Abstract
Switching between metabolically active and dormant states provides bacteria with protection from environmental stresses and allows rapid growth under favorable conditions. This rapid growth can be detrimental to the environment, e.g., pathogens in recreational lakes, or to industrial processes, e.g., fermentation, making it useful to quickly determine when the ratio of dormant to metabolically active bacteria changes. While a rapid increase in metabolically active bacteria can cause complications, a high number of dormant bacteria can also be problematic, since they can be more virulent and antibiotic-resistant. To determine the metabolic state of Escherichia coli and Salmonella Typhimurium, we developed two paper-based colorimetric assays. The color changes were based on oxidoreductases reducing tetrazolium salts to formazans, and alkaline phosphatases cleaving phosphates from nitrophenyl phosphate salt. Specifically, we added iodophenyl-nitrophenyl-phenyl tetrazolium salt (INT) and methylphenazinium methyl sulfate to metabolically active bacteria on paper and INT and para-nitrophenyl phosphate salt to dormant bacteria on paper. The color changed in less than 60 min and was generally visible at 103 CFU and quantifiable at 106 CFU. The color changes occurred in both bacteria, since oxidoreductases and alkaline phosphatases are common bacterial enzymes. On one hand, this feature makes the assays suitable to a wide range of applications, on the other, it requires specific capture, if only one type of bacterium is of interest. We captured Salmonella or E. coli with immobilized P22 or T4 bacteriophages on the paper, before detecting them at levels of 102 or 104 CFU, respectively. Determining the ratio of the metabolic state of bacteria or a specific bacterium at low cost and in a short time, makes this methodology useful in environmental, industrial and health care settings.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Explore related subjects
Discover the latest articles and news from researchers in related subjects, suggested using machine learning.References
Achbergerová L, Nahálka J, Lartigue C, Noskov V, Chuang R, Algire M, Benders G, Montague M, Ma L, Moodie M, Merryman C, Vashee S, Krishnakumar R, Assad-Garcia N, Andrews-Pfannkoch C, Denisova E, Young L, Qi Z, Segall-Shapiro T, Calvey C, Parmar P, Hutchison C, Smith H, Venter J (2011) Polyphosphate—an ancient energy source and active metabolic regulator. Microb Cell Factories 10:63. https://doi.org/10.1186/1475-2859-10-63
Adkins JA, Boehle K, Friend C, Chamberlain B, Bisha B, Henry CS (2017) Colorimetric and electrochemical bacteria detection using printed paper- and transparency-based analytic devices. Anal Chem 89:3613–3621. https://doi.org/10.1021/acs.analchem.6b05009
Almaas E, Kovács B, Vicsek T, Oltvai ZN, Barabási A-L (2004) Global organization of metabolic fluxes in the bacterium Escherichia coli. Nature 427:839–843. https://doi.org/10.1038/nature02289
Anany H, Chen W, Pelton R, Griffiths MW (2011) Biocontrol of Listeria monocytogenes and Escherichia coli O157:H7 in meat by using phages immobilized on modified cellulose membranes. Appl Environ Microbiol 77:6379–6387. https://doi.org/10.1128/AEM.05493-11
Ayyash S, Wu WI, Ravi Selvaganapathy P (2014) Fast and inexpensive detection of bacterial viability and drug resistance through metabolic monitoring. In: 2014 I.E. healthcare innovation conference. HIC 2014:22–25
Becker D, Selbach M, Rollenhagen C, Ballmaier M, Meyer TF, Mann M, Bumann D (2006) Robust Salmonella metabolism limits possibilities for new antimicrobials. Nature 440:303–307. https://doi.org/10.1038/nature04616
Berridge MV, Herst PM, Tan AS (2005) Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction. Biotechnol Annu Rev 11:127–152. https://doi.org/10.1016/S1387-2656(05)11004-7
Bessey OA, Lowry OH, Brock MJ (1946) A method for the rapid determination of alkaline phosphates with five cubic millimeters of serum. J Biol Chem 164:321–329
Blake MS, Johnston KH, Russell-Jones GJ, Gotschlich EC (1984) A rapid, sensitive method for detection of alkaline phosphatase-conjugated anti-antibody on western blots. Anal Biochem 136:175–179. https://doi.org/10.1016/0003-2697(84)90320-8
