Diverse CTX Phages among Toxigenic Vibrio cholerae O1 and O139 Strains Isolated between 1994 and 2002 in an Area Where Cholera is Endemic in Bangladesh

Abstract

PCR surveillance of the rstR genes of CTX phages in Vibrio cholerae O1 and O139 showed no relationship between the incidence of disease and changes in the rstR but showed variations in their presence in O1 and O139 strains and the occurrence of multiple types in a few strains.

Of the 209 currently recognized serogroups of Vibrio cholerae, only strains belonging to serogroups O1 and O139 can cause cholera. Two major virulence gene clusters are now known to carry key virulence genes that are essential for the pathogenicity of V. cholerae O1 and O139. These gene clusters include the CTXφ prophage (14), which carries the ctxA and ctxB genes (the genes that encode cholera toxin [CT], which is responsible for severe diarrhea), and the toxin-coregulated pilus (TCP) pathogenicity island, which carries genes for the biosynthesis of the TCP, required for colonization of the small intestinal epithelium (7).

The approximately 7-kb CTXφ genome consists of the core and the RS2 region. The core region encodes proteins needed for the assembly and secretion of viral particles (Psh, Cep, pIII^CTX, Ace, and Zot) and also encodes CT, which is not necessary for phage morphogenesis (3), while the RS2 region represents a site-specific recombination system that allows lysogenic phage to integrate at a specific site on the host chromosome (14). The RS2 region of CTX prophage encodes proteins required for replication (RstA), phage integration (RstB), and regulation (RstR) of the lysogeny of CTXφ (14). An antirepressor, rstC, is carried by a satellite phage, RS1, often present adjacent to the CTX prophage in toxigenic V. cholerae O1 El Tor and O139 strains (1, 5).

Diversity of the CTX phage repressor rstR has been described previously, and this diversity constitutes heteroimmunity among diverse CTX phages (8, 2). The difference in the rstR gene is also the only known genetic difference between any two different CTX phage types. The existence of at least four different rstR genes carried by different CTX phages, namely, CTX^ET, CTX^class, CTX^Calc, and CTX^Env, has been recognized (8, 2, 10). The epidemiological significance of the diversity of CTX phages is not clearly known, but at least two periods of explosive resurgence of cholera have been associated with strains showing changes in the rstR type of CTX phages. The first was the resurgence of V. cholerae O139 in August 1996 in Calcutta, India, which continued for a year (8, 9, 13), and the second was the resurgence of strain O139 in March to April of 2002 in Dhaka, Bangladesh (6). On the basis of their rstR genes and other phenotypic traits, genetic hybrids of classical and El Tor biotypes that cause cholera have been shown to exist, and these hybrids have been designated the Matlab variants of V. cholerae (11). To further document the distribution and temporal changes in the CTX phage contents of epidemic strains, we conducted a surveillance of CTX phage types by analyzing the types of rstR genes carried by a large collection of toxigenic V. cholerae strains.

We selected every 10th consecutive strain of V. cholerae O1 or O139 isolated from cholera patients admitted to the Matlab hospital, 50 km south of Dhaka, Bangladesh, from 1994 to 2002. A total of 169 strains of V. cholerae O1 and 95 strains of V. cholerae O139 isolated between 1994 and 2002 (with the exception of the year 1999) were included in this study. The procedure for the selective isolation of V. cholerae from stool samples of patients with acute secretory diarrhea and subsequent identification has been described in detail previously (12).

The serogroup of the strains selected were confirmed by using polyclonal O1 and O139 antisera. PCR was performed according to a previously described procedure (11). The primer sequences are shown in Table 1. V. cholerae O1 isolates (classical 154) and V. cholerae O139 (AR-196318) and V. cholerae non-O1 non-O139 (environmental SCE-188) isolates (10) were used as standard reference strains. We also used an rstC probe as described previously (4) to examine whether CTX prophage-negative strains, which show an rstR amplicon, carried RS1. The PCR products from five representative isolates (MJ1347, MM1079, MM2071, MP1950, and MP2044) were purified with a Microcon centrifugal filter device (Millipore Corporation, Bedford, Mass.), and a cycle sequencing reaction was performed with the same primers. DNA sequencing was performed by using standard conditions in an ABI PRISM 310 automated sequencer (Perkin-Elmer-Applied Biosystems, Foster City, Calif.). DNA sequence editing and analysis were performed with DNASTAR package 5.06 software.

TABLE 1.

Oligonucleotide primer sequences used in PCR assays^a

Gene	Primer sequence (5′-3′)	Amplicon size (bp)
ctxA (forward)	5′-CTCAGACGGGATTTGTTAGGCACG-3′
ctxA (reverse)	5′-TCTATCTCTGTAGCCCCTATTACG-3′	308
rstR1 (forward)	5′-CTTCTCATCAGCAAAGCCTCCATC-3′	500
rstR2 (forward)	5′-GCACCATGATTTAAGATGCTC-3′	500
rstR3 (forward)	5′-CTGTAAATCTCTTCAATCCTAGG-3′	∼300
rstR4 (forward)	5′-GTTAACGCTTCAAGCCTG-3′	400
rstA3 (reverse)	5′-TCGAGTTGTAATTCATCAAGAGTG-3′

^aPrimers were for the detection of rstR and ctxA genes in V. cholerae O1 and O139 strains isolated from hospitalized patients in Matlab, Bangladesh.

