اثرات ضدبیوفیلمی و ضد باکتریایی سویه های تولید کننده باکتریوسین اشریشیا کلی و باسیلوس سوبتلیس
محورهای موضوعی : میکروب شناسی پزشکی
محمدرضا سرجوقیان
1
(کارشناس ارشد، گروه زیست فناوری میکروبی، دانشکده بیوتکنولوژی، پردیس علوم و فناوری نوین، دانشگاه سمنان)
شکیبا درویش علیپور آستانه
2
(استادیار، گروه زیست فناوری میکروبی، دانشکده بیوتکنولوژی، پردیس علوم و فناوری نوین، دانشگاه سمنان)
کلید واژه: استافیلوکوکوس اورئوس, باکتریوسین, کلبسیلا نمونیه, کروماتوگرافی لایه نازک, سودوموناس ائروجینوسا,
چکیده مقاله :
سابقه و هدف: شیوع عفونت، به همراه گسترش باکتریهای مقاوم به آنتی‌بیوتیک، سبب شده است که ترکیبات پپتیدی تولید شده توسط باکتری‌ها (باکتریوسین‌ها) مورد توجه قرار گیرند. این مطالعه با هدف مقایسه اثر ضد میکروبی در باکتریهای دارای توانایی تولید باکتریوسین انجام شد.مواد و روش ها: فعالیت بازدارندگی باکتریهای جدا شده از خاک نواحی مختلف سمنان (کلکسیون میکروبی دانشگاه سمنان (DDBCC ، علیه باکتریهای شاخص به روش انتشار در آگار بررسی گردید. سویه‌های منتخب با آزمون‌های بیوشیمیایی و مولکولی شناسایی شدند. پس از تغلیظ باکتریوسین‌های منتخب با کمک غلظت اشباعی سولفات آمونیوم، اثرات تخریب بیوفیلم در هر باکتریوسین مطالعه و سپس به روش کروماتوگرافی لایه نازک تخلیص و فعالیت ضد میکروبی آنها بررسی شد.یافته ها: نتایج تعیین توالی نشان داد که سویه‌های منتخب DDBCC38 و DDBCC51 دارای شباهت 95 درصدی به اشریشیا کلی و سویه DDBCC46 شباهت 96 درصدی به باسیلوس سوبتلیس بود. باکتریوسین های تغلیظ شده DDBCC51 و DDBCC38 رشد باسیلوس سرئوس، باسیلوس آنتراسیس، کلبسیلا نمونیه را پس از 52 ساعت مهار کرده و بیوفیلم سودوموناس ائروجینوسا و استافیلوکوکوس اورئوس را به ترتیب 12، 40 درصد و 19.6، 40 درصد تخریب کرد. در مقابل باکتریوسین سویه DDBCC46 در 72 ساعت علیه کلبسیلا نمونیه و سودوموناس ائروجینوسا موثر بود. اما تاثیری بر تخریب بیوفیلم نشان نداد. تخلیص باکتریوسین DDBCC38 و DDBCC51، با استفاده از کروماتوگرافی لایه نازک، قطر هاله عدم رشد را علیه کلبسیلا نمونیه 2 میلی‌متر افزایش داد. اما باکتریوسین DDBCC46 قطر هاله را 4 میلی‌متر کاهش داد.نتیجه‌گیری: با توجه به ویژگی های ضد بیوفیلمی و آنتاگونیسمی جدایه های مورد بررسی، انجام مطالعات تکمیلی به منظور بهینه سازی شرایط تولید و شناسایی مولکولی نوع باکتریوسین تولیدی پیشنهاد می گردد.
Background & Objectives: The prevalence of infection, along with the spread of antibiotic-resistant bacteria has caused the antibacterial peptide, bacteriocins, to be considered. The purpose of this study was to compare the antibacterial effect of bacteriocin-producing bacteria.Materials & Methods: The inhibitory activity of bacteria isolated from different regions of Semnan soil, Dasht Desert (Semnan University Bacterial Culture Collection; DDBCC) was studied against the indicators by agar diffusion method. Candidate strains were identified by biochemical and molecular tests. Concentrating the selected bacteriocins by ammonium sulfate saturation, the effects of biofilm destruction were studied. Then, using a thin layer chromatography the selected bacteriocins were purified and their antimicrobial activity was confirmed.Results: The 16s rRNA sequencing results showed 98% similarity of DDBCC38 and DDBCC51 isolates to E. coli and 96% similarity of the DDBCC46 isolate to Bacillus subtilis. Concentrated DDBCC51 and DDBCC38 bacteriocins inhibited the growth of Bacillus cereus, Bacillus anthracis, and Klebsiella pneumonia after 52 hours. Pseudomonas aeruginosa and Staphylococcus aureus biofilms were destructed by DDBCC51 and DDBCC38 bacteriocins 12.0% and 40.0%, and 19.6% and 40%, respectively. In contrast, concentrated DDBCC46 bacteriocin was effective on K. pneumonia and P. aeruginosa in 72 hours but had no effect on biofilm destruction. The purification of DDBCC38 and DDBCC51 bacteriocins by thin layer chromatography resulted in 2 mm increasing of inhibition zone diameter against K. pneumoniae. However, purified DDBCC46 bacteriocin reduced it by 4 mm.Conclusion: Considering the anti-biofilm and antagonistic properties of the respective isolates, further studies for optimization of the production conditions and molecular identification of the produced bacteriocin are proposed.
