Abstract
Bacillus pumilus D194a formed a strong biofilm on 96-well polystyrene microtiter plates and stainless-steel surfaces. Its optimum environmental factors for growth were determined as 50°C, pH 6.0, 1.5% NaCl for 48 h, whereas, the optimum biofilm formation was measured at 50°C, pH 8.0, 0% NaCl. The isolate was observed to have peritrichous flagella and it produced rigid pellicle. Extracellular polymeric substances (EPSs) of this strain were also determined to be; carbohydrate (33.5 µg/mL), protein (790 µg/mL) and extracellular DNA (eDNA) (18.5 kb). D194a cells formed biofilms on many abiotic surfaces, such as glass, polystyrene, stainless steel, polyvinyl chloride, polycarbonate, polypropylene, wherein polycarbonate (6.23 log/cm2) and polypropylene (6.14 log/cm2) were the generally preferred ones. Biofilm samples were treated with 15 different sanitation agents, including sodium metaperiodate (96.83%), nisin (93.82%), trichloroacetic acid (93.66%) and lysozyme (94.1%) for sanitation purposes. The elimination of eDNA in its 2 h-biofilm with DNase I was 98.48%, while this results was found as 96.06% for its 12 h-mature biofilm. In conclusion, this research demonstrated that B. pumilus D194a is a strong biofilm producer with a rigid structure harboring high protein and eDNA content, which cannot be easily destroyed by various chemical agents.
Similar content being viewed by others
REFERENCES
Abee, T., Kovács, Á.T., Kuipers, O.P., and Van der Veen, S., Biofilm formation and dispersal in Gram-positive bacteria, Curr. Opin. Biotech., 2011, vol. 22, no. 2, pp. 172‒179.
Adnan, M., Alshammari, E., Patel, M., Ashraf, S.A., Khan, S., and Hadi, S., Significance and potential of marine microbial natural bioactive compounds against biofilms/biofouling: necessity for green chemistry, Peer J., 2018, vol. 6, e5049.
Allison, D.G., The biofilm matrix, Biofouling., 2003, vol. 19, no. 2, pp. 139‒150.
Almeida, M.A.N. and De Franca, F.P., Thermophilic and mesophilic bacteria in biofilms associated with corrosion in a heat exchanger, World J. Microb. Biot., 1999, vol. 15 no. 4, pp. 439‒442.
Anand, S., Singh, D., Avadhanula, M., and Marka, S., Development and control of bacterial biofilms on dairy processing membranes, Compr. Rev. Food Sci. F., 2014, vol. 13, no. 1, pp. 18‒33.
Antoniou, K. and Frank, J.F., Removal of Pseudomonas putida biofilm and associated extracellular polymeric substances from stainless steel by alkali cleaning, J. Food Protect, 2005, vol. 68, no. 2, pp. 277‒281.
Arnold, J.W. and Bailey, G.W., Surface finishes on stainless steel reduce bacterial attachment and early biofilm formation: scanning electron and atomic force microscopy study, Poult. Sci., 2000, vol. 79, no. 12, pp. 1839‒1845.
Artham, T., Sudhakar, M., Venkatesan, R., Nair, C.M., Murty, K.V.G.K., and Doble, M., Biofouling and stability of synthetic polymers in sea water, Int. Biodeter. Biodegr., 2009, vol. 63, no. 7, pp. 884‒890.
Beech, I.B., Corrosion of technical materials in the presence of biofilms—current understanding and state-of-the art methods of study, Int. Biodeter. Biodegr., 2004, vol. 53, no. 3, pp. 177‒183.
Blesa, A. and Berenguer, J., Contribution of vesicle-protected extracellular DNA to horizontal gene transfer in Thermus spp., Int Microbiol., 2015, vol. 18, no. 3, pp. 177‒187.
Bolton, N., Critchley, M., Fabien, R., Cromar, N., and Fallowfield, H., Microbially influenced corrosion of galvanized steel pipes in aerobic water systems, J. Appl. Microbiol., 2010, vol. 109, no. 1, pp. 239‒247.
Brooks, J.D. and Flint, S.H., Biofilms in the food industry: problems and potential solutions, Int. J. Food Sci. Tech, 2008, vol. 43, no. 12, pp. 2163‒2176.
