Deciphering the molecular mechanisms underlying anti-pathogenic potential of a polyherbal formulation Enteropan® against multidrug-resistant Pseudomonas aeruginosa

Sweety Parmar; Gemini Gajera; Nidhi Thakkar; Hanmanthrao S. Palep; Vijay Kothari

doi:10.33393/dti.2024.3082

Authors

Sweety Parmar Institute of Science, Nirma University, Ahmedabad - India https://orcid.org/0000-0001-9011-5191
Gemini Gajera Institute of Science, Nirma University, Ahmedabad - India https://orcid.org/0000-0002-1604-912X
Nidhi Thakkar Institute of Science, Nirma University, Ahmedabad - India https://orcid.org/0009-0005-6781-4441
Hanmanthrao S. Palep Dr. Palep’s Medical Research Foundation, Mumbai - India https://orcid.org/0000-0001-6848-8135
Vijay Kothari Institute of Science, Nirma University, Ahmedabad - India https://orcid.org/0000-0003-1035-7217

DOI:

https://doi.org/10.33393/dti.2024.3082

Keywords:

Antimicrobial resistance (AMR), Anti-virulence, Caenorhabditis elegans, Iron/Sulphur homeostasis, Metal homeostasis, Network analysis, Nitrosative stress, Polyherbal, Transcriptome

Abstract

Objective: Anti-pathogenic potential of a polyherbal formulation Enteropan® was investigated against a multidrug-resistant strain of the bacterium Pseudomonas aeruginosa.

Methods: Growth, pigment production, antibiotic susceptibility, etc., were assessed through appropriate in vitro assays. Virulence of the test pathogen was assessed employing the nematode worm Caenorhabditis elegans as a model host. Molecular mechanisms underlining the anti-pathogenic activity of the test formulation were elucidated through whole transcriptome analysis of the extract-exposed bacterial culture.

Results: Enteropan-pre-exposed P. aeruginosa displayed reduced (~70%↓) virulence towards the model host C. elegans. Enteropan affected various traits like biofilm formation, protein synthesis and secretion, quorum-modulated pigment production, antibiotic susceptibility, nitrogen metabolism, etc., in this pathogen. P. aeruginosa could not develop complete resistance to the virulence-attenuating activity of Enteropan even after repeated exposure to this polyherbal formulation. Whole transcriptome analysis showed 17% of P. aeruginosa genome to get differentially expressed under influence of Enteropan. Major mechanisms through which Enteropan exerted its anti-virulence activity were found to be generation of nitrosative stress, oxidative stress, envelop stress, quorum modulation, disturbance of protein homeostasis and metal homeostasis. Network analysis of the differently expressed genes resulted in identification of 10 proteins with high network centrality as potential targets from among the downregulated genes. Differential expression of genes coding for five (rpoA, tig, rpsB, rpsL, and rpsJ) of these targets was validated through real-time polymerase chain reaction too, and they can further be pursued as potential targets by various drug discovery programmes.

Downloads

Download data is not yet available.

References

Kunz Coyne AJ, El Ghali A, Holger D, Rebold N, Rybak MJ. Therapeutic strategies for emerging multidrug-resistant Pseudomonas aeruginosa. Infect Dis Ther. 2022;11(2):661-682. https://doi.org/10.1007/s40121-022-00591-2 PMID:35150435 DOI: https://doi.org/10.1007/s40121-022-00591-2

Craig M. CDC’s Antibiotic Resistance Threats Report 2019. Extended spectrum β-lactamase (ESBL)-producing Enterobacteriaceae. CDC; 2019.

