Screening of Natural product as an efflux pump inhibitor and synthesis of its antibiotics conjugates: a novel approach to treat MDR strains

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Among these, the upregulation of efflux pumps plays a key role in multidrug resistance. Thus, the problem is addressed by screening and using natural product-antibiotic conjugates against MDR Staphylococcus . Out of the screened natural products, the blue pigment was showed an excellent efflux pump inhibitory activity in Staphylococus aureus ATCC 6538, which again proved by fluorescence emission and real time efflux assay. The MIC of the blue pigment was found to be 64 µg/ml however, the conjugates of blue pigment with ciprofloxacin and penicillin enhanced the antibacterial activity by 1.5 to 1.9 fold respectively, against S. aureus . The synthesis of conjugates were confirmed by UV-visible spectroscopic analysis. Overall, the present study highlights the potential of blue pigment derived from Streptomyces as an efflux pump inhibitor, and its conjugates with antibiotics could offer a better option for tackling multidrug-resistance bacteria. S. auerus efflux pump Conjugates Pseudomonas sp Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Antimicrobial resistance is documented as one of the most life-threatening risks to human health, due to which it is accountable for millions of deaths every year worldwide [ 1 ]. The major cause of the development of antimicrobial resistance was heavy use of antibiotics in humans, animals, and the environment, also due to the spread of antibiotic-resistant bacteria [ 2 ]. There are six leading pathogens, including Staphylococcus aureus, Streptococcus pneumoniae, Klebsiella pneumoniae, Acinetobacter baumannii , and Pseudomonas aeruginosa , responsible for millions of deaths attributed to antimicrobial resistance. The methicillin-resistant S. aureus (MRSA) caused the highest deaths (> 100000) attributable to antimicrobial resistance in 2019 [ 1 ]. Similarly, Pseudomonas aeruginosa is highly resistant to clinically used antibiotics, causing extensive infections and leading to serious disease conditions and mortality [ 3 ]. The bacterial pathogen develops resistance to the antibiotics through various mechanisms, for example, modification of target, production of antibiotic hydrolyzing enzymes, down regulation of porin channels, and upregulation of efflux pumps [ 4 ]. In recent years, to tackle the issue of antimicrobial resistance, several strategies were employed, such as the development of novel drugs with multiple targets (levonadifloxacin-target both DNA gyrase and topoisomerase IV) [ 5 ], the combination of β-lactam- β-lactamase inhibitor (Ceftazidime + avibactam) to restore the activity of antibiotics [ 6 ] and facilitation of drug entry (Cefiderocol) into the cell through the membrane channels by ‘trojan horse’ active transport mechanism [ 7 ]. However, the upregulation of efflux pumps is still the major bottleneck in the antimicrobial resistance of bacteria. In multi-drug resistance strains, the efflux pump contributes a major role in making the drug ineffective by expelling out [ 8 ]. The two leading pathogens, S. aureus and P. aeruginosa , are well known for the expression of different efflux pumps, which mainly include the RND efflux pumps in P. aeruginosa and NorA efflux pumps in S. aureus , respectively [ 9 , 10 ]. Unfortunately, till date there is no drug or drug combination available in clinical use that effectively down-regulates the efflux pump. Thus, there is an urgent need to look for alternate options such as adjuvants, new efflux pump inhibitors, and drug enhancers to curtail such infections. Also, due to the scarcity of new antimicrobial drugs, the re-evaluation of old substances with antimicrobial activity and their combination with the existing antibiotics will be a potential approach [ 11 , 12 , 13 ]. The various plant-derived secondary metabolites have excellent potential as resistance-modifying agents. The phytochemicals display noteworthy efflux pump inhibitory potential [ 14 ]. Similarly, in microbial sources, there are two secondary metabolites of Streptomyces MF-EA-371-NS1 namely EA-371α and EA-371δ reported for specific inhibitors of the Mex AB-Opr M pump in P. aeruginosa [ 15 ]. On this premise, the present study focused on the screening of previously reported microbial and plant-origin natural products such as naringenin, prodgiosin, curcumin, and microbial pigments as an efflux pump inhibitor. The efflux pump expressing S. aureus and P. aeruginosa were selected as test organisms. Besides, the conjugate of natural product + standard antibiotic was prepared and evaluated for its antibacterial potential against multidrug resistant strains. 2. Materials and Methodology 2.1 Materials: All analytical grade chemicals were procured from the Sigma Aldrich Co.(St Louis, MO, USA). Starch casein agar was purchased from Hi-media Lab Pvt. Ltd, Mumbai, India. Pseudomonas aeruginosa and Staphyloccoucs aureus (Clinical isolates and Standard cultures procured from ATCC and MTCC (Chandigrah). 2.2 Screening of antibacterial activity of naturally occurring plant and microbial compounds for efflux pump inhibition in MDR strains : To check the mechanism of antibiotic resistance and inhibition of efflux pump in MDR Pseudomonas aeruginosa and Staphylococcus aureus was analysed by agar based Ethidium Bromide cartwheel assay [ 16 ]. For this purpose, a nutrient agar plates containing the EtBr (700 µg/L) were prepared. The 24 h old culture of P. aeruginosa clinical isolate M and MTCC 27583 and S. aureus ATCC 6538 and clinical isolates was diluted up to turbidity standard McFarland 0.5 in 0.9% saline and then inoculated in cartwheel pattern on agar plates. After 24 h of incubation the plates were observed under UV trans-illuminator for the presence of fluorescence in each organism. Similarly, to evaluate the inhibition of efflux pump activity, freshly growing cultures of Pseudomonas aeruginosa MTCC 27583 and Staphylococcus aureus (Clinical isolate denoted as M) and S. auerus ATCC 6538 treated with the piperine (positive control), curcumin, prodgiosin, narigenin and blue pigment isolated from Streptomyces sp. at 100 µg/ml concentration for 2 h at room temperature under mild shaking. Treated cultures were swabbed on EtBr containing agar plates and observed under UV trans-illuminator for the presence of fluorescence after 24 h of incubation. The intensity of fluorescence emission was measured by Image J software. 2.3 Determination of Minimum inhibitory concentration of test compounds and existing antibiotics : The procedure was carried out for all the test organisms ( Staphyloccocus aurues and Pseudomonas aeruginosa ) and each test was carried out in triplicate by micro dilution method [ 17 ]. Stock solution of the test agent curcumin (dissolved in DMSO (dimethylsulfoxide; Merck, Germany), blue pigment (BP) and piperine (Pip) according to [ 17 ]. 