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Depolymerase improves gentamicin efficacy during Klebsiella pneumoniae induced murine infection.

Bansal S, Harjai K, Chhibber S - BMC Infect. Dis. (2014)

Bottom Line: Highly significant reduction in neutrophil infiltration and levels of pro-inflammatory and anti-inflammatory cytokines was also observed.In addition, as confirmed through flow cytometry, the hyperimmune sera raised against the enzyme did not neutralize its activity.Moreover, since this decapsulating protein does not possess a lytic activity therefore there would be no chances of development of bacterial resistance against it.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Panjab University, Sector-14, Chandigarh 160014, India. sanjaychhibber8@gmail.com.

ABSTRACT

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.

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Bacterial load (Log10CFU ml-1) in the lungs of mice infected via the intranasal route withK. pneumoniaeB5055 and treated with gentamicin (1.5 mg/kg) and/or depolymerase (50 μg). No sample was taken and processed on day 1 in the treated groups. G1, G2, G3, G4 represent Groups 1, 2, 3, 4. Arrows indicate significant reduction in Log bacterial count between groups on days 2, 3, 5, 7. Error bars represent standard deviation (S.D) from four independent values.
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Fig1: Bacterial load (Log10CFU ml-1) in the lungs of mice infected via the intranasal route withK. pneumoniaeB5055 and treated with gentamicin (1.5 mg/kg) and/or depolymerase (50 μg). No sample was taken and processed on day 1 in the treated groups. G1, G2, G3, G4 represent Groups 1, 2, 3, 4. Arrows indicate significant reduction in Log bacterial count between groups on days 2, 3, 5, 7. Error bars represent standard deviation (S.D) from four independent values.

Mentions: Intranasal instillation of bacteria in mice resulted in bacterial load of 4.2 logs, 24 h post infection (Figure 1, day 1). It was similar to the intranasal dose administered to mice. Thereafter, multiplication of bacteria in lungs resulted in a peak in bacterial count on day 3 (8.849 ± 1.321) followed by subsequent decrease (Figure 1). Intraperitoneal injection of gentamicin daily (initiated 24 h post infection) was not effective in this model of experimental pneumonia as bacterial counts were similar to that observed in untreated infected animals throughout the course of infection. When bacterial depolymerase was injected 24 h post infection, although bacteria were present in the lung tissue throughout the course of infection (Figure 1) but, an average reduction of ~2 logs (P < 0.01) was observed. Co-administration of gentamicin along with bacterial depolymerase resulted in significant reduction (P < 0.01) in average bacterial count (~3.4 logs) in comparison to the untreated or gentamicin treated animals.Figure 1


Depolymerase improves gentamicin efficacy during Klebsiella pneumoniae induced murine infection.

Bansal S, Harjai K, Chhibber S - BMC Infect. Dis. (2014)

Bacterial load (Log10CFU ml-1) in the lungs of mice infected via the intranasal route withK. pneumoniaeB5055 and treated with gentamicin (1.5 mg/kg) and/or depolymerase (50 μg). No sample was taken and processed on day 1 in the treated groups. G1, G2, G3, G4 represent Groups 1, 2, 3, 4. Arrows indicate significant reduction in Log bacterial count between groups on days 2, 3, 5, 7. Error bars represent standard deviation (S.D) from four independent values.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4150946&req=5

Fig1: Bacterial load (Log10CFU ml-1) in the lungs of mice infected via the intranasal route withK. pneumoniaeB5055 and treated with gentamicin (1.5 mg/kg) and/or depolymerase (50 μg). No sample was taken and processed on day 1 in the treated groups. G1, G2, G3, G4 represent Groups 1, 2, 3, 4. Arrows indicate significant reduction in Log bacterial count between groups on days 2, 3, 5, 7. Error bars represent standard deviation (S.D) from four independent values.
Mentions: Intranasal instillation of bacteria in mice resulted in bacterial load of 4.2 logs, 24 h post infection (Figure 1, day 1). It was similar to the intranasal dose administered to mice. Thereafter, multiplication of bacteria in lungs resulted in a peak in bacterial count on day 3 (8.849 ± 1.321) followed by subsequent decrease (Figure 1). Intraperitoneal injection of gentamicin daily (initiated 24 h post infection) was not effective in this model of experimental pneumonia as bacterial counts were similar to that observed in untreated infected animals throughout the course of infection. When bacterial depolymerase was injected 24 h post infection, although bacteria were present in the lung tissue throughout the course of infection (Figure 1) but, an average reduction of ~2 logs (P < 0.01) was observed. Co-administration of gentamicin along with bacterial depolymerase resulted in significant reduction (P < 0.01) in average bacterial count (~3.4 logs) in comparison to the untreated or gentamicin treated animals.Figure 1

Bottom Line: Highly significant reduction in neutrophil infiltration and levels of pro-inflammatory and anti-inflammatory cytokines was also observed.In addition, as confirmed through flow cytometry, the hyperimmune sera raised against the enzyme did not neutralize its activity.Moreover, since this decapsulating protein does not possess a lytic activity therefore there would be no chances of development of bacterial resistance against it.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Panjab University, Sector-14, Chandigarh 160014, India. sanjaychhibber8@gmail.com.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus