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.     

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.     

Pathogenicity islands and phages in Vibrio cholerae evolution

The identification of accessory genetic elements (plasmids, phages and chromosomal 'pathogenicity islands') encoding virulence-associated genes has facilitated our efforts to understand the origination of pathogenic microorganisms. Toxigenic Vibrio cholerae, the etiologic agent of cholera, represents a paradigm for this process in that this organism evolved from environmental nonpathogenic V. cholerae by acquisition of virulence genes. The major virulence genes in V. cholerae, which are clustered in several chromosomal regions, appear to have been recently acquired from phages or through undefined horizontal gene transfer events. Evidence is accumulating that the interactions of phages with each other can also influence the emergence of pathogenic clones of V. cholerae. Therefore, to track the evolution of pathogens from their nonpathogenic progenitors, it is also crucial to identify and characterize secondary genetic elements that mediate lateral transfer of virulence genes in trans. Understanding the evolutionary events that lead to the emergence of pathogenic clones might provide new approaches to the control of cholera and other infectious diseases.     

Substantiation of the necessity of developing a single set of phages for serotyping of Vibrio cholerae

A single set of phages for typing different V. cholerae biotypes has been developed in the USSR and proposed for use instead of two sets of phages intended for this purpose. This set has been developed in view of the following facts: the similarity of the biological properties of V. cholerae biotypes and the absence of absolute criteria for their differentiation; the existence of the foci of cholera with both biotypes circulating there; the probability experimentally confirmed, of the conversion of one biotype into the other.     

CTXphi infection of Vibrio cholerae requires the tolQRA gene products

CTXphi is a lysogenic filamentous bacteriophage that encodes cholera toxin. Filamentous phages that infect Escherichia coli require both a pilus and the products of tolQRA in order to enter host cells. We have previously shown that toxin-coregulated pilus (TCP), a type IV pilus that is an essential Vibrio cholerae intestinal colonization factor, serves as a receptor for CTXphi. To test whether CTXphi also depends upon tol gene products to infect V. cholerae, we identified and inactivated the V. cholerae tolQRAB orthologues. The predicted amino acid sequences of V. cholerae TolQ, TolR, TolA, and TolB showed significant similarity to the corresponding E. coli sequences. V. cholerae strains with insertion mutations in tolQ, tolR, or tolA were reduced in their efficiency of CTXphi uptake by 4 orders of magnitude, whereas a strain with an insertion mutation in tolB showed no reduction in CTXphi entry. We could detect CTXphi infection of TCP(-) V. cholerae, albeit at very low frequencies. However, strains with mutations in both tcpA and either tolQ, tolR, or tolA were completely resistant to CTXphi infection. Thus, CTXphi, like the E. coli filamentous phages, uses both a pilus and TolQRA to enter its host. This suggests that the pathway for filamentous phage entry into cells is conserved between host bacterial species.     

Genetics of stress adaptation and virulence in toxigenic Vibrio cholerae

Vibrio cholerae, a Gram-negative bacterium belonging to the gamma-subdivision of the family Proteobacteriaceae is the etiologic agent of cholera, a devastating diarrheal disease which occurs frequently as epidemics. Any bacterial species encountering a broad spectrum of environments during the course of its life cycle is likely to develop complex regulatory systems and stress adaptation mechanisms to best survive in each environment encountered. Toxigenic V. cholerae, which has evolved from environmental nonpathogenic V. cholerae by acquisition of virulence genes, represents a paradigm for this process in that this organism naturally exists in an aquatic environment but infects human beings and cause cholera. The V. cholerae genome, which is comprised of two independent circular mega-replicons, carries the genetic determinants for the bacterium to survive both in an aquatic environment as well as in the human intestinal environment. Pathogenesis of V. cholerae involves coordinated expression of different sets of virulence associated genes, and the synergistic action of their gene products. Although the acquisition of major virulence genes and association between V. cholerae and its human host appears to be recent, and reflects a simple pathogenic strategy, the establishment of a productive infection involves the expression of many more genes that are crucial for survival and adaptation of the bacterium in the host, as well as for its onward transmission and epidemic spread. While a few of the virulence gene clusters involved directly with cholera pathogenesis have been characterized, the potential exists for identification of yet new genes which may influence the stress adaptation, pathogenesis, and epidemiological characteristics of V. cholerae. Coevolution of bacteria and mobile genetic elements (plasmids, transposons, pathogenicity islands, and phages) can determine environmental survival and pathogenic interactions between bacteria and their hosts. Besides horizontal gene transfer mediated by genetic elements and phages, the evolution of pathogenic V. cholerae involves a combination of selection mechanisms both in the host and in the environment. The occurrence of periodic epidemics of cholera in endemic areas appear to enhance this process.     

