Combined application of bacteriophages with a competitive exclusion culture and carvacrol with organic acids can reduce Campylobacter in primary broiler production.

For reducing Campylobacter (C.) in the food production chain and thus the risk to the consumer, the combined application of different measures as a multiple-hurdle approach is currently under discussion. This is the first study to investigate possible synergistic activities in vivo, aiming at reducing intestinal C. jejuni counts by administering (i) bacteriophages (phages) in combination with a competitive exclusion (CE) product and (ii) carvacrol combined with organic acids. The combined application of the two selected phages (Fletchervirus phage NCTC 12673 and Firehammervirus phage vB_CcM-LmqsCPL1/1) and the CE product significantly reduced C. jejuni loads by 1.0 log10 in cecal and colonic contents as well as in cloacal swabs at the end of the trial (33 and 34 days post hatch). The proportion of bacterial isolates showing reduced phage susceptibility ranged from 10.9% (isolates from cecal content) to 47.8% (isolates from cloacal swabs 32 days post hatch) for the Fletchervirus phage, while all tested isolates remained susceptible to the Firehammervirus phage. The use of carvacrol combined with an organic acid blend (sorbic acid, benzoic acid, propionic acid, and acetic acid) significantly reduced Campylobacter counts by 1.0 log10 in cloacal swabs on day 30 only.

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.

Inhibition of phage-resistant bacterial pathogen re-growth with the combined use of bacteriophages and EDTA.

The combined effects of ethylenediaminetetraacetic acid (EDTA) and bacteriophage (phage) treatment of foodborne pathogens were investigated. Although viable counts for Campylobacter jejuni decreased by 1.5 log after incubation for 8 h in the presence of phage PC10, re-growth was observed thereafter. The combination of phage PC10 and 1 mM EDTA significantly inhibited the re-growth of C. jejuni. The viable counts for C. jejuni decreased by 2.6 log (P < 0.05) compared with that of the initial count after 24 h. Moreover, EDTA at 0.67 or 1.3 mM, combined with the specific lytic phages, also effectively inhibited the re-growth of phage-resistant cells of Campylobacter coli, Salmonella enterica serovar Enteritidis, and Salmonella enterica serovar Typhimurium. In addition, the combined effects of lytic phages and EDTA were investigated on the viability of Campylobacter in BHI broth at low temperatures followed by the optimum growth temperature. The re-growth of C. coli was significantly inhibited by the coexistence of 1.3 mM EDTA, and the viable counts of surviving bacteria was about the same as the initial viable count after the incubation. This is the first study demonstrating the combined use of lytic phages and EDTA is effective in inhibiting the re-growth of phage-resistant bacteria in Gram-negative bacteria.

How does feedback from phage infections influence the evolution of phase variation in Campylobacter?

Campylobacter jejuni (C. jejuni) causes gastroenteritis following the consumption of contaminated poultry meat, resulting in a large health and economic burden worldwide. Phage therapy is a promising technique for eradicating C. jejuni from poultry flocks and chicken carcasses. However, C. jejuni can resist infections by some phages through stochastic, phase-variable ON/OFF switching of the phage receptors mediated by simple sequence repeats (SSR). While selection strength and exposure time influence the evolution of SSR-mediated phase variation (PV), phages offer a more complex evolutionary environment as phage replication depends on having a permissive host organism. Here, we build and explore several continuous culture bacteria-phage computational models, each analysing different phase-variable scenarios calibrated to the experimental SSR rates of C. jejuni loci and replication parameters for the F336 phage. We simulate the evolution of PV rates via the adaptive dynamics framework for varying levels of selective pressures that act on the phage-resistant state. Our results indicate that growth reducing counter-selection on a single PV locus results in the stable maintenance of the phage, while compensatory selection between bacterial states affects the evolutionary stable mutation rates (i.e. very high and very low mutation rates are evolutionarily disadvantageous), whereas, in the absence of either selective pressure the evolution of PV rates results in mutation rates below the basal values. Contrastingly, a biologically-relevant model with two phase-variable loci resulted in phage extinction and locking of the bacteria into a phage-resistant state suggesting that another counter-selective pressure is required, instance, the use of a distinct phage whose receptor is an F336-phage-resistant state. We conclude that a delicate balance between counter-selection and phage-attack can result in both the evolution of phase-variable phage receptors and persistence of PV-receptor-specific phage.

