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In vitro and in vivo activity of EDTA and antibacterial agents against the biofilm of mucoid Pseudomonas aeruginosa

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Abstract

Purpose

Refractory infection caused by bacterial biofilm is an important clinical problem. Pseudomonas aeruginosa is a common pathogen responsible for persistent and chronic biofilm infections. We aimed to explore the in vitro and in vivo activity of ethylenediamine tetraacetic acid (EDTA) in combination with antibacterial agents against mucoid P. aeruginosa biofilm.

Methods

The minimal inhibitory concentration (MIC) and minimal bactericidal concentration of ciprofloxacin, gentamicin, and ampicillin alone or with EDTA against P. aeruginosa were determined in vitro. Extracellular polysaccharides (EPS) and structural parameters of the biofilm were monitored. P. aeruginosa was aerosolized and delivered into the lungs of guinea pigs, which were treated with ciprofloxacin with or without EDTA. The colony-forming units (CFUs) of P. aeruginosa were determined from the lungs.

Results

EDTA reduced the MIC of ciprofloxacin and ampicillin by about 30-fold and that of gentamicin by twofold. EDTA reduced the biofilm EPS and the proportion of viable bacteria. The thickness, average diffusion distance, and textural entropy of EDTA-treated biofilm were significantly decreased. EDTA plus antibiotics reduced the colony counting from 107 to 103 CFU/mL. In vivo, EDTA plus ciprofloxacin had a significantly lower mean CFU/g of lung tissue (EDTA + ciprofloxacin 1.3 ± 0.19; EDTA 4.4 ± 0.57; ciprofloxacin 4.2 ± 0.47), and lung lesions were less severe compared with the single treatment groups.

Conclusions

EDTA can destroy the biofilm structures of mucoid P. aeruginosa in vitro. Moreover, EDTA and ciprofloxacin had a significant bactericidal effect against biofilm in vivo.

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References

  1. Bauer TT, Torres A, Ferrer R, Heyer CM, Schultze-Werninghaus G, Rasche K. Biofilm formation in endotracheal tubes. association between pneumonia and the persistence of pathogens. Monaldi Arch Chest Dis. 2002;57:84–7.

    CAS  PubMed  Google Scholar 

  2. Ohgaki N. Bacterial biofilm in chronic airway infection. Kansenshogaku Zasshi. 1994;68:138–51.

    Article  CAS  PubMed  Google Scholar 

  3. Kobayashi H. Biofilm disease: its clinical manifestation and therapeutic possibilities of macrolides. Am J Med. 1995;99:26S–30S.

    Article  CAS  PubMed  Google Scholar 

  4. Singh PK, Schaefer AL, Parsek MR, Moninger TO, Welsh MJ, Greenberg EP. Quorum-sensing signals indicate that cystic fibrosis lungs are infected with bacterial biofilms. Nature. 2000;407:762–4. doi:10.1038/35037627.

    Article  CAS  PubMed  Google Scholar 

  5. Stiver HG, Zachidniak K, Speert DP. Inhibition of polymorphonuclear leukocyte chemotaxis by the mucoid exopolysaccharide of Pseudomonas aeruginosa. Clin Invest Med. 1988;11:247–52.

    CAS  PubMed  Google Scholar 

  6. Prince AS. Biofilms, antimicrobial resistance, and airway infection. N Engl J Med. 2002;347:1110–1. doi:10.1056/NEJMcibr021776.

    Article  PubMed  Google Scholar 

  7. Moskowitz SM, Foster JM, Emerson J, Burns JL. Clinically feasible biofilm susceptibility assay for isolates of Pseudomonas aeruginosa from patients with cystic fibrosis. J Clin Microbiol. 2004;42:1915–22.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. O’Loughlin CT, Miller LC, Siryaporn A, Drescher K, Semmelhack MF, Bassler BL. A quorum-sensing inhibitor blocks Pseudomonas aeruginosa virulence and biofilm formation. Proc Natl Acad Sci USA. 2013;110:17981–6. doi:10.1073/pnas.1316981110.

    Article  PubMed  PubMed Central  Google Scholar 

  9. Banin E, Brady KM, Greenberg EP. Chelator-induced dispersal and killing of Pseudomonas aeruginosa cells in a biofilm. Appl Environ Microbiol. 2006;72:2064–9. doi:10.1128/AEM.72.3.2064-2069.2006.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Vaara M. Agents that increase the permeability of the outer membrane. Microbiol Rev. 1992;56:395–411.

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Hancock RE. Alterations in outer membrane permeability. Annu Rev Microbiol. 1984;38:237–64. doi:10.1146/annurev.mi.38.100184.001321.

    Article  CAS  PubMed  Google Scholar 

  12. Delcour AH. Outer membrane permeability and antibiotic resistance. Biochim Biophys Acta. 2009;1794:808–16. doi:10.1016/j.bbapap.2008.11.005.

