Campylobacter jejuni continues to be the leading cause of bacterial food-borne illness worldwide, so improvements to current methods used for bacterial detection and disease prevention are needed. We describe here the genome and proteome of C. jejuni bacteriophage NCTC 12673 and the exploitation of its receptor-binding protein for specific bacterial detection. Remarkably, the 135-kb Myoviridae genome of NCTC 12673 differs greatly from any other proteobacterial phage genome described (including C. jejuni phages CP220 and CPt10) and instead shows closest homology to the cyanobacterial T4-related myophages. The phage genome contains 172 putative open reading frames, including 12 homing endonucleases, no visible means of packaging, and a putative trans-splicing intein. The phage DNA appears to be strongly associated with a protein that interfered with PCR amplification and estimation of the phage genome mass by pulsed-field gel electrophoresis. Identification and analyses of the receptor-binding protein (Gp48) revealed features common to the Salmonella enterica P22 phage tailspike protein, including the ability to specifically recognize a host organism. Bacteriophage receptor-binding proteins may offer promising alternatives for use in pathogen detection platforms.
The ability of phages to specifically interact with and lyse their host bacteria makes them ideal antibacterial agents. The range of applications of bacteriophage can be extended by their immobilization on inert surfaces. A novel method for the oriented immobilization of bacteriophage has been developed. The method was based on charge differences between the bacteriophage head, which exhibits an overall net negative charge, and the tail fibers, which possess an overall net positive charge. Hence, the head would be more likely to attach to positively charged surfaces, leaving the tails free to capture and lyse bacteria. Cellulose membranes modified so that they had a positive surface charge were used as the support for phage immobilization. It was established that the number of infective phages immobilized on the positively charged cellulose membranes was significantly higher than that on unmodified membranes. Cocktails of phages active against Listeria or Escherichia coli immobilized on these membranes were shown to effectively control the growth of L. monocytogenes and E. coli O157:H7 in ready-to-eat and raw meat, respectively, under different storage temperatures and packaging conditions. The phage storage stability was investigated to further extend their industrial applications. It was shown that lyophilization can be used as a phage-drying method to maintain their infectivity on the newly developed bioactive materials. In conclusion, utilizing the charge difference between phage heads and tails provided a simple technique for oriented immobilization applicable to a wide range of phages and allowed the retention of infectivity.
A long history of experimental work has shown that addition of bacteriophages to a monoculture of bacteria leads to only a temporary depression of bacterial levels. Resistant bacteria usually become abundant, despite reduced growth rates relative to those of phage-sensitive bacteria. This restoration of high bacterial density occurs even if the phages evolve to overcome bacterial resistance. We believe that the generality of this result may be limited to monocultures, in which the resistant bacteria do not face competition from bacterial species unaffected by the phage. As a simple case, we investigated the impact of phages attacking one species in a two-species culture of bacteria. In the absence of phages, Escherichia coli B and Salmonella enterica serovar Typhimurium were stably maintained during daily serial passage in glucose minimal medium (M9). When either of two E. coli-specific phages (T7 or T5) was added to the mixed culture, E. coli became extinct or was maintained at densities that were orders of magnitude lower than those before phage introduction, even though the E. coli densities with phage reached high levels when Salmonella was absent. In contrast, the addition of a phage that attacked only Salmonella (SP6) led to transient decreases in the bacterial number whether E. coli was absent or present. These results suggest that phages can sometimes, although not always, provide long-term suppression of target bacteria.
Salmonella enterica and Escherichia coli O157:H7 are major food-borne pathogens causing serious illness. Phage SFP10, which revealed effective infection of both S. enterica and E. coli O157:H7, was isolated and characterized. SFP10 contains a 158-kb double-stranded DNA genome belonging to the Vi01 phage-like family Myoviridae. In vitro adsorption assays showed that the adsorption constant rates to both Salmonella enterica serovar Typhimurium and E. coli O157:H7 were 2.50 × 10−8 ml/min and 1.91 × 10−8 ml/min, respectively. One-step growth analysis revealed that SFP10 has a shorter latent period (25 min) and a larger burst size (>200 PFU) than ordinary Myoviridae phages, suggesting effective host infection and lytic activity. However, differential development of resistance to SFP10 in S. Typhimurium and E. coli O157:H7 was observed; bacteriophage-insensitive mutant (BIM) frequencies of 1.19 × 10−2 CFU/ml for S. Typhimurium and 4.58 × 10−5 CFU/ml for E. coli O157:H7 were found, indicating that SFP10 should be active and stable for control of E. coli O157:H7 with minimal emergence of SFP10-resistant pathogens but may not be for S. Typhimurium. Specific mutation of rfaL in S. Typhimurium and E. coli O157:H7 revealed the O antigen as an SFP10 receptor for both bacteria. Genome sequence analysis of SFP10 and its comparative analysis with homologous Salmonella Vi01 and Shigella phiSboM-AG3 phages revealed that their tail fiber and tail spike genes share low sequence identity, implying that the genes are major host specificity determinants. This is the first report identifying specific infection and inhibition of Salmonella Typhimurium and E. coli O157:H7 by a single bacteriophage.
