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. 2024 Feb 11;103(4):103548. doi: 10.1016/j.psj.2024.103548

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

Xiaoyan Zhang ⁎, Mengjun Tang *, Qian Zhou *, Junxian Lu *, Hui Zhang †, Xiujun Tang *, Lina Ma *, Jing Zhang *, Dawei Chen *, Yushi Gao ⁎,1
PMCID: PMC10964072  PMID: 38442560

Abstract

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.

Key words: Campylobacter, lytic phage, poultry meat, food safety

INTRODUCTION

Campylobacter, especially Campylobacter jejuni and Campylobacter coli, are major foodborne bacterial pathogens that can cause human gastroenteritis worldwide (Kaakoush et al., 2015). The transmission of Campylobacter to humans occurs primarily through the food chain, and contaminated poultry meat is considered the major source of pathogenic transfer to humans (García-Sánchez et al., 2018; Rasschaert et al., 2020; EI-Saadony et al., 2023; Heimesaat et al., 2023). Previous studies from around the world have estimated that 50 to 80% of all campylobacteriosis cases can be attributed to the poultry reservoir Asuming-Bediako et al., 2019, Hailu et al., 2021, Skarp et al., 2016, Van Gerwe, 2012. As a foodborne bacteria transmitted via foodborne routes, Campylobacter are constantly exposed to the antibiotics used in animals, agriculture, and clinical treatments (Rasschaert et al., 2020). In recent years, the resistance of Campylobacter to most clinically important antibiotics has increased significantly, thereby threatening public health (Liu et al., 2020; Tang et al., 2021). Clinical Campylobacter isolates collected from a consecutive surveillance program between 2019 and 2021 in Shanghai, China, have shown high resistance, especially to quinolones and tetracyclines, with above 30% of multidrug-resistant (MDR) strains (Gao et al., 2023). In this context, the control and prevention of MDR Campylobacter are important to public health.

Various control measures to reduce the risk of Campylobacter transmission from poultry meats to humans have been investigated, including on-farm and postharvest control programs (Taha-Abdelaziz et al., 2023). To reduce the risk of Campylobacter infection in humans, the extensive use of chemical, physical, and biological methods has been increasing in poultry-carcass treatment (Sohaib et al., 2016; Bai et al., 2022). However, due to the major drawback of these conventional methods, such as potential drug residue, bacterial resistance, unfavorable qualities for consumers concerning sensory, environment pollution, challenges in maintaining microbial safety in food matrices remain (Ge et al., 2022). Consequently, the application of bacteriophages as a novel approach to reducing the contamination of foodborne pathogens is attracting widespread attention. Bacteriophages (phages) are viruses that can specifically infect bacteria. Lytic phages can be used to effectively combat the specific pathogen in animals and on food products. Due to their ability of killing MDR pathogens, phages are currently eliciting renewed research interest (Kortright et al., 2019; Ushanov et al., 2020).

Campylobacter phages are natural “predators” that can potentially bring Campylobacter under control at different stages of the food chain (Mahony et al., 2011; Ushanov et al., 2020). They offer potential solutions to reduce the pathogen in poultry, both in on-farm and post-harvest applications. Peh et al demonstrated that phage application reduced the fecal Campylobacter excretion and Campylobacter concentrations in the colon of broilers (Peh et al., 2023). Phage treatment can also be used to inactivate Campylobacter attached to food contact surfaces (Jordá et al., 2023). Most Campylobacter phages are lytic and able to lyse bacteria upon infection. They are divided into 3 groups according to the size and morphology (Javed et al., 2014). Group I contain phages with large genomes that seem to be rare. They have not been described in detail or used for applications (Sails et al., 1998). While the application of a group II and a group III phage reduced the numbers of C. jejuni in chickens efficiently (Hammerl et al., 2014). They have also been used to reduce Campylobacter on chicken meat in previous studies (Atterbury et al., 2003). A broad host range is an important prerequisite for successful phage application. Group II and group III phages diverge in terms of their specificity. Group II phages frequently infect both C. jejuni and C. coli. However, phages of group III often lyse more C. jejuni strains than Group II phages (Hammerl et al., 2014). For that reason, Campylobacter phages should contain members of both groups to combat C. jejuni and C. coli, to optimize the application strategy and to prevent phage resistance (Jäckel et al., 2019). Although phages are already being used to control Salmonella and Listeria monocytogenes, the number of reports about Campylobacter phage application for therapeutic purposes are limited (Mahony et.al., 2011; LeLièvre et.al, 2019). Therefore, a broad selection of well-characterized phages should be available for decreasing the Campylobacter load in animals and food products (Jäckel et al., 2019).