Table 2 shows the distribution of different types of rstR genes among 169 strains of V. cholerae O1 and 95 O139 strains, isolated between 1994 and 2002 from hospitalized patients in Matlab, Bangladesh. We propose to designate the rstR genes with subscript numbers (rstR₁, rstR_2., etc.) since we anticipate that the number of such rstR genes that will be discovered in the future is likely to increase and thus a number designation is more suitable. The nucleotide sequences of 10 rstR amplicons from five isolates of V. cholerae O1 and O139 were similar to those of canonical rstR genes, with minor differences, as shown in Table 3.

TABLE 2.

Occurrence of the various rstR genes examined in this study among V. cholerae O1 and O139 strains

rstR gene(s)	Original nomenclature (reference)	No. of positive isolates
		V. cholerae O1	V. cholerae O139
rstR1	rstRclass (8)	9	0
rstR2	rstRET (8)	141a	83
rstR3	rstRCalc (8)	0	0
rstR4	rstREnv (10)	0	0
rstR1 + rstR2	Not reported	6	0
rstR2 + rstR4	Not reported	6	6
rstR2 + rstR3	Combination (8)	0	3
rstR1 + rstR2 + rstR3	Not reported	0	1
Noneb		7	2
Total		169	95

^aTwo strains were negative for the ctxA gene.

^bNone, negative for all of the rstR genes tested.

TABLE 3.

Nucleotide sequences of various rstR genes of V. cholerae O1 and O139 isolates from Matlab, Bangladesh, in comparison with corresponding sequences in GenBank

Strain (gene)	Serogroup	GenBank accession no.	Mutation comparison (accession no.)
MJ1347 (rstR1)	O1	AY704650	Identical to the V. cholerae 569B repressor rstR (AF055890)
MJ1347 (rstR2)	O1	AY704651	Silent substitution of C to T at position 1938 compared to the V. cholerae O1 biovar El Tor N16961 transcriptional repressor rstR (AE004224)
MM1079 (rstR2)	O1	AY704652	Silent substitution of C to T at position 1938 compared to the V. cholerae O1 biovar El Tor N16961 transcriptional repressor rstR (AE004224)
MM2071 (rstR2)	O1	AY704653	Silent substitution of C to T at position 1938 compared to the V. cholerae O1 biovar El Tor N16961 transcriptional repressor rstR (AE004224)
MM2071 (rstR4)	O1	AY704654	Silent substitution of A to G at position 375 and C to T at position 452 compared to the Vibrio phage CTX RSTR (rstR) (AY145127)
MP1950 (rstR1)	O139	AY704655	Identical to the V. cholerae 569B repressor rstR (AF055890)
MP1950 (rstR2)	O139	AY704656	Silent substitution of C to T at position 1938 compared to the V. cholerae O1 biovar El Tor N16961 transcriptional repressor rstR (AE004224)
MP1950 (rstR3)	O139	AY704657	Identical to the Vibrio phage CTXφ Calcutta rstR (AF133310)
MP2044 (rstR2)	O139	AY704658	Silent substitution of C to T at position 1938 compared to the V. cholerae O1 biovar El Tor N16961 transcriptional repressor rstR (AE004224)
MP2044 (rstR3)	O139	AY704659	Identical to the Vibrio phage CTXφ Calcutta rstR (AF133310)

Of the 169 O1 strains and 95 O139 strains, 9 and 2 strains, respectively, did not carry the ctxA gene and were considered nontoxigenic. Two of the nontoxigenic strains of V. cholerae O1, however, carried the rstR₂ genes. We further examined all nine V. cholerae O1 and two V. cholerae O139 strains with a probe specific for rstC to search for the presence of the RS1 element, which would explain the presence of the rstR₂ gene in the nontoxigenic V. cholerae O1 strains. However, only one of the two nontoxigenic V. cholerae O1 strains hybridized with the rstC probe; the other strain did not hybridize with the probe.

Three isolates from the year 1997 are of special interest. One of the isolates, MM004, is like the CTX-negative isolates of previous years, in that it was negative for an rstR gene of any type, ctxA, and rstC. The isolate MM1079, however, was positive for rstR₂ but negative for the ctxA gene and positive for rstC. Yet another 1997 isolate, MM2644, was rstR₂ positive but negative for both the ctxA and rstC genes. These strains might have undergone deletion in part of the CTX prophage. Overall, PCR results for the incidence of different rstR types in O1 and O139 strains showed no relationship between the temporal incidence of the disease and changes in CTX. However, the data presented do indicate variation in the incidence of rstR types, their presence in O1 and O139 strains, and the infrequent but interesting occurrence of multiple types in some strains. The rstR gene offers a window to assess the evolution of the phage.

Nucleotide sequence accession numbers.

The nucleotide sequence accession numbers of 10 rstR amplicons from five isolates of V. cholerae O1 and O139 have been submitted to GenBank under accession numbers AY704650 to AY704659.