Campylobacter spp. and bacteriophages from broiler chickens: Characterization of antibiotic susceptibility profiles and lytic bacteriophages

Abstract Bacteria of the genus Campylobacter are the most common pathogens causing zoonotic diseases in humans. Therefore, the aim of the study was to isolate Campylobacter bacteria from broiler chickens and evaluate their susceptibility to selected antibiotics by determining minimum inhibitory concentrations (MIC), followed by isolation and characterization of bacteriophages specific for Campylobacter spp. The material for the study consisted of field isolates of Campylobacter spp. obtained from the gut (cecum) of broiler chickens directly after slaughter in slaughterhouses, and bacteriophages specific for these strains. We isolated 48 strains from poultry (140 broiler chickens): 31 strains of Campylobacter jejuni and 17 of Campylobacter coli. Identification of the strains was confirmed by multiplex PCR and MALDI‐TOF mass spectrometry. Over 83% of Campylobacter strains were resistant to ciprofloxacin, and over half the isolates were resistant to erythromycin, gentamicin, and tetracycline. Resistance to three or more antibiotics was observed in 91.6% of all strains. Four bacteriophages were obtained, and on the basis of their morphological structure, they were assigned to two families of the order Caudovirales: Myoviridae and Siphoviridae. A high percentage of the Campylobacter strains were resistant to at least three of the antibiotic groups tested. All of the phages exhibited lytic activity against the Campylobacter spp. isolates, but the antibacterial effect of the phages was not observed for all strains.

years. According to reports published by the European Food Safety Authority (EFSA), campylobacteriosis is one of the most frequently reported zoonotic diseases in humans in the European Union, with 246,307 confirmed cases in 2016, which was 10.5% higher than in the previous year (EFSA & ECDC, 2014, 2015, 2016. Widespread drug resistance in bacteria and legal restrictions on the use of antibiotics in livestock farming, particularly in the EU, and since 2009 in the United States and Canada as well, have led to the need for new solutions to eliminate pathogenic (and in particular zoonotic) bacteria, in order to ensure the safety of raw materials used for food (Maron, Smith, & Nachman, 2013). According to the EU/EEA, the prevalence of multiresistant Campylobacter spp. isolates in EU countries in 2012 was over 28% (EFSA and ECDC, 2014).
One possible solution providing an alternative to antibiotics is the use of bacteriophages, a group of viruses infecting bacterial cells. Phages lack the cell structure and enzyme systems essential for food intake and protein synthesis and can replicate only in living cells (Hagens & Loessner, 2010). This is the largest group of viruses, surpassing the number of bacteria ten times (10 31 ). They are present throughout the environment, for example, in water and wastewater, soil, human and animal feces, and products of plant (fruits and vegetables) and animal origin (Andreatti Filho et al., 2007;O'Flaherty, Ross, & Coffey, 2009). Mainly on the basis of genome type and virion morphology, bacteriophages were assigned to 873 species, 204 genera, and 14 subfamilies in the 2015 taxonomy release ICTV report (International Committee on Taxonomy of Viruses, EC 48, Budapest, Hungary, August 2016). However, the vast majority (about 96%) belong to the families Myoviridae, Podoviridae, or Siphoviridae, which are phylogenetically related and comprise the order Caudovirales (Weinbauer, 2004;Wernicki, Nowaczek, & Urban-Chmiel, 2017).
The antibacterial properties of phages have found application in experimental therapies in humans and animals and in the development of disinfectants eliminating bacteria from the surfaces of foods of plant and animal origin (Abuladze et al., 2008;Carlton, Noordman, Biswas, Meester, & Loessner, 2005;Lim et al., 2011;Weber-Dąbrowska, Mulczyk, & Górski, 2000).
The aim of the study was to isolate Campylobacter spp. strains from broiler chickens and to evaluate their susceptibility to selected antibiotics by determining minimum inhibitory concentrations (MIC), and then to isolate and characterize bacteriophages specific for Campylobacter spp.

| Isolation and morphological analysis of Campylobacter spp.
The material for the study consisted of field isolates of Campylobacter spp. obtained from the gut (cecum) of 140 broiler chickens directly after slaughter in slaughterhouses in southeastern Poland in September and October. The birds were from different indoor flocks. Presumptive identification of Campylobacter spp. isolates was based on colony morphology, Gram staining, and growth in microaerobic conditions. Initial isolation was carried out in Bolton Broth (Oxoid Ltd., UK). The cultures were incubated at 37°C for 48 hr in microaerophilic conditions (5% O 2 , 10% CO 2 , 85% N) in the CampyGen system (Oxoid Ltd.). On media that showed growth of gray, flat, and moist bacterial colonies with a tendency to expand, single colonies belonging morphologically to the Campylobacter spp. type were collected, directly transferred to selective mCCDA agar, and incubated at 41.5°C for 48 hr in microaerophilic conditions (Dudzic et al., 2016). The isolates were stored at −80°C in the Microbank system for storage of micro-organisms (Biocorp, PL).

