Advertisement

Efficacy of Indolicidin, Cecropin A (1-7)-Melittin (CAMA) and Their Combination Against Biofilm-Forming Multidrug-Resistant Enteroaggregative Escherichia coli

  • Jess Vergis
  • S. V. S. Malik
  • Richa Pathak
  • Manesh Kumar
  • R. Sunitha
  • S. B. Barbuddhe
  • Deepak B. RawoolEmail author
Article
  • 82 Downloads

Abstract

The present study examined the anti-biofilm efficacy of two short-chain antimicrobial peptides (AMPs), namely, indolicidin and cecropin A (1-7)-melittin (CAMA) against biofilm-forming multidrug-resistant enteroaggregative Escherichia coli (MDR-EAEC) isolates. The typical EAEC isolates re-validated by PCR and confirmed using HEp-2 cell adherence assay was subjected to antibiotic susceptibility testing to confirm its MDR status. The biofilm-forming ability of MDR-EAEC isolates was assessed by Congo red binding, microtitre plate assays and hydrophobicity index; broth microdilution technique was employed to determine minimum inhibitory concentrations (MICs) and minimum biofilm eradication concentrations (MBECs). The obtained MIC and MBEC values for both AMPs were evaluated alone and in combination against MDR-EAEC biofilms using crystal violet (CV) staining and confocal microscopy-based live/dead cell quantification methods. All the three MDR-EAEC strains revealed weak to strong biofilm-forming ability and were found to be electron-donating and weakly electron-accepting (hydrophobicity index). Also, highly significant (P < 0.001) time-dependent hydrodynamic growth of the three MDR-EAEC strains was observed at 48 h of incubation in Dulbecco’s modified Eagle’s medium (DMEM) containing 0.45% D-glucose. AMPs and their combination were able to inhibit the initial biofilm formation at 24 h and 48 h as evidenced by CV staining and confocal quantification. Further, the application of AMPs (individually and combination) against the preformed MDR-EAEC biofilms resulted in highly significant eradication (P < 0.001) at 24 h post treatment. However, significant differences were not observed between AMP treatments (individually or in combination). The AMPs seem to be an effective candidates for further investigations such as safety, stability and appropriate biofilm-forming MDR-EAEC animal models.

Keywords

Antimicrobial peptide Biofilm Enteroaggregative E. coli Multidrug resistance 

Notes

Acknowledgements

The authors thank the Director of ICAR-Indian Veterinary Research Institute, Izatnagar, India, for providing facilities for the research. The authors are grateful to Dr. Chobi Debroy and Dr. Bhushan Jayarao, Pennsylvania State University, State College, PA, USA, for providing EAEC DNA. We thank Dr. Ravikumar G.V.P.P.S., Division of Animal Biotechnology, for his expertise for confocal microscopy. The technical assistance by Mr. K.K. Bhat and Dr. Deepa Ujjawal is acknowledged.

Funding information

The research work was supported by grants received from CAAST-ACLH (NAHEP/CAAST/2018-19) of ICAR-World Bank-funded National Agricultural Higher Education Project (NAHEP).

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.

Supplementary material

12602_2019_9589_MOESM1_ESM.ppt (1.9 mb)
ESM 1 (PPT 1951 kb)

