Bacterial strains
A total of 206 non-duplicate clinical isolates of Staphylococcus aureus were used in the study for in vitro characterization of phages. All bacterial isolates used in this study was collected from the diagnostic laboratories located in Tamil Nadu and Mizoram, India. The antibiotic resistance profiling and the clonal analysis of the bacterial isolates was performed at Antibiotic Resistance and Phage Therapy Lab, VIT, Vellore. Out of 206 isolates, 106 isolates were conformed as MRSA by antibiotic resistance profiling. This prospective phage study on Staphylococcus aureus has been approved by the Institutional Ethical Committee (IEC) of Vellore Institute of Technology, Vellore (VIT/IECH/004/January 28, 2017). Antibiotic resistance profiling experiments were conducted following the guidelines of the Indian Council for Medical Research (ICMR) with Biosafety Level II and regulations.
Phage isolation, purification, and propagation
The phages specific to Staphylococcus aureus was screened in various water and soil samples such as sewage waters collected from hospital outlets, soil from veterinary hospitals, water samples from the lake, sewage from water treatment plants, sewage sample from schools, hostels located in and around Vellore, Tamil Nadu. The collected water and soil samples were transported to Antibiotic Resistance and Phage Therapy Laboratory, VIT, Vellore, and the phage was isolated by the enrichment technique. Briefly, 2 ml of the log phase culture and
10 ml of sewage water was added to the 10 ml of the double strength BHI/CaCl2 broth and incubated at 37 ℃ overnight. The mixture was centrifuged at 5000 x g for 15 to 20 min, and the supernatant was filtered using a 0.22-micron syringe filter.
The filtrate obtained was tested for the presence of phage by spot test and double agar overlay method. Briefly, for the spot test, the bacterial lawn was prepared in BHI/CaCl2 agar plates, and 5 µl of phage filtrate was spotted on the top of the bacterial lawn. In the double agar overlay method (DAOL), 200 µl of host bacteria was mixed with 10 µl of the phage filtrate. The mixture was incubated at 37 ℃ for 5 min, and 2 mL of molten soft agar (BHI/CaCl2) was added, and overlaid onto the hard agar (BHI/CaCl2). The plates were incubated at 37 ℃ overnight. The appearance of a clear zone in the spot test and the plaques in the DAOL represents the presence of bacteriophage. Throughout the study, 1% hard agar and 0.5% soft agar were used to improve the transparency and enhance the plaque visibility. The phage was purified by picking a single, well-isolated, transparent plaque in SM buffer (Sodium-Magnesium buffer) (1 L: 5.8g, NaCl; 50 mL, 1M Tris-HCl [pH 7.5]; 2g, MgSO4.7H2O; 5 mL, 2% gelatin and 1 mM of CaCl2) and purified for three successive rounds using the double agar overlay method. Purified phage was propagated by multiplying with the host bacterium by web pattern DAOL (plate with an excessive number of plaques that are hazily defined, neither entirely clear nor distinct plaques) (Liu et al. 2020). For web pattern DAOL, 200 µl of host bacteria was mixed with 104 to 105 PFU/mL of phage (concentration varies upon the plaque size), and a double agar overlay was performed as described previously. Following incubation at 37 ℃ overnight, SM buffer of 3 mL was added onto the plate and kept at 4 ℃ for 4 hrs to remove the phage from the DAOL plate. After incubation, the buffer collected was filtered sterilized and stored at 4 ℃ for further analysis.
Lifecycle analysis – Adsorption assay
The phage adsorption onto the host bacteria was determined by analyzing the number of non-adsorbed phages at a standard time interval. Briefly, adsorption assay was performed by mixing bacterial host with phage at a multiplicity of infection (MOI) 0.001 and incubated at 37 ℃. At every 2 min interval, an aliquot of 100 µl was drawn from the interaction tube, diluted in BHI broth (4.4 mL) and chloroform (0.5 mL). The dilution was incubated at room temperature for 30 minutes, and a double agar overlay technique was performed to assess the number of non-absorbed phages.
Lifecycle analysis – latency time and burst size
A one-step growth curve experiment was carried out to determine the latency period, and burst size of the phages studied. Briefly, phage was mixed with the bacterial host to obtain an MOI 0.001, and the mixture was incubated at 37 ℃ for phage adsorption (duration as determined from adsorption assay). After phage adsorption, the mixture was centrifuged at 13,000 x g for 2 min, and BHI/CaCl2 broth was added to the pellet. The aliquots were removed at 2 min (vB_Sau_S90) and 5 min (vB_Sau_S165) intervals, and a double agar overlay was performed. The absorption efficiency was calculated by ((Control – Test) / Control) x 100, and the values are expressed as percentage (%).
Stability analysis
The viability of the phages at varying temperature and pH ranges was analyzed using the phages at 109 PFU/mL. The prepared phages were subjected to a varying temperature range of 20, 30, 40, 50, 60, 70, 80 ℃ for 60 min, and the activity was determined using the double agar overlay method. For pH stability studies, phage particles were prepared in pH-controlled buffer ranging from pH 1-14 and incubated at 37°C for 60 min, and the activity was assessed using the double agar overlay method.
Transmission Electron Microscopy
The morphology of the phage particles was determined by negative staining with 2% uranyl acetate and visualized under Transmission Electron Microscopy (TEM). Each phage of 10 µl volume (109 PFU/mL) was spotted on top of the carbon-coated copper grid, and the phage was allowed to absorb to the copper grid for 2 to 3 min. After incubation, the grid was carefully stained with 2% uranyl acetate for 2 min, and the excess stain was washed. The grid was carefully dried in a desiccator, and the phage morphology was viewed under TEM (Transmission Electron Microscope) (FEI-TECNAI G2-20 TWIN, VIT, Vellore).
