Introduction

Salmonella enteritidis (S. enteritidis), a gram-negative bacterium, is a common cause of human illnesses1. S. enteritidis is the most widespread serotype in China and other countries2. Recently, antibiotics are still the most commonly used to treat bacterial infections. However, multidrug-resistant strains of S. enteritidis are increasing year by year, especially the carbapenem resistant isolates3. As a result, the speed of new antibiotics supply is unable to keep up with that of the bacterial resistance increase. One of the reasons for antibiotic resistance is the formation of bacterial biofilm4. Once the bacterial biofilms formed, they protected bacteria from the body’s immune system, antibiotics, physical and chemical factors. Therefore, there is a need to develop new, efficient, environmental therapeutic strategies to combat S. enteritidis biofilms.

Bacteriophages (phages) are viruses of bacteria. They are widely distributed not only in the natural environment1, but also in the gastrointestinal tracts of both humans and animals5. In recent decades, phages have been reconsidered to combat multidrug-resistant bacteria infections due to their high efficacy, specificity, and safety profiles6,7,8. In addition, some phages have the ability to deal with bacterial biofilms. Among these phage genome sequence, there was at least one gene encoded depolymerase. More and more studies have shown that phage depolymerases are able to degrade bacterial extracellular polysaccharide. For instance, a novel depolymerase Dep37 derived from Klebsiella pneumoniae (K. pneumoniae) ST11 K64 CRKP phage vB_KpnP_IME1309 was reported. Dep37 not only inhibited CRKP biofilm formation, but also degraded mature biofilms9. In the mouse sepsis model, the Acinetobacter baumannii phage depolymerase DepAPK09 can protect 100% of the mice from a lethal dose of Acinetobacter baumannii (A. baumannii) infection10. The phage depolymerase Dpo36 of Salmonella abortus equi phage 4FS1 is able to reduce biofilm formation and removal significantly11. At present, researches on phage depolymerases mainly focuses on K. pneumoniae and A. baumannii phage depolymerases. Only a few S. enteritidis phage depolymerases have been studied. We want to know whether S. enteritidis phage depolymerases could both inhibit biofilm formation and degrade mature biofilms or not, just like Dep379.

In this study, we isolated a carbapenem-resistant S. enteritidis phage vB_Sen_S3P. And then we conducted whole genome sequencing and bioinformatic analysis to find out the depolymerase specific gene responsible for combating bacterial biofilms. The depolymerase Dpo52 was expressed and the biological characteristics were tested. Our study showed for the first time that the S. enteritidis phage depolymerase Dpo52 inhibited the biofilm formation of a carbapenem-resistant S. enteritidis. Furthermore, Dpo52 was stable in a wide temperature and pH range and non-cytotoxic to macrophages. Once we had also found a Escherichia coli phage depolymerase Dpo42, and we wanted to compare the two phage depolymerases.

Methods and materials

Ethics approval statement

The study was approved by the ethics committee of the First Hospital of Jilin University and all participants had signed an informed consent form. All methods were carried out in accordance with relevant guidelines and regulations. The sample collection procedures were conducted in accordance with the principles stated in the Guidelines for Collection, Transport, Receipt and Handling of Medical Laboratory Samples (GB/T 42060–2022).

Bacterial strains and drug resistance

The S. enteritidis isolates were isolated from patients at the First Hospital of Jilin University (Changchun, Jilin province, China). All bacteria strains were cultured in LB medium (LB, BioCorp, Warszawa, Poland) and were stored at -80℃ in LB broth contained with 20% v/v glycerol. The antibiotic susceptibility of the S. enteritidis isolates was evaluated using the VITEK® 2 Compact system (bioMérieux, Boston, USA). MICs results was interpreted according of the Clinical and Laboratory Standards Institute (CLSI) M100 manual12. We used Tryptic soy broth medium (TSB, bioMérieux, Marcy-l’Étoile, France) to culture bacterial biofilms. We also tested all strains to determine the host range of the phage and to obtain the antibiofilms activity of depolymerase.

