Abstract
The resistance of bacterial biofilms to antibiotic treatment has been attributed to the emergence of structurally heterogeneous microenvironments containing metabolically inactive cell populations. In this study, we use a three-dimensional individual-based cellular automata model to investigate the influence of nutrient availability and quorum sensing on microbial heterogeneity in growing biofilms. Mature biofilms exhibited at least three structurally distinct strata: a high-volume, homogeneous region sandwiched between two compact sections of high heterogeneity. Cell death occurred preferentially in layers in close proximity to the substratum, resulting in increased heterogeneity in this section of the biofilm; the thickness and heterogeneity of this lowermost layer increased with time, ultimately leading to sloughing. The model predicted the formation of metabolically dormant cellular microniches embedded within faster-growing cell clusters. Biofilms utilizing quorum sensing were more heterogeneous compared to their non-quorum sensing counterparts, and resisted sloughing, featuring a cell-devoid layer of EPS atop the substratum upon which the remainder of the biofilm developed. Overall, our study provides a computational framework to analyze metabolic diversity and heterogeneity of biofilm-associated microorganisms and may pave the way toward gaining further insights into the biophysical mechanisms of antibiotic resistance.
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Alpkvist E, Picioreanu C, van Loosdrecht M, Heyden A (2006) Three-dimensional biofilm model with individual cells and continuum EPS matrix. Biotechnol Bioeng 94:961–979
Alpkvist E, Klapper I (2007) A multidimensional multispecies continuum model for heterogeneous biofilm development. Bull Math Biol 69:765–789
Anguige K, King J, Ward J (2005) Modelling antibiotic- and anti-quorum sensing treatment of a spatially-structured Pseudomonas aeruginosa population. J Math Biol 51:557–594
Ardré M, Henry H, Douarche C, Plapp M (2015) An individual-based model for biofilm formation at liquid surfaces. Phys Biol 12:066015
Bassler BL, Wright M, Showalter R, Silverman M (1993) Intercellular signalling in Vibrio harveyi: sequence and function of genes regulating expression of luminescence. Mol Microbiol 9:773–786
Bester E, Wolfaardt G, Joubert L, Garny K, Saftic S (2005) Planktonic-cell yield of a pseudomonad biofilm. Appl Environ Microbiol 71:7792–7798
Blaser J, Vergères P, Widmer AF, Zimmerli W (1995) In vivo verification of in vitro model of antibiotic treatment of device-related infection. Antimicrob Agents Chemother 39:1134–1139
Boyd A, Chakrabarty A (1994) Role of alginate lyase in cell detachment of Pseudomonas aeruginosa. Appl Environ Microbiol 60:2355–2359
Castro SL, Nelman-Gonzalez M, Nickerson CA, Ott CM (2011) Induction of attachment-independent biofilm formation and repression of hfq expression by low-fluid-shear culture of Staphylococcus aureus. Appl Environ Microbiol 77:6368–6378
Chambless JD, Hunt S, Stewart P (2006) A three-dimensional computer model of four hypothetical mechanisms protecting biofilms from antimicrobials. Appl Environ Microbiol 72:2005–2013
Chambless JD, Stewart P (2007) A three-dimensional computer model analysis of three hypothetical biofilm detachment mechanisms. Biotechnol Bioeng 97:1573–1584
Chang I, Gilbert ES, Eliashberg N, Keasling J (2003) A three-dimensional, stochastic simulation of biofilm growth and transport-related factors that affect structure. Microbiology 149:2859–2871
Costerton JW, Cheng KJ, Geesey GG, Ladd T, Nickel J, Dasgupta M, Marrie T (1987) Bacterial biofilms in nature and disease. Annu Rev Microbiol 41:435–464
Darouiche RO, Dhir A, Miller A, Landon G, Raad I, Musher D (1994) Vancomycin penetration into biofilm covering infected prostheses and effect on bacteria. Antimicrob Agents Chemother 170:720–723