Wattal C, Kler N, Oberoi JK, Fursule A, Kumar A, Thakur A. Neonatal sepsis: mortality and morbidity in neonatal sepsis due to multidrug-resistant (MDR) organisms: part 1. Indian J Pediatr. 2020;87(2):117-121. https://doi.org/10.1007/s12098-019-03106-z PMID:31828600 DOI: https://doi.org/10.1007/s12098-019-03106-z

Sendra E, Fernández-Muñoz A, Zamorano L, et al. Impact of multidrug resistance on the virulence and fitness of Pseudomonas aeruginosa: a microbiological and clinical perspective. Infection. 2024 Aug;52(4):1235-1268. https://doi.org/10.1007/s15010-024-02313-x PMID: 38954392. DOI: https://doi.org/10.1007/s15010-024-02313-x

Diggle SP, Whiteley M. Microbe profile: Pseudomonas aeruginosa: opportunistic pathogen and lab rat. Microbiology (Reading). 2020;166(1):30-33. https://doi.org/10.1099/mic.0.000860 PMID:31597590 DOI: https://doi.org/10.1099/mic.0.000860

Parasuraman S, Thing GS, Dhanaraj SA. Polyherbal formulation: concept of Ayurveda. Pharmacogn Rev. 2014;8(16):73-80. https://doi.org/10.4103/0973-7847.134229 PMID:25125878 DOI: https://doi.org/10.4103/0973-7847.134229

Benamar-Aissa B, Gourine N, Ouinten M, Yousfi M. Synergistic effects of essential oils and phenolic extracts on antimicrobial activities using blends of Artemisia campestris, Artemisia herba alba, and Citrus aurantium. Biomol Concepts. 2024 Feb 14;15(1). doi: 10.1515/bmc-2022-0040. PMID: 38353049 DOI: https://doi.org/10.1515/bmc-2022-0040

Makhoba XH, Viegas C Jr, Mosa RA, Viegas FPD, Pooe OJ. Potential impact of the multi-target drug approach in the treatment of some complex diseases. Drug Des Devel Ther. 2020;14:3235-3249. https://doi.org/10.2147/DDDT.S257494 PMID:32884235 DOI: https://doi.org/10.2147/DDDT.S257494

Kumar M, Sarma DK, Shubham S, et al. Futuristic non-antibiotic therapies to combat antibiotic resistance: a review. Front Microbiol. 2021;12:609459. https://doi.org/10.3389/fmicb.2021.609459 PMID:33574807 DOI: https://doi.org/10.3389/fmicb.2021.609459

Ruparel FJ, Shah SK, Patel JH, Thakkar NR, Gajera GN, Kothari VO. Network analysis for identifying potential anti-virulence targets from whole transcriptome of Pseudomonas aeruginosa and Staphylococcus aureus exposed to certain anti-pathogenic polyherbal formulations. Drug Target Insights. 2023;17(1):58-69. https://doi.org/10.33393/dti.2023.2595 PMID:37275512 DOI: https://doi.org/10.33393/dti.2023.2595

Allen RC, Popat R, Diggle SP, Brown SP. Targeting virulence: can we make evolution-proof drugs? Nat Rev Microbiol. 2014;12(4):300-308. https://doi.org/10.1038/nrmicro3232 PMID:24625893 DOI: https://doi.org/10.1038/nrmicro3232

Corsi AK, Wightman B, Chalfie M. A transparent window into biology: a primer on Caenorhabditis elegans. Genetics. 2015;200(2):387-407. https://doi.org/10.1534/genetics.115.176099 PMID:26088431 DOI: https://doi.org/10.1534/genetics.115.176099

Patel H, Patel F, Jani V, et al. Anti-pathogenic potential of a classical Ayurvedic Triphala formulation. F1000Res. 2019 Jul 18;8:1126. doi: 10.12688/f1000research.19787.2. PMID: 33093941; DOI: https://doi.org/10.12688/f1000research.19787.1

Joshi C, Patel P, Palep H, Kothari V. Validation of the anti-infective potential of a polyherbal ‘Panchvalkal’ preparation, and elucidation of the molecular basis underlining its efficacy against Pseudomonas aeruginosa. BMC Complement Altern Med. 2019;19(1):19. https://doi.org/10.1186/s12906-019-2428-5 PMID:30654785 DOI: https://doi.org/10.1186/s12906-019-2428-5

