Antimicrobial and Antibiofilm Activity of Lys-[Trp6]hy-a1 Combined with Ciprofloxacin Against Gram-Negative Bacteria

Author(s): Victor Alves Carneiro*, Simone Torres de Oliveira, Rondinely Lima Silva, Humberlania de Sousa Duarte, Maria Laína Silva, Maria Nágila Carneiro Matos, Rafaela Mesquita Bastos Cavalcante, Ciro Siqueira Figueira, Esteban Nicolás Lorenzón, Eduardo Maffud Cilli, Rodrigo Maranguape Silva da Cunha

Journal Name: Protein & Peptide Letters

Volume 27 , Issue 11 , 2020


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Abstract:

Background: Ciprofloxacin (Cip) is the most commonly used quinolone in clinical practice; however large-scale use has favored the increase of multiresistant pathogenic microorganisms. Antimicrobial peptides (AMPs) appear to be a promising alternative in potentiating these conventional drugs.

Objective: The aim of this study was to evaluate the effect of the peptide Lys-[Trp6]hy-a1 (lys-a1) on the antimicrobial and antibiofilm activity of ciprofloxacin against clinically relevant gram-negative bacteria.

Methods: The antimicrobial effects of Cip and lys-a1 were assessed by determining the minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs). The synergistic action of Cip and lys-a1 was determined by checkerboard assay. The time-kill curve was constructed for the Cip/lys-a1 combination against Pseudomonas aeruginosa ATCC 9027. The antibiofilm activity of this combination was analyzed by crystal violet, colony-forming unit count and atomic force microscopy (AFM).

Results: The data demonstrated that lys-a1 was able to inhibit planktonic growth of strains of P. aeruginosa and Klebsiella pneumoniae both at 125 μg/mL. The fractional inhibitory concentration index (FICi) showed a synergistic effect between Cip and lys-a1 against P. aeruginosa, decreasing the MICs of the individual antimicrobial agents by 4- and 8-fold, respectively. This effect was also observed for the death kinetics and antibiofilm activity. Analysis of the early biofilms (6 h) as well as isolated cells by AFM images evidenced the cell perturbation caused by Cip/lys-a1 treatment.

Conclusion: These data suggest that lys-a1 has biotechnological potential as a therapeutic tool for the treatment of infections caused by clinically relevant microorganisms, especially P. aeruginosa.

Keywords: Lys-a1, antimicrobial peptide, Pseudomonas aeruginosa, ciprofloxacin, synergism, antibiofilm activity.