Salmonella enterica subspecies enterica serovar Typhimurium is a Gram-negative pathogen causing salmonellosis. Salmonella Typhimurium-targeting bacteriophages have been proposed as an alternative biocontrol agent to antibiotics. To further understand infection and interaction mechanisms between the host strains and the bacteriophages, the receptor diversity of these phages needs to be elucidated.
Twenty-five Salmonella phages were isolated and their receptors were identified by screening a Tn5 random mutant library of S. Typhimurium SL1344. Among them, three types of receptors were identified flagella (11 phages), vitamin B12 uptake outer membrane protein, BtuB (7 phages) and lipopolysaccharide-related O-antigen (7 phages). TEM observation revealed that the phages using flagella (group F) or BtuB (group B) as a receptor belong to Siphoviridae family, and the phages using O-antigen of LPS as a receptor (group L) belong to Podoviridae family. Interestingly, while some of group F phages (F-I) target FliC host receptor, others (F-II) target both FliC and FljB receptors, suggesting that two subgroups are present in group F phages. Cross-resistance assay of group B and L revealed that group L phages could not infect group B phage-resistant strains and reversely group B phages could not infect group L SPN9TCW-resistant strain.
In this report, three receptor groups of 25 newly isolated S. Typhimurium-targeting phages were determined. Among them, two subgroups of group F phages interact with their host receptors in different manner. In addition, the host receptors of group B or group L SPN9TCW phages hinder other group phage infection, probably due to interaction between receptors of their groups. This study provides novel insights into phage-host receptor interaction for Salmonella phages and will inform development of optimal phage therapy for protection against Salmonella.
Of the Salmonella enterica serovars, S. Enteritidis and S. Typhimurium are responsible for most of the Salmonella outbreaks implicated in the consumption of contaminated foods in the Republic of Korea. Because of the widespread occurrence of antimicrobial-resistant Salmonella in foods and food processing environments, bacteriophages have recently surfaced as an alternative biocontrol tool. In this study, we isolated a virulent bacteriophage (wksl3) that could specifically infect S. Enteritidis, S. Typhimurium, and several additional serovars. Transmission electron microscopy revealed that phage wksl3 belongs to the family Siphoviridae. Complete genome sequence analysis and bioinformatic analysis revealed that the DNA of phage wksl3 is composed of 42,766 bp with 64 open reading frames. Since it does not encode any phage lysogeny factors, toxins, pathogen-related genes, or food-borne allergens, phage wksl3 may be considered a virulent phage with no side effects. Analysis of genetic similarities between phage wksl3 and four of its relatives (SS3e, vB_SenS-Ent1, SE2, and SETP3) allowed wksl3 to be categorized as a SETP3-like phage. A single-dose test of oral toxicity with BALB/c mice resulted in no abnormal clinical observations. Moreover, phage application to chicken skin at 8°C resulted in an about 2.5-log reduction in the number of Salmonella bacteria during the test period. The strong, stable lytic activity, the significant reduction of the number of S. Enteritidis bacteria after application to food, and the lack of clinical symptoms of this phage suggest that wksl3 may be a useful agent for the protection of foods against S. Enteritidis and S. Typhimurium contamination.
Phage therapy is the use of bacteriophages as antimicrobial agents for the control of pathogenic and other problem bacteria. It has previously been argued that successful application of phage therapy requires a good understanding of the non-linear kinetics of phage–bacteria interactions. Here we combine experimental and modelling approaches to make a detailed examination of such kinetics for the important food-borne pathogen Campylobacter jejuni and a suitable virulent phage in an in vitro system. Phage-insensitive populations of C. jejuni arise readily, and as far as we are aware this is the first phage therapy study to test, against in vitro data, models for phage–bacteria interactions incorporating phage-insensitive or resistant bacteria. We find that even an apparently simplistic model fits the data surprisingly well, and we confirm that the so-called inundation and proliferation thresholds are likely to be of considerable practical importance to phage therapy. We fit the model to time series data in order to estimate thresholds and rate constants directly. A comparison of the fit for each culture reveals density-dependent features of phage infectivity that are worthy of further investigation. Our results illustrate how insight from empirical studies can be greatly enhanced by the use of kinetic models: such combined studies of in vitro systems are likely to be an essential precursor to building a meaningful picture of the kinetic properties of in vivo phage therapy.
