title: Aeromonas punctata derived depolymerase improves susceptibility of Klebsiella pneumoniae biofilm to gentamicin.
abstract: 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 Km value) and high turnover number (indicated by Kcat value) of the bacterial depolymerase (Km = 89.88 μM, Kcat = 285 s(-1)) over the phage derived one (Km = 150 μM, Kcat = 107 s(-1)).
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Microbiology, Panjab University, Sector-14, Chandigarh, 160014, India. shru.126@gmail.com.
authors.forename: Shruti
authors.initials: S
authors.lastname: Bansal
authors.affiliation: Department of Microbiology, Panjab University, Sector-14, Chandigarh, 160014, India. kusum_harjai@hotmail.com.
authors.forename: Kusum
authors.initials: K
authors.lastname: Harjai
authors.affiliation: Department of Microbiology, Panjab University, Sector-14, Chandigarh, 160014, India. sanjaychhibber8@gmail.com.
authors.forename: Sanjay
authors.initials: S
authors.lastname: Chhibber
mesh_terms: D000333:Aeromonas / Q000201:enzymology*; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D058491:Bacterial Load; D018441:Biofilms / Q000187:drug effects*; D005839:Gentamicins / Q000494:pharmacology*; D007700:Kinetics; D007711:Klebsiella pneumoniae / Q000187:drug effects* / Q000502:physiology; D037102:Lectins / Q000032:analysis; D018613:Microscopy, Confocal; D011133:Polysaccharide-Lyases / Q000378:metabolism*; D011135:Polysaccharides, Bacterial / Q000378:metabolism*; D011485:Protein Binding
chemical_list: D000900:Anti-Bacterial Agents; D005839:Gentamicins; D037102:Lectins; D011135:Polysaccharides, Bacterial; D011133:Polysaccharide-Lyases