Cholera Phage Research Articles

Temperate bacteriophage ΦO18P from an Aeromonas media isolate: Characterization and complete genome sequence

A group of 74 Aeromonas isolates from surface water of three ponds in Bielefeld, Germany was screened for prophage induction after UV irradiation. The phage ΦO18P was induced from the Aeromonas media isolate O18. ΦO18P belongs to the Myoviridae phage family. The complete nucleotide sequence of the double stranded DNA genome of bacteriophage ΦO18P consists of 33,985 bp. The genome has 5′ protruding cohesive ends of 16 bases. On the ΦO18P genome 46 open reading frames ( orfs) were identified which are organized in the modules integration and regulation, replication, head, packaging, tail and lysis. Additionally the phage DNA includes a methylase gene. Comparison of the genome architecture with those of other bacteriophages revealed significant similarities to the P2 phage family and especially to the prophages of Aeromonas salmonicida and the Vibrio cholerae phage K139.

Vibrio cholerae O139 Bengal—10 years on

A second aetiological agent of cholera, Vibrio cholerae O139 Bengal was identified in late 1992 when it caused large outbreaks of diarrhoea in India and Bangladesh. The new strain probably arose as a result of lateral transfer of genes encoding a novel somatic antigen and a capsule from an unknown bacterium to an O1 El Tor strain with the subsequent loss of genes encoding O1 somatic antigen. O139 produces a semi-rough type colony with a truncated lipopolysaccharide which contains a unique sugar, colitose. Otherwise, O139 and O1 are strikingly similar. The diseases produced by the two serogroups are indistinguishable. There is a moderate inflammatory response in cholera, and in spite of the possession of a capsule by O139, it is no more inflammatory than O1. A number of diagnostic tests have been developed for O139 that are modelled after tests for O1 including rapid tests for field use. O139 has spread to countries of south and south-east Asia, China and Russia. Currently, cholera is caused by both these serogroups in these countries with the O1 serogroup predominant. O139 continues to cause occasional local outbreaks in India and Bangladesh. Genotyping of isolates has shown circulation of multiple clones and their derivation from multiple progenitors. Although there have been changes in antibiogram with the resistance genes being carried on a constin, the strains remain susceptible to tetracycline, a preferred antibiotic for treatment. O139 carries a number of filamentous lysogenic and lytic phages and the latter have been utilized to develop a phage typing scheme. A number of environmental variables have been linked to the occurrence of cholera and lytic phages may determine the course of epidemics. Although the capsule confers serum resistance, vibriocidal antibody assay has been developed for O139. However, unlike in O1 cholera, serum vibriocidal antibody does not seem to correlate with protection against O139 infection. Promising O139 vaccines have been developed or are in the pipeline. It is too early to predict whether O139 will become the causative agent of 'the eighth pandemic of cholera'; it will require many years of monitoring and reporting from regions prone to cholera to find the answer.

New Wrinkles and Folds in Site-Specific Recombination

The new work of reveals a new site-specific recombination strategy to establish lysogeny, in which a double-stranded recombination substrate is assembled from the folded single-stranded DNA genome of the filamentous Vibrio cholerae phage CTXphi. This strategy allows the phage to use the host's recombinases while at the same time preventing inappropriate excision of the prophage.

Self-limiting nature of seasonal cholera epidemics: Role of host-mediated amplification of phage.

Phage predation of Vibrio cholerae has recently been reported to be a factor that influences seasonal epidemics of cholera in Bangladesh. To understand more about this phenomenon, we studied the dynamics of the V. cholerae-phage interaction during a recent epidemic in Dhaka. Because the outbreak strain causing this epidemic was resistant to multiple antibiotics, including streptomycin, we used a selective medium containing streptomycin to monitor accurately the abundance of this strain in the environment. The changing prevalence in the environment of the epidemic V. cholerae O1 strain and a particular lytic cholera phage (JSF4) to which it was sensitive was measured every 48-72 h for 17 weeks. We also monitored the incidence of phage excretion in stools of 387 cholera patients during the epidemic. The peak of the epidemic was preceded by high V. cholerae prevalence in the environment and was followed by high JSF4 phage levels as the epidemic ended. The buildup to the phage peak in the environment coincided with increasing excretion of the same phage in the stools of cholera patients. These results suggest that patients toward the end of the epidemic ingested both JSF4 phage and the outbreak V. cholerae strain. Host-mediated phage amplification during the cholera epidemic likely contributed to increased environmental phage abundance, decreased load of environmental V. cholerae and, hence, the collapse of the epidemic. Thus, in vivo phage amplification in patients and subsequent phage predation in the environment may explain the self-limiting nature of seasonal cholera epidemics in Bangladesh.

