Up to 50% of patients with myasthenia gravis (MG) without acetylcholine receptor antibodies (AChR-Abs) have antibodies to muscle-specific kinase (MuSK). Most MuSK antibodies (MuSK-Abs) are IgG4 and inhibit agrin-induced MuSK phosphorylation, leading to impaired clustering of AChRs at the developing or mature neuromuscular junction. However, IgG1-3 MuSK-Abs also exist in MuSK-MG patients, and their potential mechanisms have not been explored fully.125I-α-bungarotoxin. Transcriptomic analysis was performed on treated myotubes.C2C12 myotubes were exposed to MuSK-MG plasma IgG1-3 or IgG4, with or without purified agrin. MuSK, Downstream of Kinase 7 (DOK7), and βAChR were immunoprecipitated and their phosphorylation levels identified by immunoblotting. Agrin and agrin-independent AChR clusters were measured by immunofluorescence and AChR numbers by binding of IgG1-3 MuSK-Abs impaired AChR clustering without inhibiting agrin-induced MuSK phosphorylation. Moreover, the well-established pathway initiated by MuSK through DOK7, resulting in βAChR phosphorylation, was not impaired by MuSK-IgG1-3 and was agrin-independent. Nevertheless, the AChR clusters did not form, and both the number of AChR microclusters that precede full cluster formation and the myotube surface AChRs were reduced. Transcriptomic analysis did not throw light on the pathways involved. However, the SHP2 inhibitor, NSC-87877, increased the number of microclusters and led to fully formed AChR clusters.MuSK-IgG1-3 is pathogenic but seems to act through a noncanonical pathway. Further studies should throw light on the mechanisms involved at the neuromuscular junction. AChR clustering is induced by the release of agrin, a soluble heparan-sulfate proteoglycan from the motor nerve terminal.4 Agrin binds to low-density lipoprotein receptor-related protein 4 (LRP4) which then interacts with MuSK on the postsynaptic membrane.5 This triggers MuSK phosphorylation and recruitment of Downstream of Kinase 7 (DOK7) leading to a phosphorylation cascade that eventually reaches rapsyn and the AChR which cocluster (reviewed in references Myasthenia gravis (MG) with antibodies against muscle-specific kinase (MuSK-MG) is an autoimmune disease that impairs neuromuscular transmission leading to widespread weakness and fatigability of skeletal muscles.8 These antibodies block the binding of agrin-LRP4 to MuSK resulting in inhibition of the phosphorylation cascade and dispersal of AChRs, as studied using the mouse C2C12 cell line in vitro.10 Although not as dominant as MuSK-IgG4-Abs, however, MuSK-IgG1, 2, or 3 subclass antibodies exist in all patients with MuSK-MG and were also reported to interfere with AChR clustering.10 Moreover, divalent antibodies are pathogenic in mice immunized against purified MuSK, irrespective of mouse IgG1 antibodies .11The autoantibodies against MuSK (MuSK-Abs) are mainly of the IgG4 subclass and functionally monovalent due to “fab-arm exchange.”12 could restore the AChR clusters as it did for IgG4 MuSK-Abs.13The aim of this work was to develop further insights into the potential pathogenic role(s) of MuSK-IgG1-3-Abs (MuSK-IgG1-3) and to see whether the SHP2 phosphatase inhibitor, NSC-87877,13 Five chosen plasmas were pooled for IgG subclass fractionation and incubated overnight at 4°C with an IgG4 affinity matrix . IgG4 was eluted from the matrix with glycine buffer  and neutralized. The flow-through fractions containing IgG1-3 were then purified by binding to protein G Sepharose  and eluted with glycine buffer, neutralized, and re-exposed to the IgG4 affinity matrix to remove any residual IgG4. MuSK-Ab titres were measured in each of the purified fractions by radioimmunoassay and adjusted to 0.5 nM for all experiments.Plasmapheresis material was collected, with informed consent, from 8 MuSK-Ab positive MG patients. The total and MuSK-IgG1-3 titres were measured as previously described.13 Myotubes were then incubated with purified MuSK antibody subclasses (0.5 nM) in the presence or absence of recombinant short form agrin 1:1,000 (producing approximately 50% of maximum AChR clusters). Myotubes exposed to medium only were negative controls. AChR clusters were labeled with Alexa Fluor 594 α-bungarotoxin  and fixed in 3% formaldehyde. Twenty fields, selected with bright field, were counted for “fully formed clusters” (≥3 μm) and, in some experiments, also for “microclusters” (≥1 μm and <3 μm) using ImageJ software. When required, NSC-87877 was added with or without agrin.13C2C12 mouse myoblasts  were differentiated into myotubes with differentiation medium .Phosphorylation of MuSK, DOK7, and βAChR was assessed by precipitation of each protein and Western blotting. Myotubes were