Campylobacteriosis is the most frequently reported foodborne bacterial gastroenteritis in the European Union (EU) since 2005 (European Food Safety Authority [EFSA] and European Centre for Disease Prevention and Control [ECDC], 2021), but represents also an important zoonosis worldwide (Skarp et al., 2016). In 2019, the European Food Safety Authority (EFSA) notified 220,682 campylobacteriosis cases in the EU. While 49.8% of the reported cases were acquired in the EU, 3% were considered to originate from other countries. However, for 47.2% of the human cases, further information on the travel status or the country of infection was unknown (European Food Safety Authority [EFSA] and European Centre for Disease Prevention and Control [ECDC], 2021). In general, consumption of broiler meat is considered to be the most common cause for human infections (European Food Safety Authority [EFSA], 2010). Thermophilic Campylobacter spp. have been shown to be the most prevalent cause of campylobacteriosis. Campylobacter (C.) jejuni and C. coli are the most frequent species, while C. lari, C. fetus and C. upsaliensis have been detected less frequently in campylobacteriosis cases (European Food Safety Authority [EFSA] and European Centre for Disease Prevention and Control [ECDC], 2021). Symptoms of Campylobacter enteritis include watery to hemorrhagic diarrhea and abdominal pain. Severe long-term sequelae like the Guillain-Barré syndrome, reactive arthritis, and erythema nodosum can occur in rare cases subsequent to the infection (Nachamkin, 2002; Moore et al., 2005; Skarp et al., 2016).

Campylobacter jejuni and C. coli can colonize the intestine of various wild, domestic, and farm animals (Mulder et al., 2020; European Food Safety Authority [EFSA] and European Centre for Disease Prevention and Control [ECDC], 2021). In broiler flocks, they can usually be detected after 2–3 weeks of housing and spread rapidly until almost all animals of the flock are colonized without showing clinical symptoms (Shreeve et al., 2000; Newell and Fearnley, 2003; Newell et al., 2011). During defeathering, evisceration, and further processing, the pathogens can be transferred to the carcasses (Lee and Newell, 2006; Sasaki et al., 2013), and by cross-contamination during food preparation, contamination of other foodstuffs can occur (Luber et al., 2006; Cardoso et al., 2021). Current estimations predict that a reduction in the Campylobacter load in chicken ceca by 2 log₁₀ units can diminish the risk for human infection, arising from consumed broiler meat, by 42%. However, a 3 log₁₀ unit reduction would lead to a 58% lower risk for human infections (European Food Safety Authority [EFSA] et al., 2020; European Food Safety Authority [EFSA] and European Centre for Disease Prevention and Control [ECDC], 2021).

Bacteriophages (phages) are viruses that specifically infect bacterial genera, species or strains. Currently, the International Committee on Taxonomy of Viruses (ICTV) recognizes nine Campylobacter viruses (ICTV, 2019) that all have an AT-rich double stranded DNA and belong to the family Myoviridae (Bradley’s morphotype A1) (Sails et al., 1998) in the order of Caudovirales. Phages in this family are non-enveloped viruses, with a head-tail structure encompassing a contractile tail. Campylobacter viruses which are part of the Siphoviridae family, have recently been described, but are not yet recognized by the ICTV (Nowaczek et al., 2019). The ICTV currently recognizes a scheme based on sequence analysis that subdivides the family into the two genera Firehammerviruses, including four recognized members [type species CP220, formerly named Cp220likevirus (2014) and Cp220virus (2015–2018)], and Fletcherviruses, including five recognized members [type species CP8, formerly named Cp8unalikevirus (2014) and Cp8virus (2015–2018)] (Javed et al., 2014; Adams et al., 2016; ICTV, 2019; Jackel et al., 2019). Moreover, there are currently two unclassified Firehammerviruses, as well as one classified and 21 unclassified Fletcherviruses listed by the NCBI taxonomy browser (Schoch et al., 2020). A former typing scheme divides Campylobacter phages into three groups based on their genome sizes (group I ∼320 kb, group II ∼184 kb, and group III ∼138 kb). These groups could also be predicted according to their prevailing host specificity (Sails et al., 1998; Javed et al., 2014). Fletcherviruses were assigned to phage group III and Firehammerviruses to phage group II before ICTV classification was established (Hammerl et al., 2011; Javed et al., 2014; Jackel et al., 2019; Ushanov et al., 2020).

