Pseudomonas aeruginosa strains from clinical and environmental sources against gram-positive and gram-negative bacteria and fungi. The studies also analyzed the correlation between enzyme production and inhibitory effects to gain insights into the antimicrobial capabilities of P. aeruginosa strains; (2) Methods: Both studies employed similar methodologies, including the use of disk diffusion and well diffusion methods to assess the inhibitory effects of P. aeruginosa strains against target pathogens. Enzyme production was analyzed through various biochemical assays to determine the diversity and frequencies of enzyme secretion among the strains; (3) Results: A comparative analysis of enzyme production in P. aeruginosa strains from clinical sources revealed significant variations in enzyme production, with hemolysin and protease being the most commonly produced enzymes. Gelatinase production showed lower rates, whereas chondroitinase and hyaluronidase were absent or occurred less frequently. In contrast, a comparative analysis of enzyme production in environmental isolates showed different patterns, indicating adaptation to environmental conditions. Pyocyanin production was absent in all environmental isolates. The inhibitory effects against gram-positive and gram-negative bacteria varied among different P. aeruginosa strains, with strain-specific variations observed. Limited inhibitory effects were observed against fungi, primarily toward gram-positive bacteria; (4) Conclusions: The findings highlight the strain-specific nature of inhibitory effects and enzyme production in P. aeruginosa strains. The correlation between enzyme production and inhibitory effects against gram-positive bacteria suggest a potential role of specific enzymes, such as hemolysin and protease, in the antimicrobial activity. The complexity of the relationship between enzyme production and the inhibition of different pathogens requires further investigation. The results emphasize the potential of P. aeruginosa strains as sources for antimicrobial strategies, particularly against gram-positive bacteria. Future research should focus on understanding the mechanisms underlying these inhibitory effects and exploring their therapeutic applications.(1) Background: This study summarizes the findings of two studies investigating the inhibitory effects of  Pseudomonas aeruginosa is an opportunistic pathogen that poses a significant threat to public health and causes various infections in both clinical and environmental settings [P. aeruginosa isolates from clinical and environmental sources have provided valuable insights into the diversity and significance of enzyme production in clinical infections and the potential antimicrobial applications of P. aeruginosa [P. aeruginosa isolates from clinical and environmental sources. This manuscript aims to explore and contrast the enzyme production and inhibitory potential of P. aeruginosa isolates, shedding light on their adaptation and potential as sources for antimicrobial strategies.settings ,2. Undersettings ,4. This settings ,6. Comparuginosa ,8. HowevP. aeruginosa strains from clinical and environmental sources. By conducting a comprehensive comparative analysis, we will examine the diversity of enzyme production profiles and their potential implications for clinical infections and antimicrobial applications. Our findings will provide valuable insights into the complex interplay between enzyme production, pathogenesis, and antimicrobial activity in P. aeruginosa and pave the way for future research and therapeutic advancements in combating this notorious pathogen.In this study, we further contribute to the understanding of the enzyme production by P. aeruginosa obtained from different clinical sources: blood, sputum, urine samples, and wound swabs  isolates. Notably, blood and urine samples consistently exhibited a 100% occurrence of these enzymes, indicating their strong presence in these sources. Sputum samples and wound swabs also demonstrated relatively high frequencies, with 87% and 90% occurrence, respectively. Conversely, gelatinase production had slightly lower overall rates, with 53 (87%) isolates producing this enzyme. The occurrence of gelatinase varied across different sources, with wound swabs and urine samples exhibiting higher frequencies  than blood and sputum samples .P. aeruginosa isolates from clinical sources.Pyocyanin production was observed in 37 (61%) isolates, with blood samples displaying the highest frequency (100%). In contrast, wound swabs and urine samples had relatively lower frequencies of 15% and 67%, respectively. Fibrinolysin and lipase showed similar overall production rates, with 46 (75%) and 45 (74%) isolates, respectively. Blood samples and wound swabs exhibited the highest frequencies for both enzymes (75% for fibrinolysin and 63% for lipase), while sputum and urine samples showed lower frequencies. Coagulase production was observed in 40 (66%) isolates, with blood samples showing the highest frequency (75%), followed by wound swabs (70%). Sputum and urine samples had lower frequencies . Lecithinase production was detected in 37 (61%) isolates, with wound samples showing the highest frequency (100%). Blood and urine samples had intermediate frequencies, whereas sputum samples showed the lowest frequency. Elastase production was relatively low, with only 17 (28%) isolates producing this enzyme. Blood samples exhibited the highest frequency (56%), while sputum and urine samples had much lower frequencies. DNase production was observed in 8 (13%) isolates, mainly in blood and urine samples . No DNase production was detected in sputum samples or wound swabs. Lastly, no production of chondroitinase or hyaluronidase was observed in any of the samples, indicating their absence among the P. aeruginosa isolates revealed variations in the presence and frequency of different enzymes across blood, sputum, urine samples, and wound swabs. Hemolysin and protease exhibited the highest overall production rates, followed by gelatinase, pyocyanin, fibrinolysin, lipase, coagulase, lecithinase, elastase, DNase, chondroitinase, and hyaluronidase, which either had lower production rates or were absent in the isolates. These findings highlight the diverse enzymatic profiles of P. aeruginosa strains and provide valuable insights into their potential roles in clinical infections. Hemolysin and protease in blood and urine samples suggest their significance in systemic and urinary tract infections caused by P. aeruginosa. Additionally, the high occurrence of gelatinase in wound swabs and urine samples indicates its potential role in tissue damage and urinary tract infections.In summary, the comparative analysis of enzyme production by P. aeruginosa and its ability to use different enzymatic pathways depending on the site of infection. The absence or lower occurrence of certain enzymes, such as chondroitinase and hyaluronidase, suggests that these particular strains of P. aeruginosa may not possess the enzymatic machinery to degrade these specific components found in the clinical sources analyzed.The varying frequencies of enzyme production across different clinical sources emphasize the adaptability of P. aeruginosa is crucial for developing targeted treatment strategies and designing effective antimicrobial therapies. By identifying the enzymes that are predominantly produced in specific clinical sources, healthcare professionals can tailor their approaches to combating P. aeruginosa infections. This knowledge can aid in developing novel therapeutics, such as enzyme inhibitors or vaccines, that target the key enzymes involved in the pathogenesis of P. aeruginosa infections.Understanding the diversity of enzyme production by P. aeruginosa isolated from various environmental sources. The results presented in A comparative analysis examined the production of enzymes by The protease production exhibited a higher overall rate, with 25 (83.3%) isolates producing this enzyme. However, the frequency of protease production differed across the locations. Location 3/4 showed the highest frequency at 100%, followed by 3/2 at 83.3% and 22G at 66.7%. The remaining locations exhibited frequencies ranging from 75% to 80%.Gelatinase production was observed in 20 (66.7%) isolates, and its frequency varied among the locations. Locations 3/3 and 3/4 displayed the highest frequencies at 75% and 100%, respectively. Other locations exhibited frequencies ranging from 50% to 66.7%.Interestingly, pyocyanin production was not detected in any of the environmental isolates. On the other hand, fibrinolysin production was observed in 9 (30%) isolates, with frequencies varying among the locations. Locations 3/1, 3/2, and 3/3 exhibited 30%, 33.3%, and 25%, respectively, while the remaining locations displayed frequencies ranging from 25% to 33.3%.P. aeruginosa isolates from different environmental sources revealed variations in the presence and frequency of enzymes across distinct locations. Hemolysin, protease, and gelatinase exhibited varying frequencies, while pyocyanin production was absent. Fibrinolysin production was detected in a subset of isolates. These findings emphasize the diverse enzymatic profiles of environmental P. aeruginosa strains and highlight the influence of location-specific variations in enzyme production.To summarize, a comparative analysis of enzyme production by P. aeruginosa isolated from clinical and environmental sources, unveiling significant disparities in their enzymatic profiles. In the clinical isolates, hemolysin and protease emerged as the most prevalent enzymes, exhibiting a high production rate in 58 (95%) isolates. Notably, blood and urine samples consistently displayed a frequency of 100% for these enzymes, highlighting their robust presence in clinical sources. Sputum samples and wound swabs also showed relatively high frequencies, with 87% and 90%, respectively. Conversely, gelatinase production demonstrated slightly lower overall rates, with 53 (87%) isolates producing this enzyme. The frequency varied among different sources, with wound and urine samples exhibiting higher frequencies  than blood and sputum samples .A comparative analysis was conducted to evaluate the production of enzymes by In contrast, environmental isolates exhibited distinct patterns of enzyme production. Among the enzymes investigated, hemolysin was produced by 13 (43.3%) isolates, with varying frequencies across different locations. The highest frequency of hemolysin production was observed in locations 3/1, 3/2, and 3/3, each with 50%. The protease production displayed a higher overall rate, with 25 (83.3%) isolates producing this enzyme and location 3/4 exhibiting the highest frequency (100%). Gelatinase production was observed in 20 (66.7%) isolates, with locations 3/3 and 3/4 displaying the highest frequencies of 75% and 100%, respectively.Interestingly, pyocyanin production was absent in any environmental isolates, indicating its non-occurrence in these sources. Fibrinolysin and lipase displayed lower production rates in both clinical and environmental