Phage therapy is an antimicrobial treatment based on specific viruses which are natural predators of bacteria. This approach is being promoted as a possible alternative treatment for use against antibiotic-resistant strains of bacteria. Despite its long history and many potential benefits, adoption of phage therapy has been retarded by a variety of factors, including a poor understanding of the therapeutic consequences of the phage–bacteria relationship. In our work we bring together theory and data by testing kinetic models of phage–bacteria interactions against data for an important agent of human food poisoning, Campylobacter jejuni. Our model explicitly allows for resistant bacteria because these have not been properly accounted for in previous phage therapy theory but will be relevant to practical applications. The excellent fit of our model to the data confirms the value of such combined approaches and supports an interpretative viewpoint based on critical density-dependent thresholds that are not part of standard pharmacology. We also find that phage activity appears to be dose-dependent, and we speculate on possible causes for this. Our work illustrates how mathematical models can considerably enhance insights from empirical studies, as an important step in advancing the understanding of phage therapy.
Some bacteriophages target potentially pathogenic bacteria by exploiting surface-associated virulence factors as receptors. For example, phage have been identified that exhibit specificity for Vi capsule producing Salmonella enterica serovar Typhi. Here we have characterized the Vi-associated E1-typing bacteriophage using a number of molecular approaches. The absolute requirement for Vi capsule expression for infectivity was demonstrated using different Vi-negative S. enterica derivatives. The phage particles were shown to have an icosahedral head and a long noncontractile tail structure. The genome is 45,362 bp in length with defined capsid and tail regions that exhibit significant homology to the S. enterica transducing phage ES18. Mass spectrometry was used to confirm the presence of a number of hypothetical proteins in the Vi phage E1 particle and demonstrate that a number of phage proteins are modified posttranslationally. The genome of the Vi phage E1 is significantly related to other bacteriophages belonging to the same serovar Typhi phage-typing set, and we demonstrate a role for phage DNA modification in determining host specificity.
Bacteriophages are estimated to be the most abundant entities on earth and can be found in every niche where their bacterial hosts reside. The initial interaction between phages and Campylobacter jejuni, a common colonizer of poultry intestines and a major source of foodborne bacterial gastroenteritis in humans, is not well understood. Recently, we isolated and characterized a phage F336 resistant variant of C. jejuni NCTC11168 called 11168R. Comparisons of 11168R with the wildtype lead to the identification of a novel phage receptor, the phase variable O-methyl phosphoramidate (MeOPN) moiety of the C. jejuni capsular polysaccharide (CPS). In this study we demonstrate that the 11168R strain has gained cross-resistance to four other phages in our collection (F198, F287, F303, and F326). The reduced plaquing efficiencies suggested that MeOPN is recognized as a receptor by several phages infecting C. jejuni. To further explore the role of CPS modifications in C. jejuni phage recognition and infectivity, we tested the ability of F198, F287, F303, F326, and F336 to infect different CPS variants of NCTC11168, including defined CPS mutants. These strains were characterized by high-resolution magic angle spinning NMR spectroscopy. We found that in addition to MeOPN, the phase variable 3-O-Me and 6-O-Me groups of the NCTC11168 CPS structure may influence the plaquing efficiencies of the phages. Furthermore, co-infection of chickens with both C. jejuni NCTC11168 and phage F336 resulted in selection of resistant C. jejuni bacteria, which either lack MeOPN or gain 6-O-Me groups on their surface, demonstrating that resistance can be acquired in vivo. In summary, we have shown that phase variable CPS structures modulate phage infectivity in C. jejuni and suggest that the constant phage predation in the avian gut selects for changes in these structures leading to a continuing phage–host co-evolution.
bacteriophage; Campylobacter jejuni; capsular polysaccharide; phase variation; phosphoramidate; methylation
Poultry meat is one of the most important sources of human campylobacteriosis, an acute bacterial enteritis which is a major problem worldwide. Campylobacter coli and Campylobacter jejuni are the most common Campylobacter species associated with this disease. These pathogens live in the intestinal tract of most avian species and under commercial conditions they spread rapidly to infect a high proportion of the flock, which makes their treatment and prevention very difficult. Bacteriophages (phages) are naturally occurring predators of bacteria with high specificity and also the capacity to evolve to overcome bacterial resistance. Therefore phage therapy is a promising alternative to antibiotics in animal production. This study tested the efficacy of a phage cocktail composed of three phages for the control of poultry infected with C. coli and C. jejuni. Moreover, it evaluated the effectiveness of two routes of phage administration (by oral gavage and in feed) in order to provide additional information regarding their future use in a poultry unit.
