Campylobacter Phages Research Articles

A broad host phage, CP6, for combating multidrug-resistant Campylobacter prevalent in poultry meat

Campylobacter is a major cause of bacterial foodborne diarrhea worldwide. Consumption of raw or undercooked chicken meat contaminated with Campylobacter is the most common causative agent of human infections. Given the high prevalence of contamination in poultry meat and the recent rise of multi-drug-resistant (MDR) Campylobacter strains, an effective intervention method of reducing bird colonization is needed. In this study, the Campylobacter-specific lytic phage CP6 was isolated from chicken feces. Phage CP6 exhibited a broad host range against different MDR Campylobacter isolates (97.4% of strains were infected). Some biological characteristics were observed, such as a good pH (3-9) stability and moderate temperature tolerance (<50 ℃). The complete genome sequence revealed a linear double-stranded DNA (178,350 bp, group II Campylobacter phage) with 27.51% GC content, including 209 predicted open reading frames, among which only 54 were annotated with known functions. Phylogenetic analysis of the phage major capsid protein demonstrated that phage CP6 was closely related to Campylobacter phage CPt10, CP21, CP20, IBB35, and CP220. CP6 phage exerted good antimicrobial effects on MDR Campylobacter in vitro culture and reduced CFUs of the host cells by up to 1-log compared with the control in artificially contaminated chicken breast meat. Our findings suggested the potential of CP6 phage as a promising antimicrobial agent for combating MDR Campylobacter in food processing.

Application of Bacteriophages to Limit Campylobacter in Poultry Production.

Campylobacter is a major foodborne pathogen with over a million United States cases a year and is typically acquired through the consumption of poultry products. The common occurrence of Campylobacter as a member of the poultry gastrointestinal tract microbial community remains a challenge for optimizing intervention strategies. Simultaneously, increasing demand for antibiotic-free products has led to the development of several alternative control measures both at the farm and in processing operations. Bacteriophages administered to reduce foodborne pathogens are one of the alternatives that have received renewed interest. Campylobacter phages have been isolated from both conventionally and organically raised poultry. Isolated and cultivated Campylobacter bacteriophages have been used as an intervention in live birds to target colonized Campylobacter in the gastrointestinal tract. Application of Campylobacter phages to poultry carcasses has also been explored as a strategy to reduce Campylobacter levels during poultry processing. This review will focus on the biology and ecology of Campylobacter bacteriophages in poultry production followed by discussion on current and potential applications as an intervention strategy to reduce Campylobacter occurrence in poultry production.

Reduced Infection Efficiency of Phage NCTC 12673 on Non-Motile Campylobacter jejuni Strains Is Related to Oxidative Stress.

Campylobacter jejuni is a Gram-negative foodborne pathogen that causes diarrheal disease and is associated with severe post-infectious sequelae. Bacteriophages (phages) are a possible means of reducing Campylobacter colonization in poultry to prevent downstream human infections. However, the factors influencing phage-host interactions must be better understood before this strategy can be predictably employed. Most studies have focused on Campylobacter phage binding to the host surface, with all phages classified as either capsule- or flagella-specific. Here we describe the characterization of a C. jejuni phage that requires functional flagellar glycosylation and motor genes for infection, without needing the flagella for adsorption to the cell surface. Through phage infectivity studies of targeted C. jejuni mutants, transcriptomic analysis of phage-resistant mutants, and genotypic and phenotypic analysis of a spontaneous phage variant capable of simultaneously overcoming flagellar gene dependence and sensitivity to oxidative stress, we have uncovered a link between oxidative stress, flagellar motility, and phage infectivity. Taken together, our results underscore the importance of understanding phage-host interactions beyond the cell surface and point to host oxidative stress state as an important and underappreciated consideration for future phage-host interaction studies.