Davies DG, Parsek MR, Pearson J, Iglewski B, Costerton J, Greenberg E (1998) The involvement of cell-to-cell signals in the development of a bacterial biofilm. Science 280:295–298
De Beer D, Srinivasan R, Stewart PS (1994) Direct measurement of chlorine penetration into biofilms during disinfection. Appl Environ Microbiol 60:4339–4344
Deretic V, Schurr MJ, Boucher JC, Martin DW (1994) Conversion of Pseudomonas aeruginosa to mucoidy in cystic fibrosis: environmental stress and regulation of bacterial virulence by alternative sigma factors. J Bacteriol 176:2773–2780
Duddu R, Chopp C, Moran B (2009) A two-dimensional continuum model of biofilm growth incorporating fluid flow and shear stress based detachment. Biotechnol Bioeng 103:92–104
Eberl H, Morgenroth E, Noguera D, Picioreanu C, Rittmann B, van Loosdrecht M, Wanner O (2006) Mathematical modeling of biofilms. IWA Publishing, London
Emerenini BO, Hense BA, Kuttler C, Eberl H (2015) A mathematical model of quorum sensing induced biofilm detachment. PLoS ONE 10:e0132385
Fagerlind MG, Webb JS, Barraud N, McDougald D, Jansson A, Nilsson P, Harlén M, Kjelleberg S, Rice S (2012) Dynamic modelling of cell death during biofilm development. J Theor Biol 295:23–36
Falsetta ML, Klein MI, Colonne P, Scott-Anne K, Gregoire S, Pai C, Gonzalez-Begne M, Watson G, Krysan D, Koo Bowen WH (2014) Symbiotic relationship between Streptococcus mutans and Candida albicans synergizes virulence of plaque biofilms in vivo. Infect Immun 82:1968–1981
Fozard JA, Lees M, King J, Logan B (2012) Inhibition of quorum sensing in a computational biofilm simulation. Biosystems 109:105–114
Frederick MR, Kuttler C, Hense BA, Eberl HJ (2011) A mathematical model of quorum sensing regulated eps production in biofilm communities. Theor Biol Med Model 8:8
Fuqua C, Greenberg E (2002) Listening in on bacteria: acyl-homoserine lactone signalling. Nat Rev Mol Cell Biol 3:685–695
Gefen O, Gabay C, Mumcuoglu M, Engel G, Balaban N (2008) Single-cell protein induction dynamics reveals a period of vulnerability to antibiotics in persister bacteria. Proc Natl Acad Sci USA 105:6145–6149
Gefen O, Balaban NQ (2009) The importance of being persistent: heterogeneity of bacterial populations under antibiotic stress. FEMS Microbiol Rev 33:704–717
Grobe KJ, Zahller J, Stewart P (2002) Role of dose concentration in biocide efficacy against Pseudomonas aeruginosa biofilms. J Ind Microbiol Biotechnol 29:10–15
Guo P, Weinstein A, Weinbaum S (2000) A hydrodynamic mechanosensory hypothesis for brush border microvilli. Am J Physiol Renal Physiol 279:F698–F712
Hall-Stoodley L, Costerton J, Stoodley P (2004) Bacterial biofilms: from the natural environment to infectious diseases. Nat Rev Microbiol 2:95–108
Hunter R, Beveridge T (2005) High-resolution visualization of Pseudomonas aeruginosa pao1 biofilms by freeze-substitution transmission electron microscopy. J Bacteriol 187:7619–7630
Janissen R, Murillo DM, Niza B, Sahoo P, Nobrega M, Cesar C, Temperini M, Carvalho H, de Souza A, Cotta M (2015) Spatiotemporal distribution of different extracellular polymeric substances and filamentation mediate Xylella fastidiosa adhesion and biofilm formation. Sci Rep 5:9856
Jayaraman A, Wood T (2008) Bacterial quorum sensing: signals, circuits, and implications for biofilms and disease. Annu Rev Biomed Eng 10:145–167
Jefferson KK, Goldmann D, Pier G (2005) Use of confocal microscopy to analyze the rate of vancomycin penetration through Staphylococcus aureus biofilms. Antimicrob Agents Chemother 49:2467–2473
Keren I, Shah D, Spoering A, Kaldalu N, Lewis K (2004a) Specialized persister cells and the mechanism of multidrug tolerance in Escherichia coli. J Bacteriol 186:8172–8180
Keren I, Kaldalu N, Spoering A, Wang Y, Lewis K (2004b) Persister cells and tolerance to antimicrobials. FEMS Microbiol Lett 230:13–18
Kim J, Hahn JS, Franklin M, Stewart P, Yoon J (2009) Tolerance of dormant and active cells in Pseudomonas aeruginosa pa01 biofilm to antimicrobial agents. J Antimicrob Chemother 63:129–135