| Genetic identification of bacteria by multiplex PCR and MALDI-TOF mass spectrometry
DNA was isolated using a commercial DNA purification kit (GeneMatrix Bacterial & Yeast Genomic DNA Purification Kit; EURx, PL) according to the manufacturer's instructions. The genus and species identification of the bacterial isolates was confirmed by multiplex PCR using specific primers according to own previous study (Dudzic et al., 2016). PCR was carried out using primers amplifying the 16S rRNA gene of Campylobacter jejuni and Campylobacter coli to determine genus (MD16S1 and MD16S2l), as well as primers amplifying the mapA gene of C. jejuni (MDmapA1 and MDmapA2) and the ceuE gene of C. coli (MDCOL3 and MDCOL2). Reference strains of C. jejuni NCTC 12662 and C. coli ATCC 33559 were used as positive controls. The primer sequences and conditions are given in Table 1.
Amplification reactions were carried out in a thermal cycler (Eppendorf Mastercycler gradient, USA) using the following program: One cycle at 94°C/5 min, 30 cycles 58°C/1 min, 72°C/1 min, 94°C/1 min, and one cycle 72°C/5 min. The amplification products TA B L E 1 Sequences of primers and conditions specific for Campylobacter spp. identification in multiplex PCR

| Antibiotic susceptibility of Campylobacter spp. isolates
The susceptibility of the isolated Campylobacter strains to selected antibiotics was tested by determining the minimum inhibitory concentrations (MIC) in Mueller Hinton broth (Sigma-Aldrich). MICs were determined by the broth microdilution procedure in 96-well flat-bottomed microtiter plates according to Andrews (2001

| Isolation of bacteriophages specific for
Campylobacter spp.
Bacteriophages were isolated from 70 fecal samples collected from 20 different farms. To isolate bacteriophages, we used 10 g of chicken feces suspended in 100 ml SM buffer with 2% gelatine.
The 10% w/v suspension obtained in this manner was incubated in a rocking shaker at 4°C/120 rpm overnight. Then, the supernatant was centrifuged at 13,000 g/10 min and filtered through syringe filters 0.45 and 0.22 µm in diameter (Roth). The presence of bacteriophages in the lysate was confirmed by the double-layer plate method according to Loc Carrillo, Connerton, Pearson, and Connerton (2007). For this purpose, 200 µl of bacterial suspension was added to 4 ml of NZCYM broth supplemented with 0.7% agar at about 45°C with 1 M CaCl 2 and 1 M MgSO 4 . This was mixed thoroughly and poured onto a plate with NZCYM medium supplemented with 1.2% agar. The phage lysate was applied to the surface of the plate in the form of a 10 µl drop of cooled top agar. After 20 min of incubation at room temperature, the plates were transferred to an incubator and incubated at 42°C for 24 or 48 hr in microaerophilic conditions. The presence of bacteriophages was confirmed by observation of zones of inhibition of bacterial growth (lysis). Before further characterization, the phages were individually plaque-purified three times on agar plates according to Han et al. (2013).
To determine the lytic activity spectrum of the bacteriophages, 10 µl of bacteriophage suspension was placed on plates containing individual strains of Campylobacter spp. and incubated for 48 hr in microaerophilic conditions. The bacteriophage activity spectrum was determined on the basis of zones of inhibition of bacterial growth in the form of plaques, as the lytic effect of the phage activity. The bacteriophages were evaluated for their lytic activity against all Campylobacter strains isolated from poultry during the study.
Two bacteriophages specific for C. jejuni (φ198 and φ287), obtained from Prof. Lone Brøndsted (University of Copenhagen, Denmark), were additionally used as reference phages for comparison with the wild-type phages obtained in this study.

| Morphological analysis of bacteriophages specific for Campylobacter spp. by transmission electron microscopy
Morphological analysis of the bacteriophages was carried out by transmission electron microscopy of negatively stained slides. For this purpose, 5 ml of each phage suspension in TM buffer was adsorbed onto glow-discharged carbon-coated 200 mesh copper grids, and then, after 3 min adsorption, the excess lysate was collected and the slides were negatively stained with 2% uranyl acetate (pH 4.0) for 15 s. The slides were examined using a Philips CM 100 transmission electron microscope at 12,000-80,000× magnification, and selected phages were recorded using iTEM software (Olympus Soft Imaging Solution). The bacteriophages were classified according to , Carvalho, Susano, et al. (2010) and Furuta et al. (2017).