Chen, X. and Stewart, P.S., Biofilm removal caused by chemical treatments, Water Res., 2000, vol. 34, no. 17, pp. 4229‒4233.
Cihan, A.C., Tekin, N., Ozcan, B., and Cokmus, C., The genetic diversity of genus Bacillus and the related genera revealed by 16S rRNA gene sequences and ARDRA analyses isolated from geothermal regions of Turkey, Braz. J. Microbiol., 2012, vol. 43, no. 1, pp. 309‒324.
Cihan, A.C., Karaca, B., Ozel, B.P., and Kilic, T., Determination of the biofilm production capacities and characteristics of members belonging to Bacillaceae family, World J. Microb. Biot., 2017, vol. 33, no. 6, p. 118.
Coughlan, L.M., Cotter, P.D., Hill, C., and Alvarez-Ordóñez, A., New weapons to fight old enemies: novel strategies for the (bio) control of bacterial biofilms in the food industry, Front Microbiol., 2016, vol. 7, p. 1641.
De Beer, D. and Paul S., Microbial biofilms, in The Prokaryotes, New York: Springer, 2006, pp. 904‒937.
Domingos, D.F., Dellagnezze, B.M., Greenfield, P., Reyes, L.R., Melo, I.S., Midgley, D.J., and Oliveira, V.M., Draft genome sequence of Bacillus pumilus CCMA-560, isolated from an oil-contaminated mangrove swamp, GenomeA., 2013, vol. 1, no. 5, e00707-13.
Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., and Smith, F., A colorimetric method for the determination of sugars, Nature, 1951, vol. 168, no. 4265, p. 167.
Galiè, S., García-Gutiérrez, C., Miguélez, E.M., Villar, C.J., and Lombó, F., Biofilms in the food industry: health aspects and control methods, Front Microbiol., 2018, vol. 9, p. 898.
Flemming, H.C., Role and levels of real-time monitoring for successful anti-fouling strategies-an overview, Water Sci. Technol., 2003, vol. 47, no. 5, pp. 1‒8.
From, C., Hormazabal, V., and Granum, P.E., Food poisoning associated with pumilacidin-producing Bacillus pumilus in rice, Int. J. Food Microbiol., 2007, vol. 115, no. 3, pp. 319‒324.
Giaouris, E.D. and Nychas, G.J.E., The adherence of Salmonella enteritidis PT4 to stainless steel: the importance of the air–liquid interface and nutrient availability, Food Microbiol., 2006, vol. 23, no. 8, pp. 747‒752.
Grande, R., Di Giulio, M., Bessa, L.J., Di Campli, E., Baffoni, M., Guarnieri, S., and Cellini, L., Extracellular DNA in Helicobacter pylori biofilm: a backstairs rumour, J. Appl. Microbiol., 2010, vol. 110, no. 2, pp. 490‒498.
Herigstad, B., Hamilton, M., and Heersink, J., How to optimize the drop plate method for enumerating bacteria, J. Microbiol. Meth., 2001, vol. 44, no. 2, pp. 121‒129.
Horn, J.M., Davis, M., Martin, S., Lian, T. and Jones, D., Assessing microbiologically influenced corrosion of waste package materials in the Yucca Mountain repository, 6th Int. Conf. Nucl. Eng., San Diego, California, May, 1998.
Husmark, U. and Rönner, U., The influence of hydrophobic, electrostatic and morphologic properties on the adhesion of Bacillus spores, Biofouling, 1992, vol. 5, no. 4, pp. 335‒344.
Kilic, T., Karaca, B., Ozel, B. P., Ozcan, B., Cokmus, C., and Coleri Cihan, A., Biofilm characteristics and evaluation of the sanitation procedures of thermophilic Aeribacillus pallidus E334 biofilms, Biofouling., 2017, vol. 33, no. 4, pp. 352‒367.
Logan, N.A., Bacillus and relatives in foodborne illness, J. Appl. Microbiol., 2012, vol. 112, no. 3, pp. 417‒429.
Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 1951, vol. 193, no. 1, pp. 265‒275.
Lücking, G., Stoeckel, M., Atamer, Z., Hinrichs, J., and Ehling-Schulz, M., Characterization of aerobic spore-forming bacteria associated with industrial dairy processing environments and product spoilage, Int. J. Food Microbiol., 2013, vol. 166, no. 2, pp. 270‒279.