[1]
Limoli, D.H.; Jones, C.J.; Wozniak, D.J. Extracellular polysaccharides in biofilm formation and function. Microbiol. Spectr., 2015, 3(3), 1-30.
[http://dx.doi.org/10.1128/microbiolspec.MB-0011-2014] [PMID: 26185074]
[2]
Flemming, H-C.; Wingender, J.; Szewzyk, U.; Steinberg, P.; Rice, S.A.; Kjelleberg, S. Biofilms: an emergent form of bacterial life. Nat. Rev. Microbiol., 2016, 14(9), 563-575.
[http://dx.doi.org/10.1038/nrmicro.2016.94] [PMID: 27510863]
[3]
Santos, A.L.S.D.; Galdino, A.C.M.; Mello, T.P.; Ramos, L.S.; Branquinha, M.H.; Bolognese, A.M.; Columbano Neto, J.; Roudbary, M. What are the advantages of living in a community? A microbial biofilm perspective! Mem. Inst. Oswaldo Cruz, 2018, 113(9), e180212.
[http://dx.doi.org/10.1590/0074-02760180212] [PMID: 30066753]
[4]
Beloin, C.; Fernández-Hidalgo, N.; Lebeaux, D. Understanding biofilm formation in intravascular device-related infections. Intensive Care Med., 2017, 43(3), 443-446.
[http://dx.doi.org/10.1007/s00134-016-4480-7] [PMID: 27497588]
[5]
Arciola, C.R.; Campoccia, D.; Montanaro, L. Implant infections: adhesion, biofilm formation and immune evasion. Nat. Rev. Microbiol., 2018, 16(7), 397-409.
[http://dx.doi.org/10.1038/s41579-018-0019-y] [PMID: 29720707]
[6]
Rosenthal, V.D.; Al-Abdely, H.M.; El-Kholy, A.A.; AlKhawaja, S.A.A.; Leblebicioglu, H.; Mehta, Y.; Rai, V.; Hung, N.V.; Kanj, S.S.; Salama, M.F.; Salgado-Yepez, E.; Elahi, N.; Morfin Otero, R.; Apisarnthanarak, A.; De Carvalho, B.M.; Ider, B.E.; Fisher, D.; Buenaflor, M.C.S.G.; Petrov, M.M.; Quesada-Mora, A.M.; Zand, F.; Gurskis, V.; Anguseva, T.; Ikram, A.; Aguilar de Moros, D.; Duszynska, W.; Mejia, N.; Horhat, F.G.; Belskiy, V.; Mioljevic, V.; Di Silvestre, G.; Furova, K.; Ramos-Ortiz, G.Y.; Gamar Elanbya, M.O.; Satari, H.I.; Gupta, U.; Dendane, T.; Raka, L.; Guanche-Garcell, H.; Hu, B.; Padgett, D.; Jayatilleke, K.; Ben Jaballah, N.; Apostolopoulou, E.; Prudencio Leon, W.E.; Sepulveda-Chavez, A.; Telechea, H.M.; Trotter, A.; Alvarez-Moreno, C.; Kushner-Davalos, L. Remaining authors. International Nosocomial Infection Control Consortium report, data summary of 50 countries for 2010-2015: Device-associated module. Am. J. Infect. Control, 2016, 44(12), 1495-1504.
[http://dx.doi.org/10.1016/j.ajic.2016.08.007] [PMID: 27742143]
[7]
Khan, H.A.; Baig, F.K.; Mehboob, R. Nosocomial infections: Epidemiology, prevention, control and surveillance. Asian Pac. J. Trop. Biomed., 2017, 7, 478-482.
[http://dx.doi.org/10.1016/j.apjtb.2017.01.019]
[8]
El Zowalaty, M.E.; Al Thani, A.A.; Webster, T.J.; El Zowalaty, A.E.; Schweizer, H.P.; Nasrallah, G.K.; Marei, H.E.; Ashour, H.M. Pseudomonas aeruginosa: arsenal of resistance mechanisms, decades of changing resistance profiles, and future antimicrobial therapies. Future Microbiol., 2015, 10(10), 1683-1706.
[http://dx.doi.org/10.2217/fmb.15.48] [PMID: 26439366]
[9]
Ahmed, M.N.; Porse, A.; Sommer, M.O.A.; Høiby, N.; Ciofu, O. Evolution of antibiotic resistance in biofilm and planktonic Pseudomonas aeruginosa populations exposed to subinhibitory levels of ciprofloxacin. Antimicrob. Agents Chemother., 2018, 62(8), 1-12.
[http://dx.doi.org/10.1128/AAC.00320-18] [PMID: 29760140]
[10]
Steckbeck, J.D.; Deslouches, B.; Montelaro, R.C. Antimicrobial peptides: new drugs for bad bugs? Expert Opin. Biol. Ther., 2014, 14(1), 11-14.
[http://dx.doi.org/10.1517/14712598.2013.844227] [PMID: 24206062]
[11]
Lorenzón, E.N.; Santos-Filho, N.A.; Ramos, M.A.; Bauab, T.M.; Camargo, I.L.; Cilli, E.M. C-terminal lysine-linked magainin 2 with increased activity against multidrug-resistant bacteria. Protein Pept. Lett., 2016, 23(8), 738-747.
[http://dx.doi.org/10.2174/0929866523666160511150907] [PMID: 27165408]
[12]
