Abstract
Biofilm is a microbial population which live in a self-produced extracellular polymeric matrix by attaching to surfaces. Biofilms consist of different different types of organisms such as bacteria, fungi, protozoa, etc. Many biofilms that develop in nature consist of more than one type of organism. Biofilms protect bacteria from adverse conditions such as temperature fluctuation and disinfectants. The aim of this study was to determine the effective elimination strategies for combating biofilm and planktonic bacteria in cooling tower model system using different decontamination / disinfection techniques. In this study, 14 week-old biofilms were treated with temperatures of 4 °C, 65 °C; pH of 3, 11; 2 and 10 mg/l chlorine, 2 and 10 mg/l monochloramine; hypotonic salt (0.01% NaCl) and hypertonic salt (3% NaCl) solution. For enumeration, number of aerobic heterotrophic bacteria was determined by conventional culture method, number of live bacteria was determined by LIVE/DEAD viability kit, CTC-DAPI and Alamar blue staining methods. Temperature of 65 °C, pH of 3, 10 mg/l monochloramine and hypertonic salt solution were the most effective parameters for decontamination of biofilm and planktonic bacteria. Biofilm bacteria in the circulating water system were significantly more resistant than planktonic bacteria against stress factors. When the numbers of epifluorescence microscopy and conventional culture technique were compared, significantly higher number of live bacteria were detected using epifluorescence microscopy. Bacteria enter the viable but non-culturable phase by loosing their culturability under stress conditions. For this reason, the conventional culture method should be supported by different techniques to get more realistic numbers.
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Anderson KL, Roberts C, Disz T, Vonsteġn V, Hwang K, Overbeek R, Olson PD, Projan SJ, Dunman PM (2006) Characterization of the Staphylococcus aureus heat shock, cold shock, stringent, and SOS responses and their effects on log-phase mRNA turnover. J Bacteriol 188:6739–6756. https://doi.org/10.1128/JB.00609-06
Araya R, Tani K, Takagi T, Yamaguchi N, Nasu M (2002) Bacterial activity and community composition in stream water and biofilm from an urban river determined by fluorescent in situ hybridization and DGGE analysis. FEMS Microbiol Ecol 43:111–119. https://doi.org/10.1111/j.1574-6941.2003.tb01050.x
Asakura H, Ishiwa A, Arakawa E, Makino S, Okada Y, Yamamoto S, Igimi S (2006) Gene expression profile of Vibrio cholerae in the cold stress-induced viable but non-culturable state. Environ Microbiol 9(4):869–879. https://doi.org/10.1111/j.1462-2920.2006.01206.x
Barria C, Malecki M, Arraiano CM (2013) Bacterial adaptation to cold. Microbiology 159:2437–2443. https://doi.org/10.1099/mic.0.052209-0
Bedard E, Charron D, Lalancette C, Deziel E, Prevost M (2014) Recovery of Pseudomonas aeruginosa culturability following copper- and chlorine-induced stress. FEMS Microbiol Lett 36:226–234. https://doi.org/10.1111/1574-6968.12494
Behnke S, Parker AE, Woodall D, Camper AK (2011) Comparing the chlorine disinfection of detached biofilm clusters with those of sessile biofilms and planktonic cells in single- and dual-species cultures. Appl Environ Microbiol 77(20):7176–7184. https://doi.org/10.1128/AEM.05514-11
Besinis A, De Peralta T, Handy RD (2014) The antibacterial effects of silver, titanium dioxide and silica dioxide nanoparticles compared to the dental disinfectant chlorhexidine on Streptococcus mutans using a suite of bioassays. Nanotoxicology 8(1):1–16. https://doi.org/10.3109/17435390.2012.742935
Bloomfield SF, Arthur M, Begun K, Patel H (1993) Comparative testing of disinfectants using proposed European surface test methods. Lett Appl Microbiol 10:287–294. https://doi.org/10.1111/j.1472-765X.1993.tb01439.x
Boulos L, Prevost M, Barbeau B, Coallier J, Desjardins R (1999) LIVE/DEAD BacLight: application of a new rapid staining method for direct enumeration of viable and total bacteria in drinking water. J Microbiol Methods 37:77–86. https://doi.org/10.1016/S0167-7012(99)00048-2