PG, Gardiner GE. Assessment of the bacteriocinogenic potential of marine bacteria reveals
lichenicidin production by seaweed-derived Bacillus spp. Mar Drugs. 2012; 10(10): 2280-2299.
2. Riley MA, Wertz JE. Bacteriocins: evolution, ecology, and application. Annu Annual Rev
Microbial. 2002; 56(1): 117-137.
3. O’Connor oss ill C Co er D. n imicro ial an a onis s a ains ood pa ho ens
A bacteriocin perspective. Curr Opin Food Sci. 2015; 2: 51-57.
4. Oscariz JC, Lasa I, Pisabarro AG. Detection and characterization of cerein 7, a new bacteriocin
produced by Bacillus cereus with a broad spectrum of activity. FEMS Microbiol Lett. 1999;
178.
5. Maróti G, Kereszt A, Kondorosi E, Mergaert P. Natural roles of antimicrobial peptides in
microbes, plants and animals. Res Microbiol. 2011; 162(4): 363-374.
6. Chopra L, Singh G, Jena KK, Sahoo DK. Sonorensin: A new bacteriocin with potential of an
anti-biofilm agent and a food biopreservative. Sci Rep. 2015; 5: 13412.
7. O’ ullivan O ill C oss Co er . Bac eriocin inin in e a enomes. nc clopedia o
metagenomics: Springer; 2015. p: 54-60.
8. An J, Zhu W, Liu Y, Zhang X, Sun L, Hong P, Wang Y, Xu C, Xu D, Liu H. Purification and
characterization of a novel bacteriocin CAMT2 produced by Bacillus amyloliquefaciens
isolated from marine fish Epinephelus areolatus. Food Control. 2015; 51: 278-282.
9. Mathur H, C Rea M, D Cotter P, Hill C, Paul Ross R. The sactibiotic subclass of bacteriocins:
an update. Curr Protein Pept Sci. 2015; 16(6): 549-558.
10. Hassan M, Diep DB, Javadzadeh Y, Dastmalchi S, Nes IF, Sharifi Y, Yari S, Farajnia S,
Lotfipour F. Prevalence of bacteriocin activities and bacteriocin-encoding genes in Enterococcal
clinical isolates in Iran. Can J Microbiol. 2012; 58(4): 359-368.
11. Gálvez A, López RL, Pulido RP, Burgos MJG. Application of lactic acid bacteria and their
bacteriocins for food biopreservation. Food Biopreservation: Springer, 2014; p: 15-22.
12. ęs a adni Zielińs a D ołoż n-Krajewska D. Potential of bacteriocins from lab to
improve microbial quality of dry-cured and fermented meat products. Acta Sci Pol Technol
Aliment. 2017; 16(2): 119-126.
13. Prieto ML, O'Sullivan L, Tan SP, McLoughlin P, Hughes H, Gutierrez M, Lane JA, Hickey
RM, Lawlor PG, Gardiner GE. In vitro assessment of marine Bacillus for use as livestock
probiotics. Mari Drugs. 2014; 12(5): 2422-2445.
14. Balciunas EM, Martinez FAC, Todorov SD, de Melo Franco BDG, Converti A, de Souza
Oliveira RP. Novel biotechnological applications of bacteriocins: a review. Food Control. 2013;
32(1): 134-142.
15. Cotter PD, Ross RP, Hill C. Bacteriocins- a viable alternative to antibiotics? Nat Rev
Microbiol. 2013; 11(2): 95-105.
16. Vos P, Garrity G, Jones D, Krieg NR, Ludwig W, Rainey FA, Schleifer K-H, Whitman W.
Bergey's Manual of Systematic Bacteriology: Volume 3: The Firmicutes: Springer Science &
Business Media; 2011.