Isolation and purification of phage particles

The phage named vB_Sen_S3P was isolated with clinical isolate S. enteritidis S3P as host bacteria. vB_Sen_S3P was separated from hospital sewage samples in Changchun, China. Phage isolation and purification were performed as described previously13,14. We used a sterile 0.22-µm filter (Millex-GP Filter Unit, Bedford, Massachusetts, USA, LOT: R6MA05262) to remove these impurities and microorganisms of 100-mL water sample. Then, purified water was replaced by the filtered solution to prepare LB liquid medium (tryptone 1%, yeast extract 0.5%, NaCl 1%) instead of purified water. Host S. enteritidis S3P cells were cultured to the logarithmic phase, added to LB liquid medium and mixed. The culture was centrifuged at 4 °C and 8,000 × g for 15 min after incubation at 37℃ for 18 h. The sterile 0.22-µm filter was used to filter the supernat. The double layer agar plate method was used to purify phages. The plates were cultured at 37℃ for 12 h. Phages were purified until the plaques were uniform in size. Phage lysates were centrifuged at 4 °C at 8,000 × g for 30 min. The Suspended Medium (SM) buffer (0.01% gelatin, 100 mmol/L NaCl, 50 mmol/L Tris-HCl, and 10 mmol/L MgSO4) was used to store purified phages at 4℃. Then, we added 1 µg/mL RNase A and DNase I (Sigma-Aldrich, St. Louis, Missouri, USA) into the phage supernatant, and the mixture was incubated at room temperature for 30 min. 1 mol/L NaCl was used to separate bacterial fragments and phage particles on ice and incubated for 1 h. The supernatant was then centrifuged at 4℃ and 10,000 × g for 10 min. To precipitate the phage particles, 10% (m/v) PEG 8000 was added to the supernatant and gently mixed on ice, followed by incubation for 3 h. Samples were centrifuged at 10,000 × g for 10 min, resuspended in SM buffer, extracted with chloroform to remove bacterial debris, and centrifuged at 4℃ and 8,000 g for 10 min.

Determination of multiplicity of infection (MOI)

The MOI was detected as described by Guo et al.14. Briefly, clinical isolate S. enteritidis S3P host cells were cultured to the logarithmic phase. Then, the culture was loaded into glass test tubes at 1 × 106 CFU/mL containing liquid LB medium. Finally, phages were added at MOIs of 10, 1, 10− 1, 10− 2, 10− 3, 10− 4, 10− 5, or 10− 6 (PFU/CFU). The bacteria and phage mixtures were incubated in a table concentrator at 180 rpm at 37℃ for 12 h. Phage titers in the filtrates were measured using the double layer agar plate method and repeated three times.

One-step growth curve assay

A one-step growth curve experiment of phages was constructed as described previously by Guo et al.14. , with minor modifications. S. enteritidis S3P host cells were grown to the mid-exponential phase. Phages were added at an MOI of 10− 5, and the mixture of log-phase bacteria and an MOI of 10− 5 phages was incubated at 4℃ for 15 min. The supernatant fluid was taken out after centrifuging at 12,000 × g for 1 min and resuspended with 10 mL of fresh LB liquid medium. Then the suspension was incubated at 37℃ at 180 rpm. Sampling was done at 0, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 120, and 150 min to test phage titration usedthe double layer agar plate method. The burst size was defined as the ratio of the average number of phage in final rise phase to the average number of phage in the latent period.

Thermal and pH stability of phages

To measure the thermal and pH stability of the phage, a modification of the previously described methods was used15. For temperature sensitivity testing, the phage suspensions were incubated at frozen temperature (-20℃), refrigeration temperature (4℃), room temperature (25℃), incubation temperature (37℃), and other temperatures (50, 60, 70℃), respectively. Then samples were collected 60 min later for the thermal stability test. For pH stability testing, the phage suspensions were incubated at 37℃ (pH 1–13) for 1 h. Then, phage titers were assessed by the double-layer agar plate method.

Host range of phage vB_Sen_S3P and phage depolymerase Dpo52

The host range of vB_Sen_S3P and Dpo52 were investigated against 30 Salmonella strains, E. coli ATCC 25,922, P.aeruginosa ATCC 27,853, K. pneumoniae ATCC 13,833, S.sonnei ATCC 25,931, S.aureus ATCC 29,213, S. epidermidis ATCC 26,096, and E. faecalis ATCC 29,212 through a spot test16. Overnight-cultured the above tested bacterial strains (200 µL, 108 CFU/mL) were coated on LB solid medium plates and allowed to dry. Then, phage suspension (5 µL, 108 PFU/mL) and phage depolymerase Dpo52 (0.1 µL, 1.95 mg/mL) were dropped onto each plate, and the plates were cultured at 37 °C for an additional 10 h to observe the formation of clear spots.

Transmission electron microscopy

Phages were concentrated and purified as previously described14. The concentrated phage was dropped onto a 200-mesh copper film on a slide and negatively stained with phosphotungstic acid (PTA, 2% wt/vol, pH = 7) for 10 min. The morphology of the phages was observed by a transmission electron microscope (TEM) (JEOL JEM-1200EXII, Japan Electronics and Optics Laboratory, Tokyo, Japan) at an acceleration voltage of 80 kV.