Durai S, Vigneshwari L, Balamurugan K. Caenorhabditis elegans-based in vivo screening of bioactives from marine sponge-associated bacteria against Vibrio alginolyticus. J Appl Microbiol. 2013;115(6):1329-1342. https://doi.org/10.1111/jam.12335 PMID:24034129 DOI: https://doi.org/10.1111/jam.12335

Patel P, Joshi C, Palep H, Kothari V. Anti-infective potential of a quorum modulatory polyherbal extract (panchvalkal) against certain pathogenic bacteria. J Ayurveda Integr Med. 2020;11(3):336-343. https://doi.org/10.1016/j.jaim.2017.10.012 PMID:33012317 DOI: https://doi.org/10.1016/j.jaim.2017.10.012

Patel P, Joshi C, Funde S, Palep H, Kothari V. Prophylactic potential of a Panchgavya formulation against certain pathogenic bacteria. F1000Res. 2018 Oct 8;7:1612. doi: 10.12688/f1000research.16485.1. PMID: 30416718 DOI: https://doi.org/10.12688/f1000research.16485.1

Patel P, Joshi C, Kothari V. Anti-pathogenic efficacy and molecular targets of a polyherbal wound- care formulation (Herboheal) against Staphylococcus aureus. Infect Disord Drug Targets. 2019;19(2):193-206. doi: 10.2174/1871526518666181022112552. PMID: 30345928. DOI: https://doi.org/10.2174/1871526518666181022112552

Joshi C, Kothari V, Patel P. Importance of selecting appropriate wavelength, while quantifying growth and production of quorum sensing regulated pigments in bacteria. Recent Pat Biotechnol. 2016;10(2):145-152. https://doi.org/10.2174/1872208310666160414102848 PMID:27076088 DOI: https://doi.org/10.2174/1872208310666160414102848

Hirshfield IN, Barua S, Basu P. Overview of biofilms and some key methods for their study. In: Goldman E, Green LH, eds. Practical handbook of microbiology. 2nd ed. CRC Press; 2008:695-708:chap 42.

Trafny EA, Lewandowski R, Zawistowska-Marciniak I, Stępińska M. Use of MTT assay for determination of the biofilm formation capacity of microorganisms in metalworking fluids. World J Microbiol Biotechnol. 2013;29(9):1635-1643. https://doi.org/10.1007/s11274-013-1326-0 PMID:23515965 DOI: https://doi.org/10.1007/s11274-013-1326-0

Misko TP, Schilling RJ, Salvemini D, Moore WM, Currie MG. A fluorometric assay for the measurement of nitrite in biological samples. Anal Biochem. 1993;214(1):11-16. https://doi.org/10.1006/abio.1993.1449 PMID:7504409 DOI: https://doi.org/10.1006/abio.1993.1449

Yildirim K, Atas C, Tanyel Akcit E, et al. Nitrate reductase assay for rapid determination of methicillin-resistant Staphylococcus aureus clinical isolates. Lab Med. 2024;55(2):174-178. https://doi.org/%EF%BB%BF10.1093/labmed/lmad056 https://doi.org/10.1093/labmed/lmad056 PMID:37352501 DOI: https://doi.org/10.1093/labmed/lmad056

Barnes RJ, Bandi RR, Wong WS, et al. Optimal dosing regimen of nitric oxide donor compounds for the reduction of Pseudomonas aeruginosa biofilm and isolates from wastewater membranes. Biofouling. 2013;29(2):203-212. https://doi.org/10.1080/08927014.2012.760069 PMID:23368407 DOI: https://doi.org/10.1080/08927014.2012.760069

Simner PJ, Hindler JA, Bhowmick T, et al. What’s new in antibiograms? Updating CLSI M39 guidance with current trends. J Clin Microbiol. 2022;60(10):e0221021. https://doi.org/10.1128/jcm.02210-21 PMID:35916520 DOI: https://doi.org/10.1128/jcm.02210-21

Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193(1):265-275. https://doi.org/10.1016/S0021-9258(19)52451-6 PMID:14907713 DOI: https://doi.org/10.1016/S0021-9258(19)52451-6

