Depolymerase improves gentamicin efficacy during Klebsiella pneumoniae induced murine infection

BMC Infectious Diseases

Sanjay Chhibber¹  Kusum Harjai¹  Shruti Bansal¹ 

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

关键词: Gentamicin;    Capsule depolymerase;    Innate immune response;    Aeromonas punctata;    Klebsiella pneumoniae;   

Others  :  1125788
DOI  :  10.1186/1471-2334-14-456

 received in 2014-06-15, accepted in 2014-08-19,  发布年份 2014

【 摘 要 】

Background

Presence of capsule enhances the virulence of bacteria that cause pneumonia, meningitis, cystic fibrosis, dental caries, periodontitis. Capsule is an important virulence factor for Klebsiella pneumoniae and infections due to this pathogen have been associated with high mortality rates. In the present study, use of an Aeromonas punctata derived capsule depolymerase against K. pneumoniae, to reinstate the efficacy of gentamicin during pneumonia and septicemia was investigated.

Methods

Depolymerase was administered in mice intraperitoneally (50 μg) alone as well in combination with gentamicin (1.5 mg/kg), 24 h post infection during acute lung infection and 6 h later during septicemia. Bacterial load, neutrophil infiltration and cytokine levels were estimated. The immunogenicity of protein was also studied.

Results

In comparison to groups treated with gentamicin alone, combination treatment with depolymerase and gentamicin significantly reduced (P < 0.01) bacterial titer in the lungs, liver, kidney, spleen and blood of experimental animals. Highly significant reduction in neutrophil infiltration and levels of pro-inflammatory and anti-inflammatory cytokines was also observed. This indicated an efficient capsule removal by the enzyme, that improved gentamicin efficacy in vivo. Although the enzyme was found to be immunogenic, but no significant reduction in treatment efficacy was observed in the preimmunized as well as naïve mice. In addition, as confirmed through flow cytometry, the hyperimmune sera raised against the enzyme did not neutralize its activity.

Conclusion

The results confirm that administration of enzyme ‘depolymerase’ along with gentamicin not only checked the virulence of K. pneumoniae in vivo but it also increased its susceptibility to gentamicin at a lower concentration. Such a strategy would help to avoid exposure to higher concentration of gentamicin. Moreover, since this decapsulating protein does not possess a lytic activity therefore there would be no chances of development of bacterial resistance against it. Therefore, it should be studied further for its successful inclusion in our prophylactic/therapeutic regimes.