Extraction of phage genome

The method used to extract phage genomes has been reported previously17. Briefly, the concentrated phage was treated with DNase I (final concentration, 10 µg/mL) and RNase A (final concentration, 5 µg/mL) in SM buffer at 37℃ for 1 h. Then, EDTA was added (pH 8.0, final concentration 25 mmol/mL). Finally, a viral genome extraction kit (Omega Bio-Tek Inc., Doraville, GA, United States) was used to extract the phage genomic DNA. DNA concentration was determined through Qubit4.0 (Thermo Fisher Scientific Inc., Waltham, USA).

Sequencing and analysis of phage genome sequence

Whole-genome sequencing was performed by Sangon Biotech (Shanghai) Co., Ltd. Hieff NGS®MaxUp II DNA Library Prep Kit for Illumina® (YEASEN, Shanghai, China) was used to complete the library preparation. The libraries were sequenced through DNBseq-T7 (BGI, Shenzhen, China) sequencer by 2 × 150 bp paired end sequence kit according to the manufacture’s instructions. The sequences were assembled via SPAdes v3.15.2. GeneMarkS (Georgia Institute of Technology, Atlanta, GA, United States) was used to predicte the potential open reading frames (ORFs). PSI-BLAST (threshold = 0.0001) from the National Center for Biotechnology Information (NCBI) (https://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to annotate the functions of the encoded regions in vB_Sen_S3P. To improve genome annotations/predictions, all proteins encoded by ORFs in the genome sequence of vB_Sen_S3P were subjected to a BLASTP search against the phage protein database at NCBI (https://blast.ncbi.nlm.nih.gov/) and Pfam (http://pfam.xfam.org/family). The genome map of vB_Sen_S3P was drawn using CLC Main Workbench version 7.7.3 (CLC Bio-Qiagen, Aarhus, Denmark). The phylogenetic tree was constructed using BioEdit (7.0.4.1), MEGA 6, and PHYLIP software (version 3.697).

Phylogenetic tree analysis

The phylogenetic tree analysis of vB_Sen_S3P was performed based on terminase large subunit sequences using the Interactive Tree Of Life (iTOL) (https://itol.embl.de) as previously described18. The phylogenetic tree was generated using Parsnp with default settings, and the neighbor-joining (NJ) method with 1,000 bootstraps was employed to ensure robustness. The resulting unrooted phylogenetic tree was visualized using iTOL.

Analysis and expression of phage depolymerase

To determine the gene encoding the depolymerase, the protein structure was analyzed using Alfafold 3 (https://alphafoldserver.com/welcome) and PSIPRED (https://bioinf.cs.ucl.ac.uk/psipred/). The phylogenetic tree of Dpo52 was constructed based on the protein sequences for Dpo52 and 19 other phage proteins using BioEdit (7.0.4.1), MEGA 6, and PHYLIP software (version 3.697). ORF52, a putative phage depolymerase, was amplified from the purified phage vB_Sen_S3P by PCR using the specific primers Dpo52PF (5’-CGCGGATCCATGTCTAGTGGTTGCGG-3’) and Dpo52PR (5’-CGCCTCGAGTTATGCCAAAGTTAATCTTG-3’). Underlined nucleotides indicate the recognition sequences for BamHI and HindⅢ. The PCR fragment was excised with BamHI and HindⅢ. and inserted into the pET-28a expression vector (Novagen, Madison, Wisconsin, USA). The constructed plasmid was transformed into Escherichia coli BL21 cells. An exponentially growing culture (OD600 = 0.6) was induced with 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 16℃ overnight. The cultures were collected by centrifugation at 8,000 × g and 4℃ for 20 min. The cells were then collected and suspended in lysis buffer (500 mM NaCl, 50 mM Na2HPO4, 10 mM KH2PO4, and 2.7 mM KCl; pH 7.4) and disrupted with an ultrasonic disintegrator. The supernatant was obtained by centrifugation at 12,000 × g for 15 min at 4℃. The purified recombinant protein Dpo52 was purified from the soluble fraction using a Ni-NTA column (Genscript, Nanjing, China) according to the manufacturer’s instructions. The purified recombinant protein Dpo52 was placed in dialysis tubing (Viskase, MD34, Viskase Companies, Inc., Brazil) against PBS buffer overnight. And then, the protein was analyzed by performing 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purified recombinant protein was quickly frozen in liquid nitrogen and stored at − 80℃.

Cytotoxicity test of Dpo52

Human monocytic leukemia THP-1 cells were grown in Dulbecco’s modified Eagle medium (DMEM) medium supplemented with 10% heat-inactivated fetal bovine serum (FBS). 5 × 103 THP-1 cells were seeded into 24-well tissue culture plates, cultured for 4 h until the cells were adherent to the wall, 10 µL Dpo52 with different concentrations (0.1 µg/µL, 0.5 µg/µL, 1 µg/µL) was added to each well, and sterile PBS was used as a negative control. The experiments were performed in triplicate. Cell Counting Kit-8 (CCK-8) (Vazyme Biotech Co., Ltd., Nanjing, China) was used to detect the cytotoxicity of 24 h culture.

