The ability of two different Vibrio spp. bacteriophages to infect Vibrio harveyi, Vibrio cholerae and Vibrio mimicus

AIMS: To determine the host range of the Vibrio harveyi myovirus-like bacteriophage (VHML) and the cholera toxin conversion bacteriophage (CTX Phi) within a range of Vibrio cholerae and V. mimicus and V. harveyi, V. cholerae and V. mimicus isolates respectively. METHODS AND RESULTS: Three V. harveyi, eight V. cholerae and five V. mimicus isolates were incubated with VHML and CTX Phi. Polymerase chain reaction (PCR) was used to determine the presence of VHML and CTX Phi in infected isolates. We demonstrated that it was possible to infect one isolate of V. cholerae (isolate ACM #2773/ATCC #14035) with VHML. This isolate successfully incorporated VHML into its genome as evident by positive PCR amplification of the sequence coding part of the tail sheath of VHML. Attempts to infect all other V. cholerae and V. mimicus isolates with VHML were unsuccessful. Attempts to infect V. cholerae non-01, V. harveyi and V. mimicus isolates with CTX Phi were unsuccessful. CONCLUSIONS: Bacteriophage infection is limited by bacteriophage-exclusion systems operating within bacterial strains and these systems appear to be highly selective. One system may allow the co-existence of one bacteriophage while excluding another. VHML appears to have a narrow host range which may be related to a common receptor protein in such strains. The lack of the vibrio pathogenicity island bacteriophage (VPI Phi) in the isolates used in this study may explain why infections with CTX Phi were unsuccessful. SIGNIFICANCE AND IMPACT OF THE STUDY: The current study has demonstrated that Vibrio spp. bacteriophages may infect other Vibrio spp.     

Comparative genomic analyses of the vibrio pathogenicity island and cholera toxin prophage regions in nonepidemic serogroup strains of Vibrio cholerae

Two major virulence factors are associated with epidemic strains (O1 and O139 serogroups) of Vibrio cholerae: cholera toxin encoded by the ctxAB genes and toxin-coregulated pilus encoded by the tcpA gene. The ctx genes reside in the genome of a filamentous phage (CTXphi), and the tcpA gene resides in a vibrio pathogenicity island (VPI) which has also been proposed to be a filamentous phage designated VPIphi. In order to determine the prevalence of horizontal transfer of VPI and CTXphi among nonepidemic (non-O1 and non-O139 serogroups) V. cholerae, 300 strains of both clinical and environmental origin were screened for the presence of tcpA and ctxAB. In this paper, we present the comparative genetic analyses of 11 nonepidemic serogroup strains which carry the VPI cluster. Seven of the 11 VPI(+) strains have also acquired the CTXphi. Multilocus sequence typing and restriction fragment length polymorphism analyses of the VPI and CTXphi prophage regions revealed that the non-O1 and non-O139 strains were genetically diverse and clustered in lineages distinct from that of the epidemic strains. The left end of the VPI in the non-O1 and non-O139 strains exhibited extensive DNA rearrangements. In addition, several CTXphi prophage types characterized by novel repressor (rstR) and ctxAB genes and VPIs with novel tcpA genes were found in these strains. These data suggest that the potentially pathogenic, nonepidemic, non-O1 and non-O139 strains identified in our study most likely evolved by sequential horizontal acquisition of the VPI and CTXphi independently rather than by exchange of O-antigen biosynthesis regions in an existing epidemic strain.     

Filamentous phages linked to virulence of Vibrio cholerae

The pathogenicity of Vibrio cholerae depends upon its production of two key virulence factors: the toxin co-regulated pilus (TCP), a colonization factor, and cholera toxin, an exotoxin. Genes encoding both virulence factors were introduced into V. cholerae by horizontal gene transfer. The toxin genes are contained within the genome of CTXphi, an integrated filamentous phage identified in 1996. In the past few years, it has been shown that CTXphi relies on novel processes for phage DNA integration, replication and secretion. In addition, expression of CTXphi genes--including the toxin genes--and transmission of CTXphi were recently found to be promoted by the antirepressor RstC, which is encoded within RS1, a newly described satellite phage of CTXphi. The genetic island that encodes TCP has also been described as a filamentous phage; however, these sequences are unlike the genome of any previously characterized filamentous phage.     

Vibrio cholerae temperate phage O139: characteristics and role in changing expression of chromosomal virulence genes

Restriction analysis of temperate cholera phage 139 isolated from Vibrio cholerae P16064, serogroup 0139, showed its DNA to be double-stranded linear with cohesive terminals. DNA-DNA hybridization on nylon membranes revealed that many V. cholerae strains of serogroup 0139 isolated in different regions contained a temperate cholera phage 139 in their genomes. Southern blot hybridization of chromosomal DNA PST-fragments with phage probe showed that the temperate phage 139 was identical to the temperate phage of serogroup II V. eltor. The phage integrated in the chromosome near genes encoding motility (mot) and production of the capsule (cap) and purine (pur). Phage genome is apparently responsible for instability of cap, pur, and mot genes whose products are important for the development of an infectious process in cholera.     