starved of fetal calf/horse serum for 3 hours and incubated at 37°C with MuSK-Ab subclasses with or without agrin . After the appropriate incubation time, the myotubes were lysed in cold lysis buffer , and phosphatase  inhibitor cocktails) and centrifuged for 10 minutes at 12,600 rpm. For pulldown of MuSK exposed to MuSK-IgG1-3 or IgG4, the supernatants were incubated overnight at 4°C with Protein G Dynabeads  without addition of further MuSK-Abs. For pulldown from cells exposed to DMEM or control IgG, a polyclonal anti-MuSK antibody AF562  was used. Mouse DOK7 was precipitated with anti-DOK7 , and βAChR was first labeled with biotin-α-bungarotoxin  and precipitated with Dynabeads Streptavidin T1 . The precipitated proteins were eluted into SDS sample buffer for electrophoresis and Western blotting. The blots were first probed with anti-phosphotyrosine antibody 1:1,000  followed by secondary rat anti-mouse native IgG HRP-conjugated antibody . The nitrocellulose was stripped and reprobed for MuSK, DOK7, or βAChR expression with commercial antibodies AF562, A7, and anti-βAChR/148 , respectively. Band densitometry was performed using ImageJ software. MuSK, DOK7, and βAChR phosphorylation levels were normalized to the levels of their respective proteins.125I-α-bungarotoxin at 1 × 106 cpm/mL per well. After washing, the cells were lysed in extraction buffer , and the total cpm was counted on a γ-counter.C2C12 myotubes were exposed overnight to agrin  and MuSK-Ab subclasses (0.5 nM). Myotubes were incubated for 1 hour at room temperature with Total RNA was extracted from myotubes after exposure to the antibody preparations or NSC-87877 100 µg for 2.5 hours at 37°C. Myotubes were washed with differentiation medium, and RNA was extracted using TRIzol Reagent ; contaminating genomic DNA was removed using the TURBO DNA-free kit  following manufacturers' instructions. After purity check, 20 µL of each sample (at 50 ng/µL) were sent in triplicates to Novogene (UK) for transcriptomic library preparation and sequencing. RNA sequences were aligned to the mus musculus genome using STAR (Spliced Alignment to a Reference). Reads were counted using featureCounts and analyzed using edgeR (Bioconductor). Data were filtered to exclude genes with <1 count per million. RNA composition was adjusted using calcNormFactors, and the coefficient of variation and dispersion was estimated. TopTags was used to obtain log-fold change data. Bigwig files were created from BAM files for viewing in the UCSC genome browser.p values <0.05 were considered statistically significant.Statistical tests were performed with GraphPad Prism 6 as indicated in the figure legends. Error bars represent the standard error of the mean of 3 independent experiments unless otherwise specified. Differences with The study of archived patient samples, as in reference The authors confirm that all data supporting the findings of this study will be made available upon reasonable request to the corresponding author.links.lww.com/NXI/A890). To confirm first that MuSK-IgG1-3 as well as IgG4 inhibited agrin-induced AChR clustering, myotubes were pre-exposed overnight to DMEM alone, agrin, or agrin plus 0.5 nM of the 2 MuSK-Ab preparations. AChR clusters were counted as previously described.13Levels and specificities of pooled plasma, IgG1-3 and IgG4 fractions are shown in eFigure 1 (p < 0.001) by MuSK-IgG1-3 even in the absence of agrin (p < 0.05), suggesting a direct activation of MuSK and the downstream pathway by the antibodies . For MuSK and DOK7, phosphorylation increased sharply in the first 1–2 hours and then fell to around 50% at 8 hours . Phospho125I-α-bungarotoxin to label the membrane AChRs. Compared with DMEM or agrin alone, there were reductions  after incubation in IgG1-3 but not in IgG4 , but as expected, there were 70/field after agrin exposure  IgG1-3 v control). The only known gene in the AChR clustering pathway was Rho GTPase, upregulated 3.8 log-fold; but with an enzyme assay, agrin, IgG1-3, and IgG4 MuSK-Abs all increased Rho GTPase enzyme activity equally (data not shown).To try to identify additional proteins/pathways that could be involved in the effects of IgG1-3, myotubes were exposed for 2.5 hours to DMEM, MuSK-IgG1-3, or total IgG from a healthy individual. Intracellular RNA was extracted, and gene expression in DMEM alone compared with MuSK-IgG1-3 or healthy IgG. A total of 102 gene transcripts were upregulated and 15 were downregulated at least 2 log-fold by MuSK-IgG1-3. After subtracting transcripts in healthy IgG from those for MuSK-IgG1-3, cytokines and transcriptional factors  were highly represented in the IgG1-3–treated myotubes , the Ahrr repressor, and Sic39a10 Zinc