• Bacterial culture, Isolation, and morphology of bacteriophage
The bacterial strain was cultivated in a shaking incubator for the entire night on LB broth with shaking at 37°C. 20 ml of sewage samples were collected and centrifuged at 8000 rpm for 20 minutes and filtered using a 0.22 µm syringe filter. 100µl of filter, 100µl of overnight culture, and 3ml of soft agar were poured over the agar plate. These plates are incubated at 37°C until the plaque formation is finished. To purify bacteriophages, a plaque was selected from the plate and placed using Pasteur pipettes into a microtube filled with saline SM buffer. After centrifuging the material for ten minutes at 5000 g and syringe-filtering the lysate, the supernatant was kept for later use at 4°C[14]. After mixing 100 µl of S. maltophilia culture overnight and 100 µl of isolated phage stock (106 PFU/ml), they were incubated for 5 minutes at room temperature. 3 ml of 0.7% LB top agar was then added. The mixture was transferred to an LB plate and incubated for eighteen hours at 37°C. After a day, the preceding steps were carried out three to five more times to further purify the bacteriophages. Plaques obtained from the final purification procedures were stored at 4°C after centrifugation and filtration in SM buffer[15].
After adding 4ml of SM buffer to the plate, it was gently wiped. After being transferred to a 15 mL sterile falcon, the scraped top agar and SM buffer were centrifuged at 8000g for 10 minutes. We poured the supernatant through the syringe filter (0.22 µm). A single drop of filtrate was placed on a copper grid and subjected to negative staining using a 2% (w/v) phosphotungstic acid solution. A transmission electron microscope with 80000 magnifications was used to examine the grid[16].
• Anti-Biofilm Activity of phage.
The bacterial suspension in LB broth was prepared using a fresh culture of the strong biofilm producer S.maltophilia isolate. It was then adjusted to 0.5 MacFarland as previously reported and phage lysate was diluted to 10 1 to 10 9. 200 µL cultures were filled into each well of 96 sterile fat bottom tissue cultures, and the plates were subsequently incubated for 24 hours at 37°C. Bacteria that adhered to form biofilms were preserved and stained with 0.1% crystal violet solution[15]. Deionized water was used to remove extra stains, and three rounds of washing were completed. Furthermore, plates were stored to dry. Distilled water was used to resolubilize the pigments that were stuck to the bacterial cells. The dyed adherent biofilm's optical density (OD) was measured at 600 nm using a micro-ELISA auto-reader. As controls, wells with a medium devoid of microorganisms were employed. The biofilm production strength was measured and categorized as high (OD > 0.240), moderate (OD = 0.120–0.240), weak, and not producing biofilm (OD < 0.120). The biofilm production strength was measured in triplicate and the average was computed. It was categorized as strong (OD > 0.240), moderate (OD = 0.120–0.240), weak, and not producing biofilm (OD < 0.120).
• Host range specificity
To determine the host specificities of specific phages, the bacteria listed in Tables I and II were exposed to a spot test. The Department of Microbiology at SRM Medical College Hospital and Research Centre, located at SRM Nagar, Potheri, Chengalpattu, Tamil Nadu, India, provided twenty-four distinct bacterial cultures and six distinct strains of Stenotrophomonas maltophilia were procured from the Department of Microbiology, University of Putra, Malaysia. For each candidate, a five-microliter phage suspension was placed on the surface of the bacterial lawn. After being incubated at 37°C for 18 hours, plates were checked to see if a clear plaque had developed.[9][17]..
• Determining the optimal MOI
A standard methodology was used to identify the MOI that produced the highest titer. Stenotrophomonas maltophilia culture in the log phase was infected with the selected phage at several MOIs ranging from 0.001 to 10. For every condition, the number of plaques was determined three times[18].
• One Step growth curve
After being combined with the phage at a multiplicity of infection (MOI) of 0.1, Stenotrophomonas maltophilia was incubated for 10 minutes at 37°C. After 15 minutes of centrifugation at 5000×g to remove the unabsorbed free phage, the infected cells were moved into 50 mL of LB medium. The phage titer was measured by sampling the culture every five minutes while it was being continuously shaken at 37°C. Three duplicates of each experiment were run[14].
• Phage-antibiotic synergy
Synergy testing was performed with LB medium and various antibiotics using 96 well plates. 20µl of phage dilutions from 103 to 109 and 5µl antibiotics of different concentrations from 0 to 256µg/ml were added to 180µl of LB broth and 20µl of bacterial culture (total of 200 µl). The readings were taken after 8 hours at OD600. Bacterial culture, phage alone, and phage-antibiotics were taken as control[19].
• DNA isolation of the bacteriophages
To 8ml of phage lysate, 4 µL of RNase and DNase (RNase free) were added. Incubated at 37°C for 30 minutes. To this 2ml PEG 6000 was added. Norgen phage DNA isolation kit was used to extract the phage-encapsulated DNA of chosen phages, according to the manufacturer's instructions. Using electrophoresis, the DNA's size and purity were assessed. Genome sequencing was done using the PHASTEST tool. The phylogenetic tree and circular proteomic tree were constructed using the ViPTree tool [20].
Accession number sequence
The Phage CM2 genome's nucleotide sequence is listed in GenBank with accession number PP550502. And SRA Submission ID is SUB14372823 AND BioProject ID is PRJNA1099129.