Bacterial populations produce a small number of dormant persister cells that exhibit multidrug tolerance. All resistance mechanisms do essentially the same thing: prevent the antibiotic from hitting a target. By contrast, tolerance apparently works by shutting down the targets. Bactericidal antibiotics kill bacteria by corrupting their targets, rather than merely inhibiting them. Shutting down the targets then protects from killing. The number of persisters in a growing population of bacteria rises at mid-log and reaches a maximum of approximately 1% at stationary state. Similarly, slow-growing biofilms produce substantial numbers of persisters. The ability of a biofilm to limit the access of the immune system components, and the ability of persisters to sustain an antibiotic attack could then account for the recalcitrance of such infections in vivo and for their relapsing nature. Isolation of Escherichia coli persisters by lysing a population or by sorting GFP-expressing cells with diminished translation allowed to obtain a gene expression profile. The profile indicated downregulated biosynthetic pathways, consistent with their dormant nature, and indicated overexpression of toxin/antitoxin (TA) modules. Stochastic overexpression of toxins that inhibit essential functions such as translation may contribute to persister formation. Ectopic expression of RelE, MazF, and HipA toxins produced multidrug tolerant cells. Apart from TA modules, glpD and plsB were identified as potential persister genes by overexpression cloning of a genomic library and selection for antibiotic tolerance. Yeast Candida albicans forms recalcitrant biofilm infections that are tolerant to antibiotics, similarly to bacterial biofilms. C. albicans biofilms produce multidrug tolerant persisters that are not mutants, but rather phenotypic variants of the wild type. Unlike bacterial persisters, however, C. albicans persisters were only observed in a biofilm, but not in a planktonic stationary population. Identification of persister genes opens the way to a rational design of anti-biofilm therapy. Combination of a conventional antibiotic with a compound inhibiting persister formation or maintenance may produce an effective therapeutic. Other approaches to the problem include sterile-surface materials, prodrug antibiotics, and cyclical application of conventional antimicrobials.
Access provided by Autonomous University of Puebla. Download to read the full chapter text
Chapter PDF
We’re sorry, something doesn't seem to be working properly.
Please try refreshing the page. If that doesn't work, please contact support so we can address the problem.
Keywords
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.
References
Avery SV (2006) Microbial cell individuality and the underlying sources of heterogeneity. Nat Rev Microbiol 4:577–587
Baillie GS, Douglas LJ (2000) Matrix polymers of Candida biofilms and their possible role in biofilm resistance to antifungal agents. J Antimicrob Chemother 46:397–403
Balaban NQ, Merrin J, Chait R, Kowalik L, Leibler S (2004) Bacterial persistence as a phenotypic switch. Science 305:1622–1625
Bayles KW (2000) The bactericidal action of penicillin: new clues to an unsolved mystery. Trends Microbiol 8:274–278
Belofsky G, Percivill D, Lewis K, Tegos GP, Ekart J (2004) Phenolic metabolites of Dalea versicolor that enhance antibiotic activity against model pathogenic bacteria. J Nat Prod 67:481–484
Belofsky G, Carreno R, Lewis K, Ball A, Casadei G, Tegos GP (2006) Metabolites of the smoke tree, Dalea spinosa, potentiate antibiotic activity against multidrug-resistant Staphylococcus aureus. J Nat Prod 69:261–264
