Large-Scale Production of Cronobacter sakazakii Bacteriophage Φ CS01 in Bioreactors via a Two-Stage Self-Cycling Process.

Cronobacter sakazakii is an opportunistic pathogenic bacterium found in powdered infant formula and is fatal to neonates. Antibiotic resistance has emerged owing to overuse of antibiotics. Therefore, demand for high-yield bacteriophages as an alternative to antibiotics has increased. Accordingly, we developed a modified mass-production method for bacteriophages by introducing a two-stage self-cycling (TSSC) process, which yielded high-concentration bacteriophage solutions by replenishing the nutritional medium at the beginning of each process, without additional challenge. pH of the culture medium was monitored in real-time during C. sakazakii growth and bacteriophage CS01 propagation, and the changes in various parameters were assessed. The pH of the culture medium dropped to 5.8 when the host bacteria reached the early log phase (OD540 = 0.3). After challenge, it decreased to 4.65 and then recovered to 4.94; therefore, we set the optimum pH to challenge the phage at 5.8 and that to harvest the phage at 4.94. We then compared phage production during the TSSC process in jar-type bioreactors and the batch culture process in shaker flasks. In the same volume of LB medium, the concentration of the phage titer solution obtained with the TSSC process was 24 times higher than that obtained with the batch culture process. Moreover, we stably obtained high concentrations of bacteriophage solutions for three cycles with the TSSC process. Overall, this modified TSSC process could simplify large-scale production of bacteriophage CS01 and reduce the unit cost of phage titer solution. These results could contribute to curing infants infected with antibiotic-resistant C. sakazakii.

Phage S144, A New Polyvalent Phage Infecting Salmonella spp. and Cronobacter sakazakii.

Phages are generally considered species- or even strain-specific, yet polyvalent phages are able to infect bacteria from different genera. Here, we characterize the novel polyvalent phage S144, a member of the Loughboroughvirus genus. By screening 211 Enterobacteriaceae strains, we found that phage S144 forms plaques on specific serovars of Salmonella enterica subsp. enterica and on Cronobacter sakazakii. Analysis of phage resistant mutants suggests that the O-antigen of lipopolysaccharide is the phage receptor in both bacterial genera. The S144 genome consists of 53,628 bp and encodes 80 open reading frames (ORFs), but no tRNA genes. In total, 32 ORFs coding for structural proteins were confirmed by ESI-MS/MS analysis, whereas 45 gene products were functionally annotated within DNA metabolism, packaging, nucleotide biosynthesis and phage morphogenesis. Transmission electron microscopy showed that phage S144 is a myovirus, with a prolate head and short tail fibers. The putative S144 tail fiber structure is, overall, similar to the tail fiber of phage Mu and the C-terminus shows amino acid similarity to tail fibers of otherwise unrelated phages infecting Cronobacter. Since all phages in the Loughboroughvirus genus encode tail fibers similar to S144, we suggest that phages in this genus infect Cronobacter sakazakii and are polyvalent.

Characterization and Genomic Analysis of Novel Bacteriophage ΦCS01 Targeting Cronobacter sakazakii.

Cronobacter sakazakii is an opportunistic pathogen causing serious infections in neonates. In this study, a bacteriophage ΦCS01, which infects C. sakazakii, was isolated from swine feces and its morphology, growth parameters, and genomic analysis were investigated. Transmission electron microscopy revealed that ΦCS01 has a spherical head and is 65.74 nm in diameter with a 98.75 nm contracted tail, suggesting that it belongs to the family Myoviridae. The major viral proteins are approximately 71 kDa and 64 kDa in size. The latent period of ΦCS01 was shown to be 60 min, and the burst size was 90.7 pfu (plaque-forming units)/ infected cell. Bacteriophage ΦCS01was stable at 4-60°C for 1 h and lost infectivity after 1 h of heating at 70°C. Infectivity remained unaffected at pH 4-9 for 2 h, while the bacteriophage was inactivated at pH <3 or >10. The double-stranded ΦCS01 DNA genome consists of 48,195 base pairs, with 75 predicted open reading frames. Phylogenetic analysis is closely related to that of the previously reported C. sakazakii phage ESP2949-1. The newly isolated ΦCS01 shows infectivity in the host bacterium C. sakazakii, indicating that it may be a promising alternative to antibacterial agents for the removal of C. sakazakii from powdered infant formulas.