【 授权许可】

   
2014 Bansal et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Stanier RY, Adelberg EA, Ingraham JL: Chapter 19. In General microbiology. 4th edition. London: MacMillan; 1977:593-604.
• [2]Lesinski GB, Westernick MA: Vaccines against polysaccharide antigens. Curr Drug Targets Infect Disord 2001, 1:325-334.
• [3]Knuf M, Kowalzik F, Kieninger D: Comparative effects of carrier proteins on vaccine- induced immune response. Vaccine 2011, 29:4881-4890.
• [4]Guerry P, Poly F, Riddle M, Maue AC, Chen YH, Monteiro MA: Campylobacter polysaccharide capsules: virulence and vaccines. Front Cell Infect Microbiol 2012, 15:2-7.
• [5]Cress BF, Englaender JA, He W, Kasper D, Linhardt RJ, Koffas MAG: Masquerading microbial pathogens: capsular polysaccharides mimic host-tissue molecules. FEMS Microbiol Rev 2014, 1-38.
• [6]Kuberan B, Linhardt R: Carbohydrate based vaccines. Curr Org Chem 2000, 4:653-677.
• [7]Wilson RP, Raffatellu M, Chessa D, Winter SE, Tükel C, Bäumler AJ: The Vi-capsule prevents Toll-like receptor 4 recognition of Salmonella. Cell Microbiol 2008, 10:876-890.
• [8]Thurlow LR, Thomas VC, Fleming SD, Hancock LE: Enterococcus faecalis capsular polysaccharide serotypes C and D and their contributions to host innate immune evasion. Infect Immun 2009, 77:5551-5557.
• [9]Merino S, Toma´s JM: Bacterial capsules and evasion of immune responses. In Encyclopedia of life sciences (ELS). Chichester: John Wiley & Sons, Ltd; 2010.
• [10]Llobet E, Tomas JM, Bengoechea JA: Capsule polysaccharide is a bacterial decoy for antimicrobial peptides. Microbiol 2008, 154:3877-3886.
• [11]Wu M-C, Lin T-L, Hsieh P-F, Yang H-C, Wang J-T: Isolation of genes involved in biofilm formation of a Klebsiella pneumoniae strain causing pyogenic liver abscess. PLoS One 2011, 6:1-11.
• [12]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.
• [13]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.
• [14]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, 8:70092.
• [15]Stewart PS, Costerton JW: Antibiotic resistance of bacteria in biofilms. Lancet 2001, 358:135-138.
• [16]Stewart PS: Theoretical aspects of antibiotic diffusion into microbial biofilms. Antimicrob Agents Chemother 1996, 40:2517-2522.
• [17]Drulis-Kawa Z, Majkowska-Skrobek G, Maciejewska B, Delattre AS, Lavigne R: Learning from bacteriophages-advantages and limitations of phage and phage-encoded protein applications. Curr Prot Pept Sci 2012, 13:699-722.
• [18]Andrews JM: Determination of minimum inhibitory concentrations. J Antimicrob Chemother 2001, 48:5-16.
• [19]Bansal S, Soni SK, Harjai K, 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.
• [20]Held TK, Mielke MEA, Chedid M, Unger M, Trautman M, Huhn D, Cross AS: Granulocyte colony-stimulating factor worsens the outcome of the experimental Klebsiella pneumoniae pneumonia through direct interaction with the bacteria. Blood 1998, 91:2525-2533.
• [21]Yadav V, Sharma S, Harjai K, Mohan H, Chhibber S: Induction and resolution of lobar pneumonia following intranasal instillation with Klebsiella pneumoniae in mice. Indian J Med Res 2003, 118:47-52.
• [22]Hampton MB, Winterbourn CC: Methods for quantifying phagocytosis and bacterial killing by human neutrophils. J Immunol Met 1999, 232:15-22.
• [23]Carpenter JL: Klebsiella pulmonary infections: occurrence at one medical center and review. Rev Infect Dis 1990, 12:672-682.
• [24]Yadav V, Sharma S, Harjai K, Mohan H, Chhibber S: Lipopolysaccharide-mediated protection against Klebsiella pneumoniae induced lobar pneumonia: intranasal vs intramuscular route of immunization. Folia microbial 2005, 50:83-86.
• [25]Evrard B, Balestrino D, Dosgilbert A, Bouya-Gachancard JLJ, Charbonnel N, Forestier C, Tridon A: Roles of Capsule and Lipopolysaccharide O Antigen in Interactions of Human Monocyte-Derived Dendritic Cells and Klebsiella pneumoniae. Infect Immun 2010, 78:210-219.
• [26]Kumarasamy KK, Toleman MA, Wals TR, Bagaria J, Butt F, Balakrishnan R, Chaudhary U, Doumith M, Giske CG, Irfan S, Krishnan P, Kumar AV, Maharjan S, Mushtaq S, Noorie T, Paterson DL, Pearson A, Perry C, Pike R, Rao B, Ray U, Sarma JB, Sharma M, Sheridan E, Thirunarayan MA, Turton J, Upadhyay S, Warner M, Welfare W, Livermore DM, Woodford N: Emergence of a new antibiotic resistance mechanism in India, Pakistan, and the UK: a molecular, biological, and epidemiological study. Lancet Infect Dis 2010, 10:597-602.
