Disruption of Mycobacterium smegmatis Biofilms Using Bacteriophages Alone or in Combination with Mechanical Stress

Brendan Kiefer, John L. Dahl

Department of Biology, University of Minnesota Duluth, Duluth, MN, USA.

DOI: 10.4236/aim.2015.510073                

Abstract

Environmental mycobacteria are capable of forming biofilms in low-nutrient environments, and these biofilms may act as reservoirs for opportunistic infections. The purpose of this study was to determine if bacteriophages could disrupt existing biofilms of acid-fast staining Mycobacterium smegmatis. Using the MBEC 96-well plastic peg assay system, M. smegmatis biofilms were created and then tested for their stability in the presence of mycobacteriophages isolated from a Minnesota sphagnum peat bog. All phages tested were lytic and were observed to have weak, intermediate, and strong abilities to disrupt M. smegmatis biofilms. The formation of biofilms was severely impaired in the presence of mycobacteriophages. Phage treatment was also shown to augment M. smegmatis biofilm disruption by mechanical forces of sonication or water flow. Our study shows that, as with biofilms of Gram-positive and Gram-negative bacteria, mycobacterial biofilms are also susceptible to destruction by bacteriophages.

Keywords

Mycobacterium smegmatis, Biofilm, Mycobacteriophage, Bacteriophage

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	Tortoli, E. (2003) Impact of Genotypic Studies on Mycobacterial Taxonomy: The New Mycobacteria of the 1990s. Clinical Microbiology Reviews, 16, 319-354. http://dx.doi.org/10.1128/CMR.16.2.319-354.2003
[2]	Colville, A. (1993) Retrospective Review of Culture-Positive Mycobacterial Lymphadenitis Cases in Children in Nottingham, 1979-1990. European Journal of Clinical Microbiology & Infectious Diseases 12, 192-195. http://dx.doi.org/10.1007/BF01967110
[3]	De Groote, M.A. and Huitt, G. (2006) Infections Due to Rapidly Growing Mycobacteria. Clinical Infectious Diseases, 42, 1756-1763. http://dx.doi.org/10.1007/BF01967110
[4]	Han, X.Y., Dé, I. and Jacobson, K.L. (2007) Rapidly Growing Mycobacteria Clinical and Microbiologic Studies of 115 Cases. American Journal of Clinical Pathology, 128, 612-621. http://dx.doi.org/10.1309/1KB2GKYT1BUEYLB5
[5]	Primm, T.P., Lucero, C.A. and Falkinham, J.O. (2004) Health Impacts of Environmental Mycobacteria. Clinical Microbiology Reviews, 17, 98-106. http://dx.doi.org/10.1128/CMR.17.1.98-106.2004
[6]	Falkinham 3rd, J.O. (1996) Epidemiology of Infection by Nontuberculous Mycobacteria. Clinical Microbiology Reviews, 9, 177-215.
[7]	Stoodley, P., Sauer, K., Davies, D. and Costerton, J.W. (2002) Biofilms as Complex Differentiated Communities. Annual Review of Microbiology, 56, 187-209. http://dx.doi.org/10.1146/annurev.micro.56.012302.160705
[8]	Costerton, J., LewandowskiI, Z., Caldwell, D., Korber, D. and Lappinscott, H. (1995) Microbial Biofilms. Annual Review of Microbiology, 49, 711-745. http://dx.doi.org/10.1146/annurev.mi.49.100195.003431
[9]	Lasa, I. (2006) Towards the Identification of the Common Features of Bacterial Biofilm Development. International Journal of Food Microbiology, 9, 21-28.
