Research Article

Isolation and characterization of broad host-range of bacteriophages infecting Cronobacter sakazakii and its biocontrol potential in dairy products

Hua-xiang Li1#, Xiao-jun Yang1#, Xiao-yan Zhu2, Lu Gao1*, Sheng-qi Rao3, Lei Yuan1, Zhen-quan Yang1, 2*

1College of Food Science and Engineering, Yangzhou University, Yangzhou, Jiangsu, China;

2Jiangsu Provincial Key Construction Laboratory of Probiotics Preparation, Huaiyin Institute of Technology, Huaian, China;

3Jiangsu Key Laboratory of Dairy Biotechnology and Safety Control, Yangzhou University, Yangzhou, Jiangsu, China

Abstract

Cronobacter sakazakii (C.sakazakii) is an important pathogen contaminating dairy products (e.g., milk powder) and causes high mortality in infants. Bacteriophage as a potential biocontrol agent is a good alternative method for the control of this pathogen in dairy production and its environment. Thus, it is important to complete the C.sakazakii phage library by isolating and characterizing the broad host range of bacteriophage against C.sakazakii for control use. In this study, C.sakazakii strains from different sources were used as hosts to isolate and purify phages from human stool and sewage samples by double-layer plates. The biological characteristics, antibacterial properties, and genomes of these phages were then studied. Finally, ten virulent phages (EspYZU01–EspYZU10) infecting C. sakazakii were isolated and identified as belonging to the Myoviridae, Podoviridae, Tectivirus, and Stylovinidae families. Phage EspYZU08 presented the broadest host range and could infect all the five host strains of C.sakazakii. All 10 phages retained their infectivity at 50°C and pH 5–9. Both genomes of EspYZU05 and EspYZU08 were double-stranded DNAs with sizes of 41723 bp and 145582 bp, G+C contents of 55.69% and 46.75%, and open reading frames of 47 and 103, respectively. No toxins and antibiotic resistance genes were detected in both EspYZU05 and EspYZU08. Phage EspYZU08 and phage cocktail-3 (EspYZU01 + EspYZU03 + EspYZU08 + EspYZU09 + EspYZU10) presented excellent antibacterial efficacy for C.sakazakii in liquid broth and milk at 4°C, 25°C, and 37°C, suggesting that the phages in this study have great potential for the development of biocontrol agents against C.sakazakii in dairy and its processing environment.

Key words: Cronobacter sakazakii, bacteriophage, antibacterial effects, genome, biocontrol

*Corresponding authors: Lu Gao and Zhen-quan Yang, College of Food Science and Engineering, Yangzhou University, No. 196 West Huayang Road, Yangzhou, Jiangsu 225009, China. Emails: [email protected] and [email protected]

#These authors contributed equally to this study and shared first authorship.

Received: 31 December 2020; Accepted: 22 June 2021; Published: 6 August 2021

DOI: 10.15586/qas.v13i3.890

© 2021 Codon Publications

This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0). License (http://creativecommons.org/licenses/by-nc-sa/4.0/)

Introduction

Before 1980, Cronobacter sakazakii (C. sakazakii) was known as Enterobacter cloacae. Then, till 2007, Enterobacter cloacae was named as Enterobacter sakazakii. It was then reclassified into genus Cronobacter on the basis of its nucleotide sequence (Farmer et al., 1980; Iversen et al., 2007). The Cronobacter genus includes the following seven species: C. sakazakii, C. malonaticus, C. turicensis, C. universalis, C. muytjensii, C. dublinensis, and C. condimenti (Brady et al., 2013; Joseph et al., 2012). C. sakazakii is ubiquitous in nature and thus can be isolated from the environment and food materials and products, including dried foods and water (Healy et al., 2010; Kandhai et al., 2004). C. sakazakii has higher tolerance to desiccation, osmotic stress, and heat stress than other members of Enterobacteriaceae (Asakura et al., 2007), contributing to its survival in desiccation and osmotic stress environments, typical of powdered infant formula.

In recent years, C. sakazakii, as an emerging food-borne pathogen, has gained more and more attention. Genus Cronobacter can cause severe diseases, including bacteremia, sepsis, brain abscess, meningitis, and necrotizing enterocolitis in immunocompromised neonates, especially in pre-term and low birth weight infants (Drudy et al., 2006; Lai, 2001; Nazarowec-White and Farber, 1997; Yan et al., 2012). Besides, genus Cronobacter causes urosepsis, pneumonia, and bacteremia in immunocompromised adults, especially in the elderly population (Hawkins et al., 1991; Lai, 2001; See et al., 2007). In the United States, incidences of 1 Cronobacter infection per 100,000 infants, 8.7 Cronobacter infections per 100,000 low birth weight neonates (Himelright et al., 2001), and 1 Cronobacter infection per 10,660 very-low birth weight neonates (Stoll et al., 2004) have been reported. Although the incidence rate of C. sakazakii infection is low, fatality because of its infection is as high as 80% (Friedemann, 2009). One of the most severe outbreaks of C. sakazakii infection was in a neonatal intensive care unit of France in 1994, which lasted for more than 3 months, infecting 17 neonates and claiming three lives (Caubilla-Barron et al., 2007). Further, 18 cases of (meningitis or) bacteraemia in infants aged 1–11 months have been reported in the United Kingdom by the Food and Agriculture Organization/World Health Organization (FAO/WHO) during 1997–2007, and in 2008, 27 clinical Cronobacter isolates from young children aged 1–4 years have been reported in England and Wales (FAO/WHO, 2008). Besides, the C. sakazakii outbreaks were also reported in Belgium during 1997–1998, in Austria during 2009 –2016, and in France during 2010–2016 (Lepuschitz et al., 2009). Prevalence of infection, high mortality rates, and associated chronic neurological and developmental disorders in many survivors highlight the damaging effects of this organism on infant health (Forsythe, 2005; Lai, 2001). Thus, the International Commission for Microbiological Specifications for Foods (ICMSF), which was formed in 1962 through the action of the International Committee on Food Microbiology and Hygiene as a committee of the International Union of Microbiological Societies (IUMS) and linked to the International Union of Biological Societies (IUBS) and the World Health Organization (WHO) of the United Nations, has ranked C. sakazakii as a ‘severe hazard for restricted populations, life threatening or substantial chronic sequelae of long duration’, and has classified it with Clostridium botulinum, Cryptosporidium parvum, and Listeria monocytogenes (Abbasifar et al., 2014).

Therefore, it is important to minimize the risk of C. sakazakii contamination in foods by developing novel alternative biocontrol agents. Bacteriophages are recognized as safe, host-specific, and effective alternatives for the prevention and/or eradication of food-borne pathogens in foods and their processing environments. In fact, bacteriophages have been applied in the decontamination of livestock; sanitation of contact surfaces and equipment; and biocontrol of raw meats, fresh foods, and vegetables (Endersen et al., 2014; Goodridge and Bisha, 2014), cheese (Carlton et al., 2005), ready-to-eat foods (Bigot et al., 2011), skim milk (Ellis et al., 1973; Endersen et al., 2013), and reconstituted infant formula (Kim et al., 2007). Selected C. sakazakii phages were used to inhibit growth of C. sakazakii in the formula and show high efficiency (Kim et al., 2007). Furthermore, 67 newly isolated C. sakazakii phages have been tested, some of which have reduced C. sakazakii up to 4 log (CFU/mL) in pure broth culture, which shows that the C. sakazakii phages have a great potential of being a biocontrol agent for controlling C. sakazakii in foods (Zuber et al., 2008). In addition, C. sakazakii phages also exhibit excellent efficiency in alleviating Cronobacter-induced urinary tract infections in mice (Tóthová et al., 2011).

These studies demonstrate that bacteriophages are promising natural agents for the control of C. sakazakii. Although a total of 21 genomes of phages infecting C. sakazakii have been published in the National Center for Biotechnology Information (NCBI) database (https://www.ncbi.nlm.nih.gov/genome) so far, the library of C. sakazakii phages and their genomic information are still limited for developing biocontrol agents, because C. sakazakii strains are of high diversity and tend to become phage-resistant. Therefore, new broad host range phages must be isolated, and phage cocktails must be created as broad-spectrum food biocontrol agents against C. sakazakii.

In this study, human stool samples and sewage samples were used to isolate broad-spectrum virulent phages against C. sakazakii. In all, 10 bacteriophages infecting C. sakazakii were isolated and purified, and their morphological features, dynamics of infection, and host range were characterized. In addition, genomes of two broad-spectrum phages (EspYZU05 and EspYZU08) were completely sequenced to understand their characteristics. On the basis of these results, cocktail of virulent phages were prepared and evaluated for their potential and efficacy in the biocontrol of C. sakazakii strains in the liquid broth and milk medium under different temperatures and pH values to evaluate their possibility of being novel and efficient biocontrol agents.

Materials and Methods

All the chemicals used in this study, except for nutrient broth, were of analytical grade and purchased from Sangon Biotech Co. Ltd. (Shanghai).

Bacterial strains and cultivation

C. sakazakii strains CICC 21560, CICC 21545, CICC 21569, CICC 21673, and CICC 22919 were used as bacterial hosts for isolating phages in this study. These strains were purchased from the China Industrial Culture Collection Center. The strains were stored at -80°C and routinely based on nutrient agar and nutrient broth (Hangzhou Microbial Reagent Co. Ltd.) at 37°C.