| RE SULTS
We obtained 48 bacterial strains belonging to the genus Campylobacter spp.; species identification in MALDI-TOF mass spectrometry was confirmed for the 48 isolates, among which 31 strains were classified as C. jejuni and 17 as C. coli. For 34 strains, the identification score ranged from 2.0 to 2.299, which indicated reliable identification to the genus level and probable identification to the species level. For 11 strains, the identification score ranged from 1.7 to 1.99, and three strains had a score below 1.700 (Table 2)  The bacteriophages assigned to the family Myoviridae had elongated icosahedral heads (100-120 nm) without an envelope, and contrac-

| D ISCUSS I ON
Bacteria of the genus Campylobacter spp. appear in a few chicks at the end of the first week after hatching, and then spread very quickly through the entire flock between the ages of 2 and 3 weeks.
Campylobacter bacteria are one of the main causes of contamination of poultry carcasses in the slaughterhouse, and these carcasses are the primary source of campylobacteriosis infections in humans (Hwang et al., 2009  The growing phenomenon of bacterial resistance to commonly used antibiotics in both human and veterinary medicine necessitates the search for alternative methods to combat bacterial pathogens. Phages specific for Campylobacter spp. are used to reduce the occurrence of this pathogen in the gut of poultry, in order to limit economic losses resulting from reduced productivity, weight gains, and feed conversion, as well as to reduce the risk of contamination of meat for human consumption Loc Carrillo et al., 2005). Bacteriophages exhibiting specificity for Campylobacter spp. can be used successfully to eliminate bacteria from carcass surfaces .
In the present study, we obtained four bacteriophages exhibiting specificity for 17 strains of Campylobacter spp. This was presumably linked to the fact that the samples were obtained from indoor poultry housing systems, which probably had a significant influence on the number of bacteriophages obtained for the tested Campylobacter strains.
A similar low percentage of phages specific for Campylobacter spp. was observed in a study by Hansen, Rosenquist, Baggesen, Brown, and Christensen (2007), in which only five bacteriophages were obtained for 222 fecal samples from broiler chickens. In a study by Atterbury, Connerton, Christine, et al. (2003), phages were obtained from only 11% of all samples used for isolation of bacterial viruses. Firlieyanti, Connerton, and Connerton (2016) have also reported a low 2.7% isolation frequency of Campylobacter-specific bacteriophages. According to other authors (Carvalho, Susano, et al., 2010;Owens, Barton, & Heuzenroeder, 2013), the low activity of bacteriophages against Campylobacter spp. may be due to the origin of these bacteria. These authors suggest that the most phage-positive samples are usually obtained from birds kept in backyard farming systems rather than closed systems.
Most bacteriophages exhibiting specificity for Campylobacter bacteria have a structure typical of phages of the family Myoviridae (Hansen et al., 2007;Hwang et al., 2009;Sorensen et al., 2015). In the present study, however, in addition to the phages assigned to the Myoviridae family, we also obtained phages which were assigned to the Siphoviridae family on the basis of their morphological structure. This is interesting because the available literature concerning strains of Campylobacter spp. includes few reports describing bacteriophages of the family Siphoviridae (Ackermann, 2007;Sails, Wareing, Bolton, Fox, & Curry, 1998).
Restriction analysis of the phage material obtained using double digestion with HindIII + ClaI and MseI + TaqI confirmed the presence of single restriction fragments, which was insufficient for a compre-

| CON CLUS ION
To sum up, the results of the study indicate that bacteria of the genus Campylobacter spp. are characterized by a high frequency of strains resistant to at least three of the antibiotic groups tested. The occurrence of bacteriophages specific for Campylobacter spp. strains is very low; the use of material from the living environments of birds raised in backyard systems is much more conducive to the acquisition of bacteriophages. Restriction analysis of phages requires testing of many enzymes using the double digestion method, which makes it possible to obtain more restriction fragments. A full genetic characterization of the phages obtained in the present study will be performed as the next stage of our research and will involve the use of more bacteriophages obtained from poultry environments.

ACK N OWLED G EM ENT
The authors would like to thank Professor Lone Brøndsted (University of Copenhagen, Denmark) for the reference phages we used for comparison with the wild-type phages obtained in this study.