Structure and arrangement of the cholera toxin genes in Vibrio cholerae O139

Abstract The sequence of the ctxB gene encoding the B subunit of cholera toxin has been determined for a strain of Vibrio cholerae of the novel O139 serotype associated with recent outbreaks of severe cholera throughout South-East Asia and found to be identical to the ctxB gene in V. cholerae O1 of the E1 Tor biotype. Analyses by Southern hybridization and PCR showed that all strains of the O139 serotype V. cholerae tested carried cholera toxin genes and other gene associated with a virulence cassette DNA region at two loci identical or homologous to those identified in the Classical rather than the E1 Tor biotype of V. cholerae serotype O1 although these loci in O139 could reside on restriction fragments of variable size.     

O1群和O139群霍乱弧菌主要抗原研究进展

霍乱弧菌是引起人和动物烈性肠道传染病霍乱的病原体。在霍乱弧菌的200多个血清群中,只有O1群和O139群霍乱弧菌能引起霍乱。快速准确检测O1群和O139群霍乱弧菌是霍乱防治的关键。表面抗原在O1群和O139群霍乱弧菌检测中发挥着重要作用。简要综述了O1群和O139群霍乱弧菌的脂多糖、霍乱肠毒素、外膜蛋白W、毒素共调菌毛和甘露糖敏感血凝素等5种主要抗原的研究进展。     

霍乱弧菌检测方法的研究进展

烈性肠道传染病霍乱能引起大范围乃至世界性大流行,在我国被列为甲类传染病.霍乱弧菌是导致感染者严重腹泻、引起霍乱的病原菌.霍乱弧菌的快速、准确检测是霍乱预防、控制的重要依据.目前,国内、外针对霍乱弧菌建立了许多有效的检测方法,尤其是分子生物学相关技术的应用,为霍乱弧菌的检测提供了新的手段.本文综述了近年来霍乱弧菌检测方法...     

Cholera toxin gene-positive Vibrio cholerae O1 Ogawa and Inaba strains produce the new cholera toxin

Abstract Two strains of cholera toxin (CT) gene-positive Vibrio cholerae O1, Ogawa, isolated from patients with diarrhoea and the hypertoxigenic V. cholerae O1, Inaba (569B), were found to produce the new cholera toxin that has earlier been demonstrated to be elaborated by CT gene-negative human and environmental isolates of V. cholerae O1. The CT gene-positive strains produce the new cholera toxin simultaneously with CT, indicating that they contain the gene coding for the new cholera toxin in addition to that of CT.     

Comparative Genomic Analyses of the Vibrio Pathogenicity Island and Cholera Toxin Prophage Regions in Nonepidemic Serogroup Strains of Vibrio cholerae

Two major virulence factors are associated with epidemic strains (O1 and O139 serogroups) of Vibrio cholerae: cholera toxin encoded by the ctxAB genes and toxin-coregulated pilus encoded by the tcpA gene. The ctx genes reside in the genome of a filamentous phage (CTX), and the tcpA gene resides in a vibrio pathogenicity island (VPI) which has also been proposed to be a filamentous phage designated VPI. In order to determine the prevalence of horizontal transfer of VPI and CTX among nonepidemic (non-O1 and non-O139 serogroups) V. cholerae, 300 strains of both clinical and environmental origin were screened for the presence of tcpA and ctxAB. In this paper, we present the comparative genetic analyses of 11 nonepidemic serogroup strains which carry the VPI cluster. Seven of the 11 VPI⁺ strains have also acquired the CTX. Multilocus sequence typing and restriction fragment length polymorphism analyses of the VPI and CTX prophage regions revealed that the non-O1 and non-O139 strains were genetically diverse and clustered in lineages distinct from that of the epidemic strains. The left end of the VPI in the non-O1 and non-O139 strains exhibited extensive DNA rearrangements. In addition, several CTX prophage types characterized by novel repressor (rstR) and ctxAB genes and VPIs with novel tcpA genes were found in these strains. These data suggest that the potentially pathogenic, nonepidemic, non-O1 and non-O139 strains identified in our study most likely evolved by sequential horizontal acquisition of the VPI and CTX independently rather than by exchange of O-antigen biosynthesis regions in an existing epidemic strain.     