Meyer, B., Approaches to prevention, removal and killing of biofilms, Int. Biodeter. Biodegr., 2003, vol. 51, no. 4, pp. 249‒253.
Mukhtar, T., Afridi, M.S., McArthur, R., Van Hamme, J.D., Rineau, F., Mahmood, T., and Mehmood, S., Draft genome sequence of Bacillus pumilus SCAL1, an endophytic heat-tolerant plant growth-promoting bacterium, Genome A., 2018, vol. 6, no. 18, e00306-18.
Nieminen, T., Rintaluoma, N., Andersson, M., Taimisto, A.M., Ali-Vehmas, T., Seppälä, A., and Salkinoja-Salonen, M., Toxinogenic Bacillus pumilus and Bacillus licheniformis from mastitic milk, Vet. Microbiol., 2007, vol. 124, nos. 3‒4, pp. 329‒339.
Okshevsky, M., Regina, V.R., and Meyer, R.L., Extracellular DNA as a target for biofilm control, Curr. Opin. Biotech., 2015, 33, pp. 73‒80.
Ozel, P.B., Kilic, T., Karaca, B., Yildiz, E.D., Cokmus, C., and Cihan, A.C., Productive biofilms from mesophilic to thermophilic endospore-forming bacilli for industrial applications, J. Microbiol., Biotech. Food Sci., 2017, vol. 7, no. 1, pp. 14‒21.
Parkar, S.G., Flint, S.H., and Brooks, J.D., Physiology of biofilms of thermophilic bacilli-potential consequences for cleaning, J. Ind. Microbiol. Biotechnol., 2003, vol. 30, no. 9, pp. 553‒560.
Parkar, S.G., Flint, S.H., and Brooks, J.D., Evaluation of the effect of cleaning regimes on biofilms of thermophilic bacilli on stainless steel, J. Appl. Microbiol., 2004, vol. 96, no. 1, pp. 110‒116.
Palmer, J.S., Flint, S.H., Schmid, J., and Brooks, J.D., The role of surface charge and hydrophobicity in the attachment of Anoxybacillus flavithermus isolated from milk powder, J. Ind. Microbiol. Biotechnol., 2010, vol. 37, no. 11, pp. 1111‒1119.
Pirttijärvi, T.S.M., Wahlström, G., Rainey, F.A., Saris, P.E.J., and Salkinoja-Salonen, M.S., Inhibition of bacilli in industrial starches by nisin, J. Ind. Microbiol. Biot., 2001, vol. 26, no. 3, pp. 107‒114.
Pitts, B., Hamilton, M.A., Zelver, N., and Stewart, P.S., A microtiter-plate screening method for biofilm disinfection and removal, J. Microbiol. Methods, 2003, vol. 54, no. 2, pp. 269‒276.
Ponnusamy, K., Paul, D., Kim, Y.S., and Kweon, J.H., 2 (5H)-Furanone: a prospective strategy for biofouling-control in membrane biofilm bacteria by quorum sensing inhibition, Braz. J. Microbiol., 2010, vol. 41, no. 1, pp. 227‒234.
Ren, D., Sims, J.J., and Wood, T.K., Inhibition of biofilm formation and swarming of Escherichia coli by (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2 (5H)-furanone, Environ. Microbiol., 2001, vol. 3, no. 11, pp. 731‒736.
Ren, D., Sims, J.J., and Wood, T.K., Inhibition of biofilm formation and swarming of Bacillus subtilis by (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2 (5H)-furanone, Lett. Appl. Microbiol., 2002, vol. 34, no. 4, pp. 293‒299.
Rogers, J., Dowsett, A.B., Dennis, P.J., Lee, J.V., and Keevil, C.W., Influence of plumbing materials on biofilm formation and growth of Legionella pneumophila in potable water systems, Appl. Environ. Microb., 1994, vol. 60, no. 6, pp. 1842‒1851.
Sadekuzzaman, M., Yang, S., Mizan, M.F.R., and Ha, S.D., Current and recent advanced strategies for combating biofilms, Compr. Rev. Food Sci. F., 2015, vol. 14, no. 4, pp. 491‒509.
Sadiq, F.A., Flint, S., Yuan, L., Li, Y., Liu, T., and He, G., Propensity for biofilm formation by aerobic mesophilic and thermophilic spore forming bacteria isolated from Chinese milk powders, Int. J. Food Microbiol., 2017, 262, p. 89‒98.
