A novel pre-CTX prophage in the Vibrio cholerae serogroup O139 strain.

In Vibrio cholerae, the lysogenic bacteriophage CTXΦ carries the cholera toxin genes ctxAB, which can be transferred from toxigenic strains to nontoxigenic strains through infection and lysogenic conversion of CTXΦ. This phage also has the precursor genome which does not harbor ctxAB, named pre-CTXΦ. Based on the sequences of the transcriptional regulator-encoding gene rstR alleles in CTXΦ/pre-CTXΦ, multiple types of these prophages have been classified and identified in toxigenic and nontoxigenic V. cholerae strains. In this study, by combining the short-read and long-read sequencing approaches of next generation sequencing, we obtained the complete genome sequence of the studied V. cholerae toxigenic serogroup O139 strain and identified the CTXΦ and a pre-CTXΦ genome type encoding a novel rstR allele, pre-CTXZHJΦ. This pre-CTX prophage integrates into the small chromosome of the V. cholerae host strain and coexists with a typical CTXETΦ prophage present in the large chromosome, which is commonly present in the seventh pandemic serogroup O1 and toxigenic serogroup O139 strains. RstRZHJ could bind to the ig-2 region in the RstAB promotor in the pre-CTXZHJΦ genome, and could repress the expression of its own rstAB genes but could not repress rstAB expression in CTXETΦ and CTXclassΦ, suggesting that the V. cholerae strains carrying the pre-CTXZHJΦ prophage cannot prevent the infection of these epidemic CTXΦs, hence have the potentiality to become toxigenic by acquiring and lysogenic conversion of CTXΦs. Our study identified a novel pre-CTXΦ type, and presents the new evidence for the complexity and diversity of the CTXΦ/pre-CTXΦ family in V. cholerae.

High quality reference genomes for toxigenic and non-toxigenic Vibrio cholerae serogroup O139.

Toxigenic Vibrio cholerae of the O139 serogroup have been responsible for several large cholera epidemics in South Asia, and continue to be of clinical and historical significance today. This serogroup was initially feared to represent a new, emerging V. cholerae clone that would lead to an eighth cholera pandemic. However, these concerns were ultimately unfounded. The majority of clinically relevant V. cholerae O139 isolates are closely related to serogroup O1, biotype El Tor V. cholerae, and comprise a single sublineage of the seventh pandemic El Tor lineage. Although related, these V. cholerae serogroups differ in several fundamental ways, in terms of their O-antigen, capsulation phenotype, and the genomic islands found on their chromosomes. Here, we present four complete, high-quality genomes for V. cholerae O139, obtained using long-read sequencing. Three of these sequences are from toxigenic V. cholerae, and one is from a bacterium which, although classified serologically as V. cholerae O139, lacks the CTXφ bacteriophage and the ability to produce cholera toxin. We highlight fundamental genomic differences between these isolates, the V. cholerae O1 reference strain N16961, and the prototypical O139 strain MO10. These sequences are an important resource for the scientific community, and will improve greatly our ability to perform genomic analyses of non-O1 V. cholerae in the future. These genomes also offer new insights into the biology of a V. cholerae serogroup that, from a genomic perspective, is poorly understood.

Environmental bacteriophages active on biofilms and planktonic forms of toxigenic Vibrio cholerae: Potential relevance in cholera epidemiology.