Deoxyinosine and 7-Deaza-2-Deoxyguanosine as Carriers of Genetic Information in the DNA of Campylobacter Viruses.

Several reports have demonstrated that Campylobacter bacteriophage DNA is refractory to manipulation, suggesting that these phages encode modified DNA. The characterized Campylobacter jejuni phages fall into two phylogenetic groups within the Myoviridae: the genera Firehammervirus and Fletchervirus Analysis of genomic nucleosides from several of these phages by high-pressure liquid chromatography-mass spectrometry confirmed that 100% of the 2'-deoxyguanosine (dG) residues are replaced by modified bases. Fletcherviruses replace dG with 2'-deoxyinosine, while the firehammerviruses replace dG with 2'-deoxy-7-amido-7-deazaguanosine (dADG), noncanonical nucleotides previously described, but a 100% base substitution has never been observed to have been made in a virus. We analyzed the genome sequences of all available phages representing both groups to elucidate the biosynthetic pathway of these noncanonical bases. Putative ADG biosynthetic genes are encoded by the Firehammervirus phages and functionally complement mutants in the Escherichia coli queuosine pathway, of which ADG is an intermediate. To investigate the mechanism of DNA modification, we isolated nucleotide pools and identified dITP after phage infection, suggesting that this modification is made before nucleotides are incorporated into the phage genome. However, we were unable to observe any form of dADG phosphate, implying a novel mechanism of ADG incorporation into an existing DNA strand. Our results imply that Fletchervirus and Firehammervirus phages have evolved distinct mechanisms to express dG-free DNA.IMPORTANCE Bacteriophages are in a constant evolutionary struggle to overcome their microbial hosts' defenses and must adapt in unconventional ways to remain viable as infectious agents. One mode of adaptation is modifying the viral genome to contain noncanonical nucleotides. Genome modification in phages is becoming more commonly reported as analytical techniques improve, but guanosine modifications have been underreported. To date, two genomic guanosine modifications have been observed in phage genomes, and both are low in genomic abundance. The significance of our research is in the identification of two novel DNA modification systems in Campylobacter-infecting phages, which replace all guanosine bases in the genome in a genus-specific manner.

FlhF(T368A) modulates motility in the bacteriophage carrier state of Campylobacter jejuni.

The carrier state is an alternative bacteriophage life cycle by which virulent bacteriophage can persist in association with host bacteria. Campylobacter jejuni carrier state strains exhibit growth phase dependent motility due to a truncated flagella phenotype. Genome sequencing identified a T368A substitution in the G3 domain of the SRP-like GTPase FlhF from C. jejuni PT14CP30A carrier state strains, which we hypothesized to be the cause of the complex motility phenotype. We have analyzed the role of this mutation in C. jejuni PT14 and demonstrated that flhF(T368A) leads to a large proportion of cells unable to synthesize flagella, while the remaining cells form a single flagellum at one pole leading to significantly reduced motility. The flhF(T368A) mutation causes a reduction in the phage adsorption constant, which leads to a decrease in infection efficiency. Down-regulation of σ28 and σ54 dependent flagellar genes were observed as responses to the flhF(T368A) mutation. FlhF(T368A) protein is impaired in GTPase activity and exhibits reduced stability. C. jejuni carrying flhF(T368A) are less sensitive to bacteriophage infection and formation of the carrier state. The acquisition of flhF(T368A) in carrier state strains acts to prevent super-infection and maintain association with the bacteriophage that provoked the interaction.

