Viruses. 2014 Oct 3;6(10):3778-86. doi: 10.3390/v6103778.

Effect of bacteriophage infection in combination with tobramycin on the emergence of resistance in Escherichia coli and Pseudomonas aeruginosa biofilms.

Lindsey B. Coulter, Robert J. C. McLean, Rodney E. Rohde, and Gary M. Aron

Department of Biology, Texas State University, San Marcos, TX 78666: (Lindsey B. Coulter), (Robert J.C. McLean), (Gary Aron)

Clinical Laboratory Science Program, Texas State University, San Marcos, TX 78666 (Rodney E. Rohde)



Bacteriophage infection and antibiotics used individually to reduce biofilm mass often result in the emergence of significant levels of phage and antibiotic resistant cells. In contrast, combination therapy in Escherichia coli biofilms employing T4 phage and tobramycin resulted in greater than 99% and 39% reduction in antibiotic and phage resistant cells, respectively. In P. aeruginosa biofilms, combination therapy resulted in a 60% and 99% reduction in antibiotic and PB-1 phage resistant cells, respectively. Although the combined treatment resulted in greater reduction of E. coli CFUs compared to the use of antibiotic alone, infection of P. aeruginosa biofilms with PB-1 in the presence of tobramycin was only as effective in the reduction of CFUs as the use of antibiotic alone. The study demonstrated phage infection in combination with tobramycin can significantly reduce the emergence of antibiotic and phage resistant cells in both E. coli and P. aeruginosa biofilms, however, a reduction in biomass was dependent on the phage-host system.

PMID: 25285538



Bacterial biofilms are clusters of bacterial cells that adhere to a living or nonliving surface and grow to be several millimeters thick. Biofilms can be found growing in the lungs of cystic fibrosis patients, on medical implants such as catheters, and even on our teeth as plaque. Due to the structure of biofilms, it is often difficult to treat and cure infected individuals. While the uppermost region of the biofilm is often reduced by antibiotics, the slower-growing, innermost cells are not affected and can become resistant to the treatment [1, 2, 3]. Other treatment methods have been proposed to eradicate biofilm colonies, one in particular is bacteriophages.

Bacteriophages are bacterial viruses that only target bacterial cells. Phages were used for the treatment of various bacterial infections before the discovery of antibiotics and are still used in parts of Eastern Europe, commonly for antibiotic resistant infections. Given the world’s current situation with the rise of antibiotic resistance, many scientists have turned back to investigating phage treatment. Several studies have been published indicating the use of phage on biofilms to be more effective than antibiotics, however phages alone have not been found to eradicate biofilms and could lead to similar issues with resistance [4, 5].

To better reduce biofilm mass, combination therapy has been employed. Several studies found treating biofilms with different combinations of antibiotics was more effective at reducing biofilm mass compared to a single antibiotic [6, 7]. Similarly, pre-treating catheters with cocktails, or mixtures, of bacteriophages was found more effective at reducing the emergence of a biofilm [8]. More recently, studies have found combining antibiotic with phage was more effective at reducing biofilm mass than either antibiotic or phage alone [9, 10, 11]. We hypothesized the combination of phage and antibiotic to be more effective in reducing the overall biofilm mass and in reducing the emergence of resistant cells.

Our research focused on biofilms caused by Escherichia coli and Pseudomonas aeruginosa. The biofilms were established for 48 hours on silicone disks then treated for 24 hours with phage specific for each organism (T4 for E. coli and PB-1 for P. aeruginosa), antibiotic (tobramycin), or a combination of the two. After the 24 hour treatment, the biofilms were sonicated off the disk and plated on agar plates for colony counts. We used LB plates to detect all colonies, LB plates with tobramycin to detect tobramycin resistant colonies, and LB plates with a phage overlay to detect phage resistant colonies.

