اثر هم افزایی مهاری آنزیم آلژینات لیاز نوترکیب باکتریایی و آنتی بیوتیک های رایج درمانی بر رشد سلول های پلانکتونی سودوموناس آئروجینوسا
محورهای موضوعی : میکروب شناسی پزشکیحدیث طوافی 1 , احیا عبدی عالی 2 , پریناز قدم 3 , سارا غروی 4
1 - استادیار، دانشگاه ملایر، دانشکده ی علوم پایه، گروه زیست شناسی
2 - دانشیار، دانشگاه الزهرا، دانشکده ی علوم زیستی، گروه میکروبیولوژی
3 - دانشیار، دانشگاه الزهرا، دانشکده ی علوم زیستی، گروه بیوتکنولوژی
4 - دانشیار، دانشگاه الزهرا، دانشکده ی علوم زیستی، گروه بیوتکنولوژی
کلید واژه: سودوموناس آئروجینوسا, آلژینات لیاز, سلول های پلانکتونی, مهارآنتی بیوتیکی,
چکیده مقاله :
سابقه و هدف: آلژینات، یکی از مهمترین عوامل بیماری زایی سودوموناس آئروجینوسا است. این مطالعه با هدف سنجش اثر هم افزایی آلژینات لیاز نوترکیب باکتریایی و آنتی بیوتیک های رایج در درمان عفونت های سودوموناسی بر رشد سلول های پلانکتونی سودوموناس آئروجینوسا انجام شد.مواد و روش‌ها: در مطالعه حاضر سودوموناس آئروجینوسا TAG48 از نمونه بالینی جدا سازی و شناسایی شد. به منظور تخلیص آنزیم آلژینات لیاز و اثر آن بر سلول های پلانکتونی، ژن آلژینات لیاز (algL) جداسازی، همسانه سازی، تعیین توالی و بیان شد و با استفاده از ستون کروماتوگرافی تمایلی تخلیص گردید. اثر آنزیم خالص شده و آنتی بیوتیک های سیپروفلوکساسین، توبرامایسین و سفکسیم بر سلول های پلانکتونی سودوموناس آئروجینوسا بررسی شد و آزمون های حداقل غلظت مهاری، حداقل غلظت کشندگی و همچنین بررسی اثر توام آنتی بیوتیک ها و آنزیم بر سلول های یاد شده ارزیابی شد.یافته‌ها: بررسی آنتی بیوتیک ها و آنزیم نشان دادند که MIC مربوط به سیپروفلوکساسین، توبرامایسین، سفکسیم و آنزیم آلژینات لیاز در باکتریTAG48 به ترتیب 4، 16، 128 و 9.37 میکروگرم بر میلی لیتر می باشد. میزان MBC نیز برابر با MIC محاسبه گردید. همچنین نتایج نشان داد که آلژینات لیاز با توبرامایسین و سفکسیم بر رشد سلول های پلانکتونی سودوموناس آئروجینوسا TAG48 اثرهم افزایی مهاری دارند، اما این اثر هم افزایی با سیپروفلوکساسین دیده نشد.نتیجه گیری: با توجه به اهمیت آلژینات در بیماری زایی سودوموناس آئروجینوسا، تخریب آن می تواند در کاهش بیماری زایی موثر باشد. شناسایی ژن های جدید algL در جوامع میکروبی می تواند رویکرد جدیدی برای مطالعه و تحقیق آلژینات لیاز هایی با فعالیت های ویژه علیه آلژینات های باکتریایی در نمونه های میکروبی بالینی باشد.