transporter were upregulated >2 log-fold by NSC-87877, suggesting an induction of metabolic/detoxicating pathways. Of possible relevance to the clustering of the AChRs, however, was neuregulin-1 (Nrg1), but this was just below threshold.It is widely accepted that the IgG4 MuSK-Abs form the highest proportion of MuSK-Abs in patients with MG, inhibit agrin-induced MuSK phosphorylation (and DOK7 phosphorylation as shown here), and, consequently, the phosphorylation and clustering of the AChRs. The potential pathogenic pathway of the smaller population of MuSK-IgG1-3, however, is unclear. Here, we confirm that IgG1-3-Abs also inhibit AChR clustering, but their effects differ from those of IgG4, may involve a different pathway, and their effect can be counteracted by SHP2 inhibition. Overall, the results suggest that there is more to learn about the pathways involved in both AChR cluster formation and the pathophysiology of MuSK-MG.10 Recent studies have used engineered monovalent or divalent monoclonal antibodies, derived from MuSK-MG patient B cells, to explore the roles of subclass and valency. These found that passive transfer of monovalent MuSK-Abs rapidly induced clinical and electromyographical defects in mice, whereas only 1 of the 2 divalent antibodies produced the same phenotype and more gradually. Similarly, monovalent MuSK-Abs inhibited MuSK phosphorylation , whereas the same antibodies in divalent form increased MuSK phosphorylation and a partial increase rather than an inhibition of AChR clustering.15-17 We did not find evidence of AChR clusters in the MuSK-IgG1-3–treated myotubes but did find substantial inhibition of clustering. The differences may relate to the use of lower concentrations (0.5 nM MuSKAb activity) of native IgG1-3, rather than cloned antibodies.As known from previous studies, MuSK-IgG4 antibodies undergo Fab-arm exchange in vivo becoming functionally monovalent, and their Fab fragments inhibit agrin-induced MuSK phosphorylation and AChR clustering.7 There are some parallels between the results reported here and those found with a mutation in the tyrosine kinase domain of MuSK, A617V.18 In cells constitutively expressing MuSK A617V, MuSK phosphorylation was increased, whereas AChR clusters were reduced, but in this case, microclusters were increased over the numbers in medium alone. It was concluded that overactive MuSK phosphorylation impairs the formation of large clusters/true NMJ morphology, similar to findings here.MuSK-Abs seemed to impair the transition from unclustered AChRs to microclusters and then to fully matured clusters, but this could be due to a mechanism acting independently of the canonical clustering pathway.13 Here, we show that NSC-87877 also overcomes the inhibitory effects of IgG1-3. The drug may circumvent the effects of IgG1-3, acting through separate pathways that regulate the formation and maturation of AChR clusters. NSC-87877 was also shown to enhance AChR stabililty,19 but whether it has any effect on MuSK membrane expression is not clear.We showed earlier that NSC-87877 alone induces MuSK phosphorylation, enhances the effect of agrin, and overcomes the inhibitory effect of MuSK-IgG4.20 these were not identified in the current analysis. RNA for Rho GTPase was upregulated by MuSK-IgG1-3, but the enzyme activity was not different between agrin, IgG1-3, and IgG4 antibody-treated myotubes (data not shown). Three main pathways are now known to be involved in the formation and maintenance of the neuromuscular junction ,29 and this possibility needs to be explored in in vivo models. Divalent binding of MuSK by IgG1-3-Abs, leading to internalization by endocytosis and proteolysis, is conceptually likely . In this context, it is interesting that exposure to agrin alone was shown to cause internalization of MuSK32; the reduced phosphorylation over time in the presence of agrin alone  activated complement on MuSK-expressing HEK cells,in alone  would be16 Despite these limitations, the results should stimulate further research not only on MuSK-MG but also on the additional pathways involved in the physiology of the neuromuscular junction.There are several limitations of this study. We did not examine the MuSK epitopes of the serum MuSK-Abs that we studied, and it is possible that they differ from those of IgG4. The control IgG for the transcriptomic analysis was not IgG4-depleted, and this might affect the results. Cloned human MuSK antibodies would have been very valuable, particularly for the transcriptomic analyses, although one has to consider that patient antibodies are polyclonal, binding to various epitopes, and monoclonal antibodies may not be representative of the predominant serum antibody.


