Biofilms are surface attached communities of bacteria, fungi, protozoa, and many other microorganisms. Potential biofilm formers are known as “ESKAPE'' which includes Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter aerogenes . Among them, Pseudomonas aeruginosa is considered as the most health injurious biofilm forming organism that causes nosocomial infections like ventilator associated pneumonia, chronic wound infections, chronic rhinosinusitis etc. Efficient removal of biofilm from medical devices is a big challenge to avoid hospital acquired infections as these devices are delicate and cannot be reprocessed using harsh chemicals or high temperature. Therefore, use of mild solutions for removal of biofilm is advisable. In the present study, Pseudomonas aeruginosa PAO1 acted as potential biofilm former with 10% inoculum size in TSB medium. The biofilm of P.aeruginosa PAO1 was studied microscopically and the results revealed that with time the number of cells increased and thick biofilm formation was observed with more Exopolysacharide production. Susceptibility of P. aeruginosa PAO1   against some antibiotics and enzymes were analyzed individually as well as in combinations using microtiter plate. The efficiency of antibiotics to eradicate biofilm was higher than the enzymes but the use of antibiotics alone required higher concentration to eradicate biofilm. While combination of enzymes and antibiotics can eradicate the biofilm at sub minimal concentration as well as that can minimize the load of antimicrobials in the environment. Therefore, in this study when combination of ciprofloxacin at sub MIC of 1.56 µg/ml was applied with lysozyme and protease, 68±0.5% and 56±0.6% biofilm eradication were observed, respectively but only 40±0.5% eradication was observed when treated with ciprofloxacin alone. Similarly, when combination of levofloxacin at sub MIC of 6 µg/ml was applied with DNase, 95% eradication was observed while only 32±0.8% eradication was observed when treated with levofloxacin alone.

1.         Whitchurch CB, Tolker-nielsen T, Ragas PC, Mattick JS. Extracellular DNA required for bacterial biofilm formation. Science. 2002 Feb 22;295(5559):1487. doi: 10.1126/science.295.5559.1487, PMID 11859186.

2.         Jamal M, Tasneem U, Hussain T, Andleeb S. Bacterial biofilm: its composition, formation and role in human infections. Res Rev J Microbiol Biotechnol Bacterial Biofilm. 2015 Jul 20;4(3):1-14.

3.         Coughlan LM, Cotter PD, Hill C, 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 Oct 18;7:1641. doi: 10.3389/fmicb.2016.01641, PMID 27803696.

4.          Bogino PC, Oliva Mde L, Sorroche FG, Giordano W. The role of bacterial biofilms and surface components in plant-bacterial associations. Int J Mol Sci. 2013, Jul 30;14(8):15838-59. doi: 10.3390/ijms140815838, PMID 23903045.

5.          Bucs SS, Valladares Linares R, van Loosdrecht MC, Kruithof JC, Vrouwenvelder JS. Impact of organic nutrient load on biomass accumulation, feed channel pressure drop increase and permeate flux decline in membrane systems. Water Res. 2014 Sep 16;67:227-42. doi: 10.1016/j.watres.2014.09.005, PMID 25282091.

6.          Gule NP, Begum NM, Klumperman B. Advances in biofouling mitigation: a review. Crit Rev Environ Sci Technol. 2016;46(6):535-55. doi: 10.1080/10643389.2015.1114444.

7.          Lewis KIM. Riddle of biofilm resistance. Antimicrob Agents Chemother. 2001 Apr 12;45(4):999-1007. doi: 10.1128/AAC.45.4.999-1007.2001, PMID 11257008.

8.          Costerton JW, Stewart PS, Greenberg EP. Bacterial biofilms: A common cause of persistent infections. Science. 1999 May 21;284(5418):1318-22. doi: 10.1126/science.284.5418.1318, PMID 10334980.

9.           Mah TF, Pitts B, Pellock B, Walker GC, Stewart PS, O’Toole GA. A genetic basis for Pseudomonas aeruginosa biofilm antibiotic resistance. Nature. 2003;426(6964):306-10. doi: 10.1038/nature02122, PMID 14628055.