Wu, X.; Li, Z.; Li, X.; Tian, Y.; Fan, Y.; Yu, C.; Zhou, B.; Liu, Y.; Xiang, R.; Yang, L. Synergistic effects of antimicrobial peptide DP7 combined with antibiotics against multidrug-resistant bacteria. Drug Des. Devel. Ther., 2017, 11, 939-946.
[http://dx.doi.org/10.2147/DDDT.S107195] [PMID: 28356719]
[13]
Jaśkiewicz, M.; Neubauer, D.; Kazor, K.; Bartoszewska, S.; Kamysz, W. Antimicrobial activity of selected antimicrobial peptides against planktonic culture and biofilm of Acinetobacter baumannii. Probiotics Antimicrob. Proteins, 2019, 11(1), 317-324.
[PMID: 30043322]
[14]
Crusca, E., Jr; Rezende, A.A.; Marchetto, R.; Mendes-Giannini, M.J.S.; Fontes, W.; Castro, M.S.; Cilli, E.M. Influence of N-terminus modifications on the biological activity, membrane interaction, and secondary structure of the antimicrobial peptide hylin-a1. Biopolymers, 2011, 96(1), 41-48.
[http://dx.doi.org/10.1002/bip.21454] [PMID: 20560142]
[15]
da Silva, B.R.; de Freitas, V.A.; Carneiro, V.A.; Arruda, F.V.S.; Lorenzón, E.N.; de Aguiar, A.S.; Cilli, E.M.; Cavada, B.S.; Teixeira, E.H. Antimicrobial activity of the synthetic peptide Lys-a1 against oral streptococci. Peptides, 2013, 42, 78-83.
[http://dx.doi.org/10.1016/j.peptides.2012.12.001] [PMID: 23340019]
[16]
Clinical and Laboratory Standards Institute (CLSI). Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard, 9 eds; Wayne, PA: CLSI, 2012.
[17]
White, R.L.; Burgess, D.S.; Manduru, M.; Bosso, J.A. Comparison of three different in vitro methods of detecting synergy: time-kill, checkerboard, and E test. Antimicrob. Agents Chemother., 1996, 40(8), 1914-1918.
[http://dx.doi.org/10.1128/AAC.40.8.1914] [PMID: 8843303]
[18]
Lewis, R.E.; Diekema, D.J.; Messer, S.A.; Pfaller, M.A.; Klepser, M.E. Comparison of Etest, chequerboard dilution and time-kill studies for the detection of synergy or antagonism between antifungal agents tested against Candida species. J. Antimicrob. Chemother., 2002, 49(2), 345-351.
[http://dx.doi.org/10.1093/jac/49.2.345] [PMID: 11815578]
[19]
Hamoud, R.; Zimmermann, S.; Reichling, J.; Wink, M. Synergistic interactions in two-drug and three-drug combinations (thymol, EDTA and vancomycin) against multi drug resistant bacteria including E. coli. Phytomedicine, 2014, 21(4), 443-447.
[http://dx.doi.org/10.1016/j.phymed.2013.10.016] [PMID: 24262063]
[20]
Chang, S.C.; Chen, Y.C.; Luh, K.T.; Hsieh, W.C. In vitro activities of antimicrobial agents, alone and in combination, against Acinetobacter baumannii isolated from blood. Diagn. Microbiol. Infect. Dis., 1995, 23(3), 105-110.
[http://dx.doi.org/10.1016/0732-8893(95)00170-0] [PMID: 8849654]
[21]
O’Toole, G.A.; Kolter, R. Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signalling pathways: a genetic analysis. Mol. Microbiol., 1998, 28(3), 449-461.
[http://dx.doi.org/10.1046/j.1365-2958.1998.00797.x] [PMID: 9632250]
[22]
Kataoka, H.; Ida, T.; Ishii, Y.; Tateda, K.; Oguri, T.; Yoshida, A.; Okuzumi, K.; Oishi, T.; Tsukahara, M.; Mori, S.I.; Yoneyama, A.; Araoka, H.; Mitsuda, T.; Sumitomo, M.; Moriya, K.; Goto, M.; Nakamori, Y.; Shibayama, A.; Ohmagari, N.; Sato, T.; Yamaguchi, K. ABX Combination Therapy Study Group (ACTs) Analysis of the influence of drug resistance factors on the efficacy of combinations of antibiotics for multidrug-resistant Pseudomonas aeruginosa isolated from hospitals located in the suburbs of Kanto area, Japan. J. Glob. Antimicrob. Resist., 2013, 1(2), 91-96.
[http://dx.doi.org/10.1016/j.jgar.2013.03.005] [PMID: 27873584]
[23]
Nakamura, I.; Yamaguchi, T.; Tsukimori, A.; Sato, A.; Fukushima, S.; Mizuno, Y.; Matsumoto, T. Effectiveness of antibiotic combination therapy as evaluated by the Break-point Checkerboard Plate method for multidrug-resistant Pseudomonas aeruginosa in clinical use. J. Infect. Chemother., 2014, 20(4), 266-269.
[http://dx.doi.org/10.1016/j.jiac.2013.12.005] [PMID: 24486172]
[24]