The aim of the study was to evaluate the antibiotic resistance of Campylobacter isolates from retail raw-poultry meats. To control these bacteria, we isolated and characterized the CP6 phage that infects broad range of MDR Campylobacter. Our promising results provided effective strategies to control MDR Campylobacter in the poultry industry.

MATERIALS AND METHODS

Campylobacter Strains and Antibiotic-Susceptibility Testing

A total of 252 retail raw-poultry meat samples (191 chicken meats, 34 pigeon meats, and 27 duck meats) were collected randomly from wet markets or supermarkets located in 10 districts in Jiangsu province, eastern China, during our laboratory annual antimicrobial-resistance surveillance program in 2020. The isolation process and the identification of presumptive colony were performed as described in our previous study (Zhang et.al., 2018). The isolated Campylobacter strains were frozen at −80°C in a brain-heart infusion (BD) broth with 20% glycerol.

Campylobacter were cultured on Mueller-Hinton (MH) agar (Oxoid, Philips) supplemented with 5% sheep blood under microaerophilic conditions (85% nitrogen, 10% carbon dioxide, and 5% oxygen) at 42°C. The antimicrobial susceptibility of Campylobacter isolates was determined using the Kirby–Bauer disk-diffusion method (Bauer et al., 1996) and as recommended by the Clinical Laboratory Standards Institute (CLSI, 2012). Ten antibiotics (Oxoid) from 8 classes were tested, including ampicillin (AMP, 10 µg), gentamicin (GEN, 10 µg), tobramycin (TOB, 10 µg), ciprofloxacin (CIP, 5 µg), tetracycline (TE, 30 µg), erythromycin (E, 15 µg), azithromycin (AZM, 15 µg), sulfonamides (SXT, 25 µg), florfenicol (FFC, 30 µg), fosfomycin (FOS, 50 µg). C. jejuni ATCC 33560 was used as a quality-control strain. Susceptible, intermediate, and resistance were interpreted based on the standard of criterion of the CLSI recommendations (CLSI, 2012). Isolates resistant to ≥3 unrelated classes of antibiotics were considered as MDR isolates.

Bacteriophage Isolation, Purification, and Propagation

Chicken fecal samples were collected from a wet market in Yangzhou city, China. For sample preparation, approximately 2 g of fecal samples were dispersed in 10 mL of SM-buffer (100 mM NaCl, 8 mM MgSO4, 50 mM Tris-HCl, pH 7.5) by using sterile cotton swabs. After shaking at 4°C for 4 h, the samples were centrifuged at 10, 000 × g for 15 min, and the supernatants were passed through a 0.22 µm pore filter (Millipore, Ireland). To improve phage-isolation efficiency, samples were processed with host bacterial enrichment following the method described by Ibai Nafarrate (Nafarrate et al., 2020) with minor modifications. In a typical procedure, 5 MDR Campylobacter isolates (Table 1) from different districts in Jiangsu province were cultured and prepared to a final concentration of about 107 CFU/mL of each isolate. Equal volumes of each isolate were mixed. A 500 µL bacterial mixture was added to 5 mL of Campylobacter selective Bolton broth (Oxoid, Philips) supplemented with selective antibiotics (Oxoid, Philips). Then 1 mL of each phage sample supernatants was added to the above preparations and incubated at 37°C for about 24 h under microaerobic conditions. After centrifugation at 10, 000 × g for 15 min, the supernatants were passed through a 0.22 µm pore filter again.

Table 1.

Host range of phage CP6.