Brazire A, Diab F, Jebbar M, Haras D (2007) Influence of high salinity on biofilm formation and benzoate assimilation by Pseudomonas aeruginosa. J Ind Microbiol Biotechnol 34:5–8. https://doi.org/10.1007/s10295-006-0087-2
Chandy JP, Angles ML (2011) Determination of nutrients limiting biofilm formation and the subsequent impact on disinfectant decay. Water Res 35(11):2677–2682. https://doi.org/10.1016/S0043-1354(00)00572-8
Chang L, Craik S (2012) Laboratory simulation of the effect of ozone and monochloramine on biofilms in drinking water mains. Ozone Sci Eng 34:243–251. https://doi.org/10.1080/01919512.2012.686864
Chen X, Stewart P (2000) Biofilm removal caused by chemical treatments. Water Res 34:4229–4233. https://doi.org/10.1016/S0043-1354(00)00187-1
Chiao TH, Clancy TM, Pinto A, Xi C, Raskin L (2014) Differential resistance of drinking water bacterial populations to monochloramine disinfection. Environ Sci Technol 48:4038–4047. https://doi.org/10.1021/es4055725
Clarelli F, Di Russo C, Natalini R, Ribot M (2013) A fluid dynamics model of the growth of phototrophic biofilms. J Math Biol 66(7):1387–1408. https://doi.org/10.1007/s00285-012-0538-5
Cochran WL, McFeters GA, Stewart PS (2000) Reduced susceptibility of thin Pseudomonas aeruginosabiofilms to hydrogen peroxide and monochloramine. J Appl Microbiol 88:22–30. https://doi.org/10.1046/j.1365-2672.2000.00825.x
Csonka L (1989) Physiological and genetic responses of bacteria to osmotic stress. Microbiol Rev 53:121–147
Davies D (2003) Understanding biofilm resistance to antibacterial agents. Nature 2:114–122. https://doi.org/10.1038/nrd1008
Denkhaus E, Meisen S, Telgheder U, Wingender J (2007) Chemical and physical methods for characterisation of biofilms. Microchim Acta 158:1–27. https://doi.org/10.1007/s00604-006-0688-5
Dikici A (2009) Çevresel stres faktörlerine karşı bakteriyel adaptasyonlar ve mekanizmaları. Electron J Food Technol 4(3):59–68
Donlan RM (2002) Biofilms: microbal life on surfaces. Emerg Infect Dis 8(9):881–890
Dusserre E, Ginevra C, Hallier-Soulier S, Festoc G, Etienne J, Jarraud S (2008) A PCR based method for monitoring Legionella pneumophila in water samples detects viable but noncultivable Legionellae that can recover their cultivability. Appl Environ Microbiol 74:4817–4824. https://doi.org/10.1128/AEM.02899-07
Eguia E, Trueba A, Giron A, Rio-Calonge B, Otero F, Bielva C (2007) Optimisation of biocide dose as a function of residual biocide in a heat exchanger pilot plant effluent. Biofouling 23(3):231–247. https://doi.org/10.1080/08927010701306740
Elvers KT, Leeming K, Lappin-Scott HM (2002) Binary and mixed population biofilms: time-lapse image analysis and disinfection with biocides. J Ind Microbiol Biotechnol 29:331–338. https://doi.org/10.1038/sj.jim.7000318
Epstein AK, Wonga TS, Belisle RA, Boggs EM, Aizenberg J (2012) Liquid-infused structured surfaces with exceptional anti-biofouling performance. Proc Natl Acad Sci 109(33):13182–13187. https://doi.org/10.1073/pnas.1201973109
Fonseca P, Moreno R, Rojo F (2011) Growth of Pseudomonas putida at low temperature: global transcriptomic and proteomic analyses. Environ Microbiol Rep 3(3):329–339. https://doi.org/10.1111/j.1758-2229.2010.00229.x
Garrett TR, Bhakoo M, Zhang Z (2008) Bacterial adhesion and biofilms on surfaces. Prog Nat Sci 18:1049–1056. https://doi.org/10.1016/j.pnsc.2008.04.001
Giao MS, Wilks SA, Azavedo NF, Vieira MJ, Keevi CW (2009) Validation of SYTO 9/pripidium iodide uptake for rapid detection of vianle but noncultivable Legionella pneumophila. Microb Ecol 58:56–62. https://doi.org/10.1007/s00248-008-9472-x
Gopinath V, Priyadarshini S, Loke MF, Arunkumar J, Marsili E, MubarakAli D, Velusamy P, Vadivelu J (2017) Biogenic synthesis, characterization of antibacterial silver nanoparticles and its cell cytotoxicity. Arab J Chem 10:1107–1117. https://doi.org/10.1016/j.arabjc.2015.11.011
