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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) 24 h post infection in various organs and blood of mice infected via intraperitoneal route withK. pneumoniaeB5055 and treated with gentamicin (1.5 mg/kg) and/or depolymerase (50 μg). G1, G2, G3, G4 represent Groups 1, 2, 3, 4. Arrows indicate significant reduction in Log bacterial count in all organs and blood from mice belonging to different groups. Error bars represent standard deviation (S.D) from four independent values.
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Fig2: Bacterial load (Log10CFU/ml) 24 h post infection in various organs and blood of mice infected via intraperitoneal route withK. pneumoniaeB5055 and treated with gentamicin (1.5 mg/kg) and/or depolymerase (50 μg). G1, G2, G3, G4 represent Groups 1, 2, 3, 4. Arrows indicate significant reduction in Log bacterial count in all organs and blood from mice belonging to different groups. Error bars represent standard deviation (S.D) from four independent values.

Mentions: Intraperitoneal administration of 102 CFU/0.1 ml of K. pneumoniae in mice led to bacterial colonization of liver(s) (6.045 ± 0.325), lungs (5.778 ± 0.314), kidney(s) (6.491 ± 0.415), spleen(s) (6.934 ± 0.512) and bacteremia (6.011 ± 0.624) within 24 h (Figure 2). Owing to a massive spread of bacteria via systemic circulation, administration of gentamicin 6 h later did not provide any significant protection (P > 0.01). In contrast, intraperitoneal administration of a single 50 μg dose of bacterial depolymerase, 6 h post infection resulted in significant reductions (P < 0.01) of ~2.7 logs, ~1.5 logs, ~2.5 logs, ~ 2.94 logs and ~3.9 logs in bacterial counts in lungs, liver(s), kidney(s), spleen(s) and blood respectively (Figure 2). Simultaneous administration of gentamicin and depolymerase, led to almost complete eradication of bacteria, abolished bacteremia and prevented bacterial dissemination into various organs.Figure 2


Depolymerase improves gentamicin efficacy during Klebsiella pneumoniae induced murine infection.

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

Bacterial load (Log10CFU/ml) 24 h post infection in various organs and blood of mice infected via intraperitoneal route withK. pneumoniaeB5055 and treated with gentamicin (1.5 mg/kg) and/or depolymerase (50 μg). G1, G2, G3, G4 represent Groups 1, 2, 3, 4. Arrows indicate significant reduction in Log bacterial count in all organs and blood from mice belonging to different groups. 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

Fig2: Bacterial load (Log10CFU/ml) 24 h post infection in various organs and blood of mice infected via intraperitoneal route withK. pneumoniaeB5055 and treated with gentamicin (1.5 mg/kg) and/or depolymerase (50 μg). G1, G2, G3, G4 represent Groups 1, 2, 3, 4. Arrows indicate significant reduction in Log bacterial count in all organs and blood from mice belonging to different groups. Error bars represent standard deviation (S.D) from four independent values.
Mentions: Intraperitoneal administration of 102 CFU/0.1 ml of K. pneumoniae in mice led to bacterial colonization of liver(s) (6.045 ± 0.325), lungs (5.778 ± 0.314), kidney(s) (6.491 ± 0.415), spleen(s) (6.934 ± 0.512) and bacteremia (6.011 ± 0.624) within 24 h (Figure 2). Owing to a massive spread of bacteria via systemic circulation, administration of gentamicin 6 h later did not provide any significant protection (P > 0.01). In contrast, intraperitoneal administration of a single 50 μg dose of bacterial depolymerase, 6 h post infection resulted in significant reductions (P < 0.01) of ~2.7 logs, ~1.5 logs, ~2.5 logs, ~ 2.94 logs and ~3.9 logs in bacterial counts in lungs, liver(s), kidney(s), spleen(s) and blood respectively (Figure 2). Simultaneous administration of gentamicin and depolymerase, led to almost complete eradication of bacteria, abolished bacteremia and prevented bacterial dissemination into various organs.Figure 2

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