Strain Specie Source District Number of drug-resistance CP6
158 C. coli Chicken feces CZ 7 +
159 C. coli Chicken feces CZ 7 +
109 C. coli Chicken feces CZ 8 +
614 C. coli Chicken meat HA 6 +
82 C. coli Chicken feces LYG 8 +
480 C. jejuni Chicken feces SQ 5 +
621 C. coli Chicken meat HA 7 +
618 C. coli Chicken meat HA 7 +
432 C. coli Chicken meat XZ 8 +
524 C. coli Chicken meat SQ 7 +
146 C. coli Chicken feces CZ 8 +
619 C. coli Chicken meat HA 8 +
104 C. coli Chicken feces CZ 5 +
359 C. coli Chicken meat YZ 6 +
410 C. coli Chicken feces XZ 5 +
433 C. coli Chicken meat XZ 5 +
375 C. coli Duck meat XZ 8 +
379 C. jejuni Chicken meat XZ sensitive +
523 C. jejuni Chicken meat SQ sensitive +
22 C. coli Chicken meat LYG 5 +
363 C. coli Chicken meat YZ 5 +
28 C. coli Chicken meat LYG 8 +
512 C. coli Chicken meat SQ 8 +
514 C. coli Chicken meat SQ 8 +
345 C. coli Chicken meat YC 5 +
324 C. coli Chicken meat YC 7 +
13 C. coli Chicken meat NJ 6 +
513 C. coli Chicken meat SQ 8 +
507 C. coli Chicken meat SQ 8 +
45 C. coli Pigeon meat LYG 5 +
336 C. coli Chicken meat YC 5 +
515 C. coli Chicken meat YC 5 +
613 C. jejuni Chicken meat HA 6 -
399 C. jejuni Chicken feces XZ 5 +
361 C. coli Chicken meat YZ 6 +
411 C. coli Chicken feces XZ 5 +
479 C. jejuni Chicken feces SQ Sensitive +
429 C. coli Chicken meat XZ Sensitive +
619 C. coli Chicken meat HA 8 +
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For further isolation, the resulting filtrate was cocultured with each of the 5 Campylobacter isolates as previously described by Carrillo (Carrillo et al., 2007) with minor modifications. In a typical procedure, Campylobacter isolates were cultured on blood MH agar (Oxoid, Philips) plates for 24 to 36 h, and a suspension in MH medium was adjusted to a McFarland of 1.0. A 500 µL bacterial suspension was added to 5 mL of NZCYM broth (Sangon Biotech, Shanghai) supplemented with 0.6% agar at about 45°C. The soft agar was mixed thoroughly and poured onto NZCYM plates containing 1.2% agar. Phage presence was applied by spotting 10 µL of the sample supernatants onto the surface of the cooled top agar. After 20 min of incubation at room temperature, the plates were incubated overnight at 42°C under microaerobic atmosphere and then examined for phage plaque presence. The presence of Campylobacter phages was confirmed by observing the inhibition zones of bacterial growth. Single plaques were removed from the soft agar by using a sterile 10 µL loop and resuspended in 900 µL of SM buffer. Before further characterization, each single plaque was purified by 3 times of successive picking and plating procedure on agar plates to ensure that it represented a single clone. Subsequent propagation of phages was performed by the double-agar-layer method as described by Steffan (Steffan et al., 2021). Fresh phage lysates were then conserved in SM buffer at 4°C before use.

Bacteriophage Host-Range Determination

The host range of the phage was determined by a spotting assay using 35 MDR and 4 sensitive Campylobacter strains, isolated from poultry meat in different regions in Jiangsu province. In a typical procedure, 1 of the 39 Campylobacter (Table 1) was cultured and mixed with 6 mL of 0.6% NZCYM soft agar and poured onto a solidified NZCYM agar plate. Then, 10 µL of 10-fold serial diluted phage suspension was spotted onto the double-layer agar plate and incubated at 42°C for 24 h under microaerobic conditions. The clear inhibition zone was recorded to reflect the lysis ability of bacterial growth. The phage titer was determined on the presence of lysis zones in each dilution. The last dilution of the phage suspension which plagues were observed was the titer. Each trial was repeated three times. The absence of visible plaques was considered a negative result.

To determine the concentration of the phage, serial dilutions of the phage lysates were spotted onto the double-layer agar plate described as above.