Berg HC, Brown DA (1972) Chemotaxis in Escherichia coli analysed by three-dimensional tracking. Nature 239:500–504
Bigger JW (1944) Treatment of staphylococcal infections with penicillin. Lancet ii:497–500
Black DS, Kelly AJ, Mardis MJ, Moyed HS (1991) Structure and organization of hip, an operon that affects lethality due to inhibition of peptidoglycan or DNA synthesis. J Bacteriol 173:5732–5739
Black DS, Irwin B, Moyed HS (1994) Autoregulation of hip, an operon that affects lethality due to inhibition of peptidoglycan or DNA synthesis. J Bacteriol 176:4081–4091
Boggs AF, Miller GH (2004) Antibacterial drug discovery: is small pharma the solution? Clin Microbiol Infect 10 [Suppl 4]:32–36
Boltner D, MacMahon C, Pembroke JT, Strike P, Osborn AM (2002) R391: a conjugative integrating mosaic comprised of phage, plasmid, and transposon elements. J Bacteriol 184:5158–5169
Brooun A, Liu S, Lewis K (2000) A dose-response study of antibiotic resistance in Pseudomonas aeruginosa biofilms. Antimicrob Agents Chemother 44:640–646
Bush K (2004) Antibacterial drug discovery in the 21st century. Clin Microbiol Infect 10 [Suppl 4]: 10–17
Cao F, Lane S, Raniga PP, Lu Y, Zhou Z, Ramon K, Chen J, Liu H (2006) The Flo8 transcription factor is essential for hyphal development and virulence Candida albicans. Mol Biol Cell 17:295–307
Chandra J, Kuhn DM, Mukherjee PK, Hoyer LL, McCormick T, Ghannoum MA (2001) Biofilm formation by the fungal pathogen Candida albicans: development, architecture, and drug resistance. J Bacteriol 183:5385–5394
Christensen SK, Gerdes K (2003) RelE toxins from bacteria and Archaea cleave mRNAs on translating ribosomes, which are rescued by tmRNA. Mol Microbiol 48:1389–1400
Christensen SK, Pedersen K, Hansen FG, Gerdes K (2003) Toxin-antitoxin loci as stress-response-elements: ChpAK/MazF and ChpBK cleave translated RNAs and are counteracted by tmRNA. J Mol Biol 332:809–819
Chung JD, Stephanopoulos G, Ireton K, Grossman AD (1994) Gene expression in single cells of Bacillus subtilis: evidence that a threshold mechanism controls the initiation of sporulation. J Bacteriol 176:1977–1984
Clardy J, Walsh C (2004) Lessons from natural molecules. Nature 432:829–837
Correia FF, D’Onofrio A, Rejtar T, Li L, Karger BL, Makarova K, Koonin EV, Lewis K (2006) Kinase activity of overexpressed HipA is required for growth arrest and multidrug tolerance in Escherichia coli. J Bacteriol 188:8360–8367
Datry A, Bart-Delabesse E (2006) Caspofungin: mode of action and therapeutic applications. Rev Med Interne 27:32–39
Davis BD, Chen LL, Tai PC (1986) Misread protein creates membrane channels: an essential step in the bactericidal action of aminoglycosides. Proc Natl Acad Sci U S A 83:6164–6168
Demain AL, Fang A (2000) The natural functions of secondary metabolites. In: Fiechter IA (ed) History of modern biotechnology. Springer, Berlin New York Heidelberg, pp 1–39
Denyer SP, Hugo WB (1991) Mechanisms of action of chemical biocides: their study and exploitation. Society for Applied Bacteriology
Dougherty TJ, Barrett JF, Pucci MJ (2002) Microbial genomics and novel antibiotic discovery: new technology to search for new drugs. Curr Pharm Des 8:1119–1135
Dubnau D, Losick R (2006) Bistability in bacteria. Mol Microbiol 61:564–572
Falla TJ, Chopra I (1998) Joint tolerance to beta-lactam and fluoroquinolone antibiotics in Escherichia coli results from overexpression of hipA. Antimicrob Agents Chemother 42:3282–3284
Garcia-Sanchez S, Aubert S, Iraqui I, Janbon G, Ghigo JM, d’Enfert C (2004) Candida albicans biofilms: a developmental state associated with specific and stable gene expression patterns. Eukaryot Cell 3:536–545
Gerdes K, Rasmussen PB, Molin S (1986) Unique type of plasmid maintenance function: postsegregational killing of plasmid-free cells. Proc Natl Acad Sci U S A 83:3116–3120