Investigating the biocontrol and anti-biofilm potential of a three phage cocktail against Cronobacter sakazakii in different brands of infant formula.

In recent years, the microbiological safety of powdered infant formula has gained increasing attention due to the identification of contaminating C. sakazakii and its epidemiological link with life-threatening neonatal infections. Current intervention strategies have fallen short of ensuring the production of infant formula that is free from C. sakazakii. In this study, we describe the isolation and characterisation of three bacteriophages (phages) and their application as a phage cocktail to inhibit the growth of C. sakazakii in different brands of infant formula, while also assessing the phages ability to prevent biofilm formation. All three phages, isolated from slurry, possess a relatively broad host range, verified by their ability to infect across genera and species. When all three phages were combined and used as part of a phage cocktail, 73% coverage was obtained across all Cronobacter strains tested. Optimum thermo-tolerance and pH stability were determined between 4°C-37°C, and pH6-8, respectively, well within the normal range of application of infant formula. Genome sequencing and analysis revealed all the phages to be free from lysogenic properties, a trait which renders each favourable for phage therapy applications. As such, the combined-phage preparation (3×108pfu/mL) was found to possess a strong bactericidal effect on C. sakazakii/C. sakazakii LUX cells (≤104cfu/mL), resulting in a significant reduction in cell numbers, to below the limit of detection (<10cfu/mL). This was observed following a 20h challenge in different brands of infant formula, where samples in the absence of the phage cocktail reached concentrations of ~109cfu/mL. The phage cocktail also demonstrated promise in preventing the establishment of biofilm, as biofilm formation could not be detected for up to 48h post treatment. These results highlight the potential application of this phage preparation for biocontrol of C. sakazakii contamination in reconstituted infant formula and also as a preventative agent against biofilm formation.

Inhibition of Cronobacter sakazakii Virulence Factors by Citral.

Cronobacter sakazakii is a foodborne pathogen associated with fatal forms of necrotizing enterocolitis, meningitis and sepsis in neonates and infants. The aim of this study was to determine whether citral, a major component of lemongrass oil, could suppress putative virulence factors of C. sakazakii that contribute to infection. Sub-inhibitory concentrations of citral significantly decreased motility, quorum sensing, biofilm formation and endotoxin production. Citral substantially reduced the adhesion and invasion of C. sakazakii to Caco-2 cells and decreased bacterial survival and replication within the RAW 264.7 macrophage cells. Citral also repressed the expression of eighteen genes involved in the virulence. These findings suggest that citral has potential to be developed as an alternative or supplemental agent to mitigate the infections caused by C. sakazakii.

A Newly Isolated Bacteriophage, PBES 02, Infecting Cronobacter sakazakii.

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°C to 55°C 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.

A Novel Bacteriophage Targeting Cronobacter sakazakii Is a Potential Biocontrol Agent in Foods.