Biofilm assay

The activity of depolymerase Dpo52 was determined via a spot test as previously described with some modifications19. In brief, serial dilutions of Dpo52 (800, 400, 200, 100, 20, 2.0, 0.2, and 0.02 ng) were dropped on a bacterial lawn of S. enteritidis, and PBS was used as a negative control. The agar plates were incubated at 37 °C for 10 h before observation. The plates were incubated at 37 °C for 8 h before observation. The formation of a transparent zone halo on the bacterial lawn was a sign of the depolymerase activity of Dpo52.

The crystal violet staining assay was used to study the formation of biofim. The preventing effects of Dpo52 on biofilm formation were detected through S. enteritidis isolates S25 and S32 as described previously20. Briefly, S. enteritidis S25 and S32 were cultured to log phase, centrifuged, resuspended in equal amounts of TSB medium, mixed, and added to each well (100 µL) of 96-well micro-titre plates. These wells were then divided into seven groups: PBS (control), 1 ng/µL Dpo52, 2 ng/µL Dpo52, 4 ng/µL Dpo52, 6 ng/µL Dpo52, 8 ng/µL Dpo52, 16 ng/µL Dpo52, 32 ng/µL Dpo52 and 64 ng/µL Dpo52. Each group contained triplicate samples. The plate was covered, and bacteria were permitted to adhere and grow at 37℃ for 72 h without agitation. After incubation, the residual biofilms were stained, and the OD590 was measured.

Capsule staining

The capsule staining of S. enteritidis S3 was negatively stained with India ink solution and carbol fuchsin solution. Cells grown in the absence and presence of Dpo52 were transferred to 10 µL of 1% carbol fuchsin solution (Sigma-Aldrich, St. Louis, MO, USA) and mixed for 30 s. The mixture was spread across a glass slide to form a thin film and then air dried. Another 10 µL of India ink solution was dropped across the glass slide. Capsules are stained and observed underneath the microscope (100×, oil, Olympus CX-41, Olympus America, Center Valley, PA 18034-0610).

Statistical analysis

GraphPad Prism 5 (GraphPad Software, Inc., CA, USA) was used for all statistical analyses and chart generation. Two-way analysis of variance (ANOVA) was used to analyze the data of 3 groups and more than 3 groups. The P-value in the figure is indicated as follows: NS indicates no significant difference. ***p < 0.001; **p < 0.01; *p < 0.05. OD values in biofilm assays were normalized (Normalized Biofilm = ODbiofilm / ODinitial). Error bars represent the standard deviation of the mean.

Results

General Microbiological characteristics of vB_Sen_S3P

The clinical isolates were resistant to a variety of antibiotics, such as ampicillin (80.0%), piperacillin (76.7%), and ampicillin/sulbactam (56.7%) (Table S1). Moreover, the minimum inhibitory concentration (MIC) value of imipenem and meropenem for two strains (S25 and S27) isolates were all ≥ 16 µg/ml. According to the MIC breakpoints of the Clinical and Laboratory Standards Institute (CLSI) M100 manual12 for Salmonella spp, S25 and S27 were resistant (2/30) to imipenem and meropenem.

Among these 30 clinical isolates, S. enteritidis S3 was used as the host to isolate the lytic phage vB_Sen_S3P. The lytic spectrum of vB_Sen_S3P showed that vB_Sen_S3P lysed 22 out of the 30 tested clinical isolates of S. enteritidis, including carbapenem-resistant S. enteritidis S25 and S27 (Table 1). There were transparent zone halos around these clear phage plaques on the bacterial lawn (Fig. 1A). These halos indicated the vB_Sen_S3P had the anti-biofilm activity of depolymerases. Through transmission electron microscope, vB_Sen_S3P had an icosahedral head (approximately 25 ± 5 nm in diameter) and a short non-shrinkable tail (approximately 155 ± 5 nm in length). Based on these morphological characteristics, the phage vB_Sen_S3P belongs to the Caudoviricetes class (GenBank PV021103.1 taxonomy browser, https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/ wwwtax.cgi? id = 3401163) (Fig. 1B). The highest phage titer of vB_Sen_S3P was observed at an MOI of 10− 5 (Fig. 1C). In the one-step growth curve, vB_Sen_S3P had a latent period of approximately 20 min and a burst size of approximately 16,931 PFU/infection (Fig. 1D).