Collection of samples

A total of 100 samples of human stool were collected from patients suspected of C. sakazakii infection in the Affiliated Hospital of Yangzhou University, Yangzhou, Jiangsu, China after signing the consent. Apart from these, 12 raw sewage samples were collected from Kangyuan Dairy Co. Ltd. (Yangzhou, Jiangsu, China).

Isolation and purification of phages

In order to isolate bacteriophages, we homogenized 25 mL of fresh sewage or 5 g of stool sample with 45 mL of sodium chloride–magnesium sulfate (SM) buffer (NaCl, 5.8 g; MgSO47H2O, 2 g; Tris-HCl 1 mol/L [pH 7.5], 50 mL; gelatin, 0.1 g: dissolved in distilled water to a final volume of 1 L). After overnight incubation at 4°C, the homogenized sample was centrifuged at 5,000 × g for 10 min at 4°C, and the supernatant was filtered using sterile 0.22-μm membrane. Afterward, 5 mL of filtrate and 100 μL of host bacteria (C. sakazakii) suspension in logarithmic growth phase were aseptically added to a tube with 5-mL nutrient broth incubated overnight at 37°C with continuous shaking at a speed of 120 rpm/min. After incubation, the broth was centrifuged at 5,000 × g for 10 min at 4°C. The phage suspended in the semen was filtered through a sterile 0.22-μm membrane to remove residual bacterial cells.

Thereafter, the presence of viable infective phages in supernatant was tested by a two-layer plating method. The supernatant (100 μL) was mixed with host bacteria (100 μL) in early logarithmic growth phase and added to a tube with 5 mL of soft nutrient agar (0.7% agar), tempered to 37°C, and thereafter poured onto the surface of a nutrient agar plate (2% agar). The plates were incubated overnight at 37°C and examined for phage plaques. Extract a single phage plaque and resuspend it in SM buffer. The isolation processes were repeated for three to five times to purify individual phages.

Amplification and collection of phages

Inoculation of 5 mL of the mixture of host bacteria and bacteriophages into 500 mL of fresh nutrient broth was performed and shaken overnight at 37°C at a speed of 120 rpm/min. After phage propagation and amplification, the lysates were treated with DNase I and RNase A with a final concentration of 1 μg/mL at 37°C for 30 min and then with 1 mol/L NaCl in ice for 1 h. The host cell fragments were removed at a 10,000 × g centrifugation of 10 min at 4°C. Phage particles were concentrated in 10% (w/v) polyethylene glycol 8,000 by overnight incubation in ice. The phages were pelleted by 11,000 × g centrifugation for 15 min at 4°C and resuspended in 1 mL of SM buffer. The phages were treated with 1 mL of chloroform and centrifuged at 3,000 × g for 15 min at 4°C. Phages dispersed in the upper aqueous phase were collected. The aqueous phase was overlaid on a CsCl step gradient (density = 1.4, 1.5, and 1.7 g/mL, 1 mL each step) in 5-mL centrifuge tube and horizontal centrifuge at 22,000 × g for 2 h at 4°C. The phage band (density between 1.5 and 1.7 g/mL) was drawn through the wall of centrifuge tube by using a syringe and stored at 4°C for further experiments.

Determination of phage titer

First, the purified phages were diluted for 10 times with SM buffer. Thereafter, 100 μL of phage suspension was mixed with 100 μL of host bacterium suspension. The mixture was added to 5 mL of nutrient broth incubated at 37°C for 12 h with continuous shaking at 120 rpm/min. The culture medium was centrifuged at 5,000 × g for 10 min at 4°C, and the supernatant was filtered through a sterile 0.22-μm membrane filter. Subsequently, 100 μL of filtrate (phage suspension) was serially diluted with a gradient to 10-fold, mixed with 100 μL of host bacterium suspension (~109 CFU/mL), and incubated for 10 min at 37°C. Then the mixture was added to 5 mL of soft agar and spread on nutrient broth agar plates. The titers of the phages were determined by the soft agar overlay method. All assays were carried out in triplicate.

Transmission electron microscope

Phage morphology was observed by TEM (Phenom XL G2, the Netherlands) analysis. CsCl phage suspension was dropped on the membrane side of 400 mesh copper grid. After 10 min of adsorption, excess solution was removed with a bibulous paper. The copper grid was treated for 2 min in a drop of 2% phosphotungstic acid, and excess liquid was removed by infrared light. Finally, the specimens were examined and the morphology and dimensions of phages were recorded with a Tecnai-12 TEM.

Host range activity

Soft agar, 5 mL, containing 100 μL of host bacteria was overlaid on 1.5% nutrient agar plates. Then 10 μL of phage suspension (~109 PFU/mL) was spotted on the overlaid plates and dried with blotting paper. Petri dish was cultured for 8–12 h at 37°C. The lysis activity of the test bacteria by phages was determined by counting the plaques of spots.

Effect of heat treatment on phage infectivity

In order to determine the effect of temperature on phages, we placed 500 μL of phage suspension in water bath at 50, 60, and 70°C. Subsequently, 100 μL of phage suspension was immediately obtained for phage titer measurement for 20, 40, and 60 min as described in Section 2.5. Measurement was replicated for three times, and the average values were used for analysis.

Effect of pH on phage infectivity

Effect of pH on phage activity was determined in nutrient solution with different pH values (pH 3–11). In general, 50 μL of phage suspension was mixed with 950 μL of nutrient broth adjusted to pH of 3–11 and incubated at 37°C for 2 h. Subsequently, as described in Section 2.5, 100 μL of phage suspension was obtained immediately for phage titer measurement. All assays were performed in triplicate.

Optimization of MOI

MOI is a ratio of virus particles to potential host cells. The host cells were infected with phages in different ratios (0.001, 0.01, 0.1, 1, and 10 PFU/CFU) and incubated at 37°C for 6 h. The culture medium was centrifuged at 5,000 × g at 4°C for 10 min. The supernatant was filtered through sterile 0.22-μm membrane filter, and phage titer was measured as described in Section 2.5. The MOI resulting in the highest phage titer within 6 h was regarded as optimal MOI.

One-step growth curve of phage

First, 100 μL of phages (~108 PFU/mL) and their host bacteria (~107 CFU/mL) were mixed and allowed to adsorb for 10 min at 37°C. Here, the C. sakazakii strain of CICC 21560 was used as host for EspYZU01 and EspYZU02. The C. sakazakii strain of CICC 22919 was used as host for EspYZU03, EspYZU04, EspYZU05, EspYZU06, EspYZU07, EspYZU08, EspYZU09 and EspYZU10. The culture was centrifuged at 5,000 × g for 30 s at 4°C. The pelleted cells were washed twice with pre-warmed nutrient broth, resuspended in 5 mL of nutrient broth, and incubated at 37°C. The bacteriophage titer was determined by double agar plate method. The samples were taken every 10 min in 0–2 h and every 15 min in 2–4 hours. A one-step growth curve was drawn with phage processing time as the abscissa and phage titer as the ordinate. During the incubation period, the burst size was calculated by the ratio of the final count of released phage particles to the initial count of infected bacterial cells.

Genome sequencing and analysis

DNA extraction and purification

The DNA extraction steps are the same as provided in the instructions of Ezup Spin Column Super Plant Genomic DNA Extraction Kit (Sangon Biotech, Shanghai, China). Briefly, first, 500 μL of purified phages was mixed with 20 μL of 500 mmol/L ethylenediaminetetra acetic acid, 30 μL of 10% SDS, and 3 μL of 10 mg/mL proteinase K and incubated at 56°C for 1 h. Isovolumetric chloroform:isoamyl alcohol:phenol (25:24:1) mixture was added, mixed thoroughly, and centrifuged at 12,000 × g for 5 min at 4°C. The upper layer was carefully transferred to a new sterile tube. This step was repeated using equal volumes of chloroform:isoamyl alcohol (24:1) mixture, which was centrifuged at 12,000 × g for 5 min at 4°C. After incubation at –20°C for 1 h, DNA was precipitated from the solution with isopropanol. The solution was centrifuged at 4°C at 12,000 × g for 10 min, and the supernatant was separated. The precipitate was washed twice with 70% ethanol, dried at room temperature, resuspended in 20-μL TE buffer (pH 7.4), and dissolved at 37°C for 30 min. The quality and quantity of DNA was evaluated by GeneQuant (Eppendorf, Germany) and by running DNA on an agarose gel by electrophoresis followed by visualization.

Genome sequencing and annotation

The genomic DNA of phage was sequenced by Novogene Biology Information Technology Co. Ltd. (Beijing, China). A polymerase chain reaction (PCR)-free sequencing library of inserts of approximately 500 bp was constructed, and the Illumina HiSeqTM 2000 sequencing platform was used for sequencing. The obtained raw sequencing data were filtered, and sequences containing the linker, primer, and low-quality data were removed. The clean data were used for subsequent analysis. The phage genome sequence was assembled using the SOAPdenovo software, and assembly conditions were adjusted to obtain the best stitching results. The amino acid sequences of the target species were compared with non-redundant (NR) database, and blast technology was used to locate the target species’ genes and their corresponding functional annotation information (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Given that each sequence may have more than one alignment, an optimal alignment result was retained as an annotation for the gene in order to ensure its biological significance. Homology alignment of the target and reference genomes was performed using the MUMmer software. A genome-wide map of phage was constructed using CGView (http://wishart.biology.ualberta.ca/cgview/).

