Biofilm formation of the thermophilic Anoxybacillus rupiensis strain DSM 17127T on abiotic surfaces and removal of its biofilm structure on polystyrene surfaces

Abstract

The aim of this study was to examine the biofilm formation of Anoxybacillus rupiensis DSM 17127T on abiotic surfaces used in the industry and to remove the biofilm structure formed on polystyrene surfaces with sanitation agents. The genomic DNA (gDNA) and the extracellular DNA (eDNA) in the biofilm matrix structure of the bacteria were determined by spectrophotometric analysis and agarose gel imaging. For the biofilm formation on abiotic surfaces and the removal of the biofilm structure formed on polystyrene surfaces, crystal violet binding assay was applied. The optimum temperature, pH, and salinity for the growth and biofilm formation of the bacteria were 55 oC, 8.0, 1 % and, 60 oC, 8.0, 0 %, respectively. The molecular weight of gDNA (27.6 kb) was determined to be larger than eDNA (20.9 kb). gDNA and eDNA were partially purified and treated with DNase I, RNase A and proteinase K. The purified gDNA was completely disrupted only by DNase I., and the purified eDNA was resistant to all three enzymes. As a result of the treatment of biofilm containing eDNA with DNase I, it was observed that the eDNA was sensitive to DNase I and the biofilm mass decreased by 80 % within 2 hours. The bacterium was observed to form biofilms on polycarbonate, polypropylene, polyvinyl chloride, stainless steel, polystyrene and glass surfaces, and the most ideal surface determined as polycarbonate (5.69 log cfu/cm2). In the biofilm removal studies, protein degrading sanitation agents were found to be more effective than polysaccharide degrading agents. In conclusion, it was determined that the eDNA of the bacteria plays an important structural role in the integrity and robustness of the mature biofilm matrix. In addition, it was showed that the bacterium is capable of forming biofilms on abiotic surfaces. Dairy industrial sanitation agents had a significant effect on the removal of the biofilm mass of Anoxybacillus rupiensis.

Keywords

• Abiotic surfaces
• Anoxybacillus rupiensis
• biofilm removal
• sanitation agents

Abiyotik yüzeylerde termofilik Anoxybacillus rupiensis DSM 17127T suşunun biyofilm oluşumu ve polistiren yüzeyler üzerindeki biyofilm yapısının giderimi

Abstract

Bu çalışmanın amacı, Anoxybacillus rupiensis DSM 17127T’nin endüstride kullanılmakta olan abiyotik yüzeyler üzerinde biyofilm oluşumunu incelemek ve polistiren yüzeyler üzerinde oluşan biyofilm yapısının sanitasyon ajanları ile giderimini sağlamaktır. Bakterinin genomik DNA (gDNA)’sı ve biyofilm matriksi yapısında bulunan ekstraselüler DNA (eDNA)’sı spektrofotometrik analiz ve agaroz jel görüntülemesi ile tespit edilmiştir. Polistiren yüzeylerde biyofilm oluşumu ve biyofilm yapısının giderimi kristal viyole bağlanma yöntemi ile belirlenmiştir. Bakterinin ideal planktonik gelişimi için optimum sıcaklık, pH ve tuzluluk istekleri sırasıyla 55 oC, 8.0, % 1 ve ideal biyofilm üretimi için 60 oC, 8.0, % 0 olarak saptanmıştır. gDNA’nın (27.6 kb) molekül ağırlığının, eDNA’dan (20.9 kb) daha büyük olduğu belirlenmiştir. gDNA ve eDNA saflaştırıldıktan sonra DNaz I, RNaz A ve proteinaz K ile muamele edilmiştir. gDNA sadece DNaz I ile tamamen parçalanmıştır. Saflaştırılmış haldeki eDNA ise, üç enzime de direnç göstermiştir. Ancak, olgun biyofilmlerin DNaz I enzimi ile muamelesi sonucunda biyomasta 2 saat içerisinde % 80 oranında azalma gözlenmiştir. Bakterinin polikarbonat, polipropilen, polivinil klorür, paslanmaz çelik, polistiren ve cam yüzeylerinde biyofilm oluşturduğu gözlenmiş olup, en ideal yüzey polikarbonat (5.69 log kob/cm2) olarak belirlenmiştir. Biyofilm giderimi çalışmalarında, protein parçalayıcı sanitasyon ajanlarının polisakkarit parçalayıcı ajanlardan daha fazla etkili olduğu görülmüştür. Sonuç olarak, bakterinin eDNA’sının olgun biyofilm matriksinin bütünlüğü ve sağlamlığı adına önemli bir yapısal rol oynadığı belirlenmiştir. Ayrıca, bakterinin abiyotik yüzeylerde biyofilm oluşturma yeteneğine sahip olduğu görülmüştür. A. rupiensis’in biyofilminin giderimi için süt endüstrisinde kullanımı olan seçilmiş sanitasyon ajanlarının önemli ölçüde etki sağladığı saptanmıştır.