The results indicate that experimental colonisation of chicks was successful and that the birds showed no signs of disease even at the highest dose of Campylobacter administered. The phage cocktail was able to reduce the titre of both C. coli and C. jejuni in faeces by approximately 2 log10 cfu/g when administered by oral gavage and in feed. This reduction persisted throughout the experimental period and neither pathogen regained their former numbers. The reduction in Campylobacter titre was achieved earlier (2 days post-phage administration) when the phage cocktail was incorporated in the birds' feed. Campylobacter strains resistant to phage infection were recovered from phage-treated chickens at a frequency of 13%. These resistant phenotypes did not exhibit a reduced ability to colonize the chicken guts and did not revert to sensitive types.
Our findings provide further evidence of the efficacy of phage therapy for the control of Campylobacter in poultry. The broad host range of the novel phage cocktail enabled it to target both C. jejuni and C. coli strains. Moreover the reduction of Campylobacter by approximately 2 log10cfu/g, as occurred in our study, could lead to a 30-fold reduction in the incidence of campylobacteriosis associated with consumption of chicken meals (according to mathematical models). To our knowledge this is the first report of phage being administered in feed to Campylobacter-infected chicks and our results show that it lead to an earlier and more sustainable reduction of Campylobacter than administration by oral gavage. Therefore the present study is of extreme importance as it has shown that administering phages to poultry via the food could be successful on a commercial scale.
Campylobacter jejuni and Campylobacter coli are the most common bacterial causes of foodborne gastroenteritis which is occasionally followed by a debilitating neuropathy known as Guillain-Barré syndrome. Rapid and specific detection of these pathogens is very important for effective control and quick treatment of infection. Most of the diagnostics available for these organisms are time consuming and require technical expertise with expensive instruments and reagents to perform. Bacteriophages bind to their host specifically through their receptor binding proteins (RBPs), which can be exploited for pathogen detection. We recently sequenced the genome of C. jejuni phage NCTC12673 and identified its putative host receptor binding protein, Gp047. In the current study, we localized the receptor binding domain to the C-terminal quarter of Gp047. CC-Gp047 could be produced recombinantly and was capable of agglutinating both C. jejuni and C. coli cells unlike the host range of the parent phage which is limited to a subset of C. jejuni isolates. The agglutination procedure could be performed within minutes on a glass slide at room temperature and was not hindered by the presence of buffers or nutrient media. This agglutination assay showed 100% specificity and the sensitivity was 95% for C. jejuni (n = 40) and 90% for C. coli (n = 19). CC-Gp047 was also expressed as a fusion with enhanced green fluorescent protein (EGFP). Chimeric EGFP_CC-Gp047 was able to specifically label C. jejuni and C. coli cells in mixed cultures allowing for the detection of these pathogens by fluorescent microscopy. This study describes a simple and rapid method for the detection of C. jejuni and C. coli using engineered phage RBPs and offers a promising new diagnostics platform for healthcare and surveillance laboratories.
To understand phage infection and host cell lysis mechanisms in pathogenic Salmonella, a novel Salmonella enterica serovar Typhimurium-targeting bacteriophage, SPN9CC, belonging to the Podoviridae family was isolated and characterized. The phage infects S. Typhimurium via the O antigen of lipopolysaccharide (LPS) and forms clear plaques with cloudy centers due to lysogen formation. Phylogenetic analysis of phage major capsid proteins revealed that this phage is a member of the lysogen-forming P22-like phage group. However, comparative genomic analysis of SPN9CC with P22-like phages indicated that their lysogeny control regions and host cell lysis gene clusters show very low levels of identity, suggesting that lysogen formation and host cell lysis mechanisms may be diverse among phages in this group. Analysis of the expression of SPN9CC host cell lysis genes encoding holin, endolysin, and Rz/Rz1-like proteins individually or in combinations in S. Typhimurium and Escherichia coli hosts revealed that collaboration of these lysis proteins is important for the lysis of both hosts and that holin is a key protein. To further investigate the role of the lysogeny control region in phage SPN9CC, a ΔcI mutant (SPN9CCM) of phage SPN9CC was constructed. The mutant does not produce a cloudy center in the plaques, suggesting that this mutant phage is virulent and no longer temperate. Subsequent comparative one-step growth analysis and challenge assays revealed that SPN9CCM has shorter eclipse/latency periods and a larger burst size, as well as higher host cell lysis activity, than SPN9CC. The present work indicates the possibility of engineering temperate phages as promising biocontrol agents similar to virulent phages.