METHODS: Phages isolated from environmental waters in Bangladesh were tested for their host specificity towards V. cholerae O1 and O139, and the ability to disperse V. cholerae biofilms formed in the laboratory. Representative phages were further characterized by electron microscopy and whole genome sequencing. Selected phages were then introduced in various combinations to biofilms of toxigenic V. cholerae added to samples of river water, and the dispersion of biofilms as well as the growth kinetics of V. cholerae and the phages were monitored. RESULTS: A phage cocktail composed of three different phages isolated from surface waters in Bangladesh and designated as JSF7, JSF4, and JSF3 could significantly influence the distribution and concentration of the active planktonic form and biofilm associated form of toxigenic V. cholerae in water. While JSF7 showed a biofilm degrading activity and dispersed cells from both V. cholerae O1 and O139 derived biofilms thus increasing the concentration of planktonic V. cholerae in water, JSF4 and JSF3 showed strong bactericidal activity against V. cholerae O1 and O139 respectively. A mixture of all three phages could effectively reduce both biofilm-associated and planktonic V. cholerae in river water microcosms. SIGNIFICANCE: Besides potential applicability in phage-mediated control of cholera, our results have relevance in appreciating possible intricate role of diverse environmental phages in the epidemiology of the disease, since both biofilms and phages influence the prevalence and infectivity of V. cholerae in a variety of ways.

High prevalence and diversity of pre-CTXΦ alleles in the environmental Vibrio cholerae†O1 and O139 strains in the Zhujiang River estuary.

Toxigenic conversion of environmental Vibrio cholerae strains through lysogenic infection by the phage CTXΦ is an important step in the emergence of new pathogenic clones. The precursor form of the CTXΦ phage, pre-CTXΦ, does not carry the cholera toxin gene. During our investigation, we frequently found pre-CTXΦ prophages in non-toxigenic isolates in the serogroups of O1 and O139 strains in the Zhujiang estuary. We observed high amounts of sequence variation of rstR and gIII(CTX) in the pre-CTXΦ alleles as well as in the tcpA sequences within the strains. In addition, a new pre-CTXΦ allele, with a novel rstR sequence type and hybrid RS2, was identified. Our findings show that active, complicated gene recombination and horizontal transfer of pre-CTXΦs occurs within V. cholerae environmental strains, which creates a complex intermediate pool for the generation of toxigenic clones in the estuarine environment.

Whole genome sequencing and comparative genomic analyses of two Vibrio cholerae O139 Bengal-specific Podoviruses to other N4-like phages reveal extensive genetic diversity.

BACKGROUND: Vibrio cholerae O139 Bengal is the only serogroup other than O1 implicated in cholera epidemics. We describe the isolation and characterization of an O139 serogroup-specific phage, vB_VchP_VchO139-I (ϕVchO139-I) that has similar host range and virion morphology as phage vB_VchP_JA1 (ϕJA1) described previously. We aimed at a complete molecular characterization of both phages and elucidation of their genetic and structural differences and assessment of their genetic relatedness to the N4-like phage group. METHODS: Host-range analysis and plaque morphology screening were done for both ϕJA1 and ϕVchO139-I. Both phage genomes were sequenced by a 454 and Sanger hybrid approach. Genomes were annotated and protein homologies were determined by Blast and HHPred. Restriction profiles, PFGE patterns and data on the physical genome structure were acquired and phylogenetic analyses were performed. RESULTS: The host specificity of ϕJA1 has been attributed to the unique capsular O-antigen produced by O139 strains. Plaque morphologies of the two phages were different; ϕVchO139-I produced a larger halo around the plaques than ϕJA1. Restriction profiles of ϕJA1 and ϕVchO139-I genomes were also different. The genomes of ϕJA1 and ϕVchO139-I consisted of linear double-stranded DNA of 71,252 and 70,938 base pairs. The presence of direct terminal repeats of around 1974 base pairs was demonstrated. Whole genome comparison revealed single nucleotide polymorphisms, small insertions/deletions and differences in gene content. Both genomes had 79 predicted protein encoding sequences, of which only 59 were identical between the two closely related phages. They also encoded one tRNA-Arg gene, an intein within the large terminase gene, and four homing endonuclease genes. Whole genome phylogenetic analyses of ϕJA1 and ϕVchO139-I against other sequenced N4-like phages delineate three novel subgroups or clades within this phage family. CONCLUSIONS: The closely related phages feature significant genetic differences, in spite of being morphologically identical. The phage morphology, genetic organization, genomic content and large terminase protein based phylogeny support the placement of these two phages in the Podoviridae family, more specifically within the N4-like phage group. The physical genome structure of ϕJA1 could be demonstrated experimentally. Our data pave the way for potential use of ϕJA1 and ϕVchO139-I in Vibrio cholerae typing and control.