Genomic sequence and receptor for the Vibrio cholerae phage KSF-1phi: evolutionary divergence among filamentous vibriophages mediating lateral gene transfer

KSF-1phi, a novel filamentous phage of Vibrio cholerae, supports morphogenesis of the RS1 satellite phage by heterologous DNA packaging and facilitates horizontal gene transfer. We analyzed the genomic sequence, morphology, and receptor for KSF-1phi infection, as well as its phylogenetic relationships with other filamentous vibriophages. While strains carrying the mshA gene encoding mannose-sensitive hemagglutinin (MSHA) type IV pilus were susceptible to KSF-1phi infection, naturally occurring MSHA-negative strains and an mshA deletion mutant were resistant. Furthermore, d-mannose as well as a monoclonal antibody against MSHA inhibited infection of MSHA-positive strains by the phage, suggesting that MSHA is the receptor for KSF-1phi. The phage genome comprises 7,107 nucleotides, containing 14 open reading frames, 4 of which have predicted protein products homologous to those of other filamentous phages. Although the overall genetic organization of filamentous phages appears to be preserved in KSF-1phi, the genomic sequence of the phage does not have a high level of identity with that of other filamentous phages and reveals a highly mosaic structure. Separate phylogenetic analysis of genomic sequences encoding putative replication proteins, receptor-binding proteins, and Zot-like proteins of 10 different filamentous vibriophages showed different results, suggesting that the evolution of these phages involved extensive horizontal exchange of genetic material. Filamentous phages which use type IV pili as receptors were found to belong to different branches. While one of these branches is represented by CTXphi, which uses the toxin-coregulated pilus as its receptor, at least four evolutionarily diverged phages share a common receptor MSHA, and most of these phages mediate horizontal gene transfer. Since MSHA is present in a wide variety of V. cholerae strains and is presumed to express in the environment, diverse filamentous phages using this receptor are likely to contribute significantly to V. cholerae evolution.     

Characterization of Vibrio cholerae Bacteriophages Isolated from the Environmental Waters of the Lake Victoria Region of Kenya

Over the last decade, cholera outbreaks have become common in some parts of Kenya. The most recent cholera outbreak occurred in Coastal and Lake Victoria region during January 2009 and May 2010, where a total of 11,769 cases and 274 deaths were reported by the Ministry of Public Health and Sanitation. The objective of this study is to isolate Vibrio cholerae bacteriophages from the environmental waters of the Lake Victoria region of Kenya with potential for use as a biocontrol for cholera outbreaks. Water samples from wells, ponds, sewage effluent, boreholes, rivers, and lakes of the Lake Victoria region of Kenya were enriched for 48 h at 37 °C in broth containing a an environmental strain of V. cholerae. Bacteriophages were isolated from 5 out of the 42 environmental water samples taken. Isolated phages produced tiny, round, and clear plaques suggesting that these phages were lytic to V. cholerae. Transmission electron microscope examination revealed that all the nine phages belonged to the family Myoviridae, with typical icosahedral heads, long contractile tails, and fibers. Head had an average diameter of 88.3 nm and tail of length and width 84.9 and 16.1 nm, respectively. Vibriophages isolated from the Lake Victoria region of Kenya have been characterized and the isolated phages may have a potential to be used as antibacterial agents to control pathogenic V. cholerae bacteria in water reservoirs.     