Campylobacter phages use hypermutable polyG tracts to create phenotypic diversity and evade bacterial resistance

Phase variation is a common mechanism for creating phenotypic heterogeneity of surface structures in bacteria important for niche adaptation. In Campylobacter, phase variation occurs by random variation in hypermutable homonucleotide 7-11G (polyG) tracts. To elucidate how phages adapt to phase-variable hosts, we study Fletchervirus phages infecting Campylobacter dependent on a phase-variable receptor. Our data demonstrate that Fletcherviruses mimic their host and encode hypermutable polyG tracts, leading to phase-variable expression of two of four receptor-binding proteins. This creates phenotypically diverse phage populations, including a sub-population that infects the bacterial host when the phase-variable receptor is not expressed. Such population dynamics of both phage and host promote co-existence in a shared niche. Strikingly, we identify polyG tracts in more than 100 phage genera, infecting more than 70 bacterial species. Future experimental work may confirm phase variation as a widespread strategy for creating phenotypically diverse phage populations.

Distribution of CRISPR Types in Fluoroquinolone-Resistant Campylobacter jejuni Isolates.

To aid development of phage therapy against Campylobacter, we investigated the distribution of the clustered regularly interspaced short palindromic repeats (CRISPR) systems in fluoroquinolone (FQ)-resistant Campylobacter jejuni. A total of 100 FQ-resistant C. jejuni strains from different sources were analyzed by PCR and DNA sequencing to determine resistance-conferring mutation in the gyrA gene and the presence of various CRISPR systems. All but one isolate harbored 1–5 point mutations in gyrA, and the most common mutation was the Thr86Ile change. Ninety-five isolates were positive with the CRISPR PCR, and spacer sequences were found in 86 of them. Among the 292 spacer sequences identified in this study, 204 shared 93–100% nucleotide homology to Campylobacter phage D10, 44 showed 100% homology to Campylobacter phage CP39, and 3 had 100% homology with Campylobacter phage CJIE4-5. The remaining 41 spacer sequences did not match with any phages in the database. Based on the results, it was inferred that the FQ-resistant C. jejuni isolates analyzed in this study were potentially resistant to Campylobacter phages D10, CP39, and CJIE4-5 as well as some unidentified phages. These phages should be excluded from cocktails of phages that may be utilized to treat FQ-resistant Campylobacter.

Developing Innolysins Against Campylobacter jejuni Using a Novel Prophage Receptor-Binding Protein.

Campylobacter contaminated poultry remains the major cause of foodborne gastroenteritis worldwide, calling for novel antibacterials. We previously developed the concept of Innolysin composed of an endolysin fused to a phage receptor binding protein (RBP) and provided the proof-of-concept that Innolysins exert bactericidal activity against Escherichia coli. Here, we have expanded the Innolysin concept to target Campylobacter jejuni. As no C. jejuni phage RBP had been identified so far, we first showed that the H-fiber originating from a CJIE1-like prophage of C. jejuni CAMSA2147 functions as a novel RBP. By fusing this H-fiber to phage T5 endolysin, we constructed Innolysins targeting C. jejuni (Innolysins Cj). Innolysin Cj1 exerts antibacterial activity against diverse C. jejuni strains after in vitro exposure for 45 min at 20°C, reaching up to 1.30 ± 0.21 log reduction in CAMSA2147 cell counts. Screening of a library of Innolysins Cj composed of distinct endolysins for growth inhibition, allowed us to select Innolysin Cj5 as an additional promising antibacterial candidate. Application of either Innolysin Cj1 or Innolysin Cj5 on chicken skin refrigerated to 5°C and contaminated with C. jejuni CAMSA2147 led to 1.63 ± 0.46 and 1.18 ± 0.10 log reduction of cells, respectively, confirming that Innolysins Cj can kill C. jejuni in situ. The receptor of Innolysins Cj remains to be identified, however, the RBP component (H-fiber) recognizes a novel receptor compared to lytic phages binding to capsular polysaccharide or flagella. Identification of other unexplored Campylobacter phage RBPs may further increase the repertoire of new Innolysins Cj targeting distinct receptors and working as antibacterials against Campylobacter.

Two Distinct Modes of Lysis Regulation in Campylobacter Fletchervirus and Firehammervirus Phages.

