Isolation and characterization of bacteriophages from sewerage water targeting methicillin resistance S. aureus in burn patients

Isolation and characterization of bacteriophages from sewerage water targeting methicillin resistance S. aureus in burn patients | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Isolation and characterization of bacteriophages from sewerage water targeting methicillin resistance S. aureus in burn patients Nishat Zafar, Aamir Aslam, Sajjad Ur Rahman, Muhammad Saqib This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4554648/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The spectrum of infections caused by methicillin-resistant Staphylococcus aureus (MRSA) ranges from minor to potentially life-threatening. Due to its increased antibiotic resistance, treatment failures are frequent, necessitating novel eradication approaches. In this study, we aimed to isolate MRSA from burn patients, ascertain its antibiogram pattern, and isolate and characterize bacteriophages from sewerage water targeting MRSA. A total of 70 samples were collected from burn patients, and MRSA was identified and characterized using various biochemical and molecular tests, alongside antibiotic sensitivity profiling. MRSA prevalence was found to be 28.6%, with biochemical and molecular analyses confirming its presence. Antibiotic susceptibility testing revealed that 94% of isolates were sensitive to tobramycin and gentamycin, while lowest sensitivity (2%) was observed against vancomycin. Using the soft agar overlay method, three bacteriophages (Phage-1, Phage-2, and Phage-3) were successfully isolated from sewerage water. Among all the phages, Phage-3 demonstrated a broader host range. Furthermore, Phage-3 displayed optimal activity within a pH range of 6–8 and at temperatures between 20–40°C. Phage-3 indicated a rapid decrease in adsorption time within 0–5 minutes, while its one-step growth curve revealed a latent phase lasting up to 30 minutes, followed by a significant increase in titer from 30–50 minutes. Methicillin-resistant Staphylococcus aureus (MRSA) Bacteriophages Characterization Antibiogram Sewerage water Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 INTRODUCTION Globally, Staphylococcus aureus ( S. aureus ) stands out as the primary pathogen responsible for burn infections, predominantly acquired in hospital settings, with a tendency to infect the skin and inhabit the human nares. Approximately 20–40% of the population is affected by skin infections caused by S. aureus. The microorganism utilizes quorum sensing for cell-to-cell communication, facilitating its colonization at various sites [ 1 ]. S. aureus is renowned as one of the most significant bacteria causing diseases in humans. It is implicated in various infections of the skin and soft tissues, such as abscesses (boils), furuncles, and cellulitis, as well as more severe conditions including pneumonia, osteomyelitis, and septicemia, albeit most of these infections are typically not highly severe [ 2 , 3 ]. The bacterium is accountable for numerous serious skin infections, establishing it as one of the most notorious and prevalent bacterial pathogens. Each year, a substantial number of individuals worldwide suffer from skin diseases caused by S. aureus. Moreover, it contributes to pneumonia, respiratory tract infections, cardiovascular complications, and other hospital-acquired infections [ 4 , 5 ]. S. aureus bloodstream infections have been documented at an annual rate of 20–50 per 100,000 individuals, with a mortality rate ranging from 10–30 percent. In the United States alone, S. aureus bacteremia is responsible for an estimated 20,000 deaths annually. While less immediately life-threatening, moderately severe skin infections caused by S. aureus , such as furuncles, abscesses, and wound infections, can still result in significant morbidity [ 6 , 7 ]. The mortality and morbidity rates associated with S. aureus bloodstream infections surpass those of other diseases such as viral hepatitis, acquired immune deficiency syndrome, and tuberculosis. Additionally, S. aureus has been linked to atopic dermatitis, contributing to a substantial global public health burden [ 8 ]. Horizontal gene transfers enable Staphylococcus aureus to acquire antibiotic-resistant genes, leading to resistance against various antibiotics [ 9 ]. These genes may have evolved in antibiotic producers as a defense mechanism against inhibitory molecules, facilitating their survival in competitive environments. The prevalence of antibiotic-resistant bacteria, including those resistant to methicillin, is increasing globally, particularly in healthcare settings like burn units, where methicillin-resistant S. aureus (MRSA) has become a significant nosocomial pathogen [ 10 ]. Treatment of MRSA infections often involves antibiotics such as quinupristin, dalfopristin, and glycopeptides like vancomycin. However, resistance to these antibiotics, as well as to oxacillin, methicillin, tetracycline, and tigecycline, is observed in many strains of S. aureus. In some cases, resistance to vancomycin and linezolid may also have emerged [ 8 ]. The development of drug resistance in S. aureus is facilitated by mobile genetic elements (MGEs), which are transferred via horizontal gene transfer. These elements encode virulence factors and antibiotic resistance mechanisms, including resistance to beta-lactam antibiotics [ 11 ]. The emergence of methicillin-resistant S. aureus (MRSA) and vancomycin-resistant S. aureus (VRSA) underscores the urgent need for new antimicrobial agents. Despite the resistance of these strains to commonly used antibiotics, current standards of care recommend antibiotics such as Daptomycin and Linezolid [ 12 ]. However, the discovery of new antibiotics has been slow in recent decades, despite the effectiveness of existing drugs in most cases. MRSA infections in burn patients remain a significant concern globally, with reported prevalence rates exceeding 50%, and in some studies, surpassing 66% in recent years [ 13 ]. In the era of antibiotic resistance, phages are increasingly recognized as promising candidates for controlling bacterial infections across various domains. Their targeted mechanisms of action have led to their utilization in diverse fields including the food industry, medicine, animal husbandry, biotechnology, and agriculture. Numerous studies have documented the efficacy of phages against various pathogens, including S. aureus. Phages specifically targeting S. aureus have been identified and deployed for their potential in combating S. aureus infections. For instance, in a recent study, a cocktail of phages was assessed for its effectiveness against MRSA both in vivo and in vitro, suggesting a promising avenue for treating S. aureus infections by combining phages with conventional antibiotics [ 14 ]. Moreover, it's crucial to note that some bacteriophage genomes harbor genes encoding highly virulent bacterial toxins, rendering the host bacterium pathogenic only upon lysogenization by the toxin-encoding phage. Additionally, the transfer of antibiotic resistance genes to bacteria through phages has been documented. Consequently, research endeavors have been initiated to leverage phages for gene insertion into desired bacterial strains, with previous studies demonstrating feasibility and ongoing plans for further exploration [ 15 ]. The present research was designed to isolate Methicillin Resistant Staphylococcus aureus strains from burn patients and ascertain their antibiogram patterns. Furthermore, a central objective of this study was the isolation and characterization of bacteriophages specifically targeting Methicillin Resistant Staphylococcus aureus . METHODOLOGY This research was conducted at the Institute of Microbiology, Faculty of Veterinary Science, University of Agriculture Faisalabad, received ethical approval from the Institutional Biosafety Committee (IBC) (give reference number of letter). During this investigation, Methicillin-resistant S. aureus (MRSA) was isolated and identified from wound pus of burn patient’s samples. Subsequently, bacteriophages specific to these MRSA isolates were isolated, identified, and characterized. Sample collection Samples were collected from Allied Hospital in Faisalabad, Pakistan, specifically from patients with burn injuries (n = 70). Sterile swabs were utilized for sampling purposes. The procedure involved removing bandages and cleansing the wound area with phosphate buffered saline (PBS) before obtaining samples from the surface of the burn wound. Following collection, the samples were appropriately labeled and stored at 4°C for future analysis [ 16 ]. Isolation of S. aureus Bacteria isolation involved the utilization of commercially available culture media (Oxoid, UK). Following sample collection, swabs were streaked onto both nutrient agar (Oxoid, UK) and Staph-110 agar (Oxoid, UK) media. Incubation at 37°C for 24 hours ensued. Subsequently, the inoculum was transferred onto Mannitol Salt Agar (MSA) plates (Oxoid, UK) to facilitate the selective growth of S. aureus [ 17 ]. After a further 24 hours of incubation at 37°C, the plates were examined to assess the presence of S. aureus growth. Characterization of S. aureus The representative colonies sub-cultured on MSA were further processed for identification and confirmation of S. aureus. This was done based on Gram’s straining by following the protocol explained by [ 18 ], morphological characters and biochemical tests. For morphological identification, cultured bacterial colonies were incubated for one day and their size, texture, shape, and color were examined to identify cultural characteristics [ 19 ]. After examining their physical appearances, the required colonies (positive for Gram staining and cocci in shape) were streaked on new Petri plates to obtain selected colonies ( S. aureus ). For this, quadrant streaking was performed and pure culture was stored for further use. For biochemical characterization, tests including catalase test [ 20 ], beta hemolysis [ 21 ], coagulase test [ 19 ], Voges-Proskauer (VP) test [ 21 ] and mannitol fermentation test [ 20 ] were performed. Antibiotic sensitivity profiling The isolated strains underwent further testing for antimicrobial sensitivity using the disc diffusion method, following standard guidelines outlined by the Clinical and Laboratory Standards Institute (CLSI). Various antibiotics, including penicillin (30 µg), gentamicin (30 µg), methicillin (15 µg), vancomycin (30 µg), clindamycin (30 µg), rifampin (30 µg), cefotaxime (30 µg), azithromycin (30 µg), oxacillin (30 µg), linezolid (30 µg), amoxicillin with clavulanic acid (30 µg), and sulfamethoxazole (30 µg), were employed for the testing. To facilitate the testing, Mueller-Hinton agar media was prepared, containing beef extract (2 g/L), acid hydrolysate of casein (17.5 g/L), starch (1.5 g/L), and agar (17 g/L). The inoculum of each isolate was adjusted to ½ McFarland turbidity standard in normal saline, achieved by dissolving 2–3 freshly grown colonies and inoculating the sample onto agar plates. Antibiotic discs were then placed on the plates, which were subsequently incubated for 24 hours at 37°C. Following the incubation period, the zone of inhibition around each disc was measured. Based on these measurements, the isolates were categorized as resistant, intermediate, or sensitive, in accordance with CLSI guidelines [ 17 , 19 ]. Molecular characterization of S. aureus The identification of isolated and pure isolates was further confirmed through PCR, targeting the nuc and mecA genes, following the protocol outlined by [ 22 ]. The forward and reverse primers of nuc and mecA genes respectively, are as followes: (F = 5′-TACAAAGGTCAACCAATGACATTCAGACTA-3′; R = 5′-TAAATGCACTTGCTTCAGGGCCATAT-3′) and (F = 5′-TCAGGTACTGCTATCCACCCTCAA-3′ ; R = 5′-TGGATAGACGTCATATGAAGGTGTGCT-3′). Initially, bacterial DNA was extracted by centrifuging an overnight bacterial suspension (1.5 mL) at 8000 rpm for five minutes. This process was repeated after washing the pellet with normal saline, and the resulting pellet was dissolved in 0.5 mL of normal saline, followed by the addition of 0.5 mL of 1% SDS solution. Subsequently, 5 µL of proteinase K was added, and the suspension was incubated at 56°C for 18 hours. Following this, 1 mL of phenol chloroform isoamyl alcohol was added, agitated with a 1 mL pipette, and then centrifuged at 8000 rpm for 5 minutes. This step was repeated twice, with the second repetition using chloroform instead of phenol chloroform isoamyl alcohol. After centrifugation, 0.8 volumes of ice-chilled isopropanol and 0.2 volumes of sodium acetate were added to the supernatant, which was then incubated at -5°C for 10 minutes. The suspension was subsequently centrifuged at 13000 rpm for 15 minutes, and after discarding the supernatant, the pellet was washed using 70% ethanol. Once dried, it was dissolved in 20 µL of DNAase-free molecular biology-grade water and stored for further use. For the analysis of DNA bands on the gel, a 1% agarose gel was employed. The gel was run at a steady 175V for approximately an hour, and the resulting bands were photographed using a gel imager [ 23 ]. Isolation and enrichment of bacteriophages Bacteriophages were isolated from sewage water using the soft agar overlay method as described by [ 24 ], were obtained from sewage water. Initially, bacterial isolates were cultured in 5 mL of Staph-110 broth at 37°C for 24 hours. Subsequently, water samples were centrifuged at 5000 rpm for five minutes and filtered using a syringe filter (0.45 µm). A 500 µl aliquot of day-old culture was then mixed with 1.5 mL of Staph-110 broth and 1 mL of filtered sewage water in an Eppendorf tube, followed by incubation at 37°C for 24 hours. After incubation, the mixture was centrifuged at 10,000 rpm for five minutes, and the supernatant was filtered using a 0.22 µm syringe filter to eliminate remaining bacteria. Next, 1 mL of the amplified supernatant was incubated with 500 µl of bacterial suspension at 37°C for 40 minutes to allow for and 1.5 mL of nutrient fresh broth was added, which made this mixture to a total volume of 3 ml. This 3 ml mixture was mixed with 3 ml of 0.8% w/v of soft agar (molten at 45°C) so that final concentration of soft agar was 0.4% w/v and this mixture was poured onto an agar plate spread quickly. The plates were allowed to cool and solidify for 15 minutes without disturbance before being placed in a 37°C incubator for 24 hours. For the enrichment of isolated bacteriophages, selected plaques were circled with a lab marker on the plate base. Each plaque was picked up using sterile scalpel then mixed with 100 µL of Phage Buffer/SM buffer in a sterile, and placed in clearly labeled microcentrifuge tube followed by trituration with a pipette. Subsequently, 10 µl of chloroform was added and mixed well. Finally, the phages were stored at 4°C after proper labeling [ 25 ]. Determination of phage titer Phage titers were determined using the agar overlay method, which involved serially diluting phage samples and counting plaque numbers. To initiate the serial dilutions, 50 µL of SM buffer was added to the Eppendorf tubes. Subsequently, 5 µL of phage was introduced into the first tube, followed by gentle mixing. Then, 5 µL of the mixture from the first tube was transferred to the second tube and mixed again. This process continued to achieve dilutions ranging from 10 3 -10 8 . Afterward, 500 µL of bacterial suspension was added to each tube, and the mixtures were incubated for 45 minutes to allow for adsorption of phages. Following the incubation period, this mixture was used in agar overly method and plates were then incubated at 37°C for 24 hours to allow for plaque formation [ 26 ]. Host range analysis Host range analysis of phages against S. aureus was conducted using a spot assay method as described by [ 27 ]. A diagram illustrating the plate layout was prepared. Initially, 200 µL of the bacterial host suspension in log phase growth was evenly spread onto an agar plate using a sterile spreader and allowed to set for 15 minutes. Subsequently, 3 µL of each phage dilution was pipetted onto their respective locations on the plate using a 10 µL pipette. As a negative control, a spot of 3 µL SM buffer was placed in the final space. Following this, the plate was air dried for 10–15 minutes before being positioned upright and incubated at 37°C for 24 hours. Characterization of phage Specific pH and thermo-stability To evaluate the thermos-stability of bacteriophage suspensions, they were subjected to varying temperatures ranging from 20°C to 80°C for 15 minutes and phage titer was determined by agar overlay method [ 47 ]. For the assessment of pH stability, bacteriophage samples were dispersed into distinct tubes containing SM buffer. These samples were then subjected to a 2-hour incubation at 37°C across a pH range spanning from 3 to 11. Subsequently, phage titers were determined utilizing the agar overlay method [ 28 ]. Determination of phage adsorption time Bacteriophage suspensions with a Multiplicity of Infection (MOI) of 0.05 were incubated with a culture of S. aureus at 37°C. Subsequently, at time points of 10, 15, 20, 25, 30, 45, and 55 minutes, samples measuring 1000 µL each were subjected to centrifugation at 12000 rpm for 5 minutes. Following centrifugation, the supernatant was carefully collected, and the phage titer was determined using the agar overlay method. This allowed for the calculation of the percent decrease of free phages over time, employing the methodology with slight modifications as described by [ 29 ]. One-step growth curve The culture of Staphylococcus aureus was mixed with phage at a multiplicity of infection (MOI) of 0.0001 and allowed to adsorb for 15 minutes at 37°C, followed by centrifugation at 5000 rpm for 10 minutes. Subsequently, the bacterial pellet underwent two washes before being resuspended in pre-warmed LB broth and then incubated at 37°C. Samples were collected at various time intervals (every 10 minutes) for a total of nine times and diluted accordingly. The phage titer of each sample was determined using the agar overlay method as described by [ 30 ]. RESULTS Prevalence rate and isolation of S. aureus A total of 70 burn wound swab samples were collected for the isolation and identification of S. aureus. It was determined that 50 out of the 70 samples (71.4%) tested positive for S. aureus through culture isolation and biochemical identification. Among these S. aureus -positive samples, 20 out of 50 (40%) were identified as Methicillin-resistant Staphylococcus aureus (MRSA) (Fig. 1 a). The overall prevalence of MRSA among the study population was 28.6% (20 out of 70). Upon inoculation of burn wound samples on mannitol salt agar (MSA) and nutrient agar, positive growth was observed. The S. aureus positive isolates exhibited yellow colonies on nutrient agar (Fig. 1 b) while yellow colonies with yellow zones on MSA (Fig. 1 c). Characterization of S. aureus The yellow-colored colonies, which exhibited positivity in Gram staining, were further analyzed through biochemical characterization to confirm their identity as Staphylococcus aureus . Microscopic examination following Gram staining revealed the presence of characteristic purple-colored cocci (Fig. 2 a). Subsequent biochemical tests affirmed the identity of the isolates as S. aureus , as they tested positive for catalase (Fig. 2 b), coagulase (Fig. 2 c), and Mannitol fermentation (Fig. 2 d) tests. However, the Voges-Proskauer (VP) test yielded negative results for this bacterium. Moreover, in hemolysis assays conducted on sheep blood agar, the positive isolates exhibited beta hemolysis accompanied by inhibition zones. (Fig. 2 