We found a greater decrease in overall biofilm mass with the combination of phage T4 and tobramycin in E. coli biofilms, compared to the use of either alone. The combination of phage PB-1 and tobramycin on P. aeruginosa biofilms was just as effective in decreasing overall biofilm mass as tobramycin alone. In the treatment of both E. coli and P. aeruginosa biofilms, we found the combination of phage and antibiotic resulted in a decrease in the emergence of resistant cells compared to either treatment alone. This indicates that overall combination therapy consisting of phage and antibiotic could be beneficial in reducing the emergence of resistant cells, however the efficacy in reducing overall biofilm mass is dependent on the phage chosen for treatment. Future studies on combination therapy should consider treatment of mixed biofilm communities as well as investigation into other phage-host systems.

Importance of this study: our data, along with others’ [9, 10, 11], suggests the use of combinational therapy consisting of phage and antibiotic is effective in reducing both biofilm mass and the emergence of resistant cells and therefore should be considered for the treatment of biofilms.



  1. De la Fuente-Núñez, C.; Reffuveille, F.; Fernandez, L.; Hancock, R.E.W. Bacterial biofilm development as a multicellular adaptation: Antibiotic resistance and new therapeutic strategies. Curr. Opin. Microbiol. 2013, 16, 580–589.
  2. Nickel, J.C.; Ruseska, I.; Wright, J.B.; Costerton, J.W. Tobramycin resistance of Pseudomonas aeruginosa cells growing as a biofilm on urinary catheter material. Antimicrob. Agents Chemother. 1985, 27, 619–624.
  3. Hoyle, B.D.; Alcantara, J.; Costerton, J.W. Pseudomonas aeruginosa biofilm as a diffusion barrier to piperacillin. Antimicrob. Agents Chemother. 1992, 36, 2054–2056.
  4. Krylov, V.N. Bacteriophages of Pseudomonas aeruginosa: Long-term prospects for use in phage therapy. Adv. Virus Res. 2014, 88, 227–278.
  5. Sulakvelidze, A.; Alavidze, Z.; Morris, J.G., Jr. Bacteriophage therapy. Antimicrob. Agents Chemother. 2001, 45, 649–659
  6. Tré-Hardy, M.; Nagant, C.; El Manssouri, N.; Vanderbist, F.; Traore, H.; Vaneechoutte, M.; Dehaye, J.P. Efficacy of the combination of tobramycin and a macrolide in an in vitro Pseudomonas aeruginosa mature biofilm model. Antimicrob. Agents Chemother. 2010, 54, 4409–4415.
  7. Parra-Ruiz, J.; Vidaillac, C.; Rose, W.E.; Rybak, M.J. Activities of high-dose daptomycin, vancomycin, and moxifloxacin alone or in combination with clarithromycin or rifampin in a novel in vitro model of Staphylococcus aureus biofilm. Antimicrob. Agents Chemother. 2010, 54, 4329–4334.
  8. Fu, W.; Forster, T.; Mayer, O.; Curtin, J.J.; Lehman, S.M.; Donlan, R.M. Bacteriophage cocktail for the prevention of biofilm formation by Pseudomonas aeruginosa on catheters in an in vitro model system. Antimicrob. Agents Chemother. 2010, 54, 397–404.
  9. Bedi, M.S.; Verma, V.; Chhibber, S. Amoxicillin and specific bacteriophage can be used together for eradication of biofilm of Klebsiella pneumoniae B5055. World J. Microbiol. Biotechnol. 2009, 25, 1145–1151.
  10. Verma, V.; Harjai, K.; Chhibber, S. Structural changes induced by a lytic bacteriophage make ciprofloxacin effective against older biofilm of Klebsiella pneumoniae. Biofouling 2010, 26, 729–737.
  11. Rahman, M.; Kim, S.; Kim, S.M.; Seol, S.Y.; Kim, J. Characterization of induced Staphylococcus aureus bacteriophage SAP-26 and its anti-biofilm activity with rifampicin. Biofouling 2011, 27, 1087–1093.




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