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


As these molecules fundamentally differ in their inhibitory mode and chemical structure, our finding may open a new line for developing more effective allosteric/active site inhibitors to combat flaviviruses infection.Arboviruses such as Dengue, yellow fever, West Nile, and Zika are flaviviruses vector-borne RNA viruses transmitted biologically among vertebrate hosts by blood-taking vectors. Many flaviviruses are associated with neurological, viscerotropic, and hemorrhagic diseases, posing significant health and socioeconomic concerns as they adapt to new environments. Licensed drugs against them are currently unavailable, so searching for effective antiviral molecules is still necessary. Epigallocatechin molecules, a green tea polyphenol, have shown great virucidal potential against flaviviruses, including DENV, WNV, and ZIKV. The interaction of EGCG with the viral envelope protein and viral protease, mainly identified by computational studies, describes the interaction of these molecules with viral proteins; however, how the viral NS2B/NS3 protease interacts with epigallocatechin molecules is not yet fully deciphered. Consequently, we tested the antiviral potential of two epigallocatechin molecules (EGC and EGCG) and their derivative (AcEGCG) against DENV, YFV, WNV, and ZIKV NS2B/NS3 protease. Thus, we assayed the effect of the molecules and found that a mixture of the molecules EGC (competitive) and EGCG (noncompetitive) inhibited the virus protease of YFV, WNV, and ZIKV more effectively with IC Flaviviridae family consist of 89 virus species divided into four genera. The genus Flavivirus encompasses 53 enveloped positive-sense single RNA viruses. The virion consists of the three structural proteins (capsid (C), membrane (M), and envelope (E)) and seven nonstructural proteins presented in infected cells   according to Formula (2):θ is the ellipticity measured at a given wavelength λ (deg), c is the protein concentration (mol L−1), l is the cell path length (cm), and n is the number of amino acids.Seven repeated scans were performed for all CD measurements, and three separate scans were used to establish the baseline. The wavelength range applied for far-UV spectra was settled from 200 nm to 260 nm in a time constant of 1 s and 50 nm/min continuous scanning mode, using a Jasco J-1100 CD spectrometer . NS2B/NS3The ligand structure of EGC, EGCG, and AcEGCG was retrieved from the Zinc database . LigandsIn silico studies were carried out on X-ray models of Dengue2 (PDB code: 4M9T), yellow fever (PDB code: 6URV), West Nile (PDB code: 2IJO), and Zika (PDB code: 5LC0) NS2B/NS3 protease. Furthermore, the docking process was initiated by following a series of necessary steps such as ligands and proteins preparation, grid generation, and molecular docking.+ or Cl− counterions.All of the proteins had the amino acid side chain corrected to the protonation state of pH 7.4, determined by the H++ web server . The comThe MD and computational analysis were performed based on a previously described approach ,33. In oTo gain insight into the binding mode of the ligand, we docked the ligands to the stable models prepared previously. Docking calculations were performed using AutoDock Vina 1.1.12 ,35. The AMBER18  program α atoms, residue-by-residue over the equilibrated trajectories. Clustering analysis was performed with the k-means method ranging from 2 to 6, and Davies-Bouldin index (DBI) values and silhouette analyses were used to assess the clustering quality. The interaction energy between the proteins and the ligands was calculated using the generalized Born GB-Neck2 [The CPPTRAJ  program GB-Neck2  implicitE. coli cells (as described before), we tested the impact effect of two plant-derivative catechins (EGC and EGCG) and the modified one, AcEGCG  [50 values of 6.3 µM (DENV2) and 8 µM (YFV). Compared to EGCG, EGC does not have a trihydroxybenzoate ring, which may contribute to its inhibitory activity toward the active site. Therefore, the IC50 values increased drastically against DENV2 (145 µM), followed by YFV, WNV, and ZIKV  . The samand ZIKV , did notprotease , Table 5pro does not alter the Km but changes the Vmax, indicating that both molecules inhibit the proteases by a noncompetitive mode, which means allosterically  were mixed with a molar ratio of 1:1 .In order to determine the binding affinity of EGC, EGCG, and AcEGCG molecules, we used the SPR (surface plasmon resonance) approach. After activation of carboxylic groups via EDC/NHS, NS2B/NS3Kinetic fitting revealed dissociation constants from the low µM to low nM range for the two-state kinetic reaction, as seen in pro interaction with the epigallocatechin molecules induced a time-dependent secondary state, as demonstrated by SPR (pro). Intrinsic tryptophan (Trp) fluorescence was used to characterize the NS2B/NS3pros under the influence of the EGC, EGCG, and