e) Molecular characterization of S. aureus The suspected S. aureus isolates were subsequently confirmed through PCR targeting the nuc and mecA genes. The findings revealed that positive S. aureus isolates exhibited a band size of 279bp for the nuc gene and 310bp for the mecA gene (refer to Fig. 3 a and Fig. 3 b for visual representation). Antibiotic Susceptibility of S. aureus The antibiotic susceptibility of positive S. aureus isolates was assessed using Kirby Bauer's disc diffusion method. Following CLSI guidelines, the zone of inhibition around each disc was measured, as shown Fig. 4 a for a representative strain of S. aureus . The results, depicted in Fig. 4 b revealed that 94% of the isolates exhibited sensitivity to tobramycin and gentamycin. Vancomycin showed the least sensitivity at 2%, followed by azithromycin at 21% and ampicillin at 40%. In Fig. 3 b, it was observed that sulfamethoxazole had the least resistance at 2%, followed by fusidic acid at 5%, and clindamycin at 7%. Isolation of Bacteriophages Three phages, namely Phage-1, Phage-2, and Phage-3, were successfully isolated using the agar overlay method. These phages exhibited strong lytic activity against eleven MRSA isolates, warranting their selection for further analysis. When propagated in petri plates, these phages produced large to medium-sized plaques (3–5 mm) against the tested MRSA isolates, as the results are illustrated by one representative phage in Fig. 5 . Host range analysis The positive isolates of MRSA underwent further processing to determine their host-range against tested phages. Results revealed that phages exhibited lytic activity against all tested MRSA isolates the phages assessed, as evidenced by the inhibition zone (Fig. 6 ). Table 1 outlines the specific activity of different phages against MRSA strains. All three phages, Phage-1, Phage-2, and Phage-3 were tested for lytic activity. Notably, Phage-3 demonstrated broad lytic activity, leading to its selection for further characterization. Tell here how many percent tested strains were lysed by each of the phage. Table 1 List of host range of various MRSA isolates against the selected phages. (Colored boxes show Phage lysis and white boxes show no lysis) MRSA strains Bacteriophages Phage-1 Phage-2 Phage-3 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Temperature and pH sensitivity of phage 3 Because Phage-3 exhibited broadest lytic spectrum against tested strains, so it was chosen for further characterization. Phage-3 exhibited sustained activity levels of 10 8 at 20°C, 30°C, and 40°C. However, there was a decline in phage activity to 10 4 and 10 2 at 50°C and 60°C respectively. Subsequently, phage activity was completely lost after exposure to 70°C or 80°C for 15 minutes (Fig. 7 a). At pH 3 and 4, Phage-3 was non-viable, while at pH 5, it showed viability of 10 6 . Remarkably, consistent viability of 10 8 was observed at pH 6, 7, and 8. However, at pH 9, 10, and 11, the viability rapidly decreased to 10 4 and 10 2 pfu respectively (Fig. 7 b). Phage titers were assessed at various time points using the agar overlay method to track the decrease in free phages over time. The results revealed a sharp decline in phage-3 adsorption within the first 0 to 5 minutes. By 25 minutes, there was no further adsorption of phage-3 observed (Fig. 7 c). In the one-step growth curve analysis, the latent period concluded after 30 minutes, followed by an increase in phage titer (growth phase), which persisted until 50 minutes before reaching a plateau (Fig. 7 d). DISCUSSION Burn wound infections pose a significant challenge in clinical management, exacerbated by the presence of methicillin-resistant Staphylococcus aureus (MRSA), polymicrobial flora, and fungi. The emergence of antibiotic resistance, recognized as a global public health threat, further complicates treatment strategies [ 31 ]. MRSA, characterized by the mecA gene encoding the low-affinity penicillin-binding protein (PBP2a), conferring resistance to β-lactam antibiotics, has become endemic worldwide [ 32 ]. Thus, identifying and managing MRSA infections are critical in therapeutic settings. In our study, we aimed to isolate and characterize MRSA strains from burn wound samples and elucidate the bacteriophages targeting these strains. Utilizing a percentage prevalence approach, we determined that S. aureus accounted for 71.4% of isolates, with MRSA comprising 40% of them, indicating a substantial prevalence of MRSA within our research cohort [ 33 ]. Specifically, MRSA was prevalent in 28.6% of the total samples. This prevalence aligns with previous research by [ 32 ], who found MRSA in 28.3% of burn patients, showcasing the persistent challenge posed by MRSA in such populations. Furthermore, our findings corroborate those of [ 34 ], who reported a significant incidence of MRSA isolates through culture identification and biochemical assays, emphasizing the widespread presence of MRSA in clinical settings. In summary, our study reinforces the urgent need for vigilance in detecting and managing MRSA infections, particularly in the context of burn wounds, where the consequences of infection can be severe. In our investigation, both culture identification and biochemical assays yielded positive results for S. aureus. Colonies displaying a yellow tint, along with inhibition zones, were observed on both mannitol salt agar and nutrient agar plates, confirming the presence of S. aureus isolates. Positive Gram staining further confirmed the presence of purple cocci. Biochemical testing demonstrated the ability of S. aureus isolates to catalyze reactions, coagulate blood, and ferment Mannitol. Isolates exhibiting beta hemolysis and inhibitory zones on sheep blood agar were considered positive. These findings were consistent with those of [ 35 ], who conducted research with identical aims. Moreover, positive results for the coagulase enzyme test were in line with expectations for S. aureus. Confirmation of S. aureus isolates was also possible through catalase and mannitol salt agar assays, as noted by [ 36 ]. [ 37 ] similarly focused on S. aureus isolation and identification, reporting positive results for gram-positive cocci on mannitol salt agar, as well as positive catalase and coagulase tests. Furthermore, [ 38 ] identified S. aureus isolates through culture techniques, noting yellow growth on nutrient agar and yellow colonies with inhibition zones on mannitol salt agar plates, findings consistent with our own. These results underscore the effectiveness of traditional culture techniques in isolating and identifying S. aureus , despite their time-consuming and tedious nature. Experts in both human and animal health are alarmed by the rise of antibiotic-resistant staphylococci , likely stemming from inappropriate antimicrobial usage by healthcare professionals, inexperienced practitioners, and self-medication by consumers. Regular use of these treatments may lead to the emergence of resistant strains, rendering S. aureus particularly resilient to commonly used antibiotics [ 39 ]. Antibiotic sensitivity testing was performed in our investigation, revealing that most isolates were susceptible to gentamicin (94%) and tobramycin (94%). However, only 2% of isolates were sensitive to vancomycin, while 21% were sensitive to azithromycin. Sulfamethoxazole (2% resistance), fusidic acid (5% resistance), and clindamycin (7% resistance) exhibited favorable outcomes. All S. aureus isolates demonstrated resistance to penicillin, with varying degrees of resistance to other antibiotics such as gentamicin (53.8%), clindamycin (7.7%), and azithromycin (61.5%) [ 32 ]. In a similar study by and [ 40 ], utilizing the same methodology, 32.4% of S. aureus isolates were found to be resistant to azithromycin. Moreover, the percentage of amoxicillin-, oxytetracycline-, and sulfa-resistant MRSA isolates exceeded the overall percentage. However, kanamycin (75%), streptomycin (58.3%), and nalidixic acid (50%) showed efficacy against MRSA isolates. Discrepancies between our findings and theirs may be attributed to differences in sample sizes and geographical locations. Additionally, regional antibiotic usage patterns could potentially contribute to such differences. In our analysis, the mecA gene, which is crucial in the development of antibiotic resistance in MRSA, was identified, that is consistent with previous findings [ 41 ]. This observation aligns with the work of [ 42 ], who also discovered the mecA gene in S. aureus. Conversely, [ 43 ] did not detect the mecA gene in S. aureus isolates from frozen chicken meat. The potential of bacteriophages as alternatives to antibiotics has garnered attention. They could serve as effective treatments for drug-resistant staphylococcus infections acquired in various settings. Despite antibiotics being the primary treatment for such infections, resistance to commonly used medications like methicillin and vancomycin is increasingly prevalent [ 44 ]. In our research, bacteriophages were isolated and characterized against MRSA isolates. Phage-3 demonstrated robust lytic activity against all MRSA isolates. Additionally, we found that phage3 PK0AM1 exhibited stability at pH 6–8 and 20–40°C. Similar findings regarding temperature and pH sensitivity were also reported by [ 45 ] and [ 46 ]. An adsorption time till 25 minutes was recorded with a sharp decline in adsorption time from 0–5 minutes and phage showed latent phase till 30 minutes and phage titer at 30–50 minutes in one step growth curve. CONCLUSION The findings of this study underscore the significance of further investigations into phage 3 and its potential host range, suggesting promising avenues for phage-related research. These results hold particular relevance for the development of bacteriophage-based treatment strategies aimed at combating MRSA strains, particularly those associated with critical clonal lineages. Declarations Funding No funding was received for this study. Ethics approval All procedures performed in studies involving human participants were in accordance with the Ethical standards of the Institutional Biosafety Ethical approval from University of Agriculture, Faisalabad (Ref. No. 1292/ORIC/2021/03 dated 18.03.2021). 