Klapper I, Dockery J (2002) Finger formation in biofilm layers. SIAM J Appl Math 62:853–869
Koerber AJ, King JR, Ward J, Williams P, Croft J, Sockett R (2002) A mathematical model of partial-thickness burn-wound infection by Pseudomonas aeruginosa: quorum sensing and the build-up to invasion. Bull Math Biol 64:239–259
Koutsoudis MD, Tsaltas D, Minogue TD, von Bodman SB (2006) Quorum-sensing regulation governs bacterial adhesion, biofilm development, and host colonization in Pantoea stewartii subspecies stewartii. Proc Natl Acad Sci USA 103:5983–5988
Kreft JU, Booth G, Wimpenny J (1998) Bacsim, a simulator for individual-based modelling of bacterial colony growth. Microbiology 144:3275–3287
Kreft JU, Picioreanu C, Wimpenny J, van Loosdrecht M (2001) Individual-based modelling of biofilms. Microbiology 147:2897–2912
Kreft J, Wimpenny J (2001) Effect of eps on biofilm structure and function as revealed by an individual-based model of biofilm growth. Water Sci Technol 43:135–141
Kroukamp O, Dumitrache R, Wolfaardt G (2010) Pronounced effect of the nature of the inoculum on biofilm development in flow systems. Appl Environ Microbiol 76:6025–6031
Kussell E, Kishony R, Balaban N, Leibler S (2005) Bacterial persistence: a model of survival in changing environments. Genetics 169:1807–1814
Langebrakea JB, Dilanji GE, Hagen SJ, De Leenheer P (2014) Traveling waves in response to a diffusing quorum sensing signal in spatially-extended bacterial colonies. J Theor Biol 363:53–61
Lawrence JR, Korber DR, Hoyle B, Costerton J, Caldwell D (1991) Optical sectioning of microbial biofilms. J Bacteriol 173:6558–6567
Leisner M, Kuhr J-T, Rädler JO, Frey E, Maier B (2009) Kinetics of genetic switching into the state of bacterial competence. Biophys J 96:1178–1188
Lewis K (2007) Persister cells, dormancy and infectious disease. Nat Rev Microbiol 5:48–56
Ma L, Conover M, Lu H, Parsek MR, Bayles K, Wozniak DJ (2009) Assembly and development of the Pseudomonas aeruginosa biofilm matrix. PLoS Pathog 5:e1000354
Ma R, Liu J, Y-t Jiang, Liu Z, Z-s Tang, D-x Ye, Zeng J, Z-w Huang (2010) Modeling of diffusion transport through oral biofilms with the inverse problem method. Int J Oral Sci 2:190–197
Matz C, Bergfeld T, Rice S, Kjelleberg S (2004) Microcolonies, quorum sensing and cytotoxicity determine the survival of Pseudomonas aeruginosa biofilms exposed to protozoan grazing. Environ Microbiol 6:218–226
McDougald D, Rice SA, Barraud N, Steinberg PD, Kjelleberg S (2012) Should we stay or should we go: mechanisms and ecological consequences for biofilm dispersal. Nat Rev Microbiol 10:39–50
Mulcahy LR, Burns JL, Lory S, Lewis K (2010) Emergence of Pseudomonas aeruginosa strains producing high levels of persister cells in patients with cystic fibrosis. J Bacteriol 192:6191–6199
Ng W, Bassler B (2009) Bacterial quorum-sensing network architectures. Annu Rev Genet 43:197–222
Nystrom T (2001) Not quite dead enough: on bacterial life, culturability, senescence, and death. Arch Microbiol 176:159–164
Nystrom T (2003) Conditional senescence in bacteria: death of the immortals. Mol Microbiol 48:17–23
Picioreanu C, Van Loosdrecht M, Heijnen J (1998a) Mathematical modeling of biofilm structure with a hybrid differential-discrete cellular automaton approach. Biotechnol Bioeng 58:101–116
Picioreanu C, van Loosdrecht M, Heijnen J (1998b) A new combined differential-discrete cellular automaton approach for biofilm modeling: application for growth in gel beads. Biotechnol Bioeng 57:718–731
Picioreanu C, van Loosdrecht M, Heijnen J (2000) Effect of diffusive and convective substrate transport on biofilm structure formation: a two-dimensional modeling study. Biotechnol Bioeng 69:504–515
Picioreanu C, van Loosdrecht M, Heijnen J (2001) Two-dimensional model of biofilm detachment caused by internal stress from liquid flow. Biotechnol Bioeng 72:205–218
Picioreanu C, Kreft J, Loosdrecht MV (2004) Particle-based multidimensional multispecies biofilm model. Appl Environ Microbiol 70:3024–3040