References

  1. 1.
    Jensen BH, Olsen KE, Struve C, Krogfelt KA, Petersen AM (2014) Epidemiology and clinical manifestations of enteroaggregative Escherichia coli. Clin Microbiol Rev 27:614–630.  https://doi.org/10.1128/CMR.00112-13 CrossRefGoogle Scholar
  2. 2.
    Rogawski ET, Guerrant RL, Havt A, Lima IF, Medeiros PH, Seidman JC et al (2017) Epidemiology of enteroaggregative Escherichia coli infections and associated outcomes in the MAL-ED birth cohort. PLoS Neglected Trop Dis 11:e0005798.  https://doi.org/10.1371/journal.pntd.0005798 CrossRefGoogle Scholar
  3. 3.
    Ali MM, Ahmed SF, Klena JD, Mohamed ZK, Moussa TA, Ghenghesh KS (2014) Enteroaggregative Escherichia coli in diarrheic children in Egypt: molecular characterization and antimicrobial susceptibility. J Infect Develop Countr 8:589–596.  https://doi.org/10.3855/jidc.4077 CrossRefGoogle Scholar
  4. 4.
    Kong H, Hong X, Li X (2015) Current perspectives in pathogenesis and antimicrobial resistance of enteroaggregative Escherichia coli. Microb Pathog 85:44–49.  https://doi.org/10.1016/j.micpath.2015.06.002 CrossRefPubMedGoogle Scholar
  5. 5.
    Hall CW, Mah TF (2017) Molecular mechanisms of biofilm-based antibiotic resistance and tolerance in pathogenic bacteria. FEMS Microbiol Rev 41:276–301.  https://doi.org/10.1093/femsre/fux010 CrossRefPubMedGoogle Scholar
  6. 6.
    Lin S, Yang L, Chen G, Li B, Chen D, Li L, Xu Z (2017) Pathogenic features and characteristics of food borne pathogens biofilm: biomass, viability and matrix. Microb Pathog 111:285–291.  https://doi.org/10.1016/j.micpath.2017.08.005 CrossRefPubMedGoogle Scholar
  7. 7.
    Lebeaux D, Ghigo JM, Beloin C (2014) Biofilm-related infections: bridging the gap between clinical management and fundamental aspects of recalcitrance toward antibiotics. Microbiol Mol Biol Rev 78(3):510–543.  https://doi.org/10.1128/MMBR.00013-14 CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Batoni G, Maisetta G, Esin S (2016) Antimicrobial peptides and their interaction with biofilms of medically relevant bacteria. Biochim Biophys Acta Biomembr 1858:1044–1060.  https://doi.org/10.1016/j.bbamem.2015.10.013 CrossRefGoogle Scholar
  9. 9.
    Ribeiro SM, Felicio MR, Boas EV, Goncalves S, Costa FF, Samy RP et al (2016) New frontiers for anti-biofilm drug development. Pharmacol Therapeutics 160:133–144.  https://doi.org/10.1016/j.pharmthera.2016.02.006 CrossRefGoogle Scholar
  10. 10.
    Yazici A, Ortucu S, Taskin M, Marinelli L (2018) Natural-based antibiofilm and antimicrobial peptides from microorganisms. Curr Top Med Chem doi 18:2102–2107.  https://doi.org/10.2174/1568026618666181112143351 CrossRefGoogle Scholar
  11. 11.
    Kumar P, Kizhakkedathu J, Straus S (2018) Antimicrobial peptides: diversity, mechanism of action and strategies to improve the activity and biocompatibility in vivo. Biomolecules 8:4.  https://doi.org/10.3390/biom8010004 CrossRefGoogle Scholar
  12. 12.
    Zasloff M (2002) Antimicrobial peptides of multicellular organisms. Nature. 415:389–395.  https://doi.org/10.1038/415389a CrossRefGoogle Scholar
  13. 13.
    Anunthawan T, de la Fuente-Núñez C, Hancock RE, Klaynongsruang S (2015) Cationic amphipathic peptides KT2 and RT2 are taken up into bacterial cells and kill planktonic and biofilm bacteria. Biochim Biophys Acta Biomembr 1848:1352–1358.  https://doi.org/10.1016/j.bbamem.2015.02.021 CrossRefGoogle Scholar
  14. 14.
    De Zoysa GH, Cameron AJ, Hegde VV, Raghothama S, Sarojini V (2015) Antimicrobial peptides with potential for biofilm eradication: synthesis and structure activity relationship studies of battacin peptides. J Med Chem 58:625–639.  https://doi.org/10.1021/jm501084q CrossRefPubMedGoogle Scholar
  15. 15.
    Silva T, Claro B, Silva BF, Vale N, Gomes P, Gomes MS et al (2018) Unravelling a mechanism of action for a cecropin A-melittin hybrid antimicrobial peptide: the induced formation of multilamellar lipid stacks. Langmuir 34(5):2158–2170.  https://doi.org/10.1021/acs.langmuir.7b03639 CrossRefPubMedGoogle Scholar
  16. 16.