Background & Objectives: Alginate is one of the most important virulence factors of Pseudomonas aeruginosa. This study aimed to evaluate the synergistic effect of recombinant alginate lyase and common antibiotics in the treatment of Pseudomonas infections on the growth of planktonic cells of P. aeruginosa. Materials & Methods: In this study, P. aeruginosa TAG48 was isolated and identified from the clinical sample. To purify alginate lyase and its effect on planktonic cells, the alginate lyase gene (algL) was isolated, cloned, sequenced and expressed. The resultant enzyme was purified by affinity chromatography. The ciprofloxacin, tobramycin, and cefixime were also used to test the effectiveness of these antibiotics on planktonic cells of P. aeruginosa by carrying out MIC, MBC. The synergistic effects of these antibiotics and the recombinant alginate lyase enzyme on planktonic cells were evaluated. Results: Our results indicated that the antibiotics and the enzyme have shown MIC for ciprofloxacin, tobramycin, cefixime and enzyme in the following concentrations: 4, 16,128 and 9.37 μg/ml, respectively. MBC was also calculated equal to MIC. Also, alginate lyase exhibited synergy with tobramycin and cefixime on the growth of planktonic cells of P. aeruginosa TAG48 but not with ciprofloxacin. Conclusion: Regarding the importance of alginate in the pathogenicity of P. aeruginosa, its degradation could reduce this characteristic of the bacteria. Detection of novel algL genes in bacterial communities can also lead the way for the study of alginate lyases with specific activities against alginates from pathogenic bacteria in microbial samples.
and clinical Pseudomonas aeruginosa. Microbes Environ. 2010; 25(4): 266-274.
2. Tashiro Y, Yawata Y, Toyofuku M, Uchiyama H, Nomura N. Interspecies interaction between
Pseudomonas aeruginosa and other microorganisms. Microbes Environ. 2013; 28(1): 13-24.
3. Silby MW, Winstanley C, Godfrey SA, Levy SB, Jackson RW. Pseudomonas genomes: diverse and adaptable. FEMS Microbiol Rev. 2011; 35(4): 652-680.
4. 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.
5. Lutz L, Pereira DC, Paiva RM, Zavascki AP, Barth AL. Macrolides decrease the minimal
inhibitory concentration of anti-pseudomonal agents against Pseudomonas aeruginosa from
cystic fibrosis patients in biofilm. BMC Microbiol. 2012; 12(1): 1.
6. Ghafoor A, Hay ID, Rehm BH. Role of exopolysaccharides in Pseudomonas aeruginosa
biofilm formation and architecture. Appl Environ Microbiol. 2011; 77(15): 5238-5246.
7. Wei Q, Ma LZ. Biofilm matrix and its regulation in Pseudomonas aeruginosa. Int J Mol Sci.
2013; 14(10): 20983-31005.
8. Wong TY, Preston LA, Schiller NL. Alginate lyase: review of major sources and enzyme
characteristics, structure-function analysis, biological roles, and applications. Annu Rev
Microbiol. 2000; 54(1): 289-340.
9. Shankar T, Sivakumar T, Satya C, Ponmanickam P. Purification, characterization and
immobilization of alginase produced by Bacillus sp associated with Sargassum wightii.
Universal J Microbiol Res. 2016; 4(1): 11-22.
10.Zhu B, Yin H. Alginate lyase: Review of major sources and classification, properties, structure
-function analysis and applications. Bioengineered. 2015; 6(3): 125-131.
11. Park HH, Kam N, Lee EY, Kim HS. Cloning and characterization of a novel oligoalginate
lyase from a newly isolated bacterium Sphingomonas sp. MJ-3. Mar Biotechnol. 2012; 14(2):
189-202.
12. Kim HS, Lee C-G, Lee EY. Alginate lyase: structure, property, and application. Biotechnol
Bioprocess Eng. 2011; 16(5): 843-851.
13. Ghadaksaz A, Fooladi AAI, Hosseini HM, Amin M. The prevalence of some Pseudomonas
virulence genes related to biofilm formation and alginate production among clinical isolates.
J Appl Biomed. 2015; 13(1): 61-68.
14. Preis J, Ashwell G. Alginic acid metabolism in bacteria. J Biol Chem. 1962; 237: 309-316.
15. English BK, Gaur AH. The use and abuse of antibiotics and the development of antibiotic
resistance. Hot Topics in Infection and Immunity in Children VI: Springer; 2010. p. 73-82.
