A Newly Isolated Bacteriophage, PBES 02, Infecting Cronobacter sakazakii

Abstract

A novel bacteriophage, PBES 02, infecting Cronobacter sakazakii was isolated and characterized. It has a spherical head of 90 nm in diameter and a tail of 130 nm in length, and belongs to Myoviridae as observed under a transmission electron microscope. The major virion protein appears to be 38 kilodaltons (kDa) in size. The latent period of PBES 02 is 30 min and the burst size is 250. Infectivity of the phage remained intact after exposure to temperatures ranging from 4℃ to 55℃ for 1 h. It was also stable after exposure to pHs ranging from 6 to 10 for 1 h. The phage effectively removed contaminating Cronobacter sakazakii from broth infant formula. PBES 02 has a double-stranded DNA genome of 149,732 bases. Its GC ratio is 50.7%. Sequence analysis revealed that PBES 02 has 299 open reading frames (ORFs) and 14 tRNA genes. Thirty-nine ORFs were annotated, including 24 related to replication and regulation functions, 10 related to structural proteins, and 5 related to DNA packaging. The genome of PBES 02 is closely related to that of two other C. sakazakii phages, CR3 and CR8. Comparison of DNA sequences of genes encoding the major capsid protein revealed a wide geographical distribution of related phages over Asia, Europe, and America.

Introduction

Bacteria belonging to the genus Cronobacter, formerly known as Enterobacter sakazakii, are gram-negative pathogens with increasing clinical importance [9]. Neonates and infants could suffer from infections by these opportunistic pathogens [20]. Powdered infant formula (PIF) is the major source of such infections. Bacterial contamination can occur at any point from the manufacturing process to reconstitution of PIF, and also during improper storage of reconstituted products [11]. Enterocolitis, meningitis, and bacteremia are major symptoms of Cronobacter infection. Fatality rates reach as high as 80% and infections among immunocompromised and elderly adults have also been reported [7].

Bacteriophages are viruses infecting bacterial hosts. The antimicrobial properties of bacteriophages are suitable for application of phages as alternatives to antibiotics. Phage therapy with bacteriophages has been used since the discovery of phages and was recently revitalized owing to the increasing appearance of antibiotic-resistant pathogenic bacteria [3,17,22].

Bacteriophages infecting Cronobacter sakazakii have been previously reported, including GAP32 [1], CR3 [19], and ESP2949-1 [15]. In vitro application of bacteriophages for Cronobacter infection has been described [12,24]. The in vivo therapeutic potential of phages for Cronobacter infection was evaluated in an animal model system using mice [21] and Galleria mellonella [2].

In this report, we describe the isolation and characterization of a new phage, PBES 02, infecting Cronobacter sakazakii.

 

Materials and Methods

Isolation and Propagation of Phage

The bacteriophage was isolated from a wastewater treatment facility located in Gwangju, Gyung-gi-Do, Korea. First, 300 μl of wastewater was mixed with 300 μl of freshly grown bacteria and melt soft agar, and then poured onto an agar plate. A standard double-layer agar technique [14] was used to obtain a single plaque. The host bacterium used for phage isolation was Cronobacter sakazakii (ATCC 29544), grown at 37℃. For the concentration of phage, glycerol step gradient centrifugation was employed [18].

Host Range Test

To test the infection specificity of the phage, the following Enterobacter strains were obtained from the Korea Culture Type Collection (KCTC): Enterobacter cloacae (KCTC 1685), Enterobacter cloacae subsp. cloacae (KCTC2361), Enterobacter aerogenes (KCTC 2190), Enterobacter mori (KCTC 23898), Enterobacter asburiae (KCTC 23920), Enterobacter siamensis (KCTC 23282), and Enterobacter ludwigii (KCTC 23921).

Transmission Electron Microscopy

A phage solution containing 1010 PFU was added to a formvar carbon film (200 mesh copper grids) and negatively stained with 2% uranyl acetate. Morphology was observed under a LIBRA 120 energy-filtering transmission electron microscope (Carl Zeiss).

Whole Genome Sequencing

Phage DNA was isolated as previously described [16]. Genomic DNA was isolated and subjected to next-generation sequencing using Illumina MiSeq (LAS, Korea). Assembly was performed using CLC Genomics Workbench. A tRNA search was done using tRNAscan-SE 1.21 (http://lowelab.ucsc.edu/tRNA scan-SE1), and ORF prediction was performed using Glimmer3 version 3.02.

Genomic Tree Construction

Construction of a comparative genomic tree was performed using the Molecular Evolutionary Genetics Analysis ver. 6 tool (http://www.megasoftware.net) based on the maximum likelihood method.