isolates. Furthermore, chondroitinase, hyaluronidase, coagulase, elastase, and DNase were not detected in any environmental isolates.P. aeruginosa isolates derived from clinical and environmental sources revealed notable discrepancies in enzyme patterns and frequencies. The clinical isolates exhibited higher frequencies of enzyme production and a wider range of enzyme diversity than the environmental isolates. These findings indicate that the enzymatic profiles of P. aeruginosa strains are influenced by their source of isolation, implying the potential adaptation to specific ecological niches and selective pressures.In summary, the comparative analysis of enzyme production by P. aeruginosa strains from clinical specimens was conducted to evaluate their inhibitory effects against gram-positive bacteria and fungi. The results, presented in P. aeruginosa strains against different pathogens.Isolation of P. aeruginosa strain ATCC27853 displayed notable activity. After 24 h of incubation, it showed no inhibition against Staphylococcus aureus ATCC29213 and Bacillus cereus ATCC14579. However, after 48 h, it exhibited strong inhibition against both S. aureus ATCC29213 and B. cereus ATCC14579. Regarding Streptococcus pneumoniae ATCC49619, P. aeruginosa ATCC27853 showed weak inhibition (+) after 24 h, which increased to partial inhibition after 48 h.Regarding the inhibitory effects against gram-positive bacteria, the P. aeruginosa isolated from urine (PAurine) demonstrated significant inhibitory effects against gram-positive bacteria. After 24 and 48 h of incubation, it exhibited strong inhibition against S. aureus ATCC29213, S. pneumoniae ATCC49619, and B. cereus ATCC14579. However, no inhibition was observed against Candida albicans ATCC10231.P. aeruginosa isolated from wounds (PAwound), it displayed varying inhibitory effects against gram-positive bacteria. After 24 h of incubation, it showed no inhibition against S. aureus ATCC29213, weak inhibition against S. pneumoniae ATCC49619, and partial inhibition against B. cereus ATCC14579. After 48 h, it exhibited a partial inhibition against all three gram-positive bacteria tested. No inhibition was observed against C. albicans ATCC10231.In the case of P. aeruginosa isolated from blood (PAblood) exhibited different inhibitory patterns. It showed no inhibition against S. aureus ATCC29213 after 24 and 48 h of incubation. Similarly, no inhibition was observed against S. pneumoniae ATCC49619 after 24 h, but, after 48 h, it displayed weak inhibition. B. cereus ATCC14579 showed no inhibition after 24 h but exhibited strong inhibition after 48 h. Against C. albicans ATCC10231, PAblood displayed weak inhibition after both 24 and 48 h of incubation.P. aeruginosa isolated from sputum (PAsputum), it exhibited varying inhibitory effects against gram-positive bacteria. After 24 h of incubation, it showed no inhibition against S. aureus ATCC29213 and B. cereus ATCC14579. However, it displayed a partial inhibition against S. pneumonia ATCC49619. After 48 h, PAsputum exhibited a weak inhibition against S. aureus ATCC29213, a partial inhibition against S. pneumonia ATCC49619, and strong inhibition against B. cereus ATCC14579. No inhibition was observed against C. albicans ATCC10231.In the case of P. aeruginosa strains isolated from different clinical specimens against gram-positive bacteria and fungi. The strain ATCC27853 displayed strong inhibition against S. aureus ATCC29213 and B. cereus ATCC14579, while PAurine showed notable inhibition against all tested gram-positive bacteria. The inhibitory effects of PAwound, PAblood, and PAsputum varied across the different pathogens, with varying levels of inhibition observed. Additionally, no inhibition was observed against C. albicans ATCC10231 for most P. aeruginosa strains.The results highlight the variable inhibitory effects of urine showed inhibition against C. albicans ATCC10231 among the clinical specimens, exhibiting a weak inhibition after 24 and 48 h of incubation. The other P. aeruginosa strains isolated from wounds (PAwound), blood (PAblood), and sputum (PAsputum) did not exhibit any inhibitory effects against the tested fungi.The results from the inhibitory effects on fungi reveal interesting findings. Only PAP. aeruginosa strains isolated from clinical specimens against gram-positive bacteria and fungi. PAurine displayed the most promising inhibitory effects against gram-positive bacteria, while the inhibitory effects on fungi were limited to PAurine only. These findings suggest the potential of P. aeruginosa strains, particularly those isolated from urine, in combating certain pathogens, highlighting their importance in clinical settings and their potential for further research and exploration in antimicrobial strategies.Overall, the results demonstrate the varying inhibitory capabilities of P. aeruginosa and its inhibitory effects against gram-positive bacteria. Enzymes produced by P. aeruginosa, such as hemolysin, protease, gelatinase, fibrinolysin, lipase, coagulase, lecithinase, elastase, and DNase, have been implicated in various pathogenic processes and may contribute to the bacterium’s ability to inhibit the growth of gram-positive bacteria.There is a potential relationship between enzyme production by P. aeruginosa isolates. These enzymes damaged host tissues and interfere with the immune response, allowing P. aeruginosa to establish infections. The inhibitory effects against fungus were limited, with only one strain showing weak inhibition against C. albicans. The strong inhibitory effects observed against gram-positive bacteria, such as S. aureus and B. cereus, may be attributed to the production of these enzymes.For example, hemolysin and protease were the most frequently produced enzymes in P. aeruginosa pathogenesis and could contribute to inhibitory effects against gram-positive bacteria. These enzymes have been associated with tissue destruction, the evasion of host defenses, and the degradation of the extracellular matrix. Their production rates and inhibitory effects may vary across different clinical sources, indicating the complexity of the relationship between enzyme production and inhibition.Gelatinase, fibrinolysin, lipase, coagulase, lecithinase, elastase, and DNase also play roles in P. aeruginosa may compensate for their absence and contribute to the observed inhibitory effects.Notably, the absence of chondroitinase and hyaluronidase, which are enzymes involved in the breakdown of extracellular matrix components, suggests that these particular enzymes may not play a significant role in the inhibitory effects against gram-positive bacteria. However, other enzymes produced by P. aeruginosa may be associated with its inhibitory effects against gram-positive bacteria, further research is needed to fully understand the specific mechanisms and interactions involved. The complexity of P. aeruginosa’s enzymatic repertoire and its interplay with bacterial physiology and host–pathogen interactions necessitate comprehensive investigations to elucidate the relationship between enzyme production and the inhibition of gram-positive bacteria.Overall, while enzyme production by P. aeruginosa strains isolated from environmental specimens were evaluated against gram-positive bacteria and fungi. The results, presented in P. aeruginosa strains against different pathogens.The inhibitory effects of S. aureus ATCC29213, strong inhibition against B. cereus ATCC14579, and partial inhibition against S. pneumoniae ATCC49619. The other P. aeruginosa strains showed varying levels of inhibition, including weak, partial, and strong inhibition, against the gram-positive bacteria tested.In terms of gram-positive bacteria, strains PA ATCC27853, PA (3/1), PA (3/2), PA (3/3), PA (3/4), PA (3/5), and PA 22G were tested for their inhibitory effects. After 24 and 48 h of incubation, PA ATCC27853 displayed no inhibition against C. albicans ATCC10231, only PA ATCC27853 exhibited a weak inhibition after 24 and 48 h of incubation. The remaining P. aeruginosa strains showed no inhibitory effects against C. albicans.Regarding P. aeruginosa strains varied among different environmental sources and target pathogens. PA ATCC27853 consistently displayed strong inhibition against B. cereus ATCC14579, while its inhibition against S. aureus ATCC29213 and S. pneumoniae ATCC49619 varied from no inhibition to partial inhibition. The other P. aeruginosa strains exhibited a range of inhibitory effects on the gram-positive bacteria, with variations in the degree and consistency of inhibition. However, none of the strains, except PA ATCC27853, showed any inhibitory effects against C. albicans.Comparing the results, it is evident that the inhibitory effects of P. aeruginosa strains isolated from environmental specimens. The variations in inhibitory profiles indicate the presence of different antimicrobial mechanisms employed by the strains. Further research is necessary to understand these mechanisms and their potential applications in combating pathogens. Additionally, the limited inhibitory effects against C. albicans indicate the need for exploring alternative strategies to target fungal infections.These findings highlight the diverse inhibitory capabilities of P. aeruginosa strains in different environmental contexts and provide valuable insights for future studies and the development of antimicrobial approaches.Overall, these results contribute to our understanding of the inhibitory potential of P. aeruginosa strains from environmental specimens provide interesting insights into a potential relationship between enzyme secretion and the inhibition of gram-positive bacteria and fungi.The results of the analysis of the enzyme production and the inhibitory effects of P. aeruginosa displayed higher frequencies of enzyme production and a broader range of enzyme diversity compared with the environmental isolates. Hemolysin and protease were found to be the most commonly produced enzymes in clinical isolates, with high production rates. The clinical isolates also showed the presence of other enzymes, such as gelatinase, fibrinolysin, and lipase, albeit at lower production rates. These enzymes play a role in virulence and host tissue degradation.In the comparative analysis, it was observed that clinical isolates of P. aeruginosa showed distinct patterns of enzyme production. While hemolysin, protease, and gelatinase production were observed at varying frequencies, enzymes such as pyocyanin, chondroitinase, hyaluronidase, coagulase, elastase, and DNase were not detected. These results indicate a differential enzyme secretion profile in environmental isolates.On the other hand, environmental isolates of P. aeruginosa strains against gram-positive bacteria and fungi, it became clear that the strains exhibited diverse inhibitory capabilities. PA ATCC27853 consistently displayed strong inhibition against B. cereus ATCC14579, while its inhibition against S. aureus ATCC29213 and S. pneumoniae ATCC49619 varied. The other P. aeruginosa strains also showed varying levels of inhibition against the gram-positive bacteria