Aeromonas punctata derived depolymerase improves susceptibility of Klebsiella pneumoniae biofilm to gentamicin

BMC Microbiology

Sanjay Chhibber¹  Kusum Harjai¹  Shruti Bansal¹ 

[1]Department of Microbiology, Panjab University, Sector-14, Chandigarh 160014, India

关键词: Gentamicin;    Enzyme kinetics;    Phage depolymerase;    Bacterial depolymerase;    Anti-biofilm enzyme;   

Others  :  1221667
DOI  :  10.1186/s12866-015-0455-z

 received in 2014-08-01, accepted in 2015-05-28,  发布年份 2015

【 摘 要 】

Background

To overcome antibiotic resistance in biofilms, enzymes aimed at biofilm dispersal are under investigation. In the present study, applicability of an Aeromonas punctata derived depolymerase capable of degrading the capsular polysaccharide (CPS) of Klebsiella pneumoniae, in disrupting its biofilm and increasing gentamicin efficacy against biofilm was investigated.

Results

Intact biofilm of K. pneumoniae was recalcitrant to gentamicin due to lack of antibiotic penetration. On the other hand, gentamicin could not act on disrupted biofilm cells due to their presence in clusters. However, when depolymerase (20 units/ml) was used in combination with gentamicin (10 μg/ml), dispersal of CPS matrix by enzyme facilitated gentamicin penetration across biofilm. This resulted in significant reduction (p < 0.05) in bacterial count in intact and disrupted biofilms. Reduction in CPS after treatment with depolymerase was confirmed by confocal microscopy and enzyme linked lectinosorbent assay. Furthermore, to substantiate our study, the efficacy of bacterial depolymerase was compared with a phage borne depolymerase possessing similar application against K. pneumoniae. Although both were used at same concentration i.e. 20 units/ml, but a higher efficacy of bacterial depolymerase particularly against older biofilms was visibly clear over its phage counterpart. This could be explained due to high substrate affinity (indicated by K_m value) and high turnover number (indicated by K_cat value) of the bacterial depolymerase (K_m = 89.88 μM, K_cat = 285 s⁻¹) over the phage derived one (K_m = 150 μM, K_cat = 107 s⁻¹).

Conclusion

Overall the study indicated that, the A. punctata derived depolymerase possesses antibiofilm potential and improves gentamicin efficacy against K. pneumoniae. Moreover, it can serve as a potential substitute to phage borne depolymerases for treating biofilms formed by K. pneumoniae.