200 µL of the nutrient broth was added in each of fourteen MIC tubes per bacterial strain. In the first MIC tube containing 200 µL broth, 200 µL of stock was added. After mixing well, 200 µL was transferred to the second MIC tube. This was continued till the last (14th ) tube. From the last tube 200 µL of the final solution was discarded. By following this serial dilution, the concentrations of the aqueous extract achieved was the following – 512, 256,128, 64, 32, 16, 8, 4, 2,, 0.5, 0.25, 0.12, 0.06 µg /ml. respectively. To each of the fourteen such prepared MIC tubes with varying concentrations, 200 µL (5 X 10 5 organisms/ml) of the earlier prepared strain of organism was added such that the final volume per tube was 400 µL. The tubes were then incubated for 24 hours at 37 o C. Similarly, the MIC for standard antibiotics ciprofloxacin and penicillin against S. auerus ATCC 6538 were determined. After the incubation, the MIC values were determined by visual inspection of the tubes. In each series of tubes, the last tube with clear supernatant was considered to be without any growth and taken as MIC value. Turbidity in the MIC tube indicated growth of the bacteria implying that the bacteria are resistant to test compound and existing antibiotics 2.3 Combination study: From the above results, the synergistic effect of blue pigment and curcumin, with ciprofloxacin and penicillin against S. auerus ATCC 6538 respectively, were determined by well diffusion assay. For this purpose the ciprofloxacin and penicillin at its 2/3 MIC concentration and blue pigment and curcumin at its 1/2 MIC concentration were used. The synthesized conjugates were written as Blue pigment-Ciprofloxacin (BP-Cipr), Curcumin-Ciprofloxacin (Cur-Cipr). Similarly the conjugates with penicillin were written as Blue pigment-penicillin (BP-Peni) and Curcumin-penicillin (Cur-Peni).Fold increase in antibacterial activity was calculated as per the protocol described by Birla et al. [ 18 ]. 2.4 Fluorescence emission assay: The effect of blue pigment and its antibiotic conjugates on uptake and release of EtBr studied by fluorescence emission assay and modified spectrophotometric real time efflux assay as reported earlier respectively [ 19 , 20 ]. Overnight grown culture of Staphylococcus aureus (Clinical isolate) and S. auerus ATCC 6538 was used to prepare bacterial suspension (turbidity adjusted according to standard McFarland) in uptake buffer (110 mM NaCl, 7 mM KCl, 50 mM NH4Cl, 0.4 mM Na 2 HPO 4 , 52 mM Tris base and 0.2% glucose, adjusted to pH 7.5 with HCl). The cells were pelleted out by centrifugation at 5000 G for 5 min and suspended in uptake buffer containing blue pigment, conjugated Penicillin-blue pigment and Ciprofloxacin-blue pigment (at its MIC concentration), Piperine (at its MIC concentration) and EtBr at 10 µg/mL concentration. After 1 h of incubation at 37°C (for EtBr uptake) the cells with treatment and without treatment (control) were again separated, washed two times with uptake buffer and suspended in same buffer. Immediately, the suspension was assayed for the release of EtBr using spectrophotometer (Shimadzu, UV 1800) and accumulation of EtBr by Spectrophotoflurometer (Shimadzu, RF-5301) and TLC method. 2.5 Natural product antibiotic conjugates study On the basis of above MIC results, the minimum inhibitory concentrations of test compound and antibiotics were used for synthesis of conjugates. Simple method in which the test compound and antibiotics were mixed in 1:1 ratio in phosphate buffer (pH 7). Briefly, 2 ml of), blue pigment (10 µg/ml) and curcumin ( 200 µg/ml) mixed with 2 ml of Ciprofloxacin (0.06 µg/ml)/Penicillin (2 µg/ml) and incubate for 1h at 37°C with continuous shaking. The synthesis of conjugated antibiotic was confirmed by UV visible spectrophotometer (UV 1800, Shimadzu). Again antimicrobial activity of synthesized conjugated were checked by disc diffusion assay. 2.7 Statistical Analysis: Statistical analyses were performed using Graph Pad Prism (Version 2.1, USA). All experiments were performed in triplicates and data represents as a mean ± standard deviation. The differences among means were determined using One-Way ANOVA and multiple comparisons at significance and confidence levels of 0.05 (95% confidence interval) was determined. The p value of < 0.05 was considered significant. 3. Results and Discussion 3.1 Screening of efflux pumps inhibitory potential of natural products The all-natural products prodigiosin, naringenin, curcumin, orange and blue pigment of Streptomyces were tested for its efflux pump inhibition activity. For this purpose first we confirmed the presence of active efflux pumps in selected strains of S. aureus by simple, instrument free agar based EtBr cartwheel [ 16 ]. EtBr act as a substrate for many efflux pumps and it expel out from the bacterial cells [ 16 ]. Among the tested bacterial strains of Staphylococcus , S. aureus ATCC 6538 showed highest efflux pump activity i.e. no fluorescence was observed even at high concentration like 700 µg/ml EtBr. Meanwhile, S. aureus M (a clinical isolate) showed slight efflux pump activity, indicated by reduced fluorescence. Therefore, S. aureus ATCC 6538 was selected for the further efflux pump inhibition study (Fig. 1 ). For the confirmation of efflux pump inhibitory potential of the compounds, effluxing activity of test organism were evaluated by exposing a test organism S. aureus ATCC 6538 to natural products. The test organism S. aureus ATCC 6538 was treated with prodigiosin, naringenin, blue, orange pigment, curcumin, EtBr (negative control) and piperine (positive control) and then swabbed on EtBr agar plate. After 24 h incubation the plates were observes for the fluorescence emission (Fig. 2 ) under UV transilluminator (Camag, Swiss). The bacterial suspension without treatment was used as a growth control, which showed no fluorescence (Fig. 2 a). Amongst the tested natural products it was observed that, only blue pigment and curcumin showed an efflux pump inhibition potential in S. aureus ATCC 6538 while, in light of EtBr (negative control) and piperine (positive control) none of the other tested natural products showed efflux pump inhibition (Fig. 2 b, c, d and e). The retention of EtBr was confirmed by fluorescence emission, piperine, blue pigment and curcumin showed a higher peak (red colour intensity) as compare to growth control and EtBr (Fig. 2 f, g, h, i and j). The treatment of blue pigment leads to accumulate higher EtBr, which resulted in maximum fluorescence as compare to curcumin treatment (Fig. 2 i). This indicates the higher efflux pump inhibition potential of blue pigment against S. aureus ATCC 6538. Thus, both the products blue pigment and curcumin were shortlisted for the further studies. Multidrug resistance phenotype of bacteria is due to the active efflux activity, hence inhibition of efflux pump activity proven to be an excellent way to tackle resistance [ 20 ]. In this investigation it was proved that the blue pigment act as an efflux pump inhibitor in S. aureus ATCC 6538. Similarly, Kalia et al. [ 20 ] reported capsaicin as a Nor A efflux pump inhibitor in the Staphylococcus aureus . The copper nanoparticles also reported as potent inhibitor of efflux pumps in Staphylococcus aureus and P. aeruginosa [ 21 ]. Similarly, Martin et al. [ 16 ] reported the EtBr cartwheel assay was a very good tool for identification of MDR phenotype in clinical strains and also, for determination of MIC of antibiotics, which was a substrate for efflux pump thereby, confirmed the activity of overexpressed efflux system. Also, coumarin from Mesua ferrea showed the inhibition of EtBr efflux by arresting overexpressed Nor A efflux pump in Staphylococcus aureus [ 11 ] 3.2 Determination of Minimum inhibitory concentration of natural products and standard antibiotics On the basis of screening results of efflux pump inhibition, the minimum inhibitory concentration (MIC) of blue pigment, curcumin and standard antibiotics i.e. ciprofloxacin and penicillin was determined against S. aureus ATCC 6538 by microdilution method. MICs for all screened compound and standard antibiotics are