Seasonal epidemics of cholera inversely correlate with the prevalence of environmental cholera phages

The relationship among (i) the local incidence of cholera, (ii) the prevalence in the aquatic environment of Vibrio cholerae, and (iii) bacterial viruses that attack potentially virulent O1 and O139 serogroup strains of this organism (cholera phages) was studied in Dhaka, Bangladesh. Over nearly a 3-year period, we found that significantly more environmental water samples contained either a phage or a phage-susceptible V. cholerae strain than both (P < 0.00001). The number of cholera patients varied seasonally during this period and frequently coincided with the presence of pathogenic V. cholerae strains in water samples that otherwise lacked detectable cholera phages. Interepidemic periods were characterized by water samples containing cholera phages but no viable bacteria. Our data support the conclusion that cholera phages can influence cholera seasonality and may also play a role in emergence of new V. cholerae pandemic serogroups or clones.

Emergent Strain of Human Adenovirus Endemic in Iowa

To the Editor: We read with interest the letter by Sarkar et al. on new Vibrio cholerae phages (1). The description of new V. cholerae phages is a welcome tool for epidemiologic studies of this species. Our main concern about their work is the inaccurate picture that is presented of the cholera epidemic in Brazil. Some of the statements made in the final paragraphs are in disagreement with the official epidemiologic records and the characteristics of the Vibrio bacteria that occurred in Brazil during the 1990s epidemic (2). In 1991, the seventh cholera pandemic reached South America by the Pacific coast, spreading to Brazil in the same year (3). In Brazil, the first cholera cases were reported in the Amazon region bordering Peru; within a few months a large number of cholera cases were recorded in states facing the Atlantic Ocean in the northeastern region (2). According to the official figures of the Brazilian Ministry of Health (2), 168,598 cases of cholera caused by a V. cholerae O1 El Tor strain occurred in Brazil from 1991 to 2001. Of these, 155,363 (92.1%) occurred in the northeastern area of the country, with 2,037 deaths. From 2001 to 2003, the number of confirmed cases was 4,756, 734, and 7, respectively. Sarkar et al. (1) indicate that 60,000 cases occurred from 1991 to 2001 in Rio de Janeiro, a city localized in the southeastern region; the official records report only 349 cases. The statement that “since 1993, no cholera cases caused by O1 have been reported” is also perplexing. From 1994 to 2001, the official records report 68,583 cases of cholera in Brazil (51,324 of these in 1994, the second major year of cholera incidence). Are the authors suggesting that this number of cases was caused by non-O1 V. cholerae? The official records state that the cholera epidemic in Brazil was caused by an El Tor O1 strain (4,5).

Physicochemical characterization of vibriophage N5

Phage N5 is one of the phages of Vibrio cholerae serovar O1 biotype El Tor (Ghosh, A. N., Ansari, M. Q., and Dutta, G. C. Isolation and morphological characterization of El Tor cholera phages. J. Gen. Virol. 70: 2241–2243, 1989). In the present communication the growth curve, molecular weight and confirmation of the genome, partial denaturation map and restriction endonuclease digestion pattern have been determined. Partial denaturation map indicates that the genome has non-permuted / invariant sequence. Presence of cohesive ends has also been documented.

VGJ phi, a novel filamentous phage of Vibrio cholerae, integrates into the same chromosomal site as CTX phi.

We describe a novel filamentous phage, designated VGJ phi, isolated from strain SG25-1 of Vibrio cholerae O139, which infects all O1 (classical and El Tor) and O139 strains tested. The sequence of the 7,542 nucleotides of the phage genome reveals that VGJ phi has a distinctive region of 775 nucleotides and a conserved region with an overall genomic organization similar to that of previously characterized filamentous phages, such as CTX phi of V. cholerae and Ff phages of Escherichia coli. The conserved region carries 10 open reading frames (ORFs) coding for products homologous to previously reported peptides of other filamentous phages, and the distinctive region carries one ORF whose product is not homologous to any known peptide. VGJ phi, like other filamentous phages, uses a type IV pilus to infect V. cholerae; in this case, the pilus is the mannose-sensitive hemagglutinin. VGJ phi-infected V. cholerae overexpresses the product of one ORF of the phage (ORF112), which is similar to single-stranded DNA binding proteins of other filamentous phages. Once inside a cell, VGJ phi is able to integrate its genome into the same chromosomal attB site as CTX phi, entering into a lysogenic state. Additionally, we found an attP structure in VGJ phi, which is also conserved in several lysogenic filamentous phages from different bacterial hosts. Finally, since different filamentous phages seem to integrate into the bacterial dif locus by a general mechanism, we propose a model in which repeated integration events with different phages might have contributed to the evolution of the CTX chromosomal region in V. cholerae El Tor.