10.         Berendonk TU, Manaia CM, Merlin C, Fatta-Kassinos D, Cytryn E, Walsh F, Bürgmann H, Sørum H, Norström M, Pons MN, Kreuzinger N, Huovinen P, Stefani S, Schwartz T, Kisand V, Baquero F, Martinez JL. Tackling antibiotic resistance: the environmental framework. Nat Rev Microbiol. 2015 May 15;13(5):310-7. doi: 10.1038/nrmicro3439, PMID 25817583.

11.         Pendleton JN, Gorman SP, Gilmore BF. Clinical relevance of the ESKAPE pathogens. Expert Rev Anti Infect Ther. 2013 Jan 10;11(3):297-308. doi: 10.1586/eri.13.12, PMID 23458769.

12.         Sousa AM, Pereira MO. Pseudomonas aeruginosa diversification during infection development in cystic fibrosis lungs-A review. Pathogens. 2014 Aug 18;3(3):680-703. doi: 10.3390/pathogens3030680, PMID 25438018.

13.         Wisplinghoff H, Bischoff T, Tallent SM, Seifert H, Wenzel RP, Edmond MB. Nosocomial bloodstream infections in US hospitals: analysis of 24,179 cases from a prospective nationwide surveillance study. Clin Infect Dis. 2004 Jul 15;39(3):309-17. doi: 10.1086/421946, PMID 15306996.

14.         Morita Y, Tomida J, Kawamura Y. Responses of Pseudomonas aeruginosa to antimicrobials. Front Microbiol. 2014 Jan 8;4(1):422. doi: 10.3389/fmicb.2013.00422, PMID 24409175.

15.         Pamp SJ, Tolker-nielsen T. Multiple roles of biosurfactants in structural biofilm development by Pseudomonas aeruginosa. J Bacteriol. 2007 Sep 28;189(6):2531-9. doi: 10.1128/JB.01515-06, PMID 17220224.

16.         Allesen-holm M, Barken KB, Yang L, Klausen M, Webb JS, Kjelleberg S, Molin S, Givskov M, Tolker-nielsen T. A characterization of DNA release in Pseudomonas aeruginosa cultures and biofilms. Mol Microbiol. 2006 Feb 16;59(4):1114-28. doi: 10.1111/j.1365-2958.2005.05008.x, PMID 16430688.

17.         Gilbert P, McBain AJ, Rickard AH. Formation of microbial biofilm in hygienic situations: A problem of control. Int Biodeterior Biodegrad. 2003 Jun;51(4):245-8. doi: 10.1016/S0964-8305(03)00043-X.

18.         Simões M, Simões LC, Vieira MJ. A review of current and emergent biofilm control strategies. LWT Food Sci Technol. 2010;43(4):573-83. doi: 10.1016/j.lwt.2009.12.008.

19.         Rasamiravaka T, Labtani Q, Duez P, El Jaziri M. The formation of biofilms by Pseudomonas aeruginosa: a review of the natural and synthetic compounds interfering with control mechanisms. BioMed Res Int. 2015;2015:759348. doi: 10.1155/2015/759348, PMID 25866808.

20.         Srey S, Jahid IK, Ha S. Biofilm formation in food industries: A food safety concern. Food Control. 2013 Jun 5;31(2):572-85. doi: 10.1016/j.foodcont.2012.12.001.

21.         Flemming HC, Wingender J. The biofilm matrix. Nat Rev Microbiol. 2010 Sep;8(9):623-33. doi: 10.1038/nrmicro2415, PMID 20676145.

22.         Reid G, Kang YS, Lacerte M, Tieszer C, Hayes KC. Bacterial biofilm formation on the bladder epithelium of spinal cord injured patients. II. Toxic outcome on cell viability. Paraplegia. 1993 Aug;31(8):494-9. doi: 10.1038/sc.1993.80, PMID 8414632.