Application of phages

Preparation of phage cocktail

The method for preparing phage cocktail is as follows: 1-mL phage suspension with a titer of ~108 PFU/mL is mixed, and then stored at 4°C until take into use. Phage cocktail-1 contains EspYZU01 and EspYZU05; cocktail-2 contains EspYZU02, EspYZU03, and EspYZU07; and cocktail-3 contains EspYZU01, EspYZU03, EspYZU08, EspYZU09, and EspYZU10. The C. sakazakii cocktail was prepared by mixing 1 mL of each host bacteria at a concentration of ~107 PFU/mL, and used immediately.

Application of phage against C. sakazakii in nutrient broth

First, 100 μL of phage cocktail or EspYZU08 (~1×108 PFU/mL) was mixed with 100 μL of C. sakazakii cocktail (~1×107 CFU /mL). The mixture was then inoculated into 4.8 mL of nutrient broth and cultured at 37°C, 25°C, and 4°C with continuous shaking at a speed of 120 rpm/min. Optical density values at 600 nm (OD600 nm) were measured every 3 h at 25°C and 37°C and every 24 h at 4°C. Nutrient broth containing 100 μL of SM buffer instead of bacteriophage served as a positive control. All analyses were performed in triplicate.

Application of phage against C. sakazakii in milk

Phage cocktail or EspYZU08 (~1×108 PFU/mL), 5 mL, was mixed with 5 mL of C. sakazakii cocktail (~1×107 CFU/mL), which was mixed by five strains of CICC 21560, CICC 21545, CICC 21569, CICC 21673, and CICC 22919 in the same ratio. The mixture was then inoculated into 40 mL of milk and incubated at 37°C, 25°C, and 4°C with continuous shaking at a speed of 120 rpm/min. Standard plate count was performed on nutrient agar every 3 h at 25°C and 37°C and every 24 h at 4°C to quantify surviving cells. Milk containing 5 mL of SM buffer instead of bacteriophage was used as a positive control. All experiments were carried out in triplicate, and the bacterial concentration was expressed as mean CFU/mL count and standard deviation.

Results

Isolation and purification of phages

A total of 10 phages infecting C.sakazakii strains were isolated from human stool and sewage samples and were marked as EspYZU01–EspYZU10. All 10 phages formed visible and uniform size plaques on the host strain (Figure 1A). Among these 10 phages, EspYZU01, EspYZU02, EspYZU06, EspYZU07, EspYZU08, EspYZU09, and EspYZU10 formed clear plaques of 0.5–1 mm in diameter. EspYZU03 and EspYZU04 formed blurry plaques of approximately 1 mm in diameter, and EspYZU05 formed a clear plaque of approximately 4 mm in diameter.

Figure 1. Characterization of (A) plaque and (B) microscopic morphological features of Cronobacter sakazakii phages. 01: EspYZU01; 02: EspYZU02; 03: EspYZU03; 04: EspYZU04; 05: EspYZU05; 06: EspYZU06; 07: EspYZU07; 08: EspYZU08; 09: EspYZU09; 10: EspYZU10. (A) Images of phage plaques are magnified four times and shown with red squares. (B) Morphology and dimensions of phages were examined with a Tecnai-12 transmission electron microscope.

The phages were then purified and collected by discontinuous Cesium chloride (CsCl) density gradient centrifugation. A visible bacteriophage band of 1.45–1.50 g/mL CsCl was obtained with a titer of 1010 –1011 PFU/mL.

Morphology of phages

Phage morphology was characterized by transmission electron microscopy (TEM). The results showed that 10 Cronobacter phages exhibited four types of morphology (Figure 1B). EspYZU01 and EspYZU09 had an elongated polyhedron head with a diameter of ~70 nm × 120 nm (L/W = 1.7) and a tail length of ~120 nm, indicating that the phages belonged to the Myoviridae family. EspYZU02 and EspYZU08 had an isometric polyhedron head with a diameter of ~90 nm, tail length of ~120 nm, and a contractile tail sheath, indicating that the phages also belonged to the Myoviridae family. EspYZU03, EspYZU04, and EspYZU07 had an isometric polyhedron head but without tail sheath, indicating that the phages also belonged to the Myoviridae family. EspYZU05 had an isometric polyhedron head with a diameter of ~55 nm and a tail length of ~18 nm, indicating that the phages belonged to the Podoviridae family. EspYZU06 had an isometric polyhedron head with a diameter of ~60 nm but without a tail, indicating that the phages belonged to the Tectivirus family. EspYZU10 had an isometric polyhedron head with a diameter of ~130 nm and a tail length of ~250 nm but without tail sheath, indicating that the phages belonged to the Stylovinidae family.

Host range of phage

The results of host range of phages are listed in Table 1. EspYZU02 had the highest specificity and could only infect the CICC 21560 strain. EspYZU08 had the broadest infection spectrum and could infect all five C.sakazakii strains. The eight other phages could infect two to four C.sakazakii strains (Table 1).

Table 1. Host range of Cronobacter sakazakii phages.

Host species Strain Lysis by bacteriophages
01 02 03 04 05 06 07 08 09 10
Cronobacter sakazakii CICC 21560 + + - - - - - + + -
Cronobacter sakazakii CICC 21545 + - + + + - + + - +
Cronobacter sakazakii CICC 21569 - - + - + + + + - +
Cronobacter sakazakii CICC 21673 + - + + + - + + + -
Cronobacter sakazakii CICC 22919 - - + + + + + + + +
Cronobacter sakazakii CsYZ-01 - - - - + - - + - +
Cronobacter sakazakii CsYZ-04 - - + + + + + + + +
Cronobacter sakazakii CsYZ-06 - - - - + - - + - -
Cronobacter turicensis CtYZ-03 - - - - - - - - - -
Cronobacter malonaticus CmYZ-01 - - - - - - - - - -
Enterobacter cloacae CICC10017 - - - - - - - - - -
Enterobacter cloacae EcY02 - - - - - - - - - -
Enterobacter cloacae EcY05 - - - - - - - - - -
Escherichia coli EcJ01 - - - - - - - - - -
Escherichia coli EcJ05 - - - - - - - - - -
Escherichia coli EcJ07 - - - - - - - - - -
Enterobacter hormaechei SYZU2-5 - - - - - - - - - -
Pseudomonas fluorescens Pf5401 - - - - - - - - - -
Pseudomonas fluorescens Pf5502 - - - - - - - - - -
Pseudomonas fluorescens Pf5507 - - - - - - - - - -
Pseudomonas fluorescens Pf5608 - - - - - - - - - -
Klebsiella pneumoniae KpJ08 - - - - - - - - - -
Klebsiella pneumoniae KpJ06 - - - - - - - - - -
Klebsiella pneumoniae KpJ05 - - - - - - - - - -
Klebsiella pneumoniae KpJ03 - - - - - - - - - -
Bacillus subtilis BsJ01 - - - - - - - - - -
Bacillus subtilis BsJ02 - - - - - - - - - -
Bacillus subtilis BsJ05 - - - - - - - - - -
Bacillus subtilis BsJ07 - - - - - - - - - -
Bacillus subtilis BsJ08 - - - - - - - - - -
Number of hosts   3 1 5 4 7 3 5 8 4 5

Note: 01: EspYZU01; 02: EspYZU02; 03: EspYZU03; 04: EspYZU04; 05: EspYZU05; 06: EspYZU06; 07: EspYZU07; 08: EspYZU08; 09: EspYZU09; 10: EspYZU10.

+: having lytic activity; -: having no lytic activity.

Characterization of phages

Effect of temperature and pH on phage infectivity

The effect of temperature on phage infectivity was tested by exposing phages to a range of different extreme temperatures. When the phages were exposed to 50°C for 1 h, infectivity retained by all phages was quite well. At 60°C for 1 h, the infectivity of EspYZU01, EspYZU07, EspYZU08, and EspYZU10 declined slightly (<20%); that of EspYZU02, EspYZU03, EspYZU04, and EspYZU6 declined by 20–44.8%; and that of EspYZU05 and EspYZU9 declined by >50%. At 70°C, phage infectivity declined sharply. EspYZU01, EspYZU02, EspYZU04, and EspYZU5 lost their infectivity after 40 min; EspYZU03 and EspYZU9 lost their infectivity after 20 min; and only EspYZU06, EspYZU07, EspYZU08, and EspYZU10 retained 22.1–41.7% of their infectivity after 1 h (Figure 2A). The results indicated that EspYZU07, EspYZU08, and EspYZU10 have the best thermal stability.