Table 1 The lytic spectrum of vB_Sen_S3P and the antibiofilm activity of Dpo52.
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Fig. 1
Fig. 1
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Characteristics of phage vB_Sen_S3P: (A) Plaques formed by phage vB_Sen_S3P. Plaques formed on double-layer agar LB plates of S. enteritidis S3 cells incubated for 24 h. (B) Morphology of the phage vB_Sen_S3P. vB_Sen_S3P was observed by transmission electron microscopy (TEM). The scale bars represent 100 nm. (C) Determination of multiplicity of infection of bacteriophage. The x-axis indicates different MOIs, and the y-axis indicates the phage titers. Each dot on the graph is the average titer of assay. (D) One-step growth curve for vB_Sen_S3P. vB_Sen_S3P was added at an MOI of 10− 5, and culture samples were harvested at regular intervals. The x-axis indicates the time post-infection, and the y-axis indicates phage titers. Each dot on the graph represents an average titer.

General features of the vB_Sen_S3P genome

The genome sequence of vB_Sen_S3P was deposited in GenBank under the accession number PV021103.1. The complete genome of the bacteriophage vB_Sen_S3P was a linear, double-stranded DNA and consisted 43,620 bp with the following nucleotide composition: A (24.88%), T (25.36%), G (25.11%), and C (24.65%). The GC content was 49.76%, and the genome contained both structural and non-structural genes. Based on a comparative analysis of the complete genome, five phages show similarity to vB_Sen_S3P, specifically Salmonella phage vB-SeS-0121, Salmonella phage vB_SenS_S528 (unpublished), Pseudomonas phage vB_PsS_LDT32522, Proteus phage ABTNLP14 (unpublished), and Salmonella phage L1323 (Table 2). All ORFs were compared to the known sequences deposited in the NCBI public databases, and the best matches for the sequences were shown in Table S2. Among the 63 identified ORFs, 41 ORFs (65.08%) were matched with the known functional protein in the NCBI public databases. The remaining 22 ORFs were annotated to hypothetical protein with unknown functions. No drug-resistant, virulence, or lysogenic genes were found in the genome.

Table 2 Global phage genome comparison.
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All ORFs matching proteins in the databases with known functions were divided into morphogenesis, nucleotide metabolism and replication, DNA packaging, and host lysis coding modules (Fig. 2). Based on bioinformatics analysis, vB_Sen_S3P encoded a cluster of proteins involved in DNA replication and phage metabolism, including ORF06 (ssDNA binding protein), ORF39 (DNA-binding domain), ORF41 (replicative DNA helicase RepA), ORF42 (DNA-binding domain), ORF44 (exonuclease), ORF45 (ssDNA-binding protein), ORF46 (DNA polymerase), ORF47 (putative restriction endonuclease), ORF49 (DNA helicase), ORF50 (putative helicase), ORF61 (metallophosphoesterase) and ORF63 (Rha family transcriptional regulator). The DNA packaging proteins included ORF16 (portal protein), ORF17 (large terminase subunit) and ORF18 (putative terminase small subunit). ORF8 (o-spanin), ORF28 (phage lysozyme), ORF29 (putative class II holin), ORF30 (holin) and ORF52 were associated with host lysis. As seen from the phylogenetic trees constructing by the terminase large subunit (Fig. 3), vB_Sen_S3P has higher homology with Salmonella phage, such as JD01, vB SenS-EnJE6, vB SE126 1PH123, vB SE130 2P, PhiSEP1, F118P13, vB SpuP Spp11, SP4, vB SenS-EnJE1, SP239, vB-SeS-01.

Fig. 2
Fig. 2
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Graphical representation of the vB_Sen_S3P genome. The directions of transcription are presented as arrows. Proposed modules are based on hypothetical functions predicted from bioinformatic analysis. The genome map was drawn using CLC Main Workbench, version 7.7.3 (CLC Bio-Qiagen, Aarhus, Denmark).

Fig. 3
Fig. 3
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Phylogenetic tree analysis of vB_Sen_S3P based on terminase large subunit sequences. The numbers next to the branches is bootstrap value and represent confidence (%).

Analysis of predicted genes encoding depolymerase

There were 22 PSI-BLAST results (Table 3). However, only two PSI-BLAST iteration results indicated that Dpo52 in vB_Sen_S3P demonstrated homology to Klebsiella phage depolymerases. Dpo52 showed 41.4% sequence identity to Klebsiella phage depolymerases. Additional comparisons to tail spike proteins and structural homologs were analyzed. InterProScan analysis of the N-terminal 1–85 aa segment of Dpo52 did not detect any conserved domains in this region (E-value N/A), suggesting that despite the sequence similarity, no recognized structural domain is annotated in current databases. The N-terminal domain of Dpo52 (residues 86–132) was annotated as a canonical phage receptor-binding module (Pfam: PF18668, residues 86–132; E = 3.8 × 10−⁸; InterPro: IPR040775). While the C-terminal region of Dpo52 (residues 135–684) was annotated as a polysaccharide-degrading enzyme (SUPERFAMILY: SSF51126; residues 135–684; E = 0; InterPro: IPR011050).