tested. However, none of the strains, except PA ATCC27853, exhibited inhibitory effects against C. albicans ATCC10231.When examining the inhibitory effects of the P. aeruginosa strains. The clinical isolates that displayed higher frequencies of enzyme production may possess a broader repertoire of antimicrobial enzymes that contribute to their inhibitory effects against gram-positive bacteria and possibly fungi. In contrast, the environmental isolates, which exhibited a more limited enzyme secretion profile, may have different inhibition mechanisms, or rely on alternative antimicrobial strategies.These results indicate a potential association between enzyme secretion and the inhibitory effects of P. aeruginosa strains and their potential interactions with gram-positive bacteria and fungi. Understanding the relationship between enzyme secretion and inhibition can provide valuable insights for developing novel antimicrobial approaches and exploring the potential of P. aeruginosa as a source of antimicrobial agents.Further research is necessary to elucidate the specific role of different enzymes in the inhibitory effects of P. aeruginosa using well and disk diffusion methods are summarized in urine exhibited higher inhibition against S. pneumonia and C. albicans than against S. aureus and B. cereus using the well and disk diffusion methods. PAwound strain showed a greater inhibition against B. cereus compared to S. pneumonia and C. albicans using the good diffusion method. In contrast, the disk diffusion method displayed stronger inhibition against S. pneumonia and B. cereus compared to C. albicans. The PAblood strain displayed higher inhibition against S. pneumonia, B. cereus, and C. albicans compared to B. cereus using the good diffusion method. The disk diffusion method exhibited a stronger inhibition against S. pneumonia and C. albicans than B. cereus and S. aureus. Regarding the strains isolated from environmental sources, all P. aeruginosa strains  showed similar patterns of inhibition. They displayed higher inhibition against S. pneumonia and C. albicans compared to S. aureus and B. cereus using the well and disk diffusion methods.The results of the comparative study evaluating the inhibition degrees of pathogenic gram-positive bacteria and fungi by various strains of P. aeruginosa strains isolated from clinical specimens against gram-negative bacteria were evaluated and are presented in Escherichia coli ATCC25922, Klebsiella pneumoniae ATCC700603, and Salmonella enterica serotype Typhi ATCC13311.The inhibitory effects of E. coli ATCC25922, K. pneumoniae ATCC700603, or S. enterica serotype Typhi ATCC13311. In contrast, PAurine exhibited weak inhibition against E. coli ATCC25922 after both time intervals, while no inhibition was observed against the other two gram-negative bacteria. PAwound displayed weak inhibition against K. pneumoniae ATCC700603 after 24 h of incubation, but no inhibition was observed against the other two bacteria. PAblood did not show any inhibitory effects against any of the gram-negative bacteria tested. PAsputum, on the other hand, exhibited very strong inhibition against S. enterica serotype Typhi ATCC13311 after 24 h of incubation but no inhibition against the other two bacteria.After 24 and 48 h of incubation, strain PA ATCC27853 did not show any inhibition against P. aeruginosa strains varied among the different clinical specimens and the target gram-negative bacteria. PA ATCC27853, which is a reference strain, did not show any inhibition against the tested gram-negative bacteria. PAurine exhibited weak inhibition against E. coli ATCC25922 but no inhibition against other bacteria. PAwound displayed weak inhibition against K. pneumoniae ATCC700603 and no inhibition against other bacteria. PAsputum demonstrated strong inhibition against S. enterica serotype Typhi ATCC13311 but no inhibition against other bacteria. PAblood did not exhibit any inhibitory effects against any of the gram-negative bacteria tested.Comparing the results, it is evident that the inhibitory effects of P. aeruginosa strains isolated from different clinical specimens. The differences in inhibition indicate variations in the antimicrobial mechanisms employed by the strains against gram-negative bacteria. Further research is necessary to understand these mechanisms and their potential applications in combating gram-negative bacterial infections.These findings highlight the variability in the inhibitory capabilities of P. aeruginosa strains against gram-negative bacteria. Understanding the strain-specific variations in inhibition can help develop targeted antimicrobial strategies and explore the potential of P. aeruginosa as a source of antimicrobial agents.The results presented in P. aeruginosa isolates from various clinical sources revealed variations in enzyme secretion patterns. Hemolysin and protease were the most commonly produced enzymes, with a high production rate across all clinical sources. Gelatinase production was slightly lower but still exhibited a significant presence. Pyocyanin production was observed in most isolates, whereas fibrinolysin, lipase, and coagulase showed moderate production rates. Lecithinase, elastase, and DNase production was relatively low, and chondroitinase and hyaluronidase production was absent in the clinical isolates.A comparative analysis of the enzyme production by P. aeruginosa strains exhibited varied results. Strain PA ATCC27853, the reference strain, did not show any inhibition against the gram-negative bacteria tested. PAurine exhibited weak inhibition against E. coli ATCC25922 but showed no inhibition against K. pneumoniae ATCC700603 or S. enterica serotype Typhi ATCC13311. PAwound displayed weak inhibition against K. pneumoniae ATCC700603, while PAsputum exhibited very strong inhibition against S. enterica serotype Typhi ATCC13311 but no inhibition against the other tested bacteria. PAblood did not exhibit any inhibitory effects against any of the gram-negative bacteria tested.Regarding the inhibitory effects against gram-negative bacteria, the tested P. aeruginosa strains from clinical sources do not directly correlate with their inhibitory effects against gram-negative bacteria. Although hemolysin and protease were highly produced across clinical samples, their presence did not necessarily produce strong inhibitory effects against gram-negative bacteria. This result indicates that other factors, such as additional antimicrobial compounds or specific antimicrobial mechanisms, may contribute to the inhibitory capabilities of P. aeruginosa strains.These results indicate that producing specific enzymes by P. aeruginosa strains. These findings emphasize the complexity of microbial interactions and the need for a comprehensive understanding of the antimicrobial properties of P. aeruginosa to help develop effective therapeutic strategies.Further research is needed to elucidate the underlying mechanisms behind the inhibitory effects and explore potential relationships between enzyme secretion and the inhibition of gram-negative bacteria by P. aeruginosa strains isolated from various environmental sources against gram-negative bacteria, including E. coli ATCC25922, K. pneumoniae ATCC700603, and S. enterica serotype Typhi ATCC13311. The results, presented in A comparative analysis assessed the inhibitory effects of E. coli ATCC25922 after both time intervals, while no inhibition was observed against K. pneumoniae ATCC700603 or S. enterica serotype Typhi ATCC13311. Similarly, the strain PA (3/2) displayed weak inhibition against E. coli ATCC25922 but showed no inhibition against the other two bacteria. Strains PA (3/3), PA (3/4), and PA (3/5) all exhibited partial inhibition against E. coli ATCC25922 and K. pneumoniae ATCC700603 after both time intervals. Strains PA (3/3) and PA (3/4) also showed a partial inhibition against S. enterica serotype Typhi ATCC13311. Strain PA (22G) displayed a partial inhibition against E. coli ATCC25922 and K. pneumoniae ATCC700603 but no inhibition against S. typhi ATCC13311.Strain PA ATCC27853, the reference strain, did not show any inhibition against the tested gram-negative bacteria after 24 and 48 h of incubation. Strain PA (3/1) exhibited weak inhibition against P. aeruginosa strains isolated from environmental sources varied among different strains and target gram-negative bacteria. The reference strain, PA ATCC27853, did not exhibit any inhibitory effects. Strains PA (3/1) and PA (3/2) displayed weak inhibition against E. coli ATCC25922 but showed no inhibition against other bacteria. Strains PA (3/3), PA (3/4), and PA (3/5) demonstrated partial inhibition against E. coli ATCC25922 and K. pneumoniae ATCC700603, with PA (3/3) and PA (3/4) also showing partial inhibition against S. enterica serotype Typhi ATCC13311. Strain PA (22G) displayed a partial inhibition against E. coli ATCC25922 and K. pneumoniae ATCC700603 but no inhibition against S. enterica serotype Typhi ATCC13311.Comparing the results, it is evident that the inhibitory effects of P. aeruginosa strains from environmental sources possess variable inhibitory capabilities against gram-negative bacteria. The differences in inhibition may be attributed to variations in the strains’ antimicrobial mechanisms, the production of antimicrobial compounds, or other factors yet to be explored. Further research is necessary to understand the underlying mechanisms and assess the potential of these environmental P. aeruginosa strains in combating gram-negative bacterial infections.These findings indicate that P. aeruginosa strains isolated from environmental sources against gram-negative bacteria. Understanding the strain-specific variations in inhibition can help develop targeted antimicrobial strategies and explore P. aeruginosa’s potential as a source of antimicrobial agents.The results presented in P. aeruginosa strains from environmental sources reveal interesting observations. While there is no direct correlation between the presence of specific enzymes and the inhibition of gram-negative bacteria, the variations in enzyme production across different strains and locations could contribute to the variations in inhibitory effects.The results of the comparative analysis between enzyme secretions and the inhibition of gram-negative bacteria in P. aeruginosa isolates. Strains that exhibited higher frequencies of enzyme production, such as protease in strain 3/4, showed partial or strong inhibitory effects against the tested gram-negative bacteria. This result suggests that certain enzymes, such as protease, may contribute to the inhibitory potential of P. aeruginosa strains.Among the enzymes studied, hemolysin, protease, gelatinase, fibrinolysin, and lipase were detected at varying frequencies among the P. aeruginosa is known to produce a wide range of antimicrobial compounds and possess various mechanisms of antimicrobial action. These factors and the presence of enzymes may collectively contribute to the inhibitory effects against gram-negative bacteria.However, the inhibitory effects observed in this study may also involve other factors