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.
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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
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
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
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
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
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
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
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
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
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
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
Zasloff M (2002) Antimicrobial peptides of multicellular organisms. Nature. 415:389–395. https://doi.org/10.1038/415389a
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
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
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
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
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
Wayne PA (2018) Performance standards for antimicrobial susceptibility testing, 28th edn. Supplement M100. Clinical and Laboratory Standards Institute, USA
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
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
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
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
Wayne PA (1999) Methods for determining bactericidal activity of antimicrobial agents: approved guideline. M26-A. USA: National Committee for Clinical Laboratory Standards
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
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 37:1771–1776
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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).
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Vergis, J., Malik, S.V.S., Pathak, R. et al. Efficacy of Indolicidin, Cecropin A (1-7)-Melittin (CAMA) and Their Combination Against Biofilm-Forming Multidrug-Resistant Enteroaggregative Escherichia coli. Probiotics & Antimicro. Prot. 12, 705–715 (2020). https://doi.org/10.1007/s12602-019-09589-8
- Antimicrobial peptide
- Enteroaggregative E. coli
- Multidrug resistance