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Isolation and identification of Staphylococcus aureus from milk and milk products, associated factors for contamination, and their antibiogram in Holeta, Central Ethiopia. Veterinary Medicine International , 2022 . Neelam, Jain, V. K., Singh, M., Joshi, V. G., Chhabra, R., Singh, K., & Rana, Y. S. (2022). Virulence and antimicrobial resistance gene profiles of Staphylococcus aureus associated with clinical mastitis in cattle. Plos one , 17 (5), e0264762. Ocloo, R., Nyasinga, J., Munshi, Z., Hamdy, A., Marciniak, T., Soundararajan, M., ... & Whitelaw, A. (2022). Epidemiology and antimicrobial resistance of staphylococci other than Staphylococcus aureus from domestic animals and livestock in Africa: a systematic review. Frontiers in Veterinary Science , 9 , 1059054. Yimana, M., & Tesfaye, J. (2022). Isolation, identification and antimicrobial profile of methicillin‐resistant Staphylococcus aureus from bovine mastitis in and around Adama, Central Ethiopia. Veterinary Medicine and Science , 8 (6), 2576-2584. Khairullah, A. R., Rehman, S., Sudjarwo, S. A., Effendi, M. H., Ramandinianto, S. C., Gololodo, M. A., ... & Kurniawati, D. A. (2022). Detection of mecA gene and methicillin-resistant Staphylococcus aureus (MRSA) isolated from milk and risk factors from farms in Probolinggo, Indonesia. F1000Research , 11 . Bokharaei, N. M., Dallal, M. S., Pourmand, M. R., & Rajabi, Z. (2020). Antibiotic resistance pattern and detection of mecA gene in Staphylococcus aureus isolated from Iranian Hamburger samples. Journal of food quality and hazards control . Parvin, M. S., Ali, M. Y., Talukder, S., Nahar, A., Chowdhury, E. H., Rahman, M. T., & Islam, M. T. (2021). Prevalence and multidrug resistance pattern of methicillin resistant S. aureus isolated from frozen chicken meat in Bangladesh. Microorganisms , 9 (3), 636. Samarahan, K. (2020). Could bacteriophages isolated from the sewage be the solution to methicillin-resistant Staphylococcus aureus?. 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Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4554648","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":317000102,"identity":"a80bb566-2529-445e-8a91-0badc9ef4c76","order_by":0,"name":"Nishat Zafar","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8klEQVRIiWNgGAWjYDACZgY2KOMAA8MHBoYEIrWA1LElMDDOIEoLA5IWZh5itJizsz978PMHgz1/G/Ozz7Ztdnn87A1sEh9zcGuxbOYxN+wBWiBxjM14dm5bcrFkzwFmw5nbcGsxOMzDJsGTAHTd/QZj5tw25sQNNxIYH/Pi1cL+TPJPAgOP/DH2z8yWbfUgLQyH8WthMJMG2iJhcIzHmJmx7TAxtvCYScukMRgYHuMpZuw5dzxxZs/BZvx+OX/8meQbGwZ7uWPsmxl+lFUn9rM3H5P4iEcLFPyHUIzglMDYQFA9EvhDiuJRMApGwSgYKQAASHpKe5d4SqUAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-2855-0410","institution":"University of Agriculture Faisalabad","correspondingAuthor":true,"prefix":"","firstName":"Nishat","middleName":"","lastName":"Zafar","suffix":""},{"id":317000103,"identity":"f8ef61f6-bd71-4bcb-80ea-b5ae2c0bee89","order_by":1,"name":"Aamir Aslam","email":"","orcid":"","institution":"University of Agriculture Faisalabad","correspondingAuthor":false,"prefix":"","firstName":"Aamir","middleName":"","lastName":"Aslam","suffix":""},{"id":317000104,"identity":"c2b9a699-e623-4bd9-b789-7e3a885a3541","order_by":2,"name":"Sajjad Ur Rahman","email":"","orcid":"","institution":"University of Agriculture Faisalabad","correspondingAuthor":false,"prefix":"","firstName":"Sajjad","middleName":"Ur","lastName":"Rahman","suffix":""},{"id":317000105,"identity":"6a649795-7697-466d-ac49-bdc0f9415ca0","order_by":3,"name":"Muhammad Saqib","email":"","orcid":"","institution":"University of Agriculture Faisalabad","correspondingAuthor":false,"prefix":"","firstName":"Muhammad","middleName":"","lastName":"Saqib","suffix":""}],"badges":[],"createdAt":"2024-06-09 17:37:00","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4554648/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4554648/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":60599736,"identity":"3ad213d6-3211-4c23-a9d2-aff91dbabbeb","added_by":"auto","created_at":"2024-07-18 15:59:34","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":653414,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePrevalence rate and isolation of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS. aureus. \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e(a) Prevalence rate of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS. aureus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and MRSA (b) \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS. aureus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e growth on nutrient media (c) \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS. aureus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e growth on MSA media\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4554648/v1/6c5427e0f667efc1542210c3.png"},{"id":60599735,"identity":"c55bc9f7-cf9d-46f0-bc90-cbd23b662000","added_by":"auto","created_at":"2024-07-18 15:59:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":862889,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS. aureus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003e(a) Gram staining, (b) catalase test, (c) coagulase test, (d) Mannitol fermentation test (A) Positive (b) Negative result and (e) Beta hemolysis test\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4554648/v1/11ed0199c87cde47d4877ccc.png"},{"id":60599711,"identity":"08d46930-8fda-44dd-b679-eef8c1239abd","added_by":"auto","created_at":"2024-07-18 15:59:30","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":429167,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMolecular characterization of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS. aeureus\u003c/strong\u003e\u003c/em\u003e (a) Molecular detection of \u003cem\u003enuc\u003c/em\u003e gene by gel electrophoresis; Lane 1 is DNA ladder, Lanes 2, 3, 4, 5, 7 and 8 are positive samples. Lane 9 indicates positive control and Lane 10 indicates negative control. (b) Molecular detection of \u003cem\u003emecA\u003c/em\u003e gene by gel electrophoresis; Lane 1 is DNA ladder, Lanes 2-5, 7 and 8 are positive isolates with a band size of 310bp. Lane 9 indicates positive control and Lane 10 indicates negative control.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4554648/v1/ff1462e058b47e2b18f27206.png"},{"id":60599738,"identity":"c172214d-65a0-459c-ae27-b78dfc7ab3f5","added_by":"auto","created_at":"2024-07-18 15:59:34","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":349792,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAntimicrobial susceptibility profile of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS. aureus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003e(a) zone of inhibition (b) Antibiotic susceptibility pattern of \u003cem\u003eS. aureus \u003c/em\u003epositive isolates.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4554648/v1/4c33d1d9201d56a778e19cf3.png"},{"id":60599734,"identity":"29d53a23-863d-412a-bf8f-9feff397918d","added_by":"auto","created_at":"2024-07-18 15:59:33","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":203723,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBacteriophage isolation;\u003c/strong\u003eThe arrow line exhibits the formation of plaques formed by phages.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4554648/v1/b33babb7b9814d72116f2d72.png"},{"id":60599737,"identity":"d73f1e3f-834a-4a4b-af24-5fe410bbb60f","added_by":"auto","created_at":"2024-07-18 15:59:34","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":213703,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHost range test of phages (\u003c/strong\u003ePhage-1, Phage-2, and Phage-3\u003cstrong\u003e) against selected MRSA isolates;\u003c/strong\u003e a clear zone of inhibition indicates positive lytic activity.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4554648/v1/24a97961f874cba715d26d75.png"},{"id":60599712,"identity":"d71be2f0-eff1-4b37-b24a-930185f1f119","added_by":"auto","created_at":"2024-07-18 15:59:31","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":125679,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of Phage-3. (a) Thermostability evaluation (b) Optimum pH evaluation (c) Adsorption time evaluation (d) One step growth curve analysis\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-4554648/v1/c0eb94ebc6e92adeab72194d.png"},{"id":61725733,"identity":"533edfcc-2916-48fd-b87a-b318119f0a79","added_by":"auto","created_at":"2024-08-04 17:56:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5015792,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4554648/v1/dc7b41d4-806a-4dee-9845-c262c273dd3e.pdf"}],"financialInterests":"","formattedTitle":"Isolation and characterization of bacteriophages from sewerage water targeting methicillin resistance S. aureus in burn patients","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eGlobally, \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (\u003cem\u003eS. aureus\u003c/em\u003e) stands out as the primary pathogen responsible for burn infections, predominantly acquired in hospital settings, with a tendency to infect the skin and inhabit the human nares. Approximately 20\u0026ndash;40% of the population is affected by skin infections caused by \u003cem\u003eS. aureus.