Cademartiri R, Anany H, Gross I, Bhayani R, Griffiths M, Brook MA (2010) Immobilization of bacteriophages on modified silica particles. Biomaterials 31:1904–1910. https://doi.org/10.1016/j.biomaterials.2009.11.029
Carrilho E, Martinez AW, Whitesides GM (2009) Understanding wax printing: a simple micropatterning process for paper-based microfluidics. Anal Chem 81:7091–7095. https://doi.org/10.1021/ac901071p
Clegg SJ, Jia W, Cole JA (2006) Role of the Escherichia coli nitrate transport protein, NarU, in survival during severe nutrient starvation and slow growth. Microbiology 152:2091–2100. https://doi.org/10.1099/mic.0.28688-0
Clokie MRJ, Kropinski AM (2009) Bacteriophages: methods and protocols, Methods in molecular biology, p 307
Coates ARM (2003) Dormancy and low-growth states in microbial disease 3:289
Fernandes E, Martins VC, Nóbrega C, Carvalho CM, Cardoso FA, Cardoso S, Dias J, Deng D, Kluskens LD, Freitas PP, Azeredo J (2014) A bacteriophage detection tool for viability assessment of Salmonella cells. Biosens Bioelectron 52:239–246. https://doi.org/10.1016/j.bios.2013.08.053
Fiksdal L, Pommepuy M, Caprais MP, Midttun I (1994) Monitoring of fecal pollution in coastal waters by use of rapid enzymatic techniques. Appl Environ Microbiol 60:1581–1584
Gómez R, Bashir R, Bhunia AK (2002) Microscale electronic detection of bacterial metabolism. Sensors Actuators B Chem 86:198–208. https://doi.org/10.1016/S0925-4005(02)00175-2
Hallegraeff GM (1993) On the global increase of harmful algal bloom. Wetl 12:2–15
Hatzinger PB, Palmer P, Smith RL, Peñarrieta CT, Yoshinari T (2003) Applicability of tetrazolium salts for the measurement of respiratory activity and viability of groundwater bacteria. J Microbiol Methods 52(1):47–58. https://doi.org/10.1016/S0167-7012(02)00132-X
Hu J, Wang S, Wang L, Li F, Pingguan-Murphy B, TJ L, Xu F (2014) Advances in paper-based point-of-care diagnostics. Biosens Bioelectron 54:585–597. https://doi.org/10.1016/j.bios.2013.10.075
Jokerst JC, Adkins JA, Bisha B, Mentele MM, Goodridge LD, Henry CS (2012) Development of a paper-based analytical device for colorimetric detection of select foodborne pathogens. Anal Chem 84:2900–2907. https://doi.org/10.1021/ac203466y
Jones K (2001) Campylobacters in water, sewage and the environment. J Appl Microbiol 90:68S–79S. https://doi.org/10.1046/j.1365-2672.2001.01355.x
Ke C-J, He Y-H, He H-W, Yang X, Li R, Yuan J, Koike M, Reed LJ, Carroll WR, Neveling U, Klasen R, Bringer-Meyer S, He H-W, Yuan J-L, Peng H, Ciszak EM, Korotchkina LG, Dominiak PM, Schwartz ER, Reed LJ, Maldonado ME, Oh K-JA, Frey PA, Nemeria N, Yan Y, Zhang Z, Khailova LS, Bernkhardt R, Khiubner G, Sterk JP, William CS, Charles LH, Reid EE, Thompson P, Lyttle CR, Hinmant LM, Blass JP, Koike M, Shah PC, Reed LJ, Milne JLS, Shi D, Berg A, Westphal AH, Bosma HJ, Nemeria N, Volkov A, Brown A, Thelen JJ, Miernyk JA, Randall DD, Duggleby RG, Dennis DT, Thompson P, Reid EE, Lyttle CR, Harris RA, Bowker-Kinley MM, Huang B, Shen LC, Atkinson DE, Guo K, Qian R, Schwab MA, Kölker S, van den HLP (2014) A new spectrophotometric assay for measuring pyruvate dehydrogenase complex activity: a comparative evaluation. Anal Methods 6:6381. https://doi.org/10.1039/C4AY00804A
Lakshmanan RS, Guntupalli R, Hu J, Kim D-J, Petrenko VA, Barbaree JM, Chin BA (2007) Phage immobilized magnetoelastic sensor for the detection of Salmonella Typhimurium. J Microbiol Methods 71:55–60. https://doi.org/10.1016/j.mimet.2007.07.012
Lazcka O, Del CFJ, Muñoz FX (2007) Pathogen detection: a perspective of traditional methods and biosensors. Biosens Bioelectron 22:1205–1217. https://doi.org/10.1016/j.bios.2006.06.036
Lennon JT, Jones SE (2011) Microbial seed banks: the ecological and evolutionary implications of dormancy. Nat Rev Microbiol 9:119–130. https://doi.org/10.1038/nrmicro2504
Lewis K (2007) Persister cells, dormancy and infectious disease. Nat Rev Microbiol 5:48–56. https://doi.org/10.1038/nrmicro1557
Li L, Mendis N, Trigui H, Oliver JD, Faucher SP (2014) The importance of the viable but non-culturable state in human bacterial pathogens. Front Microbiol 5:258. https://doi.org/10.3389/fmicb.2014.00258