Satpathy, S., Sen, S.K., Pattanaik, S., and Raut, S., Review on bacterial biofilm: an universal cause of contamination, Biocatal. Agric. Biotechnol., 2016, 7, pp. 56‒66.
Saxena, P., Joshi, Y., Rawat, K., and Bisht, R., Biofilms: architecture, resistance, quorum sensing and control mechanisms, Indian J. Microbiol., 2018, pp. 1‒10.
Simmonds, P., Mossel, B.L., Intaraphan, T., and Deeth, H.C., Heat resistance of Bacillus spores when adhered to stainless steel and its relationship to spore hydrophobicity, J. Food Protect., 2003, vol. 66, no. 11, pp. 2070‒2075.
Simões, M., Simões, L.C., and Vieira, M.J., A review of current and emergent biofilm control strategies, LWT- Food Sci.Tech-Brazil., 2010, vol. 43, no. 4, pp. 573‒583.
Stepanović, S., Vuković, D., Dakić, I., Savić, B., and Š-vabić-Vlahović, M., A modified microtiter-plate test for quantification of staphylococcal biofilm formation, J. Microbiol. Methods, 2000, vol. 40, no. 2, pp. 175‒179.
Stiefel, P., Mauerhofer, S., Schneider, J., Maniura-Weber, K., Rosenberg, U., and Ren, Q., Enzymes enhance biofilm removal efficiency of cleaners, Antimicrob. Agents Chemotherap., 2016, AAC-00400.
Tabak, M., Scher, K., Hartog, E., Romling, U., Matthews, K.R., Chikindas, M.L., and Yaron, S., Effect of triclosan on Salmonella typhimurium at different growth stages and in biofilms, FEMS Microbiol. Lett., 2007, vol. 267, no. 2, pp. 200‒206.
Villanueva, J., Switala, J., Ivancich, A., and Loewen, P.C., Genome sequence of Bacillus pumilus MTCC B6033, GenomeA., 2014, vol. 2, no. 2, e00327-14.
Wilson, K., Preparation of genomic DNA from bacteria, Current Protocols in Molecular Biology, 2001, vol. 56, no. 1, pp. 2‒4.
Woodward, M J., Sojka, M., Sprigings, K.A., and Humphrey, T.J., The role of SEF14 and SEF17 fimbriae in the adherence of Salmonella enterica serotype Enteritidis to inanimate surfaces, J. Med. Microbiol., 2000, vol. 49, no. 5, pp. 481‒487.
Yu, R., Hou, C., Liu, A., Peng, T., Xia, M., Wu, X., and Qiu, G., Extracellular DNA enhances the adsorption of Sulfobacillus thermosulfidooxidans strain ST on chalcopyrite surface, Hydrometallurgy, 2018, 176, pp. 97‒103.
Yuan, D.D., Liu, G.C., Ren, D.Y., Zhang, D., Zhao, L., Kan, C.P., and Zhang, L.B., A survey on occurrence of thermophilic bacilli in commercial milk powders in China, Food Control, 2012, vol. 25, no. 2, pp. 752‒757.
Yuan, Y. and Gao, M., Genomic analysis of a ginger pathogen Bacillus pumilus providing the understanding to the pathogenesis and the novel control strategy, Sci. Rep., 2015, 5, 10259.
Zhao, C.W., Wang, H.Y., Zhang, Y.Z., and Feng, H., Draft genome sequence of Bacillus pumilus BA06, a producer of alkaline serine protease with leather-dehairing function, J. Bacteriol., 2012, vol. 194, no. 23, pp. 6668‒6669.
Funding
This research was supported by the Scientific Research Project Office of Ankara University (project number 11B4240003).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interests. The authors declare that they have no conflict of interest.
Statement on the welfare of animals. This article does not contain any studies involving animals performed by any of the authors.
Rights and permissions
About this article
Cite this article
Kilic, T., Coleri Cihan, A. Biofilm Formation of the Facultative Thermophile Bacillus pumilus D194A and Affects of Sanitation Agents on Its Biofilms. Microbiology 89, 64–73 (2020). https://doi.org/10.1134/S0026261720010087
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1134/S0026261720010087