An experimental study of the population and evolutionary dynamics of Vibrio cholerae O1 and the bacteriophage JSF4

Studies of Vibrio cholerae in the environment and infected patients suggest that the waning of cholera outbreaks is associated with rise in the density of lytic bacteriophage. In accordance with mathematical models, there are seemingly realistic conditions where phage predation could be responsible for declines in the incidence of cholera. Here, we present the results of experiments with the El Tor strain of V. cholerae (N16961) and a naturally occurring lytic phage (JSF4), exploring the validity of the main premise of this model: that phage predation limits the density of V. cholerae populations. At one level, the results of our experiments are inconsistent with this hypothesis. JSF4-resistant V. cholerae evolve within a short time following their confrontation with these viruses and their populations become limited by resources rather than phage predation. At a larger scale, however, the results of our experiments are not inconsistent with the hypothesis that bacteriophage modulate outbreaks of cholera. We postulate that the resistant bacteria that evolved play an insignificant role in the ecology or pathogenicity of V. cholerae. Relative to the phage-sensitive cells from whence they are derived, the evolved JSF4-resistant V. cholerae have fitness costs and other characters that are likely to impair their ability to compete with the sensitive cells in their natural habitat and may be avirulent in human hosts. The results of this in vitro study make predictions that can be tested in natural populations of V. cholerae and cholera-infected patients.     

Role of moderate bacteriophage 139 in change in production of cholera toxin in a classic strain of Vibrio cholerae

New data were obtained concerning cell sensitivity of pathogenic strains of cholera vibrions, which belong to the serogroup O1 of classical biovar, to the temperate bacteriophage K139, the native host of which is Vibrio cholerae O139. Molecular-genetic and biochemical studies showed that phage 139 integrated into the chromosome of strains V. cholerae O1 can change their toxigenic properties. A change in the production of cholera toxin (CT) in lysogens is associated both with an increase in the activity of the toxR regulatory gene and with a distortion of the structure of a chromosomal DNA region that contains a copy of the operon ctxAB encoding CT biosynthesis.     

Investigation of seven Vibrio virulence genes among Vibrio alginolyticus and Vibrio parahaemolyticus strains from the coastal mariculture systems in Guangdong, China

AIMS: To investigate the distribution of the virulence of two Vibrio species among different strains obtained from the mariculture systems on the coast of Guangdong in China and the correlation between the virulence strains and the virulence genes among Vibrio alginolyticus. METHODS: Besides three strains, 72 V. alginolyticus strains and seven Vibrio parahaemolyticus strains were examined by PCR or semi-nested PCR for the virulence genes (tlh, trh, tdh, toxR, toxRS, ctxA, VPI). Additionally, the virulence of 18 V. alginolyticus strains was tested. SIGNIFICANCE AND IMPACT OF THE STUDY: Virulence genes homologous to those in the V. parahaemolyticus and Vibrio cholerae are widely distributed among V. alginolyticus and V. parahaemolyticus in the coastal mariculture systems in Guangdong, China. Some of the V. alginolyticus strains are pathogenic to aquatic animals, and might have derived their virulence genes from V. parahaemolyticus or V. cholerae, representing a possible reservoir of these genes. However, there is no correlation between presence and absence of the virulence genes used to investigate V. alginolyticus and its virulent strains. In this report, we also show that tlh is distributed among V. alginolyticus.     

多重PCR方法检测霍乱弧菌的研究

霍乱弧菌是霍乱的病原体,可以分为O1群、O139群和非O1/非O139群。O1群和O139群霍乱弧菌产生的霍乱肠毒素(也称霍乱毒素)是产生霍乱的主要原因,也只有O1群和O139群霍乱弧菌可引起霍乱。其他群的霍乱弧菌毒性不高,但在食品中也不允许被检出。实验以霍乱胶原酶基因和霍乱毒素基因为目的基因,试图建立一种PCR方法对霍乱弧菌进行检测研究,结果表明此方法可以用于食品中的霍乱弧菌检测。     

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.     

Role of Temperate Bacteriophage 139 in Changing Cholera Toxin Production in Vibrio cholerae Classical Biovar

New data were obtained concerning cell sensitivity of pathogenic strains of cholera vibrions, which belong to the serogroup O1 of classical biovar, to the temperate bacteriophage 139, the native host of which is Vibrio cholerae O139. Molecular–genetic and biochemical studies showed that phage 139 integrated into the chromosome of strains V. cholerae O1 can change their toxigenic properties. A change in the production of cholera toxin (CT) in lysogens is associated both with an increase in the activity of the toxR regulatory gene and with an alteration of the structure of a chromosomal DNA region that contains a copy of the operon ctxAB encoding CT biosynthesis.     

