Related Experiment Videos
Gentamicin inactivation in purulent exudates: role of cell lysis
Abstract:
Factors contributing to the binding and reversible inactivation of gentamicin by purulent exudates were studied in a simplified in vitro model consisting of purified human polymorphonuclear leukocytes (PMNLs). Whereas intact PMNLs (10(6)-10(8)/ml) bound almost no [14C]gentamicin, freeze-thawed PMNLs showed extensive [14C]gentamicin binding, expressed as antibiotic cosedimenting with particulate material from the lysed PMNLs. Antibiotic binding could be related to the concentration of lysed PMNLs and to the amount of [14C]gentamicin added. Binding of [14C]gentamicin by lysed PMNLs was highly sensitive to DNase I but was unaffected by RNase, Triton X-100, or protease. Purified chromatin or DNA from either purulent exudates or lysed PMNLs reproduced the [14C]gentamicin-binding pattern obtained with crude PMNL lysate. These results show that gentamicin inactivation in purulent exudates can be correlated with binding of the antibiotic to lysed PMNLs; PMNL chromatin DNA is identified as one of the major binding factors.
Insights
Gentamicin is inactivated by binding to DNA released from lysed polymorphonuclear leukocytes (PMNLs) in purulent exudates. This binding explains how the antibiotic loses effectiveness in infection sites.
Area of Science:
- Microbiology
- Pharmacology
- Cell Biology
Background:
- Gentamicin is an aminoglycoside antibiotic crucial for treating bacterial infections.
- Purulent exudates, common in infections, can reduce gentamicin's efficacy.
- The precise mechanisms of gentamicin inactivation in exudates are not fully understood.
Purpose of the Study:
- To investigate the factors responsible for gentamicin binding and inactivation by purulent exudates.
- To elucidate the role of human polymorphonuclear leukocytes (PMNLs) in this process.
- To identify the specific components within PMNLs that bind gentamicin.
Main Methods:
- Utilized an in vitro model with purified human PMNLs.
- Compared gentamicin binding to intact versus freeze-thawed (lysed) PMNLs.
- Assessed the effect of DNase I, RNase, Triton X-100, and protease on binding.
- Investigated binding using purified PMNL chromatin and DNA.
Main Results:
- Intact PMNLs showed minimal gentamicin binding, while lysed PMNLs exhibited extensive binding.
- Gentamicin binding correlated with the concentration of lysed PMNLs and antibiotic added.
- Binding was highly sensitive to DNase I, indicating DNA's role, but unaffected by RNase, Triton X-100, or protease.
- Purified DNA and chromatin from PMNLs replicated the binding pattern.
Conclusions:
- Gentamicin inactivation in purulent exudates is linked to its binding by lysed PMNLs.
- PMNL chromatin DNA is a primary factor responsible for gentamicin binding and subsequent inactivation.
- Understanding this interaction is key to improving antibiotic efficacy in infections.