One-Step Growth

Ten milliliters of C. sakazakii grown to mid-exponential phase at 37℃ was infected with phage at an MOI of 0.1. Phage adsorption was allowed to progress in the mixture for 5 min, followed by centrifugation at 11,000 ×g for 5 min. The supernatant was discarded and the pellet was resuspended in 20 ml of fresh LB broth and further incubated. Two 100 μl cultures were obtained at every 5 min and chloroform (1% (v/v)) was added to one of the samples. The phage titer was measured using a standard doubleagar overlay plaque assay method. Burst size was calculated as phage out/phage in [5].

Temperature Stability of Phage for Infectivity

Phages in SM buffer were incubated at 4℃, 37℃, 45℃, 55℃, 65℃, or 80℃ for 1 h prior to infection. The infectious phage titer was checked using plaque assays.

pH Stability of Phage for Infectivity

Buffers (2 M sodium acetate at pH 3, pH 4, pH 5, or pH 6; 2 M Tris-Cl pH 7, pH 8, or pH 9; and 2M glycine, pH 10 or pH 11) were prepared. One milliliter of phage solution containing 4 × 106 PFU was mixed with the same volume of each buffer and the mixture was incubated at room temperature for 1 h prior to infection. The infectious phage titer was checked using plaque assays.

Biocontrol in a Broth Medium

Premium infant goat formula stage 1 powder (2.8 g) (IlDong Foodies, Korea) was dissolved in 20 ml of H2O. Cronobacter sakazakii (KCTC 2949) was inoculated at a concentration of 102 CFU/ml followed by addition of phage PBES 02 at the MOI of either 104 or 105. The mixture was incubated at 37℃ for 10 h. Bacteria were counted at every 2 h.

 

Results and Discussion

Purified bacteriophage PBES 02 was observed under a transmission electron microscope (Fig. 1). It belongs to the family Myoviridae of the order Caudovirales, possessing a spherical head of 90 nm in diameter, a rigid tail of 130 nm in length, and tail fibers.

Fig. 1.Transmission electron microscope image of bacteriophage PBES 02.

When the purified virion was subjected to SDS-PAGE analysis, at least four different virion proteins were observed after Coomassie blue staining (Fig. 2A). The 38 kDa protein band was seen as the most prominent of all, suggesting it is the major virion protein. An open reading frame (ORF) from nucleotides 59539 to 60537 (Table 1 and GenBank protein ID AKY04013.1, see below) of genomic DNA from PBES 02 was annotated as the gene encoding the major capsid protein. The predicted polypeptide is 332-aminoacids long and the size correlates well with the prominent protein band observed from SDS-PAGE. It also correlates well with mass spectrometry analysis (data not shown). The ProtParam-calculated molecular mass of this protein is 37.6 kDa (http://web.expasy.org/cgi-bin/protparam/protparam).

Fig. 2.Characterization of phage PBES 02. (A) SDS-PAGE analysis of virion proteins of phage PBES 02. (B) One-step growth of PBES 02 in 37℃ at the MOI of 0.1. (C) Stability of PBES 02 after exposure to various temperatures. (D) Stability of PBES 02 after exposure to various pHs. (E) Elimination of C. sakazakii from broth infant formula with the addition of phage PBES 02 at two different MOIs.

Table 1.Annotation of ORFs found in genomic DNA of PBES 02.

One-step growth of PBES 02 revealed that it has a latent period of 30 min and the complete burst took 105 min post infection (Fig. 2B). The burst size was 250.

Phages are exposed to various environmental conditions outside the laboratory. In case one intends to use this phage for therapeutic purposes, its stability under various conditions needs to be tested. We performed a phage stability test in a wide range of temperatures and pHs (Figs. 2C and 2D). Infectivity remained intact after exposure to temperatures ranging from 4℃ to 55℃ for 1 h. Infectivity decreased sharply at temperatures above 65℃ for 1 h. Seventy-five percent of the infectivity was lost after exposure to 65℃ for 1 h. Considering the fact that infant formula is rarely exposed to this temperature after production, phage PBES 02 should be suitable for application as an antibacterial agent. Infectivity also remained intact after exposure to pHs ranging from 6 to 10. Structural proteins comprising the capsid and tail seemed resistant to mildly alkaline conditions rather than acidic conditions. A sharp decrease in infectivity was observed at pHs lower than 5. Infectivity decreased as exposure time was extended to 8 h in both temperature and pH variations. When Cronobacter sakazakii was grown in a broth of infant formula, the addition of PBES 02 at the MOI of 104 inhibited bacterial growth and the addition of the phage at the MOI of 105 completely eliminated the bacteria in 6 h (Fig. 2E).