Cronobacter sakazakii is an important pathogen that causes high mortality in infants. Due to its occasional antibiotic resistance, a bacteriophage approach might be an alternative effective method for the control of this pathogen. To develop a novel biocontrol agent using bacteriophages, the C. sakazakii-infecting phage CR5 was newly isolated and characterized. Interestingly, this phage exhibited efficient and relatively durable host lysis activity. In addition, a specific gene knockout study and subsequent complementation experiment revealed that this phage infected the host strain using the bacterial flagella. The complete genome sequence analysis of phage CR5 showed that its genome contains 223,989 bp of DNA, including 231 predicted open reading frames (ORFs), and it has a G+C content of 50.06%. The annotated ORFs were classified into six functional groups (structure, packaging, host lysis, DNA manipulation, transcription, and additional functions); no gene was found to be related to virulence or toxin or lysogen formation, but >80% of the predicted ORFs are unknown. In addition, a phage proteomic analysis using SDS-PAGE and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) revealed that seven phage structural proteins are indeed present, supporting the ORF predictions. To verify the potential of this phage as a biocontrol agent against C. sakazakii, it was added to infant formula milk contaminated with a C. sakazakii clinical isolate or food isolate, revealing complete growth inhibition of the isolates by the addition of phage CR5 when the multiplicity of infection (MOI) was 10(5).

Supersize me: Cronobacter sakazakii phage GAP32.

Cronobacter sakazakii is a Gram-negative pathogen found in milk-based formulae that causes infant meningitis. Bacteriophages have been proposed to control bacterial pathogens; however, comprehensive knowledge about a phage is required to ensure its safety before clinical application. We have characterized C. sakazakii phage vB_CsaM_GAP32 (GAP32), which possesses the second largest sequenced phage genome (358,663bp). A total of 571 genes including 545 protein coding sequences and 26 tRNAs were identified, thus more genes than in the smallest bacterium, Mycoplasma genitalium G37. BLASTP and HHpred searches, together with proteomic analyses reveal that only 23.9% of the putative proteins have defined functions. Some of the unique features of this phage include: a chromosome condensation protein, two copies of the large subunit terminase, a predicted signal-arrest-release lysin; and an RpoD-like protein, which is possibly involved in the switch from immediate early to delayed early transcription. Its closest relatives are all extremely large myoviruses, namely coliphage PBECO4 and Klebsiella phage vB_KleM-RaK2, with whom it shares approximately 44% homologous proteins. Since the homologs are not evenly distributed, we propose that these three phages belong to a new subfamily.

Efficiency of bacteriophage therapy against Cronobacter sakazakii in Galleria mellonella (greater wax moth) larvae.

Cronobacter sakazakii, an opportunistic pathogen found in milk-based powdered infant formulae, has been linked to meningitis in infants, with high fatality rates. A set of phages from various environments were purified and tested in vitro against strains of C. sakazakii. Based on host range and lytic activity, the T4-like phage vB_CsaM_GAP161, which belongs to the family Myoviridae, was selected for evaluation of its efficacy against C. sakazakii. Galleria mellonella larvae were used as a whole-animal model for pre-clinical testing of phage efficiency. Twenty-one Cronobacter strains were evaluated for lethality in G. mellonella larvae. Different strains of C. sakazakii caused 0 to 98% mortality. C. sakazakii 3253, with an LD50 dose of ~2.0×10(5) CFU/larva (24 h, 37 °C) was selected for this study. Larvae infected with a dose of 5×LD50 were treated with phage GAP161 (MOI=8) at various time intervals. The mortality rates were as high as 100% in the groups injected with bacteria only, compared to 16.6% in the group infected with bacteria and treated with phage. Phage GAP161 showed the best protective activity against C. sakazakii when the larvae were treated prior to or immediately after infection. The results obtained with heat-inactivated phage proved that the survival of the larvae is not due to host immune stimulation. These results suggest that phage GAP161 is potentially a useful control agent against C. sakazakii. In addition, G. mellonella may be a useful whole-animal model for pre-screening phages for efficacy and safety prior to clinical evaluation in mammalian models.

The immune-enhancing effect of the Cronobacter sakazakii ES2 phage results in the activation of nuclear factor-κB and dendritic cell maturation via the activation of IL-12p40 in the mouse bone marrow.