Keywords

• Abiyotik yüzeyler
• Anoxybacillus rupiensis
• biyofilm giderimi
• sanitasyon ajanları

References

1. Pikuta, E., Lysenko, A., Chuvilskaya, N., Mendrock, U., Hippe, H., Suzina, N., Nikitin, D., Osipov, G. ve Laurinavichius, K., Anoxybacillus pushchinensis gen. nov., sp. nov., a novel anaerobic, alkaliphilic, moderately thermophilic bacterium from manure, and description of Anoxybacillus flavitherms comb. nov., International Journal of Systematic and Evolutionary Microbiology, 50, 6, 2109-2117, (2000).
2. Derekova, A., Sjøholm, C., Mandeva, R. ve Kambourova, M., Anoxybacillus rupiensis sp. nov., a novel thermophilic bacterium isolated from Rupi basin (Bulgaria), Extremophiles, 11, 4, 577-583, (2007).
3. Jabeen, F., Muneer, B. ve Qazi, J. I., Characterization of thermophilic bacteria Anoxybacillus rupiensis and cultivation in agroindustrial wastes isolated from hot spring in Chakwal, Pakistan, Pakistan Journal of Zoology, 51, 4, 1243-1250, (2019).
4. Al-Jailawi, M. H., Mahdi, S. M. ve Fadil, A. M., Thermophilic bacteria isolated from hydrocarbon contaminated soils in Iraq, International Journal of Biotechnology, Photon, 111, 275-283, (2013).
5. Goh, K. M., Kahar, U. M., Chai, Y. Y., Chong, C. S., Chai, K. P., Ranjani, V., Illias, R. M. ve Chan, K., Recent discoveries and applications of Anoxybacillus, Applied Microbiology and Biotechnology, 97, 4, 1475-1488, (2013).
6. Cihan, A. C. ve Yildiz, E. D., General characteristics, taxonomic studies and biotechnological importance of the genus Anoxybacillus, Communications Faculty of Sciences University of Ankara Series C Biology, 25, 1-2, 56-82, (2016).
7. Burgess, S. A., Lindsay, D. ve Flint, S. H., Thermophilic bacilli and their importance in dairy processing, International Journal of Food Microbiology, 144, 2, 215-225, (2010).
8. Burgess, S. A., Brooks, J. D., Rakonjac, J., Walker, K. M. ve Flint, S. H., The formation of spores in biofilms of Anoxybacillus flavithermus, Journal of Applied Microbiology, 107, 3, 1012-1018, (2009).