    Article  CAS  PubMed  Google Scholar 

  13. Chaudhary M, Patnaik SK, Payasi A. Evaluation of different drugs in down-regulation of efflux pump genes expression in methicillin-resistant staphylococcus aureus strains. Am J Infect Dis. 2014;10:184–9.

    Article  Google Scholar 

  14. Chaudhary M, Payasi A. Ethylenediaminetetraacetic acid: a non antibiotic adjuvant enhancing Pseudomonas aeruginosa susceptibility. Afr J Microbiol Res. 2012;6:6799–804.

    Article  CAS  Google Scholar 

  15. Finnegan S, Percival SL. EDTA: an antimicrobial and antibiofilm agent for use in wound care. Adv Wound Care (New Rochelle). 2015;4:415–21. doi:10.1089/wound.2014.0577.

    Article  Google Scholar 

  16. Gotthelf LN. Diagnosis and treatment of otitis media in dogs and cats. Vet Clin N Am Small Anim Pract. 2004;34:469–87. doi:10.1016/j.cvsm.2003.10.007.

    Article  Google Scholar 

  17. Kotsakis GA, Lan C, Barbosa J, Lill K, Chen R, Rudney J, Aparicio C. Antimicrobial agents used in the treatment of peri-implantitis alter the physicochemistry and cytocompatibility of titanium surfaces. J Periodontol. 2016;28:1–20. doi:10.1902/jop.2016.150684.

    Google Scholar 

  18. Lebeaux D, Leflon-Guibout V, Ghigo JM, Beloin C. In vitro activity of gentamicin, vancomycin or amikacin combined with EDTA or l-arginine as lock therapy against a wide spectrum of biofilm-forming clinical strains isolated from catheter-related infections. J Antimicrob Chemother. 2015;70:1704–12. doi:10.1093/jac/dkv044.

    CAS  PubMed  Google Scholar 

  19. Micek ST, Lloyd AE, Ritchie DJ, Reichley RM, Fraser VJ, Kollef MH. Pseudomonas aeruginosa bloodstream infection: importance of appropriate initial antimicrobial treatment. Antimicrob Agents Chemother. 2005;49:1306–11. doi:10.1128/AAC.49.4.1306-1311.2005.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Valgas C, Machado de Souza S, Smania EFA, Smania A, Jr. Screening methods to determine antibacterial activity of natural products. Braz J Microbiol. 2007;38:369–80.

    Article  Google Scholar 

  21. Sanchez-Gomez S, Lamata M, Leiva J, Blondelle SE, Jerala R, Andra J, Brandenburg K, Lohner K, Moriyon I, Martinez-de-Tejada G. Comparative analysis of selected methods for the assessment of antimicrobial and membrane-permeabilizing activity: a case study for lactoferricin derived peptides. BMC Microbiol. 2008;8:196. doi:10.1186/1471-2180-8-196.

    Article  PubMed  PubMed Central  Google Scholar 

  22. Rabe T, Mullholland D, van Staden J. Isolation and identification of antibacterial compounds from Vernonia colorata leaves. J Ethnopharmacol. 2002;80:91–4.

    Article  CAS  PubMed  Google Scholar 

  23. Berche P, Gaillard JL, Richard S. Invasiveness and intracellular growth of Listeria monocytogenes. Infection. 1988;16:S145–8.

    Article  PubMed  Google Scholar 

  24. Strathmann M, Wingender J, Flemming HC. Application of fluorescently labelled lectins for the visualization and biochemical characterization of polysaccharides in biofilms of Pseudomonas aeruginosa. J Microbiol Methods. 2002;50:237–48.

    Article  CAS  PubMed  Google Scholar 

  25. Beyenal H, Lewandowski Z, Harkin G. Quantifying biofilm structure: facts and fiction. Biofouling. 2004;20:1–23. doi:10.1080/0892701042000191628.

    Article  CAS  PubMed  Google Scholar 

  26. Guo SY, Sheng-Qi LI, Liu Y, Sun JM. An intrapulmonary biofilm model of Pseudomonas aeruginosa. Chin J Microbiol Immunol. 2003;23:292–5.

    Google Scholar 

  27. Otani T, Katami K, Une T, Osada Y, Ogawa H. Nonbacteremic Pseudomonas pneumonia in immunosuppressed guinea pigs. Microbiol Immunol. 1982;26:67–76.

    Article  CAS  PubMed  Google Scholar 

  28. Buckley LM, McEwan NA, Nuttall T. Tris-EDTA significantly enhances antibiotic efficacy against multidrug-resistant Pseudomonas aeruginosa in vitro. Vet Dermatol. 2013;24:519-e122. doi:10.1111/vde.12071.

    PubMed  Google Scholar 

  29. Matsukawa T, Ikeda S, Imai H, Yamada M. Alleviation of the two-cell block of ICR mouse embryos by polyaminocarboxylate metal chelators. Reproduction. 2002;124:65–71.

    Article  CAS  PubMed  Google Scholar 

  30. Alakomi HL, Saarela M, Helander IM. Effect of EDTA on Salmonella enterica serovar Typhimurium involves a component not assignable to lipopolysaccharide release. Microbiology. 2003;149:2015–21. doi:10.1099/mic.0.26312-0.