Graumann P, Wendrich TM, Weber MHW, Schröder K, Marahiel MA (1997) A family of cold shock proteins in Bacillus subtilis is essential for cellular growth and for efficient protein synthesis at optimal and low temperatures. Mol Microbiol 25:741–756. https://doi.org/10.1046/j.1365-2958.1997.5121878.x
Hall-Stoodley L, Stoodley P (2005) Biofilm formation and dispersal and the transmission of human pathogens. Trends Microbiol 13(1):7–10. https://doi.org/10.1016/j.tim.2004.11.004
Hall-Stoodley L, Stoodley P, Kathju S, Høiby N, Moser C, Costerton JW, Moter A, Bjarnsholt T (2012) Towards diagnostic guidelines for biofilm-associated infections. FEMS Immunol Med Microbiol 65(2):127–145. https://doi.org/10.1111/j.1574-695X.2012.00968.x
Islam N, Ross JM, Marten MR (2015) Proteome analyses of Staphylococcus aureus biofilm at elevated levels of NaCl. Clin Microbiol 4(5):1–15. https://doi.org/10.4172/2327-5073.1000219
Katebian L, Jiang SC (2013) Marine bacterial biofilm formation and its responses to periodic hyperosmotic stress on a flat sheet membrane for seawater desalination pretreatment. J Membrane Sci 425:182–189. https://doi.org/10.1016/j.memsci.2012.08.027
Kawarai T, Furukawa S, Narisawa N, Hagiwara C, Ogihara H, Yamasaki M (2009) Biofilm formation by Escherichia coli in hypertonic sucrose media. J Biosci Bioeng 107(6):630–635. https://doi.org/10.1016/j.jbiosc.2009.01.018
Kim J, Pitts B, Stewart PS, Camper A, Yoon J (2008) Comparison of the antimicrobial effects of chlorine, silver ion, and tobramycin on biofilm. Antimicrob Agents Chemother 52(4):1446–1453. https://doi.org/10.1128/AAC.00054-07
Kim J, Park HD, Chung S (2012) Microfluidic approaches to bacterial biofilm formation. Molecules 17:9818–9834. https://doi.org/10.3390/molecules17089818
Lee S, Elimelech M (2007) Salt cleaning of organic-fouled reverse osmosis membranes. Water Res 41:1134–1142. https://doi.org/10.1016/j.watres.2006.11.043
Lee KWK, Periasamy S, Mukherjee M, Xie C, Kjelleberg S, Rice SA (2013) Biofilm development and enhanced stress resistance of a model, mixed-species community biofilm. ISME J. https://doi.org/10.1038/ismej.2013.194
Luo J, Chen Z, Sun Y (2006) Controlling biofilm formation with an N halamine-based polymeric additive. J Biomed Mater Res A. https://doi.org/10.1002/jbm.a.30689
MacDonald R, Brözel VS (2000) Community analysis of bacterial biofilms in a simulated recirculating cooling-water system by fluorescent in situ hybridization with rRNA-targeted oligonucleotide probes. Water Res 34(9):2439–2446. https://doi.org/10.1016/S0043-1354(99)00409-1
Mansi A, Amori I, Marchesi I, Proietto AR, Marcelloni AM, Ferranti G, Magini V, Valeriani F, Borella P (2014) Legionella spp. survival after different disinfection procedures: comparison between conventional culture, qPCR and EMA-qPCR. Microchem J 112:65–69. https://doi.org/10.1016/j.microc.2013.09.017
Melada S, Coniglio MA (2015) Monochloramine for remediation of Legionella only in domestic hot water systems: an iron fist in a velvet glove. Open J Prev Med 5:143–150. https://doi.org/10.4236/ojpm.2015.53017
Molhoek EM, Dijk AV, Veldhuizen EJA, Haagsmanb HP, Bikker FJ (2011) A cathelicidin-2-derived peptide effectively impairs Staphylococcus epidermidis biofilm. Int J Antimicrob Agents 37:476–479. https://doi.org/10.1016/j.ijantimicag.2010.12.020
Momba MNB, Ndaliso S, Binda MA (2003) Effect of conbined chlorine-monochloramine process on the inhibition of biofilm regrowth in potable water systems. Water Supply 3:215–221. https://doi.org/10.2166/ws.2003.0106
Münchbach M, Nocker A, Narberhaus F (1999) Multiple small heat shock proteins in rhizobia. J Bacteriol 181:83–90
Murthy PS, Venkatesan R, Nair KVK, Ravindran M (2004) Biofilm control for plate heat exchangers using surface seawater from the open ocean for the OTEC power plant. Int Biodeter Biodegr 53:133–140. https://doi.org/10.1016/j.ibiod.2003.11.003
Neilands J (2007) Acid tolerans of Streptococcus mutans biofilm (PhD dissertation). Malmö University. Retrieved from http://urn.kb.se/resolve?urn=urn:nbn:se:mau:diva-7704