Campylobacter phages are divided into two genera; Fletchervirus and Firehammervirus, showing only limited intergenus homology. Here, we aim to identify the lytic genes of both genera using two representative phages (F352 and F379) from our collection. We performed a detailed in silico analysis searching for conserved protein domains and found that the predicted lytic genes are not organized into lysis cassettes but are conserved within each genus. To verify the function of selected lytic genes, the proteins were expressed in E. coli, followed by lytic assays. Our results show that Fletchervirus phages encode a typical signal peptide (SP) endolysin dependent on the Sec-pathway for translocation and a holin for activation. In contrast, Firehammervirus phages encode a novel endolysin that does not belong to currently described endolysin groups. This endolysin also uses the Sec-pathway for translocation but induces lysis of E. coli after overexpression. Interestingly, co-expression of this endolysin with an overlapping gene delayed and limited cell lysis, suggesting that this gene functions as a lysis inhibitor. These results indicate that Firehammervirus phages regulate lysis timing by a yet undescribed mechanism. In conclusion, we found that the two Campylobacter phage genera control lysis by two distinct mechanisms.

Bacteriophages to Control Campylobacter in Commercially Farmed Broiler Chickens, in Australia.

This study describes the development and use of bacteriophage cocktails to control Campylobacter in broiler chickens, in a commercial setting, in Queensland Australia, following the birds from farm to the processing plant. The components of the bacteriophage cocktails were selected to be effective against the maximum number of Campylobacter jejuni and Campylobacter coli isolates encountered on SE Queensland farms. Farms were identified that had suitable Campylobacter target populations and phage were undetectable 1 week prior to the intended treatment. Cocktails of phages were administered at 47 days of age. Groups of study birds were slaughtered the following day, on-farm, at the end of flock transport to the plant, and at processing (approximately 28 h post-treatment). On Farm A, the phage treatment significantly reduced Campylobacter levels in the ceca at the farm in the range of 1–3 log10 CFU/g (p = 0.007), compared to mock treated controls. However, individual birds sampled on farm (1/10) or following transport (2/10) exhibited high cecal Campylobacter counts with low phage titers, suggesting that treatment periods > 24 h may be required to ensure phage replication for effective biocontrol in vivo. At the time of the trial the control birds in Farm B were phage positive despite having been negative one week earlier. There was no significant difference in the cecal Campylobacter counts between the treatment and control groups following treatment but a fall of 1.7 log10 CFU/g was observed from that determined from birds collected the previous week (p = 0.0004). Campylobacter isolates from both farms retained sensitivity to the treatment phages. These trials demonstrated bacteriophages sourced from Queensland farms have the potential to reduce intestinal Campylobacter levels in market ready broiler chickens.

Binding of Phage-Encoded FlaGrab to Motile Campylobacter jejuni Flagella Inhibits Growth, Downregulates Energy Metabolism, and Requires Specific Flagellar Glycans.

Many bacterial pathogens display glycosylated surface structures that contribute to virulence, and targeting these structures is a viable strategy for pathogen control. The foodborne pathogen Campylobacter jejuni expresses a vast diversity of flagellar glycans, and flagellar glycosylation is essential for its virulence. Little is known about why C. jejuni encodes such a diverse set of flagellar glycans, but it has been hypothesized that evolutionary pressure from bacteriophages (phages) may have contributed to this diversity. However, interactions between Campylobacter phages and host flagellar glycans have not been characterized in detail. Previously, we observed that Gp047 (now renamed FlaGrab), a conserved Campylobacter phage protein, binds to C. jejuni flagella displaying the nine-carbon monosaccharide 7-acetamidino-pseudaminic acid, and that this binding partially inhibits cell growth. However, the mechanism of this growth inhibition, as well as how C. jejuni might resist this activity, are not well-understood. Here we use RNA-Seq to show that FlaGrab exposure leads C. jejuni 11168 cells to downregulate expression of energy metabolism genes, and that FlaGrab-induced growth inhibition is dependent on motile flagella. Our results are consistent with a model whereby FlaGrab binding transmits a signal through flagella that leads to retarded cell growth. To evaluate mechanisms of FlaGrab resistance in C. jejuni, we characterized the flagellar glycans and flagellar glycosylation loci of two C. jejuni strains naturally resistant to FlaGrab binding. Our results point toward flagellar glycan diversity as the mechanism of resistance to FlaGrab. Overall, we have further characterized the interaction between this phage-encoded flagellar glycan-binding protein and C. jejuni, both in terms of mechanism of action and mechanism of resistance. Our results suggest that C. jejuni encodes as-yet unidentified mechanisms for generating flagellar glycan diversity, and point to phage proteins as exciting lenses through which to study bacterial surface glycans.