summarized in Table S1. From the Table 1 , it was observed that blue pigment and curcumin showed high MIC values against S. aureus as compare to standard antibiotics penicillin, ciprofloxacin (Table 1 ). Table 1 MIC of different natural compounds against test organism. Sr. No. Test Compound S. auerus ATCC 6538 (MIC in µg/ml) 1. Ciprofolxacin 16 2. Peniciilin 2.0 3. Blue pigment 64.0 4. Curcumin 25 3.3 Combination study: As the blue pigment and curcumin showed comparatively less antimicrobial potential, their combinational effect was evaluated. During the combination study it was observed that both blue pigment and curcumin were significantly increased the antimicrobial potential of penicillin (Table 2 ). The conjugate of curcumin and blue pigment with penicillin showed 1.1 to 1.9 folds increase in antibacterial activity of penicillin (Cur-Peni, BP-Peni) respectively, against S. aureus ATCC 6538 (Table 2 , Fig. 3 a). Similarly, about 1.2 to 1.5 folds increase in antibacterial activity of ciprofloxacin when conjugated with curcumin and blue pigment (Cur-Cipro, BP-Cipro,) respectively (Table S2, Fig. 3 b). Table 2 Antibacterial activity of Natural product-antibiotic conjugates Sr. No Treatments Zone of Inhibition (mm) Fold increase in antibacterial activity S. auerus ATCC 6538 1. Penicillin 12 -- 2. Blue pigment-penicillin (BP-Peni) 23 1.9 3. Curcumin- Penicillin (Cur- Peni) 14 1.1 4. Ciprofloxacin 7 -- 5. Blue pigment- Ciprofloxacin (BP-Cipro) 11 1.5 6. Curcumin- Ciprofloxacin (Cur-Cipro) 9 1.2 Note: The fold increase area of different antibiotics for Staphyloccocus aureus was calculated by the equation (b 2 - a 2 ) ⁄a 2 , where a and b are zone of inhibitions for antibiotic (a) and antibiotic + natural product (b ) respectively (Birla et al., 2008). To confirm this combination effect and mode of actions, these all natural products were further screened for its efflux pump inhibition potential by fluorescence emission assay. The efflux pump inhibition was one of the major mechanism describe before for high antimicrobial potential in such combinations i.e. antibiotics and natural products [ 19 , 20 ]. 3.4 Fluorescence emission assay: To confirm the inhibitory action of blue pigment antibiotic conjugate against efflux pump of S. aureus ATCC 6538 fluorescence emission of EtBr and real time efflux assay was carried out. The increase in fluorescence intensity is proportional to accumulation of EtBr [ 19 ]. The increase in accumulation of EtBr within the cell confirmed the inhibition of efflux pump. Staphylococcus aureus ATCC 6538 showed the high fluorescence intensity which directly related to accumulation of EtBr within the cell (Fig. 4 a). The penicillin-Blue pigment (BP-Peni) conjugates showed maximum inhibition of efflux pump as compared to positive control piperine -EtBr a known efflux pump inhibitor (Fig. 4 a). Blue pigment showed significant inhibition of efflux pump in S. aureus ATCC 6538 confirmed by fluorescence emission assay, hence we checked the effect of blue pigment and its conjugate with penicillin on uptake and release of EtBr in S. aureus ATCC 6538 by real time efflux assay. The efflux of EtBr again confirmed by the spectrophotometric analysis. It was found that in control (without treatment) set the high amount of EtBr release by cells (high intensity of characteristic peak of EtBr, Fig. 4 b, peak 2) as compared with standard peak of EtBr (Fig. 4 b, peak 1). Concentration of EtBr released by penicillin treated cells was lower as peak intensity was decreased followed by blue pigment treated cells (Fig. 4 b, peak 3 and 4, respectively) as compared to control. In blue pigment-penicillin (BP-Pen) conjugate treatment the characteristic peak of EtBr was not observed, which proved that the EtBr completely retain by cells (Fig. 4 b, peak 5). Hence, it confirmed the complete inhibition of efflux pump activity due to our synthesized conjugate BP-Pen in S. aureus ATCC 6538. Similarly, the released EtBr confirmed by TLC method (inset of Fig. 4 b). It was found that in S. aureus ATCC 6538 treated with the BP-Penicillin EtBr band in TLC was not observed (less intensity as compared to control) which again confirmed that the EtBr was retained by cells due to inhibition of efflux pump and released by cells (inset of Fig. 4 b). Similarly, Christane et al. [ 21 ] quantified inhibition of efflux pump by Cu- nanoparticles in S. aureus and P. aeruginosa on the basis of residual fluorescence by real time efflux assay. The results are in accordance with the Christane et al. [ 21 ]. 3.5 Natural product antibiotic conjugates study Synthesized natural product antibiotic conjugates studied by UV-visible spectrophotometer (Shimadzu UV -1800, Tokyo, Japan). The spectra of natural product antibiotic conjugates were represented in Fig. 5 . It was observed that standard penicillin showed the maximum absorption at 264 nm. The UV visible spectra of curcumin-penicillin showed the characteristics peaks of both curcumin (at 405nm) and penicillin (at 310 and 264 nm), respectively (Fig. 5 a). Also, UV- Visible spectra Blue pigment –penicillin conjugates (BP-Peni) showed all characteristic peak of pigment (at 600 nm) and penicillin which confirmed the synthesis of natural product antibiotic conjugates (Fig. 5 b). Similarly, Blue pigment (BP)-Cipro conjugates showed the strongest peak at 600 nm and 271 nm, respectively which is the characteristics peak of blue pigment and ciprofloxacin (Fig. 5 c) which confirmed the successful synthesis of antibiotic conjugates. Also, in Curcumin-Cipro (Cur-Cipro) conjugate the characteristic peak of both curcumin (at 571 nm) and ciprofloxacin (at 271 nm) (Fig. 5 d) was observed. Such results are reported in case of silver, gold, copper, selenium and chitosan nanoparticles conjugated with the different antibiotics which enhanced the activity of antibiotics [ 21 , 22 , 23 , 24 ]. 4. Conclusions Antimicrobial resistance is recognized as one of the extreme risks to human health, which are accountable for millions of deaths every year worldwide. Hence, in present study successfully screened the natural products and its conjugates synthesized with standard antibiotic to tackle the antimicrobial resistance in bacteria. During the screening study for efflux pump inhibition, it was proved that blue pigment was act as a potential efflux pump inhibitor as they inhibits the efflux of EtBr in S. aureus ATCC 6538, this was also confirmed by EtBr cartwheel, fluorescence emission and real time efflux assay. Originally blue pigment, ciprofloxacin and penicillin showed the moderate antibacterial activity with MICs of 64, 16 and 2 µg/ml respectively, against S. aureus ATCC 6538. Interestingly, in the conjugation, the blue pigment with penicillin and ciprofloxacin showed enhanced activity by 1.5 to 1.9 fold against S. aureus. Similarly, the synthesis of natural product–antibiotic conjugates and efflux pumps inhibition was confirmed by UV-visible spectroscopic analysis. The present study of synthesis of natural product–antibiotic conjugates will be helpful in future to manage the antibiotics resistant pathogens like S. aureus in which major antibiotics resistance mechanism is efflux pump along with others. Declarations 5.0 Disclosure statement Authors do not have any conflict of interest Author Contribution K.R.M. : All experimental work done and wrote the main manuscript text, prepared figures.S,.K.: Designed the experiment and reviewed the manuscript.S.V.P.: Reviewed the manuscript and guide the experimental part.All authors reviewed the manuscript. Acknowledgements: This work was supported by Indian Council of Medical Research, Govt. of India (ICMR) RA-I fellowship (File No.45/23/2022-BIO/BMS). References Murray J, Ikuta S, Sharara F, Swetschinski L, Aguilar GR, Gray A, Han C, Bisignano C, Rao P, Wool E, Johnson SC (2022) Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. The Lancet. 