The restricted host range of the Campylobacter phages described so far allows specific targeting of pathogenic species (Richards et al., 2019), but at the same time raises the need to combine various phages in cocktails for effective applications in food production settings, where different, unknown Campylobacter strains might be encountered (Chan et al., 2013; Kittler et al., 2013). Combining phages also offers the advantage of preventing bacterial resistance development that may occur as a result of phage application (Fischer et al., 2013; Azam and Tanji, 2019). Nonetheless, while some phage-resistant bacterial cells might occur even if cocktails were used (Fischer et al., 2013), phage-resistant bacteria were shown to exhibit a reduced colonization fitness (Kittler et al., 2014; Oechslin, 2018; Azam and Tanji, 2019). Phages can be used at multiple stages of the food production process without changing the organoleptic properties of the products, such as odor or taste. They can be applied during meat production pre- or post-harvest, and for bio sanitation (Connerton et al., 2011; Polaska and Sokolowska, 2019) as mandated by a farm to fork strategy (Nauta et al., 2007). However, a lack of data and approval procedures still impedes the implementation of phages as an efficient alternative for Campylobacter biocontrol (European Food Safety Authority [EFSA], 2009; European Food Safety Authority [EFSA] et al., 2020).

Application of Campylobacter phages in food production settings has been considered as a promising technology (European Food Safety Authority [EFSA] et al., 2020; Zbikowska et al., 2020). Previous studies highlighted the need for (i) a lytic phage collection directed against current Campyobacter field strains (Carrillo et al., 2005), (ii) more precise and faster methods to identify and select useful phages (Xie et al., 2018; Zbikowska et al., 2020), and (iii) a better understanding of bacteriophage kinetics to allow for advanced considerations concerning dosing and timing of phage applications (Cairns et al., 2009; Hammerl et al., 2014; Loessner et al., 2020). Furthermore, genome determination of Campylobacter phages was also shown to be laborious due to prevailing DNA modification and extensive repetitive sequences, including hypermutable polyGtracts, resulting in low amounts of extracted DNA and/or incomplete genomes (Hammerl et al., 2011; Carvalho et al., 2012; Jackel et al., 2015; Crippen et al., 2019; Sorensen et al., 2021). However, all Campylobacter phages described to date are closely related in their genetic composition and exhibit highly conserved nucleotide sequences. To the best of our knowledge, all Campylobacter phages are free of genes encoding antimicrobial and/or virulence factors (Hammerl et al., 2015). Questions regarding the stability of the phage genomes are hard to evaluate as Campylobacter phages comprise homing endonucleases, which might be involved in adaption of the genomes according to the prevailing selection pressures (i.e., host specificity etc.). This study represents an initiative to increase the number of carefully characterized phages against C. jejuni. Furthermore, we aimed to determine differences in the performance of phages with similar host ranges or origins in liquid cultures of Campylobacter field strains and analyzed their stability upon storage, varying pH values and temperatures (Carrillo et al., 2005; Jonczyk et al., 2011) for estimating their suitability for practical application under real life settings.