Efficient isolation of Campylobacter bacteriophages from chicken skin, analysis of several isolation protocols

The application of Campylobacter specific bacteriophages appears as a promising food safety tool for the biocontrol of this pathogen in the poultry meat production chain. However, their isolation is a complicated challenge since their occurrence appears to be low. This work assessed the efficiency of seven protocols for recovering Campylobacter phages from chicken skin samples inoculated at phage loads from 5.0 × 101 to 5.0 × 106 PFU/g. The enrichment of chicken skin in selective Bolton broth containing target isolates was the most efficient procedure, showing a low detection limit of 5.0 × 101 PFU/g and high recovery rates of up to 560%. This method's effectiveness increased as phage concentration decreased, showing its suitability for phage isolation. When this method was applied to isolate new Campylobacter phages from retail chicken skin, a total of 280 phages were recovered achieving an isolation success rate of 257%. From the 109 samples 68 resulted phage positive (62%). Chicken skin could be, therefore, considered a rich source in Campylobacter phages. This method is a simple, reproducible and efficient approach for the successful isolation of both group II and III Campylobacter specific bacteriophages, which could be helpful for the enhancement of food safety by reducing this pathogen contamination in broiler meat.

Development of a Lyophilization Process for Campylobacter Bacteriophage Storage and Transport.

Bacteriophages are a sustainable alternative to control pathogenic bacteria in the post-antibiotic era. Despite promising reports, there are still obstacles to phage use, notably titer stability and transport-associated expenses for applications in food and agriculture. In this study, we have developed a lyophilization approach to maintain phage titers, ensure efficacy and reduce transport costs of Campylobacter bacteriophages. Lyophilization methods were adopted with various excipients to enhance stabilization in combination with packaging options for international transport. Lyophilization of Eucampyvirinae CP30A using tryptone formed a cake that limited processing titer reduction to 0.35 ± 0.09 log10 PFU mL−1. Transmission electron microscopy revealed the initial titer reduction was associated with capsid collapse of a subpopulation. Freeze-dried phages were generally stable under refrigerated vacuum conditions and showed no significant titer changes over 3 months incubation at 4 °C (p = 0.29). Reduced stability was observed for lyophilized phages that were incubated either at 30 °C under vacuum or at 4 °C at 70% or 90% relative humidity. Refrigerated international transport and rehydration of the cake resulted in a total phage titer reduction of 0.81 ± 0.44 log10 PFU mL−1. A significantly higher titer loss was observed for phages that were not refrigerated during transport (2.03 ± 0.32 log10 PFU mL−1). We propose that lyophilization offers a convenient method to preserve and transport Campylobacter phages, with minimal titer reduction after the drying process.

Application of Campylobacter jejuni Phages: Challenges and Perspectives.