Morphological Analysis of Phage CP6 by Transmission Electron Microscopy (TEM)

The phage suspension was prepared as described above and purified by CsCl2 density-gradient centrifugation protocols (Steffan et al., 2021). A 100 µL phage suspension (approximately 1 × 109 PFU/mL) was dropped onto a mica sheet (∼3 × 3 mm2 size) and absorbed for 10 min. The sample was stained with 2% (w/v) uranyl acetate. Excess dye was removed with filter paper, and the sample was dried for 15 min. The phage was examined with a TEM system (Tecnai 12; Philips; The Netherlands).

Phage Genome-Sequencing Analysis

Phage DNA was extracted from a high-titer lysate (109 PFU/mL) by using an ABigen λ Phage DNA Purification kit (ABigen Corporation, Beijing, China) in accordance with the manufacturer's instructions. Extracted phage genome was sequenced using Illumina Hiseq sequencer and assembled using Newbler 2.9. Genemarks software was used to predict the protein-coding genes of the genome, and the sequence alignment of the coding genes was completed by Diamond software. The putative protein function of the open reading frames (ORFs) of the phage genome was analyzed suing the NR database. To identify the function of the presumed proteins, Diamond BLASTp was used to compare the protein sequences with the protein sequences in the database. To determine the relationship between phage CP6 and other Campylobacter phages, the phylogenetic tree based on the phage major capsid protein (P6000133) were generated using MEGA6. The nucleotide sequence of CP6 phage was submitted to the CNGBdb database with accession number CNP0004549.

Thermal and pH Stability of the Phage

The thermal stability of the phage was identified by incubating 300 µL of phage suspension (108 PFU/mL) for 30 or 60 min at 30, 40, 50, or 60°C using a heating block. Samples were collected after 0, 30, and 60 min to determine the titers by double-layer agar method. pH stability was measured in pH-adjusted phosphate buffer within different pH ranges of 2 to 13, and 108 PFU/mL phage was stored at 37°C for 1 h. The suspension concentration was determined by spotting assays on double-layered 0.6 % soft agar as described above.

The stability of phages during storage was assessed by storing 10 mL of phage suspension for 6 months at 4°C. Phage concentration was then tested at regular intervals. All experiments were repeated 3 times.

Phage Lytic Activity for Liquid Culture of MDR Campylobacter

For the lytic activity of the phage, fresh Campylobacter cultures (optical density, OD600 0.6, of about 2×107 CFU/mL) were mixed with the diluted phage suspension to reach an three different multiplicities of infection (MOI) of 10, 1, and 0.1, in tissue culture bottles and incubated at 42 ℃ under microaerobic conditions with shaking (100 rpm). The lytic activity of CP6 was evaluated by measuring the OD600 of field strain Cc512, an MDR C. coli, in liquid cultures with and without exposure to the phage. OD600 was measured every 3 h to reflect the growth of Campylobacter for a total of 24 h. Bacterial growth without the appearance of phage was also determined for a period of 24 h. We prepared 2 replication tissue culture bottles, and the experiment was performed in triplicate. The average data with standard deviation were presented.

Inactivation of Campylobacter in Chicken Meat Using Phage CP6

The antimicrobial activity of CP6 was evaluated in chicken meat artificially contaminated with Campylobacter. Raw chicken meat was purchased from a market and cut aseptically into 2 × 2 cm square pieces (about 2 g) and then placed on 4-well plates. Samples were treated with UV for 30 min on each side in a biosafety cabinet, and the test results showed that they were sterile. For the experiments, Campylobacter Cc512 of 106 CFU/mL (200 µL) was used to mimic a bacterial contamination event and yielded an final inoculation level of approximately 1 × 104 CFU/g to 5 × 104 CFU/g. Nonbacterium-treated squares served as a control. Then, 200 µL of phage diluted with SM buffer (107–109 PFU/g) was dropped onto the chicken squares with a pipette. The same volume of SM buffer without phage was included as a negative control. All samples were prepared and incubated at 4°C for 6, 12, 18, 24, and 36 h under microaerobic atmosphere. At each time point, chicken squares were transferred into a 15 mL tube containing 5 mL of MH buffer and vortexed for 2 min at room temperature. The liquid portion was centrifuged at 3,000 × g for 10 min at 4°C. The pellet was resuspended with 10 mL of MH buffer and diluted to 10-fold serial suspension. The CFU reduction of each group was estimated by spotting 10 µL suspension on MH agar plates after incubating at 42°C under microaerobic atmosphere. The CFU was determined in triplicate.