9. Meyer, B., Approaches to prevention, removal and killing of biofilms, International Biodeterioration and Biodegradation, 51, 4, 249-253, (2003).
10. Simões, M., Simões, L. C. ve Vieira, M. J., A review of current and emergent biofilm control strategies, LWT-Food Science and Technology, 43, 4, 573-583, (2010).
11. Gibson, H., Taylor, J. H., Hall, K. E. ve Holah, J. T., Effectiveness of cleaning techniques used in the food industry in terms of the removal of bacterial biofilms, Journal of Applied Microbiology, 87, 1, 41-48, (1999).
12. Torres, C. E., Negro, C., Fuente, E. ve Blanco, A., Enzymatic approaches in paper industry for pulp refining and biofilm control, Applied Microbiology and Biotechnology, 96, 2, 327-344, (2012).
13. Cordeiro, A. L. ve Werner, C., Enzymes for antifouling strategies, Journal of Adhesion Science and Technology, 25, 17, 2317-2344, (2011).
14. Cihan, A. C., Karaca, B., Ozel, B. P. ve Kilic, T., Determination of the biofilm production capacities and characteristics of members belonging to Bacillaceae family, World Journal of Microbiology and Biotechnology, 33, 6, 118, (2017).
15. Bridier, A., Briandet, R., Thomas, V. ve Dubois-Brissonnet, F., Resistance of bacterial biofilms to disinfectants: a review, Biofouling, 27, 9, 1017-1032, (2011).
16. Woodward, M. J., Sojka, M., Sprigings, K. A. ve Humphrey, T. J., The role of SEF14 and SEF17 fimbriae in the adherence of Salmonella enterica serotype Enteritidis to inanimate surfaces, Journal of Medical Microbiology, 49, 5, 481-487, (2000).
17. Stepanović, S., Vuković, D., Dakić, I., Savić, B. ve Švabić-Vlahović, M., A modified microtiter-plate test for quantification of staphylococcal biofilm formation, Journal of Microbiological Methods, 40, 2, 175-179, (2000).
18. Wilson, K., Preparation of genomic DNA from bacteria, Current Protocols in Molecular Biology, 56, 1, 2-4, (2001).
19. Grande, R., Di Giulio, M., Bessa, L. J., Di Campli, E., Baffoni, M., Guarnieri, S. ve Cellini, L., Extracellular DNA in Helicobacter pylori biofilm: a backstairs rumour, Journal of Applied Microbiology, 110, 2, 490-498, (2010).
20. Herigstad, B., Hamilton, M. ve Heersink, J., How to optimize the drop plate method for enumerating bacteria, Journal of Microbiological Methods, 44, 2, 121-129, (2001).
21. Giaouris, E. ve Nychas, G. J., The adherence of Salmonella Enteritidis PT4 to stainless steel: the importance of the air-liquid interface and nutrient availability, Food Microbiology, 23, 8, 747-752, (2006).
22. Parkar, S., Flint, S. ve Brooks, J. D., Evaluation of the effect of cleaning regimes on biofilms of thermophilic bacilli on stainless steel, Journal of Applied Microbiology, 96, 1, 110-116, (2004).
23. Parkar, S., Flint, S. ve Brooks, J., Physiology of biofilms of thermophilic bacilli-potential consequences for cleaning, Journal of Industrial Microbiology and Biotechnology, 30, 9, 553-560, (2003).
24. Ponnusamy, K., Paul, D., Kim, Y. S. ve Kweon, J. H., 2 (5H)-Furanone: a prospective strategy for biofouling-control in membrane biofilm bacteria by quorum sensing inhibition, Brazilian Journal of Microbiology, 41, 1, 227-234, (2010).
25. Tabak, M., Scher, K., Hartog, E., Romling, U., Matthews, K. R., Chikindas, M. L. ve Yaron, S., Effect of triclosan on Salmonella typhimurium at different growth stages and in biofilms, FEMS Microbiology Letters, 267, 2, 200-206, (2007).
26. Pitts, B., Hamilton, M. A., Zelver, N. ve Stewart, P. S., A microtiter-plate screening method for biofilm disinfection and removal, Journal of Microbiological Methods, 54, 2, 269-276, (2003).