Pizarro GE, Garcia C, Moreno R, Sepulveda M (2004) Two-dimensional cellular automaton model for mixed-culture biofilm. Water Sci Technol 49:193–198
Postgate J, Hunter J (1962) The survival of starved bacteria. J Gen Microbiol 29:233–263
Potera C (1999) Forging a link between biofilms and disease. Science 283:1837–1839
Pu Y et al (2016) Enhanced efflux activity facilitates drug tolerance in dormant bacterial cells. Mol Cell 62:284–294
Queck SY, Jameson-Lee M, Villaruz A, Bach T, Khan B, Sturdevant D, Ricklefs S, Li M, Otto M (2008) Rnaiii-independent target gene control by the agr quorum-sensing system: insight into the evolution of virulence regulation in Staphylococcus aureus. Mol Cell 32:150–158
Quinones B, Dulla G, Lindow S (2005) Quorum sensing regulates exopolysaccharide production, motility, and virulence in Pseudomonas syringae. Mol Plant Microbe Interact 18:682–693
Rayner MG, Zhang Y, Gorry M, Chen Y, Post J, Ehrlich G (1998) Evidence of bacterial metabolic activity in culture-negative otitis media with effusion. JAMA 279:296–299
Rochex A, Lebeault J (2007) Effects of nutrients on biofilm formation and detachment of a Pseudomonas putida strain isolated from a paper machine. Water Res 41:2885–2892
Rutherford S, Bassler B (2012) Bacterial quorum sensing: its role in virulence and possibilities for its control. Cold Spring Harb Perspect Med 2:a012427
Stewart P (1993) A model of biofilm detachment. Biotechnol Bioeng 41:111–117
Stewart PS, Huang B, Hamilton MA, Hunt SM, Werner EM (2004) Hypothesis for the role of nutrient starvation in biofilm detachment. Appl Environ Microbiol 70:7418–7425
Stewart P, Franklin M (2008) Physiological heterogeneity in biofilms. Nat Rev Microbiol 6:199–210
Stickler DJ, Morris NS, McLean RJC, Fuqua C (1998) Biofilms on indwelling urethral catheters produce quorum-sensing signal molecules in situ and in vitro. Appl Environ Microbiol 64:3486–3490
Stoodley P, Debeer D, Lewandowski Z (1994) Liquid flow in biofilm systems. Appl Environ Microbiol 60:2711–2716
Stoodley P, Sauer K, Davies D, Costerton J (2002) Biofilms as complex differentiated communities. Annu Rev Microbiol 56:435–464
Stoodley P, Dodds I, Boyle J, Lappin-Scott H (2012) Influence of hydrodynamics and nutrients on biofilm structure. J Appl Microbiol 85:19S–28S
Tan CH, Koh KS, Xie C, Tay M, Zhou Y, Williams R, Ng W, Rice S, Kjelleberg S (2014) The role of quorum sensing signalling in eps production and the assembly of a sludge community into aerobic granules. ISME J 8:1186–1197
Tseng BS, Zhang W, Harrison J, Quach T, Song J, Penterman J, Singh P, Chopp D, Packman A, Parsek M (2013) The extracellular matrix protects Pseudomonas aeruginosa biofilms by limiting the penetration of tobramycin. Environ Microbiol 15:2865–2878
von Bodman SB, Majerczak D, Coplin D (1998) Quorum-sensing regulation governs bacterial adhesion, biofilm development, and host colonization in Pantoea stewartii subspecies stewartii. Proc Natl Acad Sci USA 95:7687–7692
Wanner O, Gujer W (1986) A multispecies biofilm model. Biotechnol Bioeng 28:314–328
Ward KH, Olson ME, Lam K, Costerton J (1992) Mechanism of persistent infection associated with peritoneal implants. J Med Microbiol 36:406–413
Yang X, Beyenal H, Harkin G, Lewandowski Z (2000) Quantifying biofilm structure using image analysis. J Microbiol Methods 39:109–119
Acknowledgements
This work was supported by the Start-Up Research Grant (No. SB/YS/LS-210/2013), Science and Engineering Research Board, India.
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Machineni, L., Rajapantul, A., Nandamuri, V. et al. Influence of Nutrient Availability and Quorum Sensing on the Formation of Metabolically Inactive Microcolonies Within Structurally Heterogeneous Bacterial Biofilms: An Individual-Based 3D Cellular Automata Model. Bull Math Biol 79, 594–618 (2017). https://doi.org/10.1007/s11538-017-0246-9
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DOI: https://doi.org/10.1007/s11538-017-0246-9