beyond enzyme secretions. P. aeruginosa strains from environmental sources. Understanding the relationship between enzyme production and bacterial inhibition can provide valuable insights into the antimicrobial potential of P. aeruginosa and aid in developing targeted antimicrobial strategies.Further research is needed to elucidate the specific roles of enzymes and other antimicrobial factors in the inhibitory effects of P. aeruginosa using well and disk diffusion methods are summarized in urine demonstrated higher inhibition against K. pneumonia and S. enterica serotype Typhi compared to E. coli using both the well and disk diffusion methods. The PAwound strain showed greater inhibition against S. enterica serotype Typhi than E. coli and K. pneumonia using the well diffusion method. In contrast, the disk diffusion method displayed stronger inhibition against S. enterica serotype Typhi and K. pneumonia compared to E. coli. The PAblood strain did not show any inhibitory effects against the tested gram-negative bacteria in the well and disk diffusion methods. Regarding the strains isolated from environmental sources, all PA strains  exhibited similar inhibition patterns. They displayed higher inhibition against S. enterica serotype Typhi than E. coli and K. pneumonia using the well and disk diffusion methods.The results of the comparative study evaluating the inhibition degrees of pathogenic gram-negative bacteria by various strains of P. aeruginosa. Among the strains isolated from clinical sources, PAurine showed higher inhibition against S. pneumonia and C. albicans than against S. aureus and B. cereus using the well and disk diffusion methods. On the other hand, the PAwound strain exhibited a stronger inhibition against B. cereus compared to S. pneumonia and C. albicans in the well diffusion method. In contrast, the disk diffusion method displayed stronger inhibition against S. pneumonia, and B. cereus compared to C. albicans. The PAblood strain demonstrated higher inhibition against S. pneumonia, B. cereus, and C. albicans compared with B. cereus in the well diffusion method. The disk diffusion method exhibited a stronger inhibition against S. pneumoniae and C. albicans compared to B. cereus and S. aureus. Among the strains isolated from environmental sources, all P. aeruginosa strains showed similar inhibition patterns, displaying higher inhibition against S. pneumonia and C. albicans than against S. aureus and B. cereus using the well and disk diffusion methods.This comparative study evaluated the inhibition degrees of pathogenic gram-positive bacteria and fungi using various strains of P. aeruginosa. Among the strains isolated from clinical sources, PAurine showed higher inhibition against K. pneumonia and S. enterica serotype Typhi compared to E. coli using both the well and disk diffusion methods. The PAwound strain exhibited a stronger inhibition against S. enterica serotype Typhi than E. coli and K. pneumoniae in the well diffusion method. In contrast, the disc diffusion method displayed stronger inhibition against S. enterica serotype Typhi and K. pneumoniae compared to E. coli. The PAblood strain did not show any inhibitory effects against the tested gram-negative bacteria in the well and disc diffusion methods. Among the strains isolated from environmental sources, all PA strains exhibited similar inhibition patterns, displaying higher inhibition against S. enterica serotype Typhi than against E. coli and K. pneumoniae using both the well and disk diffusion methods.In a comparative study of pathogenic gram-negative bacteria, the inhibition degrees were evaluated using various strains of P. aeruginosa. The inhibitory effects varied depending on the bacterial species and the source of P. aeruginosa isolates.In summary, comparative studies revealed variations in the inhibition degrees of pathogenic gram-positive and gram-negative bacteria by different strains of P. aeruginosa isolates from clinical sources [P. aeruginosa infections [P. aeruginosa [P. aeruginosa and its potential impact on clinical infections.Several studies have explored the enzyme production profiles of  sources ,10,11. T sources ,13. Thesfections ,15. In aruginosa ,17. ThesP. aeruginosa strains. The knowledge gained from these studies is vital for developing targeted treatment strategies and designing effective antimicrobial therapies. By identifying the key enzymes involved in the pathogenesis of P. aeruginosa infections, healthcare professionals can tailor their approaches to combat these infections. Furthermore, future research can focus on investigating the specific roles of these enzymes in P. aeruginosa pathogenicity and exploring potential therapeutic interventions targeting these enzymes.Comparing the results of these comparative analyses with previous research enhances our understanding of the diversity and clinical significance of enzyme production by P. aeruginosa isolates from clinical sources [P. aeruginosa pathogenesis [Gelatinase production, on the other hand, showed slightly lower overall rates in the clinical isolates. This finding was in line with previous studies that reported varying frequencies of gelatinase production in  sources ,19. The ogenesis ,21.P. aeruginosa [The absence or lower occurrence of certain enzymes, such as chondroitinase and hyaluronidase, in the clinical isolates, agreed with previous studies that reported the absence of these enzymes in certain strains of ruginosa ,23. ThisP. aeruginosa have also shed light on the enzymatic profiles of these strains [P. aeruginosa [P. aeruginosa strains adapt their enzymatic pathways based on the site of infection or environmental conditions. This adaptability aligns with previous studies that have emphasized the versatility and adaptability of P. aeruginosa and its ability to use different enzymatic pathways depending on the ecological niche [Comparative analyses of enzyme production in environmental isolates of  strains ,25. The ruginosa ,27. Moreal niche ,30,31,32P. aeruginosa strains. This knowledge is crucial for developing targeted treatment strategies and designing effective antimicrobial therapies. By identifying the key enzymes involved in the pathogenesis of P. aeruginosa infections, healthcare professionals can tailor their approaches to combat these infections. Future research can focus on investigating the specific roles of these enzymes in P. aeruginosa pathogenicity and exploring potential therapeutic interventions targeting these enzymes.By comparing the results of this study with previous research, we can better understand the diversity and clinical significance of enzyme production by P. aeruginosa strain ATCC27853 showed notable activity, exhibiting strong inhibition against S. aureus ATCC29213 and B. cereus ATCC14579 after 48 h of incubation. However, it showed no inhibition against S. aureus ATCC29213 and B. cereus ATCC14579 after 24 h. Similar studies have reported the varying inhibitory effects of P. aeruginosa strains against different gram-positive bacteria, highlighting the strain-specific nature of these effects [In terms of inhibitory effects against gram-positive bacteria,  effects ,34,35.P. aeruginosa isolated from urine (PAurine) demonstrated significant inhibitory effects against gram-positive bacteria, showing strong inhibition against S. aureus ATCC29213, S. pneumoniae ATCC49619, and B. cereus ATCC14579 after both 24 and 48 h of incubation. However, it exhibited no inhibition against C. albicans ATCC10231. Previous studies have also reported the inhibitory effects of P. aeruginosa against gram-positive bacteria [C. albicans is an interesting finding.bacteria ,34,35, bP. aeruginosa isolated from PAwound, PAblood, and PAsputum against gram-positive bacteria varied across different pathogens. PAwound showed a partial inhibition against S. pneumonia ATCC49619 after 24 h, while PAblood exhibited a weak inhibition against S. pneumonia ATCC49619 after 48 h. These results indicate that the inhibitory effects of P. aeruginosa strains may differ depending on the source of isolation and the specific pathogen being tested [The inhibitory effects of g tested ,37.urine displayed a weak inhibition against C. albicans ATCC10231 after 24 and 48 h of incubation. PAwound, PAblood, and PAsputum did not exhibit any inhibitory effects against the tested fungus. These findings indicate that the inhibitory effects of P. aeruginosa strains are primarily directed toward gram-positive bacteria rather than fungi [Regarding the inhibitory effects on fungi, only PAan fungi ,39.P. aeruginosa strains from environmental specimens against gram-positive bacteria and fungi, the results demonstrated varying levels of inhibition against different pathogens. The strains tested showed different degrees of inhibition against gram-positive bacteria and C. albicans. The strain PA ATCC27853 consistently displayed strong inhibition against B. cereus, while its inhibition against S. aureus and S. pneumoniae varied. None of the strains, except PA ATCC27853, showed any inhibitory effects against C. albicans.In the study comparing the inhibitory effects of P. aeruginosa strains can vary among different environmental sources and target pathogens. These data suggest the presence of different antimicrobial mechanisms employed by the strains [C. albicans indicate the need for exploring alternative strategies to target fungal infections.When comparing these results with previous studies, it is evident that the inhibitory effects of  strains ,21. FurtP. aeruginosa. The analysis revealed that clinical isolates of P. aeruginosa exhibited higher frequencies of enzyme production and a broader range of enzyme diversity compared with environmental isolates. Hemolysin and protease were the most commonly produced enzymes in clinical isolates, whereas environmental isolates showed distinct patterns of enzyme production.Interesting insights were obtained in another study that focused on the association between enzyme production and the inhibition of gram-positive bacteria and fungi by P. aeruginosa strains in this study also displayed diverse capabilities. PA ATCC27853 consistently exhibited strong inhibition against B. cereus, but its inhibition against S. aureus and S. pneumoniae varied. The other P. aeruginosa strains also showed varying levels of inhibition against gram-positive bacteria, but none exhibited inhibitory effects against C. albicans.The inhibitory effects of the P. aeruginosa strains [These findings indicate a potential association between enzyme secretion and the inhibitory effects of  strains ,23,24. CP. aeruginosa strains against gram-negative bacteria, similar variations in inhibition were observed. The strains exhibited different degrees of inhibition against the tested bacteria, indicating strain-specific differences in antimicrobial activity [In Xu et al. , that inactivity ,41. HoweP. aeruginosa isolates and their correlation with antimicrobial activity against gram-negative bacteria. The findings showed that the enzyme production varied among clinical isolates, with some enzymes more commonly produced than