Vestergaard, M.; Paulander, W.; Marvig, R.L.; Clasen, J.; Jochumsen, N.; Molin, S.; Jelsbak, L.; Ingmer, H.; Folkesson, A. Antibiotic combination therapy can select for broad-spectrum multidrug resistance in Pseudomonas aeruginosa. Int. J. Antimicrob. Agents, 2016, 47(1), 48-55.
[http://dx.doi.org/10.1016/j.ijantimicag.2015.09.014] [PMID: 26597931]
[25]
Barbosa, C.; Beardmore, R.; Schulenburg, H.; Jansen, G. Antibiotic combination efficacy (ACE) networks for a Pseudomonas aeruginosa model. PLoS Biol., 2018, 16(4), e2004356.
[http://dx.doi.org/10.1371/journal.pbio.2004356] [PMID: 29708964]
[26]
Raheem, N.; Straus, S.K. Mechanisms of action for antimicrobial peptides with antibacterial and antibiofilm functions. Front. Microbiol., 2019, 10, 2866.
[http://dx.doi.org/10.3389/fmicb.2019.02866] [PMID: 31921046]
[27]
de la Fuente-Núñez, C.; Reffuveille, F.; Mansour, S.C.; Reckseidler-Zenteno, S.L.; Hernández, D.; Brackman, G.; Coenye, T.; Hancock, R.E. D-enantiomeric peptides that eradicate wild-type and multidrug-resistant biofilms and protect against lethal Pseudomonas aeruginosa infections. Chem. Biol., 2015, 22(2), 196-205.
[http://dx.doi.org/10.1016/j.chembiol.2015.01.002] [PMID: 25699603]
[28]
Rudilla, H.; Fusté, E.; Cajal, Y.; Rabanal, F.; Vinuesa, T.; Viñas, M. Synergistic antipseudomonal effects of synthetic peptide AMP38 and carbapenems. Molecules, 2016, 21(9), e1223.
[http://dx.doi.org/10.3390/molecules21091223] [PMID: 27626405]
[29]
Choi, H.; Lee, D.G. Synergistic effect of antimicrobial peptide arenicin-1 in combination with antibiotics against pathogenic bacteria. Res. Microbiol., 2012, 163(6-7), 479-486.
[http://dx.doi.org/10.1016/j.resmic.2012.06.001] [PMID: 22705395]
[30]
Jorge, P.; Grzywacz, D.; Kamysz, W.; Lourenço, A.; Pereira, M.O. Searching for new strategies against biofilm infections: Colistin-AMP combinations against Pseudomonas aeruginosa and Staphylococcus aureus single- and double-species biofilms. PLoS One, 2017, 12(3), e0174654.
[http://dx.doi.org/10.1371/journal.pone.0174654] [PMID: 28355248]
[31]
Dosler, S.; Karaaslan, E. Inhibition and destruction of Pseudomonas aeruginosa biofilms by antibiotics and antimicrobial peptides. Peptides, 2014, 62, 32-37.
[http://dx.doi.org/10.1016/j.peptides.2014.09.021] [PMID: 25285879]
[32]
Chen, H.; Wubbolts, R.W.; Haagsman, H.P.; Veldhuizen, E.J.A. Inhibition and eradication of Pseudomonas aeruginosa biofilms by host defence peptides. Sci. Rep., 2018, 8(1), 10446.
[http://dx.doi.org/10.1038/s41598-018-28842-8] [PMID: 29993029]
[33]
Haney, E.F.; Trimble, M.J.; Cheng, J.T.; Vallé, Q.; Hancock, R.E.W. Critical assessment of methods to quantify biofilm growth and evaluate antibiofilm activity of host defence peptides. Biomolecules, 2018, 8(2), 1-22.
[http://dx.doi.org/10.3390/biom8020029] [PMID: 29883434]
[34]
Singh, S.; Singh, S.K.; Chowdhury, I.; Singh, R. Understanding the mechanism of bacterial biofilms resistance to antimicrobial agents. Open Microbiol. J., 2017, 11, 53-62.
[http://dx.doi.org/10.2174/1874285801711010053] [PMID: 28553416]
[35]
Pletzer, D.; Hancock, R.E. Antibiofilm peptides: Potential as broad-spectrum agents. J. Bacteriol., 2016, 198(19), 2572-2578.
[http://dx.doi.org/10.1128/JB.00017-16] [PMID: 27068589]
[36]
Chung, P.Y.; Khanum, R. Antimicrobial peptides as potential anti-biofilm agents against multidrug-resistant bacteria. J. Microbiol. Immunol. Infect., 2017, 50(4), 405-410.
[http://dx.doi.org/10.1016/j.jmii.2016.12.005] [PMID: 28690026]
[37]
Malanovic, N.; Lohner, K. Antimicrobial peptides targeting gram-positive bacteria. Pharmaceuticals (Basel), 2016, 9(3), 59.
[http://dx.doi.org/10.3390/ph9030059] [PMID: 27657092]


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Article Details

VOLUME: 27
ISSUE: 11
Year: 2020
Page: [1124 - 1131]
Pages: 8
DOI: 10.2174/0929866527666200416145549
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