17. Pevsner J. Analysis of genomic DNA with the UCSC genome browser. Bioinformatics for
DNA sequence analysis. Methods Mol Biol. 2009; 537: 277-301.
18. Mouloud G, Daoud H, Bassem J, Atef IL, Hani B. New bacteriocin from Bacillus clausii
strainGM17: purification, characterization, and biological activity. Appl Biochem Biotechnol.
2013; 171(8): 2186-2200.
19. Bodour AA, Miller-Maier RM. Application of a modified drop-collapse technique for
surfactant quantitation and screening of biosurfactant-producing microorganisms. J Microbiol
Method. 1998; 32(3): 273-280.
20. Dunder H. Characterization and purification of a bacteriocin produced by Leuconostoc
mesenteroides subsp. cremoris: Middle East Technical University; 2006.
21. Bakkiyaraj D, Sivasankar C, Pandian SK. Inhibition of quorum sensing regulated biofilm
formation in Serratia marcescens causing nosocomial infections. Bioorg. Med Chem Lett. 2012;
22(9): 3089-3094.
22. Daba H, Pandian S, Gosselin J, Simard R, Huang J, Lacroix C. Detection and activity of a
bacteriocin produced by Leuconostoc mesenteroides. Appl Environ Microbiol. 1991; 57(12):
3450-3455.
23. Selvendran M, Babu M. Studies on novel bacteriocin like inhibitory substance (BLIS) from
microalgal symbiotic Vibrio spp MMB2 and its activity against aquatic bacterial pathogens. J
Appl Pharm Sci. 2013; 3(2): 169.
24. Biswas A, Banerjee R. A lab originated bacteriocin and its partial purification and
demonstration of antimicrobial activity. Int J Curr Microbiol App Sci. 2016; 5(3): 728-737.
25. Cherif A, Ouzari H, Daffonchio D, Cherif H, Ben Slama K, Hassen A, Jaoua S, Boudabous A.
Thuricin 7: a novel bacteriocin produced by Bacillus thuringiensis BMG1. 7, a new strain
isolated from soil. Lett Appl Microbiol. 2001; 32(4): 243-247.
26. He Z, Kisla D, Zhang L, Yuan C, Green-Church KB, Yousef AE. Isolation and identification
of a Paenibacillus polymyxa strain that coproduces a novel lantibiotic and polymyxin. Appl
Environ Microbiol. 2007; 73(1): 168-178.
27. Hammami I, Rhouma A, Jaouadi B, Rebai A, Nesme X. Optimization and biochemical
characterization of a bacteriocin from a newly isolated Bacillus subtilis strain 14B for
biocontrol of Agrobacterium spp. strains. Lett Appl Microbiol. 2009; 48(2): 253-260.
28. Stern NJ, Svetoch EA, Eruslanov BV, Kovalev YN, Volodina LI, Perelygin VV, Mitsevich
EV, Mitsevich IP, Levchuk VP. Paenibacillus polymyxa purified bacteriocin to control
Campylobacter jejuni in chickens. J Food Prot. 2005; 68(7): 1450-1453.
29. Naghmouchi K, Paterson L, Forster B, McAllister T, Ohene-Adjei S, Drider D, Teather R,
Baah J. Paenibacillus polymyxa JB05-01-1 and its perspectives for food conservation and
medical applications. Arch Microbiol. 2011; 193(3): 169-177.
30. Motta AS, Cladera-Olivera F, Brandelli A. Screening for antimicrobial activity among bacteria
isolated from the Amazon basin. Braz J Microbiol. 2004; 35(4): 307-310.
31. Shelar SS, Warang SS, Mane SP, Sutar RL, Ghosh JS. Characterization of bacteriocin
produced by Bacillus atrophaeus strain JS-2. Int J Biol Chem. 2012; 6: 10-16.
32. Wan X, Li R, Saris PE, Takala TM. Genetic characterisation and heterologous expression of
leucocin C, a class IIa bacteriocin from Leuconostoc carnosum 4010. Appl Microbiol
Biotechnol. 2013; 97(8): 3509-3518.
33. Ahern M, Verschueren S, Sinderen D. Isolation and characterisation of a novel bacteriocin
produced by Bacillus thuringiensis strain B439. FEMS Microbiol Lett. 2003; 220(1): 127-131.
34. Kamoun F, Mejdoub H, Aouissaoui H, Reinbolt J, Hammami A, Jaoua S. Purification, amino
acid sequence and characterization of Bacthuricin F4, a new bacteriocin produced by Bacillus
thuringiensis. J Appl Microbiol. 2005; 98(4): 881-888.