Gerdes K, Christensen SK, Lobner-Olesen A (2005) Prokaryotic toxin-antitoxin stress response loci. Nat Rev Microbiol 3:371–382
Gonzalez-Pastor JE, Hobbs EC, Losick R (2003) Cannibalism by sporulating bacteria. Science 301:510–513
Guz NR, Stermitz FR, Johnson JB, Beeson TD, Wilen S, Hsiang J-F, Lewis K (2001) Flavonolignan and flavone inhibitors of a Staphylococcus aureus multidrug resistance (MDR) pump. Structure-activity relationships. J Med Chem 44:261–268
Hall-Stoodley L, Costerton JW, Stoodley P (2004) Bacterial biofilms: from the natural environment to infectious diseases. Nat Rev Microbiol 2:95–108
Harrison JJ, Ceri H, Roper NJ, Badry EA, Sproule KM, Turner RJ (2005a) Persister cells mediate tolerance to metal oxyanions in Escherichia coli. Microbiology 151:3181–3195
Harrison JJ, Turner RJ, Ceri H (2005b) Persister cells, the biofilm matrix and tolerance to metal cations in biofilm and planktonic Pseudomonas aeruginosa. Environ Microbiol 7:981–994
Hayes F (2003) Toxins-antitoxins: plasmid maintenance, programmed cell death, and cell cycle arrest. Science 301:1496–1499
Heath RJ, Rock CO (1999) A missense mutation accounts for the defect in the glycerol-3-phosphate acyltransferase expressed in the plsB26 mutant. J Bacteriol 181:1944–1946
Hegde SS, Vetting MW, Roderick SL, Mitchenall LA, Maxwell A, Takiff HE, Blanchard JS (2005) A fluoroquinolone resistance protein from Mycobacterium tuberculosis that mimics DNA. Science 308:1480–1483
Hooper DC (2002) Target modification as a mechanism of antimicrobial resistance. In: Lewis K, Salyers A, Taber H and Wax R (eds) Bacterial resistance to antimicrobials: mechanisms genetics medical practice and public health. Marcell Dekker, New York, pp 161–192
Hoyle BD, Jass J, Costerton JW (1990) The biofilm glycocalyx as a resistance factor. J Antimicrob Chemother 26:1–5
Hsieh PC, Siegel SA, Rogers B, Davis D, Lewis K (1998) Bacteria lacking a multidrug pump: a sensitive tool for drug discovery. Proc Natl Acad Sci U S A 95:6602–6606
Hu Y, Coates AR (2005) Transposon mutagenesis identifies genes which control antimicrobial drug tolerance in stationary-phase Escherichia coli. FEMS Microbiol Lett 243:117–124
Jennings BR, Ridler PJ (1983) Interaction of chromosomal stains with DNA. An electrofluorescence study. Biophys Struct Mech 10:71–79
Jesaitis AJ, Franklin MJ, Berglund D, Sasaki M, Lord CI, Bleazard JB, Duffy JE, Beyenal H, Lewandowski Z (2003) Compromised host defense on Pseudomonas aeruginosa biofilms: characterization of neutrophil and biofilm interactions. J Immunol 171:4329–4339
Kaeberlein T, Lewis K, Epstein SS (2002) Isolating uncultivable microorganisms in pure culture in a simulated natural environment. Science 296:1127–1129
Kaern M, Elston TC, Blake WJ, Collins JJ (2005) Stochasticity in gene expression: from theories to phenotypes. Nat Rev Genet 6:451–464
Keren I, Kaldalu N, Spoering A, Wang Y, Lewis K (2004a) Persister cells and tolerance to antimicrobials. FEMS Microbiol Lett 230:13–18
Keren I, Shah D, Spoering A, Kaldalu N, Lewis K (2004b) Specialized persister cells and the mechanism of multidrug tolerance in Escherichia coli. J Bacteriol 186:8172–8180
Korch SB, Hill TM (2006) Ectopic overexpression of wild-type and mutant hipA genes in Escherichia coli: effects on macromolecular synthesis and persister formation. J Bacteriol 188:3826–3836
Korch SB, Henderson TA, Hill TM (2003) Characterization of the hipA7 allele of Escherichia coli and evidence that high persistence is governed by (p) ppGpp synthesis. Mol Microbiol 50:1199–1213
Korobkova E, Emonet T, Vilar JM, Shimizu TS, Cluzel P (2004) From molecular noise to behavioural variability in a single bacterium. Nature 428:574–578