[10]	Ojha, A.K., Trivelli, X., Guerardel, Y., Kremer, L. and Hatfull, G.F. (2010) Enzymatic Hydrolysis of Trehalosedimycolate Releases Free Mycolic Acids during Mycobacterial Growth in Biofilms. Journal of Biological Chemistry, 285, 17380-17389. http://dx.doi.org/10.1074/jbc.M110.112813
[11]	Whitchurch, C.B., Tolker-Nielsen, T., Ragas, P.C. and Mattick, J.S. (2002) Extracellular DNA Required for Bacterial Biofilm Formation. Science, 295, 1487-1487. http://dx.doi.org/10.1126/science.295.5559.1487
[12]	Bardouniotis, E., Ceri, H. and Olson, M.E. (2003) Biofilm Formation and Biocide Susceptibility Testing of Mycobacterium fortuitum and Mycobacterium marinum. Current Microbiology, 46, 28-32. http://dx.doi.org/10.1007/s00284-002-3796-4
[13]	Bardouniotis, E., Huddleston, W., Ceri, H. and Olson, M.E. (2001) Characterization of Biofilm Growth and Biocide Susceptibility Testing of Mycobacterium phlei Using the MBEC Assay System. FEMS Microbiology Letters, 203, 263-267. http://dx.doi.org/10.1016/s0378-1097(01)00364-0
[14]	Hall-Stoodley, L. and Lappin-Scott, H. (1998) Biofilm Formation by the Rapidly Growing Mycobacterial Species Mycobacterium fortuitum. FEMS Microbiology Letters, 168, 77-84. http://dx.doi.org/10.1111/j.1574-6968.1998.tb13258.x
[15]	Schwartz, T., Kalmbach, S., Hoffmann, S., Szewzyk, U. and Obst, U. (1998) PCR-Based Detection of Mycobacteria in Biofilms from a Drinking Water Distribution System. Journal of Microbiological Methods, 34, 113-123. http://dx.doi.org/10.1016/S0167-7012(98)00081-5
[16]	September, S., Brozel, V. and Venter, S. (2004) Diversity of Nontuberculoid Mycobacterium Species in Biofilms of Urban and Semiurban Drinking Water Distribution Systems. Applied and Environmental Microbiology, 70, 7571-7573. http://dx.doi.org/10.1128/AEM.70.12.7571-7573.2004
[17]	Adetunji, V.O., Kehiude, A.O., Bolatito, O.K. and Chen, J. (2014) Biofilm Formation by Mycobacterium bois: Influence of Surface Kind and Temperatures of Sanitizer Treatments on Biofilm Control. BioMed Research International, 2014, Article ID: 210165. http://dx.doi.org/10.1155/2014/210165
[18]	Hall-Stoodley, L., Brun, O.S., Polshyna, G. and Barker, L.P. (2006) Mycobacterium marinum Biofilm Formation Reveals Cording Morphology. FEMS Microbiology Letters, 257, 43-49. http://dx.doi.org/10.1111/j.1574-6968.2006.00143.x
[19]	Marsollier, L., Brodin, P., Jackson, M., Kordulakova, J., Tafelmeyer, P., Carbonnelle, E., et al. (2007) Impact of Mycobacterium ulcerans Biofilm on Transmissibility to Ecological Niches and Buruli Ulcer Pathogenesis. PLoS Pathogens, 3, e62. http://dx.doi.org/10.1371/journal.ppat.0030062
[20]	Falkinham III, J., Iseman, M., Haas, P. and Soolingen, D. (2008) Mycobacterium avium in a Shower Linked to Pulmonary Disease. Journal of Water and Health, 6, 209-213.
[21]	Feazel, L.M., Baumgartner, L.K., Peterson, K.L., Frank, D.N., Harris, J.K. and Pace, N.R. (2009) Opportunistic Pathogens Enriched in Showerhead Biofilms. Proceedings of the National Academy of Sciences of the United States of America, 106, 16393-16399. http://dx.doi.org/10.1073/pnas.0908446106
[22]	Nishiuchi, Y., Maekura, R., Kitada, S., Tamaru, A., Taguri, T., Kira, Y., et al. (2007) The Recovery of Mycobacterium avium-intracellulare Complex (MAC) from the Residential Bathrooms of Patients with Pulmonary MAC. Clinical Infectious Diseases, 45, 347-351. http://dx.doi.org/10.1086/519383