Dulley JR, Grieve PA. A simple technique for eliminating interference by detergents in the Lowry method of protein determination. Anal Biochem. 1975;64(1):136-141. https://doi.org/10.1016/0003-2697(75)90415-7 PMID:1137083 DOI: https://doi.org/10.1016/0003-2697(75)90415-7

Mishra M, Tiwari S, Gomes AV. Protein purification and analysis: next generation Western blotting techniques. Expert Rev Proteomics. 2017;14(11):1037-1053. https://doi.org/10.1080/14789450.2017.1388167 PMID:28974114 DOI: https://doi.org/10.1080/14789450.2017.1388167

Suzuki J, Kunimoto T, Hori M. Effects of kanamycin on protein synthesis: inhibition of elongation of peptide chains. J Antibiot (Tokyo). 1970;23(2):99-101. https://doi.org/10.7164/antibiotics.23.99 PMID:4906633 DOI: https://doi.org/10.7164/antibiotics.23.99

Ullah H, Ali S. Classification of anti-bacterial agents and their functions. Antibacterial Agents. 2017;10:1-6. https://doi.org/10.5772/intechopen.68695 DOI: https://doi.org/10.5772/intechopen.68695

Jahn CE, Charkowski AO, Willis DK. Evaluation of isolation methods and RNA integrity for bacterial RNA quantitation. J Microbiol Methods. 2008;75(2):318-324. https://doi.org/10.1016/j.mimet.2008.07.004 PMID:18674572 DOI: https://doi.org/10.1016/j.mimet.2008.07.004

Andrews S. FastQC: a quality control tool for high throughput sequence data. 2010. http://www.bioinformatics.babraham.ac.uk/projects/fastqc

Chen S, Zhou Y, Chen Y, Gu J. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics. 2018 Sep 1;34(17):i884-i890. doi: 10.1093/bioinformatics/bty560. PMID: 30423086 DOI: https://doi.org/10.1093/bioinformatics/bty560

Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012;9(4):357-359. https://doi.org/10.1038/nmeth.1923 PMID:22388286 DOI: https://doi.org/10.1038/nmeth.1923

Liao Y, Smyth GK, Shi W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics. 2014;30(7):923-930. https://doi.org/10.1093/bioinformatics/btt656 PMID:24227677 DOI: https://doi.org/10.1093/bioinformatics/btt656

Robinson MD, McCarthy DJ, Smyth GK. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics. 2010;26(1):139-140. https://doi.org/10.1093/bioinformatics/btp616 PMID:19910308 DOI: https://doi.org/10.1093/bioinformatics/btp616

Conesa A, Götz S. Blast2GO: A comprehensive suite for functional analysis in plant genomics. Int J Plant Genomics. 2008;2008:619832. https://doi.org/10.1155/2008/619832 PMID:18483572 DOI: https://doi.org/10.1155/2008/619832

Ye J, Zhang Y, Cui H, et al. WEGO 2.0: a web tool for analyzing and plotting GO annotations, 2018 update. Nucleic Acids Res. 2018;46(W1):W71-W75. https://doi.org/10.1093/nar/gky400 PMID:29788377 DOI: https://doi.org/10.1093/nar/gky400

Szklarczyk D, Kirsch R, Koutrouli M, et al. The STRING database in 2023: protein-protein association networks and functional enrichment analyses for any sequenced genome of interest. Nucleic Acids Res. 2023;51(D1):D638-D646. https://doi.org/10.1093/nar/gkac1000 PMID:36370105 DOI: https://doi.org/10.1093/nar/gkac1000

Chin CH, Chen SH, Wu HH, Ho CW, Ko MT, Lin CY. cytoHubba: identifying hub objects and sub-networks from complex interactome. BMC Syst Biol. 2014;8(suppl 4):S11. https://doi.org/10.1186/1752-0509-8-S4-S11 PMID:25521941 DOI: https://doi.org/10.1186/1752-0509-8-S4-S11

Shannon P, Markiel A, Ozier O, et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 2003;13(11):2498-2504. http://www.genome.org/cgi/doi/10.1101/gr.1239303 https://doi.org/10.1101/gr.1239303 PMID:14597658 DOI: https://doi.org/10.1101/gr.1239303