399:629–55. https://doi.org/10.1016/S0140-6736(21)02724-0 McEwen SA, Collignon PJ (2018). Antimicrobial resistance: a one health perspective. Antimicrobial resistance in bacteria from livestock and companion animals. 521 – 47. https://doi.org/10.1128/9781555819804.ch25 . 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Copper nanoparticles as an efflux pump inhibitor to tackle drug resistant bacteria. RSC Adv 51: 2899–12909 Saha, B, Bhattacharya J, Mukherjee A, Ghosh A, Santra C, Dasgupta AK, Karmakar P (2007) In vitro structural and functional evaluation of gold nanoparticles conjugated antibiotics. Nanoscale Res Lett 2: 614–622. Nikam PB, Salunkhe J D, Marathe K R, Alghuthaymi M, Abd-Elsalam KA, Patil SV (2022) Rhizobium pusense-Mediated Selenium Nanoparticles–Antibiotics Combinations against Acanthamoeba sp . Microorg 10: 2502. Wahab S, Ali HM, Khan M, Khan T, Krishnaraj C, Yun SI (2024). Green synthesis and antibacterial assessment of chitosan/silver nanocomposite conjugated with tobramycin against antibiotic resistant Pseudomonas aeruginosa . Arab J Chem 17: 105458. https://doi.org/10.1016/j.arabjc.2023.105458 . Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5787579","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":399970366,"identity":"92aebbe1-47a9-490b-b040-57d75474c493","order_by":0,"name":"Kiran Marathe","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAklEQVRIiWNgGAWjYLACxgYGAzYQ48MPGxC38QBBLQehWhhn9qSBTSBOC4hm5mE7DBbAq0W3/ezDzx932BjzSR9+9oCH57zd2vbDQFtqbKJxaTE7k24scfBMmhkbX5q5gYTF7eRtZxKBWo6l5Tbg0nIgjUHiYNthGzYeBjMJA57byWYHgFoYGw7j1nL+GfOPg23/gVrYv0kksJ1LNjv/kICWG2lsQFsOmLHx8JhJHGA7YGd2g5AtN56xWZxtSzYGaimTbOxJTjC7AbQlAZ9fzqcx36hsszOc38O+TfrPDzt7s/PpDx98qLHBqQUDJIJVJhCrHATsSVE8CkbBKBgFIwMAAGnnYuP5KJZ+AAAAAElFTkSuQmCC","orcid":"","institution":"Kavayitri Bahinabai Chaudhari North Maharashtra University","correspondingAuthor":true,"prefix":"","firstName":"Kiran","middleName":"","lastName":"Marathe","suffix":""},{"id":399970367,"identity":"261d1ff2-41a3-4a20-9676-545a712faf28","order_by":1,"name":"Sunil Koli","email":"","orcid":"","institution":"Department of Microbiology, Yashwantrao Chavan College of Science, Vidyanagar, Karad-415124, MS, India.","correspondingAuthor":false,"prefix":"","firstName":"Sunil","middleName":"","lastName":"Koli","suffix":""},{"id":399970368,"identity":"f2524838-32c7-4491-97ab-08ed7c123a53","order_by":2,"name":"Satish Patil","email":"","orcid":"","institution":"Kavayitri Bahinabai Chaudhari North Maharashtra University","correspondingAuthor":false,"prefix":"","firstName":"Satish","middleName":"","lastName":"Patil","suffix":""}],"badges":[],"createdAt":"2025-01-08 09:23:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5787579/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5787579/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":73635517,"identity":"c1d6d7f9-c1a5-45da-a7d4-002195fc2c30","added_by":"auto","created_at":"2025-01-13 07:13:25","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":96908,"visible":true,"origin":"","legend":"\u003cp\u003eEfflux pump activity at high concentration of EtBr in \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eP. aeruginosa\u003c/em\u003e by cartwheel assay a) Control (in absence of UV illumination) b) Test (in presence of UV illumination)\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5787579/v1/0c856096ae9c11d658de0cd2.jpg"},{"id":73635518,"identity":"f6194321-661a-46be-a337-092b9fce499e","added_by":"auto","created_at":"2025-01-13 07:13:25","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":184919,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of natural products on efflux pump inhibition in \u003cem\u003eS. aureus\u003c/em\u003e ATCC 6538 by fluorescence emission assay performed in UV-transilluminator. a) Growth control of S. aureus ATCC 6538 without substance, b) EtBr only, c) Piperine + EtBr (positive control), d) Blue pigment +EtBr, e) Curcumin+ EtBr; Histogram of red colour intensity of f) Growth control, g) EtBr , h) Piperine + EtBr and i) Blue pigment +EtBr, j) Curcumin+ EtBr.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5787579/v1/20a13df25f709cf78ddbf5d8.jpg"},{"id":73635528,"identity":"a4c1ea2d-f1db-4963-9c8c-b2d079198614","added_by":"auto","created_at":"2025-01-13 07:13:25","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":210994,"visible":true,"origin":"","legend":"\u003cp\u003eAntibacterial activity of conjugated blue pigment \u0026nbsp;a) with penicillin \u0026nbsp;and \u0026nbsp;b) ciprofloxacin against \u003cem\u003eS. aureus\u003c/em\u003e ATCC 6538\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5787579/v1/4be8bc7014f7641d3330df45.jpg"},{"id":73635859,"identity":"86c3e74b-0da8-4dee-91b9-53167315aeff","added_by":"auto","created_at":"2025-01-13 07:21:25","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":406506,"visible":true,"origin":"","legend":"\u003cp\u003ea) Effect of blue pigment on efflux pump inhibition in \u003cem\u003eS. aureus \u003c/em\u003eATCC 6538 by fluorescence emission assay and b) Spectrophotometric analysis of efflux of EtBr in \u003cem\u003eS. aureus\u003c/em\u003eATCC 6538 treated with blue pigment by real time efflux assay\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5787579/v1/ed126a66e2a91bc545dcb17b.jpg"},{"id":73635538,"identity":"f1619b76-f4b2-4f36-9b5f-6a7a131dafe3","added_by":"auto","created_at":"2025-01-13 07:13:26","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":116659,"visible":true,"origin":"","legend":"\u003cp\u003eSpectroscopic analysis of synthesized antibiotic conjugates: a) \u003cdel\u003e\u0026nbsp;\u003c/del\u003eCurcumin-Penicillin, b) Blue pigment- Penicillin, c) Curcumin-Ciprofloxacin, d) Blue pigment- Cprofloxacin\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5787579/v1/50b04850ad75104aa5fbbe58.jpg"},{"id":73820860,"identity":"9fa6e172-6627-4ee6-9478-6b89401a40e3","added_by":"auto","created_at":"2025-01-15 04:16:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1822856,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5787579/v1/78614772-2e97-44ef-a264-5fd1d7ca0def.pdf"},{"id":73635855,"identity":"66e64c65-80e4-45b3-950f-9737bdae185a","added_by":"auto","created_at":"2025-01-13 07:21:25","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":186443,"visible":true,"origin":"","legend":"","description":"","filename":"GaphicalAbstract.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5787579/v1/9861d07eaa9fbf41b611e23e.