Phage types of Vibrio cholerae O1 and O139 in the past decade in India.

Cholera has been a prevalent disease worldwide since the early 19th century. Vibrio cholerae O1 and O139 are the two serogroups that have been mainly implicated in causing cholera. This study reports the results of biotyping, serotyping and phage typing of V. cholerae O1 and O139 (1998-2007) strains received from different parts of India for the identification of the trends in the occurrence and spread of cholera in the country. However, there has been a notable steep decline in the occurrence of V. cholerae O139 strains over the past few years resulting in no strain of V. cholerae O139 being received from any part of India in 2007 and 2008. Of the total strains received, 79.1% were serotyped as Ogawa and the remaining 20.9% were found to be Inaba, which indicates that Ogawa was the predominant serotype. Almost 100% typeability was observed with the new scheme of V. cholerae O1, with type 27 being the dominant phage type and V. cholerae O139 strains were clustered into the predominant phage type T-1. From the phage typing and serotyping results, it can be concluded that V. cholerae O1 (T-27) and O139 (T-1) strains circulate throughout the country at any given time.

Effect of storage and sodium chloride on excision of CTXPhi or pre-CTXPhi and CTXPhi from Vibrio cholerae O139 strains.

We examined the effect of storage and sodium chloride on excision of CTXPhi or pre-CTXPhi and CTXPhi from Vibrio cholerae O139 strains. We found that one strain of V. cholerae O139 VO146P showed loss of the complete phage array, and other strain VO170P showed partial loss of the phage array giving rise to altered strains designated as VO146N and VO170N. Results of PCR and RFLP analysis revealed that both strains (VO146P and VO170P) possessed a single copy of pre-CTX(ET)Phi and two copies of CTXPhi comprising CTX(Class)Phi and CTX(Calc)Phi arranged in tandem, and integrated in the large chromosome. The presence of classical ctxB was detected in CTX(Calc)Phi of both V. cholerae O139 strains. Nucleotide sequencing of three housekeeping genes showed no difference between parent and altered strains of V. cholerae O139.

Genetic analysis of CTX prophages with special reference to ctxB and rstR alleles of Vibrio cholerae O139 strains isolated from Kolkata over a decade.

Chronological analysis of 125 Vibrio cholerae O139 strains isolated during 1993-2005 in Kolkata revealed the prevalence of two new genotypes of cholera toxin (CT) and novel combinations of ctxB and rstR alleles resulting in variant CTX prophages. One of the new genotypes of ctxB, which first appeared in 1996 with the re-emerged V. cholerae O139 strains that had CTX Calcutta phage, was designated as genotype 4. In 1998, another new genotype, designated as genotype 5, was detected that prevailed mostly in CTX phages with El Tor rstR. The prototype El Tor CTX phage with genotype 3 gradually disappeared in O139, and since 2002 the predominant CTX prophages in O139 are Calcutta phages with genotype 4 and El Tor phages with genotype 5. Results showed that V. cholerae O139 strains of Kolkata, isolated over a decade, harboured CTX prophages in the large chromosome having no RS1 downstream of CTX prophage. During the course of its intermittent incidence over a decade, five types of O139 strains were detected based on CT genotypes. Such abrupt genetic changes in O139 strains might not favour its continued prevalence in human cases in Kolkata, India.

Surveillance of vibriophages reveals their role as biomonitoring agents in Kolkata.

