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Influence of Excipients on the Antimicrobial Activity of Tobramycin Against Pseudomonas aeruginosa Biofilms

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Abstract

Purpose

It is unknown if inactive pharmaceutical ingredients influence the activity of antibiotics they are co-formulated with. Recently it was found that materials acting as carbon nutrient sources for bacteria can promote bacterial dispersion from a biofilm and/or reverse the persister state of a subpopulation of bacteria within the biofilms. Both can make bacteria more susceptible to antibiotics. Thus, the aim was to identify potential excipients to improve antibiotic activity in Pseudomonas aeruginosa biofilms.

Methods

We screened 190 potential excipients alone, and in combination with tobramycin sulfate against P. aeruginosa (strain PAO1) grown planktonically or as biofilms. After the excipient screening stage, we investigated the effect of 10 selected excipients against a more virulent strain (luminescent strain UCBPP-PA14). Temporal changes in luminescence, as an indicator of bacterial proliferation, and surviving colony forming units (CFUs) from the treated PA14 biofilms were quantified.

Results

Forty-eight materials tested caused a reduction of PAO1 proliferation either alone or combined with tobramycin. L-alanine (p < 0.05), D-alanine (p > 0.05), and N-acetyl-D-glucosaminitol (p > 0.05) improved the activity of tobramycin measured by PA14 luminometry. Additionally, L-alanine and succinic acid significantly reduced the survival of PA14 biofilms (p < 0.05).

Conclusions

L-alanine, succinic acid, and N-acetyl-D-glucosaminitol may be useful as antibiotic adjuvants in future tobramycin anti-biofilm formulations.

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Abbreviations

BHI:

Brain heart infusion

CFU:

Colony forming units

EPS:

Extracellular polymeric substance

PA:

Pseudomonas aeruginosa

PBS:

Phosphate saline buffer

TOB:

Tobramycin sulfate

XTT:

2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H–tetrazolium-5-carboxanilide

References

  1. Driscoll JA, Brody SL, Kollef MH. The epidemiology, pathogenesis and treatment of Pseudomonas aeruginosa infections. Drugs. 2007;67(3):351–68.

    Article  CAS  PubMed  Google Scholar 

  2. WHO priority pathogens list for R&D of new antibiotics. Avalaible at: http://www.who.int/mediacentre/news/releases/2017/bacteria-antibiotics-needed/en/. Accessed on 31 Oct 2017.

  3. Micek ST, Kollef MH, Torres A, Chen C, Rello J, Chastre J, et al. Pseudomonas aeruginosa nosocomial pneumonia: impact of pneumonia classification. Infect Control Hosp Epidemiol. 2015;36(10):1190–7.

    Article  PubMed  Google Scholar 

  4. Weinstein RA, Gaynes R, Edwards JR, System NNIS. Overview of nosocomial infections caused by gram-negative bacilli. Clin Infect Dis. 2005;41(6):848–54.

    Article  Google Scholar 

  5. Fick RB Jr. Pathogenesis of the pseudomonas lung lesion in cystic fibrosis. Chest. 1989;96(1):158–64.

    Article  PubMed  Google Scholar 

  6. Bjarnsholt T, Jensen PO, Fiandaca MJ, Pedersen J, Hansen CR, Andersen CB, et al. Pseudomonas aeruginosa biofilms in the respiratory tract of cystic fibrosis patients. Pediatr Pulmonol. 2009;44(6):547–58.

    Article  PubMed  Google Scholar 

  7. Nixon GM, Armstrong DS, Carzino R, Carlin JB, Olinsky A, Robertson CF, et al. Clinical outcome after early Pseudomonas aeruginosa infection in cystic fibrosis. J Pediatr. 2001;138(5):699–704.

    Article  CAS  PubMed  Google Scholar 

  8. Murphy TD, Anbar RD, Lester LA, Nasr SZ, Nickerson B, VanDevanter DR, et al. Treatment with tobramycin solution for inhalation reduces hospitalizations in young CF subjects with mild lung disease. Pediatr Pulmonol. 2004;38(4):314–20.