Statistical Analysis

Statistical analysis was performed using PRISM software. Comparisons between control and experimental groups were analyzed using nonparametric one-way ANOVA followed by Bonferroni's multiple-comparison posttest.

RESULTS

Campylobacter Isolation and Antimicrobial Susceptibility

A total of 100 Campylobacter isolates (34 C. jejuni and 66 C. coli) were isolated from 252 retail raw-poultry meat samples (Table S1), with a total isolation rate of 39.7%. Among them, 8, 13, and 79 Campylobacter were isolated from meat of duck, pigeon, and chicken, respectively. The percentage of Campylobacter strains isolated from chicken meat samples from the wet market reached 41.5% and was higher than that obtained from meat of other sources.

Table S2 The susceptibility of the Campylobacter isolates to 10 antimicrobial agents is shown in Table S2. Analysis of drug susceptibility showed that the highest percentages of Campylobacter strains were resistant to tetracycline (60.0%), erythromycin (54%), and azithromycin (54%). The percentage of strains resistant to ciprofloxacin, gentamicin, tobramycin, and ampicillin was ranged from 40.0% to 52.0%. The lower resistance among the strains were observed for florfenicol (13.0%) and Fosfomycin (2.0%). A total of 31 different AMR patterns were identified. The main AMR pattern in the isolates was a combination of tetracycline, ampicillin, gentamicin, erythromycin, and ciprofloxacin. Sixty-five percent (65.0%) of all isolates were classified as MDR, among which 6 and 8 strains of C. coli were resistant to 9 and 8 tested antibiotics, respectively. The MDR rate of C. coli (84.8%) was significantly higher than that of C. jejuni (26.5%) (Table S3).

Isolation and Host-Range Determination of CP6 Phage

A new Campylobacter phage CP6 was isolated from chicken feces using the MDR Campylobacter Cc512 isolated from retail chicken meat as the host strain. Phage CP6 formed the clear plaque on the double-layer agar plate, with a diameter of about 1 to 2 mm (Figure 1A).

Figure 1.

Figure 1

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Morphology of CP6 phage. (A) Phage plaque on the host lawn. (B) Determination of phage titer. (C) Electron microscopy of phage CP6. The bar indicates the magnification size of 100 nm.

Phage CP6 exhibited a broad host range against Campylobacter (Table 1). The obtained results indicate that the tested bacteriophage showed lytic activity against 34 out of 35 MDR and 4 out of 4 susceptible Campylobacter isolates. No lytic activity of bacteriophage CP6 against other bacteria was observed.

Morphological and Sequence Analysis of CP6 Phage

Phage CP6 formed the clear plaque of approximately 1 to 2 mm on the host lawn (Figure 1A). The phage titer was determined on the presence of lysis zones in each dilution. The last dilution of the phage suspension which plagues were observed was the titer (Figure 1B). Based on the morphological observation with TEM, CP6 phage showed an isometric polyhedral head (80.53 ± 1.02 nm in diameter) and a noncontractile short tail (94.35 ± 1.05 nm) (Figure 1C).