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Screening of Natural product as an efflux pump inhibitor and synthesis of its antibiotics conjugates: a novel approach to treat MDR strains","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAntimicrobial resistance is documented as one of the most life-threatening risks to human health, due to which it is accountable for millions of deaths every year worldwide [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The major cause of the development of antimicrobial resistance was heavy use of antibiotics in humans, animals, and the environment, also due to the spread of antibiotic-resistant bacteria [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. There are six leading pathogens, including \u003cem\u003eStaphylococcus aureus, Streptococcus pneumoniae, Klebsiella pneumoniae, Acinetobacter baumannii\u003c/em\u003e, and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e, responsible for millions of deaths attributed to antimicrobial resistance. The methicillin-resistant \u003cem\u003eS. aureus\u003c/em\u003e (MRSA) caused the highest deaths (\u0026gt;\u0026thinsp;100000) attributable to antimicrobial resistance in 2019 [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Similarly, \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e is highly resistant to clinically used antibiotics, causing extensive infections and leading to serious disease conditions and mortality [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe bacterial pathogen develops resistance to the antibiotics through various mechanisms, for example, modification of target, production of antibiotic hydrolyzing enzymes, down regulation of porin channels, and upregulation of efflux pumps [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In recent years, to tackle the issue of antimicrobial resistance, several strategies were employed, such as the development of novel drugs with multiple targets (levonadifloxacin-target both DNA gyrase and topoisomerase IV) [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], the combination of β-lactam- β-lactamase inhibitor (Ceftazidime\u0026thinsp;+\u0026thinsp;avibactam) to restore the activity of antibiotics [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] and facilitation of drug entry (Cefiderocol) into the cell through the membrane channels by \u0026lsquo;trojan horse\u0026rsquo; active transport mechanism [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. However, the upregulation of efflux pumps is still the major bottleneck in the antimicrobial resistance of bacteria. In multi-drug resistance strains, the efflux pump contributes a major role in making the drug ineffective by expelling out [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe two leading pathogens, \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eP. aeruginosa\u003c/em\u003e, are well known for the expression of different efflux pumps, which mainly include the RND efflux pumps in \u003cem\u003eP. aeruginosa\u003c/em\u003e and NorA efflux pumps in \u003cem\u003eS. aureus\u003c/em\u003e, respectively [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Unfortunately, till date there is no drug or drug combination available in clinical use that effectively down-regulates the efflux pump. Thus, there is an urgent need to look for alternate options such as adjuvants, new efflux pump inhibitors, and drug enhancers to curtail such infections. Also, due to the scarcity of new antimicrobial drugs, the re-evaluation of old substances with antimicrobial activity and their combination with the existing antibiotics will be a potential approach [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe various plant-derived secondary metabolites have excellent potential as resistance-modifying agents. The phytochemicals display noteworthy efflux pump inhibitory potential [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Similarly, in microbial sources, there are two secondary metabolites of \u003cem\u003eStreptomyces\u003c/em\u003e MF-EA-371-NS1 namely EA-371α and EA-371δ reported for specific inhibitors of the Mex AB-Opr M pump in \u003cem\u003eP. aeruginosa\u003c/em\u003e [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOn this premise, the present study focused on the screening of previously reported microbial and plant-origin natural products such as naringenin, prodgiosin, curcumin, and microbial pigments as an efflux pump inhibitor. The efflux pump expressing \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eP. aeruginosa\u003c/em\u003e were selected as test organisms. Besides, the conjugate of natural product\u0026thinsp;+\u0026thinsp;standard antibiotic was prepared and evaluated for its antibacterial potential against multidrug resistant strains.\u003c/p\u003e"},{"header":"2. Materials and Methodology","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Materials:\u003c/h2\u003e \u003cp\u003eAll analytical grade chemicals were procured from the Sigma Aldrich Co.(St Louis, MO, USA). Starch casein agar was purchased from Hi-media Lab Pvt. Ltd, Mumbai, India. \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e and \u003cem\u003eStaphyloccoucs aureus\u003c/em\u003e (Clinical isolates and Standard cultures procured from ATCC and MTCC (Chandigrah).\u003c/p\u003e \u003cp\u003e \u003cb\u003e2.2 Screening of antibacterial activity of naturally occurring plant and microbial compounds for efflux pump inhibition in MDR strains\u003c/b\u003e:\u003c/p\u003e \u003cp\u003eTo check the mechanism of antibiotic resistance and inhibition of efflux pump in MDR \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e was analysed by agar based Ethidium Bromide cartwheel assay [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. For this purpose, a nutrient agar plates containing the EtBr (700 \u0026micro;g/L) were prepared. The 24 h old culture of \u003cem\u003eP. aeruginosa clinical isolate M and\u003c/em\u003e MTCC 27583 and \u003cem\u003eS. aureus\u003c/em\u003e ATCC 6538 and clinical isolates was diluted up to turbidity standard McFarland 0.5 in 0.9% saline and then inoculated in cartwheel pattern on agar plates. After 24 h of incubation the plates were observed under UV trans-illuminator for the presence of fluorescence in each organism. Similarly, to evaluate the inhibition of efflux pump activity, freshly growing cultures of \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e MTCC 27583 and \u003cem\u003eStaphylococcus aureus (Clinical isolate denoted as M)\u003c/em\u003e and \u003cem\u003eS. auerus\u003c/em\u003e ATCC 6538 treated with the piperine (positive control), curcumin, prodgiosin, narigenin and blue pigment isolated from \u003cem\u003eStreptomyces sp.\u003c/em\u003e at 100 \u0026micro;g/ml concentration for 2 h at room temperature under mild shaking. Treated cultures were swabbed on EtBr containing agar plates and observed under UV trans-illuminator for the presence of fluorescence after 24 h of incubation. The intensity of fluorescence emission was measured by Image J software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e2.3 Determination of Minimum inhibitory concentration of test compounds and existing antibiotics\u003c/b\u003e:\u003c/h2\u003e \u003cp\u003eThe procedure was carried out for all the test organisms (\u003cem\u003eStaphyloccocus aurues\u003c/em\u003e and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e) and each test was carried out in triplicate by micro dilution method [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Stock solution of the test agent curcumin (dissolved in DMSO (dimethylsulfoxide; Merck, Germany), blue pigment (BP) and piperine (Pip) according to [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. 