Table 3 PSI-BLAST iteration of Dpo52 in vB_Sen_S3P.
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Based on the preliminary data, Dpo52 will most likely be depolymerase from a S. enteritidis phage. Dpo52 contained 684 amino acids with a molecular weight of ~ 72.83 KDa and a theoretical pI of 5.12. The secondary structure of Dpo52 was predicted using PSIPRED (Fig. S1) and included 15 helices, 50 strands and coil regions. Through the predictive analysis of Alfafold 3, the tertiary structure modeling of Dpo52 protein appeared very confident (Fig. S2). Hence, crystal structure of ORF52 was avoided. As shown in the protein-level phylogenetic tree (Fig. S3), Dpo52 represented a different evolutionary offshoot branch from 3 other phage depolymerases or tail spike protein, while shared a same evolutionary offshoot branch from 16 other similar phage tail proteins.

Expression and activity of phage depolymerase Dpo52

The putative depolymerase Dpo52 of S. enteritidis phage vB_Sen_S3P was expressed and purified. The predicted size of Dpo52 was ~ 72.83 KDa, and the protein was expressed and purified (2 µg/µL) from supernatant liquid. SDS-PAGE analysis indicated that the size of Dpo52 was approximately 72 kDa, which was the expected size (Fig. 4A). In order to test the biological activity of Dpo52, different concentrations of Dpo52 (ranging from 0.02 ng to 800 ng) were dropped on lawns of S. enteritidis strain S3 (Fig. 4B). Compared to the negative control, 20 ng, 100 ng, 200 ng, 400 ng, and 800 ng Dpo52 could form obvious halo zones on lawns of S. enteritidis strain S3. As the concentration of Dpo52 decreased, the sizes of the halo zones on the plate also decreased. When the concentration of Dpo52 decreased to 2.0 ng, the size of the halo zone was vague resembling that of the negative control. When the concentration of Dpo52 was below 0.2 ng, the size of the halo zone was hardly visible. Thus, we supposed the activity of phage depolymerase Dpo52 exhibited a concentration-dependent effect.

Fig. 4
Fig. 4
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Dpo52 overexpression and activity: (A) An SDS-PAGE analysis of purified Dpo52. Lanes: M, 10–250 KDa protein marker; 1: uninduced E. coli BL21 cells; 2: induced E. coli BL21 cells; 3: soluble lysate of the induced E. coli BL21 cells; 4: pellet; 5: flow-through; 6: wash (20 mmol/L imidazole); 7: elute (250 mmol/L imidazole → 72 kDa). (B) Enzyme activity assay of Dpo52. Serial dilutions of Dpo52 (ranging from 0.02 ng to 800 ng) were dropped on lawns of S. enteritidis S3 strain. PBS was used as a control.

Biological characteristics of Dpo52

In all, 30 strains of S. enteritidis were utilized for activity testing, and Dpo52 formed halo zones on lawns of 13 strains (Table 1). In addition, the enzyme activity of Dpo52 was stable at pH 4–11 (Fig. 5A) and in a temperature range of 4 to 60℃ (Fig. 5B). In order to find out the cause of halo zones formed by Dpo52, the lytic activity of Dpo52 against S. enteritidis was tested. The result showed Dpo52 has no lytic activity to S. enteritidis strain S3 (Fig. S4A). Thus, we guessed Dpo52 might have an impact on the biofilm of S. enteritidis, as well as in combination with the prediction of protein structure and function.

Fig. 5
Fig. 5
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The stability of Dpo52. (A) Stability of Dpo52 at different pH values. Dpo52 at different pH values were spotted on lawns of S. enteritidis strain S3. (B) Stability of Dpo52 at different temperatures. Dpo52 at different temperatures were spotted on lawns of S. enteritidis strain S3.

The cytotoxicity test of Dpo52 was also conducted for evaluating the cytotoxicity as a candidate antibacterial agent. Whether Dpo52 was a low concentration (0.1 µg/µL), or a high concentration (1 µg/µL), the survival rate of human monocytic leukemia THP-1 cells is a 100% (Fig. S4B). This suggested that Dpo52 would not affect the survival of human macrophages.