    Vijay D, Dhaka P, Vergis J, Negi M, Mohan V, Kumar M, Doijad S, Poharkar K, Kumar A, Malik SS, Barbuddhe SB, Rawool DB (2015) Characterization and biofilm forming ability of diarrhoeagenic enteroaggregative Escherichia coli isolates recovered from human infants and young animals. Comp Immunol Microbiol Infect Dis 38:21–31.  https://doi.org/10.1016/j.cimid.2014.11.004 CrossRefPubMedGoogle Scholar
  17. 17.
    Cravioto A, Gross RJ, Scotland SM, Rowe B (1979) An adhesive factor found in strains of Escherichia coli belonging to the traditional infantile enteropathogenic serotypes. Curr Microbiol 3:95–99 https://link.springer.com/article/10.1007/BF02602439 CrossRefGoogle Scholar
  18. 18.
    Wayne PA (2018) Performance standards for antimicrobial susceptibility testing, 28th edn. Supplement M100. Clinical and Laboratory Standards Institute, USAGoogle Scholar
  19. 19.
    Di Luca M, Maccari G, Maisetta G, Batoni G (2015) BaAMPs: the database of biofilm-active antimicrobial peptides. Biofouling 31:193–199.  https://doi.org/10.1080/08927014.2015.1021340 CrossRefPubMedGoogle Scholar
  20. 20.
    Freeman DJ, Falkiner FR, Keane CT (1989) New method for detecting slime production by coagulase negative staphylococci. J Clin Pathol 42:872–874.  https://doi.org/10.1136/jcp.42.8.872 CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Bellon-Fontaine MN, Rault J, Van Oss CJ (1996) Microbial adhesion to solvents: a novel method to determine the electron-donor/electron-acceptor or Lewis acid-base properties of microbial cells. Colloids Surf B: Biointerfaces 7:47–53.  https://doi.org/10.1016/0927-7765(96)01272-6 CrossRefGoogle Scholar
  22. 22.
    Wakimoto N, Nishi J, Sheikh J, Nataro JP, Sarantuya JA, Iwashita M et al (2004) Quantitative biofilm assay using a microtiter plate to screen for enteroaggregative Escherichia coli. Am J Trop Med Hyg 71:687–690.  https://doi.org/10.4269/ajtmh.2004.71.687 CrossRefPubMedGoogle Scholar
  23. 23.
    Wayne PA (1999) Methods for determining bactericidal activity of antimicrobial agents: approved guideline. M26-A. USA: National Committee for Clinical Laboratory StandardsGoogle Scholar
  24. 24.
    Jorge P, Grzywacz D, Kamysz W, Lourenço A, Pereira MO (2017) Searching for new strategies against biofilm infections: colistin-AMP combinations against Pseudomonas aeruginosa and Staphylococcus aureus single-and double-species biofilms. PLoS One 12:e0174654.  https://doi.org/10.1371/journal.pone.0174654 CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Ceri H, Olson ME, Stremick C, Read RR, Morck D, Buret A (1999) The Calgary Biofilm Device: new technology for rapid determination of antibiotic susceptibilities of bacterial biofilms. J Clin Microbiol 37:1771–1776PubMedPubMedCentralGoogle Scholar
  26. 26.
    Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez JY, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676–682.  https://doi.org/10.1038/nmeth.2019 CrossRefGoogle Scholar
  27. 27.
    Davies J, Davies D (2010) Origins and evolution of antibiotic resistance. Microbiol Mol Biol Rev 74(3):417–433.  https://doi.org/10.1128/MMBR.00016-10 CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Ballal M, Devadas SM, Chakraborty R, Shetty V (2014) Emerging trends in the etiology and antimicrobial susceptibility pattern of enteric pathogens in rural coastal India. Int J Clin Med 5:425–432.  https://doi.org/10.4236/ijcm.2014.57058 CrossRefGoogle Scholar
  29. 29.
    Dhaka P, Vijay D, Vergis J, Negi M, Kumar M, Mohan V, Doijad S, Poharkar KV, Malik SS, Barbuddhe SB, Rawool DB (2016) Genetic diversity and antibiogram profile of diarrhoeagenic Escherichia coli pathotypes isolated from human, animal, foods and associated environmental sources. Infect Ecol Epidemiol 6:31055.  https://doi.org/10.3402/iee.v6.31055 CrossRefPubMedGoogle Scholar
  30. 30.