200 \u0026micro;L of the nutrient broth was added in each of fourteen MIC tubes per bacterial strain. In the first MIC tube containing 200 \u0026micro;L broth, 200 \u0026micro;L of stock was added. After mixing well, 200 \u0026micro;L was transferred to the second MIC tube. This was continued till the last (14th ) tube. From the last tube 200 \u0026micro;L of the final solution was discarded. By following this serial dilution, the concentrations of the aqueous extract achieved was the following \u0026ndash; 512, 256,128, 64, 32, 16, 8, 4, 2,, 0.5, 0.25, 0.12, 0.06 \u0026micro;g /ml. respectively. To each of the fourteen such prepared MIC tubes with varying concentrations, 200 \u0026micro;L (5 X 10\u003csup\u003e5\u003c/sup\u003eorganisms/ml) of the earlier prepared strain of organism was added such that the final volume per tube was 400 \u0026micro;L. The tubes were then incubated for 24 hours at 37\u003csup\u003eo\u003c/sup\u003eC. Similarly, the MIC for standard antibiotics ciprofloxacin and penicillin against \u003cem\u003eS. auerus\u003c/em\u003e ATCC 6538 were determined. After the incubation, the MIC values were determined by visual inspection of the tubes. In each series of tubes, the last tube with clear supernatant was considered to be without any growth and taken as MIC value. Turbidity in the MIC tube indicated growth of the bacteria implying that the bacteria are resistant to test compound and existing antibiotics\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Combination study:\u003c/h2\u003e \u003cp\u003eFrom the above results, the synergistic effect of blue pigment and curcumin, with ciprofloxacin and penicillin against \u003cem\u003eS. auerus\u003c/em\u003e ATCC 6538 respectively, were determined by well diffusion assay. For this purpose the ciprofloxacin and penicillin at its 2/3 MIC concentration and blue pigment and curcumin at its 1/2 MIC concentration were used. The synthesized conjugates were written as Blue pigment-Ciprofloxacin (BP-Cipr), Curcumin-Ciprofloxacin (Cur-Cipr). Similarly the conjugates with penicillin were written as Blue pigment-penicillin (BP-Peni) and Curcumin-penicillin (Cur-Peni).Fold increase in antibacterial activity was calculated as per the protocol described by Birla et al. [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Fluorescence emission assay:\u003c/h2\u003e \u003cp\u003eThe effect of blue pigment and its antibiotic conjugates on uptake and release of EtBr studied by fluorescence emission assay and modified spectrophotometric real time efflux assay as reported earlier respectively [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Overnight grown culture of \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (Clinical isolate) and \u003cem\u003eS. auerus\u003c/em\u003e ATCC 6538 was used to prepare bacterial suspension (turbidity adjusted according to standard McFarland) in uptake buffer (110 mM NaCl, 7 mM KCl, 50 mM NH4Cl, 0.4 mM Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, 52 mM Tris base and 0.2% glucose, adjusted to pH 7.5 with HCl). The cells were pelleted out by centrifugation at 5000 G for 5 min and suspended in uptake buffer containing blue pigment, conjugated Penicillin-blue pigment and Ciprofloxacin-blue pigment (at its MIC concentration), Piperine (at its MIC concentration) and EtBr at 10 \u0026micro;g/mL concentration. After 1 h of incubation at 37\u0026deg;C (for EtBr uptake) the cells with treatment and without treatment (control) were again separated, washed two times with uptake buffer and suspended in same buffer. Immediately, the suspension was assayed for the release of EtBr using spectrophotometer (Shimadzu, UV 1800) and accumulation of EtBr by Spectrophotoflurometer (Shimadzu, RF-5301) and TLC method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Natural product antibiotic conjugates study\u003c/h2\u003e \u003cp\u003eOn the basis of above MIC results, the minimum inhibitory concentrations of test compound and antibiotics were used for synthesis of conjugates. Simple method in which the test compound and antibiotics were mixed in 1:1 ratio in phosphate buffer (pH 7). Briefly, 2 ml of), blue pigment (10 \u0026micro;g/ml) and curcumin ( 200 \u0026micro;g/ml) mixed with 2 ml of Ciprofloxacin (0.06 \u0026micro;g/ml)/Penicillin (2 \u0026micro;g/ml) and incubate for 1h at 37\u0026deg;C with continuous shaking. The synthesis of conjugated antibiotic was confirmed by UV visible spectrophotometer (UV 1800, Shimadzu). Again antimicrobial activity of synthesized conjugated were checked by disc diffusion assay.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Statistical Analysis:\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using Graph Pad Prism (Version 2.1, USA). All experiments were performed in triplicates and data represents as a mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. The differences among means were determined using One-Way ANOVA and multiple comparisons at significance and confidence levels of 0.05 (95% confidence interval) was determined. The p value of \u0026lt;\u0026thinsp;0.05 was considered significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Screening of efflux pumps inhibitory potential of natural products\u003c/h2\u003e \u003cp\u003eThe all-natural products prodigiosin, naringenin, curcumin, orange and blue pigment of \u003cem\u003eStreptomyces\u003c/em\u003e were tested for its efflux pump inhibition activity. For this purpose first we confirmed the presence of active efflux pumps in selected strains of \u003cem\u003eS. aureus\u003c/em\u003e by simple, instrument free agar based EtBr cartwheel [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. EtBr act as a substrate for many efflux pumps and it expel out from the bacterial cells [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Among the tested bacterial strains of \u003cem\u003eStaphylococcus\u003c/em\u003e, \u003cem\u003eS. aureus\u003c/em\u003e ATCC 6538 showed highest efflux pump activity i.e. no fluorescence was observed even at high concentration like 700 \u0026micro;g/ml EtBr. Meanwhile, \u003cem\u003eS. aureus\u003c/em\u003e M (a clinical isolate) showed slight efflux pump activity, indicated by reduced fluorescence. Therefore, \u003cem\u003eS. aureus\u003c/em\u003e ATCC 6538 was selected for the further efflux pump inhibition study (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFor the confirmation of efflux pump inhibitory potential of the compounds, effluxing activity of test organism were evaluated by exposing a test organism \u003cem\u003eS. aureus\u003c/em\u003e ATCC 6538 to natural products. The test organism \u003cem\u003eS. aureus\u003c/em\u003e ATCC 6538 was treated with prodigiosin, naringenin, blue, orange pigment, curcumin, EtBr (negative control) and piperine (positive control) and then swabbed on EtBr agar plate. After 24 h incubation the plates were observes for the fluorescence emission (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e) under UV transilluminator (Camag, Swiss). The bacterial suspension without treatment was used as a growth control, which showed no fluorescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Amongst the tested natural products it was observed that, only blue pigment and curcumin showed an efflux pump inhibition potential in \u003cem\u003eS. aureus\u003c/em\u003e ATCC 6538 while, in light of EtBr (negative control) and piperine (positive control) none of the other tested natural products showed efflux pump inhibition (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, c, d and e). The retention of EtBr was confirmed by fluorescence emission, piperine, blue pigment and curcumin showed a higher peak (red colour intensity) as compare to growth control and EtBr (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003ef, g, h, i and j). The treatment of blue pigment leads to accumulate higher EtBr, which resulted in maximum fluorescence as compare to curcumin treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003ei). This indicates the higher efflux pump inhibition potential of blue pigment against \u003cem\u003eS. aureus\u003c/em\u003e ATCC 6538. Thus, both the products blue pigment and curcumin were shortlisted for the further studies.