Susceptibility of the tested
Bacterial cells grow and divide, replicating repeatedly to reach the large numbers present during an infection or on the surface of the body. To grow and divide, organism must synthesize or take up many types of surrounding biomolecules. Antibiotics interfere with the specific process that is essential for this growth and/or division of bacteria [
There were four major sites in bacterial cell that they serve as the basis for the action of antibiotics: the cell wall, ribosome, nucleic acids, and cell membrane [
On the other hand, the basic mechanisms by which microorganisms can resist antibiotics were (1) to alter the receptor for the drug (the molecule on which it exerted its effect) as fluoroquinolones, (2) to decrease the amount of drug that reached the receptor by altering entering or increasing removal of the drug as tetracyclines, (3) to destroy or inactivate the drug, (4) to develop resistant metabolic pathways, [
The relation between resistances of Gram-negative to different antibiotics, the outer membrane proteins, and lipids composition of these organisms was provided. Hydrophilic antibacterial agents were prevented from entering through the outer membrane by the lipopolysaccharide layer and the underlying phospholipids, whereas hydrophobic agents were excluded by outer membrane protein. Hypersusceptibility to antibiotics might occur when the lipopolysaccharide had been altered but also when the outer membrane proteins had remained constant [
On the other hand, exopolysaccharides were produced by many bacteria from clinical and environmental habitats [
Exopolysaccharides were believed to protect bacterial cells; EPSs matrix provided an effective barrier that restricted penetration of chemically reactive bioacids, cationic antibiotics, and antimicrobial agents; for that, these EPSs played an important role in bacterial resistance by imitating diffusion of antibiotics to cells [
EPSs produced by certain important bacteria such as
This research aimed to study the growth pattern of the tested
Two antibiotics were used: tetracycline TE capsule (500 mg) (Chemical Industry Development Co., Egypt) and ciprofloxacin CIP suspension (200 mg) (Amirya Pharmaceutical Industries, Egypt). Using liquid dilution method, different concentrations of each antibiotic were supplemented to 100 mL King’s B broth medium, pH 7 ± 0.2. Each treatment was inoculated with 1 mL of bacterial suspension for 24 hr and at age 0.05 McFarland. Cultures were incubated for 24 hr at 37°C. The growth was determined by measuring turbidity of liquid cultures at 600 nm using spectrophotometer. The minimum inhibitory concentration was determined in liquid cultures visually as the lowest concentration of antibiotic which prevent the visible growth [
The protein contents (as mg/mL) of both untreated and antibiotic treated liquid bacterial cultures were determined using the modified assay of Lowry et al. [
OMPs of both untreated and antibiotic treated (at sub-MIC) strains were extracted by the method [
Antibiotic treated (8.0 or 5.0 mg/100 mL TE and CIP, resp.) and untreated bacterial liquid cultures (500 mL) were centrifuged for 15 min at 5000 rpm, washed with distilled water, transferred to bottles with known weight, and then dried at 60°C up to constant weight. The dry weights were subjected to lipid analysis.
Total lipids of treated and untreated cells were extracted according to the method [
Total lipid contents of known weight of bacterial dry cells were extracted with mixture of chloroform/methanol (2 : 1 V/V) for 24 hr. The residues were reextracted again with chloroform/methanol mixture for 2 hr and evaporated under vacuum condition. This crude lipid residue was resuspended in chloroform and washed with sodium chloride solution (0.9%) in separating funnel, then dried by passing through solid anhydrous sodium sulfate, transferred to a known weight container, and evaporated at room temperature to get rid of the chloroform. Total lipids were calculated as % of dry weight. The total lipid extract was dissolved in known volume of chloroform (5 mL) and stored at −5°C in glass Stoppard volumetric flask.