• [27]Wu G, Fan X, Li L, Wang H, Ding J, Hongbin W, Zhao R, Gou L, Shen Z, Xi T: Interaction of antimicrobial peptide S-thanatin with lipopolysaccharide in vitro and in an experimental mouse model of septic shock caused by a multidrug-resistant clinical isolate of Escherichia coli. Int J Antimicrob Agents 2010, 35:250-254.
• [28]Kabha K, Nissimov L, Athamna A, Keisari Y, Parolis H, Parolis LA, Grue RM, Schlepper-Schafer J, Ezekowitz AR, Ohman DE: Relationship among capsule structure. Infect Immun 1995, 63:847-852.
• [29]Hoffman LR, D’Argenio DA, MacCoss MJ, Zhang Z, Jones RA, Miller SI: Aminoglycoside antibiotics induce bacterial biofilm formation. Nature 2005, 436:1171-1175.
• [30]Durante-Mangoni E, Grammatikos A, Utili R, Falagas ME: Do we still need the aminoglycosides? Int J Antimicrob Agents 2009, 33:201-205.
• [31]Lavender HF, Jagnow JR, Clegg S: Biofilm formation in vitro and virulence in vivo of mutants of Klebsiella pneumoniae. Infect Immun 2004, 72:4888-4890.
• [32]Kristian SA, Birkenstock TA, Sauder U, Mack D, Götz F, Landmann R: Biofilm formation induces C3a release and protects staphylococcus epidermidis from IgG and complement deposition and from neutrophil-dependent killing. J Infect Dis 2008, 197:1028-1035.
• [33]Brown DT: Carbocysteine. Drug Intell Clin Pharm 1988, 22:603-607.
• [34]Merino S, Camprubi S, Alberti S, Benedi VJ, Tomas JM: Mechanisms of Klebsiella pneumoniae resistance to complement-mediated killing. Infect Immun 1992, 60:2529-2535.
• [35]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, 11:1503-1508.
• [36]Mushtaq N, Redpath MB, Luzio JP, Taylor PW: Treatment of experimental Escherichia coli infection with recombinant bacteriophage-derived capsule depolymerase. J Antimicrob Chemother 2005, 56:160-165.
• [37]Scorpio A, Chabot DJ, Day WA, O’Brien DK, Vietri NJ, Itoh Y, Mohamadzadeh M, Friedlander AM: Poly-ÿ-glutamate capsule-degrading enzyme treatment enhances phagocytosis and killing of encapsulated Bacillus anthracis. Antimicrob Agents Chemother 2007, 51:215-222.
• [38]Scorpio A, Tobery SA, Ribot WJ, Friedlander AM: Treatment of experimental anthrax with recombinant capsule depolymerase. Antimicrob Agents Chemother 2008, 13:1014-1020.
• [39]Scorpio A, Chabot DJ, Day WA, Hoover TA, Friedlander AM: Capsule depolymerase overexpression reduces Bacillus anthracis virulence. Microbiol 2010, 156:1459-1467.
• [40]Rashel M, Uchiyama J, Ujihara T, Uehara Y, Kuramoto S, Sugihara S, Muraoka A, Sugai M, Hiramatsu K, Honke K, Matsuzaki S: Efficient elimination of multidrug-resistant Staphylococcus aureus by cloned lysin derived from bacteriophage phi MR11. J Infect Dis 2007, 196:1237-1247.
• [41]Djurkovic S, Loeffler JM, Fischetti VA: Synergistic killing of Streptococcus pneumoniae with the bacteriophage lytic enzyme Cpl-1 and penicillin or gentamicin depends on the level of penicillin resistance. Antimicrob Agents Chemother 2005, 49:1225-1228.
• [42]Witzenrath M, Schmeck B, Doehn JM, Tschernig T, Zahlten J, Loeffler JM, Zemlin M, Müller H, Gutbier B, Schütte H, Hippenstiel S, Fischetti VA, Suttorp N, Rosseau S: Systemic use of the endolysin Cpl-1 rescues mice with fatal pneumococcal pneumonia. Crit Care Med 2009, 37:642-649.
• [43]Zelmer A, Martin MJ, Gundogdu O, Birchenough G, Lever R, Wren BW, Luzio JP, Taylor PW: Administration of capsule-selective endosialidase E minimizes upregulation of organ gene expression induced by experimental systemic infection with Escherichia coli K1. Microbiol 2010, 156:2205-2215.
• [44]Loessner MJ, Kramer K, Ebel F, Scherer S: C-terminal domains of Listeria monocytogenes bacteriophage murein hydrolases determine specific recognition and high-affinity binding to bacterial cell wall carbohydrates. Mol Microbiol 2002, 44:335-349.
• [45]Baquero F, Hsueh PR, Paterson DL, Rossi F, Bochicchio GV, Gallagher G, Lantz K, Villasenor JB, McCarroll K, Abramson MA, Chow JW: In Vitro susceptibilities of aerobic and facultatively anaerobic gram-negative bacilli isolated from patients with intra-abdominal infections worldwide: 2005 results from study for monitoring antimicrobial resistance trends (SMART). Surg Infect 2009, 10:99-104.