Figure 2. Effects of (A) temperature and (B) pH on phage infectivity. 01: EspYZU01; 02: EspYZU02; 03: EspYZU03; 04: EspYZU04; 05: EspYZU05; 06: EspYZU06; 07: EspYZU07; 08: EspYZU08; 09: EspYZU09; 10: EspYZU10; (A) The effect of temperature on the viability of phage in nutrient broth at 50–70°C for 20–60 min. (●), (■), and (▲), respectively, represent the infectivity of phage exposed at 50°C for 20–60 min, 60°C for 20–60 min, and 70°C for 20–60 min. (B) The effect of pH on the viability of phage in nutrient broth at pH 3–11 and 37°C for 2 h. Each assay was conducted in triplicate, and the values were expressed as mean ± standard deviation.

The effect of pH on phage infectivity was tested by exposing phages to pH ranging from 3.0 to 11 for 2 h at 37°C. The infectivity retained by all phages was quite well if they were exposed to an environment having pH 5.0–9.0 but declined sharply at pH < 5.0. At pH < 4 or >10, EspYZU01, EspYZU06, EspYZU07, EspYZU08, and EspYZU10 retained their high infectivity. Good infectivity of EspYZU03, EspYZU04, EspYZU05, and EspYZU09 was retained in alkaline environment (pH = 7–10) but declined sharply at pH < 4 (Figure 2B). The results indicated that EspYZU01, EspYZU06, EspYZU07, EspYZU08, and EspYZU10 had the best pH stability.

Optimal multiplicity of infection (MOI) of phages

The MOI of all phages were determined as described in Section 2.10, and the results are listed in Table 2. EspYZU02, EspYZU03, and EspYZU04 had the maximal MOI of 10, and EspYZU06 and EspYZU10 showed the minimum MOI of 0.001.

Table 2. The optimum multiplicity of infection (MOI) of phages.

Phage MOI Phage/Host (CFU/mL) Titer (PFU/mL)
EspYZU01 0.1 (104/105) 5.6×1010
EspYZU02 10 (106/105) 2.1×1010
EspYZU03 10 (106/105) 2.3×104
EspYZU04 10 (106/105) 1.09×104
EspYZU05 1 (105/105) 2.35×1011
EspYZU06 0.001 (102/105) 8.7×1010
EspYZU07 0.1 (104/105) 1.97×109
EspYZU08 0.1 (104/105) 1.62×109
EspYZU09 1 (105/105) 2.58×109
EspYZU10 0.001 (102/105) 4.6×109

One-step growth curves of phages were obtained by propagation on C.sakazakii at 37°C (Figure 3). The latent periods of phages occurred from 10 to 45 min. The rising period began with average burst size of 65–439 phage particles per bacterium (Table 3). Among the phages, EspYZU09 had the shortest latent period of 10 min, and EspYZU06 showed the longest latent period of 45 min. EspYZU10 had the maximal burst size of 439, and EspYZU08 demonstrated the second maximal burst size of 366.

Figure 3. One-step growth curve of phages. 01: EspYZU01; 02: EspYZU02; 03: EspYZU03; 04: EspYZU04; 05: EspYZU05; 06: EspYZU06; 07: EspYZU07; 08: EspYZU08; 09: EspYZU09; 10: EspYZU10. Each assay was conducted in triplicate, and the values were expressed as mean ± standard deviation.

Table 3. The lysis property of Cronobacter sakazakii phages.

Phage Latent period (min) Burst period (min) Stable period (min) Burst size (phage particles per bacterium)
EspYZU01 0–20 20–80 After 80 65
EspYZU02 0–30 30–100 After 100 192
EspYZU03 0–40 40–100 After 100 112
EspYZU04 0–20 20–100 After 100 59
EspYZU05 0–20 20–180 After 180 135
EspYZU06 0–40 40–135 After 135 127
EspYZU07 0–20 20–150 After 150 73
EspYZU08 0–40 40–180 After 180 366
EspYZU09 0–10 10–110 After 110 215
EspYZU10 0–40 40–180 After 180 439

Genome analysis of phage

Among the phages, EspYZU05 and EspYZU08 infected the same host of CICC 21569 and showed broad host range. However, they presented remarkably different morphologies; hence, we sequenced and analyzed the whole genomes of EspYZU05 and EspYZU08. The genome of EspYZU05 was obtained with a size of 41723 bp, G+C content of 55.69%, gene coding percentage of 93.04%, and open reading frames (ORFs) of 47 (Figure 4A). The genome data of EspYZU05 was deposited in GenBank under the accession number MW882933; annotation of ORFs is shown in Table S1 provided in supplementary material. The genome of EspYZU08 with a size of 145582 bp, G+C content of 46.76%, gene coding percentage of 42.03%, and ORF of 103 was obtained (Figure 4B). The genome data of EspYZU08 was deposited in GenBank under the accession number MW882934; annotation of ORFs is shown in Table S2 provided in supplementary material. Genomes of both EspYZU05 and EspYZU08 were double-stranded DNAs, and no tRNA gene was analyzed. Regarding the similarity of phage proteins with those from the NCBI database, the genome of EspYZU05 was found to be highly similar to that of C. sakazakii phage vB_CskP_GAP227 (KC107834.1), with 96% identity and 98% coverage rate. The genome of EspYZU08 was very similar to Salmonella phage PVP-SE1 (GU070616.1) with 97% identity and 82% coverage rate.

Figure 4. Genome pattern of phages EspYZU05 and EspYZU08. (A) Genome pattern of phage EspYZU05 (accession number is “MW882933”); (B) Genome pattern of phage EspYZU08 (accession number is “MW882934”). Circles display (from the outside): (1) ORFs transcribed in clockwise or counterclockwise direction; (2) G+C% content. Values >42.97% (average) are outward peaks, and those <42.97% are inward peaks; (3) GC skew (G−C/G+C, in a 1-kb window and 0.1-kb incremental shift). Values >0 are inward peaks, and those <0 are outward peaks; (4) Physical map is scaled in kbp (for interpretation of references to color in this figure, the reader is referred to the web version of this article).