Cholera is a public health threat in all developing countries. Kolkata, a city in eastern India, is an endemic zone for cholera. During the course of a comprehensive investigation on the distribution of phages of Vibrio cholerae O1 and O139 in freshwater bodies in Kolkata, we were able to isolate the phages of V. cholerae O1 and O139. Vibrio cholerae O1 phages were found at all the sites and exhibited a distinct seasonal cycle, with a primary peak (13.6-17.2 PFU mL(-1)) during monsoon (June to August) in both 2006 and 2007. Vibrio cholerae O139 phages were present in the environment and were predominant during monsoon in the year 2006, except for late winter and early summer from February to April. In contrast, in the year 2007, the O139 phages could be isolated only during July to December, with the highest counts of 12.0 PFU mL(-1) determined in August. The multiplex PCR results showed that 90 samples were positive for wbe of V. cholerae O1, 32 samples for O139 (wbf) and 18 samples for both. This study shows that surveillance of vibriophages indicates the presence of V. cholerae O1 and O139 in water bodies in and around Kolkata and could therefore serve as a powerful biomonitoring agent.

Filamentous vibriophage fs2 encoding the rstC gene integrates into the same chromosomal region as the CTX phage [corrected].

The genome of the filamentous phage of Vibrio cholerae fs2 was found to contain rstC and rstB1 (truncated) genes downstream of ORF500. att-fs2-dir and att-fs2-rev sequences homologous to that of att-CTXphi were found between orf500 and rstC of the fs2 genome. This prompted us to search for the integration site of fs2 in the genomes of V. cholerae O1 and O139. The genome of fs2 was found to integrate downstream of attRS of the CTXphi phage, which integrated into chromosome I of V. cholerae O1 and O139. When infected with fs2, a fimbriate strain of V. cholerae O1 appeared to reduce fimbrial production in an adult rabbit ileal loop assay.

El Tor and Calcutta CTXPhi precursors coexisting with intact CTXPhi copies in Vibrio cholerae O139 isolates.

Restriction fragment length polymorphism analyses of the array of CTXPhi prophages in strains CRC262 and CRC266 of Vibrio cholerae O139 revealed the presence of copies of complete CTXPhi and pre-CTXPhi prophages coexisting at a single chromosomal locus in each strain. Restriction pattern and comparative nucleotide sequence analysis revealed pre-CTXPhi precursors of both the El Tor and Calcutta lineages. Thus, we hypothesize that two precursor variants independently acquired cholera toxin genes and gave rise to the current El Tor and Calcutta CTXPhi prophages. We discuss the implications of these results in terms of the evolution and origin of the current diversity of CTXPhi prophages.

Complexity of rice-water stool from patients with Vibrio cholerae plays a role in the transmission of infectious diarrhea.

At the International Centre for Diarrhoeal Disease Research, Bangladesh, one-half of the rice-water stool samples that were culture-positive for Vibrio cholerae did not contain motile V. cholerae by standard darkfield microscopy and were defined as darkfield-negative (DF(-)). We evaluated the host and microbial factors associated with DF status, as well as the impact of DF status on transmission. Viable counts of V. cholerae in DF(-) stools were three logs lower than in DF(+) stools, although DF(-) and DF(+) stools had similar direct counts of V. cholerae by microscopy. In DF(-) samples, non-V. cholerae bacteria outnumbered V. cholerae 10:1. Lytic V. cholerae bacteriophage were present in 90% of DF(-) samples compared with 35% of DF(+) samples, suggesting that bacteriophage may limit culture-positive patients from producing DF(+) stools. V. cholerae in DF(-) and DF(+) samples were found both planktonically and in distinct nonplanktonic populations; the distribution of organisms between these compartments did not differ appreciably between DF(-) and DF(+) stools. This biology may impact transmission because epidemiological data suggested that household contacts of a DF(+) index case were at greater risk of infection with V. cholerae. We propose a model in which V. cholerae multiply in the small intestine to produce a fluid niche that is dominated by V. cholerae. If lytic phage are present, viable counts of V. cholerae drop, stools become DF(-), other microorganisms bloom, and cholera transmission is reduced.

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.

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.