Phage host range and efficiency of plating
The host range of the bacteriophages against 205 non-duplicate clinical strains of S. aureus was determined using a spot test. The phage bacterial host (SA-90 and SA-165) was employed as control strains for the respective phage, and the remaining 205 isolates were taken as a test strain. The host range of the phage was validated by spotting a phage lysate of 5 µl (106 PFU/mL) onto the bacterial lawn, and the phage activity was confirmed as sensitive if a complete zone of clearance was observed, intermediately sensitive if discrete plaques were seen, and resistant for no spot. To further analyze lysis efficiency (plaque formation), phage from 1010 to 106 PFU/mL was spotted onto the test strains that showed a zone of clearance in the host range analysis. The efficiency of plating (EOP) was interpreted by a range of scoring system in comparison to the controls following the methods of Kutter et al. (Kutter 2009). High EOP - ≥ 4 spots of complete clearance and produced individual plaques as phage concentration decreased, Moderate EOP - < 4 and ≥ 2 spots of complete clearance and individual plaques in the least phage concentration, Low EOP - < 2 spots of complete clearance and individual plaques in the least phage concentration and No EOP - clearance throughout but with faintly hazy background till 101 PFU/mL with no observable plaques.
In vitro time killing assay
Time kill analysis was performed to assess the antibacterial efficacy of phages and to determine the phage dosing (MOI) as described previously (Liu et al. 2020). Briefly, S. aureus bacterial culture (108 CFU/mL) was mixed with phage at different MOIs of 0.01, 0.1, 1 and, 10 and incubated at 37 ℃. The optical density (OD600) was measured at every 2 hrs intervals for 22 hrs. The assay was performed using the host bacterium (only). The results are expressed as log fold reduction. (Log-fold reduction was calculated using the formula - log (OD before treatment / OD after treatment). All data analysis was performed using the GraphPad Prism version 8.0 software package.
Screening and elimination of phage insensitive bacterial mutants by multiple phage dose and serial passage
The behavioral response of phage-insensitive bacterial mutants to multiple phage doses and serial passage was assessed. The work of Denes et al. has been modified here to understand the behavior of phage insensitive mutants (Denes et al. 2015). This experiment was performed separately for both the phage reported in this study. Briefly, five independent tubes (A, B, C, D, E) containing the same proportion of phage (MOI 10) and their respective host bacteria (108 CFU/mL) (SA-90 with vB_Sau_S90 and SA-165 with vB_Sau_S165) was incubated at 37 ℃ for 24 hrs. After incubation for 24 hrs (period for bacterial evolution), multiple phage treatments and serial passage were performed independently on each tube at 37 ℃.
The bacterial cells that regrew (Phage insensitive cells) after the phage bacterial interaction was interpreted based on their response to subsequent phage infection and scored as– Sensitive type – bacterial cells that were susceptible to phage without any requirement of serial passage, Transient resistant type – bacterial cells that become phage susceptible only after the serial passage, Resistant type – bacteria cells that showed phage resistance even after several serial passages or multiple phage doses.
The multiple dosage and serial passage were conducted in each tube as follows - In tube A, the mixture was centrifuged at 10,000 X g for 5 min, and the pellet was washed twice with SM buffer and resuspended in 200 µl of BHI broth. The spread plate was performed in a bacteriophage-free agar plate and incubated at 37 ℃ for 24 hrs. The rate of phage resistance was determined after incubation, and a spot test was performed on random colonies to check for the bacterial cell type that evolved after single phage treatment in liquid media. In tube B, the mixture was centrifuged at 10,000 X g for 5 min, and the pellet was resuspended in 200 µl of BHI broth. The resuspended pellet was mixed with the second dose of phage (109 PFU/mL), and a double agar overlay was performed to eliminate any growing phage-sensitive cells and the rate of resistance after removing the sensitive cells was determined. The plates were incubated at 37 ℃ for 24 hrs to determine the appearance of phage-insensitive bacterial colonies. The individual colonies obtained in DAOL was picked and inoculated in 1 ml of BHI broth. For each colony, five alternative serial passage (day - 1, 3, 5, 7, 9) along with five alternative phage doses (109 PFU/mL) (day – 2, 4, 6, 8, 10) was administered to check the effect of bacterial clearance by multiple phage doses and serial passage. Simultaneously, spot test was performed after each serial passage to check, if serial passage aided in the reversion (from transient phage resistance to phage sensitive cells). In tube C, the phage insensitive cells were isolated similar to tube B and the colonies from DAOL were treated only by multiple phage treatment (109 PFU/mL) for five subsequent days. Here, serial passage was eliminated to check the effect of bacterial clearance only by multiple phage doses. Simultaneously, spot test was performed after each phage treatment to check, if reversion (from transient phage resistance to phage sensitive cells) occurred without serial passage. In tube D, the bacterial cells obtained from single phage treatment in liquid media was treated with five alternative days of serial passage and five alternative days of phage treatment (109 PFU/mL). The tube D experiment analyzes the combinatorial effect of serial passage and phage multiple doses in liquid media without eliminating uninfected phage sensitive cells. In tube E, the bacterial cells obtained from single phage treatment in liquid media was treated with only multiple phage doses (109 PFU/mL) for five subsequent days without serial passage. Tube E aided in determining the significance of the serial passage in eliminating phage insensitive and phage sensitive cells in liquid media. The experimental setup described above are depicted in supplementary figure 1. The rate of resistance was calculated using the formula: Phage resistant rate (CFU/mL) = (Number of bacterial colonies after phage treatment / Initial number of the colony).
Statistical analysis
The plaque size was analyzed manually using a ruler, and the values are provided as mean ± SD. All data were expressed as mean ± standard deviation (SD) and analyzed using the GraphPad Prism version 8.0 software package.