Nguyen TMN, Ilef D, Jarraud S, Rouil L, Campese C, Che D, Haeghebaert S, Ganiayre F, Marcel F, Etienne J, Desenclos JC (2006) A community wide outbreak of legionnaires disease linked to industrial cooling towers—how far can contaminated aerosols spread? J Infect Dis 193:102–111
Nostro A, Cellini L, Giulio MD, Darrigo M, Marino A, Blanco AR, Favaloro A, Cutroneo G, Bisignano G (2012) Effect of alkaline pH on staphylococcal biofilm formation. Acta Pathol Microbiol Immunol Scand 120:733–742. https://doi.org/10.1111/j.1600-0463.2012.02900.x
Novick RP (2003) Autoinduction and signal transduction in the regulation of Staphylococcal virulence. Mol Microbiol 48:1429–1449. https://doi.org/10.1046/j.1365-2958.2003.03526.x
Ortega-Morales BO, Lopez-Cortes A, Hernandez-Duqu G, Crassous P, Guezennec J (2001) Extracellular polymers of microbial communities colonizing ancient limestone monuments. Methods Enzymol 336:331–339. https://doi.org/10.1016/S0076-6879(01)36599-0
Osaka I, Hefty PS (2013) Simple resazurin-based microplate assay for measuring Chlamydia infections. Antimicrob Agents Chemother 57(6):2838–2840. https://doi.org/10.1128/AAC.00056-13
Otter JA, Vickery K, Walker JT, Pulcini EL, Stoodley P, Goldenberg SD, Salkeld JAG, Chewins J, Yezli S, Edgeworth JD (2015) Surface-attached cells, biofilms and biocide susceptibility: implications for hospital cleaning and disinfection. J Hosp Infect 89:16–27. https://doi.org/10.1016/j.jhin.2014.09.008
Ozcakir O (2007) Bakterilerin canlı ama kültürde üretilemeyen formları. Mikrobiyol Bult 41:477–484
Paraje MG (2011) Antimicrobial resistance in biofilms. In: Mendez-Vilas A (ed) Science against microbial pathogens: communicating current research and technological advances. Formatex research center, Spain, pp 736–744
Parsek MR, Singh PK (2003) Bacterial biofilms: an emerging link to disease pathogenesis. Annu Rev 57:677–701. https://doi.org/10.1146/annurev.micro.57.030502.090720
Pasmore M, Todd P, Pfiefer B, Rhodes M, Bowman CN (2002) Effect of polymer surface properties on the reversibility of attachment of Pseudomonas aeruginosa in the early stages of biofilm development. Biofouling 18(1):65–71. https://doi.org/10.1080/08927010290017743
Peeters E, Nelis HJ, Coenye T (2008) Comparison of multiple methods for quantification of microbial biofilms grown in microtiter plates. J Microbiol Methods 72:157–165. https://doi.org/10.1016/j.mimet.2007.11.010
Pettit RK, Weber CA, Pettit GR (2009) Application of a high throughput Alamar blue biofilm susceptibility assay to Staphylococcus aureus biofilms. Ann Clin Microbiol Antimicrob 8(28):1–7. https://doi.org/10.1186/1476-0711-8-28
Rampersad SN (2012) Multiple applications of alamar blue as an indicator of metabolic function and cellular health in cell viability bioassays. Sensors 12:12347–12360. https://doi.org/10.3390/s120912347
Rodriguez GG, Philps 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
Saby S, Sibille I, Mathieu L, Paquin JL, Block JC (1997) Influence of water chlorination on the counting of bacteria with DAPI (4,6-diamidino-2-phenylindole). Appl Environ Microbiol 63(4):1564–1569
Sanli-Yurudu NO, Kimiran-Erdem A (2015) Evaluation of the antimicrobial activity of colloidal silver-hydrogen peroxide against model cooling tower biofilm. In: Méndez-Vilas A (ed) The battle against microbial pathogens: basic science, technological advances and educational programs. Formatex Research Center, Badajoz, pp 526–533
Schaule G, Fleming HC, Ridgway HF (1993) Use of 5-cyano-2,3-ditolyl tetrazolium chloride for quantifying planktonic and sessile bacteria in drinking water. Appl Environ Microbiol 59:3850–3857
Schumann W, Hecker M, Msadek T (2002) Regulation and function of heat-inducible genes in Bacillus subtilis. In: Sonenshein AL, Hoch JA, Losick RM (eds) Bacillus subtilis and its closest relatives: from genes to cells. American society for microbiology press, Washington, pp 359–368
Serra DO, Hengge R (2014) Stress responses go three dimensional—the spatial order of physiological differentiation in bacterial macrocolony biofilms. Environ Microbiol 16(6):1455–1471. https://doi.org/10.1111/1462-2920.12483