[23]	Espeland, E. and Wetzel, R. (2001) Complexation, Stabilization, and UV Photolysis of Extracellular and Surface-Bound Glucosidase and Alkaline Phosphatase: Implications for Biofilm Microbiota. Microbial Ecology, 42, 572-585. http://dx.doi.org/10.1007/s00248-001-1023-7
[24]	Le Magrex-Debar, E., Lemoine, J., Gellé, M., Jacquelin, L. and Choisy, C. (2000) Evaluation of Biohazards in Dehydrated Biofilms on Foodstuff Packaging. International Journal of Food Microbiology, 55, 239-243. http://dx.doi.org/10.1016/S0168-1605(00)00177-X
[25]	McNeill, K. and Hamilton, I. (2003) Acid Tolerance Response of Biofilm Cells of Streptococcus mutans. FEMS Microbiology Letters, 221, 25-30. http://dx.doi.org/10.1016/S0378-1097(03)00164-2
[26]	Donlan, R.M. and Costerton, J.W. (2002) Biofilms: Survival Mechanisms of Clinically Relevant Microorganisms. Clinical Microbiology Reviews, 15, 167-193. http://dx.doi.org/10.1128/CMR.15.2.167-193.2002
[27]	Tengra, F.K., Dahl, J.L., Dutton, D., Caberoy, N.B., Coyne, L. and Garza, A.G. (2006) CbgA, a Protein Involved in Cortex Formation and Stress Resistance in Myxococcus xanthus Spores. Journal of Bacteriology, 188, 8299-8302. http://dx.doi.org/10.1128/JB.00578-06
[28]	Covert, T.C., Rodgers, M.R., Reyes, A.L. and Stelma, G.N. (1999) Occurrence of Nontuberculous Mycobacteria in Environmental Samples. Applied and Environmental Microbiology, 65, 2492-2496.
[29]	Gomez-Smith, C.K., La Para, T.M. and Hozalski, R.M. (2015) Sulfate Reducing Bacteria and Mycobacteria Dominate the Biofilm Communities in a Chloraminated Drinking Water Distribution System. Environmental Science & Technology, 49, 8432-8440. http://dx.doi.org/10.1021/acs.est.5b00555
[30]	Le Dantec, C., Duguet, J., Montiel, A., Dumoutier, N., Dubrou, S. and Vincent, V. (2002) Occurrence of Mycobacteria in Water Treatment Lines and in Water Distribution Systems. Applied and Environmental Microbiology, 68, 5318-5325. http://dx.doi.org/10.1128/AEM.68.11.5318-5325.2002
[31]	Tsintzou, A., Vantarakis, A., Pagonopoulou, O., Athanassiadou, A. and Papapetropoulou, M. (2000) Environmental Mycobacteria in Drinking Water before and after Replacement of the Water Distribution Network. Water, Air, and Soil Pollution, 120, 273-282. http://dx.doi.org/10.1023/A:1005266827726
[32]	Cerca, N., Oliveira, R. and Azeredo, J. (2007) Susceptibility of Staphylococcus epidermidis Planktonic Cells and Biofilms to the Lytic Action of Staphylococcus Bacteriophage K. Letters in Applied Microbiology, 45, 313-317. http://dx.doi.org/10.1111/j.1472-765X.2007.02190.x
[33]	Chhibber, S., Bansal, S. and Kaur, S. (2015) Disrupting the Mixed Species Biofilm of Klebsiella pneumonia B5055 and Pseudomonas aeruginosa PAO Using Bacteriophages Alone or in Combination with Xylitol. Microbiology, 161, 1369-1377.
[34]	Sass, P. and Bierbaum, G. (2007) Lytic Activity of Recombinant Bacteriophage φ11 and φ12 Endolysinson Whole Cells and Biofilms of Staphylococcus aureus. Applied and Environmental Microbiology, 73, 347-352. http://dx.doi.org/10.1128/AEM.01616-06