The genomic nucleic acid is a double-stranded DNA composed of 149,732 bases (GenBank Accession No. KT353109.1). Its GC ratio is 50.7%. Sequence analysis revealed that it has 299 ORFs and 14 tRNA genes. Thirty-nine ORFs were annotated, including 24 related to regulatory and replication functions, 10 related to structural proteins, and 5 related to DNA packaging (Table 1 and Fig. 3A). The most closely related phage found in GenBank was CR3 (Accession No. JQ691612.1). BLAST analysis revealed that the two phages have 97% identity from 91% coverage in nucleotide sequences. Phage CR3 [19] has a genomic DNA of 149,273 bases in length, and 265 ORFs and 18 tRNAs were identified. PBES 02 has a longer genomic DNA than CR3 by 459 base pairs, and it has 34 more ORFs and 4 less tRNA genes than CR3. Since CR3 was never characterized biologically [19], comparison other than those based on genomic analysis is impossible at this time. Comparison of the genomes of the two phages is shown in Fig. 3B. The length of the genomes, similarity, and numbers of ORFs and tRNA genes suggest that the two phages could have a common ancestor.

Fig. 3.Genomic analysis of PBES 02. (A) Schematic representation of the dsDNA genome of phage PBES 02. Putative ORFs are represented by arrows, with predicted functions where available. Proposed modules are based on predicted functions. White: hypothetical protein; black: replication and regulation; grey: structural protein; red: tRNA; blue: DNA packaging. (B) MAUVE analysis of genomic DNAs from phage PBES 02 (upper) and CR3 (lower).

The presence of tRNA genes in the genome of this phage is not surprising since tRNA genes have been reported from phages infecting Salmonella [13], Celeribacter [23], and Campylobacter [10], to name a few. Although production of phage tRNA has not been confirmed in these cases, one can logically think of advantages for phage production. With the high-speed production of phage tRNAs with extra supply of tRNAs in a lytic cycle, it can accelerate phage protein translation. In case phage tRNAs have better affinity for tRNA synthetases, it can give priority for phage protein translation over host protein translation, leading to very efficient production of phages. In case the codon usage for phage protein is different from that of the host, phage-encoded tRNAs can also be advantageous.

We performed a genomic tree analysis using genes encoding the major capsid protein from all Cronobacter phage genomic DNA sequences found in GenBank (Fig. 4). We were able to find complete genome sequences of 14 different Cronobacter phages. In terms of genome size, there were three classes: small size (28–41 kbp), medium size (149–223 kbp), and large size (358 kbp). Interestingly, genes encoding major capsid proteins were conserved to some extent among phages across different classes (e.g., small ENT39118 and medium S13, medium vB CsaM GAP161 and small mEp235). It may implicate mosaic genomic architectures of related phages generated by illegitimate recombination over a long period of evolutionary history [6]. In other words, the coding requirements for the virion structure and assembly genes are similar for these related phages, but the size of the nonstructural genome segments is variable. The phages have been reported from four different locations in the world: Switzerland, England, Canada, and Korea. We found that closely related phages in terms of genes encoding major capsid protein were isolated from different regions of the world (e.g., vB CsaM GAP161 from Canada, mEp235 from Switzerland, and CR5 from Korea are closely related). Although the sample size for comparing genomic DNA sequences was not large enough, we would like to conclude that distribution of these viruses is not geographically limited. Somehow, the viruses and perhaps the host bacteria traveled along human paths and/or naturally translocated across oceans.

Fig. 4.Genomic tree comparing major capsid proteins from 14 different phages infecting Cronobacter sakazakii. A genomic tree was constructed using the bootstrap test of phylogeny using MEGA 6. ENT39118 (isolated from Korea, with a 39 kbp genome), S13 (Korea, 182 kbp), CR5 (Korea, 223 kbp), vB CsaM GAP161 (Canada, 178 kbp), mEP235 (Swiss, 37 kbp), vB CsaM Gap32 (Canada, 358 kbp), vB CskP GAP227 (Canada, 41 kbp), phiES15 (Korea, 39 kbp), vB CsaM GAP31 (Canada, 147 kbp), ESSI-2 (Korea, 28 kbp), CR9 (Korea, 151 kbp), CR3 (Korea, 149 kbp), and CR8 (England, 149 kbp) were the phages used for comparison.

For biocontrol purposes, phage specificity needed to be tested. We tested eight different Enterobacter species as host bacteria for phage PBES 02 (Table 2). None of them were susceptible to the phage infection, suggesting PBES 02 would be a suitable control agent for Cronobacter sakazakii infection.

Table 2.Host-range test for phage PBES 02.

Phage PBES 02 may be a good candidate for application as an antibacterial in infant formulas, since addition of antibiotics in an infant formula would not be accepted in the era of rapidly growing antimicrobial-resistant microorganisms. Its stability at temperatures up to 65℃ makes it especially suitable considering the shelf life of the product at room temperature. As is true for other phage applications [4,7], a cocktail of different phages would be more practical for diminishing the appearance of resistant mutant Cronobacter sakazakii.