Shen Y, Stojicic S, Haapasalo M (2010) Bacterial viability in starved and revitalized biofilms: comparison of viability staining and direct culture. J Endod 36(11):1820–1823. https://doi.org/10.1016/j.joen.2010.08.029
Shimizu K (2014) Regulation systems of bacteria such as Escherichia coli in response to nutrient limitation and environmental stresses. Metabolites 4:1–35. https://doi.org/10.3390/metabo4010001
Singh VK, Syring M, Singh A, Singhal K, Dalecki A, Johansson T (2012) An insight into the significance of the DnaK heat shock system in Staphylococcus aureus. Int J Med Microbiol 302:242–252. https://doi.org/10.1016/j.ijmm.2012.05.001
Smith I, Fricker EJ, Eccles J, Searle R (2004) Laboratory observations of biocie efficacy in model cooling tower systems. ASHRAE Trans 110:314–324
Tanji Y, Nishihara T, Miyanaga K (2007) Monitoring of biofilm in cooling water system by measuring lactic acid consumption rate. Biochem Eng J 35:81–86. https://doi.org/10.1016/j.bej.2007.01.001
Taormina PJ, Beuchat LR (2001) Survival and heat resistance of Listeria monocytogenes after exposure to alkali and chlorine. Appl Environ Microbiol 67(6):2555–2563. https://doi.org/10.1128/AEM.67.6.2555-2563.2001
Thieringer HA, Jones PG, Inouye M (1998) Cold shock and adaptation. Bioassays 20:49–57. https://doi.org/10.1002/(SICI)1521-1878(199801)20:1%3c49::AID-BIES8%3e3.0.CO;2-N
Turetgen I (2004) Comparison of the efficacy of free residual chlorine and monochloramine against biofilms in model and full scale cooling towers. Biofouling 20:81–85. https://doi.org/10.1080/08927010410001710027
Turetgen I (2008) Induction of viable but nonculturable (VBNC) state and the effect of multiple subculturing on the survival of Legionella pneumophila strains in the presence of monochloramine. Ann Microbiol 58:153–156. https://doi.org/10.1007/BF03179460
Vatansever C, Turetgen I (2018) Investigating the effects of different physical and chemical stress factors on microbial biofilm. Water SA 44(2):308–317. https://doi.org/10.4314/wsa.v44i2.16
Weber MM, French CL, Barnes MB, Siegele DA, McLean RJC (2010) A previously uncharacterized gene, yjfO (bsmA), influences Escherichia coli biofilm formation and stress response. Microbiology 156:139–147. https://doi.org/10.1099/mic.0.031468-0
Wiegert T, Homuth G, Versteeg S, Schuman W (2001) Alkaline shock induces the Bacillus subtilis σW regulon. Mol Microbiol 41(1):59–71. https://doi.org/10.1046/j.1365-2958.2001.02489.x
Wouters JA, Sanders JW, Kok J, De Vos WM, Kuipers OP, Abee T (1998) Clustered organization and transcriptional analysis of a family of five csp genes of Lactococcus lactis MG1363. Microbiology 144:2885–2893. https://doi.org/10.1099/00221287-144-10-2885
Xu P, Xu Z, Wang J, Zhang Y, Zhang L (2012) MIC in circulating cooling water system. J Water Resour Prot 4:203–206. https://doi.org/10.4236/jwarp.2012.44022
Xue Z, Hessler CM, Panmanee W, Hassett DJ, Seo Y (2013) Pseudomonas aeruginosa inactivation mechanism is affected by capsular extracellular polymeric substances reactivity with chlorine and monochloramine. FEMS Microbiol Ecol 83:101–111. https://doi.org/10.1111/j.1574-6941.2012.01453.x
Yamanaka K, Fang L, Inouye M (1998) The CspA family in Escherichia coli: multiple gene duplication for stress adaptation. Mol Microbiol 27:247–255. https://doi.org/10.1046/j.1365-2958.1998.00683.x
Zhang XS, Garcia-Contreras R, Wood TK (2007) YcfR (BhsA) influences Escherichia coli biofilm formation through stress response and surface hydrophobicity. J Bacteriol 189:3051–3062. https://doi.org/10.1128/JB.01832-06
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Vatansever, C., Turetgen, I. Investigation of the effects of various stress factors on biofilms and planktonic bacteria in cooling tower model system. Arch Microbiol 203, 1411–1425 (2021). https://doi.org/10.1007/s00203-020-02116-2
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DOI: https://doi.org/10.1007/s00203-020-02116-2