Transcriptomic Analysis of the Campylobacter jejuni Response to T4-Like Phage NCTC 12673 Infection.

Campylobacter jejuni is a frequent foodborne pathogen of humans. As C. jejuni infections commonly arise from contaminated poultry, phage treatments have been proposed to reduce the C. jejuni load on farms to prevent human infections. While a prior report documented the transcriptome of C. jejuni phages during the carrier state life cycle, transcriptomic analysis of a lytic C. jejuni phage infection has not been reported. We used RNA-sequencing to profile the infection of C. jejuni NCTC 11168 by the lytic T4-like myovirus NCTC 12673. Interestingly, we found that the most highly upregulated host genes upon infection make up an uncharacterized operon (cj0423⁻cj0425), which includes genes with similarity to T4 superinfection exclusion and antitoxin genes. Other significantly upregulated genes include those involved in oxidative stress defense and the Campylobactermultidrug efflux pump (CmeABC). We found that phage infectivity is altered by mutagenesis of the oxidative stress defense genes catalase (katA), alkyl-hydroxyperoxidase (ahpC), and superoxide dismutase (sodB), and by mutagenesis of the efflux pump genes cmeA and cmeB. This suggests a role for these gene products in phage infection. Together, our results shed light on the phage-host dynamics of an important foodborne pathogen during lytic infection by a T4-like phage.

Characterization and Application of Lytic Bacteriophages against Campylobacter jejuni Isolated from Poultry in Japan.

　The aim was to isolate Campylobacter jejuni-specific lytic phages from meats on the market in Japan. These phages were effectively isolated from 13 of 15 (86.7%) retail chicken meat samples (skin and liver) by the enrichment method using Preston Campylobacter Selective Enrichment Broth and 10 host Campylobacter strains. Among the 26 phage isolates, 14 were extracted by means of C. jejuni L26 as a host strain. Phage PHC10 showed the broadest lytic spectrum: active against 67.4% of the 46 C. jejuni strains tested. The other phage isolates showed different lytic spectra. Because phages PHC5, PHC10, PHC19, PHC22, and PHC25 possess an icosahedral head and a contracted tail, they seem to be members of the Myoviridae family. Effects of 19 phage isolates on viability of C. jejuni were investigated. These phages reduced viable counts of C. jejuni by 1-3 log after 6-12 h of incubation at 42℃ as compared to the initial counts. The C. jejuni L26 was found to be suitable as a host because of the wide hosting range. The phages isolated in this study seem to be promising biocontrol agents against C. jejuni in food.

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.

Phage exposure causes dynamic shifts in the expression states of specific phase-variable genes of Campylobacter jejuni.

Phase variation (PV) creates phenotypic heterogeneity at high frequencies and in a reversible manner. This phenomenon allows bacteria to adapt to a variety of different environments and selective pressures. In Campylobacterjejuni this reversible adaptive process is mediated by mutations in homopolymeric G/C tracts. Many C. jejuni-specific phages are dependent on phase-variable surface structures for successful infection. We previously identified the capsular polysaccharide (CPS) moiety, MeOPN-GalfNAc, as a receptor for phage F336 and showed that phase-variable expression of the transferase for this CPS modification, cj1421, and two other phase-variable CPS genes generated phage resistance in C. jejuni. Here we investigate the population dynamics of C. jejuni NCTC11168 when exposed to phage F336 in vitro using a newly described method - the 28-locus-CJ11168 PV analysis. Dynamic switching was observed in the ON/OFF states of three phase-variable CPS genes, cj1421, cj1422 and cj1426, during phage F336 exposure, with the dominant phage-resistant phasotype differing between cultures. Although loss of the phage receptor was predominately observed, several other PV events also led to phage resistance, a phenomenon that increases the chance of phage-resistant subpopulations being present in any growing culture. No other PV genes were affected and exposure to phage F336 resulted in a highly specific response, only selecting for phase variants of cj1421, cj1422 and cj1426. In summary, C. jejuni may benefit from modification of the surface in multiple ways to inhibit or reduce phage binding, thereby ensuring the survival of the population when exposed to phages.