Table S1 The genome annotation of phage EspYZU05

Gene_id Database Start codon End codon Terminal codon usage Strand Length Gene product Ontology Blast hit with the highest max score (locus_tag, Query cover, E value, Ident)
569000001 NR 658 960 TAA + 303 Hypothetical protein NA. KC107834.1, Cronobacter sakazakii phage vB_CskP_GAP227 (GAP227_01, 100%, 8e-141, 97%)
569000002 NR 1493 2047 TAA + 555 Hypothetical protein NA. KC107834.1, Cronobacter sakazakii phage vB_CskP_GAP227 (GAP227_02, 99%, 0.0, 95%)
569000003 NR 2121 2237 TAG + 117 Hypothetical protein NA. KC107834.1, Cronobacter sakazakii phage vB_CskP_GAP227 (GAP227_03, 100%, 3e-46, 97%)
569000004 NR 2308 2712 TGA + 405 Hypothetical protein NA. KC107834.1, Cronobacter sakazakii phage vB_CskP_GAP227 (GAP227_04, 100%, 0.0, 98%)
569000005 NR 2712 3269 TAA + 558 Hypothetical protein NA. KC107834.1, Cronobacter sakazakii phage vB_CskP_GAP227 (GAP227_05, 100%, 0.0, 96%)
569000006 NR 3262 3504 TAG + 243 Hypothetical protein NA. KC107834.1, Cronobacter sakazakii phage vB_CskP_GAP227 (GAP227_06, 100%, 7e-101, 95%)
569000007 NR 3574 3798 TGA + 225 Hypothetical protein NA. KC107834.1, Cronobacter sakazakii phage vB_CskP_GAP227 (GAP227_07, 69%, 4e-58, 94%)
569000008 NR 3809 4336 TAA + 528 Hypothetical protein NA. KC107834.1, Cronobacter sakazakii phage vB_CskP_GAP227 (GAP227_08, 100%, 0.0, 90%)
569000009 NR 4329 4712 TGA + 384 Hypothetical protein NA. KC107834.1, Cronobacter sakazakii phage vB_CskP_GAP227 (GAP227_09, 100%, 2e-167, 95%)
569000010 NR 4709 5005 TGA + 297 Hypothetical protein NA. KC107834.1, Cronobacter sakazakii phage vB_CskP_GAP227 (GAP227_10, 100%, 4e-134, 96%)
569000011 NR 5002 5421 TAA + 420 Hypothetical protein NA. KC107834.1, Cronobacter sakazakii phage vB_CskP_GAP227 (GAP227_11, 100%, 1e-179, 94%)
569000012 NR 5423 5737 TAG + 315 Hypothetical protein NA. KC107834.1, Cronobacter sakazakii phage vB_CskP_GAP227 (GAP227_12, 88%, 5e-123, 96%)
569000013 NR 5721 5864 TAA + 144 Hypothetical protein NA. NA.
569000014 NR 5851 6108 TAA + 258 Hypothetical protein NA. LN878149.1, Cronobacter phage Dev-CD-23823 (gp14, 100%, 2e-117, 97%)
569000015 NR 6113 6337 TAA + 225 Hypothetical protein NA. KC107834.1, Cronobacter sakazakii phage vB_CskP_GAP227 (GAP227_14, 100%, 3e-104, 98%)
569000016 NR 6392 7066 TAA + 675 DNA primase biological process/molecular function KC107834.1, Cronobacter sakazakii phage vB_CskP_GAP227 (GAP227_15, 100%, 0.0, 97%)
569000017 NR/GO 7066 8313 TGA + 1248 DNA helicase molecular function/biological process KC107834.1, Cronobacter sakazakii phage vB_CskP_GAP227 (GAP227_16, 98%, 0.0, 98%)
569000018 NR 8326 8535 TGA + 210 Hypothetical protein NA. LN878149.1, Cronobacter phage Dev-CD-23823 (gp18, 77%, 2e-10, 70%)
569000019 NR/Swiss-Prot/GO 8535 9443 TAA + 909 ATP-dependent DNA ligase biological process/molecular function KC107834.1, Cronobacter sakazakii phage vB_CskP_GAP227 (GAP227_19, 100%, 0.0, 91%)
569000020 NR/GO 9509 11977 TGA + 2469 DNA-directed DNA polymerase biological process/molecular function KC107834.1, Cronobacter sakazakii phage vB_CskP_GAP227 (GAP227_20, 100%, 0.0, 98%)
569000021 NR 12045 12863 TAA + 819 Hypothetical protein NA. KC107834.1, Cronobacter sakazakii phage vB_CskP_GAP227 (GAP227_21, 100%, 0.0, 98%)
569000022 NR/GO 12863 13825 TGA + 963 DNA exonuclease biological process/molecular function KC107834.1, Cronobacter sakazakii phage vB_CskP_GAP227 (GAP227_22, 100%, 0.0, 98%)
569000023 NR 13830 14243 TAA + 414 Hypothetical protein NA. KC107834.1, Cronobacter sakazakii phage vB_CskP_GAP227 (GAP227_23, 100%, 0.0, 97%)
569000024 NR/GO 14236 14700 TAA + 465 DNA endonuclease VII biological process/molecular function KC107834.1, Cronobacter sakazakii phage vB_CskP_GAP227 (GAP227_24, 100%, 0.0, 97%)
569000025 GO 14700 15743 TAA + 1044 Hydrolase biological process/molecular function KC107834.1, Cronobacter sakazakii phage vB_CskP_GAP227 (GAP227_25, 100%, 0.0, 96%)
569000026 NR 15968 16297 TAA + 330 Hypothetical protein NA. KC107834.1, Cronobacter sakazakii phage vB_CskP_GAP227 (GAP227_26, 100%, 4e-159, 98%)
569000027 NR 16297 16605 TAG + 309 Hypothetical protein NA. KC107834.1, Cronobacter sakazakii phage vB_CskP_GAP227 (GAP227_27, 97%, 9e-141, 97%)
569000028 NR/Swiss-Prot/GO 16687 19149 TAA + 2463 DNA-directed RNA polymerase biological process/molecular function KC107834.1, Cronobacter sakazakii phage vB_CskP_GAP227 (GAP227_29, 99%, 0.0, 96%)
569000029 NR 19271 19450 TGA + 180 Hypothetical protein NA. KC107834.1, Cronobacter sakazakii phage vB_CskP_GAP227 (GAP227_30, 100%, 1e-87, 100%)
569000030 NR 19443 19871 TAA + 429 Hypothetical protein NA. KC107834.1, Cronobacter sakazakii phage vB_CskP_GAP227 (GAP227_31, 100%, 0.0, 98%)
569000031 NR 19871 20290 TAA + 420 Hypothetical protein NA. KC107834.1, Cronobacter sakazakii phage vB_CskP_GAP227 (GAP227_32, 100%, 1e-160, 91%)
569000032 NR/Swiss-Prot 20306 21826 TAA + 1521 Head-tail connector protein cellular component KC107834.1, Cronobacter sakazakii phage vB_CskP_GAP227 (GAP227_33, 100%, 0.0, 96%)
569000033 NR 21838 22674 TAA + 837 Scaffolding protein biological process KC107834.1, Cronobacter sakazakii phage vB_CskP_GAP227 (GAP227_34, 100%, 0.0, 93%)
569000034 NR/Swiss-Prot 22762 23781 TAA + 1020 Major capsid protein cellular component KC107834.1, Cronobacter sakazakii phage vB_CskP_GAP227 (GAP227_35, 100%, 0.0, 99%)
569000035 NR 23856 24461 TAA + 606 Tail tubular protein A biological process/cellular component KC107834.1, Cronobacter sakazakii phage vB_CskP_GAP227 (GAP227_36, 100%, 0.0, 97%)
569000036 NR/Swiss-Prot 24464 27097 TAA + 2634 Tail tubular protein B biological process/cellular component KC107834.1, Cronobacter sakazakii phage vB_CskP_GAP227 (GAP227_37, 100%, 0.0, 94%)
569000037 NR 27101 27895 TAA + 795 Hypothetical protein NA. KC107834.1, Cronobacter sakazakii phage vB_CskP_GAP227 (GAP227_38, 100%, 0.0, 97%)
569000038 NR 27905 30157 TAA + 2253 Hypothetical protein NA. KC107834.1, Cronobacter sakazakii phage vB_CskP_GAP227 (GAP227_39, 100%, 0.0, 98%)
569000039 NR 30160 33957 TAA + 3798 Transglycosylase molecular function/cellular component KC107834.1, Cronobacter sakazakii phage vB_CskP_GAP227 (GAP227_40, 100%, 0.0, 95%)
569000040 NR 34029 36605 TAA + 2577 Tail fiber protein molecular function/cellular component KC107834.1, Cronobacter sakazakii phage vB_CskP_GAP227 (GAP227_41, 100%, 0.0, 95%)
569000041 NR 36616 36804 TGA + 189 Hypothetical protein NA. KC107834.1, Cronobacter sakazakii phage vB_CskP_GAP227 (GAP227_41A, 100%, 5e-91, 9%)
569000042 NR 36788 37123 TAA + 336 Hypothetical protein NA. KC107834.1, Cronobacter sakazakii phage vB_CskP_GAP227 (GAP227_42, 100%, 1e-88, 99%)
569000043 NR/Swiss-Prot 37123 39048 TAG + 1926 DNA maturase B biological process KC107834.1, Cronobacter sakazakii phage vB_CskP_GAP227 (GAP227_43, 100%, 0.0, 98%)
569000044 NR 39095 39994 TAA + 900 Major tail subunit cellular component KC107834.1, Cronobacter sakazakii phage vB_CskP_GAP227 (GAP227_44, 97%, 0.0, 91%)
569000045 NR/Swiss-Prot/GO 40031 40579 TGA + 549 Lysozyme biological process/molecular function KC107834.1, Cronobacter sakazakii phage vB_CskP_GAP227 (GAP227_45, 100%, 0.0, 94%)
569000046 NR 40576 40941 TGA + 366 Hypothetical protein NA. KC107834.1, Cronobacter sakazakii phage vB_CskP_GAP227 (GAP227_46, 100%, 0.0, 99%)
569000047 NR 40919 41107 TAA + 189 Hypothetical protein NA. KC107834.1, Cronobacter sakazakii phage vB_CskP_GAP227 (GAP227_47, 100%, 2e-84, 97%)