\u003c/em\u003e The microorganism utilizes quorum sensing for cell-to-cell communication, facilitating its colonization at various sites [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eS. aureus\u003c/em\u003e is renowned as one of the most significant bacteria causing diseases in humans. It is implicated in various infections of the skin and soft tissues, such as abscesses (boils), furuncles, and cellulitis, as well as more severe conditions including pneumonia, osteomyelitis, and septicemia, albeit most of these infections are typically not highly severe [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe bacterium is accountable for numerous serious skin infections, establishing it as one of the most notorious and prevalent bacterial pathogens. Each year, a substantial number of individuals worldwide suffer from skin diseases caused by \u003cem\u003eS. aureus.\u003c/em\u003e Moreover, it contributes to pneumonia, respiratory tract infections, cardiovascular complications, and other hospital-acquired infections [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eS. aureus\u003c/em\u003e bloodstream infections have been documented at an annual rate of 20\u0026ndash;50 per 100,000 individuals, with a mortality rate ranging from 10\u0026ndash;30 percent. In the United States alone, \u003cem\u003eS. aureus\u003c/em\u003e bacteremia is responsible for an estimated 20,000 deaths annually. While less immediately life-threatening, moderately severe skin infections caused by \u003cem\u003eS. aureus\u003c/em\u003e, such as furuncles, abscesses, and wound infections, can still result in significant morbidity [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The mortality and morbidity rates associated with \u003cem\u003eS. aureus\u003c/em\u003e bloodstream infections surpass those of other diseases such as viral hepatitis, acquired immune deficiency syndrome, and tuberculosis. Additionally, \u003cem\u003eS. aureus\u003c/em\u003e has been linked to atopic dermatitis, contributing to a substantial global public health burden [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHorizontal gene transfers enable \u003cem\u003eStaphylococcus aureus\u003c/em\u003e to acquire antibiotic-resistant genes, leading to resistance against various antibiotics [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. These genes may have evolved in antibiotic producers as a defense mechanism against inhibitory molecules, facilitating their survival in competitive environments. The prevalence of antibiotic-resistant bacteria, including those resistant to methicillin, is increasing globally, particularly in healthcare settings like burn units, where methicillin-resistant \u003cem\u003eS. aureus\u003c/em\u003e (MRSA) has become a significant nosocomial pathogen [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTreatment of MRSA infections often involves antibiotics such as quinupristin, dalfopristin, and glycopeptides like vancomycin. However, resistance to these antibiotics, as well as to oxacillin, methicillin, tetracycline, and tigecycline, is observed in many strains of \u003cem\u003eS. aureus.\u003c/em\u003e In some cases, resistance to vancomycin and linezolid may also have emerged [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe development of drug resistance in \u003cem\u003eS. aureus\u003c/em\u003e is facilitated by mobile genetic elements (MGEs), which are transferred via horizontal gene transfer. These elements encode virulence factors and antibiotic resistance mechanisms, including resistance to beta-lactam antibiotics [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe emergence of methicillin-resistant \u003cem\u003eS. aureus\u003c/em\u003e (MRSA) and vancomycin-resistant \u003cem\u003eS. aureus\u003c/em\u003e (VRSA) underscores the urgent need for new antimicrobial agents. Despite the resistance of these strains to commonly used antibiotics, current standards of care recommend antibiotics such as Daptomycin and Linezolid [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. However, the discovery of new antibiotics has been slow in recent decades, despite the effectiveness of existing drugs in most cases. MRSA infections in burn patients remain a significant concern globally, with reported prevalence rates exceeding 50%, and in some studies, surpassing 66% in recent years [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the era of antibiotic resistance, phages are increasingly recognized as promising candidates for controlling bacterial infections across various domains. Their targeted mechanisms of action have led to their utilization in diverse fields including the food industry, medicine, animal husbandry, biotechnology, and agriculture. Numerous studies have documented the efficacy of phages against various pathogens, including \u003cem\u003eS. aureus.\u003c/em\u003e Phages specifically targeting \u003cem\u003eS. aureus\u003c/em\u003e have been identified and deployed for their potential in combating \u003cem\u003eS. aureus\u003c/em\u003e infections. For instance, in a recent study, a cocktail of phages was assessed for its effectiveness against MRSA both in vivo and in vitro, suggesting a promising avenue for treating \u003cem\u003eS. aureus\u003c/em\u003e infections by combining phages with conventional antibiotics [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMoreover, it's crucial to note that some bacteriophage genomes harbor genes encoding highly virulent bacterial toxins, rendering the host bacterium pathogenic only upon lysogenization by the toxin-encoding phage. Additionally, the transfer of antibiotic resistance genes to bacteria through phages has been documented. Consequently, research endeavors have been initiated to leverage phages for gene insertion into desired bacterial strains, with previous studies demonstrating feasibility and ongoing plans for further exploration [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe present research was designed to isolate Methicillin Resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e strains from burn patients and ascertain their antibiogram patterns. Furthermore, a central objective of this study was the isolation and characterization of bacteriophages specifically targeting Methicillin Resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e.\u003c/p\u003e"},{"header":"METHODOLOGY","content":"\u003cp\u003eThis research was conducted at the Institute of Microbiology, Faculty of Veterinary Science, University of Agriculture Faisalabad, received ethical approval from the Institutional Biosafety Committee (IBC) (give reference number of letter). During this investigation, Methicillin-resistant \u003cem\u003eS. aureus\u003c/em\u003e (MRSA) was isolated and identified from wound pus of burn patient\u0026rsquo;s samples. Subsequently, bacteriophages specific to these MRSA isolates were isolated, identified, and characterized.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSample collection\u003c/h2\u003e \u003cp\u003eSamples were collected from Allied Hospital in Faisalabad, Pakistan, specifically from patients with burn injuries (n\u0026thinsp;=\u0026thinsp;70). Sterile swabs were utilized for sampling purposes. The procedure involved removing bandages and cleansing the wound area with phosphate buffered saline (PBS) before obtaining samples from the surface of the burn wound. Following collection, the samples were appropriately labeled and stored at 4\u0026deg;C for future analysis [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eIsolation of\u003c/b\u003e \u003cb\u003eS. aureus\u003c/b\u003e\u003c/p\u003e \u003cp\u003eBacteria isolation involved the utilization of commercially available culture media (Oxoid, UK). Following sample collection, swabs were streaked onto both nutrient agar (Oxoid, UK) and Staph-110 agar (Oxoid, UK) media. Incubation at 37\u0026deg;C for 24 hours ensued. Subsequently, the inoculum was transferred onto Mannitol Salt Agar (MSA) plates (Oxoid, UK) to facilitate the selective growth of \u003cem\u003eS. aureus\u003c/em\u003e [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. After a further 24 hours of incubation at 37\u0026deg;C, the plates were examined to assess the presence of \u003cem\u003eS. aureus\u003c/em\u003e growth.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCharacterization of\u003c/b\u003e \u003cb\u003eS. aureus\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe representative colonies sub-cultured on MSA were further processed for identification and confirmation of \u003cem\u003eS. aureus.\u003c/em\u003e This was done based on Gram\u0026rsquo;s straining by following the protocol explained by [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], morphological characters and biochemical tests. For morphological identification, cultured bacterial colonies were incubated for one day and their size, texture, shape, and color were examined to identify cultural characteristics [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. After examining their physical appearances, the required colonies (positive for Gram staining and cocci in shape) were streaked on new Petri plates to obtain selected colonies (\u003cem\u003eS. aureus\u003c/em\u003e). For this, quadrant streaking was performed and pure culture was stored for further use. For biochemical characterization, tests including catalase test [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], beta hemolysis [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], coagulase test [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], Voges-Proskauer (VP) test [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] and mannitol fermentation test [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] were performed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eAntibiotic sensitivity profiling\u003c/h2\u003e \u003cp\u003eThe isolated strains underwent further testing for antimicrobial sensitivity using the disc diffusion method, following standard guidelines outlined by the Clinical and Laboratory Standards Institute (CLSI). Various antibiotics, including penicillin (30 \u0026micro;g), gentamicin (30 \u0026micro;g), methicillin (15 \u0026micro;g), vancomycin (30 \u0026micro;g), clindamycin (30 \u0026micro;g), rifampin (30 \u0026micro;g), cefotaxime (30 \u0026micro;g), azithromycin (30 \u0026micro;g), oxacillin (30 \u0026micro;g), linezolid (30 \u0026micro;g), amoxicillin with clavulanic acid (30 \u0026micro;g), and sulfamethoxazole (30 \u0026micro;g), were employed for the testing.