Methods for Isolation, Purification, and Propagation of Bacteriophages of Campylobacter jejuni.

Here, we describe the methods for isolation, purification, and propagation of Campylobacter jejuni bacteriophages from samples expected to contain high number of phages such as chicken feces. The overall steps are (1) liberation of phages from the sample material; (2) observation of plaque-forming units on C. jejuni lawns using a spot assay; (3) isolation of single plaques; (4) consecutive purification procedures; and (5) propagation of purified phages from a plate lysate to prepare master stocks.

Methods for Initial Characterization of Campylobacter jejuni Bacteriophages.

Here we describe an initial characterization of Campylobacter jejuni bacteriophages by host range analysis, genome size determination by pulsed-field gel electrophoresis, and receptor-type identification by screening mutants for phage sensitivity.

Genomic insights from whole genome sequencing of four clonal outbreak Campylobacter jejuni assessed within the global C. jejuni population.

BACKGROUND: Whole genome sequencing (WGS) is useful for determining clusters of human cases, investigating outbreaks, and defining the population genetics of bacteria. It also provides information about other aspects of bacterial biology, including classical typing results, virulence, and adaptive strategies of the organism. Cell culture invasion and protein expression patterns of four related multilocus sequence type 21 (ST21) C. jejuni isolates from a significant Canadian water-borne outbreak were previously associated with the presence of a CJIE1 prophage. Whole genome sequencing was used to examine the genetic diversity among these isolates and confirm that previous observations could be attributed to differential prophage carriage. Moreover, we sought to determine the presence of genome sequences that could be used as surrogate markers to delineate outbreak-associated isolates. RESULTS: Differential carriage of the CJIE1 prophage was identified as the major genetic difference among the four outbreak isolates. High quality single-nucleotide variant (hqSNV) and core genome multilocus sequence typing (cgMLST) clustered these isolates within expanded datasets consisting of additional C. jejuni strains. The number and location of homopolymeric tract regions was identical in all four outbreak isolates but differed from all other C. jejuni examined. Comparative genomics and PCR amplification enabled the identification of large chromosomal inversions of approximately 93 kb and 388 kb within the outbreak isolates associated with transducer-like proteins containing long nucleotide repeat sequences. The 93-kb inversion was characteristic of the outbreak-associated isolates, and the gene content of this inverted region displayed high synteny with the reference strain. CONCLUSIONS: The four outbreak isolates were clonally derived and differed mainly in the presence of the CJIE1 prophage, validating earlier findings linking the prophage to phenotypic differences in virulence assays and protein expression. The identification of large, genetically syntenous chromosomal inversions in the genomes of outbreak-associated isolates provided a unique method for discriminating outbreak isolates from the background population. Transducer-like proteins appear to be associated with the chromosomal inversions. CgMLST and hqSNV analysis also effectively delineated the outbreak isolates within the larger C. jejuni population structure.

A novel DNA-binding protein from Campylobacter jejuni bacteriophage NCTC12673.

We previously suggested that the double-stranded genomic DNA of Campylobacter jejuni bacteriophage NCTC12673 was complexed with proteins. Mass spectrometry of peptides obtained from tryptic digests of purified phage DNA indicated that phage protein Gp001 co-purified with the DNA. Gp001 is an acidic protein that lacks any obvious homology or conserved domains found in known DNA-binding proteins. The DNA-binding ability of recombinant Gp001 was examined using an electrophoretic mobility shift assay. Slow DNA-Gp001 complex formation was observed at pH 5.5, but not at neutral or basic pH. This nucleoprotein complex had difficulty entering agarose gels used in the assay while proteinase K pretreatment released the DNA from the complex. No mobility shift was observed when the DNA was immediately subjected to electrophoresis after mixing with Gp001, even if both components were separately pre-incubated at pH 5.5. The complexed DNA was unable to transform chemically competent Escherichia coli cells and was less susceptible to degradation by nucleases. The formation of Gp001-DNA complexes at low pH may provide a mechanism for maintaining DNA integrity while the phage pursues its host through the gastrointestinal tract. Also, this feature can potentially be used to improve DNA delivery protocols applied in gene therapy.