\u003c/p\u003e \u003cp\u003eMultidrug resistance phenotype of bacteria is due to the active efflux activity, hence inhibition of efflux pump activity proven to be an excellent way to tackle resistance [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In this investigation it was proved that the blue pigment act as an efflux pump inhibitor in \u003cem\u003eS. aureus\u003c/em\u003e ATCC 6538. Similarly, Kalia et al. [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] reported capsaicin as a Nor A efflux pump inhibitor in the \u003cem\u003eStaphylococcus aureus\u003c/em\u003e. The copper nanoparticles also reported as potent inhibitor of efflux pumps in \u003cem\u003eStaphylococcus aureus\u003c/em\u003e and \u003cem\u003eP. aeruginosa\u003c/em\u003e [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Similarly, Martin et al. [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] reported the EtBr cartwheel assay was a very good tool for identification of MDR phenotype in clinical strains and also, for determination of MIC of antibiotics, which was a substrate for efflux pump thereby, confirmed the activity of overexpressed efflux system. Also, coumarin from \u003cem\u003eMesua ferrea\u003c/em\u003e showed the inhibition of EtBr efflux by arresting overexpressed Nor A efflux pump in \u003cem\u003eStaphylococcus aureus\u003c/em\u003e [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e3.2 Determination of Minimum inhibitory concentration of natural products and standard antibiotics\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eOn the basis of screening results of efflux pump inhibition, the minimum inhibitory concentration (MIC) of blue pigment, curcumin and standard antibiotics i.e. ciprofloxacin and penicillin was determined against \u003cem\u003eS. aureus\u003c/em\u003e ATCC 6538 by microdilution method. MICs for all screened compound and standard antibiotics are summarized in Table S1. From the Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, it was observed that blue pigment and curcumin showed high MIC values against \u003cem\u003eS. aureus\u003c/em\u003e as compare to standard antibiotics penicillin, ciprofloxacin (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMIC of different natural compounds against test organism.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSr. No.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTest Compound\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eS. auerus ATCC 6538\u003c/em\u003e\u003c/p\u003e \u003cp\u003e(MIC in \u0026micro;g/ml)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCiprofolxacin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePeniciilin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBlue pigment\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e64.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCurcumin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Combination study:\u003c/h2\u003e \u003cp\u003eAs the blue pigment and curcumin showed comparatively less antimicrobial potential, their combinational effect was evaluated. During the combination study it was observed that both blue pigment and curcumin were significantly increased the antimicrobial potential of penicillin (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The conjugate of curcumin and blue pigment with penicillin showed 1.1 to 1.9 folds increase in antibacterial activity of penicillin (Cur-Peni, BP-Peni) respectively, against \u003cem\u003eS. aureus\u003c/em\u003e ATCC 6538 (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Similarly, about 1.2 to 1.5 folds increase in antibacterial activity of ciprofloxacin when conjugated with curcumin and blue pigment (Cur-Cipro, BP-Cipro,) respectively (Table S2, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAntibacterial activity of Natural product-antibiotic conjugates\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSr. No\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTreatments\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eZone of Inhibition (mm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFold increase in antibacterial activity\u003c/p\u003e \u003cp\u003e\u003cem\u003eS. auerus\u003c/em\u003e ATCC 6538\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePenicillin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBlue pigment-penicillin (BP-Peni)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCurcumin- Penicillin (Cur- Peni)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCiprofloxacin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBlue pigment- Ciprofloxacin (BP-Cipro)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCurcumin- Ciprofloxacin (Cur-Cipro)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eNote: The fold increase area of different antibiotics for \u003cem\u003eStaphyloccocus aureus\u003c/em\u003e was calculated by the equation (b\u003csup\u003e2\u003c/sup\u003e - a\u003csup\u003e2\u003c/sup\u003e) \u0026frasl;a\u003csup\u003e2\u003c/sup\u003e, where a and b are zone of inhibitions for antibiotic (a) and antibiotic\u0026thinsp;+\u0026thinsp;natural product (b ) respectively (Birla et al., 2008).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTo confirm this combination effect and mode of actions, these all natural products were further screened for its efflux pump inhibition potential by fluorescence emission assay. The efflux pump inhibition was one of the major mechanism describe before for high antimicrobial potential in such combinations i.e. antibiotics and natural products [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Fluorescence emission assay:\u003c/h2\u003e \u003cp\u003eTo confirm the inhibitory action of blue pigment antibiotic conjugate against efflux pump of \u003cem\u003eS. aureus\u003c/em\u003e ATCC 6538 fluorescence emission of EtBr and real time efflux assay was carried out. The increase in fluorescence intensity is proportional to accumulation of EtBr [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The increase in accumulation of EtBr within the cell confirmed the inhibition of efflux pump. \u003cem\u003eStaphylococcus aureus\u003c/em\u003e ATCC 6538 showed the high fluorescence intensity which directly related to accumulation of EtBr within the cell (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). The penicillin-Blue pigment (BP-Peni) conjugates showed maximum inhibition of efflux pump as compared to positive control piperine -EtBr a known efflux pump inhibitor (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003eBlue pigment showed significant inhibition of efflux pump in \u003cem\u003eS. aureus\u003c/em\u003e ATCC 6538 confirmed by fluorescence emission assay, hence we checked the effect of blue pigment and its conjugate with penicillin on uptake and release of EtBr in \u003cem\u003eS. aureus\u003c/em\u003e ATCC 6538 by real time efflux assay. The efflux of EtBr again confirmed by the spectrophotometric analysis. It was found that in control (without treatment) set the high amount of EtBr release by cells (high intensity of characteristic peak of EtBr, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, peak 2) as compared with standard peak of EtBr (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, peak 1). Concentration of EtBr released by penicillin treated cells was lower as peak intensity was decreased followed by blue pigment treated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, peak 3 and 4, respectively) as compared to control. In