others. These results align with the current study, which observed variations in enzyme secretion patterns among clinical isolates. However, the correlation between enzyme production and inhibitory effects against gram-negative bacteria was not straightforward in both studies, indicating the involvement of additional factors in the antimicrobial activity.Another study by Kunz Coyne et al.  exploredP. aeruginosa strains against specific gram-negative bacteria. The results demonstrated consistent inhibition by certain strains against the tested bacteria, which differs from the current study, where variations in inhibition were observed among different strains of P. aeruginosa. These differences may be attributed to variations in strains, bacterial targets, and methodologies employed in the studies.In contrast, using different methodologies, Voumard et al.  investigP. aeruginosa strains may differ among studies, potentially influencing the overall inhibitory effects observed.However, it is important to acknowledge that variations in methodologies, such as strain selection, bacterial or fungal targets, and incubation conditions, may exist among different studies. These variations can contribute to differences in observed inhibition patterns. Additionally, the specific clinical and environmental sources of P. aeruginosa against pathogenic bacteria. The variations in inhibition may be attributed to factors such as antimicrobial mechanisms, the production of antimicrobial compounds, or other yet unidentified factors.Despite these variances, the consistent findings across studies emphasize the strain-specific variations in the inhibitory capabilities of Further research is warranted to better understand the underlying mechanisms driving the variations in inhibitory effects. Investigating the specific antimicrobial factors and their potential applications in combating gram-positive and gram-negative bacterial infections will help develop targeted antimicrobial strategies.P. aeruginosa strains in inhibiting pathogenic gram-positive and gram-negative bacteria provide valuable insights into the variations in inhibitory effects. The findings underscore the importance of further research to unravel the underlying mechanisms and explore the potential of P. aeruginosa as a source of effective antimicrobial agents against gram-positive and gram-negative bacteria.Comparative studies of P. aeruginosa isolated from clinical sources. First, the study relied on a specific set of clinical samples, and the findings may not be representative of enzyme production in P. aeruginosa infections in general. Additionally, the analysis focused on a specific set of enzymes and did not encompass the entire range of enzymes produced by P. aeruginosa. Other enzymes that could play a role in clinical infections might have been overlooked, leading to an incomplete understanding of the diversity of the enzyme production.There are certain limitations to consider when interpreting the results of the comparative analysis of enzyme production by P. aeruginosa isolated from clinical sources, the clinical implications of these findings should be interpreted cautiously, considering the limitations and subjectivity inherent in such analyses.The interpretation of the significance of enzyme production in clinical infections is subject to various factors that could introduce subjectivity. Different studies may use different criteria to determine the clinical relevance of specific enzymes, leading to discrepancies in the reported importance of certain enzymes. Additionally, various confounding factors could influence the clinical outcomes associated with enzyme production, such as host immune response, co-infections, and underlying comorbidities. Therefore, while the comparative analysis provides insights into the prevalence of enzyme production in P. aeruginosa ATCC27853, gram-positive bacteria such as S. aureus ATCC29213, S. pneumonia ATCC49619, and B. cereus ATCC14579, as well as the fungus C. albicans ATCC10231, and gram-negative bacteria such as E. coli ATCC25922, K. pneumoniae ATCC700603, and S. enterica serotype Typhi ATCC13311. These meticulously selected bacterial strains of exceptional quality were procured from the American Type Culture Collection and, subsequently, brought to KAMC for conducting rigorous laboratory diagnostic assessments.The study includes various quality bacterial strains obtained from the Microbiology Laboratory, King Abdulaziz Medical City (KAMC), Riyadh, Saudi Arabia. These strains consist of the control strain P. aeruginosa. In total, 91 P. aeruginosa strains were isolated, 61 from clinical sources and 30 from environmental sources.A total of 510 samples were collected from environmental and clinical sources. The 251 clinical specimens collected included blood, urine, and sputum samples, wound swabs, and 259 environmental water samples collected from different locations at Wadi Hanifah, Riyadh, Saudi Arabia. Collected samples were transported immediately in an ice box to the Microbiology Laboratory, King Saud Bin Abdulaziz University for Health Sciences (KSAU-HS), College of Applied Medical Sciences, Medical Laboratory Sciences, Riyadh, Saudi Arabia, for processing within one hour of collection. All clinical and environmental samples were processed to isolate P. aeruginosa infections using appropriate aseptic techniques. These samples were labeled and transported to the Microbiology Laboratory at KSAU-HS while maintaining suitable conditions to preserve sample integrity. There was no direct contact with the patients during this process.Blood, urine, and sputum samples, and wound swabs were collected from patients suspected of having P. aeruginosa growth.Blood: Use blood culture bottles to inoculate and incubate blood samples in a suitable automated blood culture system to enhance Pseudomonas aeruginosa.Urine: Perform urine culture by streaking the samples onto selective agar plates, such as MacConkey agar , which are known to promote the growth of Sputum: Process sputum samples using standard microbiological techniques, including sample homogenization, serial dilution, and plating onto MacConkey agar plates.P. aeruginosa.Wounds: Swab the wound area with a sterile swab and streak the swab onto MacConkey agar plates to isolate and cultivate Water samples were collected from various locations along Wadi Hanifah in Riyadh, Saudi Arabia. These samples were labeled and transported to the Microbiology Laboratory at KSAU-HS while maintaining suitable conditions to preserve sample integrity.P. aeruginosa cells present and the water samples were filtered using sterile filters with appropriate pore sizes. Then, the filtered water was plated onto MacConkey agar plates to isolate and culture colonies of P. aeruginosa.Using sterile containers, water samples were collected from multiple points along Wadi Hanifah in Riyadh, Saudi Arabia, to ensure representative sampling. The concentration of any potential In the laboratory, samples from clinical and environmental sources were cultured first on selective agar, MacConkey agar; then, suspected colonies were subcultured on blood agar  and Mueller–Hinton agar  to observe hemolysis and pigmentation. All the inoculated plates were incubated at 37 °C for 18–24 h, and growth was evaluated on these media. Isolates were identified based on standard bacteriological methods such as morphology, colonial characteristics, hemolysis, and pigment production on these media . FurtherP. aeruginosa) and the control strain P. aeruginosa ATCC27853 in MacConkey agar. These cultures were incubated under optimal conditions to facilitate enzyme production.Start by inoculating quality strains  casein  and incubated at 37 °C for 30 min. Subsequently, 0.45 mL of a 10% (w/v) tri-chloroacetic acid solution at a final concentration of 5% w/v was added to stop the proteolysis. The mixture was incubated at room temperature for 1 h. After incubation, the reaction mixture was centrifuged at 12,000× g for 5 min, and the absorbance of the supernatant was measured at 280 nm. One unit of protease is defined as the amount of enzyme that hydrolyzes casein to produce equivalent absorbance to 1 µmol of tyrosine/min with tyrosine as standard [The bacterial culture was centrifuged at 24,000× standard .p-nitrophenol palmitate (pNPP) as the substrate. The reaction mixture comprised 700 µL pNPP solution and 300 µL lipase solution. The pNPP solution was prepared by adding solution A (0.001 g pNPP in 1 mL isopropanol) into solution B  with stirring until all was dissolved. Then, the absorbance was measured at 410 nm for the first 2 min of reaction. One unit (1U) was defined as the amount of enzyme that liberated 1 µmol of pNPP per min (ɛ: 1500 L/mol cm) under the test conditions [The lipase activity was determined spectrophotometrically at 30 °C using nditions .A hemolysin test was carried out using blood agar media. Isolates were planted in blood agar base with the addition of sheep blood that had been fabricated to as much as 5% of blood agar base, and then incubated at 37 °C for 18–24 h. A positive result indicates that the microbe is pathogenic if a clear zone is formed around the colony on the media .The DNase test was performed using DNase agar . Bacteria to be tested were inoculated on a DNase agar plate, added with 0.005% methyl green, and then incubated at 37 °C for 24 h. After incubation, the DNase agar plate was immersed in 1 N HCl for 5 min . A positThe gelatinase test was carried out using bacteria that had been pure-cultured using a loop needle, then inserted into the nutrient gelatin media  in the middle of the media. Bacteria inoculated into gelatin media were stored in an incubator at 37 °C for ±3 days. After waiting for ±3 days, observe using a test tube containing bacterial isolates stored in the refrigerator at 4 °C. Wait 25 min for the jar in the refrigerator. Furthermore, observations were made, including whether or not the gelatin was melted compared to the control. If there is melting of gelatin, it indicates that the bacteria can produce gelatinase exoenzymes .This test consists of inoculating a suspension of the bacterial strain in rabbit plasma in a test tube, which was incubated in a bacteriological incubator at 37 °C for 24 h. Over this period, it will be observed whether or not a clot is formed in the plasma, in which the presence of coagulation will indicate a positive result, and the opposite a negative result .g) incubation at 37 °C for 20 min. The reaction was ended by adding 100 μL of 0.12 M EDTA  (pH 8.0). Following centrifugation (8000× g for 20 min), the absorbance at 495 nm was calculated with a spectrophotometer calibrated to control the elastin-Congo red sample incubated without the enzyme. One unit (U) of elastase was equivalent to the quantity of enzyme, causing a rise in A495 by 0.01 after 1 h of incubation under standard assay conditions [Every 1 mL of elastase was mixed with 20 mg of the Congo red elastin substrate  suspended in 2 mL of 0.2 mol/L acid buffer (pH 7.4) with shake . Silica-gel-absorbed crude pigment was loaded on a column (30 cm length × 