Kumamoto CA (2005) A contact-activated kinase signals Candida albicans invasive growth and biofilm development. Proc Natl Acad Sci U S A 102:5576–5581
Kumamoto CA, Vinces MD (2005) Alternative Candida albicans lifestyles: growth on surfaces. Annu Rev Microbiol 59:113–133
LaFleur MD, Kumamoto CA, Lewis K (2006) Candida albicans biofilms produce antifungal-tolerant persister cells. Antimicrob Agents Chemother 50:3839–3846
Lee SB, Koepsel RR, Morley SW, Matyjaszewski K, Sun Y, Russell AJ (2004) Permanent, nonleaching antibacterial surfaces. 1. Synthesis by atom transfer radical polymerization. Biomacromolecules 5:877–882
Leid JG, Shirtliff ME, Costerton JW, Stoodley AP (2002) Human leukocytes adhere to, penetrate, and respond to Staphylococcus aureus biofilms. Infect Immun 70:6339–6345
Levin BR, Rozen DE (2006) Non-inherited antibiotic resistance. Nat Rev Microbiol 4:556–562
Levy SB, Marshall B (2004) Antibacterial resistance worldwide: causes, challenges and responses. Nat Med 10:S122–S129
Lewis K (2000) Programmed death in bacteria. Microbiol Mol Biol Rev 64:503–514
Lewis K (2001a) In search of natural substrates and inhibitors of MDR pumps. J Mol Microbiol Biotechnol 3:247–254
Lewis K (2001b) Riddle of biofilm resistance. Antimicrob Agents Chemother 45:999–1007
Lewis K, Klibanov AM (2005) Surpassing nature: rational design of sterile-surface materials. Trends Biotechnol 23:343–348
Lewis K, Lomovskaya O (2002) Drug efflux. In: Lewis K, Salyers A, Taber H and Wax R (eds) Bacterial resistance to antimicrobials: mechanisms genetics medical practice and public health. Marcel Dekker, New York, pp 61–90
Lewis K, Salyers A, Taber H, Wax R (2002) Bacterial resistance to antimicrobials: mechanisms genetics medical practice and public health. Marcel Dekker, New York
Lewis K, Spoering A, Kaldalu N, Keren I, Shah D (2005) Persisters: specialized cells responsible for biofilm tolerance to antimicrobial agents. In: Pace J, Rupp ME and Finch RG (eds) Biofilms infection, and antimicrobial therapy. Taylor & Francis, Boca Raton, pp 241–256
Li XZ, Nikaido H (2004) Efflux-mediated drug resistance in bacteria. Drugs 64:159–204
Lin J, Tiller JC, Lee SB, Lewis K, Klibanov AM (2002) Insights into bactericidal action of surface-attached poly(vinyl-N-hexylpyridinium) chains. Biotechnol Lett 24:801–805
Lin J, Qiu S, Lewis K, Klibanov AM (2003) Mechanism of bactericidal and fungicidal activities of textiles covalently modified with alkylated polyethylenimine. Biotechnol Bioeng 83:168–172
Lomovskaya O, Bostian KA (2006) Practical applications and feasibility of efflux pump inhibitors in the clinic - a vision for applied use. Biochem Pharmacol 71:910–918
Mack D, Becker P, Chatterjee I, Dobinsky S, Knobloch JK, Peters G, Rohde H, Herrmann M (2004) Mechanisms of biofilm formation in Staphylococcus epidermidis and Staphylococcus aureus: functional molecules, regulatory circuits, and adaptive responses. Int J Med Microbiol 294:203–212
Markham PN, Neyfakh AA (1996) Inhibition of the multidrug transporter NorA prevents emergence of norfloxacin resistance in Staphylococcus aureus. Antimicrob Agents Chemother 40:2673–2674
Markham PN, Westhaus E, Klyachko K, Johnson ME, Neyfakh AA (1999) Multiple novel inhibitors of the NorA multidrug transporter of Staphylococcus aureus. Antimicrob Agents Chemother 43:2404–2408
McCool JD, Long E, Petrosino JF, Sandler HA, Rosenberg SM, Sandler SJ (2004) Measurement of SOS expression in individual Escherichia coli K-12 cells using fluorescence microscopy. Mol Microbiol 53:1343–1357
Meyer AL (2005) Prospects and challenges of developing new agents for tough Gram-negatives. Curr Opin Pharmacol 5:490–494