blue pigment-penicillin (BP-Pen) conjugate treatment the characteristic peak of EtBr was not observed, which proved that the EtBr completely retain by cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, peak 5). Hence, it confirmed the complete inhibition of efflux pump activity due to our synthesized conjugate BP-Pen in \u003cem\u003eS. aureus\u003c/em\u003e ATCC 6538. Similarly, the released EtBr confirmed by TLC method (inset of Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). It was found that in \u003cem\u003eS. aureus\u003c/em\u003e ATCC 6538 treated with the BP-Penicillin EtBr band in TLC was not observed (less intensity as compared to control) which again confirmed that the EtBr was retained by cells due to inhibition of efflux pump and released by cells (inset of Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Similarly, Christane et al. [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] quantified inhibition of efflux pump by Cu- nanoparticles in \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eP. aeruginosa\u003c/em\u003e on the basis of residual fluorescence by real time efflux assay. The results are in accordance with the Christane et al. [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Natural product antibiotic conjugates study\u003c/h2\u003e \u003cp\u003eSynthesized natural product antibiotic conjugates studied by UV-visible spectrophotometer (Shimadzu UV -1800, Tokyo, Japan). The spectra of natural product antibiotic conjugates were represented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003e. It was observed that standard penicillin showed the maximum absorption at 264 nm. The UV visible spectra of curcumin-penicillin showed the characteristics peaks of both curcumin (at 405nm) and penicillin (at 310 and 264 nm), respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Also, UV- Visible spectra Blue pigment \u0026ndash;penicillin conjugates (BP-Peni) showed all characteristic peak of pigment (at 600 nm) and penicillin which confirmed the synthesis of natural product antibiotic conjugates (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eSimilarly, Blue pigment (BP)-Cipro conjugates showed the strongest peak at 600 nm and 271 nm, respectively which is the characteristics peak of blue pigment and ciprofloxacin (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003ec) which confirmed the successful synthesis of antibiotic conjugates. Also, in Curcumin-Cipro (Cur-Cipro) conjugate the characteristic peak of both curcumin (at 571 nm) and ciprofloxacin (at 271 nm) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003ed) was observed. Such results are reported in case of silver, gold, copper, selenium and chitosan nanoparticles conjugated with the different antibiotics which enhanced the activity of antibiotics [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eAntimicrobial resistance is recognized as one of the extreme risks to human health, which are accountable for millions of deaths every year worldwide. Hence, in present study successfully screened the natural products and its conjugates synthesized with standard antibiotic to tackle the antimicrobial resistance in bacteria. During the screening study for efflux pump inhibition, it was proved that blue pigment was act as a potential efflux pump inhibitor as they inhibits the efflux of EtBr in \u003cem\u003eS. aureus\u003c/em\u003e ATCC 6538, this was also confirmed by EtBr cartwheel, fluorescence emission and real time efflux assay. Originally blue pigment, ciprofloxacin and penicillin showed the moderate antibacterial activity with MICs of 64, 16 and 2 \u0026micro;g/ml respectively, against \u003cem\u003eS. aureus\u003c/em\u003e ATCC 6538. Interestingly, in the conjugation, the blue pigment with penicillin and ciprofloxacin showed enhanced activity by 1.5 to 1.9 fold against \u003cem\u003eS. aureus.\u003c/em\u003e Similarly, the synthesis of natural product\u0026ndash;antibiotic conjugates and efflux pumps inhibition was confirmed by UV-visible spectroscopic analysis. The present study of synthesis of natural product\u0026ndash;antibiotic conjugates will be helpful in future to manage the antibiotics resistant pathogens like \u003cem\u003eS. aureus\u003c/em\u003e in which major antibiotics resistance mechanism is efflux pump along with others.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e5.0 Disclosure statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors do not have any conflict of interest\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eK.R.M. : All experimental work done and wrote the main manuscript text, prepared figures.S,.K.: Designed the experiment and reviewed the manuscript.S.V.P.: Reviewed the manuscript and guide the experimental part.All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements:\u003c/h2\u003e \u003cp\u003eThis work was supported by Indian Council of Medical Research, Govt. of India (ICMR) RA-I fellowship (File No.45/23/2022-BIO/BMS).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMurray J, Ikuta S, Sharara F, Swetschinski L, Aguilar GR, Gray A, Han C, Bisignano C, Rao P, Wool E, Johnson SC (2022) Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. 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Arab J Chem 17: 105458. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.arabjc.2023.105458\u003c/span\u003e\u003cspan address=\"10.1016/j.arabjc.2023.105458\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"S. auerus, efflux pump, Conjugates, Pseudomonas sp","lastPublishedDoi":"10.21203/rs.3.rs-5787579/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5787579/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBacteria acquire resistance to different classes of antibiotics through various mechanisms. Among these, the upregulation of efflux pumps plays a key role in multidrug resistance. Thus, the problem is addressed by screening and using natural product-antibiotic conjugates against MDR \u003cem\u003eStaphylococcus\u003c/em\u003e. Out of the screened natural products, the blue pigment was showed an excellent efflux pump inhibitory activity in \u003cem\u003eStaphylococus aureus\u003c/em\u003e ATCC 6538, which again proved by fluorescence emission and real time efflux assay. The MIC of the blue pigment was found to be 64 µg/ml however, the conjugates of blue pigment with ciprofloxacin and penicillin enhanced the antibacterial activity by 1.5 to 1.9 fold respectively, against \u003cem\u003eS. aureus\u003c/em\u003e. The synthesis of conjugates were confirmed by UV-visible spectroscopic analysis. Overall, the present study highlights the potential of blue pigment derived from \u003cem\u003eStreptomyces\u003c/em\u003eas an efflux pump inhibitor, and its conjugates with antibiotics could offer a better option for tackling multidrug-resistance bacteria.\u003c/p\u003e","manuscriptTitle":"Screening of Natural product as an efflux pump inhibitor and synthesis of its antibiotics conjugates: a novel approach to treat MDR strains","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-13 07:13:20","doi":"10.21203/rs.3.rs-5787579/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b8fa1510-ce23-4a6b-aeb8-55931f989d23","owner":[],"postedDate":"January 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-01-18T06:38:09+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-13 07:13:20","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5787579","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5787579","identity":"rs-5787579","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}