Untergasser A, Cutcutache I, Koressaar T, et al. Primer3 – new capabilities and interfaces. Nucleic Acids Res. 2012;40(15):e115. https://doi.org/10.1093/nar/gks596 PMID:22730293 DOI: https://doi.org/10.1093/nar/gks596

Borrero-de Acuña JM, Timmis KN, Jahn M, Jahn D. Protein complex formation during denitrification by Pseudomonas aeruginosa. Microb Biotechnol. 2017;10(6):1523-1534. https://doi.org/10.1111/1751-7915.12851 PMID:28857512 DOI: https://doi.org/10.1111/1751-7915.12851

Chautrand T, Depayras S, Souak D, et al. Gaseous NO2 induces various envelope alterations in Pseudomonas fluorescens MFAF76a. Sci Rep. 2022;12(1):8528. https://doi.org/10.1038/s41598-022-11606-w PMID:35595726 DOI: https://doi.org/10.1038/s41598-022-11606-w

Mansour H, Ouweini AEL, Chahine EB, Karaoui LR. Imipenem/cilastatin/relebactam: A new carbapenem β-lactamase inhibitor combination. Am J Health Syst Pharm. 2021;78(8):674-683. https://doi.org/10.1093/ajhp/zxab012 PMID:33580649 DOI: https://doi.org/10.1093/ajhp/zxab012

Souza GHA, Rossato L, Brito GT, Bet GMDS, Simionatto S. Carbapenem-resistant Pseudomonas aeruginosa strains: a worrying health problem in intensive care units. Rev Inst Med Trop Sao Paulo. 2021 Sep 27;63:e71. doi: 10.1590/S1678-9946202163071. PMID: 34586305 DOI: https://doi.org/10.1590/s1678-9946202163071

Pragasam AK, Raghanivedha M, Anandan S, Veeraraghavan B. Characterization of Pseudomonas aeruginosa with discrepant carbapenem susceptibility profile. Ann Clin Microbiol Antimicrob. 2016;15(1):12. https://doi.org/10.1186/s12941-016-0127-3 PMID:26911874 DOI: https://doi.org/10.1186/s12941-016-0127-3

Takahashi E, Lee JM, Mon H, et al. Effect of antibiotics on extracellular protein level in Pseudomonas aeruginosa. Plasmid. 2016;84-85:44-50. https://doi.org/10.1016/j.plasmid.2016.03.001 PMID:26997534 DOI: https://doi.org/10.1016/j.plasmid.2016.03.001

Sun J, Deng Z, Yan A. Bacterial multidrug efflux pumps: mechanisms, physiology and pharmacological exploitations. Biochem Biophys Res Commun. 2014;453(2):254-267. https://doi.org/10.1016/j.bbrc.2014.05.090 PMID:24878531 DOI: https://doi.org/10.1016/j.bbrc.2014.05.090

Sultan M, Arya R, Kim KK. Roles of two-component systems in Pseudomonas aeruginosa virulence. Int J Mol Sci. 2021;22(22):12152. https://doi.org/10.3390/ijms222212152 PMID:34830033 DOI: https://doi.org/10.3390/ijms222212152

Murakami K, Ono T, Viducic D, et al. Role for rpoS gene of Pseudomonas aeruginosa in antibiotic tolerance. FEMS Microbiol Lett. 2005;242(1):161-167. https://doi.org/10.1016/j.femsle.2004.11.005 PMID:15621433 DOI: https://doi.org/10.1016/j.femsle.2004.11.005

Persat A, Inclan YF, Engel JN, Stone HA, Gitai Z. Type IV pili mechanochemically regulate virulence factors in Pseudomonas aeruginosa. Proc Natl Acad Sci USA. 2015;112(24):7563-7568. https://doi.org/10.1073/pnas.1502025112 PMID:26041805 DOI: https://doi.org/10.1073/pnas.1502025112