Milovic NM, Wang J, Lewis K, Klibanov AM (2005) Immobilized N-alkylated polyethylenimine avidly kills bacteria by rupturing cell membranes with no resistance developed. Biotechnol Bioeng 90:715–722
Morel C, Stermitz FR, Tegos G, Lewis K (2003) Isoflavone MDR efflux pump inhibitors from Lupinus argenteus. Synergism between some antibiotics and isoflavones. J Agricult Food Chem 51:5677–5679
Morgan HC, Meier JF, Merker RL (2000) Method of creating a biostatic agent using interpenetrating network polymers. US Patent No. 6, 146, 688
Moyed HS, Bertrand KP (1983) hipA, a newly recognized gene of Escherichia coli K-12 that affects frequency of persistence after inhibition of murein synthesis. J Bacteriol 155:768–775
Moyed HS, Broderick SH (1986) Molecular cloning and expression of hipA, a gene of Escherichia coli K-12 that affects frequency of persistence after inhibition of murein synthesis. J Bacteriol 166:399–403
Mukherjee PK, Chandra J, Kuhn DM, Ghannoum MA (2003) Mechanism of fluconazole resistance in Candida albicans biofilms: phase-specific role of efflux pumps and membrane sterols. Infect Immun 71:4333–4340
Osburne MS, Grossman TH, August PR, MacNeil IA (2000) Tapping into microbial diversity for natural products drug discovery. ASM News 66:411–417
Pedersen K, Christensen SK, Gerdes K (2002) Rapid induction and reversal of a bacteriostatic condition by controlled expression of toxins and antitoxins. Mol Microbiol 45:501–510
Projan SJ, Shlaes DM (2004) Antibacterial drug discovery: is it all downhill from here? Clin Microbiol Infect 10 [Suppl 4]:18–22
Ramage G, Bachmann S, Patterson TF, Wickes BL, Lopez-Ribot JL (2002) Investigation of multidrug efflux pumps in relation to fluconazole resistance in Candida albicans biofilms. J Antimicrob Chemother 49:973–980
Samaranayake YH, Ye J, Yau JY, Cheung BP, Samaranayake LP (2005) In vitro method to study antifungal perfusion in Candida biofilms. J Clin Microbiol 43:818–825
Sat B, Hazan R, Fisher T, Khaner H, Glaser G, Engelberg-Kulka H (2001) Programmed cell death in Escherichia coli: some antibiotics can trigger mazEF lethality. J Bacteriol 183:2041–2045
Schatz A, Bugie E, Waksman SA (1944) Streptomycin, a substance exhibiting antibiotic activity against Gram-positive and Gram-negative bacteria. Proc Soc Exp Biol Med 55:66–69
Scherrer R, Moyed HS (1988) Conditional impairment of cell division and altered lethality in hipA mutants of Escherichia coli K-12. J Bacteriol 170:3321–3326
Schmelzle T, Hall MN (2000) TOR, a central controller of cell growth. Cell 103:253–262
Schmid MB (2004) Seeing is believing: the impact of structural genomics on antimicrobial drug discovery. Nat Rev Microbiol 2:739–746
Severina II, Muntyan MS, Lewis K, Skulachev VP (2001) Transfer of cationic antibacterial agents berberine, palmatine and benzalkonium through bimolecular planar phospholipid film and Staphylococcus aureus membrane. IUBMB Life Sciences 52:321–324
Shah DV, Zhang Z, Kurg K, Kaldalu N, Khodursky A, Lewis K (2006) Persisters: a distinct physiological state of E. coli. BMC Microbiol 6:53
Silver LL (2006) Antibacterial drug discovery and development - SRI’s 11th Annual Summit. Antibacterial trends and current research. IDrugs 9:394–397
Singh PK, Schaefer AL, Parsek MR, Moninger TO, Welsh MJ, Greenberg EP (2000) Quorum-sensing signals indicate that cystic fibrosis lungs are infected with bacterial biofilms. Nature 407:762–764
Spoering AL, Lewis K (2001) Biofilms and planktonic cells of Pseudomonas aeruginosa have similar resistance to killing by antimicrobials. J Bacteriol 183:6746–6751
Spoering AL, Vulic M, Lewis K (2006) GlpD and PlsB participate in persister cell formation in Escherichia coli. J Bacteriol 188:5136–5144