    Cegelski L, Pinkner JS, Hammer ND, Cusumano CK, Hung CS, Chorell E, Åberg V, Walker JN, Seed PC, Almqvist F, Chapman MR, Hultgren SJ (2009) Small-molecule inhibitors target Escherichia coli amyloid biogenesis and biofilm formation. Nat Chem Biol 5(12):913–919.  https://doi.org/10.1038/nchembio.242 CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Reichhardt C, Jacobson AN, Maher MC, Uang J, McCrate OA, Eckart M et al (2015) Congo red interactions with curli-producing E. coli and native curli amyloid fibers. PLoS One 10(10):e0140388.  https://doi.org/10.1371/journal.pone.0140388 CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Doijad SP, Barbuddhe SB, Garg S, Poharkar KV, Kalorey DR, Kurkure NV, Rawool DB, Chakraborty T (2015) Biofilm-forming abilities of Listeria monocytogenes serotypes isolated from different sources. PLoS One 10:e0137046.  https://doi.org/10.1371/journal.pone.0137046 CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Perpetuini G, Tittarelli F, Schirone M, Di Gianvito P, Corsetti A, Arfelli G et al (2018) Adhesion properties and surface hydrophobicity of Pichia manshurica strains isolated from organic wines. LWT Food Sci Technol 87:385–392.  https://doi.org/10.1016/j.lwt.2017.09.011 CrossRefGoogle Scholar
  34. 34.
    Donlan RM, Costerton JW (2002) Biofilms: survival mechanisms of clinically relevant microorganisms. Clin Microbiol Rev 15:167–193.  https://doi.org/10.1128/CMR.15.2.167-193.2002 CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Sheikh J, Hicks S, Dall'Agnol M, Phillips AD, Nataro JP (2001) Roles for Fis and YafK in biofilm formation by enteroaggregative Escherichia coli. Mol Microbiol 41:983–997.  https://doi.org/10.1046/j.1365-2958.2001.02512.x CrossRefPubMedGoogle Scholar
  36. 36.
    Fjell CD, Hiss JA, Hancock RE, Schneider G (2012) Designing antimicrobial peptides: form follows function. Nat Rev Drug Discov 11:37–51.  https://doi.org/10.1038/nrd3591 CrossRefGoogle Scholar
  37. 37.
    de la Fuente-Núñez C, Reffuveille F, Mansour SC, Reckseidler-Zenteno SL, Hernández D, Brackman G, Coenye T, Hancock REW (2015) D-enantiomeric peptides that eradicate wild-type and multidrug-resistant biofilms and protect against lethal Pseudomonas aeruginosa infections. Chem Biol 22:196–205.  https://doi.org/10.1016/j.chembiol.2015.01.002 CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Falla TJ, Karunaratne DN, Hancock RE (1996) Mode of action of the antimicrobial peptide indolicidin. J Biol Chem 271:19298–19303.  https://doi.org/10.1074/jbc.271.32.19298 CrossRefPubMedGoogle Scholar
  39. 39.
    Segev-Zarko LA, Saar-Dover R, Brumfeld V, Mangoni ML, Shai Y (2015) Mechanisms of biofilm inhibition and degradation by antimicrobial peptides. Biochem J BJ20141251 468:259–270.  https://doi.org/10.1042/BJ20141251 CrossRefPubMedGoogle Scholar
  40. 40.
    Mataraci E, Dosler S (2012) In vitro activities of antibiotics and antimicrobial cationic peptides alone and in combination against methicillin resistance Staphylococcus aureus biofilms. Antimicrob Agents Chemother 56:6366–6371.  https://doi.org/10.1128/AAC.01180-12 CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Grassi L, Maisetta G, Esin S, Batoni G (2017) Combination strategies to enhance the efficacy of antimicrobial peptides against bacterial biofilms. Front Microbiol 8:2409.  https://doi.org/10.3389/fmicb.2017.02409 CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Mishra B, Wang G (2017) Individual and combined effects of engineered peptides and antibiotics on Pseudomonas aeruginosa biofilms. Pharmaceuticals 10:58.  https://doi.org/10.3390/ph10030058 CrossRefPubMedCentralGoogle Scholar
  43. 43.
    Riool M, de Breij A, Drijfhout JW, Nibbering PH, Zaat SA (2017) Antimicrobial peptides in biomedical device manufacturing. Front Chem 5:63.  https://doi.org/10.3389/fchem.2017.00063 CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Mas-Moruno C, Su B, Dalby MJ (2019) Multifunctional coatings and nanotopographies: toward cell instructive and antibacterial implants. Adv Healthcare Mat 8(1):1801103.  https://doi.org/10.1002/adhm.201801103 CrossRefGoogle Scholar
  45. 45.
    Liu J, Ling JQ, Zhang K, Wu CD (2013) Physiological properties of Streptococcus mutans UA159 biofilm-detached cells. FEMS Microbiol Lett 340:11–18.  https://doi.org/10.1111/1574-6968.12066 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Jess Vergis
    • 1
  • S. V. S. Malik
    • 1
  • Richa Pathak
    • 1
  • Manesh Kumar
    • 1
  • R. Sunitha
    • 1
  • S. B. Barbuddhe
    • 2
  • Deepak B. Rawool
    • 1
    Email author
  1. 1.Division of Veterinary Public HealthICAR-Indian Veterinary Research InstituteBareillyIndia
  2. 2.ICAR-National Research Centre on MeatChengicherlaIndia

Personalised recommendations