The bacteriophage ES2 is a virus for bacterial host cells. Unlike other phages that are known for their therapeutic effects, the ES2 phage has never been clearly examined as a therapeutic agent. To systematically and conclusively evaluate its therapeutic efficacy, the expression of the surface markers CD86, CD40, and MHCII, the production of the proinflammatory cytokines IL-6, IL-1α, IL-1ß, and TNF-α, and the underlying NF-κB signaling pathway in murine bone marrow-derived dendritic cells (BM-DCs) in response to ES2 phage infection were examined. The bacteriophage ES2, which was isolated from swine fecal samples an antigen, affected the expression of the cell surface molecules and proinflammatory cytokines that are associated with the DC maturation processes. Treatment with ES2 phage also led to NF-κBp65 activation and translocation to the nucleus, which indicates the activation of NF-κB signaling. Furthermore, the ES2 phage induced the promoter activity of IL-12p40. Our chromatin immunoprecipitation assay revealed that p65 was enriched at the IL12-p40 promoter as a direct target of chromatin. The present study demonstrates that the ES2 phage potently induces DC maturation via immune-enhancement processes.

Complete genome sequence of Cronobacter sakazakii bacteriophage vB_CsaM_GAP161.

Cronobacter sakazakii is an opportunistic pathogen that causes infant meningitis and is often associated with milk-based infant formula. We have fully sequenced the genome of a newly isolated lytic C. sakazakii myovirus, vB_CsaM_GAP161, briefly named GAP161. It consists of 178,193 bp and has a G+C content of 44.5%. A total of 277 genes, including 275 open reading frames and two tRNA-encoding genes, were identified. This phage is closely related to coliphages RB16 and RB43 and Klebsiella pneumoniae phage KP15.

Genome sequence of Cronobacter sakazakii myovirus vB_CsaM_GAP31.

Cronobacter sakazakii is a pathogen that predominantly infects immunocompromised individuals, especially infants, where it causes meningitis. The genome of lytic C. sakazakii myovirus vB_CsaM_GAP31 has been fully sequenced. It consists of 147,940 bp and has a G+C content of 46.3%. A total of 295 genes, including 269 open reading frames and 26 tRNA genes, were identified. This phage is related to Salmonella phage PVP-SE1 and coliphages vB_EcoM-FV3 and rV5.

Complete genome sequence of Cronobacter sakazakii temperate bacteriophage phiES15.

While most phage genome studies have been focused on the virulent phages, the inducible temperate bacteriophage genome study provides more detailed information about the interaction between the host strain and the phage. To study this interaction in detail, UV-induced phiES15 bacteriophage was isolated from the host strain Cronobacter sakazakii ES15 and its genome was completely sequenced. Here we announce the genome sequence of phiES15 and report major findings from the annotation.

Complete genome sequence of Cronobacter sakazakii bacteriophage CR3.

Due to the high risk of Cronobacter sakazakii infection in infants fed powdered milk formula and the emergence of antibiotic-resistant strains, an alternative biocontrol agent using bacteriophage is needed to control this pathogen. To further the development of such an agent, the C. sakazakii-targeting bacteriophage CR3 was isolated and its genome was completely sequenced. Here, we announce the genomic analysis results of the largest C. sakazakii phage known to date and report the major findings from the genome annotation.

Complete genome of temperate phage ENT39118 from Cronobacter sakazakii.

Cronobacter sakazakii infection is particularly harmful to infants, and putative virulence factors of prophage origin have been identified in C. sakazakii. In this study, the phage ENT39118 was isolated from wild-type C. sakazakii; it belongs to the family Siphoviridae. The genomic sequence of phage ENT39118 was composed of circular double-stranded DNA with a length of 39,012 bp. The sequence of ENT39118 showed weak sequence similarity to some reported regions of the prophage sequences in the C. sakazakii BAA-894 genome. To our knowledge, this is the first study of the genomic sequencing and annotation of this temperate phage, which was obtained from a C. sakazakii isolate from powdered infant formula.

Genomic analysis of bacteriophage ESP2949-1, which is virulent for Cronobacter sakazakii.