【 授权许可】

   
2015 Bansal et al.

【 图 表 】

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【 参考文献 】

• [1]Flemming HC, Wingender J. The biofilm matrix. Nat Rev Microbiol. 2010; 8:623-33.
• [2]Moscoso M, García E, López R. Pneumococcal biofilms. Int Microbiol. 2009; 12:77-85.
• [3]Rezaei E, Safari H, Naderinasab M, Aliakbarian H. Common pathogens in burn wound and changes in their drug sensitivity. Burns. 2011; 37:805-7.
• [4]Sahly H, Podschun R. Clinical, bacteriological, and serological aspects of Klebsiella infections and their spondylarthropathic sequelae. Clin Diagn Lab Immunol. 1997; 4:393-9.
• [5]Lai YC, Peng HL, Chang HY. RmpA2, an activator of capsule biosynthesis in Klebsiella pneumoniae CG43, regulates K2 cps gene expression at the transcriptional level. J Bacteriol. 2003; 185:788-800.
• [6]Murphy CN, Clegg S. Klebsiella pneumoniae and type 3 fimbriae: nosocomial infection, regulation and biofilm formation. Future Microbiol. 2012; 7:991-1002.
• [7]Podschun R, Ullmann U. Klebsiella spp. as nosocomial pathogens: epidemiology, taxonomy, typing methods and pathogenicity factors. Clin Microbiol Rev. 1998; 11:589-603.
• [8]Cano V, Moranta D, Llobet-Brossa E, Bengoechea JA, Garmendia J. Klebsiella pneumoniae triggers a cytotoxic effect on airway epithelial cells. BMC Microbiol. 2009; 9:156. BioMed Central Full Text
• [9]Russo TA, Gill SR. Draft genome sequence of the hypervirulent Klebsiella pneumoniae strain hvKP1, isolated in Buffalo, New York. Genome Announce. 2013; 1:e0006513.
• [10]Stewart PS. Theoretical aspects of antibiotic diffusion into microbial biofilms. Antimicrob Agents Chemother. 1996; 40:2517-22.
• [11]Hughes KA, Sutherland IW, Clark J, Jones M. Bacteriophage and associated polysaccharide depolymerases novel tools for study of bacterial biofilms. J Appl Microbiol. 1998; 85:583-90.
• [12]Høiby N, Bjarnsholta T, Givskov M, Molinc S, Ciofu O. Antibiotic resistance of bacterial biofilms. Int J Antimic Agents. 2010; 35:322-32.
• [13]Llobet E, Tomas JM, Bengoechea JA. Capsule polysaccharide is a bacterial decoy for antimicrobial peptides. Microbiology. 2008; 154:3877-86.
• [14]Anderl JN, Zahller J, Roe F, Stewart PS. Role of nutrient limitation and stationary-phase existence in Klebsiella pneumoniae biofilm resistance to ampicillin and ciprofloxacin. Antimicrob Agents Chemother. 2003; 47:1251-6.
• [15]Donlan RM. Biofilm elimination on intravascular catheters: important considerations for the infectious disease practitioner. Clin Infect Dis. 2011; 52:1038-45.
• [16]Thallinger B, Prasetyo EN, Nyanhongo GS, Guebitz GM. Antimicrobial enzymes: an emerging strategy to fight microbes and microbial biofilms. J Biotechnol. 2013; 8:97-109.
• [17]Azeredo J, Sutherland IW. The use of phages for the removal of infectious biofilms. Curr Pharmac Biotechnol. 2008; 9:261-6.
• [18]Pastagia M, Euler C, Chahales P, Fuentes-Duculan J, Krueger JG, Fischetti VA. A novel chimeric lysin shows superiority to mupirocin for skin decolonization of methicillin resistant and sensitive Staphylococcus aureus strains. Antimicrob Agents Chemother. 2011; 55:738-44.
• [19]Bansal S, Harjai K, Chhibber S. Depolymerase improves gentamicin efficacy during Klebsiella pneumoniae induced murine infection. BMC Infect Dis. 2014; 14:456. BioMed Central Full Text
• [20]Bansal S, Harjai K, Soni SK, Chhibber S. Aeromonas punctata derived depolymerase that disrupts the integrity of Klebsiella pneumoniae capsule: Optimization of depolymerase production. J Basic Microbiol. 2013; 53:1-10.
• [21]Hanlon GW, Denyer SP, Olliff CJ, Ibrahim LJ. Reduction in exopolysaccharide viscosity as an aid to bacteriophage penetration through Pseudomonas aeruginosa biofilms. Appl Environ Microbiol. 2001; 67:2746-53.
• [22]Miller GL. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem. 1959; 31:426-8.
• [23]Verma V, Harjai K, Chhibber S. Characterization of a T7-like lytic bacteriophage of Klebsiella pneumoniae B5055: a potential therapeutic agent. Curr Microbiol. 2009; 59:274-81.
• [24]Reiger D, Molbert EF, Stirm S. Escherichia coli capsule bacteriophages III. Fragments of bacteriophage 29. J Virol. 1975; 15:964-75.
• [25]Anderl JN, Franklin MJ, Stewarti PR. Role of antibiotic penetration limitation in Klebsiella pneumoniae biofilm resistance to ampicillin and ciprofloxacin. Antimicrob Agents Chemother. 2000; 44:1818-24.
• [26]Bedi MS, Verma V, Chhibber S. Amoxicillin and specific bacteriophage can be used together for eradication of biofilm of Klebsiella pneumoniae B5055. World J Microbiol Biotechnol. 2009; 25:1145-51.
• [27]El-Azizi M, Rao S, Kanchanapoom T, Khardori N. In vitro activity of vancomycin, quinupristin/dalfopristin and linezolid against intact and disrupted biofilms of Staphylococci. Ann Clin Microbiol Antimicrob. 2005; 4:21-9. BioMed Central Full Text
• [28]Strathmann M, Wingender J, Flemming HC. Application of fluorescently labeled lectins for the visualization and biochemical characterization of polysaccharides in biofilms of Pseudomonas aeruginosa. J Microbiol Meth. 2002; 50:237-48.