Host adaption to the bacteriophage carrier state of Campylobacter jejuni.

The carrier state of the foodborne pathogen Campylobacter jejuni represents an alternative life cycle whereby virulent bacteriophages can persist in association with host bacteria without commitment to lysogeny. Host bacteria exhibit significant phenotypic changes that improve their ability to survive extra-intestinal environments, but exhibit growth-phase-dependent impairment in motility. We demonstrate that early exponential phase cultures become synchronised with respect to the non-motile phenotype, which corresponds with a reduction in their ability to adhere to and invade intestinal epithelial cells. Comparative transcriptome analyses (RNA-seq) identify changes in gene expression that account for the observed phenotypes: downregulation of stress response genes hrcA, hspR and per and downregulation of the major flagellin flaA with the chemotactic response signalling genes cheV, cheA and cheW. These changes present mechanisms by which the host and bacteriophage can remain associated without lysis, and the cultures survive extra-intestinal transit. These data provide a basis for understanding a critical link in the ecology of the Campylobacter bacteriophage.

A receptor-binding protein of Campylobacter jejuni bacteriophage NCTC 12673 recognizes flagellin glycosylated with acetamidino-modified pseudaminic acid.

Bacteriophage receptor-binding proteins (RBPs) confer host specificity. We previously identified a putative RBP (Gp047) from the campylobacter lytic phage NCTC 12673 and demonstrated that Gp047 has a broader host range than its parent phage. While NCTC 12673 recognizes the capsular polysaccharide (CPS) of a limited number of Campylobacter jejuni isolates, Gp047 binds to a majority of C. jejuni and related Campylobacter coli strains. In this study, we demonstrate that Gp047 also binds to acapsular mutants, suggesting that unlike the parent phage, CPS is not the receptor for Gp047. Affinity chromatography and far-western analyses of C. jejuni lysates using Gp047 followed by mass spectrometry indicated that Gp047 binds to the major flagellin protein, FlaA. Because C. jejuni flagellin is extensively glycosylated, we investigated this binding specificity further and demonstrate that Gp047 only recognizes flagellin decorated with acetamidino-modified pseudaminic acid. This binding activity is localized to the C-terminal quarter of the protein and both wild-type and coccoid forms of C. jejuni are recognized. In addition, Gp047 treatment agglutinates vegetative cells and reduces their motility. Because Gp047 is highly conserved among all campylobacter phages sequenced to date, it is likely that this protein plays an important role in the phage life cycle.

Reduction of Campylobacter jejuni in broiler chicken by successive application of group II and group III phages.

BACKGROUND: Bacteriophage treatment is a promising tool to reduce Campylobacter in chickens. Several studies have been published where group II or group III phages were successfully applied. However, these two groups of phages are different regarding their host ranges and host cell receptors. Therefore, a concerted activity of group II and group III phages might enhance the efficacy of a treatment and decrease the number of resistant bacteria. RESULTS: In this study we have compared the lytic properties of some group II and group III phages and analysed the suitability of various phages for a reduction of C. jejuni in broiler chickens. We show that group II and group III phages exhibit different kinetics of infection. Two group III and one group II phage were selected for animal experiments and administered in different combinations to three groups of chickens, each containing ten birds. While group III phage CP14 alone reduced Campylobacter counts by more than 1 log10 unit, the concomitant administration of a second group III phage (CP81) did not yield any reduction, probably due to the development of resistance induced by this phage. One group of chickens received phage CP14 and, 24 hours later, group II phage CP68. In this group of animals, Campylobacter counts were reduced by more than 3 log10 units. CONCLUSION: The experiments illustrated that Campylobacter phage cocktails have to be carefully composed to achieve the best results.