2 cm diameter) equilibrated with 1% methanol in chloroform. Purified pyocyanin was eluted with 15% methanol in chloroform. The eluted fractions were examined by scanning a UV-Vis spectrophotometer ; fractions with the same λ-max were collected and dried on a rotary evaporator  at 37 °C. The purified pyocyanin was subjected to spectroscopic analysis. Ultraviolet and visible absorption spectra of purified pyocyanin dissolved in chloroform or 0.1 N HCl were recorded over 200–700 nm. Mass spectrometric analysis of purified pyocyanin was performed on Shimadzu mass spectrometry GCMS-QP 1000 EX mass spectrometry  at 70 eV .g, 30 min), and the supernatant was discarded. The precipitate was then suspended in 2 mL distilled water and sonicated for 15 s episodes. Following centrifugation at 15,000× g for 5 min, the supernatant was harvested, and its fibrinolytic activity was assessed based on plasma plate method. The supernatant  of 24 h bacterial culture at optimum pH (pH 7) and temperature (40 °C) was harvested and subjected to different treatments. The fibrinolytic activity of treated samples was evaluated based on the plasma plate method. Then, the bacterial culture supernatant was mixed separately with each prepared solution in a 1:1 (v/v) ratio and incubated at 40 °C for 60 min. Following treatment, the fibrinolytic activity was evaluated in a plasma plate assay based on the differences in halo zone formation compared to the untreated sample [The bacterial culture was centrifuged  and incubated for 24 h at 37 °C. After incubation, the cultures were centrifuged at 2500 rpm for 15 min to obtain a cell-free filtrate, and 100 μL of the filtrate was transferred into 10 mm wells made centrally in the egg-yolk agar plates and incubated for 24 h at 37 °C. Opaque zones were measured as indicators of lecithinase production, and the means were used as criterion of lecithinase activity [One ml of each bacterial isolate at a cell density of 6 × 10activity .One mg/mL of sodium hyaluronate   for 48 h) stock solution was diluted before use with an equal volume of 0.02 M phosphate buffer solution , 1.0 g of anhydrous dibasic sodium phosphate, and 8.2 g of sodium chloride in water to make 1 L). Then, 10 mg/mL of BSA solution was diluted with 4 volumes of 0.5 M acetate buffer solution  and 20.5 mL of glacial acetic acid in water to make 1 L, and then adjust with 4 M hydrochloric acid to pH 3.1). The standard solution was prepared immediately before use by dissolving standard hyaluronidase in cold hydrolyzed gelatin solution (mix 250 mL of phosphate buffer solution with 250 mL of water and dissolve 330 mg of hydrolyzed gelatin in the mixture) to obtain a solution of 10 U/mL. The sample solution was also properly diluted with cold hydrolyzed gelatin before use. The sample detection reaction temperature was 42 °C .−1 Tris-HCl, pH 7.4). CS A and CS B degradation was monitored by UV absorbance at 232 nm, and the activity was calculated using a molar extinction coefficient of 3800 L mol−1 cm−1. The protein concentration was detected using the Bradford Protein Assay Kit . One international unit was defined as the amount of protein that could form 1 mmol L−1 4,5-unsaturated uronic acid per minute at 37 °C [Chondroitinase (CS) activity was measured according to the UV 232 nm method. The enzymatic reaction was carried out at 37 °C using CS A and CS B as substrates in the same buffer . Create wells in the agar and introduce standardized inoculum of each isolated P. aeruginosa strain to be tested. After incubation, the inhibitory potential was assessed by measuring the inhibition zones around the wells.Prepare agar plates with suitable growth media and inoculate them with quality strains  plate was inoculated by spreading the swab over the entire sterile agar surface. This procedure is repeated by spreading two more times, rotating the plate approximately 60° each time to ensure an even distribution of inocula. Sterile disks impregnated with purified compounds derived from P. aeruginosa strains were placed on the agar surface. These disks contained potential antimicrobial agents. The plates were then incubated at 37 °C for bacterial and fungal growth. After the incubation period, the inhibition zones formed around the disks were measured, indicating the effectiveness of the isolated P. aeruginosa strains against the target pathogenic bacteria and fungi. The results obtained from this procedure provide valuable insights into the antimicrobial potential of isolated strains and their suitability for further exploration as potential sources of novel antimicrobial compounds.The procedure for conducting the disk diffusion method using isolated P. aeruginosa strains sourced from clinical and environmental origins, targeting both gram-positive and gram-negative bacteria as well as fungi. Through parallel methodologies using disk and well diffusion techniques, coupled with enzyme production assessments via biochemical assays, insights into the antimicrobial potential of P. aeruginosa strains were garnered. Notably, significant variations in the enzyme production were noted among clinical strains, with hemolysin and protease prevailing, while gelatinase, chondroitinase, and hyaluronidase exhibited varying frequencies. Conversely, environmental isolates exhibited distinct enzymatic patterns, indicative of adaptation. Inhibition against bacterial strains showcased strain-specificity, particularly against gram-positive bacteria, and limited antifungal effects against C. albicans. The study underscores the strain-dependent nature of the inhibitory responses and enzyme secretion, hinting at the potential contributions of specific enzymes like hemolysin and protease in antimicrobial activity. The intricate interplay between enzyme production and pathogen inhibition warrants further exploration. These findings underscore the promising reservoir of P. aeruginosa strains for antimicrobial applications, especially targeting gram-positive bacteria, urging future research into the mechanistic underpinnings and therapeutic potentials of these inhibitory phenomena.This study presents a comprehensive overview of two investigations into the inhibitory effects of diverse


Hericium erinaceus (Bull.) Pers.), Reishi (Ganoderma lucidum (Curtis) P. Karst.), Chaga (Inonotus obliquus (Ach. ex Pers.) Pilát), Cordyceps (Ophiocordyceps sinensis (Berk.) G.H. Sung, J.M. Sung, Hywel-Jones and Spatafora), Shiitake (Lentinula edodes (Berk.) Pegler), and Turkey Tail (Trametes versicolor (L.) Lloyd), are considered new-generation foods and are of growing interest to consumers. They are characterised by a high content of biologically active compounds, including -β-d-glucans, which are classified as dietary fibre, triterpenes, phenolic compounds, and sterols. Thanks to their low-fat content, they are a low-calorie product and are classified as a functional food. They have a beneficial effect on the organism through the improvement of its overall health and nutritional level. The biologically active constituents contained in medicinal mushrooms exhibit anticancer, antioxidant, antidiabetic, and immunomodulatory effects. In addition, these mushrooms accelerate metabolism, help fight obesity, and slow down the ageing processes thanks to their high antioxidant activity. The vast therapeutic properties of mushrooms are still not fully understood. Detailed mechanisms of the effects of medicinal mushrooms on the human organism still require long-term clinical studies to confirm their nutraceutical effects, their safety of use, and their dosage. Medicinal mushrooms have great potential to be used in the design of innovative functional foods. There is a need for further research on the possibility of incorporating mushrooms into food products to assess the interactions of their bioactive substances with ingredients in the food matrix. This review focuses on the properties of selected medicinal mushrooms and their effects on the human organism and presents current knowledge on the possibilities of their use in the production of functional foods.Medicinal mushrooms, e.g., Lion’s Mane ( For many years, mushrooms have accompanied humans both as food and medicine. Data from the literature indicate that, with the onset of hunting, mushrooms began to play an important role in the human diet . FruitinLentinula edodes (Berk.) Pegler) and proved that the wood-cultured fungus had a higher content of terpenoids and phenolic components and concurrently exhibited higher antioxidant and hypoglycaemic potential compared to the sack-cultured Shiitake (Lentinula edodes (Berk.) Pegler). In the case of Chaga (Inonotus obliquus (Ach. ex Pers.) Pilát), which is a parasite of various deciduous trees, only sclerotia derived from birch trunks have contained tree-specific compounds (betulin and betulinic acid) showing anticarcinogenic activity. Equally great importance for the chemical composition and health-promoting potential of medicinal mushrooms is ascribed to the world region from which they originate .,45.Ophio-glucans . It take-glucans . Its pos-glucans , breast -glucans  and live-glucans . In turng cancer .Trametes versicolor (L.) Lloyd) mushrooms is called PSP (Poly Saccharo Peptide) and activates immune cells by increasing the production of cytokines, chemokines, histamine, and prostagladin E. It reduces the detrimental effects of chemotherapy by alleviating fatigue, loss of appetite, vomiting, a dry mouth, and other related discomforts [Ophiocordyceps sinensis (Berk.) G.H. Sung, J.M. Sung, Hywel-Jones, and Spatafora). Its chemical structure resembles that of the nucleoside adenosine (ribose + adenine sugar); however, it lacks one hydroxyl group at position three of the five-membered ring of the ribose moiety. Adenosine is involved in DNA and/or RNA synthesis in cells. Thanks to its analogy to adenosine, cordycepin can build into the RNA and DNA structures of bacteria and viruses and interfere with the biosynthesis and modification of nucleic acids, thereby limiting the growth of these microorganisms. It increases the proliferation and secretion of T and B lymphocytes and has anti-inflammatory effects through a reduction in the expression of pro-inflammatory cytokines and chemokines. Additionally, it inhibits platelet aggregation and shows suppressive properties against tumour cells [Another type of glucan that is complexed with protein present in Turkey Tail (comforts . In addiur cells . The posHericium erinaceus (Bull.) Pers.), Cordyceps (Ophiocordyceps sinensis (Berk.) G.H. Sung, J.M. Sung, Hywel-Jones and Spatafora), and Shiitake (Lentinula edodes (Berk.) Pegler) have the highest protein content (more than 20%) [Ganoderma lucidum). This type of protein, named TVC, was also isolated by Li et al. [Trametes versicolor. As demonstrated by the authors, TVC increases the proliferation of human peripheral blood lymphocytes and is responsible for the increased necrosis of alpha tumour cells that are induced by mouse macrophages [Ophiocordyceps sinensis (Berk.) G.H. Sung, J.M. Sung, Hywel-Jones and Spatafora). Studies have demonstrated a protective role for this