One of the key determinants of the size, composition, structure, and development of a microbial community is the predation pressure by bacteriophages. Accordingly, bacteria have evolved a battery of antiphage defense strategies. Since maintaining constantly elevated defenses is costly, we hypothesize that some bacteria have additionally evolved the abilities to estimate the risk of phage infection and to adjust their strategies accordingly. One risk parameter is the density of the bacterial population. Hence, quorum sensing, i.e., the ability to regulate gene expression according to population density, may be an important determinant of phage-host interactions. This hypothesis was investigated in the model system of Escherichia coli and phage λ. We found that, indeed, quorum sensing constitutes a significant, but so far overlooked, determinant of host susceptibility to phage attack. Specifically, E. coli reduces the numbers of λ receptors on the cell surface in response to N-acyl-l-homoserine lactone (AHL) quorum-sensing signals, causing a 2-fold reduction in the phage adsorption rate. The modest reduction in phage adsorption rate leads to a dramatic increase in the frequency of uninfected survivor cells after a potent attack by virulent phages. Notably, this mechanism may apply to a broader range of phages, as AHLs also reduce the risk of χ phage infection through a different receptor.
To enable the successful manipulation of bacterial populations, a comprehensive understanding of the factors that naturally shape microbial communities is required. One of the key factors in this context is the interactions between bacteria and the most abundant biological entities on Earth, namely, the bacteriophages that prey on bacteria. This proof-of-principle study shows that quorum sensing plays an important role in determining the susceptibility of E. coli to infection by bacteriophages λ and χ. On the basis of our findings in the classical Escherichia coli-λ model system, we suggest that quorum sensing may serve as a general strategy to protect bacteria specifically under conditions of high risk of infection.
Acute enteric infections caused by salmonellas remain a major public health burden worldwide. Poultry, particularly chickens, are known to be the main reservoir for this zoonotic pathogen. Although some progress has been made in reducing Salmonella colonization of broiler chickens by using biosecurity and antimicrobials, it still remains a considerable problem. The use of host-specific bacteriophages as a biocontrol is one possible intervention by which Salmonella colonization could be reduced. A total of 232 Salmonella bacteriophages were isolated from poultry farms, abattoirs, and wastewater in 2004 and 2005. Three phages exhibiting the broadest host ranges against Salmonella enterica serotypes Enteritidis, Hadar, and Typhimurium were characterized further by determining their morphology and lytic activity in vitro. These phages were then administered in antacid suspension to birds experimentally colonized with specific Salmonella host strains. The first phage reduced S. enterica serotype Enteritidis cecal colonization by ≥4.2 log10 CFU within 24 h compared with controls. Administration of the second phage reduced S. enterica serotype Typhimurium by ≥2.19 log10 CFU within 24 h. The third bacteriophage was ineffective at reducing S. enterica serotype Hadar colonization. Bacteriophage resistance occurred at a frequency commensurate with the titer of phage being administered, with larger phage titers resulting in a greater proportion of resistant salmonellas. The selection of appropriate bacteriophages and optimization of both the timing and method of phage delivery are key factors in the successful phage-mediated control of salmonellas in broiler chickens.
The predominant food borne pathogen in the western world today is Campylobacter. Campylobacter specific bacteriophages (phages) have been proposed as an alternative agent for reducing the burden of Campylobacter in broilers. One concern in relation to phage biocontrol is the narrow host range often displayed by phages. To identify the potential of phages as a Campylobacter reducing agent we needed to determine their infectivity on a panel of isolates representing the Campylobacter strains found in broilers as well as humans.
In this study, Campylobacter phages were isolated from the intestines of broilers and ducks and from abattoir sewage. Twelve phages were investigated to determine their ability to infect the Campylobacter Penner serotypes commonly present in Danish poultry and patients with campylobacteriosis. A total of 89% of the Campylobacter jejuni strains and 14% of the Campylobacter coli strains could be infected by at least one of the bacteriophages. The majority of the phages infected the most common serotypes in Danish broilers (O:1,44; O:2; O:4-complex), but showed limited ability to infect 21 of the less frequent Campylobacter serotypes. Pulse field gel electrophoresis (PFGE) and restriction endonuclease analysis (REA) were used to characterize the phage genomes. Three categories of bacteriophages were observed. I: a genome size of ~194 kb and refractory to digestion with HhaI; II: a genome size of ~140 kb and digestible by HhaI; and III: a genome size undeterminable in PFGE. The categorization of the phages correlated with the host range patterns displayed by the phages. Six phages were subjected to transmission electron microscopy (TEM). They all belonged to the family of Myoviridae.
