Characterization of novel virulent broad-host-range phages of Xylella fastidiosa and Xanthomonas.

The xylem-limited bacterium Xylella fastidiosa is the causal agent of several plant diseases, most notably Pierce's disease of grape and citrus variegated chlorosis. We report the isolation and characterization of the first virulent phages for X. fastidiosa, siphophages Sano and Salvo and podophages Prado and Paz, with a host range that includes Xanthomonas spp. Phages propagated on homologous hosts had observed adsorption rate constants of ~4 × 10(-12) ml cell(-1) min(-1) for X. fastidiosa strain Temecula 1 and ~5 × 10(-10) to 7 × 10(-10) ml cell(-1) min(-1) for Xanthomonas strain EC-12. Sano and Salvo exhibit >80% nucleotide identity to each other in aligned regions and are syntenic to phage BcepNazgul. We propose that phage BcepNazgul is the founding member of a novel phage type, to which Sano and Salvo belong. The lysis genes of the Nazgul-like phage type include a gene that encodes an outer membrane lipoprotein endolysin and also spanin gene families that provide insight into the evolution of the lysis pathway for phages of Gram-negative hosts. Prado and Paz, although exhibiting no significant DNA homology to each other, are new members of the phiKMV-like phage type, based on the position of the single-subunit RNA polymerase gene. The four phages are type IV pilus dependent for infection of both X. fastidiosa and Xanthomonas. The phages may be useful as agents for an effective and environmentally responsible strategy for the control of diseases caused by X. fastidiosa.

Vibrio cholerae typing phage N4: genome sequence and its relatedness to T7 viral supergroup.

OBJECTIVES: In countries where cholera is endemic, Vibrio cholerae O1 bacteriophages have been detected in sewage water. These have been used to serve not only as strain markers, but also for the typing of V. cholerae strains. Vibriophage N4 (ATCC 51352-B1) occupies a unique position in the new phage-typing scheme and can infect a larger number of V. cholerae O1 biotype El Tor strains. Here we characterized the complete genome sequence of this typing vibriophage. METHODS: The complete DNA sequence of the N4 genome was determined by using a shotgun sequencing approach. RESULTS: Complete genome sequence explored that phage N4 is comprised of one circular, double-stranded chromosome of 38,497 bp with an overall GC content of 42.8%. A total of 47 open reading frames were identified and functions could be assigned to 30 of them. Further, a close relationship with another vibriophage, VP4, and the enterobacteriophage T7 could be established. DNA-DNA hybridization among V. cholerae O1 and O139 phages revealed homology among O1 vibriophages at their genomic level. CONCLUSIONS: This study indicates two evolutionary distinctive branches of the possible phylogenetic origin of O1 and O139 vibriophages and provides an unveiled collection of information on viral gene products of typing vibriophages.

Detection of virulence-associated and regulatory protein genes in association with phage typing of human Vibrio cholerae from several geographical regions of the world.

Vibrio cholerae O1, O139 and occasionally non-O1/non-O139 serogroups are most often responsible for epidemic and pandemic cholera. This study used genotypic patterns of PCR-based detection of virulence-associated and regulatory protein genes, along with phage typing, to characterize 86 V. cholerae strains. Thirty-eight of 53 O1 biotype El Tor strains harboured both tcpA classical and tcpA El Tor genes, and three El Tor strains lacked the V. cholerae O1-specific gene (Vc-O1); three O139 strains contained both Vc-O1 and Vc-O139 genes and seven out of ten non-O1/non-O139 strains possessed the Vc-O1 gene. The latter strains all harboured the virulence-associated genes ctxA, zot, ace, RS1, hlyA, ompU, rtxA and sxt. Two phage types, T27 and T25, were predominant in strains from different geographical regions of India, whereas more variation in phage susceptibility was observed for tetracycline-resistant strains from Kolkata. These results suggest that the pattern and distribution of virulence genes and phage types of V. cholerae are equally useful and discriminatory in tracing the origin of newly emerging strains.

Isolation and Characterization of Novel Broad Host Range Bacteriophages of Vibrio cholerae O1 from Bengal.

OBJECTIVES: We have isolated a total of five newer cholera phages which are novel broad host range to incorporate with the existing phage typing schemes for an extended typing scheme. MATERIALS AND METHODS: These newly isolated phages were well characterized including the electron micrograph. A total of 300 Vibrio cholerae strains were isolated from the different endemic region in India were included in phage typing study. RESULTS: These phages were found different from the existing phages. Electron microscopic results showed that the phages belonged to myophage and podophage group. Characterization of the phages based on pH, temperature, and organic solvent sensitivity showed differences among the phages used in this study. All the strains of Vibrio O1 were typeable (100%) with the five set of cholera phages. Of these, 40% strains were clustered under Type-1. CONCLUSION: The newer Vibrio phages are novel and broad host range and will be useful to incorporate with the existing phage typing system for more precisely discriminate the strains of Vibrio cholerae.