Simple SummaryCampylobacter jejuni is a zoonotic bacterial pathogen found in abundance, e.g., in raw poultry. C. jejuni is part of the avian gut microbiome, causing no harm to birds. When birds are slaughtered, the bacterium is released from the intestines and contaminates the meat. Cross-contaminated foods processed in parallel to the meat cause diarrhea in humans and in rare cases may cause post-infectious complications like arthritis and paralysis of peripheral nerves. Illnesses caused by C. jejuni have been on the rise in both developed and developing countries. At the same time, C. jejuni has acquired resistance to important antibiotics, which further complicates therapeutic approaches. To decrease the Campylobacter load on chicken carcasses, chemical or thermal treatment can be used. However, this may change the taste or affect the appearance of chicken meat. Alternative treatments include application of probiotics active against C. jejuni to live chickens or the use of bacteriophages (viruses killing bacteria) to decrease Campylobacter in the guts of live chickens prior to killing. This article discusses phage-based applications already in use against other bacterial pathogens and specifically highlights the challenges associated with the use of Campylobacter phages, emphasizing the ways to overcome these challenges based on the existing research.Bacteriophages (phages) are the most abundant and diverse biological entities in the biosphere. Due to the rise of multi-drug resistant bacterial strains during the past decade, phages are currently experiencing a renewed interest. Bacteriophages and their derivatives are being actively researched for their potential in the medical and biotechnology fields. Phage applications targeting pathogenic food-borne bacteria are currently being utilized for decontamination and therapy of live farm animals and as a biocontrol measure at the post-harvest level. For this indication, the United States Food and Drug Administration (FDA) has approved several phage products targeting Listeria sp., Salmonella sp. and Escherichia coli. Phage-based applications against Campylobacter jejuni could potentially be used in ways similar to those against Salmonella sp. and Listeria sp.; however, only very few Campylobacter phage products have been approved anywhere to date. The research on Campylobacter phages conducted thus far indicates that highly diverse subpopulations of C. jejuni as well as phage isolation and enrichment procedures influence the specificity and efficacy of Campylobacter phages. This review paper emphasizes conclusions from previous findings instrumental in facilitating isolation of Campylobacter phages and improving specificity and efficacy of the isolates.

Phage Biocontrol of Campylobacter jejuni in Chickens Does Not Produce Collateral Effects on the Gut Microbiota.

Bacteriophage biocontrol to reduce Campylobacter jejuni levels in chickens can reduce human exposure and disease acquired through the consumption of contaminated poultry products. Investigating changes in the chicken microbiota during phage treatment has not previously been undertaken but is crucial to understanding the system-wide effects of such treatments to establish a sustainable application. A phage cocktail containing two virulent Campylobacter phages was used to treat broiler chickens colonized with C. jejuni HPC5. Campylobacter counts from cecal contents were significantly reduced throughout the experimental period but were most effective 2 days post-treatment showing a reduction of 2.4 log10 CFU g-1 relative to mock-treated Campylobacter colonized controls. The administered phages replicated in vivo to establish stable populations. Bacteriophage predation of C. jejuni was not found to affect the microbiota structure but selectively reduced the relative abundance of C. jejuni without affecting other bacteria.

Campylobacter Phage Isolation and Characterization: What We Have Learned So Far.

Lytic Campylobacter phages, which can be used to combat this pathogen in animals and on food products, have been studied for more than 30 years. Though, due to some peculiarities of the phages, which hampered their isolation and particularly their molecular analysis for a long time, progress in this research field was rather slow. Meanwhile, the situation has changed and much more is known about the biology and genetics of those phages. In this article, we address specific issues that should be considered when Campylobacter phages are studied, starting with the isolation and propagation of the phages and ending with a thorough characterization including whole-genome sequencing. The basis for advice and recommendations given here is a careful review of the scientific literature and experiences that we have had ourselves with Campylobacter phages.

Comparative genomics of Cp8viruses with special reference to Campylobacter phage vB_CjeM_los1, isolated from a slaughterhouse in Ireland.

Campylobacter phage vB_CjeM_Los1 was recently isolated from a slaughterhouse in the Republic of Ireland using the host Campylobacter jejuni subsp. jejuni PT14, and full-genome sequencing and annotation were performed. The genome was found to be 134,073bp in length and to contain 169 predicted open reading frames. Transmission electron microscopy images of vB_CjeM_Los1 revealed that it belongs to the family Myoviridae, with tail fibres observed in both extended and folded conformations, as seen in T4. The genome size and morphology of vB_CjeM_Los1 suggest that it belongs to the genus Cp8virus, and seven other Campylobacter phages with similar size characteristics have also been fully sequenced. In this work, comparative studies were performed in relation to genomic rearrangements and conservation within each of the eight genomes. None of the eight genomes were found to have undergone internal rearrangements, and their sequences retained more than 98% identity with one another despite the widespread geographical distribution of each phage. Whole-genome phylogenetics were also performed, and clades were shown to be representative of the differing number of tRNAs present in each phage. This may be an indication of lineages within the genus, despite their striking homology.

Phase Variable Expression of a Single Phage Receptor in Campylobacter jejuni NCTC12662 Influences Sensitivity Toward Several Diverse CPS-Dependent Phages.