We have characterized and identified the host range of 12 Danish Campylobacter phages. Due to their ability to infect the majority of the common serotypes in Denmark we suggest the phages can become an effective agent in the effort to reduce the incidence of campylobacteriosis in Denmark. This study provides the basis for future experiments in Campylobacter phages and knowledge for the selection of Campylobacter phages for biocontrol in broilers.
Salmonella spp. are enteropathogenic gram-negative bacteria that use a large array of virulence factors to colonize the host, manipulate host cells, and resist the host's defense mechanisms. Even closely related Salmonella strains have different repertoires of virulence factors. Bacteriophages contribute substantially to this diversity. There is increasing evidence that the reassortment of virulence factor repertoires by converting phages like the GIFSY phages and SopEΦ may represent an important mechanism in the adaptation of Salmonella spp. to specific hosts and to the emergence of new epidemic strains. Here, we have analyzed in more detail SopEΦ, a P2-like phage from Salmonella enterica serovar Typhimurium DT204 that encodes the virulence factor SopE. We have cloned and characterized the attachment site (att) of SopEΦ and found that its 47-bp core sequence overlaps the 3′ terminus of the ssrA gene of serovar Typhimurium. Furthermore, we have demonstrated integration of SopEΦ into the cloned attB site of serovar Typhimurium A36. Sequence analysis of the plasmid-borne prophage revealed that SopEΦ is closely related to (60 to 100% identity over 80% of the genome) but clearly distinct from the Fels-2 prophage of serovar Typhimurium LT2 and from P2-like phages in the serovar Typhi CT18 genome. Our results demonstrate that there is considerable variation among the P2-like phages present in closely related Salmonella spp.
The surface plasmon resonance (SPR) technique is a well-established method for the measurement of molecules binding to surfaces and the quantification of binding constants between surface-immobilized proteins and proteins in solution. In this paper we describe an extension of the methodology to study bacteriophage-bacterium interactions. A two-channel microfluidic SPR sensor device was used to detect the presence of somatic coliphages, a group of bacteriophages that have been proposed as fecal pollution indicators in water, using their host, Escherichia coli WG5, as a target for their selective detection. The bacterium, E. coli WG5, was immobilized on gold sensor chips using avidin-biotin and bacteriophages extracted from wastewater added. The initial binding of the bacteriophage was observed at high concentrations, and a separate, time-delayed cell lysis event also was observed, which was sensitive to bacteriophage at low concentrations. As few as 1 PFU/ml of bacteriophage injected into the chamber could be detected after a phage incubation period of 120 min, which equates to an approximate limit of detection of around 102 PFU/ml. The bacteriophage-bacterium interaction appeared to cause a structural change in the surface-bound bacteria, possibly due to collapse of the cell, which was observed as an increase in mass density on the sensor chip. These results suggest that this methodology could be employed for future biosensor technologies and for quantification of the bacteriophage concentration.
There has been a recent resurgent interest in bacteriophage biology. Research was initiated to examine Campylobacter jejuni-specific bacteriophage in the Russian Federation to develop alternative control measures for this pathogen.
A C. jejuni flagellum-specific phage PV22 from Proteus vulgaris was identified in sewage drainage. This phage interacted with C. jejuni by attachment to flagella followed by translocation of the phage to the polar region of the bacterium up to the point of DNA injection. Electron microscopic examination revealed adsorption of PV22 on C. jejuni flagella after a five minute incubation of the phage and bacteria. A different phenomenon was observed after incubating the mix under the same conditions, but for twenty minutes or longer. Phage accumulated primarily on the surface of cells at sites where flagella originated. Interestingly, PV22 did not inject DNA into C. jejuni and PV22 did not produce lytic plaques on medium containing C. jejuni cells. The constant of velocity for PV22 adsorption on cells was 7 × 10-9 ml/min.
It was demonstrated that a bacteriophage that productively infects P. vulgaris was able to bind C. jejuni and by a spot test that the growth of C. jejuni was reduced relative to control bacteria in the region of phage application. There may be two interesting applications of this effect. First, it may be possible to test phage PV22 as an antimicrobial agent to decrease C. jejuni colonization of the chicken intestine. Second, the phage could potentially be utilized for investigating biogenesis of C. jejuni flagella.