\u003c/p\u003e \u003cp\u003eTo facilitate the testing, Mueller-Hinton agar media was prepared, containing beef extract (2 g/L), acid hydrolysate of casein (17.5 g/L), starch (1.5 g/L), and agar (17 g/L). The inoculum of each isolate was adjusted to \u0026frac12; McFarland turbidity standard in normal saline, achieved by dissolving 2\u0026ndash;3 freshly grown colonies and inoculating the sample onto agar plates. Antibiotic discs were then placed on the plates, which were subsequently incubated for 24 hours at 37\u0026deg;C. Following the incubation period, the zone of inhibition around each disc was measured. Based on these measurements, the isolates were categorized as resistant, intermediate, or sensitive, in accordance with CLSI guidelines [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eMolecular characterization of\u003c/b\u003e \u003cb\u003eS. aureus\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe identification of isolated and pure isolates was further confirmed through PCR, targeting the \u003cem\u003enuc\u003c/em\u003e and \u003cem\u003emecA\u003c/em\u003e genes, following the protocol outlined by [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The forward and reverse primers of \u003cem\u003enuc\u003c/em\u003e and \u003cem\u003emecA\u003c/em\u003e genes respectively, are as followes:\u003c/p\u003e \u003cp\u003e(F\u0026thinsp;=\u0026thinsp;5\u0026prime;-TACAAAGGTCAACCAATGACATTCAGACTA-3\u0026prime;;\u003c/p\u003e \u003cp\u003eR\u0026thinsp;=\u0026thinsp;5\u0026prime;-TAAATGCACTTGCTTCAGGGCCATAT-3\u0026prime;) and\u003c/p\u003e \u003cp\u003e(F\u0026thinsp;=\u0026thinsp;5\u0026prime;-TCAGGTACTGCTATCCACCCTCAA-3\u0026prime; ;\u003c/p\u003e \u003cp\u003eR\u0026thinsp;=\u0026thinsp;5\u0026prime;-TGGATAGACGTCATATGAAGGTGTGCT-3\u0026prime;).\u003c/p\u003e \u003cp\u003eInitially, bacterial DNA was extracted by centrifuging an overnight bacterial suspension (1.5 mL) at 8000 rpm for five minutes. This process was repeated after washing the pellet with normal saline, and the resulting pellet was dissolved in 0.5 mL of normal saline, followed by the addition of 0.5 mL of 1% SDS solution. Subsequently, 5 \u0026micro;L of proteinase K was added, and the suspension was incubated at 56\u0026deg;C for 18 hours. Following this, 1 mL of phenol chloroform isoamyl alcohol was added, agitated with a 1 mL pipette, and then centrifuged at 8000 rpm for 5 minutes. This step was repeated twice, with the second repetition using chloroform instead of phenol chloroform isoamyl alcohol. After centrifugation, 0.8 volumes of ice-chilled isopropanol and 0.2 volumes of sodium acetate were added to the supernatant, which was then incubated at -5\u0026deg;C for 10 minutes. The suspension was subsequently centrifuged at 13000 rpm for 15 minutes, and after discarding the supernatant, the pellet was washed using 70% ethanol. Once dried, it was dissolved in 20 \u0026micro;L of DNAase-free molecular biology-grade water and stored for further use.\u003c/p\u003e \u003cp\u003eFor the analysis of DNA bands on the gel, a 1% agarose gel was employed. The gel was run at a steady 175V for approximately an hour, and the resulting bands were photographed using a gel imager [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eIsolation and enrichment of bacteriophages\u003c/h2\u003e \u003cp\u003eBacteriophages were isolated from sewage water using the soft agar overlay method as described by [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], were obtained from sewage water. Initially, bacterial isolates were cultured in 5 mL of Staph-110 broth at 37\u0026deg;C for 24 hours. Subsequently, water samples were centrifuged at 5000 rpm for five minutes and filtered using a syringe filter (0.45 \u0026micro;m). A 500 \u0026micro;l aliquot of day-old culture was then mixed with 1.5 mL of Staph-110 broth and 1 mL of filtered sewage water in an Eppendorf tube, followed by incubation at 37\u0026deg;C for 24 hours. After incubation, the mixture was centrifuged at 10,000 rpm for five minutes, and the supernatant was filtered using a 0.22 \u0026micro;m syringe filter to eliminate remaining bacteria. Next, 1 mL of the amplified supernatant was incubated with 500 \u0026micro;l of bacterial suspension at 37\u0026deg;C for 40 minutes to allow for and 1.5 mL of nutrient fresh broth was added, which made this mixture to a total volume of 3 ml. This 3 ml mixture was mixed with 3 ml of 0.8% w/v of soft agar (molten at 45\u0026deg;C) so that final concentration of soft agar was 0.4% w/v and this mixture was poured onto an agar plate spread quickly. The plates were allowed to cool and solidify for 15 minutes without disturbance before being placed in a 37\u0026deg;C incubator for 24 hours.\u003c/p\u003e \u003cp\u003eFor the enrichment of isolated bacteriophages, selected plaques were circled with a lab marker on the plate base. Each plaque was picked up using sterile scalpel then mixed with 100 \u0026micro;L of Phage Buffer/SM buffer in a sterile, and placed in clearly labeled microcentrifuge tube followed by trituration with a pipette. Subsequently, 10 \u0026micro;l of chloroform was added and mixed well. Finally, the phages were stored at 4\u0026deg;C after proper labeling [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of phage titer\u003c/h2\u003e \u003cp\u003ePhage titers were determined using the agar overlay method, which involved serially diluting phage samples and counting plaque numbers. To initiate the serial dilutions, 50 \u0026micro;L of SM buffer was added to the Eppendorf tubes. Subsequently, 5 \u0026micro;L of phage was introduced into the first tube, followed by gentle mixing. Then, 5 \u0026micro;L of the mixture from the first tube was transferred to the second tube and mixed again. This process continued to achieve dilutions ranging from 10\u003csup\u003e3\u003c/sup\u003e-10\u003csup\u003e8\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAfterward, 500 \u0026micro;L of bacterial suspension was added to each tube, and the mixtures were incubated for 45 minutes to allow for adsorption of phages. Following the incubation period, this mixture was used in agar overly method and plates were then incubated at 37\u0026deg;C for 24 hours to allow for plaque formation [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eHost range analysis\u003c/h2\u003e \u003cp\u003eHost range analysis of phages against \u003cem\u003eS. aureus\u003c/em\u003e was conducted using a spot assay method as described by [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. A diagram illustrating the plate layout was prepared. Initially, 200 \u0026micro;L of the bacterial host suspension in log phase growth was evenly spread onto an agar plate using a sterile spreader and allowed to set for 15 minutes. Subsequently, 3 \u0026micro;L of each phage dilution was pipetted onto their respective locations on the plate using a 10 \u0026micro;L pipette. As a negative control, a spot of 3 \u0026micro;L SM buffer was placed in the final space. Following this, the plate was air dried for 10\u0026ndash;15 minutes before being positioned upright and incubated at 37\u0026deg;C for 24 hours.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCharacterization of phage\u003c/h2\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003eSpecific pH and thermo-stability\u003c/h2\u003e \u003cp\u003eTo evaluate the thermos-stability of bacteriophage suspensions, they were subjected to varying temperatures ranging from 20\u0026deg;C to 80\u0026deg;C for 15 minutes and phage titer was determined by agar overlay method [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. For the assessment of pH stability, bacteriophage samples were dispersed into distinct tubes containing SM buffer. These samples were then subjected to a 2-hour incubation at 37\u0026deg;C across a pH range spanning from 3 to 11. Subsequently, phage titers were determined utilizing the agar overlay method [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of phage adsorption time\u003c/h2\u003e \u003cp\u003eBacteriophage suspensions with a Multiplicity of Infection (MOI) of 0.05 were incubated with a culture of \u003cem\u003eS. aureus\u003c/em\u003e at 37\u0026deg;C. Subsequently, at time points of 10, 15, 20, 25, 30, 45, and 55 minutes, samples measuring 1000 \u0026micro;L each were subjected to centrifugation at 12000 rpm for 5 minutes. Following centrifugation, the supernatant was carefully collected, and the phage titer was determined using the agar overlay method. This allowed for the calculation of the percent decrease of free phages over time, employing the methodology with slight modifications as described by [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eOne-step growth curve\u003c/h2\u003e \u003cp\u003eThe culture of \u003cem\u003eStaphylococcus aureus\u003c/em\u003e was mixed with phage at a multiplicity of infection (MOI) of 0.0001 and allowed to adsorb for 15 minutes at 37\u0026deg;C, followed by centrifugation at 5000 rpm for 10 minutes. Subsequently, the bacterial pellet underwent two washes before being resuspended in pre-warmed LB broth and then incubated at 37\u0026deg;C. Samples were collected at various time intervals (every 10 minutes) for a total of nine times and diluted accordingly. The phage titer of each sample was determined using the agar overlay method as described by [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003ePrevalence rate and isolation of\u003c/b\u003e \u003cb\u003eS. aureus\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA total of 70 burn wound swab samples were collected for the isolation and identification of \u003cem\u003eS. aureus.