Campylobacter jejuni NCTC12662 is sensitive to infection by many Campylobacter bacteriophages. Here we used this strain to investigate the molecular mechanism behind phage resistance development when exposed to a single phage and demonstrate how phase variable expression of one surface component influences phage sensitivity against many diverse C. jejuni phages. When C. jejuni NCTC12662 was exposed to phage F207 overnight, 25% of the bacterial cells were able to grow on a lawn of phage F207, suggesting that resistance develops at a high frequency. One resistant variant, 12662R, was further characterized and shown to be an adsorption mutant. Plaque assays using our large phage collection showed that seven out of 36 diverse capsular polysaccharide (CPS)-dependent phages could not infect 12662R, whereas the remaining phages formed plaques on 12662R with reduced efficiencies. Analysis of the CPS composition of 12662R by high-resolution magic angle spinning nuclear magnetic resonance (HR-MAS NMR) showed a diminished signal for O-methyl phosphoramidate (MeOPN), a phase variable modification of the CPS. This suggested that the majority of the 12662R population did not express this phase variable modification in the CPS, indicating that MeOPN serves as a phage receptor in NCTC12662. Whole genome analysis of 12662R showed a switch in the length of the phase variable homopolymeric G tract of gene 06810, encoding a putative MeOPN-transferase located in the CPS locus, resulting in a non-functional protein. To confirm the role of 06810 in phage resistance development of NCTC12662, a 06810 knockout mutant in NCTC12662 was constructed and analyzed by HR-MAS NMR demonstrating the absence of MeOPN in the CPS of the mutant. Plaque assays using NCTC12662Δ06810 demonstrated that seven of our CPS-dependent Campylobacter phages are dependent on the presence of MeOPN for successful infection of C. jejuni, whereas the remaining 29 phages infect independently of MeOPN, although with reduced efficiencies. Our data indicate that CPS-dependent phages uses diverse mechanisms for their initial interaction with their C. jejuni host.

A multiplex real-time PCR for the detection and differentiation of Campylobacter phages.

Campylobacter jejuni and C. coli are important food-borne pathogens that are widespread in animal husbandry. To combat Campylobacter along the food chain, the application of lytic phages has been shown to be a promising tool. Campylobacter phages are currently classified into three groups, of which group II and group III phages are the most common. Members of each group are closely related, whereas the two groups share only little DNA similarity. Moreover, while group III phages are specific for C. jejuni, group II phages additionally infect C. coli. Phage cocktails intended to be used for applications should be composed of various phages that differ in their host range and growth kinetics. The isolation of phages is generally performed by plaque assays. This approach has the limitation that phages are merely identified by their lytic activity on certain indicator strains and that relatively high numbers of phages must be present in a tested sample. Therefore, a more sensitive molecular detection system would be beneficial, which allows a pre-screening of samples and the quick detection and discrimination of group II and group III phages. New phages can then be isolated by use of indicator strains that may be different from those typically applied. On the basis of available Campylobacter phage genome sequences, we developed a multiplex PCR system for group II and group III phages selecting the tail tube gene and the gene for the base plate wedge, respectively, as target. Phages of both groups could be identified with primers deduced from the putative tail fiber gene. Efficient release of phage DNA from capsids was achieved by an extended heat treatment or digestion of phage particles with proteinase K/SDS yielding a detection limit of 1 pfu/ml. Individual detection of group II phages, group III phages and of both groups was studied with artificially contaminated chicken skin. To recover phages that had strongly adhered to the skin, stomaching was the most efficient technique. The developed PCR protocol was employed to detect Campylobacter phages in food and environmental samples. In 50 out of 110 samples group II and/or group III phages were identified.

The Bacteriophage Carrier State of Campylobacter jejuni Features Changes in Host Non-coding RNAs and the Acquisition of New Host-derived CRISPR Spacer Sequences.