16. Chatterjee M, Anju C, Biswas L, Kumar VA, Mohan CG, Biswas R. Antibiotic resistance in
Pseudomonas aeruginosa and alternative therapeutic options. Int J Med Microbiol. 2016; 306
(1): 48-58.
17. Tavafi H, Abdi-Ali AA, Ghadam P, Gharavi S. Screening of alginate lyase-producing bacteria
and optimization of media compositions for extracellular alginate lyase production. Iran
Biomed J. 2017; 21(1): 48.
18. Tavafi H, Ali AA, Ghadam P, Gharavi S. Screening, cloning and expression of a novel
alginate lyase gene from P. aeruginosa TAG 48 and its antibiofilm effects on P. aeruginosa
biofilm. Microb Pathog. 2018; 124: 356-364.
19. CLSI. Performance standards for antimicrobial susceptibility testing; 22nd Informational
Supplement. CLSI Document M100-S22. Clinical and Laboratory Standards Institute Wayne,
Pennsylvania; 2012.
20. Lorian V. Antibiotics in laboratory medicine: Lippincott Williams & Wilkins; 2005.
21. Sabaeifard P, Abdi-Ali A, Soudi MR, Dinarvand R. Optimization of tetrazolium salt assay for
Pseudomonas aeruginosa biofilm using microtiter plate method. J Microbiol Methods. 2014;
105: 134-140.
22. Shafiei M, Ali AA, Shahcheraghi F, Saboora A, Noghabi KA. Eradication of Pseudomonas
aeruginosa biofilms using the combination of n-butanolic cyclamen coum extract and
ciprofloxacin. Jundishapur J Microbiol. 2014; 7(2):e14358.
23. May TB, Shinabarger D, Maharaj R, Kato J, Chu L, DeVault JD. Alginate synthesis by
Pseudomonas aeruginosa: a key pathogenic factor in chronic pulmonary infections of cystic
fibrosis patients. Clin Microbiol Rev. 1991; 4(2): 191-206.
24. Wozniak DJ, Wyckoff TJ, Starkey M, Keyser R, Azadi P, O'Toole GA. Alginate is not a
significant component of the extracellular polysaccharide matrix of PA14 and PAO1
Pseudomonas aeruginosa biofilms. Proc Natl Acad Sci. 2003; 100(13): 7907-7912.
25. Dong S, Yang J, Zhang XY, Shi M, Song XY, Chen XL. Cultivable alginate lyase-excreting
bacteria associated with the arctic brown alga Laminaria. Mar Drugs. 2012; 10(11): 2481-2491.
26. Hatch RA, Schiller NL. Alginate lyase promotes diffusion of aminoglycosides through the
extracellular polysaccharide of mucoid Pseudomonas aeruginosa. Antimicrob Agents
Chemother. 1998; 42(4): 974-977.
27. Eftekhar F, Speert DP. Alginase treatment of mucoid Pseudomonas aeruginosa enhances
phagocytosis by human monocyte-derived macrophages. Infect Immun. 1988; 56(11):
2788-2793.
28. Kaneko Y, Yonemoto Y, Okayama K, Kimura A, Murata K. Symbiotic formation of alginate
lyase in mixed culture of bacteria isolated from soil. J Biosci Bioeng. 1990; 69(3): 192-194.
29. May TB, Chakrabarty AM. Pseudomonas aeruginosa: genes and enzymes of alginate
synthesis. Trends Microbiol. 1994; 2(5): 151-157.
30. Kim DE, Lee EY, Kim HS. Cloning and characterization of alginate lyase from a marine
bacterium Streptomyces sp. ALG-5. Mar Biotechnol. 2009; 11(1): 10-16.
31. Thallinger B, Prasetyo EN, Nyanhongo GS, Guebitz GM. Antimicrobial enzymes: an
emerging strategy to fight microbes and microbial biofilms. Biotechnol. J. 2013; 8(1): 97-109.