Table S2 The genome annotation of phage EspYZU08

Gene_id Database Start codon Start codon usage End codon Terminal codon usage Strand Length Gene product Ontology Blast hit with the highest max score (locus_tag, Query cover, E value, Ident )
AFU63762.1 NR 3480 ATG 3641 TAA - 162 Hypothetical protein NA. JX181824, Salmonella phage SSE-121 (AFU63765.1, 79% , 4e-37, 90%)
AFU63760.1 NR 4011 GTG 4400 TAA - 390 Putative carbohydrate binding domain protein NA. KR296694, Salmonella phage 40 (SP40_123, 99%, 0.0, 97%)
AFU63758.1 NR 4729 ATG 5376 TGA - 648 Putative membrane protein NA. JX181824, Salmonella phage SSE-121 ( 100%, 0.0 ,89%)
AFU63756.1 NR 5597 ATG 5971 TGA - 375 Hypothetical protein NA. JX181824.1,Salmonella phage SSE-121( 100%, 5e-169, 96%)
AFU63754.1 NR 6537 ATG 6926 TGA - 390 Hypothetical protein NA. JX181824.1,Salmonella phage SSE-121( 98%, 2e-153, 93%)
AFU63752.1 NR 8406 ATG 8663 TGA - 258 Minor tail protein NA. None
AFU63750.1 NR 9666 ATG 10685 TAA + 1020 Hypothetical protein NA. KF550303.1,Enterobacteria phage 4MG(4MG_243,25%, 2e-27, 77%)
AFU63746.1 NR 11654 ATG 12034 TGA + 381 Hypothetical protein NA. JX181824.1,Salmonella phage SSE-121( 100%, 5e-144, 92%)
AFU63744.1 NR 13529 ATG 13723 TAA + 195 Hypothetical protein NA. JX181824.1,Salmonella phage SSE-121( 98%, 7e-55, 88%)
AFU63740.1 NR 14092 ATG 14268 TGA + 177 Hypothetical protein NA. GU070616.1,Salmonella phage PVP-SE1( 100%, 4e-72 ,96%)
AFU63738.1 Swiss-Prot/COG 15279 ATG 15770 TGA + 492 Predicted phosphatase homologous to the C-terminal domain of histone macroH2A1 NA. KR296694.1,Salmonella phage 40(SP40_91,98%, 4e-136, 85%)
AFU63734.1 NR 16315 ATG 16533 TAA + 219 Hypothetical protein NA. JX181824.1,Salmonella phage SSE-121( 100%, 1e-92 ,96%)
AFU63730.1 NR 17921 ATG 18100 TAA + 180 Hypothetical protein NA. KF550303.1,Enterobacteria phage 4MG(4MG_267,100%, 2e-29, 81%)
AFU63728.1 GO 18706 TTG 19236 TGA + 531 Hydrolase molecular function KR296694.1,Salmonella phage 40(SP40_78,100%, 0.0, 96%)
AFU63726.1 Swiss-Prot 21417 ATG 22703 TGA + 1287 Protein rIIB NA. JX181824.1,Salmonella phage SSE-121( 100%,0.0,90%)
AFU63724.1 NR 24018 ATG 24467 TAA + 450 Hypothetical protein NA. KR296694.1,Salmonella phage 40(SP40_69,46%,3e-57 ,87%)
AFU63722.1 NR 24629 ATG 24943 TAA + 315 Hypothetical protein NA. None
AFU63720.1 NR 25220 ATG 25483 TGA + 264 Hypothetical protein NA. None
AFU63718.1 NR 26191 ATG 26832 TAA + 642 Glucanases NA. None
AFU63710.1 NR/GO 29075 ATG 31774 TAA + 2700 DNA-directed DNA polymerase biological process/molecular function KR296694.1,Salmonella phage 40(SP40_57,100%, 0.0, 99%)
AFU63708.1 NR 31809 ATG 32006 TGA + 198 Hypothetical protein NA. KR296694.1,Salmonella phage 40(SP40_56,91%, 2e-55, 89%)
AFU63706.1 NR 32741 ATG 32947 TAA + 207 Hypothetical protein NA. None
AFU63704.1 NR/Swiss-Prot/GO 32959 ATG 34032 TAA + 1074 Nucleotidyltransferase biological process/molecular function JN882284.1,Cronobacter phage vB_CsaM_GAP31(GAP31_248,98% ,0.0, 83%)
AFU63702.1 NR 34386 ATG 34589 TAA + 204 Hypothetical protein NA. GU070616.1,Salmonella phage PVP-SE1( 41%, 1e-22 ,93%)
AFU63696.1 NR 36648 ATG 37019 TGA + 372 Putative membrane protein NA. JN882284.1,Cronobacter phage vB_CsaM_GAP31(GAP31_240,100%, 3e-52, 77%)
AFU63694.1 NR 37281 ATG 37775 TGA + 495 Hypothetical protein NA. None
AFU63692.1 NR 38035 GTG 38262 TAA + 228 Putative membrane protein NA. JN882284.1,Cronobacter phage vB_CsaM_GAP31(GAP31_235,63%, 8e-20, 81%)
AFU63690.1 NR 38683 ATG 39867 TAA + 1185 Hypothetical protein NA. KR296694.1,Salmonella phage 40(SP40_36,100%, 0.0, 79%)
AFU63688.1 NR 41657 GTG 43627 TAA - 1971 Tail fiber protein NA. KF550303.1,Enterobacteria phage 4MG(4MG_057,100%, 0.0 ,96%)
AFU63686.1 NR 43923 ATG 44396 TGA - 474 Putative membrane protein NA. JN882284.1,Cronobacter phage vB_CsaM_GAP31(GAP31_229,98%, 1e-150, 88%)
AFU63682.1 NR 46494 ATG 47126 TAA - 633 Hypothetical protein NA. JN882284.1,Cronobacter phage vB_CsaM_GAP31(GAP31_225,100%, 0.0, 87%)
AFU63680.1 NR/Swiss-Prot 48740 ATG 50878 TAA - 2139 Colanic acid biosynthesis protein WcaM NA. JN882284.1,Cronobacter phage vB_CsaM_GAP31(GAP31_223,100%, 0.0, 83%)
AFU63678.1 NR 53678 ATG 54352 TAA - 675 Hypothetical protein NA. JN882284.1,Cronobacter phage vB_CsaM_GAP31(GAP31_221,100%, 5e-146, 81%)
AFU63676.1 NR 54990 ATG 55691 TAA - 702 Putative baseplate assembly protein NA. None
AFU63674.1 NR 56684 ATG 57034 TAA - 351 Hypothetical protein NA. JN882284.1,Cronobacter phage vB_CsaM_GAP31(GAP31_217,100%, 5e-114 ,89%)
AFU63672.1 NR 57988 ATG 60378 TAA - 2391 Hypothetical protein NA. GU070616.1,Salmonella phage PVP-SE1( 100%, 0.0, 86% )
AFU63670.1 NR 60710 ATG 61183 TAA - 474 Hypothetical protein NA. KF550303.1,Enterobacteria phage 4MG(4MG_057,100%, 0.0 ,96%)
AFU63668.1 NR 61735 ATG 63147 TAA - 1413 Tail sheath protein NA. KF550303.1,Enterobacteria phage 4MG(4MG_059,100%, 0.0, 91%)
AFU63666.1 NR 63805 ATG 64239 TAA - 435 Hypothetical protein NA. None
AFU63664.1 NR 64781 ATG 65299 TAA - 519 Hypothetical protein NA. JN882284.1,Cronobacter phage vB_CsaM_GAP31(GAP31_207,100%, 3e-167, 88%)
AFU63662.1 NR 65859 ATG 66245 TAA - 387 Hypothetical protein NA. None
AFU63660.1 Swiss-Prot 66939 ATG 69560 TAA - 2622 Collagen-like protein 6 NA. JX181824.1,Salmonella phage SSE-121( 97%, 0.0, 79%)
AFU63658.1 NR/Swiss-Prot/GO 70236 ATG 71249 TAA - 1014 Major capsid protein cellular component JX181824.1,Salmonella phage SSE-121( 100% ,0.0, 95%)
AFU63656.1 NR 71709 ATG 72713 TAA - 1005 Hypothetical protein NA. JX181824.1,Salmonella phage SSE-121( 100%, 0.0, 96%)
AFU63654.1 NR 73406 GTG 74956 TAA - 1551 Hypothetical protein NA. JX181824.1,Salmonella phage SSE-121( 100%, 0.0 ,93%)
AFU63652.1 NR 76572 ATG 76862 TGA - 291 Hypothetical protein NA. JX181824.1,Salmonella phage SSE-121( 100% ,2e-137, 98% )
AFU63650.1 NR 77257 ATG 78066 TGA - 810 Hypothetical protein NA. JX181824.1,Salmonella phage SSE-121( 100%, 0.0, 89%)
AFU63648.1 NR 79376 ATG 79528 TAA - 153 Hypothetical protein NA. GU070616.1,Salmonella phage PVP-SE1( 100%, 9e-68, 99%)
AFU63644.1 NR 81630 ATG 81998 TAA - 369 Hypothetical protein NA. GU070616.1,Salmonella phage PVP-SE1( 84% 0.0 97%)
AFU63882.1 NR 83895 ATG 84506 TAA - 612 Hypothetical protein NA. JX181824.1,Salmonella phage SSE-121( 100%, 0.0, 98%)
AFU63880.1 NR 84682 ATG 84870 TGA - 189 Putative major capsid/head protein NA. JX181824.1,Salmonella phage SSE-121( 100% ,1e-72, 94%)
AFU63878.1 NR 85477 ATG 85644 TGA - 168 Hypothetical protein NA. JX181824.1,Salmonella phage SSE-121( 100%, 2e-39, 85%)
AFU63876.1 NR 86145 ATG 86327 TGA - 183 Hypothetical protein NA. JX181824.1,Salmonella phage SSE-121( 100%,1e-72,95%)
AFU63874.1 NR 86491 ATG 86736 TAA - 246 Conserved hypothetical protein NA. GU070616.1,Salmonella phage PVP-SE1( 100%, 3e-119, 99%)
AFU63870.1 NR/Swiss-Prot/KEGG/GO 89338 ATG 90138 TAA - 801 Ribose-phosphate pyrophosphokinase biological process/molecular function JN882284.1,Cronobacter phage vB_CsaM_GAP31(GAP31_180,100% ,2e-130, 78%)
AFU63868.1 NR 90731 ATG 91054 TGA + 324 Hypothetical protein NA. JX181824.1,Salmonella phage SSE-121( 100%, 2e-117, 91%)
AFU63866.1 NR 91447 ATG 91734 TGA + 288 Transposase-like protein NA. JX181824.1,Salmonella phage SSE-121( 100% ,9e-76 ,85%)
AFU63864.1 NR 92593 ATG 92949 TAA + 357 Hypothetical protein NA. None
AFU63862.1 NR/GO 93099 ATG 94043 TGA + 945 Ligase molecular function JX181824.1,Salmonella phage SSE-121( 98%, 0.0, 84%)
AFU63860.1 NR 94394 ATG 94801 TAG + 408 NEDD4-binding protein 2-like 1 NA. JX181824.1,Salmonella phage SSE-121( 100%, 0.0, 99% )
AFU63858.1 NR/GO 95900 ATG 97195 TGA + 1296 DNA ligase biological process/molecular function GU070616.1,Salmonella phage PVP-SE1( 85% ,0.0 ,81%)
AFU63856.1 NR 97641 ATG 98012 TAA + 372 Putative membrane protein NA. JN882284.1,Cronobacter phage vB_CsaM_GAP31(GAP31_165,100%, 2e-102, 85%)
AFU63854.1 NR 98770 TTG 99009 TAA + 240 Hypothetical protein NA. GU070616.1,Salmonella phage PVP-SE1( 100% ,1e-107, 97%)
AFU63852.1 NR 99392 ATG 99595 TAA + 204 Hypothetical membrane protein NA. JX181824.1,Salmonella phage SSE-121( 100% ,1e-72, 92%)
AFU63850.1 COG 99736 ATG 100626 TAA + 891 Membrane protease subunits NA. JX181824.1,Salmonella phage SSE-121( 100%, 0.0, 99%)
AFU63848.1 NR 100894 ATG 101097 TAA + 204 Hypothetical membrane protein NA. GU070616.1,Salmonella phage PVP-SE1( 100%, 6e-96, 99%)
AFU63846.1 NR/GO 101602 TTG 102039 TGA + 438 Endonuclease biological process/molecular function JX181824.1,Salmonella phage SSE-121( 100%, 0.0, 99%)
AFU63844.1 NR 103166 ATG 103483 TGA + 318 Hypothetical protein NA. JX181824.1,Salmonella phage SSE-121( 100%, 9e-161, 99%)
AFU63842.1 GO 104123 GTG 104653 TGA + 531 Endonuclease biological process/molecular function JX181824.1,Salmonella phage SSE-121( 100%, 0.0, 99%)
AFU63840.1 GO 104810 ATG 105784 TAA + 975 Exdonuclease biological process/molecular function JX181824.1,Salmonella phage SSE-121( 100% ,0.0, 99%)
AFU63838.1 NR 106001 ATG 106606 TAA + 606 Hypothetical protein NA. JX181824.1,Salmonella phage SSE-121( 100%, 0.0, 96%)
AFU63836.1 NR 107201 ATG 107395 TGA + 195 Hypothetical protein NA. KF550303.1,Enterobacteria phage 4MG(4MG_128, 100%, 1e-72, 93%)
AFU63834.1 NR/GO 107715 ATG 108680 TGA + 966 Thymidylate synthase biological process/molecular function JX181824.1,Salmonella phage SSE-121( 83%, 1e-173, 81%)
AFU63832.1 NR 108763 ATG 109239 TAA + 477 Hypothetical protein NA. JX181824.1,Salmonella phage SSE-121( 100%, 0.0, 96%)
AFU63830.1 NR/Swiss-Prot/GO 109479 TTG 111776 TAA + 2298 Ribonucleotide reductase of class Ia (aerobic) alpha subunit biological process/molecular function JX181824.1,Salmonella phage SSE-121( 96%, 0.0 ,97%)
AFU63828.1 NR 112917 TTG 113264 TAA + 348 Hypothetical protein NA. JX181824.1,Salmonella phage SSE-121( 100%, 2e-167, 98%)
AFU63826.1 NR/Swiss-Prot/KEGG/COG/GO 113540 GTG 115621 TGA + 2082 Ribonucleoside-triphosphate reductase biological process/molecular function JX181824.1,Salmonella phage SSE-121( 100% ,0.0 ,97%)
AFU63824.1 NR 116092 ATG 116232 TGA + 141 Hypothetical protein NA. JX181824.1,Salmonella phage SSE-121( 100%, 8e-63, 99%)
AFU63822.1 KEGG/GO 116685 ATG 117395 TAA + 711 Chitinase biological process/molecular function KF550303.1,Enterobacteria phage 4MG(4MG_139,78%, 3e-144, 84%)
AFU63820.1 NR 118206 ATG 118838 TAG + 633 Hypothetical protein NA. KF550303.1,Enterobacteria phage 4MG(4MG_141,100%, 8e-154, 83%)
AFU63818.1 NR 119141 ATG 119632 TGA + 492 Hypothetical protein NA. KF550303.1,Enterobacteria phage 4MG(4MG_143,99%, 1e-71, 78%)
AFU63816.1 GO 120563 ATG 121243 TGA + 681 Methyltransferase biological process/molecular function JX181824.1,Salmonella phage SSE-121( 100%, 0.0, 99%)
AFU63814.1 NR 121444 ATG 121719 TGA + 276 Hypothetical protein NA. JX181824.1,Salmonella phage SSE-121( 100%, 3e-139, 100%)
AFU63812.1 NR 121928 ATG 122314 TAA + 387 Hypothetical protein NA. JX181824.1,Salmonella phage SSE-12( 100%, 0.0, 99%)
AFU63808.1 NR 124195 ATG 124401 TAG + 207 Hypothetical protein NA. JX181824.1,Salmonella phage SSE-121( 100%, 1e-97, 99%)
AFU63806.1 NR 124627 ATG 125115 TAA + 489 Hypothetical protein NA. KF550303.1,Enterobacteria phage 4MG(4MG_159,100%, 3e-107 ,82%)
AFU63804.1 NR 125299 ATG 125631 TAA + 333 Hypothetical membrane protein NA. JX181824.1,Salmonella phage SSE-121( 100%, 4e-169, 99% )
AFU63802.1 GO 126012 ATG 127073 TGA + 1062 Transferase molecular function JX181824.1,Salmonella phage SSE-121( 100%, 0.0, 99%)
AFU63798.1 NR 128402 ATG 128890 TGA + 489 Hypothetical protein NA. KF550303.1,Enterobacteria phage 4MG(4MG_168,98%, 3e-107, 82%)
AFU63796.1 NR 129280 ATG 129774 TAA + 495 Hypothetical protein NA. GU070616.1,Salmonella phage PVP-SE1( 100%, 2e-90, 79%)
AFU63792.1 NR 130586 ATG 130843 TGA + 258 Hypothetical protein NA. GU070616.1,Salmonella phage PVP-SE1( 100%, 2e-71, 86%)
AFU63790.1 NR 131058 ATG 131312 TGA + 255 Hypothetical protein NA. GU070616.1,Salmonella phage PVP-SE1( 100%, 7e-111, 97% )
AFU63788.1 NR 133937 ATG 134104 TAA + 168 Hypothetical protein NA. None
AFU63784.1 NR 136193 ATG 136351 TGA + 159 Hypothetical protein NA. JX181824.1,Salmonella phage SSE-121( 100%, 4e-51, 91%)
AFU63782.1 NR 140147 ATG 140374 TAA - 228 Hypothetical protein NA. JX181824.1,Salmonella phage SSE-121( 100%, 1e-102, 97%)
AFU63778.1 NR 141387 ATG 141728 TAA - 342 Hypothetical protein NA. JX181824.1,Salmonella phage SSE-121( 100%, 9e-146, 95%)
AFU63776.1 NR 141944 ATG 142129 TAA - 186 Hypothetical protein NA. None
AFU63774.1 NR 142539 ATG 142823 TAA - 285 Hypothetical protein NA. JX181824.1,Salmonella phage SSE-121( 100%, 4e-134, 98%)
AFU63772.1 NR 143388 TTG 143735 TAA - 348 Hypothetical protein NA. JX181824.1,Salmonella phage SSE-121( 99%, 2e-168, 98%)
AFU63770.1 NR 144422 ATG 144601 TAA - 180 Hypothetical protein NA. JX181824.1,Salmonella phage SSE-121( 94%, 4e-47, 88%)
AFU63768.1 NR 145080 ATG 145430 TAG - 351 Hypothetical protein NA. JX181824.1,Salmonella phage SSE-121( 100%, 2e-167, 98%)

