serotype O1 strain which could be a progenitor strain of V. cholerae O139 (12), and AP1, an O139 strain which appeared in late 1992, were also included in the study for comparison (13). Chromosomal DNA was prepared (1), restricted with a va

Site‐specific integration of the conjugal Vibrio cholerae SXT element into prfC

“…We have found by Southern hybridization studies that these recent SXT S O139 strains harbour sequences closely related to the SXT element (data not shown). For example, our initial analysis of Calcutta 1996 isolate AS207 (Sharma et al, 1997) suggests that this strain harbours a mutation in the gene encoding trimethoprim resistance and Bangladesh 1998 isolate 2055 harbours a deletion, both resulting in SXT sensitivity. Despite these mutations in the antibiotic resistance genes, the SXT element att site PCR assay showed that in both isolates the mutated SXT elements are integrated into prfC (data not shown).…”

Genomic diversity among Vibrio cholerae O139 strains isolated in Bangladesh and India between 1992 and 1998

“…For ribotyping as well as CTX genotyping, restriction enzyme BglI (Takara Shuzo Japan or Bethesda Research Laboratories, Gaithersburg, MD, USA) was used to digest chromosomal DNA and the fragments were separated by agarose gel (0.8%) electrophoresis. The rRNA gene probe was a 7.5kb BamHI fragment of pKK3535 described previously [9,11,15]. The gene probe used for CTX genotyping was a 0.5-kb EcoRI fragment of pCVD27 [16] representing 94% of the ctxA gene encoding the A subunit of cholera toxin (CT).…”

“…A reemergence of V. cholerae O139 was noted in Calcutta from August 1996, and subsequently the O139 vibrios reappeared in both India and Bangladesh [8]. Although the reemerged strains of V. cholerae O139 had identical biochemical traits as the strains isolated during 1992^1993, molecular characterization revealed unique changes in the organization of the CTX genetic element [9,10] and ribotype of reemerged strains [11]. All these studies demonstrated the changing epidemiology of cholera, and suggested that the O139 serogroup is likely to spread over a period of time to other choleraendemic areas in the world and constitute the eighth pandemic of cholera.…”

Diversity in the arrangement of the CTX prophages in classical strains of Vibrio cholerae O1

“…This could only be possible if downstream sequences of these two CTX prophages are exactly identical i.e., two of three CTX prophages of GP13, and two of four CTX prophages of GP147 occupies exactly the same position occupied by 2 CTX prophages of the strain GP8. The arrangement of the CTX prophages with one CTX prophage in each of two replicons [2,5] is a constant pattern for the classical biotype strains unlike the El Tor O1 and O139 strains where diverse arrangements of the CTX prophages have been reported [18,19]. Even the classical O1 strains which reemerged in Bangladesh in 1979 had identical copy numbers and organization of the CTX prophages [20].…”