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

ABSTRACT: 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.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 ?g/ml), dispersal of CPS matrix by enzyme facilitated gentamicin penetration across biofilm. This resulted in significant reduction (p?

SUBMITTER: Bansal S

PROVIDER: S-EPMC4461996 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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

Bansal Shruti S Harjai Kusum K Chhibber Sanjay S

BMC microbiology 20150611

<h4>Background</h4>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.<h4>Results</h4>Intact biofilm of K. pneumoniae was recalcitrant to gentamicin due to lack of antibiotic penetration. ...[more]

PMID: 26063052

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Project description:BackgroundPresence 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.MethodsDepolymerase 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.ResultsIn 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.ConclusionThe 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.

Project description:The production of capsular polysaccharides by Klebsiella pneumoniae protects the bacterial cell from harmful environmental factors such as antimicrobial compounds and infection by bacteriophages (phages). To bypass this protective barrier, some phages encode polysaccharide-degrading enzymes referred to as depolymerases to provide access to cell surface receptors. Here, we characterized the phage RAD2, which infects K. pneumoniae strains that produce the widespread, hypervirulence-associated K2-type capsular polysaccharide. Using transposon-directed insertion sequencing, we have shown that the production of capsule is an absolute requirement for efficient RAD2 infection by serving as a first-stage receptor. We have identified the depolymerase responsible for recognition and degradation of the capsule, determined that the depolymerase forms globular appendages on the phage virion tail tip, and present the cryo-electron microscopy structure of the RAD2 capsule depolymerase at 2.7-Å resolution. A putative active site for the enzyme was identified, comprising clustered negatively charged residues that could facilitate the hydrolysis of target polysaccharides. Enzymatic assays coupled with mass spectrometric analyses of digested oligosaccharide products provided further mechanistic insight into the hydrolase activity of the enzyme, which, when incubated with K. pneumoniae, removes the capsule and sensitizes the cells to serum-induced killing. Overall, these findings expand our understanding of how phages target the Klebsiella capsule for infection, providing a framework for the use of depolymerases as antivirulence agents against this medically important pathogen. IMPORTANCE Klebsiella pneumoniae is a medically important pathogen that produces a thick protective capsule that is essential for pathogenicity. Phages are natural predators of bacteria, and many encode diverse "capsule depolymerases" which specifically degrade the capsule of their hosts, an exploitable trait for potential therapies. We have determined the first structure of a depolymerase that targets the clinically relevant K2 capsule and have identified its putative active site, providing hints to its mechanism of action. We also show that Klebsiella cells treated with a recombinant form of the depolymerase are stripped of capsule, inhibiting their ability to grow in the presence of serum, demonstrating the anti-infective potential of these robust and readily producible enzymes against encapsulated bacterial pathogens such as K. pneumoniae.

Dataset Information

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

Publications

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

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