The results of complete genome of CP6 indicated that it had a linear double-stranded DNA and comprised 178,350 bp with 27.51% total GC content, allowing its classification in group II campylobacter phage (Sails et al. 1998) (Figure S1). The genome sequence of phage CP6 had an 89.6% coding rate, and the average length of each gene was about 764 bp. Noncoding RNA was not predicted to be encoded by the genome. The genome annotation (NR database) of CP6 phage predicted 209 ORFs with high similarity to Campylobacter phage CPt10, CP21, CP20, IBB35, and CP220. Only 54 (27.0%) ORFs of phage CP6 showed homology with known functional proteins. Sixteen structural protein genes were identified, encoding products for the head or neck protein domains (CP6_032, CP6_081, CP6_112, CP6_128, CP6_132), tail proteins (CP6_062, CP6_063, CP6_069, CP6_076, CP6_095, CP6_115, CP6_127, CP6_142, CP6_164), and major capsid proteins (CP6_133 and CP6_134). Enzymes related to bacteriophage synthesis and assembly were identified, such as phage DNA methylase (CP6_006), ribonucleotide reductase (CP6_056), possible EndoVII packaging and recombination endonuclease (CP6_111), phage DNA packaging protein (terminase) (CP6_083), phage DNA ligase (CP6_114), and phage DNA topoisomerase (CP6_042, CP6_087). Furthermore, tail-fiber attachment catalyst (CP6_069) and tail lysozyme (CP6_164) (T4-gp05-like), associated with cell binding and lysis, shared high identity with those of Campylobacter virus IBB35 (96.9% identity) and CP21(98.2% identity) (Table S4).

When the identity was set to 60%, 11 Campylobacter phage proteins were classified. In the major capsid protein-based tree, CP6 formed a cluster together with phages CP20 (MK408758.1) and CPt10 (NC027996.1) (Figure 2). CP6 also did not contain any gene encoding integrase proteins, and genes for virulence factors such as toxin and antibiotic resistance were not found in the phage genome.

Figure 2.

Figure 2

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Neighbor-joining phylogenetic trees based on the major capsid protein of CP6 and other Campylobacter phages. The phylogenetic trees were generated using the neighbor-joining method with 1000 bootstrap replicates in MEGA6. (Accession number: Campylobacter phage CP220-1: FN667788.1, Campylobacter phage CP220-2: NC_027997.1; Campylobacter phage CPt10-1: FN667789, Campylobacter phage CPt10-2: NC_027996.1; Campylobacter phage CP21-1: HE815464.1, Campylobacter phage CP21-2: NC_019507.1; Campylobacter phage CP20: MK408758.1; Campylobacter phage F379: MT932329.1; Campylobacter phage CJLB-12: MW074125.1; Campylobacter phage CJLB-14: MW074126.1; Campylobacter phage vB_CcoM-IBB_35: NC_027989.1; P6000133: this study).

The complete genome sequence of Campylobacter phage CP6 was deposited into the CNGBdb database under accession number CNP0004549.

Temperature and pH Sensitivity of CP6

CP6 was thermally stable (Figure 3A). After incubation at 30°C, 40°C, and 50°C, no significant decrease in phage number was observed. However, after incubation at 60°C for 30 and 60 min, the phage number decreased by 2.3 log and 2.0 log, respectively. All phages were dead after incubation at 90°C for 30 min. These results showed that CP6 was thermally stable below 50°C. CP6 was also fairly stable over a wide pH range from 3 to 9 during 1 h of incubation (Figure 3B). The phage titer decreased by 0.9 log at pH 10 and then decreased sharply above pH 11. These findings indicated that CP6 had an average tolerance to acidic and alkaline environments.

Figure 3.

Figure 3

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Stability test of CP6 phage in various condition. (A) Temperature tolerance in range from 30°C to 90°C. (B) pH tolerance after 60 min of incubation at pH ranging from 2 to 13. The data are the mean of 3 determinations.

Inhibition of Growth of MDR Campylobacter In an In Vitro Culture

Bacterial growth was inhibited in four different concentration input liquids after 6 h compared with the untreated control (Figure 4). In the control group, Cc512 cells showed the logarithmic growth from 6 h to 24 h. However, the growth of Cc512 was obviously inhibited by CP6 in different dose inputs, causing continuous inhibition of bacterial growth. No significant difference was found in the antibacterial effects of CP6 at different MOI inputs (Figure 4).

Figure 4.

Figure 4

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Inhibition of growth of MDR Campylobacter field isolate Cc512 in a vitro culture by phage CP6. Campylobacter infected by CP6 at MOI 10, 1, 0.1 and SM buffer as control for 24 h using optical density as the indicator.