Isolation of lytic bacteriophage against Vibrio harveyi

Aims: The isolation of lytic bacteriophage of Vibrio harveyi with potential for phage therapy of bacterial pathogens of phyllosoma larvae from the tropical rock lobster Panulirus ornatus. Methods and Results: Water samples from discharge channels and grow‐out ponds of a prawn farm in northeastern Australia were enriched for 24 h in a broth containing four V. harveyi strains. The bacteriophage‐enriched filtrates were spotted onto bacterial lawns demonstrating that the bacteriophage host range for the samples included strains of V. harveyi, Vibrio campbellii, Vibrio rotiferianus, Vibrio parahaemolyticus and Vibrio proteolyticus. Bacteriophage were isolated from eight enriched samples through triple plaque purification. The host range of purified phage included V. harveyi, V. campbellii, V. rotiferianus and V. parahaemolyticus. Transmission electron microscope examination revealed that six purified phage belonged to the family Siphoviridae, whilst two belonged to the family Myoviridae. The Myoviridae appeared to induce bacteriocin production in a limited number of host bacterial strains, suggesting that they were lysogenic rather than lytic. A purified Siphoviridae phage could delay the entry of a broth culture of V. harveyi strain 12 into exponential growth, but could not prevent the overall growth of the bacterial strain. Conclusions: Bacteriophage with lytic activity against V. harveyi were isolated from prawn farm samples. Purified phage of the family Siphoviridae had a clear lytic ability and no apparent transducing properties, indicating they are appropriate for phage therapy. Phage resistance is potentially a major constraint to the use of phage therapy in aquaculture as bacteria are not completely eliminated. Significance and Impact of the Study: Phage therapy is emerging as a potential antibacterial agent that can be used to control pathogenic bacteria in aquaculture systems. The development of phage therapy for aquaculture requires initial isolation and determination of the bacteriophage host range, with subsequent creation of suitable phage cocktails.     

Biotype-specific tcpA genes in Vibrio cholerae

Abstract The tcpA gene, encoding the structural subunit of the toxin-coregulated pilus, has been isolated from a variety of clinical isolates of Vibrio cholerae , and the nucleotide sequence determined. Strict biotype-specific conservation within both the coding and putative regulatory regions was observed, with important differences between the El Tor and classical biotypes. V. cholerae O139 Bengal strains appear to have El Tor-type tcpA genes. Environmental O1 and non-O1 isolates have sequences that bind an E1 Tor-specific tcpA DNA probe and that are weakly and variably amplified by tcpA -specific polymerase chain reaction primers, under conditions of reduced stringency. The data presented allow the selection of primer pairs to help distinguish between clinical and environmental isolates, and to distinguish El Tor (and Bengal) biotypes from classical biotypes from classical biotypes of V. cholerae . While the role of TcpA in cholera vaccine preparations remains unclear, the data strongly suggest that TcpA-containing vaccines directed at O1 strains need include only the two forms of TcpA, and that such vaccines directed at (O139) Bengal strains should include the TcpA of El Tor biotype.     

Characterization of Novel Alleles of Toxin Co-Regulated Pilus A Gene (tcpA) from Environmental Isolates of Vibrio cholerae

Vibrio cholerae is causative agent of life threatening diarrheal disease, cholera. The toxin co-regulated pilus (TCP) is a critical colonization factor of V. cholerae and it also serves as receptor for CTXФ. In this study, we describe nucleotide sequence of four novel alleles of tcpA gene from toxigenic and non-toxigenic V. cholerae isolated from environmental sources. The phylogenetic analysis of tcpA revealed that it is related to tcpA of newly emerged O1 strain and unrelated to tcpA of wild type (classical and El Tor strains). All strains showed variant tcpA and also harbored intact Vibrio Pathogenicity Island (VPI). The expression of all variant alleles was demonstrated by RT-PCR.     

Construction of genetically marked Vibrio cholerae O1 vaccine strains

Abstract Attenuated Vibrio cholerae O1 vaccine strains lacking the gene encoding the A subunit of cholera toxin have proven efficacious in preventing experimental cholera. As these strains move from closed, contained testing environment to large-scale field trials, a readily assayable phenotypic trait to distinguish a vaccine strain from wild-type V. cholerae O1 is desirable. We have constructed three derivatives of the attenuated V. cholerae strain CVD 103 which carry a mercury resistance or urease marker in the hlyA gene. CVD 103-HgR was constructed using a protracted marker-exchange procedure; this strain was found to have somewhat lowered colonisation efficiency in infant mice in comparison to its parent strain, CVD 103. The insertion of the resistance marker was repeated using a suicide vector system; CVD 103-HgR2 was found to colonise infant mice as efficiently as CVD 103. Strain CVD 103-UR, in which sequences encoding urease were inserted using a suicide vector, also colonised infant mice as well as CVD 103. The genetically marked strains CVD 103-HgR, CVD 103-HgR2 and CVD 103-UR form the basis for a generation of defined oral vaccines that may give single-dose, long-lasting protection to populations at risk from cholera.