The present study was undertaken to explore the interaction of ciprofloxacin and chloramphenicol with bacterial membranes in a sensitive and in a resistant strains of
The plasmatic membrane is a chemoosmotic barrier that provides an interface between the organism and the environment. This bilayer presents an electrochemical potential (negative in the interior) which plays a basic role in the control of the exchange of solutes. Disturbances in the membrane potential can provide a rapid and sensitive indication of those stimuli that lead to physiological functionally important changes with respect to bacterial viability [
Fluorescent molecules have been extensively used as probes of biological membranes. These hydrophobic and amphiphilic probes are associated with membranes when added to cells or artificial systems, and their resultant fluorescence properties can be used to monitor a variety of membrane characteristics. In general, the addition of effectors results in the deenergization of cells, which leads to increased fluorescence from the probes present in the cell suspension, such as negatively charged 8-anilino-1-naphthalenesulfonate (ANS) [
ANS binding and fluorescence strongly respond to modulation of the surface potential, with the energy-dependent quenching being largely due to the generation of
It has been demonstrated that the determination of the membrane potential based on fluorochromes provides a useful and sensitive approximation for the monitoring of the cellular stresses in bacteria [
The effect of the oxidative stress generated by reactive oxygen species (ROS) has been described as one of the most important sources of metabolic disturbance and the cellular damage. These agents are involved in the first important changes in the plasmatic membrane, and consequently at the beginning of cellular death [
Bacterial gyrase inhibitors, including synthetic quinolone antibiotics, induce a breakdown in iron regulatory dynamics, which promotes the formation of the ROS that contribute to cell death [
Bactericidal antibiotic killing mechanisms are currently attributed to the class of specific drug-target interactions. However, the understanding of many of the bacterial responses that occur as a consequence of the primary drug-target interaction remains incomplete. It is known that oxidative stress in bacteria can be caused by exogenous agents that originate toxic effects, and our previous studies have shown that ciprofloxacin (CIP) and chloramphenicol (CMP), among others, can stimulate the induction of ROS in different bacterial species [
The aim of the present study was to explore the effects of clinically used antibiotics such as CIP and CMP on the lipid surface and to estimate the variation in the membrane potential in
The antimicrobial activities of CIP and CMP were evaluated in two strains, one standard strain
Overnight cultures of
The suspensions were centrifuged, and 1 mL of Triton 1% V/V in NaCl 10% was added to the pellet. Then, 20
The approach used to determine the dissociation constant (
The assays were carried out at least in triplicate. Data were expressed as mean ± SD and analyzed by the Student’s
Data were plotted as the reciprocal of the fluorescence signal (1/F) versus the reciprocal of the concentration of free ANS (1/ANS), as the reciprocal of the intercept gives the limit of the ANS fluorescence (
The values of bound ANS and free ANS were calculated from (
The surface potential (
The change in membrane potential (
The same procedure was performed to determine the number of binding sites for ANS when the bacterial suspensions were incubated with the antibiotics studied, where a decrease in the number of binding sites for ANS with antibiotic would suggest changes in the cell surface.
Any alteration in the binding of ANS to the membrane of
The fluorescence of ANS emission was determined at 516 nm in the presence of CIP and CMP and in the absence of antibiotic. The data were plotted as the reciprocal of the fluorescence signal (
Scatchard plots of ANS interaction with
Table
Parameters obtained from the ANS binding studies in
|
|
|
Ψ (mV) | ΔΨ (mV) | |
---|---|---|---|---|---|
| |||||
Control without antibiotic | 143 | 251 | 603028 | −306 | — |
Ciprofloxacin 256 |
70 | 927 | 52983 | −406 | −100 |
Chloramphenicol 4 |
62 | 846 | 48332 | −331 | −25 |
| |||||
Clinical strain | |||||
| |||||
Control without antibiotic | 77 | 902 | 45110 | −287 | — |
Ciprofloxacin 256 |
77 | 937 | 46865 | −302 | −15 |
Whereas the value of
The membrane potential is an important parameter that controls various cellular processes. It is a sensitive indicator of energy status and cell viability, with membrane depolarization leading to excessive production of ROS which is an indication of an advance in cellular dysfunction and precedes many other signs of cellular injury. A reduction in the potential also provides information about the feasibility of transferring an electron “
The changes in the electric potential obtained in the present work showed alterations in the bacterial membrane of
Montero et al. established that CIP interacts with neutral and charged membranes at the surface level (headgroup region). They also postulated that this could be part of the mechanism of entry of the 6-fluoroquinolones through the cytoplasmic membrane [
There are previous results on
The present results demonstrated that the strains had a particular behavior in the presence of each antibiotic, an effect that was manifested by the differences obtained in the bacterial membrane potential. Moreover, on comparing the sensitive strain with the resistant one, a higher alteration in the membrane potential was observed in sensitive bacterium that was associated with the effect of the antibiotic [
In our previous reports, we demonstrated that CIP and CMP induce oxidative stress in
Paulina L. Páez and María C. Becerra contribute equally to this work.
The authors thank Dr. Gerardo Argüello (Departamento de Físicoquímica, Facultad de Ciencias Químicas, UNC, INFIQC-CONICET) for assistance with the fluorescence measurements. The authors thank Consejo Nacional de Investigaciones Científicas y Técnicas de Argentina (CONICET), Secretaría de Ciencia y Técnica de la Universidad Nacional de Córdoba (SECyT) for financial support. We thank native English speaker Dr. Paul Hobson for revision of this manuscript. Paulina L. Páez and María C. Becerra are members of the Research Career of CONICET.