Bacteria isolates
Clinical strains of Klebsiella pneumoniae were obtained from the "MicroMir" collection. All strains were examined on a MALDI-TOF Microflex mass spectrometer (Bruker, US) and with biochemical tests (MIKROLATEST, Erba Mannheim) with further analysis on Multiskan Ascent spectrophotometer (Thermo Scientific, US) before their adding to collection. Strains were obtained from:
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Clinical isolates from Tula; Serpukhov; Moscow; Pushchino; city of Ufa;
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Kulakov Scientific Center of Obstetrics, Gynecology and Perinatology;
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Moscow City Scientific Research Institute of Emergency Care named after N. V. Sklifosovsky;
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Clinical Hospital No. 83 of FMBA, Moscow;
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City Clinical Hospital No. 67 in Moscow.
Strains are clinical and were obtained from many patients over several years, which allows us to talk about the relevance of the collection and its compliance with the current spectrum of circulating strains. More detailed information about characteristics of the bacterial strains, including MALDI-TOF and MIKROLATEST results, is included in Additional File 1.
To isolate and produce bacteriophages and to conduct experiments, four strains were used: Kl 327, Kl 325, Kl 315 and Kl 263. These strains are used in the laboratory as the most convenient for production and the most sensitive to bacteriophages. A separate strain was used for each bacteriophage: Kl 327 for vB_KpnS_FZ10, Kl 325 for vB_KpnS_FZ41, Kl 315 for vB_KpnP_FZ12 and Kl 263 for vB_KpnM_FZ14.
Isolation of phages
Klebsiella phages vB_KpnS_FZ10, vB_KpnP_FZ12, vB_KpnM_FZ14 and vB_KpnS_FZ41 were isolated in 4 separate enrichments from sewage water collected from a waste-water treatment plant near hospital №5 located in Chekhov district, Moscow region, Russia. The isolation process began with the addition of sodium phosphate buffer (pH 7.0) to the sample to a final concentration of 0.05 M, and NaCl was added to a final concentration of 1 M. The mixture was incubated on an Environmental Shaker-Incubator ES-20/60 (Biosan, Latvia) for 60 min at + 37° C and 100 rpm. The supernatant was then withdrawn and centrifuged (6800 g, 20 min.) on an Avanti J-E centrifuge with a JA 14.50 rotor (Beckman Coulter, US). The supernatant was then centrifuged for 2 h at 96,200 g in an Optima L-90 K ultracentrifuge (Beckman Coulter, US) with SW28 rotor. The supernatant was removed and the pellet dissolved in 1 ml of 0.1 M Tris–HCl buffer (pH 7.0). The resulting suspension was subsequently filtered through 1.2 μm, 0.45 μm and 0.22 μm filters (MF-Millipore, US). The resulting filtrate was stored at 4° C.
Bacteriophage amplification and purification
800 ml of Brain Heart Infusion (BHI) broth and 10 ml of an overnight culture of Klebsiella pneumoniae cells were mixed. In each shaking flask, cells of a specific Klebsiella pneumoniae strain (Kl 327, Kl 325, Kl 315 and Kl 263) were presented. The mixture was incubated on an Environmental Shaker-Incubator ES-20/60 (Biosan, Latvia) for 2 h at + 37° C and 100 rpm. Then 1 ml of bacteriophage filtrate was added to the flasks and incubated overnight. In the morning, the lysis of the bacterial culture was observed. The contents of the flasks were differentially centrifuged and the resulting was then subsequently filtered as described above. The filtrates were stored at 4° C in a refrigerator.
Bacteriophage plaque assays
A suspension of Klebsiella pneumoniae cells in physiological salt solution was prepared. The concentration of cells in suspension is approximately 109 cfu/ml (colony forming units per ml). Then, serial dilutions (from 10–1 to 10–10) of each of four obtained filtrates with bacteriophages in physiological salt solution were prepared. 0.2 ml of culture and 0.1 ml of a specific dilution of the bacteriophage were added to tubes with soft agar (0.6%), mixed. The resulting mixture was distributed on Petri dishes with solid agar. Incubated 24 h at a temperature of + 37 °C in a thermostat (Binder GmbH, Germany). After the incubation period, the obtained plaques were evaluated.