The functional ORFs of phage genomes could be classified into structure (e.g., membrane protein, scaffolding protein, capsid/head protein, head–tail connector protein, tail tubular protein, minor tail protein, tail sheath protein, and tail attachment protein), packaging (e.g., ribonucleotide reductase), DNA manipulation (e.g., DNA helicases, DNA polymerase, DNA ligase, DNA primase, DNA maturase, and DNA exonuclease), transcription (e.g., RNA polymerase and RNA ligase), and additional functions (e.g., nicotinamide–nucleotide adenylyltransferase and transposase protein). Furthermore, some host lysis-related proteins were found, such as lytic glycosylase (ORF39) and endolysin (ORF45) in EspYZU05 and colanic acid degrading protein (ORF18) in EspYZU08, which contribute to infecting and lysing the host cell. However, many products of predicted ORFs in genomes remain hypothetical proteins, these may result from the insufficient annotation data of C. sakazakii bacteriophage genomes.

Furthermore, the safety of phages was assessed on the basis of genomes, and no gene for toxins and antibiotic resistance was detected.

Bacterial challenge test of phage and cocktail in liquid broth

In order to evaluate the best antibacterial efficacy of phages, we measured the infectivity of EspYZU08 and phage cocktails in liquid broth at different temperatures (Figure 5). Cocktail-3 (EspYZU01 + EspYZU03 + EspYZU08 + EspYZU09 + EspYZU10) could inhibit the growth of C.sakazakii for 12 h at 37°C, 24 h at 25°C, and 12 days at 4°C. The inhibition ratio of I12 h, I24 h, and I12 d reached 99.20%, 98.69%, and 72.40%, respectively. Compared with other phages and cocktails, cocktail-3 presented the best antibacterial effect. Thus, cocktail-3 was used to further evaluate its antibacterial effect in food.