Spudich JL, Koshland DE Jr (1976) Non-genetic individuality: chance in the single cell. Nature 262:467–471
Stermitz FR, Lorenz P, Tawara JN, Zenewicz L, Lewis K (2000a) Synergy in a medicinal plant: antimicrobial action of berberine potentiated by 5’-methoxyhydnocarpin, a multidrug pump inhibitor. Proc Natl Acad Sci U S A 97:1433–1437
Stermitz FR, Scriven LN, Tegos G, Lewis K (2002) Two flavonols from Artemisia annua which potentiate the activity of berberine and norfloxacin against a resistant strain of Staphylococcus aureus. Planta Med 68:1140–1141
Stermitz FR, Cashman KK, Halligan KM, Morel C, Tegos GP, Lewis K (2003) Polyacylated neohesperidosides from Geranium caespitosum: bacterial multidrug resistance pump inhibitors. Bioorg Med Chem Lett 13:1915–1918
Tegos G, Stermitz FR, Lomovskaya O, Lewis K (2002) Multidrug pump inhibitors uncover the remarkable activity of plant antimicrobials. Antimicrob Agents Chemother 46:3133–3141
Tiller JC, Liao CJ, Lewis K, Klibanov AM (2001) Designing surfaces that kill bacteria on contact. Proc Natl Acad Sci U S A 98:5981–5985
Tran JH, Jacoby GA (2002) Mechanism of plasmid-mediated quinolone resistance. Proc Natl Acad Sci U S A 99:5638–5642
Vazquez-Laslop N, Lee H, Neyfakh AA (2006) Increased persistence in Escherichia coli caused by controlled expression of toxins or other unrelated proteins. J Bacteriol 188:3494–3497
Vetting MW, Hegde SS, Fajardo JE, Fiser A, Roderick SL, Takiff HE, Blanchard JS (2006) Pentapeptide repeat proteins. Biochemistry 45:1–10
Vilcheze C, Weisbrod TR, Chen B, Kremer L, Hazbon MH, Wang F, Alland D, Sacchettini JC, Jacobs WR Jr (2005) Altered NADH/NAD+ ratio mediates coresistance to isoniazid and ethionamide in mycobacteria. Antimicrob Agents Chemother 49:708–720
Von Eiff C, Heilmann C, Peters G (1999) New aspects in the molecular basis of polymer-associated infections due to staphylococci. Eur J Clin Microbiol Infect Dis 18:843–846
Vuong C, Voyich JM, Fischer ER, Braughton KR, Whitney AR, DeLeo FR, Otto M (2004) Polysaccharide intercellular adhesin (PIA) protects Staphylococcus epidermidis against major components of the human innate immune system. Cell Microbiol 6:269–275
Walker GC (1996) The SOS response of Escherichia coli. In: Neidhardt FC (ed) Escherichia coli and Samonella. Cellular and molecular biology. ASM Press, Washington DC, pp 1400–1416
Walsh C (2003a) Antibiotics. Actions, origins, resistance. ASM Press, Washington DC
Walsh C (2003b) Where will new antibiotics come from? Nat Rev Microbiol 1:65–70
Wiuff C, Zappala RM, Regoes RR, Garner KN, Baquero F, Levin BR (2005) Phenotypic tolerance: antibiotic enrichment of noninherited resistance in bacterial populations. Antimicrob Agents Chemother 49:1483–1494
Yoshida H, Maki Y, Kato H, Fujisawa H, Izutsu K, Wada C, Wada A (2002) The ribosome modulation factor (RMF) binding site on the 100S ribosome of Escherichia coli. J Biochem 132:983–989
Zurenko GE, Ford CW, Hutchinson DK, Brickner SJ, Barbachyn MR (1997) Oxazolidinone antibacterial agents: development of the clinical candidates eperezolid and linezolid. Expert Opin Investig Drugs 6:151–158
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2008 Springer-Verlag Berlin Heidelberg
About this chapter
Cite this chapter
Lewis, K. (2008). Multidrug Tolerance of Biofilms and Persister Cells. In: Romeo, T. (eds) Bacterial Biofilms. Current Topics in Microbiology and Immunology, vol 322. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-540-75418-3_6
Download citation
DOI: https://doi.org/10.1007/978-3-540-75418-3_6
Publisher Name: Springer, Berlin, Heidelberg
Print ISBN: 978-3-540-75417-6
Online ISBN: 978-3-540-75418-3
eBook Packages: Biomedical and Life SciencesBiomedical and Life Sciences (R0)