Preparation of "clean lines" of bacteriophages and evaluation of plaques morphology
Single plaques with agar fragments obtained for each of the four filtrates were excised from soft agar and placed in different tubes with 1 ml of physiological salt solution. They were incubated for 10 min at room temperature. Then the extracts were taken and amplification/plaque assays steps were repeated (3 repetitions were made). The obtained filtrates were stored at 4° C in a refrigerator. After plaque assays described above, the morphology of plaques was evaluated.
Determination of phage host range
To assess the lytic spectrum of the accumulated bacteriophage, the double-layer method was used. As a test culture, 14 strains of Klebsiella pneumoniae were used. A suspension of Klebsiella pneumoniae cells in physiological salt solution was prepared. The concentration of cells in suspension is approximately 109 cfu/ml. 0.2 ml of culture were added to tubes with soft agar (0.6%), mixed, poured on Petri dish with agar and allowed to solidify. Then, the phage suspension in several dilutions was spotted on the soft agar. Petri dishes were incubated for 24 h at + 37° C in a thermostat (Binder GmbH, Germany). After incubation, the presence of lysis spots was assessed. To verify that the formation of lysis spots was caused by the lytic action of the phage, bacteriophage plaque assays were performed as described above. Moreover, an analysis of lysis spots on a JEM-1011 electron microscope (JEOL, Japan) was performed. For this, a fragment of the agar plate was taken from the lysis spot and placed in a 0.1 ml drop of physiological saline. Incubated for 25–30 min. After that, the agar plate was removed and electron microscopy of the droplet was carried out. The presence of bacteriophages in the test material was evaluated.
Centrifugation in CsCl gradient
Solutions with different percentages of CsCl (50, 30, 20, 10, 5) were prepared. The solvent used was 0.05 M Tris–HCl buffer (pH 7.0). After that, the solutions were carefully added to the centrifuge tube from the SW 28 rotor, layering the gradient from a higher concentration to a lower one using a Pasteur pipette. The bacteriophage filtrate was added last. At each stage, phase uniformity was controlled, preventing their mixing. Tubes were equilibrated with a 5% CsCl solution and placed in an Optima L-90 K ultracentrifuge (Beckman Coulter, US). Then centrifuged for 2 h at 96,200 g. After centrifugation, the content of the zone with phage was carefully selected into eppendorf tubes using a pipette. The contents of the tubes were then centrifuged for 2 h at 96,200 g on an Optima L-90 K centrifuge (Beckman Coulter, US) with a SW 28 rotor. The pellet was resuspended in 0.05 M Tris–HCl buffer (pH 7.0). The resulting phage suspension was stored at 4° C in a refrigerator.
Electron microscopy
Electron microscopy was performed using high-titer bacteriophage filtrates, purified in a CsCl density gradient. Samples were deposited on carbon-coated nitrocellulose films, stained with 1% uranyl acetate and examined in the transmission electron microscope (TEM) JEM-1011 (JEOL, Japan). Electron micrographs were taken with the Erlangshen ES500W (Gatan, US) camera. The parameters of bacteriophages were measured using ImageJ program based on images obtained using an electron microscopy. Standards based on measurements of the tail length (114 nm) of the bacteriophage T4 were used as markers. 35 particles were measured for each phage and SD (standard deviation) was calculated.
Estimation of frequency of phage-resistant forms generation
Cells from an overnight culture were suspended in in physiological salt solution to 108 cfu/ml, 0.1 ml of culture and 0.1 ml of phage filtrate initially containing 108 pfu/ml (plaque forming units per ml) were added to tubes with soft agar (0.6%), mixed. The resulting mixture was distributed on Petri dishes with solid agar. Incubated 24 h at a temperature of + 37 °C in a thermostat (Binder GmbH, Germany). Afterwards, the number of resistant colonies was evaluated and the frequency of phage-resistant forms generation was calculated. The resulting resistant colonies were looped into a test tubes with 10 ml of BHI broth and mixed. The resulting suspensions was incubated for 2 h at 37 °C in a thermostat (Binder GmbH, Germany). Subsequently, mitomycin C was added to the test tubes to final concentrations of 0.2 μg/ml, 0.5 μg/ml and 2 μg/ml and they were incubated for 24 h at + 37 °C in a thermostat (Binder GmbH, Germany). The suspensions were then centrifuged (6800 g, 20 min) on an Avanti J-E centrifuge with a JA 14.50 rotor (Beckman Coulter, US), supernatants were taken into clean tubes, centrifuged for 2 h at 96,200 g in an Optima L-90 K ultracentrifuge (Beckman Coulter, US) with SW28 rotor and the sample was analyzed on electron microscope for phage presence in the suspension. In addition, initial suspension of resistant colonies was treated with chloroform, then centrifuged (6800 g, 20 min) on an Avanti J-E centrifuge with a JA 14.50 rotor (Beckman Coulter, US) and supernatants were spotted on phage-sensitive Klebsiella pneumoniae lawns.