Tetracycline resistant and polymyxin B sensitive vibrio cholerae O1 E1 tor isolated from the recent epidemics.

By examining 99 Vibrio cholerae O1 isolates from cholera epidemics in a variety of areas in Laos in 1998, we found two unusual characteristics of the organisms. All except 4 isolates were moderately resistant to tetracycline without having plasmids, and susceptibilities to the other drugs were as expected. Eleven isolates showed the same level of susceptibility to polymyxin B as classical V. cholerae O1. With the exception of 4 tetracycline-susceptible strains, all the isolates were resistant to or poorly sensitive to the vibriostatic agent O/129 (2, 4-diamino-6, 7-di-iso-propyl pteridine phosphate). All isolates, including those susceptible to polymyxin B, produced El Tor hemolysin which was neutralized by anti-El Tor hemolysin and were resistant to cholera phage IV, indicating that they were of the El Tor biotype.

Characterization of Vibrio cholerae O1 Isolated in Lao People's Democratic Republic.

Thirty-four strains of Vibrio cholerae O1 serotype Ogawa isolated in Lao People's Democratic Republic in 1995 were examined for their biological behaviour and cholera toxin productivity. Biotyping indicated that the 34 strains belonged to the El Tor biotype. All strains were hemolysin positive and resistant to polymyxin B and cholera phage IV. However, 4 strains were negative for the hemolysin activity as determined by the test tube method, although they were positive for the antigenic activity. Prophage typing identified 31 strains of the Celebes type, including 3 cured Celebes type, and 3 Ubol type. All the strains produced detectable levels of cholera toxin; 15 isolates produced more than 100 ng/ml, 10 strains between 10 and 100 ng/ml and 9 strains less than 10 ng/ml, determined by using AKI medium in a stationary test tube. The maximum production was 200 ng/ml.

Structural organization of the transfer RNA gene clusters of cholera phage φ l49

Vibrio cholerae phage φ l49 codes tRNAs specific for twelve different amino acids. These tRNA genes are contained in two different HindIII fragments 11 and 3.4 kb in size, of the phage genome. The 3.4 kb HindIII fragment was cloned inEscherichia coli using pBR328 as vector. The recombinant plasmid pNR347 produced nine of the twelve tRNA species (arginine, proline, serine, tyrosine, histidine, lysine, leucine, tryptophan and aspartic acid) encoded in the phage genome.

Vibrio cholerae serogroup O1 in northeast Thailand

Strains of Vibrio cholerae serogroup O1 biotype El Tor that are susceptible to Mukerjee cholera bacteriophage group IV (S. Mukerjee, Bull. W.H.O. 28:333-336, 1963) were found. Cholera vibrios isolated from epidemics in northeast Thailand were characterized, and 57 of 60 strains isolated in 1986 were susceptible to cholera phage IV. However, all 113 strains isolated in 1988 were not susceptible to the phage. All isolates in both epidemics revealed behaviors typical of El Tor vibrios, except phage IV susceptibility in the 57 strains. Although the plaques of phage IV were generally translucent, plaques on some isolates looked transparent, just like those on classical vibrios. The organisms grown in the plaques were lysogenized. If this kind of strain is frequently isolated, the biotype of V. cholerae O1 should be reconsidered.

Hemolytic Vibrio cholerae O1 that is sensitive to Mukerjee's cholera phage IV and the phage produced by the hemolytic vibrio lysogenized with the infection of Mukerjee's cholera phage IV.

Strains of hemolytic Vibrio cholerae O1 (El Tor vibrio) which are sensitive to Mukerjee's cholera phage group IV were isolated from cholera patients in North-East Thailand in 1986. Plaques of the phage on these hemolytic V. cholerae O1 were usually translucent but almost transparent on some strains, just like the plaques on non-hemolytic V. cholerae O1 (classical vibrio). These hemolytic V. cholerae O1 were lysogenized with the infection of cholera phage IV, and the lysogenized strains produced phage different from cholera phage IV. These hemolytic strains were classified into Cured type in prophage typing of V. cholerae O1, El Tor, because they were also lysogenized with Kappa phage and were hemolytic. When Cured-type V. cholerae O1, El Tor previously isolated in various countries were examined for the sensitivity to cholera phage IV, some of the isolates were sensitive.

Isolation and morphological characterization of El Tor cholera phages.

Five bacteriophages have been isolated for phage typing of Vibrio cholerae biotype El Tor. The morphology of these phages has been studied by electron microscopy.