Incorporation of self-derived CRISPR DNA protospacers in Campylobacter jejuni PT14 occurs in the presence of bacteriophages encoding a CRISPR-like Cas4 protein. This phenomenon was evident in carrier state infections where both bacteriophages and host are maintained for seemingly indefinite periods as stable populations following serial passage. Carrier state cultures of C. jejuni PT14 have greater aerotolerance in nutrient limited conditions, and may have arisen as an evolutionary response to selective pressures imposed during periods in the extra-intestinal environment. A consequence of this is that bacteriophage and host remain associated and able to survive transition periods where the chances of replicative success are greatly diminished. The majority of the bacteriophage population do not commit to lytic infection, and conversely the bacterial population tolerates low-level bacteriophage replication. We recently examined the effects of Campylobacter bacteriophage/C. jejuni PT14 CRISPR spacer acquisition using deep sequencing strategies of DNA and RNA-Seq to analyze carrier state cultures. This approach identified de novo spacer acquisition in C. jejuni PT14 associated with Class III Campylobacter phages CP8/CP30A but spacer acquisition was oriented toward the capture of host DNA. In the absence of bacteriophage predation the CRISPR spacers in uninfected C. jejuni PT14 cultures remain unchanged. A distinct preference was observed for incorporation of self-derived protospacers into the third spacer position of the C. jejuni PT14 CRISPR array, with the first and second spacers remaining fixed. RNA-Seq also revealed the variation in the synthesis of non-coding RNAs with the potential to bind bacteriophage genes and/or transcript sequences.

A Flagellar Glycan-Specific Protein Encoded by Campylobacter Phages Inhibits Host Cell Growth.

We previously characterized a carbohydrate binding protein, Gp047, derived from lytic Campylobacter phage NCTC 12673, as a promising diagnostic tool for the identification of Campylobacter jejuni and Campylobacter coli. We also demonstrated that this protein binds specifically to acetamidino-modified pseudaminic acid residues on host flagella, but the role of this protein in the phage lifecycle remains unknown. Here, we report that Gp047 is capable of inhibiting C. jejuni growth both on solid and liquid media, an activity, which we found to be bacteriostatic. The Gp047 domain responsible for bacterial growth inhibition is localized to the C-terminal quarter of the protein, and this activity is both contact- and dose-dependent. Gp047 gene homologues are present in all Campylobacter phages sequenced to date, and the resulting protein is not part of the phage particle. Therefore, these results suggest that either phages of this pathogen have evolved an effector protein capable of host-specific growth inhibition, or that Campylobacter cells have developed a mechanism of regulating their growth upon sensing an impending phage threat.

Campylobacter group II phage CP21 is the prototype of a new subgroup revealing a distinct modular genome organization and host specificity

BackgroundThe application of phages is a promising tool to reduce the number of Campylobacter along the food chain. Besides the efficacy against a broad range of strains, phages have to be safe in terms of their genomes. Thus far, no genes with pathogenic potential (e.g., genes encoding virulence factors) have been detected in Campylobacter phages. However, preliminary studies suggested that the genomes of group II phages may be diverse and prone to genomic rearrangements.ResultsWe determined and analysed the genomic sequence (182,761 bp) of group II phage CP21 that is closely related to the already characterized group II phages CP220 and CPt10. The genomes of these phages are comprised of four modules separated by very similar repeat regions, some of which harbouring open reading frames (ORFs). Though, the arrangement of the modules and the location of some ORFs on the genomes are different in CP21 and in CP220/CPt10. In this work, a PCR system was established to study the modular genome organization of other group II phages demonstrating that they belong to different subgroups of the CP220-like virus genus, the prototypes of which are CP21 and CP220. The subgroups revealed different restriction patterns and, interestingly enough, also distinct host specificities, tail fiber proteins and tRNA genes. We additionally analysed the genome of group II phage vB_CcoM-IBB_35 (IBB_35) for which to date only five individual contigs could be determined. We show that the contigs represent modules linked by long repeat regions enclosing some yet not identified ORFs (e.g., for a head completion protein). The data suggest that IBB_35 is a member of the CP220 subgroup.ConclusionCampylobacter group II phages are diverse regarding their genome organization. Since all hitherto characterized group II phages contain numerous genes for transposases and homing endonucleases as well as similar repeat regions, it cannot be excluded that these phages are genetically unstable. To answer this question, further experiments and sequencing of more group II phages should be performed.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1837-1) contains supplementary material, which is available to authorized users.