Sensitivity of phage particles to temperature and pH
In temperature and pH sensitivity assays the methods used by Jamal et al. were applied [41]. The bacteriophage filtrate was incubated for 1 h in test tubes in a water bath (GFL, Germany) at temperatures of 37, 50, 55, 60, 65 and 70 °C. Then, serial dilutions of the bacteriophage filtrate from 10–2 to 10–10 were prepared in increments of 102. Phage titer was obtained by plaque assay as described above. To study pH sensitivity, tubes with meat-peptone broth (MPB) (initial pH 7.2) were prepared with pH values from 3 to 13. To obtain the desired pH values, 6 M NaOH and HCl solutions were used. Filtered MPB through filters with a pore diameter of 0.22 μm (MF-Millipore, US). 1 ml of phage was added to the tubes with each pH value and incubated for 18 h at + 37° C in an incubator (Binder GmbH, Germany). The pH was monitored after the incubation period. Same incubation period for pH sensitivity assay was also used by Soleimani Sasani and Eftekhar [42]. Then, serial dilutions of the solution with phages from 10–1 to 10–10 were prepared and plaque assay done as described above to obtain phage titer.
Phage adsorption rate
Cells from an overnight culture were suspended in BHI broth to 109 cfu/ml. 5 ml of bacterial suspension and phage suspension diluted to 107 pfu/ml were incubated in 45 ml of BHI broth on Environmental Shaker-Incubator ES-20/60 (Biosan, Latvia) at + 37 °C and 100 rpm for 5 min. After that the supernatant was filtered through 0.22 μm pore filter and the free phages were enumerated using the plaque assay described above. The reduction in phage titer was the number of phages adsorbed to the cells. Bacteriophage filtrate was used as a control. A decrease in titer in the control was not observed. The adsorption constant was calculated by the following formula:
$$k = \left( {\frac{ - 1}{{Bt}}} \right) \times \ln \left( {\frac{P}{{P_{0} }}} \right)$$
P–concentration of free phage per ml, P0–initial concentration of phage, B–initial concentration of bacteria, k–adsorption rate constant (ml/min), t—time (min).
One-step growth curve
1 ml of BHI broth, a cell suspension with a final concentration of 109 cfu/ml and a bacteriophage filtrate with a final concentration of 107 pfu/ml were mixed. The mixture was incubated at 37 °C for 8 min in a thermostat (Binder GmbH, Germany) and then centrifuged for 2 min at 4800 g on Centrifuge 5424 (Eppendorf, Germany). The supernatant was removed and the pellet resuspended in 100 ml of BHI broth. Aliquots of 0.5 ml of the resulting suspension were taken every 5 min for 80 min and the bacteriophage titer was assessed using plaque assay described above. The latency period was defined as the the interval between adsorption of the phage to the host cell and release of phage progeny. The burst size of the phage was expressed as the ratio of the final count of released phage particles to the number of infected bacterial cells during latent period.
Phage propagation in liquid nutrient medium and evaluation of the bacteriophage titer
Cells from an overnight culture were suspended in BHI broth to 109 cfu/ml. 5 ml of bacterial suspension containing 109 cfu/ml and phage diluted to 106 pfu/ml were incubated in 45 ml of BHI broth on Environmental Shaker-Incubator ES-20/60 (Biosan, Latvia) at + 37 °C and 100 rpm for 18 h. As a control one flask had no phage added to the Klebsiella culture. Flasks were incubated for 18 h at + 37 °C and 100 rpm. After that, the phage titre in the experimental system was evaluated using plaque assays described above. Incubated for 24 h at + 37 °C in a thermostat (Binder GmbH, Germany). After incubation, the phage titer was evaluated.