32. Cotton LA, Graham RJ, Lee RJ. The role of alginate in P. aeruginosa PAO1 biofilm structural
resistance to gentamicin and ciprofloxacin. J Exp Microbiol Immunol. 2009; 13: 58-62.
33. Tseng BS, Zhang W, Harrison JJ, Quach TP, Song JL, Penterman J. The extracellular matrix
protects Pseudomonas aeruginosa biofilms by limiting the penetration of tobramycin. Environ
Microbiol. 2013; 15(10): 2865-2878.
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and clinical Pseudomonas aeruginosa. Microbes Environ. 2010; 25(4): 266-274.
2. Tashiro Y, Yawata Y, Toyofuku M, Uchiyama H, Nomura N. Interspecies interaction between
Pseudomonas aeruginosa and other microorganisms. Microbes Environ. 2013; 28(1): 13-24.
3. Silby MW, Winstanley C, Godfrey SA, Levy SB, Jackson RW. Pseudomonas genomes: diverse and adaptable. FEMS Microbiol Rev. 2011; 35(4): 652-680.
4. 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.
5. Lutz L, Pereira DC, Paiva RM, Zavascki AP, Barth AL. Macrolides decrease the minimal
inhibitory concentration of anti-pseudomonal agents against Pseudomonas aeruginosa from
cystic fibrosis patients in biofilm. BMC Microbiol. 2012; 12(1): 1.
6. Ghafoor A, Hay ID, Rehm BH. Role of exopolysaccharides in Pseudomonas aeruginosa
biofilm formation and architecture. Appl Environ Microbiol. 2011; 77(15): 5238-5246.
7. Wei Q, Ma LZ. Biofilm matrix and its regulation in Pseudomonas aeruginosa. Int J Mol Sci.
2013; 14(10): 20983-31005.
8. Wong TY, Preston LA, Schiller NL. Alginate lyase: review of major sources and enzyme
characteristics, structure-function analysis, biological roles, and applications. Annu Rev
Microbiol. 2000; 54(1): 289-340.
9. Shankar T, Sivakumar T, Satya C, Ponmanickam P. Purification, characterization and
immobilization of alginase produced by Bacillus sp associated with Sargassum wightii.
Universal J Microbiol Res. 2016; 4(1): 11-22.
10.Zhu B, Yin H. Alginate lyase: Review of major sources and classification, properties, structure
-function analysis and applications. Bioengineered. 2015; 6(3): 125-131.
11. Park HH, Kam N, Lee EY, Kim HS. Cloning and characterization of a novel oligoalginate
lyase from a newly isolated bacterium Sphingomonas sp. MJ-3. Mar Biotechnol. 2012; 14(2):
189-202.
12. Kim HS, Lee C-G, Lee EY. Alginate lyase: structure, property, and application. Biotechnol
Bioprocess Eng. 2011; 16(5): 843-851.
13. Ghadaksaz A, Fooladi AAI, Hosseini HM, Amin M. The prevalence of some Pseudomonas
virulence genes related to biofilm formation and alginate production among clinical isolates.
J Appl Biomed. 2015; 13(1): 61-68.
14. Preis J, Ashwell G. Alginic acid metabolism in bacteria. J Biol Chem. 1962; 237: 309-316.
15. English BK, Gaur AH. The use and abuse of antibiotics and the development of antibiotic
resistance. Hot Topics in Infection and Immunity in Children VI: Springer; 2010. p. 73-82.
16. Chatterjee M, Anju C, Biswas L, Kumar VA, Mohan CG, Biswas R. Antibiotic resistance in
Pseudomonas aeruginosa and alternative therapeutic options. Int J Med Microbiol. 2016; 306
(1): 48-58.
17. Tavafi H, Abdi-Ali AA, Ghadam P, Gharavi S. Screening of alginate lyase-producing bacteria
and optimization of media compositions for extracellular alginate lyase production. Iran
Biomed J. 2017; 21(1): 48.