Nineteen C. jejuni-specific bacteriophages from chicken samples were isolated in this study. The host range of all isolated phages was evaluated by testing them against a panel of well-characterized C. jejuni and C. coli isolates, comprising current field strains and using a highly reliable spot test format. Applied methods for host range analysis vary widely among currently published studies and no validation of reproducibility for these methods is currently available (Sails et al., 1998; Hansen et al., 2007; Hwang et al., 2009; Janez et al., 2014; Sorensen et al., 2015). While some host range panels include more isolates compared to this study a representation of the current epidemiological situation in broiler production (Carrillo et al., 2005) and further information on the used strains, such as origin and typing results, were considered to be more valuable compared to a broad but less defined panel.

From the newly isolated phages, CP1-4, CP1-5, CP74-2c1, and CP132-3c were selected for further characterization. The phages were chosen based on the assumption that the origin or host range of phages might influence their performance in liquid culture. Based on their morphology, the analyzed phages could be classified as members of the Myoviridae in the order Caudivirales (Sails et al., 1998). They were further classified as group III phages as their genome sizes were similar to 138 kb, their genomes were susceptible to HhaI digestion (Sails et al., 1998; Hansen et al., 2007; Javed et al., 2014), their head sizes close to 100 nm (Sails et al., 1998), and they exclusively infected C. jejuni strains (Jackel et al., 2019). Subsequent real-time PCR results confirmed this identification. Results from previous studies indicated that most group III phages use capsule polysaccharide receptors to attach to their hosts (Sorensen et al., 2015; Zampara et al., 2017). Sequenced members of this group belong to the genus Fletcherviruses (Hammerl et al., 2011; Javed et al., 2014; Jackel et al., 2019; Ushanov et al., 2020). They have been used for reducing Campylobacter on chicken meat in different studies and proved more efficient in binding to Campylobacter cells at low temperatures than the flagellotropic group II phages (Atterbury et al., 2003; Goode et al., 2003; Zampara et al., 2017). While some studies did not result in significant Campylobacter reduction on the treated products (Orquera et al., 2012), other studies reported a significant reduction by these phages (Thung et al., 2020). Experiments on reduction of Campylobacter in broiler chickens by using group III phages showed significant reductions in some of the settings and varying duration of the reducing effect (Carrillo et al., 2005; Wagenaar et al., 2005; Scott et al., 2007; Kittler et al., 2013). Additionally, first trials under commercial conditions were carried out, showing the general suitability of group III phages for use in food production settings (Kittler et al., 2013).

Whole-genome sequencing of phage genomes is mandatory for final evaluation of their suitability and approval in most countries. However, in accordance with results of our study, genome determination of Campylobacter phages has been described to be challenging due to (i) problems regarding preparation of necessary DNA amounts and (ii) the presence of DNA modification hampering DNA processing by using enzymatic procedures. Thus, the suitability of alternative sequencing procedures (i.e., use of different DNA library preparation procedures, alternative sequencing approaches) needs to be determined. It needs to be pointed out here that phage DNA pre-amplification with Phi29 polymerase has been shown as an appropriate possibility to omit enhancing effects for DNA sequencing and manipulation for some Campylobacter phages (Kropinski et al., 2011; Crippen et al., 2019). Further studies need to be conducted to check whether the phage genomes of this study can be also determined by conducting this additional amplification step. However, if possible further amplification steps (PCR) should be omitted to yield a low level of nucleotide alterations in the genomes caused by additional processing (i.e., PCR amplification).

To evaluate the efficiency of the selected phages in reducing Campylobacter, two C. jejuni field strains (Cj18 and LH86), which were susceptible to all four phages, were exposed to those phages at different MOI_input for 26 h [similar to the experiments by Carrillo et al. (2005) or Scott et al. (2007)]. All phages were able to reduce bacterial growth at an MOI_input of 10 (Figure 3), resulting in lower cell densities and reduced AUC values compared to the untreated controls (Figure 4). While significant reductions in AUC values were observed in most experiments using a lower MOI_input, the experiments using Cj18 with the phages CP74-2c1 and CP132-3c did not result in decreased AUC’s (Figure 4A). They showed a substantial decrease of the AUC in experiments using an MOI_input between 1 and 10, indicating lysis without replication (passive inundation), as was suggested for a similar case by El-Shibiny et al. (2009).