Preventive Effect of CP6 on the Campylobacter Contamination of Chicken Breast Meat

The antimicrobial activity of CP6 was evaluated in chicken meat artificially contaminated with Campylobacter. The treatment of chicken meat samples with CP6 showed successful growth inhibition of the MDR C. coli Cc512 at 4°C at 24 h. As shown in Figure 5, after incubation at 4°C for 24 h, viable counts of Campylobacter in the control of chicken meat remained stable, with only a slight decrease at 36 h. CP6 addition reduced the viability of Campylobacter in chicken meat. After phage treatment for 6 h, the viable counts of Campylobacter decreased by 1.02 to 1.23 log.

Figure 5.

Figure 5

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Inactivation of Campylobacter in chicken meat by phage CP6 at 4°C. Values correspond to the host numbers and show the mean for 3 independent experiments.

DISCUSSION

Campylobacter is a commensal of gastrointestinal tract of various domestic animals and birds and is frequently found in chicken flocks (Humphrey et al., 2007). As a commensal organism with high load (109 CFU/g) in chicken ceca, Campylobacter easily contaminates carcasses of slaughtered birds (Thibodeau et al., 2015). With the widespread use of antimicrobial agents in poultry and livestock production systems, the surveillance of AMR in Campylobacter has identified high levels of resistance to some drugs in many parts of the world (Bundurus et al., 2023). Furthermore, severe MDR increases the threat to public safety (Zhang et al., 2016). In this study, a total Campylobacter prevalence of 39.7% was recorded in retail poultry meat. Certain positive rates of Campylobacter isolation were also found in pigeon and duck meat samples (38.2% and 29.6%, respectively). Thus, retail pigeon and duck meat had a risk of carrying Campylobacter. About 65% of Campylobacter isolates were resistant to multiple antimicrobial agents in this work. Many previous studies have described the increasing rate of MDR phenotypes of Campylobacter isolates of human and chicken meat origin in China and other countries (Habib et al., 2023; Zhang et al., 2016; Santos-Ferreira et al., 2022; Wang et al., 2022). The comparatively high resistance rate of ciprofloxacin and macrolide and the high level of MDR Campylobacter isolated from poultry meat is an important public-health issue, requiring multidisciplinary-level interventions.

Given the rising concern in antimicrobial resistance, phage therapy may play an important role in combating MDR foodborne pathogens, including Campylobacter (Janež et al., 2013; Jäckel et al., 2019). Phage applications targeting Campylobacter can be used in ways like those against Listeria and Salmonella, but very few Campylobacter phage products have been approved anywhere to date (Ushanov et al., 2020). Thus, a broad selection of well-characterized Campylobacter phages should be available for controlling MDR Campylobacter in animals and food products. In this regard, the CP6 isolated and characterized in this study showed a broad host range against MDR Campylobacter prevalent in poultry meat. It infected 33 of the 34 MDR Campylobacter and 4 of the 4 sensitive Campylobacter isolates, being lytic to 97.4% (37/38) among all isolates from different districts. Many previous studies have been conducted on Campylobacter phages and their potential applications (Ushanov et al., 2020). Most studies have examined the efficacy of phages in eliminating Campylobacter in chickens (Ei-Shibiny et al., 2009; Steffan et al., 2021; Steffan et al., 2022). Compared with previous isolated phages with a relatively narrow host range (Jäckel et al., 2015; Nowaczek et al., 2019; Steffan et al., 2021), CP6 had a relatively broader host range and showed lytic properties against almost all tested MDR Campylobacter strains.

Several studies have been published that group II or group III Campylobacter phages were successfully applied EI-Shibiny et al., 2009, Javed et al., 2014, Steffan et al., 2021. However, these two groups of phages are different regarding their host ranges and host cell receptors. An important feature of Campylobacter group II phage is that they are often specific to C. jejuni and C. coli, whereas group III phages demonstrate specificity exclusively to C. jejuni (Jäckel et al., 2015). According to its genome size (178,350 bp), CP6 in this study belonged to group II, exhibiting a stronger lytic activity to C. coli. It can provide a possible successive application of phage-cocktail design to reduce Campylobacter in food products and animals.