\u003c/em\u003e It was determined that 50 out of the 70 samples (71.4%) tested positive for \u003cem\u003eS. aureus\u003c/em\u003e through culture isolation and biochemical identification. Among these \u003cem\u003eS. aureus\u003c/em\u003e-positive samples, 20 out of 50 (40%) were identified as Methicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (MRSA) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). The overall prevalence of MRSA among the study population was 28.6% (20 out of 70).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUpon inoculation of burn wound samples on mannitol salt agar (MSA) and nutrient agar, positive growth was observed. The \u003cem\u003eS. aureus\u003c/em\u003e positive isolates exhibited yellow colonies on nutrient agar (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb) while yellow colonies with yellow zones on MSA (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003cb\u003eCharacterization of\u003c/b\u003e \u003cb\u003eS. aureus\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe yellow-colored colonies, which exhibited positivity in Gram staining, were further analyzed through biochemical characterization to confirm their identity as \u003cem\u003eStaphylococcus aureus\u003c/em\u003e. Microscopic examination following Gram staining revealed the presence of characteristic purple-colored cocci (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Subsequent biochemical tests affirmed the identity of the isolates as \u003cem\u003eS. aureus\u003c/em\u003e, as they tested positive for catalase (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), coagulase (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec), and Mannitol fermentation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed) tests. However, the Voges-Proskauer (VP) test yielded negative results for this bacterium. Moreover, in hemolysis assays conducted on sheep blood agar, the positive isolates exhibited beta hemolysis accompanied by inhibition zones. (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eMolecular characterization of\u003c/b\u003e \u003cb\u003eS. aureus\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe suspected \u003cem\u003eS. aureus\u003c/em\u003e isolates were subsequently confirmed through PCR targeting the \u003cem\u003enuc\u003c/em\u003e and \u003cem\u003emecA\u003c/em\u003e genes. The findings revealed that positive \u003cem\u003eS. aureus\u003c/em\u003e isolates exhibited a band size of 279bp for the \u003cem\u003enuc\u003c/em\u003e gene and 310bp for the \u003cem\u003emecA\u003c/em\u003e gene (refer to Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb for visual representation).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAntibiotic Susceptibility of\u003c/b\u003e \u003cb\u003eS. aureus\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe antibiotic susceptibility of positive \u003cem\u003eS. aureus\u003c/em\u003e isolates was assessed using Kirby Bauer's disc diffusion method. Following CLSI guidelines, the zone of inhibition around each disc was measured, as shown Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea for a representative strain of \u003cem\u003eS. aureus\u003c/em\u003e. The results, depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb revealed that 94% of the isolates exhibited sensitivity to tobramycin and gentamycin. Vancomycin showed the least sensitivity at 2%, followed by azithromycin at 21% and ampicillin at 40%. In Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, it was observed that sulfamethoxazole had the least resistance at 2%, followed by fusidic acid at 5%, and clindamycin at 7%.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eIsolation of Bacteriophages\u003c/h2\u003e \u003cp\u003eThree phages, namely Phage-1, Phage-2, and Phage-3, were successfully isolated using the agar overlay method. These phages exhibited strong lytic activity against eleven MRSA isolates, warranting their selection for further analysis. When propagated in petri plates, these phages produced large to medium-sized plaques (3\u0026ndash;5 mm) against the tested MRSA isolates, as the results are illustrated by one representative phage in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eHost range analysis\u003c/h2\u003e \u003cp\u003eThe positive isolates of MRSA underwent further processing to determine their host-range against tested phages. Results revealed that phages exhibited lytic activity against all tested MRSA isolates the phages assessed, as evidenced by the inhibition zone (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e outlines the specific activity of different phages against MRSA strains. All three phages, Phage-1, Phage-2, and Phage-3 were tested for lytic activity. Notably, Phage-3 demonstrated broad lytic activity, leading to its selection for further characterization. Tell here how many percent tested strains were lysed by each of the phage.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eList of host range of various MRSA isolates against the selected phages.\u003c/b\u003e (Colored boxes show Phage lysis and white boxes show no lysis)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eMRSA strains\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eBacteriophages\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePhage-1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePhage-2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePhage-3\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eTemperature and pH sensitivity of phage 3\u003c/h2\u003e \u003cp\u003eBecause Phage-3 exhibited broadest lytic spectrum against tested strains, so it was chosen for further characterization. Phage-3 exhibited sustained activity levels of 10\u003csup\u003e8\u003c/sup\u003e at 20\u0026deg;C, 30\u0026deg;C, and 40\u0026deg;C. However, there was a decline in phage activity to 10\u003csup\u003e4\u003c/sup\u003e and 10\u003csup\u003e2\u003c/sup\u003e at 50\u0026deg;C and 60\u0026deg;C respectively. Subsequently, phage activity was completely lost after exposure to 70\u0026deg;C or 80\u0026deg;C for 15 minutes (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003eAt pH 3 and 4, Phage-3 was non-viable, while at pH 5, it showed viability of 10\u003csup\u003e6\u003c/sup\u003e. Remarkably, consistent viability of 10\u003csup\u003e8\u003c/sup\u003e was observed at pH 6, 7, and 8. However, at pH 9, 10, and 11, the viability rapidly decreased to 10\u003csup\u003e4\u003c/sup\u003e and 10\u003csup\u003e2\u003c/sup\u003e pfu respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePhage titers were assessed at various time points using the agar overlay method to track the decrease in free phages over time. The results revealed a sharp decline in phage-3 adsorption within the first 0 to 5 minutes. By 25 minutes, there was no further adsorption of phage-3 observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec). In the one-step growth curve analysis, the latent period concluded after 30 minutes, followed by an increase in phage titer (growth phase), which persisted until 50 minutes before reaching a plateau (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed).\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eBurn wound infections pose a significant challenge in clinical management, exacerbated by the presence of methicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (MRSA), polymicrobial flora, and fungi. The emergence of antibiotic resistance, recognized as a global public health threat, further complicates treatment strategies [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. MRSA, characterized by the \u003cem\u003emecA\u003c/em\u003e gene encoding the low-affinity penicillin-binding protein (PBP2a), conferring resistance to β-lactam antibiotics, has become endemic worldwide [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Thus, identifying and managing MRSA infections are critical in therapeutic settings.\u003c/p\u003e \u003cp\u003eIn our study, we aimed to isolate and characterize MRSA strains from burn wound samples and elucidate the bacteriophages targeting these strains. Utilizing a percentage prevalence approach, we determined that \u003cem\u003eS. aureus\u003c/em\u003e accounted for 71.4% of isolates, with MRSA comprising 40% of them, indicating a substantial prevalence of MRSA within our research cohort [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Specifically, MRSA was prevalent in 28.6% of the total samples.\u003c/p\u003e \u003cp\u003eThis prevalence aligns with previous research by [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], who found MRSA in 28.3% of burn patients, showcasing the persistent challenge posed by MRSA in such populations. Furthermore, our findings corroborate those of [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], who reported a significant incidence of MRSA isolates through culture identification and biochemical assays, emphasizing the widespread presence of MRSA in clinical settings. In summary, our study reinforces the urgent need for vigilance in detecting and managing MRSA infections, particularly in the context of burn wounds, where the consequences of infection can be severe.\u003c/p\u003e \u003cp\u003eIn our investigation, both culture identification and biochemical assays yielded positive results for \u003cem\u003eS. aureus.\u003c/em\u003e Colonies displaying a yellow tint, along with inhibition zones, were observed on both mannitol salt agar and nutrient agar plates, confirming the presence of \u003cem\u003eS. aureus\u003c/em\u003e isolates. Positive Gram staining further confirmed the presence of purple cocci. Biochemical testing demonstrated the ability of \u003cem\u003eS. aureus\u003c/em\u003e isolates to catalyze reactions, coagulate blood, and ferment Mannitol. Isolates exhibiting beta hemolysis and inhibitory zones on sheep blood agar were considered positive. These findings were consistent with those of [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], who conducted research with identical aims. Moreover, positive results for the coagulase enzyme test were in line with expectations for \u003cem\u003eS. aureus.