Efficacy of Indolicidin, Cecropin A (1-7)-Melittin (CAMA) and Their Combination Against Biofilm-Forming Multidrug-Resistant Enteroaggregative Escherichia coli
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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.
KeywordsAntimicrobial peptide Biofilm Enteroaggregative E. coli Multidrug resistance
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.
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.
- 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
- 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.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.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.Wayne PA (2018) Performance standards for antimicrobial susceptibility testing, 28th edn. Supplement M100. Clinical and Laboratory Standards Institute, USAGoogle Scholar
- 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
- 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.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
- 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
- 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.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
- 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
- 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
- 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