Transmission of phage by glassy-winged sharpshooters, a vector of Xylella fastidiosa.

Xylella fastidiosa subsp. fastidiosa (Xff) is the causal agent of Pierce's Disease (PD) of grapevines and is vectored by the glassy-winged sharpshooter (GWSS, Homalodisca vitripennis). Previously we have reported the development of a bacteriophage (phage) based biocontrol system for PD, but no information on insect transmission of phages has been reported. Here we communicate that laboratory reared GWSSs fed on cowpea plants (Vigna unguiculata subsp. unguiculata) harboring the virulent phage Paz were able to uptake of phage efficiently when the phage was present in high concentration, but were inefficient in transfer to plants.

Control of Pierce's Disease by Phage.

Pierce's Disease (PD) of grapevines, caused by Xylella fastidiosa subsp. fastidiosa (Xf), is a limiting factor in the cultivation of grapevines in the US. There are presently no effective control methods to prevent or treat PD. The therapeutic and prophylactic efficacy of a phage cocktail composed of four virulent (lytic) phages was evaluated for control of PD. Xf levels in grapevines were significantly reduced in therapeutically or prophylactically treated grapevines. PD symptoms ceased to progress one week post-therapeutic treatment and symptoms were not observed in prophylactically treated grapevines. Cocktail phage levels increased in grapevines in the presence of the host. No in planta phage-resistant Xf isolates were obtained. Moreover, Xf mutants selected for phage resistance in vitro did not cause PD symptoms. Our results indicate that phages have great potential for biocontrol of PD and other economically important diseases caused by Xylella.

Genotypic characterization of Vibrio cholerae isolates using several DNA fingerprint techniques.

Serious pandemics of cholera have occurred throughout the known history of mankind, especially in India, which is a motherland for cholera disease. For the last 20 years several DNA-based typing methods have been employed to study the clonal relatedness between Vibrio cholerae isolates irrespective of their geographical locations. Traditional typing methods, such as biochemical tests, phage typing, serotyping, biotyping and antimicrobial susceptibility tests, have produced reliable and informative data regarding V. cholerae for a long time. Gradually molecular typing techniques have taken the place of traditional typing methods because they produce the same results upon repeat testing of V. cholerae strain. In this article we focus on the discriminatory power of different DNA fingerprint techniques that are generally used to know the homogeneity and heterogeneity among different V. cholerae isolates.

Pathogenic potential of vibriophages against an experimental infection with Vibrio cholerae O1 in the RITARD model.

Cholera continues to be an important public health problem in developing countries, including India. This study concerns the feasibility of possible exploitation of bacteriophages as a biocontrol agent to eliminate the pathogen Vibrio cholerae from the gut using the removable intestinal tie-adult rabbit diarrhoea (RITARD) model. A control rabbit challenged with 10(9) colony-forming units (CFU)/mL of V. cholerae MAK 757 developed Grade II to IV diarrhoea, but the phage-treated rabbit that received 10(9)CFU/mL MAK 757 and 10(8) plaque-forming units (PFU)/mL cocktail phages produced only Grade II diarrhoea. Histological results revealed that in the control rabbit (MAK 757-treated), villi lost their normal shape and showed more inflammatory cellular infiltration in the lamina propria compared with the experimental rabbit. Our data suggest that phages could be valuable as prophylaxis against V. cholerae infection.

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.

Phenotypic and molecular typing of Vibrio cholerae O1 and O139 isolates from India.

OBJECTIVES: To determine whether Vibrio cholerae strains with similar phage types are also genetically homogeneous or heterogeneous by molecular typing methods like PFGE and RAPD-PCR employed in this study. METHODS: A total of 26 V. cholerae O1 and O139 strains received from different parts of India were analyzed by using conventional phage typing method, antibiogram and molecular typing methods such as PFGE and RAPD-PCR. RESULTS: Both O1 and O139 strains were resistant against two antibiotics (Ampicillin and Furazolidone) were detected. All of these strains were clustered in a single phage type, i.e., Type 27 for V. cholerae O1 and Type 1 for V. cholerae O139. Extensive molecular characterization by RAPD and PFGE showed that six sets of O1 and O139 strains, each comprising two strains, had identical PFGE and RAPD profiles. Only one O139 strain (PL-4) had unique RAPD and PFGE profile among all the 26 V. cholerae strains used in this study. CONCLUSION: Apart from serology, the strains of V. cholerae can be discriminated by this conventional phage typing system that offers the basic information on identification, biotyping and discrimination of strains. But, a high level of heterogeneity was observed in RAPD and PFGE profiles indicating the clonal diversity of V. cholerae O1 and O139 strains. It was concluded that these strains were phenotypically identical through phage typing system and antibiogram but genetically dissimilar, as shown in molecular typing systems.