compound in lowering blood glucose levels in alloxan-induced hyperglycaemic rats. A dose of 50–100 mg/kg of the body weight of the animals also resulted in a reduction in aglycated haemoglobin (HbA11C) levels 5 weeks after the study. The oxidative stress induced by high sugar levels and the animal body weight decreased [In addition to polysaccharides, proteins, and peptides are important bioactive components that are present in mushrooms. Their content ranges widely from 4.6 to 56.3 g/100 g and is mainly determined by the mushroom species. Of the mushrooms discussed, Lion’s Mane (han 20%) . The amihan 20%) . The moshan 20%) . Lectinshan 20%) ,66. Thesi et al.  from therophages . A charaecreased . Numerouecreased , its nutecreased  reportedecreased .Ophiocordyceps sinensis (Berk.) G.H. Sung, J.M. Sung, Hywel-Jones, and Spatafora) and Shiitake (Lentinula edodes (Berk.) Pegler) are the most abundant in fat. About 52–87% of the fat is made up of unsaturated fatty acids (UFAs) such as oleic (C18:1) and linoleic (C18:2) acids [Ophiocordyceps sinensis (Berk.) G.H. Sung, J.M. Sung, Hywel-Jones and Spatafora). The minor fatty acid in this mushroom is saturated fatty acids, e.g., palmitic (C16:0) and stearic (C18:0) acids. Guo et al. [Ophiocordyceps sinensis (Berk.) G.H. Sung, J.M. Sung, Hywel-Jones and Spatafora).The fat content in mushrooms varies depending on the species but can range from 0.1 to 5.9 g/100 g . Among m2) acids ,71,72. TOphiocordyceps sinensis (Berk.) G.H. Sung, J.M. Sung, Hywel-Jones, and Spatafora, conducted by Guo et al. [Lentinula edodes (Berk.) Pegler) mushrooms. Stearic acid, hydroxyhexacosanoic acid, linoleic acid, palmitic acid, hydroxyarachidic acid, hydroxyheneicosanoic acid, and hydroxy-tricosenoic acid are present in smaller amounts [Comparative examinations between indoor-cultivated and wild o et al. , demonst amounts .Linoleic acid is known to have anticancer effects on breast, colon, and prostate cancer; thus, as a natural source of this acid, medicinal mushrooms also exhibit such properties . FurtherMushrooms are also a source of sterols classified as bioactive compounds. The most common of these is ergosterol. This compound undergoes photolysis to vitamin D2 when exposed to UV radiation . A studyInonotus obliquus (Ach. Ex Pers.) Pilát) had the highest polyphenolic content and the highest antioxidant activity. The total phenolic content in this mushroom was at 97 µmol GAE/mg, while the content in reishi (Ganoderma lucidum (Curtis) P. Karst.), shiitake, and turkey tail (Trametes versicolor (L.) Lloyd) was 21, 13, and 0.1 µmol GAE/mg, respectively. The water-ethanol extract from Chaga (Inonotus obliquus (Ach. Ex Pers.) Pilát) exhibited approximately five times higher antioxidant activity against DPPH than other mushrooms. Mushroom polyphenols exhibit multidirectional beneficial effects on the human body: anticancer, antioxidant, hypoglycemic, slowing down the aging process, and preventing the degenerative diseases of the nervous system and cardiovascular diseases. When used as a food additive, they reduce fat oxidation processes and extend the shelf life of products [Thanks to the presence of polyphenols, including mainly phenolic acids represented by benzoic acid and cinnamic acid derivatives, medicinal mushrooms can be attributed to antioxidant activity. As reported by Ahmed et al. , gallic,products .Ganoderma lucidum (Curtis) P. Karst. and Inonotus obliquus (Ach. ex Pers.) Pilát. Data in the literature have reported that large amounts of these compounds, e.g., in reishi (Ganoderma lucidum (Curtis) P. Karst.) and chaga (Inonotus obliquus (Ach. ex Pers.) Pilát). Terpenes exert primarily anti-inflammatory effects. Triterpenes isolated from Ganoderma lucidum (Curtis) P. Karst. and Inonotus obliquus (Ach. ex Pers.) Pilát reduced the secretion of pro-inflammatory cytokines in macrophages  and the inflammatory mediators of nitric oxide (NO) and prostaglandin E2 (PGE2) [Ganoderma lucidum (Curtis) P. Karst., which, as shown by Akihisa et al. [Another group of compounds includes terpenes, with the general formula (C5H8)n, and terpenoids containing additional functional groups . Triterpenes are the main biologically active metabolites of terpenoid nature and are synthesized by 2 (PGE2) ,85. Simia et al. ,87. The a et al. .The nutritional value of medicinal mushrooms is also related to their high vitamin and micronutrient content. The vitamins present in mushrooms are mainly fat-soluble vitamins, including A and E. as well as vitamin D2  and provitamin D2 (ergosterol). Interestingly, medicinal mushrooms are considered to be the only non-animal raw material that contains vitamin D ,89. ThanInonotus obliquus (Ach. ex Pers.) Pilát), cordyceps (Ophiocordyceps sinensis (Berk.) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora), shiitake, lion’s mane (Hericium erinaceus (Bull.) Pers.) or reishi (Ganoderma lucidum (Curtis) P. Karst.) is coffee. Its consumption regulates blood pressure, prevents heartburn, stimulates mental performance, boosts energy, and strengthens the immune system and performance of the organism [Ganoderma lucidum (Curtis) P. Karst.) and shiitake (Lentinula edodes (Berk.) Pegler) have been used most commonly. In all food products, the addition of dried and powdered mushrooms resulted in an increase in protein and the total and insoluble dietary fibre and significantly increased the micronutrient content [Trametes versicolor (L.) Lloyd), cordyceps (Ophiocordyceps sinensis (Berk.) G.H. Sung, J.M. Sung, Hywel-Jones and Spatafora), lion’s mane (Hericium erinaceus (Bull.) Pers.), or Chaga (Inonotus obliquus (Ach. ex Pers.) Pilát) preparations for food enrichment. Given the high health-promoting potential of these mushrooms, further research into the possibility of developing new functional foods with the above-mentioned mushrooms is advisable. The effect on the addition of selected medicinal mushrooms on quality parameters and the chemical composition of food products is presented in Medicinal mushrooms and mushroom-derived preparations containing bioactive compounds are classified as nutraceuticals. According to the European Food Safety Agency, they can be used as supplements due to their health-promoting and disease-preventing activity . The proorganism . Some me content . The int content . In the To date, a great deal of research has already been conducted into medicinal mushrooms; however, given the diversity of species and the amount of bioactive substances contained therein, this area still appears to be incompletely explored. It seems advisable to conduct research not only to isolate and identify the bioactive substances present in mushrooms but also to conduct clinical experiments to confirm the therapeutic effect of these substances. Such studies could facilitate a determination of the dose and duration of use for mushroom nutraceuticals. Toxicological studies confirming the safety of medicinal mushrooms are also needed. In the context of using medicinal mushrooms for the development of functional foods, it is important to study the interactions between the biologically active compounds present in mushrooms and food ingredients. It is important to bear in mind that the components present in the food matrix may act synergistically or antagonistically with mycochemicals, increasing or reducing their beneficial physiological effects, respectively. Based on the analysis of available information and scientific research, it can be concluded that the addition of medicinal mushrooms to foods, especially cereal products, can make their chemical composition more attractive due to their great health-promoting properties and the presence of biologically active compounds. Medicinal mushrooms are known for their potential to improve immunity, regulate metabolism, and prevent many diseases. The abundance of polysaccharides, polyphenols, amino acids, and vitamins in medicinal mushrooms is a valuable source of biologically active compounds that can contribute to maintaining the health and well-being of the body. At the same time, further scientific research is needed to confirm these benefits and develop optimal methods for the addition of medicinal mushrooms to foods, taking into account technological, sensory, and food safety aspects.


As a result of the increasing prevalence of multidrug-resistant G+ bacteria such as methicillin-resistant Staphylococcusaureus (MRSA), novel antimicrobial agents must urgently be developed for the treatment of infections caused by G+ bacteria. Endolysins are bacteriophage (phage)-encoded enzymes that can specifically hydrolyze the bacterial cell wall and quickly kill bacteria. Bacterial resistance to endolysins is low. Therefore, endolysins are considered promising alternatives for solving the mounting resistance problem. In this review, endolysins derived from phages targeting G+ bacteria were classified based on their structural characteristics. The active mechanisms, efficacy, and advantages of endolysins as antibacterial drug candidates were summarized. Moreover, the remarkable potential of phage endolysins in the treatment of G+ bacterial infections was described. In addition, the safety of endolysins, challenges, and possible solutions were addressed. Notwithstanding the limitations of endolysins, the trends in development indicate that endolysin-based drugs will be approved in the near future. Overall, this review presents crucial information of the current progress involving endolysins as potential therapeutic agents, and it provides a guideline for biomaterial researchers who are devoting themselves to fighting against bacterial infections.Gram-positive (G Drugproblems . TherefoBacteriophages (phages) are the most abundant biological entities on earth. They are widespread all over the biosphere from the soil to marine environments, the atmosphere, and the human body. Phages are viruses that can specifically infect and rapidly kill the bacterial hosts in their lytic life cycles , 7. Afte−) bacteria effectively presents a physical protective barrier against endolysins, which can directly target the bonds in the peptidoglycan and lyse the cell wall of G+ bacteria that do not have outer membranes [+ bacterial infections. Many recombinant endolysins have already been expressed, identified, and purified; they sufficiently display potent bacteriolytic activity against G+ bacteria [K completely eliminates the staphylococcal biofilms within 4 h [K [+ bacterial infections. The endolysin-based candidate drugs such as P128 and N-Rephasin® SAL200 are being tested in phases II and IIa in the treatment of S.aureus bacteremia, respectively [+ bacterial infections.The presence of the outer membrane of Gram-negative  have a simple globular configuration, whereas most of the endolysins derived from phages targeting G+ bacteria  comprise two modular structures: an N-terminal catalytic domain (CD) joined by a flexible linker to a C-terminal cell wall-binding domain (CBD) [S.aureus phage 187, has two CDs but lacks a CBD [+ bacteria , we propose a systematic classification of these endolysins based on their domain compositions  –32. Somein (CBD) , 34. A cin (CBD) –37. Specks a CBD –40. Therbacteria , 43, 44.bacteria , 45, 46.bacteria , 48, trubacteria , 50.Fig.