However, when comparing AUC values of experiments using different MOI_input (Figure 4), they do not indicate a dose dependence. Moreover, after ∼ 20 h, many OD₆₀₀ values for MOI_input 10 were higher than those for MOI_input 0.001, indicating a potential negative correlation between MOI_input and OD₆₀₀ values, while a positive correlation was expected as found in experiments by Rajnovic et al. (2019) for E. coli bacteriophages (Rajnovic et al., 2019). In 2009, El-Shibiny et al. (2009) reported similar results for in vivo tests with the group II phage CP220. In this study, the lowest phage dose resulted in the highest reduction in C. jejuni HPC5 cell numbers in chickens.

At the start of the growth experiment, phages were added at a concentration of ∼10⁷ PFU/ml. Phage titers at the end of the experiment showed similar concentrations within 10⁶–10⁸ PFU/ml, while bacteria growth was indicated by high OD₆₀₀ value (see above). The reasons for finding high phage and bacteria numbers at the same time at an MOI_input of 10 are unclear, but mathematical models (Levin et al., 1977; Cairns et al., 2009; Bull et al., 2014; Loessner et al., 2020) have predicted that phages and bacteria could coexist at high numbers. Therefore, further studies are necessary to elucidate the relationship of Campylobacter phages and bacteria in order to improve efficacy testing and practical phage application. In experiments using the Campylobacter isolate LH86 and the phages CP74-2c1 and CP132-3c at an MOI_input below 0.1, an initial rise and subsequent decline in OD₆₀₀ values were observed (Figures 3G,H). Similar observations were made during experiments using E. coli as host bacteria in other studies (Bull et al., 2014; Tolen et al., 2018; Rajnovic et al., 2019). It was assumed that except for Cj18 with CP74-2c1 and CP132-3c, the other Campylobacter populations could show a similar growth but this was masked by the detection limit at low OD₆₀₀ values. Further tests are needed for a better understanding of these observations and their relevance for practical application.

In experiments combining Cj18 with CP74-2c1 or CP132-3c, significantly reduced AUC values were observed at an MOI_input of 10 only (Figure 4A), while an MOI_input of 1 or lower resulted in AUC values equal to or in one instance even significantly higher than the control (Figure 4A). Results from host range analysis indicated phage amplification, but showed reduced EOP values for the two phages on this strain. Experiments with an MOI_input of 10 and 0.001 resulted in reduced final phage concentrations that were 2–3 log₁₀ units lower compared to the starting concentrations. These findings indicate that the loss of virions was not compensated by virion production, potentially explaining the finding that bacterial reduction only occurred if phage concentrations exceeded bacterial concentrations. In this case, bacterial reduction would have relied on initial phage concentrations and not on phage replication. Similar results of non-lytic interactions impacting bacterial growth have been reported by a previous study (Carrillo et al., 2005).

Although the number of bacterial isolates tested for phage susceptibility was limited and carful interpretation is necessary, all tested combinations of bacteria and phages resulted in resistant bacterial colonies at the end of the experiment. Interestingly, Cj18 colonies that were resistant against CP74-2c1 and CP132-3c were not resistant against CP1-4 and CP1-5. This could indicate that these phages infected bacteria by using different recognition sites. However, a higher number of tested isolates and further tests would be necessary to confirm these results.