23.         Reid G. Applications from bacterial adhesion and biofilm studies in relation tourogenital tissues and biomaterials: a review. J Ind Microbiol. 1994 Mar;13(2):90-6. doi: 10.1007/BF01584104, PMID 7765341.

24.         Hassan A, Usman J, Kaleem F, Omair M, Khalid A, Iqbal M. Evaluation of different detection methods of biofilm formation in the clinical isolates. Braz J Infect Dis. 2011 Jul 11;15(4):305-11. doi: 10.1590/S1413-86702011000400002, PMID 21860999.

25.         CLSI subcommittee on antimicrobial susceptibility Testing [internet]. Vol. 1. p. 1-15; 2020 Jan 5. CLSI AST news update [cited 9/10/2020]. Available from: https://clsi.org/media/3486/clsi_astnewsupdate_january2020.pdf.

26.         Perez LR, Barth AL. Biofilm production using distinct media and antimicrobial susceptibility profile of Pseudomonas aeruginosa. Braz J Infect Dis. 2011 Jul–Aug;15(4):301-4. doi: 10.1016/S1413-8670(11)70196-9, PMID 21860998.

27.         Gupta PV, Nagarsenker MS. Antimicrobial and antibiofilm activity of Enzybiotic against Staphylococcus aureus. Nat Prod Res. 2015 Dec;32(18):2225-8.

28.         Shukla SK, Rao TS. An improved crystal violet assay for biofilm quantification in 96-well microtitre plate. bioRxiv. 2017:1-10. doi: DOI.

29.         Eladawy M, El-Mowafy M, El-Sokkary MMA, Barwa R. Effects of lysozyme, proteinase K, and cephalosporins on biofilm formation by clinical isolates of Pseudomonas aeruginosa. Interdiscip Perspect Infect Dis. 2020;2020:6156720. doi: 10.1155/2020/6156720, PMID 32089678.

30.         Cendra MDM, Blanco-Cabra N, Pedraz L, Torrents E. Optimal environmental and culture conditions allow the in vitro coexistence of Pseudomonas aeruginosa and Staphylococcus aureus in stable biofilms. Sci Rep. 2019;9(1):16284. doi: 10.1038/s41598-019-52726-0. PMID 31705015.

31.         Saggu SK, Jha G, Mishra PC. Enzymatic degradation of biofilm by metalloprotease from Microbacterium sp. Sks10. Front Bioeng Biotechnol. 2019 Aug 7;7(8):192. doi: 10.3389/fbioe.2019.00192, PMID 31448272.

32.         Sharma D, Saharan BS, Chauhan N, Procha S, Lal S. Isolation and functional characterization of novel biosurfactant produced by Enterococcus faecium. Springerplus. 2015;4:4. doi: 10.1186/2193-1801-4-4, PMID 25674491.

33.         Wilson C, Lukowicz R, Merchant S, Valquier-Flynn H, Caballero J, Sandoval J, Okuom M, Huber C, Brooks TD, Wilson E, Clement B, Wentworth CD, Holmes AE. Quantitative and qualitative assessment methods for biofilm growth: A mini-review. Res Rev J Eng Technol. 2017 Oct 24;6(4):1-42. PMID 30214915.

34.         Janek T, ?ukaszewicz M, Krasowska A. Identification and characterization of biosurfactants produced by the Arctic bacterium Pseudomonas putida BD2. Colloids Surf B Biointerfaces. 2013 Jun 10;110:379-86. doi: 10.1016/j.colsurfb.2013.05.008, PMID 23751417.

35.         Marques SC, Rezende JdGOS, Alves LAdF, Silva BC, Alves E, Abreu LRd, Piccoli RH. Formation of biofilms by Staphylococcus aureus on stainless steel and glass surfaces and its resistance to some selected chemical sanitizers. Braz J Microbiol;38(3):538-43. doi: 10.1590/S1517-83822007000300029.