Novel type of specialized transduction for CTX phi or its satellite phage RS1 mediated by filamentous phage VGJ phi in Vibrio cholerae

The main virulence factor of Vibrio cholerae, the cholera toxin, is encoded by the ctxAB operon, which is contained in the genome of the lysogenic filamentous phage CTX phi. This phage transmits ctxAB genes between V. cholerae bacterial populations that express toxin-coregulated pilus (TCP), the CTX phi receptor. In investigating new forms of ctxAB transmission, we found that V. cholerae filamentous phage VGJ phi, which uses the mannose-sensitive hemagglutinin (MSHA) pilus as a receptor, transmits CTX phi or its satellite phage RS1 by an efficient and highly specific TCP-independent mechanism. This is a novel type of specialized transduction consisting in the site-specific cointegration of VGJ phi and CTX phi (or RS1) replicative forms to produce a single hybrid molecule, which generates a single-stranded DNA hybrid genome that is packaged into hybrid viral particles designated HybP phi (for the VGJ phi/CTX phi hybrid) and HybRS phi (for the VGJ phi/RS1 hybrid). The hybrid phages replicate by using the VGJ phi replicating functions and use the VGJ phi capsid, retaining the ability to infect via MSHA. The hybrid phages infect most tested strains more efficiently than CTX phi, even under in vitro optimal conditions for TCP expression. Infection and lysogenization with HybP phi revert the V. cholerae live attenuated vaccine strain 1333 to virulence. Our results reinforce that TCP is not indispensable for the acquisition of CTX phi. Thus, we discuss an alternative to the current accepted evolutionary model for the emergence of new toxigenic strains of V. cholerae and the importance of our findings for the development of an environmentally safer live attenuated cholera vaccine.     

High frequency of a novel filamentous phage, VCY φ, within an environmental Vibrio cholerae population

Environmental Vibrio cholerae strains isolated from a coastal brackish pond (Oyster Pond, Woods Hole, MA) carried a novel filamentous phage, VCY, which can exist as a host genome integrative form (IF) and a plasmid-like replicative form (RF). Outside the cell, the phage displays a morphology typical of Inovirus, with filamentous particles ～1.8 μm in length and 7 nm in width. Four independent RF isolates had identical genomes, except for 8 single nucleotide polymorphisms clustered in two regions. The overall genome size is 7,103 bp with 11 putative open reading frames organized into three functional modules (replication, structure and assembly, and regulation). VCY shares sequence similarity with other filamentous phages (including cholera disease-associated CTX) in a highly mosaic manner, indicating evolution by horizontal gene transfer and recombination. VCY integrates in the vicinity of the putative translation initiation factor Sui1 in chromosome II of V. cholerae. A screen of 531 closely related host isolates showed that ～40% harbored phages, with 27% and 13% carrying the IF and RF, respectively. The relative frequencies of the RF and IF differed among strains isolated from the pond or lagoon of Oyster Pond, suggesting that the host habitat influences intracellular phage biology. The overall high prevalence within the host population shows that filamentous phages can be an important component of the environmental biology of V. cholerae.     

Preservation of cholerogenicity in cholera vibrios in the incomplete L-cycle stage]

The work presents the characteristics of the forms appearing as a result of the incomplete L cycle of Vibrio cholerae obtained in experimental conditions after the cultivation of typical V. eltor strain, serovar Ogawa, for 5 months at room temperature in sterile river water without subculturing. The culture formed shaprly changed L-similar colonies, had decreased agglutinability and was resistant to diagnostic cholera phages, but retained its cholerogenicity for suckling rabbits. A suggestion was made concerning the possible epidemiological significance of such forms and the necessity of their detection in the course of bacteriological analysis for cholera.     

Detection of toxigenic Vibrio cholerae from environmental water samples by an enrichment broth cultivation-pit-stop semi-nested PCR procedure

A pit-stop semi-nested PCR assay for the detection of toxigenic Vibrio cholerae in environmental water samples was developed and its performance evaluated. The PCR technique amplifies sequences within the cholera toxin operon specific for toxigenic V. cholerae. The PCR procedure coupled with an enrichment culture detected as few as four V. cholerae organisms in pure culture. Treated sewage, surface, ground and drinking water samples were seeded with V. cholerae and following enrichment, a detection limit of as few as 1 V. cholerae cfu ml(-1) was obtained with amplification reactions from crude bacterial lysates. The proposed method, which includes a combination of enrichment, rapid sample preparation and a pit-stop semi-nested PCR, could be applicable in the rapid detection of toxigenic V. cholerae in environmental water samples.     