    Article  PubMed  Google Scholar 

  9. Hoiby N, Bjarnsholt T, Givskov M, Molin S, Ciofu O. Antibiotic resistance of bacterial biofilms. Int J Antimicrob Agents. 2010;35(4):322–32.

    Article  PubMed  Google Scholar 

  10. Costerton JW. Cystic fibrosis pathogenesis and the role of biofilms in persistent infection. Trends Microbiol. 2001;9(2):50–2.

    Article  CAS  PubMed  Google Scholar 

  11. Stewart PS, Costerton JW. Antibiotic resistance of bacteria in biofilms. Lancet. 2001;358(9276):135–8.

    Article  CAS  PubMed  Google Scholar 

  12. Harrison-Balestra C, Cazzaniga AL, Davis SC, Mertz PM. A wound-isolated Pseudomonas aeruginosa grows a biofilm in vitro within 10 hours and is visualized by light microscopy. Dermatol Surg. 2003;29(6):631–5.

    PubMed  Google Scholar 

  13. Mihai MM, Holban AM, Giurcaneanu C, Popa LG, Oanea RM, Lazar V, et al. Microbial biofilms: impact on the pathogenesis of periodontitis, cystic fibrosis, chronic wounds and medical device-related infections. Curr Top Med Chem. 2015;15(16):1552–76.

    Article  CAS  PubMed  Google Scholar 

  14. Nichols WW, Dorrington S, Slack M, Walmsley H. Inhibition of tobramycin diffusion by binding to alginate. Antimicrob Agents Chemother. 1988;32(4):518–23.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Tseng BS, Zhang W, Harrison JJ, Quach TP, Song JL, Penterman J, et al. The extracellular matrix protects Pseudomonas aeruginosa biofilms by limiting the penetration of tobramycin. Environ Microbiol. 2013;15(10):2865–78.

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Walters MC, Roe F, Bugnicourt A, Franklin MJ, Stewart PS. Contributions of antibiotic penetration, oxygen limitation, and low metabolic activity to tolerance of Pseudomonas aeruginosa biofilms to ciprofloxacin and tobramycin. Antimicrob Agents Chemother. 2003;47(1):317–23.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Stewart PS, Franklin MJ. Physiological heterogeneity in biofilms. Nat Rev Microbiol. 2008;6(3):199–210.

    Article  CAS  PubMed  Google Scholar 

  18. Werner E, Roe F, Bugnicourt A, Franklin MJ, Heydorn A, Molin S, et al. Stratified growth in Pseudomonas aeruginosa biofilms. Appl Environ Microbiol. 2004;70(10):6188–96.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Conlon BP, Rowe SE, Lewis K. Persister cells in biofilm associated infections. Adv Exp Med Biol. 2015;831:1–9.

    Article  PubMed  Google Scholar 

  20. Percival SL, Hill KE, Malic S, Thomas DW, Williams DW. Antimicrobial tolerance and the significance of persister cells in recalcitrant chronic wound biofilms. Wound Repair Regen. 2011;19(1):1–9.

    Article  PubMed  Google Scholar 

  21. Du J, Bandara HM, Du P, Huang H, Hoang K, Nguyen D, et al. Improved Biofilm Antimicrobial Activity of Polyethylene Glycol Conjugated Tobramycin Compared to Tobramycin in Pseudomonas aeruginosa Biofilms. Mol Pharm. 2015;12(5):1544–53.

    Article  CAS  PubMed  Google Scholar 

  22. Chiang WC, Nilsson M, Jensen PO, Hoiby N, Nielsen TE, Givskov M, et al. Extracellular DNA shields against aminoglycosides in Pseudomonas aeruginosa biofilms. Antimicrob Agents Chemother. 2013;57(5):2352–61.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Alipour M, Suntres ZE, Omri A. Importance of DNase and alginate lyase for enhancing free and liposome encapsulated aminoglycoside activity against Pseudomonas aeruginosa. J Antimicrob Chemother. 2009;64(2):317–25.