[35]	Sharma, M., Ryu, J. and Beuchat, L. (2005) Inactivation of Escherichia coli O157:H7 in Biofilm on Stainless Steel by Treatment with an Alkaline Cleaner and a Bacteriophage. Journal of Applied Microbiology, 99, 449-459. http://dx.doi.org/10.1111/j.1365-2672.2005.02659.x
[36]	Sillankorva, S., Oliveira, R., Vieira, M. and Azeredo, J. (2008) Real-Time Quantification of Pseudomonas fluorescens Cell Removal from Glass Surfaces Due to Bacteriophage φS1 Application. Journal of Applied Microbiology, 105, 196-202. http://dx.doi.org/10.1111/j.1365-2672.2008.03743.x
[37]	Knezevic, P. and Petrovic, O. (2008) A Colorimetric Microtiter Plate Method for Assessment of Phage Effect on Pseudomonas aeruginosa Biofilm. Journal of Microbiological Methods, 74, 114-118. http://dx.doi.org/10.1016/j.mimet.2008.03.005
[38]	Teng, R. and Dick, T. (2003) Isoniazid Resistance of Exponentially Growing Mycobacterium smegmatis Biofilm Culture. FEMS Microbiology Letters, 227, 171-174. http://dx.doi.org/10.1016/S0378-1097(03)00584-6
[39]	Kazda, J. (2000) The Ecology of Mycobacteria. Klewer Academic Publishers, Dordrecht. http://dx.doi.org/10.1007/978-94-011-4102-4
[40]	Sauer, K. (2003) The Genomics and Proteomics of Biofilm Formation. Genome Biology, 4, 219. http://dx.doi.org/10.1186/gb-2003-4-6-219
[41]	Carson, L., Gorman, S.P. and Gilmore, B.D. (2010) The Use of Lytic Bacteriophages in the Prevention and Eradication of Biofilms of Proteus mirabilis and Escherichia coli. FEMS Immunology & Medical Microbiology, 59, 447-455. http://dx.doi.org/10.1111/j.1574-695x.2010.00696.x
[42]	Chhibber, S., Nag, D. and Bansal, S. (2013) Inhibiting Biofilm Formation by Klebsiella pneumonia B5055 Using an Iron Antagonizing Molecule and a Bacteriophage. BMC Microbiology, 13, 174.
[43]	Chibeu, A., Lingohr, E.J., Masson, L., Manges, A., Harel, J., Ackermann, H.W., Kropinski, A.M. and Boerlin, P. (2012) Bacteriophages with the Ability to Degrade Uropathogenic Escherichia coli Biofilms. Viruses, 4, 471-487.
[44]	Kelly, D., McAuliffe, O., Ross, R.P. and Coffey, A. (2012) Prevention of Staphylococcus aureus Biofilm Formation and Reduction in Established Biofilm Density Using a Combination of Phage K and Modified Derivatives. Letters in Applied Microbiology, 54, 286-291.
[45]	Siringan, P., Connerton, P.L., Payne, R.J. and Connerton, I.F. (2011) Bacteriophage-Mediated Dispersal of Campylobacter jejuni Biofilms. Applied and Environmental Microbiology, 77, 3320-3326. http://dx.doi.org/10.1128/AEM.02704-10
[46]	Zhang, Y., Hunt, H.K. and Hu, Z. (2013) Application of Bacteriophages to Selectively Remove Pseudomonas aeruginosa in Water and Wastewater Filtration Systems. Water Research, 47, 4507-4518.
[47]	Zambrano, M.M. and Kolter, R. (2005) Mycobacterial Biofilms: A Greasy Way to Hold It Together. Cell, 123, 762-764. http://dx.doi.org/10.1016/j.cell.2005.11.011
[48]	Hatfull, G.F. (2010) Mycobacteriophages: Genes and Genomes. Annual Review of Microbiology, 64, 331-356. http://dx.doi.org/10.1146/annurev.micro.112408.134233
[49]	Hatfull, G.F., Cresawn, S.G. and Hendrix, R.W. (2008) Comparative Genomics of the Mycobacteriophages: Insights into Bacteriophage Evolution. Research in Microbiology, 159, 332-339. http://dx.doi.org/10.1016/j.resmic.2008.04.008