Figure 5. Growth inhibition of C. sakazakii using phage EspYZU08 or phage cocktail in nutrient broth at different temperatures. Cocktail-1: EspYZU01 + EspYZU05; cocktail2: EspYZU02 + EspYZU03 + EspYZU07; cocktail-3: EspYZU01 + EspYZU03 + EspYZU08 + EspYZU09 + EspYZU10. (■) represents C. sakazakii grown in the absence of phage; (●) represents C. sakazakii grown in the presence of phage EspYZU08 or phage cocktail (~1 × 108PFU/mL). Growth inhibition of C. sakazakii was determined by CFU/mL counts. Each assay was conducted in triplicate, and the values were expressed as mean ± standard deviation.

Application of phage cocktail in milk

In order to verify the potential of phages as a novel biocontrol agent against C.sakazakii in food, we used cocktail-3 (EspYZU01 + EspYZU03 + EspYZU08 + EspYZU09 + EspYZU10) with a titer of ~1×108 PFU/mL to evaluate antibacterial effect in milk at different temperatures (Figure 6). After cocktail-3 was added, the total viable counts of C.sakazakii in milk decreased to the minimum value for 6 h at 37°C, 9 h at 25°C, and 5 days at 4°C and reduced by 2.92 lg CFU/mL, 2.97 lg CFU/mL, and 1.64 lg CFU/mL compared with the initial values and by 4.57 lg CFU/mL, 4.25 lg CFU/mL, and 3.43 lg CFU/mL compared with the control (no phage), respectively. In addition, addition of cocktail-3 significantly decreased the population of C.sakazakii (P < 0.001) relative to the control within a certain time frame. This result suggested that cocktail-3 could remarkably inhibit growth of C.sakazakii in milk for 6 h at 37°C–9 h at 25°C and 5 days at 4°C.

Figure 6. Growth inhibition of C. sakazakii using phage cocktail-3 in milk. Cocktail-3 was mixed with EspYZU01, EspYZU03, EspYZU08, EspYZU09, and EspYZU10. (■) represents C. sakazakii grown in the absence of phage; (●) represents C. sakazakii grown in the presence of phage cocktail (~1 × 108 PFU/mL). Growth inhibition of C. sakazakii was determined by CFU/mL counts. Assays were performed in triplicate and bacterial concentrations were expressed as mean ± standard deviation.

Discussions

C. sakazakii, generally found in contaminated infant milk formula powders, is a fatal food-borne pathogen with high mortality rates (Drudy et al., 2006). Bacteriophage is considered as an alternative and promising approach to control C. sakazakii in foods. In this study, we isolated and characterized 10 Cronobacter phages with host lysis activity from sewage and stool samples of patients suspected of C. sakazakii infection. Sewage is a primary niche for Enterobacteriaceae; thus, the isolation of Cronobacter phages from effluent environments is not uncommon (Kim et al., 2007; Zuber et al., 2008). After morphological observation through TEM, 10 phages were considered to belong to Myoviridae, Podoviridae, Tectivirus, and Siphoviridae families. The phage susceptibility was assessed using five C. sakazakii strains. EspYZU08 had the broadest host range because it infected all five strains. The broad host range capabilities of five combined C. sakazakii phages show an infection profile extending across several genera (Zuber et al., 2008). Thus, the C. sakazakii phage of EspYZU08 may have good application prospect to control multiple pathogens in food. In addition, given that phages EspYZU08 and EspYZU05 have lytic lifestyles and do not possess genes for toxic proteins, they meet the required properties of phages intended for biocontrol applications. Although a transpose was found in phage EspYZU08, the transpose was proved to be safe for human cells and even could be used for clinical trial (Magnani et al., 2018; Zhang et al., 2021).

Environmental stability is essential for phages to be used as a biocontrol in foods. The common environmental pressure includes low pH and high temperature. The environmental stability of 10 phages was tested. It was found that all phages retained their maximum infectivity after exposure to pH ranging from 5 to 9, but their infectivity declined sharply at pH < 5. The pH stability of these phages was similar to some other C. sakazakii phages. C. sakazakii phages leB, leE, and leN retain their activity after exposure to pH 6–10, but no viable phages could be observed when exposure to both pH = 2 and 4 for 1 h (Endersen et al., 2017). The C. sakazakii phage PBES 02 also retained its infectivity after exposure to pH = 6–10, but its infectivity decreased at pH < 5 (Lee et al., 2016b). However, some phages have very high pH stability, such as coliphage λ, which shows no remarkable decrease in titer at pH = 3–11 (Jepson and March, 2004).

Temperature also plays a fundamental role in the survival of phages. High optimal temperatures are thought to extend the latent period, whereas low optimal temperatures are often thought to result in reduced multiplication rate (Tey et al., 2009). In this study, phages retained their infectivity at 50°C and showed slightly decreased infectivity at 60°C following a 1-h challenge. However, at 70°C, many phages were inactivated following incubation for 1 h. The thermostability of these phages was also similar to that of some other C. sakazakii phages. The C. sakazakii phages leB, leE, and leN retained their infectivity between 4°C and 50°C, and no viable phages could be recovered from the lysates exposed to 60°C, 72°C, or 90°C for 1 h (Endersen et al., 2017). The infectivity of C. sakazakii phage PBES 02 is retained after exposure to 4–55°C for 1 h but decreased sharply (75% lost) at >65°C for 1 h (Lee et al., 2016b).

Clarifying the genomic information of each bacteriophage is essential to ensure the specificity and safety (without virulence factors) of bacteriophage as a biological therapeutic agent (Brüssow et al., 2004; Faruque and Mekalanos, 2012). Besides, genome sequencing helps to further understand the phage–host interactions and provide necessary information to further exploit their biological therapeutic properties. Thus, the genomes of EspYZU05 and EspYZU08 were sequenced and analyzed, and no gene for toxins and antibiotic resistance was detected. However, some endolysin-supporting proteins were found, such as lytic glycosylase, endolysin, and colanic acid-degrading protein, which support the infection and lysis of host cell.

In this study, the lysis activity of these phages for food application was demonstrated using phage cocktail (~1×108 PFU/mL) against C. sakazakii (~1×107 CFU/mL) in milk. The results showed that cocktail-3 (EspYZU01 + EspYZU03 + EspYZU08 + EspYZU09 + EspYZU10) presented the best efficacy and controlled C.sakazakii for 6 h at 37°C, 9 h at 25°C, and 5 days at 4°C. Relative to initial values, the population of C.sakazakii was reduced by 2.92 lg CFU/mL (44.92%) at 37°C, 2.97 lg CFU/mL (45.69%) at 25°C, and 1.64 lg CFU/mL (25.23%) at 4°C. The Cronobacter phages leB, leE, and leN were combined as a part of phage cocktail (~3×108 PFU/mL) to assess their ability to inhibit the growth of C. sakazakii (~1×104 CFU/mL) in four different brands of infant formula. The C. sakazakii concentrations were reduced to below the detection limit (<10 CFU/mL) in 5-h incubation when challenged with phage cocktail, and this level of inactivation was maintained over the 20-h challenge (Endersen et al., 2017). The Cronobacter phage CR5 at an MOI of 105 was added to a sample containing C. sakazakii, and the bacterial strain was lysed at 2 h and never recovered up to 10 h (Lee et al., 2016a). When the Cronobacter phage PBES 02 with an MOI of 105 was added to infant formula containing C. sakazakii, the bacteria were completely eliminated in 6 h (Lee et al., 2016b). The Cronobacter phage Dev2 completely killed the bacteria at a high initial MOI (102 CFU/mL bacteria and 108 PFU/mL phages) in LB medium and reconstituted milk formula; similar results were observed at 20°C, 30°C, and 37°C (Kajsík et al., 2014).

In general, we used a low MOI of 10, and the biocontrol results were similar to those of some other C. sakazakii phages. The level of C. sakazakii contamination in powdered infant formula is very low (<1 bacterial cell/100 g) (Holý and Forsythe, 2014). However, the contaminating levels of C. sakazakii used in this study were much higher than those typically found in powdered infant formula, demonstrating the efficacy of this phage cocktail to be explored further.

Conclusions

A total of 10 Cronobacter phages (EspYZU01–EspYZU10) were isolated from sewage and human stool samples. After morphological observation and characterization, the genomes of phages EspYZU05 and EspYZU08 were analyzed, and no toxins and antibiotic resistance genes were detected. The phage cocktail-3 (EspYZU01 + EspYZU03 + EspYZU08 + EspYZU09 + EspYZU10) presented antibacterial efficacy against C.sakazakii in milk for 6 h at 37°C, 9 h at 25°C, and 5 days at 4°C. These results suggest that this phage cocktail may be used to develop a novel phage biocontrol agent against C. sakazakii in dairy and its production environment.

Acknowledgements

This work was financially supported by the the National Natural Science Foundation of China (Grant numbers: 31371806 and 32001661); the Natural Science Foundation of Jiangsu Province, China (Grant number: BK20190890); Jiangsu Agricultural Science and Technology Innovation Fund, China (Grant numbers: CX(15) 1013 and CX(15)1012); and the Science and Technology Innovation Team Fund of Yangzhou University (2016).

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