Phospholipids were determined according to the method described by [
Analysis of fatty acids (as methyl esters) was conducted according to method [
Total exopolysaccharides of antibiotic treated (8.0 or 5.0 mg/100 mL TE and CIP resp.) and untreated
The obtained data were statistically analyzed to determine the means, standard deviation, and one sample
Resistance had been defined as the temporary or permanent ability of an organism and its progeny to remain viable and/or multiply under conditions that would destroy or inhibit other members of the strain. Resistance referred to instances where the basis of increased tolerance was a genetic change and where the biochemical basis was known. Antimicrobial substances target a range of cellular loci, from the cytoplasmic membrane to respiratory functions, enzymes, and the genetic material [
Through this research, resistance of the tested
Results in Table
Effect of increasing concentrations of TE and CIP on growth of the tested
Antibiotic conc. |
OD (600 nm) | |
---|---|---|
TE | CIP | |
0 | 0.67 ± 0.01 | 0.67 ± 0.01 |
1 | 0.64 ± 0.01 | 0.61 ± 0.01 |
2 | 0.59 ± 0.01 | 0.52 ± 0.01 |
3 | 0.51 ± 0.01 | 0.32 ± 0.01 |
4 | 0.40 ± 0.01 | 0.11 ± 0.01 |
5 | 0.33 ± 0.01 | 0.02 ± 0.01 |
6 | 0.23 ± 0.01 |
|
7 | 0.10 ± 0.01 | — |
8 | 0.03 ± 0.01 | — |
9 |
|
— |
|
10.023 | 6.286 |
|
<0.001*** | <0.001*** |
***more highly significant at <0.001.
In this connection, the resistance to tetracycline was normally due to the acquisition of new genes [
Antibiotics may have multiple sites of action in bacterial cell. Biochemical, cytological, molecular, biological, and genetic changes were induced as a result of antibiotic action. Through this research, changes in protein contents, OMPs pattern, lipid fractions, and EPSs of the tested
Results in Table
Effect of sub-MICs of TE and CIP on total cellular proteins and outer membrane proteins contents of the tested
Treatment | Total cellular proteins content (mg/mL) | OMPs content (mg/mL) |
---|---|---|
Untreated | 11.3 ± 0.10 | 5.60 ± 0.10 |
TE (8.0 mg/100 mL) | 8.20 ± 0.10 | 5.40 ± 0.10 |
CIP (5.0 mg/100 mL) | 7.10 ± 0.10 | 5.60 ± 0.10 |
|
14.088 | 125.484 |
|
<0.001*** | <0.001*** |
***more highly significant at <0.001.
From the previous experiment, it was found that it was very important to study the molecular characterization of OMPs for TE and CIP treated and untreated cells of the tested
Results in Table
Molecular weights and amount % of extracted outer membrane proteins of the tested
Treatment | Untreated | CIP (5.0 mg/100 mL) | TE (8.0 mg/100 mL) | M | ||||
---|---|---|---|---|---|---|---|---|
Lanes | Lane 1 | Lane 2 | Lane 3 | Lane 4 | ||||
Rows | Mol. wt. | Amount% | Mol. wt. | Amount% | Mol. wt. | Amount% | Mol. wt. | Amount% |
|
||||||||
r1 | ||||||||
r2 | 194.8 | 0.52076 | 194.8 | 1.7818 | ||||
r3 | 142.52 | 0.9757 | ||||||
r4 | 118.3 | 28.639 | 118.3 | 14.699 | 120 | 29.606 | ||
r5 | 86 | 0.79774 | ||||||
r6 | 78.95 | 8.3631 | ||||||
r7 | 71.75 | 7.3978 | 71.75 | 14.519 | 71.75 | 8.362 | ||
r8 | 54.8 | 17.071 | 54.8 | 1.3673 | 54.8 | 7.027 | ||
r9 | 47 | 0.6413 | ||||||
r10 | 40.663 | 0.56036 | 40.663 | 1.679 | ||||
r11 | 33.712 | 6.4954 | 34 | 20.721 | ||||
r12 | 31.721 | 20.327 | 31.72 | 20.264 | 31.721 | 7.3197 | ||
r13 | 30.135 | 6.2393 | ||||||
r14 | 28.635 | 32.422 | 28.635 | 20.573 | 28.635 | 30.652 | ||
r15 | 28 | 0.36329 | ||||||
r16 | 20.333 | 0.27476 | 20.333 | 8.2844 | 20.333 | 2.215 | 20 | 1.0261 |
|
||||||||
Sum | 79.03 | 94.168 | 94.8333 | 53.155 | ||||
In lane | 100 | 100 | 100 | 100 |
Outer membrane proteins banding pattern of antibiotics treated and untreated
Lipid profile is one of the most important characterizations in Gram-negative bacteria. Bacterial lipids were classified into nine groups: hydrocarbons, glycerides, waxes, steroids, phospholipids, glycolipids, lipopolysaccharides, peptidolipids, and peptidoglycolipids [
Antibiotics and different antibacterial compounds had an influence upon the lipid metabolism. Through this research the effect of both TE and CIP treatments on lipid profile and fatty acids was studied.