• [29]Hoffman LR, D’Argenio DA, MacCoss MJ, Zhang Z, Jones RA, Miller SI. Aminoglycoside antibiotics induce bacterial biofilm formation. Nature. 2005; 436:1171-5.
• [30]Saha R, Saha N, Donofrio RS, Bestervelt LL. Microbial siderophores: a mini review. J Basic Microbiol. 2013; 53:303-17.
• [31]Leslie A. Preventing biofilm formation using microbes and their enzymes. MMG 445 Basic Biotechnol. 2011; 7:6-11.
• [32]Johansen C, Falholt P, Gram L. Enzymatic removal and disinfection of bacterial biofilms. Appl Environ Microbiol. 1997; 63:3724-8.
• [33]Lu TK, Collins JJ. Dispersing biofilms with engineered enzymatic bacteriophage. Proc Natl Acad Sci. 2007; 104:11197-202.
• [34]Lewis K. Riddle of biofilm resistance. Antimicrob Agents Chemother. 2001; 45:999-1007.
• [35]Hunt BE, Weber A, Berger A, Ramsey B, Smith AL. Macromolecular mechanisms of sputum inhibition of tobramycin activity. Antimicrob Agents Chemother. 1995; 39:34-9.
• [36]Hatch RA, Schiller NL. Alginate lyase promotes diffusion of aminoglycosides through the extracellular polysaccharide of mucoid Pseudomonas aeruginosa. Antimicrob Agents Chemother. 1998; 42:974-7.
• [37]Anwar H, Strap JL, Costerton JW. Establishment of aging biofilms: possible mechanism of bacterial resistance to antimicrobial therapy. Antimicrob Agents Chemother. 1992; 36:1347-51.
• [38]Wood LF, Leech AJ, Ohman DE. Cell wall-inhibitory antibiotics activate the alginate biosynthesis operon in Pseudomonas aeruginosa: roles of sigma (AlgT) and the AlgW and Prc proteases. Mol Microbiol. 2006; 62:412-26.
• [39]Presterl E, Grisold AJ, Reichmann S, Hirschl AM, Georgopoulos A, Graninger W. Viridans Streptococci in endocarditis and neutropenic sepsis: biofilm formation and effects of antibiotics. J Antimicrob Chemother. 2005; 55:45-50.
• [40]Izano EA, Sadovskaya I, Vinogradov E, Mulks MH, Velliyagounder K, Ragunath C et al.. Poly-N-acetylglucosamine mediates biofilm formation and antibiotic resistance in Actinobacillus pleuropneumoniae. Microb Pathog. 2007; 43:1-9.
• [41]Izano EA, Wang H, Ragunath C, Ramasubbu N, Kaplan JB. Detachment and killing of Aggregatibacter actinomycetemcomitans biofilms by dispersin B and SDS. J Dent Res. 2007; 86:618-22.
• [42]Pamp SJ, Gjermansen M, Tolker-Nielsen T. The biofilm matrix—A sticky framework. In: The Biofilm Mode of Life. Horizon Bioscience. 2007.37-69.
• [43]Ymele-Leki P, Ross JM. Erosion from Staphylococcus aureus biofilms grown under physiologically relevant fluid shear forces yields bacterial cells with reduced avidity to collagen. Appl Environ Microbiol. 2007; 73:1834-41.
• [44]Stewart PS, Costerton JW. Antibiotic resistance of bacteria in biofilms. Lancet. 2001; 358:135-8.
• [45]Jun SY, Jung GM, Son JS, Yoon SJ, Choi YJ, Kang SH. Comparison of the antibacterial properties of phage endolysins SAL-1 and LysK. Antimicrob Agents Chemother. 2011; 55:1764-7.
• [46]Mizuta K, Ohta M, Mori M, Hasegawa T, Nakashima I, Kato N. Virulence for mice of Klebsiella strains belonging to the O1 group: relationship to their capsular (K) types. Infect Immun. 1983; 40:56-61.
• [47]Mushtaq N, Redpath MB, Luzio JP, Taylor PW. Prevention and cure of systemic Escherichia coli K1 infection by modification of the bacterial phenotype. Antimicrob Agents Chemother. 2004; 48:1503-8.
• [48]Scorpio A, Tobery SA, Ribot WJ, Friedlander AM. Treatment of experimental anthrax with recombinant capsule depolymerase. Antimicrob Agents Chemother. 2008; 13:1014-20.
• [49]Hsu CR, Lin TL, Pan YJ, Hsieh PF, Wang JT. Isolation of a bacteriophage specific for a new capsular type of Klebsiella pneumoniae and characterization of its polysaccharide depolymerase. Plos One. 2013; 8:e70092.
• [50]Pleteneva EA, Bourkaltseva MV, Shaburova OV, Krylov SV, Pechnikova EV, Sokolova OS et al.. TL, the new bacteriophage of Pseudomonas aeruginosa and its application for the search of halo producing bacteriophages. Rus J Gen. 2011; 47:1-5.
• [51]Vandenbergh PA: Bacteria for expressing a polysaccharide depolymerase containing a novel recombinant plasmid. 1989, US 4822740 A.
• [52]Tomlinson S, Taylor PW. Neuraminidase associated with coliphage E that specifically depolymerizes the Escherichia coli K1 capsular polysaccharide. J Virol. 1985; 55:374-8.
• [53]Projan S. Phage-inspired antibiotics? Nat Biotechnol. 2004; 22:167-8.
• [54]Lu TK, Collins JJ. Engineered bacteriophage targeting gene networks as adjuvants for antibiotic therapy. Proc Natl Acad Sci. 2009; 106:4629-34.
• [55]Hagens S, Bläsi U. Genetically modified filamentous phage as bactericidal agents: a pilot study. Lett Appl Microbiol. 2003; 37:318-23.
• [56]Hagens S, Habel A, Ahsen U, Gabain A, Bläsi U. Therapy of experimental Pseudomonas infections with a nonreplicating genetically modified phage. Antimicrob Agents Chemother. 2004; 48:3817-22.
• [57]Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 2012; 9:671-5.