\u003c/em\u003e Confirmation of \u003cem\u003eS. aureus\u003c/em\u003e isolates was also possible through catalase and mannitol salt agar assays, as noted by [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] similarly focused on \u003cem\u003eS. aureus\u003c/em\u003e isolation and identification, reporting positive results for gram-positive cocci on mannitol salt agar, as well as positive catalase and coagulase tests. Furthermore, [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] identified \u003cem\u003eS. aureus\u003c/em\u003e isolates through culture techniques, noting yellow growth on nutrient agar and yellow colonies with inhibition zones on mannitol salt agar plates, findings consistent with our own. These results underscore the effectiveness of traditional culture techniques in isolating and identifying \u003cem\u003eS. aureus\u003c/em\u003e, despite their time-consuming and tedious nature.\u003c/p\u003e \u003cp\u003eExperts in both human and animal health are alarmed by the rise of antibiotic-resistant \u003cem\u003estaphylococci\u003c/em\u003e, likely stemming from inappropriate antimicrobial usage by healthcare professionals, inexperienced practitioners, and self-medication by consumers. Regular use of these treatments may lead to the emergence of resistant strains, rendering \u003cem\u003eS. aureus\u003c/em\u003e particularly resilient to commonly used antibiotics [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAntibiotic sensitivity testing was performed in our investigation, revealing that most isolates were susceptible to gentamicin (94%) and tobramycin (94%). However, only 2% of isolates were sensitive to vancomycin, while 21% were sensitive to azithromycin. Sulfamethoxazole (2% resistance), fusidic acid (5% resistance), and clindamycin (7% resistance) exhibited favorable outcomes. All \u003cem\u003eS. aureus\u003c/em\u003e isolates demonstrated resistance to penicillin, with varying degrees of resistance to other antibiotics such as gentamicin (53.8%), clindamycin (7.7%), and azithromycin (61.5%) [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In a similar study by and [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], utilizing the same methodology, 32.4% of \u003cem\u003eS. aureus\u003c/em\u003e isolates were found to be resistant to azithromycin. Moreover, the percentage of amoxicillin-, oxytetracycline-, and sulfa-resistant MRSA isolates exceeded the overall percentage. However, kanamycin (75%), streptomycin (58.3%), and nalidixic acid (50%) showed efficacy against MRSA isolates. Discrepancies between our findings and theirs may be attributed to differences in sample sizes and geographical locations. Additionally, regional antibiotic usage patterns could potentially contribute to such differences.\u003c/p\u003e \u003cp\u003eIn our analysis, the \u003cem\u003emecA\u003c/em\u003e gene, which is crucial in the development of antibiotic resistance in MRSA, was identified, that is consistent with previous findings [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. This observation aligns with the work of [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], who also discovered the \u003cem\u003emecA\u003c/em\u003e gene in \u003cem\u003eS. aureus.\u003c/em\u003e Conversely, [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] did not detect the \u003cem\u003emecA\u003c/em\u003e gene in \u003cem\u003eS. aureus\u003c/em\u003e isolates from frozen chicken meat.\u003c/p\u003e \u003cp\u003eThe potential of bacteriophages as alternatives to antibiotics has garnered attention. They could serve as effective treatments for drug-resistant staphylococcus infections acquired in various settings. Despite antibiotics being the primary treatment for such infections, resistance to commonly used medications like methicillin and vancomycin is increasingly prevalent [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn our research, bacteriophages were isolated and characterized against MRSA isolates. Phage-3 demonstrated robust lytic activity against all MRSA isolates. Additionally, we found that phage3 PK0AM1 exhibited stability at pH 6\u0026ndash;8 and 20\u0026ndash;40\u0026deg;C. Similar findings regarding temperature and pH sensitivity were also reported by [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e] and [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. An adsorption time till 25 minutes was recorded with a sharp decline in adsorption time from 0\u0026ndash;5 minutes and phage showed latent phase till 30 minutes and phage titer at 30\u0026ndash;50 minutes in one step growth curve.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eThe findings of this study underscore the significance of further investigations into phage 3 and its potential host range, suggesting promising avenues for phage-related research. These results hold particular relevance for the development of bacteriophage-based treatment strategies aimed at combating MRSA strains, particularly those associated with critical clonal lineages.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo funding was received\u0026nbsp;for this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures performed in studies involving human participants were in accordance with the Ethical standards of the Institutional\u0026nbsp;Biosafety\u0026nbsp;Ethical approval from University\u0026nbsp;of Agriculture, Faisalabad\u0026nbsp;(Ref. No.\u0026nbsp;1292/ORIC/2021/03 dated\u0026nbsp;18.03.2021).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the author declares no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eNisar, S., Kirkpatrick, L. D., \u0026amp; Shupp, J. W. (2021). Bacterial virulence factors and their contribution to pathophysiology after thermal injury. \u003cem\u003eSurgical Infections\u003c/em\u003e, \u003cem\u003e22\u003c/em\u003e(1), 69-76.\u003c/li\u003e\n\u003cli\u003eMork, R. L., Hogan, P. G., Muenks, C. E., Boyle, M. G., Thompson, R. M., Sullivan, M. L., ... \u0026amp; Fritz, S. A. (2020). 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Staphylococcus aureus persistence properties associated with bovine mastitis and alternative therapeutic modalities. \u003cem\u003eJournal of applied microbiology\u003c/em\u003e, \u003cem\u003e129\u003c/em\u003e(5), 1102-1119.\u003c/li\u003e\n\u003cli\u003eMahmoud, E. R. A., Ahmed, H. A. H., Abo-senna, A. S. M., Riad, O. K. M., \u0026amp; Abo, M. M. A. A. R. (2021). Isolation and characterization of six gamma-irradiated bacteriophages specific for MRSA and VRSA isolated from skin infections. \u003cem\u003eJournal of Radiation Research and Applied Sciences\u003c/em\u003e, \u003cem\u003e14\u003c/em\u003e(1), 34-43.\u003c/li\u003e\n\u003cli\u003eCha, Y., Son, B., \u0026amp; Ryu, S. (2019). Effective removal of staphylococcal biofilms on various food contact surfaces by Staphylococcus aureus phage endolysin LysCSA13. \u003cem\u003eFood microbiology\u003c/em\u003e, \u003cem\u003e84\u003c/em\u003e, 103245.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Methicillin-resistant Staphylococcus aureus (MRSA), Bacteriophages, Characterization, Antibiogram, Sewerage water","lastPublishedDoi":"10.21203/rs.3.rs-4554648/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4554648/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe spectrum of infections caused by methicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (MRSA) ranges from minor to potentially life-threatening. Due to its increased antibiotic resistance, treatment failures are frequent, necessitating novel eradication approaches. In this study, we aimed to isolate MRSA from burn patients, ascertain its antibiogram pattern, and isolate and characterize bacteriophages from sewerage water targeting MRSA. A total of 70 samples were collected from burn patients, and MRSA was identified and characterized using various biochemical and molecular tests, alongside antibiotic sensitivity profiling. MRSA prevalence was found to be 28.6%, with biochemical and molecular analyses confirming its presence. Antibiotic susceptibility testing revealed that 94% of isolates were sensitive to tobramycin and gentamycin, while lowest sensitivity (2%) was observed against vancomycin. Using the soft agar overlay method, three bacteriophages (Phage-1, Phage-2, and Phage-3) were successfully isolated from sewerage water. Among all the phages, Phage-3 demonstrated a broader host range. Furthermore, Phage-3 displayed optimal activity within a pH range of 6\u0026ndash;8 and at temperatures between 20\u0026ndash;40\u0026deg;C. Phage-3 indicated a rapid decrease in adsorption time within 0\u0026ndash;5 minutes, while its one-step growth curve revealed a latent phase lasting up to 30 minutes, followed by a significant increase in titer from 30\u0026ndash;50 minutes.\u003c/p\u003e","manuscriptTitle":"Isolation and characterization of bacteriophages from sewerage water targeting methicillin resistance S. aureus in burn patients","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-18 15:59:18","doi":"10.21203/rs.3.rs-4554648/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6f6f5f3b-34c2-4675-9446-38de70a78e76","owner":[],"postedDate":"July 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-08-04T17:47:59+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-18 15:59:18","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4554648","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4554648","identity":"rs-4554648","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}