    Article  CAS  PubMed  Google Scholar 

  24. Bandara H, Nguyen D, Mogarala S, Osinski M, Smyth H. Magnetic fields suppress Pseudomonas aeruginosa biofilms and enhance ciprofloxacin activity. Biofouling. 2015;31(5):443–57.

    Article  CAS  PubMed  Google Scholar 

  25. Bandara HM, Harb A, Kolacny D Jr, Martins P, Smyth HD. Sound waves effectively assist tobramycin in elimination of Pseudomonas aeruginosa biofilms in vitro. AAPS PharmSciTech. 2014;15(6):1644–54.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Street CN, Gibbs A, Pedigo L, Andersen D, Loebel NG. In vitro photodynamic eradication of Pseudomonas aeruginosa in planktonic and biofilm culture. Photochem Photobiol. 2009;85(1):137–43.

    Article  CAS  PubMed  Google Scholar 

  27. Kim J-S, Heo P, Yang T-J, Lee K-S, Cho D-H, Kim BT, et al. Selective killing of bacterial persisters by a single chemical compound without affecting normal antibiotic-sensitive cells. Antimicrob Agents Chemother. 2011;55(11):5380–3.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Sauer K, Cullen MC, Rickard AH, Zeef LA, Davies DG, Gilbert P. Characterization of nutrient-induced dispersion in Pseudomonas aeruginosa PAO1 biofilm. J Bacteriol. 2004;186(21):7312–26.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Ross SS, Fiegel J. Nutrient dispersion enhances conventional antibiotic activity against Pseudomonas aeruginosa biofilms. Int J Antimicrob Agents. 2012;40(2):177–81.

    Article  Google Scholar 

  30. Allison KR, Brynildsen MP, Collins JJ. Metabolite-enabled eradication of bacterial persisters by aminoglycosides. Nature. 2011;473(7346):216–20.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Barraud N, Buson A, Jarolimek W, Rice SA. Mannitol enhances antibiotic sensitivity of persister bacteria in Pseudomonas aeruginosa biofilms. PLoS One 2013;8(12).

  32. FDA Inactive Ingredient Database. Available at: https://www.accessdata.fda.gov/scripts/cder/iig/index.cfm Accessed on 31 Oct 2017.

  33. Code of Federal Regulations (CFR) 21CFR210.3. Available at: https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm?fr=210.3. Accessed on 2 Nov 2017.

  34. Barker AF, Couch L, Fiel SB, Gotfried MH, Ilowite J, Meyer KC, et al. Tobramycin solution for inhalation reduces sputum Pseudomonas aeruginosa density in bronchiectasis. Am J Respir Crit Care Med. 2000;162(2 Pt 1):481–5.

    Article  CAS  PubMed  Google Scholar 

  35. Mogayzel PJ Jr, Naureckas ET, Robinson KA, Brady C, Guill M, Lahiri T, et al. Cystic Fibrosis Foundation Pulmonary Clinical Practice Guidelines C. Cystic Fibrosis Foundation pulmonary guideline. pharmacologic approaches to prevention and eradication of initial Pseudomonas aeruginosa infection. Ann Am Thorac Soc. 2014;11(10):1640–50.

    Article  PubMed  Google Scholar 

  36. Mikkelsen H, McMullan R, Filloux A. The Pseudomonas aeruginosa reference strain PA14 displays increased virulence due to a mutation in ladS. PLoS One. 2011;6(12):e29113.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Yang L, Hu Y, Liu Y, Zhang J, Ulstrup J, Molin S. Distinct roles of extracellular polymeric substances in Pseudomonas aeruginosa biofilm development. Environ Microbiol. 2011;13(7):1705–17.