36.         Grillon A, Schramm F, Kleinberg M, Jehl F. Comparative activity of ciprofloxacin, levofloxacin and moxifloxacin against Klebsiella pneumoniae, Pseudomonas aeruginosa and Stenotrophomonas maltophilia assessed by minimum inhibitory concentrations and time-kill studies. PLOS ONE. 2016 Jun 3;11(6):e0156690. doi: 10.1371/journal.pone.0156690, PMID 27257956.

37.         Nahar S, Mizan MFR, Ha AJ, Ha S. Advances and Future Prospects of Enzyme-Based biofilm Prevention Approaches in the Food Industry: enzyme-based biofilm prevention…. Compr Rev Food Sci Food Saf. 2018 Sep 4;17(6):1484-502. doi: 10.1111/1541-4337.12382.

38.         Seghal Kiran G, Nishanth Lipton A, Kennedy J, Dobson AD, Selvin J. A halotolerant thermostable lipase from the marine bacterium Oceanobacillus sp. PUMB02 with an ability to disrupt bacterial biofilms. Bioengineered. 2014;5(5):305-18. doi: 10.4161/bioe.29898, PMID 25482232.

39.         Tielen P, Rosenau F, Wilhelm S, Jaeger KE, Flemming HC, Wingender J. Extracellular enzymes affect biofilm formation of mucoid Pseudomonas aeruginosa. Microbiol (Reading). 2010, Mar 30;156(7):2239-52. doi: 10.1099/mic.0.037036-0, PMID 20360178.

40.         Banar M, Emaneini M, Satarzadeh M, Abdellahi N, Beigverdi R, Leeuwen WB, Jabalameli F. Evaluation of mannosidase and trypsin enzymes effects on biofilm production of Pseudomonas aeruginosa isolated from burn wound infections. PLOS ONE. 2016, Oct 13;11(10):e0164622. doi: 10.1371/journal.pone.0164622, PMID 27736961.

41.         Molobela IP. Cloete, TE2 and Beukes M. Protease and amylase enzymes for biofilm removal and degradation of extracellular polymeric substances (EPS) produced by Pseudomonas fluorescens bacteria. Afric. J Microbiol Res. 2010 Jul 18;4(14):1515-24.

42.         Penta J, Jannu KK, Musthyala R. Antimicrobial studies of selected antibiotics and their combination with enzymes. Int J Pharm Sci. 2010 Feb 22;2(3):43-4.

43.         Alkawash MA, Soothill JS, Schiller NL. Alginate lyase enhances antibiotic killing of mucoid Pseudomonas aeruginosa in biofilms. APMIS. 2006;114(2):131-8. doi: 10.1111/j.1600-0463.2006.apm_356.x, PMID 16519750.

44.         Tetz GV, Artemenko NK, Tetz VV. Effect of DNase and antibiotics on biofilm characteristics. Antimicrob Agents Chemother. 2009 Dec 8;53(3):1204-9. doi: 10.1128/AAC.00471-08, PMID 19064900.

45.         Fanaei Pirlar R, Emaneini M, Beigverdi R, Banar M, B van Leeuwen W, Jabalameli F. Combinatorial effects of antibiotics and enzymes against dual-species Staphylococcus aureus and Pseudomonas aeruginosa biofilms in the wound-like medium. PLOS ONE. 2020, Jun 25;15(6):e0235093. doi: 10.1371/journal.pone.0235093, PMID 32584878.

46.         Eckhart L, Fischer H, Barken KB, Tolker-Nielsen T, Tschachler E. DNase1L2 suppresses biofilm formation by Pseudomonas aeruginosa and Staphylococcus aureus. Br J Dermatol. 2007 Apr 25;156(6):1342-5. doi: 10.1111/j.1365-2133.2007.07886.x, PMID 17459041.

47.         Huki? M, Seljmo D, Ramovic A, Ibrišimovi? MA, Dogan S, Hukic J, Bojic EF. The effect of lysozyme on reducing biofilms by Staphylococcus aureus, Pseudomonas aeruginosa, and Gardnerella vaginalis: an in vitro examination. Microb Drug Resist. 2018 May 22;24(4):353-8. doi: 10.1089/mdr.2016.0303, PMID 28922066.