Dpo52 inhibited the biofilm formation of S. enteritidis

The enzymatic activity of Dpo52 on biofilm formation was further tested against clinical isolates S. enteritidis S25 and S32 using 96-well micro-titre plates. Dpo52 with different concentrations on exponential-phase bacteria S. enteritidis S25 and S32 was tested to evaluate the inhibition effect of S. enteritidis biofilm formation. The results of crystal violet staining assay in Fig. 6A showed that the OD595 values of six different Dpo52-treated groups (2 ng/µL, 4 ng/µL, 8 ng/µL, 16 ng/µL, 32 ng/µL, 64 ng/µL) decreased significantly in comparison to that of 1 ng/µL Dpo52-treated group and the untreated group (0 ng/µL Dpo52) (P < 0.001). Moreover, with the increasing concentration of Dpo52, the OD595 values decreased correspondingly. It indicated that Dpo52 inhibited the biofilm formation of S. enteritidis S25 and S32 and exhibited dose-dependent activity. However, the mature biofilms formed by S. enteritidis S25 and S32 at 72 h couldn’t be destroyed by Dpo52. The OD590 values of three different Dpo52-treated groups (0.1 µg/µL, 0.5 µg/µL, 1 µg/µL) were much higher than that of the negative control (P > 0.05; Fig. 6B).

Fig. 6
Fig. 6
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Antibiofilm activity of phage depolymerase Dpo52: (A) Dpo52 inhibited the biofilm formation of S. enteritidis. PBS (0 ng/µL, as control group) and equivoluminal Dpo52 (2 ng/µL, 4 ng/µL, 8 ng/µL, 16 ng/µL, 32 ng/µL, 64 ng/µL, respectively) was incubated with S. enteritidis S25 and S32 (~ 1 × 105 CFU/well) in 96-well plates for 48 h. The biofilms were assessed by crystal violet staining, and the absorbances were measured at 595 nm. Significant differences are indicated (***P < 0.001). Error bars = ± SD; n = 3. (B) Dpo52 had nothing to do with the mature biofilms formed by S. enteritidis S25 and S32 at 72 h. PBS (as negative control group) and equivoluminal Dpo52 (0.1 µg/µL, 0.5 µg/µL, 1 µg/µL, respectively) was added after S. enteritidis S25 and S32 (~ 1 × 105 CFU/well) in 96-well plates for 72 h. The biofilms were assessed by crystal violet staining, and the absorbances were measured at 595 nm. Error bars = ± SD; n = 3.

Degradation of extracellular polysaccharide by Dpo52

Carbol fuchsin staining assay and microscopic analyses showed that S. enteritidis S3 adhere to each other and encircled by light pink capsule around. After treatment with Dpo52, cells are separated and almost lost the light pink capsule (Fig. 7). It indicated that Dpo52 might degradate extracellular polysaccharide of S. enteritidis S3.

Fig. 7
Fig. 7
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Carbol fuchsin staining of S. enteritidis S3 in the absence and presence of Dpo52. Capsules were negatively stained with carbol fuchsin solution and appear light pink underneath the microscope.

Comparative analysis between phage depolymerases Dpo42 and Dpo52

Dpo42 was derived from E. coli phage while Dpo52 was derived from S. enteritidis phage. Although the quantity of amino acids, the molecular weight, theoretical pI were different, both Dpo42 and Dpo52 could degradate extracellular polysaccharide of their own host cells. Dpo42 included 9 α-helices and 55 β-strands, while Dpo52 included 15 α-helices and 50 β-strands (Table S3).

Discussion

The prevalence of carbapenem resistant S. enteritidis poses a big threat to the public health. In this study, phage vB_Sen_S3P was isolated and depolymerase Dpo52 targeting carbapenem resistant S. enteritidis was identified. Previous studies also reported phage vB_SalD_ABTNLS324, vB-SeS-0121, and vB_SenM_BP1307625, vB_SenS_ST1UNAM26, LPST15327, and SE202 specific for S. enterica serotypes. Phage vB_Sen_S3P showed high genome sequence similarity to Salmonella phage vB-SeS-0121, Salmonella phage vB_SenS_S528 (unpublished), Pseudomonas phage vB_PsS_LDT32522, Proteus phage ABTNLP14 (unpublished), and Salmonella phage L1323. Among these S. enteritidis phages, depolymerase activity has not been analyzed yet. To the best of our knowledge, Dpo52 is the first genetically engineered S. enteritidis phage depolymerase with the ability to prevent S. enteritidis biofilms formation. The therapeutic effect of S. enteritidis phage vB_Sen_S3P, as well as its preventive effect against food poisoning, will be explored in a subsequent study.