Virulent phage ESP2949-1, which was isolated from sewage, has an icosahedral head, a contractile tail, and a double-stranded DNA genome with a length of 49,116 bp with 50.09% G+C content. Phage ESP2949-1 showed 3% similarity to enterobacteria phage TLS. Bioinformatics analysis of the phage genome revealed 43 putative open reading frames (ORFs). Predicted protein products of the ORFs were determined and described. Based on its morphology, phage ESP2949-1 can be classified as a member of the family Myoviridae. To our knowledge, this is the first report of the genomic sequence and characterization of phage ESP2949-1 isolated from sewage.

Genomic sequence analysis of virulent Cronobacter sakazakii bacteriophage ES2.

Virulent Cronobacter sakazakii bacteriophage ES2 was isolated from swine fecal samples, and the genome sequence by was determined GS-Flx. Bacteriophage ES2 had a double-stranded DNA genome with a length of 22,162 bp and a G+C content of 50.08%. The morphological characteristics under a transmission electron microscope indicated that bacteriophage ES2 belongs to the family Myoviridae. The structural proteins, including the phage coat protein, were separated by SDS-PAGE and identified by Q-TOF. Bioinformatics analysis of the bacteriophage genome revealed 30 putative open reading frames (ORFs). The predicted protein products of the ORFs were determined and described. To our knowledge, the genome of the newly isolated bacteriophage ES2 was not significantly similar to that of any previously reported bacteriophages of members of the family Enterobacteriaceae.

[Isolation and characterization of bacteriophages of Enterobacter sakazakii].

OBJECTIVE: To isolate bacteriophage of Enterobacter sakazaki from sewage using reference and isolated strains as indicators, and to observe the biological characteristics of the bacteriophage. METHODS: Bacteriophages were isolated from sewage with double layer agar. Specificity and host ranges of the bacteriophages were determined by reference bacterium from same genus and family. Phage particles were observed by electron microscope and its molecular character was analyzed by Random amplified polymorphic DNA. RESULTS: Five bacteriophages of E. sakazakii were isolated from sewage and showed relatively narrow host ranges, only E. sakazakii could be lysised. The phage SK2 isolated from ATCC 51329 could form plaques on 24 of all 27 E. sakazakii strains (89%). All five phage particles had the hexagonal heads and tails after observing with negatively stained method by electron microscope. Random amplified polymorphic DNA analysis showed the polymorphism of the five phages. CONCLUSION: E. sakazakii phages isolated from sewage were only sensitive to E. sakazakii, and had potential usage in typing, preventing, treating E. sakazakii and entironment protection.

Decreasing Enterobacter sakazakii (Cronobacter spp.) food contamination level with bacteriophages: prospects and problems.

Enterobacter sakazakii (Cronobacter spp.) is an opportunistic pathogen, which can cause rare, but life-threatening infections in neonates and infants through feeding of a contaminated milk formula. We isolated 67 phages from environmental samples and tested their lytic host range on a representative collection of 40 E. sakazakii strains. A cocktail of five phages prevented the outgrowth of 35 out of 40 test strains in artificially contaminated infant formula. Two E. sakazakii phages represented prolate head Myoviridae. Molecular tests identified them as close relatives of Escherichia coli phage T4. The remaining three phages represented isometric head Myoviridae with large genome size of 140 and 200 kb, respectively, which belonged to two different DNA hybridization groups. A high dose of 10(8) pfu ml(-1) of phage could effectively sterilize a broth contaminated with both high and low pathogen counts (10(6) and 10(2) cfu ml(-1)). In contrast, broth inoculated with 10(4) phage and 10(2) bacteria per ml first showed normal bacterial growth until reaching a cell titre of 10(5) cfu ml(-1). Only when crossing this threshold, phage replication started, but it could not reduce the contamination level below 100 cfu ml(-1). Phages could be produced with titres of 10(10) pfu ml(-1) in broth culture, but they were not stable upon freeze-drying. Addition of trehalose or milk formula stabilized the phage preparation, which then showed excellent storage stability even at elevated temperature.