    Article  CAS  PubMed  Google Scholar 

  31. Wooley RE, Jones MS. Action of EDTA-Tris and antimicrobial agent combinations on selected pathogenic bacteria. Vet Microbiol. 1983;8:271–80.

    Article  CAS  PubMed  Google Scholar 

  32. Al-Azemi A, Fielder MD, Abuknesha RA, Price RG. Effects of chelating agent and environmental stresses on microbial biofilms: relevance to clinical microbiology. J Appl Microbiol. 2011;110:1307–13. doi:10.1111/j.1365-2672.2011.04983.x.

    Article  CAS  PubMed  Google Scholar 

  33. Lambert RJ, Hanlon GW, Denyer SP. The synergistic effect of EDTA/antimicrobial combinations on Pseudomonas aeruginosa. J Appl Microbiol. 2004;96:244–53.

    Article  CAS  PubMed  Google Scholar 

  34. Cavaliere R, Ball JL, Turnbull L, Whitchurch CB. The biofilm matrix destabilizers, EDTA and DNaseI, enhance the susceptibility of nontypeable Hemophilus influenzae biofilms to treatment with ampicillin and ciprofloxacin. Microbiologyopen. 2014;3:557–67. doi:10.1002/mbo3.187.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Zenga J, Gagnon PM, Vogel J, Chole RA. Biofilm formation by otopathogenic strains of Pseudomonas aeruginosa is not consistently inhibited by ethylenediaminetetraacetic acid. Otol Neurotol. 2012;33:1007–12. doi:10.1097/MAO.0b013e31825f249e.

    PubMed  PubMed Central  Google Scholar 

  36. Farca AM, Piromalli G, Maffei F, Re G. Potentiating effect of EDTA-Tris on the activity of antibiotics against resistant bacteria associated with otitis, dermatitis and cystitis. J Small Anim Pract. 1997;38:243–5.

    Article  CAS  PubMed  Google Scholar 

  37. Chauhan A, Lebeaux D, Ghigo JM, Beloin C. Full and broad-spectrum in vivo eradication of catheter-associated biofilms using gentamicin-EDTA antibiotic lock therapy. Antimicrob Agents Chemother. 2012;56:6310–8. doi:10.1128/AAC.01606-12.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Sarkisova S, Patrauchan MA, Berglund D, Nivens DE, Franklin MJ. Calcium-induced virulence factors associated with the extracellular matrix of mucoid Pseudomonas aeruginosa biofilms. J Bacteriol. 2005;187:4327–37. doi:10.1128/JB.187.13.4327-4337.2005.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Banin E, Vasil ML, Greenberg EP. Iron and Pseudomonas aeruginosa biofilm formation. Proc Natl Acad Sci USA. 2005;102:11076–81. doi:10.1073/pnas.0504266102.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Hachem R, Bahna P, Hanna H, Stephens LC, Raad I. EDTA as an adjunct antifungal agent for invasive pulmonary aspergillosis in a rodent model. Antimicrob Agents Chemother. 2006;50:1823–7. doi:10.1128/AAC.50.5.1823-1827.2006.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Sanchez-Fructuoso AI, Blanco J, Cano M, Ortega L, Arroyo M, Fernandez C, Prats D, Barrientos A. Experimental lead nephropathy: treatment with calcium disodium ethylenediaminetetraacetate. Am J Kidney Dis. 2002;40:59–67. doi:10.1053/ajkd.2002.33936.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

The authors are grateful to Xian-Lei Lu for donation of the strain and to Wei Sun and Zi-Guo Luo for their technical help in image processing. This work was supported by the National Natural Science Foundation of China (Grant No. 30772363).

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Authors and Affiliations

  1. Department of Neonatology, Children’s Hospital, Chongqing Medical University, Chongqing, China

    Zhenqiu Liu, Qi Lu, Fang Li, Jialin Yu, Zhengli Wang, Yu He & Chao Song

  2. Ministry of Education Key Laboratory of Child Development and Disorders, Chongqing, China

    Zhenqiu Liu, Qi Lu, Fang Li, Jialin Yu, Zhengli Wang, Yu He & Chao Song

  3. Key Laboratory of Pediatrics in Chongqing and Chongqing International Science and Technology Cooperation Center for Child Development and Disorders, Chongqing, China

    Zhenqiu Liu, Qi Lu, Fang Li, Jialin Yu, Zhengli Wang, Yu He & Chao Song

  4. Department of Neonatology, Maternal and Child Health Hospital of Xiamen City, Xiamen, Fujian, China

    Yaying Lin

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  1. Zhenqiu Liu
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Correspondence to Jialin Yu.

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Liu, Z., Lin, Y., Lu, Q. et al. In vitro and in vivo activity of EDTA and antibacterial agents against the biofilm of mucoid Pseudomonas aeruginosa . Infection 45, 23–31 (2017). https://doi.org/10.1007/s15010-016-0905-z

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