35. Al-Mathkhury HJF, Ali AS, Ghafil JA. Antagonistic effect of bacteriocin against urinary
catheter associated Pseudomonas aeruginosa biofilm. N Am J Med Sci . 2011; 3(8): 367.
36. Gupta MK, Gauri S, Shrivastava A. Assessment of animicrobial potential of Bacillus cereus
isolated from extreme environmental condition. J Microbiol Biotechnol Res. 2017; 3(2)
_||_
PG, Gardiner GE. Assessment of the bacteriocinogenic potential of marine bacteria reveals
lichenicidin production by seaweed-derived Bacillus spp. Mar Drugs. 2012; 10(10): 2280-2299.
2. Riley MA, Wertz JE. Bacteriocins: evolution, ecology, and application. Annu Annual Rev
Microbial. 2002; 56(1): 117-137.
3. O’Connor oss ill C Co er D. n imicro ial an a onis s a ains ood pa ho ens
A bacteriocin perspective. Curr Opin Food Sci. 2015; 2: 51-57.
4. Oscariz JC, Lasa I, Pisabarro AG. Detection and characterization of cerein 7, a new bacteriocin
produced by Bacillus cereus with a broad spectrum of activity. FEMS Microbiol Lett. 1999;
178.
5. Maróti G, Kereszt A, Kondorosi E, Mergaert P. Natural roles of antimicrobial peptides in
microbes, plants and animals. Res Microbiol. 2011; 162(4): 363-374.
6. Chopra L, Singh G, Jena KK, Sahoo DK. Sonorensin: A new bacteriocin with potential of an
anti-biofilm agent and a food biopreservative. Sci Rep. 2015; 5: 13412.
7. O’ ullivan O ill C oss Co er . Bac eriocin inin in e a enomes. nc clopedia o
metagenomics: Springer; 2015. p: 54-60.
8. An J, Zhu W, Liu Y, Zhang X, Sun L, Hong P, Wang Y, Xu C, Xu D, Liu H. Purification and
characterization of a novel bacteriocin CAMT2 produced by Bacillus amyloliquefaciens
isolated from marine fish Epinephelus areolatus. Food Control. 2015; 51: 278-282.
9. Mathur H, C Rea M, D Cotter P, Hill C, Paul Ross R. The sactibiotic subclass of bacteriocins:
an update. Curr Protein Pept Sci. 2015; 16(6): 549-558.
10. Hassan M, Diep DB, Javadzadeh Y, Dastmalchi S, Nes IF, Sharifi Y, Yari S, Farajnia S,
Lotfipour F. Prevalence of bacteriocin activities and bacteriocin-encoding genes in Enterococcal
clinical isolates in Iran. Can J Microbiol. 2012; 58(4): 359-368.
11. Gálvez A, López RL, Pulido RP, Burgos MJG. Application of lactic acid bacteria and their
bacteriocins for food biopreservation. Food Biopreservation: Springer, 2014; p: 15-22.
12. ęs a adni Zielińs a D ołoż n-Krajewska D. Potential of bacteriocins from lab to
improve microbial quality of dry-cured and fermented meat products. Acta Sci Pol Technol
Aliment. 2017; 16(2): 119-126.
13. Prieto ML, O'Sullivan L, Tan SP, McLoughlin P, Hughes H, Gutierrez M, Lane JA, Hickey
RM, Lawlor PG, Gardiner GE. In vitro assessment of marine Bacillus for use as livestock
probiotics. Mari Drugs. 2014; 12(5): 2422-2445.
14. Balciunas EM, Martinez FAC, Todorov SD, de Melo Franco BDG, Converti A, de Souza
Oliveira RP. Novel biotechnological applications of bacteriocins: a review. Food Control. 2013;
32(1): 134-142.
15. Cotter PD, Ross RP, Hill C. Bacteriocins- a viable alternative to antibiotics? Nat Rev
Microbiol. 2013; 11(2): 95-105.
16. Vos P, Garrity G, Jones D, Krieg NR, Ludwig W, Rainey FA, Schleifer K-H, Whitman W.
Bergey's Manual of Systematic Bacteriology: Volume 3: The Firmicutes: Springer Science &
Business Media; 2011.
17. Pevsner J. Analysis of genomic DNA with the UCSC genome browser. Bioinformatics for
DNA sequence analysis. Methods Mol Biol. 2009; 537: 277-301.
18. Mouloud G, Daoud H, Bassem J, Atef IL, Hani B. New bacteriocin from Bacillus clausii
strainGM17: purification, characterization, and biological activity. Appl Biochem Biotechnol.
2013; 171(8): 2186-2200.