Treatment of gram-negative bacteria with lethal doses of polymyxin B and colistin resulted in the formation of projections of the outer layer of the cell wall. Phages T3, T4, and T7, which use wall lipopolysaccharide as receptors, were specifically prevented from adsorbing to Escherichia coli B cells treated with polymyxin, whereas phages T1, T2, T5, and T6 were not. In the systems of phage P22C-Salmonella typhimurium LT2 and phage C21-S. typhimurium variant SL1069, the phage were prevented from adsorbing to the host cell treated with the antibiotics. Electron microscopic observations show that phage T2 adsorbed irreversibly to the normal smooth surface between the projections on the outer layer caused by the drug treatment. These results indicate that lipopolysaccharide is affected by polymyxin functionally and morphologically, but lipoprotein is not. The purified lipopolysaccharide showed a ribbon-like structure when viewed face on and showed trilamellar structure when viewed edge on. The lipopolysaccharide from E. coli B was irreversibly adsorbed by phages T3, T4, and T7, but not phage T2. Often, phage T4 adsorbed to both sides of the lipopolysaccharide strand at comparable distances. Phage P22C adsorbed through the spikes of the tail-plates to the lipopolysaccharide from S. typhimurium LT2. Lipopolysaccharide which was treated with low doses of the drug (2.5 to 6.25 μg of polymyxin B per ml to 100 μg of lipopolysaccharide per ml) turned into the coiled form and was partially broken down into short segments with coiled form. The loosely coiled lipopolysaccharide retains both its function as the receptor and its trilamellar structure. Treatment with high doses of the drug (12.5 to 25 μg of polymyxin B per ml to 100 μg of lipopolysaccharide per ml) caused the collapse of the trilamellar structure of the strand. These collapsed lipopolysaccharides became flat and fused with each other, making an amorphous mass, and finally they were broken into small collapsed fragments.
Campylobacter jejuni is a worldwide cause of human diarrhoeal disease. Clustered Repetitively Interspaced Palindromic Repeats (CRISPRs) and associated proteins allow Bacteria and Archaea to evade bacteriophage and plasmid infection. Type II CRISPR systems are found in association with combinations of genes encoding the CRISPR-associated Cas1, Cas2, Cas4 or Csn2, and Cas9 proteins. C. jejuni possesses a minimal subtype II-C CRISPR system containing cas1, cas2, and cas9 genes whilst cas4 is notably absent. Cas4 proteins possess 5′-3′ exonuclease activity to create recombinogenic-ends for spacer acquisition. Here we report a conserved Cas4-like protein in Campylobacter bacteriophages that creates a novel split arrangement between the bacteriophage and host that represents a new twist in the bacteriophage/host co-evolutionary arms race. The continuous association of bacteriophage and host in the carrier state life cycle of C. jejuni provided an opportunity to study spacer acquisition in this species. Remarkably all the spacer sequences observed were of host origin. We hypothesize that Campylobacter bacteriophages can use Cas4-like protein to activate spacer acquisition to use host DNA as an effective decoy to bacteriophage DNA. Bacteria that acquire self-spacers and escape phage infection must overcome CRISPR-mediated autoimmunity either by loss of the interference functions leaving them susceptible to foreign DNA incursion or tolerate changes in gene regulation.
Campylobacter; CRISPR; bacteriophage; Cas4; carrier state life cycle
This laboratory is investigating radiolabeled bacteriophages for specific detection of infection through gamma imaging. Previously a 99mTc-labeled M13 phage demonstrated specific binding for its host Escherichia coli in vitro and in mice through imaging.
This study was extended to phages P22, E79, VD-13, and phage 60. Each was radiolabeled with 99mTc using the chelator MAG3, labeled phages were evaluated for binding to host and non host bacteria in vitro and in a mouse model.
In vitro each 99mTc-phage bound to its host at least 4-fold higher than to non host bacteria. For example, 99mTc-E79 showed 10- to 20-fold greater binding to host Pseudomonas aeruginosa than non host Escherichia coli and Salmonella enterica, respectively. 99mTc-phage 60 showed 20-fold greater binding to host Klebsiella pneumoniae over non hosts. Mice received host or non host bacteria in one thigh and 3 h later the 99mTc-phages were administered iv. After a further 3 h the tissues were counted. Liver accumulation was highest for 99mTc-E79, averaging 39% compared to an average of 13% for the other 99mTc-phages. Animals infected with host bacteria showed infected thigh to normal thigh ratios of 14.2 for 99mTc-E79, 2.9 for 99mTc- P22, 3.5 for 99mTc- VD-13 and 2.1 for 99mTc- phage 60.
Although specific host binding was observed in vitro for each of the these four 99mTc-phages, only 99mTc-E79 showed specificity for its host in an in vivo model.