18. Tavafi H, Ali AA, Ghadam P, Gharavi S. Screening, cloning and expression of a novel
alginate lyase gene from P. aeruginosa TAG 48 and its antibiofilm effects on P. aeruginosa
biofilm. Microb Pathog. 2018; 124: 356-364.
19. CLSI. Performance standards for antimicrobial susceptibility testing; 22nd Informational
Supplement. CLSI Document M100-S22. Clinical and Laboratory Standards Institute Wayne,
Pennsylvania; 2012.
20. Lorian V. Antibiotics in laboratory medicine: Lippincott Williams & Wilkins; 2005.
21. Sabaeifard P, Abdi-Ali A, Soudi MR, Dinarvand R. Optimization of tetrazolium salt assay for
Pseudomonas aeruginosa biofilm using microtiter plate method. J Microbiol Methods. 2014;
105: 134-140.
22. Shafiei M, Ali AA, Shahcheraghi F, Saboora A, Noghabi KA. Eradication of Pseudomonas
aeruginosa biofilms using the combination of n-butanolic cyclamen coum extract and
ciprofloxacin. Jundishapur J Microbiol. 2014; 7(2):e14358.
23. May TB, Shinabarger D, Maharaj R, Kato J, Chu L, DeVault JD. Alginate synthesis by
Pseudomonas aeruginosa: a key pathogenic factor in chronic pulmonary infections of cystic
fibrosis patients. Clin Microbiol Rev. 1991; 4(2): 191-206.
24. Wozniak DJ, Wyckoff TJ, Starkey M, Keyser R, Azadi P, O'Toole GA. Alginate is not a
significant component of the extracellular polysaccharide matrix of PA14 and PAO1
Pseudomonas aeruginosa biofilms. Proc Natl Acad Sci. 2003; 100(13): 7907-7912.
25. Dong S, Yang J, Zhang XY, Shi M, Song XY, Chen XL. Cultivable alginate lyase-excreting
bacteria associated with the arctic brown alga Laminaria. Mar Drugs. 2012; 10(11): 2481-2491.
26. Hatch RA, Schiller NL. Alginate lyase promotes diffusion of aminoglycosides through the
extracellular polysaccharide of mucoid Pseudomonas aeruginosa. Antimicrob Agents
Chemother. 1998; 42(4): 974-977.
27. Eftekhar F, Speert DP. Alginase treatment of mucoid Pseudomonas aeruginosa enhances
phagocytosis by human monocyte-derived macrophages. Infect Immun. 1988; 56(11):
2788-2793.
28. Kaneko Y, Yonemoto Y, Okayama K, Kimura A, Murata K. Symbiotic formation of alginate
lyase in mixed culture of bacteria isolated from soil. J Biosci Bioeng. 1990; 69(3): 192-194.
29. May TB, Chakrabarty AM. Pseudomonas aeruginosa: genes and enzymes of alginate
synthesis. Trends Microbiol. 1994; 2(5): 151-157.
30. Kim DE, Lee EY, Kim HS. Cloning and characterization of alginate lyase from a marine
bacterium Streptomyces sp. ALG-5. Mar Biotechnol. 2009; 11(1): 10-16.
31. Thallinger B, Prasetyo EN, Nyanhongo GS, Guebitz GM. Antimicrobial enzymes: an
emerging strategy to fight microbes and microbial biofilms. Biotechnol. J. 2013; 8(1): 97-109.
32. Cotton LA, Graham RJ, Lee RJ. The role of alginate in P. aeruginosa PAO1 biofilm structural
resistance to gentamicin and ciprofloxacin. J Exp Microbiol Immunol. 2009; 13: 58-62.
33. Tseng BS, Zhang W, Harrison JJ, Quach TP, Song JL, Penterman J. The extracellular matrix
protects Pseudomonas aeruginosa biofilms by limiting the penetration of tobramycin. Environ
Microbiol. 2013; 15(10): 2865-2878.