Stability against wide ranging temperatures and pH values is an important prerequisite of successful phage application. In this study, CP6 exhibited good stability below 50°C and at pH 3.0 to 9.0, indicating that CP6 may have a prerequisite for utilization in the biocontrol of Campylobacter within a wide range of temperatures or pH values. The stability of CP220 at different pH values has been examined and found to be stable at pH above 4 (Ei-Shibiny et al., 2009). Steffan (Steffan et al., 2021) reported 3 phages that are stable only during exposure to pH 3 for at least 2 h at 42°C. These phages can combat environmental stresses during application in primary food production. Herein, we found no obvious change in phage titer when storing CP6 in SM buffer at 4°C for 6 months. This result was similar to previously reported phages (Hammerl er al., 2014; Steffan et al., 2021), guaranteeing sufficient shelf life for storage under cold conditions.

Complete genomic analyses revealed that CP6 had high homology with Campylobacter phage CP220, a lytic phage from retail chicken samples that reduced Campylobacter colonization in poultry (Ei-Shibiny et al., 2009). Claudia Jäckel analyzed the genomic sequence of group II phage CP21 that is closely related to phages CP220 and CPt10 (Jäckel et al., 2015). Campylobacter group II phages are diverse and contain numerous genes for transposases and homing endonucleases, indicating that these phages may be genetically unstable (Jäckel et al., 2015). Furthermore, no virulence, toxin production, and antibiotic resistance genes were identified in the CP6 genome, suggesting that it may not be hazardous to humans. As a virulent phage, genes encoding those associated with integrase protein and lysogen decision were not identified, confirming its lytic activity.

Regarding lysis ability, CP6 showed good antimicrobial effects on MDR Campylobacter in an in vitro culture. Thus, CP6 can be used to treat antibiotic-resistant Campylobacter and minimize the transmission of antimicrobial-resistance genes. Campylobacter is also a major foodborne pathogen and typically acquired through the consumption of undercooked contaminated poultry products (Oison et al., 2022). Therefore, future applications of Campylobacter phage as a potential way to treat raw-poultry meat infections during slaughtering and processing can enhance food safety. To date, there are very few patents on phage products relating to their use for Campylobacter load reduction in poultry flocks or on processed meat (Ushanov et al., 2020). In our study, CP6 reduced the CFUs of host cells by up to 1-log compared with the control in artificially contaminated chicken breast meat. No dose-dependent bacteriolytic activity was found in this study. More research should be conducted to evaluate CP6 as a candidate food-protection antibacterial agent against MDR Campylobacter during food processing.

CONCLUSION

C. jejuni and C. coli with high AMR were isolated from retail poultry meats. Lytic phages can serve as natural antimicrobial agents to treat antimicrobial-resistance problems in food-producing animals. We isolated and characterized CP6, a new phage infecting Campylobacter, and evaluated it as an alternative for biocontrol agents. CP6 belonged to group Ⅱ and had a broad host range of lytic effect in vitro. Furthermore, it had good pH and temperature tolerance. These are interesting properties for the development of intestinal phage applications. CP6 phage treatment can reduce the levels of MDR Campylobacter on artificially contaminated chicken meat, suggesting its potential in the biocontrol of food contamination by MDR Campylobacter. In further study, we will consider evaluating its application effect and safety, so as to lay a foundation for the development of the Campylobacter phage.

Acknowledgments

ACKNOWLEDGMENTS

This work was supported by the project of seed industry revitalization and listing in Jiangsu province (JBGS (2021)109).

Author Contribution: X. Z. participated in the study design, carried out all data analysis, and prepared the manuscript. Y. G. supervised and assisted in the manuscript preparation. M. T. and Q. Z. provided data and revised the manuscript. J. L., X. T., L. M., and J. Z. participated in the study design and provided data. H. Z. conceived the study and revised the manuscript. All authors contributed to the article and approved the submitted version. All authors have read and agreed to the published version of the manuscript.

DISCLSOURES

The authors declare that they have no conflict of interest.

Footnotes

Supplementary material associated with this article can be found in the online version at doi:10.1016/j.psj.2024.103548.

Appendix. Supplementary materials

mmc1.docx (347.2KB, docx)
mmc2.docx (35.7KB, docx)

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