    Article  CAS  PubMed  Google Scholar 

  38. Ryder C, Byrd M, Wozniak DJ. Role of polysaccharides in Pseudomonas aeruginosa biofilm development. Curr Opin Microbiol. 2007;10(6):644–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Colvin KM, Gordon VD, Murakami K, Borlee BR, Wozniak DJ, Wong GC, et al. The pel polysaccharide can serve a structural and protective role in the biofilm matrix of Pseudomonas aeruginosa. PLoS Pathog. 2011;7(1):e1001264.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Herigstad B, Hamilton M, Heersink J. How to optimize the drop plate method for enumerating bacteria. J Microbiol Methods. 2001;44(2):121–9.

    Article  CAS  PubMed  Google Scholar 

  41. Palmer KL, Aye LM, Whiteley M. Nutritional cues control Pseudomonas aeruginosa multicellular behavior in cystic fibrosis sputum. J Bacteriol. 2007;189(22):8079–87.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Boulette ML, Baynham PJ, Jorth PA, Kukavica-Ibrulj I, Longoria A, Barrera K, et al. Characterization of alanine catabolism in Pseudomonas aeruginosa and its importance for proliferation in vivo. J Bacteriol. 2009;191(20):6329–34.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Kay W, Gronlund AF. Amino acid transport in Pseudomonas aeruginosa. J Bacteriol. 1969;97(1):273–81.

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Hill KE, Malic S, McKee R, Rennison T, Harding KG, Williams DW, Thomas DW. An in vitro model of chronic wound biofilms to test wound dressings and assess antimicrobial susceptibilities. J Antimicrob Chemother. 2010; dkq105.

  45. Sun Y, Dowd SE, Smith E, Rhoads DD, Wolcott RD. In vitro multispecies Lubbock chronic wound biofilm model. Wound Repair Regen. 2008;16(6):805–13.

    Article  PubMed  Google Scholar 

  46. Palmer GC, Whiteley M. Metabolism and pathogenicity of Pseudomonas aeruginosa infections in the lungs of individuals with cystic fibrosis. Microbiol Spectr 2015;3(4).

  47. Shrout JD, Chopp DL, Just CL, Hentzer M, Givskov M, Parsek MR. The impact of quorum sensing and swarming motility on Pseudomonas aeruginosa biofilm formation is nutritionally conditional. Mol Microbiol. 2006;62(5):1264–77.

    Article  CAS  PubMed  Google Scholar 

  48. Elliott D, Burns JL, Hoffman LR. Exploratory study of the prevalence and clinical significance of tobramycin-mediated biofilm induction in Pseudomonas aeruginosa isolates from cystic fibrosis patients. Antimicrob Agents Chemother. 2010;54(7):3024–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Hoffman LR, D'Argenio DA, MacCoss MJ, Zhang Z, Jones RA, Miller SI. Aminoglycoside antibiotics induce bacterial biofilm formation. Nature. 2005;436(7054):1171–5.

    Article  CAS  PubMed  Google Scholar 

  50. Bhat R, Marx A, Galanos C, Conrad R. Structural studies of lipid A from Pseudomonas aeruginosa PAO1: occurrence of 4-amino-4-deoxyarabinose. J Bacteriol. 1990;172(12):6631–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Kong M, Chen XG, Xing K, Park HJ. Antimicrobial properties of chitosan and mode of action: a state of the art review. Int J Food Microbiol. 2010;144(1):51–63.

    Article  CAS  PubMed  Google Scholar 

  52. Tang H, Zhang P, Kieft TL, Ryan SJ, Baker SM, Wiesmann WP, et al. Antibacterial action of a novel functionalized chitosan-arginine against Gram-negative bacteria. Acta Biomater. 2010;6(7):2562–71.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Narayanaswamy VP, Giatpaiboon S, Baker SM, Wiesmann WP, LiPuma JJ, Townsend SM. Novel glycopolymer sensitizes Burkholderia cepacia complex isolates from cystic fibrosis patients to tobramycin and meropenem. PLoS One. 2017;12(6):e0179776.

    Article  PubMed  PubMed Central  Google Scholar 

  54. Shakil S, Khan R, Zarrilli R, Khan AU. Aminoglycosides versus bacteria--a description of the action, resistance mechanism, and nosocomial battleground. J Biomed Sci. 2008;15(1):5–14.