Myriam P, Braulio P, Javiera RA, et al. Insights into systems for iron-sulfur cluster biosynthesis in acidophilic microorganisms. J Microbiol Biotechnol. 2022;32(9):1110-1119. https://doi.org/10.4014/jmb.2206.06045 PMID:36039043 DOI: https://doi.org/10.4014/jmb.2206.06045

Mitchell AM, Silhavy TJ. Envelope stress responses: balancing damage repair and toxicity. Nat Rev Microbiol. 2019;17(7):417-428. https://doi.org/10.1038/s41579-019-0199-0 PMID:31150012 DOI: https://doi.org/10.1038/s41579-019-0199-0

Hajiagha MN, Kafil HS. Efflux pumps and microbial biofilm formation. Infect Genet Evol. 2023;112:105459. https://doi.org/10.1016/j.meegid.2023.105459 PMID:37271271 DOI: https://doi.org/10.1016/j.meegid.2023.105459

Deuerling E, Gamerdinger M, Kreft SG. Chaperone interactions at the ribosome. Cold Spring Harb Perspect Biol. 2019;11(11):a033977. https://doi.org/10.1101/cshperspect.a033977 PMID:30833456 DOI: https://doi.org/10.1101/cshperspect.a033977

Hamza EH, El-Shawadfy AM, Allam AA, Hassanein WA. Study on pyoverdine and biofilm production with detection of LasR gene in MDR Pseudomonas aeruginosa. Saudi J Biol Sci. 2023;30(1):103492. https://doi.org/10.1016/j.sjbs.2022.103492 PMID:36466220 DOI: https://doi.org/10.1016/j.sjbs.2022.103492

Coggan KA, Wolfgang MC. Global regulatory pathways and cross-talk control Pseudomonas aeruginosa environmental lifestyle and virulence phenotype. Curr Issues Mol Biol. 2012;14(2):47-70. PMID:22354680

Marsden AE, Intile PJ, Schulmeyer KH, et al. Vfr directly activates exsA transcription to regulate expression of the Pseudomonas aeruginosa type III secretion system. J Bacteriol. 2016;198(9):1442-1450. https://doi.org/10.1128/JB.00049-16 PMID:26929300 DOI: https://doi.org/10.1128/JB.00049-16

Schuster M, Greenberg EP. Regulatory networks in pathogenic bacteria: lessons from cell‐cell communication in Pseudomonas aeruginosa. Virulence mechanisms of bacterial pathogens. ASM Press; 2007:75-88. https://doi.org/10.1128/9781555815851.ch6 DOI: https://doi.org/10.1128/9781555815851.ch6

Häussler S, Becker T. The pseudomonas quinolone signal (PQS) balances life and death in Pseudomonas aeruginosa populations. PLoS Pathog. 2008;4(9):e1000166. https://doi.org/10.1371/journal.ppat.1000166 PMID:18818733 DOI: https://doi.org/10.1371/journal.ppat.1000166

Bradley R, Simon D, Spiga L, Xiang Y, Takats Z, Williams H. Laser desorption rapid evaporative ionization mass spectrometry (LD-REIMS) demonstrates a direct impact of hypochlorous acid stress on PQS-mediated quorum sensing in Pseudomonas aeruginosa. mSystems. 2024;9(4):e0116523. https://doi.org/10.1128/msystems.01165-23 PMID:38530056 DOI: https://doi.org/10.1128/msystems.01165-23

Xu Y, Zheng X, Zeng W, et al. Mechanisms of heteroresistance and resistance to imipenem in Pseudomonas aeruginosa. Infect Drug Resist. 2020;13:1419-1428. https://doi.org/10.2147/IDR.S249475 PMID:32523360 DOI: https://doi.org/10.2147/IDR.S249475

Fazeli-Nasab B, Sayyed RZ, Mojahed LS, et al. Biofilm production: a strategic mechanism for survival of microbes under stress conditions. Biocatal Agric Biotechnol. 2022;42:102337. https://doi.org/10.1016/j.bcab.2022.102337 DOI: https://doi.org/10.1016/j.bcab.2022.102337