AcEGCG molecules following the possible red or blue shift excitation changes. The maximum Trp emission on the proteins at pH 7.5 was centered at 338 nm; after titration of the EGCG and AcEGCG molecules, the emission band for DENV2, YFV, and ZIKV NS2B/NS3pros shifted to 360 nm  and EGC was docked at the active site of the proteases. The selected protease/ligand complex was subject to 100 ns (2 replicas) of MD simulation embedded in an octahedral TIP3P water box. The same starting atom position with different random initial velocities was used for the replicates. Based on binding energy and the frame cluster, a representative structure was chosen for each protease/molecule complex  see ; those s complex .pros backbone atoms regarding the starting structure along the 100 ns trajectories. Concerning the complex with EGC, the NS2B domain did not stabilize well in one of the replicas along the MD simulation  drastically reduces the inhibition effect of the EGCG molecule.For brevity’s sake, it is worth remembering that noncompeting molecules were suggested to bind to the pockets on the back of the protease’s active site, as described by ,25,33.pro adopts a chymotrypsin-like fold with a catalytic triad composed of His51, Asp75, and Ser135 in the cleft between two ß-barrels. Despite the low sequence identity (~50%) (NS2B/NS3y (~50%) a,b, the y (~50%) c. Nevertpros display polar and negatively charged surfaces; the same can be observed for the allosteric sites  and shows that EGC, EGCG, and its derivative EGCG octaacetate inhibit protease activity, with the EGCG showing the finest ICal agent ,20,46,47pro; however, in cell infection studies, they might interact with multiple binding sites [pro. Molecular docking and dynamics simulation studies predicted the epigallocatechin molecule’s binding pocket, showing that the EGC potentially forms interactions with the activity site of the studied proteins. On the other hand, EGCG and AcEGCG molecules interact with the viral protease allosterically.The mode of action of these molecules is not very well understood, but in this work, we can hypothesize that the molecules interact with the NS2B/NS3ng sites . In thisDespite the polymorphism identified in several viral proteins, this modification may not affect the catalytic efficiency of the enzyme ,50. TherThe molecule EGC exhibited a competitive inhibition model suggesting that even if the molecule interacts with amino acid residues in the active/binding site, it still allows the interaction between the substrate and the protein, probably regarding the size of the molecule.pro with the EGCG molecule through the active site using in silico studies, which disagrees with our experimental results, which show that this molecule is an allosteric inhibitor. In addition to their virucidal capacity, several studies have shown that EGCG can act by blocking the necessary receptors for viral attachment to the target cell, as in the case of HIV, where the drug binds to CD4 expressed on T cells and inhibits the virus binding to this site, blocking viral infection [In our in vitro assay, the EGCG molecule interacts noncompetitively with the proteases, indicating that this molecule is an allosteric inhibitor. We suggest the trihydroxybenzoate ring prevents the molecule from interacting with the active site. Yadav and colleagues  describenfection ,52. Carnnfection  describenfection  describenfection ,49, HasVnfection , HCV NS3nfection , and HIV50 value in the nM range.Here, we have demonstrated that the epigallocatechin molecules presented in this study, natural compounds found in abundance in many foods (EGC and EGCG) and its modified form (AcEGCG), inhibit in vitro the activity of the essential virus protease (NS2B/NS3). Despite these encouraging results, we have shown that combining the EGC and EGCG molecules, which displayed different interaction modes (competitive and noncompetitive), drastically increased the inhibition of the YFV, WNV, and ZIKV proteases, displaying an ICEGCG and its derivatives have a range of wide biological activity, but in the human body, these molecules can undergo multiple processes of metabolization ,54,55. TFlavivirus genus  and point to EGC and EGCG as potential scaffolds for the prospect design of mixed antiviral compounds that affect the viral protein in two different binding regions. Optimization of these bioactive molecules is essential to achieve high oral bioavailability, including chemical modifications, absorption, and penetration enhancers, as well as formulation design. It is important to emphasize that oral delivery of new lead molecules can be facilitated by a thorough understanding of the interaction of the inhibitory molecule with its target.In conclusion, our results have revealed the antiviral activity of two different epigallocatechin, EGC and EGCG, and its derivative, AcEGCG, against the protease of four clinically relevant members of the