    Article  CAS  PubMed  Google Scholar 

  55. Sousa LP, Silva AF, Calil NO, Oliveira MG, Silva SS, Raposo NRB. In vitro inhibition of Pseudomonas aeruginosa adhesion by xylitol. Braz Arch Biol Technol. 2011;54(5):877–84.

    Article  Google Scholar 

  56. Ammons MC, Ward LS, Fisher ST, Wolcott RD, James GA. In vitro susceptibility of established biofilms composed of a clinical wound isolate of Pseudomonas aeruginosa treated with lactoferrin and xylitol. Int J Antimicrob Agents. 2009;33(3):230–6.

    Article  CAS  PubMed  Google Scholar 

  57. Wolcott RD, Rhoads DD. A study of biofilm-based wound management in subjects with critical limb ischaemia. J Wound Care. 2008;17(4):145. -148, 150-142, 154-145

    Article  CAS  PubMed  Google Scholar 

  58. Garber N, Guempel U, Belz A, Gilboa-Garber N, Doyle RJ. On the specificity of the D-galactose-binding lectin (PA-I) of Pseudomonas aeruginosa and its strong binding to hydrophobic derivatives of D-galactose and thiogalactose. Biochim Biophys Acta. 1992;1116(3):331–3.

    Article  CAS  PubMed  Google Scholar 

  59. Blanchard B, Imberty A, Varrot A. Secondary sugar binding site identified for LecA lectin from Pseudomonas aeruginosa. Proteins: Struct Funct Bioinf. 2014;82(6):1060–5.

    Article  CAS  Google Scholar 

  60. Chen X, Stewart PS. Biofilm removal caused by chemical treatments. Water Res. 2000;34(17):4229–33.

    Article  CAS  Google Scholar 

  61. Meylan S, Porter CB, Yang JH, Belenky P, Gutierrez A, Lobritz MA, et al. Carbon sources tune antibiotic susceptibility in Pseudomonas aeruginosa via tricarboxylic acid cycle control. Cell Chem Biol. 2017;24(2):195–206.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Taber HW, Mueller JP, Miller PF, Arrow AS. Bacterial uptake of aminoglycoside antibiotics. Microbiol Rev. 1987;51(4):439–57.

    CAS  PubMed  PubMed Central  Google Scholar 

  63. Berridge MV, Herst PM, Tan AS. Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction. Biotechnol Annu Rev. 2005;11:127–52.

    Article  CAS  PubMed  Google Scholar 

  64. Sabaeifard P, Abdi-Ali A, Soudi MR, Dinarvand R. Optimization of tetrazolium salt assay for Pseudomonas aeruginosa biofilm using microtiter plate method. J Microbiol Methods. 2014;105:134–40.

    Article  CAS  PubMed  Google Scholar 

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ACKNOWLEDGMENTS AND DISCLOSURES

The authors thank Dr. H.M.H.N. (Nihal) Bandara and Dr. Frederic Tewes for sharing their knowledge and experience in microbiology.

Tania Bahamondez-Canas would like to thank to CONICYT (Becas Chile) for the scholarship to pursue her Ph.D. studies.

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Authors and Affiliations

  1. Division of Molecular Pharmaceutics and Drug Delivery, College of Pharmacy, The University of Texas at Austin, 2409 University Ave, A1920, Austin, Texas, 78712, USA

    Tania Bahamondez-Canas & Hugh D. C. Smyth

  2. Center for Infectious Disease, The University of Texas at Austin, Austin, Texas, USA

    Hugh D. C. Smyth

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  1. Tania Bahamondez-Canas
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  2. Hugh D. C. Smyth
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Correspondence to Hugh D. C. Smyth.

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Bahamondez-Canas, T., Smyth, H.D.C. Influence of Excipients on the Antimicrobial Activity of Tobramycin Against Pseudomonas aeruginosa Biofilms. Pharm Res 35, 10 (2018). https://doi.org/10.1007/s11095-017-2301-5

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