Phage depolymerases have been proven to prevent the bacterial biofilm formation and eradicate the mature biofilm by many researches, such as Acinetobacter baumannii phage depolymerase Dpo7128,29, Klebsiella pneumoniae phage depolymerase Depo1615, K. pneumoniae jumbo phage depolymerase gp53130, E. coli phage depolymerase Dpo1031, Staphylococcus aureus phage depolymerase Dpo732, Acinetobacter pittii phage depolymerase Dpo2733. Most of the studies related to phage depolymerases are concentrated on K. pneumoniae and A. baumannii phage depolymerases. When it comes to Salmonella phage depolymerase, it is very rare. For instance, S. Typhimurium phage depolymerase DpolP22 and endolysin LysPB32 with antibiotics can control biofilm formation on medical and food-processing equipment34. S. abortus phage depolymerase Dpo36 significantly reduced biofilm formation and removal. recover from the significant economic losses caused by S. abortus equi in donkey husbandry11. Phage vB_SalA_KFSST3, not expressed phage depolymerase, was used to control S. Typhimurium and its biofilm on cantaloupes at cold temperature35. This study investigated the efficacy of a S. enteritidis phage depolymerase activity to control S. enteritidis biofilm. In addition, all 30 clinical strains were separated from human blood, human tissue or human stool, including two carbapenem resistant S. enteritidis strains. Interestingly enough, both phage vB_Sen_S3P and depolymerase Dpo52 can target two carbapenem resistant S. enteritidis S25 and S27, which laying a solid foundation for the clinical applications in future.

Based on our bioinformatics predictions, there were 22 PSI-BLAST results related to the ORF52 of phage vB_Sen_S3P (Dpo52). However, only two PSI-BLAST iteration results indicated that Dpo52 demonstrated homology to depolymerase. And Dpo52 showed low identity (41.4% and 39.0%, respectively) to KL64 Klebsiella phage depolymerase and K64 Klebsiella phage depolymerase. Although Dpo52 showed high identity (e.g. 80.6% and 48.1%, respectively) to Salmonella phage tail spike protein and Escherichia phage tail spike protein, none of them (20 PSI-BLAST results except the KL64 and K64 Klebsiella phage depolymerase) reported the proteins with depolymerase activity. Thus, it implied Dpo52 may be a depolymerase and especially from a carbapenem resistant S. enteritidis phage.

The activity of Dpo52 was evaluated and found Dpo52 prevented biofilm formation of several S. enteritidis isolates in vitro via degradating extracellular polysaccharide. Since no report about S. enteritidis phage depolymerase was retrieved at present, we compared the Salmonella phage depolymerases with our expressed S. enteritidis phage depolymerase Dpo52. It was reported that 0.01 µg/mL S. abortus phage depolymerase Dpo36 significantly inhibited biofilm formation. Moreover, 1 µg/mL S. abortus phage depolymerase Dpo36 degraded preformed biofilms11. In our study, 2 ng/µL S. enteritidis phage depolymerase Dpo52 significantly inhibited biofilm formation, which the concentration was much more lower. The only drawback was that our results showed that Dpo52 couldn’t degraded mature biofilms. Another phage depolymerase Dpo42 found by our group also couldn’t degraded mature biofilms. Through simple comparative analysis between Dpo42 and Dpo52, we found Dpo52 had 63 fewer amino acids than Dpo42. Dpo52 had 6 α-helices acids than Dpo42, while 5 fewer β-strands. In general, Dpo52 and Dpo42 were from phage and inhibited biofilm formation but not degraded mature biofilm. In fact, majority phage depolymerases only inhibit biofilm formation but not degrade mature biofilm36. It is encouraging that, in recent years, some scientists have found several phage depolymerases both inhibit biofilm formation and degrade mature biofilm, such as Klebsiella aerogenes phage depolymerase37, A.baumannii phage depolymerase Dpo7128, Proteus mirabilis phage depolymerase38. In order to enlarge the anti-biofilm spectrum and improve the activity of phage-derived depolymerase, further studies need to be carried out.

Although Dpo52 inhibited biofilm formation, it could not degrade mature biofilms. This limits its potential therapeutic application. The mature bacterial biofilms are multicellular communities that consist of water (up to 97%), microbial cells (2%–5%), extracellular polysaccharide (1%–2%), proteins (< 1%– 2%), and DNA and RNA (< 1%–2%)38. Our results had shown Dpo52 only degradated extracellular polysaccharide, and had nothing to with microbial cells, proteins DNA and RNA. After mature biofilms had formed, this kind tight junction of cells prevented Dpo52 accessibility to extracellular polysaccharide. Moreover, different bacteria might have different extracellular polysaccharrides. It was highly probable that Dpo52 acted on specific extracellular polysaccharrides substances. In order to find out the exact type of extracellular polysaccharrides which Dpo52 acted on, further studies should analyzed the structure of extracellular polysaccharrides.

Conclusion

To our knowledge, this is the first study of a S. enteritidis phage depolymerase. Dpo52 inhibited the biofilm formation of carbapenem-resistant S. enteritidis via its enzymatic activity of extracellular polysaccharide degradation. Considering the safety and specificity of phage itself, phage depolymerase depolymerases are preferable for clinical application. Further studies need to be carried out to enlarge the anti-biofilm spectrum and improve the activity of phage-derived depolymerase.