27. Margaryan, A., Shahinyan, G., Hovhannisyan, P., Panosyan, H., Birkeland, N. K. ve Trchounian, A., Geobacillus and Anoxybacillus spp. from terrestrial geothermal springs worldwide: Diversity and biotechnological applications, In D. Egamberdieva, N. Birkeland, H. Panosyan, ve W. Li (Ed.), Extremophiles in eurasian ecosystems: ecology, diversity, and applications, pp. 119-166, (2018).
28. Cihan, A. C., Cokmus, C., Koc, M. ve Ozcan, B., Anoxybacillus calidus sp. nov., a thermophilic bacterium isolated from soil near a thermal power plant, International Journal of Systematic and Evolutionary Microbiology, 64, 1, 211-219, (2014).
29. Whitchurch, C. B., Tolker-Nielsen, T., Ragas, P. C. ve Mattick, J. S., Extracellular DNA required for bacterial biofilm formation, Science, 295, 5559, 1487-1487, (2002).
30. Tetz, G. V., Artemenko, N. K. ve Tetz, V. V., Effect of DNase and antibiotics on biofilm characteristics, Antimicrobial Agents and Chemotherapy, 53, 3, 1204-1209, (2009).
31. Nijland, R., Hall, M. J. ve Burgess, J. G., Dispersal of biofilms by secreted, matrix degrading, bacterial DNase, PloS One, 5, 12, e15668, (2010).
32. Harmsen, M., Lappann, M., Knochel, S. ve Molin, S., Role of extracellular DNA during biofilm formation by Listeria monocytogenes, Applied and Environmental Microbiology, 76, 7, 2271-2279, (2010).
33. Martins, M., Uppuluri, P., Thomas, D. P., Cleary, I. A., Henriques, M., Lopez-Ribot, J. L. ve Oliveira, R., Presence of extracellular DNA in the Candida albicans biofilm matrix and its contribution to biofilms, Mycopathologia, 169, 5, 323-331, (2010).
34. Lappann, M., Claus, H., Van Alen, T., Harmsen, M., Elias, J., Molin, S. ve Vogel, U., A dual role of extracellular DNA during biofilm formation of Neisseria meningitides, Molecular Microbiology, 75, 6, 1355-1371, (2010).
35. Shields, R. C., Mokhtar, N., Ford, M., Hall, M. J., Burgess, J.G., ElBadawey, M. R. ve Jakubovics, N. S., Efficacy of a marine bacterial nuclease against biofilm forming microorganisms isolated from chronic rhinosinusitis, PLoS One, 8, 2, e55339, (2013).
36. Flint, S., Palmer, J., Bloemen, K., Brooks, J. ve Crawford, R., The growth of Bacillus stearothermophilus on stainless steel, Journal of Applied Microbiology, 90, 2, 151-157, (2001).
37. Somerton, B., Flint, S., Palmer, J., Brooks, J. ve Lindsay, D., Preconditioning with cations increases the attachment of Anoxybacillus flavithermus and Geobacillus species to stainless steel, Applied and Environmental Microbiology, 79, 13, 4186-4190, (2013).
38. Sadiq, F. A., Flint, S., Yuan, L., Li, Y., Liu, T. ve He, G., Propensity for biofilm formation by aerobic mesophilic and thermophilic spore forming bacteria isolated from Chinese milk powders, International Journal of Food Microbiology, 262, 89-98, (2017).
39. Loiselle, M. ve Anderson, K. W., The use of cellulase in inhibiting biofilm formation from organisms commonly found on medical implants, Biofouling, 19, 2, 77-85, (2003).
40. Hedstrom, L., Serine protease mechanism and specificity, Chemical Reviews, 102, 12, 4501-4524, (2002).
41. Molobela, I. P., Cloete, T. E. ve Beukes, M., Protease and amylase enzymes for biofilm removal and degradation of extracellular polymeric substances (EPS) produced by Pseudomonas fluorescens bacteria, African Journal of Microbiology Research, 4, 14, 1515-1524, (2010).
42. Chen, X. ve Stewart, P. S., Biofilm removal caused by chemical treatments, Water Research, 34, 17, 4229-4233, (2000).