A broad host range and a high lytic efficiency are requirements for phage application under practical conditions, while stability against adverse effects in food production settings is a prerequisite for their utilization. Application of phages in primary production exposes them to room temperature at dosing and to the chicken’s body temperature of 42 °C after ingestion. Low pH values between 3 and 5 (Mabelebele et al., 2017) are encountered for 2–3 h (Svihus and Itani, 2019) when passing through the chicken’s crop, proventriculus, and gizzard. In this regard, temperature stability at 22 and 42°C in combination with different pH levels was tested as proposed by Carrillo et al. (2005). All four phage were stable in buffered solutions with pH values ranging from pH 4 to pH 11 at 22 and 42°C, while reduced stability was observed after exposure to pH 2 and 12. Thung et al. (2020) reported similar findings for the phage Cj01. Interestingly, just one bacteriophage (CP1-4), was able to retain stability at a pH of 3 at 42°C for 24 h, while the other three phages (CP1-5, CP74-2c1, and CP132-3c) proved only stable during exposure to pH 3 for at least 2 h at 42°C. At pH 3 and 22°C, the stability was less pronounced. Based on these results, the selected phages, CP1-4, CP1-5, CP74-2c1, and CP132-3c would be able to resist environmental stresses during application in primary production settings of commercial food production. CP1-4, however, showed even higher stability than Campylobacter phages from previous studies.

To guarantee a sufficient shelf life for storage under practical conditions and to evaluate stability on food products at retail level, the average reduction rate of phage activity per month was calculated. At 4.5°C the average reduction rate for all four phages ranged from 1.02 to 1.20 (PFU/ml)/month, which allowed to extrapolate that in 1 year of storage an average of not more than one log₁₀ unit of phage activity would be lost. Similar stabilities of Campylobacter-specific bacteriophages were reported by Hammerl et al. (2014). At 23.5°C, the rates increased and ranged from 1.29 to 1.82 (PFU/ml)/month. These results show a sufficient stability of the selected phages for food production settings. As the phages proved stable at temperature levels found in household refrigerators, it became important to show that heat treatment, as it would occur during food preparation, was sufficient to inactivate the bacteriophages. An exposure to 70°C for 15 min was sufficient to deactivate all four bacteriophages, while 15 min at 60°C already caused a significant reduction in phage titers.

The reductions in concentrations of CP1-4 and CP1-5 after 60 min at –20 and 50°C are unexpected (Figure 5D), as this would indicate short-term stability at –20 and 50°C. However, results presented by Figures 5A,B contradict this conclusion, as they prove a high stability of these two phages at 22 and 42°C at pH 7 for 24 h. Interestingly, the increased stability of CP1-4 against acidic pH values did not correspond to an increased temperature stability. This combination of high pH-stability and easy heat inactivation would be a favorable combination in phages used in a farm to fork approach.

Phage CP1-4 proved to be the most promising candidate for a wide variety of applications, as it provides a broad host range, high stability at low pH values, and heat inactivation at moderate temperatures. The phage CP1-5 originated from the same sample as CP1-4, however, its host range included five bacteria less than that of CP1-4. It appeared equally efficient in bacteria reduction based on growth experiments in liquid culture and displayed a significantly reduced pH stability, illustrating that phages with different characteristics can be isolated from the same sample.

On the other hand, the phages CP74-2c1 and CP132-3c have been rejected for commercial use based on missing efficiency at medium and lower phage doses in liquid cultures with one of the selected field strains. The obtained results illustrate the need for kinetic tests (optical density based or otherwise) on different Campylobacter to elucidate overall phage performance, as host range analysis with only laboratory strains does not allow for a rational phage choice. Problems during DNA extraction as well as the presence of extensive repetitive sequences on the genomes of Camplyobacter phages make the genetic evaluation laborious and force a search for better analysis protocols (Sorensen et al., 2021).

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

CK and SK did funding acquisition. SK and SS did study design and planning. Bacteriophage isolation was performed by GS and SK, DNA isolation was done by SS and CJ. Bacteriophage genome sequencing was done by JH, electron micrographs were taken by MR, host range determination was jointly done by EP and SS, while the rest the experiments and data analysis were performed by SS. SS and SK conceptualized and wrote the draft manuscript. SS, GS, JH, CK, EP, MR, CJ, MP, and SK helped with writing and editing the final manuscript. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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