Campylobacter jejuni motility is required for infection of the flagellotropic bacteriophage F341.

Previous studies have identified a specific modification of the capsular polysaccharide as receptor for phages that infect Campylobacter jejuni. Using acapsular kpsM mutants of C. jejuni strains NCTC11168 and NCTC12658, we found that bacteriophage F341 infects C. jejuni independently of the capsule. In contrast, phage F341 does not infect C. jejuni NCTC11168 mutants that either lack the flagellar filaments (ΔflaAB) or that have paralyzed, i.e., nonrotating, flagella (ΔmotA and ΔflgP). Complementing flgP confirmed that phage F341 requires rotating flagella for successful infection. Furthermore, adsorption assays demonstrated that phage F341 does not adsorb to these nonmotile C. jejuni NCTC11168 mutants. Taken together, we propose that phage F341 uses the flagellum as a receptor. Phage-host interactions were investigated using fluorescence confocal and transmission electron microscopy. These data demonstrate that F341 binds to the flagellum by perpendicular attachment with visible phage tail fibers interacting directly with the flagellum. Our data are consistent with the movement of the C. jejuni flagellum being required for F341 to travel along the filament to reach the basal body of the bacterium. The initial binding to the flagellum may cause a conformational change of the phage tail that enables DNA injection after binding to a secondary receptor.

Identification and characterisation of new Campylobacter group III phages of animal origin.

Campylobacter-specific bacteriophages (phages) are considered as an alternative intervention strategy to decrease the level of poultry contamination with Campylobacter, a leading cause of gastroenteritis worldwide. Eradication efficiency depends primarily on phage-host interaction mediated by phage tail-spike proteins and bacterial receptors. Here, this interaction was characterised using tail-spike gene sequence analysis, phage neutralisation by antiserum and host range analysis of newly isolated group III Campylobacter phages with 68 Campylobacter jejuni and Campylobacter coli strains. Three different groups of phages were obtained using antibody neutralisation assay, and they were further divided according to polymorphisms observed within tail fibre sequences and host range. Only moderate congruence was observed between these criteria with notable exception of two phages. The infection relied on capsule in all phages isolated, and flagella were found to influence phage propagation on agar plates, but not in broth. Their specificity was more C. jejuni oriented with tendency to lyse human isolates more efficiently. Additionally, natural resistance of C. jejuni to phages did not correlate with their antibiotic resistance patterns. These findings provide new insights into Campylobacter-phage interaction.

Colonisation of a phage susceptible Campylobacter jejuni population in two phage positive broiler flocks.

The pathogens Campylobacter jejuni and Campylobacter coli are commensals in the poultry intestine and campylobacteriosis is one of the most frequent foodborne diseases in developed and developing countries. Phages were identified to be effective in reducing intestinal Campylobacter load and this was evaluated, in the first field trials which were recently carried out. The aim of this study was to further investigate Campylobacter population dynamics during phage application on a commercial broiler farm. This study determines the superiority in colonisation of a Campylobacter type found in a field trial that was susceptible to phages in in vitro tests. The colonisation factors, i.e. motility and gamma glutamyl transferase activity, were increased in this type. The clustering in phylogenetic comparisons of MALDI-TOF spectra did not match the ST, biochemical phenotype and phage susceptibility. Occurrence of Campylobacter jejuni strains and phage susceptibility types with different colonisation potential seem to play a very important role in the success of phage therapy in commercial broiler houses. Thus, mechanisms of both, phage susceptibility and Campylobacter colonisation should be further investigated and considered when composing phage cocktails.