Li S, Li Y, Chen H, Horikawa S, Shen W, Simonian A, Chin BA (2010) Direct detection of Salmonella Typhimurium on fresh produce using phage-based magnetoelastic biosensors. Biosens Bioelectron 26:1313–1319. https://doi.org/10.1016/j.bios.2010.07.029
Martinez AW, Phillips ST, Butte MJ, Whitesides GM (2007) Patterned paper as a platform for inexpensive, low-volume, portable bioassays. Angew Chemie - Int Ed 46:1318–1320. https://doi.org/10.1002/anie.200603817
Murphree CA, Heist EP, Moe LA (2014) Antibiotic resistance among cultured bacterial isolates from bioethanol fermentation facilities across the United States. Curr Microbiol 69:277–285. https://doi.org/10.1007/s00284-014-0583-y
Nie Z, Nijhuis CA, Gong J, Chen X, Kumachev A, Martinez AW, Narovlyansky M, Whitesides GM (2010) Electrochemical sensing in paper-based microfluidic devices. Lab Chip 10:477–483. https://doi.org/10.1039/b917150a
Ozkanca R, Flint KP (1997) Relationship between respiratory enzymes and survival of Escherichia coli under starvation stress in lake water. J Appl Microbiol 82:301–309
Ozkanca R, Flint KP (1996) Alkaline phosphatase activity of Escherichia coli starved in sterile lake water microcosms. J Appl Bacteriol 80:252–258
Pelton R (2009) Bioactive paper provides a low-cost platform for diagnostics. TrAC - Trends Anal Chem 28:925–942. https://doi.org/10.1016/j.trac.2009.05.005
Poncet S, Milohanic E, Mazé A, Nait Abdallah J, Aké F, Larribe M, Deghmane A-E, Taha M-K, Dozot M, De Bolle X, Letesson JJ, Deutscher J (2009) Correlations between carbon metabolism and virulence in bacteria. Contrib Microbiol 16:88–102. https://doi.org/10.1159/000219374
Rodriguez GG, Phipps D, Ishiguro K, Ridgway HF (1992) Use of a fluorescent redox probe for direct visualization of actively respiring bacteria. Appl Environ Microbiol 58:1801–1808
Rompré A, Servais P, Baudart J, de-Roubin MR, Laurent P (2002) Detection and enumeration of coliforms in drinking water: current methods and emerging approaches. J Microbiol Methods 49:31–54
Smith JJ, McFeters GA (1997) Mechanisms of INT (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride), and CTC (5-cyano-2,3-ditolyl tetrazolium chloride) reduction in Escherichia coli K-12. J Microbiol Methods 29:161–175. https://doi.org/10.1016/S0167-7012(97)00036-5
Ullrich S, Karrasch B, Hoppe H, Jeskulke K, Mehrens M (1996) Toxic effects on bacterial metabolism of the redox dye 5-cyano-2,3-ditolyl tetrazolium chloride. Appl Environ Microbiol 62:4587–4593
Vella SJ, Beattie P, Cademartiri R, Laromaine A, Martinez AW, Phillips ST, Mirica KA, Whitesides GM (2012) Measuring markers of liver function using a micropatterned paper device designed for blood from a fingerstick. Anal Chem 84:2883–2891. https://doi.org/10.1021/ac203434x
Wang C, Sauvageau D, Elias A (2016) Immobilization of active bacteriophages on polyhydroxyalkanoate surfaces. ACS Appl Mater Interfaces 8:1128–1138. https://doi.org/10.1021/acsami.5b08664
Yang K, Metcalf WW (2004) A new activity for an old enzyme: Escherichia coli bacterial alkaline phosphatase is a phosphite-dependent hydrogenase. Proc Natl Acad Sci U S A 101:7919–7924. https://doi.org/10.1073/pnas.0400664101
Yetisen AK, Akram MS, Lowe CR (2013) Paper-based microfluidic point-of-care diagnostic devices. Lab Chip 13:2210–2251. https://doi.org/10.1039/c3lc50169h
Acknowledgements
The authors thank Abigail Meyer for her contribution to the colorimetric assays.
Funding
This research was funded in part by the Water Environment Research Foundation (WERF) (No. U4R13), the Leopold Center for Sustainable Agriculture (LCSA, No. ESP2014-02), the Midwest Dairy Association (MDA), and by the Center for Health Effects of Environmental Contamination (CHEEC).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no competing interests.
Ethical statement
This article does not contain any studies with human participants or animals performed by any of the authors.
Electronic supplementary material
ESM 1
(PDF 280 kb).
Rights and permissions
About this article
Cite this article
Hice, S.A., Santoscoy, M.C., Soupir, M.L. et al. Distinguishing between metabolically active and dormant bacteria on paper. Appl Microbiol Biotechnol 102, 367–375 (2018). https://doi.org/10.1007/s00253-017-8604-y
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-017-8604-y