Characterization of the phage-specific transfer RNA molecules coded by cholera phage φ 149

Aminoacylation of tRNA isolated from choleraphage φ 149-infected cells with individual 3H-labeled l-amino acids followed by hybridization with phage DNA revealed that the phage encodes tRNAs specific for arginine, proline, glycine, isoleucine, serine, valine, tyrosine, histidine, lysine, leucine, tryptophan, and aspartic acid. Aminoacylation of phagecoded tRNAs isolated from phage DNA-RNA hybrids also confirmed this observation except for tryptophan.

Ultraviolet inactivation and photoreactivation of the cholera phage 'kappa'.

The lysogenic cholera phage, 'Kappa' is some ten to twenty folds more resistant to UV (254 nm) than are most of the T. phages of E. coli, or the cholera phage PL 163/10, or the host V. cholerae strain H218 Smr, the 37% (D37) and 10% (D10) survival doses being 255.8 J/m2 and 633.6 J/m2 respectively. The UV-irradiated 'Kappa' phages could be photoreactivated in the host V. cholerae strain H218 Smr to a maximum extent of 40%. The removal of the number of lethal hits per phage by the survival-enhancement treatment (photoreactivation) with time followed an exponential relation, the constant probability of removal of lethal hit per unit time being 2.8 x 10(-2)min-1. The UV-irradiated phages could also be Weigle reactivated in the host strain H218 Smr by a small degree, the maximum reactivation factor (ratio of survivals in UV-irradiated and non-irradiated hosts) being 1.50.

Inducible reactivation of UV-irradiated cholera phage e5 in Vibrio cholerae MAK757.

The survival of UV-irradiated cholera phage e5 was found to increase when the host cells, Vibrio cholerae MAK757, were exposed to a low dose of UV irradiation before phage infection (Weigle reactivation), indicating the existence of a UV-inducible DNA repair pathway (SOS repair) in V. cholerae MAK757. The induction signal generated by UV irradiation was transient in nature and lasted about 20-30 min at 37 degrees C. Maximal Weigle reactivation of the phage was obtained when the host cells were irradiated with a UV dose of 16 J/m2. V. cholerae MAK757 was also found to possess efficient photoreactivation and host cell reactivation of UV-damaged DNA in phage e5.

Characterization of Vibrio eltor typing phages: properties of the Eltor phage e4.

Biophysical characteristics of Vibrio eltor phage e4, a key phage in the Vibrio cholerae typing scheme were studied. This icosahedral phage was found to contain 12 structural polypeptides with mol. wt. ranging from 25,000 to 120,000. One of these polypeptides of mol. wt. 50,000 accounted for most of the structural proteins present and was probably the major phage capsid protein. The phage genome comprised a single linear, double-stranded DNA molecule, 69.2 kbp in length (45.6 X 10(6) mol. wt.) as determined by electron microscopy and restriction fragment analyses. The G + C content was 34.6%. Electron microscopy data indicated that unlike the DNAs of other cholera phages, phage e4 DNA is not circularly permuted. Adsorption under normal conditions was biphasic with rate constants of 1.02 X 10(-9)/ml/min up to 60% adsorption and 3 X 10(-10)/ml/min thereafter. Intracellular phage multiplication was characterized by a latent period of 27 min. The burst size was approximately 100 phage particles per infected cell.

沖縄の都市河川から分離したコレラ菌の諸性状とその病原性について

In 1980, Vibri cholerae O1, biotype El Tor, serotype Inaba, was isolated from the Aja river, Okinawa, The 12 strains examined belonged to classical Ubol type.The original focus of the organisms could not be determined in spite of intensive survey.The organisms was characterized by an unique biochemical behaviour, i.e., no fermentation of sucrose in aerobic condition within 24 hours.The phage typing by El Tor cholera phage revealed type 1 in 11 strains and type 6 in 1 strain.On the contrary, the other 32 Ubol type strains recently isolated in various countries in the world revealed type 4 or untypable. These facts suggested that the Vibrio cholerae O1 isolated in Okinawa was not recently imported, but has been living there for a long time as native organism.The pathogenicity of the organisms were examined with De-test, toxigenicity and adhesive property to the intestinal epithelium.And these organisms appeared non or less pathogenic.

Repair of ultraviolet light-induced DNA damage in cholera bacteriophages.

DNA repair-proficient and -deficient strains of Vibrio cholerae were used to examine host cell reactivation, Weigle reactivation and photoreactivation of u.v.-irradiated cholera bacteriophages. U.v. light-induced DNA damage in phages of different morphological and serological groups could be efficiently photoreactivated. Host cell reactivation of irradiated phages of different groups was different on the same indicator host. Phage phi 149 was the most sensitive, and phi 138 the most resistant to u.v. irradiation. While phi 138 showed appreciable host cell reactivation, this was minimal for phi 149. Attempts to demonstrate Weigle reactivation of u.v.-irradiated cholera phages were not successful, although u.v.-induced filamentation of host cells was observed.