Application of duplex-PCR in rapid and reliable detection of toxigenic Vibrio cholerae in water samples in Thailand

Toxigenic Vibrio cholerae, the cause of cholera, is a native flora of the aquatic environment which is transmitted through drinking water and still remains the leading cause of morbidity and mortality in many developing countries including Thailand. The culture method (CM), which is routinely used for assessing water quality, has not proven as efficient as molecular methods because the notorious pathogen survives in water mostly in a non-culturable state. We employed duplex-polymerase chain reaction (duplex-PCR) for detection of tcpA and ctxA genes in toxigenic V. cholerae, and compared PCR detection with CM in various waters of Khon Kaen Municipality, Thailand. We also evaluated the effect of different pre-PCR conditions on the results of ctxA and tcpA detection including: 1) water filtered and enriched in alkaline peptone water (APW) for 3 h before PCR, 2) water filtered without enrichment before PCR, and 3) use of only enrichment in APW for 6 h before PCR. Of the 96 water samples (taken from waste-water, potable and waste-water from patients' houses, and from rivers) tested, 48 (50%) were positive for ctxA and tcpA by duplex-PCR, whereas only 29 (30%) were positive for V. cholerae by CM. Of the 29 V. cholerae isolated by CM, 2 (7%) were toxigenic V. cholerae belonging to serovar O1, while the rests were non-O1/ non-O139. Results revealed, therefore, that ctxA and tcpA-targeted duplex PCR is more sensitive than CM for detection of toxigenic V. cholerae from water samples because CM detected much less toxigenic V. cholerae than the non-toxigenic V. cholerae. Template DNA as low as 100 fg or 23 cells of V. cholerae in the water sample was detected in duplex PCR. Pre-PCR filtration followed by enrichment for 3 h significantly increase in the efficiency of duplex-PCR detection of toxigenic V. cholerae.     

Single multiplex polymerase chain reaction for environmental surveillance of toxigenic—Pathogenic O1 and Non-O1vibrio cholerae

A multiplex PCR assay was developed for the detection of toxigenic and pathogenic V. cholerae from direct water sources using specific primers targeting diverse genes, viz. outer membrane protein (ompW), cholera toxin (ctxB), ORF specific for O1 (rfbG), zonula occludens (zot) and toxin co-regulated pilus (tcpB); among these genes, ompW acts as internal control for V. cholerae, the ctx gene as a marker for toxigenicity and tcp for pathogenicity. The sensitivity of multiplex PCR was 5 x 10(4) V. cholerae cells per reaction. The procedure was simplified as direct bacterial cells were used as template and there was no need for DNA extraction. The assay was specific as no amplification occurred with the other bacteria used. Toxigenic V. cholerae were artificially spiked in different water samples, filtered through a 0.45 microm membrane, and the filters containing bacteria were enriched in APW for 6 h. PCR following filtration and enrichment could detect as little as 8 V. cholerae cells per mL in different spiked water samples. Various environmental potable water samples were screened for the presence of V. cholerae using this assay procedure. The proposed method is rapid, sensitive and specific for environmental surveillance for the presence of toxigenic-pathogenic and nonpathogenic V. cholerae.     

Ecological relationship between Vibrio parahaemolyticus and agar-digesting vibrios as evidenced by bacteriophage susceptibility patterns.

Twenty bacteriophages active against Vibrio parahaemolyticus and agar-digesting vibrios, isolated from oysters (Crassostrea gigas) and Dungeness crab (Cancer magister) and by induction of a lysogenic agar digester, were tested as to their host range. These phages were specific for V. parahaemolyticus and various agar-digesting vibrios, and interspecies lysis occurred only between these two groups. V. alginolyticus, V. anguillarum and related species, V. cholerae, and a group of marine psychrophilic and psychrotrophic vibrios were not affected. No correlation was observed between the O and K serotypes of V. parahaemolyticus strains and bacteriophage susceptibility patterns, and 7 of 28 strains of V. parahaemolyticus were not lysed by any of the phages. Only two of the phage isolates were capable of lysing all susceptible V. parahaemolyticus strains. No correlation was observed between the inter-and intraspecies genetic relatedness (DNA homologies) of V. parahaemolyticus and agar-digesting vibrios and susceptibility patterns to different bacteriophages. Some of the phages were capable of plaque formation on V. parahaemolyticus as well as on some strains of agar-digesting vibrios that were separated by 70 to 80% differences in their DNA homologies. The possible ecological significance of these vibrio bacteriophages, particularly those having a wide host range, is discussed.     