+ bacteria have naturally designed such distinct domain structures to better disseminate the progeny particles [S.aureus, including MRSA [S.aureus, including disruption of biofilms and protection of mice from S.aureus endophthalmitis [Sequence comparison of endolysins of the same type shows high homology within the N-terminal enzymatically active domain and low similarity within the C-terminal cell binding region , 51. Thearticles . The simarticles , 51, 52.articles . For exaing MRSA . In addihalmitis . Therefo− bacteria, the cell walls of G+ bacteria are thicker (15–80 nm) and consist of tens of layers of peptidoglycan associated with teichoic acids  of streptomycin-resistant B.cereus within 10 s [B.cereus cells resulted in a 17,000-fold decrease of bacterial numbers within 20 s and near sterilization at 2 min when compared with 50 mM Tris buffer treatment [Endolysins show bactericidal activity against certain bacterial species that are closely related to the bacterial hosts of the phages from which they were produced. Despite their strong specificity, the host range of endolysins can reach approximately two-thirds of the tested strains, and some even reach 100%  was exposed to increasing concentrations  of either agent for over 8 days in vitro [B.anthracis or the formation of dense biofilms by staphylococci or streptococci does not block endolysin lytic activity [Given the resistance crisis, phage therapy was proposed and served as a powerful regimen for clinical infections . However choline , polyrha choline , and neu choline , respectobserved , 24. Endin vitro . LysGH15in vitro . The expactivity , 79, 92.+ bacteria [+ bacteria since the discovery of endolysins, especially after treatment failures increased considerably in S. aureus, Streptococcus sp., Enterococcus sp., and B. anthracis infections treated with antibiotic alone [+ bacteria; the majority of these studies involved mouse models and targeted MRSA and streptococcal species. Thus, this section mainly focused on the endolysins (including chimeolysins) in pre-clinical and clinical trial phases. Table + bacteria, and it will be discussed further.Increasing interest in endolysins comes with emerging bacterial resistance and increasing need for novel antimicrobial agents. Given the natural structure of exposed bacterial cell wall without outer membrane barriers, endolysin therapy usually works best against infections caused by Gbacteria . Immediaic alone , 94, 95.S.aureus is a representative of G+ bacteria that can cause skin and soft tissue infections, fetal pneumonia, pericarditis, brain abscess, bacteremia, and toxic shock syndrome [S.aureus infections are caused by MRSA in 15 European countries, and the resistance rates are closer to 50% in some of these countries [syndrome , 134. StS.aureus, and MSSA strains by > 2-log10 in vitro and protected against death caused by MRSA in mouse nasal decolonization and bacteremia models [S.aureus.In the case of antibiotic treatment failure, endolysin is an effective option to control MRSA infections . For exaa models . Eight ea models . The aboS.aureus infections are close to clinical application. P128 is an engineered endolysin (chimer) developed by the Indian company Gangagen, and it is currently in the clinical development stage. P128 is active against globally prevalent drug-resistant clinical S.aureus and S.epidermidis isolates. It exerts potent activity against sinus-derived S.aureus biofilms and is developed for clearing S.aureus nasal colonization and MRSA infection in mice and dogs [S.aureus [K, has the potential to reduce S.aureus colonization in the skin; thus, it may be used as a disinfecting agent in the healthcare environment [Furthermore, several endolysins for the treatment of and dogs , 136. St.aureus . Exebaca.aureus . AnotherS.pneumoniae is another clinically important G+ bacterium that can cause different diseases ranging from a streptococcal pharyngitis to life-threatening pneumonia [S.pneumoniae and ultimately prevent the development of acute otitis media following infection with influenza virus [S.pneumoniae rat endocarditis [S.pneumoniae to undetectable titers  5 h after a single dose treatment and did not induce Pal-resistant pneumococci after extensive exposure to the enzyme [S.suis phage has broad lytic activity against group A Streptococcus, group B Streptococcus, group G Streptococcus, group E Streptococcus, and S.pneumoniae. PlySs2 (128 μg/mL) led to a 3-log reduction in the growth of S.pyogenes within 1 h, and 2 mg of PlySs2 protected 92% (22/24) of mice from bacteremia caused by mixed MRSA and S.pyogenes infections [neumonia . Endolysneumonia , 139. A neumonia . Cpl-1 cza virus . A singlza virus . Cpl-1 acarditis  and muricarditis . In a moe enzyme . Therefofections . Similarfections , 76, 125+ bacteria, Staphylococcus and Streptococcus are the most common in clinical settings. Therefore, many studies on endolysin-related treatment have been conducted, whereas research on endolysin therapy of other G+ bacteria is relatively lacking. However, an increasing number of studies on endolysin treatment of other G+ bacteria have been performed in recent years. For example, PlyV12 from an E.faecalis phage and LysEFm5 from an E.faecium phage have been described to be useful in the treatment of mucosal infections [B.cereus and B.anthracis are pathogenic bacilli that can cause serious harm to their hosts via food poisoning and anthrax toxicity, respectively [B.cereus and B.anthracis both in vitro and in vivo [C.perfringens, which can cause the infection of over 95% of chickens [C.perfringens [L.monocytogenes in soya milk and is greatly needed in food safety and food processing systems [+ bacterial colonization from mucosal epithelium of either carriers or infected individuals and systemic infections, paving the way for endolysins to be applied as alternatives to the treatment of associated diseases in humans and animals.Among Gfections , 129. B.ectively . Two rec in vivo , 127. Mochickens . The endfringens , 131. In+ bacterial infections, the antimicrobial spectrum and antibacterial capacity of endolysins can be improved through the synergistic effect between the endolysins and antibiotics or other enzymes with diverse enzymatic specificities [S.aureus 305 through checkerboard assay [In the treatment of Gficities , 144. Strd assay . Two antrd assay , 146.S.aureus, including MRSA clinical strains [The synergistic effect between different endolysins or other cell wall hydrolases  can be explained by the enhanced destructive effect generated when two different bonds were simultaneously cleaved within the 3D peptidoglycan meshwork. Alternatively, the cleavage of the first bond by one enzyme can result in better accessibility to the second target site by the other endolysin, causing a faster degradation of the substrate. A novel chimeric endolysin ClyS exhibited a typical pattern of synergistic action with both vancomycin and oxacillin in vitro. More importantly, ClyS and oxacillin at low doses that were no protective individually were found to present synergistic effects against MRSA septic death in a mouse model . The com strains . Animal  strains . Concurr strains , 149. Th strains , 151.S.aureus; it shows no toxicity and adverse effects in mice, dogs, and monkeys under pre-clinical safety evaluation [S.aureus [As for applications, endolysin safety is an inescapable issue. Numerous experimental studies have shown that endolysins are innocuous after both topical and systemic administration in mice , 69, 76.aluation , 153. Fualuation . A high-.aureus . No seve.aureus , 154. Eu.aureus . However.aureus . To dateGiven the global prevalence of multidrug-resistant bacterial infections, endolysins are considered as an attractive therapeutic option in clinical settings. Although endolysins show many advantages in the treatment of drug-resistant bacteria Table , some chEscherichiacoli is the most common organism for the production of recombinant proteins, as this expression platform is well-established, and the cellular and molecular tools needed in the process of protein expression from gene cloning to protein purification are widely accessible [S.aureus recombinant endolysins are expressed in E. coli [E. coli expression system in the aspects of host engineering, expression vector design, and culture optimization [Pichiapastoris expression system has high recombinant protein yields; even some filamentous fungi or other systems can be considered for the production of recombinant endolysins [S. pneumoniae, can be successfully expressed in chloroplasts of Chlamydomonasreinhardtii; this host has many advantages, such as the lack of endotoxins, no infectious potential, and low production costs [Cutibacteriumacnes in cyanobacteria can reduce production costs and avoid toxicity issues caused by toxic bacterial components such as endotoxin [The large-scale production of phage endolysins has attracted great attention. However, the two main challenges that need to be solved are manufacturing cost and safety. cessible , 157. Inmization . Alternadolysins . For exaon costs . MoreoveE. faecalis and S. aureus, respectively [+ and G− pathogens by at least twofold [Furthermore, new strategies are needed to develop novel and suitable endolysins that possess enhanced bactericidal activity; expanded lytic spectrum; and increased solubility, stability, and circulating half-life. The modifications of native endolysins by molecular engineering and specific designing can completely create new enzymes with several improved features. Several approaches have been applied to modify endolysin enzymes, including domain deletion, addition, shuffling, and site-directed modifications. Virion-associated lysins (VALs) are another class of phage-encoded enzymes with antimicrobial activities that have been engineered to act on certain bacteria by fusing them into a chimeric lysin. EC300 and P128 are good examples of the VAL-derived chimeolysins that efficiently target ectively , 160. Thectively . A chimeectively . A study twofold . PerhapsS. aureus and its biofilm at pH 5  (PNIPAM) nanoparticles can be released in the S.aureus infection sites at 37 °C , production process optimization, and drug delivery development can be used to enhance the potential of endolysins, making endolysins a clinically proven drug to combat the crisis of drug-resistant bacteria in the future.The unique mode of action, the rapid killing activity against bacteria (including persisters), and the low probability of resistance development are appealing features of endolysins for their application as alternatives of antibacterial agents. Many studies have shown that endolysins are effective antimicrobial agents that have synergistic effects with diverse antibiotics and antimicrobial peptides. With the high priority for the development of novel agents against multidrug-resistant bacteria, phage endolysins are promising candidates that serve as therapeutic options for controlling G