19. Bodour AA, Miller-Maier RM. Application of a modified drop-collapse technique for
surfactant quantitation and screening of biosurfactant-producing microorganisms. J Microbiol
Method. 1998; 32(3): 273-280.
20. Dunder H. Characterization and purification of a bacteriocin produced by Leuconostoc
mesenteroides subsp. cremoris: Middle East Technical University; 2006.
21. Bakkiyaraj D, Sivasankar C, Pandian SK. Inhibition of quorum sensing regulated biofilm
formation in Serratia marcescens causing nosocomial infections. Bioorg. Med Chem Lett. 2012;
22(9): 3089-3094.
22. Daba H, Pandian S, Gosselin J, Simard R, Huang J, Lacroix C. Detection and activity of a
bacteriocin produced by Leuconostoc mesenteroides. Appl Environ Microbiol. 1991; 57(12):
3450-3455.
23. Selvendran M, Babu M. Studies on novel bacteriocin like inhibitory substance (BLIS) from
microalgal symbiotic Vibrio spp MMB2 and its activity against aquatic bacterial pathogens. J
Appl Pharm Sci. 2013; 3(2): 169.
24. Biswas A, Banerjee R. A lab originated bacteriocin and its partial purification and
demonstration of antimicrobial activity. Int J Curr Microbiol App Sci. 2016; 5(3): 728-737.
25. Cherif A, Ouzari H, Daffonchio D, Cherif H, Ben Slama K, Hassen A, Jaoua S, Boudabous A.
Thuricin 7: a novel bacteriocin produced by Bacillus thuringiensis BMG1. 7, a new strain
isolated from soil. Lett Appl Microbiol. 2001; 32(4): 243-247.
26. He Z, Kisla D, Zhang L, Yuan C, Green-Church KB, Yousef AE. Isolation and identification
of a Paenibacillus polymyxa strain that coproduces a novel lantibiotic and polymyxin. Appl
Environ Microbiol. 2007; 73(1): 168-178.
27. Hammami I, Rhouma A, Jaouadi B, Rebai A, Nesme X. Optimization and biochemical
characterization of a bacteriocin from a newly isolated Bacillus subtilis strain 14B for
biocontrol of Agrobacterium spp. strains. Lett Appl Microbiol. 2009; 48(2): 253-260.
28. Stern NJ, Svetoch EA, Eruslanov BV, Kovalev YN, Volodina LI, Perelygin VV, Mitsevich
EV, Mitsevich IP, Levchuk VP. Paenibacillus polymyxa purified bacteriocin to control
Campylobacter jejuni in chickens. J Food Prot. 2005; 68(7): 1450-1453.
29. Naghmouchi K, Paterson L, Forster B, McAllister T, Ohene-Adjei S, Drider D, Teather R,
Baah J. Paenibacillus polymyxa JB05-01-1 and its perspectives for food conservation and
medical applications. Arch Microbiol. 2011; 193(3): 169-177.
30. Motta AS, Cladera-Olivera F, Brandelli A. Screening for antimicrobial activity among bacteria
isolated from the Amazon basin. Braz J Microbiol. 2004; 35(4): 307-310.
31. Shelar SS, Warang SS, Mane SP, Sutar RL, Ghosh JS. Characterization of bacteriocin
produced by Bacillus atrophaeus strain JS-2. Int J Biol Chem. 2012; 6: 10-16.
32. Wan X, Li R, Saris PE, Takala TM. Genetic characterisation and heterologous expression of
leucocin C, a class IIa bacteriocin from Leuconostoc carnosum 4010. Appl Microbiol
Biotechnol. 2013; 97(8): 3509-3518.
33. Ahern M, Verschueren S, Sinderen D. Isolation and characterisation of a novel bacteriocin
produced by Bacillus thuringiensis strain B439. FEMS Microbiol Lett. 2003; 220(1): 127-131.
34. Kamoun F, Mejdoub H, Aouissaoui H, Reinbolt J, Hammami A, Jaoua S. Purification, amino
acid sequence and characterization of Bacthuricin F4, a new bacteriocin produced by Bacillus
thuringiensis. J Appl Microbiol. 2005; 98(4): 881-888.
35. Al-Mathkhury HJF, Ali AS, Ghafil JA. Antagonistic effect of bacteriocin against urinary
catheter associated Pseudomonas aeruginosa biofilm. N Am J Med Sci . 2011; 3(8): 367.
36. Gupta MK, Gauri S, Shrivastava A. Assessment of animicrobial potential of Bacillus cereus
isolated from extreme environmental condition. J Microbiol Biotechnol Res. 2017; 3(2)