Ecology of Vibrio species, including Vibrio cholerae, in natural waters of Kent, England

The distribution of Vibrio species in water from two sites in Kent was studied between 1978 and 1980. They were counted by a most probable number technique using alkaline peptone water for enrichment followed by plating onto thiosulphate citrate bile salt sucrose agar, or by direct plating of water onto the same agar. In a freshwater stream both upstream and downstream from a human sewage works outfall V. metschnikovii was the predominant Vibrio. Vibrio anguillarum was isolated sporadically. Non-O1 serovars of V. cholerae occurred only twice. At the other site in a ditch containing static, brackish water, non-O1 V. cholerae (highest number 400 cfu/ml) was observed as present only from May to November. Vibrio anguillarum was isolated throughout the sampling period. The presence of non-O1 V. cholerae in both sites was not dependent on the input of human sewage. The hypothesis that non-toxigenic V. cholerae can survive and multiply in water was tested in the ditch by the use of submersible chambers constructed of polycarbonate membranes and Plexiglass. The seasonal incidence of non-O1 V. cholerae in the brackish water site could be explained by the multiplication of the organism when the water temperature exceeded 9°C. It was concluded that strains of V. cholerae that are unable to produce cholera toxin are indigenous to static brackish water environments and the possibility that this applies to toxigenic strains as well should be investigated.     

Ecological relationships between Vibrio cholerae and planktonic crustacean copepods.

Strains of Vibrio cholerae, both O1 and non-O1 serovars, were found to attach to the surfaces of live copepods maintained in natural water samples collected from the Chesapeake Bay and Bangladesh environs. The specificity of attachment of V. cholerae to live copepods was confirmed by scanning electron microscopy, which revealed that the oral region and egg sac were the most heavily colonized areas of the copepods. In addition, survival of V. cholerae in water was extended in the presence of live copepods. Attachment of viable V. cholerae cells to copepods killed by exposure to -60 degrees C was not observed. Furthermore, survival of V. cholerae was not as long in the presence of dead copepods as in the live copepod system. A strain of Vibrio parahaemolyticus was also seen to attach to copepod surfaces without effect on survival of the organism in water. The attachment of vibrios to copepods was concluded to be significant since strains of other bacteria, including Pseudomonas sp. and Escherichia coli, did not adhere to live or dead copepods. Attachment of V. cholerae to live copepods is suggested to be an important factor of the ecology of this species in the aquatic environment, as well as in the epidemiology of cholera, for which V. cholerae serovar O1 is the causative agent.     

霍乱传染源的微生态学研究

霍乱弧菌表面具有粘附性,并能降解甲壳质,在河口水中能牢固地粘附于浮游动物桡足类墨氏胸刺水蚤表面上分解甲壳质而延长生命活动,借此在河口底层越冬,由此提示墨氏胸刺水蚤可能成为人以外的传染源。该宿主与霍乱孤菌的微生态系,尚需进一步研究。     

Adult non-biting midges: possible windborne carriers of Vibrio cholerae non-O1 non-O139

Vibrio cholerae is a waterborne bacterium native to the aquatic environment. There are over 200 known serogroups yet only two cause cholera pandemics in humans. Direct contact of human sewage with drinking water, sea-born currents and marine transportation, represent modes of dissemination of the bacteria and thus the disease. The simultaneous cholera outbreaks that occur sometimes in distant localities within continental landmasses are puzzling. Here we present evidence that flying, non-biting midges (Diptera; Chironomidae), collected in the air, carry viable non-O1 non-O139 serogroups of V. cholerae. The association of V. cholerae with chironomid egg masses, which serve as a V. cholerae reservoir, was further confirmed. In simulated field experiments, we recorded the transfer of environmental V. cholerae by adult midges from the aquatic environment into bacteria-free water-pools. In laboratory experiments, flying adult midges that emerged from V. cholerae (O1 or O139) contaminated water transferred the green fluorescent protein (GFP)-tagged pathogenic bacteria from one laboratory flasks to another. Our findings show that aerial transfer by flying chironomids may play a role in the dissemination of V. cholerae in nature.