Results in Table
Effect of sub-MICs of TE and CIP on lipid fractions of the tested
Lipid fraction | Treatment | ||
---|---|---|---|
Untreated | TE (8.0 mg/100 mL) | CIP (5.0 mg/100 mL) | |
Total lipids (TL) (% of dry wt.) | 10.10 ± 0.10 | 12.70 ± 0.10 | 11.20 ± 0.10 |
Phospholipids (PL) (as % of TL) | 59.90 ± 0.10 | 62.30 ± 0.10 | 60.90 ± 0.10 |
Neutral lipids (NL) (as % of TL) | 40.09 ± 0.01 | 37.70 ± 0.10 | 39.10 ± 0.10 |
|
|||
|
5.070 | 5.247 | 5.154 |
|
<0.01** | <0.01** | <0.01** |
**highly significant at <0.01.
In this relation, it could be concluded that the resistant mutant of bacteria to antibiotics had more total lipids and phospholipids contents than sensitive one [
Regarding the fatty acids, results in Table
Effect of sub-MICs of TE and CIP on fatty acids percentages in the tested
Fatty acids (as % of TL) | Untreated | TE (8.0 mg/100 mL) | CIP (5.0 mg/100 mL) | |
---|---|---|---|---|
(1) Caproic | C6:0 | — | — | — |
(2) Caprylic | C8:0 | 2.33 | 3.27 | 3.57 |
(3) Capric | C10:0 | 1.96 | 10.14 | 2.47 |
(4) Lauric | C12:0 | 3.03 | 7.09 | 1.23 |
(5) Myristic | C14:0 | — | 4.49 | — |
(6) Palmitic | C16:0 | 2.70 | 6.77 | 1.03 |
(7) Stearic | C18:0 | 2.53 | 4.24 | 3.23 |
(8) Oleic | C18:1 | 19.17 | 19.03 | 16.68 |
(9) Linoleic | C18:2 | 24.69 | 12.87 | 23.47 |
(10) Linolenic | C18:3 | 13.77 | 11.66 | 14.57 |
(11) Arachidic | C20:0 | 9.24 | 8.99 | 15.56 |
(12) Eicosadienoic | C20:1 | 13.99 | 5.98 | 12.66 |
(13) Behenic | C20:0 | 6.56 | — | 5.63 |
|
||||
Unsaturation index | 2.50 | 1.1 | 2.06 |
Related observation was recorded by Hassanein [
It can be concluded that antibiotic treatment at certain antibiotic concentrations led to altering and/or misreading of genetic code for fatty acids and lipid biosynthesis which led to the disappearance of some fatty acids and induction of others [
Also, this research extended to study the production of EPSs by the tested
In the light of these important facts, this research was extended to study quantitative change in the EPSs produced by the tested
Effect of sub-MIC of TE and CIP on exopolysaccharides (EPSs) of the tested
The antibiotic treatment led to altering and/or misreading of genetic code for fatty acids and lipid biosynthesis and resulted in disappearance of some fatty acids and induction of others. The decrease in unsaturation index indicates transformation of lipids to solid state, making plasma membrane more rigid and lose its fluidity. Also, this research investigated the production of EPSs by the tested
The authors declare that there is no conflict of interests regarding the publication of this paper.
The authors would like to extend their sincere appreciation to the Deanship of Scientific Research at King Saud University for funding of this research through the Research Group Project no. RG-1435-016.