Isolation and restriction digestion of phage DNA
Phage genomic DNA was extracted from high-titre bacteriophage filtrates, purified in a CsCl density gradient. An ethylenediaminetetraacetic acid (EDTA) solution was added to 0.45 ml of the bacteriophage filtrate to a final concentration of 25 mM. Then, proteinase K was added to a final concentration of 100 μg/ml and incubated at + 50 °C for 2 h. After that, sodium dodecyl sulfate (SDS) was added to a concentration of 0.5% and incubated at + 55 °C for 2 h. Then the mixture was heated for 20 min at + 65 °C to inactivate the enzymes. Next, a double extraction with chloroform was performed. 0.5 ml of isopropyl alcohol was added to the aqueous phase, after which the DNA was extracted onto a glass rod. After washing three times in a 70% ethanol solution, the DNA was dried for 5 min on air, and then dissolved in 0.4 ml of TE buffer (pH 8.0). The quantity and quality of the extracted DNA was monitored by analysis on a NanoDrop ND-1000 spectrophotometer (ThermoFisher, US).
Phage DNA was digested with restriction enzymes according to the manufacturer's protocol for 90 min at + 37 °C (at + 30 °C for SmaI) in the buffer and conditions appropriate for restriction enzyme (in 50 μl of the reaction: 5 units of the enzyme, 1 μg of DNA, and the volume of distilled water). The following restriction endonucleases were used: HindIII, HinfI, HaeIII, SspI, BamHI, EcoRV, NotI, EcoRI, KpnI, MspI, VspI, NdeI, BgII, BgIII, PvuI, SmaI. To analyze the results, electrophoresis was performed on a 1% agarose gel at 180–200 V for 1 h at room temperature on a Sub-cell Model 92 Cell (Biorad). 15–20 μl (1–2 μl of buffer for application to 5 μl of reaction) were added to the wells. Lambda DNA/HindIII (Thermo Scientific, US) was used as markers.
Visualization of the results was carried out after staining agarose gels for 20 min in a solution of ethidium bromide (1 μg/ml). To document the results, the DOCPRINT system (Vilber Lourmat, France) was used. The obtained restriction profiles were compared with those predicted in silico using the RestrictionMapper program (restrictionmapper.org).
Genome sequencing, assembly and annotation
Phage DNA was extracted by chloroform extraction [43]. The DNA library was constructed using a Nextera DNA Library Preparation Kit (Illumina, San Diego, CA) and sequenced with an Illumina HiSeq T1500 sequencer, resulting in approximately 1 million 2 × 250 paired-end reads. The quality control and primary processing was performed using FASTQC and trimmomatic (HEADCROP:20, SLIDINGWINDOW:3:24, MINLEN:200, CROP:200) [44]. Coverage was normalized to × 50 using BBNorm [45]. De novo assembly was performed using MIRA assembler [46] version 4.9.6. Contig completion was confirmed by comparison to closely related genomes known to be complete (accession numbers are presented in Table 5). Open reading frames search was performed using MetaProdigal 2.6 [47]. Annotation was performed using all peer-reviewed phage and bacterial proteins from UniProt (https://www.uniprot.org) and all proteins from databases of determinants of antimicrobial resistance and bacterial virulence factors: VFDB [48], CARD [49], ARG-ANNOT [50] and Resfinder [51]. The remaining ORFs were annotated with hmmscan application (minimum e-value 0.001) [52], using all bacterial HMM profiles from the Pfam database (https://pfam.xfam.org). tRNA was predicted using tRNAscan-SE 2.0 [53]. All analyses, except those indicated, were performed using default parameters. BLASTn (https://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to search for similarity with other bacteriophages, and to calculate average nucleotide identity and query coverage.
The complete genome sequences of Klebsiella pneumoniae phages vB_KpnS_FZ10, vB_KpnP_FZ12, vB_KpnM_FZ14 and vB_KpnS_FZ41 have been deposited in GenBank under the accession numbers MK521904, MK521905, MK521906 and MK521907, respectively. Raw Illumina reads are available on NCBI SRA under accession numbers SRR10037530, SRR10037529, SRR10037528 and SRR10037527, respectively. The associated BioProject accession number is PRJNA562287. GenomeVx tool [54] was used for complete genome visualization. Taxonomic identification was made by GenBank (https://www.ncbi.nlm.nih.gov/genbank/) based on the phylogenetic classification scheme used in the NCBI Taxonomy Database (https://www.ncbi.nlm.nih.gov/Taxonomy).