Infection detection; bacteriophage; Tc-labeled phage
The potential of bacteriophage as an alternative biocontrol agent has recently been revisited due to the widespread occurrence of antibiotic-resistant bacteria. We isolated a virulent bacteriophage, SPC35, that can infect both Salmonella enterica serovar Typhimurium and Escherichia coli. Morphological analysis by transmission electron microscopy and analysis of its 118,351-bp genome revealed that SPC35 is a T5 group phage belonging to the family Siphoviridae. BtuB, the outer membrane protein for vitamin B12 uptake, was found to be a host receptor for SPC35. Interestingly, resistant mutants of both E. coli and S. Typhimurium developed faster than our expectation when the cultures were infected with SPC35. Investigation of the btuB gene revealed that it was disrupted by the IS2 insertion sequence element in most of the resistant E. coli isolates. In contrast, we could not detect any btuB gene mutations in the resistant S. Typhimurium isolates; these isolates easily regained sensitivity to SPC35 in its absence, suggesting phase-variable phage resistance/sensitivity. These results indicate that a cocktail of phages that target different receptors on the pathogen should be more effective for successful biocontrol.
A method was developed for oriented immobilization of bacteriophage T4 through introduction of specific binding ligands into the phage head using a phage display technique. Fusion of the biotin carboxyl carrier protein gene (bccp) or the cellulose binding module gene (cbm) with the small outer capsid protein gene (soc) of T4 resulted in expression of the respective ligand on the phage head. Recombinant bacteriophages were characterized in terms of infectivity. It was shown that both recombinant phages retain their lytic activity and host range. However, phage head modification resulted in a decreased burst size and an increased latent period. The efficiency of bacteriophage immobilization with streptavidin-coated magnetic beads and cellulose-based materials was investigated. It was shown that recombinant bacteriophages form specific and strong bonds with their respective solid support and are able to specifically capture and infect the host bacterium. Thus, the use of immobilized BCCP-T4 bacteriophage for an Escherichia coli B assay using a phage multiplication approach and real-time PCR allowed detection of as few as 800 cells within 2 h.
Phages are promising alternatives to antibodies as the biorecognition element in a variety of biosensing applications. In this study, a monolayer of bacteriophage P22 whose tailspike proteins specifically recognize Salmonella serotypes was covalently bound to glass substrates through a bifunctional cross linker 3-aminopropyltrimethoxysilane. The specific binding of Salmonella typhimurium to the phage monolayer was studied by enzyme-linked immunosorbent assay and atomic force microscopy. Escherichia coli and a Gram-positive bacterium Listeria monocytogenes were also studied as control bacteria. The P22 particles show strong binding affinity to Salmonella typhimurium. In addition, the dried P22 monolayer maintained 50% binding capacity to Salmonella typhimurium after a one-week storage time. This is a promising method to prepare phage monolayer coatings on surface plasmon resonance and acoustic biosensor substrates in order to utilize the nascent phage display technology.
bacteriophage; Salmonella typhimurium; lipopolysaccharide membrane
Campylobacter jejuni produces both lipooligosaccharide (LOS) and a higher-molecular-weight polysaccharide that is believed to form a capsule. The role of these surface polysaccharides in C. jejuni-mediated enteric disease is unclear; however, epitopes associated with the LOS are linked to the development of neurological complications. In Escherichia coli and Salmonella enterica serovar Typhimurium the waaF gene encodes a heptosyltransferase, which catalyzes the transfer of the second l-glycero-d-manno-heptose residue to the core oligosaccharide moiety of lipopolysaccharide (LPS), and mutation of waaF results in a truncated core oligosaccharide. In this report we confirm experimentally that C. jejuni gene Cj1148 encodes the heptosyltransferase II enzyme, WaaF. The Campylobacter waaF gene complements an S. enterica serovar Typhimurium waaF mutation and restores the ability to produce full-sized lipopolysaccharide. To examine the role of WaaF in C. jejuni, waaF mutants were constructed in strains NCTC 11168 and NCTC 11828. Loss of heptosyltransferase activity resulted in the production of a truncated core oligosaccharide, failure to bind specific ligands, and loss of serum reactive GM1, asialo-GM1, and GM2 ganglioside epitopes. The mutation of waaF did not affect the higher-molecular-weight polysaccharide supporting the production of a LOS-independent capsular polysaccharide by C. jejuni. The exact structural basis for the truncation of the core oligosaccharide was verified by comparative chemical analysis. The NCTC 11168 core oligosaccharide differs from that known for HS:2 strain CCUG 10936 in possessing an extra terminal disaccharide of galactose-β(1,3) N-acetylgalactosamine. In comparison, the waaF mutant possessed a truncated molecule consistent with that observed with waaF mutants in other bacterial species.